RESUMEN
[Figure: see text].
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Diafragma/metabolismo , Insuficiencia Cardíaca/metabolismo , Mitocondrias Musculares/metabolismo , Debilidad Muscular/metabolismo , Calcio/metabolismo , Diafragma/fisiopatología , Diafragma/ultraestructura , Femenino , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/ultraestructura , Proteínas Musculares/metabolismo , Debilidad Muscular/etiología , Debilidad Muscular/patología , NADPH Oxidasas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Mechanical ventilation (MV) is identified as an independent contributor to diaphragmatic atrophy and contractile dysfunction. Appropriate sedation is also essential during MV, and anesthetics may have direct adverse effects on the diaphragm. However, there is a lack of research into the effects of different anesthetics on diaphragm function during MV. OBJECTIVES: In the present study, we aim to examine the effect of midazolam, dexmedetomidine, and propofol on diaphragm function during MV. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Animals were experienced 12â¯h of MV or spontaneous breathing (SB) with continuous anesthetics infusion. Diaphragm contractile properties, cross-sectional areas, microcirculation, oxidative stress, and proteolysis were examined. MEASUREMENTS AND MAIN RESULTS: Diaphragmatic specific force was markedly reduced in the midazolam group compared with the dexmedetomidine (-60.4⯱â¯3.01%, pâ¯<â¯0.001) and propofol group (-58.3⯱â¯2.60%, pâ¯<â¯0.001) after MV. MV sedated with midazolam induced more atrophy of type II fibers compared with dexmedetomidine (-21.8⯱â¯2.11%, pâ¯=â¯0.0001) and propofol (-8.2⯱â¯1.53%, pâ¯=â¯0.003). No significant differences of these indices were found in the midazolam, dexmedetomidine, and propofol groups under SB condition (all pâ¯>â¯0.05, respectively). Twelve hours of MV resulted in a time dependent reduction in diaphragmatic functional capillary density (PB -25.1%, pâ¯=â¯0.0001; MZ -21.6%, pâ¯=â¯0.0003; DD -15.2%, pâ¯=â¯0.022; PP -24.8%, pâ¯=â¯0.0001, respectively), which did not occur in the gastrocnemius muscle. The diaphragmatic lipid peroxidation adducts 4-HNE and HIF-1α levels were significantly lower in dexmedetomidine group and propofol group compared to midazolam group (pâ¯<â¯0.05, respectively). Meanwhile, the catalase and SOD levels were also relatively lower (pâ¯<â¯0.05, respectively) in midazolam group compared to dexmedetomidine group and propofol group. CONCLUSIONS: Twelve hours of mechanical ventilation during midazolam sedation led to a more severe diaphragm dysfunction than dexmedetomidine and propofol, possibly caused by its relative weaker antioxidant capacity.
