Throughout the performance horse industry, the occurance of various muscle disorders is common and can be detrimental to the performance and longevity of equine athletes. Research has revealed effects of diet manipulation, exercise, and electrolyte supplementation on the symptoms and occurrence of disorders like exertional rhabdomyolysis (ER). However, there has been no investigation on effects of Mg on muscle function in horses during exercise. Six Quarter Horse mares were used to study the effects of varying levels of Mg on indices of muscular dysfunction during a standardized exercise test (SET) on a highspeed treadmill. Three rations were used over three 28 d periods: control (Trt 1), low Mg (Trt 2), and high Mg (Trt 3). A baseline SET was conducted prior to day 0 (Trt 0). Blood samples were taken during the SET at rest, immediately post, 1 h, 6 h, and 24 h post exercise for analyses of serum muscle enzymes and Mg concentrations. Heart rates (HR), respiration rates (RR), and rectal temperatures (RT) also were documented. No effect of Trt was observed on HR or RR at any point throughout the SET. Resting RT?s were lowest in Trt 0 (P<0.05). There was no Trt effect on blood lactate (LA) during the SET; however, blood glucose (GLU) at rest in Trt 2 was lower than Trt 0 (P<0.05). Treatment had an effect on both resting serum creatine phosphokinase (CK), as Trt 3 was higher than Trt 0, and the change in CK from rest to 24 h post SET, where Trt 1 exhibited the greatest increase in CK concentration (P<0.05). Serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were not affected by Trt (P>0.05). Serum alkaline phosphatase (AP) at rest and 6 h post exercise was lower in Trt 2 than in Trt 3 (P<0.05). Additionally, serum P was lowest at rest in Trt 0 and highest 6 h post exercise in Trt 3, also varying within each Trt. Average daily intake (ADI) of Mg was higher in Trt 1 and 3 than in Trt 2 (P<0.05). Finally, at rest, immediately post and 24 h post exercise, serum Mg was highest in Trt 3 (P<0.05). Results suggest there is an effect of dietary Mg on serum muscle enzyme and Mg concentrations and potentially, overall performance in the equine athlete.

Mathematical relationships have long been used to describe many aspects of muscle function such as the relationship between muscle force and muscle length, muscle force and velocity of contraction or the degree of muscle activation during a contraction. During this work various mathematical expressions have been employed in order to gain an insight into different aspects of muscle activity. The first part of the work examined whether performing a strength protocol on a dynamometer can lead to an increase in eccentric strength output as well as in the neuromuscular activation of the quadriceps group of muscles that appears inhibited during slow concentric and fast eccentric contractions. Neuromuscular activation was modelled via a three-parameter sigmoid function that was also tested for robustness to perturbations in the maximum activation values. During the second part of the study the "functional" hamstrings to quadriceps ratio H:Qfun was expressed as a function of two variables i.e., angular velocity and joint angle. Initially nine-parameter torque-angular velocity-angle profiles were obtained for the knee extensors and flexors from a group of participants. A theoretical 17- parameter H:Qfun function was then derived for each dataset. Subsequently, a simpler, 6-parameter function was derived, RE = aexp(bωn + cθm)-dω1/2θ2 that best reproduced the original 17-parameter fit. Finally, a six-segment subject specific torque-driven model of the Snatch lift was developed in order to investigate the optimal mechanics of the lift. The model simulated the lift from its initiation until the end of the second pull when the feet of the athlete momentarily leave the platform. The six-segment model comprised of foot, shank, thigh, torso (head + trunk), arm and forearm segments with torque generators at the ankle, knee, hip and shoulder joints respectively. The torque profiles were obtained using an isokinetic dynamometer.

