The Effects And Mechanism Of CGRP And NGF On The Expressions Of P53 And HSP70, And The Abilities Of Spatial Learning And Memory In Focal Cerebral Ischemia/Reperfusion Rats

ObjectiveTo study the effects of exogenous calcitonin gene - related peptide ( CGRP) and nerve growth factor ( NGF) on the expressions of p53, HSP70 in rats hippocampus after focal cerebral ischemia/reperfusion (I/R) and the abilities of spatial learning and memory of rats, to explore the effects and mechanism of CGRP and NGF on ischemic brain tissue.Materials and MethodsMiddle cerebral artery occlusion ( MCAO) was employed to make focal cerebral ischemia, the expressions of p53, HSP70 protein and p53, HSP70 mRNA were detected with immunohistochemical and in situ hybridization methods and analyzed by microimage analysis system; Morris water maze was used to evaluate the abilities of spatial learning and memory, to observe the effects of CGRP and NGF on the spatial learning and memory abilities.1. Animals1.1 126 healthy male Sprague - Dawley rats weighting 250 - 300g (supplied by Experimental Animal Center of China Medical Universit) were randomly divided into: ( 1) sham - operated ( sham ) group (n=6), (2) ischemia/ reperfusion ( R/I) group, ( 3 ) NGF group, ( 4 ) CGRP group, ( 5 ) CGRP&NGF group. R/I group, NGF group, CGRP group, CGRP&NGF group were subdivided into 6, 12, 24, 48 and 72h group after 2h MCAO, 6 rats in each group(n=6).1.2 30 healthy male Sprague - Dawley rats weighting 250 - 300g ( supplied by Experimental Animal Center of China Medical Universit) were randomly divided into: (1) sham group, (2) R/I group, (3) CGRP&NGF group,n = 10.2. Middle Cerebral Artery OcclusionRats were anesthetized with intraperitoneal injection of 10 % chloral hydrate (350 mg/kg). The right middle cerebral artery (MCA) was occluded u-sing previously described methods ( Kuge Y et al. , 1995 ). Briefly, the right common carotid artery (CCA) , external carotid artery (ECA) and internal carotid artery (ICA) were isolated via a ventral midline incision. A 50 mm length of monofilament nylon suture (φ0.28mm) , its tip smeared with silicone, was introduced into the ICA lumen (18 ± 1. 0mm) , the origin of the MCA was blocked. After 2 hours of MCAO, reperfusion was induced by withdrawing the suture. Sham group underwent the entire procedure except without suture inserted and infused the medicine.A trocar was inserted into left ICA through the left ECA kept in reserve until 0.9%NaCl, CGRP and NGF was infused. In I/R group, 1ml of 0.9%NaCl was infused into left ICA through left ECA trocar, finished in 30min;in NGF group, NGF (500U/ml, lml) was infused, finished in 30min; in CGRP group, CGRP (1μg/ml,1ml) was infused, finished in 30min; in CGRP&NGF group, CGRP (2(2μg/ml,0.5ml) and NGF (1000U/ml, 0.5ml) were infused, finished in 30min. The trocar was withdrawed after injection, the left ECA was ligated.0.9%NaCl, CGRP and NGF were administrated through peritoneal cavity injection in Morris water maze groups, I/R group was injected 0.9%NaCl 1ml, CGRP&NGF group was injected CGRP (2μg/ml, 0.5ml) and NGF (1000U/ ml, 0. 5ml) , once a day, lasted 10 days. The first administration was finished in 15 minutes after I/R.Evaluation of neurological function was scored on Zea Longa five - point scale (Longa EZ, et al. , 1989) : Grade 0, no observable neurologic deficit; Grade 1, failure to extend the left forepaw fully; Grade 2, failure to extend the left forepaw and intermittent circling; Grade 3, sustained circling without moving forward; Grade 4, unable to walk spontaneously and a depressed level ofconsciousness. Selecting criteria; the rat whose grade is 1, 2 or 3 was employed in the study.3. The observation of the abilities of spatial learning and memoryMorris water maze test was performed to study the ability of spatial learning and memory in each group of rats in the 11 to 16th day after reperfusion.SPSS11.5 statistical software was used to analyse the data, analysis of variance was used to compare the escape latencies and the times of crossing the former site of the removed hidden platform per minute of each group.4. Hematoxylin -Eosin (HE) stainingThe paraffin sections were stained with HE, the histopathologic changes were observed under the microscope to demonstrate if the ischemic model is successful in sham group and I/R group.5. Immunohistochemistry and in situ hybridization6. 12 ,24, 48 and 72 hours after reperfusion, the rats were anesthetized with intraperitoneal injection of 10 % chloral hydrate (350 mg/kg) and perfused transcardially with 50 ml saline followed by 4% paraformaldehyde in PBS (0. lmol/L, pH 7.4). Brains were removed out, coronally dissected into 2.0mm thick sections, 6.0mm before the line between ears, put into the same fixative for 1 hour. Washed adequately using distilled water, then the sections were put into 30% sucrose in PBS (0.1 mol/L, pH 7.4) , stored at4cC over the night, frozen slice machine was used for continuous coronal slice, the thickness is 16 jxm. The slides were detected with immunohistochemical and in situ hybridization methods respectively.The average gray values of immunoreactive products of CA1 region in each group were measured by MetaMorph ( USA) image analysis system, all values are presented as mean SD. The data was managed with paired - sample t - test between the two means of two samples and comparison between two of means of multiple samples in variance analysis.Results1. Histopathologic changes in hippocampusIn sham group, neurons were arranged orderly and intactness; the neurons in I/R group were swollen, arranged asymmetrically, the cellular interspace widened, the volume of some cells decreased with nuclear pyknosis and dyed darkly.2. p53mRNA in situ hybridizationp53mRNA positive cells were not found in sham group, but was found in hippocampus of I/R group. The average gray values of p53mRNA positive product in CGRP or NGF groups were higher compared with I/R group ( P <0.01) , and the average gray values in CGRP&NGF groups were higher compared with CGRP or NGF group (P<0.01).3. p53 protein immunohistochemistryp53 protein positive cells were not found in sham group, and were found in hippocampus after 6h reperfusion in ischemic group, the number of the positive cells increased as the time went on, and at 24h reached the peak, then decreased gradually. Under microscope, immunoreactive products were brown, mainly expressed in nuclear, slightly expressed in cytoplasm. The average gray values of p53 protein positive cells at each period in I/R group was obviously lower than those in the sham group (P <0.01) ;The average gray values of p53 protein positive cells at each period in CGRP or NGF group were higher than that of I/R group (P<0.01), the average gray values of p53 protein positive cells in CGRP&NGF group were obviously higher than that of CGRP or NGF group (P<0.01).4. HSP70mRNA in situ hybridizationHSP70mRNA positive cells were not found in sham *group, but were found in hippocampus of focal cerebral I/R group. The average gray values of HSP70mRNA positive product in CGRP or NGF groups were lower compared with I/R group (P <0. 01) , and the average gray values in CGRP&NGF groups were lower compared with CGRP or NGF group (P <0.01).5. HSP70 protein immunohistochemistryHSP70 positive cells were not found in sham group, but were found obviously in I/R group. The average optical density (OD) values of HSP70 positive product in CGRP and NGF groups were higher compared with I/R group (P <0.