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The effects of temperature change and lung expansion on lung liquid production in in vitro preparations… Garrad, E. Philippa 1990

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T H E EFFECTS OF T E M P E R A T U R E C H A N G E AND LUNG EXPANSION ON LUNG L I Q U I D P R O D U C T I O N IN IN VITRO P R E P A R A T I O N S O F L U N G S F R O M F E T A L G U I N E A P I G S (Cavia porcellus)  By  E . Philippa Garrad BSc. University of British Columbia, Canada, 1986  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE  in  T H E F A C U L T Y O F G R A D U A T E STUDIES Department of Zoology  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A September, 1990 © P h i l i p p a Garrad, 1990  1990-9-25  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  thesis for scholarly by  this  his  or  her  t-OCVf  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Q r H - W \CT  ^ 0  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  Department of %QO  the  is  that  an  advanced  Library shall make it  permission for extensive  granted  by the  understood be  for  allowed  that without  head  of  my  copying  or  my written  1. ABSTRACT  This study examined the effects of lung expansion and changes in temperature on fluid movement by the lungs in the initial period after birth. In addition, experiments with amiloride support the belief that fluid reabsorption acts via a sodium transport mechanism.  Lungs from fetal guinea pigs (56-67 days of gestation) were supported in vitro for three hours, and lung liquid production rates were measured using a dye dilution technique. The average production rate in the first hour of untreated preparations was 1.30 ±0.22 ml/kg body weight per hour, and this did not change significantly during the remainder of the experiment (n=30). This rate is comparable to secretion rates previously reported from chronically catheterized sheep.  In 36 further preparations, the lungs were transferred from 37°C to fresh Krebs-Hanseleit saline at one of the following temperatures, for one hour (an A B A design): (a) 29°C; (b) 32°C; (c) 34°C; (d) 35°C; (e) 36°C; (f) 39°C. In all cases, the temperature change resulted in an immediate and significant fall in secretion. All lungs showed a tendency towards recovery when returned to starting conditions, except those subjected to a temperature increase. Reductions of 2-3°C, those normally seen in the delivery room, had the greatest effect and caused not only a decrease in secretion, but promoted fluid reabsorption. Amiloride at 1 0 M had no effect on control preparations, but _6  completely blocked the reabsorption stimulated by a temperature drop of 2°C.  Expansion of the lungs, which occurs naturally as a newborn attempts to take its first breaths, was also examined. Thirty fetal lungs were expanded by one of the following amounts: (a) 18%; (b) 31%; (c) 43%; (d) 50%; (e) 72%. All expansions resulted in a significant fall in secretion rate, with the effect being proportional to the degree of expansion. Amiloride at 10~ M again blocked the 6  strong reabsorption occurring with 70% expansion. Further studies investigated the possibility that expansion causes reabsorption via the local release of a substance occurring in the lungs. When one set of lungs was expanded in the presence of a second, unexpanded set, both showed a significant  - ii -  1990-10-10  decrease in secretion, suggesting that the expanded lung had released some factor which affected the otherwise untreated lung. However, studies with a- and (3- adrenergic blockers showed that it is unlikely the expanded lung was liberating either adrenaline or nor-adrenaline.  The results of this study show that two changes which are likely to occur in the period immediately after birth, namely a 2-3°C decrease in core temperature, and lung expansion, may be important in promoting the vital reabsorption of fluid. They suggest that expansion may release substances locally in the lungs which stimulate this reabsorption, and that the fluid is removed from the potential air spaces via sodium transport mechanisms.  - iii -  1990-10-10  Table of Contents 1  ABSTRACT  ii  LIST OF FIGURES  vii  2  ACKNOWLEDGEMENTS  viii  3  INTRODUCTION  1  3.1  Origin of Fetal Lung Fluid  1  3.2  The Mechanism of Lung Fluid Production  2  3.3  The Secretion of Lung Fluid  3  3.4  The Reabsorption of Lung Fluid  5  3.5  Factors which Affect Fluid Movement in the Fetal Lung  8  4  S T A T E M E N T OF T H E P R O B L E M  12  5 METHODS AND MATERIALS . .  13  5.1  Animals  13  5.2  Surgical Procedures  13  5.3  Experimental Procedures  14  5.4  Statistical Methods  18  5.5  Chemical Methods  18  6 RESULTS  19  6.1  Fluid Production by in vitro Lungs from Fetal Guinea Pigs  6.2  The Effect of Temperature Change on Fluid Production  19  6.2.1  Fluid Production by Fetal Lungs Subjected to a Fall in Temperature  19  6.2.2  The Effect of a Temperature Decrease on Ion Movement  24  6.2.3  Fluid Production by Fetal Lungs Subjected to a Rise in Temperature  6.2.4  The Effect of a Temperature Increase on Ion Movement  - iv -  19  . . . .  24 27  1990-10-10  6.2.5  The Effect of Expansion on Fluid Secretion by in vitro Fetal Lungs (Figure 5) 30  6.2.7  The Effect of Expansion on Ion Movement  36  6.2.8  The Effect of Expansion on the Rate of Passage of Phospholipids into the Lung Fluid  36  The Effect of 70% Expansion on the Levels of Lactate Dehydrogenase in Lung Fluid  36  6.2.10 Pressures inside the Lung Following Expansion of 70%  37  6.2.11 The Effect of Amiloride on Fluid Secretion Following Expansion of the Lungs (Figure 7)  37  The Remote Effect of Expansion  41  6.3.1  The Remote Effect of Expansion in the Presence of Phentolamine (Figure 9)  44  6.3.2  The Remote Effect of Expansion in the Presence of Propranalol (Figure 10)  47  7 DISCUSSION 7.1  27  6.2.6  6.2.9  6.3  The Effect of Amiloride on Fluid Secretion Following a Temperature Decrease (Figure 4)  50  The Effect of Temperature Changes on Lung Fluid Secretion  51  7.1.1  The Effect of a Temperature Reduction  51  7.1.2  The Effect of a Temperature Increase  54  7.2  The Effect of Expansion on Lung Fluid Secretion  56  7.3  Is the Expansion within Physiological Limits?  58  7.3.1  Are the Lungs Damaged by Expansions of 72%?  58  7.3.2  What is the force driving the fluid out of the potential air spaces?  60  8 REFERENCES  63  APPENDIX A  69  A.l  Preparation of Krebs-Hensleleit Saline  69  A.1.1  To prepare stock solutions  69  A.1.2  To prepare 1 litre of saline from stock solutions add into a I L volumetric flask 69  - v -  1990-10-10  APPENDIX B  70  B.l  Preparation of Blue Dye  70  B.2  To Make Standard for Spectrophotometry  70  APPENDIX C  71  APPENDIX D  72  APPENDIX E  73  1990-10-10  List of Figures  1  Apparatus for the maintenance of the in vitro guinea pig fetal lung preparation 16  2  The effect of reduced temperatures on lung liquid production by in vitro lungs from fetal guinea pigs 22  3  The effect of a 2°C increase in temperature on lung liquid production by in vitro lungs from fetal guinea pigs  25  4  The effect of amiloride on lung liquid production by in vitro lungs from fetal guinea pigs, with and without a 2°C fall in temperature  28  5  The effect of expansion on lung liquid production by in vitro- lungs from fetal guinea pigs . 32  6  The relationship between expansion of the lungs and the fall in fluid production  34  7  The effect of amiloride on lung liquid production by in vitro lungs from fetal guinea pigs, with and without expansion by 70%.  39  8  The remote effect of expansion on lung liquid production by in vitro fetal guinea pig lungs. 42  9  The remote effect of expansion on lung liquid production by in vitro fetal guinea pig lungs, in the presence of phentolamine 45  10  The remote effect of expansion on lung liquid production by in vitro fetal guinea pig lungs, in the presence of propranalol 48  - vi I -  1990-10-10  2.  ACKNOWLEDGEMENTS  I would like to thank D r . Anthony Perks for his encouragement and patience during my experiments and in the preparation of this thesis. Also Birgitta Woods for her invaluable help with the early laboratory work and for contributing some of her data to this thesis. Dueul Chuang also allowed me to borrow his data for figure 2a. Many thanks to Elizabeth Vanderhorst for the ion analysis and to David McBeath for his work on the Electron Microscope. I want to thank Larry Nelson for all his support during this thesis, and Craig and Catriona Wilson for spending hours of their time helping me with the typesetting. Finally I want to say thankyou to my parents for always being there when I needed them, and for all the typing my mother did during the accomplishment of this work.  - viii -  1990-10-10  3. INTRODUCTION  The lung first appears in the human at 22-26 days after fertilization, as a shallow groove in the ventral wall of the gut, when the embryo is just 5mm long. The lungs, trachea and extra pulmonary bronchi develop from an endodermal outgrowth of the foregut. The tubular lung-bud then undergoes extensive branching to form the bronchial tree, the foundation of the lung itself. The cells which comprise the epithelial lining of the lung, such as ciliated, goblet and alveolar epithelial cells, as well as epithelial derivatives, are all derived from endoderm (Williams, 1977).  3.1  Origin of Fetal Lung Fluid  For many years it has been known that fetal lungs arefluid-filled,but even today the exact mechanisms by which this fluid is produced are not known. The earliest researchers believed that the fluid in the lung was simply inspired amniotic fluid; that the fetus made in utero respiratory movements which drew amniotic fluid into the respiratory tract and lungs (Winslow, 1786, quoted by Preyer, 1885; in Olver and Strang, 1974). Addison and How (1913) thought this unlikely, since it is improbable -that respiratory movements would be strong enough to draw amniotic fluid all the way into the terminal air sacs, and by 1939, it was generally accepted that the fetus does not undergo respiratory-like movements under normal conditions (Windle et a/,1939). However, if fetal requirements are not met, with respect to CO2 eUmination and O2 uptake, then the fetus may perform gasping movements (Windle et al, 1939). This still left unanswered the question as to the origin of fetal lung fluid.  During the course of their studies on alveolar development, Potter and Bohlender (1941) discovered a fetus with an isolated mass of lung tissue in the left pleural cavity. This tissue had no connection with the trachea, and thus amniotic fluid was unable to enter. Upon examination of the lung tissue, the alveolar spaces were found to be large, and the walls normally developed. A second fetus with a complete tracheal obstruction also had well-developed lungs. This showed that the inspiration of  -  1 -  1990-10-10  amniotic fluid was not necessary for normal alveolar development. Their studies also showed that during development, the lungs expanded gradually due to the fluid with which they were constantly filled. However, despite proving the fluid was not amniotic fluid, they were unable to suggest its origin.  In 1948, Jost and Policard ligated the trachea of rabbit fetuses and saw an increase in lung volume, suggesting that the lungs produce their own fluid.  Reynolds supported this in 1953 when he  performed an experiment in which he delivered a fetal sheep by Caesarean section, left the placental circulation intact and prevented the fetus from breathing by placing a rubber nose-bag over its head. He found an accumulation of clear, viscous fluid in the nose bag, apparently secreted by the lungs, naso-pharynx and mouth, indicating that this liquid must be a source of amniotic fluid, rather than the result of its inspiration. The lungs may be as important a source of amniotic fluid as the kidneys, especially in early gestation, since amniotic fluid is present before the fetal urethra is patent (Setnikar et al, 1959). Setnikar suggested that the inhalation of amniotic fluid could even be detrimental to the fetus, as corpuscles contained in the fluid would collect in the lung and cause respiratory impairment at birth. It was later shown that amniotic fluid is prevented from entering the lungs by a slow, but continuous flow of fluid out of the trachea (Carmel et al, 1965; Howatt et al, 1965), and possibly by means of a laryngeal sphincter which can be closed to block off the trachea, at least temporarily (Adams et al, 1969 ).  