Contribution of microglia/macrophage to the pathogenesis of TMEV infection in the central nervous system.

The infection of the central nervous system (CNS) with neurotropic viruses induces neuroinflammation and an immune response, which is associated with the development of neuroinflammatory and neurodegenerative diseases, including multiple sclerosis (MS). The activation of both innate and adaptive immune responses, involving microglia, macrophages, and T and B cells, while required for efficient viral control within the CNS, is also associated with neuropathology. Under pathological events, such as CNS viral infection, microglia/macrophage undergo a reactive response, leading to the infiltration of immune cells from the periphery into the brain, disrupting CNS homeostasis and contributing to the pathogenesis of disease. The Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination disease (TMEV-IDD), which serves as a mouse model of MS. This murine model made significant contributions to our understanding of the pathophysiology of MS following subsequent to infection. Microglia/macrophages could be activated into two different states, classic activated state (M1 state) and alternative activated state (M2 state) during TMEV infection. M1 possesses the capacity to initiate inflammatory response and secretes pro-inflammatory cytokines, and M2-liked microglia/macrophages are anti-inflammatory characterized by the secretion of anti-inflammatory cytokines. This review aims to discuss the roles of microglia/macrophages M1/M2-liked polarization during TMEV infection, and explore the potential therapeutic effect of balancing M1/M2-liked polarization of microglia/macrophages on MS.

Application of Theiler's murine encephalomyelitis virus in treatment of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) infected mice have been often used as an animal model for Multiple sclerosis (MS) due to their similar pathology in the central nervous system (CNS). So far, there has been no effective treatment or medicine to cure MS completely. The drugs used in the clinic can only reduce the symptoms of MS, delay its recurrence, and increase the interval between relapses. MS can be caused by many factors, and clinically MS drugs are used to treat MS regardless of what factors are caused rather than MS caused by a specific factor. This can lead to inappropriate medicine, which may be one of the reasons why MS has not been completely cured. Therefore, this review summarized the drugs investigated in the TMEV-induced disease (TMEV-IDD) model of MS, so as to provide medication guidance and theoretical basis for the treatment of virus-induced MS.

Longitudinal quantitative assessment of TMEV-IDD-induced MS phenotypes in two inbred mouse strains using automated video tracking technology.

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system affecting over 2.5 million people worldwide. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a murine model that reproduces the progressive form of MS and serves as a reference model for studying virus-induced demyelination. Certain mouse strains such as SJL are highly susceptible to this virus and serve as a prototype strain for studying TMEV infection. Other strains such as SWR are also susceptible, but their disease course following TMEV infection differs from SJL's. The quantification of motor and behavioral deficits following the induction of TMEV-IDD could help identify the differences between the two strains. Motor deficits have commonly been measured with the rotarod apparatus, but a multicomponent assessment tool has so far been lacking. For that purpose, we present a novel way of quantifying locomotor deficits, gait alterations and behavioral changes in this well-established mouse model of multiple sclerosis by employing automated video analysis technology (The PhenoTyper, Noldus Information Technology). We followed 12 SJL and 12 SWR female mice and their mock-infected counterparts over a period of 9 months following TMEV-IDD induction. We demonstrated that SJL and SWR mice both suffer significant gait alterations and reduced exploration following TMEV infection. However, SJL mice also display an earlier and more severe decline in spontaneous locomotion, especially in velocity, as well as in overall activity. Maintenance behaviors such as eating and grooming are not affected in either of the two strains. The system also showed differences in mock-infected mice from both strains, highlighting an age-related decline in spontaneous locomotion in the SJL strain, as opposed to hyperactivity in the SWR strain. Our study confirms that this automated video tracking system can reliably track the progression of TMEV-IDD for 9 months. We have also shown how this system can be utilized for longitudinal phenotyping in mice by describing useful parameters that quantify locomotion, gait and behavior.

The Contribution of Microglia and Brain-Infiltrating Macrophages to the Pathogenesis of Neuroinflammatory and Neurodegenerative Diseases during TMEV Infection of the Central Nervous System.

