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Quantification of the tissue changes in the human lung with chronic lung disease using a combination… Coxson, Harvey Owen 1998

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QUANTIFICATION OF THE TISSUE CHANGES IN THE HUMAN LUNG WITH CHRONIC LUNG DISEASE USING A COMBINATION OF COMPUTED TOMOGRAPHY AND STEREOLOGY by HARVEY OWEN COXSON B.Sc, The University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Experimental Medicine)  We a^ept this thesis as>GOTTfl7rfning ; t e > f h e required standard  THE U N I V E R S I T Y OF B R I T I S HC O L U M B I A April 1998 ©H a r v e yO w e n Coxson, 1998  In presenting this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT Idiopathic p u l m o n a r y fibrosis (IPF) a n dp u l m o n a r ye m p h y s e m a are chronic lung  diseases exhibiting progressive deterioration in p u l m o n a r y function as the lung architecture is  remodeled. This thesis quantifies these tissue changes using a novel combination of c o m p u t o m o g r a p h y (CT) a n d quantitative histology. Pre-operative C T scans w e r e obtained f r o m  patients with IPF, patients receiving lung v o l u m e reduction surgery for diffuse e m p h y s e m aa  f r o m patients with minimal to mild e m p h y s e m a undergoing l o b e c t o m y for a small peripheral • •• • •  •  j .  tumour. Total lung v o l u m ew a s calculated using the pixel dimensions o n the C T scan wh  airspace a n d tissue v o l u m e as well as the regional lung expansion w e r e estimated using t  ray attenuation values. Tissue samples w e r e obtained at either o p e n lung biopsy (IPF) or  surgical resection (control a n de m p h y s e m a )a n d prepared for quantitative histology. A m e t h  for correcting the histology specimens to a n in vivo level of inflation w a s developed so tha  tissue composition a n d surface area could b e estimated using stereologic techniques. T h e  data s h o w s that there is a reorganization of lung p a r e n c h y m a in IPF with a disproportionat  of airspace a n d surface area without increasing the total a m o u n t of tissue. T h e patients w e m p h y s e m as h o w evidence of a progressive proteolytic destruction of tissue v o l u m ea n d  surface area. There is a negative correlation b e t w e e n regional lung expansion a n d surface  area in e m p h y s e m aa n d a positive correlation b e t w e e n surface area a n d the diffusing cap  of the lung in both diseases. This technique should prove useful in the longitudinal assess of chronic lung diseases a n d the monitoring of response to treatment.  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF TABLES  vi  LIST OF FIGURES  vii  ACKNOWLEDGEMENTS  viii  PREFACE  ix  CHAPTER 1: INTRODUCTION TO QUANTITATIVE ANALYSIS OF THE LUNG  1  1.1 Quantitative Histology Of The Lung 1.1.1 Sampling 1.1.2B\as 1.1.3 Variance  1 1 2 3  1.2 Stereological Methods 1.2.1 Stereological Probes 1.2.2 Cavalieri's Volume Estimator 7.2.3 Volume Fraction 1.2.4 Surface Area 1.2.5 Multi-Level Sampling Design  4 4 6 7 7 11  1.3 Quantitative Gross Analysis Using Computed Tomography  12  CHAPTER 2: WORKING HYPOTHESIS. SPECIFIC AIMS AND STRATEGY  16  2.1 Working Hypothesis  16  2.2 Specific Aims  17  2.3 Strategy  17  2.4 Summary  17  CHAPTER 3: THE NORMAL HUMAN LUNG  18  3.1 Descriptions of the Lung  18  3.1.1 Gross Lung Structure 3.1.2 Cellular Lung Structure 3.1.3 Extra-cellular Matrix  20 21 23  3.2 The Clinical Measurement of Lung Function  iii  25  3.3 The Pleural Pressure Gradient  27  3.4 Experiment #1  28  3.5 Material and Methods  28  3.5.1 Pulmonary Function Studies 3.5.2 CT Studies 3.5.3 Quantitative Histology  29 29 33  3.5.4 Statistical Analysis  35  3.6 Results  35  3.7 Discussion  45  CHAPTER 4 : INTERSTITIAL LUNG DISEASE  48  4.1 Introduction to Interstitial Pulmonary Fibrosis (IPF)  48  4.1.1 Clinical Description of IPF 4.1.2 Radiological Description of IPF 4.1.3 Histological Description of IPF 4.2 Fibrotic Mechanisms 4.2.1 Cellular Mechanisms of IPF 4.2.2 Molecular Mechanisms of IPF  49 50 50 51 51 52  4.3 Quantitative Studies of IPF  53  4.4 Experiment #2  55  4.5 Material and Methods  55  4.5.1 4.5.2 4.5.3 4.5.4  Pulmonary Function Studies CT Studies Quantitative Histology Statistical Analysis  56 57 58 62  4.6 Results  62  4.7 Discussion  72  CHAPTER 5: PULMONARY EMPHYSEMA  76  5.1 Introduction to Pulmonary Emphysema  76  5.1.1 Functional Description of Emphysema 5.1.2 Radiological Description of Emphysema 5.1.3 Histological Description of Emphysema 5.2 Pathogenesis of Emphysema 5.2.1 Protease/Antiprotease Theory 5.2.2 Inflammatory-Repair Mechanism  11 78 78 80 80 81  iv  5.3 Quantitative Studies in Emphysema 5.3.1 Gross Analysis 5.3.2 Histologic Analysis 5.3.3 Radiological Analysis  82 82 83 84  5.4 Experiment #3  85  5.5 Materials and Methods 5.5.1 Pulmonary Function Studies 5.5.2 CT Studies 5.5.3 Quantitative Histology 5.5.4 Statistical Analysis  86 87 87 88 92  5.6 Results  93  5.7 Discussion  104  CHAPTER 6: SUMMARY AND DISCUSSION  108  6.1 Summary  108  6.2 Future Directions  112  6.3 Conclusion  113  REFERENCES  115  v  LIST OF TABLES Table 1. Stereologic rules for the selection of a sampling probe.  5  Table 2. Cellular composition of the lung.  21  Table 3. Pulmonary Function Data.  37  Table 4. Individual lobar volumes measured by CT in 9 patients.  38  Table 5. Lobar weight and volume.  39  Table 6. Lung volume and Gas per Gram of Tissue.  40  Table 7. Stereology.  41  Table 8. Patient Demographics.  65  Table 9. Lung Volumes and Weights.  66  Table 10. CT Estimated Regional Lung Inflation.  67  Table 11. Light Microscopy Volume Fractions (%).  68  Table 12. Patient Demographics.  96  Table 13. Lung Volumes and Weights.  97  Table 14. CT Estimated Regional Lung Inflation.  98  Table 15. Quantitative Histology.  99  Table 16. Percent Emphysema of Resected Lobe.  vi  100  LIST OF FIGURES Figure 1.  Representation of variance and bias in a sample.  4  Figure 2  Sample point counting grid on an object.  ,6  Figure 3.  Sample point counting grid on light microscopic section of human lung.  8  Figure 4.  Sample intercept counting grid on light microscopic section of human lung.  9  Figure 5.  Multi-level sampling design.  10  Figure 6.  CT scan of human lung showing segmentation of the different lobes.  31  Figure 7.  A representative CT analysis slice.  32  Figure 8.  Graph of the CT density of the lung.  42  Figure 9.  Graph of the pressure volume curves.  43  Figure 10.  Graph of the pleural pressure gradient.  44  Figure 11.  Classification of interstitial lung diseases based on pathogenesis.  48  Figure 12.  Representative electron micrograph from IPF patient biopsy.  61  Figure 13.  CT density of the lung.  69  Figure 14.  Volume fraction of the tissue in the biopsied regions of lung.  70  Figure 15.  Weight of the interstitial components.  71  Figure 16.  CT scan of human lung with emphysema using the density mask.  89  Figure 17.  Gross lung slice and CT scan.  90  Figure 18.  CT density of the lung.  Figure 19.  Mixed effects regression line for surface area per volume and lung inflation. Mixed effects regression line for surface area and diffusing capacity of the lung for carbon monoxide.  Figure 20.  Figure 21.  Figure 22.  101  102 103  Mixed effects regression line for surface area and diffusing capacity of the lung for carbon monoxide for all patients.  111  Three dimensional reconstruction of a human lung with emphysema.  114  vii  ACKNOWLEGMENTS  No scientific w o r k can be completed without the assistance and support of m a n y people.  As such, I wish to t h a n k my supervisor and m e n t o r Dr. J a m e s C. H o g g for getting me started in. this field and tortus guidance and support through all of the aspects of my career in science. I also wish to t h a n k my supervisory committee, Drs.  Peter D. Pare, Clive R. Roberts, and John  M a y o for their constructive input and instruction. This project w o u l d not h a v eb e e n possible without collaborations f r o m the University of I o w au n d e r the direction of Gary W. H u n n i n g h a k e and Dr. Robert R. Rogers at the University of Pittsburgh. I also wish to t h a n k Ms. H a y e d e h Bezad and Dr. Benard Meshi for their technical assistance with the stereology; the Histology Laboratory St. Pauls ' Hospital for processing the histological material; the c o m p u t e d t o m o g r a p h y / m a g n e t i c resonance imaging staff at St. Pauls ' Hospital for gathering and transferring the CT images; Dr. K e n n e t h P. Whittall and Mr. Don Kirkby for their wizardry with the computers; Ms. Barbara M o o r e for collecting the p u l m o n a r y function data; Messrs Joe C o m e a u and Stuart G r e e n e for their c o m p u t e r and photographic expertise; and Ms. Lorri Verburgt and Ms. Yulia D Y ' achkova for their statistical advice. Special appreciation is also extended to my g o o d friends (Paul and M a r y Lacey and Gary and Heidi Rae)  who introduced  me to fly fishing and provided me with so m u c h moral support through this time. T h a n k you also to my parents and family who always believed in me.  Finally, I wish to t h a n k my wife  M a u r e e n for her patience, faith and u n w a v e r i n g love. T h a n k you all and God bless you.  viii  PREFACE  Chapters 3 and 4 are modifications of published papers. The introduction has been rewritten to match thesis requirements, and the methods have been modified to reduce redundancies. The results, and discussion are as published. The complete publication record is listed below. Chapter 3: Coxson, H.O., J.R. Mayo, H. Behzad, B.J. Moore, L.M. Verburgt, C A . Staples, P.p. Pare and J.C. Hogg. The measurement of lung expansion with computed tomography and comparison with quantitative histology. J. Appl. Physiol. 79:1525-1530. 1995. Chapter 4: Coxson, H.O., J.C. Hogg, J.R. Mayo, H. Behzad, K.P. Whittall, D.A. Schwartz, P.G. Hartley, J.R. Galvin, J.S. Wilson and G.W. Hunninghake. Quantification of idiopathic pulmonary fibrosis using computed tomography and histology. Am. J. Respir. Crit. Care Med. 155:1649-1656. 1997.  ix  CHAPTER 1: INTRODUCTION TO QUANTITATIVE ANALYSIS OF THE LUNG  1.1  Quantitative histology of the lung Stereology is a group of statistical and geometrical procedures which yield information  about objects in three dimensions from two dimensional sections (48) and the history, theory and methods of this technique have been reviewed in a number of excellent books and review articles (16,36,40,48,49,74,144,233,251,253,254). These techniques are very powerful, not only because they allow the quantification in three-dimensions, but because they are unbiased and extremely efficient (74,144). Stereology began with investigations of geometrical probability theory in the 1700's, which was then applied to quantitative problems in geology and metallurgy in the mid 1800s and finally to histopathology starting in the mid 1900s (4). The popularization of these methods to investigations of the lung is attributed to Weibel and his classic book, Morphometry of the Human Lung (249) and was the point from which stereology began to be applied to questions of structure inhealth and disease. This has led to an explosion in the mathematical theory on which the procedures are based as well as in refinements of techniques for sampling and quantification. This chapter will cover the basic principles for a stereologic analysis of the human lung including: the sampling protocols, the basic test probes and their associated formulas. The subsequent chapters will apply these techniques to the normal lung and two disease states, a fibroproliferative disorder, idiopathic pulmonary fibrosis (IPF) and a destructive disorder, emphysema.  1.1.1  Sampling In virtually all studies, it is impractical to quantify the whole population. Therefore, the  population is sampled and estimates about the population are made from the measurements  1  made on this sample. Sampling is extremely important for the stereological analysis of very small structures because the high level of magnification that is needed to observe small structure greatly reduces the area that can be examined in one field of view. The reliability of any estimate is very dependent on the bias of the sample, and the variance inherent in the technique used to make the estimate (35).  1.1.2  Bias There is no method for calculating the bias in a sample (35). For this reason  stereological analysis has develop into what is know as "design-based" stereology (16) where the procedures used to quantify the lung rely on the sampling design of the study. The power of this approach is that it does not require any assumptions about size, shape or distribution of the structures under investigation, as is the case with the "model-based" study (233). The two main forms for reducing bias within the sample are a random sample of the lung structure, and a uniform sample of the whole organ. Random sampling gives all parts of the specimen equal opportunity to be selected (144) while uniformity allows the sampling of structures which may not be randomly distributed throughout the specimen (16). Both of these criteria are satisfied by choosing the first area, or slice, randomly, and then using a predetermined interval to choose the subsequent samples. For example, an object's volume can be estimated by cutting the object into multiple slices of a uniform thickness, randomly choosing a slice, (i.e. slice 2), and then systematically choosing every third slice until the object is completely sampled (16,144). Variations on this procedure can be used for selecting tissue biopsies, and microscopic fields of view (16,35,74,144) which yields a systematic-random sample. It is important to note here that these sections are truly random and must be differentiated from samples that are chosen arbitrarily or because they "appear interesting." These later sampling protocols introduce a bias into the sample that can dramatically effect the  2  reliability of the outcome. There are many other forms of bias within the sample mostly due to technical limitations. These include, but are not limited to: tissue processing, resolving power, section thickness, automatic edge detection algorithms, and image recognition (144). All of these biases must be taken into account when designing stereological studies and their influence on the results will be discussed later.  1.1.3  Variance The variance associated with the estimation of a structure is determined by its biological  properties and can be calculated for the sample. The first factor to consider when calculating the variance of a structure is the biological variation, which takes the form of how the structures are distributed within the organ and any between subject variability. The second area where variance can occur is due to the measuring technique used (214). The variance for the sample can be calculated by adding the variance at each sampling level to obtain the overall standard error of the mean (SEM) for the sample according to equation 1:  ni  '  SE^ = J — +  :  ;  ~ —  V Tl(sub) ft(sub)  + X  2  ~  —  -+  —  M(his) M(sub) tl(his) *ft(mic) X  ~~  '  3  ."  °>  f-  :  S<me  [1]  ?l(sub) tt(kis) tt(mic) M(mea) X  X  X  where {s b)) is the variance between subjects, histologic samples (s ^)), microscopic field of 2  2  (su  view {s ( )), and the measurements (s ( )), while n is the number of subjects, histologic 2  2  miC  mea  samples, fields of view and measurements respectively. It can be seen from this equation that the greatest effect on SEM will come from the number of subjects examii ied, even if the s ( ) 2  mea  is greater than the s ( b) (214). Therefore, a design-based stereological study attempts to 2  SU  minimize the variance of the estimator by applying the most work to the level that has the greatest impact on the overall SEM.  Figure 1 s h o w s how variance and bias affect the estimate. If the dots represent the m e a s u r e d values, and the target represents sampling bias, the ideal estimation w o u l dh a v e low bias and low variance (fig 1 A). H o w e v e r , it is possible that this estimate can h a v e too large of variance (fig 1B) or too large a bias (fig 1C) or, at worst, a combination of both (fig Therefore, care  1D).  \  m u s t be taken in  Low Variance  High Variance  the experimental design to reduce  Low Bias  the bias to a  B  m n im i u m and concentrate the quantitative effort  High Bias  to the m e a s u r e m e n t which has  D  the  greatest variance because it has the greatest  Figure 1. Representation of variance and bias in a sample. See text for full explanation.  impact on the estimation of the population  1.2  Stereological Methods  1.2.1  Stereological Probes  Classical stereology b e g a n in geology in 1842  with the w o r k of Delesse, (4) and it took  almost 100 years for the,techniques to b e c o m e developed e n o u g h to apply to histologic  4  specimens (4).  During the last thirty years the techniques h a v eb e c o m e understood by  researchers other than mathematicians and statisticians and h a v eb e e n refined for general use in estimating the size of three-dimensional structures (16,74,144). Stereology obtains three- , dimensional information by applying a geometric p r o b e to a two-dimensional section. The p r o b e can take the f o r m of a point, a line, a plane, or a v o l u m e (16),  and the intersections  b e t w e e n the probe and the sampling plane provide information a b o u t the three-dimensional relationship of the structure. The rule for the use of probes is that the dimensional quantity of the object of interest plus the dimension of the p r o b em u s t equal three. Table 1 s h o w s how p r o b e is chosen. In the first line, the object of interest is v o l u m e which has three dimensions,  therefore, the p r o b em u s th a v e zero dimensions so the sum equals three. It can be seen that for surface area (dimensions = 2) the p r o b em u s th a v e one dimension and so on for length and n u m b e r .  Table 1. Stereologic rules for the selection of a sampling probe. The object dimension plus the test grid dimension m u s t total three. See text for full explanation.  Object Dimension  Test grid dimensions  Total  V o l u m e (3)  Point (0)  3  Surface Area (2)  Line (1)  3  Length (1)  Plane (2)  N u m b e r (0)  V o l u m e (3)  5  ' •.  3  1.2.2  Cavalieri's Volume Estimator The most simple application of stereology is in the estimation of volume. The 1 7  tn  century mathematician Cavalieri proved that the volume of any object can be estimated by cutting the object into parallel slices with a constant thickness, summing the cross-sectional area for all the slices and multiplying by the slice thickness. This relationship holds true for objects of any shape or size as long as the first slice is randomly positioned within the volume (16,74). The cross-sectional area can be measured in any way, but the most simple is to apply a test probe of points to the surface and count the number of points falling on the object. Since the points on the probe represent an area of the probe (figure 2), which is simply the distance between points squared, the area of the slice can be estimated by summing the points and multiplying by the area associated with each point. Therefore, Cavalieri's volume can be estimated from equation 2: Volume  = XP  x  d  2  xh  [2]  +  +  +  ~  .  +  +  where d is the distance between points on the probe, h is the thickness of the section, and IP is the sum of the points r  falling on the object.  _.  _  . .  •  ..  ..  .  Figure 2. Sample point counting grid on an object. ^ _ ^stance between points squared. See  A  r  e  a  text for full explanation.  6  1.2.3  Volume Fraction By using the same probe of points as in Cavalieri's volume estimation, the number of  points falling on a structure of interest within the section divided by the total number of points on the section (figure 3) is an estimate of the areal fraction (A ), or fraction of the total area A  occupied by the structure (74). It has been shown that for multiple systematic-random sections through the object, the sample now represents a volume so that the areal fraction is a reliable estimate of the fraction of the volume fraction (V ), occupied by the structure (4,16,35,74,144). v  Volume fraction is calculated using equation 3:  Y •  P  _ £ j (structure) . '(^(structure) ~ V p ZJ "(total) J  . <• '  1  where IP is the sum of points falling on the structure and the total object respectively. Since volume fraction is a volume ratio, the volume of individual structures can be estimated by multiplying the volume fraction of a structure by the total volume of the organ.  1.2.4  Surface Area The surface area of the structure is estimated using a using a probe of lines and  counting the intersections between the lines and the structure of interest, along with the number of line end points that fall on the structure (figure 4) (4,74,249). The surface density (S ), or v  surface to volume ratio, is calculated from .equation 4:  s  - i  x  l L  .  f 4 l  where U is the sum of the intersects between the structure and the line probe, L P is the sum of the line end points that fall on the Structure, and / is the length of the line. As with the volume fraction, the surface area is calculated by multiplying the S by the total volume. Finally, v  7  Figure 3.  Sample point counting grid o n light microscopic section of h u m a n lung. Volume fraction = n u m b e r of points on lung structure divided b y total n u m b e r of points.  8  Figure 4.  Sample intercept counting grid on light microscopic section of h u m a n lung. Surface density = n u m b e r of line intersects with lung tissue divided b yn u m of end points on tissue multiplied b y 4 divided b y the length of the line. S text for full explanation. 9  Figure 5.  Multi-level sampling design. A: Level 1, CT scan representing gross lung structure. B: Level 2, low level light microscopy representing lung parenchyma and airspace. C : Level 3, High level light microscopy representing alveolar wall and capillary lumen. D: Level 4, electron microscopy representing cell and extracellular matrix compositon. See text for full explanation.  10  since S is an estimate of the surface area per volume, the inverse of S is an estimate of the v  v  v o l u m e per surface area, which is another expression for thickness.  