| BACKGROUNDMultiple Sclerosis (MS) is chronic inflammatory and demyelinating disorder disease involving white matter with the numerous affected areas of central nervous system possibly due to autoimmunity, which classically follows a relapsing remitting course associated with increasing neurological deficits. MS usually leads to patients experience multiple nerve dysfuntion including muscle weakness or paralysis, dysphagia, urinary or fecal incontinence, which serious effect to patient's living quality. The disease incidence of MS is higher in Northern Europe and North America than Africa and oriental area. With the increasing improvement of diagnosis instrument, the disease incidence of MS recently shows a ascending tendency obviously in China.The typical MS lesion macroscopic observation as a ocalized, sharply demarcated, grayness and translucency sclerosis maculae within the white matter of nervous system. In light microscope the pathologic characteristics for perivascular inflammatory cells infiltrating accompanied by regeneration, axon damage and astrocytes scarring. However the major cause of myelin sheath disorganization in the central nervous system is still unknow. Many studies supported that MS is an autoimmune disease. Because it was found that the immune cells such as CD4+ Th1 existed in CNS lesion, suggesting the abnormal immune responses participate the central nervous system inflammatory reaction. Molecular mimicry theory presumed that a common antigen shared by viruses which MS patients infected and host CNS myelin protein components or oligodendrocytes, these viruses or some super antigens activate T cells (mainly CD4+ Th1 cells), these activated T cells permeate blood-brain barrier under the help of adhesion molecules to combined with antigen presenting cell to cause inflammation demyelinated reaction in the central nevous system. After receiving altered peptide lligand treated, a strong induced reaction of MBP reactive CD4+T cell was found in MS patients' spinal fluid and peripheral blood, these MBP reactive T cells produce high level of TNF-αand IFN-γ, suggesting that the peripheral activated CNS itself reactive T cells really involved in the pathogenesis of MS.Currently, there is still no effective treatment protocols for MS, the existing treatments are mainly aimed at the immunological ingredients. In China, common drugs used for treating MS are corticosteroids, interferon beta and ciclosporin A. In recent years, there are new drugs approved in MS treatment include:interferon beta, acetate (GA) meters novantrone et al, but the disaffect efficacy, high price, potential side effects and toxicity of these drugs limiting their clinical promotion. Therefore, exploring new treatment for MS become a very urgent topic.Fluoxetine, one of selective serotonin reuptake inhibitor (SSRI), is commonly used drug for depression. Recently, a double-blind, placebo-controlled exploratory trial showed fluoxetine may have potential benefical effect on MS, because this study found that fluoxetine reduced the formation of new enhancing lesions in patients with MS based on brain MRIs findings Suggesting that fluoxeting fluoxetine may have myelin protection. However, the mechanism underlying the fluoxetine effect on MS is not clear.Recent studies suggested the fluoxetine had immunoregulative effect via regulating 5-HT reuptake in presynaptic membrane of the central nervous system, increasing remarkably the 5-hydroxytryptamine (5-HT) content in synaptic space. 5-HT have stimulatory or inhibitory effects on immune cells including B, T and NK cells, and monocytes/macrophages. And fluoxetine may regulate immune system via directly down-regulating the activation of T-Lymphocytes and the expression of TNF-α, IFN-γand inhibiting the activation of NF-κB. What's more, studies reported fluoxetine have neuro-protective effect by up-regulating the expression of BDNF, which plays an important role in differentiation, outgrowth and survival of peripheral and central neurons during brain development and in neuronal recovery after brain damage.In one word, fluoxetine have potential neuroprotective in the CNS, however whether fluoxetine has direct effect on MS need further further laboratory and clinical study.OBJECTIVEThis study objective was to examine whether fluoxetine can have protective effect on MS via the Experimental Autoimmune Encephalomylitis (EAE) model. And the potiental mechisms responsible for the fluoxetine effect on MS was exploredMETHOD:1. Rats were randomly divided into four groups:Model control group, Saline model control group, Low-dose fluoxetine group (10mg/kg) and High-dose fluoxetine group (20mg/kg). Each rat in four groups was received guinea pig spinal cord homogenate and pertussis toxin (PT) to prepare EAE model. Rats in Low-dose fluoxetine group (10mg/kg) and High-dose fluoxetine group were given fluoxene (10mg/kg or 20mg/kg) by daily gavage since 14 days before antigen emulsion. Rats of the saline model group were given saline instead of fluoxetine as scheduled. The drug/saline was continuously administered for 25 days. Model control group was not given either fluoxetine or saline.2. The clinical score were assigned on the basis of the presence of the following symptoms:0, normal; 1, piloerection, tail weakness; 2, tail paralysis; 3, tail paralysis plus hindlimb weakness; 4, tail paralysis plus partial hindlimb paralysis; 5, total hindlimb paralysis; 6, hind and forelimb paralysis; 7, moribund/dead.3. Histology changes in each group were messured by hematoxylin and eosin (HE) and Luxol Fast Blue (LFB) method.4. The cytokines expression in plasma samples were measured by ELISA medthod. The plasma was collected on day 0, day 16, and day 25 after immunization from rats in each group.Results1. The manifestations of our EAE rats in clinical signs, HE and myelin staining were all corresponding with the typical clinical and pathological feature of EAE.2. Each rat in four groups began to show neurological deficit 4-5 days after immunization. The first sign of those rats was presented as piloerection and distal tail weakness. Then EAE rats gradually developed complete tail paralysis, hindlimb paralysis, and hind and forelimb paralysis. Each score, per animal, reached the peak at approximately day 16 after immunization, and then began to descend, followed by gradual and partial recovery. At day 17 after immunization, the score of neurological deficit in rats began to descend; The mean clinical scores of both the low-dose fluoxetine group and high-dose fluoxetine group were significantly different from those of the model control group and saline model group at day 18 after immunization and continued to the end of the experiment;3. No significant difference on animal mortality was found among model control group, saline model control group and low-dose fluoxetine group. But the animal mortality of high-dose fluoxetine group was higher than other three groups.4. Significant decrease of inflammatory foci and demylination scores were found in low-dose fluoxetine group comparing with that in the model control group and the saline control group based on the histologic results.5. The serum IFN-γlevel in model control group, saline model control group and low-dose fluoxetine group increased on the day 16 after immunization and then decreased to normal range on the day25 after immunization. Significant difference of the serum IFN-γlevel was found between 10mg/kg fluoxetine group comparise with that in the model control and saline control groups. Those difference were not found on the day before immunization and on the day25 after immunization; In contrast, there were no significant difference among the three groups for TNF-αand BDNF levels in the three defferent time points.ConclusionOur study first proved that spinal cord inflammatory foci in histopathological analysis was attenuated by fluoxetine and down-regulated the IFN-y in relation to remission of neurological deficits-linked behaviors, and the spinal cord demyelination was also attenuated. Thus, the immnomodulatory mechanism for fluoxetine may take place in the CNS. Thus, fluoxetine may have a potential for remission of MS, while the appropriate dose remains to be determine in order to avoid serotonin-toxicity. |
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