The Effects Of NGF And CGRP On The Expressions Of CREB And Tau Phosphorylation In Rats Hippocampuses During Focal Cerebral Ischemia/reperfusion Injury

Ischemic cerebrovascular disease is a kind of severe life-threatening neurological disease. Ischemic stroke accounts for the most of ischemic cerebrovascular disease, and it seriously threatens the health and life of human. It not only torments patients themselves,but also brings the society 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 acute stage of cerebral ischemia are very crucial measurements to the recovery of cerebral ischemia. Nerve growth factor (NGF) can promote the survival and regeneration of neuronal cell,and it has protective and therapeutical effects on cerebral ischemia injury. Calcitonin gene-related peptide (CGRP) is an effective peptide to relax vessel, and it has powerful dilation to vessel and immunomodulation roles in cerebral blood circulation. Exogenous CGRP can markedly increase cerebral blood flow,reduce the volume of infarction and exert neuroprotection. While the molecular mechanism of NGF and CGRP 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. Phosphorylated CREB mediates a lot of genes expression. It has become a research focus in neuroscience field that how CREB protects the neural cell. Whether CREB involves the protective mechanism of NGF and CGRP to ischemic neuron remains unclear.Tau proteins are mainly expressed in neurons where they play major regulatory roles in the organization and integrity of the cytoskeleton network. They promote microtubule stability and assembly, maintain their structural integrity,and promote axonal transport. Their cellular functions are mainly influenced by phosphorylation. Abnormally hyperphosphorylated tau loses its biological function,which may aggregate into helical filament,and form neurofibrillary tangles—the characteristically pathological change in Alzheimer's disease. Transient cerebral ischemia induces tau hyperphosphorylation in a site-specific manner. Epidemic investigation showed that the prevalence of dementia in ischemic stroke patients is nine times higher than controls at 4 years after a lacunar infarct. These indicate that tau proteins involve the pathophysiologic mechanism in cerebral ischemia. Therefore the intervention measurement on tau protein may be the therapertic target to cerebral ischemia. Whether the protective effect of NGF and CGRP to ischemic neurons are conducted by intervention on tau hyperphosphorylation should be further explored.Methods1 . Animals146 healthy male Wistar rats weighing 250-300 grams (supplied by Experimental Animal Center of China Medical University) were randomly divided into 5 groups:(1)sham operated (Sham)group (n=31); (2) focal cerebral ischemia/ reperfusion (I/R) group(n=31); (3)NGF administration group (n=28); (4)CGRP administration group (n=28); (5) NGF& CGRP group (n=28).The above 5 groups were subdivided into reperfusion 3 h, 12 h, 24 h, 48 h and 72h groups after 2 hours'occlusion of right middle cerebral artery.2. Middle cerebral artery occlusionFocal 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 1,2 or 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 NGF(1000U/kg), CGRP(3μg/kg), NGF and CGRP(NGF,1000 U/kg;CGRP3μg/kg) was injected into right common cervical artery through the microsyringe pump within 30min in NGF group,CGRP group,and NGF&CGRP group respectively. The velocity of injection remained 2 ml/h.3. TTC staining3 rats taken from Sham group and ischemia/reperfusion group at reperfusion 24 h timepoint respectively were randomly decapitated and the coronal brain slices were stained with TTC solution. The samples were postfixed and taken pictures.4.Preparing the histological slicesThe rats from above 5 groups at different reperfusion timepoints were anesthetized with intraperitoneal injection of 10% chloral hydrate(300 mg/kg).The animals were perfused transcardially with 100 ml saline followed by 4% paraformaldehyde in PBS(0.1 M,pH7.4).The brains were removed and postfixed. Coronal frozen brain slices were cut and stored.5.Hematoxylin-Eosin(HE) staining was employed to detect the histologic changes in different groups.The frozen brain slices from different groups at 24 h reperfusion were stained with HE solution, and the histopathologic changes of hippocampuses were observed under the microscope.6.Nissl staining was used to count the number of neurons in right hippocampal CA1 region.7. In situ hybridization experiment was used to determine the expression of CREB mRNA and tau mRNA in sections.8.Detecting the expressions of CREB and phosphorylated CREB in sections by using immunohistochemical technique.9. Detecting the expressions of phosphorylated tau and tau-5 in sections by using immunohistochemical technique.10.The above sections were quantified with image analysis technology and the data were statistically analysed. The average optical density values (OD) of positive products of CREB mRNA,CREB,p-CREB and tau mRNA,p-tau(Serl99/202),tau-5 in hippocampal CA1 regions in each group were measured by image analysis system. Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by post hoc analysis with SPSS10.0 software. Significance was regarded as P<0.05.11. The expressions of CREB and phosphorylated CREB in hippocampus were also detected with Western blotting.12. The expressions of phosphorylated tau and tau-5 in hippocampus were also detected with Western blotting.Results1. Observation on experimental animal's behaviorThe rats graded 1 to 3 by Longa criterion had obvious neurological deficit symptoms. While there was no neurological deficit in sham-operated rats.2.Results of TTC stainingThere was an apparent stable infarction area in lateral area of right striatum and temporal cortex in ischemia/reperfusion rats.The infarction belonged to the supply area of ipsilateral middle cerebral artery. Thus the model was successful.3. Histopathological observation and neurons countingThe structure of right hippocampus was intact and neurons in hippocampal CA1 region had clear tigroid body in cytoplasm in Sham group. There were some ischemic changes and the normal neurons reduced in right hippocampal CA1 region in ischemia/reperfusion group with 24 h reperfusion. The normal neurons increased, and the morphology of neuron were improved in NGF group and CGRP group compared to I/R group. The survived neurons increased in CA1 region in NGF&CGRP group compared with NGF group or CGRP group (P<0.05).4.Results of CREB in situ hybridization staining and quantitative analysis.There was apparent expression of CREB mRNA in right hippocampal CA1 region in sham group. The average optical density values (OD)of CREB mRNA positive products reduced in I/R group compared to sham group. The OD of CREB mRNA in NGF group and CGRP group was higher than that in I/R group, and it further increased in NGF&CGRP group (P<0.05).5. Immunohistochemical analysis for CREB and p-CREBNeurons throughout the ipsilateral hippocampal CA1 region showed high levels of CREB immunoreactivity in sham group. The OD of CREB positive products in ipsilateral CA1 region decreased in I/R group compared to sham group. The expression of CREB increased in NGF group and CGRP group compared to I/R group. The expression of CREB in NGF&CGRP group was higher than that in NGF group and CGRP group respectively (P<0.05) .The expression of p-CREB in ipsilateral CA1 region in sham group was relatively less,but it apparently increased in I/R group.The OD of p-CREB in NGF group and CGRP group was higher than that in I/R group and sham group respectively. In NGF& CGRP group, the expression of p-CREB was higher than that in NGF group or CGRP group (P<0.05) .6.Detecting the expression of CREB and p-CREB in hippocampus with Western blot experiment.Western blot indicated that the integrated optical density value (IDV) of CREB band at 43kDa relative molecular weight in I/R group was less than that in sham group. It increased in NGF group and CGRP group compared with sham group. In NGF & CGRP group, the IDV of CREB band was higher than that in NGF group and CGRP group respectively (P<0.05).The IDV of CREB bands was higher in I/R group than that in Sham group. It increased in NGF group and CGRP group,and further increased in NGF&CGRP group. The IDV of p-CREB band was higher in I/R group than that in Sham group. It increased in NGF group and CGRP group,and further increased in NGF&CGRP group.7. Immunohistochemical analysis of p-tau (Ser199/202) and tau-5.There was some expression of p-tau in Sham group, and it apparently increased at reperfusion 3 h timepoint in I/R group,and it continued to increase at 12 h and 24 h timepoint,then decreased. It was still higher at reperfusion 72 h timepoint compared with sham group(P<0.05).NGF and CGRP could obviously decreased the immunoreactivity of p-tau(Ser199/202) in ischemic neurons in hippocampus compared with I/R group(P<0.05). The OD of p-tau(Ser 199/202) further decreased in NGF& CGRP group compared with NGF group or CGRP group(P<0.05).The OD of tau-5 positive products was higher in I/R group than that in sham group at every reperfusion timepoint(P<0.05).It decreased in NGF group and CGRP group compared with I/R group(P<0.05).The immunoreactivity of tau-5 decreased in NGF & CGRP group compared with I/R group,but there was no difference compared with NGF group or CGRP group.8.The results of expressions of p-tau(Ser199/202) and tau-5 in hippocampus with Western blotting.Western blot indicated the integrated optical density value (IDV) of p-tau band increased in I/R group compared with sham group, and it decreased in NGF group and CGRP group as compared to I/R group,but higher than that in sham group(P<0.05).The IDV of p-tau band further decreased in NGF & CGRP group compared with I/R group,NGF group and CGRP group respectively(P<0.05).The expression of tau-5 increased in I/R group compared with sham group. It reduced in NGF group and CGRP group compared to I/R group(P<0.05).The IDV of tau-5 decreased in NGF&CGRP group compared with I/R group,but there was no difference from NGF group and CGRP group.9. Results of tau in situ hybridization and quantitative analysis.The positive product of tau mRNA showed brown,and mainly expressed in cytoplasm, and some located in nuclear. There was apparent expression of tau mRNA in ipsolateral hippocampal CA1 region in every groups. There was not apparent difference in expression of tau mRNA among these groups.Conclusion1.NGF and CGRP ameliorate the morphology of ischemic neurons of hippocampus during focal cerebral ischemia/reperfusion. Both promote the recovery of ischemic neurons,which may be conducted by upregulating the expression of CREB mRNA, CREB protein and p-CREB in hippocampal ischemic neurons. Activated CREB may be involved in the crucial molecular mechanisms of NGF and CGRP to ischemic neurons.2. NGF and CGRP apparently attenuate the level of tau hyperphosphorylation and deregulate the expression of total tau proteins,but they have no obvious effect on the expression of tau mRNA in rat hippocampus during focal cerebral ischemia/reperfusion. Inhibiting tau hyperphosphorylation may be composed of one of important molecular mechanisms of NGF and CGRP in protecting ischemic neurons.3. Phosphorylated CREB and tau protein may be the cross point of the signal transduction pathways of NGF and CGRP to ischemic neurons. They may cooperate with each other to promote the recovery of the ischemic neurons. The effect of combined usage of NGF and CGRP are more effective than NGF and CGRP treatment solely.4. CREB and tau protein are probably new targets of the research in recovering the neuronal function and cerebral ischemia therapy.