Pinpointing the candidate region for muscular dystrophy in chickens with an abnormal muscle gene

Muscular dystrophies, a group of inherited diseases with the progressive weakness and degeneration of skeletal muscle, contain genetically variable diseases. Though chicken muscular dystrophy with abnormal muscle (AM) has long been known, the gene responsible has not yet been identified. In this study, a resource family for AM was established with 487 F2 individuals and 22 gene markers, including microsatellite and insertion–deletion markers, were developed. The haplotypes were analyzed with these markers for the candidate region of GGA2q described in a previous study. The candidate region was successfully narrowed down to approximately 1Mbp. The region included seven functional genes predicted as the most likely AM candidates.     

Loss of p16/Ink4a drives high frequency of rhabdomyosarcoma in a rat model of Duchenne muscular dystrophy

Rhabdomyosarcoma (RMS) is an aggressive type of soft tissue sarcoma, and pleomorphic RMS is a rare subtype of RMS found in adult. p16 is a tumor suppressor which inhibits cell cycle. In human RMS, p16 gene is frequently deleted, but p16-null mice do not develop RMS. We reported that genetic ablation of p16 by the crossbreeding of p16 knock-out rats (p16-KO rats) improved the dystrophic phenotype of a rat model of Duchenne muscular dystrophy (Dmd-KO rats). However, p16/Dmd double knock-out rats (dKO rats) unexpectedly developed sarcoma. In the present study, we raised p16-KO, Dmd-KO, and dKO rats until 11 months of age. Twelve out of 22 dKO rats developed pleomorphic RMS after 9 months of age, while none of p16-KO rats and Dmd-KO rats developed tumor. The neoplasms were connected to skeletal muscle tissue with indistinct borders and characterized by diffuse proliferation of pleomorphic cells which had eosinophilic cytoplasm and atypical nuclei with anisokaryosis. For almost all cases, the tumor cells immunohistochemically expressed myogenic markers including desmin, MyoD, and myogenin. The single cell cloning from tumor primary cells gained 20 individual Pax7-negative MyoD-positive RMS cell clones. Our results demonstrated that double knock-out of p16 and dystrophin in rats leads to the development of pleomorphic RMS, providing an animal model that may be useful to study the developmental mechanism of pleomorphic RMS.     

Nutritional muscular dystrophy in a four-day-old Connemara foal

This report describes a four-day-old, full-term Connemara colt, presented for the evaluation of a progressive inability to rise unassisted. A diagnosis of nutritional muscular dystrophy was made based on muscular weakness, elevated muscle enzymes and low vitamin E, selenium and glutathione peroxidase activity. The foal was treated with intramuscular vitamin E-selenium and made a full recovery.     

The electroretinographic phenotype of dogs with Golden Retriever muscular dystrophy

Objective To characterize the flash electroretinogram (ERG) in the Golden Retriever muscular dystrophy (GRMD) dog and to compare the results with those from a control group of Golden Retrievers. To investigate whether similar abnormalities of the ERG as those found in a majority of human patients with Duchenne muscular dystrophy (DMD) are also observed in the GRMD dog, the canine model for DMD. Animals Five GRMD dogs and five age‐matched clinically normal Golden Retrievers. Procedure An ophthalmic examination was carried out prior to performing electroretinography under general anesthesia. Rod, combined rod–cone and oscillatory potentials responses were recorded after dark adaptation. Responses to 30‐Hz‐flicker were recorded after light adaptation. The ERG responses of the GRMD dogs were compared with those of the control dogs by use of a Wilcoxon signed rank test. Results GRMD dogs had significantly reduced a and b‐wave amplitudes after dim white flash stimuli (rod response) and reduced a‐wave amplitude after bright white flash stimuli (rod–cone response). Conclusion and clinical relevance The ERG abnormalities observed in the GRMD dog suggest a dysfunction in the rod signaling pathway. These ERG alterations are different from those observed in human patients with DMD.     