The infection of the central nervous system (CNS) with neurotropic viruses induces neuroinflammation and is associated with the development of neuroinflammatory and neurodegenerative diseases, including multiple sclerosis and epilepsy. The activation of the innate and adaptive immune response, including microglial, macrophages, and T and B cells, while required for efficient viral control within the CNS, is also associated with neuropathology. Under healthy conditions, resident microglia play a pivotal role in maintaining CNS homeostasis. However, during pathological events, such as CNS viral infection, microglia become reactive, and immune cells from the periphery infiltrate into the brain, disrupting CNS homeostasis and contributing to disease development. Theiler's murine encephalomyelitis virus (TMEV), a neurotropic picornavirus, is used in two distinct mouse models: TMEV-induced demyelination disease (TMEV-IDD) and TMEV-induced seizures, representing mouse models of multiple sclerosis and epilepsy, respectively. These murine models have contributed substantially to our understanding of the pathophysiology of MS and seizures/epilepsy following viral infection, serving as critical tools for identifying pharmacological targetable pathways to modulate disease development. This review aims to discuss the host-pathogen interaction during a neurotropic picornavirus infection and to shed light on our current understanding of the multifaceted roles played by microglia and macrophages in the context of these two complexes viral-induced disease.

High throughput method for detecting murine brain atrophy using a clinical 3T MRI.

BACKGROUND: There is a lack of understanding of the mechanisms by which the CNS is injured in multiple sclerosis (MS). Since Theiler's murine encephalomyelitis virus (TMEV) infection in SJL/J mice is an established model of progressive disability in MS, and CNS atrophy correlates with progressive disability in MS, we used in vivo MRI to quantify total ventricular volume in TMEV infection. We then sought to identify immunological and virological biomarkers that correlated with increased ventricular size. METHODS: Mice, both infected and control, were followed for 6 months. Cerebral ventricular volumes were determined by MRI, and disability was assessed by Rotarod. A range of immunological and virological measures was obtained using standard techniques. RESULTS: Disability was present in infected mice with enlarged ventricles, while infected mice without enlarged ventricles had Rotarod performance similar to sham mice. Ventricular enlargement was detected as soon as 1 month after infection. None of the immunological and virological measures correlated with the development of ventricular enlargement. CONCLUSIONS: These results support TMEV infection with brain MRI monitoring as a useful model for exploring the biology of disability progression in MS, but they did not identify an immunological or virological correlate with ventricular enlargement.

C57BL/6J mice exposed to perfluorooctanoic acid demonstrate altered immune responses and increased seizures after Theiler's murine encephalomyelitis virus infection.

Introduction: Neurological diseases can stem from environmental influences such as antecedent viral infections or exposure to potential toxicants, some of which can trigger immune responses leading to neurological symptoms. Theiler's murine encephalomyelitis virus (TMEV) is used to model human neurological conditions associated with prior viral infections, with outcomes partly attributable to improper induction and regulation of the immune response. Perfluorooctanoic acid (PFOA) can alter pathologies known to influence neurological disease such as inflammatory responses, cytokine expression, and glial activation. Co-exposure to TMEV and PFOA was used to test the hypothesis that early life exposure to the potential immunotoxicant PFOA would affect immune responses so as to render TMEV-resistant C57BL/6J (B6) mice susceptible to viral-induced neurological disease. Methods: Neonate B6 mice were exposed to different treatments: non-injected, sham-infected with PBS, and TMEV-infected, with the drinking water of each group including either 70 ppt PFOA or filtered water. The effects of PFOA were evaluated by comparing neurological symptoms and changes in immune-related cytokine and chemokine production induced by viral infection. Immune responses of 23 cytokines and chemokines were measured before and after infection to determine the effects of PFOA exposure on immune response. Results: Prior to infection, an imbalance between Th1, Th2, and Treg cytokines was observed in PFOA-exposed mice, suppressing IL-4 and IL-13 production. However, the balance was restored and characterized by an increase in pro-inflammatory cytokines in the non-infected group, and a decrease in IL-10 in the PFOA + TMEV group. Furthermore, the PFOA + TMEV group experienced an increase in seizure frequency and severity. Discussion: Overall, these findings provide insight into the complex roles of immune responses in the pathogenesis of virus-associated neurological diseases influenced by co-exposures to viruses and immunotoxic compounds.

