Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Emodin attenuates inflammation and demyelination in experimental autoimmune encephalomyelitis

Emodin,a substance extracted from herbs such as rhubarb,has a protective effect on the central nervous system.However,the potential therapeutic effect of emodin in the context of multiple sclerosis remains unknown.In this study,a rat model of experimental autoimmune encephalomyelitis was established by immune induction to simulate multiple sclerosis,and the rats were intraperitoneally injected with emodin(20 mg/kg/d)from the day of immune induction until they were sacrificed.In this model,the nucleotide-binding domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome and the microglia exacerbated neuroinflammation,playing an important role in the development of multiple sclerosis.In addition,silent information regulator of transcription 1(SIRT1)/peroxisome proliferator-activated receptor-alpha coactivator(PGC-1α)was found to inhibit activation of the NLRP3 inflammasome,and SIRT1 activation reduced disease severity in experimental autoimmune encephalomyelitis.Furthermore,treatment with emodin decreased body weight loss and neurobehavioral deficits,alleviated inflammatory cell infiltration and demyelination,reduced the expression of inflammatory cytokines,inhibited microglial aggregation and activation,decreased the levels of NLRP3 signaling pathway molecules,and increased the expression of SIRT1 and PGC-1α.These findings suggest that emodin improves the symptoms of experimental autoimmune encephalomyelitis,possibly through regulating the SIRT1/PGC-1α/NLRP3 signaling pathway and inhibiting microglial inflammation.These findings provide experimental evidence for treatment of multiple sclerosis with emodin,enlarging the scope of clinical application for emodin.

Alcohol intolerance and myalgic encephalomyelitis/chronic fatigue

BACKGROUND The literature is mixed about the occurrence of alcohol intolerance among patients with myalgic encephalomyelitis/chronic fatigue syndrome(ME/CFS).Surveys that asked respondents with ME/CFS whether they experienced alcohol intolerance within a recent time frame might produce inaccurate results because respondents may indicate that the symptom was not present if they avoid alcohol due to alcohol intolerance.AIM To overcome this methodologic problem,participants in the current study were asked whether they have avoided alcohol in the past 6 mo,and if they had,how severe their alcohol intolerance would be if they were to drink alcohol.METHODS The instrument used was a validated scale called the DePaul symptom questionnaire.Independent t-tests were performed among the alcohol intolerant or not alcohol intolerant group.The alcohol intolerant group had 208 participants,and the not alcohol intolerant group had 96 participants.RESULTS Using specially designed questions to properly identify those with alcohol intolerance,those who experienced alcohol intolerance vs those who did not experience alcohol intolerance experienced more frequent/severe symptoms and domains.In addition,using a multiple regression analysis,the orthostatic intolerance symptom domain was related to alcohol intolerance.CONCLUSION The findings from the current study indicated that those with ME/CFS are more likely to experience alcohol intolerance.In addition,those with this symptom have more overall symptoms than those without alcohol intolerance.

Role of nuclear factor κB in multiple sclerosis and experimental autoimmune encephalomyelitis

The transcription factor nuclear factor κB（NF-κB） plays major roles in inflammatory diseases through regulation of inflammation and cell viability.Multiple sclerosis（MS） is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system（CNS）.It has been shown that NF-κB is activated in multiple cell types in the CNS of MS patients,including T cells,microglia/macrophages,astrocytes,oligodendrocytes,and neurons.Interestingly,data from animal model studies,particularly studies of experimental autoimmune encephalomyelitis,have suggested that NF-κB activation in these individual cell types has distinct effects on the development of MS.In this review,we will cover the current literature on NF-κB and the evidence for its role in the development of MS and its animal model experimental autoimmune encephalomyelitis.

