Experimental Autoimmune Encephalomyelitis Rats Research Articles

Glycan changes in the olfactory mucosa of rats with experimental autoimmune encephalomyelitis

Glycans are components of glycoconjugates and function in odorant recognition and cell signaling in the olfactory mucosa. However, little is known about glycan expression in the olfactory mucosa in the presence of neuroinflammatory disorders, which can influence olfaction. We evaluated the changes in glycan in the olfactory mucosa of rats with experimental autoimmune encephalomyelitis (EAE) by histochemical analyses of 21 lectins. In the olfactory mucosa of normal control rats, 16 lectins bound to olfactory sensory neurons, supporting cells, basal cells, nerve and Bowman’s glands, and their expression did not significantly change during the course of EAE. In rats with paralytic-stage EAE, five lectins showed different reactivities with the olfactory mucosa compared to those of normal control rats. Of them, Bandeiraea simplicifolia lectin (BSL)-II and BSL-I showed transiently downregulated binding to olfactory sensory neurons and supporting cells in rats with EAE. The reactivities of Lens culinaris agglutinin for the basement membrane, Vicia villosa agglutinin for Bowman’s glands and Dolichos biflorus agglutinin for all nuclei were upregulated in the olfactory mucosa of EAE rats. These results suggest that BSL-II-binding N-acetyl-glucosamine and BSL-I-binding N-acetyl-galactose are involved in transient olfactory dysfunction in EAE, which may hamper odor perception and/or signal processing in olfactory sensory neurons.

Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats

BackgroundFolate receptor-β (FR-β) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-β expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-β expression and evaluated its potential as an in vivo imaging target.MethodsFocal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund’s adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-β-targeting aluminum [18F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([18F]AlF-NOTA-folate, 18F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine (11C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-β, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent.ResultsImmunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-β positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-β correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18F-FOL and 11C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-β positivity. A blocking assay using folate glucosamine further verified the tracer’s specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18F-FOL was significantly higher than that of 11C-PBR28 (P = 0.016).ConclusionOur EAE results imply that FR-β may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-β-targeted PET imaging with 18F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Matrine alleviates astrogliosis through sphingosine 1-phosphate signaling in experimental autoimmune encephalomyelitis

Expression of sphingosine/sphingosine 1-phosphate (SPH/S1P) in resident cells of the central nervous system plays an important role in the pathogenesis of multiple sclerosis (MS). Accumulated evidence has shown the protective effects of S1P receptor modulators on MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, effective therapies to regulate SPH/S1P molecules themselves have not been well addressed. Our previous studies showed that matrine (MAT), a natural alkaloid component extracted from the Sophora root, has beneficial effects in EAE through immunomodulation. Here we demonstrate that MAT alleviated astrogliosis in the CNS of EAE rats, and downregulated levels of SPH, S1P and S1P1 expression in CNS tissues and astrocytes. Expression of SPH kinases (SPHK) 1 and 2, which splice SPH into S1P, was also inhibited by MAT treatment. In vitro studies showed a direct inhibitory effect of MAT on S1P1 expression of activated astrocytes, suggesting that MAT could function as an S1PRs antagonist. Moreover, MAT upregulated the expression of plasma gelsolin, which combines with S1P to reduce its concentration. These findings indicate that MAT could alleviate astrogliosis in EAE through diminishing the SPH/SPHK/S1P1 pathway.

Ghrelin Attenuates Neuroinflammation and Demyelination in Experimental Autoimmune Encephalomyelitis Involving NLRP3 Inflammasome Signaling Pathway and Pyroptosis.

