The Effect Of Fasudil On Expression Of Rock â…¡ In Spinal Cords In Experimental Autoimmune Encephalomyelitis Mice

Objective: Rho/Rho - kinase signaling pathway is a ubiquitous one in vivo. It involved in regulation of cell proliferation, adhesion, migration, etc. It was believed to control cell behavior in the upstream. Recent studies showed that Rock was activated in many pathological states in CNS. Rock inhibitors can improve the pathological conditions. In present study, we treat C57BL/6 experimental autoimmune encephalomyelitis (EAE) mice with Rho kinase inhibitor Fasudil in order to observe clinical effect and potential immunologic mechanism. In addition, we hope to supply experimental evidence for clinical application of Fasudil to treat MS and other CNS autoimmune disease.Method: Female C57BL /6 mice were randomly divided into EAE group,Fasudil treated group and adjuvant group. Mice in EAE and treated groups were immunized with myelin oligodendrocyte glycoprotein peptides (MOG35-55) emulsified in CFA. Mice in adjuvant group were injected with normal saline instead of MOG35-55. Fasudil was intraperitoneally injected in the dosage of 50 mg·kg-1·d-1 in treated group on day 7 post-immunization (p.i.). Mice in EAE and adjuvant group were injected with normal saline. Mice were evaluated for clinical signs every other day. Mice were sacrificed on days 28-30 p.i. Spinal cords were dissected. The pathological changes were detected by HE staining and Luxol Fast Blue staining. The expression of Rock-Ⅱin spinal cords in each animal was detected by immunofluorescence staining.Results:1. C57BL/6 mice with EAE induced by MOG35-55 were established successfully. On day 12p.i., the mice in EAE group began to show apparently clinical symptoms such as: eat less than normal state, the reduction of activity, the loss of body weight, the paralysis of tail and one side of hindlimb and so on. Mice displayed paralysis of two hindlimbs and even forelimb paralysis with the disease exacerbation. The highest scores reached 4. The body weight and the incidence in Fasudil treated group was statistically significantly decreased when compared with those of EAE group (19.56±0.66 vs 15.75±0.81, P<0.05; 35% vs 100% , P<0.05). The clinical symptom scores and mean time of disease onset in Fasudil treated group were statistically significantly decreased when compared with those of EAE group (2.24±0.26 vs 3.80±0.78, P<0.05; 15.55±1.50 vs 13.35±1.309 , P<0.01). The disease did not appear in mice administered by Freund adjuvant and the weight of the mice increased.2. HE staining showed marked inflammatory cell infiltration in spinal cords of EAE mice. The score of inflammatory cell infiltration according to Okuda standard in Fasudil treated group was statistically significantly increased when compared with those of EAE group (20.22±1.56 vs 31.78±1.04, P<0.05). Luxol Fast Blue staining showed obvious demyelination in EAE group than the EAE group. The degree of demyelination was positively correlated with the severity of the disease.3. Indirect immunofluorescence showed that Rock-Ⅱexpressed in spinal cords of mice in each group. The higher the clinical score in EAE mice, the more the expression of Rock-Ⅱ. It showed that the expression of Rock-Ⅱis obviously in vascular endothelial cells. The number of dot positioning of Rock-Ⅱin Fasudil treated group was statistically significantly decreased when compared with those of EAE group (20.22±1.56 vs 31.78±1.04, P<0.01).Conclusion:1. MOG35-55 can induce EAE successfully in C57BL / 6 mice.2. Rho/Rock signaling pathway involved in the pathogenesis of EAE.3. Fasudil could significantly decrease the incidence rate, relieve the clinical severity of EAE and lessen inflammatory cell infiltration and demyelination.4. Fasudil could significantly decrease the expression of Rock-Ⅱwhich may be one of mechanism that Fasudil can treat EAE.