Mast cells in the pathophysiology of Duchenne muscular dystrophy in Golden Retriever dogs / O papel dos mastócitos na patogênese da distrofia muscular de Duchenne em cães Golden Retriever

The Golden Retriever muscular dystrophy (GRMD) is one of the best models of Duchenne muscular dystrophy (DMD), with similar genotypic and phenotypic manifestations. Progressive proliferation of connective tissue in the endomysium of the muscle fibers occurs in parallel with the clinical course of the disease in GRMD animals. Previous studies suggest a relationship between mast cells and the deposition of fibrous tissue due to the release of mediators that recruit fibroblasts. The aim of this study was to evaluate the presence of mast cells and their relationship with muscle injury and fibrosis in GRMD dogs of different ages. Samples of muscle groups from six GRMD and four control dogs, aged 2 to 8 months, were collected and analyzed. The samples were processed and stained with HE, toluidine blue, and Azan trichrome. Our results showed that there was a significant increase in infiltration of mast cells in all muscle groups of GRMD dogs compared to the control group. The average number of mast cells, as well as the deposition of fibrous tissue, decreased with age in GRMD dogs. In the control group, all muscle types showed a significant increase in the amount of collagenous tissue. This suggests increased mast cell degranulation occurred in younger GRMD dogs, resulting in increased interstitial space and fibrous tissue in muscle, which then gradually decreased over time as the dogs aged. However, further studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis.(AU)

O cão Golden Retriever distrófico (Golden Retriever muscular dystrophy - GRMD) é um dos melhores modelos da distrofia muscular de Duchenne (DMD), com manifestações genotípicas e fenotípicas similares. A proliferação progressiva de tecido conjuntivo no endomísio das fibras musculares ocorre paralelamente ao curso clínico da doença em animais GRMD. Estudos anteriores sugerem uma relação entre os mastócitos e a deposição de tecido fibroso devido à liberação de mediadores que recrutam fibroblastos. O objetivo deste estudo foi avaliar a presença de mastócitos e sua relação com a lesão muscular e fibrose em cães GRMD de diferentes idades. Amostras de grupos musculares de seis GRMD e quatro controles, com idade entre 2 a 8 meses, foram coletadas e analisadas. As amostras foram processadas e coradas com HE, azul de toluidina e tricrômico de Azan. Nossos resultados mostraram que houve um aumento significativo na infiltração de mastócitos em todos os grupos musculares de cães GRMD em comparação com o grupo controle. O número médio de mastócitos, assim como a deposição de tecido fibroso, diminuiu com a idade em cães GRMD. No grupo controle, todos os tipos musculares mostraram um aumento significativo na quantidade de tecido colágeno. Isto sugere o aumento da degranulação de mastócitos em cães GRMD mais jovens, resultando em aumento do espaço intersticial e tecido fibroso no músculo, que então gradualmente diminuiu com o tempo à medida que os cães envelheceram. No entanto, mais estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose.(AU)

Mast cells in the pathophysiology of Duchenne muscular dystrophy in Golden Retriever dogs / O papel dos mastócitos na patogênese da distrofia muscular de Duchenne em cães Golden Retriever

The Golden Retriever muscular dystrophy (GRMD) is one of the best models of Duchenne muscular dystrophy (DMD), with similar genotypic and phenotypic manifestations. Progressive proliferation of connective tissue in the endomysium of the muscle fibers occurs in parallel with the clinical course of the disease in GRMD animals. Previous studies suggest a relationship between mast cells and the deposition of fibrous tissue due to the release of mediators that recruit fibroblasts. The aim of this study was to evaluate the presence of mast cells and their relationship with muscle injury and fibrosis in GRMD dogs of different ages. Samples of muscle groups from six GRMD and four control dogs, aged 2 to 8 months, were collected and analyzed. The samples were processed and stained with HE, toluidine blue, and Azan trichrome. Our results showed that there was a significant increase in infiltration of mast cells in all muscle groups of GRMD dogs compared to the control group. The average number of mast cells, as well as the deposition of fibrous tissue, decreased with age in GRMD dogs. In the control group, all muscle types showed a significant increase in the amount of collagenous tissue. This suggests increased mast cell degranulation occurred in younger GRMD dogs, resulting in increased interstitial space and fibrous tissue in muscle, which then gradually decreased over time as the dogs aged. However, further studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis.(AU)

