RESUMEN
A central role for vitamin D (VD) in immune modulation has recently been recognized linking VD insufficiency to autoimmune disorders that commonly exhibit sex-associated differences. Similar to other autoimmune diseases, there is a higher incidence of multiple sclerosis (MS) in women, but a poorer prognosis in men, often characterized by a more rapid progression. Although sex hormones are most likely involved, this phenomenon is still poorly understood. Oxidative stress, modulated by VD serum levels as well as sex hormones, may act as a contributing factor to demyelination and axonal damage in both MS and the corresponding preclinical models. In this study, we analyzed sex-associated differences and VD effects utilizing an animal model that recapitulates histopathological features of the progressive MS phase (PMS). In contrast to relapsing-remitting MS (RRMS), PMS has been poorly investigated in this context. Male (n = 50) and female (n = 46) Dark Agouti rats received either VD (400 IU per week; VD+) or standard rodent food without extra VD (VD-) from weaning onwards. Myelination, microglial activation, apoptotic cell death and neuronal viability were assessed using immunohistochemical markers in brain tissue. Additionally, we also used two different histological markers against oxidized lipids along with colorimetric methods to measure protective polyphenols (PP) and total antioxidative capacity (TAC) in serum. Neurofilament light chain serum levels (sNfL) were analyzed using single-molecule array (SIMOA) analysis. We found significant differences between female and male animals. Female rats exhibited a better TAC and higher amounts of PP. Additionally, females showed higher myelin preservation, lower microglial activation and better neuronal survival while showing more apoptotic cells than male rats. We even found a delay in reaching the peak of the disease in females. Overall, both sexes benefitted from VD supplementation, represented by significantly less cortical, neuroaxonal and oxidative damage. Unexpectedly, male rats had an even higher overall benefit, most likely due to differences in oxidative capacity and defense systems.
Asunto(s)
Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple , Humanos , Femenino , Masculino , Ratas , Animales , Caracteres Sexuales , Vitamina D , Vitaminas , Suplementos Dietéticos , Modelos Animales , Hormonas Esteroides GonadalesRESUMEN
Vitamin D (VD) is the most discussed antioxidant supplement for multiple sclerosis (MS) patients and many studies suggest correlations between a low VD serum level and onset and progression of the disease. While many studies in animals as well as clinical studies focused on the role of VD in the relapsing-remitting MS, knowledge is rather sparse for the progressive phase of the disease and the development of cortical pathology. In this study, we used our established rat model of cortical inflammatory demyelination, resembling features seen in late progressive MS, to address the question about whether VD could have positive effects on reducing cortical pathology, oxidative stress, and neurofilament light chain (NfL) serum levels. For this purpose, we used male Dark Agouti (DA) rats, with one group being supplemented with VD (400 IE per week; VD+) from the weaning on at age three weeks; the other group received standard rodent food. The rat brains were assessed using immunohistochemical markers against demyelination, microglial activation, apoptosis, neurons, neurofilament, and reactive astrocytes. To evaluate the effect of VD on oxidative stress and the antioxidant capacity, we used two different oxidized lipid markers (anti- Cu++ and HOCl oxidized LDL antibodies) along with colorimetric methods for protective polyphenols (PP) and total antioxidative capacity (TAC). NfL serum levels of VD+ and VD- animals were analyzed by fourth generation single-molecule array (SIMOA) analysis. We found significant differences between the VD+ and VD- animals both in histopathology as well as in all serum markers. Myelin loss and microglial activation is lower in VD+ animals and the number of apoptotic cells is significantly reduced with a higher neuronal survival. VD+ animals show significantly lower NfL serum levels, a higher TAC, and more PP. Additionally, there is a significant reduction of oxidized lipid markers in animals under VD supplementation. Our data thus show a positive effect of VD on cellular features of cortical pathology in our animal model, presumably due to protection against reactive oxygen species. In this study, VD enhanced remyelination and prevented neuroaxonal and oxidative damage, such as demyelination and neurodegeneration. However, more studies on VD dose relations are required to establish an optimal response while avoiding overdosing.
Asunto(s)
Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple , Masculino , Ratas , Animales , Vitamina D , Antioxidantes/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Vitaminas/farmacología , Vitaminas/uso terapéutico , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Modelos AnimalesRESUMEN
An increasing body of evidence associates low vitamin D levels with increased risk of multiple sclerosis (MS), suggesting the possibility of a gene-environment interaction for this environmental factor in MS pathogenesis. Moreover, it has been shown that vitamin D downregulates major histocompatibility complex (MHC) class II expression in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We here report about the impact of a dietary vitamin D supplementation on EAE in the rat strains having functionally relevant allelic variations in the CIITA (Mhc2ta) gene, a master regulator of MHC class II expression. Full length myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in DA.PVGav1-Vra4 congenic rats harboring the Vra4 locus from PVG strain in the EAE- susceptible DA background, and compared to the parental strains. The congenic rats fed with either vitamin D supplemented, deprived or regular diet developed an intermediate clinical EAE phenotype, in contrast to DA and PVG strains. Immunopathological studies revealed vitamin D dose-dependent effect on demyelination and inflammatory infiltration of the central nervous system (CNS), expression of MHC class II and CIITA, as well as downregulation of a range of pro-inflammatory genes. Taken together, our findings demonstrate an impact of vitamin D on the target tissue pathology and peripheral immune response during EAE in DA.PVGav1-Vra4 congenic strain. Thereby, our data provide evidence of a modulatory effect of vitamin D in context of genetic variances in the Vra4 locus/Mhc2ta gene in MS-like neuroinflammation, with potential relevance for the human demyelinating disease.
