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Collagen content of the human diaphragm Scott, Alexander 2003

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COLLAGEN CONTENT OF THE HUMAN DIAPHRAGM by Alexander Scott B.Sc.(PT), University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (School of Human Kinetics) We accept this thesis as conforming to the.required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 2003 © Alexander Scott 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT INTRODUCTION In limb muscle, the amount of endomysial, perimysial, and basal lamina collagen can increase as an adaptive response to endurance training or in response to exertion-induced injury. In the costal diaphragm, there is evidence for both adaptation and injury in people with chronic obstructive pulmonary disease (COPD). The present study had four objectives: (1) to quantify the proportional cross-sectional area of collagen (CSA c o n ) and the percentage of myofibres with abnormal or injured morphology (Pabn) in the post-mortem COPD diaphragm, and to compare this in individuals with no significant respiratory diagnosis (NSRD); (2) to determine the CSA c o n and P a b n in different regions of the human diaphragm (midcostal, costal insertion, central tendon region, crural); (3) to compare the CSA c o n and P a b n in the diaphragm and a non-respiratory muscle (psoas major); (4) to determine the relationship between CSA^i and fibre cross-sectional area and P a b n . METHODS Mid-costal diaphragm biopsies were obtained and formalin-fixed from 6 COPD and 6 age- and gender-matched NSRD subjects. For the regional studies, whole diaphragms were obtained and formalin-fixed from 18 subjects. CSA c o n was determined by computer-assisted point-counting of a 1.3 mm 2 area on 6 um-thick cross-sections. P a b n was calculated by categorizing all muscle fibres within an area of 1.7 mm2 as normal or abnormal based on morphological criteria with an inter-rater reliability of 0.957. Fibre areas were calculated from the means of at least 200 fibres per biopsy using computerized image analysis software. RESULTS The COPD diaphragm displayed a greater CSA^,, than NSRD (mean + SE: 24.2 + 1.0% vs 18.6 +1.1%, p< 0.001) and a greater P a b n (28.4 + 7.2% vs. 12.0 +_1.3%, p< 0.05). Abnormal areas of accumulated endomysial and perimysial collagen (i.e. fibrosis) were observed more frequently in the COPD diaphragm, often in association with small, abnormally shaped muscle fibres. The COPD diaphragm displayed features of acute and chronic injury including inflammation and necrosis. The CSA c o n and P a b n of the crural diaphragm were smaller than the mid-costal values (18.0 + 0.6% vs. 20.9 + 0.6%, p < 0.01 and 11.9 + 2.5% vs. 14.6 + 2.6%, p<0.01 respectively). The psoas major displayed a lower P a b n (8.3 + 2.2%) and CSA^,, (12.3 + 0.5%) than the mid-costal (p < 0.01) and crural diaphragm (p < 0.001). CONCLUSION The increased injury and collagen in the post-mortem COPD diaphragm is extensive. iii Table of Contents Abstract » List of Tables iv List of Figures v List of Abbreviations vi 1. Introduction Background 1 Objectives 2 Hypotheses 3 2. Methods Subjects 4 Diaphragm sampling and processing 4 Proportional cross-sectional area quantification of collagen 7 Quantification of abnormal muscle fibres and fibre size 9 Statistical analysis 11 3. Results Subject characteristics ' 11 COPD vs. no signficant respiratory diagnosis 12 Regional differences in the diaphragm 12 Diaphragm vs. psoas major 20 Correlation and fibre size analysis 20 Types of abnormal cytoplasm observed 20 4. Discussion COPD vs. NSRD 28 Regional differences in the diaphragm 32 Diaphragm vs. psoas major 33 Correlation and fibre size analysis 34 Interpretation of abnormal cytoplasm 35 Relevance 36 Summary 36 References 38 Appendix I Literature Review The diaphragm in normal breathing 45 The diaphragm in COPD .46 Exertion-induced muscle injury 47 Intramuscularconnective tissue of the diaphragm 50 Role of collagen in modulating inflammation and regeneration 52 Possible factors leading to increased Connective tissue in the diaphragm 52 Summary 57 Appendix II Raw data Calculation of collagen CSA standard error 58 Reliability testing for point-counting of collagen CSA 59 Reliability of morphological categories 60 Reliability testing for fibre size 61 Collagen CSA counts NSRD 63 COPD 65 Regional 67 Injury counts NSRD 85 COPD 87 Regional 89 iv List of Tables Table 1. Descriptive characteristics of COPD and NSRD subjects 5 Table 2. Descriptive characteristics of subjects for regional study 6 Table 3. Categories for computerized point counting of H&E-stained diaphragm cross-sections 10 V List of Figures Figure 1 Diaphragm sample sites 8 Figure 2 Photomicrographs of picrosirius red-stained diaphragm cross sections 13 Figure 3 Cross-sectional area of collagen and percentage of abnormal fibres in COPD and non-respiratory subjects 15 Figure 4 Photomicrographs of abnormal morphology in the human diaphragm 16 Figure 5 Categorization of muscle fibres in COPD and non-respiratory Subjects 18 Figure 6 Regional variation in the cross-sectional area of collagen and percentage of abnormal fibres in the diaphragm 19 Figure 7 Categorization of muscle fibres in different regions of the diaphragm 21 Figure 8 Comparison of cross-sectional area of collagen and percentage of abnormal fibres in psoas major and the diaphragm 22 Figure 9 Categorization of muscle fibres in the psoas major and the diaphragm 23 Figure 10 Correlations analysis 24 Figure 11 Diaphragm fibre size in COPD and non-respiratory subjects 26 List of Abbreviations A N O V A analysis of variance C O P D chronic obstructive pulmonary disease CSAC0|| proportional cross-sectional area of collagen CT computerized tomography CTR central tendon region E C M extra-cellular matrix FENAi forced expiratory volume in one second FVC forced vital capacity H&E haematoxylin and eosin HIV human immunodeficiency virus N S R D no significant respiratory disease Pabn percent of fibres with abnormal morphology Pint nuc percent of internally nucleated fibres Pabncyt percent of fibres with abnormal cytoplasm Pabnshape percent of fibres with abnormal shape or size P S R picrosirius red RV residual volume SD standard deviation S E standard error T L C total lung capacity A delta; difference 1 CHAPTER 1 Introduction The collagen reinforcements within skeletal muscles include the basal lamina, endomysium and perimysium, all of which are involved in active and passive force transmission (Huijing 1999). In limb muscles, the amount of collagen in these structures increases in response to acute or chronic exertion-induced muscle injury (Myllyla et al. 1986, Stauber et al. 2000). Exertion-induced injury is defined as a disruption of sarcomeres in response to repetitive contractions, and is characterized by inflammation and necrosis, typically followed by complete regeneration (Wernig et al. 1990). An acute increase in interstitial collagen plays important roles in stabilizing regenerating myotubes and facilitating the adhesion of satellite cells and macrophages involved in myofibre regeneration (Myllyla et al. 1986, Hurme & Kalimo 1992). However, a chronic or permanent increase in the amount of collagen (i.e. fibrosis) results from repeated bouts of exertion-induced injury (Stauber et al. 2000). In chronic, exertionally injured limb muscles, the increase in collagen was accompanied by numerous small fibres which may have been atrophic or incompletely regenerated (Stauber et al. 2000). Unlike exertionally injured limb muscles, respiratory muscles are prone to be overloaded for relatively long periods of time without respite in a variety of neuromuscular and pulmonary pathologies (Reid & MacGowan 1998). If the diaphragm's intramuscular collagen content were increased due to chronic exertional injury, this might decrease its efficiency of force transmission and impair its ability to generate inspiratory pressures especially in cases where higher diaphragm activation was required. Individuals with COPD show an increased area of exertion-induced diaphragm injury following inspiratory resistive loading (Orozco-Levi et al. 2001). The amount of injury, which is manifest as areas of disrupted sarcomeres, has been correlated to the degree of hyperinflation (RV7TLC). Diaphragm injury among individuals with airflow limitation has also been positively correlated with the forced expiratory volume in one second (FEVi), with internally nucleated fibres as the most common abnormal feature (MacGowan et al. 2001). In rats, low-level ventilatory loading applied over 30 days resulted in similar features of chronic injury in the diaphragm including increased variability of fibre size, internally nucleated fibres and focal areas of fibrosis (Reid & Belcastro 1999). Thus, the diaphragm appears to be prone to exertion-induced injury at relatively low loads, and particularly among individuals with COPD and hyperinflation of the chest wall. Individuals with COPD experience high levels of diaphragm 2 activation even at rest; individuals with COPD demonstrated a 45% voluntary maximal RMS of the diaphragm during quiet breathing, compared to 8% in normal individuals (Sinderby et al. 1998). The diaphragm has distinct costal and crural regions, and performs most of the work of breathing. The generation of effective inspiratory pressures by the diaphragm requires that a portion of the force generated by myofibres be transmitted by the collagens of the extra-cellular matrix, with considerable variation in muscle-fibre architecture across the regions of the diaphragm (Boriek et al. 2001 b). Despite this, the cross-sectional collagen fraction has not been reported for the various regions of the human diaphragm. Although life-time endurance training or chronic stimulation in animals has been shown to cause an increase in the intramuscular collagens, such a process has never been shown in humans (Kovanen 1989, Wright et al. 1997). The diaphragm presents a unique opportunity to determine whether a skeletal muscle adapted for endurance by a lifetime of repetitive low-level contractions would demonstrate a greater cross-sectional area of collagen than a non-respiratory muscle from the same individual. In the absence of features of injury, a greater amount of collagen in the diaphragm than in a non-respiratory muscle would support the postulate that the collagen content of human skeletal muscles may be upregulated in response to chronic loading, as appears to be the case in animals (Rodrigues et al. 1996). Objectives The objectives of the study were: 1. to determine whether individuals with COPD have a greater area fraction of intramuscular collagen and proportion of injured fibres in the diaphragm compared to those individuals with no significant respiratory disease; 2. to determine if different regions of the diaphragm displayed greater or lesser area fractions of intramuscular collagen and proportions of injured fibres; 3. to compare the area fraction of intramuscular collagen and and proportion of injured fibres in the diaphragm and in a non-respiratory muscle, the psoas major; 4. to examine the relationship between the area fraction of intramuscular collagen, the proportion of injured fibres, and the size and variability of size in costal diaphragm muscle fibres. Hypotheses 1. The diaphragm of individuals with C O P D will have a greater cross-sectional area of collagen (CSAcon) and P a b n than N S R D in the mid-costal region. 2. Because of possible regional variations in stress and strain and accumulated injury, the C S A c o n and P a b n will not be uniform across the human diaphragm. 3. CSAcon and P a b n of the costal and crural diaphragm will be greater than the C S A c o N of the psoas major. 4. Increasing variability of fibre size and increasing P a b n will be associated with an increase in CSA C 0||. A decrease in fibre size will be associated with an increase in C S A c o n . CHAPTER 2 Methods Subjects Ethical approval was obtained from the university and hospital ethical committees. Consent for autopsy was obtained in each case from next of kin by the appropriate physician. Cases were excluded if under legal proceedings; if infected with Hepatitis B or C or HIV; or if death occurred greater than 96 hours prior to autopsy. For the study of regional variation in CSA^i, diaphragms and psoas biopsies were obtained from 18 subjects. For the comparison between those with COPD and NSRD, autopsy findings and medical records were reviewed with a respirologist to confirm the diagnosis of COPD or the absence of significant respiratory pathology. Additional exclusion criteria included (in addition to the above) congestive heart failure, neuromuscular disease, myopathies, and insufficient evidence to ascertain the presence of COPD. In order to be included in the COPD group, the autopsy findings had to be corroborated by the medical records. Autopsy findings of COPD included the destruction of air spaces distal to the terminal bronchioles, hyperplasia of mucus-producing glands and the accumulation of mucus, inflammation and edema in the small airways. Corroborating evidence in some subjects included a positive CT scan of the chest, hyperinflation of the chest on X-ray, reduced FEVi / FVC ratio (< 60%), prolonged smoking history, and a history of breathlessness or chronic cough. Eight out of 59 biopsies gathered in 1996 and 1998-9 met the above criteria, as did 4 of the 18 regional biopsies gathered in 2001-2, for a total of 12 age- and gender-matched COPD and non-respiratory subjects (see Table 1). The spirometric reports for 5 of 6 COPD patients were obtained. FEV! ranged from 0.80 litres (33% predicted) to 1.91 litres (52% predicted). Spirometry was completed within 2 years prior to death. Diaphragm sampling and processing For the regional studies, 18 diaphragms and psoas biopsies were dissected during autopsy at the time of evisceration, without the necessity of additional invasive incisions. The exact method of dissection varied depending on the attending pathologist, however the orientation of the diaphragm could be preserved in every case. Whole costal diaphragms were pinned flat in anatomic orientation on paraffin wax and fixed in 10% buffered formalin. The crural diaphragm was either dissected intact along CD > 3 cr T^. 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Diaphragm biopsy sites were sampled from four sites (Figure 1); (1) right mid-costal (in the mid-axillary line); (2) right musculotendinous insertion (~3 mm from the visible beginning of the central tendon in the mid-axillary line); (3) right costal insertion (as close as possible to the costal insertion in the mid-axillary line); and (4) right crural diaphragm midway between origin and insertion. A psoas major biopsy was taken from the muscle belly distal to the lumbar origin. For the comparison between COPD and NSRD, either the right or left mid-costal diaphragm was sampled. After fixation in formalin, biopsies were oriented transversely and longitudinally under a dissecting microscope, paraffin-embedded in tissue cassettes, sectioned on a microtome at 6 pm and floated onto glass slides. Sections were de-paraffinized in Hemo-De (Fisher, Toronto ON) and stained with standard (Harris') H&E. For visualization of collagen, sections were re-hydrated in graded alcohol, rinsed for 10 minutes under running water, stained in picrosirius red (PSR) for 1 hour, rinsed in 1% acetic acid, rapidly dehydrated, cleared in Hemo-De and coverslipped with Permount (Fisher Canada, Toronto ON). In skeletal muscle, PSR specifically stains collagen fibres of the extracellular matrix (ECM) (including types I, III, and V) and the basal lamina (type IV) (Sweat et al. 1964). Cross-sectional quantification of PSR-stained tissue is commonly used to compare the amount of collagen in skeletal muscle biopsies (e.g. Wright et al. 1997). Proportional Cross-sectional Area Quantification of Collagen Cross-sections stained with PSR were used to determine CSA c ou of the mid-costal diaphragm in the two groups (COPD and NSRD) and across the different diaphragm regions and psoas muscle. Cross-sections were randomly assigned a number and digitized by an investigator who was blinded to the clinical history associated with the biopsy. The number of viewing fields per section (15) were determined from calculations based on two biopsies (see Appendix, p. 58). The standard error of CSA^i based on 20, 15, and 10 viewing fields was calculated; an acceptable standard error of 0.3% was achieved using 15 viewing fields. Viewing fields per section were digitized using a Nikon brightfield microscope with a 40x objective and 0.6x reduction lens attached to a SPOT digital camera. The viewing fields were selected in a systematically randomized way, keeping at least 100 pm from the 8 Figure 1. Diaphragm sample sites. Black dots represent sampling locations. For full description of locations, see text. Based on a diagram in Osmond (1995). 9 edges of the biopsy, and avoiding large vessels. A 63-point grid of evenly spaced crosses was projected onto each digitized field. The smallest discernible area of the upper right quadrant in each cross was classified as collagen, sarcoplasm, or no count (red blood cell, nerve, muscle spindle, empty space, artifact). The CSAcoii was defined as the number of points falling on collagen divided by the total number of points falling on collagen and sarcoplasm. Twenty fields were evaluated by two raters with an inter-rater reliability of r = 0.957. Quantification of Abnormal Muscle Fibres and Fibre Size Cross-sections stained with H&E were used to evaluate muscle fibre abnormalities in the mid-costal diaphragm of the two groups (COPD and NSRD) and across the different diaphragm regions and psoas muscle. H&E cross-sections were also used to measure mid-costal muscle fibre CSA in the two groups—COPD and NSRD. Cross-sections were randomly assigned a number and digitized by an investigator who was blinded to the clinical history associated with the biopsy. Twenty randomly selected viewing fields per section were digitized using a Nikon brightfield microscope with a 40x objective and 0.6x reduction lens attached to a SPOT digital camera to yield an image of 1315 x 1033 pixels projected onto a computer monitor. A micrometer was used to measure the resultant image size (334 x 261 pm) and to calibrate the measures of fibre area (3.9 pixels per pm). For quantification of H&E morphology, twenty digitized fields per cross-section were examined. Fibres were classified according to normal or pathological categories based on morphology, similar to those published previously (MacGowan et al. 2001) allowing the percentage of fibres with abnormal morphology to be determined (Table 3). Inter-rater reliability of the categories ranged from r = 0.94 to r = 0.96 based on categorization of > 5000 fibres on separate days. To obtain measures of fibre area, at least 200 fibres per biopsy were outlined with a mouse using image analysis software (Image Pro Plus 4). Inter-rater reliability of fibre area was r = 0.99 based on 100 independently measured fibres. Variability coefficients of muscle fibre size were calculated by multiplying the standard deviation by 1000 and then dividing by the mean fibre area as outlined by Dubowitz (1985). > O " 3 o 3 0 > 3 0 ) O fl) "o CO > C T 3 O —* 3 ffi. CO N ' 0 CO zr 0 ) " O CD ro r 1 > 3 cr i— 3 ro o —. 3 3 91 ffi. 3 £= O O CD o t= "a CO fl> CO 3 co ro z Z TJ o 3 orm erip 0 ) . zr CD CO o N ' ro o 3 fl> £= 3 fl) O C L CO CD CO zr 3 US fl) T 3 CD CD fl) ro CO fl) CO cB 3 a =» CD C T ° : 3 C T —\ CD co C L ° 3, 2 5 S 2, ro co C L — C Q fl) co <3 y. ro _ 0 ) O C T = S fl) ro CD w Q. g -C T zr 3 0 — K ro' C L "o' § o o r i 0 03 — • i-t-3 CD 0 3 C L 8 0 1 s= a-l O CD 3 C L 3 C g-3 Q . _ - O C L W CD CO CD o « < CO ro 0 3 o cn TJ fl). CD cn ffi 3| 3 ' C Q O —\ C T 0 ) cn O •o zr o' o o •a &) cn 3 C Q —\ 0 ) 3 c i i . i i . Q3 3 CD =1 •8 I S = 0 ) 2 ,3 C T CD S C Q CD -1 CD fl) cn ro TZ 3 o o % o •a 0 ) co cr ro % ro ro 3 3 c o CD c cn 0 ) 3 a. cn 0 ) 8 o ro 3 3 0 ) I o 3 o C Q CD 3 O c: cn to N ' ro TD o C Q o 3 ffi. cn zr Q) "a ro m < ro 3_ I—t-CD X CD C L CD O cn 3 ' o T J O O o T J . 0 > cn 3 z c o. ro £ = CO cn c 0 ) i—t-CD C L 3 to t: 3 o —h cn 0 ) —1 o o ro 3 3 o> a ro o 11 Statistical Analysis To test for differences in C S A c o n and P a b n in the age- and gender-matched COPD and NSRD groups, paired t-tests were used (a = 0.05). To test for differences in C S A c o n and Pabn across the central tendon, mid-costal, costal insertion, and crural regions, an ANOVA (1 within-subjects factor) was used. To test for differences in C S A c o n and P a b n among the costal and crural diaphragm and the psoas, an ANOVA (1 within-subjects factor) was used. Because of the variability of C S A c o n , the fields from each individual (15 fields) were pooled for analysis by group (COPD / NSRD) and region, as commonly done in muscle biopsy studies (e.g. Anderson et al., 1998). In the case of a significant main effect, Tukey's post-hoc was used to test for pair-wise differences. To gain further insight, the sub-categories of P a b n (percentage of internally nucleated fibres, Pintnuc; percentage of fibres with abnormal cytoplasm, Pabncyt! percentage of fibres with abnormal shape or size, PabnShape) were subjected to analysis identical to that described for P a b n . To test the null hypothesis that the COPD and NSRD fibre size distributions were from identical populations, the Kolmogorov-Smirnov test was used. All statistical analysis was performed using S P S S statistical software. Results The descriptive characteristics of the COPD and NSRD subjects are presented in Table 1. There were 4 males and 2 females in each group. Age (COPD range: 55 to 78 yr, NSRD range: 52 to 78 yr) and BMI (COPD range: 19.6 to 30.3 kg/m2, NSRD range 15.8 to 27.3 kg/m2) were similar. FEV! values were available for 5 of the 6 COPD patients, and ranged from 33% to 61% of predicted values. One of the COPD subjects also had pulmonary fibrosis associated with the COPD (Table 1, subject 11). The causes of death in the NSRD group were predominantly cardiac, whereas among COPD patients the predominant cause of death was respiratory failure. Of the 6 COPD subjects, 5 had pneumonia. One patient (subject 10) experienced prolonged mechanical ventilation (35 d), with repeated attempts to wean followed by 14 days on assist-control ventilation prior to death following a planned one-way extubation. Subjects from whom whole diaphragms were obtained (n = 18) included 11 males and 7 females with ages ranging from 27 to 82 yr (Table 2). The causes of death included acute and chronic causes. The majority (n = 12) had an acute or chronic respiratory diagnosis. None were mechanically ventilated for longer than 4 d. Two subjects were taking corticosteroids however the dose could not be determined from the medical records. 