Multiple sclerosis (MS) is a chronic autoimmune and degenerative disease of the central nervous system, and conventional treatments have limited efficacy or side effects. Ghrelin, a 28-amino acid octanoylated peptide, has been reported to have neuroprotective effects, including anti-oxidation, anti-inflammation, and anti-apoptosis. Pyroptosis, also called inflammatory cell death, is triggered by overly active inflammasomes and accompanied by the production of numerous cytokines. As immune dysfunction is primarily involved in the pathogenesis of MS, this study aimed to explore the therapeutic effects and precise functional mechanisms of ghrelin against the nod-like receptor protein 3 (NLRP3) inflammasome and pyroptosis in experimental autoimmune encephalomyelitis (EAE). Sprague Dawley rats were immunized with guinea pig spinal cord homogenates and pertussis toxin to develop an EAE model. All rats were randomly divided into four groups: normal control group, EAE group, EAE + ghrelin group, and ghrelin control group. EAE rats showed abnormal behavioral scores and body weight changes. Histologic analysis displayed severe inflammatory infiltration and demyelination in the brain and spinal cord of EAE rats. Ghrelin treatments potently restored these abnormal changes. In addition, the ghrelin-treated EAE group showed significantly downregulated expression of inflammatory cytokines. The expression of proteins involved in the NLRP3 signaling pathway and pyroptosis was decreased as well. We also found that the anti-inflammatory effect of ghrelin was associated with inhibition of nuclear factor (NF)-κB activation. Compared with rats in the healthy control group, rats in the ghrelin control group did not show statistically significant changes in histologic examinations, pro-inflammatory cytokines production, or molecules involved in the NLRP3 signaling pathway, which indicated that ghrelin induced no side effects in the animals of our study. Our findings provide more insight into the use of ghrelin as a novel candidate for MS.

Propranolol diminished severity of rat EAE by enhancing immunoregulatory/protective properties of spinal cord microglia

Sympathetic dysfunction is suggested to contribute to development of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) alike. Considering important role of microglia in development/resolution of neuroinflammation, contribution of noradrenaline, the key sympathetic end-point mediator, in modulation of microglial phenotypic and functional properties in rat EAE model was examined. The study showed that noradrenaline acting in neurocrine and autocrine/paracrine way might influence microglia during EAE. Propranolol treatment over the effector phase moderated EAE course. This was associated with the increased proportion of microglia expressing CX3CR1, the key molecule in their immunomodulatory/neuroprotective action, and upregulation of CX3CR1 downstream Nrf2 gene. This correlated with the increased heme oxygenase-1 (HO-1) expression and phagocytic capacity of microglia, and their phenotypic changes mirrored in increased proportion of CD163- and CD83-expressing cells. The enhanced HO-1 expression was linked with the decreased proportion of microglial cells expressing IL-1β and IL-23, and possibly IL-6, followed by increased proportion of IL-10–expressing microglia, and downregulated MCP-1/CCL2 expression. Consistently, spinal cord infiltration with blood-borne myeloid and CD4+ T cells, as well as CD4+ T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17+ cells co-producing IFN-γ and GM-CSF were decreased in propranolol-treated rats compared with saline-injected controls. The in vitro investigations of the effects of noradrenaline on microglia showed that noradrenaline through β-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests β-adrenoceptor–mediated neuroinflammation-promoting role of noradrenaline in EAE via modulation of microglial Nrf2 expression, and thereby forms the basis for further translational pharmacological research to improve multiple sclerosis therapy.

Do Myelin Oligodendrocyte Glycoprotein Antibodies Represent a Distinct Syndrome?

Do Myelin Oligodendrocyte Glycoprotein Antibodies Represent a Distinct Syndrome?

CCL26 and CCR3 are associated with the acute inflammatory response in the CNS in experimental autoimmune encephalomyelitis

Chemokine ligand 26 (CCL26) is a member of the eotaxin family. It works by interacting exclusively with chemokine receptor 3 (CCR3) and acts as an eosinophil-selective chemoattractant. There is an emerging role for eotaxins in autoimmune diseases. Studies have reported that chemokine ligand 11 (CCL11) and CCL26 are upregulated in patients with neuromyelitis optica spectrum disorder (NMOSD) during remission, CCL26 levels appear to be decreased in relapsing-remitting multiple sclerosis (RRMS), whereas CCL26 levels are significantly increased in secondary progressive multiple sclerosis (SPMS), indicating that CCL26 participates in the pathogenesis of multiple sclerosis (MS). We investigated the levels of CCL26, CCR3 and claudin-5 (a marker of changes in BBB (blood-brain barrier) permeability) at different stages of experimental autoimmune encephalomyelitis (EAE) to explore the underlying immune mechanisms of EAE. Our results showed that the levels of CCL26 and CCR3 in EAE rats were significantly increased compared with those in the control group. The levels of CCL26 in the serum and in brain tissues as well as the protein expression of CCR3 in brain tissues were positively correlated with the inflammatory scores of brain tissues from EAE rats and were negatively correlated with the protein expression of claudin-5. We concluded that CCL26, which in turn binds to the receptor CCR3, showed pro-inflammatory effects and aggravated tissue damage involving BBB impairment, especially in the acute stage of EAE. Our study uncovers another possible immunopathological mechanism of MS and provides a possible target for immune therapy.

