Thaís Peixoto Gaiad

Ringo, a Golden Retriever Muscular Dystrophy (GRMD) dog with absent dystrophin but normal strength

The closest model to human Duchenne Muscular Dystrophy (DMD) is the Golden Retriever Muscular Dystrophy (GRMD) dog, which carries a point mutation in the splice acceptor site in intron 6 of the orthologe X-linked dystrophin gene, leading to the absence of protein in the muscles. These dogs present clinical signs within the first weeks of life involving the limbs as well as masticatory muscles. Diaphragmatic and intercostal muscles impairment leads to progressive respiratory failure. Death occurs from bronchopneumonia and cardiac arrest, usually before 2 years of age. Here, we report the case of Ringo, an exceptional GRMD dog showing an unusually mild course. Currently, at age 4 years and 10 months he is able to run, jump and open doors while standing on his rear paws. He was also able to breed naturally, which apparently has never been reported before. Ringo is descendant of Beth, a GRMD female carrier donated by Dr. Joe Kornegay (University of North Carolina, USA), and all affected descendants carry the same original mutation. The diagnosis in all dogs was established right after birth through DNA genotyping and elevated serum creatine kinase (CK). At birth, Ringo’s serum CK level was increased 10-fold as compared to his three normal sibs. One affected brother had a comparable serum CK while the other had a 20-fold increase. At 15 days serum CK in the affected dogs was 4to 5-fold higher than in normal siblings. Pedigree analysis (Fig. 1) revealed that Ringo had two affected brothers from the same litter. One of them died at 2 weeks of age. The other one is still alive but with a

Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.

Physical therapy assessment tools to evaluate disease progression and phenotype variability in Golden Retriever muscular dystrophy

Dogs suffering from Golden Retriever muscular dystrophy (GRMD) present symptoms that are similar to human patients with Duchenne muscular dystrophy (DMD). Phenotypic variability is common in both cases and correlates with disease progression and response to therapy. Physical therapy assessment tools were used to study disease progression and assess phenotypic variability in dogs with GRMD. At 5 (T0), 9 (T1), 13 (T2) and 17 (T3)months of age, the physical features, joint ranges of motion (ROM), limb and thorax circumferences, weight and creatine kinase (CK) levels were assessed in 11 dogs with GRMD. Alterations of physical features were higher at 13 months, and different disease progression rates were observed. Passive ROM decreased until 1 year old, which was followed by a decline of elbow and tarsal ROM. Limb and thorax circumferences, which were corrected for body weight, decreased significantly between T0 and T3. These measurements can be used to evaluate disease progression in dogs with GRMD and to help discover new therapies for DMD patients.

Alterações do trato digestório de cães da raça Golden Retriever afetados pela distrofia muscular

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, Massons 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.

Motor Physical Therapy Affects Muscle Collagen Type I and Decreases Gait Speed in Dystrophin-Deficient Dogs

Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.

Effect of physical therapy on joint range of motion and muscle collagen deposition in the golden retriever muscular dystrophy (GRMD) model

OBJETIVO: Elucidar o efeito da fisioterapia na Amplitude de Movimento Articular (ADM) e na fibrose muscular em animais GRMD. METODOS: Estudo nao randomizado, com grupo controle, cego, seis meses de intervencao, avaliacao antes e depois da intervencao. Seis animais da raca Golden Retriever, distroficos, machos, media de idade 10,16±3,46 meses e peso de 17,75±6,01 kg foram separados em grupo tratado (n=3) e nao tratado. Esses grupos de animais foram nomeados: G1=grupo tratado antes do tratamento; G2=grupo tratado apos tratamento; G3=grupo nao tratado antes do tratamento; G4=grupo nao tratado apos tratamento. O G1 participou do programa de fisioterapia que consistiu em um circuito de 300 metros com obstaculos. As ADMs do joelho, tarso, cotovelo e carpo foram avaliadas com goniometro antes e apos o tratamento. A area de colageno do musculo vastus lateralis foi mensurada por histomorfometria, e a localizacao dos tipos de colageno I, III e IV foi estudada por Imuno-histoquimica (IHC). RESULTADOS: Os valores da ADM do tarso do G2 apresentaram uma tendencia a aumentar. A area de colageno muscular foi diferente entre os grupos apos o tratamento, e uma tendencia ao aumento desses valores no G4 foi observada. Os colagenos dos tipos I e III foram os mais observados, constituindo feixes largos no perimisio nos dois grupos (G2 e G4). O colageno do tipo I foi mais observado no endomisio do que o colageno do tipo III. O colageno do tipo IV foi observado apenas na lâmina basal. CONCLUSAO: A Fisioterapia parece aumentar a ADM do tarso dos animais do grupo tratado sem aumentar a fibrose muscular.