Effect of Siponimod on Brain and Spinal Cord Imaging Markers of Neurodegeneration in the Theiler's Murine Encephalomyelitis Virus Model of Demyelination.

Siponimod (Sp) is a Sphingosine 1-phosphate (S1P) receptor modulator, and it suppresses S1P- mediated autoimmune lymphocyte transport and inflammation. Theiler's murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis (MS) exhibits inflammation-driven acute and chronic phases, spinal cord lesions, brain and spinal cord atrophy, and white matter injury. The objective of the study was to investigate whether Sp treatment could attenuate inflammation-induced pathology in the TMEV model by inhibiting microglial activation and preventing the atrophy of central nervous tissue associated with neurodegeneration. Clinical disability score (CDS), body weight (BW), and rotarod retention time measures were used to assess Sp's impact on neurodegeneration and disease progression in 4 study groups of 102 animals, including 44 Sp-treated (SpT), 44 vehicle-treated, 6 saline-injected, and 8 age-matched healthy controls (HC). Next, 58 (22 SpT, 22 vehicle, 6 saline injected, and 8 HC) out of the 102 animals were further evaluated to assess the effect of Sp on brain region-specific and spinal cord volume changes, as well as microglial activation. Sp increased CDS and decreased BW and rotarod retention time in TMEV mice, but did not significantly affect most brain region volumes, except for lateral ventricle volume. Sp suppressed ventricular enlargement, suggesting reduced TMEV-induced inflammation in LV. No significant differences in spine volume changes were observed between Sp- and vehicle-treated animals, but there were differences between HC and TMEV groups, indicating TMEV-induced inflammation contributed to increased spine volume. Spine histology revealed no significant microglial density differences between groups in gray matter, but HC animals had higher type 1 morphology and lower type 2 morphology percentages in gray and white matter regions. This suggests that Sp did not significantly affect microglial density but may have modulated neuroinflammation in the spinal cord. Sp may have some effects on neuroinflammation and ventricular enlargement. However, it did not demonstrate a significant impact on neurodegeneration, spinal volume, or lesion volume in the TMEV mouse model. Further investigation is required to fully understand Sp's effect on microglial activation and its relevance to the pathophysiology of MS. The differences between the current study and previous research using other MS models, such as EAE, highlight the differences in pathological processes in these two disease models.

Chronic encephalomyelitis virus exhibits cellular tropism and evades pDCs by binding to sialylated integrins as the cell surface receptors.

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease similar to multiple sclerosis in mice. Although sialic acids have been shown to be essential for TMEV attachment to the host, the surface receptor has not been identified. While type I interferons play a pivotal role in the elimination of the chronic infectious Daniel (DA) strain, the role of plasmacytoid dendritic cells (pDCs) is controversial. We herein found that TMEV binds to conventional DCs but not to pDCs. A glycomics analysis showed that the sialylated N-glycan fractions were lower in pDCs than in conventional DCs, indicating that pDCs are not susceptible to TMEV infection due to the low levels of sialic acid. TMEV capsid proteins contain an integrin recognition motif, and dot blot assays showed that the integrin proteins bind to TMEV and that the viral binding was reduced in the desialylated αX ß2 . αX ß2 protein suppressed TMEV replication in vivo, and TMEV co-localized with integrin αM at the cell membrane and TLR 3 in the cytoplasm, suggesting that αM serves as the viral attachment and entry. These results show that the chronic encephalomyelitis virus utilizes sialylated integrins as cell surface receptors, leading to cellular tropism to evade pDC activation.

Ovalbumin-specific CD4+ and CD8+ T cells contribute to different susceptibility for Theiler's murine encephalomyelitis virus persistence.

