Focal white matter lesions induce long-lasting axonal degeneration, neuroinflammation and behavioral deficits.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with episodes of inflammatory demyelination and remyelination. While remyelination has been linked with functional recovery in MS patients, there is evidence of ongoing tissue damage despite complete myelin repair. In this study, we investigated the long-term consequences of an acute demyelinating white matter CNS lesion. For this purpose, acute demyelination was induced by 5-week-cuprizone intoxication in male C57BL/6 J mice, and the tissues were examined after a 7-month recovery period. While myelination and oligodendrocyte densities appeared normal, ongoing axonal degeneration and glia cell activation were found in the remyelinated corpus callosum. Neuropathologies were paralleled by subtle gait abnormalities evaluated using DigiGait™ high speed ventral plane videography. Gene array analyses revealed increased expression levels of various inflammation related genes, among protein kinase c delta (PRKCD). Immunofluorescence stains revealed predominant microglia/macrophages PRKCD expression in both, cuprizone tissues and post-mortem MS lesions. These results support the hypothesis that chronic microglia/macrophages driven tissue injury represents a key aspect of progressive neurodegeneration and functional decline in MS.

CD44 expression in the cuprizone model.

Numerous studies report that changes in extracellular matrix components and receptors, such as CD44, contribute to immune cell recruitment and thus lesion formation in multiple sclerosis (MS). In the present study, we used the cuprizone model to elucidate the expression pattern of CD44 in a toxin-induced MS model. Therefore, tissues of cuprizone-intoxicated mice were analyzed by real-time qRT-PCR and immunohistochemical staining against CD44. Co-localization analyses of CD44-positive cells with glial cell markers were performed by immunofluorescence labeling and in-situ hybridization. To investigate the functional importance of CD44 expression for myelination and glial cell activation, Cd44-deficient mice were used. In this study we demonstrate that CD44 expression is induced in a time-dependent manner in an autoimmune-independent model of MS. Up-regulation of CD44 expression was primarily associated to the superficial and perivascular glia limitans and demyelinated white matter structures, particularly the corpus callosum. In the demyelinated corpus callosum, CD44 was localized on GFAP+ astrocytes and IBA1+ microglial cells. Despite a robust expression induction, Cd44-deficiency did not ameliorate cuprizone-induced pathology. Although further studies will be needed to examine the functional relevance of CD44 in the cuprizone model, the spatial and temporal expression pattern of CD44 will pave the way to evaluate its precise role in different (immune and non-immune) pathological conditions.

Stereological Investigation of Regional Brain Volumes after Acute and Chronic Cuprizone-Induced Demyelination.

Brain volume measurement is one of the most frequently used biomarkers to establish neuroprotective effects during pre-clinical multiple sclerosis (MS) studies. Furthermore, whole-brain atrophy estimates in MS correlate more robustly with clinical disability than traditional, lesion-based metrics. However, the underlying mechanisms leading to brain atrophy are poorly understood, partly due to the lack of appropriate animal models to study this aspect of the disease. The purpose of this study was to assess brain volumes and neuro-axonal degeneration after acute and chronic cuprizone-induced demyelination. C57BL/6 male mice were intoxicated with cuprizone for up to 12 weeks. Brain volume, as well as total numbers and densities of neurons, were determined using design-based stereology. After five weeks of cuprizone intoxication, despite severe demyelination, brain volumes were not altered at this time point. After 12 weeks of cuprizone intoxication, a significant volume reduction was found in the corpus callosum and diverse subcortical areas, particularly the internal capsule and the thalamus. Thalamic volume loss was accompanied by glucose hypermetabolism, analyzed by [18F]-fluoro-2-deoxy-d-glucose (18F-FDG) positron-emission tomography. This study demonstrates region-specific brain atrophy of different subcortical brain regions after chronic cuprizone-induced demyelination. The chronic cuprizone demyelination model in male mice is, thus, a useful tool to study the underlying mechanisms of subcortical brain atrophy and to investigate the effectiveness of therapeutic interventions.