12 1. COPD vs NSRD In d i a p h r a g m c r o s s s e c t i o n s p r o c e s s e d f o r P S R , c o l l a g e n w a s l o c a l i z e d t o t h e e n d o m y s i u m , p e r i m y s i u m , e p i m y s i u m , b a s a l l a m i n a , a n d in v e s s e l w a l l s ( F i g u r e 2 ) . T h e o n l y o t h e r s t r u c t u r e s w h i c h s t a i n e d p o s i t i v e l y f o r c o l l a g e n w e r e n e r v e s h e a t h s - t h e s e w e r e a s s i g n e d t o t h e n o c o u n t c a t e g o r y . P o i n t c o u n t i n g r e v e a l e d a g r e a t e r p r e s e n c e o f c o l l a g e n in b i o p s i e s f r o m t h e C O P D g r o u p c o m p a r e d t o t h e N S R D g r o u p ( F i g u r e 3 ) . F o c a l a r e a s o f f i b r o s i s a s s o c i a t e d w i t h s m a l l f i b r e s o r s i g n s o f n e c r o s i s w e r e o b s e r v e d in t h e C O P D d i a p h r a g m a n d , r a r e l y , in t h e N S R D g r o u p . T h e CSAc 0 i i w a s h i g h e r in t h e C O P D d i a p h r a g m c o m p a r e d t o N S R D ( 2 4 . 2 + 1 . 9 % v s . 1 8 . 6 + 2 . 8 % , p < 0 . 0 0 1 ) ( F i g u r e 3 ) . H & E s t a i n i n g r e v e a l e d a v a r i e t y o f a b n o r m a l i t i e s w h i c h w e r e p r e s e n t in b o t h C O P D a n d N S R D d i a p h r a g m s , b u t w h i c h w e r e m o r e e x t e n s i v e in t h e C O P D b i o p s i e s ( F i g u r e 4 ) . T h e C O P D d i a p h r a g m h a d a h i g h e r P a b n ( 2 8 . 4 + 7 . 2 % v s . 1 2 . 0 + 1 . 3 % , p < 0 . 0 5 ) ( F i g u r e 3 ) . W h e n s u b - c a t e g o r i e s o f P a b n w e r e a n a l y z e d , t h e m o s t p r e d o m i n a n t f e a t u r e w a s a n e l e v a t e d Pabncyt ( C O P D = 1 6 . 0 + 1 2 . 8 % , N S R D = 2 . 5 + 1 .5%, p < 0 . 0 5 ) ( F i g u r e 5 ) . T h e p e r c e n t a g e s o f i n t e r n a l l y n u c l e a t e d f i b r e s ( C O P D = 8 . 8 + 2 . 2 , N S R D = 6 . 4 + 0 . 9 ) a n d a b n o r m a l l y s h a p e d f i b r e s ( C O P D = 3 . 6 + 0 . 9 , N S R D = 2 . 1 + 0 . 7 ) t e n d e d t o b e h i g h e r in t h e C O P D g r o u p b u t w e r e n o t s i g n i f i c a n t l y d i f f e r e n t . 2. Regional differences in the diaphragm T h e l o c a l i z a t i o n o f c o l l a g e n in a l l d i a p h r a g m r e g i o n s w a s i d e n t i c a l t o t h a t d e s c r i b e d a b o v e f o r t h e C O P D / N S R D g r o u p s , w i t h t h e e x c e p t i o n t h a t in b i o p s i e s f r o m t h e c e n t r a l t e n d o n r e g i o n , p o i n t s o c c a s i o n a l l y fe l l o n a r e a s s t a i n e d d e n s e l y r e d w h i c h c o r r e s p o n d e d t o t e n d i n o u s i n s e r t i o n s . T h e CSAcoii o f t h e d i f f e r e n t d i a p h r a g m s a m p l e s i t e s is g i v e n in F i g u r e 6 . T h e c e n t r a l t e n d o n r e g i o n h a d t h e h i g h e s t c o l l a g e n C S A c o N ( 2 3 . 1 + 0 . 8 % ) f o l l o w e d b y t h e m i d - c o s t a l ( 2 0 . 9 + 0 . 6 % ) a n d c o s t a l i n s e r t i o n ( 1 9 . 1 + 0 . 7 % ) . T h e c r u r a l r e g i o n h a d t h e l o w e s t C S A c o N ( 1 8 . 0 + 0 . 6 ) . A b n o r m a l d i a p h r a g m m o r p h o l o g y w a s o b s e r v e d in a l l r e g i o n s o f t h e d i a p h r a g m e x a m i n e d ( F i g u r e 6 ) , i n c l u d i n g a l l o f t h e f e a t u r e s l i s t e d in T a b l e 1 . T h e P a b n o f t h e m i d - c o s t a l r e g i o n ( 1 4 . 6 + 2 . 6 % ) w a s h i g h e r t h a n t h e c r u r a l d i a p h r a g m ( 1 1 . 9 + 2 . 5 % , p < 0 . 0 1 ) a n d t h e c o s t a l i n s e r t i o n ( 1 1 . 8 + 2 . 3 % , p < 0 . 0 5 ) , b u t l o w e r t h a n t h e c e n t r a l t e n d o n r e g i o n ( 2 1 . 9 + 3 . 1 % , p < 0 . 0 1 ) . A c o m p a r i s o n o f a b n o r m a l m o r p h o l o g y a m o n g s t t h o s e w i t h a n d w i t h o u t r e s p i r a t o r y c o n d i t i o n s w a s n o t p e r f o r m e d . 13 Figure 2. Photomicrographs of Picrosirius Red-stained human diaphragm cross sections. (Top panel) Normal distribution of endomysial (arrow head) and perimysial collagen (open arrows). Black arrow points to separation artifact. (Bottom panel) Increased presence of collagen fibres in the endomysium (black arrow) and perimysium (open arrow). Note the small and abnormally shaped fibres embedded in the fibrotic region. 40 35 ^ . 30 1 < 25 O 20 c cu J2 15 o ° 10 5 0 - ** T T -Non-respiratory COPD Non-respiratory COPD Figure 3. (Top panel) Cross-sectional area of collagen in COPD and non-respiratory subjects. Error bars represent SE of pooled viewing fields from 6 COPD and 6 non-respiratory subjects (90 fields per group). (Bottom panel) Pabn in COPD and non-respiratory subjects. Error bars represent SE of n = 6. * denotes p < 0.05, ** denotes p< 0.001. 16 Figure 4. Abnormal H&E morphology in human diaphragm cross sections. A: Extensive injury in the mid-costal diaphragm of a COPD subject. Black arrow; lipofuschin accumulation. Open arrow; basophilic cytoplasm. Black arrowhead; disrupted cytoplasm. Open arrowhead; hyaline (rounded) fibre. B: Peripheral basophilia, enlarged nucleus. C: Black arrow; lobulated fibre. Open arrow; angulated fibre. Black arrowhead; internally nucleated fibre. Open arrowhead; fibre with granular cytoplasm, lipofuscin accumulation, and increased nuclearity. D: Hypercellularity and expanded interstitium. Scale bars represent 20 pm. (0 c o O CD 30 25 20 15 O •£ 10 0> u Q. 0 L i • Control • COPD I n t e r n a l l y N u c l e a t e d A b n o r m a l C y t o p l a s m A b n o r m a l S i z e / S h a p e Categorization of Muscle Fibres F i g u r e 5 . C a t e g o r i z a t i o n o f m u s c l e f i b r e s in C O P D a n d n o n - r e s p i r a t o r y s u b j e c t s . E r r o r b a r s r e p r e s e n t s t a n d a r d e r r o r ( n = 6 ) . * d e n o t e s p < 0 . 0 5 . C T R M i d - c o s t a l C o s t a l C r u r a l I n s e r t i o n Diaphragm Sample Site C T R M i d - c o s t a l C o s t a l C r u r a l I n s e r t i o n Diaphragm Sample Site F i g u r e 6 . R e g i o n a l v a r i a t i o n in t h e CSAcoii a n d Pabn. (Top panel) * d i f f e r e n t f r o m C T R (p < 0 . 0 0 1 ) a n d m i d - c o s t a l ( p < 0 . 0 1 ) . * * d i f f e r e n t f r o m c o s t a l i n s e r t i o n a n d c r u r a l ( p < 0 . 0 0 1 ) . # d i f f e r e n t f r o m c r u r a l ( p < 0 . 0 1 ) . # # d i f f e r e n t f r o m C T R (p < 0 . 0 0 1 ) . (Bottom panel) * * d i f f e r e n t f r o m e a c h o t h e r r e g i o n (p < 0 . 0 1 ) . * d i f f e r e n t f r o m e a c h o t h e r r e g i o n (p < 0 . 0 5 ) . # d i f f e r e n t f r o m m i d c o s t a l (p < 0 . 0 5 ) a n d C T R ( p < 0 . 0 1 ) . C T R , c e n t r a l t e n d o n r e g i o n . 20 A n a l y s i s o f t h e s u b c a t e g o r i e s r e v e a l e d t h a t Pintnuc o f t h e m i d - c o s t a l d i a p h r a g m (8 .1 + 0 . 8 % ) w a s h i g h e r t h a n t h e c r u r a l ( 6 . 6 + 0 . 9 % ) a n d l o w e r t h a n t h e c e n t r a l t e n d o n r e g i o n ( 1 7 . 7 + 3 . 0 % ) ; ( F i g u r e 7 ) . T h e Pabnsnape o f t h e m i d - c o s t a l d i a p h r a g m ( 5 . 5 + 1 . 5 % ) w a s h i g h e r t h a n b o t h t h e c r u r a l d i a p h r a g m ( 2 . 6 + 0 . 8 % , p < 0 . 0 1 ) a n d t h e c o s t a l i n s e r t i o n ( 2 . 6 + 1 . 0 % , p < 0 . 0 5 ) . 3. Comparisons Between Psoas Major and the Costal and Crural Diaphragm T h e e n d o m y s i u m a n d p e r i m y s i u m o f t h e p s o a s m a j o r , o n a v e r a g e , a p p e a r e d l e s s d e n s e a n d l e s s t h i c k t h a n in t h e c o s t a l o r c r u r a l d i a p h r a g m . CSAc 0 n o f t h e p s o a s m a j o r ( 1 2 . 3 + 0 . 5 % ) w a s l o w e r t h a n t h e m i d - c o s t a l ( p < 0 . 0 0 1 ) a n d c r u r a l r e g i o n s (p < 0 . 0 1 ) ( F i g u r e 8 ) . T h e p s o a s m a j o r d i s p l a y e d l e s s a b n o r m a l / i n j u r e d m o r p h o l o g y , w i t h v e r y f e w s i g n s o f n e c r o s i s o r l i p o f u s c i n a c c u m u l a t i o n . P a b n o f p s o a s w a s 8 . 3 + 2 . 2 % , w h i c h w a s l o w e r t h a n b o t h t h e m i d - c o s t a l ( p < 0 . 0 0 1 ) a n d c r u r a l r e g i o n s (p < 0 . 0 1 ) . A n a l y s i s o f s u b - c a t e g o r i e s s h o w e d t h a t t h e p s o a s h a d l e s s a b n o r m a l c y t o p l a s m t h a n t h e c o s t a l a n d c r u r a l d i a p h r a g m s ( 0 . 8 + 0 . 2 % , p < 0 . 0 5 ) , a n d a t e n d e n c y t o w a r d f e w e r f i b r e s w i t h a b n o r m a l s i z e a n d s h a p e t h a n t h e m i d - c o s t a l d i a p h r a g m (2 .1 + 0 . 4 % , p = 0 . 0 6 5 ) ( F i g u r e 9 ) . T h e PabnCyt o f t h e m i d - c o s t a l r e g i o n ( 8 . 4 + 2 . 9 % ) w a s h i g h e r t h a n t h e c r u r a l r e g i o n ( 6 . 5 + 2 . 2 % , p < 0 . 0 5 ) . T h e P i n t nuc o f t h e p s o a s w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o s t a l o r c r u r a l d i a p h r a g m s . 4. Correlation and Fibre Size Analysis T h e r e w a s n o c o r r e l a t i o n b e t w e e n C S A c o n a n d Pabn. C S A c o n a n d f i b r e s i z e , o r C S A c o n a n d f i b r e s i z e v a r i a b i l i t y ( F i g u r e 1 0 ) . W h e n e x a m i n i n g t h e f i b r e s i z e d a t a , it w a s o b s e r v e d t h a t t h e r e w a s a n a p p a r e n t i n c r e a s e in t h e n u m b e r o f s m a l l f i b r e s a m o n g C O P D s u b j e c t s ( F i g u r e 1 1 ) . T o t e s t t h e n u l l h y p o t h e s i s t h a t t h e t w o d i s t r i b u t i o n s ( C O P D a n d N S R D ) w e r e f r o m t h e s a m e p o p u l a t i o n , t h e K o m o g o r o v -S m i r n o v t e s t w a s s e l e c t e d in c o n s u l t a t i o n w i t h a s t a t i s t i c i a n . T h e t e s t d e t e c t e d a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e t w o d i s t r i b u t i o n s ( p < 0 . 0 0 1 ) . T h e m e d i a n f i b r e a r e a f o r C O P D p a t i e n t s w a s 9 7 3 . 7 + 2 2 . 9 p m 2 , c o m p a r e d t o 1 1 3 6 . 7 + 1 9 . 0 p m 2 a m o n g t h e N S R D g r o u p (p < 0 . 0 0 1 ) . T h e a v e r a g e v a r i a b i l i t y c o e f f i c i e n t s o f m u s c l e f i b r e s i z e w e r e 3 6 . 9 % a n d 3 8 . 7 % f o r C O P D a n d N S R D ( n . s . ) Types of Abnormal Cytoplasm Observed In b o t h t h e C O P D / N S R D s t u d y a n d t h e r e g i o n a l s t u d y o f w h o l e d i a p h r a g m s , a b n o r m a l c y t o p l a s m w a s a p r o m i n e n t f e a t u r e in s o m e b i o p s i e s . T h e m o s t c o m m o n c y t o p l a s m a b n o r m a l i t i e s w e r e 21 • C T R U M i d - c o s t a l • C o s t a l I n s e r t i o n • C r u r a l I n t e r n a l l y n u c l e a t e d A b n o r m a l C y t o p l a s m A b n o r m a l S i z e / S h a p e Categorization of Muscle Fibres F i g u r e 7 . C a t e g o r i z a t i o n o f m u s c l e f i b r e s in d i f f e r e n t r e g i o n s o f t h e d i a p h r a g m . * * d i f f e r e n t f r o m e a c h o f t h e o t h e r r e g i o n s f o r t h a t c a t e g o r y . * d i f f e r e n t f r o m c r u r a l ( p < 0 . 0 5 ) . # d i f f e r e n t f r o m c o s t a l i n s e r t i o n (p < 0 . 0 5 ) a n d c r u r a l ( p < 0 . 0 1 ) . C T R , c e n t r a l t e n d o n r e g i o n . E r r o r b a r s d e n o t e S E ( n = 1 8 ) . 30 25 20 * 15 A < c/> o c Q) O) J 10 o o 0 * _T_ * M i d - c o s t a l C r u r a l P s o a s Diaphragm Sample Site 30 —• 25 W 20 _: 15 ra E V. O c < 10 0 I I * X M i d - c o s t a l C r u r a l P s o a s Diaphragm Sample Site F i g u r e 8 . C o m p a r i s o n o f c r o s s - s e c t i o n a l a r e a o f c o l l a g e n a n d p e r c e n t a g e o f a b n o r m a l f i b r e s in p s o a s m a j o r a n d t h e d i a p h r a g m . F o r e a c h p a n e l , * d e n o t e s a s i g n i f i c a n t d i f f e r e n c e f r o m e a c h o f t h e o t h e r 2 r e g i o n s , p < 0 . 0 1 . T o p p a n e l e r r o r b a r s r e p r e s e n t S E o f p o o l e d d a t a f r o m 1 8 s u b j e c t s (n > 2 4 5 f i e l d s p e r r e g i o n ) . B o t t o m p a n e l e r r o r b a r s r e p r e s e n t S E o f n = 1 8 . 23 30 W I 25 -I o ° 20 H i l 15 o •g 10 e 5 Q. # • Mid-costal H Crural I Psoas Internally nucleated Abnormal Cytoplasm Categorization of Muscle Fibres Abnormal Size / Shape Figure 9. Categorization of muscle fibres in the psoas major and the diaphragm. * denotes significant difference from the other 2 categories in that region. # different from crural (p < 0.05). Error bars represent SE (n = 18). Figure 10. Analysis of correlations between CSAcon and H&E morphology in COPD and non-respiratory subjects. A; percent abnormal fibres (r=0.271, n.s.): B; fibre area (r= -0.206, n.s.): C; coefficient of variation of fibre area (r= -0.027, n.s.). 60 -r 50 S 4 0 r = 0.271 25 t 30 CO £ O 20 c < 10 • • 10 20 30 Collagen CSA 40 70 -j 60 ^ 50 -I > O 40 co <u < 30 a> r =-0.027 10-1 0 0 10 20 30 40 Collagen CSA 2000 T-£ 400 -Si il 200 -0 -0 10 20 30 40 Collagen CSA Frequency h-* t o o o o o o o o 91 27 lipofuscin accumulation and disrupted cytoplasm, although these were not quantitated separately. Cytoplasm disruption included fibres where there were distinct regions of staining within a fibre cross-section such as peripheral basophilia, abnormal "clumps" of eosinophilia, and central or peripheral eosinophilia. Areas where the cytoplasm was absent within an apparently intact sarcolemma were viewed, sometimes centrally, sometimes peripherally. Areas were observed where the sarcolemma was clearly disrupted, often in association with increased cellularity, and apparent spillage of cytoplasm contents into the interstitium. Occasionally the entire fibre cross-section stained uniformly a paler or more basophilic hue. Large rounded fibres with a glassy eosiniphilic texture were also observed. 28 Discussion 1. C O P D v s N R D S The cross-sectional area of collagen and proportion of injured fibres in the post-mortem COPD diaphragm have not previously been reported. The CSA c o n in the mid-costal diaphragm was 24.2 + 1.9% in individuals with COPD. This was higher than the CSA c o n in age- and gender-matched individuals with NSRD (18.6 + 2.8%, p < 0.001), and higher than reported values for humans (16.3%) and animals (17.4 %, 18.0%) (MacGowan et al. 2001, Reid & Belcastro 1999, Gosselin et al. 1993). Under the microscope, regions of fibrillar collagen could be distinguished by their density (i.e. intensity of red staining) and their fibrous morphology. These areas of fibrosis were sometimes associated with small, abnormally shaped muscle fibres (Figure 2). Similar focal areas of fibrosis and misshapen fibres have been reported in the diaphragm following chronic resistive loading in hamsters (Reid & Belcastro, 1999). The COPD diaphragm had a higher percentage of abnormal and injured muscle fibres (28.4 + 1.9%) compared to the NSRD group (12.0 + 2.8%, p < 0.05). The COPD patients in this study were not receiving chemotherapy or sufficient corticosteroids to cause diaphragm injury or weakness, neither were they older than the NSRD group. Ventilatory overload is known to cause diaphragm injury and an acute increase in the cross sectional area of connective tissues (Jiang et al. 1998), however this is the first quantitative study to suggest that this process can occur in humans. Jiang et al. (1998a) reported a 38.5 % higher area of diaphragm connective tissue compared to control rabbits 3 days after a bout of inspiratory resistive loading in rabbits. A portion of this increase may have been due to inflammatory edema rather than an increase in collagen content. Several weeks after IRL, a third group showed similar values to those of control rabbit diaphragm. An acute increase in collagen synthesis within muscle following exertion-induced injury has been observed as early as 2 days post-injury, peaking at day 5 when regeneration predominates (Myllyla et al. 1986). By contrast, signs of a more permanent fibrosis (i.e. accumulation of dense, fibrillar collagen) and injury of the human diaphragm was reported by Kariks (1989). In this post-mortem study of the diaphragm in sudden infant death syndrome, fibrosis was observed qualitatively in H&E cross- and longitudinal sections of costal and crural diaphragm. As in the present study, fibrosis was accompanied by features of exertion-induced injury, including pale or granular cytoplasm, disruption of the sarcolemma, large rounded fibres, small angulated or rounded fibres, centrally nucleated fibres, 29 and areas of absent sarcoplasm. These features in the SIDS diaphragm are accompanied by a relative lack of fatigue-resistant fibres, which may predispose to diaphragm fatigue, injury and respiratory failure (Lamont et al. 1995). Surgically stable COPD patients showed myofibrillar disruptions at the EM level, however the diaphragm appeared normal under the light microscope, with no evidence of inflammation or fibrosis (Orozco-Levi et al. 2001). In contrast, the current study demonstrated extensive post-mortem injury, and inflammation and fibrosis at the light microscope level. The majority of COPD subjects in this study were experiencing an acute on chronic disease process. Thus, the present study may represent a "snapshot" of diaphragm morphology immediately following or during an acute on chronic increase in ventilatory loading, in many of the patients. One COPD patient was mechanically ventilated for a prolonged period (35 days), which could lead one to argue that mechanical ventilation may have been responsible for the abnormal diaphragm morphology in this patient. Mechanical ventilation for as little as 18 hours may cause diaphragm proteolysis characterized by type II fibre atrophy (Shanley et al. 2002). However, mechanical ventilation alone has not been associated with the type of morphology suggestive of exertion-induced injury which was visualized in this study. In addition, the long-term ventilated patient experienced numerous failed weaning trials, during which the respiratory rate and accessory muscle use were noted to be increased. Thus the combination of diaphragm disuse and periods of overload may have contributed to the observed injury in this patient (P a b n = 11.3%). Mechanical ventilation results in impaired diaphragm force generation (Powers et al. 2002), which would then predispose to subsequent exertion-induced injury. The extent to which injury would recover following an exacerbation is unknown. It is probable that some of the abnormal morphology observed in this study would recover or disappear as occurs following an acute ventilatory overload in animal models of diaphragm injury (Reid et al. 1994, Jiang et al. unpublished data), either via regeneration and remodeling, or by permanent loss of injured fibres. However, permanent fibrosis of limb muscle results from injuries that are extensive, that disrupt the basal lamina (Hurme & Kalimo 1992), or that repeatedly interrupt the normal process of regeneration (myoblast differentiation, proliferation, and fusion) (Stauber et al. 2000). Whereas diaphragm fatigue is defined as a loss of force-generating capacity that is reversible by rest, diaphragm injury is characterized by a structural injury and a loss of force-generation that may worsen over time. A single bout of exertion-induced injury is typically followed by collagen remodeling in the endo- and perimysium and basal lamina 30 (Han et al. 1999, Koskinen et al. 2001), likely in response to collagenases released during the inflammatory response to the injury (Kherif et al. 1999). But if an exertion-induced injury is chronically repeated, the collagen remodeling can be disturbed, resulting in fibrillar collagen and proteoglycan accumulation with small, misshapen fibres (Stauber et al. 2000). Evidence of this type of injury is still present after 3 months (Stauber et al. 2000). Collagen is the major contributor to the stiffness of skeletal muscle, and also plays a vital role in the transmission of forces in the diaphragm, via linkages with the myofibrillar cytoskeleton involving integrin-61 complexes, and dystrophin-dystroglycan complexes (Boriek et al. 2001a). The abnormal distribution and quantity of collagen observed in the COPD diaphragm likely altered its mechanical properties, over and above the loss of force which may result from the injury of myofibres itself (Jiang et al. 1998b). In addition, fibres embedded in more dense regions of connective tissue may experience impaired diffusion to and from capillaries (Josza et al. 1990). The combined effects of injury and fibrosis on the active and passive properties of the diaphragm require further study. The generalizability of the differences between the COPD and NSRD patients in this study is limited by the relatively low number of subjects (6 per group), and the lack of distinction by clinical features that would allow clinicians to identify patients at risk of diaphragm injury during exacerbations. The medical records did not consistently document indices of the work of breathing such as respiratory rate, accessory muscle use, and hyperinflation. Important distinctions between patients who are normocapnic and hypercapnic, and patients with and without hyperinflation of the thorax could not be made reliably. Hypercapnic COPD patients are more .susceptible to diaphragm fatigue following maximal ventilatory maneuvers (Rafferty et al. 1999), and experience increased diaphragm activation, placing them at higher risk of diaphragm fatigue and injury during acute exacerbations (Topeli, Laghi & Tobin 2001). Hyperinflation reduces the effectiveness of the diaphragm and, although reducing its contribution to ventilation in COPD patients (DeTroyer & Loring 1995), nonetheless accounts for a significant portion of the observed myofibrillar disruption following ventilatory loading in COPD patients (Orozco-Levi et al. 2001). Further work is needed to identify diaphragm injury in vivo in clinical populations, particularly during exacerbations or when attempting to wean from mechanical ventilation. Fibrotic areas imply that episodes of overload may have a cumulative effect on the diaphragm that could reduce its force-generating ability during future periods where maximum ventilation is required. Unlike limb muscle, 31 respiratory muscle does not normally have the opportunity to rest but must regenerate whilst continuing to contract. In vivo study of diaphragm injury using blood borne markers of myofibrillar injury would seem a promising approach, particularly when conducting ventilator weaning trials. It is possible that, in addition to fibrosis resulting from chronic injury, the CSA c o n was increased as an adaptation to chronic loading. Collagen synthesis and degradation can be modulated by the mechanical environment, independent of injury, likely through a stretch-sensitive integrin-mediated pathway in endomysial and permimysial fibroblasts (Chiquet 1999). Chronic loading is thought to cause increased proportions of type I fibres in the diaphragm of individuals with severe COPD (Levine et al. 1997). Limb muscles with a high proportion of type I fibres also have a higher collagen concentration, and a thicker basal lamina, endomysium and perimysium (Kovanen 1989). When skeletal muscles are chronically stimulated at a low frequency (which is known to induce a fast-slow transformation of skeletal muscle), the endomysium and perimysium adapt by rapidly increasing their amount of collagen. The CSAC0|| of latissimus dorsi muscle increased from 16.0 + 1.4% to 23.8 + 2.9% after 21 days of stimulation at 10 Hz, in the absence of injury (Wright et al. 1997). Future studies may examine the mechanisms of the increased collagen content, with the goal of minimizing fibrosis and facilitating regeneration of contractile tissue. In the present study the mechanisms involved in the injury and death of muscle fibres were not specifically examined. However, there was an increased presence of lipofuscin in the COPD diaphragm, although this category was not separately quantitated. Lipofuscin was present intracellulary and, very occasionally, in extracellular deposits in the endomysium. Lipofuscin results from free-radical-induced lipid peroxidation, and may be seen an index of accumulated oxidative stress (Allaire et al. 2002, Sohal & Brunk 1989). Lipofuscin also accumulates in muscle tissue with age, thus the relatively advanced age of our subjects likely contributed to the amount of lipofuscin observed. An increased presence of lipofuscin in the skeletal muscle of patients with COPD has been reported in the quadriceps (Allaire et al. 2002). In the present study, the diaphragm of individuals with COPD showed evidence of accumulated oxidative stress similar to that found in limb muscles. The presence of lipofuscin accumulation in another model of exertion-induced injury, the mdx mouse, is striking. In the mdx mouse diaphragm, the majority of apoptotic muscle fibres demonstrated an accumulation of lipofuscin (Nakae et al. 2001). Future studies 32 may attempt to examine whether the COPD diaphragm has higher numbers of apoptotic muscle fibres, and their relation to free radical formation. The presence of abnormal morphology (12% of fibres) in the diaphragm of individuals without respiratory disease deserves comment. First, the NSRD group was advanced in age (mean 65.8 y), which likely contributed to an accumulation of lipofuscin and internally nucleated fibres. Further, sarcomeric disruptions are common in the diaphragm of normal individuals, perhaps as part of a normal, ongoing cycle of degeneration and regeneration resulting in the gradual turnover of the myofibrils (Orozco-Levi et al. 2001), similar to the turnover of bone or tendon which is continually being degraded and synthesized simultaneously. The process seems to occur in the absence of inflammation (Orozco-levi et al. 2001). Fibres which have undergone a previous bout of injury and regeneration are often identified by the fact that the nucleus remains internally after regeneration is complete (Anderson et al. 1987). In individuals with no respiratory disease, the abnormal myofibres were usually isolated, i.e. surrounded by normal muscle, with an absence of inflammatory cells. This was in contrast to the COPD diaphragms in which some entire viewing fields were characterized by abnormall cytoplasm (Figure 4 panel A) or internal nucleation, and in which morphology suggestive of phagocytosis could be observed. 