Masaryk University Brno
Faculty of Medicine
Department of Anatomy
NERVE INJURY INDUCED NEUROPATHIC PAIN AND
SPREAD OF INFLAMMATORY RESPONSE TO REMOTE
STRUCTURES OF THE NERVOUS SYSTEM
Habilitation Thesis
(Collection of Articles)
MUDr. Marek Joukal, Ph.D.
2021
Acknowledgments
I would like to thank to all my co-authors and colleagues who guided my research career.
Especially, I would like to thank prof. RNDr. Petr Dubový, CSc. for mentorship and support in
my research activities. Most of all, I would like to thank my family for their support, love and
patience.
Table of contents
Commentary ...............................................................................................................................7
1. Introduction and aims.............................................................................................................8
2. Nociceptive pathway ..............................................................................................................9
2.1. Peripheral nociceptive receptors......................................................................................9
2.2. The primary nociceptive neurons ....................................................................................9
2.3. Nociceptive afferentation in the spinal cord..................................................................10
2.4. Tertiary nociceptive neurons .........................................................................................11
2.5. Cortical projections of nociceptive pathway .................................................................12
2.6. Descending modulatory pathways.................................................................................13
3. Peripheral neuropathic pain models .....................................................................................15
3.1. Experimental peripheral nerve injury............................................................................16
3.1.1. Sciatic nerve transection.........................................................................................16
3.1.2. Chronic constriction injury.....................................................................................16
3.1.3. Partial sciatic nerve ligation ...................................................................................17
3.1.4. Spared nerve injury.................................................................................................18
3.1.5. Segmental L5/L6 spinal nerve ligation...................................................................19
3.2. Experimental spinal nerve root injury ...........................................................................19
3.2.1 Dorsal root compression..........................................................................................19
3.2.2. Dorsal rhizotomy....................................................................................................20
3.2.3. Ventral rhizotomy...................................................................................................20
4. Wallerian degeneration.........................................................................................................22
4.1. Axon degeneration.........................................................................................................22
4.2. Wallerian degeneration as sterile inflammatory reaction..............................................23
5. Reaction of the DRG to peripheral nerve injury...................................................................24
5.1. Cellular reactions in DRG induced by peripheral nerve injury.....................................24
5.2. Molecular reactions in DRG induced by peripheral nerve injury .................................25
5.2.1. Cytokines and chemokines involved in neuropathic pain development and
maintenance......................................................................................................................25
5.2.2. Nerve injury-induced regeneration state in the DRG.............................................27
6. Reaction of the spinal cord and brain stem to nerve injury..................................................29
6.1. Inflammatory reactions in the spinal cord of neuropathic pain models ........................29
6.2. Pain processing and modulation in the brain stem ........................................................33
6.2.1. The gracile and cuneate nucleus.............................................................................33
6.2.2. Rostral ventromedial medulla.................................................................................33
6.2.3. Pons ........................................................................................................................35
6.2.4. Mesencephalon.......................................................................................................35
7. Diencephalon and its reaction to nerve injury......................................................................37
7.1. Thalamus .......................................................................................................................37
7.2. Hypothalamus................................................................................................................37
8. Pain processing and modulation in the telencephalon..........................................................38
8.1. Basal ganglia .................................................................................................................38
8.2. Anterior cingulate cortex...............................................................................................39
8.3. Ventrolateral orbitofrontal cortex..................................................................................39
8.4. Insular cortex.................................................................................................................40
8.5. Somatosensory cortex....................................................................................................41
9. Barriers of the nervous system, their alteration and contribution to spreading of
inflammatory reaction after nerve injury..................................................................................42
9.1. The blood-nerve barrier.................................................................................................42
9.2. The blood-DRG barrier .................................................................................................43
9.3. The cerebrospinal fluid-DRG barrier ............................................................................44
9.4. Blood-brain and blood-spinal cord barrier ....................................................................47
9.5. Blood-cerebrospinal fluid barrier ..................................................................................50
10. Conclusions and future perspectives ..................................................................................53
11. References ..........................................................................................................................55
12. Commented overview of selected publications..................................................................77
13. Full list of author´s publications in impacted journals…………………………………..178
List of abbreviations
CAP-23 Cytoskeleton-Associated Protein 23
CNS Central Nervous System
CSF Cerebrospinal Fluid
DRG Dorsal Root Ganglia
FE FluoroEmerald
GABA Gamma-Aminobutyric Acid
GAP-43 Growth-Associated Protein 43
GFAP Glial Fibrillary Acidic Protein
Iba1 Ionized Calcium Binding Adapter Molecule 1
IL Interleukin
NGF Nerve Growth Factor
RVM Rostral Ventromedial Medulla
SCG-10 Superior Cervical Ganglion 10
SPRR-1A Small Proline-Rich Repeat Protein 1A
TLR Toll-Like Receptor
TNF-α Tumor Necrosis Factor α
ZO Zonulin
7
Commentary
Peripheral nerve injury may result in neuropathic pain represented by spontaneous allodynia
and thermal hyperalgesia. It has been found that unilateral nerve injury induces cellular and
molecular changes not only in the structures anatomically related to the injured nerve but also
in structures of the nervous system unrelated to the site of injury. The Cellular and Molecular
Research Group at the Department of Anatomy, which I am a member of, was the first who
gave an evidence that sciatic nerve injury induces cellular and molecular changes in remote
cervical dorsal root ganglia (DRG). These changes correspond with induction of proregenerative
state in the remote DRG. Moreover, we found that descending modulation of pain
perception is mediated via CCL2/CCR2 signaling pathway in the periaqueductal gray and the
rostral ventromedial medulla. Therefore, there is compelling evidence indicating that
neuroinflammatory response after nerve injury may spread to structures of the nervous system
not only trans-synaptically via the sensory pathway but also through the nervous system
barriers. In the field of the nervous system barriers, our experiments revealed that subarachnoid
space filled with cerebrospinal fluid directly communicates with the DRG giving anatomical
background for spread of inflammatory molecules to remote structures of nervous system via
cerebrospinal fluid. In addition, we found that peripheral nerve injury induces immune reaction
in the choroid plexus, where it localizes the blood-cerebrospinal fluid barrier, indicating its
possible alteration after nerve injury. Presented habilitation thesis is written as a collection of
published articles with a commentary introducing the topic and describing current state of
knowledge.
8
1. Introduction and aims
Neuropathic pain is defined as a chronic pain condition that occurs and persists in a
heterogeneous group of etiologically different diseases characterized by primary lesions or
dysfunction of the peripheral or central nervous system 1
. The World Health Organization has
estimated that 22% of the world’s primary care patients have chronic debilitating pain making
chronic pain a problem to be addressed by all physicians and health professionals 2
.
Peripheral neuropathic pain is manifested by spontaneous pain, hyperalgesia and allodynia,
which arises as a result of various types of nerve damage, e.g., diabetic neuropathy, HIV
neuropathy, post-herpetic neuralgia, drug-induced neuropathy, and traumatic nerve injury 3,4
.
It is generally accepted that the unilateral injury of the peripheral nerve causes a wide range of
inflammatory reactions alongside the neuroaxis. These reactions are not only restricted to the
structures that are anatomically related to the injured sciatic nerve but also remote structures
such as cervical dorsal root ganglia (DRG) 5,6
or rostral ventromedial medulla and
periaqueductal gray 7
. These changes might be caused by spread of the pro-inflammatory
chemokines and cytokines via blood circulation and/or cerebrospinal fluid 8,9
.
The goal of presented habilitation thesis is to provide an overview of current knowledge about
the nerve injury-induced neuropathic pain and spread of the inflammatory response to the
central nervous system (CNS) and to summarize results of my experimental work in this field.
Details about design of experiments and used methods can be found in attached publications
(Chapter 12).
9
2. Nociceptive pathway
The nociceptive pathway begins on the peripheral nociceptive receptors and contains three
neurons; the primary neuron is located in the DRG, the secondary neuron in the spinal cord and
the tertiary neuron in the thalamus. The nociceptive pathway terminates in the sensory cerebral
cortex.
2.1. Peripheral nociceptive receptors
The sensation of pain begins on high-threshold receptors (nociceptors) as a reaction on tissue
damage. Nociceptors are free nerve endings different with regard to stimulus specificity. Some
nociceptors react on intense mechanical stimulus (mechanical nociceptors), others respond to
extreme heat or coldness (cold and heat nociceptors) and some nociceptors respond to chemical
stimuli, e.g. inflammatory state. However, many nociceptors are polymodal, responding to
several kinds of stimuli. Nociceptive fibers are predominantly thinly myelinated and
unmyelinated (Aδ and C fibers) with conduction velocity 5 – 30 m/s for Aδ fibers and 0.4 – 1.4
m/s for C fibers 10,11
.
2.2. The primary nociceptive neurons
The primary sensory neuron of the nociceptive pathway has its cell bodies in the DRG. Since
the bodies of primary neurons are pseudounipolar, they send their peripheral branches to the
peripheral nerves and central branches to the spinal cord forming the dorsal root. The axons of
different kinds of receptors are intermingled in peripheral nerves and the dorsal root, while they
are grouped according to their thickness as soon as they enter the spinal cord. Heavily
myelinated Aα and Aβ fibers enter the spinal cord medially while thinly myelinated Aδ fibers
and unmyelinated C fibers laterally 12
.
10
2.3. Nociceptive afferentation in the spinal cord
Afferent fibers reach the spinal cord and form the Lissauer tract to synapse with the secondorder
neurons distributed alongside the dorsal horn of spinal cord. The nociceptive neurons
respond to specific noxious stimuli and they are found in laminae I, II, V and VI. Inputs for
these neurons are high threshold Aδ nociceptive fibers and polymodal C nociceptive fibers with
somatotopic organization, mainly in lamina I 13–15
.
Axons of the secondary nociceptive neurons pass through the anterolateral funiculus and
posterior funiculus. These afferent bundles transmit nociceptive stimuli to the structures of the
brain stem and diencephalon 14,16
.
The bodies of neurons sending their axons to form the spinothalamic tract are localized mainly
in lamina I and V of the spinal cord. In addition, scattered neurons are also found more ventrally
in laminae VII and VIII 12,17,18
. Most of the neurons project to the contralateral thalamus,
although a small group of cells projects ipsilaterally. Based on the origin and the projection, the
spinothalamic tract can be divided into three bundles. The first is monosynaptic
neospinothalamic pathway or ventral spinothalamic tract directly projected to the lateral part of
the thalamus that carries fibers involved in the sensory-discriminative component of pain. The
second, dorsal spinothalamic tract, is multisynaptic paleospinothalamic pathway that terminates
on intralaminar and dorsal nuclei of the thalamus involved in the motivational-affective aspects
of pain. The third tract is projected directly to the medial central nucleus of the thalamus that is
responsible for affective component of pain 14,16
.
The spinoreticular tract starts on neurons localized in the deep layers of the dorsal horn of spinal
cord as well as in laminae VII and VIII of the ventral horn 16,19
. The tract has two components,
the first one projects to the precerebellar nucleus involved in motor control. The other
component is involved in nociception and projects to the medial pontobulbar reticular
11
formation. The pontocerebellar tract is believed to play a role in motivational-affective
characteristics and neurovegetative responses to pain 13,14
.
The spinomesencephalic tract originates in lamina I, IV and V as well as the ventral horn and
lamina X. Spinomesencephalic projections run to several areas of the midbrain including
nucleus cuneiformis, intercollicular nucleus, deep layers of the superior colliculus, nucleus of
Darkschewitsch, pretectal nuclei, red nucleus, Edinger-Westphal nucleus, interstitial nucleus of
Cajal. Moreover, the spinomesencephalic tract also terminates in periaqueductal gray that plays
important role in descending modulatory system for attenuation of pain 14,16
.
2.4. Tertiary nociceptive neurons
Tertiary nociceptive neurons are found in the thalamus that is the main relay station for
ascending tracts directed to the cortex. The thalamus receives fibers involved in perception,
integration, and transfer of the pain, mainly via the spinothalamic tract that projects to both
medial and lateral nuclear complex. Many spinothalamic fibers terminate in the ventral
posterolateral nucleus that is somatotopically organized. The ventral posterolateral nucleus has
interconnections with the primary sensory cortex responsible for localization of the pain and its
intensity.
The spinothalamic tract has also terminations on the neurons of the ventral posteromedial
nucleus that send axons to the prefrontal cortex. Since the ventral posteromedial nucleus
receives fibers from the parabrachial nucleus and has interconnections with the amygdala,
hypothalamus and periaqueductal gray, it is believed to be involved in emotional aspects and
vegetative response to the pain. In addition, the spinothalamic fibers end on the posterior
nucleus, the intralaminar nuclei (e.g. central lateral nucleus) and mediodorsal nucleus 12,14
.
12
2.5. Cortical projections of nociceptive pathway
The painful stimuli activate multiple cortical areas, namely primary sensory cortex, secondary
sensory cortex, insula, anterior cingulate cortex and prefrontal cortex. The afferent fibers from
the thalamus form the medial and lateral system of the nociceptive pathway based on the
projection sites from medial and lateral thalamic nuclear complexes (Fig. 1). Although the
division of the afferent nociceptive pathway to the lateral and medial system is oversimplified,
it is useful because of grouping of the cerebral regions that seem to have similar role in pain
perception. The lateral system is believed to play a role in perception of the location and
intensity of the pain, while the medial system in affective component of the pain 20–22
.
Figure 1. The cortical projections of nociceptive pathways 22
. VPL – ventral posterior lateral
nucleus; VPM – ventral posterior medial nucleus; VPI – ventral posterior inferior nucleus;
VMpo – posterior part of ventromedial nucleus; MDvc – ventrocaudal part of medial dorsal
nucleus; Pf – parafascicular nucleus; CL – centrolateral nucleus; SI – primary somatosensory
cortex; SII – secondary somatosensory cortex; ACC – anterior cingulate cortex.
13
2.6. Descending modulatory pathways
Pain is modulated by descending modulatory circuit that receives inputs from several areas of
the central nervous system, e.g. the hypothalamus, cingulate cortex or amygdala. However, the
periaqueductal gray in the midbrain plays a pivotal role in nociceptive modulation. The
periaqueductal gray has columnar functional organization with ventrolateral and dorsolateral
columns. It has been found that stimulation of the ventrolateral column evokes active defense
behavior and opioid-dependent analgesia (abolished by injection of naloxone). In contrast,
stimulation of the dorsolateral column causes freezing reaction and opioid-independent
analgesia 23
. Axons of the periaqueductal gray have their synapses in the rostral ventromedial
medulla on neurons of the nucleus raphe magnus and the nucleus reticularis gigantocellularis.
These neurons have connections with the spinal cord forming the raphespinal tract that directly
or indirectly modulates nociceptive afferentation 12,24
(Fig. 2).
14
Figure 2. Scheme of the nociceptive pathway on the right (purple) and descending modulatory
pathways on the left. Red neurons induce inhibition, while green mediates facilitation of
nociception. Modified from Brodal 12
.
15
3. Peripheral neuropathic pain models
Peripheral nerve injury frequently results in development of the neuropathic pain that may differ
depending upon the type of nerve damage and the individual. Our knowledge of cellular and
molecular events in the nervous system following peripheral nerve injury is based on various
types of experimental nerve lesions in animal models.
Peripheral nerve injury causes cellular and molecular changes in primary afferent neurons, their
peripheral and central branches. Severity and form of molecular and cellular changes depend
on many factors including type and lesion position. Generally, reaction of neurons in
corresponding DRG is more intensive when the nerve damage is closer to the neuronal
perikaryon and prognosis of recovery is much poorer in comparison to lesions far from the
perikaryon 25,26
. Total axotomy produces more severe reaction than peripheral nerve ligation
when continuity of the axons is not disrupted 27
. In addition, age of the organism plays an
important role in reaction to the injury. Experimental studies revealed that peripheral nerve
injury in young animals causes more intensive reaction of the nervous system than in older
animals 28,29
.
Numerous experimental models were developed to study neuropathic pain induction as a
consequence of molecular and cellular changes in the nervous system. In general, the
experimental models can be divided according to position of the lesion (spinal nerve roots,
DRG, spinal nerve or peripheral nerve) and the type of lesion (transection, compression by
loose or tight ligation, cryoneurolysis, stimulation of perineuronal inflammation, tumor cells
invasion or laser radiation) 30
.
Because of favorable anatomical organization, majority of experimental models are undertaken
on the hind limb of rodents, which is innervated from L3 – L6 spinal segments 31,32
.
16
3.1. Experimental peripheral nerve injury
The sciatic nerve is the most commonly used peripheral nerve for the nerve lesions having the
maximum amount of neuronal perikarya in rat DRG of the L4 and L5 segments 33
.
3.1.1. Sciatic nerve transection
The model of sciatic nerve transection was described by Wall with colleagues 34
to mimic the
“phantom pain” well known in patients undergoing the amputation or transverse spinal lesion.
Transection of the sciatic nerve is performed in the middle of thigh. The proximal stump is
either ligated or inserted into the polyethylene tube or a 5-8 mm long segment of the nerve is
removed to prevent reinnervation. The axotomy causes immediate loss of both sensory and
motor innervation in area innervated by the sciatic nerve. The whole distal stump undergoes
molecular and cellular changes well known as the Wallerian degeneration. Since the axotomy
results in blockade of retrograde transport and interruption of the blood-nerve barrier, the
Wallerian degeneration products (cytokines, chemokines and cellular debris) are easily
transported via blood circulation. The disadvantage of this model is impossibility of behavioral
testing including allodynia and hyperalgesia in denervated limb. In addition, deafferentation of
the limb results in “self-attack”, the autotomy. It is questionable if this phenomenon is a sign of
spontaneous pain 35
or a response to total denervation of the hind paw.
3.1.2. Chronic constriction injury
Bennet and Xie 36
for the first time described the chronic constriction injury. In its original
form, four tight ligatures of chromic gut (4-0) were placed on the sciatic nerve in the middle of
thigh. The chromic gut causes local inflammatory response and subsequent nerve edema
inducing an increased compression of axons. Nerve inflammation caused by the chromic gut is
17
accompanied by immune reaction 37
with invasion of immune cells 38
. Therefore, the chronic
constriction injury using the chromic gut is not suitable to distinguish between inflammatory
reaction induced by the chromic gut and that produced during Wallerian degeneration 30,39
.
Nevertheless, use of sterilized material (e.g. Prolen) to prevent inflammatory response and
edema of the nerve has disadvantage in non-uniform amount of injured and uninjured axons.
To prevent this disadvantage, it is possible to use polyethylene “cuff” with standardized
diameter placed on the sciatic nerve. Tissue reaction between the nerve and cuff can be
minimalized using biocompatible material 40
.
Chronic constriction injury causes loss of both sensory and motor fibers. Considerable loss of
heavily myelinated Aα and Aβ axons and smaller amount of Aδ and C fibers with partial
deafferentation of the hind limb was demonstrated following the sciatic nerve ligation 41,42
. The
DRG associated with the nerve ligation contain a mixture of neurons with injured and uninjured
axons. The uninjured axons distal to the ligation are exposed to the Wallerian degeneration
products (cytokines, chemokines) from injured fibers. These molecules can be retrogradely
transported by uninjured axons to corresponding DRG neurons. Therefore, the nerve ligation is
a suitable model for studying contribution of the Wallerian degeneration to neuropathic pain
induction.
3.1.3. Partial sciatic nerve ligation
Partial sciatic nerve ligation is also known as “Seltzer´s ligation” 43
that mimic nerve contusion.
Partial sciatic nerve ligation is performed using curved needle and silk thread (8-0) at the midthigh
with the aim to affect approximately one-third to one-half of nerve fibers. In comparison
to the chronic constriction injury, majority of heavily myelinated axons remain unaffected and
contribute to development of mechanical allodynia. Partial nerve ligation induces lower
18
inflammatory component than the chronic constriction injury model with chromic gut.
However, variability of number of affected axons, inconsistent manner of damaged endoneurial
cells by needle penetration, and random mixture of L4-L5 spinal nerve afferent injuries are
considered to be disadvantageous in this model.
3.1.4. Spared nerve injury
Spared nerve injury is a model of partial limb denervation with minimal variability of nerve
damage. Rat´s hind limb plantar area is innervated by three branches of the sciatic nerve, the
common fibular, the tibial and the sural nerves 44
. Classical spared nerve injury model described
by Decosterd and Woolf 45
in rats was based on transection of the tibial and the common fibular
nerves while sural nerve was left intact. In mice, the dynamics of mechanoallodynia and thermal
hyperalgesia after spared sural nerve does not correlate with those seen in rats. Therefore,
variations of spared nerve injury when the common peroneal or the tibial nerve is left intact
were studied 46
. Reinnervation of axotomized nerves was prevented by tight ligature of the
proximal stump and removal of 2-4 mm segment of appropriate nerve. In contrast to
aforementioned models of peripheral nerve injury, spared nerve injury model produces the most
constant degree of axonal damage. In addition, the direct contact of injured and uninjured axons
is avoided in peripheral nerve. However, there is considerable co-mingling of injured and
uninjured neurons in corresponding DRG. This situation allows studies of potential paracrine
signaling in DRG and its contribution to neuropathic pain development. It is important to note
that even only two branches out of three are axotomized, there are changes in all neurons in
DRG associated with the sciatic nerve. Advantage of this model is possibility of behavioral
testing in the skin region innervated by non-affected branch of the sciatic nerve.
19
3.1.5. Segmental L5/L6 spinal nerve ligation
The spinal nerve ligature is performed using tight (3-0) ligature distally to corresponding DRG
on the L5-L6 spinal nerves while leaving the L4 spinal nerve intact 47
. This model is based on
experimental paradigm that there is a population of axotomized and intact neurons in one DRG.
Since the L4 spinal nerve is intact, it is possible to investigate changes in sensitivity in
corresponding plantar dermatomes.
Although spinal nerve ligation produces uniform changes in associated DRG, an extensive
surgical approach to vertebral foramina and tissue damage near intact L4 spinal nerve are
disadvantageous in this model. Therefore, evaluation of molecular changes, including
inflammatory, is very problematic.
3.2. Experimental spinal nerve root injury
In contrast to peripheral nerve injury models, experimental models of spinal root injury are
more difficult to achieve. Overall range of surgical approach is the biggest limitation. Before
the spinal root lesion, it is necessary to retract the paravertebral muscles, remove the transverse
processes, provide hemilaminectomy and open the dural sac.
3.2.1 Dorsal root compression
The dorsal root of spinal nerve contains primary central arms of afferent fibers. Therefore, there
is no motor defect in case of the dorsal root injury. Model of the dorsal root compression
simulates the compressive radiculopathy. Original model was described by Tabo with
colleagues 48
where the silk ligature (7-0) was placed on the dorsal roots of L4-L6 spinal
segments. Corresponding DRG contain populations of affected and unaffected primary sensory
20
neurons. Some of the fibers proximal to the lesion undergo Wallerian degeneration, while a
mixture of injured and uninjured axons are found distal to the lesion.
There were developed alternative methods of the dorsal root compression to guarantee constant
force on the axons. The dorsal root can be compressed using the steel bar inserted to the
intervertebral foramen 49
or tube containing material that can expand following fluid absorption
50
. Temporal cellular and molecular changes during compressive radiculopathy and subsequent
decompression can be studied using combination of epidural catheter and nylon rod 51
.
3.2.2. Dorsal rhizotomy
Dorsal rhizotomy mimics an avulsion of the dorsal root nerve out of the spinal cord frequently
caused by traction mechanism. The model is based on complete transection of the dorsal root
of spinal nerve between the cervical/thoracic or lumbar/sacral segments 52
that affects all
primary sensory neurons in corresponding DRG. Moreover, there is deafferentation to
associated segments of the spinal cord resulting in various degrees of self-mutilation in
experimental animals. Although complete transection of the dorsal root causes deafferentation
of the spinal segments, allodynia and hyperalgesia are present in experimental animals in
similar degree to the dorsal root compression model 53
.
3.2.3. Ventral rhizotomy
Ventral rhizotomy is the first model of neuropathic pain where afferent sensory fibers remain
intact. The model is based on complete transection of the ventral root of spinal nerve. Motor
axons distal to the lesion undergo Wallerian degeneration and generate cellular and molecular
changes that affect intact afferent fibers. Ventral rhizotomy causes morphological and
21
metabolic changes in corresponding DRG represented by invasion of macrophages and
increased expression of tumor necrosis factor α (TNF-α) 54,55
.
Figure 3. Schematic drawing of the peripheral neuropathic pain models. 1 – Sciatic nerve
transection; 2 – Chronic constriction injury; 3 – Partial sciatic nerve ligation; 4 – Spared nerve
injury; 5 – Segmental L5/L6 spinal nerve ligation; 6 – Dorsal root compression; 7 – Dorsal
rhizotomy; 8 – Ventral rhizotomy. Modified from Klusáková and Dubový 30
.
22
4. Wallerian degeneration
In 1850, Dr. Augustus Waller described degeneration of the cranial nerves after injury in frog
56
. Therefore, cellular and molecular changes after peripheral nerve injury are well known as
Wallerian degeneration. These events form convenient environment for regeneration of axons
and thus provide an important precondition for reinnervation.
Injury of the nerve and axons causes cascade of events including proliferation of Schwann cells
57
, invasion of circulating macrophages 58
, alteration of the blood-nerve barrier 59
, changes in
the endoneurial extracellular matrix 60
and elevation of cytokine production 61
.
4.1. Axon degeneration
The axotomy induces degeneration of axoplasm, axolemma and myelin distal to nerve lesion.
Degeneration of axon is an active event independent of non-neuronal cells surrounding the
axons 62
. Degeneration of the axon is possibly triggered by impaired axonal transport 63
.
Increased free intracellular Ca2+
and activation of calpains lead to decrease of microtubular and
neurofilament protein levels 64
. These events result in axonal fragmentation in 48 hours 65
.
Schwann cells play crucial role in initiation of the Wallerian degeneration. Fragments of the
myelin sheath appear within Schwann cell cytoplasm as ovoids and small whorls of myelin
debris and the Schwann cell displayed numerous lipid droplets. Then, the myelin debris is
phagocytized by the recruited macrophages 66,67
.
Circulating macrophages infiltrate epineurium of the lesion site 3 days after injury, while
endoneurium is infiltrated 5 to 7 days after injury 68
. Then, the macrophages spread to entire
distal stump of injured nerve and reach maximum number 14 days after injury 69
. Besides
myelin phagocytosis, macrophages secrete mitogenic factors for Schwann cells and fibroblasts
58
.
23
Besides the macrophages, Wallerian degeneration is accompanied by invasion of mast cells,
neutrophils and lymphocytes to the site of nerve lesion. Mast cells degranulate within the
endoneurium of the injured nerve and thus contribute to recruitment of macrophages and
neutrophils 70
. Moreover, they participate in initiation of inflammatory reaction and
development of hyperalgesia 71
. Neutrophils are responsible for production of a wide range of
inflammatory cytokines and chemokines in injured nerve. They are attracted by factors of
degranulated resident mast cells 72
.
4.2. Wallerian degeneration as sterile inflammatory reaction
Tissue injury leads to inflammatory reaction that is characterized by increased expression of
immune mediators responsible for wound healing 73
. Nerve injury induces not only production
of neurotrophic factors but also induction of inflammatory response accompanied by production
of cytokines, chemokines and transcription factors 74
. Besides immune cells, activated glial cells
contribute to secretion of cytokines and chemokines in nervous system 61
. The first cytokines
secreted after nerve injury are TNF-α, interleukin 1α (IL-1α) and IL-1β 75
because they play
crucial role in macrophage recruitment to the site of nerve injury 76
. Cytokines produced during
Wallerian degeneration have mainly negative effect on neuropathic pain induction 77,78
.
However, in vitro experiments revealed that some cytokines like TNFα, IL-1β, IL-4 and IL-6
have a synergic effect with neurotrophic factors to induction of axon outgrowth 79
.
24
5. Reaction of the DRG to peripheral nerve injury
Peripheral nerve injury induces cellular and molecular changes in the DRG that contribute to
induction and maintenance of neuropathic pain 80
. The DRG contain bodies of primary sensory
neurons that are surrounded with satellite glial cells. Since the primary sensory neurons are
pseudounipolar, they send off peripheral and central branches. The peripheral branches are
localized within peripheral nerves and reach peripheral terminals in target tissue, while the
central branches run in the dorsal roots to the CNS.
5.1. Cellular reactions in DRG induced by peripheral nerve injury
The cellular changes induced by peripheral nerve injury are related to proliferation and
activation of the satellite glial cells 81,82
as well as invasion of macrophages and lymphocytes
to corresponding DRG 83
. Primary stimuli inducing invasion of macrophages to ipsilateral DRG
seems to be triggered by retrograde signaling from injured nerve 84
. Injury-induced invasion of
macrophages is probably triggered also by release of CX3CL1 (fractalkine) 85
and CCL2 from
axotomized DRG neurons 86
.
Numbers of macrophages immunoreactive for major histocompatibility system II increase in
the corresponding DRG 1 week after a nerve transection and remain elevated for 3 months. In
early phase, macrophages are diffusely distributed in the DRG and later are concentrated around
injured sensory neurons 55,87
. Activated ED1+ macrophages frequently invade the satellite glial
cell-neuron unit in corresponding DRG after nerve injury. Thus, the satellite glial cells together
with invaded macrophages have ideal position for modification of the neuron environment 55
.
There is evidence that activated ED1+ macrophages invade not only the ipsilateral but also
contralateral DRG 55
.
25
5.2. Molecular reactions in DRG induced by peripheral nerve injury
Molecular reactions in the DRG following the peripheral nerve injury represent double-edge
sword. On one side, these reactions participate in neuropathic pain development and
maintenance. However, on the other side, the same reactions give background for reactivation
of the neuronal regeneration program important for successful recovery of the injured peripheral
nerve. Our lab is focused on both events and published articles in highly impacted journals.
5.2.1. Cytokines and chemokines involved in neuropathic pain development and maintenance
The cytokines and chemokines are secreted in corresponding DRG after peripheral nerve injury
from immune cells, neurons and satellite glial cells. They have been implicated to play a role
in neuropathic pain process by two mechanisms. Firstly, they may act directly on primary
sensory neurons, and secondly, they can indirectly act via activation of signaling pathways in
immune cells 88
. From the pathophysiological point of view, the cytokines and chemokines
might be divided into two groups, pro- and anti-inflammatory.
The pro-inflammatory cytokine IL-1β is produced and secreted by macrophages, monocytes
and microglia as a part of stress reaction. Expression of the IL-1β receptor has been detected in
DRG suggesting IL-1β autocrine and paracrine effect on sensory processing 89,90
. It has been
found that application of endogenous IL-1 receptor antagonist reduces pain-associated behavior
suggesting the central IL-1β effect on hyperalgesia 91
.
TNF-α is a pro-inflammatory cytokine that initiates cascade of activation of other cytokines,
chemokines and growth factors. Expression of TNF-α and its receptors (TNF- α receptor 1 and
2) in DRG has been proved in different models of peripheral neuropathic pain 92–94
. Importantly,
unilateral chronic constriction injury induced enhanced expression of TNF-α not only in
ipsilateral but also in contralateral and remote cervical DRG 6
. In addition, enhanced expression
26
of TNF-α receptor in the injured nerve was detected after chronic constriction injury and nerve
crush 95
. Direct application of TNF-α to the sciatic nerve produced dose-dependent ectopic
firing in afferent axons 96
, suggesting the role of TNF-α in neuropathic pain processing.
Expression of the pleiotropic pro-inflammatory cytokine IL-6 is associated with hyperalgesia
and allodynia in models of peripheral nerve injury 97–99
. Increased IL-6 mRNA levels were
detected in DRG after chronic constriction injury. In addition, IL-6 knockout mice displayed
attenuation of hyperalgesia compared with wild type mice after chronic constriction injury 99
.
Intrathecal application of IL-6 antagonist attenuated nerve-injury induced hyperalgesia 100
.
Moreover, it has been found that expression of IL-6 is increased not only in nerve injuryaffected
DRG but also in remote DRG 101
. Upon binding the specific receptor, IL-6 brings
together the intracellular regions of gp130 to initiate signal transduction through STAT3
signaling pathway 102
. We found that intrathecal application of IL-6 caused nuclear
translocation of STAT3 and its phosphorylation at the tyrosine-705 and serine-727 in lumbar
and cervical DRG. Interestingly, similar reaction was displayed in remote cervical DRG after
chronic constriction injury. These findings suggest spread of inflammatory response from nerve
injury-affected DRG to remote DRG through cerebrospinal fluid (CSF) in subarachnoid space
5
(Chapter 12, Article A).
The IL-10 is anti-inflammatory cytokine with suppressor effect on many pro-inflammatory
cytokines, including e.g. IL-1 and TNF-α 103,104
. It has been found that peripheral nerve injury
induces increased bilateral expression of IL-10 not only in the homonymous DRG but also in
the heteronymous DRG unassociated with the injured nerve 6
.
27
5.2.2. Nerve injury-induced regeneration state in the DRG
Peripheral nerve injury induces significant changes in metabolism, survival, excitability and
neurotransmitter functions of affected neurons 105
. These events may lead to reactivation of
intrinsic regenerative program as a part of functional recovery in injured peripheral nerves. The
DRG provide a very potent in vitro and in vivo model for testing of mechanisms regulating
reactivation of silent regenerative program.
Regenerative state in the DRG neurons can be detected by expression of regenerationassociated
molecules like cortical cytoskeleton-associated protein 23 (CAP-23), growthassociated
protein 43 (GAP-43), superior cervical ganglion 10 (SCG-10) or small proline-rich
repeat protein 1A (SPRR-1A). These molecules are important intrinsic determinants of the
increased regenerative ability of the DRG neurons 106,107
. However, different populations of the
DRG neurons may respond differently with respect to their regeneration capacity and
expression of molecular markers. Therefore, we aimed in our research to describe expression
of aforementioned markers with respect to size of neurons and their phenotype.
We found that immunohistochemical detection of GAP-43 recognizing both phosphorylated
and dephosphorylated forms is not sufficient as a marker for recognition of all types of neurons
with regenerating axons. Moreover, this marker cannot be used for detection of regenerating
axons in early periods after nerve injury. Contrary to GAP-43, SCG-10 is more reliable for
detection of neurons with reactivated regeneration program and regenerating sensory axons.
Other markers that could be used as a good alternative for recognition of neurons with
regenerating axons are ATF3 and STAT3 108
(Chapter 12, Article B).
In our second experimental work, we were focused on induction of pro-regenerative state in the
DRG non-associated with injured nerve. We correlated expression of GAP-43 and SCG-10 with
length of regenerating axons in crushed ulnar nerve after prior sciatic nerve injury. Seven days
28
after sciatic nerve compression or transection, our experiments revealed increased bilateral
expression of GAP-43 and SCG-10 not only in primary sensory neurons of the lumbar DRG
(L4-L5) associated with injured nerve, but also in remote cervical DRG (C6-C8). Moreover,
increased expression of pro-regenerative proteins in the cervical DRG was associated with the
greater length of regenerated axons 1 day after ulnar nerve crush following prior sciatic nerve
injury as compared to controls with only ulnar nerve crush. To confirm axonal regeneration
capacity of the cervical DRG, we used neurite outgrowth assay of in vitro cultivated DRG
neurons. In next part of experiments, we found that the IL-6 signaling pathway plays a key role
in activating the pro-regenerative state in remote DRG. Our results revealed that the proregenerative
state induced in remote primary sensory neurons non-associated with the injured
nerve reflects a systemic reaction of these neurons to unilateral sciatic nerve injury, possibly
due to spread of inflammatory reaction via the cerebrospinal fluid 109
(Chapter 12, Article C).
The role of IL-6 in axonal regeneration in remote DRG neurons after scitic nerve lesion has
been confirmed in our third experimental work. We compared protein levels of SCG-10,
STAT3 and axon regeneration in IL-6 knockout mice and their wild type counterparts. We
found that unilateral nerve compression and transection caused upregulation of SCG-10 protein
levels and activated STAT3 in DRG neurons not only in lumbar but also in cervical segments
of wild type mice. DRG neurons from IL-6 knockout mice also bilaterally increased levels of
SCG-10 and STAT-3 in both lumbar and cervical segments after sciatic nerve lesions. However,
levels of SCG-10 protein in cervical and lumbar DRG of IL-6 knockout mice were lower when
compared to their wild type counterparts. This finding correlates with significantly shorter
regeneration of axons distal to the crushed ulnar nerve 110
(Chapter 12, Article D).
29
6. Reaction of the spinal cord and brain stem to nerve injury
Central branches of the DRG primary sensory neurons enter the posterolateral sulcus of spinal
cord through dorsal root. Heavily myelinated A fibers involved in mediation of non-noxious
somatosensory inputs enter the spinal cord medially and travel through the dorsal columns
projecting ipsilateral to second-order cells in the brainstem, including cells in the nucleus
gracilis and nucleus cuneatus 111
. Lightly myelinated (Aδ) and unmyelinated (C) fibers enter
spinal cord laterally and most of them terminate on gray matter of the homonymous or segment.
The gray matter of the dorsal horn may be divided into 10 laminae on which low-threshold
mechanoreceptors terminate in laminae III and IV, high-threshold nociceptors in laminae I, II,
V and unmyelinated C-fibers in lamina II 13,112,113
.
6.1. Inflammatory reactions in the spinal cord of neuropathic pain models
It was demonstrated that the peripheral nerve injury-induced hyperalgesia is associated with
cellular and molecular changes in the dorsal horn of the spinal cord in segments corresponding
to injured nerve. These changes are related to activation of microglial cells and astrocytes as
well as alteration of pro- and anti-inflammatory cytokines produced by neurons, microglia,
astrocytes and invading immune cells 72,114–117
.
It is generally accepted that microglial cells are considered to be immune cells of the CNS
playing a key role as scavenger cells in inflammation, infection, trauma, ischemia and
neurodegeneration in the CNS 118,119
. Since the microglial cells belong to monocytemacrophage
lineage, they form clusters around cell bodies of injured neurons, similar to
macrophages in the DRG 118,120
. The peripheral nerve injury induces robust activation of
microglia in the dorsal horn of spinal cord that precedes to activation of astrocytes. It was found
that specific molecular markers for microglial activation Toll-like receptor (TLR) 4, ITGAM
30
and CD14 are upregulated in the dorsal horn early after nerve injury 121
. Activated microglia is
immunohistochemically detected by OX-42 antibody directed against a complement receptor 3
antigen (CD11b/c) or antibody against ionized calcium-binding adapter molecule 1 (Iba1)
122,123
.
Activation of the resident microglia in the spinal cord as a response to nerve injury can be
mediated via three pathways; fractalkine (CX3CL1) mediated pathway through the CX3CR1
receptor, CCR2 pathway and TLRs. Fractalkine (CX3CL1) is a neuronal transmembrane
glycoprotein from which a soluble chemokine domain can be cleaved by proteolysis. However,
fractalkine is biologically active in both membrane-bounded and soluble form 124
. Role of the
fractalkine/CX3CR1 signaling pathway in neuropathic pain was studied by intrathecal
administration of fractalkine and CX3CR1 neutralizing antibody. Intrathecal injection of the
fractalkine induced mechanical allodynia and contrary, the CX3CR1 neutralizing antibody
decreased mechanical allodynia after nerve injury 85
. Member of the CC chemokine family
CCL2 has the ability to recruit immune cells including macrophages and microglia to the site
of neuronal injury 125
. It has been found that CCL2 is a key mediator in development of
neuropathic pain. The peripheral nerve injury induces upregulation of CCL2 in the DRG that is
transported and released in the dorsal horn of spinal cord. Moreover, lumbar application of
CCL2 induces mechanical allodynia and microglial activation in naïve rats and inhibition of
endogenous CCL2 following nerve injury attenuates both phenomena 88
. TLRs are 12
evolutionary conserved membrane receptors that initiate innate immune response by
recognizing molecules shared by different pathogens. Signaling pathway mediated by TLR
leads to activation of NF-κB, subsequently to upregulation of interferons and expression of proinflammatory
cytokines and chemokines 124,126
. It has been found that activation of TLR2 and
TLR4 plays crucial role in microglial activation and production of pro-inflammatory cytokines
31
after nerve injury. Inhibition of TLR2 and TLR4 signaling pathway lead to attenuation of
microglial reaction and neuropathic pain-like behavior in nerve-injured animals 127,128
.
Astrocytes are the most abundant glial population participating in immune reactions. They have
been investigated mainly because of their supportive functions for neurons. Astrocyte
hypertrophy and activation expressed by Glial Fibrillary Acidic Protein (GFAP) upregulation
have been found in the dorsal horn of spinal cord after nerve injury 129,130
. In contrast to
microglial reaction, activation of astrocytes is more persistent and can last for more than 150
days 86
. Activated astrocytes are a major source of pro-inflammatory cytokines that contribute
significantly to induction and maintenance of peripheral neuropathic pain 131,132
.
It has been found that peripheral nerve injury induces upregulation of pro-inflammatory
cytokines and downregulation of anti-inflammatory cytokines in activated microglial cells and
astrocytes related to induction and maintenance of peripheral neuropathic pain 72,117,133
. The
major pro-inflammatory cytokines that affect peripheral neuropathic pain are IL-1β, IL-6 and
TNF-α 134
. Expression of IL-1β following the nerve injury is enhanced in microglia and
astrocytes of the CNS structures 135
. Moreover, it has been found that intraplantar application
of IL-1β to naïve animals produces hyperalgesia 136
. Mechanical allodynia and thermal
hyperalgesia are associated with IL-6 expression. Intrathecal application of IL-6 induces onset
of neuropathic pain-like behavior in naïve animals. Conversely, intrathecal administration of
IL-6 inhibitor relieves pain in nerve-injured animals 97,100,134
. Receptors for TNF-α, TNFR1 and
2, are present in both neurons and glia 137
. There is evidence that at injured nerve sites, TNF-α
is retrogradely transported and released at the terminals in the dorsal horn of spinal cord 138
. In
contrast to IL-1β, IL-6 and TNF-α, the major cytokines with anti-inflammatory effect are IL-4
and IL-10. It has been found that the expression of both IL-4 and IL-10 is decreased in the
dorsal horn of spinal cord after nerve injury 139,140
. Since low blood levels of IL-4 and IL-10
were found in patients with chronic widespread pain, they seem to be crucial for onset and
32
maintenance of the chronic pain 141
. IL-4 is pleiotropic anti-inflammatory cytokine that is
produced by various types of immune cells including CD4+ T lymphocytes, mast cells,
eosinophils and basophils 142
. There is growing body of evidence that even IL-4 inhibits
macrophage activation it also upregulates transcription of µ- and δ-opioid receptors. Therefore,
IL-4 may attenuate nociception through endogenous opioid system 143,144
. IL-10 is a very potent
anti-inflammatory cytokine repressing the expression of pro-inflammatory cytokines (IL-1β,
IL-6 and TNFα). Acute administration of IL-10 has ability to suppress development of pain 145
.
In our research about the reaction of the spinal cord on nerve injury, we were focused on
attenuation of behavioral hyperalgesia using intrathecal application of the CD200 fusion protein
117
. CD200 is a membrane glycoprotein with immune suppression effect via its receptor
CD200R. CD200 is mainly expressed on neurons while CD200R preferentially on myeloid cells
including microglia 146
. Increased level of CD200 is associated with less activated monocytes
and increasing expression of IL-10 in the CNS in experimental model of autoimmune
encephalomyelitis 147
. Conversely, CD200-/- mice displayed immune cells dysregulation,
increased activation of microglial cells and enhanced susceptibility of autoimmune
encephalomyelitis 148,149
. In our experimental study, we found increased mRNA levels of proinflammatory
cytokine (IL-1β, IL-6 and TNFα) and decreased mRNA levels of antiinflammatory
cytokines (IL-4 and IL-10) in spinal cord of L4-5 segments after chronic
constriction injury. We proved that intrathecal application of CD200 fusion protein diminished
elevation of pro-inflammatory cytokine mRNA and simultaneously ameliorated deficit of antiinflammatory
cytokines after chronic constriction injury. Moreover, intrathecal CD200 fusion
protein application suppressed activation of microglial cells and astrocytes after nerve injury.
In agreement with cellular and molecular changes, the CD200 fusion protein also attenuated
mechanical allodynia and thermal hyperalgesia in nerve-injured animals. However, the anti-
33
inflammatory effect of CD200 fusion protein was only transient and dropped at 24h after
intrathecal application (Chapter 12, Article E).
6.2. Pain processing and modulation in the brain stem
6.2.1. The gracile and cuneate nucleus
Innocuous information such as touch, pressure and vibration are conveyed via the dorsal
columns and transmitted in the medulla oblongata. The gracile nucleus receives information
from the lower part of the body while the cuneate nucleus receives information from the upper
part of the body. It has been found that both nuclei are involved in neuropathic pain
development and maintenance 150
.
Increased Gamma-Aminobutyric Acid (GABA) synaptic uptake together with robust
astrogliosis was reported in the gracile nucleus after spared nerve injury 151
. Moreover, it has
been found that gracile nucleus plays a role in central sensitization by upregulation of
phosphorylated NMDA receptor 1 in neurons 152
and phosphorylated-p38 MAPKimmunopositive
microglia 153
. Role of the medulla oblongata in neuropathic pain processing
was also confirmed by experiments using nerve injury of the median nerve. These experiments
revealed important role of astrocyte activation expressed by increased GFAP immunoreactivity
in the cuneate nucleus after median nerve injury 154
. Furthermore, it has been demonstrated that
median nerve injury induces increased neuropeptide Y immunoreactivity and c-fos expression
in the cuneate nucleus corresponding with behavioral signs of the neuropathic pain 155,156
.
6.2.2. Rostral ventromedial medulla
Rostral ventromedial medulla (RVM) is composed of nucleus raphe magnus and
gigantocellularis. Their neurons are involved in descending attenuation of pain perception as
34
was described in chapter 2.8. According to their pharmacological and physiological properties,
the neurons of RVM can be divided into three groups, “On-cells”, “Off-cells”, and “neutral
cells”. The “On-cells” increase their firing rate before the nocifensive reflexes and enhance
response to the pain. Contrary to the “On-cells”, the “Off-cells” decrease firing rate before the
nocifensive reflexes and inhibit nociception. Remaining cells that display no changes in firing
rate are “neutral cells” 150
. In neuropathic pain conditions, balance of circuitry within the RVM
shift to domination of the “On-cells” over the “Off” and “neutral cells” 157,158
. However,
sensitisation of both “On” and “Off-cells” to innocuous and noxious mechanical stimuli was
demonstrated to facilitate allodynia and hyperalgesia 150,159
.
Since activation of glial cells affects neuronal activity via cytokines and chemokines, activation
of astrocytes and/or microglia in RVM may affect modulatory role of descending pathways in
neuropathic pain maintenance. Early transient activation of microglia and delayed reaction of
astrocytes in RVM related to mechanical hyperalgesia was reported after chronic constriction
injury of the infraorbital nerve 160
. However, after spinal nerve ligation strong activation of
microglia was displayed 10 days after injury 161
.
