Medicine (Northwest Academic Centre) - Theses
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ItemSarcopenia in Australia and New Zealand and the importance of muscle( 2023-03)Abstract Background Sarcopenia is a condition of low muscle strength, low muscle mass and poor physical performance. Sarcopenia is highly prevalent in older adults, particularly among those with comorbidities such as frailty, or those living in residential aged care. Sarcopenia is associated with increased risk for falls, fractures, hospitalisation, institutionalisation and mortality, but remains under recognised and under treated. There is no universally accepted definition of sarcopenia which creates inconsistencies between and within regions and healthcare providers. The most recent and widely cited definitions of sarcopenia are the revised European Working Group for Sarcopenia in Older People (EWGSOP2), Sarcopenia Definitions and Outcomes Consortium (SDOC), and the revised Asian Working Group for Sarcopenia in Older People (AWGS2). The absence of a universal definition has hampered research progress and contributed to limited translation of sarcopenia knowledge to clinical practice. Additionally, the importance of low muscle mass and its contribution to negative outcomes in older people remains unclear, largely owing to disagreement around the techniques used to determine muscle quantity or quality. Furthermore, in Australia and New Zealand, there have been no regionally specific consumer-informed clinical guidelines to aid health professionals in caring for people with sarcopenia, or to guide research, which has limited knowledge translation. Aims To i) understand the impact of sarcopenia definitions on its prevalence and outcomes, ii) explore the role of an accurate measure of muscle mass on mobility and disability outcomes, and iii) establish consumer- and topic expert-informed evidence-based clinical and research sarcopenia guidelines for use in Australia and New Zealand. Methods Chapter 2 employed a narrative literature review approach to critically examine current literature and identify knowledge gaps. Chapters 3, 8 and 9 used variations of the modified Delphi method with consumer expert (Chapter 8) and topic expert (Chapters 3 and 9) participants to establish regionally specific operational definitions of sarcopenia in Australia and New Zealand, and present clinical and research sarcopenia guidelines. Chapters 4 to 7 employed either factor analysis or Classification and Regression Tree (CART) analysis to understand the prevalence of sarcopenia, and associations of muscle mass (and surrogate measures), muscle strength and physical performance with adverse outcomes in older adults. Chapter 4 applied factor analysis to the longitudinal Osteoporotic Fractures in Men Study (MrOS) to determine groupings and associations of muscle strength, physical performance, body composition and muscle mass with activities of daily living limitations and disability, and mobility disability. Chapter 5 applied CART to the MrOS study data to determine which muscle strength, physical performance, body composition and muscle mass measures predict incident mobility disability. Chapter 6 applied the CART methodology to a pooled, cross-sectional cohort of eight epidemiologic studies from Australia and New Zealand to determine optimal variables and cut points predicting slow walking speed (<0.8 m/s), and compared agreement between recent sarcopenia definitions. Chapter 7 applied the CART methodology to the Dubbo Osteoporosis Epidemiology Study (DOES2) to determine which baseline muscle strength, physical performance and body size and composition variables best predict incident mortality and falls, and prevalent slow walking speed (<0.8 m/s) in older women and men. Results Chapter 3 established consensus among topic experts that the original European Working Group for Sarcopenia in Older People (EWGSOP1) operational definition was recommended for Australia and New Zealand. Chapter 8 showed that people living with sarcopenia, their carers and healthcare consumers have different assessment and outcome priorities than sarcopenia topic experts. Chapter 9 produced 17 consensus statements on sarcopenia prevention, assessment and management in Australia and New Zealand, and established the EWGSOP2 sarcopenia as the preferred definition in the region. Chapters 4 and 5 demonstrated the importance of an accurate measure of muscle mass (D3Cr muscle mass) in predicting negative outcomes in older men. Chapter 4 showed that D3Cr muscle mass is strongly correlated with measures of muscle strength and physical performance, and combined these measures are associated with negative mobility and disability outcomes in older men. Chapter 5 found that D3Cr muscle mass is an important predictor of incident mobility disability in older men, in contrast to DXA-determined lean mass. Chapters 6 and 7 examined Australian and New Zealand older adults, demonstrating poor sarcopenia definition agreement (Chapter 6) and variable predictive ability of different measures of muscle strength and physical performance for mortality and falls in women and men (Chapter 7). Chapter 6 replicated the findings of the SDOC