Empirical Study Of Preconditioning Theory On Neuro-protective

BACKGROUDStroke is associated with high morbidity and mortality.Its prevalence is continuously increasing worldwide. Acute cerebrovascular disease is one of the first three top causes of the death and the first cause of disability in the world.Among the total,the incidence of cerebral infarction is coming the. first,with percentage of 80%. Cerebral infarction causes death and disability, which bring albatross to their family and the society.Hypoxia can lead to various kinds of reactions in animal or human bodies to compensate for hypoxic influence. But the extent to which animal or human bodies fight against hypoxia,namely tolerate hypoxia is limited.Brain is the most vulnerable organ in animal and human bodies and the main limiting factor influencing tolerance of the bodies to hypoxia. In the central nerve system, injuries induced by hypoxia/ischemia affect the health of human beings seriously. However,in recent years, investigations have showed that the tolerance of the bodies to hypoxia can be enhanced through such measures as hypoxic/ischemic preconditioning(HPC) and pharmacological preconditioning(PPC). Both HPC and PPC are category of precondition theory.Researches have indicated that HPC and PPC could improve the hypoxic tolerance of animals and in vitro cells.The problem is how to apply it to clinical application.The invention of cardia-cerebral vessels health care meter(Pat-number:200710176701) is the first step of clinical application.On the base of our prophase work,in this experimental we will investigate the effects and mechanisms of HPC and PPC on cultured hippocampal neurons.OBJECTIVESFirstly, to investigate the effect and imaginabale mechanisms of cerebrospinal fluid, brain homogenate and blood plasma of hypoxic-preconditioned Wistar rat on apoptosis of hippocampal neurons. Secondly, to study the effect and possible mechanisms of adenosine preconditioning on apoptosis of hippocampal neurons.METHODS1.Adult healthy Witar rats were exposed to hypoxia cabin for 3 hours to copy the hypoxic-preconditioning model, taking the cerebrospinal fluid, brain tissue and blood, dealing them to obtain aseptic cerebrospinal fluid, brain homogenate extract and blood plasma, to determine the SOD and MDA content of them.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, normal cerebrospinal fluid/brain homogenate extrat/blood plasma and oxygen-glucose deficiency group,hypoxic- recondition-ned cerebrospinal fluid/brain homogenate extract/blood plasma and oxygen-glucose deficiency group. Neurons apoptosis was induced by oxygen-glucose deficiency Earle's solution. Apoptosis was studied by using method of AnnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofluorescence staining.2.Cultured hippocampal neurons randomized into normal control group, oxygen-glucose deficiency group, low-does adenosine and oxygen-glucose deficiency group, middle-does adenosine and oxygen- glucose deficiency group, high-does adenosine and oxygen-glucose deficiency group, Apoptosis was studied by using method of AnexinV/PI double staining. The expression of apoptosis related genes bcl-2 and bax were analized by quantitative immunofl-uorescence staining.RESULTS1.The content of SOD in hypoxic-preconditioned cerebrospinal fluid was increased (P<0.01)and content of MDA was decreased(P<0.05)compard with normal cerebrospinal fluid; The content of SOD in hypoxic-preconditioned brain homogenate was increased(P<0.01)and content of MDA was decreased(P< 0.01)compard with normal brain homogenate; The content of SOD in hypoxic-preconditioned blood plasma was increased(P<0.01)and content of MDA was decreased(P<0.01)compard with normal blood plasma.2. AnnexinV/PI double staining results showed that apoptotic cells were observed by chance in N group,and a devil of apoptotic cells in OGD group.Both normal and hypoxic-preconditioned CSF could protected cultured hippocampal neurons from oxygen- glucose deficiency(P<0.01),the later one has stronger effects(P<0.01);Normal brain homogenate could protect neurons weakly(P< 0.05),and hypoxic- preconditioned brain homogenate defended neurons obviously(P<0.01); Both normal and hypoxic-preconditioned blood plasma could protected oxygen- glucose deficiency neurons(P<0.01),the later one has greater conservation(P<0.01).3. The expression of apoptosis related genes Bcl-2 and Bax, which were analized by quantitative immunofluorescence, were both discovered seldomly in N group.Bcl-2 was up-regulated in groups,which cultured by normal CSF(P< 0.01), brain homogenate(P<0.05)and blood plasma(P<0.05), compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.01)and blood plasma(P<0.01),compared with normal CSF/brain homogenate/ blood plasma group.At the same time, Bax was down-regulated in groups,which cultured by normal CSF(P<0.01), brain homogenate(P<0.01)compared with OGD group,and hypoxic-preconditioned CSF(P<0.01), brain homogenate(P< 0.05)and blood plasma(P<0.01), compared with normal CSF/brain homogenate/ blood plasma group. There were no significant difference on the expression of Bax between normal blood plasma and OGD group and OGD only group.4.Adenosine preconditioning detectection resulted that low-does adenosine preconditioning had no conservation on OGD hippocampal neurons.Middle-does adenosine preconditioning could protecte neurons obviously, increasing the express of Bcl-2 protein and decreasing the express of Bax protein compared with OGD group.high-does adenosine preconditioning could protecte neurons and advancing the rate of Bcl-2/Bax more respectively compared with Middle-does adenosine preconditioning and OGD group.CONCLUSIONS1.hypoxic-preconditioning increase the activity of SOD and decrease the content of MDA in cerebrospinal fluid, brain homogenate and blood plasma.2.hypoxic-preconditioned CSF, brain homogenate and blood plasma could protected hippocampal neurons from OGD injury, degrade the rate of apoptosis, up-regulate the express of Bcl-2 and down-regulate the expression of Bax.3.Middle-does and high-does adenosine could enhance the hypoxic tolerance of hippocampal neurons,and advancing of the rate of Bcl-2/Bax played an important role in inhibiting apoptosis.