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Role of hyperventilation in hypoxia on lung growth in rats
Respiration Physiology, 76 (1989) 179--190 Elsevier
179
RSP 01530
Role of hyperventilation in hypoxia on lung growth in rats Edmund E. Faridy and Wen Z. Yang Department of Physiology. University of Manitoba, Winnipeg, Canada R3E OW3 (Accepted for publication 21 January 1989) Abstract. This study was conducted in an attempt to differentiate the contribution of hyperventilation, if any, from that of low Po2 on adaptive lung growth in response to hypoxia. Male albino rats were exposed to one ofthe following: (1) Room air for 7 days, as control; (2) 10% 02 in N 2 for 7 days; (3) 10% 02 for 6 h, 1 day or 2 days and air for the remaining of 7 days; (4) 10% 02 for 2 days and 7% CO2 in air for 5 days; (5) air for 2 days and 7% CO2 for 5 days; or (6) 7% CO2 in air for 7 days. Lung growth was assessed by measuring the lung weight, lung air volume, lung DNA content and rage of DNA synthesis in lung explants. Hypoxia stimulated lung DNA synthesis even when administered for only 6 h, and the effects persisted for a few days alter discontinuation of hypoxia. Hypercapnia did not stimulate DNA synthe~-~ in lung. In 2 clay hypoxic 5 day air rats the lung weight and lung DNA content increased, in 2 day air 5 day hypercapnic rats only the lung volume increased, and in 2 day hypoxic 5 day hypercapnic rats all parameters of lung growth, i.e., lung weight, DNA content and air volume increased as in 7 day hypoxic rats. The results suggest that adaptive or compensatory lung growth in hypoxia is brought about on one hand by the direct effect of low Po2 on lung cells, resulting in lung hyperplasia, and on the other hand by the mechanical stimulation of lung tissue by hyperventilation, causing lung distension.
Compensatory lung growth; Hypercapnia; Lung air volume; Lung DNA synthesis; Lung pressure-volume
Observations on humans native to high altitudes (Hurtado, 1932; Guleria et al., 1971) and on experimental animals exposed to hypoxic environment (Bartlett and Remmers, 1971; Burri and Weibel, 1971; Cunningham et ai., 1974) suggest that chronic hypoxia induces an adaptive growth response in the lung which is aimed to increase surface area for gas exchange. The mechanisms by which the lung grows in hypoxia are unclear. Furthermore, the role of hyperventilation as a consequence of low Po2 on adaptive lung growth in response to hypoxia has not been delineated. The present study, conducted in male albino rats, was undertaken in an attempt to differentiate the contribution of hyperventilation, if any, from that of low Po2 on lung growth.
Correspondence address: Edmund E. Faridy, Department of Physiology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. 0034-5687/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
180
E.E. FARIDY AND W.Z. YANG
Methods Male Sprague-Dawley rats weighing between 135 and 150 g were used for this study. The rats were kept in a room with controlled temperature and humidity and supplied with standard rat chow and water ad libitum. For studies of DNA synthesis two groups of rats were used as controls: (1)normal controls - these rats were kept in wire cages (one rat in each cage) and were sacrificed at various days from 0 to 7; and (2) experimental controls - on day 0, these rats (in groups of 12) were transferred into an environmental chamber, and exposed to air for up to 7 days. The experimental rats, on day 0, were also transferred into the environmental chamber but exposed to either: 1) hypoxia (10% 02 in N2) for 6 h or 1 day and air for the remaining of 7 days; 2)hypoxia for up to 7 days; or 3)hypercapnia (7 % CO2 in air) for 7 days. The chamber was made of plexiglass and had a volume of 98 L. A wire cage within the chamber had sufficient space to accommodate 12 rats. The chamber gas was maintained at predetermined concentrations of 02, CO 2 and N z. A continuous flow of gas from air, 02, CO2 and N2 cylinders washed out the excess CO2 and humidity from the chamber. The gas flow ranged between 5-7 L/min depending on the number of rats in the chamber. The chamber gas was continuously mixed by a noiseless fan, and passed over a temperature controlled copper coil. The inflow and the outflow gases were monitored by 02 and CO2 analyzers (Beckman). A temperature of 23 °C + 0.5 and a CO2 concentration of less than 0.2% (in air and in hypoxia experiments) were maintained over the duration of the experiment. The rats were then sacrificed on days 1, 2, 3, 4, 5 and 7 of exposure to above gases. On the day of sacrifice, the rats were exsanguinated under sodium pentobarbital anesthesia (5 mg/100 g BW) by