Thoroughbred racehorses undergo strenuous exercise which often leads to the occurrence of exercise-induced pulmonary hemorrhage (EIPH), in which capillaries rupture within the alveoli in the lungs causing bleeding. Severe cases of EIPH lead to epistaxis and may result in fatality. Presently, the loop diuretic furosemide is the only medication approved to mitigate the effects of EIPH. Often regarded in the racing industry as "performance enhancing" due to 4% weight loss ensued by its diuretic effect, it is unknown what effects furosemide may have on muscle recovery. Therefore, the objective of this study was to determine the effects various doses of furosemide may have on equine satellite cell (eqSC) myogenesis and metabolism. Mitotic index was increased (P<0.05) as a result of treatment with 100 µg/mL furosemide, a 10-fold pharmacological dose, in comparison to vehicle, but was not different (P>0.05) compared to the physiological dose of 10 µg/mL furosemide. Average cell number decreased (P<0.05) in the excess furosemide group compared to all other groups. Pax7 expression did not differ (P>0.05) between groups. Expression of the differentiation transcription factor myogenin, and embryonic sarcomeric myosin heavy chain decreased (P<0.05) when cells were treated with 100 µg/mL furosemide. Fusion index and myotube area decreased (P<0.05) as a result of treatment with excess furosemide. Glycogen concentration in myotubes was lower (P<0.05) following treatment with 100 µg/mL furosemide, while IGF-1 was unsuccessful in rescuing the effects of furosemide. Excess furosemide decreased expression of muscle creatine kinase while increasing expression of phosphoglucomutase 1, glycogen synthase 1, and glycogen branching enzyme 1 (P<0.05). Excess furosemide decreased basal oxygen consumption rate (OCR) and increased OCR after addition of oligomycin (P<0.05). Excess furosemide did not affect myotube glycolysis rates in vitro. In conclusion, furosemide inhibits muscle differentiation and oxidative metabolism in eqSCs. / Master of Science / Thoroughbred racehorses often bleed from the lungs as a result of high-intensity exercise. This condition can oftentimes be fatal depending on severity. Furosemide, is used in the industry to reduce blood pressure within the lungs during racing to prevent bleeding. Furosemide, a diuretic given four hours prior to a race, causes a horse to excrete up to 4% of its body weight. This effect of furosemide decreases the weight a horse must carry during a race, thus allowing the horse to run faster. Therefore, deemed as a performance enhancing drug due to its effects on the kidney, to our knowledge, no research has been conducted on what effects furosemide might have on muscle generation. High-intensity exercise causes massive muscle damage and therefore must be repaired to prepare for the next bout of exercise. Muscle generation is called myogenesis. Stem cells, or satellite cells, that lie within the muscle become activated, recognizing the need for muscle repair. Satellite cells divide, increasing in cell number and then fuse together, forming new muscle fibers. Satellite cells undergo different types of metabolism depending on their state of development. For example, proliferating cells require glucose for energy, while cells fusing together forming myotubes, require oxidative metabolism for long-lasting energy. Therefore, the objective of this study was to determine the effects furosemide might have on muscle formation and metabolism. The excess furosemide dose (100 µg/mL) decreased cell proliferation. The expression of regulatory factors responsible for forming myotubes at different stages of muscle development are decreased when cells were treated with the defined excess furosemide dose. Furosemide decreased the ability of satellite cells to generate myotubes. Glycogen concentration was also decreased as a result of excess furosemide treatment. Gene expression of enzymes involved in glycogen synthesis were increased from treatment with our excess furosemide dose. No effect of furosemide was seen on glycolysis, whereas oxidative metabolism suffered as a result of treatment with excess furosemide. In conclusion, furosemide does indeed affect muscle generation and oxidative metabolism.