MicroRNAs as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis

Multiple sclerosis is an autoimmune neurodegenerative disease of the central nervous system characterized by pronounced inflammatory infiltrates entering the brain,spinal cord and optic nerve leading to demyelination.Focal demyelination is associated with relapsing-remitting multiple sclerosis,while progressive forms of the disease show axonal degeneration and neuronal loss.The tests currently used in the clinical diagnosis and management of multiple sclerosis have limitations due to specificity and sensitivity.MicroRNAs(miRNAs)are dysregulated in many diseases and disorders including demyelinating and neuroinflammatory diseases.A review of recent studies with the experimental autoimmune encephalomyelitis animal model(mostly female mice 6–12 weeks of age)has confirmed miRNAs as biomarkers of experimental autoimmune encephalomyelitis disease and importantly at the pre-onset(asymptomatic)stage when assessed in blood plasma and urine exosomes,and spinal cord tissue.The expression of certain miRNAs was also dysregulated at the onset and peak of disease in blood plasma and urine exosomes,brain and spinal cord tissue,and at the post-peak(chronic)stage of experimental autoimmune encephalomyelitis disease in spinal cord tissue.Therapies using miRNA mimics or inhibitors were found to delay the induction and alleviate the severity of experimental autoimmune encephalomyelitis disease.Interestingly,experimental autoimmune encephalomyelitis disease severity was reduced by overexpression of miR-146a,miR-23b,miR-497,miR-26a,and miR-20b,or by suppression of miR-182,miR-181c,miR-223,miR-155,and miR-873.Further studies are warranted on determining more fully miRNA profiles in blood plasma and urine exosomes of experimental autoimmune encephalomyelitis animals since they could serve as biomarkers of asymptomatic multiple sclerosis and disease course.Additionally,studies should be performed with male mice of a similar age,and with aged male and female mice.

Sinomenine reduces iNOS expressionvia inhibiting the T-bet IFN-γ pathway in experimental autoimmune encephalomyelitis in rats

Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating rheumatic and arthritic diseases. In our previous studies, we found that sinomenine reduced cellular infiltration within the spinal cord and alleviated experimental autoimmune encephalomyelitis （EAE） in rats. In this study, we further investigated the mechanisms of sinomenine treatment in EAE rats. In EAE rats, treatment with sinomenine exerted an anti-inducible NO synthase （anti-iNOS） effect, which is related to the reductions of Thl cytokine interferon-y （IFN-7） and its transcription factor, T-bet, in spinal cords. Moreover, sinomenine treatment of splenocytes stimulated with anti-CD3 antibody and recombinant rat in- terleukin 12 reduced the expression of T-bet and IFN-y in vitro and also reduced the capability of supernatants of splenocyte culture to induce iNOS expression by primary astrocytes. However, sinomenine had no direct inhibito- ry effect on iNOS produced by astrocytes cultured with IFN-y and tumor necrosis factor α in vitro. In conclusion, the anti-iNOS effect of sinomenine on EAE is mediated via the suppression of T-bet/IFN-y pathway.

Effects of Yishendaluo decoction on axonal degeneration,inflammatory reaction,and neurological function in a mouse model of experimental autoimmune encephalomyelitis