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Anestesia , Dexmedetomidina/farmacología , Diafragma/fisiopatología , Midazolam/farmacología , Propofol/farmacología , Respiración Artificial , Animales , Antioxidantes/metabolismo , Autofagia/efectos de los fármacos , Fenómenos Biomecánicos , Línea Celular , Diafragma/efectos de los fármacos , Diafragma/ultraestructura , Masculino , Contracción Muscular/efectos de los fármacos , Oxidantes/metabolismo , Estrés Oxidativo/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Ratas Wistar , Signos VitalesRESUMEN
BACKGROUND: Ventilator-induced diaphragmatic dysfunction is a serious complication associated with higher ICU mortality, prolonged mechanical ventilation, and unsuccessful withdrawal from mechanical ventilation. Although neurally adjusted ventilatory assist (NAVA) could be associated with lower patient-ventilator asynchrony compared with conventional ventilation, its effects on diaphragmatic dysfunction have not yet been well elucidated. METHODS: Twenty Japanese white rabbits were randomly divided into four groups, (1) no ventilation, (2) controlled mechanical ventilation (CMV) with continuous neuromuscular blockade, (3) NAVA, and (4) pressure support ventilation (PSV). Ventilated rabbits had lung injury induced, and mechanical ventilation was continued for 12 h. Respiratory waveforms were continuously recorded, and the asynchronous events measured. Subsequently, the animals were euthanized, and diaphragm and lung tissue were removed, and stained with Hematoxylin-Eosin to evaluate the extent of lung injury. The myofiber cross-sectional area of the diaphragm was evaluated under the adenosine triphosphatase staining, sarcomere disruptions by electron microscopy, apoptotic cell numbers by the TUNEL method, and quantitative analysis of Caspase-3 mRNA expression by real-time polymerase chain reaction. RESULTS: Physiological index, respiratory parameters, and histologic lung injury were not significantly different among the CMV, NAVA, and PSV. NAVA had lower asynchronous events than PSV (median [interquartile range], NAVA, 1.1 [0-2.2], PSV, 6.8 [3.8-10.0], p = 0.023). No differences were seen in the cross-sectional areas of myofibers between NAVA and PSV, but those of Type 1, 2A, and 2B fibers were lower in CMV compared with NAVA. The area fraction of sarcomere disruptions was lower in NAVA than PSV (NAVA vs PSV; 1.6 [1.5-2.8] vs 3.6 [2.7-4.3], p < 0.001). The proportion of apoptotic cells was lower in NAVA group than in PSV (NAVA vs PSV; 3.5 [2.5-6.4] vs 12.1 [8.9-18.1], p < 0.001). There was a tendency in the decreased expression levels of Caspase-3 mRNA in NAVA groups. Asynchrony Index was a mediator in the relationship between NAVA and sarcomere disruptions. CONCLUSIONS: Preservation of spontaneous breathing using either PSV or NAVA can preserve the cross sectional area of the diaphragm to prevent atrophy. However, NAVA may be superior to PSV in preventing sarcomere injury and apoptosis of myofibrotic cells of the diaphragm, and this effect may be mediated by patient-ventilator asynchrony.
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Diafragma/lesiones , Diafragma/fisiología , Soporte Ventilatorio Interactivo/métodos , Mecánica Respiratoria/fisiología , Ventiladores Mecánicos , Animales , Diafragma/ultraestructura , Soporte Ventilatorio Interactivo/efectos adversos , Conejos , Distribución Aleatoria , Ventiladores Mecánicos/efectos adversosRESUMEN
Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.
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Diafragma/metabolismo , Endotelio/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/genética , Animales , Capilares/crecimiento & desarrollo , Capilares/metabolismo , Capilares/ultraestructura , Caveolas/metabolismo , Caveolas/ultraestructura , Diafragma/crecimiento & desarrollo , Diafragma/ultraestructura , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Endotelio/crecimiento & desarrollo , Endotelio/ultraestructura , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Hígado/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Transducción de Señal/genéticaRESUMEN
INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.
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Envejecimiento/patología , Diafragma/ultraestructura , Síndromes Miasténicos Congénitos/patología , Unión Neuromuscular/ultraestructura , Vesículas Sinápticas/ultraestructura , Acetilcolina/metabolismo , Envejecimiento/metabolismo , Animales , Diafragma/metabolismo , Diafragma/fisiopatología , Modelos Animales de Enfermedad , Endocitosis , Potenciales Postsinápticos Excitadores/fisiología , Exocitosis , Técnicas de Silenciamiento del Gen , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Placa Motora , Síndromes Miasténicos Congénitos/genética , Síndromes Miasténicos Congénitos/metabolismo , Síndromes Miasténicos Congénitos/fisiopatología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiopatología , Mecánica Respiratoria/fisiología , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/genéticaRESUMEN
Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation in local density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.
