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Histological characteristics of the vastus lateralis muscle in patients undergoing hip surgery Payne, Amelia Patterson 2009

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HISTOLOGICAL CHARACTERISTICS OF THE VASTUS LATERALIS MUSCLE IN PATIENTS UNDERGOING HIP SURGERY  by  AMELIA PATTERSON PAYNE B.Sc., West Virginia University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Rehabilitation Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2009  © Amelia Patterson Payne, 2009  ABSTRACT In aging populations, musculoskeletal changes cause a decline in the ability to perform activities of daily living, to exercise or to maintain an active lifestyle. Musculoskeletal changes may include the loss of muscle mass (sarcopenia), joint changes (osteoarthritis), or bone deterioration (osteoporosis). These types of musculoskeletal changes can also result in the need for hip surgery, a common procedure in older adults, often resulting from a hip fracture due to a non-traumatic event, or a hip replacement due to osteoarthritis. The primary risk factor for hip surgeries is the underlying musculoskeletal condition: osteoporosis (OP) or osteoarthritis (OA). Each of these conditions can lead to serious disability. In OP, bone density is low and bony microarchitecture is disrupted; in OA, the bones become hard, and develop outgrowths. Skeletal muscle plays an active role in bone density and strength, making it reasonable to assume that the structure and characteristics of skeletal muscle tissue will also affect bones and joints. The purpose of this study is to describe morphological and histological differences in muscle fibre characteristics of an important lower extremity muscle, the vastus lateralis (VL). These characteristics were compared between persons undergoing hip surgery and a control group of comparably-aged older adults. This comparison occurred by quantifying the percentage of normal and abnormal muscle fibres, as well as the percentage of adipocytes. Using an ANCOVA to examine between group differences, this study found a difference (p<O.05) between groups in percentage of abnormal fibres and inflamedJnecrotic fibres. This shows that in some histological characteristics, the muscle tissue of older adults who experience hip surgery is different from those who do not. This study also found low to moderate, positive correlations of 11  abnormal fibres, inflamed/necrotic fibres, and internally nucleated fibres with age. There are few reports of muscle structure in persons prior to undergoing hip surgery. Therefore, this knowledge will contribute to infonnation on degenerative changes that may be present in the VL in older individuals prior to hip fracture.  111  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF ABBREVATIONS GLOSSARY  x xi  ACKNOWLEDGEMENTS DEDICATION  xiv xv  CHAPTER 1: INTRODUCTION  1  1.1  Statement of the Problem  1  1.2  Study Purpose, Aims, Hypotheses and Rationale  3  1.2.1  Study Purpose  3  1.2.2  Specific Aims  3  1.2.3  Hypotheses  3  1.2.4  Rationale for the Study  4  CHAPTER 2: LITERATURE REVIEW  6  2.1  Hip Fractures  6  2.2  Reparative Hip Surgery  6  2.3  Osteoporosis and Osteopenia  7  2.3.1  Pathology and Diagnosis  7  2.4  Hip Replacement Surgery  11  2.5  Osteoarthritis  12  iv  2.5.1  Pathology and Diagnosis  .12  2.6  Muscle Characteristics During Aging  15  2.7  Muscle Biopsy  16  2.8  Characteristics of Skeletal Muscle Tissue  18  2.9  Vastus Lateralis  21  CHAPTER 3: STUDY DESIGN AND METHODS 3.1  Study Design  23 23  3.1.1  Subjects  23  3.1.2  Ethical Approval  24  3.1.3  Muscle Biopsy  25  a) Procedures  25  b) Categories and Definitions for Fibre Counting  29  Statistical Analysis  31  a) Parametric Analysis  31  b) Non- Parametric Analysis  32  3.1.4  CHAPTER 4: STUDY RESULTS  34  4.1  Sample Characteristics  34  4.2  Descriptive Tissue Features of the Muscle Biopsies  35  4.3  Comparison of Hip Surgery and Control Groups  41  4.4  a)  Parametric Analysis  41  b)  Non- Parametric Analysis  41  Correlation Between Categories of Abnormal Muscle Fibres and Age a)  Parametric Analysis  42 42  V  b) 4.5  Non-Parametric Analysis  Reliability  46  CHAPTER 5: DISCUSSION and CONCLUSION 5.1  First StudyAim 5.1.2  44  Findings Regarding Adipocytes  47 47 52  5.2  Second Study Aim  53  5.3  Study Strengths and Limitations  54  5.3.1  Study Strengths  54  5.3.2  Study Limitations  55  5.4  Future Directions and Contributions to Field of Study  57  5.5  Clinical Relevance of Study Findings  59  5.6  Conclusion  59  REFERENCES  61  APPENDICES  75  Appendix I  Criteria for the Classification of Hip Osteoarthritis  75  Appendix 2  Consent for Obtaining Muscle Biopsies  76  Appendix 3  Raw Data from Biopsy Quantification  81  Appendix 4  Compiled Data  95  vi  LIST OF TABLES Table 3.1  Categories for Quantification of Abnormal Myofibres Based on Haematoxylin- and Eosin- Stained Cross-Sections of the Vastus Lateralis  Table 4.1  28  Sample Characteristics for Subjects with OA or OP who Underwent Hip Surgery and Control Subjects (mean  *p<005  ±  standard deviation) 35  vii  LIST OF FIGURES Figure 2.1  Risk factors for osteoarthritis  14  Figure 2.2  Cross-section of normal skeletal muscle  18  Figure 2.3  Vastus lateralis in respect to lower extremity  21  Figure 4.1  Percentage total fibre count of normal muscle, abnormal muscle, and adipose tissue of H&E stained cross sections measured by the fibre counting technique for the total sample (n=35). Bars and whiskers represent mean ± SD  Figure 4.2  36  Percentage of total fibre count for subcategories of abnormal muscle H&E stained cross-sections measured by the fibre counting technique for the total sample (n=35). Bars and whiskers represent mean ± SD  Figure 4.3  37  Percentage of total fibre count of normal muscle, abnormal muscle, and adipose tissue of H&E stained cross sections measured by the fibre counting technique for the OA/OP (n= 15) and control (n=20) groups. Bars and whiskers represent mean (value label)  Figure 4.4  ±  SD  38  Percentage of total fibre count for subcategories of abnormal muscle H&E stained cross-sections measured by the fibre counting technique for the OAIOP (n=15) and control (n20) groups. Bars and whiskers represent mean (value label)  ±  SD  39  viii  Figure 4.5  Examples of H&E stained cross sections of the vastus lateralis in people undergoing hip surgery: A) inflamed necrotic fibre and adipose tissue; B) small angular fibres and internal nuclei  Figure 4.6  Parametric correlation scatterplot of percentage of abnormal muscle fibres vs. age  Figure 4.7  (r = 0.49)  43  Non-parametric correlation scatterplot of percentage of abnormal muscle fibres vs. age (r  Figure 4.9  43  Parametric correlation scatterplot of percentage of inflamed necrotic fibres vs. age (r = 0.45)  Figure 4.8  40  =  0.54)  45  Non- parametric correlation scatterplot of percentage of inflamed necrotic fibres vs. age (r = 0.63)  45  ix  LIST OF ABBREVATIONS ADL  Activities of Daily Living  BMI  Body Mass Index  BMD  Bone Mineral Density  CREB  University of British Columbia Clinical Research Ethics Board  CT  Connective Tissue  DXA  Dual-Energy X-ray Absorbtiometry  H&E  Haematoxylin and Eosin  OA  Osteoarthritis  OP  Osteoporosis  SD  Standard Deviation  VL  Vastus Lateralis  x  GLOSSARY Abnormal findings in muscle tissue: increase in connective tissue, and fatty deposits and change in typical cell configuration, including the cellular contour or outline or circumference being small, or with acute angles, as well as the position of nuclei within the cell, and/or muscle fibre atrophy. Body mass index (BMI): statistical measure of the weight of a person, scaled according to height. Measured by body weight divided by square of height (BMI  =  kg/rn ) 2 . Bone mineral density (BMD): a measurement to determine bone density, and risk for fractures due to osteoporosis. BMC  =  bone mineral content (gram)/width  ). 2 of bone (cm Fibrosis:  thickening and scarring of connective tissue.  Fibre type grouping: abnormal occurrence of large clusters of the same fibre type in skeletal muscle (compared to, in normal muscle, different histochemical fibre types distributed in mosaic checkerboard pattern). Histology:  the anatomical study of the form of structures of animals or plants, seen under the microscope. Also termed microscopic anatomy.  Histornorphometry: the quantitative study of the microscopic organization and structure of a tissue. Morphology:  the study of the configuration or structure of animals and plants.  Mosaic patterns: the typical structural distribution of fibre types in skeletal muscle, which is heterogeneous and does not consist of fibre grouping.  xi  Muscle fibre types: Type 1: Slow-twitch, oxidative, and resistant to fatigue. High amounts of myoglobin and mitochondria. Type 2a: Fast-twitch, oxidative, glycolytic, and somewhat resistant to fatigue. Can have intermediate amounts of myoglobin and mitochondria. Type 2b: Fast-twitch, glycolytic fibers that fatigue quickly. Low amounts of myoglobin and mitochondria. Muscle strength: the ability of a person to exert force upon an object. Musculoskeletal system: an organ system comprised of the human skeleton, and skeletal muscle that is attached to the skeleton by tendons. Myopathy:  a neuromuscular condition in which the muscle fibres do not function for a variety of reasons, resulting in muscular weakness. The primary defect is within the muscles, rather than in the nerves.  Osteoarthritis (OA): a disease of the bone and joint, characterized by degeneration of joint cartilage and underlying bone, causing pain and stiffness. Osteoblast:  a mononucleated cell that is responsible for bone formation.  Osteoclast:  a large, multinucleated cell that is responsible for resorption of the mineralized matrix of bone.  Osteonecrosis: the loss of blood to the bone, which causes bone death and collapse. Can lead to pain and arthritis. Osteoporosis (OP): a disease of the bone, in which the bone mineral density is reduced and micro-architecture is disrupted, causing a greater risk of fracture.  xli  The World Health Organization defines OP in women as 2.5 standard deviations below peak bone mass of an average 20-year-old healthy female. Plasmalemma: cell membrane. Semi-permeable lipid hi-layer found in all cells. Sarcopenia:  loss of muscle mass and strength that occurs with aging.  xlii  ACKNOWLEDGEMENTS  I would primarily like to acknowledge my graduate supervisor, Donna Maclntyre, for the encouragement, support and wisdom that you bestow upon me daily. Thank you for teaching me critical thinking, to be rigourous in information seeking, and to persevere through unforseen obstacles.  My thesis committee, Darlene Reid, Susan Harris, and Chris Palmer for their constructive feedback, and academic drive that has gotten me to accomplish this thesis with success.  The faculty, staff and fellow students at the Muscle Biophysics Lab, and Rehabilitation Aimed at Muscle Research group, whose hard work, and academic intellect have taught me more than I would have imagined throughout this degree. Thank you to Jonathan Berkowitz for assistance with the statistical analysis.  Finally, my friends Erika, Celine, Kristin, Erik, and Audrey, who have been my community in Vancouver since I arrived. To my fellow graduate students, specifically Marc, Sharon, and Mineko, thank you for being helping me maintain a balanced lifestyle, answering my inquiries, and for being intellectual inspirations.  xiv  Jor my parents who have taught me to pursue my passions, no matter where they Ceac(me. Thank you for believIng in me and supporting the numerous undertakings that I pursue.  4ndto those adults and athletes who surrounclme daily, who believe as I d: that strong muscles are healthy muscles. Those who believe that making healthy choices Is a key to sustaining a functiona6fufllled and long hfe: Thank you for being an inspiration, andfor encouraging me through work andschool  May you remain “young at heart”forever  xv  CHAPTER 1: INTRODUCTION 1.1  Statement of the Problem  Age-related musculoskeletal changes, such as sarcopenia (loss of muscle mass), osteoporosis (OP), or osteoarthritis (OA), cause a decline in the ability to perform activities of daily living (ADL), to exercise, or to maintain an active lifestyle.’ These changes can combine to contribute to functional loss and increased pain, common fears of living a long life. 3 In addition, an unfortunate event in older adulthood can lead to ’ 2 the possibility of musculoskeletal surgery. Most hip surgeries happen because of the need to replace the joint due to OA, or to repair the bone following hip fracture, often secondary to OP. Hip fractures in older adults commonly occur following relatively low levels of trauma, such as a fall from a standing height, and are disabling, frequently requiring extended hospitalization. 4 Some individuals who experience OA of the hip may have pre-disposing factors that  have increased their risk for severe arthritis. OA is well known as a common and primary example of a disease of aging, 6 but age alone does not cause the disease. 5 ’ 3 Risk factors include those associated with increased joint loading such as obesity, previous physical activity or joint  78 ry  Muscles and tendons protect the joint 2 by distributing loads across the entire joint ’ 9 surface, thus lessening the load applied directly to the joint. In combination with ligaments and the joint capsule, most muscles and tendons assume a protective role by limiting abnormal joint movement.’ 0 In addition, the mechanical stress from muscle that is placed on the bone during physical activity contributes to an increase in bone density 1  and strength. 13 Therefore, it is reasonable to assume that the structure and characteristics of the muscle tissue will also affect joints and bones; for example, fibre degeneration, or atrophy occurring in skeletal muscle may precede diseases of the joint and bone, such as OA or OP. A previous report showed that there is muscle fibre atrophy in older adults;’ 4 however, there are few reports of muscle structure in people who are undergoing hip surgery. Studies investigating hip fractures are limited to a single biopsy study in older women, 15 and to using dual-energy X-ray absorptiometry (DXA) to measure skeletal muscle mass and fat mass composition, as well as bone mineral density (BMD), in elderly women.’ 6 The vastus lateralis (VL) is the largest quadriceps muscle (an extensor of the leg at the knee joint)’ 7 and is active during many functional tasks such as locomotion and rising from sitting to standing.’ 8 It is possible that as individuals age and develop chronic musculoskeletal diseases, fibres of the VL may show abnormalities and degeneration. These changes may be especially common in people who experience hip surgery as a result of OA or hip fracture secondary to OP. Because the VL may be accessible when orthopedic surgeons perform hip surgery, obtaining and examining a biopsy from this muscle could lead to greater understanding of this muscle’s characteristics, as compared to the VL of older adults without a hip surgery. Tissue samples from the VL of patients with OA or OP undergoing hip surgery were compared in this study to the tissue from the VL of a control group. Describing the muscle tissue of patients undergoing surgical repair following hip fracture, or hip replacement due to OA, will add to the literature on muscle histology prior to hip surgery, and may inform clinicians of changes that occur in the muscle with these conditions. 2  1.2  Study Purpose, Aims, Hypotheses and Rationale  1.2.1 Study Purpose  The overall purpose of this study was to describe morphological and histological differences in muscle fibre characteristics of the VL in persons undergoing surgery of the hip for repair following fracture, or joint replacement as a result of OA, compared to a control group of comparably-aged older adults. 1.2.2 SpecfIc Aims  The first aim of the study was to compare the muscle tissue from the VL of patients undergoing hip surgery with similar muscle tissue from healthy control subjects of similar age, to determine if there were between-group differences. The second study aim was to examine the relationship between the percentage of abnormal muscle fibres and age among a sample comprised both of healthy older adults and older adults with OA or OP. 1.2.3 Hypotheses  It was hypothesized that patients undergoing a surgical procedure on the hip would have a higher percentage of abnormal muscle fibres and adipose tissue in the VL, than healthy, age-similar control subjects. Secondly, it was hypothesized that the percentage of abnormal muscle fibres would be positively related (r 0.75) to age in a sample comprised of both healthy older adults and those with GA or OP.  3  1.2.4 Rationalefor the Study  In 2001, Statistics Canada reported that the number of Canadians aged 65 years or older increased by 2.4 million between 1981 and 2000 (12.5% of Canadian population). Statistics Canada projected that by 2016, 16.6% of Canadians would be over 65 years of age, and by 2051, 25.4% would be over age 65.’ Although the rate at which an individual ages may be a subjective measurement depending on lifestyle choices and genetics, older adults face a variety of physiological changes that occur as well. In addition to cardiovascular, 2023 pulmonary, 25 endocrine, and neural changes, ’ 24 26 older adults experience musculoskeletal changes that affect daily function. These changes may include decreased muscle strength, 27M  RTNE\’ 1995  loss of muscle mass, 28 decreased  bone mass, 29 and changes in body composition. ° 3 Sarcopenia, which is a loss of muscle mass, occurs secondary to advancing age andJor immobility. Not only does overall muscle mass decline, but there are increases in nonmuscle mass constituents, such as adipose and connective tissues. These age-related musculoskeletal changes can combine with other risk factors to predispose adults to development of bone andlor joint disease, and have a deteriorating effect on bone and joint diseases such as OP and OA. 3 Sarcopenia is also a major contributor to frailty, a variation from nonnal aging. 