Effects Of Atorvastatin On The Experimental Autoimmune Encephalomyelitis

ObjectiveMultiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system that causes relapsing and progressive neurological impairment. The disease-modifying agents currently used in the treatment of MS are not completely effective and are associated with adverse effects and high costs. Thus, alternative treatment options are highly desirable.HMG-CoA reductase inhibitors"statin"are widely used oral cholesterol-lowing drugs. Apart from the effect of lowing cholesterol, statins have important biologic effects that may be independent of their cholesterol-reducing properties. Recent studies indicate that statins have anti-inflammatory and neuroprotective properties which may be a future treatment option for MS-either in an add-on therapy regimen or alone.In our study, we administrate atorvastatin to the rats of experimental autoimmune encephalomyelitis (EAE)-an animal model of MS, in order to investigate the therapeutic potential in treatment of neuroinflammatory diseases like MS.MethodsOne hundred healthy female Wistar rats weighing 180~200g were divided into four groups: blank control group (10 rats), EAE group (30 rats), low dose atorvastatin treatment group (30 rats), high dose atorvastatin treatment group (30 rats). EAE group and treatment groups were divided into three groups: 14-day group, 21-day group, 28-day group, separately. All experimental rats were immunized subcutaneously in the four footpads and back with emulsion 0.5 ml, which including fresh GPSCH as antigen, emulsified with an equal volume of CFA containing Mycobacterium tuberculosis 6mg/ml. Clinical signs of EAE was assessed a minimum of twice daily by two investigators. Scores were assigned on the basis of the following symptoms: 0,normal mouse; 1, piloerection, tail weakness; 2, tail paralysis; 3, tail paralysis plus hindlimb weakness; 4, tail paralysis plus partial hindlimb paralysis; 5, total hindlimb paralysis; 6, hind and forelimb paralysis; 7, moribund/dead. Atorvastatin was dissolved in 0.9% salt solution and treated rats with oral administration at a daily dose 2mg/kg and 8mg/kg per rats in low dose and high dose groups. This treatment was started on the first day of immunization and continued daily for the duration of the experiment, and EAE group left untreated. During the experiment, the mean maximal score of animals at different time point and the incidence of disease were observed as results.Rats were sacrificed after anesthesia with intraperitoneal injection. Spinal cords were removed promptly and immersed in 4% paraformaldehyde less than 7 days, then the tissue were embedded in paraffin and sectioned at 4μm thickness. Some of the sections were stained with H&E to assess leukocyte infiltration and inflammation. Some of sections were studied with immunohistochemistry as described below. The slides were incubated with anti-MMP-9 antibody (1:100, polyclonal), then the tissues were further incubated with the second antibody, avidin-biotin-perxidase complex. Among these approaches, we should wash these specimens with PBS, at last, the results were analyzed with microscopy.The sera from rats were achieved and were detected the level of IL-4 with radiative immunization assay in order to discuss whether atorvastatin effects the IL-4 production. Spinal cords from rats were obtained individual at three different phases for histological and immunohistochemistry evaluation.Results1 The incidence of disease in different groupMost rats had neurofuction deficiency at different degrees, and the clinical signs were started at about 13~14 days after induction. Comparison of the incidence of disease in three different groups, we found that the frequency of EAE attacks in EAE group were higher than low and high dose treatment groups(P=0.08).2 The profile of relapsing-remitting EAEIn 28-day group, there were 7 rats had two attacks before being sacrificed and there were 4 and 2 rats in low-dose and high-dose treatment groups. The frequency of the second attacks in EAE group were significant higher than the high dose atorvastatin treatment group(P=0.02).3 Clinical profile of EAE in different groupMost rats had neurofuction deficiency at different degrees, comparison of the neurofuction deficiency in three different groups, we found that the degree of EAE attacks in EAE group were high than the treatment groups(P=0.03).4 Neuropathological findingsWe evaluated the leukocyte infiltration and inflammation with H&E staining. The results demonstrated before the clinical signs emerging, some mononuclear infiltrates were observed in the perivascular space and leptomeninges of spinal cord, what's more, the degree of infiltration was associated with the severity of EAE. The extent of inflammation in EAE group was higher than atorvastatin treatment groups(P=0.01).5 Immunohistochemical findingsIn the early phase, the small number of cells positive MMP-9 were found in spinal cord of rats, with the progress of disease, the number of MMP-9 positive cells was increased. In the treatment groups, the number of MMP-9 positive cells was less than the EAE group, and the therapeutic potential of atorvastatin occurred in a dose-dependent fashion(P=0.001).6 Analysis of IL-4 content in serumIL-4 is one of the most crucial cytokines in multiple sclerosis pathogenesis. In the early phase, the low level of expression of IL-4 in sera was observed, and the level of it correlated with the severity and activity of EAE. With the development of disease, when the rats were in the recovery phase, the level of IL-4 increased. While when the new clinical symptom appeared, IL-4 expression was observed in lower level again. In our study, atorvastatin can significant increase the expression of IL-4(P=0.04).Conclusion1 Atorvastatin reduces the incidence and the recurrence of disease in EAE rats, and protects the rats from the severity of the disease.2 Atorvastatin can reduce the expression of MMP-9, and then reduces the leukocyte infiltration and inflammation in spinal cord of EAE rats.3 IL-4 in serum is associated with the activity of EAE. Atorvastatin can increase the level of IL-4 in serum.4 The effect of atorvastatin on the EAE is dose-dependent. Only high dose atorvastatin can be detected this effect.