environmental health perspectives
effect of lead intoxication on the
postnatal growth of the
rat nervous system by martin r krigman' and edward l hogan
lead encephalopathy was induced in developing long-evans rats by adding lead
carbonate 4 w w to the diet of nursing mother immediately after delivery the
morphological and biochemical features of cerebral ontogenesis were studied in 30-day-
old rats by the 30th postnatal day the overall effect of lead intoxication was retardation of
brain growth the mass of both the cerebral gray and white matter was appreciably
reduced in the lead rats without any reduction in cell populations while the neuronal
population was preserved the growth of neurons was reduced and their maturation
retarded the retarded neuronal growth was characterized by the limited proliferation
of processes in the neuropil and by the reduction in the number of synapses per neuron however synaptogenesis was neither delayed nor perturbed but reduced by the limited
development of neuronal dendritic fields the myelination was altered and its cerebral
content significantly reduced the effect of lead on myelination was one of hypomyelina-
tion the hypomyelination appears to be primarily related to retarded growth and mat-
uration of the neuron and is not a reflection of a defect in the myelinating glia or a
delay in the initiation of myelination introduction
the vulnerability of the nervous system
to the toxic effects of lead is most evident
in the young while the neural aspects of
tdepartment of pathology university of north
carolina school of medicine chapel hill north
carolina 27514 $department of medicine neurology university
of north carolina school of medicine chapel hill north carolina 27514 present address department
of neurology medical college of south carolina charleston south carolina 29401 studies supported by u s public health service
grant es-00481 plumbism have been the subject of numerous
studies we still have much to learn about
the extent of the effects of lead on the ner-
vous system this is particularly true for
body lead burdens in children which have
been previously considered nontoxic 1 2 the nervous system of the young is obviously
different from that of the adult the episodic
nature of the growth and maturation of the
developing nervous system may make the
young more vulnerable at selected periods 3 to a hostile environment than the adult in an attempt to define the effects of lead
on the developing nervous system we have
studied the model of lead encephalopathy
may 1974 187
described by pentschew and garro 4 the
model uses the nursing rat and is charac-
terized by selected morphological features
resembling those of clinical encephalopathy this report is based upon quantitative mor-
phometric and biochemical analyses of
postnatal brain development and contains
our findings on the effects of lead on neuro-
nal growth and maturation synaptogenesis and myelination the studies reported herein
were limited to the 30th postnatal day by
this time the nervous system is highly de-
veloped the brain weight is nearly three-
fourths that of an adult 5 neurogenesis
has stopped 5 6 synaptogenesis has near-
ly ceased 7 8 and the most active period
of myelination is over 3 also the pups
are already weaned materials and methods
a more detailed description of the animal
model morphometric procedures and chemi-
cal analyses appears in our earlier reports 9 10 animal model
lead intoxication was produced in the
long-evans strain of rats by the procedure
described by pentschew and garro 4 im-
mediately after birth the lead litters were
reduced to six pups per dam and the nursing
mother was given free access to water and
a lead-containing diet this diet was ground
purina lab chow to which 4 o lead car-
bonate by weight was added after weaning
at day 25 the lead pups received the same
diet as their mothers the control litters
were adjusted to eight pups per litter and
the maternal diet was free of lead morphometric procedures
the morphometric studies of myelin were
carried out on three control and three lead
rats brains were fixed by perfusion with
5 gluteraldehyde in 0 1m phosphate buffer ph 7 4 the pyramids with the corticospinal
tracts were isolated from the medulla at the
level of the obex the isolated pyramidal
sections were post-fixed in 1 osmium te-
troxide in the phosphate buffer stained en
bloc with uranyl acetate dehydrated initial-
ly in graded alcohols and finally in propylene
oxide and embedded in araldite the speci-
mens were so embedded that the cut sections
were perpendicular to the tracts thin gray sections were cut and the contrast was
enhanced by staining with urany acetate
and reynolds lead citrate stain a jeolco
100-b electron microscope was used in all