O cão Golden Retriever distrófico (Golden Retriever muscular dystrophy - GRMD) é um dos melhores modelos da distrofia muscular de Duchenne (DMD), com manifestações genotípicas e fenotípicas similares. A proliferação progressiva de tecido conjuntivo no endomísio das fibras musculares ocorre paralelamente ao curso clínico da doença em animais GRMD. Estudos anteriores sugerem uma relação entre os mastócitos e a deposição de tecido fibroso devido à liberação de mediadores que recrutam fibroblastos. O objetivo deste estudo foi avaliar a presença de mastócitos e sua relação com a lesão muscular e fibrose em cães GRMD de diferentes idades. Amostras de grupos musculares de seis GRMD e quatro controles, com idade entre 2 a 8 meses, foram coletadas e analisadas. As amostras foram processadas e coradas com HE, azul de toluidina e tricrômico de Azan. Nossos resultados mostraram que houve um aumento significativo na infiltração de mastócitos em todos os grupos musculares de cães GRMD em comparação com o grupo controle. O número médio de mastócitos, assim como a deposição de tecido fibroso, diminuiu com a idade em cães GRMD. No grupo controle, todos os tipos musculares mostraram um aumento significativo na quantidade de tecido colágeno. Isto sugere o aumento da degranulação de mastócitos em cães GRMD mais jovens, resultando em aumento do espaço intersticial e tecido fibroso no músculo, que então gradualmente diminuiu com o tempo à medida que os cães envelheceram. No entanto, mais estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose.(AU)

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.(AU)

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.(AU)

Relevant aspects of golden retriever muscular dystrophy for the study of Duchenne muscular dystrophy in humans / Aspectos relevantes da distrofia muscular do Golden Retriever para o estudo da distrofia muscular de Duchenne em humanos

Golden Retriever muscular dystrophy (GRMD) is the most representative model for studying Duchenne muscular dystrophy (DMD) in humans, owing its phenotypic expression. DMD is a recessive disorder linked to the X chromosome in which the loss of dystrophin induces progressive weakness and degeneration of the skeletal and cardiac muscles, which lead to replacement by connective and adipose tissues. Onset of clinical signs occurs between 2 and 5 years of age, and many patients die from heart or respiratory failure. The main studies concerning dystrophic Golden Retrievers (DGR) sought to elucidate the pathophysiology of the disease and its clinical implications to develop therapies and alternative treatments to improve the quality of life and increase longevity of DMD patients. This review presents an overview of relevant contributions of the DGR model for elucidating DMD in humans.

A distrofia muscular do Golden Retriever (DMGR) é o modelo mais representativo para o estudo da distrofia muscular de Duchenne (DMD) em humanos devido a sua expressão fenotípica. A DMD é uma desordem genética recessiva ligada ao cromossomo X onde a perda da distrofina induz fraqueza progressiva e degeneração do músculo esquelético e cardíaco conduzindo a substituição do músculo por tecido conjuntivo e adiposo. O início da doença ocorre entre 2 e 5 anos de idade e muitos pacientes morrem por insuficiência cardíaca ou respiratória. Os principais estudos realizados no Golden Retriever distrófico (GRD) buscam elucidar a fisiopatogenia da doença e suas implicações clínicas na tentativa de testar terapias e tratamentos alternativos para melhoria da qualidade de vida do paciente distrófico e aumentar sua longevidade. Esta revisão apresenta uma visão geral sobre relevante contribuição do modelo GRD para elucidar a DMD em humanos.

Relevant aspects of golden retriever muscular dystrophy for the study of Duchenne muscular dystrophy in humans / Aspectos relevantes da distrofia muscular do Golden Retriever para o estudo da distrofia muscular de Duchenne em humanos

Golden Retriever muscular dystrophy (GRMD) is the most representative model for studying Duchenne muscular dystrophy (DMD) in humans, owing its phenotypic expression. DMD is a recessive disorder linked to the X chromosome in which the loss of dystrophin induces progressive weakness and degeneration of the skeletal and cardiac muscles, which lead to replacement by connective and adipose tissues. Onset of clinical signs occurs between 2 and 5 years of age, and many patients die from heart or respiratory failure. The main studies concerning dystrophic Golden Retrievers (DGR) sought to elucidate the pathophysiology of the disease and its clinical implications to develop therapies and alternative treatments to improve the quality of life and increase longevity of DMD patients. This review presents an overview of relevant contributions of the DGR model for elucidating DMD in humans.(AU)

