| Objective Patients with acute ischemic stroke may preserveneurological function if the cerebral blood flow is restored by intravenousthrombolysis or mechanical recanalization in super acute stage. Mostpatients with ischemic stroke develop cerebral infarction, and haveneurological dysfunction. Cerebral edema is the leading cause of death inpatients with acute ischemic stroke. Most patients with ischemic stroke canbe intervened by effective treatment against cerebral edema and promotingCNS axonal regeneration, restoring neurological function. However,treatment options for against cerebral edema are very limited, and theinhibitory molecules surrounding neurons can severe impede the axonalregeneration of CNS. Extracellular matrix proteins involved in theformation of the basement membrane, the regulation of water balance inthe brain tissue, and are closely related to axonal regeneration inhibitorymolecules. Fibulin-5is an extracellular matrix glycoprotein which serves asthe extracellular matrix protein and may participate in signal transduction.The expression of fibulin-5is very rich in the embryonic neural crest, and it is reduced in the central nervous system after maturation. The expression offibulin-5is increased again during the disease states. Fibulin-5is anantagonist of angiogenesis which can inhibit the vascular sprouting,stabilize the basement membrane, and inhibit the production of matrixmetalloproteinase enzyme. Fibulin-5can inhibit the damage of tightjunctions between endothelial cells. Fibulin-5competes with thefibronectin to bind the integrins α5β1in neuron via RGD sequence.Fibulin-5overexpression enhances the binding force with the integrins ofα5β3, α5β5and α9β1, which inhibits the production of reactive oxygenspecies, and enhances the cell resistance to oxidative stress andanti-apoptotic ability. The increase of fibulin-5mRNA levels in the cellunder hypoxic conditions is closely related to the Akt/mTOR pathway.Fibulin-5overexpression can activate Akt; high levels of p-Akt can activateGSK-3β (glycogen synthase kinase-3β) of its downstream signalingmolecules. The phosphorylation of GSK-3β can promote the assembly ofmicrotubules and extending axons, promotes axonal regeneration afterischemic injury. Fibulin-5is widely distributed in the extracellular matrixsurrounding neurons, and involved in the formation of the basementmembrane, and may be closely related to the development and axonaldamage reparation. The adenoviral vector of fibulin-5overexpression wasconstructed in our study. The rats were transfected with adenovirusAd-Fbln-5by intracerebral injection seven days later, the cerebral ischemia-reperfusion models were made. The intervention of fibulin-5overexpression on the brain edema and infarct volume following focalcerebral I/R injury in rats was investigated. We invesgitated whetherfibulin-5overexpression may reduce the cerebral edema and minimize theinfarct volume following focal cerebral I/R injury in rats, whether mayinhibit expression of AQP4protein following ischemic insult. We aimed toinvestigate whether fibulin-5overexpression may enhance axonalregeneration and neurological function recovery by increasing the levels ofp-Akt and p-GSK-3β in neurons in rats after ischemic injury, as well as bysuppressing the neuron apoptosis and promoting the expression GAP43protein.MethodsPart I1. The overexpression vector of adenovirus Ad-Fbln-5wasmanufactured. Three different solution of Ad-Fbln-5, Ad-HK and normalsaline (negative control) were injected via microinjection needle into thecortex and hippocampus of ischemic side in rats by stereotactic surgery.Virulence of adenovirus Ad-Fbln-5was detected by histopathology andimmunohistochemistry approaches. The appropriate virus titer oftransfection was resolved. The cerebral cortex and hippocampus of rat wastransfected by adenovirus Ad-Fbln-5at titer of9.5×1010pfu/ml that wasverified with immunofluorescence method. The level of fibulin-5mRNA in the tissue of right middle cerebral artery territory was analyzed byquantitative PCR at7days after transfection of adenovirus Ad-Fbln-5.2. Two hundred and sixteen male adult sprague dawley rats wererandomly divided into5groups, the sham-operated group, adenovirusAd-Fbln-5invention plus sham-operated group, MCAO/reperfusion(I/R)group, adenovirus Ad-Fbln-5invention plus MCAO/reperfusion group,adenovirus Ad-HK intervention