2. Regional differences in the diaphragm The CSAC0|| and P a bn were higher in the mid-costal diaphragm than the crural diaphragm. The difference between costal and crural diaphragms is consistent with previous studies which have found distinct actions (Ward & Macklem 1995), timing (Easton et al. 1993) and morphometry (Sanchez et al. 1985) of these two regions. In individuals with normal lung function, evidence for more intense contractions in the costal than crural diaphragm comes from the larger diameters of Type I and II muscle fibres in the costal than the crural diaphragm (Sanchez et al. 1985). If the costal diaphragm is subject to more intense loading, this may explain the higher incidence of exertional injury and regeneration in this region, as diaphragm injury is known to be load-dependent (Jiang et al. 1998a). Models of diaphragm injury have also found the costal diaphragm to be more susceptible to injury than the crural (Jiang et al. 1998a, Reid et al. 1994). In addition to affecting the amount of injury, more intense loading in the costal diaphragm could lead directly to increased collagen synthesis. 33 C S A C O i i w a s g r e a t e r in t h e r e g i o n o f t h e c e n t r a l t e n d o n , l i k e l y b e c a u s e o f t h e t e n d i n o u s i n s e r t i o n s w h i c h c o u r s e t h r o u g h t h e m u s c l e a n d b l e n d w i t h t h e t h i c k e n e d e n d o m y s i u m in t h i s r e g i o n , a s r e p o r t e d p r e v i o u s l y in l i m b m u s c l e ( A n d e r s o n 1 9 8 5 ) . T h e t r a n s i t i o n f r o m t e n d o n t o m u s c l e , h o w e v e r , is l e s s e x t e n s i v e ( i .e . m o r e a b r u p t ) in t h e d i a p h r a g m t h a n in l i m b m u s c l e s s u c h a s t h e g a s t r o c n e m i u s o r b i c e p s f e m o r i s . A s r e p o r t e d p r e v i o u s l y in l i m b m u s c l e ( A n d e r s o n 1 9 8 5 ) , t h e r e w e r e a g r e a t e r n u m b e r o f i n t e r n a l n u c l e i c l o s e t o t h e t e n d o n ; a n o r m a l f e a t u r e ( a l t h o u g h c o n t r i b u t i n g t o P a b n a s d e f i n e d in t h i s s t u d y ) w h o s e s i g n i f i c a n c e is u n k n o w n . T h e a m o u n t o f o t h e r m o r p h o l o g i c a l a b n o r m a l i t i e s w a s n o t h i g h e r in t h i s r e g i o n , in c o n t r a s t t o e v i d e n c e o f i n c r e a s e d i n j u r y a t t h i s r e g i o n in t h e m d x d i a p h r a g m ( A n d e r s o n e t a l . 1 9 9 8 ) a n d in t h e d i s t a l q u a d r i c e p s f e m o r i s - t e n d o n r e g i o n f o l l o w i n g e c c e n t r i c l o a d i n g ( M a c l n t y r e e t a l . 1 9 9 6 ) . In t h e p r e s e n t s t u d y , t h e P a b n w a s h i g h e s t in t h e m i d - c o s t a l r e g i o n . A s t u d y o f r e g i o n a l d i a p h r a g m s h o r t e n i n g in a c a n i n e m o d e l f o u n d t h a t t h e m i d - c o s t a l r e g i o n w a s t h e o n l y r e g i o n t o e x p e r i e n c e e c c e n t r i c c o n t r a c t i o n s w h e n t h e a f t e r l o a d w a s i n c r e a s e d ( W a k a i e t a l . 1 9 9 4 ) . T h i s s u p p o r t s t h e p o s t u l a t e t h a t h i g h l e v e l s o f s t r e s s o r s t a i n m a y b e c o n t r i b u t i n g t o i n j u r y in t h i s r e g i o n o f t h e d i a p h r a g m . T h e o b s e r v a t i o n o f a l o w e r i n c i d e n c e o f i n j u r y a r o u n d t h e c o s t a l i n s e r t i o n h a s i m p l i c a t i o n s in t h e i n t e r p r e t a t i o n o f p r e v i o u s s t u d i e s o f d i a p h r a g m i n j u r y , a s t h i s r e g i o n h a s b e e n p r e v i o u s l y u s e d a s a s u r g i c a l b i o p s y s i t e ( O r o z c o - L e v i e t a l . 2 0 0 1 ) . S a m p l i n g f r o m t h e c o s t a l i n s e r t i o n r e g i o n in h u m a n s w o u l d a p p e a r t o u n d e r e s t i m a t e t h e e x t e n t o f i n j u r y in t h e m i d - c o s t a l r e g i o n . 3. Comparisons Between Psoas Major and the Costal and Crural Diaphragm T h i s is t h e f i r s t h u m a n s t u d y t o c o m p a r e t h e a m o u n t o f c o l l a g e n a n d a b n o r m a l m o r p h o l o g y o f t h e d i a p h r a g m w i t h a c o n t r o l ( n o n - r e s p i r a t o r y ) m u s c l e . T h e C S A c o n a n d P a b n w e r e h i g h e s t i n t h e c o s t a l d i a p h r a g m , f o l l o w e d b y t h e c r u r a l d i a p h r a g m , w i t h t h e l o w e s t C S A ^ i a n d P a b n in t h e p s o a s m a j o r . It is p o s s i b l e t h a t v e n t i l a t i o n r e s u l t s in a c h r o n i c l o a d i n g w h i c h is s u f f i c i e n t t o s t i m u l a t e t h e s y n t h e s i s o f m o r e c o l l a g e n in t h e d i a p h r a g m t h a n is f o u n d in l i m b m u s c l e s o r p o s t u r a l m u s c l e s . F r o m a n i m a l s t u d i e s , t h e r e c t u s a b d o m i n u s l i k e w i s e h a s a l o w e r c o l l a g e n c o n t e n t t h a n t h e d i a p h r a g m ( R o d r i g u e s e t a l . 1 9 9 6 ) , d e s p i t e i ts s i m i l a r f i b r e t y p e p r o f i l e t o b o t h t h e d i a p h r a g m a n d t h e p s o a s m a j o r ( J o h n s o n e t a l . 1 9 7 3 ) . T h e p s o a s m a j o r , a l t h o u g h n o t r e p r e s e n t a t i v e o f l i m b m u s c l e s s u c h a s t h e q u a d r i c e p s w h i c h h a v e a g r e a t e r p r o p o r t i o n o f t y p e II f i b r e s , w a s u s e d a s a c o m p a r i s o n w i t h t h e d i a p h r a g m w h i c h e x p e r i e n c e s m o r e c h r o n i c a c t i v a t i o n . T h e p s o a s m a j o r is i n t e r m e d i a t e in t e r m s o f i ts f i b r e t y p e p r o f i l e , 34 due to its dual role as a postural and locomotor muscle. Whereas the psoas can rest during times of illness and metabolic stress, the diaphragm must continue to contract, frequently to a greater degree than usual if the work of breathing is increased for any reason. The higher P a b n in the diaphragm than in the psoas highlights the susceptibility of the diaphragm to injury in individuals with a range of respiratory and non-respiratory diagnoses. A post-hoc sub-group analysis will be performed in consultation with a statistician to determine whether P a b n and CSA c on varied similarly across regions between respiratory and non-respiratory patients regions, and between the diaphragm and psoas. 4. Correlation and Fibre Size Analysis There were no significant correlations between CSA c on and fibre area, C S A c o n and fibre area variability, or C S A ^ i and P a b n . . Thus, the qualitative association between small fibres and fibrosis did not reach statistical significance as a correlation, probably because this association was not observed consistently in every viewing field. The lack of correlation between injury and CSA c on may be related to issues of statistical power, as it is well established that collagen remodeling is associated with exertional injury (Han et al. 1999); additionally, the CSAcon and P a b n were both increased in COPD subjects. It is possible that some fibres which are injured and unable to regenerate are simply replaced by connective tissue, thus reducing the ability to correlate these two processes morphometrically (Anderson et al. 1998). In addition, altered amounts of mechanical loading in the diaphragm may change collagen synthesis and degradation rates independently of factors released upon injury, which would further reduce the strength of the association. Because the patients were not matched by BMI, we examined the combined fibre size distributions for the whole COPD and NSRD groups, which overall had similar BMI. Using the Kolmigorov-Smirnov test in consultation with a statistician (Dr Bruno Zumbo, U.B.C), we found that the distributions of fibre size from the COPD and NSRD groups were significantly different (p < 0.001). The COPD diaphragm displayed a smaller median fibre area than in the NSRD group (973.7 + 22.9 pm 2 vs 1136.7 + 19.0 pm2). A single other post-mortem fibre area analysis of the diaphragm (Scott & Hoy 1976) found similar values for fibre area as in this study (1014.8 pm2 in 24 subjects), however BMI was not reported, which invalidated their finding of increased fibre area among emphysema patients (Scott & Hoy 1976). Fibre size of the costal diaphragm in patients with COPD was previously shown to be correlated 35 w i t h B M I , F E V i a n d F V C ( S a n c h e z e t a l . 1 9 8 5 ) . T h i s a n d o t h e r s t u d i e s r e p o r t i n g f i b r e a r e a s o f t h e d i a p h r a g m h a v e b e e n c o n d u c t e d u s i n g f r o z e n t i s s u e , a n d y i e l d m u c h l a r g e r v a l u e s ( m e a n s o f 2 3 0 0 u r n 2 - 2 7 0 0 u m 2 ) ( M i z u n o & S e c h e r 1 9 8 9 , R e i d 1 9 9 5 , O r o z c o - L e v i e t a l . 2 0 0 1 ) . T h e l a r g e r v a l u e s a r e l i k e l y d u e t o t h e l o n g i t u d i n a l c o n t r a c t i o n a n d t r a n s v e r s e b r o a d e n i n g o f s a m p l e s w h i c h o c c u r s d u r i n g s a m p l i n g o f f r e s h t i s s u e s , a n d d u e t o t h e u n a v o i d a b l e p r e s e n c e o f s h r i n k a r t i f a c t i n d e h y d r a t e d , f o r m a l i n - f i x e d s p e c i m e n s . In t h i s s t u d y , t h e f i n d i n g o f s m a l l e r f i b r e s in t h e C O P D g r o u p w a s a c c o m p a n i e d b y a n i n c r e a s e in t h e p r o p o r t i o n o f f i b r e s w i t h a b n o r m a l m o r p h o l o g y a n d a n i n c r e a s e in t h e c r o s s - s e c t i o n a l a r e a o f c o l l a g e n in t h e C O P D g r o u p . In t h e q u a n t i t a t i v e a n a l y s i s w e d i d n o t d i f f e r e n t i a t e b e t w e e n f i b r e s w h i c h m a y h a v e b e e n r e g e n e r a t i n g , o r u n d e r g o i n g a p o p t o s i s . In t h e q u a d r i c e p s m u s c l e o f i n d i v i d u a l s w i t h C O P D , m u s c l e w a s t i n g h a s b e e n a t t r i b u t e d t o a p o p t o s i s o f m y o n u c l e i r e s u l t i n g in m y o f i b r e s h r i n k a g e o r a t r o p h y ( A g u s t i e t a l . 2 0 0 2 ) . It is p o s s i b l e t h a t a s i m i l a r p r o c e s s o f a p o p t o s i s m a y b e o c c u r r i n g in t h e d i a p h r a g m . B e c a u s e d e t a i l e d i n f o r m a t i o n o n n u t r i t i o n a l s t a t u s w a s n o t a v a i l a b l e , it r e m a i n s p o s s i b l e t h a t n u t r i t i o n a l d e p r i v a t i o n c o u l d e x p l a i n t h e s m a l l e r d i a p h r a g m f i b r e s , a s in t h e ra t ( D e k h u i j z e n e t a l . 1 9 9 5 ) . H o w e v e r , n u t r i t i o n a l d e p r i v a t i o n a l o n e w o u l d n o t e x p l a i n t h e m o r p h o l o g i c a l a p p e a r a n c e o f c l u s t e r e d s m a l l f i b r e s e m b e d d e d in c o n n e c t i v e t i s s u e , a s u n d e r n u t r i t i o n c a u s e d a r e d u c t i o n o f f i b r e c r o s s - s e c t i o n a l a r e a in t h e a b s e n c e o f m y o p a t h i c m o r p h o l o g y ( D e k h u i k z e n e t a l . 1 9 9 5 ) . 5. Interpretation of Abnormal Cytoplam T h e i n t e r p r e t a t i o n o f a b n o r m a l c y t o p l a s m a s v i e w e d i n c r o s s - s e c t i o n s is h i n d e r e d b y t h e f a c t t h a t t h e t w o - d i m e n s i o n a l i m a g e m a y n o t r e p r e s e n t t h e e x t e n t o r t y p e o f i n j u r y o r r e g e n e r a t i o n o c c u r r i n g p r o x i m a l l y o r d i s t a l l y a l o n g t h e m u s c l e f i b r e . In a d d i t i o n , i n j u r y a n d f a t i g u e m a y c o - o c c u r in t h e d i a p h r a g m , s u c h t h a t t h e l o s s o f f o r c e - g e n e r a t i n g c a p a c i t y m a y e x c e e d m o r p h o l o g i c a l a b n o r m a l i t i e s . In t h i s s t u d y , l i p o f u s c i n a c c u m u l a t i o n a n d d i s r u p t e d c y t o p l a s m , a l t h o u g h n o t q u a n t i f i e d s e p a r a t e l y , w e r e a m o n g t h e m o s t c o m m o n l y o b s e r v e d a b n o r m a l i t i e s in t h e d i a p h r a g m . T h e e f f e c t s o f l i p o f u s c i n (if a n y ) o n t h e f u n c t i o n o f s k e l e t a l m u s c l e a r e n o t k n o w n . F i b r e s w i t h a b n o r m a l s t a i n i n g p a t t e r n s s u c h a s p e r i p h e r a l b a s o p h i l i a o r a b n o r m a l " c l u m p s " o f e o s i n o p h i l i a h a v e p r e v i o u s l y b e e n d o c u m e n t e d a s n o n - s p e c i f i c s i g n s o f s k e l e t a l m u s c l e p a t h o l o g y a n d i n j u r y ( A n d e r s o n , 1 9 8 5 ) . E o s i n n o r m a l l y b i n d s t o m y o s i n , t h u s a r e a s o f c l u m p e d o r a b s e n t e o s i n l i k e l y r e p r e s e n t d i s r u p t i o n o r 36 degradation of the myofibrillar structure. This often appeared to occur in the absence of inflammation. In contrast, classic features of necrosis including disruption of the sarcolemma in association with increased cellularity were also observed. The spillage of cytoplasm into the interstitium is a major contributor to the inflammatory response following cellular injury. Occasionally an entire fibre cross-section was stained uniformly a paler or more basophilic hue, perhaps indicating altered binding characteristics of the H&E stain in that local chemical environment as a result of injury or regeneration. Large rounded fibres with a glassy or fuzzy eosiniphilic texture were also observed, which appeared identical to the classic "hyaline fibres" observed following exertion-induced injury (Friden & Liber 1998). These fibres likely correspond to retracted and swollen areas just adjacent to a lesion, as observed in longitudinal section (Friden & Liber 1998). 6. Relevance It is possible that the abnormal morphology we have observed would lead to a loss of force-generation in the COPD diaphragm. A reduced volume of functioning myofibrils could be compensated by an increased activation of the muscle, or by hypertrophy. The distribution of muscle fibres in the COPD patients was in fact smaller than those with NSRD, suggesting that hypertrophy did not compensate significantly. Further, the diaphragm may already be maximally activated during acute exacerbations in some individuals with COPD (Topeli et al. 2001). In this case, the areas of disrupted sarcoplasm observed in this study may have resulted from increased diaphragm activation under conditions of increased ventilatory loading, and may have contributed to respiratoryfailure. 7. Summary, Future Directions By examining the post-mortem, mid-costal COPD diaphragm in relation to NSRD, this study has documented the most extensive respiratory muscle injury to date in patients with COPD, and has described a new phenomenon in association with this injury~the accumulation of collagen. The histochemical techniques employed to demonstrate injury and fibrosis are within the reach of any hospital pathology laboratory. Given the extent of the injury and fibrosis in some of the samples, it appears reasonable to speculate that diaphragm dysfunction and injury may be contributing to symptoms and mortality in some patients. The creation of a larger data base of biopsies would allow the 37 prevalence and clinical correlates of this phenomenon to be better determined. 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J Cell Sci 107 (Pt 2):669-81. Ward ME, Macklem PT. 1995. Kinematics of the chest wall. In: C Roussos (ed) The Thorax, 2 n d ed. Lung Biology in Health and Disease. C Lenfant (executive ed). New York: Marcel Dekker, 515-533. Warhol MJ, Siegel A, Evans WJ, Silverman LM. 1984. Skeletal muscle injury and repair in marathon runners after competition. Am J Pathol 118:331-339. Watchko JF, Johnson BD, Gosselin LE, Prakash YS, Sieck GC. 1994. Age-related differences in diaphragm muscle injury after lengthening activations. J Appl Physiol 77: 2125-33. Wernig A, Irintchev A, Weisshaupt P. 1990. Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent wheel-running. J Physiol 428: 639-652. Williams PE, Kyberd P, Simpson H, Kenwright J , Goldspink G. 1998. The morphological basis of increased stiffness of rabbit tibialis anterior muscles during surgical limb lengthening. J Anat 193:131-8. Williams PE, Catanesi T, Lucey EG, Goldspink G. 1988. The importance of stretch and contractile activity in the prevention of connective tissue accumulation in muscle. J Anat 158:109-14. Williams PE, Goldspink G. 1984. Connective tissue changes in immobilised muscle. J Anat 138: 343-50. Williams PE, Goldspink G. 1981. Changes in the connective tissue component of muscle during periods of decreased or increased activity. J Anat 133:133. Wright H, Williams P, Cox V, Goldspink D. 1997. The effect of 21 and 24 days of mechanical stimulation on collagen in the rabbit latissimus dorsi muscle. JAP 499:P114. 45 Appendix I: Literature Review The diaphragm in normal breathing Tidal breathing in healthy people involves an active inspiratory phase and a passive expiratory phase (Bradburne & McCool 1990). During quiet breathing in upright people, the chest wall is expanded by the actions of the parasternal intercostal muscles, the scalene muscles, and the diaphragm (DeTroyer & Loring 1995). These muscles contract and cause air to be drawn in through the trachea, bronchi, bronchioles, and into the alveoli where oxygen and carbon dioxide diffuse across the alveolar-capillary membranes. During passive expiration, the inspiratory muscles relax, allowing the elastic recoil of the lungs and chest wall to expel the air until the functional residual capacity is reached, at which point the passive inward and outward recoils of the lungs and chest wall are balanced. After a brief period of relaxation, the next inspiratory cycle begins. Whereas the actions of the parasternal intercostals and the scalenii are determined by their insertions on the ribs, the action of the diaphragm has both a direct (thoracic) and an indirect (abdominal) component (Tobin 1990). The diaphragm normally assumes a domed position, with a zone of apposition against the lower ribs. The costal fibres of the diaphragm originate on the lower ribs and insert into the broad central tendon. As these costal fibres contract, the lower ribs are pulled upwards and outwards in a bucket-handle motion guided by the costosternal, costotransverse and costvertebral joints. This is known as the insertional action of the diaphragm. The sternal and crural fibres of the diaphragm, respectively originating from the dorsal surface of the sternum and the anterior bodies of the upper three lumbar vertebrae, also attach to the central tendon. The combined actions of costal, sternal and crural fibres cause the central tendon to descend by rotating around the axis formed by the costal wall insertion in a way which allows the radius of curvature of the diaphragm to be maintained (Boriek et al. 1997). This causes intra-abdominal pressure to rise and exert a lateral expansion force determined by the area of the zone of apposition (Boriek et al. 1996). The extent of the diaphragm's contraction depends on firing frequency of the phrenic nerve, the preload (stretch) and afterload (abdominal pressure) conditions, and its intrinsic contractility (Wakai et al. 1994). At maximal inspiration at high lung volume, the diaphragm shortens by about 25% of its measured excised length (Boriek et. al., 1997). The crural diaphragm generates less inspiratory muscle force than the costal diaphragm, as it does not contribute to the insertional action of rib cage expansion (Ward & Macklem, 1995). The crural 46 diaphragm demonstrates a greater contribution to postural adjustments than the costal diaphragm (van Lunteren et al. 1985), and has been shown to receive less blood flow during exercise than the costal diaphragm (Poole et al. 2000). The diaphragm in COPD Although the primary pathology of chronic lung diseases involves the lung parenchyma and airways, the diaphragm can undergo secondary adaptive and pathological changes. COPD includes the pathologies of chronic bronchitis and emphysema, and often co-occurs with some degree of reversible airways reactivity. Diseases which fall under the diagnostic umbrella of COPD are characterized by decreased flow-rates, particularly the expiratory flow rate. Decreased expiratory flow-rate (i.e. F E V L the forced expiratory volume in 1 second) results from airway mucus, edema and bronchospasm, and early airways closure due to destruction of alveoli and loss of elastic tethering (Tobin 1990). The