Background: In older adults, respiratory function may be seriously compromised when a marked decrease of respiratory muscle (RM) strength coexists with comorbidity and activity limitation. Respiratory muscle training has been widely studied and recommended as a treatment option for people who are unable to participate in whole-body exercise training (WBET); however, the effects of inspiratory muscle training and yoga breathing exercises on RM function remain unknown, specifi cally in impaired older adults. Purpose: To evaluate the effects of inspiratory threshold training (ITT) and yoga respiratory training (YRT) on RM function in institutionalized frail older adults. Methods: Eighty-one residents (90% women; mean age, 85 years), who were unable to perform WBET (inability to independently walk more than 10 m), were randomly assigned to a control group or one of the 2 experimental groups (ITT or YRT). Experimental groups performed a supervised intervalbased training protocol, either through threshold inspiratory muscle training device or yoga breathing exercises, which lasted 6 weeks (5 days per week). Outcome measures were collected at 4 time points (pretraining, intermediate, posttraining, and follow-up) and included the maximum respiratory pressures (maximum inspiratory pressure [MIP] and maximum expiratory pressure [MEP]) and the maximum voluntary ventilation (MVV). Results: Seventy-one residents completed the study: control (n = 24); ITT (n = 23); YRT (n = 24). The treatment on had a signifi cant effect on MIP YRT (F 6,204 = 6.755, P <.001, η 2 = 0.166), MEP (F 6,204 = 4.257, P <.001, η 2 = 0.111), and MVV (F 6,204 = 5.322, P <.001, η 2 = 0.135). Analyses showed that the YRT group had a greater increase of RM strength (MIP and MEP) and endurance (MVV) than control and/or ITT groups. Conclusion: Yoga respiratory training appears to be an effective and well-tolerated exercise regimen in frail older adults and may therefore be a useful alternative to ITT or no training, to improve RM function in older population, when WBET is not possible.

No description available.

Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P<0.0001). At 11 wks, only WT+S EDL stress in vitro was greater than all other groups at 50-150 Hz. The in vitro relative stress-frequency relationship of mdx+S EDL was left shifted from the other treatment groups. Inflammatory genetic markers were increased in mdx+S mice. These data suggest treatment of mdx mice with 0.1% SM improves ankle flexor torque in vivo, causes a left shift of the stress-frequency relationship in vitro, and modulates the inflammatory gene signature. / Master of Science

Angiotensin Converting Enzyme (ACE) IID, and a actinin 3 (AC1N3) R577X polymorphisms have been linked to the strength and power performance of elite athletes and suggested to influence skeletal muscle function in the general popUlation. This research investigated the association of these two candidate gene polymorphisms with the muscle function of young and older men, and the distribution of these genotypes in frequent fallers compared to controls. Muscle function measurements of young and older men included isometric strength, absolute and relative isokinetic strength at high velocity (ratio of torque at 2400 ·s"; torque at 30°·s") and the time course of an evoked twitch. Additionally body composition was measured by skinfold thickness (young men) and DXA scanning (old men) to estimate fat-free mass, an index of muscularity, and fat mass. ACE and AC1N3 genotypes were determined from whole blood samples using polymerase chain reaction, and serum ACE activity using spectrophotometry. The gemtypes of frequent fallers referred to a Falls Clinic were compared to a control group of healthy men. ACE genotype was not associated with any measure of muscle function, including the time course of an evoked twitch or absolute and relative high velocity torque, or body composition in these populations (ANOVA, 0.12<P<0.97). Serum ACE activity appeared to be weakly associated with knee extensor (R = 0.19, P = 0.07) and elbow flexor (R = 0.20, P = 0.06) isometric strength in older men, and was negatively correlated with the relative torque at high velocity (R = -0.23, P = 0.03). AC1N3 genotype was associated with fat mass in older men (P = 0.04), but was not associated with any measure of muscle function or muscularity (KruskalWaIIis, 0.26<P<0.95). Finally there was no apparent difference in the distribution of ACE IID (r: = 0.54, P = 0.77) and AC1N3 RIX (r: = 0.76, P = 0.68) genotypes between frequent fallers and controls. Any influence of these individual polymorphisms seems unlikely to be of sufficient magnitude to produce genotype related differences in muscle function in young or older free living UK Caucasian men. Serum ACE activity may have a small association with the isometric and dynamic strength of older men. However, AC1N3 genotype was associated with increased fat mass in XX individuals, that suggests this polymorphism may have an association with the accumulation of body fat over the life span of older men.