Hypertrophic muscular dystrophy in a young dog

Hypertrophic muscular dystrophy was diagnosed in a 10-month-old male Rat Terrier with hypersalivation, dysphagia, gait abnormalities, and generalized weakness. Serum creatine kinase activity was high, and electromyography revealed myotonic discharges. Histologic examination of a muscle biopsy specimen revealed muscle fiber degeneration, clusters of basophilic regenerating fibers, and endomysial fibrosis. Staining for dystrophin, a sarcolemmal protein, was decreased, compared with that in muscle specimens from clinically normal dogs. Treatment with mexilitene hydrochloride and procainimide hydrochloride resulted in temporary improvement in clinical signs, but the disease became refractory to treatment, and the dog was euthanatized. Clinical and histologic characteristics of this dystrophin deficiency-related muscular dystrophy were similar to those of X-linked muscular dystrophy in dogs, hypertrophic muscular dystrophy in cats, and Duchenne muscular dystrophy in humans.     

Dystrophin-deficient muscular dystrophy in a Weimaraner

A 2-year-old, male Weimaraner with muscular dystrophy was presented with generalized muscle atrophy of the limbs; hypertrophy of the neck, infraspinatus, and lingual muscles; dysphagia; and regurgitation. Unilateral cryptorchidism, unilateral renal agenesis, and hiatal hernia were also detected. Spontaneous muscle activity was identified on myography. Serum creatine kinase was markedly elevated. Immunohistochemical staining for dystrophin was restricted to suspected revertant (characteristics of immaturity) fibers. Histologically, skeletal myofiber degeneration, endomysial fibrosis, and mineralization were present. Following euthanasia, necropsy revealed hypertrophy of the diaphragm and cardiac muscle fibrosis. This case of muscular dystrophy represents a slowly progressive form with organ agenesis.     

Muscle carbonic anhydrase III levels in normal and muscular dystrophia afflicted chickens

Background

The levels and immunohistochemical localization of muscle carbonic anhydrase III (CA-III) in healthy chickens and in muscular dystrophia affected (DA) chickens show that the muscles of diseased animal undergo a progressive increase of enzyme activity.

Methods

An enzyme-linked immunoassay was used to assess the CA-III levels in the muscles and other tissues from eight normal White Leghorn chickens and in two chickens with muscular dystrophy. Immunohistochemical localization of the enzyme in the muscles of these animals was also determined.

Results

The levels of CA-III in the tensor fasciae latae and the superficial pectoral muscles of the DA chickens were higher than the level in normal chickens. The concentrations of CA-III in erythrocytes and plasma from diseased chickens were approximately 15-fold and 1.4-fold higher than in the normal chickens, respectively. In the superficial pectoral and the tensor fasciae latae muscles of diseased chickens, the numbers of strongly stained and weakly stained fibers were greater than that in the normal chickens.

Conclusion

The levels of CA-III in the superficial pectoral muscle, the tensor fasciae latae muscle, plasma and erythrocytes from the chickens with muscular dystrophy were higher than found in normal chickens.     

Age-related thoracic radiographic changes in golden and labrador retriever muscular dystrophy

Golden retriever and Labrador retriever muscular dystrophy are inherited progressive degenerative myopathies that are used as models of Duchenne muscular dystrophy in man. Thoracic lesions were reported to be the most consistent radiographic finding in golden retriever dogs in a study where radiographs were performed at a single-time point. Muscular dystrophy worsens clinically over time and longitudinal studies in dogs are lacking. Thus our goal was to describe the thoracic abnormalities of golden retriever and Labrador retriever dogs, to determine the timing of first expression and their evolution with time. To this purpose, we retrospectively reviewed 390 monthly radiographic studies of 38 golden retrievers and six Labrador retrievers with muscular dystrophy. The same thoracic lesions were found in both golden and Labrador retrievers. They included, in decreasing frequency, flattened and/or scalloped diaphragmatic shape (43/44), pulmonary hyperinflation (34/44), hiatal hernia (34/44), cranial pectus excavatum (23/44), bronchopneumonia (22/44), and megaesophagus (14/44). The last three lesions were not reported in a previous radiographic study in golden retriever dogs. In all but two dogs the thoracic changes were detected between 4 and 10 months and were persistent or worsened over time. Clinically, muscular dystrophy should be included in the differential diagnosis of dogs with a combination of these thoracic radiographic findings.     

