| ObjectiveTo study the effects of exogenous calcitonin gene related peptide( CGRP) and nerve growth factor(NGF) on the expression of Fas mRNA / Fas, ICAM -1 mRNA / ICAM - 1 in rats hippocampus during focal cerebral ischemia / reper-fusion(I/R) and the regulation on neural dysfunction of damaged neurons and size of cerebral infarction volume to explore the effects and protective mechanism of CGRP and NGF on ischemic brain tissue.MethodsThe model of ischemia/reperfusion in rat was induced by using intraluminal suture occlusion of middle cerebral artery, ischemia for 2h and then reperfusion. The expressions of Fas protein and FasmRNA , ICAM - 1 protein and ICAM -1mRNA were detected with immunohistochemical and in situ hybridization methods and analyzed by microimage analysis system to observe the effects of CGRP and NGF on the expressions of Fas mRNA / Fas, ICAM - 1mRNA / ICAM - 1 in rats hippocampus during focal cerebral ischemia / reperfusion (I/R) and the regulation on neural dysfunction of damaged neurons. The neural dysfunction wasscored by Zea Longa' s criteria when the reperfusion was supplied for 6h after the ischemia lasted for 2h. The infarction volume was determined by TTC staining , the change in the tissue of the cerebral cortex and hippocampus of the rat was observed under light microscope by HE staining.1. Animals162 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 (1/ R) group, (3 ) NGF group, (4 ) CGRP group, (5 ) CGRP and NGF group. I/R group, NGF group, CGRP group, CGRP and NGF group were subdivided into 6, 12,24,48 and 72h group after 2h MCAO, 6 rats in each group(n =6).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 using 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 separated via a ventral midline incision. A 50 mm length of monofllament nylon suture (0. 28 mm), its tip smeared with silicone, was introduced into the ICA lumen (18 ± 1. 0mm), the origin of the MCA was blocked. 2 hours after MCAO, reperfusion was induced by withdrawing the suture. Sham group underwent the entire procedure except for inserting suture and infusing 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, lml 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 (1 |xg/ml, 1 ml) was infused, finished in 30min;in CGRP and NGF group, CGRP(2jxg/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 lml, CGRP&NGF group was injected CGRP(2ng/ml,0. 5ml) and NGF( lOOOU/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 of consciousness. Selecting criteria;the rat whose grade is 1, 2 or 3 was employed in the study.3. Detection of neural dysfunctionThe neural dysfunction was scored by Zea Longa criteria when the reperfu-sion was supplied for 6h after the ischemia lasted for 2h.4. Hematoxylin - Eosin (HE) staining and TTC stainingThe frozen sections were dried at room temperature, the histopathologic changes were observed by HE staining under the microscope to demonstrate if the ischemic model is successful in sham group and I/R group. The infarction volume was determined by TTC staining in I/R group and CGRP and NGF group.5. Immunohistochemistry and in situ hybridizationAt 6,12,24,48 and 72 hours reperfusion, rats were anesthetized with 10% chloral hydrate (350 mg/kg) and perfused transcardially with 50 mi saline followed by 4% paraformaldehyde in PBS (0. 1 mol/L , pH 7.4) to perfuse and post -fixed. Brains were removed, coronally dissected into 2. 0 mm thick sections, 6.0 mm in front of the line between ears. The sections were 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 314X1 o-ver the night, frozen slice machine was used for continuous coronal slice, the thickness is 16 fim. 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 were presented as mean ± SD. The data was managed with paired - sample t -test between the two means of two samples and compared between two of means of multiple samples in variance analysis.Results1. Neural injury changesAll the rats after focal cerebral inschemia / reperfusion showed neural injury of different levels, and the score is obviously higher than that in sham group (p <0.01). The score of CGRP and NGF group is obviously lower than that of I/R group (p<0.01).2. The changes of infarction volumeThe infarction volume in ischemia / reperfusion is 281 ±21 mm" , in CGRP and NGF group is 226 ± 13mm3.3. Histopathologic changes in hippocampusIn sham group, neurons were arranged orderly and intact in shape;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.4. Fas protein immunohistochemistryFas protein positive cells were not found in sham group, and found in hippocampus 6h after reperfusion in ischemic group, the number of the positive cells increased as the time went on, and at 24h reached the peak, then de-creased gradually. Under microscope, immunoreactive product was brown, mainly expressed in cytoplasm. The average gray values of Fas protein positive cells at each timepoint in I/R group were obviously lower than those in the sham group(P <0. 01);The average gray values of Fas protein positive cells at each timepoint in CGRP or NGF group were higher than that in I/R group (P <0. 05), the average gray values of Fas protein positive cells in CGRP and NGF group were obviously higher than that in CGRP or NGF group(P <0.05).5. FasmRNA in situ hybridizationFasmRNA positive cells were not found in sham group , but found in hippocampus of I/R group . The average gray values of FasmRNA positive product in CGRP or NGF groups were higher compared with I/R group( P <0.01) ,and the average gray values in CGRP and NGF groups were higher compared with CGRP orNGFgroup(P<0.01).6. ICAM - 1 protein immunohistochemistryICAM -1 positive cells were not found in sham group, but found obviously in I / R group. The average optical density (OD) values of ICAM - 1 positive product in CGRP and NGF groups were lower compared with I/R group(P <0. 01) , and the average OD values of ICAM - 1 positive product were lower in CGRP and NGF group than in CGRP or NGF group(P <0.01).7. ICAM - lmRNA in situ hybridizationICAM -1 mRNA positive cells were not found in sham group, but found in hippocampus of focal cerebral I/R group. The average gray values of ICAM -lmRNA positive product in CGRP or NGF groups were higher compared with 1/ R group ( P < 0. 01) , and the average gray values in CGRP and NGF groups were higher compared with CGRP or NGF group( P <0.01).ConclusionThe score of neural injury in CGRP and NGF group is obviously lower than that in I/R group, CGRP and NGF are capable of ameliorating the level of neural dysfunction and protective for ischemic neurons.The infarction volume in CGRP and NGF group is obviously lower than that in I/R group, CGRP and NGF are capable of ameliorating the level of the ischemic damage on neurons and protectve for ischemic neurons.CGRP and NGF inhibit the transcription of Fas ^ ICAM - 1 gene and down-regulate the expressions of Fas and ICAM - 1 proteins respectively in I/R rat hippocampus, which may be one of the mechanism for the protective effects of CGRP and NGF on the ischemic brain neurons.The combined usage of CGRP and NGF obviously inhibits the transcription of Fas N ICAM -1 gene and downregulates the expression of Fas ^ ICAM - 1 protein in rat hippocampus, indicates that they have coordinative effect on ischemic neurons.
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