Quantification of the Tissue Changes in the Human Lung with Chronic Lung Disease Using a Combination of Computed Tomography and Stereology
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 ;te>fhe required standard
THE UNIVERSITY OF BRITISH COLUMBIA
April 1998
© Harvey Owen 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 pulmonary fibrosis (IPF) and pulmonary emphysema are chronic lung diseases exhibiting progressive deterioration in pulmonary function as the lung architecture is remodeled. This thesis quantifies these tissue changes using a novel combination of computed tomography (CT) and quantitative histology. Pre-operative CT scans were obtained from patients with IPF, patients receiving lung volume reduction surgery for diffuse emphysema and from patients with minimal to mild emphysema undergoing lobectomy for a small peripheral
• •• • • • j. tumour. Total lung volume was calculated using the pixel dimensions on the CT scan while airspace and tissue volume as well as the regional lung expansion were estimated using the X- ray attenuation values. Tissue samples were obtained at either open lung biopsy (IPF) or surgical resection (control and emphysema) and prepared for quantitative histology. A method for correcting the histology specimens to an in vivo level of inflation was developed so that the tissue composition and surface area could be estimated using stereologic techniques. The data shows that there is a reorganization of lung parenchyma in IPF with a disproportionate loss of airspace and surface area without increasing the total amount of tissue. The patients with emphysema show evidence of a progressive proteolytic destruction of tissue volume and surface area. There is a negative correlation between regional lung expansion and surface area in emphysema and a positive correlation between surface area and the diffusing capacity of the lung in both diseases. This technique should prove useful in the longitudinal assessment of chronic lung diseases and the monitoring of response to treatment. TABLE OF CONTENTS
ABSTRACT ii
TABLE 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 1.1.1 Sampling 1 1.1.2B\as 2 1.1.3 Variance 3
1.2 Stereological Methods 4 1.2.1 Stereological Probes 4 1.2.2 Cavalieri's Volume Estimator 6 7.2.3 Volume Fraction 7 1.2.4 Surface Area 7 1.2.5 Multi-Level Sampling Design 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 20 3.1.2 Cellular Lung Structure 21 3.1.3 Extra-cellular Matrix 23
3.2 The Clinical Measurement of Lung Function 25
iii 3.3 The Pleural Pressure Gradient 27
3.4 Experiment #1 28
3.5 Material and Methods 28 3.5.1 Pulmonary Function Studies 29 3.5.2 CT Studies 29 3.5.3 Quantitative Histology 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 49 4.1.2 Radiological Description of IPF 50 4.1.3 Histological Description of IPF 50 4.2 Fibrotic Mechanisms 51 4.2.1 Cellular Mechanisms of IPF 51 4.2.2 Molecular Mechanisms of IPF 52
4.3 Quantitative Studies of IPF 53
4.4 Experiment #2 55
4.5 Material and Methods 55 4.5.1 Pulmonary Function Studies 56 4.5.2 CT Studies 57 4.5.3 Quantitative Histology 58 4.5.4 Statistical Analysis 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 11 5.1.2 Radiological Description of Emphysema 78 5.1.3 Histological Description of Emphysema 78
5.2 Pathogenesis of Emphysema 80 5.2.1 Protease/Antiprotease Theory 80 5.2.2 Inflammatory-Repair Mechanism 81
iv 5.3 Quantitative Studies in Emphysema 82 5.3.1 Gross Analysis 82 5.3.2 Histologic Analysis 83 5.3.3 Radiological Analysis 84
5.4 Experiment #3 85
5.5 Materials and Methods 86 5.5.1 Pulmonary Function Studies 87 5.5.2 CT Studies 87 5.5.3 Quantitative Histology 88 5.5.4 Statistical Analysis 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. 100
vi 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. 101
Figure 19. Mixed effects regression line for surface area per volume and lung inflation. 102 Figure 20. Mixed effects regression line for surface area and diffusing capacity of the lung for carbon monoxide. 103
Figure 21. Mixed effects regression line for surface area and diffusing capacity of the lung for carbon monoxide for all patients. 111
Figure 22. Three dimensional reconstruction of a human lung with emphysema. 114
vii ACKNOWLEGMENTS
No scientific work can be completed without the assistance and support of many people.
As such, I wish to thank my supervisor and mentor Dr. James C. Hogg 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 thank my supervisory committee, Drs. Peter D. Pare, Clive R. Roberts, and John R.
Mayo for their constructive input and instruction. This project would not have been possible without collaborations from the University of Iowa under the direction of Gary W. Hunninghake and Dr. Robert R. Rogers at the University of Pittsburgh. I also wish to thank Ms. Hayedeh
Bezad and Dr. Benard Meshi for their technical assistance with the stereology; the Histology
Laboratory St. Paul's Hospital for processing the histological material; the computed tomography/magnetic resonance imaging staff at St. Paul's Hospital for gathering and transferring the CT images; Dr. Kenneth P. Whittall and Mr. Don Kirkby for their wizardry with the computers; Ms. Barbara Moore for collecting the pulmonary function data; Messrs Joe
Comeau and Stuart Greene for their computer and photographic expertise; and Ms. Lorri
Verburgt and Ms. Yulia D'Yachkova for their statistical advice. Special appreciation is also extended to my good friends (Paul and Mary Lacey and Gary and Heidi Rae) who introduced me to fly fishing and provided me with so much moral support through this time. Thank you also to my parents and family who always believed in me. Finally, I wish to thank my wife
Maureen for her patience, faith and unwavering love. Thank you all and God bless you.
viii PREFACE
Chapters 3 and 4 are modifications of published papers. The introduction has been re• written 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, CA. 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 ' : ~ 2 ~— ~~ ' 3 SE^ = J—+ ; — + — -+ . " S
2 2 where {s (sub)) is the variance between subjects, histologic samples (s ^)), microscopic field of
2 2 view {s (miC)), and the measurements (s (mea)), while n is the number of subjects, histologic
samples, fields of view and measurements respectively. It can be seen from this equation that
2 the greatest effect on SEM will come from the number of subjects examii ied, even if the s (mea)
2 is greater than the s (SUb) (214). Therefore, a design-based stereological study attempts to
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 shows how variance and bias affect the estimate. If the dots represent the measured values, and the target represents sampling bias, the ideal estimation would have low bias and low variance (fig 1 A). However, it is possible that this estimate can have too large of a variance (fig 1B) or too large a bias (fig 1C) or, at worst, a combination of both (fig 1D).
Therefore, care \ must be taken in Low Variance High Variance the experimental Low Bias design to reduce the bias to a minimum and B concentrate the quantitative effort High Bias to the measurement D which has 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 began in geology in 1842 with the work of Delesse, (4) and it took almost 100 years for the,techniques to become developed enough to apply to histologic
4 specimens (4). During the last thirty years the techniques have become understood by researchers other than mathematicians and statisticians and have been refined for general use in estimating the size of three-dimensional structures (16,74,144). Stereology obtains three- , dimensional information by applying a geometric probe to a two-dimensional section. The probe can take the form of a point, a line, a plane, or a volume (16), and the intersections between the probe and the sampling plane provide information about 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 probe must equal three. Table 1 shows how a probe is chosen. In the first line, the object of interest is volume which has three dimensions, therefore, the probe must have zero dimensions so the sum equals three. It can be seen that for surface area (dimensions = 2) the probe must have one dimension and so on for length and number.
Table 1. Stereologic rules for the selection of a sampling probe. The object dimension plus the test grid dimension must total three. See text for full explanation.
Object Dimension Test grid dimensions Total
Volume (3) Point (0) 3
Surface Area (2) Line (1) 3
Length (1) Plane (2)
Number (0) Volume (3) ' •. 3
5 1.2.2 Cavalieri's Volume Estimator
The most simple application of stereology is in the estimation of volume. The 17tn 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 = XPxd2 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 Figure 2. Sample point counting grid on an object.
falling on the object. Area ^ _ ^stance between points squared. See
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 (AA), or fraction of the total area 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 (Vv), occupied by the structure (4,16,35,74,144).
Volume fraction is calculated using equation 3:
Y P • _ £jJ (structure) . '(^(structure) ~ V1 p . <• ' ZJ "(total) 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 (Sv), or
surface to volume ratio, is calculated from .equation 4:
s -ixlL . f4l
where U is the sum of the intersects between the structure and the line probe, LP 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 Sv by the total volume. Finally,
7 Figure 3. Sample point counting grid on light microscopic section of human lung. Volume fraction = number of points on lung structure divided by total number of points.
8 Figure 4. Sample intercept counting grid on light microscopic section of human lung. Surface density = number of line intersects with lung tissue divided by number of end points on tissue multiplied by 4 divided by the length of the line. See 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 Sv is an estimate of the surface area per volume, the inverse of Sv is an estimate of the volume per surface area, which is another expression for thickness.
1.2.5 Multi-Level Sampling Design
To estimate the volume fraction of small structures, the lung must be sampled using a very high level of magnification. However, since the sample size heeded to reliably estimate the volume fraction, goes up with the magnification a method of optimizing the sample size for small structures is needed. This sampling protocol is known as the "Multi-level", or "cascade design" (35) and makes 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 parenchyma of the lung (par). To quantify the collagen you start at the lowest level of magnification where the structure can be visualized, and then using increasing levels of magnification the object phase at one level becomes the reference phase of the next level as shown in figure 5. Finally, the volume
fraction is calculated by multiplying the object Vv by the Vv of the reference space at the previous level in a cascading manner as shown in equation 5:
Vv(col)~VY(col)Qevel4)^VV(awl)nevel5)^
In summary, 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 probe of points is then applied to these
fields of view as shown in figure 4 and the number of intersects between the sample and the
probe is counted. Equations 3 and 4 are used to obtain the volume fraction and surface
density. Volume fractions of small objects are obtained using the cascade equation 5 which is
11 then multiplied by the lung volume to estimate the structural component's volume. Surface area of lung parenchyma is estimated by multiplying the surface density by the lung volume, and the
mean parenchymal thickness is calculated by taking the inverse of Sv.
1.3 Quantitative Gross Analysis Using Computed Tomography
Computed tomography (CT) scans are obtained by projecting a beam of X-rays through the body to a detector on the opposite site which records the absorbance, or attenuation, of the
X-rays by structures within the body (38). This is performed in a complete circle around the body so that-the attenuation values are also given spatial information and the images are reconstructed on a matrix of 512 X 512 picture elements, or pixels, which have the dimensions of the field of view divided by the number of pixels. However, the X-ray beam also has a thickness, known as collimation, so the pixels of a CT scan image are more appropriately referred to as voxels, because they are actually volume elements. Therefore, the CT scan can be used to reliably estimate the volume of the lung by summing the voxel dimensions, which is analogous to the Cavalieri principle described above.
