Sodium chloride-induced changes in oxidative stress, inflammation, and dysbiosis in experimental multiple sclerosis.

Objectives: The high-salt diet (HSD) has been associated with cognitive dysfunction by attacking the cerebral microvasculature, through an adaptive response, initiated in the intestine and mediated by Th17 cells. In the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), it has been described that NaCl causes an increase in T cell infiltration in the central nervous system. NaCl also promotes macrophage response and Th17 cell differentiation, worsening the course of the disease. HSD may trigger an activation of the immune system and enhance inflammation. However, certain studies not only do not support this possibility, but support the opposite, as the effect of salt on immune cells may not necessarily be pathogenic. Therefore, this study aimed to evaluate the effect of an over intake of salt in rats with EAE, based on the clinical course, oxidative stress, markers of inflammation and the gut dysbiosis.Methods: 15 Dark Agouti rats were used, which were divided into control group, EAE group and EAE + NaCl group. Daily 0.027 g of NaCl dissolved in 300 µl of H2O was administered through a nasogastric tube for 51 days.Results: NaCl administration produced an improvement in clinical status and a decrease in biomarkers of oxidative stress, inflammation, and dysbiosis.Conclusion: The underlying mechanism by which NaCl causes these effects could involve the renin-angiotensin-aldosterone system (RAAS), which is blocked by high doses of salt.

The ultrastructure of muscle fibers and satellite cells in experimental autoimmune encephalomyelitis after treatment with transcranial magnetic stimulation.

In this study, we investigated the effect of transcranial magnetic stimulation (TMS) on the ultrastructure of muscle fibers and satellite cells in rats with experimental autoimmune encephalomyelitis (EAE). EAE-induced animals were treated with TMS (60 Hz at 0.7 mT) for 2 hours in the morning, once a day, 5 days a week, for 3 weeks, starting on day 15 post-immunization. The rats were sacrificed on day 36 post-immunization, and the soleus muscles were evaluated by light microscopy and transmission electron microscopy. Findings were compared with a non-treated EAE group. Electron microscopy analysis showed the presence of degenerated mitochondria, autophagic vacuoles, and altered myofibrils in non-treated EAE group. This correlates with the presence of acid phosphatase activity in muscle fibers and core-targetoid lesions with desmin immunohistochemistry. Most myonuclei in the EAE group showed apoptotic features. In contrast, EAE induced-TMS treated animals had less ultrastructural changes in the mitochondria and the myofibrils, together with less frequent apoptotic nuclear features. Peripheral desmin+ protrusions, as a marker of active satellite cells, were significantly increased in TMS-treated group. This correlates ultrastructurally with the presence of active features in satellite cells in the TMS group. In conclusion, the attenuation of ultrastructural alterations in muscle fibers and activation response of satellite cells caused by EAE indicated that skeletal muscle had a regenerative response to TMS.

Transcranial Magnetic Stimulation Improves Muscle Involvement in Experimental Autoimmune Encephalomyelitis.

Skeletal muscle is affected in experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis that produces changes including muscle atrophy; histological features of neurogenic involvement, and increased oxidative stress. In this study, we aimed to evaluate the therapeutic effects of transcranial magnetic stimulation (TMS) on the involvement of rat skeletal muscle and to compare them with those produced by natalizumab (NTZ). EAE was induced by injecting myelin oligodendrocyte glycoprotein (MOG) into Dark Agouti rats. Both treatments, NTZ and TMS, were implemented from day 15 to day 35. Clinical severity was studied, and after sacrifice, the soleus and extensor digitorum longus muscles were extracted for subsequent histological and biochemical analysis. The treatment with TMS and NTZ had a beneficial effect on muscle involvement in the EAE model. There was a clinical improvement in functional motor deficits, atrophy was attenuated, neurogenic muscle lesions were reduced, and the level of oxidative stress biomarkers was lower in both treatment groups. Compared to NTZ, the best response was obtained with TMS for all the parameters analyzed. The myoprotective effect of TMS was higher than that of NTZ. Thus, the use of TMS may be an effective strategy to reduce muscle involvement in multiple sclerosis.