3.2  The Mechanism of Lung Fluid Production  Once it had been established that the lungs themselves secreted fluid, the mechanism of this secretion remained to be found. Adams et al (1963) analyzed the lung fluid and found it to be different in almost every respect from amniotic and allantoic fluids, having a much higher sodium and chloride content, but being lower in concentrations of potassium, protein, sugar and urea. These workers did find, however, that except for the low protein content, lung fluid was quite similar to fetal blood plasma. They proposed that it may be the result of an active secretory process or an  - 2-  1990-10-10  ultrafiltrate produced through the pulmonary capillaries. Fluid appeared to be secreted against a hydrostatic pressure, and if the trachea was ligated, thus preventing any escape of lung fluid, the lungs were forced to expand as more and more fluid was produced (Carmel et al, 1965). In 1969, Adamson et al showed that not only did lung liquid composition differ from that of amniotic fluid, but it was also strikingly different from fetal carotid artery plasma, especially with respect to total CO2 content and p H ( p H of lung fluid = 6.27; p H of plasma = 7.29). They therefore thought it likely that the lung liquid was a special material actively elaborated by the fetal lung. They could provide no evidence as to the part of the lung in which the fluid was formed, but, since no variations in fluid composition were found anywhere in the lung, felt that the entire cellular lining must be capable of maintaining the observed electrolyte concentration differences from plasma.  3.3  The Secretion of Lung Fluid  Olver et al (1975) showed that the luminal side of canine tracheal epithelium was electrically negative with respect to the submucosal side and predicted that anions were transported actively towards the lumen or cations were moved in the reverse direction. They confirmed this by showing a net flux of C l ~ towards the tracheal lumen, and of N a outwards, although they were unable to +  specify the actual cells responsible, or in fact, whether or not this property extended to the alveolar epithelium. However, later studies by Olver et al (1981) showed that the fetal sheep lung can actively transport C l ~ into the lung lumen by 84-87 days gestation (term = 147 days), suggesting that by this stage the epithelium is already free from leaks which allow passive diffusion. However, it does appear that the secretory system of the immature fetal lung may be less efficient than in the later fetus. A n essential prerequisite for net secretion is that the epithelium has low permeability, especially to those ions which are actively transported. The alveolar capillary membrane is the barrier which separates lung fluid (or gases in the adult lung) from the blood in the pulmonary capillaries. In adults and near-term fetuses, the barrier is composed of a single layer of alveolar epithelial cells  - 3-  1990-10-10  with basement membranes, an interstitial space of varying width, and a single layer of capillary endothelial cells. In terms of pore theory, the capillary walls of the fetal lamb are equivalent to a porous membrane, containing cylindrical water-filled channels 9-15nm in radius. Alveolar walls, on the other hand, have characteristics attributable to pores with radii of only 0.5-0.6nm (Egan et al, 1976). This means that plasma and interstitial fluid cannot readily enter the alveoli because most water-soluble plasma solute molecules are too large to penetrate the alveolar barrier. The alveolar membrane is so effectively 'tight' that it is quite impermeable to proteins, with the result that alveolar liquid has a much lower protein concentration than plasma. However, if it is damaged, as may occur in artificially ventilated premature babies, this barrier may allow the leakage of proteinrich liquid into the airspaces, leading to the formation of the hyaline membranes associated with respiratory distress.  The presence of fluid in the lungs during fetal life is vital for their normal development. Although exact mechanisms are still not well understood, it appears that the secretion of C l  -  into the alveolar  space is a major driving force for the production of lung fluid (Strang, 1977). It has been proposed (Frizzell et al, 1982) that C l passes across the basolateral membrane neutrally coupled with N a . -  +  Because the interior of the cell tends to be electronegative with respect to the luminal compartment ( C l ~ is able to enter the cell against its electrochemical gradient because N a is moving down a +  favourable gradient), some of the accumulated C l moves down its electrochemical gradient across -  the apical membrane by means of a conductive path (Gatzy, 1983). The N a exits the cell back +  across the basolateral membrane via N a - K +  +  pump. This keeps intracellular N a low, so that +  its entry is favoured when coupled with C l " . The K  +  exits the cell passively through channels in  the basolateral membrane. The apical membrane also appears able to conduct N a , but this is +  usually in the reverse direction (lumen to cell) to C l . However, N a does enter the luminal fluid, -  +  possibly via a paracellular route.  -4-  1990-10-10  3.4  The Reabsorption of Lung Fluid  The lungs of the fetal lamb contain about 30ml of liquid per kg body weight, which is formed in situ at 2-3 m l / k g hr (Strang, 1977). A t birth, rapid reversal i n the direction of liquid flow is essential for a smooth transition from placental to pulmonary gas exchange. As well as being a major functional event for the lungs, birth is also a major morphological event, as alveoli are absent in many species at birth (Thurlbeck, 1977). In fact in most species the majority of alveoli develop post-natally. The number of alveoli present in the human neonate is not known, but observations suggest that the terminal airspaces are larger in newborn children than in older children, suggesting that alveoli are either absent, or poorly developed at birth. How fluid reabsorption is achieved is still not entirely understood and is an area of much controversy. The existence of two active ion transport systems working in opposition would allow fine regulation of fluid production and reabsorption. For example, locally released substances that increase chloride ion secretion and decrease sodium uptake may promote fluid production. Substances which increase sodium absorption relative to chloride secretion may reduce secretion or even cause reabsorption; such a system will be discussed in detail later. As early as 1913, Addison and How realized that the lung has a remarkable capacity for the absorption of fluids, even into adult life, when they found that pouring 25 litres of water down the trachea of a horse, over a six hour period, had no effect other than the deepening of respiration. The same authors also noted that when the fetus' lungs expanded with the first breath of air at birth, the fluid in the trachea and bronchi was drawn downward into the lungs, with part of the fluid remaining adherent to the inside of the walls. A t the same time, the structure of the alveolar epithelial cells, which were irregularly cuboidal with rounded nuclei, changed upon air breathing. W i t h increased area of the alveolar walls, the nuclei were spaced further apart, the cytoplasm was drawn out, and the cells became very thin and flat. The mesenchyme appeared denser with more compact nuclei, and contained more conspicuous and distended blood vessels. Addison and How, however, were unable to determine where the fluid went when it was reabsorbed.  - 5-  1990-10-10  Aherne and Dawkins (1964) showed that in rabbits, the lung tissue lying close to the arteries, veins and bronchi become distended with liquid in the first few hours after the start of ventilation. They suggested that the fluid may pass from the alveoli to the lung interstices very quickly, and then be removed from this site more slowly in the lymph. Humphreys et al (1967) tested this theory by mixing dye with the lung liquid and following its path during reabsorption. After the onset of ventilation, dye was found in high concentrations in the thoracic lymph duct, and to less degree in the right lymph duct. A n increase in lymph flow was seen at the start of ventilation, and the proportion of lung lymph going to the thoracic duct was larger than that under resting conditions. Whatever causes the uptake of fluid at the start of ventilation must depend on the presence of a force which displaces the fluid from the alveolar spaces. Humphreys and his co-workers (1967) suggested that this force could be due to a decrease in pulmonary capillary hydrostatic pressure (which would occur if vasodilation at the start of ventilation included a decrease in post-capillary resistance). Or, the displacing force may be produced by the movements of ventilation itself (during a positive or a negative pressure lung inflation, alveolar pressure is higher than the pressure in the adjacent lung tissue, thus alveolar fluid may be displaced). The pressure on the thorax during vaginal delivery may be partly responsible for the rapid removal of lung fluid. If this were the principle method of fluid removal, it would be expected that fetuses delivered by Caesarian section would have respiratory problems. However, despite the fact that the gross appearance of vaginally-delivered rabbit lungs seem completely aerated after ten minutes, whereas the lungs of Caesarean-delivered rabbits take several hours to reach the same state, Adams et al (1971) found fluid removal differences between the two types of delivery were not statistically significant. Bland et al (1979) supported these findings, but also observed that fetal rabbits, born either vaginally or by Caesarian section during labour, had significantly less fluid in their lungs than those born operatively before the onset of labour. This suggests that labour is extremely important in initiating the removal of fluid from the potential air-spaces at birth.  In further experiments Bland et al (1982), found that excess fluid drained after birth by the  -6-  1990-10-10  lymphatics only averaged about 11.4% of the residual liquid in the lungs at birth. These workers suggested that most of the liquid must flow into the interstitium, and then be removed by the microcirculation of the lungs. They offered several possible factors which may help this mechanism. The first is the active transport of salt and water across the pulmonary epithelium; this moves fluid into the lung interstitium. Secondly, a transepithelial hydraulic pressure difference, and a substantial difference in the protein osmotic pressure drive the fluid out of the potential air spaces.  Another factor which undoubtedly helps in reabsorption was proposed earlier by Egan et al (1976). These workers found that at the onset of breathing in the lamb, there is a marked increase in the alveolar permeability to solutes and water, which appears to be related to the expansion of the lungs following birth. It is known that the alveolar permeability of the adult lung is not fixed, but tends to vary with the degree of lung inflation. The same effect appears to occur in the newborn lung, and during birth the increase i n alveolar permeability is six to tenfold. In addition, all the forces acting across the alveolar epithelium at this time are driving fluid out of the potential air spaces, so significant leakage of large molecules into the air spaces is unlikely. During a normal birth, the epithelial permeability increases just long enough for reabsorption to occur, and by 12 60 hours after birth the pore radius is back to normal (Egan, 1983).  Experiments by Perks and Cassin (1985) have shown that significant decreases in fluid production are not seen until the lungs are expanded by more than 18.6% of their total volume. Expansions greater than 18.6% result in increased sodium transport (from lumen towards interstitium); the movement of sodium being acompanied by that of water.  Studies with amiloride, a molecule which blocks sodium channels in the apical membrane of tight epithelia (Soltoff and Mandel, 1983) have shown this molecule to prevent the adrenaline-stimulated reabsorption of fluid from the lung lumen (Brown et al, 1983). This supports the idea that whatever force initiates reabsorption does so by causing the transepithelial movement of N a from the lumen. +  -7-  1990-10-10  3.5 Factors which Affect Fluid Movement in the Fetal Lung Several hormonal factors affect secretion and reabsorption of lung liquid, although most of these may operate indirectly, possibly by changing c A M P levels (Prizzell et al, 1982; Cott et al, 1986). Studies on such mechanisms have resulted in the discovery of many compounds which promote fluid reabsorption. Much less common is the discovery of factors which promote secretion. This may be because, in vivo, secretion of lung fluid is likely to be occurring at a maximal rate until just before birth, so an increase in rate upon the addition of a supposed stimulant would not be seen. However, prolactin has been found to be effective in stimulating fluid production, together with N a  +  and  C l ~ secretion (Cassin and Perks, 1982). This is reminiscent of the effect of prolactin on ion and water movement in lower vertebrates, including fish. Infusions of saline into the fetus also seem to increase fluid production, with the amount of increase dependent on infusion rate. Experiments by Brace (1982) showed that within an hour of infusing saline into a fetal sheep less than 10% of the saline remained in the fetus, suggesting that the fetal lungs may be partially responsible for maintenance of normal body volumes and osmolality during gestation. As sodium movement appears to be involved, fetal lungs may play an important role in controlling fetal blood volume and pressure.  