early airways closure can result in chronic air trapping and hyperinflation. Hyperinflation causes the diaphragm to assume a more flattened position. In this position, both the insertional and appositional actions of the diaphragm are impaired (DeTroyer 1997). Thus, not only must the diaphragm contract more forcefully against the increased resistance of narrowed airways, but it must do so from the mechanical disadvantage of a relatively ineffective flattened position (Tobin 1990). The diaphragm in COPD shows some signs of adaptation to this chronically increased loading and decreased efficiency. The extent of diaphragm activation is chronically increased in individuals with severe COPD (Sinderby et al. 1998), with a corresponding increase in the proportion of endurance type I fibres and increased concentrations of slow myosin heavy chain in the costal diaphragm (n=6; Levine et al. 1997). Orozco-Levi et al. (1999) found a greater density of mitochondria in costal diaphragm muscle fibres in individuals with moderate COPD. The number of mitochdondria was moderately correlated with both F E V , (r=-0.55) and the degree of hyperinflation, RV/TLC (r= -0.63). When hyperinflation is corrected for (and use of steroids controlled for—see below), diaphragm pressure-generation in individuals with COPD appears to be normal (Tobin 1990). MacGowan et al. (2001) examined 21 costal diaphragm biopsies obtained during lung resection surgery for pathologies such as emphysema and cancer. FEV"! ranged from 16 to 122% of predicted values. The diaphragms of individuals with decreasing FEVi showed increasing proportional cross-47 sectional area of abnormal or injured muscle (r=-0.53, p<0.05). Internally nucleated fibres, i.e. muscle fibres which may have undergone injury and regeneration, were the most prevalent morphologic feature in this population. This was the first evidence of airflow limitation correlating significantly with features of diaphragm injury and regeneration. Orozco-Levi et al. (2001) examined the costal diaphragm in patients with or without COPD undergoing thoracotomy or laparotomy. Of the 18 COPD and 11 normal subjects, 7 with COPD and 5 normals underwent inspiratory loading prior to surgery and biopsy removal. The amount of diaphragm injury was quantified as the density of disrupted sarcomeres under the electron microscope. The density of sarcomeric disruptions was inversely correlated to FENAi (% predicted), i.e. worsening airflows correlated with increasing injury (r=-0.585) similar to MacGowan et al. (2001). The correlation between diaphragm injury and RV (as an index of hyperinflation) was even greater: increasing hyperinflation was associated with increasing diaphragm injury (r=0.723). Sarcomeric disruptions were present in all groups, including the non-loaded controls (around 10 disruptions per 100 pm2). This value was 89% greater in loaded controls, providing undisputable structural evidence of exertion-induced injury in the human diaphragm. Non-loaded COPD subjects had an even greater number of sarcomeric disruptions than the loaded control group, in support of the postulate that COPD patients are more susceptible to exertion-induced injury of the diaphragm. The inspiratory-loaded COPD patients had the highest level of exertion-induced injury (39 disruptions per 100pm2). Thus, the COPD diaphragm, in particular in subjects with hyperinflation, demonstrates an increased susceptibility to exertion-induced injury. Exertion-induced muscle injury Exertion-induced muscle injury has been well described in animal and human limb muscles, although the mechanisms responsible are still under investigation (McArdle & Jackson 1997). It typically occurs following a period of unaccustomed activity, either of an increased duration or an increased intensity (Faulkner et al. 1993). It occurs particularly after exercise involving eccentric contractions, but also occurs with purely concentric exercise of sufficient intensity (Gibala et al. 1995). Exertion-induced muscle injury in limb muscles typically fully resolves with time by full regeneration of damaged fibres (Wernig et al. 1990). 48 Hikada et al. (1983) examined serial gastrocnemius biopsies of competitive marathon runners immediately before and after a marathon, and at 1, 3, 5 and 7 days after the event. Electron microscopy revealed focal and extensive z-line streaming, disrupted sarcolemma, and swollen mitochondria. Abnormalities were greater at 1 and 3 days post-event as opposed to immediately afterwards. Neutrophils, lymphocytes, and macrophages were seen in the intracellular space and invading necrotic muscle fibres. The authors noted that the delayed peak of histologic abnormalities corresponded with the phenomenon of delayed onset muscle soreness (DOMS), which was attributed to the inflammatory process. They hypothesized that sarcolemma disruption led to influx of calcium, and activation of calcium dependent proteases. Warhol et al. (1985) examined serial gastrocnemius biopsies from marathon runners up to 12 weeks after the event. After 1 week the injury had begun to resolve, and after 1 month healing was nearly complete, with centrally located nuclei being the dominant observed abnormality. By 8-10 weeks, the injury was completely resolved, although veteran runners displayed focal areas of interstitial fibrosis which were attributed to the effects of repeat injury. The delayed peak in the soreness and the extent of injury has been repeatedly observed in various experimental models of exertion-induced muscle injury. Clarkson et al. (1992) found DOMS to peak at 2-3 days following eccentric biceps exercise in humans, which corresponds to the period of greatest inflammatory infiltration, (e.g. McCully & Faulkner 1985). This second peak in injury is known as the secondary injury (Faulkner et al. 1993), and is attributed to the action of phagocytes which are recruited to clear away debris, but which may extend the original area of injury in the process (Maclntyre et al. 1995). A current model (Benz et al. 1998) of the events of exertion-induced muscle injury describes a process involving several stages. An initial injury is caused by high focal tensions developed in the maximally, actively contracting muscle, perhaps causing disruption of the sarcolemma ort-tubules. This leads to increased concentrations of intracellular calcium and the activation of calpain, a calcium-dependent protease. Calpain degrades the intermediate filaments (such as desmin) that maintain the sarcomeric registry. A delayed, inflammatory, phagocytic response may extend the initial injury through secretion of proteases and free radicals to a degree that can even equal the original area of trauma. Regeneration then procedes to replace the damaged fibres over the ensuing weeks. This model has been supplemented by further suggestions that exercise could result in a decreased energy supply of the sarcolemma calcium channels, also leading to raised intracellular 49 calcium concentrations (McArdle & Jackson 1997). In addition to triggering calpain, calcium could then trigger apoptosis of the muscle fibre, contribute to cross-bridge rigor and further strain damage of the myofibrillar structure, and could also further inhibit mitochondrial recycling of ATP (McArdle & Jackson 1997). In addition, the generation of free radicals by muscle injury, and later by leukocytes, could further impair calcium homeostasis (McArdle & Jackson 1997). The various possible contributions of (1) the initial strain injury to the sarcolemma, sarcoplasmic reticulum or transverse tubular system, (2) impaired calcium homeostasis, (3) apoptosis and (4) free radicals have yet to be worked out (McArdle & Jackson 1997). Although blood-borne markers of muscle injury such as creatine kinase and myoglobin have traditionally been used to quantify the amount of damage following exertion, they may not provide a sensitive measure of muscle injury (Bar et al. 1997). Intracellular damage may be insufficient to cause lysis of the membrane and release of blood-borne proteins. Conversely, exertion may cause small resealable breaks in the muscle membrane that cause some spillage of proteins, but that do not progress to necrosis and structural damage (McNeil & Khakee 1992). Thus, at present microscopy remains the standard for studying certain aspects of exertion-induced muscle injury (Faulkner et al.1993). Microscopy also allows the gross identification of inflammatory infiltrates and features of regeneration (Grounds 1991). Exertion-induced muscle injury has been observed in the diaphragm in animal models of ventilatory loading. Reid et al. (1994) developed a model of ventilatory loading in which the trachea of hamsters was constricted for 6 days by the surgical application of a polyvinyl cuff around the trachea. H&E diaphragm cross-sections were analysed quantitatively using a point-counting system. This analysis demonstrated that the costal and crural diaphragms had more extensive areas of abnormal fibres than control animals. Z-band streaming and loss of sarcomeric registry were visible with electron microscopy. In addition, contractile proteins were extracted from the samples and allowed to react with calpain. Tropomyosin and alpha-actinin were degraded to a greater degree from exogenous calpain in the samples from banded animals, indicating that these samples had undergone changes that facilitated increased proteolysis by calpain. Rats subjected to tracheal banding for 1, 2, 3, or 4 days demonstrated a similar exertion-induced muscle injury. The injury was greatest in animals subjected to 3 days of banding. Jiang et al. (1998a, 1998b) also found that force-loss was greatest 3 days after an acute 50 episode of inspiratory resistive loading. Rabbits were subjected to 90-minute bouts of inspiratory loading. On day 3, the costal and crural diaphragm and the parasternal intercostals contained inflammatory cells, necrotic muscle fibres, increased fraction of abnormal muscle and an expanded extracellular matrix. The largest injury occurred in the costal diaphragm. A longer-term study induced tracheal banding of hamsters for 30 days (Reid & Belcastro 1999). A significant increase in the area fraction of abnormal muscle was observed. The dominant features were variation in fibre size, abnormal size and shape of muscle fibres, and focal areas of increased connective tissue. Intramuscular connective tissue of the diaphragm The connective tissue of skeletal muscle is composed of cellular and extracellular material. The predominant connective tissue cell is the fibroblast, which is responsible for secreting the majority of extracellular material, although myoblasts can also secrete collagen and other extracellular connective tissue components (Carrino 1998). The extracellular material is organized into a matrix (the ECM) that surrounds, supports, and anchors the muscle fibres. The ECM of muscle comprises two main categories of proteins. The first group—glycoproteins—includes collagens I, III, IV, V, fibronectin, laminin, and entactin. Small amounts of other collagens (VI, Vll, Xlll, XIV, XV, XVIII, XIX) are also present (Walchli et al. 1994, Myers et al. 1999, Bonaldo et al.1998, Saarela et al. 1998, Hurme & Kalimo 1992). The second group—proteoglycans—consists of a glycosaminoglycan bound to a core protein. The glycosaminoglycans of muscle (hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparan sulfate) can assume various aggregations and play a diversity of structural and metabolic roles beyond the scope of this review (Carrino 1998). Finally, elastin is an important determinant of the diaphragm's passive properties (Rodrigues & Rodrigues 2000). The ECM of skeletal muscle may be divided into two distinct structures: the basal lamina, and the interstitial matrix (Sanes 1986). The basal lamina lies closest to the muscle fibre surface, and forms a sheath around the muscle fibre consisting mainly of laminin-entactin-collagen IV complexes (Timpl et al. 1984). The basal lamina provides a continuous structural link between the intermediate filaments of the cytoskeleton, and the collagen fibres of the endomysium, via complexes of integrin-vinculin and dystrophin-dystroglycan (Monti et al. 1997). This continuity of intra- and extracellular structure plays an 51 important role in allowing the endomysium to disperse and transmit the forces generated by the sarcomeres. The endomysium surrounding each fibre consists of a matrix mainly of collagens I, III and V. An innermost layer of collagen consists mainly of finer strands (probably collagens VI and Vll) arranged in patterns of opposing spirals (Ohtani et al. 1988). Thicker collagen fibrils also wind their way through the endomysium, frequently branching and anastomosing with the inner sheath, occasionally bridging between adjacent myofibres. The perimysium surrounding each bundle of myofibres is continuous with the endomysium, has the same constituents, and contains muscle spindles, nerves, and vessels. In an arrangement unique among skeletal muscles, the lymphatic vessels of the diaphragm communicate directly with the pleural and peritoneal cavities (Ohtani et al. 1993). The outermost connective tissue sheath, the epimysium, consists mainly of collagen I (Sanes, 1986; McCormick, 1994). From this arrangement, it is apparent that the ECM maintains tissue boundaries and an orderly arrangement of myofibres, nerves, and vessels. It allows forces to be efficiently transmitted and dispersed through the links between the cytoskeleton and the matrix, along and across the muscle's line of pull, and from fibre to fibre (Kovanen 1989). Collagen is responsible for the majority of muscle's stiffness (change in force / change in length), although cross-bridges and intermediate filaments also play a role (Monti et al. 1997). The major contribution of cross-linked collagen to stiffness of muscle was determined in early experiments with lathyrism (Feit et al. 1989). Collagen in muscle can take fibrillar (e.g. types I, III, V) and non-fibrillar (e.g. type IV) forms. Types I and III collagen assemble into stably cross-linked fibres of larger (> 40 nm) and smaller (< 40 nm) diameters, and can also form cofibrils (Rucklidge et al. 1992). The formation of fibrous collagen involves numerous post-translational steps, both intra- and extra-cellular. In the rough endoplasmic reticulum and Golgi apparatus, proline residues are hydroxylated by prolyl hydroxylase to form a stable triple helical structure—procollagen. The hydroxylation of proline allows the formation of hydrogen bonds which maintain the pro-collagen in its helical state (Bella et al. 1994). Each procollagen triple helix is capped by an amino (N) and a carboxyl (C) propeptide. Once in the interstitium, the propeptides are cleaved enzymatically by C- and N-propeptidases. This cleavage allows end-to-end assembly of fibrils to proceed. Fibrils are then bound together into fibres by the formation of cross-links such as hydroxylysine, which contribute to collagen's tensile quality (Feit et al. 1989). As collagen matures the 52 hydroxylysine cross-links are spontaneously replaced by irreducible cross-links such as hydroxypyridinium (McCormick & Thomas 1998). Mature fibrous collagen is stiffer, contains more hydroxypyridinium cross-links, and is more resistant to proteolysis than the newly synthesized collagen typical of regenerating tissue (Gosselin et al. 1994). Role of collagen in modulating inflammation and regeneration In addition to their structural roles, collagen and other ECM components of the basal lamina and interstitial matrix play metabolically active roles in inflammation and regeneration. Following an initial exertion-induced muscle injury, light microscopy reveals focal disruptions of the bonds between the myofibres and the surrounding matrix (Stauber et al. 1990); this disruption could be partially responsible for initiating the inflammatory response, as laminin and collagen fragments are known to induce a pro-inflammatory chemotaxis of leukocytes (Steadman et al. 1993). The formation of myotubes on a gel-base of laminin I, entactin, and collagen IV is more extensive and results in a greater number of new myonuclei than myotubes grown on a standard gel (Grounds et al. 1998). The myoblasts go on to secrete a new basal lamina scaffold that either supplements or replaces the old one (Caplan et al. 1987). Thus, repetitive cycles of injury and regeneration may lead to thickened accumulations of basal lamina including collagen IV (Anderson et al. 1987). Fibroblasts are normally the most numerous cells in the connective tissue of muscle. In addition to secreting the majority of collagen and other ECM proteins, fibroblasts produce and are acted upon by inflammatory signals. IL-1 and TNF-a, produced early in the inflammatory response by macrophages, induce fibroblasts to proliferate and secrete ECM components including collagens. Fibroblastic proliferation is also encouraged by PDGF, which is produced by the fibroblasts themselves, as well as macrophages (Cannon & St Pierre 1998). TGF-B, IL-6, and PDGF are considered fibrogenic factors. Although fibronectin and collagen themselves inhibit ECM secretion by a negative feedback mechanism (Nicod & Dayer 1999), a repeated injury leading to chronic inflammation could lead to ongoing elevated levels of pro-inflammatory and fibrogenic cytokines. Possible factors leading to increased connective tissue in the diaphragm 1. Chronic injury and inflammation 53 Single and repeated bouts of exertion-induced muscle injury result in increased collagen content. After a single bout of exertion-induced muscle injury (e.g. 9 hours downhill treadmill running), collagen synthesis (prolyl hydroxylase activity) increased from days 2 to 20, with peak synthesis occurring at 5 days post-injury when signs of regeneration were most prevalent. Immunohistochemistry revealed increased distribution and staining intensity of all collagens examined (III, IV and V) from day 5 to the end of the experiment (day 20). The staining was increased in association with injured and regenerating areas, and likely played a role in stabilizing the regenerating myotubes. Hydroxyproline was increased at day 10 (by 40%) but not significantly different from baseline at. day 20 (Myllala et al. 1986). A second experiment clarified the earlier time-course of collagen synthesis in muscle after 2 hours of down-hill running; procollagen IV mRNA expression was increased by 12 hours, and collagens I and III by 24 hours. Peak mRNA levels occurred 2 to 4 days later (Han et al. 1999). The investigators did not observe an increase in hydroxyproline content, which led the authors to suggest that the increased collagen content was dependent on the severity of the injury. Relatively permanent fibrotic changes have been shown following repeated exertion-induced muscle injury. Stauber et al. (1996) subjected rats to bouts of eccentric exertion-induced injury three times weekly for 4 weeks. Rat soleus muscles were tetanically stimulated then stretched at high or slow velocity. The soleus was then examined with scanning electron microscopy. The high velocity protocol caused substantial thickening of the perimysium and the endomysium surrounding both small and normally sized fibres. To examine the duration of these changes, a similar high velocity exertion-induced injury protocol was applied in a second experiment 5 times weekly for 6 weeks (Stauber et al., 2000). The soleus was examined after 0, 1, 2, and 3 months of recovery. Non-contractile tissue area increased from baseline (15.7%) to 50.5% at 0 months, and remained elevated at 18.6% at 3 months. The molar ratio of hydoroxypyridinium cross-links to total collagen was equivalent to baseline at 0 months, moderately higher at 1 month, and leveled off to remain higher by 75% at 2 and 3 months. 2. Adaptation to training Under specific conditions, it appears that connective tissue concentration may undergo an adaptive increase in response to training or overload. Resistance training regimes result in no change of connective tissue area, i.e., connective tissue area increases proportionally with muscle fibre area 54 (Mikesky et al. 1991, MacDougall et al. 1994, Stone 1990). Other studies have examined the effect of endurance training on collagen synthesis in muscle. After the initiation of a daily endurance training program, collagen synthesis was elevated at day 1, and returned to baseline by day 20 (Takala et al. 1983). The increase was likely in response to an initial exertion-induced injury at the commencement of training. Lifetime, intensive endurance training resulted in a chronic increase of hydroxyproline and collagen IV, and an increased passive stiffness of the muscle (Kovanen et al. 1987). Chronic eccentric training on a motorized treadmill caused an increase in the stiffness of the gastrocnemius muscle (Reich etal. 2001). 3. Passive Loading Whether or not prolonged stretch induces an increased connective tissue content seems to depend on the amount of load and injury caused by the protocol. Muscles stretched with no added load (i.e. weight) do not accumulate connective tissue (Goldspink et al. 1991); conversely, the stretching of muscles under weighted loading induces considerable injury and fibrosis (Williams et al. 1998). A recent experiment induced prolonged muscular stretch of the latissiumus dorsi without causing any necrosis or overt injury, and found that connective tissue area increased from 15 to 19% after 3 weeks (Cox et al. 2000). The rabbit latissimus dorsi was stretched over 3 weeks by inflating an expandable balloon underneath the muscle. Independently of factors released by injury, matrix-secreting cells may therefore respond to loads being transmitted through the muscle in ways which have yet to be fully described, but which could involve integrin receptors (Chiquet 1999). 