Theiler's murine encephalomyelitis virus (TMEV) is the causative agent of TMEV-induced demyelinating disease (TMEV-IDD); a well-established animal model for the chronic progressive form of human multiple sclerosis (MS). In susceptible mice with an inadequate immune response, TMEV-IDD is triggered by virus persistence and maintained by a T cell mediated immunopathology. OT-mice are bred on a TMEV-resistant C57BL/6 background and own predominantly chicken ovalbumin (OVA)-specific populations of CD8+ T cells (OT-I) or CD4+ T cells (OT-II), respectively. It is hypothesized that the lack of antigen specific T cell populations increases susceptibility for a TMEV-infection in OT-mice on a TMEV-resistant C57BL/6 background. OT-I, OT-II, and C57BL/6 control mice were infected intracerebrally with the TMEV-BeAn strain. Mice were scored weekly for clinical disease and after necropsy, histological and immunohistochemical evaluation was performed. OT-I mice started to develop progressive motor dysfunction between 7 and 21 days post infection (dpi), leading up to hind limb paresis and critical weight loss, which resulted in euthanasia for humane reasons between 14 and 35 dpi. OT-I mice displayed a high cerebral virus load, an almost complete absence of CD8+ T cells from the central nervous system (CNS) and a significantly diminished CD4+ T cell response. Contrarily, only 60% (12 of 20) of infected OT-II mice developed clinical disease characterized by mild ataxia. 25% of clinically affected OT-II mice (3 of 12) made a full recovery. 5 of 12 OT-II mice with clinical disease developed severe motor dysfunction similar to OT-I mice and were euthanized for humane reasons between 13 and 37 dpi. OT-II mice displayed only low virus-immunoreactivity, but clinical disease correlated well with severely reduced infiltration of CD8+ T cells and the increased presence of CD4+ T cells in the brains of OT-II mice. Though further studies are needed to reveal the underlying pathomechanisms following TMEV infection in OT mice, findings indicate an immunopathological process as a main contributor to clinical disease in OT-II mice, while a direct virus-associated pathology may be the main contributor to clinical disease in TMEV-infected OT-I mice.

CD28-signaling can be partially compensated in CD28-knockout mice but is essential for virus elimination in a murine model of multiple sclerosis.

The intracerebral infection of mice with Theiler's murine encephalomyelitis virus (TMEV) represents a well-established animal model for multiple sclerosis (MS). Because CD28 is the main co-stimulatory molecule for the activation of T cells, we wanted to investigate its impact on the course of the virus infection as well as on a potential development of autoimmunity as seen in susceptible mouse strains for TMEV. In the present study, 5 weeks old mice on a C57BL/6 background with conventional or tamoxifen-induced, conditional CD28-knockout were infected intracerebrally with TMEV-BeAn. In the acute phase at 14 days post TMEV-infection (dpi), both CD28-knockout strains showed virus spread within the central nervous system (CNS) as an uncommon finding in C57BL/6 mice, accompanied by histopathological changes such as reduced microglial activation. In addition, the conditional, tamoxifen-induced CD28-knockout was associated with acute clinical deterioration and weight loss, which limited the observation period for this mouse strain to 14 dpi. In the chronic phase (42 and 147 dpi) of TMEV-infection, surprisingly only 33% of conventional CD28-knockout mice showed chronic TMEV-infection with loss of motor function concomitant with increased spinal cord inflammation, characterized by T- and B cell infiltration, microglial activation and astrogliosis at 33-42 dpi. Therefore, the clinical outcome largely depends on the time point of the CD28-knockout during development of the immune system. Whereas a fatal clinical outcome can already be observed in the early phase during TMEV-infection for conditional, tamoxifen-induced CD28-knockout mice, only one third of conventional CD28-knockout mice develop clinical symptoms later, accompanied by ongoing inflammation and an inability to clear the virus. However, the development of autoimmunity could not be observed in this C57BL/6 TMEV model irrespective of the time point of CD28 deletion.

Magnetic resonance imaging approaches for studying mouse models of multiple sclerosis: A mini review.

Given that multiple sclerosis (MS) is a complex disease with an unclear etiology, a single animal model is unlikely to accurately represent all aspects of pathology and clinical features of the human condition. However, the availability of three major types of murine models of MS, that is, experimental autoimmune encephalomyelitis (EAE), viral models, and toxic models, enables studies of several relevant features of this debilitating disease. Researchers have recently begun to combine magnetic resonance imaging (MRI) technologies with other experimental strategies to acquire complementary information, for example, anatomical and functional, and study the effect of experimental manipulations longitudinally in a noninvasive way. This review summarizes the latest MRI studies investigating critical aspects of MS, such as atrophy, demyelination, neuroaxonal damage, and neuroinflammation, in mouse models of MS. Advanced techniques will be briefly discussed, providing references to specialized literature for the readers. Thus, this review aims to describe different imaging protocols used to study critical aspects of MS in a research laboratory, discussing the main related findings in the most significant murine models of the disease.

