Prolonged periods of non-weightbearing activity result in a significant loss of bone mass which increases the risk of fracture with the initiation of mechanical loading. The loss of bone mass is partially driven by declines in bone formation yet the mechanisms responsible for this decline are unclear. To investigate the limitations of osteoblasts during disuse, marrow ablation was superimposed on hindlimb unloaded mice. Marrow ablation is a useful model to study osteoblast functionality as new cancellous bone is rapidly formed throughout the marrow of a long bone while hindlimb unloading is the most common method used to produce skeletal unloading. The specific hypotheses of this study were aimed at determining if changes in osteoblast functionality, differentiation, and/or proliferation were compromised in non-weightbearing bone in response to a bone formation stimulus. Additionally, the influence of having compromised osteoblast functionality at the time of stimulation was assessed in non-weightbearing bones. Key outcome measures used to address these hypotheses included static and dynamic cancellous bone histomorphometry, bone densitometry, and real-time polymerase chain reaction (PCR) analyses of gene expression. The results document similar ablation-induced increases of cancellous bone in both weightbearing and unloaded animals. Similarly, there was no influence of load on ablation-induced increases in cancellous bone forming surface or mineral apposition rate. Unloading did significantly attenuate the ablation-induced increase in bone formation rate, due to reduced levels of total surface mineralization. When osteoblast functionality was compromised prior to marrow ablation, bone formation rate increases were also attenuated in ablated animals due to reduced mineralization. Additionally, increases in forming surface were attenuated as compared to unloaded animals having normal osteoblast function at the time of ablation. Collectively, these data identify mineralization as the limiting step in new bone formation during periods of disuse. The caveat, however, is that when bone formation is stimulated after a period of unloading sufficient to compromise osteoblast functionality, increases in osteoblast recruitment to the bone surface are compromised.

Long duration spaceflight missions out of lower earth orbit, back to the lunar surface, or possibly to Mars highlight the importance of preserving muscle mass and function. Muscle atrophy occurs within days of exposure to microgravity and prevailing thought is that a primary mechanism for muscle atrophy is a reduction in skeletal muscle protein synthesis. This dissertation examines the ability of skeletal muscle to recover muscle protein synthesis with slight perturbation, such as ambulatory reloading during disuse as well as partial loading, similar to body mass seen on the moon or Mars. We use traditional precursor-product labeling to measure protein synthesis, but use a relatively novel tracer, deuterium oxide, in order to make cumulative measures of protein synthesis over 24 h. The overarching goal of this dissertation is to define the response of skeletal muscle protein synthesis to different loading parameters in order to better understand the contribution of protein synthesis to skeletal muscle mass during disuse. In the first study, we demonstrate that muscle atrophy during 5 days of hindlimb unloading is in part due to a decrease in protein synthesis. We also highlight the ability of skeletal muscle to adapt by allowing two 1 h ambulatory reloading sessions on days 2 and 4. Although this countermeasure is able to rescue protein synthesis in soleus and gastrocnemius, it is unable attenuate any losses in muscle mass. In the second study, we compare partial weight loading to traditional hindlimb unloading. Weight bearing of 1/3 or 1/6 body weight is able to attenuate losses in muscle mass seen with unloading. Protein synthesis is maintained after 21 days of the experimental protocol, suggesting that protein synthesis is responsive to load and is likely not the only mechanism for determining muscle mass. In the final study, the effects of < 1 Gy x-ray exposure and partial weight suspension are measured to better understand the complex space environment, which includes a wide variety of radiation. Surprisingly, we found no effects of radiation on muscle protein synthesis in 1 G or partial loading. Targeting only protein synthesis may not be enough of a stimulus as evidenced by the data in this dissertation. Future plans should use a multiple-systems approach to counteract atrophy by increasing protein synthesis to maintain/elevate muscle mass during periods when it is otherwise compromised.

