Research About The Theropeutic Effect Of Erythropoietin In The 4-VO Ischemia Rats

ObjectiveTo investigate whether use of erythropoietin (EPO)exerts protection on neuron in hippocampus CA1 sub-region and to explore the neuroprotective mechanism of EPO.MethodsTransient global brain ischemia model was established by using 4-VO technique. Male Sprague Dawley rats weight range from 200g to 250g were randomized into experiment group, normal saline group(compare group), and erythropoietin group. There are 30 Sprague Dawley rats in experiment group ,30 rats in erythropoietin group and 6 rats in saline group. The rats in experiment group were established global brain ischemia model by using four-vessel occlusion technique. The rats in erythropoietin group were injected intraperitoneally with EPO(4000U/kg) at the basement of success establishing global brain ischemia model. The rats in saline group were injected intraperitoneally with 1ml physiologic saline. The experiment group and erythropoietin group were randomized into 5 group by the different reperfusion time.Briefly, Male Sprague Dawley rats were prepared for forebrain ischemia under 10% chloral hydrate (300mg/kg) injected intraperitoneally anesthesia. by electrocauterization of the bilateral vertebral arteries. The common carotid artery were exposed through a midline incision of the neck. and reversible clasps placed loosely around the common carotid arteries placement of atraumatic. Twenty-four hours later, the awake rats were restrained and the carotid clasps tightened to produce 4-vessel occlusion . The carotid clasps were removed after 15 min of 4- vessel occlusion The rats appeared hyperspasmia or semisideration in the process of operation were eliminated. Body temperature was maintained at 37℃ in the process of operation.Before freezing, the region of the dorsal hippocampus containing the CAl layer was carefully dissected from the remainder of the hippocampus.All of the animals were anesthetized with an intraperitoneal injection of chloral hydrate (300 mg/kg of body weight) according to different time point. Rats were perfused transcardially with 200 ml of saline, then 300 ml of 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4), and killed by decapitation. Brains were postfixed in 4% paraformaldehyde overnight and rinsed with 75%, 85%,95%, and 100% ethanol. The issues were transparented with xylene after air drying and embedded in paraffin. Paraffin-embedded serial coronal sections were cut at a thickness of 6 um and stained by HE technique and TUNEL technique. Tissue samples were fixed and embedded for electron microscopy analysis by a software, Image Pro Plus. Significance was evaluated using SPSS 11.5 software. P<0.05 was considered significant.ResultsHE staining: there were three to four layer of pyramidal neuron in the CAl region in hippocampus in compare group, lining up in order and tight, endochylema abundance, and with one or two clear nucleoli. In the experiment group, the numbers of neuron were different in the different time point. There were distinct difference between each time point.(p<0.05). The number of normal neurons were declining with the reperfusion time raising. In the EPO group, there were only few death neuron in the CAl zone. The number of normal neurons were more than the experiment group in every ischemia time point. There were distinct difference between the EPO group and experiment group (p<0.05).TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) staining: There were no TUNEL positive cells in the CAl zone of compare group. In the experiment group, there were significantly fewer TUNEL-positive cells. The number of TUNEL positive cells were elevating with the ischemia and reperfusion time. There were distinct difference among the 48h,72h, in the EPO group and compare group (p<0.05). In the EPO group, the number of TUNEL positive cells was raising obviously until 48h after reperfusion and reaching to the climax on the 72h. There were distinct difference between the EPO group and experiment group (p<0.05).Conclusions 1, In the four-vessel occlusion rats model ,with the time extending afterreperfusion, the ischemia neurons death gradually. 2% EPO could inhibit the neuron apoptosis induced by total cerebral ischemia and EPO could have neuroprotective effects to the neurons injured by hypoxia and ischemia.