Immunophenotyping lymphocyte and acute phase proteins in canine X-linked muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is the most common X-linked muscular disease affecting humans. The Golden Retriever Muscular Dystrophy model (GRMD) is considerthe most suitable for several studies. This assay aims to quantify lymphocyte subpopulations CD4, CD5, and CD8, and standardize, the serum electrophoretic profile, to understand their contribution to the pathologic process in normal Golden Retriever dogs (GR group) and dystrophic´s (GRMD group), through the umbilical cord blood, in dogs aged from 2 to 3 months (GR II and GRMD II), and in dogs over 1 year of age (GR III and GRMD III). No significant differences were observed between the CD8+ lymphocyte subpopulations of the groups studied. The CD4+ and CD5+ lymphocyte subpopulations were significantly higher in the GRMD III group compared to the GR III group. Twenty-two different proteins in the gel were identified. The serum concentrations of the proteins belonging to the GR I and GRMD I groups were significantly lower than those of the other groups. We show that expression of acute phase proteins are worst during the aging of the dogs. We hope to expand knowledge to better understand the GRMD model and the translational data.

An improved method for studying mouse diaphragm function.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

Non-invasive evaluation of muscle disease in the canine model of Duchenne muscular dystrophy by electrical impedance myography.

Dystrophin-deficient dogs are by far the best available large animal models for Duchenne muscular dystrophy (DMD), the most common lethal childhood muscle degenerative disease. The use of the canine DMD model in basic disease mechanism research and translational studies will be greatly enhanced with the development of reliable outcome measures. Electrical impedance myography (EIM) is a non-invasive painless procedure that provides quantitative data relating to muscle composition and histology. EIM has been extensively used in neuromuscular disease research in both human patients and rodent models. Recent studies suggest that EIM may represent a highly reliable and convenient outcome measure in DMD patients and the mdx mouse model of DMD. To determine whether EIM can be used as a biomarker of disease severity in the canine model, we performed the assay in fourteen young (~6.6-m-old; 6 normal and 8 affected) and ten mature (~16.9-m-old; 4 normal and 6 affected) dogs of mixed background breeds. EIM was well tolerated with good inter-rater reliability. Affected dogs showed higher resistance, lower reactance and phase. The difference became more straightforward in mature dogs. Importantly, we observed a statistically significant correlation between the EIM data and muscle fibrosis. Our results suggest that EIM is a valuable objective measurement in the canine DMD model.

Arquitetura comparativa dos pulmões de camundongos normais e afetados pela Distrofia Muscular de Duchenne / Comparative lung architecture of normal and by Duchenne Muscular Dystrophy affected mice

A Distrofia Muscular de Duchenne (DMD) é uma doença genética de caráter recessivo que caracterizada por fraqueza muscular progressiva de cintura pélvica e escapular evoluindo para insuficiência respiratória e, ou cardíaca. O camundongo mdx é um modelo amplamente utilizado para estudos da DMD. Apesar do fenótipo destes animais serem mais suave, estes apresentam o principal músculo respiratório, o diafragma com morfologia e bioquímica semelhante à DMD humana, fato este que pode comprometer a função respiratória e consequentemente os pulmões. Foi realizado um estudo anatômico descritivo do parênquima pulmonar dos pulmões de 5 animais modelo mdx comparando estes com os pulmões de 5 camundongos BALB/C57 (Mus musculus). Os pulmões foram analisados macroscopicamente e através de microscopia de luz e eletrônica de varredura. Os achados sugerem que o modelo mdx apresenta morfologia pulmonar semelhante aos camundongos BALB/C57 e que seu uso deve ser cauteloso e criterioso em ensaios clínicos que aborde este órgão.(AU)