Prolonged periods of non-weightbearing activity result in a significant loss of bone mass which increases the risk of fracture with the initiation of mechanical loading. The loss of bone mass is partially driven by declines in bone formation yet the mechanisms responsible for this decline are unclear. To investigate the limitations of osteoblasts during disuse, marrow ablation was superimposed on hindlimb unloaded mice. Marrow ablation is a useful model to study osteoblast functionality as new cancellous bone is rapidly formed throughout the marrow of a long bone while hindlimb unloading is the most common method used to produce skeletal unloading. The specific hypotheses of this study were aimed at determining if changes in osteoblast functionality, differentiation, and/or proliferation were compromised in non-weightbearing bone in response to a bone formation stimulus. Additionally, the influence of having compromised osteoblast functionality at the time of stimulation was assessed in non-weightbearing bones. Key outcome measures used to address these hypotheses included static and dynamic cancellous bone histomorphometry, bone densitometry, and real-time polymerase chain reaction (PCR) analyses of gene expression. The results document similar ablation-induced increases of cancellous bone in both weightbearing and unloaded animals. Similarly, there was no influence of load on ablation-induced increases in cancellous bone forming surface or mineral apposition rate. Unloading did significantly attenuate the ablation-induced increase in bone formation rate, due to reduced levels of total surface mineralization. When osteoblast functionality was compromised prior to marrow ablation, bone formation rate increases were also attenuated in ablated animals due to reduced mineralization. Additionally, increases in forming surface were attenuated as compared to unloaded animals having normal osteoblast function at the time of ablation. Collectively, these data identify mineralization as the limiting step in new bone formation during periods of disuse. The caveat, however, is that when bone formation is stimulated after a period of unloading sufficient to compromise osteoblast functionality, increases in osteoblast recruitment to the bone surface are compromised.

Osteocytes (resident bone cells) are believed to sense loading-induced interstitial fluid flow in bone and transduce the signals to osteoclasts (bone resorption cells) and osteoblasts (bone formation cells) to regulate bone remodeling. Recent studies have shown that bone disuse causes osteocyte apoptosis, which precedes osteoclast activity at the local remodeling site. Although osteoclast precursors are known to travel via the circulation, the specific mechanism by which they are transported to the remodeling site is unclear. We hypothesized that lack of fluid flow induces osteocyte apoptosis. Furthermore, we hypothesized that osteocyte populations containing apoptotic osteocytes secrete cytokines that: 1) promote angiogenesis, and 2) activate the endothelium to promote osteoclast precursor adhesion to the endothelium such that osteoclast precursors can be delivered closer and directly to the remodeling site. In our in vitro studies, we found that lack of oscillatory fluid flow (mimicking mechanical disuse) promotes osteocyte apoptosis. In addition, osteocyte populations containing apoptotic cells promote endothelial cell proliferation, migration, and tubule formation. Inhibition of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), abrogated osteocyte apoptosis-mediated angiogenesis. Furthermore, we found that osteocyte populations containing apoptotic cells secrete cytokines that promoted osteoclast precursor adhesion. Upon further investigation, we found that apoptotic osteocytes secreted elevated levels of inflammatory cytokine interleukin 6 (IL-6), and its soluble receptor, sIL-6R. We demonstrated that both IL-6 and sIL-6R are required to activate the endothelium to express ICAM-1. Inhibition of ICAM-1 and IL-6 by blocking antibodies abolished apoptotic osteocyte-mediated osteoclast precursor adhesion. Our findings suggest for the first time that osteocytes communicate to endothelial cells directly to mediate angiogenesis and osteoclast precursor adhesion. Results from this study may assist in a better understanding of osteoclast precursor recruitment at the initial onset of bone resorption.

Human exposure to reduced weightbearing results in bone loss. The rate of bone loss during microgravity exposure is similar to that of a post-menopausal women. In fact, the maintenance of bone mass is intimately dependent on exercise. Therefore, exercise associated mechanical loads to bone tissue are an important countermeasure to prevent disuse-induced bone loss. However, the types of exercise modalities required to prevent such bone loss are unclear. Moreover, how mechanical loading to bone translates into molecular osteogenic signals in bone cells is unknown. Radiation exposure is another potent inducer of bone loss, namely observed on Earth in the clinical setting following radiotherapy procedures. It is expected that long duration space missions outside the protection of Earth’s magnetosphere will result in significant galactic cosmic radiation exposure. However, the magnitude of bone loss resulting from this galactic cosmic radiation exposure is unclear. Moreover, it is unknown if radiation exposure will exacerbate disuse-induced bone loss. Therefore, a series of experiments were designed to determine: 1) Will simulated galactic cosmic radiation exacerbate reduced weightbearing-induced bone loss? 2) Will pharmacological activation of the putative mechanosensing Wnt pathway enhance exercise-induced bone mass gain? To address these questions the experimental study series employed two animal models of reduced weightbearing, hindlimb unloading and partial weightbearing. These model test-beds enabled the evaluation of two novel countermeasures (simulated resistance exercise and glycogen synthase kinase-3 (GSK-3) therapeutic) and simulated exposure to space radiation environments. To test the impact of simulated space radiation (28Si) one study of the series was conducted at Brookhaven National Laboratory. To quantify the impact of the abovementioned countermeasures and space radiation on bone, mechanical testing, peripheral quantitative computed tomography, micro-computed tomography, histomorphometry, and immunohistochemistry served as primary outcome measures. The primary findings are: 1) Low-dose high-LET radiation negativity impacts maintenance of bone mass by lowering bone formation and increasing bone resorption. This impaired bone formation response is in part due to sclerostin induced suppression of Wnt signaling. 2) Combining GSK-3 inhibition with high intensity exercise mitigates cancellous bone loss and restores cortical periosteal growth during disuse.