31 Frailty is a loss of physiological reserve that puts an individual at risk for increasing disability from minor stresses. Muscle weakness, weight  loss, sarcopenia, frequent falls, exercise intolerance and immobility are common features of frailty. 33 ’ 3  4  Hip fractures are common in older adults, creating (through decreased mobility) an increased risk for mortality, decrease in lean body mass, and increase in total fat mass. 34 Often resulting from a non-traumatic event (e.g., a low-impact fall), an intertrochanteric fracture or a femoral neck fracture can cause serious disability. Those who have hip fractures often experience incomplete functional recovery, such as the ability to walk independently, or to independently carry out activities of daily living. 34 The need for hip replacement surgery is also common in older adults and often necessary when a person experiences the joint damage of OA that causes daily pain and interferes with daily activity. Although decreased function in both OP and OA may imply that there are also changes in muscle structure, there are few reports of muscle structure in persons prior to undergoing surgery for hip fracture or hip replacement. It would be valuable to know if degenerative changes in the muscle have already occurred prior to hip fracture or joint replacement by comparing the VL muscle of those undergoing surgery to that of healthy control subjects.  5  CHAPTER 2: LITERATURE REVIEW 2.1  Hip Fractures  The hip is a synovial, ball-and-socket joint (enarthrosis), and has multi-axial movement between the rounded head of the femur and the cup-like acetabulum. Two different types of hip fractures typically occur in older adults: intertrochanteric fracture and femoral neck fracture. Intertrochanteric fracture is more common but femoral neck fracture is more dangerous because of the risk of damaging the blood supply to the femoral head. 35 Intertrochanteric fractures occur along the lines between the greater and lesser trochanters, and through cancellous bone, which has a high blood supply and therefore heals well. Femoral neck fracture occurs when a fall producing a direct blow to the bone occurs. This can happen in three ways: 1) from a lateral rotation of the extremity; 2) when the head of the femur is firmly fixed by the anterior capsule while the neck rotates posteriorly; or 3) when the posterior cortex impinges on the acetabulum. Femoral neck fractures can lead to serious complications during recovery, such as osteonecrosis of the femoral head. 36 2.2  Reparative Hip Surgery  In a femoral neck fracture, surgical screws may be used, or a metal device may replace the broken part of the head of the femur (partial joint replacement; hemiarthroplasty). As a result of having a hemiarthroplasty, the patient is able to mobilize without having to wait for the bone to heal. In the case of intertrochanteric fracture, a compression screw and side plate are placed in the femur to hold the broken bone in place while the femoral head is still able to move normally in the socket. The fracture is stabilized with the  6  screw inserted into the femoral head and crossing through the fracture. The plate runs down the femoral shaft and is held in place by smaller screws. 36 Important considerations following hip fracture and the resulting surgery are patient mortality and functional outcome. The dominant factors that influence these two considerations are the patient’s age and their level of functional status prior to the injury. No matter which type of fracture occurs, data show that patients who have low functional capacity prior to hip fracture have a difficult time coping with the challenges of the fracture. 37 This has also been confirmed when investigating the factors that affect short-term rehabilitation outcomes. Hershkovitz et al. 38 recently reported that not only pre-injury functional status, but also cognitive ability, nutritional status and depression were important factors associated with the rehabilitation success of patients with hip fracture. Rehabilitation success was measured using functional gain, discharge destination and length of stay in the facility. With a large proportion of the outcome of hip fracture depending on pre-injury functional level, it is important to understand the integrity of the muscle tissue prior to injury. 2.3  Osteoporosis and Osteopenia  2.3.1  Pathology and Diagnosis  The greatest risk factor for hip fracture is an underlying diagnosis of osteoporosis (OP), a systemic metabolic bone disease characterized by low bone mineral density (BMD) and deterioration of bony micro-architecture. Consequently, there is an increase in bone fragility and susceptibility to fracture. ° OP is characterized by low bone mass, and 4 ’ 39 microarchitectural deterioration, with consequential increase in bone fragility and fracture risk. ’ The criteria for diagnosis of OP is a bone mineral density (BMD) that 4 7  lies> 2.5 standard deviations below the mean for young healthy people (T score  =  -2.5  43 As a pre-cursor to OP, osteopenia is low BMD compared to the normal ’ 42 SD). reference (1.0-2.5 SD below the mean). The loss of bone mass is measurable, but it is still possible to prevent bones from reaching the threshold criterion of OP.” 43 However, because this widely-used criterion was derived from Caucasian women, there may be differences when this criterion is applied to males or non-Caucasian females. Osteoporosis is a preventable and treatable condition. Some measures that aid in preventing bone loss are pharmacological, such as bisphosphonates, which increase BMD and decrease fracture incidence by reducing the rate of bone turnover and inhibiting osteoclasts. 44 Another method to build bone mass is physical activity, specifically resistance training and weight-bearing exercises, such as jogging or weight lifting. These exercises place stress on the bones, and result in osteogenesis and increased bone 45 strength. 4 ’ 6 Throughout life, bones are continuously going through a process of breaking down and remodeling. Osteoclasts remove bone by acidification and proteolytic digestion, and osteoblasts secrete the organic matrix of the bone into a resorption cavity. 47 Bone mass during adult years is a result of the amount that has been built prior to full maturity and that which is lost with aging. 48 Bone mass is added during childhood and teenage years, which results in larger and denser bones. This continues to happen until around the age of 30, when peak bone mass is reached. After that, bone loss slowly begins to be greater than bone formation, resulting in compromised bone strength. 49 Peak bone mass, which is the amount of bone that is present at the end of skeletal maturation, is a primary determinant for the risk of fracture due to OP if it is low. 8  One of the greatest difficulties with OP is that it may not be evident in an individual until a fracture, commonly of the hip, occurs. Therefore, it is important to identify the risk factors for OP and frailty fractures to aid in prevention, evaluation of the condition, and treatment. OP, hip fractures, and hip repair surgeries each have closely linked risks and lifestyle factors. With regards to OP, there are some non-modifiable risk factors and other modifiable lifestyle factors. ° 5 Non-Modifiable Risk Factors: •  Age: The largest factor contributing to OP is aging. In the mid-i 990s, it was estimated that in the USA, 30% of post-menopausal Caucasian women had OP and a further 54% were osteopenic; by the age of 80 years, 70% had OP, and 27% ’ 5 osteopenia.  •  Gender: Although women are at higher risk than men, men also have a risk for osteoporosis. OP affects approximately 45% of women over the age of 50 years, and about 16% of men; in men, OP occurs approximately a decade later than in women. 42 The decrease in BMD in women that follows menopause may be one reason for the larger prevalence. Both men and women reach peak bone density in their early 20’s, but men have, on average, a 10-12% higher peak bone mass. 42  •  Ethnic background: Individuals who are of Caucasian or Asian descent have a higher risk of developing OP. Those of African or Hispanic descent have a lower risk. In a study by Araujo et al., 52 Black men were found to have higher BMD and bone mineral content in the hip than Hispanic or White men. In a female sample, Black women also had the highest BMD, and Asian women had the lowest. Values of 9  BMD for First Nations and Caucasian women were similar, but slightly lower in Hispanic women. Risk for fracture in Black and Asian women was low, with Caucasian, Hispanic, and First Nations women, respectively, having higher risk for 53 fracture. Lifestyle Factors: •  Body mass index (BMI): Those with a low body weight, and tall height have a higher risk for OP; high body weight and BMI decrease the risk for hip fractures. 54  •  Physical activity: Inadequate exercise both in leisure aerobic activities and strength training are risk factors for OP, showing that there is a protective effect of physical activity in regard to hip fractures. 55  •  Occupation: Occupational activity does not markedly increase BMD in postmenopausal women; 56 however, long-term exposure to heavy-activity jobs may have a protective influence on risk of hip fracture. 57  •  Smoking: Smoking has been shown to have a negative effect on BMD, 58 with smokers having a higher relative risk of hip fracture and overall cumulative excess bone 59 loss. 6 ’ 0 However, with regard to menopausal status, there is some controversy as to whether BMD is the same or lower in smokers. In early reports, bone density was shown to be the same in smokers and non-smokers in pre menopausal women, but after menopause there is greater bone loss in people who are ° More recently, the negative effect of smoking has been 6 ’ 59 currently smoking.  10  shown to be independent of risk factors such as age, weight, sex, and menopausal 58 status.  Other factors such as inadequate nutrition (calcium or vitamin D deficiencies and lack of a balanced diet), medications such as glucocorticoids, anticonvulsants and those for hyperthyroidism, as well as excess alcohol intake have also shown to have significant influence on the prevalence of OP. ° It is noteworthy that within the lifestyle factors 5 outlined above, a common theme is loading upon the bone: the greater the load on the bone, be it through mechanical loading in physical activity, or physiological loading though a higher BMI, the lower the risk for OP and hip fractures. OP is a common disease in older adults, which may be partnered with frailty, or inadequate levels of physical activity. These characteristics suggest that muscle abnormalities may be present in individuals with OP since musculoskeletal changes, such as muscle atrophy and weakening, are also apparent in normal aging and with decreased activity; fibre type distribution and fibre size have also been shown to change between young and older persons. ’ 6 2.4  Hip Replacement Surgery  Primary OA is the most common indication for total hip replacement (THR) in adults. OA is typically viewed as a disease that occurs in older populations, suggesting that there may be age-related muscle abnormalities present. In aging, the size and power of muscle tissues decrease; 62 skeletal muscle fibres become smaller in diameter, and less elastic because of increasing amounts of connective tissue (fibrosis). 6 Since OA is an  11  indication for THR, the risk factors and predictive factors that contribute to the need for THR are the same for those of OA. 63 Primary OA exists when there is no other underlying condition found, such as congenital dysplasia, or rheumatoid arthritis. 64 In a two-year longitudinal study investigating the predictive factors for THR, Gossec et al. evaluated demographic data, patient history, clinical status and radiography of patients aged 40 years and more, with painful hip OA. It was found that among this population, patients with increased radiological grade, high perceived severity, and a need for non-steroidal anti-inflammatory drugs were more likely to be candidates for THR. 63 2.5  Osteoarthritis  2.5.1  Pathology and Diagnosis  Osteoarthritis (OA) is a joint disease that begins when the protective covering ofjoint cartilage begins to stiffen and wear away. OA is characterized by a gradual loss of articular cartilage within synovial joints, which is associated with hypertrophy (increased BMD) of the bone. The characteristic elements of OA include reduced joint space, the development of osteophytes (bony outgrowths), and subchrondral bone sclerosis (hardening of the bone beneath the cartilage) which contributes to the thickening of the joint capsule. 65 These changes are a result of three physiologic phases: Phase 1) Edema and microcracks: The first visible change in OA is swelling of the  extracellular matrix, particularly in the intermediate layer. The cartilage loses the  12  smooth aspect, developing microcracks. There is a focused loss of chondrocytes, which alternates with areas of chondrocyte proliferation. Phase 2) Fissuring and pining: The microcracks deepen in the cartilage perpendicular to the direction of forces of tangential cutting and along the fibrils of collagen. There is a formation of vertical clefts in the subchondral bone cartilage. Chondrocytes appear around the vertical clefts in clusters, as well as at the surface. Phase 3) Erosion: Finally, the fissures cause pieces of cartilage to detach and fall into the articular cavity. This causes osteo-cartilaginous loose bodies and the uncovering of the subchondral bone, and the formation of microcysts. These loose fragments cause the mild synovial inflammation characteristic of OA. Subchondral sclerosis increases with disease progression, due to the close proximity of new bone. 66 Although many joints, such as those in the hand, knee, hip, and spine, may develop OA, the clinical manifestations of the disease present differently in each joint group. There are large mechanical stresses applied to the hip joint, which can cause biochemical changes in cartilage, and inadequate repair mechanism for chondrocytes. When this occurs, articular cartilage disruption can occur. As disruption continues to proceed, an asymptomatic condition changes into OA. 9 Diagnoses and criteria for the definition and classification of OA of the hip are based on two types of classification: radiological and clinical. The American College of Rheumatology has classified hip OA and knee OA differently. Two combinations were used in order to develop classification criteria in hip OA: clinical with and without  13  laboratory criteria, and combined clinical, laboratory and radiographic criteria (see Appendix  1).12  Risk factors for OA fall into two categories: risks due to increased vulnerability to loading, and risks specifically related to loading factors. 9 In the first category, systemic factors such as age, gender, genetic predisposition, and ethnic background are included, whereas in the second group, lifestyle factors such as obesity (BMI), previous injury,  and intense physical activity are examples. These factors can act together to lead to OA or its progression (Figure 2.  Figure 2.1: Risk factors for osteoarthritis’° Figure removed due to copyright restrictions. Figure outlines the systemic factors, intrinsic joint vulnerabilities, and extrinsic factors that lead to the progression of development of osteoarthritis. Taken from: Felson DT. Risk factors for osteoarthritis: Understanding joint vulnerability. Clin Orthop Relat Res. 2004;(427 Suppi) : Si 6-21.  OP and OA are musculoskeletal disorders that are frequently present in the hip joints of older adults, especially women. Older women are at highest risk for OP and fractures,  and are also those who typically need hip replacement for OA. A recent study examining the prevalence of OP in OA patients undergoing THR or total knee replacement found that the occurrence of OP in those with OA is equal or greater than in 14  the general population. As well, those patients who have THR are at higher risk for OP both prior to and after surgery, 67 suggesting that the muscle characteristics of individuals who experience hip fracture may be similar to individuals who have OA, especially when the disease has progressed far enough to require surgery. 2.6  Muscle Characteristics During Aging  During the aging process, characteristics of skeletal muscle change. For example, size and power of muscle tissues 62 decrease and muscle fibres become smaller in diameter and less elastic because of increasing amounts of connective tissue (fibrosis). 6 OA and OP are often viewed as musculo-skeletal disorders that occur in older adulthood. Despite age-related skeletal muscle changes, few researchers have examined the histological characteristics of muscle structure of the thigh in people undergoing hip surgery due to OA or OP. Sato et a!. examined the effect of injury on the supporting muscles of the hip in women who had sustained a hip fracture due to a fall and reported that type II fibre atrophy may be an independent risk factor for hip fractures, possibly resulting from low physical activity or vitamin D deficiency.’ 