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Inflammatory cells in human diaphragm injury Sharma, Anju 2003

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I N F L A M M A T O R Y CELLS IN H U M A N D I A P H R A G M INJURY by ANJU S H A R M A B.Sc. (Honors) Physical Therapy, The University of Delhi, 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (School of Rehabilitation Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A March 2003 © Anju Sharma, 2003 UBC Rare Books and Special Col lec t ions - Thesis Authorisat ion Form Page 1 of 1 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t fre e l y available for reference and study. I further agree that permission for extensive copying of thi s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of tffiR/tf TAHqAJ £ a g A J £ £ g The University of B r i t i s h Columbia Vancouver, Canada Date http://www. l ib ra ry , ubc. ca/spcoll / thesauth. html 3/14/2003 11 ABSTRACT It has been postulated that exertion-induced diaphragm injury occurs due to overload during acute and chronic respiratory diseases. The presence of inflammatory cells in the injured diaphragm has been well documented in animals but not in humans. Purpose: To relate the number of inflammatory cells in the human diaphragm to 1. clinical factors 2. area fraction of abnormal diaphragm (AA) 3. fiber cross-sectional area (CSA). Methods: Biopsies from 59 postmortem subjects (20 F, 39 M) were examined. Subjects were divided into four groups: acute respiratory disease (ARD), chronic respiratory disease (CRD), ARD+CRD and no respiratory disease. Full-thickness biopsies collected from the lateral costal region of the diaphragm were formalin fixed, paraffin embedded, sectioned at 5 um and stained with H & E . Alkaline phosphatase-anti-alkaline phosphatase (APAAP) staining with antibodies directed to CD68 (macrophages), CD20 (B-cells), CD8 (T-lymphocytes) and neutrophil elastase (NP57 antibody) was done. Images of 20 randomly selected fields/biopsy were captured by a SPOT digital camera and inflammatory cells were counted in captured images. H & E images were point-counted to determine the A A and the CSA of 200 fibers/biopsy. A retrospective chart review was done to determine the age, gender, BMI and presence/absence of respiratory disease. Data were analyzed using non-parametric statistics. Results: Macrophages (97.5% of total inflammatory cells) and neutrophils (2.4% of total inflammatory cells) were common inflammatory cells found in the diaphragm of postmortem subjects. B-cells were rare (0.1% of total inflammatory cells) and no T-lymphocytes were detected. There was a positive low correlation between BMI and inflammatory cells (r=0.286, p=0.049). The number of the different types of inflammatory cells in the diaphragm did not differ between genders. Age was not correlated to the number of inflammatory cells in the diaphragm. Lastly, neutrophils, macrophages and neutrophils + macrophages were not correlated to A A . Conclusion: Inflammatory cells are present in the diaphragm of people with or without respiratory disease. Macrophages and neutrophils have a more important role to play in diaphragm injury and repair in people with/without respiratory disease, than T-lymphocytes and B-cells. This study also shows that overweight people have more inflammatory cells in their diaphragm. iv TABLE OF CONTENTS Abstract i i Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations x Acknowledgement xii CHAPTER I Literature Review and Introduction 1 1.1 Exertion-Induced Skeletal Muscle Injury 1 1.2 Respiratory Muscles 5 1.3 Factors Aggravating Respiratory Muscle Injury 13 1.4 Evidence of Respiratory Muscle Injury 19 1.5 Rationale and Justification of the Study 25 1.6 Research Hypothesis 29 1.7 Aims and Objectives 29 CHAPTER II Methods and Instrumentation 31 2.1 Subjects 31 2.2 Study Design 31 2.3 Reliability 32 2.4 Data Collection Method 32 2.5 Data Management 38 2.6 Data Analysis 39 CHAPTER III Results and Data Analysis 43 3.1 Subjects.. 43 3.2 Inflammatory Cell Counts 43 3.3 Relationship between Inflammatory Cells and Clinical Factors 45 3.4 Inflammatory Cells and Abnormal Diaphragm 47 3.5 T - Lymphocytes and Fiber Size Variation 47 3.6 Demographic Data 47 CHAPTER IV Discussion 67 4.1 Inflammatory Cell Counts 67 4.2 Relationship between Inflammatory Cells and Clinical Factors 69 4.3 Inflammatory Cells and Abnormal Diaphragm 75 CHAPTER V Conclusion and Future Perspectives 77 References 81 Appendix I Postmortem Subject Data - Analyzed Statistically 95 Appendix II Clinical Characteristics of Postmortem Subjects 98 vi LIST OF TABLES 1. Point-counting categories and definitions 40 2. Equations for calculation of area fractions of normal muscle, abnormal muscle, and connective tissue 42 3. Subject distribution in four groups 62 4. Inflammatory cells summary 63 5. Inflammatory cells and four groups 64 6. BMI groups and gender 65 7. r & p values for area fractions of abnormal diaphragm and inflammatory cell counts (per mm2) 66 LIST OF FIGURES 1. Light microscopic photograph of normal and injured diaphragm 4 2. Human respiratory system 6 3. Schematic representation of the rat diaphragm 7 4. Anatomical structure of the human diaphragm 9 5. Schematic representation of control and emphysematous diaphragms 12 6. Light microscopic photograph showing the inclusion and exclusion criteria of a positive cell 36 7. Light microscopic photographs of macrophages, neutrophils and B-cells 44 8. Bar graph showing number of inflammatory cells per cross-sectional area in four groups of subjects 49 9. Bar graph showing number of inflammatory cells per fiber in four groups of subjects '. 49 10. Bar graph showing number of neutrophils per cross-sectional area in four groups of subjects 50 11. Bar graph showing number of neutrophils per fiber in four groups of subjects 50 12. Bar graph showing number of macrophages per cross-sectional area in four groups of subjects 51 13. Bar graph showing number of macrophages per fiber in four groups of subjects 51 14. Bar graph showing gender and inflammatory cells per cross-sectional area 52 15. Bar graph showing gender and inflammatory cells per fiber 52 16. Scatter-plot of number of inflammatory cells per cross-sectional area and age 53 17. Scatter-plot of number of inflammatory cells per fiber and age 53 18. Bar graph showing number of inflammatory cells per CSA in the diaphragm of elderly and adult subjects 54 19. Bar graph showing number of inflammatory cells per fiber in the diaphragm of elderly and adult subjects , 54 20. Scatter-plot of number of inflammatory cells per cross-sectional area and BMI 55 21. Scatter-plot of number of inflammatory cells per fiber and B M I 55 22. Diagram showing the number of subjects in low, normal and high BMI groups 56 23. Bar graph showing number of inflammatory cells per cross-sectional area in high, low and normal BMI groups 57 24. Bar graph showing number of inflammatory cells per fiber in high, low and normal BMI groups of subjects 57 25. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of macrophages per cross-sectional area (mm2) 58 26. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of macrophages per fiber 58 27. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of macrophages + neutrophils per cross-sectional area (mm ) 59 28. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of macrophages + neutrophils per fiber 59 29. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of neutrophils per cross-sectional area (mm2) 60 30. Scatter-plot showing relationship between abnormal diaphragm area fractions and number of neutrophils per fiber 60 31. Subjects' cause of death 61 LIST OF ABBREVIATIONS A P A A P alkaline phosphatase-anti-alkaline phosphatase ARD acute respiratory disease ARDS acute respiratory distress syndrome BC British Columbia BMI body mass index BSA bovine serum albumin C a + + calcium C A D coronary artery disease CD compact disk CHF congestive heart failure COPD chronic obstructive pulmonary disease CRD chronic respiratory disease CRP C-reactive protein CSA cross-sectional area C V A cardiovascular accident F females FEVi forced expiratory volume in one second GI gastro-intestinal H & E hematoxylin and eosin HIV human immuno deficiency virus IL-1 interleukin-1 IL-6 interleukin-6 IPP4 Image Pro Plus 4 IRL inspiratory resistive loading kg kilogram M males m meter MI myocardial ischemia mm 2 millimeter square NRD no respiratory disease PVD peripheral vascular disease SD standard deviation TBS tris buffered saline TNF a tumor necrosis factor alpha WOB work of breathing ACKNOWLEDGEMENT I would like to acknowledge the assistance and direction of my supervisor Dr. W. Darlene Reid, throughout my graduate studies. Her constant encouragement and guidance was invaluable during these years. I developed research skills under her constant guidance. I also want to acknowledge the committee members Dr. W. Mark Elliott and Dr. Donna Maclntyre for their guidance and suggestions. Finally, I appreciate financial support from U B C and the Physiotherapy Foundation of Canada. 1 CHAPTER 1: LITERATURE REVIEW AND INTRODUCTION Respiration is the essence of life. It has two components: inspiration (breathing air in) and expiration (breathing air out). Inspiration is performed by inspiratory muscles which are the most important skeletal muscles in the body. The primary muscle of inspiration is the diaphragm which performs 70 to 80% of the work of breathing (WOB) (Reid and Dechman, 1995). Similar to other skeletal muscles, the diaphragm can get damaged due to overuse. Diaphragm injury may occur in patients with chronic obstructive pulmonary disease (COPD) which may, in part, be due to increased ventilatory demands imposed on their diaphragm. Excessive ventilatory loads on diaphragm can lead to diaphragm injury (Orozco-Levi et al. 2001). Due to the primary importance of this muscle in respiration, a better appreciation of the characteristics of diaphragm injury in people with acute and/or chronic respiratory diseases will facilitate prevention and treatment of this phenomenon. EXERTION-INDUCED SKELETAL MUSCLE INJURY Exertion-induced skeletal muscle injury is defined as structural damage to muscle as a result of overuse or exercise. Intense, unaccustomed and longer duration eccentric contractions may cause damage to contractile and cytoskeletal components of the muscle fiber (Road and Jiang, 1998). Structural abnormalities following exertion-induced muscle injury in animal models and humans include primary or secondary sarcolemmal disruption (Armstrong, 1990; Duan et al. 1990; Jenkins, 1988), swelling or disruption of the sarcotubular system (Armstrong, 1990), distortion of the myofibrillar contractile components (Armstrong et al. 1983; Friden, 1984; Friden et al. 1988; Friden et al. 1983; Friden et al. 1981; Jones et al. 1986; Lieber and Friden, 1988; Newham et al. 1983), and cytoskeletal damage (Friden et al. 1984). Functional consequences of this injury are temporary force reduction (Davies and White, 1981; Friden et al. 1983) and muscle soreness (Armstrong, 1984; Clarkson et al. 1986; Friden et al. 1983; Newham et al. 1983; Stauber, 1989). Clinically, there is elevation of plasma enzymes (e.g. creatine kinase) (Armstrong, 1984; Clarkson et al. 1986; Newham et al. 1983; Schwane et al. 1983; Stauber, 1989), myoglobin (Roxin et al. 1986) and protein metabolites (Kasperek and Snyder, 1985) in the damaged muscles. Mechanisms of Exertion-induced Skeletal Muscle Injury There is ample evidence indicating that exertion damages muscle fibers but less is known about the causative factors or cellular mechanisms involved in exertion-induced muscle injury (Armstrong, 1990). According to Armstrong and colleagues (1990, 1991), the muscle injury sequence is divided into four events. First, some initial event, which can be either physical or metabolic in nature, occurs in the muscle fiber. This event initiates the injury process. Second, the initiating event disturbs the calcium (Ca + +) homeostasis in the muscle fiber which is known as the Ca*4" overload phase. Due to overloading, the regulating mechanisms for free cytosolic C a + + levels are overwhelmed and, therefore, several intrinsic degradative pathways are activated in the fibers. The third step includes the Ca + * activation of degradative autogenic mechanisms at the site of the injury such as the phospholipase A 2 cascade (which produces arachidonic acid, prostaglandins, and leukotrienes), Ca** activated proteases and lysosomal proteases. Elevated intracellular C a + + levels can also disrupt normal 3 mitochondrial respiration and cause sarcomere contracture. These various autogenic events in the muscle fibers occur prior to and continue even after the influx of phagocytic and inflammatory cells into the site of injury. The next stage occurs after two to six hours of initiation of injury and is known as the phagocytic phase. In this phase the inflammatory cells ingest the damaged tissue, thereby stimulating the regenerative phase which becomes visible after four to six days of injury (Armstrong, 1990). Inflammatory Cell Response to Exertion-induced Skeletal Muscle Injury The third stage of exertion-induced skeletal muscle injury is the phagocytic stage which starts four to six hours after the initiating event and lasts through two to four days following exercise (Armstrong, 1990). It is marked by a typical "inflammatory response" in the tissue. During the inflammatory response, the inflammatory cells (neutrophils, macrophages and lymphocytes) increase in the blood circulation and later migrate into the muscle tissue (Figure 1). The purpose of the inflammatory response is to promote the phagocytosis and clearance of the damaged tissue, eliminate microbial invasion and prepare the tissue for repair (Maclntyre et al. 1995). Inflammation can be either acute or chronic. Acute inflammation is the first response of the body to injury. It is characterized by a rapid change in blood flow, vascular permeability, and the immigration of neutrophils and monocytes in the muscle (Kent and Hart, 1993; Reid, 1988). The accumulation of neutrophils in muscle is the "histological hallmark" of acute inflammation (Maclntyre et al. 1995). If the injury is severe and persists, the acute inflammatory phase is followed by a chronic inflammatory phase which is marked by the presence of lymphocytes and monocytes (Kent and Hart, 1993). 