Study Of Therapeutic Window Of Opportunity For NGF/VEGF Following Focal Cerebral Ischemia-reperfusion Injury In Rabbit By MRI

PartⅠ: Establishment of rabbit model of focal cerebralischemia-reperfusion by radiology and quality control systemObjective: To explore and quantify the quality control (QC) andevaluation system by investigating the experimental method in rabbit model offocal cerebral ischemia-reperfusion. Accordingly to establish a stable,dominated and reduplicated model of focal cerebral ischemia-reperfusion inrabbits, which was suitable for the study of MR imaging especially.Methods. For the purpose of quality control, test was randomly dividedinto six groups according to different strain rabbits (Group A), different threadmaterials(Group B), different occlusion time(Group C), different weight andage(Group D), different operation ways(Group E), different reperfusiontime(Group F). Angiography of internal carotid artery was performed in GroupA. The success rate and death rate in Group B to E Rabbits were assessed andMR scanning, neurological deficit score and gross anatomy were performed.In Group F rabbits MR scanning, neurological deficit score and2,3,5-triphenylterazolium chloride(TTC) staining were performed.Results: White New Zealand rabbits, male, weight 2.5-3.0kg and age4.5-5 mouths were selected as experimental animals. The guide wire with0.53mm tip diameter was optimal for the occlusion of the middle cerebralartery. Focal cerebral ischemia was induced by contact X-ray. In the meantime,the monitoring of physiological parameters (arterial blood gases, plasmaglucose and baby temperature) and other factors was done before, during andafter operation, which can effectively increase success rate and reduce deathrate. Imaging, neurological deficit score and TTC staining were the optimalevaluation system. Conclusions: It was necessary to select reasonably animal, quantifyinfluence factors and establish evaluation system before, during and afteroperation for setting up a stable, reliable model of focal ischemia-reperfusion.It also laid a foundation to further study pathologic changes of focalischemia-reperfusion by fundamental medicine and advanced MRItechnology.PartⅡ: A multiparametric MRI study of time and tissue characteristics ina rabbit model of focal cerebral ischemia-reperfusionObjective: To explore dynamic changes of time and tissue characteristicsin a rabbit model of focal cerebral ischemia-reperfusion at different timepoints with MRI techniques.Methods: A rabbit model of cerebral ischemia-reperfusion wasestablished. Seventy-eight white male New Zealand rabbits were randomlydivided into three groups: Group A rabbits (n=6) underwent no operation,Group B rabbits (n=12) underwent sham operation, Group C rabbits (n=60)underwent an operation of ischemic brain injury, in which the occlusion of leftmiddle cerebral artery was induced by guide wire. The time points ofreperfusion for Group C rabbits were 1h, 3h, 6h, 1d, 3d, 7d (n=10). Atdifferent time points, the following examinations were carried out: MRscanning, neurological deficit score, gross anatomy, light and electronmicroscopy observation.Results: No abnormal signal was seen on MRI in Group A and Group Brabbits. The MRI segmentation of tissue identified a gradation of cerebraltissue damage at all time points after MCAO. The histological observation ofischemic tissue from 0 hour to 7 days after ischemia and reperfusion on all theanimals was significantly correlated with MRI segmentation when amultiparametric (T2-, T1-, diffusion-, perfusion-weighted imaging) image anddata set was used.Conclusions: The application of multiparametric MRI in focal cerebralischemia-reperfusion at different time points can identify and characterize ischemic tissue and lay a foundation in diagnosis and treatment of cerebralischemia injury.PartⅢ: Study of therapeutic window of opportunity for NGF/VEGFfollowing focal cerebral ischemia-reperfusion injury in rabbits monitored byMRIExperiment 1: Study on therapeutic window of opportunity for NGFfollowing focal cerebral ischemia-reperfusion injury in rabbits monitored byMRIObjective: To study the therapeutic window of opportunity for NGFfollowing focal cerebral ischemia-reperfusion injury in rabbits and theeffectiveness was evaluated by advanced MR image technology.Methods: Focal cerebral ischemia 2h and reperfusion 72h model inrabbits was induced by transient occlusion of middle cerebral artery (MCA).The NGF was administered at Oh, 1h, 3h and 6h after reperfusion respectively.Infarct and edema volumes, neurological deficit score, apoptotic percentage,ADCR