Trilobatin contributes to the improvement of myopathy in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to a deficiency in dystrophin production and consequent progressive degeneration of skeletal muscle fibres, through oxidative stress and an exacerbated inflammatory process. The flavonoid trilobatin (TLB) demonstrates antioxidant and anti-inflammatory potential. Its high safety profile and effective action make it a potent therapy for the process of dystrophic muscle myonecrosis. Thus, we sought to investigate the action of TLB on damage in a DMD model, the mdx mouse. Eight-week-old male animals were treated with 160 mg/kg/day of trilobatin for 8 weeks. Control animals were treated with saline. Following treatment, muscle strength, serum creatine kinase (CK) levels, histopathology (necrotic myofibres, regenerated fibres/central nuclei, Feret's diameter and inflammatory area) and the levels of catalase and NF-κB (western blotting) of the quadriceps (QUA), diaphragm (DIA) and tibialis anterior (TA) muscles were measured. TLB was able to significantly increase muscle strength and reduce serum CK levels in dystrophic animals. The QUA of mdx mice showed a reduction in catalase and the number of fibres with a centralized nucleus after treatment with TLB. In the DIA of dystrophic animals, TLB reduced the necrotic myofibres, inflammatory area and NF-κB and increased the number of regenerated fibres and the total fibre diameter. In TA, TLB increased the number of regenerated fibres and reduced catalase levels in these animals. It is concluded that in the mdx experimental model, treatment with TLB was beneficial in the treatment of DMD.

Screening of voice disorders concerning anatomy teachers who use formaldehyde cadaveric parts in laboratory classes.

Voice disorders are common among teachers and, in particular, anatomy teachers are exposed to a potential enemy for dysphonia, irritating chemicals, that is, formaldehyde. We seek to verify the association between: (1) teaching time, (2) type of cadaveric conservation to which the teacher is exposed and (3) hours of exposure to cadaveric preservative related to the different categories of voice disorders screening (ITDV). The sample consisted of 111 teachers who answered to 02 data collection instruments: I - Sociodemographic Data; II - ITDV. Among participating teachers there were 71 male and 40 female, with an average age of 43 years and 11 months and an average teaching time of 16 years and 5 months. Association tests between teaching time and ITDV demonstrate a significant result in the relationship between voice failure and teaching time (p<0.05). All 111 teachers use their voices in laboratory classes and use cadaveric material. From those, 107 teachers are exposed to formaldehyde as cadaveric parts' conservative solution. There was a significant association (p<0.05) between voice failure and the type of cadaveric conservative solution but non-significant relationship (p>0.05) between ITDV and the time of exposure to formaldehyde preservative. Teachers' ITDV showed vocal signs and symptoms. In particular, voice loss due to time of teaching in anatomy, and voice failure, due to exposure to formaldehyde and combinations used in anatomical parts and cadavers, were significant.

Anatomy and Imaging of the Spinal Cord: An Overview.

The spinal cord comprises the part of the central nervous system located within the vertebral canal, extending from the foramen magnum to approximately the second lumbar vertebra. The spinal cord is covered by 3 meninges: dura mater, arachnoid mater, and pia mater (arranged from the outermost layer inward). A cross-section of the spinal cord reveals gray and white matter. Ascending and descending pathways have defined locations in the matter of the spinal cord. This article aims to review the spinal cord anatomy and demonstrate the imaging aspects, which are essential for the interpretation and understanding of spinal cord injuries.

Severe muscle damage after a short period of ischemia and reperfusion in an animal model.

