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Adaptive lung growth following exposure to simulated high altitude Sekhon, Harmanjatinder S. 1993

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ADAPTIVE LUNG GROWTH FOLLOWING EXPOSURE TOSIMULATED HIGH ALTITUDE.byHARMANJATINDER SINGH SEKHONB.Sc., Punjab University, India, 1970.M.B.B.S., Magadh University, India, 1981.M.Sc., The University of British Columbia, Canada, 1987.A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENT FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Pathology)We accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1993© Harmanjatinder Singh SekhonIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  PAL-27,046 yThe University of British ColumbiaVancouver, CanadaDate pm-goi 17, /993DE-6 (2/88)ABSTRACTAltered oxygen balance in the body at high altitude and in some physiological and pathologicalconditions may induce adaptive changes in the body which may be organ specific. Because gasexchange is the primary function of the lungs, they may undergo structural changes to adapt to theimbalance of oxygen. High altitude residents have large lungs and short body stature. Whether theseadaptive changes are caused by hypobaric pressure or hypoxia (low oxygen) or hypobaric hypoxiais not known. In hypoxic conditions, somatic growth retardation occurs due to undernutrition, but theeffect of undernutrition on growth of lung and other organs is not known. In this thesis, the influenceof hypobaric normoxia (410 mm Hg, oxygen enriched to correspond the fraction of oxygen (Fo 2) to0.21 at sea level), normobaric hypoxia (Fo 2 0.11), hypobaric hypoxia (410 mm Hg, Fo 2 equivalent to0.11 at sea level) and diminished somatic growth (equivalent to that occurs in hypobaric hypoxia, foodrestriction) on lung growth (including biochemical, morphometric, cytokinetic and functional aspects)in rats from 4 to 7 weeks of age was studied.After 3 weeks of exposure, somatic growth was diminished in hypobaric hypoxic and normobarichypoxic animals, but lung growth was accelerated. All absolute biochemical and morphometricmeasurements in hypobaric hypoxic and normobaric hypoxic rats were higher than undernourishedanimals indicating that augmented lung growth occurred by hyperplastic and hypertrophic changes withincreased accumulation of collagen and elastin. Lung weight, lung volume, DNA, RNA, protein anddesmosine were also increased compared to general controls. Maximal tritiated thymidine uptakeoccurred on day 3 and declined thereafter suggesting that lung growth stimulation occurred duringearly exposure. With the exception of endothelial cells (alveolar wall, arterial), the maximum responsein other cells (type II pneumonocytes, interstitial cells, unidentifiable cells) in the central alveoli wallcells lagged behind and was lower than in the peripheral alveoli. After 3 day recovery, lung DNAsynthesis reached the control levels. Pulmonary function tests showed that hypobaric hypoxia causeda decrease in expiratory flow rates (FEF corrected for FVC) and an increase in specific upstreamairway resistance while in normobaric hypoxia, FEV 0.1 /FVC%, expiratory flow rates (absolute and FEFiiicorrected for FVC, and PEER) decreased but both absolute and specific upstream airway resistanceincreased. However, static compliance remained unchanged. In addition to above parameters,hypobaric hypoxia also caused an increase in collagen and alveolar surface area. These changes didnot occur in normobaric hypoxia instead enlargement of airspaces occurred indicating overinflation ofthe lungs. The collagen concentration and elastic lung recoil at high lung volumes were alsodecreased in normobaric hypoxic animals.Hypobaric normoxia caused slight reduction in somatic growth which was associated withdecreased lung volume, but biochemical and morphometric parameters did not change. Morphometricunit structures were smaller. Peak lung growth stimulation occurred on day 5 in all the main celltypes, but not in endothelial cells. Undernutrition impaired both somatic and lung growth as lungweight and volume, cell number and size, accumulation of connective tissue proteins, alveolar number,and alveolar surface area were decreased compared to controls. DNA synthetic activity in all the maincell types diminished. However, body weight normalized lung weight, DNA, alveolar surface area andtotal alveolar number were higher in undernourished animals than general controls.These data suggest that lung growth stimulation occurs in normobaric hypoxia and hypobarichypoxia despite an inhibitory effect of undernutrition. Accelerated lung growth at high altitude isprimarily induced by low oxygen tension. However, the differences in hypobaric hypoxia andnormobaric hypoxia and changes in normobaric hypoxia indicate that hypobaric pressure per se mayplay a role in lung growth adaptation at high altitude. Geometrical location of the central alveoli maylimit their adaptive response compared to the peripheral alveoli. Synchronous endothelial cellstimulation in each component of the lung suggests that endothelial cells may respond tohemodynamic changes while the other cells respond to functional demand of the lungs. Although lunggrowth was increased in normobaric hypoxic and hypobaric hypoxic animals, pulmonary function testsobservations suggested that it may be dysanaptic. The response to hypoxic stress is organ specificas growth of lung, heart and spleen increased, but liver and kidney followed the growth patterns ofundernourished animals. Increased specific parameters of lung growth in undernourished animalsivsuggest that in conditions which compromise somatic growth, corrections made for body weight maylead to misinterpretation of true changes.VTABLE OF CONTENTSABSTRACT^ iiLIST OF TABLES xiiLIST OF FIGURES^ xiiiACKNOWLEDGMENTS xviiiCHAPTER 1: INTRODUCTION^ 11.1. NORMAL LUNG GROWTH ^  21.2. ADAPTIVE LUNG GROWTH AT HIGH ALTITUDE ^  81.2.1. EFFECT OF LOWER OXYGEN TENSION HYPOBARIC HYPDXIA ^ STRUCTURAL ADAPTATIONS ^ INTERSPECIES DIFFERENCES^CELL KINETICS AND MORPHOLOGICALCHANGES ^ FUNCTIONAL ADAPTATION ^ NORMOBARIC HYPDXIA ^  191.2.2. EFFECT OF REDUCED AMBIENT PRESSURE ^  241.2.3. EFFECT OF EXERCISE ^  251.2.4. EFFECT OF COLD  261.2.5.^SOMATIC AND ORGAN (other than lungs) GROWTHADAPTATION ^  281.3. HYPOTHESIS ^  291.4. RATIONALE  30CHAPTER 2: MATERIALS AND METHODS^ 342.1. PROJECT DESIGN ^  34vi2.1.2. CHEMICALS ^  342.1.3. ANIMALS  352.2. LUNG GROWTH ADAPTATION ^  352.2.1. EXPERIMENTAL DESIGN  352.2.2. EXPOSURE TO ALTERED AMBIENT CONDITIONS ^ 362.2.2.1. Exposure chambers ^  362.2.2.2. Exposure procedure  362.2.2.3. Animal termination ^  372.2.3. PRESSURE-VOLUME CURVES  382.2.4. BIOCHEMISTRY ^  392.2.4.1. DNA, RNA AND PROTEIN EXTRACTION ^ 412. Alkali soluble protein estimation  422. Estimation of DNA ^  422. Estimation of RNA  432.2.4.2. PROCEDURE FOR HYDROXYPROLINE AND DESMOSINEEXTRACTION ^  442. Estimation of hydroxyproline ^  44(i). Sample preparation for hydroxyproline quantification^44(ii). Hydroxyproline assay procedure ^  452. Estimation of desmosine  47(i). Desmosine preparation from lung samples ^ 47(ii). Desmosine radioimmunoassay ^  472.2.5. MORPHOMETRY^  502.2.5.1. Lung fixation and lung volume determination ^ 502.2.5.2. Lung sampling and tissue processing  512.2.5.3. Assessment of tissue shrinkage ^  51VII2.2.5.4. Morphometric measurements ^  512.3. ORGAN RESPONSE AND LUNG CYTOKINETICS  562.3.1. EXPERIMENTAL DESIGN ^  562.3.1.1. Termination of rats  572.3.1.2. Heart and lung preparation ^  572.3.2. LUNG BIOCHEMISTRY ^  582.3.2.1. Measurements of DNA synthesis ^  582.3.3. AUTORADIOGRAPHIC TECHNIQUES AND OBSERVATIONS ^ 582.3.3.1. Techniques ^  582. Tissue sampling ^  582. Tissue processing and emulsion coating ^ 592. Development of autoradiographic slides ^ 602.3.3.2.ANALYSIS OF AUTORADIOGRAPHS ^  602. Cell counting ^  602. Criteria for cell identification ^  612. Labelling indices ^  622.4. LUNG ADAPTATION: PHYSIOLOGICAL ASPECTS  622.4.1. EXPERIMENTAL DESIGN ^  622.4.2. PULMONARY FUNCTION TESTS  632.4.2.1. Animal preparation ^  632.4.2.2. Lung volume measurements ^  642.4.2.3. Flow-volume relationships  652.5. STATISTICAL ANALYSIS ^  66CHAPTER 3: RESULTS^ 683.1. LUNG GROWTH STUDY ^  683.1.1. GENERAL  68VIII3.1.1.1. NUTRITIONAL ASSESSMENT ^  683.1.1.2. SOMATIC GROWTH ^  703.1.2. HEMATOCRIT ^  713.1.3. LUNG GROWTH  733.1.3.1. NORMAL LUNG GROWTH (4-7 weeks of age) ^ 733. EXTENT OF LUNG GROWTH  733. BIOCHEMICAL CHANGES ^  733. MORPHOMETRIC CHANGES  773.1.3.2. EFFECT OF UNDERNUTRITION ^  783. EXTENT OF LUNG GROWTH  783. CHANGES ^  783. MORPHOMETRIC CHANGES  813.1.3.3. ADAPTATION TO LOW AMBIENT PRESSURE(HYPOBARIC NORMOXIA) ^  813. EXTENT OF LUNG GROWTH ^ 813. BIOCHEMICAL CHANGES  823. MORPHOMETRIC CHANGES ^ 823.1.3.4. ADAPTATION TO NORMOBARIC HYPDXIA  853. EXTENT OF LUNG GROWTH ^ 853. BIOCHEMICAL CHANGES  863. MORPHOMETRIC CHANGES ^ 873.1.3.5. ADAPTATION TO HYPOBARIC HYPDXIA  893. EXTENT OF LUNG GROWTH ^ 893. BIOCHEMICAL CHANGES  893. MORPHOMETRIC CHANGES ^ 903.2. LUNG CYTOKINETICS STUDY^  93ix3.2.1. SOMATIC GROWTH ^  933.2.2. ORGAN RESPONSE  943.2.2.1. NORMAL ORGAN GROWTH ^  943.2.2.2. EFFECT OF UNDERNUTRITION  943.2.2.3. EFFECT OF HYPOBARIC NORMOXIA (low ambientpressure) ^  953.2.2.4. EFFECT OF NORMOBARIC HYPDXIA ^  953.2.2.5. EFFECT OF HYPOBARIC HYPDXIA  973.2.3. BIOCHEMICAL ALTERATIONS IN THE LUNG ^  1003.2.3.1. EFFECT OF UNDERNUTRITION  1003.2.3.2. EFFECT OF HYPOBARIC NORMOXIA (low ambientpressure) ^  1013.2.3.3. EFFECT OF NORMOBARIC HYPDXIA ^ 1023.2.3.4. EFFECT OF HYPOBARIC HYPDXIA  1033.2.4. DNA SYNTHESIS ^  1053.2.4.1. EFFECT OF UNDERNUTRITION ^  1063.2.4.2.EFFECT OF HYPOBARIC NORMOXIA  1063.2.4.3.EFFECT OF NORMOBARIC HYPDXIA ^ 1073.2.4.4. EFFECT OF HYPOBARIC HYPDXIA  1083.2.5. AUTORADIOGRAPHY ^  1083.2.5.1. EFFECT OF UNDERNUTRITION ^  1093. CENTRAL ALVEOLI  1103. PERIPHERAL ALVEOLI ^  1103. NON-PARENCHYMA  1113.2.5.2. EFFECT OF HYPOBARIC NORMOXIA ^ 1123. CENTRAL ALVEOLI ^  1123. PERIPHERAL ALVEOLI ^  1123. NON-PARENCHYMA  1123.2.5.3. EFFECT OF NORMOBARIC HYPDXIA ^ 1133. CENTRAL ALVEOLI ^  1143. PERIPHERAL ALVEOLI  1163. NON-PARENCHYMA^  1173.2.5.4. EFFECT OF HYPOBARIC HYPDXIA  1183. CENTRAL ALVEOLI ^  1203. PERIPHERAL ALVEOLI  1213. NON-PARENCHYMA^  1233.3. LUNG PHYSIOLOGY ^  1303.3.1. LUNG VOLUMES  1303.3.1.1. EFFECT OF UNDERNUTRITION ^  1303.3.1.2. EFFECT OF HYPOBARIC NORMOXIA  1313.3.1.3. EFFECT OF NORMOBARIC HYPDXIA ^ 1313.3.1.4. EFFECT OF HYPOBARIC HYPDXIA  1313.3.2. FLOW-VOLUME RELATIONSHIPS AND LUNG MECHANICS^ 1313.3.2.1. EFFECT OF UNDERNUTRITION ^  1323.3.2.2. EFFECT OF HYPOBARIC NORMOXIA  1323.3.2.3. EFFECT OF NORMOBARIC HYPDXIA ^ 1333.3.2.4. EFFECT OF HYPOBARIC HYPDXIA  1363.3.3. PRESSURE-VOLUME CURVE CHARACTERISTICS IN EXCISEDLUNGS ^  138CHAPTER 4: DISCUSSION 1434.1. GENERAL POINTS ^  1434.2. FOOD CONSUMPTION AND SOMATIC GROWTH ^  144XI4.3. ORGAN GROWTH ADAPTATION: OTHER THAN LUNG ^  1474.4. LUNG GROWTH ADAPTATION ^  1514.4.1. EFFECT OF UNDERNUTRITION ^  1514.4.2. EFFECT OF HYPOBARIC HYPDXIA  1554.4.2.1. STRUCTURAL ADAPTATIONS ^  1594.4.2.2. CELL KINETICS ^  1674.4.2.3. FUNCTIONAL ADAPTATIONS ^  1704.4.3. EFFECT OF REDUCED AMBIENT OXYGEN (NORMOBARICHYPDXIA) ^  1764.4.3.1.^NORMOBARIC HYPDXIA vs WEIGHT-MATCHEDCONTROLS AND GENERAL CONTROLS^ 1764. STRUCTURAL ADAPTATIONS  1784. FUNCTIONAL ADAPTATIONS ^ 1844.4.3.2. HYPOBARIC HYPDXIA vs HYPOBARIC NORMOXIA ^ 1874.4.4. EFFECT OF REDUCED AMBIENT PRESSURE (HYPOBARICNORMOXIA) ^  1884.4.4.1. HYPOBARIC NORMOXIA vs GENERAL CONTROLS ^ 1904. STRUCTURAL ADAPTATIONS ^ 1914. FUNCTIONAL ADAPTATION  1934.4.4.2. HYPOBARIC HYPDXIA vs NORMOBARIC HYPDXIA ^ 1944.5. EFFECT OF 3-DAYS POST-EXPOSURE AND REFEEDING ^ 1974.6. RECAPITULATION AND CONCLUSIONS ^  201BIBLIOGRAPHY^ 206xiiLIST OF TABLESTable 1. Summary of previous studies: somatic and lung growth adaptation following chronicexposure. ^  19Table 2. Morphometric calculations. ^  54Table 3. Food intake, somatic growth and hematocrit of 4-7 week old rats. ^ 72Table 4. Results of lung growth adaptation. ^  74Table 5. Biochemical assessment of lung growth after three weeks of exposure to variousconditions. ^  75Table 6. Biochemical measurements normalized to 100 grams of body weight. ^ 76Table 7. Biochemical variables per gram of dry lung weight. ^  79Table 8. Relationships between biochemical changes in different experimental groups. ^ 80Table 9. Changes in morphometric parameters following exposure to different conditions. . . 83Table 10. Alterations in volume proportion of lung morphological structures following 3 weeksof exposure to different conditions. ^  84Table 11. Absolute volumes of parenchymal and non-parenchymal lung structures. ^ 92Table 12. Absolute and specific morphometric changes after 3 weeks of exposure. ^ 92Table 13. Cell composition of the walls of the peripheral alveoli. ^  122Table 14. Alterations in total lung capacity and shape constant K after 3 weeks ofexposure. ^  140Table 15. Changes in organ weights in weight-matched control (WMC), hypobaric hypoxic(HBHY), normobaric hypoxic (NBHY) and hypobaric normoxic (HBNO) animals after21 days of exposure  148Table 16. Morphometric changes in weight-matched control (WMC), hypobaric hypoxic(HBHY), normobaric hypoxic (NBHY) and hypobaric normoxic (HBNO) animals after21 days of exposure to different conditions.   157xiiiLIST OF FIGURESFigure 1. Relationship between altitude, barometric pressure and oxygen tension [West(281)]. ^  10Figure 2. Flow chart for extraction of protein, RNA and DNA. ^  40Figure 3. Flow chart for extraction of collagen (soluble and insoluble fractions) and elastin.^  46Figure 4. Illustrations of (a) the test grid qsed for light microscopic measurements and (b) theintercept counting technique. ^  52Figure 5. Illustration of a direct alveolar count 55^ 55Figure 6. Illustration of locations from which central and peripheral blocks_ were taken fromthe left lung for autoradiographic measurements^  59Figure 7. Daily food consumption of the general control (GC), weight-matched control (WMC),hypobaric hypoxic (HBHY), hypobaric normoxic (HBNO) and normobaric hypoxic(NBHY) groups  69Figure 8. The effect of undernutrition, hypobaric hypoxia, hypobaric normoxia and normobarichypoxia on body weight gain^  70Figure 9. Nose-tail length measurements of GC, WMC, HBHY, HBNO and NBHY at the timeof termination on days 1, 3, 5, 7, 10, 14, 21 and 3 days after returning to room air. .^94Figure 10. Right lung weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as animals 3 days after returning to room air. .^96Figure 11. Heart weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as 3 days after returning to room air^ 97Figure 12. Spleen weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as 3 days of recovery. ^  98Figure 13. Kidney weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as 3 days following recovery. ^ 99xivFigure 14. Liver weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as 3 days following recovery. ^ 100Figure 15. Changes which occurred in dry to wet weight ratio of the lung during exposure todifferent conditions. ^  101Figure 16. The amount of lung RNA in GC, WMC, HBHY, HBNO and NBHY rats after 1, 3,5, 7, 10, 14 and 21 days of exposure to different conditions as well as 3 daysfollowing recovery  102Figure 17. Lung protein content of GC, WMC, HBHY, HBNO and NBHY rats after days 1, 3,5, 7, 10, 14 and 21 days of exposure to different conditions as well as 3 daysfollowing recovery  103Figure 18. The amount of lung DNA content of rats subjected to different conditions for 1, 3,5, 7, 10, 14 and 21 days as well as 3 days following recovery^  104Figure 19. The lung RNA/ DNA ratio of undernourished, hypobaric hypoxic, hypobaricnormoxic and normobaric hypoxic animals as well as 3 days following recovery. . . . . 105Figure 20. The effect of undernutrition, hypobaric hypoxia, hypobaric normoxia andnormobaric hypoxia on the lung protein/DNA ratio. ^  106Figure 21. Tritiated thymidine incorporation into the lungs of rats subjected to hypobarichypoxia, hypobaric normoxia, normobaric hypoxia and undernutrition for 1, 3, 5, 7, 10,14 and 21 days as well as 3 days following recovery.   107Figure 22. 3H-TdR incorporation in combined alveolar wall cells (central+peripheral) in thelungs of GC, WMC, HBHY, HBNO and NBHY animals subjected to different conditionsfor 1, 3, 5, 7 and 21 days.   109Figure 23. The effect of undernourishment, hypobaric hypoxia, hypobaric normoxia andnormobaric hypoxia on the labelling index of free alveolar macrophages ^ 110Figure 24. The mast cell labelling index in the lungs of undernourished, hypobaric hypoxic,hypobaric normoxic and normobaric hypoxic animals. ^  111xvFigure 25. The cytokinetics of type I pneumonocytes in the walls of central alveoli. ^ 113Figure 26. Tritiated thymidine incorporation in type II pneumonocytes in the walls of centralalveoli. ^  114Figure 27. The capillary endothelial cell labelling index in the walls of central alveoli. ^ 115Figure 28. Cytodynamics of interstitial cells in the walls of central alveoli of undernourished,hypobaric hypoxic, hypobaric normoxic and normobaric hypoxic rats. ^ 116Figure 29. The labelling index of unidentifiable cells in the walls of central alveoli. ^ 118Figure 30. The mesothelial cell labelling index in the lungs of undernourished, hypobarichypoxic, hypobaric normoxic and normobaric hypoxic animals. ^  119Figure 31. Cytokinetics of type I pneumonocytes in the walls of peripheral alveoli. ^ 120Figure 32. The labelling index of type ll pneumonocytes in the walls of peripheral alveoli ofundernourished, hypobaric hypoxic, hypobaric normoxic and normobaric hypoxicrats   121Figure 33. The capillary endothelial cell labelling index in the walls of peripheral alveolifollowing exposure to different experimental conditions. ^  123Figure 34. The interstitial cell labelling index in the peripheral alveoli of undernourished,hypobaric hypoxic, hypobaric normoxic and normobaric hypoxic animals. ^ 124Figure 35. The labelling index of unidentifiable cells in the peripheral alveoli of GC, WMC,HBHY, HBNO and NBHY rats. ^  125Figure 36. Tritiated thymidine incorporation in bronchiolar epithelial cells following exposureto different experimental conditions. ^  126Figure 37. The bronchial epithelial cell labelling index after subjecting animals to differentexperimental conditions. ^  127Figure 38. 3H-TdR incorporation in the bronchial wall cells following exposure to differentconditions. ^  127Figure 39. Cytokinetics of arterial endothelial cells following exposure to hypobaric hypoxia,xvihypobaric normoxia, normobaric hypoxia and undernutrition. ^  128Figure 40. The labelling index of arterial wall cells following exposure to hypobaric hypoxia,hypobaric normoxia and normobaric hypoxia, and restricted feeding. ^ 128Figure 41. Autoradiographs showing cells heavily laden with black granules following tritiatedthymidine incorporation.   129Figure 42. Functional residual capacity (FRC), residual volume (RV), vital capacity (VC) andtotal lung capacity (TLC) of undernourished, hypobaric hypoxic, hypobaric normoxicand normobaric hypoxic animals exposed to different conditions for 21 days.   130Figure 43. FRC, RV and VC relative to TLC of undernourished, hypobaric hypoxic, hypobaricnormoxic and normobaric hypoxic rats  132Figure 44. Upstream resistance (Rus) and specific upstream resistance (sRus) at FVC 50%in GC, WMC, HBHY, HBNO and NBHY rats following 3 weeks of exposure to differentconditions.   133Figure 45. Forced expiratory volume in 0.1 second (FEVI) and percent of vital capacityexpired in 0.1 second (FEV0.1/FVCcY0) of rats following exposure to differentexperimental conditions.   134Figure 46. Peak expiratory flow rates and midexpiratory flow rates at FVC75_25% in ratssubjected to undernutrition, hypobaric hypoxia, hypobaric normoxia and normobarichypoxia.   135Figure 47. The flow-volume relationships in GC, WMC, HBHY, HBNO and NBHY ratsexposed to different experimental conditions for 21 days ^136Figure 48. The flow-volume curves using normalized forced expiratory flow rates for vitalcapacity of animals exposed to hypobaric hypoxia, hypobaric normoxia, normobarichypoxia and undernutrition for three weeks.   137Figure 49. Static compliance, specific static compliance (corrected for VC) and shapeconstant K of P-V curves of GC, WMC, HBHY, HBNO and NBHY rats following 3xviiweeks of exposure to different conditions. ^  138Figure 50. Pressure-volume curves recorded in intact animals which were subjected toundernutrition, hypobaric hypoxia, hypobaric normoxia and normobaric hypoxia for 21days. ^  139Figure 51. Pressure-volume curves of excised lungs of rats exposed to different experimentalconditions for 21 days^  141Figure 52. Absolute and corrected for FVC maximum flow-static recoil relationship in ratsfollowing exposure to different conditions. ^  173xviiiACKNOWLEDGMENTSI 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 graduatestudies. His invaluable advice, utmost patience and constructive criticism helped me to solve theriddles to made this work possible.Also my sincere gratitude to my supervisory committee memebers Dr. W.K. Milsom, Dr. D.Walker, Dr. J.L. Wright, Dr. D.F. Smith and Dr. G. Krystal for their invaluable time, guidance andsuggestions to improve and complete this work.I would like to express my deep appreciation of the friendship, support and timelyencouragement I have received while doing my research, from Kamala Cherukapalli, Bridget Milsom,Hassan Khadampore, Reiko Matsui, Wellington Cardoso and Christina Martynko. Especially, I wouldlike to thank Hilary Brown for her technical assistance in preparing autoradiographs and bundles ofencouragement when it was most needed.Finally I would like to extend my gratitude to Dr. Gulzara Singh and Manjit Sekhon, Avtar andGurmeet Nahal, Narinder and Ominder Chauhan, Lakhbir Kang, and Sarabjit Nahal for continuedencouragement, endless warmth, support and appreciation.xixDEDICATEDtomy win sAiNgmand toouR c9fILDREN91f9WVER A9k4D INEEEP1CHAPTER 1INTRODUCTIONPrenatally, the lungs have no role in gas exchange. During birth they undergo alterations infunctional status: a fluid filled organ begins to function as a gas exchanging organ. The fundamentalarchitecture of the lung is established during fetal development, but the main changes in the terminalunit occur postnatally (214). In humans and other mammalian species, various environmental,nutritional and hormonal factors have been implicated as potential modulators of structural andbiochemical changes associated with lung growth and development during postnatal life. These upand/or down, external and/or internal modulators control lung growth by stimulating variousbiochemical events. These events primarily include cellular proliferation and connective tissueaccumulation with concomitant remodelling of the existing framework of the respiratory structures toform new functional respiratory units (6, 47, 94, 151, 173, 261).Since efficient diffusion of gases between blood and alveolar air is a primary function of thelungs, a substantial role of oxygen as a regulator of postnatal lung growth has been postulated (45).A sizeable portion of world's population which resides at high elevations (>3000 m) has larger lungsand chest size, but smaller body weight and stature than sea level residents (273). An increase inaltitude is closely associated with a proportional decrease in both ambient pressure and oxygentension which may disturb a balance of oxygen supply and demand in the body. Although postnatallung development may be genetically programmed, an imbalance of oxygen supply and demand inthe body at high altitude may produce structural and functional alterations in the lung in order tofacilitate adequate gas exchange between alveolar and vascular compartments. The high altitudemodel has also been commonly used to investigate the effect of hypoxia which occurs in a numberof clinical conditions (e.g. pulmonary and cardiovascular diseases). The insight so gained by studyinghigh altitude adaptation not only has direct implications, but it can also be applied to the moreChapter 1: INTRODUCTION^2complicated clinical problems.A brief overview of normal human lung growth will be followed by a review of postnatal lunggrowth with an emphasis on rat studies. This will be followed by adaptive lung growth at high altitude.Because the effect of hypobaric hypoxia and normobaric hypoxia may or may not be similar, theireffect on lung growth adaptation will be reviewed separately. Thereafter, the influence of other highaltitude variables on lung growth and the effect of hypoxic stress on somatic growth and other organswill be discussed.1.1. NORMAL LUNG GROWTHAt birth, in various animal species, the lungs are immature. They lack alveoli and otherdefinitive components of adult lung structures. Investigators studying postnatal lung growth anddevelopment have observed that at birth, animals such as rats (47, 194, 275), mice (6), rabbits (97),cats (87, 97) and dogs (33) do not possess alveoli, which are the terminal air exchanging units in adultlungs. Instead, the lungs consist almost exclusively of primitive thick walled primary saccules (47, 275)or terminal sacs (33). These smooth walled primary saccules are large and lack surface complexity.The walls of these primary saccules have a double capillary network which runs on either side of theconnective tissue framework (47). Postnatally, considerable maturation of the lung is required to attainfull functional ability. After birth, the primary saccules undergo rapid subdivision by the formation ofsecondary crests which results in structural rearrangement to form true alveoli.In humans, some researchers (33, 34, 94, 135, 214) have suggested that alveoli are absentin the fetus at the time of birth and the structures then present are saccules (135). However Loosliand Potter (173) observed that from 71/2 months gestation until term, development of respiratorychannels into alveolar ducts produced small but definitive alveoli. Alveoli-like structures can be foundin the fetal lungs by 32 weeks gestation and definitive alveoli are present at 36 weeks of gestationalage (41). While Dunnill (91) found 20 million alveoli in one infant, Thurlbeck and Angus (262) reportedChapter 1: INTRODUCTION 371 million. Langston and Thurlbeck (159) documented a mean of 55 million and a range of 10-149million alveoli at the time of birth. The conflicting results could be due to biological variations,difference in tissue preparation or difficulty in defining and recognizing alveoli (261). Whether thecomplement of acini at birth increases postnatally by simple enlargement or due to formation of newstructures, has been controversial.In adults, there is a range of 200-600 million alveoli (7). Alveolar surface area increases 12fold and lung volume 20 fold from birth to adulthood. The constant number of alveoli per unit volumeand alveolar size up to 2-4 years of age in children is an indication of alveolar multiplication;subsequently it has been noted that little or no increase in total alveolar number occurs (263). someinvestigators have suggested that later in childhood, alveolar multiplication continues, but at a slowerrate and ceases entirely with somatic growth (261) while other have demonstrated that alveolarmultiplication ceases between age 4 and 11 years (82, 91).Postnatal lung growth and development has been more extensively studied in animals (6, 46,47, 87, 97, 151, 242, 243, 275) especially in rats (46, 47, 151, 182, 242). According to Burn i (46, 47),postnatal lung growth and development in the rat has three phases: lung expansion, tissueproliferation, and equilibrated growth. A fourth phase of lung growth has also been suggested to occurin some species (261).(i) LUNG EXPANSION PHASE: This phase spans days 1 to 4 of postnatal life. During thisperiod, lung growth occurs primarily by distension of the pre-existing structural units. Increasein lung tissue is minimal. Thus the volume proportion of air increases while the volumeproportion of the lung tissue decreases (46). An increase in lung volume is similar to bodyweight (47, 182). Tritiated thymidine incorporation into DNA decreases on the first two daysafter birth in rats, with a subsequent increase on the third day (199).In contrast, studies conducted on mice show an increase in lung tissue mass during thisphase. The elongation of secondary crests, increased collagen and elastin, and enhancedmitotic index of interstitial cells by the third and fourth day of postnatal life also suggest anChapter 1: INTRODUCTION^4increase in lung tissue (6). In another study (81), increased labelling index in the lungs ofmice during the early period of postnatal life indicated that some cellular multiplication musthave occurred. In rats, a prominent increase in the endothelial cell labelling index has beenreported which remained at elevated levels until day 10 of life (151). These studies suggestthat an increase in the lung tissue mass occurs during this growth phase in both mice andrats, but lung growth occurs predominantly by an increase in lung volume.(ii) TISSUE PROLIFERATION PHASE: (days 4-13). In this phase, the subdivision of primarysaccules occurs to form definitive alveoli (46). The rate of lung tissue growth is faster thanthat in the lung expansion phase. During this growth phase, specific lung volume is increasedbecause lung volume increases relatively more than body weight. Septation increasesalveolar surface area to the 1.6 power of increase in the lung volume (46). If the lung grewby simple expansion only, the surface area would increase to the two-thirds power of thechange in lung volume. Therefore, the greater increase in alveolar surface area during thisphase indicates the marked increase in complexity of lung structure and rapid multiplicationof alveoli. Alveolar surface area and capillary surface area increase steadily from day 4 to21(46). During this phase, the first part of increase in lung tissue is due to tissue proliferationand the second part is due to rearrangement of the tissue mass (46). The subdivision of theprimary saccules occurs by the formation of secondary crests. The secondary crests haveelastin fibers in their free margins, a single capillary layer and interstitial cells. 3H-Thymidineincorporation in the crest cells is 1.4 times that occurring in other parts of the saccule (151).Elastic tissue is thought to play an important role in the formation of alveoli. It has beenshown that by day 3 alveoli are already being formed and by day 8 the rate has quickenedso that there is a three-fold increase in alveolar density (182).(iii) EQUILIBRATED GROWTH PHASE: This is the third phase of alveolar growth and begins atapproximately 2 weeks of postnatal age (47). This phase is characterized by a slowerincrease in lung volume, resulting in a specific lung volume decrease. The rate of cellChapter 1: INTRODUCTION 5proliferation falls but there is a rapid increase in alveolar and capillary surface areas as thetissue mass undergoes redistribution. However, there is a decrease in alveolar surface areaper unit body weight. New alveoli continue to be added and the alveolar surface areaincreases directly with lung volume increase rather than to the two-thirds power as would beexpected on the basis of simple expansion. The secondary crests lengthen, and only a singlecapillary layer can be found in the walls of the airspaces. Between days 21 and 131, lungvolume increases six-fold; half of this increase occurs between days 21 and 44 of postnatallife in rats (46).(iv) FOURTH PHASE: This phase only exists in some species (261). This phase of simpleexpansion of airspaces begins when alveolar proliferation ceases. However, the time at whichthe alveolar multiplication ceases in various species is under dispute. Continued somaticgrowth and lung growth throughout the life span are characteristic of rats (243). However, ithas been claimed that alveolar multiplication is complete by 10 weeks of age in Sprague-Dawley rats (141). Whether this is true in all strains of rats is unknown.The pattern of biochemical changes that occurs during growth and development in most bodyorgans is the same. An increase in tissue mass of an organ may either occur by proliferation of cells,by hypertrophic changes in the cells or by both cellular hyperplasia and hypertrophic alterations (283).According to Winick and Noble (283), biochemical aspects of postnatal growth of an organ may alsobe divided into three phases in rats. During the first phase (birth to 17th day of life), the amount ofDNA and protein increase rapidly, while the amount of protein per nucleus remains constant. In thesecond phase (3 to 5-7 weeks), DNA synthesis is lower than protein synthesis, resulting in an increasein protein to DNA ratio. In the third phase, both DNA synthesis and protein synthesis diminishsignificantly, thus, the protein to DNA ratio remains unchanged.Besides hypertrophic and hyperplastic changes in the lung, lung weight may also increase dueto accelerated connective tissue protein accumulation. Connective tissue comprises approximately25% of the adult human lung. Collagen and elastin are the major elements of connective tissue.Chapter 1: INTRODUCTION 6Collagen represents 60-65% and elastin represents 20-25% of the total connective tissue; the restbeing comprised of proteoglycans and glycoproteins. The amount of collagen varies widely; forexample, in mice it comprises 6% of dry lung weight (162) and in humans it comprises 20% (37).Collagen is distributed throughout the extracellular space of the lung. In the lung parenchyma,collagen is present in the alveolar interstitium as well as epithelial and endothelial basementmembranes. In the adult, most lung collagen is insoluble because of the extracellular covalentbonding of the collagen molecules, as well as between collagen and other components of theextracellular matrix (76, 126). Although collagen content (amount/dry weight) in the tracheobronchialtree and pulmonary vasculature is greater than that in the lung parenchyma, the bulk of collagen ispresent in the alveolar structures because the total mass of the parenchyma is much larger (109).The bulk of elastin is its amorphous component comprising approximately 90% of total mature elastin;the rest being the microfibrillar component. Similar to collagen, elastin content also has interspeciesvariations ranging from as low as 2% in rodents to about 25% in man (212).The relationship between synthesis of connective tissue constituents and their accumulation inlung and alveolar formation is unclear. Emery has suggested that elastic tissue plays an importantrole in alveolar development (94, 95). Elastin fibers along with collagen are consistently found at thefree margins of the secondary crests which subdivide primary saccules, and around the mouths ofalveoli. The elastin and collagen network has often been referred to as the "fishnet" by investigators(95, 173). Emery and Fagan have postulated that alveolar multiplication occurs by three methods:segmentation, alveolarisation and compoundment.(i) During segmentation, the primary saccules divide to form smaller units, which finally developinto alveoli. Emery and Fagan (95) found that after birth there is a progressive developmentof a "fish-net" of elastic-collagen structure, the apertures of which form mouths of alveoli.They suggested elastic-collagen tissue formation as the beginning of the development ofalveoli. Therefore, Emery and Fagan (95) conceived of alveoli as appearing in the developinglung as though a balloon with a very pliant wall was inflated within and through a containerChapter 1: INTRODUCTION^7formed by a semirigid mesh.(ii) Loosli and Potter (173), and subsequently Emery and Fagan (95) stressed the importance ofconnective tissue in the process of alveolarisation. The process of alveolarisation is alsothought to develop in the non-alveolated walls of the airways. The respiratory bronchioles areconverted to alveolar ducts and terminal bronchioles to respiratory bronchioles. However, theduration of alveolarisation and its relative importance is uncertain.(iii) During compoundment, the elastic tissue fibers form and alveoli protrude between the elastic-collagen net, and the alveoli may grow into either of the saccules between which the wall lies.Primarily, lungs function as gas exchangers. Unlike other organs, the lungs have a uniquecharacteristic to gain air content per unit lung weight with or without an increase in weight (261). Anincrease in lung volume and finer subdivision of the structural units occur to provide larger gasexchanging area. Efficient gas diffusion across the alveolar wall is mainly determined by thicknessof tissue barrier and gas exchanging surface area. During postnatal lung growth and development,the formation and maintenance of a gas diffusion barrier requires highly coordinated regulatorymechanisms. In order to determine potential regulators for the effective control of postnatal lunggrowth, a variety of experimental models (e.g. lung resection, blood flow alteration, hormonaltreatments, exposure to various ambient gases, thoracic cage restrictions, ambient pressure changes,avulsion of nerve supply etc.) have been used. Although a number of studies have maintained thatstretch is a major determinant of postnatal lung growth, it has also been suggested that lung growthmay be largely controlled by oxygen consumption (45). To examine the effect of oxygen on lunggrowth, a variety of physiological (high altitude conditions, hyperactivity), pharmacological (drugsaffecting body metabolism) and pathological (e.g. interstitial lung diseases, chronic bronchitis, asthma,pulmonary hypertension, congestive heart diseases) conditions have been studied. A number ofexperimental models have been used to understand the adaptive response of the body to hypoxicconditions. Unfortunately, the metabolic, structural and functional alterations which occur in the lungsChapter 1: INTRODUCTION 8in pathological conditions cannot be reproduced using existing models. High altitude is a common andnaturally occurring model to study the effect of chronic hypoxia. About 400 million people reside inmountainous regions; of those, a sizeable fraction dwell at high altitude (>3000 m). At an elevationof >3000 m, the majority of the individuals who live for short or prolonged duration exhibit clinical,physiological, anatomical and biochemical changes (273). Hence, to examine the sequence ofadaptive structural and functional changes in the lungs and other body organs induced by high altitudeexposure is of a significant importance.1.2. ADAPTIVE LUNG GROWTH AT HIGH ALTITUDEAdaptation is "a change which reduces the physiological strain produced by a stressfulcomponent of the total environment" (26). Adaptive changes which occur due to translocation fromlow to high altitude vary with time. The initial responses to acute change in the environment such asquick ascent or exposure in a hypobaric chamber results in accommodation which lasts minutes tohours. The physiologic adaptation response that occurs over a period of days to weeks or monthsexposure to a single factor of high altitude conditions is referred to as acclimation, while adaptationto a natural high altitude environment is called acclimatization (31). High altitude environment is acomplex set of conditions including low ambient oxygen, low ambient pressure, cold, low relativehumidity and rugged terrain. Although hypoxia is considered as a major stressor at high altitude, eachcondition (independently or in combination with others) may potentially affect the adaptive responseof the body.With regard to lung growth adaptation to one or a combination of high altitude factors, variousapproaches have been employed, including: exposure to normobaric hypoxia(18, 77, 164, 186, 198,210, 247), hypobaric hypoxia (20), transporting species in question to high altitude (45), or studyingthe species inhabiting at high altitude over generations and comparing them to their counterparts atlow altitude or sea level (163, 204, 205, 258). The observations, however, are controversial. Asidefrom differences in conditions, the disparity in results may also occur due to differences in methods,Chapter 1: INTRODUCTION 9such as: type of exposure (continuous or intermittent), severity of conditions (mild, moderate, severe),and duration of exposure (acute, prolonged, chronic). The age of animals at the time of exposure(newborn, young or adult), different species of animals, the methods of nursing the animals, andsampling and manipulation of the tissue, can all introduce inconsistency in the results.Although conclusions made from controlled environmental studies regarding high altitude maynot provide direct answers to biological questions, high altitude simulated studies help to analyze theeffect of various conditions at high altitude in a known strain of a particular animal species. The effectof controlled environment, therefore, may be regarded as a study of metabolic adaptation. Whenanimals indigenous to high altitude are compared to their low altitude counterparts, some differencesmay be detected due to evolutionary selection. The strain of animals at high altitude may be differentfrom that at lower levels. This may be the result of genetic adaptation. Therefore, the inheriteddifferences in the data may compound the problem when results from one study are extrapolated toanother.High altitude environment comprises of a number of variables which may influence the adaptivelung growth response. In the following text, the effect of each condition on lung growth adaptation willbe reviewed separately.1.2.1. EFFECT OF LOWER OXYGEN TENSIONThe processes by which organisms alter their body structure and function to fit newenvironments have intrigued biologists since the initial finding by Babak (10). He found that whentadpoles or salamander larvae were raised in water with low oxygen tension, the gills becameenlarged. Drastich (89) studied the functional and structural changes in gills of Salamander Maculosaafter prolonged maintenance in 11% 02 for one month. The enlargement of gills was also associatedwith flattening of the epidermis and distension of blood vessels. Increased average cell area andmitosis in gills of animals exposed to low oxygen have also been reported (29, 89). Oxygen tensionfalls with an increase in altitude. It is possible that adaptive growth of gas exchanging surface atInspired P02 (mmHg)0^50^100^15040,00030,000Altitude( f t )20,0001 0,00010000Altitude(m)5,0000^200^400^600^760Barometric PressureChapter 1: INTRODUCTION^10higher elevations is solely regulated by changes in ambient oxygen.Lung adaptation to high altitude is characterized by a variety of structural and functionalchanges which collectively facilitate oxygen transport from the ambient air to the body tissue. Thetransfer of oxygen from the surrounding environment to blood is partially controlled by diffusionresistance in the lung. Thus, the larger the gas exchanging surface area the smaller the resistanceto gas diffusion. An increase in internal surface area is dependent upon an increase in lung volumeand internal partitioning of the respiratory units. Through internal partitioning, the lung attains a gasexchanging surface area approximately 40 times as great as body surface area (274). According toTenney and Remmers (257), the postnatal compartmentalization of the lung appears to be socontrolled that the metabolic requirements (which varies with body surface area) and the alveolarsurface area for 02 uptake are matched quantitatively in all mammalian species.Figure 1. Relationship between altitude, barometric pressure and oxygen tension [West (281)].Since ambient oxygen tension decreases automatically with a decrease in barometric pressure(Figure 1), both hypoxia and hypobaric pressure are considered together to study the effect of lowChapter 1: INTRODUCTION 11oxygen, but their effect may be different. Therefore, with regard to structural and functionaladaptations in the lung, the effect of hypobaric hypoxia will be considered separately from normobarichypoxia. HYPOBARIC HYPDXIA1. STRUCTURAL ADAPTATIONSIn earliest investigations, studies relating to high altitude acclimatization documented thatCholos Indians of Peru (native to high altitude), had short stature, low body weight, wide barrel-shapedchests and larger lungs compared to their counterparts at sea level (12, 134). Later, using Wistarmale rats, it was shown that in animals exposed to hypobaric hypoxia (390 mm Hg), the length of thechest was smaller, but the width was not (chest radiographs), thus giving a barrel-shaped appearanceto animals with stunted somatic growth (145). The authors suggested that this may have occurredbecause the lungs of hypobaric hypoxic rats were being held at a higher volume. Subsequent studiesperformed on humans have confirmed that high altitude residents have higher lung volumes (32, 84,107, 122, 130, 146, 215). Using animals, investigators have demonstrated that translocation to higherelevations (45) and exposure to experimental hypobaric hypoxia (20) produce increases in absoluteor specific (relative to body weight) lung weight and lung volume.An increase in lung volume may be accompanied by the addition of alveoli or enlargement ofexisting airspaces. Increase in alveolar number may occur at a higher rate compared to lung volume,thus increasing the complexity of the lung, or at a slower rate which may be proportional to or lowerthan an increase in lung volume. In the latter case, surface to volume ratio may remain unchangedor decrease. Among the earlier studies, Hurtado (146) examined the lungs of rabbit, dog and humanat high altitude, and found that alveolar dimensions were bigger than in the lungs of sea-levelcounterparts, but no units or methods of inflation and fixation of tissue were described. Later Cohn(66) observed increases in lung weight and alveolar surface area in rats following 2 weeks exposureChapter 1: INTRODUCTION^12to high altitude simulated conditions, and remarked that these structural changes occur as a part ofaccommodation to high altitude.Using comprehensive stereological techniques, Bartlett and Remmers (20), and Burn i andWeibel (45) performed quantitative assessment of lung growth in rats exposed to high altitude andexperimental hypobaric hypoxia and drew similar conclusions that exposure to hypobaric hypoxiastimulates lung growth but inhibits somatic growth. Bartlett and Remmers (20) observed that onemonth old rats exposed to simulated altitude of 4200 m (450 mm Hg) showed a significant increasein lung weight after 7 to 21 days of exposure. After 21 days of exposure, significant increases in lungvolume and alveolar surface area were also observed, but mean alveolar diameter was not influenced,suggesting that lung growth occurred by formation of new alveoli rather than by enlargement. In theauthors' opinion, the response to hypobaric hypoxia at day 7 was an increase in lung density (lungweight per unit lung volume) without alveolar proliferation. If the exposure continued, the density ofthe lung returned to the normal level and alveolar proliferation occurred at a faster rate than controls.Although they found no increase in lung blood volume following exposure to hypobaric hypoxia, theydid not rule out the possibility of residual blood contributing to the lung weight increase. Burn i andWeibel (45) transported 23 day old rats to high altitude (3450 m) for 21 days and compared theadaptive lung growth to those at low altitude (570 m). Absolute lung volume increased and bodyweight gain decreased in high altitude rats. The authors suggested that the unchanged relativevolume proportions of the lung compartments favoured the argument that increased lung volumeoccurred due to the addition of new respiratory units. In high altitude rats, the specific alveolar,capillary and tissue volume increased by approximately 20% and so did alveolar surface area.Although conditions leading to hypoxia in both studies (20, 45) were different (hypobaric hypoxia vshigh altitude), the adaptive lung growth response was similar. This may suggest that at high altitude,hypobaric hypoxia may be the main regulator of lung growth.Tenney and Remmers (258) suggested that the diffusing capacity of the lung is proportional tothe internal alveolar surface area. Since capillaries occupy a major fraction of the alveolar wall area,Chapter 1: INTRODUCTION 13a measure of the area of blood-filled pulmonary capillaries will be a more direct assessment ofincreased pulmonary diffusing capacity (258). Weibel (276) developed a model to calculate pulmonarydiffusing capacity using structural parameters: alveolar and capillary surface areas, capillary volume,and the harmonic mean thickness of the tissue barrier and the blood plasma layer (which separatesred blood cells and endothelial cells). Burn and Weibel (45) estimated morphometrically determinedpulmonary diffusing capacity for oxygen in rats exposed to hyperoxia and high altitude. They foundthat after 3 weeks, the pulmonary diffusing capacity for oxygen in high altitude rats (P02 100 mm Hg)increased from 0.6 ml.min -1.mmHg-1.100 g-1 to 0.72; whereas it decreased to 0.51 in hyperoxic rats(P02 290 mm Hg). This indicates that alterations in ambient 02 produces structural changes in thelung during the growth period.Although the information obtained from animals following translocation to high altitude orexposure to experimental hypobaric hypoxia helps to understand the process that regulates thephysiological adaptive response, information regarding genetic adaptation in populations residing athigher elevations is lacking. Lechner (163) conducted an interesting study to investigate thedifferences in genetic and metabolic adaptation. Lechner (163) studied adaptive lung growth in