Neuroprotective and antioxidant effects of docosahexaenoic acid (DHA) in an experimental model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease, whose etiology is not yet fully understood, although there are several factors that can increase the chances of suffering from it. These factors include nutrition, which may be involved in the pathogenesis of the disease. In relation to nutrition, docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (n-3 PUFA), has emerged as an important player in the regulation of neuroinflammation, being considered a pleiotropic molecule. This study aimed to evaluate the effect of DHA supplementation on clinical state and oxidative stress produced by experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Twenty-five Dark Agouti rats which were used divided into Control Group, Control+Vehicle Group, Control+DHA Group, EAE Group, and EAE+DHA Group. DHA was administered for 51 days by intraperitoneal (i.p.) injection at a dose of 40 mg/kg, once a day, 5 days a week. DHA supplementation produced a decrease in oxidative stress, as well as an improvement in the clinical score of the disease. DHA could exert a beneficial effect on the clinic of MS, through the activation of the antioxidant factor Nrf2.

Effect of the Combination of Different Therapies on Oxidative Stress in the Experimental Model of Multiple Sclerosis.

Oxidative stress is heavily involved in several pathological features of Multiple Sclerosis (MS), such as myelin destruction, axonal degeneration, and inflammation. Different therapies have been shown to reduce the oxidative stress that occurs in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Some of these therapies are transcranial magnetic stimulation (TMS), extra virgin olive oil (EVOO) and S-allyl cysteine (SAC). This study aims to test the antioxidant effect of these three therapies, to compare the efficacy of SAC versus TMS and EVOO, and to analyze the effect of combining SAC + TMS and SAC and EVOO. Seventy Dark Agouti rats were used, which were divided into Control group; Vehicle group; Mock group; SAC; EVOO; TMS; SAC + EVOO; SAC + TMS; EAE; EAE + SAC; EAE + EVOO; EAE + TMS; EAE + SAC + EVOO; EAE + SAC + TMS. The TMS consisted of an oscillatory magnetic field in the form of a sine wave with a frequency of 60 Hz and an amplitude of 0.7mT (EL-EMF) applied for two hours in the morning, once a day, five days a week. SAC was administered at a dose of 50 mg/kg body weight, orally daily, five days a week. EVOO represented 10% of their calorie intake in the total standard daily diet of rats AIN-93G. All treatments were maintained for 51 days. TMS, EVOO and SAC, alone or in combination, reduce oxidative stress, increasing antioxidant defenses and also lowering the clinical score. Combination therapies do not appear to be more potent than individual therapies against the oxidative stress of EAE or its clinical symptoms.

Experimental muscle injury: correlation between ultrasound and histological findings.

INTRODUCTION: In this study we correlated ultrasound findings with histological changes taking place during experimentally induced degeneration-regeneration in rat skeletal muscle. METHODS: Gastrocnemius muscles were injected with mepivacaine, and the progress of the muscle injury was monitored by ultrasound from day 1 to day 20. Muscles were extracted on the same days for histological examination. RESULTS: The degenerative phase was characterized by increased echogenicity in the injured area; thereafter, echogenicity gradually diminished during the regenerative phase, attaining normal levels by 20 days postinjection. By this stage, histological examination revealed that regeneration was complete. The heteroechoic texture observed from day 4 to day 10 appeared to reflect the coexistence of degenerative and regenerative processes. CONCLUSIONS: The results suggest that the degenerative and regenerative phases of muscle injury may be distinguished sonographically through differences in echogenicity and echotexture and, using Doppler ultrasound, differences in the degree of vascularization.

