Effect Of Ischemic Postconditioning On Global Cerebral Ischemia-Reperfusion Injury In Rats

Background Extensive research has been aimed at finding effective strategies and drugs to ameliorate or prevent brain ischemia and reperfusion(I/R) injury.However,few have been successfully applied in clinical practice,although several strategies and drugs have been shown to decrease ischemic damage in the brain in animal models.Ischemic preconditioning has been shown to provide powerful protection against I/R injury in both the heart and nervous system.However,its clinical application is only possible for cases in which the occurrence of I/R is predictable.Rapid initiation of reperfusion is the most effective treatment to reduce injury and the behavioral deficits caused by ischemia. However,reperfusion also has the potential to introduce additional injury;many studies have shown that overproduction of reactive oxygen species and overloading of calcium occur in the early reperfusion period and lead to reperfusion injury.Another endogenous protective strategy,termed "ischemic postconditioning," has been recently reported,in which several repeated cycles of brief reperfusion and reocclusion of the coronary artery were applied at the onset of full reperfusion.Because ischemia cannot be predicted and happens suddenly,ischemic postconditioning,which can be applied after ischemia,is attracting considerable attention.In clinical practice,many accidents lead to global ischemia,such as drowning,cardiac arrest,and marked hypotension during cardiac surgery,and the brain is intrinsically more vulnerable to ischemia than other organs.Recent studies demonstrated ischemic postconditioning can reduce infarct size after focal brain I/R injury and ameliorate neuronal injury after global cerebral I/R injury and spinal cord ischemia in rat.However,the molecular mechanisms underlying this phenomenon are not completely understood.In the current study,we estimated the protective effect of the ischemic postconditioning against neuronal loss and behavioral deficits after global cerebral I/R using an animal model modified from Pulsinelli's four-vessel occlusion model in rats. Furthermore,to explore the mechanisms,we also determined whether the change of the expression of Caspase-3,Bcl-2,NF-ΚB,TLR-4 are involved.Section One:Modifying the Four-vessel Occlusion Method to Establish the Global Brain Ischemic Model in RatsObjective:To modify the four-vessel occlusion rat model to improve its reliability.Methods:One hundred male Sprague-Dawly rats were randomly divided into 2groups:groupⅠ(Pulsinelli 4-VO model) and groupⅡ(modified 4-VO model).In group Ⅰ,the vertebral arteries were permanently electrocauterized and then the bilateral common carotid arteries were freed from surrounding tissues.On the following day,both common arteries were occluded for 10min with aneurysm clips to induce transient cerebral ischemia.In groupⅡ,after the bilateral common carotid arteries were freed,the four-vessel occlusion rat model was modified by the placement of 15-cm length of 10-gauge surgical silk through the cervical region of the rat such that one end of the suture traversed the paravertebral gap to the nape of the rat's neck and the other end traversed the gap between external jugular vein and nutator to the nape.The two ends are secured through a 8-gauge urethral catheter to the nape with adhesive tape.When occlusion of the common carotid arteries(the vertebral arteries having been permanently occluded 24 hours earlier),the cervical suture is tied and slowly tightened to encircle and occlude the common carotid arteries and the collateral blood vessels in the neck muscles.The clinical criteria for successful four-vessel occlusion is complete and lasting loss of the righting reflex even after innocuous stimulation.To detect the cause of unsuccessful four-vessel occlusion,the failures were examined with digital subtraction angiography(DSA).Under the guidance of fluoroscopy,a microduct was placed at aortic arch via left femoral artery and radiopaque contrast medium was injected.The success rate of vertebral arteries occlusion was observed.Results:①Compared with that in groupⅠ,the percentage of rats that meet the criteria for successful 4-VO in groupⅡsignificantly increased.There were no significant differences between groupⅠand groupⅡin the percentage of rats removd because of respiratory failure and seizure.②Significant differences were not noted between groupⅠand groupⅡin the percentage of rats in which one of the vertebral arteries failed to be permanently electrocauterized.Conclusions:The modified 4-VO to estabish the rat global cerebral ischmia model can improve the achievement ratio of the model.Section 2:Protective Effect of Ischemic Postconditioning on Global Cerebral Ischemia and Reperfusion(I/R) Injury in RatsObjective:To study the effects of ischemic postconditioning on global cerebral ischemia in rats.Method:Forty-three male Sprague-Dawley rats were randomly divided into 3 groups: group Sham(sham operation),group Control(ten-minute ischemia and persistent reperfusion) and group Postcond(ten-minute ischemia and postconditioning at early reperfusion consisting of 3 cycles of 30-second/10-second reperfusion/reocclusion followed by persistent reperfusion).After reperfusion 24h later,five rats per group were decapitated and brain were removed.The left brain water content were measured by wet-dry weight method and the MDA content and SOD activity in the right hippocampus were measured.After reperfusion 72h later,Nissl staning were used to campare the neuronal damage in the CA1 layer of the hippocampus。