cutting the abdominal aorta. The lungs were excised, under sterile conditions, dissected free of extrapulmonary airways and tissues, washed with chilled culture medium and chopped into explants of approximately I mm cubes with a pair of sharp surgical blades. All procedures were carried out in a laminar flow hood and completed within 10 min. The explants were transferred into multi-wall plastic culture plates (Flow Lab., Inc., McLean, Virginia) containing Waymouth MB 752/1 culture medium (Gibco Lab., Grand Island, New York) with 0.1 #Ci/ml of [3H]thymidine (specific radioactivity 6.7 Ci/mM. New England Nuclear, Inc., Boston, MA), and were incubated at 37 °C in a humidified atmosphere of 5 ~ C02 in air. At the end of 6 h of incubation, the explants were removed from the medium. The tissues from four wells were pooled as one sample and washed in 10% thrichloroacetic acid (TCA) and then in 95~ ethyl alcohol. The explants were digested by incubating in 2 N sodium hydroxide in 37 °C water bath overnight. The nucleus fraction was precipitated in 2N perchloric acid by centrifugation. DNA was dissolved from the pellets in 5~o TCA by incubating in 90 °C water bath for 15 rain. One millilitre of DNA extract was mixed with 10 ml of Biofluor liquid scintillation cocktail (New England Nuclear, Inc.) and counted in a liquid scintillation spectrephotometer. Another millilitre of DNA extract was used to measure DNA content by the method of Schneider (1957). The results are expressed as DPM/#g DNA x 10- 3
LUNG GROWTH IN HYPOXIA
181
For studies of lung growth the rats were divided into 5 groups. Each group was placed in the environmental chamber and subjected to one of the following gas mixtures for 7 days: (1)room air (Air) as control. This group consisted of two subgroups: (a)agematched rats (C~) as control for all experimental groups; and (b) body weight-matched (3 days younger) rats (C2) as control for hypoxic rats which do not grow as rapidly as normoxic rats; (2) Hypoxia (10% 0 2 in N2) (Hpo); (3) Hypoxia for 6 h, 1 day or 2 days and air for the remaining of 7 days (Hpo-Air); (4) Hypoxia for 2 days and hypercapnia (7% CO2 in air) for 5 days (Hpo-CO2); and (5)air for 2 days and hypercapnia for 5 days (Air-CO2). With the exception of some rats in C2 (body weight-matched controls) and Hpo (10~o 02 in N2) groups which were sacrificed on days 1, 2, 3, and 5, the rest were all sacrificed at the end of 7 days exposure to various gases. On the day of sacrifice, the rats were weighed, exsanguinated, as mentioned above, and a pneumothorax was produced by cutting the diaphragm adjacent to the xyphoid. The opening in the diaphragm was enlarged and the lungs were inspected under a magnifying glass. If the lungs were not uniformly pink in color and showed signs of possible infection (such as even a single red or grey spot) the rat was excluded from the study. The trachea immediately below the larynx was cannulated and the lungs, left intact in the chest, were degassed by placing the animal in a vacuum jar. The chest was then opened by bisecting the sternum, and the large vessels ligated at the base of the heart. The ca~mu|ated degassed lungs were then attached to a pressure-volume apparatus similar to that previously described (Gribetz etaL, 1959). The lungs were inflated with air to 30 cm H20 pressure. This inflating pressure was maintained until the lung air volume remained constant for 15 sec. The air volume observed at this transpulmonary pressure, considered as maximal lung air volume (MLV), was designated as 100~ and each volume subsequently observed after deflation to a predetermined transpulmonary pressure (20, 15, 10, 5 and 0 cm H20) was expressed as a percentage of MLV. These pressures were maintained for 20 sec before the volumes were read at each pressure. If, during the procedure, the lung air volume did not remain constant at high pressure, air leaks were assumed to be present, and the data were excluded from the study. All pressure-volume measurements were performed at room temperature. At the end ofak PV measurements the lungs were separated from the extrapulmonary airways, weighed and used to measure lung DNA and dry weight. The lung deoxyribonucleic acid (DNA) content was determined by the method of Schneider (1957) using 0.5 g samples of lung tissue. The remaining lung tissues were dried in an oven at 60 °C for one week. Statistical analysis of the data was carded out using a t-test of unpaired variates and a multiple range test for analysis of variance by Duncan's method, where applicable. Results
Only rats subjected to hypoxia for 7 days (Hpo) had body weights significantly lower than control (table 1).
