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Diaphragm injury in people shown post-mortem Clarke, Tyler James 2000

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DIAPHRAGM INJURY IN PEOPLE SHOWN POST-MORTEM by TYLER JAMES CLARKE B.Sc. (Biology) Trinity Western University, 1998. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Experimental Medicine) We accept this thesis as conforming to the reqjuiffcd. standard  THE UNIVERSITY OF BRITISH COLUMBIA June 2000 © Tyler James Clarke, 2000  UBC Special Collections - Thesis Authorisation Form  http://www.library.ubc.ca/spcoll/thesauth.html  I-n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the - requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s "thesis f o r s c h o l a r l y purposes may be granted-by-the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s - u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada  1 of 1  23/05/00 1:00 PM  ABSTRACT  Exertion induced diaphragm muscle injury has been shown in several animal models, however, substantial evidence in humans is still forthcoming. The purpose of this study was to describe and quantify diaphragm morphology in humans and relate injury to clinical factors including: age, body mass index (BMI), gender, acute respiratory disease, and/or chronic respiratory disease. Biopsies from 59 subjects undergoing autopsy were examined. Full-thickness diaphragm biopsies were obtained from the lateral costal region of the diaphragm and fixed in formalin. Biopsies were paraffin embedded, sectioned at 5 u.m and stained with H&E and Masson's trichrome. Diaphragm cross-sections were viewed at 400x using light microscopy and images from multiple fields for each cross-section were captured using a SPOT digital camera and the Image Pro Plus 4 computer program. A 64point grid was superimposed on each image and the points were assigned to 1) normal; 2) abnormal if the fiber had internal nuclei or abnormal cytoplasm, if it was inflamed, necrotic, degenerated, small or angular; or 3) connective tissue. The area fraction (AA) was determined by summing the points for each category, and dividing it by the total points for the cross-section. Immunohistochemistry was also performed on diaphragm cross-sections using the APAAP method for the monoclonal antibody NP57 against human neutrophil elastase molecule. A retrospective chart review was performed to determine the age, gender, BMI and  Ill  the presence of acute respiratory disease(s) and/or chronic respiratory diseases(s). Of the 59 subjects, 20 were females and 39 were males. In this group of subjects, two women were undernourished and ten men were obese considering a normal BMI range of 18.5 - 27.3 kg/m for women and 18.5 - 27.8 kg/m for men. Mean ages for 2  2  men and women were 65.5 + 13.7 yrs. and 72.4 + 24.5 yrs. (p < 0.05). Thirty-eight subjects had acute respiratory disease, 33 had chronic respiratory disease, 22 had a combination of acute respiratory disease superimposed on chronic respiratory disease, and 10 had neither acute nor chronic respiratory disease. Analysis by the non-parametric Mann-Whitney U test showed the following differences. Men had more abnormal diaphragm (31.6 + 15.8%) and less normal (51.6 + 21.2%) diaphragm than women (19.3 + 10.7% and 65.3 + 15.5%, respectively) (p< 0.015) (Figure 11, upper panel). Men (65.5 + 13.7 yrs.) were younger than women (72.4 +24.5 yrs.) (P<0.049). Men with acute respiratory disease compared to men without acute respiratory disease had more abnormal diaphragm morphology (35.69 +14.34% vs. 24.61 +16.46%) (PO.021) (Figure 11, upper panel), less normal diaphragm (45.08 +18.26% vs. 63.98 +21.6%) (p<0.006), and more connective tissue (19.23 +11.17% vs. 11.40 +6.73%) (P<0.021). Analysis of the whole group of patients (both M & F) showed trends toward more abnormal diaphragm in those with acute respiratory disease (Figure 13, lower panel) or chronic respiratory disease (Figure 11, lower panel) compared to those without (P<0.067 and P< 0.082, respectively). Patients with acute respiratory disease compared to those without showed a trend toward less normal diaphragm (PO.067) (Figure 13, upper  iv panel). Lastly, men with chronic respiratory disease were older (mean age 69.8 yrs) than men without chronic respiratory disease (mean age 58.7 yrs.) (P<0.048). Neutrophils were quantified in the diaphragm of 38 subjects (22 with chronic respiratory disease and 28 with acute respiratory disease). The mean number of neutrophils per fiber was 0.0044 (0 to 0.06) and the mean number of neutrophils per cross-sectional area was 1.46 (0 to 22.8). We conclude that the diaphragm in humans is susceptible to exertion-induced diaphragm injury represented by abnormal diaphragm morphology and inflammatory cell infiltrates. Men are more susceptible to diaphragm injury. Likewise, individuals with acute respiratory disease and/or chronic respiratory disease tend to have more abnormal diaphragm.  v  TABLE OF CONTENTS  Abstract  ii  Table of Contents  v  List of Tables  vii  List of Figures  viii  List of Abbreviations  ix  Acknowledgement  xi  Chapter 1 - Literature Review Exertion-Induced Skeletal Muscle Injury  1  Respiratory Muscles  11  Functional Tests of Respiratory Muscles  14  Acute Respiratory Diseases  16  Chronic Respiratory Diseases  21  Mechanisms of Respiratory Muscle Injury  30  Animal Studies  44  Diaphragm Injury in Humans  50  Chapter 2 - Research Study Introduction and Rationale  57  Research Hypothesis  59  Research Objectives  59  Methods  61  Study Design  70  Statistical Analysis  70  Results Clinical Histories  71 73  Quantitative Evaluation of Diaphragm Biopsies 78 Qualitative Evaluation of Diaphragm Biopsies 79 Discussion  105  Chapter 3 - Summary and Recommendations  122  References  125  Appendices A: Data on Subjects  136  B: Certificate of Ethical Approval  145  vii  LIST O F T A B L E S  1. Categories and Definitions for Point Counting  63  2. Equations to Calculate Area Fractions of Normal Muscle, Abnormal Muscle, and Connective Tissue.  67  3. Distribution of Subjects with Chronic Respiratory Disease  73  4. Summary of Respiratory Diseases  73  5. Distribution of Subjects with Acute Respiratory Diseases  74  6. Summary of Acute Respiratory Diseases and Dysfunctions  74  7. Subject's Nutritional Status  75  8. Subject's Initial Admission Diagnosis  75  9. Subjects with a History of Smoking  76  10. Subjects undergoing Mechanical Ventilation  76  11. Subjects undergoing Resuscitation  77  12. Subject's Cause of Death  77  13. Quantitative Evaluation of Diaphragm Biopsies  78  14. Quantitative Evaluation of Diaphragm Biopsies (sub-divisions)  79  List of Figures  1. Light photomicrograph of normal human diaphragm cross-section stained with H & E and IPP4 software used for point counting set up. 85 2. Data plots showing the relationship between the number of PMNs cross-sectional area of diaphragm in acute and chronic respiratory respiratory disease patients.  86  3. Light photomicrograph of normal human diaphragm morphology in H&E stained cross-section. 88 4. Light photomicrographs of abnormal diaphragm morphology (vacuolized and angulated fibers) in H&E stained diaphragm cross-sections. 89 5. Light photomicrographs of abnormal diaphragm morphology (inflammation) in H&E stained diaphragm cross-sections. 90 6. Light photomicrographs of abnormal diaphragm morphology (connective tissue) in H&E stained diaphragm cross-sections. 91 7. Light photomicrographs of abnormal diaphragm morphology (necrotic fibers) in H&E stained diaphragm cross-sections.  92  8. Light photomicrographs of abnormal diaphragm morphology (abnormal fiber shapes) in H&E stained diaphragm cross-sections.  93  9. Light photomicrographs of PMNs in human diaphragm cross-sections.  94  10. Light photomicrographs of PMNs in human diaphragm cross-sections.  95  11. Light photomicrographs of PMNs in human diaphragm cross-sections.  96  12-14. Box plots showing the relationship between the proportion of abnormal diaphragm muscle and age, gender, presence of acute respiratory disease, presence of chronic respiratory disease. 97-101 15. Scatterplots showing the relationship between the proportion of abnormal diaphragm muscle and age and body mass index. 103  LIST OF ABBREVIATIONS APAAP  alkaline phosphatase anti-alkaline phosphatase  ARD(s)  acute respiratory disease(s)  ARDS  adult respiratory disease 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  CRD  chronic respiratory disease  DLCO  diffusing capacity of carbon monoxide  DOMS  delayed onset muscle soreness  FEVi  forced expiratory volume in one second  FRC  functional residual capacity  FVC  forced vital capacity  GSH  glutathione  GSSG  oxidised glutathione  H&E  hematoxylin and eosin  IPP4  image pro plus  IRL  inspiratory resistance loading  ix  kPa  kilopascal  MIP  maximal inspiratory pressure  PaC0 Pa0  2  2  partial pressure of carbon dioxide in arterial blood partial pressure of oxygen in arterial blood  Pbreath  inspiratory pressure during tidal breath  Pdi  transdiaphragmatic pressure  Pes  esophageal pressure  Pga  gastric pressure  PMN(s)  polymorphonuclear leukocyte(s)  PTI  pressure-time index  RV  residual volume  SIDS  sudden infant death syndrome  Ti  inspiratory time  TBS  Tris-buffered saline  Ttot  total time for one breathing cycle .  TLC  total lung capacity  xi A C K N O W L E D G E M E N T  I would like to acknowledge the assistance and direction provided by my thesis supervisor, Dr. W. Darlene Reid, throughout my graduate studies. Dr. Reid was always available for consultation and provided valuable insight into the research process. Her research experience combined with her ability to network with others have definitely facilitated the completion of this thesis. As a result I had the opportunity to emerse myself in scientific research and learn countless skills. Dr Reid is to be commended for her constant guidance and hard work in the area of diaphragm injury research. I would also like to acknowledge and thank the other members of my committee, Drs. Jeremy Road, Darlene Redenbach and Mark Elliott. During my graduate work these members devoted a large amount of their time to the learning and research process. They were always available for consultation and provided constructive feedback. I would also like to thank Dr. Norman Wong and the Department of Experimental Medicine for the opportunity to explore science through a unique program that offers exciting courses in the area of human medical research. Similarly, I thank the MacDonald Research Laboratory at St. Paul's Hospital for providing an excellent environment to share knowledge and use research equipment. I am grateful for the University Graduate Fellowship Award offered by The Faculty of Graduate Studies, UBC and to the British Columbia Lung Association for research support. I am grateful to my parents, Ken and Valie Clarke for their immeasurable support during my graduate work. Lastly, thanks to my friends Raelene James, Jim McAurthur, Raphael Zydowicz and Duane Krocker.  CHAPTER 1: LITERATURE REVIEW  Diaphragm injury occurs in patients with chronic and/or acute respiratory diseases. Injury may occur through increased ventilatory demands imposed on the diaphragm through increased work of breathing as a result of disease. The clinical consequences of diaphragm injury may be severe, as respiratory muscle overload usually occurs. Consequently, patients with respiratory muscle overload may develop respiratory muscle fatigue and failure. Numerous animal and human studies have attempted to understand the causes, mechanisms and consequences of respiratory muscle injury. The literature on respiratory muscle injury in humans is expanding, however, much more research is necessary to understand this important field. The purpose of this chapter is to familiarise the reader with the relevant literature involving diaphragm injury in humans.  EXERTION-INDUCED SKELETAL MUSCLE INJURY Overview  Exercise-induced skeletal muscle injury has been observed in humans and animals (Armstrong, 1990). Injury to skeletal muscle fibers may occur through concentric, isometric and eccentric contractions (Faulkner et al., 1993). Eccentrically-induced muscle injury is responsible for greater structural damage because of the high strain associated with this type of contraction. Fiber type specific damage has also been observed, mainly in type II fibers with low oxidative capacity (Friden et al., 1992). Warren and co-workers (1994) demonstrated that the mouse extensor digitorum, a fast  1  twitch muscle, is more susceptible to eccentric-induced injury versus the slow twitch soleus muscle. A possible explanation for this is related to activity level, as the load imposed on the extensor digitorum is relatively low. Therefore, possibly recruitment and training history play a role as well. Injury may be observed immediately following exercise as disruption of the sarcolemma, intramuscular proteins and the structures in some sarcomeres (Armstrong, 1990). These forms of injury may be observed at the electron microscope level.  Mechanisms  of Injury  A brief description of cytokines and types of contraction will be discussed as potential mechanisms of skeletal muscle injury. Other possible cellular mechanisms of muscle injury may include high intramuscular tension and/or temperature, increased free radical production, reduced intracellular pH, and degradation by calcium activated proteases (Armstrong, 1990). These contributors to muscle pathology may be responsible for the decline in maximal force production and delayed-onset muscle soreness (DOMS) (Armstrong, 1990). For skeletal muscle injury to occur as a result of exercise, factors such as intensity, duration, and previous training history also play a pivotal role. A number of circulatory and locally produced cytokines may be responsible for prolonged muscle damage and protein breakdown (Bruunsgaard et al., 1997). For example, the synergistic effects of TNF-a and IL-1 enhance muscle proteolysis (Pederson et al., 1998). In a study by Jiang and co-workers (1998) loss of 2  diaphragmatic force and injury was observed in the diaphragm of rabbits undergoing inspiratory resistive loading (IRL). Injury was observed as fiber necrosis and inflammation. It is possible that partial injury and loss of force reported by Jiang and co-workers results from inflammation through the synergistic effects of various cytokines. However, there are other important implications, such as the duration and type of muscle contraction, when considering mechanisms of force loss related to injury. The type of muscle contraction and therefore the tension generated, is very important in regard to the amount of injury that occurs. Eccentrically-induced muscle injury results in greater loss of force and tissue pathology than concentrically induced muscle injury. Eccentric contractions are associated with greater damage than concentric contractions because fewer motor units are recruited during eccentric contraction; therefore, a relatively greater load is placed on each myofiber in eccentrically loaded muscle (for review Clarkson et al., 1999). Another reason eccentric muscle contractions are more damaging is related to the high strain involved. Strain is defined as the relative length change in muscle (for review Reid et al., 1998). Even though high force may contribute to muscle injury, human studies by Child and co-workers (1998) have shown that greater damage is caused by eccentric actions at long muscle lengths, lending support that high strain is a main mechanism (Clarkson et al., 1999). Eccentric contractions profoundly affect the tension placed on individual sarcomeres. Because some sarcomeres are stronger than others, weaker sarcomeres may be unable to maintain the tension placed during contractions of 3  greater length (Clarkson et al., 1999). Talbot and Morgan (1998) report that eccentric induced muscle damage is related to the amplitude of stretches and the range of sarcomere lengths, not to the velocity of or tension during the stretch (Clarkson et al., 1999). These results are consistent with the findings of Friden and Lieber (1992).  Injury and Functional  Changes  After injury to skeletal muscle, numerous cellular adaptations take place that have an affect on functional outcome. The typical pathology that follows eccentric muscle contraction is reduction in maximal force development and delayed onset muscle soreness (DOMS) (Armstrong, 1990). High intensity eccentric exercise results in a loss of muscle strength of 50 to 60%, which may not be fully restored for up to ten days (Clarkson et al., 1999). This reduction in strength is probably not due to DOMS, as strength declines immediately after exercise, well before DOMS is perceived and continues well after DOMS diminishes (Clarkson et al., 1999). Muscle fiber contractility is altered following eccentric exercise. Brown and colleagues (1996) have shown that a delay between excitation and the beginning of force development occurs immediately and is still present at three days post exercise (Clarkson et al., 1999). Warren and co-workers (1993b) suggest there is a failure of excitation-contraction coupling after eccentric-induced contractions (see review Clarkson et al., 1999). Similarly, Warren and co-workers (1993b) claim that 57 to 75% of the decrements of force from 0 to 5 days post-exercise are due to excitationcontraction failure (Clarkson et al., 1999). 4  Changes in the length tension relationship may also explain how muscle injury affects muscle function. After eccentric exercise, Saxton and Donnelly (1996) observed the greatest reduction in force generated by the elbow flexors was at the shortest muscle length (Clarkson et al., 1999). This disproportionate loss of strength may be attributed to sarcomeres overstretching during lengthening contractions. Morgan's data suggests that overstretching of weaker sarcomeres may not allow them to return to normal configuration upon relaxation (Clarkson et al., 1999). Increased muscle stiffness (force required to passively lengthen muscle) after eccentric exercise may contribute to an alteration in muscle function. Howell and colleagues (1993) found that stiffness increased immediately after eccentric exercise and continued for 4 days (Clarkson et al., 1999). In animal studies, Benz and coworkers (1998) estimated the number of crossbridges entering the force generating state after eccentric exercise is dramatically decreased. It has been suggested (Benz et al., 1998) that the crossbridges form attachments but the myosin heads do not perform the power stroke. Benz and co-workers suggests that the cross bridge formation serves as a brake to resist stretch and further injury (Clarkson et al., 1999).  Inflammation  - General Cell and Tissue Response  to Injury  It is known that acute and chronic inflammation occurs in muscles when they are subjected to crush injuries, toxins, anaesthesia or overloading (Tidball, 1995). The sequence of events that follow injury are part of the inflammatory response. Inflammation is characterised by the exodus of fluid and plasma proteins from 5  capillaries into the interstitial space and migration of leukocytes into injured tissue (Walker et al., 1994). The morphologic indicators of acute inflammation are edema, fibrin deposition and the accumulation of neutrophils. Injury to muscle results in substantial disruption of structural components and the sarcomere. Consequently, myofibrils retract away from the site of injury exposing the basement membrane and endomysial proteins (Tidball, 1995). An autolytic stage immediately follows, which may be related to loss of calcium homeostasis (Armstrong, 1990). Initial tissue injury results in a biphasic vasoactive response. A brief period of vasoconstriction (5-10 min) occurs, preceding a long period of vasodilatation and increased vascular permeability (Smith, 1991). This increase in permeability from the capillary, as a result of endothelial cell retraction, functions to allow proteins and fluid to pass from the intravascular space into surrounding tissue (Walker et al., 1994). Vasodilatation also functions to reduce the flow of blood, thereby facilitating the adherence and migration of leukocytes.  