RESUMO
The study of the population of muscle satellite cells (SC) is important to understand muscle regeneration and its involvement in the different dystrophic processes. We studied two dystrophic mouse models, Largemyd and Lama2dy2j/J, that show an intense and very similar pattern of muscle degeneration, but with differences in the expression of genes involved in the regeneration cascade. They are, therefore, interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this chapter are to describe the isolation and characterization of SC populations, evaluating the presence of myogenic and pluripotent stem cells markers in normal and dystrophic muscles.
Assuntos
Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/patologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia , Animais , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distrofias Musculares/patologiaRESUMO
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.
Assuntos
Terapia com Luz de Baixa Intensidade , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Óxidos N-Cíclicos/farmacologia , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Distrofias Musculares/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Óxidos N-Cíclicos/administração & dosagem , Diafragma/metabolismo , Diafragma/fisiopatologia , Modelos Animais de Doenças , Humanos , Injeções Intraperitoneais , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Fenótipo , Marcadores de SpinRESUMO
Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Distrofia Muscular Animal/patologia , Trifosfato de Adenosina/biossíntese , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/patologia , Consumo de Oxigênio/fisiologia , Regeneração/fisiologiaRESUMO
Duchenne muscular dystrophy is the most common and severe form of progressive muscular dystrophy. Previous results showed an increased survival in double knockout mice (dko) when treated with adipose-derived CD146+ cells. In this study, we analyzed the effect of CD146+ cells compared to mesenchymal stem/stromal cells (MSCs) derived from the same human adipose sample when injected in the dko mouse model without immunosuppression. Both CD146+ cells and MSCs increased the survival of treated mice when compared to vehicle-injected mice, with a more prominent effect of CD146+ cells than MSCs. Both CD146+ cells and MSCs suppressed peripheral blood mononuclear cell proliferation, indicating immunomodulatory properties. Co-culture experiments showed that MSCs have a more inflammatory profile expression, and angiogenesis assay showed that CD146+ cells can improve blood vessel formation. CD146+ cells can extend survival of muscular dystrophy mice more efficiently than MSCs, possibly due to immunomodulatory and angiogenic properties. Further investigations focusing on exogenous CD146+ cell role in vivo will improve cell therapy understanding and effectiveness.
Assuntos
Adipócitos/citologia , Antígeno CD146/metabolismo , Modelos Animais de Doenças , Células-Tronco Mesenquimais/citologia , Distrofia Muscular Animal/terapia , Neovascularização Fisiológica , Adipócitos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologiaRESUMO
Duchenne-like muscular dystrophy (canine dystrophinopathy) is a hereditary degenerative disease characterized by muscle changes similar to those described for Duchenne muscular dystrophy (DMD) and by alterations in the smooth muscles of the gastrointestinal tract. Some authors have suggested that these abnormalities may be associated with intestinal motility. This study analyzed the nitrergic and cholinergic neurons and P2X7 receptor expression in the myenteric plexus of the ileum and distal colon of dogs with muscular dystrophy. Immunohistochemical techniques were used to detect nitric oxide synthase (NOS) and acetylcholine transferase (ChAT) expression and to label all HuC/D- and P2X7 receptor-immunoreactive (IR) neurons. Transmission electron microscopy and basic histology were performed for further analysis. The results showed that nitrergic neurons exhibited a Dogiel type I morphology in the ileum and distal colon. The neuronal profile results showed that there were fewer NOS-, ChAT-, and HuC/D-IR neurons in the ileum than in the distal colon in the dystrophic (DT) dogs. Additionally, there were more NOS-, ChAT- and HuC/D-IR neurons per ganglion in the distal colon than in the ileum. The P2X7 receptor-expressing neurons colocalized with nitrergic and cholinergic neurons. Transmission and light microscopy revealed collagen between the muscle fibers, between the circular and longitudinal muscle layers and within the myenteric ganglia of dogs with muscular dystrophy. These findings provide a morphological description of the myenteric neurons in the ileum and distal colon of these DT dogs and may contribute to a better understanding of the gastrointestinal disorders found in patients with DMD. Anat Rec, 301:673-685, 2018. © 2017 Wiley Periodicals, Inc.