4 Figure 1: (A) Light photomicrograph of a hematoxylin and eosin (H&E) stained cross-section of the normal rat diaphragm. Note the polygonal shape of the diaphragm fibers and peripheral myonuclei and absence of inflammatory cells. (B) This figure shows the H & E stained cross-section of injured diaphragm after 6 hours. Note the influx of inflammatory cells (arrows). (C) This figure shows the diaphragm 24 h after injury induction. Note the increased influx of inflammatory cells (arrows). (D) This figure shows the diaphragm after 10 days when inflammatory infiltration was absent but central nuclei were observed in some fibers (arrows). Scale bars = 50 urn. Adoptedfrom Hayot et al. 2001 Macrophages are important killer cells which phagocytise extracellular targets and remove the neutrophils and necrotic tissue from the site of inflammation, thus, facilitating healing and regeneration (Faulkner et al. 1993; Evans and Cannon, 1991; Kent and Hart, 1993). The regenerative phase of exertion-induced skeletal muscle injury is seen after 4 to 6 days of the initiation of the inflammatory response and lasts up to 3 to 4 weeks after the initial injury is resolved (Faulkner et al. 1993). RESPIRATORY MUSCLES Anatomy and Function The primary function of respiratory muscles is "respiration" in which the chest wall is displaced rhythmically to pump gas in and out of the lungs (Figure 2). This rhythmic movement of air, termed ventilation, keeps the arterial blood gases within acceptable limits which is essential for the proper functioning of the body (De Troyer and Estenne, 1988). Respiration is divided into inspiration and expiration. Inspiration is inhaling the gas and expiration is exhaling the gas. The diaphragm is the primary muscle of inspiration recruited during quiet breathing (Reid and Dechman, 1995). It has three major parts, the sternal region originating from the posterior aspect of the xiphoid process, the costal region arising from the upper margins of the lower six ribs, and the thick crural region arising from the anterolateral aspect of the upper three lumbar vertebrae (Reid and Dechman, 1995). Fibers from all three regions radiate inwards inserting into the central tendon (Figure 3) (Reid and Dechman, 1995). 6 Figure 2: Human Respiratory System. The upward and downward piston like contractions of the diaphragm pump air in and out of the lungs. Adoptedfrom http://www.jeffersonhospital.org/eifront. dll?durki=5226 Figure 3: Schematic representation of the rat diaphragm depicting the crural, ventral, medial, and dorsal regions of the costal diaphragm inserting into a central tendon. Adopted From: POOLE: MedSci Sports Exerc, Volume 29(6).June 1997.738-754 8 The diaphragm is a skeletal muscle with two domes (Figure 4). During inspiration, the diaphragm contracts and the domes get flattened decreasing the zone of apposition which is the portion directly attached to the inner wall of the lower rib cage. This creates negative pressure inside the thoracic cavity allowing the air to come into the lungs. The diaphragm relaxes at the end of inspiration, returning to its dome-shaped structure during expiration. The levatores costarum (paravertebral muscles) (Schafer, 1983), external intercostals and scalene muscles are other primary inspiratory muscles also assisting in quiet breathing (Reid and Sharma, 2000). Accessory muscles of inspiration attach to the ribcage, shoulder girdle or vertebral column. They are recruited during increased demands of ventilation, such as in exercise or disease. Major accessory muscles are the sternocleidomastoid muscle, the pectoralis major, pectoralis minor, trapezius and subclavius muscles (Reid and Sharma, 2000). Expiration at rest is a passive phenomenon occurring by the elastic recoil of the lungs. There are four abdominal muscles that have an expiratory function. These are the transversus abdominis, the internal abdominal oblique, the external abdominal oblique and the rectus abdominis from deep to superficial (De Troyer and Estenne, 1988). These muscles assist with forced expiration especially during exercise and in respiratory disease in which expiration requires more effort. Figure 4: Diagram depicting the in situ anatomy of the human diaphragm at functional residual capacity. Note the dome - shaped structure of the diaphragm. Adopted From: POOLE: Med Sci Sports Exerc, Volume 29(6) June 1997.738-754 10 Respiratory Muscle Function in Disease Chronic low intensity inspiratory loading can produce diaphragmatic injury which can impair normal diaphragm functioning (Reid et al. 1994). In COPD patients, airway obstruction occurs due to the presence of mucus in the airways, airway inflammation, bronchospasm, narrowing of air passages and alveolar destruction which makes breathing difficult (Reid and Dechman, 1995). The function of the respiratory muscles is severely compromised due to the increased WOB. In COPD, the airway obstruction also causes dynamic compression of the airways leading to trapping of the air inside alveoli and a hyperinflated chest (Reid and Dechman, 1995). Hyperinflation alters the length-tension relationship of the diaphragm thereby, negatively affecting the capacity of the diaphragm to produce increased ventilatory effort (Reid and Dechman, 1995; Tobin, 1988). Increased ventilatory loads might lead to diaphragm injury. Lung and Chest Wall Mechanics in Chronic Obstructive Pulmonary Disease Hyperinflation is a major factor responsible for altered lung and chest wall mechanics in COPD. Hyperinflation leads to increased work of breathing in COPD which has been described well by Tobin (1988). In COPD, gas is trapped in the distal alveoli and airways by the collapse of airways during expiration and by the tonic contraction of accessory and intercostal muscles. The thorax accommodates the increased lung volume by lowering the diaphragm and/or by expanding the ribs. As lung volume increases in a hyperinflated chest, the diaphragm 11 progressively gets flattened and thus, becomes less capable of generating negative inspiratory pressure. Hyperinflation also changes the directional effect of chest wall recoil. Normally, the elastic recoil of the chest wall is directed outwards and assists the action of inspiratory muscles by inflating the lungs. At high levels of hyperinflation, the functional residual capacity increases markedly, and the thoracic elastic recoil becomes directed inwards at tidal lung volumes. Thus, the inspiratory muscles work against the inward elastic recoil of the lungs as well as the added inward elastic load of the chest wall. The zone of apposition also decreases as a result of hyperinflation, leading to less effective ribcage expansion. Diaphragmatic fibers which are normally oriented in a cephalo-caudal direction are directed medially and inwards in COPD and there is a decrease in costal fiber length (Figure 5). Therefore, in severe hyperinflation, the diaphragm can have an expiratory action detected clinically as Hoover's sign. In hyperinflation, the ribs are placed more horizontally than their normal oblique position making it difficult for inspiratory intercostal muscles to lift the ribs and expand the ribcage. Thus, hyperinflation increases the work of breathing by bringing many changes to the lung and chest wall mechanics. 12 Control Emphysematous i t 1 cm Figure 5: Schematic representation of excised diaphragms (abdominal surface) from control and emphysematous hamsters. Note the decrease in costal fiber length in the diaphragm from the emphysematous hamster. Adopted From: POOLE: Med Sci Sports Exerc, Volume 29(6). June 1997.738-754 13 FACTORS AGGRAVATING RESPIRATORY MUSCLE INJURY Respiratory muscle injury can also occur due to excessive loads that exceed the usual requirements of the muscle (Reid and MacGowan, 1998). In the respiratory system, excessive loading may occur as a result of increased WOB (increased resistive or elastic loads), non-uniformity of stress or strain, increased breathing rate (over-activity), decreased efficiency of the respiratory system and/or weakness of respiratory muscles (Reid and MacGowan, 1998). Factors like poor arterial blood gases, immobilization, starvation, corticosteroids and aging may also accentuate respiratory muscle injury or make the respiratory muscle more susceptible to injury (Reid and MacGowan, 1998). In many respiratory diseases, excessive loading is accompanied by one or more of the factors mentioned below, which contribute to weakness and accentuate respiratory muscle injury. Increased Resistive or Elastic Loads Resistive loads in the respiratory system are related to resistance to functioning of the respiratory system. In respiratory diseases such as COPD, the presence of mucus in airways, inflamed airway walls, decreased diameter of airways due to bronchospasm and dynamic compression of small airways due to destroyed adjacent lung parenchyma, contribute to increased resistive loads leading to ventilatory overloading (Reid and Dechman, 1995). This respiratory muscle overloading may lead to muscle fatigue and/or injury of the diaphragm. During exercise, acute exacerbation or lung infection, these resistive loads may increase, further increasing the potential for damage of the diaphragm (Reid and Dechman, 1995). 14 Elastic loads in the respiratory system are related to the increased pressure required to expand the lungs and chest wall. In some respiratory diseases, such as cystic fibrosis, the lungs become stiffer and the overall compliance of the respiratory system is decreased, leading to increased WOB. In COPD, asthma, and emphysema, hyperinflation of the chest places the chest wall closer to total lung capacity, thereby, making the chest wall and lungs less compliant (Reid and MacGowan, 1998). In interstitial lung disease, the increased interstitial collagen and thickened alveolar walls decrease the lung compliance. Decreased compliance of the lungs and chest wall increases the WOB and can lead to respiratory muscle fatigue and hypercapnic ventilatory failure. Decreased Efficiency of the Inspiratory Muscles In COPD sarcomere loss has been reported in animal models (Farkas and Roussos, 1983; Kelsen et al. 1983) and may also occur in humans with chronic hyperinflation. This adaptation is important for maintaining the length-tension relationships of the diaphragm to generate forces, but, this also results in decreased range of motion, thereby, adversely affecting the pumping action of the diaphragm (Reid and MacGowan, 1998). Non-uniformity of Stress and Strain Stress is the amount of force per cross-sectional area and strain in muscle is the relative change in length of the muscle fiber (Reid and MacGowan, 1998). The diaphragm has a large surface area, relative thinness and a very large central tendinous region (Reid and MacGowan, 1998). This makes the diaphragm susceptible to regional variations in stress 15 and strain (Reid and MacGowan, 1998). Stress is much greater during eccentric contractions than during concentric contractions (Benz et al. 1998). Diaphragm contractions during inspiration are primarily concentric in nature (Wakai et al. 1994). However, eccentric (lengthening) contractions during obstructed breathing in some parts of the diaphragm are considered to occur (Wakai et al. 1994). During contraction, different regions in the diaphragm may undergo variable shortening force such that a strong concentric contraction of the crural region could lengthen the sternal region (Wakai et al. 1994). Non-uniformity of regional diaphragm shortening and lengthening in some regions of the diaphragm might be caused by loss of coordination or co-contraction of the respiratory muscles during fatigue leading to its further weakness and injury (Reid and MacGowan, 1998). Weakness Many factors such as metabolic abnormalities (Gibson and Pride, 1995; Reid, 1995); endocrine disorders (Gibson and Pride, 1995); shock (Aubier et al. 1981); infection (Mier-Jedrzejowicz et al. 1988); sepsis (Boczkowski et al. 1988); malnutrition (Arora and Rochester, 1982); corticosteroids (Rochester, 1993) and disuse (Rochester, 1993) contribute to diaphragm weakness. Acute and chronic respiratory conditions accompanied by these factors may further lead to weak inspiratory muscles. 16 Arterial Blood Gases Hypercapnia (increased arterial blood carbon-dioxide level) reduces diaphragm function (Juan et al. 1984). Hypercapnia activates the kallikrein-kinin system (Larsen and Henson, 1983). The kallikrein-kinin system is one of the major groups of plasma mediators activated during inflammation. Thus, activation of kallikrein-kinin system might lead to an enhanced inflammatory response to injury. Hypoxemia (decreased arterial blood oxygen level) increases respiratory muscle metabolic demands and simultaneously reduces the oxygen content of blood supply to the muscles. Watchko and colleagues (1986) suggested that strength and endurance are decreased when arterial oxygen tension falls below 45 mm Hg. However, Bark and group (1988) demonstrated that the diaphragm is remarkably resistant to hypoxia, showing no evidence of impaired contractility for arterial oxygen tensions as low as 30 mm Hg, provided the tension-time index remains below 0.05. This suggests that at higher levels of tension development (increased WOB) hypoxia can precipitate fatigue. Immobilization Immobilization leads to muscle weakness and muscle fiber atrophy, especially of type II fibers. In many studies on animal models using interventions such as hindlimb suspension (Krippendorf and Riley, 1993; St. Pierre and Tidball, 1994; Warren et al. 1994) or space flight (Riley et al. 1990), it has been shown that reloading of the limb muscle induced muscle necrosis and infiltration of injured fibers by macrophages. Similar to limb 17 muscles the respiratory muscles may be susceptible to injury when they are reloaded following unloading due to mechanical ventilation. Starvation Starvation leads to mobilization of muscle proteins due to a decrease in protein synthesis and an increase in non-myofibrillar and myofibrillar protein breakdown (Li and ~ Goldberg, 1976; Millward and Waterlow, 1978). In people with chronic respiratory disease, muscle catabolism during malnutrition reduces respiratory muscle strength and endurance (Ryan et al. 1993; Whittaker et al. 1990). A decrease in nutrition leads to reduced muscle mass and function and, i f combined with chronic disease, may result in a greater relative overload on the respiratory muscles (Reid and MacGowan, 1998). Decreased nutrition may also affect the ability of fatigued or injured respiratory muscles to adapt and regenerate (Reid and MacGowan, 1998). Corticosteroids Steroids inhibit protein synthesis and increase protein catabolism, thus, resulting in muscle fiber atrophy and weakness in limb muscles. Very high doses of corticosteroids may result in an acute necrotizing myopathy in people with status asthmaticus (Griffin et al. 1992). Weiner and coworkers (1995) observed that patients with no underlying pulmonary disease developed reversible inspiratory muscle weakness due to high doses of steroids over several weeks. Corticosteroid therapy is commonly prescribed in patients with COPD, 18 asthma and interstitial lung disease. Thus, the adverse effects of steroid therapy on respiratory muscles in such patients may be serious. Aging There is little evidence available on the effects of aging on respiratory muscle function, however, respiratory muscle endurance and strength has been reported to decrease in elderly individuals (Tolep and Kelsen, 1993). There is a decline in muscle mitochondrial oxidative metabolism in older adults (Rifai et al. 1995). In a study done on mice by Zerba et al. (1990), muscle injury in limb muscles was reported to be greater in elderly mice compared to younger mice exposed to the same intensity of loading. Recovery from the same amount of injury also differed in different age groups, showing that elderly mice take more time to recover (Brooks and Faulkner, 1990). Thus, age seems to affect the skeletal muscles. It is possible that in older individuals with chronic respiratory conditions, the normal aging process accentuates the impact of excessive loading on respiratory muscle injury and impairs adaptation and regeneration. 