Continuous cuprizone intoxication allows active experimental autoimmune encephalomyelitis induction in C57BL/6 mice.

Oligodendrocyte degeneration is a hallmark of multiple sclerosis pathology, and protecting oligodendrocytes and myelin is likely to be of clinical relevance. Traditionally, oligodendrocyte and myelin degeneration are viewed as a direct consequence of an inflammatory attack, but metabolic defects might be equally important. Appropriate animal models to study the interplay of inflammation and metabolic injury are, therefore, needed. Here, we describe that in spite of its immunosuppressive effects, a continuous intoxication with cuprizone allows the induction of active experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG35-55) immunization. Although the clinical severity of EAE is ameliorated in cuprizone-intoxicated mice, the recruitment of granulocytes, and especially, CD3+ lymphocytes into the forebrain is triggered by the cuprizone insult. Such combined lesions are further characterized by oligodendrocyte apoptosis and microglia activation, closely mimicking type III multiple sclerosis lesions. In summary, we provide a protocol that allows to study the direct interplay of immune-mediated and metabolic oligodendrocyte injury and its consequences for the cerebral white and grey matters.

Oligodendrocyte degeneration and concomitant microglia activation directs peripheral immune cells into the forebrain.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently showed that encephalitogenic lymphocytes are recruited to the sites of active demyelination induced by cuprizone. Here, we investigated whether cuprizone-induced oligodendrocyte and myelin pathology is sufficient to trigger peripheral immune cell recruitment into the forebrain. We show that early cuprizone-induced white matter lesions display a striking similarity to early MS lesions, i.e., oligodendrocyte degeneration, microglia activation and absence of severe lymphocyte infiltration. Such early cuprizone lesions are sufficient to trigger peripheral immune cell recruitment secondary to subsequent EAE (experimental autoimmune encephalomyelitis) induction. The lesions are characterized by discontinuation of the perivascular glia limitans, focal axonal damage, and perivascular astrocyte pathology. Time course studies showed that the severity of cuprizone-induced lesions positively correlates with the extent of peripheral immune cell recruitment. Furthermore, results of genome-wide array analyses suggest that moesin is integral for early microglia activation in cuprizone and MS lesions. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

Visualization of the Breakdown of the Axonal Transport Machinery: a Comparative Ultrastructural and Immunohistochemical Approach.

Axonal damage is a major factor contributing to disease progression in multiple sclerosis (MS) patients. On the histological level, acute axonal injury is most frequently analyzed by anti-amyloid precursor protein immunohistochemistry. To what extent this method truly detects axonal injury, and whether other proteins and organelles are as well subjected to axonal transport deficits in demyelinated tissues is not known. The aim of this study was to correlate ultrastructural morphology with the immunohistochemical appearance of acute axonal injury in a model of toxin-induced oligodendrocyte degeneration. C57BL/6J mice were intoxicated with 0.25% cuprizone to induce demyelination. The corpus callosum was investigated by serial block-face scanning electron microscopy (i.e., 3D EM), immunohistochemistry, and immunofluorescence microscopy. Brain tissues of progressive MS patients were included to test the relevance of our findings in mice for MS. Volumes of axonal swellings, determined by 3D EM, were comparable to volumes of axonal spheroids, determined by anti-APP immunofluorescence stains. Axonal swellings were present at myelinated and non-myelinated axonal internodes. Densities of amyloid precursor protein (APP)+ spheroids were highest during active demyelination. Besides APP, vesicular glutamate transporter 1 and mitochondrial proteins accumulated at sites of axonal spheroids. Such accumulations were found as well in lesions of progressive MS patients. In this correlative ultrastructural-immunohistochemical study, we provide strong evidence that breakdown of the axonal transport machinery results in focal accumulations of mitochondria and different synaptic proteins. We provide new marker proteins to visualize acute axonal injury, which helps to further understand the complex nature of axonal damage in progressive MS.

Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3.

Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.

Combination of cuprizone and experimental autoimmune encephalomyelitis to study inflammatory brain lesion formation and progression.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

Cuprizone-Containing Pellets Are Less Potent to Induce Consistent Demyelination in the Corpus Callosum of C57BL/6 Mice.

The chopper chelator cuprizone serves as a valuable chemical tool to induce consistent and reproducible demyelination in the central nervous system. However, the daily preparation of fresh cuprizone powder mixed in finely ground rodent chow might well be a particular health problem. Alternative methods, such as the fabrication of cuprizone-containing pellets, are available. The effectiveness of this method is, however, not known. In the present study, we compared whether intoxication of C57BL/6 mice with 0.25% cuprizone mixed into ground rodent chow does induce demyelination to a similar extent compared to a cuprizone-pellet intoxication protocol. We found that feeding of 0.25% cuprizone in ground chow provides a strong, well-defined, and reproducible demyelination along with increased accumulation of microglia and axonal damage in the corpus callosum, whereas all analyzed parameters were significantly less distinct in mice fed with cuprizone-containing pellets at an equivalent concentration of cuprizone at week 5. Even a higher concentration of cuprizone in pellet formulation was less potent compared to do so. This study illustrates that the established protocol of cuprizone intoxication (i.e., mixed in ground rodent chow) is the gold standard method to achieve consistent and reproducible demyelination. Why cuprizone loses its effectiveness in pellet formulation needs to be addressed in subsequent studies.

Design-Based Stereology for Evaluation of Histological Parameters.

Valid quantification of organ volume and total cell numbers are crucial parameters for morphometric studies. The number of a specific cell type cannot be simply deduced from the number of its profiles found in thin tissue sections, as this parameter also depends on cell volume, tissue orientation as well as tissue atrophy. Design-based stereology has become the method of choice for unbiased, reproducible total cell number quantification. Steps described in this protocol include transcardial perfusion of mice, postfixation, and cryoprotection of the region of interest (ROI), followed by the preparation of a systematically and randomly sampled series of thick sections through the entire ROI. Furthermore, it is described how to perform immuno-histochemical staining of such thick cryo-sections, followed by providing a guidance for quantification of the ROI volume, the generation of unbiased virtual counting spaces, and steps to work with these counting spaces to obtain an unbiased estimate of total cell numbers.

Multiple sclerosis animal models: a clinical and histopathological perspective.

There is a broad consensus that multiple sclerosis (MS) represents more than an inflammatory disease: it harbors several characteristic aspects of a classical neurodegenerative disorder, that is, damage to axons, synapses and nerve cell bodies. While we are equipped with appropriate therapeutic options to prevent immune-cell driven relapses, effective therapeutic options to prevent the progressing neurodegeneration are still missing. In this review article, we will discuss to what extent pathology of the progressive disease stage can be modeled in MS animal models. While acute and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), which are T cell dependent, are aptly suited to model relapsing-remitting phases of MS, other EAE models, especially the secondary progressive EAE stage in Biozzi ABH mice is better representing the secondary progressive phase of MS, which is refractory to many immune therapies. Besides EAE, the cuprizone model is rapidly gaining popularity to study the formation and progression of demyelinating CNS lesions without T cell involvement. Here, we discuss these two non-popular MS models. It is our aim to point out the pathological hallmarks of MS, and discuss which pathological aspects of the disease can be best studied in the various animal models available.

Acute axonal damage in three different murine models of multiple sclerosis: A comparative approach.

