University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2002—pp. 49-55

Using Dynamic Foot Pressure as a Countermeasure to Muscle Atrophy

Charles S. Layne (UH), Mark S. F. Clarke (UH), Daniel A. Martinez (UH), Daniel L. Feeback (NASA-JSC), and Antonios Kyparos (UH)

Abstract
The stimulation of foot somatosensory receptors affects skeletal muscle (SKM) atrophy on hindlimb unloading rats. Mechanical stimulation of the plantar surface of the rat foot during hindlimb unloading (HU), utilizing a novel stimulation paradigm known as Dynamic Foot Pressure (DFP), will attenuate unloading-induced SKM atrophy.

MICROGRAVITY INDUCES SKELETAL MUSCLE (SKM) ATROPHY as a consequence of mechanical unloading of the musculoskeletal system. In the context of manned space flight, the subsequent loss of muscle strength and functionality poses significant operational implications that could jeopardize mission success. For example, the crew must maintain their physical condition to perform mission operations, such as extravehicular activity (EVA) and on-going construction tasks performed on the International Space Station (ISS). Optimal muscle function is also a prerequisite for the flight crew’s prompt response to any emergency situations that may arise during flight or landing. Such requirements are compounded when mission duration is increased, as is the case for ISS operations and for any future manned mission to Mars. As a consequence of such operational demands, designing and validating efficient in-flight countermeasures to microgravity-induced skeletal muscle atrophy become of paramount importance for the future of manned space flight.

Background and Significance
The neuromuscular system is one of the biological systems most affected during spaceflight. Microgravity induces SKM atrophy. It particularly affects the anti-gravity musculature of the lower limbs.^1,2 In general in rodents, slow-twitch muscles are more susceptible to spaceflight-induced SKM atrophy than the fast-twitch ones, and extensors are more affected than flexors.³ In space, contrary to the terrestrial environment, the absence of a constant muscle loading leads to decrease in neuromuscular activation.⁴ Weightlessness has been shown to cause a decrease in muscle volume, mass and strength, alterations in fiber type and myosin heavy chain (MHC) expression, as well as a decrease in neuromuscular function and muscle capillarity.^5,6 In addition, studies reveal that spaceflight hindlimb muscles of animals show significant changes in muscle collagen concentration of atrophied muscles following spaceflight, with a concomitant decrease in the concentration of mature cross-links.⁷ These data suggest that reduced load and muscle activation result in a rapid decline in non-collagenous muscle protein, which enhances the tissue concentration of collagen.

Hindlimb unloading (HU) is an accepted and a widely used model of microgravity-induced SKM atrophy since it results in many of the same basic functional, histological, and biochemical alterations detected in SKM during space flight.1 In the HU condition, the most rapid decrease in SKM mass occurs within the first week of suspension.⁸

Exercise, currently the primary on-orbit muscle degradation countermeasure, has not proven completely effective in preventing muscle atrophy. To date, the projected amount of time (as high as four hours per day) required to perform daily-prescribed exercise countermeasures to muscle atrophy on ISS will be a significant drain on productive crewmember time. Therefore, an atrophy countermeasure, designed for use as an integral part of the crewmember’s daily routine, may prove to be of great value in maintaining muscle mass and function during long-term space flight.

The purpose of this study is to investigate whether the application of mechanical stimuli to the plantar surface of the feet can counteract microgravity-induced muscle atrophy. The basic concept behind the application of mechanical stimuli to the soles of the feet is the well-established motor control principle that sensory input (i.e., pressure application) can modify motor output (i.e. neuromuscular activation). A possible explanation of this phenomenon might be the stimulation of the cutaneous mechanoreceptors in the skin (i.e. Merkel discs, Meissner corpuscles, Ruffini endings, Pacinian corpuscles).

Previous research conducted both during spaceflight⁹ and on the ground¹⁰ has demonstrated that increasing sensory input by applying pressure to the feet results in an increase in neuromuscular activation. Ground-based microgravity simulated study using hindlimb-unloaded rats showed a significant attenuation of muscle atrophy after pressure application to the soles of the rat feet.¹¹ Recent reports have found that providing mechanical stimulus to the legs of sheep resulted in a significant increase in bone density.¹² The evidence provides support to the hypothesis that external mechanical stimulus applied to the feet may, in part, counteract the microgravity-induced muscle atrophy providing a novel and an effective in-flight countermeasure as well as an effective rehabilitation technique for bedridden patients.

