| Objective: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) white matter.Its pathogenesis is not clearly,for the past few years, which were presumed as not only an autoimmunologic process caused by easy-infected heredity and environmental factors,but also a neural degeneration process which resulted in irreversible damage of myelin sheath and neuraxon.The damage was thought as the chief reason that lead to the neural functional deficits, experimental autoimmune encephalomyelitis(EAE) is acknowledged to be the ideal a paracmasia nimal model of MS,because its pathogenesis is very close to human's,which represent with the chronic process of acute episode and non-release,so it is admitted as one of the best animal models in the world.In our study we aim to induce an EAE model by MOG35-55in C57BL/6mice,with the interference of sulforaphane, and observe the safeguard effect of the sulforaphane on EAE for myelin sheath damage by the methods of electron microscope and Luxol Fast Blue myelin staining,moreover we may qualitatively observe the anti-inflammatory effect of sulforaphane by the method fo HE staining.At last by the method of immunohistochemistry we could observe whther sulforaphane could promote remyelination by increasing the expression of olig2.Methods:72female C57BL/6mice,inbred strain, aged8-10weeks, wheighted18-20g were randomly divided into four groups:normal group,EAE group,sulforaphane group, dexamethasone group. Except the normal group,the mouse of the other three groups were induced by immunized subcutaneously myelin oligodendrocyte glycoprotein (MOG)35-55emulsified with an equal volume of complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis4mg/ml to make EAE models. The mouse of sulforaphane group were given intraperitoneal injections of sulforaphane for5mg·kg-1every two days since immunized until sacrificed.The mouse of dexamethasone group were given intraperitoneal injections of dexamethasone for0.07mg every two days since immunized until sacrificed. The mouse of the other two groups were given intraperitoneal injections of physiological saline for1ml/rat at the same time. The efficacy of sulforaphane on the clinical manifestation was observed, and the clinical score was assessed in accordance with the standard generally used. Mouse of each group were randomly sacrificed on the13th day,20th day,30th day after immunized. The lumbar intumescentia of the spinal cords were stained with HE and LFB to observe the inflammation and myelin sheath in situ, and also stained with immunohistochemisty (IHC) to observe the distribution of the olig2immunopositive cells, The mouse of four groups were fixed with4%glutaraldehyde for electric microscope observation. All data were statistically analyzed by SPSS17.0software.The incidence rates were represented by percentages, the method for the comparison was chi square test.Measurement data were expressed as mean±standard deviation.The statistical method for comparisons of several groups was one-way ANOVA. LSD was used for the comparisons between groups. P value<0.05was considered statistically significant.Results:1Mouse conditions of different groups The incidence rate of sulforaphane group was33.33%,which was significantly lower than EAE group's(100%).The mean time for the mouse of sulforaphane group(18.78±3.11) to develop disease was significantly later than EAE group's(13.42±4.12).The highest clinical score of sulforaphane group(1.35±1.34) was significantly lower than EAE group's(2.82±1.14).The differences were all significantly statistical (P<0.05).2HE staining of different groups in lumbar intumescentia of spinal cords2.1Pre-disease There were scattered inflammatory cells on sections of EAE group.There was no inflammatory cells on those of sulforaphane group. 2.2crest-time There were diffuse inflammatory cells infiltrated and mass "blood vessel muffs" formed on sections of EAE group.There were less "blood vessel muffs" formed on sections of sulforaphane group and the inflammatory cells were mainly around small vessels.2.3paracmasia The "blood vessel muffs" and the inflammatory cells on sections of EAE group were diminished,while those of sulforaphane group were vanished.2.4There was no "blood vessel muff" or inflammatory cell on sections of control group.3transmission electron microscope(the lumbar intumescentia of spinal cords of different groups)3.1pre-disease The myelin sheath of lumber intumescentia white matter became loose and its shape became unregularly on the sections of EAE group,but there weren't much change for the myelin sheath and axon in the sections of sulforaphane and dexamethasone group.3.2crest-time On the sections of EAE group,the myelin sheath showed very loose layer structure,some showed disaggregated,broken,deficiency,the axon showed atrophy and degeneration,the structure became unclear;while on the sections of the sulforaphane and dexamethasone group,the myelin sheath showed a little loose,less disaggregation and broken,minority of axon degeneration,the structure was clear.3.3paracmasia On the sections of EAE group,the myelin sheath became looser,more axon showed atrophy and degeneration,the boundary become unclear,but the sulforaphane and dexamethasone group we could see myelin sheath dispost more tightly and axon's structure got more clear.4LFB staining of different groups in lumbar intumescentia of spinal cordsOn the sections of EAE group, showed some thiner myelin sheath but no demyelination area in the pre-disease, showed varying demyelination area in crest-time,more demyelination area in paracmasia;On the sections of sulforaphane and dexametasone groups, showed no myelin sheath change in pre-disease,some thiner myelin sheath and small demyelination area in crest-time,the myelin sheath became more and hicker in paracmasia.5Expression of olig2in lumbar intumescentia of spinal cords of different groups5.1Pre-disease In EAE group, the mean number of olig2immunopositive cells was (8.62±1.14).In sulforaphane group, the mean number of olig2immunopositive cells was (19.63±2.70), In dexametasone group,the mean number of olig2immunopositive cells was (40.00±4.00),sulforphane group and dexametasone group were significantly more than EAE group's (P<0.05).5.2crest-time In EAE group, the mean number of olig2immunopositive cells was (13.82±1.48), in sulforaphane group, the mean number of olig2immunopositive cells was (18.43±1.82), in dexamethasone group,the mean number of olig2immunopositive cells was (28.61±1.52), both groups'were significantly more than EAE group's(P<0.05).5.3paracmasia In EAE group, the mean number of olig2immunopositive cells was (27.81±1.92),In sulforaphane group, the mean number of olig2immunopositive cells was (17.68±1.14), in dexamethasone group,the mean number of olig2immunopositive cells was (22.02±2.01),the EAE group's was significantly more than sulforphane group's(P<0.05) and dexamethasone group's(P<0.05).Conclusions:1sulforaphane could lower the rate of EAE, delay the time of disease development, and alleviate the clinical severity of EAE2Sulforaphane could lessen the EAE animal model's inflammatory infiltration, decrease the myelin sheath deletion coused by immoderate inflammatory stimulation,promote remyelination, recover axon integrity and neural conductor function.3Sulforaphane could make olig2express abundantly in pre-disease, promote remyelination, relieve the patients'symptoms in time.While in the EAE group olig2generous expression occurred in paracmasia, couldn't promote remyelination in time, lead to myelin sheath and axon irreversible damage, so the patients'condition got more worse. |
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