Vznik a funkce mozkomíšního moku odběr mozkomíšního moku Zdeňka Čermáková OKB FN Brno Prezentace n1. Anatomie, funkce mozkomíšního moku, odběr funkce mozkomíšního moku, bariéry v CNS n2. Cytologické vyšetření mozkomíšního moku- kvantitativní a kvalitativní n3. Bariéry v CNS, bílkoviny v likvoru ,albumin v likvoru – klinický význam n4. Intratékální syntéza imunoglobulinů, kvantitativní a kvalitativní průkaz n5. Průkaz likvorey + spektrofotometrická křivka n6. Virové meningitidy n7. Bakteriální meningitidy n8. Skleróza multiplex n n Historie nZákladní vyšetřovací metoda v neurologii nPoznatky staré 2000 let nLumbální punkce –r.1891 (Quincke) nSnížení nitrolebečního tlaku u nemocného hydrocephalem nAplikace léků Hydrocephalus-Water-on-brain-225x300 Anatomie a fyziologie nČirá bezbarvá tekutina nVyplňuje komorový systém mozku, subarachnoideální prostor mozku a míchy nTři membrány n Měkká plena mozková n Arachnoidea n Tvrdá plena mozková n P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Where is CSF? nBetween arachnoid and pia mater meninges n n “Intrathecal” refers to under the arachnoid membrane i.e. in the CSF Meninges •On this diagram you can see the brain (white) covered by 3 protective membranes (meninges) the cranium (NB the skull is composed of the cranium and the mandible), the periosteum (a fibrous membrane that covers the outer surface of all bones, except at the joints of long bones) and lastly the skin. •CSF is located between arachnoid and pia mater meninges •The term “intrathecal” refers to the area under the archnoid membrane – ie in the CSF Tvorba likvoru nAktivní sekrece –choroideální plexy nPřestup intersticiální tekutiny z mozkové tkáně n nObjem likvoru 150-180 ml nDenní produkce 500-600 ml nResorpce do žilního a lymfatického systému n P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Where is CSF made? http://www.control.tfe.umu.se/Ian/CSF/CSF_diagram.jpg Production Reabsorption ~20 ml/hour •The brain produces roughly 500 ml of cerebrospinal fluid per day, this equates to around 20 ml per hour. This fluid is constantly reabsorbed, so that only ~150 mL is present at any one time. •CSF is formed in the four choroid plexuses in the brain, one in each of the ventricles. CSF is formed as plasma is filtered from the blood through the epithelial cells. leaves the brain cavity by being reabsorbed into the bloodstream in the dural sinuses. The CSF travels from the ventricles (chroid plexus – right next to cerebellum) through three foramina in the brain, emptying into the cerebrum, and ending its cycle in the venous blood via structures like the arachnoid granulations. The dural venous sinuses (also called dural sinuses, cerebral sinuses, or cranial sinuses) are venous channels found between layers of dura mater in the brain.^[1] They receive blood from internal and external veins of the brain, receive cerebrospinal fluid (CSF) from the subarachnoid space, and ultimately empty into the internal jugular vein. Funkce nMechanická ochrana mozku a míchy n nOchrana proti patogenům n nPřísun živin, hormonů n nHomeostáza Bariéry nStálá výměna látek – plocha asi 9m2 n krev – likvor n krev – mozek n nMechanismy – mechanické, enzymatické (specifické přenašeče..) n Hematolikvorová bariéra nOdděluje krev a mozkomíšní mok nTvořena epitelem choroideálních plexů nLátky přechází difuzí a aktivním transportem nUmožňuje přestup proteinů P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg TIGHT JUNCTIONS “Blood-CSF barrier” How is CSF made? Two largest interfaces between blood and brain extracellular fluids the epithelial layer has tight gap junctions between the cells on the side facing the ventricle (apical surface). These gap junctions prevent the majority of substances from crossing the cell layer into the CSF; thus the CP acts as a blood–CSF barrier. There are four choroid plexuses in the brain, one in each of the ventricles. CSF is formed as plasma is filtered from the blood through the epithelial cells. CP epithelial cells actively transport sodium, chloride and bicarbonate ions into the ventricles and water follows the resulting osmotic gradient In addition to CSF production, the CP act as a filtration system, removing metabolic waste, foreign substances, and excess neurotransmitters from the CSF. In this way the CP has a very important role in helping to maintain the delicate extracellular environment required by the brain to function optimally. CHoroid plexus- a rich network of blood vessels (derived from those of the pia mater, in each of the brains ventricles. It is responsible for the production of CSF While the largest interface between blood and brain is the BBB, this is also smaller less direct interface between blood and cerebrospinal fluid (CSF). Goldmann first demonstrated the existence of the blood-CSF barrier in 1913. Through the use of dyes with different properties it was found that the blood-CSF barrier was selectively permeable, rather than absolute (Bradbury, 1979).. Filtration is selective with low molecular mass proteins passing more