Effect of robotic gait training on muscle and bone characteristics in spinal cord transected rats.

Osteoporosis and loss of muscle mass are secondary issues with spinal cord injury. Robotic gait training has provided evidence of increasing bone density and muscle mass, but its effect on bone strength is undetermined. The purpose of this study was to determine the effect of a 6-week robotic locomotion training program on skeletal muscle mass and bone characteristics. Twelve female Sprague-Dawley rats received a mid-thoracic spinal cord transection at 5 days old and at 3 weeks old were assigned to a Control or Trained Group. The Trained Group performed 5-min sessions on the Rat Stepper 5 days a week for 6 weeks with 90% of body weight supported. At the end of the 6 weeks, body mass was obtained and right femurs and four lower extremity muscles were harvested. Femur bone mineral density was measured with DXA and mechanical characteristics of the femur were determined via 3-point bending testing. Independent t-tests, effects sizes and percent differences were computed between the two groups (p < 0.05). The Trained Group had significantly larger normalized femur mass (p = 0.007) and normalized soleus muscle mass (p = 0.033) when compared to the Control Group. There was a medium or large effect size with the Trained Groups' femurs having larger mass, bone mineral density, rupture loads, cortical wall thickness, shaft cross sectional area, soleus mass, normalized gastrocnemius mass, and smaller shaft inner diameters compared to the Control Group. These changes may contribute to decreasing osteoporosis and fracture risk in those with spinal cord injuries.

Assessment of hindlimb locomotor strength in spinal cord transected rats through animal-robot contact force.

Robotic locomotor training devices have gained popularity in recent years, yet little has been reported regarding contact forces experienced by the subject performing automated locomotor training, particularly in animal models of neurological injury. The purpose of this study was to develop a means for acquiring contact forces between a robotic device and a rodent model of spinal cord injury through instrumentation of a robotic gait training device (the rat stepper) with miniature force/torque sensors. Sensors were placed at each interface between the robot arm and animal's hindlimb and underneath the stepping surface of both hindpaws (four sensors total). Twenty four female, Sprague-Dawley rats received mid-thoracic spinal cord transections as neonates and were included in the study. Of these 24 animals, training began for 18 animals at 21 days of age and continued for four weeks at five min/day, five days/week. The remaining six animals were untrained. Animal-robot contact forces were acquired for trained animals weekly and untrained animals every two weeks while stepping in the robotic device with both 60 and 90% of their body weight supported (BWS). Animals that received training significantly increased the number of weight supported steps over the four week training period. Analysis of raw contact forces revealed significant increases in forward swing and ground reaction forces during this time, and multiple aspects of animal-robot contact forces were significantly correlated with weight bearing stepping. However, when contact forces were normalized to animal body weight, these increasing trends were no longer present. Comparison of trained and untrained animals revealed significant differences in normalized ground reaction forces (both horizontal and vertical) and normalized forward swing force. Finally, both forward swing and ground reaction forces were significantly reduced at 90% BWS when compared to the 60% condition. These results suggest that measurement of animal-robot contact forces using the instrumented rat stepper can provide a sensitive and reliable measure of hindlimb locomotor strength and control of flexor and extensor muscle activity in neurologically impaired animals. Additionally, these measures may be useful as a means to quantify training intensity or dose-related functional outcomes of automated training.

Robot applied stance loading increases hindlimb muscle mass and stepping kinetics in a rat model of spinal cord injury.

Following spinal cord injury (SCI) reduced limb usage typically results in muscle atrophy. While robotic locomotor training has been shown to improve several aspects of stepping ability following SCI, little is known regarding the effects of automated training on the preservation of muscle function. The purpose of this study was to evaluate the effects of two robotic locomotor training algorithms on hindlimb strength and muscle mass in a rat model of SCI. Eighteen Sprague-Dawley rats received a mid-thoracic spinal cord transection at 5 days of age, and were randomly assigned to one of three groups: control (no training), standard robotic training, and robotic training with a downward force applied to the shank during the stance phase of gait. Training occurred 5 days/week for 5 min/day, and animals received 90% body weight support for all sessions. Following 4 weeks of training, vertical and propulsive ground reaction force during stepping and en vitro mass of two plantarflexor muscles were significantly increased for all of the trained animals when compared to the untrained control group. Post hoc analysis revealed that standard robotic training did not appear to increase ground reaction force and muscle mass to the same extent as the loaded condition. These results indicate that automated robotic training helps to preserve hindlimb muscle function in rats following SCI. Further, the addition of a plantarflexion stance load appears to promote greater increases in muscle mass and stepping kinetics.

Vascular hypertrophy and hypertension caused by transgenic overexpression of profilin 1.