(8)
(8)
1.20 + 0.06
(8)
(8)
1.26 + 0.05
10.10 + 0.45
(8)
(8)
10.39 + 0.36
4.12 + 0.15
(8)
7.80 + 0.29
(8)
4.06 + 0.10
(8)
6.56 + 0.21
(8)
4.65 + 0.11
(8)
8.48 + 0.28
(8)
3.73 + 0.15
(8)
6.78 + 0.24
(8)
96.4 + 1.77
(8)
(8)
90.4 + 1.78
0.156 + 0.004
(8)
(8)
0.164 + 0.002
118.6 + 2.04
(8)
121.4 _+ 3.04
0.776 + 0.017
(8)
0.818 + 0.015
161.4 + 1.83
(8)
181.9 + 2.12
(8)
Air (C2)
(7)
1.44 + 0.05"*
(7)
10.31 + 0.34
(7)
6.64 + 0.26**
(7)
10.70 + 0.30**
(8)
4.66 + 0.08**
(8)
7.53 + 0.20**
(8)
133.4 + 4.14"*
(8)
0.216 + 0.008**
(8)
139.2 + 2.61"*
(8)
1.048 + 0.036**
(8)
161.5 _+2.28*
Hpo
(6)
1.09 + 0.06"*
(6)
9.52 + 0.43
(6)
4.61 + 0.10'
(6)
8.52 + 0.13'
(8)
4.17 + 0.13"*
(8)
7.71 _+0.27*
(8)
97.3 + 2.28**
(8)
0.180 + 0.005**
(S)
117.6 + 3.60*
(8)
0.903 + 0.023**
(8)
185.1 _+ 1.88'
Hpo-Air
(7)
1.34 + 0.04 s
(7)
! !.23 + 0.39 !
(7)
5.41 + 0.10 **0
(7)
9.75 + 0.20**o
(8)
4.10 + 0.11'
(8)
7.33 + 0.25
(8)
98.2 + 2.08**
(8)
0.176 + 0.005*
(8)
118.8 + 3.68*
(8)
0.864 + 0.028*
(8)
178.6 + 2.93*
Hpo-CO 2
(8)
1.44 + 0.05*
(8)
12.14 + 0.37*
(8)
5.35 + 0.14"
(8)
9.59 + 0.28*
(8)
3.74 + 0.10
(8)
6.69 + 0.16 a
(8)
90.8 + 2.43 a
(8)
0.162 +_0.003 a
(8)
119.4 + 4.51
(8)
0.794 + 0.016 a
(8)
179.1 + 1.66
Air-CO 2
Hpo = hypoxia (10~o 02 in N2) for 7 days; Hpo-Air = 2 days hypoxia, 5 days air; H p o - C O 2 = 2 days hypoxia, 5 days 7~o CO2 in air; A i r - C O 2 -- 2 days air, 5 days 7~o CO2 in air. BW = body weight at the day of sacrifice. MLV -- lung air volume at 30 cm H20 transpulmonary pressure. Data are expressed as mean + 1 SE; numbers in parentheses indicate the number of rats studied. Significantly different: *from CI (P<0.001 - P < 0 . 0 5 ) ; t H p o from C2 (P < 0.001-P < 0.007); *Hpo-Air and H p o - C O 2 from Hpo ( P < 0.001 - P < 0.02); §Hpo-CO2 from Hpo-Air ( P < 0.001 - P < 0.02); a Air-CO2 from H p o - C O 2 (P < 0.05). Comparisons are not made between C1 and C2, and between A i r - C O 2 and Hpo groups.
MLV (ml/mg DNA)
MLV (ml/g lung)
MLV (ml/100 g BW)
MLV (ml)
Lung DNA (rag/100 g BW)
Lung DNA (mg)
Lung dry wt (mg/100 g.BW)
Lung dry wt (g)
Lung wet wt (mg/mg DNA)
Lung wet wt (g)
Body weight (g)
Air (C,)
Lung measurements in control (Ci and C2) and experimental rats at the end of 7 day exposure to various gases.
TABLE 1
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LUNG GROWTH IN HYPOXIA
183
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Fig. l. The tritiated thymidine incorporation in lung tissues, in organ culture, obtained from rats previously exposed to hypoxia. Closed triangles represent lungs from rats exposed to 6 h hypoxia and 3/4 to 63/4 days air; open triangles: 1 day hypoxia and I to 6 days air; and closed circles: 1 to 7 days hypoxia. Each point and bar represents the mean + 1 SE (n -- 8). The horizontal broken lines are the mean + 1 SE (n = 60) for normal controls, i.e. rats kept singly in wire cages and exposed to room air. *Significantly different (P < 0.001-P < 0.05) from normal controls.
Figures 1 and 2 show that lung DNA synthesis as measured by tritiated thymidine incorporation was initially suppressed when rats were transferred into the environmental chamber, regardless of whether the gas within the chamber was normoxic, hypoxic or hypercapnic. Thereafter, neither air nor hypercapnia, but only hypoxia stimulated lung DNA synthesis. This phenomenon was observed even with as little as 6 h exposure to hypoxia. The stimulatory effect of hypoxia was more pronounced as exposure to hypoxia prolonged. The effects of hypoxia on lung DNA synthesis, however, persisted for a few days after discontinuation of hypoxia (6 h and 1 day hypoxic rats). g ~,
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Fig. 2. The tritiated thymidine incorporation in lung tissues, in organ culture, obtained from rats previously exposed to room air (experimental control - see text; closed circles) and hypercapnia (7 % COs in air; open circles). Each point and bar represents the mean + ! SE (n = 8). The horizontal broken lines are the mean + 1 SE (n = 60) for normal controls (see text). * Significantly different (P < 0.001) from normal controls.