plus MCAO/reperfusion. Sprague-dawleyrats were subjected to occlusion of the right middle cerebral artery at7daysafter adenovirus transfection, and suture were removed2hours later.Regional cerebral blood flow was detected by laser doppler, which assist todetermine that cerebral artery is completely blocked. Brain tissue watercontent was detected by the dry and wet weight method after reperfusion at3days, and infarct size was measured by TTC staining.3. The levels of AQp4mRNA in peri-infarct brain tissue were detectedby fluorescence quantitative PCR at1day and3days after I/R injury. Theexpression of VEGFa and MMP9protein in peri-infarct brain tissue at1day after I/R insult were analyzed by western blot, and the expression ofAQP4protein in peri-infarct brain tissue at3days after I/R injury wereanalyzed by western blot;4. The changes of AQP4expression in astrocytes in peri-infarct braintissue were observed by immunofluorescence in five groups at3days afterI/R injury; the expression of VEGFa protein at1day and7days after ischemic insult were further measured by immunohistochemistry.Part II1. One hundred and sixty eight male adult sprague dawley rats wererandomly divided into5groups as described before. TheMCAO/reperfusion models were made at7days after intracerebralinjection of recombinant adenovirus Ad-Fbln-5or adenovirus vectorAd-HK. The expression of fibulin-5, GAP43, p-AKt, AKt, p-GSK-3β andGSK-3β protein in peri-infarct brain tissue after I/R injury were analyzedby western blot, and the expression of GAP43, p-AKt and p-GSK-3βprotein in peri-infarct brain tissue between five groups at3days after I/Rinjury were also analyzed by western blot. The apoptotic neural cells inischemic penumbra were detected by tunel method at3days after I/Rinsult.2. The levels of GAP43mRNA in peri-infarct brain tissue andcontralateral corresponding region tissue were detected by fluorescencequantitative PCR at3day,7days after ischemia/reperfusion in five groups.3. The changes of the double immunostaining for fibulin-5and MAP2in peri-infarct brain tissue were detected after ischemic injury byimmunofluorescence. The comparison of double immunostaining forfibulin-5and GAP43detected by immunofluorescence were performed infive groups. The double immunostaining for p-GSK-3β and GAP43werealso detected across five groups. 4. The labeled fibers of BDA at7weeks after I/R insult were adoptedto compare the axon regeneration in the sham group, the adenovirusAd-Fbln-5intervention group, the I/R group and the Ad control group. Theproliferation of contralateral cortex efferent fibers and the number ofcollateral fibers crossing the midline constituting the cortex red nucleustract were compared between four groups. The effects of fibulin-5overexpression on the proliferation of collateral fibers from contralateralcortex efferent fibers were evaluated in red nucleus plane.5. The neurological severity score were used to assess the neurologicalfunction recovery at1,3,7,14and28days after ischemia/reperfusioninjury. The mNSS scores was compared across five groups; and the effectsof of fibulin-5overexpression on the neurological function recovery wasevaluated.ResultsPart I1. The levels of fibulin-5mRNA in the tissue of right middle cerebralartery territory in adenovirus Ad-Fbln-5transfected group were50timesmore than that in saline injection group. Brain tissue water content andinfarct volume in adenovirus Ad-Fbln-5intervention group wassignificantly minimized than that in the I/R group and the Ad control groupat3days after I/R (P＜0.05).2. The levels of AQP4mRNA in peri-infarct brain tissues in adenovirus Ad-Fbln-5intervention group were significantly lower than thatin the I/R group and the Ad control group at1day and3days after I/Rinjury; the expression of AQP4protein in peri-infarct brain tissues inadenovirus Ad-Fbln-5intervention group was similar to the changes ofAQP4mRNA at3days after I/R injury (P<0.05).3. The expression of VEGFa and MMP9protein in peri-infarct braintissues in adenovirus Ad-Fbln-5intervention group at1day after I/R injurywere significantly decreased than that in the I/R group and the Ad controlgroup (P<0.05).4. AQP4expression in the astrocytes in peri-infarct brain tissues inadenovirus Ad-Fbln-5intervention group at3days after I/R injury wassignificantly decreased than that in the I/R group and the Ad control group(P<0.05). The intensities of