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Diaphragm injury in chronic respiratory disease MacGowan, Nori A. 1998

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DIAPHRAGM INJURY IN CHRONIC RESPIRATORY DISEASE by NORI A. MACGOWAN B.Comm.(Hons.), Queen's University, 1987 B.Sc.(P.T.), The University of Western Ontario, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES School of Rehabilitation Sciences We accept this thesis as confonning to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1998 © Nori A. MacGowan, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Diaphragm injury may occur in chronic respiratory disease due to factors which increase the workload, cause weakness, or reduce the efficiency of the ventilatory pump. Diaphragm injury has been shown in many animal models of respiratory overload, but similar research in humans is limited. The purpose of this thesis was to investigate diaphragm injury in individuals who may experience diaphragm overload due to chronic respiratory disease. Diaphragm biopsies were obtained from twenty-one individuals going for thoracotomy surgery, and from thirty-three individuals post-mortem. In the thoracotomy study, biopsies were quick frozen (n=18) or formalin-fixed (n=3). Thick sections (10 pm) were stained with H & E. Macrophages were identified in thin sections (6 pm) using immunohistochemistry and monoclonal antibodies (Ber-MAC3, DAKO Canada Corp.). In the post-mortem study, biopsies were fixed in 10% formalin, then stained with H & E and Masson's trichrome. Area fractions of normal muscle, abnormal muscle and connective tissue were determined by point counting diaphragm cross-sections from both thoracotomy and post-mortem subjects. Image analysis was used to determine the number of macrophages in diaphragm cross-sections from thoracotomy subjects. Subjects in the thoracotomy study had airflow obstruction ranging from mild to severe, and had relatively homogeneous clinical characteristics. This study showed an inverse relationship between the proportion of abnormal muscle and the % predicted FEVr (r = -0.53, p < 0.01), and a direct relationship between the proportion of normal muscle and the % predicted F E V i (r = 0.37, p < 0.05). There was no relationship between the number of macrophages and the % predicted F E V i . Subjects in the post-mortem study were heterogeneous in their clinical characteristics. This study showed a trend towards a direct relationship between the proportion of abnormal muscle and the presence of chronic respiratory disease (p=.065), and no relationship between the proportion of abnormal muscle and other clinical factors (gender, age, body mass index, presence of acute Ill respiratory disease). We conclude from the first study that abnormal morphology is present in the diaphragm and is related to the severity of airflow obstruction in a fairly homogeneous sample of individuals going for thoracotomy surgery. We conclude from the second study that further study with a larger sample of individuals who have died of various causes may provide evidence of a relationship between abnormal diaphragm morphology and the presence of chronic respiratory disease. iv TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgment x Chapter 1 Diaphragm Injury In Chronic Respiratory Disease 1 Chronic Respiratory Disease 2 Acute Respiratory and Systemic Conditions 11 The Diaphragm 14 Diaphragm Dysfunction and Injury in Chronic Respiratory Disease 27 Mechanisms of Overload-Induced Diaphragm Injury 41 Research Hypotheses 47 Research Objectives 48 Chapter 2 Diaphragm Injury in Individuals with Airflow Obstruction Going for Thoracotomy Surgery Abstract 49 Introduction 50 Methods 52 Study Design 62 Statistical Analysis 63 Results 63 Discussion 74 Chapter 3 Post-Mortem Evaluation of Diaphragm Injury Abstract 81 Introduction 82 Methods 83 Study Design 90 Statistical Analysis 90 Results 91 Discussion 109 Chapter 4 Summary and Recommendations Comparison of the Two Research Studies 115 Summary 118 Recommendations 119 References 120 Appendices A. Informed Consent Form for Thoracotomy Subjects 131-3 B. Protocol for Staining Diaphragm Muscle Sections with Hematoxylin and Eosin 134 C. Protocol for Irmnvmohistochemistry (APAAP technique) to Identify Macrophages in Diaphragm Muscle Sections of Thoracotomy Subjects 135-6 D. Protocol for Counterstaining Diaphragm Muscle Sections using Meyer's Hematoxylin 137 E. Data Collection Sheet for Chart Review of Thoracotomy Subjects 138-9 F. Reference Equations for Determination of % predicted FEVi for Thoracotomy Subjects 140 G. Autopsy Consent Form (St. Paul's Hospital) 141 H. Autopsy Consent Form (Vancouver Hospital, Oak St. Site) 142 I. Data Collection Sheet for Chart Review of Post-Mortem Subjects 143-4 J. Detailed Clinical Characteristics of Post-Mortem Subjects 145-51 vi LIST OF TABLES 1. Categories and Definitions for Point Counting (Thoracotomy Study) 57 2. Equations to Calculate Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue in Thoracotomy Subjects 59 3. Descriptive Characteristics of Thoracotomy Subjects 64 4. Point Counting Results (Thoracotomy Study) 70 5. Number of Macrophages per mm2 and per Fibre in Diaphragm of Thoracotomy Subjects 72 6. Categories and Definitions for Point Counting (Post-Mortem Study) 86 7. Equations to Calculate Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue in Post-Mortem Subjects 89 8. Descriptive Characteristics of Post-Mortem Subjects 92-3 9. Point Counting Results (Post-Mortem Study) 103-4 LIST OF FIGURES 1. Gross anatomy of the respiratory muscles. 15 2. The diaphragm muscle in vivo (inferior view). 17 3. Photomicrograph of normal human diaphragm morphology. 19 4. Model of diaphragm overload and injury in chronic respiratory disease. 45-6 5. Photomicrographs of abnormal morphology in H & E stained human diaphragm cross-sections (thoracotomy study). 66 6. Photomicrographs of abnormal morphology in H & E stained human diaphragm cross-sections (thoracotomy study). 67 7. Photomicrograph of macrophages in human diaphragm cross-section (thoracotomy study). 68 8. Relationship between airflow obstruction and proportion of normal and abnormal diaphragm muscle (thoracotomy study). 71 9. Relationship between airflow obstruction and prevalence of macrophages in diaphragm (thoracotomy study). 73 10. Photomicrograph of normal human diaphragm morphology (post-mortem study). 97 11. Photomicrograph of abnormal morphology in human diaphragm (post-mortem study). 99 12. Photomicrograph of abnormal morphology in human diaphragm (post-mortem study). 100 13. Photomicrograph of abnormal morphology in human diaphragm (post-mortem study). 101 14. Boxplots showing relationships between proportion of abnormal diaphragm muscle and chronic respiratory disease, acute respiratory disease, and gender (post-mortem study). 106-7 15. Scatterplots showing relationship between proportion of abnormal diaphragm muscle and age and body mass index (post-mortem study). 108 viii L I S T O F A B B R E V I A T I O N S APAAP alkaline phosphatase anti-alkaline phosphatase ARDS adult respiratory distress syndrome ATS American Thoracic Society BMI body mass index BSA bovine serum albumin CF cystic fibrosis CHF congestive heart failure CO carbon monoxide COPD chronic obstructive pulmonary disease DLCO diffusing capacity of carbon monoxide DOMS delayed onset muscle soreness F E V i forced expiratory volume in one second FRC functional residual capacity F V C forced vital capacity H & E hematoxylin and eosin IRL inspiratory resistive loading M IP maximum inspiratory pressure PaC02 partial pressure of carbon dioxide in arterial blood P a 0 2 partial pressure of oxygen in arterial blood Pbreath inspiratory pressure during tidal breathing Pdi transdiaphragmatic pressure Pes esophageal pressure ix P g a gastric pressure PTI pressure-time index RV residual volume SIDS sudden infant death syndrome Tj inspiratory time TBS Tris-buffered saline T t o t total time for one breathing cycle TLC total lung capacity X ACKNOWLEDGMENT I would like to acknowledge the guidance and assistance I received from my supervisor, Dr: W. Darlene Reid, during my graduate work. By working with such a hard-working, fair, enthusiastic and motivated individual, I learned many research skills and I learned to enjoy and respect the research process. I truly appreciated her commitment to her work, and the time she invested in my learning exerience. I would also like to acknowledge the advice and support I received from my committee members, Drs. Jeremy Road, Darlene Redenbach and Donna Maclntyre. Throughout my program, their willingness to meet with me, to discuss ideas, and to provide feedback was wonderful. I would also like to thank the faculty and staff of the School of Rehabilitation Sciences at U.B.C. for fostering an environment which not only promotes higher learning, but which also encourages participation in activities which broaden the student's perspective on education, practice and research in physical therapy. Finally, I appreciate and acknowledge the financial support I received from The Physiotherapy Foundation of Canada and the Medical Research Services Foundation of British Columbia during my graduate studies. 1 CHAPTER 1: DIAPHRAGM INJURY IN CHRONIC RESPIRATORY DISEASE Chronic respiratory disease challenges the respiratory muscles by imposing increased ventilatory demands and/or by reducing the ability of the respiratory muscles to meet existing ventilatory demands. If inspiratory demands exceed inspiratory capacity, this may result in an inspiratory muscle overload which, if significant or sustained enough, may lead to inspiratory muscle dysfunction and/or injury. Possible clinical consequences of inspiratory muscle dysfunction or injury include dyspnea (breathlessness), exercise intolerance and respiratory failure. The extent to which the structure and function of the inspiratory muscles are impaired by ventilatory overload has been the focus of many animal studies and a few studies with humans who experience overload due to chronic or acute respiratory disease. Animal research has shown that diaphragm injury occurs in response to acute and chronic ventilatory loads. Moreover, evidence indicates that diaphragm injury is associated with functional impairments including reduced inspiratory force (Jiang et al., 1998b) and ventilatory failure (Reid et al., 1994). Human studies provide promising indications that diaphragm injury may occur in association with acute or chronic ventilatory loads (for review, see Reid & MacGowan, 1998). However, there remains a clear need for additional investigation in this area of research. A better understanding of the nature and prevalence of diaphragm injury in individuals with chronic respiratory disease may indicate important directions for future research concerning pharmaceutical interventions, respiratory muscle rest and training regimens, and protocols for weaning from mechanical ventilation. The purpose of the introductory chapter of this thesis is to review background information which is relevant to the two research studies presented in Chapters 2 and 3. The foUowing will be reviewed in this chapter: 2 1. pathophysiology of chronic respiratory diseases and acute respiratory or systemic conditions which are associated with diaphragm overload; 2. diaphragm anatomy, physiology and response to overload; 3. evidence and mechanisms of diaphragm injury in animal models and in humans; 4. additional factors which may contribute to diaphragm injury including aging, malnutrition, and corticosteroids. CHRONIC RESPIRATORY DISEASE Chronic respiratory diseases which increase the work of breathing or which cause respiratory muscle weakness or inefficiency include chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and restrictive lung or chest wall disease. The clinical features of these diseases and the nature of the challenges they impose upon the inspiratory muscles will be reviewed in this section. Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disease (COPD) is a progressive lung disease which has a significant impact upon the health care system in industrialized nations (Pride & Burrows, 1995). The American Thoracic Society (ATS) estimates that COPD afflicts approximately 14 million individuals in the United States, where it is the fifth most common cause of death (McFadden, 1991). In 1991, over 185,000 days were spent in Canadian hospitals as a result of COPD and over 8,147 Canadians died as a result of COPD, amounting to estimated costs of $62.2 million for the Canadian health care system (Canadian Lung Association, 1993). Mortality statistics and financial statements, however, capture only a portion of the significance of the impact of COPD 3 on our society. COPD has been associated with various impairments (airflow limitation, respiratory muscle dysfunction, right ventricular dysfunction, depression, anxiety, panic), with disability (ventuatoiy-limited decreased exercise capacity), and with handicap (decreased quality of life) (Stubbing, 1996). COPD is defined by the ATS as the presence of airflow obstruction which is generally progressive and which may be accompanied by partially reversible hyperreactivity of the airways (American Thoracic Society, 1995). Chronic bronchitis and emphysema are included within the definition of COPD. Asthma is an obstructive disease which is classified as COPD in individuals with chronic unremitting airflow obstruction (Jeffrey, 1994). Asthmatics who experience primarily acute reversible episodes of airflow obstruction, however, are usually classified under a separate pathology. Cystic fibrosis is a chronic obstructive disease but its pathology and pathophysiology are unique. For this reason, it is typically not classified as COPD. Airflow obstruction occurs in COPD as a result of increased airflow resistance secondary to a decrease in airway diameter or a loss of support for the airway wall. Unlike in healthy individuals, where airflow resistance is most significant in the upper airways, the airflow resistance in COPD is most significant in the small airways (American Thoracic Society, 1995). Chronic bronchitis is defined as the existence of a daily productive cough which persists for at least three months per year for two consecutive years (American Thoracic Society, 1995). In this condition, inflammation, smooth muscle hypertrophy, edema, bronchospasm and mucus plugs all may contribute to a decrease in airway diameter and a resultant increase in airflow resistance. Emphysema is a disease of the gas-exchanging regions of the lungs (respiratory bronchioles and alveoli), where airspaces become permanently enlarged due to destruction of the alveolar walls (American Thoracic Society, 1995). The loss of alveolar tethering reduces airway wall stability, leading to premature airway closure during expiration (dynamic airways 4 compression), air trapping and chest hyperinflation (Reid & Dechman, 1995). Centrilobular emphysema, which selectively involves the respiratory bronchioles and leaves the peripheral portion of the acinus relatively intact, affects primarily the upper lung zones and is most common in smokers (American Thoracic Society, 1995). Panacinar emphysema involves the entire acinus and affects primarily the lower lung zones. A rare and severe type of panacinar emphysema results from a genetic absence of a i -antitrypsin and develops at a relatively early age (4th or 5th decade) (Thurlbeck, 1990). Bronchial asthma is characterized by obstructed airflow resulting from widespread airway narrowing (bronchoconstriction) associated with increased airway responsiveness to a variety of stimuli (McFadden, 1991). The airflow obstruction associated with bronchial asthma may reverse spontaneously or following drug therapy (Clausen, 1990). In more severe asthma, chronic inflammation and thickening of the airway wall may contribute to airflow obstruction. The signs and symptoms, onset and progression of airflow obstruction vary considerably amongst individuals with COPD (Thurlbeck, 1991). Associated clinical signs and symptoms include cough, wheezing, dyspnea, frequent acute respiratory illnesses, altered breathing patterns and ventnatoiy-limited exercise intolerance (Thurlbeck, 1991). Individuals with COPD may develop poor arterial blood gases (hypercapnia with or without hypoxemia), which eventually can lead to respiratory failure (defined by PaC02 > 45 mm Hg or Pa02 < 55 mm Hg) and oxygen dependence. Emphysema and chronic bronchitis are typically characterized by a gradual onset and steady progression of symptoms (Clausen, 1990). In contrast, bronchial asthma is characterized by acute episodes of airflow obstruction with a variable progression and severity of symptoms (Jeffrey, 1994). This is an important distinction because the effects of airflow obstruction and the adaptive responses of the respiratory muscles in individuals with reversible 5 bronchial asthma may differ from those observed in individuals with unremitting chronic airflow obstruction (Jeffrey, 1994). Pulmonary function tests are used to assess the severity of COPD (American Thoracic Society, 1995). They typically include measures of forced expiratory volumes, lung capacities and diffusing capacity (gas transfer). The forced vital capacity (FVC), which is the volume of air forcibly expired following an inspiration to total lung capacity, is decreased in COPD. The volume expired during the first second (FEVi), however, is decreased to a much greater extent and is used as the gold standard measure of airflow obstruction in COPD (Ruppel, 1994). FEVi, FVC and FEVi/FVC are usually represented as a percentage of predicted, based upon reference equations which incorporate age, gender and height as independent variables (Crapo et al., 1981; Morris et al., 1971) and which have been established by studies with large numbers of healthy individuals (American Thoracic Society, 1995). The American Thoracic Society has recommended the use of the following stages to describe disease severity in COPD: I. FEVi > 50% predicted; JJ. FEVi =35 to 49% predicted; HI. FEVi <35% predicted. The FEVi/FVC ratio is another useful measure to describe the airflow obstruction, and to differentiate between obstructive and restrictive disease (see later section). Pre-and post-bronchodilator expiratory airflow measures indicate the reversibility of airflow obstruction. Expiratory airflow obstruction leads to air trapping and to increases in total lung capacity (TLC), functional residual capacity (FRC), and residual volume (RV) (Rochester, 1991c). Compared to young healthy individuals whose RV and FRC are approximately 25% and 50% of TLC respectively, RV and FRC in individuals with COPD may be elevated to 70% and 85% of TLC respectively (Rochester, 1991c). Increases in FRC and RV reduce inspiratory and expiratory reserve volumes, and vital capacity. 6 Carbon monoxide diffusing capacity (DLco) is a measure of the effectiveness of gas exchange across the alveolar-capillary interface (Forster et al., 1986). To test DLCO, a low concentration of carbon monoxide (CO) is added to the inspired air either during steady-state breathing or prior to a single 10-second breath-hold. Diffusion impairment in emphysema primarily results from a reduction in the alveolar-capillary interface area (American Thoracic Society, 1995). In other COPD conditions, diffusion may be impaired because of a reduction in functional surface area, a change in the pressure gradient associated with ventilation-perfusion inequality, or a thickened alveolar-capillary membrane (Forster et al., 1986). Since DLCO remains relatively normal in individuals with bronchial asthma (Forster et al., 1986), this test may be useful in differentiating between asthma and other COPD conditions. Although the hallmark of COPD is a limitation in expiratory airflow, the inspiratory not the expiratory muscles are most affected by the mechanical and metabolic factors associated with this condition. Since the diaphragm is the primary muscle of inspiration, the specific effects upon the diaphragm will be the primary focus of this section. Hyperinflation, resulting from air trapping in the distal airways, reduces the efficiency of inspiratory muscle mechanics (Goldberg & Roussos, 1990). As lung volumes increase, the diaphragm becomes flatter and shortened, placing it at a mechanical disadvantage and reducing its ability to generate inspiratory pressures (Rochester, 1991c). In acute hyperinflation, the diaphragm must function at a less than optimal point on its length-tension curve. During chronic hyperinflation, however, a hamster model of elastase-induced emphysema has shown the diaphragm adapts by reducing the number of sarcomeres, thus shifting the length-tension curve to the left and preserving diaphragm fibre force-generating ability (Farkas & Roussos, 1983; Supinski & Kelsen, 1982). Sarcomere number adaptation has yet to be demonstrated in the diaphragm of humans with chronic hyperinflation, possibly because of hmitations in current technology. Hyperinflation also affects diaphragm 7 mechanics by reducing the size of the zone of apposition. Since contraction of diaphragm fibres in the zone of apposition causes the upward and outward movement of the chest wall, a smaller zone of apposition may result in reduced chest wall expansion. In severe hyperinflation, the zone of apposition may be eHminated and diaphragm contraction may result in inward chest wall movement and expiration (Goldberg & Roussos, 1990). Finally, diaphragm efficiency is reduced in hyperinflation because of the decreased lung and chest wall compliance associated with higher lung volumes (Rochester, 1991c). Altered breathing patterns in COPD contribute to inspiratory muscle overload by increasing the inspiratory workload and predisposing the inspiratory muscles to fatigue. A useful index of overload is the pressure-time index (PTI). PTI is defined as the product of inspiratory pressure during tidal breathing (Pbreath) over maximum inspiratory pressure (MD?), and inspiratory time (Ti) over total time (Ttot) (Rochester, 1991c) (see below). PTI = Pbreath y Ti MIP Ttot where, PTI = pressure-time index Pbreath = inspiratory pressure (tidal breathing) MIP = maximum inspiratory pressure Ti time to inspire (tidal breathing) Ttot = total time for one breathing cycle (tidal breathing) PTI is closely correlated with energy expenditure. To compensate for an increase in airflow resistance, individuals with COPD reduce Ti, adopting a rapid shallow breathing pattern (Rochester, 1991b). Reducing Ti may represent an effort to decrease the oxygen cost of breathing in order to prevent respiratory muscle failure due to fatigue (Rochester, 1991c). However, a reduced Ti increases the required Pbreath/MTP ratio, which increases both the perceived effort of breathing and the actual inspiratory load. As a result, individuals with COPD have PTI 8 values which may be ten to twenty times greater than normal (Rochester, 199 lc). In healthy individuals or in individuals with COPD, the inspiratory muscles have been hypothesized to be prone to fatigue if PTI exceeds the 'Tatigue threshold" of 0.15 (Rochester, 1991c). Cystic Fibrosis Cystic fibrosis (CF) is a hereditary disease which is characterized by pulmonary and pancreatic abnormalities. The primary pathology in CF is duct or airway obstruction resulting from abnormally thickened mucus. Duct obstruction reduces pancreatic enzyme secretion to the gastrointestinal tract, causing malabsorption, poor appetite, failure to thrive, intestinal obstruction, abdominal pain and nutritional deficiencies (Cotton, 1989). Thick mucus which blocks airways and reduces the effectiveness of the mucociliary transport mechanism (Flkschmann & Murray, 1987), resulting in small airways obstruction, recurrent chest infections, bronchiectasis, atelectasis, cor pulmonale and ventilation-perfiision mismatching (Cotton, 1989). Clinical signs are few in infancy, but progress through childhood, adolescence and adulthood (Cotton, 1989; Hodson, 1989a). While cystic fibrosis affects most organ systems in the body, lung pathology accounts for 95% of the morbidity and mortality associated with CF (for review, see Zamora & Anzueto, 1992). Pulmonary function in cystic fibrosis is measured using the same tests as those previously described for COPD. Airflow obstruction due to mucus plugging, bronchospasm and/or edema causes reduced expiratory airflow measures including a reduction in the percentage of predicted F E V i (% predicted F E V i ) (Ruppel, 1994). Since 60 to 80 % of individuals with cystic fibrosis have airway hyperreactivity (Cotton, 1989), pre- and post-bronchodilator % predicted F E V i and % predicted FVC are important measures for treatment decisions (Ruppel, 1994). Pre- and post-9 chest physical therapy expiratory airflow measures are useful in monitoring the effectiveness of secretion removal techniques (RuppeL 1994). mdividuals with CF may be predisposed to inspiratory muscle overload due to factors which cause respiratory muscle weakness (malnutrition), increase ventilatory loading (chronic airflow obstruction) and/or result in inefficiency (hyperinflation). To the investigators' knowledge, there have been no detailed studies documenting abnormal diaphragm morphology in individuals with CF. From a functional perspective, inspiratory strength and endurance are often preserved in these individuals, likely because the ventilatory challenges serve as a training stimulus (Lands et al., 1993). Lands and co-workers showed that expiratory muscle strength is related to leg strength and lean body mass, but inspiratory muscle strength is related to neither (Lands et al., 1993). This suggests that there is selective preservation of inspiratory strength in individuals with CF (Lands et al., 1993). Inspiratory muscle strength in CF is not related to diaphragm thickness (Vlachos-Mayer et al., 1996). It is possible that the increased diaphragm thickness observed in some individuals with CF may reflect an increased amount of non-contractile connective tissue, rather than an increase or a preservation of contractile tissue. Vlachos-Mayer and co-workers suggest that preservation of inspiratory muscle strength in adults with CF may reflect improved interaction between the inspiratory muscles and the chest wall (Vlachos-Mayer et al., 1996). Restrictive Respiratory Disease Restrictive disease is characterized by reduced lung volumes which occur as a result of increased stiffness of the lung parenchyma or chest wall (West, 1992). Restrictive disease differs from obstructive disease in that all lung volumes are reduced. However, if restriction is due to muscle weakness, then RV may be increased due to expiratory muscle weakness. Individuals with 10 restrictive conditions have a reduced % predicted FEVi and a proportionate decrease in % predicted FVC. Thus their % predicted FEVi/FVC ratio is relatively normal (Ruppel, 1994). Chest wall pathology associated with restrictive disease refers to pathology which affects the bony skeleton and soft tissues including the respiratory muscles and their nerve supply. In kyphoscoliosis and ankylosing spondylosis, thoracic cage articulations are stiffer, decreasing the efficiency of inspiratory muscle contraction (Reid & Dechman, 1995). In obesity, inspiratory muscle inefficiency occurs because the increased adipose mass increases the energy required to mobilize the chest wall (Poole et al., 1997). Neuromuscular disease may impair respiratory muscle contractility through various mechanisms. These include muscle pathology (e.g. myopathic disease), neuromuscular junction pathology (e.g. myasthenia gravis), and nerve supply disruption (e.g. amyotrophic lateral sclerosis, pohomyelitis, Guillain-Barre' Syndrome, spinal cord injury or cerebral lesions) (Cumming et al., 1994; Dean, 1997). Restrictive disease resulting from lung pathology includes conditions where there is a loss of distensible lung tissue due to tissue destruction (lung resection), space-occupying lesions (tumour), or reduced elasticity of the lung parenchyma (interstitial fibrosis, sarcoidosis, asbestosis, radiation damage) (Ruppel, 1994). In restrictive disorders, the respiratory muscles may be challenged in various ways, depending upon the nature of the associated pathology. Decreased compliance and inefficient chest wall mechanics increase inspiratory workloads. Inspiratory muscle weakness or fatigue decreases their ability to handle ventilatory demands. Factors which may cause or exacerbate weakness or fatigue include metabolic abnormalities (hypoxia, hypercapnia, hypophosphatemia, hypomagnesemia, hypokalemia) (Reid, 1995), malnutrition (Arora & Rochester, 1982), steroid-related myopathy (Gallagher, 1994; Dekhuijzen & Decramer, 1992), and disuse (Rochester, 1993). 