Ischemic Postconditioning On Cerebral Ischemia-reperfusion Rats Tlr4 And Nf-kappa B Expression Effect

Part I:Establishment of animal models of focal ischemic postconditioningObjective:To establish animal model of focal cerebral ischemic postconditioning and to investigate the optical time windows of ischemic postconditioning against focal cerebral ischemic injury in rats.Methods:A total of 60 healthy, male, SD rats were randomly assigned to six groups (n=10 each):sham surgery group, I/R group and IP group with different time intervals (15s,30s, 4s and 1 min). Focal cerebral ischemic reperfusion model were formed via middle cerebral artery occlusion(MACO). IP group were established by there cycles of reperfusion/ischemia at different time intervals(10s,15s,30s,45s and 60s respectively)after middle cerebral artery occlusion(MCAO) for 2 hours. The models were evaluated with examinating neurologic deficit scores, infarct volume and nerve cell apoptosis at 24 h after reperfusion.Results:The ischemic reperfusion models in the rats were successfully established by intraluminal suture method. Compared with the I/R group, the neurologic deficit scores significantly decreased in IP-15s, IP-30s and IP-45s groups, and the neurologic deficit scores in the IP 15s group were lower than in the IP-30s and IP-45s groups. The infarct volume and the numbers of apoptosis cell decreased significantly in the IP-15s, IP-30s and IP-45s groups as compared with the I/R group, particularly in IP-15s group(p<0.05). there were no significant difference between I/R group and IP-60s group (P>0.05).Conclusions:The modified MCAO model is a cerebral ischemic tolerance model with favorable stability; IP could reduce focal cerebral ischemic reperfusion injury and the optical time window of IP may be three cycles of 15s/15s (reperfusion/ischemic). PartⅡ:Ischemic postconditioning on expression of TLR4 and NF-κB During Focal Cerebral Ischemia/Reperfusion in RatsObjective:To investigate ischemic postconditioning on the expression of Toll-like receptor 4 (TLR4) and Nuclear factor-KB (NF-κB) in rats during focal cerebral ischemic reperfusion, and explore the role of TLR4-NF-κB signaling pathway in ischemic postconditioning.Methods:One hundred and ten adult healthy male Sprague-Dawley rats were randomized into sham group (n=10), ischemic reperfusion group (I/R group)(n=50) and ischemic postconditioning group (IP group) (n=50). The latter two groups were randomized into five subgroups respectively according to different time points of the ischemic reperfusion (6,12,24,48 and 72h) (n=10). Focal cerebral ischemia was established by an intraluminal thread middle cerebral artery occlusion for 120min. Ischemic postconditioning was subjected to 3 cycles of 15s reperfusion interspersed by 15s ischemia immediately after onset of reperfusion. sham group were not imposed by ischemia and reperfusion treatment. Each group was evaluated with examinating neurobehavioral function deficit scores and infarct volume. The immunohistochemistry was used to determine the expressions of TLR4 protein and NF-κB protein. The levels of TLR4 mRNA and NF-κB mRNA were examined by In Situ Hybridization (ISH).Results:(1) The neurobehavioral function deficit and cerebral infarction were seen in the I/R group and IP group. Compared with the I/R group, the IP group showed decreased neurobehavioral function deficit scores (P< 0.05), and reduced cerebral infarct volume (P < 0.05).(2)TLR4 protein and mRNA increased at 6h, and reached the peak at 24h after reperfusion, then gradually decreased in the I/R group. TLR4 protein and mRNA expression of IP group were consistent with the I/R group, but compared with the I/R group, the corresponding subgroup of the IP group were significantly lower (P<0.05).(3) NF-κB protein and NF-κB mRNA were found at 6h, peaking at 24h after reperfusion in both I/R group and IP group, which was consistent with TLR4; In comparison with the corresponding subgroup thel/R group, the expression of NF-κB protein and mRNA expression were significantly lower in the IP group(P< 0.05)..Conclusions:Ischemia and reperfusion can cause the expression of TLR4 and NF-κB; IP could inhibit the expression of TLR4 and NF-κB, reduce ischemic reperfusion injury and improve neurobehavioral function.