BACKGROUND: Skeletal muscle ischemia-reperfusion injuries result in a loss of contractile function, leading to limb disability or amputation. Ischemia causes hypoxia and cellular energy failure, which is aggravated by reperfusion due to the inflammatory response and oxidative stress. The consequences of the injury vary according to the duration of the period of ischemia and reperfusion. Therefore, the present work aims to evaluate ischemia-reperfusion injuries induced in the skeletal muscles of Wistar rats submitted to 3 different application periods based on morphological and biochemical parameters. METHODS: For this, a tourniquet was applied to the root of the animals' hind limbs, occluding arterial and venous blood flow, and it was followed by reperfusion-the removal of the tourniquet. The groups were: control (without tourniquet); I30'/R60' (30 minutes of ischemia and 1 hour of reperfusion); I120'/R120' (2 hours and 2 hours); and I180'/R180' (3 hours and 3 hours). RESULTS: All ischemia-reperfusion groups showed characteristics of muscle injury. Microscopic analyses of the extensor digitorum longus, soleus, tibialis anterior, and gastrocnemius muscles showed a significant increase in the number of injured muscle fibers in the ischemia-reperfusion groups compared to the control group. There were also significant differences between the ischemia-reperfusion groups in all muscles, showing a progressive increase in the degree of injury. The quantification of the number of injured muscle fibers between the muscles revealed that at I30'/R60', the soleus muscles had a higher number of injuries in relation to the other muscles, with statistical significance. In the I120'/R120' group, the gastrocnemius muscles presented a significantly greater number of injured fibers. There were no significant differences in the I180'/R180' group. The serum levels of creatine kinase in the I180'/R180' group were significantly higher than in the control and I30'/R60' groups. CONCLUSIONS: Therefore, it was evident that the 3 ischemia-reperfusion models used were capable of causing cell damage, with these findings being more pronounced in the I180'/R180' group.

Protection of dystrophic muscle cells using Idebenone correlates with the interplay between calcium, oxidative stress and inflammation.

There is strong cross-talk between abnormal intracellular calcium concentration, high levels of reactive oxygen species (ROS) and an exacerbated inflammatory process in the dystrophic muscles of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). In this study, we investigated effects of Idebenone, a potent anti-oxidant, on oxidative stress markers, the anti-oxidant defence system, intracellular calcium concentrations and the inflammatory process in primary dystrophic muscle cells from mdx mice. Dystrophic muscle cells were treated with Idebenone (0.05 µM) for 24 h. The untreated mdx muscle cells were used as controls. The MTT assay showed that Idebenone did not have a cytotoxic effect on the dystrophic muscle cells. The Idebenone treatment was able to reduce the levels of oxidative stress markers, such as H2 O2 and 4-HNE, as well as decreasing intracellular calcium influx in the dystrophic muscle cells. Regarding Idebenone effects on the anti-oxidant defence system, an up-regulation of catalase levels, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity was observed in the dystrophic muscle cells. In addition, the Idebenone treatment was also associated with reduction in inflammatory molecules, such as nuclear factor kappa-B (NF-κB) and tumour necrosis factor (TNF) in mdx muscle cells. These outcomes supported the use of Idebenone as a protective agent against oxidative stress and related signalling mechanisms involved in dystrophinopathies, such as DMD.

Cilostazol attenuates oxidative stress and apoptosis in the quadriceps muscle of the dystrophic mouse experimental model.

Duchenne muscular dystrophy (DMD) is the most severe and frequent form of muscular dystrophy. The mdx mouse is one of the most widely used experimental models to understand aspects of the biology of dystrophic skeletal muscles and the mechanisms of DMD. Oxidative stress and apoptosis are present in early stages of the disease in mdx mice. The high production of reactive oxygen species (ROS) causes activation of apoptotic death regulatory proteins due to DNA damage and breakdown of nuclear and mitochondrial membranes. The quadriceps (QUA) muscle of the mdx mouse is a good tool to study oxidative events. Previous studies have demonstrated that cilostazol exerts an anti-oxidant effect by decreasing the production of reactive oxygen species (ROS). The present study aimed to evaluate the ability of cilostazol to modulate oxidative stress and apoptosis in the QUA muscle of mdx mice. Fourteen-day-old mdx mice received cilostazol or saline for 14 days. C57BL/10 mice were used as a control. In the QUA muscle of mdx mice, cilostazol treatment decreased ROS production (-74%), the number of lipofuscin granules (-47%), lipid peroxidation (-11%), and the number of apoptotic cells (-66%). Thus cilostazol showed anti-oxidant and anti-apoptotic action in the QUA muscle of mdx mice.

