Comparative study of post pneumonectomy compensatory lung response in growing male and female rats by Sekhon Harmanjat inder Singh B.Sc. Punjab University. Chandigarh. INDIA. 1971 M.B.B.S.. Magadh University. Gaya. INDIA, 1980 A THESIS S U B M I T T E D IN PARTIAL FULF ILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Academic Pathology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November. 1987 © Sekhon Harmanjatinder Singh. 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the 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 De-partment 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 Academic Pathology Faculty of Medicine The University of British Columbia 2211 Wesbrook Mall Vancouver. BC , Canada V6T 1W5 Date: November. 1987 ii A B S T R A C T Male and female Sprague-Dawley rats matched for litter and body weight, were sub-jected to left pneumonectomy and sham operations at four weeks of age. Three weeks following surgery, rats were sacrificed, and somatic and lung growth, pressure-volume curves, biochemical, and morphometric parameters were measured. Females weighed 48% less than males at the end of the experiment. Somatic growth of neither sex was effected by pneumonectomy. Following pneumonectomy, lung weight and lung volume increased significantly and matched that of both lungs of the sham-operated group in both sexes. The absolute amount of DNA and protein content also increased but was significantly less than that of both lungs of shams. Since females weighed less, absolute lung weight, lung volume, DNA and protein con-tent increased more in males but specific parameters (i. e. values/ 100 g body weight) increased significantly less compared to females. This occurred because specific lung weight and volume decreased with increasing body weight. Mean linear intercept and mean chord length of alveoli were increased. Alveolar surface area increased by 51% in males and 31% in females, and matched that of both lungs of shams in males but not in females. The total number of alveoli increased 15% and 18% in males and females respectively and was significantly less compared to both lungs of shams in both sexes. After pneumonectomy, the post-caval lobe increased in volume 70% and 73% in males and females respectively as compared to a 60% and 47% increase in total lung volume. The mean linear intercept and mean chord length of alveoli increased less in the upper and lower lobes compared to the middle and post-caval lobes in males as i i i well as in females. The number of alveoli per unit volume decreased more in middle and post-caval lobes compared to the upper and lower lobes in both sexes. In sham-operated male rats the upper and lower lobes had a smaller mean linear intercept and mean chord length of alveoli compared to the post-caval lobe. Postpneumonectomy, loss of elastic lung recoil at mid-volumes was observed in females. It was inferred that compensatory response following pneumonectomy was in gen-eral similar in males and females. While there was an evidence of alveolar multiplica-tion, simple dilation of airspaces occurred and this was the dominant effect especially in females. In certain aspects (weight, volume) compensatory growth was complete but in most (DNA. protein, morphometry) was not. Male and female differences could not account for differing results in the literature concerning completeness or otherwise of lung compensatory growth. Contents A B S T R A C T i i L I S T O F T A B L E S v i i i L I S T O F F I G U R E S x i A C K N O W L E D G E M E N T S x i i 1 I N T R O D U C T I O N 1 1.1 Objectives 1 1.2 Normal postnatal lung growth 1 1.3 Compensatory growth 4 1.4 Pneumonectomy and compensatory growth 5 1.4.1 Extent of compensatory lung response 5 1.4.2 Nature of response 6 1.4.3 Alveolar multiplication 8 1.4.4 Effect of age 10 1.4.5 Duration of response 11 1.4.6 Effect of sex 12 1.4.7 Lobar response 12 1.4.8 Location of response 14 1.4.9 Physiological studies 14 iv V 1.4.10 Control of compensatory lung growth 16 a. Mechanical stretch 16 b. Hypoxia and hypoxemia 17 c. Oxygen consumption 18 d. Specific growth factors 19 e. Hormones 20 f. Ozone ( O s ) 21 g. Endotoxins 21 1.5 Rationale 21 2 M A T E R I A L S A N D M E T H O D S 22 2.1 Materials 22 2.1.1 Animals 22 2.1.2 Chemicals 22 2.2 Methodology 22 