showing that hand grip strength (with body size adjustments) is the strongest predictor of prevalent slow walking speed (<0.8 m/s) and that the SDOC and EWGSOP2 sarcopenia definitions have poor agreement and produce different sarcopenia prevalence estimates. Chapter 7 found that age and walking speed adjusted for height were the most important predictors for mortality in women, and quadriceps strength (with adjustments) was the most important predictor for mortality in men. In both sexes, sit-to-stand test (with adjustments) was the most important predictor for incident falls, and timed-up-and-go test was the most important predictor for prevalent slow walking speed (<0.8 m/s). Chapter 10 critically discussed the findings, examined limitations, and outlined future directions. Conclusions This thesis demonstrated that i) different sarcopenia definitions and measures produce highly variable prevalence estimates and outcome predictions between women and men, ii) an accurate measure of muscle mass (D3Cr muscle mass) is a strong predictor of mobility and disability outcomes in older men, and iii) established consumer- and topic expert-informed sarcopenia guidelines in Australia and New Zealand. Further study is required to determine the role of the D3Cr method in clinical practice and to establish a global definition of sarcopenia.
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ItemInflammatory Bowel Disease-associated Osteosarcopenia: identification of mechanisms and therapeutic targets( 2022)Chronic inflammatory disorders such as inflammatory bowel disease (IBD) of the intestine cause an appreciable risk of musculoskeletal disorders, mainly affecting children and youth. Osteosarcopenia in IBD patients develops with generalized muscle weakness, reduced muscle mass (60%), sarcopenia (42%), osteopenia (77%) and osteoporosis (42%). Osteosarcopenia remains silent until it manifests by physical disability due to fatigue, falls and fractures, increased hospital admissions, and substantially reduced quality of life. Multifactorial etiologies comprise direct effects of underlying inflammatory disease processes, nutritional deficits, and therapeutic effects. Understanding the complex interplay between various factors that lead to IBD-induced osteosarcopenia is a formidable challenge. Murine models are widely used to explore gut-bone-muscle interactions because of the challenges and limitations inherent to human studies. Studies investigating bone and muscle status have been mainly conducted in the acute models of TNBS or DSS-induced colitis. These models enriched our understanding of bone and muscle pathology in colitis. However, the chemical damage to the gut epithelium in these models results in self-limiting inflammation in these models rather than replicating specific chronic immunopathology present in inflamed colons of IBD patients. Therefore, the association between long-term chronic inflammation and musculoskeletal health status cannot be studied in these models. Winnie mice model of colitis with single point missense mutations in the mucin gene show both innate and adaptive immune response. Colitis in Winnie mice exacerbates with age, similar to inflammation progression in IBD patients. The epithelial mucosal dysfunction, colon morphology, motility and faecal microbial and metabolomic profiles in Winnie mice are also analogous to UC patients. The objectives of our study are to define whether there are musculoskeletal alterations in Winnie mice with the onset and progression of chronic colitis, to resolve whether GDS levels are associated with underlying changes and to elucidate associated molecular mechanisms. We analyzed the phenotypic, cellular, and functional characteristics of cortical and trabecular bone in Winnie mice from prior (6 weeks old, w.o.) and during the development of inflammation (14w.o. and 24 w.o.) and these changes were compared to age and sex-matched control C57BL/6 mice. We observed skeletal deterioration with advancing age starting from the onset of colitis symptoms after 6 weeks, and gradually increasing from 14 weeks (progressive) to 24 weeks (severe colitis). Our results indicate significant and reproducible defects in bone structure, formation, and mechanical properties. Winnie mice showed deteriorated bone microstructure leading to fewer, thinner, and more separated trabeculae, and reduced cortical thickness. Paradoxically, we observed loss of some bone parameters in Winnie mice at 6w.o. without any clinical symptoms of colitis. Winnie mice at 14w.o. and 24w.o. were more susceptible to bone loss. This was evidenced by a more significant difference in deterioration of some bone parameters (trabecular thickness, elastic modulus) in Winnie mice at 14w.o. and 24w.o.. In this study, we also attempted to deduce mechanisms associated with bone loss, mediated by gut-derived serotonin (GDS) in our model. GDS levels progressively increase in Winnie mice, starting from 6w.o. to drastically higher levels at 14w.o.. The degree of bone deterioration observed in our model goes in parallel with increasing GDS levels with age. Simultaneous with increasing GDS levels, the molecular regulators FOXO1 and ATF4 genes were augmented in