Another important aspect of CT scans is that the voxels contain information about the linear attenuation of X-rays (v), where v is dependent on the density of the structure, the atomic number of the structure, and the energy of the electron beam 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 Hi-fi CT value^ = xa [6] Mifi where a is equal to 1000 for the HU scale (43). From this scale, water would have a HU of zero, air of -1000, and bone of +1000 HU (43). It has been shown that for objects with atomic numbers in the biological range such as polyethylene foam (116), epoxy (41,242), bread (41), wood (242), cork (123), and body 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):
CT„n:, + 1000 Density (g/ml) = ^ [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 (2,8,11,32,41,51,63-
66,78,85,123,134,155,156,167,193,195,204,242,270). 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) Specifi° Volume(iuni> ~ Specific 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 volume of gas per gram of tissue estimated by CT can then be expressed as a percentage of TLC, which will allow the comparison of CT scans from different patients because we know at what level of inflation the scans were performed
(32). .",<:•
Another piece of useful data for the CT density is the ability to estimate volume fractions. As Was mentioned previously, the volume fraction of tissue can be estimated histologically by applying a grid of points to a two-dimensional sample and dividing the number of points falling on tissue by the total number of points within the lung. As has been shown, a volume fraction, is simply a volume divided by a volume, so a volume fraction can be calculated using volumes that are derived using any method. Therefore, since we have an estimate of the specific volume of tissue from the literature, and an estimate of the specific volume of the whole lung calculated from equation 8 using the CT scan, a volume fraction of tissue can be estimated from the CT according to the following equation:
Specific Volume(lissue)
Volume Fraction(lissue) = . . — [9] In summary, there is clear evidence that the CT scans obtained from a properly calibrated, modern scanner can be used to estimate lung volume, density, volume of gas per gram of tissue and the volume fraction of tissue and airspace of the lung. The purpose of this thesis is to use these parameters to assess the normal lung and the changes that occur in diseases such as fibrosis and emphysema. 15 CHAPTER 2: WORKING HYPOTHESIS. SPECIFIC AIMS AND STRATEGY Chapter 1 provides background for two techniques useful for the quantitative assessment of lung structure. Stereology allows three-dimensional information to be obtained from 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 makes re-inflation to an in vivo level very difficult. Quantitative analysis of the X-ray attenuation data obtained by computed tomography is minimally invasive and provides estimates of gross lung characteristics such as volume, density and weight, as well as estimates of the volume fraction of tissue and airspace. The goal of this thesis is to combine these two techniques with a view to validate the less invasive computed tomography in terms of the more invasive stereology. 2.1 Working Hypothesis The working hypothesis of this thesis developed from a need for a non-invasive method for quantifying the structural changes in the lung in chronic disease: The combination of computed tomography scans and stereoloaic quantification of histological specimens allows the assessment of lung tissue changes in the chronic lung diseases idiopathic pulmonary fibrosis and emphysema with minimal destructive impact on the patient. The goal of this study,is to move analysis of CT scans beyond the qualitative observations currently made by diagnostic radiologists and make them more 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 measure 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 CHAPTER 3: THE NORMAL HUMAN LUNG 3.1 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 extra• cellular 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 m2 which led him to conclude that this enormous surface area made it very 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 Theodor Aeby used this approach in the 1870s to make painstaking measurements of the bronchi and their branching pattern (256), and even though his conclusions about 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 improvements in the quality of microscopes used,to image the lung and nowhere is this more evident than with the advent of the electron microscope which has allowed investigators to visualize structures down to the macro- molecular level. However, the intrinsic problem of quantification with the microscope was still that they were very time consuming, labour intensive and reduced the three dimensional structure of the lung to, two dimensional sections. Perhaps the greatest advance in the quantification of the lung came with the application of •stereological methods, as outlined in Chapter 1, which began in 1961 when Hans Elias assembled the International Society of Stereology. This society brought together investigators from biology, geology, metallurgy and mathematics to develop and refine techniques for the reliable and efficient quantification of three dimensional structures (256). Ewald Weibel in his book Morphometry of the Human Lung (249) applied these new methods to a quantification of the human lung and from this point on quantitative morphology of the lung has produced unbiased estimates of virtually all aspects of lung structure. The physiological description of the lung has been under intense scrutiny for hundreds of years but many of the advances have come since the Second World War (151,262). During this time investigators have developed techniques to quantify the functional aspects of the lung and described the important principles of airflow, the pleural pressure gradient and gas exchange. % This chapter provides a brief overview of lung structure and function and then uses modern techniques to quantify the human lung. 3.1.1 Gross Lung Structure The human lung consists of two separate lungs located (anatomically) on the left and right side of the thoracic cavity connected to the trachea by the main stem bronchus. The right lung has three lobes: the upper, middle and lower, while the left side has two lobes with the left upper lobe containing the lingula which is analogous, but smaller in volume, to the right middle lobe. The lobes are separated from each other by a wrapping of visceral pleura which forms a major fissure on the left and a major and minor fissure on the right but this separation is often incomplete allowing collateral ventilation between lobes. The lung is further partitioned into smaller units towards the periphery using the bronchial anatomy as a basis for division and nomenclature. A lobar segment is the next major division of the lung and consists of the lung that is supplied by the second division of the main stem bronchus. The secondary lobule measures 1-2.5 cm in diameter and can be visualized either on the cut surface of the lung, or by CT, and consists of a bronchiole and artery in the lobular core bounded by tissue septae that are continuous with the visceral pleura and contain the pulmonary veins and lymphatics (247). Each secondary lobule contains three to five acini which are described as the complex of all 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 exchange <75). The final region of the lung is the alveoli which are blind air sacs containing the greatest surface area to volume ratio in the lung and 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, which is in contact with the airspace of the external environment, the endothelial layer, which is in contact with the blood, and the interstitial compartment which both separates and binds these layers together. In addition, a fourth group of cells, the blood cells, move through the lung and transport the oxygen to the tissues, remove the carbon dioxide from the body, and combat infection. The red blood cells are responsible for transporting the oxygen to the tissues. The red colour is produced by the hemoglobin molecules which bind or release oxygen depending on the gradient surrounding the cells. The cells responsible for the elimination of foreign substances are the white blood cells which include the polymorphonuclear cells (PMN), monocytes, lymphocytes, eosinophils, and Table 2. Cellular composition of the lung (250) basophils. All of these cells Cells develop from a common Structure Cells % of Total Lung progenitor cell in the bone Cells Parenchyma Total 86 marrow and then differentiate Alveolar Type I . 4 AlveolarType.il 6 into specialized cells. For Endothelium 33 Mesenchymal 43 example, the lymphocytes are Nonparenchyma Total 14 Airways 5 the primary immune cells Ciliated 2-3 Glandular <1 which produce cytokines to Blood Vessels 9 Connective Tissue direct the host response and Structure Component % of Total Lung Connective Tissue antibodies to combat foreign Parenchyma Total 62 Collagen 46 particles. Eosinophils are Elastin 16 Nonparenchyma Total 38 particularly effective against Collagen 28 Elastin 10 parasitic infections and 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 (3,112,125,136) so that they are postulated to be important for wound contraction (125,136). Their exact role within the normal lung has been postulated as regulators for capillary blood flow, compliance of the interstitial space, and tissue elasticity of the lung parenchyma as well as turnover and maintenance 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 smooth muscle ceils, or of a completely different cell line but these cells have been shown to have contractile properties which may be responsible for regulating capillary blood flow (110,215). The major cell line of the airway and blood vessel interstitium is the smooth 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 summarizes the state of knowledge up into the 1970's when elastin and collagen were considered the major components of the extracellular matrix; The last twenty years have seen an explosion in the identification and description of other important molecules within the interstitium, most notably the proteoglycans (PG) and laminin (13,18,19,50,77,163,200,234,235,268). At least 19 different collagens have been 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 basement membrane (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 where 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 form into the extra-cellular matrix (139,143,277) where 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 FEVi, 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 (DLco) which determines the movement of a trace amount of carbon 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 CO 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 CO 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 CO concentration indicates the amount of CO 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 air- blood interface (257,261). 26 3.3 the Pleural Pressure Gradient Investigation of the lung with radioactive gases established.that ventilation of the lung is uneven (109,154,261). Milic-Emili and coworkers showed that when lung volume was increased the apical regions of the lung showed an initial greater change in volume compared to the basal regions, which then reversed at higher lung volumes (109,154,261). These and other studies established that regional ventilation was influenced by a gravity dependent pleural pressure gradient which causes upper regions of the lung to be expanded more fully than lower lung regions due to a higher trans-pulmonary pressure. It has been hypothesized that this pressure gradient is caused by the weight of the lung below the given region which must be supported by the lung parenchyma, somewhat like a spring which is suspended at the top (68,91,109). The distance between the coils of the spring is largest at the top and decreases towards the bottom because to the weight of the spring on each coil which is greatest at the top and minimal at the bottom. Since the upper regions are exposed to this higher pressure, they are more inflated at lower lung volumes and inflate first upon inspiration (156). This hypothesis is supported by Glazier and colleagues who undertook a morphometric analysis of alveolar size to show that alveoli in the upper regions of the lung are larger than alveoli in the lower regions at FRC (68) and Hogg and Nepszy who obtained similar results using density measurements of frozen dog lungs to show that the non-gravity dependent regions have a greater volume of gas per weight of tissue than the dependent regions (91). Gravity also influences pulmonary perfusion (109,263,265) and led West to describe lung perfusion in terms of specific zones. In Zone 1 the alveolar pressure is greater than the arterial pressure and the venous pressure, resulting in compressed capillaries and reduced blood flow. In Zone 2 the arterial pressures are greater than the alveolar pressure which is greater than the venous pressure and blood flow is now determined by the arterial and the alveolar pressure with the venous pressure being irrelevant. In Zone 3, the arterial pressure is greater than the venous 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 (DLCo) and total lung capacity (TLC) 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 image to its size in the resected specimen (Group 1). In the nine remaining patients, in whom the CT X-ray attenuation data were available, regional lung volumes were calculated. 