2.2.1 General experimental design 22 2.2.2 Experimental protocol 23 a. Surgery 23 b. Termination 24 2.2.3 Pressure-volume curve manuever 24 2.2.4 Biochemistry 25 a. D N A and protein extraction 25 b. Alkali soluble protein estimation 26 c. Deoxyribonucleic acid ( D N A ) estimation 26 2.2.5 Morphometry 28 a. Lung fixation and volume determination 28 c. T issue shrinkage factor 29 VI 2.3 Statistical analysis of data 32 3 R E S U L T S 36 3.1 Expression of results 36 3.2 Somatic growth 38 3.3 Compensatory response 39 3.3.1 Extent of response 39 a. Males 39 b. Females 39 c. Difference in males and females 42 3.3.2 Nature of response (Lobar response) 42 a. Males 42 b. Females : 43 3.3.3 Nature of response (Global response) 46 a. Males 46 b. Females 46 c. Difference in males and females 47 3.3.4 Biochemical changes 48 3.4 Lung mechanics 49 3.5° Morphometric interlobar differences in shams _ 52 3.5.1 Males 52 3.5.2 Females 52 4 D I S C U S S I O N 56 4.1 Somatic growth 57 4.2 Extent of response 57 4.3 Nature of response 59 v i i 4.4 Global response 61 4.4.1 Males 61 4.4.2 Females 62 4.5 Lobar response • • • 63 4.5.1 Males 63 4.5.2 Females 65 4.6 Lung mechanics 65 4.7 Lung structure at age 7 weeks 67 4.8 Difference between males and females following pneumonectomy . . . . 70 4.9 Intersex response and differences in the literature 71 4.10 Conclusions 76 B I B L I O G R A P H Y 78 A A P P E N D I X 89 L i s t o f Tab le s 2.1 Number of animals used per group per analysis (morphometric and biochemical.) 23 2.2 Morphometric calculations 35 3.1 Abbreviations 37 3.2 Somatic growth results for male sham-operated vs female sham-operated group, and male pneumonectomy vs female pnuemonectomy group. . . 38 3.3 Extent of compensatory lung growth results for male pneumonectomy group compared to male sham-operated group 40 3.4 Extent of compensatory lung growth results for female pneumonectomy group compared to female sham-operated group 41 3.5 Lobe volumes (ml): Male pneumonectomy group compared to male sham-operated group and female pneumonectomy group compared to female sham-operated group 43 3.6 Lobar morphometric response results for male pneumonectomy group compared to male sham-operated group 44 3.7 Lobar morphometric response results for female pneumonectomy group compared to female sham-operated group 45 3.8 Global morphometric response results for male pneumonectomy group compared to male sham-operated group 47 3.9 Global morphometric response results for female pneumonectomy group compared to female sham-operated group 48 v i i i IX 3.10 Nature of lung growth, a biochemical aspect: Male sham-operated vs male pneumonectomy group, and female sham-operated vs female pneumonectomy group 49 3.11 Static pressure-volume curve results for male sham-operated vs male pneumonectomy group, and female sham-operated vs female pneu-monectomy group 52 3.12 Morphometric results for interlobar difference 54 4.1 Global morphometric response in males following pneumonectomy. . . . 62 4.2 Global morphometric response in females following pneumonectomy. . . 63 4.3 Lobar morphometric response in males following pneumonectomy. . . . 64 4.4 Lobar morphometric response in females following pneumonectomy. . . 66 4.5 Results for hypothetical experiment [A] using data from the present study 72 4.6 Results for hypothetical experiment [B] using data from the present study 73 A . l Somatic growth results for male sham-operated vs male pneumonec-tomy, female shame-operated vs female pneumonectomy groups. . . . 89 A.2 Extent of compensatory lung growth results for male sham-operated group compared to female sham-operated group 90 A.3 Extent of compensatory lung growth results for male pneumonectomy group compared to female pnemonectomy group 90 A.4 Lobar morphometric results for male sham-operated group compared to female sham-operated group 91 A.5 Lobar morphometric results for male pneumonectomy group compared to female pnemonectomy group 92 A.6 Global morphometric response for male sham-operated group compared to female sham-operated group 93 X A.7 Global morphomteric response results for male pneumonectomy group compared to female pnemonectomy group 93 A.8 Nature of lung growth, a biochemical aspect: male sham-operated vs female sham-operated group, and male pnemonectomy vs female