11 ACUTE RESPIRATORY AND SYSTEMIC CONDITIONS Acute respiratory and systemic conditions which may initiate or intensify inspiratory muscle loading, or which may predispose to inspiratory muscle fatigue or weakness include pneumonia, acute pulmonary edema, adult respiratory distress syndrome (ARDS), and sepsis. The clinical features of these conditions and the nature of their associated inspiratory muscle challenges will be reviewed in this section. Pneumonia Pneumonia refers to an inflammation in the parenchyma of the lung (Hirschmann & Murray, 1987) which is usually caused from infection with a microorganism The inflammation may be located within the alveoli {alveolar pneumonia), in the conducting airways (bronchopneumonia), or in the alveolar septa (interstitial pneumonia) (Hirschmann & Murray, 1987; Damjanov, 1996). Clinical signs and symptoms include fever, dyspnea, tachypnea, pleuritic chest pain, cough and production of sputum (Hhschmann & Murray, 1987). In alveolar pneumonia, alveolar air is replaced by exudate, causing the lung parenchyma to become consolidated. Consolidation is visible as dense pulmonary infiltrates on chest x-rays (Damjanov, 1996). In interstitial pneumonia, chest x-rays reveal a reticular appearance, which is reflective of alveolar septal inflammation. When there is no underlying cardiopulmonary disease, pneumonia causes impaired gas exchange and arterial blood gas abnormalities characterized by hypoxemia (Pa02 < 80 to 100 mm Hg) due to right-to-left shunting (Hirschmann & Murray, 1987). Individuals with pre-existing cardiopulmonary disease are more susceptible to pneumonia (Hirschmann & Murray, 1987) and to the development of hypercapnia (PaC02 > 35 to 45 mm Hg) in addition to hypoxemia (Hodson, 1989b). Persistent or repetitive bouts of interstitial 12 pneumonia may result in permanent alveolar damage, characterized by interstitial fibrosis (Damjanov, 1996) and an associated restrictive deficit. Respiratory muscles in pneumonia are challenged by increased ventilatory demands due to an increased respiratory rate (tachypnea) and by decreased efficiency due to reduced lung compliance. They are also challenged by a metabolic load characterized by an increase in the oxygen cost of breathing (Field et al., 1982) coupled with a reduction in oxygen supply (due to a reduction in PaO )^. Acute Pulmonary Edema Acute pulmonary edema increases small airway resistance and decreases lung compliance (Ingram & Braunwald, 1987). Alveolar fluid impairs gas exchange, resulting in hypoxemia and possibly hypercapnia (Braunwald, 1987). Respiratory muscle challenges may be related to an elevated respiratory rate (increased activity requirements), reduced lung compliance (increased force requirements) and/or metabolic challenges associated with impaired gas exchange. Furthermore, if the edema is due to a reduced cardiac output, such as occurs in congestive heart failure (CFJF), respiratory muscle strength or endurance may decrease due to reductions in respiratory muscle blood flow (McParland et al., 1995). McParland and co-workers showed that inspiratory muscle weakness was selectively reduced in individuals with CHF, independent of nutritional or electrolyte abnormalities (McParland et al., 1995). Adult Respiratory Distress Syndrome Adult respiratory distress syndrome (ARDS) is an acute condition where there is widespread alveolar and/or pulmonary capillary membrane injury, resulting in increased capillary permeability, interstitial and pulmonary edema, atelectasis, and fibrosis of the lung parenchyma 13 (Morris, 1992). ARDS is characterized by impaired ventilation, impaired diffusion, and progressive hypoxemia (Morris, 1992). Mortality is approximately 50% and it is most often caused by multiple organ failure rather than respiratory failure alone (Dorinsky & Gadek, 1990). Early clinical manifestations of ARDS include dyspnea, tachycardia, restlessness, and a rapid shallow breathing pattern with accessory muscle use (Morris, 1992). The work of breathing increases with the decreased lung compliance, eventually becoming exhaustive and resulting in (hypercapnic) respiratory failure (Morris, 1992). Inspiratory muscles are challenged by increased ventilatory demands (tachypnea), poor arterial blood gases, and decreased lung compliance associated with ARDS. Inspiratory muscle effectiveness may be further impaired by weakness resulting from increased nutritional requirements due to a catabolic state (Morris, 1992). In individuals who survive ARDS, lung function may return to normal within four to six months, however some individuals will be left with fibrosis (Ingram & Braunwald, 1987) and a restrictive defect. Sepsis and Septic Shock Sepsis refers to the systemic response to severe infection (Bone, 1996). It is a complex process in which the body's inflammatory response is initiated. Clinically, sepsis progresses from fever, leukocytosis and tachycardia to shock, organ hypoperfusion and dysfunction (Sessler et al., 1996). Sepsis is frequently associated with ARDS (Sessler et al., 1996) and mortality from sepsis is approximately 40% (Marsh & Wewers, 1996). Septic shock is associated with hypoxemia, increased pulmonary shunting and decreased lung compliance (Hussain, 1998). Respiratory muscle performance may be impaired in septic shock due to increased ventilatory demands, increased metabolic demands and/or decreased contractility of the ventilatory muscles (Hussain, 1998). Decreased contractility may be related to 14 mediators (endotoxin, cytokines, reactive oxygen species or nitric oxide), to hypoperfusion of the ventilatory muscles, or to the increased oxygen consumption associated with critical illness (for review, see Hussain, 1998). Pathologic oxygen supply dependency, where oxygen consumption does not rise with the increase in oxygen supply, may occur in septic shock and may be related to abnormal oxygen extraction in the tissues (Clark Mims, 1992). It is noted that skeletal muscle oxygen extraction appears to remain largely intact (Clark Mims, 1992). The effect of septic shock upon diaphragm morphology is unknown. THE DIAPHRAGM The Respiratory Muscles In the mammalian respiratory system, the respiratory muscles act upon the chest wall to serve as the ventilatory pump, and the lungs serve to exchange gas (Poole et al., 1997). The gross anatomy of the respiratory muscles is shown in Figure 1. Inspiration is always an active event, where the inspiratory muscles contract in a coordinated manner to increase thoracic cavity volume, reduce thoracic pressure and draw air into the lungs. The primary muscles of inspiration are those which contract during normal quiet inspiration; these include the diaphragm, the scalenes and the parasternal intercostal muscles (for review, see Reid & Dechman, 1995). The diaphragm is responsible for 70% to 80% of the work of inspiration during normal tidal breathing (for review, see Reid & Dechman, 1995). During exertion or in circumstances when diaphragm function is impaired, recruitment of accessory inspiratory muscles (sternocleidomastoid, internal intercostals, external intercostals) is increased to meet ventilatory demands, with the sternocleidomastoid providing the most significant contribution (De Troyer & Estenne, 1988). 15 Expiratory Muscles Inspiratory Muscles Figure 1: Gross anatomy of the respiratory muscles. Expiratory and inspiratory muscles are represented on the left and right side of the figure respectively. Reprinted from (with permission of primary author): Reid W.D. & Dechman G. (1995). Considerations when testing and training the respiratory muscles. Physical Therapy. 75(11). 971-982. 16 During tidal breathing, expiration is usually a passive event which occurs due to elastic recoil of the lung parenchyma and chest wall. The major muscles of expiration are the abdominal muscles (rectus abdominus, transversus abdominis, internal and external oblique muscles), which facilitate expiration by pulling downwards on the lower six ribs thus decreasing thoracic volume. Abdominal muscle contraction also facilitates inspiration by increasing intra-abdominal pressure and end-expiratory diaphragm length (De Troyer & Estenne, 1988). Diaphragm Structure The diaphragm is a thin, musculotendinous structure located between the thoracic and abdominal body cavities and comprising only approximately 0.5% of the body weight in adult humans (for review, see Reid, 1995) (Figure 1). In vivo, the diaphragm is dome-shaped, with the muscular regions apposed to the chest wall and the tendinous regions forming the top of the dome. Diaphragm fibres originate from the lower ribcage and vertebral bodies, and radiate inwards to insert into a non-contractile aponeurosis known as the central tendon (Figure 2). The central tendon comprises approximately 15-20% of diaphragm surface area and approximately 3-7% of diaphragm weight (Poole et al., 1997). There are three distinct regions within the diaphragm (Figure 2). Sternal fibres originate from the posterior aspect of the xiphoid process. Costal fibres originate from the upper margins of the lower six ribs and costal cartilages. Crural fibres originate from the arcuate ligaments and from the anterolateral surfaces of the first three lumbar vertebrae (De Troyer & Estenne, 1988). Although the diaphragm is classified as parallel-fibred muscle (De Troyer & Estenne, 1988), the details of its architecture are considerably more complex. There are regional differences in thickness (Wait et al., 1995), fibre length (Leak, 1979), and number of fibres (Boriek & Rodarte, 1994). There are also regional differences in innervation (Hammond et al., 1989) and blood flow 17 Figure 2: The diaphragm muscle in vivo (inferior view). Note the three distinct regions of the diaphragm: sternal, costal, and crural. Reprinted from (with permission from primary author): Reid W.D. & Dechman G. (1995). Considerations when testing and training the respiratory muscles. Physical Therapy. 75(11). 971-982. 18 (Sexton & Poole, 1995). These structural differences (thickness, fibre length, fibre number, innervation and vascularization) may cause regional variation in force generation and contraction characteristics within the diaphragm (Reid & MacGowan, 1998). Cellular and ultrastructural features of the diaphragm are typical of skeletal muscle. Diaphragm muscle fibres are long and cylindrical in shape. They are packed closely together into bundles (fascicles) which are in turn packed closely together to form the whole muscle. In a typical 10 pm frozen cross-section stained with hematoxylin and eosin (Figure 3), the sarcolemma is observed as a distinct boundary between the acidophilic (deeper pink) sarcoplasm and the extracellular matrix (lighter pink). Nuclei are basophilic (blue) and they are normally located just beneath the sarcolemma. Two to three nuclei may be observed per fibre in a typical 10 pm cross-section (Cumming et al., 1994). In human limb muscle, the proportion of fibres containing internally located myonuclei has been estimated to be approximately 2-3 % (Heffher, 1989) or 4% (Cumming et al., 1994) in normal muscle. Internal muscle nuclei represent a non-specific reaction to muscle injury or necrosis, although they are reportedly observed in increased numbers in regenerating or degenerating fibres (Heffher, 1989). To the investigator's knowledge, the proportion of internally located nuclei has not been established in the normal human diaphragm In normal limb muscle cross-sections, the fibres are polyhedral in shape and their diameter varies by less than or equal to 25% (Cumming et al., 1994). Diaphragm fibres are also polyhedral in shape but their size may vary considerably in cross-section (Leak, 1979). Longitudinal sections observed with light microscopy show a regular pattern of striatums. Electron microscopy reveals the highly ordered interdigitating pattern of thin and thick filaments within each contractile unit (sarcomere). Each sarcomere is delimited by a thin electron dense band composed of proteins, known as the z-band (Burkitt et al., 1993) Thin actin- containing filaments project from either side of the z-band, forming the lighter staining I-band. The darker 19 Figure 3: Photomicrograph of normal human diaphragm morphology in H & E stained cross-section. Note polygonal-shaped fibres, homogeneous acidophilic cytoplasm, peripheral muscle nuclei (arrows), and scant endomysium with spindle-shaped fibroblast nuclei (arrowhead). Scale bar = 12 pm staining A-band represents the region where thick myosin-containing filaments are present alone or overlap with the thin filaments. Additional proteins form the cytoskeleton of the muscle fibre, which is important in maintaining the organization and integrity of the sarcomere (Trotter & Purslow, 1992). The extracellular matrix of skeletal muscle consists of collagen and other proteins, connective tissue cells (fibroblasts, mast cells, macrophages), small blood and lymphatic vessels, and small nerve branches. The matrix is organized into three layers, each of which act to maintain structural organization of the tissue, deliver nutrients and remove waste materials. The endomysium, a loose delicate connective tissue layer composed of collagen and reticular fibrils, surrounds individual muscle fibres (Sanes, 1994). The thicker perimysium surrounds individual fascicles. The denser collagenous epimysium surrounds the muscle and becomes continuous with the regular connective tissue of the tendinous junction. Diaphragm muscle tissue is well-vascularized; there are approximately eight to ten capillaries surrounding each myofibre (Leak, 1979). Diaphragm Function Although it is a voluntary muscle innervated by the phrenic nerve (C3-C5), diaphragm action is more autonomic in nature. Compared to other skeletal muscles, the diaphragm has a unique activation pattern, with its daily duty cycle, defined as the ratio of active to inactive times, reported to be 40-45% in most species (Sieck, 1994). This is considerably greater than a daily duty cycle of 2% and 14% reported for the cat extensor digitorum longus and soleus muscles respectively (Sieck, 1994). The diaphragm is a mixed muscle in humans, with relatively more type I and JJA fibres than type ITB fibres (for review, see Reid, 1995). Its high oxidative capacity reflects its role as the primary muscle of inspiration and the prolonged nature of its activity. 21 During diaphragm contraction, the dome descends against the abdominal contents in a piston-like fashion, increasing the vertical chest cavity dimensions. Further diaphragm contraction results in lateral expansion of the ribcage (Reid & Dechman, 1995). The increase in chest cavity volume reduces the alveolar pressure below atmospheric pressure, causing air to flow into the alveoli. During expiration, the diaphragm relaxes, allowing the lungs and chest wall to return to their previous dimensions by elastic recoil. Details of diaphragm contraction, including force production, excursion and type of contraction, are difficult to measure directly. Diaphragm force is measured indirectly, by measuring pressure changes during inspiratory efforts. Maximum inspiratory pressure (MD?), defined as the greatest subatmospheric (negative) pressure that can be developed at the mouth during inspiration against an occluded airway (RuppeL 1994), is used clinically as a global estimate of inspiratory muscle strength. It is used as an indicator during weaning from mechanical ventilation (Clanton & Diaz, 1995) and as a predictor for the development of hypercapnia in patients with COPD (Rochester & Braun, 1985). Predictive reference equations include age as the independent variable for adults 20 to 65 years in age (Black & Hyatt, 1969), and weight and age as independent variables for elderly adults 65 to 85 years in age (Enright et al., 1994). Predicted ranges of normal values for MLP within each population vary within + 40% of the mean (Clanton, 1994), reducing the value of MIP as an accurate research tool. Additionally, MIP is measured during a static maneuver against a closed system, causing the chest wall and respiratory muscles to become distorted and reducing its validity as a measure of dynamic inspiratory muscle strength. Transdiaphragmatic pressure (Pdi) is the most specific estimate of diaphragm muscle strength. It is assessed by measuring the pressure in air-filled balloon-tipped catheters inserted into the lower third of the esophagus (Pes) and the stomach (Pga). During attempted inspiration against an occluded airway, the pressure in each catheter is recorded and the 22 difference (Pga - Pes) represents the pressure change caused by diaphragmatic contraction, the Pa;. Reliability and validity of Pa; are affected by similar factors previously noted for MD?. Measures of diaphragm and inspiratory muscle force are affected by the lung volumes at which inspiratory efforts are generated, because lung volumes affect both lung and chest wall recoil and the tension-generating potential of the contractile apparatus. FRC is the most valid volume at which to measure inspiratory strength because at this volume, inward elastic lung recoil is equal to outward elastic chest wall recoil (Clanton & Diaz, 1995). At other volumes, the effects of lung and chest wall recoil are reflected in the inspiratory pressures (Clanton & Diaz, 1995; Black & Hyatt, 1969). In practice, unless inspiratory pressures are measured in a body plethysmograph, MD? and Pdi are usually measured at a lung volume that is more reliably achieved, the RV (Clanton, 1994). In addition to their effects upon lung and chest wall recoil, lung volumes also affect diaphragm force-generating potential. In skeletal muscle, the length-tension relationship shows that each muscle has an optimal length (LQ) at which there is optimal filament overlap and optimal potential for tension development (Lieber, 1992). Thus, the decrease in MD? or Pd; with increasing lung volumes is at least partially related to the degree of filament overlap and to the position on the length-tension curve (Rochester & Braun, 1985) and may also be related to changes in thoracic configuration such as flattening of the diaphragm (Clanton & Diaz, 1995). In individuals who breathe at higher lung volumes due to hyperinflation, the inspiratory muscles may be disadvantaged for these reasons. Unlike some limb muscles, where muscle excursion can be estimated and reproduced using standard limb positioning and joint range of motion measures, diaphragm excursion and type of contraction are difficult to measure. Sonomicrometry has been used to study length changes during diaphragm contraction (Wakai et al., 1994). Small transducers (crystals) are placed into the canine diaphragm, which is stimulated to contract via electrical stimulation of the phrenic 23 nerve. Wakai and co-workers demonstrated the complexity of diaphragm excursion and demonstrated regional variability in the type of diaphragm contraction (Wakai et aL 1994). Inspiration was primarily concentric in nature, but eccentric contractions occurred, primarily in the costal regions and particularly during loaded breathing (Wakai et al., 1994). In the diaphragm, lengthening contractions may result from side-to-side differences in diaphragm recruitment and contraction, whereby costal fibre shortening on one side causes costal fibre lengthening on the other side (Reid & MacGowan, 1998). Alternatively, lengthening contractions may result from differences in contraction velocity between adjacent sarcomeres. In an in vitro frog limb muscle model (Friden & Lieber, 1992), lengthening contractions were observed in the middle sarcomeres, and shortening contractions at the ends. Similar lengthening contractions could occur in the central regions (mid-costal) of the human diaphragm, perhaps during initiation of inspiration when co-contraction of the abdominal muscles occurs. In summary, although diaphragm contraction during inspiration appears to be concentric in nature, it may indeed involve both concentric and eccentric contractions. Response to Overload Skeletal muscle plasticity and its cellular and biochemical responses to increases or decreases in loading are well-known properties (Lieber, 1992). Increased loading of skeletal muscle may be achieved through increases in exercise frequency, intensity, or duration (Kenney et al., 1995). Increased loading may be further distinguished as overload or overactivity. Overload is defined as a condition where the force requirement is greater than usual (Stauber & Smith, 1998). Overactivity is defined as a condition where the required work rate is increased, resulting in increased motor neuron firing beyond physiological levels (Stauber & Smith, 1998). The diaphragm may be subjected to both overload and overactivity during very high intensity exercise 24 or in chronic respiratory disease when there is an increase in the required inspiratory force or respiratory rate. Similar to other skeletal muscles, the diaphragm exhibits plasticity in its response to overload or overactivity. Clinical studies have shown increased strength or endurance in response to inspiratory muscle resistive (strength or endurance) training regimes (Faulkner, 1995; Pardy & Rochester, 1992). Animal studies have shown type II to type I fibre type conversion (Keens et al., 1978), improvements in endurance at the expense of contractility consistent with an increase in the number of type I fibres (Prezant et al., 1983), increases in the concentration of endogenous glycogen (Green et al., 1989), and decreases in fibre size resulting in increased relative capillarization (Green et al., 1989). Levine and co-workers found that individuals with severe COPD ( F E V i 33 + 4% predicted) had a greater proportion of type I fibres and a lower proportion of type JJ fibres as compared to controls ( F E V i 69 + 3% predicted) (Levine et al., 1997). This adaptation may reflect an increase in diaphragm fatigue resistance (Levine et al., 1997) in this population. Of interest, endurance training in animals results in increased costal diaphragm oxidative capacity which is independent of training intensity (Powers & CriswelL 1996). It is therefore postulated that recruitment of the costal diaphragm plateaus at moderate training intensities, with further increases in workload being met by increased recruitment of the crural diaphragm and/or the accessory muscles (Powers & Criswell, 1996). The importance of respiratory muscle plasticity becomes exceedingly apparent when its response to disuse is observed. Weakness may result when the respiratory muscles are rested for prolonged periods of time, such as when they are unloaded during mechanical ventilation (Tobin, 1998). This is an important consideration when the respiratory muscles are reloaded during weaning from a ventilator (Tobin, 1998), as the diaphragm may be particularly sensitive to disuse because of its continuous and repetitive activation history (Sieck, 1994). 25 Exercise-Induced Injury Exercise-induced muscle injury is known to occur in response to overload or overactivity in animal and human limb muscles. Skeletal muscle is most susceptible to such injury when the loads involve eccentric, high force, prolonged and/or unaccustomed exercise (Armstrong, 1990). Effects of exercise-induced limb muscle injury in humans range from mild to severe, but they are usually self-limiting and no medical treatment is usually required (Maclntyre et al., 1995). There is typically an immediate force loss and some investigators have observed a secondary force loss which peaks in severity at 24 to 48 hours (Faulkner et al., 1993; Maclntyre et al., 1996). Depending on the particular model used, force loss gradually recovers over 7 to 14 days (Clarkson et al., 1992). Usually there is no residual functional impairment (Lieber, 1992) and, indeed, the result is often a muscle which is more resistant to injury (Clarkson et al., 1992). An associated clinical sign in exercise-induced injury is delayed onset muscle soreness (DOMS) which develops approximately 24 hours post-exercise, peaks at 2 to 3 days, and gradually improves thereafter (Clarkson et al., 1992). Other clinical signs include elevations in plasma levels of creatine kinase, stiffness, swelling and decreased muscle shortening ability (Clarkson et al., 1992). Ultrastructural evidence of damage has been described immediately post-exercise. This evidence includes z-band streaming, loss of distinct A- and I-bands and myofibrillar disorganization (for review, see Reid & MacGowan, 1998). Cytoskeletal disruption, mitochrondrial swelling and an increased number of ribosomes have been described at three days post-exercise (Friden et al., 1983). Light microscopic evidence of injury, including inflamed and necrotic fibres and internal myonuclei, may not be evident until about 24 to 48 hours post-injury (Lieber, 1992; Ebbeling & Clarkson, 1989). Exercise-induced skeletal muscle injury has been described as progressing through four distinct stages: initial event, the autogenic, phagocytic and regenerative stages (Armstrong, 1990). 26 Armstrong proposes that the initial event involves metabolic (high temperature, insufficient mitochondrial respiration, free radical production and/or acidosis) or mechanical (high specific tension) factors which result in a loss of calcium homeostasis and initiate the cascade of events associated with subsequent stages (Armstrong, 1990). Initial damage may involve disruptions in the plasma membrane (McNeill & Khakee, 1992) or disruptions in the sarcotubular system (Byrd, 1992). During the autogenic stage, myofibrillar proteins and cell membranes are degraded by factors which are intrinsic to the muscle fibres, including proteases and phospholipases (Armstrong et al., 1991). The phagocytic stage is characterized by a cellular response, with an influx of inflammatory cells (Tidball, 1995). Neutrophils are observed during earlier stages of inflammation; their function is to release degradative enzymes and initiate phagocytosis of cellular debris. Macrophages are observed during later stages of inflammation; their functions include phagocytosis of cell debris and release of mediators and growth factors (TidbalL 1995). The regenerative stage is a complex process during which satellite cells are stimulated to divide, proliferate and fuse to form new myotubes which differentiate into muscle fibres (Carlson & Faulkner, 1983). Macrophages are also important at this stage because they may secrete growth factors (TidbalL 1995). Events and stages associated with exercise-induced diaphragm muscle injury may be similar. However, an important difference is the inability of the diaphragm to rest completely since, even with mechanical ventilation, it is difficult to fully unload the diaphragm Indeed, mechanical ventilation may increase diaphragm loading if the individual does not breathe in synchrony with the ventilator (Tobin, 1998). The proposed mechanisms involved in overload-induced diaphragm injury will be discussed in a later section. 