Both coupled sodium-chloride entry into cells, and C l exit into the lumen of the lungs may be -  inhibited by loop diuretics, thus decreasing or reversing fluid secretion. Bumetanide at 1 x 1 0 ~ M 4  produces immediate reabsorption, and furosemide causes reabsorption at high concentrations (1 x 1 0 M ) , although it is less effective than bumetanide (Cassin et al, 1986). Infusions of arginige - 3  vasopressin ( A V P ) and arginine vasotocin ( A V T ) also cause significant decreases in tracheal fluid production ( Perks and Cassin, 1982, 1989; Ross et al, 1984), but the mechanisms by which they act are hard to elucidate. Possibly A V P works in conjunction with several other factors to produce slow, prolonged responses, probably more adapted to long-term drainage of the lungs than to the rapid reabsorption which occurs at birth (Perks and Cassin, 1989).  -8-  1990-10-10  The uterine contractions of the mother at birth are usually reflected by changes in fetal arterial blood pressure, and there may be some fetal asphyxia and acidosis towards the end of delivery. This usually results in the widespread stimulation of the sympathetic system, causing a rapid increase in fetal heart-rate and a large rise in plasma lactate, free fatty acids and glucose - all occurring within fifteen minutes of delivery (Comline and Silver, 1960). This has led Olver (1977) to suggest that catecholamines may be the main stimulus to convert the lungs for air-breathing. Olver and Walters (1977) found adrenaline to be particularly effective in causing reabsorption in the chronic sheep preparation, with nor-adrenaline being ineffective in equimolar amounts, suggesting that a beta-effect was operating. The response to adrenaline was also found to increase with increasing gestational age (Olver, 1981). Until 130 days gestation, infusions of adrenaline slowed or halted fluid secretion, while after 130 days they stimulated reabsorption. Because of the experiments by Olver, adrenaline became recognized as the single most important stimulus for promoting fluid reabsorption at birth. However, this has met with some dispute. For example, Bland et al (1982) found no significant increase in plasma concentrations of adrenaline with labour, and experiments by Woods (unpublished observations) have shown that, in the guinea pig, adrenaline has a much less powerful effect on fluid reabsorption than in the sheep, and that nor-adrenaline appears to be the more effective stimulus. This suggests that the effect of catecholamines may vary among species, and that the effects found in sheep by Olver and his co-workers may not necessarily be those occurring in humans. From the observations of Wurtman and Axelrod (1966) that phenylethanol -amine-N-methyltransferase (PNMT) (the enzyme which converts nor-adrenaline to adrenaline) is stimulated by adrenal glucocorticoids, Kitterman et al (1979) propose that the increase in levels of plasma Cortisol, which occurs 48 hours before the decrease in lung fluid, may stimulate PNMT activity. This results in an increased endogenous formation of adrenaline, which in turn may cause the decreased production of fluid just before the onset of labour. This idea, while agreeing with the importance of adrenaline as a control of lung fluid secretion, fits the role of this hormone into a mechanism where it is just the end-point in a series of important steps. Therefore, there are many  -9-  1990-10-10  other factors equally as necessary as adrenaline, which need to be elucidated to fully understand the mechanisms of fluid secretion and reabsorption by the fetal lung.  As well as the hormonal surges occurring in the fetus at the time of delivery, there are also many other changes it experiences, which have been less well studied. One such change is a rapid fall in body temperature.  Under normal delivery-room conditions, human fetal core temperature drops  by 2-3°C, and may remain low for several hours after birth (Gandy et al, 1964; Adamsons and Towell, 1965; Adamsons, 1966). The small size, wet skin and reduced thermogenic capacity of the newborn make it harder to maintain a stable body temperature, and temperature fluctuations are common (Adamsons and Towell, 1965; Dahm and James, 1972; Briick, 1978). It has been suggested that the initial fall in temperature at birth may be important in promoting the onset of breathing (Adamsons, 1966; Dahm and James, 1972), but until this point, there has been no investigation into a possible effect on lung fluid secretion. It seemed important, therefore, to extend the study of lung fluid production to the effects of temperature.  Another influence acting on the lungs at birth is expansion. As soon as the fetus is delivered, it must establish air-breathing, and in an attempt to do so, it makes several deep, gasping movements which expand the lungs. Studies into the effects of expansion have been limited almost entirely to ruminants. However, the physiology of ruminants often shows dissimilarities to that of other mammal groups, and therefore ruminants may not provide the best model for human lung mechanics.  It was decided to extend pilot studies performed by Perks et al (1990), which used a dye dilution technique on in vitro lungs from fetal guinea pigs (Cavia porcellus) to study these mechanical effects. The use of guinea pigs offers results from a mammal rarely used for lung fluid studies, although it has been suggested that they are the the preferred model for human lung development (Stith and Das, 1982). The size of fetal guinea pig lungs is useful, because they are large enough to study by the methods established in ruminants, yet are an ideal size for in vitro studies.  -10-  1990-10-10  In vitro preparations are also relatively uncommon in the field of fetal lung research. Most work has been performed on intact animals, and while these have provided valuable observations, they have the disadvantage that substances infused into the blood stream may be broken down in the body, or lost via the placenta, and so are at an unknown concentration by the time they reach the lungs. Certain substances may also be toxic to the whole animal and so may not be tested for their effect on the lungs. Although this problem may be solved by using tissue culture, this involves the loss of organization of the lung and so is not a particularly satisfactory solution. An in vitro preparation maintains the integrity of the lungs and allows the direct application of drugs, toxic to the intact animal, at a known concentration.  The work presented here is an extension of a relatively new technique which uses in vitro guinea pig lungs. It examines the effects of temperature change and lung expansion on fluid production by fetal guinea pig lungs.  - 11 -  1990-10-10  4. STATEMENT OF THE PROBLEM  The purpose of this study is to examine the effect of temperature change and expansion on fetal lungs at around the time of birth, using a relatively new technique and a species of animal not commonly used in fetal lung studies. In addition, studies using amiloride test for a sodium-based reabsorption similar to that already found in sheep and goats. The study also examines the possibility that lung expansion causes local release of a factor which promotes the lungs' own reabsorption.  -12-  1990-10-10  5. METHODS AND MATERIALS 5.1  Animals  Pregnant albino guinea pigs of an inbred departmental stock were given food and water ad libitum (guinea pig chow, Ralston-Purina; supplemented by fresh vegetables and vitamin C ) . Gestational length of the animals was between 56 and 66 days (term 67 days) as calculated from previous delivery dates (guinea pigs enter oestrus immediately after delivery). Where this was unknown, fetal ages were calculated from the average weight and size of the Utter, according to the methods of Ibsen, 1928.  5.2  Surgical Procedures  Pregnant guinea pigs were anaesthetized with halothane until the corneal reflex was extinguished. The fetuses were removed by Caesarean section with their amniotic sacs intact and transferred to Krebs-Henseleit saline (see Burton, .1975) at 37°C. Ligatures were placed around the amnion at the level of the neck in order to maintain a pool of amniotic fluid about the head, and to prevent fetal breathing. The fetal thorax was opened by a midline incision to expose the lungs and trachea, and the trachea was ligated rostrally. A small incision was made just below this ligation, and the trachea was cannulated with 1.5-2.0 cm polyethylene tubing ( P E 50; Intramedic, Clay Adams, Parsippany, N . J . ) . The cannula was attatched to a 1.0ml tuberculin syringe via an 18G hypodermic needle and a 3-way stop-cock (K75, Pharmaseal, Puerto Rico). The cannula was tied in place, with double ligatures, just above the bifurcation of the bronchi, and the trachea was cut rostral to the point of cannulation. The lungs and heart were then dissected carefully out of the body cavity. The heart was removed and the preparation suspended in a 50ml bath of Krebs-Henseleit saline at 37°C, where it was maintained at pH7.4 with 95% O2 and 5% C O 2 (in experiments in which two lungs were incubated together, a 100ml bath of saline was used to ensure that nutrients were not limiting). The entire dissection took 3-4 minutes and the lungs were kept moist and warm during this time by frequent washes with saline at 37°C.  f3 _  innn -in i n  5.3 Experimental Procedures As soon as the preparation was set up, approximately 0.35ml of lung fluid was drawn into the reservoir syringe, and a 10/xl sample was removed from the upper cup of the 3-way stop-cock (1701 N C H gas-tight fixed volume syringe; Hamilton Co. Reno, Nevada) as a blank for spectrophotometry. ThenO.lml of Blue Dextran 2000 (50mg/ml in 0.9% NaCl) was added via the upper cup and thoroughly mixed with the lung fluid in the syringe before being passed into the lungs.  The  preparation was left to equilibrate for 30 minutes (supporting saline was renewed at 15 and 30 minutes). During this time, fluid was withdrawn from and returned to the lungs at five minute intervals to ensure an even distribution of the dye throughout the lung fluid.  Following the equilibration period, the experiment proceeded for three hours, with the supporting saline being renewed at the start of each hour. Every ten minutes, fluid was passed into the upper cup of the 3-way stopcock and a 10/xl sample was taken. Fluid was also withdrawn and returned at five minute intervals to maintain the even distribution of dye. The samples were placed in polyethylene micro test-tubes (250/d Eppendorf C3515-7, Brinkman Instruments(Canada)Ltd., Rexdale, Ont.), diluted 1:20 with distilled water, sealed and vortexed (Vortex-Genie, Fisher Scientific, N . Y . ) . Samples were then centrifuged at 250G for 10 minutes (clinical centrifuge, Model C L , International Equipment Co., Needham Heights, Mass.). The supernatants fluid was estimated for Blue Dextran concentration by spectrophotometry (Guilford 250, Oberlin, Ohio; 250/xl quartz microcells, Type 10972, N S G Precision cells Inc. Farmington, N . Y . ; wavelength=620nm ). Experiments generally followed an A B A design. Samples taken from the first hour provided the basic rate of secretion by the lungs. During the second hour, the lungs were transferred to fresh saline and sampled to test their reaction to various treatments. In hour three, they were returned to their first-hour conditions (fresh saline at 37°C) to test for recovery from the treatment.  The first series of experiments tested the reaction of the lungs to a change in temperature. For the middle hour they were transferred to saline at one of the following temperatures: 29° C, 32° C, 34°C,  - 14 -  1990-10-10  35°C, 36°C, 37°C (control), or 39°C (±0.02°C; D l circulators with W13 insulatedbaths; Haake, Karlsruhe, West Germany). In further studies, amiloride hydrochloride (Sigma, St. Louis, Mo.) at 1 0 M was placed in the lung fluid (apically) in conjunction with transfer of the lungs to saline at - 6  35°C or 37°C (control for amiloride).  