In the light of these findings, we focused our research on changes of glial cells in RVM
following different types of peripheral nerve damage, namely chronic constriction injury and
transection of the sciatic nerve. We used OX-42 immunostaining for detection of activated
microglia and GFAP for activated astrocytes. Our results showed that both types of the nerve
injury induced microglial activation while only nerve transection caused strong astrogliosis in
PAG. Consequently, we wanted to answer what causes activation of glia in RVM after nerve
injury. Therefore, we focused on candidate chemokine CCL2 with its receptor CCR2 (see
chapter 6.1.). Our results revealed that CCL2 immunoreaction was present in neurons and
activated GFAP+ astrocytes while the CCR2 was displayed in OX-42 positive microglial cells
7
(Chapter 12, Article F).
35
6.2.3. Pons
The main structure involved in pain processing in the pons is the locus coeruleus. Neurons of
the locus coeruleus are capable to produce noradrenaline that is known to act on α2adrenoreceptors
in the spinal cord with antinociceptive effect 162
. Noradrenaline reuptake
inhibitors and gabapentin are used as approved treatments of chronic pain 163,164
. It has been
demonstrated that gabapentin acts directly within the locus coeruleus via glutamate dependent
mechanism to alleviate pain behavior after spinal nerve ligation 150,165
. On the other hand, there
are experimental works that revealed opposite role of the locus coeruleus in pain processing
166,167
. It has been found that lesion of the locus coeruleus prevented autotomy in animals after
sciatic and saphenous nerve transection 167
.
6.2.4. Mesencephalon
Two structures are involved in pain processing in the midbrain, the periaqueductal gray and the
red nucleus. The red nucleus is well known as an important structure of the extrapyramidal
system participating in e.g. motor learning, postural corrections and recovery after spinal nerve
injury 168,169
. However, experimental studies demonstrated its role in neuropathic pain
regulation. It has been shown that glutamate and IL-6 in red nucleus are related to induction
and maintenance of mechanical allodynia after spared nerve injury 170,171
. Moreover, it has been
demonstrated that cytokine levels including TNF-α, IL-β and nerve growth factor (NGF) in red
nucleus correlate with severity of behavioral changes after spared nerve injury 150,172–174
.
Periaqueductal gray plays very important role in descending modulatory pathways with its
connections in rostral ventromedial medulla (see chapter 2.8.). Reynolds 175
demonstrated that
36
electrical stimulation of the periaqueductal gray produces analgesia in rats undergoing surgery.
Further experiments revealed that local application of opiates to the periaqueductal gray
produces pain relief after spared nerve injury 176
. It is well known that glial activation
contributes to development and maintenance of chronic pain after nerve injury 145
. However,
most of the experimental studies about glial contribution on pain states were observations
performed in the dorsal horn of the spinal cord 177,178
. Therefore, the main idea of our research
was focused on reaction of astrocytes and microglia after chronic constriction injury and
transection of the sciatic nerve in the periaqueductal gray. Our results revealed more extent
activation of astrocytes in periaqueductal gray of nerve-injured animals when compared with
naïve and sham groups. Increased astrocyte activation was found on ipsilateral side of
ventrolateral periaqueductal gray than on contralateral side. More robust activation of astrocytes
was found after nerve transection than chronic constriction injury. However, in contrast to
astrocyte activation, microglial cells displayed bilateral activation after nerve injury 7
. In the
light of these results, we wanted to answer a question: what is the signaling pathway that
contributes to the glial activation in the periaqueductal gray? One of the candidate signaling
pathway may be the CCL/CCR2 pathway (reviewed in chapter 6.1.) with its ability to recruit
microglial cells to the site of nerve injury 125
. Similar to the rostral ventromedial medulla (see
chapter 6.2.1.), we found CCL2 immunoreaction in neurons and astrocytes while the CCR2 was
displayed in OX-42 positive microglial cells. Taken together, our results revealed that
CCL2/CCR2 signaling is involved in glia-glia and neuron-glia interactions in the descending
modulatory system including the RVM and the periaqueductal gray after nerve injury related
to chronic pain 7
(Chapter 12, Article F).
37
7. Diencephalon and its reaction to nerve injury
7.1. Thalamus
The thalamus contains the third-order neurons receiving information from the spinal cord about
the pain and temperature via the spinothalamic tract. Therefore, thalamic nuclei play a crucial
role in chronic pain development and processing. It has been found that nerve injury induces
decreased neuronal activity in contralateral thalamus in patients with chronic neuropathic pain
179
. However, increased spontaneous and mechanically evoked activity of neurons in the ventral
posterolateral nucleus in different neuropathic pain models 180,181
. Electrical stimulation of the
ventral posterolateral nucleus alleviates mechanical allodynia in rats after partial sciatic nerve
ligation 182
. Moreover, lesions or blocks of the intralaminar or the medial thalamic nuclei
attenuated neuropathic pain-like behavior in rats after spared nerve injury. These results suggest
involvement of these nuclei in neuropathic pain processing 183
.
It has been found that not only neurons but also glia in thalamus contribute to neuropathic pain
development. Increased glial activation in thalamus was found in patients with chronic low back
pain suggesting a role of thalamic glia in neuropathic pain development 184
. Moreover,
increased thalamic activity was also found in patients after peripheral nerve injury 185
. These
results suggest that thalamus and glia activation play a key role in rearrangement of cortical
representational maps.
7.2. Hypothalamus
The hypothalamus receives afferent nociceptive fibers and sends off efferent projections to the
brainstem and the spinal cord 150
. It has been found that chronic constriction injury induces
decreased expression of substance P and activation of microglia in the hypothalamus suggesting
38
their role in development of neuropathic pain 186
. Conversely, stimulation of the posterior part
of the hypothalamus exhibit analgesic effect in animals after chronic constriction injury 150,187
.
Magnocellular part of the paraventricular nucleus and the supraoptic nucleus produce oxytocin
playing physiological role in parturition and milk ejection reflex. However, there is compelling
evidence indicating participation of oxytocin in endogenous analgesia. Electrical stimulation of
the paraventricular and supraoptic nuclei inhibits pain perception 188
. Moreover, intrathecal
injection of oxytocin attenuates nociception after spinal nerve ligation 189
.
8. Pain processing and modulation in the telencephalon
Painful stimuli are processed in different areas of the telencephalon including primary and
secondary somatosensory cortices, the insular cortex, the anterior cingulate cortex and the
orbitofrontal cortex. It is believed that primary ad secondary somatosensory cortices play a role
in sensory-discriminative aspect of pain. The insular cortex and anterior cingulate cortex are
involved in the affective-emotional aspects of pain, while cognitive aspects of pain are
associated with activation of the prefrontal areas. Furthermore, changes in basal ganglia activity
are also present in painful states 190
.
8.1. Basal ganglia
It is well known that basal ganglia are mainly involved in motor functions. However,
experimental and clinical studies revealed also their role in processing of non-noxious and
noxious somatosensory information 191
.
The amygdala is formed by group of nuclei that are counted as a part of limbic forebrain system.
Therefore, these nuclei play a role in emotional-affective aspects of pain 150
. It has been reported
that activation of GABA-A receptors in amygdala attenuates affective processing after
39
peripheral nerve injury 192
. Spared nerve injury induces neuroplasticity in the amygdala
resulting in increased total volume of amygdalar nuclei. These findings suggest a role of the
amygdala in depressive-like symptoms well known in cases of patients with chronic pain 193
.
Striatum (putamen and the caudate nucleus) and striatal dopamine D2 receptors play a role in
modulation of pain sensation both in clinical conditions and experimental models 194
.
Microinjection of dopamine D2 receptor agonist quinpirole attenuates neuropathic
hypersensitivity in spared nerve injury-operated animals 195
. In addition, it was demonstrated
that the striatum plays dual role in neuropathic sensitivity in animals after spinal nerve ligature.
Activation of noradrenergic α2-adrenoceptors and downregulation of dopamine D receptors
reduce hypersensitivity, while activation of striatal NMDA receptors promotes hypersensitivity
196
.
8.2. Anterior cingulate cortex
The anterior cingulate cortex responds to painful stimuli both in humans and animals 150
. Using
functional magnetic resonance imaging it was found that neuronal activity within the anterior
cingulate cortex is increased in chronic pain states 197
. Electrical stimulation of the anterior
cingulate cortex produces analgesic effect in rats subjected to spared nerve injury 198
. However,
nerve injury induces neuronal caspase-dependent apoptosis triggered by NMDA receptor the
anterior cingulate cortex 199
. In addition, spared nerve injury causes astrocyte activation, which
contributes to painful state 200
.
8.3. Ventrolateral orbitofrontal cortex
The ventrolateral orbitofrontal cortex is involved in sensory integration and pain modulation. It
has connections with the thalamic nucleus submedius, hypothalamus, periaqueductal gray and
40
RVM 150,201,202
. Functional imaging techniques revealed that the ventrolateral orbitofrontal
cortex is activated in patients with chronic pain 203
. Block of neuronal activity in the
ventrolateral orbitofrontal region induces attenuation of allodynia and hyperalgesia in animals
after spared nerve injury 204
. Chemical inhibition induced by local application of morphine,
GABA or lidocaine to the ventrolateral orbitofrontal attenuated pain-like behavior in rats after
formalin injection to the hind-paw 205
. In addition, increased expression of pro-inflammatory
and proapoptotic genes correlated with allodynia and hyperalgesia were found after spared
nerve injury 206
.
8.4. Insular cortex
The insular cortex is involved in emotion, cognitive functions, motor control as well as
processing and modulation of pain 150
. It receives afferent fibers mainly from the thalamus and
sends efferent fibers to different regions including the amygdala, hypothalamus, periaqueductal
gray and rostral ventromedial medulla 202,207
. It has been found that regional cerebral blood flow
in the insular cortex correlates with pain experience and noxious stimuli 208
and lesion in the
insular cortex alleviate neuropathic pain-like behavior after sciatic nerve injury 209
. There is
growing body of evidence that opioid, GABA and dopamine are key neurotransmitters playing
a role in nociceptive modulation in the insular cortex 202,210
. Local administration of morphine
to the insular cortex induced analgesia in animals after formalin injection to the hind-paw 211
.
Locally increased presence of GABA produces lasting analgesia by enhancing inhibition of the
spinal nociceptive neurons. However, activation of GABAB-receptor-bearing insular neurons
induces hyperalgesia via projection to the amygdala 212
. Moreover, local application of the
dopamine reuptake inhibitor to the insular cortex produces inhibition of the spinal nociceptive
neurons and analgesia 213
.
41
8.5. Somatosensory cortex
The primary and secondary somatosensory cortices are involved in the sensory-discriminative
aspect of pain 202,214
. Imaging techniques revealed that painful heat stimuli induced significant
activation of both primary and secondary somatosensory cortices 215
. Electrical stimulation of
the secondary somatosensory cortex reduced formalin-induced nociceptive behavior in
experimental animals 216
. Spinal cord injury-induced neuropathic pain causes contralateral
somatosensory cortex reorganization and its amount is dependent on pain intensity 217
. It has
been found that modulation of pain perception relates to metabotropic glutamate receptors and
opioid receptors in both primary and secondary somatosensory cortices 218
.
42
9. Barriers of the nervous system, their alteration and contribution to spreading of
inflammatory reaction after nerve injury
Barriers of the nervous system play an essential role in maintaining of neural
microenvironment. However, the barriers within the nervous system react during different
diseases with alteration of their protective function. In this chapter, I would like to focus on
changes of the nervous system barriers induced by nerve injury that may play a role in spread
of inflammatory reaction alongside the neuroaxis.
9.1. The blood-nerve barrier
The blood-nerve barrier forms interface between the endoneurial compartment, surrounding
tissue and blood. The barrier is localized at the innermost layer of the perineurium and
endoneurial microvasculature in the peripheral nerve. The microvessels are continuous
capillaries where the endothelial cells are sealed with tight junctions and covered by the basal
lamina with surrounding pericytes 219
.
It has been shown that the blood-nerve barrier is impermeable for large molecules in
physiological conditions. However, the endoneurial microvessels become permeable for
labeled albumin after nerve injury. The main site of the barrier alteration is found in distal stump
of injured nerve undergoing Wallerian degeneration 220,221
. Breakdown of the blood-nerve
barrier after nerve injury is associated with changes of tight junctions proteins, namely claudin-
1, claudin-5, occluding, VE-cadherin and connexin 43 222
. However, the alteration of bloodnerve
barrier is only temporary with the highest permeability 7 days after nerve injury and
approximately one month after injury the barrier is reconstituted 223
. Alteration of the bloodnerve
barrier is double-edged sword. On the one hand, it is positive allowing macrophage
43
invasion to remove myelin debris and regenerate damaged axons. On the other hand, Wallerian
degeneration products may enter blood and thus spread to remote tissue including the CNS.
9.2. The blood-DRG barrier
The DRG are supplied with fenestrated capillaries with fenestration size between 60 to 80 nm
224
. Microvascular density in the DRG is 7 times higher than in peripheral nerve and nerve roots.
It has been found that the highest capillary density within the DRG is present in the neuronal
body-rich area 225
. In contrast to axonal compartment of the DRG, the neuronal body-rich area
contains capillaries with high density of tight junctions 226
. Experimental studies on intact
animals revealed penetration of intravenously injected tracers including horseradish peroxidase,
Evans blue albumin and fluorescein to the DRG 226–228
. Therefore, the blood-DRG barrier is
incomplete in physiological conditions and is opened for large molecules mainly because of
fenestrated capillaries (Fig. 4). However, nerve injury induces proliferation of capillaries in the
DRG forming nests around neurons with damaged axon 229
.
44
Figure 4. A simplified scheme of the blood-DRG barrier. The DRG are supplied with
fenestrated capillaries that allow free movement of solutes, molecules and cells between blood
and the DRG stroma (A). Detail of the barrier organization on B. SGCs – satellite glial cells.
9.3. The cerebrospinal fluid-DRG barrier
The barrier between the cerebrospinal fluid and the DRG is formed by meninges, especially
arachnoid that limits subarachnoid space filled with the cerebrospinal fluid, and by capsule of
the DRG. The lateral border of the subarachnoid space localized at transition of the dorsal and
ventral roots and the DRG is called subarachnoid angle. At this place, perineurium of the
peripheral nerve leaves surface of the nerve and runs between the dura mater and arachnoid.
45
Microscopic organization of the subarachnoid angle and communication with the peripheral
nerve explains a pathway for spread of inflammation to the CNS 230
.
The arachnoid villi were found in the subarachnoid angle giving anatomical substrate for
communication with the epidural space. These findings may correspond with spread of
anesthetics in epidural anesthesia 231
. In addition, important site for penetration of anesthetics
is the spinal nerve root cuff forming lateral prolongations of the dura mater and the arachnoid
membrane enclosing spinal nerve roots in their way through the epidural space to the vertebral
foramina 232
. At this place, layers of leptomeninges fuse and continue as the perineurium of
peripheral nerves while the dura mater corresponds with the epineurium 233
(Fig. 5).
Figure 5. Schematic drawing of the subarachnoid angle (SA) position with respect to meninges,
spinal nerve roots (ventral root – VR, dorsal root – DR) and the DRG. Dura mater is depicted
in black, arachnoid membrane in green and pia mater in yellow.
46
It has been found that the cerebrospinal fluid-DRG barrier is permeable for tracer fluorescein
after its intrathecal injection. Fluorescein was accumulated in the subarachnoid angle and
diffused to the DRG with proximo-distal gradient. Nerve injury induced accumulation of
fluorescein in the neuronal body-rich area of the DRG 228
.
In our experiments, we used intrathecal administration of fluorescent-conjugated dextran
FluoroEmerald (FE; MW=10 kDa) to investigate whether the cerebrospinal fluid-DRG barrier
is permeable for molecules of a size of cytokines. We found particles of FE in the cells of the
DRG capsule, satellite glial cells, interstitial space, as well as in small and medium-sized
neurons. Penetration of lumbar-injected FE into the cervical DRG was greater than that into the
lumbar DRG after intrathecal injection of FE into the cisterna magna. Moreover, FE induced
an immune reaction in the DRG with presence of the particles in antigen-presenting cells
(MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activated satellite glial
cells (GFAP+). Since we found FE inside the DRG, we wanted to answer whether there is
morphologic background for direct communication of the subarachnoid space with the DRG.
Therefore, we used intrathecal injection of methylene blue and DPP-IV immunostaining to
visualize position of the subarachnoid space and arachnoid in relation to the DRG. The
subarachnoid space delimited by the arachnoid mater was extended up to the capsule of DRG
in a fold-like recess and reached approximately half of the DRG length. The arachnoid was
found in direct contact with the neuronal body-rich area in the angle between dorsal root and
DRG as well as between spinal nerve roots at DRG. Our results clearly demonstrated direct
communication between the DRG and cerebrospinal fluid that can create another pathway for
possible propagation of inflammatory and signaling molecules from DRG primary affected by
peripheral nerve injury into DRG of remote spinal segments 8
(Chapter 12, Article G).
47
9.4. Blood-brain and blood-spinal cord barrier
Both the blood-brain barrier and the blood-spinal cord barrier are composed of continuous type
of microvessels. The endhotelia are sealed with tight junctions, enveloped by the basal lamina
with surrounding pericytes and astrocytic end-feet 234
. Compared to the blood-nerve barrier, the
complex of the endothelia/basal lamina/pericytes is surrounded by the second basal lamina
called glia limitans perivascularis 219,235
(Fig. 6).
The main component of these barriers are endothelial tight junctions composed of several
proteins including occludin, many subtypes of claudins, zonulin. Claudins and occludin are the
main transmembrane molecules mediating the epithelial contact 236
. Occludin is a tetraspan
integral protein of tight junctions that is functionally important for barrier function 237,238
. There
is evidence that occludin seals the tight junctions because its downregulation leads to disruption
of tight junction permeability 239
. Claudins form a large family of membrane proteins that have
four transmembrane domains forming several subgroups. It has been found that the brain
endothelial cells express claudin 1, 3 and 5 240–242
. Zonulin (ZO) is the cytoplasmic protein
associated with transmembrane proteins. The blood-brain endothelia display positivity for ZO-
1, -2 and -3 238
. Integrity of the blood-brain barrier after peripheral nerve injury was assessed
using intravenous injection of Evans blue albumin and horseradish peroxidase. Chronic
constriction injury, sciatic nerve transection as well as electrical stimulation of peripheral nerve
induced increased permeability of the blood-brain barrier. Interestingly, increased permeability
can be prevented by injecting local anesthetic lidocaine to the site of the nerve injury or prior
to electrical stimulation 243
.
Tight junctions of the blood-spinal cord barrier are mainly constituted of occludin, claudin-1
and -5 as well as ZO-1. In contrast to the blood-brain barrier, the blood-spinal cord barrier is
more permeable for cytokines and tracers. Increased permeability would be explained by lower
level of occludin and ZO-1 as well as less number of pericytes in comparison with the brain
48
244,245
. It has been found that peripheral nerve injury induces opening of the blood-spinal cord
barrier for small and large molecules associated with downregulation of ZO-1 and claudin-5
245
.
Interaction of astrocytes and endothelia plays a key role in correct function of the blood-brain
and the blood-spinal cord barrier. The astrocytes are capable to secret humoral agents that might
increase permeability of the blood-brain barrier e.g. endothelin-1, glutamate, IL-1β, IL-6, TNFα,
MIP-2 and nitric oxide 246,247
. However, role of astrocytes in regulation of the blood-brain
barrier permeability after peripheral nerve injury is still unclear.
49
Figure 6. A simplified scheme of anatomical organization (A) and detail (B) of the bloodbrain
barrier 219
. The endothelial cells (light blue) are linked with tight junctions (cross-links)
and embedded together with pericytes in basal lamina (purple). The second basal lamina
(pink; glia limitans perivascularis) serves for attachment of astrocytic end-feet and surrounds
50
the complex of endothelia, pericytes and the first basal lamina. Glia limitans perivascularis
and the first basal lamina limit the perivascular space.
9.5. Blood-cerebrospinal fluid barrier
The blood-cerebrospinal fluid barrier is localized in the choroid plexus of brain ventricles that
is epithelio-endothelial convolute composed of highly vascularized core, connective tissue
stroma and epithelial cells 248–250
(Fig. 7). The vascularized core is formed by fenestrated
capillaries with fenestrations ranged from 60 to 80 nm and is surrounded by connective tissue
stroma with immune cells. They were recognized as resident (ED2+) and activated (ED1+)
macrophages expressing major histocompatibility complex class II antigen as well as dendritic
OX 62+ cell 251,252
. The ventricular side of the stroma is covered by a single layer of cuboidal
epithelial cells 248,249
. Epiplexus (Kolmer) cells adhere on the ventricular side of epithelial cells
253
. Kolmer cells have various shapes from cells with fibrous processes radiating from the
centrally located cell body to cells with a few pseudopod-like processes. These cells have ability
to phagocyte and they seem to be scavengers in the cerebrospinal fluid 254
. There is evidence
that the choroid plexus supports trafficking of leukocytes to the cerebrospinal fluid 255–258
.
Apical tight junctions of cuboid cells that are responsible for regulation of paracellular diffusion
of water-soluble molecules play the major role for regulation of the blood-cerebrospinal fluid
barrier permeability 234,259
. Tight junctions are composed of proteins associated with the cellular
membrane 260
. The first described integral protein of tight junctions in the cuboidal cells of
choroid plexus was occludin 262,263
. In addition, the choroid plexus epithelial cells express also
claudin-1-6, 9-12, 19, 22 261,263,264
and ZO proteins, ZO-1265,266
, ZO-2 267,268
and ZO-3 269,270
that bind occludin and claudin to actin filaments 271,272
.
It has been found that the choroid plexus plays a key role in pathophysiology of many disorders
including inflammatory, neurodegenerative, infectious, traumatic, neoplastic and systemic
51
diseases 248,273
. However, it is unknown whether barrier function of the choroid plexus is altered
after nerve injury. Therefore, we focused on cellular changes of the choroid plexus after
peripheral nerve injury. We used ED1 and ED2 immunostaining to investigate epiplexus cell
changes in rat choroid plexus after chronic constriction injury and sham-operated animals in
comparison with the choroid plexus of naïve animals. We found significantly increased
numbers of ED1+ cells in the choroid plexus of sham-operated rats in all periods of survival
when compared with naïve animals, while the number of ED2+ increased only at 3 days from
operation. However, the number of ED1+ and ED2+ cells in the epiplexus position increased
gradually with the duration of nerve compression. We detected no or negligible cell
proliferation in the choroid plexus after sham- or nerve injury operation. This suggests that
increased number of ED1+ and ED2+ cells in the epiplexus position of the choroid plexus is
derived from peripheral monocytes passing through altered blood–cerebrospinal fluid barrier.
The changes in epiplexus cells indicate that the choroid plexus reacts to tissue injury after the
surgical approach itself and that the response to peripheral nerve lesion is greater. This suggests
a role for an altered blood-cerebrospinal fluid barrier allowing propagation of signal molecules
produced by damaged tissue and nerve to the CNS 9
(Chapter 12, Article H).
52
Figure 7. Scheme of anatomical organization (A) and detail (B) of the blood-cerebrospinal fluid
barrier. The blood-cerebrospinal fluid barrier is localized in the choroid plexus of the brain
ventricles composed of fenestrated capillaries surrounded with connective tissue stroma and
cuboidal epithelial cells with adhering Kolmer cells (A). The main site of the barrier is on the
level of the cuboidal epithelial cells that are linked by tight junctions.
53
10. Conclusions and future perspectives
My habilitation thesis integrates important findings in the field of spreading the inflammatory
reaction after nerve injury to the CNS. Spread of inflammatory response from the site of injury
may happen via two pathways or their combination. Firstly, “on-wire” pathway count with
trans-synaptic spread via sensory pathway to the centers of the CNS. Secondly, “wire-less”
pathway involves spread of pro-inflammatory cytokines and chemokines via bloodstream or
cerebrospinal fluid. This pathway represents my main research direction emphasizing
generalized reaction of the nervous system on peripheral nerve injury. This reaction may play
important role not only in neuropathic pain induction but also in nerve regeneration after nerve
injury. However, the full range of mechanisms and reasons of generalized reaction of nervous
system still remains to be elucidated.
In summary, we provided the first evidence that:
• Peripheral nerve injury induces activation of STAT3 bilaterally in the DRG neurons of
both lumbar and cervical spinal cord segments.
• IL-6 released to the subarachnoid space has ability to penetrate to the DRG and activate
STAT3.
• Intrathecal application of CD200 fusion protein has ability to attenuate gliosis and
inflammatory reaction in the spinal cord after peripheral nerve injury.
• CD200 fusion protein induces rapid but temporary suppression of mechanical allodynia
and thermal hyperalgesia in neuropathic pain model.
• Peripheral nerve injury induces pro-regenerative state in primary sensory neurons nonassociated
with injured nerve.
• IL-6 signaling pathway plays a key role in activating the pro-regenerative state in the
DRG non-associated with injured nerve.
54
• Peripheral nerve injury induces activation of astrocytes and microglia in the
periaqueductal gray and the rostral ventromedial medulla.
• Microglial activation in the periaqueductal gray and the rostral ventromedial medulla
following nerve injury is mediated via CCR2 due to neuronal as well as astrocytal
release of CCL2.
• Subarachnoid space reaches the dorsal root ganglion as a fold-like recess that gives
anatomical background for changes of molecules between the dorsal root ganglion and
the cerebrospinal fluid.
• Intrathecal application of the fluorescence-conjugated dextran FE penetrates to the DRG
and induces immune reaction.
• Peripheral nerve induces cellular changes in the choroid plexus represented by increased
number of activated and resident macrophages in the epiplexus position.
• Not only nerve injury but also surgical approach itself induces cellular changes in the
choroid plexus.
In future research, I would like to continue with experiments focused on changes in the bloodcerebrospinal
fluid barrier after nerve injury emphasizing role of tight junctions. Moreover, the
choroid plexus seems to be a candidate structure of CNS suitable for gene manipulation. This
would allow manipulation with choroid plexus permeability rate or secretory function.
In addition, I would like to continue with electrophysiological experiments of ex vivo model of
skin and preserved nerve. I learned this model in the lab of prof. Christopher Honda during my
Fulbright scholarship at the University of Minnesota.
55
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77
12. Commented overview of selected publications
(Arranged in order of appearance in text)
A. Dubový P, Hradilová-Svíženská I, Klusáková I, Kokošová V, Brázda V, Joukal M.
Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-
727 (S727) positions and its nuclear translocation in primary sensory neurons following
unilateral sciatic nerve injury. Histochem Cell Biol. 2018:1-11; doi:10.1007/s00418-018-
1656-y
B. Dubový P, Klusáková I, Hradilová-Svíženská I, Joukal M. Expression of RegenerationAssociated
Proteins in Primary Sensory Neurons and Regenerating Axons After Nerve
Injury-An Overview. Anat Rec Hoboken NJ 2007. 2018; doi:10.1002/ar.23843
C. Dubový P, Klusáková I, Hradilová-Svíženská I, Brázda V, Kohoutková M, Joukal M. A
conditioning sciatic nerve lesion triggers a pro-regenerative state in primary sensory
neurons also of dorsal root ganglia non-associated with the damaged nerve. Frontiers in
Cellular Neuroscience. 2019;13. doi:10.3389/fncel.2019.00011
D. Dubový P, Hradilová-Svíženská I, Klusáková I, Brázda V, Joukal M. Interleukin-6
contributes to initiation of neuronal regeneration program in the remote dorsal root
ganglia neurons after sciatic nerve injury. Histochemistry and Cell Biology. 2019;
152:109-117; doi:10.1007/s00418-019-01779-3
E. Hernangómez M, Klusáková I, Joukal M, Hradilová-Svíženská I, Guaza C, Dubový P.
CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity
immediately after its intrathecal application in a rat neuropathic pain model. J
Neuroinflammation. 2016;13:43. doi:10.1186/s12974-016-0508-8
F. Dubový P, Klusáková I, Hradilová-Svíženská I, Joukal M, Boadas-Vaello P. Activation
of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and
Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following
Different Types of Sciatic Nerve Injury. Front Cell Neurosci. 2018;12:40.
doi:10.3389/fncel.2018.00040
G. Joukal M, Klusáková I, Dubový P. Direct communication of the spinal subarachnoid space
with the rat dorsal root ganglia. Ann Anat - Anat Anz. 2016;205:9-15.
doi:10.1016/j.aanat.2016.01.004
78
H. Joukal M, Klusáková I, Solár P, Kuklová A, Dubový P. Cellular reactions of the choroid
plexus induced by peripheral nerve injury. Neurosci Lett. 2016;628:73-77.
doi:10.1016/j.neulet.2016.06.019
A. Dubový P, Hradilová-Svíženská I, Klusáková I, Kokošová V, Brázda V, Joukal M.
Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-
727 (S727) positions and its nuclear translocation in primary sensory neurons following
unilateral sciatic nerve injury. Histochem Cell Biol. 2018:1-11; doi:10.1007/s00418-018-
1656-y
Commentary: In this publication, we demonstrated that sciatic nerve injury induced
activation of STAT3 in both lumbar and cervical DRG by phosphorylation at the tyrosine-705
and serine-727. Moreover, we found that STAT3 activation is closely related to IL-6 level in
cerebrospinal fluid. In addtion, using IL-6 intrathecal application we showed that released IL-
6 into the subarachnoid space can penetrate the DRG and subsquently activate STAT3.
IF (WOS, 2017): 2.164
Quartile (WOS, 2017): Microscopy Q1; Cell Biology Q4
Contribution of the author: Co-authorship; approximate participation 20 %. The author
participated in performing of in vivo experiments, preparing samples for
immunohistochemistry, western blot and writing of the corresponding texts.
Vol.:(0123456789)1 3
Histochemistry and Cell Biology
https://doi.org/10.1007/s00418-018-1656-y
ORIGINAL PAPER
Bilateral activation of STAT3 by phosphorylation at the tyrosine-705
(Y705) and serine-727 (S727) positions and its nuclear translocation
in primary sensory neurons following unilateral sciatic nerve injury
Petr Dubový1
   · Ivana Hradilová‑Svíženská1
 · Ilona Klusáková1
 · Viktoria Kokošová1
 · Václav Brázda1
 · Marek Joukal1
Accepted: 24 February 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve
injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical
(C6–C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727)
positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with
the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of
STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve
indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following
nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that
this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased
bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve
injury may reflect a systemic reaction of the DRG neurons to nerve injury.
Keywords  Dorsal root ganglia · Neuroinflammation · Systemic reaction · Peripheral nerve injury
Introduction
Primary sensory neurons with their cell bodies in the dorsal
root ganglia (DRG) sendoff peripheral and central branches
of afferent axons that display different responses to neuronal
injury. While injury to peripheral axonal branches results in
profound molecular alterations involving activation of transforming
factors (Patodia and Raivich 2012), no such changes
are induced after intraspinal injury of central branches of
afferent axons (Schwaiger et al. 2000).
In response to unilateral sciatic nerve injury, protein and
mRNA levels of IL-6 and its receptors were enhanced bilaterally
in primary sensory neurons not only in DRG of lumbar
segments (L4-L5) associated with the injury, but also in
cervical segments (C7–C8) not associated with the injured
nerve (Dubovy et al. 2013; Brazda et al. 2013). Upon binding
to its specific membrane-bound receptor (IL-6R), IL-6
brings together the intracellular regions of gp130 to initiate
a signal transduction cascade via activation of signal transducer
and activator of transcription 3 (STAT3) (Aaronson
and Horvath 2002; Eulenfeld et al. 2012). Activation of
STAT3 is by JAK2-dependent phosphorylation at the tyrosine-705
(Y705), and JAK2-independent phosphorylation
mediated by mitogen-activated protein kinases (MAPKs)
at the serine-727 (S727) position (Aaronson and Horvath
2002). Activation of STAT3 by phosphorylation at the Y705
position occurs in DRG neurons very early after a nerve
*	 Petr Dubový
	pdubovy@med.muni.cz
	 Ivana Hradilová‑Svíženská
	isvizen@med.muni.cz
	 Ilona Klusáková
	iklusak@med.muni.cz
	 Viktoria Kokošová
	435583@mail.muni.cz
	 Václav Brázda
	vabdna@gmail.com
	 Marek Joukal
	mjoukal@med.muni.cz
1
	 Department of Anatomy, Cellular and Molecular Research
Group, Faculty of Medicine, Masaryk University, Kamenice
3, Brno 62500, Czech Republic
	 Histochemistry and Cell Biology
1 3
lesion and is mediated by neuropoietic cytokines including
IL-6 (Miao et al. 2006; Schwaiger et al. 2000; Sheu et al.
2000; Qiu et al. 2005) and neurotrophins (Pellegrino and
Habecker 2013; Ng et al. 2006b). Phosphorylated STAT3
dimers translocate to the nucleus and initiate transcription
of target genes (Taga 1996; Eulenfeld et al. 2012).
Activated STAT3 is a molecular mediator of IL-6 signaling,
but it is not yet known if STAT3 is also activated bilaterally
in both lumbar and cervical DRG neurons after unilateral
sciatic nerve injury. Thus far, only activation of STAT3
by phosphorylation at the Y705 position has been detected in
DRG neurons associated with injured nerve (Schwaiger et al.
2000; Sheu et al. 2000; Qiu et al. 2005). The goal of our
present experiments was, therefore, to investigate activation
of STAT3 by monitoring phosphorylation at both the Y705
and the S727 positions in DRG neurons associated and not
associated with unilateral sciatic nerve injury. To explore if
the IL-6 produced and released by lumbar DRG affected by
sciatic nerve injury can induce activation of STAT3 in cervical
DRG through cerebrospinal fluid of the subarachnoid
space, we analyzed activation and nuclear translocation of
STAT3(Y705) and STAT3(S727) in DRG after intrathecal
IL-6 delivery.
Materials and methods
Animals and surgical treatment
The experiments were performed in 32 adult male rats
(Wistar, 250–280 g, Anlab, Brno, Czech Republic) housed
in 12 h light/dark cycles at a temperature of 22–24 °C under
specific pathogen-free conditions in the animal housing
facility of Masaryk University. Sterilized standard rodent
food and water were available ad libitum. All experimental
procedures were carried out under sterile conditions by the
same person according to protocols approved by the Animal
Investigation Committee of the Faculty of Medicine, Brno,
Czech Republic. All surgical procedures were performed
under anesthesia using a mixture of ketamine (40 mg/ml)
and xylazine (4 mg/ml) administered intraperitoneally
(0.2 ml/100 g body weight).
The right sciatic nerve was exposed in the mid-thigh,
ligated using 2 ligatures and cut with a pair of sharp scissors
(complete sciatic nerve transection, CSNT, n = 8). The
proximal nerve stump was buried and fixed in muscles to
shield the distal stump from reinnervation. Alternatively,
a longitudinally slit 2-mm silicone tube of 1 mm internal
diameter was placed around the right sciatic nerve of rats
to reduce nerve diameter (sciatic nerve constriction, SNC,
n = 8) (Schmid et al. 2013). The tube was tied in place with
a sterile thread and closed to prevent the tube from opening.
The muscles and skin were closed with 5/0 sutures. The
right sciatic nerve of sham-operated rats (n = 8) was carefully
exposed without any lesion. All operated and shamoperated
rats were left to survive for 7 days. Eight other rats
without any surgical treatment were used as naive control.
Quantitative immunohistochemical analysis
Naïve, sham-, SNC- and CSNT-operated rats (n = 4 for each
group) were deeply anesthetized with a lethal dose of sodium
pentobarbital (70 mg/kg body weight, i.p.) and perfused
transcardially with 500 ml phosphate-buffered saline (PBS,
10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M
NaCl) followed by 500 ml of Zamboni’s fixative (Zamboni
and Demartin 1967). The L4-L5 and C6–C8 DRG from both
sides were detected within their intervertebral foramina following
total laminectomy and foraminotomy. The DRG
were removed, immersed separately in Zamboni’s fixative at
4 °C overnight, and then divided into samples of ipsilateral
(L-DRGi) and contralateral (L-DRGc) lumbar DRG as well
as ipsilateral (C-DRGi) and contralateral (C-DRGc) cervical
DRG separately for each group of rats (naïve, sham-, SNC-,
and CSNT-operated). The DRG samples were washed in
20% phosphate-buffered sucrose for 12 h, blocked in TissueTek®
OCT compound (Miles, Elkhart, IN) and cut to prepare
serial longitudinal cryostat sections (12 µm). The DRG
sections were mounted on chrome-alum covered slides and
processed for indirect immunohistochemical staining.
Briefly, DRG sections of lumbar and cervical segments
of naïve, sham-, SNC- and CSNT-operated rats were immunostained
simultaneously under the same conditions. Sections
were washed with PBS containing 0.05% Tween 20
(PBS-T) and 1% bovine serum albumin (BSA) for 10 min,
treated with 5% normal donkey serum in PBS-T for 30 min,
then incubated with 25 µl of rabbit polyclonal antibody
against STAT3(Y705) (1:100; Santa Cruz, USA) or rabbit
polyclonal antibody against STAT3(S727) (1:200; Abcam,
USA) in a humid chamber at room temperature (21–23 °C)
for 12 h. The immunohistochemical reaction was visualized
using TRITC-conjugated and affinity-purified donkey
anti-rabbit secondary antibody (1:100; Millipore, USA)
for 90 min at room temperature. The control sections were
incubated either without the primary STAT3(Y705) or
STAT3(S727) antibodies or by substituting the primary
antibodies with the donkey IgG isotype. The sections were
stained with Hoechst 33,342 to detect cell nuclei, mounted
in aqueous mounting medium (Vectashield; Vector Laboratories,
USA) and analyzed using an epifluorescence microscope
(Nikon Eclipse) equipped with a Nikon DS-Ri1 camera
(Nikon, Prague, Czech Republic) and a stabilized power
supply for the lamp housing.
The STAT3(Y705) and STAT3(S727) immunofluorescence
intensities in the nuclei of DRG neurons were measured
using the NIS-Elements image analysis system (Nikon,
Histochemistry and Cell Biology	
1 3
Czech Republic) according to our previously published
protocols (Dubovy et al. 2002, 2013). At least 200 nuclei
of DRG neuronal profiles were measured for each animal
group. The immunofluorescence intensities were expressed
as mean intensities ± SD.
Double immunostaining
A portion of the sections was incubated with a mixture (1:1)
of rabbit STAT3(Y705) polyclonal antibody (1:100; Santa
Cruz, USA) and mouse monoclonal antibody against ATF3
(1:100; Santa Cruz, USA) as a marker of axotomized neurons
(Tsujino et al. 2000). Another portion of DRG sections
was incubated with mouse monoclonal (1:150, Acris, USA)
or rabbit polyclonal antibody (1:200; Abcam, USA) against
STAT3(S727) and rabbit monoclonal anti–Rab7 antibody
(1:100, Cell Signaling Technology, The Netherlands) or
mouse monoclonal anti-EEA antibody (1:50, Santa Cruz,
USA) to detect STAT3(S727) in late or early endosomes,
respectively.
A mixture (1:1) of affinity purified TRITC-conjugated
donkey anti-rabbit and FITC-conjugated donkey anti-mouse
secondary antibodies (1:100; Millipore) was applied at room
temperature for 90 min. Control sections were incubated
without individual primary antibodies.
Western blot analysis
To validate the STAT3(Y705) and STAT3(S727) levels in
DRG neurons obtained by quantitative immunohistochemical
analysis, protein levels were analyzed by Western blot. In
addition, total non-phosphorylated STAT3 protein was quantified.
The naïve rats, sham-, SNC- and CSNT-operated rats
(n = 6 for each group) were deeply anesthetized with a lethal
dose of sodium pentobarbital (70 mg/kg body weight, i.p.)
and DRG of both sides were removed under aseptic conditions,
washed in protease and phosphatase inhibitor cocktails
(both Roche, Germany), fast frozen in liquid nitrogen and
stored at − 80 °C until analyzed further. The DRG of lumbar
(L4-L5) and cervical (C6–C8) segments were separated into
samples of ipsilateral (L-DRGi) and contralateral (L-DRGc)
lumbar DRG as well as ipsilateral (C-DRGi) and contralateral
(C-DRGc) cervical DRG for each group of rats (naïve,
sham-, SNC- and CSNT-operated). The DRG samples were
homogenized in PBS containing 0.1% Triton X-100 and
cocktails of protease and phosphatase inhibitors (Roche,
Germany) and centrifuged at 10,000 g for 5 min at 4 °C.
Proteins were separated by SDS–polyacrylamide gel electrophoresis
(Brazda et al. 2006) and transferred to nitrocellulose
membranes by electroblotting (BioRad). Blots were
blocked using 1% BSA in PBST (3.2 mM ­Na2HPO4, 0.5 mM
­KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH
7.4) for 1 h and incubated with rabbit polyclonal antibody
against STAT3(Y705) (1:100; Santa Cruz, USA), rabbit
polyclonal antibody against STAT3(S727) (1:200; Abcam,
USA) or mouse monoclonal antibody against STAT3
(1:1000; Cell Signaling, UK) overnight. After washing in
PBST, blots were incubated with peroxidase-conjugated
anti-rabbit or anti-mouse IgG (1:1000; Sigma, USA) at room
temperature for 1 h. Protein bands were visualized using
the ECL detection kit (Amersham, USA) on the chemiluminometer
reader LAS-3000 (Fuji, Japan) and analyzed using
image densitometry software. The levels of proteins were
normalized to the value of naïve lumbar and cervical DRG
which were arbitrarily set to one.
Quantitative assay of IL‑6 protein
in the cerebrospinal fluid
Naïve rats and those subjected to SNC or sham-operation
for 7 days (n = 6 for each group) were sacrificed by ­CO2
inhalation. Samples of the cerebrospinal fluid (CSF) were
obtained by puncture of the cisterna magna and collected
into tubes and stored at − 80 °C until analysis; care was
taken to avoid blood contamination. The total protein concentration
was measured by Nanodrop ND-1000 (Thermo
Scientific) and the level of IL-6 protein was assessed by
Quantibody® Rat Inflammation Array 1 (RayBiotech,
GA, USA) according to the manufacturer’s instructions.
Each sample collected from the individual groups of animals
was measured in quadruplicate and the data were
expressed as mean ± SEM of IL-6 protein (pg per ml of
CSF or per µg of total protein).
Intrathecal IL‑6 administration
Rats were anesthetized using a mixture of ketamine (40 mg/
ml) and xylazine (4 mg/ml) administered intraperitoneally
(0.2 ml/100 g body weight). IL-6 protein (R&D Systems)
was dissolved in artificial cerebrospinal fluid (ACSF; Hylden
and Wilcox 1980) at 20 ng/10 µl concentration. Solution of
IL-6 (10 µl) or ACSF (10 µl) with a further 10 µl ACSF was
injected via a micro syringe into the subarachnoid space
of the cisterna magna (cervical, n = 4) or between L2-L3
vertebrae (lumbar, n = 4). Animals were sacrificed in ­CO2
and perfused transcardially with Zamboni´s fixative solution
(Zamboni and Demartin.C 1967). Lumbar (L4-L5) and cervical
(C6–C8) DRG were removed following laminectomy
and foraminotomy, immersed in the Zamboni’s fixative overnight
and longitudinal cryostat sections (12 µm) were prepared.
Activation and nuclear translocation of STAT3(Y705)
and STAT3(S727) were detected and evaluated using image
analysis of immunofluorescence staining.
	 Histochemistry and Cell Biology
1 3
Statistical analyses
The Mann–Whitney U test was used to assess statistical differences
in the data of immunofluorescence intensities and
western blot analysis between naïve DRG neurons and DRG
neurons of sham-operated rats or DRG neurons after sciatic
nerve injury. Statistical significance was considered to be
indicated by a P value of < 0.05. All statistical analyses were
performed using STATISTICA 9.0 software (StatSoft, Inc.,
USA).
Results
Lumbar and cervical DRG neurons from naïve rats displayed
very low intensity of STAT3(Y705) immunofluorescence in
both nuclei and the cytoplasm (Fig. 1a). The neuronal nuclei
of both lumbar and cervical segments revealed a significant
increase in STAT3(Y705) intensity after sham-operation
compared to naïve rats (Fig. 1b; Table 1). In comparison to
DRG from naïve and sham-operated rats, a marked intensity
of STAT3(Y705) immunofluorescence was induced bilaterally
in the nuclei of lumbar and cervical DRG neurons after
both SNC and CSNT (Fig. 1c–j; Table 1). In addition, the
cytoplasm of some small- and medium-sized DRG neurons
showed a diffuse immunofluorescence after both types of
sciatic nerve injury (Fig. 1c–j). A moderate STAT3(Y705)
immunofluorescence was induced in the satellite glial cells
surrounding mainly large-sized neurons as was described
previously (Dubovy et al. 2010). We observed STAT3(Y705)
immunostaining also in the plasma membrane of large- and
medium-sized DRG neurons and their satellite glial cells
(Figs. 1c, d, i, j, 5c, d).
The nuclei of DRG neurons of both lumbar and cervical
segments removed from naïve and sham-operated rats
displayed a moderate intensity of STAT3(S727) immunofluorescence
with significantly higher levels in the neuronal
nuclei of DRG from sham-operated rats (Fig. 2a, b; Table 2).
The intensity of STAT3(S727) immunofluorescence was
significantly increased in neuronal nuclei 7 days after both
SNC and CSNT in comparison to sham-operated controls
(Fig. 2c–j; Table 2). In contrast to STAT3(Y705) immunostaining
results, the nuclei of DRG neurons of both lumbar
and cervical segments displayed a higher intensity of
STAT3(S727) immunofluorescence in the ipsilateral than
contralateral side after CSNT, while the bilateral intensity of
immunostaining following SNC was similar in the neuronal
nuclei (Table 2).
No apparent difference in the pattern of nuclear immunostaining
for STAT3(Y705) and STAT3(S727) was
observed between DRG of lumbar and cervical segments
in reaction to SNC and CSNT, even though only a part of
the neurons (about 20%) of ipsilateral lumbar DRG were
Fig. 1  Representative pictures of cryostat sections through the dorsal
root ganglia (DRG) from naïve (Naïve), sham-operated (Sham)
rats and rats with unilateral sciatic nerve compression (SNC) or
complete sciatic nerve transection (CSNT) for 7 days. The sections
of ipsilateral and contralateral DRG of the L4 spinal segment
(L4-DRGi, L4-DRGc) as well as ipsilateral and contralateral DRG of
the C7 spinal segment (C7-DRGi, C7-DRGc) were incubated under
the same conditions with rabbit polyclonal antibody recognizing
STAT3 phosphorylated in the Y705 position. In contrast to DRG of
naïve and sham-operated rat (a, b), bilateral activation and nuclear
translocation of STAT3Y705 was detected in the neurons of both
L4- and C7-DRG 7 days after unilateral SNC or CSNT (c–j). Along
with immunostained nuclei, a diffuse immunofluorescence was found
in the cytoplasm of medium- and small-sized neuronal bodies (long
arrows). In addition, STAT3(Y705) immunostaining was observed in
the plasma membrane of large- and medium-sized DRG neurons and
their satellite glial cells (arrowheads, c, d, i, j). Scale bars: 50 µm
Histochemistry and Cell Biology	
1 3
simultaneously immunopositive for ATF3 - a marker of axon
injury following SNC (Fig. 3a, b).
