| Many diseases in clinic can develop cerebral ischemia, in turn, a serial of complicated pathophysiologic changes ensue. Some neurons involved in ischemia/ reperfusion(I/R) develop degeneratin, necrosis and apoptosis, which gives rise to different neural sequela, including discord of locomotion as well as disturbance of language and cognition. Nerve growth factor , one of the most important biological molecule in central nerve system, can contribute to neuronal differentiation, keep neuronal growth and development and promote recovery of injuried neuron. Calcitonin gene relative peptide, which is first synthetised through the gene recombination, containing 37 amino acids, acts as the function of relaxing vessels, preventing and relieving vasospasm. A lot of genes, such as P38MAPK, CHOP and IL-β, are iuduced and expressed during the cerebral ischemia/reperfusion(I/R), and overexpressions of these genes may induce and accelerate apoptosis of neurons. Whether or not exogenous nerve growth factor and Calcitonin gene relative peptide can regulate the expressions of P38MAPK, CHOP and IL-βis still unknown after focal cerebral ischemia/reperfusion (I/R). So the model of focal cerebral ischemia/reperfusion (I/R) was made to detect expressions of P38MAPK, CHOP and IL-βwith the methods of in situ hybridization, immunohistochemistry and Western blot. The present study is to aim at systemically reseach on regulatory effect of NGF and CGRP on the expression of P38MAPK, CHOP and IL-βby means of the model of focal cerebral ischemia/reperfusion (I/R). These are all for further exploring effective therapeutic measure on cerebral ischemia and laying foundation on exploiting new and special drug .Materials and Methods1. AnimalsAll animal procedures were approved by the Animal Care and Use Committee at China medical University (SYSC 2003—009). 210 healthy male Wistar rats that weighted 250-280g (supplied by Experiment Animal Center of China Medical University) were used for the study and were randomly divided into: (1) sham-operated group(n=10); (2) ischemia/reperfusion (I/R) group, (3) NGF group; (4) CGRP group; (5) CGRP&NGF group. (I/R)group, NGFgroup, CGRP group and CGRP& NGF group were subdivided into 3h, 12h, 24h, 48h and 72h group after 2h MCAO, 10 rats in each group(n=10).2. Model of Middle Cerebral Artery OcclusionRats were anesthetized with intraperitoneal injection of 10% chloral hydrate (350mg/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) and internal carotid artery(ICA) were separated via a ventral midline incision. A 40 mm length of monofilament nylon suture (φ0.28mm), its tip smeared with silicone, which was introudued into the ICA lumen(18±1.0 mm), and the origin of the MCA was blocked. 2h after MCAO, reperfusion was induced by withdrawing the suture. Sham group underwent the entire proceduce 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, 1ml of 0.9%NaCl was infused into left ICA through left ECA trocar, finished in 30 minutes; in NGF group, NGF (500U/ml, 1ml) was infused, finished in 30 minutes; in CGRP group, CGRP (1μg/ml,lml) was infused, finished in 30 minutes; in NGF& CGRP group, 1ml NGF and CGRP (NGF500U/ml, CGRP 1μg/ml) were infused, finished in 30 minute. The trocar was withdrawed after injection, the left ECA was ligated.Evaluation of neurological was scored on Longa five-point scale: 0, no observed neurologic deficit; 1, failure to extend the left forepaw fully when the tail was suspended; 2, failure to extend the left forepaw and intermittent circling; 3, sustained circling without moving forward; 4, unable to walk spontaneously and a depressed level of consciousness. Selecting criteria: the rat whose grade is 1, 2 or 3 was applied in the study.3. Histopathologic changes of ischemia neuronsThe frozen sections were dried at room temperature, the histopathologic changes were observed by HE staining under the microscope to demonstrate if the ischemia model is successful in sham group and I/R group. The infarction volume was determined by TTC staining after I/R.4. In situ hybridization and immunohistochemistry3, 12, 24, 48 and 72 h reperfusion , rats were anesthetized with 10% chloral hydrate(350mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde in PBS(0.1mol/L, pH 7.4) and post-fixed. Brains were removed, coronally dissected into 15 mm thick sections, 6.0 mm in front of the line between ears. The sections were put into the same fixative for 2 h, then washed adequately using distilled water, and put sections into 30% sucrose in PBS(0.1mol/L, pH7.4), stored at 4°C over the night, frozen slice machine was used for continuous coronal slice, the thickness is 20μm. The slides were detected with in situ and immunohistochemical methods respectively.5. Western blot AnalysisThe animals were anesthetized with intraperitoneal injection of 10% chloral hydrate (350mg/kg). The brain was removed, and brain tissue was homogenized in ice-cold buffer. Then tissue bomogenate was Centrifuged (15000rpm for 1h). Proteins were separated by SDS-PAGE, and then transferred onto the nitrocelluose filter. The expression of P38MAPK, CHOP, IL-1βwere detected by ECL kit.6.Statistical AnalysisData are presented as mean±SD. Differences between groups were analyzed with an unpaired t test and SPSS11.5 analysis software . Values of P<0.05 were regarded as statistically significant.Results1. Histopathologic changes of ischemia neurons In sham group , neurons were arranged orderly and intact in shape; the neurons in I/R group were