Effect Of Erythropoietin On Hypoxia-inducible Factor-1αand Survivin Expression In Newborn Rats After Hypoxic-ischemic Brain Damage

Objective: Hypoxic-ischemic encephalopathy (HIE) is a main cause of newborn acute death andchronic nervous system damage. Study has indicated that erythropoietin (EPO) has a protectiveeffect on the hypoxic-ischemic brain tissue. This experiment by creating neonatal ratshypoxic-ischemic brain damage (HIBD) model, to observe the effect of EPO on HIF-1 andSurvivin expression after hypoxic-ischemic brain damage (HIBD) in neonatal rats, exploring itsantiapoptosis possible mechanisms, providing the theoretical basis for clinical therapy ofneonatal HIBD.Methods: 120 seven-day-old neonatal Wistar rats were randomly divided into 3 groups: shamoperated group (n=8), hypoxic-ischemic brain damage (HIBD) group (n=56) and rhEPO treatedgroup (n=56), the latter two groups were divided into seven subgroups (n=8 each) based ondifferent time points after HIBD (3h, 6h, 12h, 24h, 48h, 3d, 7d). In sham operated group, givenmedian neck incision and free left common carotid artery didn’t hypoxic-ischemic. In HIBDgroup free and ligate left common carotid artery, then given hypoxic 2h. In rhEPO treated group,immediately, 24h, 48h give a single intraperitoneal injection of rhEPO (5000IU/kg/d) afterhypoxic-ischemic brain damage. Observing the morphological changes in brain tissue after eachgroup of neonatal rats were decapitation and collecting the brain tissue at a corresponding timepoints. Observing the brain tissue paraffin sections under the HE dyed light microscope todetermine the pathological changes. Immunohistochemical technique was used to determine theexpression of HIF-1 and Survivin in brain tissues, and terminal deoxyribonucleotide transferasemeditated dUTP-biotin nick end labeling assay was applied to determine the apoptosis index(AI).Results: (1) Varying degrees of edema at different time points generally can be seen on the leftbrain tissue after HIBD, rhEPO treatment reduced certain extent of edema. (2) HE dyed: In shamgroup, brain structure and the cellular level are clear, neurons arranged closely, only see theindividual cell volume shrinkage, nucleus deeply stained. HIBD group at different time pointswith varying degrees of nerve cell edema, karyopyknosis, nuclear dissolution, fragmentation,reduction in the number of cells. RhEPO treated group compared to the HIBD group, the degreeof brain tissue injury was reduced. (3) Immunohistochemical staining results:①HIF-1 expression: HIF-1 positive stained was brown fine particle deposition, the positive cells were mainly expressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and(or) nuclei of neurons were seen, in the sham group showed a low level of expression; theexpression of HIF-1 in HIBD group increased at 3h after HIBD, reached the peak at 12h thengradually decreased (P＜0.01); in rhEPO treatment group, the expression of HIF-1 wassignificantly lower than HIBD group in 12h, 24h, 48h, 3d (P＜0.05).②Survivin expression:Survivin positive stained was brown fine particle deposition, the positive cells were mainlyexpressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and (or)nuclei of neurons were seen, Survivin expression in the sham group showed a low level; theexpression of Survivin in HIBD group was increased at 12h after HIBD, increased significantlyat 7d, and compared with the sham group, differences were statistically significant (P＜0.01)except HIBD 3h and 6h groups; in rhEPO treated group was significantly higher than HIBDgroup in 12h, 24h, 48h, 3d (P＜0.05). (4) Nerve cell apoptosis tendency: Cell apoptosis index insham group showed a low level, in HIBD group was increased at 6h after HIBD, increasedsignificantly at 7d, and compared with the sham group, differences were statistically significant(P＜0.01) except HIBD 3h group; cell apoptosis index in rhEPO treated group was significantlylower than HIBD group in 24h, 48h, 3d, 7d (P＜0.05).Conclusions: (1) It was conformed that brain histopathological damage after EPO treatment wasobviously lighter than HIBD group by observing HE dyeing results, and demonstrated that EPOtreatment can reduce the nerve damage caused by hypoxic-ischemic from pathology. (2) Brainhypoxic-ischemic can cause neuron apoptosis in cerebral cortex and the hippocampus, HIBDgroup of HIF-1 and Survivin expression compared with the sham-operated group was increased,indicating that HIF-1 and Survivin participate in the occurrence and development process ofHIBD of neonatal rat. (3) Overexpression of HIF-1 was inhibited after applying EPO treatment,while the expression of Survivin was increased, so as to delay and reduce apoptosis. This may beone of brain protection mechanisms of EPO.