Thiamine and benfotiamine: Focus on their therapeutic potential.

Thiamine, also known as vitamin B1, is an essential nutrient that plays a crucial role in energy metabolism and overall health. It is a water-soluble vitamin that plays an important role in the conversion of carbohydrates into energy in the body. Thiamine is essential for the proper functioning of the nervous system, heart and muscles. Thiamine deficiency is a life-threatening disease that leads to various disorders and lesions in the nerves and brain, at least in vertebrates. Several thiamine precursors with higher bioavailability have been developed to compensate for thiamine deficiency, including benfotiamine. Benfotiamine is more bioavailable and has higher tissue penetration than thiamine. Studies have shown its antioxidant and anti-inflammatory potential in activated immune and glial cells. It also improves complications observed in type 2 diabetes and has beneficial effects in mouse models of neurodegenerative disease. Benfotiamine represents an off-the-shelf agent used to support nerve health, promote healthy aging and support glucose metabolism. Accordingly, the present review aimed to provide an overview of the neuroprotective effects of thiamine/benfotiamine in the context of inflammation and oxidative stress.

Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

The Function of the Hypothalamic-Pituitary-Adrenal Axis During Experimental Autoimmune Encephalomyelitis: Involvement of Oxidative Stress Mediators.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic-pituitary-adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Testicular steroidogenesis is suppressed during experimental autoimmune encephalomyelitis in rats.

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

Benfotiamine Reduces Dendritic Cell Inflammatory Potency.

BACKGROUND: Benfotiamine is a synthetic liposoluble derivative of vitamin B1 that has been shown to have anti-inflammatory properties. OBJECTIVE: To study the effects of benfotiamine on dendritic cells. METHODS: Dendritic cells were obtained from murine bone marrow precursor cells in the presence of GM-CSF. Benfotiamine was applied to the cell culture during the process of bone marrow cell differentiation into dendritic cells. Dendritic cells were stimulated with lipopolysaccharide (LPS) and expression of MHC class II molecules and CD86 was determined by flow cytometry, while levels of tumor necrosis factor (TNF) and interleukin (IL)-1ß in cell culture supernatants were measured by ELISA. F-Actin, NF-κB and Nrf2 were visualized by immunofluorescent staining and microscopy. RESULTS: Benfotiamine potently reduced LPS-induced expression of MHC class II molecules and CD86, in addition to suppressing the release of pro-inflammatory cytokines TNF and IL-1ß. It also prevented LPS-imposed morphological changes of dendritic cells, i.e. enlargement and intensified protrusions. The effects were paralleled with the reduction of NF-κB translocation to the nucleus, but not of Nrf2 activation inhibition. CONCLUSION: Having in mind the importance of dendritic cells for the configuration of the immune response, our results imply that benfotiamine has the ability to regulate the immune response through inhibition of inflammatory properties of dendritic cells.

Astrocyte phenotypes: Emphasis on potential markers in neuroinflammation.

Astrocytes, the most abundant glial cells in the central nervous system (CNS), have numerous integral roles in all CNS functions. They are essential for synaptic transmission and support neurons by providing metabolic substrates, secreting growth factors and regulating extracellular concentrations of ions and neurotransmitters. Astrocytes respond to CNS insults through reactive astrogliosis, in which they go through many functional and molecular changes. In neuroinflammatory conditions reactive astrocytes exert both beneficial and detrimental functions, depending on the context and heterogeneity of astrocytic populations. In this review we profile astrocytic diversity in the context of neuroinflammation; with a specific focus on multiple sclerosis (MS) and its best-described animal model experimental autoimmune encephalomyelitis (EAE). We characterize two main subtypes, protoplasmic and fibrous astrocytes and describe the role of intermediate filaments in the physiology and pathology of these cells. Additionally, we outline a variety of markers that are emerging as important in investigating astrocytic biology in both physiological conditions and neuroinflammation.

The sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis.

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Voltage Gated Potassium Channel Kv1.3 Is Upregulated on Activated Astrocytes in Experimental Autoimmune Encephalomyelitis.