A Distrofia Muscular de Duchenne (DMD) é uma importante e severa doença músculo degenerativa causada pela mutação do gene da distrofina. Na ausência da distrofina, o sarcolema das células torna-se vulnerável devido a danos induzidos por ciclos contínuos de degeneração e regeneração. Consequentemente, a força muscular diminui e as miofibras são substituídas por tecido fibrótico. Dentre os músculos esqueléticos afetados, o diafragma, destaca-se por ser o principal músculo respiratório acometido na DMD. Similarmente a DMD humana, o modelo mdx, apesar de exibir um fenótipo suave, este apresenta um severo acometimento no músculo diafragma, assim como observado em nosso estudo prévio, aqui descritos. Entretanto, este é considerado um modelo pobre, porque ele não consegue reproduzir, o fenótipo severo observado nos pacientes. O camundongo mdx/utrn+/- (dystrophin-null heterozygous urtrophin mice) com haploinsuficiência da utrofina, tem sido hipotetizado como um modelo com um fenótipo intermediário, ganhando popularidade nos laboratórios. Porém, infelizmente, até os dias de hoje, não existe evidências fisiológicas e funcionais suficientes que justifiquem a escolha deste modelo para as terapias experimentais. Portanto, neste estudo objetivou-se esclarecer a real contribuição do camundongo mdx/utrn+/- para as terapias experimentais. Para testar esta hipótese, elegeu-se analisar a morfologia e a função muscular do músculo diafragma do mdx e mdx/utrn+/- com 2 e 6 meses de idade. Para elucidar a morfopatologia do diafragma foi utilizado microscopia de luz e análises de imunohistoquímica. A função muscular do diafragma foi analisada através da avaliação das propriedades contráteis e passivas. A forma de como executar a função muscular do diafragma foi atualizada através da criação de dois clips, os quais permitiram avaliar o músculo de forma segura. A adaptação de um novo protocolo de avaliação da função muscular mostrou-se eficaz e capaz de ser utilizada para avaliar as propriedades contráteis e passivas. Aos 2 meses de idade, as colorações de Hematoxilina e eosina, Tricômio de Masson e Alizarina vermelha revelaram que o mdx/utrn+ apresentou maior quantidade de infiltrado inflamatório, tecido conectivo e áreas de calcificação do que o mdx. Já aos 6 meses de idade, não houve diferença morfológica entre o mdx e o mdx/utrn+/-. Na análise de imunohistoquímica para eMyHC (miosina embrionária de cadeia pesada eMyHC), não foi observada diferença entre o mdx e o mdx/utrn+/-aos 2 e 6 meses de idade. Entretanto, os resultados obtidos em ambas as idades, demonstraram a presença de regeneração muscular. Na marcação da proteína distrofina e utrofina, as células inflamatórias e os tipos de fibras musculares também foram detectados por imunohistoquímicas. A marcação da proteína distrofina, mostrou-se ausente no mdx e mdx/utrn+/- com 2 e 6 meses de idade. A marcação da proteína utrofina mostrou-se mais evidente no mdx do que no mdx/utrn+/-, videnciando a haploinsuficiência nos mdx/utrn+/-. Macrófagos foram encontrados em maior quantidade no camundongo mdx/utrn+/- com 2 e 6 meses do que no mdx, mostrando que o processo de inflamação encontrou-se aumentado nos mdx/utrn+/- Neutrófilos encontraram-se aumentados no mdx/utrn+/- com 2 meses, evidenciando a fase aguda da inflamação. Entretanto nos animais de 6 meses, quantidades semelhantes de neutrófilos foram detectadas no mdx/utrn+/- e mdx. Fibras marcadas pelas isoformas MyHC- I, IIa e IIx foram detectadas em maior porcentagem no BL10 do que no mdx e mdx/utrn+/- aos 2 meses de idade, demonstrando que a diminuição destas, no mdx e mdx/utrn+/- pode colaborar para a diminuição da força nestes animais. Aos 6 meses, porcentagem similares de MyHC- I foram detectadas no BL10, mdx e mdx/utrn+/-. MyHC-IIa não foram encontradas nos animais de 6 meses. MyHC-IIx encontraram-se em maior porcentagem no BL10 do que no mdx e mdx/utrn+/- com 6 meses de idade. Baixa porcentagem de isoformas de MyHC- I/IIa foram detectadas no mdx e mdx/utrn+/- aos 2 e 6 meses de idade e encontraram-se ausentes no BL10 de mesma idade. Fibras marcadas pela isoformas MyHC-IIa/IIx apresentaram-se aumentadas no mdx e mdx/utrn+/- aos 2 e 6 meses, podendo este aumento auxiliar na manutenção da força muscular. MyHC-IIx-IIb foram detectadas em maior porcentagem no mdx, reduzidas no BL10 e ausentes no mdx/utrn+/- com 2 meses de idade. Estas não foram identificadas nos animais de 6 meses. As propriedades contráteis do camundongo mdx/utrn+/- aos 2 meses de idade apresentaram-se mais comprometidas do que no mdx. Pt (contração isométrica máxima), sPt (força específica de Pt), Po (Força tetânica máxima) e sPo (força específica de Po) exibiram-se