Study On Neuroprotective Effect Of Erythropoietin In Neonatal Rats With Hypoxic-Ischemic Brain Damage

Objectives:1. To explore the neuroprotective effect of application of exogenous recombinant human erythropoietin (rhEPO) in neonatal rats with hypoxic ischemic brain damage (HIBD), and to investigate the possibility of rhEPO treatment and the possible mechanisms.2. To compare the differences of neuroprotective effect and side effect between different administration and dosage, looking for the safer and easier treatment.Methods:1507-day-old Sprague-Dawley rats (SD rat) were randomly divided into 5 groups (n=30 in each group):①,sham operation group;②,EPO1 treatment group;③,EPO2 treatment group;④,HIBD1 group; and⑤,HIBD2 group. Left common carotid artery (CA) were ligated and cut off in the group②③④⑤, and then the models were keeped under hypoxia environment for 2 hours. In sham-operation group, the median neck incision was performed, and left common carotid artery was isolated, but not hypoxia-ischemia. In EPO1 treatment group, the model rats were instantly given single intraperitoneal injection of human gene recombination erythropoietin (rhEPO) 5000 IU/kg. In EPO2 treatment group, the model rats were instantly given intraperitoneal injection of rhEPO 1000 IU/kg for 5 straight days. Same dosage Physiological saline was given in HIBD 1 and 2 groups at same time by intraperitoneal injection.The pathological changes and the expression of the neuron specific enolase (NSE) were measured after HIBD modeling 48h,4d,8d and 18d by morphological method and immunohistochemistry method. The NSE positive cells were counted under microscope by using Image-Pro Plus 6.0. Brainstem auditory evoked potentials (BAEP) were recorded after HIBD modeling 7d,14d,21 d and 28d with an evoked potential recorder. Red blood cells were counted afte HIBD modeling 48h,8d and 18d m every group..Results:1. HIBD Modeling rats had abnormal behavior with various degrees such as twitch, respiratory disorders, disequilibrium and abnormal rotation. After modeling 2 days, edema and bleeding appeared at the left cerebral hemisphere in the rats of HIBD1 and HIBD2 group.After modeling 8 days, the left brain atrophied and the cord-like hollow structure disorder appeared; after modeling 18 days, the atrophy of left cerebral is more obviously in the same groups. There are similar changes in EPO1 and EPO2 group, but relatively much minor, the gross and tissue morphological changes of them are similar to normal rats.2. The total brain weight, the left brain weight of HIBD1 and HIBD2 groups were significantly lighter than the sham operation group (P<0.05) at every sampling point. The total brain weight, the left brain weight of EPO1 and EPO2 group were significantly heavier than the HIBD1, HIBD2 group (P<0.05). The percentage of brain damage in EPO1 and EPO2 group were smaller than group HIBD1 and HIBD2 (P<0.05), but the total brain weight, the left brain weight, the percentage of the brain damage had no stastical significance between EPO1 and EPO2 group.3. The expression of NSE was detected by immunohistochemistry method. After modeling 48h, NSE expressions were observed in intercellular spaces in the HIBD1 and HIBD2 groups. However, in the EPO1 and EPO2 group, NSE expressions were observed only in cytoplasm of neurons. After modeling 96h, NSE expression increased sharply in intercellular spaces in the HIBD 1 and 2 groups, but only a few NSE expressions were observed in intercellular spaces in the EPO1 and EPO2 groups. After modeling 8d, the NSE expression was rare in the HIBD1 and HIBD2 groups. The numbers of NSE-positive cells in the HIBD1 and HIBD2 groups were less than the sham operation group (P<0.05) at every sampling point. There were no significant differences of the NSE-positive cells in the EPO1, EPO2 group and the sham operation group at every sampling point.4. In 14-day-old rats, there were significant longer of peak latency (peak latency, PL) of wave I and wave III in the HIBD1 and HIBD2 groups than the sham operation group (P<0.05); In 21d,28d,35d, there were significant longer of PL of wave I to wave V in the HIBD1 and HIBD2 groups than the sham operation group (P<0.05); there were no significant difference (except V wave in 28,35 days rats and III wave in 21 days rats) of PL of BAEP in EPO treatment group and sham control group; there were significant longer of PL in the EPO2 group after 28 days (P<0.05); there were significant longer (P<0.05) of PL in EPO2 group compared EPO1 group after 35 days (except V wave). In 21d,28d,35d, there were significant longer of most inter-peaklatency (inter-peak latency, EPL) in the HIBD1 and HIBD2 groups than the sham operation group (P<0.05); there were no significant difference of IPL in the EPO1 group and sham operation group (P<0.05); there were significant longer (P<0.05) of IPL in EPO2 treatment group compared sham operation group after 28 and 35 days; there were significant longer (P<0.05) of IPL of waveⅢ-Ⅴand waveⅠ-Ⅴin EPO2 treatment group compared EPO1 group after 28 days.5. Red blood cell count of the EPO2 group was higher than other groups (P<0.01 or P<0.05) only in 25-day-old rats. There were no significant differences of red blood cell count in every group at other sampling point.Conclusions:1. In the early stage of HIBD, the administered immediately with EPO can play a neuroprotective role. It suggested that EPO could probably stabilize neuronal membrane to reducing neuronal necrosis and apoptosis;2. In the early stage of HIBD, the administered immediately and adequately with EPO can improve brain function, which is beneficial to the development of the brain;3. A single administration with adequate dosage of EPO treatment is superior to 5 sequential low-dose EPO treatment in brain function improvement of HIBD;4. There are no obvious side effects by using rational EPO therapy.