Winnie mice at 14w.o. compared with 6w.o.. Our data showed disease severity–dependent alteration of bone microarchitecture in Winnie mice model of colitis. Furthermore, analysis of the cellular mechanisms of bone loss revealed that the number of osteoblasts was reduced at all ages, with a concomitant elevation in osteoclasts. Chronic colitis associates with increased GDS availability, which could negatively impact osteoblastogenesis in this model. Our earlier study in Winnie mice reported that GDS is at the maximum high level after 14 weeks and ceases to rise after 24 weeks. To pin-point mechanisms potentially responsible for bone loss in colitis, we observed association of high GDS levels and FOXO1 molecular partners to regulate bone metabolism. Our study reports a high level of GDS in Winnie mice, which is paralleled with increased transcriptional activity of FOXO1. Our immunohistochemical analysis revealed dissociation of FOXO1-CREB complex in bone marrow derived mesenchymal stromal cells (BM-MSCs) of 14 weeks Winnie mice. This releases the inhibiting effect of CREB on FOXO1, leading to suppression of proliferation of osteoblasts. In contrast, no significant changes in association/dissociation of complexes with FOXO1 were observed at 6 w.o. Winnie mice compared with age-matched controls. This is in agreement with no differences in the levels of 5-HTR1B expression at 6 w.o. Winnie mice and age- matched controls. These findings may have implications for FOXO1 as the molecular node of intricate transcriptional machinery that may be associated with inhibition of bone formation. There is a possibility that in the presence of high GDS levels, most of the 5-HTR1B on bone is bound by GDS, which upregulates the expression of 5-HTR1B and FOXO1. Concomitantly, there is a shift in FOXO1 target genes from CREB to ATF4- dependent responses. Our findings suggest that the interaction of FOXO1 with ATF4 or CREB can shift to ATF4-dependent responses in the Winnie mice. In conclusion, our findings support the hypothesis that changes in GDS signaling might be one of the contributing factors to the bone loss associated with colitis. Immune cells have GDS receptors on them; therefore, we cannot exclude the role of systemic inflammation in IBD-induced osteopenia/osteoporosis. For muscle phenotype study, experimental mice were obtained from heterozygote breeders (Winnie+/-) to obtain Winnie-/- and control mice from the same breeding pair. Winnie-/- mice were compared with littermate controls (Winnie+/-). Our previous studies showed that Winnie mice develop mild spontaneous inflammation in the colorectum after they are 6w.o. in pathogen-free conditions; it progresses over time and results in severe colitis by 12 to 16w.o.. Therefore, we chose pre- and post-inflammatory stages: 6w.o. (no colitis) and 14w.o. (progressive colitis) to study colitis induced skeletal muscle phenotype alterations. We studied physiological and functional properties of muscles located in the hind limbs (TA and soleus) of Winnie mice compared to controls as declining muscle mass in the lower extremities is most significant in age-related sarcopenia. The mouse soleus has been reported to express the closest molecular resemblance to several human skeletal muscles. Human soleus muscle mediate interaction between its fascicle and tendon to generate force and mechanical work during walking and running. In this study, we tried to elucidate the phenotypic effects on skeletal muscle in Winnie mouse model of spontaneous chronic colitis. Physical parameters, namely, body weight, muscle wet weights, muscle fibre size, grip strength and voluntary locomotion and running were assessed to demonstrate functional deficits of sarcopenia. Mitochondrial oxidative activity was also assayed in muscle tissue to evaluate the possible role of oxidative stress in the development of the sarcopenic state. In fact, the explicit loss of both type 1 and 2 fibers as shown by ATPase staining demonstrated the sarcopenic state of our mouse model. Our results indicate significant and reproducible defects in muscle mass, size, oxidative capacity, and physical performance at inflammatory 14w.o. Winnies compared to age- and sex-matched controls. Therefore, Winnie mouse model of spontaneous chronic colitis can be used as a robust model to study phenotypic and functional alterations of muscle in IBD. To validate the systemic impact of colitis on muscle, we initially confirmed decreased muscle weights in Winnie mice. The most frequent phenotype in IBD patients was reduced muscle mass and weight loss. Colitis induced decreased body and muscle mass at 14w.o. Winnies undergoing colitis compared to controls. Following normalization with body weight, female and male Winnie mice displayed significantly decreased muscle (both TA and soleus) wet weights at 14w.o. vs age and sex-matched controls. This suggests that the deleterious effects of intestinal inflammation on muscle mass in Winnie mice are not solely attributable to impairment in general body weight gain. Our results are in accordance to previous studies which showed colitis associated decrease in muscle mass. However, there was no decline in skeletal muscle mass (soleus and