Baseline Gait and Motor Function Predict Long-Term Severity of Neurological Outcomes of Viral Infection.

Neurological dysfunction following viral infection varies among individuals, largely due to differences in their genetic backgrounds. Gait patterns, which can be evaluated using measures of coordination, balance, posture, muscle function, step-to-step variability, and other factors, are also influenced by genetic background. Accordingly, to some extent gait can be characteristic of an individual, even prior to changes in neurological function. Because neuromuscular aspects of gait are under a certain degree of genetic control, the hypothesis tested was that gait parameters could be predictive of neuromuscular dysfunction following viral infection. The Collaborative Cross (CC) mouse resource was utilized to model genetically diverse populations and the DigiGait treadmill system used to provide quantitative and objective measurements of 131 gait parameters in 142 mice from 23 CC and SJL/J strains. DigiGait measurements were taken prior to infection with the neurotropic virus Theiler's Murine Encephalomyelitis Virus (TMEV). Neurological phenotypes were recorded over 90 days post-infection (d.p.i.), and the cumulative frequency of the observation of these phenotypes was statistically associated with discrete baseline DigiGait measurements. These associations represented spatial and postural aspects of gait influenced by the 90 d.p.i. phenotype score. Furthermore, associations were found between these gait parameters with sex and outcomes considered to show resistance, resilience, or susceptibility to severe neurological symptoms after long-term infection. For example, higher pre-infection measurement values for the Paw Drag parameter corresponded with greater disease severity at 90 d.p.i. Quantitative trait loci significantly associated with these DigiGait parameters revealed potential relationships between 28 differentially expressed genes (DEGs) and different aspects of gait influenced by viral infection. Thus, these potential candidate genes and genetic variations may be predictive of long-term neurological dysfunction. Overall, these findings demonstrate the predictive/prognostic value of quantitative and objective pre-infection DigiGait measurements for viral-induced neuromuscular dysfunction.

The Dual PDE7-GSK3ß Inhibitor, VP3.15, as Neuroprotective Disease-Modifying Treatment in a Model of Primary Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune and degenerative disease with axonal damage and demyelination as its main features. Its dual neurological and autoimmune nature makes it a disease that is difficult to treat. Treatments that simultaneously stop the immune response while protecting and repairing the nervous system are urgent. That is of utmost importance for the primary progressive multiple sclerosis (PPMS), a rare and severe variant of MS, characterized by worsening neurological function from the onset of symptoms. In this sense, inhibitors of glycogen synthase kinase 3ß (GSK3ß) and phosphodiesterase 7 (PDE7) have recently shown great therapeutic potential for the treatment of demyelinating diseases. Here we investigated a dual inhibitor of these two targets, the small molecule VP3.15, in a preclinical model, which resembles primary-progressive MS (PPMS), the Theiler's mouse encephalomyelitis virus-induced demyelinated disease (TMEV-IDD). In our study, VP3.15 ameliorates the disease course improving motor deficits of infected mice. Chronic treatment with VP3.15 also showed significant efficacy in the immunomodulation process, as well as in the proliferation and differentiation of oligodendroglial precursors, improving the preservation of myelin and axonal integrity. Therefore, our results support a treatment with the safe VP3.15 as an integrative therapeutic strategy for the treatment of PPMS.

