Experimental Autoimmune Encephalomyelitis Scores Research Articles

Correlation of Visual System Biomarkers With Motor Deficits in Experimental Autoimmune Encephalomyelitis-Optic Neuritis.

Experimental autoimmune encephalomyelitis (EAE) scoring, the most commonly used primary outcome metric for an in vivo model of multiple sclerosis (MS), is highly variable and subjective. Here we explored the use of visual biomarkers in EAE as more objective and clinically relevant primary outcomes. Motor impairment in myelin oligodendrocyte glycoprotein-immunized C57BL/6J mice was quantified using a five-point EAE grading scale. Pattern electroretinography (pERG) and retinal ganglion cell/inner plexiform layer (RGC/IPL) complex thickness were measured 60 days after induction. Optic nerve histopathology was analyzed at endpoint. EAE mice displayed motor impairments ranging from mild to severe. Significant correlations were seen between pERG amplitude and last EAE score, mean EAE score, and cumulative EAE score. Optical coherence tomography (OCT) analysis demonstrated a significant correlation between thinning of the RGC/IPL complex and both EAE score and pERG amplitude. Optic nerve histopathology showed significant correlations between demyelination and cumulative EAE score, pERG amplitude, and RGC/IPL complex thickness, as well as between immune cell infiltration and cumulative EAE score, pERG amplitude, and RGC/IPL complex thickness in EAE mice. Unlike EAE scoring, pERG and OCT show direct measurement of retinal structure and function. Therefore we conclude that visual outcomes are well suited as a direct assessment of optic nerve involvement in this EAE model of MS while also being indicative of motor impairment. Standardizing directly translatable measurements as primary outcome parameters in the murine EAE model could lead to more rapid and relevant testing of new therapeutic approaches for mitigating MS.

Siponimod treatment response shows partial BDNF dependency in multiple sclerosis models

So far, only a small number of medications are effective in progressive multiple sclerosis (MS). The sphingosine-1-phosphate-receptor (S1PR)-1,5 modulator siponimod, licensed for progressive MS, is acting both on peripheral immune cells and in the central nervous system (CNS). So far it remains elusive, whether those effects are related to the neurotrophin brain derived neurotrophic factor (BDNF). We hypothesized that BDNF in immune cells might be a prerequisite to reduce disease activity in experimental autoimmune encephalomyelitis (EAE) and prevent neurotoxicity. MOG35–55 immunized wild type (WT) and BDNF knock-out (BDNFko) mice were treated with siponimod or vehicle and scored daily in a blinded manner. Immune cell phenotyping was performed via flow cytometry. Immune cell infiltration and demyelination of spinal cord were assessed using immunohistochemistry. In vitro, effects on neurotoxicity and mRNA regulation were investigated using dorsal root ganglion cells incubated with EAE splenocyte supernatant. Siponimod led to a dose-dependent reduction of EAE scores in chronic WT EAE. Using a suboptimal dosage of 0.45 µg/day, siponimod reduced clinical signs of EAE independent of BDNF-expression in immune cells in accordance with reduced infiltration and demyelination. Th and Tc cells in secondary lymphoid organs were dose-dependently reduced, paralleled with an increase of regulatory T cells. In vitro, neuronal viability trended towards a deterioration after incubation with EAE supernatant; siponimod showed a slight rescue effect following treatment of WT splenocytes. Neuronal gene expression for CCL2 and CX3CL1 was elevated after incubation with EAE supernatant, which was reversed after siponimod treatment for WT, but not for BNDFko. Apoptosis markers and alternative death pathways were not affected. Siponimod exerts both anti-inflammatory and neuroprotective effects, partially related to BDNF-expression. This might in part explain effectiveness during progression in MS and could be a target for therapy.

PEPITEM Treatment Ameliorates EAE in Mice by Reducing CNS Inflammation, Leukocyte Infiltration, Demyelination, and Proinflammatory Cytokine Production.