A hemorragia pulmonar induzida por esforço (HPIE) ocorre com freqüência em cavalos atletas acarretando prejuízo econômico já que estes deverão ficar temporariamente afastados dos treinamentos e provas, além de receber tratamento adequado. O Puro-Sangue Inglês (PSI) utilizado em provas de corrida é o animal mais afetado devido ao grande esforço ao qual é submetido, porém animais de outras raças utilizados em outras modalidades de esportes hípicos também podem apresentar HPIE. O presente estudo teve como objetivo quantificar a ocorrência de casos de HPIE em animais utilizados em provas de salto, através do exame endoscópico e da análise citológica de amostras do lavado traqueobrônquico (LTB). Para a realização do presente experimento foram utilizados 50 cavalos adultos residentes nos clubes hípicos de São Paulo, que foram divididos em dois grupos, A e B, conforme as diferentes alturas dos obstáculos utilizados nas provas em que participaram. No grupo A foram inseridos os cavalos que saltam obstáculos de 1,00m a 1,20m de altura, e no grupo B, os cavalos que saltam obstáculos de 1,30m a 1,50m de altura. Foram realizados exames físicos destes animais, antes e depois da participação em uma prova. Após 1 hora do final da prova, os mesmos foram submetidos a um exame endoscópico em que eram observadas as possíveis alterações do trato respiratório anterior do animal sendo a principal delas a hemorragia pulmonar induzida por esforço. Foi realizada citologia do lavado traqueobônquico (LTB) por microscopia após citocentrifugação das amostras e posterior confecção das lâminas que foram então coradas por Rosenfeld. A principal observação durante a leitura das lâminas foi à presença de hemossiderófagos. No trabalho realizado são apresentados além da citologia do trato respiratório dados como os valores médios de freqüência cardíaca, freqüência respiratória e temperatura corpórea dos animais após a participação em uma prova. Estes dados foram relacionados às alturas dos obstáculos existentes em cada prova realizada pelos animais. Em relação à ocorrência de HPIE observamos que no grupo A 50% dos animais apresentaram HPIE e no grupo B a ocorrência foi de 66,67%. Foram observados todos os graus de HPIE, porém somente no grupo B houve ocorrência de graus IV e V, demonstrando assim a correlação entre o nível de esforço realizado pelos cavalos de salto e o grau de HPIE apresentado por eles. / Effort induced pulmonary hemorrhage (EIPH) frequently occurs in athlete horses causing economic losses not only due to temporary removal from training and competition, but also due to the cost of adequate treatment. Thoroughbreds used in racing are the most frequently affected breed due to the great effort to which they are submitted; however other breeds and crossbred sport horses can also have EIPH. The present study´s objective is to quantify the occurrence of cases of EIPH in animals used in show jumping, through the endoscopic examination and cytological analysis of tracheobronchial lavage (TBL) samples. For the present experiment fifty adult horses, housed in maneges in the city of São Paulo, were used. Horses were divided into two groups, named A and B according to the different heights of the obstacles. Group A included horses that jump heights of 1.0 to 1.2 m and group B, obstacles of 1.3 to 1.5 m. Physical examinations of these animals were carried through, before and after participation in each test. One hour after the end of each series, the same ones had been submitted to an endoscopic examination where the possible alterations of previous the respiratory treatment of the animal were observed being main of them the induced pulmonary hemorrhage for effort. Cytology of tracheobronchial lavage (TBL) was performed in cytocentrifuge slides prepared on the same day, slides were stained using Rosenfeld Romanov and observed examined under light microscopy. The major focus during the observation of the slides was the presence of hemosiderophages and grading of EIPH intensity (I through V) Besides the cytological evaluation of the respiratory tract, this study includes clinical data such as the average values of cardiac frequency, respiratory frequency and body temperature of the animals after the each competition. These data were correlated with the heights of the existing obstacles jumped by each athlete horse. In relation to the EIPH occurrence we observed that in the group A 50% of the animals presented EIPH and in group B the occurrence was of 66,67%. All grades of EIPH were observed when both groups were considered, however only animals in group B showed intense hemorrhage levels (IV or V) thus demonstrating and the correlation between the levels of effort the intensity of EIPH in jumpers.