adultsea-level native fossorial pocket gophers (Thomomys bottae at 265 m, weight-matched, age unknown)following exposure to normobaric hypoxia (equivalent to the oxygen concentration at 3400 m) for threemonths. Both sea-level and normobaric hypoxic pocket gophers were compared with high altitudenative pocket gophers (Thomomys umbrinus melanotis at 3200 m). Compared to sea-level gophers,the alveolar fraction and specific alveolar surface area were greater in high altitude native andnormobaric hypoxic pocket gophers. Although these findings suggest that lung growth in high altitudenatives is influenced mainly by decreased oxygen, the author concluded that the mechanism ofadaptation at the tissue and blood level differed in high altitude natives and normobaric hypoxia-acclimated animals. Hematocrit increased only in normobaric hypoxic gophers, whereas blood oxygencarrying capacity and skeletal muscle myoglobin concentration were higher in high altitude nativegophers. The variable response may either be attributed to difference in environment [hypobaricChapter 1: INTRODUCTION 14hypoxia (high altitude) and normobaric hypoxiaj or to difference in genetic and metabolic adaptations.In another study, lung volume of high altitude (4660 m) native mice (Phyllotis Darwin!) was greaterthan the same species at sea level (204). Since the number of Type II pneumonocytes, interstitialcells and alveolar macrophages, and capillary leucocytes nuclei per unit alveolo-capillary volume wasequivalent in both groups, the authors speculated that higher lung volume in high altitude native micemay have resulted from a greater number of structural units in the lung.Most of the information available relating to high altitude adaptation in humans has beenobtained from pulmonary function tests. Only one group of investigators has performed morphometricanalysis on lungs of highlanders to determine lung structural alteration at high altitude. Saldana andGarcia-Oyola (230) studied pulmonary structural differences in males who lived at high altitude(altitude 12771 feet) and compared them to males of same age at sea level. They observed thatmales at high altitude had a greater number of alveoli and alveolar surface area. The mean alveolardiameter in high altitude subjects was also larger than in sea level subjects. The authors concludedthat a larger residual volume of the lung and hyperventilation at high altitude may be associated witha greater number of alveoli, larger alveolar surface area and larger alveoli. INTERSPECIES DIFFERENCESThe capability to acclimatize to high altitude conditions varies from species to species and evenwithin species (interstrain) due to genetic anatomical and physiological differences. For example,guinea pigs are unique animals to study for lung growth adaptation to high altitude. They haveinhabited the Andean mountains for thousands of years at elevations up to 4500 m (244) and havealso been raised at sea level for many generations. Compared to guinea pigs, the lungs of rats areimmature at birth. Furthermore, guinea pigs have the advantage of higher oxygen affinity for arterialblood than rats. At an altitude of 5000 meters, the arterial blood oxygen saturation in guinea pigs ismore than 75%, but in rats, it is about 55% (16). Arterial blood of llamas at an altitude of 5000 m ismore than 90% saturated with oxygen (16). Such structural and functional variations between theChapter 1: INTRODUCTION^15species may determine the ability of an animal to adapt to variable ambient conditions.Postnatal lung growth adaptation in newborn rats and guinea pigs to simulated conditionsequivalent to 3000 m and 5000 m elevation was studied until the age of 50 days (16). In rats,absolute lung weights increased at 3000 m but were decreased at 5000 m at the age of 21, 35 and49 days. However, compared to the controls, the specific lung weight in both 3000 and 5000 mgroups was lower on day 21 and higher on days 35 and 49. On the other hand, in guinea pigsexposed to 3000 meters, lung weight increased on days 11 and 23 but body weight remainedunaffected; whereas lung weight of guinea pigs exposed to the equivalent of 5000 m remainedunchanged, but body weight decreased. In another study, when interspecies comparisons were made,the llama (inhabits at high altitude) had smaller mean alveolar diameters compared to other speciesof similar body size such as the domestic cow, western pronghorn antelope, and domestic goat (258).Therefore, it appears that animals such as guinea pigs and llamas, perhaps due to their anatomicaland physiological differences, are able to withstand stress of severe simulated high altitude conditionsbetter than rats. CELL KINETICS AND MORPHOLOGICAL CHANGESThe lung tissue mass may increase due to hyperplastic and/or hypertrophic changes in thecells, enhanced accumulation of connective tissue matrix proteins and lung water content. Noinformation is available about biochemical alterations which occur in lung structure during the highaltitude adaptive response. Cellular kinetics and morphological changes in the lung have been studiedbut information is limited. The heterogeneous cell population of the lung of approximately 40 differentcell types is mainly distributed in three compartments: conducting airways, pulmonary vasculature andthe gas exchanging apparatus. Besides migratory cells, the cellular composition of the gasexchanging surface mainly consists of alveolar epithelium (type I pneumonocytes, type IIpneumonocytes), alveolar wall capillary endothelium and interstitial cells (fibroblasts, smooth musclecells, monocytes, plasma cells, macrophages, pericytes and a number of indeterminant cell types).Chapter 1: INTRODUCTION 16The alveolar epithelial type I cells are anatomically specialized to provide a minimum barrierto gas diffusion as their very thin cytoplasmic extensions line approximately 97% of the alveolarsurface area. They are considered as terminal cells and thus do not have the ability to proliferate andreplicate. The type II epithelial alveolar cells which represent —15% of total lung cells in theparenchyma have two major functions that are critical in maintaining the integrity of alveoli. They havethe ability to readily proliferate and differentiate into type I cells, and they produce surfactant whichprevents alveolar collapse. These cells provide approximately 3% of the alveolar surface area andhave a relatively long turnover time which ranges from 20 to 84 days (100, 245). Pearson andPearson (205) demonstrated that aside from having larger nuclei and greater volume of mitochondria,type II pneumonocytes also have more and larger lamellar bodies in mice (Phyllotis Darwin!) nativeto high altitude (4660 m) compared to those at sea level, resulting in four times more surfactant.Species having a high respiratory frequency rate have more surfactant. Increased surfactant in micenative to high altitude may occur due to hyperventilation.Endothelial cells line the extensive intra-alveolar capillary network and make up —36%(distribution of cells in the alveolar wall differs among various species) of total lung cells, and have ahigher turnover rate compared to epithelial cells. These cells are permeable to water and electrolytes,but to some extent form a barrier to high molecular weight solutes. Mesenchymal cells are foundwithin the interstitial compartment of the alveolar wall and represent —38% of the total lung cells.These cells are important to the synthesis of the major connective tissue proteins found in the alveolarwall.The kinetics of cells is generally studied either by pulse labelling DNA with a radio-labelledthymidine such as tritiated thymidine (3H-TdR), or using stathmokinetic agents such as colchicine andvinca alkaloids (Vinblastine, Vincristine) to arrest dividing cells in metaphase. Thymidine isincorporated into cellular DNA while the cell is in S-phase of the cell cycle preceding mitosis. Theuptake and incorporation of 3H-TdR by the cell nuclei, however, does not necessarily reflect celldivision, but is mainly considered an indicator of DNA synthesis. Increased DNA synthesis per seChapter 1: INTRODUCTION 17does not indicate cellular proliferation or an increase in cell number. Binucleate cells appear after birthin certain organs and increase in frequency with age (287). Multinucleate cells and polyploidy mayoccur as a result of increased DNA synthesis without an increase in cell replication. Tritiatedthymidine incorporation can be evaluated by radiochemical or autoradiographic techniques. Thegeneration cycle and duration of individual phases of the cell cycle can be analyzed by 3H-TdR pulselabelling and treatment with stathmokinetic agents simultaneously. Volkel et al. (271) studied the onsetand kinetics of cellular proliferation following exposure to hypobaric hypoxia (440 torr) in adult femaleWistar rats after 3, 6, 9, 12, 20 and 35 days of exposure, and then compared this with the results of3, 12 and 35 day controls of similar starting age. They found that maximum in vitro DNA synthesisoccurred on day 9 of exposure and measured 220-345% of controls extrapolated from days 3 and 12.Lung DNA synthesis reached control levels between day 15 and 20 of exposure.The various cell populations in the lung may respond to the hypobaric hypoxic stimulusdifferently. Niedenzu and coworkers (193) observed an 85% increase in 3H-TdR incorporation in ratsexposed to 440 mm Hg for 9 days. The labelling index of bronchial epithelial cells and vascularsmooth muscle cells increased in hypobaric hypoxic animals compared to the normobaric animals.The labelling index of alveolar interstitial cells remained unchanged. In the authors' opinion, the cellswith normally very low labelling index (bronchial epithelial cells) were stimulated, whereas the cells withalready high labelling index (interstitial cells) remained unaffected. Since DNA synthesis was onlyincreased in the lung and not in the liver and kidney, the effect of hypoxia, in the authors' opinion wasorgan specific. Another group of investigators (181) observed that in adult rats, hypobaric hypoxiainduced maximal stimulation in cells of arteries and alveolar walls on days 3 and 5 of exposure. Onthe other hand, Hunter and coworkers (145) found no significant change in lung cells labelled with 3H-TdR in young mice or rats exposed to hypobaric hypoxia (390 mm Hg) for 1 and 4 weeks. Bycontrast, the labelling index of lung cells decreased in old rats (56 days old) compared to young rats(35 days old). Therefore, the sequence of cytokinetic events in the lung following exposure tohypobaric hypoxic stress remains unclear.Chapter 1: INTRODUCTION^ FUNCTIONAL ADAPTATIONAs stated earlier, highlanders have large thoracic cages relative to sea-level residents (12, 134).Lung growth adaptation to high altitude has been assessed by using a variety of physiologicaltechniques in humans and animals born and raised at high altitude and then comparing the resultsto their counterparts at sea level. Others have observed the effect of sojourn at high altitude onlowlanders. However, the results described in the literature are controversial.A number of investigators have reported that high altitude natives have higher vital capacities(32, 39, 84, 107, 122, 130, 146, 215) whereas others (56, 75, 153, 256) observed reduction in vitalcapacity. Cerretelli (56) found that vital capacity (VC) was reduced by 10% during a Himalayanexpedition, after 30 days sojourn between 3800 and 5000 meters. The reduction in VC was notinfluenced by oxygen breathing. In the author's opinion, this reduction in VC could be attributed to anincreased inspiratory tone as once described by Peyser et al. (209), which could also have increasedresidual lung volume. Changes in FRC that take place in humans following exposure to high altitudehave been controversial; while some investigators have reported an increase in FRC (75, 146, 232,256), others did not find any change (11, 70, 153).Brody et al. (39) studied lung adaptation in 17 to 20 year old residents of the Peruvianmountains (3850 m), and found that the lung volume of highlanders was 30-35% greater thanlowlanders (600 m) of similar race, age and body size. The vital capacity of Peruvian lowlanders wasonly 84% of Caucasians but vital capacity of mountain dwellers was 116% of Caucasians. Lungelastic recoil at functional residual capacity and size-corrected pressure-volume curves were similarin Peruvian highlanders, lowlanders and Caucasian subjects of the same age, suggesting that neitherincreased muscle strength nor alterations in connective tissue properties of the lung were primarilyresponsible for large vital capacities in highlanders. Despite higher lung volumes in highlanders,absolute maximum expiratory flow rates were not increased and, when expressed as a function of lungvolume, flow rates were actually decreased. In the authors' opinion, the crossectional area or volumeof the airways did not participate in lung growth enlargement in highlanders. The authors suggested1 2Previous studies3^4Experimental conditionsAmbient Po2 (Torr) 78 95 105 91-98Ambient PB (Torr) 750 450 497 760Species Rats Rats Rats RatsAge before exposure (weeks) 4 4 3 9Age after exposure (weeks) 6 7 6 12ObservationsBody weight 4 11 It IILung weight 11 II n/a ifLung volume 11 SO SpitAlveolar surface area if Sp11 itCapillary surface area n/a SO n/aAlveolar multiplication No Yes Yes No5^680^80630^630Guinea pigs3^35 17n/a^n/a11ififn/a^n/aChapter 1: INTRODUCTION^19that the difference in flow rates may have been an expression of the manner in which hypoxiastimulates the growth of the lung. Brody et al. (39) concluded that greater lung volumes observed inPeruvian mountain dwellers resulted from postnatal acclimatization to the hypoxic environment, ratherthan from genetically determined lung growth. Other investigators have found no difference in lungcompliance between high and low altitude residents, or when either lowlanders translocated to highaltitude or highlanders moved to sea level (39, 75). Lahiri et al. (157) studied respiratory control andvital capacity in highland infants, children, and adults and in the authors' opinion, the environment isthe major determinant of pulmonary acclimatization at high altitude.Table 1. Summary of previous studies: somatic and lung growth adaptation following chronicexposure.References: 1. Bartlett (18); 2. Bartlett and Remmers (20); 3. Burn and Weibel (45); 4.Cunningham and coworkers (77); 5-6. Lechner and Banchero (164, 167). Sp (per unit bodyweight), n/a (not available). NORMOBARIC HYPDXIAAlthough yet to be precisely determined, it is widely believed that the adaptive lung growthChapter 1: INTRODUCTION 20response to decreased ambient oxygen is the same, whether low oxygen is delivered bydecompression or by gas mixture. Aside from studying lung growth adaptation at high altitude,normobaric hypoxia has also been used as a model to study lung growth alterations in variousconditions which may result in hypoxia and/or hypoxemia. While studying the adaptive response tonormobaric hypoxia, some investigators have concluded that it accelerates lung growth (77, 101, 102,164, 167) whereas others have either found no change in lung growth (18, 247) or conversely, thatit was inhibited (187). The controversies may exist due to the reasons mentioned earlier.Bartlett (18) made a quantitative assessment of lung growth in month old male Sprague-Dawleyrats after 15 days of exposure to normobaric hypoxia (10.4% 02) and normobaric hyperoxia (45.8%02). The author found that except for depressed somatic growth and increased specific lung weight,lung growth in hypoxic conditions remained unaffected. The investigator (18) attributed the increasein specific lung weight to an increased residual volume of blood remaining in the pulmonary vesselsof the low oxygen rats. On the other hand, somatic growth of hyperoxic animals was not affected, butlung growth was diminished. The author commented that prolonged normobaric hypoxic exposurecould have caused structural changes in the lung. However, this hypothesis was not supported byCunningham and associates (77), who showed that the adaptive lung response to normobaric hypoxia(12-13% oxygen) is dependent upon the age of the animal at the time of exposure, and that exposurebeyond 3 weeks did not cause further increase in lung growth parameters. The authors (77) foundthat whereas three weeks of exposure caused significant increase in size and number of alveoli innewborn rats, it produced an increase in alveolar size but not number in adult rats. An increase inalveolar surface area was also less in adults than in young animals. It has been demonstrated thateven a short exposure of six days produced an increase in mean chord length of alveoli (198). Itappears that normobaric hypoxia causes an increase in size of alveoli in both adult and young animalsbut it produces an increase in number of morphometric unit structures only in growing animals. Thissuggests that the type of adaptive response to normobaric hypoxia varies with age, but it remains tobe shown whether there is a lower limit of environmental oxygen below which lung growth regulationChapter 1: INTRODUCTION^21no longer functions.The observation made by Cunningham and associates (77) that the adaptive lung growthresponse is limited despite continuous exposure to low oxygen, was further confirmed by Lechner andBanchero (164). It is possible that hypoxic stress accelerates lung growth to reach adult lungdimensions, and that physical restrictions may limit further lung growth. Lechner and Banchero (164)tested this hypothesis and showed that male guinea pigs acclimated to normobaric hypoxia (Po 2 80Torr) for 3 weeks had significantly increased lung volume (32%) and alveolar surface area (27%)compared to controls of similar body mass. The differences between the normobaric hypoxic andnormoxic guinea pigs progressively reduced with an increasing exposure period. The hypoxia-inducedeffect on lung growth adaptation ceased despite continued hypoxic exposure when the body weightwas equivalent to or more than 900 g. The authors concluded that maximal size of the chest whichis achieved at an age of 16-20 weeks ultimately constrained accelerated lung growth because nothoracic space was available for further lung development.Morphometrically calculated pulmonary diffusion capacity remains unchanged following acute(7 days) exposure to normobaric hypoxia in rats (247) or chronic (14 weeks, Fo 2 0.126) exposure(164) in guinea pigs. The interstitial tissue thickness increased after acute exposure (164, 247) butit was no longer evident if exposure to hypoxia was prolonged. This change in the interstitium mayhave occurred due to the appearance of small electron-lucent areas indicating fluid accumulation insubendothelial locations (247). It is likely that initial thickening of interstitium is followed by thinningof tissue barrier as the lung undergoes reconstruction and structural remodelling.Low oxygen has been shown to inhibit DNA, RNA and protein synthesis (57, 191, 252) inorgans other than lung and heart. Sjostrom and Crapo (247) reported that rats maintained in 10-11%02 and mice in 10% 02 for 7 days showed increases in lung weights of 31% and 28% respectively.Even though morphometrically no differences were found, in rats the total amount of protein and DNAwas increased by 17% and 21% respectively. Increase in protein in part may be due to pulmonaryedema, but the investigators (247) found increased endoplasmic reticulum and free polyribosomes inChapter 1: INTRODUCTION 22the capillary endothelium suggesting increased protein metabolism. These findings may indicate thatnormobaric hypoxia increased protein synthesis and cellular proliferation. Another group ofinvestigators (101) also found that an exposure to normobaric hypoxia for 14 days produced asignificant increase in lung DNA in pregnant rats indicating hypoxia-induced lung growth stimulationin both young and adults. However, the other changes in the biochemical parameters such asconnective tissue proteins are yet to be investigated. On the contrary, lung DNA decreased innewborn rats exposed to normobaric hypoxia, indicating that newborns may not have developed therequired mechanisms to tolerate the hypoxic stress. This requires further investigation (187).An increase in FRC in rats (13), rabbits (209), cats (209), dogs (30) has been reported followingexposure to acute or chronic normobaric hypoxia. After exposing 4 week old male Wistar rats tohypoxia of 8%, 10%, and 12% 02 in isobaric chambers for 3 weeks, Barer et al (13) observed anincrease in FRC of 34-62%. In the authors'(13) opinion, chronic hypoxia did not increase FRC bymaking the lungs more compliant because static compliance did not change. They speculated thatstructural factors in the chest wall could have been responsible for the increase in FRC. In anotherstudy (77), lung compliance also remained unchanged regardless of age of the animals at the timeof exposure to normobaric hypoxia. These findings suggest that normobaric hypoxia may not causeany structural changes in lung tissue.Why decreased ambient oxygen stimulates growth only in some organs and not in othersremains still an enigma. With regard to initiation and control of lung growth in a reduced oxygenenvironment, various biological and physical factors have been proposed but the mechanisms bywhich these factors regulate lung growth are yet to be understood. Hypoxic exposure also results inhyperventilation as the rate of breathing and tidal volume are increased (197, 200). In normalconditions as well as in conditions which stimulate lung growth, stretch has been implicated as a majorregulator of lung growth. In hypoxic conditions hyperventilation may induce lung growth due tomechanical distortion of the lung tissue. Although other investigators (20, 207) studying lung growthin hypercapnic animals have ruled out the role of hyperventilation, Faridy and Yang (102)Chapter 1: INTRODUCTION 23demonstrated that the effect of hypoxia produces lung growth both by direct and indirect stimulation.The authors showed that while normobaric hypoxia enhanced DNA synthesis prior to an increase inlung volume, hyperventilation produced by higher ambient CO2 increased only lung volume. In theiropinion, lung tissue growth stimulation in hypoxia occurs by a direct effect of low P02, but an increasein lung volume occurs as a consequence of hyperventilation due to mechanical stimulation. Thissequence of events is in sharp contrast to those proposed by Berger and Burni (23). The authorsclaim that after partial pneumonectomy, air space distension is followed by cell proliferation, tissueremodelling, and finally restoration of normal tissue architecture. This suggests that mechanicaldistortion of the tissue induced the cellular stimulation. Therefore, the role of hyperventilation andstretch in lung growth in hypoxic stress remains unclear.It has been well documented that an exposure to low oxygen produces hemodynamicalterations (e.g. increased heart rate, cardiac output, central blood volume, hemopoiesis, hematocrit,hypertension). Since blood flow is increased in hypoxic conditions, alterations in organ blood flowcharacteristics have been suggested as regulating organ growth in hypoxic stress. Lechner andBanchero (164) suggest that a transient increase in pulmonary blood flow may occur during hypoxicexposure until hematological changes are achieved to compensate for reductions in ambient or arterialoxygen. Oxygen carrying capacity of blood increased by only 40% (166); this would not by itselfaccount for increased lung growth. They suggested that increased pulmonary blood flow during earlyexposure may have enhanced lung growth. Another study supports the view that increased blood flowinfluences lung growth because lung weight increased (48%) four weeks subsequent to ligation to theright main pulmonary artery (254). Tucker and Horvath (266) subjected Sprague-Dawley albino ratsto 440 mm Hg from 31 to 68 days of age. Increases in hematocrit and hemoglobin concentration, andincreases in absolute weights of ventricles of the heart, lungs, spleen and adrenals occurred, whilekidneys remained unaffected. Blood flow per g tissue and total organ blood flow increased in thelungs, heart and adrenals, but remained unchanged in kidneys. The authors inferred that an increasein organ weight is associated with circulatory adaptations in order to prevent the hypertrophied organsChapter 1: INTRODUCTION^24from hypoperfusion during hypoxic exposure. Alternatively, augmented growth in organs which receiveincreased blood flow in hypoxia may support the hypothesis that increased blood flow removes orlowers the concentration of local tissue-specific growth inhibitors or chalones (103). Consequently,growth of a respective organ is stimulated. It has been suggested that increased pulmonary bloodflow and pressure act directly or indirectly to stimulate endothelial cells to release endothelial-derivedgrowth factor(s). These act as paracrine factors to increase mitogenic activity and connective tissuesynthesis in the pulmonary arteries (221).1.2.2. EFFECT OF REDUCED AMBIENT PRESSUREIt has been well known from balloon ascents that barometric pressure decreases as the heightincreases. This decrease in pressure of air on body was once described by De Saussure in 1786 asa mechanism for acute mountain sickness by causing "relaxation of blood vessels". More than acentury ago, Bert ruled out the possibility that low ambient pressure plays any significant role incausing acute mountain sickness (characterized by headache, fatigue, dizziness, palpitation, nausea,loss of appetite, and insomnia (282)) as it could be relieved by oxygen breathing. However, it hasrecently been shown that a rapid descent, or use of Gamow bag (patient is exposed to increasedpressure by means of a pump) is more effective in relieving mountain sickness than simpleadministration of supplemental oxygen (124, 282). This implies that low ambient pressure plays asignificant role in the etiology of mountain sickness along with hypoxia and hypocapnic alkalosis (121).The effect of hypobaria per se is not known, but it appears that it causes hypoxemia. Levine andassociates (171) found low arterial Po 2 in hypobaric normoxic and hypobaric hypoxic sheep comparedto normobaric normoxic and normobaric hypoxic animals respectively. The same group ofinvestigators later observed that hypobaric hypoxic sheep required 65% oxygen to make themhypobaric normoxic instead of 49.7% oxygen (137). How hypobaria affects the oxygenation of bloodis not known.No direct evidence is available that hypobaria per se at high altitude induces change in lungChapter 1: INTRODUCTION 25or body growth adaptation. A group of investigators using the same rat species have observed thatwhile normobaric hypoxia (18) produced no significant change in any of the morphometric parametersof the lung, hypobaric hypoxia (20) accelerated lung growth because lung weight, lung volume andalveolar surface area increased. Although ambient Po2 used and duration of exposure in both studieswere not the same, one may speculate from the variable lung growth response in normobaric hypoxiaand hypobaric hypoxia that hypobaric pressure may have induced changes in the lungs by itself orin association with hypoxia. However, the effect of low ambient pressure on lung growth remains tobe investigated (Table 1).1.2.3. EFFECT OF EXERCISEThe rugged terrain at higher elevations may increase work load and whole body oxygenconsumption. The internal alveolar surface area of the lung correlates in a linear fashion with the rateof resting 02 consumption of mature animals (257). In mammals, lung volume is proportional to bodyweight, but the extent of internal partitioning is directly proportional to the metabolic rate (257). Thus,animals with greater oxygen demand have the smallest alveoli and the internal surface area per unitof lung volume is large.The influence of exercise on human and animal lung structure and function has beeninvestigated (17, 65, 108, 112, 265). Tiemann (265) postulated that physical exercise led toenlargement of the chest cage and accompanying lung inflation, resulting in chronic distension of thestructural units, which in turn stimulated proliferation of interalveolar septa. It has been shown thatafter subjecting 10 month-old rats and guinea pigs to strenuous swimming (210 minutes/day) for 122days, extensive pulmonary changes occurred, including increases in total lung weight and number ofalveoli attributed to finer subdivisions, and thickening of the alveolar walls (112). By contrast, ratssubjected to exhaustive exercise on a treadmill for 20 days showed no change in lung growth orcompartmentalization as all parameters of lung growth (lung weight, lung volume, alveolar surface areaor alveolar number) remained unchanged (17). In another study (108), investigators showed that ratsChapter 1: INTRODUCTION 26subjected to swimming during the second month of postnatal life had greater alveolar densities andalveolar surface area. They concluded that swimming induced alveolar proliferation. Alveolarproliferation was not related to the intensity of training but was influenced by the age of the animal atthe time of training.The effect of increased oxygen demand on lung growth has also been studied by usinghyperactive animal models. Geelhaar and Weibel (111) demonstrated that increased alveolarizationin Japanese Waltzing Mice was influenced by hyperactivity (JWM, exhibit a continuous waltzing motiondue to a genetic defect in their vestibular apparatus) compared to control mice which were of differentspecies. Contradicting that hypothesis, Bartlett and Arenson (21) suggested that increased lunggrowth in JWM is genetic rather than induced by sustained hyperactivity because they found nodifferences in lung dimensions between JWM and their phenotypically normal litter mates. Using IDPN(imino-BB'-dipropionitrile) which is known to cause hyperkinesia, Burn i et al. (48) and later Hugonnaudet al. (142) showed increased lung growth in hyperactive mice, thus supporting the hypothesis putforth by Geelhaar and Weibel (111). They found increased specific lung volume and morphometricallycalculated pulmonary diffusion capacity. However, one might argue that these results might just aswell reflect a direct influence of this drug on lung development, rather than a true adaptation toincreased oxygen consumption. Hence, experimental evidence is yet insufficient to establishconvincingly that altered oxygen consumption can induce significant changes in lung growth pattern.1.2.4. EFFECT OF COLDAlthough various experimental approaches such as drugs, hormones, or genetically determinedhyperactivity have been undertaken to study the effect of modified oxygen consumption and demand,cold is considered as more of a "physiological" experimental model to assess the adaptive responseto increased oxygen consumption (3, 131, 203). Cold is an important parameter at high altitude whichis often overlooked. The temperature falls by about 0.6° C for every 100 meter increase in altitude,regardless of latitude (132, 273). Somatic growth observations made following exposure to cold inChapter 1: INTRODUCTION 27small mammals are controversial. Some investigators have observed decreased somatic growth inlower temperatures (14, 58, 133, 250), whereas others have not (8, 113, 129, 167, 196). It has beensuggested that skeletal growth retardation possibly occurs due to reduction of blood flow to theskeletal tissue during exposure to cold (168).Increased oxygen utilization during exposure to cold acts as a stimulus to cause acceleratedlung growth in order to meet the body's oxygen consumption requirements by increasing thepulmonary diffusing capacity. Gehr et al. (113) tested this hypothesis and showed an increase inoxygen consumption without any change in body growth in seven week old rats exposed to 11° C forthree weeks. The specific lung volume of cold-exposed rats increased by 24% and was alsoassociated with larger alveolar and capillary surface area, but the capillary volume density and thebarrier thickness remained unchanged. In the authors' opinion, this may occur by an increase in thenumber of structural units and their complexity in proportion to lung volume. They suggested thatincreased stress, to which the growing lung tissue is subjected, may directly or indirectly cause lunggrowth alterations.Guinea pigs which have been inhabitants of higher altitude for generations also showedaccelerated lung growth following chronic exposure to cold temperature (5° C) from 2-18 weeks of agewhen body weight was <600 g (165). In the authors' opinion, lung growth occurred because of acontinuously elevated oxygen consumption and the absence of anatomic limitations on thoracic cagesize while the animals were in the rapid growth phase. Although lower oxygen and cold temperaturego hand in hand at high altitude, the lung growth response mechanism to these variables appears tobe common (167). The body growth rate of guinea pigs exposed to cold (6° C) and normobarichypoxia (Po2 85 Torr) remained unaffected. Following 3 weeks of exposure, specific lung volume ofcold plus hypoxia animals increased, but by 16 weeks that increase disappeared. Similar relationshipswere observed in specific alveolar epithelial and capillary endothelial surface areas. The authorsclaimed that lung growth adaptation to cold plus hypoxia is similar to that of hypoxia or cold aloneindicating that both cold and hypoxia operate through a common, rather nonspecific mechanism.Chapter 1: INTRODUCTION^281.2.5. SOMATIC AND ORGAN (other than lungs) GROWTH ADAPTATIONA stressor which potentially disturbs the functional capacity of body organs mediated by itsdirect or indirect adverse effects, eventually disrupts the normal rate of body growth. In an attemptto adjust to the altered state of homeostasis, the body undergoes various adaptive changes and thenet result is reflected in body weight. Body growth retardation has been observed in humans andanimals following exposure to high altitude, acute or chronic hypobaric hypoxia or normobaric hypoxia(18, 20, 45, 85, 125, 143, 144, 146, 185, 191, 267).It has been claimed that at high altitude, hypophagia and hypodipsia are mainly produced bylower oxygen (156). Reduced food consumption results from decreased appetite because anexposure to hypobaric hypoxia lowers caloric intake related to metabolic body weight (Appetitequotient = Cal/day/body weight°13) (5). The efficiency of protein utilization (daily weight gain/ dailyprotein intake) remains unchanged (5), but glucose utilization is impaired in hypoxic conditions (31).Besides decreased food and water intake, somatic growth retardation in response to high altitude hasalso been attributed to increased water loss (211), changes in food digestibility and utilization (59,238), alteration in body composition primarily due to decreased deposition, or loss of body fat (59, 125,237) and disturbances in protein metabolism (154). The underlying mechanisms which result insomatic growth changes are unknown, but a possible role of reduced body metabolic rate in anattempt to decrease oxygen utilization in the body in hypoxic stress has been suggested (144). Inhypoxic conditions, initial body weight loss due to decreased food and water intake occurs within thefirst 24 hours (62). Subsequently, low body weight gain or weight loss may occur due to depletion offat depots and decreased lean body mass. These differences further complicate the interpretationsof the adaptive body response.It has been shown that while the rate of somatic growth slows down in younger animals, olderanimals lose body weight after translocation to high altitude (104), or following exposure to hypoxicconditions (144, 145, 274). Besides body weight reduction, skeletal growth reduction has also beenreported in animals subjected to hypoxic stress, but its effect on growth of various bones is not uniformChapter 1: INTRODUCTION^29(143). Long bones grow relatively more than the axial skeleton. This may occur due to blood flowdifferences, skeletal maturity at the time of exposure or individual bone growth regulation (143).A number of investigators (16, 55, 64, 93, 125, 148, 185, 191, 207, 223, 253, 267, 266) havestudied the effect of exposure to high altitude, hypobaric hypoxia or normobaric hypoxia on quantitativeor qualitative growth response in various organs such as lungs, heart, spleen, adrenals, liver, kidneys,thymus, thyroid, pituitary and reproductive organs, but the observations reported are inconsistent. Inspite of inconsistent observations reported in the literature, it appears that the adaptive response tohypoxic stress in all body organs is variable. It has been demonstrated that despite decreased bodyweight gain in hypobaric hypoxic or normobaric hypoxic conditions, absolute and/or specific (per unitbody weight) weights of organs such as lungs, heart, spleen and adrenals increased (16, 55, 64, 125,148, 185, 191, 267, 266), while absolute or specific weights of liver and kidney decreased or remainedunchanged (55, 64, 93, 125, 148, 185, 191, 207). Some investigators have even reported that hypoxiaincreased absolute and/or specific weights of liver and kidney (101, 267, 266). It appears that whilehypoxic stress induces positive allometric growth of organs like the heart, lungs, spleen and adrenals,it either reduces or has no effect on growth of other organs.1.3. HYPOTHESISIt is evident that following exposure to hypoxic conditions, lung growth is either increased orremains unchanged while somatic growth is diminished. However, at high altitude, a decrease inoxygen tension is closely associated with a drop in barometric pressure. The effect of hypobarichypoxia and normobaric hypoxia on lung growth may not be the same, and the effect of low ambientpressure on the adaptive response of the body is not known. Somatic growth retardation in hypoxicconditions complicates interpretations of results regarding organ growth and specific (per unit bodyweight) observations may not provide reflections of true changes in organ growth. As a consequencethe following hypotheses were tested:MT^In hypobaric hypoxia, low oxygen may be the main operating factor in producing adaptiveChapter 1: INTRODUCTION^30structural changes in body organs, but decreased ambient pressure may alter the adaptivelung growth response.OW^The effect of hypobaric hypoxia may be immediate. If exposure is prolonged, lung growthstimulation may continue. All types of cells in the lung may participate equally in the adaptiveresponse to hypobaric hypoxia.sa,^Decreased food intake and associated diminished somatic growth in hypobaric hypoxia mayproduce structural alteration in the lung and growth of other organs.far^Lung growth in hypobaric hypoxia may not be proportional in all compartments of the lung(parenchyma growth vs conducting airway growth) and lung function may be affected.1.4. RATIONALEIn humans and animal species, acclimatization at high altitude is associated with structural andphysiological changes in the body in order to adapt to new environment. High altitude environmentis a complex set of variables. Since body metabolism is oxygen dependent, low ambient oxygen athigh altitude has been considered as a main stressor. Exposure to high altitude or hypoxic conditionsresults in somatic growth retardation which occurs due to undemutrition. Despite decreased somaticgrowth, positive allometric growth occurs in some organs such as lungs heart and spleen, and as aresult, specific weights (per unit body weight) of these organs show increases. In conditions whichsuppress somatic growth, interpretation of findings of specific results become rather complicated.Increase in specific parameters may occur either due to direct or indirect growth stimulation of someorgans, while body growth is normal, or due to diminished somatic growth, while organ growth is lessimpaired. Therefore, the effect of high altitude or hypoxic conditions may be under or overestimated.In addition, undernutrition equivalent to that which occurs in hypoxic conditions may produce changesin the structure of the lung and other organs and its effect may be variable in different organs. Hence,organ growth should ideally be assessed by comparing the experimental group with age and weight-matched cohorts.Chapter 1: INTRODUCTION 31The adaptive lung growth response at high altitude has been evaluated by exposing animalspecies in question to either normobaric hypoxia, hypobaric hypoxia or high altitude, but results werecontroversial. In some studies, the results of highlanders have been compared with lowlanders.Beside these differences in conditions, differences in the age of animals, duration of exposure, severityof conditions and different species of animals may be attributed to the controversies. In utero cellularproliferation is high in the lung, but following birth, it declines rapidly (199). In utero, normallyoccurring low oxygen tension may act as a potent stimulant for cellular multiplication. An abruptincrease in arterial oxygen tension occurs at the time of birth; spontaneous respiration may largely beresponsible for this change. Decreased oxygen at high altitude may be the leading stimulus of lunggrowth, but the effects of normobaric hypoxia (18) and hypobaric hypoxia (20) on lung growth appearto be different. This may suggest that low ambient pressure may play some role in the adaptive lunggrowth response by itself, or when delivered with hypoxia, which has not yet been examined.Previous studies regarding adaptive lung growth in normobaric hypoxia or hypobaric hypoxiahave been primarily limited to morphometric observations. Conclusions regarding alveolarmultiplication at high altitude, hypobaric hypoxia and normobaric hypoxia have been drawn based onindirect observations, rather than direct alveolar count. Little is known about biochemical aspects ofthe adaptive lung growth response to high altitude conditions and no information is available regardingquantitative changes connective tissue compartment of the lung. The sequence of events with regardto cytokinetics and cellular biochemical changes following exposure to hypobaric hypoxia, normobarichypoxia and hypobaric normoxia as well as in undernourished conditions have not been studied. Ifhyperplastic changes do occur in high altitude conditions, it is appropriate to assess cellular dynamicsof various lung cell populations that participate in the adaptive response. Due to geometrical location,lung structures in the central part of the lung may respond differently to the stimulus or the inhibitorin comparison to the peripheral part of the lung.Lung growth alteration in high altitude inhabitants has been assessed by lung functiontechniques, but the relevance of functional changes in the lung to biochemical or morphometricChapter 1: INTRODUCTION^32changes has not been studied. Therefore, once the biochemical and morphological changes havebeen determined, it will be appropriate to establish the relevance between structural and functionalchanges in normobaric hypoxia, hypobaric normoxia, hypobaric hypoxia and undernutrition byperforming lung function tests.The effect of hypoxic stress appears to be organ specific. However, the time course of organgrowth in hypoxia and the effect of undernutrition on various organs during hypoxia is not known. Anumber of mechanisms may play a part in ameliorating the effect of hypoxia. Lack of somatic growthmay reduce oxygen consumption and changes in pulmonary, cardiac and hematological parametersmay increase oxygen supply. Once the hypoxic stimulus is removed, structural changes may taketime to reverse. By contrast, changes at the cellular level may show an immediate effect. This alsoremains to be studied.Specific aims of present research are:oar^To determine the alterations in somatic and lung growth (extent and nature) adaptation tonormobaric hypoxia, hypobaric normoxia and hypobaric hypoxia in young rats usingbiochemical and morphometric techniques.Kir^To study the effects somatic growth retardation (equivalent to that occurring in normobarichypoxia and hypobaric hypoxia) on lung growth in normobaric hypoxia and hypobaric hypoxia.tar^To determine the onset and persistence of lung growth stimulation and biochemical alterationsin normobaric hypoxia, hypobaric normoxia, hypobaric hypoxia and undernourishment.air^To evaluate the dynamics of various cell populations involved in the lung growth adaptiveresponse in the central and the peripheral part of the lung in normobaric hypoxia, hypobaricnormoxia, hypobaric hypoxia and undernutrition.Bar^To determine if lung growth in normobaric hypoxia, hypobaric normoxia, hypobaric hypoxiaand undernutrition is normal or dysanaptic (disproportionate growth of lung parenchymacompared to conducting airways) by performing lung function tests.Chapter 1: INTRODUCTION^33oar^To examine the effect of hypobaric hypoxia, normobaric hypoxia, hypobaric normoxia andundernutrition on the growth of various organs.sr^To study the effect of recovery on lung growth after returning hypobaric hypoxic, normobarichypoxic and hypobaric normoxic rats to room air, and providing food ad libitum toundernourished rats.Chapter 2: MATERIALS AND METHODS^34CHAPTER 2MATERIALS AND METHODS2.1. PROJECT DESIGNThe study consisted of three sections:I. Lung growth adaptationII. Organ response and lung cytokineticsIII. Lung adaptation: physiological aspects2.1.1. MATERIALS2.1.2. CHEMICALSAll the reagents used in this study were of analytical grade and were obtained from FisherScientific Limited (Fairlawn, New Jersey, USA), Sigma Chemical Company (St. Louis, Miss., USA) orJ.T. Baker Chemical Company (New Jersey, USA), unless otherwise mentioned. Elastin anddesmosine standards were purchased from Elastin Products Limited (Pacific, Miss., USA). IodinatedBolton Hunter Reagent, which was used to label the desmosine for desmosine radioimmunoassay,was bought from New England Nuclear Laboratories (Boston, Massachussetts, USA). The desmosineantibody raised in rabbits for the use in radioimmunoassay was received as a gift from Dr. Shiv Y. Yu,then at Veterans Administration Hospital, St. Louis, Miss., USA. Tritiated thymidine was purchasedfrom Amersham Canada Limited (Oakville, Ontario, Canada) and autoradiography emulsion (NBT 2)Chapter 2: MATERIALS AND METHODS^35was received from Kodak (Toronto, Ontario).2.1.3. ANIMALSOne hundred, three week old male Sprague-Dawley rats were obtained from Charles RiverBreeding Laboratories (Guelph, Ontario, Canada) separated by litters. They were housed in theAcademic Pathology Animal Care Unit, UBC, and were given free access to standard Purina R rat chowand water. They were allowed to adapt to the laboratory environment for one week until used at fourweeks of age.2.2. LUNG GROWTH ADAPTATION2.2.1. EXPERIMENTAL DESIGNFour week old male, litter and body weight-matched Sprague-Dawley rats were randomlydivided into six groups, each consisting of 12 animals.