Inflammatory  Cell  Response  Endothelial cells assist in the adherence of leukocytes and platelets by inhibiting anticoagulant properties upon insult and secreting local cytokines to attract neutrophils. After leukocytes adhere to the vascular wall, they migrate along a chemical gradient - a process termed chemotaxis. Through surface receptors, leukocytes migrate toward increased concentrations of plasma or cell derived mediators. Activated PMNs (polymorphonuclear leukocytes) may respond to chemotactic factors by degranulation and production of free radical species (Walker et 6  al., 1994). This activation of neutrophils results in a substantial release of cytotoxic products: stimulation of phospholipid and arachidonic metabolism, generation of oxygen free radicals, and release of granule degradative enzymes (Walker et al., 1994). These reactions have dire consequences for surrounding tissues. Various cell populations respond to the site of injury to remove cellular debris from damaged tissue. The cellular phase of inflammation involves mainly PMNs and monocytes (Smith, 1991). The number of PMNs peak at the site of injury between 1-4 hours post-injury, and thereafter substantially declines (Smith, 1991). Following the initial phase of neutrophil accumulation, other inflammatory cells such as monocytes, macrophages and lymphocytes accumulate at the site of injury. Monocyte migration occurs hours after the initial insult, and will continue through 48 h or more depending on the severity of injury (Smith, 1991). Acute inflammation is typically characterised by the ubiquitous presence of macrophages (Smith, 1991). These cells function to degrade and remove necrotic tissue, while also promoting growth and healing (St. Pierre etal., 1994). In uninjured skeletal muscle there are few mononuclear cells and virtually no lymphocytes (Round et al., 1987). Orimo and co-workers (1991) used immunohistochemical analysis to characterise inflammatory cells in rat skeletal muscle after bupivacaine-induced necrosis. Thirty minutes post-injection, PMN's appeared and peaked in number at 12 hours. Macrophage number peaked 2 days postinjection. In contrast, T-cells and B-cells constituted only a small population of identified cells. Two weeks after the initial injury, all inflammatory cells returned to basal levels (Orimo et al., 1991). In exercise-induced muscle injury the macrophage 7  was previously found to be the predominant cell type (Round et al., 1987). It appears from these studies that PMNs play an initial role in myonecrosis, whereas the macrophage dominates the later phase of the inflammatory response.  Recovery  and  Remodelling  Numerous cellular adaptations such as fiber type, satellite cell number, and myosin heavy chain fragmentation (MHC) have been shown to take place in skeletal muscle in response to loading, injury, and repair. Holloszy and co-workers (1984) describe major changes in skeletal muscle after endurance training, including increased mitochondrial density, increased mitochondrial enzymes, increased capillarization, fiber type changes (type lib to lla and type II to I), and reduced lactate levels. In the human diaphragm, Levine and colleagues (1997) demonstrated that patients with severe COPD had a higher percentage of type I (slow twitch) muscle fibers compared to type II. Similarly, slow myofibrillar-protein isoforms (MHC, troponin, tropomyosin) were found to be in greater percentage than fast isoforms (Levine et al., 1997). Reid and co-workers (1999) also observed a small increase in type I fibers in hamsters tracheal banded for 30 days. This adaptation may be explained as the diaphragm muscles of patients with COPD and congestive heart failure undergo cellular changes that resemble endurance trained limb muscle (Levine et al., 1997). However, if the myocyte is unable to adapt to the given load fatigue and/or injury may occur. There are various markers of muscle injury and regeneration. Satellite cell concentration may be a good indicator of injury and regeneration is occurring. 8  Elevated satellite cell mitotic activity may reveal damage even though abnormal morphological muscle characteristics are not present (Schultz et al., 1989). However, a large concentration of satellite cells within a muscle may be partially due to its oxidative capacity, as oxidative muscles like the diaphragm contain the largest population of these cells (Schultz et al., 1989). Similarly, it is necessary to perform immunohistochemical staining or electron microscopy when identifying satellite cell populations, as these cells are indistinguishable from myonuclei using the light microscope (White, 1989). It is known that slow twitch skeletal myosin heavy chain (MHC) is released into the circulation post-exercise (Mair et al., 1992). Plasma concentrations of MHC fragments were found to increase on day 2 post-exercise (Mair et al., 1992). The fact that this protein was observed in circulation is indicative of myofilament degradation and plasma membrane disruption (Mair et al., 1992). The release of creatine kinase (CK) in the first 48 hours may be due to plasma membrane rupture; however, the detection of MHC may be representative of greater muscle injury as the contractile apparatus must be disrupted (Sorichter et al., 1997). Serum MHC normally rises 2 days after eccentric exercise-induced injury and peaks around 6-9 days (Mair et al., 1992). This late phase of release of MHC, an integral part of the contractile apparatus, is important as it demonstrates that substantial injury has occurred, and their release time into the plasma is relatively long. For myosin to be released from the cell, two major events must occur. First, the plasma membrane must be ruptured, and second, the contractile apparatus must be degraded. This most likely explains why elevated levels of CK are observed hours 9  after a strenuous exercise bout, as it resides in the cytoplasm. A normal cytoplasmic pool for the myosin heavy chains has not been reported to date; however myosin light chains have been reported in unpublished data as 1% of total (Mair et al., 1992). The delay in increased serum levels of MHC as opposed to CK is likely due to the way proteins are transported in the circulation. Proteins leaking from myofibrers enter the circulation by way of the lymph nodes rather than directly into the muscle capillaries, as muscle capillary permeability is low (Mair et al., 1992). Calcium leakage could also play a role in disrupting the contractile components if the sarcoplasmic reticulum (SR) is ruptured (Mair et al., 1992). Muscle repair is similar to the early development stage in embryonic myogenesis and is characterised by the activation of satellite cells (Lin et al., 1998) (Schiaffino et al., 1998). Ongoing muscle degeneration and regeneration may be observed in acute or chronically loaded muscle. Regeneration is associated with inflammation or secondary injury reactions. Inflammation is important for the activation of growth factors and recruitment of macrophage subpopulations. The secondary injury events are essential for satellite cell activation, muscle re-vascularisation, and motorneuron adaptation (Husmann et al., 1996). It is proposed that proteins, such as the developmental isoform of myosin heavy chain (dMHG), may be used to identify regenerating fibers in the adult muscle (Lin et al., 1998) (Whalen et al, 1990). dMHC may be used as a marker of myogenic injury, as it disappears shortly after birth and is expressed again only during injury (Lin et al., 1998).  10  RESPIRATORY MUSCLES Anatomy and Function  of Inspiratory  Muscles  The muscles of the respiratory system are essential for life. Respiratory muscle function is profoundly compromised by the presence of respiratory diseases such as chronic obstructive pulmonary disease (COPD). The diaphragm is a skeletal muscle which functions in inspiration. This muscle performs about 70-80% of the work associated with breathing (for review Reid et al, 1995). The anatomical architecture of the diaphragm is unique as its fibers radiate from a central tendinous structure, and insert into fixed structures (De Troyer et al., 1988). This muscle is composed of three main segments: the costal, crural, and sternal regions. Wakai and co-workers (1994) demonstrated the mid-costal region is most susceptible to damage because it has been shown to contract eccentrically during resistive or obstructive breathing (in Reid et al., 1998). The fibers of the costal region insert on the zyphoid process of the sternum and the upper margins of the lower six ribs (De Troyer et al., 1988). From these insertions, the fibers run cranially as they are apposed to the inner structure of the lower rib cage. This area of apposition is commonly known as the "zone of apposition" (De Toyer et al., 1988). It has been shown that diaphragmatic force for a given contraction is greater when the zone of apposition and rise in abdominal pressure is larger (De Toyer et al., 1988). Upon inspiration the muscle fibers of the diaphragm contract and shorten. Concurrently, the axial length of the apposed diaphragm shortens and the dome of the diaphragm descends relative to its costal insertions (De Toyer et al., 1988). The 11  abdominal visceral mass acts as a fulcrum against the contracting diaphragm which pushes to lift the lower ribs through insertion of the costal portion of the diaphragm. This decent of the dome during inspiration expands the thoracic cavity which causes negative intrathoracic pressure (Reid et al., 1995). Consequently, air fills the lungs to equalise atmospheric pressure with thoracic pressure. If the diaphragm becomes fatigued through inefficiency, increased work, or increased activity, diaphragm weakness as well as increased accessory muscle recruitment may result. Other primary muscles of inspiration include the scalenes and parasternal intercostals. The scalenes originate on the transverse process of the lower five cervical vertebrae and insert on the upper surface of the first and second ribs (for review Reid et al., 1995). These muscles serve to expand the rib cage during inspiration. The parasternal muscles attach to the sternum and run between the costal cartilages in a down and outward direction. Upon inspiration the rib cage rises and the anterior-posterior diameter of the rib cage increases (Reid et al., 1995). Accessory muscles such as the sternocleidomastoid are very important during impaired inspiration. This muscle originates from the mastoid process and inserts along the medial third of the clavicle and the ventral surface of the manubrium sterni. During inspiration this muscle functions to lift the sternum and increase the anteroposterior diameter of the upper rib cage (for review Reid et al., 1995). Damage to the respiratory muscles has been demonstrated in animal and human models (Jiang et al., 1997; Road et al., 1998; Reid et al., 1998; Campbell et al., 1980) and damage may contribute to respiratory muscle dysfunction. Dysfunction of respiratory muscles may be observed clinically as a result of various pulmonary diseases. 12  Respiratory  Muscles  in  Disease  The presence of COPD and other respiratory diseases adversely affect the function of respiratory muscles, as they increase the work of breathing and decrease the muscle's capacity to cope with increased ventilatory load (for review Tobin et al., 1988). At rest, patients with COPD have an increased minute ventilation, approximately 9 L per minute compared to 6 L per minute in healthy subjects (Tobin et al., 1988). Moreover, there is an increased work in breathing due to increased airway resistance and hyperinflation. This increase in work is primarily placed on the diaphragm; consequently, accessory muscles are recruited to aid with breathing. Hyperinflation is a dominant characteristic of COPD patients that drastically affects inspiratory muscle function. Hyperinflation is partially due to gas trapping as a result of small airway collapse and loss of elastic recoil. Moreover, the diaphragm's geometry has been shown to change in patients with COPD, as hyperinflation causes the inspiratory muscles to operate in an ineffective position (for review Tobin et al., 1998). Upon contraction, the diaphragm muscle fibers translate force into a pressure difference between the abdomen and pleural surfaces of the diaphragm (transdiaphragmatic pressure, P i). The tension developed in a tightly curved d  diaphragm (small R i) creates a greater transdiaphragmatic pressure than the tension d  of a flattened diaphragm. However, flattening of the diaphragm only occurs in congenital cases or at very high lung volumes. Hyperinflation also affects the elastic recoil of the thoracic cage. Thoracic elastic recoil normally assists in the action of inspiration, however when FRC is increased due 13  to hyperinflation, thoracic elastic recoil of the rib cage becomes directed inward. This forces the inspiratory muscles to work against the elastic recoil of the lungs and thoracic cage (for review Tobin et al., 1988). Moreover, as lung volume or hyperinflation increases, the size of the "zone of apposition" decreases (Tobin et al., 1988). Diaphragm fibers usually are oriented in a cephalocaudal direction, so that contraction pulls upward causing a "bucket-handle" expansion of the rib cage. As the diaphragm is oriented low and flat in hyperinflation, fibers are directed medially and diaphragmatic action may actually have an expiratory function (Tobin et al., 1988). In patients with CRD,  the "bucket handle" expansion of the rib cage via the diaphragm is  diminished, and the accessory muscles function in a "pump handle" motion to expand the upper portion of the thoracic cavity.  FUNCTIONAL TESTS OF RESPIRATORY MUSCLES  Reduced respiratory muscle function as a result of chronic respiratory disease is difficult to measure because of the inaccessibility of the respiratory muscles. Various methods have been devised to clinically assess the respiratory muscles, however, functional tests are rarely diagnostic when considered in isolation. The F  E V i  has become the most clinically useful test of airway function (Gibson,  1995). As airway disease increases in severity the  F E V i  is reduced (Gibson, 1995).  The factors involved in this test are complex, as measurement depends on many variables including the size and elastic properties of the lung, calibre of the bronchial tree, collapsibility of airway walls and patient motivation (Gibson, 1995). The FEV-iA/C 14  or FEV-i/FVC ratios are commonly used as indices of presence or absence of airflow limitation (Gibson, 1995). Maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) are the most common procedures used to evaluate the force production of the respiratory muscles (for review Clanton et al., 1995). These tests are used whenever respiratory muscle weakness, hyperinflation or exercise intolerance is suspected (Clanton et al., 1995). MIP is the most clinically important of the two as inspiration demands the greatest amount of work, whereas expiration is largely a passive function. However, MEP becomes more clinically important as lung disease progresses because coughing requires the utilisation of the expiratory muscles. Moreover, the two tests are important in differentiating respiratory muscle weakness from neuromuscular weakness. For example, diaphragmatic weakness would result in decreased MIP, whereas, neuromuscular weakness would result in reduced MIP and MEP (Clanton et al., 1995). An alternate procedure to MIP is the measurement of "sniff pressure" (SNIP). This procedure consists of maximally inspiring with a sniff manoeuvre through the nose and/or mouth. Many patients appear able to co-ordinate their musculature better with the SNIP procedure than the MIP procedure (Clanton et al., 1995). Maximal transdiaphragmatic pressure is a measure of pressure developed by the diaphragm. This measurement is made by taking the difference between pleural pressure (Pes) and gastric pressure (Pab). This procedure is useful in evaluating extreme diaphragm weakness and paralysis, and results should be approximately 1 to 2 kPa greater then the predicted MIP (Clanton et al., 1995). 15  Maximum voluntary ventilation (MVV) is a measurement of one's ability to ventilate the lungs as deeply and quickly as possible (Clanton et al., 1995). Common guidelines for this test are described by Dillard and co-workers (1993) (see Clanton et al., 1995). This test is an indirect measurement of combined respiratory muscle function, and reflects a global measurement of lung expansion and muscle contraction Similarly, vital capacity (VC) is a reliable measure of the respiratory muscle's ability to shorten against an elastic load. However, this test is not a valid means of assessing respiratory muscle weakness. A reliable test of diaphragm weakness is the comparison of VC measurements in the upright and supine positions. The diaphragm must be able to expand against abdominal contents during supine breathing, and a decline in VC by 20% in the supine position suggests diaphragm dysfunction (Clanton etal., 1995).  ACUTE RESPIRATORY DISEASES  Infections of the lower respiratory tract (below the larynx) are common in the very young and elderly. These infections include bronchial infections and various forms of pneumonia. Acute respiratory diseases have serious implications on the function of the respiratory muscles, and diaphragmatic injury may accompany various forms of acute respiratory disease.  16  Pneumonia  Pneumonia is one of the leading causes of morbidity and death in the UK and North America (Macfarlane, 1995). Pneumonia refers to inflammation of the lung parenchyma associated with alveolar filling from exudate (West, 1995). Typical pathology is characterised by PMNs in the alveoli. Pneumonia is commonly categorised according to radiographic appearance and microbial cause. Lobar pneumonia describes multiple patchy shadows in an entire lobe on the chest x-ray. Bronchopneumonia defines a more diffuse and widespread infection that originates in the bronchi and spreads to alveolar tissue. The term atypical pneumonia was coined to describe a type of pneumonia that did not respond well to penicillin, but responded to non-p-lactam antibiotics. Respiratoryfailure is one of the most prominent causes of death in patients with acute pneumococcal pneumonia. Respiratory failure may result when inflammatory exudate fills alveoli causing a loss of typical lung function (for review Light, 1999). At higher transpulmonary pressures the intra-alveolar space does not inflate easily, therefore at higher lung volumes the oxygen exchanging space is proportionally less (Light, 1999). Overall, total lung compliance is reduced and the work of breathing is increased (Light, 1999). Pulmonary function is greatly compromised as a result of acute lower respiratory tract infections such as pneumonia. The pneumonic area is not ventilated, thereby resulting in shunting and hypoxemia (for review West, 1995). Blood flow may also be drastically affected as a result of the disease process itself or through hypoxic 17  vasoconstriction (West, 1995). Moreover, chest movement may become restricted by pain or pleural effusion (West, 1995). The presence of pneumonia in individuals with underlying lung disease such as COPD is even more severe, as any infection may cause an exacerbation, with increased work of breathing leading to respiratory failure (Light, 1999).  Acute Pulmonary  Edema  Pulmonary edema is defined by Zlatnik (1997) as the accumulation of fluid in the alveoli, pulmonary interstitium, and airways. In order to keep fluid in the pulmonary vasculature, a balance must be maintained between hydrostatic pressure (driving fluid out of vessels), colloid osmotic pressure (holding fluid in vessels), the osmotic draw of the interstitial fluid, and the endothelial and epithelial layer permeability (Zlatnik, 1997). If the hydrostatic pressure or permeability increase, or if the osmotic balance is altered, pulmonary edema may result. A variety of conditions such as, congenital or ischemic heart disease, transient cardiac dysfunction, sepsis, ARDS, pneumonia, steroids, toxic fume inhalation, airway obstruction, pulmonary hypertension or pneumothorax may cause pulmonary edema (Zlatnik, 1997). The result of pulmonary edema is increased airway resistance, reduced lung compliance and improper gas exchange, possibly causing hypoxemia and hypercapnia (Braunwald, 1997). The respiratory muscles may in turn become challenged through an increase in respiratory rate, increased load and/or metabolic factors common with impaired gas exchange.  18  Acute Respiratory  Distress Syndrome  (ARDS)  ARDS, formerly known as adult respiratory distress syndrome, is a condition characterised by acute hypoxemic respiratory failure as a result of pulmonary edema (Honig et al., 1999). ARDS is the most serious complication in response to acute lung injury and a systemic response to acute inflammation or injury (Honig et al., 1999). Injury to the lung is a consequence of overexpressed systemic inflammatory responses. Injury involves both the alveolar epithelium and pulmonary capillary endothelium. Vascular permeability increases causing interstitial and alveolar edema. Consequently, this increase in the ratio of lung tissue to gas results in alveolar closing pressures that exceed transpulmonary pressures, leading to alveolar closure and collapse (Honig et al., 1999). Overall there is decreased lung compliance; therefore, respiratory muscles must generate large inspiratory pressures, thereby increasing the work of breathing. This large mechanical load imposed on the respiratory muscles may lead to fatigue and consequently, reduce tidal volumes and gas exchange and increase respiratory rate (Hosig et al., 1999). Moreover, airway resistance may be increased due to excess fluid and bronchospasm. This excess fluid in turn impairs pulmonary gas exchange which leads to hypercapnia, hypoxia, and increased breathing effort.  