4. Disuse, Immobilization and Denervation Increased collagen concentration occurs in disused limb muscles (Williams & Goldspink 1984), despite a decrease in the overall rate of collagen synthesis (Savolainen et al. 1988). Collagen content begins increasing within a few days of immobility. A portion of this relative "accumulation" of collagen may therefore be explained by the slower rate of degradation of collagen than muscle; this hypothesis is supported by the fact that electrical stimulation of disused muscle prevents the increase of collagen from occurring, perhaps by slowing the process of disuse proteolysis (Goldspink et al. 1991, Williams et al. 1988). However, when disuse is combined with immobilization, distinctive remodelling of the connective 55 tissue matrix appears to occur. The angle formed between collagen and muscle fibres in the endomysium was more acute following immobilization in a shortened position (Williams & Goldspink 1984). The normal occasional inter-myofibre collagen struts were greatly increased in number in immobilized muscle, even after 1 week of immobilization (Josza et al. 1990). The endomysium and perimysium were also abnormally thickened, as visualized qualitatively using scanning electron microscopy. Reintroduction of movement led to resorption of some of the accumulated collagen, with the efficacy of resorption dependent on the intensity of activity; free activity and low-intensity running led to only minor resorption, whereas high intensity running resulted in nearly full resorption of accumulated collagen after 11 weeks (Josza et al. 1990). This data suggests that the excessive accumulation of collagen following immobilization may be reversible with intensive rehabilitation. The relevance of limb muscle immobilization to the diaphragm is questionable, as the diaphragm is never immobilized; even during mechanical ventilation or unilateral denervation, the diaphragm is subject to some passive movement and stress. However, the disuse of the diaphragm that occurs during mechanical ventilation may be a significant contributor to diaphragm injury and subsequent fibrosis, because disused and atrophied muscle is more vulnerable to exertion-induced injury (St Pierre & Tidball 1994). In future, models of diaphragm disuse may be developed to examine the effects of deconditioning on the connective tissues of the diaphragm. Denervation may influence the connective tissue of skeletal muscle in ways that are distinct from the processes of disuse or immobilization. However, parallel studies of denervated and innervated disused muscle have not been conducted to this writer's knowledge. The accumulation of collagens I, III and fibronectin (Salonen et. al. 1985) and an increased concentration of hydroxypyridinium (Miller et al. 1999) appear to be similar to changes brought on by disuse and immobilization. The endomysium and perimysium of denervated muscle quickly become fibrotic to the point where individuals myofibres are completely encapsulated (Borisov et al. 2000). In denervated diaphragm, procollagen I and III mRNA were increased by 15- and 6-fold after only 3 days (Gosselin et al. 1995). The longer-term outcome of diaphragm denervation on connective tissue content has not been described. 56 5. Age There is a normal age-related loss of elastin and accumulation of collagens and mature cross-links in skeletal limb muscle (Gosselin et al. 1994). Collagen in aged muscle accumulates in the endomysium in particular (Kovanen et al. 1987). The diaphragm shows a similar but smaller age-related increase in collagen content (Gosselin et al. 1994). However, collagen synthesis in muscle at the levels of mRNA and enzymatic cross-linking are known to decline with age (Gosselin et al. 1994, Kovanen 1989). This supports the hypothesis that the slowing of collagen breakdown is responsible for the normal age-related accumulation of collagen in the diaphragm (Bou-Resli et al. 1991). In addition, aged diaphragm is more susceptible to contraction-induced injury (Watchko et al. 1994), and regeneration is less effective in aged skeletal muscle (Grounds et al. 1998). The dynamic of collagen synthesis and breakdown with age has not been adequately examined in the normal and injured diaphragm. However, it appears that increasing age could predispose to injury and fibrosis during ventilatory loading. 6. Corticosteroids The direct effect of corticosteroids on fibroblasts is to suppress the secretion of collagen and ground substance (Dannenberg 1979). Likewise, prednisone treatment results in decreased levels of TGF-3 in the diaphragm (Hartel et al. 2001). In the mdx mouse (which usually develops progressive fibrosis of the diaphragm), prednisone treatment reduced the amount of collagen (hydroxyproline) in the diaphragm. However, it caused a higher level of hydroxypryridinium cross-links (Hartel et al. 2001). The authors speculated that, while suppressing the fibrotic response, prednisone treatment also suppressed collagen turnover in the mdx diaphragm, allowing the time-dependent build-up of relatively stiff, permanent cross-links to occur. Acute administration of high doses of corticosteroids leads to nonspecific, widespread skeletal muscle atrophy and degeneration, including the respiratory muscles (Dekhuijzen & Decramer 1992). The inflammation and regeneration phases following myotoxic injury result in the deposition of granulation connective tissue, and could result in permanent scarring if high doses of the myotoxin were administered repeatedly. Longer-term moderate doses of corticosteroids lead to specific atrophy of type lib fibres in both proximal limb and respiratory muscles (van Balkom et al. 1997, van Balkom et al. 1994). 57 Summary The diaphragm in COPD may become mechanically disadvantaged due to hyperinflation of the chest wall. The hyperinflated diaphragm is prone to exertion-induced injury during periods of ventilatory loading. Hyperinflation and airflow limitation are two major determinants of diaphragm injury in people with COPD. Exertion-induced injury results in remodeling of the extracellular matrix, which in turn guides the processes of inflammation and regeneration. If injury is repeated chronically, the extracellular matrix is eventually characterized by a pathological increase in the amount of collagen, i.e. fibrosis. In addition to injury, factors which may lead to an increase in collagen content include chronic inflammation, myopathies such as corticosteroid myopathy, increasing age, disuse, immobilization, and denervation. Increased collagen content is usually associated with a loss of contractile tissue and an increase in muscle stiffness, which may impair the function of the muscle. 58 Determination of the number of fields per tissue cross-section to be sampled Field % Collagen Biopsy 5 Biopsy 1 1 35.3 10.2 2 21.1 20.5 3 12.5 14.0 4 26.8 11.9 5 21.6 22.0 6 14.3 10.7 7 17.9 16.7 8 31.7 17.6 9 16.7 8.2 10 22.0 19.6 11 21.3 21.3 12 34.1 19.2 13 13.6 28.3 14 17.4 26.0 15 0.0 14.0 16 23.1 22.0 17 18.2 21.4 18 29.2 25.0 19 21.7 17.9 20 25.7 18.2 MEAN 21.2 18.2 SD 8.2 5.5 # Fields Mean % Collagen Biopsy 5 Biopsy 1 15 21.9 17.6 15 20.5 19.1 15 22.7 18.2 15 20.3 18.3 15 19.5 19.5 SE 0.330 0.201 SE (% of mean) 1.55 1.10 10 22.0 15.1 10 20.4 21.7 10 18.9 17.9 10 23.5 14.2 10 18.5 20.6 SE 0.669 1.03 SE (% of mean) 3.16 5.67 Calculation of collagen CSA standard error. Prior to beginning data collection, two biopsies were selected, one with a normal amount of collagen (Biopsy 1) and one which had a slight increase (Biopsy 5). Twenty fields were point-counted, representing the maximum number of fields which could feasibly be counted, and the mean CSAcoll calculated. Next, 15 fields were repeatedly, randomly sampled from the set of 20 counted fields, and the standard error calculated. This was repeated with 10 fields. Note how for Biopsy 1 the CSAcoll ranges from 14.2 to 21.7 when 10 fields are counted, compared to 17.6 to 19.5 when 15 fields are counted. 59 Inter- & Intra-rater Reliability of Point-Counting Picrosirius Red-stained cross-sections Field Cytoplasm Collagen No Count % Collagen No. A1 W A2 A1 W A2 A1 W A2 A1 W A2 1 44 43 45 5 4 6 1 4 16 12 1 1 . 4 9 . 3 1 3 . 3 2 3 5 37 36 9 7 8 1 9 19 19 2 5 . 7 1 8 . 9 22.2 3 4 3 42 41 7 6 5 1 3 15 1 7 1 6 . 3 1 4 . 3 1 2 . 2 4 5 2 51 52 7 8 8 4 4 3 1 3 . 5 1 5 . 7 1 5 . 4 5 3 9 40 40 1 1 12 1 3 1 2 12 10 2 8 . 2 3 0 . 0 3 2 . 5 6 5 0 50 51 6 6 5 7 7 7 1 2 . 0 1 2 . 0 9 . 8 7 4 5 4 5 4 5 9 8 8 9 10 10 2 0 . 0 1 7 . 8 1 7 . 8 8 4 2 41 4 3 9 9 9 1 2 1 3 11 2 1 . 4 2 2 . 0 2 0 . 9 9 4 3 44 4 5 4 4 6 1 6 1 5 12 9 . 3 9 . 1 1 3 . 3 10 3 5 3 5 5 5 2 3 2 3 1 4 . 3 1 4 . 3 11 3 2 3 0 3 4 2 8 2 9 9 . 4 1 3 . 3 12 3 2 3 0 7 8 2 5 2 4 2 1 . 9 2 6 . 7 13 3 4 33 6 7 2 3 2 3 1 7 . 6 2 1 . 2 14 3 8 3 9 7 7 1 8 1 7 1 8 . 4 1 7 . 9 15 4 0 3 9 5 6 1 8 1 8 1 2 . 5 1 5 . 4 16 3 9 3 6 7 7 1 7 2 0 1 7 . 9 1 9 . 4 1 7 3 1 33 6 6 2 6 2 4 1 9 . 4 1 8 . 2 18 2 8 2 5 9 1 1 2 6 2 7 3 2 . 1 4 4 . 0 19 33 33 1 5 1 6 1 5 1 4 4 5 . 5 4 8 . 5 20 22 22 23 1 4 1 5 12 2 7 2 6 28 6 3 . 6 6 8 . 2 5 2 . 2 21 2 6 2 7 28 8 7 9 2 9 2 8 27 3 0 . 8 2 5 . 9 3 2 . 1 22 3 0 29 5 7 2 8 26 1 6 . 7 2 4 . 1 23 2 9 28 1 1 11 23 23 3 7 . 9 3 9 . 3 24 2 7 25 7 8 29 28 2 5 . 9 3 2 . 0 25 3 2 32 1 3 12 18 19 4 0 . 6 3 7 . 5 26 2 4 25 9 9 30 30 3 7 . 5 3 6 . 0 27 3 5 35 2 2 26 26 5 . 7 5 . 7 28 2 7 28 7 7 29 29 2 5 . 9 2 5 . 0 r (A1 vs W) 0.98 0.95 0.98 0.96 r (A1 vs A2) 0.98 0.91 0.98 0.95 Cytoplasm, collagen, and no-count represent the 3 categories used during computer-assisted pointcounting. The right-hand column is the % of collagen, excluding the no-count category. A1 and A2 represent counts made on separate days by the main investigator (Alexander Scott). W represents counts made independently by a second investigator (Dr. Wang). Pearson correlation values at the bottom of the table represent the inter-rater (A1 vs W) and intra-rater (A1 vs A2) reliability. 60 o c o c o c o o i c o T - n n c o o o n w c o N T - c n o o < M T - T - n i M i - i n i - N c o ( o ^ ^ ^ c o CM cvi i - r - c o a > c o o c o w c \ J o c M 1- CVJ CNJ CO CO C\J •55 5 i » N S i : c o o ) o p c o n ( v i i > i m p o c o N ' t O T -W i - T - ^ c o i - c j T - w c o c o ^ i n c o ^ T - CVJ T - T -E CM o < c XI ^ r O O < D S N T - 0 ) N ( D n i O ' < t i - ( 0 < O O O C O T - n O < CM T - 1- ^ CO W i - C M C O C D t ^ f C O C O C M T - T -W O l ^ O C O O l W t D N i - CM CM T - CO CM CM o c o c o o t c o c o c M T -T- CM T - CM CM CM > - I - T - O C O C > J T - ( N O ^ - I - C 3 ) U J O C O - * C N C O - * O T - C J O C M O O O O O C M O > i - CM O O - ^ C M T - C M - r - - < t T - o r - - - ^ - h ~ O O C M - i - O C M C M O C M - i - O O - i - O C 0 i - CM T -a> Q. 55 CM (0 ^ c < < i - T - O ^ W i - W T - i n o i C O N ^ O ) ( O O W O O W W W T - T - O O O O T - W T - CM <0 > a > Q. o •e CM ^ C N T - C M C O - ^ - C M C O - t - O C N J O O T - O O T - C O O O O O - i - O C O ' ^ l v . O O C M C M C M C O i n T - C O W O W O T - T - T - O C O W i - O W O I M O N ^ i n i D S C \ j T - C M C ^ < N C M C O C M C V l - i - O C M - ^ - i - C \ J O O O ^ - i - O O O C M O C O C O U J h - r « -^ W T - T - I M T - O W C M W C O m o [ ) \ f T t i - O C M 4 0 N T - C M i - « O W ^ W O O ^ C M W T - W T - o t T - T - N i n ( D t o R i c n o i - n o O i - ( M T - ^ o ( M i - i n T - i n z ,~ £ < C M i - i - C \ | T - 0 ^ ( M I M N ( 0 1 f l O J i - ( B O O C O O O ' T - T - i - f M O M a l ( M O r -^ I - ^ I - I - T - O J O I W C O T - C O C O C O C O C M ' t - C N J C M C M C M C O C O C M C O C M C M - r - C M C O N n ^ w r o i - s N c o n c o a i ^ O ) ( D o o ! 0 0 ) e o s i f i O ) ( N W i - T - i n o o ) < D _ < i - i - i - -i- C M C M C M C O T - C O C N J C O O C M C M T - C M C M C X I C M C O C M C O C O C M C M C M C O (0 E 2 5 ^ ^ ^ T - CJ CM CM (0 T - C O C O C O C O C M C M C M C M C M C M C M C O C M C O C O C M C M C M C O D - i-CMCO^lfllDNflOCJ) O i - C M C O ' j m c O S C O C S O i - C M n ' T i n c D N C O f f l O r i - T - r - T - T - T - i - i - C M C M C M C M C M C M W C M C M C M C O c o O) CD d d co co O) o d d c o oo o d d CM CO CD CD d d oo co OJ O) d d C M > < > Reliability of morphological categories. Int nuc = internally nucleated fibres, abn cytoplasm = abnormal cytoplasm, abn shape = fibres with abnormal shapes or sizes, % abnormal = Pabn, the total percentage of fibres with abnormalities. Thirty fields were counted, twice by Alexander on separate days (A1 and A2) to determine intra-rater reliability (Pearson correlation of A1 vs. A2), and once independently by Dr. Wang to determine inter-rater reliability (W vs A1). The overall inter- and intra-rater reliability is 0.94 and 0.96. 61 Fibre Area Inter-rater Reliability Testing (square microns) Fibre # Tester A (%) Fibre # Tester A (%) A S A S 1 1690.1 1715.5 1.5 48 1582.6 1557.5 -1.6 2 2322.6 2314.9 -0.3 49 1565.2 1617.4 3.2 3 1894.7 1905.1 0.5 50 249.8 317.6 21.3 4 2301.8 2203.7 -4.5 51 2754.0 2815.6 2.2 5 1185.2 1175.9 -0.8 52 1586.6 1598.4 0.7 6 1771.1 1769.4 -0.1 53 1981.1 1975.2 -0.3 7 1802.3 1773.7 -1.6 54 2002.4 2023.9 1.1 8 1743.0 1772.0 1.6 55 1550.2 1578.2 1.8 9 1288.4 1242.1 -3.7 56 1797.8 1776.7 -1.2 10 1483.9 1469.5 -1.0 57 2637.6 2661.5 0.9 11 2123.3 2118.7 -0.2 58 2580.2 2508.7 -2.8 12 2243.3 2227.7 -0.7 59 880.9 867.5 -1.5 13 2303.6 2215.5 -4.0 60 1242.0 1313.1 5.4 14 4565.7 4538.9 -0.6 61 2639.7 2569.0 -2.8 15 3142.5 3188.6 1.4 62 1722.6 1641.2 -5.0 16 2217.9 2208.6 -0.4 63 1294.9 1243.9 -4.1 17 1799.3 1794.9 -0.2 64 2633.9 2576.0 -2.2 18 2656.1 2660.3 0.2 65 2391.7 2354.0 -1.6 19 4581.9 4524.6 -1.3 66 3144.5 3175.6 1.0 20 2357.9 2411.6 2.2 67 3639.4 3666.9 0.7 21 2037.8 1991.8 -2.3 68 2002.8 1959.4 -2.2 22 378.2 380.4 0.6 69 1064.4 1017.0 -4.7 23 2262.9 2229.9 -1.5 70 2268.5 2400.0 5.5 24 294.6 296.9 0.8 71 1934.0 1887.8 -2.4 25 1079.9 1129.2 4.4 72 2259.2 2360.6 4.3 26 1749.8 1730.8 -1.1 73 2127.9 1954.0 -8.9 27 1662.7 1666.4 0.2 74 1705.4 1754.3 2.8 28 1295.7 1316.4 1.6 75 2001.8 1931.7 -3.6 29 2263.6 2280.0 0.7 76 176.5 202.6 12.9 30 3316.8 3293.2 -0.7 77 3739.9 3775.1 0.9 31 932.9 907.6 -2.8 78 3210.5 3218.5 0.2 32 1810.2 1835.7 1.4 79 2392.0 2366.4 -1.1 33 1942.5 1914.7 -1.4 80 2577.4 2618.7 1.6 34 1443.9 1463.8 1.4 81 5562.0 5538.9 -0.4 35 2022.8 2004.3 -0.9 82 2071.4 2032.2 -1.9 36 2036.9 2004.2 -1.6 83 288.2 312.8 7.9 37 2408.2 2344.0 -2.7 84 163.3 317.5 48.6 38 2824.1 2788.6 -1.3 85 202.4 180.1 -12.4 39 2357.5 2268.2 -3.9 86 3856.6 3943.9 2.2 40 3651.3 3758.8 2.9 87 3048.4 3085.7 1.2 41 3238.9 3219.1 -0.6 88 2494.5 2451.3 -1.8 42 1830.0 1903.4 3.9 89 2976.7 2985.7 0.3 43 2573.5 2555.6 -0.7 90 823.9 809.6 -1.8 44 2973.9 3170.6 6.2 91 2359.9 2371.2 0.5 45 1666.3 1711.5 2.6 92 2215.0 2111.4 -4.9 46 2855.2 2897.0 1.4 93 2812.8 2834.9 0.8 47 1550.0 1685.5 8.0 94 2274.6 2274.4 0.0 62 Inter-rater reliability of fibre area measurement. A: Alexander, S: Sunita Mathur. All fibres on a field were traced independently, then the values for individual fibres compared. The values in each column represent the cross-sectional area of individual muscle fibres. The A % column refers to the difference between A and S for each individual fibre. The average difference (94 fibres) was 0.32%; r=0.998. CO CO —* o o •g_ co cn 3 O O o co" co CD ZI O Z o O o t z O CO > o o co cn cn CD o 6" n 9L cu —i CD CU o o oT CO CD Zi SD Mean U l 4 * CO r o o CO 0 0 v l CD U l 4*. CO r o Field # Subject # CO vj CO o CD CO r o CO oo r o 0 0 r o U l r o CO CO U l r o co r o CO r o CO CO CO r o CO CO r o CO r o CO CO CO CO v l o k o CO CO L 4 ^ cn 4 ^ co oo CD C D v l -L O CO 4 ^ r o r o CO r o o U l r o CD ro co r o o r o CO r o CO r o o r o r o 4 * r o U l r o r o U l r o -N co z o oo r o r o 4 ^ CD r o cn CD r o o bo r o J i . CO r o CD U l CO r o CD r o U l r o v l U l CO r o CD 4*. U l fo r o o U l U l bo r o C D f o U l bo •vl co r o U l b o CO > io r o 4 ^ U l r o r o r o -N r o J * r o 4 ^ • CO r o r o 4 * r o v l CO o CO CO CO CO r o 0 0 r o vJ r o o CO o j ^ U l CO CO o o CD U l o r o o CO oo 4*. zt C D oo •vl co r o U l bo r o oo v l co r o CO CO CO CO 4 ^ co 4=> r o r o r o oo r o co r o U l r o oo co CO r o - t i . vJ r o CO CO j i . z o 11.0 r o oo CD co CO r o CO r o o b v l f o CO 4>. U l 4 ^ oo bo CO 4 ^ CO r o U l r o 4*. U l bo U l 4 ^ CO co r o o CD CO b o CO > CO b CO 0 0 So CO CD J i . CO oo CO v l CO v l CO oo CO CD co CO co CD CO CO 4 ^ CO 4 i . CO CO U l CO co j ^ r o CO r o t o -P» r o CO U l o U l U l CO v l U l CO CO J i . j i . J i . o CO U l r o o C D r o 4 i . vJ U l r o ro r o r o r o o r o CO r o r o r o v l v l CD r o 4 ^ r o CO v l z o CO 4 ^ v l CO bo v l v l O co r o o bo co co v l CO CD CO r o U l r o f o 0 0 CO CD U l 0 0 U l o bo r o U l 0 0 v l o CO > TJ O rf o o t z j o 5' c o O o oT c o CD -o O CO > CO TJ a £9 CO CO 0) 3 o T3_ &) CO 3 O O o oT CQ CD o o o o c 3 O CO > O o CO CO I CO CD o l—t-o" 3 CD O o ST CQ CD 3 SD Mean Field # Subject • c n 4 ^ CO r o —JL o CO o o vl Oi c n CO r o CO CO Oi vi CO o o CO c o CO CO 4*. O 4 ^ CO o o CO Oi CO CO CO 0 0 CO 0 0 CO vl CO vJ CO r o CO o CO c n CO o r o CD Oi 4 ^ 4 ^ c n v l 4 ^ 4*. CO o CO V l c n 4 ^ c o o r o r o I O o h o r o r o o c o CD o o r o r o 4 i . r o o r o r o o o N> r o r o I O r o r o CO —v Oi -z. o b> c o bi CO CO L 4 ^ 4 ^ CD 0 0 CO CO c n v l v l r o CO 0 0 vl CO c n CD CO c o b o r o r o b r o c n b r o c n c n o CO > 4 * ro CO L. CO 4*. CO CO CO CO CO 4*. o CO c n r o Oi CO o CO CO o> r o c n r o c o CO r o Oi r o vl CO o CO CO a> c n c n c n c n Oi o 4 * c n o o o _,. c n vl r o Oi o o c n CO r o ro c n r o 4 ^ r o c n r o v l r o 4 ^ r o CO ro 4 ^ CO r o r o CO r o 4*. r o Oi r o CO r o vl r o o CO r o 0 0 z. o CO vJ r o b o CO h o CO CO c n 4 ^ o b o CO Oi _x r o c n b r o o c n r o vl CO vl c n c o 4 * r o vl CO o o b o r o r o c o o CO > to vl c n CO CO r o vl CO CO r o 4 ^ r o o o r o CO r o r o c n CO r o r o 4*. CO CO r o CD CO o L c o r o Oi CO CO b c n 4*. r o CO r o vJ v l c n r o CO 4 ^ 0 0 c n c n CO o c n i o CO o CO r o CO CO r o 0 0 CO o CO CO CO c n r o 4 ^ CO CO r o c o CO Oi r o CD r o CO r o o o CO CO CO 4 i . o Oi vi b o L c n c n r o CO r o c o r o Oi vl r o v l CO Oi vl r o c n b •vl CO CD vJ c n c o O b o r o CO bi 4 i -CO r o o b o L o CO o CO > TJ o 3 ' .— O o c 3 5' CQ O — K o o co" CQ CD 3 o CO > ± CO TJ a fr9 CO D Mean cn CO ro L o co oo •vl CT) cn CO ro Field # Subject # CD 4*. ro cn CO CO o> o> ro •vl CO cn ro 00 CO o ro co ro v j •vl 4^ ro CT) ro CD ro ro 4^ ro CO CO •vl CO CD co bo o ro CT) cn ro CT) oo CO •vl co o co -A o co -L o O i CD ro •vl co v j CO cn CO o CO ro CO ro •vl ro O) CO CO CO co CO o ro v j ro oo CO ro CO o ro CD z o 11.3 ro oo ro ro v j ro co oo ro CO o d CO o d CTi v j ro L. CD o d ro CO ro cn •vl CT) ro •vl bo ro cn v j CO ro CO ro v j CO CO ro 4*. o CO > cn ro co v j v j CO cn 4^ cn CO CO o> CO cn CO CO CO o> 4*. 4*. CO en CO 4*. -P>. 4*. co CO oo CO v j CO 4^ CO oo ro v j oo cn o cn v j ro -L 00 O) •vl ro 4*. v j CT) oo •vl v j o CD CT) bo oo CO ro cn cn v j ro ro ro ro CD cn ro oo v j co ro ro z o cn v j oo cn ro ro ro o d oo 4^ ro cn d ro CO co CO bi CO —L CO v j ro cn cn ro co ro CO v j o CD v j 4*. cn co v j o CO > ro CO o cn CO cn CO O) ro CD ro CD ro 00 CO CD CO ro CO o ro oo ro v j ro oo ro ro CO CO CO ro CO o CO co CO 4*. CO cn co •v) _^ oo CD 00 ro 00 o 00 ro o o O) o o CO CD ro ro co co ro o ro CO ro CO ro v j cn ro CO ro ro •vl ro CT) ro v j ro ro o ro cn ro v j z o •vl co ro CO v j ro o cn CT) CO ro •vl cn ro •vl cn ro ro Jo oo bo ro o d ro oo CT) ro ro ro ro v j d ro ro ro •vl CT) ro CO CO cn bo —L CD v j o CO > £9 CO CO CB —1 o o TJ fl) cn 3 O O o oT CQ CD 3 o o o o c: 3 O CO > o -\ o cn cn • cn CD o o 3 to CD to o o oT CQ CD 3 CO 03 00 CO cn co CD 03 cn 03 CO CO CO CO cn oo co co oo co oo co co cn co co CD 00 CO cn CD CD oo CD ro ro cn co cn co ro ro co oo co cn co ro co co CD cn CO O O CO O O CO O O ro TJ o 3 O o c 3 i—t-3 ' CQ O o oT CQ CD 3 o co > o O TJ D 99 CO o p H 30 CO Q> O 3 CD Q. 3 0) r-t-~T 0) Q . a. H CD CD < 3 iat do 6" 3 p XJ CD cn 5" 3 CO "co B) T O o "O 0) CA 3 o b o 5T CO CD 3 z .9 z o o o c 3 o CO > o o CA CA (0 CD O b" 3 SL CD CD 0) o -* o g_ oT CQ CD 3 SD Mean Ul j » CO ro —JV. o CO oo v l o cn j i CO ro —j. Fields: cn ro CO 00 CD CO CO j i Ul CO J i v l J i CO J i o J i CO j i o CO J i CO ro 4*. ro CO cn CO CO 4*. -ti CO Ul CO o H 3D CO o Ji. CD co CO co v l o 00 CD ro v l CO CD CO co 00 O CO v l oo co CD v l CO CO ro v l v l j i oo ro v l o ro o z o v l ro ro fo CO CO CD vJ ro CO Ul CD J i b ro o b cn v l CO b ro CD CO j i v l ro CO CD CO J i IO 00 CO v l b 00 CD o CO > CO I -CD — o o CO p* tu o z o o CO > 4^  CO CO b CO CO o ro co CO CO CO j i . co CO ro CO Ul ro oo CO ro CO v l ro CD CO cn CO CO ro ro CO 3D (5* 3" o o CO »-*• 01 ro b v l co co CD 00 CD CD CD CD co O co vJ Ul CO o J* CO ro CO cn ro CO ro v l ro CD 00 ro CO ro ro o ro ro ro co ro ro L. CD IO 00 IO ro Ul co ro z o cn ro p ro ro cn o> v l ro CD CO CO Ul b ro CD fo ro Ul CD j i CD v l CD ro ro b ro CO IO Ul v l CD v l CO fo ro CO b o CO > cn CO oo CD j ^ vJ Ji. L CO CO CO J i co CO CO co CO CO CO co ro CO 4^  CO CO CD j i 4^  CO CO CD 4^  o CO O o CA 0) 3 CA CD a. ro ro f° CT> oo v l J i o v l 00 ro o o vJ o v l co o CO v l cn bo O j i v l Ul ro o CO v l CD ro ro o v l Ul o ro o j i z o cn CO fo CO CD bo Ul So ro CO fo ro CO 00 ro ro b Ul KD v l b ro CO CO oo CO ro v l J i CD oo co CD 00 oo j i o CO > cn v l co o j i J>. o CO v l CO v l CO J i ro j i . ro 00 ro v l ro Ul ro v l ro CD co CD CO Ul ro CD co ro ro ro CO o c 2. CO j i . co j>. o Ul oo oo Ul CD Ul cn j i co ro oo v l oo o cn bo ro CO So —k CO ro oo ro ro j i ro CD CO ro v l ro —i. ro CO oo CD ro co ro 4^  CO CO z o oo ro CO v l ro o b CD v l bo CO b CO 00 cn v l CD Ul CD CO o CD CO cn vJ CO Ul b ro o b ro cn cn ro CO Ul vJ co ro CD vJ o CO > cn CD CO CD cn CO CT) j * ro J i CD CO v l CO CO j i j i CO J i j i o ro v l ro co j i CO v l CO 00 CO v l ro CD CO TJ (A o CD CO bo CO v l 4^  j * CO Ul CD ro CD CO CD ro ro ro CO CD o cn CO ro ro CO ro CO v l J i ro —k ro j i v l ro CO ro o CO o CO ro ro o ro j i ro ro ro o CO CD z o j * CD CD ro - j . o b oo v l CD CO Ul j i j i CO cn b v l b 00 fo CD Ul j i v l Ul vJ bo j i b CO v l o CO > CO TJ C O Z 3 CD ~ a o " o c 3 L9 co o p H TJ CO » - r fl) O 3 CD Q _ 3 0) r - r ~ T — T 0) a a H CD CD < 3 iat do 5' 3 3 TJ CD C Q o' 3 CO CO fl) o o TJ 0) CO 3 o b o 5T C Q CD 3 z p z o o o c 3 p+ o CO > o o CO CO CO CD o 1-P 5' 3 SL 0) CD fl) o — o o 5" C Q CD 3 SD Mean cn J i to ro o co 00 vl o cn j i CO ro Fields: j * ro j i CO CD CO ro ro oo ro CO ro ro j i ro CO ro CD co ro ro ro CO ro —k ro o ro o ro CO CO o H TJ ro bo CO CO cn cn cn co cn o o oo o cn cn co oo oo O cn cn CO o v l ro ro CD CO o CO CO CJ) ro CO CO o IV) co CO CO CO CO cn CO v l CO j i CO cn CO ro z o v l b ro cn CD ro fo co CO cn fo ro 00 ro ro fo ro co j i CO o CO ro co cn CO CT) v l CO co k v l CD k CD fo CO b ro 00 b ro cn bo o CO > CO bo CO ro CO CO j i CO ro CO j i ro • v l CO v l ro j i CO CD CO j i CO co CO CO CO ro CO cn CO J i CO CO ro CO CO r -CD —p. 1-P o o CO 1-P 0) CO bo o v l co co oo v l - CO CO J i k o 00 oo co o ro ro o cn j i co cn ro ro CO ro ro CO cn ro oo J i CD o ro o ro co ro o ro o ro o ro z o CT> j> ro j i fo CD b ro o b co b ro o b ro ro co CO j i ro CD cn ro v J v J ro CD j i ro CO CO ro o b oo b ro o CD ro CO CO j ^ CO o CO > cn ro CT) b CO j i ro CO ro v l co CO ro ro cn ro CO ro v l 00 ro cn ro CO ro CD co v l cn j i v l o 00 oo co CO co o j i o JU C Q ' 3" p+ o o CO p+ 0) -ti ro v l bo ro j=. ro o ro co CO J i ro CO CO o ro cn ro v l co ro ro c» ro oo z o co CO ro v l b ro bo CO ro cn ro o CT) CO j i cn ro o b ro j i fo ro CO v l ro cn b j i CD ro oo b j i . o b o CO > CO o o CO p+ CO 3 CO CD o z o o CO > CO co CO IO cn CO CO ro CT> CO j i CO CO CO 00 ro v l CO CO CO CD CO v l CO v l CO CO CD ro CO ro oo CO o c SL cn b k o co 0 ) ro o oo v l CO k —k oo ro o oo CD ro o CD o J i v J CD CT> k Ji-k v l ro k cn ro CO j i oo oo CD CD 00 ro CD ro cn ro j i ro co z o co co ro j i b CO ro v J J i CO cn co b CO cn j i . ro ro cn ro ro j i j i o b ro CD v l ro CO j i IO CO • v l bo CD io cn bo ro cn b v l b o CO > j i CO CO p CO cn CO ro cn ro CT> ro CT> ro v l CO v l ro v l CO k CO CD ro cn ro • V l ro CO CO j i CO CO CO TJ CO o fl) CO v J cn v l j i j i CT> CT> CO CO j i j i v l oo v J j i co CD o J i j i ro v l bo ro ro cn CO J i CO CO CO CO ro CO CO ro ro oo ro o CO o ro co CO o ro CD ro j>. z o J i bo j i cn _^ cn v l k o cn CO bo oo bo 00 bo o b v l cn ro CO j i . CD CO ro j i io ro o b ro k co cn j i o CO > CO TJ C O |H 3 CD * * ro ° 89 (fl o o H DO CO CD o 3 CD Q . 3 Q> #-»• a SL a H CD CD < 3 iat do o' 3 3 31 CD CQ 5' 3 (fl (fl 0) o o TJ fl) CO 3 O b o 5T CO CD 3 z o z o o o c 3 f-t-o (fl > o o CO (0 (0 CD o 5' 3 SL fl> CD 0) o — o o 5T CQ CD 3 CO o Mean cn 41 CO ro ml o co 00 Vl O) cn CO ro Fields: cn io ro OJ CO CO CO CO ro ro ro 4^ ro CO ro ro CO cn ro co ro ro CO ro ro o CO CJJ ro cn ro 41. v j (fl o ro cn p> OJ cn 4*. CD •vl CO cn CT) 41. co 41. 