Experimental Autoimmune Encephalomyelitis Animals Research Articles

Ferritin loss in astrocytes reduces spinal cord oxidative stress and demyelination in the experimental autoimmune encephalomyelitis (EAE) model.

Demyelinating diseases such as multiple sclerosis (MS) cause myelin degradation and oligodendrocyte death, resulting in the release of toxic iron and iron-induced oxidative stress. Astrocytes have a large capacity for iron transport and storage, however the role of astrocytic iron homeostasis in demyelinating disorders is not completely understood. Here we investigate whether astrocytic iron metabolism modulates neuroinflammation, oligodendrocyte survival, and oxidative stress following demyelination. To this aim, we conditionally knock out ferritin in astrocytes and induce experimental autoimmune encephalomyelitis (EAE), an autoimmune-mediated model of demyelination. Ferritin ablation in astrocytes reduced the severity of disease in both the acute and chronic phases. The day of onset, peak disease severity, and cumulative clinical score were all significantly reduced in ferritin KO animals. This corresponded to better performance on the rotarod and increased mobility in ferritin KO mice. Furthermore, the spinal cord of ferritin KO mice display decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes. Correspondingly, the size of demyelinated lesions, iron accumulation, and oxidative stress were attenuated in the CNS of ferritin KO subjects, particularly in white matter regions of the spinal cord. Thus, deleting ferritin in astrocytes reduced neuroinflammation, oxidative stress, and myelin deterioration in EAE animals. Collectively, these findings suggest that iron storage in astrocytes is a potential therapeutic target to lessen CNS inflammation and myelin loss in autoimmune demyelinating diseases.

Impaired olfactory performance and anxiety-like behavior in a rat model of multiple sclerosis are associated with enhanced adenosine signaling in the olfactory bulb via A1R, A2BR, and A3R.

The present study shows that animals with experimental autoimmune encephalomyelitis (EAE) exhibit olfactory dysfunction and impaired general cognitive abilities, as well as anxiety-like behavior. Olfactory dysfunction occurs on average at 2 dpi, well before the onset of the first motor signs of EAE (8-10 dpi). After the initial olfactory dysfunction, the EAE animals show a fluctuation in olfactory performance that resembles the relapsing-remitting course of human MS. The study also shows severe neuroinflammation in the olfactory bulb (OB), with numerous infiltrated CD4+ T cells and peripheral macrophages in the superficial OB layers, marked microgliosis, and massive induction of TNF-α, IL-1β, and IL-6. Reduced tyrosine hydroxylase activity in the glomerular layer, pronounced granule cell atrophy, and reduced numbers of type B neuroblasts in the rostral migratory stream also indicate altered plasticity of the neuronal network in the OB. Considering the exceptionally high purinome expression in the OB, the possible involvement of purinergic signaling was also investigated. The study shows that macrophages infiltrating the OB overexpress A3R, while highly reactive microglia overexpress the adenosine-producing enzyme eN/CD73 as well as A2BR, A3R, and P2X4R. Given the simultaneous induction of complement component C3, the results suggest that the microglial cells develop a functional phenotype of phagocytizing microglia. The study also demonstrates transcriptional and translational upregulation of A1R in mitral and tufted cells, which likely influence resting network activity in OB and likely contribute to olfactory dysfunction in EAE. Overall, our study shows that olfactory dysfunction and altered social and cognitive behavior in EAE are associated with increased adenosine signaling via A1R, A2BR, and A3R.

Intraperitoneal injection of mesenchymal stem cells-conditioned media (MSCS-CM) treated monocyte can potentially alleviate motor defects in experimental autoimmune encephalomyelitis female mice; An original experimental study