1.2.5  Multi-Level Sampling Design  To estimate the v o l u m e fraction of small structures, the lung m u s t be sampled using a very high level of magnification. H o w e v e r , since the sample size heeded to reliably estimate the v o l u m e fraction, goes up with the magnification a m e t h o d of optimizing the sample size for small structures is needed. This sampling protocol is k n o w n as the "Multi-level", or "cascade design" (35) and m a k e s use of fact that small structures are located within a larger structure which can be quantified at a lower level of magnification with a smaller sample size. For example, collagen fibrils (col) are contained within the alveolar wall (awl), which is contained within parenchymal tissue (tis), which is contained within p a r e n c h y m a of the lung (par). To quantify the collagen you start at the lowest level of magnification w h e r e the structure can be visualized, and then using increasing levels of magnification the object phase at one level b e c o m e s the reference phase of the next level as s h o w n in figure 5. Finally, the v o l u m e fraction is calculated by multiplying the object V by the V of the reference space at the v  v  previous level in a cascading m a n n e r as s h o w n in equation 5: V ~V Qevel4)^V v(col)  Y(col)  nevel5)^  V(awl)  In s u m m a r y , the lung can be reliably quantified by applying a simple design-based  sampling protocol to obtain systematic-random microscopic fields of view of increasing levels of magnification. A probe of lines with end points or a p r o b e of points is then applied to these fields of view as s h o w n in figure 4 and the n u m b e r of intersects b e t w e e n the sample and the probe is counted. Equations 3 and 4 are used to obtain the v o l u m e fraction and surface density. V o l u m e fractions of small objects are obtained using the cascade equation 5 which  11  then multiplied by the lung v o l u m e to estimate the structural component s ' volume. Surface area of lung p a r e n c h y m a is estimated by multiplying the surface density by the lung volume, and m e a n parenchymal thickness is calculated by taking the inverse of  1.3  the  S. v  Quantitative Gross Analysis Using Computed Tomography  C o m p u t e dt o m o g r a p h y (CT)  scans are obtained by projecting a b e a m of X-rays through  the b o d y to a detector on the opposite site which records the absorbance, or attenuation, of the X-rays by structures within the b o d y (38).  This is p e r f o r m e d in a complete circle a r o u n d the  b o d y so that-the attenuation values are also given spatial information and the i m a g e s are reconstructed on a matrix of 512 X 512 picture elements, or pixels, which h a v e the dimensions of the field of view divided by the n u m b e r of pixels. H o w e v e r , the X-ray b e a m also has a thickness, k n o w n as collimation, so the pixels of a CT scan i m a g e are m o r e appropriately referred to as voxels, because they are actually v o l u m e elements. Therefore, the CT scan can be used to reliably estimate the v o l u m e of the lung by s u m m i n g the voxel dimensions, which is analogous to the Cavalieri principle described above. Another important aspect of CT scans is that the voxels contain information a b o u t the linear attenuation of X-rays (v), w h e r e v is d e p e n d e n t on the density of the structure, the atomic n u m b e r of the structure, and the e n e r g y of the electron b e a m of the scanner. The attenuation value is then converted to a Hounsfield Unit (HU)  scale which is based on the attenuation of  water, according to the following equation (43): .  Htructure  CT value^ =  Hi-fi  xa  [6]  Mifi w h e r e a is equal to 1000  for the HU scale (43).  zero, air of -1000, and b o n e of + 1 0 0 0 HU (43).  F r o m this scale, water would h a v e a HU of  It has b e e ns h o w n that for objects with atomic  n u m b e r s in the biological r a n g e such as polyethylene f o a m (116), e p o x y (41,242), bread (41), w o o d (242), cork (123), and b o d y tissue (41,123,156,242), the HU can be converted to 12  gravimetric density by assuming a linear relationship between X-ray values and density (41). Density is then calculated by the following equation (43): Density (g/ml) =  CT„ , + 1000 ^ n:  [7]  For this reason, many studies have focused on the correlation of CT densitometry measurements to physiologic properties, such as lung inflation and density gradients, and how to these properties change in response to disease processes (2,8,11,32,41,51,6366,78,85,123,134,155,156,167,193,195,204,242,270). There are, however, artifacts in the CT values that have effects on CT densitometry due to scanner properties such as: absorbance of low energy electrons by dense structures (beam hardening) (38,148,276), non-linear partial volume artifact near lung boundaries and transversing structures with markedly different densities such as blood vessels (69,115,148,216,242,276), and even differences associated with different manufactures and generations of GT scanners (116,117,148). Most of these concerns were raised in the early 1980's (69,138,148) when.CT densitometry was in its infancy and investigators found that there were differences in CT values associated with scanner manufacture (138), slice thickness, and reconstruction filter (69,138,148). Kemerink and associates have recently studied modern scanners and have reported that a properly calibrated scanner yields a reliable estimate of density (114,116,117) and that differences between scanners can be minimized by daily calibration using water and air phantoms (116,117). They have shown that results obtained by different scanners are within the reproducibility of clinical practice associated with lung inflation (117). They also found that for modern scanners, there is no difference in mean density due to magnification, slice thickness or reconstruction filter (116,117). However, there are differences in density resolution, the ability to discriminate materials of a different density within a histogram, that are dependent on slice thickness and reconstruction filter (114). Even though high resolution CT scanning, which makes use of thin sections and sharp reconstruction algorithms, produce an image that is qualitatively easier to  assess with the un-aided eye due to better edge discrimination and the removal of overlapping structures, the density resolution is poor. This is due to the increase in the signal to noise ratio of this technique which produces quantitative information with too large a variance for reliable density resolution. Therefore, they recommend the use of sections thicker than one millimeter without a high resolution reconstruction algorithm to differentiate different densities with the lung (114). There are also artifacts associated with patient characteristics, most notably, the size of breath that the patient takes during the scan. The density of the lung is very dependent oh the level of inflation (155,156,198,248) and differences in density can be seen between expiratory and inspiratory scans, as well as along the pleural pressure gradient (32,41,155,156,198,242,248). There have been attempts to standardize the inflation level during the scan, using a spirorpetrically gated scan which assures that all images are acquired at the same level of inspiration (10,108,195). However, this is a technically demanding procedure and is not routinely used on clinical scans. In the clinical setting, the scans are reported at either full inspiration (78,155,156,198,248), or at a tidal volume above FRC in which the patient has been asked to take a normal breath and hold it during the scan. In the 1960's Hogg and Nepzy measured the volume of gas per gram of lung tissue in frozen exsanguinated dogs using the following equation: ml (gas) g (tissue)  =  S p e c i  fi°  ~ Specific Volume^  (iuni>  Volume  [8]  where specific volume is the inverse of density, the density of blood free tissue was measured to be 1.065 g/ml and the density of lung was measured from its weight and volume. Since CT scans yield an estimate of lung density, we can calculate the volume of gas per gram of tissue in these lungs during the CT scan by applying the above equation (32). The volume of gas per gram of tissue at total lung capacity (TLC) can be estimated by dividing the patients' measured  14  TLC  by the CT estimated lung weight. The v o l u m e of gas per g r a m of tissue estimated by  can then be expressed as a percentage of TLC,  CT  which will allow the comparison of CT scans  f r o m different patients because we k n o w at w h a t level of inflation the scans w e r e performed (32).  . " , < : • Another piece of useful data for the CT density is the ability to estimate v o l u m e  fractions. As Was m e n t i o n e d previously, the v o l u m e fraction of tissue can be estimated histologically by applying a grid of points to a two-dimensional s a m p l e and dividing the n u m b e r of points falling on tissue by the total n u m b e r of points within the lung. As has b e e n shown, a  v o l u m e fraction, is simply a v o l u m e divided by a volume, so a v o l u m e fraction can be calculated using volumes that are derived using any m e t h o d . Therefore, since we h a v e an estimate of specific v o l u m e of tissue f r o m the literature, and an estimate of the specific v o l u m e of the w h o l e  lung calculated f r o m equation 8 using the CT scan, a v o l u m e fraction of tissue can be estimated f r o m the CT according to the following equation:  Volume Fraction  (lissue) <nssue)  =  Specific  Volume  . . Specific  — Volume  (lissue)  [9]  (lung)  In s u m m a r y , there is clear evidence that the CT scans obtained f r o m a properly calibrated, m o d e r n scanner can be used to estimate lung volume, density, v o l u m e of gas  per  g r a m of tissue and the v o l u m e fraction of tissue and airspace of the lung. The purpose of this thesis is to use these parameters to assess the n o r m a l lung and the changes that occur in diseases such as fibrosis and e m p h y s e m a .  15  CHAPTER 2: WORKING HYPOTHESIS. SPECIFIC AIMS AND STRATEGY  Chapter 1 provides b a c k g r o u n d for two techniques useful for the quantitative assessment of lung structure. Stereology allows three-dimensional information to be obtained f r o m systematic-random histologic samples of the lung. It is unbiased, and efficient, but very invasive in that it relies on lung tissue obtained at autopsy. Although resected lung lobes can be evaluated by this technique it is not suitable for analysis of lung biopsies because of the collapse of the specimen which m a k e s re-inflation to an in vivo level very difficult. Quantitative analysis of the X-ray attenuation data obtained by c o m p u t e dt o m o g r a p h y is minimaly invasive and provides estimates of gross lung characteristics such as volume, density and weight, as well as estimates of the v o l u m e fraction of tissue and airspace. The goal of this thesis is to  c o m b i n e these two techniques with a view to validate the less invasive c o m p u t e dt o m o g r a p h y in terms of the m o r e invasive stereology.  2.1  Working Hypothesis  The working hypothesis of this thesis developed f r o man e e d for a non-invasive m e t h o d for quantifying the structural changes in the lung in chronic disease: The combination of c o m p u t e dt o m o g r a p h y scans and stereoloaic quantification of histological specimens allows the assessment of lung tissue changes in the chronic lung diseases idiopathic p u l m o n a r y fibrosis and e m p h y s e m a with minimal destructive impact on the patient. The goal of this study,is to m o v e analysis of CT scans b e y o n d the qualitative observations currently m a d e by diagnostic radiologists and m a k et h e mm o r e useful to clinical physiologists and physicians in quantifying structural defects in a way that wi|l be useful to establishing the natural history of the disease and m e a s u r e the effect of treatment.  16  2.2  Specific Aims  1. To use lung volume and density measurements obtained by CT to measure differences in _ ;  regional lung expansion and calculate the pleural pressure gradient. 2. To validate measurements of lung structure observed on CT with the structural studies based on quantitative histology. 3. To quantify the structural defects in chronic interstitial lung disease and emphysema using the combined CT and quantitative histological approach  2.3  Strategy Specific Aim 1 and 2 will be accomplished on patients undergoing lung resection for  bronchogenic carcinoma. Pre-operative study of these patients established that their lung function was within normal limits. Specific aim # 3 will be accomplished using open lung biopsy specimens from patients with idiopathic pulmonary fibrosis (IPF), chapter 4, and surgically resected specimens from patients with emphysema (Chapter 5).  2.4  Summary The analysis;of X-ray attenuation values from a CT scan is combined with the estimates  of lung structure obtained from a histologic quantification of the lung specimen, this procedure allows the correlation of lung structure and function using techniques thatare minimally invasive to the patient. It also adds information about the process of lung re-modeling caused by chronic lung disease.  17  C H A P T E R 3: THE  3.1  N O R M A LH U M A NL U N G  Descriptions of the Lung A quantitative analysis of the lung is an extremely complex problem because of the  sizes of the tissue components which range from several centimeters to only a few micrometers and their intricate three dimensional arrangement, the early studies of the lung were descriptive and as imaging techniques improved with the refinement of light microscopes and the invention of the electron microscope, the structure of the lung and the cellular and extracellular composition have been described in great detail (159,256). One of the first quantitative histologic studies of the lung was published in 1731 by the Reverend Stephen Hales (256) who reported calf lung alveoli to be cuboid boxes about 1/100 part of an inch in diameter (254 pm). From these measurements he was able to estimate the surface area of the lung to be approximately 27 m which led him to conclude that this enormous surface area made it very 2  probable that oxygen entered the blood through the lung rather than through food (256). This was a major shift in how people viewed the function of the lung and led to many more quantitative studies which attempted to relate lung structure to function. However, the biggest problem with any quantitative study is how to maintain the three dimensional structure of the lung microanatomy while obtaining accurate and reliable measurements. This was first undertaken by cutting serial sections of the lung and then making three dimensional reconstructions of the images, as described by William Snow-Miller in his book The Lung, which is one of the first.complete quantitative studies of the human lung (159). Another technique was to dry the lung so the walls would become opaque and then use a microscope to look through the pleural surface at the internal structures (159). Still another approach for the study of the airways was to create casts of them using a material such as Woods Metal which could be poured into the airways of the lungs and then polymerized and the surrounding material  18  removed. The Swiss anatomist Christoph T h e o d o rA e b y used this approach in the 1 8 7 0 s to m a k e painstaking m e a s u r e m e n t s of the bronchi and their branching pattern (256), and even t h o u g h his conclusions a b o u t the monopodia! branching pattern ran counter to Kolliker, who was the first to describe the complete epithelium in the alveoli, Aeby s ' study is considered the first quantitative analysis of the airway structure (256). The last century, has seen great i m p r o v e m e n t s in the quality of microscopes used,to i m a g e the lung and n o w h e r e is this m o r e evident than with the advent of the electron microscope which has allowed investigators to visualize structures d o w n to the macromolecular level. H o w e v e r , the intrinsic p r o b l e m of quantification with the microscope was still that they w e r e very time consuming, labour intensive and reduced the three dimensional structure of the lung to, two dimensional sections. Perhaps the greatest a d v a n c e in the quantification of the lung c a m e with the application of •stereological m e t h o d s , as outlined in Chapter 1, which b e g a n in 1961  w h e nH a n s Elias assembled the International Society of  Stereology. This society b r o u g h t together investigators f r o m biology, geology, metallurgy and m a t h e m a t i c s to develop and refine techniques for the reliable and efficient quantification of  three dimensional structures (256). Ewald Weibel in his b o o k Morphometry of the Human Lung (249)  applied these new m e t h o d s to a quantification of the h u m a n lung and f r o m this point on  quantitative m o r p h o l o g y of the lung has p r o d u c e d unbiased estimates of virtually all aspects of lung structure. The physiological description of the lung has b e e nu n d e r intense scrutiny for hundreds  of years but m a n y of the advances h a v ec o m e since the Second World War (151,262). Durin  this time investigators h a v e developed techniques to quantify the functional aspects of the lung and described the important principles of airflow, the pleural pressure gradient and exchange.  gas  %  This chapter provides a brief overview of lung structure and function and then uses  m o d e r n techniques to quantify the h u m a n lung.  3 . 1 . 1 Gross Lung Structure  T h eh u m a n lung consists of t w o separate lungs located (anatomically) o n the left a n  right side of the thoracic cavity connected to the trachea by the m a i ns t e m bronchus. T h e  lung has three lobes: the upper, middle a n d lower, while the left side has t w o lobes with  u p p e r lobe containing the lingula which is analogous, b u t smaller in volume, to the right m  lobe. T h e lobes are separated f r o m each other b y a wrapping of visceral pleura which fo  m a j o r fissure o n the left a n dam a j o ra n dm i n o r fissure o n the right b u t this separation i incomplete allowing collateral ventilation b e t w e e n lobes. T h e lung is further partitioned into  smaller units towards the periphery using the bronchial a n a t o m y as a basis for division a n d  nomenclature. A lobar s e g m e n t is the next m a j o r division of the lung a n d consists of the  that is supplied b y the second division of the m a i ns t e m bronchus. T h e secondary lobule  m e a s u r e s 1-2.5 c m in diameter a n d can b e visualized either o n the cut surface of the lun  by CT, a n d consists of a bronchiole a n d artery in the lobular core b o u n d e db y tissue sept  are continuous with the visceral pleura a n d contain the p u l m o n a r y veins a n d lymphatics (24 Each secondary lobule contains three to five acini which are described as the c o m p l e x of airways distal to the terminal bronchiole (75). This structural definition is compatible with a  functional definition of the largest lung unit in which all airways participate in gas e x c h a n g e  T h e final region of the lung is the alveoli which are blind air sacs containing the greatest  area to v o l u m e ratio in the lung a n d are surrounded by thin walled capillaries which is the primary site of gas exchange.  20  3.1.2  Cellular Lung Structure  There are 24 different cell types in the lung (table 2) (255)  which are arranged into three  layers: the epithelial layer, w h i c h is in contact with the airspace of the external environment, the  endothelial layer, which is in contact with the blood, and the interstitial c o m p a r t m e n t which both separates and binds these layers together. In addition, a fourth g r o u p of cells, the blood cells, m o v e through the lung and transport the o x y g e n to the tissues, r e m o v e the carbon dioxide f r o m the body, and c o m b a t infection. The red blood cells are responsible for transporting the o x y g e n to the tissues. The red colour is produced by the h e m o g l o b i n molecules which bind or release o x y g e n depending on the gradient surrounding the cells. The cells responsible for the elimination of foreign substances are the white blood cells w h i c h include the polymorphonuclear cells (PMN), monocytes, lymphocytes, eosinophils, and basophils. All of these cells  Table 2. Cellular composition of the lung (250) Cells  develop f r o mac o m m o n progenitor cell in the b o n e m a r r o w and then differentiate into specialized cells. For example, the lymphocytes are the primary m i m u n e cells which produce cytokines to direct the host response and antibodies to c o m b a t foreign particles. Eosinophils are particularly effective against parasitic infections and  Structure  Cells  % of Total Lung Cells  86 Total Alveolar T y p eI .4 AlveolarType.il 6 Endothelium 33 43 M e s e n c h y m a l N o n p a r e n c h y m a Total 14 Airways 5 Ciliated 2-3 Glandular <1 9 Blood Vessels Connective Tissue % of Total L u n g C o m p o n e n t Structure Connective Tissue 62 P a r e n c h y m a Total Collagen 46 Elastin 16 38 N o n p a r e n c h y m a Total Collagen 28 Elastin 10 P a r e n c h y m a  21  basophils are capable of generating leukotrienes and supplement mast cells in immediate hypersensitivity reactions (1,106). However, the first line of defense are the PMN which are the most abundant white blood cells in the blood and migrate quickly from the blood in response to a chemotactic stimulus to phagocytose the foreign particles, and release proteolytic enzymes and chemotactic substances which direct the rest of the immune response. The monocytes follow the PMN into the tissue from the blood but then they differentiate into the phagocytic alveolar macrophages which are long lived in the lung and release important cytokines that direct the host response to both living and inert material entering the airspaces. . The first of the two types of epithelium cells are the lining cells, which are ciliated in the central airways for the movement of the mucus. In the periphery of the lung, the alveoli are lined by the alveolar type I cells which cover the majority of the alveolar surface area even though they only account for a small percentage of the total number of cells in the lung (table 2) (253,256). The second cell type are the glandular (secretory) cells which include the goblet cells in the trachea and the bronchi, and the Clara cells in the bronchioles (76,105,266) whose function is to secrete mucus which lines the airways and traps inhaled foreign particles (76,105,266). In the alveoli, the type II cells secrete surfactant which lowers the surface tension thereby preventing the collapse of the alveoli and reducing the work required for lung inflation (76,105,253,266). The endothelium is a simple squamous layer of cells which is extremely thin in the alveolar capillaries to allow optimal exchange of gases (76,105,253,266). The interstitium is the space between the basement membranes of the epithelium and the endothelium and consists of fibroblasts and pericytes as well as cells of the immune system such as mast cells and plasma cells and the extra-cellular matrix. The fibroblasts in the lung are responsible for the synthesis of the extra-cellular matrix which gives the lung its structural properties (125,139,149,178,277). During a fibroproliferative response, the fibroblasts have been shown to stain positive for smooth muscle actin and have contractile properties  22  ( 3 , 1 1 2 , 1 2 5 , 1 3 6 ) so that they are postulated to be important for w o u n d contraction (125,136). Their exact role within the n o r m a l lung has b e e n postulated as regulators for capillary blood flow, compliance of the interstitial space, and tissue elasticity of the lung p a r e n c h y m a as well as turnover and m a i n t e n a n c e of the extra-cellular matrix (110,112). Pericytes are cells which are associated with the alveolar capillaries and may be a special differentiation of the s m o o t h muscle ceils, or of a completely different cell line but these cells h a v eb e e ns h o w n to h a v e contractile properties which may be responsible for regulating capillary blood flow (110,215). The m a j o r cell line of the airway and blood vessel interstitium is the s m o o t h muscle cell and their contractile ability is responsible for the conducting properties of the airways and vessels by reacting to nerve and chemical stimuli to dilate or constrict the caliber of the airway or vessel they surround.  