41. o O OJ CO o CD ro 4*. ro v j CO •vl CO cn CO CO 41. ro cn ro CO CO cn ro •vl CO •F* ro CO CO 41. ro CD CO cn z o zo co CD 0 0 cn 4^ CO CO cn 4^ 41. bo ro 0 0 ro 41. v j CO 41. v 4 ro 41-- a . CO b o b CO bo ro CD 41. CO co CO o (fl > cn bo CO co CO co CO CO CO cn CO o ro CO CO cn ro co oo CO -n. CO CO CO -n. CO •vl CO CO ro v j CO ro (fl I -CD —•» »-*• o o (0 0) ro So cn So O) v j ro CJJ 41. cn oo 41- CO cn cn cn oo CO o cn 4^ ro cn CO oo ro CO ro •vl CO CO 41. ro -n. ro co CO v j ro cn ro ro 4^ ro CD ro oo ro oo z o v j ro J i . 4=>. CO bo v j cn ro bo OJ CO ro o v j o bo 41. v j CO o bo o cn ro 41. ro bo CD 41-ro ro CO oo CT) o (A > CO CO O CD CO ro CO oo ro oo ro cn ro CO CO cn ro co ro CD ro CO CO CO ro CO CO ro CO ro CO CO (0 zo to" 3" o o (0 SL CO v j O o CD co 41- cn 41. CO oo OJ CT) v j o CO 41. ro ro CO ro o cn ro CT) ro CD ro o ro CO ro CO ro o ro ro 41-ro cn ro ro cn ro 41. co z o oo fo ro 4^ b ro cn CD ro o bo ro 4^ CO ro CJJ cn CO ro CD ro cn ro v j cn co ro CD 41. cn to ro o cn cn bo ro OJ ho cn bo v j co ro cn b o (fl > 4^ CO ro v j CO ro cn ro oo CO ro CO CO CO CO ro oo CO CO co 41. o CO oo CO 41. CO ro CO ro CO cn (fl o o co SL 5" (0 CD 3. o' 3 CO 4^ oo CO o CD cn CD CJJ v j cn v j OJ v j CO cn o CO CD ro CD ro ro ro ro ro cn ro OJ ro CJJ v j oo oo ro CO ro 41-ro oo ro CO z o •vl v j ro o ro ro CT) ro CO o CT) CO CO CO ro CO bo ro 41. CO CO ro 41. CO OJ ro cn fo cn OJ cn b v j CO oo CT) ro cn o (fl > 4^ CO ro OJ v j ro ro ro CJJ CO oo ro CD ro -n. CO CO ro 41-ro oo ro 41-ro oo ro oo ro 41-ro 41. ro v j ro ro (fl o c -\ SL ro cn CO CT) ro ro ro cn v j oo o 4^ 41. CO o o 4^ . v j CO CO KJ CO cn CO CJJ ro CO CO ro CO oo ro oo CO oo CO o CO ro ro •vl CO cn CO cn CO cn CO CO 41. z o v j CD CD CD ro 4* CO v j cn b CJJ cn 41. b cn v l 41. b cn ro ro ro CT) ro ro ho o b 41. CO 41-bo o b O b o (fl > OJ b CO -n-cn CO co CO v j 4^ o v j CO CJJ CO OJ CO o ro OJ CO oo CO CO CO 41-ro 41. CO v j CO -n. ro v j (fl TJ (0 O 0) CO cn CO b cn CO CO ro OJ ro CO CO ro ro ro OJ CO ro o o> cn ro cn cn ro CO ro _ L ro o CO ro cn ro CO CO 4 i ro ro ro 0 0 ro •vl CO •vl ro o ro OJ CO 4 i z o CO CO •vl co ro cn L CO v j b OJ b cn bo 4^ co OJ CO O CO v j co cn v j cn CJJ v j v j -H. b oo OJ CD o (fl > (fl TJ C O I S 3 CD ~ CO ° 69 OT o o H 3J CO Oi O 3 CD Q . 3 fl) #-»• a SL Q . H CD CD < 3 iat do 5' 3 3 3J CD C Q 6" 3 C O OT fi) o o TJ fi) (0 3 O b o 5T to CD 3 z .9 z o o o c 3 r-t-o OT > O ^ O (0 CO CO CD o o' 3 SL 0) CD fi) O —H o o 5T C Q CD 3 SD Mean cn 41 CO ro O CO 00 o> cn 41 CO ro Fields: CD ro oo CO ro co CO ro CO CO ro oo CO v j CO CO ro v j ro O J ro O J ro v j _L v j ro ro ro v j CO ro ro co OT o H ZO CO co co CO cn O J ro co o 41. co co co 4 i cn O O l v j ro 4* bo ro ro o cn ro 4 i ro o oo ro v j ro O J ro v j ro ro CO v j CO ro ro v j ro v j ro co zo v j So ro cn CD CO b ro cn b CO CO ro 0 0 So 41. b ro CJJ v j ro cn b ro CO v j ro v j bo CO 41. _^ co 41. b ro CO b ro cn b i L 41. v j o OT > 4* CO CO CO ro CJJ CO 4* ro co CO 41. 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CD ro 41. ro o ro o ro •vl ro v j ro cn ro oo CO ro ro O J ro ro ro 4 i Z ocn v j bo ro CO bo ro So co CO CD •vl v j b O J CO oo CO ro bo cn bo v j CO v j ro v j v j ro bo o OT > OT TJ C O \2. 3" CD S ? 41 ? OL CO O O H -• TJ co -• ST O D 5 Q. 3 fl) ~ 3. — Q. H CD 2 I I 3 TJ CD CQ 5' 3 CO CO fl) o o TJ 0) CO 3 O O o 5T (Q CD 3 z o o o o c 3 o (0 > o o co CO CO CD o r - r 5" 3 fl) CD fl) O o oT CQ CD 3 (0 o Mean Ul ji CO ro o co oo v l CD Ul ji CO ro Fields: Ol CO CO b ro oo CO CJ) CO co j> ro CO oo ro CD CO v l CO o CO co CO co CO J i CO J i CO Ul CO Ul ro CO o H TJ v l CO o bo CO Ul CD v l CD •vl ro co co i ro oo ro CD CD v l v l O co co b o oo ro CO Ul oo ro ro v l ro j i oo CD v l ro CO ro ro ro CO Ul z o 10.2 ro CO co Ul Ul b ro o b v l Ul ro v l b cn b ro co co co b ro CO ro CD v l v l b ro CD — L Ul b j i b CD v l ro Ul b o CO > CO r-CD p* o o CO «-P 0) o z o o CO > CR co CO j i j i CO v l CO j i j i j i j i CO co ro co j i CO J i CO ro ro CO j i ro CO CO j i CO TJ io" 3" i—f o O CO 1-P fl) ro b CD j i 00 j i 00 CO j i v l j i j i 00 o Ul CD vJ CD o v l CO ro ro io oo ro oo j i co Ul v l CO o CO ro ro CO v l ro CO CO CO ro CO z o j i v l CD b v J bo j i CD CO oo b oo CO cn io ro oo co co b ro CO CD co io cn b ro CO CO Ul b o CO > CO O o CO 1-P 0) o z o 3 CO CD 3. o CO > CD b CO CO CO CO ro co J i co oo CO J i CO Ji. ro Ul CO CD j i co CO CD ro co CO co ro v l ro CO ro 00 CO o CO O c SL ro j i CO CO J i Ul v l v l oo ro j i CO CO CO v l j i CO —JL o CO ro cn j i ro oo ro Ul ro o ro ro ro ro CO o ro cn o ro j i CO ro ro co CO o CO ro CO ro z o cn CD j i oo b o cn b v J v l ro j i io Ul CO v l Ul v l V I co j i v l ro o b ro co v l CO io o CO > J i CO j i CO CO j i CO cn CO CD CO o j i o j i CO v l CO o CO 00 CO ro ro oo CO oo CO cn CO o CO o CO TJ CO o 0) CO ro cn CO b j i ro v l ro j i CD o CO j i CO CO CO o CO bo ro en ro Ul ro CD ro CD ro CD ro 00 ro o ro CO ro j i ro 00 CO ro ro ro v l CO o CO o z o O) ro CD Ul o Ul Ul i i ro vJ 00 CO j i co 00 b j i b ro Ul b ro b 00 b ro in v l CO ro CO CO CO o CO > CO TJ C O I S = CD ~ a P a. i CO o p H 3J to OJ O 3 da ntr a SB a . H (D fD < 3 iat do 5' 3 3 ZO CD CQ 5" 3 CO O) Q> —i O  TJ 0) (0 3 O b O 5T CQ CD 3 z p z o o o c 3 r-t-o CO > o o (0 (0 (0 CD o 5" 3 SL CD CD CD O o o 57 CQ CD 3 SD Mean cn 41 CO ro o co 00 -4 OJ cn •u CO ro Fields: CO KJ CO o b CO -n. CO CO o ro oo CO CO CO ro CD CO OJ CO 41. CO ro oo ro 00 CO ro CO ro CO CO 0 H zo CO 4^ 00 bo o oo •vl oo 41 U l O) CD O) OJ oo -n. v j CO O CO ro CO b co ro 41. ro O J ro •vl ro OJ ro •vl oo oo ro CO ro OJ ro v j ro ro o ro U l ro v j z0 v j ro ro ro ro v j ro o U l —k 00 CO ro ro KJ o bo CO co CO cn b ro o b cn b OJ KJ ro ro KJ CO CO CO ro U l b 00 41. CO C J 0 CO > O l ro U l v j oo U l ro CO ro CO ro oo ro oo ro oo CO 41-CO ro ro O J ro OJ CO ro ro OJ ro ro ro •n. CO 1— CD 0 0 CO 1—* 0) ro CO KJ o co 4 i 41. v j v j oo v j co 41. 41. CO co co 0 0 O l KJ CO p CO U l CO CD CO o> CO o> ro 00 ro 00 ro v j ro ro ro ro CO CO CO CO ro oo ro -n-CO ro ro CD z0 co b to ro CO U l v j CO •vl U l 4^ bo 4 i bo ro o b ro o b ro ro KJ •vl ro ro b CO CO — L CO CO 00 b co CO CO ro co b ro co 41 0 CO > OJ ro CO o bo CO CO CO OJ ro cn ro •vl 4* CO CO ro ro CD ro cn CO U l CO o ro CO CO 4 i ro o CO CO CO CO CO 03 O ) bo cn co CO —k CO ro co OJ U l U l o oo OJ CD v j 0 JU th" ZT 0 0 CO DJ O l O l ro cn 4=> CO oo CO cn ro cn •vl ro co CO CO ro ro CO ro oo CO o ro ro v j ro ro U l z 0 10.3 oo KJ L 4^ ro o b _ L O v l ro 00 co OJ U l U l CD co 4* co 4 i ro U l -n-CO CO o CO CO b 41. 4 i "41 ro L 41 00 41. 0 CO > OJ KJ CO cn bo 4* CO CO CO •vl CO •vl CO CO CO oo -n. U l CO CO CO o> 41 o ro U l ro OJ 4* ro ro U l CO CO 0 0 CO a ro KJ pj OJ CO oo cn OJ OJ 4 i OJ v j 00 v j ro OJ 0 b ro o OJ ro 00 ro ro 41. _ A co 4* oo ro o U l CO — i . ro OJ co ro CO z 0 3 CO CD a. O) CO 41. b ro bo v l v j bo CO U l 41. CO b oo KJ CO bo OJ CO OJ v j ro I CO ro CD v j 41. U l cn b 0 CO > CO cn CO 41. CO 00 CO OJ ro cn CO CO CO ro CO ro CO o ro oo CO cn CO 41. CO ro ro CD ro CO CO 0 ro 00 CO 0 c SL O l 4 i 41. OJ OJ •vl CO U l CO ro CO 4 i OJ 4 i U l U l ro cn 0 CO CD ro v j KJ co ro CO ro v j ro OJ ro 00 CO CO ro ro 4 i ro CO ro v j ro co ro CO CO CO 0 z0 ro KJ CO b 4 i CO ro co 00 00 CO U l 00 b OJ bo CO v j O bo cn b L >. 4>. v l L 41 v j OJ CO cn ro 0 CO > CO cn CO oo bo 41 CO CO CO O CO v j CO co 4 i CO ro 4 i CO 4 i ro CO v j CO OJ 41. O CO OJ 4 i O CO cn CO TJ CO 0 0) CO CO U l KJ CO 00 U l CO OJ U l v j CO CO OJ cn U l 41 OJ CD 0 ro b co U l •vl ro ro oo ro CO oo v4 ro 41. v j 00 ro o ro ro 00 ro CO •vl co z0 cn ro b OJ U l CD U l •vl U l CO 00 o CO v j CD OJ cn OJ v l -n. b ro KJ 0 b CO b ro 0 U l 0 CO > CO TJ C O |2 =• CD ~ a o 3 .-»• 3' CQ o o 5T CQ CD 3 o co > ZL 73 CO o p H XJ (A I-T 0) O 3 CD Q . 3 0) i - T - T a ffl a —1 (D fD < 3 iat do 5" 3 3 XJ (D (Q 5' 3 CO CO ffl o o TJ ffl CO 3 o b o ffl CQ CD 3 z p z o o o c 3 o CO > o o (0 CO CO CD o r - r 5" 3 ffl fl) CD ffl o - 4 * o o ffl CQ CD 3 SD Mean Ol - t i CO ro o CO oo v l CD Ul J i CO ro Fields: CO ho CO CO CO CO J i CO ro 00 ro CD CO ro CO ro CO ro CO CO CD ro co CO vl CO CO oo ro vl CO H c_ ro co CO b CO ro vl o vJ co oo Ul Ul co vl oo Ul CO O CO ro ro co —A. vl ro Ul ro j i ro j i ro j i ro ro ro CO •vl ro ro ro Ul co ro j i . ro o ro vl z o CD ro ro b ro co Ul ro p> oo - t i ro oo ho ro Ul b vJ b ro ro b IV) o b CO ro b ro ro ro CO vl _ J . 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I-T o o CO r4> ffl o z o o CO > co CO CD - t i - t i co o ro vl j i o CO oo j i ro CO 00 j i o CO v l CO vl CO CO J i ro oo CO CD CO Ul CO XJ CQ' 3" I-T o o CO I-T ffl co io vl v l - t i - Ul ro o oo o CO vJ v l ro CO o o oo o -•>. bo oo b oo ro ro CO ro Ul CO Ul ro o co co L oo L, CO ro Ul — i . j i ro o z o CD bo vl Ul oo b ro CT> bo Ul b j i co ro o bo CD b ro o CO vl b • Ul b Ul b ro CD vl oo b ro CD CO ro o j i oo b o CO > CO CO CO Ul CO CO L CO vl CO ro CO j i CO CO CO oo CO CD j i ro co CD j i o CO ro co ro co j i . co j i co Ul CO o o CO r * -SL 3 CO CD 3. ro CO oo Ul v l ro ro o Ul v| CD vl v l o 00 00 vl o ro b - A CO ho ro CO co vl ro o ro o vJ Ul ro o CD ro i . ro o ro ro ro —L. z o cn CO CD b ro CD ho Ul b ro v l CO ro CD ro CO CO b Ul ro ro Ul CD CO j i b ro CO co ro Ul b co b co b CD v l o CO > J i vl co o b CO CO CO ro CO Ul ro CD ro oo CO ro CO CO ro ro vl CO j i j i o CO CO ro co ro o CO J i CO o - 1 c — T ffl co Ol co Ul CD CO o j i 00 o CD o vl 00 vJ Ul vl o Ul b ro CO b Ul ro o ro ro ro j ^ ro Ul ro oo ro CD ro ro o CO CD ro ro ro vl CO oo ro ro z o vi ho ro ro b CO w ro Ul b - t i b CO CO b ro CD CO j i ro b ro CO co CO vl ho ro ro vl J i b CD Ul co j i ro o b vl o CO > CO TJ CO o ffl CO o z o o CO > CO TJ C O 12". 3 CD a o 3 i-r 5" CQ O o ffl CQ CD 3 o CO > 74 CO o o H 3J CO Q) O 3 CD da ntr -t a a . 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O J ro b cn b cn co oo fo ro O J bo v j co ro o bo CO v j v j b CO b i ro j i ro b o CO > 41. bo ro CJJ bo ro O J ro o ro O l ro j i CO CO CO o CO o v j ro cn CO ro ro ro ro O l CO CO CO o CO o CO r-CD — I-i-o o CO -+ 0) CO O J b O ) ro ro v j v j 41 CO ji j i v j j i 00 CO O l o CO co CO o fo CO CO CO CT) co ro ro O J ro O J ro CO CO co CO Jl ro v j CO j i CO Jl ro ro CO o ro oo z o 10.2 00 cn 00 bo CO v j cn v j 41. ro ro b o bo 00 CO bo j i CO CO CO bo _ i ro j i —L CO bo co b i CD j i CO o CO > 41 b i ro v j b i co CO ro v j CO CO ro j i ro 00 ro CO ro ro cn co ro ro JI CO CO ro O ) CO o co ro o> CO zo IQ" 3-l-i-o o CO F* SL CO b O l 41. ro CD ro v j v l cn ro CO 4^ oo O l O l co o cn k> CO o CO ro CD CO ro 00 CO o CO CO CO O l ro j i CO CO ro co CO O ) ro O J ro CO ro 00 CO CD CO Jl z o v j bo cn b i ro co ro co ro o v j ro v j CO CT) v l ro O l b v j co O J v j O l co o bo ro CO b i j-bo ro o bo o CO o CO > CO O o co l-i-CD 3 CO CD 3. o z o CD ro ro o 41-o b v j 41 CO b CO ji ro co ro ro b O J v j CO v j •vl b Jl co rv> —L b ro O J bo ro b i ro CO cn bo o CO > cn b i CO CO CO 41. -n-CO 41-ro oo CO ro O J CO •vl CO o j i o CO O l IV) CO CO O l CO CO CO CO CO ro CO ro CO O c SL ro bo oo b CO O l o v j v j —1. o O l O ) oo ro ro cn CO o o cn b ro v j CT) ro -n. ro O l ro cn CO o cn ro CO oo ro ro CO ro O J oo co ro ro ro z o CT) v j _ k CO b O J 41. L ro bo ro O J CO oo j i ro KJ ro ro CO ro cn b j . -A ji b ro cn bo ro O l b i ro O J v j j i ro ro b ro CO bo o CO > CJ) CO 41 CO 41. 4 i 4 i O CO cn ro v j j*. o b ji ro CO O J CO v j ji co ji o j i 00 j i O J j i O ) j i oo CO O l CO TJ CO o 0) CO CO bo O l — 1 cn o 41- j i ji — t ji CO oo ro o ro j i cn o oo b —L. 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Ul Ul o CO c o Ul r o j i CO CO Ul J i 4^  CO v l to 3D io" 3 " l - T o o CA ffl r o b o o b CO Ul o o CD j i CO c o CD k v l v l c o Ul c o o -Pi b r o b _Jk c o _^ o k k CT) k V l k j i r o c o v l CO J i CO CO J i v l z o Ul b o v l v l r o CT) b r o o o CO CO h o r o CO CD b o o r o v l b CD v l O v l r o r o b b J i b r o o Ul o h o CO b o to > •fi b j i c o Ul J i CO J i j i CO CT) J i Ul J i CO j i j i r o J i Ul J i Ul Ul Ul j i 00 j i r o CO Ul j i r o J i to o o CA l-T ffl r o CO v l J i o o CO CO o v l Ul v l v l CO j i o 00 00 Ul o - N Ul —k r o r o o o o o o v l v l k j i k k Ul j i Ul CO v l CO v l z o 3 CO CD 3-J i v i j i b ui v l v J b r o o b 00 h o r o Ul o b j i CO CO Ul O ) c o CD co v l i o CD b r o CO b CD b o b o to > Ul CO j i CO j i J i CO c o CO Ul j i r o CO 00 j i Ul CO 00 J i 00 CO v l j i 00 CO CO CO CO Ul r o Ji. r o j i o to O c » CO b j i b o Ul o o vJ o o J i Ul o v l j i o O ) j i o j i —L v l vJ CO r o CO r o CO CO - a o o k v l k v l r o c n r o CO r o o Ul c o z o CD b o o b o b r o Ul r o Ul CD b —k Ul b r o r o v l J i v l v l b o b • V l Ul CD CO o b r o Ul CD o to > CO j i v l v l j i o j i o o j i CO j i CD j i CT) j i v l J i CO J i o o Ul o j i v l Ul o Ul j i CO Ul r o Ul o to TJ CO o ffl CO b J i CO CO r o r o O ) Ul r o CO j i CO Ul J i CD j i J i o c o Ul I O o r o r o Ul r o r o CO v l o o o o J i vJ CD z o CO j i v l b vJ b Ul b CO b j i h o Ul CO b CO b Ul b v l J i CD P v l CO I O h o v | v l j i o to > tO TJ C O CD ~ a o co ° 3 CQ o o ffl CQ CD 3 o to > 76 to o D H -• XJ CO -• ffl O 3 2 Q. 3 ffl =? 3. 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H CD 2 I I 3 33 CD CQ 6" 3 CO W fl) - i o o •o 0) CO 3 O "6 o_ oT CQ CD 3 z o o o o c 3 «-»• o CO > o o CO CO CO CD o 5' 3 SL 0) CD 0) o o 5T CQ CD 3 CO o CD < Mean OI 41 CO ro o co 00 vj O) cn 41 CO ro Fields: v j ro oo 4* cn CO o CO CO CO ro CO o CO oo CO o CO o CO oo CO co CO oo CO CO ro ro ro cn CO o H 31 CJ) ro CO v j — i •vi 4 i OJ O) cn j i O ) cn •vl ro O l 00 v j cn o O b ro 41. b CO — J . ro CO ro 4 i ro O l ro oo ro ro v j ro oo ro v j ro o ro ro oo co IV) CD ro oo z o 16.1 ro O l v j cn CO CD L. cn 4^ _ j . O l bo L. J l CO CD cn O) v j j i CO j i v j ro O l 00 CD b cn b ro O l b j i o cn ro 00 b o CO > J i b CO v j 41 CO oo CO 4 i CO 41 4 i 4 i CO co CO CO CO OJ j i oo j i O) CO CO CO oo CO 41. CO CO CO cn CO OJ CO r-CD — i-i-o o CO l-i-0) ro 4^ co v j O O l co co CO oo co O) CO co CO CO CO oo o 4^ b OJ b i co co ro 41- o cn v j CD O) — L ro i . 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CO CO 41. 4 i j i ro CO oo j i o CO OJ j i o CO 00 j i CO j i CO o CO ro ro ro O J CO O o CO l-i-0) v j cn v j •vl O l cn OJ v j ro j i cn co OJ v j cn v j O l o OJ 41 ro co ro CO 4 i O J co — L O) ro O l co ro o CO ro CO IV) O J ro •vl CO j i CO ro z o 5" CO CD a. 41. v j 41. ro •vl b i O ro o b co b J l b O l CO CD b oo b O l b oo b CO b ro j i —k O) o CO > CO 4 i CO ro CO CO ro 4 i ro oo CO CO CO ro CO j i CO v j CO CD CO CO CO ro CO ro CO ro CO ro CO o CO CO o c SL CO O l 00 b O l •vl •vl CO O) •vl OJ CD v j ro ro cn CD O l o o 4 i v j ro ro b ro O l CO ro ro oo ro ro O l ro ro ro o cn ro CO CD CD ro O) cn oo ro ro z o OJ v j ro CO CO KJ ro ro b ro o b ro J i O l bo v j L J l b 00 CD v4 b i ro v j bo ro v j CO CO b i CO CO w CO CO b ro 4 i j i o CO > 4 i 41 ro 00 b CO o ro O) CO cn ro cn CO CO ro 00 CO — L ro cn ro ro ro ro ro co rv) OJ CO •vl CO o CO o CO TJ CO O 0) (0 CO b 00 •vl cn CO o oo L ro CO v j j i v j CO O O) 4^ o 41 b ro CO CO ro cn CO o ro CO ro cn ro o ro v j ro o ro O l ro j i ro v j ro •vl ro j i CD — 1 v j L co z o oo b ro oo b ro — L ro KJ ro b i CO J i fo ro CO bo ro ro ro ro v j b CO j i KJ j i CO b CO oo b L co J i CO CO CO ro CO CO 41. co CO CO o CO > CO TJ C O \S. =' CD ~ a o l-i-5' CQ O o 57 CQ CD 3 o CO > 78 co o o —I XJ CA - • ST O 3 2 fl> ~ S. SL o. —l CD 2 II 3 XJ CD CO 5' 3 CO CO fl) o o •o 0) CA 3 o b o 5T CQ CD 3 o o o o c 3 r - r o CO > o CA CA CO CD O r - r 5' 3 BL fl> CD fl) O o 5T CQ CD 3 SD Mean Ul ji CO ro o CO oo vl CD Ul ji CO ro Fields: CO ro L ro oo ro ro ro j i co ro ro o ro o v l ro CO ro o L oo ro U l v l ro CO o —i XJ CO bo V l v l ro co U l v l oo CD CO oo CD CO U l co CD O CO vj CO J i So CO CO CO ro CO J i CO v l CO j i CO v j CO ro CO CO CO ro CO v l j i ro CO CO CO v j ro CD z o 10.6 ro CD So CD v l ro co b v l io ro CD b ro v l bi ro co CO U l U l J ^ CO co ro U l co ro CO j i CO CD v l CO J i b J i CO io o CO > CO o r~ CD — r4> o o CO i—r SL z o o CO > So ro J i U l ro ro ro - t i ro j ^ ro j i C D ro ro j i ro v l CO o CO CO CO CO ro oo ro v j ro o CO J i U l co CD o o CD U l o ro o - co oo j i CD oo v l co o XJ CQ' 3" « - r o o CO SL U l bo ro 00 v l CO ro co CO CO CO j i CO j i ro ro ro oo ro co ro U l ro oo CO co ro j i v l ro CO CO j i z o 11.0 ro oo CD co CO ro co j i ro o b v l fo co j i U l j i oo co CO j> . 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U l co U l j i CO CD ro o b CO b o CO > U l co ro po co ro CO ro U l CO o CO CD to ro o CO ro CO ro ro v l ro ro ro v l CO J i CO CO j i CO o o CO l-T SL 3 CA CD 3-o 3 ro io CD CO CO U l U l • v l CO v l CO U l v l U l U l co U l CD o o CD So ro oo CD CO U l ro CD CO CO ro CD ro CO U l j i o ro CD ro j i CO CO CD ro v l ro j i ro CO co z o CD So k oo j i co ro _ ^ - t i v j _JL C D v l oo b v l ro U l b CO b CO U l v l b U l bi oo U l ro U l b ro co ro ro U l ro ro v l o CO > CO U l ro CD CO ro - f i L CD ro oo CO oo CO CO ro ro ro ro CO CO v l CO o ro v l ro CD ro 00 CO o CO CO o c SL CO —X U l bo -CO j i CO CO CD j i j i U l ro CD CO CD U l CD o co So CO o CO ro oo CO CO ro ro ro v j CO U l U l v l co o ro CO CO o ro oo ro co ro 00 ro CD z o 15.8 ro o v l CO j i J i O b ro 'cn v l CO 00 CO ro j i CD CD v j ro b CD CO 00 ro ro U l v l v l b j i CO C D io o CO > en CO co CO fo CO o CO 00 CO o CO CD CO j i . ro oo CO CO CO CO CO v l j i 00 CO J i ro ro ro U l CO oo CO TJ CO o 0) CO ro CO CD b o CD oo CO j i oo ro oo j i ro CD CO v l CD CO o CD j i ro CO co ro CO CO ro U l ro ro U l ro v l ro oo ro j i ro oo j i co ro CD CO j i CO ro ro ro z o vi U l U l ro U l b CO b ro v l o U l ro ro So U l v l ro o U l j>. ro j i U l • . 00 ro j i CD v l CO o CO > CO TJ C O nr. = CD ~ a o H ° ro 3 CQ O o 5" CQ CD 3 o CO > 79 CO o D H ST p • 2 o. = o> =n 3. SL Q. H CD » II 5' 3 = 33 CD CO 5" 3 CO "co fl) O o •o fl) CO 3 o "6 o 5T co CD 3 z p z o o o c 3 o CO > o o CO CO CO CD o l-T 5' 3 0) CD fl) o o_ 57 CO CD 3 SD Mean Fields: Ul J i CO ro o CO oo vl Ol Ul j i CO ro CO O o H z o XJ o CO 3> CT) bo ro CD b CO ro oo CO o ro oo CO v l ro oo ro oo co ro co CO o CO ro o ro co ro ro CO J i CO •fi b 00 'co ro 00 o co CO ro 00 oo oo o v l J i o r -CD —T. CD CO ro vj CO j i ro ro v l ro CO ro CD ro CO ro j i ro CO CO CD ro CO ro Ul ro o CO Ul ro j i CO j i ro Ul z o o o CO T—• 0) 13.1 ro Ul b CO bi ro ro So ro Ul b ro j i CO v l Ul ro oo So co o b ro co b ro v l Ul IO L L ro Ul b CD J i CO ro Ul b ro j i L o b o CO > j i bo ro j i vj ro oo ro o ro CO ro ro co CO ro ro ro ro co CO o ro oo CO CO CO v i ro j i o co o Ul I CD v l j i CO * o o XJ io' 3" T T j i b ro Ul b ro Ul ro j i CO o ro CD CO CO ro Ul ro j i ro o ro o ro j i CO j i z o o o CO T » ffl co b CO co j i IO CD 'co j i oo v l CO o co j i o Ul CO CD v l Ul co j i CO b CO ro b co o So ro oo So CO J i Ul o CO > CO bo CO CD co o co Ul j>. ro J i j>. o co ro co oo CO oo co CD CO CO ro CO CO Ul CO Ul CO Ul CO v l CO o o CO CO CO o CO 00 CD CD CD 00 CO CD J=> i v l Ul CD co o CO i - r ffl j i So Ul ro co Ul CO CO v l ro v l ro oo o v l CO ro co Ul z o 3 CO CD 3. CD b ro j i j i ro Ol b ro v l CR b oo b CO b ro ro b v l j i ro Ul Ul CO J i Ul ro CD j i CO v l b CO o b CO j i ro o Ul ro ro b o CO > o 3 CO ro co So CD ro Ul ro CD ro oo ro j i CO CO oo CO CD CO CO o CO ro ro j i CO v l ro CD CO COr° Ul vj CD Ul CD co oo CD vj CO CD co j i CD ro Ul CD o o c ffl J i b ro oo CO co CO CO CO CO ro co ro CD oo ro j i ro CD ro j i . ro vj co o ro j i CO ro ro CD z o CD J i CD b ro j i b CD v l 00 co CO v l ro Ul b CD So Ul b v l vj CD fo ro CO i ro v l CO Ul CD CD So o CO > Ul v l ro Ul ro CO ro ro ro ro CD j i co ro CO IO ro ro L co CO co k ro j i CO CO ro CO CO CO So v l So j i j i CD Ul Ul Ul oo CO 00 j i ro o TJ CO CD b CO o vj CO 00 ro v l CO Ul CO ro 00 00 ro ro CO CO v l CO v l CO J i CO ro co CO ro CD ro 00 z o o ffl CO co vj ro ro Ul L CD b • . ro j i CD —L J i J i b ro CD fo CO ro j i _J. co ro —L CO So ro •vl b ro Ul b oo co ro Ul b -JL o co CO j i CO o CO > CO TJ C O nr. 5-CD ** a o " 9 CO 3 CO O o ffl CO CD 3 o CO > 80 CO O O —i 3J CO f - » 0) O 3 CD Q_ 3 0) —* fl) a a H CD CD < 3 iat do o' 3 ZO CD CQ 5" 3 CO (O 0) o o TJ 0) (0 3 o b o 57 IQ CD 3 z p z o o o c 3 o CO > o o (0 CO (0 CD O 5" 3 SL 0) CD 0) o — o o 57 CQ CD 3 SD Mean 41 CO ro o CO oo vj CD cn 4i CO ro Fields: 4 i 4M CO v j b 4 i 41 CO CO CD CO co CO -n. CO CO CO 4 i ro CO oo ro CO J> 4 i CO CO U l CO CO oo CO CO v j CD b v j CD U l ro 41 CD CO v j CO CD CD - H CO •vl O o ro v j cn U l 4 i ro ro oo -n 41 ro U l U l 41 41. co oo z o H ZO CD ro o CO 4 i b ro ro ro co 4^ ro CO U l CO ro fo ro j>-41 co ro cn U l ro U l ro CD fo ro fo ro oo b ro CO cn U l b o CO > 4 i b co 41. b - n CO CO 41 CO CD CO oo 41- CO U l co v j CO 4 i CO CO ro 00 CO o CO 4 i CO U l CO CD CO o CO ro b cn '•vl v j CD CD •vl CD CD CO CO 4 i v j cn CD -n. J> o r-CD — 4 i ro ro ro j i CO ro CO ro L 00 CD ro ro ro co ro CD ro CD ro oo ro oo ro co ro 4 i ro CO ro ro z o O O (0 SL -Fi co CO b -n b cn b 4 i b cn b ro 'co b —i U l oo o co ro o b 4 i co cn b o b o b ro CD co o CO > U i - f i b CO 4 i co 4 i 4 i ro CO cn -n - n o CO co o - n CD 41 •vl 4 i v j 4 i CO 4 i CO 4 i 4 i ro CO ro v j 4^ b v j v j 4 i CO v j 4 i v j ro U l CD CO ro cn o ZO (Q 3" -n. v j CT) ro ro ro ro ro v j CD ro CO ro CO CD -JL U l L v j -JL CO L L ro o L CD z o o o (0 SL cn b o 41. v j v j oo v j ro o U l CO v j co ro U l -n b 4 i fo CD b CD ro b U l co J> v j O b oCO > 41. v j CO CO b CO CO CO CD ro v j ro oo CO 4 i CO •vl CO CD CO CD CO co CO U l ro CO CO CD CO 4 i CO oo 4 i o CO O o CO l-i-0) CO •vl U l CD oo -JL oo 4 i •vl CO U l 00 CD CO ro ro -n. v j o 4 i b ro U l ro CD ro cn ro •vl ro cn co oo co ro ro ro ro ro v j cn ro v j ro CD z o 3 CO CD 3. v j b 00 CO ro ii oo fo ro oo b ro ro fo o U l cn b ro o b 41 co U l 4 i b CO cn ro cn b cn b CD U l L b o CO > CO b 4>-ro 4i. CO 4 i 4 i -n. CO U l o CO CO o ro CO co CO CD 41 U l 4 i U l CO CD 4 i CO 41 CD 4 i CO ro b CD CO oo CD U l ro CD cn 4 i ro •vl •vl oo CD co o O —% c SL 41 b 41 ro CO U l 41 oo •vl ro •vl •vl CD 41. CO z o -n. b CO b U l v j ro b o 41 CO 'co U l ii ? 00 v j ro CO cn U l fo CD b CO cn v j b ro fo cn oo b o CO > CD 4 i CO CD b 4 i —L ro v j CO CD IV) U l CO 00 CO ro CO 00 4 i 4 i - t l v j CO 4 i co 4 i 4 i ro 4 i 4 i CO CO CO CO b CD b U l v j 00 •vl cn v j CD cn CO CD oo CD U l U l o TJ Vt ro CD b v j ro CD co CO ro o ro 4 i CD 4 i U l ro o ro o CO CD U l v j z o o a vt v j —L CD b -JL o b ro o b oo fo ro b b v l b CO b o fo ro ro o b ro o b CD b ro CO o ii CO ro b o CO > CO TJ C O 12". 3 CD - * a o 3 CQ o O 57 CQ CD 3 O CO > 81 CO o o H 3J CO I-* fl) O 3 ro Q. 3 0) - t a M a H CD CD < 3 iat do 5" 3 3 33 CD CQ 5" 3 CO "to fi) o o TJ fi) CO 3 O " 6 O 5T CQ CD 3 z . 