* Group 1. Baseline Controls (BC) (4 weeks old, sacrificed at the beginning of theexperiment)* Group 2. General Controls (GC) Exposure: Sea level, room air, food and water ad libitum * Group 3. Hypobaric Normoxic (HBNO) Exposure: Ambient pressure 410 mm Hg with enriched oxygen to correspondthe fraction of oxygen (Fo2) to 0.21 at sea level, food and water ad libitum * Group 4. Normobaric Hypoxic (NBHY) Exposure: At sea level, air mixed with nitrogen to decrease Fo 2 to 0.11, foodand water ad libitum Chapter 2: MATERIALS AND METHODS^36* Group 5. Hypobaric Hypoxic (HBHY) Exposure: Ambient pressure 410 mm Hg with Fo2 equivalent to 0.11 at sealevel, food and water ad libitum * Group 6. Weight-matched Controls (WMC) Exposure: Sea level, room air, restricted food to approximate the body weightto those of the hypobaric hypoxic group, water ad libitum 2.2.2. EXPOSURE TO ALTERED AMBIENT CONDITIONS2.2.2.1. Exposure chambersFour hypobaric chambers, each with a capacity of 3 cages, were designed (AcademicPathology Workshop, UBC) from polyvinyl chloride cylinders. Each of the hypobaric chambers wasequipped with an air intake for individual cages, a pressure gauge, a temperature and hygroscopicsensor hygrometer unit (Airguide, Chicago, Illinois) and an on-line port for carbon dioxide and oxygentension measurements. Similarly, the normobaric chambers were also partitioned into compartmentsfor every cage with a separate air inlet and outlet. The air inflow was adjusted to achieve 12-15 airchanges per hour. An on-line vacuum was used for hypobaric conditions (hypobaric hypoxia andhypobaric normoxia). Exposure procedureThe baseline group of rats was sacrificed at the beginning of the experiment at 4 weeks of ageto obtain baseline somatic and lung parameters. Groups 2-6 were exposed to respective ambientconditions and all rats were kept in individual cages. All animals were fed standard PurinaR rat chow.The general control rats (GC) were housed in continuously air flushed chambers and were given foodand water ad libitum. The chambers of the hypobaric normoxic (HBNO) group were connected to theon-line vacuum source to reduce the ambient pressure to 410 mm Hg. Simultaneously the air inflowinto the HBNO chambers was supplemented with 100% oxygen to correspond Fo2 to 0.21 at sea levelChapter 2: MATERIALS AND METHODS 37which was identical to that of the general control group. The oxygen tension was monitored with apolarographic oxygen analyzer (Hudson Vantronics, Temecula, CA, USA), pre-calibrated to the roomenvironment as 0.21 fraction of oxygen. The HBNO rats were given free access to food and water.For normobaric hypoxic (NBHY) animals, the chambers were flushed with a mixture of air and nitrogento obtain 0.11 Fo2. In the case of hypobaric hypoxic (HBHY) rats, the ambient pressure wasdecreased to 410 mm Hg which decreased Fo2 equivalent to 0.11 at sea level. Except for the weight-matched control group, all animals were given food and water ad libitum. The weight-matched groupof rats were pair-matched for body weight to the hypobaric hypoxic group. They were allowedrestricted food access but free access to water. On the first day, they were given 50% of the foodconsumed by them one day before the experiment was commenced. Later the weight-matchedanimals were given approximately the amount of food consumed by the hypobaric hypoxic rats theprevious day, but depending upon their body weight gain some adjustments in the amount of foodgiven were required.All groups of rats were exposed to a 12 hour light/dark cycle. Temperature and relativehumidity were monitored continuously. The gas flow through the chambers was adequate to maintainthe ambient temperature at 22 +1° C and relative humidity at 60 +5 %. No condensation of waterinside the chambers was noticed. Because water was placed in the chambers in open containers,calcium chloride was also placed in the chambers to control the humidity as a precautionary measure.02 and CO2 concentrations were measured 3 times a day. The CO2 concentration was controlled byplacing soda lime inside the chambers. Using a Fryrite carbon dioxide analyzer (BacharachInstrument Co., Pittsburgh, PA., USA) CO2 did not increase to a detectable level of 0.5%. Thechambers were opened daily for 15-20 minutes to change the shavings and to replenish food andwater. The food intake and body weight of each rat was measured daily. Animal terminationFollowing 3 weeks of continuous exposure to respective ambient conditions, the rats wereChapter 2: MATERIALS AND METHODS 38anaesthetized by intraperitoneal injections of sodium pentobarbital (5 mg/100 g body weight). Oxygenabsorption atelectasis has been shown to be a more effective procedure to remove trapped lung gasthan vacuum degassing the lung (248). The rats were given 100% oxygen to breathe for 3-5 minutesto remove gases from the lungs for pressure-volume measurements [as 02 is readily absorbed givingthe atelectic lungs a liver-like appearance (79)]. The abdomen was opened and the diaphragm wasincised to cause pneumothorax and lung collapse. The thoracic cavity was opened and a sample ofblood was taken in a capillary tube from the right ventricle of the heart for hematocrit measurement.Immediately, the trachea was clamped and cannulated. The rats were sacrificed by exsanguination,severing the abdominal aorta to reduce the contribution of pulmonary blood volume to lung weight andbiochemical measurements. Lungs, heart and extrapulmonary tissue were dissected from the thoraciccavity.2.2.3. PRESSURE-VOLUME CURVESThe lungs and heart en bloc preparation was placed in a plethysmograph with a heat sinkdesigned for lungs. The cannulated trachea was attached to a connector in the lung chamber,connected to a pre-calibrated pressure-volume curve plotting equipment (Validyne, Model MC; HewlettPackard, Model 7041 A X-Y recorder). The tracheal clamp was then removed. When the pressurechanges within the lung chamber stabilized, the lungs were inflated in a step-wise fashion with amotor-driven air syringe to a transpulmonary pressure of 25 cm water and then deflated to atranspulmonary pressure of 0 cm water. During this maneuver, if the pressure did not stay constantat 25 cm of water after inflation for 15-20 seconds, air leakage was assumed and the lungs were notincluded for analysis. Following the initial hysteresis, two pressure-volume curves were recorded foreach animal and the deflation limb of the second curve was used for lung elastic recoil analysis.Maximal lung volume was defined as the amount of air in the lung at a transpulmonary pressure of25 cm of water. Recoil pressure was calculated from the pressure-volume plots at 10 percentiles ofmaximal lung volume. According to the method of Colebatch and associates (68), all the data pointsChapter 2: MATERIALS AND METHODS^39over 30% of maximal lung volume were analyzed by fitting a single exponential to the pressure-volumecurve with the help of a digital computer. The single exponential expression was:V = Vmax -Bewhere V is the volume at pressure P. Vmax is the theoretical volume of air at infinite transpulmonarypressure, B the difference between V, the intercept on the volume axis and Kis a constant thatdescribes the shape of the curve. In this equation V max is unknown, therefore, it was necessary torepeat the computations using variable values of V max. Since it is well known that the slope of thepressure-volume curve is near zero in the region of Vmax (206), the starting value for Vmax wasassumed to be close to the observed maximum lung volume. The value V max was then increasedpositively or negatively until r2 no longer increased with the tenth of the unit change of V max. Thenthe change of V max was further reduced to the 1/100th and iterative computations were performeduntil the best fit of the curve was achieved. The best fit was determined by no further increase in thevalue of r2. The quality of the fit of the function is assessed from the r2 value, which represents theproportion of total variance of points attributable to the mathematical regression (206). The r2 valuein the present study was >0.98 in all cases.2.2.4. BIOCHEMISTRYFollowing pressure-volume curve maneuvers, the extrapulmonary tissue was removed. The rightlung was ligated and separated from the left lung. Our preliminary studies indicated that the timelapse during pressure-volume procedure did not affect DNA, RNA and protein estimations of the lungs.Lungs were washed with phosphate buffered saline to remove blood. After blotting, the wet lungweights of the right lung and the cannulated left lung (after taring the balance with a cannula and aportion of trachea) were recorded. The right lungs were frozen in liquid nitrogen and stored at -70°C until used. When required, the frozen lung samples were lyophilised (VirTis Preservator model 10-pr, Freezermobile 12 Freezedryer) until constant weights were attained and then the dry lung weightswere measured. The lung samples were thinly sliced, rehydrated in 2 ml phosphate-buffered salineLung Homogenate 1I homogenized In 4 ml PBSChapter 2: MATERIALS AND METHODS^40Freeze Dried Lung^IPrecipitation with 50% TCAVortex : keep @4 C; 1 hr.centrifuge @ 2000 rpm, 10 min.I SupernatantDiscardI^PrecipitateEthanol Extractionwith sodium acetatesaturated ethanol SupernatantDiscardDigestion with 1M NaOH@37 C. 3hrs.IPROTEIN(Lowry's method) 14^IAcidification with 0.3 M PCAVortex, keep @ 4C , 45min.centrifuge @ 2000 rpm, 10 min.I 1^ 1Supernatant Precipitatelif^ IDigestion with 1M PCARNA @95 C, 40 mins.centrifuge @ 2000 rpm, 10 min.ISupernatant1 DNABurton's Diphenylamine methodFigure 2. Flow chart for extraction of protein, RNA and DNA.Chapter 2: MATERIALS AND METHODS 41(PBS), and stored in a refrigerator overnight. The samples were homogenized with a homogenizer(Caframo, Wiarton, Ontario) and the final volumes were made up to 4 ml with phosphate bufferedsaline (PBS). One ml of the final lung homogenates were used for extraction of DNA, RNA and alkalisoluble protein, and 3 ml were used for extraction and estimation of hydroxyproline and desmosine. DNA, RNA AND PROTEIN EXTRACTIONThe procedure of Schmidt-Thanhauser (236) as modified by Wannemacher (272) was used forextraction of DNA, RNA and alkali soluble protein (Figure 2). In order to precipitate themacromolecules, one ml of the sample homogenate was mixed with 50% trichloroacetic acid (TCA)to achieve the final concentration of 15% of TCA. The TCA precipitated lung samples were vortexedand placed in a refrigerator at 4° C for one hour. The lung samples were then centrifuged (Beckman,model J-68) at 2000 rpm at 4° C for 10 minutes. The supernatants were discarded. The TCAprecipitates were then washed 3 times with sodium acetate saturated ethanol to remove lipids. Twoml of 1M sodium hydroxide (NaOH) was added to each of the residues and were placed in a 37° Cwater bath for one hour. After centrifugation at 2000 rpm for 10 minutes, the supernatants were thendecanted into clean tubes and digestion of the residues was repeated with 2 ml of NaOH. Thesupernatants of the first and second digestion of samples were pooled. One ml aliquots of pooledsupernatants were taken for protein estimation. Cold (4° C) 6 ml perchloric acid (PCA) was addedto the remainder of the NaOH digest, to give a final concentration of 0.3M PCA. The samples werevortexed and kept at 4° C for 45 minutes. The PCA treated samples were then vortexed andcentrifuged at 2000 rpm for 10 minutes and the supernatants were then decanted into clean tubes andwere used for RNA estimation. 1 ml of PCA was added to the residues and digested at 90° C for 20minutes. The digests were then cooled and centrifuged. The supernatants were removed and theresidues were digested again. After pooling the supernatants from the first and second digests, analiquot of each sample was used to determine the total amount of lung DNA and assessment of DNAsynthesis.Chapter 2: MATERIALS AND METHODS^422. Alkali soluble protein estimation Alkali soluble protein content was measured by the method of Lowry and coworkers (174).Bovine serum albumin (BSA, Sigma A-4378, fraction V) dissolved in 0.1M NaOH was used as astandard stock solution (500 pg BSA/m1). The stock solution was divided into aliquots and stored at -70° C until required for an assay. For the assay, one aliquot was thawed and pipetted in duplicatesin assay tubes to obtain a standard curve ranging from 10-200 lig of BSA. From the diluted samples150 ill aliquots were taken and total volumes of standards and samples were made to 500 111. Fiveml of freshly prepared Lowry's solution (2% sodium carbonate in 0.1M NaOH solution, 1% coppersulphate solution, 2% sodium tartrate solution and all mixed in 100:1:1 ratio) was added to the tubes,vortexed and allowed to stand at room temperature for 15-20 minutes. A half milliliter of freshlyprepared 1M Folin's phenol reagent (2N Folin-Coicalteau phenol reagent solution diluted with distilledwater and 1M NaOH solution to obtain final pH of 1.8) was added to each assay tube, vortexed andleft at room temperature for half an hour. The absorbance was read at 660 nm in aspectrophotometer (Philips Pye Unicam SP6-550 UV/Vis). The absorbance reading of all lungsamples were within the linear range of the standard curve. By approximating the absorbance on thestandard curve, protein content per sample was calculated.Protein/lung (mg) = Protein 1.1q from the standard curve X DF (dilution factor) 1000Dilution factor (DF)= Total volume of the aliquot after dilution X Total volume of NaOH soluble supernatantFraction of sample taken^Volume of aliquot taken2. Estimation of DNA DNA content estimation was done according to the method described by Burton (43) using calfthymus DNA (Sigma D-1501) as standard. A standard stock solution (0.4mg/m1) was prepared,divided into 1 ml aliquots and stored in -70° C. An aliquot was thawed, diluted 1:1 with 1.7M PCA andincubated at 90° C for 10 minutes. After cooling, aliquots were pipetted in duplicate to obtainChapter 2: MATERIALS AND METHODS^43standards containing 16, 32, 48, 64, 80, 100 and 120 jig of DNA. All standards and sample volumeswere made up to 1 ml with 1.7M PCA. Two milliliters of freshly prepared diphenylamine reagent (1g diphenylamine, 100 ml of glacial acetic acid, 2.73 ml concentrated sulphuric acid) were added to thesamples and standards and then vortexed. The samples and standards were then incubated in awater bath at 90° C for 10 minutes. After cooling, the absorbance of the mixture was read at 600 nmusing a spectrophotometer (Philips Pye Unicam SP6-550 UV/VIS). The standard curve used for DNAestimation was linear and all the samples fell within the limits of the standard curve. The DNA contentper lung was calculated as follows:DNA /lung (mg) = tilq DNA calculated from standard curve X DF 1000Dilution factor (DF) = Total volume of supernatant after PCA incubation XVolume of fraction taken for DNA assayTotal volume of NaOH supernatant X 4 Volume taken for DNA precipitation X Estimation of RNA The amount of RNA was measured by using a method of Wannemacher (272). Calf liver (typeIV) ribonucleic acid (RNA, Sigma R-7250) was dissolved in distilled water to prepare a stock solution(400 jig RNA/ml) and stored at -70° C until required for estimation. One ml of stock solution wasthawed and diluted to 10 ml with distilled water to obtain a final concentration of 40 jig/ml for assay.Standards were pipetted in duplicate concentrations with a range of 5, 10, 15, 20, 25 and 30 jig ofRNA. Distilled water was added to 500 pl of the diluted samples and standard duplicates to make finalvolume up to 5 ml. The light absorbance of standards and samples was read in a spectrophotometer(Philips Pye Unicam SP6-550 UVNis) at 260 nm. The absorbance of standards and samples wasalso read at 280 nm to correct for protein interference. The absorbance reading of standards waslinear and all sample readings fell within the range of the standards. By matching the absorbance onthe standard curve, the RNA content per sample was calculated as follows:RNA /lung (mg) = Lip RNA calculated from standard curve X DF Chapter 2: MATERIALS AND METHODS^441000Dilution factor (DF) = Total volume of diluted supernatant after PCA precipitation (RI) X500Total volume of NaOH supernatant X 4 Volume taken for DNA precipitation X PROCEDURE FOR HYDROXYPROLINE AND DESMOSINE EXTRACTIONHydroxyproline and desmosine were extracted by using the method of Laurent and associates(161), in which cyanogen bromide essentially solubilized all the lung collagen and left behind aninsoluble residue with amino-acid content similar to elastin (Figure 3). Three milliliters of lunghomogenate were centrifuged at 5000 rpm for 10 minutes and the supernatants retained. Theresidues were washed twice with 1.5 ml of phosphate buffered saline and centrifuged. The washingwas repeated twice with 1.5 ml of 2% sodium dodecyl sulphate (SDS) and centrifuged. Finally, theresidues were washed again with PBS in order to remove excess SDS. All the supernatants werethen pooled and kept for the soluble fraction of hydroxyproline estimation. The residues were thenwashed with 2 ml acetone and left overnight in the fume hood to dry. The residues were againhomogenized in 2 ml of 70% (w/v) formic acid. 0.5 ml of a 0.1 g/ml solution of cyanogen bromidein 70% formic acid was added to the residues to achieve a final cyanogen bromide concentration of20 mg/ml. The solutions were vortexed and nitrogen gas was bubbled through the samples for 15seconds. The tubes were then sealed and incubated in a shaker water bath at 37° C and the reactionwas allowed to take place for 24 hours. The resulting digests were centrifuged at 5000 rpm for 20minutes. The supernatants were decanted into 50 ml wide mouthed tubes and the residues werewashed 4 times with 4 ml of distilled water. All supernatants were pooled in the 50 ml tubes and thefinal volumes were made up to 30 ml with distilled water. Both the residues and supernatants werefrozen at -70° C and lyophilized till constant weights were achieved. Estimation of hydroxyproline (i). Sample preparation for hydroxyproline quantificationChapter 2: MATERIALS AND METHODS 45The samples recovered from the SDS/PBS extraction represented soluble collagen andcyanogen bromide soluble samples represented insoluble cross-linked collagen. In order to releasehydroxyproline from peptide linkages, both soluble and insoluble collagen samples were hydrolysedin 6N hydrochloric acid. Hydrolysis was performed at 110° C for 24 hours. After hydrolysis, two dropsof 0.02% methyl red indicator were added to the hydrolysates and 2.5M NaOH was added slowly andvortexed until a faint straw color was obtained. The hydrolysates were then filtered with Whatman #1filter paper and the total volume was made up to 15 ml with distilled water.(ii). Hydroxyproline assay procedureHydroxyproline content was measured in the hydrolysates by using the method of Woessner(285). Series of standards containing 0, 2, 4, 6, 8 and 101.19 of hydroxyproline and a known volumeof sample hydrolysates were added to the assay tubes. The total volume of standards and samplehydrolysates were made to 2 ml with distilled water. One ml of freshly prepared chloramine-T solutionwas added to each tube , vortexed and allowed to stand at room temperature for 20 minutes. In orderto eliminate excess of chloramine-T and to stop the oxidizing reaction, one ml of 3.15M PCA wasadded to the tube in the same order. The assay tubes were then vortexed and let stand sit foranother 5 minutes. Finally, one ml of Ehrlich's reagent (20 g of p-dimethylamine benzaldehyde in 100ml of n-propanol) was added, vortexed and incubated for 20 minutes in a water bath at 60° C. Theassay tubes were cooled in tap water and the absorbance of the samples were read in aspectrophotometer at 561 nm. The standard curve prepared was linear to 6 jig hydroxyproline, andthe absorbance of the lung samples were all within the linear range of the curve. The hydroxyprolinecontents of the lung samples were estimated directly from the standard curve.Hydroxyproline content of soluble and insoluble collagen fractions was estimated separately andthe sum of both was referred to as the total hydroxyproline content present in the lung. The percentof soluble or insoluble content was also calculated. Laurent and coworkers (161) have indicated thatlung collagen contains 12.2% (w/w) hydroxyproline based on amino acid analysis of collagenPrecipitateTCA precipitation^ 2% SOS washes x 2centrifuge @ 5000 rpm, 10 min.Supernatant^Precipitate(discarded)I SupernatantSOLUBLE PROTEINS(for HyPro Assay)Chapter 2: MATERIALS AND METHODS^46I^Freeze Dried Lung^Ihomogenized in 4 ml PBSI Lung Homogenate'centrifuge @ 5000 rpm, 10 min.homogenize, 70% formic acidCNBr Digestion0.5m1CNBr so1n_ (0.1g/m1 formic acid)0 37 C. 24 hrs.jrCNBr Digestcentrifuge0 5000 rpm. 15 min.Suplriatant^PrecipitateCOLLAGEN^f ^ELASTINFigure 3. Flow chart for extraction of collagen (soluble and insoluble fractions) and elastin.Chapter 2: MATERIALS AND METHODS^47standards and lung tissue collagen extracted in cyanogen bromide. We did not convert ourhydroxyproline content into collagen content. Estimation of desmosine Desmosine is unique to elastin. Measurement of the elastin content in the lung was done bymeasuring the desmosine by radioimmunoassay procedure. Peptide bound desmosine does not reactwith the antibody, therefore, samples must first be hydrolysed in 6N hydrochloric acid in order torelease the bound desmosine.(i). Desmosine preparation from lung samplesTwo ml of 6N hydrochloric acid was added to the cyanogen bromide treated residues and werehydrolysed at 110° C for 48 hours to liberate desmosine. Following hydrolysis of the samples, aliquotsof 200 gl were taken, diluted 10 times and were lyophilized to dryness. The dried samples were thenreconstituted in 1 ml of pH 7.6 potassium-phosphate buffer. The final Ph of the solution rangedbetween Ph 7.2-7.4. These solutions were further diluted to required dilutions so that they fell on thelinear part of the desmosine radioimmunoassay curve. One hundred gl aliquots of these dilutedsamples were used for measurements of desmosine by radioimmunoassay.(ii). Desmosine radioimmunoassayA. Preparation of 1251-labelled desmosine (Conjugation of desmosine with iodinated Bolton HunterReagent) The labelled desmosine molecule which is used for the quantitation of desmosine samples,initially requires conjugation with radioactive iodinated reagent. The Bolton Hunter reagent (125I-BHR)is unstable if left unused for more then 48 hours, therefore, the conjugation procedure needs to beconducted immediately. Once the conjugation is done, the labelled product is stable for 4-6 weeksChapter 2: MATERIALS AND METHODS^48at 4° C.The reaction procedure was performed in a well ventilated fume hood in accordance with theradiation safety measures. The di-iodo (1251) Bolton Hunter Reagent {N-succinidy1-3 (4 hydroxy, 3, 5[1251] diiodo-phenyl) proprionatel, total activity of 0.5 mCi (Specific activity 4400 Ci/mmol) waspurchased in two 2-250 uCi combi-V-vials in 250 gl anhydrous benzene, from New England Nuclear(NEN) Research Products, Boston, MA., USA. Both combi-V-vials were placed on ice and twohypodermic needles were inserted in through the rubber stopper. One needle was connected to thecharcoal trap for any radioactive material which might escape during the evaporation procedure andthe other needle was connected to a dry nitrogen gas supply. The benzene was carefully evaporatedto dryness with a very gentle stream of nitrogen. Then 30 pi of 1 mg/ml desmosine (Elastin Products,Pacific, Miss., USA) solution was added to each Bolton Hunter reagent combi-V-vial. The reactionmixture was then transferred to the cold room and was left overnight on a gentle shaking platform withproper shielding. Next day, the conjugation reaction was stopped by adding 0.5 ml of 0.2M glycinesolution. The reaction mixture was then transferred to the top of a 100 cm long Bio-Gel P2 (200-400mesh) (Bio-Rad laboratories, Richmond, CA) packed column (which was packed the previous day) andwas eluted with 0.1M acetic acid. One hundred 3 ml fractions were collected in siliconized vials. Twoill of solution from each vial was removed and placed in marked polypropylene (12 mm) tubes, andradioactivity was measured using a LKB gamma counter. Once the peak with the desmosineconjugates was determined, all the fractions from the peak were pooled and divided into 0.5 mlfractions and stored at 4° C. For each assay one aliquot was taken and diluted 12 times to get a totalradioactivity in 50 pi that ranged between 20,000 and 30,000 cpm.B. Desmosine standards preparation The standard desmosine solution of 11.4.g/nnl concentration was prepared in distilled water andone ml aliquots were then stored at -70° C. For each assay, one tube was thawed and serial dilutionswere made of 500, 250, 125, 62.5, 31.25, 15.62, 7.81 and 3.9 ng/ml with 7.2 pH potassium-phosphateChapter 2: MATERIALS AND METHODS 49buffer. Internal standard preparations from rat lung elastin hydrolysates, desmosine recovery samplesand control lung samples were used following every 10 samples to assess variations and precisionwithin and between the assays.C. Desmosine assay procedure Desmosine in the lung tissue hydrolysates was assayed by the method of Harel and associates(128) with some modifications. One hundred ptl of the lung hydrolysates, serially diluted standards(ranging from 3.9-500 ng/m1), internal elastin standards, lung controls and desmosine recoverysamples were added to the assay tubes. They were diluted with 100 gl 7.2 pH phosphate buffer toget a total volume of 200 j.tl. The solution was mixed with 100 gl of anti-desmosine rabbit antibody(diluted 1:100 with 7.2 phosphate buffer). The mixture was gently vortexed and then incubated at 30°C for 30 minutes (this allows for the antigen-antibody complexes to form between desmosine from thelung samples and the anti-desmosine antibody). Fifty l of the radioactive desmosine (labelled with125I-BHR (containing 20,000-30,000 cpnn in 50 RI of phosphate buffer solution) was then added to theassay tubes. The assay tubes were vortexed and then further incubated at 30° C for one hour. Thisallows the competitive binding of the radioactive desmosine to the desnnosine antibody in the reactionmixture. Therefore the reaction mixture at the end of second incubation contains Ab-Des complexes,Ab-Des(1251) complexes, free desmosine 1251 and free desmosine. Fifty IA of a 10% (w/v) solution ofpansorbin (Staphylococcus aureus cells, binding: 2.2 mg of human IgGiml of cell suspension) wasadded to the assay tubes, vortexed and incubated at 30° C for one hour [Pansorbin cells adsorb onthe surface of the antibody which is already bound to the antigen {Des or Des(125I)} and precipitatesthe Ag-Ab complexes while the free desmosine and radioactive desmosine remains in thesupernatant]. The tubes were then centrifuged at 4° C for 30 minutes at 5000 rpm. The supernatantswere carefully aspirated. The residues were washed with 0.5 ml of 7.2 pH phosphate buffer andradioactivity of the precipitates was counted in an LKB gamma counter. All the assays were carriedout in duplicate. From the results obtained, a standard curve was plotted with the assistance of aChapter 2: MATERIALS AND METHODS^50computer and the average of the duplicate cpm (counts per minute) was automatically read from thestandard curve. The sample dilutions were made such that all the readings fell within the linear partof the standard curve. Three assays were done for each sample and the average of all threeobservations was used for analysis.The sensitivity of the desmosine radioimmuno assay was optimal and linear between 15.62 to250 ng. Isodesmosine was only weakly cross-reactive (<0.1%). In order to check the non-specificinterference from the other proteins, varying amounts of hydrolysates of collagen and bovine serumalbumin were added to a measured amount of desmosine. There was no significant interference inthe assay by any of the hydrolysates up to 200 ig in concentration. The dilution of the lunghydrolysates used for the assay had desnnosine concentrations of less than 100 p.g.2.2.5. MORPHOMETRY2.2.5.1. Lung fixation and lung volume determinationAfter plotting the pressure-volume curves, the left lung with the cannulated trachea wassubmerged and distended gently with a 5 cc syringe using 10% neutral buffered formaldehyde. Thecannula was then connected to the instillation system for 72 hours at a constant transpulmonarypressure of 25 cm of water. After fixation, the trachea was clamped (to prevent fixative leakage) anddetached from the instillation system. The hilum of the lung was ligated and the trachea wasremoved. A beaker full of water was placed on a balance and the lung was suspended andsubmerged completely in the water without touching the sides or the bottom of the beaker. The lungvolume (cm3) due to water displacement was estimated as equal to the weight in grams as describedby Scherle (235). Archimedes principle states:"A body partially or totally submerged in a body of fluid experiences a buoyant force (FB)equal to the weight of the fluid displaced by the volume of that body".Chapter 2: MATERIALS AND METHODS^51Weight of fluid displaced = Buoyant force (FB)Volume of lung (VI) = ^FB Specific gravity of fluid2.2.5.2. Lung sampling and tissue processingTwo mid-sagittal blocks, one from cephalic and the other from the caudal end of each left lungwere cut. The edges of the blocks were trimmed at right angles for accurate digitization which wasused for tissue shrinkage assessment that occurred during tissue processing and embedding. Theblocks were then photographed and contact prints were made from the negatives. The blocks wereembedded in paraffin. Two 5 micron sections were cut from each block and stained with hematoxylinand eosin for light microscopic morphometric measurements. Assessment of tissue shrinkageThe areas of the blocks were measured by a computer assisted digitizer (Apple Ile, BioquantII, R. & M. Biometrics) from the contact prints of the blocks before tissue processing and embedding(pre-processed). After tissue processing and embedding (post-processed), the area of the stainedsection was also digitized in a similar fashion. The shrinkage factor was determined by dividing thearea of the tissue sections (post-processed) by the area of the pre-processed lung tissue. Areashrinkage and linear shrinkage factors were calculated as follows:Area shrinkage factor (asf) =Linear shrinkage factor (1st) =Area of post-processed tissue Area of pre-processed tissueAus_f_Eoprocessed tissue V Area of pre-processed tissueNo corrections were made for the thickness of the sections. Morphometric measurementsAll the slides were coded to eliminate bias in morphometric measurements. A microscope(Wild, M501, Heerbrugg, Switzerland) equipped with an automated stage and an overhead screenChapter 2: MATERIALS AND METHODS^52Figure 4. Illustrations of (a) the test grid used for light microscopic measurements and (b) theintercept counting technique.Chapter 2: MATERIALS AND METHODS^53fitted with a square grid (a 42 equidistant test points and two diagonally placed cross hair test lines)was used for morphometry (Figure 4a).Using a X40 objective, the area and the total length of the two diagonally placed cross hair testlines was determined to be 0.0625 mm2 and 0.64 mm respectively. Leaving a 1 mm distance alongthe margins, the rest of the area was used to choose 20 predetermined equidistant fields on a 4 X 5matrix. Each of the 42 test points were counted according to their placement on the histologicalstructures, i.e. alveolar air, alveolar duct air, alveolar wall, bronchial air and non-parenchymatoustissue. The smallest discrete structures surrounded by alveolar walls were considered as alveoli.Alveolar duct air was considered to be the cylindrical core of air within the alveolar duct and sacsinternal to the mouth of alveoli. Conducting airway air was referred as bronchial air.The counting principle used in measuring random sections was based on Delesse principle (86)which states:"Area proportions are equivalent to volumetric proportions" and "the planimetric fractions ofa section occupied by sections of a given component correspond to the fraction of the tissuevolume occupied by this component".Thus, number of test points for each of the tissue components were expressed as a fraction of thenumber of test points. This represents volume fraction of the tissue structure.Vv, = Number of test points falling on a structure x 42 or total number of test pointsIntercepts were counted either as wall intercepts (1w) or duct intercepts (Id) and the sum of the two(1w+Id) represented total number of intercepts (la). If the wall of the alveoli fell across the test line,two intercepts were counted (2 air exchanging surfaces), but if the alveolar wall touched the upper orthe right side of the test lines or the end of the test line lodged in the wall of the alveolus or in anyother tissue structure, it was counted as one intercept (Figure 4b). For the duct intercept (Id), aninterception of the test line by an imaginary line across the mouth of the alveolus was counted as twointercepts. The total number of intercepts per case was used to calculate the mean linear interceptChapter 2: MATERIALS AND METHODS^54Table 2. Morphonnetric calculations.Parameters^CalculationsAlveolar wall intercept^ =1wDuct mouth intercept = IdTotal intercepts (la) = lw+IdVolume fraction of Alveolar air^ = VValvAlveolar duct air = VVductAlveolar wall = VVwallBronchial air^ = VVbrNon-parenchyma = \NapNumber of alveoli in a field^ =NTotal length of the test line(corrected for linear shrinkage factor {Isf})^= LATArea of the grid (corrected for shrinkage fast)) = AMean linear intercept (MLI)^ =L1lwMean chord length of alveoli (laiv)^ = 2LT X VvpivlaAlveolar surface area (Sw)^ = 4 X VMLIAlveoli per unit area (NA)^ =NANumber of alveoli per unit volume (Nv)^ = J X /NA3 B X iVvalvDistribution constant (J)^ = 1Shape constant (B) = 1.55Total number of alveoli (Nat) = Nv X VTAverage alveolar volume (Valvave)^ = VT X VVah,Natas follows:Nfi * LT * 2Mean linear intercept (MLI) =^11(w or a)where Nfi is number of fields on slide i, LT is total length of test line after correcting for linear shrinkageduring tissue processing, 2 is used because every wall or duct intercept was counted as 2 intercepts,Ii is total of intercepts on slide i (w is alveolar intercept and a is sum of alveolar wall and alveolar ductChapter 2: MATERIALS AND METHODS^55Figure 5. Illustration of a direct alveolar count.Chapter 2: MATERIALS AND METHODS^56intercept.From these morphometric values, the mean linear intercept of airspaces or average interalveolarwall distance (MLI), mean chord length of alveoli (1 )sand alveolar surface area (Sw) were calculated.The total length of the test line on the grid was corrected for tissue shrinkage in order to project ontothe fixed tissue. The mean linear intercepts were calculated by dividing the total projected length oftest lines by the number of wall intercepts (Table 2).The alveoli that lay within the grid and on the upper left and right sides of the grid were counted(Figure 5). The known area (A) of the grid was corrected for tissue shrinkage during processing. Thecounted number of alveoli were used to calculate the number of alveoli per unit area (NA), number ofalveoli per unit volume (Nv), total number of alveoli (Nat) and average volume of an alveolus (Valvave).The distribution constant of the characteristic linear dimension of alveoli (J) was considered as oneand shape constant (B) of the alveolus was taken as 1.55 (278).2.3. ORGAN RESPONSE AND LUNG CYTOKINETICS2.3.1. EXPERIMENTAL DESIGNA total of 240 male Sprague-Dawley rats were used in this study. At 4 weeks of age, body weightand litter-matched rats were randomly divided into 5 groups:* Group 1.^General Control Group (GC)* Group 2.^Hypobaric Normoxic Group (HBNO)* Group 3.^Normobaric Hypoxic Group (NBHY)* Group 4.^Hypobaric Hypoxic Group (HBHY)* Group 5.^Weight-matched Control Group (WMC)Chapter 2: MATERIALS AND METHODS 57The rats were exposed to ambient conditions as detailed earlier. Six rats from each group 1-5were sacrificed on days 1, 3, 5, 7, 10, 14 and 21 of exposure. Six rats in each of the groups 2-4 werethen returned to room air for three days, and group 5 was given free access to food and water after21 days for the subsequent 3 days. This was done to study the effect of post-exposure and refeeding.On days 1, 3, 5, 7, 10, 14 and 21 of exposure, and day 3 of post-exposure, the rats wereanaesthetized with halothane. A 5 mm long skin incision was made at the mid groin and then femoralvein was carefully exposed by excising the femoral sheath. Using a 300 needle, 21..LCi/g body weightof tritiated thymidine (3H-TdR) (specific activity 23 mCi/mmol) was injected very slowly (a fast injectionappeared to be lethal to the rats). Pressure was applied at the injection site for approximately twominutes, which was found to be adequate to stop any further bleeding and no attempt was made tosuture the wound. Termination of ratsOne hour after intravenous injections of 3H-TdR, injections of sodium pentobarbital (50 mg/Kg bodyweight) were given intraperitoneally to anaesthetize the animals. Body weight and nose-tail lengthwere recorded. The rats were terminated by exsanguination from the abdominal aorta. The spleen,kidneys and liver were removed, washed, blot dried and weighed. The spleen, the right kidney anda part of the ileum (positive controls for autoradiography) were fixed with Karnofsky's solution andstored for future use. The thoracic cage was opened and the trachea was cannulated. The right lungwas clamped and ligated at the hilum and removed. After weighing of the right lung, liver and leftkidney, they were immediately frozen in liquid nitrogen and stored at -70° C until further use. Heart and lung preparationThe cannulated trachea was then connected to the lung instillation system in situ. The left lungwas distended with Karnofsky's solution (150) containing 4% glutaraldehyde at 12 cmH20transpulmonary pressure. Simultaneously, the left lung vascular bed was also perfused through theChapter 2: MATERIALS AND METHODS^58heart via the right ventricle with Karnofsky's solution containing 1% glutraldehyde at 20 cm waterpressure (this was done to flush excess 3H-TdR in the pulmonary blood volume and leukocytes, andto fix the distended pulmonary capillaries). Following 2 hours fixation of the left lung, the heart of eachanimal was dissected out, blot dried and weighed. Along with the left lung, the heart was also keptsubmerged in Karnofsky's solution and stored until required.2.3.2. LUNG BIOCHEMISTRYThe right lungs were freeze-dried to constant weights. DNA, RNA and total alkali soluble proteinextraction and amount estimation was done by similar methods as have been explained earlier. Measurements of DNA synthesisSpecific radioactivity of DNA was estimated by adding 0.5 ml of DNA extracts to 9.5 ml ofscintillation medium contained in standard scintillation vials and counting the [3H] activity using a j3-counter (Beckman, LS-6800). The incorporation of 3H-TdR into DNA (dpm/gg DNA) was consideredto be equivalent to the net rate of DNA synthesis during injected 3H-TdR.2.3.3. AUTORADIOGRAPHIC TECHNIQUES AND OBSERVATIONS2.3.3.1. Techniques2. Tissue sampling An approximately 1 mm thick mid-sagittal slice of the whole left lung was cut. From that slice,two central (one cephalic and one caudal) and two peripheral (one cephalic and one caudal) blockswere cut as shown in Figure 6. The peripheral blocks of 3-4 mm in width were cut from the lateraledge of the lung slice such that they had a visceral pleural cell layer on one side and contained nomajor vessels or bronchi. The central blocks were about 3-4 mm in width and 5-6 mm in length andconsisted of main bronchi and vessels. All blocks were stored in Karnofskys solution until processed.Chapter 2: MATERIALS AND METHODS^592. Tissue processing and emulsion coatingFigure 6. Illustration of locations from which central and peripheral blocks were taken from the leftlung for autoradiographic measurements.All blocks from each lung were processed and then embedded in methacrylate (JB4 embeddingkit, Polysciences inc., Warrington, PA., USA). Two, one micron thick sections from each block werecut and mounted on a clean slide. All slides were coated by dipping in autoradiographic emulsion(Kodak NTB 2, cat# 165 4433) which was maintained at 47° C in a water bath in a darkroom (any lightsource can increase the background noise on a developed slide). The slides were then placed inracks to drain excess emulsion. The slides were allowed to dry for an hour and then wrappedcarefully in aluminum foil and black paper to avoid any light exposure. The sealed slides wereincubated in a cold room at 4° C for 3 weeks (3 weeks of incubation was found to be optimal, as itproduced high resolution of labelled cells and low background).Chapter 2: MATERIALS AND METHODS^602. Development of autoradiographic slides The incubated slides were removed from the cold room and allowed to warm up for 4 hours toroom temperature. The slides were then immersed in D19 developer for 4 minutes at 16° C and weregently agitated. The developing process was stopped by transferring the slides in distilled water. Thedistilled water was changed twice, each time for 20-30 seconds duration. Finally, the developedsections were fixed by submersion in Kodak fixative at 16° C for 5 minutes and with gentle agitation.The fixative was washed with cold distilled water for 5 minutes and then with cold tap water for 30minutes. The slides were washed with tap water and allowed to dry. The dry slides werecounterstained with 1% toluidine blue stain prepared in 1% sodium tetraborate. The radiolabelled cellsappeared to be heavily laden with black granules with sporadic single granules in the background(Figure 41). OF AUTORADIOGRAPHS2. Cell counting Autoradiographs were examined using a Nikon microscope (100X objective) fitted with an eyepiece graticule dividing the test area into four quarters. This made keeping accurate record of countedcells possible. Two sections mounted on the same slide were used for Vitiated thymidine incorporationanalysis. After discarding 2 test fields along the margins of the sections, every fifth adjacent field onthe sections was counted until a total of 40 test fields on a slide was achieved. A field with anyartifacts was discarded and the next adjacent field was included for analysis. From both the centraland peripheral sections, labelled and unlabelled cells in the alveolar wall and free alveolarmacrophages were identified and counted. The alveolar wall cells included type I pneumonocytes,type ll pneumonocytes, alveolar wall capillary endothelial cells, interstitial cells, mast cells andunidentifiable cells. Apart from the central and peripheral alveolar wall cells, approximately 1000 cellsin the walls of subpleural alveoli were also counted from the peripheral sections. An arbitrary distanceChapter 2: MATERIALS AND METHODS^61of about 100 micron along the pleural surface was defined as the subpleural zone of the lung. Fromthe peripheral sections labelled and unlabelled mesothelial cells (<200> cells) were also recorded.On a separate occasion, the non-parenchymal labelled and unlabelled cells were also counted.In the central sections, the major arteries and bronchi with an internal diameter of more than 400 1..twere included in analysis. In the arteries, more than 150 labelled and unlabelled endothelial cells and500 arterial wall cells were counted. In case of bronchi, more than 300 bronchial epithelial cells andmore than 500 bronchial wall labelled and unlabelled cells were analyzed for cytokinetics. Withregards to peripheral sections, 200-300 bronchiolar epithelial cells were counted along approximately300-400 j.t of terminal bronchiolar surface (internal diameter <200 j.t) starting at the junction of alveolarduct and terminal bronchioles. A total of 6,000-8,000 labelled and unlabelled cells were counted ineach animal for cytodynamic study. Criteria for cell identification The alveolar wall capillary endothelial cells were identified as cells lining and often protrudinginto the capillary lumen with attenuated cytoplasm adjacent to the nucleus. Very few nucleated cellswere present within the lumen as the capillaries were flushed during the fixation process of thevascular bed. Type ll pneumonocytes were identified as polygonal cells with vacuoles and furredsurface and were often located at the corners of the alveoli. Type I pneumonocytes were located atthe alveolar surface, distinguished by elongated nuclei with long cytoplasmic extensions and had a"fried egg" appearance. All cells in the interstitium (fibroblasts, septal cells, pericytes, monocytes,lymphocytes) were counted as interstitial cells because it was difficult to identify various cellpopulations. Leukocytes in capillary lumen were not taken into account. This was done to avoid theinfluence of the labelling index of leukocytes on the analysis. The mast cells were distinct withmetachromatic staining. The bulk of mast cells were located in the subpleural area or in the walls ofvessels and bronchi. The cells which were either in cross section (tiny nucleus with lots of cytoplasmor a large nucleus with no cytoplasm) or difficult to categorize were classified as unidentifiable cells.Chapter 2: MATERIALS AND METHODS^62Free alveolar macrophages were easy to recognize in airspaces. Labelling indices The cells which were, or entered, the s-phase of the cell generation cycle at the time of 3H-TdR injection, incorporated labelled thymidine into the cell DNA, showed more than 10-20 granuleson the nuclear area on the histological slides. No other criterion was considered necessary for thenumber of the granules present over the labelled cells because the background was low and thelabelled cells were heavily laden with granules. The percent labelling index for each cell typepopulation was calculated as follows:% Labelling index = ^Total number of labelled cells * 100 Total number of cells counted (labelled + unlabelled)A total of 4000-6000 alveolar wall cells were counted in the central and peripheral sections. Thecombined alveolar wall cell number was computed as the sum of type I pneumonocytes, type IIpneumonocytes, alveolar wall capillary endothelial cells, interstitial cells, unidentifiable cells andmesothelial cells from central, peripheral and subpleural area. The labelling index for each categoryof cells was calculated by dividing the labelled cells of a particular category by the total number of thatcategory of alveolar wall cells separately for the central, peripheral and subpleural alveoli. Since thelabelling indices of the alveolar wall cells in the peripheral and subpleural areas of the lung were thesame, the cells from both areas were pooled for respective cell categories and the results werereferred as alveolar wall cells in the peripheral part of the lung.2.4. LUNG ADAPTATION: PHYSIOLOGICAL ASPECTS2.4.1. EXPERIMENTAL DESIGNChapter 2: MATERIALS AND METHODS^63Forty, four week old male Sprague-Dawley littermate rats were randomly sorted into five groupsas described earlier:* Group 1.^General Control Group (GC)* Group 2.^Hypobaric Normoxic Group (HBNO)* Group 3.^Normobaric Hypoxic Group (NBHY)* Group 4.^Hypobaric Hypoxic Group (HBHY)* Group 5.