3.1.3  Extra-cellular Matrix  Table 2 s u m m a r i z e s the state of k n o w l e d g e up into the 1970 s ' w h e n elastin and collagen w e r e considered the m a j o rc o m p o n e n t s of the extracellular matrix; The last t w e n t y years h a v e seen an explosion in the identification and description of other important molecules within the interstitium, m o s t notably the proteoglycans (PG)  and laminin  ( 1 3 , 1 8 , 1 9 , 5 0 , 7 7 , 1 6 3 , 2 0 0 , 2 3 4 , 2 3 5 , 2 6 8 ) .  At least 19 different collagens h a v eb e e n described to date, of which three are important in the interstitium of the lung, the fibrillar collagens type I and III and the sheet forming collagen type IV of the b a s e m e n tm e m b r a n e (139,191,236). The collagens are a trimer assembled in an  a-helix with each molecule of the helix consisting of repeating chains of glycine-X-Y w h e r e X is predominately proline and Y is often hydroxyproline (139,236). The individual collagen molecules are assembled within the Golgi of the fibroblasts, and secreted in a pro-collagen f o r m into the extra-cellular matrix ( 1 3 9 , 1 4 3 , 2 7 7 )w h e r e they are cleaved and assembled into  23  collagen fibers (139,143). For example, the type III collagen is a cylindrical fiber 40-200 nm thick which shows a characteristic banding pattern of 64 nm periodicity, by electron microscopy. This pattern is due to the fiber consisting of the collagen fibrils arranged in a quarter stagger pattern resulting in the negative charges of the fibrils overlapping to produce a high affinity  ,  binding of the positively charged heavy metal stains used in electron microscopy (139,236). The collagen fibers have a very high elastic modulus that gives the lung its tensile strength (132,139). Elastin fibers contain a core of polymeric insoluble elastin molecules with a mantle of microfibrils (152,211). The two identified microfibrils are the glycoproteins: microfibrillar associated glycoprotein and fibrillin (152). The protein structure of elastin contains a high concentration of hydrophobic amino acids like valine with a low content of acidic and basic amino acids and large numbers of lysine derivatives that provide cross linkage (152,211). This composition makes elastin extremely insoluble and the extensive cross linking renders it resistant to degradation (152). Elastin is usually found as amorphous bundles of dense staining material due to its large negative charge and hydrophobic characteristics (152,211). It is very likely that elastin and associated microfibrils are synthesized early in the development of the . organ and then remain relatively constant throughout the life of the organism, although the number of microfibrils does appear to decrease with age (128,152,189,211). There are reports of new elastin synthesized in disease conditions such as atherosclerosis (220) and drug induced pulmonary fibrosis (22). In contrast to collagen, elastin has high extensibility and low tensile strength (152). The proteoglycans (PG) are a large group of molecules defined by a protein core with attached side chains of glycosaminoglycans (GAG) which are repeating disaccharides (77,268). These molecules have been shown to be important for tissue hydration (77,200,220,268), cell migration (13,29,77,268,274), and the binding, and possible regulation,  24  of growth factors within the tissue matrix (18,50,268). The early descriptions of how lung structure was related to function focused mostly on the collagens and the elastin since these are the largest of the extra-cellular molecules and the. easiest to characterize. However, it has become apparent that these molecules are not the whole story for the lung and it is now clear that proteoglycans play a large role in lung structure, especially in the formative and the repair phases.  3.2  The Clinical Measurement of Lung Function Lung function is measured in terms of static lung volumes, recorded at predetermined  points in the respiratory maneuver, and dynamic lung volumes, recorded during the forced expiration. Total lung capacity (TLC) is defined as the volume of gas in the lung at full inflation and the residual volume (RV) is the point where no more air can be forced from the lung by the •  •  •  • • ' ) • '  action of the expiratory muscles (261). Functional residual capacity (FRC) on the other hand is the equilibrium point reached at the end of a quiet expiration where the inward force of the lung recoil is balanced by the outward force of the chest wall (261). These volumes are most accurately measured by seating the subject in an airtight body plethysmograph and measuring the patient's FRC using Boyle's law (44), which states that at a constant temperature the volume of any gas-varies inversely as the pressure to which the gas is subjected. For this study, the subject inhales maximally and then fully exhales to RV. The measured volume of gas that was inspired is referred to as the inspired capacity (IC) and the exhaled volume the vital capacity (VC). TLC is then calculated by adding the IC to the FRC, while RV is the TLC minus the VC (261). The dynamic lung volumes are recorded during a forced expiratory maneuver where the subject inhales to TLC and then forcibly expires as quickly as possible to RV. The expired volume is referred to as the forced vital capacity (FVC) and the volume expelled during the first  25  one second of the maneuver is referred to as the forced expiratory volume in one second (FEV^. In normal subjects, the ratio of the FEVt to FVC is approximately 80%, and this ratio can be decreased by airway obstruction that decreases the F E V i , and increased by restricted lung volume that reduces the FVC. The characteristics of obstructive lung disease are: decreased dynamic lung volumes and FEV^FVC ratio due to airflow obstruction or reduced driving pressure, and increased static volumes due to gas trapping because of the reduction of expiration. Another form of lung dysfunction is referred to as restrictive lung disease in which, as the name suggests, the lung is restricted by a stiffening of either the chest wall or lung parenchyma so that the static lung volumes and the FVC are decreased which causes an increase in the FEVt/FVC ratio even if the FEVi is minimally reduced (261). The ability of gas to diffuse from the alveolar air into the blood is measured by the diffusing capacity (D co) which determines the movement of a trace amount of carbon L  monoxide (CO) from air to blood. In this test, the subject inhales a mixture of CO and He (0.3% and 10% respectively) and breath holds for 10 seconds (157,261). The subject then exhales and after discarding the volume of dead space gas in the central airways, the concentrations of C O and He are measured. Since He is not absorbed by the blood, the difference between the inspired and expired concentration of He is used to calculate the initial alveolar concentration of C O by gas dilution. Carbon monoxide, on the other hand, is absorbed into the blood at approximately the same rate as oxygen so that the difference between the initial alveolar and expired C O concentration indicates the amount of C O absorbed by the blood. The diffusing capacity can be influenced by many factors including a change in the lung surface area, the volume of the blood in the alveolar capillaries.and the time spent by the erythrocytes at the airblood interface (257,261).  26  3.3  the Pleural Pressure Gradient Investigation of the lung with radioactive gases established.that ventilation of the lung is  u n e v e n (109,154,261). Milic-Emili and coworkers s h o w e d that w h e n lung v o l u m e was increased the apical regions of the lung s h o w e d an initial greater c h a n g e in v o l u m ec o m p a r e d to the basal regions, which then reversed at higher lung v o l u m e s (109,154,261). T h e s e and  other studies established that regional ventilation was influenced by a gravity d e p e n d e n t pleural  pressure gradient which causes u p p e r regions of the lung to be e x p a n d e dm o r e fully than low lung regions due to a higher trans-pulmonary pressure. It has b e e n hypothesized that this pressure gradient is caused by the weight of the lung b e l o w the given region which m u s t be supported by the lung p a r e n c h y m a ,s o m e w h a t like a spring which is suspended at the top (68,91,109). The distance b e t w e e n the coils of the spring is largest at the top and decreases  towards the b o t t o m because to the w e i g h t of the spring on each coil which is greatest at the top  and minimal at the bottom. Since the u p p e r regions are exposed to this higher pressure, they  are m o r e inflated at lower lung v o l u m e s and inflate first u p o n inspiration (156). This hypothe is supported by Glazier and colleagues who u n d e r t o o kam o r p h o m e t r i c analysis of alveolar size  to s h o w that alveoli in the u p p e r regions of the lung are larger than alveoli in the lower regions at FRC  (68) and H o g g and Nepszy who obtained similar results using density m e a s u r e m e n t s of  frozen dog lungs to s h o w that the non-gravity d e p e n d e n t regions h a v e a greater v o l u m e of gas per weight of tissue than the d e p e n d e n t regions (91).  Gravity also influences p u l m o n a r y  perfusion ( 1 0 9 , 2 6 3 , 2 6 5 ) and led W e s t to describe lung perfusion in terms of specific zones. In Z o n e 1 the alveolar pressure is greater than the arterial pressure and the v e n o u s pressure,  resulting in compressed capillaries and reduced blood flow. In Z o n e 2 the arterial pressures are greater than the alveolar pressure which is greater than the v e n o u s pressure and blood flow now determined by the arterial and the alveolar pressure with the v e n o u s pressure being  irrelevant. In Z o n e 3, the arterial pressure is greater than the v e n o u s pressure which in turn is  27  greater than the alveolar pressure and results in maximal dilatation of the vessels and maximum blood flow. These factors all contribute to a difference in lung density which all investigators state can not be appreciated unless the lung is fixed  in vivo (closed chest) so that  the pleural pressure gradient is intact. The introduction of CT in the 1980's allowed differences in regional lung volume to be appreciated from measurements of lung density (82,107,155,156). These investigators report that the non-dependent portions of the lung are less dense than the dependent regions, and that this relationship is dependent on body position during the scan.  3.4  Experiment #1 In this study we have used CT densitometry to measure the pleural pressure gradient in  human subjects undergoing surgery for bronchogenic carcinoma. We have combined the CT measurements of total and regional lung volume with quantitative histology to measure the structure of the human lung and to develop a technique to correct biopsy specimens to an appropriate level of inflation within the thorax to alleviate the problems associated with collapse of histologic specimens.  3.5  Material and Methods Studies were performed on 19 subjects who are part of an ongoing study of lung  structure and function at the University of British Columbia in which patients requiring lung resection for a small peripheral tumor are studied just prior to surgery. To be included in the study the patient's forced expiratory volume in one second (FEVi), forced vital capacity (FVC), FEVt/FVC ratio, diffusing capacity for carbon monoxide (D o) and total lung capacity (TLC) LC  had to be within the normal range. In a preliminary study, the images from 10 patients were used to evaluate the point counting technique by comparing the size of the tumor on the CT  28  i m a g e to its size in the resected specimen (Group 1). In the nine remaining patients, in the C T X-ray attenuation data w e r e available, regional lung v o l u m e sw e r e calculated.  3 . 5 . 1 Pulmonary Function Studies Spirometry, lung volumes a n d lung pressure-volume curves w e r em e a s u r e d in the  patients from both g r o u p 1 and 2 seated in a v o l u m e displacement b o d y plethysmograph.  Functional residual capacity (FRC) w a sm e a s u r e d using the Boyles ' L a w technique. TLC w  calculated b y adding inspiratory capacity (IC) to FRC. Residual v o l u m e (RV) w a s calculated  subtracting vital capacity (VC) f r o m TLC. In six of the nine patients in g r o u p 2, trans-pulm  pressure w a sm e a s u r e d using a differential pressure transducer (45 m p±1 0 0 cmH 0; Va 2  Northridge, CA) to c o m p a r em o u t h pressure to intra-thoracic pressure, m e a s u r e d with an esophageal balloon and P V curves w e r e constructed b y comparing these values to the  simultaneously m e a s u r e d lung v o l u m e (146). In the remaining three patients in G r o u p 2, t subdivisions of lung v o l u m ew e r e determined using a dry rolling seal spirometer and the multiple breath Helium (He) dilution technique. Spirometry and H e dilution FRC w e r e  performed o naP . K . M o r g a n computerized p u l m o n a r y function testing system (P.K. Morgan  Boston, Mass.). Dco for all 9 subjects w a sm e a s u r e db y the single breath m e t h o d of Mil L  associates (157) o n the P . K .M o r g a na u t o m a t e d diffusing capacity analyzer. T h e results a corrected for alveolar v o l u m e (V) and reported as D o A/ . A  L C  A  3 . 5 . 2 C T Studies  All the patients h a d a conventional C T scan (10 m m thick contiguous slices) withou  contrast o n a GE 9 8 0 0 Highlight A d v a n t a g eC T scanner (General Electric Medical Systems,  Milwaukee, Wl) approximately o n ew e e k prior to resection. All scans w e r e performed with subject supine during breath holding following inspiration. Conventional scanning parameters  2 9  in use at our institution w e r e used ( 1 2 0 kVe, 1 0 0m A , 2 sec, reconstructed o n standard  algorithm). T h e images for all 1 9 patients w e r e printed o n t o standard radiological film using  w i n d o w of 1 2 0 0a n d a level of -700 HU. For the nine patients studied completely, the C w a s transferred to a Sparc2 Workstation ( S u n Microsystems, M o u n t a i n View CA) a n d the w e r es e g m e n t e d out of the chest using the p r o g r a m Medical Image Viewer (Arkansas  Childrens ' Hospital Little Rock, AR, G E Medical Systems) using X-ray attenuation values of  1 0 0 0 to -500 HU. T h ev o l u m e of the w h o l e lung a n d the individual lobes w a s calculated the Cavalieri principle (153,183). This w a s accomplished b ys u m m i n g the s e g m e n t e d pixel  area in each slice a n d multiplying b y the slice thickness to get total lung volume. T h e ho  and oblique fissures w e r e noted o n each slice a n d the lung v o l u m ew a s apportioned to u  lower and middle lobes (figure 6). T h es e g m e n t e di m a g e sw e r e then passed to the num  analysis package, PV-Wave (Visual Numerics, Boulder CO), w h e r e a pixel w i d e strip a r o u n d  lung w a s subtracted a w a y to eliminate partial v o l u m e artifact, d u e to the curvature of the  a n d the chest wall, a n d each lung slice w a s divided into 1 6m l sections (40 X 4 0X1 0 figure 7). T h em e a n X-ray attenuation of each of these sections w a s calculated, as well  vertical distance f r o m the middle of each section to the m o s t gravity d e p e n d e n t (posterior)  portion of the lung. T h eC T values w e r e converted to true lung density ( g / m l )b y adding  the C T value a n d dividing b y1 0 0 0 (equation 7) (82). T h e weight of the lung or lobe to  resected w a s calculated b y multiplying the m e a n density of the lung (or lobe), b y its v o l u m  calculated b y the Cavalieri principle. T h em e a n total lung gas v o l u m e s per g r a m of tissue  TLC, FRC a n dR Vw e r e determined b y dividing the physiologically m e a s u r e d values of lun  v o l u m e at TLC, FRC a n d R Vb y the total lung w e i g h t calculated f r o m the C T scan. T h e  attenuation for each 1 6 ml cubes w a s then used to calculate the v o l u m e of gas per g r a m tissue for that cubic region of the lung according to equation 8.  3 0  Figure 6.  CT scan of h u m a n lung showing segmentation of the different lobes. A: unsegmented CT scan, B: segmented CT scan. RUL: right upper lobe, RML: right middle lobe, RLL: right lower lobe, LUL: left upper lobe (contains the lingula), LLL: left lower lobe. 31  Figure 7.  A representative CT slice showing the 40 X 40 X 1 0 (slice thickness) m m sampling regions drawn on the segmented lung. The m e a n X-ray attenuation for each of these regions, as well as the distance to the middle of the regi from the m o s t posterior (gravity dependent) region w a s calculated.  32  3.5.3  Quantitative Histology The lobectomy specimens were obtained directly from the operating room and taken to  the laboratory where they were weighed, inflated with Optimal Cutting Temperature (OCT) compound (Miles Laboratories, Elkhart, IN) diluted 1:1 with normal saline, re-weighed, and frozen in liquid nitrogen. Once frozen, the specimen was cut into 2 cm thick slices in the transverse plane on a band-saw and cores of lung 2 cm in diameter were sampled with a power driven hole-saw. These frozen cores of lung tissue were stored at -70 °C for other purposes. The remainder of the specimen was transferred to 10% formalin and fixed at room temperature for at least 24 hours. Samples were taken from these specimens and processed into paraffin, sectioned at 5 pm and stained with hematoxylin and eosin for quantitative histologic analysis. The histologic slides of the lobectomy specimens from the nine Group 2 patients were quantified using a cascade-design technique to estimate the volume fraction (V ) of airspace, v  parenchymal tissue and blood vessels, as well as the surface density (S ) and thickness of the v  parenchyma as described in Chapter 1.  In summary, this technique allows the volume fraction  of very small components to be estimated by using increasing levels of magnification so that larger objects are subdivided into their components at each successive level (35). Volume fractions are estimated by casting a grid of regular points on the field of view and counting the number of points that fall on each lung component and the total number of points on the lung (16). Equation 3 is then used to calculate the volume fractions of each lung component. In the classical description of the technique, the first level was determined using the unmagnified gross specimen (35). Therefore, to determine if the CT image would substitute for the gross specimen, the volume fractions of parenchyma, bronchovascular bundle and the tumour in the lobes of Group 1 patients was compared with the resected gross specimen. A grid of points was cast on each slice of the frozen fixed lobes and the CT images of that lobe of the 10 Group 1 patients. Once this technique was verified, the full cascade design was  33  i m p l e m e n t e d on the G r o u p 2 patients using the CT scan i m a g e for level 1. Levels 2 and 3 w e r ep e r f o r m e d at the light microscopy level using the point counting p r o g r a m Gridder (Wilrich Tech, Vancouver, B.C.)  which generated r a n d o m fields of view,  projected a grid on to the field of view via a camera-lucida a t t a c h m e n t on a Nikon L a b o p h o t light microscope and tabulated the counts. Level 2 used 100x magnification with a grid of 80 points (d = 0.11 mm ) and 40 lines (I = 0.11 mm).  The n u m b e r of points falling on airspace,  tissue (lung parenchyma), and m e d u im sized blood vessels (50-1000 urn) as well as the n u m b e r of intersects b e t w e e n the grid lines and the parenchymal-airspace interface w e r e tabulated. The surface density of the p a r e n c h y m a was estimated using equation 4. Since surface density is the surface area in a given volume, the surface area of the p a r e n c h y m a is calculated by multiplying the surface density by the v o l u m e of the lung (16). The m e a n p a r e n c h y m a thickness is calculated by taking the inverse of the surface density. Level 3 was p e r f o r m e d on 10 r a n d o m fields of view per slide at 400x magnification and the n u m b e r of points falling on airspace c o m p o n e n t s (Alveolar macrophages, alveolar PMN, other objects in the airspace, and e m p t y space) as well as the tissue c o m p o n e n t s (alveolar wall, capillary lumen, and small blood vessels (20-50 urn)) w e r e counted using a 100 point grid. The v o l u m e fractions for all the lung c o m p o n e n t s at level 2 and 3 w e r e calculated using equation 3, and the overall V for the individual lung c o m p o n e n t s was calculated by multiplying the V of that lung v  v  c o m p o n e n t by the V  v  of the c o m p o n e n t that contained it in the previous levels using equation 5  as described earlier. The v o l u m e fraction of the lung that is gas and tissue was also estimated by dividing the specific v o l u m e of tissue by the specific v o l u m e of the lung obtained f r o m the CT scans according to equation 9. The v o l u m e fraction of tissue and gas estimated using CT (V (CT)) V  c o m p a r e d to the V of tissue estimated at Level 2 using the m o r p h o m e t r i c technique to v  determine the validity of this technique.  34  Statistical Analysis  3.5.4  All data w e r e analyzed using i n d e p e n d e n t t-tests or the one way analysis of variance. Transformations w ' ere m a d e on certain variables to normalize distributions and to m a k e variances h o m o g e n e o u s . A Bonferroni sequential rejective procedure was used to correct for multiple comparisons (94).  A corrected p-value of less than 0.05 was considered significant.  Results  3.6  Table 3 s h o w s anthropometric and lung function data for all 19 patients w h e r eg r o u p1 ( n = 1 0 ) represents the patients in the preliminary study d o n e to evaluate the point counting m e t h o d and g r o u p 2 are the 9 patients in which the lungs w e r e completely analyzed for v o l u m e and density. The data for these 9 patients (table 4) s h o w s that on average the right lung accounted for 55 ± 2 % and the left lung 45 ± 2 % of the total lung v o l u m e with the right u p p e r lobe (RUL)  accounting for 21 ± 5 % , right middle lobe (RML)  ± 3 % , left u p p e r lobe (LUL)  9 ± 3 % , right lower lobe (RLL)  including the lingula 24 ± 4 % and left lower lobe (LLL)  25  22 ± 5 % .  