Kv1.3 is a voltage gated potassium channel that has been implicated in pathophysiology of multiple sclerosis (MS). In the present study we investigated temporal and cellular expression pattern of this channel in the lumbar part of spinal cords of animals with experimental autoimmune encephalomyelitis (EAE), animal model of MS. EAE was actively induced in female Dark Agouti rats. Expression of Kv1.3 was analyzed at different time points of disease progression, at the onset, peak and end of EAE. We here show that Kv1.3 increased by several folds at the peak of EAE at both gene and protein level. Double immunofluorescence analyses demonstrated localization of Kv1.3 on activated microglia, macrophages, and reactive astrocytes around inflammatory lesions. In vitro experiments showed that pharmacological block of Kv1.3 in activated astrocytes suppresses the expression of proinflammatory mediators, suggesting a role of this channel in inflammation. Our results support the hypothesis that Kv1.3 may be a therapeutic target of interest for MS and add astrocytes to the list of cells whose activation would be suppressed by inhibiting Kv1.3 in inflammatory conditions.

Immunoglobulins G from Sera of Amyotrophic Lateral Sclerosis Patients Induce Oxidative Stress and Upregulation of Antioxidative System in BV-2 Microglial Cell Line.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with a very fast progression, no diagnostic tool for the presymptomatic phase, and still no effective treatment of the disease. Although ALS affects motor neurons, the overall pathophysiological condition points out to the non-cell autonomous mechanisms, where astrocytes and microglia play crucial roles in the disease progression. We have already shown that IgG from sera of ALS patients (ALS IgG) induce calcium transients and an increase in the mobility of acidic vesicles in cultured rat astrocytes. Having in mind the role of microglia in neurodegeneration, and a well-documented fact that oxidative stress is one of the many components contributing to the disease, we decided to examine the effect of ALS IgG on activation, oxidative stress and antioxidative system of BV-2 microglia, and to evaluate their acute effect on cytosolic peroxide, pH, and on reactive oxygen species (ROS) generation. All tested ALS IgGs (compared to control IgG) induced oxidative stress (rise in nitric oxide and the index of lipid peroxidation) followed by release of TNF-α and higher antioxidative defense (elevation of Mn- and CuZn-superoxide dismutase, catalase, and glutathione reductase with a decrease of glutathione peroxidase and glutathione) after 24 h treatment. Both ALS IgG and control IgG showed same localization on the membrane of BV-2 cells following 24 h treatment. Cytosolic peroxide and pH alteration were evaluated with fluorescent probes HyPer and SypHer, respectively, having in mind that HyPer also reacts to pH changes. Out of 11 tested IgGs from ALS patients, 4 induced slow exponential rise of HyPer signal, with maximal normalized fluorescence in the range 0.2-0.5, also inducing similar increase of SypHer intensity, but of a lower amplitude. None of the control IgGs induced changes with neither of the indicators. Acute ROS generation was detected in one out of three tested ALS samples with carboxy-H2DCFDA. The observed phenomena demonstrate the potential role of inflammatory humoral factors, IgGs, as potential triggers of the activation in microglia, known to occur in later stages of ALS. Therefore, revealing the ALS IgG signaling cascade in microglial cells could offer a valuable molecular biomarker and/or a potential therapeutic target.

Down-regulation of NTPDase2 and ADP-sensitive P2 Purinoceptors Correlate with Severity of Symptoms during Experimental Autoimmune Encephalomyelitis.

The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset ( Eo ), peak ( Ep ) and recovery ( Er ) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive (ir) astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er . The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5'-nucleotidase (eN/CD73) was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4- cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which slightly increased in number with progression of the disease, particularly in CD4- cells, and then decreased in the recovery. Finally, CD4+ NTPDase2+ cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.

Extracellular ATP induces graded reactive response of astrocytes and strengthens their antioxidative defense in vitro.

It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1ß release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc.

Low-dose ribavirin treatments attenuate neuroinflammatory activation of BV-2 Cells by interfering with inducible nitric oxide synthase.

Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 µM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 µM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 µM ribavirin promoted LPS induced apoptosis. We determined that 1 µM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 µM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells.

Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes-catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and (·)O(-) 2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells.

Benfotiamine attenuates inflammatory response in LPS stimulated BV-2 microglia.

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Expression of a second ecto-5'-nucleotidase variant besides the usual protein in symptomatic phase of experimental autoimmune encephalomyelitis.