mais afetadas no mdx/utrn+/- do que no mdx. Porém aos 6 meses de idade não houve diferença significativa de força entre o mdx/utrn+/- e o mdx. As propriedades passivas do mdx/utrn+/- com 2 meses apresentou mais acometida do que no mdx. Entretanto, aos 6 meses, esta propriedade não se diferenciou entre o mdx/utrn+/- e o mdx. Em suma, conclui-se que o mdx/utrn+/- representa um modelo superior ao mdx com 2 meses de idade, uma vez que este apresentou morfologia, propriedades contráteis e passiva mais comprometidas do que no mdx. Aos 6 meses as propriedades contráteis e passivas e morfológicas do camundongo mdx/utrn+/- não se diferenciou do mdx. Sugerimos que o uso do mdx/utrn+/- com 2 meses de idade pode potencializar os testes pré-clinicos / Duchenne Muscular Dystrophy (DMD) is an important and severe muscle wasting disease caused by a dystrophin mutation. In the absence of dystrophin, sarcolemma becomes vulnerable to damage due a damage caused by continuous cycles of degeneration regeneration. Consequently, the muscle force reduces and the myofibers are replaced by fibrotic tissue. Between the skeletal muscles, the diaphragm is main affected muscle in DMD. Similarly, to a human DMD, despite the mdx model exhibit a milder phenotype, he presents the diaphragm muscle severely affected, as it was observed in our previous study, here described. However, it is also considered a poor model because it cannot reproduce the severe dystrophic phenotype seen in patients. An utrophin heterozygous utrophin mice (mdx/utrn+/- ), has been hypothesized as an intermediate model and they are gaining popularity in many laboratories. However unfortunately there is currently, no physiological enough evidence to justify the choice of this model for experimental therapies. Therefore, in this study, we aimed to elucidate the real contribution of the mdx/utrn+/- for the experimental therapies. To test this hypothesis, we evaluated the diaphragm muscle morphology and muscle function of the mdx and mdx/utrn+/- with 2 and 6 months. To elucidate the diaphragm morphology, we used light microscopy techniques and immunostaining analysis. Muscle function was evaluated through the active and passive properties. The clamps allowed to safe evaluate the diaphragm function. The update of the new protocol was efficient and able to evaluate the active and passive properties. At 2 months, the Hematoxylin and eosin, Masson Thrichome, Alisarine red, revealed that the mdx/utrn+ showed more inflammatory infiltrate, connective tissue and more areas with calcification than mdx model. At 6 months, there was no significant differences between mdx and mdx/utrn+. In the immunohistochemical analysis for eMyHC (embryonic myosin heavy chain), there was no difference between mdx and mdx/utrn+/- at 2 and 6 months. However, the results obtained in both ages, showed muscle regeneration Marking for dystrophin and utrophin protein, inflammatory cells and fiber type were also detected by immunohistochemistry. Marking for dystrophin was absent in mdx and mdx/utrn+/- with 2 and 6 months. Marking for utrophin protein was more evident in mdx than in mdx/utrn+/- mice, evidencing the utrophin haploinsufficiency in mdx/utr+/-. Macrophages were increased in mdx/utrn+/- than in mdx mice with 2 and 6 months, showing an inflammation. Neutrophils were increased in mdx/utrn+/- at 2 month-old, evidencing the acute phase of inflammation. However, at 6 months similar amounts of neutrophil were detected in mdx and mdx/utrn+/-. Fibers marked by MyHC-I, IIa and IIx were detected in a higher percentage in BL10 than in mdx and mdx/utrn+/- at 2 months, showing that this change could collaborate with force decrease in these animals. At 6 months, similar percentage of MyHC-I was detected in BL10, mdx and mdx/utrn+/-. MyHC-IIa animals were not found at 6 months. Higher percentage of MyHC-IIx was found in BL10 than in mdx and mdx/utrn+/- with 6 months. Low percentage of MyHC- I/IIa was detected in mdx and mdx/utrn+/- at 2 and 6 months and it was absent in BL10 in the same age. Fibers marked by MyHC-IIa/IIx isoforms were increased in mdx and mdx/utrn+/- with 2 and 6 months. These changes could help to maintain the muscle force. MyHC-IIx-IIb was detected in higher percentage in mdx, reduced the BL10 and it was absent in mdx/utrn+/- with 2 months. MyHC-IIx-IIb was not identified in the animals with 6 months. Contractile properties in mdx/utrn+/- at 2 months were more affected than in mdx mice. Pt (maximal twitch force), sPt (specific twitch force), Po (maximal tetanic force) and sPo (specific tetanic force) showed more severely affected in mdx/utrn+/- than in mdx mice. At 6 months there were no significant