Table 5 c o m p a r e s the calculated w e i g h t and v o l u m e of the lobe to be resected with the fresh weight of the resected specimen and its v o l u m e after inflation with cryo-protectant material.  This s h o w s that the fresh w e i g h t of the resected specimen was 76 ± 19 % of CT calculated lobe weight and that the inflated v o l u m e of the specimen was 94 ± 36 % of the v o l u m e determined in  vivo. The v o l u m e of gas per g r a m of tissue was calculated f r o m the physiologically determined TLC,  FRC,  and RV gas v o l u m e divided by the lung w e i g h t (1069  ± 327 g) and  is  s h o w n in table 6. The lung w e i g h t was calculated by dividing the CT estimated lung density by the CT estimated lung volume. The average v o l u m e was 6.0 ± 1.1 m l / g r a m at TLC, FRC,  3.6 ± 0.9 at  and 2.4 ± 0.8 at RV and the lung v o l u m e at which the CT was obtained was 3.8 ± 0.8  35  m/ lg  or 65.5 ± 11.9% of TLC. Regional lung expansion calculated in ml of gas per gram of tissue from the CT density was expressed as a percent of TLC. When this measure of lung expansion is plotted against lung height (figure 8) the regions of the lung lower in the gravitational field (posterior in a supine scan) were less well inflated than the upper (anterior) regions with a mean slope of 1.8 ± 0.09 %TLC/cm. Figure 9a shows the individual pressure volume curves for the six patients in Group 2 where the volume component is expressed as the volume of gas per gram of tissue and CT derived values of volume of gas per gram of tissue calculated from the 16 ml cubes of the CT scan are shown as individual points. Figure 9b shows the same data expressed as a percent of each patient's TLC. Figure 10 shows the mean pleural pressure gradient calculated using the PV curve in figure 9b and the lung volume data in figure 8 and has a slope of 0.24 ± 0.08 cmH 0/cm. 2  Comparison of volume fraction of lung parenchyma, large blood vessels and tumor obtained by point counting on the CT scan and directly on the resected specimen (table 7a) showed an over estimation of blood vessel volume fraction on the CT scan compared to the lobectomy specimen, with a corresponding decrease in the tissue and tumor fractions. The full cascade-design stereologic analysis of the whole lungs (table 7b) estimated a mean surface area of lung parenchyma to be 101.8 ± 35.1 m , which gives a mean thickness of 7.2 ± 2  1.0 pm. At this level of inflation, the lung is composed of 60 ± 4 % airspace, 26 ± 4 % blood vessels (including capillaries), and 14 ± 3 % parenchymal tissue. The fraction of the lung occupied by parenchyma that includes tissue, capillaries, small and medium sized blood vessels estimated by the CT was slightly higher at 22 ± 5 % than the 17 ± 5 % estimated from the level 2 quantitative histology.  36  .2 u c c  Ui _l OQ <  CO  CL  Q  3 u.  (0  11  CD  2 O  §  (%Pred) o  Ul >  > 1  Q.  1.0 ±0.1  TLC >|  93 ±25 118 ± 14  £  102 ± 9 106 ± 11  (%Pred)  FVC  CL  68 ±12  (%Pred)  o Q  Mean ± SD 3M/6F 60 ±10 162 ±8  CM  (Kg)  LL  (cm)  Ul  (yr)  £  HEIGHT WEIGHT  >I  AGE  u.  95 ± 19 0.69 ±0.13 86 ±14 100 ± 15  ^  SEX  TLC  £  83 ±23  (%Pred)  FVC CL  72 ± 17  (Kg)  Ul  (cm)  HEIGHT WEIGHT >l U.I  (yr)  AGE  CO (0  o Q  Mean ± SD 5M/5F 61 ± 11 169 ± 8  SEX  .O CO  o  O)  c CD  O  100 ± 0  CD O  ±i  0)  45 ± 2  TJ C CO  JO 3 D) C O)  55 ± 2  4873 ±1360 2218 ±698 2655 ± 670  LUNG LUNG LUNG  TOTAL LEFT RIGHT  CD JD O  c _3 O  c 22 ± 5  1071 ±423  LLL  CD  n o  CD CL CL 3  c .2  24 ± 4  k_ CD 3 (/) CO  25 ± 3  1218 ±391  o> c  rr bj o CD  RLL  (0 Q.  1147 ±358  LUL  CD  -4—' T3 C CO  3  O > (0  x> o  w LU -I  m <  3  TJ >  '•5 c  CD  9±3  o .O CO CD "D _3 O  c  21 ± 5  E  CO D) O  _CD TJ TJ  'E  -*—>  x:  O)  CD  E o >  _3  %TLV** ± SD  M  RUL  E  427±127  CC  1010 ±326  k. 3 (0 CB 0>  _i  Volume ± SD*  TJ <D  O)  co  O A  o  E  c.  c  CD 3  "co o  co CO  >  3  3 CC CD X> O  i_  CD CL CL 3  -•—<  x:  O) be  CO CO  T A B L E 5: Lobar weight and volume Resected specimen vs. CT WEIGHT (g) CASE  LOBE  SPECIMEN**  CT**  VOLUME (ml) SPEC/CT SPECIMEN*  CT  (%)  SPEC/CT  (%)  1  LUL  195  304  64  1363  1503  91  2  LLL  273  351  78  1258  1711  74  3  LUL  107  130  82  719  685  105  4  LL  555  717  77  2216  2528  88  5  LLL  184  214  86  996  653  152  6  LUL  299  412  73  1158  1598  72  7  RML  52  112  46  208  567  37  8  RLL  226  197  115  929  643  145  9  RUL  129  198  65  632  770  82  Mean ± SD  76 ± 1 9  94 ± 3 6  * calculated from inflated weight ** prior to inflation with OCT Left upper lobe: LUL, left lower lobe:LLL, right upper lobe: RUL, right middle lobe:RML, right lower lobe:RLL, and left lung:LL. ± Significantly greater than the lobectomy weights, P < 0.05.  39  T A B L E 6: Lung volume and Gas per Gram of Tissue: Liters  ml/g  % TLC  TLC  6.1 ± 1 . 4  6.0 ± 1.1  100  FRC  3.7 ± 1.0  3.6 ± 0 . 9  59.6 ± 7.2  RV  2.5 ± 1.0  2.4 ± 0.8  40.2 ± 10.9  Total Lung Weight = 1069 ± 327 g The CT scans were obtained at 65.5 ± 11.9 % TLC, i.e. just above FRC where the lung contained an average of 3.8 ± 0.8 ml of gas per gram of tissue. Total lung capacity: TLC, functional residual capacity: FRC, and residual volume: RV.  40  TO c  CD  = 1 "CO  8 8I  CM  £  >»  JE  5  il O ~D <D CO "co C CO 0 C &_ CD V) — . CO c 0 CD CA CD  o  1 E 2> TO  CO  CO  L k  CD Q. C co *S X 3 c CD CD __ us Q12 -CDC CO CO CL CO O CD c CD _ c C CO O ' c CO CO CD CO £  o  S  Csl  o  g  x: TO  "5 s.* CD£ E .2 •*= CO CO fe ™ CD CO  10  E u O)  S  x  TO C 3  L  o  u  ^"5  ns =  oo  < j2  CO o " ° c CO Q. TO ^ CD CO 4 CO E  5  !• oci £ o  CO +-* "D E CD CO £ •«= E co CO CD "O = "O Q. 2? TO c CD co CD nj O § E * - CD  o  »  o CD  .E  — CD = 5  fc  TO£  CD E £ u S ^—»  o -  — (D  | 8 g -D. «£ w  c  CO 0) M_ O ° a) g w CD ^ -C5O CO CO > T3  in  &=  5 i i CO > c  O CO CD "(0T3 "CO CD CD §5 M_ CO -C "O O TO CD C ^ £= O 9 - 0 ^ CD O to _j TO C „  £ -  TO> E ~ Jt < d £2 T3 00  8  o  o oo  o  o  o  CD  lO  (enssij B/seB |iu) 011% 42  o  o  CO  3 to ll  10 O)  Figure 9.  A graph of the pressure volume curves of the 6 patients with transpulmonary pressure measured, plotting the CT derived volume measurement of ml of g a s / g r a m of tissue against the transpulmonary pressure ( c m of H0). The individual curves are s h o w n in A and the s a m e curves are s h o w n in B w h e n corrected for percent of individual TLC. 2  43  0)  1E .E w jD  CM  co o  CD  0)  « 1  2> E =3  o  CO "O CO OJ CO  c  2 £ O C M  -9Q> 5 2 co• CD 2> wC ° > -D CD CD  LL-  E  S a,  — I CO • CD  00 C CU ~ 1 — CD £ 3 x: c TO O  «t E  o O)  X O)  E  o  CD P co  co t  ^  CD T3 CD XI CD  I  .E CD _ .E o  CD .TO ^ CD W CD  o ^  CD CD 0 5  c  CD "O  | » 8 O  -c  3  CD  TO CD  < l i  3 TO  (O Hui ) ajnssajd z  3  44  Discussion  3.7  T h e values obtained for lung a n d lobe v o l u m e (table 4 a n d 5) using this technique  similar to reported values f r o m autopsy specimens (159,188,249), radiographic, bronchoscopic  scintigrams a n d inert gas dilution (188). T h e technique also provides the a d v a n t a g e that it  minimaly invasive to the patient a n dn o assumptions are m a d ea b o u t lung shape. T h e fa  the weight of the lobe calculated f r o m the C T density w a s significantly greater than that o  resected specimen, can b e partially attributed to the loss of blood f r o m the specimen du  a n d after resection. H o w e v e r , as the v o l u m e of the O C T inflated specimens w a s9 4±3  the v o l u m e calculated f r o m the CT, the specimen w a s inflated close to it's v o l u m e within intact thorax.  T h e technique described here is less technically d e m a n d i n g than those that seek to track lung v o l u m e during C T using spirometry a n d the results s h o w that o n average the  lung w a s inflated to 6 5 . 5 ± 11.9% of TLC during the scan. T h e regional lung data also  linear relationship b e t w e e n lung v o l u m e expressed as a percent of TLC a n d height within  thorax (figure 8) with a slope of 1 . 8± 0 . 1 % T L C / c m calculated using the restricted m a  likelihood analysis (53). This confirms the classic findings of Milic-Emili et al (154) a n dK a n a etal {109) using Xenon. This difference in lung v o l u m e is d u e to a gradient in pleural 1 3 3  pressure w h e r e the d e p e n d e n t portions of the lung are at a lower transmural pressures th u p p e r regions (figure 9). O u r data s h o w s that this pressure gradient w a s0 . 2 4±0 . 0 8  c m H 0 / c m (figure 1 0 ) which c o m p a r e s very favorably to the 0 . 2c m H 0 / c m that Milic-Emili 2  2  reported o n healthy volunteers (154). This m e a n s that at a n y level of inflation, the u p p e r  o f the lung are m o r e inflated than the lower regions. As Millar a n d co-workers (156) fou  tissue v o l u m e including vessels u n d e r5m m in diameter w a s uniform f r o m the top of th  the bottom, the observed c h a n g e in lung attenuation m u s tb e d u e to regional differences inflation. These observations confirm others in the literature ( 1 5 5 , 1 5 6 , 1 9 8 , 2 4 2 , 2 4 8 )a n d  4 5  extends t h e mb y providing a simple quantitative m e t h o d for estimating the regional difference  in lung expansion (91). T h e individual P V curves can also b e used to determine the local  transmural pressure at each lung height a n d calculate the pleural pressure gradient within th  thorax. Furthermore comparisons of the regional lung v o l u m e s at a given lung height allow  determination o fw h e t h e r the lung v o l u m e of each region is appropriate for that lung height  not. This approach should b e useful in defining e m p h y s e m a t o u s destruction w h e r e the regio  v o l u m e will b e shifted u pa n d chronic interstitial disease w h e r e the regional v o l u m e should shifted d o w n .  T h ev o l u m e fraction of blood vessels obtained b y point counting the C Ti m a g e sw a s  overestimated c o m p a r e d to point counting directly f r o m the lung surface (table 7a). This co b e related to t w o factors. Firstly, the v o l u m e fraction estimates rely o n a slice with zero  thickness, such as the cut surface of a physical slice, b u t the C Ti m a g e is an average o  c o m p o n e n t s within the slice. Therefore, the orientation of the vessel in the slice, m a y cau  v o l u m e fraction to b e overestimated. This is k n o w n as the H o l m e s effect in the stereologic  literature (4), a n dv o l u m e averaging in the radiological literature (165). Secondly, part of th difference b e t w e e n the C Ta n d specimen results could also b ed u e to the inevitable loss  blood f r o m the specimen as a result of surgery. T h e large size a n d relatively spherical sh  the t u m o r in relation to the slice thickness reduces the H o l m e s effect a n d accounts for the a g r e e m e n tb e t w e e n the estimate of t u m o rv o l u m ef r o m both the C Ta n d the resected specimen. Therefore, w e attribute the overestimate of v o l u m e fraction of blood vessels to  problem of point counting of objects that are significantly smaller than the thickness of the  slice. This p r o b l e m can b e minimized with high-resolution C T which w a sn o t available for study.  T h e surface area of the entire lung of the nine patients in G r o u p 2 (table 7b) w a  estimated at Level 2 o f the histologic analysis b y counting the grid line intersects with lung  4 6  p a r e n c h y m aa n d multiplying b y the v o l u m e of the lung obtained f r o m CT. This s h o w sa  surface area of 1 0 1 . 8±3 5 . 1 ma n dam e a np a r e n c h y m a tissue thickness is 7 . 2±1 . 0 2  7b). These values are consistent with published data o np o s t m o r t e m lungs (249) a n d pre  studies f r o m ourlaboratory (89). At the level of lung inflation achieved during the C T scan  (65.5% of TLC) 60.1% of the specimen consisted of air. T h e parenchymal V o l u m e fraction  which is the s u m of the capillary, m e d u im sized blood vessels ( < 1 m m ) a n d actual lun  v o l u m e fraction accounted for 17% of the total v o l u m ew h i c h also c o m p a r e s favorably with  published data (249). T h e fraction of the lung that is occupied b y air a n dp a r e n c h y m a ca  b e determined f r o m the C T density b y dividing the specific v o l u m e of tissue (assuming a  of 1 . 0 6 5g / m l (91)) b y the calculated specific v o l u m e of the lung (tissue a n d air). This p  a slightly higher p a r e n c h y m a lv o l u m e fraction (22%) than the histology (17%) which sugges that the C T estimate m a y include larger blood vessels than w e r e observed histologically.  H o w e v e r , these C T estimates o f the fraction of tissue a n d air can b e used to correct th  histology to the appropriate air a n d tissue fraction in the intact thorax. Although this correc is small in the present study because the specimen w a s inflated, it should b e particularly in quantitative studies of lung biopsies w h e r e lung inflation is a n uncontrolled variable.  T h e results of this study confirm that the C T can b e used to obtain accurate estim the total lung v o l u m ea n dw e i g h t as well as regional lung expansion  ( 8 2 , 9 1 , 1 4 6 , 1 5 5 , 1 5 6 , 1 8 3 , 1 9 8 , 2 4 2 , 2 4 8 , 2 7 0 ) . T h e y also extend those observations b y providing a  simple m e t h o d of converting lung density to the v o l u m e of gas per g r a m of tissue. This  approach allows the C T data to b e integrated with physiologic m e a s u r e m e n t s of the pressu  v o l u m e characteristics o f the s a m e lung; to calculate the v o l u m e fractions, surface area a n  tissue thickness appropriate for the entire lung; a n d to correct these values to the appropr intra-thoracic lung volume.  4 7  CHAPTER 4: INTERSTITIAL LUNG DISEASE  4.1  Introduction to Pulmonary Fibrosis Idiopathic pulmonary fibrosis (IPF) forms what is probably a heterogeneous group of  disorders within the broad category of interstitial lung disease. The original description of IPF was based upon a pathologic description of the fibroproliferative expansion of the interstitium seen on autopsy or at open lung biopsy (25,46,113,217). However, it has become evident that the disease process involves an inflammatory infiltrate into the airspaces with subsequent reorganization of both the infiltrate and the interstitium and not the interstitium alone (88,113,124,126). Therefore, the term infiltrative lung disease has been proposed for the group rather than interstitial lung disease because it is more descriptive of the process (165). Hogg suggested that the entire group of diseases should be reclassified in terms of their pathogenic origin instead of descriptive histology (88) and his  Inflammatory Process  Lung Injury  classification divides  Neoplastic Process  r-\ Eosinophilic Granuloma  Return to Normal  the diseases into  rH  two main groups, or  Acute Inflammation  Lymphangiomyomatosis  Chronic Inflammation  Lymphoma  pathways, Usual Response  depending on whether the process  Variant Response.  U I P = Fibrosing Alveolitis  Granulomatous Response  DIP  is based on the  BOOP  End Stage Lung  inflammatory or neoplastic process  Figure 11. Classification of interstitial lung diseases based on  (figure 11). This is  pathogenesis (88)  48  an important distinction because it elevates the classification beyond qualitative descriptions into a grouping of mechanisms that are responsible for the tissue changes observed. It is important to note that the end result of all of these conditions is the end stage lung which, on , both CT and gross examination, has the appearance of dense fibrotic lung filled with "honeycomb" cysts at which point the origin of the process is lost within the massive fibrotic changes seen in the lung tissue. The most common form of IPF is the result of the nongranulomatous inflammatory processes referred to as the usual form of interstitial pneumonia (UIP) in North American (88,113), or cryptogenic fibrosing alveolitis in Europe (46,88). These terms are based on a histologic description of the tissue changes within the lung, but when this description is combined with the radiological and clinical features it is known as idiopathic pulmonary fibrosis (96,113). The etiology responsible for IPF is unknown but auto immune disorders (46,99,113,172,232), occupational exposure (34,102,113), and genetic abnormalities (34,113) have all been suggested.  4.1.1  Clinical Description of IPF Most patients with IPF die within five years of diagnosis and less than 20% of patients  respond to any sort of treatment (52,104,184,206,208,209). It usually begins with an insidious onset of breathlessness with lung function tests showing a reduction in static lung volumes (TLC, FRC, RV) with a normal, or even elevated, FEV^FVG ratio due to a greater decrease in the FVC than FEV, because the conducting airways are relatively normal (184,261). The diffusing capacity of the lung is also reduced and this was first thought to be due to the fibrosis creating an anatomic barrier for the diffusion of oxygen. However, subsequent studies showed that the major problem is ventilation-perfusion mismatching and that the barrier to diffusion only becomes important during exercise (37,54,62,84,98,118,240). The pressure volume curve of  49  patients with IPF is shifted downward and to the right signifying that the lungs in IPF are less compliant, or stiffer, than normal lungs (37,62,261,275).  4.1.2  Radiological Description of IPF  Radiologically IPF shows reticular, reticulonodular, and "ground glass" pattern on chest X-ray and CT that usually involves both lungs, but is predominantly located in the lung bases (164,165,231).  The subpleural distribution of the disease is best appreciated with the use of  high-resolution computed tomography (HRCT) which has greatly increased visualization of the lung. HRCT uses narrow beam collimation to minimize volume averaging and a high spatial frequency reconstruction algorithm to show the lung parenchyma clearly. As a result, small changes in lung density can be appreciated. As the disease advances, the cystic, "honeycomb" pattern becomes evident with most of the normal lung being replaced by dense connective tissue (164,165).  4.1.3  Histologic Description of IPF  The hallmark of UIP is the variegated appearance of the lung on biopsy, with regions of marked inflammatory and fibrotic changes right next to normal appearing regions (34,37,46,113). The affected regions show several stages of the disease starting from signs of lung injury and repair that include epithelial necrosis and alveolar collapse with exudation of fluid and inflammatory cells (34,37,113). The subsequent interstitial and intra-alveolar fibrosis is characterized by an increase in fibroblast numbers and a large increase in the ECM (34,37,113). The interstitium is thickened in these regions and there is often hyperplasia of alveolar type II cells consistent with the repair process (34,37,113). Associated with these interstitial changes are proliferation and thickening of smooth muscle surrounding both the arteries and the bronchi.  50  Histologic examination of the peripheral lung s h o w s that the inflammatory exudate  contains predominately lymphocytes, plasma cells and alveolar macrophages, with s o m e P M N s (37,46), while Bronchoalveolar lavage (BAL)  s h o w s a large proportion of PMN,  m a c r o p h a g e s-  and eosinophils t37,46,205), all of which are increased in patients who s m o k e (205).  4.2  Fibrotic Mechanisms  4.2.1  Cellular Mechanism of IPF  L u n g tissue injury results in a proteinaceous exudate into the air space associated with a recruitment of P M N s followed by m o n o c y t e sf r o m the microvasculature (126). The neutrophil response is acute and their primary purpose is to destroy infectious agents. After migration, the m o n o c y t e s differentiate into alveolar m a c r o p h a g e s that phagocytose and kill pathogenic  organisms, scavenge tissue debris and release cytokines. These cytokines include interleukin-  1 (IL-1), platelet derived g r o w t h factor (PDGF), epidermal g r o w t h factor, t u m o u r necrosis fact (TNF), and transforming g r o w t h factors -a and -(3 (TGF-oc, TGF-p). The replacement of surfactant by this exudate increases the surface tension causing alveoli to collapse onto the alveolar ducts (34,88,126). Fibroblast proliferation, differentiation into myofibroblasts and migration into the region organises the exudate into a fibrotic scar (15,29,34,88,113,125,126,160,187,191). The fibroblasts initially synthesize type III collagen, which in later stages b e c o m e s predominantly type I, fibronectin, and proteoglycans (9,15,29,125,149,191). The new ECM  is then re-epithelialized by migrating type II epithelial  cells and the end result is a cystic, fibrotic lung, with a reduction in alveolar surface area and total lung v o l u m e (34,37,88,126). The m o s t obvious histological and biochemical c h a n g e in areas of fibrosis is the increased a m o u n t of collagen. There are several m e c h a n i s m s for the increased collagen aside f r o m the obvious increase due to m o r e fibroblasts ( 9 , 4 2 , 1 2 6 , 1 4 9 , 1 9 0 , 1 9 9 ) and the increased synthetic ability of the fibroblasts (9,126,149).  51  Fibroblasts within the gingiva h a v eb e e ns h o w n to h a v e a reduced ability to phagocytose  collagen (147) although analogous m e c h a n i s m sh a v en o tb e e ns h o w n in the lung. Addition there is evidence for decreases in the synthesis of the collagen degradation e n z y m e s (i.e.  collagenase a n d stromeiysin) which are k n o w n as metalloproteinases, in conjunction with an  increase in the production of molecules that function as tissue inhibitors of metalloproteinase  (TIMP) (20,34,190). Therefore, the increase in cell n u m b e r , synthetic activity, a n d decrease degradation leads to an increase in the E C Mc o m p o n e n t s .  4 . 2 . 2 Molecular Mechanisms of IPF  TGF-p is the most well studied of the cytokines released by the process, and appe play the central role in controlling the response of the m e s e n c h y m a l cells in terms of cell differentiation, proliferation, synthesis of matrix c o m p o n e n t s ,a n d secretion of the other cytokines responsible for the propagation of the response (18,20,119,147,190,196).  TGF-p is  released initially b ym a c r o p h a g e sa n d platelets a n d is a very strong chemoattractant for phagocytes a n d fibroblasts ( 1 1 9 , 1 4 7 , 1 9 0 , 1 9 6 , 2 6 8 ) . O n c e stimulated, fibroblasts synthesize  TGF-p which has an autocrine effect on itself so that the process can continue without th of inflammatory cells (20).  