Ecto-5'-nucleotidase/cluster of differentiation 73 (CD73) (eN) is a 70-kDa glycoprotein expressed in several different mammalian tissues and cell types. It is the rate-limiting enzyme of the purine catabolic pathway, which catalyzes the hydrolysis of AMP to produce adenosine with known anti-inflammatory and immunosuppressive actions. There is strong evidence for lymphocyte and endothelial cell eN having a role in experimental autoimmune encephalomyelitis (EAE), but the role of eN in cell types within the central nervous system is less clear. We have previously shown that eN activity significantly increased in the lumbar spinal cord during EAE. The present study is aimed to explore molecular pattern of the eN upregulation over the course of the disease and cell type(s) accountable for the induction. EAE was induced in Dark Agouti (DA) rats by immunization with the spinal cord tissue homogenate and adjuvant. Animals were sacrificed 8, 15, and 28 days following immunization (D8, D15, and D28), i.e., at time points which corresponded to the presymptomatic, symptomatic, and postsymptomatic phases of the disease, respectively. Significant increase in eN activity and its upregulation at the gene and the protein levels were demonstrated at D15 and less prominently at D28 in comparison to control. Additionally, reactive astrocytes abundantly present in the lumbar spinal cord parenchyma were identified as principal cell type with significantly elevated eN expression. In all experimental groups, eN was expressed as a 71-kDa protein band of uniform abundance, whereas the overexpression of eN at D15 and D28 was associated with the expression of a second 75-kDa eN variant. The possible outcome of eN upregulation during EAE as a part of protective astrocyte repertoire contributing to the resolution of the disease is discussed.

The effect of ribavirin on reactive astrogliosis in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of CNS inflammatory and demyelinating disease multiple sclerosis. Microglia and astrocytes represent two related cell types involved in the brain pathology in EAE. Accumulations of hypertrophic reactive astrocytes, intensely stained with glial fibrillary acidic protein (GFAP), which also expressed vimentin, are prominent features of EAE lesions. Recent studies from our laboratory reported that ribavirin attenuated the disease process in EAE by reducing clinical and histological manifestations. EAE was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freund's adjuvant. Real time PCR and immunohistochemistry were used for determination of GFAP and vimentin gene and tissue expression. We have observed the increased gene and tissue expression of GFAP and vimentin in EAE rats. Ribavirin treatment significantly decreased the number of reactive astrocytes at the peak of disease. At the end of the disease, we have observed reactive GFAP(+) and vimentin(+) astrocytes in both immunized and ribavirin-treated groups, accompanied by increased level of GFAP mRNA. The present study indicates that ribavirin may have the ability to attenuate astrocyte proliferation and glial scaring at the peak of the disease and modulate the astroglial response to EAE during the time-course of the disease.

Hyperbaric oxygenation alters temporal expression pattern of superoxide dismutase 2 after cortical stab injury in rats.

AIM: To evaluate the effect of hyperbaric oxygen therapy (HBOT) on superoxide dismutase 2 (SOD2) expression pattern after the cortical stab injury (CSI). METHODS: CSI was performed on 88 male Wistar rats, divided into control, sham, lesioned, and HBO groups. HBOT protocol was the following: pressure applied was 2.5 absolute atmospheres, for 60 minutes, once a day for consecutive 3 or 10 days. The pattern of SOD2 expression and cellular localization was analyzed using real-time polymerase chain reaction, Western blot, and double-label fluorescence immunohistochemistry. Neurons undergoing degeneration were visualized with Fluoro-Jade®B. RESULTS: CSI induced significant transient increase in SOD2 protein levels at day 3 post injury, which was followed by a reduction toward control levels at post-injury day 10. At the same time points, mRNA levels for SOD2 in the injured cortex were down-regulated. Exposure to HBO for 3 days considerably down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. HBOT significantly increased mRNA levels for SOD2 at both time points compared to the corresponding L group, but they were still lower than in controls. Double immunofluorescence staining revealed that 3 days after CSI, up-regulation of SOD2 was mostly due to an increased expression in reactive astrocytes surrounding the lesion site. HBOT attenuated SOD2 expression both in neuronal and astroglial cells. Fluoro-Jade®B labeling showed that HBOT significantly decreased the number of degenerating neurons in the injured cortex. CONCLUSION: HBOT alters SOD2 protein and mRNA levels after brain injury in a time-dependent manner.