9 z o o o c 3 o CO > o " i o CO CO CO CD o 5" 3 m ts CD fi) O — • » o o oT CQ CD 3 SD Mean cn J> CO ro o CO oo Vl CD cn 4* CO ro ml Fields: v j CO co co ro cn CO CD CO o ro ro v j 4* ro ro co 4 i CO 41. O ro v j CO 4* -n. CO 4 i o CO o H 33 J > CO CD 'co cn cn o cn oo CD v j v j co oo o cn O o b CO b 00 00 00 00 00 oo oo oo oo oo oo oo oo oo z o 10.4 ro CO CD v j ro ro ro cn b CO j> CO cn v j CD b 4 i O b ro v j cn 4 i b 41 b 4 i b co b 00 b o CO > CO r-CD — o o CO 0) o z o o CO > b CO cn b CO CO CO cn CD CO ro J> o CO CO CO 4=-CO o CO cn CO 00 4 i CO CO -N CO cn CO 33 to" ZT o o CO I-* fi) ro b v j b CD J>-L oo •vl cn o 41 o 00 o CD CD o CO b ro o cn ro ro J > - 00 ro o CD ro ro cn ro 4 i ro CO ro 4 i cn 4 i ro ro ro o ro ro z o CD b v j ro L o b oo b ro cn b CO co b oo 4 i ro co ro cn b o b ro o b CD b ro 4 i 41 ro o b 4 i b o CO > CO O o CO s. 3 CO CD 3. o 3 o z o o CO > cn co CO cn b CO CD CO CD CO ro CD CO cn CO oo CO v j 4 i 4 i ro 4 i 4 i ro ro co 4 i CO CO ro 4 i CO o c —\ ta_ ro J > -cn CO ro 4* •vl cn oo 4 i o cn CD v j CO CD cn o L ro ro ro ro ro ro oo ro v j ro CO v j ro cn 00 v j cn ro v j •vl ro CD CO 4 i z o cn b ro cn •vl v j b L. CD ro cn •vl J > ro b 00 b CD ro o b ro cn CD 4 i CD in CD ro v j ro o CO > v i CO CD CO CD ro cn ro CO ro •vl CO J > CO ro ro oo CO CD 4 i —L CO CD 4 i cn CO ro -Pi CO CO TJ (0 O 0) CO b CO v j ro CD cn cn ro CD ro cn ro CD o CO 4 i o CD ro ro ro J > ro ro ro 00 ro •vl ro CO •vl ro cn —L oo —L v j cn ro v j v j ro CD CO 4 i z o cn cn o b oo b b CD 4>- co cn b ro CO cn b v j b cn b O b cn b CO o b CD CD 4 i o CO > CO TJ C O I S 3 CD ~ a o cn 3 CQ O O 5T CQ CD 3 O CO > 82 co o 0 H a O 3 2 a 3 01 =r a . SL a H ro 2 II 5' = 3 XJ CD CQ 5' 3 CO CO 0) o o TJ_ 0) (0 3 O " 6 o 5T CQ fl) 3 o o O o c o CO > o o CA CO CO CD O F* 5' 3 0) CD £4 O o 57 CQ CD 3 SD Mean u i J - CO r o o CO oo v l CD Ul j i CO r o - J L Fields: v i CO ro CO CO CO ro o CO J i Oi ro oo ro CO ro 00 ro Ul CO ro CO cn v | ro Ul ro CO CO CO b CO b t CD CO CO Oi j i co J i CO o CD ro CO Ul O o cn co ro cn b ro ro v| ro o ro 00 co ro Oi CO ro CO ro Ul ro o oo CO O CO o CO Ul ro Ul z o XJ 11.7 co co b ro CD ro j i j i j i ro o b Ul j i CO CO Oi j i CO v l CO CD b J i CO co CO j i fo ro cn b ro ro io J i 00 b CO CD j i J i CD j i . 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CO CO CO o CO > j i b CO co CO J i J i ro cn CO Oi CO v l j i o> j i o j i ro j i o CO CO j i o j i o j i cn CO co CO v l CO CO CO 'co CO b ro CO j ^ J i o Ul CO CD j i j i Ul Ul cn o T J CA j i b ro o CO v l CO cn ro CO ro ro L V l L oo ro o ro o oo CO CO CO ro CO ro CO z o o fi) (A j i b co io j i 'co o v j o b CO CO O b — J L ro b v i b co CO CO CO o b ro b L b L j i o CO > CO T J C O I S 3 CD "* a o CD 3 CQ O O sr CQ CD 3 o CO > 83 co o O H •• 33 CO - • Sf O 3.5L 5. a fl) o 5" ^ 3 a? to 5" 3 co co 0) o o TJ_ fi) CO 3 O b o 67 CQ CD 3 z o o o c 3 o CO > o o CO co CO CD O —+ o 3 0) CD 0) o o 57 CQ CD 3 CO o Mean Ol J> u ro o co oo vj OJ cn 41 CO ro Fields: CO p OJ 00 U l 4 i OJ 41 00 OJ 4 i O 4 i O l OJ v j 4 i O 00 4 i 4 i 4 i co CO v j CO CO CO o CO b o b 00 O) 00 OJ 4 i OJ oo co 4 i OJ CO OJ OJ U l 00 O o U l CO O) b ro ro ro co co O) v j o •vl co ro CO OJ 00 ro o ro U l ro O l z o zo O J bo b CO b co 4 i CD oo b OJ b U l co b CO U l b oo b o b 4 i b CO KJ ro o CO > O ) b 4>-b U l O l o OJ O l ro O J O J 4 i ro O l ro CO CO -n. ro CO oo 4 i —L O l ro cn ro -n o CO ro OJ v j 'vj L oo OJ •vl oo oo o O) — L OJ o oo CO co o r-CD — bo o b v j —L v j L OJ OJ ro ro O l OJ U l O l L 4* 00 00 ro z o o o CO M U l U l U l co b U l 4 i —X o v j —L 4 i b _x OJ b co b co ro o b ro ro b — j . ro b ro o co —L O) b O l b U l b ro b o CO > U l b o b U l J> 00 41 4 i OJ 00 CO OJ O l 4 i OJ 41 OJ OJ CJJ -n. 4 i CO 4^ 4 i 4 i 4 i ro CO ro CO v j CO oo b 00 00 co v j OJ U l oo ro O l 00 ro O OJ U l o 33 IQ' 3 " U l b —L OJ j > - J L o v j OJ OJ 00 co O l ro o> •vl CD U l ro O J ro cn z o o o CO SL b V l 'J> U l 4 i b 00 b 4 i b ro U l b O J o b —L b ro o v j ro co 41 OJ 4 i ro p 00 b ro b CO b ro b o CO > b OJ co b 41 oo 41. 4 i 4* 4 i 4 i O J OJ v j O J CJJ -n ro 4 i OJ 41 4 i O J CO CO CO CD CO oo CO v j 41 41 CO o o CO «-»• 0) 00 b v j 41 O) oo 4 i •vl ro •vl U l o v j •vl —L O J oo o OJ b p OJ U l OJ ro —L ro ro o CO OJ L 4> ro ro ro CO L v j J> ro o z o 3 CO CD OJ fo U l 00 b 00 b ro b O l v j ro v j b OJ b 4 i b 4 i b o fo ro 4 i 41 v j b O l fo ro ro 4 i b U l 4 i o CO > 3-v j ro •vl b OJ o OJ OJ ro ro ro CD OJ OJ ro CO ro ro ro OJ OJ ro CO oo CO ro •vl ro -vl ro CO OJ O) b OJ ro U l U l 41 •vl •vl ro 4*. 41 cn J > 4 i L o o o c 51 ro b 00 o b ro v j ro oo OJ o iv> co ro U l OJ o OJ 41 O J O J OJ o OJ o CO ro v j co ro CO ro CO ro z o 10.8 oo b OJ v j U l v j OJ OJ b 4* v j 00 fo ro ro -n ro OJ b O J ro 4 i CO CO CO b ro b ro b CO ro b o CO > OJ b OJ U l v j OJ co 00 ro OJ — j . ro U l OJ co 4* OJ CO 4 i ro 00 o ro —L 4 i CO CO v j CO v j 4 i O l CO 4 i CO ro b U l b 00 U l OJ v j oo O OJ ro v j o O l oo oo U l o TJ CO 00 fo ro CO OJ ro OJ ro O) 00 OJ ro ro co ro OJ 4 i ro cn oo ro U l o ro 4 i z o o 0) to OJ b _ t 00 b v j b OJ b O) fo ro — j . b v j b CD b v j 4 i b oo b o b o 4 i •vl co ro b cn ro b o CO > CO TJ C O CD ~* a o H 2 3 CQ o o 67 CQ CD 3 o CO > 84 co o a H TJ CO QJ O 3 CD Q . 3 fl) — T a tt a H CD CD < 3 iat do 5" 3 3 TJ CD CO o' 3 ( 0 " ( 0 tt o o TJ 0) CO 3 O " 6 o flT CQ CD 3 z o z o o o c 3 o CO > o o CO CO CO CD o 5" 3 tt tt CD tt o — T l o o oT CD CD 3 SD Mean Ol J i CO ro - a o CO 0 0 v l CD cn j i CO IO Fields: J>. CO co —JL J i ro o L oo ro cn ro ro o ro ro ro ro ro j i . 00 CO CD bo j i b CO CD ro CO o CD CO vl j i j i CO CD 00 O b CO o io co o ro j i ro CO CO cn CO ro ro co ro CD CO vl CO oo ro oo ro CO CO vl z o H c_ 16.1 j i J i cn vj b j i 00 vl v l ro b CO cn v l co j i CO 00 io j i cn b cn j * cn CD b 00 vj j i o b CO o co o CO > b CO o b CO o ro CO CO O ) ro cn CO j i ro cn ro co CO ro co CO ro ro •vl ro oo ro v l CO CD CO j i CO CO b co b ro _ l . CO O l CO oo CO o j i . ro oo vl o o oo j i o I -CD —p. > - T CO b ro CO b ro ro L ro IO ro co ro ro cn ro j i ro oo ro ro ro CO ro co ro cn ro CD CD ro cn z o o o CO SL vl j i ro ro b ro oo b CO L b ro io ro CD b CO b CO j i fo ro cn b j i . ro CO b ro o b ro o b ro CD CO ro v l b vl b o b o CO > b ro O l j i CR ro O l ro o CO o ro cn ro vl ro cn co ro j i ro j i ro cn co CO ro j i ro C D ro CO CO CO b vl b oo ro cn co vl cn j i . C D cn CO j i o JU co" 3 " . - T j i b CO p —JL CO co ro vl CO ro oo ro CO ro CO ro •vl ro vl CO 00 CO cn CO ro ro cn ro 00 CO ro CD z o o o co —— tt 10.8 ro ro j i CO CO co CO o b CO vl b j i CO ro CD b ro o b CO o b CO b J i b j i CO C D j i CO io co L j i co j i CO vl CO o CO > O l b ro o ro CO CO ro ro oo IO j i CO ro co CD CD ro vl ro j i C D o CD CO O o CO l - T u b ro co L oo —JL O l L J i L _ i o J i . ro to vl ro CD cn ro oo v l o O l b CO o b ro co ro j i ro O ) CO ro oo j i o ro o ro cn J i o CO o ro vl CO j i CO ro CO cn CO v l z o 3 CO CD ^ 13.1 CO co b CO ro j i ro o b j i o b J i CO CO CO j i J i CO b CO ro b cn vj b CO o j i CO CD j i cn ro co L •vl fo CO oo vj CD j i CO ro CD b o CO > 3 , U l ro CD b ro CD CO o ro oo CO cn CO o ro ro ro o ro CO CD CO o ro vj ro C D ro CD ro CO ro to ro b j i b ro j i j i j i J i CO o ro o cn CD vj CO cn CO o o io CO b CO ro ro co CO —L ro j i ro co CO oo CO CO co oo co vl ro oo co o co o CO j i CO vl ro 00 z o c tt CO fo L O l b CJ) b —L CO ro b —JL O CO L CO —JL ro b CO CO CO 00 b CO 00 b j i CO 00 fo IO io o b CD fo oo b o CO > j i vl CO ro CO ro CO CO o j i U l ro CO ro co CO o CO cn CO CO oo CO j i ro oo ro 00 CO vl CO ro CO o CO —x b CO CO ro CO ro j i cn cn CO J i CD ro J=L CO j i o TJ (A o fl) CO O l ro vl j i CO CO CO O l CO ro CO o ro oo ro CO ro co ro ro CO CO CO CO ro cn ro oo ro C D z o CO co CO CO co CJ) co CJ) co CD b ro j i CO ro b 00 co co b cn b CD vl ro b ro b 00 b co o to > CO TJ C O I S 3 CD ** a o ~ o o- 5 3 CQ o O flT CQ CD 3 o CO > 85 3 3 - i 3 0) 3 C O (D B> p * CD a > cr 3 o » o >< t-p o •a 0) (A 3 > or o —T 3 D) N CD (A - T 0) T J <D CO o o o o o o o o o o o CO CO CO CO CO o o o o o o o o o o o o CO < o o c 3 f-r (A z CO 30 D 86 3 0) 3 n •< 3 C o_ (D a> —* CD Q . 3 M O >< o •o 0) (0 3 > cr 3 o 0) N ID in ZT fi) U (I) to CO ro CO CO ro|O|O|O|C0|O|^|O|O|-H-MO|-MO|O|O|O|O|O|-MO|G0 ro ro ro <3_ c" •< O o c 3 (0 z CO 3D O 87 z o 3 OJ 3 0) < 3 rt> B) CD a > a -3 o » o " D B> CO 3 > CX 3 N CD (t> CO CO ^ c o o ^ o o - ^ r o o o - L - ' o o o o o o o o r o w ro co co co o o c 3 O o •a o 88 3 a) «< 3 CD 01 <D O. > or 3 m o •< o 0) CO 3 > C T 3 o 3 cu N <D CO 3" 01 •a CD O * CO c CO CO O) 01 CO re a * CO c CT re' o ro ro co ro ro ro CO IS 3_ c' •5 o o c 3 CO 6 o T J o CO CO CO 89 o H JD O CD 3 CD 3 a. o 3 —\ CD CQ 5' 3 o 3 CD 3 CD 3 SL >< 3 C O CD fi) i-r CD Q . ro > ! T 3 O 3 SL o < o •o fi) CA 3 co > O" 3 o 3 SL CO N' CD O ^ CO 3 " 0) T J CD Total # Fibres: |Category subtotals: ro o CO 00 Vl CD cn CO ro o CO 03 Vl CD Ul 4> CO ro Fields: 1 88.4 ro CO ro co CO o ro ro CO o CO ro CO v j CO -n. o -d o ro co ro co CO v j CO 4 i CD ro CD 41. 4 i O l CO 4 i CO O l 4 i CO 4 i z CD oo O l O l ro ro ro CO ro co v j O l J i CO ro 4 i ro ro CD ro O H CO 00 -n. o ro CD CO O l ro o ro ro ro ro o t ro ro • ro CO ro Z0 ro bo ro CO o ro O l o o ro o o o ro o CO ro CO 87.3 v j CO oo ro v j CO CO CD CO v j O l J> ro CD CO ro o -ti-CO 00 CO co CO o co 4* 4 i ro CO CO v j ro co CO O l 4* co CO CO z ^ v j bi oo J>-O) CO ro 41 O l ro O) co ro ro CO ro ro O 41 ro O l 4 i o O l ro O l J>- •vl ro o —A J>- o o ro •vl CO -n. ro ro o CO SL O l CO o o _,, o o _^  _,, o o o - j . ro o o _^  CO 90.3 co co o 41 •vl oo CO •vl oo v j Oi O l O l O l 41. 4 i ro 4 i co 4 i v j 41 CO O l •vl ro 41 CO CD o O l CO O l v j z zo O l en L o co CT> O) o 41 o CO o O ro CO CO O l CO CO o CO ro CO ro ro CO ro to" 3 -o o CO —¥ fi) J> ro O) . . co o co co co 4* O l ro 4^ O l o ro O l ro b> 00 o O l CO o o ro o o o O ro o . o o CO z o o CO 0) 3 CO CD z\ 6" 3 ro CO 87.4 •vl ro ro 41 00 v j co CO o O l v j 4 i O l ro CD 41 00 41 o 4 i O l oo O l CD CD o O l CO z oo IV) CD J>- O l ro o O l L o CO oo CO CO CO 4 i CD 4 i CO o c bo O) 4 l o o CO ro •vl ro ro O 41 4 i CO ro 4 i ro -\ SL CO CD CO o - t i CO _^ o ro ro N> ro CO O l o ro CO 96.0 CD 00 O l ro CO o ro CO ro L o o CO CO CO •vl ro ro CO oo CO O l CO 4 i CO O l CO o ro 4 i ro CD 4 i o CO 4 i ro 4 i CO CD 4 i o z CO O) v | co ro ro o o o o o . o o ro o o O ro CO TJ CO o fi) CO bi O l o o O l o ro o o o o o o o o o oo o o o o o ro ro 00 v j ro o —t. o o o o CO o o o o o o o CO = 1 o _ -* o _i o c 3 90 o H JD O CO 3 SL CD 3 Q. O 3 CD CQ 5' 3 Z o -I 3 at 3 CD 3 0) 3 C o CD CD Q. ro > cr 3 O 3 SL o •s o 0) (A 3 co > CT 3 O 3 N CD O (0 3" 0) TJ CD V ? 0^ Total # Fibres: Category subtotals: ro o CO oo v l CD cn J> CO ro o CO oo v l cn cn CO ro Fields: 1 | Category 90.6 O) 00 J>. CO ro J>. Ol 03 ro CO Ol CO J>- cn CO ro CO ro CO CD J > CO CO Ol ro co ro J > ro CO 03 v j 03 CO ro ro oo z 03 CO Ol ro CO ro J>. CO CO 00 ro o o o ro O ro —\ Ol b Ol ro ro ro o CO 03 o v j CO ro CO O ro . Ol ro e_ ro 03 ro ro 4>- o 4 > J > I o _,, ro L o o ro O cn ro _,, CO 82.8 Ol O) o ro v j ro Ol 00 ro CO ro v j oo ro 03 J > CO o CO ro ro J > ro CD oo ro CO CO ro CD ro oo oo ro ro v j ro oo ro v j z j— Ol CO o> v j J > o ro ro ro 03 CO 00 ro o ro o v j ro ro J>- ro o CD -+» —+ O 11.2 o> v l OJ v j oo v j CD cn ro CO CO CO ro ro oo CO ro CD CO CO ro o co SL b ro v j o ro CO O o o ro _^ o ro o CO o ro t _L CO 88.2 v j CO o CO CD Ol J > o CO co CO co Ol cn CO Ol Ol CO oo ro CO CO 03 ro oo o -n. o 00 o CO co CO Ol CO ro z 3j Ol j> oo CD O) J > oo CO CO CO J>. J > o o o L CO co CO ro oo o ro CO J > to" 3" o O co CD O) b Ol oo —X CO CO CD o J > CO J > cn v j CO ro CO o ro ro ro 03 b CO Ol ro o ro o O CO ro ro o CO ro o co o CO ro Ol CO #### o z o o CO 0) ### Q o —J. 3 CO CD 3. 5' 3 ### o ro #### o CO 92.2 v j CO 00 Ol ro Ol CO cn Ol CO 00 cn J > Ol ro J > Ol ro Ol J > co •vl CO v j oo z 00 •vl v l ro O) ro ro ro ro o CO ro Ol ro o c J > CO _^ J > o J>. o _^ ro CO CO ro CO CO CO ro SL o b cn o o _^ o o —1. o L _^ o o o o o CO 95.1 L o cn Ol ro v j CO CD J>. cn cn J > 03 O CD oo Oi oo cn oo CD CO 03 CO CD J>. •vl cn J > CD Ol ro cn cn cn ro Ol CD Ol J>. z o b - cn ro o _^  o _^ ro o o O _^ o O o o o o o o _ j _ TJ CO o D) CO o b Ol CO o _,, o o o o o o o O o o O o o o o CO o o ro ro J>. O) v j o L ro Ol cn 4>- ro v j ro ro ro L ro CO ro ro CO = 1 rp C i f 3 a o r o g 3 CO 91 o H XJ o fD 3 fD 3 a. o 3 CD IQ 5" 3 o 3 CO 3 r - r CD 3 SL •< 3 C O CD fl) r * CD Q. ro > cr 3 O 3 SL o * o co (A 3 w > cr 3 O 3 SL CA N" CD O CA 3" 0) TJ CD ^0 Total # Fibres: Category subtotals: ro ro o co oo v l Oi Ol -fc- CO to o co oo v l Oi Ul CO to Fields: 1 85.9 J>. Ul v l ro oo ro ro CO CO o ro co ro CO 00 CO ro CO CO ro oo —k oo ro -fc. ro v j CO ro CO ro ro v l -fc. ro z 11.8 Ul CO Oi CO CO ro -fc- Oi -fc. -fc. co ro -fc. Ul o CO CO ro CO O H ro co ro ro o o o o o o o ro o o ro o o ro ro o ro XJ co v l _,, o o o _^ CO o _^ o o o o o o o o o o o co 91.6 ro CO CO ro ro ro ro o ro o v l ro ro Ul ro v l —k co CO CO Ul ro -fc. ro v l z r~ Oi b CO ro o ro CO -fc. o o Ul ro o o ro ro CD — r . r* o o CO r * Q> CO Oi o o o CO o _^  _^  o o o o o ro k CO Oi o o o o o o o ro o _^  _^ CO 86.9 -fc-co CO CO v l ro -fc. CO CO ro ro ro Ul ro o ro o ro v l ro o ro o CO ro v l v j ro oo CO o CO ro Ul ro o ro cn ro co z XJ CO ro Ul •vl -fc-v l ro 00 .fc. -fc. -fc. Ul ro ro ro o -fc. o ro CO -fc. CO o (Q 3" r* o o CO r+ SL -fc-CO .fc. ro oo o ro co . ro o _^ CO —k CO k o ^ ro ro CO ro o o ro ro CO ro o ro CO o Ul o ro ro o CO 96.8 co co -fc. cn CD Ul •t-Ul v l v j ro -fc. CO Ul -fc. CO Ul J> - j . Ul CO Ul ro cn •fc. J>. o -fc. ro .fc. oo •fc. Oi -fc. Oi -fc. cn -fc. oo •fc. ro z O o CO r -T 0) bo o oo o o o ro o ro ro o k o o o CO ro 3 CO CD 3. 5' Ul v l Ul o ro o _^ ro o ro o o o IO _± o o o o co o ro r 1 ro o o o o o o o ro o o CO o o o o CO 3 86.4 Ul o v l ro Ul ro oo ro ro IO ro ro ro CO CO -fc. ro ro v l CO CO ro Ul ro ro ro CO CO ro ro v l ro CO ro co ro Ul z Oi bo Ul 00 -fc-O ro ro IO ro CO ro CO CO CO o CO J>. k CO ro o c Oi bo v l •fc. o ro ro CO Ul ro o -fc- o Ul o ro ro CO o -fc. ro ro o ro SL ro b ro o o o o o ro . o . o o ro o o o ro CO 98.3 co 00 CO Ul v l .fc. v l Ul co Ul .fc. -fc. v j Ul co Ul co -fc. Oi -fc. -fc. -fc. o CO o -fc. Ul Ul CO Ul CO Ul v l Ul o -fc. Ul -fc. Ol •fc. o Ul ro z • . O o O o k o o o o CO o o o ro ro o o —k k _ j . TJ CO o 0) CO o j> <_) o o O o o o o o o o o o o o o o o k ro o ro ro o o o o o _^ o o o o o o o o o o o o CO = 1 Ifl o r* O CO o 3 92 o H TJ O CD 3 CD 3 Q . O 3 CD C Q 5" 3 3 0) 3 r - r CD 3 SL >< 3 £ O CD fl) #-r CD Q . ro > cr 3 O 3 SL o < r f o TJ_ 0) CA 3 CO > cr 3 O 3 SL CA N' CD o —i CA 3" fl) T J CD Total # Fibres: |Category subtotals: ro o CO 00 v l Oi Ul 4> CO ro o co oo v l o> Ul 4* CO ro Fields: 1 53.2 CO oo Ul ro o Ul L -fc. Ul Ul v l Oi Oi v l v l -fc. 00 •fc. ro O ro v j 00 cn CO CO z CTR 42.3 Oi CO -fc. ro v l oo v l Oi oo Ul CO oo 00 —X o cn v l V l v l CD -fc. cn CTR -r--t- v j o o o ro _^  o o L ro ro o co o L L L o ro CTR b o o o o o o O o o o o o o o ro o o o ro o CO CTR 61.6 J > v l CD ro CD Ul ro Ul Oi -fc. cn CO oo -fc. co v l cn Ul co oo oo o oo Ul Ul ro co z Left Costal 17.1 oo ro CO CO Ul cn Ul oo •fc. Ul Ul -fc- -fc. Oi Ul cn ro v l Left Costal 21.3 o ro CO ro o cn CO ro CO oo co CO v l o Oi Oi co Ul cn ro ro Left Costal o io _,. o o o o o o o o o o o o o o o _L o o o o CO Left Costal 54.0 CD CO CO CO CO v l - J L ro ro oo v l oo ro ro Ul ro cn CO co -fc. ro CO ro v l Ol -fc. z Right Costal 12.9 v l co ro CO -fc. ro Ul ro -fc- ro -fc. oo cn Ul ro -fc. ro CO v l cn Ul CO _x Right Costal 33.1 ro o CO CO —X CO ro oo oo -fc- v l CO CO o v l Ul -fc. ro ro ro o oo Ul oo TO Right Costal CO bo ro CO o o ^ o CO CO o ro o o o ro CO o ro CO CO Right Costal 60.9 -fc. CO CO CO o o ro ro ro ro oo -fc. CO co o v l ro v l cn o 00 CO Ul z Costal Insertion 18.7 co ro -fc. -fc. ro Ul ro Ul -fc. -fc- v l o> CO cn v l cn CO -fc. Ul ro ro Costal Insertion 20.5 o CO oo o v l Ul CO -fc- CO ro Ul cn oo Ul ro cn -fc. ro L L ro Ul ro Costal Insertion 11.6 Ul v l CO oo ro o ro ro -fc. ro -fc. Ul o CO o Ul Ul ro Ul ro ro CO Costal Insertion 58.6 Ul co o CO -fc. o ro o ro ro ro ro —X o oo cn -fc- oo CO oo -fc. ro ro o ro -fc. ro ro co o z Crural 15.9 CD ro Ul CO CO Oi co ro CO oo Oi Ul oo CO co -fc. cn CO oo •fc. CO cn Crural 25.5 -fc. oo co ro Oi CO CD •fc. v l co CD 00 Ul v l cn cn o Oi -fc. CO -fc. ro Crural Ul CO CO o o _^ o o o ro ro -fc- -fc. CO -fc. CO o CO . o ro CO Crural 72.0 v l Oi -fc. Ul Ul o ro v l ro co ro co CO Ul 00 ro CO CD ro v l ro -fc- ro co ro Ul ro ro ro Ul CO ro -fc. ro -fc. ro v l ro CO ro co CO CO z Psoas 22.4 v l Oi o Ul v l co 00 CO cn Oi v l oo oo cn Oi o CO cn ro ro Psoas v i CO o o o o o o O o o o v l _,, o o ro o ro o o ro Psoas CO CD CO o ro _,. o ro _,. o _ i -fc. _^  ro o -fc. CO ro ro -fc. o CO ro co Psoas CD >< O _ r* O ^ o 3 r + CA 93 o H TJ O fD 3 CD 3 a o 3 CD CQ 5" 3 O 3 fl) 3 CD 3 SB <• 3 C O CD a> CD a. ro > cr 3 o 3 w o o TJ fl) 0) 3 CO > ET 3 O 3 a) N CD O Vt ZT fi) TJ CD V ? Total # Fibres: Category subtotals: ro o CO oo •vl CD cn CO ro o co oo Vl CD cn J> CO ro Fields: 1 73.7 00 o CD U l co J> o J> co CO CD o ro co J> U l ro o k oo ro CD ro J> ro CO ro CO CO U l J> o U l CO U l U l CO z CTR 24.3 co CD 00 ro ro ro v i ro U l oo v j ro CO CD ro CO v j J> CO v j U l CTR ro b CD co o O o o CO CO o ro ro o o o o o o ro CTR o b o o o o o o o o _ L o o o o o o _ l . o CO CTR 86.4 oo CD V j J> •vl CO oo J> ro CO co oo co o J> U l 4>- CO CD CO J> CO v j ro CO CO CO CO U l ro oo CO co ro co CO U l z Left Costal 12.7 L o co oo U l U l CD J> U l v j ro v j v j ro ro CD CD v j co Left Costal o b 00 o o o o o O ro _L o o o o _,. o o _,, o ro Left Costal —k v j U l o o _,. o U l _,, o _,, o o o o o o o CO ro o CO Left Costal 83.1 1074 oo co CO cn CO ro U l ro U l CD U l v j U l v j J> ro U l CO U l ro J> co CO U l ro •vl o CO co ro U l ro cn CD CD U l co v j z Right Costal 15.6 1074 CD oo U l CD U l oo o CO k oo oo CO 00 v j —J. o oo Right Costal ro 1074 CO o o o o ro o o _l o o CO o o o L ro —. ro Right Costal b 1074 ro o o o o ro o . o _ ^ _ ^ o CD o o ro o CO CO Right Costal z Costal Insertion Costal Insertion ro Costal Insertion CO Costal Insertion 86.9 co CD 00 CO U l U l CO -Pv o CO CO v l •t> CO U l CD U l o CD co U l ro CO J> CO CO CO CD cn o U l CD oo CO v j z Crural 11.7 ro o 00 ro U l U l U l 00 U l IO CD 00 U l 00 J> CD •vl v j CO CD •vl Crural b o o o ro O o ro O _^ o _^ o _± _^ ro Crural o v j v j o o o ro o o o O o o o o o o o _^ k CO Crural 96.6 1035 1000 CO ro U l CD J>- CO CD ro v j U l CD CO v j CD CD •vl CO J> J> U l U l oo U l CO U l CD J> v j CD J> U l CD CD •vl z Psoas ro v j 1035 ro oo o _^ 00 CO CO ro CO ro o o o ro o —k J> O o Psoas p 1035 _± o o o o o o o o o o o o o o o o O o ro Psoas o b 1035 CD . o o ro ro o o o o o o o o o O o CO Psoas CD >< O r* O - i o UI c 3 94 o —i TJ O fD 3 r - r ffl CD 3 a o 3 CD CQ 5' 3 Z o 3 0) 3 fD 3 ffl >< 3 C O (D ffl r f (D Q . ro > cr 3 O 3 o < i—r o TJ_ ffl (0 3 co > cr 3 O 3 u (0 N" fD O co 3 " ffl TJ (D ^ 0 Total # Fibres: Category subtotals: ro o CO oo v l o> cn CO ro o co 0 0 v l OT ui jfc. CO ro Fields: 1 57.7 ro -fc. OT J > ro k OT OT vl oo CO •fc. oo oo OT vl ro o 00 OT ro z O H XJ 33.7 oo co -fc- ro CO vl CO vl co 00 vl ro O -fc. -fc- cn oo cn ro o CO ro O o O ro ro — J L o -fc- co O ro ro 15.9 CO co CO — J L -fc. ro vl cn CO CO CO o CO CO z 1 -CD - r t r * o o CO l-T ffl ro CO 84.7 CO cn ro ro CO 00 OT OT vl OT oo cn ro o CO o ro ± -fc. co ro o cn co -fc- ro o co z XJ to' 3 " r f o o CO r f ffl CO CO CO O OT CO CO o -fc. ro o ro o CO ro o ro o ro OT b ro -fc- o o o o ro o ro o ro ro ro ro o o ro OT co ro -fc. O o o O L -fc. ro . ro ro ro -fc. o ro o o CO 91.4 -fc-oo oo •r--fc. OT CO co ro CO ro ro -fc. ro o ro vl ro cn CO OT CO OT ro o ro ro ro vl ro ro —L oo ro vj ro ro co z o o CO r - f ffl 3 CO CD 3 5' 3 cn b ro OT o o ro •fc. IO o ro ro o o ro CO CO o co 'co OT o o o o co ro o o ro ro ro o o o o ro b cn o o o o o o o o o o o o o ro o o o CO 92.9 OT CO ro cn 00 vi CO o ro CO ro vl ro -fc- CO -fc. -fc-ro CO vl CO cn ro o ro ro ro co ro -fc. ro -fc. L OT CO ro ro oo ro vj CO -fc. CO -fc. -fc. cn z o c tt CO co ro -fc. o o ro co CO o CO CO ro ro L o o o o CO b ro . . o o . ro —L ro CO ro o ro ro o o o ro ro CO o o o cn ro ro o o o o o o o o o o _^ CO 89.8 oo oo o vl co o cn CO cn OT -fc. CO CO J > ro ro CO ro co ro OT vl -fc. .fc. CO OT CO -fc. CO cn CO cn cn CO ro -fc--fc-•fc. cn -fc. CO co ro z TJ (0 O ffl CO OT b OT o OT vl cn J > . CO OT OT -fc. vl cn cn CO •fc. o •fc. ro OT b _L o o ro o O — J L o ro o o _^ o o ro ro ro io CO CO ro ro o CO o ro ro _^ o _^ o ro CO = 1 i f l 0 _ -* o 0 1 o 3 r f CO 95 o H TJ O CD 3 CD 3 Q . O 3 CD CQ 5' 3 3 co 3 CD 3 SL •<" 3 C o CD 0) CD Q . J O > cr 3 O 3 SL o •s o TJ fi) CA 3 co > D" 3 O 3 SL CA N' (D CO 3" fi) TJ CD O I-* SL * T l —i CD CO oo ro CD co v i • CO v j v J l v j oo CD bo v j ro oo 4 > CD CD b CD b oo co j> CD b co oo v j jo cn b v j b Q> CO c er| p+ o SL CO co CD 4 > CO i J>. CD cn •vl oo ro CD ro on co cn CD ro co oo co co ro ro v j co o co Ol oo O) O) CO co cn CD 2! I CD Q-l CO ro o CO ro ro co-co oo I ro Ol Ol oil CO CO cn Ol co oo oi ro ro cn ro ro CD I CO CO co co I CD I CO CO ro CD to CD CD Ol co O) CD Oi CO O. Ol CD CO CO CD CO CD ro o ro ro ro ro cn O) I CO oo O) cn oil CO CO CO CO CO Ol cn ro CO to ro cn CO ro co Q co ro irol co cn oo CO CD CO Ol cn ro co co ro I ro co I col CO ro co ro o co ro o ro to ro ro ro ro ro ro ro co ro ro ro co co to ro 4 > ro co CD ro co ro ro cn cn to to CO ro cn to ro to ro ro to ro CQ 3" L ~* - o o CO ro co CO 00 to ro ro CO co ro co I oil Ol CD Ol ro O o co !-»• fi) ro co ro co co CO CO CO ro CO CO ro co ro co ro o co CD co cn cn CD CD CO ro ro I col I to I "I Ifl o -* o CD O 3 i—F CO CO 96 o H TJ O CD 3 CD 3 a o 3 CD CO 5' 3 o 3 r~¥ CD 3 SL <" 3 C o_ CD CD i - r CD a ro > cr 3 O —i 3 SL o «< i-fr O •D fl) CO 3 w > cr 3 O 3 cu N CD CO 3" fl) TJ CD Total # Fibres: Category subtotals: ro o CO oo v l CD Ul CO ro o co oo v l CD Ul CO ro Fields: 1 81.6 cn CO o v l CO ro CO ro CO _!. ro cn CO cn CO cn ro CD ro o ro ro ro CO ro ro - J k Ji ro o — L oo — L oo ro CD — k CO ro CD ro CO z O —t JJ 15.3 co CO Ji v l _4k co CO CO _J. CD ro ro CO Ji v l J> CD CD CO CO CO — k co o . o . ro o o ro o ro o CO O t ro o IO ro CD v l o ro o o o ro o ro CO ro o o O ro o o CO z r-CD — p . f - p O O CO SL ro CO 91.5 CD v l CO CD CD co cn CO k co v l CO CO cn CO ro CD ro co CO Ul oo ro CO co v l CO ro ro v l ji co CO CO ro CD z TJ co' 3" r + o o CO CD cn CO CO O) CO o CO o CO CO co CO ro ro ro o o Ul ro — J . CO ro ro o CO ro ro k k k o o o CO o o ro o ro o ro ro v i oo o o CO o o ro ro _^ o o co o ro o o o _^ ro CO 92.9 cn CO CO cn o ro co ro cn ro CD ro v l ro ro ro oo ro ro ro ro cn ro o CO ro ro ro CD ro co CO ro CD v l ro ji CO o z o O CO 1 - P 0) 3 CO CD -1 5' 3 cn ro J> o CO o o o ro o ro cn ro ro o ro CD J> ro ro o o o o o o o o ro CD o o o o o o o o o O o o o o ro o CO 91.2 v l Ji CD cn ji CO ji J> J> cn CO CD ro oo CO J>. ro cn CO CO ro ro cn CO CD ro cn ro cn CO ro CO v l ro Ul ro CO CO CO CO z o c SL CD CO Ji cn ro ro CO o CO ro CO cn o CO o J> CO ->>. CD . ro cn oo o o o o o O o o J> CO -r- o o ro CO k ro ro o o o o o o o o o o o ro ro ro ro o CO z TJ CO o fl) CO ro CO CD «< -* o v l O 3 .