Virus-triggered spinal cord demyelination is followed by a peripheral neuropathy resembling features of Guillain-Barré Syndrome.

Theiler's murine encephalomyelitis virus (TMEV)-induces a demyelinating disease in the spinal cord (SC) of susceptible but not in resistant (B6) mouse strains. The aim of the present study was to induce SC demyelination and a peripheral neuropathy in resistant mice by switching the infection site from cerebrum to SC. B6 mice were intraspinally inoculated with TMEV. Infected mice showed clinical signs starting at 7 days post infection (dpi). Histopathology revealed a mononuclear myelitis, centred on the injection site at 3 dpi with subsequent antero- and retrograde spread, accompanied by demyelination and axonal damage within the SC. Virus protein was detected in the SC at all time points. SC inflammation decreased until the end of the investigation period (28 dpi). Concurrent with the amelioration of SC inflammation, the emergence of a peripheral neuropathy, characterized by axonal damage, demyelination and macrophage infiltration, contributing to persistent clinical sings, was observed. Intraspinal TMEV infection of resistant mice induced inflammation, demyelination and delayed viral clearance in the spinal cord and more interestingly, subsequent, virus-triggered inflammation and degeneration within the PN associated with dramatic and progressive clinical signs. The lesions observed in the PN resemble important features of Guillain-Barré syndrome, especially of acute motor/motor-sensory axonal forms.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.

Microarray-Based Gene Expression Analysis for Veterinary Pathologists: A Review.

High-throughput, genome-wide transcriptome analysis is now commonly used in all fields of life science research and is on the cusp of medical and veterinary diagnostic application. Transcriptomic methods such as microarrays and next-generation sequencing generate enormous amounts of data. The pathogenetic expertise acquired from understanding of general pathology provides veterinary pathologists with a profound background, which is essential in translating transcriptomic data into meaningful biological knowledge, thereby leading to a better understanding of underlying disease mechanisms. The scientific literature concerning high-throughput data-mining techniques usually addresses mathematicians or computer scientists as the target audience. In contrast, the present review provides the reader with a clear and systematic basis from a veterinary pathologist's perspective. Therefore, the aims are (1) to introduce the reader to the necessary methodological background; (2) to introduce the sequential steps commonly performed in a microarray analysis including quality control, annotation, normalization, selection of differentially expressed genes, clustering, gene ontology and pathway analysis, analysis of manually selected genes, and biomarker discovery; and (3) to provide references to publically available and user-friendly software suites. In summary, the data analysis methods presented within this review will enable veterinary pathologists to analyze high-throughput transcriptome data obtained from their own experiments, supplemental data that accompany scientific publications, or public repositories in order to obtain a more in-depth insight into underlying disease mechanisms.

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

Central Nervous System Demyelination and Remyelination is Independent from Systemic Cholesterol Level in Theiler's Murine Encephalomyelitis.

High dietary fat and/or cholesterol intake is a risk factor for multiple diseases and has been debated for multiple sclerosis. However, cholesterol biosynthesis is a key pathway during myelination and disturbances are described in demyelinating diseases. To address the possible interaction of dyslipidemia and demyelination, cholesterol biosynthesis gene expression, composition of the body's major lipid repositories and Paigen diet-induced, systemic hypercholesterolemia were examined in Theiler's murine encephalomyelitis (TME) using histology, immunohistochemistry, serum clinical chemistry, microarrays and high-performance thin layer chromatography. TME-virus (TMEV)-infected mice showed progressive loss of motor performance and demyelinating leukomyelitis. Gene expression associated with cholesterol biosynthesis was overall down-regulated in the spinal cord of TMEV-infected animals. Spinal cord levels of galactocerebroside and sphingomyelin were reduced on day 196 post TMEV infection. Paigen diet induced serum hypercholesterolemia and hepatic lipidosis. However, high dietary fat and cholesterol intake led to no significant differences in clinical course, inflammatory response, astrocytosis, and the amount of demyelination and remyelination in the spinal cord of TMEV-infected animals. The results suggest that down-regulation of cholesterol biosynthesis is a transcriptional marker for demyelination, quantitative loss of myelin-specific lipids, but not cholesterol occurs late in chronic demyelination, and serum hypercholesterolemia exhibited no significant effect on TMEV infection.

The antiviral drug ganciclovir does not inhibit microglial proliferation and activation.

Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS.

Transcriptional analysis of glial cell differentiation in the postnatal murine spinal cord.

Postnatal murine spinal cord represents a good model system to study mammalian central nervous system myelination in vivo as a basis for further studies in demyelinating diseases. Transcriptional changes were analyzed in SJL/J mice on postnatal day 0, 14, 49 and 231 (P0, P14, P49, P231) employing Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Additionally, marker gene signatures for astrocyte and oligodendrocyte lineage-stages were defined to study their gene expression in more detail. In addition, immunohistochemistry was used to quantify the abundance of commonly used glial cell markers. 6092 differentially regulated genes (DEGs) were identified. The up-regulated DEGs at P14, P49 and P231 compared to P0 exhibited significantly enriched associations to gene ontology terms such as myelination and lipid metabolic transport and down-regulated DEGs to neurogenesis and axonogenesis. Expression values of marker gene signatures for neural stem cells, oligodendrocyte precursor cells, and developing astrocytes were constantly decreasing, whereas myelinating oligodendrocyte and mature astrocyte markers showed a steady increase. Molecular findings were substantiated by immunohistochemical observations. The transcriptional changes observed are an important reference for future analysis of degenerative and inflammatory conditions in the spinal cord.

Transcriptomic meta-analysis of multiple sclerosis and its experimental models.

BACKGROUND: Multiple microarray analyses of multiple sclerosis (MS) and its experimental models have been published in the last years. OBJECTIVE: Meta-analyses integrate the information from multiple studies and are suggested to be a powerful approach in detecting highly relevant and commonly affected pathways. DATA SOURCES: ArrayExpress, Gene Expression Omnibus and PubMed databases were screened for microarray gene expression profiling studies of MS and its experimental animal models. STUDY ELIGIBILITY CRITERIA: Studies comparing central nervous system (CNS) samples of diseased versus healthy individuals with n >1 per group and publically available raw data were selected. MATERIAL AND METHODS: Included conditions for re-analysis of differentially expressed genes (DEGs) were MS, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in rats, proteolipid protein-induced EAE in mice, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and a transgenic tumor necrosis factor-overexpressing mouse model (TNFtg). Since solely a single MS raw data set fulfilled the inclusion criteria, a merged list containing the DEGs from two MS-studies was additionally included. Cross-study analysis was performed employing list comparisons of DEGs and alternatively Gene Set Enrichment Analysis (GSEA). RESULTS: The intersection of DEGs in MS, EAE, TMEV-IDD, and TNFtg contained 12 genes related to macrophage functions. The intersection of EAE, TMEV-IDD and TNFtg comprised 40 DEGs, functionally related to positive regulation of immune response. Over and above, GSEA identified substantially more differentially regulated pathways including coagulation and JAK/STAT-signaling. CONCLUSION: A meta-analysis based on a simple comparison of DEGs is over-conservative. In contrast, the more experimental GSEA approach identified both, a priori anticipated as well as promising new candidate pathways.