Thais Borges Lessa

Diaphragm: A vital respiratory muscle in mammals

The diaphragm is a respiratory muscle that is primarily responsible for the respiratory function in normal individuals. In mammals, the diaphragm muscle has been studied from the early days of zoology, comparative and experimental anatomy, physiology, medicine, physics, and philosophy. However, even with these early advances in knowledge pertaining to the diaphragm, comprehensive morphological data on the diaphragm are still incomplete. In this review, we summarize the beginnings of the morphological description of the diaphragm, and we describe the current status of the known morphological and embryological features. In addition, we correlate how the impairment of the diaphragm muscle in Duchenne muscular dystrophy (DMD) can lead to patient deaths. DMD is the most common X-linked muscle degenerative disease and is caused by a lack of dystrophin protein. Dystrophin is an important muscle protein that links the cellular cytoskeleton with the extracellular matrix. In the absence of dystrophin, the muscle becomes susceptible to damage during muscle contraction. This review allows researchers to obtain an overview of the diaphragm, transcending the morphological data from animals described in conventional literature.

Morphological and ultrastructural evaluation of the golden retriever muscular dystrophy trachea, lungs, and diaphragm muscle.

Duchenne muscular dystrophy (DMD) is a genetic disease, characterized by atrophy and muscle weakness. The respiratory failure is a common cause of early death in patients with DMD. Golden retriever muscular dystrophy (GRMD) is a canine model which has been extensively used for many advances in therapeutics applications. As the patients with DMD, the GRMD frequently died from cardiac and respiratory failure. Observing the respiratory failure in DMD is one of the major causes of mortality we aimed to describe the morphological and ultrastructural data of trachea, lungs (conductive and respiratory portion of the system), and diaphragm muscle using histological and ultrastructural analysis. The diaphragm muscle showed discontinuous fibers architecture, with different diameter; a robust perimysium inflammatory infiltrate and some muscle cells displayed central nuclei. GRMD trachea and lungs presented collagen fibers and in addition, the GRMD lungs showed higher of levels collagen fibers that could limit the alveolar ducts and alveoli distension. Therefore, the most features observed were the collagen areas and fibrosis. We suggested in this study that the collagen remodeling in the trachea, lungs, and diaphragm muscle may increase fibrosis and affect the trachea, lungs, and diaphragm muscle function that can be a major cause of respiratory failure that occur in patients with DMD. Microsc. Res. Tech. 77:857–861, 2014.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

BackgroundThe diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.MethodsA total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.ResultsBoth the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.ConclusionWe were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

Mice embryology: a microscopic overview.

In this work, we studied the embryology of mice of 12, 14, and 18 days of gestation by gross observation, light microscopy, and scanning electron microscopy. Grossly, the embryos of 12 days were observed in C‐shaped region of the brain, eye pigmentation of the retina, first, second, and third pharyngeal arches gill pit nasal region on the fourth ventricle brain, cervical curvature, heart, liver, limb bud thoracic, spinal cord, tail, umbilical cord, and place of the mesonephric ridge. Microscopically, the liver, cardiovascular system and spinal cord were observed. In the embryo of 14 days, we observed structures that make up the liver and heart. At 18 days of gestation fetuses, it was noted the presence of eyes, mouth, and nose in the cephalic region, chest and pelvic region with the presence of well‐developed limbs, umbilical cord, and placenta. Scanning electron microscopy in 18 days of gestation fetuses evidenced head, eyes closed eyelids, nose, vibrissae, forelimb, heart, lung, kidney, liver, small bowel, diaphragm, and part of the spine. The results obtained in this work describe the internal and external morphology of mice, provided by an integration of techniques and review of the morphological knowledge of the embryonic development of this species, as this animal is of great importance to scientific studies. Microsc. Res. Tech. 2012.

Descrição morfológica do diafragma do sagui-de-tufo-branco (Callithrix jacchus)

ABSTRACT.- Lessa T.B., Constantino M.V.P., Silva L.C.S., Santos S.R.P., Assis Neto A.C., Miglino M.A, Bombonato P.P. & Ambrosio C.E. 2012. [ Morphological description of diaphragm of white-tufted-ear-marmoset ( Callithrix jacchus ). ] Descricao morfologica do diafragma do sagui-de-tufo-branco ( Callithrix jacchus ). Pesquisa Veterinaria Brasileira 32(6):553-558. Departamento de Cirurgia, Programa de Pos-Graduacao em Anatomia dos Animais Domes-ticos e Silvestres, Faculdade de Medicina Veterinaria, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508 270, Brazil. E-mail: thaisblessa@usp.brThe diaphragm muscle found only in mammals is the main muscle in the respiratory pro-cess, serving as the border between the thoracic and abdominal cavities. Its signiicance 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

Arquitetura comparativa dos pulmões de camundongos normais e afetados pela Distrofia Muscular de Duchenne

A Distrofia Muscular de Duchenne (DMD) e uma doenca genetica de carater recessivo que caracterizada por fraqueza muscular progressiva de cintura pelvica e escapular evoluindo para insuficiencia respiratoria e, ou cardiaca. O camundongo mdx e um modelo amplamente utilizado para estudos da DMD. Apesar do fenotipo destes animais serem mais suave, estes apresentam o principal musculo respiratorio, o diafragma com morfologia e bioquimica semelhante a DMD humana, fato este que pode comprometer a funcao respiratoria e consequentemente os pulmoes. Foi realizado um estudo anatomico descritivo do parenquima pulmonar dos pulmoes de 5 animais modelo mdx comparando estes com os pulmoes de 5 camundongos BALB/C57 (Mus musculus). Os pulmoes foram analisados macroscopicamente e atraves de microscopia de luz e eletronica 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 clinicos que aborde este orgao.

Laparoscopic guided local injection in the X-linked muscular dystrophy mouse (mdx) diaphragm. An advance in experimental therapies for Duchenne Muscular Dystrophy

PURPOSE To investigate the development of a laparoscopy technique for local injection into the X-linked muscular dystrophy (mdx) diaphragm. METHODS It was used 10 mice Balb/C57 and 5 mdx mice and three differents decubitus type were tested: the right lateral, supine, and supine decubitus with 20 degrees elevation of the forelimb. Abdominal caudal face and the 10 intercostal space were tested as spot to introduce the needle into the diaphragm. RESULTS Supine position with elevation of 20 degrees forelimb and the 10th intercostal space are the beneficial position to apply a local injection. CONCLUSION It was proved to be possible to perform the laparoscopy technique in the X-linked muscular dystrophy diaphragm and this requires a specific position and technique during the surgery.

Diaphragm morphology of guinea pig (Cavia porcellus).

The diaphragm is the main respiratory muscle. Along with other respiratory muscles, the diaphragm is responsible for the muscular contraction that generates the respiratory cycle and, as a consequence, the gaseous interchanges in the lungs. Guinea pigs (Cavia porcellus Linnaeus 1758) are largely used as experimental animals in many biology applications due to their easy management, low cost, and docile behavior. As the diaphragm exerts important effects on lung physiology and function, this study aimed at investigating the morphological characteristics of the muscle, through macroscopic, microscopic, and scanning electron microscopy to add reference data for future studies. We observed a “U”‐shaped tendineous center and its morphology was similar to other mammals. These results cooperate with the descriptive and comparative anatomy of mammals, besides can be used as control data for areas of surgery and stem cells. Microsc. Res. Tech. 76:316–320, 2013.

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.

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.