3.5.1 Pulmonary Function Studies Spirometry, lung volumes and lung pressure-volume curves were measured in the patients from both group 1 and 2 seated in a volume displacement body plethysmograph. Functional residual capacity (FRC) was measured using the Boyle's Law technique. TLC was calculated by adding inspiratory capacity (IC) to FRC. Residual volume (RV) was calculated by subtracting vital capacity (VC) from TLC. In six of the nine patients in group 2, trans-pulmonary pressure was measured using a differential pressure transducer (45 mp ± 100 cmH02 ; Validyne; Northridge, CA) to compare mouth pressure to intra-thoracic pressure, measured with an esophageal balloon and PV curves were constructed by comparing these values to the simultaneously measured lung volume (146). In the remaining three patients in Group 2, the subdivisions of lung volume were determined using a dry rolling seal spirometer and the multiple breath Helium (He) dilution technique. Spirometry and He dilution FRC were performed on a P.K. Morgan computerized pulmonary function testing system (P.K. Morgan, Boston, Mass.). DLco for all 9 subjects was measured by the single breath method of Miller and associates (157) on the P.K. Morgan automated diffusing capacity analyzer. The results are corrected for alveolar volume (VA) and reported as DLCo A/A. 3.5.2 CT Studies All the patients had a conventional CT scan (10 mm thick contiguous slices) without IV contrast on a GE 9800 Highlight Advantage CT scanner (General Electric Medical Systems, Milwaukee, Wl) approximately one week prior to resection. All scans were performed with the subject supine during breath holding following inspiration. Conventional scanning parameters 29 in use at our institution were used (120 kVe, 100 mA, 2 sec, reconstructed on standard algorithm). The images for all 19 patients were printed onto standard radiological film using a window of 1200 and a level of -700 HU. For the nine patients studied completely, the CT data was transferred to a Sparc2 Workstation (Sun Microsystems, Mountain View CA) and the lungs were segmented out of the chest using the program Medical Image Viewer (Arkansas Children's Hospital Little Rock, AR, GE Medical Systems) using X-ray attenuation values of - 1000 to -500 HU. The volume of the whole lung and the individual lobes was calculated using the Cavalieri principle (153,183). This was accomplished by summing the segmented pixel area in each slice and multiplying by the slice thickness to get total lung volume. The horizontal and oblique fissures were noted on each slice and the lung volume was apportioned to upper, lower and middle lobes (figure 6). The segmented images were then passed to the numerical analysis package, PV-Wave (Visual Numerics, Boulder CO), where a pixel wide strip around the lung was subtracted away to eliminate partial volume artifact, due to the curvature of the lung and the chest wall, and each lung slice was divided into 16 ml sections (40 X 40 X 10 mm; figure 7). The mean X-ray attenuation of each of these sections was calculated, as well as the vertical distance from the middle of each section to the most gravity dependent (posterior) portion of the lung. The CT values were converted to true lung density (g/ml) by adding 1000 to the CT value and dividing by 1000 (equation 7) (82). The weight of the lung or lobe to be resected was calculated by multiplying the mean density of the lung (or lobe), by its volume calculated by the Cavalieri principle. The mean total lung gas volumes per gram of tissue at TLC, FRC and RV were determined by dividing the physiologically measured values of lung gas volume at TLC, FRC and RV by the total lung weight calculated from the CT scan. The X-ray attenuation for each 16 ml cubes was then used to calculate the volume of gas per gram of tissue for that cubic region of the lung according to equation 8. 30 Figure 6. CT scan of human 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 10 (slice thickness) mm sampling regions drawn on the segmented lung. The mean X-ray attenuation for each of these regions, as well as the distance to the middle of the region from the most posterior (gravity dependent) region was 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 (Vv) of airspace, parenchymal tissue and blood vessels, as well as the surface density (Sv) and thickness of the 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 implemented on the Group 2 patients using the CT scan image for level 1. Levels 2 and 3 were performed 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 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 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. The surface density of the parenchyma 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 (16). The mean parenchyma thickness is calculated by taking the inverse of the surface density. 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. The volume fractions for all the lung components at level 2 and 3 were calculated using equation 3, and the overall Vv for the individual lung components was calculated by multiplying the Vv of that lung component by the Vv of the component that contained it in the previous levels using equation 5 as described earlier. The volume fraction of the lung that is gas and tissue was also estimated by dividing the specific volume of tissue by the specific volume of the lung obtained from the CT scans according to equation 9. The volume fraction of tissue and gas estimated using CT (VV(CT)) compared to the Vv of tissue estimated at Level 2 using the morphometric technique to determine the validity of this technique. 34 3.5.4 Statistical Analysis All data were analyzed using independent t-tests or the one way analysis of variance. Transformations'were made on certain variables to normalize distributions and to make variances homogeneous. 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. 3.6 Results Table 3 shows anthropometric and lung function data for all 19 patients where group 1 (n=10) represents the patients in the preliminary study done to evaluate the point counting method and group 2 are the 9 patients in which the lungs were completely analyzed for volume and density. The data for these 9 patients (table 4) shows that on average the right lung accounted for 55 ± 2 % and the left lung 45 ± 2 % of the total lung volume with the right upper lobe (RUL) accounting for 21 ± 5 %, right middle lobe (RML) 9 ± 3 %, right lower lobe (RLL) 25 ± 3 %, left upper lobe (LUL) including the lingula 24 ± 4 % and left lower lobe (LLL) 22 ± 5 %. Table 5 compares the calculated weight and volume of the lobe to be resected with the fresh weight of the resected specimen and its volume after inflation with cryo-protectant material. This shows that the fresh weight of the resected specimen was 76 ± 19 % of CT calculated lobe weight and that the inflated volume of the specimen was 94 ± 36 % of the volume determined in vivo. The volume of gas per gram of tissue was calculated from the physiologically determined TLC, FRC, and RV gas volume divided by the lung weight (1069 ± 327 g) and is shown in table 6. The lung weight was calculated by dividing the CT estimated lung density by the CT estimated lung volume. The average volume was 6.0 ± 1.1 ml/gram at TLC, 3.6 ± 0.9 at FRC, and 2.4 ± 0.8 at RV and the lung volume at which the CT was obtained was 3.8 ± 0.8 ml/g 35 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 cmH20/cm. 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 m2, which gives a mean thickness of 7.2 ± 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 .O CO o O) c CD O TL C TL C (%Pred ) 10 0 ± 1 5 (%Pred ) 11 8 ± 1 4 o CL o CL 9 3 ±2 5 Q £ 8 6 ±1 4 Q £ >| o >l ^ Ul > U.I u. 1. 0 ±0. 1 0.6 9 ±0.1 3 FV C FV C 9 5 ± 1 (%Pred ) 10 6 ± 1 (%Pred ) > I > 1 Ul Q. Ul £ LL 10 2 ± 9 8 3 ±2 (Kg ) (Kg ) 6 8 ±1 2 7 2 ± 1 WEIGH T WEIGH T (cm ) (cm ) 16 9 ± 8 16 2 ± 8 HEIGH T HEIGH T CO (yr ) (0 (yr ) AG E AG E 6 1 ± Q 6 0 ±1 c .2 u SE X c SE X 5M/5 F 3 3M/6 F u. CO (0 CM Ui _l § Mea n ± S D OQ Mea n ± S D 2 < 1 O 1CL CD CD JD O CD O LUN G 10 0 ± TOTA L ±i 0) 487 3 ±136 0 TJ C CO LEF T LUN G 4 5 ± 2 JO 221 8 ±69 3 D) C O) c _3 LUN G 5 ± 2 RIGH T O 265 5 ± 67 0 c nCD o CD CL LL L CL 2 ± 5 3 107 1 ±42 3 CD CO rr CO LU L bj 2 4 ± o 114 7 ±35 8 k_ CD c CD 3 .2 (/) o (0 CO Q. -4—' O) T3 o> RL L 2 5 ± 3 C c CO 121 8 ±39 1 co O CO D) A CD _i TJ o O 3 2 1 ± 5 CD 3 O 101 0 ±32 6 CC > E (0 _3 CD o X> x> > O o E i_ c. c CD w co 3 CL 3 CL LU CD "co 3 -I TJ 3 -•—< m > CO o %TLV* * ± S D x: < '•5 Volum e ± SD * c > bOe) TABLE 5: Lobar weight and volume Resected specimen vs. CT WEIGHT (g) VOLUME (ml) CASE LOBE SPECIMEN** CT** 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 ±19 94 ±36 * 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 TABLE 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 >» £ CM "CO JE 8 8I 5 il O 00 o o 8 oo o o o o o 3 CD lO CO to (enssij B/seB |iu) 011% ll 42 Figure 9. A graph of the pressure volume curves of the 6 patients with trans- pulmonary pressure measured, plotting the CT derived volume measurement of ml of gas/gram of tissue against the transpulmonary pressure (cm of H20). The individual curves are shown in A and the same curves are shown in B when corrected for percent of individual TLC. 43 jD 0) 1 E CM .E w co o CD 0) «1 2> E =3 o CO "O CO c OJ CO 2 £ O CM -9> 5 • Q- 2 co CD 2> wC °> -D S CD CD a, LL- —I CO E • CD O 00 C «t CU ~ 1— CD £ 3 x: E c TO o E o CD P co O) co t ^ X CD T3 CD O) XI CD .E I CD _ .E o CD .TO o ^ CD ^ W CD05 CD c CD CD "O |» 8 O 3 CD -c TO CD z 3 3 (O Hui ) ajnssajd TO 44 3.7 Discussion The values obtained for lung and lobe volume (table 4 and 5) using this technique are similar to reported values from autopsy specimens (159,188,249), radiographic, bronchoscopic, scintigrams and inert gas dilution (188). The technique also provides the advantage that it is minimally invasive to the patient and no assumptions are made about lung shape. The fact that the weight of the lobe calculated from the CT density was significantly greater than that of the resected specimen, can be partially attributed to the loss of blood from the specimen during and after resection. However, as the volume of the OCT inflated specimens was 94 ± 36 % of the volume calculated from the CT, the specimen was inflated close to it's volume within the intact thorax. The technique described here is less technically demanding than those that seek to track lung volume during CT using spirometry and the results show that on average the entire lung was inflated to 65.5 ± 11.9% of TLC during the scan. The regional lung data also show a linear relationship between lung volume expressed as a percent of TLC and height within the thorax (figure 8) with a slope of 1.8 ± 0.1 %TLC/cm calculated using the restricted maximum likelihood analysis (53). This confirms the classic findings of Milic-Emili et al (154) and Kanako etal {109) using Xenon133. This difference in lung volume is due to a gradient in pleural pressure where the dependent portions of the lung are at a lower transmural pressures than the upper regions (figure 9). Our data shows that this pressure gradient was 0.24 ± 0.08 cmH0/c2 m (figure 10) which compares very favorably to the 0.2 cmH0/c2 m that Milic-Emili reported on healthy volunteers (154). This means that at any level of inflation, the upper regions of the lung are more inflated than the lower regions. As Millar and co-workers (156) found that tissue volume including vessels under 5 mm in diameter was uniform from the top of the lung to the bottom, the observed change in lung attenuation must be due to regional differences in gas inflation. These observations confirm others in the literature (155,156,198,242,248) and 45 extends them by providing a simple quantitative method for estimating the regional differences in lung expansion (91). The individual PV curves can also be used to determine the local transmural pressure at each lung height and calculate the pleural pressure gradient within the thorax. Furthermore comparisons of the regional lung volumes at a given lung height allows a determination of whether the lung volume of each region is appropriate for that lung height or not. This approach should be useful in defining emphysematous destruction where the regional volume will be shifted up and chronic interstitial disease where the regional volume should be shifted down. The volume fraction of blood vessels obtained by point counting the CT images was overestimated compared to point counting directly from the lung surface (table 7a). This could be related to two factors. Firstly, the volume fraction estimates rely on a slice with zero thickness, such as the cut surface of a physical slice, but the CT image is an average of all the components within the slice. Therefore, the orientation of the vessel in the slice, may cause its volume fraction to be overestimated. This is known as the Holmes effect in the stereologic literature (4), and volume averaging in the radiological literature (165). Secondly, part of the