A second series of experiments tested the effect of expansion on the lungs. Expansion was achieved using Krebs-Hanseleit saline (37°C), with lungs being expanded by one of the following amounts at the start of hour two: 0% (control), 18%, 31%, 43%, 50% or 72%. Although the lungs were placed in fresh saline in hour three, the expansion fluid remained in the lungs for the duration of the experiment. A n extension of this study involved the addition of 1 0 M amiloride hydrochloride - 6  as part of the expansion fluid during expansions of 70%.  In some experiments two sets of lungs were incubated together in a 100ml bath of saline. As a control, both lungs were maintained at 37°C (saline renewed each hour) for three hours without treatment. In further experiments, one set of lungs was expanded by 68% in hour 2; the other remained untreated, and samples were taken from both for three hours. This study was extended by expanding one set of lungs in the presence of a second set in supporting saline containing a) phentolamine (1.78xlO~ M), or b) propranalol (10~ M). 5  7  - 15 -  1990-10-10  Figure 1: Apparatus for the maintenance of the in vitro guinea pig fetal lung preparati  -16-  1990-10-10  3-WAY STOP—COCK OXYGEN  ^1  CLAMP l c c TUBERCULIN SYRINGE  /  33  TRACHEA ( w i t h d o u b l e 1 igaturesI  LUNGS  5.4  Statistical Methods  Fluid production rates were calculated from the fall in concentration of Blue Dextran (see Cassin and Perks, 1982; Perks and Cassin, 1989). They were estimated from plots of the total volume of secretion against time. Appropriate sequential adjustments were made every 10 minutes for addition of Blue Dextran at the onset of the experiment, and for the removal of fluid and Dextran during incubation. The rates of production of fluid over one hour intervals were calculated from the volume plots, using the slopes of their regressions, fitted by the method of least squares ( Steel and Torrie, 1970; Hewlett-Packard program SD-03A, or by Apple II Plus computer). The significance of changes in rate were estimated from changes in slope, analysed by a t-test for differences between two regressions (Steel and Torrie, 1970; for details, see Cassin and Perks,1982). Rates for different hours were also calculated by analysis of variance, followed by Newman-Keul's test (Zar,1984). Statistical significance accepted at p<0.05.  5.5  Chemical Methods  After estimating Blue Dextran concentration, samples were analyzed for sodium and potassium concentrations, by atomic absorption spectrophotometry (Model A A 120, Varian Technicon Pty, L t d . , Melbourne, Australia). The rates of secretion of these ions were calculated as shown above, but using total miUiequivilants rather than total volume. In certain cases, activity of lactate dehydrogenase (/nn/min) and concentrations of dipalmitoyl lecithin (mg/ml) (a component of surfactant) were analyzed using a Perkin Elmer Lambda 2 Spectrophotometer and a P E C S S program.  -18-  1990-10-10  6. RESULTS 6.1  Fluid Production by in vitro Lungs from Fetal Guinea Pigs  Untreated lungs from 30 late-term fetal guinea pigs (56-64 days gestation; body weight 90.17 ± 18.30g) secreted fluid for the first hour of the experiment at a rate of 1.30±0.22 m l / k g body weight per hour, and continued to secrete fluid for the remainder of the experiments (a further two hours) without any significant change in rate (supporting saline was renewed at the start of each hour) (see figure 2a). This was confirmed by both regression analysis and by analysis of variance. In all cases, sodium and potassium ion movement closely followed that of water, and there was no significant change in the rate of entry into the lung fluid of either ion throughout the experiment.  6.2  The Effect of Temperature Change on Fluid Production  Lungs from 36 fetal guinea pigs were divided into six experimental groups. During the middle hour of the experiment, each group was subjected to one of the following temperature changes: -1°C, -2°C, -3°C, -5°C, -8°C and + 2 ° C (see Figure 2).  6.2.1  Fluid Production by Fetal Lungs Subjected to a Fall in Temperature  A Reduction of 1°C (Figure 2b)  Lungs from six fetal guinea pigs ( 6 3 ± 2 days gestation; average body weight 105.4±22.3g) underwent a 1°C fall in temperature for hour 2 of the experiment. A l l six showed marked reductions in secretion rate (one showed a slight reabsorbtion of fluid). The average drop in rate was 68.2±17.1%, and this was found to be significant by both regression analysis (p<0.01) and by analysis of variance, Newman-Keul's test ( A N O V A : p<0.005). Once they were returned to their original temperature for hour 3, the lungs showed a tendency towards recovery, and their rate of secretion almost doubled that of hour 2, although it did not reach the values seen in hour 1.  - 19 -  1990-10-10  A Reduction of 2°C (Figure 2c)  The six lungs subjected to a 2°C drop showed the greatest effect seen in any of the temperature experiments. A l l displayed large reductions in fluid secretion and three showed strong reabsorptions. The average fall in secretion was 125.5± 30.1% (where 100% represents a complete halt in secretion, and values higher than this indicate the reabsorption of fluid). This fall was judged significant by regression analysis (p<0.001) and by A N O V A (p<0.005). Again, there was the start of recovery in hour 3 when the lungs were returned to their normal temperature (fetuses 6 3 ± 2 days gestation; body weight 105.7±17.5g).  A Reduction of 3°C (Figure 2d)  A temperature decrease of 3°C also had a strong effect on the lungs. Of the six lungs tested at this temperature, three reabsorbes fluid, with one having a reabsorption rate of 1.1 m l / k g per h. Despite the fact that this individual effect was greater than any seen with a 2°C decrease, the average fall in secretion was less (103.8±32.8%). This fall is significant by regression analysis (p<0.001) and by A N O V A (p<0.025). Hour 3 showed a return to secretion as the lungs began to recover, although the rate did not reach that of hour 1 (fetuses 6 2 ± 2 days gestation; body weight 96.2±23.4g).  A Reduction of 5°C (Figure 2e)  A reduction of 5°C, which is outside the normal range reported for birth, caused all six lungs to reduce their secretion, but the overall effect was less than that seen following a 2°C or a 3° C decrease.  The average fall in secretion rate was 82.7 ±16.6% (significant by regression analysis  (p<0.001) and by A N O V A (p<0.005)). It appears that, while a 5°C reduction greatly affects the secretion rate, it does not tend to promote reabsorption. It is interesting that such a large decrease in temperature does not damage the lungs permanently, since all showed an increase in secretion  -20-  1990-10-10  when returned to their original temperature for hour 3 (fetuses 6 2 ± 2 days gestation; body weight 92.5±12.7g).  A Reduction of 8°C (Figure 2f)  Although an 8°C decrease in core temperature is extremely unlikely to occur naturally, it was decided to test this extreme situation. Similar effects to those seen with a 5°C decrease were observed, and the results were remarkably consistent. In all six cases, there was an almost complete halt in secretion in hour 2, but never a reabsorption.  The average fall in secretion rate was  94.7±1.8% (significant by regression analysis (p<0.001) and A N O V A (p<0.005).  Surprisingly,  the lungs showed a tendency to recover in hour 3 when returned to 37°C, showing that irreparable damage to the secretion process had not occurred (fetuses 6 2 ± 1 days gestation; body weight 108.1 ±29.3g).  - 21 -  1990-10-10  Figure 2: The effect of reduced temperatures on lung liquid production by from fetal guinea pigs.  in vitro lungs  Based on 60 fetuses, 6 2 ± 2 days of gestation, 99.5±20.2g body weight (SD). During the middle hour only, temperatures were reduced from 37°C by one of the following amounts: (a) 0°C change (controls: 37°C throughout; n = 30); (b) -1°C (n = 6); (c) -2°C (n = 6); (d) -3°C (n = 6); (e) -5°C (n = 6); (f) -8°C (n = 6).  Ordinates: total volume of lung liquid expressed as a percentage of  that present at the end of the first hour (immediately before temperature reduction), where 100% is: (a) 0.99±0.35 ml; (b) 0.77±0.15 ml; (c) 0.64±0.14 ml; (d) 0.64±0.12 m l ; (e) 1.06±0.33 ml; (f) 0.95±0.27 ml (SD). Abscissae: time in hours. A l l regressions are lines of best fit (method of least squares; Steel and Torrie, 1970). The slopes represent production rates; the values below the lines give the mean rates in m l / k g body weight per hour. Asterisks above the lines show significant changes from the original slope (dotted lines) (significance accepted at p<0.05). Standard errors of the means (of the percent values) are omitted for clarity. They average: graph (a) 0.88±0.09; graph (b) 1.28±0.20; graph (c) 1.99±0.20; graph (d) 4.32±0.44; graph (e) 1.19±0.14; graph (f) 1.69±0.17. Corresponding coefficients of variation are: graph (a) 4.67±0.44%; graph (b) 3.19±0.55%; graph (c) 5.03±0.51%; graph (d) 10.59±0,99%; graph (e) 2.95±0.38%; graph (f) 4.08±0.49%.  -22-  1990-10-10  6.2.2  The Effect of a Temperature Decrease on Ion Movement  The movement of sodium and potassium ions closely followed that of water in all experiments involving a temperature decrease. Where the rate of secretion of water fell significantly, the rates of N a and K +  +  movement also decreased significantly, as judged by regression analysis, and both  ions tended to recover their secretion rates during the final hour of the experiments (see Appendix C ) . The only difference between water and ion movement was that ions tended to recover their secretion rates more fully i n hour 3, to rates comparable to those seen in hour 1. This suggests that water lags behind ion movement and that, given more time, the rate of water secretion would return to its original value once the lungs are put back to their normal temperature.  6.2.3  Fluid Production by Fetal Lungs Subjected to a Rise in Temperature  An Increase of 2°C (Figure 3)  Lungs from six fetal guinea pigs (62±2 days gestation; body weight 96.8± 20.Og) underwent a 2°C increase during hour 2. The result in all six cases was a complete and immediate halt in secretion (average fall 100.7±12.6%) which is significant by both regression analysis (p<0.001) and by A N O V A (p<0.025).  Unlike lungs subjected to a temperature decrease, none of the lungs showed any sign of recovery when returned to the normal temperature. Apparently, even relatively small increases in temperature can prevent both secretion and reabsorption permanently, thus impairing the function of the lungs.  -24-  1990-10-10  Figure 3: T h e effect of a 2°C increase in temperature on lung liquid production by in vitro lungs from fetal guinea pigs. Based on six fetuses, 6 2 ± 2 days of gestation; 96.8±20.0g body weight. During the middle hour only, the temperature was increased +2°C (37 to 39°C). Ordinates: total volume of lung liquid expressed as a percentage of that present at the end of the first hour (immediately before the temperature increase), where 100% is 0.84±0.16 m l . Abscissae: time in hours. A l l data presented as in Figure 1; standard errors averaged: 1.84±0.26.  Corresponding coefficients of variation: 4.72±0.71%.  Controls are shown in Figure 1(a).  -25-  1990-10-10  n  =6  6.2.4  The Effect of a Temperature Increase on Ion Movement  Both N a and K +  +  followed the movement of water. The rates of movement of both ions into the  lung fluid decreased significantly in hour 2 (shown by regression analysis) and neither showed any signs of recovery in the third hour (see Appendix C).  6.2.5  The Effect of Amiloride on Fluid Secretion Following a Temperature Decrease (Figure 4).  The above experiments have shown that a temperature decrease, especially one of 2°C, causes reabsorption of fluid by the lungs. It is commonly believed that fluid reabsorption involves sodium channels in the apical membrane. To test this postulate, amiloride, a molecule which blocks N a  +  channels i n tight epithelia (Soltoff and Mandel, 1983), was added in conjunction with a 2°C decrease, to see i f the reabsorption was prevented.  Control experiments showed that amiloride, when added to the apical surface of the lung at a concentration of 1 x 1 0 ~ M , had no significant effect on secretion when the temperature of the 6  saline was maintained at 37°C throughout the experiment (Figure 4b; fetuses 63 ± 1 days gestation; body weight 90.6±14.2g).  In six further preparations, amiloride was added apically at the start of hour 2 as the lungs were transferred to saline at 35°C. The result was a signifcant fall in secretion of 71.1 ±11.6% (regression analysis, p<0.001; A N O V A , p<0.05). However, no reabsorption was seen in any of the experiments.  -27-  1990-10-10  Figure 4: The effect of amiloride on lung liquid production by in vitro lungs from fetal guinea pigs, with and without a 2°C fall in temperature. Based on 18 fetuses, 6 3 ± 2 days of gestation, 87.9±18.9 body weight ( SD). Graph (a) shows the effect of a 2°C temperature drop. In both graphs (b) and (c), amiloride was added to the fluid in the lungs at the end of the first hour, to give a final concentration of 1 x 1 0 ~ M . Graph (a), 6  temperature remained constant at 37°C throughout. In graph (c), temperature was reduced by 2°C during the middle hour as in Figure 1(c).  Ordinates: total volume of lung liquid expressed as  a percentage of that present at the end of the first hour (immediately before addition of amiloride), where 100% is: (a) 0.64± 0.14ml; (b) 0.72± 0.20ml; (c) 0.78± 0.02(SD). A b s c i s s a e : time in hours. A l l data presented as in Figure 1, standard errors averaged: graph (a) 1.99±0.20; graph (b) 4.43±0.59; graph (c) 2.64 ± 0 . 3 3 . Corresponding coefficients of variation: graph (a) 5.03 ± 0 . 5 1 % ; graph (b) 8.95 ±1.07%; graph (c) 5.76 ±0.64%.  -28-  1990-10-10  Furthermore, there was a clear recovery during the third hour when the lungs were returned to 37°C, to a secretion rate comparable to that in hour 1. These experiments suggest that the reabsorption normally seen following a 2°C decrease is dependent on sodium movement.  6.2.6  The Effect of Expansion on Fluid Secretion by in vitro Fetal Lungs (Figure 5 )  The lungs from 30 late-term guinea pigs were divided into five groups. At the start of the second hour, the lungs were expanded with Krebs-Hanseleit saline at 37°C by one of the following amounts: 18%, 31%, 43%, 50% and 72%. The saline had been matched in optical density to the fluid already in the lungs, in order to avoid mixing artifacts; the supporting saline was renewed each hour.  Expansion by 18% (Figure 5b)  Six lungs were expanded with saline by 18.4 ± 3 . 5 % . Five of the lungs showed a decrease (one showed a slight, non-significant increase).  The average fall in secretion rate was 57.0 ±16.4%,  which was found to be significant by regression analysis (p<0.001) but not by ANOVA. Secretion continued to decrease in hour 3, but the rate was not significantly different from hour 2 (fetuses 63 ± 1 days gestation; body weight 105.1 ±8.3g).  Expansion by 31% (Figure 5c)  Six lungs expanded with saline by 30.9 ± 3 . 5 % showed an average decrease in secretion of 61.0 ±13.7%. One of the lungs showed an increase in secretion in hour 2, but this was not significant. The average fall was judged significant by regression analysis (p<0.01) but not by ANOVA. The decrease in secretion continued in hour 3, but was not significantly different from hour 2 (fetuses 63 ± 1 days gestation; body weight 96.0 ±9.9g).  -30-  1990-10-10  Expansion by 43% (Figure 5d)  Lungs which were expanded by 42.7 ± 3 . 4 % showed an average decrease in secretion of 88.4 ±17.7% (significant by regression analysis (p<0.005) and by A N O V A (p<0.005)).  Two of the six lungs  displayed slight reabsorption of fluid following their expansion. Secretion rate continued to decline in hour 3, but was not significantly different from hour 2 (fetuses 63 ± 2 days gestation; body weight 99.3 ± l l . l g ) .  Expansion by 50% (Figure 5e)  The expansion of six fetal lungs by 50.3 ± 2 . 9 % caused a complete halt in secretion. The average decrease in secretion rate upon expansion was 106.91 ± 21.3% (significant by regression analysis (p<0.001) and by A N O V A (p<0.005)).  The effect was continued in hour 3 (with no significant  difference from hour 2). Fetuses 63 ± 1 days gestation; body weight 111.6 ±32.Og).  Expansion by 72% (Figure 5f)  Six lungs were expanded by 71.7 ± 1 . 7 % . The result was a strong reabsorption in every lung, with an average, significant decrease of 199.6 ±30.9% (regression analysis (p<0.001); A N O V A (p<0.01)). The reabsorption was maintained in hour 3, and was slightly (but not significantly) greater than in hour 2 (fetuses 63 ± 1 days gestation; body weight 96.9 ±19.4g).  Figure (6) shows an increased response by the lung with increasing expansion.  There is little  difference between expansions of 20 - 30% but above this, there is an almost linear response as secretion rate falls with increasing volume of expansion.  -31 -  1990-10-10  Figure 5: The effect of expansion on lung liquid production by in vitro lungs from fetal guinea pigs. Based on 60 fetuses, 62 ± 2 days of gestation; 99.95 ±14.28g body weight (SD). At the start of the middle hour, lungs were expanded with Krebs-Hansleit saline by one of the following amounts; (a) 0% (controls; remained unexpanded throughout; n = 30); (b) 18.4 ±3.5% (n = 6); (c) 30.9 ± 3.5% (n = 6); (d) 42.7 ± 3.4% (n = 6); (e) 50.3 ± 2.9% (n = 6); (f) 71.7 ± 1.7% (n = 6).  Ordinates: total volume of lung liquid expressed as a percentage of that present at the end of the first hour (immediately before expansion) where 100% is: (a) 0.88 ±0.31ml; (b) 1.04 ±0.54rnl;(c) 0.66 ± 0.11ml; (d) 0.74 ± 0 . 17ml; (e) 0.88 ±0.15ml; (f) 1.09 ±0.24ml. Abscissae: time in hours. All data presented as in Figure 1. Average standard errors: graph (a) 0.88 ±0.09; graph (b) 2.48 ±0.20; graph (c) 2.11 ±0.25; graph (d) 2.26 ±0.22; graph (e) 3.06 ±0.42; graph (f) 2.47 ±0.43. Corresponding coefficients of variation: graph (a) 4.67 ± 0.44%; graph (b) 6.35 ±0.52%; graph (c) 5.23 ±0.60%; graph (d) 5.50 ±0.53%; graph (e) 7.67 ±1.02%.  -32-  1990-10-10  Figure 6:  tion.  The relationship between expansion of the lungs and the fall in fluid produc-  Based on 30 fetuses, 63 ± 2 days gestation; 103.23 ±17.7g body weight(SD). Expansion was acheived using Krebs-Hanseleit saline at 37°C at the start of hour 2. The mean fall in secretion in hour 2 is given for each level of expansion (each point being an average of six values), and the standard errors are shown.  -34 -  1990-10-10  200  H  CD 150  H  c o o CO  03  M—  100  50  H  20  60  40  % expansion IS  80  100  6.2.7  The Effect of Expansion on Ion Movement  Soduim ion concentrations were analyzed from the expansion experiments. The rate of their secretion appeared to follow the movement of water closely in all cases. In expansions of 20-30%, regression analysis showed no significant change in N a secretion following expansion of the lungs. +  However, with expansions of 40% and above, a significant fall in the rate was observed, corresponding to the fall in total water production. This effect was continued in hour 3. Following expansions of 50%, the rate of secretion of K ions also decreased as the fluid was reabsorbed from the lungs, +  and this rate continued to decline in hour 3 (see Appendix D).  6.2.8  The Effect of Expansion on the Rate of Passage of Phospholipids into the Lung Fluid  The amount of phospholipid in the samples was also measured throughout the experiment, in order to see whether expansion caused its entry into the alveoli. The results were analyzed by the same methods used for water and sodium. Regression analysis showed that there was no significant change in the amount of phospholipid entering the fluid in the hour following any of the expansions (see Appendix E).  6.2.9  The Effect of 70% Expansion on the Levels of Lactate Dehydrogenase in Lung Fluid  In order to test for possible cellular damage to the lungs during the maximum expansions (70%), samples were tested for the presence of the intracellular enzyme lactate dehydrogenase in the fluid during the experiments. The concentrations were analyzed by the same methods used for water, sodium and phospholipids (above). In the three preparations studied there was no evidence for any significant release of the enzyme into the lung fluid, as judged by regression analysis or ANOVA. If the cells been stretched to the point of rupture by the expansions, their lactate dehydrogenase would probably have been released into the lung fluid. The absence of increased entry of K ions +  into the fluid also indicates cellular damage did not occur.  - 36 -  1990-10-10  6.2.10  Pressures inside the Lung Following Expansion of 70%  As a further test for the likelihood of damage, the pressure inside the lung was recorded during the experiments, by means of a water manometer. The pressures were taken at the time of addition of the expansion fluid and during regular mixing of the lung fluid. During mixing, pressure increased only 1mm H2O in the three lungs studied, and when the expansion fluid was added, the pressure rose only 5- 10mm H2O. These small and transient increases were well below values found during normal expansion of the lungs at birth (Egan et al, 1976). It is therefore assumed that the extra volume added to the lungs caused no adverse pressure effects.  6.2.11  The Effect of Amiloride on Fluid Secretion Following Expansion of the Lungs (Figure 7)  It was decided to test the effect of amiloride on lungs which reabsorbed following expansion. This would provide extra evidence that the lungs were not damaged, since amiloride only blocks sodium channels in tight epithelia; therefore if reabsorption occurred due to a leaky membrane, amiloride would be unlikely to prevent it. It would also test to see whether the reabsorption seen fohowing expansion uses 6odium transport mechanisms similar to those used with a temperature decrease.  Amiloride was added at a concentration of 1 x 10~ M to the apical surface, as part of the expansion 6  fluid as the lungs were expanded by 70.2 ± 2.8%. The result was a complete halt in secretion, but the reabsorptions normally seen following expansions of this magnitude were greatly reduced or completely blocked. Figure 7c shows that the average fall in secretion rate in hour 2 was 103.7 ±16.8%; this is significant by regression analysis (p<0.001) and by ANOVA (p<0.0005). Secretion increased slightly in hour 3, but this was not significantly different from hour 2 (fetuses 63 ± 2 days gestation; body weight 100.8 ±14.0g). These experiments show that it is likely that the reabsorption seen following expansions around 70% utilizes the same sodium-based transport system as reabsorptions seen following a 2°C decrease, and that reported after adrenaline infusions (sheep;  -37 -  1990-10-10  Brown et al, 1983). They also support the fact that damage to the epithelium is unlikely after expansions of this magnitude.  -38-  1990-10-10  Figure 7: The effect of amiloride on lung liquid production by in vitro lungs f r o m fetal  guinea pigs, with and without expansion by 70%.  Based on 18 fetuses, 63 ± 2 days of gestation, 95.7 ±15.0g body weight (SD). Grapha (a) shows the effect of 70% expansion alone. In both graphs (b) and (c) amiloride was added to the lung fluid at the end of the first hour, to give a final concentration of 1 x 1 0 M . In graph (b), the lungs remained - 6  unexpanded throughout the experiment. In graph (c) lungs were expanded with Krebs-Hanseleit saline by 70.2 ± 2 . 8 % . Ordinates: total volume of lung liquid expressed as a percentage of that present at the end of the first hour (immediately before the addition of amiloride), where 100% is : (a) 1.09 ± 0.24ml; (b) 0.72 ± 0.20ml; (c) 0.79 ± 0.16ml (SD). A b s c i s s a e : time in hours. All data presented as in Figure 1, with average standard errors being: graph (a) 2.47 ± 0.43; graph (b) 4.43 ± 0.59; graph (c) 2.52 ± 0.24. Corresponding coefficients of variation: graph (a) 6.64 ± 1.19%' graph (b) 8.95 ± 1.07%; graph (c) 6.54 ± 0.61%  -39-  1990-10-10  110 100  0 75  90  t  80  UJ  2  -0-66  -0-87  70% e x p a n s i o n  130  b 120 n = 6 0-98  110  < O  0-88  100  110 100  Amiloride 1X10" M  0-86  90  6  *  c -n = 6  *  002  90r  I 92 80  •  0-13  / 1 I 70% e x p a n s i o n s  A m i l o r i d e 1X10~ M 6  70  •  TREATMENT  SALINE  TIME IN HOURS  i-o  6.3  The Remote Effect of Expansion  The mechanism by which reabsorption was promoted by expansion was unknown. However, it seemed possible that the change might activate some internal mechanism within the lungs, possibly releasing an agent capable of influencing the transport processes involved in the secretion and reabsorption of lung fluid. It was decided to test these possibilities by expanding one set of lungs by 70%, and determining whether there was any effect on a second set of unexpanded lungs suspended in the same bath of saline.  Ten preliminary studies showed that two sets of lungs placed in the same 100ml bath of saline, with no treatment to either, continued to secrete fluid at a constant rate, with no significant change, for the duration of the experiments, as judged by regression analysis or ANOVA ( Figure 8a; fetuses 63 ± 3 days gestation; body weight 92.1 ±21.2g).  In ten further studies (figure 8B), one set of lungs was expanded by 68.0 ±10.1% at the start of hour 2. As expected, the expanded lungs began to reabsorb fluid immediately after expansion. The average fall in secretion rate was 166 ±44.2% (significant by regression analysis (p<0.005) and by ANOVA (p<0.005)). However, the second, unexpanded, set of lungs also showed a significant fall in secretion to such an extent that production was almost completely halted.(94.0 ±22.3% decrease; regression analysis (p<0.005); ANOVA (p<0.01)) ( fetuses 63 ± 1 days gestation; body weight 99.4 ± 1 5.5g).  