Insulin Prevents Ischemia/reperfusion-induced Injury In Cortical Neuron Of Rat

Background and objectiveWith the aging of our population,the incidence of cerebral vascular diseases is increasing,and it shows a prominent trend of getting younger.Acute cerebral vascular disease has highly mortality and disability,it is one of the most harmfull diseases which places heavy burden to families and society.Thus,investigating its pathogenesis and looking for effective treatment constitute our most urgent problem. The lesions of acute ischemic stroke comprise ischemic area and ischemic penumbra around.The brain injury in the ischemic penumbra is reversible,it is important to recuperate the damaged nervous system through improving the blood supply of this region.Apoptosis is an important mechanism for brain ischemia reperfusion injury, and the apoptosis of brain cell in ischemic penumbra is one of the causes for the irreversible injury.Growth associated protein-43(GAP-43),a calcium-dependent calmodulin-binding protein on the nerve cell membrane,plays importmant role on nervous development,neurite regeneration,neurotransmitters release and the maintenance of synaptic function.It is a molecular marker of nerve regeneration and replasticity,Current thinking is that GAP-43 is correlated with the plasticity of central nervous system and nervous rehabilitation after brain ischemia injury,and it has been a hot point in recent years.It has been found GAP-43 is expressed in high level during the neuronal development and regeneration,and closely related to synaptic plasticity. GAP-43 regulate the axon elongation and change the cell morphology.As an intracellular signal,it also can greatly enhance the role of G-protein-coupled receptors transference.Brain-derived neurotrophic factor(BDNF),a member of the neurotrophic factor family,influences neuronal development in many aspects,such as promoting early neuronal proliferation and differentiation,maintaining the survival of mature neuron, regulating the microenvironment of neurons regeneratation,and enhancing the survival and growth of motoneurons,protecting them from damage and death.Thus BDNF has a wide range of clinical application in treatment of nervous system disease. It is induced at early stage of cerebral ischemia,and acts on every aspects of brain ischemia reperfusion injury,plays important role on brain protection.Insulin has a wide range of biological effects.It can promote the synthesis of glycogen,lipid and protein,induce gene transcription and cell growth,stimulate DNA synthesis and cell proliferation of osteoblasts,fibroblasts and so on.Insulin can be synthesized in peripheral blood and brain cells,and insulin,insulin-like growth factor (IGF) and their receptors also widely distribute in the brain.In this study,we used oxygen and glucose deprivation/reoxygenation(OGD/R) to simulate acute cerebral ischemia/reperfusion injury in in-vitro cultured neurons, detected the impact of insulin on apoptosis and expression of GAP-43 and BDNF,and further explored the relationship between insulin and nerve regeneration,synaptic plasticity and apoptosis.The work is usefull to understand the mechanism of insulin's neuroprotective effect,and provide new theoretical basis both for the clinical application of insulin and prophylaxis and treatment of ischemic cerebrovascular disease.Materials and methodsCortical neurons cultured for 7 days were randomly divided into A(normal control Group),B(OGD/R alone) and C(pretreatment with insulin) 3 groups.Group A always was cultured in the 37℃,95%O₂+5%CO₂ incubator after its medium replaced by sugar earle's.Group B was deal with OGD/R:put into constant temperature of 37℃hypoxia box,after the culture medium was replaced by earle's sugar-free,slowly filling 95%N₂ +5%CO₂(flow rate of 1 L/min) for 30 min, fully enclosing box,and leaving it alone in the culture incubator 1 h,then the restoration of serum-free medium.Group C:pretreated with the final concentration of 1MU insulin 1 h prior to OGD.Then observe the cell morphology under the inverted microscope;detect cell survival rate by MTT colorimetry,count the apoptosis neurons by TUNEL(TdT mediated dUTP nick end labeling),observe the expression of GAP-43,BDNF using immunocytochemical on each time point after sugar-reoxygenation(RO) 0h,1h,6h,12h,24h of each group.Results1.The results of cell morphology under the inverted microscope Group A: normal 7 d neuron is active,the cell body has refractive index and strong three-dimensional sense,and the neurite interlaced into nerve cell networks. Compared with group A,group B has poor shape,rounded cell body,Shorten or broken neurite after OGD/R,and the cell body became narrow or deformed is apoptosis neuron.With the time of RO prolonged,damaged and apoptotic cells increased.Group C:the damage of OGD/R cell was lighter,less apoptosis after In pretreated.2.The results of cell survival rate by MTT colorimetry Compared with group A, the cell survival rate of group B decreased obviously,and the differences were significant(P＜0.05) on each time point after RO;and later,survival rate was lower.The survival rate was increased in group C due to pretreatment of In; compared with group B,the differences were significant(P＜0.05) on 12h,24h after RO.3.The results of apoptosis neurons by TUNEL There are several apoptosis neurons in group A.Group B has much more apoptosis neurons from 6h after RO;while the quantity was less on 12h,24h after RO in group C due to pretreatment of In.4.The results of Immunocytochemical for GAP-43 The expression of GAP-43 in endochylema of normal neuron was low in group A.The expression in group B was higher than group A from 6h after RO,with the time of RO prolonged,the quantity increased;the differences was significant(P＜0.05) on 6h,12h,24h after RO,compared to group A.The expression of GAP-43 increased in Group C from 0h,and reach summit at 24h;compared to group A,B,the differences was significant(P＜0.05) on each time point after RO.5.The results of Immunocytochemical for BDNF The expression of BDNF in endochylema of normal neuron was all very low in group A.The expression in group B increased from lh after RO,with the time of RO prolonged,the quantity increased;the differences was significant(P＜0.05) compared to group A.The expression of BDNF increased obviously in Group C,the differences was significant(P＜0.05) on each time point after RO.Conclusions1.It can be observed that the survival rate of neurons decreased after RO,the apoptosis neurons increased,and the expression of GAP-43,BDNF was low.2.Insulin could relieve the damage caused by ischemia/reperfusion in cortical neuron of rat,and inhibit apoptosis.raise the expression of GAP-43,BDNF.3.Insulin may play a protective role by up-regulate the expression of GAP-43,BDNF.