difference between the CT and specimen results could also be due to the inevitable loss of blood from the specimen as a result of surgery. The large size and relatively spherical shape of the tumor in relation to the slice thickness reduces the Holmes effect and accounts for the good agreement between the estimate of tumor volume from both the CT and the resected specimen. Therefore, we attribute the overestimate of volume fraction of blood vessels to the problem of point counting of objects that are significantly smaller than the thickness of the CT slice. This problem can be minimized with high-resolution CT which was not available for this study. The surface area of the entire lung of the nine patients in Group 2 (table 7b) was estimated at Level 2 of the histologic analysis by counting the grid line intersects with lung 46 parenchyma and multiplying by the volume of the lung obtained from CT. This shows a mean surface area of 101.8 ± 35.1 m2 and a mean parenchyma tissue thickness is 7.2 ± 1.0 urn (table 7b). These values are consistent with published data on post-mortem lungs (249) and previous studies from ourlaboratory (89). At the level of lung inflation achieved during the CT scan (65.5% of TLC) 60.1% of the specimen consisted of air. The parenchymal Volume fraction, which is the sum of the capillary, medium sized blood vessels (<1 mm) and actual lung tissue volume fraction accounted for 17% of the total volume which also compares favorably with published data (249). The fraction of the lung that is occupied by air and parenchyma can also be determined from the CT density by dividing the specific volume of tissue (assuming a density of 1.065 g/ml (91)) by the calculated specific volume of the lung (tissue and air). This produces a slightly higher parenchymal volume fraction (22%) than the histology (17%) which suggests that the CT estimate may include larger blood vessels than were observed histologically. However, these CT estimates of the fraction of tissue and air can be used to correct the histology to the appropriate air and tissue fraction in the intact thorax. Although this correction is small in the present study because the specimen was inflated, it should be particularly useful in quantitative studies of lung biopsies where lung inflation is an uncontrolled variable. The results of this study confirm that the CT can be used to obtain accurate estimates of the total lung volume and weight as well as regional lung expansion (82,91,146,155,156,183,198,242,248,270). They also extend those observations by providing a simple method of converting lung density to the volume of gas per gram of tissue. This. approach allows the CT data to be integrated with physiologic measurements of the pressure volume characteristics of the same lung; to calculate the volume fractions, surface area and tissue thickness appropriate for the entire lung; and to correct these values to the appropriate intra-thoracic lung volume. 47 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 Neoplastic Process Lung Injury classification divides r-\ Eosinophilic Granuloma Return to Normal the diseases into rH Acute Inflammation Lymphangiomyomatosis two main groups, or Chronic Inflammation Lymphoma pathways, Usual Response depending on Variant Response. Granulomatous Response whether the process UIP= Fibrosing Alveolitis DIP BOOP is based on the 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 shows that the inflammatory exudate contains predominately lymphocytes, plasma cells and alveolar macrophages, with some PMNs (37,46), while Bronchoalveolar lavage (BAL) shows a large proportion of PMN, macrophages - and eosinophils t37,46,205), all of which are increased in patients who smoke (205). 4.2 Fibrotic Mechanisms 4.2.1 Cellular Mechanism of IPF Lung tissue injury results in a proteinaceous exudate into the air space associated with a recruitment of PMNs followed by monocytes from the microvasculature (126). The neutrophil response is acute and their primary purpose is to destroy infectious agents. After migration, the monocytes differentiate into alveolar macrophages that phagocytose and kill pathogenic organisms, scavenge tissue debris and release cytokines. These cytokines include interleukin- 1 (IL-1), platelet derived growth factor (PDGF), epidermal growth factor, tumour necrosis factor (TNF), and transforming growth 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 becomes 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 volume (34,37,88,126). The most obvious histological and biochemical change in areas of fibrosis is the increased amount of collagen. There are several mechanisms for the increased collagen aside from the obvious increase due to more fibroblasts (9,42,126,149,190,199) and the increased synthetic ability of the fibroblasts (9,126,149). 51 Fibroblasts within the gingiva have been shown to have a reduced ability to phagocytose collagen (147) although analogous mechanisms have not been shown in the lung. Additionally there is evidence for decreases in the synthesis of the collagen degradation enzymes (i.e. collagenase and stromeiysin) which are known as metalloproteinases, in conjunction with an increase in the production of molecules that function as tissue inhibitors of metalloproteinases (TIMP) (20,34,190). Therefore, the increase in cell number, synthetic activity, and decreased degradation leads to an increase in the ECM components. 4.2.2 Molecular Mechanisms of IPF TGF-p is the most well studied of the cytokines released by the process, and appears to play the central role in controlling the response of the mesenchymal cells in terms of cell differentiation, proliferation, synthesis of matrix components, and secretion of the other cytokines responsible for the propagation of the response (18,20,119,147,190,196). TGF-p is released initially by macrophages and platelets and is a very strong chemoattractant for phagocytes and fibroblasts (119,147,190,196,268). Once stimulated, fibroblasts synthesize TGF-p which has an autocrine effect on itself so that the process can continue without the aid of inflammatory cells (20). TGF-p has been shown to increase the production of collagen, proteoglycans, fibronectin, TIMPs, and decrease the synthesis of collagenase (20,190). It also inhibits endothelial cell division, and the proliferation of IL-1 dependant inflammatory cells (190). The proteoglycan, decorin, has been shown to bind to TGF-p and inactivate it (20) and the increase in decorin in the early stages of fibrosis led to the postulate that decorin binds TGF-p and directs it to the mesenchymal cells to potentiate the migratory and synthetic effects (196). Another growth factor, PDGF, is released from damaged endothelium, platelets and fibroblasts and has also been shown to cause proliferation of fibroblast cell lines (20,122,19.0). IL-1 is a cytokine that stimulates the immune system as well as acting as a mitogen for 52 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 (60,68,86,141,145,170,182,186,194,203,205,221,223,243,245,278). However, BALand other biochemical studies have the same intrinsic problem in that local acting cytokines and inflammatory cells can all get mixed up in the same test tube with no regard to their location within the tissue so that functional or pathogenic conclusions are difficult at best. Also, BAL washes cells predominately from the larger airways and may not be truly representative of the alveolar situation. To date there are very few quantitative studies on IPF lungs. The histologic studies are either qualitative descriptions of the disease process (113,124) or the extracellular matrix changes (9,21,23,30,55,61,86,95,100,111,125,137,143,161,169,171,177,185,191,201,203,221,269) or semi-quantitative analysis involving the use of complicated scoring systems. Several scoring systems have been derived but these are very complex and for best results involve the use of a panel of trained observers (25-27,101,244). Hyde has reported a comparison between a stereologic quantification of biopsy specimens and a panel grading system and reports that there is good correlation between the two arguing that the time consuming quantitative approach is not justified for an analysis of the tissue changes in IPF (26,101). CT analysis has fallen into this same arena as histology. Semi-quantitative scoring systems are reported to be faster and less expensive than the computer and time intensive quantitative studies (5,6,8,10,51,63,67,79,80,85,133,140,167,175,180,193,204,225,238,259,260,267). Also, quantitative studies of the CT scans in IPF are relatively new and the results are still open to interpretation as to what aspect of the CT scan should be quantified, or what aspect gives a reliable estimate of the disease process. Investigators have reported an increase in overall lung density and changes in the frequency distribution curves of the voxel attenuation values of the CT scan (31,78,155,156). Hartley studied the changes in the frequency distribution curve in 54 interstitial lung disease and showed a correlation between the moments (mean, median, mode, kurtosis, skewness) of the frequency curves and other markers 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 method for quantifying structural changes in the lungs of living patients with IPF using data obtained from CT scans and quantitative stereology (32). Total and regional lung volumes are estimated from X-ray attenuation values on CT. The surface area of the lung parenchyma and the volume fraction of each of the components of the lung is quantified by line intercept and point counting techniques using both the light and electron microscope. This study shows that a combination of the CT and histologic data provides accurate information about 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 were approved by the ethical review boards of the University of Iowa Hospitals, St. Paul's Hospital, and the Universities of Iowa 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 were enrolled by the National Institutes of Health Specialized Center of Research in Interstitial Lung Disease at the University of Iowa. To be included in this group patients had to have a clinical history, chest X- ray, and pulmonary function data suggestive of IPF, and a pathological diagnosis of usual interstitial pneumonia on open lung biopsy. The control subjects were enrolled in the lung registry of the University of British Columbia Pulmonary Research Laboratory and were part of an ongoing study of lung structure and function. These patients required either a lobectomy or 55 pneumonectomy for a small, non-obstructing, peripheral bronchogenic carcinoma whose lung function was measured a few days prior to surgery. The patients selected had normal lung function and were matched for age, sex and smoking history with the IPF patients. 4.5.1 Pulmonary Function Studies Control Patients: Spirometry, lung volumes and lung compliance were measured with subjects seated in a volume displacement body plethysmograph at the Pulmonary Research Laboratory, in Vancouver. 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. Residual volume (RV) was calculated by subtracting vital capacity (VC) from TLC. Measurements of lung volume and its subdivisions were obtained on a P.K. Morgan computerized pulmonary function testing system (P.K. Morgan, Boston, Mass.) using a dry rolling seal spirometer and multiple breath helium dilution techniques on subjects who were unwilling to enter the plethysmograph. Diffusing Capacity (DLco) by the single breath method as described by Miller and associates (157) on the P.K. Morgan automated diffusing capacity analyzer. The results are corrected for alveolar volume (VA) and reported as both DLco and DLCCVVA- The predicted normal values for FEV1f FVC, and DLCo were those of Crapo et al (33) and TLC was predicted using Goldman's values (70). IPF Patients: The lung function for this patient group was studied at the University of Iowa. Spirometry was obtained using a Medical Graphics 1070 system (St. Paul, MN) and the lung volumes were obtained with the patients seated in a body plethysmograph (Medical Graphics 1085 system). The DLco was measured using the single breath technique on the Medical 56 Graphics 1070 system. The predicted normal values were those of Morris et al (162) for FEV,, FVC, Goldman et al (70) for TLC , and Van Ganse et al (237) for DLCo (78). 