A distrofia muscular do Golden Retriever (DMGR) é o modelo mais representativo para o estudo da distrofia muscular de Duchenne (DMD) em humanos devido a sua expressão fenotípica. A DMD é uma desordem genética recessiva ligada ao cromossomo X onde a perda da distrofina induz fraqueza progressiva e degeneração do músculo esquelético e cardíaco conduzindo a substituição do músculo por tecido conjuntivo e adiposo. O início da doença ocorre entre 2 e 5 anos de idade e muitos pacientes morrem por insuficiência cardíaca ou respiratória. Os principais estudos realizados no Golden Retriever distrófico (GRD) buscam elucidar a fisiopatogenia da doença e suas implicações clínicas na tentativa de testar terapias e tratamentos alternativos para melhoria da qualidade de vida do paciente distrófico e aumentar sua longevidade. Esta revisão apresenta uma visão geral sobre relevante contribuição do modelo GRD para elucidar a DMD em humanos.(AU)

Noninvasive evaluation of respiratory muscles in pre-clinical model of Duchenne Muscular Dystrophy / Avaliação não invasiva dos músculos respiratórios no modelo pré-clinico da Distrofia Muscular de Duchenne

Since respiratory insufficiency is the main cause of death in patients affected by Duchenne Muscular Dystrophy (DMD), the present study aims at establishing a new non-invasive method to evaluate the clinical parameters of respiratory conditions of experimental models affected by DMD. With this purpose in mind, we evaluated the cardiorespiratory clinical conditions, the changes in the intercostal muscles, the diaphragmatic mobility, and the respiratory cycles in Golden Retriever Muscular Dystrophy (GRMD) employing ultrasonography (US). A control group consisting of dogs of the same race, but not affected by muscular dystrophy, were used in this study. The results showed that inspiration, expiration and plateau movements (diaphragm mobility) were lower in the affected group. Plateau phase in the affected group was practically non-existent and showed that the diaphragm remained in constant motion. Respiratory rate reached 15.5 per minute for affected group and 26.93 per minute for the control group. Expiration and inspiration movements of intercostal muscles reached 8.99mm and 8.79mm, respectively, for control group and 7.42mm and 7.40mm, respectively, for affected group. Methodology used in the present analysis proved to be viable for the follow-up and evaluation of the respiratory model in GRMD and may be adapted to other muscular dystrophy experimental models.(AU)

Uma vez que, a insuficiência respiratória é a principal causa de morte em pacientes afetados pela Distrofia Muscular de Duchenne (DMD), o presente estudo avaliou as condições clínicas cardiorrespiratórias, o movimento dos músculos intercostais, a mobilidade diafragmática, os ciclos respiratórios e a expansão da cavidade torácica em cães Golden Retriever com Distrofia Muscular (GRMD) por ultrassonografia (US) a fim de estabelecer um novo método não invasivo para avaliar os parâmetros de avaliação clínica de doenças respiratórias de modelos experimentais afetados por DMD. Um grupo controle constituído por cães da mesma raça e espécie, mas não afetados pela distrofia muscular também foram utilizados neste estudo. Os resultados mostraram que os movimentos de inspiração, expiração e platô (mobilidade do diafragma) foram menores no grupo afetado. A fase de platô no grupo afetado foi praticamente inexistente e mostrou que o diafragma destes animais permaneceu em constante movimento. A frequência respiratória atingiu 15,5 por minuto para o grupo afetado e 26,93 para o controle. Movimento de expiração e inspiração dos músculos intercostais atingiu 8,99 milímetros e 8,79 milímetros, respectivamente para o grupo controle e 7,42 milímetros e 7,40 milímetros, respectivamente para o grupo afetado. A metodologia utilizada nesta análise foi viável para o acompanhamento e avaliação do modelo respiratória em modelo GRMD e pode ser adaptado para outros modelos experimentais de distrofia muscular.(AU)

Multidrug therapy for muscular dystrophy in a GRMD (Golden Retriever muscular dystrophy) model

Golden Retriever muscular dystrophy (GRMD) is a degenerative, progressive and lethal genetic disease, homologous to human Duchenne muscular dystrophy (DMD). For both there is no cure and clinical interventions are limited to treating clinical signs. As potential treatment, we did a pilot study in GRMD and administered a cocktail of frequently used drugs that might work synergistically. Biochemical blood values were investigated and histology and real-time PCR of muscle biopsies were performed. Significant improvements occurred in experimental Golden Retrievers dogs in regard to the levels of alanine aminotransferase, neutrophils, lymphocytes and CK, which began to show values closer to normal ones when compared to control animals, as well as moderate inflammatory infiltrates in the musculature, according to histology. The drug cocktail seemed to work systemically, but did not interfere with the gene defect, thus having potential value for maintaining the health status and preventing the progression of muscular dystrophy in GRMD model.

