Research About The Neuroprotective Effect Of Erythropoietin For Transient Cerebral Hypoxia Neonatal Rats

ObjectiveA neonatal rat model of anoxia was established to mimic the pathological process of hypoxic-ischemic encephalopathy(HIE). Erythropoietin(EPO) was injected intraperitoneally to rats that had undergone anoxia. The damage of white matter and cognitive function was assessed to investigate the neuroprotective role of EPO. The vascular density in the hippocampus was dectected, and the Western blot analysis was used to evaluate the expression of related proteins in VEGF/VEGFR2 signaling pathway to confirm the possible neuroprotective mechanism of EPO. Methods1. On postnatal day seven(P7), male SD(Sprague Dawley) rats were exposed to 100% N2 environment for 10 min to establish the model of HIE, as described by Nyakas. To assess the model above and the damage degree of the brain after the anoxia, general neurobehavioral manifestations between control group and anoxia group were observed. Meanwhile, damage of neurons in hippocampus was observed by Nissl staining and brain water content(BWC) was estimated 48 h and 72 h after the anoxia.2. According to the design of experiment, P7 rats were randomly divided into five groups: control, anoxia+vehicle, anoxia+rhEPO, anoxia+SU5416+rhEPO and anoxia+SU5416. The general development of each group was assessed(including eyelid opening time, ear erecting time, incisor exposing time and body weight). The white matter damage within the corpus callosum and vascular density of the hippocampus was examined by staining with Luxol Fast Blue(LFB) and fluorescein lycopersicon esculentum(Lectin) respectively. The cognitive function was assessed by the Morris Water Maze test(MWM). The expression of EPOR, VEGF, Raf1, and ERK1/2 was evaluated by Western blot analysis. Based on the above results, overall evaluations of rhEPO was made and the role of VEGF/VEGFR2 pathway was detected. Results1. During the anoxia, rats in anoxia group present brain anoxia and neurological dysfunction as restlessness, cyanosis, asphyxia and coma. Compared to control group, the BWC of the anoxia group was increased, the damage levels of neurons in hippocampus was more serious, and the learning and memory ability together with the spatial orientation ability was poorer.2. After the anoxia treatment, physical characteristics including eyelid opening, ear erecting and incisor exposing, did not manifest any significant differences among groups. Compared with the control group, body weight of anoxia groups did not show any significant differences before postnatal day 40(P40), while after P40, the body weight difference between control and anoxia groups became significant, anoxia groups had lower body weight than the control group.3. The LFB staining showed that rats with anoxia exhibited significant myelin loss, while the white matter damage of rhEPO group was less severe compared to other anoxia groups. The staining of Fluorescein lycopersicon esculentum(Lectin) indicated that vascular density in the hippocampus CA1 area of rhEPO group was higher than other anoxia groups. Rats that suffered from anoxia exhibited significantly poor performance in the MWM test compared to the control group, whereas the administration of rhEPO could improve the cognitive function of anoxia rats.4. The result of Western blot analysis shows that the expression of EPOR and VEGF were increased after the anoxia, and they were increased in a higher level after the administration of rhEPO. The expression of t-Raf1 and t-ERK1/2 kept stable, while p-Raf1 and p-ERK1/2 levels were significantly increased after the anoxia or rhEPO administration. While, compared to anoxia+rhEPO group, the level of p-Raf1 and p-ERK1/2 in anoxia+SU5416+rhEPO group was significantly reduced. ConclusionIt confirmed that the HIE model was successfully established by exposing newborn rats to 100% N2 condition. Intraperitoneally administration of high dose rhEPO(5000U/Kg) could ameliorate brain injure caused by anoxia that resulted in less damage level of white matter, better outcome of cognitive function and angiogenesis. The beneficial effects of rh EPO may be relevant to the activation of VEGF/VEGFR2 signaling pathway.