4.5.2 CT Studies Control Patients: The control subjects in the study received a conventional CT scan (10 mm thick contiguous slices) on a GE 9800 Highlight Advantage CT scanner (General Electric Medical Systems, Milwaukee, Wl) approximately one week prior to resection. All scans were performed with the subject supine during breath holding at full inspiration. Conventional clinical scanning parameters in use at St. Paul's Hospital were used (120 kVe, 100 mA, 2 sec scan time). The images were reconstructed using a standard algorithm, and printed onto radiologic viewing film at a window of 1200 and a level of -700 HU. The image data was transferred to a Silicon Graphics Indy Workstation (Mountainview, CA) for analysis of the X-ray attenuation values. JPF Patients: The CT scans of the IPF patients were obtained at the University of Iowa on an Imatron C-100 ultrafast scanner during inspiration with the patient prone. These high resolution CT (HRCT) scans were obtained using 3 mm sections spaced by 20 mm gaps from the lung apex to the diaphragm (130 kVe, 640 mA, 0.6 s scan time) and were reconstructed using a high spatial frequency algorithm. The images were printed onto standard radiologic film at a window of 2000 and a level of -500 HU, and sent, along with the image data, to the Pulmonary Research Laboratory in Vancouver. The image data was transferred to a Silicon Graphics Indy Workstation (Mountainview, CA) for attenuation analysis. A program to segment the lungs from the chest and the large central blood vessels, and analyze the X-ray attenuation was written for the numerical analysis package PV-Wave (Visual 57 Numerics, Boulder CO). The lungs were segmented using threshold settings of -1000 to -500 HU. The volume of the whole lung (tissue and airspace) was calculated by summing the pixel dimensions in each slice and multiplying by the slice thickness. The mean CT attenuations in HU of each pixel was calculated and converted to gravimetric density (g/ml) by adding 1000 to the HU value and dividing by 1000 (equation 7) (82). The weight of the lung was calculated by multiplying the mean density of the lung by its volume. Regional volumes of gas per gram of tissue were calculated according to equation 8. To eliminate the affect of body position during the scan (supine versus prone) on the distribution curves of lung inflation, the volume of gas per gram of tissue was expressed as percent of the patient's TLC which was obtained by dividing the TLC measured in the upright position (were all the alveoli are evenly inflated) by the measured lung weight. Because of the inflation artifact associated with the biopsy specimens in the IPF patients, the VV(CT) of the biopsy was estimated by identifying the segment 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 VV(CT) from equation 9 (32). This value was then used as the level 2 VV in the stereological analysis below. 4.5.3 Quantitative Histology Control Patients: The resected lung 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 without clamping the airways (32). Once frozen, the specimen was cut into 2 cm thick slices on a band-saw and the tissue sampled with a power driven hole-saw. These samples were stored at -70 °G for other purposes. The remainder of the specimen was 58 transferred to 10% buffered formalin and fixed at room temperature for at least 24 hours. Random samples were taken for light microscopy from the fixed slices, embedded in paraffin and stained with hematoxylin and eosin. Small samples were taken for electron microscopy (EM) from the peripheral lung of five of the patients prior to OCT inflation and freezing and fixed by inflating with 2.5% glutaraldeyhyde in 0.1 M sodium cacodylate buffer using an 18 gauge needle. These specimens were post fixed in 1% Os04 in 0.1 M sodium cacodylate, dehydrated through graded ethanol and infiltrated with LR-White. The tissue blocks were sectioned with a diamond knife on an ultra-microtome ( Reichert Ultra-Cut or RMC MT-6000 XL) at 60-90 nm, picked up on formvar coated 200 mesh copper grids and stained with Uranyl Acetate, and Sato's Lead solutions. IPF Patients: At thoracotomy the lung was biopsied from at least 2 regions, one that appeared normal, and one that appeared diseased on HRCT. The site of the biopsy was confirmed by post• operative chest X-rays in the PA and the lateral position. The biopsy specimens were split and a small portion was prepared for EM as above, and the remainder was processed for light microscopy. To optimize the sampling for the stereologic analysis, a cascade design technique was used as described in Chapter 1 and represented in figure 5. In this design the lung is quantified in a series of steps that increase in magnification so that what is quantified at one level can be further sub-divided into it's components at the successive level (35). Level 1 was performed on the CT scans of both groups, using all of the available image slices by counting the number of points falling on normal lung, densely fibrotic lung, "ground- glass opacification", bronchovascular bundle, and tumor (figure 5A). Levels 2 and 3 were performed at the light microscopy level using the point counting program Gridder (Wilrich Tech, 59 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 (Vv) of each of the lung components, (VV(iC)), were estimated at each level according to equation 3, and the surface density (SV(par)) 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 Sv is an estimate of parenchymal thickness. The overall Vv is calculated by multiplying the Vv of the lung component at the highest level by the Vv that contained it in the 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 were analyzed using independent t-tests or the one way analysis of variance. Transformations were made on certain variables to normalize distributions and to make variances homogeneous. 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. 4.6 Results Table 8 shows anthropometric and lung function data for the patients studied. The control patients have normal lung function are of similar sex distribution but are slightly younger and lighter than the IPF group. The IPF group show the pattern characteristic of restrictive lung disease with a reduction in FEVY FVC, DLCp and in the subdivisions of lung volume (RV, FRC, and TLC), and an increased FEV^FVC ratio. The CT estimates of lung volume and weight (table 9) show a reduction in total lung volume in IPF compared to controls due to a reduction in the volume of the airspace. The tissue volume and therefore the lung weight was not different between the two groups. The frequency distribution in ml of gas per gram of tissue present in each voxel (figure 13) was different between the two groups (p < 0.001). The control lungs showed a normal distribution (table 10) with mean, median and mode values that are closely similar (4.6 ± 0.3,4.5 ± 0.2, and 4.3 ± 0.2 ml/g) and a relatively small variance (18.0 ± 8.2 ml/g) and skew (1.6 ± 0.6 ml/g), while the IPF lungs show a left shifted distribution with a positive skew (6.9 ± 3.4 ml/g) and a very large variance (116.8 ± 30.8 ml/g). The mode (1.4 ± 0.3 ml/g) for the IPF lungs was reduced from control levels, whereas the mean (5.7 ± 0.7 ml/g) and the median (4.1 ± 0.4 ml/g) were not different from the controls due to the large variance. When the median values were expressed as percentage TLC, the values were 72.1 ± 2.5 in control patients, and 80.6 ± 5.2 in IPF 62 patients (p > 0.05) indicating that there was little effect of body position on the degree of lung inflation. Figure 14 compares the volume fraction of tissue estimated from CT to that obtained ; with histology. This shows that in the control cases the resected lung lobes were inflated to approximately the same level during both CT and histologic analysis (figure 14a). However, the biopsies obtained from the IPF lungs were under-inflated during histologic preparation compared to the CT studies (figure 14b). The ability to measure this difference and use the CJ data to correct the biopsies to the correct level of inflation on an individual basis is critical to the subsequent analysis Of these biopsy specimens. The light microscopic data (table 11) show that after the correction to the level of lung inflation obtained during the CT scan, the volume fraction of tissue is increased and the airspace is decreased in the regions of the JPF lungs where there was radiologic evidence of disease, compared to the regions of the IPF lungs that appeared normal and the control lungs. The regions of the IPF lungs thought to be normal on CT showed an increase in the volume fraction of tissue compared to controls and a decrease in the capillary volume fraction from controls, while the medium sized blood vessels were decreased in both the normal and diseased regions of the IPF lungs. A most striking finding was the difference in surface area which was 113 ± 12 m2 in the control lungs and 30 ± 7 m2 in the diseased lungs. This reduction in surface area was associated with an increase in the mean parenchymal thickness from the control level of 8 ± 1 urn to 40 ± 11 urn in the regions of the IPF lungs judged to be normal on CT, and 97 ± 22 urn in the diseased regions. The airspace of both regions of the IPF group contained a significant amount of inflammatory exudate consisting of both proteinaceous fluid and cells. The percentage of the alveolar airspace occupied by fluid was significantly increased in the diseased regions of the IPF lungs when compared with the control lungs. The fraction of the airspace occupied by 63 macrophages was also increased from control levels in both regions of the IPF lungs and when these are expressed as number of cells per square meter of surface area, the macrophage number increased from 12±3X 108 cells/m2 to 30 ± 22 X 108 cells/m2 in "normal" regions of the IPF patients and was significantly higher, 150 ± 100 X 108 cells/m2 (p < 0.001), in the lung regions judged to be diseased on CT. Figure 15 summarizes the results of the stereological analysis of the tissue compartment of the lung based on electron microscopy and are normalized to the lung weight obtained by CT and expressed per 100 grams of lung. The data show that the electron-lucent space tended to be lower in the normal regions and higher in the diseased regions of the IPF lungs compared to control lungs. The collagen content showed a progressive increase from control to diseased IPF regions, whereas the elastin content of the tissue was decreased in the "normal" regions, and there was a trend for an increase in the inflammatory cells which did not reach statistical significance owing to the large variance in number in the different biopsies (p=0.08). 64 CO CO c •g CO co 3 ^CD "co d c > g o c O CO d > ? Q. CO 7 4 ±5 * O 13 0 ± 9 CO "> o "O CO CD Q. FR C CO (%P ) O 12 9 ± 7 O 7 9 ± 11 " O) c > "co TL C (%P ) LU o 7 3 ±4 * 11 5 ± 4 LL. TJ < tT c 81 O O o -J $ > d 8 3 ± 6 8 4 ± 5 CD CO O CD E 8 tT CD E O 9 0 ± 4 5 3 ±4 * _g > o > LO O CO CD o _> CO CO FV C FEV i I— Q. 0.7 2 ± 0.0 X 0.8 4 ± 0.02 * CD "O "O CD CD O o CD FV C (%P ) O C 6 8 ±5 * 10 5 ± 4 o o o > 6? 0 o If CO °s 9 5 ± 8 2 ±5 * E CD CD •g 'x o olO (cm ) c > O Heigh t 16 9 ± 3 16 8 ± 3 o cr E d CO CD c V o E Q. X} (Kg ) 2 o 7 0 ± 3 8 ±5 " Weigh t CO > LU c CO o "co CO 3 o o •g lc -H CO o- Ag e CO (yrs ) co E Q. 5 8 ± 2 6 7 ±2 ' c c CD o 2 CO O) o CD CD d E '£ c Se x iCD_ CD 9M/6 F 9M/6 F o CD Q CO 00 CO & T3 a> c CD O .92 IP F _3 CO o Q. .co co CO CO CO Contro l Q_ Grou p > cQo. O o CO CD (g ) Lun g Weigh t 90 6 ±7 1 108 7 ±5 5 (ml ) Tissu e Volum e 85 1 ± 6 102 1 ±5 2 o o d v Ai r (ml ) a. CO Volum e 395 2 ±29 6 241 5 ±250 * LU c 'CD o CO O CO (ml ) Tota l CD CD Volum e E c E 497 3 ± 33 8 _3 326 5 ± 284 * CD CD o CO CD o> CO TJ « > IP F CD Li. co CL Contro l £• 3 Grou p > 1 7 2 ± 3 8 1 ± 5 %TL C a. lia n m Me c d •H O LO (ml/g ) 4. 5 ± 0. 2 4. 1 ± 0. 4 CO CD c o Mod e (ml/g ) CO 4. 3 ±0. 2 1. 4 ± 0.4 * d •H CO CD (ml/g ) 1. 6 ±0. 6. 9 ± 3.4 * Skewnes s c g O O ro O H— d . c c ro v cn Q. (ml/g ) CD Varianc e c' 18. 0 ±8. 2 3 116. 8 ±30.8 * _1 ro c LU o o CO o CO +1 E © c o CC ro Mea n CD T3 (ml/g ) 5. 7 ±0. CD 4. 6 ±0. 3 E c ro CD CD oT E V- CD "" •*= ro IP F CO CD Contro l J2 Grou p ro > CM g CM CM ~ s •H -H -H CO o o i s CO LO o CO CM «u -H -H o O CO co LO CO (0 CM CM •H a> -H co +1 'I o CD a> CM u CO -H -H +1 CO CO Co +-» T- LO CM d T-• LO o S -H "H -H w "55 X O o o LU co CO i ca 05 co o CO 3 (0 o ci O o C o +) CO E o •H -H o t x: I co CL 00 o d d o CO LO E o LO E « O 0) 0) 3. d d d CL to +i CO E ° V (0 CO CM -C CL _i © CL CM d d d O CO "co CD E O) o c CM CO c re d d o x: OLO cu CL LO a +1 +) * d E LO co T 2 o o o "D v d *~ CO d c CL E CO v k_ CL CO co LO _co c cu o •cK o c o 3 +1 -H CD o o CO +i c o CO CO O 00 .