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Diafragma/ultraestructura , Matriz Extracelular/ultraestructura , Pulmón/ultraestructura , Microscopía Acústica/métodos , Andamios del Tejido , Animales , Diafragma/química , Diafragma/citología , Diseño de Equipo , Matriz Extracelular/química , Pulmón/química , Pulmón/citología , Masculino , Microscopía Acústica/instrumentación , Ratas , Ratas Wistar , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Based on the data of morphological analysis, we performed histological evaluation of rat tissue reaction to subcutaneous implantation of decellularized matrices of intrathoracic organs and tissues. Cell composition of the inflammatory infiltrate was analyzed, and the dynamics of macrophage and T and B lymphocyte content was assessed on days 7 and 14 of the experiment. It was found that the reaction to implantation depended not only on the quality of decellularization and efficiency of removal of antigen molecules, but also on the original histological structure and quality of preimplantation processing of the transplant.
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Diafragma/ultraestructura , Matriz Extracelular/ultraestructura , Pulmón/ultraestructura , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Biomarcadores/metabolismo , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacología , Desoxirribonucleasas/química , Desoxirribonucleasas/farmacología , Diafragma/citología , Diafragma/efectos de los fármacos , Diafragma/trasplante , Matriz Extracelular/química , Corazón/efectos de los fármacos , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Macrófagos/citología , Macrófagos/inmunología , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Ratas , Ratas Wistar , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Piel , Linfocitos T/citología , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
Neonates with congenital diaphragmatic hernia often require surgical defect closure with a patch. Alternatives to native diaphragmatic tissue are critically needed for this paediatric surgery. The clinical efficacy of mesh patches is limited by complications associated with residual foreign material and by hernia recurrence. In this study, we used a novel bio-3D printer method to generate large scaffold-free tissue patches composed of human cells. The resulting large tissue constructs had high elasticity and strength. Cellular patches were transplanted into rats with surgically created diaphragmatic defects. Rats survived for over 710 days after implantation of tissue constructs. CT confirmed complete tissue integration of the grafts during rat growth. Histology revealed regeneration of muscle structure, neovascularization, and neuronal networks within the reconstructed diaphragms. Our results demonstrate that created cellular patches are a highly safe and effective therapeutic strategy for repairing diaphragmatic defects, and thus pave the way for a clinical trial.
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Diafragma/fisiología , Hernias Diafragmáticas Congénitas/terapia , Regeneración , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Línea Celular , Diafragma/citología , Diafragma/ultraestructura , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Impresión Tridimensional , Ratas , Ratas Endogámicas F344RESUMEN
Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients.
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Diafragma/patología , Distrofia Muscular de Duchenne/patología , Factores de Edad , Envejecimiento/patología , Animales , Diafragma/anatomía & histología , Diafragma/ultraestructura , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía ConfocalRESUMEN
Hoxa5 is essential for development of several organs and tissues. In the respiratory system, loss of Hoxa5 function causes neonatal death due to respiratory distress. Expression of HOXA5 protein in mesenchyme of the respiratory tract and in phrenic motor neurons of the central nervous system led us to address the individual contribution of these Hoxa5 expression domains using a conditional gene targeting approach. Hoxa5 does not play a cell-autonomous role in lung epithelium, consistent with lack of HOXA5 expression in this cell layer. In contrast, ablation of Hoxa5 in mesenchyme perturbed trachea development, lung epithelial cell differentiation and lung growth. Further, deletion of Hoxa5 in motor neurons resulted in abnormal diaphragm innervation and musculature, and lung hypoplasia. It also reproduced the neonatal lethality observed in null mutants, indicating that the defective diaphragm is the main cause of impaired survival at birth. Thus, Hoxa5 possesses tissue-specific functions that differentially contribute to the morphogenesis of the respiratory tract.