pnemonectomy group 93 A.9 Static pressure-volume curve results for male sham-operated vs female sham-operated group, and male pneumonectomy vs female pneumonec-tomy group 94 List of Figures 2.1 D N A and protein extraction 27 2.2 Test grid used for light microscopic morphometry 31 2.3 Illustration of intercept counting 33 2.4 Illustration of alveolar counting 34 3.1 Static pressure-volume curve of female pneumonectomy group plotted against female sham-operated group 50 3.2 Static pressure-volume curve of male pneumonectomy group vs. male sham-operated group 51 3.3 Static pressure-volume curve of male sham-operated group vs. female sham-operated group 53 4.1 Lung weight in males at the time of 177 g body weight 68 4.2 Lung volume in males at the time of 177 g body weight 69 xi xu Acknowledgements I specially and sincerely thank my supervisor, Dr. W. M. Thurlbeck for his endless support, enormous encouragement and precious time which he spent to guide me throughout my graduate studies. His invaluable advice, utmost patience and con-structive criticism helped me to solve the riddles to made this work possible. Also my sincere gratitudes to my supervisory committee. Dr. J. B. Hudson, Dr. D. E. Brooks, Dr. D. F. Smith and Dr. J. Emerman for their invaluable time to understand my unique situation and invaluable guidance and suggestions. I would like to express my deep appreciation of the friendship , support and en-couragement I have received while doing my research, from Craig Smith, Felix Ofulue, Hassan Khadampour, Jan Larson, Kamala Sudhakar and Mary Battell. Especially, I am indebted to Dr. Sudhakar Cherukupalli for his invaluable assistance in processing this work on the computer. Finally I would like to extend my gratitude to Ominder and Narinder Chauhan , Manohar Patara. Lakhbir. Satnam and Mypinder for encouragement, endless warmth and appreciation. Dedicated to My PAZSMTS Gulzara Singh and Manjit Kaur Sekhon AND TO MY IN-LAWS Avtar Singh and Gurmeet Kaur Nahal Chapter 1 INTRODUCTION 1 . 1 O b j e c t i v e s T h e intent of this experiment was to study structural, physiological and biochemical aspects of compensatory response following pneumonectomy in growing male and fe-male rats. T h e specific objectives of this investigation were; 1. T o test the hypothesis that the postpneumonectomy compensatory response in young male and female rats, is different. 2. T o test the hypothesis that all lobes of the right lung are morphologically similar. 3. To study the difference in the interlobar compensatory response after left pneu-monectomy in growing males and females. 1 . 2 N o r m a l p o s t n a t a l l u n g g r o w t h T h e transition of the fetus from intra-uterine to extra-uterine life is sudden, the fetal-placental circulation is interrupted and adaptation to extra-uterine life is crucial. At this stage the gas exchanging surface in many species is immature without the defini-tive components of the adult lungs. It has been agreed that at the time of birth rodents such as rats (Newhauser and Dingier. 1962; Weibel. 1967; Burri et al., 1974). mice (Wilson, 1928; A m y et al., 1977) and mammals such as rabbits (Engel. 1953). cats (Engel. 1953; Dingier. 1958) and dogs (Boyden and Tompsett , 1961) do not possess 1 9 alveoli, the terminal air exchanging units in adult lungs. They contain almost exclu-sively primitive thick walled primary saccules (Weibel, 1967; Burri, 1974) or terminal sacs (Boyden and Tompsett . 1961), which undergo rapid subdivision postnatally by secondary crests which results in structural rearrangement to form true alveoli. These smooth walled primary saccules are large and lack surface complexity. T h e wall has double capillary network which runs on either side of the connective tissue framework (Burri et a l . . l974) . In humans, some investigators (Emery and Mithal . 1960; Mithal and Emery. 1961; Boyden and Tompsett . 1965; Boyden. 1967; Wi lcock. 