Lactose and Casein Cause Changes on Biomarkers of Oxidative Damage and Dysbiosis in an Experimental Model of Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Experimental Autoimmune Encephalomyelitis (EAE) in rats closely reproduces Multiple Sclerosis (MS), a disease characterized by neuroinflammation and oxidative stress that also appears to extend to other organs and their compartments. The origin of MS is a matter for discussion, but it would seem that altering certain bacterial populations present in the gut may lead to a proinflammatory condition due to the bacterial Lipopolysaccharides (LPS) in the so-called brain-gut axis. The casein and lactose in milk confer anti-inflammatory properties and immunomodulatory effects. The objectives of this study were to evaluate the effects of administration of casein and lactose on the oxidative damage and the clinical status caused by EAE and to verify whether both casein and lactose had any effect on the LPS and its transport protein -LBP-. METHODS: Twenty male Dark Agouti rats were divided into control rats (control), EAE rats, and EAE rats, to which casein and lactose, EAE+casein, and EAE+lactose, respectively, were administered. Fifty-one days after casein and lactose administration, the rats were sacrificed, and different organs were studied (brain, spinal cord, blood, heart, liver, kidney, small, and large intestine). In the latter, products derived from oxidative stress were studied (lipid peroxides and carbonylated proteins) as well as the glutathione redox system, various inflammation factors (total nitrite, Nuclear Factor-kappa B p65, the Rat Tumour Necrosis Factor-α), and the LPS and LBP values. RESULTS AND CONCLUSION: Casein and lactose administration improved the clinical aspect of the disease at the same time as reducing inflammation and oxidative stress, exerting its action on the glutathione redox system, or increasing GPx levels.

Histology of skeletal muscle reconstructed by means of the implantation of autologous adipose tissue: an experimental study.

The purpose of this study was to determine the histological characteristics of a skeletal muscle reconstructed by means of the implantation of autologous adipose tissue following an experimentally-induced volumetric muscle loss. A cylindrical piece in the belly of the rat anterior tibial muscle was removed. In the hole, inguinal subcutaneous adipose tissue of the same rat was grafted. Animals were sacrificed 7, 14, 21, 28 and 60 days posttransplantation. Histological, histochemical, immunohistochemical and morphometric techniques were used. At all times analyzed, the regenerative muscle fibers formed from the edges of the muscle tissue showed histological, histochemical and immunohistochemical differences in comparison with the control group. These differences are related to delays in the maturation process and are related to problems in reinnervation and disorientation of muscle fibers. The stains for MyoD and desmin showed that some myoblasts and myotubes seem to derive from the transplanted adipose tissue. After 60 days, the transplant area was 20% occupied by fibrosis and by 80% skeletal muscle. However, the neo-muscle was chaotically organized showing muscle fiber disorientation and centronucleated fibers with irregular shape and size. Our results support the hypothesis that, at least from a morphological point of view, autologous adipose tissue transplantation favors reconstruction following a volumetric loss of skeletal muscle by combining the inherent regenerative response of the organ itself and the myogenic differentiation of the stem cells present in the adipose tissue. However, in our study, the formed neo-muscle exhibited histological differences in comparison with the normal skeletal muscle.

Denervated muscle extract promotes recovery of muscle atrophy through activation of satellite cells. An experimental study.

PURPOSE: The objective of the present study was to determine whether a denervated muscle extract (DmEx) could stimulate satellite cell response in denervated muscle. METHODS: Wistar rats were divided into 4 groups: normal rats, normal rats treated with DmEx, denervated rats, and denervated rats treated with DmEx. The soleus muscles were examined using immunohistochemical techniques for proliferating cell nuclear antigen, desmin, and myogenic differentiation antigen (MyoD), and electron microscopy was used for analysis of the satellite cells. RESULTS: The results indicate that while denervation causes activation of satellite cells, DmEx also induces myogenic differentiation of cells localized in the interstitial space and the formation of new muscle fibers. Although DmEx had a similar effect in nature on innervated and denervated muscles, this response was of greater magnitude in denervated vs. intact muscles. CONCLUSION: Our study shows that treatment of denervated rats with DmEx potentiates the myogenic response in atrophic denervated muscles.

Extra-Virgin Olive Oil Modifies the Changes Induced in Non-Nervous Organs and Tissues by Experimental Autoimmune Encephalomyelitis Models.