TA) from 6w.o. to 14w.o. unlike decreased muscle masses shown from 7w.o. to 14w.o. in pathogenic bacteria infested gene-deficient interleukin 10 (IL-10-/-) mice. Consistently, decreased muscle mass of hind limb muscles (e.g. quadriceps and gastrocnemius) was noticed in DSS-model of colitis. However, longissimus and soleus muscles were only slightly affected in DSS-treated mice. It has also been shown that skeletal muscle mass and proteins were reduced in murine models of TNBS-induced colitis, suggesting the linkage between IBD and muscle wasting. CD patients show lower muscle mass due to poor nutritional status whereas the incidence of nutrient deficiencies is less significant in patients with UC, because CD can affect any part of the gastrointestinal tract, mainly, the small bowel. To investigate the influence of impaired mucin in goblet cells on the morphology of skeletal muscles, we observed muscle phenotype to ascertain muscle fiber sizes in Winnie mouse model of colitis. At non-inflammatory 6w.o. females and males Winnies, it was found that Muc2 knock outs didn’t affect TA fiber size. Our previous results showed that Winnie mice at 6w.o. had no lipocalin-2 (an inflammatory marker) expression, thereby, no disease activity detected at non-inflammatory 6w.o. Winnies. Consistently, we observed no muscle phenotype alterations at 6w.o. Winnies vs age and sex-matched controls. Evidently, there is chronic progression of skeletal muscle phenotypic alterations. Concomitant to reduced whole muscle mass, average TA and soleus muscles’ fibre sizes were significantly decreased at 14w.o. female and male Winnies undergoing colitis compared to age- and sex-matched controls. Male Winnie mice at 14w.o. showed more reduced muscle fiber size in TA and soleus muscle compared to age- and sex-matched controls. In summary, advanced inflammation in Winnie mice at 14w.o. induced loss of muscle mass and muscle individual fibres atrophy in both slow-twitch (soleus) and fast-twitch (TA) muscles. Muscle fibre frequency distribution showed that colitis in female Winnies at 14w.o. shifted the TA and soleus muscle fiber sizes towards more number (or increased frequency) of small-sized muscle fibers while lesser number (or decreased frequency) of large-sized fibers. Muscle fibre frequency distribution showed that colitis in male Winnies at 14w.o. shifted the TA and soleus muscle fiber sizes towards more number of smaller fibers but particularly towards lesser number of large-sized fibers. This implies atrophic signaling pathways induce large individual fibres to smaller fibres in Winnies at 14w.o. compared to controls. A delicate balance between oxidative and anti-oxidant enzymes activity get disturbed in chronic inflammation to generate mitochondrial-derived oxidative stress. We tested for SDH enzyme activity, which a common marker of mitochondrial oxidative metabolism. TA muscles showed decrease mitochondrial oxidative activity in both female and male Winnies at 14w.o. compared to age- and sex-matched controls. Oxidative and metabolic stress act as a compensatory anti-inflammatory response to re-establish homeostasis. Male Winnies showed reduced oxidative activity in soleus muscle at 14w.o. compared to age- and sex-matched controls. TA muscles in male Winnies showed more oxidative damage (51%) than female Winnies (47%). Particularly, TA muscle is more prone to the detrimental effects exerted by the enhanced oxidative stress than soleus muscle. It has been evidenced that soleus remain more oxidative than the TA and expressed higher levels of markers of mitochondrial biogenesis. Female Winnies did not show reduced oxidative activity in soleus muscle at 14w.o. compared to age- and sex-matched controls. From physical activity data, we concluded notable effect of on physical activity, particularly voluntary wheel running performance by 14w.o. Winnie mice with inflammation. A relative lack of physical activity also explains a reduction in muscle mass. Even individuals with quiescent CD have reduced physical activity. Low skeletal muscle mass, decreased oxidative capacity of TA muscle in inflammatory Winnies at 14w.o. is linked to muscle fatigue, which may be associated/causative for low voluntary wheel running activity. Short-term muscle inactivity severely reduces muscle mass and strength even in young individuals. It is reasonable to assume that exercise could be more arduous for Winnies with advanced inflammation. Skeletal muscle atrophy in inflamed Winnie mice with colitis is characterized by low muscle mass and size. We propose that in response to elevations in systemic inflammation and physical inactivity, lead to the onset of muscle atrophic responses in Winnie mice with colitis. Noticeably, Winnie mice showed reduced muscle mass/size without fiber attrition. We can conclude that Winnie mice is at the ‘sarcopenia’ stage, which is characterized by low muscle mass and size, plus low physical performance. As Winnies do not show low muscle strength, Winnies do no exhibit phenotype of ‘severe sarcopenia’. Further studies are warranted to decipher whether sarcopenia is because of impaired muscle protein synthesis and/or suppressed inhibition of muscle protein breakdown or both.