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Proteínas de Homeodominio/metabolismo , Fosfoproteínas/metabolismo , Sistema Respiratorio/embriología , Sistema Respiratorio/metabolismo , Animales , Animales Recién Nacidos , Tipificación del Cuerpo/genética , Cartílago/embriología , Cartílago/metabolismo , Diferenciación Celular/genética , Cruzamientos Genéticos , Diafragma/inervación , Diafragma/metabolismo , Diafragma/ultraestructura , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genotipo , Proteínas de Homeodominio/genética , Masculino , Mesodermo/embriología , Mesodermo/metabolismo , Modelos Biológicos , Neuronas Motoras/metabolismo , Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Especificidad de Órganos/genética , Fosfoproteínas/genética , Mucosa Respiratoria/metabolismo , Factor de Transcripción SOX9/metabolismo , Transducción de Señal/genética , Análisis de Supervivencia , Tráquea/embriología , Tráquea/metabolismo , Factores de TranscripciónRESUMEN
PURPOSE:: To evaluate the effects of duodenal-jejunal bypass (DJB) on the diaphragm muscle of obese rats fed on a western diet (WD) . METHODS:: Eighteen male Wistar rats were fed a standard rodent chow diet (CTL group) or WD ad libitum. After 10 weeks, WD rats were submitted to sham (WD SHAM) or duodenal-jejunal bypass (WD DJB). The structure, ultrastructure, collagen content and the morphometry of the neuromuscular junctions (NMJs) were analyzed two months after surgery. RESULTS:: WD SHAM rats displayed an increase in body weight, the Lee index and retroperitoneal and peri-epididymal fat pads compared to the CTL group. DJB did not alter these parameters. The muscle fiber structure and NMJs were similar in the WD SHAM and CTL groups. However, the WD SHAM group showed alterations in the fiber ultrastructure, such as loosely arranged myofibrils and Z line disorganization. In addition, WD SHAM animals presented a considerable amount of lipid droplets and a reduction in the percentage of collagen compared to the CTL group. DJB did not affect the structure or ultrastructure of the muscle fibers or the NMJs in the diaphragm of the WD DJB animals. CONCLUSION:: Duodenal-jejunal bypass did not improve the alterations observed in the diaphragm of western diet obese-rats.
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Diafragma/ultraestructura , Dieta Occidental , Duodeno/cirugía , Yeyuno/cirugía , Unión Neuromuscular/ultraestructura , Obesidad/cirugía , Anastomosis Quirúrgica , Animales , Masculino , Fibras Musculares Esqueléticas/ultraestructura , Obesidad/metabolismo , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Abstract Purpose: To evaluate the effects of duodenal-jejunal bypass (DJB) on the diaphragm muscle of obese rats fed on a western diet (WD) . Methods: Eighteen male Wistar rats were fed a standard rodent chow diet (CTL group) or WD ad libitum. After 10 weeks, WD rats were submitted to sham (WD SHAM) or duodenal-jejunal bypass (WD DJB). The structure, ultrastructure, collagen content and the morphometry of the neuromuscular junctions (NMJs) were analyzed two months after surgery. Results: WD SHAM rats displayed an increase in body weight, the Lee index and retroperitoneal and peri-epididymal fat pads compared to the CTL group. DJB did not alter these parameters. The muscle fiber structure and NMJs were similar in the WD SHAM and CTL groups. However, the WD SHAM group showed alterations in the fiber ultrastructure, such as loosely arranged myofibrils and Z line disorganization. In addition, WD SHAM animals presented a considerable amount of lipid droplets and a reduction in the percentage of collagen compared to the CTL group. DJB did not affect the structure or ultrastructure of the muscle fibers or the NMJs in the diaphragm of the WD DJB animals. Conclusion: Duodenal-jejunal bypass did not improve the alterations observed in the diaphragm of western diet obese-rats.