Multidrug therapy for muscular dystrophy in a GRMD (Golden Retriever muscular dystrophy) model

Golden Retriever muscular dystrophy (GRMD) is a degenerative, progressive and lethal genetic disease, homologous to human Duchenne muscular dystrophy (DMD). For both there is no cure and clinical interventions are limited to treating clinical signs. As potential treatment, we did a pilot study in GRMD and administered a cocktail of frequently used drugs that might work synergistically. Biochemical blood values were investigated and histology and real-time PCR of muscle biopsies were performed. Significant improvements occurred in experimental Golden Retrievers dogs in regard to the levels of alanine aminotransferase, neutrophils, lymphocytes and CK, which began to show values closer to normal ones when compared to control animals, as well as moderate inflammatory infiltrates in the musculature, according to histology. The drug cocktail seemed to work systemically, but did not interfere with the gene defect, thus having potential value for maintaining the health status and preventing the progression of muscular dystrophy in GRMD model.(AU)

Recelularização da matriz extracelular de músculo de cães saudáveis e distróficos

A Distrofia Muscular de Duchenne (DMD) é uma doença hereditária de caráter progressivo causada por mutação no gene produtor da proteína distrofina, responsável por ligar o citoesqueleto da célula muscular a matriz extracelular (MEC). Além de humanos, outros animais podem apresentar distrofias musculares, podendo ser utilizados como modelos para o estudo de distrofias musculares em humanos. O modelo animal mais utilizado é o modelo de Distrofia Muscular do Golden Retriever (GRMD). Os estudos de distrofias musculares podem ser associados a diversas áreas, como a Bioengenharia de tecidos, área que visa a utilização de MEC descelularizadas (scaffolds) para reparação/substituição de órgãos e tecidos, porém estes scaffolds também podem servir como ferramenta para estudo de MEC em doenças. Portando, o objetivo deste trabalho foi a caracterização de alguns componentes da MEC distrófica, sugerir se uma MEC distrófica pode ser um possível biomaterial para medicina regenerativa e a recelularização de MEC distróficas para observação da relação célula- matriz. Para realização deste trabalho, músculos bíceps femorais distróficos e não distróficos foram descelularizados com protocolo de utilização de SDS a 1% e passaram por análises de quantificação de DNA e fluorescência por DAPI para validação da descelularização e análises histológicas (colorações), imunohistoquímica e de Microscopia Eletrônica de Varredura para avaliação da composição, ultraestrutura e preservação das MEC distróficas e não distróficas. Como resultados, obtivemos as quantificações de DNA que alcançaram quantidade inferior a 50ng de DNA por mg de tecido e na fluorescência por DAPI não foram visualizados núcleos celulares nos tecidos descelularizados. Pelas análises histológicas foi possível observar a ausência de células nos tecidos descelularizados e a preservação de fibras colágenas da MEC. Pela imunohistoquímica, não foram observadas grandes diferenças nos componentes da MEC de músculos distróficos quando comparados com músculos não distróficos. Quando observadas, as células utilizadas para recelularização dos scaffolds apresentaram maior taxa de proliferação quando em contato com o scaffold muscular e o protocolo de esterilização do scaffold foi eficiente, já que os testes para detecção de micoplasma foram negativos. Foi observado o aumento da quantidade de DNAg após a recelularização dos scaffolds e pela MEV são visíveis camadas de células sobre a matriz extracelular. Concluímos que a MEC de músculo distrófico não interfere na diferenciação e adesão celular quando comparadas às MEC de músculo não distrófico. Embora o aumento de distrofina tenha sido observado em alguns processos de recelularização, mais estudos precisam ser realizados para comprovação deste dado.