IntroductionMultiple sclerosis (MS), as a destructive pathology of myelin in central nervous system (CNS), causes physical and mental complications. Experimental autoimmune encephalomyelitis (EAE) is laboratory model of MS widely used for CNS-associated inflammatory researches. Cell therapy using macrophage M2 (MPM2) is a cell type with anti-inflammatory characteristics for all inflammatory-based neuropathies. This experimental study investigated the probable therapeutic anti-inflammatory effects of intraperitoneal (IP) injection of MPM2 on alleviation of motor defect in EAE-affected animals. Materials and methods24 C57/BL6 female mice were divided into four groups of EAE, EAE + Dexa, EAE + PBS, and EAE + MP2. EAE was induced through deep cervical injection of spinal homogenate of guinea pigs. MPM2 cells were harvested from bone marrow and injected (106cells/ml) in three days of 10, 13 and 16 post-immunizations (p.i). Clinical score (CS), anti-inflammatory cytokines (IL-4, IL-10), pro-inflammatory gene expression (TNF-α, IL-1β) and histopathological investigations (HE, Nissl and Luxol Fast Blue) were considered. Data were analyzed using SPSS software (v.19) and p < 0.05 was considered significant level. ResultsDuring EAE induction, the mean animal weight was decreased (p < 0.05); besides, following MPM2 injection, the weight gain was applied (p < 0.05) in EAE + MPM2 groups than control. Increased (p < 0.05) levels of CS was found during EAE induction in days 17–28 in EAE animals; besides, CS was decreased (p < 0.05) in EAE + MPM2 group than EAE animals. Also, in days 25–28 of experiment, the CS was decreased (p < 0.05) in EAE + MPM2 than EAE + Dexa. Histopathological assessments revealed low density of cell nuclei in corpus callosum, microscopically. LFB staining also showed considerable decrease in white matter density of corpus callosum in EAE group. Acceleration of white matter density was found in EAE + MPM2 group following cell therapy procedure. Genes expression of TNF-α, IL-1β along with IL-4 and IL-10 were decreased (p < 0.05) in EAE + MPM2 group. ConclusionIP injection of MPM2 to EAE-affected female mice can potentially reduce the CNS inflammation, neuronal death and myelin destruction. MPM2 cell therapy can improve animal motor defects.

Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination

Disturbance in neuroendocrine signaling has been consistently documented in multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS) representing the main cause of non-traumatic brain injury among young adults. In fact, MS patients display altered hormonal levels and psychiatric symptoms along with the pathologic hallmarks of the disease, which include demyelination, neuroinflammation and axonal injury. In addition, we have recently shown that extensive transcriptional changes take place in the hypothalamus of mice upon the MS model experimental autoimmune encephalomyelitis (EAE). We also detected structural and functional aberrancies in endocrine glands of EAE animals. Specifically, we described the hyperplasia of adrenal glands and the atrophy of ovaries at disease peak. To further expand the characterization of these phenotypes, here we profiled the transcriptomes of both glands by means of RNA-seq technology. Notably, we identified fatty acid and cholesterol biosynthetic pathways as the most dysregulated molecular processes in adrenals and ovaries, respectively. Furthermore, we demonstrated that key genes encoding neuropeptides and hormone receptors undergo distinct expression dynamics in the hypothalamus along disease progression. Altogether, our results corroborate the dysfunction of the neuroendocrine system as a major pathological event of autoimmune demyelination and highlight the crosstalk between the CNS and the periphery in mediating such disease phenotypes.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

The presence of Mott cells in the lymph nodes of rats with experimental autoimmune encephalomyelitis.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis

Multiple sclerosis (MS) is characterized by a compromised blood-brain barrier (BBB) resulting in central nervous system (CNS) entry of peripheral lymphocytes, including T cells and B cells. While T cells have largely been considered the main contributors to neuroinflammation in MS, the success of B cell depletion therapies suggests an important role for B cells in MS pathology. Glial cells in the CNS are essential components in both disease progression and recovery, raising the possibility that they represent targets for B cell functions. Here, we examine astrocyte and microglia responses to B cell depleting treatments in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). B cell depleted EAE animals had markedly reduced disease severity and myelin damage accompanied by reduced microglia and astrocyte reactivity 20 days after symptom onset. To identify potential initial mechanisms mediating functional changes following B cell depletion, astrocyte and microglia transcriptomes were analyzed 3 days following B cell depletion. In control EAE animals, transcriptomic analysis revealed astrocytic inflammatory pathways were activated and microglial influence on neuronal function were inhibited. Following B cell depletion, initial functional recovery was associated with an activation of astrocytic pathways linked with restoration of neurovascular integrity and of microglial pathways associated with neuronal function. These studies reveal an important role for B cell depletion in influencing glial function and CNS vasculature in an animal model of MS.