1966; Reid, 1967: Hislop and Reid. 1974) have stated that alveoli are absent in the fetus at the time of birth and suggested that the structures in question were saccules (Hislop and Reid, 1974). How-ever, Loosli and Potter (1959) observed that from seven and one-half months gestation until term, there was further development of respiratory channels into alveolar ducts which produced small but definitive alveoli. Langston and Thur lbeck (1982) found a mean of 55 million and a range of 10-149 million alveoli at the time of birth. Dunhill (1962) reported 20 million in one infant, Hieronymi (1960; 1961) found 70 million and Thur lbeck and Angus (1975) counted 71 million alveoli at birth. It has been pointed out that by 32 weeks gestation, alveoli-like structures can be found in the fetal lungs and definitive alveoli are present at 36 weeks of gestational age (Brody and Thur l -beck, 1986). T h e conflicting results could be due to biological variations, difference in tissue preparation or difficulty of defining and recognizing alveoli (Thurlbeck. 1975). Whether the complement of acini at birth, increases postnatally by simple enlargement (Kolliker. 1881) or due to formation of additional structures (Browman, 1923; Heiss, 1923) has been a matter of controversy for the past century. In adults, there is a range of 200-600 million alveoli (Angus and Thurlbeck, 1972). T h e alveolar surface area increases twelve fold and lung volume twenty-eight fold from birth to adult life. T h e constant number of alveoli per unit volume and alveolar size up to 2-4 years of postnatal age in children is an indication of alveolar multiplication and subsequent to that no or little increase in total alveolar number occurs (Thurlbeck, 1982). Later in chi ldhood, it is thought that alveolar multiplication continues but at a slower rate and does not cease until somatic growth stops (Thurlbeck, 1975). However, some investigators have suggested that alveolar multiplication ceases between age of 4 and 3 11 years (Dunhil l . 1962; Davies and Reid, 1970). After 8-9 years of age, minimal multiplication of alveoli occurs and change in lung structure is related to chest wall growth and body stature (Thurlbeck and Haines, 1976). Boys have bigger lungs in comparison to girls of same age and height (Thurlbeck. 1982). Postnatal lung growth in animals has been studied in detail (Engel, 1953; Dingier, 1958; Weibel, 1967; Burri, 1974; Burri et al.. 1974; Kauffman et al.. 1974; A m y et al., 1977). In rats and mice during the first day of life there is little change in lung weight (Amy et al., 1977) or lung volume (Short. 1951). Trit iated thymidine incorporation into D N A for the first 24 hours is also negligible (Kauffman et al., 1974). T h e lungs then increase rapidly in weight (Amy et al.. 1977; Nijjar, 1979) and the rate of tritiated thymidine incorporation into D N A is also increased (Das and Thurlbeck, 1979). In rats, postnatal lung growth has been divided into three distinct phases (Burri, 1974). During the first phase (lung expansion phase, days 1-4), the lung primarily grows by expansion due to a gain in the volume of airspaces. The second phase (tissue proliferation phase, days 5-13) is identified with an increase in lung weight and lung volume. Lung weight doubles and lung volume triples during this period. D N A synthesis increases intensively (Kauffman et al., 1974). T h e third phase (equilibriated lung growth, day 14- ) comprises a slow but proportional increase in lung weight and lung volume. New alveoli continue to be added but the rate of cellular proliferation decreases (Kauffman et al.. 1974). Alveolar surface area increases directly with lung volume. T h e number of alveolar type I cells increase 3-4 times up to 6 weeks (Brody and Thurlbeck, 1986), and the thickness of the alveolar wall decreases primarily due to diminished cellular components of the interstitial compartment. A fourth phase (Thurlbeck. 1975) of simple expansion is present in some species. It starts when alveolar multiplication ceases between 6 and 10 weeks of age (Holmes and Thurlbeck, 1979), but it has been reported that alveolar multiplication continues even up to 133 days of age of rats (Weibel. 1967). In the past century, various techniques have been employed to manipulate the lungs to alter or stimulate lung growth in postnatal life. Several maneuvers i.e. pneu-monectomy, adrenalectomy, exposure to hypobaric hypoxic conditions, administration of growth hormone etc.. have been used to study altered lung growth. However, inter-pretation of results of these manipulations is hard to extrapolate to normal postnatal 4 lung growth, yet could serve as a useful model to understand this complex process. 