27 DIAPHRAGM DYSFUNCTION AND INJURY IN CHRONIC RESPIRATORY DISEASE Individuals with chronic respiratory disease may experience diaphragm overload due to many factors related to the disease process. These individuals may thus be predisposed to diaphragm dysfunction and/or injury. In this section, the following will be reviewed: diaphragm dysfunction and associated clinical manifestations, diaphragm injury in animals and in humans, and clinical factors which may contribute to diaphragm dysfunction and injury in individuals with chronic respiratory disease. Diaphragm Dysfunction and Associated Clinical Consequences Diaphragm dysfunction essentially refers to an impaired ability of the diaphragm to fulfill its role as the primary muscle of inspiration. This may be related to diaphragm fatigue and/or weakness. Fatigue is defined as the loss of force-generating ability which is reversible by rest (NHLBI Workshop Group, 1990). Weakness, in contrast, is defined as the loss of force-generating ability which persists in the rested muscle (NHLBI Workshop Group, 1990). Diaphragm force loss may be measured in vivo with specific tests for respiratory muscle strength and endurance (Clanton & Diaz, 1995). However, because it is difficult to completely rest the diaphragm, differentiating between a fatigued and a weak diaphragm muscle is extremely difficult (Reid & Dechman, 1995). Clinical impairments resulting from diaphragm fatigue or weakness include reduced lung volumes, persistent dyspnea, decreased exercise tolerance, and respiratory failure. Dyspnea refers to breathlessness which occurs when an individual is aware of an increased ventilatory effort (Sweer & Zwillich, 1990). Dyspnea is considered normal when it occurs during high-intensity exercise, and present when it occurs at a level of activity below where it would be expected (Jones & KiJJian, 1991). Although the mechanisms underlying dyspnea are not fully understood, it 28 is hypothesized to result when expected muscle shortening does not occur in response to the tension generated (Sweer & Zwillich, 1990), resulting in a sensation referred to as 'length-tension inappropriateness" (Campbell & Howell, 1963). In this manner, hyperinflation or other factors which impair the contractility and/or reduce the efficiency of the inspiratory muscles may play a role in the etiology of dyspnea. Dyspnea which ceases with mechanical ventilation is likely related to abnormalities in the lung such as inspiratory muscle dysfunction, while dyspnea which persists with mechanical ventilation may be related to airway inflammation, pneumothorax, pulmonary embolism or pneumonia (Rochester, 1991c). In a recent study by Banzett and co-workers, "air hunger" occurred in healthy subjects during mechanical ventilation in response to increased PaC02 levels, but was not associated with a sensation of increased inspiratory effort (Banzett et al., 1996). Exercise capacity is not usually limited by ventilatory factors in healthy individuals. However, there are many differences between the ventilatory challenges and response of healthy individuals and of those with chronic respiratory disease. Respiratory muscle oxygen consumption, which is approximately 5% of the total V 0 2 in healthy individuals, may increase to as high as 40% of the total V 0 2 in individuals with COPD (Field et al., 1982). During exercise, increased ventilation requirements increase the both the frequency of breathing and the required speed of inspiratory muscle contraction. In individuals with COPD, this may lead to worsening of the hyperinflation and a reduction in inspiratory muscle strength and/or efficiency. Exercise capacity may also be limited in these individuals because they use a greater proportion of the available oxygen to breathe, reducing the oxygen available for other muscles (Rochester, 1991c). Other factors associated with chronic respiratory disease which may limit exercise capacity by causing respiratory muscle weakness include corticosteroids, prolonged inactivity, chronic hypoxemia and chronic hypercapnia (Jones & Killian, 1991). 29 Respiratory failure is defined as a reduction in the partial pressure of oxygen in arterial blood (Pa02) below 55 mm Hg and/or an increase in the partial pressure of carbon dioxide in arterial blood (PaC02) above 45 mm Hg (Rochester, 1993). Hypoxic respiratory failure, which is characterized by hypoxemia that responds poorly to oxygen therapy, may be caused by lung pathology mvolving alveolar infiltrates, such as pneumonia or pulmonary edema (Rochester, 1993). Hypercapnic respiratory failure, also referred to as ventilatory failure, is characterized by hypercapnia (Rochester, 1993). This type of respiratory failure may result from increased respiratory workloads (interstitial lung disease, COPD), reduced respiratory muscle efficiency (kyphoscoliosis, hyperinflation), respiratory muscle weakness (neuromuscular disease, steroid myopathy) (Rochester, 1993), or diminished drive to breathe (primary alveolar hypoventilation). Diaphragm Injury in Animal Models Diaphragm injury refers to structural defects in the muscle which may be observed upon direct examination of muscle biopsies with the light or electron microscope. Because of its internal location and its essential role in ventilation, human diaphragm biopsies are difficult to obtain. With the advancement of nuclear imaging techniques, studying diaphragm injury may become less cumbersome in the future (Dechman et al., 1996). Much of the evidence to date concerning the nature and extent of diaphragm injury resulting from overload has been obtained from experiments involving animal models. Since animal experimentation allows for the control of many potential sources of variation (nutrition, age, activity levels, past medical history, medication history), animal models have begun to be used to study the mechanisms of overload-induced diaphragm injury, as well as the association between structural impairment (injury) and clinical impairments (force loss, respiratory failure). 30 Animal models which have been particularly useful in studying diaphragm injury in response to overload will be reviewed in this section. These include the mdx mouse model of human Duchenne muscular dystrophy (DMD), phrenic nerve stimulation, inspiratory resistive loading via an endotracheal tube, and resistive loading by tracheal banding. Animal models which have examined the effects of overload on diaphragm fibre size and fibre type distribution will not be reviewed. Mdx Mouse The mdx mouse lacks the gene which normally encodes for the protein dystrophin, resulting in a dystrophin deficiency similar to that in Duchenne muscular dystrophy (DMD). Dystrophin is located on the cytoplasmic surface of the sarcolemma and its structural connections with the cytoskeleton and with the extracellular matrix assist in maintaining sarcolemmal integrity (Petrof, 1998). During contraction, limb muscles of mdx mice are more susceptible to sarcolemmal rupture, as compared to limb muscles of control mice. Because sarcolemmal rupture is followed by fibre necrosis, satellite cell activation, regeneration and restoration of function, mdx limb muscles do not serve as a good model of clinical outcome in DMD. The mdx diaphragm muscle, however, is interesting in that it also undergoes sarcolemmal rupture, necrosis and regeneration, but the necrosis is greater and the regeneration is less than in mdx limb muscles (Dupont-Versteegden & McCarter, 1992). Mdx diaphragm abnormalities include central nuclei, increased connective tissue, increased variation in fibre diameter, abnormal fibre shape and fibre atrophy (for review, see Reid & MacGowan, 1998). The mechanisms leading to the abnormal morphology in mdx diaphragm do not directly reflect those involved in loaded breathing. However, this model is useful because it demonstrates the unique response of the diaphragm as compared to limb muscles with the same genetic profile. 31 The greater prevalence of abnormal morphology in the diaphragm may be related more to its activation history than to a particular susceptibility to injury (for reviews: see Reid & MacGowan, 1998; Petrol 1998). Phrenic Nerve Stimulation Phrenic nerve stimulation usually involves applying electrical stimulation to the phrenic nerve as it passes through the cervical region, although it can also be performed by applying stimuli to the nerves in the thoracic cavity. Phrenic nerve stimulation causes maximal diaphragm contraction, in response to the applied activation pattern. Reid and MacGowan (1998) describe an experiment in which phrenic nerve stimulation was applied to a rabbit model for 2 hours. After a recovery period, diaphragm injury and inflammation were observed and the following abnormalities were noted: internal nuclei, non-homogeneous or degenerating cytoplasm, and an influx of inflammatory cells within the muscle fibres or the interstitium The phrenic nerve stimulation model does not reflect the gradual onset of disease and chronic increase in workload which is characteristic of chronic obstructive or restrictive disease. It also differs in motor unit recruitment characteristics. However, this model may be particularly useful in studying the nature and time course of injury associated with acute moderate-to-high-intensity respiratory muscle loading which leads to respiratory muscle failure. Clinical conditions which may possibly be reflected by this model include acute respiratory exacerbations of COPD and the severe respiratory distress associated with status asthmaticus. Inspiratory Resistive Loading Inspiratory resistive loading (UAL) is achieved by attaching a valve which imposes increased resistance at the inspiratory port of an endotracheal tube. In a study by Jiang and co-32 workers, significant diaphragm injury occurred in rabbits following inspiratory resistive loading above the 'Tatigue threshold" (a PTI of 0.15), but not in rabbits subjected to lower inspiratory loads (Jiang et al., 1998a). Inspiratory loads were elevated such that PTI was 0.23 + 0.05 in the "high IRL" group, and 0.17 + 0.01 in the "moderate IRL" group. Loads were applied for 90 minutes and rabbits were allowed to recover for 3 days. "High IRL" loads, which were well-above the fatigue threshold, resulted in diaphragm injury which was most pronounced in the costal region. In contrast, "moderate IRL" loads, which were close to the fatigue threshold, did not result in significant injury. The results of this study suggest that inspiratory resistive loading may cause diaphragm injury, but only if it is greater than the "fatigue threshold". In another study where similar IRL loads were applied to rabbits for 90 minutes, the amount of diaphragm injury was related to force loss (Jiang et al, 1998b). In the "high IRL" (0.23 ± 0.05) group, there was significant injury as well as significant force loss (measured by a reduction in tetanic tension). In the "moderate IRL" group, in contrast, there was neither muscle injury nor force loss (Jiang et al., 1998b). Zhu and co-workers found that moderate intensity intermittent inspiratory resistive breathing (Pbreath = -30 to -50 cm H20) for two hours per day over four consecutive days resulted in a significant increase in the proportion of fibres showing an uptake of Procion orange (Zhu et al., 1997), an indication of sarcolemmal damage. The relevance of these sarcolemmal disruptions, and their relationship to histological markers of injury such as inflammation and necrosis, remains uncertain at present. Sarcomere disruption was described in this study, identified using phase contrast and electron microscopy. However, while sarcomere disruption was observed to be greater in animals subjected to inspiratory resistive breathing, and greater in the diaphragm as compared to the external intercostal muscle, it was not described using quantitative techniques. 33 The strength of its possible association with IRL intensity and with evidence of sarcolemmal damage (uptake of Procion orange) could therefore not be defined clearly. In summary, diaphragm injury associated with acute inspiratory resistive loading may be a useful model to study the respiratory muscle injury associated with acute exacerbations of COPD, acute bronchospasm or the respiratory distress associated with weaning from mechanical ventilation (Zhu et al., 1997). Tracheal Banding Tracheal banding has been used to impose resistive respiratory loads in animals at a lower intensity but for longer time periods. Tracheal banding involves the surgical insertion and tightening of a polyvinyl cuff around the trachea, thus increasing the resistance to airflow. The load is increased to a certain percentage of the maximal esophageal pressure (previously measured against an occluded airway) and may be maintained for periods ranging from a few days up to 30 days (Reid et al., 1994; Reid, 1995). Reid and co-workers tracheal banded hamsters, increasing esophageal pressure (Pes) to 20% of the maximum Pes (8.0 cm H20) (Reid et al., 1994). PTI tended to be higher in the tracheal banded hamsters, but the increase was not significant. After six days of banding, ventilatory failure was confirmed by severe hypoxemia and hypercapnia. Histologic examination of diaphragm biopsies revealed abnormal features including internal nuclei, flocculent degeneration of the cytoplasm and an influx of mononuclear inflammatory cells. Point counting revealed a significantly greater area fraction of abnormal and inflamed muscle in the costal and crural regions of the diaphragm of banded animals, and a significantly smaller area fraction of normal muscle in the crural regions of the diaphragm of banded animals. Ultrastructural damage included Z-band streaming and myofibrillar (Usorganization including loss of A- and I-bands. This study was notable for its finding that diaphragm injury at the light 34 microscopic level was associated with ventilatory failure. Another study examining the time course of injury associated with tracheal banding showed inflammation to be most marked following three days of banding, with the response involving primarily mononuclear cells (Reid, 1995). After 30 days of banding, an increase in abnormal muscle fibres and a trend towards an increased presence of connective tissue were noted, while little inflammatory response was observed (Reid, 1995). The tracheal banding model is useful as a model of obstructive disease. However, it differs from COPD in that the airflow resistance is increased during both the inspiratory and expiratory phases, thus imposing increased loads to both the inspiratory and expiratory muscles. Additionally, the resistance is increased in the trachea, a large extrathoracic conducting airway, rather than in the small airways. The low intensity and prolonged nature of the loading may reflect the chronic load experienced by individuals with moderate to severe COPD, or the increased loading experienced during an exacerbation in individuals with mild to moderate COPD. Evidence of Diaphragm Injury in Humans There is relatively little documentation of human respiratory muscle injury in response to overactivity or overload. A limited number of quantitative experimental studies describing abnormalities in the accessory muscles of respiration have been conducted to date (Campbell et al., 1980; Hards et al., 1990). Some qualitative studies describing diaphragm muscle injury have been reported (Silver & Smith, 1992; Kariks, 1989). Investigation into the mechanism of diaphragm injury and its relationship to ventilatory loading in humans is lacking. Campbell and co-workers described abnormal intercostal muscle morphology in seventeen of twenty-two subjects with mild to moderate obstructive lung disease (mean FEVi /FVC: 64 %; range: 43 to 79 %) undergoing thoracotomy surgery. Biopsies were rated as normal or abnormal, 35 based upon the absence or presence of the following abnormal features in a sample of 100 fibres: fibre splitting, fibre grouping, type II fibre atrophy, targeting, central nuclei and variation in fibre size. Abnormal features were more often present in the internal intercostals as compared to the external intercostals, and in subjects with confirmed lung cancer as compared to those without. Type II fibre atrophy was significantly related to FEVi/FVC in the internal intercostals (r = - .7852, p < 0.001), suggesting that abnormal morphology could be reflective of respiratory muscle dysfunction. However, relationships between the extent of abnormal morphology and pulmonary or respiratory muscle function were not investigated. Hards and co-workers examined biopsies from the internal and external intercostals and the latissimus dorsi muscle of 68 subjects undergoing thoracotomy surgery (Hards et al., 1990). A "qualitative score" indicated the presence or absence of the following abnormal features: necrotic fibres, basophilic fibres, fibre splitting, inflammatory cells, increased presence of internal nuclei (more than 3% of total fibres), ragged red fibres and Z-band streaming. Necrotic fibres, basophilic fibres and inflammatory cells were interpreted to represent degeneration or regeneration. In contrast to the study by Campbell and co-workers (Campbell et al., 1980), abnormal features were observed in all three accessory muscles, and they were most frequent in the external intercostal muscle. The incidence of morphologic abnormalities did not correlate with age, nutritional status, respiratory muscle strength or pulmonary function measures. A significant correlation was found between the incidence of ragged red fibres in the external intercostal fibres and malignancy, a finding which suggested that systemic factors may be important in the etiology of this particular abnormality. The incidence of moth eaten fibres and Z-band streaming were not related to malignancy. Silver and Smith (1992) reported the presence of a specific morphological abnormality, contraction band necrosis, in post-mortem costal diaphragm biopsies from infants who had died 36 suddenly. Contraction band necrosis was described as muscle fibres with either solid ("block-like") or ribbon-like ("shredded") masses of sarcoplasm. "Recent" lesions were described as hypereosinophilic and hyaline in appearance, while "older" lesions were described as being granular in appearance. Contraction band necrosis was described as being distinct from hypercontraction bands (which occur due to contraction during fixation), since these lesions lacked visible cross-striations in longitudinal sections. Of note, there was no polymorphonuclear response associated with contraction band necrosis. The amount of contraction band necrosis was not quantified, although its extent was described as ranging from absent to being present in virtually every muscle fibre in the cross-section. Lesions were described as being most extensive in infants who had died of birth asphyxia or sudden infant death syndrome (SIDS). Evidence of healing was observed in eight infants who were either resuscitated or maintained on life support for some time following the respiratory insult. Healing features, which included muscle or satellite cell nuclei with prominent nucleoli, macrophage invasion, myoblast proliferation and basophilic sarcoplasm, were interpreted to indicate regeneration. In the same paper, the authors described unpublished observations of similar necrosis in the diaphragm of 12 out of 13 adults (ages 15 to 79 years) who had died suddenly, with the most pronounced necrosis occvirring in an individual who died in status asthmaticus (Silver & Smith, 1992). The authors concluded that an acute respiratory dysfunction may serve as a sudden injury-provoking event in the diaphragm. The authors suggested that a catecholamine surge which triggers sarcomere spasm and causes local cell membrane damage may be involved in the etiology of contraction band necrosis. Kariks (1989) also described contraction band necrosis lesions in 82% of infants (198 out of 242) who died of SJDS. The lesions were focal in nature, with only a portion of the fibre affected when examined in longitudinal section. Cross-sections showed eosinophilia with a "glassy" appearance. Atrophic fibres were also described, some of which had either a round or an angulated shape and internally located nuclei. In six cases, leukocyte infiltration and scarring were noted. Vacuolization with widespread fibre disintegration was present in some biopsies. Lesions were described as "early" or "late" stages of acute, terminal muscle tissue changes. "Early" or 'late" stages were reportedly based upon the length of time the diaphragm was operating under hypoxic stress, however details regarding specific time periods of hypoxic stress in the infants were not reported. "Late stages" were characterized by (1) central vacuolization, perhaps due to myocytolysis, (2) phagocyte infiltration, (3) calcification, perhaps due to mineral deficiency, (4) fibrosis and scar formation. The author referred to end-stage "dead" muscle fibres, but did not describe this feature in detail. Lloreta and co-workers presented a case report describing mitochondrial abnormalities in the diaphragm of an mdividual with moderate COPD (FEVi 51% predicted ) (Lloreta et al., 1996). Subsarcolemmal accumulations of mitochondria and abnormal paraciystalline mitochrondrial inclusions were described in the diaphragm, with fewer being described in the intercostal muscles and the latissimus dorsi muscle. The authors suggested that these abnormalities may occur as an adaptive response by an exercise-burdened diaphragm Lindsay and co-workers described histological abnormalities in the limb and respiratory muscles of seventeen subjects with heart failure (Lindsay et al., 1996). Abnormalities were classified as major or minor, upon examination of individual muscle biopsies. Abnormalities, which included internal nuclei, tubular aggregates, moth-eaten fibres and abnormal (neonatal) myosin expression, were more prevalent in the diaphragm than in other respiratory or limb muscles. The authors concluded that internal nuclei and abnormal myosin expression were features suggestive of regeneration or fibre type transformation. The authors suggested that 38 abnormal respiratory muscle morphology may contribute to dyspnea in individuals with chronic heart failure. Other Clinical Contributing Factors Aging, malnutrition and corticosteroid ingestion will be reviewed briefly, since these factors may have a significant influence upon skeletal muscle function and structure and they are often features present in individuals with chronic respiratory disease. Aging Animal models have shown some age-related changes in the diaphragm muscle, including type lib atrophy, myosin heavy chain composition changes, decreased capillary density, and fibre grouping (Tolep & Kelsen, 1993). There also appears to be an increased susceptibility to injury in older animals. A greater amount of Z-band streaming and sarcomere disruption was observed in the diaphragm of 84 day old mice as compared to 15 day old mice in an in vitro study by Watchko and co-workers (Watchko et al.,1994). Brooks and co-workers reported that young and adult mice recovered from contraction-induced injury within two weeks, but elderly mice had not recovered completely by two months (Brooks & Faulkner, 1990). In aging, respiratory muscle energy requirements are increased but respiratory muscle strength is decreased (Tolep & Kelsen, 1993). Thus aging may contribute to the etiology of diaphragm injury by accentuating the already increased ratio of respiratory demands to respiratory capacity in individuals with chronic respiratory disease. Aging may also accentuate the effects of overload in chronic respiratory disease by impairing regenerative and adaptive responses (for review, see Reid & MacGowan, 1998). 39 Malnutrition It is estimated that 30 to 40% of individuals with COPD are underweight (Rochester, 1991c). Weight loss may occur because of decreased caloric intake in conjunction with increased metabolic demands. Malnutrition in COPD is directly related to acute respiratory failure, impaired pulmonary function and mortality (American Thoracic Society, 1995), and malnutrition in CF is directly related to decreased lung function (Heijerman, 1993). Whether weight loss is related to respiratory muscle injury is not known. The primary observation following nutritional deprivation is muscle atrophy, which is most pronounced in type II fibres (for review, see Reid & MacGowan, 1998). Diaphragm force is decreased, but specific force (force divided by cross-sectional area) remains the same, which suggests that diaphragm force loss is related more to a loss of muscle mass than to contractile protein disruption (for review, see Reid & MacGowan, 1998). While models of nutritional deprivation have not been used to study respiratory muscle injury specifically, it is possible that weight loss results in diaphragm atrophy and weakness, thereby increasing diaphragm susceptibility to overload-related injury (Reid & MacGowan, 1998). An energy imbalance, which predisposes the respiratory muscles to failure, may be involved (Rochester, 1992). Electrolyte imbalance may also impair respiratory muscle function, increasing the risk of respiratory failure (American Thoracic Society, 1995). Corticosteroids Corticosteroids inhibit protein synthesis and increase protein catabolism. This may lead to skeletal muscle structural (atrophy) and functional (weakness) changes. Although they were once thought to be spared from corticosteroid-related impairments, there is evidence to suggest that the respiratory muscles may be affected in a similar manner (Dekhuijzen & Decramer, 1992). 40 Respiratory muscle weakness has been shown in mdividuals who were prescribed intermittent bursts of steroid therapy for COPD or asthma (Decramer et al., 1994). Various morphological abnormalities have been observed in the rat diaphragm following corticosteroid ingestion, including vacuolization, necrosis and atrophy (Ferguson et al., 1990), and angulated fibres (Wilcox et al., 1989). Selective atrophy of type II fibres was observed in the diaphragm of animal models in two studies (Wilcox et al., 1989; Nava et al., 1996). Short-term high-dose corticosteroids may cause acute necrotising myopathy (Curnming et al., 1994). Although this is a relatively rare complication, acute myopathy has been reported to occur in individuals treated for status astmnaticus (Griffin et al., 1992). Three individuals treated with intravenous methylprednisone and neuromuscular blocking agents had difficulty weaning from mechanical ventilation (15-31 days of intubation), showed marked elevation in creatine kinase and myoglobin, and showed diffuse necrosis and degeneration of all fibre types in limb muscle biopsies (Griffin et al., 1992). Bom proximal and limb muscles were affected. Chronic steroid-related myopathy due to prolonged low-dose corticosteroids is a more common complication of corticosteroid ingestion. It is characterized by proximal limb weakness, type II fibre atrophy, reduced muscle mass and increased susceptibility to muscle fatigue (Dekhuijzen & Decramer, 1992). The contribution of corticosteroids to diaphragm injury likely depends upon the particular steroid used (fluorinated versus non-fluorinated), as well as the dose and the duration of treatment (Reid & MacGowan, 1998). The combined effect upon the respiratory muscles of corticosteroid ingestion, excessive loading and chronic respiratory disease is presently unknown (Reid & MacGowan, 1998). 