This indicates that the lungs release some substance which brings about their own reabsorption. This substance must potent and small enough enough to penetrate the tissue readily, since it is able to reach the supporting saline quickly enough to have an almost immediate effect on the second lung.  -41  -  1990-10-10  Figure 8: The remote effect of expansion on lung liquid production by in vitro fetal guinea pig lungs. Based on 40 fetuses, 63 ± 2 days of gestation, 94.4 ±18.4g body weight (SD). Two lungs were incubated in the same 100ml bath of saline throughout the experiment. A t the start of the middle hour, one of the two lungs was expanded by the following amount: ( A ) 0% (control, neither lung expanded during the experiment, n = 10). (B) 67.9 ± 1 0 . 1 % (SD) expansion (n = 10). O r d i nates:total volume of lung liquid expressed as a percentage of that present at the end of the first hour, where 100% is: graph (Ai) 0.91 ± 0.25ml; graph (Aii) 0.90 ± 0.33ml; graph (Bi) 0.71 ± 0. 21ml; graph (Bii) 0.86 ± 0.32ml(SD).  Abscissae: time in hours. A l l data presented as in Figure  1. Standard errors average: graph (Ai) 1.66 ± 0.18; graph (Aii) 1.93 ± 0.20; graph (Bi) 2.47 ± 0.23; graph (Bii) 1.75 ± 0.18. Corresponding coefficients of variation: graph (Ai) 5.13 ± 0.53%; graph (Aii) 5.87 ± 0.56%; graph (Bi) 8.17 ± 0.75%; graph (Bii) 5.65 ± 0.60%.  42 -  1990-10-10  no expansion  " i  T"  TIME IN HOURS  6.3.1  The Remote Effect of Expansion in the Presence of Phentolamine (Figure 9 )  The above experiments show that expansion of the lungs appeared to release some factor which caused a significant fall in secretion in lungs which were otherwise untreated. Although this factor may be one of many possibilities, it was decided to test for local catecholamine release, since catecholamines are believed to be important in promoting the onset of fluid reabsorption (Olver, 1977).  Again, two sets of lungs were incubated together for the entire experiment. A t the start of hour two, both sets of lungs were transferred to a bath containing the a-adrenergic blocker, phentolamine, at 1.78 x 1 0 - M . ) . 5  Control experiments had shown this drug to have no significant effect on fluid secretion when applied on its own at this concentration, but it does block nor-adraenaline induced reabsorbtions (Woods, unpublished observations). One set of lungs was then expanded by 70.1 ± 2 . 4 % . The result was the usual fall in secretion rate by the expanded lung (108.8 ±14.2%; significant by regression analysis (p<0.001) and A N O V A (p<0.0025); figure 9Bi). There was a slight recovery in hour three, although this was not significant. The unexpanded lungs showed a fall in secretion rate of 66.1 ± 2 4 . 1 % (significant by regression analysis (p<0.05) but not by A N O V A ) and there was no recovery in hour three (Figure 9Bii) (fetuses 64 ± 1 days gestation; body weight 88.5 ±8.98g).  Clearly, the a- blocker was unable to prevent the effects of expansion on the first lung, and did not appear to prevent responses in the untreated lung.  - 44 -  1990-10-10  Figure 9: The remote effect of expansion on lung liquid production b y in vitro fetal  guinea pig lungs, in the presence of phentolamine. Based on 18 fetuses, 64 ± 1 day6 of gestation, 88.5 ±8.98g body weight (SD). Graph A shows the effect on the lungs of 70% expansion alone. In graph B ( i and i i ) , two lungs were incubated together throughout the experiment. A t the start of the middle hour, both lungs were transferred to phentolamine at 1.78 x 1 0 ~ M , for the middle hour only, and one lung was expanded by 70.1 5  ± 2 . 4 % (SD). Ordinates: total volume of lung liquid expressed as a percentage of that present at the end of the first hour (immediatley before transfer to phenotolamine), where 100% is: (A) 1.09 ± 0.24ml; (Bi) 0.81 ±0.23ml; (Bii) 0 .78 ±0.15ml (SD). Abscissae: time in hours. All data presented as in Figure 1. Standard errors average: graph (A) 2.47 ± 0.43; graph (Bi) 3.04 ± 0.30; graph (Bii) 1.98 ± 0.26. Corresponding coefficients of variation: graph (A) 6.64 ± 1.19%; graph (Bi) 7.82 ± 0.78%; graph (Bii) 4.77 ± 0.66%  -45-  1990-10-10  110  100  0-75  00-  •0-66  80  UJ J5  120  single incubations  •0-87  70% expansion  r  B(i) n s6  110  100 •  < o  • 90 •  1-50  T  60 •  — - — i — - 0 13  009  70% expansion phentolamine 178x10''M  _  120  110  B(ii) ns6 0 41  100  90  0-49 no expansion  1 44  phentolomine 1 78«10" M S  60  TREATMENT  SALINE  1 TIME  2 IN  HOURS  joint incubations  6.3.2  The Remote Effect of Expansion in the Presence of Propranalol (Figure 10)  Two sets of lungs were incubated together and transferred to propranalol (/^-adrenergic blocker) at 1.0 x 1 0 M for the 6econd hour of the experiment. Earlier control experiments with this drug - 7  showed it to have no effect on fluid secretion by the lung when added on its own, although it was able to block the effects of adrenaline-stimulated reabsorptions (Woods, unpublished observations). Following this transfer, one set of lungs was expanded by 72.2 ±1.79% and their secretion rate fell 104.0 ±19.5% during the second hour. This was judged significant by regression analysis (p<0.05) and by ANOVA (p<0.025)(figure lOBi). There was a slight but non-significant recovery in the third hour when the lungs were returned to saline. The unexpanded lung showed a fall in secretion rate of 51.8 ± 11.5% but this was not judged significant by regression analysis or by ANOVA (figure lOBii). (fetuses 63 ± 2 days gestation; body weight 88.0 ±12.2g). Again it appears that the /3-blocker was unable to prevent the effects of expansion on the expanded preparation. It is probable that this was also true for the unexpanded preparation, but this conclusion must be given with reservation and further work is needed.  -47-  1990-10-10  Figure 10:'The remote effect of expansion on lung liquid production b y in guinea pig lungs, in the presence of propranalol.  vitro fetal  Based on 12 fetuses, 63 ± 2 days of gestation, 88.0 ±12.2g body weight (SD). Graph A shows the effect on the lungs of 70% expansion. Graph B (i and ii) shows two lungs incubated together throughout the experiment. A t the start of hour 2, both lungs were transferred to propranalol at 1 x 1 0 ~ M , for the middle hour only, and one lung was expanded by 72.2 ± 1 . 8 (SD). O r d i n a t e s : 7  total volume of lung liquid expressed as a percentage of that present at the end of the first hour (immediately before transfer to propranalol), where 100% is: ( A ) 1.09 ± 0.24ml; (Bi) 0.67 ± 0.13ml; (Bii) 0.74 ± 0.22ml(SD). Abscissae: time in hours. A l l data presented as in Figure 1. Standard errors average: graph ( A ) 2.46 ± 0.54; graph (Bi) 2.70 ± 0.24; graph (Bii) 2.52 ± 0.27. Corresponding coefficients of variation: graph ( A ) 6.64 ± 1.19%; graph (Bi) 6.54 ± 0.57%; graph (Bii) 5.93 ± 0.59%  -48 -  1990-10-10  110r  100  0 75  90  •0-66  80  •0 87  single incubations  7 0 % expansion  110r 100-  013  -006  90 •  1 38  ^  70%exponsion propranolol 1 0 K 1 0 " ' M  80  joint incubations 120r 110  B(ii) n=6 045  100 •  90  058 1 21  no expansion propranolol 1 0 * 1 0 " ' M  80  TREATMENT  SALINE  1  2  TIME  IN H O U R S  41  7. DISCUSSION  Earlier research on fetal lungs has involved the use of tn vivo experiments, using either sheep or goats. The series of experiments presented here offers a different approach to the study of lung fluid movement. The use of an tn vitro technique allows us to isolate the lungs so that treatment can be directly applied at a known concentration, while removing the possibility of influences from other body organs.  The average rate of lung fluid secretion seen in these tn vitro preparations (1.24 ±0.09 ml/kg per h) is not significantly different from that found in early studies by Setnikar (1959)(1.96 ± 0.71 m l / k g per h) (t-test assuming unequal variances; Parker, 1973, p.19-20) who used a direct collection method from ten intact fetal guinea pigs. It also compares favourably with rates found in late-term, intact fetal sheep by Scarpelli et al, 1975 (1.5 ± 0.27 ml/kg per h), Normand et al, 1974 (2.16 ± 0.02 m l / k g per h), Platzker et al, 1975 (3.2 ± 1.6 m l / k g per h) and by Perks and Cassin (1985a) (3.1 ± 2.2 m l / k g per h), who used the same dye dilution technique as that used in the present study.  Isolated lungs appear to remain in good condition for at least the three hour duration of the experiment. Evidence for this comes from the fact that (1) there is no significant change in secretion rate throughout control experiments; (2) there is no increase in the rate of entry of potassium ions into the lung fluid with time, as would be expected if tissue damage were occurring; (3) there is no rise in an intracellular enzyme with time; (4) there is no evidence for any unusual production of lactate in these preparations, since the concentration in the lung fluid is similar to that in blood plasma, and the rate of loss to the alveolar space and supporting saline shows no significant rise through incubation (Perks et al, 1990; Ruiz and Perks, 1990).  The use of guinea pigs for these experiments offers a model rarely used in studies of the neonatal lung. Despite the fact that most work has been performed on ruminants, it has been suggested that  -50 -  1990-10-10  the guinea pig may be the preferred model for human lung development (Stith and Das, 1982). Both the guinea pig and human are similarly developed at birth, and regulation of gluconeogenesis in both species is similar, whereas glucose metabolism in ruminants has many important differences (Bergman, 1973). Guinea pigs are also well suited for in vitro work, since their lungs are of a useful size for such experiments.  Their gestation period is relatively short (67 days), and they  are easy to maintain in colonies, allowing more rapid results and larger sample sizes than those usually obtained using ruminants. Their main disadvantage is that, compared to sheep and goats, only small volumes of lung fluid can be obtained. It must also be recognized that while in vitro experiments can be extremely useful, they do not represent the true physiological state of the lungs, and it is impossible to tell how they may be affected by other organs in the body.  It is commonly believed that a series of events triggered at birth is responsible for fluid clearance from the lungs of the neonate. Most studies in this field have concentrated on the drastic hormonal changes which occur in the fetus at this time. However, the fetus undergoes many physical changes at birth, which are less well studied but perhaps equally important in promoting the onset of breathing (Dahm and James, 1972). The purpose of the studies presented here is to examine the effects of some of these physical changes on fluid movement in the perinatal lung.  7.1 7.1.1  The Effect of Temperature Changes on Lung Fluid Secretion The Effect of a Temperature Reduction  Under normal conditions, the variability in core tissue temperature of adults is only 0.3% (Adamsons and Towell, 1965). However, during the birth process, deep body temperature of a healthy newborn human falls rapidly by 2-3°C and remains low for several hours (Gandy et al, 1964; Adamsons and Towell, 1965; Briick, 1978.) Thermographic studies show that the temperature drops particularly in the area of the scalp, the extremities, and the anterior thoracic cage corresponding to the region of the lungs (Tahti et al, 1972).  -51 -  1990-10-10  Several factors contribute to this rapid heat loss. Heat escapes the body mainly by convection and radiation, due to evaporation of amniotic fluid from the skin and a high surface area to body weight ratio (Adamsons and Towell, 1965; Adamsons, 1966). Thinner skin and less subcutaneous fat aid in causing the newborn to lose heat four times faster than the adult (Briick, 1961). Newborns also seem particulary sensitive to the temperature gradient between their skin and the environment. In utero the fetus has a skin temperature which is considerably higher than it will have after birth (Adamsons, 1966). Therefore, at birth, the infant experiences a temperature gradient with the environment which is unusually large. This gradient appears to be the most important factor in determining the metabolic rate of the newborn (Briick, 1978; Hull et al, 1986). Despite early ideas that the neonate behaves like a poikilotherm, it is now well established that it is a true homeotherm at birth (Schroder et al, 1987), as it is able to respond to thermal stimuli by changing its metabolic rate. Cold exposure increases metabolic rate in full-term infants by 100% within the first 15-30 minutes after birth. Even a premature baby shows metabolic and vasomotor control responses, when delivery of the infant's face causes vasoconstriction in its heel (Briick, 1961; 1978). The thermal instability seen i n the newborn is not the result of a problem with heat production, but rather a problem of uncontrollable heat loss. Although the baby increases vasoconstriction and changes its posture to conserve heat loss by radiation, it may 6till lose heat at a rate comparable to that suffered by an adult male undergoing severe cold stress (Adamsons, 1966). Even warming the baby with a radiant heat source does not prevent a drop in its core temperature, although the drop is less than that seen in infants left at room temperature (Dahm and James, 1972). Because a fetus is unlikely to experience temperature decreases as great as 2-3°C in utero, it is not known whether its thermoregulatory ability exists before birth. Since slight changes in maternal temperature tend to be paralleled by the fetus (Adamsons and Towell, 1965), it is unlikely that the fetus is able to exercise control over its own temperature, but rather is dependent on its mother for thermoregulation. Nevertheless, fetal thermoregulatory responses must be mature by the end of gestation, as they are able to operate immediately after birth. However, cooling the late-term fetus  -52-  1990-10-10  in utero does not result in a significant thermoregulatory response (Gunn et al, 1986; Schroder et al, 1987). There is apparently some factor of the birth process which acts to "switch on" the fetus' own thermoregulatory system, possibly connected with the clamping of the umbilical cord (Power et al, 1989).  It is believed that the thermal stimuli encountered at birth may be important in the initiation of breathing (Adamsons and Towell, 1965; Dahm and James, 1972). Evidence for this comes from the fact that both human and guinea pig fetuses delivered into a warm bath gasp less frequently and may even cease to breathe (Karlberg, 1963; Sarcia and James, quoted by Dahm and James, 1972). Experiments on isolated lung preparations show the lung to be greatly affected by decreases in temperature.  A fall of 1°C reduces secretion rate by almost 70%, and a fall of 2-3°C, that  normally seen in the delivery room (Adamsons and Towell,1965), stops secretion altogether and promotes reabsorption. It appears that there are two processes working in the lung, both of which are affected differently by a temperature drop. The secretion process is decreased or halted by a 2-3°C fall, whereas the process of reabsorption is apparently less affected.  Secretion of lung fluid is based on a chloride transport system, at least in sheep and guinea pigs (Olver, 1977; 1983; Cassin et al, 1986; Walters and Ramsden, 1987; Thorn and Perks, 1990), whereas fluid reabsorption is thought to involve a sodium transport mechanism (Brown et al, 1983; Cassin, Perks and Cooper, 1984 unpublished observation; Walters and Ramsden, 1987). Evidence for this reabsorption mechanism comes from experiments with amiloride, which blocks sodium channels in the apical membrane of tight epithelia (Soltoff and Mandel, 1983). The work shown here supports this theory, since amiloride at 1 0  - 6  M effectively blocks the reabsorption seen following a 2°C  decrease.  Dropping the temperature by more than 3°C stops both secretion and reabsorption, although it does not appear to damage the lungs, as there is a tendency towards recovery when the temperature is returned to normal. Premature babies, despite having thermoregulatory control, cannot equal the  -53 -  1990-10-10  metabolic response to cold seen in mature infants, due to their small body size . As a result, they suffer a greater heat loss (Adamsons and Towell, 1965). If mature infants undergo a reduction in core temperature of 2-3°C at birth, then it is probable that premature babies suffer a greater decrease. The in vitro experiments show that 2-3°C is the optimum decrease for maximum fluid clearance; below this temperature reabsorption is hindered. Perhaps one reason, therefore, that premature babies commonly experience difficulty in lung fluid clearance is that they lose too much heat at birth. However, immaturity of their responses to hormones and poor production of surfactant are generally accepted to be important factors in these problems.  The temporary fall in temperature experienced at parturition appears to be important in helping the neonate adapt to its new environment, especially with regard to the onset of breathing. It may also be useful in other ways: (1) exposure to lower temperatures after birth has been found to help stimulate lung growth in guinea pigs (Lechner et al, 1982); (2) it may play an important role in the development of thyroid function (Adamsons, 1966); and (3) a large, artificial decrease in body temperature may lessen brain damage in asphyxiated newborns by lowering the metabolic needs of the tissues (Miller et al, 1964). However, a temperature decrease is less beneficial to infants which are depressed at birth. These infants are unable to make adequate circulatory and respiratory responses to cold stress, resulting in lowered arterial oxygen tension (Stephenson et al, 1970) and severe metabolic acidosis, from which they may be unable to recover (Gandy et al, 1964).  7.1.2  The Effect of a Temperature Increase  There is little known regarding the effect of increased temperature on the neonate. Indeed, this situation is uncommon, since fetal temperature is carefully regulated in utero by the mother; the main thermal challenge to the newborn is the decrease in temperature it experiences at birth . However, the relatively common use of incubators, to keep premature babies from losing too much heat, introduces the possibility of accidental overheating of the newborn. Case studies have reported several sudden deaths among babies which were overdressed, or overwrapped with blankets  -54-  1990-10-10  and left in a warm room, and a healthy premature baby died within minutes after a fault caused his incubator to overheat (Stanton, 1980). While a healthy neonate can survive quite drastic falls in core temperature, its tolerance to even slight increases is very poor (Agate and Silverman, 1963). In fact, the dangers from overheating are so great that some pediatricians have been known to maintain premature babies at a body temperature slightly below normal, in order to prevent any chance of hyperthermia (Briick, 1978).  In adults, the most important defence against overheating is sweating. In newborns, despite having more sweat glands than the adult (Hey, 1972), sweating is inefficient. Sweating is not initiated until the rectal temperature is almost 1°C above normal, and premature babies (less than 36 weeks gestation) are unable to sweat at all until several days after birth (Rutter and Hull, 1979; Harpin and Rutter, 1982). Therefore, little heat is lost via evaporation, leaving vasodilation as the only effective response to increases in body temperature.  A newborn may react to overheating by adopting a position known as the "sunbathing posture" (Harpin et al, 1983), where it exposes as much surface area as possible by lying on its back with outstretched limbs.  Overheated babies also become less active, although their heart-rate may  increase slightly, and some will fall into a deep sleep (Harpin et al, 1983),thus it is very hard to know when a baby is suffering stress from overheating.  The most obvious sign is a slight  reddening of the skin, and even this is not always apparent. However, it is extremely important to monitor newborns for possible over-heating, as prolonged apnea is common at high environmental temperatures (Daily et al, 1969; Hey, 1972; 1975; Stanton, 1980); this may lead to brain damage.  Experiments on isolated fetal guinea pig lungs show that it is not only the brain which is susceptible to damage at elevated body temperatures. A rise of 2°C stops both secretion and reabsorption by the lung, within ten minutes. The lungs show no sign of recovery in hour three when they are returned to saline at normal temperature.  Therefore it can be assumed that while they may  recover from a temperature fall as great as 8°C, a rise of only 2 C damages the lungs irreparably. 9  -55-  1990-10-10  7.2  The Effect of Expansion on Lung Fluid Secretion  As the baby passes through the birth canal, part of the fluid in the lungs is forced out by compression of the thorax. However, this only amounts to about 25-30% of the total fluid volume (Saunders and Milner, 1978; Hand et al, 1990). It is obvious from this, and from the survival of babies born by Caesarean section, that most of the lung fluid is rapidly disposed of in some other way. This disposal appears to coincide with the expansion of the newborn's lungs during its first few breaths. Immediately after birth, a healthy fetus takes a strong gasp, and expands its lungs with air in a first step towards efficient gas exchange without the placenta. The lungs are not collapsed prior to this, but rest at functional residual capacity (Strang,1977). The fetal alveoli and alveolar ducts are well expanded by fluid, which is believed to be maintained under slight pressure during transient closures of a laryngeal sphincter (Adams et al, 1969). When the sphincter is open it allows fluid to pass out via the nose and pharynx; when closed it helps to prevent amniotic fluid from entering the lungs. However, despite their expanded state in utero, the alveoli are not fully open. The alveolar epithelium is pleated (Egan, 1982), thus allowing room for the extra expansion occuring during the first few breaths of the newborn. The large expansions with air seen at birth will remove the folds in the lung epithelium, but only massive expansions will cause the epithelium to stretch to the point of rupture.  X-ray techniques have shown that air expansion of the lungs causes a rapid redistribution of fluid and an increase in thoracic volume (Maloney et al, 1989). The expanded lung volume allows gas exchange to begin while some fluid still remains in the lungs. This allows respiration to continue relatively efficiently during the slow reabsorption of the residual fluid. As the fluid disappears, there is a corresponding decrease in thoracic volume. A healthy baby normally takes several large breaths immediately after birth, and it is common for the infant to display spells of breath-holding (Strang, 1977). Between breaths it appears to make respiratory efforts against a closed glottis, and it is possible that this force aids in pushing the  -56-  1990-10-10  fluid out of the airways (Karlberg et al, 1962); the large expansions brought about by the deep inspirations may alter the permeability of the alveolar epithelium, facilitating outward movement.  The epithelial lining which separates the alveoli from interstitial fluid in the fetus is believed to contain water-filled pores with a radius of 0.5-0.6nm (Egan, 1982). W i t h expansion of the lungs there is an increase in the water permeability of the epithelium. Pore radius increases to approximately 4.0nm, although by 12-60 hours after birth it returns to 0.9nm. (Egan et al, 1976). This transient increase in epithelial permeability allows fluid to pass from the alveolar spaces to the interstitium. Egan and his co-workers found a positive correlation between inflation volume and pore radius. Radius did not exceed 1.5nm until the lungs were expanded by more than 60% of their total volume, and did not reach 4.0nm in diameter until the expansion was 90% (Egan et al, 1975; Egan et al, 1976; Egan, 1982). A t 100% expansion, free diffusion of solutes was observed, indicating that the epithelium had become damaged and unable to regulate the flow of water and solutes. These results found by Egan fit well with those from the expansion experiments performed on t u vitro guinea pig lungs. He found expansions of 70% to increase pore radius to 3.0nm. From the in vitro preparations presented here it was found that 72% expansion caused strong reabsorption, perhaps because an increased pore radius allowed easy movement of fluid into the interstitium.  Although increased pore radius probably allows the passage of fluid through the epithelium, it is less easy to identify the force which pushes the flow in the direction of the interstitium. In Egan's studies, preparations were always intact, and therefore there was the presence of a protein osmotic gradient to drive the fluid from the alveoli. However, it is unlikely that a protein osmotic gradient exists in vitro, since the blood supply to the lungs has been cut off, and frequent changes of the surrounding saline probably washes out any residual protein; yet fluid reabsorption still occurs with expansions of 72%. Therefore, an osmotic gradient cannot be the only force driving reabsorption.  