1 . 3 C o m p e n s a t o r y g r o w t h Partial extirpation of an organ is a common surgical procedure to limit or cure a disease. Organs such as kidney, liver, adrenal, gut, and lung show compensatory growth of the remaining tissue after partial resection. However, this occurrence is not true in all organs and organs that do not show compensatory growth include ovaries, testes, brain, or the medullary portion of the adrenals (Addis, 1928). Restoration of the organ mass may take place by cellular proliferation (hyperplasia) as in the liver, or by increase in cell size (hypertrophy) as in heart, or both as in the kidney. A hyperplastic cellular response is observed by rapid increase in amount of D N A content without apparent change in cell size, while hypertrophic changes are characterized by increase in protein per nucleus and R N A to D N A ratio. In most organs, the original morphology is maintained with slight alteration in the architecture. In rats after resection of 2/3 of liver mass, the residual tissue restores the original mass within 10 days (Becker. 1969; Tsanev, 1975). Cellular proliferation is more rapid in younger animals and mitotic activity starts in the liver cells within 24 hours of hepatectomy. T h e lobes after resection are not reformed. T h e new functional units are formed by proliferation of new acini within the old lobules which ultimately increase the size of the liver. T h e architecture of the restored liver differs slightly from the original tissue (Lewan, 1977). Similarly in rats after nephrectomy soon after birth, compensatory response in the contralateral kidney is hyperplastic, but if resection is done after 40 days of age. hypertrophic changes take place (Krap et al., 1971) Complete renal function restoration takes place within 6 weeks after 75% ablation of renal mass at birth in puppies, while surgery performed at the age of 8 weeks results in only 45% restoration over the same t ime period (Archinberg, 1978). Unilateral nephrectomy results in an increase of glomerular number, if resection is done before the age of 50 days in rats, but not thereafter (Bonvalet et al. . 1972). In growing rats, removal of 50% and 70% of renal mass resulted in an increase of 81% and 168% respectively in comparison to 31% in control rats (Kauffman et al.. 1974). Thus , it is evident that compensatory growth 5 of kidney is qualitatively and quantitatively age dependant and also depends upon the quantity of the mass removed. 1 . 4 P n e u m o n e c t o m y a n d c o m p e n s a t o r y g r o w t h After resection of a lung (pneumonectomy) or a part of a lung ( lobectomy), a signifi-cant space is created within the thoracic cavity. How does the residual lung respond and how is the interdependence of function and structures maintained, were the ques-tions put forth by Hassler in 1892. He suggested that in young developing rats after ablation of a lung, the contralateral lung grew by hyperplastic regeneration. Later Mohlgaard and Rovsing (1910) and Kawamura (1914) stated that compensatory lung growth in the adults occurs by dilatation of existing structures. Since then, contro-versy has existed whether the postpneumonectomy or post lobectomy compensatory response in the contralateral or ipsilateral lung is complete (equal to both lungs of the controls), or incomplete (less than both lungs of the controls), and if it occurs by formation of additional new structures or by enlargement of the already existing ones. 1 . 4 . 1 Extent of compensatory lung response Lungs are different in comparison to the other organs of the body which have been studied for compensatory growth. They receive the entire cardiac output and after pneumonectomy or lobectomy, the whole blood flow is directed to the residual lung tissue. T hus , increased blood volume load and loss of lung volume and parenchyma has enormous impact on the remaining tissue. After pneumonectomy, a marked increase in the lung size of rats (Addis. 1928; Cohn, 1939; Romanova, 1960; Buhain and Brody, 1973; Nattie et al.. 1974; Holmes and Thurlbeck, 1979: Burri and Sehovic. 1979). rabbits (Sery et al.. 1969; Boatman. 1977: Langston et al., 1977; B o a t m a n et al., 1983), guinea pigs (Gnavi et al., 1970), cats (Bremer, 1936). and dogs (RienhofF. 