5 Nakamura and Suzuki compared a group of women with OA of the hip, and a group of women with OA of the knee, to a control group of women who had hip fractures of the proximal femur with an absence of OA.’ 4 Type II fibre muscle atrophy and abnormal arrangement of muscle fibres were found in all three groups; however, no abnormal mosaic patterns, such as fibre type grouping and grouped atrophy of fibre types, were found in the muscles of the group with hip OA or in the control group. In addition, another study 68 indicated that fibre type atrophy and abnormal arrangement of the patterns in the muscular mosaic (which may be a consequence of interruption of 15  peripheral nerve supply) may contribute to decreased muscle strength and increased functional disability in persons with joint disorders. Therefore, it may be reasonable to expect similar responses and the presence of muscle abnormalities in individuals who undergo hip surgery. Investigation of the histology of muscles of the thigh may lead to a greater understanding of the integrity of the muscles in patients undergoing surgery to treat a musculoskeletal disorder of the hip.  2.7  Muscle Biopsy  Muscle biopsies are essential in the evaluation and diagnosis of patients with possible muscle disease or myopathy. Biopsies can also provide a basis for comparison between normal and abnormal muscle tissue. There are a number of benefits of performing enzyme histochemistry on a muscle biopsy. Tissue fixation techniques in histochemistry  allow the use of fresh, unfixed, snap-frozen tissue to preserve muscle and cellular detail, without sacrificing enzyme activity. As well, histochemistry allows the determination of muscle fibre types when classifying muscular disorders. 69 However, there are also some limitations to obtaining and using muscle biopsies. First, collection of the sample itself may cause muscle damage. Second, a biopsy represents a small sample of a much larger muscle and may not reflect the abnormalities distributed in other regions of the muscle or muscle group. There is also the possibility of inadequate biopsy processing, in which the specimen becomes unusable or deficient for interpretation. Finally, a wide range of histological findings can be present in myopathies and each individual muscle characteristic may be non-specific or reflective of a variety of pathologies. 69 Therefore, it is important to be thorough and meticulous  16  when handling biopsies, as well as to understand precisely what the histological findings indicate.  17  2.8  Characteristics of Skeletal Muscle Tissue  When evaluating a biopsy of normal skeletal muscle, the muscular architecture is evident in cross-sections, as well as in longitudinal sections. Skeletal muscle is made up of bundles (fascicles) of fibres, surrounded by connective tissue. The fibre is composed of a number of myofibrils, each of which contributes to the development of force as a result of repetitive attachment and Figure 2.2: Cross section of normal skeletal muscle  reattachment of myo sin and actin filaments, which slide past each other during this process. ° 7 When observing a cross-section of a normal fascicle, muscle fibres in humans have polygonal shapes and diameters of about 5O.im. This configuration allows for tight packing of the fibres and enables the maximum number of fibres to be contained in a fascicle (Figure 2.2).71 The interstitial space between fibres is usually not wider than 1 tm. 72 Connective tissue (CT) surrounding the fibres provides structure to the muscle and transmits serial and lateral forces to the tendon. CT is organized at three levels, the epimysium, perimysium, and endomysium, which cover the complete muscle body, fascicles, and fibres, respectively. CT holds muscle fibres together, and provides a channel for blood vessels and nerves that supply the fibres. CT resists excessive passive stretching of the muscle while also distributing forces. ’ Finally, the muscle fibres 7 18  contain numerous nuclei, situated peripherally along the inner surface of the plasmalemma. In normal muscle, fewer than 3%-4% of fibres should have internally located nuclei. 69 The function of the nuclei is to direct protein synthesis. ’ The number 7 of nuclei situated within each cell is not constant and will increase if the cell grows in length or diameter or decrease if the cell atrophies. ° 7 There is some evidence indicating that people who sustain a hip fracture also have muscle 41 weakness. 7 ’ 2 As well, people who have OA in the hip often experience joint pain, considerable muscular weakness and atrophy. 73 However, in both hip fracture and hip OA, there is an absence of data regarding hip muscle morphology and histochemistry in the muscles surrounding the hip joint.’ 74 There is some evidence that muscle fibre ’ 5 atrophy (particularly type II), fibre-type grouping, and increase of fatty tissue and connective tissue (fibrosis) are prevalent in older ’ 33 adults. 7 5 In certain myopathies, 6 there are proportional histopathological changes of fibres, including the following: •  Necrosis, with infiltration of macrophagic cells surrounding degenerating fibres. Cell death (necrosis) and degeneration may be the result of trauma 5 In addition, macrophages are the main or, importantly, inflammation.’ cell type evident after muscle injury, playing a significant function in the inflammatory and regenerative stages of skeletal muscle injury. 7779  •  The presence of small, angulated fibres with dense nuclei. When muscle fibres are no longer regular, tightly packed and of polygonal shape, it is 68 an indicator of muscle fiber atrophy.  19  •  An increase in centrally nucleated fibres. Internally nucleated fibres may represent a trail of a formerly injured fibres, and have been also proposed to characterize regenerating fibers. 8 ’ 80  •  Fatty infiltration: greater amounts of fatty tissue within the muscle may be an important aspect of the loss of muscle mass and 8 62 ’ 33 strength. 9  These abnormalities are consistent with deterioration of structures within muscle, and are characteristic of myopathic processes. 83 Deterioration of muscular structures will ’ 69 lead to an increased loss of function, which may be related to diseases, such as OP or OA. The function of bones is not unrelated to the function of muscles; for instance, movement of muscle, and the resultant stressful torque on bone leads to increased osteoblast action, and therefore, bone formation.’ 3 Consequently, it is important to understand if any of the above abnormalities exist within the muscles of patients who have sustained a hip fracture or hip replacement. To quantify the characteristics of skeletal muscle in a biopsy, two methodologies can be used: fibre counting or point counting. Both methods identify ratios of abnormal or normal muscle tissue within a sample and use similar definitions in these categories. In fibre counting, the researcher will identify individual myofibres as normal or abnormal. 84 Fibre counting also focuses directly upon the individual fibres or fat cells, and categorizes them into percentages of myofibres with normal or abnormal morphology.  20  2.9  Vastus Lateralis  Because there is little literature on the structure  lateralis in respect to lower  of thigh muscles prior to hip surgery, we investigated the histology of the vastus lateralis (VL) muscle, the largest of the quadriceps femoris group. The three other quadriceps muscles are the vastus medialis, vastus intermedius, and rectus femoris. The origin of the VL is a broad aponeurosis which is attached to the superior part of the intertrochanteric line on the anterior and inferior borders of the greater trochanter, the lateral lip of the linea aspera of the femur, and the lateral aspect of the gluteal tuberosity. The VL inserts via the patellar ligament into the lateral base of the patella and tibial tuberosity (Figure 2.3). The VL is innervated by the femoral nerve with its blood supply from the femoral artery. The major actions of the VL are to extend the lower leg at the knee joint and to stabilize the knee. 84 The quadriceps muscles are important in the performance of important functional tasks such as walking, sit-to-stand (and vice versa), and ascending and descending stairs. 18 Weakness in the quadriceps has substantial impact on movement patterns during these tasks. 85 Therefore, the purpose of this study was to examine morphological and histological differences in muscle fibre characteristics of the yE in persons undergoing hip surgery compared to a control group of similar-age older adults without OA or OP. Impairments, which are considered in the International Classification of Function as an 21  abnormality of body structure and function, 86 and which are evident in persons who experience hip fracture or OA, were defined in this study as changes in muscle structure. Examining the tissue of the VL at the histological level contributes to knowledge about muscles of patients undergoing hip surgery compared to older adults without OA or OP.  22  CHAPTER 3: STUDY DESIGN AND METHODS 3.1  Study Design  This was a two-part exploratory study. The first part was a two-group comparison to examine histological differences in the vastus lateralis (VL) muscle of a group of patients undergoing hip surgery compared to those of a control group. The second part explored the relationship between percentage of abnormal fibres and age in a combined group of healthy older adults and those with OA or OP. Biopsies of the VL were taken from individuals who underwent hip surgery (see Appendix 2 for consent form) and from healthy individuals in a similar age range. Age and gender of the participants was provided from information obtained at the time of surgery or, in the case of the control group, when the biopsy was taken. 3.1.1  Subjects  Sixteen individuals who had hip surgery and 20 control subjects were recruited for this study. The group from which the control samples were obtained was the control group in a previous study. 87 Inclusion and exclusion criteria for the subjects were established prior to the start of this study, as part of ongoing research at Vancouver Coastal Health Research Institute. Inclusion Criteria: 1) Adults aged 50 to 85 years. 2) Surgery group Those scheduled for a hip repair following hip fracture, or -  those undergoing total hip replacement for OA.  23  3) Control group Otherwise healthy individuals recruited from the general -  population, as part of a previous study, who had not had hip surgery. Control subjects had been age, gender and BMI matched to the experimental group of the previous study. Subjects were free of respiratory disease, and other co morbid conditions such as cardiovascular disease, neurological conditions, or lower extremity musculoskeletal disorders. 87 Exclusion Criteria: Individuals who had: 1) Fracture related to neoplasm 2) Systemic inflammatory disease 3) Equivocal diagnoses of chronic respiratory disease or cardiovascular disease 4) Uncertain medication history that included medications that might be myopathic in action. 3.1.2  Ethical Approval  Recruitment occurred via orthopedic surgeons from the orthopedic trauma and reconstructive teams at Vancouver General Hospital. Surgeons received consent from the patient prior to surgery, and then informed the research team when the biopsy was available to collect (see Appendix 2). The biopsies for the control group were collected from a sample recruited from the general population using newspaper advertisements and posters at community centres. 87 The experimental protocol to obtain biopsies in patients undergoing hip surgery received ethical approval from the University of British Columbia Clinical Research Ethics Board (CREB). The biopsies for the control group were collected previously as part of a  24  completed study that was also approved by CREB; each participant provided written, informed consent prior to participation. 87 3.1.3  Muscle Biopsy  a) Procedures Biopsy Collection, Tissue Processing and Staining Biopsies of the VL were taken from participants in both the OA!OP group, and the control group. For the OA/OP group, the biopsy was obtained during surgery, through the site accessed for the surgery and did not exceed total muscle volume of 1.0 cm . 3 Following the surgery, the samples were immediately quick frozen for later histological processing. All biopsy preparation, storage and viewing took place at the Muscle Biophysics Lab, at the Research Pavilion, which is part of the Vancouver Coastal Health Research Institute. All procedures were carried out by lab staff and project investigators. The biopsies for the control group were taken using a 5-mm diameter biopsy needle, from the VL at approximately 15cm superior to the patella. Participants were resting and a local anesthetic was inserted into the skin and tissue surrounding the VL; a small incision was made, and using the needle and suction, a biopsy of approximately 80-100 mg was obtained. 87 The procedures for biopsy preparation, storage, and viewing of the control group muscle samples was the same as for the OAIOP group. Tissue fixation and freezing was done via a standard  Biopsy  procedure using gum tragacanth, liquid nitrogen and 88 To start, an appropriate amount of gum isopentane. tragacanth was placed onto a cork block that had been  25  labeled with a patient number; the storage container in which the specimen was stored was pre-chilled and labeled with the same patient number. The tissue was placed transversely onto the mound of gum tragacanth, so that the longitudinal axis of the muscle fibre was perpendicular to the surface, and then inverted and placed into a container of isopentane that had been pre-cooled in liquid nitrogen. The sample stayed in the isopentane until freezing was complete, approximately 10 seconds. The frozen biopsy was then removed, wrapped in cooled paraflim and quickly placed into a pre chilled airtight storage container to be stored at —70°C. or below. Staining for morphology was done using a common procedure of haematoxylin and eosin (H&E) staining comprised of serial steps of placing the tissue section in Baker’s formalin, Harris’ haematoxylin, 1% HC1 in 70% ETOH, and eosin. H&E is a routine histological stain that is used for the evaluation of basic tissue organization and cellular structure. It provides a clear stain of the muscle showing the nuclei to stain dark blue and cytoplasm to stain a consistent pink or red. Following storage, transverse sections of the muscle were cut and stained using H&E staining. Muscle samples were treated with commercially available reagents using the following procedure: To begin, the muscle tissue was fixed in Baker’s formalin for two minutes and then rinsed in tap water. It was then treated with Harris’ haematoxylin for one minute and rinsed in tap water again. Third, the muscle was treated with 1% HC1 in 70% ETOH for five seconds and rinsed in tap water. Next, it was treated with a solution to enhance the blue staining by dipping in 1.5% NaCO 3 and washing well in tap water. Finally, it was counterstained in eosin for two to five seconds, dehydrated in absolute ethanol and cleared in hemo-de and mounted on a glass slide. 26  Viewing and Image Capturing Procedures  Viewing the muscle tissue and quantifying muscle abnormalities using fibre counting was done by a graduate student (AP). Digital images were captured using the Eclipse program, attached to an IBM computer and Axiophot Photomicroscope (Nikon, Japan). Kohler illumination was used to optimize resolution while viewing the slides. In counting fibres individually, the numbers of normal and abnormal muscle fibres were recorded and the percentages of normal and abnormal fibres quantified, in relation to the total fibre count. Fibre counting proceeded using a transparency placed over a digital image, magnified at x20, on a computer monitor. Each specimen slide and digital image was identified by a random number and blinded to the observer as to whether the sample  was from the control group or OAJOP group. Healthy muscle cells are usually closely packed, polygonal in shape, and with no or few acute angles. Using H&E staining, the normal cytoplasm is a consistent pink colour with the nuclei located along the edges of the cell, staining blue. Fibres of the VL were categorized as follows: no count, normal, abnormal, and adipocytes. Sub-categories were abnormalities that included cell configuration (including small or angulated fibres), position of nuclei, inflammation or necrosis, or abnormal cytoplasm characteristics. The individual fibres were classified into one of these seven categories and sub-categories. The fibre-counting categories and sub-categories are shown in Table 3.1.  27  Table 3.1  Categories for Quantification of Abnormal Myofibres Based on 89-91  Haematoxylin- and Eosin- Stained Cross-Sections of the Vastus Lateralis.  1) Normal Muscle Fibre  2) Abnormal Muscle Fibre  3) No  Count  4) Adipocyte  Polygonal fibre with acidophilic cytoplasm (the stain is consistent throughout the cytoplasm), plasma membrane and peripheral nuclei or capillary (small blood vessel with endothelium only). If light basophilic stain is continuous from a distinct nucleus, the fibre is considered as normal. Internally nucleated fibres: A fibre with> 1 internally located nucleus (sarcoplasm between nucleus and sarcolemma). There must be at least 8 pixels of sarcoplasm between nucleus and sarcolemma. Also included in this category, light basophilic stain that is far from the edges of the fibre. Small angulated fibre: (a) small fibre ( 1/3 lesser fibre diameter of the five largest fibres in the field) or b) fibres with “spear like” extensions or extensions that are less than 45 degrees or c) fibre with?: 2 acute angles (<90 degrees) Abnormal cytoplasm: includes 1) fibres with pale acidophilic peripheral cytoplasm and enlarged peripheral nuclei with or without visible nucleoli, or 2) fibres with pale acidophilic peripheral cytoplasm and deep acidophilic “fuzzy” cytoplasm in the central region, or 3) fragmented, whirled or split cytoplasm, or 4) vacuoles, or 5) uneven cytoplasm unrelated to processing, or 6) fibre with dull or light gray staining, or 7) cytoplasmic fragmentation, or 8) lipofuscin (brown-yellow pigmentation > area of muscle nucleus). Inflamed or necrotic: Fibre with more than one inflammatory cell, or necrotic mass of inflammatory cells, and muscle debris without plasma membrane Unidentifiable muscle fibre, space or artifact. < 1/3 of the fibre can be seen in the field. Empty space surrounded by cell membrane consistent with size and shape of adipocyte.  