Experimental Design and Methods
Experimental plan. Forty adult male Wistar rats (6-month-old) were randomly assigned to four groups of ten rats each, as follows:

• ambulatory controls (AMBU),
• hindlimb unloaded only (HU),
• hindlimb unloaded wearing an inflatable boot (HU-IFL), and
• hindlimb unloaded rats wearing a non-inflatable boot (HU-NIFL).

The stimulation of mechanoreceptors is achieved by applying pressure to the plantar surface of the foot during the 10-day period of HU using a custom-built boot. The anti-atrophic effects of DFP application are quantified directly by

• morphological (muscle wet weight, myofiber cross-sectional area, neuromuscular junction size/density) analysis,
• histochemical (myofiber type distribution) analysis, and
• biochemical (myosin heavy chain-MHC isoform content, muscle collagen concentration and maturation) analysis techniques in the
• soleus (Sol), predominantly slow-twitch ankle extensor muscle,
• medial gastrocnemius (MG), predominantly fast-twitch ankle extensor muscle, and
• tibialis anterior (TA), antagonist, fast twitch ankle flexor muscle.

Hindlimb unloading procedure. Researchers are able to unload the hindlimbs by using a tail-suspended rat model.¹³ This model allows the animals to move freely about the cage using their forelimbs as their only mechanism of movement, while the hindlimbs are suspended at a 25° angle from the cage floor. The muscles of the hindlimbs do not support the weight of the animal and, hence, undergo muscle atrophy. The hindlimb unloading condition lasted ten days.

Dynamic foot pressure application. A custom-built rat inflatable boot was used to stimulate the mechanoreceptors of the soles of the foot. Under isoflurane (5%) gas anesthesia, the boot, outfitted with an inflatable/deflatable latex air bladder, was attached to the foot of one leg chosen at random in HU animals, this leg being termed the "treatment leg"; the other leg was termed the contra-lateral control leg. Pressure was applied to the foot of the treatment leg by inflation/deflation of the latex bladder using an air pump attached to a hose leading to the bladder. Pressure application parameters (i.e., inflation/deflation/rest timing) were controlled by a microprocessor. The boot was maintained on the foot only during the application of the pressure and was removed every day after the termination of the protocol.

The inflatable boot, illustrated in Fig. 1, is fabricated with a very thin and extremely light, yet durable, plastic with an attached inflatable/deflatable latex air bladder (1). The boot fits comfortably on the foot without restricting the natural movement of the ankle joint. Velcro restraint straps (2) secure the boot to the sole of the foot of the treatment leg and around the ankle joint (3) during HU. The air bladder is connected to an extremely quiet air pump by a single air/vacuum line (4). The bladder is inflated by pumping air down the line and deflated by removing the air from the bladder by reversing the direction of the pump.

The pressure stimulation protocol consisted of a 5 sec inflation/5 sec deflation of the air bladder for a total of 20 min followed by a 10-min rest interval. This cycle was repeated eight times over a four-hour period during each day of the ten-day HU period. Pressure in the bladder during the inflation was maintained at 104 mmHg. Pump cycling time and duration were controlled by a microprocessor.

Tissue Collection and Processing. Sol, MG, and TA muscles were collected from control and hindlimb unloaded animals using a procedure previously described.¹⁴ Briefly, the animals were deeply anesthetized with an i.p. injection of an anaesthesia mixture (ketamine 40-80 mg/kg body wt and xylazine 5-10 mg/kg body wt), the hindlimbs were shaved, and the muscles were exposed and carefully dissected from the limb. The excised Sol, MG, and TA muscles were attached to wooden rods by pins inserted through the tendon attachments so that the muscle could be elongated without being stretched. The muscle was then divided using sharp blades into smaller pieces, which were processed for subsequent analysis. In preparation for morphological and histochemical analysis, muscle samples were immersed in TissueTek OCT mounting medium, frozen in liquid nitrogen-cooled isopentane and stored at -80°C.

In analysis, frozen cross sections (5 m) from the Sol, MG, or TA muscle were cut using a Zeiss Microm HM 500 OM microtome cryostat and placed upon Superfrost Plus glass slides (Erie Scientific, Portsmouth, NH). Sections were allowed to air dry for one hour before histochemical staining, whereas sections destined for immuno-histochemical staining were immediately placed in fresh D-PBS buffer (pH 7.2). In preparation for biochemical analysis, muscle samples were snap-frozen in liquid nitrogen and stored at -80°C.

