The Dynamic Expression Of INOS And OPN In Experimental Autoimmune Encephalomyelitis And The Protection Of Edaravone

Objective: Multiple sclerosis is an inflammatory cell infiltration andwhite matter demyelination autoimmune diseases. Now the pathogenesis ofMS is unknown, maybe it related to immune, hereditary, infection andenvironment. There is no efficacious drug for treatment at present. Ourobjective is establishing animal mode experimental autoimmuneencephalomyelitis(EAE), discussing the dynamic expression of induciblenitric oxide synthase(iNOS) and osteopontin(OPN) in EAE, then useEdaravone for treatment, discuss its protection.Methods:1Establish animal modelWe anesthetize the animals, take the whole spinal cord, then weight it. Theweight of spinal cord and the volume of brine ice is1:10, is made to guineapig spinal cord homogenate (GPSCH). It is mixed with the complete Freud`sadjuvant with6mg/ml of bcg vaccine. Each animal is0.5ml for limbs foot padand the back subcutaneous injection.2The group of experiment and intervention70female wistar rats are divided into three groups randomly,10is for thecontrol group,30are for the EAE group,30are for the Edaravone group.According to the result of the pre-experiment,5rats of each group are killedrandomly on the tenth day after immunization. then5rats of each group arekilled randomly on the third day and the seventh day after morbidity as thepeak group and the remission group. The rats of the edaravone group are givenEdaravone for10mg·kg-1·d-1intraperitoneal injection since immunized untilkilled. The rats of the other two groups are given intraperitoneal injectionsphysiological saline intraperitoneal injections for1ml/rat at the same time. 3The clinical scoresThe rats are weighed everyday after immunization, and the situations areobserved such as mental state, eating and drinking, urine and shit, and theactivities. The clinical scores are assessed in accordance with the standardfrom Kono.4The observation of indexWe anesthesia the animals, then take the spinal cord to the4%formalin. Thelumbar enlargements are made to paraffin blocks. We observe theinflammatory cells infiltration with HE, we count the immunepositive cells ofiNOS and OPN with immunohistochemical(IHC).5Statistical methods.All data are statistically analyzed by SPSS13.0software. Measurement dataare expressed as mean±standard deviation. The comparison of several groupsis one-way ANOVA. SNK-q is used for the comparisons between each twogroups. The comparison of enumeration data is chi-square test. P value <0.05is considered statistically significant.Results:1The observation of morbidityThe rats are listlessness, fur unsmooth, decreased appetite, weight loss afterimmunization gradually. When onset tail feeble appear at first, then hind limbsand fore limbs, incontinence of urine and feces.(1) The comparison of morbidity timeThere is no morbidity in the control group, the EAE group is (12.50±1.52)days, and the Edaravone group is (15.67±1.37) days, there is statisticallysignificant(P<0.05).(2) The comparison of morbidity24rats morbidity in EAE group, the morbidity is80%,16rats morbidity in theEdaravone group, the morbidity is53.33%, there is statistically significant(P<0.05).(3) The comparison of mean clinical scoresThe mean clinical score of the EAE group is (3.20±0.84) in the third day, it ishigher than the seventh day (1.60±0.55), there is statistically significant (P<0.05). The mean clinical score of the Edaravone group i（s2.00±1.00）in thethird day, it is higher than the seventh day (0.40±0.55), there is statisticallysignificant (P<0.05). There is statistically significant of the two groups at thesame time (P<0.05).2The observation of histopathology(1) There is no inflammatory cells infiltration or “blood vessel muff” in thecontrol group.(2) Pre-disease there are a little inflammatory cells in the EAE group, there arefewer inflammatory cells in the Edaravone groups, there is no “blood vesselmuff” in the two groups.(3) The third day after onset There are diffuse inflammatory cells infiltratedand surrounding blood vessels around form “blood vessel muff” in the EAEgroup. There are fewer “blood vessel muffs” formed in the Edaravone group.(4)The seventh day after onset The “blood vessel muffs” and the inflammatorycells in the EAE group are diminished, while those of Edaravone group arefewer than before.3The observation of immunohistochemical(IHC).3.1iNOSiNOS is immunopositive on inflammatory cells, such as macrophages,monocytes, and a few astrocytes, neurons, we mainly observe theinflammatory cells.(1)Pre-disease the immunepositive cells in the EAE group is (20.53±2.67),while in the Edaravone group is (14.13±2.88), there is statistically significant(P<0.05).(2)The third day the immunepositive cells in the EAE group is (22.60±2.72),while in the Edaravone group is (16.93±2.89), there is statistically significant(P<0.05).(3)The seventh day the immunepositive cells in the EAE group is(11.33±2.26), while in the Edaravone group is (10.60±1.64), there is nostatistically significant (P>0.05).(4)The EAE group three time points: there is no statistically significant between pre-disease and the third day (P>0.05), there is statisticallysignificant between the seventh day and the third day (P<0.05).(5)The Edaravone group three time points: there is no statistically significantbetween pre-disease and the third day (P>0.05), there is statisticallysignificant between the seventh day and the third day (P<0.05).3.2OPNOPN is immunopositive on inflammatory cells, astrocytes, microglial cells,and a few neurons in white matter and the grey white matter junction, wemainly observe the inflammatory cells.(1)Pre-disease the immunepositive cells in the EAE group is (19.87±2.92),while in the Edaravone is (10.53±2.23), there is statistically significant(P<0.05).(2)The third day the immunepositive cells in the EAE group is (29.60±5.03),while in the Edaravone group is (22.73±1.62), there is statistically significant(P<0.05).(3)The seventh day the immunepositive cells in the EAE group is(11.27±2.58), while in the Edaravone group is (8.00±1.56), there isstatistically significant (P<0.05).(4)The EAE group three time points: there is statistically significant whenmultiple comparisons (P<0.05).(5)The Edaravone group three time points: there is statistically significantwhen multiple comparisons (P<0.05).Conclusions:1Edaravone can delay morbidity time, reduce morbidity, alleviate clinicalsymptoms.2Edaravone can relieve inflammatory cells infiltration of the spinal cord inEAE rats.3Edaravone can reduce the expression of iNOS and OPN of the spinal cord inEAE rats. 4We conjecture that Edaravone has a therapeuticaleffect on EAE.