BACKGROUND： Yishendaluo decoction reduces production of inflammatory mediators, relieves damage due to inflammatory reactions, and improves neural functions during experimental autoimmune encephalomyelitis. OBJECTIVE： To investigate the effects of Yishendaluo decoction on a mouse model of experimental autoimmune encephalomyelitis. DESIGN, TIME AND SETTING： The randomized, controlled, neuropathological, and molecular biological animal study was performed at the Key Laboratory of Chinese Internal Medicine, Ministry of Education, Dongzhimen Hospital of Beijing University of Chinese Medicine and Center for Neuroinformatics, General Hospital of Chinese PLA from 2005 to 2006. MATERIALS： Yishendaluo decoction pieces consisting of prepared rehmannia root, colla comus cervi, cape jasmine fruit, and grassleaf sweetflag rhizome were purchased from the Dongzhimen Hospital of Beijing University of Chinese Medicine. Rabbit anti-mouse β-amyloid precursor protein and p38 polyclonal antibody （Zhongshan Goldenbridge Biotechnology, China）, as well interferon-y and interleukin-4 ELISA kit （Boster, China）, were used in this study. METHODS： A total of 96 healthy, female, SJL/J mice, aged 8 12 weeks, were equally and randomly assigned to normal, model, hormone, and Chinese medicine groups. A total of 0.2 mL antigen preparation, supplemented with 150 μg PLP 139-151 and 400 μg H37RA, was subcutaneously injected into the upper abdomen of mice from the model, hormone, and Chinese medicine groups. Mouse models of experimental autoimmune encephalomyelitis were established by intravenous injection of 0.1 mL Bordetella pertussis solution containing 0.6 × 10^6 Bordetella pertussis at days 1 and 3. Mice from the model, Chinese medicine, and hormone groups were respectively subjected to 0.2 mL saline, 2 g/kg Yishendaluo decoction, and 0.078 mg/kg prednisone acetate, once daily for 14 consecutive days. Mice from the normal group were left intact. MAIN OUTCOME MEASURES： Pathological changes were observed using hematoxylin-eosin staining and Luxol fast blue staining. Expression of β-amyloid precursor protein and p38 protein was determined by immunohistochemistry. Levels of interferon-y and interleukin-4 were detected by ELISA. Behavioral changes were assessed in mice according to scores of neurological function. RESULTS： A few inflammatory cell infiltration, nerve fiber breakage and slight demyelination were detected in the central nervous system of mice from the Chinese medicine and hormone groups compared with the model group. Expression of β-amyloid precursor protein and p38 protein was significantly diminished in the central nervous system of mice from the Chinese medicine and hormone groups compared with the model group （P 〈 0.05 or P 〈 0.01）, and the decrease was greatest in the Chinese medicine group. The decrease in mouse weight was not significant, and neurological function scores were less in the Chinese medicine and hormone groups compared with the model group （P 〈 0.05 or P 〈 0.01）. Interferon-y levels were significantly reduced （P 〈 0.01）, and interleukin-4 levels were significantly increased （P 〈 0.01） in the brains of the Chinese medicine and hormone groups, compared with the model group. CONCLUSION： Yishendaluo decoction improved neurological function in mice with experimental autoimmune encephalomyelitis by downregulating β-amyloid precursor protein expression, resistingaxonal degeneration, and relieving inflammatory reaction. The anti-inflammatory mechanism was regulated by inhibition of the p38 mitogen-activated protein kinase signal pathway.

Effect of erhuangfang on cerebral and spinal demyelination and regeneration as well as expression of glial fibrillary acidic protein in rats with experimental allergic encephalomyelitis

BACKGROUND： It demonstrates that erhuangfang can improve clinical symptoms of multiple sclerosis and relieve side effects of hormone. However, whether erhuangfang can improve experimental allergic encephalomyelitis （EAE） or not needs a further study. OBJECTIVE： To observe the effect of erhuangfang on neuro-pathology and astrocyte in EAE rats and compare with the effect of hormone. DESIGN： Randomized controlled animal study. SETTINGS： Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University; College of Traditional Chinese Medicine, Capital Medical University. MATERIALS： The experiment was carded out in the Laboratory Center of Capital Medical University from August to October 2005. Ten adult guinea pigs （SPF grade, weighing 400 - 450 g） and 70 adult Lewis rats （SPF grade, weighing 200- 220 g） were selected in this study. Erhuangfang consisted of fiudahuang, shengdi, shuizhi, dabeimu, etc. METHODS： ①Experimental intervention： Rats were randomly divided into normal group （n=10）, model group （n=20）, western medicine group （n=20） and Chinese herb group （n=20）. Mixed emulsion, which was consisted of Freund＇s adjuvant and spinal cord homogenate of guinea pigs, was subcutaneously injected into palms of the two hindfeet of rats in the latter three groups to establish EAE models. Foot pads were injected with saline and then rats were perfused with saline in the normal group.in the model group, models were established as the same as those mentioned above, and rats were also perfused with saline. Rats in the western medicine group were perfused with saline and then 5 mg/kg prednisone acetate suspension. Rats in the Chinese herb group were perfused with erhuangfang decoction （15 g raw materials per kilogram） at 5 days before model establishment. The dosage in the four groups was 3 mL/day per rat. ②Experimental evaluation： At 28 days after model establishment, rats were randomly selected for cerebral （mainly surrounding cerebral ventricle） and spinal cord （cervical enlargement and lumbar enlargement） collections, and then haematine-eosin （HE） staining and SLG myelin staining were used to observe demyelination and regeneration; meanwhile, immunohistochemical staining was used to observe the expression of glial fibriliary acidic protein （GFAP）. MAIN OUTCOME MEASURES： Cerebral and spinal demyelination and regeneration as well as expression of GFAP in EAE rats. RESULTS： All 70 Lewis rats were involved in the final analysis. ①Demyelination and regeneration： Infiltration of inflammatory cells surrounding cerebrum and small venous vessels of spinal cord white matter, demyelination surrounding vessels and plentiful foam cells at myelinolysis sites were observed in the model group. Symptoms were relieved in the western medicine group and the Chinese herb group as compared with those in the model group. While, numbers of inflammatory infiltrated cells and vascular cuffs were decreased in focal region as compared with those in the model group; in addition, areas of softening focus and demyelination were decreased. ②Expression of GFAP： Volumes and numbers of positive cells of GFAP in white matter region were respectively bigger and higher than those of normal cells in the model group. Plentiful positive cells of GFAP were disorderly aggregated in hippocampus and surrounding small vessel cuffs. While, expression of GFAP was mildly increased surrounding focus in the Chinese herb group; however, GFAP did not express surrounding focus in the western medicine group. In addition, expressions of GFAP were not increased in non-focal region in both Chinese herb group and western medicine group. CONCLUSION： Both erhuangfang and hormone can relieve inflammatory reaction of central nervoussystem and demyelination of EAE rats. On one hand, erhuangfang can regulate reaction of astrocyte in two ways, relieve reaction and proliferation of astrocyte in non-focal region and maintain the protective effect of astrocyte on brain tissue in focal region; on the other hand, hormone can overall inhibit reaction of astrocyte.