Histochemical and Electrophoretical analysis. Researchers carried out morphometric analysis of myofiber dimensions in frozen cross-sections of Sol, MG, and TA muscles, as previously described.¹⁴ Fiber typing on frozen sections was performed utilizing the metachromatic dye-ATPase myofibrillar stain using the method originally described by Ogilvie and Feeback¹⁵ as modified by Bamman et his colleagues.⁵ Immuno-histochemical staining of MHC isoforms on a per myofiber basis was completed, as previously described.⁵ The relative amounts of MHC isoforms (Type I, Type IIa, Type IIb and Type IIx) were determined using glycerol/SDS-polyacrylamide gel electrophoresis as described.⁵ Neuromuscular junction (NMJ) size/density measurements were carried out on perfusion-fixed frozen sections histochemically stained using a standard non-specific esterase stain followed by morphometric analysis of digitized images.

Collagen Analysis. Researchers performed muscle collagen biochemistry according to a published method.¹⁶ Briefly, skeletal muscle mid belly cross-sections (~3-5 mg dry wt.) were hydrolyzed for 24 hours in 6M HCl, subjected to CF1 partition chromatography and solid phase extraction prior to elution on a RP-HPLC system. Collagen cross-link analysis of HP (hydroxylysylpyridinoline) and LP (lysylpyridinoline) were monitored fluorometrically at an excitation wavelength of 295 nm and emission wavelength of 390 nm. Cross-links were expressed as moles of cross-link per moles of collagen. Skeletal muscle collagen was quantitated using an index of collagen concentration, hydroxyproline, an amino (imino) acid. Using Waters Pico-tag (pre-column derivatization method), hydroxyproline-PITC was eluted isocratically, monitored on an absorbance detector at 254 nm and expressed as g Hyp/mg dry wt tissue.

Statistical Analysis
Data were analyzed using the SPSS program. To evaluate the differences among groups for each dependent variable researchers applied one-way analysis of variance (one way ANOVA); when the univariate F tests were significant, Tukey’s post hoc test was used to further identify differences between group pairs. To evaluate the differences between the "treatment leg" and the contralateral "control leg" in the same animal, researchers applied a paired student t-test. Statistical significance level is set at P < 0.05.

Results
Investigators noted significant differences (P < 0.05) in the Sol myofiber cross sectional area (CSA) of the "treatment leg" between the HU group and the other three groups (Fig. 2). Ten days after the HU, the Sol CSA of the HU group decreased by 34% in the right leg and 45% in the left leg compared to the same muscles of AMBU control (2955 ± 428 vs. 4478 ± 746 um2 for the right leg and 2600 ± 257 vs. 4735 ± 778 um2 for the left leg). This muscle mass loss was recovered with the application of DFP during the ten-day period of HU. The myofiber CSA in the HUIFL group increased by 35.1% compared to the HU group (4556 ± 899 vs. 2955 ± 428 um2) reaching AMBU values. A large increase (28.5%) in Sol myofiber CSA compared to HU values was also found in the HU-NIFL group (4138 ± 1003 vs. 2955 ± 428 um2). There was no difference in Sol CSA of the "treatment leg" among AMBU, HU-IFL, and HU-NIFL groups.

With respect to the "non treatment leg," there were significant differences in Sol CSA between the AMBU group and the other three groups (Fig. 3). In comparison with AMBU, the CSA decreased by 45%, 39%, and 41% for HU (2600 ± 257 vs. 4735 ± 778 um2), HU-IFL (2887 ± 617 vs. 4735 ± 778 um2) and HUNIFL (2792 ± 484 vs. 4735 ± 778 um2) group respectively. No significant differences among HU, HU-IFL, and HU-NIFL groups were found.

There was a significant difference in Sol CSA between the "treatment leg" and the "non treatment leg" in both the HU-IFL and HU-NIFL groups (Figs. 4 & 5). The CSA of the "treatment leg" in HU-IFL group increased by 36.6% compared to the "non treatment leg" (4556 ± 899 vs. 2887 ± 617 um2). Respective values for the HU-NIFLgroup were 4138 ± 1003 vs. 2792 ± 484 um2 which represents a 32.5% increase in CSA.