28  b) Categories and Definitions for Fibre Counting Categories and definitions for fibre counting were established through previously used schemes in recent literature,  89-91  and by comparing the schemes to images of normal and  abnormal skeletal muscle cross-sections, as defined in Table 3.1. Fibre counting results were recorded in an Excel database program by a graduate student (AP), with the guideline to analyze 20 images per biopsy. There were some biopsies that did not contain a total of 20 images; therefore, the number available was used. In other instances, the field of view had significant artifacts due to technical issues, and thus fibres may not have been quantified. These included: •  longitudinally or obliquely cut fibres;  •  freezer or cutting artifact;  •  indistinguishable fibre borders due to staining or artifact;  •  folding of the biopsy on the slide;  •  field was out of focus, or staining was fuzzy, smudged, or contained an air bubble.  Finally, in some fields, fibres were only partially quantified for these same reasons. On occasion, a fibre would be quantified as “no count” if characteristics were indistinguishable. If a difficult field was still quantified, a footnote was recorded to explain how much of the field was not counted, why it was not counted, or why it was a difficult field to quantify. As well, there were a number of fields that contained considerable connective tissue, open space, or red blood cells in the image. Fibre  29  counting is unable to quantify these characteristics. In this case, a footnote was made of the observation. The percentage of total fibre count of normal muscle, abnormal muscle, and adipose tissue was calculated, using the following formulas: Total count  =  Percentage of normal muscle  fibres in categories 0-6 counts in category 1 x 100 total count  Percentage of abnormal muscle  counts in category 2-5 x 100 total count  Percentage of adipose tissue =  counts in category 6 x 100 total count  Before conducting inter-observer reliability of the fibre counting, definitions, categories, and fields for the muscle imaging were established to ensure that all observers were evaluating the same operationally-defined and standardized abnormalities. Inter-observer reliability of at least twelve fields was examined before fibre counting of the study biopsies began. Intra-observer reliability of nine fields was performed within one week of the first fibre counting procedure, and re-assessed at monthly intervals during the fibre counting procedure. Biopsy images for three separate subjects were analyzed at each interval, with three fields being analyzed per subject. All subjects and fields were chosen from the combined subject group by the graduate student’s supervisor, allowing the student to be blinded to the subjects being chosen for reliability measurements. This also ensured that no subject or field was analyzed twice.  30  3.1.4  Statistical Analysis  Statistical analysis was done using SPSS version 17.0. A descriptive analysis [mean ± standard deviation (SD)] of the muscle biopsies was completed by reporting the percentage (%) of abnormal, normal, no count fibres, and adipocytes. The abnormal count was further described by evaluating the fibres according to the sub-categories of small/angulated fibres, internally nucleated fibres, inflamed necrotic fibres and fibres with abnormal cytoplasm. To assess normality of the data from the descriptive analysis, a Shapiro-Wilk test was performed. a)  Parametric Analysis  To address the first aim of the study, the percentage of normal fibres, abnormal fibres, and adipocytes between the patients who had undergone hip surgery and the healthy control subjects were analyzed. An analysis of covariance (ANCOVA) was performed to control for age as a covariate between the groups. Then, a post hoc univariate analysis was done on all categories and sub-categories. To do this, a t -test was  performed and a Bonferroni’s correction was applied because seven categories existed. As a result, alpha was set at 0.007. Finally, the patients with OA were removed from the OA!OP group in order to focus solely on patients with OP; some additional subjects  were removed from both the control group and OP group in order to make the age of the two groups equivalent. These stratified groups were re-analyzed using a t-test with n12. To examine the potential effects of aging on muscle for the second aim of the study, we combined the OA/OP and control groups, and analyzed muscle samples from all subjects. This analysis was done to provide a baseline percentage of abnormal muscle  31  fibres in a general sample of older adults, since there is no previous literature that gives any indication of this number. In order to have a larger sample size for this measurement, the two groups were combined. The relationship between the percentage of muscle fibres in each category and age in all subjects was examined using a simple linear regression. The Pearson product moment correlation was used to examine interobserver and intra-observer reliability of fibre counting. b)  Non- Parametric Analysis  Since the data violated two important assumptions for parametric tests, normal distribution and homogeneity, equivalent, non-parametric statistical tests were performed. To begin, addressing the first aim of the study, a Kruskal-Wallace test was performed to examine differences between the groups. Then, a Mann-Whitney U test was performed and the Bonferroni’ s correction was applied for the post hoc univariate analysis on all categories and sub-categories. Finally, stratified groups with OA patients removed were re-analyzed using a Mann-Whitney test with n 12. To examine the potential effects of aging on muscle for the second aim of the study, the relationship between the percentage of muscle fibres in each category and age in all subjects was examined using Spearman’ s rho.  In summary, there are strengths to the use of either parametric or non-parametric tests in analyzing data from this study. The parametric tests are generally more robust, with greater statistical power. In addition, specifically for this study, the ANCOVA controlled for age as a covariate. In regard to the non-parametric tests, such as the Kruskal-Wallace and the Mann-Whitney U, there are fewer assumptions made about the 32  population data. Most importantly, non-parametric tests can be used when the data are not normally distributed, and there is not homogeneity in the variance.  33  CHAPTER 4: STUDY RESULTS 4.1  Sample Characteristics  Sample characteristics of the participants in the study are outlined in Table 4.1. The OAIOP group was significantly older than the control group, and there were more  females than males in both groups. The subjects in the study ranged in age from 50-85 years, with a mean age of 68.7 ± 9.3 years. In the OA/OP group, the number of subjects originally equaled 16; however, while fibre counting, one subject was lost due to inability to accurately quantify the fibres due to longitudinally or obliquely cut fibres. In patients with OP, the surgical procedure would occur on day of admission or up to three days following admission.  34  Table 4.1  Sample Characteristics for Subjects with OA or OP who Underwent Hip Surgery and Control Subjects (mean ± standard deviation)  Number Age (yrs): mean Sex  +  SD*  OA/OP 15 74.5±7.4 11 women, 4 men  Control 20 64.3±8.1 11 women, 9 men  No. of Subjects Osteoarthritis Femoral neck fracture (OP) Intertrochanteric fracture (OP)  2 8 5  Time to Surgery (related to patients with hip fracture)  Same day as admission 1 day post admission 2 days post admission 3 days post admission + Unknown  2 7 2 1 1  Surgical Procedure  Total hip replacement Open reduction, internal fixation Closed reduction, internal fixation Replacement of femoral head + Unknown  2 3 1 8 1  Diagnosis  *p< 0 . 0 5 + Data on date of admission, surgical date, and type of surgery were not available for one of the OP subjects. 4.2  Descriptive Tissue Features of the Muscle Biopsies  When presenting the results, tables and statistics do not include the “no count” category, even though this category was included in preliminary data collection. When categorizing the fibres, a “no count” fibre represented a fibre which was present in the field, but not able to be categorized, whereas a count of 0 indicated that no existing fibre was observed in the image with that specific categorization. Therefore, this resulted in the tables and charts adding up to less than 100% of the total fibre count. Raw data of  35  the muscle fibres counted in each of the categories, as well as the footnotes pertaining to fields which contained indistinguishable fibres, are found in Appendices 3 and 4. When data from all the older adults (OA/OP and healthy controls) were combined, normal muscle represented the largest total percentage of muscle fibres, followed by abnormal muscle, while the category with the smallest total percentage was adipocytes (Figure 4.1).  Figure 4.1 Percentage total fibre count of normal muscle, abnormal muscle, and adipose tissue of H&E stained cross sections measured by the fibre counting technique for the total sample (n=35). Bars and whiskers represent mean ± SD.  Total Sample 70  62.95  I 60  F  50  m 40  F  a .0  iZ 30 U U,  20 10 172 0 Normal Muscle  Abnormal muscle  Adipose Ilunue  36  In regard to the sub-categories of abnormal muscle tissue, internally nucleated fibres comprised the greatest percentage, followed by fibres with abnormal cytoplasm, inflamedJnecrotic fibres and small angular fibres (Figure 4.2).  Figure 4.2 Percentage of total fibre count for subcategories of abnormal muscle H&E stained cross-sections measured by the fibre counting technique for the total sample (n=35). Bars and whiskers represent mean ± SD. Total Sample 10  >1  a a, U,  Subjects who had undergone hip surgery had a higher percentage of abnormal muscle fibres than the control group, within the total fibre count. This was also true for each sub-category of abnormal fibres. However, control group subjects had a higher percentage of total adipose tissue, than the OA/OP group. The percentage of total fibre count of normal muscle fibres, abnormal muscle fibres, and adipose tissue of H&E stained cross-sections of hip surgery subjects and control subjects is shown in Figure 4.3.  37  Figure 4.3  Percentage total fibre count of normal muscle, abnormal  muscle, and adipose tissue of H&E stained cross sections measured by the fibre counting technique for the OAIOP (n=15) and control (n=20) groups. Bars and whiskers represent mean (value label)  80.0  ±  SD.  ——-——---—----  70.0 —-  62J52 60.0  —-------———--——  50.O  jControl  —--—-_—.-_------  8 o.o  •HipSsrgery  H---—  ANCOVA 8  30.0  20.0  I  * —  p<O.05 --  --  —  -  14J07 8.5  100  -  [  -••  26  09  _•____  Normal Muscle  Abnormal muscle  Adipose tissue  38  The percentage of total fibre count for sub-categories of abnormal muscle is shown in Figure 4.4. Figure 4.4 Percentage of total fibre count for subcategories of abnormal muscle H&E stained cross-sections measured by the fibre counting technique for the OAIOP (n=15) and control (n=20) groups. Bars and whiskers represent mean (value label) ± SD. 12.0  10.0  8.0 C 0 U  6.0 0  JContr 4.0  •Hip Surgery  *ANCOVA 2.0  P<O.05 0.0 Internally nucleated fibres  Small angular fibres  lnflamrned necrotic fibres  Fibres with abnormal cytoplasm  The most prominent features of abnormal muscle tissue in hip surgery subjects were fibres with internal nuclei, followed by fibres with abnormal cytoplasm, inflamed necrotic fibres and small angulated fibres. Examples of muscle tissue with abnormal features observed in the cross sections of the VL are shown in Figure 4.5.  39  Figure 4.5 Examples of H&E stained cross sections of the vastus lateralis in people undergoing hip surgery: A) inflamed necrotic fibre (white arrow) and adipose tissue (black arrow); B) small angular fibres (white arrows) and internal nuclei (black arrow). A)  B)  40  4.3 Comparison of Hip Surgery and Control Groups To assess normality of the data, analysis of histograms and a Shapiro-Wilk test was performed. Normality was not achieved for every outcome except for age, and normal muscle fibres. a)  Parametric Analysis  To remove age as a confounder, the control group and OA!OP group were compared using an ANCOVA 5 (p<O.O ) . There were significant between-group differences in the category of abnormal fibres, and the sub-category of inflamed/necrotic fibres (Figures 4.3 and 4.4). Subsequently, a univariate post hoc analysis of results was performed on all categories using a one-tailed, two-sampled, unequal variance, student’s t-test of significance. When performing a Bonferroni’s correction with all seven categories (p<O.O07), percentage of abnormal muscle fibres and inflamed/necrotic fibres were the categories shown to meet the requirement of the correction. Finally, when the two OA subjects were removed from the OAIOP group, there were no between-group differences in age but a significant difference (p<O.O ) in abnormal fibres and inflamed necrotic 5 fibres. b)  Non- Parametric Analysis  Since the data did not meet the requirement for normality or homogeneity of variance, the control group and OA/OP group were also compared using a non-parametric Kruskal-Wallace test 5 (p<O.O ) . Confirming the results of the ANCOVA, there were significant between-group differences in the category of abnormal fibres, and the sub 41  category of inflamed/necrotic fibres (p<O.O5). Subsequently, the non-parametric univariate post hoc analysis of results was performed on all categories using a twotailed, two-sampled, unequal variance, Mann-Whitney U test of significance. Again, a Bonferroni’s correction was applied (p<O.007), finding significance between percentage of abnormal muscle fibres and inflamed/necrotic fibres. Finally, when the two OA subjects were removed from the OA/OP group, a significant difference (p<O.O ) in 5 abnormal fibres and inflamed necrotic fibres remained. 4.4  Correlation Between Categories of Abnormal Muscle Fibres and Age  a)  Parametric Analysis  A simple linear regression was performed to determine the relationship between age and the categories of abnormal muscle fibres that were found to be significantly different between OA/OP and control groups following the Bonferroni’s correction (p<O.007): abnormal fibres, inflamed/necrotic fibres, and internally nucleated fibres. Correlations were r = 0.49 for abnormal fibres vs. age (Figure 4.6), and r = 0.45 for inflamed/necrotic fibres vs. age (Figure 4.7). According to Domholdt, 92 the magnitude of these correlations would be classified as low. When a single outlier was removed in the abnormal fibre category, a stronger correlation (r = 0.64) was found, with a magnitude considered as moderate.  42  Parametric correlation scatterplot of percentage of abnormal Figure 4.6 muscle fibres vs. age (r = 0.49) Abnormal Muscle Fibres vs. Age 30  25  a 20 U U)  15  E 0 .0  10  5 0  45  55  65  75  85  95  Age  Figure 4.7 Parametric correlation scatterplot of percentage of inflamed necrotic fibres vs. age (r = 0.45) Inflamed Necrotic Fibres vs. Age  In  9  w I  0  .0 U 41  0  z  0  E E 1  -1 45  55  75  65  85  95  Age  43  b)  Non-Parametric Analysis  Spearman’ s rho was also performed to determine the relationship between age and the categories of abnormal muscle fibres, on the same categories that were found to be significantly different between OA/OP and control groups following the Bonferroni’ s (p<O.OO ) . Correlations were r=.54 (p=.OOl) for abnormal fibres vs. age correction 7 (Figure 4.8), and r=.63 (p.OOO) for inflamed/necrotic fibres vs. age (Figure 4.9). According to Domholdt, 92 the magnitude of these correlations would be classified as moderate. When a single outlier was removed in the abnormal fibre category, a stronger correlation r  0.67 (p=O.000) was found, with a magnitude considered as moderate.  44  Non-parametric correlation scatterplot of percentage of Figure 4.8 abnormal muscle fibres vs. age (r 0.54)  .0  ‘C  E 0 .0  Age Non- parametric correlation scatterplot of percentage of Figure 4.9 inflamed necrotic fibres vs. age (r = 0.63) Iriflammed Necrotic Fibres vs. Age ir  0  U  U 0 4  z  9 o:OHL;fZUl o 6D  70  90  Age  45  4.5  Reliability  Inter-observer reliability of the categories of fibre counting between the graduate student (AP) and research assistant was r = 0.966. The correlation for intra-observer reliability for the three testing sets of identical image fields ranged from r  =  0.88 to r = 1.00, for  normal muscle, abnormal muscle and adipose tissue. In summary, although both parametric and non-parametric tests were used to analyze the data, the results show the same outcomes: in individuals who have undergone a hip surgery due to OA or OP, there is a greater amount of abnormal muscle fibres, than in a control group of healthy older adults. The greater amount of abnormal fibres is most likely due to a greater amount of inflamed and necrotic fibres in the same group. There are strengths to the use of either set of tests; in this study we will disuss the results relative to the parametric analysis.  