Sepsis  Sepsis, although associated with many clinical conditions, is broadly defined as a state of reduced vascular perfusion with the underlying theme of bacterial infection 19  (Hussain, 1998). Respiratory impairment is considered to be the leading cause of death in patients with septic shock (Hussain, 1998). Cohen and colleagues (1982) demonstrated clinical and electromyographic evidence that patients with acute respiratory failure exhibited diaphragmatic contractile failure and could not be weaned from a ventilator (in Hussain, 1998). Friman (1977) showed maximal force and endurance declined significantly in limb muscles during acute infections, suggesting a central role for infection in acute respiratory failure (Hussain, 1998). Similarly, Leon and colleagues (1992) showed that endotoxin infusion in mechanically ventilated rats reduced diaphragmatic endurance by about 25% (Hussain, 1998). Other factors such as metabolic and hemodynamic changes (inflammatory cytokines, reactive oxygen species, products of arachidonic acid metabolism and nitric oxide) may also play a role (Hussain, 1998). The cardiopulmonary complications of sepsis and septic shock are profound as ventilatory-perfusion mismatching causes a fall in arterial Pa02. Increased alveolar capillary permeability results in pulmonary edema, reduced lung compliance and interferes with adequate gas exchange (Munford, 1999). ARDS may also result as 20 to 50% of patients with sepsis develop this syndrome (Munford, 1999). The cumulative effects of these symptoms have profound effects upon the respiratory muscles, specifically the diaphragm, as ventilatory demands increase.  20  CHRONIC RESPIRATORY DISEASES  Respiratory diseases are classified according to diagnostic categories. The main categories are obstructive and restrictive. Obstructive respiratory diseases are COPD (chronic bronchitis, emphysema), bronchiectasis, cystic fibrosis, asthma and bronchiolitis. Obstructive diseases are characterised by airway obstruction and increased resistance to airflow. This increased resistance to airflow may be caused inside the lumen, wall, airway or in the peribronchial region (West, 1995). Restrictive diseases are further divided into parenchymal and extraparenchymal (Weinberger et al., 1999). Restrictive - parenchymal respiratory diseases are idiopathic pulmonary fibrosis, sarcoidosis, pneumoconiosis and drug or radiation induced interstitial lung disease. Restrictive - extraparenchymal include neuromuscular disorders such as diaphragmatic weakness/paralysis, myasthenia gravis, muscular dystrophies and chest wall disorders such as kyphoscoliosis and obesity (Weinberger et al., 1999).  21  Obstructive Respiratory Diseases COPD  COPD afflicts between 10 to 20% of the United States population and is the fourth leading cause of death (Honig et al. 1999). In 1991 alone, 8,147 Canadians died from COPD, costing the nation an estimated $62.2 million (Canadian Lung Association, 1993). COPD is a progressive disease in which patients have reduced expiratory airflow (Honig et al., 1999). Both chronic bronchitis and emphysema result in the narrowing of airways, which in turn leads to increased airway resistance and increased work of breathing (Honig et al., 1999).  Chronic  Bronchitis  Chronic bronchitis is defined as excessive cough with chronic production of sputum. Inclusion criteria are: presence of cough on most days, for a period of at least three months in a given year, for two or more successive years (Thurlbeck, 1991). Moreover, this excess sputum production should not be due to any specific disease. Asthma is generally not included under the definition of COPD, even though a significant number of individuals with COPD experience asthmatic bronchitis or asthma (Thurlbeck, 1991).  22  Emphysema  Emphysema is a permanent, abnormal enlargement of air spaces, such that the appearance of the acinus is altered in the absence of fibrosis (Thurlbeck, 1991). The loss of alveolar tethering that occurs decreases airway wall stability, causing premature airway closure, air trapping, and chest hyperinflation upon expiration (for review Reid & Dechman, 1995). The average age of individuals with emphysema is 60, and males typically predominate (Thurlbeck, 1991). Smokers usually have a higher severity, however, emphysema may be found in non-smokers, particularly in their eighth and ninth decades of life (Thurlbeck, 1991). In centriacinar emphysema the destruction is confined to the central portion of the lobule, while the peripheral alveolar ducts and alveoli may remain unaffected (West, 1995). This type of emphysema typically affects the apex of the upper lobe and spreads downward as the disease continues. Two forms of centriacinar emphysema exist - industrial and non-industrial. Non-industrial is the commonest form, and it is virtually found only in smokers. In contrast panacinar emphysema reveals destruction to the whole lobule (West, 1995). This type of emphysema is not restricted regionally, but it may be more common in the lower lobes (West, 1995). Persons with emphysema generally exhibit symptoms such as coughing, wheezing, dyspnea, exercise intolerance, altered breathing patterns and frequent acute respiratory tract infections (Thurlbeck, 1991). Acute exacerbations of COPD are typically related to superimposed bacterial infection, evidenced by increased numbers of bacteria, neutrophils and inflammatory cytokines. (Honig et al., 1999) Furthermore, 23  when given antibiotic therapy patients tend to improve clinically and physiologically a small but statistically significant amount (for review Honig et al., 1999). This is important in patients that have compromised respiratory reserve. Cigarette smoking is regarded as a dominant risk factor for COPD, even though it is not in the formal definition. COPD may occur in smokers and non-smokers (otr antitrypsin protease deficiency), however, chronic bronchitis is only found in about 4% of non-smokers (Pride, 1995). Interestingly, smokers demonstrate almost double the annual decline of FEV^ (50 ml/year) when compared to non-smokers (30 ml/year) (Pride, 1995). Cigarette smoking, similar to viral infection, destroys the mucociliary system, thereby allowing bacteria to adhere to mucosal surfaces.  Environmental  Factors Associated  with Lung  Disease  Environment and occupation have been investigated to determine if a correlation exists between air quality, work place and COPD. For example photochemical pollutants such as oxides of nitrogen, ozone and acid aerosols are of concern, because studies of these agents in Los Angeles was associated with increased respiratory symptoms, and decreased and accelerated declines in F E V i (Pride, 1995). Similarly, occupational hazards may play a minor role, as studies suggest a definite additive effect in coal and gold miners, cement and cotton workers, farmers and grain handlers (Pride, 1995). The impact of occupational hazards are difficult to establish because of the long history of COPD and the dominating effect of smoking.  24  Cystic Fibrosis  (CF)  CF is a single gene defect of the chlorine channel manifesting in many body systems as COPD and other disorders (Thomas, 1997). This pulmonary obstructive disease also affects exocrine glands and organs such as the pancreas. In the pulmonary system it is expressed as duct or airflow obstruction secondary to increased mucosity resulting in productive cough, recurrent chest infections, and decreased exercise tolerance (West, 1995). Individuals with CF usually experience respiratory failure and malabsorption of nutrients (Thomas, 1997). In early life, the chest radiograph reveals areas of consolidation, fibrosis and cystic changes. Furthermore, individuals with CF show an abnormal distribution of ventilation and an increased arterial-alveolar 0 difference. Furthermore, decreased 2  FEV-i,  increased FRC and loss of elastic recoil may be observed (West, 1995). As a  result respiratory muscles such as the diaphragm are forced to work against an increased load. However, the CF patient's respiratory muscles may respond much differently to increased work than those with COPD. In a study by Lands and coworkers (1993), respiratory muscle strength and endurance appear to be maintained or increased in CF patients (n=11). The subjects for this study were mildly malnourished and had mild to moderate lung disease, yet maintained similar inspiratory muscle strength as controls. The investigators believe this response of the respiratory muscles is due to a training effect (Lands et al., 1993). This may be due to the lack of hyperinflation observed in CF patients. The respiratory muscles in CF patients would have a chance to contract and relax to their full range of motion which may optimise the training effect observed. 25  Bronchiectasis  This disease is characterised by dilatation of the bronchi accompanied by local suppuration. Ciliated epithelium are lost from the mucosal surface of the bronchi and squamous metaplasia and inflammation are observed. Furthermore, the surrounding lung may show fibrosis and chronic inflammation (West, 1995). In mild cases no loss of lung function occurs, however in advanced cases a reduction in FEV, and FVC due to fibrosis and inflammation may be observed (West, 1995). Moreover, hypoxia may result due to reduced blood flow to the affected area.  Asthma  Asthma is a common disease, as 4 to 5% of the US population is affected (McFadden Jr., 1999). Currently there is difficulty in achieving global consensus on the subclassifications of asthma, however, two broad types are allergic and nonallergic. In general, asthma affects individuals early in life and has a strong allergic component, whereas asthma developed later in life tends to be non-allergic and varied in etiology,(McFadden Jr., 1999). In asthma, individuals present with widespread narrowing of the bronchial airways (Burney, 1995). The pathology of asthma typically represents overdistention of the lungs, with gelatinous plugs of exudate in most bronchial branches (McFadden Jr., 1999). Histological examination reveals bronchial muscle hypertrophy, mucosal and submucosal vessel hyperplasia, mucosal edema, denudation of surface epithelium, basement membrane thickening and eosinophil infiltration (McFadden Jr., 1999). This 26  episodic disease is characterised by increased responsiveness of the tracheobronchial tree to a variety of stimuli (McFadden Jr., 1999). Patients usually experience an exacerbation from minutes to hours, however, some experience a condition (status asthmaticus) in which airway obstruction occurs for days or weeks occasionally leading to death (McFadden Jr., 1999). The clinical outcomes of this pathology are hyperinflation, reduced airway diameter, reduced forced expiratory volumes and flow rates, and increased airway resistance. Pulmonary function tests verify reduced lung function, as asthmatics on average tend to reduce V C < 50%, F E V i 30% of predicted, and MIPS and MEPS by 20% expected (McFadden Jr., 1999). Gas trapping is also a predominant feature, as R V may reach 400% normal predicted values and functional residual capacity may double (McFadden Jr., 1999). Furthermore, hypoxia, hypercapnia and respiratory alkalosis are common findings and ventilatory failure is observed in 10 to 15 % of patients reporting for therapy. Overall, there is an increased work of breathing, reduced efficiency of the respiratory muscles, abnormal ventilation and perfusion, and altered arterial blood gases (McFadden Jr., 1999).  27  Restrictive - Parenchymal Diseases  Restrictive diseases are characterised by restricted lung expansion because of alterations in the lung parenchyma or from disease of the pleura, chest wall, or neuromuscular apparatus (West, 1995). Restrictive diseases usually have reduced vital capacity and a small resting lung volume, however, airway resistance is not increased as seen in obstructive pulmonary diseases in their pure form (West, 1995).  Pulmonary  Fibrosis  The alveolar structure, including alveolar walls is affected in pulmonary fibrosis (Reynolds, 1999). The principle feature of this disease is thickening of the interstitium of the alveolar wall (West, 1995). The pathology does not affect conducting airways, but bronchiolitis may occur in respiratory bronchioles and alveolar units are always affected (Reynolds, 1999). Individuals with advanced interstitial lung disease, such as the chronic fibrotic lung diseases, present with reduced TLC, VC, FRC and RV (West, 1995). Airway obstruction is normally minimal and the FEV-i/FVC ratio is normal or increased. Hallmark symptoms during lung function tests are restrictive ventilatory patterns, characterising stiff, non-compliant lungs and fibrosis (Reynolds, 1999). Respiratory work is increased in patients with fibrotic lung diseases, however, individuals typically breathe in a rapid shallow pattern to reduce respiratory work (West, 1995).  28  Asbestosis  Asbestosis is a generic term used to describe pulmonary fibrosis and cancers of the respiratory tract, pleura and peritoneum caused by mineral silicates (asbestos) (Speizer, 1999). This diffuse interstitial fibrosing disease of the lung is related to the intensity and duration of asbestos exposure (Speizer, 1999). It resembles other forms of diffuse interstitial fibrosis, revealing restrictive lung patterns and reduced lung volumes (Speizer, 1999).  Pleural  Effusion  The pleural space between the lung and chest wall is lubricated by a very thin layer of fluid. Pleural effusion refers to excess fluid in the pleural space which often results when fluid formation exceeds absorption (West, 1995). Individuals with pleural effusions typically report dyspnea and may complain of pleuritic pain (West, 1995). Pleural effusions are divided into exudates and transudates and are typically the result of malignancies and infections or severe heart failure and edematous states, respectively (West, 1995). Pulmonary function is reduced as a result of pleural effusions, and is characterised by reduced FEVi and FVC (West, 1995).  29  MECHANISMS OF RESPIRATORY MUSCLE INJURY  The potential mechanisms of diaphragmatic injury are important to understand, as this will aid researchers in diagnosis and prevention of injury. The actual injury may occur at the tissue, cellular and molecular levels. Therefore diagnosis of injury may be made at a variety of levels through an assortment of techniques. The injury inducing event may be due to exertion through increased load or mechanical strain, overactivity, or a reduction in efficiency (for review Reid et al., 1998). Other contributing factors may include fatigue and weakness, immobilisation, change in metabolic activity, inflammation, poor gas exchange, malnutrition, ageing and corticosteroids. It is important to understand how these factors impact the diaphragm muscles of individuals with chronic and/or acute respiratory diseases if injury and tissue dysfunction in the diaphragm is to be understood.  Altered Resistive  and Elastic  Loads  Resistive loads are related to the flow of air through the respiratory system. These loads can increase as a result of excess mucus or edema in the airway, or from conditions such as bronchospasm which reduce airway diameter (Reid et al., 1995). Exercise or an acute exacerbation from lung infection can also increase resistive loads by increasing the work of breathing (Reid et al., 1995). These loads force the respiratory muscles to increase their work and may in turn lead to fatigue and/or injury.  30  Elastic loads in the respiratory system are related to the work involved in inflating the lungs and expanding the chest wall. When compliance is reduced and the ability to inflate the lungs and expand the chest is compromised, the work of breathing increases. Conditions such as kyphoscoliosis, which increase the anterior-posterior and lateral curvatures of the spine, inhibit the rib cage from expansion (for review Reid et al., 1995). Similarly, compliance is decreased in diseases such as interstitial lung disease, as collagen replacement reduces the elasticity of the alveolar walls (Reid et al., 1995). Overall, reduced lung compliance or elasticity may result in respiratory muscle fatigue and injury from muscle overload. The diaphragm may adapt in response to fatigue and thereby compensate for the increased load via cellular adaptations such as conversion to slow twitch fibers (Levine et al., 1997).  Mechanical  Stress and  Strain  High stress or strain may cause mechanical disruption of ultrastructural elements in muscle (Friden et al., 1992). Stress is the amount of force per cross-sectional area, whereas strain is the relative length change (for review Reid et al., 1998). According to Benz and co-workers, the amount of stress imposed on a muscle is greater during an eccentric contraction than a concentric contraction (see Reid et al., 1998). This is considered to be a fundamental reason why greater injury is observed during eccentric contractions (Reid et al., 1998). The diaphragm contracts concentrically under spontaneous breathing, however, during loaded conditions, such as obstructive  31  breathing, the diaphragm has been shown to contract eccentrically primarily in the mid-costal region (Wakai et al., 1994). Variability in fiber shortening has also been shown in the diaphragm (Wakai et al., 1994) . Because the diaphragm fibers radiate into the central tendon, asymmetrical concentric contraction may result in eccentric contraction on the contrasting side (Wakai et al., 1994). This non-uniformity in contraction may result from a loss in coordination or co-contraction in the respiratory muscles causing fatigue and/or weakness (Reid et al., 1998).  Fatigue and  Weakness  Fatigue has important implications related to diaphragm muscle injury. Fatigue has been defined as the reduction in force which is reversible through rest (for review Reid et al., 1995). Injury may result as a consequence of fatigue. If the diaphragm has reduced force, efficiency, or increased work, muscle overload will result. This overload in turn produces muscle fatigue which may result in injury and/or recovery (Reid et al., 1995) . Respiratory diseases like chronic obstructive pulmonary disease (COPD) may overload the diaphragm and consequently produce muscle injury. Superimposed episodes of acute exacerbations may result in cycles of injury and recovery on an already overloaded system. Numerous investigators have attempted to understand the exact mechanism in the "command chain" responsible for respiratory muscle fatigue. It is believed that individuals experiencing chronic respiratory muscle fatigue alter their breathing pattern 32  to combat respiratory failure (for review see Roussos, 1985). During fatigue, tidal volume decreases along with total minute ventilation and therefore V A / increases D  T  (Roussos, 1985). These changes are responsible for the CO2 retention in acute stages of hypercapnic respiratory failure. Conversely, in stages in chronic C 0  2  retention it is believed that fatigue affects the central nervous system (CNS) output. Individuals with chronic lung disease may experience changes in CNS output to prevent the mechanical disadvantage of inspiring against a greater load. Therefore, as a result of the CNS these individuals increase their frequency of breathing to minimise the shortening of the inspiratory muscles (Roussos, 1985). This may occur in patients weaning from a ventilator as they develop rapid and shallow breathing patterns, but at the expense of hypercapnia and acidosis (Moxham, 1990). This failure of the CNS to drive the respiratory muscles is known as "central fatigue" (for review see Moxham, 1990). However, it remains to be observed if this type of fatigue is a factor in ventilatory failure. Various factors may be responsible for weakness in the diaphragm, such as neurological disorders, myopathies, connective tissue disorders or systemic abnormalities (for review see Reid et al., 1998). Weakness is defined as a loss of force in the rested muscle (for review Reid et al., 1995). Weakness reduces the ability of the diaphragm to cope with imposed loads and in turn makes the muscle more susceptible to injury and dysfunction. Metabolic dysfunction may compound muscle weakness by inhibiting muscle adaptation and regeneration (Reid et al., 1998). Weakness may also result through respiratory muscle disuse, such as disuse during mechanical ventilation. 