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.

Cell therapy procedure using anti-inflammatory macrophage M2 can potentially reduce Clinical Score in animals with Experimental Autoimmune Encephalomyelitis: A preclinical systematic review and meta-analysis study.

Macrophage M2 (MP2)-based cell therapy is a novel medicinal treatment for animals with Experimental Autoimmune Encephalomyelitis (EAE) as an experimental model of multiple sclerosis (MS). This systematic review and meta-analysis study was designed to assess the overall therapeutic effects of MP2 cell therapy on Clinical Score and motor impairment in EAE-induced animals. All experiments on MP2 cell therapy in animals with EAE were gathered (by October 2, 2022) from English (PubMed, Scopus, WoS, Science Direct, and ISC) and Persian (MagIran and SID) databases. The searching strategy was designed using "Experimental Autoimmune Encephalomyelitis," "Multiple Sclerosis," and "Macrophage M2" keywords. Following primary and secondary screenings, eligible papers were selected based on the PRISMA 2020 guideline, and the study quality was assessed using the Animal Research: Reporting of In Vivo Experiments (ARRIVE) checklist. The difference in means of Clinical Score (score 0-5) as the effect size (ES) was analyzed based on the random effect model (CMA software, v.2). Subgrouping (EAE phases of Onset, Peak, and Recovery) was applied, and I2 index was used to assess the heterogeneity index. Publication bias and sensitivity indices were also evaluated. P < 0.05 was considered significant, and the confidence interval (CI) was determined 95%. Among 22 gathered papers, medium to high quality studies were selected for meta-analysis. Difference in means, P value, and I2 for Onset, Peak, and Recovery phases were 0.082 (CI95%: -0.323-0.159, P value: 0.504, I2 : 67.961%), -0.606 (CI95%: -1.518 to -0.305, P value: 0.192, I2 : 96.070%), and -1.103 (CI95%: -1.390 to -0.816, P value: 0.000, I2 : 30.880%), respectively and Overall Effect was found -0.509 (CI95%: -0.689 to -0.328, P value < 0.001). Also, P value (two-tailed) indices for publication bias were 0.366 and 0.583 for Egger's regression intercept and Begg rank correlation, respectively. The P value for sensitivity was detected 0.003. Cell therapy procedure using MP2 can potentially alleviate the Clinical Scores Index and correct the motor defects in Recovery phase of EAE animals. In healthy mice, the brain and myelin surrounding neurons are in a healthy and physiological state (1). To evaluate MS in humans, it is necessary to model this type of disease in animals using EAE procedure through subcutaneous injection of CFA, MOG35-55 , MT, and Pert. Thus, inflammation and autoimmunity occur, which finally lead to myelin destruction and motor symptoms (2). By aspiration of progenitor cells available in bone marrow, the MP2 can be isolated and cultured. By activation of these types of cells, a rich collection of MP2 can be prepared for the cell-therapy process (3). After injection through the tail vein or intra-peritoneal procedure, these cells can be located in CNS through crossing from the BBB. They begin their anti-inflammatory activities and help repair the damaged myelin (4). Eventually, the clinical symptoms can be modified considerably, and the animal motor function improves (5). CFA, complete Freund's adjuvant; MOG35-55 , myelin oligodendrocyte glycoprotein; MT, Mycobacterium tuberculosis; Pert, pertussis; EAE, Experimental Autoimmune Encephalomyelitis; BM, bone marrow; MP2, macrophage M2; and BBB, blood brain barrier.

