Effect Of Exogenous Erythropoietin On Caspase-9 And Caspase-3 Expression In Newborn Rats After Hypoxia-ischemia Brain Damage

Objective: Neonatal Hypoxic-Ischemic Brain Damage (HIBD) is caused by perinatal asphyxia often life-threatening newborns, it is also a common cause of children nervous system damage. Recent study has found that EPO (erythropoietin, EPO) has a protective effect on the hypoxic-ischemic injured brain tissue. This experiment by creating neonatal rats hypoxic-ischemic brain damage (HIBD) model. Intraperitoneal injection of exogenous recombinant human erythropoietin to observe the pathological change of brain tissue in HIBD neonatal rats and the effects of exogenous erythropoietin (EPO) on expression of Caspase-9 and Caspase-3,so as to explore the possible mechanism of the neuroprotective effect of EPO in HIBD neonatal rats, our experiment may provide an effective treatment for clinical therapy of neonatal HIBD.Methods: (1) Preparation of neonatal rat HIBD model. (2)A total of 120 seven-day-old neonatal rats were randomly divided into three groups: sham-operated group, HIBD group and rhEPO-treated group. Each group was further divided into five sub-groups (n=8 each) based on different time points after HIBD (6h, 12h, 24h, 48h, 72h) respectively. In sham-operated group, given median neck incision and free left common carotid artery didn't hypoxic-ischemic. In HIBD group free and ligate left common carotid artery, then given hypoxic 2h. In rhEPO-treated group, immediately give a single intraperitoneal injection of rhEPO(5000U/kg/d) after hypoxic-ischemic brain damage. Observed the morphological changes in brain tissue after each group of neonatal rats were decapitation and collected the brain tissue at a corresponding time points. Observed the left side of brain tissue paraffin sections under the HE dyed light microscope to determine the pathological changes. Immunohistochemical technique was used to determine the expression of Caspase-9 and Caspase-3 in brain tissues.Results: (1) Varying degrees of edema generally can be seen on the left brain tissue after HIBD, rhEPO treatment reduced certain extent of edema. (2)HE dyed: In sham-group, brain structure and the cellular level are clear, neurons arranged closely, only see the individual cell volume shrinkage, nucleus deeply stained. HIBD group at different time points with varying degrees of nerve cell swelling, degeneration, necrosis, nuclear fragmentation, dissolution, reduction in the number of neurons, neuroglial cell proliferation. rhEPO treatment group compared to the HIBD group, the degree of injury was reduced, most of nerve cell survived, cell arrangement still in rules, only see a small amount of nerve cell degeneration and necrosis. (3)Immunohistochemical staining results:①Caspase-9 expression: Caspase-9 positive stained was brown fine particle deposition, the positive cells are widely expressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and nuclei of neurons were seen, in the sham group showed a low level of expression, no significant changes at each time point; The expression of Caspase-9 in HIBD group increased at 6h after HIBD, 12h gradually increased, 24h～72h maintain the peak level(P<0.05); in rhEPO-treatment group at each time points Caspase-9 positive stained particle compared to the HIBD group were shallow, numbers were reduced, average gray value higher than HIBD group, the difference was significant (all p<0.01).②Caspase-3 expression: Caspase-3 positive staining showed brown fine particle deposition, the positive cells are widely expressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and nuclei of neurons were seen, Caspase-3 Expression in the sham group at the low level; The expression of Caspase-3 in HIBD group increased at 6h after HIBD also, 12h gradually increased, the peak reached on 24h～48h and 72h gradually decreased(P<0.05); in rhEPO-treatment group at each time points Caspase-3 positive stained particle compared to the HIBD group were shallow, numbers were reduced, average gray value higher than HIBD group, the difference was significant (all p<0.01).Conclusion: (1)Through the general concept and pathological observation of the HIBD neonatal rat brain tissue, confirmed that HIBD model is established successfully, HIBD group of Caspase-9 and Caspase-3 expression compared with the sham-operated group was increased, indicating that Caspase-9 and Caspase-3 participate in the occurrence and development process of HIBD of neonatal rat. (2) rhEPO reduce the expression of Caspase-9 and Caspase-3, which can protect the HIBD brain tissue, for clinical treatment of neonatal hypoxic-ischemic brain injury provide a new way.