The Duchenne Muscular Dystrophy (DMD) is a recessive genetic disease characterized by progressive muscle weakness of the pelvic and scapular girdle and progressing to respiratory or heart failure. The mdx mouse is a model widely used for studies. Although they possess a milder phenotype, the morphology and biochemistry of the diaphragm are similar to human DMD. We performed a descriptive anatomical study of the pulmonary parenchyma of five mdx animal models and compared these with the lungs of 5 mice BALB/C57 (Mus musculus). The findings suggest that the mdx model has morphological features similar to BALB/C57 mice and it must be used with caution in clinical trials which involve the lung.(AU)

Descrição morfológica do diafragma do sagui-de-tufo-branco (Callithrix jacchus) / Morphological description of diaphragm of white-tufted-ear-marmoset (Callithrix jacchus)

O músculo diafragma, encontrado apenas nos mamíferos, é o principal músculo no processo respiratório, servindo de fronteira entre as cavidades torácica e abdominal. Sua importância também ganha destaque em pesquisas realizadas no âmbito dos enxertos, empregando-se diversos tipos de membranas biológicas para o reparo de defeitos diafragmáticos, os quais podem gerar hérnias diafragmáticas. Apesar de muitos estudos já conduzidos para com os primatas não humanos, especialmente no que tange a espécie do novo mundo Callithrix jacchus (Sagui-de-tufo-branco), oriundo do nordeste brasileiro, as pesquisas envolvendo o uso do diafragma em tal espécie é inexistente. Deste modo objetivou-se caracterizar a morfologia e a biometria do diafragma na espécie Callithrix jacchus de ambos os sexos, analisando possíveis divergências estruturais entre machos e fêmeas. Para tal foram utilizados quatros animais, 2 machos e 2 fêmeas, adultos, que vieram a óbito por causas naturais, provenientes de um criadouro comercial. Após fixação em solução de formaldeído 10% os animais foram devidamente dissecados para fotodocumentação e em seguida o diafragma coletado para efetuação da biometria (comprimento e largura) com o uso de um paquímetro e para o processamento histológico por meio da coloração de hematoxilina-eosina e tricrômio de masson, da porção muscular. As mensurações feitas permitiram concluir que não houve diferenças signifcativas entre machos e femeas. A topografia e a presença de três aberturas (forame da veia cava caudal, hiato aórtico e esofágico) na extensão do diafragma corroboram com descrições na literatura classica para outros mamíferos. A presença de um centro tendíneo em "V" difere do encontrado para animais como o peixe-boi e porquinho-da-india, mas é similar ao encontrado para o gambá-de-orelhas-brancas e rato albino. No que diz respeito aos achados histológicos conclui-se que as fibras musculares estão dispostas de forma organizada, apresentam diâmetro grande e núcleos basais, tendo, portanto, características similares do músculo estriado esquelético tanto nos animais machos como nas fêmeas.

The diaphragm muscle found only in mammals is the main muscle in the respiratory process, serving as the border between the thoracic and abdominal cavities. Its significance also is highlighted in research conducted with grafts using various types of biological membranes for the repair of diaphragmatic defects which may cause diaphragmatic hernias. In spite of many studies already conducted in non-human primates, especially in regard to the new world species Callithrix jacchus (white-tufted-ear-marmoset) from northeastern Brazil, research involving use of the diaphragm in such species is nonexistent. Thus, the objective was to characterize the morphology and biometry of the diaphragm in C. jacchus of both sexes, analyzing possible structural differences between males and females. To this end, we used four adult white-tufted-ear-marmosets from a commercial breeder, two males and two females that had died of natural causes. After fixed in 10% formaldehyde solution, the animals were dissected for photo documentation, the diaphragm was collected for biometrics (length and width) with a caliper, and for histological processing by hematoxylin-eosin and Trichrome Masson. The measurements showed that there were no significant differences between males and females. The topography and the presence of three openings (foramen vena cava, aortic et esophageal hiatus) in the extension of the diaphragm corroborate descriptions in classical literature for other mammals. Regarding the histological findings, it was concluded that the muscular fibers are arranged in an organized shape, with a large diameter and basal nuclei, and showed similar characteristics of skeletal muscle in males and females.