the studies a random selection of fields was
obtained the plate magnification was
x13 000 and this was enlarged threefold in
printing a calibration picture was taken at
each sitting and this never varied more than
3 xo the actual morphometric analyses of
myelin were carried out upon these cali-
brated electron photomicrographs these
myelin studies included counts of myelinated
and nonmyelinated fibers measurement of
axon size circumference and counts of the
number of myelin lamellae per axon from
these studies the percent age of myelinated
fibers the ratio between axon circumference
and number of myelinated lamellae and
mean width of myelin lamellae were cal-
culated the significance of the findings was
determined by the appropriate statistical
test 11 the morphometric studies of the cortex
were carried out on four control and four
lead-intoxicated 30-day-old rats each of the
rats was from a different litter the brains
were fixed by perfusion as described above the fixed brains were cut and a coronal
section 1 cm thick was removed starting at
the level of the origin of the middle cerebral
artery from a given coronal section two
1-mm wedges of somatosensory cerebral
cortex were dissected from each cerebral
hemisphere starting at a point 6 mm lateral
to the interhemispheric fissure this proce-
dure yielded four full cortical wedges of
somatosensory parietal cortex area 3 of
krieg 12 the cortical wedges were pre-
pared for ultramicrotomy as above thick 0 5 pt araldite sections of the full cortical
mantle were cut and stained with toluidine
blue the araldite blocks were further trim-
environmental health perspectives188
med to the molecular layer or cortical layer
i and cut and stained for electron microscopy the thick sections of cortex were photo-
graphed and a montage encompassing a
segment of the entire width of a cortical
mantle was prepared the montages were
analyzed for the width of the cortex and
for the average density of neurons glia and blood vessels per area of cortex all
nuclei not related to either neuron or blood
vessels were identified as glia all identi-
fiable vascular structures irrespective of
their size were categorized as blood vessels the analysis of a cortical montage is limi-
ited in that it is only a qualitative assess-
ment it does not yield the number of struc-
tures or the size of a structure stereologic
procedures are available for light and elec-
tron microscopic studies which provide a
morphometric analysis of neurons neuropil and terminal boutons the basis for these
stereologic procedures and the arguments for
their validity have been presented by weibel 13 the relative size of the neurons in the
control and lead-intoxicated rats was esti-
mated by comparing comparable neuronal
-nuclei the pyramidal neurons in layer v
were identified long and short axes of their
nuclei were measured with an eyepiece mi-
crometer and their areas were calculated
from the measured major and minor axes the numercal-density of neurons the number
of neurons per unit volume of cortex was de-
termined by the method of weibel and gomez 14 this study was done at the light micro-
scopic level and used the 0 5 jf thick araldite
sections the volume density of neuropil the
volume of neuropil per unit volume of cere-
bral cortex was determined by the point-
count method 13 again this was a light
microscopic determination based upon the
0 5 fa thick sections the numerical density of terminal boutons
and the surface density of the terminal
bouton and of their dense zone were analyzed
at the ultrastructural level in the cortical
layer i the cortical layer i being almost
entirely neuropil random selection of the
fields to be studied was obtained by taking
an electron photomicrograph from the center
of a grid square if the field to be photo-
graphed appeared to contain less than 50 neuropil it was adjusted six adjacent cen-
tral grid squares were photographed per
grid the plate magnification was 13 000
and was consistently enlarged threefold in
printing and the magnification monitored
as before the numerical density of the
terminal boutons was determined by the
same principles employed for the neuronal
density 14 by assuming that the boutons
were elipses presenting oval profiles in sec-
tion the long and short axes were meas-
ured and the ratio obtained the criteria
used to define terminal boutons were the
presence in a membranous profile of three
or more small membrane-bound vesicles and
the presence of an electron-dense specialized
apposing membrane at the junction of a cell
process the average size of terminal bouton
was computed by dividing the volume density
of terminal boutons by the numerical dens-
ity the surface density of the terminal
boutons and their specialized electron dense
zones were determined by the procedures of