^Weight-matched Control Group (WMC)Exposure protocol was the same as has been detailed in the first section of methods.2.4.2. PULMONARY FUNCTION TESTSPulmonary function tests were performed as described by Wright and coworkers (286). Animal preparationAfter three weeks of chronic exposure to the respective conditions, the rats were anaesthetizedby injecting urethane (100 mg/100 g body weight, i.p.). After recording the somatic parameters, anapproximately 5 mm long skin incision was made along the median plane distal to the larynx. Thetrachea was exposed by dissecting the muscles and a tracheostomy was done. The trachea wascannulated with a polyethylene cannula (1.5 mm internal diameter). A gag was placed in the mouthto protect the esophageal catheter. To measure pleural pressure, an eight cm long (internal diameter1 mm, polyethylene) water-filled, multiple side-holed catheter was carefully inserted into the esophagusjust proximal to the heart (premarked) of the anaesthetized rat. The animal was then placed supineinside a pressure sensitive whole body plethysmograph consisting of a 7.5 liter chamber connectedto a bottle containing copper mash used as a body heat sink. All the holes for connections to theplethysmograph were plugged to prevent air leaks. The esophageal catheter was flushed with a smallamount of water and was then connected to the pressure transducer.Chapter 2: MATERIALS AND METHODS^642.4.2.2. Lung volume measurementsThe rats were allowed to breathe on their own. If the respiration frequency was irregular or toohigh, they were attached to a respirator and were hyperventilated for 1-2 minutes. This helped toreduce the respiratory rate to within the desired range (40-60 breaths/minute). At the end of expirationthe tracheal opening was occluded with a finger and the rat was allowed to exert effort against theocclusion (end-expiration could be monitored by carefully watching the thorax movement or therecording pen). The difference between esophageal and airway opening pressure was recorded withthe help of a differential transducer (DP 42-32, Validyne Corp., Northridge, CA) and the volume signalwas obtained by using a pressure sensitive transducer (Validyne DP 45-16). For FRC measurementsairway pressure was used. FRC was measured using Boyle's Law:"At a constant temperature, the volume of any gas varies inversely as the pressure to whichthe gas is subjected."P1 V1 = P2V2where P1 is the initial pressure, V 1 is the initial volume, P2 is changed pressure and V2 is changedvolume, while temperature remains constant. With the help of a digital computer, a pressure/volumeloop was obtained. The slope of this loop was obtained with the assistance of a computer. The slopewas then matched to read the functional residual capacity (FRC) of the rat from a calibrated chart.FRC was taken as the mean calculated from 3-4 consecutive maneuvers.Following FRC measurements, an intraperitoneal injection of succinylcholine was given to paralysethe rat (0.1 m1/100 g body weight). The animal was connected to the respirator and a tidal volumeof 2 ml was set at 60 breaths/minute frequency. When the rat was completely paralysed (the rat didnot resist the ventilator), all the ports of the plethysmograph were sealed and the pressure-volumecurves were recorded. First the lungs were deflated to -30 cm water transpulnnonary pressure andthe lung volume at this pressure was defined as residual volume (RV). The decrease in volumerecorded after deflation was assumed as expiratory reserve volume (ERV). The residual volume wasthen calculated by subtracting ERV from FRC. The lungs were then inflated to +30 cm waterChapter 2: MATERIALS AND METHODS 65transpulmonary pressure and this volume from residual volume was defined as vital capacity (VC).Total lung capacity (TLC) was calculated as a sum of VC (obtained from the pressure volume curverecording) and RV (calculated as shown above). Following an inflation at +30 cmH20 transpulmonarypressure, the lungs were deflated to -30 cm water pressure. This whole maneuver was performedonce without recording and then two pressure-volume curves were plotted. The deflation limb of thepressure-volume recording was used for pressure-volume analysis. From the deflation limbrecordings, static lung compliance (Cst) was calculated as change in lung volume per unit change intranspulmonary pressure at functional residual capacity at +5 cmH20 transpulmonary pressure. Flow-volume relationshipsAfter recording the pressure-volume curves, the lungs were inflated to +30 cmH20 pressureand were then rapidly deflated at an airway pressure of -50 cmH20 pressure until expiratory air flowceased. Using this procedure forced expiratory volume and simultaneous transpulmonary pressurechanges were recorded. From these recordings peak expiratory flow (PFER), forced expiratory flow(FEF) at percentiles of forced vital capacity (FVC), FEE corrected for vital capacity at percentiles offorced vital capacity, maximum midexpiratory flow (FEF25_75%) at 25%-75% of forced vital capacity,absolute forced expiratory volume in 0.1 second (FEV0.1) and FEV0.1 percent of forced vital capacity(FEV01/FVC°/0) were computed. Upstream resistance (Rus) was calculated (transpulmonary pressure/flow) at 50% FVC.In a normal lung, the lung compliance and airway resistance are influenced by the size of thelung (71). Therefore, in order to eliminate the effect of lung size, compliance and airway resistancewere corrected for lung volume at which these measurements were made. Specific lung compliance(sCst) was calculated by dividing the static lung compliance by lung volume and specific upstreamairway resistance (sRus) was calculated by multiplying upstream resistance with lung volume at 50%forced vital capacity (71).From the expiratory flow rates and forced vital capacity recordings, flow-volume curves wereChapter 2: MATERIALS AND METHODS 66constructed. Using the pressure-volume curve tracings, pressure at percentiles of lung volume (FRCto TLC) was calculated and then pressure-volume characteristics for elastic lung recoil were analyzed.As described in section I, the pressure-volume curves were also used to fit a monoexponential functionin the range of lung volume from FRC to TLC (68).2.5. STATISTICAL ANALYSISGroup means ±standard errors of the means (SEM's) were calculated. For section I (Lung growthadaptation) and III (Lung adaptation: physiological aspects) of the project, the statistical evaluation ofthe groups was performed by using a single factor analysis of variance to test the hypothesis that themeans of all groups were equal. If the hypothesis was rejected at p<0.01, then the Student NewmanKeuls (SNK) multiple range test was used in order to locate a significant difference at p<0.05 levelbetween the groups. Statistical calculations were done using SPSS:X and Number CruncherStatistical System.For section II (Organ response and lung cytokinetics) of the project, a two-way analysis of variancewas applied to detect the effect of treatments (undernutrition, hypobaric normoxia, normobaric hypoxiaand hypobaric hypoxia) and duration of exposure (days) and their interaction on somatic variables,organ weights, lung biochemical measurements and 3H-TdR incorporation into various types of cellsin the lung. If an interaction between the two factors was detected for a variable, then further analysiswas done separately for treatments and days using linear contrast test to locate a significantdifference. In the case of groups, eight possible contrasts (GC vs WMC, GC vs HBHY, GC vs HBNO,GC vs NBHY, WMC vs HBHY, WMC vs NBHY, HBNO vs HBHY and HBHY vs NBHY) were made todetect the effect of various conditions. The rate of organ growth was evaluated by comparing thevalues of 3, 5, 7, 10, 14 and 21 days in each group (GC, WMC, HBNO, NBHY and HBHY) to thevalue of day 1 of general controls. This was done to determine the duration of time required in eachexperimental condition to produce a significant change in organ weights. Depending upon the numberof contrasts between the groups or the days, the Bonferroni correction was applied to keep the totalChapter 2: MATERIALS AND METHODS 67chance of erroneously rejecting the hypothesis that no change occurred between two groups or twotime intervals below 5 percent (288). This required significant levels of p<0.006 between the groupsand p<0.008 between the days before applying Bonferroni correction to achieve a significance ofp<0.05. The p-values shown in the results are p-values obtained by linear contrast times number ofcontrasts made in that category. To analyze the effect of the recovery period, a t-test was performedto compare the values of body weight, nose-tail length, organ weights, lung biochemical variables andradiochemically measured 3H-TdR uptake on day 21 and 24 (3 days after returning the animals toroom air and allowing free access to food) of experimental conditions. The effect of post-exposureand refeeding among the groups was analyzed as described above.Chapter 3: RESULTS^ 68CHAPTER 3RESULTS3.1. LUNG GROWTH STUDY3.1.1. GENERALSince changes in somatic growth occurred in the experimental groups compared to thegeneral controls, and the experiments described here are complicated, the data will be presented inthe following way. The general control group will be compared to the weight-matched control groupto assess the effect of undernutrition. To evaluate the effect of low ambient pressure, the hypobaricnormoxic rats will be compared to general control rats, and the hypobaric hypoxic rats will becompared with normobaric hypoxic rats. For the effect of low oxygen, comparisons will be madebetween the nornnobaric hypoxic group and the weight-matched control group, the normobaric hypoxicgroup and the general control group, and the hypobaric hypoxic group and the hypobaric normoxicgroup. Adaptive response to hypobaric hypoxia will be determined by comparing the hypobarichypoxic group to the general control group, but more emphasis will be placed on the comparison withweight-matched controls.The normobaric hypoxic and hypobaric hypoxic rats appeared to be less active on days 0 and1 of the experiment relative to general control, weight-matched control and hypobaric normoxic ratsbut after day 1 regardless of the condition they were subjected to, no obvious difference in activityamong rats was noticed. NUTRITIONAL ASSESSMENTAs shown in Figure 7, during the first week of exposure, the hypobaric normoxic and generalcontrol animals consumed similar amounts of food, but thereafter the food intake of the hypobaric27.5-25-22.5-20-17.5-1512.5-10-7.55 ^Chapter 3: RESULTS^ 69Figure 7. Daily food consumption of the general control (GC), weight-matched control (WMC),hypobaric hypoxic (HBHY), hypobaric normoxic (HBNO) and normobaric hypoxic (NBHY) groups.normoxic animals gradually decreased and was significantly lower on day 10 and afterwards (excepton days 13, 16, 20 and 21). The total food intake of hypobaric normoxic rats during 21 days ofexposure was also significantly less compared to general controls (Table 3).The exposure to hypobaric hypoxia or to normobaric hypoxia caused a significant drop in foodconsumption on day 1, which appeared to be the point of divergence. Thereafter the daily foodconsumption of hypobaric hypoxic and normobaric hypoxic rats increased gradually but it neverreached the amount of daily food intake of general control rats during the experimental period(Figure 7). During three weeks of exposure, the total amount of food consumed by the hypobarichypoxic and normobaric hypoxic groups was significantly less, (25% and 27% respectively) than thatof the general control group (Table 3). No difference in food consumption was found between thenormobaric hypoxic and hypobaric hypoxic groups.Body Weight (g)Chapter 3: RESULTS 70The weight-matched control and hypobaric hypoxic groups were paired for body weight gain.The weight-matched control rats were allowed limited access to food to keep their daily body weightgain similar to the body weight gain of paired hypobaric hypoxic rats (Figure 7). During three weeks,the total food consumed by the weight-matched control group was significantly lower (12%) than thatof the hypobaric hypoxic group (Table 3).Figure 8. The effect of undernutrition, hypobaric hypoxia, hypobaric normoxia and normobaric hypoxiaon body weight gain. SOMATIC GROWTHBody weight was recorded as a measure of body size and nose-tail length as a determinantof skeletal growth of rats during the experimental period. The general 'control rats gained 214% bodyweight and 48% nose-tail length during 4 to 7 weeks of age (Table 3).Chapter 3: RESULTS 71During the first week of exposure, no difference in body weight gain was detected betweenthe hypobaric normoxic and general control groups, but thereafter, the body weight of the hypobaricnormoxic group lagged behind the general control group. After day 12, the hypobaric normoxicanimals were significantly smaller than the general control rats (Figure 8). At termination, thehypobaric normoxic rats were significantly lighter in body weight (9%) and shorter in nose-tail lengthcompared to those measured in the general control group (Table 3).As illustrated in Figure 8, in normobaric hypoxic and hypobaric hypoxic rats, the body weightdecreased slightly on day 1 of exposure. However, from day 3 until the end of exposure, a consistentbut slow body weight increase was observed in nornnobaric hypoxic and hypobaric hypoxic animalsbut they remained significantly smaller compared to the general control animals (Figure 8). During 3weeks of exposure, both the normobaric hypoxic and the hypobaric hypoxic groups gained 142% inbody weight. At the time of termination, the normobaric hypoxic and hypobaric hypoxic rats weresignificantly smaller in body weight (23%) and nose-tail length (7% and 9% respectively) comparedto the general control group (Table 3). When the results for body weight and nose-tail length ofweight-matched control, hypobaric hypoxic and normobaric hypoxic groups were compared, nodifferences were found among three groups (Table 3).3.1.2. HEMATOCRITBefore terminating the animals, blood samples were taken from the right ventricle andhematocrit was measured. After 21 days of exposure, the hematocrit values increased significantlyin the normobaric hypoxic and hypobaric hypoxic groups compared to both the general control andweight-matched control groups (Table 3). Hematocrit in weight-matched animals remained unchangedcompared to general controls. No difference was found between the hematocrit of hypobaric hypoxicand normobaric hypoxic rats. A slight but significant increase of 9% was also observed in rats afterexposure to hypobaric normoxia compared to general controls (Table 3).Table 3. Food intake, somatic growth and hematocrit measurements.VARIABLE BC GC WMC HBNO NBHY HBHYTOTAL FOOD 402.2+3.7a 274.7+4.2b 376.9+8.1c 292.1+3.3d 307.0+5.1 dINTAKE (g)Wb g 83.2+0.6a 261.0+2.6b 199.0+2.3c 238.0+3.2d 203.0+2.2c 202.0+1.5c -.1N)NTL cm 26.0+0.2a 38.5+0.2b 35.1+0.2° 37.2+0.3d 35.7+0.2c 34.6+0.3°Hct (`)/0) 39.0+0.9a 41.4+0.4a 41.4+0.6a 45.1+0.6b 59.0+0.7c 60.1+1.0cMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Wb (body weight), NTL (nose-tail length), Hct (hematocrit), BC (baseline controls, 4 week old), GC (general controls), WMC (weight-matchedcontrols), HBNO (hypobaric normoxic), NBHY (normobaric hypoxic) and HBHY (hypobaric hypoxic).Chapter 3: RESULTS^ 733.1.3. LUNG GROWTHThe morphometric results related to size of the airspaces or alveoli {mean linear intercept(MLI), mean chord length of alveoli (laiv), average alveolar volume (Valvave)) and structures per unitvolume or area {number of alveoli per unit area (Na), number of alveoli per unit volume (Nv), alveolarsurface area to lung volume ratio (Sw/VL)) will be referred to as unit structure morphometricparameters. The total lung weight was used to calculate the total lung volume from left lung volume. NORMAL LUNG GROWTH (4-7 weeks of age) EXTENT OF LUNG GROWTHThe lung weight and lung volume increased by 87% and 146% respectively during 4 to 7weeks of age general controls (Table 4), but specific (relative to per unit body weight) lung weight andlung volume decreased significantly. Since lung volume increased more than lung weight, a significantincrease in lung volume per unit lung weight was observed at 7 weeks of age compared to 4 weekold animals (Table 4). BIOCHEMICAL CHANGESA significant increase in the dry lung weight (Wldry) (89%), DNA (57%), RNA (69%) and alkalisoluble protein (73%) content was found between 4 weeks to 7 weeks of age (Table 5). The amountof total hydroxyproline which comprised SDS-soluble and SDS-insoluble (cyanogen bromide soluble)fractions also increased by 120% (Table 5). An increase in the insoluble fraction of hydroxyprolinewas greater (130%) than that of the soluble fraction (53%), thus causing a significant decrease in theratio of soluble to insoluble fraction of hydroxyproline at 7 weeks of age compared to 4 weeks of age.A greater increase in the amount of insoluble fraction of hydroxyproline at 7 weeks of age indicatedincreased formation of mature collagen. A marked increase (86%) occurred in desmosine contentduring 4-7 weeks of age (Table 5). Specific amounts of DNA, RNA, protein, hydroxyproline andTable 4.^Results of lung growth adaptation.VARIABLE BC GC WMC HBNO NBHY HBHYWL g 0.604+0.011a 1.130+0.024b 0.948+0.014c 1.063+0.029b 1.307+0.02d 1.326+0.028dVL m I 4.64+0.12a 11.40+0.52b 9•30+0.24c 10.57+0.17d 12.94+0.21a 12.26+0.268WL/100 g Wb g 0.725+0.011a 0.433+0.010b 0.476+0.006c 0.445+0.009b 0.646+0.008d.._. 0.656+0•014dVL/100 g Wbml5.58+0.19a 4.35+0.17b 4.70+0•12b 4.43+0.09b 6.38+0.08c 6.07+0.12°VL/WL (ml/g) 7.72+0.24a 10.10+0.44b 9.87+0.31 b 9.85+0.24b 9.87+0.13b 9.29+0.29bMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.WL (total lung weight), VL (total lung volume), BC (baseline controls), GC (general controls), WMC (weight-matched controls), HBNO (hypobaricnormoxic), NBHY (normobaric hypoxic) and HBHY (hypobaric hypoxic).Table 5. Biochemical assessment of lung growth after three weeks of exposure to various conditions.VARIABLE BC GC WMC HBNO NBHY HBHYDry/Wet WL % 21.35+0.18a 21.23+0.23a 21.43+0.12a 21.82+0.33a 21.02+0.33a 21.48+0.16aWIdry mg 74.0+1.5a 139+3.0b 120+2.0c 138+3.0b 168+4.0d 173+3.0dDNA mg 247+0.07a 3•88+0•07b 3.37+0.07c 3.73+0.09b 4.41+0.11d 4.39+0.11dRNA mg 2.34+0.07a 396011 b 2.82+0.11c 3.76+0.09b 4.52+0.12d 4.45+0.12dPROTEIN mg 37.57+0.66a 65.08+1.37b 52.94+0.91c 64•99+1.48b 80.55+2.51d 84.97+1.97dOHP(SOL) mg 0.079+0.002a 0.121+0.004b 0.105+0.004c 0.124+0.004b 0.141+0.006d 0.155+0.003dOHP(INS) mg 0.525+0.029a 1.209+0.029b 0.985+0.030c 1.210+0.030b 1 258+0.05b 1.376+0.03dOHP mg 0.605+0.030a 1•334+0.033b 1.090+0.033c 1•334+0.0351' 1.400+0.051 b 1.531+0.033dDES ug 12.05+0.47a 23.21+1.57b 20.42+1.15b 26.41+2.28b 33.14+2.18c 40.15+2.52dMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Dry/Wet ratio (dry lung weight to wet lung weight ratio), Wld (dry weight of the right lung), OHP (hydroxyproline [SOL] soluble fraction, [INS]insoluble fraction), DES (desmosine)cnTable 6. Lung biochemical measurements normalized to 100 grams of body weight.VARIABLE BC GC WMC HBNO NBHY HBHYDNA mg 2.96+0.08a 1.47+0.03" 1.70+0.030 1.56+0.03" 2.17+0.05d 2.17+0.06dRNA mg 2.99+0.08a 1.52+0.03c 1.41+0.04" 1.58+0.03° 2.23+0.07d 2•19+0.06dPROTEIN mg 45.12+0.67a 24.94+0.45b 26.60+0.41 b' 27.21+0.66c 39.60+1.26d 42.07+1.07dOHP mg 0.725+0.038a 0.510+0.014" 0.547+0.016b 0.558+0.011 b 0.689+0.024a 0.757+0.017aDESMOSINE ug 14.5+0.5a 8.9+0.6" 10.2+0.5" 11.1+0.9b 16.4+1.1a 19.9+1.3°Means +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Chapter 3: RESULTS^ 77desmosine were significantly higher in 4 week old compared to those in 7 week old rats (Table 6).The concentrations (biochemical measurements per unit dry weight of the lung tissue) of DNAand RNA were significantly less at 7 week of age compared to 4 week old animals (Table 7). Theconcentration of hydroxyproline increased significantly as age increased, but the desmosineconcentration remained constant indicating that a dynamic equilibrium of elastin was achieved before4 weeks of age (Table 7). Except for a significant increase in 7 week old animals for thehydroxyproline to DNA (OHP/DNA) ratio, no differences in other ratios [RNA/DNA, desmosine to DNA(DES/DNA), desmosine to hydroxyproline (DES/OHP)] were observed between the two age groups(Table 8). MORPHOMETRIC CHANGESResults of unit structure morphometric measurements showed that between 4 and 7 weeksof age, significant increases in mean linear intercept of airspaces (MLI), mean chord length of alveoli(lab) and average volume of alveoli (Valvave), and associated decreases in the number of alveoli perunit area (Na), number of alveoli per unit volume (Nv) and surface to volume ratio (Sw/VL) wereobserved (Table 9).Seven week old rats had a significantly increased volume fraction of alveolar air (Vvaiv)compared to the 4 week old rats (Table 10). Although the volume fraction of duct air (Vvdept), volumefraction of alveolar wall (Vvwan), volume fraction of non-parenchymatous tissue (Vv) and volumefraction of bronchial air (Vvbr) did not change (Table 10), absolute volume of alveolar air (Vaiv), alveolarduct ( Yduct), alveolar wall (Vwan), non-parenchymal tissue (Vnp) and bronchial air (Vbr) increasedsignificantly in 7 week old animals (Table 11).The total number of alveoli (Nat) obtained by direct alveolar count and internal surface area(Sw) increased significantly (70% and 133% respectively) between 4 and 7 weeks of age (Table 12).Despite significant increases in absolute values of the Sw and Nat in 7 week old rats, body weightnormalized values were significantly smaller in 7 week old animals compared to 4 week old animalsChapter 3: RESULTS^ 78(Table 12).The results showed that during 4 to 7 weeks of postnatal age, lung air volume increased morethan the lung weight. Absolute biochemical and morphometric measurements increased with age butdid not keep up with body growth. A decrease in DNA concentration and an increase inhydroxyproline concentration indicated that with age, the intercellular matrix increased more than thecellular component of the lung. Interestingly, elastin appeared to plateau before 4 weeks of age. Theunit structure morphometric results showed that even though enlargement of the airspaces occurredduring 4 to 7 week of age, alveolar number continued to increase. EFFECT OF UNDERNUTRITION3. EXTENT OF LUNG GROWTHThree weeks of undernutrition caused reductions in lung weight (16%) and lung volume (18%)compared to the general control group (Table 4). On the other hand, specific lung weight of theundernourished group was significantly higher than that of the general control group. Lung volume perunit lung weight remained unchanged in undernourished rats (Table 4). CHANGESThe rats given restricted food had significantly lower WIthy, DNA, RNA, protein andhydroxyproline content (14%, 13%, 29%, 19% and 18% respectively) compared with those found inthe general control group (Table 5). When biochemical parameters were corrected for body weight,except for a significant increase in DNA content, other parameters in the weight-matched control groupremained similar to those in the general control group (Table 6).The concentrations of DNA, hydroxyproline and desmosine remained unaffected inundernourished animals, but the concentrations of RNA and protein decreased significantly (Table 7).None of the biochemical ratios changed except for RNA/DNA ratio which was significantly decreased(Table 8).Table 7.^Biochemical variables per gram of dry lung weight.VARIABLE BC GC WMC HBNO NBHY HBHYDNA mg 32.2+0.5a 28.0+0.5b 28.4+0.6b 27.0+0.6b,c 26.4+0.9b'b 25.3+0.4bRNA mg 31.7+0.60a 28.5+0.35b 23.5+0.53b 27.3+0.54b 27 0+0 5b_ 25.7+0.53"PROTEIN mg 501.4+9.7a 470.0+6.3a 443.6+5.5b 471.1+8.5a 480.1+7.6a 491•0+7.7aOHP mg 8.12+0.33a 9•60+0•16b 9.12+0.20b'b 9•65+0.12b 8.36+0.26a 8.84+0.12a'eDESMOSINE ug 163.0+6.0a 166.2+11.4a 170.8+8.5a 192.5+15.7a 196.4+9.2a 232.9+13.1"Means +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Table 8.^Relationships between biochemical changes in different experimental groups.VARIABLE BC GC WMC HBNO NBHY HBHYRNA/DNA(mg/mg)1.00+0.03a 1.02+0.02a 0.83+0.03b 1.01+0.02a 1.03+0.03a 1.02+0.02aPROTEIN/DNA(mg/mg)15.27+0.47a 16.81+0.34b 15.65+0.31a 17.43+0.63b'c 18.41+0.65c 19.39+0.57cOHP/DNA(ug/mg)243.2+11.9a 342•2+6•8b'c 320.0+10.3b 358.1+6.8c 318.0+9.64- 349.3+5.1cDES/DNA(ug/mg)4.91+0.20a 5.98+0.4a'b 6.22+0.34a'b 7.18+0.49b 7.44+0.5° 9.25+.51cDES/OHP(ug/mg)21.O+1 .5' 17.5+1.5a 18.8+1.0a 20.1+1.7a'b 23.7+1.5b'c 26.5+1.8c'dOHP (Sol) % 15.57+0.92a 10•03+0.34b 10•71+0•47b 10•29+0•29b 11•46+0•55b 11.27+0.47bMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.OHP (hydroxyproline), DES (desmosine), OHP(sol) °X, (percent of soluble fraction of total hydroxyproline content).ODaChapter 3: RESULTS^ 813. MORPHOMETRIC CHANGESUndernutrition did not cause changes in the unit structure morphometric parameters such asMLI, lalv, NA, Nv and Valvave, when compared to those in the general control group (Table 9), but theSw/VL ratio was higher in undernourished animals. The volume fractions of the lung structuresremained unchanged (Table 10) but absolute volumes of alveolar air, alveolar duct air and alveolarwall decreased significantly (Table 11). Even though, the morphometric measures of the lung unitstructures remained unchanged, S, and Nat which are functions of lung volume were significantly lower(16% and 12% respectively) and the specific values for these parameters were significantly higher inthe weight-matched group compared to the general control group (Table 12).The above results showed that undernutrition diminished somatic growth as well as lunggrowth assessed by lung weight and volume, and also by biochemical and morphometric analyses.Somatic growth was affected more than lung growth. On the other hand, lung structure based onbiochemical measurements (except for RNA) and morphometric results remained unchanged by foodrestriction, therefore, indicating diminished lung growth without altering the structure of the lung. ADAPTATION TO LOW AMBIENT PRESSURE (HYPOBARIC NORMOXIA) EXTENT OF LUNG GROWTHAs recorded in Table 4, in low ambient pressure conditions, the lung volume decreasedsignificantly compared to the general control group, but lung weight did not. No differences betweenthe general control and hypobaric normoxic groups were found when the values for lung weight andlung volume were normalized for body weight (Table 4). No change in lung volume per unit lungweight was found in hypobaric normoxic rats.When the hypobaric hypoxic and normobaric hypoxic groups were compared to assess theeffect of hypobaric pressure neither absolute nor specific lung weight and lung volume showed anyChapter 3: RESULTS^ 82changes (Table 4). BIOCHEMICAL CHANGESNo significant differences between the general control and hypobaric normoxic groups werefound in any of the lung biochemical parameters (DNA, RNA, alkali soluble protein, hydroxyproline anddesmosine) measured (Table 5). To observe the effect of low ambient pressure, the normobarichypoxic and hypobaric hypoxic groups were compared and it was found that the biochemicalparameters in normobaric hypoxic and hypobaric hypoxic groups were also similar, except for amarked increase (21%) in desmosine content in hypobaric hypoxic animals, which was significant(Table 5).The specific amount of lung DNA, RNA, protein, hydroxyproline or desmosine were also thesame in the hypobaric normoxic and general control groups. No differences were found for specificbiochemical estimations between the normobaric hypoxic and hypobaric hypoxic groups, except thatthe amount of desmosine was higher in the hypobaric hypoxic group (Table 6).The concentrations of all biochemical measurements remained unchanged following exposureto hypobaric normoxia (Table 7). When the hypobaric hypoxic and normobaric hypoxic groups werecompared, only the concentration of desmosine was different and significantly higher in the hypobarichypoxic group. DES/DNA was also significantly increased in the hypobaric hypoxic group comparedto the nornnobaric hypoxic group (Table 8). MORPHOMETRIC CHANGESThe hypobaric normoxic rats had significantly smaller MLI and Valvave, and greater NA, Nv andSw/VL ratio compared to those in general controls (Table 9). Absolute volume of alveolar air, alveolarduct air and alveolar wall decreased significantly (Table 11). When the hypobaric hypoxic andnormobaric hypoxic groups were compared, the values of MLI and Valvave were significantly lower andNA and Nv were higher in hypobaric hypoxic animals. No differences were found in absolute valuesTable 9. Changes in morphometric parameters following exposure to different conditions.VARIABLE BC GC WMC HBNO NBHY HBHYMLI (4m) 78.6+1.2e 85.4+1.5b 81.9+1.3" 76.5+1.1a 91.1+1.6° 84.3+1.8"laiv (ilm) 34.9+0.6e 40.9+0.8" 38.6+0.6b 38.4+0.6b 45.1+0.7c 43.5+1.3°NA (10+3) 22.6+0.41a 18.1+0.38" 19.1+0.32" 19.7+0.37c 16.7+0.33d 18.4+0.42"Nv (10+5) 30.0+0.85e 20.9+0.73b 23.0+0.55" 24.2+0.67c 18.3+0.51d 20.9+0.71 bValVave (10-8) 18.1+0.6e 27.3+1.3" 24.4+0.7b'c 23.9+0.7c 32.0+0.8d 28.7+1.1e;NI_ ratio 0.095+0.002e b0.082+0.002 0.089+0.002e 0•091+0•002e 0.075+0.001°4 0.080+0.002"Means +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.MLI (mean linear intercept), laiv (mean chord length of alveoli), NA (number of alveoli per unit area), Nv (number of alveoli per unit volume), Valvave(average volume of alveoli), SwNI ratio (surface to volume ratio of alveoli {SwNL*Vvair}).0:■Table 10. Alterations in volume proportions of lung morphological structures following 3 weeks of exposure to different conditions.VARIABLE BC GC WMC HBNO NBHY HBHYVValv 0.539+0.008a 0.568+0.008b'c 0.553+0.007° 0.576+0.00813'c 0.578+0.006bc 0.595+0.012cVVduct a0.217+0.007 0.204+0.008° 0.202+0.009° 0.182+0.008b 0.178+0.007b 0.176+0.008bVVwali 0.161+0.005a 0.151+0.004a 0.163+0.005a 0.158+0.005a 0.160+0.003a 0.161+0.005aWnp 0.050+0.004a'b 0.036+0.006a 0.049+0.006a'b 0.048+0.005a'b 0•061+0•003b 0.055+0.005a'bVVbr 0.029+0.005a 0.037+0.006a 0.031+0.006a 0.030+0.004a 0.020+0.003a 0.023+0.004aWduct/VVair 0.403+0.017a 0.361+0.01 7a'b 0.367+0.020a'b 0.316+0.016b 0.3084.016b 0.297+0.016bVVwall/VVair 0.301+0.011a 0.266+0.010a 0•2924.010a 0.277+0.012a 0.276+0.005a 0.271+0.010aMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.VValv (Volume proportion of alveolar air), Vvdupt (volume proportion of alveolar duct air), Vvwan (volume proportion of alveolar wall), Vvproportion of non-parenchymatous tissue), Vvbr (volume proportion of conducting airway air).Chapter 3: RESULTS^ 85of 5 ^Nat, but the specific values were significantly higher in the hypobaric normoxic groupcompared to those in the general control group (Table 12).In short, a decrease lung volume in hypobaric normoxia indicated that lung growth wasdiminished. On the other hand, in absolute terms biochemical analysis indicated that lung growthremained unaffected by hypobaric pressure. Despite decreased lung volume the internal surface areaand total number of alveoli were equivalent to that of general controls and this appeared to occurbecause of smaller unit structures and increased complexity of the lung in hypobaric normoxicconditions.When hypobaric hypoxic and normobaric hypoxic animals were compared for the effect oflower ambient pressure in conjunction of low oxygen, hypobaric pressure did not appear to affect lunggrowth pattern, except that desmosine accumulation was increased in the presence of decreasedpressure in hypobaric hypoxic animals. Compared to the normobaric hypoxic animals, the changesin unit structure morphometric parameters in the hypobaric hypoxic animals were similar to thoseobserved in hypobaric normoxic rats compared to general controls. These findings suggested thatelastin accumulation increased and the size of airspaces decreased in hypobaric pressure whendelivered with low oxygen. ADAPTATION TO NORMOBARIC HYPDXIA3. EXTENT OF LUNG GROWTHThree weeks of exposure to normobaric hypoxia caused significant increases in lung weight(38%) and fixed lung volume (39%) compared to weight-matched controls. Lung weight and lungvolume were also significantly higher (16% and 14% respectively) in normobaric hypoxic animals whencompared with those in general controls (Table 4). No change in fixed lung volume per unit lungweight was observed in the normobaric hypoxic group compared to weight-matched controls or generalcontrols. Since normobaric hypoxic animals had a lower body weight than the general control animals,specific lung weight and volume increased markedly in the normobaric hypoxic group (Table 4).Chapter 3: RESULTS^ 863. BIOCHEMICAL CHANGES(i). NBHY vs WMC: The results of biochemical analysis showed that normobaric hypoxia producedmarked increases in WI dry (40%) and lung DNA (31%), RNA (60%) and protein (52%) compared toweight-matched control rats (Table 5). DNA concentration remained unchanged but RNA and totalprotein concentrations were significantly higher in normobaric hypoxic animals compared to thoseobserved in weight-matched controls (Table 7). This resulted in significant increases in RNA/DNA andprotein/DNA ratios in animals exposed to nornnobaric hypoxia compared to the animals subjected tofood restriction (Table 8).The total amount of hydroxyproline which is a sum of soluble and insoluble fractions ofhydroxyproline increased significantly (28%) following three weeks of exposure to normobaric hypoxiacompared with weight-matched controls (Table 5). Although an increase in the soluble fraction ofhydroxyproline which is a measure of immature or newly synthesized collagen (34%) was greater thanthe insoluble fraction which represents mature or cross-linked collagen (28%), it did not raise thesoluble/insoluble hydroxyproline ratio. The amount of desmosine increased markedly (62%) innormobaric hypoxic rats than that of weight-matched controls. Despite increased accumulation inconnective tissue proteins in normobaric hypoxic animals, the amount of hydroxyproline relative to drylung weight fell significantly but desmosine concentration remained undisturbed (Table 7). Thisunequal effect of normobaric hypoxia on connective proteins caused a significant elevation in thedesmosine/hydroxyproline ratio over that found in weight-matched controls whereas OHP/DNA andDES/DNA ratio remained unchanged (Table 8).(ii). NBHY vs GC: The amounts of WIthy, DNA, RNA, protein and desmosine were significantly higherin the normobaric hypoxic group (21%, 13%, 14%, 24% and 43% respectively) than those observedin the general control group (Table 5). Since the amount hydroxyproline did not increase equivalentto the increase in dry lung weight, its concentration fell significantly compared to general controls butconcentration of other biochemical measurements (DNA, RNA, protein, and desmosine) remained theChapter 3: RESULTS^ 87same (Table 7). The Protein/DNA, DES/DNA and DES/OHP ratios were elevated in normobarichypoxic animals compared to those observed in general controls (Table 8).The biochemical measurements expressed relative to body weight were significantly higherin normobaric hypoxic group compared to those in general controls (Table 6).(iii). HBHY vs HBNO: When a comparison was made between hypobaric normoxic and hypobarichypoxic groups to assess the effect of low oxygen tension, increases in all biochemical parameterswere similar to those found between the general control and normobaric hypoxic groups (Table 5).In addition, the amounts of hydroxyproline and desmosine also increased significantly in hypobarichypoxic animals compared to hypobaric normoxic animals.Compared to the hypobaric normoxic animals, the concentrations of lung DNA, RNA andprotein in the hypobaric hypoxic animals did not change but that of hydroxyproline concentrationdecreased whereas desmosine increased significantly (Table 7). These changes led to significantincreases in the DES/DNA and DES/OHP ratios (Table 8). MORPHOMETRIC CHANGES(i). NBHY vs WMC: As shown in Table 9, following three weeks of exposure to normobaric hypoxia,MLI, laiv and Valvave increased while NA, Nv and Sw/VL ratio decreased significantly compared to theweight-matched control group. Since fixed lung volume was significantly larger in normobaric hypoxicanimals and the volume fractions of lung structures did not change, absolute volumes of alveolar air,alveolar wall and non-parenchymal tissue increased significantly compared with weight-matchedcontrols (Table 11). Despite larger unit structures and fewer alveoli per unit volume in normobarichypoxic animals, the Sw and Nat increased significantly (17% and 12% respectively) compared tothose in weight-matched control rats (Table 12).(ii). NBHY vs GC: When normobaric hypoxic animals were compared for unit structure morphometricChapter 3: RESULTS 88parameters to those of general controls, MLI, laiv and Valvave increased and NA, Nv and Sw/VLdecreased significantly in normobaric hypoxic animals. Except for a significant increase in the Vvnpin the normobaric hypoxic group compared to the general control group, other volume fractions of lungstructures did not differ (Table 10). Absolute volumes of alveolar air, alveolar wall and non-parenchyma also increased significantly in normobaric hypoxic rats (Table 11). No differences werefound between the normobaric hypoxic and general control groups for absolute Sw and Nat (Table 12).Specific Sw and Nat measurements were greater in normobaric hypoxic rats compared to generalcontrol rats.(iii). HBHY vs HBNO: In order to examine the effect of hypoxia, the morphometric results of hypobarichypoxic and hypobaric normoxic rats were also compared. The changes dimensions of unit structures,and volume fractions and absolute volumes of lung structures were similar to those observed bycomparing the normobaric hypoxic and general control groups (Table 9-Table 11). No differenceswere found between hypobaric hypoxic and hypobaric normoxic rats for Sw and Nat (Table 12). Onlyspecific Sw was higher in the hypobaric hypoxic group compared to the hypobaric normoxic group(Table 12).In summary, these observations demonstrated that all absolute biochemical as well asmorphometric variables of lung growth increased during adaptation to nornnobaric hypoxia comparedto the animals of similar body weight suggesting that lung growth was enhanced by low oxygen. Theamount of hydroxyproline fell relative to an increase in lung weight but concentrations of RNA andprotein were elevated in normobaric hypoxic animals. Increased DNA content indicated a hyperplasticresponse, but increases in RNA/DNA and protein/DNA ratio showed that cellular hypertrophy alsooccurred in normobaric hypoxia. Unit structures enlarged along with addition of alveolar structures.Compared to general controls, with the exception of hydroxyproline, all biochemicalparameters increased along with lung weight and volume, indicating accelerated lung growth.Morphometric results indicated enlargement of unit structures with no changes in alveolar surface areaChapter 3: RESULTS^ 89and alveolar number. Except for a significant decrease in the concentration of hydroxyproline, otherbiochemical concentrations remained unchanged. On the other hand, increases in DES/DNA andDES/OHP ratios indicated changes in connective tissue equilibrium with increased elastinaccumulation. Comparison between hypobaric hypoxia and hypobaric normoxia also revealed similarchanges, suggesting that effect of hypoxia was similar whether delivered with or without hypobaricpressure. ADAPTATION TO HYPOBARIC HYPDXIA3. EXTENT OF LUNG GROWTHDuring three weeks of hypobaric hypoxic exposure, the adaptive changes in lung growthparameters were quite similar to those observed in normobaric hypoxia. Significant increases in lungweight and fixed lung volume were recorded in hypobaric hypoxic rats (40% and 32% respectively)compared to those in the weight-matched control group and they were also higher (17% and 8%respectively) than those in the general control group (Table 4). Lung weight per unit volume did notchange compared to both weight-matched and general controls. When the results were expressed perunit body weight, significant increases in lung weight and lung volume were found in hypobaric hypoxicrats compared to those in general controls (Table 4). BIOCHEMICAL CHANGES(i). HBHY vs WMC: The rats exposed to hypobaric hypoxic conditions displayed a large increase inWIthy, DNA, RNA and protein content (44%, 30%, 58% and 60% respectively) compared to theirweight-matched controls (Table 5). The amount of DNA per unit of dry lung weight was significantlylower in hypobaric hypoxic rats, whereas RNA and protein concentrations were higher than thoseobserved in weight-matched controls (Table 7) and as result of this the RNA/DNA and protein/DNAratios elevated significantly (Table 8).The connective tissue proteins were also increased in hypobaric hypoxic rats compared toChapter 3: RESULTS 90weight-matched controls. Significant accumulation of hydroxyproline (soluble and insoluble fractions)and desmosine occurred which increased 41% and 97% respectively (Table 5). The soluble fractionof hydroxyproline increased slightly more (48%) than the insoluble fraction (40%) but thesoluble/insoluble fraction ratio remained unchanged. Since an increase in desmosine content wasgreater than an increase in lung weight, its concentration was elevated, whereas the concentration ofhydroxyproline did not change (Table 7). As a result of these alterations in connective tissueaccumulation, the DES/OHP ratio increased in hypobaric hypoxic animals compared to weight-matchedcontrols (Table 7). Hypobaric hypoxia increased the DES/DNA and OHP/DNA ratios (Table 8).(ii). HBHY vs GC: Significant increases of 13%, 12% and 30% were found in DNA, RNA and proteincontent respectively compared to those in general control rats (Table 5). The amounts ofhydroxyproline and desmosine also increased significantly (15% and 73% respectively) in thehypobaric hypoxic group. Hypobaric hypoxia caused a drop in the concentrations of DNA andhydroxyproline but the concentration of desnnosine was elevated while RNA and protein concentrationsremained unchanged compared to general controls (Table 7). Since DNA and hydroxyprolineconcentration decreased following three weeks of exposure to hypobaric hypoxia, the protein/DNA,DES/DNA and DES/OHP ratios increased significantly (Table 8).When all biochemical measurements (DNA, RNA, protein, hydroxyproline and desmosine)were expressed per unit body weight, specific values in the hypobaric hypoxic group were significantlygreater than those calculated for the general control group (Table 6). MORPHOMETRIC CHANGES(i). HBHY vs WMC: Three weeks of hypobaric hypoxic exposure resulted in significant increases laivand Valvave and a decrease in Sw/VL compared to the weight-matched control group but MLI, NA andNv remained unchanged (Table 9). The volume fraction of alveolar air increased significantly, but inaddition to the absolute volume of alveolar air, the volumes of alveolar wall and non-parenchymalChapter 3: RESULTS 91tissue also increased significantly in hypobaric hypoxic animals compared to weight-matched controls(Table 10-Table 11). Since the values of MLI and Nv did not alter, but lung volume increased, thealveolar surface area and total number of alveoli were increased (28% and 21% respectively) in ratsexposed to hypobaric hypoxia (Table 12).(ii). HBHY vs GC: With regards to morphometric changes in hypobaric hypoxia compared to generalcontrols, with the exception of mean chord length of alveoli which increased significantly, the valuesof unit structure morphometric parameters remained the same as those in general controls (Table 9).Absolute volumes of alveolar air, alveolar wall and non-parenchymal tissue were greater in hypobarichypoxic rats (Table 11). Exposure to hypobaric hypoxia also caused a significant increase (10%) inalveolar surface area but not in total alveolar number (Table 12).The Sw and Nat expressed per unit body weight were found to be higher in the hypobarichypoxic group than those in the general control group (Table 12).In brief, marked increases in absolute lung weight, lung volume, and biochemical andmorphonnetric measurements in hypobaric hypoxic rats compared with weight-matched controlsshowed that hypobaric hypoxia produced enhanced lung growth. Unlike normobaric hypoxia, exceptfor slight enlargement of alveolar size, the unit structure dimensions remained unaltered. Connectivetissue proteins accumulated markedly and accumulation of elastin was greater than accumulation ofcollagen. The results showed that hypobaric hypoxia produced hyperplastic as well as hypertrophicalterations in the lung compared to weight-matched controls.Although the percentage increases of absolute lung growth parameters in hypobaric hypoxicanimals were small compared to the general control group, this indicated that lung growth wasaccelerated by hypobaric hypoxia. Increase in lung growth occurred by increase in cell number andcell size without disturbing the dimensions of morphomatric unit structures. However, disturbance inconnective tissue protein balance was found as accumulation of elastin was relatively greater thancollagen as estimated by measuring hydroxyproline and desmosine contents.Table 11. Absolute volumes of parenchymal and non-parenchymal lung structures.VARIABLE BC GC WMC HBNO NBHY HBHYalv 2.50+0.07 6.48+0.37 5.13+0.13 6.10+0.16 7.52+0.12 7.29+0.19 •Vduct 1•00+0•04a 2.37+0•15b 1.93+0.12c 1.92+0.100 2.27+0.1113'c 2.14+0.09"cVwall 0.75+0.03a 1.70+0.07b 1.50+0.05° 1.67+0.051' 2.08+0•05d 1.98+0.08dVflp 1111 0•23+0•02a 0.44+0.06b 0.47+0.0613 0•50+0•05b 0.79+0.06° 0.67+0.07cVbr ml 0.13+0.02a 0.37+0.06" 0.26+0.05" 0•32+0•04b 0.25+0.04° 0.27+0.04°Vaiv (volume of alveolar air), Vduct (volume of duct air), Vwali (volume of alveolar wall), Vflp (volume of non-parenchymatous tissue), Vbr (volume ofbronchial air)Table 12. Absolute and specific morphometric parameters after 3 weeks of exposure.VARIABLE^BC^GC^WMC^HBNO^NBHY^HBHYSw (1112)^0.236+0.011a^0.527+0.018"^0.454+0.007c^0.553+0.011 b'd^0.566+0.01 b'd^0.582+0.012dNat (10+6)^13.9+1.2a^23.7+0.8"^21.1+0.6c^25.5+0.8"^23.6+0.9"^25.6+0.9"V100 g Wb (m2)^0.284+0.01a^0.202+0.006"^0.229+0.004c^0.232+0.005c^0.279+0.006a^0.288+0.007aNat/100 g Wb (10+5)^16.7+1.1a^9.1+0.3"^10.7+0.3°^10.7+0.5c'd^11.6+0.5c'd^12.6+0.4dMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Sw (internal alveolar surface area), Nat (total number of alveoli).Chapter 3: RESULTS^ 933.2. LUNG CYTOKINETICS STUDYTwo normobaric hypoxic rats died during injection of tritiated thymidine. The histologicalsections of the lungs and gross appearance of other organs showed no evidence of infection in controlor experimental animals used in this study.3.2.1. SOMATIC GROWTHDuring three weeks of exposure, body weight changes in all groups were similar to thoseobserved in the first experiment (Figure 8). In this experiment, the recovery from hypobaric normoxia,hypobaric hypoxia, normobaric hypoxia and undernutrition was also studied. Hypobaric nornnoxic,normobaric hypoxic and hypobaric hypoxic animals were returned to room air and weight-matchedcontrols were fed ad libitum for 3 days. During three days of recovery period (day 24 of theexperiment), body weight increased significantly in normobaric hypoxic (214.0 ±4.0 g), hypobarichypoxic (217.2 ±3.7 g) and weight-matched control (218.8 ±6.4 g) rats compared to their body weightson day 21 (196.2 ±5.7, 193.0 ±3.0 and 189.8 ±2.7 g respectively) of the experiment. In generalcontrols, body weight increased form 252.1 ±3.9 g to 273.6 ±7.2 g during three days of the recoveryperiod. Despite significant increases in body weights, the normobaric hypoxic , hypobaric hypoxic andweight-matched control groups had smaller body weight compared to the general control group. Thehypobaric normoxic rats which also weighed less after 21 days of exposure (231.5 ±5.0 g) failed tocatch up (251.3 ±3.5 g) to the general control group after 3 day recovery.Compared to the general control group, the nose-tail lengths of the hypobaric hypoxic,normobaric hypoxic and weight-matched control groups were also smaller but not until day 10 ofexposure was a significant difference (p<0.05) found (Figure 9) and they remained smaller even after3 days of recovery period. Although, the hypobaric normoxic group also had smaller nose-tail lengthmeasurements, the difference was not significant compared to the general control rats at any timeinterval studied (Figure 9).• GC • WMC^ HBHY^0 NBHYO HBNONose—ail length (cm)40 —38 —36 —34 —32 —30 —28 —2624..11^13^15^17^19^21 23 25Days of exposure_Chapter 3: RESULTS^943.2.2. ORGAN RESPONSENose—tail lengthFigure 9. Nose-tail length measurements of GC, WMC, HBHY, HBNO and NBHY at the time oftermination on days 1, 3, 5, 7, 10, 14, 21 and 3 days after returning to room air. NORMAL ORGAN GROWTHIn general controls, a significant increase in lung weight was found by day 7 (Figure 10), heartweight by day 10 (Figure 11) and spleen weight by day 7 compared to the values on day 1 of theexperiment (Figure 12). The weights of both kidney and liver appeared to increase more rapidly asthey were significantly higher on day 5 of experimental period (Figure 13 and Figure 14). EFFECT OF UNDERNUTRITIONThe results of right lung, heart, spleen, kidney and liver growth are shown in Figure 10-Figure 14. Significant increases in weights of the lung, kidney and liver did not occur until day 14 inweight-matched animals while heart and spleen weights increased significantly by day 21 comparedto day 1 of general controls. After 21 days of undemutrition, free access to food for 3 days resultedChapter 3: RESULTS^ 95in significant increases in lung, heart, kidney and liver weights but not in spleen weight (Figure 10-Figure 14).Compared to general controls, lung weights of weight-matched controls decreased on days3 and 5 but a significant difference was first noted on day 7 (p=0.008) and thereafter (Figure 10). Theheart and kidney weights were affected as early as day 5 and day 3 respectively of the experimentand were significantly smaller (p=0.008 and p=0.01 respectively) and continued to be smallerafterwards (Figure 11, Figure 13). Spleen weight also decreased during the early period ofundernutrition but a significant difference was not observed until day 14 (Figure 12), however, liverweight reduced significantly on day 1 (p=0.001) and remained so thereafter (Figure 14). After day 21of the experiment, food ad libitum for 3 days produced significant increases in liver and kidney weightsbut failed to reach the values of general control rats. The increases in the weights of lung, heart andspleen did not reach significant levels (Figure 10-Figure 14). EFFECT OF HYPOBARIC NORMOXIA (low ambient pressure)Gradual increases in weights were observed in all organs during exposure to hypobaricnormoxic conditions. Lung and liver weights increased significantly by day 7, heart and spleen by day10 while kidney weight increased by day 5 (Figure 10-Figure 14).The pattern of organ growth in hypobaric normoxic animals was similar to that in generalcontrols. No significant differences were found between hypobaric normoxic rats and general controlrats at any time interval during the exposure (Figure 10-Figure 14).Comparisons made between the hypobaric hypoxic group and normobaric hypoxic group toassess the effect of low ambient pressure also did not reveal any changes in growth of organs. Whenthe animals were returned to room air following chronic hypobaric normoxic exposure, no significantincreases in organ weights were observed3.2.2.4. EFFECT OF NORMOBARIC HYPDXIAChapter 3: RESULTS^96Figure 10. Right lung weights of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as animals 3 days after returning to room air.Compared to the values of day 1 general controls, the weights of lung and spleen increasedsignificantly on day 3 of exposure to normobaric hypoxia while a significant increase in heart weightwas found on day 7 (Figure 10-Figure 12). Growth of kidneys and liver appeared to be slow assignificant increases in weights were found on day 14 of normobaric hypoxic exposure (Figure 13 andFigure 14).When comparison was made between the normobaric hypoxic and weight-matched controlgroups, an increase in lung growth was evident by day 3 of exposure as lung weight was significantlyhigher in normobaric hypoxic rats (Figure 10). This increase continued during the rest of the exposure.The weights of spleen and heart were significantly greater, on days 5 and 7 respectively in thenormobaric hypoxic group compared to those in weight-matched controls and continued to be higherthereafter until the end of the experiment (Figure 11, Figure 12). However, neither kidney nor liverHeart weight (g)• GC^V HBHY 0 NE2Hyt3^5^7^9^11^13 15 17 19 21 23 25Days of exposure1 —0 9 —ID 8 —O 7 —O 6 —O 5-O 4O 3-O 2• Wk4C^0 BONOChapter 3: RESULTS^97weight differed from those recorded in weight-matched control rats (Figure 13, Figure 14).Heart weightFigure 11. Heart weights of undernourished, hypobaric hypoxic, hypobaric normoxic and normobarichypoxic animals as well as 3 days after returning to room air.Compared to general controls, lung weight showed a significant increase from day 3 ofexposure and thereafter (Figure 10). Heart and spleen weights also increased compared to thegeneral control group but never reached a significant level (Figure 11, Figure 12). As displayed inFigure 13 and Figure 14, the kidneys and liver gained less weight and were significantly smaller onday 3 of exposure and remained significantly smaller during the rest of the period compared to generalcontrols.Following removal of normobaric hypoxic stress for three days, an increase in growth of lung,heart and spleen was no longer apparent (Figure 10-Figure 12). Although slight increases in kidneyand liver weights occurred during the recovery period, they did not reach a significant level andremained significantly smaller than those of general controls. EFFECT OF HYPOBARIC HYPDXIAThe changes in organ growth rate were similar to those that occurred in normobaric hypoxicSpleen weight (g)O . 