LEDT and Idebenone treatment modulate autophagy and improve regenerative capacity in the dystrophic muscle through an AMPK-pathway.

PURPOSE: Considering the difficulties and challenges in Duchenne muscular dystrophy (DMD) treatment, such as the adverse effects of glucocorticoids, which are the main medical prescription used by dystrophic patients, new treatment concepts for dystrophic therapy are very necessary. Thus, in this study, we explore the effects of photobiomodulation (PBM; a non-invasive therapy) and Idebenone (IDE) treatment (a potent antioxidant), applied alone or in association, in dystrophic muscle cells and the quadriceps muscle, with special focus on autophagy and regenerative pathways. METHODS: For the in vitro studies, the dystrophic primary muscle cells received 0.5J LEDT and 0.06µM IDE; and for the in vivo studies, the dystrophic quadriceps muscle received 3J LEDT and the mdx mice were treated with 200mg/kg IDE. RESULTS: LEDT and IDE treatment modulate autophagy by increasing autophagy markers (SQSTM1/p62, Beclin and Parkin) and signaling pathways (AMPK and TGF-ß). Concomitantly, the treatments prevented muscle degeneration by reducing the number of IgG-positive fibers and the fibers with a central nucleus; decreasing the fibrotic area; up-regulating the myogenin and MCH-slow levels; and down-regulating the MyoD and MHC-fast levels. CONCLUSION: These results suggest that LEDT and IDE treatments enhance autophagy and prevented muscle degeneration in the dystrophic muscle of the experimental model. These findings illustrate the potential efficacy of LEDT and IDE treatment as an alternative therapy focused on muscle recovery in the dystrophic patient.

Low-Level Photobiomodulation Therapy Modulates H2O2 Production, TRPC-6, and PGC-1α Levels in the Dystrophic Muscle.

Objective: This study evaluated photobiomodulation therapy (PBMT) effects on the factors involved in mitochondrial biogenesis, on the mitochondrial respiratory complexes, and on the transient receptor potential canonical channels (such as TRPC-1 and TRPC-6) in in vitro (mdx muscle cells) and in vivo studies (gastrocnemius muscle) from mdx mice, the dystrophin-deficient model of Duchenne muscular dystrophy (DMD). Background: There is no successful treatment for DMD, therefore demanding search for new therapies that can improve the muscle role, the quality of life, and the survival of dystrophic patients. Methods: The dystrophic primary muscle cells received PBMT at 0.6 J and 5 J, and the dystrophic gastrocnemius muscle received PBMT at 0.6 J. Results: The dystrophic muscle cells treated with PBMT (0.6 J and 5 J) showed no cytotoxicity and significantly lower levels in hydrogen peroxide (H2O2) production. We also demonstrated, for the first time, the capacity of PBMT, at a low dose (0.6 J), in reducing the TRPC-6 content and in raising the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) content in the dystrophic gastrocnemius muscle. Conclusions: PBMT modulates H2O2 production, TRPC-6, and PGC-1α content in the dystrophic muscle. These results suggest that laser therapy could act as an auxiliary therapy in the treatment of dystrophic patients.

Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy.

Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 µg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H2O2 levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-κB levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1α and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway.

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.

Multiple LEDT wavelengths modulate the Akt signaling pathways and attenuate pathological events in mdx dystrophic muscle cells.

This study is aimed at investigating the effects of LEDT, at multiple wavelengths, on intracellular calcium concentration; on transient receptor potential canonical channels; on calcium-binding protein; on myogenic factors; on myosin heavy chains; on Akt signaling pathway; on inflammatory markers; and on the angiogenic-inducing factor in dystrophic muscle cell culture experimental model. Dystrophic primary muscle cells were submitted to LEDT, at multiple wavelengths (420 nm, 470 nm, 660 nm, and 850 nm), and evaluated after 48 h for cytotoxic effects and intracellular calcium content. TRPC-1, TRPC-6, Calsequestrin, MyoD, Myogenin, MHC-slow, MHC-fast, p-AKT, p-mTOR, p-FoxO1, Myostatin, NF-κB, TNF-α, and VEGF levels were evaluated in dystrophic primary muscle cells by western blotting. The LEDT, at multiple wavelengths, treated-mdx muscle cells showed no cytotoxic effect and significant lower levels in [Ca2 +]i. The mdx muscle cells treated with LEDT showed a significant reduction of TRPC-1, NF-κB, TNF-α and MyoD levels and a significant increase of Myogenin, MHC-slow, p-AKT, p-mTOR, p-FoxO1 levels, and VEGF levels. Our findings suggest that different LEDT wavelengths modulate the Akt-signaling pathways and attenuate pathological events in dystrophic muscle cells, and a combined multiwavelength irradiation protocol may even provide a potentially therapeutic strategy for muscular dystrophies.

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.

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.

Morphofunctional characteristics of skeletal muscle in rats with cerebral palsy.

Knowledge of skeletal muscle adaptations is important to understand the functional deficits in cerebral palsy (CP). This study aimed to investigate the morphofunctional characteristics of skeletal muscle in a CP animal model. Initially, pregnant Wistar rats were injected intraperitoneally with saline or lipopolysaccharide over the last five days of pregnancy. The control group (n = 8) consisted of male pups born to females injected with saline. The CP group (n = 8) consisted of male pups born to females injected with lipopolysaccharide, which were submitted to perinatal anoxia [day of birth, postnatal day 0 (P0)] and sensorimotor restriction (P1-P30). The open-field test was undertaken on P29 and P45. On P48, the animals were weighed, and the plantaris muscle was collected and its weight and length were measured. Transverse sections were stained with haematoxylin-eosin, NADH-TR, Masson's trichrome and non-specific esterase reaction for analysis. and transmission electron microscopy was performed. In the CP group, reductions were observed in mobility time, number of crossings and rearing frequency, body weight, muscle weight and length, and nucleus-to-fibre and capillary-to-fibre ratios. There was a statistically significant increase in the percentage area of the muscle section occupied by collagen; reduction in the area and increase in the number of type I muscle fibres; increase in myofibrillar disorganization and Z-line disorganization and dissolution; and reduction in the area and largest and smallest diameters of neuromuscular junctions. Thus this animal model of CP produced morphofunctional alterations in skeletal muscle, that were associated with evidence of motor deficits as demonstrated by the open-field test.

Delirium during Weaning from Mechanical Ventilation.

Background. We compare the incidence of delirium before and after extubation and identify the risk factors and possible predictors for the occurrence of delirium in this group of patients. Methods. Patients weaned from mechanical ventilation (MV) and extubated were included. The assessment of delirium was conducted using the confusion assessment method for the ICU and completed twice per day until discharge from the intensive care unit. Results. Sixty-four patients were included in the study, 53.1% of whom presented with delirium. The risk factors of delirium were age (P = 0.01), SOFA score (P = 0.03), APACHE score (P = 0.01), and a neurological cause of admission (P = 0.01). The majority of the patients began with delirium before or on the day of extubation. Hypoactive delirium was the most common form. Conclusion. Acute (traumatic or medical) neurological injuries were important risk factors in the development of delirium. During the weaning process, delirium developed predominantly before or on the same day of extubation and was generally hypoactive (more difficult to detect). Therefore, while planning early prevention strategies, attention must be focused on neurological patients who are receiving MV and possibly even on patients who are still under sedation.