Toward a Combination of Biomarkers for Molecular Characterization of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system associated with chronic inflammation, demyelination, and axonal damage. MS is a highly heterogeneous disease that leads to discrepancies regarding the clinical appearance, progression, and therapy response of patients. Therefore, there is a strong unmet need for clinically relevant biomarkers capable of recapitulating the features of the disease. Experimental autoimmune encephalomyelitis (EAE) is a valuable model for studying the pathophysiology of MS as it recapitulates the main hallmarks of the disease: inflammation, blood-brain barrier (BBB) disruption, gliosis, myelin damage, and repair mechanisms. In this study, we used the EAE-PLP animal model and established a molecular RNA signature for each phase of the disease (onset, peak, remission). We compared variances of expression of known biomarkers by RT-qPCR in the brain and spinal cord of sham and EAE animals monitoring each of the five hallmarks of the disease. Using magnetic cell isolation technology, we isolated microglia and oligodendrocytes of mice of each category, and we compared the RNA expression variations. We identify genes deregulated during a restricted time frame, and we provide insight into the timing and interrelationships of pathological disease processes at the organ and cell levels.

Role of the intestinal microbiota in the pathogenesis of multiple sclerosis. Part 1. Clinical and experimental evidence for the involvement of the gut microbiota in the development of multiple sclerosis

The review discusses the complex role of the intestinal microbiota in the pathogenesis of multiple sclerosis, summarizes data from studies of changes in the composition of the intestinal microbiome in patients with multiple sclerosis, and provides evidence of the involvement of the intestinal microbiota in the development of experimental autoimmune encephalomyelitis in animals, a valid model of multiple sclerosis.

Comparative effectiveness of preventive treatment with dimethyl fumarate-loaded solid lipid nanoparticles and oral dimethyl fumarate in a mouse model of multiple sclerosis

BackgroundOrally administered dimethyl fumarate (DMF) presents gastrointestinal adverse effects, such as pain and diarrhea, in addition to flushing and lymphopenia. ObjectiveSolid lipid nanoparticles (SLNs) with DMF were developed for subcutaneous administration. MethodsDMF-incorporated SLNs and free DMF were tested in mice induced with experimental autoimmune encephalomyelitis (EAE). ResultsPreventive treatment of free or incorporated DMF were able to reduce the EAE clinical scores, increase the weight of the animals, reduce the lesion area (demyelination and infiltration), reduce microglial fluorescence intensity and reduce the number of microglial cells and astrocytes, when compared to untreated EAE animals. Groups that received DMF had reduced numbers of T cells, B cells and natural killer (NK) cells in the blood, when compared to the non-induced group. ConclusionsDMF incorporated in SLNs was as effective as free DMF in reducing the clinical scores of the animals, but with reduced administrations when given subcutaneously. In addition, SLN-DMF preventive treatment partially prevented a reduction in the percentages of T and B cells, in the blood, when compared to preventive treatment with free DMF (oral), which suggests reduction of lymphopenia.

Gliosis attenuation in experimental autoimmune encephalomyelitis by a combination of dimethyl fumarate and pregabalin.

Dysregulated microglia and astrocytes have been associated with progressive neurodegeneration in multiple sclerosis (MS), highlighting the need for strategies that additionally target intrinsic inflammation in the central nervous system (CNS). The objective of the present study was to investigate the glial response in experimental autoimmune encephalomyelitis (EAE)-induced mice treated with a combination of dimethyl fumarate (DMF) and pregabalin (PGB). For that, 28 C57BL/6J mice were randomly assigned to the five experimental groups: naïve, EAE, EAE-DMF, EAE-PGB, and EAE-DMF + PGB. Pharmacological treatments were initiated with the beginning of clinical signs, and all animals were euthanized at 28 dpi for the lumbar spinal cord evaluation. The results demonstrated a stronger attenuation of the clinical presentation by the combined approach. DMF alone promoted the downregulation of Iba-1 (microglia/macrophages marker) in the ventral horn compared with the non-treated EAE animals (P < 0.05). PGB treatment was associated with reduced Iba-1 immunofluorescence in both the dorsal (P < 0.05) and ventral horn (P < 0.05) compared to EAE vehicle-treated counterparts. However, the combined approach reduced the Iba-1 marker in the dorsal (P < 0.05) and ventral (P < 0.01) horns compared to non-treated EAE animals and further reduced Iba-1 in the ventral horn compared to each drug-alone approach (P < 0.05). In addition, the combination of DMF and PGB reduced activated astrocytes (GFAP) in both the dorsal and ventral horns of the spinal cord to a naïve-like level and upregulated Nrf-2 expression. Taken together, the data herein suggest robust attenuation of the glial response in EAE mice treated with DMF and PGB.