We have overexpressed either the cDNA of human profilin 1 or expressed the mutant (88R/L) in the blood vessels of transgenic FVB/N mice. Reverse transcription-PCR indicated selective overexpression of profilin 1 and 88R/L in vascular smooth muscle cells. Polyproline binding showed increased profilin 1 and 88R/L proteins in transgenic mice compared with control (~30%, p < 0.05). Rhodamine-phalloidin staining revealed increase stress fiber formation in vascular smooth muscle cells of profilin 1 compared with 88R/L and control. Hematoxylin and eosin staining showed clear signs of vascular hypertrophy in the aorta of profilin 1 mice versus 88R/L and control. However, there were no differences between 88R/L and control mice. Western blotting confirmed the activation of the hypertrophic signaling cascades in aortas of profilin 1 mice. Phospho-ERK1/2 was significantly higher in profilin 1 than 88R/L and control (512.3 and 361.7%, respectively, p < 0.05). Profilin 1 mice had significant increases in phospho-JNK as compared with 88R/L and control (371.4 and 346%, respectively, p < 0.05). However, there were no differences between 88R/L and control mice in both kinases. There was a significant increase in ROCK II kinase in the aorta of profilin 1 mice compared with controls (>400%, p < 0.05). Tail cuff and circadian monitoring of blood pressure showed significant increases in systolic and mean arterial blood pressures of profilin 1 mice starting at age 6 months compared with controls (~25 mm Hg, p < 0.05). These results suggest that increased actin polymerization in blood vessels triggers activation of the hypertrophic signaling cascades and results in elevation of blood pressure at advanced age.

Hypertension caused by transgenic overexpression of Rac1.

Reactive oxygen species, including superoxide, are important mediators of the pathophysiology of hypertension. In the vasculature, superoxide antagonizes nitric oxide (NO*), resulting in increased vascular tone. The GTP binding protein Rac regulates a wide variety of cellular functions, including the activation of NADPH oxidase, the major source of O2*-in the blood vessel wall. An hypothesis is that Rac1 may act as an important regulator of vascular O2*- production, contributing to the balance between O2*- and NO* and maintaining consequent homeostasis of blood pressure. To alter the activity of vascular NADPH oxidase, the authors developed a transgenic animal model that overexpresses the human cDNA of the constitutively active mutant of Rac1 (RacCA) in smooth muscle cells using the smooth muscle +/--actin promoter. The RacCA transgenic had excessive amounts of O2*- in the vessel wall that, which led to heightened production of peroxynitrite, as detected by increased protein nitration and reduced NO* levels. RacCA mice developed moderate hypertension, which was corrected by N-acetyl-L-cysteine (NAC). RacCA transgenic mice also developed left ventricular hypertrophy as a secondary effect of pressure overload. The data suggest that Rac1 is a critical regulator of the redox state of blood vessels and homeostasis of blood pressure.

Endurance exercise training attenuates cardiac beta2-adrenoceptor responsiveness and prevents ventricular fibrillation in animals susceptible to sudden death.

Enhanced cardiac beta(2)-adrenoceptor (beta(2)-AR) responsiveness can increase susceptibility to ventricular fibrillation (VF). Exercise training can decrease cardiac sympathetic activity and could, thereby, reduce beta(2)-AR responsiveness and decrease the risk for VF. Therefore, dogs with healed myocardial infarctions were subjected to 2 min of coronary occlusion during the last minute of a submaximal exercise test; VF was observed in 20 susceptible, but not in 13 resistant, dogs. The dogs were then subjected to a 10-wk exercise-training program (n = 9 susceptible and 8 resistant) or an equivalent sedentary period (n = 11 susceptible and 5 resistant). Before training, the beta(2)-AR antagonist ICI-118551 (0.2 mg/kg) significantly reduced the peak contractile (by echocardiography) response to isoproterenol more in the susceptible than in the resistant dogs: -45.5 +/- 6.5 vs. -19.2 +/- 6.3%. After training, the susceptible and resistant dogs exhibited similar responses to the beta(2)-AR antagonist: -12.1 +/- 5.7 and -16.2 +/- 6.4%, respectively. In contrast, ICI-118551 provoked even greater reductions in the isoproterenol response in the sedentary susceptible dogs: -62.3 +/- 4.6%. The beta(2)-AR agonist zinterol (1 microM) elicited significantly smaller increases in isotonic shortening in ventricular myocytes from susceptible dogs after training (n = 8, +7.2 +/- 4.8%) than in those from sedentary dogs (n = 7, +42.8 +/- 5.8%), a response similar to that of the resistant dogs: +3.0 +/- 1.4% (n = 6) and +3.2 +/- 1.8% (n = 5) for trained and sedentary, respectively. After training, VF could no longer be induced in the susceptible dogs, whereas four sedentary susceptible dogs died during the 10-wk control period and VF could still be induced in the remaining seven animals. Thus exercise training can restore cardiac beta-AR balance (by reducing beta(2)-AR responsiveness) and could, thereby, prevent VF.