Axonal damage has been identified as a significant contributor to permanent clinical disability in multiple sclerosis. In the context of demyelinating disorders, this destructive event can be the result of inflammation, demyelination and/or the activation of innate defense cells such as microglia or monocytes. The relative contribution of each of these variables to acute axonal injury is, however, unknown. In the present study, we compared the extent of acute axonal damage in three different murine demyelination models using anti-amyloid precursor protein (APP) immunohistochemistry. T cell dependent (MOG35-55-induced experimental autoimmune encephalomyelitis (EAE)) as well as T cell independent demyelination models (cuprizone- and lysolecithin-induced demyelination) were used. APP+ spheroids were present in all three experimental demyelination models. The number of APP+ spheroids was highest within LPC-induced lesions. Equal amounts were found in the spinal cord of MOG35-55-EAE animals and the corpus callosum of cuprizone-intoxicated animals. Moreover, we detected increased immunoreactivity of the pre-synaptic protein vesicular glutamate transporter 1 (VGluT1) in demyelinated foci. VGluT1-staining revealed long stretched, ovoid-like axonal structures which co-localized with APP. In summary, we showed that acute axonal damage is evident under various experimental demyelination paradigms. Furthermore, disturbed axonal transport mechanisms, which are responsible for intra-axonal APP accumulation, do not only disturb APP, but also the transport of other synaptic proteins. These results indicate that, despite differences in their characteristics, all three models may serve as valid and suitable systems for investigating responsible mechanisms of axonal damage and potential protective strategies.

Thalamus Degeneration and Inflammation in Two Distinct Multiple Sclerosis Animal Models.

There is a broad consensus that multiple sclerosis (MS) represents more than an inflammatory disease: it harbors several characteristic aspects of a classical neurodegenerative disorder, i.e., damage to axons, synapses, and nerve cell bodies. While several accepted paraclinical methods exist to monitor the inflammatory-driven aspects of the disease, techniques to monitor progression of early and late neurodegeneration are still in their infancy and have not been convincingly validated. It was speculated that the thalamus with its multiple reciprocal connections is sensitive to inflammatory processes occurring in different brain regions, thus acting as a "barometer" for diffuse brain parenchymal damage in MS. To what extent the thalamus is affected in commonly applied MS animal models is, however, not known. In this article we describe direct and indirect damage to the thalamus in two distinct MS animal models. In the cuprizone model, we observed primary oligodendrocyte stress which is followed by demyelination, microglia/astrocyte activation, and acute axonal damage. These degenerative cuprizone-induced lesions were found to be more severe in the lateral compared to the medial part of the thalamus. In MOG35-55-induced EAE, in contrast, most parts of the forebrain, including the thalamus were not directly involved in the autoimmune attack. However, important thalamic afferent fiber tracts, such as the spinothalamic tract were inflamed and demyelinated on the spinal cord level. Quantitative immunohistochemistry revealed that this spinal cord inflammatory-demyelination is associated with neuronal loss within the target region of the spinothalamic tract, namely the sensory ventral posterolateral nucleus of the thalamus. This study highlights the possibility of trans-neuronal degeneration as one mechanism of secondary neuronal damage in MS. Further studies are now warranted to investigate involved cell types and cellular mechanisms.

Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves.

Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro.

Female sex steroids and glia cells: Impact on multiple sclerosis lesion formation and fine tuning of the local neurodegenerative cellular network.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease that shows a female-to-male gender prevalence and alleviation of disease activity during late stage pregnancy. In MS-related animal models, sex steroids ameliorate symptoms and protect from demyelination and neuronal damage. Underlying mechanisms of these protective avenues are continuously discovered, in part by using novel transgenic animal models. In this review article, we highlight the regulation of glia cell function by female sex steroids. We specifically focus on the relevance of glia cells for immune cell recruitment into the central nervous system and show how estrogen and progesterone can modulate these cell-cell communication pathways. Since MS is considered to have a strong neurodegenerative component, principal neuroprotective mechanisms, exerted by sex-steroids will be discussed as well. Activation of steroid receptors might not just act as immunosuppressant but at the same time harmonize brain-intrinsic networks to dampen neurodegeneration and, thus, disease progression in MS.