Dystrophin‐deficient muscular dystrophy in a Norfolk terrier

A six‐month‐old male entire Norfolk terrier was presented with a 3‐month history of poor development, reluctance to exercise and progressive and diffuse muscle atrophy. Serum creatine kinase concentration was markedly elevated. Magnetic resonance imaging of the epaxial muscles revealed asymmetrical streaky signal changes aligned within the muscle fibres (hyperintense on T2‐weighted images and short‐tau inversion recovery with moderate contrast enhancement on T1‐weighted images). Electromyography revealed pseudomyotonic discharges and fibrillation potentials localised at the level of the supraspinatus, epaxial muscles and tibial cranialis muscles. Muscle biopsy results were consistent with dystrophin‐deficient muscular dystrophy. The dog remained stable 7 months after diagnosis with coenzyme Q10 and l ‐carnitine; however after that time, there was a marked deterioration and the owners elected euthanasia. This case report describes the clinical presentation, magnetic resonance imaging, electrodiagnostic and histopathological findings with immunohistochemical analysis in a Norfolk terrier with confirmed dystrophin‐deficient muscular dystrophy, which has not been previously described in this breed.     

RADIOGRAPHIC FEATURES OF GOLDEN RETRIEVER MUSCULAR DYSTROPHY

Golden Retriever muscular dystrophy is an inherited, degenerative myopathy due to the absence of dystrophin and is used as a model of Duchenne muscular dystrophy of young boys. This report describes the radiographic abnormalities of Golden Retriever muscular dystrophy in 26 dogs. The thoracic abnormalities included diaphragmatic asymmetry (18/26), diaphragmatic undulation (18/26), and gastro-esophageal hiatal hernia (6/26). Pelvic abnormalities included narrowing of the body of the ilia (14/19), ventral deviation and curvature of the tuber ischii (14/19), elongation of the obturator foramen with a decrease in opacity of the surrounding bone (12/19), and lateral flaring of the wings of the ilia (12/19). Abdominal abnormalities consisted of hepatomegaly (14/22) and poor serosal detail (12/22). The unique thoracic abnormalities were a consistent finding in affected Golden Retriever muscular dystrophy dogs. The diagnosis of muscular dystrophy should be included in the differential list if the combination of diaphragm undulation and asymmetry, and gastro-esophageal hiatal hernia are identified. These diaphragmatic abnormalities are related to hypertrophy and hyperplasia of the diaphragm. Additionally, the skeletal changes of pelvic tilt, elongation of the pelvis, widening of the obturator foramina and thinning of the ischiatic tables appear to be specific to Golden Retriever muscular dystrophy in dogs. These pelvic abnormalities are most likely secondary to bone remodeling associated with the progressive skeletal myopathy and subsequent contracture/fibrosis.     

Sarcocystis calchasi encephalitis in a rock pigeon

A rock pigeon (Columba livia) caught in Akihabara, Tokyo, showed neurological symptoms, such as head tilt and circling. Pathological examinations revealed abundant Sarcocystic cysts in the skeletal muscle and myocardium with mild myositis, and numerous schizonts and sarcocysts with severe multifocal granulomatous T-lymphocytic infiltration in the central nervous system. A Sarcocystis calchasi-specific gene was detected in the muscle and brain. This case indicates S. calchasi was distributed in Japan and caused severe encephalitis to rock pigeons.     