Results from in vitro lung studies are also consistent with those of Perks and Cassin (1985) who used the dye-dilution method on intact fetal goats. These researchers found that all expansions  -57-  1990-10-10  over 18.6% caused a significant fall in secretion. Expansion over 57% consistently resulted in fluid reabsorption, with the rate of absorption increasing at higher expansions. In the present experiments, amiloride at 1 0  - 6  M was added to the in vitro preparations in conjunction with expansions  of 70%, to see i f reabsorption was reduced. The result was a consistent blocking of the reabsorption, although secretion was still decreased significantly. As with the reabsorption brought about by a temperature decrease, that seen after expansion appears to involve sodium channels, and if these sodium channels increase in radius due to expansion, amiloride is still apparently able to block them.  7.3  Is the Expansion within Physiological Limits?  It is possible that expanding the lungs with saline may not produce the same effects as expansion with air for a number of reasons: (1) as air passes into the lungs it must overcome the frictional resistance of the fluid and a high surface tension at the gas-liquid interface (Hull, 1969; Saunders and Milner, 1978). This does not occur with saline expansion; (2) expansion with saline does not cause the change in tissue elasticity seen when the lungs go from a fluid-filled to an air-filled state (Avery and Normand, 1965); (3) air may not be distributed about the lungs in the same way as saline when the lungs are expanded. However, using the dye dilution technique with an in vitro preparation precludes the use of air as an expansion medium as it becomes impossible to remove fluid from the lung for sampling. Perks and Cassin (1985) measured the natural expansion of the lungs in the first breath of spontaneously breathing fetal goats and found it to be at least 62%. Other researchers (Karlberg et al, 1962; Saunders and Milner, 1978) have calculated that expansions of 70% are not uncommon during the first breath of healthy human newborns. Therefore, it is assumed that the average expansion of 72% in the in vitro preparations presented here is within physiological limits.  7.3.1  Are the Lungs Damaged by Expansions of 72%?  It is unlikely the tn vitro preparations were damaged by expansion, even those as great at 72%. Following the experiment the lungs were examined using scanning electron microscopy, and were  -58-  1990-10-10  compared to control lungs and to lungs removed directly from the fetus without being used experimentally. Scanning electron microscopy shows only external views of the cells, but can reveal evidence of tearing in the cell walls or intercellular junctions. No such damage was seen in expanded lungs, and there was apparently no difference between expanded and control lungs, or those removed from the fetus with no experimental study. Measurements of pressure were also taken as the expansion fluid was initially added to the lungs. The average pressure produced was a transitory l c m H2O, considerably below that of 70cm H2O recorded by Karlberg et al (1962) in spontaneously breathing human infants. Pressures as high as those found by Karlberg would perhaps damage the lung if applied statically to an isolated region. However, pressures during respiration are transitory, and much of the high pressure in the upper airways is dispersed by the branching of the alveoli and in moving the fluid out of the airspaces (Strang, 1977). Despite high pressures recorded by Karlberg and his co-workers, other researchers have found quite different results.  Egan et al (1975) reported that pressures above 41cm H2O caused epithelial  breakdown, although those below 35cm H2O only caused a very slight increase in pore radius. They suggested that pressures between 35-40cm H2O probably occur in spontaneous ventilation and promote fluid reabsorption without causing damage to the lungs. Peterson et al (1988) found that inflation pressures of only 10cm H2O increased clearance of diethylentriamine pentaacetate from the alveolar space by 56% in adult humans. These are far greater than the transitory pressures of l c m H2O seen in the in vitro preparations. Similar mechanisms probably occurr in adults and neonates, although lower pressures are required to promote clearance of solutes in adults probably because there is no large resistance to overcome from fluid within the lung. Pressures as high as those seen in intact animals are unlikely to occur in an isolated preparation, since the opposing resistance of the comparitively stiff thorax has been removed. Further evidence that the lungs are not damaged by expansions around 70% comes from the fact that amiloride blocks the reabsorption of fluid normally seen with high expansions. A t 1 0 ~ M 6  amiloride is specific to sodium channels (Soltoff and Mandel,1983) and therefore would be unlikely  -59-  1990-10-10  to prevent fluid leaking through a damaged epithelium. Three preparations subjected to 70% expansion were also tested for the amount of lactate dehydrogenase in the lung fluid. In all three cases there was no significant increase in lactate dehydrogenase during the experiment. This too supports the fact lungs were not damaged by expansion of this magnitude.  It is extremely important to understand the point at which expansion of the lungs and high pressures will cause damage, as it is often necessary to mechanically ventilate human newborns suffering asphyxiation. Some workers have observed that chronically high pressures from mechanical ventilators can cause epithelial damage in the lungs (ElKady and Jobe, 1988), while others have found that injury is more often caused by the frequent repetitive changes in lung volume brought about by a ventilator (Bowton and Kong, 1989). Trang et al (1988) have suggested that the artificial ventilation of a newborn suffering respiratory distress impairs cardiac output, so that despite an increase in arterial oxygen there may be a decrease in systemic oxygen transport. Damage to the lungs in a newborn is also dependent on chest wall compliance and elastic recoil forces, such as that from the diaphragm, which oppose volume expansion (Egan, 1982). These set limits on the volume which the thorax can contain. While the lungs of an infant are actually no more delicate than those of the adult (Avery and Normand, 1965), the thicker, less compliant chest wall of adults offers more protection against over-expansion than the fragile wall of a newborn infant (Hernandez et al, 1989).  7.3.2  What is the force driving the fluid out of the potential air spaces?  As pointed out earlier, in an m vitro preparation, there is not likely to be a strong protein osmotic gradient across the alveolar epithelium. If a protein osmotic gradient does exist at the start of the experiment, in seems unlikely that it could be maintained for any length of time in such a small preparation. However, the tn vitro lungs have shown reabsorptions that last for at least an hour, and sometimes even two. This raises the question as to what drives the reabsorption in the absence of a protein osmotic gradient. A s the lungs have been separated from the body, reabsorbtion must  -60-  1990-10-10  be initiated by the lungs themselves. Certain stimuli to the lungs may release some factor, perhaps neural or hormonal, which promotes the reabsorption of their own fluid. This was shown to be a possibility following the experiments with two lungs, when expansion of one apparently had an effect on a second lung suspended in the same bath. The factor released upon expansion must be potent and small enough to permeate the tissue of the unexpanded lung very quickly, since this lung is affected within ten minutes of the other lung being expanded. The release of local catecholamines upon expansion is a possibility, based upon the ultrastructure of the lungs. Electron microscopy and pharmacological techniques have shown the presence of adrenergic nerves in guinea pig respiratory smooth muscle, although the density of these nerves is significantly less in the terminal airways than around the trachea (O'Donnell et al, 1978; Jones et al, 1980). The guinea pig lung also possesses nerve endings containing mitochondria, which are believed to be mechanosensitive and perhaps indicate the location of stretch receptors in the muscle (Hoyes and Barber, 1980). The pattern of innervation in the guinea pig lung and trachea is similar to that of humans (Richardson, 1983), and both species also possess alpha and beta receptors on the smooth muscle within the lungs. Wurtman and Axelrod (1966) observed the presence of phenylethanolamine-N-methyltransferase ( P N M T ) , an enzyme within the lung which converts nor-adrenaline to adrenaline. Therefore it is possible that stimulation of stretch receptors within the lung, by expansion, causes local release of catecholamines which then act via receptors to drive fluid out of the potential air spaces. However, the studies using phentolamine and propranalol indicate that the factor released following expansion is probably not a catecholamine, since neither drug was able to prevent the reabsorption caused by 70% expansion, or the change in fluid production seen in a second co-incubated lung (although the evidence from the second lung is not clear in the case of propranolol) These studies show that some of the physical changes experenced at birth, such as a 2-3°C drop in core temperature and expansion of the lungs with the first few breaths, are important in helping fluid clearance from the air spaces of the lungs. The exact mechanisms responsible for this removal of fluid are still not clear, although sodium channels are apparently involved. 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Z A R , J . H . 1984. Biostatistical Analysis. 2nd ed. Prentice-Hall Inc. Englewood Cliffs, N.J. pp 718.  -68-  1990-10-10  APPENDIX A.  A.l A.1.1  Preparation of Krebs-Hensleleit Saline To prepare stock solutions  Stock Solutions 1. 69.2g NaCl and 3.5g KCI in IL distilled water 2. 36.8g CaCl in IL distilled water 2  3. 15.0g MgS0 in IL distilled water 4  4. 16.0g KH P0 in IL distilled water 2  A.1.2  4  To prepare 1 litre of saline from stock solutions add into a IL volumetric flask  1. 10 ml MgS04 stock solution 2. 100 ml NaCl/KCl stock solution 3. 2.1g NaHC0  3  4. 2.0g Glucose Fill the volumetricflaskat least half-full with distilled water and make sure the solids have completely dissolved before adding: 6. 10 ml K H 2 P O 4 stock solution 7. 10 ml CaCb stock solution Fill theflaskto 1.0 litre mark with distilled water. Bubble with oxygen for at least 30 minutes.  -69-  1990-10-10  A P P E N D I X B. B.l  Preparation of Blue Dye  1. Add 9.0g NaCl to IL distilled water 2. Weigh out 5.0g Blue Dextran 2000 dye crystals 3. Add dye to 100 ml 0.9% NaCl 4. Add magnetic stirrer and mix for at least 1 hour  B.2  To Make Standard for Spectrophotometry  1. Put 1 ml of dye (from (A) above) in 100 ml distilled water 2. Remove 25 ml and add 25 ml distilled water  -70 -  1990-10-10  APPENDIX C. The Effect of a Temperature Decrease on Ion Movement Ion Na+ Na Na+ Na Na+ K+ K+ K+ K+ K+ +  +  Temp drop 1°C 2°C 3°C 5°C 8°C 1°C 2°C 3°C 5°C 8°C  Rate Hrl 0.212±0.13 0.171±0.11 0.206±0.15 0.361±0.21 0.400±0.24 0.051±0.02 0.031±0.01 0.055±0.02 0.035±0.02 0.063±0.03  Rate Hr2 0.072±0.06 0.078±0.06 0.031±0.08 0.082±0.09 0.054±0.03 0.011±0.01 -.002±0.02 0.010±0.02 0.003±0.02 -0.007±0.01  Rate Hr3 0.098±0.06 0.083±0.06 0.089±0.08 0.236±0.15 0.128±0.10 0.019±0.02 0.003±0.01 0.020±0.02 0.023±0.03 0.033±0.02  Significance Hrl vs 2 Hrl vs 3 p<0.01 p<0.05 ns p<0.05 p<0.001 p<0.01 p<0.001 ns p<0.01 p<0.01 p<0.02 p<0.01 p<0.01 p<0.05 p<0.001 p<0.001 p<0.001 ns p<0.001 p<0.01  Hr2 vs 3 ns ns p<0.01 p<0.02 ns ns ns p<0.01 p<0.001 p<0.001  The Effect of a Temperature Increase on Ion Movement Ion Na K+  +  Temp rise Rate Hrl 2°C 0.312±0.20 2°C 0.067±0.04  Rate Hr2 0.069±0.07 0.019±0.01  Rate Hr3 0.038±0.08 -0.012±0.01  -71 -  Significance Hrl vs 2 Hrl vs 3 Hr2 vs 3 p<0.001 p<0.001 ns p<0.001 p<0.001 p<0.001  1990-10-10  APPENDIX D. The Effect of Expansion on Ion Movement Ion Na+ Na+ Na+ Na Na+ K+ K+ +  % Expansion 20% 30% 40% 50% 70% 50% 70%  Rate Hrl 0.322±0.17 0.146±0.06 0.109±0.07 0.186±0.06 0.166±0.09 0.146±0.24 0.033±0.04  Rate Hr2 0.273±0.04 0.121±0.11 0.042±0.06 0.043±0.10 -0.041±0.07 0.027±0.03 0.012±0.03  Rate Hr3 0.097±0.13 0.080±0.04 0.017±0.03 0.031±0.09 -0.157±0.12 0.008±0.02 -0.012±0.02  - 72 -  Significance Hrl vs 2 Hrl vs 3 ns p<0.05 ns p<0.01 p<0.005 p<0.002 p<0.001 p<0.001 p<0.005 p<0.001 p<0.02 p<0.001 ns p<0.05  Hr2 vs 3 ns ns ns ns p<0.05 p<0.02 p<0.01  1990-10-10  APPENDIX E. The Effect of Expansion on Phospholipid Movement  DPL DPL DPL DPL  %Expanison 20% 30% 40% 70%  Rate Hrl 0.026±0.03 0.0008±0.02 0.040±0.06 0.007±0.05  Rate Hr2 0.021±0.03 0.0005±0.02 -0.277±0.57 0.004±0.04  -73-  Rate Hr3 0.022±0.03 0.0003±0.01 0.345±0.69 -0.003±0.02  Significance Hrl vs 2 Hrl vs 3 ns ns ns ns ns p<0.001 ns p<0.001  Hr2 vs 3 ns ns p<0.001 p<0.001  1990-10-10  

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