Besides nuclear translocation of STAT3(S727), the
immunoreaction corresponding to the activated form of
STAT3 was observed in vesicular structures in the cytoplasm
of DRG neurons of both lumbar and cervical segments after
SNC and CSNT (Fig. 2). Double immunostaining with
mouse monoclonal STAT3(S727) and rabbit monoclonal
Rab7 antibodies revealed activated STAT3 (phosphorylated
at the S727 position) in late endosomes (Fig. 3c–e). However,
no double immunofluorescence was observed when
the DRG sections were immunostained simultaneously with
STAT3(S727) antibody and antibody against the early endosomal
antigen EEA (data not shown).
To verify the results of bilateral STAT3 activation in DRG
neurons of both lumbar and cervical segments after unilateral
sciatic nerve injury, STAT3(Y705) and STAT3(S727)
protein levels were analyzed by Western blot. STAT3(Y705)
protein levels were increased bilaterally in both lumbar and
cervical DRG 7 days after unilateral SNC and CSNT when
compared with controls from naïve or sham-operated rats.
Only SNC induced a significantly higher STAT3(Y705) protein
level in the ipsilateral rather than contralateral lumbar
DRG, while cervical DRG of the same animals as well as
both lumbar and cervical DRG after CSNT revealed bilaterally
similar protein levels (Fig. 4a).
Protein levels of STAT3(S727) were also significantly
increased in cervical and lumbar DRG of both sides after
SNC and CSNT in comparison to controls from naïve or
sham-operated rats. In contrast to differences between ipsilateral
and contralateral lumbar and cervical DRG after
CSNT in STAT3(S727) as measured by image analysis, no
differences in whole STAT3(S727) protein levels were found
by Western blot analysis (Fig. 4b).
Table 1  Results of STAT3(Y705) immunofluorescence intensity
measured in the neuronal nuclei of lumbar (L-DRG) and cervical
(C-DRG) dorsal root ganglia of naïve rats (Naive), ipsilateral (i) and
contralateral (c) to sham-operation (Sham) and sciatic nerve compression
(SNC) or complete sciatic nerve transection (CSNT) for 7 days
(n = 6 for each group)
a
 Significant difference (p < 0.05) compared to Naïve
b
 Significant difference (p < 0.05) compared to the ipsilateral side
*Significant difference (p < 0.05) compared to Sham
L-DRGi L-DRGc C-DRGi C-DRGc
SNC 27.1 ± 6.0*a
33.3 ± 3.9*a
24.6 ± 7.3*a
25.2 ± 6.1*a
CSNT 22.5 ± 4.0*a
29.5 ± 5.8*ab
25.2 ± 4.1*a
28.7 ± 4.3*a
Sham 12.5 ± 2.6a
12.7 ± 1.3a
13.2 ± 2.6a
13.9 ± 2.1a
Naive 7.6 ± 1.3 8.7 ± 1.7
Fig. 2  Representative pictures of cryostat sections through the dorsal
root ganglia (DRG) showing immunofluorescence staining of activated
STAT3 by phosphorylation in the S727 position and its nuclear
translocation. The DRG were from naïve (Naïve), sham-operated
(Sham) rats and rats with unilateral sciatic nerve compression (SNC)
or complete sciatic nerve transection (CSNT) for 7 days. The sections
of ipsilateral and contralateral L4-DRG (L4-DRGi, L4-DRGc) as well
as ipsilateral and contralateral C7-DRG (C7-DRGi, C7-DRGc) were
incubated under the same conditions with rabbit polyclonal antibody
specific to STAT3 phosphorylated at the S727 position. Scale bars:
50 µm
	 Histochemistry and Cell Biology
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In contrast to STAT3(Y705) or STAT3(S727), protein
levels of total STAT3 recognized by antibody against nonphosphorylated
domains did not change in any DRG of
naïve, sham-, SNC- or CSNT-operated rats (Fig. 4c).
IL‑6 protein in the cerebrospinal fluid
The levels of IL-6 protein in CSF of naïve and sham-operated
rats were undetectable with the method used, but IL-6
protein was increased to 450.8 ± 142.3 or 4331.8 ± 132.3 pg/
µg total protein in rats with unilateral sciatic nerve injury.
Effect of intrathecal IL‑6 injection on STAT3
activation and nuclear translocation in DRG neurons
Activation of STAT3 is implicated in the IL-6 signaling
pathway. We, therefore, tested the nuclear translocation of
activated STAT3 in the DRG neurons of both lumbar and
cervical segments following intrathecal injection of IL-6. In
comparison to ACSF injection (Fig. 5a, b, e, f), intrathecal
application of IL-6 resulted in increased immunostaining for
STAT3(Y705) (Fig. 5 c, d) and STAT3(S727) (Fig. 5g, h) in
neuronal DRG nuclei of both lumbar and cervical segments.
These results demonstrate the activation and nuclear translocation
of STAT3 by IL-6 diffusing from the CSF in the
subarachnoid space along the spinal cord into DRG. No significant
differences were observed in nuclear translocation of
Table 2  Results of STAT3(S727) immunofluorescence intensity
measured in the neuronal nuclei of lumbar (L-DRG) and cervical
(C-DRG) dorsal root ganglia of naïve rats (Naive), ipsilateral (i) and
contralateral (c) to sham-operation (Sham) and sciatic nerve compression
(SNC) or complete sciatic nerve transection (CSNT) for 7 days
(n = 6 for each group)
a
 Significant difference (p < 0.05) compared to Naïve
b
 Significant difference (p < 0.05) compared to the contralateral side
*Significant difference (p < 0.05) compared to Sham
L-DRGi L-DRGc C-DRGi C-DRGc
SNC 19.2 ± 2.9*a
22.5 ± 4.2*a
19.7 ± 3.5*a
18.8 ± 3.2*a
CSNT 18.1 ± 2.3*ab
15.9 ± 2.0*a
19.6 ± 3.1*ab
16.4 ± 1.7*a
Sham 13.5 ± 1.6a
13.7 ± 2.8a
15.2 ± 1.8a
14.6 ± 2.0a
Naive 11.8 ± 1.5 11.7 ± 1.7
Fig. 3  a, b A cryostat section through the L4-DRG ipsilateral to
SNC after double immunostaining for STAT3(Y705) (red) and ATF3
(green). Merged red and green immunofluorescence (a) indicates that
only a portion of the neuronal nuclei displayed both STAT3(Y705)
and ATF3 immunostaining (arrows) while rest are labeled only by
STAT3(Y705) immunofluorescence and they did not express ATF3 as
a marker of axon injury. The nuclei of the same section were stained
with Hoechst (b). c–e A cryostat section through the L4-DRG neuron
ipsilateral to SNC for 7 days following double immunostaining
for detection of STAT3(S727) by mouse monoclonal antibody (red,
c) and Rab7 by rabbit monoclonal antibody (green, d) with Hoechst
staining of nuclei (blue). Merged immunofluorescence (e) indicates
that STAT3(S727) is localized in late endosomes (arrowheads)
besides the neuronal nucleus. Scale bars: 50 µm for a, b 20 µm for
c–e 
Histochemistry and Cell Biology	
1 3
activated STAT3 between IL-6 delivery into CSF of cisterna
magna or lumbar spinal cord segments.
Discussion
DRG neurons with the peripheral branches of their afferent
axons in the peripheral nerve are frequently used as an
in vivo model to study molecular mechanisms of neuronal
reaction to injury. Nerve lesion injury of peripheral axonal
branches induces upregulation of IL-6 and activation of
STAT3 in the corresponding DRG (Dubovy et al. 2013;
Miao et al. 2006; Murphy et al. 1999a).
We showed earlier that unilateral sciatic nerve injury can
induce increased levels of IL-6 protein and mRNA bilaterally
not only in DRG neurons associated with L4-L5 spinal
cord segments but also in remote DRG of cervical segments
(Dubovy et al. 2013). A similar bilateral increase of IL6R protein
and mRNA was detected in DRG after SNC (Brazda et al.
2013). Therefore, a similar pattern of STAT3 activation was
expected following unilateral SNC or CSNT.
Fig. 4  Results of Western blot analysis of STAT3Y (a), STAT3S
(b) and STAT3 (c) protein levels in DRG of L4-L5 (l) and C6–C8
(c) segments removed from ipsilateral (i) and contralateral (c) sides
of Naïve as well as sham-, SNC- and CSNT-operated rats for 7 days.
Upper panels illustrate representative Western blots of DRG from 6
naïve rats and 6 rats for each period of survival. Equal loading of proteins
was confirmed by actin levels (Actin). Lower panels show densitometry
of individual STAT3Y, STAT3S and STAT3 protein bands
after normalization to actin; the intensities of the STAT3Y, STAT3S
and STAT3 bands from naïve DRG were taken as 1
	 Histochemistry and Cell Biology
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Activation and translocation of STAT3Y705
and STAT3S727 in DRG after nerve injury
STAT3 has two conserved amino-acid residues (Y705 and
S727), which are phosphorylated during STAT3 activation
(Heinrich et al. 2003). Thus far, activation of STAT3 was
detected only at the Y705 position in DRG neurons ipsilateral
to peripheral nerve injury (Qiu et al. 2005; Wu et al.
2007; Bareyre et al. 2011). We present here, for the first
time, activation of STAT3 also at the S727 position with
the subsequent nuclear translocation in DRG neurons after
two types of sciatic nerve injury. The coincident activation
of STAT3 in the Y705 and S727 positions after two types
of sciatic nerve injury in the DRG neurons was shown by
immunohistochemical staining and Western blot analysis.
Total STAT3 protein levels remained unaffected when compared
to naïve, sham-, SNC- and CSNT-operated animals.
The results suggest that the functional effect of STAT3 is
elicited through post-translational modification and/or translocation,
cytoplasmic translation and nuclear transcription
(Nicolas et al. 2012; Haghikia et al. 2014).
Activation of STAT3 by phosphorylation at both the
Y705 and S727 positions was also detected in motor neurons
in vivo (Schwaiger et al. 2000) and PC12 line cells
and sympathetic neurons in vitro (Pellegrino and Habecker
2013; Ng et al. 2006a). In contrast to STAT3(Y705) translocated
into the nuclei of PC12 line cells, STAT3(S727) was
found in axons and their growth cones (Ng et al. 2006a).
Similarly, STAT3(S727) was detected in the axons and their
growth cones of sympathetic neurons, and in their nuclei which
was not the case for PC12 line cells (Pellegrino and
Habecker 2013).
As was expected, the STAT3(Y705) immunostaining
was found predominantly in the neuronal nuclei of DRG
indicating nuclear translocation of STAT3 activated in the
Y705 position after sciatic nerve injury or intrathecal IL-6
injection. In addition, diffuse STAT3(Y705) immunofluorescence
was present in the cytoplasm of some DRG neurons.
STAT3(Y705) immunostaining was found also in the plasma
membrane of some DRG neurons and their satellite glial
cells—mainly after intrathecal injection of IL-6. Activated
STAT3 has been reported to affect cell adhesion (Silver et al.
2004; Shah et al. 2006; Hombria and Sotillos 2008) and
the plasma-membrane location of STAT3(Y705) in DRG
is likely associated with the mutual contact of neurons and
their satellite glial cells. Nevertheless, the precise functional
involvement of membrane distribution of activated STAT3
in the DRG requires further experimental evidence. Besides
neuronal nuclei, STAT3(S727) vesicular immunofluorescence
was also found after both types of sciatic nerve injury
and intrathecal IL-6 injection. Double immunostaining
revealed that at least a part of vesicular immunoreactivity
for STAT3 phosphorylated in the S727 position is present in
late endosomes which also contain MAPKs (German et al.
2011). Thus, our immunohistochemical results showing different
intracellular locations of STAT3 phosphorylated at
the Y705 and S727 positions are consistent with a model
of cytoplasmic and endocytic regulation of IL-6-induced
STAT3 activation (Shah et al. 2006; German et al. 2011).
Functional involvement of STAT3(S727) is not completely
clear until now. Activated STAT3 phosphorylated
Fig. 5  Representative pictures of cryostat sections through the dorsal
root ganglia (DRG) of L4 and C7 spinal segments from rats treated
with intrathecal injection of artificial cerebrospinal fluid (a, b, e,
f) or IL-6 (c, d, g, h) for 1 day. The cryostat sections were immunostained
with rabbit polyclonal antibody recognizing STAT3 activated
by phosphorylation at the Y705 (A-D) or S727 (E-H) positions.
In contrast to sections through DRG of animals treated with artificial
cerebrospinal fluid (ACSF), intrathecal IL-6 injection induced a
strong increase of STAT3(Y705) and STAT3(S727) immunofluorescence
in neuronal nuclei. Similarly to the DRG after SNC and CSNT,
STAT3(S727) immunostaining was present also in vesicular structures
of neurons (arrowheads) besides nuclear staining. The plasma
membrane localization of STAT3(Y705) immunostaining was obvious
in DRG neurons and their satellite glial cells after intrathecal
injection of IL-6 (arrowheads, c, d,). Scale bars: 50 µm
Histochemistry and Cell Biology	
1 3
at the S727 position may facilitate interaction with the
transcriptional co-activators CBP and P300, maximizing
the transcriptional activity of STAT3(Y705) (Schuringa
et al. 2001). In addition, phosphorylation of the S727 position
may modulate the duration of STAT3 transcriptional
activity by promoting dephosphorylation of STAT3(Y705)
(Wakahara et al. 2012). However, STAT3(S727) has also
been suggested to mediate transcription activation without
any detectable STAT3 phosphorylated at the Y705 position
(Decker and Kovarik 2000). However, maximal sympathetic
axon outgrowth requires phosphorylation of STAT3 at both
positions (Pellegrino and Habecker 2013).
Activation of STAT3 in lumbar and cervical DRG
following unilateral sciatic nerve injury and its
possible functional involvement
The bilateral activation of STAT3 at both the Y705 and S727
positions and their nuclear translocation were detected in
DRG neurons of both lumbar and cervical segments following
unilateral SNC or CSNT. Double immunostaining for
STAT3(Y705) and ATF3 in sections through lumbar DRG
ipsilateral to SNC showed STAT3 activation also in the DRG
neurons without any axonal injury. Moreover, activation and
nuclear translocation of activated STAT3 in lumbar DRG
neurons contralateral to nerve injury as well as in cervical
segments of both sides, i.e., in DRG neurons without anatomical
relation to the injured nerve, suggests the diffusion
of signal molecules into DRG along the spinal cord. The
cytokine IL-6 is a candidate signal molecule for spreading
neuroinflammation after nervous system injury (Erta et al.
2012) and its increased synthesis was detected in the associated
DRG but also in DRG not associated with the injured
nerve (Dubovy et al. 2013). Although the expression of IL-6
in DRG associated with injured sciatic nerve correlated well
with the duration of hypersensitivity (Murphy et al. 1999b),
the possible involvement of increased IL-6 levels in remote
DRG neurons in the induction or maintenance of neuropathic
pain has not yet been demonstrated. The results seem
to reflect rather a general neuroinflammatory reaction of
DRG along the neural axis (Dubovy et al. 2013).
What is still unclear is the mechanism for the activation
of STAT3 in DRG neurons not associated with unilateral
peripheral nerve injury. We have detected a robust penetration
of dextran molecules of molecular weight similar to
IL-6 from CSF of the spinal subarachnoid space into DRG
(Joukal et al. 2016). Intrathecal injection of IL-6 with subsequent
activation of STAT3 presented here suggested
that IL-6 in the CSF of perispinal subarachnoid space is
a possible mediator of STAT3 activation in DRG neurons
not associated with the injured nerve. If there is no barrier
between DRG and the CSF of subarachnoid space, a
nerve injury-induced increase of IL-6 in the corresponding
DRG can release IL-6 molecules into the subarachnoid space
and their transport via CSF into remote DRG can activate
STAT3 in their neurons. This is supported by the observation
of increased IL-6 protein levels in CSF of rats with
sciatic nerve injury. Moreover, STAT3 activated primarily by
nerve injury-induced IL-6 may trigger further synthesis and
release of IL-6 through the amplified feedback loop detected
in various cell models (Murakami and Hirano 2012).
A further possible mechanism for the bilateral regulation
of the IL-6/STAT3 pathway in both lumbar and cervical
DRG neurons after unilateral sciatic nerve injury is
suggested by published results from exosomes containing
microRNAs (miRNAs) released after nerve injury. Various
miRNAs were significantly increased in DRG neurons and
released by exosomes after peripheral nerve injury (Wu et al.
2011; Hori et al. 2016; Chang et al. 2017). Notably, miR-21
was gradually increased in DRG in an IL-6-dependent manner
and significantly increased in exosomes extracted from
the blood of nerve-ligated mice (Hori et al. 2016). Moreover,
it was demonstrated that miR-21 expression is strictly upregulated
via an IL-6/STAT3 pathway (Loffler et al. 2007) and
miRNAs may regulate transcription of STAT target genes
(Pencik et al. 2016). However, any direct functional relationship
between miR-21 and activated STAT3 in DRG neurons
after nerve injury remains unconfirmed.
In conclusion, we detected STAT3 activation by phosphorylation
at the Y705 and S727 positions in both lumbar and
cervical DRG neurons after unilateral sciatic nerve injury
of various types. We showed that IL-6 from the CSF in the
perispinal subarachnoid space can mediate STAT3 activation
in remote DRG neurons. These results indicate a systemic
reaction of DRG neurons alongside the spinal cord induced
by unilateral spinal nerve injury.
Acknowledgements We thank Ms. Dana Kutějová, Ms. Jitka
Mikulášková, Ms. Marta Lněníčková, Mgr. Jana Vachová and Mr.
Lumír Trenčanský for their skillful technical assistance. Supported by
grant 16-08508S of The Czech Science Foundation.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no competing
interests.
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B. Dubový P, Klusáková I, Hradilová-Svíženská I, Joukal M. Expression of RegenerationAssociated
Proteins in Primary Sensory Neurons and Regenerating Axons After Nerve
Injury-An Overview. Anat Rec Hoboken NJ 2007. 2018; doi:10.1002/ar.23843
Commentary: In this publication, we summarized the critical issues of immunohistochemical
detection of regenaration associated proteins in primary sensory neurons and axons. The
overview was supplemented with our own results of GAP43 and SCG10 immunostaining in
DRG after nerve injury.
IF (WOS, 2017): 1.373
Quartile (WOS, 2017): Anatomy and Morphology Q3
Contribution of the author: Co-authorship; approximate participation 20%. The author
participated in performing of in vivo experiments, preparing samples for
immunohistochemistry and writing of the corresponding texts.
Expression of Regeneration-Associated
Proteins in Primary Sensory Neurons
and Regenerating Axons After Nerve
Injury—An Overview
PETR DUBOVY ,* ILONA KLUSAKOVA,
IVANA HRADILOVA-SVIZENSKA, AND MAREK JOUKAL
Department of Anatomy, Cellular and Molecular Research Group, Masaryk University,
Brno, Czechia, Czech Republic
ABSTRACT
Peripheral nerve injury results in profound alterations of the affected
neurons resulting from the interplay between intrinsic and extrinsic molecular
events. Restarting the neuronal regenerative program is an important
prerequisite for functional recovery of the injured peripheral nerve. The primary
sensory neurons with their cell bodies in the dorsal root ganglia provide
a useful in vivo and in vitro model for studying the mechanisms that
regulate intrinsic neuronal regeneration capacity following axotomy. These
studies frequently need to indicate the regenerative status of the corresponding
neurons. We summarize the critical issues regarding immunohistochemical
detection of several regeneration-associated proteins as markers for the
initiation of the regeneration program in rat primary sensory neurons and
indicators of axon regeneration in the peripheral nerves. This overview also
includes our own results of GAP43 and SCG10 expression in different DRG
neurons following double immunostaining with molecular markers of neuronal
subpopulations (NF200, CGRP, and IB4) as well as transcription factors
(ATF3 and activated STAT3) following unilateral sciatic nerve injury. Anat
Rec, 00:000–000, 2018. VC 2018 Wiley Periodicals, Inc.
Key words: peripheral nerve; regeneration; degeneration;
therapies
Peripheral nerve injury results in profound alterations
of metabolism, survival, excitability, and transmitter
functions of the affected neurons. These processes result
from the interplay between intrinsic and extrinsic molecular
events (Fawcett and Keynes, 1990; Doron-Mandel
et al., 2015); restarting the regenerative program and the
capacity to survive injury are important prerequisites for
functional recovery in injured peripheral nerves.
Primary sensory neurons with their cell bodies in the
dorsal root ganglia (DRG) provide a useful in vivo and in
vitro model to study the mechanisms regulating intrinsic
neuronal regeneration capacity following axotomy. These
studies frequently need to indicate the regenerative status
of specific neurons using a simple and reliable method.
The aim of the present overview is to critically summarize
recent knowledge, combined with our own experience,
regarding the immunocytochemical detection of various
regeneration-associated proteins to be used as markers
labeling the initiation and progress of the regeneration
Grant sponsor: The Czech Science Foundation; Grant number:
16-08508S.
*Correspondence to: Petr Dubovy, Professor and Head,
Department of Anatomy, Faculty of Medicine, Kamenice 3, CZ-
625 00 Brno, Czech Republic. Tel.: (420) 54949-1333 E-mail:
pdubovy@med.muni.cz
Received 16 October 2017; Revised 9 November 2017;
Accepted 8 December 2017.
DOI 10.1002/ar.23843
Published online 00 Month 2018 in Wiley Online Library
(wileyonlinelibrary.com).
THE ANATOMICAL RECORD 00:00–00 (2018)
VVC 2018 WILEY PERIODICALS, INC.
Rec, 301:1618–1627, 2018. © 2018 Wiley Periodicals, Inc.
Published online 8 May 2018 in Wiley Online Library
(wileyonlinelibrary.com).
THE ANATOMICAL RECORD 301:1618–1627 (2018)
© 2018 WILEY PERIODICALS, INC.
program in primary sensory neurons as well as indicators
of axon regeneration in peripheral nerves.
CLASSIFICATION OF NEURONS
IN THE DRG
The DRG contain the bodies of primary sensory neurons
that are markedly diverse as to their physiological
function in afferent signaling and their reactions to a
peripheral nerve injury. The subpopulations of DRG neurons
can be classified according their size, cytological,
chemical, and physiological properties (Lawson, 2002).
Classically, DRG neurons are divided into large-, medium,
and small-sized (Lawson et al., 1974) and may be identified
with further molecular phenotyping. The large neurons
that sendoff heavy, myelinated axons also display
strong immunostaining for NF200, while medium- and
small-sized neurons give off lightly myelinated Ad and
unmyelinated C axons, respectively (Lawson, 2002). The
medium- and small-sized neurons that express substanceP
and calcitonin gene-related peptide (CGRP) are classified
as peptidergic, while small neurons free of CGRP but
stained with Griffonia simplicifolia isolectin B4 (IB4) are
denoted as nonpeptidergic nociceptive neurons (Lawson
et al., 1993; Hunt and Mantyh, 2001). The IB41 subpopulation
differs from other neurons in expressing the receptor
for glial cell line-derived neurotrophic factor (GDNF)
rather than other neurotrophins (Molliver et al., 1997). A
number of in vivo studies of the neuronal regenerative
program were based on rat or mouse sciatic nerve injuries
where nearly all (98%–99%) sciatic DRG neurons were
located in the L4-L5 DRG of rat (Swett et al., 1991) or the
L3-L4 DRG of mice (Rigaud et al., 2008).
REGENERATION-ASSOCIATED PROTEINS
Besides extrinsic factors produced by cells distal to
the nerve injury, reactivation of a silent regenerative
program of DRG neurons is needed for successful axon
regeneration. The reactivation of the neuronal regeneration
program can be detected by the expression of
various regeneration-associated proteins like growth
associated protein 43 (GAP43), cortical cytoskeleton
associated protein 23 (CAP23), superior cervical ganglion
10 (SCG10), or small proline-rich repeat protein 1A
(SPRR1A) that are important intrinsic determinants of
the increased regenerative ability of DRG neurons
(Bonilla et al., 2002; Mason et al., 2002).
Growth-Associated Protein 43
The prototypical growth-associated protein, GAP43, is
a membrane- and cytoskeletal-associated phosphoprotein
that is expressed at high levels in neurons during development
and is concentrated in the axonal growth cone
(Skene and Willard, 1981). Correlation between the
expression of GAP43 and axonal growth has led to it
being used as a marker for the regenerative state of neurons
(Verge et al., 1990). GAP43 regulates the neuronal
actin cytoskeleton and is a substrate for protein kinase
C (PKC; Larsson, 2006) in promoting axonal growth
(Laux et al., 2000). Removal of the distal nerve stump or
blocking axonal transport does not alter GAP43 mRNA
levels in DRG neurons indicating that molecular
inducers produced by nerve injury are not critical for
GAP43 upregulation (Skene, 1989).
The DRG neurons of na€ıve rats display a low basal
level of GAP43 immunostaining (Stewart et al., 1992;
Schreyer and Skene, 1993) that is significantly increased
mainly in large and medium-sized neurons after sciatic
nerve injury (Table 1; Fig. 1A,D). In contrast to large
NF2001 and medium sized CGRP1 neurons, small
IB41 neurons indeed did not display GAP43 after experimental
nerve injury (Fig. 2). The absence of GAP43 protein
in small IB41 neurons is in accord with in vitro
experiments that IB4-labeled primary sensory neurons
do not express GAP43, and this is related to their
impaired intrinsic axonal regeneration capacity (Leclere
et al., 2007). Another explanation for these findings is
that other proteins or signaling molecules, in addition to
GAP43, are required for stimulation of axonal outgrowth.
Moreover, axonal regeneration of IB4-labeled
neurons can be intrinsically different from that of other
classes of DRG neurons where SPRR1A is upregulated
after sciatic nerve injury and that is also associated with
axonal regeneration (Bonilla et al., 2002). Increased
immunostaining for GAP43 can be stimulated over a
long period also in DRG neurons that do not or cannot
successfully regenerate their injured axons after nerve
transection (Woolf et al., 1990). This indicates that the
mere presence of GAP43 protein is not sufficient as proof
of neuronal regeneration status.
It was found that only GAP43 immunoreactive neurons
supported axon regrowth of 7 mm or longer within
the first week following dorsal root injury. At later time
points, axon regrowth was observed from neurons both
with and without GAP43 immunoreactivity. The results
suggest that axon regeneration is not absolutely dependent
on the presence of GAP43 but rather its expression
is correlated with a capacity for rapid outgrowth (Andersen
and Schreyer, 1999).
GAP43 is activated by PKC-mediated phosphorylation
at serine 41 (S41) that promotes the polymerization and
stabilization of filamentous actin (F-actin) related to
axon growth and sprouting (Tsai et al., 2007). The phosphorylated
form is dephosphorylated by calcineurin,
thus blocking F-actin polymerization and stabilization
(Lautermilch and Spitzer, 2000). In contrast to immunostaining
predominantly of large-sized neurons in na€ıve
DRG by antibodies, recognizing both phosphorylated and
unphosphorylated GAP43, an S41-GAP43-specific antibody
immunostained mainly medium- and small-sized
DRG neurons (Table 1; Fig. 1B). However, a more
intense S41-GAP43 immunoreaction was induced by
axotomy in all types of DRG neurons (Fig. 1C) indicating
that immunostaining with S41-GAP43 antibody is a reliable
indicator of the neuronal regeneration program.
Superior Cervical Ganglion 10 Protein
Superior cervical ganglion 10 (SCG10) protein, also
known as stathmin 2 (STMN2), is a neuron-specific member
of the stathmin family (Sugiura and Mori, 1995)
whose members influence microtubule dynamics by promoting
microtubule depolymerization (Riederer et al.,
1997; Antonsson et al., 1998). Overexpression of SCG10
protein in neurons enhances axon outgrowth under
in vitro (Riederer et al., 1997) as well as under in vivo conditions
(Mason et al., 2002).
2 DUBOVY ET AL.PROTEINS ASSOCIATED WITH NEURON REGENERATION 1619
Upregulation of SCG10 mRNA could not be detected
in ipsilateral axotomized sensory neurons one day after
nerve transection, but became apparent only later during
the first week after surgery (Voria et al., 2006). This
is consistent with the results of Mason et al. (2002) who
found increased SCG10 mRNA levels in DRG neurons
and spinal motor neurons 3 days after nerve crush, but
not at 1 day. In contrast, the intensity of SCG10 immunostaining
was significantly increased in most neurons
of the rat DRG within 1 day-crushed nerve (Table 1; Fig.
3A,B). This is in agreement with published results in
mice (Shin et al., 2014). The discrepancy in the onset of
SCG10 expression after nerve injury is probably due to
differences in experimental animals, procedure for nerve
crush and methods of SCG10 detection. It is important
to note that in contrast to GAP43, all DRG neurons with
activating transcription factor 3 (ATF3) immunopositive
nuclei displayed SCG10 immunostaining 7 days after
complete sciatic nerve transection (CSNT; Fig. 3C–E).
Differences of GAP43 and SCG10 as
Markers of Neuronal Regenerative
Status and Regenerating Axons
It was demonstrated that GAP43, CAP23, and SCG10
are coregulated in axotomized neurons and are correlated
with the neuronal ability to regenerate their axons
under in vivo conditions (Mason et al., 2002). However,
other results of in vivo experiments also revealed upregulation
of GAP43 in the DRG neurons without axotomy
suggesting that increased GAP43 may not always be
related to activation of the regeneration program of neurons,
but is rather a metabolic sign of neuronal potential
for plasticity and rapid axon regeneration (Benowitz and
Routtenberg, 1997; Andersen and Schreyer, 1999). Moreover,
as was mentioned above, injured IB41 nociceptive
neurons do not express GAP43 although the neurons
displayed other molecules like SCG10 indicating their
regenerative status (Bonilla et al., 2002). Double
TABLE 1. Antibodies used for immunostaining of representative sections and summary of their cellular
expression in DRG neurons or regenerated axons of the peripheral nerves
Marker Host Specificity Dilution Supplier Immunofluorescence staining
GAP43 Mousea
Phosphorylated and
dephosphorylated
1:500 Sigma GAP43c
in large and medium
sized DRG neurons of the
regenerative state, small
IB4c
neurons are GAP43–
,
GAP43c
in regenerated
axons and activated
Schwann cells
S41-GAP43 Rabbitb
Phosphorylated
S41-GAP43
1:500 Thermo Fisher S41-GAP43c
in all types of
DRG neurons of the regenerative
state
ATF3 Rabbitb
C-terminal peptide 1:200 Santa Cruz ATF3c
in the nuclei of axotomized
DRG neurons
ATF3 Mousea
Internal region of
ATF3
1:200 Santa Cruz ATF3c
in the nuclei of axotomized
DRG neurons
NF200 Rabbitb
Neurofilament H
(200kD)
1:200 Sigma NF200c
in large sized DRG
neurons with heavy myelinated
axons
CGRP Rabbitb
CGRP 1:500 Acris CGRPc
in peptidergic subpopulation
of medium and
small sized DRG neurons
IB4-FITC – Isolectin B4 1:50 Vector IB4c
in nonpeptidergic subpopulation
of small sized
DRG neurons
SCG10 Rabbitb
C-terminal peptide 1:1000 LSBio SCG10c
in all types of DRG
neurons of the regenerative
state, SCG10c
in sensory
regenerated axons also
after a short period of nerve
injury
Anti-bIII tubulin Mousea
C-terminal peptide 1:500 Exbio For double immunostaining
with GAP43 or SCG10 to
detect regenerated axons
SPRR1A Rabbitb
Internal 40–89
amino acids
1:300 Abcam SPRR1Ac
in all types of DRG
neurons of the regenerative
state
STAT3 Rabbitb
Phosphorylated at
Y705
1:100 Santa Cruz STAT3c
as a marker of regenerative
state of DRG
neurons
a
Monoclonal antibody.
b
Polyclonal antibody.
c
Positive immunoreaction and –
negative immunoreaction.
The binding of primary antibodies was visualized with donkey anti-mouse or anti-rabbit TRITC- or FITC-conjugated secondary
antibodies (1:400; Millipore).
PROTEINS ASSOCIATED WITH NEURON REGENERATION 3DUBOVÝ ET AL.1620
immunofluorescence staining of DRG neurons after sciatic
nerve injury demonstrated a higher amount of
SCG101 than GAP431 neurons without differences in
DRG subpopulations (Fig. 3F–H).
GAP43 is a widely used marker for regenerating
axons, but besides regenerating axons, it is also upregulated
in Schwann and Schwann-derived cells (Plantinga
et al., 1993; Dubovy and Aldskogius, 1996). This GAP43
immunostaining of Schwann cells associated with regenerated
axons make their detection difficult, especially
during early periods of axon regeneration (Fig. 4A).
It was demonstrated that SCG10 is transported from
the soma to the proximal axonal stump very early after
axotomy, whereas it is rapidly lost in distal axon stumps
due to phosphorylation by cJun N-terminal kinase (JNK;
Tararuk et al., 2006; Shin et al., 2012, 2014). Thus,
SCG10 immunostaining is rapidly increased in proximal
axonal stumps and regenerating sprouts within 1 hr
after axotomy (Shin et al., 2012, 2014) in contrast to
GAP43 labeling regenerated axons after 3 days (Shalom
and Yaron, 2014). Therefore, detection of regenerated
axons by SCG10 immunostaining is more useful mainly
during early periods after nerve injury, for example, 1
day after nerve crush (Fig. 4B–D). In addition, SCG10 is
a good marker for regenerated sensory axons, as its levels
increase only in the proximal stumps of afferent but
not motor axons of the injured sciatic nerve (Shin et al.,
2014).
Small Proline-Rich Repeat Protein 1A
SPRR1A or Cornifin-A is one of a group of epithelial
differentiation proteins that are induced in axotomized
neurons. A very low level of SPRR1A is found in intact
DRG neurons, but peripheral nerve injury induces a 60fold
increase of its expression in axotomized DRG
Fig. 1. Representative sections of L4-DRG of na€ıve rat (A, B) and from rat with CSNT for 7 days (C–F). The sections (A, D–F) were immunostained
with mouse monoclonal anti-GAP43 antibody recognizing both phosphorylated and dephosphorylated forms. For double immunostaining
(D–F), the section was further processed for immunostaining with rabbit polyclonal antibody against ATF3 and cell nuclei were stained by
Hoechst 33342 (blue). Low intensity GAP43 immunofluorescence is seen mainly in large DRG neurons of na€ıve rat (A, arrowheads). Increased
intensity of GAP43 is detected in large-sized neurons (arrowheads), while medium- (thick arrows) or small-sized neurons (slim arrows) display
weak or no immunopositivity, respectively (D). Most neuronal nuclei were simultaneously decorated by ATF3 immunopositivity indicating injury to
their axons (E), but the merged picture (F) illustrates that neurons with weak or no GAP43 immunostaining showed axon injury (thick and slim
arrows). The sections of L4-DRG from na€ıve (B) and CSNT–operated (C) rat were immunostained for S41-GAP43 and cell nuclei were labeled by
Hoechst 33342 (blue). Large neuronal bodies lack S41-GAP43 immunofluorescence, but they are enveloped by S41-GAP431 satellite glial cells
(B, arrowheads). In addition, medium- and small-sized neuronal bodies of na€ıve DRG displayed a low intensity of S41-GAP43 immunofluorescence
(B, arrows). In contrast to GAP43, all DRG neurons including those with large (arrowheads) and medium or small bodies (arrows) display
increased S41-GAP43 immunostaining after axotomy by CSNT (C). Scale bars: 135 mm for A, B; 77 mm for C–F.
4 DUBOVY ET AL.PROTEINS ASSOCIATED WITH NEURON REGENERATION 1621
neurons. In addition, overexpression of SPRR1A colocalizes
with F-actin in membrane folds of DRG neurons
and significantly increases axon outgrowth in vitro,
while knockdown of SPRR1A gene inhibits axon regeneration
(Bonilla et al., 2002). Since SPRR1A is not normally
present but is expressed de novo in DRG neurons
following peripheral nerve injury at a time corresponding
to rapid regeneration, it has been proposed as good
marker for a reactivated neuronal regeneration program
(Bonilla et al., 2002; Starkey et al., 2009).
However, immunohistochemical staining for SPRR1A in
L4-DRG of mice undergoing sciatic nerve transection
shows a smaller increase in immunofluorescence intensity
in large neurons compared to small ones (Table 1; Fig. 5).
MOLECULAR MARKERS INVOLVED
IN THE STRESS-RELATED RESPONSE OF
AXOTOMIZED DRG AND THE ONSET OF THE
AXON REGENERATION PROGRAM
In contrast to the regeneration-associated proteins
mentioned above, another unique set of molecular
markers can be used for monitoring the regenerative
status of DRG neurons. This group of markers induced
by axon injury includes transcription factors involved
either in the stress-related response of axotomized neurons
or contributing to the onset of axon regeneration.
These markers include injury-induced molecules like
ATF3 (Tsujino et al., 2000) or activated signal
Fig. 2. Representative sections of L4-DRG ipsilateral to CSNT for 7 days double immunostained for GAP43 and NF200 (A–C) or CGRP (D–F)
or stained with FITC labeled IB4 (G-I). The highest intensity GAP43 immunostaining is limited to NF2001 neurons with large-sized bodies (A–C,
arrowheads). A weak GAP43 reaction is present in CGRP1 neurons of medium-sized bodies (D–F, arrowheads), but CGRP1 neurons of smallsized
bodies are free of GAP43 immunostaining (D–F, arrows). In contrast to large neurons (arrowheads), no GAP43 immunofluorescence is
observed in nonpeptidergic IB41 neurons (G-I, arrows). Scale bars: 135 mm for A–C, G–I; 77 mm for D–F.
PROTEINS ASSOCIATED WITH NEURON REGENERATION 5DUBOVÝ ET AL.1622
transducer and activator of transcription 3 (STAT3)
known to be among the molecular determinants associated
with the onset of neuronal regeneration (Zigmond,
2012a).
Activating Transcription Factor 3
ATF3 is a member of the activating transcription factor/cAMP-responsive
element binding protein (ATF/
CREB) family of transcription factors that is induced in
a variety of stressed tissues (Hai et al., 1999, 2010).
Since ATF3 is not expressed in intact DRG neurons and
its occurrence is induced by nerve injury, it is regarded
as a marker for identifying axotomized neurons. The
time course of ATF3 induction depends on the distance
between the injury site and the cell body (Tsujino et al.,
2000). ATF3 represses transcription as a homodimer and
Fig. 3. Representative sections of L4-DRG from na€ıve rat (A) and rat operated on nerve crush for one day (B) or CSNT for 7 days (C–H). The
sections were immunostained for detection of SCG10, and double immunostained with ATF3 (C–E) or GAP43 antibody (F–H). Cell nuclei were
stained by Hoechst 33342 (blue). No SCG10 immunofluorescence can be seen in the L4-DRG of na€ıve rat (A). Increased SCG10 immunostaining
in neuronal bodies is already seen one day after nerve crush (B). Most large (arrowheads), medium, and small (arrows) neuronal bodies show
immunopositivity for both SCG10 and ATF3 indicating that neurons undergoing axotomy display the marker for neuronal regeneration (C-E). A
merged picture (H) after double immunofluorescence staining for SCG10 (F) and GAP43 (G) shows that SCG10 immunostaining is present in
most large neurons that are also GAP431 (arrowheads), as well as in most medium and small neurons that display a negative reaction to
GAP43 (arrows). In the L4-DRG after CSNT for 7 days only about one third (33%) of the neurons displayed positive immunostaining for both
SCG10 and GAP43. Scale bars: 135 mm.
6 DUBOVY ET AL.PROTEINS ASSOCIATED WITH NEURON REGENERATION 1623
activates transcription as a heterodimer when coexpressed
with its heterodimeric partners or other proteins
like cJun (Hai et al., 1999). For ATF3 activation, cJun
needs to be phosphorylated by JNK on serines at both
positions 63 and 73 (Waetzig et al., 2006).
It was reported that the axon regeneration program is
initiated when both ATF3 and phosphorylated cJun
(pcJun) are upregulated in neurons in response to axotomy
(Pearson et al., 2003; Lindwall et al., 2004). In contrast,
there are data indicating that activation of cJun is
not coincident with induction of ATF3 in injured DRG
neurons and that it is unlikely therefore that pcJun is
necessary for ATF3 action in axotomized neurons
(Seijffers et al., 2006). Moreover, experiments using
ATF3-transgenic mice suggested that ATF3 overexpression
results in an increase in axon outgrowth in vitro
and regeneration after nerve injury in vivo. However,
ATF3 alone in uninjured DRG neurons leads to a slight
upregulation of some growth-associated gene targets,
and ATF3 upregulation on its own is not enough to fully
recapitulate the peripheral nerve regeneration program
(Seijffers et al., 2007). Recent results indicate that the
Fig. 5. Representative sections of L4-DRG from na€ıve mouse (A) and mouse operated on CSNT for 7 days (B, C) immunostained for SPRR1A.
Cell nuclei were stained by Hoechst 33342 (C, blue). No SPRR1a immunopositivity can be seen in the section from na€ıve L4-DRG (A), while section
of L4-DRG ipsilateral to CSNT demonstrates clear immunostaining in large (arrowheads), medium and small (arrows) neuronal bodies. Scale
bars: 90 mm for A, B; 45 mm for C.
Fig. 4. Longitudinal cryostat sections of the rat ulnar nerve distal to one-day crush with clip immunostained for GAP43 (A) and SCG10 (B–D).
The sections of ulnar nerve stump (A) were incubated with GAP43 antibody (A) or double immunostained for SCG10 and bIII tubulin (B–D). It is
impossible to detect a position of regenerating axons that are GAP43 immunopositive together with Schwann cells (A). In contrast, only regenerated
axons are precisely decorated with SCG10 immunofluorescence (B, C). This is verified by colocalization with bIII tubulin immunopositivity
(D). Scale bars: 135 mm.
PROTEINS ASSOCIATED WITH NEURON REGENERATION 7DUBOVÝ ET AL.1624
ATF3 transcriptional activity might induce “effector”
regeneration-associated genes like SPRR1A, Galanin,
and GAP43 facilitating peripheral axon regeneration
(Gey et al., 2016).
Besides the proregenerative role, ATF3 immunostaining
of neuronal nuclei is a useful marker for axotomized
DRG neurons. Colocalization of ATF3 with GAP43 or
SCG10 in DRG neurons after nerve injury (Figs. 1 and
4) enables precise studies of axon regeneration following
axotomy.
Signal Transducer and Activator
of Transcription 3
STAT3, a member of the STAT family, is activated as
part of Janus kinase (JAK)–STAT signaling pathway
(Aaronson and Horvath, 2002). Activation of STAT3 is
due to JAK2- dependent phosphorylation at the tyrosine-
705 (Y705), and JAK2-independent phosphorylation at
the serine-727 (S727) positions (Tsai et al., 2007). Phosphorylated
STAT3 dimers translocate to the nucleus and
initiate transcription of target genes (Eulenfeld et al.,
2012). STAT3 activation occurs in DRG neurons very
early after a nerve lesion and the influence of neuropoietic
cytokines (Zhong et al., 1999; Cafferty et al.,
2001, 2004; Miao et al., 2006; Zigmond, 2012b) and neurotrophins
(Pellegrino and Habecker, 2013) suggests the
involvement of STAT3 signaling in the promotion of
axon growth (Lee et al., 2004; Zigmond, 2012b).
The activation of STAT3 associated with restoration of
neuronal regeneration capacity is also supported by in
vitro experiments with suppressor of cytokine signaling
3 (SOCS3), which provides feedback inhibition of STAT3
signaling. Overexpression of SOCS3 in primary sensory
neurons blocks nuclear translocation of STAT3 and axonal
growth while neutralization of SOCS3 stimulates
axonal outgrowth (Miao et al., 2006).
Some studies demonstrate that STAT3 can transactivate
GAP43 expression in vivo and in vitro (Wu and Bradshaw,
1996; Schwaiger et al., 2000). As was mentioned
above, GAP43 promotes axon regeneration when phosphorylated
at the S41 position by PKC that is also able to
phosphorylate STAT3 at S727 (Tsai et al., 2007).
The DRG neurons of all size categories displayed
nuclear translocation of immunostaining for STAT3-Y705
after nerve transections. The results of STAT3-Y705 colocalization
with GAP43 indicate reactivation of the axon
regeneration program even in DRG neurons with low or
no immunoreactivity towards GAP43 (Table 1; Fig. 6).
SUMMARY AND CONCLUSION
Reactivation of neuronal regeneration is one of the
most important conditions for successful reinnervation of
injured peripheral nerves. DRG neurons are very useful
Fig. 6. Representative sections of L4-DRG from na€ıve rat (A) and rat treated with CSNT for 7 days (B–D). The sections were immunostained
antibody recognizing STAT3 phosphorylated at Y705. For double immunostaining, the section was further incubated with GAP43 antibody recognizing
both phosphorylated and dephosphorylated forms. Cell nuclei were stained by Hoechst 33342. Neurons and their nuclei in the section
from na€ıve L4-DRG do not display STAT3Y immunostaining (A). Immunopositivity indicating nuclear translocation of STAT3Y705 in both large
(arrowheads) and medium or small neuronal bodies (arrows) is detected in the section from L4-DRG ipsilateral to CSNT. However, only large neuronal
bodies display a distinct GAP43 immunofluorescence. Scale bars: 135 mm.
8 DUBOVY ET AL.PROTEINS ASSOCIATED WITH NEURON REGENERATION 1625
in studying the reactivation of the neuronal regeneration
program, and are frequently used as in vivo and in vitro
models. However, different populations of DRG neurons
behave differently with respect to their regeneration
capacity as well as the expression of molecular markers.
This presents a problem for effective and reliable experimentation.
Both published and original results presented
here suggest that immunohistochemical detection
of GAP43 by antibodies recognizing both phosphorylated
and dephosphorylated forms fail to identify all types of
DRG neurons that regenerate their axons. Moreover,
GAP43 immunostaining is also unsuitable to detect
regenerated axons during early periods after nerve
injury. SCG10, another protein related to the cytoskeletal
dynamics of regenerating axons is a more reliable
marker than GAP43 for visualization of the reactivating
neuronal regeneration program with the added advantage
of enabling the specific detection of regenerated
sensory axons. Immunostaining for selected transcription
factors closely associated with the induction of the
regeneration program like ATF3 or STAT3 is a good
alternative for detecting DRG neurons that regenerate
their axons.