Amyloid precursor protein and growth-associated protein 43 expression in brain white matter and spinal cord tissues in a rat model of experimental autoimmune encephalomyelitis

Studies have demonstrated that amyloid precursor protein （APP） expression increases in multiple sclerosis tissues during acutely and chronically active stages. To determine the relationship between axonal injury and regeneration in multiple sclerosis, an animal model of experimental autoimmune encephalomyelitis was induced using different doses of myelin basic protein peptide. APP and growth-associated protein 43 （GAP-43）, which is considered a specific marker of neural regeneration, were assessed by western blot analysis. Expression of APP and GAP-43, as well as the correlation between these two proteins, in brain white matter and spinal cord tissues of experimental autoimmune encephalomyelitis rats at different pathological stages was analyzed. Results showed that APP and GAP-43 expression increased during the acute stage and decreased during remission, with a positive correlation between APP and GAP-43 expression in brain white matter and spinal cord tissues. These results suggest that APP and GAP-43 could provide nutritional and protective effects on damaged neurons.

Nogo receptor expression in microglia/macrophages during experimental autoimmune encephalomyelitis progression

Myelin-associated inhibitory factors within the central nervous system（CNS） are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1（NgR1） has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis（MS）. In this study, the animal model of MS, experimental autoimmune encephalomyelitis（EAE） was induced in ngr1^＋/＋ and ngr1^–/– female mice following injection with the myelin oligodendrocyte glycoprotein（MOG_（35–55）） peptide. A fatemap analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively（increasing locomotor disability） from both genotypes, using the CD11 b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1^＋/＋ and ngr1^–/– mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1^＋/＋ and ngr1^–/– mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1^＋/＋ and ngr1^–/– mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of Ng R on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1^–/– mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1^–/– mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1^–/– mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.

The next-generation sphingosine-1 receptor modulator BAF312(siponimod) improves cortical network functionality in focal autoimmune encephalomyelitis

Autoimmune diseases of the central nervous system(CNS) like multiple sclerosis(MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease, whereas progressive MS(PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with relapsing MS have limited success in PMS. BAF312(siponimod) is a novel sphingosine-1-phosphate receptor modulator shown to delay progression in PMS. Besides reducing inflammation by sequestering lymphocytes in lymphoid tissues, BAF312 crosses the blood-brain barrier and binds its receptors on neurons, astrocytes and oligodendrocytes. To evaluate potential direct neuroprotective effects, BAF312 was systemically or locally administered in the CNS of experimental autoimmune encephalomyelitis mice with distinct grey-and white-matter lesions(focal experimental autoimmune encephalomyelitis using an osmotic mini-pump). Ex-vivo flow cytometry revealed that systemic but not local BAF312 administration lowered immune cell infiltration in animals with both grey and white matter lesions. Ex-vivo voltage-sensitive dye imaging of acute brain slices revealed an altered spatio-temporal pattern of activation in the lesioned cortex compared to controls in response to electrical stimulation of incoming white-matter fiber tracts. Here, BAF312 administration showed partial restore of cortical neuronal circuit function. The data suggest that BAF312 exerts a neuroprotective effect after crossing the blood-brain barrier independently of peripheral effects on immune cells. Experiments were carried out in accordance with German and EU animal protection law and approved by local authorities(Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen;87-51.04.2010.A331) on December 28, 2010.