TGF-p has been shown to increase the production of collagen,  proteoglycans, fibronectin, TIMPs, a n d decrease the synthesis of collagenase (20,190). It a  inhibits endothelial cell division, a n d the proliferation of I L 1 d e p e n d a n t inflammatory cells (1 T h e proteoglycan, decorin, has b e e ns h o w n to bind to  TGF-p and inactivate it (20) and  increase in decorin in the early stages of fibrosis led to the postulate that decorin binds  T  a n d directs it to the m e s e n c h y m a l cells to potentiate the migratory a n d synthetic effects (1 Another g r o w t h factor, PDGF, is released f r o md a m a g e d endothelium, platelets and  fibroblasts a n d has also b e e ns h o w n to cause proliferation of fibroblast cell lines ( 2 0 , 1 2 2 , 1 9  I L 1 is a cytokine that stimulates the m i m u n e system as well as acting as a m i t o g e n for  5 2  endothelial cells causing them to proliferate and migrate over the new connective tissue (190). Studies of the kidneys have shown that collagen breakdown products stimulate fibroblasts to proliferate and produce collagen in the absence of any inflammatory response (212) suggesting that a prolonged inflammatory response is not necessary fOr fibrosis (20,122). In summary, the remodelling of the lung matrix to produce the changes in IPF requires input from many sources, all of which stimulate fibroblasts through the production of TGF-p\ This response is an expression of the normal response to injury that is necessary for the elimination of harmful agents and the repair of the damaged tissue (15,34,258). However, the fibrotic response in IPF is maladaptive in that it does not abate and the tissue is never returned to a normal state (15,34).  4.3  Quantitative Studies of  IPF  • There are several problems with quantitative studies of fibrosis in human lungs. The first problem is obtaining suitable tissue for analysis because autopsy specimens have often reached the end-stage with only large masses of connective tissue remaining. Open lung biopsy specimens are better than autopsy specimens, but because of the variable distribution of, the disease it may be difficult to obtain enough specimens to get a truly representative sample of the lung. Also, the biopsy collapses during the surgical removal and must be reinflated to an appropriate level to perform a quantitative analysis. However, since the biopsy is usually from peripheral lung the airway structure is no longer available for instillation of fixative and the pleural surface is disrupted so that it does not contain the fixative at an appropriate pressure within the lung parenchyma to inflate the airspaces* BAL is a very popular method for assaying the extent and the progression of IPF. Many attempts have been made to correlate the cells and molecules retrieved with BAL to pulmonary function (37,39,62,78,176,186,207) or.disease activity.  53  ( 6 0 , 6 8 , 8 6 , 1 4 1 , 1 4 5 , 1 7 0 , 1 8 2 , 1 8 6 , 1 9 4 , 2 0 3 , 2 0 5 , 2 2 1 , 2 2 3 , 2 4 3 , 2 4 5 , 2 7 8 ) . H o w e v e r , BALand other biochemical studies h a v e the s a m e intrinsic p r o b l e m in that local acting cytokines a n d  inflammatory cells can all get m i x e du p in the s a m e test tube with n o regard to their loc  within the tissue so that functional or pathogenic conclusions are difficult at best. Also, B A  w a s h e s cells predominately f r o m the larger airways a n dm a yn o tb e truly representative of alveolar situation.  T o date there are very f e w quantitative studies o n IPF lungs. T h e histologic studies either qualitative descriptions of the disease process ( 1 1 3 , 1 2 4 ) or the extracellular matrix changes ( 9 , 2 1 , 2 3 , 3 0 , 5 5 , 6 1 , 8 6 , 9 5 , 1 0 0 , 1 1 1 , 1 2 5 , 1 3 7 , 1 4 3 , 1 6 1 , 1 6 9 , 1 7 1 , 1 7 7 , 1 8 5 , 1 9 1 , 2 0 1 , 2 0 3 , 2 2 1 , 2 6 9 ) or semi-quantitative analysis involving the use of complicated scoring systems. Several scoring  systems h a v eb e e n derived b u t these are very c o m p l e xa n d for best results involve the u panel of trained observers (25-27,101,244). H y d e has reported a comparison b e t w e e na  stereologic quantification of biopsy specimens a n d a panel grading system a n d reports that there is g o o d correlation b e t w e e n the t w o arguing that the time consuming quantitative approach is n o t justified for an analysis of the tissue changes in IPF (26,101). C T analysis has fallen into this s a m e arena as histology. Semi-quantitative scoring  systems are reported to b e faster a n d less expensive than the c o m p u t e ra n d time intensiv quantitative studies ( 5 , 6 , 8 , 1 0 , 5 1 , 6 3 , 6 7 , 7 9 , 8 0 , 8 5 , 1 3 3 , 1 4 0 , 1 6 7 , 1 7 5 , 1 8 0 , 1 9 3 , 2 0 4 , 2 2 5 , 2 3 8 , 2 5 9 , 2 6 0 , 2 6 7 ) . Also,  quantitative studies of the C T scans in IPF are relatively n e wa n d the results are still o p e  interpretation as to w h a t aspect of the C T scan should b e quantified, or w h a t aspect give  reliable estimate of the disease process. Investigators h a v e reported an increase in overall  lung density a n d changes in the frequency distribution curves of the voxel attenuation value  the C T scan (31,78,155,156). Hartley studied the changes in the frequency distribution curv  5 4  interstitial lung disease and s h o w e d a correlation b e t w e e n the m o m e n t s( m e a n , median, m o d e  kurtosis, skewness) of the frequency curves and other m a r k e r s of disease activity such as lung function and BAL results (78).  4.4  Experiment # 2  In this study, we report results obtained using a new m e t h o d for quantifying structural changes in the lungs of living patients with IPF using data obtained f r o m CT scans and quantitative stereology (32). attenuation values on CT.  Total and regional lung v o l u m e s are estimated f r o m X-ray The surface area of the lung p a r e n c h y m a and the v o l u m e fraction of  each of the c o m p o n e n t s of the lung is quantified by line intercept and point counting techniques using both the light and electron microscope. This study s h o w s that a combination of the  CT  and histologic data provides accurate information a b o u t the changes present in the lung and could provide a basis for measuring the progression of disease in subsequent CT studies.  4.5  Material and Methods  The procedures used in this study w e r e approved by the ethical review boards of the University of I o w a Hospitals, St. Pauls ' Hospital, and the Universities of I o w a and British  Columbia. All the patients in this study signed informed consent forms that allowed the use of  physiologic data, CT scans, and the surgical tissue. The patients with IPF w e r e enrolled by the National Institutes of Health Specialized Center of Research in Interstitial L u n g Disease at the  University of Iowa. To be included in this g r o u p patients had to h a v e a clinical history, chest Xray, and p u l m o n a r y function data suggestive of IPF, and a pathological diagnosis of usual interstitial p n e u m o n i a on o p e n lung biopsy. The control subjects w e r e enrolled in the lung registry of the University of British Columbia P u l m o n a r y Research Laboratory and w e r e part of an ongoing study of lung structure and function. These patients required either a lobectomy or  55  p n e u m o n e c t o m y for a small, non-obstructing, peripheral bronchogenic carcinoma w h o s e lung function was m e a s u r e d a few days prior to surgery. The patients selected had n o r m a l lung function and w e r em a t c h e d for age, sex and s m o k i n g history with the IPF patients.  4.5.1  Pulmonary Function Studies  Control Patients:  Spirometry, lung v o l u m e s and lung compliance w e r em e a s u r e d with subjects seated in v o l u m e displacement b o d yp l e t h y s m o g r a p h at the P u l m o n a r y Research Laboratory, in Vancouver. Functional residual capacity (FRC) (44,146,181). Total lung capacity (TLC) FRC.  Residual v o l u m e (RV)  was m e a s u r e d using the Boyles ' Law technique  was calculated by adding inspiratory capacity (IC)  was calculated by subtracting vital capacity (VC)  to  f r o m TLC.  M e a s u r e m e n t s of lung v o l u m e and its subdivisions w e r e obtained on a P.K. M o r g a n computerized p u l m o n a r y function testing s y s t e m (P.K.  Morgan, Boston, Mass.) using a dry  rolling seal spirometer and multiple breath helium dilution techniques on subjects who w e r e unwilling to enter the plethysmograph. Diffusing Capacity (D co) by the single breath m e t h o d L  as described by Miller and associates (157)  on the P.K.  M o r g a na u t o m a t e d diffusing capacity  analyzer. The results are corrected for alveolar v o l u m e (V ) and reported as b o t h D co and A  DLCCVVA-  The predicted n o r m a l values for FEV FVC,  and TLC  was predicted using Goldman s ' values (70).  1 f  L  and D o w e r e those of Crapo et al LC  (33)  IPF Patients: The lung function for this patient g r o u p was studied at the University of Iowa. Spirometry was obtained using a Medical Graphics 1070  s y s t e m (St. Paul, MN) and the lung  v o l u m e sw e r e obtained with the patients seated in a b o d yp l e t h y s m o g r a p h (Medical Graphics 1085  system). The Dco was m e a s u r e d using the single breath technique on the Medical L  56  Graphics 1 0 7 0 system. T h e predicted n o r m a l values w e r e those of Morris et al (162) for FVC, G o l d m a n et al (70) for TLC , a n dV a nG a n s e et al (237) for Do (78). L C  4 . 5 . 2 C T Studies Control Patients: T h e control subjects in the study received a conventional C T scan (10 m m thick  contiguous slices) o naG E9 8 0 0 Highlight A d v a n t a g eC T scanner (General Electric Medical  Systems, Milwaukee, Wl) approximately o n ew e e k prior to resection. All scans w e r e perform  with the subject supine during breath holding at full inspiration. Conventional clinical scannin  parameters in use at St. Pauls ' Hospital w e r e used ( 1 2 0 kVe, 1 0 0m A , 2 sec scan tim i m a g e sw e r e reconstructed using a standard algorithm, a n d printed o n t o radiologic viewing  at a w i n d o w of 1 2 0 0a n d a level of 7 0 0 HU. T h ei m a g e data w a s transferred to a Si Graphics I n d y Workstation (Mountainview, CA) for analysis of the X-ray attenuation values.  JPF Patients:  T h eC T scans of the IPF patients w e r e obtained at the University of I o w ao n an  C 1 0 0 ultrafast scanner during inspiration with the patient prone. These high resolution C T  ( H R C T ) scans w e r e obtained using 3 m m sections spaced b y2 0m mg a p sf r o m the lu  to the d i a p h r a g m( 1 3 0 kVe, 6 4 0m A ,0 . 6 s scan time) a n dw e r e reconstructed using a  spatial frequency algorithm. T h e images w e r e printed onto standard radiologic film at a w i n  of 2 0 0 0a n d a level of 5 0 0 HU, a n d sent, along with the i m a g e data, to the P u l m o n a  Research Laboratory in Vancouver. T h ei m a g e data w a s transferred to a Silicon Graphics Workstation (Mountainview, CA) for attenuation analysis.  Ap r o g r a m to s e g m e n t the lungs f r o m the chest a n d the large central blood vesse  analyze the X-ray attenuation w a s written for the numerical analysis p a c k a g e P V - W a v e (Visua  5 7  Numerics, Boulder CO).  The lungs w e r es e g m e n t e d using threshold settings of -1000  to  -500  HU. The v o l u m e of the w h o l e lung (tissue and airspace) was calculated by s u m m i n g the pixel dimensions in each slice and multiplying by the slice thickness. The m e a n CT attenuations in HU of each pixel was calculated and converted to gravimetric density ( g / m l ) by adding 1000 the HU value and dividing by 1000  (equation 7) (82).  to  The weight of the lung was calculated by  multiplying the m e a n density of the lung by its volume. Regional v o l u m e s of gas per g r a m of tissue w e r e calculated according to equation 8. To eliminate the affect of b o d y position during the scan (supine versus prone) on the distribution curves of lung inflation, the v o l u m e of gas  per  g r a m of tissue was expressed as percent of the patients ' TLC which was obtained by dividing the TLC m e a s u r e d in the upright position ( w e r e all the alveoli are evenly inflated) by the m e a s u r e d lung weight. Because of the inflation artifact associated with the biopsy specimens in the patients, the  V  V(C  T)  IPF  of the biopsy was estimated by identifying the s e g m e n t on CT that was  biopsied using the surgical report, and the position of the surgical clips on the post-operative chest X-rays (PA and lateral) and calculating the  V  V ( C  T)  from equation 9 (32).  This value was  then used as the level 2 V in the stereological analysis below. V  4.5.3  Quantitative Histology  Control Patients: The resected lung specimens w e r e obtained directly f r o m the operating r o o m and taken to the laboratory w h e r e they w e r e weighed, inflated with Optimal Cutting T e m p e r a t u r e (OCT) c o m p o u n d (Miles Laboratories, Elkhart, IN) diluted 1:1 with normal saline, re-weighed, and frozen in liquid nitrogen without clamping the airways (32).  O n c e frozen, the specimen was  into 2 cm thick slices on a b a n d s a w and the tissue sampled with a p o w e r driven hole-saw. These samples w e r e stored at -70 °G for other purposes. The remainder of the specimen was  58  cut  transferred to 10% buffered formalin a n d fixed at r o o m temperature for at least 2 4 hours.  R a n d o m samples w e r e taken for light microscopy f r o m the fixed slices, e m b e d d e d in para  a n d stained with hematoxylin a n d eosin. Small samples w e r e taken for electron microscop  (EM) f r o m the peripheral lung of five of the patients prior to O C T inflation a n d freezing a  b y inflating with 2.5% glutaraldeyhyde in 0 . 1 Ms o d i u m cacodylate buffer using a n 1 8g a  needle. These specimens w e r e post fixed in 1 % Os0 in 0 . 1 Ms o d i u m cacodylate, deh 4  through graded ethanol a n d infiltrated with LR-White. T h e tissue blocks w e r e sectioned w  d i a m o n d knife o n an ultra-microtome ( Reichert Ultra-Cut or R M CM T 6 0 0 0 XL) at 6 0 9 0  picked u po n formvar coated 2 0 0m e s h copper grids a n d stained with Uranyl Acetate, a n Satos ' Lead solutions.  IPF Patients:  At t h o r a c o t o m y the lung w a s biopsied f r o m at least 2 regions, o n e that appeared n  a n do n e that appeared diseased o nH R C T . T h e site of the biopsy w a s confirmed b y po  operative chest X-rays in the P Aa n d the lateral position. T h e biopsy specimens w e r e spl  a small portion w a s prepared for E M as above, a n d the remainder w a s processed for lig microscopy.  T o optimize the sampling for the stereologic analysis, a cascade design technique w  used as described in Chapter 1 a n d represented in figure 5. I n this design the lung is  in a series of steps that increase in magnification so that w h a t is quantified at o n e level further sub-divided into it's c o m p o n e n t s at the successive level (35).  Level 1 w a sp e r f o r m e do n the C T scans of both groups, using all of the availab  slices b y counting the n u m b e r of points falling o nn o r m a l lung, densely fibrotic lung, "grou  glass opacification", bronchovascular bundle, a n dt u m o r (figure 5A). Levels 2 a n d3w e r e  performed at the light microscopy level using the point counting p r o g r a m G r i d d e r (Wilrich T  5 9  Vancouver, B.C.) which generated random fields of view, projected a grid on to the field of view via a camera-lucida attachment on a Nikon Labophot light microscope and tabulated the counts. Level 2 usedTOOx magnification with a grid of 80 points (d = 0.11 mm ) and 40 lines (I = 0.11 mm). The number of points falling on airspace, tissue (lung parenchyma), and medium sized blood vessels (50-1000 urn) as well as the number of intersects between the grid lines and the parenchymal-airspace interface were tabulated (figure 5B-C). Level 3 was performed on 10 random fields of view per slide at 400x magnification and the number of points falling on airspace components (Alveolar macrophages, alveolar PMN, other objects in the airspace, and empty space) as well as the tissue components (alveolar wall, capillary lumen, and small blood vessels (20-50 urn)) were counted using a 100 point grid. Level 4 was performed using TEM images. 10 systematic area weighted fields per grid (35) were photographed onto 35 mm slide film at a magnification of 1080x using a Phillips 300 transmission electron microscope. These slides were projected onto a grid of 120 points with a magnification of 10x to give a final magnification 10800x and the number of points falling on electron-lucent space, collagen fibers, elastin fibers, interstitial cells, inflammatory cells and unidentifiable substances were tabulated using Gridder  (figure 5D and figure 12).  The volume fraction (V ) of each of the lung components, v  at each level according to equation 3, and the surface density  (S ( r)) V  pa  (V (i )), V  C  were estimated  was estimated using  equation 4. Since surface density is the surface area in a given volume, the surface area of the parenchyma is calculated by multiplying the surface density by the volume of the lung. The inverse of S is an estimate of parenchymal thickness. The overall V is calculated by v  v  multiplying the V of the lung component at the highest level by the V that contained it in the v  v  previous levels according to equation 5.  60  Figure 12.  Representative electron micrograph from IPF patient biopsy. IC: interstitial cell, Col: collagen, El: elastin, ES: electron-lucent space.  61  4.5.4  Statistical Analysis  All data w e r e analyzed using independent t-tests or the one way analysis of variance. Transformations w e r em a d e on certain variables to normalize distributions and to m a k e variances h o m o g e n e o u s . A Bonferroni sequential rejective procedure was used to correct for multiple comparisons (94).  4.6  A corrected p-value of less than 0.05 was considered significant.  Results  Table 8 s h o w s anthropometric and lung function data for the patients studied. The control patients h a v en o r m a l lung function are of similar sex distribution but are slightly y o u n g e r and lighter than the IPF group. The IPF g r o u ps h o w the pattern characteristic of restrictive lung disease with a reduction in F E V Y FVC, and TLC),  D p LC  and in the subdivisions of lung v o l u m e (RV,  FRC,  and an increased FEV^FVC ratio.  The CT estimates of lung v o l u m e and weight (table 9) s h o w a reduction in total lung v o l u m e in IPF c o m p a r e d to controls due to a reduction in the v o l u m e of the airspace. The tissue v o l u m e and therefore the lung weight was not different b e t w e e n the two groups. The frequency distribution in ml of gas per g r a m of tissue present in each voxel (figure 13)  was  different b e t w e e n the two g r o u p s (p < 0.001). The control lungs s h o w e d a normal distribution (table 10) with m e a n ,m e d i a n and m o d e values that are closely similar (4.6 ± 0.3,4.5 ± 0.2, 4.3 ± 0.2 ml/g)  and a relatively small variance (18.0  ± 8.2 ml/g)  and s k e w (1.6 ± 0.6 m l / g ) , while  the IPF lungs s h o w a left shifted distribution with a positive s k e w (6.9 ± 3.4 ml/g) large variance ( 1 1 6 . 8 ± 30.8  m l / g ) . The m o d e (1.4 ± 0.3 ml/g)  f r o m control levels, w h e r e a s the m e a n (5.7 ± 0.7 ml/g)  and  and a very  for the IPF lungs was reduced  and the m e d i a n (4.1 ± 0.4 ml/g)  w e r e not  different f r o m the controls due to the large variance. W h e n the m e d i a n values w e r e expressed as percentage TLC,  the values w e r e 72.1 ± 2.5 in control patients, and 80.6  62  ± 5.2 in IPF  patients (p > 0 . 0 5 ) indicating that there w a s little effect of b o d y position o n the degree o inflation.  Figure 1 4c o m p a r e s the v o l u m e fraction of tissue estimated f r o mC T to that obtained with histology. This s h o w s that in the control cases the resected lung lobes w e r e inflated  approximately the s a m e level during b o t hC Ta n d histologic analysis (figure 14a). H o w e v e r , biopsies obtained f r o m the IPF lungs w e r e under-inflated during histologic preparation c o m p a r e d to the C T studies (figure 14b). T h e ability to m e a s u r e this difference a n d use  data to correct the biopsies to the correct level of inflation o n an individual basis is critica subsequent analysis O f these biopsy specimens.  T h e light microscopic data (table 1 1 )s h o w that after the correction to the level of lu inflation obtained during the C T scan, the v o l u m e fraction of tissue is increased a n d the  airspace is decreased in the regions of the JPF lungs w h e r e there w a s radiologic evidence disease, c o m p a r e d to the regions of the IPF lungs that appeared n o r m a la n d the control  T h e regions of the IPF lungs t h o u g h t to b en o r m a lo nC Ts h o w e d an increase in the v o  fraction of tissue c o m p a r e d to controls a n d a decrease in the capillary v o l u m e fraction f r o m controls, while the m e d u im sized blood vessels w e r e decreased in both the n o r m a la n d  diseased regions of the IPF lungs. A m o s t striking finding w a s the difference in surface a  which w a s1 1 3±1 2 m in the control lungs a n d3 0 ± 7 m in the diseased lungs. T 2  2  in surface area w a s associated with a n increase in the m e a np a r e n c h y m a l thickness f r o m  control level of 8 ± 1 u r n to 40 ± 1 1 u r n in the regions of the IPF lungs judged to CT, a n d9 7±2 2u r n in the diseased regions. T h e airspace of both regions of the IPF g r o u p contained a significant a m o u n t of  inflammatory exudate consisting of both proteinaceous fluid a n d cells. T h e percentage of th  alveolar airspace occupied b y fluid w a s significantly increased in the diseased regions of the  IPF lungs w h e nc o m p a r e d with the control lungs. T h e fraction of the airspace occupied b  6 3  m a c r o p h a g e sw a s also increased f r o m control levels in both regions of the IPF lungs a n d  these are expressed as n u m b e r of cells per square m e t e ro f surface area, the m a c r o p h a g e  n u m b e r increased f r o m 12±3X 10 cells/m to 3 0±2 2 X 10 cells/m in "normal" regions 8  2  8  2  the IPF patients a n dw a s significantly higher, 1 5 0±1 0 0 X 10 cells/m ( p < 0.001), in 8  2  regions judged to b e diseased o n CT. Figure 1 5 summarizes the results of the stereological analysis of the tissue  c o m p a r t m e n t of the lung based o n electron microscopy a n d are normalized to the lung w  obtained b yC Ta n d expressed per 1 0 0g r a m s of lung. T h e data s h o w that the electron  space tended to b e lower in the n o r m a l regions a n d higher in the diseased regions of th  lungs c o m p a r e d to control lungs. T h e collagen content s h o w e d a progressive increase f r o m control to diseased IPF regions, w h e r e a s the elastin content o f the tissue w a s decreased  "normal" regions, a n d there w a s a trend for an increase in the inflammatory cells which d  reach statistical significance o w i n g to the large variance in n u m b e r in the different biopsies ( p = 0 . 0 8 ) .  64  CO  c  CO 3  o  '£ a> .co  Q  c .92 co Q_  Group  00  74 ±5* 79 ± 11" 73 ±4* 53 ±4* 88 ±5"  169 ± 3  82 ±5* 68 ±5* 0.84 ± 0.02*  84 ± 5  129 ± 7 115 ± 4 83 ± 6 90 ± 4 105 ± 4 0.72 ± 0.02  67 ±2'  2O)  (yrs)  Q.  Sex  o lc  Age  CO  95 ± 5  (%P)  FVC  °s  IPF 9M/6F  Weight  Height  If  6?  168 ± 3  >  tT  (cm)  FVC  FEVi  8  d  70 ± 3  >  tT  (Kg)  $  <  58 ± 2  (%P)  FRC TLC  81  Control 9M/6F  ? (%P)  >  130 ± 9  d >  O CO Q. CO O CO CO "> "O CD O  > LU LL.  