| Objective :Multiple sclerosis(MS) is a common demyelination disease of the central nervous system(CNS). For a long time, multiple sclerosis has been considered a primary demyelinating disease of the CNS extensive demyelination and axonal structure is relatively preserved. Until the late nineties of the last century ,foreign scholars confirmed the existence of axonal injury in acute and chronic MS lesions;Impaired axonal was found in multiple sclerosis plaques or on its edges and in the general form of the normal white matter also had a small amount of axonal damage.The axonal injury of Lesions can cause wanerina degeneration and lead to axonal loss.Currently considered Ms axonal degeneration or damage, one of the main pathological changes,started to occur early in the course of the disease and may have contributed to the clinical neurological dysfunction.Degree of axonal injury associated with the severity of the disease.Therefore,currently the exploration on mechanism and related protective effect of axonal injury in multiple sclerosis is becoming a research hotspot.The cause and exact mechanism of axonal damage is currently still unclear.At present, some studies at home and abroad have shown that: oxidative stress play a very important role in the pathogenesis of MS. Oxygen free radicals and other metabolites generated by Oxidative stress can oxidize biological macromolecules in vivo,induce free radical chain reaction,generate a large number of lipid peroxidation, cause membrane damage, a variety of subcellular structural damage, enzyme activity decreased or disappeared, cause cell autolysis.α-lipoic acid as a strong antioxidant, has attracted wide attention of people. It belongs to vitamin B compounds, is a natural anti-oxidants. It is mainly through scavenging free radicals, chelating metal ions, recycled from other anti-oxidants play its antioxidant activity. At abroad, it through intervention the process of oxidative stress, reducing free radical damage of blood vessels and nerves has been used to treat diabetic peripheral neuropathy and has achieved good efficacy. There is little research onα-lipoic acid and multiple sclerosis at home and abroad,However,α-lipoic acid on axonal injury in MS whether the protective effect is unclear.In this study, by establishing a model of multiple sclerosis - experimental autoimmune encephalomyelitis (EAE), related indicators of axonal injury were detected in the pathogenesis of rats , to understand axonal injury in the pathogenesis of MS. Byα-lipoic acid on the intervention of EAE in rats to observe the corresponding changes in indicators of axonal injury, understanding of protective effect for axonal injury, and thus for clinical treatment of MS to provide new ideas and experimental evidence.Methods :1 Animal group: A total of 80 adult healthy female Wistar rats with body weighing between 180 and 200g were divided randomly into different groups: normal control group(NC,6 rats), other rats were immunized by fresh guinea pig spinal cord homogenate(GPSCH). After they first paroxysm,divided randomly out 6 rats as disease group(DIS),others divided randomly into EAE group, dexamethasone(DXM 2 mg·kg-1·d-1) group, alpha-lipoic acid (LA 100mg·kg-1·d-1 ) group, and the 3 groups were divided into Remission group(REM ), relapse group(REL) two sub-groups according to the course of the disease, each sub-group 6 rats . No recurrence in DXM group and; LA group, according to the separately mean days of remission and relapse of EAE in preliminary experiments,the rats were divided into 7 days group after intervention (DXM7d group; LA7d group)and 14 days group after intervention (DXM14d group; LA14d group).2 Model induction: Fresh whole guinea pig spinal cord homogenate and an equivalent complete Freund's adjuvant ( CFA) is made completely immune antigen. 0.5ml/rat,hypodermic injection in five points of foot pads and the back. This treatment was started on the first day of EAE outbreak and continued daily for 7 day.3 Neural function assessment: Clinical signs of EAE were assessed the mean of twice daily by two observations. Scores were assigned on the basis of the following symptoms: 1, tail weakness; 2, tail weakness plus limb asthenia; 3, mild limb paralysis; 4, severe limb paralysis; 5,moribund/dead.4 Histopathology:Rats were sacrificed after anesthesia with intraperitoneal injection. Tissues of the spinal cord were fixed with 4﹪formalin, then the tissues were embedded in paraffin. The sections were stained with modified trichrome staining and modified Bielschowsky silver staining, pathological changes were observed under light microscope.5 immunohistochemistry: Tissues of the spinal cord were embedded in paraffin and sectioned at 6μm thickness. The slides were respectively incubated with anti-NF and anti-β-APP , then the tissues were further incubated with the second antibody, avidin-biotin-perxidase complex. Among these approaches, we should wash these specimens with PBS, DAB color, at last, the results were analyzed with microscopy.6β-APP in Western blot: Quick to take spinal cord tissue placed in -80℃refrigerator spare. Extract protein, electrophoresis, transfer film, closed, plus first antibody ,washing film, plus second antibody, washing film, color.At last, image acquisition, data analysis.Results :1 The relapse of disease in different groupComparison of the relapse of disease in three groups, we found the frequency of EAE attacks in EAE group were higher that the treatment groups (P <0.05).2 Clinical profile of EAE in different groupNeurological deficits scores of DXM group,LA group were lower than those of EAE group (P <0.01).3 Histopathology3.1 trichrome staining In the normal control group, the vast majority of axonal thickness uniformity, course direction of a more consistent, the continuity of axonal integrity. Varying degrees axonal changes appeared in the other groups.The axon with inhomogeneous chromatosis was swollen and beaded-discontinued;The myelin was porous and partly disaggregated.3.2 modified Bielschowsky silver stainingIn the normal control group, the vast majority of axonal thickness uniformity, course direction of a more consistent, the continuity of axonal integrity. Varying degrees axonal changes appeared in the other groups. Axon appeared swelling, uneven thickness, deformed, pale staining vacuoles, fracture,retraction ball change.4 Immunohistochemistry4.1 Expression of NFIn normal group, the expression of NF was most; expression of NF decreased in the disease period, an increase in the expression of remission, expression again decreased in the relapse period; by DXM, and LA intervention,NF expression increased.4.2 Expression ofβ-APPβ-APP was expressed in normal group, but a very small number, expression ofβ-APP increased in the disease period, an decreased in the expression of remission, expression again increased in the relapse period; by DXM, and LA intervention,β-APP expression decreased.5β-APP in western blotβ-APP was expressed in normal group, but a very small number, expression ofβ-APP increased in the disease period, an decreased in the expression of remission, expression again increased in the relapse period; by DXM, and LA intervention,β-APP expression decreased. The results consistented with the results ofβ-APP immunohistochemistry.Conclusions :1 LA reduces the morbidity, and protects the rats from the severity of the disease. 2 Through the modified trichrome staining and modified Bielschowsky silver staining,axonal injury in pathological changes are observed in the EAE rats.3β-APPand NF in spinal cord are associated with the activity of EAE, with the course of development has been a definite trend. Drug intervention on various indicators have an impact. alpha-lipoic acid on EAE in rats have protective effect on axonal injury and its protective effect may be adopted to achieve by anti-oxidative stress.
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