| Objective: To observe pathological changes of optic nerve,brain,spinal cord through light microscope and electron microscope and variations of ordinary photographs in different times for understanding the state and degree of the pathological changes of them in EAE. To observe the corresponding changes in indicators of optic nerve and axonal injury byα-lipoic acid on the intervention of EAE in rats for understanding of the protective effect on optic nerve and axonal injury, and thus may provide new ideas and experimental evidence for clinical treatment of MS.Methods: A total of 72 adult healthy female Wistar rats, were divided randomly into different groups: control group (NC, 12 rats) immunized by physiological saline and CFA. Other rats were immunized by 1:10 fresh guinea pig spinal cord homogenate (GPSCH). After first paroxysm, they were divided randomly into EAE group and LA group, and the 2 groups were divided into 7d after the first attack group and half a year group. LA group were injected intraperitoneal LA 100mg·kg-1·d-1. This treatment was started on the first day of EAE outbreak and continued daily for 7 days.Weigh the whole guinea pig spinal cord, mix spinal cord and icy salt water separately with the proportion W/V 1:10 to produce homogenate, then a 1:1 mixture of the homogenate and complete Freund's adjuvant (CFA) resulted in the encephalitogenic emulsion,is made completely immune antigen. 0.5ml/rat, hypodermic injection in five points of foot pads and the back.Clinical signs of EAE were assessed once 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.Blood were taken from heart in rats in each group after anesthesia with intraperitoneal injection at 7th after the first attack and half a year of the different group within after immunization. The optic nerve,brain and spinal cord tissues were got and embedded in paraffin. The sections were stained with HE staining, pathological changes were observed under light microscope. The ultrathin sections were observed under electron microscope for pathological changes of myelin sheath and axon.Quickly took spinal cord tissue to detectβ-APP in western blot. Ordinary photographs were carried out on 7d after first paroxysm and half a year in each group. The MDA and SOD level activity in the tissues of rats was measured by MDA kit and SOD kit.Results:1 The long-term course of disease in different group50 of the rats were firstly attacked after immunization. 15 of them were randomly taken as LA group. Each 7 of EAE group and LA group were carried out on 7d after the first attack. 36 of the others were observed, 29 of EAE group, 7 of LA group. At half a year, rats in long-term course of EAE group were observed the highest 5 times of EAE only 1(3.45%). At half a year, rats in long-term course of LA group were observed the highest 2 times of diseases 3(42.86%), 1 times were 4(57.14%). Comparison of the long-term course of disease in three groups, we found the frequency of EAE attacks in EAE group were higher than the treatment groups (P <0.05).2 Histopathologic examination2.1 HE staining7d after the first attack: the infiltration of inflammatory cells inside the optic nerve,brain tissue,spinal cord and aroud the choroid plexus and the perivascular cuffings of the disease. The degree of the LA group is much slighter than the EAE group in brain and spinal cord. At half a year: the infiltration of inflammatory cells inside the optic nerve,brain and spinal cord tissue and aroud the choroid plexus of the disease. The structure of the brain and spinal cord of the LA group loosed but almost normal, without the infiltration of inflammatory cells at half a year.2.2 Electron microscope7d after the first attack: the myelin sheath appearance of the optic nerve,brain and spinal cord tissue of the disease and the LA group were inordinate, with conspicuous rarefaction,disaggregation,confluens or absence. The destruction of axon were obvious, with indefinite construction or even absence. The mitochondria inside the axon were swollen, even with vacuolization. At half a year: the destruction of myelin sheath of optic nerve,brain and spinal cord of the disease were obviously, including conspicuous rarefaction,disaggregation. The destruction of axon was obvious, with degeneration,atrophy, even absence of the axon. The myelin sheath of optic nerve,brain and spinal cord tissue in the LA group showed a compact lamellar structure, without conspicuous rarefaction,disaggregation.3β-APP in western blotβ-APP was expressed in control group, but very little; the expression ofβ-APP increased in the first attack period, the expression decreased at half a year; by LA intervention,β-APP expression decreased.4 Ordinary photographsThe normal boundary of papilla optica was clear at the seventh day of the first attack and half a year in the control group and the LA group.The papilla optica was atrophied, and their borders were unclear at the seventh day of the first attack and half a year of the disease, the optic nerve heads became more atrophiy in half a year.5 The MDA level in the tissuesMDA level in the tissues of rats in EAE of half a year group is higher than other group(P<0.01). MDA level in the tissues of rats in EAE at half a year group is higher than the seventh day of the first attack of the disease (P<0.01).6 The SOD level in the tissuesSOD level in the tissues of rats in EAE at the seventh day of the first attack is higher than other group(P<0.01). SOD level in the tissues of rats in EAE and LA of half a year group is higher than control group (P<0.01). SOD level in the tissues of rats in LA group of half a year is higher than of the seventh day after the first attack (P<0.01).Conclusions:1 Optic neuropathy is obvious in EAE rats, inflammation,demyelination and axonal damage are the main changes.2 LA can protect the rats from the severity of the disease and reduce the morbidity.3 Through the HE staining and transmission electron microscope observation, axonal injury in pathological changes are observed in the EAE rats.4 MDA,SOD in serum of EAE rats,β-APP 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.5 Alpha-lipoic acid on EAE in rats has protective effect on optic nerve and axonal injury, its protective effect may be adopted to achieve by anti -oxidative stress.
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