> o o o CO CO d E E CM o o o E CD H— o LU +-» E CO u CO c Ci re c CD CD c O -H +1 +1 " i— Q. CD CD CD > 00 CO CO CM Q_ CO c CD oo CO •a T3 O CO CD CD CD co CO o "CO "co CO CO CO CD E E CD ' E T3 CO. o a. c: o CD CO o 3 cu CO • • 1 o z Q O w CO 1- o LL CO cu CD XJ IS- o Q. (0 _3 CO OL CO > CL zz a s o CO CO o CO LL CM c A CO sz aga i c CO 03 c— O * CO CO 1- oCO sCO ' c o c LO to CO CO X ol o o C>O CO -C tro l o c c o h e 0 rc e 3 CO +—» 10 CL to V> CO c CO CO D) (0 tCoO W se d (0 rep r 05 to CO OLCO c e x e d CO c CO x: CO h- x: CO to o to LO CO ! c O d e E co i- i_ o CO CO CO JLZ CL -*-» „, o CO CO o o i3 wvo sz o CO > Q. co CO CN 3 S|exoAP% CO 69 i ! ! I : 1 • : I 3 4 i 5 5 • 8 9 4 ^ ^ \ \ \ Patient # >» o> 0.20 o • 2 0.10 - w - X 0.00 | H 1- H f- H 1 I) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (C T -0.10 - © - o 1 c -0.20 1 (0 • I £ -0.30 - a - c -0.40 - o - o -0.50 2 UL -0.60 CO - E -0.70 - o3 - > -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 ii m I If 15 9= CO CO to c CO CO XI io O CO o 'oi2 o' 0 XJ v 5 da .2 3 t1 2^ c o c TJ C -H H- CO ° O LJ- £ O 9; •2* T" CD l*S «ti8 x: co co *- to X5 CO 00 -> 5 fl) I I g£ anssji Bun-| Booi Jad ii|6iajy\ 3 71 4.7 Discussion This study provides new quantitative information on the composition of human lungs with IPF obtained using a combination of CT and histologic analysis. The CT scans show a reduction in total lung and airspace volume in IPF (table 9), which correlates with the reduction of static and dynamic lung volumes obtained by pulmonary function tests (table 8). The distribution of volume of gas per gram of lung in IPF lungs, (figure 13) shows that the larger proportion of the lung has lower volume of gas per gram of tissue than controls, and that there are also over-inflated regions indicating larger airspaces. The lung regions with low gas volumes per weight are diseased with reduced airspace, surface area and a reorganization of the tissue components. The regions with a high gas volume per gram are probably the result of the formation of cystic spaces in the diseased areas that are thought to result from a collapse of alveoli onto alveolar ducts with distortion of the ducts by the repair process (88,126). However, it is also possible that some of the overinflation is due to a concomitant emphysematous process (207). Differences in total and regional lung volume will be influenced by the degree of lung inflation during the CT scan, however, the estimates of lung weight should be reliable because it is based on density of the lung which reflects the degree of lung expansion (volume). The effect of body position on lung inflation was accounted for by expressing the volume of gas per gram of tissue of each of the voxels as a percentage of the total lung weight divided into the TLC measured in the upright position where the alveoli are know to be evenly inflated throughout the lung (154). As there was no statistically significant difference in the median values of percentage TLC between the two groups, we conclude that the lung parameters estimated from CT can be compared between the two groups even though some CT scans were conducted prone and others in the supine position. There may have also been differences introduced due to the different scanners that were used for the study, and the slice 72 thickness and reconstruction algorithms used to acquire the images. However, Kemerink and colleagues have shown that the reconstruction algorithms and slice thickness have a negligible effect on densitometry measurements (116). Further, the same group tested numerous scanners for densitometry measurements and concluded that comparisons between properly calibrated scanners is possible (117). The largest difference in densitometry measurements will come from inhomogeneous lungs and since normal human lungs contain a more homogeneous parenchyma than the diseased condition, thicker slices will be appropriate but thinner slices should be used for the changes associated with fibrotic lung disease. Therefore, considering all of the variances associated with different human lungs, which includes the varying degrees of disease, we believe that the differences in scanners and scan acquisition are of minimal consequence. As we have previously shown, there was a good correlation between the air to tissue fraction calculated by both CT and histology in the control cases where the resected specimens were fixed in inflation. However, in the IPF lungs the histologic fraction of air to tissue was markedly reduced because the lung biopsies collapse (figure 14). By locating the segments of the lung that were biopsied on the pre-operative CT scan, the volume fraction of tissue and airspace can be estimated in the intact chest using equation 9. These values can then be used to correct the histologic estimates to the appropriate inflation at the time of the CT scan and is a very important advance in quantitative histology. Surprisingly the data show that lung weight and tissue volume was the same in IPF and control lungs which establishes that the CT appearance reflects a reduction in airspace more than an increase in tissue. The histology suggests that the reduction in airspace is due to a collapse of alveoli on ducts and that this accounts for the marked reduction in surface area. Hogg (88) has previously postulated that the exudate of fluid through the tissue and into the airspaces increases surface tension and causes the alveoli to collapse onto alveolar ducts. Kuhn et al (126) showed that the 73 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 m2 in control patients to 30 ± 7 m2 in IPF and a ten fold increase in mean parenchymal thickness from 8 ± 1 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 DLcoA/A remained within normal limits in these cases. 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 where the diseased regions of the lung showed an increase in electron-lucent space, collagen and interstitial cells per 100 grams of tissue, compared to the control lungs and regions of the IPF lungs not grossly involved by disease. The normal appearing lung regions in the patients . with IPF were ndt significantly different from the controls for any of the variables but did have less inflammatory cells than the diseased regions. Interestingly the amount of elastin present was decreased in the normal regions of the IPF lungs and tended to be intermediate in the diseased regions. The observed reduction in elastin content in the IPF lungs is consistent with reports of minimal synthesis of elastin in mature human lungs (211), and we postulate that proteolytic mechanisms associated with migration inflammatory cells may also result in the reduction in elastin content. The increase in the estimated collagen content within the interstitium has been well documented in IPF (46,126,191), and our observations suggest that the fibrotic process is well advanced in these areas. Bensadoun and co-workers have recently shown that in there is an increase in the proteoglycan content of the interstitium in regions of IPF lungs where there is active disease (13). Proteoglycans play an important role in the fibrotic response by acting as receptors for growth factors (18), directing effects on the synthesis and degradation of elastin (150) and collagen (13). Proteoglycans are highly negatively charged molecules that require hydration to maintain their shape and hence are important for the modulation of tissue hydration (13). As the EM fixation used in our study does not preserve these molecules (234), we speculate that they account for the increase in electron-lucent space present in the diseased areas of the lung. We conclude that the CT scan can be combined with histology to provide a quantitative estimate of the tissue changes occurring within the lungs of patients with IPF. The CT density provides the ratio of tissue and air which can be used to correct open lung biopsies to the level of inflation at which the CT was performed. This approach could provide a method of following the progress of disease using the CT analysis to estimate the histologic changes. 75 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 enlargement that occurs in Down's syndrome. Therefore, in order to be defined as emphysema there must be destruction of the normal tissues, defined as "the reduction of that tissue to a useless form or nothingness" (219). The qualification "without obvious fibrosis" was added to the definition to exclude the airspace enlargement seen in interstitial lung diseases such as sarcoidosis, eosinophilic granuloma and in the end stage fibrotic lung where the predominant feature is the fibroproliferative response which is due to a completely different pathogenic process than the emphysematous destruction. It should be noted here that for a diagnosis by this definition the lung tissue must be directly observed and any other method of obtaining this diagnosis is presumptive. However, there are many features of the disease that when combined enable the physician to make a diagnosis of the disease without having to obtain lung tissue. 5.1.1 Functional Description of Emphysema The most obvious functional characteristic of COPD is the classic airflow limitation shown by a reduced FEV1t FVC and FEVVFVC ratio as well as increases in all of the subdivisions of lung volume (197,261). The pressure volume curve of these patients is shifted up and to the left and there is a reduction in the elastic recoil of the lung (93,181,197,261) signifying a more compliant lung than the normal condition so that at any trans-pulmonary pressure the lung will have a higher volume. The airflow limitation in these patients, shown by a reduction in the FEVL can be due to obstructions within the airways, or to dynamic limitations of the airways as the equal pressure point migrates towards the periphery of the lung and becomes located within the small airways (197,261). The FEVVFVC ratio is decreased due to the reduction in the FEVi. the subdivisions of lung volume are elevated in this condition partially due to the airflow limitation and partially due to the increased compliance of the lung. The greatest change is seen in the RV resulting in a greatly increased RV/TLC ratio and an RV 77 that approaches the VC volume (197). There is often a decrease in DLco which is attributed to the loss of alveolar surface area for gas exchange and the reduction in the bipod volume of the lung (261). 5.1.2 Radiological Description of Emphysema The chest X-ray is an imperfect tool for visualizing emphysema because the overlapping structures hide the emphysematous changes in the lung parenchyma (56,202). Hyperinflation of the chest is shown 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 emphysema on chest X-rays does not correlate very well with pathologic scores and only has a 60-80% diagnostic accuracy with significant false positive rates (202). The( introduction of CT scans advanced in the visualization of emphysema because it removes the overlapping structures allowing the lung parenchyma to be visualized in cross section. On CT emphysema shows hypodense regions and is usually associated with pruning or obliteration of the pulmonary vessels (202). CT has shown high sensitivity and accuracy in the diagnosis of emphysema 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 means 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 main forms of emphysema that can be identified on the gross specimen: centrilobular, panlobular and paraseptal. The most common 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 were the first to show that widespread emphysema was present in younger patients with od-antitrypsin (aPIr ) deficiency (131). At about the same time Gross et al (73) showed that emphysema could be produced by instilling the enzyme papain into the lung. These observations led to the theory that an imbalance between the release of proteases from stimulated PMN and macrophages and the inactivation by serum components could result in emphysema (17,87,103,272). It has been hypothesized that this balance can be upset through a global inactivation of inhibitors, an overwhelming influx of proteases, or through the creation of a local environment where the proteases can be released and the antiproteases would be excluded, such as a "pocket" formed between the PMN and the endothelial cell (57,87,131,272). Originally this theory was developed for the human neutrophil elastase which degrades collagen, fibronectin and proteoglycans as well as elastin but has since been extended to take into account other proteases such as the metalloproteases of the alveolar macrophages and cathepsin G from the PMN (17,57,87,103,218,272). As well as proteases, activated PMN and macrophages release reactive oxygen products through a transfer of electrons from NADPH to oxygen to form the superoxide ion 02" (57,87). Superoxide reacts with hydrogen ions to form hydrogen peroxide (H202) that in presence of transition metal ions can generate the highly reactive hydroxyl radical OH" (57,87). PMN granules also contain myloperoxidase which combines with H202 in the presence of chloride ions to form the hypochlorous acid OHCI" (57,87). These free radicals have very damaging consequences for living tissue by oxidizing membrane 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 have their effects on the host tissue (57). Cigarette smoke is considered the major cause of centrilobular emphysema because it 80 is known to contain a multitude of chemicals, some of which are powerful oxidants with long half lives, and others inactive 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 phase with partially destroyed lung architechure could account for the emphysematous lesions. 