readiy into CSF than larger ones. There is also much active transport of substances into, and out of, of the CSF as it is made. The choroid plexus and the arachnoid membrane act together at the barriers between the blood and CSF. On the external surface of the brain the ependymal cells fold over onto themselves to form a double layered structure, which lies between the dura and pia, this is called the arachnoid membrane. Within the double layer is the subarachnoid space, which participates in CSF drainage. Passage of substances from the blood through the arachnoid membrane is prevented by tight junctions (Nabeshima et al., 1975). The arachnoid membrane is generally impermeable to hydrophilic substances, and its role is forming the Blood-CSF barrier is largely passive. The choroid plexus forms the CSF and actively regulates the concentration of molecules in the CSF. The choroid plexus consist of highly vascularized, "cauliflower-like" masses of pia mater tissue that dip into pockets formed by ependymal cells. The preponderance of choroid plexus is distributed throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemispheres. The cells of the choroidal epithelium are modified and have epithelial characteristics. These ependymal cells have microvilli on the CSF side, basolateral interdigitations, and abundant mitochondria (Segal, 1999). The ependymal cells, which line the ventricles, form a continuous sheet around the choroid plexus. While the capillaries of the choroid plexus are fenestrated, non-continuous and have gaps between the capillary endothelial cells allowing the free-movement of small molecules, the adjacent choroidal epithelial cells form tight junctions preventing most macromolecules from effectively passing into the CSF from the blood (Brightman, 1968). However, these epithelial-like cells have shown a low resistance as compared the cerebral endothelial cells, approximately 200 W ·cm2, between blood and CSF (Saito and Wright, 1983); Figure NB: The blood-brain barrier (BBB) is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that allow relatively free exchange of substance across / between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. Thus the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain. Additionally, BBB breakdown is theorized to be a key component in central nervous system (CNS) associated pathologies. BBB investigation is an ever growing and dynamic field studied by pharmacologists, neuroscientists, pathologists, physiologists, and clinical practitioners. P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Na+ Cl-HCO3- Waste products Excess neurotransmitters etc. TIGHT JUNCTIONS “Blood-CSF barrier” Two largest interfaces between blood and brain extracellular fluids the epithelial layer has tight gap junctions between the cells on the side facing the ventricle (apical surface). These gap junctions prevent the majority of substances from crossing the cell layer into the CSF; thus the CP acts as a blood–CSF barrier. There are four choroid plexuses in the brain, one in each of the ventricles. CSF is formed as plasma is filtered from the blood through the epithelial cells. CP epithelial cells actively transport sodium, chloride and bicarbonate ions into the ventricles and water follows the resulting osmotic gradient In addition to CSF production, the CP act as a filtration system, removing metabolic waste, foreign substances, and excess neurotransmitters from the CSF. In this way the CP has a very important role in helping to maintain the delicate extracellular environment required by the brain to function optimally. CHoroid plexus- a rich network of blood vessels (derived from those of the pia mater, in each of the brains ventricles. It is responsible for the production of CSF While the largest interface between blood and brain is the BBB, this is also smaller less direct interface between blood and cerebrospinal fluid (CSF). Goldmann first demonstrated the existence of the blood-CSF barrier in 1913. Through the use of dyes with different properties it was found that the blood-CSF barrier was selectively permeable, rather than absolute (Bradbury, 1979).. Filtration is selective with low molecular mass proteins passing more readiy into CSF than larger ones. There is also much active transport of substances into, and out of, of the CSF as it is made. The choroid plexus and the arachnoid membrane act together at the barriers between the blood and CSF. On the external surface of the brain the ependymal cells fold over onto themselves to form a double layered structure, which lies between the dura and pia, this is called the arachnoid membrane. Within the double layer is the subarachnoid space, which participates in CSF drainage. Passage of substances from the blood through the arachnoid membrane is prevented by tight junctions (Nabeshima et al., 1975). The arachnoid membrane is generally impermeable to hydrophilic substances, and its role is forming the Blood-CSF barrier is largely passive. The choroid plexus forms the CSF and