To investigate the effect of the therapeutic treatment of the immunopeptide, peptide inhibitor of trans-endothelial migration (PEPITEM) on the severity of disease in a mouse model of experimental autoimmune encephalomyelitis (EAE) as a model for human multiple sclerosis (MS), a series of experiments were conducted. Using C57BL/6 female mice, we dosed the PEPITEM in the EAE model via IP after observing the first sign of inflammation. The disease was induced using MOG35-55 and complete Freund's adjuvants augmented with pertussis toxin. The EAE score was recorded daily until the end of the experiment (21 days). The histological and immunohistochemistry analysis was conducted on the spinal cord sections. A Western blot analysis was performed to measure the protein concentration of MBP, MAP-2, and N-Cadherin, and ELISA kits were used to measure IL-17 and FOXP3 in the serum and spinal cord lysate. The therapeutic treatment with PEPITEM reduced the CNS infiltration of T cells, and decreased levels of the protein concertations of MBP, MAP-2, and N-Cadherin were observed, in addition to reduced concertations of IL-17 and FOXP3. Using PEPITEM alleviated the severity of the symptoms in the EAE model. Our study revealed the potential of PEPITEM to control inflammation in MS patients and to reduce the harmful effects of synthetic drugs.

A Novel Sensory Wave (P25) in Myelin Oligodendrocyte Glycoprotein-induced Experimental Autoimmune Encephalomyelitis Murine Model

Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is a murine model for multiple sclerosis. This model is characterized by chronic and progressive demyelination, leading to impairment of motor function and paralysis. While the outcomes of the disease, including impaired motor function and immunological changes, are well-characterized, little is known about the impact of EAE on the electrophysiology of the motor and sensory systems. In this study, we assessed evoked potentials as a quantitative marker for in vivo monitoring of nervous system damage. Motor-evoked potentials (MEPs) and sensory-evoked potentials (SEPs) were first standardized in naïve C57BL mice and studied thoroughly in EAE mice. The duration of MEPs and the number of connotative potentials increased significantly alongside an increase in temporal SEP amplitudes. Moreover, a new SEP wave was identified in naïve animals, which significantly increased in MOG-induced EAE animals with no or mild symptoms (clinical score 0–2, 0–5 scale). This wave occurred ∼25 milliseconds poststimulation, thus named p25. P25 was correlated with increased vocalization and was also reduced in amplitude following treatment with morphine. As the EAE score progressed (clinical score 3–4, 0–5 scale), the amplitude of MEPs and SEPs decreased drastically. Our results demonstrate that desynchronized neural motor activity, along with hypersensitivity in the early stages of EAE, leads to a complete loss of motor and sensory functions in the late stages of the disease. The findings also suggest an increase in p25 amplitude before motor deficits appear, indicating SEP as a predictive marker for disease progression. PerspectiveThis article assesses p25, a new sensory electrophysiology wave that correlates with pain-related behavior in MOG-induced EAE mice and appears prior to the clinical symptoms. Motor electrophysiology correlates with traditional motor behavior scoring and histology.

Hsp65-producing Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into spinal cord.

Multiple sclerosis is an autoimmune disease that affects the central nervous system. Because of its complexity and the difficulty to treat, searching for immunoregulatory responses that reduce the clinical signs of disease by non-aggressive mechanisms and without adverse effects is a scientific challenge. Herein we propose a protocol of oral tolerance induction that prevented and controlled MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The genetically modified strain HSP65-producing Lactococcus lactis was orally administered for 5 consecutive days either before or during disease development in mice. Both protocols of feeding HSP65 resulted in significant reduction in the clinical score of EAE. Frequencies of LAP+CD4+Foxp3- regulatory T cells were higher in spleens and inguinal lymph nodes of fed mice. In addition, intravital microscopy showed that adherence of leukocytes to venules in the spinal cord was reduced in orally treated mice. Oral treatment with HSP65-producing L.lactis prevented leukocytes to leave the secondary lymphoid organs, therefore they could not reach the central nervous system. Despite the inhibition of pathological immune response that drive EAE development, activated T cells were at normal frequencies suggesting that oral tolerance did not induce general immunosuppression, but it led to specific control of pathogenic T cells. Our results indicate a novel therapeutic strategy to prevent and control autoimmune diseases such as multiple sclerosis.

PD-L1 is required for estrogen-induced protection against severe EAE in IL-10 deficient mice1.