difference in Pt, sPt, Po and sPo between mdx/utrn+/- and mdx mice. Passive properties of mdx/utrn+/- with 2 months presented more affected than the in mdx mice. However, at 6 months, this property did not differ between mdx/utrn+/- and mdx mice. In summary, we concluded that the mdx/utrn+/- at 2 month represent a superior model than the mdx with matched-age, since they presented morphology and contractile and passive properties more compromised than mdx mice. At 6 months, the mdx/utrn+/-contractile and the passive properties and morphology did not differ from the mdx mice age-matched. We suggest that the use of mdx/utrn+/- with 2 months-old would represent would represent a better model to test the potential of the therapies than mdx mice

The human foot contributes significantly to the function of the whole lower extremity during standing and locomotion. Nevertheless, the foot and ankle often suffer injuries and are affected by many musculoskeletal and neurological pathologies. The overall aim of this thesis was to evaluate gait parameters and muscle function change due to foot and ankle injury, malalignment and co-contraction. Using 3D gait analysis, analytical analyses and computational simulations, biomechanical consequences of gait impairment at the ankle and foot were explored in ablebodied persons and in patient groups with disorders affecting walking. We have characterized gait patterns of subjects with ankle fractures with a modified multi-segment foot model. The inter-segmental foot kinematics were determined during gait in 18 subjects one year after surgically-treated ankle fractures. Gait data were compared to an age- and gender-matched control group and the correlations between functional ankle score and gait parameters were determined. It was observed that even with fairly good clinical results, restricted range of motion and malalignment at and around the injured area were found in the injured limb. Moment-angle relationship (dynamic joint stiffness) - the relationship between changes in joint moment and changes in joint angle - is useful for demonstrating interaction of kinematics and kinetics during gait. Ankle dynamic joint stiffness during the stance phase of gait was analyzed and decomposed into three components in thirty able-bodied children, eight children with juvenile idiopathic arthritis and eight children with idiopathic toe-walking. Compared to controls, the component associated with changes of ground reaction moment was the source of highest deviation in both pathological groups. Specifically, ankle dynamic joint stiffness differences can be further identified via two subcomponents of this component which are based on magnitudes and rates of change of the ground reaction force and of its moment arm. And differences between the two patient groups and controls were most evident and interpretable here. Computational simulations using 3D musculoskeltal models can be powerful in investigating movement mechanisms, which are not otherwise possible or ethical to measure experimentally. We have quantified the effect of subtalar malalignment on the potential dynamic function of the main ankle dorsiflexors and plantarflexors: the gastrocnemius, soleus and tibialis anterior. Induced acceleration analysis was used to compute muscle-induced joint angular and body center of mass accelerations. A three-dimensional subject-specific linkage model was configured by gait data and driven by 1 Newton of individual muscle force. The excessive subtalar inversion or eversion was modified by offsetting up to ±20˚ from the normal subtalar angle while other configurations remain unaltered. We confirmed that in normal gait, muscles generally acted as their anatomical definitions, and that muscles can create motion in many joints, even those not spanned by the muscles. Excessive subtalar eversion was found to enlarge the plantarflexors’ and tibialis anterior’s function. In order to ascertain the reliability of muscle function computed from simulations, we have also performed a parametric study on eight healthy adults to evaluate how sensitive the muscle-induced joints’ accelerations are to the parameters of rigid foot-ground contact model. We quantified accelerations induced by the gastrocnemius, soleus and tibialis anterior on the lower limb joints. Two types of models, a ‘fixed joint’ model with three fixed joints under the foot and a ‘moving joint’ model with one joint located along the moving center of pressure were evaluated. The influences of different foot-ground contact joint constraints and locations of center of pressure were also investigated. Our findings indicate that both joint locations and