actively regulates the concentration of molecules in the CSF. The choroid plexus consist of highly vascularized, "cauliflower-like" masses of pia mater tissue that dip into pockets formed by ependymal cells. The preponderance of choroid plexus is distributed throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemispheres. The cells of the choroidal epithelium are modified and have epithelial characteristics. These ependymal cells have microvilli on the CSF side, basolateral interdigitations, and abundant mitochondria (Segal, 1999). The ependymal cells, which line the ventricles, form a continuous sheet around the choroid plexus. While the capillaries of the choroid plexus are fenestrated, non-continuous and have gaps between the capillary endothelial cells allowing the free-movement of small molecules, the adjacent choroidal epithelial cells form tight junctions preventing most macromolecules from effectively passing into the CSF from the blood (Brightman, 1968). However, these epithelial-like cells have shown a low resistance as compared the cerebral endothelial cells, approximately 200 W ·cm2, between blood and CSF (Saito and Wright, 1983); Figure NB: The blood-brain barrier (BBB) is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that allow relatively free exchange of substance across / between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. Thus the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain. Additionally, BBB breakdown is theorized to be a key component in central nervous system (CNS) associated pathologies. BBB investigation is an ever growing and dynamic field studied by pharmacologists, neuroscientists, pathologists, physiologists, and clinical practitioners. Hematoencephalická bariéra nBariéra mezi krevními vlásečnicemi a mozkovou tkání nJe tvořena endotelem a basální membránou kapilár a vrstvou astrocytů nPřestup látek z krve do mozku se uskutečňuje na podkladě jejich rozpustnosti v tucích nebo pomocí přenašečových systémů nSnadno prostupuje alkohol, nikotin,plyny P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg Blood-brain barrier ENDOTHELIAL CELL ASTROCYTE FOOT TIGHT JUNCTIONS •Endothelial cells n P:\Technical Directors shared documents\Wikilite\Main page design\TBS logo black JPG.jpg 2 major barriers that separate blood and brain extracellular fluids Blood-CSF barrier Blood-brain barrier ENDOTHELIAL CELL ASTROCYTE FOOT Two largest interfaces between blood and brain extracellular fluids 1. Epithelium choroid plexus 2. Endothelial cells Odběr nLumbální punkce nSubokcipitální, ventrikulární nRychlé doručení do laboratoře n (do 1 hod. od odběru) nKrvavý likvor (nutno stočit do 10 min.) n nOdběr likvoruVYŠETŘENÍ LIKVORU KATEŘINA MRÁZOVÁVYŠETŘENÍ VYŠETŘENÍ LIKVORU MRÁZOVÁKATEŘINA MRÁZOVÁ33Místo vpichu je na spojnici vrcholů kostí kyčelních a křížení s páteří v místě L4Místo vpichu je na Místo vpichu je na spojnici vrcholů kostí spojnici vrcholů kostí kyčelních a křížení s kyčelních a křížení s páteří v místě L4páteří v místě L4Lumbální punkceLumbální punkceOdběr se provádí pomocí jehly s mandrenemdo sterilních zkumavek. Množství u dospělého činí 10-15 ml. n n Soubor:Thisisspinaltap.jpg lumbalni_punkce_technika ilu_35 ilu_34 Komplikace lumbální punkce nSuchá punkce – nesprávná poloha jehly, artrotické změny nTraumatická lumbální punkce - poranění epidurální žilní pleteně, komplikace při vyšetření, může komplikovat stanovení diagnózy subarachnoideálního krvácení Soubor:Spinal needles.jpg Indikace odběru likvoru nInfekční onemocnění - zánět mozkových blan (meningitis) a zánět mozku (encephalitis) n nAutoimunitní onemocnění - sclerosis multiplex (poškození myelinových obalů), Guillain-Baré syndrom, sarkoidóza n nSubarachoideální krvácení, které není prokazatelné jinými zobracovacími metodami. n nOnkologická onemocnění centrálního nervového systému nebo průkaz metastáz. Kontraindikace lumbální punkce nHemokoagulační poruchy n nU nemocných se zánětlivými afekcemi kůže nebo dekubity v oblasti bederní páteře n nZvýšený nitrolebeční tlak Zevní komorová drenáž n n Indikace nSnížení nitrolebního tlaku (akutní hydrocefalus, dekompenzovaný chronický hydrocafalus, trauma CBS) n nOdvod zánětlivého likvoru (riziko akutní obstrukce vývodných cest, riziko rozvoje pozánětlivého obstrukčního hydrocefalu) n nOdvod krvavého likvoru po krvácení nebo operaci (riziko obstrukce vývodných cest, riziko pozdějšího rozvoje obstrukčního hydrocefalu) n nOdklonění přirozeného toku likvoru (hojení rány v oblasti zadní jámy lební) n Komorový katetr •Spojovací set •Antirefluxní sběrná komora •Sběrný sáček •Měřítko s posunem •Laserové ukazovátko Odběr vzorku z komorové drenáže n n nNa kultivace každých 24-72 hodin nSterilní rukavice, sterilní čtverce nDesinfekce (chlorhexidin, Softa sept) nDesinfekce odběrového místa, snětí krycí čepičky, odběr vzorku, desinfekce, nová krycí čepička n Hydrocephalus, odběr z V-P shuntu ventrikuloperitonealni_drenaz Odběr z Ommaya rezervoáru http://upload.wikimedia.org/wikipedia/commons/thumb/6/63/Ommaya_01.png/800px-Ommaya_01.png