41 MECHANISMS OF OVERLOAD-INDUCED DIAPHRAGM INJURY Animal studies have demonstrated clear evidence that diaphragm injury occurs in response to overload. Animal studies have also demonstrated an association between diaphragm injury and functional impairment including force loss and ventilatory failure. Human studies have provided preliminary evidence that respiratory muscle abnormalities occur in individuals who experience ventilatory loading due to chronic respiratory muscle disease, but neither association nor causation have been clearly demonstrated in humans. Although the mechanisms of overload-induced diaphragm injury in chronic respiratory disease have not been fully elucidated, some of the factors proposed to be involved will be reviewed in this section. The diaphragm may be predisposed to injury by a combination of mechanical and metabolic factors. Mechanical factors such as hyperinflation (COPD, CF) and chest wall abnormalities (restrictive disease) may severely impair the efficiency and effectiveness of diaphragm function by reducing diaphragm excursion, by causing the diaphragm to work at less-than optimal lengths and/or by impairing transmission of force. By causing a mechanical disadvantage and impairing the capacity of the diaphragm to meet ventilatory demands, these factors may result in a 'relative' diaphragm overload. Another mechanical factor which has been proposed to increase diaphragm vulnerability to injury is perpendicular shearing stress during diaphragm muscle contraction. Although diaphragm architecture lends itself to such stresses, two reports (Margulies et al., 1994; Boriek et al., 1994) concluded that the stress associated with diaphragm contraction in dogs occurred in parallel to the muscle fibres, and that significant shearing stress did not occur. Boriek and co-workers suggested that perhaps the diaphragm is not extensible in a perpendicular direction in order to minimize the elastic work required during contraction (Boriek et al., 1994). Stiffness in direction perpendicular to the direction of muscle force could increase diaphragm susceptibility to injury by increasing 42 sarcolemrnal shearing forces during acute increases in workload. There is no evidence, however, to support or refute this possible mechanism of overload-induced diaphragm injury. Finally, overload or overactivity may predispose certain regions of the diaphragm to injury by accentuating regional differences in contraction characteristics. Lengthening contractions are more likely to occur in the mid-costal regions of the diaphragm (Wakai et al., 1994). Since lengmening contractions are known to increase skeletal muscle susceptibility to injury, this may explain the greater injury noted in the costal diaphragm in some animal models of resistive ventilatory loading (Reid et al., 1994; Jiang et al., 1998a). Metabolic factors could predispose the diaphragm to injury and dysfunction. Decreases in pH which occur during exercise may be worsened by acidosis due to ventilatory impairments and poor gas exchange (for review, see Reid & Samrai, 1995). The pH may be further lowered by increased lactic acid production, resulting from increased recruitment of type Ub fibres. Some type Ha fibres are likely recruited in the diaphragm during normal quiet breathing, but type Ub fibres are held in reserve for more forceful diaphragmatic efforts (Sieck, 1988). If the increased inspiratory demands in chronic respiratory disease increase type lib fibre recruitment, this would increase the reliance upon anaerobic metabolism and the production of lactic acid. The increased energy cost of breathing associated with an elevated resistance to airflow may also be an important metabolic factor. In contrast to healthy individuals who breathe at approximately 65% of their maximal voluntary ventilation during maximal exercise, individuals with chronic airflow obstruction may breathe at 90% of their maximal voluntary ventilation during maximal exercise (Rochester, 1991). Increased respiratory metabolic demands may result in insufficient mitochondrial respiration and insufficient ATP production in the diaphragm This may cause some fibres to develop rigor, possibly increasing their susceptibility to injury during subsequent contractions (Friden & Lieber, 1992). 43 Hypoxia due to ischemia has also been proposed as a potential injury-provoking event (Kariks, 1989; Silver & Smith, 1992). The diaphragm is well-vascularized (Leak, 1979), however, leading Silver and Smith (Silver & Smith, 1992) to suggest that diaphragm injury is more likely to result from widespread ischemia such as occurs in hypoperfusion, rather than from focal ischemia. The medial costal region has the highest mass specific blood flow (Sexton & Poole, 1997) and the greater apparent susceptibility to injury in animal models. The greater vascularity reflects a higher metabolic demand (Poole et al., 1997), which implies that perhaps it is the increased demands which initiate injury rather that inadequate blood supply. Alternatively, it is possible that when diaphragm contraction reaches a certain activation threshold, local ischemia occurs (Campbell et al., 1980). Autolysis following initial injury appears to be related to an elevated intracellular calcium concentration (Armstrong, 1990). In vivo activation of calpain, a calcium-activated non-lysosomal protease, has been observed in the limb muscles of rats run to exhaustion (58 +11 minutes) (Belcastro, 1993) and in the diaphragm of tracheal banded rats (Reid et al., 1994). If an increased activity level of calpain occurs in the diaphragm in response to overload or overactivity in individuals with chronic respiratory disease, it could enhance subsequent degradation and impair the processes of recovery and adaptation. Whether this occurs in the human diaphragm in response to chronic ventilatory loading is unknown. Clinical symptoms including soreness, stiffness and swelling have not been studied in association with diaphragm injury. These may constitute important protective mechanisms during the phagocytic and regenerative stages of exercise-induced limb muscle injury, by ensuring that adequate rest or reduced activity is enforced so that recovery and a positive adaptation may occur (Smith, 1991). Due to the essential nature of its contraction, the diaphragm may not be afforded these protective mechanisms. It would be difficult but intriguing to investigate whether localized 44 shoulder pain (referred from the phrenic nerve) occurs in association with overload-induced diaphragm injury in humans. The theoretical model presented in Figure 4 incorporates some of the concepts associated with diaphragm overload, injury, and adaptive responses in chronic respiratory disease. This model and its proposed relationships will not be tested. However, it is included in this thesis to provide a theoretical context for the two research studies to be presented in Chapters 2 and 3. Positive adaptations such as improved strength and endurance in response to strength and endurance training are known to occur in the respiratory muscles of animals and humans (for review, see Reid & Samrai, 1995). To achieve these gains, diaphragm overload is imposed, leading to fatigue, weakness, and/or injury followed by recovery and a training effect. If injury occurs, regeneration and recovery may follow. Alternatively, since diaphragm workload requirements are continual, its activity level may not be adequately reduced in order for regeneration and recovery to occur. In fact, the unrelenting overload may cause the fatigued and/or weak diaphragm, which continues to contract, to become injured and subsequently repaired by a process of connective tissue replacement rather than by regeneration with viable contractile elements. This could result in fibre degeneration, with an associated loss of contractile tissue elements and an increase in connective tissue content. Connective tissue will provide the repaired muscle with increased tensile strength, but with reduced contractile ability. This adaptation would be considered as a 'negative adaptation", with an associated impaired ability to generate force and function in its role as a key muscle of the ventilatory pump. 45 Figure 4: Model of diaphragm overload and injury in chronic respiratory disease. Note this is a theoretical model which indicated proposed factors and relationships. Adapted from: Reid W.D. & MacGowan N.A. (1998). Respiratory muscle injury in animal models and humans. Molecular and Cellular Biochemistry. 170(1-2). 63-80. 46 DECREASED FORCE INCREASED WORKLOAD DECREASED EFFICIENCY • malnutrition • disuse atrophy • corticosteroids • abnormal blood gases • electrolyte imbalance • sepsis • altered breathing pattern • bronchospasm • mucus • infection »hyperinflation • flail chest > kyphoscoliosis (") DIAPHRAGM OVERLOAD (+) cycle of diaphragm training, recovery and adaptation fatigue and/or weakness cycle of diaphragm injury, \ repair and impairment RECOVERY regeneration of muscle tissue training effect ADAPTATION • increased strength • improved endurance • protection from injury J INJURY repair by connective tissue replacement IMPAIRMENT • reduced lung volumes • dyspnea • exercise intolerance • respiratory failure 47 RESEARCH HYPOTHESES Because diaphragm injury occurs in animals with loaded breathing, and because mdividuals with chronic respiratory disease experience ventilatory loading, we predicted that there would be evidence of diaphragm injury in individuals with chronic respiratory disease. We also postulated that the extent of this injury would be greater in those with more severe airflow obstruction, and that various clinical factors may affect the extent of diaphragm injury. In the first research study, we hypothesized that, in a sample of mdrviduals with mild to severe airflow obstruction, 1. the proportion of abnormal diaphragm muscle is directly related to the severity of airflow obstruction and thus inversely related to the % predicted FEVi; 2. the proportion of normal diaphragm muscle is inversely related to the degree of airflow obstruction and thus directly related to the % predicted FEVi; 3. the number of macrophages per fibre and per unit area in the diaphragm is directly related to the severity of airflow obstruction and thus inversely related to the % predicted FEVi. In the second research study, we hypothesized that in a sample of individuals who died of various causes, the proportion of abnormal diaphragm is related to one or more of the following clinical factors: presence of chronic respiratory disease, presence of acute respiratory disease, age, body mass index (BMI), gender. 48 RESEARCH OBJECTIVES The objectives of the first research study were: 1. to describe the structural changes, at the light microscopic level, in diaphragm biopsies obtained during thoracotomy surgery; 2. to determine the proportion of normal muscle, abnormal muscle and connective tissue in the diaphragm biopsies; 3. to relate the proportions of abnormal and normal diaphragm in the biopsies to a clinical measure of airflow obstruction (% predicted FEVi); 4. to determine the number of macrophages per muscle fibre and per mm2; 5. to relate the number of macrophages per muscle fibre and per mm2 to a clinical measure of airflow obstruction (% predicted FEVi). The objectives of the second research study were: 1. to describe the structural changes, at the light microscopic level, in diaphragm biopsies obtained during autopsy; 2. to determine the proportion of normal muscle, abnormal muscle and connective tissue in the diaphragm biopsies; 3. to determine the contribution of the following clinical factors to the proportion of abnormal diaphragm muscle: chronic respiratory disease, acute respiratory disease, age, body mass index (BMI), gender. 49 CHAPTER 2: DIAPHRAGM INJURY IN INDIVIDUALS WITH AIRFLOW OBSTRUCTION GOING FOR THORACOTOMY SURGERY ABSTRACT In chronic obstructive pulmonary disease (COPD), the respiratory muscles are subjected to increased loading as airflow obstruction progresses. Although ventilatory overload has been shown to result in diaphragm injury in animal models, similar injury has not been shown in humans. The purpose of this study was to describe the nature and the prevalence of diaphragm injury in individuals with airflow obstruction. Partial-thickness diaphragm biopsies were obtained from twenty-one subjects going for thoracotomy surgery. Mean FEVi was 74 + 34 % predicted (range: 16 to 122 % predicted). Biopsies were quick frozen (n=18) or fixed in formalin (n=3), and stained with H & E. In frozen sections, macrophages were labeled using monoclonal antibodies (Ber-MAC3, DAKO Corp.). The area fractions of normal muscle, abnormal muscle and connective tissue were determined using point counting. The number of macrophages per fibre and per mm2 was determined using image analysis. Abnormal features in H & E sections included internal myonuclei, hpofuscin pigmentation, small angular fibres, and small basophilic fibres. Inflamed and necrotic fibres were observed but were not a common feature. The area fractions of normal muscle, abnormal muscle and connective tissue were 66.2 + 9.0 %, 17.6 + 7.2 %, and 16.3 + 4.2 %, respectively. The area fraction of abnormal muscle was inversely related to % predicted FEVi (r = -0.53, p<0.01). The area fraction of normal muscle was directly related to % predicted FEVi (r = 0.37, p<0.05). Macrophages were observed in the endomysium and perimysium of the diaphragm Mean number of macrophages per fibre was 0.41 ± 0.18, and per mm2 was 52 + 19. There was no relationship between number of macrophages and % predicted FEVi. This study showed that as severity of airflow obstruction increases, the proportion of abnormal diaphragm increases and the proportion of normal diaphragm decreases. The 50 prevalence of macrophages in the diaphragm is not related to airflow obstruction and may be affected by many factors. INTRODUCTION The diaphragm is a unique muscle with an essential role in inspiration. It has adapted structurally and functionally in order to maximize its efficiency of force generation and transmission (Poole et al., 1997). The diaphragm responds to increased ventilatory demands and healthy individuals rarely exceed their ventilatory reserve capacity, even during maximal exercise (Rochester, 1991c). Chronic obstructive pulmonary disease (COPD) is a disease characterized by increased resistance to airflow (Snider, 1995). COPD specifically challenges the diaphragm by increasing inspiratory muscle demands and by contributing to inspiratory muscle inefficiency or weakness (Reid, 1995). Similar to limb muscles, the diaphragm has been shown to respond to overload challenges with cellular (Faulkner, 1995; Poole et al., 1997) and functional (for review, see Reid & Samrai, 1995) adaptations. In contrast to models of limb muscle overload, where rest and recovery are permitted, diaphragm overload associated with COPD can be unrelenting and prolonged. The diaphragm in COPD may respond with gains in strength and/or endurance. Its adaptive capacity may however be impaired by factors which accentuate muscle weakness or limit regeneration. These include poor nutritional status, corticosteroids and poor arterial blood gases (for review, see Reid, 1995). If the unrelenting overload and clinical contributing factors exceed diaphragm capacity, diaphragm dysfunction or injury may be the result. Functional consequences may range from dyspnea and exercise intolerance to more severe ventilatory-related impairments including respiratory failure and death. In limb muscles, overload is used as a training stimulus to achieve improved strength and endurance (Kenney et al., 1995). Ffigh-intensity, eccentric and/or prolonged exercise in limb 51 muscles may constitute a 'pathological' overload stimulus, leading to impaired function (force loss) or structural damage (muscle injury). Diaphragm injury has been shown in animal models of resistive ventilatory loading, including acute high-intensity loading over a few hours (Jiang et al., 1998a; Jiang et al., 1998b) and chronic low-intensity loading over several days (Reid et al., 1994). Sarcolemmal defects in diaphragm muscle fibres have been shown to occur in animals subjected to moderate intensity ventilatory loads repeated over several days (Zhu et al, 1997). A few studies (Hards et al., 1990; Campbell et al., 1980) have described respiratory muscle abnormalities in human subjects who experience ventilatory loading due to chronic airflow obstruction, but there were several limitations in these studies which the present study has addressed. Both Hards and co-workers (Hards et al., 1990) and Campbell and co-workers (Campbell et al., 1980) examined accessory respiratory muscle biopsies. Although injury may occur in other respiratory muscles in response to ventilatory overload, animal studies indicate that the diaphragm is affected to a greater degree (Jiang et al., 1998a). Moreover, the costal diaphragm has been shown to incur greater injury than the crural diaphragm (Reid et al., 1994; Jiang et al., 1998a). A second limitation is that previous human studies were limited to subjects with mild (Hards et al., 1990) or mild to moderate (Campbell et al., 1980) levels of chronic airflow obstruction. A third hrnitation is that abnormal morphology was described qualitatively rather than quantitatively. Both Hards and co-workers (Hards et al., 1990) and Campbell and co-workers (Campbell et al., 1980) described muscle injury as being either present or absent, and did not quantitatively evaluate the proportion of injured muscle. Finally, although some studies have described inflammatory cells (Hards et al., 1990), necrosis (Silver & Smith, 1992; Kariks, 1989), and phagocyte infiltration (Kariks, 1989) in the respiratory muscles of individuals with chronic or acute respiratory disease, no studies to date have described the location nor the prevalence of macrophages in respiratory muscle tissue. Macrophages have important roles in both the 52 inflarnmatory and the regenerative stages of skeletal muscle injury (TidbalL 1995) and they likely have a significant role in overload-induced diaphragm injury. However, at the present time, their prevalence in the diaphragm of individuals who experience ventilatory loading due to airflow obstruction has yet to be described in the literature. We hypothesized that, in the presence of mild to severe airflow obstruction, (1) the proportion of abnormal diaphragm muscle increases with severity of airflow obstruction, (2) the proportion of normal diaphragm muscle decreases with severity of airflow obstruction, and (3) the prevalence of macrophages in the diaphragm increases with severity of airflow obstruction. The purpose of this study was to describe the nature and prevalence of diaphragm injury, using the light microscope and quantitative techniques, in individuals with mild to severe airflow obstruction. The specific aims were to: (1) describe normal and abnormal diaphragm morphology; (2) determine the relationship between the area fraction of abnormal diaphragm muscle and % predicted FEVi; (3) determine the relationship between the area fraction of normal diaphragm muscle and % predicted FEVi; (4) determine the relationship between the number of macrophages in the diaphragm and % predicted FEVi. METHODS Subjects Thirty-one subjects undergoing thoracotomy surgery consented to participate in this study. Reasons for surgery included lung resection for cancer (n=27), bullectomy for bullous disease (n=T), lung transplantation for emphysema (n=2), and lung volume reduction for emphysema (n=l). Informed consent was obtained by one of the thoracic surgeons, Dr. Ken Evans or Dr. 53 Guy Fradet. The consent form has been included in Appendix A. Approval was granted by the Ethics Committee at the University of British Columbia (reference number: C95-0292). Inclusion criteria were as follows: (1) fitness for surgery and (2) completion of pre-operative pulmonary function tests. Exclusion criteria were as follows: (1) acute exacerbation requiring hospitalization within four weeks prior to surgery, (2) chronic respiratory disease of a restrictive nature including interstitial lung disease, sarcoidosis, tuberculosis, neuromuscular disease or chest wall abnormality, (3) previous lower lobectomy or pneumonectomy, and/or (4) inadequate muscle biopsy size or quality (minimum of 5 viable fields at 500x). Muscle Sampling and Preparation Ehiring surgery, the surgeon removed a partial-thickness muscle biopsy from the thoracic surface of the costal diaphragm lateral to the central tendon and medial to the costal insertion. The biopsy was removed with a sharp scalpel and the site was secured with a suture. The hemi-diaphragm selected for sampling was determined by surgical details including the location of the incision. Muscle biopsies were placed in sterile specimen containers in a few millilitres of saline or on saline-soaked gauze, and immediately delivered to the operating room front desk. Within thirty minutes, biopsies were retrieved and transported by the investigator, Nori MacGowan, or by the supervisor, Dr. W. Darlene Reid, to laboratory facilities in the Department of Anatomical Pathology at Vancouver Hospital (Oak St. Site). The first three obtained biopsies were fixed in 10% formalin, then transferred into 70% alcohol after 48 hours. Rermining biopsies (n=28) were quick frozen using the following technique. Biopsies were oriented using a dissecting light microscope and placed in a small polyvinyl mold (5 mm x 15 mm x 15 mm) containing an embedding medium (O.C.T. Compound, Tissue-Tek, Torrance, CA). A rectangular piece of paper was placed beside the biopsy to indicate 54 the direction of the longitudinal axis of the muscle fibres. As an exception, one larger biopsy was mounted on a cork using gum tragancanth prior to freezing. Biopsies were submerged in isopentane cooled to just above freezing in liquid nitrogen, wrapped with a paraffin sheath, and transferred into a cooled vial labeled with a two-digit random number. Vials were stored at -70° Celsius in a freezer until further processing. Histology Biopsies fixed in formalin (n=3) were processed in laboratory facilities in the Department of Academic Pathology, University of British Columbia. They were dehydrated, embedded, sectioned and stained with hematoxylin and eosin (H & E). Prior to processing, frozen biopsies (n=28) were removed from the freezer and placed in a cryostat cooled to -22° Celsius for a minimum of twenty minutes, to warm the tissue to optimal sectioning temperature (Curnming et al., 1994). Excess frozen O.C.T. Compound (Tissue-Tek, Torrance, CA) was trimmed and the frozen block was mounted on the cryostat chuck using O.C.T. Compound (Tissue-Tek, Torrance, CA) such that the longitudinal axis of the muscle fibres in the biopsy was perpendicular to the surface of the microtome blade. Thick transverse cryosections (10 pm thickness) were cut, allowed to air dry for 60 minutes, and stained with H & E (see protocol in Appendix B). Thin transverse cryosections (6 pm thickness) were cut, mounted on slides coated with 3-aminopropylethoxysilane, and air-dried for 30 minutes. Thin sections were processed using immunohistochemistry and anti-macrophage monoclonal antibodies (see next section). All slides were labeled with the appropriate two-digit random number, so that the investigator, Nori MacGowan, would be blinded to biopsy identity during subsequent quantitative evaluation. 55 hnmunoMstochemistry Monoclonal antibodies were used to label macrophages in frozen diaphragm cross-sections, using irnmunohistochemistry and the alkaline phosphatase anti-alkaline phosphatase (APAAP) method (Appendix C). Thin cryosections mounted on coated slides were fixed in acetone for 10 minutes, allowed to air dry for 30 minutes, then rehydrated with Tris-buffered saline at pH 7.6 (TBS). Sections were incubated in 5% normal rabbit serum diluted in TBS buffer containing 1% bovine serum albumin (1% BSA) for 15 minutes, to block non-specific binding of antibodies. Excess solution was drained and sections were incubated in the primary antibody for one hour at room temperature. The primary antibody was a monoclonal mouse anti-human macrophage clone (Ber-MAC3, DAKO Corp.), diluted to a concentration of 1:50 in 1% BSA. Following incubation with the primary antibody, sections were washed twice with TBS, for five minutes per wash. The secondary antibody (rabbit anti-mouse IgG, DAKO Corp.) was applied for thirty rrrinutes at room temperature, diluted to a concentration of 1:20 in 1% BSA, followed by two frve-minute washes with TBS. Sections were incubated with APAAP complex (DAKO Corp.) for thirty minutes at room temperature, diluted to a concentration of 1:50 in 1% BSA, followed by two five-minute washes with TBS. The alkaline phosphatase substrate, Naphthol AS-B1 phosphate in 1% New Fuchsin, was prepared and immediately applied to the sections for ten minutes. The reaction was terminated by rinsing with TBS followed by tap water. Sections were lightly counterstained using Meyer's Hematoxylin (Appendix D), and mounted with coverslips using a permanent mounting medium. Positive controls consisted of sections of human lymph node processed as per the above technique. Negative controls were muscle sections processed as per the above technique, using 56 mouse anti-human IgGi (DAKO Corp.) as the primary antibody, diluted to the same mouse IgG concentration as Ber-MAC3 (DAKO Corp.). Prior to processing the muscle sections for immunohistochemistry, pilot experiments were conducted to choose the most effective primary antibody and determine appropriate concentrations. Ber-MAC3 (DAKO Corp.), which reacts with the macrophage differentiation antigen (Mr 140 000) located on the plasma membrane and in the cytoplasm of stimulated monocytes and resident macrophages (Backe'et al., 1991), delineated macrophage cell boundaries better than EBMn, an anti-human macrophage CD68 clone (DAKO Corp.). This was likely because EBMn recognizes only intracytoplasmic proteins (Drossos et al., 1995). In addition, EBMn is not specific to macrophages, but also reacts with peripheral blood monocytes, large lymphocytes, basophils and mast cells. EBMn also reacts weakly with neutrophils. Three dilutions of Ber-MAC3 (DAKO Corp.) were utilized in our pilot experiments to determine the most appropriate concentration. Qualitative Evaluation of H & E Sections Sections stained with H & E were examined using a Nikon light microscope at a magnification of 500x (eyepiece 10x, optivar 1.25x, objective 40x). Sections were evaluated for biopsy quality, normal and abnormal morphology, and artifact. Structural features were classified into nine categories (Table 1). One category represented normal muscle, five categories represented abnormal muscle, and three categories represented connective tissue elements. A tenth category represented empty space, nervous tissue, artifact, and epimysium 57 TABLE 1: Categories and Definitions for Point Counting (Thoracotomy Study) CATEGORIES 1. Normal 2. Internal myonucleus 3. Collagen 4. Fibroblast 5. Inflammatory cell 6. Lipofuscin 7. Abnormal but viable 8. Inflamed or necrotic 9. Blood vessel No count = space, nerve, artifact, epimysial connective tissue DEFINITIONS 1. Fibre with polygonal shape, homogeneous acidophilic cytoplasm, intact plasma membrane, peripheral nuclei 2. Fibre containing > 1 internally located nuclei (sarcoplasm between nucleus and sarcolemma) 3. Protein fibrils of endomysial or perimysial connective tissue 4. Spindle-shaped interstitial nucleus 5. Round-shaped interstitial nucleus 6. Fibre containing brown-yellow pigmentation > area of a muscle nucleus 7. Small fibre with > 2 oblique angles or with basophilic peripheral sarcoplasm 8. Fibre containing > 1 inflammatory cell or necrotic mass of inflammatory cells and muscle debris without plasma membrane 9. Blood vessel, lymphatic vessel or capillary 58 Quantitative Evaluation of H & E Sections •' Computer-assisted point counting was used to quantify the proportion of normal muscle, abnormal muscle and connective tissue in diaphragm biopsy cross-sections. The set-up consisted of an IBM compatible computer with a stereology software package (The Gridder, WillRich Technologies, American Megatrends Inc.) and a Nikon light microscope with a camera lucida. Using the software program, a grid consisting of 63 point-intercepts arranged in a 7 x 9 rectangular pattern was projected from the computer monitor via the camera lucida onto the image of the muscle cross-section. The top left field of view of the muscle cross-section was brought into focus at a rrragnification of 500x (eyepiece lOx, optivar 1.25, objective 40x). The tissue occupying the smallest discernible region in the top left quadrant of each point-intercept was sequentially identified and assigned to a specific category (see Table 1) using the number keyboard. Photographs were used for reference when assigning point-intercepts to categories. The software program caused point-intercepts to flash one at a time, beginning with the top left point-intercept on the grid and ending with the lowest right point-intercept. Once assigned, point-intercepts ceased flashing, thus ensuring that all points were counted once. Once all 63 point-intercepts on the grid had been assigned in this manner, the software program tabulated the total count for each category and saved this information on a floppy diskette. The muscle section was manually advanced to an adjacent field of view in a horizontal or a vertical direction. For each muscle section, all possible non-overlapping fields were counted in this standardized manner. Area fractions of normal muscle, abnormal muscle and connective tissue were calculated using the equations presented in Table 2. Prior to point counting, it was detemiined that, if a fibre contained two abnormal features, the point-intercepts would be assigned to a particular category using the following protocol. If an 59 TABLE 2: Equations to Calculate Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue in Thoracotomy Subjects Total Count £ Categories 1 to 8 Area Fraction Normal Muscle = SCategorvl x^nn Total Count Area Fraction Abnormal Muscle Y Categories 2. 