prescribed degrees-of-freedom of models affect the predicted potential muscle function, wherein the joint locations are most influential. The pronounced influences can be observed in the non-sagittal plane. Excessive muscle co-contraction is a cause of inefficient or abnormal movement in some neuromuscular pathologies. We have identified the necessary compensation strategies to overcome excessive antagonistic muscle cocontraction at the ankle joint and retain a normal walking pattern. Muscle-actuated simulation of normal walking and induced acceleration analysis were performed to quantify compensatory mechanisms of the primary ankle and knee muscles in the presence of normal, medium and high levels of co-contraction of two antagonistic pairs (gastrocnemiustibialis anterior and soleus-tibialis anterior). The study showed that if the co-contraction level increases, the nearby synergistic muscles can contribute most to compensation in the gastrocnemius-tibialis anterior pair. In contrast, with the soleus-tibialis anterior co-contraction, the sartorius and hamstrings can provide important compensatory roles in knee accelerations. This dissertation documented a broad range of gait mechanisms and muscle functions in the foot and ankle area employing both experiments and computational simulations. The strategies and mechanisms in which altered gait and muscles activation are used to compensate for impairment can be regarded as references for evaluation of future patients and for dynamic muscle functions during gait. / QC 20120514

Unilateral, below-knee amputees have altered gait mechanics, which can significantly affect mobility. For example, amputees often have asymmetric leg loading as well as higher metabolic cost and an increased risk of falling compared to non-amputees. Below-knee amputees lose the functional use of the ankle muscles, which are critical in non-amputee walking for providing body support, forward propulsion and leg-swing initiation. The ankle muscles also regulate angular momentum in non-amputees, which is important for providing body stability and preventing falls. Thus, compensatory mechanisms in amputee walking are developed to accomplish the functional tasks normally provided by the ankle muscles. In Chapters 2 and 3, three-dimensional forward dynamics simulations of amputee and non-amputee walking were generated to identify compensatory mechanisms and their effects on joint loading and metabolic cost. Results showed that the prosthesis provided body support, but did not provide sufficient body propulsion or leg-swing initiation. As a result, compensations by the residual leg gluteus maximus, gluteus medius, and hamstrings were needed. The simulations also showed the intact leg tibio-femoral joint contact impulse was greater than the residual leg and that the vasti and hamstrings were the primary contributors to the joint impulse on both the intact and residual legs. The amputee simulation had higher metabolic cost than the non-amputee simulation, which was primarily due to prolonged muscle activity from the residual leg gluteus maximus, gluteus medius, hamstrings, vasti and intact leg vasti and ankle muscles. In Chapter 4, whole-body angular momentum in amputees and non-amputees was analyzed. Reduced residual leg propulsion resulted in a smaller range of sagittal plane angular momentum in the second half of the gait cycle. Thus, to conserve angular momentum, reduced braking was needed in the first half of the gait cycle. Decreased residual leg braking appears to be an important mechanism to regulate sagittal plane angular momentum in amputee walking, but was also associated with a greater range of angular momentum that may contribute to reduced stability in amputees. These studies have provided important insight into compensatory mechanisms in below-knee amputee walking and have the potential to guide rehabilitation methods to improve amputee mobility. / text

A Distrofia Muscular de Duchenne (DMD) é uma importante e severa doença músculo degenerativa causada pela mutação do gene da distrofina. Na ausência da distrofina, o sarcolema das células torna-se vulnerável devido a danos induzidos por ciclos contínuos de degeneração e regeneração. Consequentemente, a força muscular diminui e as miofibras são substituídas por tecido fibrótico. Dentre os músculos esqueléticos afetados, o diafragma, destaca-se por ser o principal músculo respiratório acometido na DMD. Similarmente a DMD humana, o modelo mdx, apesar de exibir um fenótipo suave, este apresenta um severo acometimento no músculo diafragma, assim como observado em nosso estudo prévio, aqui descritos. Entretanto, este é considerado um modelo