Studies On The Therapeutic Effect Of MCI-186 On Experimental Autoimmune Encephalomyelitis And Its Mechanism

Mutiple sclerosis (MS) is an autoimmune demyelinating disease of central nervous system (CNS) with preponderence in young females and high disability. Despite the progress in recent years, no effective therapeutic method can halt the disease. The etiology and pathogenesis of MS still are the hot points for researchers in the field of neurology. We don't have systemic study in this area due to the limitation of our technology. Our topic began with inducing the animal model of MS (experimental autoimmune encephalomyelitis, EAE). So we could study the mechanism of free radical in EAE and observe the therapeutic effect of free radical scavenger MCI-186 on treating EAE.In the future, it could provide the basis of theory in MS treated with MCI-186.Objectives: (1) To determine whether there were any differences in the days of disease onset and the maximum clinical scores in first attack of EAE induced with PLP139-151 among SJL/J mice with different ages; (2) To determine whether MRI scan could be used as a new method to assess the blood brain barrier (BBB) permeability in EAE; (3) To explore the change of Ca²⁺ fluorescence intensity in brain slices at the onset and the peak of EAEand determine the relationship between them; (4) To determine the effect of MCI-186 on the progression of the disease, BBB permeability, inflammatory infiltration,free radical generation, cell apoptosis of EAE, and to draw a conclusion about the potential mechanism of them. At the meanwhile we could find the best dose and time of MCI-186 for the model. Methods: (1) EAE was induced in female SJL/J mice by subcutaneous immunization with PLP139-151. The mice were divided into four groups according to their ages(6w,8w,10w and 12w). To compare the differences in the days of disease onset and the maximum clinical scores in first attack of EAE among SJL/J mice with different ages; (2) Mice were anesthetized with chloral hydrate by i.p. followed being injected constrast medium Gd-DTPA at tail veins. EAE mice were separately given routine and Gd-DTPA enhanced MRI examination in relapse and remission.The change of BBB permeability was judged by comparing the relative singal intensity at the relapse with the remission of EAE on the clearest scan in axle plane or coronal plane on Gd-DTPA enhanced MRI T1WI; (3) After the brain slices from EAE loaded with fluo-3/AM, the relative Ca2+ fluorescence intensity of cortices and parenchyma at the onset and peak of EAE and control goup were detected and compared by using laser confocal scanning microscope; (4) mice were immunized with PLP139-151 emulsified in complete freund' adjuvant at bilateral foot pad. Control group was injected with saline at tail veins.Another group was injected with MCI-186 at tail veins and i.p.. Eight days after immunization, bilateral inguinal lymphoid nodes were collected and the lymphocytes were suspended with the same volume RPMI 1640 and counted. The proliferation responses of lymphoid node cells stimulated by different concentration of MCI-186 with PLP139-151 were detected by 3H-thymidine incorporation. To observe the effect of MCI-186 on the course of EAE, different doses of MCI-186 were used before and after EAE onset. The BBB permeability was evaluated by Gd-DTPA enhanced MRI examination. The sections were stained with hematoxylin-erosin for histopathological examination. The severity of inflammation was graded in a double-blind fashion. Specific antibodies of NT and iNOS were used to detect their expression by immuohistochemistry and Western blot. To determine whether MCI-186 treatment modulates the extent of cell apoptosis in EAE, spinal cord or brain sections were assessed for the presence of cells with fragmented DNA by TUNEL assay.Results: (1) There were significant differences in the days of disease onset among four goups. The time of EAE onset in low age group was significantly earlier than that in old age group. The maximum scores in six weeks group were higher than those in twelve weeks group; (2) The relative signal intensity of focus on Gd-DTPA enhanced MRI TIWI in replase was much higher than that in remission. Both of them were higher than that of controlgroup; (3) Ca2+ fluorescence intensity on the cortices of brain slices was higher than that in parenchyma at the onset of EAE and in control. Ca2+ fluorescence intensity at the peak of EAE was significantly higher than that at the disease onset on cortices and the local areas of parenchyma ; (4) MCI-186 treatment could significantly suppress inflammation. The severity of inguinal lymphadenectasis in MCI-186-treated group was inferior to sham treatment group and the number of lymphocytes in the former was fewer than that in the latter. The proliferation response of lymphoid node cells in MCI-186-treated group was suppressed when compared with merely PLP139-151 stimulated group and control group. MCI-186 could delay and even prevent EAE onset and protect BBB from being damaged when administered before EAE onset, and suppress inflammatory infiltration and production of free radicals.Cell apoptosis was rare in MCI-186-treated group before EAE onset. If administered after EAE onset,MCI-186 could effectively prevent the progression of course and promote remission. It could restore BBB integrity and suppress inflammation and production of free radicals in EAE, but it did not play a role in inducing cell apoptosis. Conclusions:(l) There were differences in the days of onset and severity of EAE induced in SJL/J mice with different ages.The age of SJL/J mouse was an important effector in inducing EAE. EAE would better be induced in female SJL/J mice which ages are eight weeks to ten weeks. If intervention ofEAE was performed, span of the ages of female SJL/J mice used to induce EAE would better be lower than two weeks. Only that the experimental results would be reliable; (2) Gd-DTPA enhanced MRI scan could be used as an objective method to assess the BBB permeability; (3) In EAE mice there was obvious influx of Ca2+ in neural cells on the cortices and the local area in parenchyma and its increase was accompanied with the progression of EAE. Influx of Ca2+ in EAE might be the main reason of causing axonal degeneration and permanent deletion of nervous function; (4) The effect of MCI-186 treatment on EAE was certain before and after the onset of EAE. MCI-186 played an important role in EAE via protecting or restoring the BBB integrity and suppressing inflammation and production of free radicals. It also could stop antigenic drift to reduce the relapses of EAE.