46  CHAPTER 5: DISCUSSION and CONCLUSION 5.1  First Study Aim  The first aim of the study was to compare the muscle tissue from the VL of patients undergoing hip surgery with similar muscle tissue from healthy control subjects of similar age, to determine if there were between-group differences. It was hypothesized that the OA/OP group would have a higher percentage of abnormal muscle fibres than the healthy control group. This hypothesis was supported, i.e., a significant difference was found between the percentage of abnormal muscle fibres in the OA/OP group and the percentage of abnormal fibres in the control group. To remove age as a confounding factor (because the two groups differed significantly in  mean age), an ANCOVA (p<O.O ) was performed. The total percentage of abnormal 5 muscle fibres is a sum of the sub-categories of small/angulated fibres, inflamed/necrotic fibres, internally nucleated fibres, and fibres with abnormal cytoplasm. When combined, the sub-categories contributed to the significant differences found in the percentage of abnormal fibres between the groups. When these sub-categories were analyzed individually, however, only inflamed/necrotic fibres were found to be significantly different between the two groups. When the hip surgery (OA/OP) and control groups were combined, inflamed necrotic fibres ranked third in total percentage of abnormal fibres present in the muscle tissue of older adults, behind internally nucleated fibres and fibres with abnormal cytoplasm, and  followed only by small angular fibres. Inflamed necrotic fibres represented 0.53% of 47  fibres in the control group, and 2.6 1% in the OAIOP group. No previous studies have examined the presence of inflamed or necrotic fibres in individuals undergoing hip surgery. However, my study found a difference in this group compared to a control group of healthy adults. This difference between groups could be explained by a number of uncontrolled factors, including wait time for surgery. In some patients who experienced hip fracture, surgery occurred on the day of hospital admission whereas for others, the wait time was up to three days. It is possible that the impact of a fall that resulted in hip fracture or the trauma from obtaining the biopsy might have been factors partly responsible for the inflammation that occurred in the muscle tissue. In muscle fibre histology, it is difficult to distinguish between inflamed fibres and necrotic fibres. Even though inflamed and necrotic fibres have a similar presentation when viewing a digital image of the muscle cross-section, a difference exists in the time course in which these characteristics would appear in skeletal muscle. Acute inflammation increases greatly 1-12 hours following muscle damage, 93 whereas myofibre necrosis generally occurs approximately 1-3 days following overloading protocol, i.e., lengthening contractions, or hindlimb suspension in animal models. 94 The role of inflammation and fibre regeneration in skeletal muscle has been examined in the literature, without a clear outcome. In the present study, the significant difference in the presence of inflamed necrotic fibres between the OA/OP group and the control group could indicate the phagocytic phase of acute inflammation, 96 the initiation of muscle ’ 95 repair phase,  95,96  or, conversely, might be a sign of impaired regenerative capacity of  the muscle. 95  48  Inflammation in muscle fibres has both positive and negative aspects. Inflammation benefits the muscle by being a protective response to tissue injury, 95 helping with recovery and repair from injury, and to promote the clearance of damaged tissue. 96 As a response to skeletal muscle injury, there are three stages of inflammation: phagocytosis, repair and remodeling. 98 In the first stage of inflammation, there is a marked increase ’ 97 of neutrophils, followed by a decline approximately 24 hours after injury, when the number of macrophages increase, sequentially rising in ED1+ and ED2+ macrophages. ’ 95 97,99  Inflammation and prominence of inflammatory cells in muscle fibres are thought to be responsible for the removal of cellular debris. For example, neutrophils play a role in inflammation by the phagocytosis of damaged tissue, and release of pro-inflammatory cytokines to recruit circulating macrophages. 97 However, neutrophils have also been shown to promote tissue injury (“killing” healthy tissue), and play a role in secondary muscle 9597 damage. 9 ’ 9 This damage may be caused by oxidative modification of skeletal muscle proteins, and also may impair or delay the events associated with resolution of skeletal muscle injury.’ 00 Following the invasion of neutrophils in injured skeletal muscle, a prominent invasion of macrophages occurs. There are two sub-types of macrophages prominent in skeletal muscle, ED 1 + and ED2. 99 ED 1 + macrophages are more abundant during the early stages of inflammation, where they play a role in phagocytosis. ED2 macrophages become abundant later, in the repair and regeneration phase of inflammation. 97 This ’ 79 invasion of macrophages (specifically ED2) often represents the initiation of the repair phase of inflammation. It has been reported that macrophages do not contribute to 49  muscle membrane injury, 98 or secondary damage. 97 Instead, they have a role in recovery and may have a number of roles in this phase; these include removing debris through phagocytosis, changing to an anti-inflammatory phenotype in regenerating muscle, preventing apoptosis of muscle fibres, releasing factors to promote cell activation and growth, and secreting cytokines and growth factors to promote muscle fibre repair. 97 Following inflammation and repair, remodeling of muscle occurs. Macrophages, particularly type ED2+, are important in satellite cell activation and satellite cellmediated muscle 95 repair. 101 Satellite cells, involved in the normal growth of muscle ’ tissue, contribute to the regeneration of muscle tissue following injury or  02  the regenerative stage, satellite cells proliferate and can replace the previously damaged 98 However, it has been proposed that systemic inflammation of muscle tissue muscle. may be related to the function of satellite cells. Changes in environment, or circulating substances surrounding the satellite cells, may be factors in impaired differentiation, particularly in older ages. Therefore, systemic inflammation may be a factor limiting myogenic 3 regulation,’° and may also contribute to impaired regenerative capacity of older muscle, by limiting availability of growth factors that promote muscle 95 regeneration. The infiltration of inflammatory cells, such as phagocytes that surround fibres, may represent the negative aspect of degeneration, leading to cell death (necrosis). Necrosis may represent a pathological process, which may result in muscle deterioration. However, the presence of inflammatory cells, such as ED2 macrophages, may represent the positive aspect of muscle fibre regeneration. Because of the difficulty in 50  distinguishing between an inflamed fibre and a necrotic fibre, in this study, we cannot determine if the fibres in any of our sample groups are in the process of inflammation, necrosis, or the degeneration/regeneration cycle. A post hoc analysis was performed on all categories of muscle fibres and adipose tissue, to further support the ANCOVA. To apply a more conservative measure, a Bonferroni’s correction was performed, confirming that the percentage of abnormal fibres was significantly greater in the OA/OP group than in the control group. In individual sub categories, percentage of inflamed/necrotic fibres was significantly greater in the OAJOP group than in the control group; the percentage of small angulated fibres, internally nucleated fibres, fibres with abnormal cytoplasm, and adipose tissue were not significantly different between the groups The internally nucleated sub-category of abnormal muscle fibres represented the greatest percentage of abnormal muscle fibres present in the total sample of older adults. Internally nucleated fibres represented 4.25% of fibres in the control group and 6.85% in the OA/OP group. Internally nucleated fibres represent a trail of formerly injured fibres, which characterizes regenerating fibres. ’ 104, 105 Presence of internal nuclei in 81 more than 3% of the muscle fibres is considered abnormal, ’ 107 and can be considered 106 a sign of muscle damage and slow recovery from 00 injury’ 104,105 Therefore, an ’ abnormal percentage of internally nucleated fibres in the overall sample (comprised of both the OA/OP subjects and healthy controls) may represent a measure of muscle damage or degeneration, suggesting the possibility that as individuals age and/or undergo surgery, there is a greater amount of immature myofibres and impaired muscle Overall, the presentation of histological characteristics of degenerated 51  muscle fibres (the combination of inflamed necrotic and increased internally nucleated fibres) may imply that there is a functional deficit in the VE muscle of people with features of abnormal muscle fibres. Following the post hoc test, two patients with OA were removed from the OAIOP group as well as the oldest subjects from this group, and the youngest subjects from the control group so that each group had 12 subjects. The other subjects were removed in order to make the age of the two groups equivalent. Although this step reduced the subject numbers and decreased statistical power, it allowed us to do an analysis focusing on the subjects with OP. The percentage of abnormal fibres, and inflamedlnecrotic fibres were once again found to be significantly different between these two smaller groups comprised of 12 subjects each. Our results confirmed that for people in this study with hip fracture there were changes in the VL suggestive of muscle degeneration. Because the surgery occurred within 1-3 days of the fracture, we cannot speculate if the degeneration was present in the muscle before the fracture or as a result of the fracture. 5.1.2  Findings Regarding Adipocytes  In an unexpected finding, the control group biopsies contained more adipocytes than those of the OA/OP group, albeit not significantly more. Prior to data collection and analysis, while addressing the first aim of the study, we hypothesized that there would be a greater percentage of adipose tissue in individuals with OA or OP who underwent hip surgery, as compared to a control group. A greater presence of fatty tissue within the  muscle may be an important aspect in the loss of muscle mass and strength.  52  There are conflicting findings in studies examining the relationship between bone mineral density (BMD) and fat mass. Travison et al. 108 demonstrated that the positive relationship between relative weight of an individual and proximal femur strength was accounted for by lean mass, not adipose tissue. However, Di Monaco et al.’ 6 showed that fat mass, not skeletal muscle mass, is a pivotal detenninant of BMD. Finally, a study by Nguyen et a!. 72 showed lower abdominal fat may moderately contribute to increased risk of hip fracture; however, this risk factor was not independent from those of femoral neck BMD or weight of the person. Considering that extra fat tissue may provide cushioning from the impact of a fall, the only study examining force attenuation shows that “force attenuation in trochanteric soft tissues alone is insufficient to prevent hip fracture.”° 9 The current body of conflicting literature may suggest that further investigation in this area should be performed, specifically in investigating the relationship between fat and bone. 5.2  Second Study Aim  To address the second aim of the study, evaluating the relationship between age and percentage of muscle fibres, a simple linear regression was performed on the total sample of OAIOP patients and control subjects, comparing subjects’ age to the percentage of muscle fibre characteristics. The analysis demonstrated that, as individuals’ age, there is a greater percentage of abnormal muscle fibres (r = .49, p .003). In addition, relationships between age and inflamed/necrotic fibres (r =.45, .007), and age and internally nucleated fibres (r = .47, p  =  p  =  .004) were found.  53  While a high percentage of abnormal muscle fibres may suggest a lack of integrity of the muscle, there has been limited research describing the presence of abnormal muscle fibres in people undergoing hip surgery. My findings support those of Sato et al. 15 who examined the middle gluteal muscle of fractured hips in older women, and found that the muscle of those who had hip fracture showed degenerating and regenerating fibres, fibre necrosis, and inflammatory reaction. In two other studies’ 68 examining histological ’ 4 characteristics of the VL in people with osteoarthritis, undergoing total hip or knee replacements, the authors reported that fibre type atrophy and abnormal arrangement of patterns in the muscular mosaic may contribute to decreased muscle strength and increased functional disability. However, an increased percentage of fibre atrophy was not found in the current study when comparing the biopsies of the OAIOP group to those of the healthy controls. Because muscle strength and functional disability were not examined in the present study, it is not possible to compare the current findings to this previous research.’ 68 In essence, the results of the study contribute information related ’ 4 to the percentage of abnormal muscle fibres present in the VL of people with hip surgery. 5.3  Study Strengths and Limitations  5.3.1  Study Strengths  This was a feasibility study aimed at further understanding the baseline levels of normal and abnormal fibres in healthy older adults and those with OA or OP. We investigated the differences in percentage of normal and abnormal muscle fibres between a control group and a group of individuals with OAJOP who underwent hip surgery. Being amongst the first of its kind, this study provides a starting point and direction for future 54  studies, as well as providing initial knowledge of relative percentages of normal and abnormal muscle fibres in older adults. Throughout the course of the study, high interand intra-observer reliability in fibre counting were maintained, suggesting that data collection was consistent throughout the course of the study. As the study progressed, a number of different statistical analyses were employed. As we proceeded through those analyses, we began to understand our findings more thoroughly, and become more confident with the results. Although the results did not confirm all of our hypotheses, they facilitate a better understanding of differences in muscle structure between two different groups of older adults. 5.3.2  Study Limitations  There were a number of limitations within this study. Both sample and group sizes were small for the number of variables that were investigated. In addition, when the groups were combined for the linear regression analysis, the combined group did not represent a completely healthy sample of older adults, because 15 out of 35 individuals had undergone a hip surgery. Therefore, the sample used to examine the relationship between age and muscle fibre characteristics was a mixed group. There were a number of limitations in the techniques used in this study. In each technique requiring an individual to learn new skills, a learning curve occurs. For example, even careful preparation and storing procedures can damage the biopsy. Obtaining the biopsy may lead to damage that did not exist prior to the biopsy being taken. Specifically in the control group where a needle was used, the size of the biopsy, and poor alignment with the muscle may lead to technical disruptions of the biopsy. In 55  the preparation of the biopsy, cutting and staining techniques may lead to the cells within the digital biopsy image being difficult to quantify, leading to inaccurate counting of the muscle fibres. Also, freezing artifacts may lead to inaccurate quantification of the individual muscle fibres. To minimize this possible inaccurate quantification, when quantifying difficult fields with large amounts of cutting or freezer artifact, the investigator (AP) made notes of these unusual observations. The biopsies in the two groups were taken from different locations in the VL. Furthermore, the VL is not a true muscle of the hip but rather of the knee. Although the VL is a muscle that is important in locomotion and actions such as sit-to-stand, a number of limitations arise. First, biopsies from the VL may not be entirely representative of the structure of the muscles surrounding the hip. Secondly, since the biopsy location was high on the VL (at the site of the surgical incision and in close proximity to the hip) in the OA/OP group, it is possible that in some of the patients with OP the VL was injured during a fracture-related fall and might have been contused. The presence of red cells outside of blood vessels in some of the biopsy images also indicates this possibility. It is possible that the impact of falling might have caused changes in the muscle fibres, such as acute inflammation, that were not present prior to a fall. Finally, the structure of the proximal VL may be different than that of the distal VL. In the control group, the biopsy was obtained distally, i.e., 15 cm superior to the patella, a notably different part of the muscle than the proximal portion. This study would have been stronger if it had been possible to access and examine a muscle from the hip, such as the gluteus medius, or quadratus femoris, instead of the VL. This would increase the strength and clinical relevance of the study by reflecting the characteristics of a true muscle of the hip. A true 56  muscle of the hip may be more affected by the musculoskeletal condition of the hip joint, compared to a muscle of the leg, such as the VL. With the hip fracture surgery (OP) group, the wait times between admission and surgery varied among subjects. Because the wait time to surgery was beyond the control of the current investigator, the different lengths of time might have led to differences in the muscle fibres among the OP subjects within the OAIOP group. Wait time until surgery may cause changes to the muscle, such as atrophy, that did not exist prior to hip fracture. In addition, in subjects with OP who may have experienced a hip fracture due to a fall, the inflammatory process may be at different stages across subjects, depending on their wait time until surgery, therefore contributing to differences in the muscle fibres. With regard to the fibre counting technique, it is possible that the investigator became more efficient as she proceeded through data collection and likely more accurate. Although the reliability of fibre counting remained high throughout data collection, a learning curve presents a possibility that the fibre counts at the beginning of the study were different than those at the end of the study. 