RADIOGRAPHIC AND ULTRASONOGRAPHIC FEATURES OF HYPERTROPHIC FELINE MUSCULAR DYSTROPHY IN TWO CATS

Hypertrophic fellne musculer dystrophy has been reported as an X-linked inherited deficiency of a cytoskeletal myofiber protein called dystrophin. This report deserlbes the radiographic and ultrasonographic abnormalities of two male littermate domestic short-hair cats and reviews the previous reported findings assoclated with hypertrophic feline muscular dystrophy. The thoracic radiographic abnormalities included: progressive cardiomegaly, large convex, scalloped irregularities associated with the vetral aspect of the diaphragm, and variable degrees of esophageal dilation (megaesophagus) with associated cranioventral aspiration pneumonia. Echocardiographic features included: concentric left vetricular wall thickening, increased left ventricular and diastolic and systolic dimensions, and an increase in endocardial echogenicity. Abdominal radiographic abnormalities included: hepatosplenomegaly, peritoneal effusion, renomegaly, adrenal gland mineralization, and paralumbar and diaphragmatic musculature enlargement. Abdomlnal ultrasonographic abnormalities included: irregularly thickened muscular portion of the diaphragm; hypoechogenicity of the liver; peritoneal effusion; hepatosplenomegaly; renomegaly with hyperechoic cortex and medulla; and adrenal gland mineralization. The irregular scalloped appearance of the diaphragm (particularly along the ventral/sternal margin) was a consistenl radiographic abnormlity in the two cats with hypertrophic feline muscular dystrophy after the age of 7 months. This finding was confirmed by ultrasound as a thickened irregular, hyperechoic diaphragm. A diagnosis of hypertrophic feline muscular dystrophy should be strongly suspected if this abnormality is identified.     

Elevated level of microRNA-210 at the initiation of muscular regeneration in acetic acid-induced non-ischemic skeletal muscular injury in mice

The alteration in microRNA-210 level, a hypoxia-inducible microRNA, is not well known in non-ischemic tissue injury. In this study, we characterized the histopathological time course of acetic acid-induced skeletal muscle injury as a non-ischemic tissue injury model and investigated the expression of microRNA-210, hypoxia-inducible factor 1α, and growth factors using quantitative polymerase chain reaction analysis. After a single intramuscular dose of 3% (v/v) acetic acid to C57BL/6J mice, focal coagulative necrosis of muscle fibers was noted from 3 h after dosing and infiltration of F4/80 and Galectin-3 positive M2 macrophage was noted at 1 d after dosing. Muscular regeneration was initiated from 3 d, when M2 macrophage infiltration was most prominent, till 14 d after dosing. Hif1α and Hgf expression increased from 3 h onwards, and microRNA-210 level increased after 3 d after the treatment. However, no clear elevation in the levels of Igf1 or Vegf was observed. The infiltrative macrophages and regenerative muscle fibers were positive for hypoxia-inducible factor 1α, microRNA-210, and hepatocyte growth factor as assessed by immunohistochemistry or in situ hybridization. In this study, dominant infiltration of M2 macrophages at muscular necrosis and subsequent regeneration after a single intramuscular injection of acetic acid in mice were observed. The increase in hif1α level was observed just after the muscular injury in this non-ischemic tissue injury model, and the elevation in microRNA-210 level was noted at the initiation of tissue regeneration, indicating its effects on tissue protection and repair.     

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.     

Dystrophin-deficient muscular dystrophy in a Labrador retriever

Sex-linked muscular dystrophy associated with dystrophin deficiency has been reported in several breeds of dogs and is best characterized in the golden retriever. In this case report, a young, male Labrador retriever with dystrophin-deficient muscular dystrophy is presented. Clinical signs included generalized weakness, lingual hypertrophy, and dysphagia. Electromyographic abnormalities including complex repetitive discharges were present. Serum creatine kinase concentration was dramatically elevated. Histopathological changes within a muscle biopsy specimen confirmed a dystrophic myopathy, and dystrophin deficiency was demonstrated by immunohistochemical staining. While X-linked muscular dystrophy has not previously been reported in the Labrador retriever, a hereditary myopathy with an autosomal recessive mode of inheritance has been characterized. A correct diagnosis and classification of these two disorders are critical for breeders and owners since both the mode of inheritance and the prognosis differ.     