VEGFa immunostaining in adenovirusAd-Fbln-5intervention group at1day after I/R injury were significantlylower than that in the I/R group and the Ad control group, and there wereno difference between the Ad-Fbln-5intervention group, the I/R group andthe Ad control group at7days as measured by immunohistochemistry.Part II1. The expression of fibulin-5protein in peri-infarct brain tissue wasgradually increased and peaked at3days after I/R injury. The expression offibulin-5protein was detected in neurons and its surroundings inperi-infarct brain tissue after I/R, the coexpression of fibulin-5and GAP43 were obviously detected in peri-infarct brain tissues at7days after I/R. Thenumber of the double immunostaining for fibulin-5and GAP43in ischemicbrain tissues in adenovirus Ad-Fbln-5intervention group was much morethan that in the I/R group at7days after I/R (P<0.05).2. The expression of p-AKT, p-GSK-3β, GAP43protein in peri-infarctbrain tissues began to increase after I/R, the expression of p-AKT andp-GSK-3β protein in peri-infarct brain tissues peaked at3days after I/R;the expression of GAP43protein in peri-infarct brain tissues peaked valueat7days after I/R (P<0.05). The expression levels of p-AKT, p-GSK-3β,and GAP43protein in peri-infarct brain tissues in adenovirus Ad-Fbln-5intervention group were significantly higher than that in the I/R group andthe Ad control group at3days after I/R. The expression levels of GAP43protein in peri-infarct brain tissues in adenovirus Ad-Fbln-5interventiongroup was also significantly higher than that in the I/R group and the Adcontrol group at7days after I/R. The apoptotic neural cells in ischemicpenumbra in adenovirus Ad-Fbln-5intervention group were alsosignificantly less than that in the I/R group and the Ad control group at3days after I/R (P<0.05).3. The levels of GAP43mRNA in peri-infarct brain tissue inadenovirus Ad-Fbln-5intervention group were significantly higher thanthat in the I/R group and the Ad control group at3day,7days after I/R;The levels of GAP43mRNA in contralateral corresponding region tissue in adenovirus Ad-Fbln-5intervention group were significantly higher thanthat in the I/R group and the Ad control group at3day and7days after I/R(P<0.05).4. The collateral branches of nerve fibers were stained by using BDAin red nucleus plane. We found that adenovirus Ad-Fbln-5interventiongroup formed dense webs. The number of collateral fibers crossing themidline in adenovirus Ad-Fbln-5intervention group was significantly morethan that in the I/R group and the Ad control group. The ipsilateral rednuclear was stained much darker in adenovirus Ad-Fbln-5interventiongroup.5. The adenovirus Ad-Fbln-5intervention group had a significantlylower mean score (7.3±1.70) than did the I/R group and the Ad controlgroup (9.5±1.60and9.10±1.92) at3days after I/R. At7days to28days,the difference of score between the three groups was more evident.Conclusions1. Our findings suggest that fibulin-5overexpression might inhibit theexpression of AQP4protein in prei-infart brain tissue following I/R injuryand attenuate the brain edema and infarct volume in rats, which might bemediated by down-regulation of VEGF and MMP9expression.2. The expression of fibulin-5protein in prei-infart brain tissue wasgradually increased in rats after cerebral I/R injury, peaked the highest levelat3days. Fibulin-5was expressed not only in the vascular tissues, but also in the neuronal cell body and its surroundings. The expression of fibulin-5in neurons needs further validation.3. The expression level of p-AKt, p-GSK-3β protein in neurons inprei-infart brain tissue were promoted by fibulin-5overexpression thatenhanced the anti-apoptotic ability of nerve cells, and fibulin-5overexpression could promote the expression of GAP43in neurons inprei-infart brain tissues.4. The overexpression of fibulin-5could promote the proliferation ofcontralateral cortex efferent fibers, and increase the number of collateralfibers crossing the midline which constitutes the cortex red nucleus tractdominating ipsilateral red nucleus. The axonal regeneration of centralnervous system was promoted by fibulin-5overexpression, and therecovery of neurological function was promoted after ischemia/reperfusion brain injury. Fibulin-5may be a potential new molecule fortreatment of cerebral ischemic injury. |
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