Unsupervised quantification of tissue immunofluorescence in animal models of multiple sclerosis - Instructions for use.

BACKGROUND: In the analysis of animal models of CNS diseases such as experimental autoimmune encephalomyelitis (EAE), immunostaining and histopathology are important readouts. However, the complex morphological features of a tissue staining are often reduced to a single measure which relies on tedious manual planimetry. Furthermore, the measure itself and co-variables such as the region being analysed are chosen in a human decision-making process, which introduces bias. NEW METHOD: First aim of the present study is to provide an open-source workflow for the high-throughput, unsupervised quantification of different stainings in the spinal cord. We evaluate different EAE models, spinal cord regions and different time points of disease. By applying random forest classification, we compare different measures. RESULTS: Exemplified for glial reactivity, we show that measures and variables interact and that their values are non-normally distributed, hampering the common use of parametric tests. Furthermore, we demonstrate that one-dimensional measures are insufficient descriptors for immunofluorescence data in EAE and thus need to be considered as partly invalid. COMPARISON WITH EXISTING METHODS: We show in a systematic analysis of EAE studies that currently published immunohistological outcomes are highly incompatible regarding methodology and statistics. Furthermore, they lack the report of important information necessary for reproducibility and do not use unsupervised automatic analysis. CONCLUSIONS: Our results discover relevant caveats in the currently used methods of immunofluorescence analysis. The provided step-by-step instructions and open-source code are intended to serve as a framework for sensitive, unbiased immunofluorescence analysis of tissue sections in translational research.

Anticoagulation with warfarin and rivaroxaban ameliorates experimental autoimmune encephalomyelitis.

BACKGROUND: In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE). METHODS: Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP139-151. Stable anticoagulation was maintained throughout the entire experiment. Mice without anticoagulation treated with the vehicle only were used as controls. The neurological deficit was recorded during the course of EAE, and histopathological analyses of inflammatory lesions were performed. RESULTS: In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed. CONCLUSION: We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.

Reliability of infarct volumetry: Its relevance and the improvement by a software-assisted approach.

Despite the efficacy of neuroprotective approaches in animal models of stroke, their translation has so far failed from bench to bedside. One reason is presumed to be a low quality of preclinical study design, leading to bias and a low a priori power. In this study, we propose that the key read-out of experimental stroke studies, the volume of the ischemic damage as commonly measured by free-handed planimetry of TTC-stained brain sections, is subject to an unrecognized low inter-rater and test-retest reliability with strong implications for statistical power and bias. As an alternative approach, we suggest a simple, open-source, software-assisted method, taking advantage of automatic-thresholding techniques. The validity and the improvement of reliability by an automated method to tMCAO infarct volumetry are demonstrated. In addition, we show the probable consequences of increased reliability for precision, p-values, effect inflation, and power calculation, exemplified by a systematic analysis of experimental stroke studies published in the year 2015. Our study reveals an underappreciated quality problem in translational stroke research and suggests that software-assisted infarct volumetry might help to improve reproducibility and therefore the robustness of bench to bedside translation.

"Disease modifying nutricals" for multiple sclerosis.

The association between vitamin D and multiple sclerosis has (re)-opened new interest in nutrition and natural compounds in the prevention and treatment of this neuroinflammatory disease. The dietary amount and type of fat, probiotics and biologicals, salmon proteoglycans, phytoestrogens and protease inhibitor of soy, sodium chloride and trace elements, and fat soluble vitamins including D, A and E were all considered as disease-modifying nutraceuticals. Studies in experimental autoimmune encephalomyelitis mice suggest that poly-unsaturated fatty acids and their 'inflammation-resolving' metabolites and the gut microflora may reduce auto-aggressive immune cells and reduce progression or risk of relapse, and infection with whipworm eggs may positively change the gut-brain communication. Encouraged by the recent interest in multiple sclerosis-nutrition nature's pharmacy has been searched for novel compounds with anti-inflammatory, immune-modifying and antioxidative properties, the most interesting being the scorpion toxins that inhibit specific potassium channels of T cells and antioxidative compounds including the green tea flavonoid epigallocatechin-3-gallate, curcumin and the mustard oil glycoside from e.g. broccoli and sulforaphane. They mostly also inhibit pro-inflammatory signaling through NF-κB or toll-like receptors and stabilize the blood brain barrier. Disease modifying functions may also complement analgesic and anti-spastic effects of cannabis, its constituents, and of 'endocannabinoid enhancing' drugs or nutricals like inhibitors of fatty acid amide hydrolase. Nutricals will not solve multiple sclerosis therapeutic challenges but possibly support pharmacological interventions or unearth novel structures.