CO  •g co CD ^  "co c g o c d  o  CO Q. CO O O)  c "co o  TJ  c  O O CD CO  o  -J  O  CD CD  E _g o > o CO I—  Q. X CD "O CD O O  0  E O > LO CO  O CD _> CO  "O CD  o  CD C  o o  oo  CO  E  CD  CO  2  LU CO -H  co c CO CD  E  CD i_ CO CO CD  _3  CO >  CD  •g 'x o c o E c o X} CO  o  CO  c CD  > cr CD  E o > "co 3 •g CO CD  d  o  CO Q.  co o  O  d V Q.  c o  oE o c  CD CD  o  &  lO  o  O CO Q. CO O  T3  o>  «  >  o  £• 3  E  IPF 3265 ± 284*  4973 ± 338  _3  (ml)  'CD  Control  <Z Air  2415 ±250*  3952 ±296  (ml)  Volume  CO  Volume  Total  TJ C CO CO CD  Group  Lung Weight (g) 1087 ±55 906 ±71  Tissue Volume (ml) 1021 ±52 851 ± 66  CO CD  o o d  a.  v  CO LU  •H  CO CD  co >  co c  c o O E o  E  c  CD CO CO CD  CD CD TJ Li. CL  © CC  T3  CD  o  T  ""  ro E •*=  5.7 ±0.7  4.6 ±0.3  18.0 ±8.2  (ml/g) 1.6 ±0.6  (ml/g)  Skewness  116.8 ±30.8* 6.9 ± 3.4*  3 _1  IPF  CO  Control  ro c o  (ml/g)  c' Variance  cn  Mean  H—  Group  c g ro . c 1.4 ± 0.4*  4.3 ±0.2  (ml/g)  Mode  4.1 ± 0.4  4.5 ± 0.2  (ml/g)  Meclian  81 ± 5  72 ± 3  %TLC  1  a.  m d  •H O LO  CO  CD  c o  CO  d  •H CO CD  O  c ro  CD  LU CO  c ro  +1  CD  E  CD  V-  ro  J2 ro >  CO CD  O O  d  Q. v  o o E o  c  CD  CD  s i  s  CM •H CO  CM CM -H  o  CO  g~ -H  o  LO  o  u«  -H  o CO  CO  (0  a>  •H  'I  co  ua>  Co  o  CO CM -H O  co LO  CM CM -H  o  +1 CD  CM  +-»  -H CO  -H  T-  LO  S  d  X LU  O  -H  T• -  "H  co  CO +1 CO  CM LO -H  o CO  w  "55  o i  ca 05  CO  (0  CO  E  o o o  ci  +)  o  LO  to  (0  E 0)  E _i  3.  ° © CM  re a o  CO  cu  o  CO CO  c o  3  o -H t  d «  d+i d  c  CM  cu  +1  E  O •H  co d  I  0)  o  co  d  LO  +1 CO  d  d  CO  CM  d  d  d o + ) * co T d *~ co  LO  -H  +i 00  O  CO  d  3  C  o x: o E o C L  C L C O  -C C L O CO CD O ) CO  x:  E Ci O  ure Q. CO  > Q _  CM CO  O  o  1 -  XJ  o 1  o CO  oo  a. 3  O  IS-  c:  o  o  E  o  z  L L Q .  L O  V C L  "co E o c  2 o E k_ _co o CO  CD  .> CO  c  " D  CO  •K c  o o E o  LU CO  +1  +1  CD " i— CD  CO  c CO CD  T3  cu w CO c u (0  OL  E CD CO CO CD  _3 CO >  o d  v  O d  v  C L  C L  CO  CO  LO  LO  H—  "CO  CO • •  -H 00  O  C L  CM CD  00 CO  +-»  c  •a CD  co CO CD CO  o c o  o  E o c CD CD  "co 'E o  c o o E o c CD CD  T3 CD CO CO CD CO.  Q  CL  as  zz  o  CO  o  CO  CO L L  c  agai  CM  A  CO sz  c CO 03 * c O CO CO 1—  s  o CO CO '  c o CO to c C O ol X  LO  o o >  10  V> D)  rce he  C O +—» C L to CO c O CO C (0 to CO to CO C O OL c  ex ed  W (0 05  o c o c o  sed repr  0 3  CO  trol  CO  -C  CO  c CO x: CO hx: CO  o to to L O  CO  de  c  ! O  E  i - ciC_oO  o  CO CO  JLZ  -*-»  CL  „, o  CO o o 3 vo sz o CO > Q. CO  co CO  CN  3  S|exoAP%  CO  69  i  w  i ! ! I i : 3  4  5  5  •  8  4• :^ I^ \ \ \  1 9  Patient #  •  2 w  0.10 -  (CT  >» 0.20 o> o  I) -0.10 -  X  ©  -  0.00  |  H 2  1-  3  4  5  6  H 7  f8  10  -  o  c -0.20  1 •  (0  £  9  11  12  13  H 14  1  15  1 I  -0.30 -  a  -  c -0.40 o o -0.50  2  UL -0.60 CO E -0.70 3 o  >  -0.80 -0.90 -1.00  Patient # Figure 14. Shows the volume fraction of the tissue in the biopsied regions of lung estimated from CT minus the volume fraction estimated from histology. A: control group, B: IPF group.  70  TJ  m  1  2 eof  i  i  I If m  15 9= CO CO  to c CO CO XI i o  O CO o ' o i 2 o' 0 XJ v  5 d a .2 3 c <D c o  CL  E o  o ©  12^co  t  TJ C co  -H  8 " « c  C  CO co  « co  CD  1£  i  E  to co c _o  I Jfl <°  in  (A °  • Q.  I§  1  .+=" CO L1 "55 52 °> «- * *  TJ  CO ~  CP  c c (0 •- 3 CO H-  CO  ° O LJ£ O 9; •2* T" CD  l« *t i S8  x : co co *- to X 5 CO 00 ->  5  I  I g£ a n s s j i  Bun-|  Booi Jad ii|6iajy\  71  3  fl)  4.7  Discussion  This study provides n e w quantitative information o n the composition of h u m a n lungs  with IPF obtained using a combination of C Ta n d histologic analysis. T h eC T scans s h o w  reduction in total lung a n d airspace v o l u m e in IPF (table 9), which correlates with the red o f static a n dd y n a m i c lung v o l u m e s obtained b yp u l m o n a r y function tests (table 8). T h e  distribution of v o l u m eo f gas per g r a m of lung in IPF lungs, (figure 1 3 )s h o w s that the l  proportion of the lung has lower v o l u m e of gas per g r a m of tissue than controls, a n d tha are also over-inflated regions indicating larger airspaces. T h e lung regions with l o w gas  v o l u m e s per weight are diseased with reduced airspace, surface area a n d a reorganization the tissue components. T h e regions with a high gas v o l u m e per g r a m are probably the  the formation of cystic spaces in the diseased areas that are thought to result f r o m a col  alveoli onto alveolar ducts with distortion of the ducts b y the repair process (88,126). H o w it is also possible that s o m e of the overinflation is d u e to a concomitant e m p h y s e m a t o u s process (207).  Differences in total a n d regional lung v o l u m e will b e influenced b y the degree of lu  inflation during the C T scan, h o w e v e r , the estimates of lung weight should b e reliable bec it is based o n density of the lung which reflects the degree of lung expansion (volume).  effect of b o d y position o n lung inflation w a s accounted for b y expressing the v o l u m e of ga  g r a m of tissue of each of the voxels as a percentage of the total lung weight divided int TLC m e a s u r e d in the upright position w h e r e the alveoli are k n o w to b e evenly inflated  throughout the lung (154). As there w a sn o statistically significant difference in the m e d i a n  values of percentage TLC b e t w e e n the t w o groups, w e conclude that the lung parameters  estimated f r o mC T can b ec o m p a r e db e t w e e n the t w o groups even t h o u g hs o m eC T sca w e r e conducted p r o n ea n d others in the supine position. There m a yh a v e also b e e n  differences introduced d u e to the different scanners that w e r e used for the study, a n d the  72  thickness a n d reconstruction algorithms used to acquire the images. H o w e v e r ,K e m e r i n ka n  colleagues h a v es h o w n that the reconstruction algorithms a n d slice thickness h a v e a negligib effect o n densitometry m e a s u r e m e n t s (116). Further, the s a m eg r o u p tested n u m e r o u s scanners for densitometry m e a s u r e m e n t sa n d concluded that comparisons b e t w e e n properly  calibrated scanners is possible (117). T h e largest difference in densitometry m e a s u r e m e n t sw  c o m ef r o mi n h o m o g e n e o u s lungs a n d since n o r m a lh u m a n lungs contain a m o r eh o m o g e n  p a r e n c h y m a than the diseased condition, thicker slices will b e appropriate b u t thinner slices  should b e used for the c h a n g e s associated with fibrotic lung disease. Therefore, considerin  of the variances associated with different h u m a n lungs, w h i c h includes the varying degrees disease, w e believe that the differences in scanners a n d scan acquisition are of minimal consequence.  As w eh a v e previously s h o w n , there w a sag o o d correlation b e t w e e n the air to tiss  fraction calculated b y both C Ta n d histology in the control cases w h e r e the resected specim  w e r e fixed in inflation. H o w e v e r , in the IPF lungs the histologic fraction of air to tissue w  m a r k e d l y reduced because the lung biopsies collapse (figure 14). B y locating the s e g m e n t s  the lung that w e r e biopsied o n the pre-operative C T scan, the v o l u m e fraction of tissue a  airspace can b e estimated in the intact chest using equation 9. T h e s e values can then b  to correct the histologic estimates to the appropriate inflation at the time of the C T scan a  very important advance in quantitative histology. Surprisingly the data s h o w that lung weight  a n d tissue v o l u m ew a s the s a m e in IPF a n d control lungs which establishes that the C T  appearance reflects a reduction in airspace m o r e than a n increase in tissue. T h e histology suggests that the reduction in airspace is d u e to a collapse of alveoli o n ducts a n d that  accounts for the m a r k e d reduction in surface area. H o g g (88) has previously postulated tha  the exudate of fluid through the tissue a n d into the airspaces increases surface tension and causes the alveoli to collapse o n t o alveolar ducts. K u h n et al (126) s h o w e d that the  7 3  subsequent organization of this process with epithelial growth over the exudate incorporates the material present in the airspace into tissue. These events change the relative fraction of tissue to air (table 11) but does not significantly increase in the volume of tissue or lung weight. The . major consequence is a decrease in the alveolar surface area from 113 ± 12 m in control 2  patients to 30 ± 7 m in IPF and a ten fold increase in mean parenchymal thickness from 8 ± 1 2  to 97 ± 22 pm because the alveoli account for the majority of the lung surface area (249). The large variance in these values is the result of regions of dense fibrosis being located geographically dose to normal regions (113). This reduction in surface area contributes to the reduction of the diffusing capacity of the lung (table 8), but because the alveolar volume is also reduced, the D coA/ remained within normal limits in these cases. L  A  There was an increase in the volume of alveolar airspace occupied by proteinaceous and cellular components in the IPF biopsy specimens (table 11). Although we cannot discount the effect of surgical trauma on the efflux of edema into the airspace, it is unlikely that this is the sole cause of the cellular infiltrate as the time period between surgical removal of the tissue and fixation is relatively short. Therefore, the increase in volume of alveolar exudate with increased numbers of PMN in the fibrotic regions of the lung is consistent with the hypothesis described previously, and the literature concerning the histologic appearance of IPF (88). The increase in macrophage number in both regions of the IPF lungs is also of interest in that these cells are considered to be key players in directing the fibrotic response (191,213). They are capable of synthesizing large amounts of growth factors including transforming growth factor-p (191), as well as platelet derived growth factor (213) and interleukin-1 (121). Our data demonstrate the variability of the disease process where some regions contain numerous cells, while others are similar to controls. This provides a more accurate picture of the peripheral lung condition than that represented in bronchoalveolar lavage. The degree of tissue reorganization is best appreciated on EM examination (figurel 5)  74  w h e r e the diseased regions of the lung s h o w e da n increase in electron-lucent space, collag  a n d interstitial cells per 1 0 0g r a m so f tissue, c o m p a r e d to the control lungs a n d regions o  IPF lungs n o t grossly involved b y disease. T h en o r m a l appearing lung regions in the patie with IPF w e r en d t significantly different f r o m the controls for a n y of the variables b u t did  less inflammatory cells than the diseased regions. Interestingly the a m o u n t of elastin presen  w a s decreased in the n o r m a l regions of the IPF lungs a n d tended to b e intermediate in  diseased regions. T h e observed reduction in elastin content in the IPF lungs is consistent  reports of minimal synthesis of elastin in m a t u r eh u m a n lungs (211), a n dw e postulate th proteolytic m e c h a n i s m s associated with migration inflammatory cells m a y also result in the reduction in elastin content. T h e increase in the estimated collagen content within the  interstitium has b e e n well d o c u m e n t e d in IPF (46,126,191), a n d our observations suggest th the fibrotic process is well a d v a n c e d in these areas.  B e n s a d o u na n d co-workers h a v e recently s h o w n that in there is a n increase in the  proteoglycan content of the interstitium in regions of IPF lungs w h e r e there is active diseas (13). Proteoglycans play a n important role in the fibrotic response b y acting as receptors  g r o w t h factors (18), directing effects o n the synthesis a n d degradation of elastin (150) a n d collagen (13). Proteoglycans are highly negatively charged molecules that require hydration maintain their s h a p ea n dh e n c e are important for the modulation of tissue hydration (13).  the E M fixation used in our study does n o t preserve these molecules (234), w e speculate  they account for the increase in electron-lucent space present in the diseased areas of the  W e conclude that the C T scan can b ec o m b i n e d with histology to provide a quant  estimate of the tissue changes occurring within the lungs of patients with IPF. T h eC Td provides the ratio of tissue a n d air which can b e used to correct o p e n lung biopsies to  of inflation at w h i c h the C Tw a s performed. This approach could provide a m e t h o d of fo the progress of disease using the C T analysis to estimate the histologic changes.  7 5  CHAPTER 5: PULMONARY EMPHYSEMA  5.1  Introduction to Pulmonary Emphysema Chronic obstructive pulmonary disease refers a group of disorders presenting with  chronic airflow limitation and are usually placed in one of two basic classes. Type A patients have hyperinflation of their lungs without cough and wheezing and are considered to have predominantly emphysema (14,197,261). The type B patients are considered to have chronic bronchitis and they present with cough and sputum and are often more hypoxic and hypercapnic than the type A patients (14,197,261). However, this classification actually represents the extremes of these groups and there is a great degree of overlap between these extreames. The pathologic studies of COPD lungs show even more overlap between the groups with all patients having a degree of emphysema and some airway disease (14,197,229). Emphysema is a pathologic diagnosis, and while there are several established criteria that must be met, it is still a subjective diagnosis. An early definition of emphysema published by the Ciba Guest symposium in 1959 defined emphysema as "enlargement of the acinus that might or might not be accompanied by destruction of the respiratory tissue" (28). In the early 1960's, the World Health Organization (271) and the American Thoracic Society (7) adopted a. similar but different definition that limited emphysema to "enlargement of any part of the acinus with destruction of the respiratory tissue". The current definition: "the abnormal permanent enlargement of airspaces distal to the terminal bronchioles, accompanied by destruction of their walls, without obvious fibrosis," was published in 1985 in an attempt to define the destruction term of the ATS definition and to exclude diseases where there is airspace enlargement but the predominant feature is fibrosis (219). Each of these terms was carefully chosen to differentiate between the enlargement of airspaces which is a natural process in the aging (senile) lung and the compensatory overinflation of the remaining lung following a lobectomy or pneumonectomy,  76  or the generalized airspace e n l a r g e m e n t that occurs in D o w n s ' syndrome. Therefore, in order to be defined as e m p h y s e m a there m u s t be destruction of the normal tissues, defined as  "the  reduction of that tissue to a useless f o r m or nothingness" (219). The qualification "without obvious fibrosis" was a d d e d to the definition to exclude the airspace e n l a r g e m e n t seen in interstitial lung diseases such as sarcoidosis, eosinophilic g r a n u l o m a and in the end stage fibrotic lung w h e r e the p r e d o m i n a n t feature is the fibroproliferative response which is due to a completely different pathogenic process than the e m p h y s e m a t o u s destruction. It should be noted here that for a diagnosis by this definition the lung tissue m u s t be directly observed and  any other m e t h o d of obtaining this diagnosis is presumptive. H o w e v e r , there are m a n y feature of the disease that w h e nc o m b i n e d enable the physician to m a k e a diagnosis of the disease without having to obtain lung tissue.  5.1.1  Functional Description of Emphysema  The m o s t obvious functional characteristic of C O P D is the classic airflow limitation s h o w n by a reduced FEV FVC and FEVVFVC ratio as well as increases in all of the 1 t  subdivisions of lung v o l u m e (197,261). The pressure v o l u m e curve of these patients is shifted up and to the left and there is a reduction in the elastic recoil of the lung ( 9 3 , 1 8 1 , 1 9 7 , 2 6 1 ) signifying a m o r e compliant lung than the n o r m a l condition so that at any trans-pulmonary  pressure the lung will h a v e a higher volume. The airflow limitation in these patients, s h o w nb reduction in the  FEVL  can be due to obstructions within the airways, or to d y n a m i c limitations of  the airways as the equal pressure point migrates t o w a r d s the periphery of the lung and b e c o m e s located within the small airways (197,261). The FEVVFVC ratio is decreased due to the reduction in the FEVi. the subdivisions of lung v o l u m e are elevated in this condition partially due to the airflow limitation and partially due to the increased compliance of the lung. The greatest c h a n g e is seen in the RV resulting in a greatly increased R V / T L C ratio and an  77  RV  that approaches the VC v o l u m e (197). There is often a decrease in Dco which is attributed to L  the loss of alveolar surface area for gas e x c h a n g e and the reduction in the bipod v o l u m e of the lung (261).  5.1.2  Radiological Description of Emphysema  The chest X-ray is an imperfect tool for visualizing e m p h y s e m a because the overlapping structures hide the e m p h y s e m a t o u s changes in the lung p a r e n c h y m a (56,202). Hyperinflation of the chest is s h o w n as the flattening of the diaphragm, best seen on the lateral view, and  an  increased retrosternal airspace (192,202,230). There is also a decrease in the caliber or presence of vessels in the outside third of the lung (192,202,230). The detection of e m p h y s e m a on chest X-rays does not correlate very well with pathologic scores and only has 60-80% diagnostic accuracy with significant false positive rates (202). The introduction of CT scans advanced in the visualization of e m p h y s e m a because it (  r e m o v e s the overlapping structures allowing the lung p a r e n c h y m a to be visualized in cross section. On CT e m p h y s e m as h o w sh y p o d e n s e regions and is usually associated with pruning or obliteration of the p u l m o n a r y vessels (202). CT has s h o w n high sensitivity and accuracy in the diagnosis of e m p h y s e m a with better than 80% sensitivity and only 2-3%  false positives  (202,229). A further advantage of CT over chest X-rays is that CT scans contain the information on the X-ray attenuation values of the lung. These values provide the m e a n s for quantification of lung disease in a sensitive and reproducible fashion that visual grading systems can never achieve.  5.1.3  Histologic Description of Emphysema  There are three m a i n forms of e m p h y s e m a that can be identified on the gross specimen: centrilobular, panlobular and paraseptal. The m o s tc o m m o n form is centrilobular  78  (centriacinar) emphysema which is predominately located in the upper lobes but becomes more diffuse with increasing severity (226,272). Centrilobular emphysema affects the center of the pulmonary lobule and is surrounded by normal lung parenchyma so that on gross examination: the affected lobule collapses in on itself below the level of the bronchi and vessels in the.center of the lobule. Histologically, airspace enlargement is observed in the center of the lobule and is often associated with a distorted respiratory bronchiole (272). Panlobular (panacinar) emphysema is primarily located in the lower lobes and grossly affects the whole lobule so that the lobular septae, the airways and the vessels are elevated above the parenchymal surface (226,272). Microscopically, there is enlargement of airspaces throughout the entire lobule. As the severity of the emphysema increases, it becomes more difficult to differentiate between panlobular and centrilobular emphysema, especially histologically, and the observer must try and find a relatively normal region on the lung to determine the type of emphysema. The third type of emphysema is paraseptal (distal acinar) which is located subpleurally and is characterized by bullae in the upper lobes (272), the walls of which may be fibrotic, while the surrounding airspace is appears normal (272). Emphysema is differentiated from simple airspace enlargement by the anatomical location of the tissue changes, which are focally located in emphysema, and more uniformly distributed in the senile lung. Histologically, the senile lung is usually associated with enlargement of the alveolar ducts and saccules rather than any changes in the alveoli while Down's syndrome shows widened alveolar ducts and enlarged alveoli in suggesting an incomplete alveolar development rather than a destructive process (272).  79  5.2  Pathogenesis of Emphysema  5.2.1  Protease/Antiprotease Theory  Laurell and Ericksson w e r e the first to s h o w that widespread e m p h y s e m a was present in  y o u n g e r patients with od-antitrypsin (aPI) deficiency (131). At a b o u t the s a m e time Gross et r  al (73) s h o w e d that e m p h y s e m a could be produced by instilling the e n z y m e papain into the lung. These observations led to the theory that an imbalance b e t w e e n the release of proteases f r o m stimulated PMN  and m a c r o p h a g e s and the inactivation by s e r u mc o m p o n e n t s could result  in e m p h y s e m a (17,87,103,272). It has b e e n hypothesized that this balance can be upset through a global inactivation of inhibitors, an o v e r w h e l m i n g influx of proteases, or through the creation of a local e n v i r o n m e n tw h e r e the proteases can be released and the antiproteases w o u l d be excluded, such as a "pocket" f o r m e db e t w e e n the PMN  and the endothelial cell  (57,87,131,272). Originally this theory was developed for the h u m a n neutrophil elastase which degrades collagen, fibronectin and proteoglycans as well as elastin but has since b e e n extended to take into account other proteases such as the metalloproteases of the alveolar m a c r o p h a g e s and cathepsin G f r o m the PMN activated PMN  ( 1 7 , 5 7 , 8 7 , 1 0 3 , 2 1 8 , 2 7 2 ) . As well as proteases,  and m a c r o p h a g e s release reactive o x y g e n products through a transfer of  electrons f r o mN A D P H to o x y g e n to f o r m the superoxide ion 0" (57,87). Superoxide reacts 2  with h y d r o g e n ions to f o r mh y d r o g e n peroxide (H0) that in presence of transition metal ions 2  2  can generate the highly reactive hydroxyl radical OH" (57,87). PMN  granules also contain  myloperoxidase which c o m b i n e s with H0 in the presence of chloride ions to f o r m the 2  2  hypochlorous acid O H C I " (57,87). These free radicals h a v e very d a m a g i n g consequences for living tissue by oxidizing m e m b r a n e lipids, oxidizing, cleaving and cross-linking proteins, cleaving DNA,  and changing cell permeability. While they are usually targeted to foreign  substances (bacteria), they can also h a v e their effects on the host tissue (57). Cigarette s m o k e is considered the m a j o r cause of centrilobular e m p h y s e m a because it  80  is known to contain a multitude of chemicals, some of which are powerful oxidants with long half lives, and others inactive <Xi-PI by oxidizing the active site (57,272). Cigarette smoke has also been shown to cause the sequestration of PMN arid monocytes within the lung (142), and promote the release of their granules which contain the proteases and oxidants (87).  