Dissertation
Dissertation > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke)

Study the Role of the JAK/STAT Signal Pathway on the Brain Injury after Focal Cerebral Ischemia and Reperfusion in Rats

Author XieHuiFang
Tutor XuRuXiang
School First Military Medical University
Course Neurosurgery
Keywords Cerebral ischemia and reperfusion Protein tyrosine kinase Signal transducer and activator of transcription AG490 Edaravone caspase-3 Apoptosis
CLC R743.3
Type PhD thesis
Year 2007
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BackgroundThe ischemic cerebrovacular disease is a common disease gravely affectinghuman health. It has such characteristics as high incidence rate, high death rate, highmutilation and high recurrence rate. It had been reported that the incidence rate wasabout 109.7~217 per 100,000, the occurrence rate about719~745.6 per 100,000 andthe death ratc about 116~141.8 per 100,000 in China. 50~70% storke survivorssuffer from scrious disabilities such as paralysis and aphasia, bringing heavy burdenson their families and the society.The pathophysiological process of cerebral ischemia particularly duringischemia-reperfusion injury is very complex. The pathogcncsis involves energyfailure, cellular toxicity of excitatory amino acids, calcium ovcrload, acidosis,toxicity of monoaminc neurotransmitters, oxygen free radicals and manyinflammatory factors so on. Recent studics show that necrosis and apoptosis cancoexist in different regions of an ischemic lesion, necrosis mainly exist in the core ofischemic brain region, and apoptosis cxist in the penumbra. There is a pressing needto find how to rescue the ischemic neuronal cell in the penumbra. Therefore, furtherstudies on the cellular signal transduction mechanisms of the neuronal apoptosismight give a new insight on the treatment of cerebral ischemia. During cerebral ischemia, apoptosis is an improtment cell death mechanism.Many studies have demonstrated that ischemia modulates the expression of severalsignaling molecules and transcription factors, including protein kinase (PKC),extracellular signal-regulated mitogen-activated protein kinase(ERK), c-jun, c-junNH2 terminal kinase(JNK), phosphoinositide-3 kinas(P13-K), serine/threoninekinase(Akt), nuclear factor-κB(NF-κB), signal transducers and activator oftranscription(STAT), and so on. Such changes may play a major role in the neuronalinjury or the recovery process. But the signal transduction mechanisms involved inapoptotic cell death remain unclear.JAK/STAT signal transduction pathway had been identified recently years. Thispathway consists of the JAKs family(JAK1,JAK2,JAK3,TYK2) and STATsfamily(STAT1,STAT2,STAT3,STAT4,STAT5a,STAT5b,STAT6). Binding of cytokinsto their member receptors induces transphosphorylation of the receptor-associatedJAKs,which in turn leads to phosphorylation of the down-stream STAT family oftranscription factor, phosphorylated STAT forms homo-or hetrodimers andtranslocates into the nucleus,where their binding to conserved genomic regulatorysequences controls the expression of a wild array of genes.Therefore,the JAK/STATpathway provides cells with a vital mechanism for responding to various extrcellularstimuli including ischemia stress.JAK/STAT pathway is now recognized as an important menberane-to-nucleussignaling pathway for a variety of cytokines(IFN、IL-2、IL-4、IL-6、CNTF),growthfactors(EGF、PDGF、CSF) and oxidative stress,Which is involved in the cellularproliferation,differentiation and/or apoptosis.STAT3 is one of the important elementsof signal transducers and activator of transcriptions, wildly expressed in differentceils and tissues, participating in the pathophysilogical process of cell growth,differentiation and apoptosis.It has been demonstrated that STAT3 activation can induce Bcl-2 overexpression and anti-apoptosis in human tumors.Another recentstudy has demonstrated that heart ischemia-induced STAT3 activation providesprotection to the infracted heart. The precise function of ischemia-induced STAT3activation remains currently unknown.Cerebral ischemia-reperfusion produced many reactive oxygen species andinflammatory cytokines such as interleukin-1(IL-1),interleukin-6(IL-6),tumornecrosis factor-a(TNF-a), which play important roles contributing to ischemicneuronal damage. Recent studies shows overpression of STAT1 and STAT3phosphorytation contributes to neuronal injury following cerebralischemia-reperfusion, down-regulation of anti-apoptotic gene Bcl-2, Bcl-x expression.STAT1 knockout mice showed small infracts, less neurological deficits and fewer thenumber of TUNEL positive neurons compared wild-typt mice. However, somestudies showed that ischemia-induced STAT3 activation might be neuronal protectionand prevent neuronal apoptosis. Now the function significance of STAT3 activation toischemia brain damage is not evaluated. It is still poorly understood whetherJAK/STAT signal pathway induced