—p CO 97 o H TJ O CD 3 CD 3 a o 3 —i CD CQ 5' 3 3 01 3 r* CD 3 SL •< 3 C O CD 03 p+ CD Q. ro > cr 3 o 3 SL o •< r-f o •o 0) CO 3 co > cr 3 o 3 SL co N' CD o CO 3" 0) TJ CD 0^ Total # Fibres: Category subtotals: ro o CO oo v l o> cn j i CO ro o co oo v l O) Ol J i CO ro Fields: 1 86.8 Ul Ul -r-v l co ro CO CO J i CO ro ro —L Oi Oi ro j i ro j i ro Ul CO ro CO ro j i ro ro CO —L oo ro o ro o CO ro j i z O , | 10.7 Ul Ul ro CO L ro CO o -ti ro ro j i CO CO •vl ro o CO CO ro oo ro Ul CO o o o o o o o ro ro ro o o o o ro ro TJ CO CO v l o o ro —L ro O o o o o CO CO 89.5 CO IO co CD -N cn ro CO -t-oo cn -ti Ul -ti o j i CO Ul j i j i J i CO j - . ro j i v l Ul Oi j i oo ro CD ro CD j i j i Ul CO v l co o z r-CD — r . »-r o o CO r * SL CO b ro v l CO ^ o Ul o o o o ro co ro ro ro v i b CD CO CO -T-. vj oo -ti CO Oi j i CO CO CO j>. j i CD ro ro Ul io J > v l co co oo ro o O j i ro v l o o o o o ro CD CO 92.3 oo Ul v l Ul ro CO CO J > . Oi ro oo co v l J i ro Ul o CO co o j i cn j i o ro CO co j i CO CD CO O) CO Ul j i o J i CO j i o co v l co J i z TJ CQ* 3" o O CO r r a b CO Ul o IO o ro ro CO o o o ro CO J i o ro Ul Oi CO ro oo ro o co o CO o o o ro o J i ro j i o ro ro ro Ul b 00 o o o ro ro ro Oi o ro ro ro oo cn o CO o CO 92.3 CD CO oo Ul oo CD Ul J i J i CO vj CO CO oo ro CD CO ro oo ro oo CO CO ro j i j i j i ro CO CO ro ro CO ro CD ro Oi CO o ro CO CO z O o CO r * 0) 5" CO CD a. o" 3 Ul b CO ro v l o ro o ro ro o ro o ^ j i CO o o J i ro v l v l CO o o o CO o o o o o o CO ro ro fo J > . o o CO ro ro o o o o o o CO o o o o CO 94.7 v l o o CD Oi CO CO ro v l cn ro oo v l co ro CO co CD CO Ol ro co ro j i CO CD CO J i J i v l J i J i CO Ul CO ro CO o ro v l CO CD z o c SL CO b ro CO ro O ro ro o o ro o O ro b Oi o o O o ro o ro O o ro o J i o o ro ro b 00 ro L o o o o o O CO o o o ro CO 96.1 oo CO ro v l Ol o o ro vj oo co co o v l CD co CO v l Ul Oi Ol CD ro o CO cn oo co CO CO CO — J L CD ro vj vj Oi v l CO CO CD ro oo Ol z TJ CO O 0) CO p o o o O o O o o o o o o o o O o o CO _& b co o ro _,, ro O CO _,, o o o cn o ro CO o O ro ro ro b Ul ro ro CO ro -ti ro o o J i co J i J i J > . ro o J i ro CO 2 2. cr c rcD«2 a o 00 O 3 98 o H 30 O CD 3 i^-tt CD 3 Q. O 3 T CD ta o 3 3 m 3 CD 3 tt «<" 3 C O CD Q> —+ CD Q. ro > 3 O 3 » o >< o TJ_ ffl CO 3 CO > CT 3 O 3 N CD O CO 3" u TJ CD V ? Total # Fibres: Category subtotals: ro o CO oo CD Ol CO ro _ 1 o CO oo -j CD Ol J> CO ro Fields: 1 71.6 CD CO J> CO ro ro CO ro o CO CD ro o ro co ro CO ro 00 CO ro ro ro oo ro O l cn CO ro I oo ro O l J > z O 21.0 CO o U l CO CO J> CD o U l ro co co •n v j co CO J> CD —L. CD oo ro CD CT) L. o L ro ro U l o o ro 30 12.0 v j J > -~^  ro ro O l ro ro CD CO O l CO ro CO ro ro CD . CO 91.8 v j ro CO CD CD ro J>-J> CO O l ro oo ro ro IO co O l CO J>-O l o CO CD ro CT) o J> ro co ro O l ro ro CO CD ro 00 z Left Costal ro bo ro o CO ro ro o o O ro o o o ro o _ k ro Left Costal CO CD ro ro -n ro CO o CO CT) J> ro ro ro Left Costal o U l o o o o o _,. o _,, O o o o o o _,, o o o CO Left Costal 89.0 co CD J > . ro CO CO ro ro •vl ro CD co J> ro J> oo CO •vl v j ro o CO o ro CD ro ro ro ro CO ro oo ro o z Right Costal bo CO co ro o ro O l ro o ro o ro CO CO CO CO CO o o Right Costal CO ro CD ro o o CO ro o o o ro . o o . o . ro Right Costal 23.0 U l v j O l O l o oo CO v j o CO CO ro ro CD J>- CD co CO CO Right Costal 89.8 J > -CO CO CD 00 ro CO ro CO ro CO ro CO ro ro ro cn ro CO ro o CO o CO oo ro o CD ro L CD —L CD CO - J L CO z Costal Insertion v j ro CO ro o CO ro CO ro ro CO CO CO O CO o — J . Costal Insertion ro CO CO o o o o - J L L o ro o o L o O ro O o o ro Costal Insertion 11.5 U l CO CO CD CO CO ro o O l CD o CO o L CO Costal Insertion 84.5 v j co CD CD •vl CO ro O l CO CO CO U l ro co CO CO CO CD ro CO ro v j CO o ro v l CO CO ro v j ro ro J>-CD ro CD CD v j CD J> CO CD ro U l z Crural 10.3 00 ro ro CO v j U l ro CD U l J>. ro CO O ) ro U l o 00 O l oo Crural U l fo ro CD o o o o CD CO o ro O l J > co o ro J > ro Crural 11.3 CD o ro co U l J>- o CO o 00 ro CO o co ro o J> CO CO CO Crural 94.8 CD ro CO U l CD CD CO CD co CD CO 00 ro - v l CO 00 CO CD CO CD ro CD ro 00 CO CO CO CD CD ro cn ro o ro CO ro 00 ro J> ro o v j z Psoas b ro U l o o ro L o ro ro ro o ro ro ro J > o CO o Psoas o b CO o L o o o o o o o o o o o o o o o _,. _^  ro Psoas o b U l o o o ro o o _,. o o o o o ro _,. o o o o o CO Psoas CD >< O _ -* o ID O 3 99 o H XJ o CD 3 p* SL r - f (0 3 a o 3 CD CQ 5" 3 O 0) 3 r - f CD 3 SL 3 C O CD a r f CD Q. ro > cr 3 O -r 3 SL o O TJ_ CD CA 3 CO > cr 3 o ^ 3 SL to u CD O -t CO 3" a> TJ CD ^0 Total # Fibres: Category subtotals: ro o CO oo v l cn cn Ji CO ro — J . o CO oo v l O) oi j i CO ro Fields: 1 70.2 CO CO CO ro 00 o ro CO CO ro v l CO O) -ti cn CO oo J i oo o co 0) ro o cn co v l o o z O H XJ 25.6 o ro CO CO co CO o co ro v l Ol cn Ol co O) v l cn o v l Ol O) J > CO v l o o ro o ro . o o o ro o o ro ro ro b cn o o ro o o o o ro ro ro t o o CO 79.0 CO J > ro co CJ) ro o ro cn J > . CO ro ro co j i CO Ol v l CO z I -CD - 4 t r f o o CO r f SL 14.0 J > ro ro -ti v j -ti ro o o CO o CO CO ro j i CO v i b ro ro ro o — L o o o -Pi o ro o - J L cn ro o O o ro b CO o o o o _,, o o _^ o o o o o o o _L o o O o CO 81.3 CO no o CO o co CO v l v l CO cn oo oo ro -ti oo ro cn cn co o cn Ol ro o Ol v l CO z XJ CQ' 3" r— o o CO r— 0) 12.1 j i O) ro CO co ro co -ti j i o o co ro j i J i O) CD b ro Ol o ro ro o ro CO o o ro ro o ro CO ro CO io ro ro . o o o o — L o o o o CO o - J L o o — L CO 88.6 J > . ro ro CO v l J > . — J L CJ) L J i CJ) CO ro o ro ro v l CO ro o ro ro o ro Gi- 00 00 Ol ro ro o CO ro CO ro cn Z O O CO i - T ffi 3 co CD 5" 3 v l b CO CO cn -ti o ro CO ro ro o j i ro ro o o o ro CO b Ol o o o o o o o o o o co J i ro o io - J L o o o o o o o o o o o o o o o o o o o CO 85.2 j i co — L j -o ro o ro cn IO CO o ro o -ti ro o CO - J L V l ro cn ro v l ro J i - J L cn ro j i ro CO ro ro Ji. L cn ro _ J . z o c SL 10.0 -ti oo ro co CO ro ro CO CO co CO J > ro o J i CO CO b ro CO o ro - J L — L -ti o ro L _ J L o J i o ro o o ro o b o o o o o o o o o o o ro ro o o o o o o o CO 97.9 ro J> ro ro 0) v l O) •ti J i CO ro -ti CO CO v l CO v l •vl cn Ol v l CD 00 J i J i Ol j i cn Ol CO O) CO Ol CO v l J i O) v l cm j i cn •vl 0) 00 z TJ (A O ffi CO o b o o CO o o o o ro o o o ro o o o o o ho CO o o o o o o o o o o o o o ro o o o o o ro b CO o - J L o ro o Ol o o o o ro o _,, o o o CO cr Q a o _L O o c 3 100 o H TJ O CD 3 CD 3 a. o ZJ -1 CD CQ 5" 3 o 3 ca 3 CD 3 fi) 3 C o CD fi) i—* CD Q . ro > ZT 3 O -r 3 SL o * o TJ fi) CO 3 CO > CT 3 O 3 SL co N' CD (0 3" 0) TJ CD Total # Fibres: Category subtotals: ro o CO oo v | OJ Ol J> CO ro o co oo v l O) cn *- CO ro Fields: 1 89.2 v j O) CJ) -J. CO CD CO CO CO OJ ro ro co CO v j CO oo CO co J> J> CO J> ro o ro oo ro J> CO OJ CO co CO L CO -d J>-o CO v j z CTR 10.7 v j CO CJJ O l o O J CO CO cn o O l co o ro CTR o L o o o o o o o o o o o o o o o o _ o o o ro CTR ro j> 00 CO o o IO o o J>- _,, o o ro o — L o o CO CTR 95.4 OJ co O) cn co ro CD ro co co ro CO ro ro oo CO CO CO O l O l ro CO J>-ro ro co ro CO CO o CO v j CO co o ro co CO z Left Costal 2.89 ro o CO o ro o o o o o ro ro ro ml Left Costal L ro o o o ro o o o o . o o ro Left Costal o CO ro o o o o o —1. o o o o o o o o o o o o o CO Left Costal 93.1 CO ro CD v i v j ro ro CD 00 O l ro O l oo o CO ro 4> O l ro oo 4 > CO CO CO CO CO k CO OJ o o CO v j J>-v j J>- ro v j z Right Costal cn v j •vl _± CO ro 00 _,, ro o CO _,. CD CO o _i OJ _± ro CO Right Costal Ks o o ro o o o o o o o o o o ro o . to Right Costal CO CO CO ro o ro CO o o o CO ro ro o ro v j o oo CO CO Right Costal 94.7 J> no v j -d OJ CO CO ro ro v j ro O l co ro OJ CO o ro oo CO o ro v j CO ro ro O J ro CO ro ro ro J> ro ro v j ro co z Costal Insertion CO v j co ro o _L o t o o ro o _^  ro ro co o Costal Insertion CD CO o o o o o o o ro ro o o _L o o o ro Costal Insertion O j>- ro o o o o o o o o o o o o o o o o CO Costal Insertion 95.6 cn co cn OJ co CO ro CO OJ 00 CO CO OJ CO CO 00 O) CO CO ro v j ro cn ro ro l \ J o ro ro v j ro O l ro J>. CO J> ro ro ro CO ro oo ro oo z Crural CO fo CD o o ro ro ro o ro o o ro o _ j . _ k Crural fo v j o o _i o o o ro o o o o ro o o o o o o ro Crural o cn CO o o o o o o o o o _ ^ o o o o o o o o CO Crural 97.9 v j co oo •vl 00 J> o O l CO v j -d oo v j CO ro cn 4>- O l o ro J>. o -d ro CO cn CO v j J> o CO co oo CO CO oo CO ro CO o z Psoas o cn o o . o o o o o . o o o o o o Psoas b 00 o o — L . o o ro k o o o o o o o ro Psoas 0.63 O l o o o o o o o o o o o o ro ro o o o o o CO Psoas s S. CD •< O _ ** o - i o - i c 3 »-*• CO 101 o H 3J O CD 3 a 3 a. o 3 CO CQ 5" 3 Z o 3 CO 3 r* CD 3 CO •<" 3 C o CD fl) r f CD Q . IO > CT 3 O —\ 3 SL o < o T J fl) CO 3 w > cr 3 O 3 co N CD CO 3" CO TJ CD 0^ Total # Fibres: Category subtotals: ro o CO oo v l cn cn ji CO ro o CO oo v l O) cn ji CO ro Fields: 1 74.4 CO cn v l ro CO CO ro 00 - cn ro CO ro o ro —j. k ro v l 00 ro j i CO cn ro v j oo z O 23.5 j i 03 O CO v l v l ro v l CO j i v j cn v l CO co J i CO cn v l CO CO cn V l v l ro k o o o O ro ro O CO o o o o O o o o o o ro XJ b> cn o O O o O o o o k o o o o o o o CO 89.4 cn cn co cn o o ro oo co Ji. CO CO v l ro CD CO co ro ro ro CO o ro 00 ro CO CO o ro co ro cn co ro ro ro cn cn ro cn z Left Costal 10.2 cn v l cn ro cn cn ro CO oo ro o CO cn cn j i ro Left Costal o ro o o o o o o o o o o o o o k o o o o o ro Left Costal ro b j i o ro o j i to o k o o o o o o o o CO Left Costal 86.2 cn rn cn cn v l j>. CO CO ro ro CO ro ro cn oo CO J i CO ro co ro v l co v l CO J i ro ro j i ro cn co cn co o co v l co cn ro v l cn z Right Costal co b cn o co j i ro ro ro o oo ro ^ cn cn CO cn j i cn j i Right Costal co CO ro ro ro o o ro CO co ro CO o co j i ro Right Costal Ol co CO cn . CO o o —k k v j o ro k ro o ro oo o CO CO Right Costal 83.6 oo o CO cn v l CO CO CO ro CO CO CO 00 CO cn CO ro 00 j i ro j i o CO CO CO o co CO j i 00 CO J* CO CO k co o J i CO CO z Costal Insertion 14.4 cn CO o j i J i j i oo CO cn cn cn CO CO oo co co cn ro oo Costal Insertion ro b cn L o o o o o o o J i ro o o o j i o ro Costal Insertion —A. co o o o o o ro k o o o o o o o CO Costal Insertion 91.1 CO cn cn oo oo o CO 00 J i cn j i cn cn ro cn CO cn j i o cn o j i CO j i ro J i v l CO CO CO cn 00 cn CO j i 00 j i j i j i cn z Crural v i io v l o J i cn CO j i CO CO ro CO ro CO CO CO cn v l ro cn v l CO Crural V 4 cn o o o co o o ro o o o ro O ro ro Crural CO b CO j i —k. o cn CO o cn o j - . CO o . o j i o CO o O CO CO Crural 84.5 cn v | CO cn j i ro CO o ro v l ro CO ro co ro CO CO CD ro j i CO 00 CO cn CO cn ro cn CO o ro ro cn ro 00 ro v l CO 00 z Psoas 11.8 cn v l cn oo o v l j i v l k CO 00 oo j i ro CO ro o oo ji. CO CO j i cn _Jk Psoas o v j cn o j i o o o o o o _^ o o o o o _^  o ro Psoas co b ro o ro o o CO ro o o o o o _,. o _ i o _,, o o CO Psoas = 1 S -i CD < a o - i o ro c 3 r f (0 102 o H TJ O CD 3 CD 3 a o 3 —i CD CQ 5' 3 3 ffl 3 r 4 > CD 3 0) 3 C O CD ffl r-r CD Q. ro > cr 3 O 3 po o o ffl CO 3 w > cr 3 O 3 CO N' CD CO 3" ffl TJ CD 0^ Total # Fibres: | Category subtotals: ro o CO oo v l o> cn ji CO ro o CO oo V l Ol cn ji CO ro Fields: 1 89.1 v l oo 00 cn J i J i 00 j i 00 J i v l ro CO ro co ro cn CO cn o j i CO co co ro j i O J CO J i ro v l j i oo j i CO cn CO — L J i CO CO Z O C J b oo O J ro cn v l ro J i J i J i j i ro cn CO CO j i O j - . J i . V l ro co co —i - J L ro . ro cn o o - J L ro o CO ro j i ro . ro J i CO ro TJ CO b ro oo ro o o o ro ro j i o o CO co o ro o ro ro CO 91.2 cn CO ro ro 00 CO cn j i o J i o CO cn ro co CO cn CO O J CO ro CO CO o ro v l O ) CO co cn ro ro co CO 00 ro oo z 1-CD —•» o o CO f - F SL CD b o> - t i j i j i . ro <y> o ro ro o CO j i . CO ro Ol o ro ro oo - J . ro b oo —1. CO o o o ro o CO o o o o ro o o ro v l o o o o o o o o o ro IO o _,. _L o o o o CO 87.9 co O J 00 co ro j i cn co 00 CO CO CO j i cn o j i v l j i j i oo CO O J j i . ro cn CO oo 00 v l CO oo cn v l cn ro cn v l j i CO co ro ro o z TJ IQ 3J o o (0 F T ffl -P-v i j i j i ro ro co j i ro O ro —1 o j i O J co o ro o CO v i j i v l o co co ro j i j i ro j i CO ro - J L cn ro ro j i CO co o cn ro 4^  co o o o ro ro o o ro L o o o o . o CO 94.1 co cn CD CO o ro j i CO cn ro cn CO cn CO co co 00 v l CO CO j i v l j i oo J i o CO oo j i . CO cn J i j i v l CO co j i CO v j j i j i CO oo CO O J z o o CO F r ffl 3 CO CD 5' 3 cn b cn CO CO ro ro v l CO CO IO cn O J co ro co CO o o o b OO o o o o o o CO O o o o o o o ro o o o o ro ro ro ro ro ro ro o o ro o ro j i o o o o CO 90.5 oo v l v l v l co j i j i . j i CO O ) CO oo ro v l cn J i J i ro CO oo CO j>. J i co J i co cn oo co o co ro v l CO oo ro o CO J i oo o j i 00 Ji. v l z o c ffl Ol j i cn . ro ro ro CO cn ro ro ro ro . j i cn . cn ro b CO 00 j i o CO o 00 CO ro ro j i IO ro ro ro ro j> ro CO o o o o o ro o ro ro _^ o o o o _^ _ ^ CO 88.9 oo oo CO v j 00 cn CO 00 ro co j i o CO o CO j i o cn o Ji. o co j i . CO o j i o CO ro CO j i O J J i ro j>. v l CO v l CO cn j i CO v l cn z TJ CO o ffl CO 00 v l cn co o J i J i o j i cn oo v l ro J i ro j i J i CO v l 00 ro ro b ro oo o o o ro ro ro cn ro CO _,, _,, o _,. o CO 00 o ro b CO o o ro o o _,. L o _,. o ro o - J L CO - J L ro o o o CO c°-5. r ^ J CD •< F * O - i o CO c 3 CO 103 o H TJ O CD 3 —+ —i SL n 3 a o 3 a> CQ 5" 3 O cu 3 CD 3 SL •<• 3 C O CD 0) #-»• CD Q> t o > CT 3 O 3 SL o < o T J 0) CO 3 co > CT 3 O 3 SL co N" CD O (0 3 " 0) T J CD V ? 0^ Total # Fibres: Category subtotals: t o o co 03 O) cn J> CO ro _ i o CO oo vj CD O l J d CO r o Fields: 1 92.5 —L r o cn o cn o cn o O l co J d co CD cn • d •vl cn O l O l r o O l co O) •vl O l CD cn cn J > CD o J d CD CD O t o oo cn 03 j d CO O) z O H CD CD CD r o r o J>- o J d v j CD 03 o v l r o CO co j d CD CO co —L. cn o O o O CO o o O r o TJ r o b r o CO CO _^ o o o O O cn O . O o oo o O CO 92.2 v j CO v j CO co J d CO CO oo - d J d CO co oo j d o co oo d -03 CO •vl J d v j CO cn J d o CO CO t o co J d CO r o co j d o r o co CO o z J>-b CO co o r o o r o r o o o O J d o CO CO o CD CD 1 CD — . - » • O O (0 — * 0) b r o v j o j d o o r o o 03 O CO r o o CO CO t o O r o r o f o v j CO o o o o o CO O _,, O o t o o CO o _^ CO 87.0 v j co CO cn CO O l CO - d o CO CO J d CO CO CO O l r o v j CO J d r o CO CO 00 CO r o CO ^1 O l CO CO 00 CO o t o v j co r o z TJ CQ 3 -i - r o o CO o f v j oo IY> CD CD r o r o o CO r o cn cn r o CD r o cn cn CD r o J d oo J d j d _^ O l f o J> CO J>- r o r o o r o CO o o CO J> r o CO j d r o o r o j d J d r o b 03 t o _^ _^ o J>- _^ o r o o r o o o o o o o o o CO 88.0 oo 03 co CO CO CO v j t o co - d CD CO co - d r o O l CO O l o j d J>-O l r o J d v j - d CD co J I co cn j d oo cn CD CO j d j d cn J d J d r o oo z O o CO 3" 3 CO CD ? , 5' 3 10.3 CD cn CO CD oo CD O l - d O l oo o O) O l CO J>- O) j d oo O l r o CD O) J d j d v j CD O o o 00 CO o r o o o o o L r o o o r o o b 03 O o o o o t o o o o o o o o O o o CO 91.6 CD o cn oo t o CD J d o O l v j O l t o O l o J d v j oo co r o r o r o j * . CO r o J d O l J d v j t o o CO oo j d O) r o CD O l cn CO O l r o v j CO cn z o c SL cn b J d O l r o r o CO O r o r o 00 ro O l cn o r o r o 03 o CO ml CO j d CO cn O) r o t o t o r o o o r o o 03 o o r o o b CO o o o o r o o o o o o o o o o o o O o o CO 92.6 00 CD CD oo CO o CO o J d t o O l o J d 03 O l r o j -r o CO 03 CD CO cn CO r o CO - d CD CO oo cn CO O l oo cn cn cn CO —L r o oo CO v j z T J CO o 0) CO r o b oo t o CO r o L o O o o 03 - d o o o o r o j d CO f o L o r o o o o _^ o j d o r o o o o o o r o - d CO v j o o o CO o _,. r o r o r o CO CD CO j d 03 CO t o CO CD v3 o ^ -* o ti 3 F * CO 104 o -4 =0 3 I sr i i o 3" 3 3 sr •t 3 At 3 C O 8 JO > cr •1 at I 3 • • > or SL (0 N* <o o - 1 «o 3* at •o as 1 * 1 • • Category subtotals: 8 CO 0 0 cn * CO to a * o CO oo -4 at 4d CO to "Tl a s » •a 73.71 24.31 2.01 0.51 • fe fe CO CO fe j*. ro CO 4* Ol o —Ik 00 ro ca ro CO oi fe - X Ol 8 - J k on CO —x 2 3 73.71 24.31 2.01 0.51 oo fo - J i to ro _x. -4 ro cn CO -4 to « J k CO CO -4 CO -vl on - A 73.71 24.31 2.01 0.51 o> CO o —x O o O CO CO o ro ro o o O o - J k O o ro #0 73.71 24.31 2.01 0.51 © o o ~x. O o o o o o o o o — X © o - J X O CO 86.4112.71 0.91 171 - J CO CO 4fc 00 4> CO JV O *> J d t w CO K ro — X CO - J X CO co 8 8 CO <o ro CO CO Ol Z r -86.4112.71 0.91 171 — X o CO 00 cn Ol - A . J> cn J - ~v| ro - X —X -vl "-J ro ro CO CD -vl CO -JX I 86.4112.71 0.91 171 CO o o o o o © ro - J X o o o O —1. o • X o ^x o ro I 86.4112.71 0.91 171 - J X cn o o o cn _x o _± - x o O O o o o -JX a CO o CO 83.1115.61 1.21. 1.91 1074 I § co cn CO J> ro cn ro 8 cn CO cn ro t fe 'ti to -4 & CO CO ro Ol ro cn fe CD on CO -4 Z 1 Right Costal 83.1115.61 1.21. 1.91 1074 I - A cn — k ~x a> cn CO a O co —X CO CO CO —X —X - J X —x co a J d -~4 i t - J k o Co —ft . _* 1 Right Costal 83.1115.61 1.21. 1.91 1074 I co O o o o ro o o - J . o — k o CO O o o ro —X ro 1 Right Costal 83.1115.61 1.21. 1.91 1074 I o © o O ro o o - X —X - J k O —x o> O o ro O CO to 1 Right Costal — • Z Costal Insertion -JX Costal Insertion to Costal Insertion CO Costal Insertion SI co -* —X vj —X cn o § cn CO fe CO -vl J> 8 fe § $ J> CO ft ro — X fe 8 on o HJ 4s. 00 z I Crural - J X Is) —x o 00 ro cn cn cn CO cn ro o> CO on 00 4d o> -4 -v| CO OJ •vl I Crural -mX • J X J x o _^  * j k _^  o o -4k ro o o •Jx ro •Jk o —X O . j k o —JX -Jk to I Crural - J k o o o ro o o o o ~x p o o _x o O o o *Jk CO I Crural $ OJ M Vi p o 1000 CO ro J> - J k o> ro 4> -vl 8 CO 4> . v. 8! fe cn CO Ol CO ^ % K CD V j Z i Psoas 1 o to CO o CO CO CO ro CO o O o 4- ro o 4V o O •Jx i Psoas 1 o o o o o o o o o o 9 O o o o O o o to i Psoas 1 o> o J d o ro ro| o o o o O o o o O o o CO i Psoas 1 So o • i 105 o H TJ O CD 3 r f » r f CD 3 a o 3 -n CD CD 5' 3 O 3 CD 3 r f CD 3 SL «<" 3 C O CD fi) r f CD a. ro > cr 3 o 3 SL o r f o TJ fi) CO 3 to > c r 3 O 3 0) N CD O CO 3" 0) TJ CD Total # Fibres: Category subtotals: to o CO oo vl oo cn j i CO ro o CO 00 vl Oi cn J i CO ro Fields: 1 81.7 Oi CD Ji. Ol CD •vl v l ro j i ro v l ro CO ro oo ro Oi CO CO ro ro CO CO J i CO ro ro 00 ro 00 ro o CO Ol co vj ro CO J i ro CO z O —i 15.0 O J i ro to v l oo Ol cn ro cn v l CO J i cn Ol 00 Ol J i Ol v l oo _^  co CO ro CO _,. ro _,. 00 o — J L j i o o o o o _,. _,. ro o IO 33 CD CO j i ro _^  ro o ro j i ro o o v l ro ro CO o co o CO 90.3 00 v l 00 v l CD CO J i Ol CO 00 J i co Ol ro CO Ol co CO ro Ji. j i v l Ol j i j i j i CO o CO v l ro CO j i CD CO •vl j i v l CO j i CO CD CO ro z v i fo 00 CO co co ro ro ro j i CO j i cn CO cn Ol oo j i ro j i ro ro CD r f o o CO r f fi) ro Ol ro ro o o ro o o o o ro ro o j i CO o ro CO o ro J i co co cn v l o o ro cn ro o CO o ro cn ro o . CO 85.5 v l 00 CD j i ro v l co Ol ro oo CO CO cn CO ro ro CO CO ro j i Oi j i co CO j i . ro CO CO CO CO ro oo co oo co ro ro oo ro CO z TJ ID' 3" r f o o CO r f fi) 11.8 00 cn Ol oo CD vj v l o j i oo o CO v l ro ro CO v l ro b co o ro ro o o CO o o ro o CO o ro O ro o — L — J L ro Ol bo j i ro CO o o ro CO . CO ro oo cn CO oo ro CO 84.0 OO Ol CO ro o ro o j i oo ro o ro Oi ro oo ro v l CO J i ro Ol CO o oo ro co ro co ro oo ro cn ro j i CO CO o z o o CO r f fi) 3 CO CD a. 5" 3 14.2 00 v l oo CD j i j i j i 00 ro o v l CO Oi cn j i CO j i oo j i Ol CO co r j . bo o o o o o o o o ro o o ro o Ol CO o o o o o o o o o o o o o o o o o CO 89.2 v l O J i Oi ro oo CO ro CO CO CO ro oo ro 00 CO v l j i CO v l CO 00 j i ro CO o CO CO ro v l CO v l CO o ro j i ro oo ro j i ro CO ro co z o —I c SL CD b j i ro CO ro ro j i j i ro o ro CO ro ro CO o CO o cn ro _ i b CO j i ro o CO ro o CO CO J i ro - J L o j i CO ro ro b Oi CO o o _ ^ o o o CO o o o o . ro ro o CO 93.4 v l J i 00 Oi CD v l co ro CO CO ro ro v l CO CO co CO co CO oo ro CO CO v l j i ro CO CO j i J i CO co j i CO J i Ol j i CO v l CO ro z TJ CO O fi) CO j*. io CO J > . — J L o Ol ro CO o ro v l ro ro ro o ro o ro o v l Ol o o o o o o ro o o o o o o o o o o o ro ro v l - J L CO CO _,. o _,. ro o o o _1 o o _,. _,. CO o CO o o CO CD «< o _ r* O o O) c 3 r f CO 106 o H TJ O CD 3 CD 3 a o 3 CD CQ 5' 3 O 3 CD 3 i-i-CD 3 51 >< 3 C O CD 0) i-p CD a. to > o-3 O 3 cu_ o o TJ fi) (A 3 co > cr 3 O —i 3 fi> (A N" CD O (A 3" tt TJ CD V ? Total # Fibres: Category subtotals: ro o CO oo 0) cn J l CO ro o co 00 -sl O) cn J> CO ro Fields: 1 86.7 oo J> vj CO Jd CO Oi CO J l ro CO cn o CO cn ji CO J l ro J l Ul CO vj 00 Ul J l CO Ul CO CO CO vj CO CO J l o J l Ul ro Ul CO o CO Ul z O H 11.0 co 00 o Oi CO ro cn oo CO Ul •vl L J l CO Ul Ul ro CO co J l —J. N> jd ro o ro _x ro o ro L o _ l —lx ro ro o o ro o o ro TJ 00 cn o ro o CO o o ro o o o . o o o o CO CO 93.1 CD cn cn ji vj ji 00 cn o cn o Ul vj Ul CO ro Ul J l Vl Ul Ul o ji CO Jd cn co CD TO CD J l o ji L J l Ul J l Oi z J l CO CD v| jd ro o J l ro CO •vl oo vj o ro ro o o ro o o 1 CD —•» i-p O O CO tt ro b oo ro cn , ji . ro ro O o ro o o ro o ro ro ro ro o bo oo o o o CO o O o o _^ o o o o o o o CO z TJ to" 3" o O CO fi) _j> ro co z O O CO r-i-fi) 3 CO CD z\ o 3 ro CO 90.6 Oi o Jx cn jd •vl ro •vl ro ro IO •vl ro 00 ro CO ro Ul ro CO ro ji CO o ro CO CO J l ro vj co ro o oo CO CO ro Oi J l Ul ro co CO ro z o c —t tt 00 bo ro oo o CO CO . CO ro o o . o CO o o cn CD 00 Jd CO o ro o o Ul o ro ro o o Ul J l ji o CO ro O Vl Jd o o o o o _ i o o ro o o o o o o o o o o CO 98.6 L oo Ol cn CO cn CD v j ro Oi Oi Oi Oi Oi CO Oi CO J l oo CD Oi Oi cn oo Oi CO Oi o Ul CO Oi Ul 00 Oi v j ji CO J l vj J l Oi 00 Oi z TJ CO O 0) CO b ro o O o o ro ro o L o o ro o O o o ro ro o o O o ro o o o O o o o o o o o o o O o ro o b CO o o O o o o o o o o _,. o o o o _L o O o CO o ->• o - i o "sl C 3 CO 107 o H JD O CD 3 CD 3 a o 3 CD CQ 5' 3 O 3 CD 3 CD 3 co •< 3 C o_ CD co #-f CD Q . > CT 3 O 3 SL o o •o CO CO 3 co > CT 3 O -r 3 SL co N" CD o CO 3" 0) "D CD Total # Fibres: Category subtotals: ro o co 03 v l cn *» CO ro o CO oo v l 00 Ul 4- CO ro Fields: 1 77.4 o CO CO r o r o r o r o o o r o CD 4* k CO o —X o 4*. c n c n k CD r o CO 4=-CO c n c n z CTR 21.8 oo oo c o CO v l CD r o c n v l c n c n c o CO c n 4*. c n o r o CD CTR o v i CO o o O O o o O o o _,. _,. o o _,. o o o o ro CTR CO b r o O r o o _^ k o r o o o o o o CO CTR 95.11 c o o v l v j o r o c o c o 4* o CD CO v l 4* CO -t-oo CO c n CO c n CO v l 4=-4*. 4*. k 4=-CO CO o 4* r o CO —Jk CO CO CO r o 4i 4*. 4^  CO z CO CO CO r o r o —k o r o O r o o r o CO v l r o Left Costal O b v j o o o O o o o r o o o o o o o o r o o ro Left Costal r o v l r o r o o CO o o O o o o o r o r o o CO Left Costal 91.4 00 00 c n oo CO v l r o r o r o r o v l r o CD r o c o CO 4* r o r o r o v l r o c n r o CD 4* O r o 4>. CO CO CO CO 4^  r o CD CO o CO 4i CO c n z T l c n b c o J > c n —k o r o o r o r o 00 r o CO r o r o o o O CO light Costal CO L CO o r o o CO CO o o o r o o r o _ x o o o r o ro light Costal b oo o o L o o o o o k r o _^  o o o o o o CO light Costal 89.3 oo CO v l r o CO oo r o v j r o c o CO CO c o 4i CO CD CO CD c o o CO v l CO r o oo 4i v l 4-r o c n c o •vl CO c n 4^  v l c o c n z Costal Insertion v l b 00 CO o J > . CO o 4i c n oo c n CO CO r o CO r o c n v l CO CO Costal Insertion co r o 00 o o r o o 4^  CO CO r o o r o o o o o o o ro Costal Insertion b CO o r o o o o o o o r o o k CO o r o o k o o CO Costal Insertion 94.4 1096 1035 J > oo c n o c n o c n CD 4* oo c n r o 4^  CO c n c n c n CO c n oo c n r o CD r o 4* 4* 4*. oo 4* CO c n v l 4* v l c n 4^  c n CO CD -i>. z Crural 4^  b 1096 c n o c n c n o r o CO r o —k CO r o CO IO 4^  CO CO r o CO 4i Crural b 1096 r o o o o o o o o o O o r o o r o O ro Crural o b 1096 v l o o o o o o o o o o o o O CO Crural 97.8 1266 1238 c n oo CD v l CD 4- CO c n v l c n CD CD v l CD c n o> r o CO CO CD CD v l v l O c n oo c n c o CD 4* 00 4^  oo CO v j c n v l z Psoas b 1266 v l ro r o r o r o 4* o o O o o O O - X r o Psoas o b 1266 O o o o o o o O o o o o o O o o O O o o o ro Psoas o b 1266 _ x _,. o o o o o r o k r o o o O o r o o O r o _ x o CO Psoas = 1 f l o — -* o _ i o oo £ 3 r+ CO 

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