5.2.2  Inflammatory-Repair Mechanism The second theory for the degradation of tissue in emphysema is the inflammatory  repair mechanism (57,87,174,272) which operates similarly to the fibrotic response described in the previous chapter. In this mechanism, the inflammatory cells are activated and recruited to the site of an insult where they release their proteases and oxidants which have an initial local degradative effect on the tissue (272). This step is followed by activation of the repair process which increases the extracellular matrix components in the damaged area (58,59). There are numerous reports on the ultra-structural changes in emphysema which show thickened alveolar walls (58,59,168), rearrangement of collagen and elastin fibers (12), initial collagen destruction followed by collagen synthesis (273) and increases in the amount of collagen per unit area of alveolar wall (24,128,129). These data all support the theory that emphysema is not a simple destructive mechanism, but has an initial degradation response that is followed by a fibroproliferative repair phase. It is important to note that these fibrotic changes are all at the microscopic level and the predominant feature of emphysema is destruction and not fibrosis as seen in interstitial lung disease. Therefore, while the distinction of "without obvious fibrosis" is debated by some investigators (272), because this fibrosis is mild it is argued that the qualification should remain as part of the definition to exclude the end stage lung of interstitial lung disease (229). Clearly there is a potential for overlap between the two theories as both mechanisms involve elements of the inflammatory process which has an exudative and proliferative (i.e.  81  repair) phase. Proteolytic destruction occurring during the period of exudation of fluid and cells  followed by a proliferative p h a s e with partially destroyed lung architechure could account for the e m p h y s e m a t o u s lesions.  5.3  Quantitative Studies in Emphysema  Quantitative anatomical studies on lungs with e m p h y s e m ar a n g ef r o m those using sem quantitative scoring systems to detailed stereologic and other m o r p h o m e t r i c analysis of the lung. The introduction of CT resulted in r e n e w e d interest in these areas w h e r e several investigators developed semi-quantitative scoring systems for the CT scans and detailed analysis of the X-ray attenuation values to quantify the extent of the e m p h y s e m a t o u s changes within the lung. M o s t of these studies a t t e m p t to correlate the observed morphologic changes with the changes in the physiology of the lung.  5.3.1  Gross Analysis:  The original analysis of the e m p h y s e m a t o u sc h a n g e was initiated by G o u g h and W e n t w o r t h (71) who pioneered the use of p a p e rm o u n t e d thin sections and led to the original description of the extent and severity of the tissue changes in the disease process (135,229,272). The preparation of the lung for this m e t h o d is extremely tedious and time consuming so Thurlbeck and colleagues developed a modification of this technique which uses a picture grading system w h e r e lung slices are c o m p a r e d to a panel of photographs and assigned a score, or grade, b e t w e e n 0 and 100.  Scores of 5-25  indicate mild e m p h y s e m a , 30-  50 m o d e r a t ee m p h y s e m a and 60 or greater are defined as severe (227,229). It has been s h o w n that the semi-quantitative panel grading system provides a fast, efficient and reliable estimate of the extent and severity of e m p h y s e m a (227,229), but was never designed to be used with lobectomy specimens (272)  and does not provide quantitative data in terms of  82  absolute v o l u m e s or v o l u m e fractions of the lung involved by disease (227). Therefore, a  quantitative analysis Of the lung m u s t use techniques that provide three-dimensional information on the lung specimen and not comparisons to a picture. Quantitative analysis can be achieved using the standard stereologic point counting grid (227)  or a grid of squares ( 1 5 8 , 2 2 4 )  superimposed over the gross specimen and the n u m b e r of points, or squares, over e m p h y s e m a is divided by the n u m b e r over n o r m a l lung.  5.3.2  Histologic Analysis:  A gross analysis gives data on the extent and the severity of the changes in the lung tissue while a histologic analysis attempts to quantify the changes in the tissue at the cellular and molecular level. Since the definition of e m p h y s e m a centers on enlargement of airspaces  with destruction of lung tissue, the m o s tc o m m o n analysis is quantification of the airspace size p e r f o r m e d using the m e a n linear intercept (Lm) technique (4,45,70,93,249). This procedure is p e r f o r m e d by counting intercepts b e t w e e n a test grid of lines and lung p a r e n c h y m a on systematic r a n d o m sections of the lung and is a derivation of the surface area method, described in the opening chapter ( 4 5 , 2 4 9 , 2 5 2 , 2 5 3 , 2 5 7 ) . A variation by Lang and coworkers ( 1 2 8 , 1 2 9 ) uses an a u t o m a t e di m a g e analysis s y s t e m to m e a s u r e the length of the alveolar  walls and then calculates the surface area per unit v o l u m e( A W U V ) . Point counting technique h a v e also b e e n used at this level to estimate the tissue and airspace v o l u m e changes as a result of the destruction (83,227).  These data all s h o w that in e m p h y s e m a there is a reduction in the tissue v o l u m e fractio (83),  with a corresponding decrease in the surface area (S or A W U V ) (56,72,128,129), and v  an  increase in the size of the airspaces as m e a s u r e d by Lm (93,181,229). H o w e v e r , in h u m a n  subjects these results are not straight forward as s h o w n by the w i d er a n g e of alveolar sizes and  surface areas with s o m e estimates of severe e m p h y s e m a t o u s lungs within the normal range  83  (229). Undoubtedly s o m e of this variation is due to the s a m p l e used, which tend to be patients in their sixth decade with significant smoking histories who w o u l dh a v e significant age related changes as well as varying degrees of e m p h y s e m a (229,239). Another analysis is the destructive index (127,229), which records breaks in the parenchymal tissue, and bronchial attachments to peripheral airways. This technique does not provide three-dimensional lung m e a s u r e m e n t s and does not s e e m to be very sensitive to mild changes in the lung p a r e n c h y m a (229). There h a v e also b e e n attempts to quantify the n u m b e r of inflammatory cells within the vasculature and airspaces of e m p h y s e m a t o u s lungs (83,89,241). These studies h a v es h o w n that there is an increase in the n u m b e r of m a c r o p h a g e s within the airspaces of patients with e m p h y s e m a , which is further increased in patients classified as current s m o k e r s (241). T h e y also s h o w that the n u m b e r of PMN  in the microvasculature increases in subjects that are  actively s m o k i n g (142). Studies h a v e also s h o w n that here is an. increase in both the degradation and synthesis of collagen and elastin producing changes in the parenchymal tissue thickness indicative of a fibroproliferative response (12,24,128-130,179).  5.3.3  Radiological Analysis  The CT scan has proven to demonstrate the presence, extent and severity of the e m p h y s e m a and to correlate better with pathology and p u l m o n a r y function tests ( 7 2 , 1 2 0 , 2 2 2 , 2 2 9 ) than the chest film (173,192,202,228). CT scans also yield an i m a g e of the lung slice that is similar in appearance to the gross slice and has led investigators to m o d i f y the picture grading system to be used on gross sections cut in the transverse plane so that comparisons can be m a d e to the CT i m a g e (66).  While these correlations h a v eb e e n good,  they consistently underestimate the extent of the mild e m p h y s e m a( 6 6 , 1 5 8 , 1 6 6 ) which is  attributed to v o l u m e averaging within the slice because the thinner slices used for H R C T yield  84  better correlation with pathology (64,66,246).  Am a j o r source of artifact with C T is d u e to the variable inflation v o l u m e s of the lu  during the scan which can differ f r o m slice to slice a n df r o m scan to scan. This has le  investigators to design apparatus that enable scans to b e obtained at a k n o w na n d reprod  spirometric level ( 1 0 , 1 0 8 , 1 9 5 ) or to develop correction criteria which express the lung inflatio  as a percent of the individuals ' T L C (31,32). This latter technique enables the C T scan to c o m p a r e d to the gross specimen a n d to use stereologic techniques o n biopsy material.  Investigators h a v e also s h o w n that expiratory C T scans are m o r e reproducible intra a n d int  scan, a n d very efficient at demonstrating the hyperinflated regions of the lung d u e to gas trapping (47,65). As previously mentioned, the C T scan data contains information o n the attenuation  rays within the lung p a r e n c h y m a ,a n d Hayhurst s h o w e d that patients with e m p h y s e m ah a d  m o r e voxels in the -900 to 1 0 0 0H U r a n g e (81). I n another study Gould used a c o m p assess the frequency distribution of the X-ray attenuation values a n d found the lowest 5  t h  percentile of the voxels correlated with the extent of the e m p h y s e m aa n dh a d a negative  correlation with m e a s u r e m e n t s of A W U V (72). Muller et al used a similar technique to c o m  the extent of e m p h y s e m a quantified b y a "density m a s k " at different H U cutoff values with extent of e m p h y s e m a quantified o n the gross lung specimen (120,166). H es h o w e d that  best correlation for conventional C T scans used a density m a s k of -910 H Ua n d Geveno followed this study b y demonstrating that -950 H Uw a s the best cut off for H R C T (64).  5.4  Experiment #3  Chapters 3 a n d 4 of this thesis h a v e described a technique which combines C T  m e a s u r e m e n t s with quantitative histology to provide information a b o u t the structure of n o r m a  lungs a n d lung with idiopathic p u l m o n a r y fibrosis (31). This technique uses X-ray attenuatio  8 5  values to estimate lung density a n d calculate lung weight, tissue a n d gas volumes. T h eC  estimates of the proportion of tissue a n d air in the total v o l u m e is then used to correct th  histologic m e a s u r e m e n t sm a d eo n resected lung tissue to the level of lung inflation present during the C T scan.  This chapter c o m p a r e s the lung structure of h e a v ys m o k e r sw h oh a d maintained nea normal lung function with minimal e m p h y s e m a , as defined b y a "density m a s k " technique, that present in patients with similar s m o k i n g histories a n d advanced e m p h y s e m a t o u s lung  destruction. This procedure provides quantitative data o n lung structure that will b e useful i following the natural history of the developing e m p h y s e m a t o u s process, correlating these  structural changes with function a n d assessing the benefit of lung v o l u m e reduction surgery.  5.5  Materials and Methods  T h e procedures used in this study w e r e approved b y the ethical review boards of S  Pauls ' Hospital, the University of Pittsburgh Hospital, a n d the Universities of British Columbia  a n d Pittsburgh. All of the patients signed informed consent forms that allowed the use of  physiologic data, C T scans a n d the surgically resected tissue. T h e yw e r e divided into contr  m i l d e m p h y s e m a , and s e v e r e e m p h y s e m a groups according to the severity of e m p h y s e m a t o u s  destruction of lung tissue. The patients in the control a n dm i l d e m p h y s e m a groups required either a lobectomy or p n e u m o n e c t o m y for a small, non-obstructing, peripheral bronchogenic  carcinoma a n dw e r e part of an ongoing study of lung structure a n d function at the Unive British Columbia P u l m o n a r y Research Laboratory in Vancouver. T h es e v e r e e m p h y s e m a  g r o u pw e r e selected for lung v o l u m e reduction surgery at the University of Pittsburgh. The patients w e r e separated into their groups based o n the percent of the lung defined as e m p h y s e m a t o u s using the "density m a s k " technique (166).  8 6  5.5.1  Pulmonary Function Studies Spirometry and lung volumes were measured pre-operatively with the subjects seated in  a volume displacement body plethysmograph using previously described techniques (31,32,93,210).Functional residual capacity (FRC) was measured using the Boyle's Law technique.(44,146,181). Total lung capacity (TLC) was calculated by adding inspiratory capacity (IC) to FRC, and residual volume (RV) was calculated by subtracting vital capacity (VC) from TLC. Diffusing Capacity (D o) was measured by the single breath method as LC  described by Miller and associates (157). The predicted normal values for FEV! and FVC were those of Crapo etal(33),  for D co; Miller etal(157) L  and TL€ was predicted using Goldman's  values (70).  5.5.2  CT Studies The subjects in the study received a conventional, non-contrast CT scan (10 mm thick  contiguous slices) on a G E 9800 Highlight Advantage CT scanner (General Electric Medical Systems, Milwaukee, Wl) approximately one week prior to surgery. AN scans were performed with the subject supine during breath holding after an inspiration. The image data was transferred to a Silicon Graphics Indy Workstation (Mountainview, CA) for analysis of the X-ray attenuation values. The CT scan analysis used to evaluate the lung has been described in detail elsewhere (31,32). Briefly it was performed using a program written for the numerical analysis package PV-Wave  (Visual Numerics, Boulder CO). The lung parenchyma was segmented from the  chest and the large central blood vessels, and the volume of the whole lung (tissue and airspace) was calculated by summing the voxel dimensions in each slice. The density of the lung (g/ml) was estimated using equation 7 (31,82). Lung weight was estimated by multiplying the mean lung density by the volume. The volume of gas per gram of tissue for each voxel was  87  calculated according to the equation 8 (31,32). The frequency distribution of the individual voxels was plotted and the m o m e n t s of the curve w e r e obtained. V o l u m e fraction (V) of tissue and airspace in the resected portions of lung for the v  s e v e r e e m p h y s e m a cases and specific regions identified on the l o b e c t o m y specimen was calculated according to equation 9, which has b e e n fully discussed a b o v e (31,32). This CT estimation of v o l u m e fraction is used to correct the histologic estimates of the tissue and airspace to the level of inflation the patient achieved during the CT scan (31). The extent of the e m p h y s e m a in each patient was estimated by applying a "density m a s k " to the CT scans using the settings of Muller et al (166)  (figure 16).  This procedure  identifies all of the voxels within the lung that h a v e a HU value of -910 or less and expresses t h e m as a percent of the total. An HU value of -910  represents a lung inflation value of 10.2  gas per g r a m of tissue (equation 2). This is three standard deviations a b o v e the 6.0  ml  m/ lg  established for TLC in patients of similar age and smoking history which h a v e normal lung function (32).  The patients with mild disease had greater than five percent but less than 20  percent of their lung v o l u m e inflated to v o l u m e sa b o v e 10.2 ml/g, while patients with severee m p h y s e m a all had greater than 20 percent of their lung inflated a b o v e this volume. This technique for measuring e m p h y s e m a was validated by comparing the CT m e a s u r e m e n t of the percent e m p h y s e m a in a lobe to the quantitative histological estimates of the actual a m o u n t of e m p h y s e m a present in that lobe after it was resected.  5.5.3  Quantitative Histology  Resected Lobes: The specimens w e r e prepared for quantitative histology as previously described (31,32). Briefly, this was p e r f o r m e d by inflating the fresh surgical specimen with Optimal Cutting T e m p e r a t u r e (OCT)  c o m p o u n d (Miles Laboratories, Elkhart, IN) diluted 1:1 with normal saline,  88  A: CT scan of h u m a n lung. B. CT scan with density m a s k ( 9 1 0 H U = 10.2 m l / g ) applied, attenuation values less than -910 is s h o w n in red and values greater than -910 is in blue. 89  90  and freezing in liquid nitrogen. The frozen specimens were cut into 2 cm thick slices in the transverse plane on a band-saw and fixed in 10% buffered formalin at room temperature for at least 24 hours. The volume fraction of normal and emphysematous lung was estimated from . the gross lung slices by floating them in water and overlaying a grid of points. The number of points falling on emphysematous holes (severe, >5 mm diameter, moderate, 2-5 mm, mild <2 mm) and normal lung parenchyma was counted using a magnifying lens to estimate the volume fraction of emphysema and normal lung (figure 17). Hematoxylin and eosin stained sections were prepared from random samples of the lobectomy specimens for the stereologic analysis. A second set of slides were prepared from a subset of the patients where the site of the biopsy could be identified on the CT scan.  Lung Volume Reduction Surgery Specimens: The tissue from the patients with severe-emphysema was received fresh from the operating room following the lung volume reduction surgery procedure and fixed, as received, in 10% formalin. Representative samples were embedded in paraffin and stained with hematoxylin and eosin.  Stereology: To optimize the sampling for the stereologic analysis, a cascade design technique was used as has been previously described in chapter 1 (31,32,35). Level 1 was performed on the fixed slices of the lobectomy specimens from the subset of patients where the histology sample site could be identified on the CT scan. Levels 2 and 3 were performed on all available sections at the light microscopy level using the point counting program Gridder  (Wilrich Tech, Vancouver, B.C.) which generated random fields of view,  projected a grid on to the field of view via a camera-lucida attachment on a Nikon Labophot  91  light microscope and tabulated the counts. Level 2 used 100x  magnification with a grid of 80  points and 40 lines. The n u m b e r of points falling on airspace, tissue (lung parenchyma), and  m e d u im sized blood vessels ( 5 0 1 0 0 0 pm) as well as the n u m b e r of intersects b e t w e e n the grid lines and the parenchymal-airspace interface w e r e tabulated. Level 3 was p e r f o r m e d on 10 r a n d o m fields of view per slide at 4 0 0 x magnification and the n u m b e r of points falling on airspace c o m p o n e n t s (Alveolar m a c r o p h a g e s , alveolar PMN,  other objects in the airspace, and  e m p t y space) as well as the tissue c o m p o n e n t s (alveolar wall, capillary lumen, and small blood vessels (20-50 pm) w e r e counted using a 100 point grid. The v o l u m e fraction (V ) of each of the lung components, (V (i )), w e r e estimated at v  V  C  each level according to equation 3, and the surface density S(par) was estimated using V  equation 4. Since surface density is the surface area in a given volume, the surface area of the p a r e n c h y m a is calculated by multiplying the surface density by the v o l u m e of the lung. This analysis was performed on the r a n d o m sections for an estimate of the total lung surface area as well as the biopsies f r o m the specific sites to estimate the surface density in specific regions of the lung that could be identified on CT.  The surface density m e a s u r e m e n t s for all patients  w e r e pooled and tested for correlation with the CT m e a s u r e m e n t s of ml gas per g r a m of tissue  f r o m the s a m e region using a m i x e d effects regression analysis. The overall V is calculated b v  multiplying the V  v  of the lung c o m p o n e n t at the highest level by the V that contained it in the v  previous levels according to equation 5.  5.5.4  Statistical Analysis  All data w e r e analysed using the one way analysis of variance and the multivariate  analysis of variance. Transformations w e r em a d e on certain variables to normalise distributions and to m a k e variances h o m o g e n e o u s . The correlation b e t w e e n CT m e a s u r e m e n t s of lung expansion and surface area per v o l u m e as well as the diffusing capacity of the lung and surface  92  area w e r e analysed using a m i x e d effects regression analysis. A p-value o f less than 0 . 0 considered significant.  5.6  Results T h e control g r o u p( N = 2 3 )h a s less than five percent o f their lung v o l u m e in the  e m p h y s e m a t o u s category ( greater than 1 0 . 2m l gas per g r a mo f tissue). T h o s e with m  disease ( N = 7 )h a v eam e a no f1 3 percent ( r a n g e5 2 0 % ) in this category a n d those wit e m p h y s e m a( N = 1 4 ) h a v eam e a no f4 6 percent ( r a n g e 24-60%) in this category. T h e patient d e m o g r a p h i c s (table 1 2 )s h o w that the control g r o u p is slightly y o u n g e r  h a v e a similar sex distribution, a n db o d y size to the e m p h y s e m a groups. T h es m o k i n gh  is n o t statistically different b e t w e e n the three g r o u p sb u t the patients with mild e m p h y s e m a  tended to s m o k e less. T h o s e with m i l d e m p h y s e m ah a v e FEV^ FVC, T L Ca n dR V value  are similar to the controls b u t the FEV^FVC ratio a n d D co are reduced while the FRC is L  elevated. Those with severe e m p h y s e m ah a v e grossly a b n o r m a l lung function with all value s h o w i n g the classic obstructive pattern o fr e d u c e d FEV^ FVC, FEV.i/FVC a n d D co a n d L  elevated TLC, FRC, a n d RV.  T h eC T estimates o f lung v o l u m e (table 1 3 )s h o wa n increase in total lung a n da v o l u m e in the s e v e r e e m p h y s e m ag r o u pc o m p a r e d to the m i l d e m p h y s e m ag r o u p which in  is greater than the controls. T h e tissue v o l u m ea n d therefore the lung w e i g h t is decrease  s e v e r e e m p h y s e m a ,b u t there is n o difference in lung w e i g h tb e t w e e n the m i l d e m p h y s e m a g r o u pa n d the control subjects.  