Excision Repair Cross Complementing 1 Protects Brain Against Ischemic Injury In Adult Rats Following Middle Cerebral Artery Occlusion

CNS injury from ischemia and reperfusion is proposed to occur via multiple interrelated mechanisms including excessive extracellular accumulation of the excitotoxin glutamate,an increase in intracellular Ca2+, and oxidative stress.These processes contribute to the generation of reactive oxygen and nitrogen species that damage protein,lipid,and DNA.Damaged DNA triggers the apoptotic process and deteriorates brain injury.Meanwhile, damaged DNA activates endogenous repair mechanisms.Nucleotide excision repair(NER) is one of the most important DNA repair mechanisms and plays roles in repairing a wide variety of DNA lesions.Excision repair cross complementing 1(ERCC1) is a key factor of NER,which acts in a complex with XFP to make the 5' incision during the process of NER.Previous study has been shown that the ERCC1 null mice were severely runted and died before weaning.In vitro study indicated that the cells with ERCC1 deficiency lost capacity to repair damaged DNA.In the contrast,ERCC1 transgene can restore DNA repair function.These results demonstrated that ERCC1 is one of the limiting factors in NER pathway.Therefore,we would ask whether ERCC1 has any effect on the pathophysiological process of brain after stroke. In this thesis,we used middle cerebral artery occlusion(MCAO) model to induce a transient cerebral ischemia.With MCAO model,we observed the changes of ERCC1 expression in the brain after a transient ischemia.With molecular techniques,we further studied functional significance of ERCC1 in the ischemic injured brain.The results of this thesis as followed:1.Expression and cellular distribution of ERCC1 in the brain of adult ratsImmunohistochemical staining was used to show the location of ERCC1 in rat brain.We observed that the immuno-positive cells widely distributed in various brain regions,including the striatum and the cortex,which are the injured territories following MCAO.Furthermore,immunostaining was used to detect cellular and subcellular distribution of ERCC1 in the brain.The results showed that ERCC1 positive signals were only observed in the nuclei of cells,and the cells stained with ERCC1 could further co-labeled with microtubule associated protein-2(MAP-2),a marker of mature neuron,or glial fibrillary acidic protein(GFAP),a marker of astrocyte,indicating that ERCC1 mainly located in the nuclei of neuron and astrocyte in the rat brain.2.Time-dependent expression of ERCC1 in the cortex and striatum of rat brains after MCAO.Western blot analysis was used to detect the dynamic changes of ERCC1 in the cortex and striatum of rat brain following MCAO.The results showed that,in the cortex,ERCC1 significantly reduced at 1 d,then increased at 3 d and lasted to 14 d after MCAO.In the striatum,ERCC1 reduced at 1 d, increased at 3 d and further enhanced after then.Compared to the controls,the contents of striatal ERCC1 were significantly higher at 7 d and 14 d after MCAO.Immunohistochemical study showed the same pattern of ERCC1 expression in the frontoparietal cortex and medial striatum of brains after ischemia.The results suggest that cerebral ischemia induced the time-dependent up-regulation of ERCC1 expression in the brain of rats.3.Deterioration of ischemic brain injury by ERCC1 antisense treatmentThe pcDNA3-ERCC1-antisense was injected into the lateral ventricle of rat brain to reduce the endogenous expression of ERCC1 after ischemia.The results showed that administration of 5μg or 10μg ERCC1 antisense plasmids significantly reduced ERCC1 level in the brain at 3 d and 7 d after ischemia.Meanwhile,antisense treatment increased the number of cells with single strand DNA breaks and enlarged ischemic infarct volume.The results reflect that the induction of endogenous ERCC1 expression may play an important role in the pathophyiological process of brain after injury.4.Protection of ERCC1 overexpression against ischemic brain injuryp-ERCC1-EGFP-N1 plasmids and p-EGFP-N1 plasmids(control) were intraventricularly injected into rat brain after MCAO operation and immunohistochemical staining was used to detect the expression of EGFP,a reportor gene product.We observed that the EGFP positive signals could be detected in ipsilateral hemisphere at 7 d of reperfusion after ischemia.Under this experimental condition,we found that 10 and 15μg,but not 5μg of ERCC1 antisense treatment significantly reduced the infarct volume.The results suggest that overproduction of ERCC1 plays neuroprotection against ischemic neuronal injury.Conclusion:Ischemia induced the time-dependent expression of ERCC1 in the injured regions of brain.ERCC1 Knock-down by antisense increased DNA damage and infarct volume of rat brain.Overexpression of ERCC1 in the brain reduced ischemic infarct volume.Putting together,the results indicate that endogenous ERCC1 in the brain may be an important neuroprotective factor.Our results also suggest that exogenous introducing of ERCC1 into brain may be a new strategy to protect neuronal injury after stroke.