IL-10 knockout (KO) mice can be protected against experimental autoimmune encephalomyelitis (EAE) with low-dose estrogen (E2) treatment similar to wild type (WT) mice, indicating that IL-10 is not required for E2-induced EAE protection. Our previous study demonstrated that E2 treatment induced an increase in programmed death ligands 1 (PD-L1) and 2 (PD-L2) on monocytes and macrophages in the periphery and within the CNS. In this study, we selectively inhibited the function of PD-L1 and PD-L2 to evaluate their critical role in maintaining E2-induced protection against EAE in IL-10-KO mice. This study used female IL-10 KO mice pre-treated with either E2 or sham pellets seven days prior to induction of EAE and subsequently treated with Vehicle or antibodies to PD-L1, PD-L2 or respective isotype controls. Mice were scored daily for EAE severity over 21 days post-EAE induction. Cells from the spleen and brain were evaluated by flow cytometry. Differences in EAE severity were assessed in E2 and sham pre-treated IL-10-KO mice treated with α-PD-L1 or α-PD-L2 antibodies over the course of disease compared to treatment with Vehicle or isotype control antibodies. The results revealed real-time development of severe EAE in E2-pre-treated IL-10-KO mice treated with α-PD-L1 but not α-PD-L2 antibodies, mediated in part by increased percentages of activated CD74+CD11b+ myeloid cells in spleen and brain as well as splenic B-cells, T-cells and CD73+ cells. These results demonstrate unequivocally that PD-L1 but not PD-L2 was required to retain the inhibitory effects of E2 on clinical EAE scores in female IL-10-KO mice and further implicate the emergence of the MIF/CD74 axis as a contributing pathogenic mechanism.

HMGB1 from Astrocytes Promotes EAE by Influencing the Immune Cell Infiltration-Associated Functions of BMECs in Mice.

High mobility group box 1 (HMGB1) has been reported to play an important role in experimental autoimmune encephalomyelitis (EAE). Astrocytes are important components of neurovascular units and tightly appose the endothelial cells of microvessels by their perivascular endfeet and directly regulate the functions of the blood-brain barrier. Astrocytes express more HMGB1 during EAE while the exact roles of astrocytic HMGB1 in EAE have not been well elucidated. Here, using conditional-knockout mice, we found that astrocytic HMGB1 depletion decreased morbidity, delayed the onset time, and reduced the disease score and demyelination of EAE. Meanwhile, there were fewer immune cells, especially pathogenic T cells infiltration in the central nervous system of astrocytic HMGB1 conditional-knockout EAE mice, accompanied by up-regulated expression of the tight-junction protein Claudin5 and down-regulated expression of the cell adhesion molecules ICAM1 and VCAM1 in vivo. In vitro, HMGB1 released from astrocytes decreased Claudin5 while increased ICAM1 and VCAM1 expressed by brain microvascular endothelial cells (BMECs) through TLR4 or RAGE. Taken together, our results demonstrate that HMGB1 derived from astrocytes aggravates EAE by directly influencing the immune cell infiltration-associated functions of BMECs.

“Near Cure” treatment of severe acute EAE in MIF-1-deficient female and male mice with a bifunctional MHCII-derived molecular construct

Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a “near cure”) with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue.

Development of organ-specific autoimmunity by dysregulated Aire expression.

Deficiency for AIRE/Aire in both humans and mice results in the development of organ‐specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self‐tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen‐specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire‐knockin mice: 3xAire‐KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire‐KI were impaired in a T‐cell receptor‐transgenic system. Furthermore, 3xAire‐KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild‐type littermates, suggesting that augmented Aire expression exacerbates organ‐specific autoimmunity under disease‐prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3–CD19– cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ‐specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ‐specific autoimmune disease and dysregulated AIRE expression in clinical settings.

Does physical exercise improve or deteriorate treatment of multiple sclerosis with mitoxantrone? Experimental autoimmune encephalomyelitis study in rats