Effect of oxymatrine on interferon-gamma and tumor necrosis factor-alpha serum levels in an experimental rat model of autoimmune encephalomyelitis

BACKGROUND： Studies have demonstrated that experimental autoimmune encephalomyelitis （EAE） onset correlates with increased interferon-v （IFN-γ） and tumor necrosis factor-α （TNF-α） expression. Oxymatrine （OM） has been shown to inhibit autoimmune responses, but there are no reports showing that it could prevent the development of EAE. OBJECTIVE： To observe the effect of OM on serum levels of IFN-γ and TNF-α in a rat model of EAE.DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Experimental Animal Center of Henan Academy of Chinese Medicine and at the Key Disciplines Laboratory Clinical Medicine of Henan Province between July and December 2008. MATERIALS： OM was purchased from Chia-tai Tianqing Pharmaceutical, China; complete Freund＇s adjuvant was purchased from Sigma, USA. METHODS： Forty female Wistar rats were randomly assigned to four groups： EAE model （M）, low-dose OM treatment （OM-L）, high-dose OM treatment （OM-H）, and normal control （N, no immunization）, with 10 rats in each group. EAE was established in the M, OM-L, and OM-H groups following immunization with Guinea pig spinal cord homogenate and complete Freund＇s adjuvant. The M and N groups were intraperitoneally injected with normal saline （6.7 mL/kg per day）, the OM-L group received an intraperitoneal injection of OM （100 mg/kg per day）, and the OM-H group received OM （150 mg/kg per day）. MAIN OUTCOME MEASURES： At 16 days after immunization, the degree of histopathological changes in the spinal cord was assessed by hematoxylin-eosin stanining. Enzyme-linked immunosorbent assay was used to detect serum levels of IFN-γ, and radioimmunoassay was utilized to determine serum TNF-α level. Neurological scores were measured on a daily basis according to a 0-5 scale. RESULTS： Daily injections of OM, both high and low doses, resulted in decreased neurological scores in EAE rats （P〈0.01）, as well as reduced cellular infiltration in the spinal cord and decreased levels of serum IFN-γ and TNF-α （P〈 0.01）. CONCLUSION： OM reduced the onset and severity of EAE, which correlated with decreased IFN-γ and TNF-α expression.

A (H1N1) Influenza Pneumonia with Acute Disseminated Encephalomyelitis:A Case Report

INTRODUCTION A 56-year-old Chinese female patient with A （H1N1） influenza pneumonia accompanied by acute disseminated encephalomyelitis （ADEM） of the Central Nervous System （CNS） is described in this article. The patient had typical clinical manifestation, and the diagnosis was reached after MRI and other examinations. From this case, we can conclude that the virus of A （H1N1） influenza can infect CNS, and we should pay more attention to patients of A （H1N1） influenza pneumonia with neurological complications.

Zuogui pills for myelinolysis in a rat model of experimental autoimmune encephalomyelitis

Zuogui pills have been shown to attenuate the inflammatory reaction in a rat model of experimental autoimmune encephalomyelitis （EAE）. The present study attempted to investigate the pathology underlying the influence of Zuogui pills on myelinolysis in EAE rats. Hematoxylin-eosin and Luxol fast blue staining showed that the myelinolysis foci in the cerebrum, cerebellum, brain stem, and the spinal cord of EAE rats were significantly decreased, along with serum myelin basic protein content following treatment with Zuogui pills.