T h e cumulative frequency distribution curves (figure 1 8 )o fm l gas per g r a mo f tissu  present in each voxel are different b e t w e e n the three groups. T h e vertical a r r o w indicates  cut off at 9 1 0H U in the density m a s k technique w h i c h is equivalent to 1 0 . 2m l gas pe  tissue. I n the control g r o u p9 9 ± 0 percent o f the voxels are b e l o w this cut off, c o m p a  9 3  • ± 1 percent of the m i l d e m p h y s e m aa n d5 4 ± 3 percent of the severe cases o fe m p h y  (table 13). T h o s e with s e v e r e e m p h y s e m ah a v e 20% o f their lung inflated b e y o n d2 0m / lg  which is m o r e than three times the a m o u n t of air contained in the n o r m a lh u m a n lung at  (32). Further information about the frequency distribution of the m / lg values is s h o w n in ta  1 4w h e r e the control lungs s h o wan o r m a l distribution with m e a n ,m e d i a na n dm o d e valu  are closely similar (4.5 ± 0.2, 4 . 4 ± 0.1, a n d4 . 4±0 . 2m l / g )a n d a relatively small vari  2 . 3 ml/g). T h em i l d e m p h y s e m ag r o u ps h o w a distribution which is slightly shifted to the r  around a m e a n of 7 . 1 ±0 . 3m l / g , m e d i a n of 5 . 8 ±0.3 m l / g ,a n dm o d e of 5 . 3±0 . 6 very large variance ( 2 0 7 . 8±1 1 1 . 9m l / g ) . T h es e v e r e e m p h y s e m ag r o u p has a flattened  distribution which is shifted to the right with a m e a n of 1 4 . 0±1 . 2m l / g ,am e d i a n of 9  m l / g ,m o d e of 8 . 1 ±0 . 5m l / ga n d the largest variance ( 5 7 8 . 5±1 9 9 . 6m l / g ) . W h e n th values are expressed as percentage of m e a s u r e d TLC, the control a n dm i l d e m p h y s e m a  patients are n o t different (66.0 ± 2.2% versus 7 4 . 6±3 . 2 % )h o w e v e r , the m e d i a n value fo  s e v e r e e m p h y s e m a patients w a s significantly greater ( 9 9 . 7 ± 4.9%) than the other t w o group indicating that these patients lungs are hyper-inflated.  Table 1 5s h o w s the stereology data for all three groups corrected to the level o f lun  inflation present during the C T scan. This s h o w s a decrease in the percentage of the lun  occupied b y tissue, a n d an increase in the percentage occupied b y airspace in the g r o u p  severe-emphysema. There is a small b u t significant decrease in the surface area per v o l u m  of lung in m i l d e m p h y s e m aa n d a very m a r k e d reduction in this value in the resected lung regions of the g r o u p with s e v e r e e m p h y s e m a .  T h e airspace of these lungs contains an inflammatory exudate. T h e cellular c o m p o n e n  of the exudate varies with P M N increasing f r o m 1 ± 0 X 10 cells/m in the control g r o 8  2  X 10 cells/m in the mild disease a n d 1 9 7±3 4 X 10 cells/m in the lungs with severe 8  2  8  2  e m p h y s e m a . T h e macrophages, o n the other hand, w e r en o t increased in the mild e m p h  9 4  and showed a large but variable increase in the group with severe emphysema. The fluid volume present in the exudate was increased only in the mild-emphysematous group. Figure 19 shows the mixed effects regression analysis between lung volume (ml gas/g tissue) and surface area per volume which has a negative slope (-3.7) and an intercept of 124 cm /ml both of which are significantly different from zero (p=0.001). The mixed effects 2  regression analysis of surface area versus measured D o (figure 20) shows a positive LC  correlation with a slope of 0.1 ml/min/mmHgm and an intercept of 7.1 ml/min/mmHg. 2  The amount of emphysema detected in the same lobe by either CT or histological analysis is compared in table 16. This shows that the volume fraction of the lesions greater than 5 mm in diameter was similar using both techniques. However, the histologic analysis shows that a large fraction of the lobe in both groups of specimens contains lesions smaller than 5 mm in diameter which were not detected by the CT scan.  95  2  C M  C D  cn o E co Q 66 ±3*  Mild-Emphysema 6M/1F 1155 ±185  558 ±111 69 ± 3  77 ± 4 168 ± 3  175 ± 3  167 ± 2 0.73 ± 0.01 0.64 ± 0.04*  102 ± 3 102 ± 6  FVC  FEVi  32 ± 2** 74 ± 4** 0.30 ±0.01**  84 ± 5  93 ± 3  (%P)  a  70 ± 2  FVC  >  892 ±124  (cm)  Height  122 ± 6  113 ± 3  (%P)  TLC  157 ±9*  128 ± 5  (%P)  FRC  154 ±10  133 ± 8  46 ± 5** 137 ±5" 185 ±8" 222 ± 13"  78 ±13*  89 ± 3  £  59±2  Q .  ?  Control 15M/8F  o lc (Kg)  Weight Q  (cig yrs)  Smoking 8  ?  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Those with mild emphysema have a lower FEV^FVC ratio and D|_co with a slightly higher FRC than the control group, whereas those with severe disease have reduced dynamic lung volumes combined with elevation in all of the subdivisions of lung volume, indicating that they have a marked reduction in the ability to empty their lungs. These patients also have a reduction in their diffusing capacity which is greatest in the severe group. Table 13 shows that severe-emphysema is associated with an increase in gas volume and a reduction in lung tissue volume and weight. Some of this decrease in tissue volume could be due to a reduced blood volume associated with the shift of the lung into zone 1 and 2 caused by airway closure and high alveolar gas pressure relative to pulmonary venous and arterial pressure (197,264,265). However, further analysis of the data (table 15) shows that there is a very large decrease in the tissue volume fraction and the surface area per volume consistent with lung destruction. The mild-emphysema cases show an increased total and airspace volume without a significant decrease in the tissue volume, lung weight (table 13) or lung surface area (table 15). These data suggest that early emphysema is associated with minimal destruction of surface area associated with an increase in lung volume to produce a significant reduction in surface to volume ratio whereas severe emphysema is dominated by a destruction of the parenchymal surface. This interpretation is consistent with early lesions in the peripheral airways that would prolong the time constants of peripheral lung units and increase lung volume before the onset of the destruction of the lung surface area (90). There is a marked increase in the volume of exudate onto the alveolar surface in the cases with mild emphysema and this fluid contains excess numbers of PMN. In the more  104  severe cases the fluid v o l u m e returns to control levels b u t the n u m b e r of P M Na n d alveola m a c r o p h a g e s present on the surface are both increased. A c h a n g e in the nature of the  exudate in severe disease could b e important in the pathogenesis of lung destruction. T h e relative importance of the proteolytic e n z y m e s produced b yP M Na n d those produced b y alveolar m a c r o p h a g e sa n d other cells in the pathogenesis of e m p h y s e m a is controversial  (17,89). T h e cigarette s m o k i n g habit increases the n u m b e r of P M Na n dm a c r o p h a g e s in lu  tissue a n d airspaces ( 2 0 5 , 2 4 1 )a n d active s m o k i n g increases P M N concentration in the lung microvessels (142). O u r data s h o w that mild lung destruction is only associated with an  increase in P M Nw h e r e a s advanced lung destruction is associate with large n u m b e r s of bot  P M Na n dm a c r o p h a g e so n the alveolar surface. As the controls in this study h a d a signif  smoking history, their m a c r o p h a g e level is probably already elevated f r o m that in non-smoke  T h e alveolar m a c r o p h a g ea n d the P M N are linked through a n e t w o r k of cytokine a n dg r o w  factors (57) a n d the increased m a c r o p h a g e sm a yk e e p the recruitment of P M N high e n o u g  that the P M N degradative e n z y m e s can destroy the lung tissue to produce the severe dise Other investigators h a v e estimated the severity of e m p h y s e m a using either a "density  m a s k " with a single cut off value ( 1 5 8 , 1 6 6 ) or the lowest fifth percentile (72) of the x-ray attenuation values expressed in Hounsfield Units (HU). T h e simple calculation w e use to  convert H U obtained f r o m conventional C T scans to a m o r e physiologically meaningful unit,  of gas per g r a m of tissue (32), allows us to relate regional to total lung v o l u m ea n d dete  the overall lung v o l u m e at which the C T scan w a s obtained. This data s h o w (table 14) t  w h e n the lung v o l u m e at the time of the C T scan is expressed as a percent of m e a s u r e  the control g r o u pw a s at 6 6 . 0 ± 2.2% of TLC the mild e m p h y s e m a t o u sg r o u pw a s at 7 4 .  3.2% and the severely e m p h y s e m a t o u sg r o u pw a s at 9 9 . 7 ± 4.9% of TLC. This tendency  those with severe disease to reach a higher overall lung v o l u m e is consistent with the fact e m p h y s e m a t o u s lesions reach full inflation at very l o w transpulmonary pressures (92).  1 0 5  Further analysis of frequency distributions of regional lung volumes (ml/g) show that the control patients have a normal distribution of lung inflation with a similar mean, median and mode and 99% of the lung being below the density mask cutoff value (table 14, figure 18). Emphysema shifts this curve away from normality with increasing proportion of the lung being above the cut off value in the severely affected group (table 14, figure 18). This value (-910 HU = 10.2 ml/g) is three standard deviations above that obtained by dividing measured TLC by measured lung weight in the control group (6.0 ml/g) (32). Therefore, some of the lung between 6.1 and 10 ml/g should also be abnormal. This is confirmed by the anatomic studies of the resected lobes which show a large percentage of emphysematous holes less than five mm in diameter are not detected by CT (table 16). This confirms previous reports (158,166) showing that the CT technique accurately identifies holes larger than 5 mm in diameter but fails to identify the smaller lesions. These smaller lesions are probably represented by values between normal TLC (6.0 ml/g) and the cut off (10.2 ml/g) in the density mask technique. The inability to detect these small lesions with CT is the result of volume averaging on 10 mm thick slices which affects the segmentation of objects less than 5 mm in diameter (38,276). Thin slices of a high resolution scans allow better visualization of these smaller lesions (64,97) but Kemerink and associates have shown that the signal to noise ratio is so high that it diminishes the ability to discriminate different lung densities within the same slice (114). This means that the thinner slice provides better visualization of small structures but the thicker slice and a lower spatial frequency reconstruction algorithm provides better lung density discrimination and more reliable estimates of the extent of the emphysema in the lung. In earlier studies, Gould et al also showed a negative correlation between the EMI measurement of X-ray attenuation and a histologic measurement of surface area of alveolar wall per unit lung volume (AWUV) (72). The relationship between surface area and volume of a sphere is one of radius squared divided by radius cubed which means that the surface to  106  volume ratio will decrease as the sphere enlarges. Our data (figure 19) shows the predicted negative relationship between lung surface area per volume (cm /ml) and lung volume (ml/g) 2  with lung expansion. The regression line for the mean value has quite tight 95% confidence limits in the range of the normal values for RV, FRC and TLC and this relationship persists up to the cut off for the detection of holes greater than 5 mm in diameter (10.2 ml/g). The confidence limits widen at higher lung volume presumably because of a variable destruction of the lung surface. This relationship allows lung surface to volume rations and lung surface area to be predicted form the CT and these measurements could be used to track the progression of lung destruction in individual patients. There was a good correlation between the surface area calculated from lung histology and the diffusing capacity of the lung for carbon monoxide (figure 20) that is in agreement with published data (72). The ability to relate lung surface area to CT measurements of lung volume per gram and to a functional assessment of gas exchange impairment provides a powerful tool for the analysis of the structural and functional changes in chronic lung diseases. The algorithms currently used to assess the CT could be easily modified to make these calculation available to clinicians who might use them to assess the progress of lung destruction in emphysema and to evaluate the impact of lung volume reduction surgery and possibly other therapies on lung structure. In summary, our data show values obtained for the CT scan can provide an accurate assessment of the tissue and airspace changes in emphysema that should be useful in the longitudinal assessment of emphysematous lung destruction.  107  C H A P T E R 6: S u m m a r y and Discussion  6 . 1 S u m m a r y  T h e data presented in this thesis h a s established that the C T scan can b ec o m b i n 1  with quantitative histology to quantify the tissue changes in chronic lung diseases. T h eC T  scan provides a powerful tool for the estimation of total a n d regional lung v o l u m e sa n dw  a n d provides a m e t h o d of correcting o p e n lung biopsy material to the level of lung inflatio the intact thorax.  T h e data f r o m the IPF studies presented in chapter 4 s h o w that the decrease in t  lung v o l u m e which is d u e to a loss of the airspace v o l u m e with minimal changes in the  volume. In e m p h y s e m a , there is a n increase in the total lung v o l u m e which is due, first  an increase in the airspace volume, with m i n o rc h a n g e s in the tissue volume, which is th  followed b y a decrease in the tissue c o m p o n e n t through proteolytic destruction. T h e freque distribution curves of these studies s h o w thatthe control lungs h a v e a normal distribution  around a m e a na n dm e d i a n of 4 . 5m lg a s / g tissue which is 66% of the patients ' TLC d  lung weight (6.0 m l / g ) . I n IPF the curve is grossly shifted to the left (figure 1 3 ) indicatin  there is an increase in the proportion of the lung occupied b y tissue, a n d the long tail s  that there are regions of hyperinflation probably d u e to the cystic changes of the e n d stag  lung, concomitant e m p h y s e m a t o u s changes, or a combination of both. In e m p h y s e m a (figu  18), the curve is shifted toward m o r e gas v o l u m e per g r a m of tissue a n d this is especia  in the severe cases w h e r e over half of the lung has a density greater than 1 0 . 2m l / g ,w  b e e ns h o w n to b eag o o d estimate of the e m p h y s e m a present o n conventional C T scans  (120,158,166). Also, the C T density provides data o n the tissue a n d airspace v o l u m e frac  which is essential for the correction of lung biopsy specimens to an appropriate level of in for comparisons b e t w e e n individuals a n d even different biopsy f r o m the s a m e lung.  1 0 8  T h e frequency distribution data in the C T analysis provides an important tool for the longitudinal studies of lung disease because it is sensitive to c h a n g e s in lung composition.  present study confirms the w o r k of Hayhurst (81) in e m p h y s e m aa n d Hartley (78) in interst  lung disease, w h os h o w e d that the properties of the attenuation curve correlate with chang  lung structure. W eh a v e modified their technique to express the x-ray attenuation values in  terms of m l of gas per g r a m of tissue which w e propose is a m o r e physiologically useful  expression because it allows the analysis of the lung in terms of the pleural pressure gradie  a n d regional lung inflation. T h e greatest p r o b l e m with this analysis is that it does n o t sepa  airspace infiltration f r o m fibrotic remodeling a n dm o r e investigations n e e d to b ed o n e in this area with careful correlations b e t w e e n the frequency distribution curves of the radiological categories "ground-glass" attenuation a n d "honey-combing" with the histological estimates of the lung structure. A quantitative histological analysis of the lung assumes that the specimen has been  inflated to a n appropriate level. W eh a v es h o w n in chapter 3 that this is a valid assump  the gross specimen is able to b e inflated through the airways. H o w e v e r , figure 1 4s h o w s w h e n the inflation is not controlled, the histologic information is n o t comparable b e t w e e n individuals, or even different biopsies. This thesis describes a technique w h e r eC T  m e a s u r e m e n t s of lung density can b e used to calculate the v o l u m e fraction of tissue (equa 9) in specific regions of the lung. These values can then b e used to correct o p e n lung w h e r e the inflation cannot b e controlled, to an appropriate level of inflation for quantitative anatomic analysis (chapter 4).  T h e stereology data s h o w s that there is an increase in the tissue v o l u m e fractions o  lungs with IPF a n d a decrease in e m p h y s e m a . There is, h o w e v e r , a loss of surface area both conditions which is d u e to a reorganization of the lung p a r e n c h y m a in IPF a n da  destruction of alveolar walls in e m p h y s e m a . Figure 2 0s h o w s that the surface area of the  1 0 9  is related to the diffusing capacity in the e m p h y s e m a t o u s patients. W h e n the surface area DLCO  m e a s u r e m e n t sf r o m the patients with IPF are a d d e d to the analysis, a statistical differ  is n o t detected to the e m p h y s e m a t o u s cases so the g r o u p s can b e combined. T h em i x e d  effects regression line for all of the patients (figure 21) has a slope of 0 . 0 9m / lm n i/ m m H g a n d an intercept of 9 . 4m / lm n i/ m m H g , both of w h i c h are highly significantly different f r o m  ( p < 0.001). This is a n important finding because C Tm e a s u r e m e n t s of lung inflation h a v  s h o w n to h a v e a negative correlation with the surface area (figure 19, (72)) a n d this pro  tool for the clinician to quantitatively follow the patient with chronic lung disease a n d to as the changes in lung structure a n d function over the course of the disease.  T h e inflammatory response in the lung is very important in the pathogenesis of both a n de m p h y s e m aa n d this study has s h o w n that there are increased inflammatory cells I n  airspaces of both diseases. These cells consist mostly of alveolar m a c r o p h a g e s because of  the chronic condition of the process, a n d the central role that m a c r o p h a g e s play in terms modulating the inflammatory a n d the fibroproliferative response. It is very tempting to  characterize these changes as different o u t c o m e s to the s a m e process because it has b e e n  s h o w n that there is a fibroproliferative response in the early stages of e m p h y s e m a that res  in modifications to the elastin a n d collagen content o f the p a r e n c h y m a which is characterize fibrosis. H o w e v e r , this fibrosis is mild a n d the p r e d o m i n a n t feature of e m p h y s e m a is the destruction of the lung tissue. T h e exact molecular events of the fibrosis still n e e dm o r e  clarification, b u t it is n o w obvious that the larger molecules of collagen a n d elastin are n o t  only players in the g a m ea n d that proteoglycans are a very d y n a m i c constituent of the fib  response. Proteoglycans appear very early in the fibroproliferative response a n dh a v eb e e n  linked to k e y roles in fibrosis such as tissue hydration, cytokine binding a n d cellular adhesi  1 1 0  o o  co  CM  g  ^•v CD CO C CO Z5  I « CD  £  3  <*-  CO.  ff J=  Zj  O TJ  CD c CL C CD O CO O O o CO c CO TJ  o LO  LP  o o  11  T J J= XJ § c > CD E LO OJ CO TJ CD CO C CD CO E LL TJ c CO 0.  §•  co O O  < o o  -s CO  CD  oT-g 2 C =  S CO  cc  TJ CJ) o CD C — ' ^2 TJ CD CD  .g -f= -g O <o 8 2 £ S! (5 ~  o  LO  CD CO CO CO O CL CO jo t o  ° 3  CD C O t t) CD CD  co  Q_  3  O  IR =• °» CD CO X  m  -=  <° CO CD  E •=  CO O CD -c .g CO  o*1 o o c o  i s JZ CO  CO Z LO CO  o  CN  CO  2  (6HLULU/UJUI/|IJU) 0 3 H Q  111  E  (77,200,268). Their exact role in e m p h y s e m a is still to b e investigated, b u t the increased  alveolar wall thickness seen early o n in the e m p h y s e m a t o u s lesions is an attractive site for  synthesis. W h yo n e process proceeds to the proliferative stage while the other o n e elevate the degradative response still n e e d s to b e delineated.  6 . 2  Future Directions Quantitative three dimensional analysis of the lung in chronic lung diseases is an  important step. Correlations with semi-quantitative scoring systems a n d functional  characteristics has proven to b ew e a ka n d of limited value aside f r o m a quick process fo assessing the severity of the disease. It is time to m o v eb e y o n d these semi-quantitative analysis a n d describe the lung in t e r m s of the three-dimensional structural parameters that  possesses. T h eC T scan avails itself for quantitative longitudinal studies of the lung becaus  is minimaly invasive a n d easy to perform. A f e w simple calculations of the data m a y pro  important information that allows the assessment o f the lung in terms of airspace enlargem  loss of surface area a n d tissue destruction or reorganization. This m a yb e c o m e very impo  in the long t e r m follow u p of patients with IPF to assess the time course of the disease assess treatment protocols a n d in e m p h y s e m a for the assessment of the lung structure in  terms of selecting patients for the palliative surgical treatment of lung v o l u m e reduction surg  L u n gv o l u m e reduction surgery is a controversial a n d very expensive procedure a n d the ab  to choose patients pre-operatively that will benefit f r o m the intervention has great ramification  for both the patients ' health a n d the resources of the health care provider. A quantitative analysis of the lung m a yb e able to delineate the structural factors in the lung that are responsible for the i m p r o v e m e n t or n o n i m p r o v e m e n t of these patients. Also, b y use of a  density m a s ka n d three-dimensional reconstruction, a m a p can b e provided to the surgical  clearly demonstrating the extent a n d location of the e m p h y s e m a to help decide w h e r e the  1 1 2  site for reduction surgery (figure 22).  6.3  Conclusion  I n conclusion, it has b e e ns h o w n that the combination of quantitative C Ta n d stereo  provides reliable quantitative data o n the lung structure in chronic lung disease. These stud  h a v e detailed the procedure that enables the C T to m e a s u r e the total a n d regional v o l u m e  changes within the lung a n d to quantify the ultra-structural changes responsible for the path physiological changes seen b y the clinician.  1 1 3  Figure 22.  Three dimensional reconstruction of a h u m a n lung with e m p h y s e m a using CT scan images. E m p h y s e m a is s h o w n in red, and normal lung parenchyma in blue, while dense tissue is green.  114  REFERENCES  1.  Abbas, A.K., A.H. 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