weibel 13 the surface density is a ratio
between the surface of a bouton and a unit
volume of neuropil the average surface area
of a single terminal bouton or its electron-
dense line was calculated by dividing the
surface density by the numerical density of
the terminal boutons the size distribution of the cellular pro-
cesses in the neuropil was analyzed from the
electron photomicrographs a line was drawn
across a print and the maximum diameter
of all processes subtended by the line was
measured the results of the light and electron mi-
croscopic morphometric studies were com-
bined for each rat four thick sections and
24 electron micrographs and the average
for the control and the lead-intoxicated
groups determined the significance of the
differences between the groups was analyzed
by student's t test 11 biochemical analyses
single brains were employed for most
studies but pools of two to four brains were
may 1974 189
necessary for the sphingolipid fatty acid
analyses and the fractionation studies the
animals were sacrificed by decapitation and
the brains were removed and weighed the
specimens were generally analyzed immedi-
ately however a few specimens were stored
at 20°c for less than 1 month prior to
analysis lead determinations were made of
homogenized whole brains by the method
of bessman and layne 15 lipids were
extracted by the method of folch-pi lees and sloane-stanley 16 phospholipid was
determined by the procedure of fiske and
subbarow 17 individual phospholipids
were resolved by thin-layer chromatography tlc 18 with silica gel h and quanti-
tated by phosphorus content 19 choles-
terol was determined by using the method
of sperry and brand 20 and cerebrosides
and sulfatides by the orcinol reaction 21 protein and dna were extracted from the
brain tissue 22 and measured by the me-
thods of lowry et al 23 and burton 24 respectively gangliosides were extracted
by the method of suzuki 25 resolved into
major species by tlc with silica gel h
and the neuranimic acid nana content
determined by the resorcinol reaction 26 cerebrosides sulfatides and ceramides were
separated and purified by florisil column
chromatography and tlc with silica gel 27 these sphingolipids were separated
into hydroxy- and n-fatty acid-containing
species by tlc cleaved by methanolysis 28 and the fatty acid methyl esters ex-
tracted into hexane the composition of
these methylated fatty acids was determined
by gas-liquid chromatography the myelin and synaptosome subfractions
were prepared by centrifugation on a dis-
continuous sucrose-ficoll gradient as de-
scribed by kurokawa et al 29 the purity
of the isolated myelin and synaptosome frac-
tions were confirmed by electron microscopy the gangliosides were extracted as above 25 resolved in their major subspecies by
tlc with silica gel h and quantified by
the resorcinol method 26 results
the lead-intoxicated rats showed a regular
sequence of functional changes fig 1 be-
tween days 22 and 24 there urinary incon-
tinence occurred and 2-4 days later caudal
paraplegia which rapidly evolved over a
24-hr period was noted if water and the
lead-supplemented food were made easily
available during this period of disability many of the paraplegic rats survived to day
30 the brains of the experimental animals
sacrificed on day 30 showed the brown dis-
coloration of the cerebellum fig 1 the
brains of these lead animals were smaller
and paler fig 1 than those of the con-
trols but otherwise no differences
were noted histological preparations reveal-
ed focal hemorrhages in the cerebellar folia
with endothelial proliferation of small blood
vessels and cysts in the medullary cores the
cysts did not have a lining and were sur-
rounded by reactive astrocytes and micro-
glia few gitter cells were noted in the
remaining regions of the brain rare cysts
were found in the corpus callosum a luxol
fast blue stain revealed many well myeli-
nated tracts and there were no discernible
differences between the control and lead-
intoxicated rats glia in the white matter
of both groups were predominantly mature
interfascicular oligodendroglia in multiple
representative sections of cerebrum cortex
was well preserved in the experimental rats
and the cyto- and the myelo-architecture
were comparably developed in both groups morphomretric studies
with electron microscopy structures of
the white matter myelin sheaths and
axons were indistinguishable in experimen-
tal and control animals however the quanti-
tative analysis of the pyramidal tract re-
vealed a number of significant differences the results of the morphometric analysis
of the myelin are presented in table 1 the
ratio of myelinated to nonmyelinated axons
was essentially the same in lead and control
rats 41 and 43 o respectively the analy-