19 EVIDENCE OF RESPIRATORY MUSCLE INJURY Human Studies Research on human diaphragm injury can be extremely challenging due to the anatomical location of the diaphragm and the ethical complexities involved in obtaining muscle biopsies. Inspite of these challenges, diaphragm injury is an ever growing field of research. Mostof the research performed to date on respiratory muscle injury in humans has been performed on patients undergoing surgeries (laparotomy and thoracotomy) or those with myopathies. A recent study on 21 subjects (8 males, 13 females; mean FEVi = 74+ 34%) undergoing thoracotomy was performed by MacGowan and colleagues (2001). The purpose of this study was to describe and quantify diaphragm injury, count macrophages and determine the association of diaphragm injury to airflow obstruction. Eighteen subjects in this study were diagnosed with lung cancer and the other three had bullous disease, bronchiolitis obliterans and emphysema respectively. In this study the abnormal features found in the diaphragm of subjects were internal nuclei, lipofuscin pigment, small angulated fibers (definition in Table 1), inflamed fibers and inflammatory cells in the endomysial and perimysial connective tissue. Macrophages were identified to be present in the connective tissue which was a similar finding to Kariks (1989). Kariks in 1989 examined diaphragms from 242 infants who died of sudden infant death syndrome. Biopsies taken from the right and left costal diaphragm of these subjects 20 showed macrophages in the interstitial and perivascular spaces in diaphragms undergoing fiber atrophy and disintegration. Hards and group (1990) and Campbell et al. (1980), looking at external intercostals (a group of primary inspiratory muscles other than the diaphragm) of patients undergoing thoracotomy observed "moth-eaten" fibers {termed "targeting" by Campbell et al. (1980)}, fiber size variation and Z-band streaming in external intercostals. These abnormalities were reported to be greater in the external intercostals than the control muscle (latissimus dorsi). Diaphragm injury has also been documented in COPD patients (Orozco-Levi et al. 2001). The purpose of this study was to determine if the diaphragm from COPD patients is more susceptible to injury and also i f an acute inspiratory overload in COPD patients results in more injury as compared to non loaded COPD patients. In this study, subjects were divided into four groups: COPD patients not loaded (n=T 1), control patients with normal pulmonary function not loaded (n=6), COPD patients loaded (n=7) and control patients with normal pulmonary function loaded (n=5). The age of control subjects was reported to be 62 + 10 years, however, the age of COPD patients was unreported. Biopsies were taken from the costal diaphragm of all subjects while undergoing thoracotomy or laparotomy. A l l diaphragm samples showed signs of sarcomeric disruptions in both COPD and non-COPD groups, however the COPD group showed a higher area fraction of sarcomeric disruption. The loaded COPD group showed a 38% greater sarcomeric disruption density and 32% greater abnormal area fraction compared to non-loaded COPD group. The loaded control group showed 89% greater sarcomeric disruption density and 83% greater abnormal area fraction compared to non-loaded control group. Surprisingly, signs of necrosis and inflammatory cells were not observed in the diaphragm of any of the subjects. 21 Also, no relationship was found between the sarcomeric disruption and age, anthropometrical or biochemical nutritional parameters in this study. Abnormalities in the diaphragm of COPD patients were also shown in a study by Lloreta and colleagues (1996). They reported only light microscopic changes in the diaphragm, unlike Orozco-Levi et al. (2001) who reported sarcomeric disruptions seen electron-microscopically. The study by Lloreta and colleagues (1996) was based on a single case. The subject was a 46 year old man with a 100-pack per year smoking history, who had marked COPD and was diagnosed with stage I squamous cell carcinoma. Biopsies were obtained from his diaphragm, latissimus dorsi, and intercostal muscles during a surgical procedure. This group reported variable proportions of angulated fibers and occasional internal nuclei in the diaphragm of the COPD subject along with mitochondrial abnormalities. Histological abnormalities have also been reported in the diaphragm of people with heart disease. The aim of the study by Lindsay and coworkers (1996) was to compare histological findings in limb and respiratory muscles from control subjects and patients with heart failure of two different etiologies. Seventeen subjects in total having dilated cardiomyopathy (n=10) and ischemic heart failure (n=7) with severe impaired left ventricular function were chosen for the study purposes. Seventeen control subjects with normal left ventricular function undergoing surgery for different reasons were also included in the study. Biopsies, taken from all subjects during surgery, were processed using histological and immunohistochemical analyses. Significant structural abnormalities such as internal nuclei, tubular aggregates and evidence of abnormal expression of myosin isoforms 22 suggestive of fiber type transformation and/or regeneration were found to be more prevalent in the diaphragm than in the limb muscles of the experimental group. Animal Studies Due to better control of variables such as nutrition, age and body mass index (BMI) animals have been widely used in diaphragm injury research. Extensive research on diaphragm injury has been done by Reid et al. (1994) and Jiang and co-workers (1998b) who used hamsters or rabbits for their studies. They used tracheal banding and inspiratory resistive loading (IRL), as the interventions to induce injury of the diaphragm. Reid and co-workers (1994) induced ventilatory failure in 14 hamsters by tightening a polyvinyl band around the trachea for six days. This resulted in a severe respiratory acidosis, hypoxemia and increased pulmonary resistance. Diaphragm tissue was stained by hematoxylin and eosin (H&E), and the point counting technique was used to quantify the area fraction of abnormal muscle. The investigators observed larger areas of abnormal muscle and inflammatory cells in the costal and crural regions of the diaphragm in the tracheal-banded hamsters as compared to the control group. Electron micrographs also revealed sarcomeric disruption and Z-band streaming in the diaphragm of tracheal-banded hamsters. This group further reported that mononuclear cells were the most common inflammatory cells found in the tracheal-banded hamsters. Histochemical analysis of diaphragm cross-sections in yet another study done on hamsters tracheal banded for 30 days, showed a five-fold greater area fraction of abnormal muscle, a greater fiber size variation 23 and also a 3% higher proportion of type I fibers in tracheal banded hamsters than controls (Reid and Belcastro, 1999). A loss of diaphragm force production has been reported in a study on rabbits undergoing IRL (Jiang et al. 1998a). The purpose of this study was to find the effect of delayed diaphragm injury produced by IRL on diaphragm force production. In this study three groups of rabbits (n=7 in each group) were subjected to high, moderate and no IRL for 1.5 hours. Marked diaphragm injury was found in the high IRL groups (p<0.01), but no significant diaphragm injury was found in the other two groups. It was also found that the baseline trans-diaphragmatic pressure frequency values in the high IRL group were significantly reduced at most frequencies suggesting that the diaphragm injury induced by high IRL has a significant impact on diaphragm force production and the attendant force loss produced by IRL is dependent on the intensity of inspiratory loading. Another study with a similar animal model (Jiang et al. 1998b) was done to examine whether an acute episode of IRL could produce secondary diaphragm inflammation and injury. The authors hypothesized that application of intense IRL might lead to delayed diaphragm injury and inflammation and that the development of the respiratory muscle injury would be load dependent. Under light microscopy, it was found that the rabbits exposed to high IRL showed marked costal diaphragm injury characterized by necrotic diaphragm fibers, flocculent degeneration, and a profound influx of inflammatory cells both in necrotic fibers and in the interstitial tissues. The inflammatory cells observed were neutrophils and mononuclear cells. The finding of inflammatory cells in this study was similar to Reid et al. (1994), Jiang et al. (1998b, 2001). 24 Similar results were obtained by yet another study with the same animal model of inducing diaphragm injury by IRL (Jiang et al. 2001). This study was done to determine the role of free radical scavengers in preventing diaphragm injury produced by IRL. The rabbits were divided into three groups (IRL, control and scavenger group infused with free radical scavengers; n=6 in each group). Light microscopic examination of the hematoxylin and eosin (H&E) stained diaphragm cross sections taken from the IRL group showed marked diaphragm injury characterized by necrotic diaphragm fibers, flocculent degeneration, and an influx of inflammatory cells both in necrotic fibers and in interstitial tissues. The inflammatory cells were both neutrophils and mononuclear cells. The area fraction of normal muscle in the IRL groups was significantly lower than those in other groups. Also, the area fractions of abnormal muscle and interstitial space in the IRL group were significantly higher than in the other two groups. Diaphragm injury has also been shown in rats (Hayot et al. 2001). Hayot and coworkers (2001) induced diaphragm injury in anesthetized rats by applying a caffeine solution to their right abdominal diaphragm surface. Diaphragms from different (nine in total) sets of rats (each set n=6) were removed at different days for analyses. It was found that compared with control diaphragms, the percentage of fibers with membrane damage was significantly higher in caffeine treated diaphragms at 1, 4, 6, 12 and 24 hours. Slight infiltration of inflammatory cells was also observed after 6 hours of injury induction. At 24 hours, more interstitial infiltration was evident with myofiber infiltration and at 10 days post injury interstitial infiltration was decreased and centrally nucleated myofibers were noticed. The above mentioned animal studies suggest that diaphragm injury occurs as a result of ventilatory overloading, but in humans it is still to be proved. Consistently, inflammatory 25 cells were observed in the injured diaphragm along with other histological abnormalities in animal and human studies. In the current study, further investigation of the type of inflammatory cells present in the injured diaphragm fibers in humans and their relationship with various clinical factors was done. RATIONALE AND JUSTIFICATION OF THE STUDY The diaphragm is the major muscle of inspiration which contracts continuously throughout life. In this state of continuous work, even a small prolonged ventilatory load can cause diaphragm injury which is seen as structural abnormalities under the light microscope. As a result of injury, there is an influx of inflammatory cells in the injured muscle (Maclntyre et al. 1996). Most commonly observed inflammatory cells in muscle as a result of exertion-induced injury are neutrophils and mononuclear cells (Maclntyre et al. 2000; Kent and Hart, 1993). In the current study, neutrophils, macrophages and lymphocytes were observed and counted in the diaphragm. Neutrophil infiltration in the muscle occurs within hours of muscle injury (Orimo et al. 1991; Robertson et al. 1993). There is evidence that circulating neutrophils get activated during exercise (Gray et al. 1993; Pyne, 1994) and enter the injured muscle tissue within 30 minutes of the exercise stimulus (Reid, 1988). These are the principal inflammatory cells during the acute vascular phase of injury. If the injury is sufficiently severe and persists, a chronic cellular response may follow the acute vascular reaction and monocytes infiltrate from the capillaries resulting an increased number of macrophages at the site of injury. At the site of injury, macrophages clear up cellular and 26 tissue debris, thus, performing a key role in regeneration and remodeling of tissues. Neutrophils may also play a role in the amplification of injury and have been