1935; Fisher and Simnett, 1973; Thur lbeck et al.. 1981; Arnup et al. 1984) has been described. However, the extent of the postpneumonectomy response of lung is still controversial. Previous studies have shown that after pneumonectomy or lobectomy, equal or near equal (differences which were statistically insignificant) values (lung weight, lung volume. D N A and protein content) were achieved in comparison to both 6 lungs of controls (Sery et al.. 1969; Romanova, 1971; Nattie et al., 1974; Langston et al. . 1977: Boatman, 1977; Burri ad Sehovic, 1979: Thur lbeck et al.. 1981; Yee and Hyatt. 1983; Rannels et al., 1984). Hence, the restitution was considered to be complete. O n the other hand, some investigators have documented an incomplete response. M c B r i d e (1985) found that in ferrets, the volume of the contralateral lung after pneu-monectomy was 17% less than controls. Long-Evans rats after left pneumonectomy attained only 85% lung volume and lung weight in comparison to the controls (Buhain and Brody, 1973). Holmes and Thur lbeck (1979) observed that Sprague-Dawley rats pneumonectomized at 4, 8, and 12 weeks of age achieved equal lung weight but a smaller lung volume than controls. Brody et al. (1978) reported that in mice, increase in lung weight preceeded the increase in lung volume, but Das and Thur lbeck (1979) observed these changes in reverse order in rabbits. Increase in lung volume may not be due to an increase of tissue volume. It may be due to overexpansion of the lung, a unique characteristic of that organ (Thurlbeck, 1975). In rats, the response appears to be complete by about one week, in mice by two weeks and in rabbits by three weeks (Cagle and Thurlbeck. 1988). 1 . 4 . 2 N a t u r e o f r e s p o n s e T h e increase in tissue mass of the remaining lung after pneumonectomy may be due to hypertrophy (increase in cell size as assessed by an increased P r o t e i n / D N A ratio and/or R N A / D N A ratio), hyperplasia (as assessed by an increase in D N A content), of the parenchyma, or a combination of both. T h e lung has the unique characteristic that it may be able to increase in volume by overexpansion of airspaces to reach a new size without an increase in tissue volume. T h i s increase in size should not be interpreted as hypertrophy and the terms hypertrophy and hyperplasia should be confined to the cellular response within the lung. In the early postoperative period, increase in lung weight is also observed due to edema and congestion (Romanova et al., 1967). She also found a small increase in the wet/dry lung weight ratio ascribed to edema. Addis (1928) reported that, based on nitrogen content, the 40% increase in the weight of the 7 remaining lung in albino female rats 61 days after pneumonectomy, was not due to the presence of blood (he removed the blood by flushing the lungs), but due to increase in the actual tissue mass. In rats (Fisher and Simnett, 1973; Rannels et al.. 1979). mice (Simnett. 1974; Brody et al., 1978) and rabbits (Cowan and Crysta l . 1975; Das and Thurlbeck. 1979) after pneumonectomy, cellular hyperplasia has been noticed by observing increased mitotic indices or increased tritiated thymidine incorporation of parenchymal cells and total D N A content. Romanova et al. (1967) observed that the mitotic index doubled on the 3rd or 4th day postpneumonectomy and 5 fold on day 5. By day 7, it returned to a 2 fold increase. D N A synthesis was found to be significantly increased in alveolar wall cells by day 5 and peaked on day 11 in rabbits. After 21 days, the total D N A content was equal to control (both lungs) values, and D N A synthesis were the same as in controls (Das and Thurlbeck, 1979). R N A and protein content increase in parallel to lung weight and cease to increase as soon as the restoration is complete (Rannels et al., 1984). A n increase in R N A synthesis reached m a x i m u m on day 4 and 5 in contralateral lungs of rats (Romanova et al.. 1967). In rats after pneumonectomy, an increase in D N A and R N A content was noticed but no increase in R N A / D N A ratio was observed. Th us , it was considered that a hyperplasia took place (Buhain and Brody, 1973). Rannels et al. (1979) found an increase in protein to D N A ratio (statistically insignificant) and a significant increase in R N A / D N A ratio. T h e y