5. 6. 7. 8 * inn Total Count Area Fraction Connective Tissue £ Categories 3 and 4 x inn Total Count Note: See Table 1 for category definitions. 60 even number of point-intercepts had been projected upon the fibre, an equal number of the point-intercepts would be assigned to the two "abnormal" categories reflected by the two observed abnormal features. In the case where an uneven number of point-intercepts had been projected upon the fibre, certain features were to dominate others, in the following order of importance: internal nuclei, lipofuscin pigmentation, small angulated or small basophilic fibres. If inflammatory cells were present in the muscle fibre, all point-intercepts which had been projected upon that fibre were classified in the inflamed/necrotic category, regardless of whether other abnormal features were present. Reliabihty was established prior to point counting. Identical categories, definitions and point counting protocols were used by the investigator, Nori MacGowan, and the supervisor, Dr. W. Darlene Reid, during determination of inter-rater reliability. Inter-rater reliability was established to be 0.84 for the area fraction of normal muscle and 0.87 for the area fraction of abnormal muscle. Intra-rater reliability was established by the investigator, Nori MacGowan, to be 0.96 for the area fraction of normal muscle and 0.94 for the area fraction of abnormal muscle. Quantitative Evaluation of Macrophages Macrophages were quantified using the multipurpose image analysis system, Bioview (Infrascan, Richmond, BC). Muscle cross-section images were viewed at a magnification of 125x using a light microscope (Nikon, Microphot-FX, Tokyo, Japan). High resolution colour images of sequential fields of view were captured using a video camera (25.4 mm Vidicon, 60 Hx resolution, 81 series, DAGE-MTI Inc., Michigan City, IN) and displayed on a 20-inch colour monitor (1024 x 1024 pixels, 24 bit) (Sony Multiscan HG, Tokyo, Japan). Using Bioview, a rectangular large unbiased counting frame was superimposed on the image of the muscle section displayed on the monitor. To avoid double counting features which were located on the edges of 61 the counting frame, two edges were designated as "inclusion" edges (dashed lines), and the other two were designated as "exclusion" edges (solid lines). A stage micrometer (Nikon), delineated in tenths of a millimetre, was used to check the calibration of the counting frame prior to set-up each day. The area of the counting frame was calculated to be 0.24 mm2. Macrophages and muscle fibres were counted if they were located within the counting frame, or if they touched an "inclusion" edge of the counting frame. Macrophages and muscle fibres located outside the counting frame or touching an "exclusion" edge were excluded from the counts, regardless of whether they also touched an "inclusion" edge. All fields of view were captured on the monitor and evaluated in this manner until at least fifteen fields had been analyzed per section. Total numbers of macrophages, muscle fibres and fields were tabulated for each biopsy. The number of macrophages per fibre was calculated by dividing the total number of macrophages by the total number of muscle fibres. The number oi macrophages per mm2 was calculated by dividing the total number of macrophages by the total area evaluated. Total area was calculated as the product of the number of fields evaluated and the area of the counting frame. Chart Review A chart review was completed for each subject. The Data Collection Sheet has been included in Appendix E. Each chart was assigned a random three-digit number, which was matched to the random two-digit biopsy numbers and the subject identities on a master list. The master list was retained in a secure filing cabinet to ensure confidentiality. This also ensured that the investigator, Nori MacGowan, was blinded to biopsy identities and to their respective clinical data during biopsy evaluation and subsequent analysis. 62 Spirometry values were obtained for each subject from routine pre-operative pulmonary function testing reports. All pulmonary function tests were conducted in a pulmonary function laboratory by a registered respiratory therapist utilizing standard protocols as per ATS Standards of Care (American Thoracic Society, 1995). Percentage of predicted forced expiratory volume in one second (% predicted FEVi) values were obtained from the laboratory reports. The reference equations utilized by the laboratories have been included in Appendix F. Validity of spirometric measures was ensured through the use of routine calibration procedures in the respective pulmonary function laboratories. As per recommendations by the American Thoracic Society (1995), spirometric measures were repeated until a minimum of three acceptable forced expiratory curves were achieved and the best two out of three forced expiratory volume curves were within 5% of each other. Rehability was ensured through the use of standard testing protocols recommended by the American Thoracic Society (1995). These protocols included the use of nose clips, standard subject positioning, and standard instructions. STUDY DESIGN Study design was a descriptive post-hoc non-experimental analysis of relationships between histological evidence of diaphragm injury and clinical measures of airflow obstruction. Histological evidence of diaphragm injury was obtained by examining muscle biopsies excised during thoracotomy surgery. Clinical measures of airflow obstruction were obtained from pulmonary function testing conducted prior to surgery. STATISTICAL ANALYSIS The Pearson product moment correlation coefficient (r) was used to determine the relationship between the % predicted FEVi and four variables: area fraction of abnormal muscle, area fraction of normal muscle, number of macrophages per fibre, number of macrophages per mm2. Significance of the correlation coefficients was tested using one-tailed t-tests. A significance level of p<0.05 was chosen. RESULTS Subject Descriptive Characteristics The sample was one of convenience. No subjects were excluded by the investigator as a result of the first three exclusion criteria, all of which related to clinical characteristics. Ten subjects were excluded because of the fourth criteria, inadequate biopsy size or quality (see next section). Descriptive characteristics of the twenty-one subjects included in the study are presented in Table 3. Subjects included eight females and thirteen males. Included subjects were undergoing thoracotomy surgery for lung resection (n=18), bullectomy (n=l), or lung transplant (n=2). Diagnoses included lung cancer (n=17), emphysema (n=2), bullous disease (n=l), and bronchiolitis obliterans (n=l). Mean age was 62 +10 years (range: 43 to 81 years). Mean body mass index (BMI), defined as the ratio of the weight (kg) over the square of the height (m2) (Heimburger & Weisnier, 1997), was 24.6 ± 3.9 kg/m2 (range: 16.7 to 31.1 kg/m2). Mean % predicted FEVi was 74 ± 34 % (range: 16 to 122 %). 64 TABLE 3: Descriptive Characteristics of Thoracotomy Subjects Subject Age l l l l l Gender B M I l i i l l l l Surgery Diagnosis % predicted F E V i 1 59 F 23.1 R pneumonectomy lung cancer 111 2 71 M 25.1 L pneumonectomy lung cancer 113 3 66 F 20.4 R pneumonectomy lung cancer 120 4 71 M 29.8 LLL resection lung cancer 67 5 49 M 24.9 L pneumonectomy lung cancer 45 6 67 M 18.2 RLL resection, RUL wedge lung cancer 60 7 62 M 19.0 RUL & chest wall resection lung cancer 78 8 55 M 16.7 lung transplant emphysema 16 9 81 M 24.9 RLL resection, RUL wedge lung cancer 104 10 54 F 31.1 RML resection lung cancer 86 11 76 M 25.7 LLL resection lung cancer 62 12 68 M 24.2 L bullectomy bullous disease 27 13 55 F 22.0 LUL wedge bronchiolitis obliterans 30 14 46 M 29.8 lung transplant emphysema 16 15 72 F 25.2 RUL resection lung cancer 120 16 71 M 25.2 RLL resection lung cancer 89 17 48 F 23.9 RML & RUL resection lung cancer 122 18 43 M 24.9 R pneumonectomy liing cancer 64 19 59 M 26.9 RLL resection lung cancer 67 20 67 F 30.8 RUL wedge lung cancer 72 21 58 F 24.3 R pneumonectomy lung cancer 80 Mean 62 8M 24.6 74 SD 10 13 F 3.9 34 Min 43 16.7 16 Max 81 31.1 122 A B B R E V I A T I O N S : BMI - body mass index; % pred F E V i - % predicted forced expiratory volume in one second; yrs - years; M - male; F - female; R - right; L - left; L L - lower lobe; ML -middle lobe; UL - upper lobe; wedge - wedge resection; SD - standard deviation; Min - minimum; Max - maximum. 65 Qualitative Evaluation All biopsies were relatively small and contained a portion of epimysium There was marked variation in the amount of muscle tissue in the biopsies. Ten biopsies contained large amounts of fat and connective tissue, with few or no muscle fibres; these biopsies were excluded from further analysis due to poor muscle biopsy quality. In the twenty-one biopsies retained for quantitative evaluation, biopsy size ranged from approximately 1 mm x 1 mm x 1 mm (1 mm3) to approximately 5 mm x 8 mm x 1 mm (40 mm3). Normal features in H & E diaphragm cross-sections included polygonal-shaped fibres, fibres with peripherally-located muscle nuclei, fibres with homogeneously stained acidophilic cytoplasm, and closely packed fibres with scant endomysial connective tissue containing spindle-shaped fibroblast nuclei (Figure 3). Artifact included folding, tearing, ice crystal formation, and widened interstitial spaces. Abnormal features included fibres with one or more internally-located muscle nuclei (Figure 5, upper panel), fibres with subsarcolemmal or perinuclear hpofuscin pigmentation (Figure 5, upper and lower panels), small angulated fibres (Figure 6, upper panel), inflamed fibres (Figure 6, lower panel), and inflammatory cells in the endomysium or perimysium (Figure 6, lower panel). Necrotic fibres and small basophilic fibres were rarely observed. In diaphragm cross-sections processed using monoclonal antibodies and immunohistochemistry, macrophages were distinguished by their deep pink cytoplasm and plasma membrane, which contrasted sharply with the pale basophilic muscle fibres and the moderately basophilic muscle nuclei and nuclei in the extracellular space (Figure 7). Macrophages were scattered throughout the endomysial and perimysial connective tissue layers. A few regions contained two or more closely approximated macrophages in widened regions of the interstitial space. No macrophages were identified within muscle fibres. Figure 5: Photomicrographs of abnormal morphology in H & E stained human diaphragm cross-sections (thoracotomy study). Note internal nuclei (arrows) and hpofuscin pigmentation (arrowheads). Scale bar = 12 urn 6 7 Figure 6: Photomicrographs of abnormal morphology in H & E stained human diaphragm cross-sections (thoracotomy study). Note small angulated fibre (arrow), inflamed fibre (arrowhead), and inflammatory cells in endomysium (asterisk). Scale bar = 12 pm 68 Figure 7: Photomicrograph of macrophages in human diaphragm cross-section (thoracotomy study). Note macrophages (arrows) stained using immunohistochemistry and anti-macrophage monoclonal antibodies (Ber-MAC3, D A K O Corp.). Note large unbiased counting frame in the field of view. Scale bar = 12 urn. 69 Quantitative Evaluation Point counting results for H & E sections (n=21) are presented in Table 4. Mean number of fields analyzed per biopsy was 22 ± 13 fields (range: 5 to 52 fields). The area fractions of normal muscle, abnormal muscle and connective tissue were 66.2 + 9.0 % (range: 43.0 to 81.4%), 17.6 ± 7.2 % (range: 4.2 to 33.6 %), and 16.3 ± 4.2 % (range: 10.4 to 25.3 %), respectively. The largest abnormal muscle category was the internal nuclei category, with a mean area fraction of 14.7 ± 6.9 % (range: 2.7 to 31.7 %). Statistical analysis showed an inverse correlation between % predicted FEVi and the area fraction of abnormal muscle (r = -0.53, p<0.01) (Figure 8, upper panel), and a direct correlation between % predicted FEVi and the area fraction of normal muscle (r = 0.37, p<0.05) (Figure 8, lower panel). The results of macrophage counting (n=18) are presented in Table 5. The mean number ofmacrophages per fibre was 0.41 + 0.18 (range: 0.08 to 0.74). The mean number of macrophages per mm2 was 52 + 19 (range: 18 to 85). There was no correlation (r=-0.05, p>0.05) between the number of macrophages per fibre and the % predicted FEVi (Figure 9, upper panel). There was no correlation (r=0.01, p>0.05) between the number of macrophages per mm2 and the % predicted FEVi (Figure 9, lower panel). 70 TABLE 4: Point Counting Results (Thoracotomy Study) i n It-<U > W 3 5 «!!§ t--m I 00 rf ro CN oo CN m VO rf CN CN rr o CN CN ro o vo vo CN m VO VO 00 ON ro CN rr o rr o ro m CN i a S *S I a I o ° » - i a ° s 11 1/1 *o S CO O ' 2 ° cs S * .S3 * 2 is CD « a o § W O ^ ° 2 1=1 — o o <« •• fe 2 3 H .a | NO o 3' O VO ro ro O O CN CN CN O CN 00 VO VO ON o CN o ON rr CN ro rf CN CN r-m rf CN , rf oo »n ON VO CN r-o CN rf O VO ro ro l l l l i i i i CN ro VO O ro rf VO VO VO in o ON r> o o CN rf vo O in vo ON vo vo 00 VO VO CN VO ON r-» CN rf VO oo o vo 00 CN VO VO O ON O O CO rf 00 mmm fa "° CN o o CN o CN o ON O o ON o l S1 o rf O O rf CN o o o ro O oo o o VO o o 00 o o 00 o o o in o in o 00 o o rf O O r-ro o iKttiffiSigji o ro CN rf CN o CN CN CN in m rf o CN VO ro 81 rf rf t-» ON o oo o ro O N ro O ro rf rf ON rf o ro oo oo rf o rf O ON o CN rf CN he VO o o o o o ON O O m o o o in O m o o o o o CN CN o © VO O O CN O ro O vo o o o o o CN CN o I in >^5 iai o o o o o o o o o o o o CN o VO o o CN o o CN CN o ON in o o oo o o o o o o o o o o o o o o rf o o o o o o o CN CN o 2 a o o o o o o o o o o o o vo o o vo s1 £ 1 o VO t--o o o o rf O ro o VO o o o o o CN o o o o o o O N O o o o o ro O mm III * ll o o o o o o o o o o o ON ro O m o o 00 o o o o o in o o ro © ro o O O o o o o o o o o o o ro o o 00 o o o o o ON ro o mmm :iS5i CN O CN O 00 0511 CN ro VO CN CN ro o ro rf oo CN vo VO VO VO vo vo m r-m VO ON CN 00 o |S' ON CN ON O O CN rf vo ro O CN O m VO CN m VO ON VO VO CN ro oo VO VO VO m o ON oo CN rf VO 00 r-rf CN ON 00 o vo o CN O N CN O 00 CN rf CN VO VO c es I ON VO o o O N O CN o o ro rf ro oo ca 3 71 « S u o u < S 0.90 -r 0.80 • -0.70 • -0.60 - -0.50 - -0.40 •• 0.30 - -0.20 - -0.10 •-0.00 - -r=0.37, p<0.05 1 1 1 50 100 150 % predicted FEVi Figure 8: Relationship between airflow obstruction and proportion of normal and abnormal diaphragm muscle (thoracotomy study). There is an inverse relationship (r = - 0.53, p<0.01) between % predicted FEVi and area fraction abnormal muscle (upper panel), and a direct relationship (r = 0.37, p<0.05) between % predicted FEVi and area fraction normal muscle. TABLE 5: Number of Macrophages per mm2 and per Fibre in Diaphragm of Thoracotomy Subjects Subject % predicted llliiiiiiffllillili!!!!! Macrophages per mm2 Macrophages per Fibre 4 67 47 0.74 5 45 18 0.22 6 60 47 0.29 7 78 86 0.48 8 16 61 0.23 9 104 47 0.43 10 86 67 0.40 11 62 40 0.33 12 27 59 0.63 13 30 62 0.43 14 16 55 0.34 15 120 24 0.08 16 89 41 0.72 17 122 78 0.37 18 64 52 0.51 19 67 39 0.46 20 72 34 0.46 21 80 82 0.23 Mean 74 52 0.41 SD 34 19 0.17 Min 16 18 0.07 Max 122 86 0.74 ABBREVIATIONS: SD - standard deviation; Min - minimum; Max - maximum 73 0.8 Qi u 0.7 • • Xi to 0.6 • r=-0.05, ;es per 0.5 • p>0.05 'nnhnfl Upil<l£ 0.4 0.3 • • • • w w a 0.2 • • • =tfc 0.1 • 0 i i i i i i 0 50 100 150 % predicted FEV, Figure 9: Relationship between airflow obstruction and prevalence of macrophages in diaphragm (thoracotomy study). There is no relationship (r = 0.01) between % predicted F E V i and number of macrophages per mm2 (upper panel). There is no relationship (r = -0.05) between % predicted F E V i and number of macrophages per fibre (lower panel). 74 DISCUSSION In this study we found that an increased severity of airflow obstruction is associated with an increased proportion of abnormal diaphragm muscle and a decreased proportion of normal diaphragm muscle (Figure 8). These relationships were found in a group of individuals fit enough for thoracotomy surgery but with a large range of airflow obstruction (% predicted FEVi range: 16 to 122 % predicted). This study is the first to report a quantitative description showing the proportion of abnormal diaphragm morphology in individuals with COPD. Similar abnormal morphology was found in the diaphragm, compared to abnormal features previously described in other muscles of respiration, including internal nuclei (Campbell et al., 1980; Hards et al., 1990, Lindsay et al., 1996), inflammatory cells (Hards et al., 1990), necrotic fibres (Hards et al., 1990), basophilic fibres (Hards et al., 1990), and small angular fibres (Campbell et al., 1980). Similar morphological features have been described previously in the diaphragm, including internal nuclei (Lindsay et al., 1996), inflammatory or phagocytic cells (Silver & Smith, 1992; Kariks, 1989), necrotic fibres (Silver & Smith, 1992), basophilic fibres (Silver & Smith, 1992), and small angular fibres (Silver & Smith, 1992; Kariks, 1989). None of the previous studies, however, quantified the extent of the abnormal respiratory muscle morphology, thus increasing the difficulty in comparing and contrasting the results of the present study with previous research. In the present study, approximately 15% of the cross-sectional area represented muscle fibres with internal nuclei. Previous research has not quantified the area fraction of fibres with internal nuclei, but expressed their prevalence as a percentage of total fibre counts. Thus our results may not be directly compared with the published normal values of 2 to 3% (Heffher, 1989) and 4% (Cumming et al., 1994) in normal limb muscles, and 5 to 10% in the Hmb muscles of subjects with neuromuscular disease (Heffher, 1989). Our results do reveal, however, that of the 75 point-intercepts which projected upon muscle fibres during point-counting (versus elements of the extracellular matrix), approximately 22% projected upon muscle fibres with internal nuclei, indicating a relatively large cross-sectional area of fibres with internal nuclei in our study. Muscle fibres with internal nuclei may be fibres which are undergoing or which have undergone regeneration. Chronic low-intensity ventilatory loading associated with chronic airflow obstruction may stimulate diaphragm fibre nuclei to assume an internal location in order to direct ongoing regenerative efforts in response to muscle damage. Alternatively, some diaphragm fibre nuclei may remain internally located following cessation of regeneration. Internal nuclei may also be reflective of pending muscle fibre degeneration. Finally, it is possible that internal nuclei may reflect fibre type transformation, a suggestion proposed by Lindsay and co-workers (Lindsay et al., 1996), who observed internal nuclei and abnormal (neonatal) myosin expression in the diaphragm of individuals with heart failure. This study showed a relatively low percentage of the diaphragm cross-sectional area to be represented by inflamed or necrotic fibres. There were also few inflammatory cells (round nuclei) in the interstitium and diaphragm morphology was not characterized by an inflammatory response. These findings contrasted with those of the animal models of resistive ventilatory loading, in which diaphragm injury was characterized by necrotic fibres, flocculent degeneration of the cytoplasm, and an influx of inflammatory cells in the interstitium and in necrotic fibres (Reid et al., 1994; Jiang et al., 1998a). The difference in our study was not altogether unexpected, however, since subjects did not experience acute increases in ventilatory loading. Hards and co-workers reported necrotic fibres and inflammatory cells (Hards et al., 1990), but they did not describe the morphology nor extent in detail, and thus it is difficult to compare our results. The necrotic fibres observed in the present study differed from the necrotic fibres previously described by Silver and Smith (1992), and Kariks (1989). These investigators described fibres with a loss of cross-76 striatums, hypereosmophilia and a hyaline appearance. In contrast, the necrotic fibres observed in this study were denned as a mass of inflammatory cells and cellular debris, with breakdown of the plasma membrane. Previous investigators have also described macrophage (Silver & Smith, 1992) or phagocyte (Kariks, 1989) invasion in muscle fibres, a finding which has been interpreted to indicate regeneration (Silver & Smith, 1992) or healing (Kariks, 1989). The rare incidence of necrosis in our study, as compared to these two previous reports (Silver & Smith, 1992; Kariks, 1989), may be related to the difference in subject characteristics. In the reports by Silver and Smith (1992), and Kariks (1989), subjects experienced acute respiratory distress and eventually died, while in our study, subjects did not experience acute respiratory distress and biopsies were obtained during surgery. Deposits of hpofuscin pigmentation have been described in human vastus lateralis three days post-eccentric exercise, a finding which was interpreted to indicate an acute increase in lysosomal protein degradation (Friden, 1984). To the investigators knowledge, our study is the first to report hpofuscin in diaphragm muscle. Lipofuscin pigments are residual bodies which result from lysosomal autolysis of lipids (Groer, 1989). Lipofuscin is sometimes referred to as an "age pigment", since it is most common in the long-lived cells of the nervous system (Groer, 1989). An increased presence of hpofuscin in skeletal muscle may be reflective of an increased lysosomal activity over the lifetime of the involved fibre, but whether this relates primarily to age or to overuse of a muscle fibre is unknown. Abnormal diaphragm morphology in our study was not characterized by regenerating fibres, which are typically characterized by basophilic sarcoplasm and enlarged nuclei with visible nucleoli (Heffher, 1989). Small basophilic fibres likely represented regenerating fibres, which may have been similar to the regenerating basophilic fibres described by Silver and Smith (1992) 77 in the diaphragm of eight infants who survived an acute respiratory insult for some time. However, as previously stated, they were not a prominent morphological feature in our study. Small angular fibres described in our study may represent fibres which have atrophied due to malnutrition, disuse or steroid myopathy (Heffher, 1989). Similar atrophic fibres were described in previous reports (Kariks, 1989; Campbell et al., 1980), but their significance was not discussed. Since our study did not control for nutrition, past history of mechanical ventilation, nor corticosteroid ingestion, the relevance of this feature in our study is uncertain. The results of our study contrast those of Hards and co-workers, who found no relationship between the incidence of accessory muscle abnormalities and pulmonary function measures (Hards et al., 1990). This difference may be attributed to the possibility that more injury is present in the diaphragm than in the accessory muscles of respiration, a finding which has been noted in animal models of resistive ventilatory loading (Jiang et al., 1998a) and in individuals with heart failure (Lindsay et al., 1996). Indeed, the external and intercostal muscles are believed to be mainly postural muscles which are inactive or minimally active during resting or stimulated breathing. The parasternal intercostal muscles are more active but were not sampled by Hards and co-workers (Hards et aL 1990). Our findings may also have differed because a broader range of subjects was evaluated. Specifically, the inclusion of subjects with severe airflow obstruction may have improved the power of our correlation analysis. Lastly, our study evaluated "abnormal muscle" as a ratio variable (proportion) rather than as an ordinal variable (absence or presence). It is possible that injury severity is reflected better by a continuum of severity, rather than by its presence or absence, and that diaphragm injury may have been more effectively evaluated in our study because subtle as well as more pronounced abnormalities were included in the quantitative description. 