ACKNOWLEDGEMENTS
The authors thank Ms. Dana Kutejova, Ms. Marta
Lnenıcˇkova, Ms. Jitka Mikulaskova, and Mgr. Jana Vachova
for their skillful technical assistance.
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10 DUBOVY ET AL.PROTEINS ASSOCIATED WITH NEURON REGENERATION 1627
C. Dubový P, Klusáková I, Hradilová-Svíženská I, Brázda V, Kohoutková M, Joukal M. A
conditioning sciatic nerve lesion triggers a pro-regenerative state in primary sensory
neurons also of dorsal root ganglia non-associated with the damaged nerve. Frontiers in
Cellular Neuroscience. 2019;13. doi:10.3389/fncel.2019.00011
Commentary: In this publication, we demonstrated that sciatic nerve injury induced
increased expression of SCG10 and GAP43 in the cervical DRG. The increase of
regeneration-associated proteins corresponded with greater length of regenerating axons in the
ulnar nerve 1 day after crush. In addition, we showed IL-6 signaling pathway as crucial for
pro-regenerative state in remote DRG.
IF (WOS, 2017): 4.3
Quartile (WOS, 2017): Neurosciences Q1
Contribution of the author: Co-authorship; approximate participation 20%. The author
participated in performing of in vivo experiments, preparing samples for
immunohistochemistry, western blot, RT-PCR and writing of the corresponding texts.
ORIGINAL RESEARCH
published: 04 February 2019
doi: 10.3389/fncel.2019.00011
Edited by:
Thomas Fath,
Macquarie University, Australia
Reviewed by:
Simone Di Giovanni,
Imperial College London,
United Kingdom
Stefania Raimondo,
University of Turin, Italy
*Correspondence:
Petr Dubový
pdubovy@med.muni.cz
Received: 08 October 2018
Accepted: 14 January 2019
Published: 04 February 2019
Citation:
Dubový P, Klusáková I,
Hradilová-Svíˇzenská I, Brázda V,
Kohoutková M and Joukal M (2019) A
Conditioning Sciatic Nerve Lesion
Triggers a Pro-regenerative State in
Primary Sensory Neurons Also of
Dorsal Root Ganglia Non-associated
With the Damaged Nerve.
Front. Cell. Neurosci. 13:11.
doi: 10.3389/fncel.2019.00011
A Conditioning Sciatic Nerve Lesion
Triggers a Pro-regenerative State in
Primary Sensory Neurons Also of
Dorsal Root Ganglia Non-associated
With the Damaged Nerve
Petr Dubový*, Ilona Klusáková, Ivana Hradilová-Svíˇzenská, Václav Brázda,
Marcela Kohoutková and Marek Joukal
Department of Anatomy, Laboratory of Cellular and Molecular Neurobiology, Faculty of Medicine, Masaryk University,
Brno, Czechia
The primary sensory neurons of dorsal root ganglia (DRG) are a very useful model
to study the neuronal regenerative program that is a prerequisite for successful axon
regeneration after peripheral nerve injury. Seven days after a unilateral sciatic nerve injury
by compression or transection, we detected a bilateral increase in growth-associated
protein-43 (GAP-43) and superior cervical ganglion-10 (SCG-10) mRNA and protein
levels not only in DRG neurons of lumbar spinal cord segments (L4-L5) associated
with injured nerve, but also in remote cervical segments (C6-C8). The increase in
regeneration-associated proteins in the cervical DRG neurons was associated with
the greater length of regenerated axons 1 day after ulnar nerve crush following prior
sciatic nerve injury as compared to controls with only ulnar nerve crush. The increased
axonal regeneration capacity of cervical DRG neurons after a prior conditioning sciatic
nerve lesion was confirmed by neurite outgrowth assay of in vitro cultivated DRG
neurons. Intrathecal injection of IL-6 or a JAK2 inhibitor (AG490) revealed a role for
the IL-6 signaling pathway in activating the pro-regenerative state in remote DRG
neurons. Our results suggest that the pro-regenerative state induced in the DRG neurons
non-associated with the injured nerve reflects a systemic reaction of these neurons to
unilateral sciatic nerve injury.
Keywords: unilateral nerve injury, primary sensory neurons, pro-regenerative state, GAP-43, SCG-10, IL-6, ulnar
nerve crush, neurite outgrowth assay
INTRODUCTION
It is well-known that besides extrinsic factors, activation of a neuronal regenerative program is
necessary for successful axon regeneration after peripheral nerve injury. The primary sensory
neurons, whose bodies are in the dorsal root ganglia (DRG), are a useful model to study the
mechanisms regulating the neuronal regeneration program after axotomy. The DRG neurons have
peripheral and central branches of afferent axons with different responses to injury. Injury to
peripheral axonal branches induces transcription-dependent changes of regeneration-associated
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 February 2019 | Volume 13 | Article 11
Dubový et al. Pro-regenerative State of Remote Neurons
genes and proteins that promote axon regeneration by enhancing
the regeneration potential of DRG neurons (Liu et al., 2011).
In contrast, central axonal branches extending into the dorsal
columns of the spinal cord fail to regenerate when injured
because insufficient activation of the neuronal regeneration
program (Schwaiger et al., 2000; Qiu et al., 2005). However, a
conditioning lesion of the peripheral nerve, where peripheral
axonal branches are injured beforehand, triggers a regenerative
program in DRG neurons that is sufficient to allow regeneration
also of central axonal branches (Neumann and Woolf, 1999).
This phenomenon of a conditioning peripheral nerve lesion with
the activation of the pro-regenerative state in DRG neurons is
at least partly associated with upregulation of some neuropoietic
cytokines including IL-6 (Cafferty et al., 2001; Zigmond, 2011,
2012). It was also shown that increased axon regeneration
was conditioned in the homologous nerve contralateral to
the injured nerve (Yamaguchi et al., 1999; Ryoke et al.,
2000).
In our previous experiments we have found bilateral increased
levels of IL6, as well as its receptor mRNA and protein, not only in
DRG associated with the injured sciatic nerve, but also in remote
cervical DRG (Brázda et al., 2013; Dubový et al., 2013). Moreover,
unilateral sciatic nerve injury induced bilateral activation of
STAT3 by phosphorylation at the tyrosine-705 (Y705) position
in DRG neurons of both lumbar and cervical segments (Dubový
et al., 2018a) that is a critical transforming factor of the neuronal
pro-regenerative state (Bareyre et al., 2011; Zigmond, 2011).
Based on our own and other previously published results, we
hypothesize that nerve injury may stimulate the pro-regenerative
state not only in the DRG neurons associated with damaged
axons, but also in DRG neurons not directly associated with the
injured nerve.
Activation of the neuronal pro-regenerative state is
characterized by upregulation of various transforming factors,
regeneration-associated genes and proteins which are important
intrinsic determinants of neuronal regeneration capacity (Mar
et al., 2014; Rishal and Fainzilber, 2014; Chandran et al., 2016).
For example, phosphorylation and activation of the transcription
factor cJun (Itoh et al., 2009; Frey et al., 2015) and the mitogenactivated
protein kinase p38 (p38 MAPK; Verma et al., 2005; Nix
et al., 2011) is associated with induction of the pro-regenerative
state in DRG neurons following nerve injury.
Regeneration-associated proteins like growth-associated
protein-43 (GAP-43) or superior cervical ganglion-10 (SCG-10)
are generally used as markers for detecting the neuronal
pro-regenerative state (Bonilla et al., 2002; Mason et al.,
2002). GAP-43 is the prototypical GAP, is expressed at high
levels in neurons during development and concentrated in
the axonal growth cone (Skene and Willard, 1981). DRG
neurons of naïve rats display a low basal level of GAP-43
that is significantly increased after nerve injury (Stewart
et al., 1992; Schreyer and Skene, 1993; Liabotis and Schreyer,
1995). The SCG-10 protein, also known as stathmin 2, is
a neuron-specific member of the stathmin family (Sugiura
and Mori, 1995) that is upregulated specifically in primary
sensory neurons regenerating their axons (Shin et al.,
2014).
The goal of the present study was to investigate whether a
sciatic nerve injury can activate the pro-regenerative state of
DRG neurons not only in the corresponding lumbar but also
in remote cervical spinal cord segments. The pro-regenerative
state of DRG neurons was shown by the upregulation of GAP-43
and SCG-10 mRNAs and proteins as well as activation of cJun
and p38 in correlation with the extent of regenerated axons after
ulnar nerve crush following prior sciatic nerve injury. Increased
axon regeneration capacity in cervical DRG neurons initiated
by the conditioning sciatic nerve lesion was also confirmed by
neurite outgrowth assay of in vitro cultivated DRG neurons.
Moreover, intrathecal injection of IL-6 revealed that this cytokine
can mediate this systemic reaction of DRG along the neuroaxis
after unilateral sciatic nerve lesion.
MATERIALS AND METHODS
Animals and Surgical Treatment
The experiments were performed in 194 adult male rats (Wistar,
250–280 g, Anlab, Brno, Czechia) housed in 12 h light/dark
cycles at a temperature of 22–24◦C under specific pathogen-free
conditions in the animal housing facility of Masaryk University.
Sterilized standard rodent food and water were available
ad libitum. Animals for surgical treatments were anesthetized
using a mixture of ketamine (40 mg/ml) and xylazine (4 mg/ml)
administered intraperitoneally (0.2 ml/100 g body weight). All
surgical procedures were carried out under sterile conditions
by the same person according to protocols approved by the
Animal Investigation Committee of the Faculty of Medicine,
Brno, Czechia.
The right sciatic nerve of rats was exposed in mid-thigh,
ligated with two ligatures and cut (complete sciatic nerve
transection, CSNT). The proximal nerve stump was fixed in
muscles to protect the distal stump from reinnervation. A
longitudinally slit silicone tube of 2-mm length and 1 mm
internal diameter was placed around the right sciatic nerve of rats
to reduce the nerve diameter (sciatic nerve compression, SNC;
Schmid et al., 2013). The tube was tied in place with a sterile
thread to close and prevent tube from opening. The muscles
and skin were closed with 5/0 sutures. The right sciatic nerve of
sham-operated rats was carefully exposed without any lesion.
To determine the progression in time of the pro-regenerative
state in cervical DRG after sciatic nerve lesion, a set of
SNC-operated rats from the pilot study was left to survive for 1,
3, 7, and 14 days and compared with naïve rats or sham-operated
rats surviving for 1 and 3 days (n = 3 for each group). Based
on the pilot study in which GAP-43 peaked at 7 days after
unilateral sciatic nerve lesion (Figure 1), further groups of
operated and sham-operated rats were left to survive for 7 days
after surgical treatment and used for bilateral analysis of the
neuronal regeneration program in both lumbar and cervical
DRG.
Quantitative Immunohistochemical
Analysis
Naïve rats and sham-, SNC- and CSNT-operated rats (n = 6 for
each group) were deeply anesthetized with a lethal dose of
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 1 | Results of western blot analysis of growth-associated
protein-43 (GAP-43) protein levels in cervical dorsal root ganglia (DRG;
C6-C8) removed from naïve rats as well as sham- and sciatic nerve
compression (SNC)-operated rats. Sham-operated rats were left to survive for
1 and 3 days, SNC-operated rats for 1, 3, 7 and 14 days (n = 3 for each
group). Upper panel (A) shows a representative western blot with GAP-43
protein in cervical DRG ipsilateral (i) and contralateral (c) to unilateral SNC.
Equal loading of proteins was confirmed by actin levels (Actin). Lower panel
(B) shows densitometry of the individual protein bands after normalization to
actin; the intensities of the bands from naïve cervical DRG were as arbitrarily
set to 1. ∗
Significant difference (p < 0.05) compared to naïve or
sham-operated rats in a Mann-Whitney U-test.
sodium pentobarbital (70 mg/kg body weight, i.p.) and perfused
transcardially with 500 ml phosphate-buffered saline (PBS, pH
7.4) followed by 500 ml of Zamboni’s fixative (Zamboni and
Demartin, 1967). The L4-L5 and C6-C8 DRG from both sides
were detected in their intervertebral foramina following total
laminectomy and foraminotomy. The DRG were removed,
immersed separately in Zamboni’s fixative at 4◦C overnight, and
then collected separately into samples of ipsilateral (L-DRGi) and
contralateral (L-DRGc) lumbar as well as ipsilateral (C-DRGi)
and contralateral (C-DRGc) cervical DRG for each group of rats
(naïve, sham-, SNC-, and CSNT-operated).
The DRG samples were washed in 20% phosphate-buffered
sucrose for 12 h, blocked in Tissue-Tek OCT compound
(Miles, Elkhart, IN, USA) and cut to prepare serial longitudinal
cryostat sections (12 µm). The DRG sections were mounted
on chrome-alum covered slides and processed for indirect
immunohistochemical staining for GAP-43 and SCG-10. Briefly,
DRG sections of lumbar and cervical segments of naïve,
sham-, SNC- and CSNT-operated rats were immunostained
simultaneously under the same conditions. Sections were
washed with PBS containing 0.05% Tween 20 (PBS-T) and
1% bovine serum albumin (BSA) for 10 min, treated with 5%
normal donkey serum in PBS-T for 30 min, then incubated
with 25 µl of mouse monoclonal antibody against GAP-43
(1:500; Sigma, Ronkonkoma, NY, USA) and rabbit polyclonal
antibody against SCG-10 (1:1,000; LSBio, Seattle, WA, USA),
phospho-cJun (1:100; Cell Signaling, New York, NY, USA)
or phospho-p38 (1:200; Chemicon Int., Temecula, CA, USA)
in a humid chamber at room temperature (21–23◦C) for
12 h. The immunohistochemical reaction was visualized by
treatment with FITC-conjugated and affinity-purified donkey
anti-mouse or anti-rabbit secondary antibody (1:100; Millipore,
USA) for 90 min at room temperature. The control sections
were incubated without the primary antibody. Sections were
stained with Hoechst 33342 to detect cell nuclei, mounted in
aqueous mounting medium (Vectashield; Vector Laboratories,
Burlingame, CA, USA) and analyzed using an epifluorescence
microscope (Nikon Eclipse, Nikon, Czechia) equipped with a
camera (DFC-480; Leica Microsystems) and a stabilized power
supply for the lamp housing.
The neuronal diameter, GAP-43 and SCG-10
immunofluorescence intensities were measured using a
NIS-Elements image analysis system (Nikon, Czechia) as
previously (Dubový et al., 2002, 2013). At least 100 neuronal
profiles containing nuclei were measured for each animal group.
The sizes of DRG neurons in sections for immunofluorescence
were categorized as small (<25 µm), medium (25–40 µm), and
large (>40 µm) according to their diameters calculated from the
areas of neuronal profiles. The immunofluorescence intensities
were expressed as mean intensity ± SD.
Western Blot Analysis
In the pilot study, the cervical DRG (C6-C8) of naïve rats and
rats surviving 1 and 3 days after sham-operation and 1, 3, 7 and
14 days after SNC-operation (n = 3 for each group) were removed
bilaterally, flash-frozen in liquid nitrogen and stored at −80◦C
until western blot analysis of GAP-43.
The results of GAP-43 and SCG-10 levels obtained by
quantitative immunohistochemistry in DRG neurons 7 days after
sciatic nerve lesions were verified by western blot analysis. The
DRG from lumbar (L4-L5) and cervical (C6-C8) levels (n = 3 for
each group in three independent experiments) were collected
as described under aseptic conditions. Samples of ipsilateral
(L-DRGi) and contralateral (L-DRGc) lumbar DRG as well as
ipsilateral (C-DRGi) and contralateral (C-DRGc) cervical DRG
from each group of rats were flash-frozen in liquid nitrogen and
stored at −80◦C until analysis.
The DRG samples were homogenized in PBS containing
protease inhibitors (LaRoche, Switzerland) with 0.1% Triton X-
100, and centrifuged at 10,000 g for 5 min at 4◦C. Proteins were
separated by SDS-polyacrylamide gel electrophoresis (Brazda
et al., 2006) and transferred to nitrocellulose membranes by
electroblotting (BioRad). Blots were blocked using 1% BSA
in PBST (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM
KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4) for 1 h and
incubated with anti-GAP-43 mouse monoclonal (1:1,000; Sigma,
Ronkonkoma, NY, USA), rabbit polyclonal anti-phosphorylated
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Dubový et al. Pro-regenerative State of Remote Neurons
S41-GAP43 (1:500; Thermo Fisher Scientific, Waltham, MA,
USA) or anti-SCG-10 (1:500; LSBio, Seattle, WA, USA)
antibodies overnight. Blots were washed in PBST and incubated
with peroxidase-conjugated anti-mouse or anti-rabbit IgG
(1:1,000; Sigma, Seattle, WA, USA) at room temperature for
1 h. Protein bands were visualized by the ECL detection kit
(Amersham, USA) on the chemiluminometer reader LAS-3000
(Fuji, Japan) and analyzed using densitometry image software.
The levels of proteins were normalized to the value of naïve DRG,
which was arbitrarily set as one.
Real Time RT-PCR
The expression of GAP-43 and SCG-10 mRNAs in DRG was
analyzed by real-time PCR (RT-PCR). Whole DRG from each
group of rats (n = 3 for each group in three independent
experiments) were removed under aseptic conditions from
lumbar (L4-L5) and cervical (C6-C8) segments of both sides,
collected as ipsilateral and contralateral samples and stored
in RNA later (Thermo Fisher Scientific, Inc., Waltham, MA,
USA) at 4◦C. First-strand synthesis was performed using
TaqMan High Capacity RNA-to-cDNA Kit and the quality
and concentration evaluated by optical density using NanoDrop.
PCR amplification, in triplicate for each sample, was performed
using ABI Prism 7300, TaqMan Gene Expression Master
Mix, and TaqMan Gene Expression Assay Probes FAMTM
(Thermo Fisher Scientific, Inc., Waltham, MA, USA) for the
target genes GAP-43 (assay ID-Rn01474579_m1) and SCG-10
(assay ID—Rn00584886_m1). Determinations were made with
reference to the reporter gene encoding rat actin (actin,
beta—Rn00667869_m1) Endogenous Control (VIC ). The
polymerase activation step at 95◦C for 15 min was followed by
40 cycles of 15 s at 95◦C and 60 s at 60◦C. The validity of the
results was checked by running appropriate negative controls
(water instead of cDNA by for PCR amplification; omitting
reverse transcriptase for cDNA synthesis). Specific mRNA levels
were calculated after normalizing to actin mRNA in each sample.
Relative expression was determined using the Comparative Ct
Model (∆∆Ct) with actin as the housekeeping gene. Data were
presented as relative mRNA units compared with control values
(expressed as fold over naive value).
In vivo Assay of Axon Regeneration in
Crushed Ulnar Nerve
Rats with prior operation on SNC (n = 6) or CSNT (n = 6) for
7 days were re-operated to expose and mobilize a short segment
of the right ulnar nerve. The right ulnar nerve was also mobilized
in a control group without any previous sciatic nerve injury
(n = 6). The ulnar nerve was then crushed using a clamp of a
defined force of 1.9 N for 2 × 1 min (Ronchi et al., 2009) under
a stereoscopic microscope. The distal margin of the crush injury
was indicated with a 10-0 epineurial suture and the skin wound
was closed with 5/0 sutures.
To investigate peripheral axon regeneration, the ulnar nerves
were removed 1 day after the crush injury following pericardial
infusion with Zamboni fixative solution and ulnar nerve samples
were fixed by immersion in Zamboni fixative solution overnight.
After washing with 10% sucrose in PBS, longitudinal cryostat
sections of 10 µm thickness were cut and immunostained for
SCG-10, which is a more selective marker of regenerating sensory
axons than GAP-43 (Shin et al., 2014). SCG-10 fluorescence
intensity was analyzed along the length of the nerve distal to the
crush; the regeneration index was determined by measuring the
length of the longest SCG-10 decorated axons from the crush site
(Abe et al., 2010). The length of SCG-10+ axons was measured
by a person blind to the experimental conditions in every third
section using a NIS-Elements image analysis system (Nikon,
Czechia).
In vitro Assay of Increased Axonal
Outgrowth of Cervical DRG Neurons
Induced by Sciatic Nerve Lesion
An in vitro culture of dissociated DRG neurons was prepared
according to a modified protocol (Christie et al., 2010).
Sham-operated rats (n = 4) and rats operated by SNC (n = 4)
or CSNT (n = 4) for 7 days were deeply anesthetized by an
intraperitoneal application of sodium pentobarbital (70 mg/kg)
and killed by decapitation. The DRG of lumbar (L4-L5) and
cervical (C6-C8) segments were removed bilaterally under
aseptic conditions following laminectomy. The samples were
collected in ice-cold Ca2+/Mg2+-free Hank’s buffered salt
solution (CMF-HBSS, Sigma Aldrich), and the spinal roots and
connective tissues were removed.
DRG were then dissociated by incubation in medium
containing 0.1% collagenase type I (5,000 U/ml) for 90 min
followed by 0.25% trypsin/EDTA at 37◦C for 25 min. The
DRG suspension was prepared by triturating through glass
pipette tips, washed twice with Dulbecco’s Modified Eagle’s
Medium/Nutrient F-12 Ham (DMEM/F12) supplemented with
10% fetal bovine serum (FBS; all from Sigma Aldrich) and
the suspension was spun for 5 min at 1,500 rpm at 4◦C. The
cells were resuspended and placed into a culture medium of
DMEM/F12 supplemented with 2 mM glutamine, 100 U/ml
Penicillin and 100 µg/ml Streptomycin (all from Sigma Aldrich),
N2 and B27 (ThermoFisher Sci., diluted according to the
manufacturer’s instructions). The cells were re seeded at a density
of 200 cells on glass cover slips previously coated with Geltrex
(ThermoFisher Sci.).
Cultures were incubated at 37◦C in a humidified atmosphere
containing 5% CO2 for 2 days, washed in PBS, fixed by Zamboni
solution for 20 min and immunostained with mouse anti-β
tubulin III primary antibody (Sigma, 1:500) overnight at 4◦C.
The cells were then washed in PBS followed by incubation with
goat anti-mouse TRITC-conjugated secondary antibody (Life
Technologies, 1:1,000) for 90 min. Finally, cells were washed
in PBS, stained with Hoechst 33342 to detect cell nuclei and
mounted in a Vectashield aqueous mounting medium (Vector
Laboratories, Burlingame, CA, USA). Three slides for each
experimental group were analyzed under a Nikon Eclipse NI-E
epifluorescence microscope equipped with a Nikon DS-Ri1
camera (Nikon, Prague, Czechia) using a 10× objective by a
person blind to the experimental conditions.
To analyze neurite outgrowth and length, digital images of
at least 100 randomly selected nucleate neurons per cover slip
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Dubový et al. Pro-regenerative State of Remote Neurons
were acquired. Neurites longer than the diameter of the neuronal
bodies were analyzed in digital pictures converted to grayscale for
better visualization. Neurite outgrowth initiation was quantified
as the number of neurites per neuron counted using the Count
and Taxonomy module of NIS- Elements software (Nikon,
Prague, Czechia). Axonal elongation was analyzed by taking the
total length of neurites per neuron when axons of individual
neurons were traced and measured using the Neurite Tracer
Plugin for ImageJ (Pool et al., 2008). The mean number of
neurites per neuron and total neurite length per neuron were
calculated from triplicate experiments and data were present as
mean ± SD.
In vivo Assay of Axon Regeneration in the
Crushed Ulnar Nerve and Changes in the
Pro-regenerative State of Cervical DRG
Neurons After Intrathecal Injection of IL-6
or JAK2 Inhibitor AG490
Recombinant rat IL-6 protein (R&D Systems) was dissolved in
artificial cerebrospinal fluid (ACSF; Hylden and Wilcox, 1980) at
20 ng/10 µl. AG490 (Sigma), an inhibitor of JAK2, was dissolved
in ACSF at a concentration of 5 µM.
A solution of IL-6 (10 µl) or ACSF (10 µl) along with
a further 10 µl ACSF was injected via a micro syringe into
the lumbar subarachnoid space of intact rats (n = 2 for each
group). Animals were left to survive for 1 day and cervical
DRG (C6-C8) were removed following pericardial infusion
with Zamboni fixative solution. Longitudinal DRG sections
were double immunostained with mouse monoclonal antibody
against GAP-43 (1:500; Sigma, USA) and rabbit polyclonal antiSTAT3
(Y705) antibody (1:100; Santa Cruz, CA, USA). The
immunofluorescence reaction was visualized by treatment with
FITC-conjugated donkey anti-mouse and TRITC-conjugated
donkey anti-rabbit secondary antibodies (1:100; Millipore, USA)
for 90 min at room temperature. Activation and nuclear
translocation of STAT3 as well as GAP-43 immunofluorescence
intensity were measured using a NIS-Elements image analysis
system (Nikon, Czechia) as described previously.
To investigate in vivo the role of IL-6 in triggering the
pro-regenerative state in cervical DRG, the right ulnar nerve was
crushed as described (n = 8). A solution of IL-6 (10 µl) or ACSF
(10 µl) along with a further 10 µl ACSF was then injected via
a micro syringe into the lumbar subarachnoid space (n = 4 for
each group). The ulnar nerves were removed 1 day later (after
the crush and intrathecal administration of IL-6) and fixed with
Zamboni fixative solution. Axon regeneration was assessed on
longitudinal cryostat sections (10 µm thick) immunostained for
SCG-10 and analyzed as described.
To test whether the JAK2/STAT3 signaling pathway is
involved in inducing the pro-regenerative state of cervical DRG
neurons after prior sciatic nerve injury, the right sciatic nerve of
8 rats was cut (CSNT). After 7 days, the rats with prior CSNT
were re-operated to expose and crush the right ulnar nerve.
10 µl of AG490 solution (5 µM) or ACSF (10 µl), along with
a further 10 µl ACSF was then injected via a micro syringe
into the subarachnoid space of the cisterna magna (n = 4 for
each group). The length of regenerated SCG10+ axons was
assessed on longitudinal cryostat sections (10 µm thick) 1 day
after the ulnar nerve crush and intrathecal injection of ACSF
or AG490 as described above. In addition, cryostat sections of
cervical DRG (C6-C8) ipsilateral to the ulnar nerve crush were
double immunostained for GAP-43 and STAT3 and analyzed
using a NIS-Elements image analysis system (Nikon, Czechia) as
described above.
Enzyme-Linked Immunosorbent Assay
(ELISA) of IL-6 in Rat Plasma
Three naïve rats and those operated on to create SNC or
CSNT for 1, 3, and 7 days (n = 3 for each group), as
well as sham-operated rats for 3 (n = 3) and 7 (n = 3)
days were sacrificed by CO2 inhalation. Blood samples were
obtained by intracardiac puncture and collected into tubes
containing heparin and protease inhibitor cocktail (LaRoche,
Switzerland). Plasma was separated by centrifugation (2,500 g
for 12 min) and stored at −60◦C until analyzed. Total protein
was measured by Nanodrop ND-1000 (Thermo Scientific) and
the level of IL-6 protein was assessed using an ELISA kit
with a sensitivity of 5 pg/ml (BioSource International, USA)
according to the manufacturer’s instructions. Measurement was
carried out on a SUNRISE Basic microplate reader (Tecan,
Salzburg, Austria) and the data were normalized as picograms
of IL-6 protein to 100 µg of total protein. IL-6 protein levels
were compared to baseline values in plasma from naïve rats,
which was arbitrarily set as one. Data were expressed as
mean ± SD.
Statistical Analyses
Statistical differences between data of immunofluorescence
intensities, western blot analysis and RT-PCR of naïve DRG
neurons and DRG neurons of sham-operated rats or rats with
SNC and CSNT were tested using a Mann-Whitney U-test
(p < 0.05). The same statistical analysis was used to compare
IL-6 protein levels in plasma of naïve and sham-operated or
SNC- and CSNT-operated rats. The mean number of neurites
per neuron and total neurite length per neuron were compared
between DRG neurons of sham- and SNC- or CSNT-operated
rats using one-way ANOVA and Tukey’s post hoc test to
determine statistical significance. All statistical analyses were
performed using STATISTICA 9.0 software (StatSoft, Inc., Tulsa,
OK, USA).
RESULTS
Immunohistochemical and Western Blot
Analysis of GAP-43 and SCG-10 Proteins
Western blot analysis of GAP-43 in cervical DRG of rats
surviving 1, 3, 7, and 14 days after SNC showed the protein
level peaked at 7 days with a drop at 14 days when compared
to cervical DRG of naïve or sham-operated rats (Figure 1).
Therefore, subsequent analyses illustrating the pro-regenerative
state of cervical DRG neurons after sciatic nerve lesions were
performed in rats 7 days after SNC or CSNT.
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Dubový et al. Pro-regenerative State of Remote Neurons
The lumbar and cervical DRG neurons of naïve and
sham-operated rats displayed a low basal level of GAP-43
immunostaining. GAP-43 immunofluorescence increased
strongly in large and medium-sized neurons of both ipsilateral
and contralateral lumbar DRG 7 days after SNC and CSNT.
Some medium- and small-sized neurons displayed only weak
GAP-43 immunopositivity. Sections of cervical DRG also
demonstrated a similar pattern of bilaterally increased GAP-43
immunofluorescence (Figure 2).
Image analysis revealed a bilateral increase of mean GAP-43
immunofluorescence intensity in all size-classes of neurons
in both lumbar and cervical DRG after unilateral SNC
and CSNT in comparison with naïve and sham-operated
animals. Sciatic nerve injury induced a greater increase
in GAP-43 immunofluorescence intensity in large neurons
than in medium- and small-sized neurons of both lumbar
and cervical DRG. Further, a more significant increase
in GAP-43 immunofluorescence was measured bilaterally
in lumbar (3.3–2.6 times) than in cervical DRG neurons
(2.5–1.9 times) compared to naïve or sham-operated rats.
However, no significant differences were found between
individual size-classes of neurons in the SNC and CSNT
experimental groups (Figure 2M).
Basal SCG-10 immunofluorescence was very low or absent
in the lumbar and cervical DRG neurons of naïve rats. Most
neurons of all sizes in lumbar and cervical DRG of both sides
displayed significantly enhanced SCG-10 immunofluorescence
intensity 7 days after SNC and CSNT compared to DRG
FIGURE 2 | Representative pictures of cryostat sections through the DRG from naïve (A,G), sham-operated (B,H) rats and rats with unilateral sciatic nerve
compression (SNC; C,D,I,J) or complete sciatic nerve transection (CSNT; E,F,K,L) for 7 days. The sections of ipsilateral (C,E) and contralateral (D,F) DRG of the
L4 spinal segment as well as ipsilateral (I,K) and contralateral (J,L) DRG of the C7 spinal segment were incubated under the same conditions with mouse
monoclonal antibody recognizing GAP-43. Scale bars = 75 µm. (M) Graph illustrating the mean intensity of GAP-43 immunofluorescence in individual DRG
neuron-size classes of cervical (C6-C8) and lumbar (L4-L5) spinal segments ipsilateral (i) and contralateral (c) to unilateral SNC and transection (CSNT) for 7 days;
n = 6 for each group. Neurons A ≥ 40 µm; Neurons B 25–40 µm; Neurons C ≤ 25 µm. ∗
Significant difference (p < 0.05) compared to naive or sham-operated rats
in a Mann-Whitney U-test.
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 3 | Representative pictures of cryostat sections through the DRG from naïve (A,G), sham-operated (B,H) rats and rats with unilateral SNC (C,D,I,J) or
CSNT (E,F,K,L) for 7 days. The sections of ipsilateral (C,E) and contralateral (D,F) DRG of the L4 spinal segment as well as ipsilateral (I,K) and contralateral (J,L)
DRG of the C7 spinal segment were incubated under the same conditions with rabbit polyclonal antibody recognizing superior cervical ganglion-10 (SCG-10). Scale
bars = 75 µm. (M) Graph illustrating the mean intensity of SCG-10 immunofluorescence measured in individual DRG neuron-size classes of cervical (C6-C8) and
lumbar (L4-L5) spinal segments ipsilateral (i) and contralateral (c) to unilateral SNC and transection (CSNT) for 7 days; n = 6 for each group. Neurons A ≥ 40 µm;
Neurons B 25–40 µm; Neurons C ≤ 25 µm. ∗
Significant difference (p < 0.05) compared to naive or sham-operated rats in a Mann-Whitney U-test.
from naïve or sham-operated rats. However, the increase in
SCG-10 immunofluorescence intensity was lower than for
GAP-43 (approximately 1.8–2 times in lumbar and 1.4–2.3 times
in cervical DRG; Figures 3A–L). Moreover, image analysis
of SCG-10 immunofluorescence intensity did not show any
preferential increase among individual size-classes of DRG
neurons (Figure 3M).
The increased levels of GAP-43 and SCG-10 proteins induced
by SNC and CSNT and detected in the lumbar and cervical
DRG of both sides by quantitative immunohistochemistry were
verified by western blot analysis. Total GAP-43 and SCG-10
proteins were also significantly increased bilaterally in both
lumbar and cervical DRG after unilateral SNC and CSNT for
7 days compared to naïve or sham-operated controls. Seven
days after SNC or CSNT, the levels of GAP-43 protein were
increased bilaterally in the lumbar and cervical DRG about twoto-three
times compared to naïve and sham-operated controls.
As was expected, GAP-43 protein levels shot up significantly in
lumbar DRG after CSNT than SNC. Levels of GAP-43 protein in
cervical DRG also increased significantly compared to controls,
but the elevation was not to the extent seen in lumbar DRG
(Figures 4A,B).
GAP-43 is activated by protein kinase C-mediated
phosphorylation at serine 41 (S41) that promotes the
polymerization and stabilization of filamentous actin (F-actin)
related to axon growth and sprouting (Tsai et al., 2007).
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 4 | Results of western blot analysis of GAP-43, pGAP-43 and
SCG-10 protein levels in DRG of L4-L5 (L) and C6-C8 (C) segments removed
from ipsilateral (i) and contralateral (c) sides of naïve as well as sham-, SNCand
CSNT-operated rats for 7 days. Upper panel (A) illustrates representative
western blots of DRG from three rats for each group. Equal loading of proteins
was confirmed by actin levels (Actin). The same Actin controls were used for
(Continued)
FIGURE 4 | Continued
analysis of GAP-43, pGAP-43 and SCG-10 protein levels in this set
representative western blots. Lower panels (B) show densitometry of
individual protein bands after normalization to actin from three independent
experiments; the intensities of the bands from naïve DRG were as arbitrarily
set to 1. ∗
Significant difference (p < 0.05) when compared to sham-operated
rats; †
Significant difference (p < 0.05) compared to contralateral DRG in a
Mann-Whitney U-test.
Although in our experiments we used an antibody recognizing
both unphosphorylated and phosphorylated GAP-43, the
western blot analysis verified levels of activated GAP-43 using a
specific antibody against phosphorylated GAP-43 (pGAP-43).
The results demonstrated that activated pGAP-43 was increased
bilaterally not only in lumbar but also in cervical DRG after
SNC or CSNT compared to naïve or sham-operated controls.
The magnitude of pGAP-43 increase was very similar to the
increased GAP-43 levels. However, in contrast to significantly
higher GAP-43 protein levels in ipsilateral than contralateral
lumbar DRG after both SNC and CSNT, pGAP-43 protein level
was significantly higher only in ipsilateral lumbar DRG after
CSNT (Figures 4A,B).
In contrast to GAP-43 and pGAP-43, the increase in levels of
SCG-10 was less marked (about 1.5–1.8-fold) and the increase
was more or less the same on both sides. Moreover, no
significant differences in SCG-10 levels were found in lumbar
DRG from SNC- and CSNT-operated animals. Similar protein
levels of SCG-10 were also measured in cervical DRG after
SNC and CSNT, as in the case of GAP-43 and pGAP-43
(Figures 4A,B).
RT-PCR Analysis of GAP-43 and SCG-10
mRNA
RT PCR was used to determine the levels of the relevant GAP-43
and SCG-10 mRNA in cervical and lumbar DRG 7 days after
unilateral sciatic nerve injury by SNC or CSNT. Samples of
cervical and lumbar DRG from sham-operated animals displayed
no significant changes in SCG-10 and GAP-43 mRNA levels
compared to naïve controls.
Levels of GAP-43 and SCG-10 mRNAs were significantly
increased bilaterally in both cervical and lumbar DRG 1 week
following sciatic nerve injury by SNC or CSNT compared to
naïve or sham-operated controls. GAP-43 mRNA increased
bilaterally in lumbar DRG to a higher level after CSNT than SNC.
However, a statistically significant difference between ipsilateral
and contralateral lumbar DRG was found only in the CSNT
group. In contrast to lumbar DRG, cervical ones displayed a
bilateral increase of GAP-43 mRNA that was greater after SNC
than after CSNT.
Similar to the GAP-43 and SCG-10 protein results, RT-PCR
revealed a substantially smaller increase in SCG-10 mRNA
levels in lumbar DRG (only up to 2.6-fold of sham-operated
controls) than was measured for GAP-43 mRNA. Levels of
SCG-10 mRNA were higher bilaterally in lumbar DRG after
CSNT than after SNC, but the differences were not statistically
significant. Both SNC and CSNT induced an approximately
similar elevation of SCG-10 mRNA in cervical DRG of
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 5 | Results of real-time PCR (RT-PCR) of relative GAP-43 (A) and
SCG-10 (B) mRNA levels in DRG of lumbar (L4-L5) and cervical (C6-C8)
spinal segments of removed from ipsilateral (i) and contralateral (c) sides of
Naïve as well as sham-, SNC- and CSNT-operated rats for 7 days; n = 6 for
each group. Relative expressions were determined using Actin as the
housekeeping gene and normalized to naïve controls. ∗
Significant difference
(p < 0.05) compared to sham-operated rats in a Mann-Whitney U-test.
both sides compared to naïve and sham-operated controls
(Figure 5).
Sciatic Nerve Lesion Induced Activation of
cJun and p38 MAPK in Cervical DRG
Neurons
To confirm the sciatic nerve injury-induced pro-regenerative
state of cervical DRG neurons demonstrated by GAP-43
and SCG10 analyses, activation of p-cJun and p-p38 MAPK
was also investigated. Basal p-cJun and p-p38 MAPK
immunofluorescence was very low in cervical DRG neurons of
naïve and sham-operated rats. Sciatic nerve lesion increased
immunofluorescence intensity of both p-cJun and p-p38 MAPK
in the nuclei of cervical DRG neurons. Further, a higher intensity
of p-p38 MAPK immunofluorescence was seen in the soma of
neurons (Figure 6).
FIGURE 6 | Representative pictures of cervical (C7) DRG neurons of intact
rats (A–D) and rats 7 day after sham- (B,E) or SNC-operation (C,F). DRG
sections were immunostained for p-cJun (A–C) and p-p38 (D–F). Scale
bars = 40 µm. (G) Graph illustrating mean immunofluorescence intensity of
activated p-cJun (red columns) and p-p38 (green columns) in cervical DRG
neurons of naïve, sham, and SNC groups. SNC induced activation of p-cJun
and p-p38 (arrowheads, C,G). ∗
Significant difference (p < 0.05) compared to
naïve or sham-operated rats in a Mann-Whitney U-test.
Axon Regeneration Assay in Crushed
Ulnar Nerve After Prior Sciatic Nerve Injury
The axonal regeneration capacity of cervical DRG neurons was
investigated in longitudinal sections of one-day-old crushed
ulnar nerves by immunostaining for SCG-10—a specific marker
for regenerated axons of primary sensory neurons (Shin et al.,
2014). Axon regeneration index expressed as SCG-10+ axon
extension from the point of nerve crush was greater in SNC- and
CSNT-operated rats compared to control rats with only ulnar
nerve crush (Figures 7A,B).
In vitro Assay of Axonal Outgrowth
Capacity of Cervical DRG Neurons After
Sciatic Nerve Injury
The increased capacity of cervical DRG neurons to regenerate
their axons after prior sciatic nerve injury was verified by an
in vitro assay of neurite outgrowth in DRG neurons taken from
sham-, SNC- and CSNT-operated rats (Figures 8A–F). The
diameter of neuronal bodies cultivated in vitro was between
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 7 | (A) Representative longitudinal sections through the ulnar
nerves distal to the crush site (solid line) showing SCG-10 positive
regenerated axons. Arrows indicate the tip of the longest SCG-10 positive
axons. The ulnar nerves were removed after 1 day from control rat (only ulnar
nerve crush) and from rats 7 days after prior SNC or CSNT. Scale bars =
100 µm. (B) The top portion of the graph illustrates mean length of
regenerated SCG10+ axons ± SD in the ulnar nerve 1 day after crush in rats
without a sciatic nerve injury (Control) and following 7 days from SNC or
CSNT; n = 6 for each group. ∗
Significant difference (p < 0.05) compared to
control. The middle portion illustrates mean length of regenerated SCG10+
axons ± SD in the ulnar nerve 1 day after crush and intrathecal injection of
10 µl of artificial cerebrospinal fluid (ACSF) or IL-6 (20 ng/10 µl); n = 4 for
each group. ∗∗
Significant difference (p < 0.05) compared to control. The
bottom portion illustrates mean length of regenerated SCG10+ axons ± SD in
the ulnar nerve 1 day after crush 7 days from prior CSNT and intrathecal
application of ACSF (CSNT+ACSF) or JAK2 inhibitor (CSNT+AG490);
n = 4 for each group. ‡
Significant difference (p < 0.05) compared to
CSNT+ACSF group in a Mann-Whitney U-test.
25 and 45 µm, thus encompassing all morphological classes of
DRG neurons.
The mean number of neurites per neuronal body of cervical
and lumbar DRG taken from sham-operated rats was very
similar. Compared to cervical DRG neurons of sham-operated
rats, the number of neurites sent off by cervical DRG neurons
taken from SNC- and CSNT-operated rats was significantly
higher. Surprisingly, cervical and contralateral lumbar DRG
neurons displayed higher neurite outgrowth than lumbar DRG
neurons ipsilateral to CSNT (7.0 ± 2.1 and 7.0 ± 1.7 compared
to 4.6 ± 1.5; Figure 8G).
The total length of neurites per neuron was significantly larger
in neurons from lumbar DRG of both sides and those cultivated
from cervical DRG 7 days after SNC. Seven days after CSNT, the
total neurite length per neuron was significantly larger in neurons
cultivated from cervical and contralateral lumbar DRG, but not
in neurons cultivated from ipsilateral lumbar DRG (Figure 8H).
IL-6 Protein Level in Plasma
IL-6 protein levels increased significantly in the plasma of
sham-operated rats at 3 days but returned to normal by 7 days
after treatment. Plasma IL-6 levels were elevated also in SNC and
CSNT rats at 1 and 3 days but dropped back close to normal
7 days after sciatic nerve lesions (Figure 9). These IL-6 plasma
level measurements are consistent with our previous results
following SNC (Dubový et al., 2013) and indicate that IL-6 in the
blood is likely not inducing the pro-regenerative state in cervical
DRG neurons 7 days after sciatic nerve lesion.
Axon Regeneration Assay in Crushed
Ulnar Nerve and Changes in the
Pro-regenerative State in Cervical DRG
Neurons After Intrathecal Application of
IL-6 and JAK2 Inhibitor
To investigate if IL-6 is responsible for activation of the
pro-regenerative state of rat cervical DRG neurons, we
intrathecally applied IL-6. We showed previously that intrathecal
application of IL-6 induced activation and nuclear translocation
of STAT3 in DRG neurons (Dubový et al., 2018a). In the
present experiments, we observed that intrathecal injection of
IL-6 increased not only the activation of STAT3 but also the
expression of GAP-43 in intact DRG neurons. In contrast,
application of AG490 after CSNT and ulnar nerve crush resulted
in decreased STAT3 activation and expression of GAP-43 in
cervical DRG (Figure 10).
In addition, we measured the lengths of SCG-10+ regenerated
axons distal to the ulnar nerve crush following intrathecal IL-6
application. The regenerated axons were significantly longer
compared to control ulnar nerve crush or nerve crush and
subsequent intrathecal injection of ACSF (Figure 7B, the middle
portion). The pro-regenerative state of neurons is mediated
by phosphorylation of STAT3 at the Y705 position by JAK2
(Schwaiger et al., 2000; Qiu et al., 2005; Niemi et al., 2016). When
an inhibitor of JAK2 (AG490) was applied intrathecally in rats
with ulnar nerve crush for a day following prior CSNT, SCG-10+
regenerated axons were significantly shorter compared to those
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 8 | (A–F) Representative pictures of cervical (C6-C8) and lumbar (L4-L5) DRG neurons of the ipsilateral side dissociated and cultured in vitro after removing
of DRG from sham-, SNC- or CSNT-operated rats for 7 days. The DRG neurons were fixed and immunostained for βIII-tubulin after 2 days of in vitro incubation.
Scale bars = 75 µm. The graphs illustrate mean number of neurites (G) and total neurite length (H) per neuronal body of in vitro cultivated DRG neurons of cervical
(C6-C8) and lumbar (L4-L5) spinal segments from the ipsilateral (i) and contralateral (c) sides removed from sham-, SNC- and CSNT-operated rats (n = 4 for each
group). At least 100 randomly selected neurons with nuclei per cover slip were measured. ∗
Significant difference (p < 0.05) compared to sham-operated controls in a
Mann-Whitney U-test.
of the rats subjected to CSNT and subsequent ulnar nerve crush
and intrathecal injection only of ACSF (Figure 7B, the bottom
portion).
DISCUSSION
Activation of the neuronal regenerative program after axon
injury is an important prerequisite for useful reinnervation and
functional recovery. This pro-regenerative status of the neurons
is linked with the upregulation of regeneration-associated
molecules (Ma and Willis, 2015). For example, regenerationassociated
proteins like GAP-43 and SCG-10 are frequently used
as molecular markers of the pro-regenerative state of neurons
induced by experimental nerve injury. The DRG neurons are
a useful model for studying the induction of the regenerationassociated
program marked by increased levels of GAP-43 and
SCG-10 mRNA and protein (Mason et al., 2002; Shin et al.,
2014). The DRG neurons of naïve rats display a low basal
level of GAP-43 or SCG-10 immunostaining that is significantly
increased in the neuronal bodies over a long period after sciatic
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 9 | IL-6 protein levels in the plasma of naïve, sham-, SNC- and
CSNT-operated rats (n = 3 for each group). Blood samples of sham-operated
rats were obtained 3 (3D) and 7 (7D) days after surgical treatment and those
from SNC- and CSNT-operated rats were obtained 1 (1D), 3 (3D), and 7 (7D)
days after operation. ∗
Significant difference (P < 0.05) compared to baseline
levels of naive rats in a Mann-Whitney U-test.
nerve injury (Stewart et al., 1992; Schreyer and Skene, 1993; Shin
et al., 2014; Dubový et al., 2018b).
GAP-43 immunostaining is a widely used marker for
regenerating axons in experimental models of peripheral
nerve injury. Apart from regenerating axons, intense GAP-43
immunoreactivity is also present in Schwann and Schwannderived
cells (Plantinga et al., 1993; Dubovy and Aldskogius,
1996). This GAP-43 immunostaining of Schwann cells associated
with regenerated axons makes their detection difficult, especially
during early periods of axon regeneration (Dubový et al., 2018b).