Duchenne Muscular Dystrophy (DMD) is a progressive hereditary disease caused by a mutation in the gene producing the dystrophin protein, responsible for linking the cytoskeleton of the muscle cell to the extracellular matrix (ECM). In addition to humans, other animals may have muscular dystrophies and can be used as models for the study of muscular dystrophies in humans. The most used animal model is the Golden Retriever Muscular Dystrophy (GRMD) model. Muscular dystrophy studies can be associated with several areas, such as tissue bioengineering, an area that aims to use decellularized ECM (scaffolds) for repairing / replacing organs and tissues, but these scaffolds can also serve as a tool for studying the ECM in diseases. Therefore, the objective of this work was to characterize some components of the dystrophic ECM, to suggest whether a dystrophic ECM may be a possible biomaterial for regenerative medicine and the recellularization of dystrophic ECM to observe the cell-matrix relationship. To perform this work, dystrophic and non-dystrophic femoral biceps muscles were decellularized using a 1% SDS protocol and underwent DNA quantification and fluorescence analyzes by DAPI to validate decellularization and histological (staining), immunohistochemistry and Scanning Electron microscopy analyzes to evaluate the composition, ultrastructure and preservation of dystrophic and non-dystrophic ECM. As a result, we obtained the DNA quantifications that reached a quantity of less than 50ng of DNA per mg of tissue and in the fluorescence by DAPI, cell nuclei were not seen in the decellularized tissues. Through histological analyzes it was possible to observe the absence of cells in the decellularized tissues and the preservation of collagen fibers of the ECM. Due to immunohistochemistry, no major differences were observed in the ECM components of dystrophic muscles when compared with non-dystrophic muscles. When observed, the cells used to recellularize the scaffolds showed a higher proliferation rate when in contact with the muscle scaffold and the scaffold sterilization protocol was efficient, since the tests for mycoplasma detection were negative. An increase in the amount of DNAg was observed after the scaffolds were recellularized and by SEM, layers of cells are visible on the extracellular matrix. We conclude that the ECM of dystrophic muscle does not interfere with cell differentiation and adhesion when compared to the ECM of non-dystrophic muscle. Although the increase in dystrophin has been observed in some recellularization processes, more studies need to be carried out to prove this data.

Avaliação de Pericitos no modelo GRMD (Golden Retriever Muscular Dystrophy)

A Distrofia Muscular de Duchenne (DMD) é uma doença genética decorrente de uma mutação ou deleção da distrofina, uma proteína que se expressa através de um gene de caráter recessivo relacionado ao cromossomo X. Através do modelo animal canino Golden Retriever Muscular Dystrophy (GRMD), pesquisas em busca de novas alternativas terapêuticas são objeto de estudo para uma melhor compreensão da doença, por apresentarem um fenótipo similar aos pacientes humanos com DMD. O estudo visa avaliar a morfologia macro e microscópica, quantificar a área de fibrose e identificar células perivasculares (pericitos) nos músculos gastrocnêmio, diafragma, miocárdio e língua de cães Golden Retriever afetados com Distrofia Muscular (n=4) comparados com cães normais (n=4). Foram realizadas análises da morfologia, pelas características anatômicas macroscópicas, microscopia eletrônica de varredura e histologia (colorações de Hematoxilina e Eosina e Picrossírius). Os principais achados macroscópicos foram contratura muscular e hipertrofia (diafragma); infiltrado de tecido adiposo (gastrocnêmio); e a macroglossia. Microscopicamente, as principais alterações foram necrose e degeneração, observada por áreas de hipertrofia e atrofia miofibrilar, acúmulo de gordura e presença de fibrose na musculatura estriada esquelética. Concluiu-se que a musculatura estriada esquelética dos cães GRMD sofreram significativa variação na morfologia, com maior deposição fibrose comparado ao músculo cardíaco. A identificação dos pericitos em animais GRMD, demonstrou uma imunomarcação menos intensa em relação aos cães controle.

Duchenne Muscular Dystrophy (DMD) is a genetic disease caused by a mutation or deletion of the dystrophin, protein which is expressed through a recessive gene associated to X chromosome. The Golden Retriever Muscular Dystrophy (GRMD) canine model serves to new alternatives of therapies and to test better understand the disease pathogenesis, as they present a phenotype similar to the patients with DMD. The aim of this study was to evaluate the macro and microscopic morphology, to quantify the fibrotic area and to identify perivascular cells in the gastrocnemius, diaphragm, myocardium and tongue muscles of Golden Retriever dogs affected with Muscular Dystrophy (n = 4) compared to normal dogs (n = 4). Morphology macroscopic anatomical characteristics and scanning electron microscopy, histological (Hematoxylin and Eosin staining and Picrossírius staining). The main macroscopic findings were muscle contracture and hypertrophy (diaphragm); adipose tissue infiltrate (gastrocnemius); and a macroglossia. Microscopically, the main stages were necrosis and degeneration, observed by areas of hypertrophy and myofibrillar atrophy, fat accumulation and presence of fibrosis in skeletal muscles. After the extended muscles of the GRMD parenchyma were more morphologically varied, with greater deposition of fibrosis to the cardiac muscle. The identity of pericitos in animals GRMD, demonstrated an immunostaining in relation to the remote control.