observed in the injured diaphragm and interstitium (Jiang et al. 1998b). Both macrophages and neutrophils are also implicated in the pathogenesis of COPD (Tetley, 2002; Stockley, 2002). Lymphocytes have an essential role in cell-mediated immunity. They are the major inflammatory cells to be recruited in the circulation during exercise (Gannon et al. 2001). They are also considered to play a key role in apoptosis in dystrophic muscles and in muscle necrosis (Spencer et al. 1997). Thus, inflammatory cells (neutrophils, macrophages and lymphocytes) have a considerable role in the immune response to exertion-induced muscle injury. Quantifying inflammatory cells (neutrophils, macrophages and lymphocytes) in human diaphragm is an interesting area of research which was one of the major aims of the current investigation. There are many clinical factors that might affect the amount and type of diaphragm injury, such as the presence of acute and/or chronic respiratory disease, gender, BMI and age. The relationship of these clinical factors and inflammatory cells was also analyzed in the current study. Respiratory disease such as COPD, induces ventilatory overloading of the diaphragm. COPD patients have been reported to be more susceptible to diaphragm injury (Orozco-Levi et al. 2001). Due to the complexities involved in research on human diaphragm, the ventilatory overloading due to COPD is being simulated in laboratories by placing a polyvinyl band around the trachea of animals (tracheal banding) or by decreasing the airway opening leading to resistive breathing (acute IRL). In both models of exertion-induced diaphragm injury, a higher percentage of abnormal diaphragm and inflammatory 27 cells were found in the diaphragm of the animals involved in the experiments (Reid et al. 1994; Jiang et al. 1998b; Jiang et al. 2001). Investigation of gender differences in response to exertion-induced muscle injury is an interesting field of research. Schneider et al. (1999) and Maclntyre et al. (2000) have reported a difference in the time inflammatory cells peak in the circulation between males and females. Also, females have been shown to experience less skeletal muscle injury compared to males (Tarnopolsky et al. 1995). It has also been reported that estrogen and gender can amplify macrophage and neutrophil function in endotoxic rats (Spitzer and Zhang, 1996). The female sex hormone estrogen has antioxidant properties and antioxidants diminish the muscle damage caused by exercise. Therefore, logically it can be deduced that estrogen has a protective role against exercise-induced muscle injury (Tiidus, 1999). In addition, it has been reported that females in the luteal phase (menstrual cycle phase with high estrogen and progesterone levels) have more IL-1 (interleukin-1) compared to males (Cannon and Dinarello, 1985). IL-1 is capable of increasing macrophage mediated superoxide production. Superoxides and free radicals along with release of proteases and hypochlorous acid by neutrophils and macrophages are critical in removing damaged tissue following exertion-induced injury and thus, facilitating repair (Maclntyre et al. 2000). A l l these studies are suggestive that gender seems to affect the inflammatory response to exertion-induced skeletal muscle injury. This factor was examined in the current study. Aging is associated with a decrease in muscle mass, strength, motor units and an increase in connective tissue (Campbell et al. 1973; Danneskiold-Samsoe et al. 1984). Diaphragmatic maximal tetanic force production in senescent rats is reported to decrease with aging (Polkey et al. 1997). It has also been reported that elderly people (65-75 yrs) 28 have less diaphragm strength when compared to young adults (19-20 yrs) (Tolep et al. 1995). A study by Roos et al. (1997) showed that there is a decrease in diaphragm strength of 20 to 40% during isometric and concentric work in the elderly. Also, a decline in the absolute number of T-lymphocytes, in the blood of older dogs has been reported by Greely et al. (2001). A n age-related decrease in diaphragm strength might predispose elderly people to diaphragm fatigue in the presence of conditions that impair inspiratory muscle function or increased ventilatory load. Due to increased ventilatory demands the diaphragm may suffer from an exertion-induced injury resulting in the invasion of the site of injury by inflammatory cells. The relationship of these inflammatory cells (macrophages, neutrophils and lymphocytes) with age, in the diaphragm muscle of people with respiratory diseases was investigated in this study. Body mass index is the ratio of weight to height squared (BMI=kg/m ). It is an anthropometric measurement of the body fat with respect to height giving an estimate of obesity or malnutrition. Malnutrition frequently accompanies the later stages of COPD and may affect 50% of patients hospitalized (Ryan et al. 1993). Malnutrition has also been shown to be an independent risk factor for mortality in COPD. It causes diaphragm atrophy altering muscle function (Whittaker et al. 1990). Underweight asthma patients tend to have more significant respiratory problems with a higher prevalence of symptoms, reduced lung function, and increased airway responsiveness (Schachter et al. 2001). It has also been reported that refeeding malnourished COPD patients improves respiratory muscle strength and endurance (Whittaker et al. 1990). On the other extreme, obesity might be a risk factor for asthma (Shaheen et al. 1999), although this is controversial (Schachter et al. 2001). Obesity also causes dyspnea increasing the work of breathing (Sin et al. 2002; Powers et al. 29 1996). Adipose tissue around the chest wall causes a reduction in chest wall compliance making inspiration increasingly more difficult and resulting in lower static volumes and flows (Ladosky et al. 2001). Obesity can also profoundly alter pulmonary function and diminish exercise capacity by its adverse effects on respiratory mechanics. It can cause resistance within the respiratory system and affect respiratory muscle function, lung volumes, work and energy cost of breathing, control of breathing, and gas. exchange (Koenig, 2001). Obesity also places the patient at risk of respiratory failure (Koenig, 2001). These were the reasons for examining a correlation between BMI and inflammatory cells (a histological abnormality suggesting diaphragm injury) in the diaphragms of people with acute and/or chronic respiratory disease. RESEARCH HYPOTHESIS We hypothesized that a higher number of inflammatory cells would be associated with subjects who had both acute and chronic respiratory disease and those with only acute respiratory disease as compared to subjects with no acute or chronic respiratory disease. Further, we postulated that greater diaphragm injury would be associated with a higher number of neutrophils and macrophages. Lastly, we postulated that a greater fiber size variation would be associated with subjects having a greater number of T-lymphocytes in their diaphragms. AIMS AND OBJECTIVES The specific aims and objectives of this study were as follows. . 30 a) To quantitate the number of inflammatory cells (neutrophils, macrophages, B-cells and T-lymphocytes) in the diaphragm of postmortem subjects. b) To determine the relationship between these inflammatory cells and clinical factors. These clinical factors were: presence of acute respiratory disease and/or presence of chronic respiratory disease, gender, age, and body mass index. c) To determine if there is a relationship between the number of neutrophils, macrophages, combined neutrophils + macrophages, and the area fraction of abnormal diaphragm. d) To determine the relationship between fiber size variation and T-lymphocytes in the diaphragm. 31 CHAPTER 2: METHODS AND INSTRUMENTATION SUBJECTS Fifty-nine subjects who underwent autopsy for different reasons were included in this study. The diaphragm samples were obtained from the mortuaries of Vancouver Hospital and St. Paul's Hospital, Vancouver, BC, Canada. The mean age of subjects was 68 +13 years. There were 20 females and 39 males in this study with mean ages 72 + 12 and 66 +14 years, respectively. Inclusion criteria of the subjects included signed consent from the subject's next-of-kin for an unrestricted autopsy. Individuals who died more than 96 hours prior to autopsy, had hepatitis B or C virus, HIV, Creutzfeldt-Jakob disease, or who were forensic cases were not included in the study. STUDY DESIGN This was a retrospective descriptive study, in which medical charts of 59 postmortem subjects were reviewed to get demographic information. Later inflammatory cells were counted in their diaphragms to determine the relationships of inflammatory cells with the subjects' age, gender, BMI and the presence of acute and/or chronic respiratory disease. 32 RELIABILITY Inter-rater reliability in cell counting was performed by the investigator, Anju Sharma, and her thesis supervisor Dr. W. D. Reid in identical fields. The correlations between the supervisor and investigator were r=0.980 for fiber counting and r=0.991 for cell counting in 15 images. DATA COLLECTION METHOD Biopsy Sampling and Preparation The diaphragm biopsies were obtained with the assistance of the autopsy staff at St. Paul's Hospital and Vancouver Hospital. Left, right or the entire diaphragm was removed during the autopsy, placed in a plastic container and fixed with formalin for later processing. A scalpel blade was used to obtain a 4 cm x 4 cm diaphragm biopsy, taken midway between the costal origin and central tendon insertion of the lateral costal region. For histological processing, 1 cm x 1 cm biopsies were cut from the 4 cm x 4 cm biopsies and placed in tissue cassettes. The cassettes were kept in formalin until histological processing took place. Processing was done in the laboratory facilities of the Department of Academic Pathology, University of British Columbia. During the processing, biopsies were dehydrated, embedded in paraffin, sectioned at 5 um thickness, stained with H & E, and mounted on glass slides. 33 Immunohistochemistry Diaphragm cross-sections were de-paraffinized in Hemo-De and rehydrated in a graded series of isopropyl alcohols. The alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (Cordell et al. 1984) for paraffin embedded formalin fixed sections was employed and the staining was carried out using an automatic autostainer (Universal Staining System Version 2.0, D A K O , Carpinteria, CA). A l l incubations were carried out at room temperature. Briefly, sections were rinsed in TBS (tris buffered saline, pH 7.6) before incubation in 5% normal rabbit serum diluted in TBS buffer containing 1% bovine serum albumin (BSA) for 15 minutes to block any nonspecific binding of antibodies. After blocking, excess solution was removed from the sections and the sections were then incubated with the primary antibody for one hour. Sections were washed with TBS prior to application of the secondary antibody (rabbit anti-mouse IgG: DAKO) for 30 minutes. The sections were then washed in TBS again followed by incubation with A P A A P complex (DAKO) for 30 minutes. After washing with TBS, the substrate was applied. The alkaline phosphatase substrate, Napthol AS-B1 phosphate in 1% New Fuschin, was prepared immediately prior to applying to the sections. This substrate gives a strong deep pink precipitate at the reaction site. Sections were automatically counterstained with Mayer's hematoxylin and then manually dehydrated in isopropanol, Hemo-de and xylene. Lastly, sections were mounted with cover slips using a permanent mounting medium (Entellan, Merck). The monoclonal antibody, NP57 (DAKO) diluted to a concentration of 1:40 TBS/BSA was used to label neutrophils in human diaphragm cross-sections. This antibody selectively stains for the neutrophil granule protein elastase. Monoclonal mouse antibody CD68 (DAKO) was used to label macrophages. The CD68 antigen is located in the lysosomes of macrophages. Monoclonal mouse antibody CD20 (DAKO) which recognizes the intracytoplasmic epitope located on CD20 antigen present on a majority of B-lymphocytes was used to identify B-cells. Monoclonal antibody CD8 (Vector Labs, Burlington, Ontario) was used to identify cytotoxic T-lymphocytes. CD8 antibody recognizes glycoproteins CD8 on the surface of cytotoxic T-lymphocytes. Positive controls consisted of sections of human tonsil and lungs processed similarly to the experimental slides. Diaphragm cross-sections also served as negative controls, with mouse IgGi (BD Biosciences, Toronto, Ontario) replacing the primary antibody, diluted to the same concentration as the respective primary antibodies. Image Capturing Images of twenty randomly selected fields from each biopsy were captured by a SPOT digital camera (Diagnostic Instruments, Inc. Michigan: Version 2.2.) connected to an IBM compatible computer and microscope (Nikon Microphot, Japan) and saved to a CD. Images were captured for H & E , CD68, CD20, CD8 and NP57 slides. The magnification of H & E and CD68 slides was double the magnification of CD20, CD8 and NP57 slides. The area of each CD68 image was 91800 urn2 (0.0918 mm2) and the area of CD20, CD8 and NP57 images was 357000 urn2 (0.357 mm2). 35 Quantitative Evaluation of Inflammatory Cells Fibers and inflammatory cells were counted manually in the captured images. The left and bottom sides of the image were the inclusion sides and the other two sides were exclusion sides. An overhead transparency was taped on the computer monitor screen. Cells and fibers were marked with red and blue overhead pens on the transparency to ensure the accuracy of the counts. Cells within the image or touching the inclusion sides were counted. Cells touching the exclusion sides were not counted. Partially viewed fibers and inflammatory cells were excluded from the total counts if they fell on the corners where an exclusion side meets an inclusion side. However, cells falling on the corner where two inclusion sides met were counted. The number of the inflammatory cells was expressed per fiber and per millimeter square (mm2). Any cell around or inside a blood vessel.or in epimysial connective tissue was excluded from the count. Also i f a cell lay outside the line touching the edges of the two outermost and nearest fibers, it was excluded. However, i f the cell lay on the line or inside the line it was counted (Figure 6). The total number of inflammatory cells per fiber was calculated by dividing the total number of inflammatory cells by the total number of fibers in the section. The total number of inflammatory cells per cross sectional area was determined by dividing the total number of inflammatory cells by the total area viewed. The mean, standard deviation, maximum and minimum values were calculated using an E X C E L spreadsheet (Microsoft, 2000). 