The Effect Of BFGF On The Expression Of CREB In Rat Hippocampus And Parietal Cortex During Focal Cerebral Ischemia/reperfusion

Objective:Ischemic cerebrovaseular disease is a kind of severe life-threatening neurological disease. Along with the elevationof standard of living and aging of the global population,cerebrovasculardisease,heart disease and malignancy are the three deadly pathogeny inhuman being,therefore,the rate of incidence, disability,mortality andrelapse rate of cerebrovaseular disease are high altogether. Inrecent year,the epidemiologic data indicates that the rate of incidence andmortality of cerebrovaseular disease is higher than that of heart disease. Cerebrovascular disease not only effect the health of sufferer,but also effect the quality of living and subsistence,which bring heavy mental andeconomic burden to the family and society. The credibility and stabilityof experimental result is directly affected by the ideal animal model whichis close to cerebral ischemia, and it seriously threatens the health and life of human. It not only torments patients themselves, but also brings the soeiety and the patients' families heavy burdens. It has been a very pressing issue to resolve in current neuroscience field. A number of experiments have showed that enchancing neuroprotective effect,rescuing the neurons in penumbra area of infarction and vulnerable area to ischemia,and reducing the death of ischemic neurons in the aeute stage of cerebral ischemia are very erueial measurements to the recovery of cerebral ischemia. Fibroblast growth factor receptor (bFGF) can promote the survival and regeneration of neuronal cell,and it has protective and therapeutical effects on cerebral ischemia injury. Exogenous bFGF can exertneuroprotection. While the molecular mechanism of bFGF to ischemic neuron remains somewhat unclear. Cyclic-AMP response element binding protein(CREB) is a key modulator in many signal transduction pathways. CREB is also an intracellular signal molecule. It has become a research focus in neuroscience field that how CREB protects the neural cell. Whether CREB involves the protective mechanism of bFGF to ischemic neuron remains unclear.Methods: 1. Animals: 60 healthy Wistar rats weighing 230-270 grams (supplied by Experimental Animal Center of Shenyang Medical College) were randomly divided into 3 groups:(l)sham operated (Sham)group (n=12);(2)focal cerebral ischemia/reperfusion (I/R) group(n=24); (3)bFGF administration (bFGF) group(n=24).2.Middle cerebral artery occlusion:Focal cerebral ischemia/reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. Longa criterion was used to grade the behavior of the experimental animals when the animals recovered from anesthetization. The rats whose grade was 2,3 were employed in this experiment. Reperfusion was induced by carefully withdrawing the suture after 120 min of middle cerebral occlusion. In I/R group, 1 ml 0.9% saline was infused into right common cervical artery through the microsyringe pump within 30min. 1 ml of bFGF (500IU/ml; 2000IU/kg),was injected into right common cervical artery through the microsyringe pump within 30 min in bFGF group respectively .The velocity of injection remained 2ml/h.3.Preparing the histological slices:The rats from above 3 groups at 48h reperfusion timepoints were anesthetized with intraperitoncal injection of 4% paraformaldehyde(40mg/kg). The animals were perfused transcardially with 250 ml saline followed by 4% paraformaldehyde in PBS(0.1M, PH7.4). The brains were removed and postfixed. Coronal frozen brain slices were cut and stored.4.Hematoxylin-Eosin(HE) staining was employed to detect the histologicchanges in different groups. The frozen brain sliecs from different groups at 48h reperfusion were stained with HE solution, and the histopathologic changesof hippocampuses and parietal cortex region were observed under themicroscope.5. TUNEL staining was used to count the number of neurons in righthippocampal CA1 and parietal cortex regions.6.Detecting the expressions of CREB in sections by using immunohisto-chemical technique.7.The above sections were quantified with image analysis technology and thedata were statistically analysed. The average optical density values(OD) ofpositive products of CREB in hippocampal CA1 and parietal cortex regions ineach group were measured by image analysis system. Statistical analysis wascondueted by post hoc analysis with SPSS10.0 software. Significance wasregarded as P<0.05.Results: 1.Histopathological observation and neurons counting:In Sham group,the morphology of nervous cells in hippocampal CAI and parietal cortexregions were normal,the structure were entire,the arrange of cells were tidyand clear,the boundary were clear,the cytoplasm were light pink and thenucleus were blue,the contour of nucleus were clear which presents oval orround. There were some ischemic changes and the normal neurons reduced inright hippocampal CAI and parietal cortex regions in isehemla/reperfusiongroup with 48 h reperfusion. The normal neurons increased, and themorphology of neuron were improved in bFGF group compared to I/R group(P<0.05).2.Immunohistochemical analysis for CREB:Neurons throughout the ipsilateralhippocampal CAI and parietal cortex regions showed high levels of CREBimmunoreactivity in sham group. The OD of CREB positive products inipsilateral hippocampal CAI and parietal cortex regions decreased in I/R groupcompared to sham group.The expression of CREB increased in bFGF groupcompared to I/R group (P<0.05).Conclusion: 1. BFGF decreased significantly the number of apoptotic cells inrat hippocampal CA1 and parietal cortex during focal cerebralischemia/reperfusion, and ameliorate the morphology of ischemic neurons inischemic neuronal protection role.2. bFGF can significantly upregulated the expression of CREB in theipsilateral hippocampal CA1 and parietal cortex during focal cerebralischemia/reperfusion and CREB may be the cross point of signal transductionpathways of bFGF to ischemic neurons. CREB may be involved in theprotective mechanism of bFGF to ischemic neurons.