Disuse of weight bearing bones has been shown to cause bone loss. This poses a health concern for people exposed to microgravity, such as astronauts. Animal studies are used to study factors related to bone loss and countermeasures to prevent bone loss. This study used a hindlimb unloaded (HU) rat model to simulate microgravity and a muscle stimulation countermeasure to simulate resistive exercise. Uniaxial strain gages were implanted on the antero-medial aspect of the proximal tibia to measure the mechanical strain during a typical exercise session. In a separate but parallel study, the exercise was shown to be an effective countermeasure to disuse related bone loss. The current study sought to understand the loading of the bone during the exercise. To determine if the strain response changes during a protocol using this countermeasure, strains were measured on a group of weight bearing animals and a group that were hind limb unloaded and received the countermeasure for 21 days. Strain magnitudes and rates were considered and related to torques at the ankle joint. No significant differences in strain magnitudes were noted between the baseline control group and the hindlimb unloaded group that received the countermeasure. The two kinds of contractions used in an exercise session are isometric and eccentric. The isometric contractions are used to adjust the stimulation equipment for the eccentric contractions, which constitute the exercise. Peak strain levels during the isometric contractions ranged from 900 to 2200 microstrain while the eccentric were 38% lower and ranged from 600 to 1400. Eccentric strain rates were 62% lower than the isometric contractions strain rates. These results indicate that the strain environment during the isometric contractions may be causing more of the osteogenic response than the eccentric contractions, which have previously been thought to be the primary part of the countermeasure.

Disuse of weight bearing bones has been shown to cause bone loss. This poses a health concern for people exposed to microgravity, such as astronauts. Animal studies are used to study factors related to bone loss and countermeasures to prevent bone loss. This study used a hindlimb unloaded (HU) rat model to simulate microgravity and a muscle stimulation countermeasure to simulate resistive exercise. Uniaxial strain gages were implanted on the antero-medial aspect of the proximal tibia to measure the mechanical strain during a typical exercise session. In a separate but parallel study, the exercise was shown to be an effective countermeasure to disuse related bone loss. The current study sought to understand the loading of the bone during the exercise. To determine if the strain response changes during a protocol using this countermeasure, strains were measured on a group of weight bearing animals and a group that were hind limb unloaded and received the countermeasure for 21 days. Strain magnitudes and rates were considered and related to torques at the ankle joint. No significant differences in strain magnitudes were noted between the baseline control group and the hindlimb unloaded group that received the countermeasure. The two kinds of contractions used in an exercise session are isometric and eccentric. The isometric contractions are used to adjust the stimulation equipment for the eccentric contractions, which constitute the exercise. Peak strain levels during the isometric contractions ranged from 900 to 2200 microstrain while the eccentric were 38% lower and ranged from 600 to 1400. Eccentric strain rates were 62% lower than the isometric contractions strain rates. These results indicate that the strain environment during the isometric contractions may be causing more of the osteogenic response than the eccentric contractions, which have previously been thought to be the primary part of the countermeasure.

Introduction: Despite the advantages of using a hearing aid (HA), only 1 out of 5 individuals who could benefit from a (HA) actually use one (World Health Organization, 2012). If an individual does not use a HA then it may impact on their quality of life, as well as others around them (Chia et al., 2007). Therefore it is important to understand why individuals do not use HAs after obtaining them. To date, there has been no study that investigates the reasons for HA disuse in the New Zealand population. Methods: Two groups of adults with hearing impairment were recruited: HA users (N = 35) and HA disusers (N = 35). Six self-report questionnaires, three audiometric tests and two other body function measures were compared between the groups. Results: Several variables differentiated HA users from disusers, these significant variables were: cognition, understanding speech in noise, acceptance of noise, age at testing, education, hearing assistance technology (HAT) use, HA satisfaction, self-efficacy, accepted need, application for HA subsidy, HA outcomes, stages-of-change, perceived environmental influence, follow-up support and hearing related activity limitations/participation restrictions (AL/PR). Discussion: The clinical value of identifying factors related to HA disuse is so clinicians can identify “red flags” for disuse before the client stops using their HAs. By identifying these red flags, rehabilitation can be tailored around the clients’ needs; before the negative consequences of an untreated hearing impairment is felt.

