Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS.

Golden retriever muscular dystrophy (GRMD) provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD). The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR) versus GRMD-gene carrier (CaGR) and affected female dogs (AfCR). Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

Kinematic gait analyses in healthy Golden Retrievers / Análise cinemática da marcha de cães Golden Retriever saudáveis

Kinematic analysis relates to the relative movement between rigid bodies and finds application in gait analysis and other body movements, interpretation of their data when there is change, determines the choice of treatment to be instituted. The objective of this study was to standardize the march of Dog Golden Retriever Healthy to assist in the diagnosis and treatment of musculoskeletal disorders. We used a kinematic analysis system to analyse the gait of seven dogs Golden Retriever, female, aged between 2 and 4 years, weighing 21.5 to 28 kg, clinically normal. Flexion and extension were described for shoulder, elbow, carpal, hip, femorotibialis and tarsal joints. The gait was characterized lateral and had accepted hypothesis of normality for all variables, except for the stance of hip and elbow, considering a confidence level of 95%, significance level α = 0.05. Variations have been attributed to displacement of the stripes during movement and the duplicated number of reviews. The kinematic analysis proved to be a consistent method of evaluation of the movement during canine gait and the data can be used in the diagnosis and evaluation of canine gait in comparison to other studies and treatment of dogs with musculoskeletal disorders.

A análise cinemática relaciona-se com o movimento relativo entre corpos rígidos e encontra aplicação na análise da marcha e de outros movimentos do corpo. A interpretação de seus dados, quando há alteração, determina a escolha do tratamento a ser instituído. O objetivo deste estudo foi padronizar a marcha do cão Golden Retriever saudável visando auxiliar no diagnóstico e tratamento de afecções músculo esquelética. Neste estudo utilizou-se um sistema de análise cinemática para analisar a marcha de sete cães da raça Golden Retriever, fêmeas, idade entre 2 e 4 anos, peso variando de 21.5 a 28 kg, clinicamente sadias. Dados morfométricos foram coletados para descrever a população estudada. Variáveis de tempo e distâncias foram mensuradas para descrever a marcha, movimentos de flexão e extensão foram descritos para as articulações do ombro, cubital, cárpica, do quadril, femorotibial e társica. A marcha foi caracterizada lateral e teve hipótese de normalidade aceita para todas as variáveis, exceto para o apoio de quadril e apoio de cúbito, considerando um grau de confiança de 95%, ou seja, nível de significância α = 0.05. As variações foram atribuídas ao deslocamento das tarjas durante o movimento e ao repetido número de avaliações. A análise cinemática provou ser um consistente método de avaliação do movimento durante a marcha canina e os dados obtidos podem ser utilizados no diagnóstico e na comparação em avaliações de marcha para outros estudos e tratamento de cães com afecções musculoesqueléticas.

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord.

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.

Existem diferenças nos parâmetros hematológicos e bioquímicos séricos entre fêmeas normais e portadoras do modelo experimental GRMD (golden retriever muscular dystrophy)? / There are differences in hematological and biochemical parameters between carriers and not carriers of experimental model of GRMD (golden retriever muscular dystrophy)?

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.

The purpose of this study was to evaluate whether there are alterations in hematological and biochemical patterns of female Golden Retriever dogs carrying the gene for progressive muscular dystrophy compared to not carriers dogs of the same breed, age and gender. We analyzed 33 animals, divided into two groups; one consisting of 13 not carriers dogs Golden Retrievers (GRNP) and the other composed of 14 dogs Golden Retrievers carrying the gene for muscular dystrophy (GRP). Animals of both groups underwent biochemical and hematological tests with the same frequency and at the same time interval. Although there is a statistically significant difference between groups for some hematological parameters evaluated, all results were in line with the benchmarks used. In the assessment of serum biochemical parameters in the determination of ALT GRS group was slightly above average, but without major clinical significance CK also showed high levels in GRP group, due to muscle degeneration and necrosis characteristic of the disease, the changes found in this analysis were already expected. The other parameters did not change.

Alterações do trato digestório de cães da raça Golden Retriever afetados pela distrofia muscular / Changes of the digestive tract of Golden Retriever dogs affected by muscular dystrophy

O modelo experimental canino Golden Retriever portador da Distrofia Muscular (GRMD) é o melhor substituto entre os modelos animais para estudar a Distrofia Muscular de Duchenne. Além da musculatura estriada, a doença pode afetar a musculatura estriada cardíaca e a musculatura lisa, e desta forma, o funcionamento do trato digestório, já que o músculo liso é o elemento primário dos órgãos tubulares. Através de estudo morfológico descritivo, o objetivo deste trabalho foi verificar se a distrofia muscular afeta a arquitetura geral do trato digestório e como se dispõe sua estrutura muscular em animais afetados. Foram realizadas avaliações descritivas macro e microscópicas com colorações de Hematoxilina-Eosina, Tricrômio de Masson e Picrosirius. Entre os resultados apresentados, verificou-se que o esôfago e o fígado dos animais afetados encontraram-se alterados, assim como o estômago não ocupava seu lugar habitual. O músculo diafragma apresentava-se atrofiado e diferenças histológicas foram encontradas na camada muscular do sistema gastrointestinal, em geral. Outras estruturas do tubo digestório de GRMDs apresentaram-se de maneira similar a de um animal normal.

The experimental canine model Golden Retriever carrier of Muscular Dystrophy (GRMD) is the best substitute of animal models to study Duchenne Muscular Dystrophy. Above striated muscle, the disease can affect the heart and smooth muscle, so the functioning of the digestive tract, as the smooth muscle is the primary element of tubular organs. Through morphological description, the purpose of this study was to determine whether the muscular dystrophy affects the overall architecture of the digestive tract and how is willing this muscular structure. Were evaluated macroscopic and microscopic optical description staining with hematoxylin-eosin, Masson's trichrome and Sirius. The esophagus and liver of affected animals were altered. The stomach of the animals did not occupy the usual space. The diaphragm muscle had atrophied. The general histological structure of the digestive tract presented in a manner similar to a normal animal. Changes and histological differences were found in the muscle layer.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

BACKGROUND: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. METHODS: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. RESULTS AND DISCUSSION: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. CONCLUSION: Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.