This study reveals the existence of oxidative stress (reactive oxygen species (ROS)) in non-nervous organs and tissues in multiple sclerosis (MS) by means of a model of experimental autoimmune encephalomyelitis (EAE) in rats. This model reproduces a similar situation to MS, as well as its relationship with intestinal microbiota starting from the changes in bacterial lipopolysaccharide levels (LPS) in the outer wall of the gram-negative bacteria. Finally, the administration of extra-virgin olive oil (EVOO), hydroxytirosol (HT), and oleic acid (OA) exert beneficial effects. Twenty-five Dark Agouti two-month-old male rats, weighing around 190 g, were distributed into the following groups: Control, EAE (experimental autoimmune encephalomyelitis group), EAE + EVOO, EAE + HT, and EAE + OA. The glutathione redox system with the EAE was measured in heart, kidney, liver, and small and large intestines. The LPS and the correlation with oxidative stress in the small and large intestines were also investigated. The results showed that (1) the oxidative damage in the EAE model affects non-nervous organs and tissues; (2) The LPS is related to inflammatory phenomena and oxidative stress in the intestinal tissue and in other organs; (3) The administration of EVOO, HT, and OA reduces the LPS levels at the same time as minimizing the oxidative damage; (4) EVOO, HT, and OA improve the disease's clinical score; and (5) on balance, EVOO offers a better neuroprotective effect.

Comparative of transcranial magnetic stimulation and other treatments in experimental autoimmune encephalomyelitis.

The effects of transcranial magnetic stimulation (TMS), natalizumab (nata), dimethyl fumarate (DMF) and dexamethasone (DEX) on clinical score and oxidative stress produced by a single dose of myelin oligodendrocyte glycoprotein (MOG) in tail of Dark Agouti rats was studied. TMS (60Hz and 0.7 mT), nata (5mg/kg), DMF (15mg/kg) and DEX (300µg/kg) was applied for 21 after the administration of MOG (150µg). We estimated clinical score, as well as lipid peroxides, carbonylated proteins and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio content in brain, spinal cord and blood. MOG triggered significant increase in clinical score and in the levels of lipid peroxides and carbonylated proteins levels, but reduced GSH/GSSG ratio in brain, spinal cord and blood. Both TMS and clinical treatments, although TMS more significantly, decreased the changes caused by MOG administration. These results support the antioxidant and neuroprotective action of TMS, as well as an activity higher than other clinical treatments.

Effects of transcranial magnetic stimulation on oxidative stress in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) reproduces a multiple sclerosis (MS)-like experimental model. The main objective was to evaluate the effect of extremely low-frequency electromagnetic fields (EL-EMF) application, like a paradigm of transcranial magnetic stimulation (TMS) in the development of EAE. Rats were injected with a single dose of 150 µg of myelin oligodendrocyte glycoprotein (MOG, fragment 35-55) to produce experimental MS. To assess the effect of TMS application in EAE, the rats were treated with TMS (60 Hz and 0.7 mT) for 2 h in the morning, once a day, 5 days a week, during 3 weeks. TMS was applied to the head. The effect of TMS on EAE was evaluated as motor symptoms and, oxidative and cell damage. The data showed that MOG induced motor symptoms as tail paralysis and limb paresis/paralysis, oxidative stress and cell death similar to MS when compared with control animals. Importantly, TMS application attenuated motor symptoms, oxidative and cell damage, whereas it increased antioxidant system. Our findings suggest that: (i) MOG reproduces an experimental model of MS characterised by oxidative and cell damage; and (ii) TMS application decreases oxidative stress and cell death induced by MOG.

Transcranial magnetic stimulation modifies astrocytosis, cell density and lipopolysaccharide levels in experimental autoimmune encephalomyelitis.