Cessation of anti-VLA-4 therapy in a focal rat model of multiple sclerosis causes an increase in neuroinflammation

BackgroundPositron emission tomography (PET) can be used for in vivo evaluation of the pathology associated with multiple sclerosis. We investigated the use of longitudinal PET imaging and the 18-kDa translocator protein (TSPO) binding radioligand [18F]GE-180 to detect changes in a chronic multiple sclerosis-like focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) rat model during and after anti-VLA-4 monoclonal antibody (mAb) treatment. Thirty days after lesion activation, fDTH-EAE rats were treated with the anti-VLA-4 mAb (n = 4) or a control mAb (n = 4; 5 mg/kg, every third day, subcutaneously) for 31 days. Animals were imaged with [18F]GE-180 on days 30, 44, 65, 86 and 142. Another group of animals (n = 4) was used for visualisation the microglia with Iba-1 at day 44 after a 2-week treatment period.ResultsAfter a 2-week treatment period on day 44, there was a declining trend (p = 0.067) in [18F]GE-180-binding in the anti-VLA-4 mAb-treated animals versus controls. However, cessation of treatment for 4 days after a 31-day treatment period increased [18F]GE-180 binding in animals treated with anti-VLA-4 mAb compared to the control group (p = 0.0003). There was no difference between the groups in TSPO binding by day 142.ConclusionsThese results demonstrated that cessation of anti-VLA-4 mAb treatment for 4 days caused a transient rebound increase in neuroinflammation. This highlights the usefulness of serial TSPO imaging in the fDTH-EAE model to better understand the rebound phenomenon.

Novel Phloroglucinol Derivative Compound 21 Protects Experimental Autoimmune Encephalomyelitis Rats <i>via</i> Inhibiting Th1/Th17 Cell Infiltration

Multiple Sclerosis (MS) is a chronic autoimmune disease characterized by inflammatory infiltration and demyelination in the central nervous system (CNS). Among all the factors that are involved in the immunological mechanisms of MS, T helper 1 (Th1) cells and T helper 17 (Th17) cells play a critical role. Compound 21, a novel phloroglucinol derivative, has shown remarkable effects on protecting myelin damage in our previous study. However, whether it has effects on MS remains unclear. In this study, the experimental autoimmune encephalomyelitis (EAE) rat model was established to mimic the pathological process of MS and to evaluate the neuroprotective effect of Compound 21. The results illustrated that Compound 21 treatment notably attenuated the neurological deficits, immune infiltration and demyelination of EAE rats. Mechanistic investigation revealed that Compound 21 treatment reduced the population of Th1/Th17 cells and inhibited their infiltration into the CNS. Further study found that the inhibition of Th1/Th17 cell infiltration was related to the direct suppression of Th1/Th17 cell differentiation and inhibition of pro-inflammatory microglial cells. Collectively, all these results proved that Compound 21 treatment suppressed infiltrated Th1/Th17 cells to alleviate the demyelination of EAE rats, suggesting its potential role as a novel candidate for MS treatment. Funding Statement: This investigation was supported by grants from National Sciences Foundation of China (81630097, 81773718), CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-3-011), CAMS The Fundamental Research Funds for the Central Universities (2018RC350002), National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Thirteenth Five-year Plan Period (2018ZX09711001-003-005, 2018ZX09711001-008-005, 2018ZX09711001-003-020). Declaration of Interests: The authors declare no competing financial or other interests. Ethics Approval Statement: All the animal experiments were carried out according to the guidelines of the Beijing Municipal Ethic Committee for the care and use of laboratory animals.

Natural killer cells as participants in pathogenesis of rat experimental autoimmune encephalomyelitis (EAE): lessons from research on rats with distinct age and strain.