environmental health perspectives190
figure 1 montage of 30-day old control and lead rats a disparity in body size between the control and lead
rats the discoloration of the hind limbs of the lead rat is due to urinary incontinence b photograph
showing not only the discoloration due to the incontinence but atrophy of the hind limb musculature c dorsal view of the brains in the lead rat the pallor of the cerebral hemispheres and the brown discolora-
tion of the cerebellum are evident d ventral view of the brains other than the pallor there are no ap-
parent differences sis of the number of lamellae in the sheaths
of the myelinated axons revealed that there
were significantly fewer lamellae p 0 01 in the experimental animals the mean
for the lead-intoxicated and controls was
7 8 and 8 9 lamellae respectively in the
axons with but one or two myelin lamellae the myelin was not compact these were
invariably promyelinating fibers and in both
groups comprised less than 0 3 o of the
myelinated fibers the regression analysis
of the number of myelin lamellae per unit
width of the myelin sheath showed that the
distance between a single compact myelin
lamella was 107 a for the controls and 117
a for the lead-intoxicated rats this dif-
ference was not significant and the values
were comparable to the 106 a reported by
karlson 30 for aldehyde-fixed material distribution analysis of the axon size re-
vealed that on the average the axons were
larger in the control animals p 0 05 than in the lead-intoxicated rats the mean
axon circumference was significantly re-
duced p 0 05 in the lead rats and the
values for control and lead-intoxicated rats
may 1974 191
table 1 morphometric analysis of myelin and axons
of 30-day rats a
control lead
unmyelinated fibers 43 41
promyelinating fibers of myelinated axons 0 3 0 3
mean number of myelin
lamellae per myelinated
axon 8 9 7 8
width of myelin lamella a 107 117
mean diameter of myelinated
axons p 1 64 1 52
diameter of axon when
initial myelin lamella
appears p 0 41 0 32
'the values are based upon the analysis of the
pyramidal tract in the medulla of three lead and
three control rats were 11 8 and 10 6 units respectively if
we assume the axon profiles to be circular the diameters are 1 64 for controls and
1 52 jz for the experimental animals the
relationship between axon size and number
of myelin lamellae was calculated from the
population of axons with 8-20 lamellae the
regression analysis showed that for the re-
lationship between axon diameter and num-
ber of myelin lamellae the coefficient of cor-
relation was highly significant p 0 001 for both the experimental and control
groups the slope of the regression curve which is a measure of the rate at which
the number of myelin lamellae are added
per unit increment in axon size was es-
sentially identical for the two groups there
was a significantly different p 0 05 value
for the axon size on which a compact myelin
lamella would theoretically first appear in
the control rat this was equivalent to a cir-
cular axon with a diameter of 0 41 while
in lead intoxication the corresponding diam-
eter was 0 32 al myelination was thus ini-
tiated on a smaller axon in the lead-intoxi-
cated rat and the number of myelin lamellae
for any given axon diameter was greater
in the lead-intoxicated than in the control
rats however the axons were larger in the
control group and there were more myelin
lamellae per brain changes in the cerebral cortex of the lead-
intoxicated rats were apparent in all levels
of analysis the results of the analyses of
the montages of the somatosensory cortex
are presented in table 2 the cortical mantle
was thinner p 0 01 in the lead group
and there was also a hypercellularity of the
cortex p 0 01 both the glia and neurons
contributed to the hypercellularity how-
ever the increase of glia was greater than
that of neurons most of the glia were
astrocytes this astrocytosis was also evident
in other regions of cortex the area density
or number of vessels per unit area of cortex
was essentially the same for both groups however if the blood vessels were considered
in terms of the neurons the ratio of blood
vessels to neurons was 0 341 and 0 386 for
the lead and control groups respectively while this decrease in the number of blood
vessels per neuron was marginally signifi-
cant 0 05 p 0 10 this difference may
be important in terns of the observations of
an obliteration of selective capillaries in
concordance with lead encephalopathy 31 the quantitative morphological analysis
of the thick plastic sections are presented
in table 3 there was an increase in the
numercial density of neurons p 0 05 ni
the lead rats the neuropil comprised a smal-
ler proportion p 0 05 of the cortex in
the lead rats the nuclei of the pyramidal
neurons in layer v were significantly smal-