Duchenne Muscular Dystrophy: Experimental models on Physical Therapy

Neuromuscular disorders are a heterogeneous group of genetic diseases. Nowadays, more than 30 genetically defined forms are recognized and, in the last decade, mutations in several genes have been reported to result in the deficiency or loss of function of a variety of important muscle proteins (Shelton & Engvall, 2005). Defects in components of the dystrophin-glycoprotein complex (DGC) are known to be an important cause of different forms of muscular dystrophies (Ervasti & Campbell 1993; Yoshida & Ozawa 1990). Lack of dystrophin protein in muscle cells is characteristic of Duchenne muscular dystrophy (DMD), which is a progressive and fatal X-linked genetic disorder. Many animal models have been studied to identify an efficient treatment for this disease in humans. Two mammalian models of DMD have been widely used in preclinical trials to understand the pathogenesis of the disease and development of efficient therapeutic strategies for humans. Mdx-mouse is the most used animal model for DMD, followed by the Golden Retriever Muscular Dystrophy (GRMD) canine model. Mdx-mouse morphology displays some features of muscle degeneration, but the pathogenesis of the disease is comparatively mild. This model has a slightly shorter life spam as compared to wild-type controls (Banks & Chamberlain, 2008) and muscle degeneration is different from the one seen in DMD patients. An important degeneration and regeneration of muscle fibers is observed at a young age in the mdx-mouse (2 to 4 weeks), which results in the muscle morphological changes of centralized nuclei and heterogeneity of fiber size. Necrosis is also observed at this early age but decreases around sixty days. Loss of muscle tissue is slow and muscle weakness is not evident until later in life. Fibrosis, a marked feature of DMD muscle, is less pronounced in mdx-mouse, with the exception of diaphragm muscle (Hueber et al., 2008). Dystrophin deficiency has also been reported in cats as hypertrophic feline muscular dystrophy (HFMD), in which diaphragmatic hypertrophy is often fatal (Shelton & Engvall, 2005). Similar to mdx pathology, the skeletal muscle of the HFMD cats undergoes repeated cycles of degeneration and regeneration but does not develop the debilitating fibrosis that is

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.

Low Intensity Training Provokes Adaptations on Muscle Fibrosis of a Muscular Dystrophy Model

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder with progressive clinical signs until death, around the second decade of life. Mdx is the most used animal model to pre-clinical studies of DMD. Parameters of exerci se n this muscular disease are still unknown. This research aimed to investigate if the low intensity treadmill training would exace rbate the markers of muscle injury, fibrosis, and the composition of the extracellular matrix by type I and III collagens of the mdx mode l. Dystrophic 11-week-old male mice were separated in exercised (mdxE, n=8) and sedentary (mdxC, n=8) groups. Wild-type mice were used as control (WT, n=8). Exercised group underwent a LIT protocol (9 m/min, 30min, 3days/week, 60 days) on a horizontal treadmill. Gastrocnemius muscle was collected at day 60 and processed to morphological and morphometric analyzes. Sedentary mdx animals presented inflammatory infiltrate and necrotic fibers. Histochemical analysis revealed that the perimysium of the mdxC group is or anized into thick and clustered collagen fibers, which generates a larger area of intramuscular collagen fibers for these animals. His tomorphometry attested that fraction area of collagen fibers of mdxC group was higher than mdxE group (p=0.04) and mdxE group values similar to WT group (p=1.00). Centrally located nuclei fibers and the variance coefficient (VC) of minimal Feret’s diameter was similar in md xE a d mdxC groups (p=1.00) and both groups presented higher mean values than WT group (p<0.00). Immunohistochemistry revealed the presence of type I collagen mainly in the mdxC group. LIT protocol had not exacerbated muscle injuries resulting from the dystr op indeficiency membrane fragility at the same time that had reduced the intramuscular collagen deposition. LIT had positively influ enced these markers of dystrophic muscle injury on gastrocnemius muscle of mdx model.

Low-intensity training provokes adaptive extracellular matrix turnover of a muscular dystrophy model

Recommendations of therapeutic exercise in Duchenne muscular dystrophy are still controversial. The hypothesis that a low-intensity training (LIT) protocol leads to muscle adaptations on mdx mice model was tested. Dystrophic male mice with 8 weeks old were separated in exercised (mdxE, n= 8) and sedentary (mdxC, n= 8) groups. Wild-type mice were used as control (WT, n= 8) group. Exercised group underwent a LIT protocol (9 m/min, 30 min, 3 days/wk, 60 days) on a horizontal treadmill. At day 60 all animals were analyzed regarding parameters of markers of muscle lesion and extracellular matrix turnover of muscle tissue by collagens fibers on tibial anterior muscle. Histomorphometry attested that centrally located nuclei fibers and the coefficient of variance of minimal Feret’s diameter was similar in mdxE and mdxC groups (P= 1.000) and both groups presented higher mean values than WT group (P< 0.001). Fraction area of collagen fibers of mdxE group was lower than mdxC group (P= 0,027) and similar to WT group (P= 0,751). Intramuscular area of Col3 of the mdxE group was higher than mdxC and WT groups (P<0.001). Intramuscular area of Col1 on the mdxE group was similar to the mdxC group (P= 1.000) and both groups were higher than WT group (P< 0.001). LIT protocol had not influenced muscle injuries resulting from the dystrophin-deficiency membrane fragility. Although, LIT had provoked adaptations on extracellular matrix bringing higher elastic feature to dystrophic muscle tissue.