Nogo-A and Nogo-A receptor expression in periventricular white matter of experimental autoimmune encephalomyelitis rats

BACKGROUND：Previous studies have focused on the correlation between Nogo-A expression and multiple sclerosis or between Nogo-A receptor （NgR） expression and multiple sclerosis in the central nervous system. Expression patterns of Nogo-A and NgR remain poorly understood in rat models of experimental autoimmune encephalomyelitis （EAE）.OBJECTIVE：To observe dynamic changes in Nogo-A and NgR protein expression, and to verify the correlation between Nogo-A and NgR protein, as well as expression patterns at various time points, in periventricular tissue of EAE rats.DESIGN, TIME AND SETrlNG：A neuroimmunological, randomized, controlled experiment was performed at the Clinical Institute of Hunan People＇s Hospital of China from September to November 2008.MATERIALS：Immunohistochemistry （streptavidin-biotin-peroxidase complex method） kit was purchased from Boster, China.METHODS：A total of 60 female, Wistar rats, aged 6-8 weeks, ware randomly assigned to EAE and control groups （n = 30, respectively）. Guinea pig spinal cord homogenate, self-made complete Freund＇s adjuvant （0.2 mL/100 g）, and pertussis vaccine （0.2 mL） were subcutaneously injected into the hindlimb foot pad of rats from the EAE group to create rat models of EAE. Complete Freund＇s adjuvant （0.2 mL） was infused into rats from the control group.MAIN OUTCOME MEASURES：Nogo-A and NgR protein expression was determined in periventricular white matter using immunohistochemical methods. Neurological scores ware determined in all rats.RESULTS：Rats from the EAE group developed acute-onset EAE following immunization. The pathogenetic symptoms reached a peak on day 15, and neurological scores ware also greatest at this time point. Neurological scores decreased with recovery of the illness. Nogo-A was shown to be expressed in neuronal cells and oligodendrocytes, and expression increased 11 days after immunization （P 〈 0.01）, decreased by day 13 （P 〈 0.01）, and then increased again by day 15. Nogo-A expression remained greater in the EAE group compared with the control group at day 30 （P 〈 0.01）. In the EAE group, NgR protein was primarily expressed on the surface of neuronal bodies and axons. NgR expression increased 13-18 days after immunization （P 〈 0.01 or P 〈 0.05）.CONCLUSION：Nogo-A and NgR protein expression altered with disease course in periventdcular white matter of EAE rats. Results suggested that Nogo-A and NgR were involved in EAE occurrence.

Effects of Yishendaluo decoction on blood-brain barrier integrity in mice with experimental autoimmune encephalomyelitis

This study investigated the effects of Yishendaluo decoction on the loss of blood-brain barrier integrity in mice exhibiting experimental autoimmune encephalomyelitis.To this end,we used real-time fluorescent quantitative PCR to measure the levels of mRNAs specific to the T cell markers CD4 and CD8,and the monocyte marker CD11b.In addition,we used Evans blue dye extravasation in the spinal cord and brain tissues to assess blood-brain barrier permeability.The results indicated that an increase in blood-brain barrier permeability was associated with an increase in CD4,CD8 and CD11b mRNA expression in experimental autoimmune encephalomyelitis mice.Yishendaluo decoction administration significantly reversed inflammatory cell accumulation in cerebral tissues of experimental autoimmune encephalomyelitis mice.

Axonal damage in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in a C57BL/6 mouse model may be not secondary to inflammatory demyelination

The present study established a chronic experimental autoimmune encephalomyelitis model in C57BL/6 mice induced by myelin oligodendrocyte glycoprotein peptides and complete Freund＇s adjuvant. Onset latency was 12 days, with an incidence rate of 100%. Neuropathological characteristics included perivascular inflammatory cell infiltration, demyelination, neuronal degeneration, and axonal damage within cerebral and myelic white matter. Electron microscopy revealed swollen mitochondria, complete organ disappearance, and fused or broken myelin sheath structure, which were accompanied by myelin sheath reconstruction. Moreover, axonal damage was not consistent with demyelination distribution, and severity of axonal damage did not correlate with demyelination. Results suggested that axonal damage in an experimental autoimmune encephalomyelitis model is not secondary to inflammatory demyelination.

