Effect Of Recombinant Human Erythropoietin On The Expression Of MBP And GluR4 In The Neonatal Rat Model Of Periventricular Leukomalacia

Objective: To investigate the neuroprotective effects of recombinant human erythropoietin(r EPO)on neonatal rat periventricular leukinalacia(PVL)model by detecting the expression of myelin basic proteinotein(MBP)and glutamate receptor 4(Glu R4)in periventricular white matter.Methods: 3-day-old neonatal SD rats were randomly divided into sham operation group,PVL model group and r EPO intervention group.PVL model was established by ligation of the left common carotid artery and fellow hypoxia after operation.In sham operation group,only left common carotid artery was separated neither ligation nor hypoxia.r EPO(5000U/kg)was injected intraperitoneally in r EPO intervention group after hypoxia,once every 24 h for consecutive 7 days.The sham operation group and PVL model group were injected intraperitoneally with the same capacity of normal saline.Brain tissue was collected at 3d,7d and 14 d after modeling.Hematoxylin-eosin staining(HE)and Luxolfastblue staining(LFB)were used to observe the pathological changes of brain tissues and myelin injury in the three groups at different time points,and Hoechst staining was used to observe the apoptosis of brain tissues.The expression of myelin basic proteinotein(MBP)and glutamate receptor 4(Glu R4)in the white region around ventricle were detected by immunohistochemical staining and image quantitative analysis at each time point after operation.Result:1.Pathological change of brain tissue of three groups under optical microscopeThe width of the ventricle in three groups:At 3d,7d and 14 d after operation,the width of the left ventricle was similar to right ventricle in the sham operation group;at3th day after surgery,the width of the left ventricle in the PVL model group and r EPO intervention group were no significant change compared with right ventricle;the left ventricle of the PVL model group and r EPO intervention group was significantly enlarged compared with the opposite side on days 7 and 14 after surgery,but the left ventricle of r EPO intervention group enlarged seriously than the PVL model group.Pathological change of brain tissue:At 3d,7d and 14 d after operation,no pathological changes were found in white matter around the lateral ventricles in the sham operation group,and the cells were arranged tightly and regularly with clear boundaries.Three day after operation,PVL model group had edema,disorder and loose structure of periventricular white matter cells,inflammatory cell infiltration,and round softening lesions of different sizes.On the 7th day after operation,the pathological change of white matter around the lateral ventricle in PVL model group were worse than before,coagulative necrosis and cystic changes were obvious.On day 14 after surgery,glial scar formation,cell necrosis in periventricular white matter area and decreased number of neurons were observed in PVL model group;compared with PVL model group,the injury degree of r EPO intervention group was significantly relieve,the degree of white matter looseness and cerebral softening focus were significantly improved,and the enlargement of left lateral ventricle was alleviated.2.LFB staining of myelin sheath of white matter in three groupsThree days after surgery,the myelin arrange was disorder and irregular,with myelin edema and vacuole formation myelin fiber uneven staining was observed in the PVL model group and r EPO intervention group.On the 7th day after surgery,the lesions of PVL model group and r EPO intervention group were most serious,including myelin sparseness and edema,numerous vacuoles,partial myelin fracture and loss of myelin fibers,and myelin staining was lighter than that of the 3rd day after surgery.On postoperative day 14,the nerve fibers in PVL model group were become regularly than those on postoperative day 7.The myelin injury in r EPO intervention group was mild compared with the PVL model group.Myelin sheath staining in sham operation group showed dense fibers and regular arrangement at different time points.3.Apoptosis staining results of neonatal rats in three groupsIn the sham operation group,the apoptotic cell was rarely at 3d,7d and 14 d after surgery;In the PVL model group and r EPO intervention group,the number of apoptotic cells in white matter area were increased from day3 to day7,at 14 th day,the number of apoptotic cells decreased compared with 7d after surgery.At the same time point,the number of apoptotic cells in the PVL model group was the most,the r EPO intervention group was second,the sham operation group was least.4.MBP and Glu R4 immunohistochemical staining4.1 The expression of MBP in periventricular white matterThere was almost no MBP expression in periventricular white matter of the three groups of newborn rats 3 d after operation.MBP positive expression was found in the periventricular white matter area of newborn rats in the three groups at 7 and 14 days after operation.at the same time point,the expression of MBP of the sham operation group was highest,r EPO intervention group was medium,and PVL model group was lowest,P<0.05;Compared with the express of MBP at the 7d and 14 d after operation,the express of MBP at 14 d was increased than 7d,there have a significantly difference in each groups,P < 0.05.4.2 The expression of GluR4 in periventricular white matter of three groupsGlu R4 was positively expressed in periventricular white matter area was observed in three groups.At 3d,7d and 14 d after operation,the expression of Glu R4 in the sham operation group was least,r EPO intervention group was medium,and PVL model group was maximum,P<0.05.The Glu R4 express in each groups showed a downward trend accompany with the age increased,the Glu R4 express was the highest on day 3,and then decreased on day 7,at 14 d after operation,the Glu R4 express was minimum,P<0.05.Conclusion:1.r EPO can relieve the pathological damage of periventricular white matter and myelin sheath injury and decreased the number of apoptotic cells in the neonatal rat model of periventricular leukomalacia.These results suggest that rEPO has neuroprotective effect on the neonatal rat model with PVL.2.rEPO can enhance the expression of MBP and decrease the express of GluR4 in periventricular white matter,r EPO may can relieve oligodendrocyte damage due to cerebral ischemia and cerebral hypoxia,relieve excitatory toxic damage to developing white matter in the neonatal rat model of periventricular leukomalacia,promote oligodendrocyte precursor differentiation and myelin sheath repair,its suggest r EPO was involved in the protection of neonatal PVL model through the pathophysiological mechanism mentioned above.