5.3 Quantitative Studies in Emphysema Quantitative anatomical studies on lungs with emphysema range from those using semi• quantitative scoring systems to detailed stereologic and other morphometric analysis of the lung. The introduction of CT resulted in renewed interest in these areas where 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 emphysematous changes within the lung. Most of these studies attempt 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 emphysematous change was initiated by Gough and Wentworth (71) who pioneered the use of paper mounted 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 method is extremely tedious and time consuming so Thurlbeck and colleagues developed a modification of this technique which uses a picture grading system where lung slices are compared to a panel of photographs and assigned a score, or grade, between 0 and 100. Scores of 5-25 indicate mild emphysema, 30- 50 moderate emphysema and 60 or greater are defined as severe (227,229). It has been shown that the semi-quantitative panel grading system provides a fast, efficient and reliable estimate of the extent and severity of emphysema (227,229), but was never designed to be used with lobectomy specimens (272) and does not provide quantitative data in terms of 82 absolute volumes or volume fractions of the lung involved by disease (227). Therefore, a quantitative analysis Of the lung must 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 (158,224) superimposed over the gross specimen and the number of points, or squares, over emphysema is divided by the number over normal 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 emphysema centers on enlargement of airspaces with destruction of lung tissue, the most common analysis is quantification of the airspace size performed using the mean linear intercept (Lm) technique (4,45,70,93,249). This procedure is performed by counting intercepts between a test grid of lines and lung parenchyma on systematic random sections of the lung and is a derivation of the surface area method, described in the opening chapter (45,249,252,253,257). A variation by Lang and coworkers (128,129) uses an automated image analysis system to measure the length of the alveolar walls and then calculates the surface area per unit volume (AWUV). Point counting techniques have also been used at this level to estimate the tissue and airspace volume changes as a result of the destruction (83,227). These data all show that in emphysema there is a reduction in the tissue volume fraction (83), with a corresponding decrease in the surface area (Sv or AWUV) (56,72,128,129), and an increase in the size of the airspaces as measured by Lm (93,181,229). However, in human subjects these results are not straight forward as shown by the wide range of alveolar sizes and surface areas with some estimates of severe emphysematous lungs within the normal range 83 (229). Undoubtedly some of this variation is due to the sample used, which tend to be patients in their sixth decade with significant smoking histories who would have significant age related changes as well as varying degrees of emphysema (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 measurements and does not seem to be very sensitive to mild changes in the lung parenchyma (229). There have also been attempts to quantify the number of inflammatory cells within the vasculature and airspaces of emphysematous lungs (83,89,241). These studies have shown that there is an increase in the number of macrophages within the airspaces of patients with emphysema, which is further increased in patients classified as current smokers (241). They also show that the number of PMN in the microvasculature increases in subjects that are actively smoking (142). Studies have also shown 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 emphysema and to correlate better with pathology and pulmonary function tests (72,120,222,229) than the chest film (173,192,202,228). CT scans also yield an image of the lung slice that is similar in appearance to the gross slice and has led investigators to modify the picture grading system to be used on gross sections cut in the transverse plane so that comparisons can be made to the CT image (66). While these correlations have been good, they consistently underestimate the extent of the mild emphysema (66,158,166) which is attributed to volume averaging within the slice because the thinner slices used for HRCT yield 84 better correlation with pathology (64,66,246). A major source of artifact with CT is due to the variable inflation volumes of the lung during the scan which can differ from slice to slice and from scan to scan. This has led investigators to design apparatus that enable scans to be obtained at a known and reproducible spirometric level (10,108,195) or to develop correction criteria which express the lung inflation as a percent of the individual's TLC (31,32). This latter technique enables the CT scan to be compared to the gross specimen and to use stereologic techniques on biopsy material. Investigators have also shown that expiratory CT scans are more reproducible intra and inter scan, and very efficient at demonstrating the hyperinflated regions of the lung due to gas trapping (47,65). As previously mentioned, the CT scan data contains information on the attenuation of X- rays within the lung parenchyma, and Hayhurst showed that patients with emphysema had more voxels in the -900 to -1000 HU range (81). In another study Gould used a computer to assess the frequency distribution of the X-ray attenuation values and found the lowest 5th percentile of the voxels correlated with the extent of the emphysema and had a negative correlation with measurements of AWUV (72). Muller et al used a similar technique to compare the extent of emphysema quantified by a "density mask" at different HU cutoff values with the extent of emphysema quantified on the gross lung specimen (120,166). He showed that the best correlation for conventional CT scans used a density mask of -910 HU and Gevenois followed this study by demonstrating that -950 HU was the best cut off for HRCT (64). 5.4 Experiment #3 Chapters 3 and 4 of this thesis have described a technique which combines CT measurements with quantitative histology to provide information about the structure of normal lungs and lung with idiopathic pulmonary fibrosis (31). This technique uses X-ray attenuation 85 values to estimate lung density and calculate lung weight, tissue and gas volumes. The CT estimates of the proportion of tissue and air in the total volume is then used to correct the histologic measurements made on resected lung tissue to the level of lung inflation present during the CT scan. This chapter compares the lung structure of heavy smokers who had maintained nearly normal lung function with minimal emphysema, as defined by a "density mask" technique, to that present in patients with similar smoking histories and advanced emphysematous lung destruction. This procedure provides quantitative data on lung structure that will be useful in following the natural history of the developing emphysematous process, correlating these structural changes with function and assessing the benefit of lung volume reduction surgery. 5.5 Materials and Methods The procedures used in this study were approved by the ethical review boards of St. Paul's Hospital, the University of Pittsburgh Hospital, and the Universities of British Columbia and Pittsburgh. All of the patients signed informed consent forms that allowed the use of physiologic data, CT scans and the surgically resected tissue. They were divided into control, mild-emphysema, and severe-emphysema groups according to the severity of emphysematous destruction of lung tissue. The patients in the control and mild-emphysema groups required either a lobectomy or pneumonectomy for a small, non-obstructing, peripheral bronchogenic carcinoma and were part of an ongoing study of lung structure and function at the University of British Columbia Pulmonary Research Laboratory in Vancouver. The severe-emphysema group were selected for lung volume reduction surgery at the University of Pittsburgh. These ' patients were separated into their groups based on the percent of the lung defined as emphysematous using the "density mask" technique (166). 86 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 (DLCo) was measured by the single breath method as described by Miller and associates (157). The predicted normal values for FEV! and FVC were those of Crapo etal(33), for DLco; Miller etal(157) 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 GE 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 moments of the curve were obtained. Volume fraction (Vv) of tissue and airspace in the resected portions of lung for the severe-emphysema cases and specific regions identified on the lobectomy specimen was calculated according to equation 9, which has been fully discussed above (31,32). This CT estimation of volume 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 emphysema in each patient was estimated by applying a "density mask" 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 have a HU value of -910 or less and expresses them as a percent of the total. An HU value of -910 represents a lung inflation value of 10.2 ml gas per gram of tissue (equation 2). This is three standard deviations above the 6.0 ml/g established for TLC in patients of similar age and smoking history which have normal lung function (32). The patients with mild disease had greater than five percent but less than 20 percent of their lung volume inflated to volumes above 10.2 ml/g, while patients with severe- emphysema all had greater than 20 percent of their lung inflated above this volume. This technique for measuring emphysema was validated by comparing the CT measurement of the percent emphysema in a lobe to the quantitative histological estimates of the actual amount of emphysema present in that lobe after it was resected. 5.5.3 Quantitative Histology Resected Lobes: The specimens were prepared for quantitative histology as previously described (31,32). Briefly, this was performed by inflating the fresh surgical specimen with Optimal Cutting Temperature (OCT) compound (Miles Laboratories, Elkhart, IN) diluted 1:1 with normal saline, 88 A: CT scan of human lung. B. CT scan with density mask (-910 HU = 10.2 ml/g) applied, attenuation values less than -910 is shown 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 number of points falling on airspace, tissue (lung parenchyma), and medium sized blood vessels (50-1000 pm) as well as the number of intersects between the grid lines and the parenchymal-airspace interface were tabulated. 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 pm)) were counted using a 100 point grid. The volume fraction (Vv) of each of the lung components, (VV(iC)), were estimated at each level according to equation 3, and the surface density SV(par) 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. This analysis was performed on the random sections for an estimate of the total lung surface area as well as the biopsies from the specific sites to estimate the surface density in specific regions of the lung that could be identified on CT. The surface density measurements for all patients were pooled and tested for correlation with the CT measurements of ml gas per gram of tissue from the same region using a mixed effects regression analysis. The overall Vv is calculated by multiplying the Vv of the lung component at the highest level by the Vv that contained it in the previous levels according to equation 5. 5.5.4 Statistical Analysis All data were analysed using the one way analysis of variance and the multivariate analysis of variance. Transformations were made on certain variables to normalise distributions and to make variances homogeneous. The correlation between CT measurements of lung expansion and surface area per volume as well as the diffusing capacity of the lung and surface 92 area were analysed using a mixed effects regression analysis. A p-value of less than 0.05 was considered significant. 5.6 Results The control group (N=23) has less than five percent of their lung volume in the emphysematous category ( greater than 10.2 ml gas per gram of tissue). Those with mild disease (N=7) have a mean of 13 percent (range 5-20%) in this category and those with severe emphysema (N=14) have a mean of 46 percent (range 24-60%) in this category. The patient demographics (table 12) show that the control group is slightly younger but have a similar sex distribution, and body size to the emphysema groups. The smoking history is not statistically different between the three groups but the patients with mild emphysema tended to smoke less. Those with mild-emphysema have FEV^ FVC, TLC and RV values that are similar to the controls but the FEV^FVC ratio and DLco are reduced while the FRC is elevated. Those with severe emphysema have grossly abnormal lung function with all values showing the classic obstructive pattern of reduced FEV^ FVC, FEV.i/FVC and DLco and elevated TLC, FRC, and RV. The CT estimates of lung volume (table 13) show an increase in total lung and airspace volume in the severe-emphysema group compared to the mild-emphysema group which in turn is greater than the controls. The tissue volume and therefore the lung weight is decreased in severe-emphysema, but there is no difference in lung weight between the mild-emphysema group and the control subjects. The cumulative frequency distribution curves (figure 18) of ml gas per gram of tissue present in each voxel are different between the three groups. The vertical arrow indicates the cut off at -910 HU in the density mask technique which is equivalent to 10.2 ml gas per gram of tissue. In the control group 99 ± 0 percent of the voxels are below this cut off, compared to 87 93 •± 1 percent of the mild-emphysema and 54 ± 3 percent of the severe cases of emphysema (table 13). Those with severe-emphysema have 20% of their lung inflated beyond 20 ml/g which is more than three times the amount of air contained in the normal human lung at TLC (32). Further information about the frequency distribution of the ml/g values is shown in table 14 where the control lungs show a normal distribution with mean, median and mode values that are closely similar (4.5 ± 0.2, 4.4 ± 0.1, and 4.4 ± 0.2 ml/g) and a relatively small variance (2.9 ± 2.3 ml/g). The mild-emphysema group show a distribution which is slightly shifted to the right around a mean of 7.1 ± 0.3 ml/g, median of 5.8 ±0.3 ml/g, and mode of 5.3 ± 0.6 ml/g with a very large variance (207.8 ± 111.9 ml/g). The severe-emphysema group has a flattened distribution which is shifted to the right with a mean of 14.0 ± 1.2 ml/g, a median of 9.8 ± 0.4 ml/g, mode of 8.1 ± 0.5 ml/g and the largest variance (578.5 ± 199.6 ml/g). When the median values are expressed as percentage of measured TLC, the control and mild-emphysema patients are not different (66.0 ± 2.2% versus 74.6 ± 3.2%) however, the median value for the severe-emphysema patients was significantly greater (99.7 ± 4.9%) than the other two groups indicating that these patients lungs are hyper-inflated. Table 15 shows the stereology data for all three groups corrected to the level of lung inflation present during the CT scan. This shows a decrease in the percentage of the lung occupied by tissue, and an increase in the percentage occupied by airspace in the group with severe-emphysema. There is a small but significant decrease in the surface area per volume of lung in mild-emphysema and a very marked reduction in this value in the resected lung regions of the group with severe-emphysema. The airspace of these lungs contains an inflammatory exudate. The cellular component of the exudate varies with PMN increasing from 1 ± 0 X 108 cells/m2 in the control group to 9 ± 6 X 108 cells/m2 in the mild disease and 197 ± 34 X 108 cells/m2 in the lungs with severe emphysema. The macrophages, on the other hand, were not increased in the mild emphysema 94 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 cm2/ml both of which are significantly different from zero (p=0.001). 