Cross-talk between TRPC-1, mTOR, PGC-1α and PPARδ in the dystrophic muscle cells treated with tempol.

Background Ca2+ dysregulation and oxidative damage appear to have a central role in Duchenne muscular dystrophy (DMD) progression. The current study provides muscle cell-specific insights into the effect of Tempol on the TRPC 1 channel; on the positive and negative regulators of muscle cell differentiation; on the antioxidant enzymatic system; on the activators of mitochondrial biogenesis; and on the inflammatory process in the dystrophic primary muscle cells in culture. METHODS: Mdx myotubes were treated with Tempol (5 mM) for 24 h. Untreated mdx myotubes and C57BL/10 myotubes were used as controls. RESULTS: The Trypan Blue, MTT and Live/Dead Cell assays showed that Tempol (5 mM) presented no cytotoxic effect on the dystrophic muscle cells. The Tempol treated-mdx muscle cells showed significantly lower levels in the fluorescence intensity of intracellular calcium; TRPC-1 channel; MyoD; H2O2 and O2•- production; 4-HNE levels; SOD2, CAT and GPx levels; and TNF levels. On the other hand, SOD, CAT and GR mRNA relative expression were significantly higher in Tempol treated-mdx muscle cells. In addition, higher levels of Myogenin, MHC-Slow, mTOR, PGC-1α and PPARδ were also observed in Tempol treated-mdx muscle cells. CONCLUSION: Our findings demonstrated that Tempol decreased intracellular calcium and oxidative stress in primary dystrophic muscle cells, promoting a cross-talk between TRPC-1, mTOR, PGC-1α and PPARδ.

Oxidative Stress, Inflammation, and Activators of Mitochondrial Biogenesis: Tempol Targets in the Diaphragm Muscle of Exercise Trained-mdx Mice.

The mdx mouse phenotype aggravated by chronic exercise on a treadmill makes this murine model more reliable for the study of muscular dystrophy. Thus, to better assess the Tempol effect on dystrophic pathways, the analyses in this study were performed in the blood samples and diaphragm muscle from treadmill trained adult (7-11-weeks old) mdx animals. The mdx mice were divided into three groups: mdxSed, sedentary controls (n = 28); mdxEx, exercise-trained animals (n = 28); and mdxEx+T, exercise-trained animals with the Tempol treatment (n = 28). The results demonstrated that the Tempol treatment promoted muscle strength gain, prevented muscle damage, reduced the inflammatory process, oxidative stress, and angiogenesis regulator, and up regulated the activators of mitochondrial biogenesis. The main new findings of this study are that Tempol reduced the NF-κB and increased the PGC1-α and PPARδ levels in the exercise-trained-mdx mice, which are probably related to the ability of this antioxidant to scavenge excessive ROS. These results reinforce the use of Tempol as a potential therapeutic strategy in DMD.

Ixazomib, an oral proteasome inhibitor, exhibits potential effect in dystrophin-deficient mdx mice.

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm2 in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF-ß in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

Tempol improves redox status in mdx dystrophic diaphragm muscle.

Oxidative stress is a critical element in relationship to the pathophysiology of Duchenne muscular dystrophy (DMD). In the mice the diaphragm (DIA) is most resembles the dystrophic human pathology. In this study we have evaluated the consequences of a synthetic antioxidant (tempol) on oxidative stress parameters in the DIA muscle of mdx mice. The mdx mice were separated into two groups: mdx, the control group receiving intraperitoneal (i.p.) injections of saline solution (100 µL), and mdxT, the treated group receiving i.p. injections of tempol (100 mg/kg). The tempol-treated group showed reduced oxidative stress markers, decreasing the dihydroethidium reaction (DHE) area; autofluorescent lipofuscin granules; and 4-hydroxynonenal (4-HNE)-protein adduct levels. DIA muscle of mdx mice. At the same time, the manganese-superoxide dismutase 2 (SOD2) levels were increased in the tempol-treated group. In addition, the tempol-treated group showed reduced levels of glutathione-disulphide reductase (GSR), glutathione peroxidase 1 (GPx1) and catalase (CAT) in immunoblots. The tempol-treated group has also shown lower relative gene expression of SOD1, CAT and GPx than the non-treated group. Our data demonstrated that tempol treatment reduced oxidant parameters and increased anti-oxidant SOD2 levels in the DIA muscle of mdx mice, which may contribute to the normalization of the redox homeostasis of dystrophic muscles.

