Karla Patrícia Cardoso Araújo

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.

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.

Estudo morfofuncional dos rins de cães da raça Golden Retriever afetados pela distrofia muscular (GRMD)

Duchenne muscular dystrophy (DMD) is a severe myopathy of recessive X-linked character and the most relevant animal study model is the Golden Retriever muscular dystrophy (GRMD). In addition to the severe changes occurring in the striated musculature, several studies show that other structures, including viscera, may prove to be altered in this pathology. Thus, this study aimed to analyze and compare possible structural and functional alterations of the kidney in GRMD dogs. In this study model, it was possible to observe the presence of convex and concave faces, the renal hilum, and the cranial and caudal poles of the kidneys. The organ was surrounded by a fibrous capsule. In a sagittal section of the organ, the presence of the cortical and medullary regions and the renal pelvis were noticed. On microscopic examination, it was possible to identify the medullary and cortical zones and their structures: the renal corpuscles formed by the glomerulus and Bowmans capsule, the proximal and distal convoluted tubules, the collecting ducts, the blood vessels, and the segments of the loops of Henle. The serum creatinine and urea were within normal limits. Thus, according to our results, we may conclude that the affected animals under study showed no structural or functional changes in the kidneys, something which allows us to suggest that, despite the impaired water intake, renal structure remains preserved in GRMD animals.

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

Morphological Tools for Describing the Male External Genitalia of Sapajus apella

Sapajus apella is a wild monkey of South America distributed across almost all of Brazil. This species adapts to domesticated life and reproduces easily. The present study describes the macro- and microscopic morphology of male genital organs (penis, penis bone, glans penis, prepuce, bulb of penis, and urethra) of Sapajus apella. Four male monkeys were used in this study. For macroscopic description, the genitals were dissected, examined and photographed. For microscopic analysis, samples were stained by HE and Tricom Masson and analyzed by scanning electron microscopy and light microscopy. The penis has a gutter shape with numerous spines on the free part of the penis and glans, and showed cavernous body elements in which mesenchymal cells appear. The glans penis is well developed with a broad crown shape. The prepuce does not cover the free part of the penis. The bulb displays well-developed muscle structure and the membranous urethra is very elongated. These results reveal that Sapajus apella shows specific male genital features, different from other primates.

Comportamento dos nervos glossofaríngeo e vago, na região retrofaríngea de ovinos: origem aparente no crânio, trajeto, ramificação e distribuição

In 60 hemiheads of sheep of the Santa Ines breed the apparent origin in the skull of itinerary, ramification and distribution of the glossopharingeal nerve (Gf) and the vagus nerve (Vg) in the retropharyngeal region (Rr) were studied. By fixation and dissection of the specimens it was seen that the glossopharyngeal nerve and the vagus nerve arise from the jugular foramen in 100% of the cases. The right and the left glossopharingeal nerve (Glde) are frequently (86.6%) located more medially to the tympanic bulla, pass caudally to the stylohyoid bone, bypass the margin of the caudal stylopharyngeal muscle, the tonsilla, of the pharyngeal and the lingual mucous membrane. These branches are distributed, respectively, in the carotid sinus, pharyngeal musculature, soft palate, stylopharyngeal muscle, palatine tonsil, pharyngeal mucosa and the caudal third of the tongue (100%). The right and the left vagus nerve run caudally in association with the accessory nerves (Ac) up to the atlas (70%) and go then medially to the occipital artery, dorsally to the common carotid and the sympathetic trunk in the cervical region (80%). The vague nerves have pharyngeal (86.6%) and cranial laryngeal (100%) branches.

Study of the cardiac left atrioventricular valvar complex in water buffaloes (Bubalus bubalis) of the Jafarabadi breed.

Atrioventricular valve complex of 30 Jafarabadi water buffaloes, adult males were studied in this research with no heart diseases. The animals were obtained from a slaughterhouse in Brazilian State of Parana. The hearts were opened at the third portion affording access to the valve complex. The complexes had its area, number and type of tendinous cords submitted to analysis. The results showed that the complex is composed by two cusps and four accessory cusps, two or three papillary muscles in which 10-25 tendinous cords fix on the cusps that face the ventricle wall. The total area of the complex was on average 38.56cm 2 , with a minimum of 24.96cm 2 and a maximum of 55.54cm 2 . Statistically, no relation between the number of cords and the cusps’ area where they are inserted or with the number of papillary muscle where they originated from was observed.

Morfofisiologia da inervação do diafragma de ovinos

Thirty diaphragms of sheep of Santa Ines breed were studied regarding their origin, division and arrangement of the right and left phrenic nerves (Fde), and the participation of other nerves in the innervation of the diaphragm. By fixing and dissecting pieces, it was found that phrenic nerves (F) frequently come from the ventral branches of the 5th (C5) and 6th (C6) cervical spinal nerves (Ec), at right (46.67%) and at left (43.33%). The F often form a lumbocostal trunk, sternal branches at right (40.00%) and lumbar, costal and esternal branches at left (36.68%). The lumbar branches of F innervate frequently at left (96.67%) the homolateral pillar of the diaphragma, and at right (50.00%) they give fillets to Vena cava caudalis. The costal branches of the F innervate at left (90.00%) and at right (76.66%) the dorsal and ventral regions of the pars costalis. The sternal branches of the F innervate at right (100.00%) and at left (83.33%) the pars sternalis and the ventral region of the pars costalis at the same side. The intercostal nerves (VII to XII pairs, 63.33%) contribute to innervate the diaphragm of Santa Ines sheep.