33  Immobilisation  The diaphragm muscle is continuously contracting because of its obligatory role in inspiration. A highly active and well-trained muscle will exhibit a training effect if given ample time to regenerate. Consequently, trained muscles are less susceptible to injury (Road et al., 1998). Conversely, excessive rest has been shown to make a muscle more susceptible to injury (Warren, 1994). Excessive rest in the diaphragm occurs in mechanically ventilated patients which could predispose the diaphragm to injury (Road et al., 1998). Furthermore, mechanical ventilation is much more complex than previously understood and if the ventilator settings are inappropriate, respiratory work may be greater than under normal breathing (Tobin et al., 1998). When mechanical ventilation is properly implemented, reduced respiratory effort is achieved through assisted breathing. This results in a partial or complete reduction in respiratory muscle loading. Muscle immobilisation has been shown to result in weakness and fiber atrophy, specifically in fast-twitch muscle fibers (St. Pierre, 1994). Similarly, prolonged bed rest (4 to 6 weeks) results in reduced muscle strength from 6 to 40% (Bloomfield, 1997). Microscopically, muscle biopsies from bed-rested subjects demonstrate Z-line streaming, myofibrillar protein disorganisation and cellular edema (Bloomfield, 1997). These cellular changes may occur in the diaphragm of mechanically ventilated patients as respiratory muscle rest is occurring. Respiratory muscles may be even more susceptible to injury during reloading associated with ventilator weaning (Tobin et al., 1998). Respiratory muscles may have reduced strength or fatigue resistance due to fiber and enzymatic changes. 34  Furthermore, it is not uncommon for patients to develop tachypnea and dynamic hyperinflation after weaning from a ventilator (Tobin et al., 1998). This in turn may result in respiratory muscle fatigue and injury.  Metabolic Abnormalities  (calpain, Free  radicals)  High intracellular calcium may also be a potential mechanism of injury, as it can activate degradative enzymes. A rise in calcium has been shown to induce phospholipases, proteases, and endonucleases (Belcastro et al., 1998). Similarly, a rise in calcium can activate the non-lysosomal cysteine protease (calpain) which cleaves a variety of protein substrates, such as cytoskeletal and myofibrillar proteins (Belcastro et al., 1998). Calpain activity increased in rabbits after short periods of moderate intensity inspiratory resistive loading (IRL) (Jiang et al., 1998). Conversely, the high intensity IRL group, which had greater diaphragm injury, did not show a rise in calpain activity. Unfortunately, calpain activity was not measured at different intervals; and normal calpain levels in the high intensity group may be related to increased protein turnover and autolysis due to intensity (Jiang et al., 1998). Changes in calpain activity were also observed in chronic low intensity loading by Reid and co-workers (1994). In this study hamsters were tracheal banded for six days until the esophageal pressure was 8.0 cm H 0 during normal breathing. After six days 2  of loading, increased rates of tropomyosin and a-actinin degradation were observed upon exposure to exogenous calpain (Reid et al., 1994). 35  In conditions such as respiratoryfailure, free radical induced muscle injury may be partially responsible for respiratory muscle dysfunction (Supinski, 1998). The role free radicals may have in muscle injury is vast, as free radicals may trigger other injurious pathways, through cytokines or proteolytic enzymes (Supinski, 1998). In a study by Anzueto and colleagues (1992) large inspiratory resistive loads were placed on rats. They found that generation of free radicals is associated with a decline in glutathione (GSH), and increases in oxidised glutathione (GSSG) concentrations and GSSG/GSH ratios (Supinski, 1998). Results were an increase in GSSG concentrations and an decrease in GHS levels in the diaphragm (Supinski, 1998). These changes were closely correlated with significant diaphragmatic fatigue, as judged by the force generating capacity of diaphragm muscle strips in vitro (Supinski, 1998). While many studies mention the production of free radicals during muscle contraction, it is important to understand the effects that free radicals have on cellular function and muscle contraction. In a study by Shindoh and co-workers (1990), N-acetylcysteine (NAC) was administered to rabbits (Supinski, 1998). NAC scavenges detrimental superoxide anions, hydroxyl radicals, and hydrogen peroxide while providing cysteine for protective glutathione synthesis (Supinski, 1998). Rabbits that received NAC had a substantial reduction in diaphragm fatigue induced through electrical stimulation in situ (Supinski, 1998). If free radicals partially contribute to fatigue, and fatigue contributes to respiratoryfailure during loading situations, administration of free radical scavengers such as NAC should delay the time to respiratory failure (Supinski, 1998).  36  In an earlier experiment by Supinski and co-workers (1994) NAC or saline was administered to rats undergoing a large inspiratory resistive load (see Supinski, 1998). NAC treated animals tolerated loading better than controls by maintaining higher inspiratory pressures and volumes and increasing the time to respiratory arrest (Supinski, 1998). These findings suggest that compounds such as NAC reduce the development of respiratory failure and may have important implications for critical care patients with respiratory dysfunction.  Inflammation  Inflammation has been shown to be accompanied by severe force loss in limb muscles following eccentric-induced exercise (for review Clarkson et al., 1999). The presence of PMNs is characteristic of acute inflammation and upon activation they release proteolytic enzymes and free radicals that result in proteolysis (Pizza et al., 1995). Furthermore, PMNs have the ability to destroy normal cells and connective tissue in inflammatory muscle disease (Pizza et al., 1995). As a consequence of PMN infiltration, macrophages increase in number and invade damaged muscle fibers and engulf injurious debris (Tidball, 1995). This in turn results in greater muscle damage through secondary muscle injury. Diaphragmatic injury and inflammation have been shown to occur after short (1.5 hours) periods of intense inspiratory resistive loading and long periods of low intensity loading (Road et al., 1998). A reduction in force by 48% observed by Road and colleagues (1995) after high intensity IRL is profound as inflammation and secondary 37  injury may have impeded diaphragm contractility (in Jiang et al., 1998). Moreover, this is significant as patients with respiratory disease are subjected to prolonged increased respiratory loads.  Atypical Arterial Blood  Gases  Atypical arterial blood gas levels may systemically affect individuals with respiratory disease. It has been shown that respiratory rate (RR) is higher in hypercapnic COPD patients (RR=23) compared with normal subjects (RR=17) (Tobin et al., 1988). This increase in RR leads to increased respiratory work, and may lead to fatigue and/or injury. Larson and colleagues (1983) demonstrated an increase in pC0 can activate the 2  kallikrien-kinin system leading to bradykinin production and hypoxia can impede the removal of bradykinin (for review see Reid et al., 1998). Overall this increases the inflammatory response which is believed to be a contributing factor to diaphragm injury (Jiang et al., 1998). However, the contribution of altered arterial blood gases and respiratory acidosis that occurs as a result of loaded breathing may not play a significant role in muscle injury. In a study by Jiang and co-workers (1998) short intense inspiratory resistive loads were imposed on the diaphragms of rabbits. Respiratory acidosis was induced through loaded breathing, however, direct injury was observed in the respiratory muscles (mainly costal diaphragm) but not to the gastrocnemius muscle as metabolically-induced damage (Jiang et al., 1998). Because injury was not observed on the gastrocnemius muscle, it is believed that atypical arterial blood gas levels do not directly cause muscle injury (Jiang et al., 1998). 38  Contrary to the observations of Jiang and co-workers (1998), Juan and colleagues (1984) observed that hypercapnia reduces the capacity of the unfatigued diaphragm to generate force. In the study by Juan and colleagues (1984) electromyographic changes analogous to diaphragmatic fatigue were observed in four men during inspiration of C 0 . Hypercapnia resulted in reduced diaphragmatic generating 2  pressure by 10 to 30 percent, when C 0 levels increased 7.5% (arterial C 0 tension of 2  2  54mm Hg) (Juan et al., 1984). The authors believe that contractility is affected by reduced muscle pH, which in turn makes the diaphragm more susceptible to fatigue and the respiratory patient potentially susceptible to acute respiratory failure (Juan et al., 1984). Reasons for the discrepancy between Jiang and co-workers and Juan and co-workers may be that hypercapnia does not cause systemic muscle injury in nonmobile skeletal muscle, rather, hypercapnia predisposes a working muscle to fatigue and later injury.  Starvation  Starvation results in a breakdown of muscle proteins as a result of reduced protein synthesis (for review see Reid et al., 1998). This catabolic state has been shown to reduce respiratory muscle strength and endurance in patients with chronic lung disease (Ryan et al., 1993). The major effect appears to be a reduction in muscle mass, which in turn could make the diaphragm more susceptible to injury and less able to regenerate (Reid et al., 1998). Several other factors related to nutrition adversely affect the diaphragm in COPD patients. Malnutrition is common in COPD 39  patients, as approximately 20% have a body weight of less than 85% predicted (Tobin et al., 1988). This weight loss and malnutrition has been associated with diaphragmatic muscle atrophy (Tobin et al., 1988). Starvation and malnutrition may have a number of important implications in the development of diaphragm fatigue and injury. Antioxidants such as vitamins C and E are naturally found in a balanced diet. A number of experiments have looked at fatigue and muscle degeneration and the absence of natural antioxidants. Jarger and colleagues (1972) found muscle degeneration in vitamin E deficient rats, and Davies and colleagues (1982) determined that vitamin E deficient rats were more susceptible to fatigue (Supinski, 1998). Similarly, Anzueto and co-workers (1993) studied inspiratory resistive breathing in rats with vitamin E deficiency. Both non-loaded and loaded vitamin E deficient animals had reduced force in vitro, with loaded vitamin E deficient animals having the least force overall (Supinski, 1998). This is an important consideration for patients malnourished with respiratory disease.  Ageing  Many metabolic and cellular changes within muscle are either directly or indirectly correlated with sarcopenia, the age-related loss of muscle mass. Muscle unit remodelling, changes in fiber type and number, alterations in enzymes and aerobic capacity, decreases in myosin heavy-chain synthesis, reduced hormone levels (growth hormone [GH], insulin growth factor 1 [IGF-1], testosterone), increased oxygen free radicals, increased insulin resistance (due to tyrosine kinase activity in insulin 40  receptors), and reduced cardiovascular function all contribute to the degeneration of skeletal muscle. Decrease in strength ranges from 20 to 40% during isometric and concentric contractions in the elderly (Roos et al., 1997). Individuals beyond their eighth decade of life demonstrate an even greater loss in strength up to 50% or more (Roos et al., 1997). Many factors contribute to the age related atrophy that occurs. Motor unit remodelling plays a significant role even though this process is the consequence of a natural cycle. It has been estimated that 1% of the total number of motor units are lost per annum, beginning in the third decade (Roos et al., 1997). The ageing process in skeletal muscles varies between muscle groups and individuals. With age many alterations in fiber type have been observed. Subjects between the ages of 20 to 29 had 39% type I muscle fibers, whereas the 60 to 65 year old group had 66% type I fibers (for review see Thompson, 1994). Moreover, the distribution of type II fibers was reported to decrease linearly from the third to seventh decades of life. The vastus lateralis (VL) muscle was studied in two age groups (mean age 30 and 72) from human autopsy samples. The size of the VL muscle in the older group were 18% smaller and the percentage of fibers were reduced by 25% (Thompson et al., 1994). A decline in fiber size, mainly type II, was also observed. Overall with age, the loss in muscle mass is greater in type II fibers. This may be partially due to the muscle compensating for the changes in activity that occur with age. In individuals with respiratory diseases like COPD, the greater proportion of type I muscle fibers reported by Levine and co-workers (1997) support these findings.  41  Balagopal and co-workers (1997) demonstrated that an age-related decline in myosin heavy-chain synthesis rate occurred by 31% and 44% in middle and older aged subjects, respectively (Proctor et al., 1998). This reduction in myosin heavychain was correlated with a declines in strength, insulin growth factor I (IGF-I), and dehydroepiandrosterone sulphate (DHEAS), and free testosterone in men (Balagopal et al., 1997). The decrease in the above mentioned hormones would have detrimental effects in regards to protein synthesis, as they are anabolic compounds. It was further determined that the correlation between myosin heavy-chain synthesis and hormones was stronger in males (Balagopal et al., 1997). It is known that insulin resistance in the tissues occurs with age and this results in a reduction of insulin's growth promoting effects (Carvalho et al., 1996). The insulin resistance that occurs is associated with decreased glucose tolerance. This especially affects skeletal muscle, an important target for insulin. IGF-1 regulates cell growth and metabolism, stimulates DNA synthesis, cell multiplication, amino acid uptake, and protein synthesis and increases glucose transport and metabolism (Willis, 1996). During ageing, levels of IGF-1 decrease, and response in skeletal muscle to IGF-1 is reduced due to decreased IGF-1 receptors (Oldham et al., 1996). Decreases in IGF-1 during the ageing process are partially due to decreased levels of growth hormone and decreased expression of IGF-1 mRNA (Willis, 1996). Oxygen free radicals are found naturally in the body at all times and can produce irreversible damage to cells such as enzyme inactivation, DNA damage, lipid peroxidation, and alteration of intracellular oxidation-reduction states. During ageing 42  the production of oxygen reactive species increases as a result of mitochondrial deterioration (Leeuwenburgh et al., 1994). This deterioration causes leakage of damaging  H2O2.  Consequently, neutrophil infiltration and lysosomal responses occur  as a result of mitochondrial deterioration, which may lead to secondary free radical production (Leeuwenburgh et al., 1994). These combined events put the cell under additional oxidative stress. When injury is inflicted in the diaphragm, ageing reduces the ability to repair and regenerate damaged tissue. In a study involving the same eccentric-induced muscle injury in young and old mice, the young mice recovered after two weeks, whereas the old mice had still not recovered after two months (Brooks et al.,1990). Therefore, ageing not only results in reduced muscle mass and increased injury, but also an inability to repair and regenerate injured tissue when compared to younger controls.  Corticosteroids  Therapy involving corticosteroids has been shown to adversely affect skeletal muscle (for review see Reid et al., 1998). Furthermore, the administration of corticosteroids has been documented to contribute to respiratory muscle weakness (for review Decramer et al., 1994). This is because steroids are believed to inhibit protein synthesis and increase muscle catabolism (Reid et al., 1998). Corticosteroids are commonly prescribed to patients with COPD, asthma and interstitial lung disease. Numerous animal models have been employed to determine the contribution of corticosteroids and other injury inducing events on the diaphragm. Muscle atrophy, 43  especially in type lib fibers, has been observed, however, many studies conflict as a result of different steroids, doses, subjects, administration techniques and background conditions (Reid et al., 1998).  ANIMAL STUDIES  The application of animal models has been helpful in understanding the etiology, possible mechanisms and functional outcomes in diaphragm injury. Animal models allow for greater control of confounding variables such as age, nutritional state, and pharmacological and medical histories. Important animal models of diaphragm injury include acute inspiratory resistive loading, tracheal banding, experimental emphysema, phrenic nerve and direct muscle stimulation, corticosteroid administration, nutritional deprivation, phrenic nerve section and ageing.  Acute Inspiratory  Resistive  Loading  Diaphragm injury and force reduction have been observed in rabbits after inspiratory resistive loading for 1.5 hrs. It had been shown that intense IRL for short periods (1.5 hrs) produces significant diaphragm injury, including necrotic fibers and inflammation (Road et al., 1998). Furthermore this injury is translated into a reduction in diaphragmatic force by 48% (Road et al., 1998). Injury was observed in the parasternal intercostals and crural diaphragm, but to a lesser degree than the costal region of the diaphragm (Jiang et al., 1998). This study demonstrates that short 44  periods of high intensity diaphragm loading results in injury and inflammation three days later, and that injury is load dependant (Jiang et al., 1998). These short periods of high intensity IRL require activation of the diaphragm well above the fatigue threshold to produce secondary injury (Jiang et al., 1998). Inflammatory cells have previously been identified in non-limb skeletal muscle, such as the diaphragm. During periods of IRL, an acute inflammatory process occurs, and muscle fibers are invaded predominantly by PMNs and macrophages. Morphological evidence by Jiang and co-workers (1998) determined that diaphragm injury and inflammation occurred after 1.5 hours of high-intensity IRL in rabbits. Injury was identified by the presence of necrotic diaphragm muscle fibers and a profound influx of inflammatory cells in the necrotic fibers and interstitial spaces (Jiang et al., 1998). These researchers suggest that the surplus of PMNs, mononuclear cells and mediators of inflammation contributed to the muscle injury observed three days postloading (Jiang et al., 1998). The reasoning behind this conclusion is based on a study demonstrating reduced muscle injury after administration of anti-inflammatory medication (cited in Jiang et al., 1998).  Tracheal Banding  Reid and co-workers (1994) demonstrated that chronic low intensity loading through tracheal banding in hamsters resulted in diaphragm injury. Hamsters were subjected to an increased breathing load via a cuff around the trachea that increased esophageal pressure to 8.0 cm H 0. After six days of inspiratory resistive loading, 2  45  point counting of respiratory muscle was performed on H&E stained cross-sections of diaphragm. A greater proportion of abnormal muscle was observed in the costal and crural regions of the diaphragm in banded hamsters than in the diaphragm of healthy hamsters (Reid et al., 1994). Furthermore, electron micrographs of longitudinal sections of diaphragm showed disorganised myofibrils, disrupted A- and l-bands, and Z-band streaming (Reid et al., 1998). In a 30 day study of tracheal banded hamsters, reduced arterial blood gases and abnormal fiber morphology were observed (for review Reid et al., 1998). Moreover, quantitative analysis showed small increases in type I fibers and the area fraction of abnormal muscle tissue (Reid et al., 1998). This animal model is similar to some aspects of overloading observed in chronic respiratory diseases; therefore, one may hypothesise that similar diaphragm injury occurs in patients with acute or chronic respiratory diseases.  Experimental  Emphysema  Reid and co-workers (1994) induced experimental emphysema in hamsters through transtracheal injections of elastase and intraperitoneal injections of (3aminoproprionitrile. This model resulted in profound hyperinflation, and some abnormalities were observed in H&E stained cross-sections of diaphragms from some hamsters; however, changes in area fractions of normal and abnormal diaphragm tissue were not significantly different compared to controls (Reid et al., 1994). Furthermore, in vitro analysis of diaphragm muscle strips did not show any difference 46  in force output over a range of frequencies (Reid et al., 1994). Supinski and coworkers (1980) also observed normalised force for the cross-sectional area in the costal region of the diaphragm after elastase-induced emphysema (for review Reid et al., 1998). Thus far, significant injury in the diaphragm has not been correlated with the experimental model of emphysema. Possibly too much normal diaphragm muscle confounds the changes.  