pobre, porque ele não consegue reproduzir, o fenótipo severo observado nos pacientes. O camundongo mdx/utrn+/- (dystrophin-null heterozygous urtrophin mice) com haploinsuficiência da utrofina, tem sido hipotetizado como um modelo com um fenótipo intermediário, ganhando popularidade nos laboratórios. Porém, infelizmente, até os dias de hoje, não existe evidências fisiológicas e funcionais suficientes que justifiquem a escolha deste modelo para as terapias experimentais. Portanto, neste estudo objetivou-se esclarecer a real contribuição do camundongo mdx/utrn+/- para as terapias experimentais. Para testar esta hipótese, elegeu-se analisar a morfologia e a função muscular do músculo diafragma do mdx e mdx/utrn+/- com 2 e 6 meses de idade. Para elucidar a morfopatologia do diafragma foi utilizado microscopia de luz e análises de imunohistoquímica. A função muscular do diafragma foi analisada através da avaliação das propriedades contráteis e passivas. A forma de como executar a função muscular do diafragma foi atualizada através da criação de dois clips, os quais permitiram avaliar o músculo de forma segura. A adaptação de um novo protocolo de avaliação da função muscular mostrou-se eficaz e capaz de ser utilizada para avaliar as propriedades contráteis e passivas. Aos 2 meses de idade, as colorações de Hematoxilina e eosina, Tricômio de Masson e Alizarina vermelha revelaram que o mdx/utrn+ apresentou maior quantidade de infiltrado inflamatório, tecido conectivo e áreas de calcificação do que o mdx. Já aos 6 meses de idade, não houve diferença morfológica entre o mdx e o mdx/utrn+/-. Na análise de imunohistoquímica para eMyHC (miosina embrionária de cadeia pesada eMyHC), não foi observada diferença entre o mdx e o mdx/utrn+/-aos 2 e 6 meses de idade. Entretanto, os resultados obtidos em ambas as idades, demonstraram a presença de regeneração muscular. Na marcação da proteína distrofina e utrofina, as células inflamatórias e os tipos de fibras musculares também foram detectados por imunohistoquímicas. A marcação da proteína distrofina, mostrou-se ausente no mdx e mdx/utrn+/- com 2 e 6 meses de idade. A marcação da proteína utrofina mostrou-se mais evidente no mdx do que no mdx/utrn+/-, videnciando a haploinsuficiência nos mdx/utrn+/-. Macrófagos foram encontrados em maior quantidade no camundongo mdx/utrn+/- com 2 e 6 meses do que no mdx, mostrando que o processo de inflamação encontrou-se aumentado nos mdx/utrn+/- Neutrófilos encontraram-se aumentados no mdx/utrn+/- com 2 meses, evidenciando a fase aguda da inflamação. Entretanto nos animais de 6 meses, quantidades semelhantes de neutrófilos foram detectadas no mdx/utrn+/- e mdx. Fibras marcadas pelas isoformas MyHC- I, IIa e IIx foram detectadas em maior porcentagem no BL10 do que no mdx e mdx/utrn+/- aos 2 meses de idade, demonstrando que a diminuição destas, no mdx e mdx/utrn+/- pode colaborar para a diminuição da força nestes animais. Aos 6 meses, porcentagem similares de MyHC- I foram detectadas no BL10, mdx e mdx/utrn+/-. MyHC-IIa não foram encontradas nos animais de 6 meses. MyHC-IIx encontraram-se em maior porcentagem no BL10 do que no mdx e mdx/utrn+/- com 6 meses de idade. Baixa porcentagem de isoformas de MyHC- I/IIa foram detectadas no mdx e mdx/utrn+/- aos 2 e 6 meses de idade e encontraram-se ausentes no BL10 de mesma idade. Fibras marcadas pela isoformas MyHC-IIa/IIx apresentaram-se aumentadas no mdx e mdx/utrn+/- aos 2 e 6 meses, podendo este aumento auxiliar na manutenção da força muscular. MyHC-IIx-IIb foram detectadas em maior porcentagem no mdx, reduzidas no BL10 e ausentes no mdx/utrn+/- com 2 meses de idade. Estas não foram identificadas nos animais de 6 meses. As propriedades contráteis do camundongo mdx/utrn+/- aos 2 meses de idade apresentaram-se mais comprometidas do que no mdx. Pt (contração isométrica máxima), sPt (força específica de Pt), Po (Força tetânica máxima) e sPo (força específica de Po) exibiram-se mais afetadas no mdx/utrn+/- do que no mdx. Porém aos 6 meses de idade não houve diferença significativa de força entre o mdx/utrn+/- e o mdx. As propriedades passivas do mdx/utrn+/- com 2 meses apresentou mais acometida do que no mdx. Entretanto, aos 6 meses, esta propriedade não se diferenciou entre o mdx/utrn+/- e o mdx. Em suma, conclui-se que o mdx/utrn+/- representa um modelo superior ao mdx com 2 meses de idade, uma vez que este apresentou morfologia, propriedades contráteis e passiva mais comprometidas do que no mdx. Aos 6 meses as propriedades contráteis e passivas e morfológicas do camundongo mdx/utrn+/- não se diferenciou do mdx. Sugerimos que o uso do mdx/utrn+/- com 2 meses de idade pode potencializar os testes pré-clinicos / Duchenne Muscular Dystrophy (DMD) is an important and severe muscle wasting disease caused by a dystrophin mutation. In the absence of dystrophin, sarcolemma becomes vulnerable to damage due a damage caused by continuous cycles of degeneration regeneration. Consequently, the muscle force reduces and the myofibers are replaced by fibrotic tissue. Between the skeletal muscles, the diaphragm is main affected muscle in DMD. Similarly, to a human DMD, despite the mdx model exhibit a milder phenotype, he presents the diaphragm muscle severely affected, as it was observed in our previous study, here described. However, it is also considered a poor model because it cannot reproduce the severe dystrophic phenotype seen in patients. An utrophin heterozygous utrophin mice (mdx/utrn+/- ), has been hypothesized as an intermediate model and they are gaining popularity in many laboratories. However unfortunately there is currently, no physiological enough evidence to justify the choice of this model for experimental therapies. Therefore, in this study, we aimed to elucidate the real contribution of the mdx/utrn+/- for the experimental therapies. To test this hypothesis, we evaluated the diaphragm muscle morphology and muscle function of the mdx and mdx/utrn+/- with 2 and 6 months. To elucidate the diaphragm morphology, we used light microscopy techniques and immunostaining analysis. Muscle function was evaluated through the active and passive properties. The clamps allowed to safe evaluate the diaphragm function. The update of the new protocol was efficient and able to evaluate the active and passive properties. At 2 months, the Hematoxylin and eosin, Masson Thrichome, Alisarine red, revealed that the mdx/utrn+ showed more inflammatory infiltrate, connective tissue and more areas with calcification than mdx model. At 6 months, there was no significant differences between mdx and mdx/utrn+. In the immunohistochemical analysis for eMyHC (embryonic myosin heavy chain), there was no difference between mdx and mdx/utrn+/- at 2 and 6 months. However, the results obtained in both ages, showed muscle regeneration Marking for dystrophin and utrophin protein, inflammatory cells and fiber type were also detected by immunohistochemistry. Marking for dystrophin was absent in mdx and mdx/utrn+/- with 2 and 6 months. Marking for utrophin protein was more evident in mdx than in mdx/utrn+/- mice, evidencing the utrophin haploinsufficiency in mdx/utr+/-. Macrophages were increased in mdx/utrn+/- than in mdx mice with 2 and 6 months, showing an inflammation. Neutrophils were increased in mdx/utrn+/- at 2 month-old, evidencing the acute phase of inflammation. However, at 6 months similar amounts of neutrophil were detected in mdx and mdx/utrn+/-. Fibers marked by MyHC-I, IIa and IIx were detected in a higher percentage in BL10 than in mdx and mdx/utrn+/- at 2 months, showing that this change could collaborate with force decrease in these animals. At 6 months, similar percentage of MyHC-I was detected in BL10, mdx and mdx/utrn+/-. MyHC-IIa animals were not found at 6 months. Higher percentage of MyHC-IIx was found in BL10 than in mdx and mdx/utrn+/- with 6 months. Low percentage of MyHC- I/IIa was detected in mdx and mdx/utrn+/- at 2 and 6 months and it was absent in BL10 in the same age. Fibers marked by MyHC-IIa/IIx isoforms were increased in mdx and mdx/utrn+/- with 2 and 6 months. These changes could help to maintain the muscle force. MyHC-IIx-IIb was detected in higher percentage in mdx, reduced the BL10 and it was absent in mdx/utrn+/- with 2 months. MyHC-IIx-IIb was not identified in the animals with 6 months. Contractile properties in mdx/utrn+/- at 2 months were more affected than in mdx mice. Pt (maximal twitch force), sPt (specific twitch force), Po (maximal tetanic force) and sPo (specific tetanic force) showed more severely affected in mdx/utrn+/- than in mdx mice. At 6 months there were no significant difference in Pt, sPt, Po and sPo between mdx/utrn+/- and mdx mice. Passive properties of mdx/utrn+/- with 2 months presented more affected than the in mdx mice. However, at 6 months, this property did not differ between mdx/utrn+/- and mdx mice. In summary, we concluded that the mdx/utrn+/- at 2 month represent a superior model than the mdx with matched-age, since they presented morphology and contractile and passive properties more compromised than mdx mice. At 6 months, the mdx/utrn+/-contractile and the passive properties and morphology did not differ from the mdx mice age-matched. We suggest that the use of mdx/utrn+/- with 2 months-old would represent would represent a better model to test the potential of the therapies than mdx mice