postulated that the increase of R N A / D N A ratio might be due to newly synthesized protein for collagen formation. Ultrastructural morphometric studies have demonstrated an increase in cell number of type II pneumocytes and interstitial cells, and an increase in cell size of type I pneumocytes and endothelial cells (Thet and Law. 1984). In rabbits there was rapid accumulation of collagen in the lung which was preceded by an increase in the rate of collagen synthesis (Cowan and Crystal , 1975). During the second week following pneumonectomy, the rate of collagen synthesis (calculated as nmoles of 1 4 C-pro l ine incorporation into 1 4 C-hydroxypro l ine per mg of D N A per hour) increased 3 times that of controls and after 4 weeks the collagen content per dry lung weight was constant (Cowan and Crystal , 1975). A n increase of 216% in lysyl oxydase activity (an enzyme responsible for initiating the extracellular crosslinking of collagen and elastin) within 24 hours after pneumonectomy was observed in Golden 8 Syrian hamsters (Brody et al., 1979). Light microscopic studies have shown that after pneumonectomy, no thickening of the alveolar septa occurred (Cowan and Crystal , 1975). However in rats, initial transient septal thickening which later on returned to normal has been demonstrated by a scanning electron microscopic study (Burri et al., 1982). It is assumed to be due to increased collagen synthesis (Burri et al.. 1982). Other studies (Burri and Sehovic, 1979; Thet and Law, 1984) found no change in volume proportion of interstitial matrix by light and electron microscopy. 1.4.3 A l v e o l a r m u l t i p l i c a t i o n O l d as well as modern morphometric techniques have not solved the riddle of the most debated area of compensatory lung growth, whether postpneumonectomy com-pensatory growth is accompanied by formation of new alveoli (Addis, 1928; Bremer. 1936; Longacre and Johansmann. 1940; Cohn. 1940; Romanova. 1960; Gnavi et al., 1970; Nattie et al.. 1974; Langston et al., 1977; Thur lbeck et al., 1981) or by an increase in the size of already existing structures by dilatation (Reinhoff et al., 1935; Sery et al.. 1969; Buhain and Brody, 1973; B o a t m a n . 1977; Boatman et al.. 1983). Before modern morphometric techniques, dimensional studies were done by count-ing and measuring surface features of alveoli and evaluation of alveolar multiplication by comparing histological sections. Reinhoff et al. (1935) made histological compar-isons of the excised lung with the remaining lung 6 months following pneumonectomy. He deduced that compensatory changes were accompanied by dilatation of already existing respiratory units, without an increase in number of conduct ing airways and blood vessels. Cohn (1940) studied the effect of pneumonectomy in rats after 30 days and re-ported a prior increase in alveolar septal thickening and then alveolar multiplication. Kaszler (1955) observed alveolar dilatation and increased vascularization in dogs 3 weeks after pneumonectomy. Modern morphometric techniques are more comprehensive and precise. By using these techniques, mean linear intercept (the average interalveolar wall distance), mean chord length of alveoli, alveolar surface area, number of alveoli per unit volume, and total number of alveoli can be est imated. In the case of an increase in lung volume due 9 to overinflation, mean linear intercept and mean chord length of alveoli will increase to the cube root of the increase in the lung volume. The number of alveoli per unit volume will decrease inversely to the increase in the lung volume. Surface area will increase to the two-thirds power of the increase in the lung volume. In the case of increased complexity due to complete alveolar multiplication, mean linear intercept, mean chord length of alveoli and number of alveoli per unit volume will remain constant, whereas alveolar surface area will increase directly with the increase in the lung volume. T h e relative ratio of volume proportion (density) of alveolar duct air (V„ (g) 0.430 ± 0.009 0.496 ± 0.008' W d r y / r / 1 0 0 g WZ> (g) 0.084 ± 0.001 o.ioi ±o.oor V/ r /100 g W6 (ml) 4.01 ±0.11 4.83 ±0.18** DNA/ r /100 g WZ> (mg) 2.41 ±0.05 2.95 ± 0.04* Pro/r/100 g WZ> (mg) 36.79 ± 0.66 51.18 ±0.87* Values are means ±SEM's . *P < 0.0005; * * P < 0.005. Table A.3: Extent of compensatory lung growth results for male pneumonectomy group compared to female pneumonectomy group. Variables Male pneumo Female pneumo WIR (g) 1.094 ± 0.03 0.893 ± 0.02* W d r y Z * (g) 0.207 ± 0.006 0.167 ±0.005* VIR (ml) 10.49 ±0.34 8.57 ±0.40** DNA/* (mg) 5.25 ±0.14 4.31 ±0.14* ProZR (mg) 87.36 ± 2.77 80.19 ± 1.90*-* WZfl/100 g Wb (g) 0.433 ± 0.011 0.525 ±0.015* VWW '100 g W6 (g) 0.082 ± 0.001 0.098 ± 0.002* MIRI 100 g W6 (ml) 4.17 ±0.14 5.13 ±0.16* DIMAZtf/100 g W6 (mg) 2.07 ± 0.03 2.54 ±0.05* ProZ^/100 g VV6 (mg) 34.47 ± 0.82 47.31 ±0.81-Values are means ±SEM's . *P < 0.0005. * * P < 0.005. * * *P < 0.05. 