Phrenic Nerve and Direct Muscle Stimulation  Electrical stimulation of the phrenic nerve and diaphragm have been shown to induce injury. In a rabbit model, unilateral phrenic nerve stimulation (duty cycle: 0.70, 30 Hz, 200 usee pulse duration) was conducted 60 times/minute for 2 hours. Mononuclear cells were observed one and two days later, and the greatest area fraction of abnormal muscle was in the sternal region (Dechman et al., 1996). Direct stimulation of the diaphragm has also been shown to result in injury. In this animal model, electrodes were placed directly on the hamster diaphragm with stimulation (duty cycle: 0.30, 30 Hz, 200 usee pulse duration, 80 repetitions/min) continuing until Pdi decreased 50%. PMNs in the interstitium were observed within 30 minutes and invaded muscle fibers within a few hours (for review Reid et al., 1998).  47  Corticosteroid Administration  Corticosteroids have been linked to numerous abnormalities in the diaphragm. Dekhuijzen and colleagues (1993) observed vacuolisation, necrosis and atrophy after administration of cortisone acetate, prednisolone and triamcinolone (for review Reid et al., 1998). These same researchers also demonstrated prolonged mild atrophy in type lib fibers after ceasing triamcinolone treatment (Reid et al., 1998). Conversely, Nava and co-workers (1996) did not observe myogenic changes in the diaphragm after prednisolone and triamcinolone administration (Reid et al., 1998). Contrasting results are common in the studies of corticosteroids in diaphragm injury. Some studies report reductions in fatigue resistance and specific tension per cross-sectional area, whereas others show no association (Reid et al., 1998). Similar controversy exists when comparing fluorinated and non-fluorinated steroids to diaphragm injury (Reid et al., 1998). The inconsistency in results may be due to variation in dose, administration, and experimental species. In humans, the impact of steroid use is further complicated by a heterogeneous population that may have other diseases and pharmaceutical requirements.  Nutritional Deprivation  A lack of consistency exists in animal studies involving nutritional deprivation and morphological abnormalities in the diaphragm. Lewis and co-workers (1986) observed that the main feature of reduced nutritional intake over time is diaphragm muscle atrophy, especially in type lib fibers (for review Reid et al., 1998). Nutritional 48  deprivation does not appear to be correlated with diaphragm injury of in vitro functional assessments. Atrophy and loss of muscle mass are the main observations in nutritional deprivation, and these outcomes could result in reduced relative strength, and increased fatigue and injury upon respiratory overload (Reid et al., 1998).  Phrenic Nerve  Section  Animal models involving phrenic nerve section have been implemented to understand diaphragm injury. Initially after phrenic nerve section, type I fibers undergo hypertrophy, whereas type II fibers atrophy (Zhan et al., 1992). Prolonged periods after phrenic nerve section result in greater muscle atrophy, and the deposition of fat and connective tissue until the diaphragm is completely fibrotic (for review see Reid et al., 1998). These animal models are important for identifying possible changes that may occur in clinical diaphragm injury under mechanical ventilation.  Ageing  The age related changes in the diaphragm include alterations in myosin heavy chain composition, increased fiber grouping, reduced capillary density and increased atrophy in type Mb fibers (for review see Reid et al., 1998). In older animals, both diaphragmatic force and endurance are reduced. If injury is induced in the diaphragm of animals, older animals might be more susceptible to injury and regeneration time might be greatly increased. 49  DIAPHRAGM INJURY IN HUMANS  Respiratory muscle injury in humans is a limited yet growing field of research. The majority of information is from patients undergoing thoracotomy, or with myopathies. Research involving thoracotomy patients is limited because only individuals with mild lung disease are suitable for surgery. Recently, MacGowan and co-workers (1998) studied diaphragm injury in two patient groups: patients with chronic respiratory disease undergoing thoracotomy surgery and post-mortem subjects with various respiratory diseases. Mid-costal biopsies of the diaphragm were investigated using light microscopy. Different protocols were used to sample and examine the two patient groups. Common morphological findings were the presence of collagen, and internal and enlarged nuclei in the diaphragms of patients with chronic respiratory diseases. This may be indicative of previous and ongoing muscle regeneration (see MacGowan, 1998). Similarly, necrotic and inflamed cells were quantified and found to be present in both studies. In the thoracotomy group, diaphragm biopsies were obtained from 21 people undergoing thoracotomy surgery (FEV-i: 74 + 34% predicted). The relationship between abnormal diaphragm and airflow obstruction was consistent with data from animal research (MacGowan, 1998). This study was the first to quantify and describe the area fraction of abnormal diaphragm in individuals with airflow limitation. Point counting was used to determine the proportion of normal muscle, abnormal muscle and connective tissue. The area fraction of normal muscle, abnormal muscle and connective tissue were 66.2 + 9.0%, 17.6 + 7.2%, and 16.3 + 4.2%, respectively, 50  (MacGowan, 1998). Furthermore, the monoclonal antibody Ber-MAC3 was used to label macrophages and image analysis was used to quantify the number of macrophages/mm . The number of macrophages was not related to the percent 2  predicted FEV-|. However, it is concluded that increased severity of airflow obstruction, as determined via FEV-i, is associated with an increased proportion of abnormal diaphragm and decreased proportion of normal diaphragm (MacGowan, 1998). In a second study, McGowan (1998) investigated the diaphragm of 33 post-mortem subjects. Thirteen subjects had chronic respiratory disease and twenty-two had acute respiratory disease. The area fraction of normal muscle, abnormal muscle and connective tissue were determined using a point counting set up on H&E stained cross-sections. The proportion of abnormal diaphragm was not significantly correlated to the clinical factors reviewed (age, gender, BMI, presence of chronic and/or acute respiratory disease). Further research is required in this area to determine if there is a significant relationship between abnormal diaphragm morphology and the presence of chronic respiratory disease, as statistics showed p=0.065 for these two variables (MacGowan, 1998). Campbell and co-workers (1980) used biochemical analysis to investigate the external intercostals, internal intercostals and latissimus dorsi muscles of obstructive lung disease patients undergoing thoracotomy. Thoracotomy subjects ranged in pulmonary function from none to moderate airflow obstruction, and 17 of the twentytwo patients showed morphological changes in both intercostal muscles (Campbell et al., 1980). Fourteen of 22 external intercostal samples, and 16 of 22 internal intercostal samples had variation in fiber size, angular fibers, targeting, split fibers and 51  internal nuclei (Campbell et al., 1980). Fiber atrophy (type II) was greater in the internal intercostals and was significantly related to airflow obstruction, but not to age, weight loss or malignancy (Campbell et al., 1980). Hards and co-workers (1990) examined the relationship between pulmonary function, nutritional intake, respiratory muscle strength and respiratory muscle morphology. These investigators obtained muscle biopsies from the internal intercostals, external intercostals and latissimus dorsi in 68 patients undergoing thoracotomy surgery. Muscle fibers were qualitatively scored for the presence or absence of inflammatory cells, necrotic fibers, basophilic fibers, fiber splitting, internal nuclei, ragged red fibers, z-band streaming and fiber type grouping. However, similar to Campbell and colleagues (1980) the authors did not quantify their observations. Contrary to Campbell and colleagues (1980) results showed greater qualitative changes in the external intercostals. No relationship was found between pulmonary function and respiratory muscle strength and fiber proportions and diameter. Conversely, a significant correlation was found between the percentage of ideal body weight, and type I and II fiber diameters (Hards et al., 1990). The authors also concluded that sex and nutrition influence the morphometry of accessory respiratory muscles. Silver and Smith (1992) performed a morphologic study of the anterolateral diaphragm in 125 newborns and infants who died suddenly. Diaphragmatic contraction band necrosis (D-CBN) was the most common finding, and was especially prominent in cases where acute asphyxia was the mode of death (Silver and Smith, 1992). D-CBN was believed to occur at or shortly before the time of death, and 52  reflected a systemic injury possibly caused by a catecholamine response rather than a focal injury (Silver and Smith, 1992). Kariks (1989), observed similar abnormalities in the diaphragm in 80% of 242 infants that died suddenly. Scott and co-workers (1982) concluded that diaphragm weakness was a cause of ventilatory muscle fatigue in infants that survived sudden infant death syndrome (SIDS) (see Kariks, 1989). The most common pathological feature in the diaphragms was focal acute muscle fiber coagulative necrosis (Kariks, 1989). This lesion was present in 198 cases. Similar to Silver and Smith (1992) contraction band necrosis was observed, however, contraction bands varied from singlely affected fibers to groups of fibers. Muscle fiber atrophy was also identified in 35 cases, however, most atrophy was restricted to single fibers or groups of fibers in single fascicles (Kariks, 1989). Atrophic fibers were small, uneven, possessed a round or angulated appearance, and in some cases contained internal nuclei (Kariks, 1989). Furthermore, fibers within the same fascicle were hypertrophied and round in appearance. Kariks (1989) also observed that central vacuoles and fiber splitting were commonly present within the injured infant diaphragm, however, splitting could be due to shrinkage artifact as a result of rigor mortis. In some cases entire fibers were completely disintegrated, and in one case 40 to 50% of fibers appeared disintegrated (Kariks, 1989). In ten cases a fibrous scar was present in the diaphragm. This is in contrast to Silver and Smith (1992) who observed recent injury in the diaphragm in the form of contraction band necrosis.  53  It is known that most infants with SIDS have acute pulmonary congestion which causes reduced cardiac output, hypoxemia and increased work of breathing (Kariks, 1989). These factors combined could result in increased fatigue and damage to the diaphragm. The scarring in 10 cases may represent repair of injury due to a repetitious load on the diaphragm followed by incomplete regeneration. Lindsay and co-workers (1996) studied two etiologies of patients with heart failure (n=17) and compared histological findings in limb and respiratory muscles to similar muscles in control patients. Control subjects were undergoing either surgical ablation of electrical pathways (n=7) or coronary artery surgery (n=10). All control subjects had normal ventricular function, whereas patients with heart failure had severely compromised left ventricular function and all but one were undergoing cardiac transplantation. Biopsies were taken from the quadriceps femoris, sternothyroid, pectoralis major and anterior diaphragm muscles. Histological features were classified as major or minor, and common histological abnormalities found were tubular aggregates, internal nuclei, unusual myosin staining and cores (Lindsay et al., 1996). It is known that skeletal muscle has both reduced strength and resistance to fatigue in individuals with heart failure. Possible causes may be reduced blood flow, decreased exercise, altered muscle metabolism, presence of toxins and myopathies related to cardiac failure (Lindsay et al., 1996). The multitude of cytokines and various drugs for heart disease have not been examined for their role in skeletal muscle injury. Lindsay and co-workers concluded that histological abnormalities were greatest in the diaphragm compared to other muscles examined, and were greatest in patients with idiopathic dilated cardiomyopathy. Histological observations, although inconsistent, 54  suggest that muscle fiber regeneration and transformation was occurring (Lindsay et al., 1996). Levine and co-workers (1997) studied the cellular adaptations of the costal diaphragm in patients with COPD. Six individuals with severe COPD (mean  F E V i  =  33 + 4 percent of predicted) undergoing lung-volume-reduction surgery and 10 controls (4 with mild pulmonary impairment and 6 terminally comatose organ donors) were chosen. The proportions of various myosin heavy and light chains, troponin and tropomyosin were determined by SDS gel electrophoresis, and immunohistochemistry was used to determine the proportion of various muscle fibers. However, these authors did not mention if injury was present in the diaphragms of study subjects. Results showed the diaphragm biopsies from COPD patients had a greater proportion of type I slow myosin heavy chain (64 + 3% vs. 45 + 2%, P<0.001) and lower proportions of type I la fast myosin heavy chains ( 29 + 3% vs. 39 + 2%, P<0.001) and lib (8 + 1% vs. 17 + 1%, P<0.001)than controls (Levine etal., 1997). Immunohistochemistry revealed similar results in fiber typing. Furthermore, COPD patients had a greater proportion of slow myosin light chains, troponins, and tropomyosin, whereas controls had greater proportions of fast isoforms (Levine et al., 1997). The authors concluded the greater proportions of slow twitch characteristics in the diaphragms of COPD patients is an adaptation to increase fatigue resistance (Levine et al., 1997). These features observed in the diaphragms of COPD and congestive heart failure patients are similar to those in endurance trained limb muscles, as the diaphragm may be undergoing constant moderate exercise (Levine et al., 1997). 55 •  Research from Lloreta and co-workers (1996) supports these findings, as an individual with COPD (FEVi 51% predicted) had mitochondrial abnormalities in the diaphragm. Subsarcolemmal accumulations of mitochondria along with paracrystalline mitochondrial inclusion were noted in the diaphragm, and may be the consequence of an exercise effect (Lloreta et al., 1996). The inflammatory response is known to contribute to muscle injury through the release of free radicals and cell degradation. Inflammation has been observed in the human diaphragm, however, the degree of injury to which it contributes is unknown. Kariks (1989) observed macrophages in the diaphragms of infants with SIDS. In cases where muscle atrophy and disintegration occurred, macrophages were usually noted in either the interstitial or perivascular spaces. In 1998, MacGowan immunostained for macrophages in frozen human diaphragm tissue taken from thoracotomy patients suffering from COPD and other chronic respiratory diseases. Image analysis was used to quantify the number of macrophages in cross-sections taken from subjects. No relationship was found between the number of macrophages and the severity of COPD, as classified by % predicted FEV1. In summary, abnormal diaphragm features and inflammatory cells have been observed in the human diaphragm.  56  CHAPTER 2: RESEARCH STUDY INTRODUCTION AND RATIONALE  The diaphragm is the most important muscle of inspiration. In healthy subjects, muscles such as the diaphragm will possibly respond to an increased work load through hypertrophy and fiber changes to optimise efficiency. In subjects with acute and/or chronic respiratory diseases the diaphragm may become injured. Injury can be observed as morphological changes under light microscopy. Furthermore, these morphological changes in the diaphragm may be measured as functional respiratory changes that make the diaphragm less efficient for its role in inspiration. There are numerous causes and mechanisms that may be responsible for the observed morphological changes in the diaphragm. A possible cause responsible for injury in the diaphragm is exertion. Patients with respiratory disease may overload the diaphragm so that fatigue and/or weakness develops. Because of the obligatory role of the diaphragm, rest is not possible except during mechanical ventilation. Consequently, injury may result in the diaphragm of these subjects. To compound this injury, other potential factors such as corticosteroids, atypical blood gases, mechanical ventilation, resuscitation, malnutrition, and cardiac dysfunction may be present in the heterogeneous sample population. Diaphragm injury has been investigated in numerous animal studies and fewer human studies (for review Reid et al., 1998). Evidence is still forthcoming and a variety of studies are necessary to further understand this important topic.  57  It has been shown that sarcopenia (age related change in muscle) is an important variable when considering muscle morphology. For this reason age has been chosen as a variable that will be statistically analysed to determine if the proportion of abnormal muscle (%) is related to the mean age of subjects. Gender differences in muscle have also been shown. In particular females tend to experience less skeletal muscle injury when compared to males (Tarnopolsky et al., 1995). This may be related to a hormonal protective effect (Komulainen et al., 1999). Therefore, we will determine if gender is correlated with the proportion of abnormal diaphragm morphology (%). Obesity and undernutrition have important implications when considering respiratory muscle injury. Obese individuals have more adipose tissue around the thorax and as a result must work harder to perform the act of respiration. Individuals that are starved or malnourished may experience impaired regeneration or decreased fatigue resistance. Proper nutrition is important for expedient regeneration, and a balance between lipids, carbohydrates and proteins is essential for proper fuel utilisation in muscle. For these reasons we have decided to analyse the relationship between body mass index and the proportion of abnormal diaphragm (%). Lastly, the presence of acute and/or chronic respiratory disease will be analysed to determine if either of these disease states are related to the proportion of abnormal diaphragm morphology (%). It is known that the respiratory muscles becomes injured in exertioninduced animal models. Acute and/or chronic respiratory diseases may injure the diaphragm through a variety of mechanisms, including exertion. Therefore, five variables (age, gender, BMI, presence of acute respiratory disease, presence of  58  chronic respiratory disease) will be determined through a retrospective patient chart review and correlated to the proportion of abnormal diaphragm injury (%). It is also known that inflammatory cell infiltrates and the mediators of inflammation contribute to tissue injury through a secondary reaction. Even though inflammation is a tightly controlled response, collateral and systemic injury may occur in the respective inflamed tissue. It has been shown that PMNs are the initial cell infiltrates in acute inflammation. We will use immunohistochemistry to quantitatively and qualitatively describe the presence of PMNs in the human diaphragm. Virtually no inflammatory cells should be observed in the healthy diaphragm muscle. If PMNs are observed, their presence in the diaphragm may correlate with the proportion of abnormal diaphragm tissue, as a secondary reaction may be occurring.  RESEARCH HYPOTHESIS AND OBJECTIVES  We hypothesize that diaphragm injury occurs in individuals with COPD and other chronic respiratory diseases. Injury is represented by inflammation, connective tissue replacement, or abnormal fiber morphology in the human diaphragm. The purpose of this study was to describe abnormal diaphragm morphology quantitatively and qualitatively in patients with and without respiratory disease. Our objectives were to determine: 1) the presence and contribution of chronic and/or acute respiratory diseases and other relevant demographic factors such as age, gender, and body mass index and their relationship to indicators of diaphragm injury through a retrospective chart review of 59 post-mortem subjects; 2) the proportion of abnormal 59  muscle, normal muscle and connective tissue in the diaphragm by point counting H&E stained cross-section samples; 3) the proportion of PMNs in a cross-sectional area of the diaphragm using the monoclonal antibody NP57 against human neutrophil elastase molecule; and 4) whether more abnormal diaphragm was present in: a) those with acute respiratory disease; b) those with chronic respiratory disease; c) those with a BMI outside the normal range; d) older individuals; e) greater quantities in either gender.  60  METHODS Subjects  Fifty-nine subjects undergoing autopsy were recruited for this study. Inclusion criteria required signed consent from the subject's next-of-kin for an unrestricted autopsy. Exclusion criteria included: individuals that died more than 96 hours prior to autopsy, had Hepatitis B or C virus, HIV, Creutzfeldt-Jakob Disease, forensic cases.  