BackgroundMitoxantrone has proved efficacy in treatment of multiple sclerosis (MS). The fact that physical exercise could slow down the progression of disease and improve performance is still a debatable issue, hence; we aimed at studying whether combining mitoxantrone with exercise is of value in the management of MS.MethodsThirty-six male rats were divided into sedentary and exercised groups. During a 14-day habituation period rats were subjected to exercise training on a rotarod (30 min/day) before Experimental Autoimmune Encephalomyelitis (EAE) induction and thereafter for 17 consecutive days. On day 13 after induction, EAE groups (exercised &sedentary) were divided into untreated and mitoxantrone treated ones. Disease development was evaluated by motor performance and EAE score. Cerebrospinal fluid (CSF) was used for biochemical analysis. Brain stem and cerebellum were examined histopathological and immunohistochemically.ResultsExercise training alone did not add a significant value to the studied parameters, except for reducing Foxp3 immunoreactivity in EAE group and caspase-3 in the mitoxantrone treated group. Unexpectedly, exercise worsened the mitoxantrone effect on EAE score, Bcl2 and Bax. Mitoxantrone alone decreased EAE/demyelination/inflammation scores, Foxp3 immunoreactivity, and interleukin-6, while increased the re-myelination marker BDNF without any change in tumor necrosis factor-α. It clearly interrupted the apoptotic pathway in brain stem, but worsened EAE mediated changes of the anti-apoptotic Bcl2 and pro-apoptotic marker Bax in the CSF.ConclusionsThe neuroprotective effect of mitoxantrone was related with remyelination, immunosuppressive and anti-inflammatory potentials. Exercise training did not show added value to mitoxantrone, in contrast, it disrupts the apoptotic pathway.

Treatment With CD52 Antibody Protects Neurons in Experimental Autoimmune Encephalomyelitis Mice During the Recovering Phase.

Multiple sclerosis (MS) is a chronic autoimmune disease driven by T and B lymphocytes. The remyelination failure and neurodegeneration results in permanent clinical disability in MS patients. A desirable therapy should not only modulate the immune system, but also promote neuroprotection and remyelination. To investigate the neuroprotective effect of CD52 antibody in MS, both C57BL/6J and SJL mice with experimental autoimmune encephalomyelitis (EAE) were treated with CD52 antibody at the peak of disease. Treatment with CD52 antibody depleted T but not B lymphocytes in the blood, reduced the infiltration of T lymphocytes and microglia/macrophages in the spinal cord. Anti-CD52 therapy attenuated EAE scores during the recovery phase. It protected neurons immediately after treatment (within 4 days) as shown by reducing the accumulation of amyloid precursor proteins. It potentially promoted remyelination as it increased the number of olig2/CC-1-positive mature oligodendrocytes and prevented myelin loss in the following days (e.g., 14 days post treatment). In further experiments, EAE mice with a conditional knockout of BDNF in neurons were administered with CD52 antibodies. Neuronal deficiency of BDNF attenuated the effect of anti-CD52 treatment on reducing EAE scores and inflammatory infiltration but did not affect anti-CD52 treatment-induced improvement of myelin coverage in the spinal cord. In summary, anti-CD52 therapy depletes CD4-positive T lymphocytes, prevents myelin loss and protects neurons in EAE mice. Neuronal BDNF regulates neuroprotective and anti-inflammatory effect of CD52 antibody in EAE mice.

The Efficacy of Fecal Microbiota Transplantation in Experimental Autoimmune Encephalomyelitis: Transcriptome and Gut Microbiota Profiling.

Objective To study the protective effect of fecal microbiota transplantation (FMT) on experimental autoimmune encephalomyelitis (EAE) and reveal its potential intestinal microflora-dependent mechanism through analyses of the intestinal microbiota and spinal cord transcriptome in mice. Method We measured the severity of disease by clinical EAE scores and H&E staining. Gut microbiota alteration in the gut and differentially expressed genes (DEGs) in the spinal cord were analyzed through 16S rRNA and transcriptome sequencing. Finally, we analyzed associations between the relative abundance of intestinal microbiota constituents and DEGs. Results We observed that clinical EAE scores were lower in the EAE+FMT group than in the EAE group. Meanwhile, mice in the EAE+FMT group also had a lower number of infiltrating cells. The results of 16S rRNA sequence analysis showed that FMT increased the relative abundance of Firmicutes and Proteobacteria and reduced the abundance of Bacteroides and Actinobacteria. Meanwhile, FMT could modulate gut microbiota balance, especially via increasing the relative abundance of g_Adlercreutzia, g_Sutterella, g_Prevotella_9, and g_Tyzzerella_3 and decreasing the relative abundance of g_Turicibacter. Next, we analyzed the transcriptome of mouse spinal cord tissue and found that 1476 genes were differentially expressed between the EAE and FMT groups. The analysis of these genes showed that FMT mainly participated in the inflammatory response. Correlation analysis between gut microbes and transcriptome revealed that the relative abundance of Adlercreutzia was correlated with the expression of inflammation-related genes negatively, including Casp6, IL1RL2 (IL-36R), IL-17RA, TNF, CCL3, CCR5, and CCL8, and correlated with the expression of neuroprotection-related genes positively, including Snap25, Edil3, Nrn1, Cpeb3, and Gpr37. Conclusion Altogether, FMT may selectively regulate gene expression to improve inflammation and maintain the stability of the intestinal environment in a gut microbiota-dependent manner.