ler in the lead rats the electron microscopic studies revealed
no apparent effects of lead of cortical neuro-
pil development however the morphometric
analysis of the diameter of the cellular proc-
esses in the neuropil brought out an ap-
preciable difference the cellular processes
of the lead-intoxicated rats were larger p 0 01 the mean diameters were 0 374 and
table 2 area density of cells in montages of soma-
tosensory cortex of 30-day rats control lead
thickness of cortical
mantle mm 1 88 1 56
cells mm-' 1107 1623
glia mm' 514 871
neurons mm' 581 685
blood vessels mm' 224 233
environmental health perspectives192
table 3 quantitative analysis of somatosensory
cortex of 30-day rats control lead
neurons x 101 per mm 7 05 8 98
pyramidal neurons layer v
nuclear area a' 78 67
neuropil of cortex 66 57
0 334 fz for the lead and control rats respec-
tively in both groups the synapses analyzed
were almost entirely of the axon-dendritic
type the synaptic vesicles were not quanti-
fied per bouton examined however there
were no qualitative differences in the num-
ber or the types of vesicles per terminal
bouton all the vesicles were of the hollow
spherical type the morphometric analysis
of the terminal boutons table 4 revealed
no effect of lead intoxication on the numeri-
cal density volume density or surface den-
sity of synapses in the neuropil there was
also no discernible difference in the surface
density of the terminal bouton dense line the values in table 4 were utilized in cal-
culating the volume total surface and dense
line surface of an average terminal bouton and these latter data are presented in table
5 there were no apparent differences be-
tween the lead and the control groups in
any of the values obtained for an average
bouton chemical studies the general analyses of the brain are
summarized in table 6 lead concentration
was of course greater in the lead animals
than in the controls 0 30 ppm and 9 2 ppm respectively the dna concentration nor-
table 4 quantitative analysis of synapses in mole-
cular layer of neuropil of somatosensory cortex
of 30-day rats control lead
terminal boutons x 10'
per mm' 9 35 10 3
volume density mm' mm' 0 156 0 181
total surface density x 10' mm' mm 3 89 5 12
dense line surface density x
10-9 mm' mm' 0 871 1 06
table 5 morphometric features of an average
terminal bouton in molecular layer control lead
axial ratio 0 66 0 68
volume ' 0 167 0 175
total surface a' 4 17 4 96
dense line surface a' 0 933 1 03
mally decreases while the whole brain con-
tent of dna increases during the initial
20-30 postnatal day 5 6 there were no
significant differences in dna content be-
tween groups the dna concentration was
increased but not significantly in the lead
rats and this probably reflected the increased
cellularity of a diffuse gliosis which was
apparent in the histological preparations in addition the brain protein content was
significantly p 0 05 reduced in the lead
rats the contents in brain of galactolipids cholesterol plasmalogens and total phos-
pholipids normally rise rapidly during the
first 20 postnatal days 3 however by
the day 30 table 7 the lead-exposed ani-
mals had significantly less p 0 02 of all
of these structural lipid constituents the
partition of the phospholipids into their
main classes table 8 showed no appre-
ciable differences in composition between
the experimental and control animals membranes and especially myelin are part-
icularly rich in cerebrosides and sulfatides as well as the ceramides which are their
likely metabolic precursors the composition
of the fatty acid moieties isolated from these
galactolipids table 9 showed several fea-
tures previously reported as indicative of
maturing myelin there is a higher propor-
tion of long-chain hydroxy fatty acids hfa than of normal or nonhydroxy fatty acids nfa in cerebrosides and sulfatides 32 in the ceramides there is a predominance
table 6 general composition of 30-day rat brains control lead
lead concn ppm 0 30 9 2
dna content tg 1757 1665
protein content mg 164 142
nana content jug 1551 1884
may 1974 193
table 7 lipid composition of 30-day rat brains control lead
total lipid phosphorus
content umole 77 6 51 0
conen umole g wet weight 53 0 43 2
cholesterol
content jsmole 92 8 52 6
concn jsmole g wet weight 65 2 43 3
cerebroside hexose
content mole 32 3 21 1
concn mole g wet weight 22 7 16 1
plasmalogens
content umole 25 5 14 4
conen umole g wet weight 17 2 12 2
of short-chain saturated normal fatty 33 in these sphingolipids there is a
ler percentage of monounsaturated
acids
smal-
fatty
acids in hfa than in nfa 20 27 34 the distributions of fatty acid species in
the brain cerebrosides and sulfatides of both
groups of rats are comparable in general
pattern no unique fatty acids were identi-
