The Effect Of Penehyclindine Hydrochloride On The Neonatal Brain Ischemia-reperfusion Injury

Children with cerebral palsy, seizures, mental retardation and ataxia is caused by neonatal cerebral ischemia-reperfusion injury. Timely restoration of blood supply to ischemic tissue is the key to treatment of the disease, but at the same time, the researchers found reperfusion tissue damage aggravate, this phenomenon is called ischemia-reperfusion injury. The pathogenesis of Ischemia reperfusion is the result of the interaction of the many factors, but cell apoptosis in ischemia-reperfusion injury plays an important role. Therefore, effective inhibiting cell apoptosis, restore cell functions, is the key to a cure.Cytochrome-c (Cyt-c) is a water-soluble protein which located at the outer surface of mitochondrial inner membrane, it is not only the mitochondria electron-transport chain complex components, while in oxidation respiratory chain transmission plays the role of electronics. Research shows that Cyt-c of a large release can lead to the nucleus appear apoptosis. Cyt-c release from the mitochondria is the key research to cell apoptosis mechanisms. Protease family of caspases is the key to mammalian cell apoptosis, Caspase-3 is considered the most key protein. Mitochondrial pathway of apoptosis is through by Caspase-3 activate downstream substrate, making the cell present apoptosis characteristics.Research shows that M acetylcholine receptor blocker to cerebral ischemia reperfusion injury has protective effect, the mechanisms include Ca2+ overload,free radical generation and excitability amino acids released, cell energy metabolization obstacle, acetylcholine increased and such as doctrines. The penehyclindine hydrochloride not only restrains oxygen free radical generation, stable lysosome and cell membrane, also can inhibit the release of inflammatory cytokines, improve microcirculation, but the specific mechanism needs to be studied further.ObjectiveThis study intends to adopt neonatal rats ischemia-reperfusion injury model, through the preoperative give penehyclindine hydrochloride intervention, to observed the brain pathological changes by light-microscopy, and using immunohistochemical method to detective Cyt-c and Caspase-3 changes, discusses the mechanism of penehyclindine hydrochloride for neonatal rats ischemia-reperfusion injury.MethodsThirty-two 7-day-old Sprague-Dawley pups were randomly divided into 4 groups (n=8). Group NS, which was treated by intraperitoneal inject NS before preoperation 30min.Then separated the bilateral carotid artery of the pups, without occlusion and reperfusion. Group IR, which was treated by intraperitoneal inject NS before preoperation 30min.Occlued the bilateral carotid artery 15min and followed reperfusion 24h. Group PL, which was intraperitoneal inject penehyclindine hydrochloride 0.1mg·kg before surgery. Group PH, which was intraperitoneal inject penehyclindine hydrochloride 1mg·kg before surgery. After the neonatal brain ischemia-reperfusion 24h, the pups were anesthesia with ether, take off the brain quickly, then fixed the brain with 4% formaldehyde solution. Take the HE staining, observed the brain under light microscope to determined the pathological changes. Meanwhile, using immunohistochemical technology to detect the Cyt-c protein and Caspase-3 protein.Results1.By microscopy:group NS had normal structure;the neural cells which in group IR were arranged disorder, not only had serious pyknosis and apoptosis in brain tissue, but also had a large number of ghost cells. Between cells, there were many loosely tissue and cell-free area; group PL showed more apoptotic cells, the cells were arranged neat. The pyknosis and ghost cells were significantly less than group IR; The neurons in group PH had closely-arranged, there had less apoptotic cells and pyknosis,but the extent and scope were lighter than group PL, heavier than group N/S.2. Brain Cyt-c protein expression:Compared with group NS, the expression of Cyt-c protein in brain tissues in group IR,PL and PH increased obviously(P <0.05).Compared to group IR, the expression of Cyt-c protein were lower in group PL and PH (P<0.05).Compared to group PL, the expression of Cyt-c protein reduced significantly.3. Brain Caspase-3 protein expression:Compared with group NS, the expression of Caspase-3 protein in the group IR,PL and PH increased distinctly(P<0.05).Compared with group IR, the expression of Caspase-3 protein were lower in group PL and PH (P<0.05).Compared to group PL, the expression of Caspase-3 protein decreased significantly.ConclusionThe penehyclindine hydrochloride could reduce the neonatal pups brain ischemia-reperfusion injury, reduced neuronal apoptosis, and lighten the development of the brain ischemia-reperfusion injury. The mechanism related to the inhibition of Cyt-c protein and Caspase-3 protein expression in mitochondrial.