AIMS: Experimental autoimmune encephalomyelitis (EAE) is considered a valid experimental model for multiple sclerosis, a chronic neuroinflammatory condition of the central nervous system. Additionally, some evidence has shown that some microbial products such as the bacterial lipopolysaccharide could lead to the activation of reactive immune cells, triggering neuroinflammation. Several studies have found that transcranial magnetic stimulation (TMS) may exert a neuroprotective effect. Therefore, we aimed to assess the effect of TMS on the neuroinflammation occurring in EAE. MATERIALS AND METHODS: A total of 44 male Dark Agouti rats were used. EAE induction was performed administering subcutaneously at the dorsal base of the tail a single dose of myelin oligodendrocyte glycoprotein. Clinical evaluation of motor symptoms was performed. Brain and spinal cord were collected and analyzed for nitric oxide, bacterial lipopolysaccharide and lipopolysaccharide-binding protein. We also carried out a histologic exam, which included an astrocyte immunostaining and Nissl staining for the assessment of brain cell density and pyknotic nuclei. KEY FINDINGS: TMS effectively ameliorated motor impairment secondary to EAE. This form of magnetic field was capable of decreasing the proliferation of astrocytes as a response to the autoimmune attack, reducing the content of nitric oxide, bacterial lipopolysaccharide and lipopolysaccharide-binding protein in central nervous system. Moreover, in treated animals, brain cell density was improved and the number of pyknotic nuclei was decreased. SIGNIFICANCE: Transcranial magnetic stimulation modifies astrocytosis, cell density and lipopolysaccharide levels in EAE. These results suggest that TMS could be a promising treatment for neuroinflammatory conditions such as multiple sclerosis.

Lipopolysaccharide Binding Protein and Oxidative Stress in a Multiple Sclerosis Model.

Recent findings in experimental autoimmune encephalomyelitis (EAE) suggest that altering certain bacterial populations present in the gut may lead to a proinflammatory condition, that could result in the development of multiple sclerosis (MS). Also, Reactive Oxygen Species seem to be involved in the course of MS. In this study, it has been aimed to relate all these variables starting from an analysis of the lipopolysaccharide (LPS) and LPS-binding protein (LBP) with the determination of parameters related to oxidative stress in the blood, brain and spinal cord. For this purpose, samples obtained from EAE rats and relapsing-remitting (RRMS) MS patients were used. In addition, EAE rats were treated with Natalizumab, N-acetyl-cysteine and dimethyl fumarate. Natalizumab was also employed in RRMS. The results of this study revealed an improvement in the clinical symptoms of the EAE and MS with the treatments, as well as a reduction in the oxidative stress parameters and in LBP. Correlations between the clinical variables of the disease, i.e. oxidative damage and LBP, were established. Although the conclusions of this research are indeed relevant, further investigation would be necessary to establish the intrinsic mechanisms of the MS-oxidative stress-microbiota relationship.

Muscular hypertrophy and atrophy in normal rats provoked by the administration of normal and denervated muscle extracts.

This study was conducted to determine the effects of extracts obtained from both normal and denervated muscles on different muscle types. Wistar rats were used and were divided into a control group and four experimental groups. Each experimental group was treated intraperitoneally during 10 consecutive days with a different extract. These extracts were obtained from normal soleus muscle, denervated soleus, normal extensor digitorum longus, and denervated extensor digitorum longus. Following treatment, the soleus and extensor digitorum longus muscles were obtained for study under optic and transmission electron microscope; morphometric parameters and myogenic responses were also analyzed. The results demonstrated that the treatment with normal soleus muscle and denervated soleus muscle extracts provoked hypertrophy and increased myogenic activity. In contrast, treatment with extracts from the normal and denervated EDL had a different effect depending on the muscle analyzed. In the soleus muscle it provoked hypertrophy of type I fibers and increased myogenic activity, while in the extensor digitorum longus atrophy of the type II fibers was observed without changes in myogenic activity. This suggests that the muscular responses of atrophy and hypertrophy may depend on different factors related to the muscle type which could be related to innervation.

Natalizumab Modifies Catecholamines Levels Present in Patients with Relapsing- Remitting Multiple Sclerosis.