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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 volume returns to control levels but the number of PMN and alveolar macrophages present on the surface are both increased. A change in the nature of the exudate in severe disease could be important in the pathogenesis of lung destruction. The relative importance of the proteolytic enzymes produced by PMN and those produced by alveolar macrophages and other cells in the pathogenesis of emphysema is controversial (17,89). The cigarette smoking habit increases the number of PMN and macrophages in lung tissue and airspaces (205,241) and active smoking increases PMN concentration in the lung microvessels (142). Our data show that mild lung destruction is only associated with an increase in PMN whereas advanced lung destruction is associate with large numbers of both PMN and macrophages on the alveolar surface. As the controls in this study had a significant smoking history, their macrophage level is probably already elevated from that in non-smokers. The alveolar macrophage and the PMN are linked through a network of cytokine and growth factors (57) and the increased macrophages may keep the recruitment of PMN high enough that the PMN degradative enzymes can destroy the lung tissue to produce the severe disease. Other investigators have estimated the severity of emphysema using either a "density mask" with a single cut off value (158,166) or the lowest fifth percentile (72) of the x-ray attenuation values expressed in Hounsfield Units (HU). The simple calculation we use to convert HU obtained from conventional CT scans to a more physiologically meaningful unit, ml of gas per gram of tissue (32), allows us to relate regional to total lung volume and determine the overall lung volume at which the CT scan was obtained. This data show (table 14) that when the lung volume at the time of the CT scan is expressed as a percent of measured TLC the control group was at 66.0 ± 2.2% of TLC the mild emphysematous group was at 74.6 ± 3.2% and the severely emphysematous group was at 99.7 ± 4.9% of TLC. This tendency for those with severe disease to reach a higher overall lung volume is consistent with the fact that emphysematous lesions reach full inflation at very low transpulmonary pressures (92). 105 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 (cm2/ml) and lung volume (ml/g) 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 CHAPTER 6: Summary and Discussion 6.1 Summary The data1 presented in this thesis has established that the CT scan can be combined with quantitative histology to quantify the tissue changes in chronic lung diseases. The CT scan provides a powerful tool for the estimation of total and regional lung volumes and weight, and provides a method of correcting open lung biopsy material to the level of lung inflation in the intact thorax. The data from the IPF studies presented in chapter 4 show that the decrease in total lung volume which is due to a loss of the airspace volume with minimal changes in the tissue volume. In emphysema, there is an increase in the total lung volume which is due, first of all, to an increase in the airspace volume, with minor changes in the tissue volume, which is then followed by a decrease in the tissue component through proteolytic destruction. The frequency distribution curves of these studies show thatthe control lungs have a normal distribution around a mean and median of 4.5 ml gas/g tissue which is 66% of the patient's TLC divided by lung weight (6.0 ml/g). In IPF the curve is grossly shifted to the left (figure 13) indicating that there is an increase in the proportion of the lung occupied by tissue, and the long tail suggests that there are regions of hyperinflation probably due to the cystic changes of the end stage lung, concomitant emphysematous changes, or a combination of both. In emphysema (figure 18), the curve is shifted toward more gas volume per gram of tissue and this is especially true in the severe cases where over half of the lung has a density greater than 10.2 ml/g, which has been shown to be a good estimate of the emphysema present on conventional CT scans (120,158,166). Also, the CT density provides data on the tissue and airspace volume fraction which is essential for the correction of lung biopsy specimens to an appropriate level of inflation for comparisons between individuals and even different biopsy from the same lung. 108 The frequency distribution data in the CT analysis provides an important tool for the longitudinal studies of lung disease because it is sensitive to changes in lung composition. This present study confirms the work of Hayhurst (81) in emphysema and Hartley (78) in interstitial lung disease, who showed that the properties of the attenuation curve correlate with changes in lung structure. We have modified their technique to express the x-ray attenuation values in terms of ml of gas per gram of tissue which we propose is a more physiologically useful expression because it allows the analysis of the lung in terms of the pleural pressure gradient and regional lung inflation. The greatest problem with this analysis is that it does not separate airspace infiltration from fibrotic remodeling and more investigations need to be done in this area with careful correlations between the frequency distribution curves of the radiological categories "ground-glass" attenuation and "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 an appropriate level. We have shown in chapter 3 that this is a valid assumption if the gross specimen is able to be inflated through the airways. However, figure 14 shows that when the inflation is not controlled, the histologic information is not comparable between individuals, or even different biopsies. This thesis describes a technique where CT measurements of lung density can be used to calculate the volume fraction of tissue (equation 9) in specific regions of the lung. These values can then be used to correct open lung biopsies, where the inflation cannot be controlled, to an appropriate level of inflation for quantitative anatomic analysis (chapter 4). The stereology data shows that there is an increase in the tissue volume fractions of lungs with IPF and a decrease in emphysema. There is, however, a loss of surface area in both conditions which is due to a reorganization of the lung parenchyma in IPF and a destruction of alveolar walls in emphysema. Figure 20 shows that the surface area of the lung 109 is related to the diffusing capacity in the emphysematous patients. When the surface area and DLCO measurements from the patients with IPF are added to the analysis, a statistical difference is not detected to the emphysematous cases so the groups can be combined. The mixed effects regression line for all of the patients (figure 21) has a slope of 0.09 ml/min/mmHg/m2 and an intercept of 9.4 ml/min/mmHg, both of which are highly significantly different from zero (p < 0.001). This is an important finding because CT measurements of lung inflation have been shown to have a negative correlation with the surface area (figure 19, (72)) and this provides a tool for the clinician to quantitatively follow the patient with chronic lung disease and to assess the changes in lung structure and function over the course of the disease. The inflammatory response in the lung is very important in the pathogenesis of both IPF and emphysema and this study has shown that there are increased inflammatory cells In the airspaces of both diseases. These cells consist mostly of alveolar macrophages because of the chronic condition of the process, and the central role that macrophages play in terms of modulating the inflammatory and the fibroproliferative response. It is very tempting to characterize these changes as different outcomes to the same process because it has been shown that there is a fibroproliferative response in the early stages of emphysema that results in modifications to the elastin and collagen content of the parenchyma which is characterized as fibrosis. However, this fibrosis is mild and the predominant feature of emphysema is the destruction of the lung tissue. The exact molecular events of the fibrosis still need more clarification, but it is now obvious that the larger molecules of collagen and elastin are not the only players in the game and that proteoglycans are a very dynamic constituent of the fibrotic response. Proteoglycans appear very early in the fibroproliferative response and have been linked to key roles in fibrosis such as tissue hydration, cytokine binding and cellular adhesion 110 o o co g CM ^•v CD CO C CO Z5 I « CD £ 3 <*- Zj O TJ CO. ff J= CD c CL C CD O O O o CO o LO c CO CO TJ LP 11 o o TJ J= XJ § c> CD E LO OJ § • CO TJ CD CO C CD CO E LL TJ c CO 0. co CD O oT-g 2 O < C o = SCO c o TJ CJ) o -s CD C —' CO ^2 TJ CD CD .g -f= -g O LO CO o CO CN (6HLULU/UJUI/|IJU) 03HQ 2 111 (77,200,268). Their exact role in emphysema is still to be investigated, but the increased alveolar wall thickness seen early on in the emphysematous lesions is an attractive site for PG synthesis. Why one process proceeds to the proliferative stage while the other one elevates .... the degradative response still needs to be 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 and functional characteristics has proven to be weak and of limited value aside from a quick process for assessing the severity of the disease. It is time to move beyond these semi-quantitative analysis and describe the lung in terms of the three-dimensional structural parameters that it possesses. The CT scan avails itself for quantitative longitudinal studies of the lung because it is minimally invasive and easy to perform. A few simple calculations of the data may provide important information that allows the assessment of the lung in terms of airspace enlargement, loss of surface area and tissue destruction or reorganization. This may become very important in the long term follow up of patients with IPF to assess the time course of the disease and to assess treatment protocols and in emphysema for the assessment of the lung structure in terms of selecting patients for the palliative surgical treatment of lung volume reduction surgery. Lung volume reduction surgery is a controversial and very expensive procedure and the ability to choose patients pre-operatively that will benefit from the intervention has great ramifications for both the patient's health and the resources of the health care provider. A quantitative CT analysis of the lung may be able to delineate the structural factors in the lung that are responsible for the improvement or non-improvement of these patients. Also, by use of a density mask and three-dimensional reconstruction, a map can be provided to the surgical team clearly demonstrating the extent and location of the emphysema to help decide where the best 112 site for reduction surgery (figure 22). 6.3 Conclusion In conclusion, it has been shown that the combination of quantitative CT and stereology provides reliable quantitative data on the lung structure in chronic lung disease. These studies have detailed the procedure that enables the CT to measure the total and regional volume changes within the lung and to quantify the ultra-structural changes responsible for the patho• physiological changes seen by the clinician. 113 Figure 22. Three dimensional reconstruction of a human lung with emphysema using CT scan images. Emphysema is shown in red, and normal lung parenchyma in blue, while dense tissue is green. 114 REFERENCES 1. Abbas, A.K., A.H. Lichtman, and J.S. Pober. Cells and tissues of the immune system. 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