Biopsy Sampling and Preparation  During autopsy the left, right, or both hemi-diaphragms was removed by a pathologist, pathology resident, or autopsy staff at St. Paul's Hospital or Vancouver General Hospital. Full-thickness diaphragm biopsies were collected from the midcostal region of the diaphragm. Effort was made to avoid stretching the muscle tissue upon removal. A scalpel was used to make incisions in the diaphragm muscle, and biopsy samples ranging from 4 cm x 4 cm to full-diaphragms were taken. Biopsy samples were immediately placed in plastic containers and formalin fixed for later processing. Sections of approximately 1 cm x 3 cm were taken from the flattest part of the costal region for histological processing. These sections were placed in a tissue cassette. Cassettes were kept in formalin until histological processing took place. Processing took place in the laboratory facilities in the Department of Academic Pathology, University of British Columbia. Biopsies were dehydrated, embedded in 61  paraffin, sectioned at 5 Lim intervals and stained with hematoxylin and eosin (H&E). Cross and transverse sections were mounted on glass slides and labelled with the respective autopsy number. Image capturing  A SPOT digital camera (Diagnostic Instruments, Inc. Michigan) Version 2.2 was used to capture images of diaphragm samples. This camera was connected to an IBM compatible computer and a microscope (Nikon Microphot, Japan). Images captured were transferred to the computer monitor via the SPOT digital camera. All images captured were of diaphragm cross-sections at 400x (optivar 1 .Ox, objective 40x). Image magnification was determined using a 50 micrometer calibration slide.  Quantitative Evaluation of H&E Stained Diaphragm Cross-Sections  All twenty fields randomly captured per section were evaluated to determine normal and abnormal muscle morphology and artifact. Numerous pathologists were consulted to ensure true and artefactual abnormalities were distinguished from normal tissue morphology. Tissue features were classified in 1 of 10 categories. Normal muscle morphology (1), abnormal features (2-6 & 8), connective tissue (7 & 9), and non-muscle (0) as itemized and defined in Table I.  62  Table I: C a t e g o r i e s a n d Definitions for Point C o u n t i n g  Categories 0.  N o C o u n t : s p a c e , nerve, artefact, epimysial c o n n e c t i v e t i s s u e  1.  Normal  2.  Internal m y o n u c l e u s  3.  Inflamed / Necrotic  4.  S m a l l / A n g u l a t e d / R o u n d Fibers  5.  Abnormal Cytoplasm  6.  Inflammatory C e l l  7.  C o l l a g e n / Fibroblast  8.  D e g e n e r a t e d / Atrophic Fibers  9.  Capillary  63  Definitions  0. Space, nerve, artefact, epimysium, wall or lumen of vessel larger than capillary. 1. Fiber with polygonal shape, homogeneous acidophilic cytoplasm, intact plasma membrane, peripheral nuclei. 2. Fiber containing > 1 internally located nuclei (8 pixels of sarcoplasm between nucleus and sarcolemma). 3. Fiber containing > 1 inflammatory cell or necrotic mass of inflammatory cells and muscle debris without plasma membrane. 4. Small fiber (< 1/3 the lesser fiber diameter (LFD) of the five largest fibers in the field ) (b) fibers with "spear-like" projections or extension that are less than 45 degrees and (c) fibers that have lost polygonal shape and are large and rounded. 5. Includes: (a) fiber with pale acidophilic peripheral cytoplasm and enlarged peripheral nuclei with or without visible nucleoli, or (b) fiber with pale acidophilic peripheral cytoplasm and deep acidophilic "fuzzy" cytoplasm in the central region, or (c) lipofusion, or (d) split or whirled fibers, or (e) vacuoles, or (f) uneven cytoplasmic staining unrelated to processing, or (g) fiber with dull or light grey staining, or (h) cytoplasmic fragmentation. 6. Round nucleus located in interstitium, with morphological features of mononucleated or multinucleated inflammatory cells. 7. Includes: (a) fibrils of endomysium or perimysium, or (b) spindle-shaped nucleus in interstitium. 8. Includes: (a) fiber with withering appearance undergoing degeneration without inflammatory cells (b) fiber undergoing atrophy with normal appearance. 9. Endothelial cell, lumen, or contents of a capillary.  64  Q u a n t i t a t i v eE v a l u a t i o n o f H & E  S e c t i o n s  Quantification of the proportion of normal muscle relative to abnormal muscle or to connective tissue was accomplished through point counting. The computer program Image Pro Plus (IPP4) (Media Cybernetics, LP. Silver Spring MD) version 4.0 was used to assign computer projected points on the diaphragm tissue to one often tissue categories. Using the IPP4 software program, a grid was superimposed onto one of twenty fields captured using the digital camera. The 64 point computer grid consisted of 8 x 8 points (Figure 1). The grid was outlined by a margin that excluded partially viewed fibers. Points were only counted on fibers in which the majority of the fiber could be seen. Fibers that were only partially in view were excluded from counts (Figure 1). The tissue occupying the smallest discernible region in the top right quadrant of the point - intercept was designated to one of the ten categories. A confirmation summary of all points in each category and the total points counted was available on the program before moving onto the next field. After all 64 points were counted, the summation of data was performed and saved to a floppy diskette. The area fractions of normal muscle, abnormal muscle and connective tissue were determined using the equation in Table II and the categories listed in Table I. If a fiber contained two abnormal features, the point-intercepts were assigned to a respective category using the following protocol. If an even number of points were contained within a fiber, the points would be evenly distributed to the two abnormal  65  categories. If the fiber contained an uneven number of points, certain features would predominate. Abnormal features were ranked in the following order of importance from greatest to least: necrotic inflamed, internal nuclei, abnormal cytoplasm, contracted / degenerated / atrophic, and small /angulated / round. The priority of abnormal features was determined by ranking the most injurious features as most important (ie) fiber necrosis. Other features were placed in order by considering their definitions as most commonly mentioned in the literature related to muscle injury. Inter-rater reliability of classification of points was performed in identical fields based on established definitions for each criteria. Using identical categories, definitions and fields a correlation was determined between the investigator, Tyler Clarke, and the thesis supervisor Dr. W.D. Reid. Correlation between the investigator, and supervisor for normal, abnormal and connective tissue was r= 0.97, 0.87 and 0.93, respectively. The overall correlation was determined to be r = 0.97 between the two investigators.  66  Table II: Area Fraction Equations for Calculation of Normal Muscle, Abnormal Muscle, and Connective Tissue.  Total Count  =  S Counts in Categories 1-9  Area Fraction of Normal Muscle  =  S Counts in Category 1 x 100 Total Count  Area Fraction of Abnormal Muscle =  S Counts in Categories 2 - 6 & 8 x 100 Total Count  Area Fraction of Connective Tissue = I Counts in Categories 7 & 9 x 100 Total Count  Immunohistochemistry  Diaphragm cross-sections were de-paraffinzed in Hemo-De and a graded series of isopropyl alcohols. Slides were then kept in de-ionised H 0 until loaded into 2  a Autostainer: Universal Staining System Version 2.0 (DAKO, Carpinteria CA). The alkaline phosphatase-anti-alkaline phosphatase (APAAP) method for paraffin embedded formalin fixed sections was employed. The alkaline phosphatase substrate, Naphthol AS-B1 phosphate in 1% New Fuchsin, was prepared immediately before loading into the autostainer application to cross-sections. Sections were automatically counterstained with Meyer's Hematoxylin and then manually dehydrated 67  by the investigator in isopropanol, Hemo-De and Xylol. Sections were lastly mounted with coverslips using a permanent mounting medium. The monoclonal antibody, NP57 (DAKO Corp., Carpenteria, CA.) diluted to a concentration of 1:40 in 1% bovine serum albumin (BSA) was used to label neutrophils in human diaphragm cross-sections. This antibody selectively stains for neutrophil granule protein elastase. Positive controls consisted of sections of human tonsil processed in the same manner. Diaphragm cross-sections also served as negative controls, with mouse Igd (DAKO, Corp., Carpenteria, CA.) replacing the primary antibody, diluted to the same concentration as NP57. Quantitative Evaluation of PMNs The SPOT digital camera was used to capture images. This camera was connected to an IBM compatible computer and a microscope. Diaphragm crosssections were brought into focus at 200x and muscle fibers and positive cells (PMNs) in ten random fields were quantified on the computer monitor. A transparent overhead was taped to the computer monitor and a red pen was used to mark each fiber, ensuring accuracy of counts. The top and left sides of the screen were inclusion frames, in which fibers and PMNs that were partially out of view were included in the counts. The bottom and right sides of the screen were exclusion frames. Partially viewed fibers and PMNs were excluded from the total counts if they fell on the corners where the exclusion frame meets the inclusion frame. It was determined that the area  68  of 1 counting frame was 0.356 mm . The total area (3.56 mm ) was determined by 2  2  multiplying the number of fields (n=10) by the area per counting frame 0.356 mm . 2  The total number of PMNs per fiber was calculated by dividing the total number of PMNs by the total number of muscle fibers. The total number of PMNs per crosssectional area (CSA) was determined by dividing the total number of PMNs by the total area viewed (3.56mm ). 2  Chart Reviews  A detailed retrospective chart review for all subjects was completed. Presence or absence of respiratory disease (Table lll-VI) was determined by the investigator (Tyler Clarke) and a respirologist (Dr. J Road) after a detailed examination of the subjects clinical history through a retrospective chart review. The chart review usually contained a pathologist's autopsy report, detailing pathological findings that correlated with previous clinical presentations. This included demographic information such as gender, age, body mass index (BMI) presence or absence of chronic and/or acute respiratory diseases, medication and disease history, cause of death, date of death, date of autopsy. Confidentiality was maintained by keeping all records in a locked filing cabinet, referring to patients in this study by a random three-digit code. This code corresponds to a four to five digit autopsy code on a master list in a locked cabinet. Only Tyler Clarke (investigator), Dr. W. Darlene Reid (supervisor) and Dr. J. Road (respirologist) had access to chart information.  69  Study Design  This study was a retrospective non-experimental descriptive analysis of the relationship between potential clinical factors (age, gender, BMI, presence or absence of chronic respiratory disease, and presence or absence of acute respiratory disease) and abnormal diaphragm morphology.  Statistical Analysis  The non-parametric Mann-Whitney U test was used to determine the contribution of clinical factors (age, gender, BMI, presence of acute respiratory disease, presence of chronic respiratory disease) to the proportion of abnormal diaphragm morphology. The dependent variable, area fraction of abnormal diaphragm, was a continuous variable. Two ratio variables (age and BMI) and three ordinal variables (gender, presence or absence of acute respiratory disease, presence or absence of chronic respiratory disease) were the independent variables. A significance level of p<0.05 was selected for the Mann-Whitney U test. A correlation analysis was performed to determine significance and which variables would be selected for the Mann-Whitney U test.  70  RESULTS Clinical Characteristics of Post-Mortem Patients:  Twenty female and thirty-nine male subjects were included in this study. Thirtythree of the 59 subjects had a chronic respiratory disease, 38 had acute respiratory disease, 22 had acute respiratory disease superimposed on chronic respiratory disease and 10 had no respiratory diseases (Tables III & V). Age for both sexes ranged from 26 to 87 years, with females ranging in age from 48 to 86 years and males ranging from 26 to 87, years. The mean age for females was 72.4 (SD 11.9) and males 65.6 (SD 13.7). Of the 58 subjects age was accounted for, 37 were considered elderly (ages 65 to 87) (Enright et al., 1994), whereas 21 were considered adults (ages 26 to 64) (Black & Hyatt, 1969). Of the 37 elderly subjects, 23 were classified as having a chronic respiratory disease, whereas 8 of the 21 adults had a chronic respiratory disease (Table III). Body mass index (BMI = kg/m ) was used to access weight to height ratios in 2  subjects. According to Naidu & Rao (1994) the lower limit of normal BMI is 18.5 kg/m . 2  Williamson (1993), defined the upper limit for normal BMI to be 27.3 kg/m for women 2  and 27.8 kg/m for men. In this study the BMI was available for 40 subjects; values 2  ranged from 17.3 to 38.8 kg/m , with females and males exhibiting mean BMIs of 2  24.5kg/m and 26.5kg/m , respectively. All of the 10 subjects over the normal BMI 2  2  were males, while the 2 individuals under the normal BMI were females. Subjects were classified according to demographic data (see Appendix A) and summarised by nutritional status (Table VII), initial diagnosis (Table VIII), smoking 71  history (Table IX), use of mechanical ventilation (Table X), resuscitation history (Table XI), and cause of death (Table Xll).  72  Table III: Distribution of Subjects with Chronic Respiratory Disease (CRD)  Subjects with CRD  33  Subjects with CRD and ARD  22  Neither ARD nor CRD  10  Males with CRD  24  Females with CRD  9  Table IV: Summary of Chronic Respiratory Diseases  COPD  20  IPF  4  Pleural Effusion  2  Asthma  1  Asthma and COPD  1  Chronic Bronchitis  1  COPD and IPF  1  Chronic Pulmonary Edema  1  Asbestosis  1  Veno-occulsive Disease  73  1  Table V: Distribution of Subjects with Acute Respiratory Disease (ARD)  Subjects with ARD  38  Males with ARD  25  Females with ARD  13  Table VI: Summary of Acute Respiratory Diseases and Dysfunctions (ARD subjects may have had > 1 concurrent diseases or dysfunctions).  Pneumonia  25  Pulmonary Edema  12  Respiratory Failure  8  Hypoxia  6  Hypercapnia  3  Sepsis  2  ARDS  2  Interstitial Pneumonitis  1  Pleural Effusion  1  74  Table VII: Subject's Nutritional Status  Reported malnourished (n=14)  Reported obese (n=12)  (2 with BMIs under normal range)  (10 with BMIs over normal range)  2 had CRD  2 had CRD  3 had ARD  4 had ARD  7 had CRD and ARD  0 had CRD and ARD  Table Vlll: Subject's Initial Admission Diagnosis  Cardiac dysfunction (n=17)  Respiratory dysfunction (n=17)  6 had CRD  1 had CRD  2 had ARD  8 had ARD  4 had CRD and ARD  8 had CRD and ARD  75  Table IX: Subjects with a History of Smoking n=21 6 had CRD 1 had ARD 8 had CRD and ARD  Table X: Subjects undergoing Mechanical Ventilation n=22 4 had CRD 9 had ARD 6 had CRD and ARD  76  Table XI: Subjects undergoing Resuscitation  4 had CRD 2 had ARD 1 CRD and ARD  Table Xll: Subject's Cause of Death  Cardiac Related (n=24)  Respiratory Related (n=19)  7 had CRD  1 had CRD  5 had ARD  8 had ARD  6 had CRD and ARD  8 had CRD and ARD  77  Table XIII: Quantitative Evaluation of Diaphragm Biopsies  Distribution of Mean Area Fractions (A ) of 3 main Categories (Normal and Abnormal Muscle and Connective Tissue in the Diaphragm (%)). A  Normal Muscle  56.29 % SE 2.65 %(16.5 to 83.5%)  Abnormal Muscle  27.42 % SE 2.00 % (5.4 to 61.4%),  Connective Tissue  16.28 % SE 1.25 % (3.4 to 57.6%)  Collagen is included as part of connective tissue. Ranges shown in brackets.  78  Table XIV: Quantitative Evaluation of Diaphragm Biopsies continues.  Distribution of Mean Area Fractions (A ) of Sub-divisions of 3 main Categories. A  Collagen / Fibroblast  15.88 % SE 1.26 % (2.9 to 57.2%)  Abnormal Cytoplasm  11.62 % SE 1.57 % (0 to 49.6%)  Fibers with Internal Nuclei  11.06 % SE 0.92 % (1.0 to 29.9%)  Small Angulated or Rounded Fibers  1.81 % SE 0.36 % (0 to 16.8%)  Degenerated or Atrophic Fibers  1.60 % SE 0.24 % (0 to 11.2 %)  Inflammatory Cells  1.17 % S E 0.10 %(0 to 4.1%)  Capillaries  0.40 % SE 0.05 % (0 to 1.8%)  Inflamed / Necrotic Fibers  0.17 % SE 0.04% (0 to 1.6%)  Ranges shown in brackets. See Tables I & II for description of each category.  Quantification of PMNs  The results of PMN counting (n=38) are represented in Figure2. Of the 38 samples quantified, 22 had CRD and 28 had ARD. The mean number of PMNs per fiber was 0.0044 (range: 0 to 0.06). The mean number of PMNs per cross-sectional area was 1.46/mm (range: 0 to 22.8/mm ). 2  2  Qualitative Description of Diaphragm Cross-sections  Table XIII provides a summary of point counting results. The most common category observed was the normal muscle category (Table XIII). Normal muscle 79  architecture reveals closely packed polygonal fibers arranged in fascicles, surrounded by perimysial connective tissue (Figure 3). Within normal muscle limited endomysial connective tissue free of leukocytes is observed (Figure 3). In the transverse section, multiple nuclei are located in the periphery of the muscle fibers, but normal muscle may contain 4% internally located nuclei (Cumming et al., 1994). Fibers vary in size according to age, sex, and biopsy site, however, the coefficient of variation for fiber diameter normally does not exceed 25% (Cumming et al., 1994). Fibers observed as normal in the present study maintained these characteristics, however any fibers with internal nuclei were categorised as abnormal (Figure 1 dark arrow). The second most common category was the abnormal tissue category (Table XIII). Within the abnormal category the sub-category collagen / fibroblast was the most commonly observed feature (Table XIV). Collagen is a typical feature of skeletal muscle as it is observed around individual muscle fibers and fascicles. Collagen was identified as endomysium or perimysium fibrils and stained a light pink with H&E stain. In many diaphragm biopsies the amount of collagen was dramatically increased and appeared abnormal (Figures 4-8). All diaphragm biopsies contained some abnormal features. The most common abnormal features in this study, ranging from most to least common, were the presence of: a) collagen (Figures 4-8); b) abnormal cytoplasm (Figures 4); c) internal nuclei (Figures 1,4,6-10); d) small, angulated or rounded fibers (Figures 4,6-8); e) degenerated, or atrophic fibers (Figure 4 & 5); f) inflammatory cell (Figure 5); g) and inflamed / necrotic fibers (Figure 7 & 9-11).  80  Abnormal cytoplasm includes fibers that had pale acidophilic peripheral cytoplasm and enlarged peripheral nuclei; pale acidophilic peripheral cytoplasm with "fuzzy" cytoplasm in the central region; lipofusion; split; "whorled" fibers; vacuoles; and uneven cytoplasmic staining. These features were distinguished from processing artifact, and when in question were brought to the attention of a pathologist to ensure proper categorisation. Some of these features are observed in figure 4. Approximately 4% of normal muscle cross-sections contain internal nuclei, which may increase closer to the myotendinous junction (Cumming et al., 1994). In order for a count to enter this category there must be at least one internal nuclei and a clear distinction must be made between the nucleus and sarcolemma. Upon magnification in the Image Pro Plus computer program, this distance must be at least 8 pixels. Internal nuclei were typically observed singularly (Figure 1, dark arrow) or in pairs (Figure 8) within a muscle fiber. The greatest amount of internal nuclei observed by the investigator within a cross-sectional diaphragm fiber is shown in figure 8. Small fibers were defined as < 1/3 the lesser fiber diameter (LFD) of the five largest fibers with normal morphology within the field. Angulated fibers contained sprouting extension or concave sides, whereas rounded fibers had lost their polygonal appearance and were spherical in appearance. The angulated fibers observed had sharp spindle-like appearances and spear-like projections. The majority of these projections contained small angles, less than 45°, and many contained concave sides (Figure 7). The rounded fibers had lost their normal polygonal appearance and were almost circular. These fibers were usually larger and at times stained darker with H&E than other surrounding fibers (Figure 7). 