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Application of a triple‐helical peptide inhibitor of MMP‐2/MMP‐9 to examine T‐cell activation in experimental autoimmune encephalomyelitis

AbstractMultiple sclerosis (MS) is an inflammatory demyelinating disease whereby pathology includes multifocal perivascular mononuclear cell infiltration into the central nervous system, oligodendrocyte loss and demyelination, and variable axonal loss. Little is known about the causative agent in MS, but the matrix metalloproteinase (MMP) family of proteolytic enzymes has been implicated in disease progression and resolution. Recent studies revealed that MMP‐2 and/or MMP‐9 are required for an effective T‐cell‐mediated immune response to antigenic stimulation. In the present studies, we used an MMP‐2/MMP‐9 selective triple‐helical peptide inhibitor (THPI) to examine the role of these enzymes in antigen specific T‐cell responses. We found that treatment of murine CD4+ T‐cells with the THPI, followed by CD3/CD28 stimulation of the T‐cell receptor (TCR), resulted in reduced cell proliferation and production of interleukin 2 (IL‐2), interferon gamma (IFN‐γ), and tumor‐necrosis factor alpha (TNF‐α). Application of the THPI in experimental autoimmune encephalomyelitis (EAE), a widely accepted animal model of MS, reduced clinical severity of the disease and weight loss. Splenocytes isolated from THPI‐treated EAE animals had reduced proliferation compared with untreated animals. In an in vitro recall experiment, splenocytes isolated from EAE mice were treated with the THPI, followed by CD3/CD28 stimulation of the TCR. Treated splenocytes had reduced production of IL‐2, IL‐6, IL‐10, IFN‐γ, and TNF‐α compared with untreated splenocytes isolated from EAE mice. Overall, the MMP‐2/MMP‐9‐targeting THPI modulates T‐cell response to antigenic stimulation, including proliferation and immune cell production of specific cytokines, providing insight as to why the THPI is effective in reducing the clinical severity of EAE.

Healthy Properties of a New Formulation of Pomegranate-Peel Extract in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

A new formulation of a pomegranate-peel extract (PEm) obtained by PUAE (Pulsed Ultrasound-Assisted Extraction) and titrated in both ellagic acid (EA) and punicalagin is proposed, characterized and then analyzed for potential health properties in mice suffering from the experimental autoimmune encephalomyelitis (EAE). PEm effects were compared to those elicited by a formulation containing EA (EAm). Control and EAE mice were chronically administered EAm and Pem dissolved in the drinking water, starting from the day 10 post-immunization (d.p.i.), with a “therapeutic” protocol to deliver daily 50 mg/kg of EA. Treated EAE mice did not limit their daily access to the beverage, nor did they show changes in body weight, but they displayed a significant amelioration of “in vivo” clinical symptoms. “Ex vivo” histochemical analysis showed that spinal-cord demyelination and inflammation in PEm and EAm-treated EAE mice at 23 ± 1 d.p.i. were comparable to those in the untreated EAE animals, while microglia activation (measured as Ionized Calcium Binding Adaptor 1, Iba1 staining) and astrocytosis (quantified as glial fibrillar acid protein, GFAP immunopositivity) significantly recovered, particularly in the gray matter. EAm and PEm displayed comparable efficiencies in controlling the spinal pathological cellular hallmarks in EAE mice, and this would support their delivery as dietary supplementation in patients suffering from multiple sclerosis (MS).

Dopaminergic Receptors as Neuroimmune Mediators in Experimental Autoimmune Encephalomyelitis.