36 Figure 6: Figure shows the inclusion and exclusion criteria of a positive cell. If the cell lies at the periphery adjacent to the epimysium of the cross-section, then it is counted i f it lies on the line or inside the line joining the two outermost fibers (shown by a star). If the cell lies outside the line joining the two outermost fibers at the periphery of the cross-section, it is excluded (shown by arrow). Scale bar = 50|i. 37 Quantitative Evaluation ofH & E Stained Diaphragm Cross-sections Quantitative evaluation of H & E stained diaphragm cross-sections was previously performed by an MSc student, Tyler Clarke (Clarke, 2000). Captured images were evaluated to determine the area fractions of normal and abnormal muscle morphology according to the classification given in Table 1 (Clarke, 2000). Quantification of the proportion of normal muscle relative to abnormal muscle or to connective tissue was done by point counting (Cruz-Orive and Weibel, 1990). The computer program Image Pro Plus (IPP4, Media Cybernetics, LP. Silver Spring M D version 4.0) was used to sum the number of points on the diaphragm tissue cross-sections assigned to one of ten categories. Using the IPP4 software program, a grid consisting of 64 points was superimposed on one of twenty captured images. Points were counted on only those fibers which could be entirely seen in the field. Partially viewed fibers were excluded. The area fractions of normal muscle, abnormal muscle and connective tissue were determined using the equation in Table 2 and categories listed in Table 1 (Clarke, 2000). Fiber Cross-Sectional Area For determining the cross-sectional area of a muscle fiber, images of the H & E stained cross-sections were viewed on a computer monitor using IPP4. Using a digital image of a stage micrometer captured at the same magnification as H & E slides, a known distance (in microns) as determined from the stage micrometer was calibrated to the length (in pixels) for the digital image. Fiber size was calculated by outlining the perimeter of the cross-38 sectional profile of each fiber using a mouse connected to the computer. Each fiber was numbered and IPP4 was used to compute the area of each fiber. For each biopsy, fiber size of - 200 fibers was calculated and the mean was obtained. This data was transferred to E X C E L for further computations. Chart Reviews Clinical factors (presence of acute and/or chronic respiratory disease, gender, age and BMI were determined through a retrospective patient chart review and were examined for their relationship to the number of inflammatory cells (macrophages, neutrophils and B-cells) in the injured diaphragm. In this study subjects between 26-64 years were classified as adults and 65 or above 65 years were classified as elderly (Polkey et al. 1997). The 49 subjects for which BMI values were available were divided into three groups (normal, high and low BMI) according to lower limit of normal BMI (18.5 kg/m ) defined by Naidu and Rao (1994) and upper limit 2 2 defined by Williamson (1993) (27.3 kg/m for females and 27.8 kg/m for males). DATA MANAGEMENT A l l records were kept locked in a filing cabinet to maintain confidentiality. A l l biopsies were given random code numbers from 1 to 59 and subjects were referred to by using this random number. Only the investigator and the thesis supervisor had access to the data. 39 DATA ANALYSIS Non-parametric statistical tests were done to analyze the collected data. The statistical tests used for analyses are as follows: 1) Kruskal Wallis one way A N O V A was performed to find out the difference between the inflammatory cell, neutrophil and macrophage counts of the four subject groups: acute respiratory disease (ARD), both acute respiratory disease + chronic respiratory disease (ARD+CRD), chronic respiratory disease (CRD) and no respiratory disease (NRD). 2) Mann Whitney U test was done to find out the differences between inflammatory cell counts in females and males. 3) Spearman correlation coefficient by ranks was done to find out the correlation between: (a) Number of inflammatory cells and BMI (b) Number of inflammatory cells and age (c) Number of neutrophils and abnormal diaphragm area fraction (d) Number of macrophages and abnormal diaphragm area fraction (e) Number of neutrophils + macrophages combined and abnormal diaphragm area fraction A significance level of p<0.05 was selected for all tests. 40 Table 1: Point-counting Categories and Definitions (Clarke, 2000) Categories 0. No count 1. Normal 2. Internal nucleus 3. Inflamed/Necrotic 4. Small/Angulated/Round Fibers 5. Abnormal Cytoplasm 6. Inflammatory Cell 7. Collagen/Fibroblast 8. Degenerated/Atrophic Fibers 9. Capillary Definitions 0. No count: Space, nerve, artefact, epimysium, wall or lumen of vessel larger than capillary. 1. Fiber with polygonal shape, homogeneous acidophilic cytoplasm, intact plasma membrane, peripheral nuclei. 2. Fiber containing > 1 internally located nuclei (8 pixels of sarcoplasm between nucleus and sarcolemma). 41 3. Fiber containing > 1 inflammatory cell or necrotic mass of inflammatory cells and muscle debris without plasma membrane. 4. Small fiber (< 1/3 the lesser fiber diameter of the five largest fibers in the field) (b) fibers with "spear like" projections or extensions that are less than 35 degrees and . (c) fibers that have lost the normal polygonal shape and are large and rounded. 5. Includes: (a) fiber with pale acidophilic peripheral cytoplasm and enlarged peripheral nuclei with or without visible nucleoli, or (b) fiber with pale acidophilic peripheral cytoplasm and deep acidophilic "fuzzy" cytoplasm in the central region, or (c) lipofuscin, or (d) split or whirled fibers, or (e) vacuoles, or (f) uneven cytoplasmic staining unrelated to processing, or (g) fiber with dull or light grey staining, or (h) cytoplasmic fragmentation. 6. Round nucleus located in interstitium, with morphological features of mononucleated or multinucleated inflammatory cells. 7. Includes: (a) fibrils of endomysium or perimysium, or (b) spindle-shaped nucleus in interstitium. 8. Includes: (a) fiber with withering appearance undergoing degeneration without inflammatory cells (b) fiber undergoing atrophy with normal appearance. 9. Endothelial cell, lumen, or contents of a capillary. 42 Table 2: Equations for Calculation of Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue (Clarke, 2000) Total Count = £ Counts in Categories 1 -9 Area Fraction of Normal Muscle = 7, Counts in Category 1 x 100 Total Count Area Fraction of Abnormal Muscle = X Counts in Categories 2-6 & 8 x 100 Total Count Area Fraction of Connective Tissue = X Counts in Categories 7 & 9 x 100 Total Count 43 CHAPTER 3: RESULTS & DATA ANALYSIS SUBJECTS The fifty-nine subjects included in the study were divided into four groups according to the presence or absence of respiratory disease, as found in the medical charts from respective hospitals. The four groups were acute respiratory disease (ARE); n=20), chronic respiratory disease (CRD; n=7), acute and chronic respiratory disease (ARD+CRD; n=22) and no respiratory disease (NRD; n=10). Subjects with various medical conditions classified under each group are shown in Table 3. INFLAMMATORY C E L L COUNTS Macrophages, neutrophils and B-cells were observed in the biopsies (Figure 7). The number of macrophages was found to be far greater than that for neutrophils and B-cells. Macrophages constituted 97.5% of the total inflammatory cells counted as compared to 2.4% of neutrophils and 0.1% B-cells. Means, ranges and the number of biopsies evaluated for macrophages, neutrophils, B-cells and T-lymphocytes are shown in Table 4. 44 Figure 7: (A) Upper figure shows a large number of macrophages in the diaphragm of a postmortem subject identified by CD68 antibody (B) Middle figure shows neutrophils in a fiber of the diaphragm of a postmortem subject identified by NP57 antibody (C) Lower figure shows a fiber with B-cell on periphery in the diaphragm of a postmortem subject identified by CD20 antibody. Note the smaller number of neutrophils and B-cells compared to macrophages. Arrows indicate the cells in each figure. Scale bars = 50um. Picture B and C were taken at the same magnification. 45 RELATIONSHIP BETWEEN INFLAMMATORY CELLS AND CLINICAL FACTORS Inflammatory Cells and Respiratory Disease Kruskal Wallis one way A N O V A by ranks showed that there were no differences in the number of inflammatory cells present in the diaphragms of people in the four subject groups (ARD, ARD+CRD, CRD, NRD) (see table 4 for p values). Mean inflammatory cells (macrophages + neutrophils + B-cells), neutrophils and macrophages in the four subject groups (ARD, ARD+CRD, CRD, NRD) are shown in Figures 8, 9 (inflammatory cells), Figures 10, 11 (neutrophils), Figures 12, 13 (macrophages) and Table 5. Only three subjects out of 56 showed B-cells in their biopsies, therefore, no statistical analysis was performed for B-cells. Slides for 40 subjects were processed for T-lymphocytes, however, no T-lymphocytes were found in any of the biopsies. Thus, no statistical analysis was performed. Inflammatory Cells and Gender There were 20 females and 39 males included in the study. Comparison of the two gender groups by Mann Whitney U test showed no statistically significant differences in the number of inflammatory cells in the diaphragms of males and females (p= 0.934 for inflammatory cells per CSA; p= 0.747 for inflammatory cells per fiber). Mean inflammatory cells per CSA and per fiber in males and females are shown in Figures 14 and 15. 46 Inflammatory Cells and Age Spearman correlation by ranks showed no correlation between the age of the subjects and the inflammatory cells found in their diaphragm (Figures 16, 17). The mean age for all subjects was 68 + 13 years ranging from 26 to 87 years. There were 38 elderly and 21 adult subjects included in the study. The Mann Whitney U test showed that the number of inflammatory cells present in the diaphragms of elderly and adult subjects were not significantly different (p = 0.381 for inflammatory cells per mm ; p = 0.665 for inflammatory cells per fiber). Mean inflammatory cells in adults and elderly subjects are shown in Figures 18 and 19. Inflammatory Cells and Body Mass Index Body mass index (BMI) values were available for 49 subjects. The mean BMI values of the 49 subjects, males and females are shown in Table 6. BMI and the number of inflammatory cells showed a significant positive correlation {p-0.049, r=0.286 (per CSA); p= 0.05, r= 0.284 (per fiber); Figure 20, 21}. The 49 subjects, for which BMI values were available, were divided into three groups (normal, high and low BMI) (Figure 22). Kruskal Wallis one way A N O V A showed no significant difference in the presence of inflammatory cells in the diaphragm of subjects with normal, high or low BMI (p - 0.448 for inflammatory cells per mm ; p = 0.425 for inflammatory cells per fiber). The mean values of inflammatory cells in these three groups 47 are shown in Figures 23 and 24. Distribution of males and females in three BMI groups and inflammatory cells in each group is shown in Table 6. INFLAMMATORY CELLS AND ABNORMAL DIAPHRAGM No correlation between the number of macrophages, combined neutrophils + macrophages and neutrophils with abnormal area fraction of diaphragm was found in the postmortem subjects (see Figures 25-30). The "r" and "p" values of correlation between the number of neutrophils, macrophages and neutrophils + macrophages are shown in Table 7. T - LYMPHOCYTES AND FIBER SIZE VARIATION No T - lymphocytes were found in any of the biopsies, therefore no analysis was done. DEMOGRAPHIC DATA Demographic data of all subjects was collected by reviewing medical charts from the respective hospitals to obtain the age, BMI, gender and information about presence or absence of acute and/or chronic respiratory disease as shown in Appendix I. Information for each subject about the cause of death, smoking history, mechanical ventilation, differential diagnosis, and resuscitation history collected from medical charts is shown in Appendix II. Figure 31 shows the breakdown of the number of subjects with A R D , ARD+CRD, CRD and 48 NRD who died from causes of death that were cardiac related, respiratory related and other causes. 49 t o R E E S a Inflammatory Cel ls per Cross-Sectional Area 200.0 150.0 100.0 50.0 0.0 42.7 79.8 78.0 66.8 ARD ARD+CRD CRD NRD Presence of Respiratory Disease Figure 8: Mean number of inflammatory cells per cross-sectional area (mm2) in four groups of subjects. A R D - acute respiratory disease, ARD + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, N R D - no respiratory disease. Bars indicate the SD. Inflammatory Cells per Fiber ? 