Results:①The rats in I/R group,in comparison with in sham and postcond,had a higher brain water content.Significant differences were noted in the brain water content between sham and Postcond group.②Significant differences were noted in the level of SOD and MDA among sham,I/R group,and Postcond group.③Compared with that in sham group,the live neurons in hippocampal CA1 in I/R group and Postcond group significantly decreased after ischemia-reperfusion.The change was relieved in group Postcond.Conclusions:Ischemic postconditioning have a neuroprotective effect against global cerebral ischemia-reperfuion injury in rats.It can decrease the damage of hippocampal.Its neuroprotective effects may be related to the inhibited lipid peroxidation.Section 3:Influence of Ischemic Postconditioning on Neurological and Behavioral Outcomes after Global Cerebral Ischemia and Reperfusion(I/R) Injury in RatsObjective:To study the immediate and delayed influence of ischemic postconditioning on neuroethology after global cerebral ischemia and reperfusion injury in rats.Methods:Ninety-six male Sprague-Dawlay rats were randomly allocated to 3 groups: sham group,I/R group and Postcond group.Neurological severity scores(NSS) were tested at 1hr,6hr,12hr,24hr,48hr,96hr after reperfusion.Five and 28 days after reperfusion,Morris water maze test was emplyed to assess spatial learning and memory per group respectively and to analyze the effects of postconditioning.Results:①Compared to the sham group,the NSSs increased in I/R group and Postcond group,and the change was relieved in Postcond group.②Cognition impairment was found in the I/R and Postcond groups,and the statistic analysis showed that there were significant differences between I/R and Postcond groups in ANOVA.Shortened mean escape latency was detected in the Postcond group compared with the the I/R group during the same trial times. Conclusions:Ischemic postconditioning exerted an immediate and delayed protective effect on cognitive impairment caused by global cerebral ischemia-reperfusion injury in rats。Section 4:Influence of Ischemic Postconditioning on Neural Apoptosis after Global Cerebral Ischemia and Reperfusion(I/R) Injury in RatsObejective:To study the influence of ischemic postconditioning on neural apoptosis and to explore the role of caspase-3,bcl-2 in the mechanism of neural apoptosis after global cerebral ischemia and reperfusion(I/R) injury in rats.Methods:The levels of neural apoptosis were measured by transferase dUTP-bition nick end labeling(TUNEL) and the expression of caspase-3 and bcl-2 were measured by immunohistochemical method and Western-Blot after reperfusion 24hr later.Results:①Compared to the sham group,the levels of neural apoptosis increased in I/R group and Postcond group,and the change was relieved in Postcond group at reperfusion 72hr.②The expression of caspase-3 and bcl-2 increased in I/R group and Postcond group at reperfusion 24h,and compared to that in I/R group,the expression of caspase-3 and bcl-2 decreased and the expression of bcl-2 increased in Postcond group.Conclusions:Ischemic postconditioning can relieve neural apoptosis and the mechanism may be related to the change of the expression of caspase-3 and bcl-2.Section 5:Effects of Ischemic Postconditioning on Inflammatory Reaction and NF-κB,MPO,and TLR-4 after Global Cerebral Ischemia and Reperfusion(I/R) Injury in RatsObjectives:To investigate the effect of ischemic postconditioning on inflammatory reaction after ischemia-reperfusion injury in rats and possible mechanism.Methods:MPO activity in hippocamp was detected by MPO kit after reperfusion 24h later.The activity of NF-κB were measured by EMSA.The expression of TLR4 mRNA were detected by Real-Time PCR after reperfusion 4h,12h,24h,and 72h.Results:Significant increase of activity of MPO and NF-κB were shown after reperfusion in I/R group and Postcond group.The expression level of TLR4 mRNA increased after reperfusion,came to the highest point after 24h,then decreased in the following time.Postconditioning could inhibit activity of MPO and NF-κB,and down-regulate the expression of TLR4 mRNA.Conclusions:Postconditioning can inhibit the activity of MPO and NF-κB and down-regulate the expression of TLR4 mRNA and attenuated inflammatory injury after ischemia- reperfusion injury in rats.The mechanism of its neuroprotective effect against global cerebral ischemia-reperfusion injury may be by inhibiting inflammatory reaction.