fied in the lead rats the ganglioside content normally in-
creases rapidly during early postnatal life and a significant difference p 0 01 was
already evident in the experimental group
as early as day 30 the gangliosides from
whole brain were resolved into major sub-
species table 10 neither a significant
difference in the whole brain ganglioside
composition of the two groups nor an in-
crease in minor gangliosides of the experi-
mental animals was observed the isolated
myelin subfraction reflected a very signifi-
cant enrichment in gm1 as suzuki has es-
tablished 35 however no difference in
ganglioside composition of myelin from lead-
intoxicated and control animals was found in addition no differences between the
groups were found in the isolated syna-pto-
some fraction discussion
the cerebral growth was impaired in the
lead-intoxicated rat both the wet weight of
brain and the total brain protein were signif-
icantly diminished by the 30th postnatal
day on the other hand the number of cells
in the brain as estimated by the dna con-
tent of brain was smaller for both groups in view of the decreased brain weight and
protein content with the preservation of an
table 8 composition of phospholipids in brains of lead-intoxicated and control rats phosphatidyl serine
phosphatidyl phosphatidyl phosphatidyl
ethanolamine choline sphingomyelin inositol control 2 32 5 36 2 6 1 20 3
lead 2 37 0 36 3 5 4 21 6
a phospholipids were obtained from brains of control and lead-intoxicated rats at 30 days postnatal age the values are expressed as percentage of total lipids the number of experiments is in parentheses table 9 selected aspects of fatty acid composition of ceramides cerebrosides and
sulfatides of control c and lead-intoxicated pb rat brain cerebrosides sulfatides ceramides
normal fatty a-hydroxy normal fatty a-hydroxy normal
acids fatty acids acids fatty acids fatty acids
c pb c pb c pb c pb c pb
ratio 16 0-20 0 8 0 2 8 20 28 1 6 1 4 20 28 0 2 0 4
monounsaturated of total 41 44 15 16 33 35 18 19 15 19
total fa nmole g wet weight 1088 1013 n n 687 410 n n 190 209
a lipids were obtained from brains of control and lead-intoxicated rats at 30 days postnatal age n = not detected environmental health perspectives194
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apparently normal number of brain cells we deduce that postnatally lead intoxication
leads to restricted cellular growth and re-
tarded maturation 36 this is in keeping
with our findings of altered cortical develop-
ment and myelination there was a generalized reduction' in the
mass of gray matter which was most ap-
parent in the cortex the lead brains weighed
less and the measured cortical mantle was
thinner this reduction in tissue mass was
quite complex considering there was actually
an increased density of neurons and glia in
these gray structures however the post-
natal cortical ontogenesis of the rat like
other mammals is one of progressive corti-
cal mantle enlargement secondary to the
growth and differentiation of neurons 37 38 specifically the neuronal changes con-
sist of growth of soma dendrites and axons a progressive subdivision of axon terminals
and of dendrites and synaptogenesis in the
process of cortical maturation there is a
decrease in the average size of the cell proc-
esses constituting the neuropil this reduc-
tion in the size of cellular processes is as
noted above the result of the subdivision
of proximal dendrites into distal twigs and
the growth of fine axon sprouts 38 39 thus the larger mean size of the cellular
processes noted in the neuropil of the lead
rats may be due to a restriction of the sub-
division of the axons and the dendrites a
reduction in the subdivision of dendrites
and axons in the neuropil readily accounts
for part of the reduced cortical growth it
also accounts for the fact that the neuropil
constituted a smaller percentage of the cortex
in the intoxicated rats than in the control
rats there was also an overall reduction in
the size of neuronal nuclei and soma which
adds to the overall reduction in cortex on
the basis of a reported constant relationship
between neuronal nuclear size and soma
size 40-42 we are assuming that the
homologous neurons were consistently smal-
ler in the lead rats in terms of the increased neuronal density studies by altman 6 and others 5 36 have shown that there is essentially no post-
natal neurogenesis in the rat neocortex it
thus appears reasonable to assume there
was a stable population of neurons in the
lead-intoxicated rats and the paucity of the
neuropil and the overall reduction in neuron
size accounts for the increased neuronal
density in studying the ultrastructure of the neu-
ropil there were no apparent abnormalities
of synaptogenesis however a morphometric