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Animales , Masculino , Ratas , Diafragma/ultraestructura , Duodeno/cirugía , Dieta Occidental , Yeyuno/cirugía , Unión Neuromuscular/ultraestructura , Obesidad/cirugía , Anastomosis Quirúrgica , Distribución Aleatoria , Ratas Wistar , Fibras Musculares Esqueléticas/ultraestructura , Obesidad/metabolismoRESUMEN
We recently reported that skeletal muscle fibers of obscurin knockout (KO) mice present altered distribution of ankyrin B (ankB), disorganization of the subsarcolemmal microtubules, and reduced localization of dystrophin at costameres. In addition, these mice have impaired running endurance and increased exercise-induced sarcolemmal damage compared with wild-type animals. Here, we report results from a combined approach of physiological, morphological, and structural studies in which we further characterize the skeletal muscles of obscurin KO mice. A detailed examination of exercise performance, using different running protocols, revealed that the reduced endurance of obscurin KO animals on the treadmill depends on exercise intensity and age. Indeed, a mild running protocol did not evidence significant differences between control and obscurin KO mice, whereas comparison of running abilities of 2-, 6-, and 11-mo-old mice exercised at exhaustion revealed a progressive age-dependent reduction of the exercise tolerance in KO mice. Histological analysis indicated that heavy exercise induced leukocyte infiltration, fibrotic connective tissue deposition, and hypercontractures in the diaphragm of KO mice. On the same line, electron microscopy revealed that, in the diaphragm of exercised obscurin KO mice, but not in the hindlimb muscles, both M-line and H-zone of sarcomeres appeared wavy and less defined. Altogether, these results suggest that obscurin is required for the maintenance of morphological and ultrastructural integrity of skeletal muscle fibers against damage induced by intense mechanical stress and point to the diaphragm as the skeletal muscle most severely affected in obscurin-deficient mice.
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Diafragma/fisiología , Diafragma/ultraestructura , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Musculares/metabolismo , Condicionamiento Físico Animal/métodos , Sarcómeros/fisiología , Sarcómeros/ultraestructura , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Ancirinas/metabolismo , Tolerancia al Ejercicio/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Proteínas Musculares/genética , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.
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Venenos de Crotálidos/toxicidad , Diafragma/efectos de los fármacos , Fosfolipasas A2 Grupo II/toxicidad , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Nervio Frénico/efectos de los fármacos , Animales , Bothrops , Diafragma/ultraestructura , Masculino , Ratones , Nervio Frénico/ultraestructuraRESUMEN
Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.
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Diafragma/efectos de los fármacos , Oximas/uso terapéutico , Piperidinas/uso terapéutico , Respiración Artificial/efectos adversos , Animales , Diafragma/patología , Diafragma/ultraestructura , Femenino , Unidades de Cuidados Intensivos , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteómica , RatasRESUMEN
OBJECTIVE: To investigate the effects of hydrogen sulfide (H2S) on contraction capacity of diaphragm in type 1 diabetic rats.â© METHODS: Thirty-two male SD rats were randomly divided into a normal group (NC), a diabetic group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8). Intraperitoneal injection of streptozotocin was utilized to establish diabetic rat model. After the modeling, the rats in the DM+NaHS and the NaHS groups were intraperitoneally injected with 28 µmol/kg NaHS solution. 8 weeks later, the diaphragm contractility was assessed by isolated draphragm strips perfusion. The peak twitch tension (Pt), maximum tetanic tension (Po) and maximal rates of contraction/relaxation (±dT/dtmax) were determined. The alterations in diaphragm ultrastructure were observed under electron microscopy. The diaphragm weight/body weight (DW/BW) was measured. The activities of succinic dehydrogenase (SDH), lactate dehydrogenase (LDH) and sarcoplasmic reticulum Ca2+ ATPase (SERCA) were analyzed by spectrophotometric method. The mRNA levels of SERCA and prospholamban (PLB) in diaphragm were detected by RT-PCR.