81  Degenerated or atrophic fibers were typically observed. These fibers may be of normal or reduced size and appeared undergoing degeneration or atrophy. Fibers in this category had degenerating, "withering" or "shrinking" appearances (Figure 5). Inflammatory cells, most commonly occurring as macrophages or PMNs are associated with abnormal morphology. These cells are difficult to distinguish from cell nuclei with light microscopy and immunohistochemistry or electron microscopy is usually required. These cells were identified by either their round dark or multilobulated nucleus in the interstitium. These cells were normally observed singularly, but at times multiple inflammatory cells were present in focal areas (Figure 5). Capillaries were seldom observed in cross-sectional fields of view. Capillaries were observed by their lumen or by the presence of exclusive contents such as erythrocytes. The investigator was careful to distinguish capillaries from muscle spindles that contain small diameter fibers enclosed by a connective tissue capsule. The least common abnormal feature that was observed was the presence of inflamed or necrotic muscle fibers. These fibers had at least 1 inflammatory cell or necrotic mass of inflammatory cells and muscle debris without a plasma membrane. The inflammatory lesions were usually focal and rarely diffuse. Inflamed diaphragm muscle fibers usually had an obvious loss of myofibrillar material (Figure 6).  82  Relationship between Clinical Factors and Diaphragm Morphology  Box plots were used to illustrate the relationship between independent variables and the dependant variable (area fraction of abnormal diaphragm). The ordinal independent variables (presence of chronic respiratory disease, presence of acute respiratory disease and gender) are shown in figures 2-14. The relationship between the ratio of independent variables (age and body mass index) and dependant variable (area fraction of abnormal diaphragm) are represented through scatterplots (Figure 5). PMNs were seldom observed in the diaphragm. The antibody NP-57 (DAKO Corp.) was used to label PMNs in diaphragm cross-sections. PMNs were typically observed in the endomysial connective tissue (Figures 9 & 11) and at times were observed in necrotic and inflamed fibers (Figures 9-11). The non-parametric Mann-Whitney U test was used in this study to analyse the correlation between all six variables in 59 subjects (39 males and 20 females). The test showed men had more abnormal diaphragm than women (Figure 13, lower panel) (P=0.015). Male subjects with acute respiratory disease had a significantly greater percentage of abnormal diaphragm muscle than male subjects without acute respiratory disease (Figure 12, upper panel) (p=.021). The percent difference of abnormal diaphragm tissue for males with and males without acute respiratory disease compared to those without is 35.69% and 26.61%, respectively. The mean age of male subjects was significantly lower than female subjects (M: mean 65.6 yrs. + SD 13.7; F: mean 72.4 yrs. + SD 11.9) (Figure 13, upper panel) (p=.049). Men with chronic respiratory disease (mean 70.0 yrs. + SD 10.3) were significantly older than 83  men without (58.7 yrs. + SD 15.7) (p=0.048). Males and females with acute respiratory disease had a greater proportion of abnormal diaphragm tissue than those without acute respiratory disease (Figure 14, lower panel) (p=.067). The percent difference of abnormal diaphragm for males and females with acute respiratory disease compared to those without was 29.61% and 23.47%, respectively. Similarly, all subjects (M & F) with chronic respiratory disease tended to have a greater proportion of abnormal diaphragm tissue than those without chronic respiratory disease (Figure 12, lower panel)(p=.082). The percent difference of abnormal diaphragm for males and females with chronic respiratory disease compared to those without was 30.39% and 23.66%, respectively. There was no significant relationships between BMI nor age and the proportion (A ) of abnormal diaphragm (Figure 15, A  upper and lower panels, respectively).  84  Figure 1:  Image of a computer monitor screen showing a light photomicrograph of H & E stained human diaphragm crosssection and Image Pro Plus 4 software used for the point counting set up.  85  Figure 2:  Data plots showing the relationship between the number of PMNs per cross-sectional area and the presence of acute respiratory disease (upper panel) or chronic respiratory disease (lower panel). No relationship was found between the number of PMNs and the presence of acute or chronic respiratory disease.  86  PMNs in Diaphragm  5 h 0  4-  Absent Present (n=10) (n=28) Presence of Acute Respiratory Disease  PMNs in Diaphragm  Absent Present (n=16) (n=22) Presence of Chronic Respiratory Disease  87  Figure 3 :  Light photomicrograph of normal morphology in H & E stained human diaphragm cross-section. Note normal polygonal appearance or muscle fibers with nuclei in the periphery (arrow) and minimal connective tissue between fibers. Scale bar = 50 urn  88  Figure 4:  Light photomicrograph of vacuolized and angulated fibers in a H & E stained human diaphragm cross-section. Note vacuolization (arrow), small angulated fiber with internal nuclei (asterisk), and connective tissue (arrowhead). Scale bar = 50 Lim.  89  Figure 5:  Light photomicrograph of inflammatory cell infiltration in area with extensive connective tissue in a H & E stained human diaphragm cross-section. Note inflammatory cells in endomysial connective tissue (arrow), small atrophic fibers (arrowhead), and large adjacent fibers (asterisk). Scale bar = 50 pm.  90  Figure 6:  Light photomicrograph of extensive connective tissue in a H & E stained human diaphragm cross-section. Note connective tissue (arrow) and internally located nuclei (arrowhead). Scale bar = 50 um.  91  Figure 7:  Light photomicrograph demonstrating 2 necrotic cells in a H & E stained human diaphragm cross-section. Note necrotic inflamed fibers (arrows), small angulated fiber (arrowhead), and connective tissue (asterisk). Scale bar = 50 u.m.  92  Figure 8:  Light photomicrograph showing a variety of fiber shapes in a H & E stained human diaphragm cross-section. Note large rounded fiber with multiple internal nuclei (large arrow), angulated fibers (small arrow and arrowhead), connective tissue (asterisk). Scale bar = 50 urn.  93  Figure 9:  Light photomicrograph of human diaphragm cross-section labeled with NP-57 antibody to PMNs. Note PMN in endomysial connective tissue (arrow) and an inflamed fiber (arrowhead). Scale bar = 50 urn  94  *  1 ,  0 m  »  61 I  4  Figure 10:  Light photomicrograph of necrotic and inflamed fibers in a human diaphragm cross-section labeled with NP-57 antibody to PMNs. Note inflamed fibers (arrows and arrowhead). Scale bar = 50  urn.  95  Light photomicrograph of necrotic and inflamed fibers in a human diaphragm cross-section labeled with NP-57 antibody to PMNs. Note PMN in endomysial connective tissue (small arrow), inflamed fiber (large arrow), and internally located nuclei (arrowhead). Scale bar = 50 urn  96  Figure 12:  Boxplots showing the relationship between the area fraction of abnormal diaphragm muscle (%) and gender and acute respiratory disease (upper panel) and chronic respiratory disease (lower panel). Males with acute respiratory disease had more abnormal diaphragm than females with acute respiratory disease (upper panel). Shaded area indicates interquartile range, midline indicates median, lines projecting from ends indicate the range of values.  97  (n=7)  (n=14)  (n=13)  (n=25)  Gender & Acute Respiratory Disease  <  oJ N=  Present (n=33)  Absent (n=26)  Chronic Respiratory Diseases  98  Figure 13:  Boxplots showing the relationship between age and gender (upper panel) and the area fraction of abnormal diaphragm muscle (%) and gender (lower panel). Females were older than males (upper panel) and males had more abnormal diaphragm (lower panel). Shaded area indicates inter-quartile range, midline indicates median, lines projecting from ends indicate the range of values.  99  100 90 H  N=  F  M  (n=20)  (n=39) G E N D E R  100  Figure 14: Boxplots showing the relationship between the presence of acute respiratory disease and the area fraction of normal diaphragm morphology (%) (upper panel) and abnormal diaphragm morphology (%) (lower panel). Shaded area indicates inter-quartile range, midline indicates median, lines projecting from ends indicate the range of values.  101  _  .7  <  0.0 Absent (n=21)  Present (n=21)  Acute Respiratory Diseases  102  Figure 15:  Scatterplots showing the relationship between the area fraction of abnormal diaphragm muscle (%) and BMI (upper panel) or age (lower panel). No relationship was found between the area of abnormal diaphragm morphology and BMI or age.  103  VP  LU -J  .6-  O W .53  <  .4 -  or  o .3z CD  < .2 • < LU < .1 20  10  30  40  Body Mass Index (Kg/m )l 2  20  30  40  50  60  AGE (years)  104  70  80  90  DISCUSSION  In humans, gender differences have been documented for skeletal muscle strength, fiber size and type, and motor unit number (Miller et al., 1993). It has also been observed that asthmatic males experience reduced strength and efficiency of the respiratory muscles compared to females (Weiner et al., 1990). Surprisingly, our study showed that male subjects had a greater proportion of abnormal diaphragm morphology than female subjects (p=.015). Furthermore, male subjects with acute respiratory disease had a significantly greater proportion of abnormal diaphragm morphology and connective tissue than female subjects with acute respiratory disease (p=.021). The differences in the area fraction measurements (A ) of abnormal A  diaphragm morphology between males and females was not an anticipated result, as this study is the first to show gender differences in diaphragm injury in humans. In contrast, little information on gender differences of the diaphragm in human and animal models is available. MacGowan (1998) observed significant gender differences in the presence of abnormal diaphragm morphology using a point counting system in a small sample of post-mortem patients (n=26) but significance was lost when all subjects in the study (n=33 included the 7 subjects lacking BMI) were included. Gender differences in ventilatory response to hypoxia in rats has been observed (Mortola et al., 1996). During short periods of hypoxia, female rats have lower respiratory resistance and greater respiratory system compliance than male rats. These findings are consistent with those by Yokoyama and co-workers (1984) who 105  found lung compliance to be 65% greater and pulmonary resistance to be 31% less in female rats than males (Mortola et al., 1996). These differences in female rats would lead to less ventilatory work, however, these differences may change in animals with lung disease. Interestingly, ovariectomy did not alter the female's responses observed (Mortola et al., 1996). Whether these gender differences exist in humans and protect against diaphragm injury in female respiratory patients remains to be reported. Gender differences in diaphragm injury may be partially attributed to insulin resistance. Insulin is necessary for maintenance of muscle protein mass, and the inability to use insulin in metabolism has been shown in male Sprague-Dawley rats. Furthermore, insulin resistance has been shown to increase as a function of body weight (Lawler et al., 1994). Interestingly, adult male rats have less protein content and oxidative enzyme activity in the diaphragm than female rats (Lawler et al., 1994). The researchers hypothesise that this is related to greater peripheral insulin resistance in the fatter male rats (Lawler et al., 1994). A decrease in oxidative enzymes and protein content would likely lead to reduced fatigue resistance and reduced regeneration if observed in humans. Unfortunately, we were not able to consistently document the presence or absence of diabetes in our retrospective chart review. Body mass index was examined in 40 of 59 subjects, however, only 10 were regarded as obese. Further studies examining the relationships between diabetes, obesity and diaphragm injury may demonstrate similar findings in humans. Gender differences have also been observed in blood flow distribution in the diaphragm of rabbits (Lublin et al., 1995). Blood flow was reported to be two to three fold higher in muscles of active respiration (diaphragm and intercostals) in females 106  than males. These differences were observed in both controls and heat-stress animals, leading the authors to propose that sex hormones (estrogens and progesterone) play a role. The human diaphragm is well vascularised, however, blood flow distribution in the diaphragm may change in individuals with cardiac dysfunction. If gender differences in blood flow existed in the human diaphragm, diaphragmatic ischemia and reduced regeneration may occur in males. This is because reduced blood flow would inhibit the transport of essential elements of regeneration such as oxygen and amino acids. It is known that malnutrition may contribute indirectly to diaphragm fatigue and prolong regeneration time. Malnutrition may affect the diaphragm differently in males and females. In a study by Prezant and co-workers (1994) the effects of malnutrition (10 weeks) were studied on diaphragm contractility, fatigue and fiber types in male and female rats. Only in males did malnutrition reduce costal diaphragm weight and result in costal diaphragm atrophy of both type I and II fibers (Prezant et al., 1994). However, in spite of these changes the male rats preserved diaphragm contractility and fatigue resistance. Similar to Komulainen and co-workers (1999) and Lublin and co-workers (1995) the authors concluded that the lack of significant effect of malnutrition on female diaphragm morphology and function may be the result of a protective hormonal environment. It is possible that females have an innate protective effect against muscle injury due to differences in hormone levels. It has been shown that after downhill running, females rats have reduced (5-glucuronidase (lysosomal enzyme of muscle injury) activity in the quadriceps 4 days post-exercise compared to the male rats (Komulainen 107  et al., 1999). Furthermore, male rats showed reduced dystrophin staining in some fibers post-exercise, whereas female rats did not (Komulainen et al., 1999). The loss of staining indicates disruption of the cytoskeletal system. The authors also noted that histopathological changes (swollen fibers, inflammation and fibers necrosis) occurred later and to a lesser extent in the female rats. They concluded that oestrogen levels may stabilise the sarcolemma or provide protection against oxidative stress in female rats (Komulainen et al., 1999). Females with acute respiratory disease showed significantly less abnormal diaphragm than males with acute respiratory disease. Therefore, it is reasonable to propose that female subjects in our study could be protected from diaphragm muscle injury, especially in acute respiratory disease. Possible explanations could be related to a hormonal protective effect, or the way females use fuels in endurance exercise. Conversely, it is equally possible that males with acute respiratory disease have something that decreases their resistance to diaphragm injury. Tarnopolsky and co-workers (1995) observed that female athletes oxidised more lipid and less glycogen and protein during submaximal exercise. If this is true in the diaphragm, women may be less susceptible to fatigue, and consequently injury through different fuel preferences during exercise. This may partially explain the observed lack of significant diaphragm injury in female ARD subjects, and less abnormal diaphragm morphology in female CRD subjects. It is unclear what gender differences, if any, may be present in the diaphragm of patients with respiratory disease. Our observation that males show more diaphragm injury warrants an investigation into other possible gender differences, including 108  ventilatory responses, diaphragm mass, enzyme function, perfusion, atrophy, injury, cause of death, fuel utilisation and genetic influences. Medical histories and clinical presentation could reveal other possible factors that would explain the greater proportion of abnormal diaphragm in males. A similar number of males (41%) and females (40%) died of cardiac related causes, however, a greater number of males died of respiratory related causes (36%) than females (25%). A greater number of males were also initially diagnosed with respiratory related problems (33%) than females (15%). Furthermore, a greater number of males were found to have a smoking history (41%) than females (25%). What is similar and perplexing is that males were prescribed corticosteroids (21%) and resuscitated (15%) roughly the same amount when compared to females (20%) and (15%), respectively. Furthermore, males were less often mechanically ventilated (33%) compared to females (45%). In a recent study of 221,306 mechanical ventilation cases (52.8% males) gender differences have been observed in the use of mechanical ventilation (MV) for various underlying illnesses (Clermont et al., 2000) (p<0.0001). These authors speculated this difference exists for numerous reasons including a causal link between gender and a predisposition to acute respiratory failure (Clermont et al., 2000). The similarities reported in our study between the use of corticosteroids and resuscitation in males and females and the observation that females were mechanically ventilated more is surprising. We would have expected more diaphragm injury in the females that were mechanically ventilated, but for unknown reasons males with acute respiratory disease still had greater diaphragm injury than females with acute respiratory disease. 109  The combination of medical histories and clinical presentations in our study may be related to the explanations as to why males are more susceptible to diaphragm injury than females. Our study was comprised of 66.1% males and 33.9% females. For this reason alone more data from medical charts was available, therefore, more assumptions as to why injury is greater in males may have been made. At the same time, more males (36%) died of respiratory related causes than females (25%). Therefore, it is reasonable to propose that males would have more diaphragm injury. Age was a significant variable in this study, as men (65.5 ±13.7 yrs) were younger than women (72.4 ±11.9 yrs) (p<0.049). The observation that men were younger and a majority in this post-mortem study is not surprising as many gender related causes of death are known. According to the National Centre for Health Statistics, 1992 age adjusted death rates per 100,000 for a variety of causes were published. Male life expectancy (73.2 yrs) was 6.6 years less than female life expectancy (79.8 yrs) (Virginia et al., 1999). Furthermore, the ratio of male to female deaths were: heart disease 1.9; COPD 1.7; malignant neoplasms 1.4; cerebrovascular disease 1.2; motor vehicle crashes 2.3; pneumonia and influenza 2.0; suicide 5.9; chronic liver disease or cirrhosis 2.5; alcohol induced causes 3.5; drug induced 2.9; and all causes 1.8 (Virginia etal., 1999). In our study, men with chronic respiratory disease (n=24) were older (69.8 yrs) than men without chronic respiratory disease (n=15) (58.7 yrs) (p<0.048). This is not a surprising finding as most of the mortality associated with COPD is in individuals over the age of 65 (Pride, 1995). This increased age of death in chronic respiratory patients versus non respiratory patients could also be due to recent advances in the 110  management of respiratory disease and the decline of smoking in the last two decades. In countries such as the US and UK where smoking peaked two decades ago, death from COPD has fallen except in those over 75 years of age (Pride, 1995). The relationship between abnormal diaphragm morphology and the presence of chronic respiratory diseases was shown to be a trend p=0.082. This relationship did not show significance as outlined by our hypothesis that the presence of chronic respiratory disease would contribute significantly to abnormal diaphragm morphology. A similar relationship was also observed by MacGowan (1998) in a post-mortem study consisting of 33 subjects. MacGowan observed a slightly stronger trend that abnormal diaphragm morphology is related to the presence of chronic respiratory disease (p=.065). These observations are important as diaphragm injury is occurring in individuals with chronic respiratory disease. The greatest abnormal feature quantified in our study was the presence of collagen/fibroblast (A 15.88 + SE 1.26%, range: 2.9 to 57.2% ). This was an A  expected finding, as similar research by MacGowan (1998) also found the greatest abnormal feature to be the increased presence of collagen/fibroblast (A 12.3 + SD A  4.1%, range: 4.6 to 22.4%). Extensive connective tissue has been observed in the diaphragms of two individuals with respiratory disease (Reid et al., 1998). Moreover, fibrosis was observed in the diaphragms of infants that died from SIDS (Kariks, 1989). Furthermore, phrenic nerve section in animals and some of the diaphragms of hamsters tracheal banded for 30 days showed increased presence of connective tissue (Reid et al., 1998). Rabbits that underwent high IRL for 90 minutes