Alterações morfofuncionais renais em cães Golden Retriever Distróficos (GRMD) / Kidneys morphological changes in Golden Retriever Muscular Dystrophy dogs (GRMD)

A Golden Retriever Muscular Dystrophy (GRMD) é geneticamente homóloga à distrofia muscular de Duchenne (DMD) que acomete seres humanos. É uma doença genética que gera degeneração progressiva da musculatura esquelética. Considerando-se as intensas alterações musculares, é natural pensar em uma possível lesão renal decorrente da intensa lesão muscular. Foram avaliados seis cães machos da raça Golden Retriever afetados pela distrofia muscular (GRMD) e três cães machos clinicamente sadios. A concentração de creatinina foi determinada e as proteínas urinárias foram avaliadas por eletroforese em gel de poliacrilamida. Os resultados mostraram que a proteinúria patológica não está diretamente associada à Distrofia Muscular de Duchenne, porém diversos parâmetros apresentaram concentrações aumentadas para animais afetados, como a razão proteína/albumina, que foi maior em cães distróficos, podendo ser indício de microalbuminúria e conseqüente lesão renal precoce. Estes resultados visam embasar avaliações clínicas e futuros estudos considerando-se as patologias decorrentes ou associadas a esta doença genética.(AU)

The Golden Retriever Muscular Dystrophy (GRMD) is genetically homologous to Duchenne muscular dystrophy (DMD) that affects humans. It is a genetic disease that causes progressive degeneration of skeletal muscle. Considering the intense muscle changes, it is natural to think in possible kidney damage caused by intense muscle injury. We evaluated six male Golden Retriever dogs affected by Duchenne muscular dystrophy (GRMD) and three clinically healthy male dogs. The urinary proteins and creatinine concentration were determined. The proteins were analyzed by polyacrylamide gel electrophoresis. The results showed that pathological proteinuria is not directly associated with Duchenne muscular dystrophy, but several parameters showed increased concentrations for affected animals, as the ratio protein/albumin, which was higher in dystrophic dogs, probably a consequence of microalbuminuria a sign of early kidney damage. These results aim to base future studies and clinical evaluations considering the pathologies arising from or associated with this genetic disease.(AU)

Immunohistochemical and Morphometric Evaluation of Gastrocnemius Muscle and Myotendinous Junction of Golden Retriever Dogs with Muscular Dystrophy

In this research we studied the alterations in the muscles and myotendinous junction of dogs with muscular dystrophy. Gastrocnemius muscle (GM) and myotendinous junction (MTJ) samples of 3 controls and 6 dystrophic dogs were subjected to histopathological and immunohistochemical assays. Histopathological/histomorphometric analysis showed that the lesions in the GM were more pronounced and showed the highest percentage of Fiber Type II (FTII) and low values for the minimum diameters and areas of Fiber Type I (FTI) and FTII than those in the MTJ. FTII in dystrophic MTJ was morphologically similar to the controls. There was a significant difference (p 0.07) in the MHCI antigen compared to the control. We conclude that the preservation of the morphological features of the MTJ can be directly related to a better stabilization of FTII, lower expression of the MHCI complex and less cytotoxic activity of CD8.

Immunohistochemical and Morphometric Evaluation of Gastrocnemius Muscle and Myotendinous Junction of Golden Retriever Dogs with Muscular Dystrophy

In this research we studied the alterations in the muscles and myotendinous junction of dogs with muscular dystrophy. Gastrocnemius muscle (GM) and myotendinous junction (MTJ) samples of 3 controls and 6 dystrophic dogs were subjected to histopathological and immunohistochemical assays. Histopathological/histomorphometric analysis showed that the lesions in the GM were more pronounced and showed the highest percentage of Fiber Type II (FTII) and low values for the minimum diameters and areas of Fiber Type I (FTI) and FTII than those in the MTJ. FTII in dystrophic MTJ was morphologically similar to the controls. There was a significant difference (p 0.07) in the MHCI antigen compared to the control. We conclude that the preservation of the morphological features of the MTJ can be directly related to a better stabilization of FTII, lower expression of the MHCI complex and less cytotoxic activity of CD8.(AU)

Estudo morfofuncional dos rins de cães da raça Golden Retriever afetados pela distrofia muscular (GRMD) / Morphofunctional study in kidneys of Golden Retriever dogs affected by muscular dystrophy (GRMD)