Photobiomodulation Therapy for Attenuating the Dystrophic Phenotype of Mdx Mice.

This study analyzed photobiomodulation therapy (PBMT) effects on regenerative, antioxidative, anti-inflammatory and angiogenic markers in the dystrophic skeletal muscle of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD), during the acute phase of dystrophy disease. The following groups were set up: Ctrl (control group of normal wild-type mice; C57BL/10); mdx (untreated mdx mice); mdxPred (mdx mice treated with prednisolone) and mdxLA (mdx mice treated with PBMT). The PBMT was carried out using an Aluminum Gallium Arsenide (AIGaAs; IBRAMED® laserpulse) diode, 830 nm wavelength, applied on the dystrophic quadriceps muscle. The mdxLA group showed a degenerative and regenerative area reduction simultaneously with a MyoD level increase, ROS production and inflammatory marker reduction and up-regulation in the VEGF factor. In addition, PBMT presented similar effects to prednisolone treatment in most of the parameters analyzed. In conclusion, our results indicate that PBMT in the parameters selected attenuated the dystrophic phenotype of mdx mice, improving skeletal muscle regeneration; reducing the oxidative stress and inflammatory process; and up-regulating the angiogenic marker.

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 µM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

Tempol treatment shows phenotype improvement in mdx mice.

Considering potential Tempol effects on mdx muscle fibers, in this study we evaluated its effects on relevant dystrophic phenotypic characteristics, such as muscle degeneration, inflammatory process and angiogenesis, which as yet have not been investigated. Mdx mice were randomly assigned into three groups: mdxS, the control group receiving intraperitoneal (i.p.) injections of saline solution (100µL); mdxP, positive control group receiving prednisolone (1mg/kg) by oral gavage; and mdxT, treated group receiving i.p. injections of tempol (100 mg/kg). C57BL/10 mice were also used as controls. Tempol treatment promoted gain in muscle strength and reduced myonecrosis and inflammatory response in the dystrophic diaphragm (DIA) and biceps brachii (BB) muscles. No evidence of Tempol's beneficial performance on angiogenesis in DIA and BB mdx muscles was found. The findings presented here show that Tempol treatment improves dystrophic phenotype, supporting its use as a potential therapeutic strategy in DMD.

Sex influences diaphragm muscle response in exercised mdx mice.

Physical exercise promotes increased muscle damage in the mdx mice, the experimental model of Duchenne muscular dystrophy. Studies suggest that the estrogen level in females makes them less susceptible to muscle injuries. The aim of this study was to characterize the diaphragm (DIA) muscle response to physical exercise in male and female mdx mice. The animals were divided into four groups: female sedentary mdx; male sedentary mdx; female mdx submitted to exercise; and male mdx mice submitted to exercise. Blood samples were used to determine creatine kinase (CK). Regenerated muscle fibers were indicated by the presence of central nucleus and also inflammation areas were determined in DIA muscle sections. The alpha and beta estrogen receptors (ER) were determined by means of immunohistochemistry evaluation in the dystrophic DIA muscle. Male mdx animals submitted to exercise showed increased CK levels and inflammatory area. The quantification of regenerated fibers was higher in male animals, submitted or not to physical exercise. Greater alpha and beta ER expression was verified in the females submitted to exercise in the DIA muscle than in the other experimental groups. Therefore, estrogen may have contributed to the prevention of increased inflammatory process and DIA injury in females submitted to exercise.