revealed widening of the interstitium in the diaphragm (Road et al., 1998) but this is likely 111  related to edema rather than increased collagen. Conversely, other investigators who studied respiratory muscle injury such as Hards and co-workers (1990) observed moth-eaten fibers and ragged red fibers as the most common abnormalities. Increased endomysial, perimysial and fatty connective tissue occurs when muscle fibers degenerate. As severity of injury increases in the affected muscle so does the deposition of connective tissue (Banker and Engel, 1994). The initial loss of muscle fibers may be due to degeneration, necrosis or atrophy and the surplus of connective tissue represents impaired regeneration. The regeneration of myofibrils with extracellular matrix expansion and increased collagen is commonly observed in muscles that have been overloaded or subjected to exertion-induced injury (Stauber et al., 1998). According to Kariks (1989) fibrosis may be regarded as the end stage of regeneration as a result of re-occurring injury. Chronically over-loaded muscles result in existing myofibers to become surrounded by increased extracellular matrix, and fibrosis may result from the deposition of Type I collagen around myofibers (Stauber et al., 1998). The main factors governing the deposition of connective tissue are the size of the damaged area and the severity of muscle architectural damage (Banker and Engel, 1994). As the area of damage increases in size, so does the proliferation of collagen (Banker and Engel, 1994). The presence of collagen not only impairs the contractile function of muscle, but also inhibits future muscle regeneration. Regenerating muscle sprouts extensions to neighbouring fibers which guide the shape and size of the new fiber. It is known that regenerating fibers circumvent the fibrotic barrier by aborting regeneration (Banker and Engel, 1994). The investigator in this 112  study observed that areas of increased connective tissue density was usually focally located and varied greatly between fields within a sample. Collagen was infrequently the dominant morphological feature in an overall field of view. The second most common abnormal feature was the presence of abnormal cytoplasm (A 11.62 ± SE 1.57%, range: 0 to 49.6%). Features such as acidophilic, A  basophilic or "fuzzy" cytoplasm, lipofuscin, whorled fibers, vacuolisation, cytoplasmic fragmentation and uneven staining are included in this category. The most common abnormal cytoplasm sub-characteristics were acidophilic or basophilic cytoplasm, patches of "fuzzy" or densely staining cytoplasm, hyaline fibers and "loose" cytoplasm. Basophilic fibers were commonly observed and quantified as abnormal cytoplasm. These fibers stain more intensely and appear as a deeper purple colour when stained with H & E . These fibers are indicative of regeneration as the deeper stain is likely due to the concentration of polysomes and ribosomes that are the prerequisites of new contractile filaments (Banker and Engel, 1994). Moreover, regenerating fibers usually contain numerous nuclei and nucleoli that are large, dark and prominent (Banker and Engel, 1994) which were observed at times in our study. Basophilia also presented in the form of ragged-red fibers, but these fibers were seldom observed. Ragged-red fibers increase with age and may be due to abnormal mitochondria, however this must be confirmed by subsequent histochemical staining using a trichrome stain. Another common abnormal feature that was quantified under the category abnormal cytoplasm are the heavily stained hyaline fibers. The appearance of hyaline features and loose "stream-like" cytoplasm are present in regenerating fibers 113  (Cummings et al., 1994). These brightly stained fibers are the result of hypercontraction bands, similar to the observations by Silver and Smith (1992) in the diaphragms of infants that died suddenly. We observed these bands to partially transverse a cross-section of a fiber, and at times consume the entire fiber area. Silver and Smith attributed these bands to a catecholamine surge that induced a sudden sarcomeric spasm (Silver and Smith, 1992). At the electron microscope level it is evident that loss of l-bands, Z-band streaming, overstretching of the sarcomere and degeneration of the myofilaments occurs (Cummings et al., 1994). Consequently, these hyaline fibers may undergo necrosis as debris is removed by phagocytic cells. The surplus of connective tissue observed in this study may be due to the degeneration of these hyaline fibers, as fibroblastic processes circumscribe the degenerating fiber (Banker and Engel, 1994). Fibers with a "fuzzy" appearance within the cytoplasm were occasionally observed. This denser staining "fuzzy" appearance may be representative of a fiber with zones of Z-band streaming that could be observed with electron microscopy (Cummings et al., 1994). Split or branching fibers were also occasionally observed. Fiber splitting is indicative that focal damage has occurred at the fiber separation (Banker and Engel, 1994). Furthermore, similarities have been observed between fibronectin extensions from a central nucleus preceding fiber splits (Stauber et al., 1998). It is believed that somehow fibronectin is involved in the splitting of fibers (Stauber etal., 1998). Other features less commonly observed were delta lesions. These fibers are represented by pale areas lacking cytoplasmic detail and they appear partially 114  digested. These delta lesions are representative of "focal subsarcolemmal dissolution of the myofilaments" accompanied by the loss of the plasma membrane (Cummings et al., 1994). Lipofuscin was occasionally observed, but tended to be present in a few select biopsies. This yellow-brown pigment that accumulates in fibers is the end product of lipid or lipoprotein peroxidation. These lesions were typically observed under the sarcolemma, irregular in outline, and approximately the size of nuclei. This abnormal feature that increases with age is non-specific in pathology and may be found in normal muscle (Banker and Engel, 1994). Interestingly, these deposits are accentuated in vitamin E deficiency (Banker and Engel, 1994). Vitamin E deficiency has been shown in animal studies to induce fatigue and decrease force during IRL (Supinski, 1998). Furthermore, vitamin E deficiency may be present in those subjects that are malnourished. Lobulated fibers were seldom observed and were categorised as abnormal cytoplasm. This feature is a non-specific change in muscle that usually occurs in type 1 fibers (Banker and Engel, 1994). Fibers with a multicore or moth-eaten appearance were also rare in appearance. These fibers have reduced oxidative enzyme activity and some show myofibrillar degeneration (Banker and Engel, 1994). However, without further histochemical staining it is not possible to determine if these features truly existed in our study. Vacuolisation was occasionally observed and categorised in the abnormal cytoplasmic category. Vacuoles were observed as an empty space within the fiber usually in a central location. Although the vacuole size varied, it was most commonly 115  solitary and comprised one-third the total fiber area in a cross-section. Vacuoles are thought to contain numerous different substances, including the contents of cytoplasmic degradation (Banker an Engel, 1994). The presence of internal nuclei was common in all biopsies and ranged from 10 to 261 internal nuclei per 20 fields viewed. The mean area fraction (AA) for internal nuclei was 11.06 + SE 0.92%, range: 1 to 29.9%. This is much greater than the number of internal nuclei (4%) usually encountered in skeletal muscle biopsies (Cummings et al., 1994). The presence of internal nuclei are a non-specific abnormal response but are a characteristic finding in primary muscle diseases such as muscular dystrophy (Banker and Engel, 1994). In a similar study by MacGowan (1998) internal nuclei were observed as the second most common abnormal feature in the diaphragm of patients with and without chronic and/or acute respiratory disease. MacGowan (1998) observed a mean area fraction of internal nuclei of 6.9 + SD 5.0%, range: 1.2 to 19.0%. This is substantially less than our observed counts of internal nuclei. Possible explanations are related to the differences in the point-counting system used in each study. Our study used an Image Pro Plus computer program that allowed for images to be saved and zoomed when features were in question. Conversely, MacGowan used a microscope at 500x and projected a grid from the computer monitor via the camera lucida onto the image of the muscle cross-section. Internal nuclei have been observed in numerous studies and have been shown to appear after 24 hours and peak at 48 hours post-injury in mice skeletal muscles after eccentric-induced muscle injury (Lowe et al, 1995). Increased myonuclear number associated with functional overload may be due to satellite cells activated to 116  proliferate during overload (McCall et al, 1998) following injury (Stauber et al., 1998) heavy exercise, denervation or drug treatment (steroids) (Kasper & Xun, 1996). These additional myonuclei may facilitate skeletal muscle hypertrophy, as increased myonuclei expand the amount of DNA available for protein synthesis (McCall et al, 1998). The number of myonuclei is thought to be related to "nuclear domain", or "DNA unit" which is the theoretical volume of cytoplasm associated per myonucleus (Allen et al., 1995). The addition of new myonuclei maintains a constant cytoplasmic-tomyonuclear volume during hypertrophy. Conversely, the number of myonuclei decline during atrophy (Allen et al., 1995). Increases in internal myonuclei are interpreted as a regenerative feature, whereby the internal nuclei orchestrates the repair process (Carlson & Faulkner, 1983). In our study it is reasonable to assume the surplus of internal nuclei in the diaphragms of patients with respiratory disease is related to functional overload and may be involved in the repair or hypertrophy of the muscle fibers. Small angulated or rounded fibers were occasionally observed and rounded fibers usually appeared singly in the field (A 1.81 + SE 0.36%, range: 0 to 16.8%). A  Round fibers were large and had lost their polygonal appearance. This rounding appearance in a cross-section is a non-specific myopathy, however rounding and angulation typically accompany denervation atrophy (Banker and Engel, 1994). Possible explanations for the combination of small angulated fibers in the same area as large rounded fibers may be due to selective denervation of the small angulated fibers. In these situations only fibers of the denervated motor unit atrophy, whereas hypertrophy of the nondenervated fibers occurs (Banker and Engel, 1994). This 117  hypertrophy is most likely due to increased workload placed on the nondenervated fibers (Banker and Engel, 1994). A result of denervation is that other motor units may reinnervate by collateral sprouting and there is an increase in fibers of the same histochemical type. This may explain the increase in type I fibers observed by Levine and co-workers (1997) in the diaphragms of patients with COPD. Degenerated and atrophic fibers were commonly observed (A 1.6 + SE 0.24%, A  range: 0 to 11.2). Fibers in this category were withering and many possessed a grey degenerating appearance. Myogenic atrophy may be responsible for the loss of fiber size. In this type of atrophy repeated cycles of degeneration followed by incomplete regeneration or repair occur. Furthermore, myogenic atrophy may be caused by focal cytoplasmic degeneration or corticosteroids (Banker and Engel, 1994). Denervation atrophy (outlined above) may also be responsible for the observed atrophy of many diaphragm fibers. Inflammatory cells were rarely observed in the interstitium (A 1.17 + SE 0.10%, A  range 0 to 4.1%). Without immunohistochemical analysis it is not possible to confirm or speculate on the type of cell observed. Macrophages have been observed in the diaphragm, however, their appearance was not related to the severity of airflow obstruction (FEV -i) observed in thoracotomy patients (MacGowan, 1998). Inflammatory cells are believed to contribute to the secondary injury observed in rabbits undergoing high IRL (Jiang et al., 1998). However, their role in human diaphragm injury is not known at this time. Similarly, inflamed or necrotic fibers were rarely observed in the diaphragms of our subjects (A 0.17 ± SE 0.04 %, range: 0 to 1.6%). This may be because the injury A  118  induced was not sufficient enough to result in necrosis. Chronic low intensity injury may be occurring which would explain the surplus of collagen observed. Another explanation may be that acute inflammation may occur in the diaphragm and biopsies were obtained after acute inflammation and during the chronic inflammation phase. The numerous amounts of morphological abnormalities observed are not to be confused with post-mortem autolysis. One of the inclusion requirements was that diaphragms were only taken from subjects that died < 96 hours prior to autopsy. To ensure that no post-mortem autolysis was occurring during this period MacGowan (1998) studied rats that died at various times up to 96 hours. Post-mortem autolysis (PMA) is distinct from all other abnormalities in our study, as PMA shows reduced staining in the central region of H&E diaphragm cross sections (MacGowan, 1998). PMA was not observed in our human samples as the majority of subjects were recruited one day after death. Counts were excluded in samples where this type of staining was observed. Age and BMI were not shown to contribute to diaphragm injury as determined by the proportion of abnormal diaphragm morphology. Males had a mean BMI of 26.46 kg/m (S.D. 4.64) whereas, females had a mean BMI of 24.48 kg/m (S.D. 5.24). 2  2  Both genders had BMIs within the normal range. In obese individuals, body fat is stored near the chest wall and obesity increases respiratory muscle work while also impairing pulmonary function (Poole et al., 1997). In a rat model of obesity there is an increase in oxidative fibers in the diaphragm, and increased diaphragmatic mass. Furthermore, force generated by the costal diaphragm is reduced by 15% when compared to leaner rats (Poole et al., 1997). Conversely, malnutrition which is 119  represented by a reduced BMI may affect respiratory muscles from adapting to the increased workload imposed by acute and/or chronic respiratory disease. Malnutrition may depress respiratory muscle function and regeneration in patients with acute and/or chronic respiratory disease, as diaphragm mass is reduced in underweight patients (Poole et al., 1997). The number of obese (n=10) and underweight subjects (n=2) as indicated by the BMI showed no correlation to abnormal diaphragm morphology. Our population was very diverse in clinical presentation and BMI may prove to be significant in association to diaphragm injury in the future when a larger population is studied. Subjects with acute respiratory disease showed a trend toward abnormal diaphragm morphology. This is surprising that significance was not shown because individual cases demonstrated marked diaphragm injury. It is possible that the range in clinical presentations in our heterogeneous sample were too great to show significance. The infrequent finding of PMNs in the diaphragm in this study was surprising because of their initial role in inflammation and muscle injury. Neutrophils have been observed almost immediately after direct diaphragm muscle stimulation in hamsters (Reid et al., 1989) and after IRL in rabbits (Jiang et al., 1998). This study is important as it quantifies the presence of PMNs in the human diaphragm in patients with and without chronic an/or acute respiratory diseases. The relatively small number of PMNs observed, except in two cases, may be due to the time course of injury that was uncontrollable in this study. Furthermore, the interaction between the patient's drugs and PMN presence in the muscle was not analysed. Likewise, other factors such as 120  age, blood flow, radiation, chemotherapy, malnutrition, and concurrent diseases were not analysed. It is interesting that three of the five individuals with the most PMNs per muscle fiber had acute respiratory disease only, and none had chronic respiratory disease. This may be due to the time course of the injury-inducing event, as individuals with chronic respiratory disease may receive partial injury from other inflammatory cells such as T and B-cells.  121  Chapter 3: SUMMARY AND RECOMMENDATIONS  SUMMARY  The most important observation of this study is the significant finding of gender differences in the diaphragms of subjects with acute respiratory diseases. All males, and males with acute respiratory disease had more diaphragm injury than all females and females with acute respiratory disease, respectively. Numerous reasons may explain this finding, but it is possible that females have a protective hormonal effect to muscle injury. Furthermore, the observation that injury tends to occur in patients with chronic and/or acute respiratory diseases is important. Other important characteristics of this study are the large sample size and qualitative and quantitative descriptions made. This study should prove useful for future research into the many possible factors contributing to diaphragm injury. Morphological abnormalities were observed in the diaphragms of patients with and without respiratory disease. Morphological indicators of injury observed from greatest to least were: increased deposition of collagen, abnormal cytoplasm, internal nuclei, small angulated or rounded fibers, degenerative or atrophic fibers, inflammatory cells, necrotic or inflamed fibers. Since the most common abnormality was increased collagen it is reasonable to assume that incomplete regeneration is occurring in diaphragms that are overloaded due to respiratory diseases. Age, body mass index and the presence of PMNs in the diaphragm were not related to diaphragm injury as determined by abnormal diaphragm morphology. A 122  larger or more defined sample size may show different findings for the relationship between obesity and malnutrition, and diaphragm injury.  RECOMMENDATIONS  This study is limited by its heterogeneity of subjects possibly masking significant findings that would be present in a more defined subject population. Studies in the future should include a similar or larger sample size and investigate other important variables such as diabetes, protease activity, diaphragm mass, muscle fiber type and size, fuel utilisation and the response of various inflammatory cells. Most importantly, studies in the future should have a more defined sample population. This study and a similar study by MacGowan (1998) both showed abnormal diaphragm in subjects with chronic and/or acute respiratory disease, however, both failed to show significant relationships. Subjects in the future for the experimental group should have stricter inclusion criteria. For example, only subjects with chronic and/or acute respiratory disease that were smokers or had idiopathic dilated cardiomyopathy would be included. This type of experimental protocol should draw out the group that is consistently demonstrating diaphragm injury in a significant manner, but is diluted by another portion of respiratory disease patients that have minimal diaphragm injury. For example we found that 14 individuals with abnormal diaphragm equal to or greater than 40% (range: 41.5 to 61.4%). Of these 14, all were males and of the thirteen we had chart information for, 12 had cardiac dysfunction and 6 were smokers. Furthermore, 11 had acute respiratory disease, 9 had chronic 123  respiratory disease, and 7 had both. In this entire study of 59 subjects, 36% had a smoking history, however, upon examination of our sub-population of 14 subjects (13 with extensive chart information available) 43% had a smoking history. This type of focussing may reveal that certain individuals within the respiratory disease group are more susceptible to significant diaphragm injury. Therefore, future studies that successfully demonstrate diaphragm injury in patients with chronic and/or acute respiratory disease(s) may find that only a sub-population of these patients show significant injury worthy of intervention. It is also recommended that more information on subject respiratory function is included in future studies. This may help to explain the gender differences observed. It is also recommended that stains for muscle regeneration (dMHC and desmin) are incorporated into future studies. This analysis would confirm our observation that extensive muscle regeneration is occurring in the diaphragms of patients with acute and/or chronic respiratory diseases.  124  References  Allen D.L., Monke S.R., Talmadge R.J., Roy R.R., & Edgerton V.R. (1995). Plasticity of myonuclear number in hypertrophied and atrophied mammalian skeletal muscle fibers. Journal of Applied Physiology. 78(5). 1969-1976. American Thoracic Society (1995). 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