| Objective: Neonatal hypoxic-ischemic brain damage (HIBD) is caused in most cases by perinatal asphyxia. It endangers the lives of newborns, also was a common cause of nervous system disability in childhood. In recent years, some studies showed erythropoietin (EPO) had some protective effect on hypoxic-ischemic brain damage. This experiment have prepared neonatal hypoxic-ischemic brain damage (HIBD) rats model. Through intraperitoneal injection of exogenous recombinant human erythropoietin, pathological changes of brain tissue in HIBD neonatal rats have been observed. And the impacts of rhEPO on expression of Bcl-2 and Bax proteins of brain tissues of HIBD neonatal rats have also been observed. To probe into the protective effect of rhEPO on HIBD neonatal rats and its possible molecular mechanism, our experiment may provide an effective way as clinical therapy for neonatal HIBD.Methods: (1) Preparation of neonatal HIBD rat model. (2) Detection of behavior ability of rats: To observe the behavior changes of each rat before experiment and after hypoxia. (3) Seven-day-old Wistar rats were randomly divided into three groups: Sham-operated group, HIBD group, and rhEPO therapy group,n=40 in each group. Then each group was randomly divided into five subgroups (n=8 in each subgroup) based on different time points at 6h, 12h, 24h, 48h, and 72h after HIBD. In sham-operated group, rats were performed operation by taking median neck incision and separated the left common carotid artery, and hypoxic-ischemic was not conducted. In HIBD group, the left common cartoid artery of rats were separated and ligated. Then they were put under hypoxic environment for 2h. In rhEPO therapy group, rats were instantly given single dose of recombinant human erythropoietin (5000IU/Kg) by intraperitoneal injection following the hypoxic-ischemic brain damage. All rats were decapitated at different time points and brain tissues were collected. Then general morphological change of the brain was observed. The pathological changes of brain tissue were observed under light microscope by HE stain. And the expressions of Bcl-2, Bax in cerebral cortex were detected by immunohistochemical method and image analyses were performed at different time points.Results: (1) Neonatal rats had showed various abnormal behaviors after HIBD.(2)After HIBD, general morphological changes of brain showed abnormal at different degrees.After HIBD,the brain tissue edema was apparent at HIBD12h, the brain tissue significant edema was apparent at HIBD 24-48h, the brain tissue necrosis was apparent at HIBD 72h, However, above-mentioned changes weakened after accepting rhEPO therapy. (3)HE stain and histological studies showed: In sham-operated group, brain tissues were in normal size and morphology. Brain tissue structure and cellular layer were clear, neurons were aligned closely. Only the volumes of individual cells were shrinked and nuclus of cells were deeply stained. In HIBD group, brain tissue edema was apparent at HIBD12h. After 24h of HIBD, neurons aligned in disorder. Cell gaps enlarged. After HIBD 72h, much degeneration and necrosis of neurons developed. In rhEPO therapy group, the large number of nerve cells survived, the degree of injury was decreased. Only a small number of nerve cells showed degeneration and necrosis. The majority of nerve cells in hippocampus area was normal in morphology, and showed normal pyramidal cells. (4) Immunohistochemical stain results:â‘ Expression of Bcl-2, Bcl-2 positive staining showed buffy fine grain deposition, Bcl-2 positive cells were found in cerebral cortex and hippocampus areas, and positive staining was mainly in cytoplasm. Bcl-2 protein in sham-operated group showed a low level of expression. There were no statistical significant changes at different time points(P>0.05). The expression of Bcl-2 at different time points in HIBD group rats was increased compared with that of sham-operated group. There was statistical significant difference between them (P<0.01). The expression of Bcl-2 at different time points in rhEPO therapy group was increased compared with that of HIBD group, there was statistical significant difference between them (P<0.01). Brown granules in cytoplasm of nerve cells increased and Bcl-2 protein expression increased significantly in rhEPO therapy group. The peak value was at the 24h in HIBD group and rhEPO therapy group.â‘¡Expression of Bax protein: Bax positive staining showed buffy fine grain deposition, Bax positive cells were found in cerebral cortex and hippocampus, and positive staining was mainly in cytoplasm. Bax in sham-operated group rats expressed weakly. The expression of Bax at each time point in HIBD group was increased compared with that of rhEPO therapy group, there was statistical significant difference between them (P<0.01). The expression of Bax at each time point in rhEPO therapy group was increased compared with that of sham-operated group, there was statistical significant difference between them (P<0.01). The peak value was at the 24h in HIBD group and rhEPO therapy group.â‘¢Changes of the ratio of Bcl-2 to Bax,at 24h points, the ratio was lower after HIBD, but it increased after rhEPO therapy.Conclusions: (1) This study confirmed that hypoxic-ischemic brain injury model have been successfully prepared. Through observing behavioral ability and histopathology of brain tissue of rats, it demonstrated rhEPO therapy can reduce hypoxic ischemic brain damage from general morphology and pathology brain.(2) rhEPO can up-regulate the expression of Bcl-2 and down-regulated the expression of Bax of brain tissues. It can up-regulate the ratio of Bcl-2/ Bax, alleviate pathological injuries in the course of HIBD. It may inhibit neuronal apoptosis and provide a protective effect for hypoxia-ischemia-induced neuronal injury of neonatal rats. Further, our conclusions may provide new path for clinical treatment of newborns with HIBD.
|
PDF Full Text Request