the neuronal apoptosis through activation ofcaspase-3 or not. There will be far-reching significance to further study therelationship between the JAK/STAT pathway and neuronal apoptosis and to developmethods to cure cerebral ischemia using neuronal protectors as agent targets.So as to establish the reliable focal cerebral ischemia-reperfusion model.Toinvestigate the cellular localization of JAK2 and STAT3 phosphorylation and thechange of neuronal apoptosis as a time of reperfusion following transient focalischemia. Furthermore, the effect of inhibition of JAK2 and STAT3 phosphorylation,a JAK2 inhibition and antioxidant, on neuronal deficits, infract volume, pro-apoptoticgene and neuronal apoptosis.To explore the functional significance of JAK2 andSTAT3 excess activation and possible ways causing the death of neuronal cells. Objective1. To observe the activation and changeful pattern of JAK2 and STAT3 as a timeof reperfusion following transient focal ischemia.2. To explore whether JAK2/STAT3 signal pathway induced the neuronalapoptosis through activation of caspase-3 or not.3. To study the effect of inhibition of JAK2 and STAT3 phosphorylation, a JAK2inhibition and antioxidant, on neuronal deficits, infract volume, pro-apoptotic geneand neuronal apoptosis.and to explore the functional significance of JAK2 and STAT3excess activation on neuronal cell apotosis after cerebral ischemia.PartⅠStudy on Expression of P-JAK2, P-STAT3 Protein and CellApoptosis after Focal Cerebral Ischemia and Reperfusion Injury inRatsMiddle cerebral artery occlusion/reperfusion rat’s models were performed byusing modified filament method. To observe the expression of P-JAK2 and P-STAT3protein and cell apoptosis after focal cerebral ischemia and reperfusion injury in rats.Methods1. Adult male Sprague-Dawley rats weighing 250~300g were randomly dividedinto sham-operated group, 3h, 6h, 12h,24h, 72h, 168h of reperfusion after 2h ofMCAO. Each group had 10 animals.2. Focal cerebral ischemia-reperfusion rat’s models were performed by usingmodified filament method.3. Animals were anesthetized at 3h,6h,12h,24h,72h,168h of reperfusion after 2hof MCAO.They were perfused with saline, then 4% paraformaldehyde in 0.1Mphosphate buffer(PB). The brains were removed and sectioned for H.E staining,P-JAK2, P-STAT3 immunohistochemistry and TUNEL staining of apoptosis.The P-JAK2, P-STAT3 immunoreactive cells and apoptosis cells ot each group werequantified.4. Western Blot Analysis were used to detect the expression of P-JAK2 andP-STAT3 protein.Result1. Immunohistochemical analysis show in the sham animals and 3h ofreperfusion after MCAO animals, no P-JAK2 and P-STAT3 was detected in anyregions of the cerebral cortex. The expression of P-JAK2 and P-STAT3 protein wasslightly upregulated in ischemic cerebral cortex and striatum at 6h of reperfusion afterMCAO. The immunoreactivity increased significantly at 12h of reperfusion,prominent at 24h of reperfusion in the peri-ischemia region, and thereafter decreased.2. Western blot analysis of P-JAK2 and P-STAT3 protein after cerebral ischemiaand reperfusion: Western blot analysis with anti-P-JAK2 and anti-P-STAT3 detected asingle band with a molecular mass of 131KD and 92KD respectively. In the shamanimals, no band was detected. The band of anti-P-JAK2 and anti-P-STAT3 wereslightly detected in ischemic cerebral cortex at 3h of reperfusion after MCAO,increased significantly at 12h of reperfuaion, peaked at 24h of reperfusion, andthereafter decreased.3. In the sham animal and the contralateral cerebral cortex of MCAO animals,no TUNEL positive cell was deteted. The apoptosis cells were deteted in the core ofischemic brain region at 3h of reperfusion after MCAO, increased significantly in theperi-ischemia at 6h of reperfuaion, peaked at 24h~48h of reperfusion, and thereafterdecreased.ConclusionsCerebral ischemia-reperfusion injury can activated the JAK2/STAT3 signalingtransduction pathway. The expression of P-JAK2 and P-STAT3 protein were increased significantly after cerebral ischemia and reperfusion injury in rats. Theimmunoreactivity was prominent in the pere-ischemia region, peaked at 24 hourreperfusion and thereafter decreased slightly, The apoptotic cells were also increasedobviously after cerebral ischemia and reperfusion injury in rats. peaked at 24 hourreperfusion.Up-regulation of JAK2 and STAT3 phosphorylation may be involved inthe ischemic cellular events including apoptosis. JAK2/STAT3 signal pathway playeda role in the pathophysiological process of cerebral ischemia/reperfusion cell injuryand renovation.PartⅡThe Influence of AG490,a Cytokine Signaling Inhibitor, onJAK2/STATs Signal Pathway and Apoptosis after Focal CerebralIschemia-Reperfusion in RatsAG490,a JAK2 phosphoralation inhibitor, prevented the signal pathway ofinflammatory mediators, inhibited JAK2 and STAT3 phosphorylation,down-regulatedthe expression of the post-ischemia P-JAK2 