RESUMEN
Cortical demyelination is a common finding in patients with chronic multiple sclerosis (MS) and contributes to disease progression and overall disability. The exact pathomechanism that leads to cortical lesions is not clear. Research is limited by the fact that standard animal models of multiple sclerosis do not commonly affect the cortex, or if they do in some variants, the cortical demyelination is rather sparse and already remyelinated within a few days. In an attempt to overcome these limitations we implanted a tissue-compatible catheter into the cortex of Dark Agouti rats. After 14days the rats were immunized with 5µg myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's Adjuvant, which did not cause any clinical signs but animals developed a stable anti-MOG antibody titer. Then the animals received an injection of proinflammatory cytokines through the catheter. This led to a demyelination of cortical and subcortical areas starting from day 1 in a cone-like pattern spreading from the catheter area towards the subarachnoid space. On day 3 cortical demyelination already expanded to the contralateral hemisphere and reached its peak between days 9-15 after cytokine injection with a widespread demyelination of cortical and subcortical areas of both hemispheres. Clinically the animals showed only discrete signs of fatigue and recovered completely after day 15. Even on day 30 we still were able to detect demyelination in subpial and intracortical areas along with areas of partial and complete remyelination. Loss of cortical myelin was accompanied with marked microglia activation. A second injection of cytokines through the catheter on day 30 led to a second demyelination phase with the same symptoms, but again no detectable motor dysfunction. Suffering of the animals appeared minor compared to standard Experimental Autoimmune Encephalomyelitis and therefore, even long-term observation and repeated demyelination phases seem ethically acceptable.
Asunto(s)
Corteza Cerebral/patología , Citocinas/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/patología , Lateralidad Funcional/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/inmunología , Fibrina/metabolismo , Adyuvante de Freund/efectos adversos , Lateralidad Funcional/efectos de los fármacos , Inmunización/efectos adversos , Lípidos/efectos adversos , Masculino , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Actividad Motora , Proteína Proteolipídica de la Mielina/metabolismo , Glicoproteína Asociada a Mielina/efectos adversos , Glicoproteína Asociada a Mielina/sangre , Proteínas del Tejido Nervioso/metabolismo , Ratas , Estadísticas no ParamétricasRESUMEN
Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Vitamina D/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Genómica/métodos , Activación de Linfocitos/efectos de los fármacos , Esclerosis Múltiple/tratamiento farmacológico , Ratas , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study investigated the histological tissue reaction to long-term implanted cerebral open flow microperfusion (cOFM) probes in the frontal lobe of the rat brain. Most probe-based cerebral fluid sampling techniques are limited in application time due to the formation of a glial scar that hinders substance exchange between brain tissue and the probe. A glial scar not only functions as a diffusion barrier but also alters metabolism and signaling in extracellular brain fluid. cOFM is a recently developed probe-based technique to continuously sample extracellular brain fluid with an intact blood-brain barrier. After probe implantation, a 2 week healing period is needed for blood-brain barrier reestablishment. Therefore, cOFM probes need to stay in place and functional for at least 15 days after implantation to ensure functionality. Probe design and probe materials are optimized to evoke minimal tissue reaction even after a long implantation period. Qualitative and quantitative histological tissue analysis revealed no continuous glial scar formation around the cOFM probe 30 days after implantation and only a minor tissue reaction regardless of perfusion of the probe.
Asunto(s)
Encéfalo/citología , Electrodos Implantados/efectos adversos , Perfusión/instrumentación , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Lóbulo Frontal/citología , Lóbulo Frontal/metabolismo , Proteína Ácida Fibrilar de la Glía , Masculino , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Diseño de Prótesis , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The association of vitamin D deficiency with higher prevalence, relapse rate and progression of multiple sclerosis (MS) has stimulated great interest in using vitamin D supplementation as a preventative measure and even a therapy for established MS. However, there is a considerable lack of evidence when it comes to an age/developmental stage-dependent efficacy of vitamin D action and a time-window for the most effective prophylactic treatment remains unclear. We studied the effect of vitamin D supplementation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS, at three different developmental stages in rats. Supplementation treatment was initiated: i) prior to gestation and maintained throughout pre- and early postnatal development (gestation and lactation); ii) after weaning, throughout juvenile/adolescence period and iii) in adult age. We observed a marked attenuation of EAE in juvenile/adolescent rats reflected in a less severe CNS inflammation and demyelination, accompanied by a lower amount of IFN-γ producing MOG-specific T cells. Moreover, the cytokine expression pattern in these rats reflected a more anti-inflammatory phenotype of their peripheral immune response. However, the same supplementation regimen failed to improve the disease outcome both in adult rats and in rats treated during pre- and early post-natal development. Our data demonstrate a developmental stage-dependent efficiency of vitamin D to ameliorate neuroinflammation, suggesting that childhood and adolescence should be the target for the most effective preventive treatment.
Asunto(s)
Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Vitamina D/administración & dosificación , Envejecimiento/fisiología , Animales , Suplementos Dietéticos , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Inflamación/tratamiento farmacológico , Inflamación/patología , Inflamación/fisiopatología , Esclerosis Múltiple/fisiopatología , Ratas , Resultado del TratamientoRESUMEN
Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.