5.4  Future Directions and Contributions to Field of Study  This feasibility study provides a strong foundation for future research directed toward the relationship of muscle structure and bone strength. Representing the beginning of an important area of research for coming years, this study contributes to the basic understanding of the histology and structure of skeletal muscles that, in turn, play an important role in degenerative conditions such as OA and OP. Assuming that skeletal muscle characteristics and fibre structure affect joints and bones, this study facilitates  57  discussion of the relationship between muscle and bone, which may lead to the research question of whether a disease of the bone may be preceded by a disease of the muscle? Future studies should include larger samples and provide the BMI for all the subjects. A larger sample would enhance generalizability to the population of older adults, and BMI would provide information regarding physiological loading of the joint. The sample groups should be matched for age, so that age would not be a confounding variable. The biopsy collection should be taken from multiple hospital sites, in order to be representative of a larger, and more diverse, population. Finally, the biopsies should be taken from the same area of the muscle, and be taken from a true muscle of the hip, which would better reflect the relationship between the bones of the hip joint and the muscles surrounding. In categorizing abnormal fibre types, it would be beneficial to separate inflamed fibres from necrotic fibres. Because inflammation is an acute response, whereas necrosis occurs over a longer period of time, the presence of inflammatory cells and necrotic fibres in skeletal muscle can represent different processes that are occurring. Greater understanding of the progression of inflammation in skeletal muscle or necrosis of muscle tissue would occur, if a clearer histological difference could be made between these two different fibre types. Studies that include monitoring and history of physical activity by the subjects involved would contribute valuable information to this area of research. Physical activity, specifically weight-bearing activities, could minimize the need for hip surgery as a result of a fall. It would be interesting to investigate the relationship of physical activity to 58  histological muscle characteristics in a sample of older adults undergoing hip surgery for OA or OP. 5.5  Clinical Relevance of Study Findings  Strongly based in basic science, this study may seem to be distantly related to any clinical concerns. However, understanding cellular differences of a lower extremity muscle in a group of patients with OA or OP, as compared to a healthy group of a similar age range, might be important in the treatment of older adults. Impairments, which are defined in the International Classification of Function as a change in body structure and function, may be directly related to the changes of muscle structure in patients with OA or OP. Adequate functioning of the lower extremities could be related to specific characteristics of skeletal muscle, e.g., the structure of the fibres, the percentage of abnormal vs. normal fibres. As well, muscle loading (which is influenced by muscle structure) contributes to the maintenance of bone architecture, decreasing the amount of bone loss in aging. Although we are not able to speculate on the changes of functional status in people with OA or OP, prior to or following hip surgery, this study provides an understanding of muscle pathophysiology that is essential for clinicians working with older populations. In order to focus on the maintenance of healthy muscles, this study can help clinicians to continue to link body structure to health conditions, activity, and participation. 5.6  Conclusion  In conclusion, the most significant finding in this study was the difference between groups in percentage of abnormal fibres, specifically the increased presence of inflamedlnecrotic fibres in the OA!OP group. This confirmed the first hypothesis, that 59  there would be a greater percentage of abnormal fibres in a group with OA or OP compared to a healthy control group. This shows that in some histological characteristics, the muscle tissue of older adults who experience hip surgery is different from those who do not. As well, there were low to moderate, positive correlations of abnormal fibres, inflamedJnecrotic fibres, and internally nucleated fibres with age. 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Acta Orthop. 2007;78:505-510. 74. Rasch A, Dalen N, Berg HE. Test methods to detect hip and knee muscle weakness and gait disturbance in patients with hip osteoarthritis. Arch Phys Med Rehabil. 2005;86:237 1-2376.  69  75. Lexell J, Taylor CC, Sjostrom M. What is the cause of the ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men. JNeurol Sd. 1988;84:275-294. 76. Kirkendall DT, Garrett WE,Jr. The effects of aging and training on skeletal muscle. Am JSports Med. 1998;26:598-602. 77. Orimo S, Hiyamuta E, Arahata K, Sugita H. Analysis of inflammatory cells and complement C3 in bupivacaine-induced myonecrosis. Muscle Nerve. 1991;14:515-520. 78. Round TM, Jones DA, Cambridge G. Cellular infiltrates in human skeletal muscle: Exercise induced damage as a model for inflammatory muscle disease? J Neurol Sci. 1987;82:1-1 1. 79. Tidball JG. Inflammatory cell response to acute muscle injury. Med Sci Sports Exerc. 1995;27: 1022-1032.  80. Macgowan NA, Evans KG, Road JD, Reid WD. Diaphragm injury in individuals with airflow obstruction. Am JRespir Grit Care Med. 2001;163:1654-1659. 81. Charge SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev. 2004;84:209-238. 82. MacIntosh BR, Gardiner PF, McComas AJ. Aging. In: Skeletal Muscle Form and Function. 2nd ed. Windsor: Human Kinetics; 2006:328. 83. Wortman RL. Myopathic dieseases. Bulletin on the Rheumatic Diseases [serial online]. ;5 1: 14-Dec-2007. 70  84. Moses KP, Nava P, Banks J, Petersen D. Atlas of Clinical Gross Anatomy. New York: Elsevier Mosby; 2005 :495. 85. Mizner RL, Snyder-Mackler L. Altered loading during walking and sit-to-stand is affected by quadriceps weakness after total knee arthroplasty. J Orthop Res. 2005;23: 1083-1090.  86. Darzins P, Fone S, Darzins S. The international classification of functioning, disability and health can help to structure and evaluate therapy. Aust Occup Ther J. 2006;53:127-131.  87. Mathur S. Skeletal Muscle Dysfunction in People with COPD and Recipients ofLung Transplants. [Ph.D]. Vancouver: University of British Columbia; 2005. 88. Muscle biopsy general instructions. Available at: http://www.medicine.uiowa.edulpath_handbook/appendix/anatomicpath/ex muscle bio psy.html. Accessed 12/10, 2007. 89. Scott A, Wang X, Road JD, Reid WD. Increased injury and intramuscular collagen of the diaphragm in COPD: Autopsy observations. Eur Respir J. 2006;27: 51-59. 90. Reid WD, Sharma A, Elliott WM, Davis JE, Road JD. Diaphragm morphology at postmortem in people with acute and chronic respiratory disease. Cardiopulmonary Physical Therapy Journal. 2007; 18:3--12.  91. MacGowan N. Diaphragm injury in chronic respiratory disease. [M. Sc.J. Vancouver: University of British Columbia; 1998.  71  92. Domholdt E. Analysis of relationships. In: Physical Therapy Research Principles and Applications. 1st ed. Philadelphia: Saunders; 1993:275. 93. Clarkson PM, Hubal MJ. Exercise-induced muscle damage in humans. Am JPhys Med Rehabil. 2002;81 :S52-69. 94. Cannon JG. Intrinsic and extrinsic factors in muscle aging. Ann N YAcad Sd. 1 998;854:72-77. 95. Toumi H, F’guyer S, Best TM. The role of neutrophils in injury and repair following muscle stretch. J Anat. 2006;208 :459-470. 96. Maclntyre DL, Reid WD, McKenzie DC. Delayed muscle soreness. the inflammatory response to muscle injury and its clinical implications. Sports Med. 1 995;20:24-40. 97. Smith C, Kruger MJ, Smith RM, Myburgh KH. The inflammatory response to skeletal muscle injury: Illuminating complexities. Sports Med. 2008;38:947-969. 98. Tidball JG, Berchenko E, Frenette J. Macrophage invasion does not contribute to muscle membrane injury during inflammation. JLeukoc Biol. 1999;65:492-498. 99. Tidball JG. Interactions between muscle and the immune system during modified musculoskeletal loading. Clin Orthop Relat Res. 2002;(403 Suppl):S100-9. 100. Pizza FX, Peterson TM, Baas TH, Koh TI. Neutrophils contribute to muscle injury and impair its resolution after lengthening contractions in mice. JPhysiol. 2005;562:899-91 3. 72  101. Merly F, Lescaudron L, Rouaud T, Crossin F, Gardahaut MF. Macrophages enhance muscle satellite cell proliferation and delay their differentiation. Muscle Nerve. 1 999;22:724-732.  102. Aiway SE, Siu PM. Nuclear apoptosis contributes to sarcopenia. Exerc Sport Sci Rev. 2008;36:51-57. 103. Degens H. Age-related skeletal muscle dysfunction: Causes and mechanisms. J Musculoskelet Neuronal Interact. 2007;7 :246-252. 104. Sasaki A, Aizawa T, Tomiya A, Matsubara Y, Kokubun S, Itoi E. Effect of resting interval for muscle regeneration in mice. Ups JMedSci. 2007;1 12:175-181. 105. Thaloor D, Miller KJ, Gephart J, Mitchell P0, Pavlath GK. Systemic administration of the NF-kappaB inhibitor curcumin stimulates muscle regeneration after traumatic injury. Am JPhysiol. 1999;277:C320-9.  106. Grobler LA, Collins M, Lambert MI, et al. Skeletal muscle pathology in endurance athletes with acquired training intolerance. Br J Sports Med. 2004;3 8:697-703. 107. Lexell J, Jarvis J, Downham D, Salmons S. Quantitative morphology of stimulation-induced damage in rabbit fast-twitch skeletal muscles. Cell Tissue Res. 1992;269: 195-204. 108. Travison TG, Araujo AB, Esche GR, Beck TJ, McKinlay TB. Lean mass and not fat mass is associated with male proximal femur strength. JBone Miner Res. 2008;23:189198.  73  109. Robmovitch SN, McMahon TA, Hayes WC. Force attenuation in trochanteric soft tissues during impact from a fall. J Orthop Res. 1995;13:956-962.  74  APPENDICES Appendix 1  Criteria for the Classification of Hip Osteoarthritis  12  “Clinical Criteria: A classification tree was developed, without radiographs, for clinical and laboratory criteria or for clinical criteria alone. A patient was classified as having hip OA if pain was present in combination with either 1) hip internal rotation 15°, pain present on internal rotation of the hip, morning stiffness of the hip for 60 minutes, and age> 50 years, or 2) hip internal rotation < 15° and an erythrocyte sedimentation rate (ESR) 45 mm/hour; if no ESR was obtained, hip flexion 115° was substituted (sensitivity 86%, specificity 75%). Clinical plus radiographic criteria: the traditional format combine pain with at least 2 of the following 3 criteria: osteophytes (femoral or acetabular), joint space narrowing (superior, axial, and/or medial), and ESR <20 mm/hour (sensitivity 89%; specificity 91%). The radiographic presence of osteophytes best separated OA patients and controls by the classification tree method (sensitivity 89%; specificity 91%).”  75  Appendix 2  Consent for Obtaining Muscle Biopsies  THE UNIVERSITY OF BRITISH COLUMBLA OEPARIMENT OF ORTHOPAEDICS # llO828Weit10’A’eni Vcouvr, BC, V5Z 116 CANADA OflIce (604) 675-5239 F (604) 875-4438  Consent Form Thigh Muscle From Middle Aged and Older Adults Principal Investigator: Dr W.D. Reid, PhD, Dept of Physical Therapy, University of Bntish Columbia. Vancouver General Hospital, (604) 875-5239 Co-Investigator(s): Drs. P.J, O’Brien, MD, FRCSC, Robert N. Meek. FRCSC P. Blachut, MD. FRCSC. H. Broekhuyse, MO, FRCSC; and P. Guy, MD, FRCSC, Orwiopaedic Trauma, Drs. C. Duncan, MO, FRCSC, B. Masri, MD, FRCSC, D. Garbuz, MD, FRCSC, N. Greidanus. MD, FRCSC, Reconstructive Orthopaedics, Vancouver General Hospital Contact Telephone # (Available 24 Hours): (604) 875-5239 Background: We are inviting subjects such as yourself who have been diagnosed with a thigh fracture that is unrelated to any underlying bone disease, or Osteoporosis (a reduction in the amount of bone mass, leading to fractures after minimal trauma) or Osteoarthritis (a form of arthntis that results in the destruction of the articular cartilage that lines the joints and seen most often in the larger weight bearing jOnt5 of the hips, knees and spine, but may also be evident in the small joints of the hands) to participate In a research study. In order to tietter understand the role of muscle in Osteoporosis and more generally, the dysfunction or deterioration of muscle overall, we are initiating a study that will compare the muscle from three separate groups. These groups are 1. Subjects diagnosed with a traumatic fracture of the thigh that requires surgical treatment 2. Subjects diagnosed with a hip fracture related to Osteoporosis that requires surgical treatment 3. Subjects diagnosed with Osteoarthritis that requires surgical treatment. There is a Canadian national orthopaedic fundraising group called Hip Hip Hooray. Our study was reviewed in the past by a multidisciplinary Orthopaedic professional committee of Hip Hip Hooray, charged with distributing funds for research. We applied to this committee for a grant and were successful In getting a grant to cover the operating costs of this research project. Clinical studies and observation indicate that subjects with Osteoporosis have weak muscles as well as weak bones. Subjects of the same age with Osteoarlhritis have generally strong muscles and strong bones. It is well known that people who have CF Version 9: October 10, 2007  Page 1 of 5  76  Go To fext Page  weak muscles duo to musoo diseases (such as muscular dystrophy. pa’alys.s from polio) go or to develop wea’ bones. It is thought that cores beconie weak duo to a lack of use. It s not clearly understood yet, wny people vtn weak bones (wrich are goneraly riot painful) also develop weak muscles. It wou’d be more ogcal, based on t9e infornatior’ to date, to say that peooo witri panful oirits (such as in Osteoartriritis) would have weak muscles as a result of trying to avoid pain cy riot using the joints. Purposa: The purpose of ths study Is to compare the muscle bSsuo in peooe ‘rem tnreo groups (T’aumatic fracture. Ostooarthrtc and Osteopootc) to determine whether there a’e dfforlng characteristics within the muscle tissue. Alternatives: if you choose not to participate in this study, your planned surgice procedure wit be carried out as scheduled. The choice not to oartciOate will smply moan that the muscle sample will not be harvested.  Inclusion Criteria • SubecLs with either ostcoarthrts or OStoopo’osiS who are undergoing a cor’ective hip oporaton • Subjects w’o are undergoing repai’ of a traumatic fracture of the thigh whore the injury is unrelatea to an underyirig bone osease • Sub; ecLs wrio a’o between 40 and 85 years of age Exclusion Criteria Factors which will exclude you from pa”tiooation in ti’s study inciudo.  •  D agnoss of lri’larnma!ory Arthrtis ( nf’matiori o the ;oirt lining as ri Rrioumatoid Arthritis). Diagnosis of a cotagen disease (any aisOrder affecting the connective tissue, with rheumaUc symptoms inudng muscle stiffness, soreness and pain in the joirits ane associated st’uctures). Infection at the operative ste  • • • •  Fractures related to a noopasm HIV positive status Hepatitis B or C positive status ilepatitis B virus, Hepatitis C virus, human  •  • • • •  mmurodeficioncy  vrus  (HIV),  Creutzfodt-Jakob Disease Excessive use of alcohol Prolonged Corticostoroid (Prodnisone) use Musclo paralytic condtion (such as mi.iscular dystroøhy) Patients under 41) years of age Patients ovo’ 85 years of age  CF Version 9: October10. 2007  Page 2o’5  77  Exclusion specific to Ostoporosis Patients: —  Hip (Proxma femur. femoral reck. Or ntertrochantorc rract.jes) in tho past on the same side as the current hip fracture  Study Procedures: If you agree to participate in the study you will have surgery on your nip as you normally would i you wore not particioating in tn’s study. If you enter trio stjcy, a smal muscle sample (may DO taken from two ciffereni m USCOS but will not exceed 2.5 x 11.0 cm in total size) w.ll be taken from either the vastus lateralis (the a’go muscle codorng your upper outor leg), trio gluteus modus (muscle at the outer surface of pelvis), or trio cuadratis femons (muscle located oetwoen trio lower bony OiViS and the upoer portion of me thigh bone). The muscle sample wI be taker from trio area triat will oo accessed for your surgcal treatment. Since trio muscle sampo{s) will be taken from the same area to be accessed for your operation, it wik be easily acccss:blo at the same time as your operation. You will not be aware of any effect from the bioøsy such as pain or functonal loss. The taking of the samplc( will not lengthen the time iooaod to perform yoo surgery. The foilow-up is the same whether you choose to part ci aato in the study or rot. The samole( ‘etnovod. will be taon to the Muscle Rosea’ch Lao at Vancouver Genera Hospital wrie’e t wil either be testea immediately with various chemicals aria equiemont to assess its compositor at the col’uiar level or it will Co storoc for later analysis. The sample wit be aDoled with a uncue Study identi’er wflch s not inked in any way to information that could identify you. This sample wi i be oct in a locked storage facility, in a locked laboratory space wtbr Vancouver Genoa Hospital. Tissue samples will be d sposod o accordng to the established protocol for safe arid confdential nardling of human specimens at Vancouver Genera Hospital. Medical Records Will be reviewed and relevant clinical nforrnation will be collectea aria matched to donor tissue. Al information collected will DO assigned to a unique Study a which is not Inked ri any way to information that could Identify you.  