9NE1Hy........................O.8^ HEIHY O HEIN°O.70.6_-eO.5O.41 1 1 1 1 1 1 1 19 11 13 15 17 19 21 23 25O.25Chapter 3: RESULTS^98pleen weightDays of exposureFigure 12. Spleen weights of undernourished, hypobaric hypoxic, hypobaric normoxic and normobarichypoxic animals as well as 3 days of recovery.conditions. A significant increase in lung weight occurred by day 3 of exposure to hypobaric hypoxiacompared to general control day 1 lung weight (Figure 10). Hypobaric hypoxia produced increasesin heart and spleen weights on days 10 and 5 respectively (Figure 11-Figure 12). Significant increasesin the weights of liver and kidney were found on day 14 of hypobaric hypoxic exposure compared tothe day 1 values of general controls (Figure 13, Figure 14).The results of lung growth are displayed in Figure 10. Compared to weight-matched controls,lung weight increased significantly in hypobaric hypoxic rats on day 3 of exposure and continued tobe greater thereafter (Figure 10). The heart and spleen weights in hypobaric hypoxic rats weresignificantly higher in hypobaric hypoxic rats from days 7 and 5 respectively onwards compared toweight-matched animals (Figure 11, Figure 12). On the other hand, growth of both kidney and liverdeclined and the weights were equivalent to those noted in weight-matched control rats throughoutthe exposure (Figure 13, Figure 14).Compared with general controls, the lung weight increased on day 5 of hypobaric hypoxicexposure and continued to be higher until the end of the experiment (Figure 10). Relative to lungChapter 3: RESULTS^ 99Figure 13. Kidney weights of undernourished, hypobaric hypoxic, hypobaric normoxic and normobarichypoxic animals as well as 3 days following recovery.growth, heart and spleen growth adaptation occurred at a slower rate in hypobaric hypoxic rats as theirweights increased gradually and were significantly higher (p=0.008 and p=0.04 respectively) only onday 21 (Figure 11, Figure 12). On the other hand, the kidney and liver weights fell on day 1 ofhypobaric hypoxic exposure and were significantly lower on day 3 onwards (Figure 13, Figure 14).During three days of recovery following chronic hypobaric hypoxic exposure, lung and spleengrowth stopped while the rate of heart growth remained unaffected (Figure 10-Figure 12). On theother hand, the weights of kidney and liver increased markedly during 3 days of recovery periodcompared to those on day 21 of exposure, but did not reach the values of general controls (Figure 13,Figure 14).In short, it was evident that liver and kidney growth was diminished in normobaric hypoxic andhypobaric hypoxic conditions during the early period of exposure. These changes Were similar tothose occurred in weight-matched controls and never showed an adaptive response during theexposure. On the contrary, despite a decrease in somatic growth, the heart, spleen and lung weightsincreased markedly compared to weight-matched control animals. The lungs appeared to adapt earlierChapter 3: RESULTS^ 100Figure 14. Liver weights of undernourished, hypobaric hypoxic, hypobaric normoxic and normobarichypoxic animals as well as 3 days following recovery.than heart and spleen to both hypobaric hypoxia and normobaric hypoxia. Low ambient pressure didnot show a specific growth effect on any of the organs studied. In hypobaric hypoxic rats, 3 days ofrecovery period enhanced kidney and liver growth, but appeared to diminish spleen and lung growthrates and heart growth remained unaffected. In normobaric hypoxic rats, during the recovery period,the increase in growth of lung, heart and spleen stopped while liver and kidney showed growthstimulation. When food was provided ad lib to the weight-matched control rats, growth of all organsexcept the spleen was augmented.3.2.3. BIOCHEMICAL ALTERATIONS IN THE LUNG3.2.3.1. EFFECT OF UNDERNUTRITIONDry to wet weight ratio of the lung remained unchanged (Figure 15). Food restriction causeda significant decrease in RNA and protein on day 3 (p=0.05 and p=0.016) and these measurementsstayed lower than those of general controls until the end of the experiment (Figure 16, Figure 17). TheDry/Wet weight ratio of the lungDry/Wet weight ratio0.251111111^.13^5^7^9^11^13 15 17 19 21 23 25Days of exposure0.—0.20 • GC ^^ HBHY___^0 NBHY• WMC^O HEM_Chapter 3: RESULTS^ 101Figure 15. Changes which occurred in dry to wet weight ratio of the lung during exposure to differentconditions.amount of DNA also decreased on day 7, but a significant difference only appeared on day 14(p=0.004) and remained lower until day 21 (Figure 18). The RNA/DNA ratio fell significantly on day5 and remained consistently lower for the rest of the undernourished treatment (differences were notsignificant on days 10 and 21) (Figure 19). The protein/DNA ratio decreased significantly on days 3(p=0.003) also remained low afterwards (differences were not significant on day 10)(Figure 20).When weight-matched control rats were given free access to food for three days, the amountof DNA, RNA and protein content increased markedly (Figure 16-Figure 18). Increases in theRNA/DNA and protein/DNA ratios were apparent but the RNA/DNA ratio stayed significantly lower. EFFECT OF HYPOBARIC NORMOXIA (low ambient pressure)Dry to wet weight of the lung fell in hypobaric normoxic animals on day 1 but a significantdifference was observed on day 3 (Figure 15). No significant differences existed between hypobaricnormoxic rats and general controls for absolute (RNA, DNA and protein) or relative (RNA/DNA,protein/DNA) parameters (Figure 16-Figure 20). When hypobaric hypoxic animals and nornnobaricChapter 3: RESULTS^102Figure 16. The amount of lung RNA in GC, WMC, HBHY, HBNO and NBHY rats after 1, 3, 5, 7, 1014 and 21 days of exposure to different conditions as well as 3 days following recovery.hypoxic animals were compared for biochemical estimations of lung growth adaptation, the changeswere not different. EFFECT OF NORMOBARIC HYPDXIACompared with the weight-matched group, the RNA and protein content increased significantly(p <0.0008) on day 3 of exposure and continued to be higher thereafter (Figure 16, Figure 17).Normobaric hypoxia also produced an increase in the amount of DNA of the lung but it occurred atlater than RNA or protein. The amount of DNA was found to be significantly elevated on day 5(p=0.0008) and remained higher until the end of the exposure (Figure 18). The RNA/DNA andprotein/DNA ratios were significantly greater in hypobaric hypoxic animals from day 3 to 14 (Figure 19,Figure 20). On day 21, only the protein/DNA ratio was significantly higher in normObaric hypoxicanimals.Compared to the general control rats, dry to wet weight ratio of the lung fell on day 3 and 5but did not reach a significant level (Figure 15). The amount of RNA and protein in normobaricProtein content (mg)• GC • WMC^ HBHY^0 NBHYO HBNO........................10095908580757065605550454035303^5^7^9^11 13 15 17 19 21 23 25Days of exposureChapter 3: RESULTS^103Protein contentFigure 17. Lung protein content of GC, WMC, HBHY, HBNO and NBHY rats after days 1, 3, 5, 7, 1014 and 21 days of exposure to different conditions as well as 3 days following recovery.hypoxic rats increased significantly (p=0.002 and p=0.04 respectively) on day 3 of exposure andafterwards,but an increase in the amount of DNA was only noted on days 7-14 (Figure 18). As resultof the delayed increase in DNA content, a significant increase in the RNA/DNA ratio occurred on days3, 5 and 7 (Figure 19), and in the protein/DNA ratio on days 3, 5, 7 and 14 (Figure 20).Following removal of the rats from normobaric hypoxic conditions, no further increase in theamount of RNA, DNA and protein occurred (Figure 16-Figure 18). The RNA/DNA and protein/DNAratios fell and reached general control levels (Figure 19 and Figure 20). EFFECT OF HYPOBARIC HYPDXIAExposure to hypobaric hypoxia resulted in marked increases in biochemical parameterscompared to weight-matched animals. Significant increases were observed in RNA and proteincontent in hypobaric hypoxic animals on day 3 (p<0.0008) and in DNA content on day 5 (p=0.04), andthese increases continued thereafter until the end of exposure (Figure 16-Figure 18). The RNA/DNAChapter 3: RESULTS^104Figure 18. The amount of lung DNA content of rats subjected to different conditions for 1, 3, 5, 7, 10,14 and 21 days as well as 3 days following recovery.and protein/DNA ratios were also elevated on day 3 and continued to be so afterwards.When results were compared to those noted in general controls, dry/wet weight ratio of thelung decreased on day 3 and 5 but not significantly (Figure 15). RNA and protein content increasedsignificantly on day 3 (p=0.1 and p=0.45 respectively) and thereafter (Figure 16, Figure 17). Theabsolute amount of DNA also increased and reached a significant level on day 7 (p=0.008) andcontinued to be greater (Figure 18). As illustrated in Figure 19 and Figure 20, the RNA/DNA andprotein/DNA ratios were significantly increased on days 3 and 5.After three days of recovery, the rate of increase in the amount of total protein and DNA inhypobaric hypoxia appeared undisturbed but marked decrease in the amount of RNA was found(Figure 16-Figure 18). Hence a fall in RNA content produced a sharp drop in the RNA/DNA ratio,which reached the level found in weight-matched control rats (Figure 16, Figure 19). The protein/DNAratio also fell and reached the general control level (Figure 20).In brief, it was evident that lung growth adaptation in normobaric hypoxia and hypobarichypoxia occurred earlier during the exposure in terms of RNA and protein contents and this wasChapter 3: RESULTS^105Figure 19. The lung RNA/ DNA ratio of undernourished, hypobaric hypoxic, hypobaric normoxic andnormobaric hypoxic animals as well as 3 days following recovery.followed by hyperplastic changes as an increase in which DNA content lagged behind the changes'that occurred in RNA and protein. On the other hand, a reduction in all biochemical parameters wasfound in undernourished rats compared to general controls rats. Therefore, increases in biochemicalmeasurements were much greater in hypobaric hypoxic and normobaric hypoxic groups compared tothe weight-matched group than compared to the general control group. The RNA/DNA andprotein/DNA ratios, which are considered as indicators of cellular dimensions, also increased duringthe first week of exposure and remained slightly higher for the remaining period. The amount of RNA,protein and DNA did not alter following exposure to low ambient pressure. The three day recoveryperiod appeared to stimulate lung growth in the undernourished rats, whereas it appeared to reduceincreased lung growth in hypobaric hypoxic and normobaric hypoxic rats.3.2.4. DNA SYNTHESISTritiated thymidine (3H-TdR) incorporation in DNA was estimated biochemically and expressed2322212019181716 —15 —14 —13 —12PRO-MIN/DNAI^IIIIIIIII• GC • WMC^ H8HY^0 NBHYO HBNO____-----------Chapter 3: RESULTS^106PROTEIN/DNA9^11^13 15 17 19 21 23 25Days of exposureFigure 20. The effect of undernutrition, hypobaric hypoxia, hypobaric normoxia and normobarichypoxia on the lung protein/DNA ratio.as counts per minute/ .t.g of DNA (Figure 21). EFFECT OF UNDERNUTRITION3H-TdR incorporation was significantly decreased in the weight-matched control rats on dayone of the experiment. DNA synthesis fell further between day 3 and 5 and it remained depressedfrom then onwards. When the weight-matched control rats were given free access to food after 21days of undernutrition, a three fold increase in the rate of DNA synthesis was noted on day 24 of theexperiment. OF HYPOBARIC NORMOXIAThe hypobaric normoxic rats, showed an increase in the DNA synthetic rate on days 1 to 7,but a significant change was only found on day 5 of exposure compared to that of general controlanimals. The rate of 3H-TdR incorporation reached general control levels on day 10 of exposure.When hypobaric hypoxic and normobaric hypoxic animals were compared for the effect of reducedDNA SYNTHESISCPM/ug of DNA content550 -500 -450 -400 -350 -300-250-200 -^\*4`.150 - 100 / 50• GC• WMC• HH O HNO..... ................... ...........111111111[115^7^9^11^13^15^17^19^21^23^25Days of exposure1^3Chapter 3: RESULTS^ 107Figure 21. Tritiated thymidine incorporation into the lungs of rats subjected to hypobaric hypoxia,hypobaric normoxia, normobaric hypoxia and undernutrition for 1, 3, 5, 7, 10, 14 and 21 days as wellas 3 days following recovery.ambient pressure, no differences were found between the groups for DNA synthetic activity. OF NORMOBARIC HYPDXIAWhen the results of normobaric hypoxic group were compared to those found in the weight-matched controls, 3H-TdR incorporation was similar on day 1, increased more than twelve times onday 3, seventeen times on day 5 (because DNA synthesis dropped further on day 5 than on day 3 inthe weight-matched control group), nine times on day 7 and remain significantly higher for rest of theexposure (Figure 21).Compared to the general control animals, the rate of DNA synthesis in normobaric hypoxicanimals was significantly diminished on day one. It increased sharply (six and a half fold) on day 3Chapter 3: RESULTS 108of exposure. The increase fell to four fold on day 5, two fold on day 7 and then declined further.Although the rate of 3H-TdR incorporation reached general control levels, a significant increase wasobserved on day 21 in nornnobaric hypoxic animals.When the normobaric hypoxic animals were returned to room ambient conditions for threedays, 3H-TdR incorporation declined and reached normal levels. EFFECT OF HYPOBARIC HYPDXIAThe results of 3H-TdR incorporation in the hypobaric hypoxic rats were similar to those noticedin the normobaric hypoxic rats. Following a drop in the rate of DNA synthesis on day 1, it increasedtwelve fold on day 3, seventeen fold on day 5, eight fold on day 7, four fold on day 10, three fold onday 14 and four fold on day 21 compared to that in weight-matched controls.To summarize, exposure to hypobaric hypoxic or normobaric hypoxic conditions stimulatedDNA synthesis in the lungs. After an initial fall on day 1, it peaked on day 3 followed by a gradualdecline until day 10 when it reached close to normal levels but stayed consistently higher until the endof exposure compared to weight-matched animals. On day 21, DNA synthesis in hypobaric hypoxicand normobaric hypoxic animals showed a significant increase. After returning the hypobaric hypoxicand normobaric hypoxic animals to room air, 3H-TdR incorporation reached normal levels (generalcontrols). An increase in DNA synthesis in hypobaric hypoxic and normobaric hypoxic rats was muchgreater when comparison was made with those of weight-matched control rats, where it wasdepressed throughout the exposure. 3H-TdR incorporation increased in weight-matched control ratswhen they were given food ad libitum. DNA synthesis also increased in the rats exposed to lowambient pressure alone and peaked on day 5, but the percentage increase was lower than thatobserved in the normobaric hypoxic or hypobaric hypoxic rats.3.2.5. AUTORADIOGRAPHYCombined alveloar wall cellsV H8HY^0 N8HY0 H8NO_7^9^11^13^15^17^19^21Days of exposure54 . 543 . 532 . 521 . 51. 50 % labelled cellsChapter 3: RESULTS 109Autoradiographs of general controls and rats subjected to hypobaric normoxia, normobarichypoxia, hypobaric hypoxia and undernutrition for 1, 3, 5, 7 and 21 days were analyzed for pulmonarycell kinetics. 3H-TdR labelled and unlabelled alveolar wall cells were counted in three different regions(central, peripheral and subpleural) of the lung (see materials and methods). Alveolar wall cellsincluding, type I pneumonocytes, type II pneumonocytes, alveolar wall capillary endothelial cells,interstitial cells and unidentifiable cells were counted and labelling indices were computed. Since theresults of subpleural and peripheral alveolar wall cells were similar, the observations of both sites werepooled and referred as peripheral alveolar wall cells.Figure 22. 3H-TdR incorporation in combined alveolar wall cells (central+peripheral) in the lungs ofGC, WMC, HBHY, HBNO and NBHY animals subjected to different conditions for 1, 3, 5, 7 and 21days. EFFECT OF UNDERNUTRITIONThe combined alveolar wall cell labelling index (LI) declined on day 1, but a significantdecrease were found on days 3, 5, 7 and 21 of exposure (p=0.0016, p=0.0008, p=0.016 and p=0.016respectively) in undernourished rats compared to general control rats (Figure 22). The LI of freeTotal free alveolar macrophages2018161412108642-0lobetIod fro. civookr nwocchopea5^7^9^11^13^15^17^19^21Days of exposureChapter 3: RESULTS^ 110alveolar macrophage and mast cells remained unaffected (Figure 23, Figure 24). CENTRAL ALVEOLINo detectable changes were found in the LI of type I pneumonocytes (Figure 25). Althoughthe LI of type II pneumonocytes was diminished during the experimental period, no significantdifference was detected between the weight-matched control group compared to the general controlgroup (Figure 26). The LI of endothelial cells fell and reached a significant level only on day 5(p=0.0008) (Figure 27). Compared to general controls, except for day 1, 3H-TdR incorporation ininterstitial cells was significantly decreased throughout the experiment (Figure 28). The LI ofunidentifiable cells remained unchanged (Figure 29).Figure 23. The effect of undernourishment, hypobaric hypoxia, hypobaric normoxia and normobarichypoxia on the labelling index of free alveolar macrophages. PERIPHERAL ALVEOLINo changes were found in the labelling indices of mesothelial cells or type I pneumonocytes(Figure 30, Figure 31) compared to general controls. The labelling indices of type II pneumonocytes,endothelial cells and interstitial cells fell in weight-matched animals and were significantly lower on19.^217^9^11^13^15^17Days of exposure% labelled most cells1210• GC • WMC^ HBHY O HBNO_0 NBHY.............^.......................................Chapter 3: RESULTSmast cellsFigure 24. The mast cell labelling index in the lungs of undernourished, hypobaric hypoxic, hypobaricnormoxic and normobaric hypoxic animals.days 5 and 7 (Figure 32, Figure 33, Figure 34) but the decline in the LI of unidentifiable cells did notreach a significant level (Figure 35). NON-PARENCHYMAA significant decrease in 3H-TdR incorporation in bronchiolar epithelium was observed on day7 and in bronchial epithelium on day 3 and 7 in animals subjected to limited food access comparedto the free fed animals (Figure 36-Figure 37). The labelling indices of bronchial wall cells, arterialendothelium and arterial wall cells declined on day 3 of the experiment and remained low thereafter(Figure 38, Figure 39, Figure 40).In short, the cytokinetic results showed that 3H-TdR incorporation in combined alveolar wallcells fell due to undernutrition in the weight-matched animals which was mainly 'a reflection ofdepressed DNA synthesis in type ll pneumonocytes, endothelial cells and interstitial cells. The effectof undernutrition was apparent on day 1 but became more apparent on day 5 and 7 of theexperimental period.Chapter 3: RESULTS^ 1123.2.5.2. EFFECT OF HYPOBARIC NORMOXIACompared to the general control group, the rats exposed to the low ambient pressure, showeda significant increase in the combined alveolar wall cell labelling index on days 5 and 7 of theexperiment (Figure 22). However, when the hypobaric hypoxic and normobaric hypoxic groups werecompared to observed the effect of low pressure, no significant difference was found. The LI of freealveolar macrophage and mast cells did not change in hypobaric normoxic rats (Figure 23, Figure 24). CENTRAL ALVEOLICompared to the general control group, except a significant increase in the interstitial cells onday 7 (p=0.0008) (Figure 28), the labelling indices remained unchanged in all cell types. The LI of thetype II pneumonocytes peaked on day 7 but never reached a significant level (Figure 26). PERIPHERAL ALVEOLICompared with the general control group, the number of labelled nnesothelial cells in thehypobaric normoxic group was significantly higher (p=0.04) on day 3 of hypobaric normoxic exposure(Figure 30). The LI of type II pneumonocytes was also increased and reached a significant level onday 5 (Figure 32) whereas the interstitial cell labelling index was significantly higher on days 5 (p=0.03)and 7 (p=0.0008) of exposure to hypobaric normoxia (Figure 34). NON-PARENCHYMAThe LI of bronchial wall cells increased significantly on day 5 (Figure 38) whereas the LI ofarterial wall cells (Figure 40) increased on days 3 and 5 in hypobaric normoxic rats compared togeneral controls. The labelling indices of bronchiolar and bronchial epithelium (Figure 36, Figure 37)and arterial endothelial cell did not change (Figure 39). On the other hand, low ambient pressure didnot cause any significant change in cellular dynamics of non-parenchymal cells when the hypobarichypoxic and normobaric hypoxic groups were compared (Figure 36-Figure 40).Central type I pneumonocytes01^3^5^7^9^11^13^15^17^19^21Days of exposure% labelled cells54 5 —143 . 53 —2 . 521 . 510 . 5• GC ^^ H8HY^0 NBHY• WMC O HBNO_Chapter 3: RESULTS 113In summary, the combined alveolar wall cell labelling index appeared to peak on day 5 of lowambient pressure exposure. Mesothelial cell 3H-TdR incorporation was maximum on day 3.Peripheral type II pneumonocytes, interstitial cells and unidentifiable cells showed maximum 3H-TdRincorporation on day 5, whereas central type II pneumonocytes and interstitial cells reached theirmaximum 3H-TdR incorporation on day 7. 3H-TdR incorporation in endothelial cells remainedunaffected both in the central and peripheral regions of the lung. DNA synthesis in arterial andbronchial wall cells was stimulated by lower ambient pressure while in arterial endothelial andconducting airways epithelial cells it remained unaltered.Figure 25. The cytokinetics of type I pneumonocytes in the walls of central alveoli. EFFECT OF NORMOBARIC HYPDXIAAs displayed in Figure 22, 3H-TdR incorporation in combined alveolar wall cells wassignificantly decreased on day 1 of exposure to normobaric hypoxia compared with the general controlrats and reached the value of weight-matched control rats. On day 3 of exposure to normobarichypoxia, DNA synthesis in alveolar wall cells increased markedly and was nine times higher comparedChapter 3: RESULTS^ 114Figure 26. Tritiated thymidine incorporation in type II pneumonocytes in the walls of central alveoli.to weight-matched controls and four times higher than general controls. After day 3, the labellingindex of combined alveolar wall cells in normobaric hypoxic rats began to decline gradually butremained consistently higher than weight-matched control animals. On day 21 of exposure, their LIremained higher compared to both weight-matched controls (p=0.0008) and general controls(p=0.024). The LI of free alveolar macrophage cells increased significantly on day 5 (p=0.04)compared to weight-matched animals (Figure 23). The mast cell labelling index in normobaric hypoxicrats was significantly elevated on day 1 and remained higher on days 3 and 5 compared to that ofweight-matched controls but not compared to general controls (Figure 24). CENTRAL ALVEOLINo change in the proportion of labelled type I pneumonocytes was observedt in normobarichypoxic animals (Figure 25). As shown in Figure 26, the rate of 3H-TdR incorporation in type IIpneumonocytes fell on day 1 (p=0.05) compared to general controls and was indistinguishable fromthat of weight-matched controls. It was followed by a sharp rise on day 3 (p=0.016) which sustainedlabelled cells6 -5.5 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0 . 50 I^I^I it21• GC • WMC^ HBHY^0 NBHYO REMO_•3^5^7^9^11^13^15^17^19'Days of exposureChapter 3: RESULTS^115Central endothelial cellsFigure 27. The capillary endothelial cell labelling index in the walls of central alveoli.until day 5 (p=0.0008) of exposure (the % increase was greater on day 5 than day 3) compared to thatof the weight-matched control group. Thereafter the labelling index of type II pneumonocytes fell onday 7 but was significantly higher compared to the weight-matched control (p=0.003) and reachednormal levels on day 21. Endothelial cell 3H-TdR incorporation also dropped after one day innormobaric hypoxic conditions but a significant increase was observed on day 3 (11.5 times weight-matched controls and 4.5 times general controls) and on day 5 it began to decline (Figure 27). Aprecipitous fall in the labelling index of endothelial cells did not occur until day 7 when it reachedgeneral control levels but remained higher than weight-matched controls.Following a small drop on day one of exposure to normobaric hypoxia, 3H-TdR incorporationin interstitial cells rose significantly on day 3. After maximal stimulation on day 5 it fell on day 7 ofexposure but was significantly higher compared to both weight-matched and general controls(Figure 28). On day 21, the rate of 3H-TdR incorporation was similar as that of general and weight-matched controls. The LI of the unidentifiable cells was significantly higher on day 3 (p=0.048) and5 (p=0.002) compared to the weight-matched control group, and day 5 compared to the general controlChapter 3: RESULTS^ 116(p=0.008) group but thereafter it reached normal levels (Figure 29). PERIPHERAL ALVEOLIUpon exposure to normobaric hypoxia, the labelling indices of all cells fell on day 1 ofexposure and such changes were identical to those found in weight-matched animals. Although themaximal increase in the proportion of labelled mesothelial cells was found on day 3, this change,however, was not significant (because of the large variance). A significant change was only observedon day 5 (p=0.03) compared to weight-matched animals (because of a continued decrease in labellingindex of mesothelial cells in the weight-matched group) but not compared to general controls(Figure 30). After day 3, the mesothelial cell labelling index fell gradually,and was similar to that ofweight-matched or general controls on days 7 and 21 of exposure.Figure 28. Cytodynamics of interstitial cells in the walls of central alveoli of undernourished, hypobarichypoxic, hypobaric normoxic and normobaric hypoxic rats.Autoradiographs showed that maximal incorporation. of 3H-TdR into the peripheral type IIpneumonocytes occurred on day 3 compared to both weight-matched-(p=0.007) and general control(p=0.01) groups (Figure 32). On day 5, the type II pneumonocyte LI fell sharply and this was followedChapter 3: RESULTS 117by a further decrease on day 7 and finally reached the general control level at day 21 of exposure.Similar to the central alveoli, on day 3 of exposure, the LI of the capillary endothelial cells showed aten fold increase compared to weight-matched controls and a six fold increase compared generalcontrols. It fell sharply on day 5 but was significantly higher on day 21 compared to both weight-matched and general control rats (Figure 33).The pattern of the rate of 3H-TdR incorporation in the peripheral interstitial cells was alsosimilar to those of peripheral type II pneumonocytes and endothelial cells. Following a decrease onday 1, the LI of interstitial cells peaked on day 3 of exposure and then declined gradually to reach thelevel of general controls on day 21 of exposure but was significantly higher compared to that in weight-matched animals (Figure 34). The proportion of unidentifiable labelled cells also peaked on day 3 andthen decreased sharply on day 5 and continued to fall and reached the level of general controls onday 7 but was significantly higher compared to weight-matched controls (p=0.03). No difference wasfound between both control groups and normobaric hypoxic rats for the LI of unidentifiable cells on day21 of exposure (Figure 35). NON-PARENCHYMAThe proportion of bronchiolar and bronchial epithelial cells, bronchial wall cells, arterialendothelial cells and arterial wall cells which incorporated 3H-TdR in normobaric hypoxic animalsincreased significantly on days 3, 5, 7 and 21 of exposure compared to weight-matched controls(Figure 36-Figure 40). The maximal response was observed on day 3 and thereafter it declinedgradually. Compared to general controls, significant increases in the labelling indices of bronchiolarepithelial cells on day 3 (280%), bronchial epithelial cells on days 3 (88%) and 5, bronchial wall cellson days 3 (262% )and 5, arterial endothelial cells on days 3 (247%), 5 and 7, and arterial wall cellson days 3 (483%), 5 and 7 were also observed (Figure 36-Figure 40).In summary, it was evident from the results that DNA synthesis activity in all cells wasdepressed on the first day of exposure to normobaric hypoxia which was similar to that of weight-Central miscellaneous cells% labelled cellsV MBHY0 1-181Y 0 HBNCL............... ..... ..... ..... ......5^7^9^11^13^15Days of exposureI^I I^I^I^17^19^2154.543.532.521.510.50Chapter 3: RESULTS^ 118Figure 29. The labelling index of unidentifiable cells in the walls of central alveoli.matched control animals but on day 3 a dramatic increase in the proportion of 3H-TdR labelled cellsoccurred. Thereafter 3H-TdR incorporation in the combined alveolar wall cells gradually decreasedbut stayed slightly higher during the rest of the exposure period. 3H-TdR incorporation in peripheraltype ll pneumonocytes, interstitial cells and unidentifiable cells peaked on day 3 of exposure. The LIof the central type II pneumonocyte, interstitial cells and unidentifiable cells reached a peak on day5, but the LI of both central and peripheral alveolar wall endothelial cells peaked on day 3. At the timeof maximal stimulation, the percentage increase in the LI in the wall of peripheral alveoli was slightlyhigher compared to the central alveoli. The results of autoradiographs showed that peak stimulationmesothelial cells occurred on day 3 while free alveolar macrophage were stimulated on day 5 ofnormobaric hypoxic exposure. The mast cells appeared to be affected by normobaric hypoxia fromthe very first day of exposure and remained consistently higher before sharply falling after day EFFECT OF HYPOBARIC HYPDXIAThe results of combined alveolar wall cell 3H-TdR incorporation is shown in Figure 22. AsNBHY^ HOMY O HMO_...............................9^11^13^15^17^19^21Days of exposure• GC • WMCChapter 3: RESULTS^119Mesothelial cells% labelled cellsFigure 30. The mesothelial cell labelling index in the lungs of undernourished, hypobaric hypoxic,hypobaric normoxic and normobaric hypoxic animals.noted in normobaric hypoxic animals, the number of labelled combined alveolar wall cells droppedsignificantly (p=0.02) on day 1 of exposure to hypobaric hypoxia compared to that of general controlsand was equivalent to that of weight-matched controls. On day 3, a sharp and significant increase(331%) in the labelling index was observed which dropped to 295% on day ,, 142% on day 7 and84% on day 21 of exposure compared to the general control group. Since the labelling index ofcombined alveolar wall cells was depressed throughout the experimental period in weight-matchedanimals, except for day 1 an increase in the labelling index of alveolar wall cells in the hypobarichypoxic animals was highly significant compared to the weight-matched group. The labelling indexof free alveolar macrophages was significantly increased in hypobaric hypoxic animals on day 5compared to both weight-matched controls (p=0.008) and general controls (p=0.02) (Figure 23). Theproportion of mast cells which incorporated 3H-TdR increased significantly on days 1, 3 and 5compared to weight-matched controls (Figure 24).Peripheral type I pneumonocytes% labelled cells1^3^5^7^9^11^13^15^17^19^21Days of exposure• GC • WMC54 . 543 . 532 . 521 . 510 . 50.V HBHY ^0 NBHY0 HBNOChapter 3: RESULTS^120Figure 31. Cytokinetics of type I pneumonocytes in the walls of peripheral alveoli. CENTRAL ALVEOLINo change in the labelling index of type I pneumonocytes was noticed (Figure 25). Comparedto weight-matched controls, except for day 1, the labelling index of type II pneumonocytes wassignificantly higher (p<0.007) throughout the exposure in hypobaric hypoxic animals (Figure 26). Whencomparison was made with the general control group, a significant increase in the labelling index oftype II pneumonocytes was observed on days 3 (p=0.008) and 5 (p=0.002) and following a sharp dropon day 7, it eventually reached at the general control group levels on day 21. Except on days 1 and21, the endothelial cell labelling index was significantly higher (p <0.007) compared to the weight-matched rats (Figure 27). Compared to general controls, endothelial cell 3H-TdR incorporation wassignificantly lower (p=0.048) on the first day but increased markedly on day 3 (256%), 184% on day5 and dropped thereafter to reach the control levels on days 7 and 21 (Figure 27).Compared to general controls, apart from slightly less 3H-TdR incorporation in the interstitialcells on day 1, a significant increase on day 3 (p=0.0008) and further increment on day 5 wereobserved. On day 7, the rate of increased 3H-TdR incorporation declined but remained significantlyPeripheral type II pneumonocytes% labelled cells-• CC ^V H8HY ^0 NBHY• WMC 0 H8NO: _____ .....^......................................9^11Days of exposure3^5^7 13 1554 . 543 . 532 . 521 . 510 . 50Chapter 3: RESULTS^121Figure 32. The labelling index of type II pneumonocytes in the walls of peripheral alveoli ofundernourished, hypobaric hypoxic, hypobaric normoxic and normobaric hypoxic rats.higher (p=0.04) and reached the general control group values on day 21 (Figure 28). On the otherhand, compared to the weight-matched group, the labelling index of the interstitial cells in hypobarichypoxic group remained significantly higher from day 3 through day 21 of exposure (Figure 28). TheLI of the unidentifiable cells were also significantly greater on day 5 than general controls, and on days3 and 5 compared to the weight-matched controls (Figure 29). PERIPHERAL ALVEOLISimilar to normobaric hypoxic animals, 3H-TdR incorporation in all cells in the walls ofperipheral alveoli was depressed on day 1 of exposure and labelling indices were identical to thoserecorded in weight-matched controls. The LI of mesothelial cell increased on days 3, 5 and 7, andmaximal 3H-TdR incorporation occurred on day 3 but a significant increase (p=0.003) was achievedonly on day 5 (Figure 30) compared to weight-matched controls. After day 1 of exposure to hypobarichypoxia, the labelling indices of type ll pneumonocytes, alveolar wall endothelial cells, interstitial cellsand unidentifiable cells increased markedly on day 3 of exposure and remained significantly highChapter 3: RESULTS^ 122Table 13. Cell composition of the walls of the peripheral alveoli.VARIABLE GC WMC HBHY HBNO NBHYType I pneumonocytes (°/0)Day 1 9.98+0.37 10.78+0.59 9.96+0.4 10.33+0.59 10.16+0.37day 3 10.01+0.17 9.54+0.38 10.01+0.58 10.31+0.34 10.02+0.16Day 5 10.11+0.39 9.85+0.57 9.53+0.46 10.01+0.51 9.77+0.24Day 7 10.08+0.36 9.59+0.35 9.43+0.47 9.62+0.22 9.06+0.45Day 21 10.08+0.25 9.88+0.22 10.58+0.32 10.09+0.4 10.07+0.17Type ll pneumonocytes (%)Day 1 14.47+0.55 15.49+0.47 14.22+0.43 15.09+0.39 14.85+0.57Day 3 15.51+0.20 14.51+0.65 16.04+0.48 15.66+0.6 14.57+0.87Day 5 15.53+0.4 15.37+0.2 14.32+0.46 14.73+0.6 14.03+0.27Day 7 14.56+0.77 13.75+0.93 16.65+0.76 14.80+0.55 15.28+0.73Day 21 14.69+0.66 15.46+0.56 15.83+0.49 15.64+0.28 15.05+0.51Capillary endothelial cells (°/0)Day 1 29.93+0.28 28.62+1.37 27.14+1.89 29.17+1.94 30.58+0.74Day 3 30.59+0.45 31.27+0.87 29.23+1.31 30.28+0.92 30.66+0.69Day 5 30.43+0.3 30.92+0.25 30.14+0.75 30.76+1.89 33.56+2.11Day 7 29.96+0.53 29.91+0.62 28.99+0.75 28.69+0.26 30.49+1.07Day 21 31.25+1.52 29.87+1.84 29.72+1.35 28.52+1.07 31.85+1.25Interstitial cells (%)Day 1 34.00+0.46 33.27+1.06 33.79+0.85 33.76+1.56 31.85+0.55Day 3 32.60+0.31 34.14+2.11 32.84+0.36 30.94+0.47 32.11+0.82Day 5 32.24+0.38 32.44+0.71 34.71+1.21 32.66+1.3 32.27+1.76Day 7 31.22+1.64 32.85+0.46 32.68+0.83 35.64+0.51 31.58+1.12Day 21 31.19+1.04 32.63+1.22 33.25+0.7 33.31+1.12 31.02+0.75Unidentifiable cells (%)Day 1 11.25+0.22 11.81+1.52 14.89+1.58 11.64+1.19 12.54+0.78Day 3 11.29+0.36 10.53+0.54 11.86+0.25 11.80+0.6 12.62+0.73Days 11.67+0.43 11.40+0.32 11.29+0.49 12.83+1.06 10.35+0.55Day 7 15.18+0.56 13.89+0.99 12.24+1.29 12.25+0.36 12.59+0.36Day 21 12.79+0.71 12.17+0.84 10.61+0.29 12.42+0.44 12.01+0.35Type I/Type ll pneumonocyte ratioDay 1 0.69+0.05 0.70+0.04 0.71+0.04 0.70+0.05 0.69+0.04Day 3 0.65+0.02 0.66+0.03 0.62+0.03 0.66+0.02 0.70+0.05Day 5 0.65+0.02 0.64+0.03 0.67+0.04 0.67+0.04 0.70+0.03Day 7 0.69+0.04 0.71+0.06 0.57+0.06 0.65+0.03 0.59+0.06Day 21 0.69+0.02 0.64+0.03 0.67+0.03 0.65+0.02 0.67+0.02No differences were found between the groups.if-----D.-------. --iv ^ •1^1^i^1^r^1^1^i^I,^i3^5^7^9^11^13^15^17^19^21Doys of exposure6 -5 . 5 -5 -4 . 5 -4 -3 . 5 -32 . 5 -2 -1 . 5 -10 . 50• GC • WMC^ HBHY O HBNO_NBHY -------------------------------------------------------------------Chapter 3: RESULTS^123Peripheral endothelial cells% labelled cellsFigure 33. The capillary endothelial cell labelling index in the walls of peripheral alveoli followingexposure to different experimental conditions.(p<0.01) until the end of exposure (except unidentifiable cells were not significantly different on days7 and 21) compared to weight-matched animals (Figure 32-Figure 35).When hypobaric hypoxic animals were compared to general controls, 3H-TdR incorporationin type ll pneumonocytes was enhanced in hypobaric hypoxic animals on day 3 by 449%, and on day5 by 303%. It decreased to 95% on day 7 and reached at a normal level on day 21 (Figure 32). Theendothelial and interstitial cell labelling indices also declined significantly (p=0.02 and p=0.05respectively) on day 1, but they rose sharply on day 3 and remained significantly higher on days 5,7and 21 (p <0.03) in hypobaric hypoxic rats (Figure 33). 3H-TdR incorporation in unidentifiable cellsalso increased but a significant change was only found on days 3 (p=0.016) and 5 (p=0.0008)(Figure 35). NON-PARENCHYMAThe number of bronchiolar and bronchial epithelial cells, bronchial wall cells, arterialendothelial cells and arterial wall cells which incorporated 3H-TdR in hypobaric hypoxic animals peakedlabelled cells76 . 565 . 554 . 543 . 532 . 521 . 510 . 50 ^1^3^5• GC • WMC•T' --------- .......... ..7^9^11^13^15Days of exposureNBHY17^19^21• HBHY O HBNO_11^1^1^I,^IChapter 3: RESULTS^124Peripheral interstitial cellsFigure 34. The interstitial cell labelling index in the peripheral alveoli of undernourished, hypobarichypoxic, hypobaric nornnoxic and normobaric hypoxic animals.on day 3 of exposure and afterwards declined but remained significantly higher than those of weight-matched animals (Figure 36-Figure 40).Compared to the general control group, the labelling indices of bronchiolar and bronchialepithelial cells increased by 340% and 133% respectively on day 3. The arterial endothelial cell andarterial wall cell labelling indices increased significantly on days 3 (222% and 550% respectively), 5and 7 while the bronchial wall cell labelling index increased on days 3 (287%) and 5 in hypobarichypoxic rats compared to general controls (Figure 38-Figure 40). At the end of exposure, the labellingindices of all cells reached at the normal level.In brief, these findings have demonstrated that alveolar wall cells or cells in the non-parenchymal compartment of the lung were stimulated during the first week of exposure to hypobarichypoxic conditions. On day one, the labelling indices of all cells decreased and the change wassimilar to that observed in the undernourished animals. Thereafter, the labelling indices of cells inweight-matched animals continued to fall but in hypobaric hypoxic animals increased dramaticallyresulting in marked increases. Although the combined alveolar wall cell labelling index peaked on dayPeripheral miscellaneous cells% labelled cellsIII GC • WMCV HBHY 0 HBNO._0 NBHY 0,19^11^13^15^17^19^21Days of exposure54 . 543 . 532 . 521 . 510 . 50Chapter 3: RESULTS^125Figure 35. The labelling index of unidentifiable cells in the peripheral alveoli of GC, WMC, HBHY,HBNO and NBHY rats.3, the percentage increase in the labelling index on day 5 was greater than day 3 when compared toweight-matched controls. This occurred because the labelling index in weight-matched controlscontinued to fall until day 7 of the experiment. Following a peak stimulation of combined alveolar wallcell on day 3, DNA synthetic activity begun to declined gradually thereafter to reach the general controllevels on day 7 and stayed at a slightly higher level until the end of the exposure period. Similar tonormobaric hypoxia, the mesothelial cells, type ll pneumonocytes, alveolar capillary endothelial,interstitial and unidentifiable cells in the walls of peripheral alveoli also had maximum incorporation of3H-TdR on day 3 of exposure to hypobaric hypoxia. In the walls of central alveoli, the labelling indexof endothelial cells peaked at day 3 but the labelling index of type II pneumonocytes, interstitial andunidentifiable cells peaked on day 5. The overall assessment revealed that 3H-TdR incorporation intype II pneumonocytes, interstitial and unidentifiable cells in peripheral alveoli: and type llpneumonocytes and interstitial cells in central alveoli in hypobaric hypoxic animals was highercompared to those in normobaric hypoxic animals. The labelling indices of endothelial cells andunidentifiable cells in the walls of peripheral and central alveoli were lower in the hypobaric hypoxic1 0% labelled cells-9 -8 -7 -6 -5 -4 -3 -2 -III GC • WMCV HBHY 0 HBNO_0 NBHY 09^11^13^15^17^19^21Days of exposureChapter 3: RESULTS^126Bronchiolar epithelial cellsFigure 36. Tritiated thymidine incorporation in bronchiolar epithelial cells following exposure todifferent experimental conditions.group than in the normobaric hypoxic group. The results also showed that DNA *thesis in freealveolar macrophage cells and mast cells increased during early exposure to hypobaric hypoxia.• GC ^^ HBHY ^0 NBHY• WMC^O HEM_% labelled cells10.--1•^f I^I^I^I^I^I^1^I3^5^7^9^11^13Days of exposure987654321015 17 19 21Chapter 3: RESULTS^127Bronchial epithelial cells1 0 —9 —8 —7 —6 —5 —4 —3 —2 —1% labelled cellsi: _4--• GC V HBHY^0 NBHY0 H81,10• WMC___---._—-r—r.-------Ij0 f11r—i3^5 719i11113Ii^i^I^415^17^19^21Days of exposureFigure 37. The bronchial epithelial cell labelling index after subjecting animals to different experimentalconditions.Bronchial wall cellsFigure 38. 3H-TdR incorporation in the bronchial wall cells following exposure to different conditions.III GC • WMC0 NBHYH81-1Y 0 HEN°4 . 543 . 5 —3—2 . 5 —112 —^5:\. 5 — f13^157^9^11Days of exposure050 I^^I17^19^213^5: Lc^ov HHeNHY0_7 —6 — .-5 —4 —113 —^\ ------2 — ---- -------- ----- ----- .....1• 1111 0% labelled cells—9 —8 —0 NBHY............. ....Chapter 3: RESULTS^128Pulmonary arterial endothelial cells5 —% labelled cellsFigure 39. Cytokinetics of arterial endothelial cells following exposure to hypobaric hypoxia, hypobaricnormoxia, normobaric hypoxia and undernutrition.Arterial wall cells0 1^ •II^3^5^7^9^11^13^15^17^19^21Days of exposureFigure 40. The labelling index of arterial wall cells following exposure to hypobaric hypoxia, hypobaricnormoxia and normobaric hypoxia, and restricted feeding.A. Labelled cells in the peripheral part of the lung of hypobaric hypoxic rats after 3 days of exposureB. Labelled bronchiolar epithelial cells and alveolar wall cells in hypobaric hypoxic animals after 3days of exposure (X800)(X200).Chapter 3: RESULTS^129Figure 41. Autoradiographs showing cells heavily laden with black granules following tritiatedthymidine incorporation.15E'.-10LUNG VOLUMES AND CAPACITIESE2:21 GC^Ell HBHY^NBHY20 —^ WMC^HBNOI^I• BIhiLIE111 .^••05•::FRC^RV^VC^TLCChapter 3: RESULTS^ 1303.3. LUNG PHYSIOLOGY3.3.1. LUNG VOLUMES3.3.1.1. EFFECT OF UNDERNUTRITIONThe weight-matched control rats were 22% smaller in body weight compared to the generalFigure 42. Functional residual capacity (FRC), residual volume (RV), vital capacity (VC) and total lungcapacity (TLC) of undernourished, hypobaric hypoxic, hypobaric normoxic and normobaric hypoxicanimals exposed to different conditions for 21 days.control group (201 +6 and 257±8 g respectively) . The functional residual capacity (FRC), residualvolume (RV), vital capacity (VC), FRC/TLC and RV/TLC % remained unchanged compared to generalcontrols, but total lung capacity (TLC) was significantly lower in undernourished animals (Figure 42,Figure 43).Chapter 3: RESULTS^ 1313.3.1.2. EFFECT OF HYPOBARIC NORMOXIAThe rats exposed to low ambient pressure were smaller in size (234 +8 g) compared with thegeneral control group (257±8 g) but not significantly. The values of FRC, RV, VC, TLC, FRC/TLCand RV/TLC of hypobaric normoxic animals did not differ from those of general control rats (Figure 42,Figure 43). When hypobaric hypoxic and normobaric hypoxic animals were compared to detect theeffect of low ambient pressure, no differences were found between the groups. EFFECT OF NORMOBARIC HYPDXIAThe hypoxic rats were significantly smaller (198±5 g) in body weight than general controls(257±8 g). FRC, RV, VC and TLC were significantly increased (19%, 18%, 16%, 17% respectively)during 3 weeks of normobaric hypoxic exposure compared to those in the weight-matched controlgroup (Figure 42). Significant increases in FRC (17%) and TLC (8%) were also observed comparedto general control values. FRC/TLC°/0 or RWTLCcY0 did not alter compared to those in both weight-matched and general controls (Figure 43).When comparison was made between hypobaric hypoxic and hypobaric normoxic rats, FRCand TLC were significantly greater in hypobaric hypoxic animals. EFFECT OF HYPOBARIC HYPDXIAThe adaptive response to hypobaric hypoxia resulted in a significant increase in FRC, RV, VCand TLC (21%, 18%, 21% and 20% respectively) compared to weight-matched controls (Figure 42).Increases in FRC (19%) and TLC (11%) in hypobaric hypoxic rats also reached a significant level(p<0.009) when compared to the general control group. FRC/TLC°/o and RV/TLC% remained thesame as in weight-matched control and general control rats (Figure 43).3.3.2. FLOW-VOLUME RELATIONSHIPS AND LUNG MECHANICSFlow resistance and static compliance are lung size dependent (71). Therefore, specificI^ I WMC^HBNOFRC/TLC% RV/TLC% VC/TLC%Chapter 3: RESULTS^ 132PROPORTION OF LUNG VOLUMES AND CAPACITIES[22 GC^EMI HBHY^NBHYFigure 43. FRC, RV and VC relative to TLC of undernourished, hypobaric hypoxic, hypobaricnormoxic and normobaric hypoxic rats.upstream resistance (sRus) and specific static compliance (sCst) were calculated as described inmaterials and methods. EFFECT OF UNDERNUTRITIONNeither Rus nor sRus in undernourished animals differed from those in general controls(Figure 44). Forced expiratory volume in 0.1 second (FEVol ) and forced expiratory volume/forced vitalcapacity % (FEVol/FVC %) also remained unchanged in weight-matched animals compared to generalcontrols (Figure 45). The peak expiratory flow rate (PEFR), forced expiratory flow rates (FEF) andFEF corrected for FVC were also similar to those in the general control group (Figure 46, Figure 47,Figure 48). Cst as well as sCst remained unchanged (Figure 49). As shown in Figure 50, thepressure-volume characteristics and exponential shape constant K in the weight-matched control andgeneral control groups were also the same. EFFECT OF HYPOBARIC NORMOXIA0.14 —I172 GCI WMC0 . 7 -0.12 — 0.6 —V'0.10 — 0.5 —UMISMIMEM=MOMMIUUMMI0a,0.08 — 0.4 —EMIMIME.IMMOMI0.06 — 0.3 —0CE00.04 -- IE(.) 0 . 2 —MMOM:0.02 — 0.1 —WEIEMI0.00 — 0.0 —Rus (FVC50%)tral HBHY^NBHYHBNOMangEMIWEI1111111011NMIMINIMNMIMEMEMINMINMI11111.111MM.MENMEIUMWMM.EMINMIMOOSEEMIMEEEMINMIEMII^Specific RusChapter 3: RESULTS^133Figure 44. Upstream resistance (Rus) and specific upstream resistance (sRus) at FVC 50% in GC,WMC, HBHY, HBNO and NBHY rats following 3 weeks of exposure to different conditions.The Rus, sRus, flow rates and the flow-volume relationship in hypobaric normoxic animalswere similar to those in general controls (Figure 44, Figure 47, Figure 48). The Cst, sCst and thepressure-volume curve characteristics in hypobaric animals also remained unchanged (Figure 49,Figure 50). When comparison between hypobaric hypoxic and normobaric hypoxic rats was madeto evaluate the effect of hypobaric pressure, FEV01/FVC°/0, FEF25_75%, and PEFR were significantlyhigher in hypobaric hypoxic animals. No differences in pressure-volume characteristics, Cst and sCstwere detected between the two groups. EFFECT OF NORMOBARIC HYPDXIANBHY vs WMC: Compared to weight-matched controls, the Rus and sRus were significantly increased(Figure 44). The FEV0.1 of normobaric hypoxic animals was equivalent to that in weight-matchedcontrol animals (Figure 45), but FEV1/FVC°/0 decreased significantly in normobaric hypoxic animalsChapter 3: RESULTS^134Figure 45. Forced expiratory volume in 0.1 second (FEV1) and percent of vital capacity expired in 0.1second (FEV01/FVC°/0) of rats following exposure to different experimental conditions.(Figure 45). The peak expiratory flow rate and FEF (275%) decreased significantly in normobarichypoxic animals compared to those in weight-matched animals (Figure 46). Significant reductions inFEF at 50% and 70% of FVC and predicted FEF at 50-90% of FVC were observed in normobarichypoxic rats compared to weight-matched control rats (Figure 47, Figure 48). No significantdifferences were found in the values of Cst and sCst between normobaric hypoxic animals (0.303+0.013 ml.cmH20-1 and 0.0306 +0.0011 cmH20-1 respectively) and weight-matched controls (0.25+0.02 ml.cmH20-1 and 0.0291 +0.0011 cmH20-1 respectively). Pressure-volume curves and the shapeconstant K of normobaric hypoxic rats also did not differ from those of weight-matched controls(Figure 49, Figure 50).NBHY vs GC: Compared to general controls, Rus and sRus were significantly higher in normobarichypoxic animals (Figure 44). FEV01/FVC°/0, PEFR and FEF25_75, were significantly lower innormobaric hypoxic animals than general controls (Figure 45, Figure 46). Absolute and corrected (forEZ21 GC^MO HBHY^NBHYLiii WMC^HBNO140120••••Il••••11••••ISWAMIranOMR.PEFR^FEF (25-75%)••••-•■•06040Chapter 3: RESULTS^135Figure 46. Peak expiratory flow rates and midexpiratory flow rates at FVC75_25% in rats subjected toundernutrition, hypobaric hypoxia, hypobaric normoxia and normobaric hypoxia.FVC) forced expiratory flow rates also decreased significantly at 50-90% of FVC following exposureto normobaric hypoxia (Figure 47, Figure 48). Increases in Cst, sCst and the constant K in normobarichypoxic animals (0.303 +0.013 ml.cmH20-1, 0.0306±0.0011 cmH20-1 and 0.209 ±0.011 respectively)did not reach levels of significance compared to general controls (0.208 +0.014 ml.cmH20-1, 0.0272+0.0014 cmH20-1 and 0.18 ±0.008 respectively) (Figure 49). However, transpulmonary pressure at50-90% of lung volume (FRC to TLC) was significantly decreased compared to that in general controlrats (Figure 50).HBHY vs HBNO: To assess the effect of low oxygen, the hypobaric hypoxic and hypobaric normoxicanimals were also compared. Rus was significantly higher (Figure 44) and FEF25_75% was lower inhypobaric hypoxic animals than hypobaric normoxic animals (Figure 46). Hypobaric hypoxia alsoproduced a significant reduction in expiratory flow rates corrected for FVC at 60-80% of FVC(Figure 48). No differences in the pressure-volume relationships between the two groups were noticedChapter 3: RESULTS^136Figure 47. The flow-volume relationships in GC, WMC, HBHY, HBNO and NBHY rats exposed todifferent experimental conditions for 21 days.