Theiler's virus-induced demyelinating disease as an infectious model of progressive multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease of unknown etiology. However, several studies suggest that infectious agents, e.g., Human Herpes Viruses (HHV), may be involved in triggering the disease. Molecular mimicry, bystander effect, and epitope spreading are three mechanisms that can initiate immunoreactivity leading to CNS autoimmunity in MS. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a pre-clinical model of MS in which intracerebral inoculation of TMEV results in a CNS autoimmune disease that causes demyelination, neuroaxonal damage, and progressive clinical disability. Given the spectra of different murine models used to study MS, this review highlights why TMEV-IDD represents a valuable tool for testing the viral hypotheses of MS. We initially describe how the main mechanisms of CNS autoimmunity have been identified across both MS and TMEV-IDD etiology. Next, we discuss how adaptive, innate, and CNS resident immune cells contribute to TMEV-IDD immunopathology and how this relates to MS. Lastly, we highlight the sexual dimorphism observed in TMEV-IDD and MS and how this may be tied to sexually dimorphic responses to viral infections. In summary, TMEV-IDD is an underutilized murine model that recapitulates many unique aspects of MS; as we learn more about the nature of viral infections in MS, TMEV-IDD will be critical in testing the future therapeutics that aim to intervene with disease onset and progression.

Viral Clearance and Neuroinflammation in Acute TMEV Infection Vary by Host Genetic Background.

A wide range of viruses cause neurological manifestations in their hosts. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the brain, depending in part on host genetic background. The interaction between host genetic background, neurological response to viral infection, and subsequent clinical manifestations remains poorly understood. In the present study, we used the genetically diverse Collaborative Cross (CC) mouse resource to better understand how differences in genetic background drive clinical signs and neuropathological manifestations of acute Theiler's murine encephalomyelitis virus (TMEV) infection. For the first time, we characterized variations of TMEV viral tropism and load based on host genetic background, and correlated viral load with microglial/macrophage activation. For five CC strains (CC002, CC023, CC027, CC057, and CC078) infected with TMEV, we compared clinical signs, lesion distribution, microglial/macrophage response, expression, and distribution of TMEV mRNA, and identified genetic loci relevant to the early acute (4 days post-infection [dpi]) and late acute (14 dpi) timepoints. We examined brain pathology to determine possible causes of strain-specific differences in clinical signs, and found that fields CA1 and CA2 of the hippocampal formation were especially targeted by TMEV across all strains. Using Iba-1 immunolabeling, we identified and characterized strain- and timepoint-specific variation in microglial/macrophage reactivity in the hippocampal formation. Because viral clearance can influence disease outcome, we used RNA in situ hybridization to quantify viral load and TMEV mRNA distribution at both timepoints. TMEV mRNA expression was broadly distributed in the hippocampal formation at 4 dpi in all strains but varied between radiating and clustered distribution depending on the CC strain. We found a positive correlation between microglial/macrophage reactivity and TMEV mRNA expression at 4 dpi. At 14 dpi, we observed a dramatic reduction in TMEV mRNA expression, and localization to the medial portion of field CA1 and field CA2. To better understand how host genetic background can influence pathological outcomes, we identified quantitative trait loci associated with frequency of lesions in a particular brain region and with microglial/macrophage reactivity. These QTL were located near several loci of interest: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1), and transmembrane protein 106 B (Tmem106b). Together, these results provide a novel understanding about the influences of genetic variation on the acute neuropathological and immunopathological environment and viral load, which collectively lead to variable disease outcomes. Our findings reveal possible avenues for future investigation which may lead to more effective intervention strategies and treatment regimens.

Divergent complement system activation in two clinically distinct murine models of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease featuring neuroinflammation and neurodegeneration in young adults. So far, most research has focused on the peripheral immune system, which appears to be the driver of acute relapses. Concurrently, the mechanisms underlying neurodegeneration in the progressive forms of the disease remain unclear. The complement system, a molecular component of the innate immunity, has been recently implicated in several neurological disorders, including MS. However, it is still unknown if the complement proteins detected in the central nervous system (CNS) are actively involved in perpetuating chronic inflammation and neurodegeneration. To address this knowledge gap, we compared two clinically distinct mouse models of MS: 1) proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (rEAE) resembling a relapsing-remitting disease course, and 2) Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) resembling a progressive disease. Real-time PCR was performed in the spinal cord of rEAE mice, TMEV-IDD mice and age-matched sham controls to quantify gene expression for a broad range of complement components. In both experimental models, we found significantly increased expression of complement factors, such as C1q, C3, CfB, and C3aR. We showed that the complement system, specifically the classical complement pathway, was associated with TMEV-IDD pathogenesis, as the expression of C1q, C3 and C3aR1 were all significantly correlated to a worse disease outcome (all P≤0.0168). In line with this finding, C1q and C3 deposition was observed in the spinal cord of TMEV-IDD mice. Furthermore, C1q deposition was detected in spinal cord regions characterized by inflammation, demyelination, and axonal damage. Conversely, activation of the classical complement cascade seemed to result in protection from rEAE (C1q: P=0.0307). Interestingly, the alternative pathway related to a worse disease outcome in rEAE (CFb: P=0.0006). Overall, these results indicate potential divergent roles for the complement system in MS. The chronic-progressive disease form is more reliant on the activation of the classic complement pathway, while protecting from acute relapses. Conversely, relapsing MS appears more likely affected by the alternative pathway. Understanding the functions of the complement system in MS is critical and can lead to better, more targeted therapies in the future.

