| Background and purposeMultiple Sclerosis(MS)is an inflammatory,demyelinating autoimmune disease that infects the central nervous system(CNS),which is specific in youngster.Pathological features are wide range of perivascular inflammatory cell infiltration,blood-brain barrier(BBB)damage and demyelination of white matter within the CNS,a relapse-mitigation process are shown in the lesions.Experimental autoimmune encephalomyelitis(EAE)is an autoimmune disease that is induced by neuronal myelin antigens and is specifically sensitized of CD4~+T cells mediator.It is the ideal animal model to study MS immune pathogenesis and to guide the treatment of clinical medicine both at home and abroad.At present,specific etiology and pathogenesis of MS is still not very clear,an important feature of MS and EAE:a large number of non-specific white blood cells are existed in inflammatory sites of CNS parenchymal parenchymal to identify myelin antigen,clinical symptoms of EAE and a large number of non-special allogeneic inflammatory cells in the CNS are presented simultaneously.In recent years,with the further understanding of the disease,studies have found that chemokines and their receptors involved in all aspects of MS pathogenesis,by understanding the role of chemokines and their receptors in MS,it provides a good prospect in the study of MS pathogenesis,in order to seek new therapeutic targets.Eotaxin is an eosinophil(EOS)selective chemotaxis and activator,it plays a role by specifically binding to the CCR3 receptor,which is significant at the effect of chemotactic,activating and collecting eosinophil,Eotaxin is currently known as the EOS chemotaxis which has the highest selectivity and largest effect.Brain microvascular endothelial cells and their tight junctions are the first barrier to BBB.Changes in tight junctions and their associated proteins may lead to changes in BBB permeability,Claudin-5 is an important component of BBB,changes of it can reflect damage in BBB or not,which is the specific indicator to monitor the integrity of BBB.In this study,by examination of the concentration of CCL26 in the early stage,acute stage and remission stage of experimental autoimmune encephalomyelitis(EAE)rats in serum and in brain tissue,The protein expressions of CCR3 and Claudin-5 in brain tissue,to explore its correlation with the severity of EAE and its possible mechanism,thus providing a theoretical basis for the treatment of MS.Methods:1.The establishment of EAE rats models:60 female Wistar rats weighing 180-220 g,6-8 weeks old were randomly divided into two groups:control group and EAE group,30 rats in each group.EAE group was divided into early stage group(11 days after immunization),acute group(18 days after immunization),remission group(24 days after immunization),10 rats in each group.Establishment of EAE rat model:A guinea pig spinal cord homogenate(GPSCH)was prepared in a sterile environment and incubated in a 1:1 volume ratio with complete freund adjuvant(CFA)containing 6 mg/ml Bacillus Calmette-Guérin(BCG),followed by repeated bolting under ice-bath conditions for at least 1 h to prepare a stable water-in-oil type antigen emulsion,the experimental rats were anesthetized and emulsion was injected into the rat limbs foot subcutaneously at four points.Each rat was given 0.5 ml antigen emulsion to establish the EAE model.2.Collection of animal samples:On the day of immunization,the behavioral characteristics,activity status,dietary mental status,body weight change and neurological score of wistar rats were observed and recorded in detail.Animals were anesthetized with 10%chloral hydrate on the 11th day(early stage),18th day(acute stage)and 24 days(remission stage)of EAE rats.Then,the apical blood was collected and the supernatant was collected after centrifugation,stored at-80℃refrigerator for usage.Pre-cooling saline was used to rapidly perfuse the heart.Under the condition of sterile condition,the brain tissue and spinal cord of the experimental rats were collected and were put into liquid nitrogen.Expanded parts of the brain and spinal cord were made into paraffin sections.3.Detection of relative indicators:The infiltration of inflammatory cells in brain tissue was assessed directly by hematoxylin-eosin(HE).The concentration of CCL26 in serum and in brain tissue was detected by enzyme-linked immunosorbent assay(ELISA).The expression of CCR3 protein in brain tissue was detected by immunohistochemistry.The expression of Claudin-5 protein in brain tissue was detected by immunofluorescence method.The data were analyzed by spss 17.0 software.Results:1.Changes in body weight:Since the second day of immunization,the body weight in the control group showed a steady growth,the EAE group showed a tendency to increase first and then decreased.2.Neurological score:No neurological deficits were seen in the control group,the neurological score was kept as 0,and the neurological score in the EAE group was significantly higher than that in the normal group(p<0.01).There was significant difference in neurological score among the acute stage group,the early stage group and the remission group(p<0.05).3.Histopathological changes:There was no inflammatory cell infiltration in rats in the control group.The inflammatory score of rats in the EAE group was more severe than that in the control group(p<0.01).The inflammatory score of rats in the acute stage group was significantly higher than that in the early stage group(p<0.05)and the remission stage group(p<0.05).4.The expression of Claudin-5 in brain tissue of the EAE group was significantly lower than that in the control group,the difference was statistically significant(p<0.01).Compared with the early stage group and remission stage group,the intensity of Claudin-5 in the brain of the acute stage group was significantly lower(p<0.05).5.The serum level of CCL26 in the EAE group was significantly higher than that in the control group(p<0.01).Compared with the early stage group and the remission stage group,the level of CCL26 in the acute stage group was significantly higher(p<0.05).6.The expression of CCR3 positive cells in brain tissue:the expression of CCR3in the EAE group was significantly higher than that in the control group(p<0.01).Compared with the early stage group and remission stage group,the expression of CCR3 in the brain of the acute group was significantly higher(p<0.05).7.In brain tissue,the level of CCL26 in the EAE group was significantly higher than that in the control group(p<0.01).Compared with the early stage group and the remission stage group,the level of CCL26 in the acute stage group was significantly higher(p<0.05).8.In the EAE group,the levels of CCL26 in serum and in brain tissue and the expression levels of CCR3 protein in brain tissue were positively correlated with inflammatory score,and were negatively correlated with the expression levels of claudin-5 protein.9.in the EAE group,the levels of CCL26 in serum was positively correlated with the levels of CCL26 in brain tissue.10.in the EAE group,the expression levels of claudin-5 protein was negatively correlated with inflammatory score.Conclusion:1.CCL26 and CCR3 significantly increased in the acute phase of the EAE rats,suggesting that CCL26 and CCR3 may participate in the pathogenesis of the EAE rats.2.CCL26 and CCR3 were positively correlated with inflammatory score in the EAE rats,suggesting that CCL26 and CCR3 may be related to the severity of the disease,and the higher the expression,the heavier the disease.3.CCL26 and CCR3 are negatively correlated with the expression level of claudin-5 protein,suggesting that CCL26 and CCR3 may be entered into the brain tissue through the damaged blood-brain barrier and thus cause inflammatory response.4.In the pathogenesis of the EAE rats,there was the damage of blood-brain barrier,and its inflammation score was related to the severity of the damage of blood-brain barrier. |
PDF Full Text Request