A Distrofia Muscular de Duchenne (DMD) é uma miopatia severa de caráter recessivo ligada ao cromossomo X e o modelo animal de estudo mais relevante é o Golden Retriever Muscular Dystrophy (GRMD). Além das severas alterações que ocorrem na musculatura estriada, muitos estudos mostram que outras estruturas, inclusive viscerais, podem se mostrar alteradas nesta patologia. Desta forma, este trabalho objetivou análisar e comparar possíveis alterações estruturais e funcionais do rim em cães GRMD. Neste modelo de estudo, foi possível observar a presença das faces convexa e côncava, do hilo renal e dos pólos craniais e caudais dos rins. O órgão mostrou-se envolto por uma cápsula fibrosa. Em um corte sagital do órgão, notou-se a presença das regiões cortical e medular e da pelve renal. Na análise microscópica foi possível identificar a zona medular e cortical com suas estruturas: os corpúsculos renais formados pelo glomérulo e pela cápsula de Bowman, os túbulos contorcidos proximais e distais, os ductos coletores, vasos sanguíneos e os segmentos das Alças de Henle. As dosagens séricas de creatinina e uréia encontram-se dentro dos limites de normalidade. Desta forma, de acordo com os nossos resultados, podemos concluir que os animais afetados estudados, não apresentaram alterações estruturais ou funcionais dos rins, o que nos permitir sugerir que apesar da ingestão hídrica comprometida, a estrutura renal, mantem- se preservada nos animais GRMD.(AU)

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 Bowman's 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.(AU)

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.(AU)

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.(AU)

Estudo morfológico do Sistema Nervoso Central de cães com Distrofia Muscular do Golden Retriever (GRMD)

Distrofia muscular de Duchenne é uma desordem neuromuscular causada pela mutação ou deleção do gene da distrofina, a qual é ligada ao cromossomo X. Estudos recentes têm demonstrado o importante papel da distrofina no SNC, sendo sua deficiência relacionada com uma variedade de anormalidades na função do SNC, como comportamento e disfunção cognitiva. Os modelos animais mais adequados para esses estudos são os que apresentam o quadro clinico mais semelhante ao da DMD encontrada em humanos, como cães Golden Retriever com distrofia muscular (GRMD). Por não haver ainda estudos a respeito do SNC de animais GRMD, o objetivo deste trabalho foi analisar a morfologia do encéfalo dos GRMD e o de animais não distróficos, através de análise macroscópica, utilizando métodos de medição e registro fotográfico, e análise microscópica, utilizando a técnica de coloração de violeta cresil modificada. Entretanto, usando a metodologia proposta, não foi possível verificar diferenças significativas no encéfalo quando comparados os animais distróficos e os não distróficos, o que está em concordância com a literatura para a DMD usando os mesmos parâmetros. Em tempo, existe uma variação individual na morfologia do encéfalo do cão, independente de serem animais do grupo de distróficos ou controles. Outras técnicas devem ser aplicadas a fim de elucidar as consequências da ausência total ou parcial da distrofina no SNC.

Duchenne muscular dystrophy is a neuromuscular disorder caused by the mutation or deletion of the dystrophin gene, which is linked to chromosome X. Recent studies have shown the important role of dystrophin in the CNS, and its related defect with a variety of abnormalities in the function of CNS, such as behavior and cognitive dysfunction. The most suitable animals models for these studies are those with the most similar clinical picture to DMD found in humans, as Golden Retriever dogs with muscular dystrophy (GRMD). There are no further studies on the GRMD animal CNS, and the aim of this study was to analyze the morphology of the brain of GRMD and not dystrophic animals through macroscopic analysis using measurement and photographic registration methods, and microscopic analysis using the modified cresyl violet staining technique. However, using the proposed methodology, we could not find significant differences in the brain when comparing the dystrophic animals and non-dystrophic, which is in agreement with the literature for DMD using the same parameters. In time, there is individual variation in dog brain morphology, whether they are animals of the dystrophic group or controls. Other techniques should be applied in order to elucidate the consequences of the total or partial absence of dystrophin in the CNS.

