Hypoxic-ischemic Brain Injury On The Pineal Gland Function And Exogenous Neuroprotective Effect Of Melatonin

Partâ… Revising HIBD rat model and check upObjectiveIn this study,we improve Rice's HIBD model of neonatal rat to construct Stable and reliable neonatal rat model with moderate and severe hypoxic-ischemic brain damage. Various methods were applied to identify whether the model is successful.We reduced the mortality of experimental rat model with traditional method,provided reliable experimental rat model of HIBD for futher research.Method7-day-old neonatal SD rats were divided randomly into three groups,improved HIBD model(G group),traditional Rice model(C group) and control group(S group),samples were obtained at the time of 6 hours,48 hours,72 hours after the model were contributed. Behavioral changes of rat model,HE stain,Immunohistochemistry and apoptosis of brain anatomy specimens were employed to identify whether the model is successful.Result1.G group had more behavioral abnormalities include systemic or right side libms spasm and left-side rolling than C group.2.Left-side hemisphere of G group had severe edema,pallor and necrosis.That of the C group only had mild edema.3.The NSE staining cells of G group at the left cortex decreased at 6 hrs,48 hrs the lowest,then return to the normal at 72 hrs.At the same time,the apoptosis of that was obvious at 48 hrs and still lower at 72,which was different from C group significantly(p ＜0.01).4.No death in G group,but it had 4 dead during anaesthesia,another 8 dead of hypoxia.(p＜0.01)ConclusionThe improved Rice's HIBD model of neonatal rat have the merit of low mortality and high success rate,and were identified with various methods.On this basis,we constructed moderate and severe hypoxic-ischemic brain damage animal model successfully.Partâ…¡The effect on pineal function in HIBD rat modelObjectiveThis study by observing the expression of Melatonin(MLT) synthetic enzyme,aromatic alkyl amine-N-acetyltransferase(AANAT),Hydroxyindole-O-methyltransferase (HIOMT) message RNA(mRNA) in pineal gland,the relationship of induce Nitric Oxide Synthase(iNOS) with apoptosis to understand the relationship between pineal function and HIBD;Radioimmunoassay to evaluate the level of peripheral blood MLT.MethodSPF-class 7-day-old neonatal Sprague-Dawley(SD) rats were divided randomly into 10 groups,6 every group,improved HIBD model T₁:6 hours after HIBD,T₂:12 hours after HIBD,T₃:24 hours after HIBD,T₄:48 hours after HIBD,T₅:72 hours after HIBD, corresponding sham-operated group:S₁,S₂,S₃.S₄,S₅.Electron microscopic observing morphological changes of pineal gland,radioimmunoassay to evaluate the level of peripheral blood MLT.Result1.The level of AANATmRNA in pineal gland:at the point of 12 hours,24hours,48 hours after HIBD,the level of AANATmRNA were lower then sham-operated group; Vertical compare the level of AANATmRNA between sham-operated group,there has no significant change,at the point of 12 hours after HIBD,it started to descend,24 hours up to a low peak,48 hours picked-up,72 hours recovered.2.The level of HIOMTmRNA in pineal gland:compared with sham-operated group, the level of HIOMTmRNA after HIBD has no significant change,Vertical compare the level of HIOMTmRNA,not only sham-operated group,but also HIBD group has no manifest alteration.3.iNOS:6 hours and 48 hours after neonatal rat brain damage,pineal gland content of iNOS heightened obviously.4.Apoptosis:at the early period of brain damage,the pineal apoptosis significantly increased,especially the 6 hours and 48 hours.5.Electron microscopic observing:after brain damage,obviouse change of pineal morphology was also at the time of 6 hours and 48 hours,they are significant swelling of the mitochondria,extremely rough endoplasmic reticulum expansion,cell degeneration.6.The level of peripheral blood MLT:compared with sham-operated group,the level of MLT drawdowned after HIBD 24 and 48 hours.Vertical compare the level of MLT of sham group,no significant changes were observed.24 hours after HIBD,it decreased,and was equal to 48 hours,recoverd to normal 72 hours.Conclusion1.HIBD can induce the gene expression of key enzyme of MLT synthesis(AANAT) to decrease slightly 12 hours after HIBD,24 hours up to a low peak,48 hours elevated,72 hours recovered to the virgin quantity.The gene of HIOMT that is the last enzyme of MLT synthesis expression had no change in pineal gland after HIBD.2.HIBD can induce peripheral blood MLT density degraded,start to decrease at 12 hours,lasting to 48 hours,recoverd at 72 hours.Cut down the protective effect of endogenous of MLT,and it may be involved in the pathogenesis of early HIBD.3.In the early period of HIBD,pineal function brokendown,down regulated expression of AANAT gene is the "engine" of it.4.After brain damaged,the expression of iNOS of pineal gland cell improved at 6 hours and 48 hours,then fall-off,72 hours restored basically.With the method of TUNEL,we detected the apoptosis in situ,its change in sync with iNOS.iNOS expression can promote the role of apoptosis,iNOS expression and apoptosis involved in the pineal gland cells morphological changes in the hypoxic-ischemic brain injury of neonatal rat.Partâ…¢Neuroprotective effects of melatonin against hypoxic-ischemic brain damage in neonatal rats.ObjectiveTo study the neuroprotective effects of melatonin against hypoxic-ischemic brain damage in neonatal rats and the medication time of melatonin.MethodsPostnatal 7-day-old Sprague-Dawley rats were randomly divided into six groups: sham operation group,model group(HIBD group),melatonin treated groups(according to the administration time divided into before HIBD group,after HIBD 0 hour group,after HIBD1 hour group and after HIBD 2 hours group).The brains were obtained in different times after HIBD,hematoxylin-eosine staining was used to observe the tissues pathological changes,terminal deoxynucleotidyl transferase mediated dUDP-biotin nick end labeling was used to detect the apoptosis of neurons in cortex and hippocampus, immunohistochemistry was used to detect the expression of neurone specific enolase, Blood was obtained by heart puncture,the serum was detected by enzyme-linked immunosorbentassay for neurone specific enolase.ResultsThe neuronal apoptosis in cortex and hippocampus appeared at 6 hours after HIBD, peaked at 24 hours,and then decreased gradually,There was a remarkable lower quantity in melatonin treated groups compared with HIBD group at 6h,12h,24h,48h and 72h(p＜0.01)though there were differences among melatonin treated groups.The changes of neurone specific enolase in brain tissue and serum have significant correlation(r=-0.92), they appeared at 6 hours after HIBD,peaked at 24 hours,and then decreased gradually, The activity of neurone specific enolase in melatonin treated groups has a significant difference in statistics compared with HIBD group at 6h,12h,24h,48h and 72h(p＜0.01) though there were differences among melatonin treated groups.There weren't remarkable differences in the changes of cell apoptosis and neurone specific enolase among before HIBD group,after HIBD 0 hour group and after HIBD1 hour group(p＞0.05),but there were significant differences between theirs and after HIBD2 hours group(p＜0.01).Conclusion1.Melatonin has protective and therapeutical effects against hypoxic-ischemic brain damage in neonatal rats.2.The changes of NSE in brain tissue and serum have better correlation,The changes of NSE in serum can demonstrate the severity of brain damage as well.3.There has a time limited in using melatonin,preventive medication and medication within 2 hours after HIBD could obtain good effects,and medication within 1 hour group is better than 2 hours after HIBD group.