Assuntos
Colo/patologia , Doenças do Cão/patologia , Íleo/patologia , Distrofia Muscular Animal/patologia , Plexo Mientérico/patologia , Animais , Colina O-Acetiltransferase/metabolismo , Colo/metabolismo , Doenças do Cão/metabolismo , Cães , Íleo/metabolismo , Distrofia Muscular Animal/metabolismo , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Óxido Nítrico Sintase/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMO
INTRODUCTION: In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to increased calcium influx and muscle necrosis. Patients suffer progressive muscle loss, and cardiomyopathy is an important determinant of morbidity. P2 purinergic receptors participate in the increased calcium levels in dystrophic skeletal muscles. METHODS: In this study, we evaluated whether P2 receptors are involved in cardiomyopathy in mdx mice at later stages of the disease. RESULTS: Western blotting revealed that P2Y2 receptor levels were upregulated (54%) in dystrophic heart compared with a normal heart. Suramin reduced the levels of P2Y2 to almost normal values. Suramin also decreased heart necrosis (reduced CK-MB) and the expression of the stretch-activated calcium channel TRPC1. CONCLUSIONS: This study suggests that P2Y2 may participate in cardiomyopathy in mdx mice. P2-selective drugs with specific actions in the dystrophic heart may ameliorate cardiomyopathy in dystrophinopathies. Muscle Nerve 55: 116-121, 2017.
Assuntos
Antinematódeos/farmacologia , Diafragma/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Miocárdio/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Suramina/farmacologia , Animais , Cálcio/metabolismo , Creatina Quinase/sangue , Diafragma/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Receptores Purinérgicos P2Y2/genética , Canais de Cátion TRPC/metabolismoRESUMO
Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.
Assuntos
Adipogenia , Biomarcadores/metabolismo , Diferenciação Celular , Tecido Conjuntivo/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miofibroblastos/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Contagem de Células , Doença Crônica , Denervação , Fibrose , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Músculo Esquelético/inervação , Distrofia Muscular Animal/patologia , Necrose , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição 4 , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Congenital muscular dystrophies 1D (CMD1D) present a mutation on the LARGE gene and are characterized by an abnormal glycosylation of α-dystroglycan (α-DG), strongly implicated as having a causative role in the development of central nervous system abnormalities such as cognitive impairment seen in patients. However, in the animal model of CMD1D, the brain involvement remains unclear. Therefore, the objective of this study is to evaluate the cognitive involvement in the Large(myd) mice. To this aim, we used adult homozygous, heterozygous, and wild-type mice. The mice underwent six behavioral tasks: habituation to an open field, step-down inhibitory avoidance, continuous multiple trials step-down inhibitory avoidance task, object recognition, elevated plus-maze, and forced swimming test. It was observed that Large(myd) individuals presented deficits on the habituation to the open field, step down inhibitory avoidance, continuous multiple-trials step-down inhibitory avoidance, object recognition, and forced swimming. This study shows the first evidence that abnormal glycosylation of α-DG may be affecting memory storage and restoring process in an animal model of CMD1D.
Assuntos
Comportamento Animal , Distrofia Muscular Animal/congênito , Distrofia Muscular Animal/patologia , Animais , Aprendizagem da Esquiva , Modelos Animais de Doenças , Habituação Psicofisiológica , Aprendizagem em Labirinto , Camundongos Knockout , Distrofia Muscular Animal/fisiopatologia , NataçãoRESUMO
In Duchenne muscle dystrophy (DMD) and in the mdx mouse model of DMD, a lack of dystrophin leads to myonecrosis and cardiorespiratory failure. Several lines of evidence suggest a detrimental role of the inflammatory process in the dystrophic process. Previously, we demonstrated that short-term therapy with eicosapentaenoic acid (EPA), at early stages of disease, ameliorated dystrophy progression in the mdx mouse. In the present study, we evaluated the effects of a long-term therapy with omega-3 later in dystrophy progression. Three-month-old mdx mice received omega-3 (300 mg/kg) or vehicle by gavage for 5 months. The quadriceps and diaphragm muscles were removed and processed for histopathology and Western blot. Long-term therapy with omega-3 increased the regulatory protein MyoD and muscle regeneration and reduced markers of inflammation (TNF-α and NF-kB) in both muscles studied. The present study supports the long-term use of omega-3 at later stages of dystrophy as a promising option to be investigated in DMD clinical trials.