swollen, arranged asymmetrically, the cellular inter-space widened, the volume of some cells decreased with nulear pyknosis and dyed darkly. Conspicuous infarct was observed at 24h after I/R, but not foung in sham group.2. P38MAPK mRNA in situ hybridization detectionIn cortex , the number of positive neurons for P38MAPK mRNA was modest in sham group, but increased as the time went on in ischemic group, reaching the peak at 12h after reperfusion, then decreased gradually. P38MAPK mRNA positive product was brown under microscope, and mainly expressed in cytoplasm. The average optical density of positive cells for P38MAPK mRNA at each timepoint in I/R group were obviously higher than those in sham group(P<0.01); The average optical density of positive cells for P38MAPK mRNA in NGF or CGRP group were lower than those in I/R group(P<0.05); The average optical density of positive cells for P38MAPK mRNA in NGF&CGRP group were lower than those in NGF or CGRP group respectively(P<0.05).3. P38MAPK protein assayThe number of positive neurons of cortex for P38MAPK protein was modest in sham group, but increased as the time went on in ischemic group, reaching maximum at 24h after reperfusion , then decreased gradually . Positive product for P38MAPK protein was brown under microscope, and mainly expressed in cytoblast. The average optical density of positive cells for P38MAPK protein at each timepoint in I/R group were obviously higher than those in sham group(P<0.01); The average optical density of positive cells for P38MAPK protein in NGF or CGRP group were lower than those in I/R group(P<0.05); The average optical density of positive cells for P38MAPK protein in NGF&CGRP group were lower than those in NGF or CGRP group respectively (P<0.05). Western blot assay showed the similar result.4. CHOP in situ hybridization detectionCHOP mRNA positive cells were not found in sham group, but obviously found in cortex in I/R group. The number of positive cells increased as time went on in ischemic group, and reached the peak at 24h after reperfusion. CHOP mRNA positive product was brown under microscope, and mainly expressed in cytoplasm. Except for 3h and 72h, the average optical density of positive cells for CHOP mRNA at each timepoint in I/R group were obviously higher than those in sham group(P<0.01); The average optical density of positive cells for CHOP mRNA in NGF or CGRP group were lower than those in the I/R group(P<0.05); The average optical density of positive cells for CHOP mRNA in NGF&CGRP group were lower than those in NGF or CGRP group respectively (P< 0.05).5. CHOP protein assayCHOP protein positive cells were not found in sham group, but found obviously in cortex in I/R group. Expression of CHOP protein increased gradually and reached maximum at 24h after reperfusion, then decreased. Positive product for CHOP protein was brown under microscope, and mainly expressed in cytoblast. The average optical density of positive cells for CHOP protein at each timepoint in I/R group were obviously higher than those in sham group(P<0.01); The average optical density of positive cells for CHOP protein in NGF or CGRP group were lower than those in I/R group(P<0.01); The average optical density of positive cells for CHOP protein in NGF&CGRP group were lower than those in NGF or CGRP group respectively(P< 0.01). Western blot assay showed the similar result.6. IL-βmRNA in situ hybridization detectionIL-βmRNA positive cells were not found in sham group, but obviously found in I/R group. The number of positive cells increased as time went on in ischemic group, and reached the peak at 24h after reperfusion. IL-βmRNA positive product was brown under microscope, and mainly expressed in cytoplasm. The average optical density of positive cells for IL-βmRNA at each timepoint in I/R group were obviously higher than those in sham group(P<0.05); The average optical density of positive cells for IL-βmRNA in NGF or CGRP group were lower than those in the I/R group(P<0.05); The average optical density of positive cells for IL-βmRNA in NGF &CGRP group were lower than those in NGF or CGRP group respectively (P<0.05).7. IL-βprotein assayIL-βprotein positive cells were not found in sham group, but obviously found in I/R group. Expression of IL-βprotein increased gradually and reached maximum at 48h after reperfusion, then decreased. Positive product for IL-βprotein was brown under microscope, and mainly expressed in cytoblast. The average optical density of positive cells for IL-βprotein at each timepoint in I/R group were obviously higher than those in sham group(P<0.01); The average optical density of positive cells for IL-βprotein in NGF or CGRP group were lower than those in I/R group(P<0.05); The average optical density of positive cells for IL-βprotein in NGF&CGRP group were lower than those in NGF or CGRP group respectively(P<0.05). Western blot assay showed the similar result.Conclusion1. The expressions of mRNA and protein for P38MAPK, CHOP, IL-1βare upregulated after MCAO in rat cortex, suggesting that P38MAPK, CHOP, IL-1βinvolved neuronal apoptosis induced by I/R.2. NGF and CGRP downregulate the expreesions of mRNA and protein for P38MAPK, CHOP, IL-1βin rat cortex, which might be one of the molecular mechanisms and a target for improving function of the ischemic neurons.3. The combined usage of NGF and CGRP obviously regulates the expressions of P38MAPK, CHOP, IL-1βin rat cortex, both can enhance the neuronal protective effects respectively.
|
PDF Full Text Request