analysis reveals that the average number of
synapses per neuron is reduced based upon
the results presented in table 4 this is not
evident the numerical density of synapses
was increased in the lead rats however this datum is misleading as it is expressed
in terms of neuropil as also noted in the
lead rats table 3 the neuropil comprises
a smaller proportion of the cortex and the
neuronal density is greater when the synap-
tic density was recalculated in terms of
cortex and the neuronal density factors are
taken into account the average number of
synapses per lead neuron was less p 0 05 than per control neuron table 11 the basic mechanisms whereby lead in-
toxication affects synaptogenesis in the de-
veloping rat were not apparent however on another level the effect of lead was in
the reduction of the available dendritic
sites and or terminal boutons as has been
noted the lead neurons are relatively smaller
than the control neurons in the cortex the
direct relationship between neuronal size
and the extent of dendritic branching has
been demonstrated by bok 43 moreover based upon the inverse relationship between
size of neurite processes in the neuropil and
the progressive subdivision of the dendrites
and axons we have further evidence for a
reduction in the size of the dendritic tree in addition valverde 44 has pointed out
that the further a dendritic twig is from the
soma the greater is the density of dendritic
table 11 synaptic-neuronal relationship in cortex control lead
synaptic density in cortex
x 10-' per mm' 6 17 5 81
synapses per neuron x 10-' 8 75 6 46
environmental health perspectives196
spines per unit length of dendrite this
further emphasizes the significance of the
restricted dendritic field in reducing the
average number of synapses per neuron however the neuropil analysis also suggests
that axon subdivision was also retarded whether the latter reflects a primary in-
hibition of proliferation of axon terminals
or an inhibition secondary to the absence of
dendritic sties is not clear at this time by 30 days of age the lead-intoxicated
rats also had a generalized reduction in
myelin while this was not apparent in the
histologic preparation even with myelin
stains there was a significant reduction in
brain content of the constituents of mem-
branes phospholipids galactolipids plas-
malogens and cholesterol the decrease of
myelin content was also consistent with the
observed decrease in the mean size of an
axon plus the decrease in the mean number
of myeline lamellae per axon in the lead rats quantitatively the decreases of galactolipids
and cholesterol were of a similar magnitude
for those compounds in which myelin is
enriched 45-47 the gangliosides which
are concentrated in neurons 48 49 were
similarly decreased a study of the molecular
subspecies of the brain phospholipids and
gangliosides did not reveal any apparent
changes in their composition the same was
true for the detailed ganglioside analysis
of the myelin subfraction all these data
suggest that the myelin deficiencies reflect a
selective disorder of cellular metabolism similarly the fatty acid analyses of the
isolated cerebrosides and sulfatides indicate
there was no molecular variation in the
myelin membrane of the lead rats in the
same way our ultrastructural studies showed
that the myelin formed in the face of
lead intoxication was morphologically in-
distinguishable from that of the normal rat moreover the relationship between myelin-
ation and axon diameter was preserved while the axons were smaller in the lead
rats and myelination added to the axon was
initiated on a smaller axon there was no
difference in the percentage of myelinated
fibers or of fibers undergoing myelination when exposed to excessive dietary lead
during postnatal brain development which
includes active myelination and neuronal
maturation the brain of the rat manifested
significant deficiencies in myelin lipids and
gangliosides from a temporal study of my-
elination 9 we know that the deficit of
ganglioside content occurred prior to the
period of most active myelination in the rat
and thu's preceded the age at which the re-
duction of myelin lipid content was most
apparent clearly the neuron and most
likely the myelinating glial cell were com-
promised by the exposure to this heavy metal we believe it is reasonable to suggest that
the decreased myelination represents a sec-
ondary hypomyelination following retarded
neuronal growth and maturation further-
more while the average axon size was de-
creased in the lead-poisoned rat there was
a normal investment of myelin lamellae for
a given size this suggests that the initiation
and timing of myelination were not signifi-
cantly affected though the total prolifera-
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