(Figure 49, Figure 50). EFFECT OF HYPOBARIC HYPDXIAHBHY vs WMC: As shown in Figure 44, Rus (0.0953±0.0062 cmH20.m1-1.S-1) in hypobaric hypoxicanimals was not different from that in weight-matched controls (0.0857 +0.0041 cmH20.m11.S-1).However, sRus (0.489 +0.034 cmH20.S-1) was significantly increased compared to weight-matchedcontrols (0.362 ±0.018 cmH20.S-1). The changes occurred in FEV.i, FEV1/FVC°/0, PEFR and FEF inhypobaric hypoxic animals during the three weeks of exposure were not significant (Figure 45,Figure 46, Figure 47). When the FEF were normalized for VC, it was significantly lower at 60-80%Chapter 3: RESULTS^137Figure 48. The flow-volume curves using normalized forced expiratory flow rates for vital capacity ofanimals exposed to hypobaric hypoxia, hypobaric normoxia, normobaric hypoxia and undernutritionfor three weeks.of FVC in hypobaric hypoxic rats compared to those found in weight-matched control rats (Figure 48).HBHY vs GC: Compared with general control animals, sRus increased following exposure tohypobaric hypoxia (Figure 44). A significant reduction in FEF25_75% was found in hypobaric hypoxicanimals (Figure 46). Expiratory flow rates were also decreased but significant decreases were onlydetected when expiratory flow rates were corrected for FVC at 60-80% FVC (Figure 48).As shown in Figure 49, the Cst and sCst were not effected by exposure to hypobaric hypoxicconditions. There was no change in the constant K value (Figure 49) or pressure-volumecharacteristics of rats exposed to hypobaric hypoxia compared to both weight-matched and generalcontrols (Figure 50)./221 GC^MI HBHY^NBHYWMC^HBNO 0. 400.05 —C s t s Cs0.350.300. —0.03 —0.00 — Cons ant K0.5 —0.4 —0.3 —0.2 —a3 0 . 1 —E0.0 —Chapter 3: RESULTS^138Figure 49. Static compliance, specific static compliance (corrected for VC) and shape constant K ofP-V curves of GC, WMC, HBHY, HBNO and NBHY rats following 3 weeks of exposure to differentconditions.3.3.3. PRESSURE-VOLUME CURVE CHARACTERISTICS IN EXCISED LUNGSTotal lung capacity and static pressure-volume curves were recorded in the excised lungs.As shown in Table 14, the total lung capacity (TLC) increased by 114% between 4 weeks to 7 weeksof age. Compared to the baseline control group (BC), TLC in the normobaric hypoxic and hypobarichypoxic group increased by 134% during three weeks of exposure and was not different from thegeneral control group. On the other hand, the TLC gain in the weight-matched animals during threeweeks was only 66%, which was significantly lower than that in the normobaric hypoxic and hypobarichypoxic groups. The TLC in the hypobaric normoxic group was significantly smaller compared to thegeneral control group. The values of specific TLC in general controls, weight-matched controls andhypobaric normoxic rats were similar and were significantly lower compared to the normobaric hypoxicand hypobaric hypoxic groups (Table 14). When TLC calculated per unit lung weight was compared,Chapter 3: RESULTS^ 1391 0 0 —9 0 —8 0 —70 —.6 0 —50 —40 —30 —20 —10—0 1111111111111110 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Transpulmonary pressure (cm water)Figure 50. Pressure-volume curves recorded in intact animals which were subjected to undemutrition,hypobaric hypoxia, hypobaric normoxia and normobaric hypoxia for 21 days.no differences were detected among the groups. The exponential shape constant K of pressure-volume curves also remained unchanged following exposure to the various experimental conditions(Table 14). As illustrated in Figure 51, the transpulmonary pressure was calculated at 10 percentilesof total lung capacity and pressure-volume curves were constructed and were similar in all groupssuggesting that elastic lung recoil remained unaltered after subjecting animals to hypobaric normoxia,normobaric hypoxia, hypobaric hypoxia and undernutrition for three weeks.In summary, following exposure to normobaric hypoxia, FEV01/FVC°/0, PEFR, FEF(25_75%), andabsolute and FVC corrected FEF at 50-90% of FVC were significantly reduced compared to weight-matched controls. In addition to above parameters, FEV0.1 was also decreased in normobaric hypoxicanimals compared to general controls. Upstream resistance and specific upstream resistanceincreased. Elastic lung recoil decreased at high lung volumes in normobaric hypoxic animalscompared to general controls. The hypobaric hypoxic group showed similar changes in their flow-Table 14. Alterations in total lung capacity and shape constant k after 3 weeks of exposure.VARIABLE BC GC WMC HBNO NBHY HBHYTLC (ml) 4.21+0.23a 9.01+0.45b 6.98+0.45c 7.27+0.34c 9.86+0.43b 9.85+0.73bTLC/100 g Wb 5.00+0.31a 3.46+0.20b 3•47+0•28b 3.17+0.13b 4.85+0.18a 4.85+0.35aTLC/g WLT 6.99+0.54a 8.19+0.50a 7.37+0.59a 7.02+0.30a 7.47+0.30a 7.80+0.67aConstant k 0.124+0.005a 0.124+0.005a 0.123+0.006a 0.123+0.003a 0.127+0.007a 0.119+0.007aMeans +sem's are shown and those without a common superscript in a horizontal row are significantly different at p<0.05.Total lung capacity (TLC), Body weight (Wb), Lung weight (WLT), Constant K (shape constant of pressure-volume curve)Chapter 3: RESULTS^141100 —9080 1 3,70 — a60 —50 7.40 —30 —2010—ec • GC 0 HBNO0 II Ii Ill I III0^5^10^15^20^25Transpulmonary Pressure (cm water)100 —90 — •80 — o70 —60 —50 — nil40 —30 —20 — • GC V t1P-H.Y. N e HYW1AC•100 1111111^I I I^I II0^5^10^15^20^25Transpulmonary Pressure (cm water)Figure 51. Pressure-volume curves of excised lungs of rats exposed to different experimentalconditions for 21 days.Chapter 3: RESULTS 142volume relationship as those found in the normobaric hypoxic rats but these changes did not reachlevels of significance except for the FEF 25_75% and FEF at 60-80% of FVC corrected for FVC. Inhypobaric normoxic or weight-matched control rats neither flow-volume relationships nor staticpressure-volume characteristics showed any alteration suggesting that lung structure was not affectby low ambient pressure or undernutrition.Chapter 4: DISCUSSION^ 143CHAPTER 4DISCUSSION4.1. GENERAL POINTSThe main emphasis of this research was to examine the process of acclimation to several ofthe changes associated with high altitude in growing animals, with particular interest in lung growthand development. Thus, we studied structural and functional aspects of adaptive lung growth tohypobaria, hypoxia and hypobaric hypoxia. The structural aspects were studied using biochemical andmorphometric techniques. Functional adaptation of the lungs was assessed by pulmonary functiontests. The adaptive lung growth was further studied using radiochemical and autoradiographictechniques after administration of a radiolabelled DNA precursor. Through such analysis, weattempted to quantify the cellular response in various compartments of the lung. The effects ofhypobaria and hypoxia on food consumption and somatic growth were also studied. In addition, weevaluated the effect of hypobaria, hypoxia and hypobaric hypoxia on heart, spleen, liver and kidneygrowth, to see whether the effect of high altitude on growth was organ specific.The results of organ growth are often normalized for body size in experiments which producechanges in somatic growth. With regards to body dimensions, however, no definitive parameter isavailable as an indicator of body size. An increase in body weight with time is a widely acceptedmeasure of somatic growth, but body weight may also change because of altered fat and watermetabolism without any associated change in skeletal dimensions or lean body mass. Some studieshave used nose-tail, or tail and femur length as assessments of body size. Although a goodcorrelation between stature and lung size exists (264), skeletal growth measurements may not provideaccurate information of body growth and size in some circumstances. A study conducted by Hunterand Clegg (143) revealed that skeletal growth was non-uniform in rats exposed to 390 mm Hg as longbones grew relatively more than the axial skeleton. Similar findings have also been reported byChapter 4: DISCUSSION 144Acheson and MacIntyre (1). Thus, caution should be used while interpreting allometric results. Toavoid complications of corrections for body size, we used weight-matched animals as appropriatereference controls for estimating the specific effects of hypoxia and hypobaria. This made itunnecessary to normalize our data for body weight. The deleterious effect of acute or chronichypobaric hypoxia and normobaric hypoxia on somatic growth (31) has never been debated, but itraises an important question; does diminished somatic growth, equivalent to that expected to occurin hypobaric hypoxia, also cause identical reductions in growth of all body organs? In order todetermine if lung growth in undernourished animals was different from that of control animals, wecompared specific (per unit body weight) variables (i.e. lung weight, volume, alveolar surface area,total alveolar number) between the two control groups.Even though it would have been interesting to examine adaptive changes in suckling rats, wechose not to use rats yet to be weaned for the following reasons: (1) It is difficult to effectively controlbody weight of individual suckling rats to use as weight-matched controls. (2) Diminished milkproduction in hypobaric hypoxic conditions has also been reported (185). (3) In addition, the mothersmay secrete some growth-related factors in milk. We preferred to study lung growth in rats from 4 to7 weeks of age as during this time a marked increase occurs in lung weight and lung volume (46),alveolar multiplication continues (141), and lung growth has been extensively studied in this age group(261).4.2. FOOD CONSUMPTION AND SOMATIC GROWTHA fall in daily food intake of hypobaric hypoxic and normobaric hypoxic rats was observed onthe first day, but food intake began to increase after day 3. This may indicate that within 2 days ofexposure the rats were able to adjust to the new environment. After day 3, food intake increasedgradually but the amount of food consumed by both hypobaric and normobaric hypoxic rats remainedlower than general controls. Similar observations have been made by other investigators (5, 62, 59,92, 125, 237, 274).Chapter 4: DISCUSSION 145At the end of three weeks exposure, the body weight gain relative to the total food intake(referred to as the efficiency of food utilization (total weight gain/total food consumed)) fell in ratsexposed to normobaric hypoxia (0.411 ±0.008) and hypobaric hypoxia (0.392 ±0.005) relative togeneral controls (0.447 ±0.006). The efficiency of food utilization was also lower in hypobaric hypoxicand normobaric hypoxic animals compared to the weight-matched animals (0.430 ±0.012). Glosterand coworkers (114) also found that when the amount of food consumed by experimental animals wasgiven to pair-fed adult male Sprague-Dawley rats, they were bigger than the rats exposed to 400 mmHg for two weeks. These observations indicate that decreased oxygen availability not only decreasesappetite and food intake, but also the efficiency of food utilization.Carbohydrate and lipid metabolism may be impaired more than protein metabolism inhypobaria. Evidence for this was shown by Alippi and associates (5) as the efficiency of proteinutilization in Wistar rats exposed to 342 mm Hg did not change for 23 days. Hochachka et al. (138)hypothesized that some amino acids may function as an anaerobic source of energy but on the otherhand, oxygen is required for utilization of fats and carbohydrates (31). Barrie and Harris (15) founddecreased glucose utilization in the hearts of guinea pigs kept at a simulated altitude of 5000 m for28 days. Similar observations have been made in the liver (61). Furthermore, reducedgastrointestinal absorption of carbohydrates and fats has been found in humans following exposureto high altitude (36). Despite a lower food intake, the weight-matched controls gained the same weightas hypobaric hypoxic animals. Enforced food restriction results in meal eating (eating all food in onemeal) which is known to cause lipogenesis (170, 259) rather than nibbling (eating small amounts offood throughout the day) which occurs in hypobaric hypoxic animals. This may be one of the reasonsfor lower efficiency of food utilization in hypoxic animals.Previous studies have documented that chronic hypobaric hypoxic and normobaric hypoxicconditions lead to a decrease in body weight and this occurs in animals and humans of all age groups(5, 18, 45, 57, 77, 92, 267). It has often been postulated that at high altitude or in high altitudesimulated conditions, low oxygen concentration is responsible for poor body weight gain. However,Chapter 4: DISCUSSION 146no direct evidence is available. In the present experiment we observed that hypobaric hypoxic andnormobaric hypoxic animals lost weight slightly on day 1 but they recovered by day 3 of exposure.Similar changes have been reported by Clegg and Harrison (62), who conclude that animals adaptto hypobaric hypoxic conditions after 24 hours. The initial body weight loss at high altitude orsimulated conditions has been attributed to anorexia (106, 238, 274), food absorption (106),digestibility (59), disturbances in protein metabolism (154) and dehydration due to hypohydration,hyperventilation and other water loss (211).Following 3 days of exposure, both normobaric hypoxic and hypobaric hypoxic rats showed asign of acclimatization as they started to gain weight. Although the rate of weight gain increasedprogressively with time, it lagged behind general control rats, and thus the animals weighed less thangeneral controls at the end of the exposure. These findings implied that even though the rats beganto acclimatize to hypobaric and normobaric conditions earlier during the exposure, they did not adaptfully to the altered conditions. Although our findings are in agreement with other investigators (5, 18,45, 57, 77, 92, 267), we cannot rule out the argument that further exposure might have resulted incomplete somatic adaptation. The few studies which explored adaptation to extended exposureperiods have reported conflicting results. It appeared that while rats kept at high altitude for 10months failed to maintain a normal rate of growth (267), the rate of body growth of normobaric hypoxicguinea pigs remained unchanged for 14 weeks (164). The discrepancy may be related to biologicalvariations between the species.Nose-tail length is often used as a measure of skeletal growth. Concomitant with body weightdecrease, a decrease in nose-tail length was noticed after 10 days of exposure to hypobaric ornormobaric hypoxia. Thereafter the skeletal size of both hypobaric hypoxic and normobaric hypoxiagroups remained stunted and was similar to that of the weight-matched group. This observation isin contrast to Cunningham and coworkers (77), who exposed rats to nornnobaric hypoxic conditions,but it is in agreement with Hunter and Clegg (143). They found that reduction in axial growth occurredwithin one week of exposure to hypobaric hypoxia.Chapter 4: DISCUSSION 147Our results showed that skeletal growth was identical in both hypobaric hypoxia and normobarichypoxia. Therefore, our observations demonstrated that low body weight gain and reduced skeletalgrowth leading to stunting of somatic growth occurred under both hypobaric hypoxia and normobarichypoxia. The adverse effect of these conditions on body weight appeared immediately followingexposure while skeletal retardation occurred slowly. These findings also indicated that reducedsomatic growth in hypobaric hypoxia is mainly caused by decreased oxygen.Animals exposed to hypobaric normoxia ate less food (7%) than general controls but thisdecrease was less compared to hypobaric hypoxia (24%). Low ambient pressure also appeared toaffect the efficiency of food utilization because it was lower in hypobaric normoxic animals(0.418±0.005) compared to general controls (0.447±0.006). Similarly the hypobaric hypoxic animalshad a lower efficiency of food utilization (0.392±0.005) than normobaric hypoxic animals (0.411±0.008).Hypobaric normoxia slowed the rate of body weight gain and at the end of exposure hypobaricnormoxic rats weighed less than general controls. Following the first week of exposure to hypobaricnormoxia, skeletal growth was also stunted, but significant reduction in nose-tail length was apparentonly after three weeks of exposure compared to the general control group. On the other hand, nodifferences were found in body weight and nose-tail length between hypobaric hypoxic and normobarichypoxic animals. Studying the response of hypobaric pressure per se, Epstein and Saruta (98) founddecreased body weight of 8 men kept for 9 days in hypobaric normoxia. Therefore, the findings ofthe present study implied that low ambient pressure itself interfered with somatic growth but its rolein the presence of hypoxia (in hypobaric hypoxia) was not clear. It is possible that the effect of lowambient pressure was masked by a greater effect of low ambient oxygen in hypobaric hypoxia.Alternatively, decreased oxygen concentration and hypobaric pressure may act through the commonmechanism.4.3. ORGAN GROWTH ADAPTATION: OTHER THAN LUNGWe found that both hypobaric hypoxia and normobaric hypoxia stunted somatic growth.Chapter 4: DISCUSSION^ 148Table 15. Changes in organ weights in weight-matched control (WMC), hypobaric hypoxic (HBHY),normobaric hypoxic (NBHY) and hypobaric normoxic (HBNO) animals after 21 days of exposure.Organs^WMC^HBHY^NBHY^HBNOA. Compared to general controls. B. Compared to weight-matched controls. Heart^ ftSpleenHematocritLiverKidneyHeartSpleenHematocritLiverKidneyftftftftftftUndernourishment equivalent to that observed in hypobaric hypoxia and normobaric hypoxia, producedliver, heart, spleen and kidney growth retardation in weight-matched rats. It was of interest that inundernourished animals, significant decrease in weight occurred in liver on day 1, kidney on day 3,heart on day 5 and spleen on day 14, and subsequently remained lower thereafter compared togeneral controls. Following diminished growth of all organs during the first week of food restriction,organ weights increased steadily thereafter but remained lower compared to animals fed ad libitum.It showed that a decrease in somatic growth, equivalent to that in hypobaric hypoxic or normobarichypoxic animals, was associated with suppression of the growth rate of all the organs studied.However, the time course of the effect of food restriction on growth of organs was not identical. If weChapter 4: DISCUSSION 149consider day 1 values of general controls as a baseline, after 21 days heart, spleen, kidney and liverweights in undernourished animals increased 14%, 21%, 33% and 60% respectively while body weightincreased 93%. This suggested that, not only was the effect of undernutrition on growth of bodyorgans variable, organs such as the liver which showed an immediate effect also has a capacity toadapt and grow better than other organs. The variable effect of undernutrition on organ growth hasbeen also reported by other investigators (191, 284). Winick and noble (284) also concluded that theeffect of malnutrition is age dependent.The weight of heart and spleen increased significantly by day 7 of hypobaric hypoxic exposure,and remained higher compared to weight-matched controls until the end of exposure. The absoluteweights of heart and spleen also increased compared to general controls but significant differenceswere only found in hypobaric hypoxic animals on day 21. Although we exsanguinated the animals toreduce the amount of residual blood in the organs, a contribution of blood to the increase in the weightof spleen due to higher hematocrit in hypobaric hypoxia and normobaric hypoxia cannot be ruled out.Our heart and spleen growth observations are in agreement with those reported in previous studies(16, 55, 64, 93, 125, 148, 185, 191, 207, 223, 253, 267, 266). We have shown that the level of dietarydeficiency equivalent to that in hypobaric hypoxia and normobaric hypoxia suppressed growth of heartand spleen. This provided evidence that heart and spleen growth in hypobaric hypoxia andnormobaric hypoxia was greater when compared to the general control animals. Hunter andcoworkers (145) have shown that tritiated thymidine ( 3H-TdR) incorporation into DNA increased in theheart after one week when four week old rats were subjected to 390 mm Hg. In our study, nodifferences in heart and spleen growth response were found between hypobaric hypoxia andnormobaric hypoxia. This suggested that heart and spleen growth adaptation in hypobaric hypoxiawas primarily induced by reduced oxygen.Contrary to heart and spleen growth, the weights of kidney and liver in rats kept in hypobarichypoxia and normobaric hypoxia were reduced on day 3 of exposure compared to general controlsand were equivalent to those of weight-matched animals. Thereafter, kidney and liver growth patternChapter 4: DISCUSSION 150in hypobaric hypoxia and normobaric hypoxia were similar to that in undernourished rats. Results ofour experiment clearly indicated that the effect of hypobaric hypoxia or normobaric hypoxia is organspecific. Regardless of hemodynamic changes which occur in hypobaric hypoxia or normobarichypoxia, liver and kidney growth appeared to be influenced by undernourishment and followed thepattern of somatic growth. Our findings are in agreement with some investigators (57, 77, 64, 93, 125)but are in contrast with others (101, 185, 207) who found no difference or an increase in weights ofliver and kidney. Reduced or no change in the rate of 3H-TdR incorporation in kidney and liver after9 days of exposure to hypobaric hypoxia has been reported (193). However, it has been pointed outthat undernutrition and normobaric hypoxia appear to affect liver growth differently (191) in thatnormobaric hypoxia reduced cell number, whereas undernutrition produced a greater decrease incytoplasmic mass than cell number. Therefore, an effect on liver growth in hypoxic conditions maynot be purely alimentation dependent but could also be induced by a direct effect of hypoxia.Hypobaric hypoxia and normobaric hypoxia are known to produce changes in hemodynamicssuch as increased hematopoietic activity (125), blood volume (31), hematocrit (31), heart rate andconcomitant hypertension. It is conceivable that such alterations increase cardiac work load and inturn produce cardiac hypertrophy. Since the spleen is mainly a storage organ, the increasedhematocrit in hypobaric hypoxia or normobaric hypoxia can increase spleen weight in these conditions.Tucker and Horvath (266) postulated that altitude-induced changes in organ weights were related toconcomitant alterations in organkplood flow. They showed that blood flow increased in lungs, heartand spleen but was reduced in the kidney in hypobaric hypoxia. Besides the effects of hemodynamicalterations, decreased intracellular oxygen may also act directly to stimulate or suppress growth oforgans. Hypoxia has been shown to induce cellular proliferation in culture but its affect does notappear to be cell specific (140). Therefore, the mechanism by which hypoxia stimulates organ growthremains elusive. The liver being the main organ involved in enzyme synthesis for carbohydrate, fatand protein metabolism has higher RNA/DNA than other tissues and is very sensitive to nutrient intake(234). Cheek and associates (57) suggested that hypoxia not only prevented DNA accumulation inChapter 4: DISCUSSION 151the liver but also reduced protein synthesis and interfered with RNA production. In another study,hypoxia reduced liver ATP by 40% (231). It is conceivable that liver growth reduction in the hypoxicenvironment may occur because of decreased food consumption.Hypobaric normoxia did not show any striking changes in growth of either heart, spleen, liveror kidney. When results of hypobaric hypoxia and normobaric hypoxia were examined for the effectof hypobaric pressure, organ growth remained unchanged. These findings suggested that hypobaricpressure by itself or in the presence of hypoxia did not affect growth of the organs which wereexamined in the present study.4.4. LUNG GROWTH ADAPTATIONAdaptive lung growth response will be discussed as follows. First, the effect of food restrictionon lung growth will be discussed. The effect of hypobaric hypoxia will be assessed mainly bycomparing it to weight-matched controls. Where appropriate, the results will also be compared to thegeneral controls. Lung growth response may vary in decreased oxygen tension delivered by loweringthe ambient pressure and by mixing the oxygen with nitrogen. Therefore, the effect of normobarichypoxia will be evaluated both by comparing the normobaric hypoxic group to weight-matched andgeneral controls, and by comparing the hypobaric hypoxic animals to hypobaric normoxic animals.Later, the role of low ambient pressure at high altitude in regulating the adaptive lung growth responsewill be discussed by comparing the hypobaric normoxic rats to general controls and hypobaric hypoxicrats to normobaric hypoxic rats. Finally, the residual effect of all conditions after a 3 day recoveryperiod will be analyzed.4.4.1. EFFECT OF UNDERNUTRITIONThis study provided an opportunity to examine the effect on lung growth of the degree ofundernutrition which was similar to that in hypobaric hypoxia. The effect of food deprivation on lunggrowth was assessed by comparing the weight-matched control group with the general control groupChapter 4: DISCUSSION^ 152which was given free access to food.During 4-7 weeks of age, lung weight and lung volume increased less in undernourishedanimals (57% and 100% respectively) compared to general controls (87% and 145% respectively).The absolute amounts of lung DNA, RNA, total protein, hydroxyproline and desmosine increased lessduring 4-7 weeks of age in undernourished rats (36%, 20%, 41%, 80% and 69% respectively)compared to general controls (57%, 69%, 73%, 120% and 92% respectively). Increases in absolutealveolar surface area and total alveolar number during the experimental period were also lower inweight-matched controls (92% and 52% respectively) compared to general controls (123% and 70%respectively). Undernutrition suppressed the rate of DNA synthesis in the lung. This was furtherconfirmed by autoradiographic results as 3H-TdR incorporation fell following diet restriction and all themain cell types in the lung tissue were equally affected. Although total lung capacity measured in vivo in undernourished rats decreased, lung function remained unaffected.DNA is confined almost entirely to cell nuclei, and its amount within the diploid cell of anyspecies is constant (96). A reduction in DNA content in undernourished rats indicated that the numberof cells in the lung decreased at day 21. This occurred as a result of decreased DNA synthesis. 3H-TdR incorporation into DNA dropped 50% on day one and continued to fall until day 7 before showinga small increase. Autoradiography results also showed that except for cells which normally undergominimal proliferation (mesothelial cells, free alveolar macrophages and mast cells), 3H-TdR into othercell types (type II pneumonocytes, endothelial cells, interstitial cells and bronchial epithelial cells) fellon day one and continued to fall until 5-7 days of food restriction. This may imply that even thoughbody weight began to increase by day 3 of food restriction, the effect of undernutrition on lung growthwas prolonged. A decrease in DNA occurs due to interference with cell division which is timedependent. DNA did not appear to be affected during the early period of food deprivation and did notdecrease significantly until day 14. Since lung weight was reduced, cell density (DNA relative to perunit dry lung weight) increased during the earlier period but it approached the control level at the endof the experiment. This may have occurred due to greater decrease in other parameters of the lungChapter 4: DISCUSSION^ 153(see below). We did not starve our rats, but similar observations have been reported in food deprivedrats (118, 228, 229).The rate of protein synthesis in the lungs of undernourished rats appeared to slow down as totalRNA and soluble protein content fell on day 3 and reached their lowest level on day 7 and then slowlyincreased but never reached the control levels. As mentioned earlier, DNA content did not decreasesignificantly until day 14, the RNA/DNA and protein/DNA ratios dropped gradually until day 7 andremained consistently lower than general controls. No changes were found in dry/wet weight ratio ofthe lung in undernourished animals. It appeared that reduced lung growth in undernourished rats didnot occur due to variable hydration but due to reduction in the normal increases in cellular cytoplasmicconstituents. During the first week, lack of lung growth resulted primarily from diminished cell size andlater from both hypoplasia and decreased cell size. Our findings with regards to decreased cell sizein response to undernourishment are consistent with those reported by other investigators (118, 195,228, 284).Previous studies have shown significant reduction in collagen and elastin in undernourished andstarved animals (195, 228). In the present study, there was no difference in soluble and insolublefractions of hydroxyproline. Since the collagen equilibrium remained undisturbed, a reduction in theamount of total hydroxyproline may have occurred because of diminished collagen synthesis. Theamount of desmosine did not change in undernourished animals compared to general controls. Usingweanling rats, Myers and associates (189) studied the influence of restricted food. Although the extentof food deprivation was more severe than ours, the results for collagen and elastin were similar. Ithas been documented that the bulk of elastin is synthesized during the first few weeks of postnatallife in rats and later reaches a plateau, whereas collagen synthesis extends well into adulthood (90,190). Moreover, lung elastin turnover is slow (90). Neither hydroxyproline nor desmosineconcentration changed due to undernutrition indicating that the integrity of lung tissue structure wasnot disrupted. This was further supported by hydroxyproline/DNA, desmosine/DNA anddesmosine/hydroxyproline ratios as they also remain unchanged.Chapter 4: DISCUSSION 154Morphometric results showed that a trend towards smaller size and increased number ofstructural units per unit lung volume was apparent in undernourished animals compared to generalcontrols. Although undernourished animals had decreased alveolar surface area and fewer alveoli,both parameters increased (92% and 52% respectively) between 4-7 weeks of age. These findingssuggested that alveolar multiplication continued at a reduced rate in undernourished animals, but theunit structures in the lung were smaller than the general controls. This may have occurred due todecreased cell number (reduced cellular proliferation) and cell size. With regards to morphometricassessment, our results were partly in agreement with those observed by other investigators (195,177, 226, 227) but we did not find emphysematous changes in the lung due to starvation (226, 227).Volume per unit lung weight did not change. This indicated that food deprivation did not alterthe distensibility of the lung. This was further substantiated by the results obtained from the excisedand in vivo static pressure-volume curve characteristics and the flow-volume relationship of the lung,as those remain unchanged. In contrast, loss of elastic lung recoil has been reported while studyingthe effect of starvation (229) or a low protein diet (177). The undernourished rats in the present studydid not lose weight but the rate of weight gain was slower than controls. Therefore, physiologicalassessment further supported the evidence obtained from biochemical measurements that, in thepresent investigation, undernutrition did not alter the structure of the lung. It was interesting toobserve that the degree of inanition in hypobaric hypoxia in the present study did not alter normal lungstructure, but rather offset the rate of normal lung growth.In previous studies, the effect of decreased food consumption leading to somatic and lunggrowth retardation following exposure to normobaric hypoxia, hypobaric hypoxia or high altitude haseither been ignored or an attempt has been made to correct lung growth relative to somatic growth.Using weight-matched control animals to evaluate the influence of ambient environment on lunggrowth, we avoided introduction of an assumption of linear correlation of lung growth to body size.Body weight is often considered as a reference variable for body size to compare lung growth withinor between species. Although it was not necessary in the present study to normalize lung growthChapter 4: DISCUSSION 155parameters to that of body weight, we did so to explore whether a relationship between lung andsomatic growth is maintained in conditions where food consumption is compromised. We found thatall specific (relative to per unit body weight) lung growth variables were higher in undernourished ratscompared to the general control rats, but only the increases in specific lung weight, DNA, alveolarsurface area and total alveolar number reached significant levels. This is not unexpected since it hasbeen shown that with increasing age and increasing body weight, specific lung parameters decrease(240). There is no definitive explanation for this disturbance in somatic and lung growth due toundernourishment. One may argue that body growth was more affected by inanition or that lunggrowth was less compromised. It was evident from the results that while body weight decreased onday one, significant decreases in lung weight and absolute lung DNA were not found until days 7 and14 respectively. This showed that a divergence in the normal relationship between lung growth andbody size occurred during the early period of food restriction. This also may have occurred due torelatively changed water content or decreased fat deposits in the body. The effect of undernutritionmay be time dependent. Short term food deprivation may result from decreased body water contentand depletion of fat depots while prolonged undernutrition may also decrease lean body mass.Whatever the reason, it was evident from our findings that body weight may not be an appropriaterepresentation of body size and normalization of lung growth variables to body weight may lead tomisinterpretation of results. On the other hand, the observations made in undernourished animalsimply that conditions which diminish somatic growth also have associated reduction in growth of bodyorgans and such reductions should be accounted for when conclusions are drawn for organ specificresponse.4.4.2. EFFECT OF HYPOBARIC HYPDXIADecreased food consumption and diminished somatic growth are common findings followingtranslocation to high altitude or exposure to hypobaric hypoxia. However, lung growth adaptation hasnever been compared to animals which have been subjected to a similar degree of undernutrition.Chapter 4: DISCUSSION 156The effect of decreased food intake on somatic and lung growth in hypobaric hypoxia can be debatedand may not be exactly similar to animals subjected to undernutrition at room air, but weight-matchedcontrols are probably better reference than general controls. We compared our results of hypobarichypoxic animals to both weight-matched animals and general control animals.With regards to adaptive lung growth to chronic hypobaric hypoxia for three weeks, comparedto weight-matched animals, lung weight (40%), lung volume (32%), DNA (30%), RNA (58%), totalsoluble protein (60%), hydroxyproline (40%), desmosine (97%), alveolar surface area (28%) and totalalveolar number (21%) increased significantly. The mean linear intercept of airspaces and numberof alveoli/unit volume remained unchanged, but mean chord length of alveoli and the volume fractionof alveolar air increased in hypobaric hypoxic animals compared to weight matched controls. Thecytokinetic study revealed that biochemically analyzed 3H-TdR incorporation showed a sharp increaseon day 3 of exposure and gradually declined thereafter but remained higher than weight-matchedcontrols during the rest of the exposure. Analysis of autoradiographs confirmed the above findingsas alveolar wall cell 3H-TdR incorporation peaked on day 3 of exposure. The results of pulmonaryfunction tests showed that FRC, RV, VC and TLC increased significantly (21%, 18%, 21% and 20%respectively) in animals exposed to hypobaric hypoxia for three weeks compared to weight-matchedcontrols. Except for a significant increase in specific airway resistance and a decrease in forcedexpiratory flow rates corrected for forced vital capacity (FRC), no other changes in pulmonary functiontests were found in hypobaric hypoxic animals.Absolute variables of lung growth in hypobaric hypoxic animals were also significantly increasedcompared to general controls but the percentage changes were lower than those found by comparingthe hypobaric hypoxic and weight-matched control groups. Compared to general controls, exposureto hypobaric hypoxia produced significant increases in lung weight (17%), lung volume (13%), DNA(13%), RNA (12%), total soluble protein (30%), hydroxyproline (15%), desmosine (73%) and alveolarsurface area (10%). Except for a significant increase in mean chord length of alveoli, no othermorphometric parameter changed in hypobaric hypoxia. Alveolar surface area (11%) increasedChapter 4: DISCUSSION 157Table 16. Morphometric changes in weight-matched control (WMC), hypobaric hypoxic (HBHY),normobaric hypoxic (NBHY) and hypobaric normoxic (HBNO) animals after 21 days of exposure todifferent conditions.Variables^WMC^HBHY^NBHY^HBNOA. Compared to general controls. MLIlalvValvaveNvNatB. Compared to weight-matched controls VLMLIlalvValvaveNvNatSwVL (lung volume), MLI (mean linear intercept), lalv (mean chord length of alveoli), Valvave(average alveolar volume), Nv (number of alveoli/unit volume), Nat (total number of alveoli), Sw(alveolar surface area.significantly in hypobaric hypoxic animals but the increase in total alveolar number (8%) did not reacha significant level. FRC and TLC in intact hypobaric hypoxic animals increased. Expiratory flow ratescorrected for FVC (60-80% FVC) decreased and specific airway resistance increased in animalsfollowing exposure to hypobaric hypoxia for three weeks. Some investigators have observedChapter 4: DISCUSSION 158increases in absolute lung weight and volume in hypobaric hypoxia (20, 218) whereas others havefound increases only in specific lung weight and volume in animals raised at (258) or translocated to(45) high altitude, or exposed to simulated high altitude (136, 219).An increase in lung growth in terms of lung weight may be brought about by hyperplastic orhypertrophic changes in lung tissue, accumulation of intercellular connective tissue matrix inparenchyma or non-parenchyma, extracellular or intracellular water content, and blood volume in thepulmonary vasculature. Bartlett and Remmers (20) observed that after 7 days of exposure tohypobaric hypoxia, the non-blood lung water increased to 35%, compared to 20% after 21 days ofexposure. In the present study, a trend of decreased dry/wet weight ratio of the lung was apparentonly during the first week of exposure but did not reach a significant level. This ruled out thepossibility that water accumulation was responsible for an increase in lung weight after three weeksof exposure. In our study, we observed an increase in hennatocrit which is one of the commonlyobserved adaptive changes that occur at high altitude (31). Increases in central blood volume in highaltitude natives (184), and in central and pulmonary blood volume in lowlanders who have resided athigh altitude for prolonged periods (224) have been reported. It is conceivable that an increase inblood volume contributed to the observed increase in lung weight of hypobaric hypoxic animals.The animals in this experiment were exsanguinated prior to the removal of lungs to minimizethe contribution of residual pulmonary blood volume in lung weight measurements. In this study, thelungs were not flushed free of blood for two reasons. Firstly, a recent study (88) has revealed thatexsanguination reduces the residual blood volume in the lungs to 15% and further flushing with salinedecreased the contribution of blood volume ranging from 5-9%. This suggested that flushing cannotcompletely remove the blood as it may remain sequestered in certain areas of lung. In addition,flushing also results in a variable amount of fluid retention in the pulmonary vasculature which mayalso alter wet lung weight measurements. Secondly, the excised lungs were also used for pressure-volume curves and total lung capacity measurements before using them for biochemical andmorphometric estimations. Flushing may also alter mechanics of the lung as fluid may leak into theChapter 4: DISCUSSION^ 159airspaces.If the contribution of blood volume to lung weight is considered to be substantial, then anincrease in lung weight could be misinterpreted. If that is so, then the contribution of increasedhennatocrit can be corrected for. Hematocrit in the present study increased by 50% following exposureto hypobaric hypoxia. In that case, the contribution of residual blood volume following exsanguinationto lung weight will be 22% instead of 15%. However, lung weight increased by 40% and 17%compared to those of the weight-matched controls and general controls. If a 7% of an increase in lungweight is attributed to the hematocrit increase, lung weights were still higher than weight-matched andgeneral controls. Furthermore, an increase in hematocrit at high altitude is in part, due to decreasedplasma volume (4, 252). In addition, Bartlett and Remmers (20) did not find any change in residuallung blood volume in rats sacrificed by exsanguination from the abdominal aorta following three weeksof exposure to hypobaric hypoxia. It seems unlikely that increased hematocrit, in the range observedin the present studies would contribute significantly to the observed lung weight increase. Thus, anincrease in lung weight may be presumed as a true change in tissue weight that occurred in hypobarichypoxic conditions. STRUCTURAL ADAPTATIONSAlterations that occur in freeze-dried lung weight most likely represent changes in the numberand/or size of cells and/or contributions of intercellular connective tissue proteins. From biochemicalmeasurements in the lung, cell size is often determined by calculating the RNA/DNA and protein/DNAratios (225, 261, 283). The use of protein/DNA ratio as a measure of cell size may not be an accurateone as the amount of protein includes both cellular and extracellular proteins such as blood and newlysynthesized intra- and extra-cellular connective tissue proteins. Thus, the extracellular proteincontribution may complicate the interpretations of protein/DNA ratio as an indicator of cell size. TheRNA/DNA ratio, on the other hand, may provide a more precise index of cell size as it is not affectedby extracellular factors.Chapter 4: DISCUSSION 160DNA synthetic activity fell on day one of hypobaric hypoxic exposure and reached the level ofweight-matched controls and this coincided with a decrease in body weight. A sudden change inambient environment may have produced this adverse effect while animals were struggling to adaptto new conditions. On day three, 3H-TdR incorporation in hypobaric hypoxic rats increased sharplyand was twelve fold higher than weight-matched controls. Following a DNA synthesis peak on day3, the difference on day 5 increased to seventeen fold (because the rate of DNA synthesis in weight-matched group fell further) and then fell to eight fold on day 7. Thereafter, it dropped gradually toreach the general control level but remained higher than that in weight-matched controls. Theseobservations indicated that a brief depression in DNA synthesis in hypobaric hypoxic rats was followedby maximum lung growth stimulation and the bulk of the cellular adaptive response was completedwithin the first week. It was noteworthy that the lungs showed a remarkable potential to adapt tohypobaric hypoxic stress in the presence of the secondary stress of undernutrition which diminishedDNA synthesis. Although DNA synthesis fell after an initial surge, continued stimulation of lung growthwas evident as the rate of 3H-TdR incorporation remained high during the rest of the exposure.However, an increase in synthetic activity of DNA does not reflect accelerated cellular multiplicationto produce greater number of cells. Cells may go through the S-phase of the cell generation cycleand not enter into the G2 and M-phase (mitotic period), and result in multinucleated cells or increasedploidy. Although decreased oxygen tension induces mitochondrial proliferation, mitochondrial DNAaccounts for less than 1% of the total DNA (119). Despite exsanguination of animals prior to sacrifice,accumulation and margination of leukocytes and their contribution to 3H-TdR incorporation in the lungof hypobaric hypoxic animals cannot be ruled out. In spite of the early elevation of DNA synthesis,the amount of lung DNA did not increase significantly until day 7. This lag between maximum DNAsynthesis and an increase in the amount of DNA could be expected. An increase in DNA isdependent upon the duration of the cell cycle which may vary in various cell types in the lung. In thepresent study, increased 3H-TdR incorporation and concomitantly higher amount of DNA comparedto general controls suggested that chronic hypobaric hypoxia not only protected lung growth at theChapter 4: DISCUSSION^ 161normal level but induced a hyperplastic response.In our study, maximum 3H-TdR incorporation occurred on day 3 of exposure to hypobarichypoxia which is in conflict with the results of Voelkel and associates (271) who found that cellularproliferation peaked (345% increase) on day 9 of exposure to hypobaric hypoxia. They (271) studiedin vitro 3H-TdR incorporation in the middle lobe of right lung slices in adult female Wistar ratspreviously exposed for 3, 6, 9, 12, 20 and 35 days to hypobaric hypoxia compared with the resultsextrapolated from 3, 12 and 35 day controls. The authors suggested that DNA synthesis may be theterminal event in a chain of other metabolic processes that result in a lag between the hypoxicstimulus and DNA synthesis. Later, in the same laboratory, using similar conditions, age, sex andstrain of rats, Niedenzu and coworkers (193) found only an 85% increase in DNA synthesis on day9 after injecting 3H-TdR intraperitoneally.The discrepancy between the above studies and our's regarding timing of maximal DNAsynthesis may be due to various reasons. The route of injection of radioactive tracer is of importance(208). Oral administration of labelled thymidine leads to incorporation into DNA but is only about 20%of that found following intravenous injection. In our laboratory, we observed that DNA incorporationof 3H-TdR following intraperitoneal injection was less than half of that obtained by intravenous routeand the variation was also greater. On the other hand, one can argue against in vitro radioactivetracer incorporation because physiological conditions are disturbed. Baserga (22) reported that 80%cells in vitro can be stimulated to synthesize DNA by changing the culture medium. According toNiedenzu and coworkers (193), higher specific DNA activities are obtained in lungs if the in vitromethod is used. However, Voelkel and coworkers also used approximately 20 times as much 3H-TdRas used by Niedenzu and associates. We used young growing male animals and it has beendemonstrated by Cunningham and associates that the adaptive response to hypoxia is not similar inyoung and adult animals (77). It has been reported that males and females of the same species showa different hemodynamic adaptive response to hypoxic conditions (219), thus lung growth adaptationto hypobaric hypoxic stress may also be variable in the two sexes. Lung growth is increased followingChapter 4: DISCUSSION^ 162pneumonectomy and recently we have found that compensatory lung growth is also different in maleand female rats (240).From our results, it is evident that hypobaric hypoxia increased production of RNA precedingand during the phase of maximal DNA synthetic activity. The amount of RNA increased slightly onday one of exposure when DNA synthesis was depressed. The amount of RNA increased significantlyon day 3 and then continued to be higher than both weight-matched and general controls. Therefore,maximal increase in RNA and the relatively smaller increase in the amount of total DNA produced amarked increase in RNA/DNA