Effect Of Erythropoietin On Hypoxic-ischemic Brain Damage In Neonatal Rat And The Mechanism

Backgrounds and aimsHypoxic-ischemic brain damage can result from perinatal asphyxia and is an importantcause of neonatal mortality and subsequent sequelae. Due to the complicated pathogenesis, theassociated clinical therapeutics is always disputable. EPO is a hematopoietic cytokine which hasrecently been found in the nervous system, recent studies suggest that EPO has neuroprotectiveeffects. The aim of this study was to investigate the pathological change of hypoxic-ischemicneonatal brain in rats and evaluate whether EPO treatment, as a novel approach, would reducethe brain damage against hypoxic-ischemie damage.Methods(1)7-day-old Sprague-Dawley rats were randomly divided into 3 groups: sham-operatedgroup, HIBD group and HIBD+EPO group. The last two groups underwent left carotid arteryligation followed by hypoxia (8ï¼…O₂) for 2h on day 7 and giving either vehicle or EPO (1000U/kg i.p. qd×3).(2)Water content of the brain, changes of brain histopathology, and bcl-2 proteinexpression in the cortex and hippocampi were observed 3 days after hypoxia-ischemia.(3)At 6h,24h,72h,120h after hypoxia-ischemia, Intracellular free Ca²⁺ was measured byfluorospectrophotometry with fluorescent indicator Fura-2/AM; the malondialdehyde (MDA),the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) weretested.(4)Long-term behavioral outcomes was assessed by Morris water-maze(place navigationtest and spatial probe test)after 4 weeks, rats were killed 6 days later, the extent of atrophy of thehypoxic-ischemic hemisphere and changes of brain histopathology were observed.Results1) Representative brain edema, pathological lesions early 3 days after hypoxic-ischemicby specimen weight and histological section, suggest the HIBD model was successfullyprepared. Erythropoietin treatment significantly mitigated brain edema, pathologicallesions early 3 days after hypoxic-ischemic.2) 3 days after hypoxic-ischemic, the expression of bcl-2 protein in cortex andhippocampi was showed by immunohistochemistry: HIBD+EPO group＞HIBD group＞sham-operated group, comparison of the three groups is with significant difference. This result certified that the brain damages of hypoxic-ischemic encephalopathy hadclose relationship with cell apoptosis, and up-regulating bcl-2 expression mightcontribute to the EPO protective mechanism.3) The spectrophotography showed that the content of MDA increased significantly andthe activity of SOD and GSH-PX decreased markedly in brain tissue of HIBD rats ascompared with sham-operated group. The result showed that hypoxia-ischemia induceslosing balance of the oxidation - oxidation prevention system so that oxygen freeradicals generated excessively and brain damages happened. MDA decreased theactivity of SOD and GSH-PX enhanced significantly after EPO treatment, suggest thatEPO may repress production of oxygen free radicals, and enhance activity ofantioxidant enzyme.4) The fluorospectrophotometry with fluorescent indicator Fura-2/AM showed thatintracellular free Ca²⁺ level of isolated brain cells from neonatal rats increasedsignificantly after hypoxic-ischemic and can be brought down by EPO treatment,suggested that intracellular free Ca²⁺ overload may participate in the hypoxic-ischemicpathogenesis process, and EPO might lessen the free Ca²⁺ overload injury.5) Morris water-maze(place navigation test) showed that 5 days total escape latency were:HIBD group＞HIBD+EPO group＞sham operation group, comparison of the threegroups at different time were with significance; Spatial probe test showed that bothcomparison of HIBD rats with HIBD+EPO group and sham operation group weresignificant for the swimming time and distance in platform surrounding area 1, butthere is no difference between HIBD+EPO group and sham operation group. The resultdemonstrated HIBD rats had learning and memory function damage, and early EPOintervention could improve long-term neurobehavioral achievements.6) Specimen and histopathological evaluation discovered that HIBD rats probably hadbrain cavities, cerebromalacia and cerebral atrophy 34 days after hypoxic-ischemic,successfully prepared HIBD model was confirmed. And EPO treatment mightsignificantly decrease the degree of brain atrophy, diminish the spared hippocampalCA1 neurons deletion, and the cavity formation, demonstrated that EPO alsosignificantly attenuated neonatal hypoxic-ischemic long-term pathological change. Conclusion1) Exogenous EPO treatment significantly attenuated neonatal hypoxic-ischemic early andlong-term pathological lesions, palliated the hypoxic-ischemic pathogenesis process,demonstrated that Erythropoietin is neuroprotective against hypoxic-ischemic braininjury in neonatal rat.2) Suppressing free intracellular Ca²⁺ overload, lessening production of oxygen freeradicals, enhancing activity of antioxidant enzyme, and up-regulating bcl-2 expressionand reducing number of apoptosis cell might contribute to the exogenous EPOprotective mechanism in neonatal hypoxic-ischemic pathogenesis.3) Exogenous EPO intervention could improve the hippocampal learning and memoryfunction, suggested that early EPO intervention could improve long-termneurobehavioral achievements.