0.50 w u « 2 jg 0.30 S a. M a 0.00 0.40 0.10 A R D A R D + C R D C R D N R D Presence of Respiratory Disease Figure 9: Mean number of inflammatory cells per fiber in four groups of subjects. A R D -acute respiratory disease, A R D + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, NRD - no respiratory disease. Bars indicate the SD. 50 7.00 6.00 -5.00 E E h s 4.00 fit 3 3.00 a a 2.00 1.00 0.00 -J N e u t r o p h i l s per C r o s s - S e c t i o n a l A r e a 1.55 1.65 1.26 0.56 ARD ARD+CRD CRD NRD Presence of Respiratory Disease Figure 10: Mean number of neutrophils per cross-sectional area (mm2) in four groups of subjects. A R D - acute respiratory disease, A R D + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, N R D - no respiratory disease. Bars indicate the SD. 0.020 0.018 2 0.016 e 0.014 s | 0.012 -I | 0.010 t 0.008 Or S 0.006 | 0.004 S 0.002 Z 0.000 Neutrophils per Fiber 0.005 0.005 0 004 0.002 ARD ARD+CRD CRD NRD Presence of Respiratory Disease Figure 11: Mean number of neutrophils per fiber in four groups of subjects. A R D - acute respiratory disease, A R D + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, NRD - no respiratory disease. Bars indicate the SD. 51 & E u a a 01) « n . o Macrophages per Cross-Sectional Area 200.0 175.0 150.0 125.0 100.0 75.0 50.0 25.0 H 0.0 41.0 71.4 76.7 76.6 A R D A R D + C R D C R D N R D Presence of Respiratory Disease Figure 12: Mean number of macrophages per cross-sectional area (mm2) in four groups of subjects. A R D - acute respiratory disease, A R D + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, N R D - no respiratory disease. Bars indicate the SD. Macrophages per Fiber .o IS QJ a. a o 0.45 i 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.13 0.17 0.18 0.16 A R D A R D + C R D C R D N R D Presence of Respiratory Disease Figure 13: Mean number of macrophages per fiber in four groups of subjects. A R D - acute respiratory disease, A R D + CRD- acute respiratory disease + chronic respiratory disease, CRD - chronic respiratory disease, NRD - no respiratory disease. Bars indicate the SD. E E o « E E 5= Gender and Inflammatory Cells per Cross-Sectional Area 175.0 150.0 125.0 100.0 75.0 50.0 25.0 0.0 70.8 58.6 FEMALE MALE Figure 14: Mean number of inflammatory cells per cross-sectional area (mm2) in females (n=20) and males (n=39). Bars indicate the SD. . o IS Gender and Inflammatory Cells per Fiber 0.40 0.30 t 0.20 o w E E J 0.10 0.00 0.18 FEMALE 0.15 MALE Figure 15: Mean number of inflammatory cells per fiber in females (n=20) and males (n=39). Bars indicate the SD. 53 E 350.0 i S u 300.0 -Q. Vi 250.0 -"3 U 200.0 ->, 1_ 150.0 -o eg 100.0 -mm 50.0 -a 55 0.0 -Age and Inflammatory Cells per CSA r= -0.119; p=0.375 • * • • 0.0 20.0 40.0 60.0 80.0 100.0 Age (years) Figure 16: Scatter-plot showing no relationship between inflammatory cells per cross-sectional area (mm ) and age. u 0? 0.6 -1 XI IS k. 0.5 -lis p< 0.4 -u 0.3 -O w 0.2-E 0.1 -E Infl: 0.0 Age and Inflammatory Cells per Fiber r=-0.103;p=0.442 t 0.0 20.0 * t f ^ M * > 40.0 60.0 80.0 100.0 Age (years) Figure 17: Scatter-plot showing no relationship between inflammatory cells per fiber and age. 54 E E U a. o 58 E E Inflammatory Cells per CSA in Elderly and Adult Subjects 180.0 160.0 140.0 4 120.0 100.0 80.0 --60.0 40.0 20.0 4 0.0 59.5 71.9 ELDERLY A D U L T Figure 18: Mean number of inflammatory cells per cross-sectional area (mm2) in the diaphragm of elderly (n= 34; >65 years) and adult (n= 18; <65 years) subjects. Bars indicate the SD. Inflammatory Cells per Fiber in Elderly and Adult Subjects 0.40 0.30 0.20 L, a a B S -0.17 ELDERLY 0.15 ADULT Figure 19: Mean number of inflammatory cells per fiber in the diaphragm of elderly (n= >65 years) and adult (n= 18; <65 years) subjects. Bars indicate the SD. 34; 55 E 350.0 i celb per m 300.0 -250.0 -200.0 -o 150.0 -+j a g 100.0 -E 50.0 -C5 c 0.0 -BMI and Inflammatory Cells per CSA r=0.286; p=0.049 0.0 10.0 20.0 30.0 40.0 50.0 Body Mass Index (BMI) 60.0 Figure 20: Scatter-plot showing a significant positive correlation between inflammatory cells per cross-sectional area (mm2) and body mass index (BMI). BMI and Inflammatory Cells per Fiber ,_ „ , r=0.284; p=0.05 « 0.6 ] 0.0 10.0 20.0 30.0 40.0 50.0 60.0 Body Mass Index (BMI) Figure 21: Scatter-plot showing a positive correlation between inflammatory cells per fiber and body mass index (BMI). 56 BMI Balance Figure 22: BMI balance showing the number of subjects in low, normal and high body mass index (BMI) group. The figure also shows the number of subjects having acute respiratory disease (ARD), acute and chronic respiratory disease (ARD+CRD), chronic respiratory disease (CRD) and no respiratory disease (NRD) in low, normal and high BMI group. 57 BMI Groups and Inflammatory Cells per CSA „ 200.0 -i l „ 1 5 0 . 0 -i a t 1 a ioo.o ] I a s .... r i r-^i _ L H I G H L O W N O R M A L B o d y M a s s Index (BMI) Figure 23: Mean number of inflammatory cells per cross-sectional area (mm2) in high, low and normal BMI group of subjects. Bars indicate the SD. BMI Groups and Inflammatory Cells per Fiber E E « c 0.50 0.40 0.30 0.20 0.10 0.00 0.21 0.20 I 0.14 H I G H L O W N O R M A L B o d y M a s s Index ( B M D Figure 24: Mean number of inflammatory cells per fiber in high, low and normal BMI group of subjects. Bars indicate the SD. 58 Abnormal diaphragm and macrophages per CSA r=0.02 l;p=0.0.876 | 300.0 Z 250.0 °- 200.0 E 150.0 100.0 50.0 0.0 D. O 0.00 20.00 40.00 60.00 80.00 A b n o r m a l d i a p h r a g m area fract ion (%) Figure 25: Scatter-plot showing no relationship between abnormal diaphragm area fraction and macrophages per cross-sectional area (mm ) of diaphragm. Abnormal diaphragm and macrophages per fiber r=0.048; p=0.727 0.60 V « £ 0.40 •a a. C P i. a 0.30 0.20 ^  0.10 0.00 0.00 20.00 40.00 60.00 80.00 A b n o r m a l d i a p h r a g m a r e a fract ion (%) Figure 26: Scatter-plot showing no relationship between abnormal diaphragm area fraction and macrophages per fiber of diaphragm. 59 Abnormal diaphragm and macrophages+neutrophils per CSA 350.0 -I r=0.097; p=0.468 300.0 - • + mm 250.0 -Q> M « M s per 200.0 -o 1-u is c. 150.0 -• • • • • • cs neutro 100.0 -50.0 -0.0 -' • • * 0.0 20.0 40.0 60.0 80.0 Abnormal diaphragm area fraction (% ) Figure 27: Scatter-plot showing no relationship between abnormal diaphragm area fraction and macrophages + neutrophils per cross-sectional area (mm2) of diaphragm. Abnormal diaphragm and macrophages+neutrophils per fiber r=0.095;p=0.480 0.0 20.0 40.0 60.0 80.0 Area fraction of abnormal diaphragm (%) Figure 28: Scatter-plot showing no relationship between abnormal diaphragm area fraction and macrophages + neutrophils per fiber of diaphragm. 60 Abnormal diaphragm and neutrophils per CSA 20.00 i per mn 15.00 -per mn 1 10.00 -1 5.00 -0.00 -r = 0 . 1 2 1 ; p = 0 . 3 9 4 0.00 20.00 40.00 60.00 80.00 A b n o r m a l d i a p h r a g m a r e a f r a c t i o n (% ) Figure 29: Scatter-plot showing no relationship between abnormal diaphragm area fraction and neutrophils per cross-sectional area (mm2) of diaphragm Abnormal diaphragm and neutrophils per fiber 0.060 £ 0.050 a. a. © 0.040 0.030 0.020 5 0.010 0.000 r=0.130;p=0.357 m 4 • - • - • r - 4 » -0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Abnormal diaphragm area fraction (%) Figure 30: Scatter-plot showing no relationship between abnormal diaphragm area fraction and neutrophils per fiber of diaphragm. 61 Cause of Death Figure 31: This figure shows the subjects' cause of death classified as either respiratory related, cardiac related or other causes. The figure also shows the number of subjects having A R D - acute respiratory disease, ARD+CRD- acute respiratory disease + chronic respiratory disease, CRD- chronic respiratory disease and NRD- no respiratory disease under each of the three categories of causes of death. 62 Table 3: Subject Distribution in Four Groups Total number of subjects = 59 A R D = 2 0 A R D + C R D = 2 2 C R D = 7 N R D = 1 0 Bronchopneumonia C O P D C O P D Tuberculosis Pulmonary emboli Pulmonary edema Asthma Grand-mal seizure Pulmonary edema Aspiration pneumonia Chronic bronchitis Shock Aspiration Pneumonia Interstitial pulmonary Post-herpetic pneumonia Respiratory failure fibrosis neuralgia A R D S Bronchopneumonia Chronic pulmonary edema P V D Respiratory failure Paraseptal emphysema Centrilobular emphysema Small intestinal Pleural effusion A R D S infarction Atyp ica l pneumonia Cardiac arrest Pulmonary fibrosis Cardiac arrythmia Bronchioli t is obliterans Abdominal aortic Pleural effusion bradycardiac collapse Pleurisy Acute M I Interstitial acute Multisystem failure pneumonitis E . col i sepsis Abbreviations: A R D - acute respiratory disease, ARD+CRD- acute respiratory disease + chronic respiratory disease, ARDS- acute respiratory distress syndrome, COPD- chronic obstructive pulmonary disease, CRD- chronic respiratory disease, MI- myocardial ischemia, NRD- no respiratory disease, PVD- peripheral vascular disease. * * * o o 13 ft a* CD o g ft) 13 O o o CD 3 CD CD O cr (/> CD CD O CD O CD* C/3 o 8L Eo' Q. O 3 CD cr CD O s cn CD t3 O t/3 CD O CD 3 CD CD O cr CD CD a. 3" o Ef CD CD cr o" 1 3 tn i-* • CD w T3 CD o > 13 CD Eft cr CD i S3 < & 0 3 5 3" S o g ° o ^ «3 CO JJL E ^ o CD H ST O > CD C/! r-f cr 8 •t o T3 cr P3 OQ CD C/5 + (3 CD & >-! O cr + o CD Inflammatory cells # T-lymphocytes B-cells Neutrophils Macrophages Inflammatory Cells Type 63.8 + 74.8 © 0.03 ±0.14 1.54 ±4.32 62.6 + 72.6 Mean per CSA 810 + 910 © 0.0001 ±0.0004 0.00 ±0.01 0.16±0.17 Mean per Fiber 0-301.1 o 0 - 0.84 0 - 24.79 0 - 282.9 Range per CSA 0-0.6 o 0-0.0019 0-0.04 0-0.56 Range per Fiber 0.764; 0.725 * * * * * 0.450; 0.372 0.724; 0.725 p values* Cells per CSA; Cells per Fiber to o OS to U\ OS No. of Biopsies Evaluated o to OS 4^  00 No. of Biopsies Cells found in os 64 Table 5: Inflammatory Cells and Four Groups Inflammatory cells per cross-sectional area (mm2) Subject Group Macrophages Neutrophils B-cells T-lymphocytes Inflammatory Cells A R D 41.0+51.9 1.55+3.69 No analysis* No analysis** 42.7 ± 51.5 ARD+CRD 71.4+76.8 1.65±4.52 79.8 ±83.5 CRD 76.7± 104.6 1.26+2.02 78.0 ± 103.5 NRD 76.6±82.1 0.56± 1.13 66.8 + 79.8 Inflammatory cells per fiber Subject Group Macrophages Neutrophils B-cells T-lymphocytes Inflammatory Cells A R D 0.13 ±0.19 0.005 ±0.012 No analysis* No analysis** 0.13 ± 0.18 ARD+CRD 0.17 + 0.15 0.004 ±0.011 0.17 ± 0.15 CRD 0.18 ±0.24 0.005 ± 0.008 0.18 ±0.24 NRD 0.16±0.17 0.002 ± 0.005 0.16±0.17 * No analysis done because positive cells were observed in only three biopsies ** No analysis done because no positive cells were observed in any of the biopsies 65 Table 6: BMI Groups and Gender BMI group No. of Females No. of Males Total Low 3 0 3 Normal 10 23 33 High 3 10 13 Total 16 33 49 Mean BMI in Females and Males BMI group Mean BMI Females Mean BMI Males Mean BMI Total Low 17.1 ± 1.3 17.1 ± 1.2 Normal 24.2+2.7 24.1 ±2 .2 24.0 ±2 .3 High 36.9 + 9.6 32.9 ±4 .0 33.4 ±5 .6 Total 25.3 ±7 .6 26.7 ±5 .0 26.3 ±5 .9 Mean Inflammatory Cells (per mm ) in Females & Males of BMI Groups BMI group No. of inflammatory cells in Females No. of inflammatory cells in Males Total Low 74.1 ±95.5 74.1 ±95.5 Normal 102.8 ±97.2 51.0±63.2 58.5 ±70.0 High 50.1+61.7 84.0 + 93.5 86.2 ± 90.9 Total 84.9 ±85.1 62.0 ± 74.7 68.50 ±77.4 Mean Inflammatory Cells (per fiber) in Females & Males of BMI Groups BMI group No. of inflammatory cells in Females No. of inflammatory cells in Males Total Low 0.20 ±0.26 0.20 ±0.26 Normal 0.16 ± 0.18 0.13 ±0.15 0.14 ± 0.16 High 0.24± 0.25 0.24± 0.25 0.21 ±0.18 Total 0.19 ±0.20 0.15± 0.16 0.16± 0.17 A l l values represent only for 49 subjects for which BMI values were available 66 Table 7: r & p values for Area Fraction of Abnormal Diaphragm and Inflammatory Cells (per mm ) Inflammatory cell Type Area Fraction of Abnormal Diaphragm Area Fraction of Abnormal Diaphragm per CSA* per Fiber Neutrophils r= 0.121, p=0.194 r=0.130, p=0.357 Macrophages r= 0.021, p=0.876 r= 0.048. p=0.727 Neutrophils+Macrophages r=0.097, p=0.468 r=0.134, p=0.315 2 * Cross - sectional area measured in mm 67 CHAPTER 4: DISCUSSION Exertion-induced skeletal muscle injury is a type of muscle injury characterized by an acute inflammatory response occurring at the initial stages of the repair process (Maclntyre et al. 2000). The inflammatory reaction is phylogenetically and ontogenetically the oldest defense mechanism (Stvrtinova et al. 1995). The cells of the immune system are widely distributed throughout the body, but in the case of injury they migrate out of the capillaries into the surrounding tissues (Tiidus, 1999). The acute post-exercise inflammatory reaction is a complex series of events associated with the influx of various fluids, proteins and inflammatory cells in the damaged skeletal muscle fibers (Tiidus, 1999). In the current study, the inflammatory cells (macrophages, neutrophils and B-cells) were counted in the diaphragms of 59 patients who underwent autopsy due to a variety of medical conditions. The relationship of inflammatory cells with the presence of respiratory disease, gender, age and BMI of these subjects will be discussed in the subsequent sections. INFLAMMATORY C E L L COUNTS Macrophages and neutrophils constitute the major set of inflammatory cells found in the damaged muscle as a result of injury (Malm et al. 2000; Tiidus, 1999). This study which is the first study to quantitate macrophages, neutrophils, B-cells and T-lymphocytes in the human diaphragm, shows similar findings. In the current study, macrophages, neutrophils, B-cells and T-lymphocytes were quantified in the diaphragms of 59 postmortem subjects 68 suffering from various diseases and it was observed that macrophages (97.5% of total inflammatory cells) were the most common inflammatory cells. The next most common inflammatory cells were neutrophils (2.4% of total inflammatory cells) followed by B-cells (0.1% of total inflammatory cells). No cytotoxic T-lymphocytes were detected in the diaphragms of postmortem subjects. The findings of this study are consistent with the previous studies quantifying inflammatory cells (Malm et al. 2000; Orimo et al. 1991; Round et al. 1987; MacGowan et-al. 2001). Malm and coworkers (2000) quantified macrophages, neutrophils, B-cells and T-lymphocytes in the vastus lateralis of healthy humans after exercise and in a control (non-exercise) group. They found that the major inflammatory cells in the biopsies of vastus lateralis of their subjects were macrophages and neutrophils. B cells and T-lymphocytes were found in minute quantities in the human muscle tissue both at rest and after exercise. They also reported that macrophages were more common than neutrophils. Orimo et al. (1991) in an animal study found similar results with a different model of muscle injury. They found that the two major subsets of inflammatory cells at different stages of bupivacaine-induced acute muscle fiber necrosis detected were macrophages and neutrophils. They also reported that T-lymphocytes and B-cells comprised a small population among all inflammatory cells counted. Similarly, Round et al. (1987) also reported that macrophages were the predominant cell type observed in the human limb muscles. They also reported very few B-cells and no T-lymphocytes in the examined biopsies. MacGowan et al. (2001) quantified macrophages in 21 subjects undergoing thoracotomy. They found 52 +19 macrophages per mm and 0.41+0.18 macrophages per 69 fiber, which is comparable to the number of macrophages counted (62.6 + 72 per mm 2; 0.16 + 0.17 per fiber) in the current study. Although there have been very few studies quantifying inflammatory cells in human skeletal muscle, the findings are in harmony with each other. Therefore, it appears that macrophages and neutrophils are the two major types of inflammatory cells present in damaged human skeletal muscle and diaphragm is no exception. RELATIONSHIP BETWEEN INFLAMMATORY CELLS AND CLINICAL FACTORS Inflammatory Cells and Respiratory Disease Neutrophils are the principal inflammatory cells to invade the site of injury during the acute phase of exertion-induced skeletal muscle injury and macrophages follow the neutrophils, being the key inflammatory cells in the chronic phase of the injury (Stvrtinova et al. 1995). In this study we expected to find more neutrophils and macrophages in the diaphragm of people with acute and chronic respiratory disease respectively. Inflammatory cell, macrophage and neutrophil counts, found in the diaphragm of people in the four groups (ARD, CRD, ARD+CRD and NRD), were not significantly different from each other. Surprisingly, it was observed that the subjects in the N R D group had a comparable number of inflammatory cells in their diaphragms compared to those found in the diaphragms of the other three groups having acute and/or chronic respiratory disease. Clarke (2000) used the same subject data as the current study and reported morphological abnormalities in the NRD 70 group. The reason for this unexpected finding might be the effect of other medical conditions and diseases on the diaphragm of the N R D subjects. The N R D subjects had a variety of diseases as listed in Table 3. Although, their medical charts did not show the presence of any kind of respiratory disease as documented in the data sheets, it is possible that an ongoing respiratory problem was not diagnosed or documented. The distribution of subjects in the four groups was very uneven and the sample size of individual groups were small (ARD+CRD=22; ARD=20; CRD=7; NRD=10). In addition, the 59 subjects included in the study were an extremely selected group of people who underwent autopsy in Vancouver Hospital and St. Paul's Hospital in Vancouver, BC, due to a wide variety of diseases which were not controlled for in this study. These are some of the factors to be considered while generalizing the findings of the current study. Inflammatory Cells and Gender Gender differences in strength, fiber size, fiber type, motor unit number and efficiency of respiratory muscles have been reported (Miller et al. 1993), however, experimental evidence on gender differences in the inflammatory response as a result of diaphragm injury in people with acute and/or chronic respiratory disease is limited. In the present study, no differences in the inflammatory cell counts of males and females were observed. These findings are similar to a longitudinal study carried out on dogs in which blood samples were taken and analyzed in individual dogs at their different age. No significant differences between the circulating white blood cell count in males and females were found (Greely et al. 2001). In contrast, gender differences in humans have 71 been reported by Maclntyre et al. (2000). They reported a greater number of technetium-99 labeled neutrophils in the quadriceps of the exercised leg of women compared to men two hours post eccentric exercise (p= 0.03). Also, Stupka et al. (2000) in a study on humans, found a greater number of inflammatory cells in the vastus lateralis of males than females. Higher numbers of inflammatory cells in male mice than female mice were also reported in a study by Schneider et al. (1999). Many other studies (Tarnopolsky et al. 1995; Spitzer and Zhang, 1996; Tiidus, 1999) have also shown gender differences in the response to exertion-induced skeletal muscle injury. Estrogen has been reported to affect the function of macrophages and neutrophils in rats (Spitzer and Zhang, 1996) and to reduce the post-exercise muscle damage in females (Tiidus, 1999). The above discussion suggests that gender influences the inflammatory response in muscle injury and female sex hormones might be one of the factors responsible for the difference. In the current study the females had an average age of 72 + 12 years (range 48-87 years), therefore, none of the females were undergoing menstrual cycle. Unless, the females in the study were on hormonal therapy which was an uncontrolled factor, it can be speculated that the diminished influence of estrogen in these females might have prevented any gender differences in the current study. However, the effects of a non-homogeneous selected subject group and many uncontrolled factors such as smoking history, use of corticosteroids, duration of mechanical ventilation and resuscitation, on the results of the current study cannot be ignored. 72 Inflammatory Cells and Age In the current study, no correlation between age and number of inflammatory cells in the diaphragm of 59 postmortem subjects was found. Similar results were found by Greely et al. (2001) who reported no significant change in inflammatory cell count in blood with aging in dogs. In a study on young and old subjects by Clarkson and Dedrick (1988), a significant increase in serum creatine kinase and muscle soreness, and a decrease in strength following novel or strenuous exercise in all subjects were reported. They had hypothesized that the changes in muscle with age may result in a reduced ability of the muscle to repair and adapt to exercise. However, they found no difference in the damage and repair process among old and young subjects. Reduction in diaphragm strength in elderly subjects has been shown by Polkey et al. (1997), who assessed diaphragm strength in 15 young and 15 elderly but, they further reported that the reduction in diaphragm strength of the elderly was minimal (13%). They also found considerable overlap between the young and elderly group and the magnitude of the reduction in elderly diaphragm strength was described as being of no functional importance. A study by Tolep et al. (1995) reported that trans-diaphragmatic pressure developed during a maximum inspiratory effort was approximately 25% lower in normal elderly men compared to young adult male subjects. This age difference might have been the effect of the small sample size (elderly, n= 10; young adults, n= 9) of this study, therefore, generalization of the results cannot be done. In another study on elderly people it was found that isometric and concentric muscle strength linearly declines with aging, whereas, eccentric muscle strength was relatively preserved (Hortobagyi et al.T995). The diaphragm injury in people with respiratory disease appears to occur mostly in the regions 73 where eccentric contractions occur. This might be, therefore, one explanation why no differences in diaphragm injury and hence the inflammatory response occurred between adults and elderly in the current study. The majority of the research work on age related changes has been done to assess muscle mass, strength, connective tissue and motor unit functioning, but, the literature correlating age and inflammatory cells in human diaphragm is inadequate. This study stands alone in being the only one to quantify inflammatory cells in the diaphragm of human subjects and correlate them to age in a group of subjects with very diverse and complicated medical conditions, which might be one more reason for not finding a relationship between age and inflammatory cells. Inflammatory Cells and Body Mass Index It has been suggested that obesity is associated with a state of chronic low inflammation (Das, 2001).Inflammatory cells along with plasma mediators are the key component of inflammation, therefore, it is interesting to find out how inflammatory cells vary with BMI in humans. The current study related B M I with inflammatory cells in human diaphragm. In this study it was observed that BMI is significantly correlated to the number of inflammatory cells in the diaphragm of postmortem subjects (r= 0.286; p= 0.049). This implies that subjects having greater BMI had more inflammatory cells in their diaphragm in the current study. The findings of this study are similar to Visser et al. (2001) who found that in children 8-16 years of age, being overweight is associated with higher white blood cell 74 counts. Similarly, it has been documented that in Pima Indians white blood cell count is positively correlated with adiposity (Weyer et al. 2002). Although, the patho-physiology of association of obesity and inflammation is unclear, it has been reported that adipose tissue expresses a variety of pro-inflammatory cytokines such as IL-6, TNF-a and complement C3 which stimulates the production of acute phase proteins such as C - reactive protein (CRP) (Weyer et al. 2002). CRP is a humoral marker of inflammation which is present in higher levels in people with obesity (Weyer et al. 2002). Cross-sectional studies have revealed that elevated CRP level is positively correlated with BMI {a clinical indicator of obesity (Visser et al. 2001)} (Weyer et al. 2002). Also, respiratory diseases have been found to be associated with elevated plasma CRP levels (Visser et al. 2001). In the light of above discussion, it can be logically deduced that, chronic inflammation is present in higher BMI people (indicated by higher plasma levels of CRP), hence there could be a possibility of finding a higher number of inflammatory cells in the muscle of obese people which justifies the finding of the current study. In conclusion, BMI was found to be positively correlated to inflammatory cells in this study. This finding of the current study along with the findings of Visser et al. (2001) suggest that obese people with respiratory disease might have more inflammatory cells in their diaphragm than non-obese people. However, further studies correlating inflammatory cells with BMI in human diaphragm in larger number of patients are required to be carried out to confirm these results. 75 INFLAMMATORY CELLS AND ABNORMAL DIAPHRAGM In the current study the possibility of using neutrophils, macrophages or their combined number as an indicator of diaphragm injury in people with or without respiratory diseases was tested. It was found, however, that neutrophils, macrophages and the combined number of neutrophils + macrophages were not significantly correlated to the area fraction of abnormal diaphragm in this group of subjects with diverse medical conditions. Neutrophils form the first line of defense in the plasma where they eliminate antigens, however, they have cytotoxic capabilities too. They possess microbicidal, bactericidal and viricidal activities which are critical to the host defense (Pyne, 1994). They phagocytize pathogens and release an array of cytotoxic factors including elastase, lysozyme and oxygen radicals. If released in an uncontrolled manner these agents can also damage healthy surrounding tissue (Evans and Cannon, 1991). This argument suggests that neutrophils might have a positive correlation with the abnormal area fraction of diaphragm in diaphragm injury but the results of the present study clearly show that this was not happening in the selected group of subjects. On the other hand, Robertson et al. (1993) have shown sufficient evidence that macrophages produce a factor or group of factors that strongly attract muscle precursor cells to the site of injury, thus, facilitating muscle regeneration. Further, St. Pierre and Tidball (1994) showed that different populations of macrophages were present in exercise-injured limb muscle of rats at different time points of injury and repair. Therefore, macrophages have an important role to play for repair and regeneration of injured muscle fibers. Their 76 role in repair might be a reason for there being a lack of correlation of macrophage numbers with abnormal diaphragm area fraction. 77 CONCLUSION & FUTURE PERSPECTIVES The major finding of this study is that macrophages are the inflammatory cells present in the diaphragm of people with acute and/or chronic respiratory disease. Since macrophages are involved in tissue repair and regeneration it can be deduced from the results that extensive regeneration occurs after injury in the diaphragm of patients with acute and/or chronic respiratory disease. B cells were very rare and no T-lymphocytes were detected in the diaphragm of postmortem subjects. These findings suggest that cytotoxic T-lymphocytes and B-cells may not have an important role to play in the diaphragm injury in these patients. Interestingly, inflammatory cells were also found in the diaphragm of subjects in NRD group. This suggests the damaging effect of the other medical conditions these subjects were suffering from on the diaphragm. Another important finding of this study was the significant correlation of BMI with the number of inflammatory cells in the diaphragm of these patients. Since influx of inflammatory cells is a histological abnormality shown in and around damaged fibers, it seems that overweight people might be more susceptible to diaphragm injury as a result of overloading. The findings of this study also suggest that age is not a factor in the increase in inflammatory cell numbers in the diaphragm of the people suffering with acute and/or chronic respiratory disease. Gender differences in the number of inflammatory cells found in the diaphragm of males and females as a result of exertion-induced diaphragm, was not evident in the selected subject group. Lastly, the findings of the current study suggest that 78 there is no correlation of neutrophils, macrophages and neutrophils + macrophages with the percentage of abnormal diaphragm. Future Research Recommendations Large sample size is an important characteristic of this study, however, when 59 subjects were divided into four groups according to the presence or absence of acute and/or chronic respiratory disease the subject number in each group was very unevenly distributed (ARD+CRD=22; ARD=20; CRD=7; NRD=10): Further research with a comparatively homogeneous, larger group sample size and more defined groups of subjects is recommended for more definite answers. Smoking history, mechanical ventilation, resuscitation and use of corticosteroids might have some effect on the number of inflammatory cells in the human diaphragm which were not controlled for in this study. Finding out the relationship of these factors with the number of inflammatory cells in the diaphragm can be another prospective study. None of the females in this study were undergoing menstrual cycle, therefore, the effect of female sex hormones cannot be studied in this sample. A study looking at the effect of female sex hormones on the number of inflammatory cells in diaphragm in a new subject group should be done in the future to examine for gender differences of the inflammatory response to exertion-induced diaphragm injury. This can be done by selecting age matched groups of females in different menstrual cycle phases and quantifying the inflammatory cells in their diaphragm biopsies. 