Curcumin β-D-Glucuronide Modulates an Autoimmune Model of Multiple Sclerosis with Altered Gut Microbiota in the Ileum and Feces.

We developed a prodrug type of curcumin, curcumin monoglucuronide (CMG), whose intravenous/intraperitoneal injection achieves a high serum concentration of free-form curcumin. Although curcumin has been reported to alter the gut microbiota and immune responses, it is unclear whether the altered microbiota could be associated with inflammation in immune-mediated diseases, such as multiple sclerosis (MS). We aimed to determine whether CMG administration could affect the gut microbiota at three anatomical sites (feces, ileal contents, and the ileal mucosa), leading to suppression of inflammation in the central nervous system (CNS) in an autoimmune model for MS, experimental autoimmune encephalomyelitis (EAE). We injected EAE mice with CMG, harvested the brains and spinal cords for histological analyses, and conducted microbiome analyses using 16S rRNA sequencing. CMG administration modulated EAE clinically and histologically, and altered overall microbiota compositions in feces and ileal contents, but not the ileal mucosa. Principal component analysis (PCA) of the microbiome showed that principal component (PC) 1 values in ileal contents, but not in feces, correlated with the clinical and histological EAE scores. On the other hand, when we analyzed the individual bacteria of the microbiota, the EAE scores correlated with significant increases in the relative abundance of two bacterial species at each anatomical site: Ruminococcus bromii and Blautia (Ruminococcus) gnavus in feces, Turicibacter sp. and Alistipes finegoldii in ileal contents, and Burkholderia spp. and Azoarcus spp. in the ileal mucosa. Therefore, CMG administration could alter the gut microbiota at the three different sites differentially in not only the overall gut microbiome compositions but also the abundance of individual bacteria, each of which was associated with modulation of neuroinflammation.

Radiosynthesis of [11C]Ibrutinib via Pd-Mediated [11C]CO Carbonylation: Preliminary PET Imaging in Experimental Autoimmune Encephalomyelitis Mice.

Ibrutinib is a first-generation Bruton's tyrosine kinase (BTK) inhibitor that has shown efficacy in autoimmune diseases and has consequently been developed as a positron emission tomography (PET) radiotracer. Herein, we report the automated radiosynthesis of [11C]ibrutinib through 11C-carbonylation of the acrylamide functional group, by reaction of the secondary amine precursor with [11C]CO, iodoethylene, and palladium-NiXantphos. [11C]Ibrutinib was reliably formulated in radiochemical yields of 5.4% ± 2.5% (non-decay corrected; n = 9, relative to starting [11C]CO2), radiochemical purity >99%, and molar activity of 58.8 ± 30.8 GBq/μmol (1.55 ± 0.83 Ci/μmol). Preliminary PET/magnetic resonance imaging with [11C]ibrutinib in experimental autoimmune encephalomyelitis (EAE) mice showed a 49% higher radioactivity accumulation in the spinal cord of mice with EAE scores of 2.5 vs. sham mice.

(R)-ketamine ameliorates the progression of experimental autoimmune encephalomyelitis in mice

Multiple sclerosis (MS) is an immune-mediated neurological disease that attacks the central nervous system, including spinal cord and brain. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model for MS. Depression is the most prevalent comorbidity in MS patients. We previously demonstrated that (R)-ketamine would be a novel antidepressant without side effects of ketamine. This study was undertaken to investigate whether (R)-ketamine could attenuate disease progression in EAE mouse model. (R)-ketamine (10 mg/kg/day for 15 days) significantly attenuated the reduction of body weight in EAE model mice compared to saline-treated mice. Furthermore, (R)-ketamine ameliorated the clinical EAE scores compared to saline-treated mice. Moreover, (R)-ketamine significantly attenuated the marked increases in the pathological scores, microglial activation, and blood-brain barrier integrity in the spinal cord compared to saline-treated mice. In conclusion, the current study suggests that (R)-ketamine could ameliorate EAE clinical scores and pathological changes in the spinal cord of EAE mice. Therefore, it is likely that (R)-ketamine would be a new potential prophylactic drug for MS.