Natural killer (NK) cells, influencing dendritic cell (DC)-mediated CD4+ lymphocyte priming in draining lymph nodes (dLNs) and controlling spinal cord (SC) infiltration with encephalitogenic CD4+T lymphocytes, modulate EAE (multiple sclerosis model). This study examined their putative contribution to age-related differences in EAE development in Dark Agouti (DA) (exhibiting age-related decrease in EAE susceptibility) and Albino Oxford (AO) (becoming susceptible to EAE with aging) rats. Aging increased NK cell number in dLNs from rats of both strains. In AO rats, but not in DA ones, it also increased the numbers of IFN-γ-producing NK cells (important for DC activation) and activated/matured DCs, thereby increasing activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated CD25+Foxp3-CD4+ cell number. Aging in DA rats diminished activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated Foxp3-CD4+ cell number. However, MBP-stimulated CD4+ cell proliferation did not differ in dLN cell cultures from young and aged AO rats (as more favorable activated/matured DC/Foxp3-CD4+ cell ratio was abrogated by lower intrinsic CD4+ cell proliferative capacity and a greater regulatory CD25+Foxp3+CD4+ lymphocyte frequency), but was lower in those from aged compared with young DA rats. At SC level, aging shifted Foxp3-CD4+/cytotoxic CX3CR1+ NK cell ratio towards the former in AO rats, so it was less favorable in aged AO rats exhibiting prolonged neurological deficit compared with their DA counterparts. The study showed strain and age differences in number of IFN-γ-producing NK cells in EAE rat dLNs, and suggested that their pathogenetic relevance depends on frequency and/or activity of other cells involved in CD4+ T cell (auto)immune response.

Exosomes derived from mesenchymal stem cells attenuate inflammation and demyelination of the central nervous system in EAE rats by regulating the polarization of microglia

Multiple sclerosis (MS) is a chronic demyelinating disease caused by central nervous system (CNS) inflammation and immune dysfunction, which often leaves patients with severe physical disabilities. Microglia function in the surveillance of the CNS, and an imbalance in the M1/M2 phenotypes of microglia contribute to the progression of MS. Recent studies indicate that exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) play therapeutic roles in many autoimmune diseases and aid in tissue repair. However, it is not clear whether BMSC-derived exosomes can attenuate MS-associated inflammation and immune dysfunction, or how BMSC exosomes protect neurons. The experimental autoimmune encephalomyelitis (EAE) rat model was used to investigate the effect of exosomes on microglia polarization and inflammation in CNS. The results showed that exosome treatment significantly decreased neural behavioral scores, reduced the infiltration of inflammatory cells into the CNS, and decreased demyelination in comparison to untreated EAE rats. In addition, exosome treatment resulted in significant increases in the levels of M2-related cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β, whereas M1-related tumor necrosis factor (TNF)-α and IL-12 levels decreased significantly. Moreover, compared with the untreated EAE group, the exosome group displayed significantly increased protein and mRNA expression levels of M2 phenotype markers, whereas M1 marker expression decreased. Our findings were further confirmed in an in vitro HAPI microglia cell line model. In conclusion, these findings indicate that BMSC-derived exosomes can attenuate inflammation and demyelination of the CNS in the EAE rat model by regulating the polarization of microglia. Therefore, the use of BMSC-derived exosomes may be a potential therapeutic approach for the treatment of autoimmune and inflammatory diseases.

Effect of CXCR2 Inhibition on Behavioral Outcomes and Pathology in Rat Model of Neuromyelitis Optica.

Objective To reduce immune-mediated damage in a rat model of neuromyelitis optica (NMO) by blocking neutrophil migration using SCH527123, a drug that inhibits CXCR2. Background Neuromyelitis optica is a relapsing autoimmune disease that preferentially targets the optic nerves and spinal cord leading to blindness and paralysis. Part of the immunopathogenesis of this disease is thought to involve neutrophils, which are present within NMO lesions. We tested the effect of blocking neutrophil migration in an NMO rat model. Methods The Lewis rat model of NMO uses a myelin-reactive experimental autoimmune encephalomyelitis (EAE) background with passive transfer of pooled human antibody from patients with aquaporin-4 (AQP4) seropositive NMO at onset of EAE symptoms. We treated rats early in the course of EAE with CXCR2 inhibitor and assessed the extent of neutrophil infiltration into the spinal cord and the extent of AQP4 depletion. Results CXCR2 inhibitor decreased neutrophil migration into the spinal cord of AQP4 IgG-treated EAE rats. However, there was no difference in the acute behavioral signs of EAE or the extent and distribution of AQP4 lesions. This suggests that neutrophils are not centrally involved in the immunopathogenesis of the Lewis rat NMO disease model. Conclusions CXCR2 inhibitor blocks neutrophil migration into the spinal cord during EAE but does not significantly reduce inflammation or AQP4 lesions in the Lewis rat model of NMO.