and activation of caspase-3 were assessed by MRI, molecular biologyand flow cytometry methods (FCM) at 72h after reperfusion.Results: NGF reduced infarct volumes compared with saline-treatedcontrols by 50.1%, 48.4% and 37.6% when administered at 0h, 1h and 3h afterreperfusion. The percentage of apoptotic cell and activation of caspase-3 in theperifocal regions were significantly lower than that in control group whenNGF administered was within 3h after reperfusion (P＜0.01). In the meantime,ADCR in the perifocal regions was significant increase. There was nodifference when administered NGF at 6h after reperfusion. Infarct volumesand ADCR were correlated significantly with activation of caspase-3 in theperifocal regions (P＜0.01).Conclusions: The therapeutic window of opportunity for NGF followingfocal cerebral ischemia-reperfusion injury in rabbit is not more than 3h afterreperfusion. The effect of NGF on the expression of caspase-3 in penumbra isrelated with the therapeutic time window. The NGF can reduce the incidenceof neuronal apoptosis during the cerebral ischemia-reperfusion injury by reducing the expression of caspase-3 protein.Experiment 2: Neuroprotective properties of VEGF in transient focalcerebral ischemia-reperfusion in rabbits: dose-response and therapeutictime-windowObjective: To study the dose-effectiveness and the therapeutic window ofthe vascular endothelial growth factor (VEGF) for focal cerebral ischemia-reperfusion injury of rabbits.Methods: Rabbit model of focal cerebral ischemia was induced by 2-hourocclusion of the middle cerebral artery. The VEGF was administeredimmediately after reperfusion at the different doses (1.25ng/μL, 2.5ng/μL,5.0ng/μL). The administration of the best dose was delayed for 1h, 3h, 6h,12h after reperfusion. We measured infarct volumes, apoptosis rate, activationof caspase-3, ADCR in the perifocal regions and neurological deficit score at72h after reperfusion.Results: When VEGF immediately administered was at the dose of2.5ng/μL and 5.0ng/μL after reperfusion, the cerebral edema, the percentageof apoptotic cell and activation of caspase-3 in the perifocal regionssignificantly decreased while the ADCR increased. And the number of bloodvessels increased markedly, which was more significant in the 2.5ng/μL groupthan that in the 5.0ng/μL. In the meantime the infarct volume decreasedsignificantly in the 2.5ng/μL group(P＜0.05). When administered VEGF withthe dose of 2.5ng/μL was delayed for 1h after reperfusion, MRI-derivedinfarct volume was significantly lower than that in control group[(393.0±19.6)mm~3 vs (468.6±29.7)mm~3, P＜0.01]. In the meantimeneurological deficit score and the percentage of apoptotic cell decreasedsignificantly than that in control group (P＜0.01). There were no statisticallysignificant differences when VEGF administered was at 3h, 6h and 12h afterreperfusion.Conclusions: The VEGF at a dose of 2.5ng/μL has displayed excellenteffect on vessel density and neuroprotective efficacy for focal cerebralischemia-reperfusion injury of rabbits. Therefore, the dose of 2.5ng/μL was used in the following treatments to further evaluate the effect of VEGF. Thetherapeutic window of opportunity for VEGF following focal cerebralischemia-reperfusion injury in rabbit is not more than 1h after reperfusion.Experiment 3: Combined administration of NGF and VEGF provides anenhanced neuroprotective time window in rabbits subjected to focal cerebralischemia reperfusionObjective: To determine the protective effect of NGF and VEGF onneural function in rabbits with focal ischemia/reperfusion and study thegenesis mechanism.Methods: Thirty-four white male New Zealand rabbits were randomlydivided into four groups: sham-operation group(n=6), saline-treated group(n=8), factor-treated 3h after reperfusion group (n=10), factor-treated 6h afterreperfusion group(n=10). Focal cerebral ischemia-reperfusion model in rabbitswas induced by transient occlusion of middle cerebral artery (MCA).Sham-operation rabbits underwent identical surgery except the depth at whichthe suture was inserted. Factor-treated rabbits received microinjection of 50μLVEGF 165(2.5ng/μL) and NGF400AU(16μg/L) at 3h and 6h after reperfusionrespectively by the route of perifocal region, which was replaced with physicalsaline of the same volume at the same time point in saline-treated rabbits. Wemeasured infarct volumes, neural cell apoptosis, caspase-3 and apparentdiffusion coefficient (ADC) ratio in the perifocal regions of cerebral ischemiaat 72h after reperfusion by MRI, 2,3,5-ttiphenyltetrazolinm chloride(TTC)staining and flow cytometry methods(FCM). Neurological evaluations werecarried out at 24h and 72h after reperfusion, using a Purdy score (rating scale:2, no neurological deficit score and 11, unconscious or dead).Results: Totally 34 rabbits entered the final analysis without death.