78 The location of macrophages in the endomysium was not an unexpected finding, since tissue macrophages are classified in histology as connective tissue cells (Burkitt et al., 1993). However, our study confirms their presence in the diaphragm and demonstrates an effective technique to identify macrophages, using immunohistochemistry and anti-macrophage monoclonal antibodies. Our study did not show a relationship between the number of macrophages and the degree of airflow obstruction (% predicted FEVi) (Figure 9). While conclusions cannot be definitively drawn, there are two ways to interpret the lack of relationship: either the relationship between airflow obstruction and prevalence of macrophages does not exist or, alternatively, it may be more complex than this analysis allowed. The latter explanation is certainly possible, given the multiple roles of macrophages and their complex interactions within the body, and also given that the time-course of injury was not controlled in this study. Further analysis of distinct subpopulations of macrophages may show the evolution of their expression with increased airflow obstruction, similar to the distinct subpopulations found in rat limb muscle after acute muscle injury (St. Pierre et aL 1994). Antibodies have been developed to identify these subsets of macrophages in rat skeletal muscle (St. Pierre et al., 1994). To the investigator's knowledge, however, antibodies to identify distinct subsets of macrophages have not been developed in human models. Thus in the present study, it was not possible to identify resident macrophages with different functions, nor to distinguish them from macrophages which may have been attracted to the site of injury as circulating monocytes. Macrophages proliferate in response to various factors secreted by fibroblasts and platelets and the extent of such a response may be significantly affected by many factors including blood supply, age, malnutrition and medications. Since none of these factors were controlled in this study, it is possible that they were a source of variability and interaction, resulting in inconclusive results. Other variables including the presence of malignancy, recent weight loss, length of illness 79 and history of radiation or chemotherapy treatment were not controlled. These may have also contributed to variability in inflammatory events and the inconclusive results concerning the presence of macrophages. There were some limitations in the present study. The sample size was relatively small. It was unfortunate that ten of the thirty-one biopsies were excluded due to inadequate size and/or quality. However, given the difficulty of sampling the diaphragm, the risks associated with causing a diaphragm defect due to a cut or a tear, and the need to obtain the biopsy quickly so as not to prolong surgery in individuals with increased surgical risk due to respiratory disease, this was not an unreasonable outcome. As noted, there was also a certain amount of uncontrolled variability in subject clinical characteristics, including age, nutritional status, medication history, presence of malignancy, length of illness, and medical treatments received. Restricting subjects based upon these clinical measures would not only have limited recruitment, but also would likely have resulted in a sample that was not representative of the population of interest. Defining subjects solely by their % predicted FEVi values may have resulted in the exclusion of relevant clinical information during definition of disease severity. However, even if measures of some of the other variables had been available for all subjects, some of these likely would have had unknown or unacceptable reliability. In the present study, the % predicted FEVi was available for all subjects and it allowed for the evaluation of severity of airflow obstruction as a continuous variable. Histology allows for direct examination of diaphragm muscle structure and identification of pathological reactions in muscle biopsies, but it is limited in that it provides insight only into structures present at a given point in time, without any indication of previous or pending events. The structAffe-function relationship is a key concept within histology, but inferences regarding function cannot be made based upon structural evidence alone (Burkitt et al., 1993). Thus, while 80 the abnormal diaphragm muscle features observed in this study may have been suggestive of diaphragm dysfunction, further study would be required to elucidate the role and significance of the observed diaphragm abnormalities, including their relationship with clinical variables. Examining single cross-sections in this study was another limitation because structural features of three- dimensional cells were identified from examination of two-dimensional images. Since the orientation, size and shape of a morphological structure affect their appearance on cross-section, morphological features may have been over- or under-counted. For example, since serial sections were not studied, focal or segmental injury may have been missed. Fibres which were identified as small angular fibres may have in fact been normal fibres which were tapering. Finally, since multiple diaphragm biopsies were not obtained, our data is representative of the sampled region of the diaphragm only (the costal diaphragm), and not the diaphragm muscle as a whole. This study showed that as severity of airflow obstruction increases, the proportion of abnormal diaphragm increases and the proportion of normal diaphragm decreases. The prevalence of macrophages in the diaphragm was not related to the severity of airflow obstruction, and may have been affected by many uncontrolled factors. Diaphragm injury in this study was described using systematic, quantitative and reproducible methodology. Directions for future research will be discussed in the final chapter of the thesis, Chapter 4. 81 CHAPTER 3: POST-MORTEM EVALUATION OF DIAPHRAGM INJURY ABSTRACT Many factors associated with chronic respiratory disease may overload the diaphragm muscle and possibly predispose it to dysfunction and/or injury. The nature and prevalence of diaphragm injury in humans who experience increased ventilatory loading remains unknown, as does its proposed role in the etiology of ventilatory failure. The objectives of this study were to describe diaphragm morphology in individuals who died of various causes, and to determine the contribution of key clinical factors to the amount of abnormal diaphragm morphology. Full-thickness diaphragm biopsies were obtained from tliirty-three subjects during autopsy (16 females and 17 males). Thirteen subjects had chronic respiratory disease and twenty-two had acute respiratory disease. Age ranged from 29 to 85 years. Mean body mass index was 25.7 kg/m2 (range: 14.1 to 38.8 kg/m2). Subjects were heterogeneous in clinical presentation and cause of death. Biopsies were fixed in formalin, embedded in paraffin, and stained with H & E and Masson's trichrome. Area fractions of normal muscle, abnormal muscle and connective tissue were determined by point counting diaphragm cross-sections. Step-wise multiple regression was used to determine the relationships between the proportion of abnormal diaphragm muscle and the following clinical factors: chronic respiratory disease, acute respiratory disease, age, body mass index, and gender. Abnormal features included internal muscle nuclei, necrotic or inflamed fibres, small angulated fibres, fibres with abnormal nuclear or cytoplasmic morphology, and fibres with a contracted or degenerating appearance. The area fraction of abnormal muscle was not significantly correlated with any clinical factors. However, there was a trend towards a direct relationship with the presence of chronic respiratory disease. Additional investigation with a 82 larger sample size is warranted to further define this relationship and to identify other contributing clinical factors. INTRODUCTION Diaphragm injury has been shown to occur in animal models when the workload of breathing is increased for a few hours (Jiang et al., 1998a) or for a few days (Reid et al., 1994). Diaphragm injury in animal models has been associated with ventilatory failure (Reid et al., 1994) and with reduced force-generating ability (Jiang et al., 1998b). To date, few investigators have studied diaphragm injury in humans, although two studies have described morphological abnormalities in the accessory muscles of mdfviduals with COPD (Hards et al., 1990; Campbell et al., 1980). Two reports have described diaphragm injury in newborns post-mortem (Kariks, 1989; Silver et al., 1992). Silver and Smith (1992) described contraction band necrosis in about one-third of the infants, with the most frequent lesions occurring in infants who died of asphyxia or sudden infant death syndrome (SIDS). Similar contraction band necrosis was described in the diaphragm in twelve out of thirteen adults who died of various causes, with the most extensive injury observed in an mdfvidual who had died of status asthmaticus (Silver & Smith, 1992). Kariks (1989) described contraction band necrosis in 80% of infants who died of SIDS. In a few cases, where the infant survived the respiratory insult for some time, there were features which indicated healing, including inflammatory cells (predominantly macrophages) and fibrous scarring (Kariks, 1989). Previous studies have described diaphragm injury using descriptive terms and qualitative methods. The contribution of various clinical factors has been described but not evaluated systematically. To the investigators knowledge, the extent of diaphragm injury has not been compared between mdividuals with and without chronic respiratory disease. Similarly, the 83 relationships between the amount of abnormal diaphragm and clinical factors have not been defined. The overall objective of this study was to describe diaphragm morphology in individuals who died of various causes and to determine the contribution of key clinical factors to the proportion of abnormal muscle. The specific aims were to: (1) describe normal and abnormal diaphragm morphology, (2) determine the proportion of normal muscle, abnormal muscle, and connective tissue in the diaphragm, (3) determine the contribution of the following clinical factors to the proportion of abnormal diaphragm muscle: presence of chronic respiratory disease, presence of acute respiratory disease, age, body mass index (BMI), and gender. We hypothesized that (1) there would be abnormal morphology in the diaphragm of these individuals, and (2) the proportion of abnormal muscle would be related to one or more of the identified clinical factors. METHODS Subjects Thirty-three subjects undergoing autopsy were recruited with the assistance of the pathologists and morgue assistants at St. Paul's Hospital and at Vancouver Hospital (Oak St. Site). The only inclusion criterion was that the subject's next-of-kin had consented to an unrestricted autopsy. Consent to autopsy forms for St. Paul's Hospital and for Vancouver Hospital (Oak St. Site) have been included in Appendices G and H respectively. Exclusion criteria were as follows: (1) individuals who had died more than 96 hours prior to autopsy, (2) individuals with known or suspected infection with Hepatitis B virus, Hepatitis C virus, human immunodeficiency virus (HTV), or Creutzfeldt-Jakob Disease, (3) forensic autopsy cases. Muscle Sampling and Processing Routine autopsy procedures occurred in the morgue at the two hospital faculties. Standard procedures included a midline Y-shaped incision and removal of a portion of the anterior chest wall by cutting through the sternum and the antero-lateral ribcage. The stomach and intestines were ligated and removed from the abdominal cavity. The contents of the thoracic cavity were removed in a "block" after severing their attachments at the cranial and caudal ends, and after severing the crural and costal attachments of the diaphragm muscle. At St. Paul's Hospital full-thickness diaphragm biopsies (n=26) were obtained by the morgue assistant using a sharp scalpeL prior to removing the thoracic cavity contents. At Vancouver Hospital (Oak St. Site), full-thickness diaphragm biopsies (n=7) were obtained by the investigator, Nori MacGowan, in a likewise manner but after the thoracic contents had been removed from the body. With both techniques, the diaphragm was maintained in its usual anatomic orientation with respect to the body organs and specific effort was made to avoid stretching the muscle tissue. In each case, the biopsy was obtained from the mid-costal region, lateral to the central tendon and medial to the ribcage insertion. The region near the central tendon was avoided because myotendinous regions have abnormal features including numerous internal nuclei, increased collagen between muscle fibres and increased variation in fibre size (Heffher, 1989). Biopsy thickness varied from a few millimetres to approximately 8 millimetres. Length and width dimensions were approximately 2 cm x 2 cm for all biopsies. Each muscle biopsy was placed flat in a small storage cassette, then fixed in 10% formalin after ten minutes. Biopsies were transferred into 70% alcohol after 48 hours. They were embedded in paraffin, sectioned at 5 pm thickness, and stained with H & E, and Masson's trichrome. Biopsies obtained from St. Paul's Hospital were processed in laboratory facilities in the Department of Academic Pathology at the University of British Columbia. Biopsies obtained from Vancouver Hospital (Oak St. Site) were processed on-site by the Department of Anatomic Pathology. Cross-sections and longitudinal sections were mounted on slides and each slide was labeled with a coded two-digit random number. An animal study was conducted, with the purpose of studying post-autolytic changes in skeletal muscle. Six male Sprague Dawley rats were sacrificed by injection with sodium pentobarbital and placed in a 4 °Celsius refrigerator two hours post-mortem. Muscle biopsies were excised from the rats at the following intervals: 2, 24, 48 and 72 hours. One-half of each biopsy was fixed in formalin, dehydrated, embedded, sectioned, and stained with H & E and with Masson's trichrome. The other half of each biopsy was quick frozen, sectioned and stained with H & E. Muscle sections were examined in a similar manner to that described below for qualitative evaluation of human tissue. Qualitative Evaluation of Diaphragm Cross-sections Diaphragm cross- and longitudinal sections were exarnined under the light microscope at 125x and 500x magnification. Overall morphology, biopsy quality, normal features and abnormal features were noted. Artifact was observed and documented. An experienced pathologist was consulted to provide an unbiased opinion regarding "real" and "artifactual" abnormalities prior to description and analysis of the muscle morphology. Structural features were classified into nine categories (Table 6). Categories were defined and detailed criteria were established for later use during quantitative evaluation (see next section). Descriptors used to define the observed features were obtained from surgical pathology textbooks (Hefmer, 1989) and atlases (Cumming et al., 1994). 86 TABLE 6: Categories and Definitions for Point Counting (Post-Mortem Study) CATEGORIES 1. Normal 2. Internal Myonucleus 3. Abnormal Cytoplasm 4. Small Angular Fibre 5. Inflamed or Necrotic 6. Collagen or Fibroblast 7. Inflammatory Cell 8. Degenerating or Contracted °. Capillary 0. No count = space, nerve, artifact, epimysium, wall or lumen of vessel larger than capillary, post-mortem autolysis DEFINITIONS 1. Fibre with homogeneous acidophilic cytoplasm, intact plasma membrane, peripheral nuclei, polygonal in shape 2. Fibre containing > 1 internally located myonucleus 3. Includes: (a) fibre with pale acidophihc peripheral cytoplasm and enlarged peripheral nuclei with or without visible nucleoli, or (b) fibre with pale acidophilic peripheral cytoplasm and deep acidophihc "fuzzy" cytoplasm in central region 4. Small fibre (< one-tenth diameter of adjacent fibres) with > 2 oblique angles 5. Includes: (a) fibre with intact plasma membrane containing at least one inflammatory cell nucleus, or (b) necrotic mass of inflammatory cells and muscle debris 6. Includes: (a) protein fibrils of endomysium or perimysium, or (b) spindle-shaped nucleus in interstitium 7. Round nucleus located in interstitium, with morphological features of mononucleated inflammatory cell 8. Includes: (a) normal-sized or large fibre with deep acidophihc 'Tiizzy" cytoplasm, some with central basophilia, cytoplasmic fragmentation, or vacuolization; or (b) fibre with dull light- or grey-staining cytoplasm, cytoplasmic fragmentation, and few or no muscle nuclei 9. Endothelial cell, lumen or contents of a capillary 87 The nine categories included one category representing normal muscle, six categories representing abnormal muscle, and two categories representing connective tissue components. Prior to quantitative evaluation (see next section), it was determined that, if a fibre contained more than one abnormal feature, certain features would dominate other features when fibres were assigned to a particular category. Fibres containing inflammatory cells were to be assigned to the inflamed or necrotic category, regardless of other abnormalities present. Likewise, fibres containing internal muscle nuclei (not inflammatory cells) were to be assigned to the internal myonucleus category regardless of other abnormalities present. The no count category was defined to include empty space, nerve, vessel wall or lumen (other than capillary), connective tissue surrounding a vessel wall (up to twice the thickness of the wall), epimysial connective tissue, artifact (folding, tearing, edge), and post-mortem autolysis (described in the results section). Rat muscle sections were examined under the same conditions, and abnormal features were described and defined using specific categories. Quantitative Evaluation of Diaphragm Cross-sections Computer-assisted point counting was used to quantify the amount of normal and abnormal morphology in diaphragm cross-sections. The set-up consisted of an IBM compatible computer with a stereology software package (The Gridder, WillRich Technologies, American Megatrends Inc.) and a Nikon light microscope with a camera lucida. Using the software program, a grid consisting of 90 point-intercepts arranged in a 9 x 10 rectangular pattern was projected from the computer monitor via the camera lucida on the image of the diaphragm muscle section. 88 A field of view in the top left region of the muscle section was brought into focus at a magnification of 500x (eyepiece lOx, optivar 1.25x, objective 40x). The tissue occupying the smallest discernible region in the top left quadrant of each point-intercept was sequentially identified and assigned to a specific category (see Table 6). Photographs and specific criteria listed in Table 6 were utilized when assigning point-intercepts to categories. Using the software program, each point-intercept flashed in turn, beginning with the top left point-intercept and ending with the lowest right point-intercept. Once assigned, the point-intercept stopped flashing and the point-intercept to be categorized next began to flash, thus ensuring that all point-intercepts were counted once. Once all 90 point-intercepts had been "counted "in this manner, the muscle section was manually advanced to the adjacent field of view in either a horizontal or a vertical direction, to a region just adjacent to the previously counted field. For each section, all possible non-overlapping fields were counted in this standardized manner, up to a maximum of 35 fields. The total counts for each category were tabulated by the software program and saved on a floppy diskette. The area fractions of normal muscle, abnormal muscle, and connective tissue were calculated utilizing the equations listed in Table 7 and the categories listed in Table 6. Prior to point counting, intra-rater reliability was calculated for normal and abnormal muscle categories using identical definitions (Table 6), calculations (Table 7) and protocols. Intra-rater reliability was r = 0.96 for abnormal muscle and r = 0.93 for normal muscle. Chart Review A detailed chart review was completed for each subject. The information sought from the clinical records and the autopsy reports is presented in the Data Collection Sheet (Appendix I). Information included date of birth, gender, height, weight, history of respiratory disease, 89 TABLE 7: Equations to Calculate Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue in Post-Mortem Subjects Total Count = X Counts in Categories 1 to 9 Area Fraction of Normal Muscle = ^Counts in Category 1 x ] nn Total Count Area Fraction of Abnormal Muscle = Y, Counts in Categories 2 through 7 x inn Total Count Area Fraction of Connective Tissue = X Counts in Categories 8 and 9 x ion Total Count Note: Categories have been defined in Table 6. 90 medication history, admission diagnosis, cause of death, pre-mortem clinical findings and post-mortem pathological findings. Confidentiality was maintained by assigning a random three-digit identifying number to each chart review. These numbers were matched to subject identities and the two-digit biopsy numbers on a master list. The master list was kept in a secure filing cabinet to which only the investigator, Nori MacGowan, and the supervisor, Dr. W. Darlene Reid, had access. STUDY DESIGN The study design was a post-hoc non-experimental descriptive analysis of relationships between histological evidence of diaphragm injury and potential contributing clinical factors. The proportion of abnormal diaphragm was determined by examining post-mortem diaphragm biopsies. Clinical characteristics relating to pre-mortem health status were obtained from a review of the subjects' medical records and autopsy report. STATISTICAL ANALYSIS Step-wise multiple regression analysis (SPSS, Chicago, EL) was performed to determine the contribution of clinical factors (presence of chronic respiratory disease, presence of acute respiratory disease, age, gender, body mass index) to the proportion of abnormal diaphragm muscle. The area fraction of abnormal muscle, a continuous variable, was the dependent variable. Two ratio variables (age, body mass index) and three ordinal variables (presence or absence of chronic respiratory disease, presence or absence of acute respiratory disease, gender) were the 91 independent variables. A significance level of p<0.01 was selected for the multiple regression model. Initially, all six variables were included in the regression model. In this model, only the subjects for whom all information was available (n=26) were included in the analysis. Forward selection and backward elimination were used to verify the results of the step-wise analysis. Variables which were found to contribute significantly to the step-wise regression model were then entered into a simple linear regression model, so that the most complete data set for that independent variable could be evaluated. Significance for simple regression was selected to be p<0.05. To rmximize the sample size using step-wise multiple regression, body mass index (BMI) was excluded as a variable in the second multiple regression model, since BMI had the least complete data set (n=26). Forward and backward step-wise regression were performed using the remaining four independent variables on all thirty-three subjects. Power analysis was conducted on variables which were not significant but indicated a strong trend, in order to determine the sample size which would be required to achieve significance. RESULTS Clinical Profiles Comprehensive clinical profiles of the subjects are presented in Appendix J and a summary of the key clinical variables utilized during statistical analysis is presented in Table 8. Sixteen females and seventeen males were included in the study. Age ranged from 29 to 85 years; fifteen subjects were between 65 and 85 years, fourteen subjects were between 50 and 64 years 92 TABLE 8: Descriptive Characteristics of Post-Mortem Subjects Subject Gender Age (>TS) BIN I (kc/m2) CRD Description ARD Description 1 F 29 16.9 Yes CF Yes pneumonia (7d) 2 F 77 24.8 No Yes post-obstructive pneumonitis (4d) 3 F 73 21.4 No No 4 M 52 30.7 No No 5 M 64 20.0 Yes COPD Yes bronchopneumonia (2d) with hypoxemia 6 M 37 n/a No No 7 F 63 35.4 No Yes ARDS, hypoxemic & hypercapnic RF 8 M 72 22.1 Yes COPD, emphysema No 9 M 84 n/a No No 10 F 57 15.2 No Yes bronchopneumonia, atelectasis, hypoxemic RF 11 F 84 22.0 Yes COPD No 12 F 78 32.5 Yes severe emphysema Yes hypercapnic RF 13 M 64 30.8 Yes asthma Yes pneumonia, hypoxemic RF 14 F 72 n/a Yes asthma, emphysema Yes hypoxemic RF 15 F 74 25.3 Yes emphysema Yes ARDS, hypoxemic RF 16 M 50 31.5 No Yes bronchopneumonia, pulmonary edema 17 M 66 n/a Yes chronic pulmonary edema No 18 M 84 24.8 Yes chronic bronchiolitis Yes bronchopneumonia (lid), hypoxemia 19 F 82 n/a No Yes acute pulmonary edema 93 TABLE 8 (continued): Descriptive Characteristics of Post-Mortem Subjects Subject Gender Age (yrs) BMI (kg/m2) CRD Description ARD Description 20 M 54 38.8 No No 21 M 82 n/a No No 22 M 64 33.1 No Yes ARDS, hypoxemic & hypercapnic RF 23 F 85 27.2 No Yes bronchopneumonia 24 M 76 27.0 Yes COPD Yes acute pulmonary edema 25 F 48 18.3 No Yes pneumonia (7d) 26 M 52 32.7 No Yes acute pulmonary edema, hypoxemia 27 F 60 26.9 Yes asthma Yes pneumonia, hypoxemic & hypercapnic RF 28 F 68 15.2 No No 29 F 51 30.1 No Yes severe CHF (24h), pulmonary infarct 30 M 57 29.1 No Yes ARDS 31 F 53 14.1 No No 32 M 52 n/a No Yes pneumonia 33 M 60 22.5 Yes COPD Yes bronchopneumonia Mean 16F, 17M 25.7 13 22 SD 6.7 Min 29 14.1 Max 85 38.8 ABBREVIATIONS: yrs - years; F - female; M - male; BMI - body mass index; CRD - chronic respiratory disease; ARD - acute respiratory disease; d - days; h - hours; RF - respiratory failure; ARDS - adult respiratory distress syndrome; COPD - chronic obstructive pulmonary disease; CHF - congestive heart failure; SD - standard deviation; Min - minimum; Max - maximum; n/a -not available. 