Osteocytes (resident bone cells) are believed to sense loading-induced interstitial fluid flow in bone and transduce the signals to osteoclasts (bone resorption cells) and osteoblasts (bone formation cells) to regulate bone remodeling. Recent studies have shown that bone disuse causes osteocyte apoptosis, which precedes osteoclast activity at the local remodeling site. Although osteoclast precursors are known to travel via the circulation, the specific mechanism by which they are transported to the remodeling site is unclear. We hypothesized that lack of fluid flow induces osteocyte apoptosis. Furthermore, we hypothesized that osteocyte populations containing apoptotic osteocytes secrete cytokines that: 1) promote angiogenesis, and 2) activate the endothelium to promote osteoclast precursor adhesion to the endothelium such that osteoclast precursors can be delivered closer and directly to the remodeling site. In our in vitro studies, we found that lack of oscillatory fluid flow (mimicking mechanical disuse) promotes osteocyte apoptosis. In addition, osteocyte populations containing apoptotic cells promote endothelial cell proliferation, migration, and tubule formation. Inhibition of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), abrogated osteocyte apoptosis-mediated angiogenesis. Furthermore, we found that osteocyte populations containing apoptotic cells secrete cytokines that promoted osteoclast precursor adhesion. Upon further investigation, we found that apoptotic osteocytes secreted elevated levels of inflammatory cytokine interleukin 6 (IL-6), and its soluble receptor, sIL-6R. We demonstrated that both IL-6 and sIL-6R are required to activate the endothelium to express ICAM-1. Inhibition of ICAM-1 and IL-6 by blocking antibodies abolished apoptotic osteocyte-mediated osteoclast precursor adhesion. Our findings suggest for the first time that osteocytes communicate to endothelial cells directly to mediate angiogenesis and osteoclast precursor adhesion. Results from this study may assist in a better understanding of osteoclast precursor recruitment at the initial onset of bone resorption.

PRMT biology during skeletal muscle disuse. / Protein arginine methyltransferase 1 (PRMT1), PRMT4 (also known as co-activator-associated arginine methyltransferase 1; CARM1), and PRMT5 are critical components of a diverse set of intracellular functions. Despite the limited number of studies in skeletal muscle, evidence strongly suggests that these enzymes are important players in the regulation of phenotypic plasticity. However, their role in disuse-induced muscle remodelling is unknown. Thus, we sought to determine whether denervation-induced muscle disuse alters PRMT expression and activity in skeletal muscle within the context of early signaling events that precede muscle atrophy. Mice were subjected to 6, 12, 24, 72, or 168 hours of unilateral hindlimb denervation. The contralateral limb served as an internal control. Muscle mass decreased by ~30% following 168 hours of disuse. Prior to atrophy, the expression of muscle RING finger 1 and muscle atrophy F-box were significantly elevated. The expression and activities of PRMT1, CARM1, and PRMT5 displayed differential responses to muscle disuse. Peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase expression and activation were altered as early as 6 hours after denervation, suggesting that adaptations in these molecules are among the earliest signals that precede atrophy. AMPK activation also predicted changes in PRMT expression and function following disuse. Our study indicates that PRMTs are important for the mechanisms that precede, and initiate muscle remodelling in response to neurogenic disuse. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that is capable of adapting to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important players in the regulation of skeletal muscle remodelling. However, their role in disuse-induced muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the role of PRMTs within the context of early, upstream signaling pathways that mediate disuse-evoked muscle remodelling. We found differential responses of the PRMTs to muscle denervation, suggesting a unique sensitivity to, or regulation by, potential upstream signaling pathways. AMP-activated protein kinase (AMPK) was among the molecules that experienced a rapid change in activity following disuse. These alterations in AMPK predicted many of the modifications in PRMT biology during inactivity, suggesting that PRMTs factor into the molecular mechanisms that precede neurogenic muscle atrophy. This study expands our understanding of the role of PRMTs in regulating skeletal muscle plasticity.