A hemorragia pulmonar induzida por esforço (HPIE) ocorre com freqüência em cavalos atletas acarretando prejuízo econômico já que estes deverão ficar temporariamente afastados dos treinamentos e provas, além de receber tratamento adequado. O Puro-Sangue Inglês (PSI) utilizado em provas de corrida é o animal mais afetado devido ao grande esforço ao qual é submetido, porém animais de outras raças utilizados em outras modalidades de esportes hípicos também podem apresentar HPIE. O presente estudo teve como objetivo quantificar a ocorrência de casos de HPIE em animais utilizados em provas de salto, através do exame endoscópico e da análise citológica de amostras do lavado traqueobrônquico (LTB). Para a realização do presente experimento foram utilizados 50 cavalos adultos residentes nos clubes hípicos de São Paulo, que foram divididos em dois grupos, A e B, conforme as diferentes alturas dos obstáculos utilizados nas provas em que participaram. No grupo A foram inseridos os cavalos que saltam obstáculos de 1,00m a 1,20m de altura, e no grupo B, os cavalos que saltam obstáculos de 1,30m a 1,50m de altura. Foram realizados exames físicos destes animais, antes e depois da participação em uma prova. Após 1 hora do final da prova, os mesmos foram submetidos a um exame endoscópico em que eram observadas as possíveis alterações do trato respiratório anterior do animal sendo a principal delas a hemorragia pulmonar induzida por esforço. Foi realizada citologia do lavado traqueobônquico (LTB) por microscopia após citocentrifugação das amostras e posterior confecção das lâminas que foram então coradas por Rosenfeld. A principal observação durante a leitura das lâminas foi à presença de hemossiderófagos. No trabalho realizado são apresentados além da citologia do trato respiratório dados como os valores médios de freqüência cardíaca, freqüência respiratória e temperatura corpórea dos animais após a participação em uma prova. Estes dados foram relacionados às alturas dos obstáculos existentes em cada prova realizada pelos animais. Em relação à ocorrência de HPIE observamos que no grupo A 50% dos animais apresentaram HPIE e no grupo B a ocorrência foi de 66,67%. Foram observados todos os graus de HPIE, porém somente no grupo B houve ocorrência de graus IV e V, demonstrando assim a correlação entre o nível de esforço realizado pelos cavalos de salto e o grau de HPIE apresentado por eles. / Effort induced pulmonary hemorrhage (EIPH) frequently occurs in athlete horses causing economic losses not only due to temporary removal from training and competition, but also due to the cost of adequate treatment. Thoroughbreds used in racing are the most frequently affected breed due to the great effort to which they are submitted; however other breeds and crossbred sport horses can also have EIPH. The present study´s objective is to quantify the occurrence of cases of EIPH in animals used in show jumping, through the endoscopic examination and cytological analysis of tracheobronchial lavage (TBL) samples. For the present experiment fifty adult horses, housed in maneges in the city of São Paulo, were used. Horses were divided into two groups, named A and B according to the different heights of the obstacles. Group A included horses that jump heights of 1.0 to 1.2 m and group B, obstacles of 1.3 to 1.5 m. Physical examinations of these animals were carried through, before and after participation in each test. One hour after the end of each series, the same ones had been submitted to an endoscopic examination where the possible alterations of previous the respiratory treatment of the animal were observed being main of them the induced pulmonary hemorrhage for effort. Cytology of tracheobronchial lavage (TBL) was performed in cytocentrifuge slides prepared on the same day, slides were stained using Rosenfeld Romanov and observed examined under light microscopy. The major focus during the observation of the slides was the presence of hemosiderophages and grading of EIPH intensity (I through V) Besides the cytological evaluation of the respiratory tract, this study includes clinical data such as the average values of cardiac frequency, respiratory frequency and body temperature of the animals after the each competition. These data were correlated with the heights of the existing obstacles jumped by each athlete horse. In relation to the EIPH occurrence we observed that in the group A 50% of the animals presented EIPH and in group B the occurrence was of 66,67%. All grades of EIPH were observed when both groups were considered, however only animals in group B showed intense hemorrhage levels (IV or V) thus demonstrating and the correlation between the levels of effort the intensity of EIPH in jumpers.