Differential changes in platelet reactivity induced by acute physical compared to persistent mental stress.

Platelets are important in hemostasis, but also contain adhesion molecules, pro-inflammatory and immune-modulatory compounds, as well as most of the serotonin outside the central nervous system. Dysbalance in the serotonin pathways is involved in the pathogenesis of depressive symptoms. Thus, changes in platelet aggregation and content of bioactive compounds are of interest when investigating physiological stress-related mental processes as well as stress-related psychiatric diseases such as depression. In the present study, a characterization of platelet reactivity in acute physical and persistent mental stress was performed (aggregation, serotonin and serotonin 2A-receptor, P-selectin, CD40 ligand, matrix metalloproteinase-2 and -9 (MMP-2 and -9), platelet/endothelial adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), ß-thromboglobulin (ß-TG) and platelet factor 4 (PF-4). Acute physical stress increased platelet aggregability while leaving platelet content of bioactive compounds unchanged. Persistent mental stress led to changes in platelet content of bioactive compounds and serotonin 2A-receptor only. The values of most bioactive compounds correlated with each other. Acute physical and persistent mental stress influences platelets through distinct pathways, leading to differential changes in aggregability and content of bioactive compounds.

Bioprofiling of platelets in medicated patients with depression.

BACKGROUND: Changes in platelet bioactivity and aggregation are of interest when studying patients with depression as this could help to explain the statistically observed association of depression and chronic somatic, especially cardiovascular disease. This link could potentially be mediated through serotonergic signaling or immunological changes. METHODS: 38 medicated patients with major depressive disorder (MDD) and 30 mentally healthy controls, both without a diagnosis of cardiovascular disease, were included in this naturalistic study. Demographic and psychometric data were obtained. Platelet aggregability was measured by PFA-100 and bioactive compounds and serotonin levels were quantified in platelet sonicate. RESULTS: The comparison of patients with controls revealed no changes in platelet aggregability, but significant differences in platelet content of several bioactive compounds. In a second analysis, patients were grouped according to the receptors and transporters influenced by their medication and again compared to controls. A significant effect of MDD was found for platelet content of serotonin, CD40L, interleukin-1ß, and platelet factor-4, independent of medication. These markers can thus be classified as sensitive to MDD. The effect of medication on platelet parameters was also evaluated. Platelet content of matrix metalloproteinase-2 and ß-thromboglobulin was normalized in MDD patients by medication acting on the serotonin transporter. LIMITATIONS: Owing to the naturalistic study design, patients were on a variety of different medications and combination therapies. This was accounted for by a novel analysis method. CONCLUSION: Platelet serotonin levels and content of immunomodulatory compounds are significantly altered in patients with MDD, even if treatment effects are taken into account.

No changes in cerebellar microvessel length density in sudden infant death syndrome: implications for pathogenetic mechanisms.

Sudden infant death syndrome (SIDS) is the leading cause of mortality in infants younger than 1 year in developed countries, but its primary cause remains unknown. Some studies suggest that there may be hypoxia in the cerebellum in SIDS subjects, but mean total Purkinje cell numbers in SIDS versus controls was recently found not to be different. Probably the best marker for chronic hypoxia in a brain region is the microvessel length per unit volume of tissue, that is, the microvessel length density (MLD). Here, we investigated MLDs using a rigorous design-based stereologic approach in all cell layers and white matter in postmortem cerebella from 9 SIDS cases who died between ages 2 and 10 months and from 14 control children, 9 of which were age- and sex- matched to the SIDS cases. We found no differences either in mean MLDs in the cerebellar layers between the SIDS cases and the controls or between controls with a low likelihood of hypoxia and those with a higher likelihood of hypoxia. Immunohistochemical detection of the astrocytosis marker glial fibrillary acidic protein showed no differences between the SIDS and the matched control cases. These data indicate that there is no association of chronic hypoxia in the cerebellum with SIDS.