The dopaminergic system plays an essential role in maintaining homeostasis between the central nervous system (CNS) and the immune system. Previous studies have associated imbalances in the dopaminergic system to the pathogenesis of multiple sclerosis (MS). Here, we examined the protein levels of dopaminergic receptors (D1R and D2R) in different phases of the experimental autoimmune encephalomyelitis (EAE) model. We also investigated if the treatment with pramipexole (PPX)-a dopamine D2/D3 receptor-preferring agonist-would be able to prevent EAE-induced motor and mood dysfunction, as well as its underlying mechanisms of action. We report that D2R immunocontent is upregulated in the spinal cord of EAE mice 14days post-induction. Moreover, D1R and D2R immunocontents in lymph nodes and the oxidative damage in the spinal cord and striatum of EAE animals were significantly increased during the chronic phase. Also, during the pre-symptomatic phase, axonal damage in the spinal cord of EAE mice could already be found. Surprisingly, therapeutic treatment with PPX failed to inhibit the progression of EAE. Of note, PPX treatment inhibited EAE-induced depressive-like while failed to inhibit anhedonic-like behaviors. We observed that PPX treatment downregulated IL-1β levels and increased BNDF content in the spinal cord after EAE induction. Herein, we show that a D2/D3 receptor-preferred agonist mitigated EAE-induced depressive-like behavior, which could serve as a new possibility for further clinical trials on treating depressive symptoms in MS patients. Thus, we infer that D2R participates in the crosstalk between CNS and immune system during autoimmune and neuroinflammatory response induced by EAE, mainly in the acute and chronic phase of the disease.

Downregulation of CD73/A2AR-Mediated Adenosine Signaling as a Potential Mechanism of Neuroprotective Effects of Theta-Burst Transcranial Magnetic Stimulation in Acute Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease caused by autoimmune-mediated inflammation in the central nervous system. Purinergic signaling is critically involved in MS-associated neuroinflammation and its most widely applied animal model—experimental autoimmune encephalomyelitis (EAE). A promising but poorly understood approach in the treatment of MS is repetitive transcranial magnetic stimulation. In the present study, we aimed to investigate the effect of continuous theta-burst stimulation (CTBS), applied over frontal cranial bone, on the adenosine-mediated signaling system in EAE, particularly on CD73/A2AR/A1R in the context of neuroinflammatory activation of glial cells. EAE was induced in two-month-old female DA rats and in the disease peak treated with CTBS protocol for ten consecutive days. Lumbosacral spinal cord was analyzed immunohistochemically for adenosine-mediated signaling components and pro- and anti-inflammatory factors. We found downregulated IL-1β and NF- κB-ir and upregulated IL-10 pointing towards a reduction in the neuroinflammatory process in EAE animals after CTBS treatment. Furthermore, CTBS attenuated EAE-induced glial eN/CD73 expression and activity, while inducing a shift in A2AR expression from glia to neurons, contrary to EAE, where tight coupling of eN/CD73 and A2AR on glial cells is observed. Finally, increased glial A1R expression following CTBS supports anti-inflammatory adenosine actions and potentially contributes to the overall neuroprotective effect observed in EAE animals after CTBS treatment.