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-05Bitstream added on 2014-06-13T20:50:59Z : No. of bitstreams: 1 biava_js_me_botfmvz.pdf: 642582 bytes, checksum: 7fc291f591db538f894f33f392dc36b4 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Hemorragia Pulmonar Induzida por Exercício (EIPH) é um processo caracterizado por apresentar sangue de origem pulmonar nas vias aéreas dos cavalos, em particular após exercício físico intenso. A presença de sangue na árvore traqueobrônquica é o principal sinal clínico desta síndrome, e afeta comumente cavalos atletas que participam de exercícios de curto percurso e alta intensidade. A síndrome está bem documentada em cavalos de corrida, onde a principal queixa é a incapacidade de manter desempenho atlético satisfatório durante a competição esportiva. Embora o grau de sangramento nesses animais seja variável, até 90% dos cavalos podem apresentar EIPH. Apesar de sua importância em cavalos de corrida o problema é ainda pouco investigado em outros cavalos de esporte como pólo, provas de três tambores, seis balizas, salto, laço em dupla e rédeas, entre outros. Com o objetivo de melhor conhecer os sinais clínicos e a prevalência de EIPH em cavalos da raça Quarto de Milha, bem como os potenciais fatores que predispõem o seu aparecimento, foram realizados os exames clínico, endoscópico e citológico do lavado broncoalveolar, colhido às cegas em tubo de silicone. Foram incluidos no presente estudo, 60 cavalos da raça Quarto de Milha da região metropolitana de Curitiba/PR, sendo 40 em provas de Seis Balizas e Três Tambores e 20 em provas de Laço em Dupla. Esses animais, após treino de uma hora nas atividades de laço em dupla, de três tambores e seis balizas, foram submetidos imediatamente ao exame clínico, e após 30 a 60 minutos ao exame endoscópico e citológico do lavado broncoalveolar (BAL). A presença e o grau de sangramento da EIPH foi determinada à inspeção, pelo exame endoscópico, utilizando-se a escala de Eppinger (1990) e pela contagem total de hemácias e percentual de hemossiderófagos, ao exame citológico / Exercise-Induced Pulmonary Hemorrhage (EIPH) is a process characterized by presence of pulmonary origin blood in upper airway of horses, particularly after intense physical exercise. The presence of blood in the tracheo-broncheal tree is the main sign of this syndrome, and it often affects horses which participate in short length and high intensity exercises. The syndrome is well reported in racing horses, in which the main complaining is the lack of capacity of athletic performance maintenance during sportive competition. Although the level of bleeding in these animals varies, up to 90% of horses may present EIPH. Despite its importance in racing horses, the problem is still under investigated in other sports horses such as polo, three barrels, six poles, jumping, roping, among others. To better understand clinical signs and prevalence of EIPH in Quarter horses of such activities, as well as the potential factors which predispose its appearance, clinical, endoscopic and cytological exams of broncho-alveolar lavage, was blinded sampled in silicon tubes. A total of 60 Quarter horses from Curitiba, Parana and surroundings were evaluated, with 40 horses of six poles and three barrels competition, and with 20 horses of double roping competition were included in the present study. Theses animals, after training of one hour in the respective activity, were immediately submitted to physical examination, and after 30 and 60 minutes to endoscopic and bronco-alveolar lavage (BAL) cytologic exams. EIPH presence and degree of bleeding was determinated by inspection, endoscopic exam, using the Eppinger scale and by the total erythrocyte count and hemosiderophage percentage on the cytologic smears. Slides of BAL were stained with Prussian blue for adequate evaluation of hemosiderophages, and with Giemsa and Papanicolau for differential cellular counting of BAL. The results showed absence of blood in the respiratory upper airway

Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / Exercise-induced pulmonary hemorrhage (EIPH) or epistaxis has been recognized in racehorses since the 16th century. Since this time, great strides have been made in terms of identifying the lungs as the source of the hemorrhage via the endoscope, utilization of bronchoalveolar lavage to quantify the hemorrhage, and the discovery of successful treatments such as furosemide and the nasal strip that ameliorate, but do not abolish EIPH. It has been determined that, in addition to extremely high pulmonary arterial pressures and the negative intrapleural pressures being the major physiologic forces causing pulmonary capillary stress failure, other factors have the potential for influencing the severity of EIPH including locomotory impact trauma, inflammatory airway disease (IAD), upper airway obstruction, coagulation anomalies, and high blood viscosity. It has been hypothesized that EIPH is detrimental to performance and this was recently confirmed by Hinchcliff et al. in 2004. EIPH is a complex multi-factorial condition with much still unknown about the etiology, best method for diagnosis, and most effective form of treatment. Chapter one of this dissertation determined the effectiveness of a novel treatment, concentrated equine serum, in ameliorating EIPH via reduction of IAD. Chapter two refuted the hypothesis that herbal formulations commonly used in the field with anecdotal success would decrease EIPH by correcting coagulation deficits during exercise, as scientific efficacy was not evident, at least at the dose and duration used in our investigation. Chapter three addressed the dogma that EIPH only occurs during maximal intensity exercise, and in demonstrating significant EIPH during sub-maximal exercise, emphasized the role that the airways play in contributing to the initiation and severity of EIPH. Chapter four examined the occurrence and severity of EIPH in the horse’s canine counterpart, the racing Greyhound. The demonstrated presence of mild EIPH in the Greyhound, a physiologically similar yet different athlete in comparison to the horse sheds new light on the etiology of this condition in both species. The results of these investigations have advanced the frontiers of our knowledge concerning EIPH. Specifically, they have generated novel information on the mechanistic bases of EIPH and have provided evidence supporting additional treatment options for reducing the severity of EIPH in horses.