Effect of olfactory ensheathing cell transplantation on myelin repair and motor function in a rat model of experimental allergic encephalomyelitis

BACKGROUND： Olfactory ensheathing cell transplantation can activate axonal regeneration and enhance myelin repair, which are beneficial for treating demyelinating diseases. OBJECTIVE： To explore the effects of olfactory ensheathing cell transplantation on myelin repair, synaptophysin expression, and motor function in a rat model of experimental allergic encephalomyelitis. DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Laboratory of Provincial Hospital affiliated to Shandong University between August 2006 and September 2007. MATERIALS： Dibenzylamine （Hoechst 33342）, luxol fast blue, and rabbit anti-rat synaptophysin antibody were provided by Sigma, USA. METHODS： Olfactory ensheatbing cells extracted from neonatal Wistar rats were cultured for 10-14 days and labeled with dibenzylamine. Spinal cord extracted from a healthy guinea pig was homogenized and equally mixed with complete Freund＇s adjuvant; thereafter, the mixture was intracutaneously injected into two posterior voix pedis of healthy male Wistar rats to establish models of experimental allergic encephalomyelitis. Rats were randomly divided into a control encephalomyelitis group and an olfactory ensheathing cell transplantation group, 36 rats in each group. Physiological saline （2 μ L） or an olfactory ensheathing cell suspension （2 μ L） was separately injected along lateral cerebral ventricle at day 7 post-model induction. MAIN OUTCOME MEASURES： The migration and distribution of olfactory ensheathing cells were observed under fluorescence microscopy; myelin repair was detected using hematoxylin-eosin staining and luxol fast blue staining; synaptophysin expression was measured using immunohistochemical staining; motor function was evaluated using a motor function scale. RESULTS： Olfactory ensheatbing cells could survive in vivo and migrate to the distal end of the transplant focus and spinal cord, and survived 21 days. Hematoxylin-eosin staining and luxol fast blue staining indicated that myelin in the transplantation group was intact, and the inflammatory focus gradually disappeared. Transplantation increased synaptophysin expression （P 〈 0.05 versus control） and motor function （P 〈 0.05）. CONCLUSION： Olfactory ensheathing cell transplantation can promote myelin repair, increase synaptophysin protein expression, and ameliorate motor function in a rat model of experimental allergic encephalomyelitis.

Immunity phenomena following olfactory ensheathing cell transplantation into experimental allergic encephalomyelitis rat brain

Olfactory ensheathing cells （OECs） can promote axonal regeneration and remyelination for the treatment of spinal cord injury. OECs can also treat experimental allergic encephalomyelitis （EAE）, but it remains unclear whether OECs might be rejected by the immune system in the brain including the destruction of the blood-brain barrier under inflammation, the release of inflammatory factors, the activation of local antigen-presenting cells （e.g., microglia cells） and antigen drainage. We found that OECs expressed major histocompatibility complex （MHC）-I molecules on the cell surface, barely expressed MHC-II, but MHC-II could be induced by interferon-v, suggesting that OECs have certain immunogenicity. When OECs were transplanted into normal animal brains, no OECs were phagocytosed by dendritic cells in the cervical lymph node, and OECs did not induce lymphocyte proliferation, which indicates that OECs share some immune privilege under normal conditions. However, OECs in the rat EAE brain were phagocytosed by dendritic cells in the cervical lymph node and enhanced lymphocyte proliferation. These findings suggest that OECs are rejected because of increased immunogenicity in EAE brain, and that brain inflammation, in particular activated dendritic cells, may be a prerequisite for rejecting OECs.

PD-L1 is increased in the spinal cord and infiltrating lymphocytes in experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis is a mouse model of human multiple sclerosis with similar pathology and pathogenesis. Thl cells play an important role in the pathogenesis of experimental allergic encephalomyelitis. This study determined the potential effect of programmed cell death 1 ligand 1 in the pathogenesis of experimental allergic encephalomyelitis induced by injecting myelin oligodendrocyte glycoprotein, complete Freund＇s adjuvant and Bordetella pertussis toxin into C57BL/6J mice. Experimental allergic encephalomyelitis mice developed disease and showed in- flammatory changes in the central nervous system by hematoxylin-eosin staining of spinal cord pathological sections, demyelination by Luxol fast-blue staining and clinical manifestations. The expression of programmed cell death 1 ligand 1 in mice was detected by immunohistochemistry, flow cytometry and western blot anatysis. The expression of programmed cell death 1 ligand 1 in the spinal cord and splenocytes of mice was significantly increased compared with normal mice. Our findings suggest the involvement of programmed cell death 1 ligand 1 in the pathogenesis of ex- perimental allergic encephalomyelitis and suggest this should be studied in multiple sclerosis.