Involvement of Organic Systems in Golden Retriever X-linked Muscular Dystrophy

Duchenne muscular dystrophy is a lethal genetic disease characterized by progressive muscle degeneration that usually had been used the Golden Retriever as a model for studying the disease (GRMD - Golden Retriever Muscular Dystrophy). A total of 16 male dystrophic Golden Retrievers dogs between 5 to 51 months of age were examined in the present study. The animals were classified as dystrophic according to two simultaneous complementary criteria: genotypic analysis and serum creatine kinase levels. The macroscopic abnormalities of the different organs and tissues and histopathological features were described using hematoxylin-eosin. The lesions in the skeletal muscles associated with the digestive problems resulted in cachexia with different intensities in all the dystrophic dogs. Cardiac muscle involvement was found in 87,5% of the GRMD dogs resulting, however, in cardiac failure in only 18,8% of the animals. The musculature of the diaphragm was hypertrophic in all affected animals resulting in progressive respiratory muscle weakness and at later stages in respiratory failure (81,25%). The liver abnormalities found in dystrophic dogs were originated mainly from heart disease and developed progressively. Hyperemia of mucosa and granular material indicated changes in the functioning and emptying of bladder. The germinative lineage cells presented moderate to severe degeneration probably due to degeneration of the scrotum and cremaster muscle which prevented the proper thermo-regulation of the testicle. Our results highlight the fact that there is significant impairment of the cardiac, respiratory and skeletal muscle systems in GRMD dogs since the age of five months. In addition, significant alterations of the gastrointestinal tract, urinary and reproductive systems are indicating the presence of degenerative lesions in the smooth musculature.(AU)

Evaluation of Intracellular Calcium in Golden Retriever Muscular Dystrophy

The present study had the objective of evaluating calcium accumulations in muscle fibers and their correlation with the canine muscular dystrophy. After the deaths of the animals (13 dystrophic and 3 non-dystrophic), samples of the skeletal muscles were collected. The material was stained with hematoxylin-eosin, Gomori's modified trichrome and alizarin red S technique (pH 4.3). The histopathological changes were analyzed and the proportions of calciumpositive (CPF) and negative muscle fibers were evaluated. Histopathological changes such as muscle fiber diameter changes, necrosis, hyalinization, presence of inflammatory infiltrate and fatty atrophy were identified in all the dystrophic muscles. Statistically significant differences in numbers of CPF between dystrophic muscles and nondystrophics were observed for the masseter (6%), brachial biceps (5%) and triceps, sartorius and femoral biceps (4%) muscles. The identifying calcium is of interest as a parameter for helping in diagnostic screening.(AU)

Fetal membranes stem cells application in pets

Background: Stem cells are precursor cells that have the capacity for self-renewal and could generate cells with characteristics similar to cells and differentiation, generating varied cell lines. Considering the plasticity of cells can be classified into totipotent, pluripotent or multipotent. According to the isolation period, the stem cells can be classified as embryonic, fetal and adult. In the embryo stage are considered totipotent because they can rebuild any tissue in the body and adulthood are considered multipotent, since they have a more limited plasticity. The fetal tissues and the fetus is a potential source for stem cells, since they expand more rapidly compared to the cells after birth. Stem cells of fetal membranes are derived from extraembryonic tissues with high capacity to differentiate into various tissues. The cord blood stem cells have mesenchymal and hematopoietic, and mesenchymal cells have the potential to proliferate and differentiate into multiple cell lineages. The yolk sac in dogs is morphologically composed of three layers: a single layer of endoderm, a simple mesothelium, and intermediate to them, the vascular mesenchyme. Work identified a population of pluripotent cells in the yolk sac can differentiate into hematopoietic cells, however, can be isolated mesenchymal stem cells. In this review we aim to focus new isolations of cells from umbilical cord blood and yolk sac of dogs, reviewing the main literature on this species. The importance of using dogs out of work has intensified in recent years, since many diseases can manifest itself in a similar way to humans. Additionally, the dog is a pet, and interest in the treatment of diseases and improved quality of life of this species has been accentuated in veterinary medicine. Thus, identifying the cellular sources in the dog opens new horizons for preclinical studies and new therapies for veterinary medicine. Review: This study is related to morphological biology multipotent stem cells, focusing its expansion and use in cell therapy in animal models that have different pathologies. A widely studied model for muscular dystrophy is the GRMD (Golden Retriever Muscular Dystrophy), which is homologous to DMD (Duchenne Muscular Dystrophy) that affects humans. It is a recessive genetic disease, X chromosome which affects approximately 1 in every 3500 boys. It is characterized by a progressive muscle degeneration, resulting from the absence or reduction in the production of dystrophin protein present in the sarcoplasmic membrane of muscle fibers. Conclusion: The use of cells derived from fetal tissue are strong candidates for veterinary regenerative medicine, since they have high capacity for cellular differentiation. The use of fetuses and fetal tissues of humans still has limitations, so the dog is a viable alternative for studies of fetal stem cells. Thus, it is extremely important to know the characteristics of morphology and proliferation of cells derived from fetuses and fetal annexes canines, including yolk sac and umbilical cord as well as know the feasibility of clinical application of these cells in preclinical testing in animal models and eventually in human medicine, thus contributing to regenerative medicine.