Brain-Derived Neurotrophic Factor Inhibits the Function of Cation-Chloride Cotransporter in a Mouse Model of Viral Infection-Induced Epilepsy.

Well over 100 different viruses can infect the brain and cause brain inflammation. In the developing world, brain inflammation is a leading cause for epilepsy and often refractory to established anti-seizure drugs. Epilepsy generally results from an imbalance in excitatory glutamatergic and inhibitory GABAergic neurotransmission. GABAergic inhibition is determined by the intracellular Cl- concentration which is established through the opposing action of two cation chloride cotransporters namely NKCC1 and KCC2. Brain-derived neurotrophic factor (BDNF) signaling is known to regulate expression of KCC2. Hence we hypothesized that viral induced epilepsy may result from aberrant BDNF signaling. We tested this hypothesis using a mouse model of Theiler's murine encephalomyelitis virus (TMEV) infection-induced epilepsy. We found that BDNF levels in the hippocampus from TMEV-infected mice with seizures was increased at the onset of acute seizures and continued to increase during the peak of acute seizure as well as in latent and chronic phases of epilepsy. During the acute phase of epilepsy, we found significant reduction in the expression of KCC2 in hippocampus, whereas the level of NKCC1 was unaltered. Importantly, inhibiting BDNF using scavenging bodies of BDNF in live brain slices from TMEV-infected mice with seizures normalized the level of KCC2 in hippocampus. Our results suggest that BDNF can directly decrease the relative expression of NKCC1 and KCC2 such as to favor accumulation of chloride intracellularly which in turn causes hyperexcitability by reversing GABA-mediated inhibition. Although our attempt to inhibit the BDNF signaling mediated through tyrosine kinase B-phospholipase Cγ1 (TrkB-PLCγ1) using a small peptide did not change the course of seizure development following TMEV infection, alternative strategies for controlling the BDNF signaling could be useful in preventing seizure generation and development of epilepsy in this model.

Serum Cytokines Predict Neurological Damage in Genetically Diverse Mouse Models.

Viral infections contribute to neurological and immunological dysfunction driven by complex genetic networks. Theiler's murine encephalomyelitis virus (TMEV) causes neurological dysfunction in mice and can model human outcomes to viral infections. Here, we used genetically distinct mice from five Collaborative Cross mouse strains and C57BL/6J to demonstrate how TMEV-induced immune responses in serum may predict neurological outcomes in acute infection. To test the hypothesis that serum cytokine levels can provide biomarkers for phenotypic outcomes of acute disease, we compared cytokine levels at pre-injection, 4 days post-injection (d.p.i.), and 14 d.p.i. Each strain produced unique baseline cytokine levels and had distinct immune responses to the injection procedure itself. Thus, we eliminated the baseline responses to the injection procedure itself and identified cytokines and chemokines induced specifically by TMEV infection. Then, we identified strain-specific longitudinal cytokine profiles in serum during acute disease. Using stepwise regression analysis, we identified serum immune markers predictive for TMEV-induced neurological phenotypes of the acute phase, e.g., IL-9 for limb paralysis; and TNF-α, IL-1ß, and MIP-1ß for limb weakness. These findings indicate how temporal differences in immune responses are influenced by host genetic background and demonstrate the potential of serum biomarkers to track the neurological effects of viral infection.