In contrast to GAP-43, SCG-10 is transported from the soma
to the proximal axonal stump very early after axotomy, whereas
it is rapidly lost in distal axon stumps (Tararuk et al., 2006;
Shin et al., 2012, 2014). In contrast to GAP-43, which labels
regenerated axons after 3 days (Sar Shalom and Yaron, 2014),
SCG-10 immunostaining is rapidly increased in proximal axonal
stumps and regenerating sprouts within hours after axotomy
(Shin et al., 2012, 2014). Moreover, SCG-10 is a marker for
regenerated sensory axons of injured peripheral nerves (Shin
et al., 2014). Therefore, we monitored GAP-43 and SCG-10
mRNA and protein to prove the pro-regenerative state of DRG
neurons, and SCG-10 was used to assess axon regeneration distal
to ulnar nerve crush after 1 day.
Unilateral Sciatic Nerve Lesion Induced
Pro-regenerative State in Both Lumbar and
Cervical DRG
Subpopulations of rat DRG neurons can be classified according
their size, their cytological, chemical and physiological properties
(Lawson, 2002) and behave differently with respect to their
regeneration capacity as well as the expression of molecular
regeneration markers (Bonilla et al., 2002; Leclere et al., 2007;
Shin and Cho, 2017). Classically, DRG neurons are divided
into large-, medium- and small-sized (Lawson et al., 1974)
FIGURE 10 | Representative pictures of cervical (C7) DRG neurons of intact
rats (A–D) and rats 1 day after the ulnar nerve crush following 7 days from
CSNT (E–H). In intact rats, 10 µl of ACSF (A,C) or IL-6 (B,D) were
intrathecally applied for 1 day. Cervical DRG were also removed from rats with
CSNT for 7 days, a subsequent ulnar nerve crush for 1 day and intrathecal
injection of 10 µl of ACSF (E,G) or AG490 (F,H). DRG sections were
immunostained for STAT3 (A,B,E,F) or double immunostained (C,D,G,H) for
STAT3 (red fluorescence) and GAP-43 (green fluorescence). Intrathecal
injection of IL-6 induced activation and nuclear translocation of STAT3
(arrowheads, B,D) as well as increased immunostaining for GAP-43 in
neurons (arrows, D) when compared with ACSF treatment (C). Cervical DRG
(Continued)
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Dubový et al. Pro-regenerative State of Remote Neurons
FIGURE 10 | Continued
neurons of rats following the ulnar nerve crush 7 days after CSNT displayed
activation and nuclear translocation of STAT3 (E,G) and intense GAP-43
immunofluorescence in the neurons when injected with ACSF (G, arrowheads
and arrows, respectively) while intrathecal application of AG490 resulted in a
marked reduction of STAT3 activation and GAP-43 immunostaining (F,H).
Scale bars = 40 µm. (I) Graph illustrating mean immunofluorescence intensity
of activated STAT3 and GAP-43 in cervical DRG neurons of intact rats
following intrathecal application of ACSF or IL-6 for 1 day and rats with CSNT
for 7 days and intrathecal injection of ACSF or AG490. ∗
Significant difference
(P < 0.05) compared with ACSF-treated rats in a Mann-Whitney U-test.
and may also be identified by further molecular phenotyping.
Quantitative immunohistochemical staining revealed increased
GAP-43 and SCG-10 immunofluorescence in both lumbar and
cervical DRG neurons ipsilateral as well as contralateral to
the SNC or CSNT. Detailed image analysis showed that the
predominant increase of GAP-43 immunofluorescence intensity
was seen in the large-sized neuronal subpopulation of both
lumbar and cervical DRG. In contrast, a similar elevation
of SCG-10 immunofluorescence intensity was observed in all
size-classes neurons of DRG in the lumbar and cervical spinal
cord segments. Principally, bilateral increase of GAP-43 and
SCG-10 proteins detected by quantitative immunohistochemical
staining in both lumbar and cervical DRG neurons after sciatic
nerve lesion was verified by measuring whole GAP-43 and
SCG-10 protein levels using western blot analysis. Further, it
also revealed that unilateral sciatic nerve lesion induced bilateral
elevation of activated (phosphorylated) GAP-43 in both lumbar
and cervical DRG.
The pro-regenerative state of DRG neurons in both lumbar
and cervical spinal cord segments was confirmed by increased
levels of GAP-43 and SCG-10 mRNA. Although SCG-10 is
considered a better marker of the pro-regenerative state of DRG
neurons (Shin et al., 2014; Dubový et al., 2018b), increases in
the levels of GAP-43 mRNA and protein were more distinct
compared to naïve or sham-operated rats in both lumbar and
cervical DRG after SNC or CSNT for 7 days. Our observation
of a bilateral increase in GAP-43 and SCG-10 mRNA and
proteins correlates well with published results indicating that
NGF mRNA is bilaterally increased in both cervical and lumbar
DRG after unilateral sciatic nerve crush (Heumann et al., 1987;
Wells et al., 1994). These results suggest that upregulation of
growth-associated molecules is not restricted to axotomized
DRG neurons and probably reflects a systemic response of DRG
neurons to unilateral sciatic nerve injury (Dubový et al., 2013,
2018a).
The expression of regeneration-associated proteins like
GAP-43 or SCG10 is operated by transcription factors, such as
c-Jun (Raivich et al., 2004; Frey et al., 2015; Valakh et al., 2015)
or members of the MAPK family, e.g., p38 (Verma et al., 2005;
Temporin et al., 2008; Nix et al., 2011; Law et al., 2016). Our
results showing increased levels of GAP-43 and SCG10 as well
as activation of p-cJun and p-p38 in cervical DRG after sciatic
nerve injury suggested that the pro-regenerative program can
be induced in remote cervical DRG neurons 7 days after sciatic
nerve lesion.
Increased in vivo and in vitro Axon
Regeneration Capacity of Cervical DRG
Neurons Confirmed Their Pro-regenerative
State Induced by Prior Sciatic Nerve
Lesion
While SCG-10 increases only in the proximal stumps of
afferent axons (Shin et al., 2014), we also measured SCG-10 in
regenerated peripheral arms of afferent axons associated with
cervical DRG neurons distal to the ulnar nerve crush. SCG-10
immunopositive axons displayed a significantly greater length
from the point of ulnar nerve crush after prior SNC or CSNT
than in controls without the conditioning lesion. These results
demonstrated in vivo the enhanced pro-regenerative state of
cervical DRG neurons corresponding with increased levels of
GAP-43 or SCG-10 induced by the conditioning sciatic nerve
lesion.
Neurite outgrowth assays are used to assess the effects of
in vivo manipulations performed prior to removing DRG from
animals (Frey et al., 2015; Al-Ali et al., 2017). In our in vitro
assay, the number of neurites and their total lengths per neuron
were significantly higher in cervical DRG neurons removed from
rats subjected to SNC or CSNT than those from sham-operated
controls. The results of the in vitro assay confirmed the enhanced
capacity of cervical DRG neurons to regenerate their neurites
after the conditioning sciatic nerve lesion.
Thus, the axon outgrowth tested in vivo and in vitro
proves the initiation of pro-regenerative state in cervical DRG
neurons non-associated with sciatic nerve lesion. Our present
results extend previously published results that unilateral nerve
injury affects the uninjured contralateral nerve with respect to
expression of inflammatory mediators (Ruohonen et al., 2002)
including in vivo promotion of axonal regeneration in the
contralateral nerve associated with enhanced cytokine expression
in the contralateral DRG (Ryoke et al., 2000).
IL-6 Is a Candidate for Signaling the
Pro-regenerative Neuronal State in
Remote DRG After Sciatic Nerve Lesion
In response to unilateral sciatic nerve injury, mRNA and protein
levels of IL-6 and its receptors were enhanced bilaterally in
primary sensory neurons not only in DRG of lumbar segments
(L4-L5) associated with the injury, but also in cervical segments
(C7-C8) not associated with the injured nerve (Brázda et al.,
2013; Dubový et al., 2013). Upon binding to its membranebound
receptor (IL-6R), IL-6 connects the intracellular regions
of gp130 to initiate a signal transduction cascade by activating
signal transducer and activator of transcription 3 (STAT3;
Eulenfeld et al., 2012). Activation of STAT3 by JAK2-dependent
phosphorylation at the tyrosine-705 (Y705) position occurs
in DRG neurons after a nerve lesion and is mediated by
neuropoietic cytokines including IL-6 (Schwaiger et al., 2000;
Sheu et al., 2000; Qiu et al., 2005; Miao et al., 2006) and
neurotrophins (Ng et al., 2006; Pellegrino and Habecker,
2013). We found STAT3 activation and nuclear translocation
bilaterally in the DRG neurons of both lumbar and cervical
spinal cord segments after unilateral SNC or CSNT. Moreover,
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Dubový et al. Pro-regenerative State of Remote Neurons
we also proved increased levels of IL-6 protein in the CSF
following nerve injury as well as activation and nuclear
translocation of STAT3 in DRG neurons along the neuroaxis
after intrathecal injection of IL-6 (Dubový et al., 2018a). It
is known that activation of STAT3 by JAK2 phosphorylation
is a prerequisite for the synthesis of regeneration-associated
proteins like GAP-43 or SCG-10 (Patodia and Raivich, 2012)
and axon regeneration (Bareyre et al., 2011; Niemi et al.,
2016). Intrathecal application of IL-6 in our present experiments
increased STAT3 activation and GAP-43 expression in cervical
DRG neurons, while a JAK2 inhibitor (AG490) decreased
STAT3 activation and nuclear translocation as well as expression
of GAP-43.
Intrathecal delivery of IL-6 promotes regeneration of the
central arms of afferent axons into the spinal dorsal cord.
This then activates the neuronal regeneration program in DRG
neurons to overcome inhibitors of axon regeneration present
in myelin (Cao et al., 2006). Therefore, in our experiments we
tested in vivo the effect of intrathecal IL-6 and JAK2 inhibitor
injection on axon regeneration after an ulnar nerve crush to
illustrate the pro-regenerative state of cervical DRG neurons.
Intrathecal IL-6 injection significantly increased the lengths of
SCG-10+ regenerated axons distal to the ulnar nerve crush. In
contrast, intrathecal application of AG490 reduced the lengths
of regenerated axons after the ulnar nerve crush following prior
unilateral SNC or CSNT. These results suggested a critical
role for the JAK/STAT signaling pathway activated by IL-6 in
inducing the pro-regenerative state in remote DRG neurons after
unilateral sciatic nerve lesion.
What remains unclear is the mode of activation of remote
DRG neurons following a conditioning unilateral sciatic nerve
lesion. Axonal transport of local WD signaling molecules would
be implicated only on the ipsilateral side of the compressed
nerve (SNC) but can be excluded when the similar bilateral
changes were found in CSNT group of rats. We observe bilateral
changes in DRG of both lumbar and cervical segments, and
this rather points towards a systemic pathway through either
the blood stream or the CSF. The low level of IL-6 in the
plasma of rats 7 days after SNC or CSNT suggests that it is
more likely to be via the CSF. We found previously that rat
DRG have no barrier to the CSF of the spinal subarachnoid
space (Joukal et al., 2016). Therefore, intrathecal injection of IL-6
with subsequent activation of STAT3 in cervical DRG neurons
(Dubový et al., 2018a) can trigger upregulation of GAP-43
and SCG-10 associated with the pro-regenerative state of these
neurons. A unilateral sciatic nerve lesion induces increased IL-6
synthesis in the corresponding lumbar DRG (Murphy et al., 1995;
Dubový et al., 2013) and the cytokine molecules can be released
into the subarachnoid space and transported via CSF into the
remote DRG to activate STAT3 (Dubový et al., 2018a) and this
then induces the neuronal pro-regenerative state.
Finally, our results indicate that the pro-regenerative state of
cervical DRG neurons illustrates a systemic reaction along the
neuroaxis to unilateral sciatic nerve injury, and that this reaction
can be mediated by IL-6 and JAK2/STAT3 signaling. Moreover,
the results suggest a role for inflammatory mediators in activating
the neuronal pro-regenerative state without direct retrograde
axonal transport of signaling molecules from the injured nerve.
CONCLUSION
In the present study, we have demonstrated that sciatic nerve
lesion for 7 days triggers bilateral activation of a pro-regenerative
state not only in the primary sensory neurons of lumbar DRG
(L4-L5) associated with injured nerve but also in remote cervical
DRG. The bilateral activation of the pro-regenerative state in
DRG neurons anatomically non-associated with the injured
nerve correlates well with our previous observation of bilateral
expression of IL-6 and activation of STAT3. Taken together,
these results indicate that the systemic reaction of DRG neurons
to a unilateral nerve lesion along the neuroaxis can be mediated
by IL-6 and JAK2/STAT3 signaling. This phenomenon can be
activated also by other molecules released into the CSF of the
perispinal subarachnoid space by neurons and non-neuronal
cells in DRG associated with the injured nerve.
AUTHOR CONTRIBUTIONS
PD designed the research and wrote the article. IK, IH-S and
MJ performed in vivo experiments and prepared samples for
immunohistochemistry, western blot and RT-PCR and wrote
the corresponding texts. MK performed in vitro experiments
and prepared samples for immunohistochemistry and wrote the
corresponding text. VB performed and analyzed western blot and
RT-PCR data and wrote the corresponding text.
FUNDING
This work was supported by grant No. 16-08508S of The Czech
Science Foundation (Grantová Agentura ˇCeské Republiky).
ACKNOWLEDGMENTS
We thank Ms. Dana Kutˇejová, Ms. Marta Lnˇeníˇcková, Ms. Jitka
Mikuláˇsková, Mgr. Jana Vachová and Mr. Lumír Trenˇcanský for
their skillful technical assistance.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Dubový, Klusáková, Hradilová-Svíˇzenská, Brázda, Kohoutková
and Joukal. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original author(s) and the copyright owner(s)
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Cellular Neuroscience | www.frontiersin.org 16 February 2019 | Volume 13 | Article 11
D. Dubový P, Hradilová-Svíženská I, Klusáková I, Brázda V, Joukal M. Interleukin-6
contributes to initiation of neuronal regeneration program in the remote dorsal root
ganglia neurons after sciatic nerve injury. Histochemistry and Cell Biology. 2019;
152:109-117; doi:10.1007/s00418-019-01779-3
Commentary: In this publication, we demonstrated a crucial role of IL-6 in axonal
regenaration. We found that unilateral nerve compression and transection caused upregulation
of SCG-10 protein levels and activated STAT3 in DRG neurons not only in lumbar but also in
cervical segments of wild type mice. DRG neurons from IL-6 knockout mice also bilaterally
increased levels of SCG-10 and STAT-3 in both lumbar and cervical segments after sciatic
nerve lesions. However, levels of SCG-10 protein in cervical and lumbar DRG of IL-6
knockout mice were lower when compared to their wild type counterparts. This finding
correlates with significantly shorter regeneration of axons distal to the crushed ulnar nerve.
IF (WOS, 2019): 3.418
Quartile (WOS, 2019): Microscopy Q1, Cell Biology Q3
Contribution of the author: Corresponding author; approximate participation 40%. The
author conceived, designed and performed experiments, participated in acquiring and
analyzing the presented data and wrote the manuscript.
Vol.:(0123456789)1 3
Histochemistry and Cell Biology (2019) 152:109–117
https://doi.org/10.1007/s00418-019-01779-3
ORIGINAL PAPER
Interleukin-6 contributes to initiation of neuronal regeneration
program in the remote dorsal root ganglia neurons after sciatic nerve
injury
Petr Dubový1
 · Ivana Hradilová‑Svíženská1
 · Ilona Klusáková1
 · Václav Brázda1
 · Marek Joukal1
 
Accepted: 19 March 2019 / Published online: 29 March 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
To assess the potential role of IL-6 in sciatic nerve injury-induced activation of a pro-regenerative state in remote dorsal
root ganglia (DRG) neurons, we compared protein levels of SCG-10 and activated STAT3, as well as axon regeneration in
IL-6 knockout (IL-6ko) mice and their wild-type (WT) counterparts. Unilateral sciatic nerve compression and transection
upregulated SCG-10 protein levels and activated STAT3 in DRG neurons not only in lumbar but also in cervical segments
of WT mice. A pro-regenerative state induced by prior sciatic nerve lesion in cervical DRG neurons of WT mice was also
shown by testing for axon regeneration in crushed ulnar nerve. DRG neurons from IL-6ko mice also displayed bilaterally
increased levels of SCG-10 and STAT3 in both lumbar and cervical segments after sciatic nerve lesions. However, levels of
SCG-10 protein in lumbar and cervical DRG of IL-6ko mice were significantly lower than those of their WT counterparts.
Sciatic nerve injury induced a lower level of SCG-10 in cervical DRG of IL-6ko than WT mice, and this correlates with
significantly shorter regeneration of axons distal to the crushed ulnar nerve. These results suggest that IL-6 contributes, at
the very least, to initiation of the neuronal regeneration program in remote DRG neurons after unilateral sciatic nerve injury.
Keywords  Primary sensory neuron · Sciatic nerve lesion · Ulnar nerve crush · SCG10 · STAT3
Introduction
The primary sensory neurons of dorsal root ganglia (DRG)
send off afferent axons with their peripheral branches in the
peripheral nerves and central branches that run through the
dorsal roots into the spinal cord. The bodies of primary sensory
neurons react to peripheral nerve injury by upregulating
a large spectrum of genes, transforming factors and regeneration-associated
proteins that regulate the neuronal regeneration
program (Mason et al. 2002; Liu et al. 2011; Bareyre
et al. 2011; Mar et al. 2016; Martin et al. 2019). Under permissive
extrinsic conditions, this results in promoting axonal
regrowth distal to the peripheral nerve damage. Therefore,
DRG neurons are frequently used as a reliable model for
in vivo and in vitro studies of the intrinsic factors coordinating
the neuronal regenerative program.
It has been found that axotomy induces a brisk transient
increase of IL-6 synthesis in sensory neurons (Murphy et al.
1995). In our previous experiments we have found bilaterally
increased levels of IL-6 and its receptor mRNA and protein
not only in DRG neurons associated with the injured sciatic
nerve, but also in remote cervical DRG (Dubový et al.
2013; Brázda et al. 2013). Unilateral sciatic nerve injury also
induced bilateral activation of signal transducer and activator
of transcription 3 (STAT3) through tyrosine-705 (Y705)
phosphorylation in rat DRG neurons of both lumbar and
cervical segments. Moreover, we detected increased IL-6
protein levels in the CSF following sciatic nerve injury and
found activation of STAT3 in cervical DRG after intrathecal
injection of IL-6 (Dubový et al. 2018a). It is well documented
that activated STAT3 is a critical transforming factor
of the neuronal pro-regenerative state (Smith et al. 2011;
Bareyre et al. 2011; Zigmond 2012a). These published
results suggest that IL-6 is a candidate molecule involved
in initiating the pre-regenerative changes in remote DRG
neurons after sciatic nerve lesion.
*	 Marek Joukal
	mjoukal@med.muni.cz
1
	 Department of Anatomy, Cellular and Molecular Research
Group, Faculty of Medicine, Masaryk University, Kamenice
3, 62500 Brno, Czech Republic
110	 Histochemistry and Cell Biology (2019) 152:109–117
1 3
Superior cervical ganglion 10 (SCG-10) protein, also
known as stathmin 2, is a neuron-specific member of the
stathmin family (Sugiura and Mori 1995), that is significantly
increased in most DRG neurons very early following
a nerve lesion (Shin et al. 2014; Dubový et al. 2018b). SCG-
10 immunostaining is also a good marker for regenerating
sensory axons (Shin et al. 2012, 2014). Therefore, SCG-10
immunodetection is useful in monitoring the pro-regenerative
state of DRG neurons and assessing axon regeneration
distal to the nerve lesion.
In the results presented here, we used a mice model to
confirm sciatic nerve injury-induced activation of a regenerative
program in remote cervical DRG neurons. We compared
SCG-10 protein levels and activation of STAT3 in
lumbar and cervical DRG neurons of interleukin-6 knockout
(IL-6ko) with wild-type (WT) mice to investigate if IL-6
is implicated in initiating the pro-regenerative program in
remote cervical DRG neurons after sciatic nerve injury. In
addition, the pro-regenerative state of cervical DRG neurons
induced by sciatic nerve lesion was tested using an axon
regeneration assay in crushed ulnar nerve.
Materials and methods
Mouse strains and surgical treatment
Thirty male mice (6–8 weeks old) of wild-type (WT) and 30
IL-6 knock out (IL-6ko) were used in all experiments. IL-
6ko mice in the C57BL/6 genetic background were obtained
from Jackson Labs. (Bar Harbor, ME) and C57BL/6J WT
mice were purchased from the Masaryk University breeding
facility as reference counterparts. Mice were housed in
individually ventilated cages with a maximum of five animals
per cage in 12 h light/dark cycles at a temperature of
22–24 °C under specific pathogen-free conditions in the
animal facility of Masaryk University. Sterilized standard
rodent food and water were available ad libitum. The
experiments were approved by the Animal Care Committee
of the Faculty of Medicine, Masaryk University, Czech
Republic and Czech Governmental Animal Care Committee,
in compliance with the Czech Animal Protection Act no.
246/1992. All surgical procedures were performed under
anesthesia using a mixture of ketamine (100 mg/kg) and
xylazine (20 mg/kg) administered intraperitoneally. Animals
were randomly allocated to experimental groups. Seven WT
and seven IL-6ko mice without any surgical treatment were
used as naïve controls.
The left sciatic nerve of ten WT and ten IL-6ko mice was
exposed in mid-thigh between the gluteus maximus and the
anterior head of the biceps femoris, ligated with two ligatures
and cut with a pair of sharp scissors (complete sciatic
nerve transection, CSNT). The proximal nerve stump was
buried in muscle to prevent the distal stump from reinnervation.
The left sciatic nerve of a further ten WT and ten
IL-6ko mice was exposed and three ligatures with 6-0 silk
suture (Ethicon) were tied around the sciatic nerve to reduce
its diameter (sciatic nerve constriction, SNC). The muscles
and skin were closed with 5/0 sutures and animals were left
to survive for 7 days.
Quantitative immunohistochemical analysis
Naïve, SNC- and CSNT-operated WT and IL-6ko mice
(n = 3 for each group) were deeply anesthetized with a lethal
dose of sodium pentobarbital (80 mg/kg body weight, i.p.)
and perfused transcardially with 100 ml phosphate-buffered
saline (PBS, pH 7.4) followed by 100 ml of Zamboni’s
fixative (Zamboni and de Martino 1967). The L3–L4 and
C6–C8 DRG from both sides were removed and immersed in
Zamboni’s fixative at 4 °C overnight and then collected into
samples of ipsilateral (L-DRGi) and contralateral (L-DRGc)
lumbar as well as ipsilateral (C-DRGi) and contralateral
(C-DRGc) cervical DRG for each group of mice.
The DRG samples were washed in 20% phosphate-buffered
sucrose for 12 h, blocked in Tissue-Tek®
OCT compound
(Miles, Elkhart, IN) and cut to prepare serial longitudinal
cryostat sections (10 µm). The sections were mounted
on chrome-alum covered slides and processed under the
same conditions for indirect immunohistochemical staining
for SCG-10 and STAT3(Y705). Briefly, sections were
washed with PBS containing 0.05% Tween 20 (PBS-T) and
1% bovine serum albumin (BSA) for 10 min, treated with 5%
normal donkey serum in PBS-T for 30 min, then incubated
with 25 µl of rabbit polyclonal antibody against SCG-10
(1:1000; LSBio, USA) or STAT3(Y705) (1:100; Santa Cruz,
USA) in a humid chamber at room temperature (21 to 23 °C)
for 12 h. SCG-10 and STAT3Y705 immunoreactions were
visualized by treatment with FITC- or TRITC-conjugated
and affinity-purified donkey anti-rabbit secondary antibodies
(1:100; Millipore, USA), respectively. Sections were stained
with Hoechst 33342 to detect cell nuclei, mounted in aqueous
mounting medium (Vectashield; Vector Laboratories,
USA) and analyzed using an epifluorescence microscope
(Nikon Eclipse, Nikon, Czech Republic) equipped with a
camera (DFC-480; Leica Microsystems) and a stabilized
power supply for the lamp housing. The control sections
were incubated either without the primary antibodies or by
substituting the primary antibodies with the donkey IgG
isotype.
Neuronal diameter and STAT3(Y705) nuclear immunofluorescence
intensities were measured using a NIS-Elements
image analysis system (Nikon, Czech Republic) as
described previously (Dubový et al. 2002, 2018a). At least
60 neuronal profiles containing nuclei were measured for
111Histochemistry and Cell Biology (2019) 152:109–117	
1 3
each animal group. The immunofluorescence intensities
were expressed as mean intensity ± SD.
Western blot analysis
SCG-10 and STAT3(Y705) protein levels in DRG were
analyzed by Western blot. Naïve, SNC- and CSNT-operated
WT and IL-6ko mice (n = 4 for each group) were deeply
anesthetized with a lethal dose of sodium pentobarbital
(80 mg/kg body weight, i.p.) and DRG of both sides were
removed under aseptic conditions from lumbar (L3–L4)
and cervical (C6–C8) positions, washed in protease and
phosphatase inhibitor cocktails (Roche, Germany), flashfrozen
in liquid nitrogen and stored at − 80 °C until being
analyzed further. The DRG samples were homogenized in
TRIS-buffered saline (pH 7.2) with 0.1% Triton X-100 and
a cocktail of protease and phosphatase inhibitors (LaRoche,
Switzerland) and centrifuged at 10,000g for 5 min at 4 °C.
Proteins were separated by SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes by
electroblotting (BioRad). After blocking with 5% bovine
serum albumin (BSA) in TRIS-buffered saline (pH 7.2)
for 2 h, the membranes were incubated with rabbit polyclonal
antibody against SCG-10 (1:500; LSBio, USA) or
STAT3(Y705) (1:100; Santa Cruz, USA) overnight. Blots
were washed in TRIS-buffered saline (pH 7.2) and incubated
with peroxidase-conjugated anti-rabbit IgG (1:1000; Sigma,
USA) at room temperature for 1 h. Protein bands were visualized
using the ECL detection kit (Amersham, USA) on
the chemiluminometer reader LAS-3000 (Fuji, Japan) and
analyzed using densitometry image software. Equal loading
of proteins was confirmed by β-actin levels (Actin). The
protein levels were compared to the value from naïve DRG,
which was arbitrarily set as one.
In vivo assay of axon regeneration in crushed ulnar
nerve
Mice that had undergone prior SNC or CSNT (n = 3 for each
group of both WT and IL-6ko) for 7 days were re-anesthetized
and the left ulnar nerve was exposed and crushed using
a clamp with a defined force of 1.9 N for 5 s at two clicks
at the same site (Ronchi et al. 2010) under a stereoscopic
microscope. The left ulnar nerve was also crushed in a control
group without any previous sciatic nerve injury (n = 3
for both WT and IL6ko). The distal margin of the crush
injury was marked with a 10-0 epineurial stitch (Ethicon),
the skin wound was closed with 5/0 sutures, and the animals
were allowed to recover for 24 h.
To investigate initiation of peripheral axon regeneration,
the left ulnar nerves were removed following pericardial
infusion with Zamboni fixative solution and nerve
samples were fixed by immersion in Zamboni fixative
solution overnight. After washing with 10% sucrose in
PBS, 10 µm-thick longitudinal cryostat sections were cut
and immunostained with rabbit polyclonal antibody against
SCG-10 (1:1000; LSBio, USA) and FITC-conjugated,
affinity-purified donkey anti-rabbit secondary antibody as
described above. SCG-10 fluorescence intensity was analyzed
along the length of the nerve distal to the crush; the
regeneration index was determined by measuring the length
of the longest SCG-10 decorated axons from the crush site
(Abe et al. 2010). The length of SCG-10 immunopositive
(SCG-10+) axons was measured in every third section by a
person blind to the experimental conditions.
Statistical analyses
Statistical differences between data of STAT3 immunofluorescence
intensities, Western blot and axon regeneration
analysis of naïve DRG neurons and DRG neurons of SNCor
CSNT-operated mice were tested using a Mann–Whitney
U test (p < 0.05). All statistical analyses were performed
using STATISTICA 9.0 software (StatSoft, Inc., USA).
Results
Immunohistochemical and Western blot analysis
of SCG‑10 and STAT3 proteins in mouse DRG
Sections immunostained under the same conditions revealed
weak SCG-10 immunoreactivity in both lumbar and cervical
DRG neurons of naïve WT and IL-6ko mice (Figs. 1a,
b, 2a, b). Unilateral SNC or CSNT of WT mice induced a
substantial increase of SCG-10 immunofluorescence intensity
in lumbar DRG neurons both ipsilateral (Fig. 1e, i)
and contralateral (Fig. 1f, j) to the sciatic nerve lesions and
bilaterally in cervical DRG (Fig. 1c, d, g, h). In contrast to
WT mice, SNC or CSNT in IL-6ko mice induced increased
SCG-10 immunofluorescence intensity only in lumbar DRG
ipsilateral to the sciatic nerve lesions (Fig. 2e, i). However,
the contralateral lumbar DRG neurons and the neurons of
cervical DRG of both sides displayed only moderate SCG-10
immunofluorescence (Fig. 2c, d, f–h, j).
Alongside increased SCG-10 immunostaining intensity,
higher STAT3 activation by phosphorylation at Tyr-705
(Y705) and its nuclear translocation was observed bilaterally
in both lumbar and cervical DRG neurons 7 days after
SNC or CSNT operated WT mice when compared with
naïve ones (Figs. 3, 5). Sections of DRG from IL-6ko mice
immunostained under the same conditions as those of WT
mice revealed only moderate activation and nuclear translocation
of STAT3(Y705), but distinct activation and nuclear
translocation of STAT3(Y705) was observed in lumbar DRG
neurons ipsilateral to the sciatic nerve lesions. Only CSNT
112	 Histochemistry and Cell Biology (2019) 152:109–117
1 3
induced the bilateral increase of nuclear STAT3(Y705)
translocation in lumbar DRG neurons with significant
enhancement on the contralateral side (Figs. 4, 5).
The in situ changes in SCG-10 and STAT3(Y705) proteins
seen as alterations of immunofluorescence intensity
in DRG neurons of unoperated WT and IL-6ko mice and
Fig. 1  Representative pictures of cryostat sections through lumbar
(L4) and cervical (C7) dorsal root ganglia (DRG) from naïve (a, b)
WT mice and those with unilateral sciatic nerve compression (SNC;
c–f) or complete sciatic nerve transection (CSNT; g–j) for 7  days.
The sections of ipsilateral and contralateral lumbar (DRG-L4i, DRGL4c)
and cervical (DRG-C7i, DRG-C7c) DRG were incubated under
the same conditions with rabbit polyclonal antibody against SCG-10
and FITC-conjugated donkey anti-rabbit secondary antibody. Unilateral
SNC or CSNT of WT mice induced a substantial increase of
SCG-10 immunofluorescence intensity in lumbar neurons both ipsilateral
(e, i) and contralateral (f, j) to the sciatic nerve lesions and
bilaterally in cervical DRG (c, d, g, h). Scale bars = 30 µm
Fig. 2  Representative pictures of cryostat sections through lumbar
(L4) and cervical (C7) dorsal root ganglia (DRG) from naïve (a, b)
IL-6ko mouse and IL-6ko mice with unilateral sciatic nerve compression
(SNC; c–f) or complete sciatic nerve transection (CSNT; g–j) for
7 days. The DRG sections were incubated under the same conditions
with rabbit polyclonal antibody against SCG-10 and FITC-conjugated
donkey anti-rabbit secondary antibody. SNC or CSNT in IL-6ko mice
induced increased SCG-10 immunofluorescence intensity only in
lumbar DRG ipsilateral to the sciatic nerve lesions (e, i). The contralateral
lumbar DRG neurons (f, j) and the neurons of cervical DRG
of both sides (c, d, g, h) displayed only moderate SCG-10 immunofluorescence.
Scale bars = 30 µm
113Histochemistry and Cell Biology (2019) 152:109–117	
1 3
following SNC or CSNT were confirmed by Western blot
analysis (Fig. 6). Levels of SCG-10 protein increased bilaterally
in both cervical and lumbar DRG of WT mice after
SNC and CSNT when compared to DRG from naïve mice.
Lumbar DRG ipsilateral to the sciatic nerve lesions showed
a greater increase of SCG-10 levels than those from the contralateral
side, but this was not statistically significant. The
increases in SCG-10 protein levels in cervical DRG from
both sides were approximately similar. SCG-10 protein was
also bilaterally increased in both lumbar and cervical DRG
of IL-6ko mice after SNC or CSNT when compared to naïve
mice. Further, CSNT—but not SNC—induced a substantially
bigger increase of SCG-10 levels in both lumbar and
cervical DRG. Generally, the values of SCG-10 protein were
significantly lower in DRG of IL-6ko than WT mice.
Compared to naïve controls, SNC and CSNT induced a
bilateral increase in STAT3(Y705) protein levels in both
lumbar and cervical DRG of both WT and IL-6ko mice
(Fig. 5). However, the extent of STAT3(Y705) increase
in IL-6ko mice was not as strong as in WT. In contrast to
WT mice, CSNT induced a more significant elevation of
STAT3(Y705) protein than SNC (Fig. 6).
Axon regeneration assay in crushed ulnar nerve
after prior sciatic nerve injury in wild‑type
and IL‑6ko mice
We tested initiation of the pro-regenerative state in cervical
DRG neurons of WT and IL-6ko mice by monitoring axonal
regeneration in longitudinal sections of crushed ulnar nerves
after SCG-10 immunostaining. The axon regeneration index
was expressed as the longest SCG-10+ axon from the point
of nerve crush. The length of SCG-10+ axons was greater
in SNC- and CSNT-operated WT mice compared to control
WT mice with only ulnar nerve crush (Fig. 7). These
results indicate that prior sciatic nerve injury was able to
induce increased regeneration capacity in cervical DRG of
WT mice.
Generally, the length of the longest SCG-10+ axons distal
to ulnar nerve crush was significantly shorter in IL-6ko mice
compared to those in WT controls. Besides, we found no
significant differences in the length of SCG-10+ regenerated
axons in the crushed ulnar nerve of IL-6ko mice with prior
SNC or CSNT (Fig. 7).
Discussion
We described previously in a rat model that sciatic nerve
lesion induced a temporary upregulation of IL-6 and its
receptor not only in DRG of the lumbar (L4–L5) but also
in remote DRG of cervical segments (Dubový et al. 2013).
Downstream signaling of IL-6 is mediated via activation
and nuclear translocation of STAT3 (Aaronson and Horvath
2002; Eulenfeld et al. 2012). Recently, we demonstrated
activation and nuclear translocation of STAT3 bilaterally in
Fig. 3  Representative pictures of cryostat sections through lumbar
(L4) and cervical (C7) dorsal root ganglia (DRG) from naïve (a, b)
WT mice and WT mice with unilateral sciatic nerve compression
(SNC; c–f) or complete sciatic nerve transection (CSNT; g–j) for
7 days. The sections of ipsilateral and contralateral lumbar (DRG-L4i,
DRG-L4c) and cervical (DRG-C7i, DRG-C7c) DRG incubated under
the same conditions show immunofluorescence staining of STAT3
activated by Y705 phosphorylation and its nuclear translocation
(arrowheads) bilaterally in lumbar (e, f, i, j) and cervical (c, d, g, h)
DRG neurons 7 days after SNC or CSNT when compared with naïve
ones (a, b). Scale bars = 30 µm
114	 Histochemistry and Cell Biology (2019) 152:109–117
1 3
DRG neurons of both lumbar and cervical segments after
unilateral sciatic nerve injury. In addition, increased levels of
IL-6 protein were detected in the cerebrospinal fluid (CSF)
following sciatic nerve injury and activation of STAT3 was
found in cervical DRG after intrathecal injection of IL-6
(Dubový et al. 2018a). Since IL-6 and activated STAT3 participate
in initiating the axonal regeneration program (Qiu
et al. 2005; Dziennis and Alkayed 2008; Smith et al. 2011;
Bareyre et al. 2011; Zigmond 2012b; Quarta et al. 2014), we
used IL-6ko mice and compared them to WT mice to see if
IL-6 is a potential mediator of the pro-regenerative stage in
DRG neurons non-associated with the injured sciatic nerve.
The induction of the regeneration-associated program can
be indicated by increased SCG-10 that is also a marker for
regenerated sensory axons of injured peripheral nerves (Shin
et al. 2014). The DRG neurons of naïve WT and IL-6ko mice
displayed a low basal level of SCG-10 that was significantly
increased in the neuronal bodies of lumbar DRG ipsilateral
to SNC or CSNT. In addition, both types of sciatic nerve
lesion in WT mice induced a bilateral elevation of SCG-
10 not only in lumbar (L3–L4), but also in cervical DRG
(C6–C8). This initiation of the pro-regenerative state in cervical
DRG neurons of WT mice by sciatic nerve lesion was
also illustrated by STAT3 activation and axon regeneration
Fig. 4  Representative pictures
of cryostat sections through
dorsal root ganglia (DRG) from
naïve (a, b) IL6ko mice and
those with unilateral sciatic
nerve compression (SNC;
c–f) or complete sciatic nerve
transection (CSNT; g–j) for
7 days. The sections of ipsilateral
and contralateral lumbar
(DRG-L4i, DRG-L4c) and
cervical (DRG-C7i, DRG-C7c)
DRG were incubated under the
same conditions with rabbit
polyclonal antibody specific
to Y705-phospho-STAT3 and
TRITC-conjugated donkey
anti-rabbit secondary antibody.
Activated and nuclear translocation
of STAT3 (arrowheads)
was observed in lumbar DRG
ipsilateral (e, i) and contralateral
(less intense; f, j) to the sciatic
nerve lesion. Scale bars = 30 µm
115Histochemistry and Cell Biology (2019) 152:109–117	
1 3
distal to ulnar nerve crush compared to mice without and
with prior SNC or CSNT. These results demonstrate for the
first time that the pro-regenerative state of cervical DRG
after prior sciatic nerve injury described in rats (Dubový
et al. 2019) is also found in the mouse model.
In contrast to WT, sciatic nerve lesions in IL-6ko mice
significantly increased SCG-10 and STAT3(Y705) levels
only in lumbar DRG neurons ipsilateral to the sciatic nerve
injury. However, contralateral DRG neurons and DRG neurons
of cervical segments on both sides displayed lower
levels of these two proteins. Corresponding to the expected
lower activation of STAT3 in cervical DRG neurons of IL-
6ko mice, the length of regenerated axons distal to ulnar
nerve crush was significantly shorter in IL-6ko than WT
mice. These results demonstrate that IL-6 deficiency leads
to reduced initiation of the pro-regenerative state in cervical
DRG neurons after prior sciatic nerve injury.
The results published regarding a role for IL-6 in activating
DRG neurons to regenerate their central afferent
branches following conditioning by a peripheral nerve injury
are controversial (Cafferty et al. 2004; Cao et al. 2006). In
addition, it has been demonstrated that in situ deactivation
of IL-6 enhanced peripheral nerve regeneration (Koulaxouzidis
et al. 2015). However, a pro-regenerative state in
DRG neurons non-associated with the lesioned nerve can be
initiated indirectly via CSF of the paraspinal subarachnoid
space including DRG along the spinal cord (Joukal et al.
2016). A role for IL-6 in the initiation of the pro-regenerative
state in cervical DRG via CSF of the subarachnoid space
Fig. 5  Results of STAT3(Y705) immunofluorescence intensity measured
in the neuronal nuclei of lumbar (L-DRG) and cervical (C-DRG)
dorsal root ganglia of naïve, WT and IL-6ko mice. DRG ipsilateral (i)
and contralateral (c) to sciatic nerve compression (SNC) or complete
sciatic nerve transection (CSNT) were collected after 7 days. *Significant
difference (p < 0.05) compared to naïve control, †
significant
difference (p < 0.05) compared to IL-6ko (Mann–Whitney u test)
Fig. 6  Results of Western
blot analysis of SCG-10 and
STAT3(Y705) protein levels
in DRG of L3-L4 (L) and
C6-C8 (C) segments collected
from ipsilateral (i) and
contralateral (c) sides of naïve,
SNC- and CSNT-operated
mice for 7 days. Upper panels
show representative blots of
SCG-10 and STAT3(Y705)
from DRG of WT and IL6ko
mice. Equal loading of proteins
was confirmed by actin levels
(Actin). Lower panels show
densitometry of SCG-10 and
STAT3(Y705) protein bands
after normalization to actin; the
intensities of the SCG-10 and
STAT3(Y705) bands from naïve
DRG were taken as 1. *Significant
difference (p < 0.05)
compared to naïve control,
†
significant difference (p < 0.05)
compared to SNC, +
significant
difference (p < 0.05) compared
to WT counterparts (Mann–
Whitney u test)
116	 Histochemistry and Cell Biology (2019) 152:109–117
1 3
is also supported by the activation and nuclear translocation
of STAT3 after intrathecal injection of IL-6 (Dubový et al.
2018a). Since DRG lack the blood-nerve barrier (Anzil et al.
1976), it is possible that remotely acting IL-6 may also be
serum-derived.
Apart from lumbar DRG after CSNT, activation of
STAT3 was lower in DRG after SNC of IL-6ko mice compared
to WT mice suggesting the involvement of other
neuropoietic cytokines including leukemia inhibitory factor
(LIF). LIF may also have a role in initiating the proregenerative
state in DRG neurons associated with peripheral
nerve injury (Sun and Zigmond 1996; Cafferty et al.
2001). However, it is still unclear whether LIF upregulated
in DRG neurons after nerve injury can be released into the
subarachnoid space and subsequently diffuse into remote
DRG as has been shown for IL-6.
Conclusion
Monitoring SCG10 and activated STAT3 levels in cervical
DRG neurons after prior sciatic nerve lesions in WT and
IL-6ko mice revealed a role for IL6 in activating the neuronal
regeneration program in DRG neurons non-associated
with the injured nerve. This was confirmed by significantly
shorter regenerated axons distal to ulnar nerve crush following
sciatic nerve lesions in IL-6ko mice compared to
WT controls.
Acknowledgements  We thank Ms. Marta Lněníčková, Ms. Jitka
Mikulášková, Mgr. Jana Vachová and Mr. Lumír Trenčanský for their
skillful technical assistance.
Funding  This work was supported by Grant no. 16-08508S of The
Czech Science Foundation.
Compliance with ethical standards 
Conflict of interest  The authors declared no potential conflicts of interest
with respect to the research, authorship, and/or publication of this
article.
Ethical approval  All procedures performed in studies involving animals
were in accordance with the ethical standards of the institution
or practice at which the studies were conducted.
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Histochemistry & Cell Biology is a copyright of Springer, 2019. All Rights Reserved.
E. Hernangómez M, Klusáková I, Joukal M, Hradilová-Svíženská I, Guaza C, Dubový P.
CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity
immediately after its intrathecal application in a rat neuropathic pain model. J
Neuroinflammation. 2016;13:43. doi:10.1186/s12974-016-0508-8
Commentary: In this publication, we demonstrated that intrathecal application of the
CD200R1 agonist CD200Fc induces rapid supression of behavioral signs of neuropathic pain
after chronic constriction of the sciatic nerve. This was followed with supression of glial
activation, decreased proinflammatory and incresed anti-inflammatory cytokine mRNA
levels.
IF (WOS, 2016): 5.102
Quartile (WOS, 2016): Immunology Q1, Neurosciences Q1
Contribution of the author: Co-authorship; approximate participation 20%. The author
conceived, designed, and carried out intrathecal administration and participated in acquiring
and analyzing the presented data.
RESEARCH Open Access
CD200R1 agonist attenuates glial activation,
inflammatory reactions, and
hypersensitivity immediately after its
intrathecal application in a rat neuropathic
pain model
Miriam Hernangómez1
, Ilona Klusáková1,2†
, Marek Joukal2
, Ivana Hradilová-Svíženská1,2†
, Carmen Guaza3
and Petr Dubový1,2*
Abstract
Background: Interaction of CD200 with its receptor CD200R has an immunoregulatory role and attenuates various
types of neuroinflammatory diseases.
Methods: Immunofluorescence staining, western blot analysis, and RT-PCR were used to investigate the modulatory
effects of CD200 fusion protein (CD200Fc) on activation of microglia and astrocytes as well as synthesis of
pro- (TNF, IL-1β, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines in the L4–L5 spinal cord segments in relation to
behavioral signs of neuropathic pain after unilateral sterile chronic constriction injury (sCCI) of the sciatic nerve.
Withdrawal thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were measured
in hind paws 1 day before operation; 1, 3, and 7 days after sCCI operation; and then 5 and 24 h after intrathecal
application of artificial cerebrospinal fluid or CD200Fc.
Results: Seven days from sCCI operation and 5 h from intrathecal application, CD200Fc reduced mechanical and
thermal hypersensitivity when compared with control animals. Simultaneously, CD200Fc attenuated activation of
glial cells and decreased proinflammatory and increased anti-inflammatory cytokine messenger RNA (mRNA) levels.
Administration of CD200Fc also diminished elevation of CD200 and CD200R proteins as a concomitant reaction of the
modulatory system to increased neuroinflammatory reactions after nerve injury. The anti-inflammatory effect of
CD200Fc dropped at 24 h after intrathecal application.
Conclusions: Intrathecal administration of the CD200R1 agonist CD200Fc induces very rapid suppression of
neuroinflammatory reactions associated with glial activation and neuropathic pain development. This may constitute a
promising and novel therapeutic approach for the treatment of neuropathic pain.
Keywords: Rat neuropathic pain model, Sterile nerve constriction, Neuroinflammation, Activated glial cells, Cytokines,
Modulation
* Correspondence: pdubovy@med.muni.cz
†
Equal contributors
1
Central European Institute of Technology (CEITEC), Masaryk University,
Kamenice 3, 62500 Brno, Czech Republic
2
Department of Anatomy, Division of Neuroanatomy, Faculty of Medicine,
Masaryk University, Kamenice 3, 62500 Brno, Czech Republic
Full list of author information is available at the end of the article
© 2016 Hernangómez et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43
DOI 10.1186/s12974-016-0508-8
Background
Peripheral neuropathic pain (PNP), manifesting as
spontaneous pain, arises as a result of many forms of
nerve damage, including traumatic nerve injury, diabetic
neuropathy, HIV neuropathy, and drug-induced
neuropathy [1, 2]. Among other effects, nerve injuryinduced
PNP is associated with inflammatory reaction
and activation of glial cells in the corresponding spinal
cord segments [1, 3, 4].
