Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Study protocol for the description and evaluation of the "Habit Coach" - a longitudinal multicenter mHealth intervention for healthy habit formation in health care professionals.

BACKGROUND: The adoption of a healthy lifestyle plays a crucial role for the health and well-being of health care professionals. Previous e- and mHealth interventions relied on deliberative psychological processes (e.g., intention, planning) to target lifestyle changes, while revealing mixed efficacy. The additional potential of non-deliberative, automatic processes (i.e., habits) for behavior change has been understudied in interventions so far. The Habit Coach mHealth intervention combines deliberative and non-deliberative processes to support health care professionals in forming healthy physical activity, nutrition and mindfulness habits in daily life. The aim of this paper is to outline the study protocol including a detailed description of the mHealth intervention, evaluation plan, and study design. The purpose of this trial is to understand healthy habit formation in health care professionals over time. METHODS: A one-arm, multicenter mHealth intervention study will be conducted. Behavioral and psychosocial predictors will be collected via within-app questionnaires across a 100-day period at baseline, post, as well as at weekly assessments. To understand habit formation across time, linear mixed models will be used. DISCUSSION: This trial aims to unravel the role of motivational and volitional determinants for healthy habit formation across multiple health behaviors in health care professionals embedded in a mHealth intervention. TRIAL REGISTRATION: This trial is registered in the German Clinical Trials Register, DRKS-ID DRKS00027156. Date of registration 17 November 2021.

An optimized and validated protocol for inducing chronic experimental autoimmune encephalomyelitis in C57BL/6J mice.

BACKGROUND: Myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis. However, variations in the induction protocol can affect EAE progression, and may reduce the comparability of data. OPTIMIZED METHOD: In the present study, we investigated the influence of the different components used for EAE induction in C57BL/6J mice on disease progression. In the present study, MOG35-55-induced chronic EAE in C57BL/6J mice has been applied as a model to challenge optimal pertussis toxin (PTx) dosing, while considering variations in batch potency. RESULTS: We demonstrate that the dosage of PTx, adjusted to its potency, influences EAE development in a dose-dependent manner. Our data show that with our protocol, which considers PTx potency, C57BL/6J mice consistently develop symptoms of EAE. The mice show a typical chronic course with symptom onset after 10.5 ± 1.08 days and maximum severity around day 16 postimmunization followed by a mild remission of symptoms. COMPARISON WITH EXISTING METHODS: Previously studies reveal that alterations in PTx dosing directly modify EAE progression. Our present study highlights that PTx batches differ in potency, resulting in inconsistent EAE induction. We also provide a clear protocol that allows a reduction in the number of mice used in EAE experiments, while maintaining consistent results. CONCLUSION: Higher standards for comparability and reproducibility are needed to ensure and maximize the generation of reliable EAE data. Specifically, consideration of PTx potency. With our method of establishing consistent EAE pathogenesis, improved animal welfare standards and a reduction of mice used in experimentation can be achieved.

Platelet Inhibition by Low-Dose Acetylsalicylic Acid Reduces Neuroinflammation in an Animal Model of Multiple Sclerosis.

Aside from the established immune-mediated etiology of multiple sclerosis (MS), compelling evidence implicates platelets as important players in disease pathogenesis. Specifically, numerous studies have highlighted that activated platelets promote the central nervous system (CNS)-directed adaptive immune response early in the disease course. Platelets, therefore, present a novel opportunity for modulating the neuroinflammatory process that characterizes MS. We hypothesized that the well-known antiplatelet agent acetylsalicylic acid (ASA) could inhibit neuroinflammation by affecting platelets if applied at low-dose and investigated its effect during experimental autoimmune encephalomyelitis (EAE) as a model to study MS. We found that oral administration of low-dose ASA alleviates symptoms of EAE accompanied by reduced inflammatory infiltrates and less extensive demyelination. Remarkably, the percentage of CNS-infiltrated CD4+ T cells, the major drivers of neuroinflammation, was decreased to 40.98 ± 3.28% in ASA-treated mice compared to 56.11 ± 1.46% in control animals at the disease maximum as revealed by flow cytometry. More interestingly, plasma levels of thromboxane A2 were decreased, while concentrations of platelet factor 4 and glycoprotein VI were not affected by low-dose ASA treatment. Overall, we demonstrate that low-dose ASA could ameliorate the platelet-dependent neuroinflammatory response in vivo, thus indicating a potential treatment approach for MS.

One Brain-All Cells: A Comprehensive Protocol to Isolate All Principal CNS-Resident Cell Types from Brain and Spinal Cord of Adult Healthy and EAE Mice.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice-applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.

The STING-IFN-ß-Dependent Axis Is Markedly Low in Patients with Relapsing-Remitting Multiple Sclerosis.

Cyclic GMP-AMP-synthase is a sensor of endogenous nucleic acids, which subsequently elicits a stimulator of interferon genes (STING)-dependent type I interferon (IFN) response defending us against viruses and other intracellular pathogens. This pathway can drive pathological inflammation, as documented for type I interferonopathies. In contrast, specific STING activation and subsequent IFN-ß release have shown beneficial effects on experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Although less severe cases of relapse-remitting MS (RRMS) are treated with IFN-ß, there is little information correlating aberrant type I IFN signaling and the pathologic conditions of MS. We hypothesized that there is a link between STING activation and the endogenous production of IFN-ß during neuroinflammation. Gene expression analysis in EAE mice showed that Sting level decreased in the peripheral lymphoid tissue, while its level increased within the central nervous system over the course of the disease. Similar patterns could be verified in peripheral immune cells during the acute phases of RRMS in comparison to remitting phases and appropriately matched healthy controls. Our study is the first to provide evidence that the STING/IFN-ß-axis is downregulated in RRMS patients, meriting further intensified research to understand its role in the pathophysiology of MS and potential translational applications.