Small Molecule Antagonists of NAADP-Induced Ca2+ Release in T-Lymphocytes Suggest Potential Therapeutic Agents for Autoimmune Disease

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date, but the precise NAADP/Ca2+ signalling mechanisms are still controversial. We report the synthesis of small-molecule inhibitors of NAADP-induced Ca2+ release based upon the nicotinic acid motif. Alkylation of nicotinic acid with a series of bromoacetamides generated a diverse compound library. However, many members were only weakly active or had poor physicochemical properties. Structural optimisation produced the best inhibitors that interact specifically with the NAADP/Ca2+ release mechanism, having no effect on Ca2+ mobilized by the other well-known second messengers d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or cyclic adenosine 5′-diphospho-ribose (cADPR). Lead compound (2) was an efficient antagonist of NAADP-evoked Ca2+ release in vitro in intact T lymphocytes and ameliorated clinical disease in vivo in a rat experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Compound (3) (also known as BZ194) was synthesized as its bromide salt, confirmed by crystallography, and was more membrane permeant than 2. The corresponding zwitterion (3a), was also prepared and studied by crystallography, but 3 had more desirable physicochemical properties. 3 Is potent in vitro and in vivo and has found widespread use as a tool to modulate NAADP effects in autoimmunity and cardiovascular applications. Taken together, data suggest that the NAADP/Ca2+ signalling mechanism may serve as a potential target for T cell- or cardiomyocyte-related diseases such as multiple sclerosis or arrhythmia. Further modification of these lead compounds may potentially result in drug candidates of clinical use.

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis.

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis

Microglia-mediated neuroinflammation accompanies many central nervous system (CNS) diseases, including multiple sclerosis (MS), and is strongly dependent on the purinergic P2X7 receptor. The nature of the inflammatory response in MS is studied for decades indicating, that proinflammatory microgliosis is involved in advanced stages of MS and is associated with active tissue damage and neurological dysfunctions. Evidence on the role of microgliosis in initial stages of the disease is scarce. Thus, in the present study, we investigated the time course of microglial activation in rat brain subjected to experimental autoimmune encephalomyelitis (EAE) which is the animal model of MS. We show that activation of microglia occurs in brains of immunized rats at a very early stage of EAE, well before the development of neurological symptoms of the disease. Enhanced immunoreactivity of microglia/macrophage-specific protein Iba-1, together with morphological features of microgliosis, was identified beginning at day 4 post immunization. Concomitantly, microglial expression of P2X7R was also examined. Moreover, our results reveal that administration of Brilliant Blue G, an antagonist of P2X7R, delays the onset of the disease and partially inhibits development of neurological symptoms in EAE rats. Blockage of P2X7R significantly reduces activation of microglia as confirmed by decreased Iba-1 immunoreactivity and suppresses neuroinflammation in EAE rat brains, as indicated by decreased protein levels of investigated proinflammatory cytokines: IL-1β, IL-6 and TNF-α. Our results indicate that microglia are involved in inducing neuroinflammation at a very early stage of MS/EAE via a P2X7R-dependent mechanism.

Prenatal stress-induced sex differences in the incidence and course of experimental autoimmune encephalomyelitis in rats

Aim. The research is aimed at investigating the effect of prenatal stress on the incidence and course of experimental autoimmune encephalomyelitis (EAE) in four-month-old rats, offspring of females with different sensitivity to EAE induction. Methods. The incidence and severity of EAE (duration of latent period (LP)), the maximum level of neurological deficit (KSmax) and the duration of the period from the first to the maximum manifestations of neurological deficit (T)) were analyzed in rats with induced EAE. Results. A decrease in the incidence of EAE in prenatally stressed four-month-old females, whose mothers were sensitive to induction of EAE, was found. Differences in the dynamics of EAE course in males from the control and prenatally stressed group were revealed. Conclusions.The EAE incidence and EAE course traits in four-month-old rats depend on a complex of factors: mother's sensitivity to induction of EAE, prenatal stress and offspring sex.
 Keywords: experimental autoimmune encephalomyelitis, prenatal stress, sex, sensitivity to induction of EAE.

Comparison of the effects of 17β- estradiol treated and untreated mesenchymal stem cells on ameliorating animal model of multiple sclerosis.