Neurological Benefits, Clinical Challenges, and Neuropathologic Promise of Medical Marijuana: A Systematic Review of Cannabinoid Effects in Multiple Sclerosis and Experimental Models of Demyelination.

Despite current therapeutic strategies for immunomodulation and relief of symptoms in multiple sclerosis (MS), remyelination falls short due to dynamic neuropathologic deterioration and relapses, leading to accrual of disability and associated patient dissatisfaction. The potential of cannabinoids includes add-on immunosuppressive, analgesic, neuroprotective, and remyelinative effects. This study evaluates the efficacy of medical marijuana in MS and its experimental animal models. A systematic review was conducted by a literature search through PubMed, ProQuest, and EBSCO electronic databases for studies reported since 2007 on the use of cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) in MS and in experimental autoimmune encephalomyelitis (EAE), Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and toxin-induced demyelination models. Study selection and data extraction were performed by 3 reviewers, and 28 studies were selected for inclusion. The certainty of evidence was appraised using the Cochrane GRADE approach. In clinical studies, there was low- and moderate-quality evidence that treatment with ~1:1 CBD/THC mixtures as a nabiximols (Sativex®) oromucosal spray reduced numerical rating scale (NRS) scores for spasticity, pain, and sleep disturbance, diminished bladder overactivity, and decreased proinflammatory cytokine and transcription factor expression levels. Preclinical studies demonstrated decreases in disease severity, hindlimb stiffness, motor function, neuroinflammation, and demyelination. Other experimental systems showed the capacity of cannabinoids to promote remyelination in vitro and by electron microscopy. Modest short-term benefits were realized in MS responders to adjunctive therapy with CBD/THC mixtures. Future studies are recommended to investigate the cellular and molecular mechanisms of cannabinoid effects on MS lesions and to evaluate whether medical marijuana can accelerate remyelination and retard the accrual of disability over the long term.

IFN-ß Deficiency Results in Fatal or Demyelinating Disease in C57BL/6 Mice Infected With Theiler's Murine Encephalomyelitis Viruses.

Type I Interferons (IFN-I) are important inducers of the antiviral immune response and immune modulators. IFN-ß is the most highly expressed IFN-I in the central nervous system (CNS). The infection of SJL mice with the BeAn or the DA strain of Theiler's murine encephalomyelitis virus (TMEV) results in a progressive demyelinating disease. C57BL/6 mice are usually resistant to TMEV-induced demyelination and eliminate these strains from the CNS within several weeks. Using C57BL/6 IFN-ß knockout (IFN-ß-/-) mice infected with TMEV, we evaluated the role of IFN-ß in neuroinfection. Despite the resistance of C57BL/6 wild type (WT) mice to TMEV infection, DA-infected IFN-ß-/- mice had to be killed at 7 to 8 days post infection (dpi) due to severe clinical disease. In contrast, BeAn-infected IFN-ß-/- mice survived until 98 dpi. Nevertheless at 14 dpi, BeAn-infected IFN-ß-/- mice showed a stronger encephalitis and astrogliosis, higher viral load as well as higher mRNA levels of Isg15, Eif2ak2 (PKR), Tnfa, Il1b, Il10, Il12 and Ifng in the cerebrum than BeAn-infected WT mice. Moreover, the majority of IFN-ß-/- mice did not clear the virus from the CNS and developed mild demyelination in the spinal cord at 98 dpi, whereas virus and lesions were absent in the spinal cord of WT mice. Persistently infected IFN-ß-/- mice also had higher Isg15, Eif2ak1, Tnfa, Il1a, Il1b and Ifng mRNA levels in the spinal cord at 98 dpi than their virus-negative counterparts indicating an activation of IFN-I signaling and ongoing inflammation. Most importantly, BeAn-infected NesCre+/- IFN-ßfl/fl mice, which do not express IFN-ß in neurons, astrocytes and oligodendrocytes, only developed mild brain lesions similar to WT mice. Consequently, IFN-ß produced by neuroectodermal cells does not seem to play a critical role in the resistance of C57BL/6 mice against fatal and demyelinating disease induced by TMEV strains.