AIMS: The main aim of this study was to verify the effect of natalizumab on the levels of circulating catecholamines and indolamine and their possible relation with MS. METHODS: For this purpose, 12 healthy individuals (control group) and 12 relapsing-remitting multiple sclerosis patients (RR-MS) were selected. The patients were treated with 300 mg of natalizumab during 56 weeks (1 dose/4 weeks) (MS-56). This selection was based on the McDonalds revision criterion and scheduled to star treatment with natalizumab. Blood samples were taken before treatment (basal level) and after 56 weeks of using natalizumab. Melatonin was measured in serum and in plasma, catecholamines (dopamine, epinephrine, and norepinephrine), carbonylated proteins, 8-hydroxy-2'deoxyguanosine (8OH-dG) and the ratio reduced glutathione/oxidised glutathione (GSH/GSSG). RESULTS: The epinephrine and dopamine levels diminished in the basal group with respect to the control and did not recover normal levels with the treatment. The melatonin was decreased in RR-MS patients and went back to its normal levels with natalizumab. Norepinephrine was increased in RR-MS and decreased in MS-56 until it equalled the control group. CONCLUSION: Natalizumab normalizes altered melatonin and norepinephrine levels in MS.

Skeletal muscle findings in experimental autoimmune encephalomyelitis.

INTRODUCTION: Skeletal muscle is a target organ in multiple sclerosis, a chronic debilitating disease of the central nervous system caused by demyelination and axonal deterioration. Since the experimental autoimmune encephalomyelitis model reproduces the relapsing-remitting course found in most multiple sclerosis patients, this model was used to compare the histological features of skeletal muscle at onset with those observed at the start of the second relapse. MATERIAL AND METHODS: Histological, histochemical and ultrastructural changes, as well as biochemical oxidative damage and antioxidant-system markers, were examined in the soleus and extensor digitorum longus muscles of Dark Agouti rats in which experimental autoimmune encephalomyelitis had been induced by active immunization using myelin oligodendrocyte glycoprotein. RESULTS: Histological examination at disease onset revealed ragged-red fibers and ultrastructural evidence of mitochondrial degeneration. At the second relapse, neurogenic changes included a wide range of cytoarchitectural lesions, skeletal muscle atrophy and the appearance of intermediate fibers; however, differences were observed between soleus and extensor digitorum longus lesions. Biochemical tests disclosed an increase in oxidative stress markers at onset, which was more pronounced at the second relapse. CONCLUSIONS: Microscopic findings suggest that two patterns can be distinguished at disease onset: an initial phase characterized by muscle mitochondrial alterations, and a second phase dominated by a histological muscle pattern of clearly neurogenic origin.

Elevated melatonin levels in natalizumab-treated female patients with relapsing-remitting multiple sclerosis: relationship to oxidative stress.

Natalizumab is currently the most successful clinical treatment for multiple sclerosis. The use of this drug is associated with the reduction in the number of relapses and a slowing in disease progression, as well as an improvement in signs and symptoms displayed by the patients. To evaluate the effect of natalizumab on melatonin and its relationship with peripheral oxidative damage, we studied the serum melatonin levels in 18 patients with relapsing-remitting multiple sclerosis. Natalizumab caused significant increases in serum melatonin concentrations. This change was associated with a rise in increase of antioxidants and a reduction in oxidative stress biomarkers. In conclusion, these data may explain, at least in part, some of the beneficial effects exhibited by disease antibody such as its antioxidant capacity.

Melatonin improves 3-nitropropionic acid induced behavioral alterations and neurotrophic factors levels.

OBJECTIVE: This study sought to determine whether melatonin causes changes in neurotrophic factors and it protects against the mycotoxin 3-nitropropionic acid (3-NP) in brain tissue. METHODS: Rats were given 3-NP over four consecutive days (20 mg/kg BW), while melatonin was administered over 21 days (1 mg/kg/BW), starting after the last injection of 3-NP. RESULTS: Rats treated with 3-NP displayed significant changes in neurotrophic factor (BDNF and GDNF) levels, together with alterations in behavior; they also displayed extensive oxidative stress and a massive neuronal damage. CONCLUSIONS: Melatonin improved behavioral alterations, reduced oxidative damage, lowered neurotrophic factor levels and neuronal loss in 3-NP-treated rats. These results suggest that melatonin exerts a neuroprotective action.