Semi-Automated Live Tracking of Microglial Activation in CX3CR1GFP Mice During Experimental Autoimmune Encephalomyelitis by Confocal Scanning Laser Ophthalmoscopy.

Confocal scanning laser ophthalmoscopy (cSLO) is a non-invasive technique for real-time imaging of the retina. We developed a step-by-step protocol for the semi-automatic evaluation of myeloid cells in cSLO images from CX3CR1GFP mice, expressing green fluorescent protein (GFP) under control of the endogenous CX3C chemokine receptor 1 locus. We identified cSLO parameters allowing us to distinguish animals with experimental autoimmune encephalomyelitis (EAE) from sham-treated/naïve animals. Especially cell count (CC) and the total microglial area (SuA) turned out to be reliable parameters. Comparing the cSLO results with clinical parameters, we found significant correlations between the clinical EAE score and the SuA and of the inner retinal layer thickness, measured by optical coherence tomography, with the CC as well as the SuA. As a final step, we performed immunohistochemistry to confirm that the GFP-expressing cells visualized by the cSLO are Iba1 positive and validated the step-by-step protocol against manual counting. We present a semi-automatic step-by-step protocol with a balance between fast data evaluation and adequate accuracy, which is optimized by the option to manually adapt the contrast threshold. This protocol may be useful for numerous research questions on the role of microglial polarization in models of inflammatory and degenerating CNS diseases involving the retina.

Brief report: Enhanced DRα1-mMOG-35-55 treatment of severe EAE in MIF-1-deficient male mice

Macrophage migration inhibitory factor (MIF-1) and its homologue d-dopachrome tautomerase (MIF-2) share the common CD74 receptor and function innately to enhance severity of multiple sclerosis (MS) as well as the experimental autoimmune encephalomyelitis (EAE) model for MS. We previously demonstrated that genetically high-MIF-expressing male subjects with relapsing MS had a significantly greater risk of conversion to progressive MS (PMS) than lower-MIF-expressing males. To expand on this observation, we utilized MIF-1, MIF-2, and MIF-1/2-DUAL-deficient male mice to discern if there would be a greater contribution of these inflammatory factors in EAE mice with severe vs. moderate clinical disease signs. As shown previously, mice deficient in either MIF-1 or MIF-2 each had a ∼25% reduction of moderate EAE compared to WT mice, with significant differences in disease onset and trajectory. However, EAE induction in mice deficient in both MIF-1 and MIF-2 genes did not result in a further reduction in EAE severity. This result suggests that the two MIF homologues were likely affecting the same pathogenic pathways such that each could partially compensate for the other but not in an additive or synergistic manner. However, MIF-1-KO, MIF-2-KO, and MIF-1/2-DUAL-KO mice with severe EAE did not exhibit a significant reduction in cumulative EAE scores compared with WT mice, but the MIF-1-KO and, to a lesser extent, MIF-1/2-DUAL-KO mice did show a significant reduction in daily EAE scores over the last 3 days of observation, and MIF-2-KO mice showed a more modest but still consistent reduction over the same span. Furthermore, deletion of MIF-1 resulted in a massive reduction in the expression of EAE- and Complete Freund’s Adjuvant-associated inflammatory factors, suggesting delayed involvement of the MIF/CD74 axis in promoting disease expression. To further explore modulation of MIF-1 and MIF-2 effects on EAE, we treated WT mice with moderate EAE using DRα1-mMOG-35-55, an inhibitor of CD74 that blocks both MIF-1 and MIF-2 action. This treatment reduced ongoing moderate EAE severity in excess of 25%, suggesting efficient blockade of the MIF/CD74 axis in disease-enhancing pathways. Moreover, DRα1-mMOG-35-55 treatment of mice with severe EAE strongly reversed EAE- and CFA-associated expression of inflammatory cytokines and chemokines including Tnf, Ccr7, Ccr6, Ccl8, Cxcr3, and Ccl19 in MIF-deficient mouse genotypes, and also exceeded innate MIF-1 and MIF-2 EAE enhancing effects, especially in MIF-1-KO mice. These results illustrate the therapeutic potential of targeting the disease-enhancing MIF/CD74 pathway in male mice with moderate and severe EAE, with implications for treatment of high-MIF-expressing RRMS human males at risk of conversion to progressive MS as well as those that have already transitioned to PMS.