Experimental PNP models are predominantly based on
injury to the sciatic nerve, wherein the maximum number
of neuronal perikarya (98–99 %) is localized at the
L4 and L5 segments [5]. Sterile chronic constriction injury
(sCCI) of the sciatic nerve is a model for study of
cellular and molecular changes inducing PNP after traumatic
nerve injury with dominant molecular signaling
from Wallerian degeneration [6]. It is well documented
that hypersensitivity and ongoing pain due to peripheral
nerve injury are associated with cellular and molecular
changes in the dorsal horn (DH) of the spinal cord related
to activation of microglial cells and astrocytes and
alteration of pro- and anti-inflammatory cytokines produced
by neurons, activated glia, and invaded immune
cells [7–12]. There is a growing body of evidence that
unilateral nerve injury results in bilateral neuroinflammatory
reaction in the dorsal root ganglia and spinal
cord DH [10, 13–15], thus illustrating signaling from the
site of Wallerian degeneration to other compartments of
the nervous system [16].
CD200 is a membrane glycoprotein of the immunoglobulin
superfamily with immune suppression effect via
its receptor CD200R. CD200 has an extracellular portion
with two immunoglobulin domains, typical of proteins
involved in cell-to-cell interaction. The CD200 receptor
CD200R1 has a similar structure but with an additional
intracellular domain that is susceptible to phosphorylation
and involved in signal transduction [17, 18]. CD200 is
highly expressed on neurons while CD200R is confined
mainly to myeloid cells like macrophages and microglia
[19–23]. In addition, CD200 is expressed in oligodendrocytes
[23] and astrocytes [23, 24]. The interaction of
CD200 with its receptor CD200R plays a significant role
in maintaining microglia in a quiescent or resting state
and attenuates various types of neuroinflammatory diseases
[19, 25, 26]. It has been demonstrated that mice with
levels of CD200 increased by spontaneous mutation in the
Wld gene have less activated monocytes and increasing
expression of IL-10 in the central nervous system following
induction of experimental autoimmune encephalomyelitis
[27]. Conversely, CD200−/− mice have been shown
to display myeloid cell dysregulation, enhanced susceptibility
to experimental autoimmune encephalomyelitis [28],
and microglial activation [29]. Furthermore, CD200R expression
can be modulated by IL-4 and IL-13 [30, 31].
Experimental studies have demonstrated that soluble
CD200 fusion protein (CD200Fc), containing the ectodomain
of CD200 bound to a murine IgG2a module, attenuates
inflammatory diseases and reduces microglial
activation [32–36]. Nevertheless, our knowledge remains
limited as to the effects of CD200Fc in attenuation of glial
cell activation and alteration of pro- and anti-inflammatory
cytokines in PNP induced by a peripheral nerve injury.
Therefore, the goal of our present study was to explore
whether intrathecal application of CD200Fc might attenuate
activation of glial cells in the spinal cord, modify synthesis
of pro- and anti-inflammatory cytokines, and reduce
behavioral signs of PNP in the sCCI model. Our results
provide the first evidence that CD200Fc administration
induces rapid attenuation of glial activation and proinflammatory
cytokine synthesis of the spinal cord in
relation to reduction of neuropathic pain signs after
experimental nerve injury.
Methods
Animals and surgical procedures
The experiments were carried out on 120 male Wistar
rats weighing 240–250 g at the beginning of experiments.
The animals were housed on 12 h light/dark cycles at
temperature 22–24 °C under specific pathogen-free conditions
in the animal housing facility of Masaryk University.
All surgical procedures were performed by one person
under aseptic conditions and deep anesthesia induced by
a xylazine and ketamine cocktail injected intraperitoneally
(xylazine 1.6 mg/kg; ketamine 64 mg/kg). Sterilized food
and water were available ad libitum. Treatment of the
animals was in accordance with the European Convention
for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes and was
supervised by the institutional Ethics Committee of
Masaryk University in Brno (Czech Republic).
Six naïve rats treated with CD200Fc were used to
demonstrate that the CD200R agonist has no effect on
basal skin sensitivity, and 18 naïve rats were utilized for
comparison of glial activation by immunohistochemistry,
western blot analysis, and relative cytokine messenger
RNA (mRNA) levels. The left sciatic nerve was exposed
at mid-thigh and three silk ligatures (Ethicon 3–0) were
applied to reduce the nerve diameter by one third in the
rats undergoing sCCI (n = 72). The left sciatic nerves of
24 sham-operated rats were only exposed without any
nerve lesion. Sham- and sCCI-operated animals were left
to survive for 7 days.
Administration of CD200Fc and vehicle
At day 7 of sCCI operation, the rats were randomly
divided into groups with intrathecal administration of
CD200Fc for 5 h (n = 18; sCCI + CD200Fc5h) and 24 h
(n = 18; sCCI + CD200Fc24h) as well as a control group
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 2 of 15
with intrathecal application of artificial cerebrospinal
fluid (ACSF) for 5 h (n = 18; sCCI + ACSF5h) and 24 h
(n = 18; sCCI + ACSF24h). CD200Fc (Cat. No. 3355-CD;
R&D Systems, Minneapolis, MN, USA) was freshly prepared
in sterile ACSF [37]. The single intrathecal injection
was administered by introducing a hypodermic needle
into the subarachnoid space of the cisterna magna for diffusion
of CD200Fc (5 μl; 2 μg/μl) or sterile ACSF (5 μl)
throughout the spinal fluid over 30 s with another 30-s
delay before removing the needle. To explore the effect of
surgical approach on inflammatory reaction in the spinal
cord, sham-operated rats were treated with ACSF (5 μl)
for 5 h (n = 18) and 24 h (n = 6, only for behavioral test). A
direct effect of CD200Fc on basal sensitivity was tested in
six naïve rats treated with CD200Fc (5 μl; 2 μg/μl) for 5
and 24 h.
Behavioral tests
Withdrawal thresholds for mechanical and thermal
hypersensitivity were measured in ipsilateral hind paws
using a dynamic plantar esthesiometer and plantar test
(Ugo Basile), respectively. Rats were first acclimated in
clear Plexiglas boxes for 30 min prior to testing. The
paws were tested alternately with 5-min intervals between
tests 1 day before operation; 1, 3, and 7 days after
sCCI operation; and then 5 and 24 h after intrathecal
ACSF or CD200Fc treatment. Six naïve rats were tested
at 5 and 24 h after intrathecal administration to investigate
a possible CD200Fc effect on basal sensitivity of animals.
Five measurements were taken for each paw and
test session. In the case of thermal hypersensitivity, withdrawal
time was measured and the intensity radiance
was set at value 50. Data for mechanical and thermal
hypersensitivity were expressed as mean ± SE of withdrawal
thresholds in grams and withdrawal latency in
seconds, respectively. All behavioral tests were conducted
in a blind manner.
Immunohistochemical staining
The naïve rats and sCCI-operated rats treated with ACSF
or CD200Fc for 5 and 24 h, as well as sham-operated and
ACSF-treated rats (n = 6 for each group) were deeply
anesthetized with a lethal dose of sodium pentobarbital
(70 mg/kg body weight, i.p.) and perfused transcardially
with 500 ml of heparinized (1000 units/500 ml)
phosphate-buffered saline (PBS; 10 mM sodium phosphate
buffer, pH 7.4, containing 0.15 M NaCl) followed
by 500 ml of Zamboni’s fixative [38]. The L4–L5 spinal
cord segments were removed, immersed separately in
Zamboni’s fixative at 4 °C overnight, and then collected
for each experimental group. The samples were washed in
20 % phosphate-buffered sucrose for 12 h and blocked in
Tissue-Tek® OCT compound (Miles, Elkhart, Ind.).
Serial transverse sections (12 μm) of the L4–L5 spinal
cord segments were cut (Leica 1800 cryostat; Leica
Microsystems, Wetzlar, Germany), collected on gelatincoated
microscopic slides, air-dried, then processed for
immunohistochemical staining.
Briefly, the sections were washed with PBS containing
0.05 % Tween 20 (PBS-TW20) and 1 % bovine serum
albumin for 10 min, then treated with 3 % normal donkey
serum in PBS-TW20 for 30 min. The spinal cord
sections were incubated with 25 μl of mouse monoclonal
anti-CD11b/c antibody (OX42, 1:50; AbD Serotec,
Kidlington, UK) or rabbit polyclonal anti-glial fibrillary
acidic protein (GFAP, 1:250; Dako, Glostrup, Denmark)
in a humid chamber at room temperature (21–23 °C)
overnight or for 180 min to identify activated microglial
cells and astrocytes, respectively. The immunoreaction
was visualized by treatment with FITC-conjugated
affinity purified donkey anti-mouse or anti-rabbit secondary
antibodies (1:400; Merck Millipore) for 90 min
at room temperature. A part of sections was incubated
overnight with mouse monoclonal anti-CD200 antibody
(OX2, 1:100; Abcam) and treated with FITC-conjugated
donkey anti-mouse (1:400; Merck Millipore) secondary
antibody. Spinal cord distribution of CD200R was detected
by immunostaining of sections with goat polyclonal
anti-CD200R (OX2R, 1:200; Santa Cruz Biotechnology,
Inc. USA) antibody and biotinylated donkey anti-goat
secondary antibody (1:400; Santa Cruz Biotechnology,
Inc. USA). Immunoreaction was visualized by TRITCconjugated
streptavidin (1:100; Jackson Laboratories,
Inc. USA). The control sections were incubated with
omission of the primary antibodies (data not shown). The
cell nuclei were stained using Hoechst 33342 (Sigma; St.
Louis, MO, USA). Sections were mounted in Vectashield
aqueous mounting medium (Vector Laboratories;
Burlingame, CA, USA) and then observed and analyzed
using a Leica DMLB epifluorescence microscope equipped
with a Leica DFC-480 camera (Leica Microsystems
GmbH, Wetzlar, Germany).
Double immunofluorescence staining
To detect cellular localization of CD200 and CD200R
proteins in the spinal cord, simultaneous immunostaining
of CD200 or CD200R with corresponding cellular markers
was carried out. Activated astrocytes and microglial cells
were identified by immunostaining for GFAP and OX42,
respectively (see above). Immunofluorescence staining
with rabbit polyclonal NeuN antibody (1:500; Merck
Millipore) was used to identify neurons of the spinal
DH. Briefly, the spinal cord sections were immunostained
for CD200 (see above), and after washing, the
sections were immunolabeled with rabbit polyclonal
anti-GFAP or NeuN antibody and TRITC-conjugated
donkey anti-rabbit (1:400; Merck Millipore) secondary
antibody. Other sections were incubated at first to
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 3 of 15
visualize distribution of CD200R and then for detection of
activated microglial cells using mouse monoclonal OX42
antibody and FITC-conjugated donkey anti-mouse secondary
antibody (see above). The control sections of the
double immunostaining were incubated as described
above but with omission of CD200 or CD200R antibodies.
In the controls, no immunostaining for CD200 or CD200R
was detected in the spinal cord sections.
To detect CD200R in activated astrocytes, the sections
immunostained for CD200R using goat polyclonal antibody
and biotin-streptavidin TRITC were next immunolabeled
with rabbit anti-GFAP polyclonal antibody and
FITC-conjugated donkey anti-rabbit secondary antibody
(see above). The control sections were incubated with
the goat anti-CD200R polyclonal antibody and FITCconjugated
donkey anti-rabbit secondary antibody or
with rabbit anti-GFAP polyclonal antibody, and next, a
biotin-streptavidin procedure was used for visualization
of CD200R. No cross reaction between the secondary
and primary antibodies was detected in these control
sections. Double immunostained sections were mounted
in Vectashield aqueous mounting medium and analyzed
using a Nikon Eclipse epifluorescence microscope equipped
with a DS-Ri1 camera (NIKON, Czech Republic).
Because detection of CD200R expression in activated
astrocytes is not routine, the colocalization of
CD200R and GFAP immunofluorescence was analyzed
by colocalization module of NIS Elements software
(Nikon, Czech Republic).
Image analysis
At least 10 sections (each separated from the next by an
interval of about 80 μm) from each of the removed L4–
L5 spinal cord segments for each animal per experimental
group were selected for image analysis. Immunostaining
area for OX42 or GFAP in spinal cord sections was
measured using an NIS elements image analysis system
(Laboratory Imaging Ltd, Prague, Czech Republic).
Briefly, a box (4 × 104
μm2
) was placed over the lateral,
central, and medial areas of DH and OX42 or GFAP
immunostained structures were detected by thresholding
technique after subtraction of background. The area
of immunostaining for OX42 or GFAP in corresponding
DH was related to the area of interest (4 × 104
μm2
)
and expressed as the mean of relative area (%) ± SD.
Western blot analysis
Rats were deeply anesthetized with a lethal dose of sodium
pentobarbital (70 mg/kg body weight, i.p.). The
L4–L5 spinal cord segments were detected following
total laminectomy and rapidly removed, frozen in dry
ice, then stored at −70 °C until elaboration for western
blot or reverse transcription (RT) and real-time polymerase
chain reaction (PCR).
The fresh tissue samples of L4–L5 spinal cord segments
from six animals for each group were homogenized
in PBS containing 0.1 % Triton X-100 and
protease inhibitors (LaRoche, Switzerland) and then
centrifuged at 10,000g for 5 min at 4 °C. Proteins were
separated by SDS-polyacrylamide gel electrophoresis
[39] and transferred to nitrocellulose membranes by
electroblotting (Bio-Rad). Blots were blocked by 5 %
milk-TBST for 1 h and incubated with mouse monoclonal
OX42 antibody (1:300; AbD Serotec), rabbit polyclonal
anti-GFAP (1:250; DAKO), goat polyclonal anti-CD200
(1:100; Santa Cruz Biotechnology), or anti-CD200R antibody
(1:100; Santa Cruz Biotechnology) at 4 °C for 18 h.
Blots were washed in PBS-TW20 and incubated with
secondary antibody (goat anti-mouse or anti-rabbit, 1:1000,
Immunotech; anti-goat, 1:8000, Bio-Rad), at room
temperature for 1 h. Protein bands were visualized using
the ECL detection kit (Amersham) on an LAS-3000 chemiluminometer
reader (Bouchet Biotech) and analyzed using
densitometry image software. The blots were stripped in
62.5 mM Tris–HCl, pH 6.8, containing 2 % SDS and 0.7 %
β-mercaptoethanol, and they were then reprobed with a
monoclonal antibody against β-tubulin (1:5000; Exbio).
Reverse transcription and real-time polymerase chain
reaction
The fresh tissue samples of L4–L5 spinal cord segments
from six animals for each group were collected. Total
RNA was extracted using RNeasy mini columns (Qiagen,
Hilden, Germany). Contaminating genomic DNA was
degraded by a treatment with DnaseI (Qiagen). The
yield of RNA was determined using a Nanodrop® spectrophotometer
(NanoDrop Technologies, Wilmington,
DE, USA). Total RNA (1 μg in 20 μL) was reverse transcribed
into complementary DNA (cDNA) using poly-dT
primers and the Promega reverse transcription kit
(Promega, Madrid, Spain). The oligonucleotide primer
sequences used are given in Table 1. SYBR® PCR was
performed using 1 μL of cDNA (corresponding to
50 ng RNA input) with 200 nM of the primers listed
above (Invitrogen, Barcelona, Spain) in a Power SYBR®
PCR Mastermix (Applied Biosystems, Foster City, CA,
USA). Cycling conditions were as follows: 50 °C for 2 min,
95 °C for 10 min, and 40 amplification cycles of 95 °C for
15 s and 60 °C for 1 min. Samples were assayed using the
Applied Biosystems PRISM 7500 sequence detection
system, assaying each sample in triplicate and running a
six-point standard curve in parallel. To ensure the absence
of contamination with genomic DNA, a control sample
using RNA as the template was run for each set of extractions.
Relative quantification was obtained by calculating
the ratio between the values obtained for each gene of
interest and those of the 18S housekeeping gene with respect
to the naïve animals.
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 4 of 15
Statistical analyses
Behavioral data were evaluated using Kruskal-Wallis
one-way analysis with Bonferroni post hoc test and p
values less than 0.05 were considered to be significant.
To verify differences of immunostaining area, western
blot and RT-PCR, a one-way ANOVA with subsequent
post hoc t tests employing a correction of alpha according
to Bonferroni for repeated measures was run with p < 0.05
as the level of significant differences between tested
samples. Statistical differences between data of relative
immunostaining area, western blot, and RT-PCR were
tested by Mann-Whitney U test (p < 0.05). All statistical
analyses were made using STATISTICA-12 software
(StatSoft, Tulsa, OK, USA).
Results
CD200Fc reduces mechanical and thermal
hypersensitivity of nerve-injured rats
As CD200Fc appears to be beneficial when administered
in cases of various diseases with an inflammatory component
[34, 36, 40], we investigated whether the CD200R1
agonist has effects on PNP behavioral signs in the sCCI
model. No statistically significant changes of thresholds
and withdrawal latencies were measured in the hind paws
of naïve rats treated with CD200Fc for 5 and 24 h, thus
indicating no influence of the CD200R agonist on basal
sensitivity (data not shown). Sham-operated rats displayed
a small but not statistically significant mechanical and
thermal hypersensitivity at days 1 and 3 with no behavioral
changes after 7 days and ACSF treatment for 5 or 24 h
(Fig. 1a, b). All hind paws of rats subjected to sCCI of the
sciatic nerve displayed significantly decreased thresholds of
mechanical (Fig. 1a) and withdrawal latencies of thermal
hypersensitivity (Fig. 1b) at days 1, 3, and 7 post-operation
when compared with 1 day before operation.
Rats operated on sCCI with intrathecal administration
of CD200Fc for 5 h displayed significant attenuation of
mechanical and thermal hypersensitivity when compared
with levels before CD200Fc injection or with ACSFtreated
rats. Thresholds of mechanical and withdrawal
latencies of thermal hypersensitivity did drop 24 h after
CD200Fc administration, but the values remained still
significantly higher when compared with levels before
CD200Fc treatment (Fig. 1a, b).
CD200Fc attenuates activation of microglial cells and
astrocytes in the spinal dorsal horn of nerve-injured rats
We investigated the regulatory effect of CD200Fc administration
on microglial activation in the sCCI model
of PNP because interaction of CD200 with CD200R decreases
microglial activation [20, 33]. No significant difference
of OX42 immunostaining was found between
naïve and sham-operated rats (Fig. 2a, d, h). The OX42
immunoreactive area indicating activated microglial cells
was markedly larger in ipsilateral DH of the spinal cord
sections from sCCI-operated and ACSF-treated rats
when compared with naïve or sham-operated animals
(Fig. 2b, e). Microglial activation expressed by the OX42
immunoreactive area was significantly attenuated in ipsilateral
DH sections prepared from the spinal cord of
sCCI-operated rats and treated with CD200Fc for 5 h
(Fig. 2c, f). However, 24 h after CD200Fc injection, the
OX42 immunostaining area was enlarged, but it
remained smaller than in control group of sCCIoperated
and ACSF-treated rats (Fig. 2g). In contrast to
ipsilateral DH, no significant extension of OX42 immunoreactive
area was detected in the contralateral DH of
sCCI-operated rats treated with ACSF or CD200Fc in
comparison to naïve or sham-operated rats (Fig. 2i–k).
The microglial cells immunostained for OX42 displayed
the typical activated, amoeboid morphology in sections
of L4–L5 spinal cord segments from rats sCCI-operated
and treated with ACSF, whereas those in sections from
CD200Fc-treated rats exhibited a ramified morphology
similar to microglia of the naïve animals (insets in
Fig. 2d–f). Quantitative changes of microglial activation
detected by OX42 immunostaining area in the spinal
cord sections of naïve, sham- and sCCI-operated, and
ACSF- or CD200Fc-treated rats (Fig. 2l) were confirmed
by western blot analysis of CD11b/c protein (Fig. 2m).
As CD200Fc appears to attenuate microglial activation
in the sCCI-operated rats, we investigated whether intrathecal
application of CD200Fc would also affect astroglial
activation. Lumbar spinal cord sections prepared
from rats 7 days after sCCI operation and treatment
with ACSF displayed a bilateral enlargement of GFAP
immunostaining area in DH when compared to the
spinal cord sections of naïve or sham-operated rats
(Fig. 3a–b, d–e, h–i). In comparison to ACSF treatment,
Table 1 Rat primer sequences used in quantitative polymerase chain reactions
Genes Forward Reverse
IL-1β 5′-TTGCTTCCAAGCCCTTGACT-3′ 5′-CTCCACGGGCAAGACATAGG-3′
TNF-α 5′-CATGAGCAC GGAAAGCATGA-3′ 5′-CCACGAGCAGGAATGAGAAGA-3′
IL-6 5′-AAGAAAGACAAAGCCAGAGTC-3′ 5′-CACAAACTGATATGCTTAGGC-3′
IL-4 5′-CCTTGCTGTCACCCTGTTCTG-3′ 5′-TGCATGGAGTCCCTTTTTCTG-3′
IL-10 5′-CAGTCAGCCAGACCCACAT-3′ 5′-GCTCCACTGCCTTGCTTT-3′
18S 5′-ATGCTCTTAGCTGAGTGTCCCG-3′ 5′-ATTCCTAGCTGCGGTATCCAGG-3′
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 5 of 15
intrathecal administration of CD200Fc bilaterally reduced
the extent of astroglial GFAP immunostaining in L4–L5
spinal cord sections from sCCI-operated rats (Fig. 3c, f, j).
The GFAP-immunostained area of activated astrocytes
was enlarged at 24 h after CD200Fc treatment, but it still
remained lower than in the spinal cord of sCCI-operated
and ACSF-treated rats. The quantitative changes of
GFAP-immunoreactive areas (Fig. 3l) indicating activation
state of astrocytes were confirmed by western blot analysis
of GFAP protein in the spinal cord of naïve, shamoperated,
and sCCI-operated and treated rats (Fig. 3m).
Cellular distribution and regulation of CD200 and
CD200R1 levels in the spinal cord of nerve-injured and
CD200Fc-treated rats
To determine precisely whether exogenous soluble
CD200Fc might modify membrane-bound CD200 and
CD200R1 expression in the spinal cord after sCCI
operation, we analyzed their proteins by immunofluorescence
staining and western blotting in naïve
and sham-operated rats in comparison with those of
sCCI-operated animals after ACSF and CD200Fc administration
for 5 h. The CD200 immunostaining was
dominantly observed in DH with a higher intensity in
the spinal cord sections from sCCI-operated and
ACSF-treated rats when compared with that from
sham-operated or sCCI-operated and CD200Fc-treated
rats (Fig. 4a–c). Similarly, a distinctly increased immunostaining
for CD200R1 was found in DH of spinal cord
sections of sCCI-operated and ACSF-treated rats when
compared with sections of the spinal cord from shamoperated
rats (Fig. 4d–f). The increased CD200 and
CD200R1 proteins in the spinal cord of sCCI-operated
and ACSF-treated as well as the protein decrease after
CD200Fc injection were confirmed by western blot
analysis (Fig. 4g, h).
Fig. 1 Results of behavioral tests. Mechanical (a) and thermal hypersensitivity (b) in sCCI-operated rats treated with ACSF or CD200Fc. Withdrawal
thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were significantly decreased in the ipsilateral hind paws
following sCCI when compared with 1 day before surgery, and CD200Fc treatment for 5 h was able to reverse these. Both behavioral signs of
PNP were nevertheless significantly weaker 24 h from CD200Fc application, although they remained still higher than 7 days after sCCI operation but before
CD200Fc application. All values in a and b represent mean ± SE from six rats per group. Asterisk indicates statistically significant difference (p < 0.001) when
compared with measurements 1 day before operation; Plus sign indicates statistical significant difference (p < 0.01) when compared to values
of sCCI-operated animals before and after CD200Fc application. Kruskal-Wallis ANOVA followed by Mann-Whitney U test.
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 6 of 15
Double immunostaining of spinal cord sections of sCCIoperated
and ACSF-treated rats revealed CD200 immunoreaction
in both NeuN+ neurons and GFAP+ astrocytes
(Fig. 5a–f). The CD200R immunofluorescence was detected
in neuropil of the spinal cord sections and double
immunostaining displayed colocalization of CD200R and
OX42 to illustrate presence of CD200R in activated
microglial cells (Fig. 5g–i). When GFAP immunostaining
corresponding with astrocyte activation was reduced after
CD200Fc treatment, we sought to observe whether activated
astrocytes could express CD200R1. Surprisingly,
CD200R immunolabeling was colocalized with GFAP
Fig. 2 Immunofluorescence staining and quantification of CD11b/c protein. OX42 (CD11b/c protein) immunofluorescence to detect microglia in
the dorsal horn (DH) of sections through L4–L5 spinal cord segments from naïve and sham-operated rats as well as sCCI-operated rats treated
with ACSF or CD200Fc. Representative pictures of OX42 immunofluorescence in ipsilateral DH of the spinal cord from sham- (a) and CCI-operated
(b, c) rats 5 h after ACSF (a, b) or CD200Fc (c) injection. Scale bars = 75 μm. A higher power magnification of OX42 immunofluorescence in DH of
naïve (d) and sham-operated (h) rats and the ipsi- and contralateral DH (DH-ipsi, DH-contra) of sCCI-operated and ACSF-treated rats (e, i) as well
as 5 (f, j) or 24 h (g, k) after injection with CD200Fc. Scale bars = 50 μm. Insets in d–f show typical shape of OX42-labeled microglia in DH of naïve,
ACSF-, and CD200Fc-treated rats, respectively. Scale bars for insets = 33 μm. l Bar graph shows quantification of OX42 immunostaining area in DH
of L4–L5 spinal cord segments from naïve and sham-operated rats as well as sCCI-operated rats for 7 days and 5 or 24 h after administration of
CD200Fc or ACSF. m Western blot shows increased CD11b/c protein level in the spinal cord of sCCI-operated in comparison to naïve or shamoperated
rats, whereas CD200Fc administration reduced this elevation. The data represent the mean ± SD optical density normalized to tubulin
from six animals in each group. *p < 0.05 when compared to DH of naïve or sham-operated rats; #p < 0.05 when compared to DH of sCCI + ACSF
rats; ++p < 0.05 when compared to 5-h treatment. Mann-Whitney U test
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 7 of 15
immunostaining, indicating expression of the receptor in
activated astrocytes of the spinal cord from sCCI-operated
rats (Fig. 6a–f). The unexpected colocalization was
verified by image analysis and measurement of indexes
using NIS Elements. Mander’s overlap measured in double
immunostained cells indicated by arrows (Fig. 6d–f) was
0.965265, 0.929336, and 0.960253. In addition, profile intensity
of red (CD200R) and green (GFAP) channels in the
cells also indicated colocalization of CD200R and GFAP
immunostaining (Fig. 6g).
Intrathecal CD200Fc administration reduces
proinflammatory and enhances anti- inflammatory cytokine
mRNAs in lumbar spinal cord segments of nerve-injured rats
To determine the immediate effect of CD200Fc treatment
on cytokine synthesis in the spinal cord 7 days
Fig. 3 Immunofluorescence staining and quantification of GFAP protein. GFAP-immunoreactive astrocytes in the dorsal horn (DH) of sections
through L4–L5 spinal cord segments from naïve rats and sham-operated rats as well as sCCI-operated rats treated with ACSF or CD200Fc.
Representative pictures of GFAP immunostaining in ipsilateral (ipsi) and contralateral (contra) DH of the spinal cord from sham- (a) and
CCI-operated (b, c) rats 5 h after ACSF (a, b) or CD200Fc (c) injection. Scale bars = 100 μm. A higher power magnification of GFAP immunostaining in
DH of naïve (d) and sham-operated (h) rats and the ipsi- and contralateral DH (DH-ipsi, DH-contra) of sCCI-operated and ACSF-treated rats (e, i) as well
as rats 5 (f, j) or 24 h (g, k) after injection with CD200Fc. Scale bars = 50 μm. l Bar graph shows quantification of GFAP-immunoreactive area in DH of
L4–L5 spinal cord segments from naïve and sham-operated rats as well as sCCI-operated rats for 7 days and 5 or 24 h after administration of CD200Fc
or ACSF. m Western blot confirmed the increase of GFAP protein level in the spinal cord of sCCI-operated in comparison to naïve or sham-operated
rats. The GFAP elevation was reduced by CD200Fc administration. The data represent the mean ± SE optical density normalized to tubulin from six
animals in each group. *p < 0.05 when compared to DH of naïve or sham-operated rats; #p < 0.05 when compared to DH of sCCI + ACSF rats.
Mann-Whitney U test
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 8 of 15
after sCCI operation, RT-PCR was carried out using
primers specific to pro- (TNF, IL-1β, IL-6) and antiinflammatory
(IL-4, IL-10) cytokines.
As expected, L4–L5 segments of the spinal cord from
sCCI-operated and ACSF-treated rats displayed robust
elevation of relative mRNAs of proinflammatory (TNF,
IL-1β, IL-6) and a decrease of anti-inflammatory cytokines
(IL-4, IL-10) when compared with spinal cord segments of
naïve or sham-operated rats. After 5 h, intrathecal administration
of CD200Fc produced normalization of TNF
mRNA expression (Fig. 7a) and significant reduction in
relative expression of IL-1β and IL-6 mRNAs (Fig. 7b–c)
in comparison with ACSF-treated rats. Conversely, relative
levels of IL-4 and IL-10 mRNAs were simultaneously
increased close to (in the case of IL-4) or exceeding (for
IL-10) those levels for spinal cord segments from naïve or
sham-operated rats (Fig. 7d–e).
Discussion
Many experimental models of PNP are principally based
on peripheral nerve injury providing a partial disconnection
of axons from their neuronal bodies [6, 41]. The
original model of CCI using four ligatures of chromic
gut (4–0) loosely tied around the sciatic nerve [42] is
not suitable for distinguishing neuroinflammatory reaction
induced by a thread material [43] and/or Wallerian
degeneration of injured axons [6]. Therefore, sCCI of
the sciatic nerve in our experiments was prepared using
Fig. 4 Effect of CD200Fc on CD200 and CD200R1 protein levels. Effect of CD200Fc on CD200 and CD200R1 protein levels of the L4–L5 spinal
cord segments from sCCI-operated rats. sCCI of the sciatic nerve and ACSF treatment increased intensity of CD200 and CD200R immunofluorescences
in DH of spinal cord sections when compared with those from sham-operated rats. Intrathecal CD200Fc administration significantly attenuated
elevation of CD200 and CD200R immunofluorescences in the spinal cord sections of sCCI-operated rats (a–f). Scale bars = 100 μm. The
CD200 and CD200R immunofluorescence changes were confirmed by western blot analysis of CD200 and CD200R proteins (g, h). Upper
panels of g and h illustrate representative western blotting bands for CD200 and CD200R in the L4–L5 spinal cord segments from sham-operated
(Sham), sCCI-operated and ACSF-treated (sCCI + ACSF5h) rats, as well as rats operated for sCCI and 5 (sCCI + CD200Fc5h) or 24 h (sCCI + CD200Fc24h)
after administration of CD200Fc. Lower panels show mean density ± SE of individual CD200 and CD200R1 protein bands in triplicate analysis of six animals
after normalization to tubulin. *p < 0.05 when compared to naïve rats; #p < 0.05 when compared to sCCI + ACSF rats; ++p < 0.05 when compared to 5 h
after administration of CD200Fc. Mann-Whitney U test
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 9 of 15
3–0 sterilized thread (Ethicon) under aseptic conditions
to study the effect of CD200Fc on spinal glial activation
and cytokine reactions in consequent Wallerian degeneration
induced by traumatic nerve compression.
It is well known that PNP induced by nerve injury is
related to activation of glial cells and upregulation of
proinflammatory cytokines in the spinal cord, and
their reduction has been associated with alleviation of
behavioral signs [44–47].
CD200/CD200R is a regulatory system of inflammation
that plays a relevant role in various diseases
when CD200 ligand binding with its receptor is impaired
[22, 25, 29]. In contrast, enhanced signaling of
CD200R by CD200Fc treatment alleviates pathological
effects of inflammation [32, 34–36]. However, the
anti-neuroinflammatory effects of CD200Fc in neuropathic
pain have not heretofore been investigated. The
present study provides the first evidence that CD200Fc
attenuates activation of glial cells and proinflammatory
cytokines, as well as mechanical and thermal hypersensitivity
in an experimental model of PNP immediately after
its intrathecal administration.
CD200Fc has been applied intraperitoneally [35], subcutaneously
[34], or intracerebroventricularly [32] in
various experimental models of inflammatory diseases.
As CD200Fc has a half life of just a few hours [48], we
applied the CD200R soluble ligand intrathecally to
achieve direct and rapid diffusion of CD200Fc into DH
of the rat spinal cord. We cannot exclude the possibility
that a small portion of the drug administered into the
cisterna magna will retrogradely reach the ventricular
system and surrounding structures playing a role in the
effects described here. However, decreased glial activation
and modulation of cytokine mRNAs and CD200/
CD200R changes revealed that CD200Fc diffused into
the lumbar level of spinal cord.
CD200Fc attenuates glial activation of the spinal cord and
neuropathic pain behavioral signs
Activation of microglial cells and astrocytes after nerve
injury is strongly associated with induction and maintenance
of mechanical and thermal hypersensitivity, and depression
of the glial activities alleviates the behavioral
signs of PNP [46, 49–51]. Microglial cells are accumulated
in the spinal cord at the ipsilateral side to nerve injury
and change their morphology from a “resting state”
in which the cell bodies are small with long and thin
processes into an “activated state” in which the cells
present an amoeboid shape with their bodies enlarged
and fine processes retracted [52, 53]. Microglial activation
after peripheral nerve injury is generally detected by
immunocytochemical staining using the OX42 antibody,
Fig. 5 Double immunostaining to detect CD200 protein in astrocytes and neurons as well as CD200R in microglia. Double immunostaining of
sections through DH of L4–L5 spinal cord segments from sCCI-operated and ACSF-treated rats. Immunofluorescence for CD200 (a) and GFAP (b)
are colocalized (c), evidencing that CD200 protein is expressed by astrocytes (arrows). In addition, double immunostaining for CD200 (d) and
NeuN (e) detected a presence of CD200 protein also in neurons (f, arrows). Immunolabeling for CD200R (g) and OX42 (h) was colocalized,
illustrating that activated microglial cells display CD200R protein (i, arrow). Merged figures (c, f, i) contain also blue channel of Hoechst
stained nuclei. Scale bars = 30 μm
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 10 of 15
directed against a complement receptor 3 antigen
(CD11b/c) or antibody against ionized calcium binding
adapter molecule 1 (Iba1) [54, 55]. However, there is
convincing evidence that enlarged OX42 immunostaining
area measured by image analysis is related with
microglial activation after nerve injury [53].
Ipsilateral DH of the spinal cord from sCCI + ACSF in
contrast to naïve or sham-operated rats displayed enlarged
areas of OX42 immunofluorescence and appearance of
OX42+ microglial cells with the shapes indicating their activated
state. The OX42 immunoreaction area was significantly
reduced in DH of sCCI-operated and CD200Fctreated
rats, thus showing very rapid effect on the marker
of activated microglia. The changes of microglial cell
activation induced by CD200Fc were confirmed by
western blot analysis of the CD11b/c protein. Our results
are in agreement with other experimental studies in
that CD200Fc rapidly reduced the markers of microglial
activation [32, 34, 56].
Astrocytes are more dominant than microglia in the
spinal cord, and they have been investigated predominantly
in relation to their supportive functions for neurons
[57]. Moreover, activated astrocytes are a major
source of cytokines and may contribute significantly to
induction and maintenance of PNP [45, 49]. Astrocytes
respond to various physiological or pathological stimuli
with the increased expression of GFAP that is generally
considered to be a marker for astrocyte activation [58].
It has been experimentally demonstrated that GFAP upregulation
in the spinal cord correlates well with nerve
injury-induced PNP [49] and that suppression of astrocyte
activation by fluorocitrate alleviates PNP symptoms
[59]. In our experiments, as demonstrated by GFAP immunofluorescence
areas and western blot analysis of
GFAP protein, astrocytes became activated in sCCIoperated
and ACSF-treated rats and were reduced bilaterally
in DH of the spinal cord after treatment with
CD200Fc. This suppression of activated astrocytes was
much less than microglial cells which is in accordance
with treatment by selective inhibitors of glial activation
[55]. Our results of western blot analysis were obtained
from whole L4–L5 segments containing possible changes
also in the ventral horn of the spinal cord. However,
OX42 and GFAP protein levels correlated with quantitative
alterations of corresponding immunofluorescence
were measured only in DH of the spinal cord.
Our present results evidence for the first time that
intrathecal application of CD200Fc for 5 h diminished
Fig. 6 Double immunostaining to detect CD200R protein in astrocytes. Double immunostaining of section through DH of L4–L5 spinal cord
segments from sCCI-operated and ACSF-treated rats to detect CD200R protein in GFAP positive astrocytes. Immunofluorescence staining for GFAP
(a, d) and CD200R (b, e) was colocalized as shown in merged figure (c, f). Scale bars = 100 μm. d–f illustrate a higher power magnification of area
limited by a box in c. Evidence of CD200R in activated astrocytes was confirmed by image analysis of three cells in the section using NIS Elements
software. Mander’s overlap was 0.965265, 0.929336, and 0.960253 for cells indicated by arrows. Scale bars = 30 μm. g illustrates representative
profile intensity of red (CD200R) and green (GFAP) channels in cell tagged 1
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 11 of 15
activation of both microglia and astrocytes in DH of
the spinal cord after sciatic nerve injury. This rapid
but reversible suppression of microglia and astrocyte
activations runs in parallel with alleviation of both
mechanical and thermal hypersensitivity. Because both activated
microglial cells and astrocytes displayed CD200R,
its activation by CD200Fc explains attenuation of the glial
activation and consequent alleviation of nerve injuryinduced
PNP like in experimental studies using other
modulators of glial activation [34, 46, 60]. Very rapid
suppression of activated glial cells by CD200Fc was
not surprising inasmuch as intrathecal injection of
other drugs has been shown to abolish glial activation
within hours [46, 61]. The CD200Fc effect was
already weakened 24 h from single injection, however,
thus indicating the necessity of repeated administration
for longer PNP attenuation, as is usual for
other molecules modulating neuroinflammation or
pain [62, 63].
Expression of CD200 and CD200R in the dorsal horn of
spinal cord
CD200 protein is constitutively expressed in neurons
and to a lesser extent in reactive astrocytes while
CD200R is expressed by microglia [21, 23]. The changes
of CD200 and CD200R1 protein levels in the spinal cord
after nerve injury and following administration of
CD200Fc have not previously been described. The
CD200 and CD200R immunostaining was predominantly
found in the spinal dorsal horn and both immunofluorescence
staining and western blot analysis revealed
significantly higher levels of CD200 and CD200R proteins
in the spinal cord after sCCI of the sciatic nerve. In
contrast to neurodegenerative diseases [19, 25, 64],
sciatic nerve injury probably activated endogenous regulatory
mechanisms including upregulation of CD200/
CD200R. Intrathecal CD200Fc treatment following sciatic
nerve injury suppressed CD200 and CD200R proteins
in the spinal cord to levels close to those of naïve
Fig. 7 RT-PCR analysis of cytokine RNAs in the spinal cord after CD200Fc administration. Effect of CD200Fc on TNF, IL-1β, IL-6, IL-4, and IL-10
mRNAs expression in L4–L5 segments of spinal cord from sCCI-operated rats for 7 days. sCCI of the sciatic nerve and ACSF treatment increased
levels of TNF (a), IL-1β (b), and IL-6 (c) as well as decreased IL-4 (d) and IL-10 (e) mRNA expression in the lumbar spinal cord segments. Intrathecal
CD200Fc administration prevented the increase of TNF, IL-1β, and IL-6 as well as the decrease of IL-4 and IL-10 mRNAs expression in the lumbar
spinal cord segments. The mRNA expression of each gene (n = 6 per group) was normalized to that of the 18S gene and the data represent the
mean ± SD. *p < 0.05, **p < 0.001 when compared to naïve rats; +p < 0.05, ++p < 0.001 when compared to sham-operated rats; #p < 0.05 when
compared to sCCI + ACSF rats. Mann-Whitney U test
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 12 of 15
or sham-operated rats. The rapid regulation of the
CD200 and CD200R proteins was parallel with activation
or deactivation of microglia cells and astrocytes detected
by CD11b/c or GFAP markers, respectively.
Using double immunostaining, we confirmed a presence
of CD200 protein in neurons and astrocytes as well as
CD200R in microglial cells as described earlier [21, 23].
However, there are no uncompromising results about expression
of CD200R in astrocytes. We detected colocalization
of GFAP and CD200R immunostaining in the spinal
DH of sCCI-operated and ACSF-treated rats, thus indicating
a presence of CD200R protein also in astrocytes activated
by nerve injury. Activation of both microglia and
astrocytes might therefore be directly modulated by
CD200Fc through stimulation of CD200R. Intense cross
talk between glial cells in the spinal DH is induced in the
initiation phase of injury-induced PNP [8, 55, 65]. CD200/
CD200R-mediated suppression of neuroinflammation may
occur not only via neuron-microglia interactions but also
via glia-glia interactions [23]. In addition to neurons,
increased expression of CD200 in reactive astrocytes may
contribute to control of microglial activation, while
CD200R upregulation probably allows a modulation of
astrocyte activation by contact with CD200+ neurons.
CD200Fc decreases proinflammatory and increases
anti-inflammatory cytokine mRNAs in the spinal cord of
nerve-injured rats
In the present time, there is no evidence about direct
operation of CD200/CD200R dysbalance in neuropathic
pain induction. It is generally accepted that CD200/
CD200R modulatory system is involved in suppression of
inflammation and glial activation. Although the recruitment
of neutrophils, macrophages, and lymphocytes from
blood might participate in the neuroimmune response of
the spinal cord to nerve injury, the activation of microglia
and astrocytes undoubtedly plays a pivotal role in production
of cytokines and chemokines [10, 12, 54].
A nerve injury induces elevation of proinflammatory
cytokine levels and their synthesis (mRNA) in the spinal
cord associated with development and maintenance of
PNP [66–68]. Experimental studies have demonstrated
that suppression of proinflammatory cytokines like TNF,
IL-1β, and IL-6 is necessary for efficacious attenuation
of mechanical and thermal hypersensitivity [67, 69, 70].
In contrast, increased synthesis or delivery of IL-10 has
been shown to attenuate proinflammatory cytokine
levels and alleviated PNP [71]. Similarly, other antiinflammatory
cytokines, such as IL-4, also have been decreased
in the CCI model of PNP and IL-4 deficiency
has been associated with mechanical hypersensitivity
[72]. Accumulating evidence indicates that nerve injury
induces upregulation of pro- and anti-inflammatory cytokines
in activated microglia and astrocytes in relation
with development and maintenance of PNP [7, 10, 44].
Our results from RT-PCR showed increase of proinflammatory
cytokine (TNF, IL-1β, IL-6) and decrease of antiinflammatory
cytokine (IL-4, IL-10) mRNAs in the spinal
cord of nerve-injured rats in comparison with naïve and
sham-operated animals. Intrathecal CD200Fc treatment
diminished elevation of proinflammatory cytokine (TNF,
IL-1β, IL-6) mRNAs and simultaneously ameliorated deficit
of IL-4 and IL-10 mRNAs induced by unilateral sCCI
of the sciatic nerve. This modulation of pro- and antiinflammatory
cytokine mRNAs by CD200Fc treatment
was simultaneous with glia deactivation and attenuation
of mechanical and thermal hypersensitivity.
CD200R1 stimulation suggests that CD200/CD200R1
interaction is involved in suppression of the proinflammatory
phenotype of microglial cells [32–34, 73] and astrocytes.
In addition, an in vitro study showed how microglia
incubation with CD200-bearing astrocytic membrane
preparations mitigates the LPS-induced increase in
mRNA expression of the proinflammatory cytokines TNF,
IL-1β, and IL-6 [74]. In line with our findings, CD200Fc
treatment significantly decreased the production of TNF
and IL-6 but not of IL-10 or TGF-β in microglial cells of
mice with experimental autoimmune encephalomyelitis
[34]. Referring to anti-inflammatory cytokines, it has been
pointed out that there is a possible involvement of
CD200Fc in increasing IL-4 and IL-10 in microglia cells
[33, 56]. Moreover, an increased expression of IL-10 in relation
to enhanced neuronal levels of CD200 has been also
found in the central nervous system of experimental autoimmune
encephalomyelitis mice [27].
The present results indicate that sciatic nerve injuryinduced
upregulation of proinflammatory cytokines in
parallel with elevation of the CD200/CD200R regulatory
system. An enhanced signaling of CD200R1 by CD200Fc
treatment attenuated activation of microglial cells and
astrocytes simultaneously with a decrease of proinflammatory
and increase of anti-inflammatory cytokine synthesis.
Due to reduction of the inflammatory reaction by
CD200Fc, expression of the CD200/CD200R regulatory
system was diminished.
In conclusion, our results suggest that support of the
CD200/CD200R regulatory system by administration of
the CD200R1 agonist CD200Fc induces very rapid suppression
of neuroinflammatory reactions associated with
glial activation and PNP development. This may constitute
a promising and novel therapeutic approach for the
treatment of PNP. Additional experiments with nerve
injury models are nevertheless needed, and particularly
with repeated CD200Fc treatment to prolong its effect.
Conclusions
The present results indicate that sCCI of the sciatic nerve
induced upregulation of proinflammatory cytokines in
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 13 of 15
parallel with elevation of the CD200/CD200R regulatory
system. An enhanced signaling of CD200R1 by administration
of the CD200R1 agonist CD200Fc induces very
rapid suppression of glial activation in the spinal cord
simultaneously with a decrease of proinflammatory and
increase of anti-inflammatory cytokine synthesis associated
with attenuation of PNP development. Due to reduction
of the inflammatory reaction by CD200Fc, expression
of the CD200/CD200R regulatory system was diminished.
Support of the CD200/CD200R regulatory system
by administration of the CD200R1 agonist CD200Fc
may constitute a promising and novel therapeutic approach
for the treatment of PNP. Additional experiments
with nerve injury models are nevertheless needed and
particularly with repeated CD200Fc treatment to prolong
its effect.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MH conceived, designed the experiments, carried out behavioral tests,
western blot, and RT-PCR analyses, and wrote the manuscript. IK, MJ, and IHS
conceived, designed, and carried out intrathecal administration and
participated in acquiring and analyzing the presented data. CG conceived and
participated in acquiring and analyzing the RT-PCR data. PD conceived,
designed and coordinated the study, and wrote the manuscript. All
authors gave final approval to the version to be published.
Acknowledgements
We thank Dana Kutějová, Jitka Mikulášková, Marta Lněníčková, Jana Vachová,
and Lumír Trenčanský for their skillful technical assistance. This work was
supported by the projects CEITEC from European Regional Development
Fund (CZ.1.05/1.1.00/02.0068) and Employment of Newly Graduated Doctors
of Science for Scientific Excellence (CZ.1.07/2.3.00/30.0009).