â© RESULTS: Compared with the NC group, there was no significant change in all measured index in the NaHS group (P>0.05), while Pt, Po and ±dT/dtmax were significantly decreased in the DM group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the diaphragm. The DW/BW ratio and the activities of SDH, LDH and SERCA were decreased. The SERCA mRNA was decreased, while PLB mRNA was increased. Compared with the DM group, the diaphragm contractility and ultrastructure damage were improved in the DM+NaHS group. The DW/BW ratio and the activities of SDH, LDH and SERCA were increased. The SERCA mRNA was increased, while PLB mRNA was decreased (all P<0.05).â© CONCLUSION: H(2)S can enhance the contraction capacity of diaphragm in type 1 diabetic rats, which is involved in regulating the activities of biological enzymes and the gene expressions of calcium regulatory proteins.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Diafragma/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Contracción Muscular/efectos de los fármacos , Animales , Peso Corporal , Diafragma/ultraestructura , L-Lactato Deshidrogenasa/metabolismo , Masculino , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Succinato Deshidrogenasa/metabolismo , Sulfuros/farmacologíaRESUMEN
Mechanical ventilation (MV) with hyperoxia is required for providing life support to patients with acute lung injury (ALI). However, MV may cause diaphragm weakness through muscle injury and atrophy, an effect termed ventilator-induced diaphragm dysfunction (VIDD). Src protein tyrosine kinase and class O of forkhead box 1 (FoxO1) mediate acute inflammatory responses and muscle protein degradation induced by oxidative stress. Induced pluripotent stem cells (iPSCs) have been reported to improve hyperoxia-augmented ALI; however, the mechanisms regulating the interactions among VIDD, hyperoxia, and iPSCs are unclear. In this study, we hypothesized that iPSC therapy can ameliorate hyperoxia-augmented VIDD by suppressing the Src-FoxO1 pathway. Male C57BL/6 mice, either wild-type or Src-deficient, aged between 6 and 8 weeks were exposed to MV (6 or 10 mL/kg) with or without hyperoxia for 2-8 h after the administration of 5 × 10(7) cells/kg Oct4/Sox2/Parp1 mouse iPSCs or iPSC-derived conditioned medium (iPSC-CM). Nonventilated mice were used as controls. MV during hyperoxia aggravated VIDD, as demonstrated by the increases in Src activation, FoxO1 dephosphorylation, malondialdehyde, caspase-3, atrogin-1 and muscle ring finger-1 production, microtubule-associated protein light chain 3-II, disorganized myofibrils, disrupted mitochondria, autophagy, and myonuclear apoptosis; however, MV with hyperoxia reduced mitochondrial cytochrome C, diaphragm muscle fiber size, and contractility (P < 0.05). Hyperoxia-exacerbated VIDD was attenuated in Src-deficient mice and by iPSCs and iPSC-CM (P < 0.05). Our data indicate that iPSC therapy attenuates MV-induced diaphragmatic injury that occurs during hyperoxia-augmented VIDD by inhibiting the Src-FoxO1 signaling pathway.
Asunto(s)
Diafragma/fisiopatología , Proteína Forkhead Box O1/antagonistas & inhibidores , Hiperoxia/metabolismo , Células Madre Pluripotentes Inducidas/citología , Transducción de Señal , Trasplante de Células Madre , Ventiladores Mecánicos/efectos adversos , Familia-src Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Medios de Cultivo Condicionados/farmacología , Diafragma/metabolismo , Diafragma/patología , Diafragma/ultraestructura , Proteína Forkhead Box O1/metabolismo , Heterocigoto , Hiperoxia/complicaciones , Hiperoxia/tratamiento farmacológico , Hiperoxia/patología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Proteínas Musculares/metabolismo , Músculos/metabolismo , Músculos/patología , Músculos/ultraestructura , Estrés Oxidativo/efectos de los fármacos , Quinolonas/farmacología , Quinolonas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Mutations in peripheral myelin protein 22 (PMP22) result in the most common form of Charcot-Marie-Tooth (CMT) disease, CMT1A. This hereditary peripheral neuropathy