94 and four subjects were between 29 and 49 years. From previously published definitions, adults are considered to be between 20 and 65 years (Black & Hyatt, 1969), and elderly adults to be between 65 and 85 years (Enright et al., 1994). Using this age classification scheme, the age distribution in this study was relatively even, with fifteen "elderly" subjects and eighteen "adult" subjects. When considered as a ratio variable, however, age was not normally distributed and was therefore not described using the mean and standard deviation. Body mass index (BMI) was distributed symmetrically, with thirteen subjects above and thirteen subjects below the mean of 25.7 kg/m2 (+ 6.7 kg/m2). Mean BMI was within the defined 'normal" range, where the lower bruit is 18.5 kg/m2 for men or women (Naidu & Rao, 1994) and the upper limit is 27.3 kg/m2 for women or 27.8 kg/m2 for men (Williamson, 1993). BMI values for subjects in the present study showed a broad range, with the lowest value (14.1 kg/m2) representing malnutrition and the highest value (38.8 kg/m2) representing moderate obesity. Using BMI as an index, five subjects were malnourished and ten subjects were obese. It is noted that BMI values were available for only twenty-six of the thirty-three subjects. Of the tjiirty-three subjects, thirteen were classified with chronic respiratory disease, twenty-two with acute respiratory disease, ten with acute superimposed upon chronic respiratory disease, and eight with neither chronic nor acute respiratory disease. Chronic respiratory disease encompassed the following diagnoses: cystic fibrosis (n=l), COPD (n=7), chronic bronchiolitis (n=T), asthma (n=3), chronic pulmonary edema (n=l). Presence or absence of chronic respiratory disease was determined by the investigator after extensive review of each subject's clinical history and pathological findings during autopsy. In order for a subject to be classified with chronic respiratory disease, clinical symptoms characteristic of chronic respiratory disease were required to be present unless there was strong pathological evidence suggestive of clinically significant chronic respiratory disease. In these cases, a pathologist was consulted to clarify and confirm the 95 significance of the pathological findings. Clinical symptoms characteristic of chronic respiratory disease included a history of frequent chest infections, altered chest wall mechanics in clinical examination notes (barrel chest, hyperinflation), abnormal pulmonary function tests, and/or a medication history which included bronchodilators or inhaled corticosteroids. Acute respiratory disease encompassed the following diagnoses: pneumonia or bronchopneumonia (n= 10), ARDS (n= 4), multisystem organ failure with hypercapnic respiratory failure (n=l), hypoxemic respiratory failure (n= 1), post-obstructive pneumonitis resulting from carcinoma of the lung (n=l), acute pulmonary edema (n=3), severe congestive heart failure with acute pulmonary infarct (n=l), bronchopneumonia with sepsis (n=l). The presence or absence of acute respiratory disease was determined by the investigator following a review of each subject's clinical history and pathological findings during autopsy. For all subjects in this study, adequate clinical evidence was available to determine the presence or absence of acute respiratory disease. In all cases, therefore, the pathological findings served only to confirm the clinical symptoms. The clinical symptoms characteristic of acute respiratory disease included an elevated respiratory rate, poor arterial blood gases, difficulty weaning from a ventilator, and chest x-ray indicative of active chest disease. Additional clinical information was obtained for most of the subjects and some of this data has been presented in Appendix J. It includes information such as admission diagnosis, cause of death, cardiac history, mechanical ventilation, smoking history, resuscitation history and corticosteroid medication history. Subjects were noted to be extremely heterogeneous in these clinical variables. Cause of death included cardiac causes (myocardial infarct, arrhythmia, cardiogenic shock, coronary artery disease with electrolyte imbalance, acute coronary insufficiency) (n=13), respiratory causes (respiratory failure, inoperable lung carcinoma, ARDS, pneumonia/bronchopneumonia, pulmonary embolism) (n= 9), infection (sepsis, pancreatitis, 96 peritonitis, ruptured diverticulitis, acute viral hepatitis) (n=5), cancer (inoperable lung carcinoma, carcinomatosis peritonei, prolymphocytic leukemia) (n=3), and other (liver failure, anoxic brain damage, multifactorial) (n=3). Thirteen subjects were mechanically ventilated for time periods ranging from 1 hour to 21 days. Some subjects were paralyzed with neuromuscular blocking agents during ventilation and others were reported to experience mtermittent periods of asynchrony with the ventilator. Fifteen subjects were resuscitated at least once during the three days prior to their death, for a period ranging from 17 minutes to 2 hours. Ten patients had been prescribed significant amounts of corticosteroid medications, including low-dose short-term regimes (n=2), low-dose long-term regimes (n=5) and high-dose short-term regimes (n=2). Smoking histories included lifetime non-smokers (n=12), previous smokers who had quit more than 10 years ago (n=5), and present smokers (n=9) whose smoking history ranged from 20 to 60 pack years. Cardiac disease was the most frequent comorbidity, including severe coronary artery disease (n=8), mild to moderate coronary artery disease (n=6) and other significant cardiac history (n=9). Qualitative Evaluation of Diaphragm Cross-sections In general, the biopsies were well-preserved and all sections contained at least 15 viable well-oriented cross-sectional fields, as observed using a light microscope (Nikon) at 500X magnification. The most common preparation artifact was shrinkage of the tissue resulting in widened interstitial spaces and connective tissue fibrils that were spread apart. Other artifacts included folding and tearing of the tissue. Normal muscle features included polygonal fibres with flattened peripherally located nuclei. This has been described in Table 6 (Category 1) and illustrated in Figure 10. The most frequently observed abnormal feature was muscle fibres which contained internal myonuclei. 97 Figure 10: Photomicrograph of normal human diaphragm morphology in H & E stained cross-section (upper panel) and cross-section stained with Masson's trichrome (lower panel). Note polygonal shaped fibres, peripheral muscle nuclei, and scant endomysium with capillaries and fibroblasts. Scale bar =12 urn This is described in Table 6 (Category 2) as the internal myonucleus category and it is illustrated in Figure 11. The criteria for this category was that there was visible cytoplasm between the muscle nucleus and the plasma membrane. Nearly all biopsies contained some fibres with a distinctly abnormal-appearing cytoplasm (Table 6, Category 3), although this feature was much less common than the internal nuclei. In these fibres, the plasma membrane appeared to be intact and the muscle nuclei were peripherally located. However, the cytoplasm and muscle nuclei exhibited unusual morphology. All fibres in this category exhibited a pale granular acidophihc peripheral cytoplasm Muscle nuclei were peripherally-located but distinctly enlarged; most nuclei contained visible nucleoli. Other fibres in this category exhibited a central region with deep acidophilic "smudged-looking" cytoplasm and a pale granular peripheral cytoplasm Occasionally hpofuscin pigmentation was observed in these fibres. These features are illustrated in Figure 12. Another abnormal feature was defined as degenerating or contracted fibres (Table 6, Category 8; Figure 13). Fibres in this category were often large (greater than twice the size of adjacent fibres) and exhibited a deeply acidophihc "fuzzy" or 'nyaline-appearing"cytoplasm throughout. Some of these fibres had central purplish-grey regions and some fibres had regions of empty space. The latter regions resembled cytoplasmic fragmentation or vacuolization. Finally, there were some fibres in this category which were normal or slightly small in size and which showed evidence of disintegrating or disintegrated sarcoplasm which was slightly grey and "fuzzy" in appearance. Other fibres showed even more pronounced evidence of cytoplasmic fragmentation; these fibres had few or no muscle nuclei and a "dull" grey- staining cytoplasm It was noted that the latter group of fibres in this category were often located adjacent to relatively large, otherwise normal-appearing muscle fibres. Figure 11: Photomicrograph of abnormal morphology in cross-section of human diaphragm stained with Masson's trichrome. Note internal nuclei (arrows) and angulated fibre (arrowhead)(lower panel). Scale bar = 12 pm 100 Figure 12: Photomicrograph of abnormal morphology in cross-section of human diaphragm stained with Masson's trichrome. Note necrotic fibre (arrow) and fibre with enlarged nucleus and granular peripheral cytoplasm (arrowhead). Scale bar = 12 urn Figure 13: Photomicrograph of abnormal morphology in cross-section (upper panel) and longitudinal section (lower panel) of human diaphragm stained with Masson's trichrome. Note fibres with contracted and degenerating appearance (arrows). Scale bar =12 urn. 102 Less common abnormal features were muscle fibres which were small and angulated but appeared otherwise viable (Table 6, Category 4) (Figure 11), and necrotic or inflamedfibres which were rarely noted (Figure 12). Biopsies from the animal study showed only one morphologic abnormality. This abnormality, defined as post-mortem autolysis, was distinct from the abnormalities observed in the human tissue. Fibres with this abnormality showed decreased staining in central regions of H & E cross-sections and no abnormalities were observed in longitudinal sections. This abnormality was not observed in biopsies obtained within two hours of death, and it was observed occasionally in biopsies obtained 72 hours post-mortem All human diaphragm biopsies were exarnined for the presence of this abnormahty. It was observed occasionally in a few diaphragm cross-sections, and excluded from the counts (recorded as a no count). Quantitative Evaluation Results of point counting are presented in Table 9. The mean area fraction ofnormal muscle was 74.6 ± 7.1 % (range: 60.1 to 88.9 %). The mean area fraction oi abnormal muscle was 11.4 ± 5.3 % (range: 2.4 to 21.3 %). The mean area fraction oi connective tissue was 14.0 + 4.3 % (range: 6.9 to 23.6 %). The most common abnormal feature was internal myonuclei, which had a mean area fraction of 6.9 ± 5.0 %. Internal myonuclei was the only abnormal feature which was present to some extent in all muscle biopsies, however its presence ranged from an area fraction of 1.2 % to an area fraction of 19.0 %. The features represented in the abnormal cytoplasm category (see Table 6 for definition) showed a mean area fraction of only 1.4 %, but again, the range was broad (range: 0 to 10.9 %). Similarly, the degenerating or contracted category showed a mean area fraction of only 1.3 %, and a broad range (range: 0 to 9.5 %). 103 TABLE 9: Point Counting Results (Post-Mortem Study) O N + Conn-ective Tissue ro 00 o i n CN CN 00 V O CN V O CN t—i O N 1—< O N i n O N O N O O N O o CO CN l> m r f O N ro CN o CN O N V O O V O f - r> ro r f O N o O N V O O r f V O m 00 o r -ro m 2 to 7 1 « i s CN O N O o r f CN O o O N O o ro 00 CN r f CO O CN o O N CN O r f CN V O O 00 O N m o CN CO o 00 r-o CO CN ro m o V O o 00 00 CN r f O i n o ro m r— 00 o B « = 3 i n CN r-ro ro r> r f 00 CN O N r-rr m V O V O r f r-t--r f OO O N ro t~-CN r-00 O i n r-O N V O r> 00 V O i n r f V O O V O m r f oo CN O 00 oo r-^  r— V O ro o 00 r- O N 00 oo O N t— V O m oo r-oo r f r-CN V O r-Q i n ro O CN O V O o CN o i n CN o ro o vo o m CN o o CN O o o V O o ro r f O r f O ro CN O i n o o o o ro CN O m o o O N O O oo O o r f CN o CN CN o oo CN O r f O O OO ? fe 3 u £ O N r f r f r f V O O V O o O N i n O N o ro O r f ro O N t--o O N O N O ro O N O O r-ro r f O N o V O r f O V O o CN V O 00 O V O o o r f ro vo O O N O r-r f r> i o £ g Ml V SI | r f O O O o o o o 00 ro o o o o i n O N o O O o CN O o o o o O O O r f r-o o o o o o o o o o o o 00 ro o O N O O N O O i n o o m r f O r-o o r-o o O o o O O O O N o o tt 1 st s a (S r 41 O O O o CN o o m o o O N O r f O o CN o o o o CN o o O O O N O O o r-o ro CN O o o o V O o o ro O O V O O O m o o r-o o o o o m CN O o o CN O o m o <y~> i i O O r f O r-o o O N o o r f O o o o o o o o CN o o o o o O O o O O r f O o r f O o ro o O o o o o o o O O CN O o o o 00 o o o o o ro O ro o o ro O o r f O o *+ £ « s a < tt N O o o O O o o o m o o I T ) o ro o o r f o o O N o o r f o o m o o CN O o O N o o 00 o o vo o CN o o CN o o ro O O ro CN O o o ro O o r f O O O N o i n o o ro O o ro o CO F o S g >. « J U a o O O o o o O N o o o o o o o o o o CN o o o O N O o o r f o o CN o o o o r f o o CN o o CN O CO o o o ro o O N o r f O O i n o r-o 00 O O vo o CN I s = z o N O o r f C-O o ro CN O ro O N o r> CN o 00 CN o 00 m O CN o o V O CN O 00 t-o 00 m ro vo r f CN o o ro O i n o O N o t--o O CN o vo o r f O O N O N o o ro o •g c fc m CN CO ro r-» r f I T ) 00 CN O N r~~ r f m V O V O r f r--r f 00 O N ro t--CN f -00 o m r-O N V O oo V O m r f vo o V O m r f 00 CN O oo 00 r> V O ro o 00 r- O N 00 00 O N r-V O m 00 t-00 r f r-CN V O t-•** if s CN ro rr V O oo O N o CN ro r f m V O f- 00 O N o CN CN CN CN ro CN r f CN m CN 104 T A B L E 9 (continued): Point Counting Results (Post-Mortem Study) m cN v a © t> ~ U £ H a & 553 a. <3 SP 1 — ;•"* *g fe S i I © .5 2 +3 — » 5 S i mmmm twi S mmm © • I on 1 2 fe ~ 'S ON CN ON CN O I a o • »H t> 03 03 o ^ 5 s § •« J -s << ns ji ^ O t « (A 3 ' a o 0 cn fl . 2 os § 2 8 1 a • o <U • rt 73 o co 03 a * 2 s 2 «8 CO l i s 511 105 Box plots (Figure 14) show the relationship between each of the ordinal independent variables (presence of chronic respiratory disease, presence of acute respiratory disease, gender) and the dependent variable (area fraction of abnormal muscle). Two-way scatterplots (Figure 15) show the relationship between each of the ratio independent variables (age, body mass index) and the dependent variable (area fraction of abnormal muscle). Step-wise regression analysis using all six variables and twenty-six subjects (14 females and 12 males) showed that male subjects had a greater area fraction of abnormal diaphragm muscle than females (p<0.01); however, simple linear regression showed no significant gender effect (p=0.117) (Figure 14, lower panel). Step-wise regression analysis using five variables and all thirty-three subjects showed no significant relationships for any of the other clinical factors except for a trend towards a direct relationship between the presence of chronic respiratory disease and the proportion of abnormal diaphragm (p=.067) (Figure 14, upper panel). Power analysis indicated that a sample which included at least thirty-four subjects with chronic respiratory disease and thirty-four subjects without chronic respiratory disease would be necessary in order to achieve significance (p<0.05) in the relationship between the proportion of abnormal muscle and the presence of chronic respiratory disease. An additional twenty-one subjects with chronic respiratory disease, and fourteen subjects without chronic respiratory disease, would therefore be required. It would also require that the variability in the dependent and independent variables in this larger sample was equivalent to the variability in these variables in the present study. J> 106 Figure 14: Boxplots showing relationships between proportion of abnormal diaphragm muscle and chronic respiratory disease (upper panel), acute respiratory disease (middle panel), and gender (lower panel). Note the trend towards a direct relationship between the area fraction of abnormal muscle and the presence of chronic respiratory disease (upper panel). Shaded area indicates inter-quartile range, midline indicates median, lines projecting from ends indicate the range of values. C? 30 JD I O CO 3 2 m 20 h CO E o c < c g o ro ro <D co cu 10 h Absent (n=20) Present (n=13) Chronic Respiratory Disease Co 30 JD O V) 3 ro 20 E u. o c .Q < C o o ro 1 0 Absent (n=11) Present (n=22) Acute Respiratory Disease Female (n=16) Male (n=17) Gender 108 25 -I (0 Area Fraction Abnorrr Muscle (%) 20 -15 -10 -5 -A A A A ^ * AAV. AA 1 1 1 1 1 1 1 1 u 1 1 1 1 1 1 1 1 20 30 40 50 60 70 80 90 100 Age (years) Figure 15: Scatterplots showing no relationship between area fraction of abnormal diaphragm and age (upper panel), and no relationship between area fraction of abnormal diaphragm and body mass index (lower panel). 109 DISCUSSION This study showed that there were morphological abnormalities in the diaphragm, and that the prevalence of these abnormalities varied considerably within a heterogeneous sample of thirty-three subjects. Diaphragm abnormahties were interpreted to represent pre-mortem structural changes, since they were distinct from the post-mortem autolytic changes observed in an animal study where other variables were controlled. We found fibres with internal myonuclei in all diaphragm biopsies (mean: 6.0 + 5.0 %; range: 1.23 to 19.03 %). Our results may not be directly compared with the reported 'normal" values of 2 to 3 % (Heffher, 1989) or 4 % (Cumming et al., 1994) internally nucleated fibres, since these values represent the percentage of total fibres in limb muscles. However, the wide range in the area fraction of internally nucleated fibres (range: 1.23 to 19.03 %) in the diaphragm of our subjects reflects marked between-subject differences and may indicate that internal nuclei are an important marker in some ongoing pathological process. The presence of internal myonuclei in mature muscle fibres is classified as an abnormal response with nonspecific etiology in pathology textbooks (Banker & Engel, 1994). This feature is often interpreted to reflect regenerated muscle, where the muscle nuclei or satellite cell nuclei have assumed a central position in order to direct regenerative processes (Carlson & Faulkner, 1983). Internal nuclei are also prominent features in dystrophic (Banker & Engel, 1994) and chronic myopathic (Cumming et al., 1994) conditions. Internal nuclei have been described as markers of injury (Reid et al., 1994), degeneration (Kuipers et al., 1983) or regeneration (Faulkner et al., 1993) in healthy skeletal muscle subjected to acute overload. In the present study, the greater proportion of internally nucleated fibres in the diaphragm of some subjects may indeed reflect either a pronounced regenerative response or the residual effects of repetitive muscle injury. 110 The clinical significance of the other abnormal diaphragm features in our subjects is difficult to ascertain, mostly because very little has been reported on these features. Abnormal features such as enlarged nuclei, visible nucleoli, and pale or basophilic sub sarcolemmal cytoplasmic staining have been described as possibly representative of fibres which have been challenged and have mounted a regenerative response (Carlson & Faulkner, 1983). Enlarged peripheral nuclei may in fact be satellite cells (Carlson & Faulkner, 1983). Basophilic peripheral cytoplasm likely reflects increased polyribosomal activity (Cumming et al., 1994), which could be indicative of protein synthesis and replenishment of the myofibrillar and/or cytoskeletal framework. Sub sarcolemmal regions with decreased staining may also reflect an increase in the size (due to swelling) and/or number of cytoplasmic organelles, although there is no evidence to support this . Such a response by mitochondria could occur in response to increased metabolic demands either due to increased contractile activity or possibly to regenerative activity. In contrast, muscle fibres described as contracted and/or degenerating (Table 6, Category 8) may represent fibres which have been challenged and have apparently been unable to mount a successful response. These fibres may be similar to those described by Kariks (1989), in which terminal acute changes had occurred to the muscle fibres. Fibres with ''fuzzy" cytoplasmic staining may represent regions of Z-band streaming (Cumming et al, 1994). Such abnormalities have also been described as comprising regions of coagulated protein with myofibrillar disorganization, and classified as "hyaline degeneration" of skeletal muscle fibres (Sifverberg, 1990). The loss of internal structural detail in these fibres perhaps was similar to the "hyahnzation" defined by Banker and Engel (1994). Fibres which appeared thus but which also contained inflammatory cells may have represented either an earlier or, more likely, a later response which resulted in fibre invasion by leukocytes and subsequent necrosis. Finally, some fibres appeared entirely non-viable, with no visible muscle nuclei and evidence of cytoplasmic I l l degradation. These may have been similar to the fibres described by Carlson and Faulkner (1983), in which severe muscle damage had resulted in myonuclei "death" and "dissolution". The trend towards a direct relationship (p=.065) between the proportion of abnormal diaphragm and the presence of chronic respiratory disease (Figure 14, upper panel) is interesting because it is consistent with our hypothesis, and with the results reported in Chapter 2 of this thesis. It is also consistent with the animal studies which have clearly demonstrated an abnormal response in the diaphragm of animal subjected to ventilatory loading (Reid et al., 1994). Based upon the power analysis, sample size would have to be slightly more than doubled in order to obtain statistical significance at a level of p<0.05. Given the heterogeneity of other clinical characteristics in our sample, this trend is encouraging and it warrants further investigation. The initial significant relationship between gender and abnormal morphology was an unexpected finding. It is possible that there was a true significant relationship between gender and abnormal muscle in the sample of twenty-six subjects (this excluded the seven subjects for whom height and weight were not available), but since significance was lost when all thirty-three subjects were included in the analysis, it must be concluded that there was no relationship between gender and abnormal muscle for the sample population. This conclusion was drawn because the smaller sample size was selected based upon missing data rather than upon any clinically relevant criteria. The lack of a relationship between gender and proportion of abnormal muscle (Figure 14, lower panel) neither supports nor refutes current evidence, since the prevalence of overload-induced muscle injury in males versus females has not been described in the literature. The present study did not show any relationship between the proportion of abnormal diaphragm muscle and some clinical factors which have been proposed to have a role in the etiology of diaphragm dysfunction and/or injury. These factors included: age, nutritional status, and acute respiratory disease. Aging has been associated with many functional deficits in 112 respiratory muscles, including decreases in strength and endurance and increases in the predisposition to injury. In the present study, there was no relationship between age and the proportion of abnormal diaphragm muscle (Figure 15, upper panel). Age was evaluated as a ratio variable, but perhaps it is a factor which either has a significant role only at a certain threshold level, or which interacts with other factors to exacerbate injury but does not act alone. Given the heterogeneity of the sample, it is also possible that an aging effect was masked by other confounding variables which could have significantly affected the findings and prevented the discovery of a true relationship. However, since the scatterplot (Figure 15, upper panel) shows no evidence to indicate a trend, it must be concluded that either there is no relationship or that the aging effect is more complex than the present analysis allowed. Nutritional status has also been identified as an important factor in the pathogenesis of respiratory muscle dysfunction and ventilatory failure (Poole et al., 1997). Respiratory muscle dysfunction could result from a loss of contractile elements or from a derangement in their composition. These changes could result in observable structural damage at the light microscopic level. In the present study, however, there was no relationship between an index of nutritional status (body mass index) and the proportion of abnormal muscle (Figure 15, lower panel). Similar to age, nutrition may perhaps be more effectively evaluated as an ordinal variable, where values below a certain threshold levels are evaluated for their effect upon diaphragm morphology. It is possible that values above a certain threshold level contribute abnormal diaphragm morphology, since the restrictive deficit associated with obesity may significantly increase the burden upon the ventilatory pump (see Chapter 1 for explanation). The scatterplot (Figure 15, lower panel), however, is suggestive of neither low nor high threshold values. As with aging, it is possible that nutritional status is a factor with significant interactive effects, such that its effect alone may be difficult to identify. 113 The lack of a relationship between the proportion of abnormal muscle and the presence of acute respiratory disease (Figure 14, middle panel) was initially surprising, since many factors associated with acute respiratory disease may either increase diaphragm workload (increased respiratory rate), reduce diaphragm efficiency (acute hyperinflation), or cause diaphragm weakness (mechanical ventilation). Additional challenges associated with acute respiratory disease include oxidative stress, electrolyte imbalance, catecholamine surges during resuscitation attempts, and high-dose corticosteroid ingestion. However, it is possible that because these factors were extremely variable within the sample of tmrty-three subjects, the sample may have been too heterogeneous and too small for a true effect to have been shown. The most important strengths of the present study are the evidence it provides regarding the presence and the diversity of morphological abnormahties in the diaphragm of individuals who died of various causes, and the interesting trend towards an increased prevalence of these abnormahties in mdividuals with chronic respiratory disease (Figure 14, upper panel). Another important strength of this study is that it provides a detailed qualitative and quantitative description of diaphragm morphology. Since this has been poorly defined using only qualitative measures and non-systematic methodology in previous studies, the present study has provided useful information for further research. This study is hmited by the small and heterogeneous sample. Because the nature and prevalence of the morphological abnormahties in our subjects were extremely varied, and because their clinical characteristics were also extremely varied, extraneous variables may have limited the ability to describe the relationships between the variables of interest. Some of these variables may have included: ingestion of neuromuscular blocking agents during mechanical ventilation (possibly contributing to neuropathic- or myopathic-type skeletal muscle changes), metabolic factors such as poor arterial blood gases, electrolyte imbalance and sepsis. 114 Additional investigation should he directed towards further defining the potential relationship between chronic respiratory disease and abnormal diaphragm morphology in human subjects. Conclusions and recommendations will be discussed in Chapter 4 of this thesis. 