Author details
1
Central European Institute of Technology (CEITEC), Masaryk University,
Kamenice 3, 62500 Brno, Czech Republic. 2
Department of Anatomy, Division
of Neuroanatomy, Faculty of Medicine, Masaryk University, Kamenice 3,
62500 Brno, Czech Republic. 3
Department of Functional and Systems
Neurobiology, Neuroimmunology Group, Cajal Institute, Consejo Superior de
Investigaciones Científicas (CSIC), Madrid, Spain.
Received: 19 November 2015 Accepted: 10 February 2016
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peptide, FGL, attenuates lipopolysaccharide-induced changes in glia
in a cd200-dependent manner. Neurosci. 2013;235:141–8.
Hernangómez et al. Journal of Neuroinflammation (2016) 13:43 Page 15 of 15
F. Dubový P, Klusáková I, Hradilová-Svíženská I, Joukal M, Boadas-Vaello P. Activation
of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and
Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following
Different Types of Sciatic Nerve Injury. Front Cell Neurosci. 2018;12:40.
doi:10.3389/fncel.2018.00040
Commentary: In this article, we demonstrated that chronic constriction injury and sciatic
nerve transection induce activation glia and astrocytes in the periaqueductal gray and rostral
ventromedial medulla. This activation is triggered by CCL2/CCR2 signaling involved in the
neuron-glia and glia-glia interactions in both supraspinal regions suggesting its role in
neuropathic pain maintenance.
IF (WOS, 2017): 4.3
Quartile (WOS, 2017): Neurosciences Q1
Contribution of the author: Co-authorship; approximate participation 15%. The author
participated in designing, performing the experiments as well as acquiring and analyzing the
presented data.
ORIGINAL RESEARCH
published: 19 February 2018
doi: 10.3389/fncel.2018.00040
Activation of Astrocytes and
Microglial Cells and
CCL2/CCR2 Upregulation in the
Dorsolateral and Ventrolateral Nuclei
of Periaqueductal Gray and Rostral
Ventromedial Medulla Following
Different Types of Sciatic Nerve Injury
Petr Dubový1
, Ilona Klusáková1†
, Ivana Hradilová-Svíženská1†
, Marek Joukal1†
and Pere Boadas-Vaello1,2
*
1
Department of Anatomy, Division of Neuroanatomy, Faculty of Medicine, Masaryk University, Brno, Czechia, 2
Research
Group of Clinical Anatomy, Embryology and Neuroscience (NEOMA), Department of Medical Sciences, Universitat de Girona,
Girona, Spain
Edited by:
Flavia Trettel,
Sapienza Università di Roma, Italy
Reviewed by:
Ji Zhang,
McGill University, Canada
Guilherme Lucas,
University of São Paulo, Brazil
*Correspondence:
Pere Boadas-Vaello
pere.boadas@udg.edu
†
These authors have contributed
equally to this work.
Received: 21 October 2017
Accepted: 01 February 2018
Published: 19 February 2018
Citation:
Dubový P, Klusáková I,
Hradilová-Svíženská I, Joukal M and
Boadas-Vaello P (2018) Activation of
Astrocytes and Microglial Cells and
CCL2/CCR2 Upregulation in the
Dorsolateral and Ventrolateral Nuclei
of Periaqueductal Gray and Rostral
Ventromedial Medulla Following
Different Types of Sciatic Nerve Injury.
Front. Cell. Neurosci. 12:40.
doi: 10.3389/fncel.2018.00040
Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in
supraspinal structures possibly involved in neuropathic pain (NPP) maintenance.
Activated glial cells in specific supraspinal subregions may affect the facilitatory role of
descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic
nerve transection (CSNT) in rats were used as NPP models to study the activation
of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial
medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein,
GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry
and western blotting. The cellular distribution of CCL2/CCR2 was monitored using
immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity
from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks
induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and
ventrolateral PAG (vlPAG) compared to naïve or sham-operated rats. More extensive
astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG
and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was
also found in RVM after CSNT than sCCI. The CD11b immunopositive region,
indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of
both sides from sCCI- and CSNT-operated rats compared to naïve or sham-operated
controls. No significant differences in microglial activation were detected in dlPAG
or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no
significant differences in microglial activation in the RVM. Neurons and activated
GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial
cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT.
Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial
cell activation was similar in the supraspinal structures in both injury nerve models.
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 February 2018 | Volume 12 | Article 40
Dubový et al. Supraspinal Structures after Nerve Injury
Activated astrocytes in PAG and RVM may sustain facilitation of the descending system
maintaining NPP, while microglial activation may be associated with a reaction to
long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and
astrocytal release of CCL2 in PAG and RVM following injury.
Keywords: activated glial cells, CCL2/CCR2, neuroinflammation, periaqueductal gray, rostral ventromedial
medulla, nerve injury, neuropathic pain model
INTRODUCTION
Peripheral nerve injuries (PNIs) due either to trauma, disease or
surgical intervention, usually result in neuroplastic changes in the
central nervous system (CNS) and pain (Berger et al., 2011). Such
changes include the activation of endogenous glial cells of the
CNS, like microglia (Streit et al., 1999; Hanisch and Kettenmann,
2007; Graeber and Streit, 2010) and astrocytes (Ridet et al.,
1997; Pekny and Nilsson, 2005; Sofroniew and Vinters, 2010;
Anderson et al., 2014; Pekny and Pekna, 2014). The activated
glial cells facilitate pain neurotransmission and induce the central
sensitization of spinal neurons located in the dorsal horn. Briefly,
PNI causes the generation of ectopic discharges in the lesioned
afferent nerve fibers and their neurons (Omana-Zapata et al.,
1997; Woolf and Mannion, 1999; Liu et al., 2000; Schaible, 2007),
triggering an increased release of excitatory neurotransmitters
and neuromodulators onto spinal cord neurons and glial cells.
These changes induce central sensitization, glial cell activation,
and hyperexcitability of nociceptive spinal cord neurons that
consequently increase their discharges through the ascending
pain pathways (Zimmermann, 2001; Schaible, 2007; Latremoliere
and Woolf, 2009; Woolf, 2011; Burnstock, 2016).
The cellular and molecular changes described till now have
been extensively studied mainly in the dorsal horn of the spinal
cord and its circuitry (Vranken, 2009, 2012; Boadas-Vaello
et al., 2016), but much less is known about supraspinal changes
associated with the induction and maintenance of neuropathic
pain (NPP) and the underlying mechanisms. Neuroplastic
processes in the spinal cord facilitate the generation and
conduction of action potentials by the ascending pathways up
to supraspinal structures, contributing to both sensory and pain
behavior alterations. The periaqueductal gray (PAG) and rostral
ventromedial medulla (RVM) are particularly interesting given
their pivotal role in nociceptive modulation (Fields et al., 2006).
The PAG has a columnar functional organization with discrete
ventrolateral PAG (vlPAG) and dorsolateral PAG (dlPAG)
columns that have differences in the antinociceptive effects with
respect to their dependence on opiate mechanisms (Lane et al.,
2004; Lovick and Bandler, 2005; Eidson and Murphy, 2013;
Wilson-Poe et al., 2013) and the spinal projection of sciatic nerve
(Keay et al., 1997).
Pre-clinical studies of nerve injury models demonstrated
glial activation in PAG (Mor et al., 2010; Ni et al., 2016)
and RVM (Wei et al., 2008; Guo et al., 2012) associated with
changes in cytokines/chemokines (Wei et al., 2008; Norman
et al., 2010; Chu et al., 2012; Guo et al., 2012). Moreover, the
chemokine CCL2, also known as monocytes chemoattractant
protein 1 (MCP-1), has been demonstrated to play a critical
role in NPP facilitation via its preferred receptor, CCR2 (Gao
et al., 2009; Jung et al., 2009; Gao and Ji, 2010). It was
demonstrated that PNI increases the release of CCL2 in the
dorsal horn of the spinal cord (Van Steenwinckel et al., 2011;
Clark et al., 2013) triggering the activation of microglial cells
(Zhang and De Koninck, 2006; Thacker et al., 2009). The role
of CCL2 signaling in supraspinal structures involved in pain
modulation has still not been completely elucidated. Reactive
microglia cells may synthesize and secrete more inflammatory
mediators, which elicit increasing excitability of superficial
dorsal horn neurons, and enhance secretion of CCL2 from
spinal astrocytes (Clark et al., 2013). Similar processes may
occur also in both PAG subregions as well as in the RVM
which are involved in the alteration of descending pain
modulation.
Overall, despite the available data regarding neuroplastic
changes in supraspinal structures associated with nervous system
injury-induced pathological pain (Boadas-Vaello et al., 2017), the
cellular and molecular processes occurring in these structures as
a consequence of PNI are not yet fully understood. Particularly,
since distinct subregions of PAG may play different or specific
roles in the descending modulation of pain (McMullan and
Lumb, 2006; Eidson and Murphy, 2013), it is of interest
to investigate whether dlPAG and vlPAG show different
pathophysiological responses after PNI, and whether these
responses appear in parallel with RVM glial activation, and to
do so in distinct PNI models. To this end, the present work was
designed to study glial activation in both PAG nuclei and RVM
after two models of sciatic nerve injury, namely, sterile chronic
constriction injury (sCCI) and complete sciatic nerve transection
(CSNT). Moreover, considering the pivotal role of chemokines in
CNS sensitization and NPP maintenance, the aim of experiments
was also to explore associated chemokine signaling through
monitoring the expression of CCL2/CCR2 which could be
involved in supraspinal glial cross-talk after sciatic nerve
injury.
MATERIALS AND METHODS
Animals and Surgical Procedures
The experiments were performed in 28 adult male rats
(Wistar, 200–250 g, Anlab, a.s. Brno, Czech Republic). Animals
were kept at 22◦C and maintained on a 12 h light/dark
cycle under specific pathogen-free conditions in the animal
housing facility of the Masaryk University. Sterilized food and
water were available ad libitum. All surgical treatments were
carried out under sterile conditions by the same person in
accordance with the European Convention for the Protection of
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Dubový et al. Supraspinal Structures after Nerve Injury
Vertebrate Animals Used for Experimental and Other Scientific
Purposes and the protocol was approved by the Animal
Investigation Committee of the Faculty of Medicine, Brno, Czech
Republic.
Animals were anesthetized by intraperitoneal (i.p.) injection
of ketamine (60 mg/kg) and xylazine (7.5 mg/kg) and the right
sciatic nerve was exposed at the level of the mid-thigh by blunt
dissection and separated from adhering tissue just proximal to
its trifurcation. Three ligatures (3-0 Ethicon) were tied around
the nerve with 1 mm spacing to reduce nerve diameter by
approximately one third of its original diameter in the sCCI
group (n = 7).
The right sciatic nerve was transected and the proximal stump
was tightly ligated and turned back to prevent spontaneous
reinnervation in the CSNT group (n = 7). The skin of the
right paws of CSNT group animals was covered with picric acid
solution to prevent autotomy. The right sciatic nerve was only
exposed without any lesion in rats (n = 7) subjected to a sham
operation. Retracted muscles and skin incision were closed with
3-0 silk sutures and the operated rats were left to survive for
3 weeks. Seven intact rats were used as naïve controls.
Behavioral Tests
Behavioral responses to noxious mechanical (paw withdrawal
threshold, PWT) and thermal (paw withdrawal latency, PWL)
stimuli were measured in both ipsi- and contralateral hind
paws by Dynamic plantar esthesiometer and Plantar test (UGO
BASILE), respectively. The location of measurement was in the
glabrous skin of the hind paws in the portion of the dermatome
innervated by the tibial nerve. Rats were first acclimated in
clear Plexiglas boxes for 30 min prior to testing. In the case
of thermal hyperalgesia, withdrawal time was measured and
the intensity radiance was set to a value of 50. The paws were
tested alternately with a 5 min interval between tests. Six PWT
and PWL measurements were taken for each paw during each
test session 1 day before and 1, 3, 7, 14 and 21 days after
operation.
Data for mechanical allodynia and thermal hyperalgesia were
expressed as mean ± SD of PWT in grams and PWL in seconds,
respectively. All behavioral tests were conducted in a blind
manner.
After last behavioral tests at day 21 from operation, animals
were sacrificed with a lethal dose of anesthetic and tissue
samples were removed for immunohistochemical and western
blot analysis.
Immunohistochemical Staining
Three naïve rats and three rats from the sCCI, CSNT and
sham operation groups were deeply anesthetized with a lethal
dose of sodium pentobarbital (70 mg/kg body weight, i.p.) and
perfused transcardially with 500 ml phosphate-buffered saline
(PBS, 10 mM sodium phosphate buffer, pH 7.4, containing
0.15 M NaCl) followed by 500 ml of Zamboni’s fixative (Zamboni
and Demartin, 1967).
Brainstem tissue between the superior and inferior colliculi
was removed and immersed in Zamboni’s fixative at 4◦C
overnight. The samples were washed in 20% phosphate-buffered
sucrose for 12 h and blocked in Tissue-Tek OCT compound
(Miles, Elkhart, IN, USA). Serial PAG coronal sections (12 µm)
from the central part of the superior colliculi to the upper
edge of the inferior colliculi corresponding to PAG between
6.72–8.04 mm from the bregma (Paxinos and Watson, 1997)
were cut (Leica 1800 cryostat; Leica Microsystems, Wetzlar,
Germany). In the same brainstem tissue, coronal sections
(12 µm) through the medulla 1 mm from posterior edge
of the inferior colliculi corresponding to RVM between
−10.8 mm and −11.4 mm from the bregma (Paxinos and
Watson, 1997) were also prepared. The sections were collected
on gelatin-coated microscopic slides, air-dried and processed for
immunohistochemical staining.
Increased expression and extent of GFAP and
OX42 immunostaining are widely accepted markers of activated
astrocytes (Pekny and Pekna, 2004) and microglial cells
(Blackbeard et al., 2007), respectively. Therefore, indirect
immunofluorescence staining for GFAP and OX42 was used
to detect activation of astrocytes and microglial cells in PAG
and RVM after sciatic nerve injury. Briefly, the sections
were washed with PBS containing 0.05% Tween 20 (PBSTW20)
and 1% bovine serum albumin for 10 min, and
then treated with 3% normal donkey serum in PBS-TW20
for 30 min. The sections were incubated with 50 µl of
mouse monoclonal anti-CD11b/c antibody (OX42), rabbit
polyclonal anti-glial fibrillary acidic protein (GFAP) or
rabbit polyclonal anti-CCL2 antibody in a humid chamber
at room temperature (21–23◦C) for 12 h to identify activated
microglial cells, astrocytes or CCL2 expression, respectively.
The immunoreaction was visualized by treatment with FITCor
TRITC-conjugated, affinity purified, donkey anti-mouse or
anti-rabbit secondary antibodies for 90 min at room temperature.
Cell nuclei were stained using Hoechst 33342 (Sigma, St. Louis,
MO, USA) and the sections were mounted in Vectashield
aqueous mounting medium (Vector Laboratories, Burlingame,
CA, USA).
A set of PAG and RVM sections was double immunostained
to detect cell types producing CCL2 or expressing CCR2. The
sections were incubated with rabbit or chicken anti-GFAP
antibody to detect activated astrocytes, with mouse monoclonal
OX42 for co-localization in activated microglial cells and
polyclonal or monoclonal NeuN antibody to identify neurons.
The first incubations were combined with immunostaining
with rabbit polyclonal anti-CCL2 antibody or goat polyclonal
anti-CCR2 antibody. The binding of primary antibodies
were visualized by appropriate FITC-, FITC- or AlexaFluor
647-conjugated secondary antibodies (Table 1).
Control sections were incubated by omitting the primary
antibodies (data not shown). Cell nuclei were stained using
Hoechst 33342 and sections were analyzed using a Nikon Eclipse
NI-E epifluorescence microscope equipped with a Nikon DS-Ri1
camera driven by NIS-Elements software (Nikon, Prague, Czech
Republic).
Image Analysis
At least 12 sections (separated from one another by an interval
of about 80 µm) of PAG or RVM for each animal were selected
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Dubový et al. Supraspinal Structures after Nerve Injury
TABLE 1 | List of primary and secondary antibodies used for immunofluorescence detection.
Antibody Source Product Dilution
GFAP pAb Rabbit Dako 1:250
GFAP pAb Chicken Abcam 1:500
OX42 mAb Mouse Serotec 1:50
NeuN pAb Rabbit Millipore 1:500
NeuN mAb Mouse Chemicon 1:500
CCL2 pAb Rabbit Serotec 1:250
CCR2 pAb Goat ThermoFisher 1:100
Anti-chicken pAb Goat Abcam 1:200
Anti-rabbit pAb Donkey Millipore 1:400
Anti-mouse pAb Donkey Millipore 1:400
Anti-mouse pAb Goat Millipore 1:400
pAb, polyclonal antibody; mAb, monoclonal antibody.
for image analysis. Immunopositive area for OX42 or GFAP
was measured using the NIS-elements image analysis system
(Laboratory Imaging Ltd., Prague, Czech Republic). Briefly, the
area of interest (40,000 µm2 for PAG; 70,000 µm2 for RVM)
was placed over the dlPAG and vlPAG) or over RVM and
the GFAP- or OX-42-immunostained structures were detected
by a thresholding technique after subtraction of background.
The boundaries of dlPAG and vlPAG columns were defined
according to the rat PAG map (Paxinos and Watson, 1997) and
other anatomical criteria (Keay and Bandler, 2001). The area of
immunostaining for OX42 or GFAP was related to the area of
interest and expressed as the mean of relative area (%) ± SD.
Western Blot Analysis
Naïve, sham-, sCCI- and CSNT-operated rats were used for
western blot analysis (four rats for each group). Rats were
killed with CO2, decapitated and the brainstem was removed.
A 2 mm thick coronal slice of the mesencephalon was rapidly
removed at the position from the central part of the superior
colliculi to the upper edge of the inferior colliculi corresponding
to PAG between 6.72 mm and 8.04 mm from the Bregma
(Paxinos and Watson, 1997). Radial segments corresponding
approximately with ipsilateral and contralateral dlPAG and
vlPAG were dissected under a stereomicroscope (see Figure 2E)
according to the boundaries defined using anatomical criteria
(Keay and Bandler, 2001).
A 2 mm coronal slice was cut though the medulla, 1 mm
from posterior edge of the inferior colliculi corresponding
approximately to 1 mm from the interaural line. This section
corresponds to the RVM between −10.8 mm and −11.4 mm
from the bregma (Paxinos and Watson, 1997). A tissue triangle
was then dissected under a stereomicroscope to isolate the RVM
area, including the nucleus raphe magnus, gigantocellularis,
and gigantocelularis pars alpha (see Figure 3E). The RVM
samples were not divided into ipsi- and contra-lateral sides
because no differences were seen during image analysis
of immunostained sections. Tissue samples were collected,
frozen in liquid nitrogen, and stored at −80◦C until further
processing.
The PAG and RVM tissue samples of individual animals were
homogenized in PBS containing 0.1% Triton X-100 and protease
inhibitors (LaRoche, Switzerland), and centrifuged at 15,000 g
for 20 min at 4◦C. Protein concentration was measured in the
tissue supernatant by Nanodrop ND-1000 (Thermo Scientific)
and normalized to the same levels. Each sample, containing
50 µg of protein, was separated by SDS-polyacrylamide gel
electrophoresis (Wei et al., 2008) and transferred to nitrocellulose
membranes by electroblotting (BioRad).
The membranes were blocked with 1% BSA in PBST (3.2 mM
Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl,
0.05% Tween 20, pH 7.4) for 1 h and incubated with mouse
monoclonal anti-CD11b/c antibody (OX42; 1:50, AbD Serotec,
Kidlington, UK) or rabbit polyclonal anti- GFAP (1:250, Dako,
Glostrup, Denmark) overnight. Blots was washed in PBST and
incubated with peroxidase-conjugated anti-mouse or anti-rabbit
secondary antibodies (Sigma, 1:1000) at room temperature
for 1 h. Equal loading of proteins was confirmed by b-actin
staining. Protein bands were visualized using the ECL detection
kit (Amersham) on an LAS-3000 chemiluminometer reader
(Bouchet Biotech) and analyzed using densitometry image
software. No differences were measured between samples of
naïve and sham-operated rats, therefore, OX42 and GFAP
protein data after normalization to actin were expressed as
fold change relative to sham PAG or RVM, which were
set to 1.
Statistical Analyses
Behavioral data among groups were evaluated using two-way
analysis of variance (ANOVA) with repeated measurements.
One-way ANOVA with Student–Newman–Keuls post hoc test
was used for comparison at each time point and p values
less than 0.05 were considered to be significant. Statistical
differences between data for staining area and western blot
analysis were tested using a Mann-Whitney U-test (p < 0.05).
All statistical analyses were performed using STATISTICA
9.0 software (StatSoft, Inc., Tulsa, OK, USA).
RESULTS
Behavioral Tests
All rats operated on to create sCCI of the sciatic nerve displayed
as signs of NPP, decreased thresholds of mechanical allodynia
(PWT) and withdrawal latencies of thermal hyperalgesia (PWL)
restricted to the hind paws ipsilateral to the nerve ligatures.
Decreased PWT and PWL was induced at day 1 and maintained
throughout the period of survival up to 3 weeks when compared
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 1 | Results of behavioral tests in rats operated upon to create unilateral sterile chronic compression injury (sCCI) of the sciatic nerve and sham-operated
rats. Development and maintenance of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) was measured in the ipsilateral hind paws of
sCCI-operated rats indicating mechanical allodynia and thermal hyperalgesia, respectively. Transient thermal hyperalgesia was measured bilaterally 3 days following
sCCI. Data are expressed as mean ± SD of PWT in grams and PWL in seconds for mechanical allodynia and thermal hyperalgesia, respectively. Symbols below the
curves indicate a statistically significant difference (p < 0.05) compared to sham-operated rats.
with sham- operated rats. Hind paws contralateral to sCCI
did not exhibit statistically significant changes of PWT and
PWL when compared with 1 day before operation or with
hind paws of sham-operated rats (Figure 1). In hind paws
of sham-operated animals, no significant changes in PWT
and PWL data were measured compared to 1 day before
operation. No autotomy was found in animals of the CSNT
group.
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 2 | Representative sections through dorsolateral periaqueductal gray (dlPAG) and ventrolateral PAG (vlPAG) of naïve (A), sham- (B), sCCI- (C) and complete
sciatic nerve transection (CSNT)-operated (D) rats immunostained for glial fibrillary acidic protein (GFAP). The figures show a bilateral increase in the area containing
activated GFAP+ astrocytes in both dlPAG and vlPAG after sCCI or CSNT for 3 weeks. Scale bars = 80 µm. (E) Schematic representation of the location of dlPAG
and vlPAG and their sampling for western blot analysis. (F) The top panel shows representative western blot of GFAP protein in PAG of naïve and sham-operated
rats and 3 weeks after sCCI or CSNT. (i) indicates ipsilateral and (c) contralateral segments of dorsolateral (dl) and ventrolateral (vl) PAG. Graph below the blot
illustrates density data obtained from three blots after normalization to actin, expressed as fold-change relative to those of sham-operated rats (set as 1). ∗
Indicates a
statistically significant difference (p < 0.05) compared to the value from sham-operated rats; # indicates a statistically significant difference (p < 0.05) between the
values from ipsilateral and contralateral vlPAG.
Activation of Astrocytes in PAG and RVM
after Chronic Compression Injury
Compared to Complete Transection
Increased immunostaining for GFAP is frequently used to
detect activation of astrocytes following various types of
nervous system injury (Pekny and Pekna, 2004). We found
a significantly larger GFAP+ area corresponding to astrocytes
in vlPAG than dlPAG in both naïve and operated rats.
Sciatic nerve injury by sCCI or CSNT for 3 weeks induced
increased GFAP intensity and a significant enlargement of
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Dubový et al. Supraspinal Structures after Nerve Injury
GFAP+ area bilaterally in both dlPAG and vlPAG when
compared to naïve or sham-operated controls. In addition,
CSNT gives rise to a larger GFAP+ area in bilateral dlPAG and
only ipsilateral vlPAG when compared with sCCI. However,
a significantly larger GFAP+ area was seen in vlPAG of
the ipsilateral than the contralateral side (Figures 2A–D,
Table 2).
Sections through RVM prepared from the same rats surviving
for 3 weeks with sCCI or CSNT also displayed an increase
in GFAP intensity and robust enlargement of the GFAP
immunopositive area in comparison to RVM sections of naïve
or sham-operated rats (Figures 3A–D). Similarly to PAG, CSNT
gives rise to a larger GFAP+ area than in the same structures of
animals subjected to sCCI (Table 2).
The increased activation of astrocytes in PAG and RVM
after sCCI and CSNT detected by image analysis of GFAP
immunofluorescence areas was confirmed by western blot
analysis of the GFAP protein (Figures 2F, 3F).
Microglia Activation in PAG and RVM after
Chronic Compression in Comparison to
Transection
Activated microglial cells were identified by increased
immunofluorescence staining for CD11b, detected using
the antibody OX42 and by changes in their shape. Weak
OX42 immunofluorescence and microglial cells with limited
ramification occupied a similar proportion of immunostained
areas in both vlPAG and dlPAG of naïve and sham-operated
animals. The OX42 immunoreactive area indicating activated
microglial cells was remarkably larger in vlPAG and dlPAG of
both sides in PAG sections from sCCI- and CSNT-operated
rats when compared to naïve or sham-operated ones. In
contrast to GFAP+ astrocytes, no significant expansion of the
OX42 immunoreactive area was detected in either dlPAG or
vlPAG after CSNT when compared to sCCI of the sciatic nerve
(Figures 4A–H, Table 3). Besides increased immunostaining
intensity and enlargement of the OX42+ area, microglial
activation was recognized by their changed morphology.
In contrast to only a few processes in sections from naïve
and sham-operated rats, activated microglial cells in PAG
displayed more vigorous ramification along with upregulation
of the constitutively expressed marker OX42 (Figures 4A–H,
insets).
Increased OX42 immunostaining intensity and enlargement
of the immunopositive area compared to RVM sections of naïve
or sham-operated rats was also observed in sections through
RVM prepared from the same brainstems as for OX42 analysis
in PAG. No significant increase in OX42+ areas was found in
RVM after CSNT than sCCI of the sciatic nerve (Figures 5A–D,
Table 3). Activated microglial cells in RVM after both types of
sciatic nerve injury were not only highly ramified but some were
also transformed morphologically to ‘‘bushy’’ type microglia
(Figures 5A–D, insets).
Increased activation of microglial cells in PAG and
RVM after sCCI and CSNT detected by image analysis of
OX42 immunofluorescence areas was confirmed by western blot
analysis of the OX42 protein (Figures 4I, 5E).
Neurons and Activated GFAP+ Astrocytes
Display Immunostaining for CCL2 While
Activated OX42+ Microglial Cells Are
Immunopositive for CCR2 in Both PAG and
RVM after Sciatic Nerve Injury
Sections through PAG from naïve and sham-operated rats
show only weak CCL2 immunofluorescence in the cells present
in the dlPAG and vlPAG columns. However, some cells in
the narrow region at the Sylvian canal (aqueduct) displayed
a higher intensity of CCL2 immunofluorescence than in the
dlPAG and vlPAG area (Figures 6A–D). Sciatic nerve injury
by sCCI and CSNT for 3 weeks induced a bilateral increase
of CCL2 immunofluorescence intensity in cells of vlPAG and
ipsilateral dlPAG, while the cells of contralateral dlPAG showed
an intensity like that of naïve or sham-operated rats. Intense
immunofluorescence intensity of CCL2 was found in the cells
of vlPAG than dlPAG 3 weeks after sciatic nerve injury
(Figures 6E–H). RVM sections of naïve and sham-operated
rats displayed very faint CCL2 immunoreaction, but the
intensity increased in the cells 3 weeks after sCCI or CSNT
(Figures 7A–D).
To reveal the cellular origin of CCL2 protein, one set of
sections were double immunostained for CCL2 and GFAP as
marker of activated astrocytes or NeuN, a molecular marker
of neurons. Double immunostaining showed the location of
CCL2 protein in all GFAP+ astrocytes as well as NeuN+
neurons of PAG and RVM (Figures 7E, 8A–F), while OX42+
microglial cells were free of any CCL2 immunofluorescence
(Figures 7F, 8G–I). The same cellular distribution of increased
immunostaining for CCL2 was observed in PAG and RVM
from animals subjected to both sCCI and CSNT for 3 weeks.
TABLE 2 | Percentage of glial fibrillary acidic protein (GFAP+) area ±SD in rostral ventromedial medulla (RVM) and dorsolateral (dl) and ventrolateral (vl) periaqueductal
gray (PAG) of naïve rats as well as in dlPAG and vlPAG of ipsilateral (ipsi) and contralateral (contra) sides from sham-operated rats and rats with sterile chronic
compression injury (sCCI) and complete sciatic nerve transection (CSNT) for 3 weeks (n = 6 for each group).
Naive Sham sCCI CSNT
ipsi contra ipsi contra ipsi contra
dlPAG 4.3 ± 1.8 5.1 ± 2.6 5.3 ± 2.8 8.6 ± 2.5∗
7.9 ± 2.3∗
12.5 ± 1.8∗‡
11.3 ± 2.4∗‡
vlPAG 10.3 ± 2.2+
10.8 ± 2.9+
10.9 ± 2.4+
17.1 ± 2.2+∗†
13.8 ± 2.8+∗
23.6 ± 3.5+∗†‡
15.2 ± 2.8+∗
RVM 3.8 ± 1.0 4.1 ± 1.9 17.5 ± 1.3∗
20.8 ± 2.1∗‡
+
Significant difference (p < 0.05) compared to dlPAG. ∗
Significant difference (p < 0.05) compared to Naïve or Sham. †
Significant difference (p < 0.05) compared to
contralateral side. ‡
Significant difference (p < 0.05) compared to sCCI.
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 3 | Representative medullary sections showing rostral ventromedial medulla (RVM) from naïve and sham-operated controls (A,B) and 3 weeks after sCCI or
CSNT (C,D). The pictures illustrate increased intensity and expansion of GFAP immunostaining in RVM after sCCI and CSNT in comparison to RVM from naïve or
sham-operated rat. Scale bars = 90 µm. (E) The drawing illustrates dissection of RVM samples for western blots including the nucleus raphe magnus and
gigantocellularis pars alpha (triangle). (F) The top panel illustrates representative western blot of GFAP protein in RVM of naïve and sham-operated rats and 3 weeks
after sCCI or CSNT. ∗
Indicates a statistically significant difference (p < 0.05) compared to the value from sham-operated rats.
Immunostaining for CCR2, a receptor of CCL2, was observed
only in OX42+ microglial cells of both PAG and RVM
(Figures 8J–L), but no CCR2 immunoreaction was found in
GFAP+ astrocytes.
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 4 | Representative sections illustrating OX42 immunofluorescence
staining in dlPAG (A–D) and vlPAG (E–H) of naïve, sham-, sCCI- and
CSNT-operated rats. Insets show the typical morphological types of OX42+
microglial cells from the respective animal groups. Scale bars = 90 µm.
(I) Western blot analysis of OX42 (CD11b/c) protein. The top panel illustrates
representative western blot in PAG of naïve and sham-operated rats and rats
3 weeks after sCCI or CSNT. (i) indicates ipsilateral and (c) contralateral
segments of dorsolateral (dl) and ventrolateral (vl) PAG. Graph below the blot
illustrates density data obtained from three blots after normalization to actin,
expressed as fold-change relative to those of sham-operated rats (set as 1).
∗
Indicates a statistically significant difference (p < 0.05) compared to the value
from sham-operated rats.
DISCUSSION
The supraspinal modulatory system is differentially involved
in descending facilitatory and inhibitory pathways to
influence transmission of nociceptive inputs from the
dorsal horn of the spinal cord to the upper structures of
the CNS (Heinricher et al., 1989; Porreca et al., 2002). A
fine equilibrium between the opposing descending controls
exists under normal conditions. Sensitization may durably
alter this balance in favor of facilitations that consequently
give rise to a NPP state. The RVM and PAG of the brainstem
are the principal (and the best studied) structures of the
endogenous modulatory system that alter spinal dorsal
horn processing of sensory input (Heinricher et al., 2009).
Despite a growing body of evidence indicating a role for
PAG and RVM in descending pain modulation, the precise
underlying cellular and molecular mechanisms involved in
descending facilitation and inhibition of the dorsal horn remain
elusive.
Several animal models based on sciatic nerve injury are
used to investigate NPP mechanisms and test novel analgesics.
The chronic constriction injury of the sciatic nerve is most
frequent model e included transection. The original CCI model
created by four ligatures of chromic gut (4–0) loosely tied
around the sciatic nerve results in inflammatory swelling
with subsequent compression of the nerve (Bennett and Xie,
1988). However, Wallerian degeneration distal to traumatic
nerve injury is considered to be neuroinflammation, and it is
impossible to distinguish between nerve inflammation induced
by chromic gut (Maves et al., 1993; Clatworthy et al., 1995) and
proper inflammatory reactions as a consequence of Wallerian
degeneration (Klusáková and Dubový, 2009). Therefore, we used
sCCI of the sciatic nerve to study NPP and glial activation
induced by traumatic nerve injury when damaged axons are
mixed with spared ones influenced with inflammatory mediators
produced only by Wallerian degeneration (Dubový, 2011). The
CCI model is associated with symptoms of chronic nerve
compression (Bennett and Xie, 1988), while the CSNT model is
rather an experimental model to study neuropathic symptoms,
such as ‘‘anesthesia dolorosa’’, pain with reference to the
area in the absence of any sensory signals (Devor, 1994). In
the present study, we used the CSNT model primarily to
compare glial reactions after total and partial (sCCI) nerve
disconnection.
It is well-known that sensitization of the CNS at different
levels by activation of glial cells can contribute to the
development and/or maintenance of chronic pain states after
a PNI (Suzuki et al., 2004). Most experimental results relating
to the role of glial activation in induction and maintenance
of NPP were observations made in the dorsal horn of the
spinal cord (Blackbeard et al., 2007; Milligan and Watkins,
2009). Less attention has been paid to the activation of
glial cells in supraspinal structures. For example, activated
astrocytes were found in RVM after unilateral CCI of the rat
infraorbital nerve (Wei et al., 2008) and in PAG following
CCI of the sciatic nerve (Mor et al., 2010, 2011). In contrast
to these separate studies in PAG and RVM of different
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Dubový et al. Supraspinal Structures after Nerve Injury
TABLE 3 | Percentage of OX-42+ area ±SD in RVM and dorsolateral (dl) and ventrolateral (vl) PAG of naïve rats as well as in dlPAG and vlPAG of ipsilateral (ipsi) and
contralateral (contra) sides from sham-operated rats and rats with sCCI and CSNT for 3 weeks.
Naive Sham sCCI CSNT
ipsi contra ipsi contra ipsi contra
dlPAG 6.9 ± 0.9 7.2 ± 1.5 7.0 ± 1.2 13.1 ± 1.8∗
12.9 ± 2.6∗
13.9 ± 2.5∗
13.2 ± 3.7∗
vlPAG 7.9 ± 2.7 8.3 ± 2.6 8.1 ± 2.9 13.4 ± 2.4∗
12.3 ± 3.7∗
13.2 ± 2.4∗
12.6 ± 2.2∗
RVM 0.7 ± 0.2 0.8 ± 0.2 3.6 ± 1.2∗
3.4 ± 0.7∗
∗
Significant difference (p < 0.05) compared to Naïve or Sham (n = 6 for each group).
FIGURE 5 | Representative medullary sections showing RVM from naïve and sham-operated controls (A,B) and rats with sCCI or CSNT (C,D). The pictures illustrate
increased intensity and enlargement of OX42 immunostaining after sCCI and CSNT when compared to RVM from naïve or sham-operated rats. Insets show the
typical morphological types of OX42+ microglial cells from the respective animal groups. Scale bars = 40 µm. (E) Western blot analysis of OX42 (CD11b/c) protein.
The top panel illustrates representative western blot in RVM of naïve and sham-operated rats and rats 3 weeks after sCCI or CSNT. Graph below the blot illustrates
density data obtained from three blots after normalization to actin, expressed as fold-change relative to those of sham-operated rats (set as 1). ∗
Indicates a
statistically significant difference (p < 0.05) compared to the value from sham-operated rats.
experimental models with varying times of survival, we
used the sCCI and CSNT models for 3 weeks to compare
long-lasting activation of astrocytes and microglial cells in
both RVM and PAG depending on the extent of nerve
injury.
Activation of Glial Cells in Ventrolateral
and Dorsolateral Subregions of PAG
The vlPAG with the spinal projection of sciatic nerve (Keay et al.,
1997) has a significant role in descending control of noxious
afferentation via connections with RVM (Eidson and Murphy,
2013). In addition to descending control, vlPAG columns have
projections to many CNS structures critical for the normal
expression of sleep-wake cycles and social behaviors (Monassi
et al., 2003; Mor et al., 2011). An increased number of GFAP+
astrocytes in vlPAG was mainly detected in a subset of rats
which displayed both NPP and disability following CCI of the
sciatic nerve (Mor et al., 2011). In contrast, dlPAG column
has projections primarily to the dorsolateral pons and the
ventrolateral medulla that are implicated in autonomic control
(Cameron et al., 1995). Moreover, descending control from
dlPAG has different effects on nociceptive reflexes evoked by
activation of C- and A-delta fibers (McMullan and Lumb,
2006).
Sciatic nerve injury by sCCI or CSNT for 3 weeks induced
activation of astrocytes bilaterally in both dlPAG and vlPAG
when compared to naïve or sham-operated control rats.
Significantly more extensive activation of astrocytes was
observed in vlPAG on the ipsilateral than the contralateral
side in both models of unilateral sciatic nerve injury that
can be related with the spinal projection of sciatic nerve
(Keay et al., 1997). In contrast, ipsilateral dlPAG displayed
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 6 | Representative sections showing immunofluorescence staining for CCL2 in dlPAG and vlPAG of naïve (A,C), sham- (B,D) and CSNT-operated (E–H)
rats. Weak CCL2 immunofluorescence was observed in the cells of both dlPAG and vlPAG from naïve and sham-operated controls (arrows in A–D). Only some cells
located close to the ependymal layer displayed intense CCL2 immunostaining (broad arrows in A–D). Sciatic nerve injury for 3 weeks induced increased
CCL2 immunofluorescence intensity in the cells (arrows) mainly in vlPAG of both ipsilateral (vlPAGi) and contralateral (vlPAGc) sides (G,H). Sections through dlPAG of
the same animal (E,F) displayed distinct CCL2 immunofluorescence in a larger number of cells close to the ependymal layer (broad arrows) compared to naïve and
sham-operated animals. Scale bars = 80 µm.
a more extensive activation of astrocytes when compared
to the contralateral column but this change was not
significant. Our results also revealed a higher induction of
astroglial activation in PAG columns by CSNT than sCCI
indicating a dependence on the extent of the sciatic nerve
injury.
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 7 | (A–D) Representative medullary sections showing RVM from naïve (A) and sham-operated (B) controls as well as rats 3 weeks from sCCI (C) or CSNT
(D) after immunostaining for CCL2. Compared to RVM of naïve and sham-operated rats (A,B), CCL2 immunofluorescence intensity was significantly increased in
cells (arrows) of RVM from rats with injured sciatic nerve (C,D). (E,F) Representative pictures of double immunofluorescence for GFAP or OX42 and CCL2 in RVM
after sCCI of the sciatic nerve for 3 weeks. (E) The section was double immunostained for GFAP (red) and CCL2 (green). The merged picture shows expression of
CCL2 protein in all GFAP+ astrocytes of RVM; nuclei (blue) were stained with Hoechst. (F) The section was double immunostained for OX42 (green) and CCL2 (red).
The merged picture shows the absence of CCL2 protein in OX42+ microglial cells (arrows); nuclei (blue) were stained with Hoechst. Scale bars = 90 µm in (B,E);
40 µm in (A,C,D,F).
In contrast to astrocytes, activation of microglial cells was
similar in dlPAG and vlPAG of both sides in the sCCI and CSNT
models of the sciatic nerve injury. This suggests that signaling
leading to microglial activation is maintained bilaterally in both
PAG columns 3 weeks after different types of sciatic nerve injury
(sCCI and CSNT).
Taken together, these findings of activated astrocytes and
microglial cells in vlPAG after sciatic nerve injury suggest
a role for activated glial cells in the modulation of activity
of this PAG column with respect to descending inhibition
or facilitation of nociceptive input following nerve injury (Ni
et al., 2016). It is clinically important that vlPAG and its
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Dubový et al. Supraspinal Structures after Nerve Injury
FIGURE 8 | Double immunofluorescence staining in vlPAG of rats 3 weeks from sCCI. (A–F) Sections illustrating double immunofluorescence staining for CCL2 and
GFAP or NeuN as markers of activated astrocytes or neurons, respectively. The merged pictures (C,F) show CCL2 protein in activated astrocytes (arrows) and
neurons (arrowheads). (G–L) Double immunofluorescence staining for OX42 as a marker for microglial cells and CCL2 (G–I) or CCR2 (J–L). The merged picture (I)
shows the absence of CCL2 protein in microglial cells. Compare CCL2 positive astrocyte-like cells (arrows) with the absence of CCL2 immunoreaction in microglial
cells (arrowheads). However, OX42+ microglial cells displayed CCR2 immunostaining (arrowheads in L). Scale bars = 40 µm for (A–F); 75 µm for (G–I); 15 µm for
(J–L).
descending projections to RVM comprise a neural circuit
for opioid-mediated analgesia (Lane et al., 2004; Eidson and
Murphy, 2013; Wilson-Poe et al., 2013). Increased activation of
microglial cells and astrocytes in vlPAG was observed only in
those animals made tolerant to morphine (Eidson and Murphy,
2013).
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Dubový et al. Supraspinal Structures after Nerve Injury
Unilateral sciatic nerve injury (sCCI and CSNT) induced
bilateral activation of astrocytes and microglial cells in both
dlPAG and vlPAG. These results are probably related with the
bilateral projections from the spinal cord to PAG responses of
which appear to be predominantly bilateral and non-somatotopic
(Yezierski and Mendez, 1991; Jones et al., 2003).
Activation of Glial Cells in RVM
The RVM provides the major common output of descending
modulatory system that is critical in the maintenance of chronic
pain states (Pertovaara et al., 1996; Porreca et al., 2002; Dubner
and Ren, 2004; Gebhart, 2004; Vanegas and Schaible, 2004;
Vera-Portocarrero et al., 2006; Bee and Dickenson, 2007). The
RVM is a relay in the pathways from both vlPAG and dlPAG
columns (Hudson and Lumb, 1996) with an important dlPAG
connection involved in the modulation of endocannabinoid
stress analgesia (Suplita et al., 2005). Early (1 and 3 days)
and transient activation of microglia and prolonged reaction
of astrocytes (14 days) were found in RVM in relation to
long-lasting mechanical hyperalgesia after CCI of the rat
infraorbital nerve (Wei et al., 2008). In contrast, a bilateral
increase of astrocyte activation was demonstrated after 3 days
with a decrease by day 10 following spinal nerve ligation
(SNL). Conversely, although a weak immunofluorescence was
observed at day 3, markedly stronger OX42 immunostaining
was found at day 10 in rats operated on SNL (Leong et al.,
2011). In our experiments, both types of sciatic nerve injury
induced a strong activation of astrocytes and microglial cells
in RVM, but no differences were found between the astrocyteand
the microglial activation induced by sCCI and CSNT.
The results indicated that the intensity of glial activation
in RVM after long–lasting nerve injury was not related to
the extent of axotomy; this is consistent with the status of
the RVM as a common output of descending modulatory
system.
Retrograde neuronal death can be assumed as the initial signal
for activation of glial cells after PNI (Flügel et al., 2001), but
we did not investigate neuronal loss of RVM neurons in our
experimental animals in relation to glial activation. However,
there is some controversy about neuronal loss in RVM in
different NPP models. While neuronal loss was found in RVM
following spinal nerve ligature (Leong et al., 2011), it was absent
after CCI or spared nerve injury (SNI) of the sciatic nerve (Leong
et al., 2016). Therefore, other mechanisms of glial activation
should be considered. Since CCL2 has a pivotal role in microglial
cell activation in the dorsal horn after PNI (Zhang and De
Koninck, 2006; Thacker et al., 2009; Van Steenwinckel et al.,
2011; Clark et al., 2013), it is worth exploring the critical role
of this chemokine also on supraspinal glial cross-talk which may
exert NPP facilitation after sciatic nerve injury.
Expression of CCL2 and CCR2 in Activated
Glial Cells of PAG and RVM
Activated glial cells upregulate synthesis and secretion of
numerous cytokines and chemokines that are involved in the
exchange of signals between neurons and the glial cells of CNS
structures. It was also demonstrated that these inflammatory
mediators modulate nociceptive transmission and may influence
NPP induction (DeLeo et al., 1996; Coyle, 1998; Colburn et al.,
1999; DeLeo and Yezierski, 2001; Watkins et al., 2003).
Several lines of evidence suggest that CCL2 and its receptor
CCR2 are expressed in both neurons and glial cells in the dorsal
horn of the spinal cord in animal NPP models (White et al.,
2005; Dansereau et al., 2008; Abbadie et al., 2009; Jung et al.,
2009). CCL2 was detected in primary sensory neurons and
their afferents in the dorsal horn of the spinal cord (Tanaka
et al., 2004; Dansereau et al., 2008; Jeon et al., 2009). Besides
primary afferents, CCL2 protein was also found in GFAP+
astrocytes activated by SNL (Gao et al., 2009). In contrast,
immunohistochemical staining localized the CCR2 protein in
spinal microglia after partial sciatic nerve injury (Abbadie et al.,
2003), whereas the CCR2 mRNA signal was also present in
deep dorsal horn neurons 3 days after SNL (Gao et al., 2009).
Upregulation of CCL2/CCR2 in the spinal dorsal horn of
NPP models suggests an important role for signaling through
this chemokine in the modulation of chronic pain induction
(reviewed in White et al., 2007; Gosselin et al., 2008; White
and Miller, 2010). This was also confirmed by attenuation of
behavioral signs of NPP in CCR2 knock-out mice (Abbadie et al.,
2003).
With respect to surpraspinal structures, neuronal CCL2 and
its receptor CCR2 associated with microglia were selectively
upregulated in the RVM following SNL. Furthermore, injection
of CCL2 into the RVM induced a dose-dependent hyperalgesia
that was prevented by pretreatment with the CCL2 inhibitor
RS-102895 (Guo et al., 2012). This showed that increased levels
of CCL2 in RVM are related with descending facilitation of NPP.
However, the precise cellular distribution of CCL2/CCR2 in PAG
following PNI still remains unclear.