ratio during the first week of exposure. Low oxygen concentrationsproduce an increase in 3H-uridine incorporation into RNA 6-8 hours prior to 3H-TdR incorporation intonuclear DNA and incorporation of radiolabelled amino acids into proteins in cultured chick embryoheart cells (139). The authors suggested that this occurred as a result of a nuclear transcriptionalprocess rather than a cytoplasmic event. Similar phenomena may have taken place in our hypobarichypoxic animals The total amount of RNA increased on day 3 but the amount of DNA did not increaseuntil day 7 compared to general controls. This provided evidence that the initial adaptive lung growthresponse occurred by cellular hypertrophy which was followed by hyperplasia. An increase in theRNA/DNA ratio during the early exposure period may have accelerated protein synthesis. Increasedprotein production was apparent as the amount of total protein also increased on day 3 and continueduntil the end of exposure. This may also explain why enhanced lung growth continued long after 3H-TdR incorporation declined and may in part have occurred by cellular hypertrophy. A pattern ofcellular response to hypobaric hypoxia was suggested from these findings. Initially the hypobarichypoxic stimulus triggered RNA production which was followed by DNA synthesis. Once adequatecellular adaptation was achieved, DNA synthesis fell but cellular hyperactivity continued to maintainelevated lung growth which largely occurs by protein accumulation. Ultrastructural studies have alsoshown an increase in cell size in the lungs of mice at high altitude (204). According to Winick andNoble (283), tissues that are actively engaged in protein synthesis are rich in RNA and a highRNA/DNA ratio is reached long before the high protein/DNA ratios are reached in the same tissue.Chapter 4: DISCUSSION 163High RNA/DNA ratios have also been shown in tissue subjected to stress such as cardiac hypertrophywhich indicate the capacity to produce more RNA per cell (115). It is interesting to note that even inthe presence of undernutrition when lung growth is retarded and cell size in the lung is decreased,hypobaric hypoxia causes an increase in lung growth by enhancing cell replication and increasing cellsize. One may conclude that lung growth in hypobaric hypoxia appears to be different from that whichoccurs in undernourished animals. It not only makes up for the lung growth that may occur inundernourishment but also results in an increase in absolute terms.Connective tissue proteins, particularly collagen, are heterogeneous in type and anatomicallocalization. After three weeks of hypobaric hypoxic exposure, increased amounts of hydroxyprolineand desmosine indicated accumulation of connective tissue proteins which would probably occur asa result of shift in the protein synthesizing machinery towards more collagen and elastin production.We did not measure hydroxyproline during the exposure. The soluble/insoluble hydroxyproline ratiodid not differ from general or weight-matched controls on day 21 of exposure. One may conclude thata dynamic equilibrium in collagen metabolism was achieved by three weeks of exposure or it remainedundisturbed in hypobaric hypoxia. In pneumonectomized rats DNA synthesis peaks after 3-5 days (38,53) but collagen synthesis does not increase until the second week (72). This may suggest thatconnective tissue protein production occurs after cell replication. Similar connective tissue proteinchanges may have occurred in hypobaric hypoxic animals.Interestingly, the concentration of hydroxyproline decreased following exposure to hypobarichypoxia compared to the general control group. This may suggest that collagen production laggedbehind lung growth. A similar decrease in hydroxyproline concentration in the right ventricle of theheart has been reported in rats exposed to simulated high altitude (175). The hydroxylation of prolineto hydroxyproline is an aerobic reaction in vitro (60). It has further been shown that the oxygenincorporated into the hydroxyl group originates from atmospheric oxygen rather than from water.Thus, the inspired oxygen may be sufficiently reduced in our animals to interfere with this reaction.The effect of undernutrition in hypobaric hypoxic conditions cannot be ruled out. It has been reportedChapter 4: DISCUSSION 164that food deprivation reduces hydroxyproline and lysyl oxidase activity (a cross-linking enzyme forcollagen and elastin) (181). Therefore, the effect of undernutrition, decreased hydroxylation of prolinedue to low oxygen and increased non-connective tissue proteins in hypobaric hypoxia may havecaused a decrease in hydroxyproline concentration.Unlike collagen, elastin per unit dry weight of the lung increased. It was apparent from ourresults that elastin increased relatively more than collagen in hypobaric hypoxia. The amount ofhydroxyproline and elastin relative to DNA content increased compared to weight-matched controlssuggesting that structural changes in the lung tissue may have occurred following exposure tohypobaric hypoxia. However, compared to general controls only the desmosine/DNA ratio increased.The possible reasons for such an imbalance in elastin and collagen will be discussed later. It istedious to estimate collagen and elastin in various components of the lung, but it has been shownhistologically that elastic tissue increases in the walls of small blood vessels in hypobaric hypoxia (69,145, 182). It is possible that the bulk of elastin accumulation may have occurred in vessel walls.Although we do not know the location of enhanced collagen and elastin accumulation in the lungtissue, such alterations in connective tissue dynamics could be expected to cause functional andstructural changes in the lung.Collagen and particularly elastin which are major components of the extracellular matrix in thelung are thought to have a critical role in defining lung structure, especially in the process ofalveolarization. The morphometric analysis showed that hypobaric hypoxia increased the volumefraction and absolute volume of alveolar air compared to those of weight-matched controls. Theabsolute volume of alveolar air also increased compared to general controls. Increases in the volumefraction and the volume of alveolar air may occur if more alveolar units are added, or the alveoli haveenlarged with or without alveolar multiplication. On the other hand, the volume fraction of parenchymalair (alveolar air + alveolar duct air) remained unchanged suggesting that rearrangement within theparenchymal structures took place as the volume fraction of alveolar duct air decreased and alveolarair increased. This remodelling of respiratory units may result from the conversion of former alveolarChapter 4: DISCUSSION 165ducts to bronchioles by a distal extension of bronchiolar epithelium (see below: the frequency oflabelled epithelial cells increased in terminal bronchioles), or alveolarization of terminal bronchioles.From our data it is uncertain which mechanism could have operated during the exposure to hypobarichypoxia. The absolute volume of alveolar wall increased compared to both weight-matched andgeneral controls. This may occur as a result of alveolar multiplication or thickening of the alveolarwalls. However, the volume fraction of alveolar wall remained unchanged, indicating that thickeningof alveolar wall was unlikely. We cannot rule out the possibility that the restructuring of alveolar wallsmight have taken place during the exposure and was completed by the end of three weeks ofexposure .With regards to dimensions of unit structures, mean linear intercept (MLI) which is an indirectmeasure of airspace size, and the number of alveoli/unit volume (Nv) remained unchanged but meanchord length (Jahr) which is a measurement of alveolar size, increased in hypobaric hypoxic ratscompared to weight-matched controls. Similar changes were also found when compared to generalcontrols. The average volume of alveoli (Valvave) increased and the surface to volume ratio of alveolidecreased compared to those of weight-matched controls. These observations coupled with a largerfraction of alveolar air led us to believe that enlargement of alveoli with no or little increase in alveolarduct dimensions occurred in hypobaric hypoxia, more so compared to weight-matched than generalcontrols. Bartlett and Remmers in simulated high altitude (20) and Tenney and Remmers at highaltitude (258) found a slight increase in mean alveolar diameter of animals (this did not reachsignificant level). Burni and Weibel (45) also observed enlargement of alveoli by indirect measurementas the surface to volume ratio of alveoli was decreased in rats at high altitude. A significant changein our study could be due to more severe hypobaric hypoxic conditions and a larger sample size.Interestingly, the only morphometric study conducted on humans by Saldana and Garcia-Oyola (230)also found a greater mean chord length in highlander males than age-matched sea-level males.Alveolar surface area increased (28%) in hypobaric hypoxic animals compared to weight-matched animals. An increase in gas exchange capacity of the lung can be brought about by threeChapter 4: DISCUSSION 166mechanisms (113): 1) enlargement of lung volume while size of the alveoli remains constant, 2)increase in the density of gas exchanging surface by finer subdivisions of the air spaces, and 3)thinning of the air-blood barrier. An increase in alveolar surface area in this study was likely areflection of increased fixed lung volume (32%). The surface/volume ratio of alveoli did not change,therefore, it was indicative that equilibrated lung growth occurred. However, the unchanged MLI mayhave been the result of altered volume fractions of alveolar air and alveolar duct air because the meanchord length of alveoli increased. Burn i and coworkers (49) proposed that increased gas exchangingsurface can occur by more initial anisotropic enlargement of alveolar ducts compared to alveoli andwhich was then followed by lengthening of alveolar walls, and thus restoring the original relationshipin volume fractions. In the present study, one can speculate that prolonged hypobaric hypoxicexposure might have restored the original relationship in volume fractions of alveolar duct air andalveolar air. Compared to weight-matched controls, the unchanged Nv and increased total alveolarnumber (21%) assessed by a direct alveolar count supported the argument that alveolar multiplicationindeed took place during hypobaric hypoxic adaptation. An increase in mean chord length of alveoliwas observed indicating enlargement of alveoli. This could be due to overexpansion of alveoli or dueto lengthening of alveolar walls. Therefore, an increase in gas exchanging area may be a combinationof alveolar multiplication, overinflation of alveoli and/or lengthening of alveolar walls.Alveolar surface area was also increased compared to general controls, indicating that lunggrowth was indeed accelerated in hypobaric hypoxia. High altitude male residents also have greateralveolar surface area and total alveolar number compared to age-matched sea level males (230).Rabinovitch and associates (218, 219, 220) found that alveolar concentration (alveoli/mm2) remainedunchanged with or without an increase in lung volume following exposure to hypobaric hypoxia inyoung or old Sprague-Dawley rats compared to room air controls. They suggested that hypobarichypoxia either accelerated or maintained the normal rate of alveolar multiplication. In the presentstudy, although alveolar surface area and total alveolar number increased, this change was lower thanthe observed increases in the amount of collagen and elastin. In a recent study, it has been shownChapter 4: DISCUSSION 167that 64% of the lung tissue is alveolar and 36% is nonalveolar (249). It is possible that the bulk ofconnective tissue protein accumulation most likely occurred in non-gas exchanging components (i.e.vascular and bronchial wall) of the lung. CELL KINETICSAutoradiography showed in hypobaric hypoxic rats, maximal 3H-TdR incorporation in alveolarwall cells occurred on day 3 and then declined slowly to reach the general control values by day 7 butremained higher than weight-matched controls. These findings matched the lung synthetic activitydetermined by biochemical analysis. Increased uptake of 3H-TdR in hypobaric hypoxic animals maynot necessarily imply that cellular multiplication occurred, but the unchanged percentage distributionof all cell types and increased total lung DNA in hypobaric hypoxic animals compared to generalcontrols support the argument that cellular multiplication occurred. One would perhaps expect achange in the percentage distribution of cells in the alveolar wall, since the rate of 3H-TdRincorporation was not identical in these cells. One explanation may be that increased 3H-TdRincorporation does not necessarily reflect cell replication. The labelling index of type ll pneumonocytesincreased but the labelling index of type I pneumonocytes did not. If we assume that the increasedlabelling index of cells is an indicator of cell replication then the type litype II pneumonocyte ratioshould have decreased. Such a change was not observed. One explanation may be that since typeI pneumonocytes are in immediate contact with alveolar air, alterations in ambient environment mayinduce functional changes and ultimately affect the life span of the cell. One can also argue thathypobaric hypoxic stress may have accelerated the differentiation of type ll pneumonocytes to typeI pneumonocytes. Alternatively, changes in type litype II pneumonocyte ratio may have occurredbetween days 7 and 21 of exposure which we did not study.Cellular kinetics of various cell populations in the central and peripheral parts of the lungshowed a variable response to hypobaric hypoxic stress. The labelling indices of type IIpneumonocytes, capillary endothelial cells, interstitial cells and unidentified cells in the walls of centralChapter 4: DISCUSSION 168alveoli increased on day 3 in hypobaric hypoxic animals compared to both weight-matched andgeneral controls. On day 5, the labelling index either increased slightly (interstitial cells andunidentified cells), stayed at the same level (type II pneumonocytes), or slightly decreased (alveolarwall endothelial cells). Unlike the central alveoli, all types of cells in the walls of peripheral alveoli andmesothelial cells showed maximal 3H-TdR incorporation on day 3 of exposure to hypobaric hypoxia.From the results it was apparent that in hypobaric hypoxia maximal DNA synthetic activity inthe peripheral part of the lung occurred earlier than in the central part. 3H-TdR incorporationhomogeneity in various cell types in the walls of peripheral alveoli and the heterogeneity in centralalveoli of the lung were interesting observations. The reason for this discordant effect in the peripheraland central region of the lung is uncertain but it suggested that the response to hypobaric hypoxia maynot be the same in all parts of the lung. Close examination of the data also revealed that regardlessof the time, the percentage increase at maximal 3H-TdR incorporation in the wall cells of the peripheralalveoli (type ll pneumonocytes 449%, alveolar wall endothelial cells 447%, interstitial cells 574%,unidentified cells 307%) also appeared to be greater than the central alveoli (type II pneumonocytes312%, alveolar wall endothelial cells 256%, interstitial cells 342%, unidentified cells 246%). Thisindicated that hypobaric hypoxic stress produced a greater effect on the peripheral part of the lungthan the central part. The peripheral part of the lung is mainly comprised of pure respiratory unitstructures without rigid axial non-parenchymatous structures while the unit structures in the central partare arranged in a more complex three dimensional geometrical pattern which may restrict growth inthe central portion of the lung. Therefore, it is conceivable that the geometrical location may facilitatemore growth in the peripheral part of the lung than the central part.Mesothelial cell 3H-TdR incorporation was synchronous with incorporation into peripheral cells.Recently a study conducted by Cagle and coworkers reported that following pneumonectomy, thepleural mesothelial cell 3H-TdR incorporation peaked two days prior to other cells in the lungparenchyma (53). They suggested that early stimulation of mesothelial cells may have occurred dueto closer proximity of these cells to the factors which stimulate lung growth or to their greater sensitivityChapter 4: DISCUSSION 169to such factors. Nattie and associates (192) documented that compensatory lung response followingpartial lung resection was not similar in central and peripheral parts of the lung in young rats. Surfacealveoli showed evidence of multiplication whereas internal alveoli dilated. A similar type of responsemay have occurred in hypobaric hypoxic animals.Autoradiographic observations of alveolar wall endothelial cells showed some unique featuresin the peripheral and central part of the lung. First, the maximal alveolar wall endothelial cellstimulation was found on day 3 of exposure regardless of central or peripheral alveoli. It was alsonoteworthy that the sequence of 3H-TdR incorporation in arterial endothelial cells was also identicalto that of alveolar wall endothelial cells. In the walls of central alveoli, the endothelial cells peakedearlier than the other cells. These observations suggest that the endothelial cell stimulation wasrelated to changes in hemodynamics (discussed in normobaric hypoxia). Second, the hypobarichypoxic stress appeared to induce a bimodal adaptive response in the alveolar wall endothelial cells.3H-TdR incorporation increased sharply and peaked on day 3 of exposure. Thereafter it fell graduallyand reached control levels on day 7 but another significant increase in the endothelial wall labellingindex was noted on day 21. Rabinovitch and coworkers (218) showed that the mean pulmonary arterypressure increased significantly after three days of exposure to hypobaric hypoxia and remained highuntil day 14. They believed that an initial increase in pulmonary artery pressure may have occurreddue to changes in hemodynamics whereas a later sustained increase was a result of structuralalterations in the pulmonary vasculature. It is possible that in our hypobaric hypoxic rats, the initialsurge in endothelial cell DNA synthesis may have been mediated by immediate changes inhemodynamics (increased cardiac output, hematocrit) and the second increase occurred due tosustained hypertension secondary to vascular structural changes.Peak stimulation of arterial wall cells did not synchronize with the arterial endothelial cells. Inhypobaric hypoxic rats, maximum stimulation of arterial wall cells occurred on day 5 of exposure andit coincided with the response in bronchial wall cells. The reason for this variable response betweenarterial endothelial cells and arterial wall cells is unclear but geometrical location and delayedChapter 4: DISCUSSION^ 170availability of humoral mediators may be responsible. FUNCTIONAL ADAPTATIONSWhether functional changes in the lung occurred prior to or after structural adaptation tohypobaric hypoxia is uncertain. Observations made by Bartlett and Remmers (20) suggest that lungmass increases before lung volume. They found that dry lung weight/lung volume was higher on day7 of exposure but not on day 20-21. We studied lung function alterations three weeks after exposureto hypobaric hypoxia. In hypobaric hypoxia, despite reduced somatic growth, biochemical,morphometric and cytokinetic evaluations provided evidence of enhanced lung growth. A wide andbarrel-shaped chest has been regarded as characteristic of residents of high altitude. In our study,the TLC in intact and excised lungs was significantly increased compared to the undernourished rats.The TLC in intact hypobaric hypoxic animals was also higher than general controls. Our results arecompatible with those found in humans by other investigators (146, 273).FRC, RV and VC increased compared to both weight-matched and general controls. Suchincreases in lung volumes and capacities also suggested that lung growth was augmented inhypobaric hypoxia. An increase in FRC has also been reported in humans (39, 75, 146, 232, 256).Human data from Andean residents have shown that most of the increased total lung capacity is dueto larger residual volume (270). It has been suggested that FRC may increase due to a sustainedinspiratory position of ribs as a result of hyperventilation (13). Our findings did not support thishypothesis. The FRC/TLC, RV/TLC and VC/TLC ratios in hypobaric hypoxic animals were similar toweight-matched and general controls suggesting that increased lung growth in hypobaric hypoxiaoccurred by a proportional increase in all fractions of volume.As in other organs, elastin and collagen are found in association with proteoglycans in the lung.The elastic fibers can stretch to 140% of their resting length before breaking while the tensile strengthof the lung tissue is attributed to collagen which can stretch only about 2%. According to Weibel (280)the axial connective tissue of the bronchi, bronchioles and pulmonary vessels, the peripheralChapter 4: DISCUSSION 171connective tissue of the pleura and the interlobar and interlobular septa, and the parenchymalconnective tissue of the alveolar septa are three functional components of the connective tissue of thelung. The axial connective tissue represents the immobile center while the peripheral connectivetissue represents the oscillating part. Parenchymal connective tissue forms the elastic link betweenthe oscillating and immobile part and thus any force exerted on the parenchyma is distributedthroughout the lung.Static lung compliance which is a measure of lung distensibility is a change in lung volume perunit change in the pulmonary transpulmonary pressure. Besides connective tissue forces and surfacetension, static compliance may also be affected by pulmonary blood volume and viscosity, smoothmuscle tone or thickening of the visceral pleura (71). In the present study, static compliance andpressure-volume characteristics in hypobaric hypoxic rats were indistinguishable from both weight-matched and general controls. Similar observations have been made in highlanders compared tolowlanders (39, 75). Static compliance may be influenced by lung size but when corrected for lungvolume, called specific compliance, this also stayed unchanged. This suggested that in hypobarichypoxia, elastic lung recoil at lower or higher lung volumes did not alter, and these largely reflectchanges in connective tissue properties of the lung. Our biochemical analysis showed an increasein elastin concentration and an increase in elastic recoil was expected. However, elastin depositionmight have occurred more in the vascular component of the lung and thus its predicted effect on thelung recoil was not observed.The pressure-volume curves were also analyzed by using the exponential curve fitting technique(68) and the K value obtained in hypobaric hypoxic rats was similar to that of weight-matched andgeneral controls. In rats as well as in other species, the shape constant K of the pressure-volumecurve has been claimed to be directly related to the mean size of the airspaces as assessed by themean linear intercept (123, 201). The mean linear intercept in the hypobaric hypoxic group did notchange compared to the weight-matched and general controls. This further supported the evidencethat in hypobaric hypoxia, the structural integrity of the lungs was maintained at a normal level.Chapter 4: DISCUSSION 172Furthermore, high altitude native mice have type ll pneumonocytes with larger lamellar bodies,resulting in four times more surfactant (205) but surfactant is decreased in undernutrition (83). Sinceundernourishment is a part of the adaptive response to high altitude, the equilibrium of surfactant maystay unaltered and may not exert any change in lung recoil properties.The resistance to the respiratory apparatus in vivo is attributable to the chest wall, lung tissueand gas in the airways. The airways account for more than 90% of the total resistance of the lungand the remainder may be represented by the tissue component which is difficult to measure (71).We measured the upstream resistance (Rus) under conditions of maximal flow when the driving forceis the static elastic recoil of the lung. The resistance is then independent of the expiratory effort. Theanimals were paralysed by an injection of succinylcholine. Gas flow is also dependent upon the sizeof the lungs. Therefore, expiratory rates and upstream resistance were corrected for lung volume (asdescribed in materials and methods).Three weeks after exposure to hypobaric hypoxia, the flow-volume curves revealed thatabsolute forced expiratory flow (FEF) rates at all percentiles of FVC (forced vital capacity) were similarcompared to weight-matched and general controls, but when FEF was corrected for lung volume, asignificant reduction was noticed at 60-80% of FVC in hypobaric hypoxic rats compared to both controlgroups. The FEF25_75% (forced expiratory flow between 25-75% of forced vital capacity) was alsodecreased. These findings suggested air flow obstruction. Rus increased and forced expiratoryvolume relative to FVC (FEV01/FVC%) decreased, but the changes were not significant. However,the specific Rus increased significantly compared to both weight-matched and general controls. Weperformed pulmonary function tests in room air and animals were exposed to room air for more thantwo hours before tests. This helped us to avoid inheritent assumptions for air density if tests areperformed in hypobaric conditions. Brody and coworkers performed tests at high altitude and lowaltitude and made correction for decreased air density in high altitude subjects. Changes in flow ratesin their highlanders compared to lowlanders were similar to our results (39). In another study, airwayresistance increased when residents of high altitude were moved to sea level (75). It is interesting• WMC^V MBHY 0 NBMY0^3^6^9^12^15Transpulmonary pressure (cm water)130120 -110C 1000`I"E 90-80a 70 -g. 60-ti 50-at; 40 -T) 30 -08 20 -LL.10 -0 ^• WMC^V HEIM^D NEIHY1^1^I^Io^3^6 9^12^15TRANSPULMONARY PRESSURE (cm water)0Chapter 4: DISCUSSION 173to note that three weeks of exposure to hypobaric hypoxia in growing rats produced similar alterationsin lung function to that of humans living at high altitude. This may imply that adaptations which occurat high altitude are phenotypic rather than genotypic.Figure 52. Absolute and corrected for FVC maximum flow-static recoil relationship in rats followingexposure to different conditions.In mammals, airway formation and alveolar partitioning takes place at different times (261). Inhumans, bronchial tree development occurs before 16 weeks of fetal gestational age, while alveolarformation continues during childhood (261). Therefore, it is possible that factors influencing one stageof development could cause dysanaptic growth (disproportionate growth pattern between constituentparts on an organ) of the lung, resulting in lung function changes. Green and coworkers (117)suggested that variability in maximum expiratory flow-volume measurements in healthy individuals mayoccur due to dysanaptic lung growth because variability in measurements could not be attributed todifferences in bronchomotor tone or size of lungs. In the present study, static compliance andChapter 4: DISCUSSION 174pressure-volume characteristics did not alter in hypobaric hypoxic rats. When absolute and lungvolume normalized expiratory flow rates at percentiles of vital capacity were plotted againsttranspulmonary pressure, predicted (corrected for FVC) flow-static recoil curve of hypobaric hypoxicanimals shifted right (Figure 52). We presume that this shift in flow-static recoil curve and a decreasein predicted expiratory rates in hypobaric hypoxic animals would have occurred if airways failed togrow in size proportional to total lung growth.Using morphometric techniques, the volume fraction of airway lumen was also assessed. Inthe present study, the volume fraction of conducting airways lumen has been referred to as the volumefraction of bronchial air (bronchial and bronchiolar air) and it was relatively small (ranged from 0.005to 0.075). Stone and associates have recently reported that only 4% of lung air is occupied by thenonalveolar region of the lung (249). Only one group of investigators (50) has been able to find asignificant decrease in volume fraction of conducting airways following partial pneumonectomy whilethe volume fraction of parenchyma did not change. The investigators suggested that such animbalance in growth of lung structures may have functional implications. We found a trend to a slightlylower volume fraction of bronchial air in hypobaric hypoxic animals (0.023 ±0.004) compared to bothweight-matched (0.031 ±0.006) and general controls (0.037 ±0.006) whereas the volume fraction ofalveolar air increased in hypobaric hypoxic animals. At the time of maximal response, the centralairway epithelium DNA synthesis increased by 130% compared to 330% in alveolar wall cells. Aninference can be drawn from these observations that lung growth in hypobaric hypoxia was dysanapticas airways grew relatively less than the gas exchanging surface.It was of interest that the labelling index of bronchiolar epithelium increased 210% more thanbronchial epithelium. During childhood, distal airways increase in diameter more than proximalairways and the length of distal airways increases more than the diameter (261). As mentioned earlierwe found a slight decrease in the fraction of alveolar duct air in hypobaric hypoxic animals. Oneexplanation may be that part of the alveolar ducts were converted into bronchioles by extension ofbronchiolar epithelium. Rabinovitch and coworkers (220) observed that hypobaric hypoxia producedChapter 4: DISCUSSION 175extension of muscularization of peripheral arteries which are not normally muscularized at the alveolarduct or wall level. The other explanation may be that peripheral airway growth stimulation occurredmore than that of central airways. The peripheral airway growth may have been triggered by thesimilar mechanism which mediated growth of the gas exchanging surface, while geometric restrictionsmay also have limited the growth of central airways. McBride (178) found an increase in cross-sectional areas of both central and peripheral airways in pneumonectomized ferrets, whereas Boatman(28) noticed an increase in axial length of airways in rabbits. If peripheral airways increased in cross-section, then the resistance in the airways would have decreased but resistance increased inhypobaric hypoxic rats. Therefore, it is probable that hypobaric hypoxia induced lengthening ofperipheral airways.The present study affirmed the findings of previous investigations (20, 45, 204) that hypobarichypoxia enhances specific lung growth. We also found evidence that absolute lung growth isincreased even compared to the general controls and it is much greater than weight-matchedcounterparts. Since specific alveolar surface area and total alveolar number was higher in weight-matched controls than in general controls, corrections of morphometric results made for body weightmay overestimate response of high altitude or hypobaric hypoxia in growing animals. Thus, cautionmust be used while interpreting specific data. Our data for morphometric analysis did not show anychange in dimensions of lung structure in undernourished rats, and hence, they served as validreference controls.In brief, despite diminished somatic growth, lung growth was accelerated in hypobaric hypoxia.Lung growth occurred both by hyperplastic and hypertrophic changes. Although hypobaric hypoxiaproduced accumulation of collagen and elastin, collagen increase lagged but elastin increased morethan the increase in lung weight. This may have occurred due to lack of oxygen to facilitate thehydroxylation of proline. Biochemical, morphometric and physiological observations showed that lungstructure remained unaltered in hypobaric hypoxia. Along with other adaptive changes in the body(increased hematocrit, decreased somatic growth) hypobaric hypoxia also induced an increase in gasChapter 4: DISCUSSION 176exchanging surface area, perhaps sufficient to meet body oxygen requirements. Lung growth occurredboth by alveolar enlargement and multiplication. Expiratory flow rates decreased and airwaysresistance increased. These changes may have occurred due to dysanaptic lung growth in hypobarichypoxia.4.4.3. EFFECT OF REDUCED AMBIENT OXYGEN (NORMOBARIC HYPDXIA)This thesis provided us an opportunity to compare adaptive lung growth response to two typesof hypoxia, either by dilution of oxygen or by lowering the ambient pressure. Dilution of oxygen withnitrogen reduces the concentration of oxygen content while the density of total gases remainunchanged, but a reduction in atmospheric pressure lowers the density of gases without changing therelative oxygen concentration. In this part of the discussion, the effect of low oxygen concentrationand low oxygen density on lung growth adaptation will be discussed separately. We did not haveweight-matched controls for the normobaric hypoxic group but their somatic growth was equivalent tohypobaric hypoxic animals. Therefore, the weight-matched group served as a suitable control groupto study the effect of normobaric hypoxia without dealing with specific lung growth parameters. NORMOBARIC HYPDXIA vs WEIGHT-MATCHED CONTROLS AND GENERALCONTROLSCompared to the weight-matched group, three weeks of exposure to normobaric hypoxiaproduced significant increases in absolute lung weight (38%), lung volume (39%), excised total lungcapacity (41%), DNA (31%), RNA (60%), total soluble protein (52%), hydroxyproline (28%), desmosine(62%), alveolar surface area (24%) and total alveolar number (12%). Mean linear intercept (MLI) andmean chord length of alveoli (lay) increased and number of alveoli/unit volume (Nv) decreased innormobaric hypoxic animals. Biochemical analysis and autoradiographic measurements showed thatnormobaric hypoxia induced peak DNA synthetic activity in the lungs on day 3 of exposure.Thereafter, it fell gradually but remained consistently higher than weight-matched animals. PulmonaryChapter 4: DISCUSSION 177function tests performed after three weeks of normobaric hypoxic exposure also showed significantchanges as functional residual capacity, residual volume, vital capacity and total lung capacityincreased significantly. Although no differences were found in the elasticity of the lung, the peakexpiratory flow rate decreased and upstream resistance increased in animals kept in normobarichypoxic conditions. Our findings are not consistent with the only other study that used pair-weighedanimals to assess the adaptive lung response. Naeye exposed suckling mice to normobaric hypoxia(12% oxygen) for 3 and 41/2 weeks (191). Although undernourished and normobaric hypoxic (12%oxygen) mice were smaller (44% and 42% respectively) in body weight than controls, lung weights ofundernourished and normobaric hypoxic mice were similar. This may suggest that the effect ofundernutrition and normobaric hypoxia on lung growth in suckling mice may be different from that ingrowing rats.Investigators in the past have usually studied the adaptive lung growth response to hypoxia bycomparing the normobaric hypoxic animals and controls kept in room air and fed ad libitum. We alsocompared normobaric hypoxic rats to general controls and found significant increases in absolute lungweight (16%), lung volume (13%), DNA (13%), RNA (14%), total protein (24%) and desmosine (43%)On the other hand, the excised total lung capacity, total amount of hydroxyproline, alveolar surfacearea and total alveolar number remained unchanged. Significant increases in functional residualcapacity and total lung capacity were also noticed in normobaric hypoxic animals. In addition,decreases in expiratory flow rates, increased flow resistance and decreased elastic lung recoilbetween 50-90% of vital capacity were observed. With regards to augmented lung growth innormobaric hypoxia, our results for lung weight and/or lung volume were in agreement with someinvestigators (13, 77, 101, 164, 247) but were in conflict with others (18, 191). Part of the controversyin the literature regarding lung growth response to decreased oxygen tension may be related to ageand species of the animals, duration and severity of exposure, and techniques used to estimate lunggrowth.In the present study, the unchanged dry/wet lung weight ratio and a significant increase in dryChapter 4: DISCUSSION 178lung weight in normobaric hypoxic animals compared to both weight-matched and general controlsindicated that increased water retention was not responsible for the marked increase in lung mass.Increased hematocrit may have contributed to an observed increase in dry lung weight of normobarichypoxic animals. The possibility of such a contribution was minimal in this study and the reasons havebeen discussed before. Therefore, an increase in lung weight in nornnobaric hypoxia was real.Fixed lung volume and lung weight in normobaric hypoxic rats not only increased comparedto weight-matched controls but also to general controls. Lechner and Banchero (164) found anincrease in absolute lung volume in guinea pigs after three weeks of exposure to normobaric hypoxiaand once guinea pigs reached 900 g body weight, an increase in lung volume no longer existed. Theysuggested that the maximum size of the thoracic cage may be the critical factor restraining further lunggrowth. In their view, lung growth accelerated to reach adult dimensions faster in hypoxic conditions.The effect of prolonged normobaric hypoxia may be different in rats, since in guinea pigs, normobarichypoxia did not appear to affect somatic growth. However, besides mechanical restrictions, otherfactors such as oxygen requirements, growth inhibitors and directly (autocrine or paracrine) orhumorally mediated factors may also help regulate growth of lungs in hypoxic conditions. STRUCTURAL ADAPTATIONSLung DNA synthetic activity in normobaric hypoxic rats increased markedly on day 3 ofexposure and dropped thereafter to reach the general control levels on day 7 but remained elevatedcompared to weight-matched controls. The total amount of DNA increased significantly on day 5compared to weight-matched controls and on day 7 compared to general controls. The RNA/DNAratio increased by day 3 in normobaric hypoxia and then declined gradually but remained slightlyelevated until the end of exposure compared to weight-matched animals. These observationssuggested that lung growth adaptation in normobaric hypoxia was brought about by both hyperplasticand hypertrophic changes and the bulk of adaptive response occurred during the first week ofexposure. Compared to general controls, it was apparent that the initial adaptive response occurredChapter 4: DISCUSSION 179by an increase in cell size. As the number of cells increased, cell size began to decrease. One mayspeculate that the initial adaptive lung response to a hypoxic stimulus is an increase in synthesis ofcytoplasmic constituents and if the stimulus is removed at this stage, the hypertrophic cells may returnto normal size (see post-exposure recovery). On the other hand, if the stimulus persists, cellularproliferation occurs which may fulfill the functional demand. Thereafter the cytoplasmic enlargementis no longer required and cell size reaches the normal levels. Faridy and coworkers (101), andSjostrom and Crapo (247) have also reported elevated DNA content in adult animals subjected tonormobaric hypoxia. On the contrary, normobaric hypoxia produced a hypoplastic lung response innewborn rats (187). The differing lung growth adaptive response in newborn and adult animals mayimply that lower oxygen availability may in fact compromise lung growth during the rapid growth phase(because tissue proliferation is maximal) but stimulate it when the lung growth rate is slow. A similarconclusion may be drawn from observations made by Cunningham and coworkers (77) as three weekold rats showed no increased in lung weight following three weeks of exposure to normobaric hypoxiabut in 6 and 12 week old rats lung weights increased by 16% and 22% respectively.Connective tissue proteins showed a variable response to nornnobaric hypoxia. Despiteincreased lung tissue mass in normobaric hypoxia, the total amount of hydroxyproline did not increasecompared to general controls. This led to a significant drop in hydroxyproline concentration comparedto both general and weight-matched controls suggesting that a decrease in hydroxyproline/unit drylung weight in normobaric hypoxia did not result from undernutrition. As has been mentioned earlier,the hydroxylation reaction of proline to hydroxyproline is aerobic and oxygen incorporation into thehydroxyl group comes from atmospheric oxygen rather than from water (213). This may imply thatin this study, the decreased ambient oxygen tension may have been the main factor interfering withcollagen formation and accumulation.In contrast to collagen, the amount of elastin increased following exposure to normobarichypoxia compared to both weight-matched and general controls indicating a greater effect ofnormobaric hypoxia on elastin than on collagen. Compare to general controls, the desmosineChapter 4: DISCUSSION 180concentration did not change, but the desmosine/DNA ratio increased significantly. This indicated thatnormobaric hypoxia induced excessive accumulation of elastin relative to cellular proliferation. It hasbeen reported that lysyl oxidase, which is a key enzyme for cross-linking newly formed elastin andcollagen, increased four to six fold in hamsters after 24 hours of exposure to 12-13% 02 and remainedhigh until the end of the exposure of 5 days (40). This may support the view that elastin formationand maturation was stimulated by acute normobaric hypoxic exposure. Since the total amount ofelastin increased and collagen did not, it is possible that the mechanisms which regulate the dynamicequilibrium of these two connective proteins in decreased ambient oxygen tension may be different.In the gas exchanging area, the connective tissue proteins are responsible for the frameworkof the lung structures. Compared to weight-matched controls, significant increases in absolute alveolarsurface area and total alveolar number signifies that normobaric hypoxia might have increased gasexchanging area by subdivision of airspaces. Other investigators (164, 167) have also foundsignificant increases in alveolar surface area in normobaric hypoxic guinea pigs compared to guineapigs of similar weight. However, alveolar surface area and total alveolar number are functions of lungvolume and did not increase in normobaric hypoxic animals compared to general controls. Thesefindings did not support the view that normobaric hypoxia increased the surface complexity of the lung.Our observations are in agreement with Bartlett (18) who found no alterations in lung morphometricparameters of normobaric hypoxic rats compared to general controls but are in conflict withCunningham and associates (77). They found an increase in total alveolar number when newborn ratswere exposed to 12-13% 02 for 3 weeks but not in those exposed from 9-12 weeks of age. It isconceivable that the nature of the adaptive response to normobaric hypoxia may vary with age andseverity of hypoxia.In the present study, it is unlikely that normobaric hypoxia induced an increase in lung surfacecomplexity. The dimensions of unit structures indeed favoured the thesis that normobaric hypoxiacaused enlargement of gas exchanging structures rather than multiplication. MLI, laiv and Valvaveincreased and Nv decreased significantly compared to the weight-matched control animals. ChangesChapter 4: DISCUSSION 181in MLI, Ialv, average alveolar volume and Nv were also significant compared to general controls. Iflung growth occurs only by enlargement of unit structures, then the linear dimensions of airspaces willincrease with the cube root of the increase in volume and the number of alveoli per unit volume willdecrease directly to the increase in volume (261). Increases in MLI and laiv and a decrease in Nv inour normobaric hypoxic animals reached the dimensions expected by simple expansion of airspaces[observed (91.1 gm, 45.1 gm and 16.7 (105) respectively) vs those predicted from general controls(89.1 gm, 42.61.Im and 16.0 (105) respectively), and vs those predicted from weight-matched controls(91.4 gm, 43.1 gm and 13.7 (105) respectively)]. No differences were found in the fractions of alveolarair, alveolar duct air and alveolar wall suggesting that enlargement of both alveolar ducts and alveoliwas equivalent. The increase in alveolar surface area was also the same as predicted by simpleexpansion of the airspaces (observed 0.566 m2 vs 0.573 m2 predicted from general controls and vs0.566 m2 predicted from weight-matched controls) supporting the view that morphometric unitstructures underwent simple enlargement in normobaric hypoxia. Regardless of age, increases inalveolar size (186, 198) and in airspaces (alveolar and alveolar duct size) (77) in rats followingexposure to normobaric hypoxia have also been reported by other investigators.Ventilatory adaptation to hypoxia is commonly regarded as a progressive hyperventilation ofincreasing magnitude which persists even after removal of hypoxic stimulus (31). Piazza andcoworkers observed that normobaric hypoxia produced sustained hyperventilation accompanied byhigher tidal volume (210). Hyperventilation may cause expansion of the lung unit structures.However, Bartlett (unpublished study in 20) after keeping young rats in 5% CO2 for 20 days ruled outthe possibility of hyperventilation being a stimulatory factor of lung growth in hypoxic conditions.Faridy and Yang (102) showed that lung hyperplasia in nornnobaric hypoxia resulted from the directeffect of low oxygen tension, whereas lung distension occurred due to mechanical stimulation of lungtissue by hyperventilation.One can consider the possibility that the prolonged hyperventilation (210) might have acted asa mechanical stimulus to produce consistently greater response in the peripheral than centralChapter 4: DISCUSSION 182airspaces. This notion was supported by the evidence that 3H-TdR incorporation increased markedlyin mesothelial cells and cells in the walls of peripheral alveoli on day 3 of exposure whereas the bulkof the cells in the walls of central alveoli showed maximum 3H-TdR incorporation on day 5. Thissuggested that stimulation of cells in the walls of peripheral alveoli occurred before stimulation of cellsin the wall of central alveoli. In addition, at the time of peak stimulation, the percentage increase in3H-TdR incorporation in the walls of peripheral alveoli was also relatively higher than in the centralalveoli. Reasons for such changes which also occurred in hypobaric hypoxia have been discussed.The percentage distribution of cells in normobaric hypoxic rats also remained similar to those ofgeneral controls. This observations in conjunction with increased lung DNA supported the idea thathypoxic stress indeed increased cell division. From biochemical analysis it was evident that cellularhypertrophy occurred. However, the question remains whether hypertrophic changes occurred in allcells or whether the hypertrophic response in normobaric hypoxia was cell specific.The pattern of kinetics in the walls of central and peripheral alveoli was also similar to hypobarichypoxic rats. The maximal response in the walls of central alveoli (type II pneumonocytes, interstitialcells and unidentified cells) was delayed except in endothelial cells. Irrespective of location (in thewalls of central or peripheral alveoli, or arteries), all endothelial cells showed maximal stimulation onday 3. Synchronous stimulation of all endothelial cells suggest that it was triggered by an endothelialcell specific mediator.Tissue-specific mitotic Inhibitors, called chalones, have been proposed as being involved intissue growth in the lung (42). It has been postulated that increased blood flow dilutes the localconcentration of these chalones and thus diminishes their inhibitory effect (103). In part, alterationsin hemodynamics in hypoxic conditions may also be responsible. A significant increase in hematocritoccurs by 36 hours of exposure to normobaric hypoxia (210). Since lungs handle the whole cardiacoutput, alterations in hemodynamics in hypoxic conditions may facilitate the removal of endothelial celldivision inhibitors. One may argue that increased blood flow and volume may also supply a humoralpromoter of cell division or produce a mechanical stress which may alter physical properties of theChapter 4: DISCUSSION 183cells to induce cell proliferation. Recently, an in vitro study has demonstrated that a 2% concentrationof oxygen stimulated endothelial cell proliferation by 102% compared to a 20% 02 concentration andit was mediated by increased adenosine release (180). Studies using models such as wound healing(155) and malignant tumors (105) which lack adequate oxygen supply have shown that reducedoxygen availability induces neovascularisation by releasing angiogenic factors. It has been suggestedthat increased blood volume in hypoxia produces pulmonary capillary recruitment by opening newcapillaries in the lung tissue (54). These studies suggest that along with hemodynannic alterations,oxygen may also exert a direct chemical stimulus to regulate the proliferation of endothelial cells inhypoxic