79 Muscle fibers with internal nuclei represent regenerating muscle fibers after injury. Since the macrophage number found in the diaphragm of the selected group of subjects in this study is very high (97.5% of total inflammatory cells) and macrophages are known to play a key role in repair and regeneration, a study finding a correlation between internal nuclei and number of macrophages can be conducted in future. In conclusion, the findings of the current study will provide a wider picture of the inflammatory response in the human diaphragm occurring as a result of the presence of acute and/or chronic respiratory disease. Limitations of the Study There are some limitations of this study to consider when reflecting on the clinical importance of the findings of the study. These limitations may also have contributed to the inability to reach a significance level in most of the results found in the study. The 59 subjects included in the study were not representative of the population of people having respiratory diseases. The subject group of this study was an extremely selected group of people who underwent autopsy in hospitals in Vancouver, B.C. Although, the population of Vancouver is multicultural, a sample size of 59 subjects cannot represent all ethnic groups. Hence, the generalization of the results of this study requires caution. The 59 subjects were divided into four groups according to the presence or absence of respiratory disease. The subject number in each group was unevenly distributed (ARD+CRD=22; ARD=20; CRD=7; NRD=10). The subjects in the four groups were not matched for age, B M I and gender. This might have contributed to some results failing to 80 reach a significance level. The sample size of females was nearly half the size of males (F= 20, M= 39) and the males and females were not age and BMI matched, this might have masked some significant gender differences. Furthermore, in this study, control for subjects' medical conditions other than respiratory disease, smoking history, mechanical ventilation, resuscitation and use of corticosteroids was not done. These factors alone or collectively might have an impact on the diaphragm in people suffering from acute and/or chronic respiratory disease. There was a lot of problem while staining T-lymphocytes. Different protocols were tested and only 40 slides could be stained. This might have affected the T-lymphocyte count. Lastly, the inclusion and exclusion criteria of this study were not very strict. A similar study with an age matched, homogenous subject group controlled for medical conditions, smoking, mechanical ventilation, resuscitation, use of corticosteroids and with stricter inclusion and exclusion criteria is suggested for future work. 81 REFERENCES Armstrong, R.B. (1984). Mechanisms of exercise-induced delayed onset muscular soreness: a brief review. 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Group BMI Age Gender Fibre size mean % Normal diaphragm % Abnormal diaphragm % Connective tissue rN E E 2 on « •s a. 2 U ca s Macrophages/fiber Neutrophils/mm2 Neutrophils/fiber (S E E 1 w o J2 a. E >. -1 Pi tm J= IS w >. o JS a. E B5 E E js >> u o .s a. E J H T-Lymphocytes/fiber fN E E "3 b o « s s « c 'Inflammatory cells/fiber 1 C 21.4 73 F 922.89 82.72 7.88 9.40 2 A 30.7 52 M 564.07 28.52 53.77 17.71 16.3 0.07 0.0000 0.00000 0.0000 0.0000 16.3 0.07 3 AC 20.0 64 M 541.98 20.68 61.43 17.91 0.0 0.00 0.0000 0.00000 0.0000 0.0000 0 0 0.0 0.00 4 A 47.9 63 F 1012.12 79.84 9.38 10.78 10.3 0.02 0.2801 0.28011 0.84 0.0018 11.5 0.02 5 C 22.1 72 M 946.41 52.73 33.65 13.61 33.8 0.07 0.0000 0.00000 0.0000 0.0000 0 0 33.8 0.07 6 AC 84 M 1400.67 82.45 9.67 7.87 0.0 0.00 0.1401 0.14006 0.0000 0.0000 0 0 0.1 0.00 7 AC 22.0 84 F 1160.39 73.37 12.95 13.68 10.9 0.03 0.0000 0.0000 0 0 8 AC 32.5 78 F 2239.96 35.15 32.90 31.95 121.2 0.43 0.0000 0.00000 0.0000 0.0000 0 0 121.2 0.43 9 AC 30.8 64 M 1690.14 60.16 23.06 16.78 37.0 0.13 0.9804 0.9804 0.0000 0.0000 0 0 38.0 0.13 10 AC 72 F 963.17 46.49 33.61 19.90 11.4 0.15 0.4202 0.4202 0.0000 0.0000 0 0 11.9 0.15 11 AC 25.3 74 F 446.24 74.86 9.24 15.90 143.2 0.25 0.0000 0.00000 0.0000 0.0000 0 0 143.2 0.25 12 A 38.6 50 M 490.02 21.73 20.65 57.62 26.3 0.07 0.0000 0.00000 0.0000 0.0000 0 0 26.3 0.07 13 A 66 M 1386.61 79.54 14.30 6.16 16.0 0.04 0.2001 0.20008 0.0000 0.0000 0 0 16.2 0.04 14 A 24.8 84 M 2044.79 36.44 45.33 18.22 0.0 0.00 1.1534 1.1534 0.0000 0.0000 0 0 1.2 0.00 15 A 82 F 2594.58 83.46 11.80 4.74 0.0 0.00 0.0000 0.0000 0.0000 0.0000 0.0 0.00 16 A 37.5 54 M 2741.36 47.83 39.84 12.33 13.2 0.07 0.5602 0.5602 0.42 0.0019 0 0 14.1 0.07 17 AC 33.1 64 M 2435.99 31.52 47.69 20.79 282.9 0.29 18.1494 18.1494 0.0000 0.0000 301.1 0.33 18 N 27.2 85 F 1217.98 70.55 14.47 12.97 0.0 0.00 3.3613 3.3613 0.0000 0.0000 3.4 0.01 19 AC 23.6 60 M 1627.42 67.11 17.11 15.77 20 A 18.3 48 F 816.02 61.50 10.00 28.50 21 AC 36.3 69 M 2755.91 77.48 15.45 7.08 29.1 0.12 7.9365 7.9365 0.0000 0.0000 0 0 37.1 • 0.16 22 N 23.6 60 F 1352.04 83.25 11.20 5.55 26.3 0.04 0.0000 0.00000 0.0000 0.0000 26.3 0.04 23 N 24.6 51 M 1002.32 79.91 13.58 6.51 78.9 0.15 0.0000 0.00000 0.0000 0.0000 78.9 0.15 24 N 26.2 79 F 1234.61 78.66 14.94 6.40 170.2 0.30 0.0000 0.0000 0 0 25 N 84 F 840.49 73.20 5.72 21.08 2.2 0.01 0.0000 0.00000 0.0000 0.0000 0 0 2.2 0.01 26 c 76 M 1293.87 32.09 43.31 24.60 183.6 0.50 0.0000 0.00000 0.0000 0.0000 0 0 183.6 0.50 27 AC 64 F 1521.16 83.24 5.50 11.26 99.7 0.35 0.7003 0.7003 0.0000 0.0000 0 0 100.4 0.35 28 A 25.1 45 M 1320.28 77.38 10.74 11.88 49.6 0.10 0.6464 0.6464 0.0000 0.0000 50.2 0.10 29 A 21.1 86 F 1796.15 37.50 33.60 28.90 0.0 0.00 0.3112 0.3112 0.0000 0.0000 0.3 0.00 30 AC 23.0 70 M 1254.07 37.04 49.31 13.65 92.1 0.26 0.0000 0.0000 0.0000 0.0000 0 0 92.1 0.26 31 N 21.0 70 M 1857.69 79.67 11.28 9.05 9.8 0.04 0.4309 0.4309 0.0000 0.0000 0 0 10.2 0.04 32 A 30.4 78 F 650.52 49.83 26.91 23.26 15.7 0.02 1.8674 1.8674 0.0000 0.0000 0 0 17.6 0.02 33 A 25.3 43 M 1456.77 58.19 23.78 18.03 5.2 0.02 0.6225 0.6225 0.0000 0.0000 0 0 5.8 0.02 34 AC 28.4 48 M 895.22 48.61 24.25 27.15 125.9 0.20 0.0000 0.00000 0.0000 0.0000 125.9 0.20 35 A 19.0 75 M 1684.91 34.98 32.70 32.32 10.9 0.04 1.8674 1.8674 0.0000 0.0000 0 0 12.8 0.05 36 c 24.9 82 M 1456.97 23.81 44.70 31.49 1.6 0.00 4.5518 4.5518 0.0000 0.0000 0 0 6.2 0.02 37 A 69 M 1072.82 51.37 20.52 28.11 91.3 0.56 0.7003 0.7003 0.0000 0.0000 92.0 0.57 38 AC 27.3 83 M 622.07 23.28 53.90 22.82 43.6 0.09 0.0000 0.0000 0 0 39 AC 25.5 68 M 1748.22 59.35 18.70 21.95 8.2 0.03 0.9337 0.9337 0.0000 0.0000 0 0 9.1 0.03 40 AC 69 M 1437.75 16.50 52.90 30.60 28.1 0.05 0.0000 0.0000 0.0000 0.0000 0 0 28.1 0.05 41 N 25.4 49 M 1058.26 83.11 5.43 11.47 107.8 0.22 0.0000 0.0000 0.0000 0.0000 0 0 107.8 0.22 42 A 21.1 80 F 855.57 59.40 28.81 11.79 100.6 0.16 0.0000 0.0000 0.56 0.0015 0 0 101.1 0.16 43 AC 66 M 662.22 58.66 24.85 16.49 3.8 0.01 0.0000 0.0000 0.0000 0.0000 0 0 3.8 0.01 44 A 20.5 79 M 1385.69 26.13 52.58 21.29 46.1 0.15 3.0558 3.0558 0.0000 0.0000 0 0 49.1 0.17 45 A 24.0 26 M 1445.25 41.86 41.48 16.67 9.3 0.02 24.8 24.7899 0.0000 0.0000 0 0 25.6 0.08 46 C 28.0 87 M 1889.29 29.91 51.86 18.23 0.0 0.00 2.80 2.8011 0.0000 0.0000 3.0 0.01 47 C 25.1 75 M 1334.98 76.33 15.23 8.45 234.7 0.48 0.00 0.0000 0.0000 0.0000 0 0 234.7 0.48 97 Subject no. Group BMI M <; Gender Fibre size mean % Normal diaphragm % Abnormal diaphragn % Connective tissue Macrophages/mm2 Macrophages/fiber (S E S (/) Is a 2 = w Z Neutrophils/fiber M E E Vi u u O JS a. E >, -1 A B-Lymphocytes/fiber E E 1 u o JZ a. E >, -1 r- T-Lymphocytes/fiber *InfIammatorycells/mm "Inflammatory cells/fibe 48 A 25.3 76 M 1528.24 ' 37.84 46.93 15.23 180.3 0.53 0.00 0.0000 0.0000 0.0000 0 0 180.3 0.5 49 N 27.0 73 M 1277.52 77.65 18.95 3.40 1.9 0.00 0.00 0.0000 0.0000 0.0000 1.9 0.0 50 A 36.8 69 M 1024.34 41.70 42.91 15.39 144.9 0.51 1.8 0.0050 0.0000 0.0000 146.7 0.5 51 N 27.1 83 F 877.18 54.69 25.71 19.60 227.4 0.46 1.2 0.0029 0.0000 0.0000 0 0 228.7 0.5 52 N 15.8 52 F 1143.03 68.69 22.29 9.03 141.6 0.39 0.0000 0.0000 0.0000 0.0000 0 0 141.6 0.4 53 AC 24.1 65 M 1354.40 47.26 38.29 14.44 7.5 0.02 0.0000 0.0000 0.0000 0.0000 0 0 7.5 0.0 54 AC 25.9 46 M 1263.95 74.27 22.22 3.51 38.8 0.13 0.0000 0.0000 0 0 55 AC 24.9 73 M 53.52 39.93 6.56 68.6 0.25 0.0000 0.0000 0.0000 0.0000 0 0 68.6 0.2 56 AC 27.3 55 F 771.28 55.10 29.42 15.48 216.8 0.48 0.0000 0.0000 0.0000 0.0000 0 0 216.8 0.5 57 A 24.8 82 M 80.51 13.64 5.85 43.2 0.12 0.0000 0.0000 0.0000 0.0000 43.2 0.1 58 c 17.3 60 F 1332.60 55.19 36.75 8.06 6.5 0.02 0.0000 0.0000 0.0000 0.0000 6.5 0.0 59 AC 29.3 68 M 1404.00 57.54 37.01 5.45 130.7 0.42 0.42 0.0012 0.0000 0.0000 0 0 131.1 0.4 N 59 49 59 59 57 59 59 59 56 56.00 52 52 56 56 40 40 52 52 Mean 26.3 68 1317.62 56.29 27.39 16.28 62.6 0.16 1.54 0.00 . 0.03 0.0001 0 0 63.9 0.16 Stdev 6 13 537.57 20.37 15.41 9.58 72.6 0.17 4.32 0.01 0.14 0.0004 0 0 74.7 0.18 Max 47.9 87 2755.91 83.46 61.43 57.62 282.9 0.56 24.79 0.04 0.84 0.0019 0 0 301.1 0.57 Min 15.8 26 446.24 16.50 5.43 3.40 0.0 0.00 0.00 0.00 0.00 0.0000 0 0 0.0 0.00 1 Abbreviations: A- Acute respiratory disease, AC- Acute and chronic respiratory disease, C- Chronic respiratory disease, | | |F-Females, M-Males, N-No respiratory disease * Inflammatory cells= number of macrophages+neutrophils+B-cells Appendix II Clinical Characteristics of Postmortem Subjects 99 Subject no. Respiratory group BMI Age |Gender | Smoking history Mechanical ventilation Resuscitation Cause of Death Differential diagnosis 1 C 21.4 73 F NA NA Yes Acute MI Cardiac arrest 2 A 30.7 52 M Yes NA Yes Cardiac arrest Mitral valve regurgitation 3 AC 20.0 64 M Yes NA No Severe acute pancreatitis Sepsis, abdominal pain 4 A 47.9 63 F No Yes No Respiratory diseases Post operative pulmonary thromboembolism 5 C 22.1 72 M Yes NA Yes Acute MI - due to CAD Acute MI 6 AC NA 84 M Yes No No Acute myocardial infarct Acute MI 7 AC 22.0 84 F No Yes Yes Cardiac arrest Aortic valve stenosis, CAD 8 AC 32.5 78 F Yes NA NA Multiple factors Rectal mass, abdominal pain, hypotension 9 AC 30.8 64 M Yes Yes Yes Recent MI Unstable angina 10 AC NA 72 F Yes No No NA Acute pancreatitis 11 AC 25.3 74 F NA Yes NA Not identified Cardiogenic shock 12 A 38.6 50 M No Yes No Acute fulminant viral hepatitis Renal transplant 13 A NA 66 M Yes Yes Yes Severe coronary atherosclerosis, acute MI Post cardiac arrest 14 A 24.8 84 M No NA NA b/1 bronchopneumonia Pneumonia, respiratory distress 15 A NA 82 F No No Yes Multifactorial (no pinpointing) Hematoma 16 A 37.5 54 M Yes Yes Yes Aspiration pneumonia Post bypass recurrent renal stones 17 AC 33.1 64 M Yes Yes Yes Respiratory failure Pneumonia 18 N 27.2 85 F No No No Cardiac (severe triple disease) Post-herpatic neuralgia 19 AC 23.6 60 M Yes Yes NA Pneumonia, COPD, toxic colitis NA 20 A 18.3 48 F NA No No Severe b/l pneumonia diabetic ketoacidosis 21 AC 36.3 69 M NA Yes NA Multiorgan failure Double bypass angina 22 N 23.6 60 F NA NA NA Acute massive small intestine infarction NA 23 N 24.6 51 M NA NA NA Cardiac arrest NA 24 N 26.2 79 F No NA NA Arrythmia - cardiac arrest NA 25 N NA 84 F NA NA NA Ruptured abdominal aortic aneurysm Syncopal episode 26 c NA 76 M NA NA NA Abdominal aortic aneurysm NA 27 AC NA 64 F NA NA NA Pulmonary fibrosis, aspiration pneumonia Idiopathic pulmonary fibrosis 28 A 25.1 45 M NA Yes NA Subaracnoid bleed, CVA Subarachnoid hemorrhage 29 A 21.1 86 F NA NA No Cardiac related Abdominal pain 30 AC 23.0 70 M NA NA NA COPD, pulmonary edema, aspiration pneumonia CHF, COPD 31 N 21.0 70 M Yes NA Yes Bradycardiac collapse NA 32 A 30.4 78 F NA Yes NA Peritoneal carcinomatosis, shock NA 33 A 25.3 43 M NA NA No Respiratory failure NA 34 AC 28.4 48 M NA NA NA Necrotizing septic fascitis, CHF' . Chills with b/l leg ulceration 35 A 19.0 75 M NA No NA Acute GI bleed NA 36 C 24.9 82 M NA NA NA Cardiac arrythmia, rheumatic heart disease NA 37 A NA 69 M NA NA Yes Cardiac arrest NA 38 AC 27.3 83 M NA NA NA Severe coronary atherosclerosis with extensive MI NA 39 AC 25.5 68 M NA NA NA Respiratory failure NA 40 AC NA 69 M Yes Yes NA Exacerbation of COPD Idiopathic 41 N NA 49 M NA NA NA Grand mal seizure, hypersensitivity to drugs RF, ischemic cardiomyopathy 42 A 21.1 80 F NA Yes NA Acute MI, CHF NA 43 AC NA 66 M NA NA NA Esophageal bleed NA 44 A 20.5 79 M NA NA NA CHF NA 45 A 24.0 26 M NA NA Yes Aspiration pneumonia NA 46 C 28.0 87 M No No No Cardiac rupture NA 47 C 25.1 75 M Yes Yes Yes Cardiac arrythmia, pulmonary edema, aspiration pneumon CAD, aortic valvular stenosis 48 A 25.3 76 M NA Yes NA ARDS, b/l pneumonia NA 49 N 27.0 73 M NA NA NA Acute MI NA 50 A 36.8 69 M NA Yes Yes Sepsis NA 51 N 27.1 83 F NA NA NA Pulmonary emboli, PVD NA 52 N 15.8 52 F NA Yes NA Multisystem organ failure, E.coli sepsis Sepsis (E. coli) 53 AC 24.1 65 M NA NA NA Brain & spinal cord cancer NA 54 AC 25.9 46 M NA Yes Yes Cardiac arrest+RF d/t bronchiolitis obliterans NA 55 AC 24.9 73 M NA NA NA Pneumonia, multiple myeloma, RF with ARDS NA 100 tion dno. t ntila BO o *-> > s t V) _o c o -*-» a La 61 c 'S M Subjec Respir BMI Age Gende Smokii Mecha Resusc Cause of Death Differential diagnosis 56 A C 27.3 55 F Yes N A N A RF, multiorgan failure secondary to sepsis N A 57 A 24.8 82 M Yes N A N A Multiple aortic aneurysms N A 58 C 17.3 60 F Yes N A N A N A N A 59 A C 29.3 68 M N A Yes No Cardiac arrest N A Abbreviations: A- Acute respiratory disease, A C - Acute and chronic respiratory disease, ARDS- Adult respiratory distress syndrome, b/1- bilateral, C- Chronic respiratory disease, CAD-Coronary artery disease, CHF- congestive heart failure, COPD- Chronic obstructive pulmonary disease, C V A - Cardiovascular accident, d/t - due to, GI- gastro -intestinal, MI- Myocardial ischemia, N- No respiratory disease, NA- Not available, PVD- Peripheral vascular disease, RF - respiratory failure 

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