Objectives:The current investigation was undertaken to evaluate the effects of 17β- estradiol (17β-ED) on the potential of the mesenchymal stem cells (MSCs) for modulation of immunity responses in an animal model of multiple sclerosis (MS).Materials and Methods:After isolation of MSCs, cells were cultured in presence of 100 nM 17β-ED for 24 hr. Modeling of experimental autoimmune encephalomyelitis (EAE) was achieved by using guinea pig spinal cord homogenate, in addition to complete Freund’s adjuvant in male Wistar rats. The processes of cell therapy were started following 12 days post-immunization. This duration allows all animals to develop a disability score. The achieved EAE clinical symptoms were regularly monitored every day until day 36, when all of examined rats were euthanized. Results:Cell therapy in the EAE rats with 17β-ED-primed MSCs exhibited more desirable consequences, which in turn lead to regression of the cumulative clinical score and neuropathological changes that are more than the therapy with untreated MSCs. The serum measures of myeloperoxidase (MPO), nitric oxide (NO) as well as splenocytes-originated pro-inflammatory interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) were significantly decreased in EAE rats treated by 17β-ED primed-MSCs compared to EAE rats that received untreated MScs. Conclusion:Combination of 17β-ED and MSCs more effectively improved the signs and symptoms of EAE.

Innovations in molecular biology and proteomics adapted to in vivo peptide phage display define active protein domains as biomarkers in multiple sclerosis neuroinflammation

With the double goal to streamline biomarker discovery of Multiple Sclerosis neuroinflammatory pathology entangled by non-affected Central Nervous System tissue and to better characterize the complex molecular interactions at the altered blood brain barrier (BBB) that becomes permissive to extravasation of blood derived immune cells and compounds, we choose to identify peptide ligands reacting with inflammatory CNS lesions. With no “a-priori” for targets by applying in vivo the phage displayed 12aa peptide screening in CNS neuroinflammation of the experimental autoimmune encephalomyelitis (EAE) rat model of MS and healthy controls, we generated two complex phage displayed peptide repertoires. By developing molecular DNA subtraction of both phage repertoires we generated a subtraction phage repertoire of EAE specific peptides. In a bioinformatics approach aligning the 270,612 EAE peptides to the human proteome the formed peptide clusters allowed the ranking of proteins being potential “mimicked” indicators of functional protein interactions. Experimentally, among the most high score mimicked proteins, derived synthesized short peptides (22aa) of mimicked domains were tested for labeling of BBB alterations in CNS sections of MS and other chronic neurodegenerative diseases. In vitro studies using the BBB model hCMEC/D3 confirmed their binding to human endothelial cells under proinflammatory stimulation. Finally, with a derived peptide covalently linked to a nanocarrier contrast agent, we could target in vivo and monitor by MRI, experimentally induced inflammatory CNS lesions. Our strategy of combining the developed molecular biology and bioinformatics approaches adapted to phage display is applicable to many disease models to identify biomarkers and functional proteins/domains.

Administration of an antagonist of P2X7 receptor to EAE rats prevents a decrease of expression of claudin-5 in cerebral capillaries

Purinergic P2X receptors, when activated under pathological conditions, participate in induction of the inflammatory response and/or cell death. Both neuroinflammation and neurodegeneration represent hallmarks of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the current study, we examined whether P2X7R is expressed in brain microvasculature of rats subjected to experimental autoimmune encephalomyelitis (EAE) and explore possible relationships with blood-brain barrier (BBB) protein—claudin-5 after administration of P2X7R antagonist—Brilliant Blue G (BBG). Capillary fraction isolated from control and EAE rat brains was subjected to immunohistochemical and Western blot analyses. We document the presence of P2X7R in brain capillaries isolated from brain tissue of EAE rats. P2X7R is found to be localized on the abluminal surface of the microvessels and is co-expressed with PDGFβR, a marker of pericytes. We also show over-expression of this receptor in isolated capillaries during the course of EAE, which is temporally correlated with a lower protein level of PDGFβR, as well as claudin-5, a tight junction-building protein. Administration of a P2X7R antagonist to the immunized rats significantly reduced clinical signs of EAE and enhances protein expression of both claudin-5 and PDGFβR. These results indicate that P2X7 receptor located on pericytes may contribute to pathological mechanisms operated during EAE in cerebral microvessels influencing the BBB integrity.

Withdrawn: Effects of Tofacitinib on Tfr/Tfh Balance and Expression of CXCL13 and TGF-β1 in Experimental Autoimmune Encephalomyelitis Rats

The article entitled, “Effects of Tofacitinib on Tfr/Tfh Balance and Expression of CXCL13 and TGF-β1 in Experimental Autoimmune Encephalomyelitis Rats” submitted in CNS & Neurological Disorders - Drug Targets (CNS&NDDT) by Dr. Zuoxiao Li has been withdrawn from the journal in accordance with BSP Editorial Policies.