Tissue Banking:  The banking of you’ tissue sample is not a requrement of your participation. You may dec’ine any banking (storing for later use arid analysis) of tissjo by leaving the chock box at trio end of this document blank. Our purpose In storing some of the tissue samples is to aiow for later aria ysis. Results from intial analyses may prompt the ieee for further analysis Cut the cotais regarding the type and reasons ‘or further anatyss are not yet predictable. The tissue wi not be used for any commercial urooses. Any banked tissue will be labeled with a unique study iaent.fer wriich is not linked in any way to information that CF Version 9: October 10. 2007  Page 3 of 5  78  coud identity you. This sample wUl be koet in a locked storage facility, ri a locked abo’atoy space within. Vancouver General Hospital. Tissje samp:os wi I oe diSpoSOd of accordrg to trio established pfotoco to’ safe arc co’ridcritial handlirg & human specimens at Varcouve’ Gerio’al Hosota Reimbursement for Participation: will not receive caymort for your pa’tópation.  Yo.j  Benefits: Yi will not onofit ‘ro’ri trio study. However, tn.s study may provide nfo’mation for futi.ro treatment o t people witi Osteopoross and oOssibIy owe’ diseases involving muscle. Risks: There are genera  ‘is..s associated wth bavrg surgery,  nciucng the stancard  treatment fo’ a trio’ your Tauatic fracture, Ostooporotic rip fractu’o, or you’ Osteoa’th’itic hib replacement. and these are not ciariged or inc’easod by your  earticipatior in this stucy Study Withdrawal:  Your oartcipation ri this study s entrely voluntary. You may withdraw from tis study at any timo witriout oroviding any reasons. I’ yoi. decdo to enter tie study and to wit’raraw at any ume ri the future there will oo no ocrialty or loss of ben&its to whch you arc otherwise ort’tloa, arid yoi.r future medical ca’e will rot  oo  affected  If you chooso to enter the study and then aeco to wtrrdraw at a later time, all data collected about you during your enrollment ri the study witi e retained ‘or analysis. By law, this data cannot be destroyed.  Confidentiality: Your cor’ficentialty will be rosooctec. No iriformatori that dsscloses ye-Jr identity will be released or pubished withojt you’ spee-fic consent to the dse-osuro. Howovo’, research records aria medical recoras identifying you may be inspected in the presence of the nvostgator or us designate by representatives of Health Canada, and the UBC Resoarc-r Et.nics Board ‘or trio purpose of rnontoring research However, rio records whici identity you by came or iriitals will oc allowed to leave the Investigators offices. Contact: If you have any questions or desire further informaton will respect to tri.s Study, please contact the research coordinator, Ms. Raman Johal at (604) 875-5239, (24 hours). If there are any concerns about your treatment or rgrrts as a ‘esea’ch subject you may contact the Research Subject information Lne at the University of Brtsh Columbia, Office of Research Sorvicos, at (604) 822-8598. Signing triis consent form in no way limits your legal rights aganst thO sponso’, investigators, or anyone else.  CF Version 9: October 10, 2007  Page 4 of 5  79  Subject Consent: I understand tnat participation in this stucy is ertiroly voluntary and tnat may refuse to particioato or I may wtharaw fron, the study at any time witho’jt any consequences to my continuing medical caro  I nave rocevod a signea and (latod copy of this consent form fo my own rocords. The Authorzoc Thiro Party and inc invostgator are satisfied that inc information contained n this consent form was oxolairied to the sub)ect to the extent that h&sho is able to understand t, that !l questions nave boon answered, arid that trio subject assents to participating in the research  LI By checking this box I consent to my tissue ceig banked for later usc. By signing below you consent to participate in this study.  Suojoct Name (p ease Print)  Autflor.zocl Third Party Name (Please Pr nt)  Subject Signature  Authorized Third Party Signature  Wtnoss Name (please Print)  Witness Signature  Investigator Name (please Print) Investigator Signatu’o  Date  Date  Date  Date  Subjects Assent to Part)clpate: I nave had the opporturity to read this consent form, to ask questions about my particioation in tuis research, anc to discuss my partcpation with my authorized tnirc party. All my questions nave been answe’ed. I understand that I may withdraw f’om this researcri at any time, and that ths will not interfere with the availability to me of other health care. I have received a copy of this consent form. I assent to oartcioatc ri this study.  Suoject Name (pease Print) CF VersIon 9: October 10, 2007  Subject Signature  Date Page 5 o’ 5  80  Appendix 3  Raw Data from Biopsy Quantification  Subtext is aligned with the field that it refers too  Subjectl NoCount Normal Internal Nuclei Small angulated lnflamedlnecrotic Abnormalcytoplasm Adipocyte Total  Subject2 NoCount Normal Internal Nuclei Smallangulated Inflamedlnecrotic Abnomial cytoplasm Adipocyte Total  1 5 22 0 0 0 2 3 32  2 5 18 2 0 0 8 4 37  3 7 17 1 1 0 1 2 29  4 9 20 1 3 0 4 2 39  5 10 18 0 0 0 5 5 38  6 6 21 1 0 0 1 0 29  7 8 20 1 0 0 2 3 34  8 4 19 0 0 0 3 0 26  9 4 23 0 0 0 8 0 35  10 3 10 3 1 0 6 1 24  11 10 7 3 0 0 7 0 27  12 0 16 1 0 0 5 1 23  13 9 13 0 0 0 8 2 32  14 6 13 0 0 0 8 5 32  15 6 13 3 0 0 6 5 33  16 5 12 1 0 0 3 1 22  17 5 20 1 0 1 6 1 34  18 5 17 1 1 0 2 0 26  19 6 17 0 0 0 3 0 26  2OTotal %Total 6 119 19.5082 19 335 54.91803 2 21 3.442623 0 6 0.983607 0 1 0.163934 5 93 15.2459 0 355.737705 32 610 100  12 3 4 5 6 7jJ 9 10 11 1213 14 15 16 17 18 19 2OTotaI %Total 234710585410491326625610 12121.68459 21 17 16 21 18 20 22 14 17 22 24 22 20 27 21 15 17 21 20 21 396 70.96774 0 0 1 0 2 1 0 0 1 1 1 1 0 0 0 1 3 0 1 1 14 2.508961 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0.358423 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0.179211 1 3 1 0 0 2 0 1 0 1 0 1 0 0 1 1 0 0 0 0 12 2.150538 0 5 0 1 0 0 2 2 0 0 0 0 0 0 0 0 0 0 0 2 12 2.150538 24 28 22 29 30 29 33 22 22 34 29 33 33 29 28 23 22 27 27 34 558 100 2 fibres on outer most right side of the field are disrupted, unable to categorize. 3 cells dassified as no count, because of staining artifactlfolding/fuzzy-ness Some fibres appear ‘folded” over; and have some staining artifact; 3 dassified as no count fibres, 2 dassified as normal One fibre with “gunk” in it. Classified as normal.  81  Subject 3 No Count Normal Internal Nuclei Small angulated lnflamedInecrotic Abnormal cytoplasm Adipocyte Total  7% 9 Total %Total 5541027 35 16.66667 16 20 21 19 20 27 132 62.85714 100300 4 1.904762 000000 0 0 000000 0 0 000000 0 0 7714442 39 18.57143 0 29 32 39 36 26 36 210 100  2 9 0 0 0 0 1 0 12  Freezer artifact; unable to quantify for lack of being able to distinguish cell borders. 2/3 of the field contains Freezer artifact; unable to quantify for lack of being able to distinguish cell borders. Freezer artifact; unable to quantify for lack of being able to distinguish cell borders. One cell with “gunk” and freezer artifact. Classified as normal. Some freezer artifact: Cell on right hand border classified as no-count, as well as cell on top left border. 2 cells along top border classified as no count: however, no distinguishable cell border b/w the two b/c of freezer artifact. Quantified bottom 2/3 of field to distinguishable cell border; top 1/3 has freezer artifact, with indistinguishable cell borders  Subject 5 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  —  0  2 42512 13 13 10 12 13 00100 00000 00000 00000 10003 18 15 16 13 18  7 8 9Total %Total 431 22 14.86486 14 17 17 109 73.64865 020 3 2.027027 121 4 2.702703 000 0 0 000 0 0 510 10 6.756757 24 25 19 148 100  Fibres are oblique and longitudinal. Unable to classify. Lots of freezer artifact; difficult to distinguish cell borders in bottom left 1/4 of field. Did not quantify these fibres. Lots of freezer artifact Lots of freezer artefict; cut almost oblique. Difficult to quantify. Lots of freezer artifact; cut almost oblique. Difficult to quantify.  82  Subject 6 No Count  Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  42i6 7 9O1j!3 392633715  13 14 15 16 17 18 19&2i’ 2 ‘;Total 3357784685  16 11 9 16 16 11 15 16 15 12 17 21 15 25 16 15 16 12 15 0210000102100020000 0000100000000200000 0000000000000000000 0010000010010100000 0000000000000000000 19 22 13 22 20 14 22 18 21 17 21 27 22 35 26 19 22 20 20  95  %Total 23.75  289 9 3 0 4 0 400  72.25 2.25 0.75 0 0 100  Lots of freezer artifact... Lots of freezer artifi lots of freezer artifact Lots of freezer artifact... lots of freezer artifact...  0  0  Too much freezer artifact for accurate counting. Not quantified. Too much freezer artifact for accurate counting; not quantified  lots of freezer artifact, somewhat out of focus. Freezer artifact, and “gunk on some fibres;” those fibres classified as normal. lots of freezer artifact. Lots of freezer artifact Lots of freezer artifact Lots of freezer artifact  Subject4 NoCount Normal Internal Nuclei Smallangulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 2 3 4 5 6 7 8 9iO1 11 12 13 14 16 17 18 Total %Total 5 4 3 4 2 2 3 6 6 3 4 4 5 4 2 4 9 70 20.23121 4 12 6 12 17 16 17 15 12 10 21 17 18 20 20 20 17 254 73.4104 0 2 0 1 3 1 0 0 1 1 0 0 0 0 0 0 0 9 2.601156 00000000000000000 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 2 0 0 0 3 0.867052 0 1 3 0 0 0 0 0 0 0 0 1 1 0 0 0 0 6 1.7341 04 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 4 1.156069 9 19 12 17 22 19 20 21 20 14 25 22 24 28 22 26 26 100 346  One fibre on far left with lots of freezer artifact; classified as no count’- as well, less than 1/3 of fibre in image.  Subject 7 No Count Normal Internal Nuclei Small angulated Inflamed!necrotic Abnormal cytoplasm Adipocyte Total  1 2 3 4 5 6 78 9 .10 Total %Total 54166310 31 30 21.58273 12 11 8 9 8 8 8 14 99 96 69.06475 10100301 00 6 4.316547 0000000010 1 0.719424 0000100000 1 0.719424 0000 000010 1 0.719424 1300 00 0000 4 2.877698 19 18 10 15 15 14 9 15 14 10 139 100 Lots of freezer artifact. Lots of freezer artifact. 83  Subject 8 No count Normal Internal Nuclei Small angulated lnflamedlnecrotic Abnormal cytoplasm Adipocyte Total  4 5 6 7 8 9 10 11 12 13 %Total 49587397775466 24335 104 25.4902 20 15 17 17 17 23 19 13 11 19 17 9 22 22 7 9 11 8 5 281 68.87255 10000003101102 11011 133.186275 01100000000100 00000 30.735294 00000000000000 00011 20.490196 01010010110000 00000 51.22549 00000000000000 00000 0 0 25 26 23 26 24 26 29 23 20 27 23 15 28 30 0 10 14 14 13 12 408 100 114 of field has staining artifact. This area was not quantified. Has a splitting fibre, but not complete. Quantified as a single, normal fibre. Also has a fibre which appears as “halfmoon” Quantified as normal. Some fibres on left side of field have a lot of freezer artifact, and “gunk;” quantified as normal. Fibres cut obliquelyllongitudinaL Unable to quantiI with accuracy. Lots of cutting fibres appear to be longitudinalloblique. Still quantified. Lots of cutting artifact present on field. Still quantified. iots of cutting artifact present on field, and some fibres appear to be longitudinafoblique. Still quantified. Lots of cutting artifact present on field, and some fibres appear to be longitudinalloblique. Still quantified.  Subject 9 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total Subject 10 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 0 9 I 0 0 0 0 10  2 1 10 0 0 0 1 0 12  3 4 13 0 0 0 0 0 17  4 3 15 0 0 0 0 0 18  5 7 13 0 1 0 0 0 21  6 Total 6 10 1 0 0 2  0 19  21 70 2 1 0 3 0 97  %Total 21 .64948 72.16495 2.061856 1.030928 0 3.092784 0 100  123456 7 89 10 11 12 1414 15 16 17 l8Total %Total 6 5 4 5 7 10 10 8 4 9 6 7 10 9 3 4 11 10 128 18.85125 28 34 19 33 33 24 38 33 21 31 24 35 30 14 25 19 28 14 483 71.13402 011112 0 2 1 1 1 1 3 1 2 6 2 3 29 4.270987 010000 000000000000 10.147275 100000 0000010001 03 60.883652 123000 1 3 1 1 1 0 1 2 1 5 0 2 24 3.53461 006000 000000000002 81.178203 36 43 33 39 41 36 49 46 27 42 32 44 44 26 31 35 41 34 679 100 Approximately 114 of field not quantified due to fibres that are folded. Approximately 4 cells of field not quantified as no count due to fibres that are folded. Field is very disrupted, with lots of connective tissue, and gunk on image. 84  Subject 11 No Count Normal Internal Nuclei Small angulated lnflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 2 8 0  o  0 0 0 10  2 3 10 0 0 0 0 0 13  3 2 12 0 0 0 0 0 14  4 4 14 0 0 0 1 0 19  5 4 6 0 0 0 2 0 12  6 4 7 0 0 0 0 0 11  7 4 9 0 0 0 1 0 14  8 0 11 0 0 0 0 0 11  9 Total 4 9 0 0 0 0 0 13  27 86 0 0 0 4 0 117  %Total 23.07692 73.50427 0 0 0 3.418803 0 100  Lots of freezer and cutting artifact Lots of freezer and cutting artifact  Subject 12 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  Subject 13 No Count Normal Internal Nuclei Small angulated lnflamed!necrotic Abnormal cytoplasm Adipocyte Total  I 2 45 5 7 1 0 00 00 00 00 10 12  4 5 6 7 8 9 10 11 13 14 15 16 17 18 19 20 Total %Total 57242544 53235446 74 26.14841 8 8 12 10 15 12 11 10 11 10 12 12 9 13 15 13 193 68.19788 0001 001 0 0001 1 000 5 1.766784 00000000 001 20000 3 1.060071 00000000 00000000 0 0 000121 1 0 0001 0020 8 2.826855 00000000 00000000 0 0 0 13 15 14 16 19 18 17 14 0 16 13 15 19 15 17 21 19 283 100 Fibres cut longitudinally; unable to quantify. Lots of freezer artifact; difficult to distinguish lots of freezer artifact; difficult to distinguish cell borders, and quantify. Fibres cut longitudinally, with lots of freezer artifact; unable to quantify. Lots of freezer artifact that disrupt cell borders Lots of freezer artifact and connective tissue that disrupts cell borders Lots of freezer artifact and connective tissue.  2 3 4 5 6 8 9 10 11 12 13 14—————— 17 18 Total %Total 32242 2134653 41 22 25 7 8 8 10 6 3 7 4 10 12 7 8 6 3 99 60.36585 00011 3212020 13 169.756098 00000 0000000 00 0 0 00000 0000000 01 10.609756 00002 0000000 21.219512 00 00100 3001000 00 53.04878 0 10 10 11 15 11 0 11 10 8 17 18 14 11 0 0 9 9 164 100 Fibres are cut longitudinally, and contain lots of cutting artifact. Unable to quantify accurately. Same image as field #8 Fibres are cut longitudinally, and contain lots of cutting artifact. Unable to quantify accurately. Fibres are cut longitudinally, and contain lots of cutting artifact. Unable to quantify accurately.  85  Subject 14 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  Subject 15 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  I 7 15 I 0 0 0 0 23  2 2 14 0 0 0 0 0 16  3 6 15 I 1 0 0 0 23  4 4 14 1 0 0 0 0 19  5 4 11 0 1 0 1 0 17  6 Total %Total 8 31 26.05042 10 79 66.38655 0 3 2.521008 I 3 2.521008 0 0 0 2 3 2.521 008 0 0 0 21 119 100  1 2 3 4 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total 5 6 %Total 6 11 6 2 5 5 6 8 4 6 7 8 7 4 7 5 2 4 4 7 114 23.65145 15 13 14 19 19 18 16 20 21 10 14 18 18 7 18 15 21 2 3 3 284 58.92116 1 1 2 1 1 0 1 0 1 4 2 1 0 1 0 1 2 2 3 3 27 5.60166 1 0 0 0 0 0 1 0 1 0 2 2 0 0 1 1 1 1 0 11 2.282158 151000201 1000021 1200 173.526971 323101000152132011 11 285.809129 00000010000000000000 10.207469 27 32 26 23 25 24 26 29 27 23 28 31 28 15 29 23 28 12 12 14 482 100  Subject 16 123 456 789 10 11 12 %Total 15 16 Total No Count 6 11 6 255 684 672 12 7121.64634 Normal 15 13 14 19 19 18 16 20 21 10 14 6 4 14 203 61.89024 Internal Nuclei 112 110 101 4 2 1 2 4 21 6.402439 Small angulated 1 0 000 010 1 0 0 4 1.219512 0 1 lnflamedlnecrotlc 151 000 201 1 0 0 11 3.353659 0 0 Abnormal cytoplasm 323 101 000 1 5 0 17 5.182927 0 1 Adipocyte 000 000 100 0 0 0 0 0 1 0.304878 Total 27 32 26 23 25 24 26 29 27 23 28 900722 328 100 1/3 of field has fibres cut longitudinally/obliquely; unable to quantify. As well, there is a lot of freezer artifact, and many red blood cells. Fibres highly disrupted and cut longitudinallylobliquely. Unable to quantify with accuracy. Lots of red blood cells, and the appearance of internally nudeated cells. Fibres highly disrupted and cut longitudinally/obliquely. Unable to quantify with accuracy. Lots of red blood cells. Fibre cross sectional ama is huge— quite disrupted, with lots of red blood cells, and artifact Quanfified as if it is regular cells, but unsure if this is accurate. 1/3 of field has fibres cut longitudinally/obliquely; unable to quantify. As well, there is a lot of freezer arfifact, and many red blood cells.  86  Subject 17  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 20 21 Total %Total NoCount 2 13 7 7 4 2 5 4 6 2 6 4 9 5 2 5 7 8 11 109 19.42959 Nomial 17 14 22 21 18 12 27 20 28 15 17 16 19 25 19 31 30 30 25 406 72,37077 IntemalNudel 0200012001140010100 132,317291 Small angulated 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 1 0 0 3 0,534759 Inflamedlnecrotic 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 1 0 0 0 0 1 0.178253 Abnormal cytoplasm 3 3 4 5 1 6 0 1 0 0 5 0 0 1 0 0 0 0 0 29 5.16934 Adipocyte 0000000000080000000 0 0 Total 22 32 33 33 23 21 34 36 36 18 29 24 28 31 23 36 39 38 57 561 100 0 Staining uses difficultly in distinguhing a staining lots of empty space on th fie. Lots of red blood cel present; unaeto quantifyth Lots of freezer aifa. Slightly fozzy. Difficutlto quantity. Lots of space between fibres; staining sometimes does don provele dear dhndion between cell borders. Staining is fuzzy, in somewhat indistind, ceusing cell borders to be hard to disboguish. Staining is fuzzy, wifh lots of freezer artifad, making Odifficoltto distinguish between fibre boarders; uoableto quantity  Subject 18 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte  Total  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2OTotal %Total 34162534322463322234 64 25.19685 7 10 9 6 9 8 5 5 9 6 9 10 6 8 6 9 6 6 9 9 152 59.84252 10131003010110003101 17 6.692913 00000000002000000010 3 1.181102 10010010000000100000 4 1.574803 0 0 0 0 1001221000101000 9 3.543307 0 2 0 0 0000000010000110 5 1.968504 12 16 11 16 13 10 12 14 14 11 14 15 14 11 11 11 12 10 14 14 254 100  87  Subjectl9 NoCount Normal Internal Nuclei Smallangulated lnflamedlnecrotic Abnormal cytoplasm Adipocyte Total  Subject 20 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 2 4 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total %Total 7 7 3 8 2 3 4 7 2 3 5 5 3 5 6 8 6 6 90 24.19355 1771010119 91119141115201215151214 23162.09677 2 3 4 3 2 3 3 3 2 3 5 2 2 0 1 0 1 2 41 11.02151 000000001000000001 20.537634 0 0 1 0 1 0 0 1 0 0 1 0 0 1 1 1 0 0 7 1.88172 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0.268817 000000000000000000 0 0 26 17 18 21 17 14 16 26 26 20 22 22 25 18 23 24 19 23 372 100  1 6 23 0 0 0 0 0 29  2 10 12 1 0 0 0 0 23  3 3 16 0 0 0 0 0 19  4 7 17 2 0 0 0 0 26  5 9 19 2 0 0 0 0 30  6 5 21 0 0 0 0 3 29  7 7 23 2 0 1 3 0 36  8 12 20 2 0 0 0 0 42  9 9 23 1 0 0 0 1 34  10 5 18 0 0 0 0 2 25  11 6 18 0 0 0 0 0 24  12 Total %Total 8 87 25.14451 29 239 69.07514 0 10 2.890173 0 0 0 0 1 0.289017 0 3 0.867052 0 6 1.734104 37 346 100  On left hand slide of field, it is difficult to distinguish cells; quantified as 2 no count fibres. On top right side of slide, unable to distinguish muscle fibres, due to staining. Could not quantify. Fibre in centre, right hand side of field appears to be splitting. Quantified as a single normal cell.  