and P-STAT3 protein in pneumbra.Toinvestigate the influence of AG490 on neurological behavior, the infarct volume, thenumble of apoptotic cells and the expression of caspase-3 protein. Which to futherexprole the role and mechanism of JAK2/STAT3 signal pathway on focal cerebralischemia/reperfusion injury in rats.Methods1. Adult male Sprague-Dawley rats weighing 250-300g were randomly dividedinto sham-operated group, ischemia-reperfusion group, saline treatment group(salineip immediately and 12hs after ischemia-reperfusion) and AG490 treatment group(AG490 1mg.kg-1·ip immediately and 12hs after ischemia-reperfusion). Each grouphad 16 animals. TTC staining (n=6), immunohistochemistry (n=6), and Western BlotAnalysis (n=4).2. Focal cerebral ischemia-reperfusion rat’s models were performed by using modified filament method.3. The neurological deficits were evaluated at 24 h of reperfusion after MCAOby classical Zea Longa method. Infract volumes were detected by TTC staining.4. Animals were anesthetized and perfused with saline,then 4% paraforma-ldehyde. The brains were removed and sectioned for P-JAK2, P-STAT3 and caspase-3immunohistochemistry and TUNEL staining of apoptosis.5. Western Blot Analysis were used to detect the expression of P-JAK2、P-STAT3、caspase-3 protein in the peri-ischemia region.Results1. The AG490 treatment group significantly improved the neurological deficitsand decreased the infact volume, Compared with the ischemia-reperfusion group andsaline treatment group (P<0.05).2. The AG490 treatment group significantly decreased the expression of P-JAK2P-STAT3 and caspase-3 protein,the number of apoptotic cells were also significantlydecreased in ischemic penumbra after focal cerebral ischemia reperfusion. Comparedwith the ischemia-reperfusion group and saline treatment group (P<0.001).ConclusionAG490, a JAK2 phosphoralation inhibitor, significantly decreased the infractvolume and improved the neurological deficits after cerebral ischemia-reperfusion.AG490 prevented the post-ischemia JAK2 and STAT3 phosphorylation, partlyblocked the signaling transduction of inflammatory cytokine, Which leads to influentthe down-stream gene caspase-3,down-regulated the expression of caspase-3 in theperi-ischemia region,reduced the number of poptotic cells, and ameliorates thecerebral ischemic injury.This results suggest that excess JAK2 and STAT 3 activationafter focal ischemia is determental to brain. Prevention of ischemic-induced JAK2and STAT3 phosphorylation is neuroprotective. JAK2/STAT3 participates in the intracellular signaling pathway of the apoptosis after cerebral ischemia reperfusion:which might contribute to modulating caspase-3 protein expression.PartⅢThe Influence of Edaravone on JAK2/STAT3 SignalPathway and Neurological Deficits after Focal CerebralIschemia-Reperfusion in RatsTo exprole the effectiveness of edaravone on JAK2/STAT3 signal pathway andneurological deficits after cerebral ischemia-reperfusion in rats and study on therelevant mechanism.Methods1. Adult male Sprague-Dawley rats weighing 250~300g were randomly dividedinto sham-operated group,ischemia-reperfusion group, saline treatment group(salineip immediately and 12hs after ischemia-reperfusion) and edaravone treatmentgroup(edaravone 3mg.kg-1·ip immediately and 12hs after ischemia-reperfusion).Eachgroup had 16 animals. TTC staining (n=6), immunohistochemistry (n=6), andWestern Blot Analysis (n=4).2. Focal cerebral ischemia-reperfusion rat’s models were performed by usingmodified filament method.3. The neurological deficits were evaluated at 24 h of reperfusion after MCAOby classical Zea Longa method. Infract volumes were detected by TTC staining.4. Animals were anesthetized and perfused with saline, then 4%paraformaldehyde. The brains were removed and sectioned for P-JAK2, P-STAT3 andcaspase-3 immunohistochemistry and TUNEL staining of apoptosis.5. Western Blot Analysis were used to detect the expression of P-JAK2,P-STAT3,caspase-3 protein in the peri-ischemia region.Results1. Edaravone could significantly improved the neurological deficits and decreased the infact volume, Compared with the ischemia-reperfusion group andsaline treatment group (P<0.01).2. Edaravone significantly decreased the expression of P-JAK2 P-STAT3 andcaspase-3 protein, the number of apoptotic cells were also significantly decreased inischemic penumbra after focal cerebral ischemia reperfusion. Compared with theischemia-reperfusion group and saline treatment group, (P<0.001).ConclusionEdaravone could down-regulated the expression of caspase-3 protein in theperi-ischemia region, reduced the number of apoptotic cells, significantly decreasedthe infract volume, improved the neurological deficits and ameliorates the cerebralischemic injury.This study suggests that edaravone could be a potent neuroprotector:involvement of the JAK2/STAT3 signal pathway.

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