Apamin as a BBB Shuttle and Its Effects on T Cell Population During the Experimental Autoimmune Encephalomyelitis-Induced Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system presented by autoimmune manifestations. This study aimed at investigating the effects of apamin administration on the activated T cell population in an experimental autoimmune encephalomyelitis (EAE) MS model. Thirty mice underwent EAE induction and were then randomly divided into 5 groups. Three groups received 10, 50, and 100µg/kg apamin; the fourth group received 1mg/kg dexamethasone; and the fifth group received the equivalent amount of PBS (phosphate-buffered saline) intraperitoneally. Peripheral CD4 + cell and memory T cell distribution was measured with a flow cytometer every week. Also, CD4 + and CD8 + cell infiltration to the brain was assessed with immunohistochemistry. It was observed that the group receiving 50µg/kg apamin had a lower EAE score in comparison with the groups receiving 100µg/kg apamin (p 0.014). Also, peripheral blood memory cells with CD44 + , CD62L - , and CD4 + markers were decreased in apamin-administered groups. Regarding the infiltrated CD8 + cells, a significant decrease (p 0.002) was observed in the group receiving 50µg/kg apamin compared with the control group. These results indicate that 50-µg/kg doses of apamin had an effective treatment over 14days; it reduced both the severity of symptoms and the infiltration of CD8 + cells into the CNS. Moreover, it increased myelin density and decreased the circulation of CD62L - , CD44L - , and CD44 + memory T cells. So, it appears that apamin plays a critical role in regulating immunity and reducing the complications of autoimmune MS.

Diosgenin ameliorates cellular and molecular changes in multiple sclerosis in C57BL/6 mice

Multiple sclerosis (MS) is especially known as a demyelinating disease of the central nervous system. Current treatments for MS are mostly based on controlling neuroinflammation and there are no treatments to promote the remyelination process at present. Diosgenin is a known herbal anti-inflammatory and antioxidant agent, which has also been shown to stimulate the growth of myelin in vitro. However, there is no or little evidence about diosgenin effects; specially on myelination, neuroprotection and its corresponding mechanisms in vivo in experimental autoimmune encephalomyelitis (EAE) as the most valid experimental model of MS. In this study, the therapeutic effect of diosgenin on clinical signs of EAE, and the corresponding cellular and molecular mechanisms have been examined with emphasis on myelination and neuroprotection mechanisms. EAE was induced using myelin oligodendrocyte glycoprotein (MOG) antigen in C57BL/6 mice. Diosgenin was gavaged (100 mg/kg) daily with the onset of paralysis signs (half tail paralysis) until the 18th post-immunization day in the treatment group. Blood and spinal cord tissue sampling was performed on post-immunization day 18. Lumbar spinal cord inflammation, demyelination, and axonal degeneration were assessed using Hematoxylin and Eosin (H & E), Luxol Fast Blue (LFB), and Bielschowsky's silver staining methods, respectively. Serum and spinal cord tissue level of tumor necrosis factor alpha (TNFα) and tissue levels of matrix metalloproteinase 9 (MMP-9) and interleukin 17 (IL-17) as inflammatory markers, microtubule-associated proteins 1A/1B light chain 3A (MAP1LC3A), and activity dependent neuroprotector homeobox (ADNP) as neuroprotective markers were assayed using enzyme linked immunosorbent assay (ELISA) method. The clinical score of EAE in the diosgenin treatment group was significantly reduced compared to the EAE group on days 15 to 18 after induction of the EAE (p < 0.001). Inflammation, demyelination and axonal loss scores also decreased significantly in the diosgenin treatment group compared to the EAE group (p < 0.05). Serum and spinal cord tissue level of TNFα and tissue level of MMP-9 considerably decreased in the diosgenin treatment group in comparison with the EAE group (p < 0.01). Diosgenin treatment had no significant effects on the tissue levels of IL-17, ADNP and MAP1LC3A. Therefore, diosgenin improved the clinical signs of EAE through lowering neuroinflammation, demyelination and axonal degeneration, but did not significantly affect the neuroprotective factors in this study. As a result, diosgenin could be a good candidate for new MS treatment strategies that, in addition to their anti-inflammatory effects, also enhance myelination.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.