conditions. However, it remains speculative that low oxygen tension may stimulate andregulate growth of different cells through different mechanisms.The number of free alveolar macrophages which incorporated 3H-TdR increased in normobarichypoxic rats on day 5 compared to the weight-matched controls. Similar changes were found inhypobaric hypoxic rats. This shows that macrophages which were until recently considered terminalcells (2), also respond to hypoxic stress by increased 3H-TdR incorporation. What induces anincrease in DNA synthesis in free alveolar macrophages is not understood. One may speculate thathypoxia stimulates the release of chemotactic substances such as macrophage activating factor whichmay stimulate the alveolar macrophages. It has been reported that hypoxia increased metabolicactivity in alveolar wall endothelial cells (247), therefore, hypoxia may also directly activate the freealveolar macrophages. Alveolar macrophages are known to release two mediators, fibronectin andmacrophage derived growth factor. Fibronectin stimulates resting fibroblasts to proliferate (25).Therefore, interstitial cell stimulation may also occur due to the paracrine effect of macrophages.Previous studies have reported increased number of peribronchial and perivascular mast cellsin animals exposed to normobaric hypoxia (152, 202). Thus, hypoxia induced hyperplasia of theamine containing cells in the lung may provide a pool of available mediators which could conceivablyaffect the pulmonary vasculature and airways. However, no direct relationship between mast cellhyperplasia and vasoconstriction in hypoxic conditions has been established. To our knowledge, 3H-Chapter 4: DISCUSSION 184TdR incorporation into mast cells in the lung in normobaric hypoxic or hypobaric hypoxic conditionshas not been studied. Our autoradiographic results showed an increased mast cell labelling index inresponse to normobaric hypoxia in the lung. We found that the bulk of the increase in the number of3H-TdR incorporated mast cells occurred in the subpleural part of the lung. The significance ofsubpleural proliferation of mast cells is not clear. It has been suggested that pulmonary vascularresistance is more closely related to alveolar than to arterial oxygen tension and it is alveolar hypoxiarather than hypoxemia that causes structural changes in pulmonary mast cells (152). Therefore, anincrease in subpleural mast cell DNA synthesis may have occurred due to the direct effect of alveolarhypoxia. Our findings showed that mast cell stimulation occurred immediately following exposure tohypobaric hypoxia or normobaric hypoxia suggesting that mast cells were responsive to low oxygentension before any other cells in the lungs. Azizkhan and associates (9) have demonstrated that mastcells stimulate angiogenesis. It is possible that mast cell proliferation may be one of the regulatorymechanisms of endothelial proliferation to the increased blood volume load in hypoxic conditions. Inaddition to histamine release, mast cells are also associated with interstitial edema and connectivetissue formation (188). Therefore, it may be that increased lung connective tissue proteinaccumulation and moisture content (during the early period of exposure) in hypobaric hypoxia andnormobaric hypoxia were also partly mediated by mast cells. FUNCTIONAL ADAPTATIONSRats exposed to three weeks of normobaric hypoxia showed significant increases in functionalresidual capacity, residual volume, vital capacity and total lung capacity compared to weight-matchedanimals. These findings suggest that normobaric hypoxia produced an increase in functional capacityof the lungs. An increase in functional capacity of the lungs in normobaric hypoxia may occur due toa raised inspiratory position of the thoracic cage, airway obstruction or due to loss of elasticity of thelung. Barer and associates (13) concluded that the raised inspiratory position of the thoracic cage wasthe possible reason for an increase in FRC of normobaric hypoxic rats as pressure-volumeChapter 4: DISCUSSION 185characteristics remained unchanged. We did not find differences among any experimental and controlgroups for FRC, RV and VC relative to TLC suggesting that all components of the lung volumeincreased proportionally. An increase in FRC in our normobaric hypoxic rats was not due topersistently hyperinflated lungs.Pressure-volume characteristics of excised normobaric hypoxic lungs were similar to those ofweight-matched and general controls. Our findings are compatible with other investigators whostudied elastic properties of excised lungs following chronic normobaric hypoxic exposure (13, 77,101). Absolute and specific lung compliance in intact normobaric hypoxic animals also remainedunchanged. This may suggest that decreased oxygen tension did not disturb structural integrity of thelungs. However, we found loss of elastic lung recoil in intact normobaric hypoxic animals between 50-90% of VC and a significant increase in airspace size compared to general controls. These alterationssupported the view that normobaric hypoxia might have caused changes in lung structure.Setnikar (241) and Mead (179) postulated that the function of collagen and elastin in lung werelargely independent of each other. The coiled collagen fibers determine lung distension characteristicsat higher lung volumes or near total lung capacity. The readily extendable elastin fibers aredeterminant of lung distensibility at low lung volumes, giving the steep part of the P-V curve. Collagenconcentration in our normobaric hypoxic animals decreased compared to general controls and thisdecrease may have been responsible for the loss of lung elastic recoil at higher lung volume. Lungcompliance was measured at low volumes where elastin fibers determine the lung recoil. Despite anincrease in the amount of elastin in normobaric hypoxia, the elastin concentration did not change.Therefore, variability in elastin and collagen equilibrium in normobaric hypoxia may in part account fordifferences in elastic lung recoil at lower and higher lung volumes.Besides tissue forces, elastic lung recoil in air-filled lungs is also influenced by surface forces ofthe lung. In order to assess the effect of tissue forces, fluid-filled P-V curve measurements couldprovide more definitive information. Unfortunately, we did not perform fluid-filled P-V curves. Foodconsumption diminished in normobaric hypoxic rats. Restricted food intake has been reported toChapter 4: DISCUSSION 186decrease disaturated phosphotydylcholine in the lung (83). It is possible that the changes in P-Vcurves of normobaric hypoxic animals may in part be the result of altered surface forces. Alterationsin P-V curves were only found in the intact animals and not in excised lungs, therefore, a loss of lungelastic recoil may in fact reflect changes in the mechanical properties of the chest wall.The FEVol expressed as percentage of FVC (FEV%) may decrease due to obstruction suchas narrowing of airways, to chest wall abnormalities and loss of elastic recoil. Our results showed thatFEW°, PEFR and FEF25_75% decreased significantly in normobaric hypoxic animals compared to bothweight-matched and general controls suggesting that ventilatory capacity fell in normobaric hypoxicanimals. Absolute and volume normalized forced expiratory flow rates decreased at 50-90% VC innormobaric hypoxic rats compared to general controls. The absolute expiratory flow rates were alsodiminished at 50-70% of vital capacity compared to weight-matched controls. In addition, absolute andspecific upstream airway resistance increased markedly following exposure to normobaric hypoxia.Airway obstruction may have contributed to such changes in flow-volume characteristics in normobarichypoxic rats. As described by Green (117), reductions in air flow rates in the lung may occur due todysanaptic lung growth where airway growth would be less than the gas exchange surface. It is likelythat decreased flow rates in normobaric hypoxic rats resulted mainly from structural alterations inairways. Since elastic lung recoil decreased at high lung volume, one can argue that reductions inmaximum expiratory flow rates may be attributed to decreased static recoil of the lung. However,maximum (corrected for FVC) flow-static recoil curve of normobaric hypoxic animals shifted far rightcompared to weight-matched controls (Figure 52). Although one can not rule out the possibility thatdecreased elastic recoil may have affected expiratory flow rates, flow-static recoil relationship supportsthe argument that expiratory flow rates may also have reduced due to dysanaptic lung growth innormobaric hypoxia. Autoradiographic results further confirmed these observations as the rate of DNAsynthesis in the central airways was lower than that in the alveolar wall cells. Implications of suchobservations related to dysanaptic lung growth have been discussed in the preceding section.Chapter 4: DISCUSSION^ 1874.4.3.2. HYPOBARIC HYPDXIA vs HYPOBARIC NORMOXIAComparison between hypobaric hypoxic and hypobaric normoxic rats allowed us to evaluatethe effect of lower oxygen density. In both low oxygen density and low oxygen concentration, thenumber of oxygen molecules remains the same but it has been postulated that decreased gas densityfacilitates ventilation (75). Therefore, it is possible that lower oxygen tension achieved by loweringambient pressure (similar to high altitude) may affect lung growth differently than low oxygenconcentration.Compared to hypobaric normoxia, lung weight, lung volume, DNA, RNA, protein, hydroxyprolineand desmosine increased significantly in hypobaric hypoxic rats. With the exceptions of lung weightand connective tissue proteins which were greater in hypobaric hypoxia compared to hypobaricnormoxia, increases in other lung growth parameters were almost equal to those found by comparingnormobaric hypoxic animals to general controls (see page 177). This suggested that except for lungweight and connective tissue proteins, the adaptive lung growth response to reduced oxygen contentwas similar whether it is delivered by dilution or by lowering the ambient pressure.Hydroxyproline content increased significantly in hypobaric hypoxic rats compared to hypobaricnormoxic rats but such an increase was not found in normobaric hypoxic animals compared to generalcontrols. This suggests that low oxygen, when delivered by reducing pressure can cause collagenaccumulation. Although, hydroxyproline concentration (relative to the dry weight of the lung) was alsolower in hypobaric hypoxic animals than general controls, the change was less dramatic than thatfound in normobaric hypoxic animals. Since hydroxylation of proline is oxygen dependent, betterventilation due to decreased gas density in lower ambient pressure may facilitate collagen formation(this will be discussed later). An increase in elastin was also greater in hypobaric hypoxic animals.Therefore, it is likely that low oxygen delivered by lowering pressure had a variable effect onconnective tissue protein equilibrium.Despite an increase in lung volume, alveolar surface area and total alveolar number did notchange. This occurred because of significant increases in MLI, laiv and average alveolar volume, andChapter 4: DISCUSSION 188decreases in Nv and surface/volume ratio in hypobaric hypoxic rats. This indicated that lung growthin decreased ambient oxygen density also occurred primarily by enlargement of airspaces. The datafrom pulmonary function tests showed that FRC and in vivo TLC were also higher in hypobaric hypoxicanimals compared to hypobaric normoxic animals. FEF 25_75% and expiratory flow rates normalizedto lung volume decreased at 60-80% of FVC, and upstream resistance increased in hypobaric hypoxicanimals compared to hypobaric normoxic animals. Similar observations were made in normobarichypoxic rats compared to general controls suggesting that structural alterations in the lung in hypobarichypoxia were produced by decreased oxygen.In summary, hypoxia produced by dilution or by lowering the ambient pressure, accelerated lunggrowth by increasing lung weight and lung volume. Compared to weight-matched controls, lunggrowth increased markedly in normobaric hypoxia. However, the pattern of lung growth was notnormal. Although lung growth in normobaric hypoxia occurred by hyperplastic and hypertrophicchanges, accumulation of collagen was hampered which may be the cause for loss of elastic lungrecoil at higher lung volumes. Morphometric analysis showed that normobaric hypoxia induce simpleexpansion of airspaces without an increase in surface complexity of the lung. DNA synthesis in thewalls of central alveoli increased less and was delayed relative to the peripheral alveoli. A fall inexpiratory flow rates suggested that normobaric hypoxia might have induced dysanaptic lung growth.This was further supported by autoradiographic results. One can infer that low oxygen tension mayhave produced a decrease in collagen concentration, overinflation of alveoli and dysanaptic lunggrowth in hypobaric hypoxia.4.4.4. EFFECT OF REDUCED AMBIENT PRESSURE (HYPOBARIC NORMOXIA)Although our results have demonstrated that increased lung growth (i.e. lung weight, volume,DNA, RNA and protein) in hypobaric hypoxia was mainly induced by decreased oxygen, the effect ofhypobaric pressure cannot be ignored. Our results showed that there were some differences in lunggrowth adaptation between hypobaric hypoxic and normobaric hypoxic animals which may beChapter 4: DISCUSSION 189attributed to lower ambient pressure. We assessed the effect of low ambient pressure by comparingthe hypobaric nornnoxic group with the general control group and the hypobaric hypoxic group with thenormobaric hypoxic group. The former provided information related to the hypobaric pressureacclimation without altering the ambient oxygen and the later comparison helped us to assess theadaptive response in low ambient pressure with decreased oxygen.Three weeks of exposure to hypobaric normoxia produced a small but significant increase (9%)in hematocrit compared to general controls. In the present study, our objective was to study lunggrowth response to hypobaric normoxia rather than hypobaric normoxemia, but the increasedhematocrit raised the important suggestion that hypobaric normoxia may have caused arterialhypoxemia. In preliminary studies, we did blood gas analysis of rats exposed to acute (30 minutes)hypobaric normoxia. Arterial Po2 (88.5 ±4.5 mm Hg) and 02 saturation (94.0 ±1.4 %) were notsignificantly different than normobaric normoxic animals (92.0 ±4.3 mm Hg and 96.2 ±1.9 %respectively).A question naturally arises with regards to the accuracy and sensitivity of the measurements.It is unlikely that measurements of blood gases were inaccurate and insensitive since these wereperformed in a quality controlled clinical laboratory. Our oxygen measurements were not precise toa decimal point and had an accuracy of ±1%. The chambers were opened daily to replenish food andfor general maintenance, and oxygen was measured three times a day. The possibility of consistentlylower oxygen settings inside hypobaric normoxic chambers were minimal. It has been reported thatan exposure to chronic mild normobaric hypoxia (19%) did not affect hematocrit values (187).Therefore, it is unlikely that a small variation in oxygen of ±1% may have produced consistenthypoxemia but an increased hematocrit in our hypobaric normoxic rats was surprising. However, otherinvestigators have found hypoxemia following exposure to hypobaric normoxia. Levine and coworkers(171) found that following two hours of exposure to hypobaric normoxia, hypobaric hypoxia andnormobaric hypoxia (simulated to 6600 m or 326 torr), arterial P02 decreased in awake sheep whichwere subjected to hypobaric normoxia. Hirai and associates (137) reported that it required 65%Chapter 4: DISCUSSION 190ambient oxygen concentration to make hypobaric sheep nornnoxemic instead of 49.7% ambient oxygenconcentration measured in similar hypobaric normoxic conditions. This issue is further complicatedby the fact that an increase in hematocrit may also occur due to a decrease in plasma volume (251)which we did not measure. Thus, we have no convincing explanation for an increase in hematocritin hypobaric normoxic rats. HYPOBARIC NORMOXIA vs GENERAL CONTROLSLung volume and the TLC of excised lungs decreased significantly in rats exposed to hypobaricnormoxia for three weeks. Dry weight of the lung, DNA, RNA, total soluble protein, hydroxyproline anddesmosine remained the same in hypobaric normoxic rats compared to general controls. Despite adecrease in lung volume, alveolar surface area and total alveolar number did not differ from those ofgeneral controls. However, dimensions of morphometric unit structures were smaller in hypobaricnornnoxic animals. Biochemically estimated 3H-TdR incorporation into DNA increased on day 5 ofexposure and thereafter reached the general control level. Autoradiographs also revealed thatmaximal DNA synthetic activity occurred on day 5 in alveolar wall cells. Lung volumes and capacitiesmeasured in vivo hypobaric normoxic animals were the same as those in general controls. Hypobaricnormoxia did not produce changes in expiratory flow rates and in pressure-volume or flow-volumecharacteristics.Somatic growth of hypobaric normoxic animals was diminished. Hypobaric normoxic animalsate less and were significantly smaller on day 10 of exposure, and on day 21 their body weight wasalmost midway between general controls and hypobaric hypoxic animals. We did not have weight-matched controls for hypobaric normoxic animals and thus results in lung growth are hard to interpret.Although it is not appropriate, we also normalized our results for body weight. Despite diminished lungvolume, specific lung weight and volume of hypobaric normoxic animals were equivalent to that ofgeneral controls. These findings suggested that lung growth was relatively less affected in hypobaricnornnoxia than somatic growth.Chapter 4: DISCUSSION 191One may argue that in the present study, hypoxemia may have stimulated lung growth inhypobaric normoxic animals. This cannot be ruled out. However, Monola and associates (187) foundthat mild (19% FIO2) normobaric hypoxia for one week caused no effect on somatic or lung growthin rats. In addition, if we delivered a higher concentration of oxygen to make hypobaric normoxicanimals normoxemic then, hyperoxia would have affected lung growth. Since we designed ourexperiments to study the effect of high altitude conditions, hypobaric normoxia was an appropriateexperimental condition for the effect of low ambient pressure. STRUCTURAL ADAPTATIONSEven though hypobaric normoxic rats gained less body weight compared to general controls,biochemically estimated lung parameters were the same. RNA/DNA, protein/DNA, hydroxyproline/DNAand desmosine/DNA ratios also remained unchanged. These findings suggested that in spite ofdiminished somatic growth, hypobaric normoxia did not produce alterations in lung structure.3H-TdR incorporation measured by biochemical analysis doubled on day 5 of exposure tonorrnobaric hypoxia indicating that decreased ambient pressure did stimulate lung growth. Thesefindings were confirmed by analysis of autoradiographs as the labelling index of cells in the alveolarwalls also peaked on day 5 of exposure. Cytodynamic heterogeneity was observed in various cellpopulations. Pleural mesothelial cell stimulation peaked on day 3 while type II pneumocytes, interstitialcells and unidentified cells in the peripheral part of the lung showed maximal stimulation on day 5.In the walls of central alveoli, 3H-TdR incorporation in type II pneumonocytes and interstitial cellspeaked on day 7. These results indicated that the peak effect of hypobaric normoxia was alsodelayed in the central part compared to the peripheral part of the lung. The reasons for relatively earlystimulation of mesothelial cells are not clear.Interestingly, a maximal increase was noticed in interstitial cells while alveolar wall and arterialendothelial cells remained unaffected by hypobaric normoxic stress. Although a small increase wasfound in hematocrit of hypobaric normoxic rats, it perhaps was not sufficient to cause hemodynamicChapter 4: DISCUSSION 192changes which could have stimulated endothelial cell DNA synthesis by removal of local cell-specificgrowth inhibitors or by supplying the cell growth promoters. On the other hand, acute exposure tohypobaria increases transvascular fluid flux due to a hydraulic pressure difference between capillarypressure and interstitial pressure (171). We also found an increase in dry/wet lung weight ratio ondays 1 and 3 of exposure to hypobaric normoxia (0.2156 ±0.0038 and 0.2113 ±0.0019 respectively)compared to general controls (0.2212 ±0.0015 and 0.2218 ±0.0021 respectively)but it reached asignificant level only on day 3. Transvascular fluid flux may be the reason for an increase in lungwater during the early period of exposure and this may stretch the interstitial space. With alteredhydrostatic pressure, changes in membrane shape and function, and cellular protein synthesis (158)have been documented. In vitro periodic stretch of the membrane on which fibroblasts were culturedstimulated cell proliferation (78). Therefore, changes in interstitial cells might have been induced byphysical forces in the interstitium of the lung under hypobaric normoxia. Alternatively, with themovement of transvascular fluid into the interstitial space, it is possible that some growth promotingfactors may also increase within the interstitium and hence stimulate growth related alterations.Therefore, results of autoradiographs of hypobaric nornnoxic animals support the notion that endothelialcells and other alveolar wall cells are stimulated through different mechanisms.Bartlett (18) did not find convincing changes in morphometric lung growth variables in ratsexposed to normobaric hypoxia for 15 days, whereas an exposure to hypobaric hypoxia for 21 daysproduced significant increases in lung volume and alveolar surface area (20). These studies (18, 20)suggest that hypobaric pressure may play an important role in lung growth adaptation. Although inthe present study, biochemical changes suggested that hypobaric normoxia did not induce structuralalteration in the lung, the nnorphometric results showed that it produced an increase in the surfacecomplexity (increased surface/volume ratio and Nv, and decreased MU I and Valvave) of the lung. Thissupported the concept that hypobaric pressure may have a definitive role in high altitude lung growthadaptation.Interestingly, despite decreased lung volume, alveolar surface area and total alveolar numberChapter 4: DISCUSSION 193remained unchanged in hypobaric normoxic rats. This may suggest that even though lung growthdiminished in hypobaric normoxia, alveolar proliferation was maintained at normal levels. Thissupports the hypothesis that alveolar number is genetically programmed. How low ambient pressuremay bring about a decrease in the size of the respiratory units is not known. One explanation maybe that alveolar proliferation occurred at the normal rate but concomitant enlargement of airspaces didnot follow as lung volume did not increase. This may have occurred due to lack of mechanicalstimulation such as hyperventilation that occurs in hypoxia (31, 210). Alternatively, reduced ambientpressure may have induced a direct effect on size of morphometric unit structures. It has beensuggested that alveolar pressure is directly transmitted to the interstitium (27). Theoretically, thealveolar pressure equilibrates with ambient pressure as it falls in hypobaric conditions and such achange in ambient pressure should not produce structural alterations in the lung. However, the pleuralcavity is a potential space between the lungs and the chest wall and the chest wall is a rigid structure.It is thus conceivable that pleural pressure may become more negative and that transpulmonarypressure may also become more negative. It can be speculated that an increased pressure gradientbetween the pleural cavity and respiratory units may in part be responsible for smaller dimensions ofairspaces in hypobaric conditions. FUNCTIONAL ADAPTATIONSAlthough the values of absolute FRC, RV, VC, TLC were lower than those of general controls,the differences were not significant. Studies conducted during Skylab 4 mission showed that vitalcapacity of crew members exposed to hypobaric normoxia for 84 days was decreased without affectingother lung functions and upon recovery vital capacity returned to the normal level (233). The authorsconcluded that these changes in vital capacity may have occurred due to a direct effect of reducedambient pressure. Similar observation have been made by other investigators (222, 268). In thepresent study, FRC/TLC, RV/TLC and VC/TLC ratios in hypobaric normoxic rats did not differ fromthose in general controls. These findings also suggested that lung growth in hypobaric normoxicChapter 4: DISCUSSION^ 194animals was not affected.As mentioned earlier, connective tissue proteins such as collagen and elastin play a major rolein elastic lung recoil and thus can influence lung flow rates. The amount of collagen and elastin andtheir concentration in the lung tissue remained unaltered in hypobaric normoxic conditions comparedto general controls. FEV01/FVC (Yo, PEFR, FEF25_75%, absolute or specific upstream resistance, andpressure-volume and flow-volume characteristics in hypobaric normoxic animals did not differ fromthose of general controls. Theoretically, the decreased mean linear intercept of airspaces in hypobaricnormoxic rats would have reduced the value of the shape constant K of pressure-volume curves butthe constant K and static lung compliance remained unchanged. These observations supported thebiochemical results suggesting that chronic hypobaric normoxia did not alter the integrity of the lungstructure. HYPOBARIC HYPDXIA vs NORMOBARIC HYPDXIASome differences were observed in the hypobaric hypoxic and normobaric hypoxic rats butotherwise the lung growth response was similar. The amount of desmosine and desmosine/unit drylung weight increased significantly in hypobaric hypoxic animals compared to normobaric hypoxicanimals. The changes that occurred in hydroxyproline were not as dramatic as desmosine. Althoughan increase in the amount of hydroxyproline in hypobaric hypoxia did not reach a significant level,hydroxyproline/DNA ratio increased significantly compared to normobaric hypoxic animals. Thesefindings suggested that elastin and collagen accumulation was increased when low oxygen wasdelivered by lowering the ambient pressure. Hydroxyproline/unit dry weight of the lung fell innormobaric hypoxic animals compared to weight-matched controls but remained unchanged inhypobaric hypoxic animals suggesting that hypobaric pressure may have counterbalanced theinhibitory effect of low oxygen on hydroxyproline equilibrium. One may suggest that increasedtransvascular fluid flux in hypobaric hypoxia (171) may have stimulated connective tissue proteinsynthesis. Leung and associates (169) found that rhythmic stretch increased collagen and proteinChapter 4: DISCUSSION 195synthesis in smooth muscle cells grown on an elastic membrane. In the present study, peripheralinterstitial cell labelling index was significantly higher on day 5 (p=0.04) in hypobaric hypoxic animalsthan normobaric hypoxic animals. It suggests that low ambient pressure may have enhanced collagensynthesis by stimulating interstitial cells in hypobaric hypoxic animals. The other explanation is thatlow ambient pressure may facilitate hydroxylation of proline.The density of air is directly proportional to the barometric pressure, thus, in hypobaricconditions the mass of air decreases with a fall in ambient pressure. It has been postulated thatdecreased mass of air in hypobaric conditions helps to reduce the resistance to turbulent flow of airin the airways (75). However, hypoxia is known to cause bronchoconstriction (31). Cruz (75)suggested that increased airway resistance due to bronchoconstriction may counterbalance the effectof the decrease in air flow resistance due to decreased air density at high altitude. It is possible thatdecreased airway resistance in hypobaric conditions may also help a greater mass of air to move intothe lungs and thus establish better ventilation in hypobaric hypoxic animals compared to normobarichypoxic animals. Therefore, relatively increased alveolar oxygenation may have helped thehydroxylation of proline and in essence, increased the collagen accumulation in the lung in thehypobaric hypoxic animals. In hypobaric normoxia, an adequate supply of oxygen was available andthat may be the reason that no obvious change in the amount of hydroxyproline was observed. Inhypobaric hypoxic conditions where oxygen was deficient, better ventilation might have increased thequantity of oxygen available for various processes. Therefore, with regards to collagen equilibrium,the role of hypobaric pressure remains elusive.Morphometric analysis showed that changes occurring in hypobaric hypoxic animals comparedto normobaric hypoxic animals were similar to those found in hypobaric normoxic animals comparedto general controls. MLI and Valvave increased and Nv decreased significantly in hypobaric hypoxicanimals compared to normobaric hypoxic animals. Normobaric hypoxia produced simple enlargementof airspaces and hypobaric hypoxia did not. Airspaces were enlarged in nornnobaric hypoxia, reducedin hypobaric normoxia and remained unchanged in hypobaric hypoxia. Therefore, low ambientChapter 4: DISCUSSION^ 196pressure in hypobaric hypoxia may have counterbalanced the airspace enlargement of hypoxia, thuskeeping the dimensions of airspaces at normal levels.FEVo.i/FVC%, PFER and FEF25_75% were higher in hypobaric hypoxic animals than normobarichypoxic animals. It was interesting to note that the values for FEV0.1/FVC%, PFER, FEF25_75%, andabsolute and corrected FEF of hypobaric hypoxic animals fell in between normobaric hypoxic rats andgeneral controls. Elastic recoil of the lung was decreased at higher lung volumes in nornnobarichypoxic animals compared to general controls but not in hypobaric hypoxic animals. Definitivereasons for these differences in hypobaric hypoxia and normobaric hypoxia are not known butalterations in lung structure (biochemical and morphometric) may be responsible. These observationslead us to believe that hypobaric pressure in hypobaric hypoxic conditions may have improved thelung function alterations produced by hypoxia alone. Compared to weight-matched controls, theamount of collagen and elastin increased more in hypobaric hypoxic rats than normobaric hypoxic rats.Postnatally, the bulk of collagen and elastin is present in lung parenchyma and is related to theformation of respiratory units (41). We do not know where in the lung the accumulation of collagenand elastin occurred in hypobaric hypoxia. Airways are the site of elastin and collagen fibers and actas guy lines (70), therefore increased collagen and elastin accumulation may assist expiratory flowrates in hypobaric hypoxic animals by making airways less compliant. Formation of new elastic laminaoccurs in peripheral vessels in hypobaric hypoxia (145, 182) and these vessels run in close proximityof airways. They may also provide support to airways and reduce the airway resistance in hypobarichypoxic animals.In brief, hypobaric normoxia stunted somatic growth and diminished lung growth. Specific lungweight and volume in hypobaric normoxia did not differ from general controls suggesting lung growthwas affected less than somatic growth. Biochemical assessment revealed that hypobaric normoxiadid not induce any changes in lung structure but morphometric analysis showed that it causedreduction in size of unit structures of the lung. Hypobaric normoxia produced no functional alterationsin the lung. Hypobaric normoxia stimulated lung growth which peaked on day 5. Lung growthChapter 4: DISCUSSION^ 197stimulation was relatively less and was also delayed compared to hypobaric hypoxia or normobarichypoxia. Endothelial cells were least stimulated by hypobaric normoxia which perhaps correlated witha small increase in hematocrit. Low ambient pressure in the presence of low oxygen tensionincreased accumulation of elastin more than collagen. Low pressure appeared to reduce airspaceenlargement and improve lung function changes caused by hypoxia alone.The similarities in hypobaric hypoxia and normobaric hypoxia may be due to two reasons.One possibility is that greater effect of hypoxic stress may have masked the effect of low ambientpressure. Alternatively, hypoxia and low ambient pressure may stimulate lung growth through acommon mechanism. Our results did not show a clear synergistic effect of hypobaric pressure andhypoxia on lung growth in hypobaric hypoxic animals. However, with regards to elastin accumulationsome additive effect of hypobaric pressure and hypoxia was apparent in hypobaric hypoxia. Thedesmosine concentration increased 16% in hypobaric normoxic rats, 18% in normobaric hypoxic ratsand 40% in hypobaric hypoxic rats compared to general controls. Similar changes were found in theamount of total desmosine and in the desmosine/DNA ratio. In other lung parameters such ascollagen accumulation and airspace size, hypobaric pressure in hypobaric hypoxia appeared to helpkeep values approximately the same as in general controls. Flow-volume characteristics of hypobaricnormoxic animals did not alter but expiratory flow rates fell and upstream resistance increased innormobaric hypoxic animals. In hypobaric hypoxic animals, the flow-volume characteristics showeda shift towards the general controls. These observations support the notion that low ambient pressureplays a significant role in lung growth adaptation at high altitude. One can speculate that in somerespects such as connective tissue protein accumulation and size of morphometric unit structures, themechanism of the effect of low ambient pressure may not be similar to that of low oxygen tension.4.5. EFFECT OF 3-DAYS POST-EXPOSURE AND REFEEDINGAdaptive response to a stress includes quantitative or qualitative alterations in organs whosefunctions are directly or indirectly affected. If that stress is removed, structural and functional changesChapter 4: DISCUSSION 198which occur as a result of adaptation may or may not return to normal levels. The recovery ofadaptive response in various organs may also be variable. We aimed to study somatic and organgrowth changes following chronic exposure to hypobaric hypoxia, normobaric hypoxia and hypobaricnormoxia, and restricted feeding. After subjecting the animals to respective experimental conditionsfor three weeks, they were returned to room air and were allowed free access to food for three days.We chose a three day recovery for following reasons: (1) hemodynamics and heart weight return tonormal levels within one week after returning to room air (197), (2) changes at cellular level may occurearly while the time course for structural remodelling following recovery may be prolonged, and (3) inthe present study, maximal lung growth stimulation and cellular biochemical changes occurred after3 days of exposure.Since undernourishment caused impairment of body weight gain and axial skeletal growth, foodad libitum for three days produced substantial increase in body weight (16%) compared to generalcontrols (8%) but skeletal growth appeared to be unaffected. Except for the spleen, growth of allorgans (heart, lung, liver and kidney) showed a considerable increase. This suggested that organswhose metabolic needs and functions were affected by undernutrition showed accelerated recovery.Winick and Noble (284) showed that recovery in body and organ weights is dependent upon the ageof the animals following undernourishment. They observed that while the body and organ weights ofadult rats subjected to restricted food intake recover fully within seven weeks, young rats subjectedto the same length of period to undernutrition did not.When food restricted animals were fed ad libitum the rate of 3H-TdR incorporation into lungtissue DNA more than doubled. In addition, the amount of lung DNA also increased slightly. Theamounts of lung RNA and protein increased suggesting enhanced protein synthesis after refeedingthe undernourished animals ad libitum. Cell size also increased as DNA/RNA and protein/DNA ratioswere increased. Similar observations have been made by other investigators following refeeding thestarved animals (228).Hunter and Crapo reported that somatic growth recovery may vary depending upon the severityChapter 4: DISCUSSION 199and duration of hypobaric hypoxia conditions (144). Our results revealed that the rate of body weightchange increased but nose-tail length was not affected by returning hypobaric hypoxic and normobarichypoxic animals to room air for three days. Hunter and Clegg (144) showed that 28 day old miceexposed for 4 or more days to hypobaric hypoxia (390 mm Hg) caused permanent stunting of bodygrowth.Interestingly, the weights of liver and kidney increased during the three day recovery in animalspreviously exposed to hypobaric hypoxia and normobaric hypoxia for 3 weeks. During exposure, liverand kidney growth were similar to those of undernourished animals suggesting that their growth wasnot directly affected by hypoxia but was related to diminished food intake. Once the inhibitory effectof a down modulator (hypoxia) was removed, increased liver and kidney growth appeared to beassociated with increased food consumption. On the other hand, following recovery, the weights oflung, heart and spleen either decreased slightly or remained unaffected in hypobaric hypoxic andnormobaric hypoxic animals. This suggests that after removal of the growth stimulus (hypobarichypoxic or nornnobaric hypoxic stress), augmented level of lung, heart and spleen growth was nolonger maintained. This may have occurred due to an immediate fall in increased cellular proliferationand/or connective tissue protein accumulation during recovery.We observed that after 3 day recovery, heart weight reached the general control levels whilelung weight remained high. Our findings matched with those made by Okubo and Mortola (197) whofound that in newborn rats, an exposure to 10% F102 for six days produced changes in ventilation,oxygen consumption, hematocrit, body weight, heart, and specific lung weight. After returning to roomair for one week, hematocrit and heart weight were back to control values but lung weight andventilation were not. The authors suggested that differences in heart and lung growth recovery mayhave occurred due to long-term effects on the regulation of breathing. In addition, heart growthadaptation in hypoxic conditions primarily occurs by hypertrophic changes in myocardial fibers (191)whereas we found that lung growth occurred both by hyperplastic and hypertrophic changes. Thismay be the reason that heart weight returned to normal levels within three days of recovery period andChapter 4: DISCUSSION^ 200lung weight did not.It has been shown that normobaric hypoxic exposure for 6 days produced permanent changesin neonatal rat lungs as lung weight, lung volume and mean chord length of alveoli remain increasedafter 44 days of recovery (198). However, the authors found that somatic growth recovery wascomplete. In the present study, 3H-TdR incorporation into the lung tissue decreased during the threeday recovery period in hypobaric hypoxic and normobaric hypoxic animals indicating that lung growthwas no longer stimulated after removal of stimulus. During recovery, in hypobaric hypoxic andnormobaric hypoxic rats, the RNA content fell, and RNA/DNA ratio decreased and reached the controllevel. These findings suggested that enhanced lung growth that occurred by continued stimulation,returned immediately to normal levels in some respects (i.e. cell size, DNA synthesis) when thehypoxic stress was discontinued. However, increased lung growth dimensions (i.e. lung weight, DNA,protein) did not appear to decrease, thus the residual effect of hypobaric hypoxia or normobarichypoxia may be permanent. We did not study long-term recovery and it is possible that unlike theheart, structural changes in the lungs which occur as a part of the adaptive response to hypoxic stressmay be permanent or require a prolonged period to recover.Chapter 4: DISCUSSION^ 2014.6. RECAPITULATION AND CONCLUSIONSIt has been postulated that besides other factors, oxygen plays a substantial role in determininggrowth and development of the lungs as gas exchange is the primary function of the lungs. Loweroxygen tension has been shown to stimulate lung growth while somatic growth is diminished.However, results are controversial. Because the reduction in somatic growth occurs due toundernutrition, it is important to know the effect of impaired nutrition on the adaptive growth responsein lungs or other organs in hypoxic conditions (hypobaric hypoxia and normobaric hypoxia) and for thefirst time it has been assessed systematically. Oxygen tension at high altitude is determined mainlyby a decrease in ambient pressure, but the role of low ambient pressure in the absence (hypobaricnormoxia) or presence of hypoxia (hypobaric hypoxia) on lung (or any other organ) growth adaptationhas not been investigated. Lung connective tissue proteins which may alter in contents followingexposure to hypobaric hypoxia and normobaric hypoxia have not been quantified before. The presentstudy also provided us the opportunity to compare the adaptive lung response to normobaric hypoxiaand hypobaric hypoxia which has not been done so far. Furthermore, the influence of normobarichypoxia and hypobaric hypoxia on lung growth variables including biochemical (both cellular andconnective tissue proteins), morphometric, cytokinetic and physiological parameters have not beenstudied in a single experiment.We found that stunting of somatic growth (body weight and axial skeleton) in both hypobarichypoxia and normobaric hypoxia occurred due to diminished food consumption which resulted mainlydue to decreased oxygen. Low ambient pressure alone also reduced body size but this reduction wasless than that occurring in hypobaric hypoxia or normobaric hypoxia. Hypobaric hypoxia andnormobaric hypoxia produced substantial increases in hematocrit. Despite retarded somatic growthin hypobaric or normobaric hypoxia, the growth of lungs, heart and spleen increased whereas in theweight-matched animals their growth decreased. This indicated that growth of these organs increasedprofoundly in hypobaric and normobaric hypoxia when compared to weight-matched animals. Theeffect of hypobaric hypoxia and normobaric hypoxia on organ growth was the same, suggesting thatChapter 4: DISCUSSION^ 202augmented growth of lungs, heart and spleen at high altitude is induced by low oxygen.Undernutrition not only diminished somatic growth but also adversely affected lung growthbecause lung weight and volume, and all quantitative biochemical and morphometric parameters weredecreased. DNA synthetic activity was inhibited by undernourishment in all the compartments of thelung. Despite reduced lung growth, pressure-volume and flow-volume characteristics remainedunchanged in undernourished animals, indicating that the structural integrity of the lungs was notdisturbed by undernutrition.Since there were no visible differences in the structure of the lungs of undernourished andgeneral controls, we can virtually rule out the effect of malnutrition at least in our hypobaric andnormobaric hypoxic animals. Therefore, the changes occurred in lung structure and function inhypobaric hypoxia or normobaric hypoxia reflected the specific effect of respective environment.Adaptive lung growth response was similar in most aspects in hypobaric hypoxia and normobarichypoxia compared to weight-matched controls.^lar^Striking increases in lung weight and volume were indicative of considerable lung growth inhypobaric hypoxia and normobaric hypoxiaor^Increases in DNA indicated hyperplastic changes while the increase in RNA suggestedenhanced protein synthesis. Cell size increased during the first week of exposure but reachednormal levels by the end of exposure.si.^Radiochemical analysis showed that maximal lung growth stimulation occurred on day 3 ofexposure as 3H-TdR incorporation into DNA increased 12-fold. Thereafter, the rate of 3H-TdRincorporation fell gradually till day 7 but remained high until the end of exposure suggestinglow but persistent cellular proliferation.^sr^Connective tissue protein accumulation was substantial, but an increase in collagen was lessthan cellular proliferation in normobaric hypoxia. On the other hand, the accumulation ofelastin was higher relative to DNA in hypobaric hypoxia suggesting a shift in connective tissueprotein dynamics.Chapter 4: DISCUSSION^ 203IV Airspace dimensions increased and surface complexity decreased more in normobaric hypoxiathan hypobaric hypoxia. Alveolar enlargement was thus a more prominent feature in lunggrowth adaptation in normobaric hypoxia compared to hypobaric hypoxia. Furthermore,alveolar surface area and total alveolar number increased more in hypobaric hypoxia thannormobaric hypoxia, thus indicating that lung growth occurred by addition of new structuralunits along with alveolar enlargement.lir Autoradiographic results showed that a high proportions of all endothelial cells (alveolar walls,or arterial) incorporated 3H-TdR on day 3 of exposure. The rate of DNA synthesis ofperipheral type ll pneumonocytes, interstitial cells and unidentified cells, and mesothelial cellspeaked on day 3 but the central cells (type II pneumonocytes, interstitial cells and unidentifiedcells) showed maximal stimulation on day 5. This suggested that endothelial cells stimulationmay be secondary to hemodynamic changes but other cells react to the direct effect of thestress. In addition, the peripheral part of the lung responds earlier than the central part andthis variation may be due to geometrical considerations. These are also new findings.Kr The connective tissue framework of the lung was not affected by hypobaric hypoxia aspressure-volume characteristics and static lung compliance remained unchanged. Upstreamairway resistance increased and expiratory flow rates at higher volumes decreased more innormobaric hypoxia than hypobaric hypoxia. This evidence combined with higher cellproliferation in the alveolar wall than conducting airways suggested dysanaptic lung growthduring acclimatization to hypobaric hypoxia and normobaric hypoxia.With regards to adaptation to hypobaric hypoxia and normobaric hypoxia, there was a clearindication of enhanced lung growth compared to general control animals. Absolute lung weight andvolume, DNA, RNA, total protein and elastin increased in normobaric hypoxia. In addition,hydroxyproline and alveolar surface area also increased in hypobaric hypoxia. In addition to the abovephysiological changes (compared to weight-matched controls), elastic lung recoil at higher volumesin normobaric hypoxic rats decreased which may be attributed to alterations in tissue forces. TheChapter 4: DISCUSSION^ 204similarities in lung growth response in hypobaric hypoxia and normobaric hypoxia provided evidencethat low ambient oxygen is the main driving stimulus to enhance lung growth at high altitude.In some aspects, lung growth differed in hypobaric hypoxia compared to normobaric hypoxia.Hydroxyproline and desmosine increased more in hypobaric hypoxia than normobaric hypoxia. Withregards to morphometric analysis, airspace size and average volume of alveoli were smaller and thenumber of alveoli per unit volume was greater in hypobaric hypoxia demonstrating that surfacecomplexity of the lung may have increased in hypobaric hypoxic animals compared to normobarichypoxic animals. Physiological measurements (flow rates, upstream airway resistance) decreased lessin hypobaric hypoxia than normobaric hypoxia. One can infer from these differences that lung growthdiverged less in hypobaric hypoxia than in normobaric hypoxia. These differences may also beattributed to low ambient pressure in hypobaric hypoxic conditions.In hypobaric normoxic animals, hematocrit increased. Apart from decreased lung volume, lunggrowth was similar to general control animals. DNA synthetic activity doubled on day 5.Autoradiographic results showed that the endothelial cells showed a minimal increase in 3H-TdRincorporation and since there was a smaller increase in hematocrit suggesting that it is possible thatendothelial cell stimulation depended upon alterations in hemodynamics. The size of airspaces wassmall and a greater number of alveoli were present per unit volume indicating increased surfacecomplexity of the lung. Specific total protein, alveolar surface area and total alveolar numberincreased. Lung structure was not disturbed as lung function tests did not show any alterationsfollowing chronic hypobaric normoxic exposure. In addition to the differences noted from comparisonof hypobaric hypoxia and normobaric hypoxia, the alterations caused by hypobaric normoxia suggestthat, although low ambient oxygen is the major determinant of lung growth adaptation at high altitude,low ambient pressure also plays a role. Connective tissue protein accumulation increased anddimensions of morphometric unit structures remained smaller in hypobaric hypoxia than normobarichypoxia.Finally, specific lung growth, DNA, alveolar surface area and total alveolar number increasedChapter 4: DISCUSSION 205in weight-matched animals compared to the general controls. 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