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

Background

The 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.

Methods

A 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.

Results

Both 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.

Conclusion

We 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.     

Dystrophin‐deficient muscular dystrophy in two lurcher siblings

Two cases of dystrophin‐deficient muscular dystrophy in 16‐week‐old male lurcher siblings are reported. The myopathies were characterised by regurgitation, progressive weakness and muscle wastage. The dogs had generalised weakness in all four limbs, with more pronounced weakness in the pelvic limbs. Reduced withdrawal in all limbs, muscle contracture and lingual hypertrophy were noted. Serum creatine kinase activities were markedly elevated. Electromyographic abnormalities included fibrillation potentials. Histopathological and immunohistochemical staining were consistent with dystrophin‐deficient muscular dystrophy. Clinical improvement was noted in one of the cases with l ‐carnitine supplementation and supportive therapy. Genetic transmission of the disease was postulated as the dogs were siblings.     

Radiographic,MRI, and CT findings in a young dog with Becker-like muscular dystrophy

An 8-month-old, male, crossbreed dog was presented for macroglossia, reduced mandibular extension, ptyalism, dysphagia, and regurgitation. Serum creatine kinase and aspartate aminotransferase activity were markedly increased. Thoracic radiographs showed an axial gastro-esophageal hiatal hernia, diaphragmatic thickening, and asymmetry. Magnetic resonance imaging of the head showed a severely enlarged tongue, symmetric increase in size of the geniohyoid and mylohyoid muscles, and diffuse masticatory hypomyotrophy. Whole-body CT ruled out other musculoskeletal abnormalities and further characterized the radiographic and MRI findings. Muscular histopathology was consistent with Becker muscular dystrophy.     

Dysphagia in Bouviers associated with muscular dystrophy; evaluation of 24 cases

Twenty-four Bouviers with dysphagia were examined between October 1986 and October 1988. The type of dysphagia was characterised by the results from the clinical examination, the videofluorographic examination and the electromyographic recordings from the oral, pharyngeal, and esophageal muscles. Electromyography indicated neurogenic as well as myogenic causes of dysphagia. Tissues from 10 dogs were available for histopathologic examination. In nine dogs there was a progressive muscular degeneration of the pharyngeal and/or esophageal muscles, resembling muscular dystrophy. In two of these dogs the same abnormalities were also noticed in the masseter and temporalis muscles and in the intrinsic laryngeal muscles. In one dog small areas with hyalin degeneration and fragmentation of muscle fibres were found in the cricopharyngeal muscle. No abnormalities in nerve tissue were found. Muscular dystrophy is a hereditary disease. The mode of transmission in these Bouviers is not yet known.     

Muscular dystrophy in female dogs

The most common form of muscular dystrophy in dogs and humans is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome, and, therefore, disease-causing mutations in dystrophin occur most often in males. Therefore, females with dystrophin deficiency or other forms of muscular dystrophy may be undiagnosed or misdiagnosed. Immunohistochemistry was used to analyze dystrophin and a number of other muscle proteins associated with muscular dystrophy in humans, including sarcoglycans and laminin alpha2, in muscle biopsy specimens from 5 female dogs with pathologic changes consistent with muscular dystrophy. The female dogs were presented with a variety of clinical signs including generalized weakness, muscle wasting, tremors, exercise intolerance, gait abnormalities, and limb deformity. Serum creatine kinase activity was variably high. One dog had no detectable dystrophin in the muscle; another was mosaic, with some fibers normal and others partly dystrophin-deficient. A 3rd dog had normal dystrophin but no detectable laminin alpha2. Two dogs could not be classified. This study demonstrates the occurrence of dystrophin- and laminin alpha2-associated muscular dystrophy and the difficulty in clinical diagnosis of these disorders in female dogs.