Our results revealed that neurons and activated astrocytes
in both PAG and RVM displayed immunostaining for
CCL2 protein, while activated microglial cells expressed
CCR2 after chronic sciatic nerve injury. The results presented
here and previously published studies of the cellular distribution
of CCL2 and CCR2 suggest that CCL2/CCR2 signaling is
involved in the neuron-glia and glia-glia interactions in both
PAG and RVM after long-lasting nerve injury related to
persistent pain. Based on the observed earlier activation of
microglia, i.e., preceding astrocyte activation (Zhang and De
Koninck, 2006; Hu et al., 2007), we can assume that the injuryassociated
nociceptive inputs trigger neuronal responses in PAG
and RVM. Among these responses is the release of neuronal
CCL2 that binds to CCR2 of microglia and promotes their
activation (Zhang and De Koninck, 2006). Activated microglia
may further stimulate astroglial activation through IL-18 and
increase neuronal activity via CXCL1 (Abbadie et al., 2009; Gao
and Ji, 2010). Furthermore, activated astrocytes may contribute
to increased levels of CCL2 resulting in the augmentation of
descending pain facilitation (Guo et al., 2012).
CONCLUSION
Partial sciatic nerve injury by sCCI results in early and more
distinct mechanical and thermal hypersensitivity up to 3 weeks.
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Dubový et al. Supraspinal Structures after Nerve Injury
While CSNT induced a robust activation of astrocytes in
both PAG and RVM, the activation of microglial cells was
similar in these supraspinal structures after both types of
sciatic nerve injury. This suggests that activated astrocytes in
PAG and RVM may sustain the facilitation of the descending
system to maintain NPP states, while activation of microglial
cells may be associated with the reaction to long-lasting
PNI. Furthermore, CCL2/CCR2 signaling may be involved in
the neuron-glia and glia-glia interactions in both PAG and
RVM after either sCCI or CSNT, triggering persistent NPP
after PNI.
AUTHOR CONTRIBUTIONS
PD conceived, designed and coordinated the experiments,
ensured quantitative immunohistochemical and western blot
analyses and wrote the manuscript. PB-V conceived, designed
and coordinated the study and wrote the manuscript. IK, IH-S
and MJ designed and performed the experiments and also
participated in acquiring and analyzing the presented data. All
authors have approved the final version for publication.
FUNDING
This work was supported by grant No. 16-08508S of the Czech
Science Foundation and grants from the Vice-Chancellorship of
Research of the University of Girona to PB-V (MOB2016 and
MPCUdG2016/087).
ACKNOWLEDGMENTS
We thank Dana Kutˇejová, Jitka Mikuláˇsková, Marta Lnˇeníˇcková,
Jana Vachová and Lumír Trenˇcanský for their skillful technical
assistance.
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Conflict of Interest Statement: The authors declare that the research was
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G. Joukal M, Klusáková I, Dubový P. Direct communication of the spinal subarachnoid
space with the rat dorsal root ganglia. Ann Anat - Anat Anz. 2016;205:9-15.
doi:10.1016/j.aanat.2016.01.004
Commentary: In this article, we demonstrated for the first time that the subarachnoid space
anatomically reaches the DRG. We proved that intrathecal applied fluorescence-conjugated
dextran diffuses to the DRG and induces inflammatory response expressed by presence of
macrophages, dendritic cells and activation of satellite glial cells.
IF (WOS, 2016): 1.579
Quartile (WOS, 2016): Anatomy and Morphology Q1
Contribution of the author: First Author; approximate participation 60 %. The author
designed and performed the experiments, analyzed the data and wrote manuscript.
Annals of Anatomy 205 (2016) 9–15
Contents lists available at ScienceDirect
Annals of Anatomy
journal homepage: www.elsevier.com/locate/aanat
Research article
Direct communication of the spinal subarachnoid space with the rat
dorsal root ganglia
Marek Joukal, Ilona Klusáková, Petr Dubov´y∗
Department of Anatomy, Division of Neuroanatomy, Faculty of Medicine, Masaryk University, CZ-625 00 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 26 October 2015
Received in revised form
21 December 2015
Accepted 13 January 2016
Keywords:
Subarachnoid space
Cerebrospinal fluid
Dorsal root ganglia
Fluoro-emerald
Immunohistochemistry
Macrophages
a b s t r a c t
The anatomical position of the subarachnoid space (SAS) in relation to dorsal root ganglia (DRG) and
penetration of tracer from the SAS into DRG were investigated. We used intrathecal injection of methylene
blue to visualize the anatomical position of the SAS in relation to DRG and immunostaining of
dipeptidyl peptidase IV (DPP-IV) for detecting arachnoid limiting the SAS. Intrathecal administration of
fluorescent-conjugated dextran (fluoro-emerald; FE) was used to demonstrate direct communication
between the SAS and DRG. Intrathecal injection of methylene blue and DPP-IV immunostaining revealed
that SAS delimited by the arachnoid was extended up to the capsule of DRG in a fold-like recess that may
reach approximately half of the DRG length. The arachnoid was found in direct contact to the neuronal
body-rich area in the angle between dorsal root and DRG as well as between spinal nerve roots at DRG.
Particles of FE were found in the cells of DRG capsule, satellite glial cells, interstitial space, as well as
in small and medium-sized neurons after intrathecal injection. Penetration of FE from the SAS into the
DRG induced an immune reaction expressed by colocalization of FE and immunofluorescence indicating
antigen-presenting cells (MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activation
of satellite glial cells (GFAP+). Penetration of lumbar-injected FE into the cervical DRG was greater than
that into the lumbar DRG after intrathecal injection of FE into the cisterna magna. Our results demonstrate
direct communication between DRG and cerebrospinal fluid in the SAS that can create another
pathway for possible propagation of inflammatory and signaling molecules from DRG primary affected
by peripheral nerve injury into DRG of remote spinal segments.
© 2016 Elsevier GmbH. All rights reserved.
1. Introduction
The subarachnoid space (SAS) filled by cerebrospinal fluid (CSF)
is much discussed clinically in the field of chronic pain. It is a common
site for application of anesthetics (Czernicki et al., 2014) and
sampling of CSF when assessing chronic pain (Zin et al., 2010).
Moreover, neuroinflammatory reaction expressed by upregulation
of cytokines (TNFa, IL-6 and IL-10) and chemokines (SDF1/CXCL12)
has been detected in dorsal root ganglia (DRG) both associated
and non-associated with damaged sciatic nerve. These findings
suggest that signaling of the molecular changes might be propagated
alongside the neuraxis from affected to remote DRG (Jancalek
et al., 2010, 2011; Dubov´y et al., 2010, 2013). Moreover, various
types of nerve injury-induced neuroinflammatory reactions have
also been found in the central nervous system structures (Vania
Apkarian et al., 2006). We hypothesize that direct communication
∗ Corresponding author. Tel.: +420 549 493 701; fax: +420 549 491 320.
E-mail address: pdubovy@med.muni.cz (P. Dubov´y).
between the SAS and DRG could provide a pathway for propagation
of signaling molecules alongside the neuraxis among DRG
of different spinal segments via CSF. The first goal of our study,
therefore, was to visualize the morphological position of the SAS
in relation to rat DRG. Second, we investigated the penetration of
10 kDa fluorescent-conjugated dextran (fluoro-emerald; FE) from
the SAS into DRG.
2. Materials and methods
2.1. Animals and surgical procedures
The experiments were performed on 20 adult male rats (Wistar,
200–250 g; Animal Breeding Facility of Masaryk University, Czech
Republic). The animals were kept at 22 ◦C and maintained on a 12 h
light/dark cycle under specific pathogen-free conditions in the animal
housing area of Masaryk University. Sterilized food and water
were available ad libitum. All experimental procedures were carried
out aseptically and according to protocols approved by the Ethical
Committee of Masaryk University, Brno and the Departmental
http://dx.doi.org/10.1016/j.aanat.2016.01.004
0940-9602/© 2016 Elsevier GmbH. All rights reserved.
10 M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15
Committee of the Ministry of Education, Youth and Sports of the
Czech Republic.
2.2. Anatomical study of the SAS in relation to DRG
2.2.1. Intrathecal methylene blue injection
Although fusion of the meninges with DRG has been described
(Shanthaveerappa and Bourne, 1962; McCabe and Low, 1969), a
position of SAS in relation to rat DRG remained unclear. Therefore,
we injected Methylene blue (MB) into SAS to macroscopically visualize
the precise position of SAS in relation to DRG. Animals (n = 5)
were anesthetized using a mixture of ketamine (40 mg/ml) and
xylazine (4 mg/ml) administered intraperitoneally (0.2 ml/100 g
body weight). MB was dissolved in artificial cerebrospinal fluid
(ACSF; Hylden and Wilcox, 1980) at 1% concentration and 50 ␮l
of this solution was injected into the SAS of the cisterna magna.
The micro syringe was inserted through the posterior atlantooccipital
membrane after skin incision and blunt reflection of
paravertebral muscles. Animals were sacrificed in CO2 and perfused
transcardially with 4% paraformaldehyde. Lumbar (L4–L5) and cervical
(C7–C8) DRG were detected in situ following laminectomy
and foraminotomy conducted with great care not to damage the
meninges. Microdissection and analysis were performed under a
stereomicroscope (Leica M50 LED) equipped with a digital camera
(Leica DFC 480, Leica Microsystems, Wetzlar, Germany).
2.2.2. Detection of the arachnoid by DPP-IV immunostaining
Five rats were sacrificed in CO2 and perfused transcardially with
Zamboni’s fixative solution (Zamboni and de Martino, 1967). DRG of
cervical (C7–C8) and lumbar (L4–L5) segments were removed and
then immersed in Zamboni’s fixative overnight. The DRG were then
washed in 20% phosphate-buffered sucrose (pH 7.2) and embedded
in Tissue-Tek OCT compound (Miles; Elkhart, IN, USA). Longitudinal
cryostat sections (12 ␮m) were then cut (Leica 1800 cryostat) and
collected on gelatin-coated microscope slides. The sections were
processed for indirect immunofluorescence staining to detect DPPIV
as a marker of the arachnoid (Haninec and Grim, 1990). Affinity
purified secondary antibody conjugated with FITC was used for
visualization of DPP-IV immunostaining (Table 1). Cell nuclei were
stained with Hoechst 33342 (Sigma; St. Louis, MO, USA), the sections
were mounted in a Vectashield aqueous mounting medium
(Vector Laboratories; Burlingame, CA, USA) and analyzed using a
Nikon Eclipse NI-E epifluorescence microscope equipped with a
Nikon DS-Ri1 camera operated by a NIS-Elements software (Nikon,
Prague, Czech Republic).
2.3. Penetration of labeled dextran (fluoro-emerald) from the SAS
into DRG and immunodetection of loaded cells
Ten rats were deeply anesthetized using a mixture of ketamine
(40 mg/ml) and xylazine (4 mg/ml) administered intraperitoneally
(0.2 ml/100 g body weight). Fluorescent-conjugated dextran
(fluoro-emerald, FE, MW = 10 kDa, lysine fixable, Molecular
Probes, Eugene, OR, USA) was dissolved in ACSF (1.5 mg in 100 ␮l).
The solution (50 ␮l) was injected into the SAS using the micro
syringe through the atlanto-occipital membrane to the cisterna
magna (cervical, n = 5) or between the L2 and L3 vertebrae (lumbar,
n = 5). All rats were left to survive for 20 h. They were then sacrificed
in CO2 and perfused transcardially with Zamboni’s fixative solution
(Zamboni and de Martino, 1967). Cervical (C7-C8) and lumbar (L4L5)
DRG were removed, immersed in Zamboni’s fixative overnight.
The samples were then washed in 20% phosphate-buffered sucrose
for 12 h and longitudinal cryostat sections (12 ␮m) were prepared.
A part of the sections was stained using Hoechst 33342 (Sigma;
St. Louis, MO, USA), mounted in a Vectashield aqueous mounting
medium (Vector Laboratories; Burlingame, CA, USA), then analyzed
using a Nikon epifluorescence microscope to examine FE penetra-
tion.
2.3.1. Immunofluorescence staining
To identify FE-loaded cells or cell reaction following FE penetration
into DRG, the sections were processed for standard indirect
immunofluorescence staining with ED1 and ED2 antibodies to
detect activated and resident macrophages, respectively. In addition,
MHC-II antibody was used to detect antigen-presenting cells
(APC), GFAP antibody was applied as a marker of activated satellite
glial cells (SGC), and Iba1 antibody was used to detect phagocytic
activity of DRG cells. In addition, Ki-67 immunostaining was used
to visualize proliferation activity of the cells. Primary and secondary
antibodies and their concentrations are listed in Table 1.
Briefly, the sections were washed with phosphate-buffered saline
(PBS) containing 0.3% bovine serum albumin (BSA) and 0.1% Tween-
20; treated with 3% normal goat or donkey serum for 30 min;
then incubated with primary antibody at room temperature for an
appropriate time and affinity purified secondary antibodies conjugated
with TRITC were applied at room temperature for 90 min.
The sections were washed in PBS, stained with Hoechst 33342,
mounted in a Vectashield aqueous mounting medium, and then
analyzed using a Nikon epifluorescence microscope. The control
sections were incubated while omitting the primary antibodies.
2.3.2. Double immunostaining for GFAP and ED1
A part of DRG sections was double immunostained for GFAP
and ED1 to detect positions of activated macrophages in relation
to sensory neuron-SGC units. Sections were treated with a mixture
of acetone and methanol (1:1) at −20 ◦C for 5 min, washed with
PBS containing 0.3% BSA and 0.1% Tween 20, then treated with 3%
normal goat serum for 30 min. The sections were then incubated
with mouse monoclonal anti-ED1 antibody at room temperature
and TRITC-conjugated goat anti-mouse secondary antibody for
90 min. Next, simultaneous immunostaining was done by incubating
the sections with rabbit polyclonal anti-GFAP antibody at room
temperature. After thorough washing in PBS, the sections were
treated with AlexaFluor 350-conjugated goat anti-rabbit secondary
antibody for 90 min. Sections were mounted in a Vectashield aqueous
mounting medium (Vector Laboratories; Burlingame, CA, USA)
after washing in redistilled water.
Table 1
Primary and secondary antibodies, their dilutions and producers.
Primary antibody Type of antibody Dilution; incubation time Producer Secondary antibody Dilution Producer
DPP-IV Mouse monoclonal 1:200; 240 min Santa Cruz, USA Goat anti-mouse 1:100 Chemicon
ED1 Mouse monoclonal 1:200; 240 min Serotec, UK Goat anti-mouse 1:50 Chemicon
ED2 Mouse monoclonal 1:200; 16 h Serotec, UK Goat anti-mouse 1:100 Chemicon
GFAP Rabbit polyclonal 1:250; 180 min Dako, Denmark Goat anti-rabbit 1:100 Chemicon
Donkey anti-rabbit 1:400 Jackson ImmunoResearch
Iba1 Goat polyclonal 1:50; 16 h Abcam, UK Biotinylated Donkey anti-goat 1:400 Santa Cruz
Ki-67 Rabbit polyclonal 1:500; 240 min Vector, USA Goat anti-rabbit 1:100 Chemicon
MHC-II Mouse monoclonal 1:100; 240 min Serotec, UK Goat anti-mouse 1:100 Chemicon
M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15 11
Fig. 1. Representative pictures of lumbar DRG after intrathecal injection of MB (A–C). Arrow indicates the SAS filled by MB as a blue stripe at the surface of DRG. The SAS
was in some cases localized at approximately half of DRG length (B). Representative cryostat sections through lumbar DRG immunostained for DPP-IV as a marker of the
arachnoid show the SAS delimited by the arachnoid as a fold-like recess reaching DRG (D and E, arrows). Furthermore, the SAS is in direct contact with DRG between the spinal
nerve roots (D and E, arrowheads) and in the angle between the dorsal root and surface of DRG (E, open arrow). The schematic drawing (F) summarizes the aforementioned
position of the SAS delimited by the arachnoid (dotted green line) in relation to DRG. Continuous black and dashed red lines indicate dura and pia mater, respectively. A
possible route of molecule penetration from CSF into DRG is depicted by the arrows. Scale bar = 3 mm (A), 1 mm (B), 500 ␮m (C), 200 ␮m (D and E).
2.3.3. Double immunostaining for GFAP and Iba1
Phagocytic activity of GFAP+ SGC was proved by simultaneous
reaction with Iba1 antibody as a marker for ionized calciumbinding
adaptor molecule 1. A part of sections was pretreated
(see above) and incubated with rabbit polyclonal anti-GFAP antibody
at room temperature and Cy5-conjugated donkey anti-rabbit
secondary antibody for 90 min. Then, goat polyclonal anti-Iba1
antibody at room temperature was applied overnight following
inhibition of endogenous streptavidin–biotin interaction. After
thorough washing in PBS, the sections were treated with biotinylated
donkey anti-goat secondary antibody and TRITC-conjugated
streptavidin (1:100; Jackson ImmunoResearch, USA) for 90 min.
The sections were washed in PBS, stained with Hoechst 33342,
mounted in a Vectashield aqueous mounting medium. TRITC and
Cy5 fluorescence were detected by combination of C-FL Epi-Fl TRITC
and Cy5 filter blocks, respectively. In the case of Iba1 immunostaining,
monochromatic images were converted into purple pseudo
color.
2.3.4. Quantitative analysis of FE penetration into DRG after
lumbar and cervical administration
The FE+ areas were measured in neuronal body compartments
of cervical and lumbar DRG after cervical or lumbar FE injection.
Every third section was used to protect against measuring the same
area twice. Two or three irregular areas of interest per section were
circumscribed in at least 10 longitudinal sections of each DRG in
neuronal body compartment and measured using NIS-Elements AR
Analysis (version 4.20.00, Nikon, Prague, Czech Republic). The FE+
area was selected by thresholding technique (Dubov´y et al., 2002). A
percentage ratio of the FE+ area to total area of neuronal body compartment
was counted and calculated for each picture. Results from
four cervical and lumbar DRG were expressed as mean ± SD and
compared using Mann–Whitney U test (StatSoft, Tulsa, OK, USA)
with p < 0.05 as the level of significance.
The categories of neuronal body sizes were determined by
diameter calculated from areas measured using NIS-Elements AR
Analysis. Only those neuronal profiles containing nuclei were
counted. The sizes of DRG neurons were categorized as small
(<25 ␮m), medium (25–40 ␮m), and large (>40 ␮m).
3. Results
3.1. Anatomical study of subarachnoid space in relation to DRG
Intrathecal application of MB was performed to investigate the
in situ position of the SAS in relation to DRG. We found the SAS filled
with MB as the blue color stripes indicating SAS extension to the
surface of DRG in all spinal cord segments (Fig. 1A–C). Longitudinal
cryostat sections of cervical and lumbar DRG immunostained
for DPP-IV revealed a blind, fold-like recess delimited by the arachnoid
and protruding up to the capsule surface of DRG. The extent
of this recess varied from short in some sections to approximately
half of DRG length in most cases. Nevertheless, the contact of the
arachnoid with DRG capsule did not seem to be a place for communication
between CSF and DRG. The place for direct communication
between the SAS and DRG parenchyma is probably localized at the
12 M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15
Fig. 2. Representative cryostat sections illustrate the amount of FE diffused into cervical (A and C) and lumbar (B and D) DRG after cervical (A and B) and lumbar application
(C and D). Note the greater amount of FE particles in cervical DRG after lumbar application (C) compared to lumbar DRG after cervical application (B). Fluorescent particles
penetrated into DRG and are dispersed in interstitial space (arrowheads) or loaded by interstitial cells (opened arrow). In addition, FE particles were found in small neurons
(full arrow) in contrast to large neuronal bodies free of FE fluorescence (asterisks). Scale bar = 60 ␮m.
angle between the dorsal root and DRG surface where the arachnoid
was in close contact with the surface of the neuronal body-rich
area. In addition, the SAS of triangular shape limited by the DPPIV+
arachnoid between the ventral and dorsal roots was also in
close contact with DRG, thus forming another possible communication
point between the SAS and DRG (Fig. 1D and E). The position
of meninges and points of a possible communication between the
SAS and DRG are shown schematically in Fig. 1F.
3.2. Penetration of FE from the SAS into DRG and
immunophenotyping of loaded cells
A robust green fluorescence was found indicating diffusion of
FE molecules from the subarachnoid space into DRG (Fig. 2A–D).
The penetration of lumbar-injected FE into the cervical DRG was
higher than into the lumbar DRG after intrathecal injection of FE
into the cisterna magna (Fig. 2B and C; Table 2). Particles of FE were
dispersed in the extracellular spaces of DRG, where they induced a
strong immune reaction as expressed by macrophage invasion.
Immunostaining for ED1 suggested that most of the FE+ cells
invading the interstitial space of both cervical and lumbar DRG
were activated macrophages (Fig. 3A–C). As demonstrated by
double immunostaining for GFAP and ED1 (Fig. 3D–G), some
of the ED1+ cells additionally invaded the sensory neuron-SGC
units. In comparison to ED1, only a few ED2+ macrophages were
observed in the interstitial space of DRG. Round-shaped resident
ED2+ macrophages were predominantly located in the neuronal
Table 2
Percentage value of FE+ area to the neuronal body-rich area of cervical (C-DRG) and
lumbar DRG (L-DRG) after cervical or lumbar FE administration.
C-DRG (x− ± SD) L-DRG (x− ± SD)
Cervical FE administration 37.13 ± 13.46 6.94 ± 1.80
Lumbar FE administration 50.42 ± 5.48*
69.30 ± 11.29
*
Significant difference (p < 0.05) when compared to L-DRG after cervical admin-
istration.
body-rich area of DRG and all of them were filled with FE (Fig. 3H–J).
The cells immunolabeled for MHC-II+ found in the interstitial space
of DRG were divided into a major population loaded with FE particles
and a minor population without such particles (Fig. 4A–C). The
FE particles were also observed in GFAP-immunostained SGC. Double
immunostaining revealed that GFAP+ SGC loaded by FE particles
also displayed immunofluorescence staining for Iba1 (Fig. 4D–F).
In addition, the FE particles penetrated into the bodies of small
and medium-sized neurons whereas the large neurons were free
of FE fluorescence (Fig. 2D). A green FE fluorescence was also
present in the capsule of DRG and attached meninges. The capsule
of DRG and attached meninges displayed FE fluorescence and
contained the cells loaded by FE particles that were recognized
as activated (ED1+) and resident macrophages (ED2+) and APC
(MHCII+) (Figs. 3A–C, H–J; 4A–C). Diffusion of FE particles induced
a minimal proliferation activity of cells inside DRG and attached
meninges, as illustrated by rare Ki-67 immunostaining (Fig. 4G–I).
All Ki67+ cells were found to be free of FE particles.
4. Discussion
The relationship between arachnoid and DRG had been investigated
by McCabe and Low (1969), who used the term “subarachnoid
angle” to describe the place where meninges reach the surface of
DRG. In addition, Shanthaveerappa and Bourne (1962) had postulated
that the perineural epithelium of spinal nerve is in fact a
prolongation of leptomeninges.
Our results from MB injection showed the blue stripes indicating
enlargement of the SAS up to the surface of DRG. The SAS
extension in the form of a fold-like recess was also detected by
immunohistochemical staining for DPP-IV, used as a marker of the
arachnoid (Haninec and Grim, 1990). Moreover, the SAS delimited
by the DPP-IV+ arachnoid was found between the dorsal and ventral
roots and as a sharp angle between the dorsal root and the neuronal
body-rich area of DRG. These regions constitute a probable pathway
for penetration of molecules from CSF of the SAS into DRG.
M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15 13
Fig. 3. Cryostat sections through lumbar DRG after lumbar application of FE illustrate the presence of activated (ED1) and resident (ED2) macrophages in DRG. Robust
invasion of ED1+ macrophages is seen in DRG parenchyma, capsule and attached meninges (A–C). A part of activated macrophages (ED1+) are loaded (arrows) or free of
FE particles (arrowheads). Double immunostaining for GFAP and ED1 reveal that some of activated macrophages invaded the sensory neuron-SGC unit and have become
“satellite macrophages” (arrows, D–G). Resident macrophages (ED2+) loaded with FE were found in DRG capsule and attached meninges (H–J, arrowheads) as well as in DRG
neuronal body-rich area (H–J, arrows). Cell nuclei were stained using Hoechst (C and J). Scale bar = 60 ␮m (A–C; H–J), 40 ␮m (D–G).
This is in accordance with Abram et al. (2006), who found fluorescein
penetration into DRG near the subarachnoid angle 30 min after
intrathecal injection but apparently paid little attention to this finding.
Hu and McLachlan (2002) indirectly demonstrated diffusion of
molecules from CSF into DRG in a region between the spinal nerve
roots where the aggregation of APC was found. These previously
published and our present results suggest that possible structural
positions for communication between the SAS and DRG are localized
in the area where the arachnoid attaches directly to the surface
of neuronal body rich area at the angle between DRG and dorsal root
and among the spinal nerve roots and DRG.
The SAS is a common site for intrathecal administration of anesthetics
used in the treatment of neuropathic pain and for surgery
(Hawksworth and Serpell, 1998; Czernicki et al., 2014). In addition,
the SAS may communicate with epidural space and anesthetics
injected epidurally cross the arachnoid lamina and reach the SAS
(Ohtani et al., 1990; Angel Reina et al., 2008). The analgesic effect
of anesthetics after intrathecal application is explained by their
penetration into the dorsal roots (Hamber and Viscomi, 1999).
However, our results after intrathecal FE application suggest a possible
penetration of molecules including anesthetics from the SAS
into DRG, where the primary sensory neurons could be affected.
The effect of anesthetics on neurons has been investigated using
patch clamp technique and explained by selective involvement of
sodium and potassium channels (Safronov et al., 1996; Scholz et al.,
1998; Komai and McDowell, 2001).
We used FITC-conjugated dextran (fluoro-emerald) as a tracer
for studying direct communication between the SAS and DRG.
Fluoro-emerald is lysine fixable conjugated dextran, and it is
used extensively as a retrograde tracer to label neurons (e.g.
after peripheral nerve lesions) or to study axonal sprouting after
surgical nerve anastomoses (Kubek et al., 2004; ˇZele et at., 2010).
In addition to neuronal tracing, dextrans are used in a wide variety
of other applications, including fluid transport, intracellular communication,
tracing cell lineage, probing membrane permeability
and endocytosis (Nasomphan et al., 2013; Dai et al., 2014; Hu et al.,
2015). Furthermore, dextrans have been used in clinical practice
(Lamke and Liljedahl, 1976) despite the fact that they can induce
anaphylactic reactions (Zinderman et al., 2006).
Although robust green FE fluorescence was found inside DRG
after both cervical and lumbar intrathecal injection, the amount
of FE diffused into cervical DRG after lumbar administration was
obviously greater than that entering lumbar DRG after cervical
injection. This is in agreement with the findings of Kusmirek
et al. (1997), who found that intrathecal injection of opioid
antagonist DAMGO administered both to the cervical and lumbar
regions spreads alongside the neuraxis. However, identical doses of
DAMGO administered to the lumbar SAS produced a suppression of
both hind- and forepaw withdrawals in contrast to cervical administration
where the withdrawals of hind paw were not suppressed.
Diffusion of FE into the arachnoid and DRG induced an immune
reaction as demonstrated by immunostaining of FE-loaded cells
for MHC-II, thus indicating APC, as well as for ED1 and ED2
indicating activated and resident macrophages, respectively
(Dijkstra et al., 1985; Dijkstra and Damoiseaux, 1993; Hu and
McLachlan, 2003). The presence of immune cells loaded by FE
in the meninges corresponds with findings of Wieseler-Frank
et al. (2007), who postulated that meninges are responsible for
the immune reaction, including secretion of proinflammatory
cytokines to the CSF after inflammatory stimulus. Intrathecal FE
injection did not induce increased proliferation activity (Ki-67+)
of the cells in the meninges and DRG of our experimental rats,
thus indicating that most of activated ED1+ macrophages were
blood-derived. Furthermore, the ED1+ macrophages loaded by
14 M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15
Fig. 4. Representative cryostat sections of lumbar DRG after lumbar FE injection. A–C illustrate presence of MHCII+ cells (APC) in DRG capsule and attached meninges (arrows)
as well as in DRG neuronal body-rich area. The majority of APC were simultaneously loaded with FE (arrowhead) and a minority is without FE particles (open arrow). D–F
demonstrate presence of FE particles in SGC double immunostained for GFAP and Iba1 (arrows) and in macrophages (arrowheads). G–I illustrate a minimal proliferation
activity detected using immunostaining for Ki67 within DRG (arrow) and attached meninges (arrowheads). All Ki67+ cells were found free of FE particles. Cell nuclei were
stained using Hoechst (C–F, I). Scale bar = 60 ␮m (A–C; G–I), 40 ␮m (D–F).
FE penetrated into the sensory neuron-SGC units to become the
“satellite macrophage,” as has been described in an experimental
neuropathic pain model (Dubov´y et al., 2007).
Particles of FE diffused from the SAS into DRG also labeled SGC,
which protect DRG neurons against foreign molecules (Hanani,
2005). FE induced activation of SGC which was confirmed by immunostaining
for GFAP. As Iba1 is involved in regulation of membrane
ruffling and phagocytosis (Imai and Kohsaka, 2002), positive double
immunostaining for GFAP and Iba1 revealed phagocytic activation
of SGC by FE particles. It is noteworthy that we found FE-loaded
SGC mainly around large neurons the bodies of which were free
of FE fluorescence. In contrast, FE particles penetrated and were
observed within bodies of small and medium-sized neurons. This
can be explained by differing numbers of SGC and their lamellae per
neuron, which increase in proportion to neuronal volume (Pannese,
1980; Ledda et al., 2004). Therefore, smaller neurons have poor
protection from fewer SGC and their cytoplasmic lamellae which
would prevent penetration of FE molecules from interstitial space
into DRG neurons.
Chronic constriction of the sciatic nerve has been shown to
evoke cellular and molecular changes not only in DRG related
to damaged nerve but also in contralateral and cervical DRG
(Dubov´y et al., 2013; Jancalek et al., 2011, 2010). There are several
mechanisms for propagating these changes, which spread to the
contralateral and remote structures after unilateral nerve damage.
It has been strongly suggested that these changes are propagated
via the commissural interneurons present in spinal cord and brainstem
(Koltzenburg et al., 1999). However, molecules involved in
Wallerian degeneration could alternatively be disseminated via
the blood stream to the remote structures (Dubov´y et al., 2007).
The present results suggest direct communication between the
SAS filled with CSF and DRG alongside the neuraxis to make possible
another non-synaptic pathway for exchange and spreading
of molecules between primary affected DRG and those in remote
spinal segments.
5. Conclusions
The anatomical position of the arachnoid and intrathecal
injection of fluorescent-conjugated dextran indicate direct communication
between the SAS and DRG and that the molecules of
CSF could be directly diffused into DRG. A penetration of dextran
molecules into DRG was confirmed also by loaded immune cells
and activated SGC around large neurons. In addition, diffusion of
dextran into small and medium-sized neurons suggests a possible
modulation of their activity by molecules present in CSF or
exogenous drugs after their intrathecal administration. Direct communication
between the SAS and DRG creates another pathway
for possible propagation of inflammatory and signaling molecules
from DRG primary affected by peripheral nerve injury into DRG of
remote spinal segments.
Acknowledgements
The authors thank D. Kutˇejová, J. Mikuláˇsková, L. Trenˇcansk´y
and J. Vachová for their skillful technical assistance. This work was
supported by grant MUNI/A/1215/2014.
M. Joukal et al. / Annals of Anatomy 205 (2016) 9–15 15
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.aanat.2016.01.
004.
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H. Joukal M, Klusáková I, Solár P, Kuklová A, Dubový P. Cellular reactions of the choroid
plexus induced by peripheral nerve injury. Neurosci Lett. 2016;628:73-77.
doi:10.1016/j.neulet.2016.06.019
Commentary: In this article, we demonstrated increased number of activated and resident
macrophages in epiplexus position in rat choroid plexus after peripheral nerve injury. We
found no proliferation in the choroid plexus after nerve injury suggesting that macrophages
were recruited from circulated monocytes passing through altered blood-cerebrospinal fluid
barrier.
IF (WOS, 2016): 2.180
Quartile (WOS, 2016): Neurosciences Q3
Contribution of the author: First Author; approximate participation 60%. The author
designed and performed the experiments, analyzed the data and wrote manuscript.
Neuroscience Letters 628 (2016) 73–77
Contents lists available at ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Research article
Cellular reactions of the choroid plexus induced by peripheral nerve
injury
Marek Joukal, Ilona Klusáková, Peter Solár, Adéla Kuklová, Petr Dubov´y∗
Department of Anatomy, Division of Neuroanatomy, Faculty of Medicine, Masaryk University, CZ-625 00 Brno, Czech Republic
h i g h l i g h t s
• Numbers of ED1+ and ED2+ epiplexus macrophages increased with duration of CCI.
• Sham-operated CP also displayed an increase in the number of epiplexus macrophages.
• No significant CP cell proliferation was found after either sham or CCI operation.
• Inflammatory reaction of CP was observed after both nerve injury as well as surgery.
a r t i c l e i n f o
Article history:
Received 11 April 2016
Received in revised form 7 June 2016
Accepted 8 June 2016
Available online 9 June 2016
Keywords:
Blood–cerebrospinal fluid barrier
Kolmer cells
Macrophages
Chronic constriction injury
a b s t r a c t
The choroid plexus (CP) of brain ventricles forms the blood–cerebrospinal fluid (blood–CSF) barrier that
is involved in many diseases affecting the central nervous system (CNS). We used ED1 and ED2 immunostaining
to investigate epiplexus cell changes in rat CP after chronic constriction injury (CCI). In contrast
to naïve CP, the CP of sham-operated rats showed an increase in the number of ED1+ cells of a similar
magnitude during all periods of survival up to 3 weeks, while the number of ED2+ increased only at 3 days
from operation. In comparison to naïve and sham-operated animals, the number of ED1+ and ED2+ cells
in the epiplexus position increased with the duration of nerve compression. We detected no or negligible
cell proliferation in the CP after sham- or CCI-operation. This suggests that increased number of ED1+
and ED2+ cells in the epiplexus position of the CP is derived from peripheral monocytes passing through
altered blood–CSF barrier. The changes in epiplexus cells indicate that the CP reacts to tissue injury after
the surgical approach itself and that the response to peripheral nerve lesion is greater. This suggests a
role for an altered blood-CSF barrier allowing for propagation of signal molecules from damaged tissue
and nerve to the CNS.
© 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The choroid plexus (CP) in the brain ventricles consists
of vascularized stroma and epithelial cells that constitute the
blood–cerebrospinal fluid (blood–CSF) barrier. The vascularized
stroma contains loose connective tissue, fenestrated capillaries,
and macrophages. The ventricular side of the stroma is covered by
epithelial cells that are responsible for secretion of cerebrospinal
fluid (CSF). Choroidal epithelial cells are connected by tight junctions
that are the main component of the blood-CSF barrier. The
epiplexus or Kolmer cells adhere on the ventricular side of the CP
epithelial cells and are regarded as part of the CP. These epiplexus
cells display diverse morphologies ranging from round to polar
∗ Corresponding author.
E-mail address: pdubovy@med.muni.cz (P. Dubov´y).
and stellate. They also have the typical cytological features of activated
macrophages including vacuoles and lysosomes [1–3]. There
is a growing body of evidence for a key CP role in many disorders
including inflammatory, neurodegenerative, infectious, traumatic,
neoplastic, as well as systemic diseases [1,2]. It has been found
that peripheral inflammation caused by lipopolysaccharide induces
molecular and cellular changes in the CP [4]. Wallerian degeneration
of damaged nerve is considered to be aseptic inflammation [5]
and produces damage associated molecular patterns (DAMPs) [6].
Moreover, Apkarian et al. [7] found an increased level of IL-1 beta
in supraspinal structures after peripheral nerve injury. We hypothesize
that Wallerian degeneration molecules, such as DAMPs, can
affect the CP via the bloodstream. The aim of our study was to investigate
whether a peripheral nerve injury can alter the epiplexus cell
composition of the CP. Changes in the number of epiplexus cells
may indicate a disruption of blood–CSF barrier and its role in the
propagation of molecules from damaged peripheral nerve to CNS.
http://dx.doi.org/10.1016/j.neulet.2016.06.019
0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
74 M. Joukal et al. / Neuroscience Letters 628 (2016) 73–77
Fig. 1. Representative images showing immunostaining with ED1 antibody in the CP from naïve rats and operated rats at 3, 7, 14 and 21 days (3D, 7D, 14D and 21D) after
CCI. Arrows indicate ED1+ epiplexus cells. The insets on the upper right show, at higher magnification, the regions indicated by the box in the main part of the image. Scale
bars = 80 ␮m (main image); 20 ␮m (insets).
2. Material and methods
2.1. Animals and surgical procedures
The experiments were performed on 52 adult male rats (Wistar,
200–250 g; Animal Breeding Facility, Masaryk University, Czech
Republic). All experimental procedures were carried out aseptically
and according to protocols approved by the Ethical Committee of
Table 1
Primary antibodies used, their dilutions, time of incubation, and suppliers.
Name Type of antibody Dilution Incubation time Supplier
ED1 Mouse monoclonal 1:200 240 min Serotec
ED2 Mouse monoclonal 1:200 16 h Serotec
Ki-67 Rabbit polyclonal 1:500 240 min Vector Laboratories
Masaryk University, Brno and the Departmental Committee of the
Ministry of Education, Youth and Sports, Czech Republic.
Rats were anesthetized with a mixture of 5% ketamine
(100 mg/kg) and 2% xylazine (10 mg/kg) administered intraperitoneally.
The chronic constriction injury (CCI) of the left sciatic
nerve was created using three ligatures (3-0; Ethicon, Somerville,
NJ) that reduced the nerve diameter by approximately one-third.
The retracted muscles and skin incision were closed with 3-0 silk
sutures and the animals were exposed to CCI for 3 (n = 8), 7 (n = 8),
14 (n = 8), and 21 (n = 8) days. The left sciatic nerve was merely
exposed but left without any lesion in sham-operated rats surviving
for 3 (n = 4), 7 (n = 4), 14 (n = 4), and 21 (n = 4) days.
The CCI- and sham-operated rats as well as naïve rats (n = 4)
were sacrificed using CO2, then perfused transcardially with 500 ml
heparinized (1000 units/500 ml) phosphate-buffered saline (pH
7.4) followed by 500 ml of Zamboni’s fixative [8]. Brains were
M. Joukal et al. / Neuroscience Letters 628 (2016) 73–77 75
Fig. 2. Representative sections showing immunostaining with ED2 antibody in the CP from naïve rats and operated rats at 3, 7, 14 and 21 days (3D, 7D, 14D and 21D) after
CCI. Arrows indicate ED2+ epiplexus cells. The insets on the upper right show, at higher magnification, the regions indicated by the box in the main part of the image. Scale
bars = 80 ␮m (main image); 20 ␮m (insets).
removed and immersed in Zamboni’s fixative for 3 days, then
washed in 10% sucrose and embedded in Tissue-Tek OCT compound
(Miles; Elkhart, IN). Coronal cryostat sections (20 ␮m) were
cut (Leica 1800 cryostat; Leica Microsystems, Wetzlar, Germany)
and mounted onto chrome-alum covered microscopic slides.
2.2. Immunohistochemical staining
The brain sections of naïve, sham- and CCI-operated animals
were immunostained under identical conditions with ED1 (antiCD68)
and ED2 (anti-CD163) antibodies to detect macrophages
with activated phagocytosis and resident tissue macrophages,
respectively [9]. The proliferation of CP cells was assessed using
Ki-67 immunostaining [10]. Sections were washed with phosphatebuffered
saline containing 0.3% bovine serum albumin and 0.1%
Tween-20, treated with 3% normal goat serum for 30 min, and
then incubated with the primary antibody at room temperature
(Table 1). Affinity purified TRITC-conjugated goat anti-mouse or
goat anti-rabbit secondary antibodies were applied at a final dilution
of 1:100 at room temperature for 90 min. Control sections were
incubated in parallel by omitting the primary antibodies. Immunostained
sections were rinsed, stained with Hoechst 33342 (Sigma;
St. Louis, MO) to detect positions of cell nuclei, and mounted in
a Vectashield aqueous mounting medium (Vector Laboratories;
Burlingame, CA). Immunofluorescence was observed and analyzed
using a Nikon Eclipse NI-E epifluorescence microscope equipped
with a Nikon DS-Ri1 camera (Nikon, Prague, Czech Republic).
2.3. Image analysis
At least 10 sections cut at 60 ␮m intervals through the brain
ventricles containing the CP were analyzed in each group. The CP
76 M. Joukal et al. / Neuroscience Letters 628 (2016) 73–77
Fig. 3. Number of ED1+ (A) and ED2+ (B) macrophages per mm2
of the CP area from naïve rats, sham- and CCI-operated rats at 3, 7, 14 and 21 days after operation.
*
Indicates significant difference (p < 0.05) when compared with the CP from naïve rats.
+Indicates significant difference (p < 0.05) when compared with the CP from sham-operated rats.
++Indicates significant difference (p < 0.05) when comparing individual times of survival after CCI.
area was determined, manually edited when needed, and measured
using the NIS-Elements AR Analysis (version 4.20.00, Nikon,
Prague, Czech Republic). Numbers of ED1+ and ED2+ cells were
manually counted in the defined area of the CP. The number of positive
cells calculated for 1 mm2 of CP area was presented as mean
plus or minus standard deviation. Two independent investigators,
who were blind to the group of animals, counted the number of
immunofluorescence positive epiplexus cells and no differences
were observed between the two sets of values. The data were compared
for CP sections from naïve, sham- and CCI-operated rats. To
verify differences between groups, a Mann–Whitney U-test was
performed using STATISTICA 5.5 software (StatSoft, Tulsa, OK, USA)
with a significance level of p < 0.05 for differences between the
tested samples.
3. Results
Immunostaining for ED1 and ED2 showed activated and resident
macrophages, respectively in the epiplexus position in all
groups of animals. The numbers of ED1+ macrophages in the CP
of sham-operated animals surviving for all investigated survival
periods were significantly increased compared to the CP of naïve
rats. In comparison with the CP of sham-operated rats, significantly
increased numbers of ED1+ macrophages were found 7, 14
and 21 days after the CCI operation. At 3 days after sham and CCI
surgery, the CP displayed similar numbers of ED1+ macrophages.
The numbers of ED1+ cells were higher than naïve rats in the CP of
CCI-operated and increased with time of survival (Figs. 1 and 3 A).
No changes in ED1+ and ED2+ macrophages were observed in the
CP stroma as well as in the meninges and blood vessels close to the
CP in animals of all groups.
Epiplexus cells immunostained for ED2 were significantly more
numerous in the CP 3 days after sham operation when compared
with the CP of naïve animals. In contrast to ED1+ cells however,
the number of ED2+ cells dropped back to values close to naïve CP
over time (7, 14 and 21 days of survival). After CCI, the numbers of
both ED1+ and ED2+ macrophages gradually increased with time
of survival (Figs. 2 and 3 B).
Immunostaining for Ki-67 showed no or very low proliferation
of CP cells in all animal groups and periods of survival (results not
shown).
4. Discussion
The presented results reveal that mere surgery including skin
incision and retraction of muscles of sham-operated as well as
peripheral nerve injury of CCI-operated rats affected the number
of macrophages in the epiplexus position (Kolmer cells) of the CP
with higher numbers of ED1+ than ED2+ cells. This indicates that the
surgical approach itself (even under aseptic conditions) may elicit
recruitment of ED1+ and ED2+ cells into the epiplexus position, but
a larger increase was induced by nerve injury starting from 7 days.
The cellular and molecular changes in nervous structures induced
by a surgical approach itself have been described by other authors
also [11,12].
A peripheral inflammatory stimulus by lipopolysaccharide
induced the synthesis of pro-inflammatory cytokines in the CP, thus
indicating that the structure must be considered a relevant mediator
of immune signals between the periphery and the brain [13].
Moreover, an increased number of macrophages in the epiplexus
position of the CP was also found in the same model for peripheral
inflammation [4].
Animal experiments revealed that nerve injury can induce
inflammatory reactions in dorsal root ganglia unrelated to the
injured nerve as well as in remote structures of the brainstem,
thalamus, and prefrontal cortex [7,14–16]. The results presented
here demonstrate that a nerve injury causes increased numbers of
ED1+ and ED2+ epiplexus cells and that this cell augmentation is
not the result of their local proliferation. The question then arises as
to what are the signaling pathways leading to these inflammatory
cellular and molecular changes. Wallerian degeneration distal to a
nerve injury is a source of DAMPs and inflammatory mediators. It
is believed that the inflammatory reaction of remote structures not
related to the primary injury can be induced by DAMPs released
from damaged tissue and distributed via the bloodstream [15,17].
Increased number of immunoreactive CP cells could be due
to de novo or enhanced synthesis of molecules recognized by
ED1 and ED2 antibodies. However, it is more likely that these
enhanced numbers of epiplexus cells are the result of proliferation
of local epiplexus cells and/or infiltration of cells considered
to be their precursors—circulatory monocytes [4]. Inasmuch as Ki-
67 immunostaining in the CP of our experimental animals was
not increased, we presume the enhanced numbers of epiplexus
macrophages both after surgery itself and after nerve injury are
M. Joukal et al. / Neuroscience Letters 628 (2016) 73–77 77
derived from recruited monocytes. Migration of nerve injuryactivated
macrophages from the dorsal root ganglia via CSF [18–20]
or through an altered blood-brain barrier [21] might be another
source of the increased number of epiplexus macrophages.
Alteration of tight junctions in the CP has been detected in various
models of neuroinflammation [22]. There is speculation that
cells from the bloodstream can reach the epiplexus position via
altered tight junctions and/or through the cytoplasm of epithelial
cells by “emperipolesis,” as has been described in monkey [23]. We
suggest that a peripheral nerve injury could alter tight junctions of
CP epithelial cells, thereby resulting in penetration of circulating
monocytes into the epiplexus position. The monocyte recruitment
may be stimulated by chemokines released by epithelial cells [24]
as well as T-lymphocytes of the CP [25]. However, further experiments
are needed to elucidate the precise mechanisms of the
increase in epiplexus macrophage numbers induced by surgical
treatment or nerve injury.
5. Conclusion
To summarize, we demonstrated significant quantitative
changes in ED1+ and ED2+ epiplexus cells in the rat CP following
chronic constriction injury of the sciatic nerve. A limited increase
of the epiplexus cells was found also after surgical treatment of
sham-operated rats. The absence of accompanying changes in Ki-67
immunostained cells showed that enhanced epiplexus cell number
is not derived from local cell proliferation but points to a
recruitment of cells derived from blood monocytes. We suggest that
DAMPs released by damaged cells after surgery (sham-operation)
and during Wallerian degeneration of injured nerve, are responsible
for the alteration of the blood–CSF barrier leading to increased
numbers of epiplexus cells.
Acknowledgements
The authors thank M. Lnˇeníˇcková, J. Mikuláˇsková, D. Kutˇejová,
L. Trenˇcask´y, and J. Vachová for their skillful technical assistance.
This work was supported by grant MUNI/A/1268/2015 (Faculty of
Medicine, Masaryk University).
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fistula: suicide or chronic wound? Forensic Sci Med Pathol 16, 377–378.
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