Endogenous BFGF In Brain Ischemia: Its Expression & Modulation And Exogenous BFGF: Its Optimal Administration Time Window & Its Signal Transduction

Purpose: basic fibroblast growth factor (bFGF) is a multifunctional cytokines, involved in the proliferation and differentiation during the development of central nervous system. It is beneficial to improve the survival of grafted neurons in transplantation. bFGF is also effective on protecting neurons from all kinds of insulting factors such as the excitotoxic damage of glutamate. Several studies have demonstrated that the intravenous administration of bFGF is effective on reducing the infract volume due to brain ischemia and promotes the recovery of nervous functions of several species of animals. But all the papers only described this phenomena, without exploring the mechanism involved in the effect of exogenous bFGF. The concentration of bFGF in central nervous system (CNS) may reach to 30-50μg/Kg. bFGF could be produced in a variety of cells in CNS, but mainly from astrocytes(As). Studies in vitro and in vivo have approved that under all kinds of insults, As could be activated and synthesizes more bFGF. But the following points still need elucidation : Are endogenous bFGF increased and to what extent in brain ischemia? Why the endogenous bFGF shows no protection against ischemia insult? And what are the influences of exogenous bFGF administrated on endogenous bFGF expression in brain ischemia? We would unshelter the protection mechanism of exogenous bFGF in brain ischemia by studying the points mentioned above.Egr-1 (egrly growth response protein 1), a protein with three zinc finger motifs, could increase the transcription activity through directly interaction with DNA. NAB2 could specially bind to the conserved R1 domain of Egr-1, thus inhibiting the transactivating protential of Egr-1. We intend to study if Egr-1 and NAB2 involved in regulating the expression of endogenous bFGF after brain ischemia, which remains unclear.bFGF is one of the heparin binding growth factors and heparan sulfhate proteoglycans (HSPGs) is considered to be its low affinity receptors. bFGF also has high affinity receptors, FGFR, which is widely expressed on many cells' surface. bFGF can bind to bFGFR to form a specific side by side heparin-induced bFGF dimer. The latter is required for dimerisation and activation of the FGF receptor tyrosine kinase and consequently for the subsequent initiation of biological responses. Some data have indicated that the function of bFGF is remarkably mediated by the mitogen-activated protein kinase(MAPK) pathway. The expression of Egr-1 could also be regulated by MAPK pathways. We would observe if MAPK pathway was involved in the actions of exogenous bFGF on anoxic cultured astrocytes and its influences on endogenous bFGF expression.BBB is compromised with ischemia, but the time window for administration of the exogenous material through the destroyed BBB is controversial. There is even no any special data regarding time window for exogenous bFGF. Our study was aimed to explore the dynamic expression of endogenous bFGF and its high affinity receptor, bFGFR, in rat after permanent middle cerebral artery occlusion (pMCAO), to study the protection mechanism of exogenous bFGF and its influence on the expression of endogenous bFGF with anoxic primary cultured astrocyte; also to test the time window, special for exogenous bFGF , of compromised BBB with ischemia in pMCAO rat, in order to provide theoretical basis for treatment of ischemic stroke with exogenous bFGF.Methods: The wistar rats were used to study the dynamic expression of Egr-1, endogenous bFGF, bFGFR from 0.5h to 14d after permanent middle cerebral artery occlusion (pMCAO); the interaction of the different cell types that expression the markers above, with immunohistochemistry, western-blot and RT-PCR techniques. The dynamic expression of Egr-1 and NAB2 from 10 minutes to 1 day, on ischemic-anoxic primary cultured astrocytes, was detected by western-blot and double immunofluorescence. Electrophoratic mobility shift assay (EMSA) was employed to evaluate the combining act...