Pathological Study On Axonal Injury Of A Rat Model Of Experimental Allergic Encephalomyelitis

BackgroudMultiple sclerosis (MS) is a disease of the central nervous system. Clinical manifestations of the disease alternate between remission and relapse, the patients of MS will be physically disabled in the later period of the disease. The histopathology of MS is complex and individualized, including different levels of reactive astrocytosis, macrophage activation, demyelination and axonal injury. As an ideal animal model for human MS. experimental allergic encephalomyelitis (EAE) is also an autoimmune disease, which mainly mediated by CD4positive T cells. The main histopathological features of the disease are demyelination and mononuclear cells infiltration around the small blood vessels in the central nervous system. With different encephalitogens. immunological methods and animals we can induce various EAE models, such as acute EAE. relapsing-remitting EAE, and chronic EAE. In this study, we induce the EAE animal model in femal Wistar rat with Cavy’s spinal cord homogenate to observe the course, clinical symptoms and pathological changes of EAE, especially to explore the characteristics of axonal injury in EAE.Historically, the inflammation and demyelianation has been focused on in the MS study. In recent years, there are considerable evidences to show that the irreversible neurological impairment of MS in the late stage is correlated with the increasing axonal injury. As an important pathological feature of MS, axonal injury may occur in the early stage of the disease, which may finally impact on the clinical manifestations and prognosis of MS. Different stages of axonal injury in MS may have different mechanisms, acute axonal injury may be caused by acute inflammatory demyelination, which can result in neurological dysfunction in the early stage of the disease. The accumulation of a variety of inflammatory factors, nitric oxide, glutamate and other toxic substances in the inflammatory condition can cause demyelination and axonal injury. When the axonal injury develops up to a threshold, it will trigger the pre-programmed neurological degeneration, and result in distinct neurological dysfunction. Therefore, we should initiate the treatment of anti-inflammation and axonal protection in the early stage of MS. in order to prevent and postpone the neurological disability effectively. The neurological dysfunction in the late stage of MS may be associated with the progressive axonal injury in the chronic demyelinating plaques, and the more severe axonal injury occurs, the more worse the disease’s prognosis comes up. Thus it can be seen, axonal injury can occur in the early stage of MS; permanent disability associates with axonal injury’in MS patients; axonal injury can play an important role in the MS; It is crucial to ascertain the mechanisms of axonal injury in MS in order to provide evidences for the clinical management in MS.As a specific hallmark of acute axonal injury, amyloid precursor protein (APP) accumulation may be found in the demyelination plaques in MS patients, which demonstrated that the axonal injury has occurred as well as demyelination. Neurofilament is the basic constituent of the axon cytoskeleton. Its phosphorylative states may be dynamically impacted by demyelination and axonal pathologic changes. Nonphosphorylated neurofilament (SMI-32) exists in the neuron cell bodies and dendrites. Neurofilament is phosphorylated in the normal axonal which wrapped by the myelin sheath, which should be negative-stained with the SMI-32stainning. So when amount of SMI-32positive-stained fibers is found, we can speculated that axonal injury has already occurred. In this study, we intend to establish EAE animal model with female Wistar rat, to observe the inflammation, demyelination and axonal injury of EAE, to ascertain the pathogenesis of MS, especially for the characteristics of the axonal injury, in order to provide evidences for the clinical management in MS.Method1. Guinea pigs were sacrificed under sterile condition, separated the spinal cord subsequently and grinded with a small amount of saline to prepare fresh spinal cord homogenate, mixed with an equal volume of complete Freund adjuvant to make dubbed antigen-adjuvant emulsion (GPSCH-CFA) for the use of preparing EAE animal model.2. Wistar rats as subjects to induce EAE model, which separated into4groups: One control group and three EAE model groups as EAE4d group, EAE14d group, and EAE24d group. Each rat of the EAE model group was injected into double foot pad subcutaneously with0.4ml GPSCH-CFA, and400ng pertussis toxin in100ul PBS was administrated intraperitoneally on the day0h and after48h. Each rat in the control group was injected with NS or CFA only at the same position.3. We carried out behavioral study, post-onset clinical symptom evaluation after injection with GPSCH. All the experimental data are expressed by the mean±standard deviation, using the statistical software SPSS13.0analysis. The brain and spinal cord of EAE rats were processed with HE. luxol fast blue and. Afterwards, we proceeded with pathological study with HE staining, LFB myelin staining, Bielschowsky axonal silver staining, as well as double-labeling indirect immunofluorescence on SMI-32and MBP, APP and MBP.Result1. Behavior changes The EAE rats emerged neurological deficits8to12days after immunization. Early symptoms included limp tail, turning back difficultly from the supine position, waddling gait or mild ataxia, hindquarter weakness, severe hindleg paralysis and urinary incontinence and weight loss. With the progression of the disease, the neurological deficits gradually worsened and peaked14to16days after immunization. And then, these symptoms started to relieve. Some rats could recover absolutely, only a few of them remained symptom of limp tail. The disease course of EAE manifested as an acute process, and the incidence rate was100%. The average neurological score in the peak was3.84points. The body weight of the rats in the EAE groups generally declined in the early stage of EAE. With the remission of clinical symptoms during the recovery period, body weight gradually recovered. The rats in the control group didn’t manifested any neurological deficits, and the body weight continued to rise.2. Pathological changes①HE staining:There were not significant lymphocyte infiltration, neurocytes necrosis, demyelination. and axonal injury in the brain and spinal cord in the group of EAE4d. The rats of group EAE14d had lots of cuffing lesions of inflammatory cell infiltration in brain and spinal tissue accompanied by multiple lesions of demyelination, axons disarranged with vesicular loss. Compared to rats in the group EAE14d, the inflammation was much serious in the brain and spinal cord of rats in the group of EAE24d, inflammatory cell infiltration in the demyelinating lesions reduced. The control rat has no obvious pathological changes in the tissue.②LFB myelin staining:Demyelination was observed with inflammatory cell infiltration.③Bielschowsky axonal silver staining:Axonal rupture, serious disorganization, and vacuolar changes were revealed.④Immunofluorescence: We observed the distribution of SMI-32and MBP with double-labeling indirect immunofluorescence under confocal microscopy, there was no obvious abnormalities in the rats of group EAE4d. The positive staining of SMI-32in the spinal cord of the rats in the group EAE14d revealed that there was a lot of nonphosphorylated neurofilament, indicating the existence of axonal injury. There were axon oval balls in the white matters of spinal cord, formed by APP accumulation in the axon. It was indicated that there was neuraxial damage occurred nonsynchronously with Myelin loss. These changes still existed in the rats in the group EAE24d.Conclusion1. The EAE rat model can be successfully induced with GPSCH-CFA, with a high incidence, good stability and feasibility. The EAE animal model manifested as acute process, as an ideal animal model of MS.2. Different levels of inflammation appearred at different stages of the EAE, the most obvious inflammatory cell infiltration occurred from the peak stage of the EAE.3. Demyelination was observed with inflammatory cell infiltration from the peak stage of the EAE.4. The neuraxial damage occurred in the early stage of EAE and covered the entire course, revealed as axonal rupture, serious disorganization, and vacuolar changes.5. The neuraxial damage occurred nonsynchronously with Myelin loss.