115 CHAPTER 4: SUMMARY AND RECOMMENDATIONS COMPARISON OF THE TWO RESEARCH STUDIES Both research studies presented in this thesis addressed the issue of diaphragm injury in humans with chronic respiratory disease. Both studies examined mid-costal diaphragm morphology at the light microscope level, and used quantitative methodology (point counting) to describe the findings. There were, however, some methodological differences between the two studies. In the thoracotomy study, relatively small partial-thickness biopsies were obtained, and they were quick frozen. In the post-mortem study, larger full-thickness biopsies were obtained, and they were fixed in formalin. The other significant difference between the two studies was in the clinical characteristics of the subjects. Although some subjects in the thoracotomy study presented with severe airflow obstruction, all had been deemed healthy enough for surgery. In contrast, subjects in the post-mortem study had either been gravely ill prior to death, or had died suddenly of cardiac or respiratory arrest. There was greater heterogeneity of clinical characteristics in the post-mortem subjects. Both studies provided descriptive histological qualitative and quantitative evidence that morphological abnormalities are present in the diaphragm of these individuals. Some morphological abnormalities were similar (internal nuclei, small angular fibres, necrotic or inflamed fibres). Of these, an increased presence of internal nuclei was the most commonly observed abnormality in both studies (14.7 + 6.9% in thoracotomy study; 6.9 + 5.0% in post-mortem study). This abnormality may reflect past or present regenerative efforts of the involved muscle fibres. The lower prevalence of this abnormality in the post-mortem study may have reflected differences in the respiratory status of the subjects. In contrast to the subjects in the thoracotomy study, all of whom had clinically significant COPD and/or carcinoma of the lung, 116 only tiiirteen of the tJiirty-three post-mortem subjects had clinically significant chronic respiratory disease. The prevalence oi small angular fibres and necrotic or inflamedfibres was nearly the same in both studies. Round nuclei in the interstitium was similar (1.0 + 0.6% in thoracotomy study; 0.7 + 0.7% in post-mortem study). Lipofuscin appeared to be more prevalent in the thoracotomy study (0.9 + 5.0%), warranting its inclusion as a separate category for quantitative evaluation. This feature, in contrast, was rarely observed in the post-mortem biopsies and it was not quantified as a separate category. Thus this feature cannot be compared across the two studies, other than by commenting that it was more prevalent, as a 'qualitative' observation, in the thoracotomy subjects. Degenerating or contracted fibres were only observed in the post-mortem biopsies. The overall prevalence of this feature was low (1.3 + 2.2%), but it was extremely prevalent in some biopsies (maximum 9.5%) and not present at all in other biopsies (minimum 0.0%). This feature had similar morphological characteristics to the contraction band necrosis lesion defined and described by others who had conducted post-mortem studies (Silver & Smith, 1992; Kariks, 1989). The observation of this feature only in biopsies from individuals who had died supported the suggestion that this abnormality may be related to critical illness, cardiopulmonary resuscitation, catecholamine surges, or other pre-mortem events. Both research studies tested the association between quantitative histological evidence of diaphragm injury and one or more clinical variables. Since both studies considered injury as a ratio variable, injury was described as a "proportion of abnormal muscle", rather than as the presence or absence of''injury" per se. The analysis in the thoracotomy study was simpler than that of the post-mortem study, as the only relationships tested were between the proportion of abnormal diaphragm, normal diaphragm, and severity of airflow obstruction. The significant 117 direct correlation between the proportion of abnormal diaphragm and airflow obstruction, and the significant inverse relationship between the proportion of normal diaphragm and airflow obstruction supported the results of animal research which has shown a greater prevalence of diaphragm injury in animals with elevated airflow resistance as compared to animals with normal airflow resistance. These results (thoracotomy study) contrasted with previous human research, in which no relationship was found between morphological abnormalities in the accessory respiratory muscles and measures of airflow obstruction (Hards et al., 1990). A possible explanation for the difference is that the diaphragm may have a greater response than the accessory muscles. Another possible explanation is that our study included subjects with more severe airflow obstruction, and thus perhaps a greater predisposition to injury. In addition, the quantitative methods in our (thoracotomy) study may have allowed for the identification of subtle but important diaphragm "injury" which may be more effectively described as a proportion rather than as a threshold value (i.e.: present versus absent). The analysis and interpretation in the post-mortem study was more complex due to the population studied and the number of relationships examined. The trend towards a direct relationship between an increased proportion of abnormal diaphragm and the presence of chronic respiratory disease is encouraging, given that there were many extraneous variables related to clinical status which could have affected the analysis. Although the analysis was more complex than the thoracotomy study, it is unlikely that it captured all extraneous variables which may have influenced diaphragm morphology in these individuals. 118 SUMMARY 1. Both studies have shown that morphological abnormalities are present in the human diaphragm Some may be indicative of acute injury {inflamed or necrotic fibres), while others (lipofuscin) may represent evidence that acute injury resulting in autolysis of muscle components has occurred at some time in the past. Other abnormalities (internal nuclei, pale peripheral cytoplasm, enlarged nuclei with visible nucleoli) are likely indicative of regenerative efforts, but whether they reflect past or present efforts may not be determined given the descriptive design of the two studies in this thesis. Finally, one interesting abnormality (contracting or degeneratedfibres) may be reflective of terminal acute changes in the muscle fibres associated with critical events, where earlier stages are reflected by condensed and disorganized sarcoplasm and later stages are reflected by cell breakdown and myonuclei death. 2. Due to the post-hoc descriptive research designs, neither study may draw conclusions regarding mechanisms or stages of such diaphragm "injury". However, the thoracotomy study clearly indicates that the extent of diaphragm "injury" is greater in individuals with more severe airflow obstruction (an indicator of severity of COPD). The post-mortem study provides additional, though statistically inconclusive, indications that more "injury" may occur in the diaphragm of individuals with chronic respiratory disease. 3. The evidence from the thoracotomy study indicates clearly that macrophages are present in the connective tissue layers of the diaphragm Their lack of presence within the muscle fibres of the thoracotomy subjects is consistent with the finding of few necrotic fibres in the diaphragm of these subjects (0.5 + 0.7 %) and suggests that diaphragm "injury" characterized 119 by cell breakdown and death was not a common finding. In the connective tissue, macrophages may be directing regeneration of the muscle fibres or they may be involved in turnover of connective tissue elements. In any case, the presence of macrophages and chronic respiratory disease is either not related, or its relationship is too complex as to be discerned with the research design of this (thoracotomy) study. RECOMMENDATIONS 1. 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Vlachos-Mayer H., Smountas A.A., Sinsky A., & Lands L.C. (1996). Respiratory muscle strength and diaphragm muscle thickness in patients with cystic fibrosis (abstract). American Journal of Respiratory and Critical Care Medicine. 153(4). A789. Wait J.L., Staworn D., & Poole D.C. (1995). Diaphragm thickness heterogeneity at functional residual capacity and total lung capacity. Journal of Applied Physiology. 78(3). 1030-1036. Wakai Y., Leevers A.M., & Road J.D. (1994). Regional diaphragm shortening measured by sonomicrometry. Journal of Applied Physiology. 77(6). 2791-2796. Watchko J.F., Johnson B.D., Gosselin L.E., Prakash Y.S., & Sieck G.C. (1994). Age-related differences in diaphragm muscle injury after lengthening activations. Journal of Applied Physiology. 77. 2125-2133. West J.B. (1992). Pulmonary Pathophysiology - the Essentials (4th ed.). Baltimore, MD: Williams &Wilkins. Wilcox P.G., Hards J.M., Bockhold K , Bressler B., & Pardy R.L. (1989). Pathologic changes and contractile properties of the diaphragm in corticosteroid myopathy in hamsters: comparison to peripheral muscles. American Journal of Respiratory Cell & Molecular Biology 1(3). 191-199. Williamson D.F. (1993). Descriptive epidemiology of body weight and weight change in U.S. adults. Annals of Internal Medicine. 119(7 Pt 2). 646-649. Zamora C.A.& Anzueto A.A. (1992). Cystic fibrosis. In S.G. Jenkinson (Ed.) Obstructive Lung Disease, (pp.97-125). New York, NY: Churchill Livingstone. 130 Zhu E., Petrof B.J., Joaguin G.E.A., Comtois N. & Grassino A.E. (1997). Diaphragm muscle fibre injury after inspiratory resistive brealmng. American Journal of Respiratory and Critical Care Medicine. 155. 1110-1116. 131 A P P E N D I X A T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A School of Rehabilitation Sciences Faculty of Medicine T325 - 2211 Wesbrook Mall Vancouver. B.C. Canada Vf>T 2B5 Tel: (604) 822-7392 Fax: (MM! 822-7624 Website: http://www.rehab.uhc.ea/srs.hlml Informed Consent Form Respiratory Muscle Pathology Principal Investigator: Dr. Darlene Reid, School of Rehabilitation Sciences, University of British Columbia Phone Number: 822-7402 Co-Investigators: Dr. Guy Fradet, Department of Surgery, Vancouver Hospital Phone Number: 875-8298 Dr. Ken Evans, Department of Surgery, Vancouver Hospital Phone Number: 877-1244 Dr. Jeremy Road, Respiratory Medicine, Vancouver Hospital Phone Number: 875-4122 Dr. Blair Walker, Pathology, St. Paul's Hospital Phone Number: 631-5346 Ext. 2704 Nori MacGowan, School of Rehabilitation Sciences, Phone Number: 822-7171 Background: The work of breathing can be increased in many different conditions that affect the lungs, the muscles that we breathe with, and/or the rib cage. These include conditions such as chronic bronchitis, emphysema, quadriplegia, and severe obesity. In some of these conditions, the breathing muscles may become exhausted and injured because they have to work so hard. The main breathing muscle is the diaphragm which is a dome-shaped muscle located between my stomach and my lungs. This muscle is usually very difficult to study because of its location. Other muscles used to breathe with include the internal and external intercostals which are short little muscles located between each rib. During my surgery, small samples of the diaphragm and intercostal muscles can be excised. After the muscle samples are excised, they can be specially processed in a research laboratory to illustrate different features of the muscle cells. During this processing, the muscle samples will be cut into very thin sections, and then stained with different chemicals to illustrate features that can be examined under a microscope. The investigators of the study can determine if the diaphragm and intercostal muscles have injured cells or abnormal features that may interfere with the normal functioning of these breathing muscles. Purpose: The purpose of this study is to determine the frequency and type of abnormal features and muscle injury in the breathing muscles of people with and without different conditions that can overload these muscles. You have been selected for this study because you have an underlying lung condition requiring further investigation. We hope to 132 Page 2 of 3 determine whether or not this condition is contributing to injury of your breathing muscles. Small samples of muscle will be excised from the diaphragm and the intercostal muscles during my surgery. Following the excision of the muscle samples, they will be processed and then examined under a light microscope. The observations of the muscle samples will be related to different aspects of my medical history including my lung function tests, weight, height, sex, and age. Study Procedures: If I decide to participate in the study, my surgery will be performed in the usual fashion except during the surgery, small samples of muscle will be shaved from the diaphragm and intercostal muscles. A small stitch will be placed in the diaphragm to support the excision she. The sampling of the muscle will only take a few minutes and will not interfere with the progress and success of my surgery. I will not experience any more pain during my post-operative recovery phase than that usually experienced after this type of surgery. No other additional tests or examination, besides those normally performed for preparation for my surgery, are required of me to participate in this study. The investigators will, however, access my charts for information from my medical history. Exclusions: Individuals who do not understand sufficient English to comprehend the Informed Consent Form and to provide informed consent will be excluded from the study. In addition, any individual to whom the surgeons, Dr. Ken Evans or Dr. Guy Fradet, believe that sampling of the diaphragm muscle would cause undue risk will be excluded from the study. Risks: The muscle biopsies will be very small. The only potential risk is a small amount of bleeding which should be easily treated with electrocautery. Benefits: The subject will not have any immediate benefits from the results of this study. A better understanding of the frequency and type of abnormahties and injuries in the breathing muscles will help formulate better treatment for people with breathing problems in the future. Alternative Treatments: not applicable. Confidentiality: Any information resulting from this research study will be kept strictly confidential. All documents will be identified only by a code number and no number or initials that are related to the subject will be used to identify information collected. Only the primary 133 Page 3 of 3 investigator, Dr. Darlene Reid, will have access to the decoded number and patient identification and she will keep this record in a locked cabinet. Remuneration/Compensation: There will be no remuneration or compensation for participation in this study. C o n t a c t : I understand that if I have any questions or desire further information with respect to this study, or if I experience any adverse effects, I should contact Dr. Ken Evans at 877-1244, Dr. Jeremy Road at 875-4122, Dr. Darlene Reid at 822-7402, or Dr. Guy Fradet at 875-8298. If I have any concerns about my treatment or rights as a research subject I may contact the Director of Research Services at the University of British Columbia, Dr. Richard Spratley at 822-9252. Patient Consent: I understand that participation in this study is entirely voluntary and that I may refuse to participate or I may withdraw from the study at any time without any consequences to my continuing medical care. I have received a copy of this consent form for my own records. I consent to participate in this study Patient Signature Date Witness Signature Date Investigator's Signature Date Technique: APPENDIX B 1. Fix in Baker's formalin for 2 rninutes. 2. Rinse three times in tap water. 3. Place in Harris' Hematoxylin for 1 minute. 4. Dip in acid alcohol for 5 seconds. 5. Rinse in tap water. 6. Quickly dip in and out of base just until it turns blue. 7. Rinse well in tap water. 8. Counterstain in eosin for 10 seconds. 9. Dehydrate in two sequential dips in absolute ethanol, 2 seconds each. 10. Clear in HEMO DE, for two rinses. Solutions: Baker's Formalin: Formalin 10 ml Calcium acetate 2 g Water to 100 ml Acid Alcohol: Base: Absolute ethanol 210 ml Distilled water 81.9 ml Concentrated HC1 (37%) 8.1 .ml Sodium bicarbonate (NaHCOs) 1.5 g Distilled water 100 ml Eosin: Stock Solution Eosin Y 1 g Water 20 ml 95% Ethanol 80 ml Working Solution 80% Ethanol 300 ml Stock Eosin 100 ml Glacial Acetic Acid 2 ml 135 APPENDIX C Section Preparation: frozen sections 6 um thick, 2 sections per slide (use slides coated with 3-aminopropylethoxysilane) Fixation: fix in acetone for 10 minutes at room temperature, air dry 30 minutes Method: • 2 drops Tris Buffered Saline (TBS) on shdes, drain & wipe around tissue • add 5% normal rabbit serum, incubate for 15 minutes in humid chamber • drain & wipe around tissue to prevent spreading • add primary monoclonal antibody (Ber-MAC3, DAKO Corp.) or negative control (Igd, DAKO Corp.) at correct dilution in TBS plus 1% bovine serum albumin (1% BSA), incubate for 1 hour in humid chamber, drain • wash with TBS for 5 minutes, drain, repeat • add secondary antibody (rabbit anti-mouse IgG, DAKO Corp.), at correct dilution in TBS plus 1% BSA), incubate for 30 minutes in humid chamber, drain • wash with TBS for 5 minutes, drain, repeat • add APPAP complex (DAKO Corp.), incubate for 30 minutes in humid chamber, drain • wash with TBS for 5 minutes, drain, repeat • prepare substrate (Naphthol AS-B1 phosphate and New Fuchsin), apply immediately to shdes, incubate for 10 minutes • wash with TBS, then tap water Solutions: Tris Buffered Saline (TBS): Mix: 20 ml stock TRIS 20 ml stock NaCl 160 ml distilled water Adjust pH to 7.6. 1% Bovine Serum Albumin (1% BSA): bovine serum albumin 0.05 g TBS 5 ml 5% Normal Rabbit Serum: rabbit serum 50 pL TBS 950 pL 136 Dilution (1:50) for Primary Antibody (Ber-MAC3. DAKO Corp.): Ber-Mac3 (DAKO Corp.) 0.7 uL 1% BSA 999.3 pX Dilution (1:50) for Negative Control Antibody (IgGu DAKO Corp.): IgGl (DAKO Corp.) 1 uL 1% BSA 499 uL Dilution (1:20) for Secondary Antibody (rabbit anti-mouse IgG. DAKO Corp.): IgG (DAKO Corp.) 50 uL 1% BSA 950 uL Dilution (1:50) for APAAP complex (DAKO Corp.): APAAP (DAKO Corp.) 20 uL 1% BSA 980 pX APPENDIX D Method: 1. Dip in Meyer's Hematoxylin for 10 seconds. 2. Rinse several times with tap water until water is clear. 3. Dip in and out of base just until it turns blue. 4. Rinse in tap water. 5. Air dry. 6. Coverslip using a permanent mounting medium Solutions: Meyer's Hematoxylin: Hematoxylin I g Distilled water 1000 ml Ammonium alum 50 g Citric acid I g Chloral hydrate 50 g Sodium iodate 0.2 g Dissolve hematoxylin, alum and sodium iodate in water, heating i f necessary. Add chloral hydrate and citric acid, boil mixture for 5 minutes. Cool and filter. Base: Sodium bicarbonate (NaHCOs) 1.5 g Distilled water 100 ml 138 APPENDIX E Data Collection Sheet: Thoracotomy Study DATE: (d/m/y) GENERAL INTO: ID. Number: Height: cm D O B . : / / Age: Weight: kg Gender: B.M.I.: P A S T M E D I ^ A T . H T S T O R Y . Respiratory: COPD: • Emphysema • Chronic Bronchitis ^ Asthma Smoking Hx Other Medical: cardiac, cancer, athritis, neuromuscular, hepatic, endocrine, etc... Musculoskeletal: Medications: RECENT MEDICAL HISTORY: Sx: Date of Surgery: (d/m/y) / / @ hrs (to hrs) Diagnosis: Pre-op Post-op Comments: Biopsy Site L R Incision: 139 LAB/TEST RESULTS. Pulmonary Function Tests: <_/_/_) FEVi FVC TLC RV FRC % pred FEVi % pred ratio % pred FVC DC L O : DLung/Va: Respiratory Muscle Strength Tests: % pred MIP MEP CXR/CTscan: Arterial Blood Gases: Electrolytes: Stress Test/Exercise Capacity: 140 A P P E N D r X F Test Prediction Equation R 2 95% Confidence Interval * Men: FVC 0.0600H - 0.0214A - 4.650 0.54 1.115 FEVi 0.0414H-0.0244A-2.190 0.64 0.842 FEVi/FVC -0.1300H- 1.152A+ 110.49 0.26 8.28 Women: FVC 0.0491H- 0.0216A- 3.590 0.74 0.676 FEVi 0.0342H - 0.0255A - 1.578 0.80 0.561 FEVi/FVC -0.2020H - 0.252A + 126.58 0.43 9.06 * calculated from a one-tailed t-test. Reference: Crapo et al, 1981 Abbreviations: FVC = forced vital capacity; FEVi = forced expiratory volume in one second; H = height (cm); A = age (years); R2 = coefficient of variation. 141 A P P E N D I X G St. Paurs Hospital 1081 Burrarti Street Vancouver. B.C. V62 1Y6 (604) 682-2344 AUTOPSY CONSEiNT FORM rOVTPt FTFD FORM MUST RF FORWARDED TO ADIVTTTTTNr. DFPARTMFNT I, the undersigned hereby consent to an autopsy being performed by the Hospital on the body of the late I understand that, at times, the removal of tissues and organs for diagnostic evaluation may include permanent retention of particular tissues and organs in order to enhance the ability of physicians to make diagnoses, assist families, provide education and perform research studies. Rotrierion5: Signed: ^ _ — (patient or person legally authorized to give consent) Relaooasbip to deceased: Wirncsj: Date: ; F O R M NO M R C M ' (R£V |CV«4) 142 APPENDIX H » » » 4 » V A N C O U V t H C t N C H A I . MOSPTTA1. B R I T I S H C O L U M B I A ' S M C A I . T H S C C N C E S C C N T W A U T H O R I T Y F O R A U T O P S Y N A M C U N I T N O .w»»o D*Tt O* O C A T H . A C E . Sex AUTHORITY FOR AUTOPSY I _ bearing the relationship of to ' : : Hereby authorize the representative of the Vancouver General Hospital to make a postmortem examination of the body and to retain tissues for microscopic examination, or teaching. R e s t r i c t i o n s , (If a n y ) — tS*rntur» oiw,tr*u, IPrion Autnonna 10 9"» Conmnt; Date Address — — Copies To: Has organ or tissue donation been discussed? Yes • No • Please see Post-Mortem Consent for Donation of Organs and Tissue Form WITNESSING TELEPHONE CONSENT We acknowledge that we have read the above statement to. who is the patient's legal representative ' (state relationship _ and he/she has given verbal consent for the above to be carried out. Signed - . Signed: Print name and position: Print name and position: See Human Tissue Gift Act Pari II Paragraph 5. 1979 for definition of legal representative. PART l • WHITE - M E O l C A l RECORDS ORIGINAL PART 2 - CANARY . PATHOLOGY COPY 143 APPENDIX I Data Collection Sheet: Post-Mortem Study DATE: (d7m/y)_ GENERAL INFORMATION I.D. Number: Date of Birth: / , / Age: Gender: Date of Admission: / / @ Date of Death: / / @ PAST MEDTCAT. T f T S T O R Y Respiratory: C F COPD: E Emphysema ^ Chronic Bronchitis ^ Asthma Other Smoking Hx Medical: (Cardiac, Cancer, Arthritis, Neuromuscular, Hepatic, Endocrine,...) Height: cm Weight: kg ( I I ) B.M.I.: hrs Adm Dx: hrs Musculoskeletal: Medication History: 144 RECENT MEDICAL HISTORY Date of AmopsyfriVm/y): / / @ hrs (Dr. ) Clinical Hx : Pathology: Cause of Death: Resuscitation: Mechanical Ventilation: Biopsy Site: Diaphragm L R LAB/TEST RESULTS Pulmonary Function Tests: Respiratory Muscle Strength Tests: Chest X-ray/CT scan: Arterial Blood Gases: 1 Electrolytes: Stress Test/Exercise Capacity: 145 APPEND DC J Acute Respiratory Disease pneumonia (7 days) post-obstructive pneumonitis (4 days) chest pain, SOB, arrested immediately no bronchopneumonia, hypoxemia (2 days) o cz hypoxemic and hypercapnic RF, sepsis, ARDS no developed labored breathing, arrested immediately Chronic Respiratory Disease cystic fibrosis, hyperinflation no no no COPD, hyperinflation o cz lung carcinoma, no respiratory signs or symptoms centrilobular emphysema, multiple bullae, COPD, low diaphragm no Cause of Death respiratory failure extensive lung carcinoma acute MI, severe CAD massive pulmonary emboli acute pancreatitis, sepsis anoxic brain damage pulmonary embolism, cor pulmonale acute MI, severe CAD acute MI Admission Diagnosis exacerbation of cyctic fibrosis RF cardiac arrest for mitral valve replacement surgery sepsis cardiac arrest for lung carcinoma resection for sigmoid carcinoma resection acute MI Nutritional Status frail, malnourished well-nourished thin, little muscle bulk obese markedly cachectic & emaciated N/A moderately obese N/A moderately obese Body Mass Index 16.9 24.8 21.4 30.7 20.0 N/A 35.4 22.1 N/A < O N CS r- — i cs m rr N O C I N O cs rr 00 _ tu a u. u-V X? p CO — cs rr N O r- 00 O N 146 Corticosteroid Medication no prednisone 4 days N/A o cz no no no o cz no Resucttation History o cz no 17 minutes 37 minutes o cz 50 minutes no 29 minutes no Smoking History no no N/A quit 13 years ago 25 pack years N/A no 55 pack years quit many years ago Mechanical Ventilation 30.5 hours no no no no 32 hours 45 hours, Pavulon no no Cardiac History no no severe triple-vessel disease CHF, mitral valve regurgitation, severe CAD mild CAD no mild CAD severe CAD severe triple-vessel CAD Subject — rr i> oo o 147 Acute Respiratory Disease atelectasis, acute bronchopneumonia, hypoxemic RF O C multisystem organ failure with hypercapnic RF pneumonia, hypoxemic RF hypoxemic RF 10 days hypoxemic RF, ARDS bronchopneumonia, pulmonary edema O c bronchopneumonia 11 days, hypoxemia 36 hours acute pulmonary edema, RR 28-36 Chronic Respiratory Disease o c COPD, low flat diaphragm severe emphysema asthma asthma, centrilobular emphysema emphysema upper lobes o c chronic pulmonary edema chronic bronchiolotis o c Cause of Death liver failure cardiac arrest carcinomatosis peritonei, severe emphysema MI, acute brain infarct RF MI acute viral hepatitis acute MI, severe CAD broncho-pneumonia multifactorial Admission Diagnosis GI bleed aortic valve stenosis rectal mass unstable angina acute pancreatitis cardiogenic shock renal transplant post-cardiac arrest pneumonia hematoma Nutritional Status malnourished good anorexia 7 days overweight 6% body weight loss Body Mass Index 15.2 22.0 32.5 30.8 N/A 25.3 31.5 N/A 24.8 N/A Age r-m oo oo T vO CN f- o m vo vo 00 CN 00 Gender UL, Lu UH s s Uu Subject O — CN in VO 00 Os 148 Corticosteroid Medication o cz o cz o cz O c methyl prednisolone 2d, hydorcortisone 12d, prednisone 10d o cz prednisone 18 days o cz no no Resucitation History o c 43 minutes o cz 18 minutes o cz o cz o _ 17 minutes no 20 minutes i 2 if) x no no 50 pack years quit 1982 quit 30 years ago no no N/A no no Mechanical Ventilation no 72 hours no 264 hours no 180 hours, Pavulon 38 hours 95 minutes no no Cardiac History no heart murmur no triple-vessel disease no severe CAD moderate CAD severe'CAD atherosclerotic heart disease CHF, mitral valve replacement : : , : : : u <w 2? s BO o — CN rr VO CO ON 149 Acute Respiratory O C o c hypoxemic and hypercapnic RF 22 days, pneumonia, ARDS bronchopneumonia pulmonary edema, RR 32-40 pneumonia 7 days hypoxemia, acute pumonary edema pneumonia, hypoxemic and hypercapnic RF o c Chronic Respiratory Disease o c o c o c o c COPD o c o c asthma o • c •<•» SB O 8 a 6 peritonitis ruptured diverticulutis CAD, hyponaturemia cardiogenic shock sepsis, severe pneumonia myocardial ischemia pneumonia, atrial septal defect acute coronary insufficiency Admission Diagnosis recurrent renal stones fever, headache pneumonia post-herpatic neuralgia diabetic ketoacidosis ischemic foot pulmonary hemorrhage malabsorption Nutritional Status well-nourished moderately obese 1 mild to moderately obese cachexic obese profound malnutrition Body Mass Index 38.8 N/A m C I 27.2 27.0 m 00 32.7 26.9 15.2 Age CN 00 vO m 00 vO 00 rr CN IT) o VO 00 VO f_ annor Vjretiuci Pu U H U H Subiect CN CN CN CO CN CN m CN VO CN CN 00 CN 150 Corticosteroid Medication no O ii c o I— CO 1 ^  • g ea c cu ca >-> amiodarone 2 months, hydrocortisone high-dose 7 days no no no no hydorcortisone high-dose 3 days Resucitation History 26 minutes 19 minutes nn no 18 minutes no 2 hours 17 minutes no Smoking History 35 pack yea N/A quit many years ago no no N/A 60 pack years 25 pack years 20 pack years Mechanical Ventilation 5 hours no 499 hours no no no no 1 hour no Cardiac History i no recent MI, angina 10 years atrial fibrillation severe triple-vessel disease severe CAD no MI, angioplasty atrial septal defect severe CAD Subject o CN CN CN CN m CN T f CN i n CN *o CN r -CN 00 CN Acute Respiratory Disease severe CHF (24 hours), acute pulmonary infarct ARDS O c pneumonia, marked SOB, accessory muscle use respiratory arrest, bronchopneumonia, sepsis Chronic Respiratory Disease o c o c o c o c end-stage COPD, FEVI 51% predicted Cause of Death acute MI, transplant vasculopathy severe CAD, ARDS prolymphocytic leukemia arythmia pneumonia, COPD s o *#* •5 g heart failure cardiogenic shock prolymphocytic leukemia heart block SOB Nutritional Status extemely cachectic Body Mass Index o co CN N/A 22.5 CU < m r » m CO in CN m o NO Grander Lu s s Subject ON CN o CO CO CN CO CO CO 

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