Subject2l NoCount Normal Internal Nuclei Smallangulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 9 20 0 0 0 3 0 32  2 2 14 0 0 0 5 0 21  3 4 13 1 0 0 4 0 22  4 5 15 0 0 0 3 0 23  5 4 14 0 0 0 6 0 24  6 5 16 0 0 0 3 0 24  7 1 11 0 0 0 5 0 17  8 1 20 0 0 0 4 0 25  9 5 14 1 0 0 5 0 25  10 4 13 0 0 0 0 0 17  11 4 11 2 0 0 0 0 17  12 5 17 2 0 0 0 0 24  j3 14 15 16 l7Total %Total 7 2 5 5 9 7720.20997 14 14 12 12 13 243 63.77953 1 1 1 0 0 9 2.362205 0 0 0 0 0 0 0 0 1 0 1 2 0.524934 0 2 2 0 4 2 48 12. 59843 2 20.524934 0 0 0 0 22 25 25 19 19 381 100 Lots of artifact, and smudged cell borders on this slide.  88  Subject 22 No Count Normal Internal Nuclei Small angulated lnflamedlnecrotic Abnormal cytoplasm Adipocyte Total  3 4. 2 6 10 7 8 11 12 13 14 15 4 1 7 3 4 4 7 6 4 4 2 5 12 7 14 15 14 7 6 13 6 12 14 15 3 0 4 2 4 0 4 3 5 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1 0 1 1 1 0 1 0 1 1 3 0 1 0 0 2 0 0 4 0 0 1 0 0 4 0 2 0 17 0 25 14 0 23 23 0 16 0 22 19 21 20 23 Longitudinal cut fibres; unable to quantify accurately Bubble on left hand side of field; therefore quantified single fibre underneath as no count. Some obliquely cut fibres, but still able to quantify as normal. Some obliquely cut fibres, but still able to quantify as normal. Longitudinal cut fibres; unable to quantify accurately Longitudinal cut fibres; unable to quantify accurately  Subject 23  Total  12  No Count  5  3  5  Normal  9  7 1  3 1  Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  16 Total %Total 5420.76923 3 18 153 58.84615 0 26 10 0 0 0 0 5 1.923077 1 11 4.230769 0 114.230769 22 100 260  -  0  0  o  o  0  2  0  0  o o  o o  13  9  16  0  0  0  0  0  0  0  %Total  4 7  7  24  37.5  3 3  0 1  o o  0 0 0 0  29 7 0 3  45.3125 10.9375 0 4.6875  13  13  1 0 64  1.5625 0 100  0  0  0  0  0  0  Biopsy cut longitudinally and obliquely; unable to quantify Biopsy cut longitudinally; unable to quantify Field is highly disrupted, with lots of artifact; some fibres are cut longitudinally. In this case, quantified as no count. Biopsy cut obliquely; unable to quantify Field is highly disrupted, with lots of artifact; some fibres are cut longitudinally or obliquely. Unable to quantify. Biopsy cut longitudinally; unable to quantify Biopsy cut longitudinally; unable to quantify Biopsy cut longitudinally; unable to quantify Field is highly disrupted, with lots of adifact difficult to quantify. Field is highly disrupted, with lots of artifact; some fibres are cut longitudinally. In this case, quantified as no count Biopsy cut longitudinally; .mable to quantify Biopsy cut longitudinally, and has lots of artifact that makes obscure cell bofders; unable to quantify Biopsy cut longitudinally; unable to quantify Biopsy cut longitudinally; unable to quantify Field is highly disrupted, with lots of artifact; some fibres are cut obliquely. In this case, quantified as no count Field is highly disrupted, with lots of artifact; some fibres are cut obliquely. in this case, quantified as no count Biopsy cut longitudinally; unable to quantify Biopsy cut longitudinally; unable to quantify Biopsy cut longitudinally; unable to quantify  89  Subject24 NoCount Normal Internal Nuclei Small angulated Inflamed/necrotic Abnonnal cytoplasm Adipocyte Total  1 2 5 3 0 0 1 0 11  2 3 14 2 0 0 0 0 19  4 4 6 5 0 1 0 0 16  3 3 13 3 1 1 0 0 21  6 4 13 3 0 1 0 0 21  5 4 13 2 0 1 0 0 20  7 4 13 2 0 0 0 0 19  9 3 13 3 1 1 0 0 21  8 7 14 3 2 0 0 0 26  10 5 12 1 1 0 0 0 19  11 4 12 3 0 3 0 0 22  12 2 9 1 0 0 1 0 13  13 3 9 2 0 1 0 0 15  14 4 9 1 0 3 2 0 19  15 4 11 3 0 4 2 0 24  16 8 5 2 1 0 1 0 17  17 7 15 5 0 3 0 0 30  Total  0  0  0  71 186 44 6 19 7 0 333  %Total 21.32132 55.85586 13.21321 1.801802 5.705706 2.102102 0 100  Lots of freezer artifact and indistinguishable cell borders. Unable to quantify 1/2 of field, unable to quantify, fibres cut longitudinally Lots of freezer artifact and difficult to distinguish cell borders, unable to quantify, fibres cut longitudinally Lots of freezer artifact and difficult to distinguish cell borders. Lots of space between some fibres. Unable to quantify this characteristic, unable to quantify, fibres cut longitudinally Lots of freezer artifact. Lots of space between some fibres. Unable to quantify this characteristic. Lots of freezer artifact Lots of space between some fibres. Unable to quantify this characteristic. Lots of space between fibres, and lots of red blood cells. Unable to quantify this characteristic. Lots of space between fibres, and lots of red blood cells. Unable to quantify this characteristic. Lots of space between fibres, and lots of red blood cells. Unable to quantify this characteristic. lots of space between fibres, and lots of red blood cells. Unable to quantify this characteristic. lots of space between fibres, and lots of red blood cells. Unable to quantify this characteristic.  Subject 25 NoCount Nomial Internal Nuclei Smallangulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 1 7 4 0 0 3 0 15  2 6 10 1 0 0 2 0 19  3 5 14 3 0 0 2 0 24  4 3 16 1 0 0 1 0 21  5 9 6 2 0 0 2 0 19  6 2 12 2 0 0 0 0 16  7 6 15 0 0 1 0 0 22  8 2 14 2 0 1 0 0 19  9 4 17 0 0 0 0 0 21  10 3 18 1 0 0 1 0 23  11 8 8 0 0 0 0 0 16  12 4 17 0 0 0 1 2 24  13 5 13 0 0 0 3 0 21  14 3 14 1 0 0 1 0 19  15 4 9 3 0 0 1 0 17  16 4 10 1 0 0 0 0 15  17 4 11 3 0 0 0 0 18  18 5 12 0 1 0 1 0 19  19 7 11 2 0 0 1 0 21  20 Total 3 13 3 0 0 1 0 20  %Total 8822.62211 247 63.49614 29 7.455013 10.257069 20.514139 20 5.141388 20.514139 389 100  90  Subject 26 No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 2 4 3 5 9 10 6 7 11 12 8 14 15 13 16 17 18 19 20 Total 65653546740383365547 9 11 15 8 11 14 12 12 14 14 13 14 17 13 17 11 12 14 11 15 1 1 1 3 1 1 1 0 2 1 0 2 2 0 2 1 3 4 1 2 0 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 1 0 1 4 3 3 1 1 3 2 2 2 5 0 2 0 1 1 2 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 20 20 23 19 19 22 23 26 20 17 19 20 24 19 24 20 23 28 20 20  Subject27 NoCount Normal lntemalNuclei Smallangulated Inflamedlnecrotic  1 9 25 5 0 0  2 8 23 0 0 0  3 5 22 2 0 0  4  5  4  6  23 3 0 0  Abnormal cytoplasm Adipocyte Total  0 0 39  1 0 32  2 0 31  Subject 28 NoCount Normal Internal Nuclei  2 8 24 2  3 9 22 3  Small angulated  0 1 1 0 36  2 0 0 0 36  0 0 0 0 37  lnfiamedlnecrotic  Abnormal cytoplasm Adipocyte Total  %Total 9522.30047 25760.32864 296.807512 40.938967 51.173709 358.215962 10.234742 426 100  20 1 0 1  6 3 21 5 0 1  7 10 21 0 0 1  8 6 25 3 0 1  9 5 21 2 0 0  10 4 19 0 0 0  11 12 19 3 0 1  12 10 19 2 0 1  13 8 18 3 0 0  14 9 18 4 0 1  15 6 19 3 0 0  16 6 13 5 0 1  17 6 22 3 0 0  18 5 19 2 1 0  19 3 20 3 0 0  2OTotal %Total 6 13120.85987 20 407 64.80892 2 518.121019 0 10.159236 1 9 1.433121  2 0 32  5 0 33  1 0 31  0 0 32  1 0 36  1 0 29  1 0 24  0 0 35  1 0 33  3 0 32  2 0 34  0 0 28  0 0 25  1 0 32  2 0 29  1 0 27  1 4 34  4  5  10  11  12  13  14  15  16  17  18  19  20  7 20 2  8 9 28 3  9  9 30 1  6 11 23 2  7  12 23 2  8 25 2  9 33 1  8 32 1  8 28 0  5 33 1  5 14 6  6 24 1  7 25 3  6 31 2  8 27 3  7 32 2  7 31 1  21 Total %Total 15720.54974 8 23 528 69.10995 1 39 5.104712  1 0 0 0 41  0 0 0 0 36  3 0 3 0 35  1 0 3 1 45  1 1 0 0 37  1 0 2 0 46  2 0 0 0 43  3 0 0 0 39  2 1 0 0 42  0 0 0 0 38  2 0 0 0 43  0 0 1 0 40  1 0 0 0 33  1 1 1 1 0 0 1 0 1 1 0 0 0 0 0 0 33 37 41 26 field is quite fuzzy, and out of focus.  25 3.980892 40.636943 628 100  23 3.010471 4 0.52356 12 1.570681 10.13089 764 100 91  Subject 29 NoCount Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  1 1 9 1 0 1 0 4 16  2 3 8 0 0 0 0 5 16  3 4 10 1 0 0 0 0 15  4 1 11 1 0 0 0 1 14  5 2 9 2 0 0 0 3 16  *  Subject3O No Count Normal Internal Nuclei Small angulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total Subject3l NoCount Normal lnternalNuclei Smallangulated Inflamedlnecrotic Abnomial cytoplasm Adipocyte Total  1 2 7 1 0 0 0 0 10  6 3 9 0 1 1 1 1 16  12 2 6 1 0 0 0 0 9  13 14 15 16 17 19 20 21 24 Total %Total 2 3 3 4 3 4 4 3 6 4823.18841 7 4 6 4 7 3 7 8 7 11555.55556 0 3 3 2 0 2 1 1 2 20 9.661836 0 0 0 0 0 0 0 0 0 1 0.483092 0 0 0 0 1 0 0 1 0 4 1.932367 1 0 0 0 0 0 0 0 0 2 0.966184 2 1 0 0 0 0 0 0 0 178.21256 12 11 12 10 11 9 12 13 15 207 100 Lots of freezer and cutting artifact. Dark staining. Hard to distinguish between cell borders. Difficult to quantify. Lots of artifact on one area of field. Unable to quantify this area accurately, so labeled fibres as “no counf’ Lots of freezer artifact Fields 7-11, 18, 25: Lost of freezer and cutting artifact; Dark staining and inability to distinguish cell borders. Unable to quantify.  2 0 5 1 0 0 0 0 6  3 5 11 0 0 0 3 0 19  4 5 12 1 0 0 2 0 20  5 0 15 1 0 0 0 0 16  6 6 12 1 0 1 0 0 20  I 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 4 9 5 4 4 6 6 5 6 7 4 8 8 8 4 12 8 21 19 22 27 24 23 28 23 21 23 22 18 28 35 25 25 33 0 1 1 2 1 0 2 2 1 1 2 3 0 3 0 2 0 2 2 3 2 5 3 3 2 2 2 2 3 4 3 4 0 2 1 4 2 4 6 4 2 1 3 2 6 6 2 4 5 3 0 0 2 0 1 1 2 0 2 1 0 0 0 1 0 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 37 33 40 43 36 41 33 33 37 37 38 42 54 38 44 44 2 fibres have staining smudging-S quanitified as “no count” 3 fibres have staining smudging; quantified as “no count” because not able to distiguish cell borders. Also, lots of basophilia  7 Total %Total 2 20 18.51852 12 74 68.51852 2 7 6.481481 0 0 0 0 1 0.925926 1 6 5.555556 0 0 0 17 108 100 18 12 30 3 2 3 0 1 51  19 9 28 0 5 2 0 0 44  20 Total 11 40 0 0 6 0 0 57  140 515 24 51 66 13 1 810  %Total 17.28395 63.58025 2.962963 6.296296 8.148148 1.604938 0.123457 100  92  Subject 32 No Count Normal Internal Nuclei Small angulated lnflamedInecroc Abnormal cytoplasm Adipocyte Total  i1Total  Subject 34 NoCount Normal lnternalNuclei Smallangulated Inflaniedlnecrodic Abnonnal cytoplasm Adipocyte Total  %Total #DIV/O! #DlVIOl #DlVIOl #DlVIOl  #DIVIOI  0 #DIVIO! 0 #DIV/0! 0  0  0 *  Subject33 NoCount Normal Internal Nuclei Smallangulated Inflamedlnecrotic Abnormal cytoplasm Adipocyte Total  o o o o o  1 10 20 0 0 2 0 0 32  2 8 15 2 0 2 0 0 27  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  13 7 23 0 0 0 1 0 31  14 2 16 1 0 2 0 0 21  15 2 13 1 0 1 0 0 17  16 6 21 3 0 0 0 0 30  17 6 17 1 0 0 0 0 24  18 6 13 1 0 0 0 0 20  19 6 18 0 0 1 1 0 26  0  O#DIVIO!  Fibres in all fields are cut longitudinally, or obliquely. Unable to Quantify all Fields  3 10 25 1 0 1 0 0 37  4 7 6 3 0 0 0 0 16  5 5 10 2 0 0 0 0 17  6 7 13 1 0 0 0 0 21  7 6 12 0 0 0 0 0 18  8 5 15 0 0 0 0 0 20  9 11 19 1 0 0 0 0 31  10 4 20 1 0 2 0 0 27  11 5 17 1 0 0 0 0 23  12 8 12 1 0 0 1 0 22  20 Total %Total 12726.18557 6 17 322 66.39175 1 21 4.329897 1 10.206186 11 2.268041 0 0 3 0.618557 0 0 0 25 485 100  1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 20 21 23 Total %Total 7 7 10 7 4 5 2 4 5 9 6 4 5 5 7 5 6 3 6 5 11219.21098 23 27 24 25 23 26 19 17 11 16 24 22 18 16 18 13 15 18 16 14 385 66.03774 1 0 0 0 2 1 1 5 1 2 2 2 2 2 3 3 2 0 1 1 315.317324 0 0 0 1 5 2 1 2 0 3 1 1 0 1 2 0 1 0 1 1 223.773585 0 0 0 1 1 0 0 0 1 1 1 1 1 0 0 1 2 0 2 1 132.229846 1 0 1 2 2 1 0 2 0 1 2 1 1 1 1 0 2 0 2 0 20 3.430532 000000000000000000 00 0 0 31 35 35 36 37 35 23 30 18 32 36 31 27 25 31 22 28 21 20 22 583 100 Staining is messy and inconstent lots of basophifia. Haff of field cut longitudinally; unable to quantify those fibres. *eld #15,19: Fibres cut longitudinally, Unable to Quantify Small seclion in top dght of field that is not in focus with a staining bubble. These fibres were not quantified. 93  Subject 35 NoCount Normal IntemalNuclel Smallangulated lnflamedlnecrotic Abnormal cytoptasm Adipocyte Total  1 5 15 4 0 0 4 0 28  Subject36 NoCount Normal Internal Nuclei Smallangulated Inflamedlnecrotic Abnomialcytoplasm Adipocyte Total  1 4 19 3 0 1 2 0 29  2 9 20 4 0 1 0 0 34  2 11 24 0 0 0 4 0 39  4 2 26 3 0 2 0 0 33  5 10 24 3 0 0 1 0 38  3 6 14 2 0 0 2 0 24  6 3 21 2 0 0 1 0 27  4 5 10 4 0 0 0 0 19  7 6 14 3 0 3 5 0 31  5 2 20 0 0 0 0 0 22  8 3 13 4 0 2 3 0 25  6 11 24 0 0 0 3 0 38  Ii 12 13 14 16 17 18 15 %Totai 19 20 21 Total 9 6 4 6 8 12 6 5 108 18.0602 3 3 8 22 17 12 26 19 24 13 23 343 57.35786 22 10 22 3 5 1 2 4 7 5 1 4 2 4 6110.20067 0 1 0 0 2 0 122.006689 1 4 0 0 4 1 0 1 1 0 0 1 14 2.341137 0 0 0 2 2 2 8 6 5 5 5 1 2 2 57 9.531773 5 0 0 0 0 0 0 30.501672 1 0 2 0 0 37 0 0 30 39 34 29 34 598 44 20 100 39 31 45 Staining is messy and inconsistent; lots of basophilia, and hpoftsdn. Unable to quantify accurately. Staining is messy and inconsistent; lots of basophilia, and lipofuscin. Unable to quantify accurately. Lots of connective tissue present; unable to quantify this charactesfic. Top 113 of field has obliquely-cut fibres; did not quantify this section.  7 3 19 0 0 0 3 0 25  8 6 16 3 0 0 4 0 29  9 10 19 2 0 0 0 0 31  10 5 22 3 0 0 1 0 31  11 2 19 1 0 0 1 0 23  12 4 11 6 0 0 0 0 21  13 4 15 2 0 1 0 4 26  15 5 22 2 0 0 1 1 31  16 7 20 3 0 0 0 0 30  17 4 12 2 0 0 2 0 20  18 6 23 2 0 0 0 0 31  19 5 21 3 0 1 0 0 30  20 6 19 1 0 0 0 2 28  22 Total 8 15 1 1 1 0 0 26  %Total 11420.61483 364 65.82278 40 7.233273 10.180832 40.723327 234.159132 71.265823 100 553  94  0(11)  LAOd!PV  mod.m.,oqo Ipo. o1q4  04 n s  .111LA001(Il  .0uo  041W 4!IU.00( pUmmoulIl  LU4qg  oqp PILfiLIlfl(I  UILALIO 101001(qV  ‘P10I) I104IN  1109  #04010  N LA * 0 N 0 N 00 N 0 .4 (0 (0 0 N 00 (I LII N LA 0 0 N 00 N .4 LA .4 1% 0 W 0 0 tnt L_j’jC (N 00 N 0* LA 0 LA 00 LA 0 N * 0 N 00 *00 00 -(0000 INn 0  LA LA IL) CI 0 ( 0 * On 0 N  S  01 N 00,1 *00 o * (N no o .4 LA N 00 LA  N CI LA LA 0 * 000 10 * (0 0 N On o 00.4 nfl Al .4 ON o 000 N (0 ,-( N 0 .-( 00 0 LI) C C  LA LII ‘0 (N  ‘-I 0 N * N 01 N Al LA N 01 N  ot00000LA.4oo.-(.-(nnt.4NLAnN000((0LALALALA0(0P,Nto  d .-  I  I  N.  IN I  I  I  1  -  I,-  (9.4111....  ‘4_.  ...  —1  1..  L....J .r  *NON N * * N  (0 Al N Al 01 (‘4 0(0 N  tm $  -  -  10.1  1001 NLII  LAOl0IDO.ION NO - ON N.1010 *.4 NLL1LAOI  010 AlAlNNN mt.  r—,r—--,r-me--,-—---,—--)-,—-1  inlr  10 N. LA.4  0 — t .-(N ID N Al lAO t 01 t Al ID LA 1% ‘-IN .40 nO.400 LA — Al LII NO * AlAlAl N LA LAO LA 01 — Al N LAO .4 Nfl N Al N 10 10 N 4 N LA LAO( 0(.4ON N Al N Al  Al 00 N * .4N LA LA Al .400 ‘400 nOt Al Al 0  I..4.  0100 .400 t * N (0 LA LA LI) 0 — 01 nfl LA 0* N 0 .4Al 01 LAID .4 LAO 0 LII LA N .4(0 * LIInoOoncnn.pmrn P1.4P1(41(SIflfll (%000(%J.4(fl.4fl(fl(fl.(mO.4(,.(flP.. LA NO.40 .-  -  Al — 0 LA 0 0 (N 0 0 LA 00 LA ON (0 LII ON N Al LA 01 NO 000* NO (0 00 NO 0110 N LA NO 0(0 Al 01 ON 00 01 00 AlO 0 01 0 10 Al LA * ‘-1 01 t Al ID NO 10 00.-I 01 0 LA 0000 NON N LA LA N N 010 00000 Al .4LA LA NO 01 C  -  I  .4 .40 (000 N.  .1.3.....  X  I  .-(LAIDNONION.4AltNLA*l0*NLAO.-IIDNN(0* LA N Ill LII LA nfl 000(0.-LID NO LA LA N 1010 N * * OAl *NOI0000AO1LAQINOIIOOAl4NNAl*QIAlNN NAlLAAl.-IIDLAIO.4LAOIQIOtLAO LA0.-(OO*O N0nlOrN00l0 * .4 N N 10(0 N P1*0(0 NAl.IAlOAl NO’  I  10010 LA Al  -  01O lDOrlAl.-IOrILO N NO D t P1)10 LA nOON 4LA .-(.-II0  IOAlI0.-IN 0.4 N flO IONINN AlOIOON 0lr4, 010.-I 01  I.  .4  -  I  -  -  -  -  lrr 0100(0(0.4  .-IIIIIA—I0LOlOr.IIIAlIIIAIDII.4Al(C...-19.N.N.I-—.IflAl,.r1flI..—lr.ln000.%LP%.nn rr  .1 N Al * LAID N LAO  ON  i..  ‘  ..  • 4”  05 . to 00 0 — — VI IS.? LI — • a’ 0 N — Ui  N N I-’  “4  05(0  I-’  — ‘0 0  IS.?  F.  ----  .  ‘.4 ‘.1 05 ‘.4 to to to NO . 0’ a’ (0 0 . — to a’ — 00 0 “4 “4 a’  .  .  .  .  (0 55 (0 0?  0 0 N 0 0 0 0 “.4 “.4 ‘.4 00 (s2t0 to (Si 000 to a’ ‘00 N 0000 to 0 ‘-400 a? N 0000 N to  a ‘.4  to  ‘.J 0 0 (0 to NN’.Ja’ ‘Oto’.J(-’0 ‘.JOiUJa’ ‘r’  0 N to .F’. N N N — N 00 to bi N 0 05 (0 to  00 C’  to  — totoUi(0  .  V  V  00000 I-’ 0 (-‘0 to 0000 N 00’ to (0 N to 0 N to Ui N 0 (0000000000 N to (Si toN 0 — N to 0000 0000 N 0 to to N (Si to 0 N . . 0000000000 N 00505 I-’ 0’.4 0toto(Oto0to0N0000N0N0Ui’.J 05 (.4 00000000000000 N to . (-‘ 9  to 0 Vi N 00 to N to N (5 (.4 to 0 to 0 (-‘ 0 N Ui Vito I-’ to a’ N to to N 00 . 0 N N OON 0 (-‘ N to 05 . (00505 . N — N to(0 N to 00 to 0 to . o to a’ N a’ to toto 0500 N to (000 . 00 to a •.toto0a’to0toa’a’N.00t-’0to(0 fiS (55 (5( 4-? r’ 4” r’ r’ .0. -‘. ,s 0tO totoN to to to  ‘FF’?4”’flr’’’’F”C’4’iL-’I,  (0(0 to to N a’ Ui 0 05 000 ‘-.4 00000 4- I-’ N to to N 0000 “-4 00000(0 (-‘0 0 05 0 ‘-.4 05 .t0 to . 0 (00000(0 00 N 0 (0 to to N a a (0 to to ‘00 N 000 N 000 N 0000 N (0  -  •ISstto55.  a  ..  ‘.1 N N to 0  U? U? (5 — U? Li? LI ‘.4 — — ‘.4 0 00 4.3 (510 0 I—’  05 to ‘.4 ‘.4  a’ N 40  N0’”4 C(  05 —  age  Group  Adipose tissue  abnormal cytoplasm  inflammed necrotic  small angular  internal nuclens  otal percentage abnormal muscle  Normal Muscle  BMI  ““B”’’ sex  ‘  a’to0.to  toUitSi  1  B  ‘ ‘ ‘  05 ‘.4 ‘.4 05 a’ a’ (U N 05(0 — 0 — 00 0 ‘0 Ui 05 “4 a’ “4 0 . N .  Nto’.(UsoS(O I  ç’  B B B  5 totoS  — ‘.Jfl’  —  (S.(eUi’.JCSU.F’(O  II  —,  to4toto  — — —  —,  05 Ui 05 05 N tO 000 0 00 (.4 (5 ‘0 . a’ 05 05 N 05 to  J  OSOS  0:cnJ  ...  o Ui 04 04 05 a’ to to to to to to ‘.4 0 fi’ Ui ‘0 ‘.4 “4 a’ to 05(0 to  ..  ‘  0 00 0 0 0 ? N 0 — 0 ( 0 0 . N 00 Ui ( to 0000 ‘.4 000 to 00 to a’ N 0000 to to 0 to N 00 ‘.1 (-‘(-‘0 000 Ui a a 0000 005L-’ 00000N 0(0 ‘.1 00 to 0 a( co 0 “4 0 to (0  a  —  O©  g’  Subject  0  -t  CD  ‘-4  C,)  I-.  —a*  i ii  -  -  L  ngni..r  dnnn ti.n.n  4-tplns.n  nflnnnndnn,.ii  nail  .——.‘..—  t.,....ZM...k  Ulm  ‘LS_  -  4*  I.  C  

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