Targeted Blood Brain Barrier Opening With Focused Ultrasound Induces Focal Macrophage/Microglial Activation in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis (MS). EAE reflects important histopathological hallmarks, dissemination, and diversity of the disease, but has only moderate reproducibility of clinical and histopathological features. Focal lesions are less frequently observed in EAE than in MS, and can neither be constrained to specific locations nor timed to occur at a pre-specified moment. This renders difficult any experimental assessment of the pathogenesis of lesion evolution, including its inflammatory, degenerative (demyelination and axonal degeneration), and reparatory (remyelination, axonal sprouting, gliosis) component processes. We sought to develop a controlled model of inflammatory, focal brain lesions in EAE using focused ultrasound (FUS). We hypothesized that FUS induced focal blood brain barrier disruption (BBBD) will increase the likelihood of transmigration of effector cells and subsequent lesion occurrence at the sonicated location. Lesion development was monitored with conventional magnetic resonance imaging (MRI) as well as with magnetic resonance elastography (MRE) and further analyzed by histopathological means. EAE was induced in 12 6–8 weeks old female C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG) peptide. FUS-induced BBBD was performed 6, 7, and 9 days after immunization in subgroups of four animals and in an additional control group. MRI and MRE were performed on a 7T horizontal bore small animal MRI scanner. Imaging was conducted longitudinally 2 and 3 weeks after disease induction and 1 week after sonication in control animals, respectively. The scan protocol comprised contrast-enhanced T1-weighted and T2-weighted sequences as well as MRE with a vibration frequency of 1 kHz. Animals were sacrificed for histopathology after the last imaging time point. The overall clinical course of EAE was mild. A total of seven EAE animals presented with focal T2w hyperintense signal alterations in the sonicated hemisphere. These were most frequent in the group of animals sonicated 9 days after immunization. Histopathology revealed foci of activated microglia/macrophages in the sonicated right hemisphere of seven EAE animals. Larger cellular infiltrates or apparent demyelination were not seen. Control animals showed no abnormalities on MRI and did not have clusters of activated microglia/macrophages at the sites targeted with FUS. None of the animals had hemorrhages or gross tissue damage as potential side effects of FUS. EAE-animals tended to have lower values of viscoelasticity and elasticity in the sonicated compared to the contralateral parenchyma. This trend was significant when comparing the right sonicated to the left normal hemisphere and specifically the right sonicated compared to the left normal cortex in animals that underwent FUS-BBBD 9 days after immunization (right vs. left hemisphere: mean viscoelasticity 6.1 vs. 7.2 kPa; p = 0.003 and mean elasticity 4.9 vs. 5.7 kPa, p = 0.024; right vs. left cortex: mean viscoelasticity 5.8 vs. 7.5 kPa; p = 0.004 and mean elasticity 5 vs. 6.5 kPa; p = 0.008). A direct comparison of the biomechanical properties of focal T2w hyperintensities with normal appearing brain tissue did not yield significant results. Control animals showed no differences in viscoelasticity between sonicated and contralateral brain parenchyma. We here provide first evidence for a controlled lesion induction model in EAE using FUS-induced BBBD. The observed lesions in EAE are consistent with foci of activated microglia that may be interpreted as targeted initial inflammatory activity and which have been described as pre-active lesions in MS. Such foci can be identified and monitored with MRI. Moreover, the increased inflammatory activity in the sonicated brain parenchyma seems to have an effect on overall tissue matrix structure as reflected by changes of biomechanical parameters.

Trichinella spiralis excretory-secretory products downregulate MMP-9 in Dark Agouti rats affected by experimental autoimmune encephalomyelitis

Matrix metalloproteinases (MMPs), are implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Our aim was to investigate whether amelioration of EAE in Dark Agouti (DA) rats, induced by Trichinella spiralis muscle larvae excretory-secretory products (ES L1), could be related to the level and activity of gelatinases, MMP-9 and MMP-2. Serum levels of MMP-9, MMP-2, NGAL/MMP-9, TIMP-1, and cytokines, evaluated by gel-zymography or ELISA, as well as gelatinases and TIMP-1 expression in the spinal cord (SC), were determined in: i) EAE induced, ii) ES L1-treated EAE induced animals. Milder clinical signs in ES L1-treated EAE induced DA rats were accompanied with lower serum levels of MMP-9 and NGAL/MMP-9 complex. However, the correlation between the severity of EAE and the level of serum MMP-9 was found only in the peak of the disease, with MMP-9/TIMP-1 ratio higher in EAE animals without ES L1 treatment. Lower expression of MMP-9 in SC of ES L1-treated, EAE induced rats, correlated with the reduced number of SC infiltrating cells. In SC infiltrates, in the effector and the recovery phase, production of anti-inflammatory cytokines IL-4 and IL-10 was higher in animals treated with ES L1 prior to EAE induction, compared to untreated EAE animals. Reduced expression of MMP-9 in SC tissue, which correlated with the reduced number of infiltrating cells, might be ascribed to regulatory mechanisms, among which is IL-10.