①AnMRI-derived ischemic lesion was created within the territory of the middlecerebral artery (MCA), such as cortex and subcortex. When NGF and VEGFwere administered at 3h and 6h after reperfusion, infarct volumes reduced by44% and 33% compared with saline-treated group (P＜0.01).②When NGFand VEGF were administered at 3h and 6h after reperfusion, neurological deficit score was significantly lower than that in saline-treated group after 24hand 72h after reperfusion[(4.8±0.8), (5.4±0.5), (6.4±0.5), P＜0.01;(2.8±0.4), (3.2±0.8), (4.6±0.5), P＜0.01].③When NGF and VEGF wereadministered at 3h and 6h after reperfusion, brain edema was significantlylower than that in saline-treated group at 72h after reperfusion [(79.2±0.5),(79.9±0.6), (81.8±0.3) %, P＜0.01].④When NGF and VEGF wereadministered at 3h and 6h after reperfusion, ADC ratio in the perifocal regionswas significantly higher than that in saline-treated group[(89±3)%, (83±3)%, (74±4)%, P＜0.01], and the percentage of apoptotic cell and caspase-3were significantly lower than that in saline-treated group[(10.4±0.7),(15.5±1.2), (20.2±1.3)%, P＜0.01; 17.4±1.3, 26.1±1.0, 54.1±6.9, P＜0.01].Conclusions: Combined administration of NGF and VEGF provides anoverall enhanced neuroprotective effect on rabbits subjected to focal cerebralischemia reperfusion, which increased the therapeutic window compared with1 agent alone. The therapeutic window of the new combination therapy is atleast 6 hours after reperfusion injury. This combination therapy may be apromising therapeutic option.PartⅣ: Extracellar signal-regulated kinases involved inNGF/VEGF-induced neuroprotective effect after focal cerebral ischemiaObjective: To investigate the effect of the activation of extracellularsignal-regulated kinase 1 (ERK1) on cerebral ischemic injury, temporospatialalterations of ERK1 immunoreactivity in hippocampus and perifocal cortexand the expression involved in NGF/VEGF-induced neuroprotective effectwas examined.Methods: Focal cerebral ischemia 2h and reperfusion 72h model inrabbits was induced by transient occlusion of middle cerebral artery (MCA).Here we investigated the dynamic changes of expression of ERK1 inhippocampus and perifocal cortex of the rabbit brain exposed to 2h ischemiaand different periods of reperfusion by immuohistochemistry. In the meantime,we investigated the peak of the expression of caspase-3 and apoptosis status by immunohistochemistry.Results: ERK1 activation was first increased in hippocampal CA3/DG atdifferent time of reperfusion. At 6h of reperfusion in other brain region, with apeak 1 to 3day, and gradually decreased to basal level at 14-day of reperfusion.The expression of caspase-3 also was rapidly and strongly activated at 1h ofreperfusion, with one active peak at 3d. NGF/VEGF significantly inhibitedERK signaling and the activation of caspase-3.Conclusions: Together, these results suggest that ERK signaling pathwaywas involved in neuronal cell death by controlling death receptor pathway inthe hippocampus and perifocal cortex. Inhibition of the ERK signalingpathway might therefore represent an efficient way of preventing neuronal celldeath following ischemic injuries in the brain.PartⅤ: A pilot study of establishment of the animal model forcerebral-cardiac syndromeOur previous studies have established a stable, dominated andreduplicated model of focal cerebral ischemia-reperfusion by occludingmiddle cerebral artery with guide wire. However, in the course of experiments,we found that arrhythmias and acute congestive heart failure occur at 4 to 6hours after reperfusion and 24 to 30 hours after reperfusion in severalexperiments about focal cerebral ischemia in rabbits weight 3.5-4.0kg and age5-6 mouths. The information coming out of these experiments was discussed.Considering the data coming out of other experiments and the data of theliterature we regard the case as the pathogenesis of "the cerebral-cardiacsyndrome". It is linked to several biohumoral alterations occurring after thestroke. On the basis of these experiments, we have investigated the feasibilityof establishing the experimental animal model of cerebral-cardiac syndrome,and indicated that this model possesses the objective conditions for study ofcerebral-cardiac syndrome. Furthermore we also point out it should be a newdirection in studying mechanism and treatment of cerebral-cardiac syndromein the future.