91 T a b l e A . 4 : L o b a r m o r p h o m e t r i c resul ts for ma le sham-opera ted g r o u p c o m p a r e d to female s h a m - o p e r a t e d g roup . Variables Male sham Female sham MM) Upper lobe 74.6 ± 2.6 72.1 ± 2.9 Middle lobe 77.0 ±3.0 72.8 ±2.1 Post-caval lobe 82.9 ±2.1 75.4 ± 1.1* Lower lobe 74.4 ± 2.2 71.3 ± 1.8 L / „ (M) Upper lobe 38.4 ±0.9 37.3 ±0.7 Middle lobe 40.4 ± 0.9 36.6 ± 0.7' Post-caval lobe 41.4 ± 1.0 38.9 ± 0.4 Lower lobe 40.2 ± 1.2 37.5 ± 1.0 SvjLOBE{m2) Upper lobe 0.057 ± 0.002 0.049 ± 0.001* Middle lobe 0.071 ±0.003 0.068 ± 0.003 Post-caval lobe 0.061 ± 0.002 0.059 ± 0.001 Lower lobe 0.155 ±0.008 0.147 ± 0.006 Nv ( x l O 6 ) Upper lobe 3.39 ±0.20 3.38 ±0.16 Middle lobe 3.31 ±0.18 3.41 ±0.11 Post-caval lobe 3.17 ±0.12 3.24 ±0.13 Lower lobe 3.90 ±0.18 3.57 ±0.14 Values are means ±SEM's . *P < 0.05. 92 Table A.5: Lobar morphometric results for male pneumonectomy group compared to female pneumonectomy group. Variables Male pneumo Female pneumo Upper lobe 74.3 ± 2 . 0 80.0 ± 2.6 Middle lobe 83.1 ± 2.2 81.8 ± 2.0 Post-caval lobe 88.8 ± 2 . 2 86.2 ± 2.1 Lower lobe 81.0 ± 1.6 78.8 ± 2.2 Llv (M) Upper lobe 41.0 ± 1.1 41.9 ± 1.2 Middle lobe 44.8 ± 0 . 8 43.9 ± 0 . 8 Post-caval lobe 46.2 ± 1.2 46.1 ± 1.1 Lower lobe 42.7 ± 0 . 7 42.7 ± 1.3 S w i 0 B £ ( m 2 ) Upper lobe 0.100 ± 0.004 0.067 ± 0.002* Middle lobe 0.100 ± 0 . 0 0 4 0.089 ± 0.003 Post-caval lobe 0.098 ± 0.005 0.088 ± 0.004 Lower lobe 0.216 ± 0.009 0.176 ± 0 . 0 0 8 * * Nv ( x l O 6 ) Upper lobe 2.84 ± 0 . 1 2 2.95 ± 0 . 1 5 Middle lobe 2.51 ± 0.09 2.62 ± 0 . 1 1 Post-caval lobe 2.03 ± 0.09 2.04 ± 0.07 Lower lobe 2.83 ± 0 . 1 4 3.125 ± 0.13 Values are means±SEM's. *P < 0.0005. **P < 0.005. 93 Table A.6: Global morphometric response results for male sham-operated group compared to female sham-operated group. Variable Male sham Female sham 76.6 ±2 .0 72.6 ±1.5 Liu (M) 39.9 ±0.6 37.6 ± 0.6** Nv (x lO 6 ) SwR(m2) 3.64 ±0.15 3.44 ±0.07 0.340 ± 0.010 0.320 ±0.010 Swj(m 2 ) NaR ( x l O 6 ) 0.485 ± 0.020 0.462 ±0.017 23.2 ±0.4 19.8 ± 0.5* N a r ( x l O 6 ) 33.0 ±0.7 28.6 ± 0.9* S W R / 1 0 0 g W6(m 2 ) 0.146 ±0.003 0.184 ± 0.008* Sw r /100 g W6(m 2 ) 0.208 ± 0.006 0.266 ±0.013* Na H /100 g W 6 ( x l 0 6 ) 10.07 ±0.31 11.42 ±0.48** NaT/100c? Wrj(xl0 6 ) 14.33 ±0.52 16.47 ± 0.73** Values are means±SEM's. *P < 0.002. * * P < 0.05. Table A.7: Global morphometric response results for male pneumonectomy group compared to female pneumonectomy group. Variable Male pneumo Female pneumo _Lm (/*) Nv (x lO 6 ) S W R (m 2 ) Na* ( x l O 6 ) SVJR /100 g W6(m 2 ) Na H /100 g W 6 ( x l 0 6 ) . 82.0 ± 1.2 43.6 ±0.5 2.59 ±0.09 0.514 ±0.018 26.6 ±0.7 0.204 ± 0.008 10.66 ±0.47 81.6 ± 1.7 43.6 ± 0.9 2.82 ±0.08 0.418 ±0.014* 23.3 ±0.5* 0.251 ± 0.007* 14.66 ±0.38* Values are means±SEM's *P < 0.003. Table A.8: Nature of lung growth, a biochemical aspect: Male sham-operated vs female sham-operated group, and male pneumonectomy vs female pneumonectomy group. Variables Male Female Male Female sham sham pneumo pneumo DNA/g \Ndryl (mg) 28.68 29.12 25.33 25.82 ±0.36 ±0.36 ±0.37 ±0.33 Protein/DNA (mg/mg) 15.2 17.3* 16.6 18.6* ±0.25 ±0.17 ±0.22 ±0.29 Values are means ±SEM's . *P < 0.003. 94 Table A.9: Static pressure-volume curve results for male sham-operated vs female sham-operated group, and male pneumonectomy vs female pneumonectomy group. Variables male female male female sham sham pneumo pneumo T L C (ml) 8.1 6.6* 8.1 6.8* ±0.63 ±0.18 ±0.49 ±0.3 T L C /100 g W6(ml) 3.45 3.83 3.23 4.05* ±0.25 ±0.12 ±0.17 ±0.19 T L C / g Wt (ml) 7.31 7.3 6.79 7.14 ±0.47 ±0.3 ±0.38 ±0.34 Constant K 0.125 0.114 0.139 0.139 ±0.005 ±0.005 ±0.006 ±0.004 Values are means ± SEM's. *P < 0.05.