is characterized by dysmyelination of peripheral nerves, reduced nerve conduction velocity, and muscle weakness. APMP22 point mutation in L16P (leucine 16 to proline) underlies a form of human CMT1A as well as the Trembler-J mouse model of CMT1A. Homozygote Trembler-J mice (Tr(J)) die early postnatally, fail to make peripheral myelin, and, therefore, are more similar to patients with congenital hypomyelinating neuropathy than those with CMT1A. Because recent studies of inherited neuropathies in humans and mice have demonstrated that dysfunction and degeneration of neuromuscular synapses or junctions (NMJs) often precede impairments in axonal conduction, we examined the structure and function of NMJs in Tr(J)mice. Although synapses appeared to be normally innervated even in end-stage Tr(J)mice, the growth and maturation of the NMJs were altered. In addition, the amplitudes of nerve-evoked muscle endplate potentials were reduced and there was transmission failure during sustained nerve stimulation. These results suggest that the severe congenital hypomyelinating neuropathy that characterizes Tr(J)mice results in structural and functional deficits of the developing NMJ.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Proteínas de la Mielina/genética , Enfermedades de la Unión Neuromuscular/etiología , Enfermedades de la Unión Neuromuscular/patología , Animales , Animales Recién Nacidos , Diafragma/patología , Diafragma/ultraestructura , Estimulación Eléctrica , Potenciales Evocados/genética , Homocigoto , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microscopía Electrónica , Conducción Nerviosa/genética , Unión Neuromuscular/patología , Unión Neuromuscular/ultraestructura , Enfermedades de la Unión Neuromuscular/genética , Mutación Puntual/genéticaRESUMEN
BACKGROUND: Mechanical ventilation (MV) is associated with atrophy and weakness of the diaphragm muscle, a condition termed ventilator-induced diaphragmatic dysfunction (VIDD). Autophagy is a lysosomally mediated proteolytic process that can be activated by oxidative stress, which has the potential to either mitigate or exacerbate VIDD. The primary goals of this study were to (1) determine the effects of MV on autophagy in the diaphragm and (2) evaluate the impact of antioxidant therapy on autophagy induction and MV-induced diaphragmatic weakness. METHODS: Mice were assigned to control (CTRL), MV (for 6 h), MV + N-acetylcysteine, MV + rapamycin, and prolonged (48 h) fasting groups. Autophagy was monitored by quantifying (1) autophagic vesicles by transmission electron microscopy, (2) messenger RNA levels of autophagy-related genes, and (3) the autophagosome marker protein LC3B-II, with and without administration of colchicine to calculate the indices of relative autophagosome formation and degradation. Force production by mouse diaphragms was determined ex vivo. RESULTS: Diaphragms exhibited a 2.2-fold (95% CI, 1.8 to 2.5) increase in autophagic vesicles visualized by transmission electron microscopy relative to CTRL after 6 h of MV (n = 5 per group). The autophagosome formation index increased in the diaphragm alone (1.5-fold; 95% CI, 1.3 to 1.8; n = 8 per group) during MV, whereas prolonged fasting induced autophagosome formation in both the diaphragm (2.5-fold; 95% CI, 2.2 to 2.8) and the limb muscle (4.1-fold; 95% CI, 1.8 to 6.5). The antioxidant N-acetylcysteine further augmented the autophagosome formation in the diaphragm during MV (1.4-fold; 95% CI, 1.2 to 1.5; n = 8 per group) and prevented MV-induced diaphragmatic weakness. Treatment with the autophagy-inducing agent rapamycin also largely prevented the diaphragmatic force loss associated with MV (n = 6 per group). CONCLUSIONS: In this model of VIDD, autophagy is induced by MV but is not responsible for diaphragmatic weakness. The authors propose that autophagy may instead be a beneficial adaptive response that can potentially be exploited for therapy of VIDD.
Asunto(s)
Autofagia , Diafragma/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Animales , Antioxidantes/farmacología , Atrofia , Autofagia/genética , Cistina/análogos & derivados , Cistina/farmacología , Diafragma/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Fagosomas/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils.