Protective Effect Of Trihexyphenidyl On Focal Cerebral Ischemia-reperfusion Injury In Rats

Objective:This project mainly explored the protective effect of trihexyphenidyl（THP）on focal cerebral ischemia-reperfusion injury in rats,and established a pharmacological foundation for the application of THP in stroke.Methods:The rat model of middle cerebral artery occlusion reperfusion（MCAO/R）was induced by modified Longa suture method in 250-300 g SD male rats.The successful model rats（Longa score≥2 points,about 150）were included in the experiment;At the same time,the rats were administrated High-dose trihexyphenidyl(THP HD,0.5 mg·kg^-1),Medium-dose trihexyphenidyl(THP MD,0.25 mg·kg^-1),Low-dose trihexyphenidyl(THP LD,0.125 mg·kg^-1),Edaravone(control group,3 mg·kg^-1)and Sham operation group（Sham,1 ml PBS）by intraperitoneal injection 30 minutes before MCAO surgery.24 hours after MCAO,rats were hocussed and brain tissue were taken out for pathological TTC staining and Nissl staining.24 hours and72 hours after MCAO,the brain tissue of the rats was taken out for immunohistochemistry and Western blot analysis.The long-term experiment,after MCAO/R,the rats were administrated once a day,and the mNSS score was monitored for 16 consecutive days,and the water maze test was performed until the 9th day.Results:The pathological analysis of TTC staining at 24 hours after MCAO showed that the infarct size of the injured brain tissue of the pre-administered Edaravone and high-doses THP were 90871.5±26225.13and 104577±23075.60,respectively.Compared with the model group,the infarct size was significantly reduced by 236839.25±35492.94（P?0.05）.The percentage relative infarction volume of pre-administered Edaravone and high-dose THP group were 27.6±7.6%and 26.4±8.03%,respectively.Compared with MCAO/R group（43.24±6.28%）,there was significantly decreased（P?0.05）;Compared with MCAO/R group,Nissl staining showed pre-administered Edaravone and high-dose THP group the number of Nissl bodies in the cerebral cortex and hippocampal CA1 region were significantly increased,and neuro cells tightly arranged;modified neurological severity score（mNSS）monitoring showed that pre-administered Edaravone and high-dose THP significantly reduced neurological deficit scores after 72 hours of MCAO/R,improve limb movement and behavior;MORRIS water maze test performed on the 9th day after MCAO/R in rats,the results showed that rats in the space exploration experiment,the average latency on day 3（latency of Edaravone and THP high-dose groups were 54.88±38.32 seconds,52.8±28.87 seconds,respectively）and day 4（latency of Edaravone and THP high-dose group 26.46±24.61 seconds,20.12±14.35 seconds,respectively）was significantly lower than MCAO/R group（day 3 latency 91.46±29.91 seconds,day 4latency 51.75±26.30 seconds）（P?0.05）;In positioning cruise test,the times of crossing platform of Edaravone and high-dose THP group respectively were 3±1.41 and 3.5±1.70,which was significantly difference comparing with the MCAO/R group（1.36±0.75,P?0.05）.Immunohistochemical and Western blot results 24 hours after MCAO showed that pre-administration of Edaravone and diphenhydramine increase the expression of Nrf2 protein in the nucleus（the IOD value of immunohistochemical Nrf2 positive cells and the protein of Nrf2 by Western blot）,Comparing with the MCAO/R group（P?0.05）,meanwhile,the activation of Nrf2 induce nuclear translocation and activation of the downstream antioxidant HO-1protein expression.The TOD values of HO-1 protein expression in Western blots were significantly higher than MCAO/R group,P?0.05).72 hours after MCAO immunohistochemistry and Western blot results showed that pre-administration Edaravone and high-dose THP significantly reduce the expression of p-p38,NF-kB,NLRP3 and caspase-1 related proteins in brain tissue.（The IOD values of immunohistochemical p-p38,NF-kB and NLRP3 positive cells were significantly higher in the two groups than MCAO/R group,P?0.05）.Conclusion:High-dose of trihexyphenidyl(THP HD,0.5 mg·kg^-1)and Edaravone can alleviate the neuropathology damage induced by cerebral ischemia-reperfusion;long-term observation after cerebral ischemia-reperfusion in rats,THP HD can improve the neurobehavioral of rats,improve exercise and learn memory.In the early stage of MCAO/R injury,pre-administered Edaravone and THP HD in rats significantly up-regulate the Nrf2/HO-1 signaling pathway and inhibit the secondary inflammatory response mediated by p38/NF-kB/NLRP3 signaling pathway.