Vitamin E treatment decreases muscle injury in mdx mice.

OBJECTIVE: Oxidative stress, in addition to the absence of the dystrophin protein, has been considered an important regulator of Duchenne muscular dystrophy (DMD). Vitamin E presents an important role as a potent antioxidant and in preserving the integrity of the cell membrane. In this study, we evaluated the effects of vitamin E therapy on some physiological pathways that can contribute to muscle injury in the diaphragm muscle of mdx mice (the experimental model of DMD) such as CK levels, inflammatory response, oxidative stress, and the enzymatic antioxidant system. METHODS: Mdx mice (14 d old) received 40 mg vitamin E/kg daily by oral gavage for 14 d, followed by the removal of the diaphragm muscle. Control mdx mice and C57BL/10 mice received saline only for the same period and were used as controls. RESULTS: Vitamin E reduced the muscle fiber damage, oxidative stress, and inflammation process in the diaphragm muscle of mdx mice. CONCLUSIONS: Vitamin E improves skeletal muscle injury in mdx mice, promoting membrane repair and exhibiting antioxidant and antiinflammatory effects. These vitamin E effects suggest that this antioxidant therapy may be a relevant approach for dystrophinopathies.

Dystrophic phenotype improvement in the diaphragm muscle of mdx mice by diacerhein.

Chronic inflammation and oxidative stress are striking features of Duchenne muscular dystrophy disease. Diacerhein is an anthraquinone, which exhibits anti-inflammatory and antioxidant properties. Based on their actions, the present study evaluated the effects of diacerhein against myonecrosis, oxidative stress and inflammatory response in the diaphragm muscle of mdx mice and compared these results to current treatment widely used in DMD patients, with a main focus on the impact of prednisone. The results demonstrated that diacerhein treatment prevented muscle damage indicated by a decrease in the IgG uptake by muscle fibers, lower CK levels in serum, reduction of fibers with central nuclei with a concomitant increase in fibers with peripheral nuclei. It also had an effect on the inflammatory process, decreasing the inflammatory area, macrophage staining and TNF-α and IL-1ß content. Regarding oxidative stress, diacerhein treatment was effective in reducing the ROS and lipid peroxidation in the diaphragm muscle from mdx mice. Compared to prednisone treatment, our findings demonstrated that diacerhein treatment improved the dystrophic phenotype in the diaphragm muscle of mdx mice similar to that of glucocorticoid therapy. In this respect, this work suggests that diacerhein has a potential use as an alternative drug in dystrophinopathy treatment and recommends that its anti-inflammatory and antioxidants properties in the dystrophic muscle should be better understood.

Beneficial cilostazol therapeutic effects in mdx dystrophic skeletal muscle.

This study evaluated the possible protective effects of cilostazol against myonecrosis in dystrophic diaphragm muscle in vivo, focusing on oxidative stress, the inflammatory response and angiogenesis. Young mdx mice, the experimental animal for Duchenne muscular dystrophy, received cilostazol for 14 days. A second group of mdx mice and a control group of C57BL/10 mice received a saline solution. In the mdx mice, cilostazol treatment was associated with reduced loss of muscle strength (-34.4%), decreased myonecrosis, reduced creatine kinase levels (-63.3%) and muscle fibres stained for immunoglobulin G in dystrophic diaphragm muscle (-81.1%), and a reduced inflammatory response, with a decreased inflammatory area (-22%), macrophage infiltration (-44.9%) and nuclear factor-κB (-24%) and tumour necrosis factor-α (-48%) content in dystrophic diaphragm muscle. Furthermore, cilostazol decreased oxidative stress and attenuated reactive oxygen species production (-74%) and lipid peroxidation (-17%) in dystrophic diaphragm muscle, and promoted the up-regulation of angiogenesis, increasing the number of microvessels (15%). In conclusion, the present results show that cilostazol has beneficial effects in dystrophic muscle. More research into the potential of cilostazol as a novel therapeutic agent for the treatment of dystrophinopathies is required.

Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress.

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.