184
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Fig. 3. Lung weight (upper panel), lung DNA content (middle panel) and lung air volume at 30 cm H20 pressure (lower panel) of rats after 1, 2, 3, 5 and 7 days of exposure to hypoxia (10~o 02 in N2; broken line) or room air (C,, i.e. body weight-matched experimental controls; solid line). Each point and bar represents the mean ± 1 SE (n = 5 to 9). * Significantly different (P < 0.001-P < 0.05) from experimental controls.
Daily alterations in lung weight, lung DNA content and lung air volume in rats exposed to hypoxia compared to controls (body weight=matched rats, C2) are shown in fig. 3. While lung weight progressively increased from the second day of exposure to hypoxia, lung DNA content and lung air volume remained below or near the control values for about 5 days and exceeded those of controls on day 7. As shown in table 1, with 7 day continuous hypoxia, lung weight, lung DNA content and lung air volume increased not only above those of body weight=matched controls (C2) but also of age-matched controls (C~). Figure 4 shows that these parameters ,of lung growth, measured at day 7, changed in proportion to the duration of exposure to hypoxia but did not change simultaneously. For example, lung DNA content increased with prolongation of hypoxia from 6 h to 2 days (air for the remaining of 7 days) but thereafter remained unchanged, while lung weight continued to increase and lung air volume enlarged only with more than 2 days hypoxia. It is obvious from figs. 3-5 that increase in lung weight is not mainly brought about by lung cell multiplicatioll. It is interesting to note (table 1) that the differences between Hpo-Air (2 days hypoxia and 5 days air) and Hpo rats (7 days hypoxia) are in lung weight and lung air volume,
LUNG GROWTH IN HYPOXIA
185
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Fig. 4. Lung weight (upper panel), lung DNA content (middle panel) and lung air volume at 30 cm H20 pressure (lower panel) of rats at the end of 7 day exposure to either air (C1: see text) (point at far leA), or hypoxia of short duration (6 h, I day, 2 days) followed by air for the remaining of 7 days (Hpo-Air), or hypoxia for 7 days (Hpo; point at far right). Each point and bar represents the mean + 1 SE (n = 6-8). The lines drawn through the corresponding points are hand-drawn approximations.
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Hpo-AIr
7.6
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0
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Lung Wet Weight (g)
1:1
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o.'16 o118 o;2o o122 Lung Dry Weight (g)
Fig. 5. Relationship between lung wet weight and DNA content (let~ panel) and lung dry weight and DNA (right panel). Each point and bar represents mean +_1 SE for a group of rats, namely from bottom to top (closed circles), C2, Air-CO2, CI, Hpo(6 h)-Air, Hpo(1 day)-Air, Hpo-CO2, and Hpo-Air (see text for explanation). Line drawn through these points is hand-drawn approximation. Open circle is for rats subjected to hypoxia for 7 days. (For statistical significance of lung wet weight/DNA see table 1).
186
E.E. FARIDY AND W.Z. YANG
both significantly lower in Hpo-Air, but not m lung DNA content. Because of this the ratio of MLV/DNA is significantly lower in Hpo-Air rats than that in control and Hpo rats. When 7 ~o CO2 in air instead of air was given to 2 days hypoxic rats (Hpo-CO2) the lung air volume significantly increased above that of Hpo-Air rats but the lung DNA content and lung weight remained the same as in Hpo-Air rats. The ratio of MLV/DNA, therefore, approached that of Hpo rats. In Air-CO2 rats neither the lung weight nor the lung DNA content changed but the lung air volume increased as it did in Hpo-CO2 rats. The ratio of MLV/DNA in this case resembled that of Hpo rats. In summary, 2 days hypoxic rats had larger lung weight and DNA content than control, and hypercapnic rats had larger lung air volume. Finally no significant differences in the deflation pressure-volume curves of lungs were noted among five groups of rats studied. Discussion
It is well known that rats raised in hypoxic environment have smaller body weight than their counterpart in normoxia (Timiras et al., 1957; Bartlett, 1970; and Pepelko, 1970). Since in most studies the effects of hypoxia on lung growth were assessed by correcting for body weight this has raised a doubt that perhaps hypoxia does not stimulate lung growth but suppresses body growth instead (Thurlbeck, 1975). For this reason in our study we included age-matched and body weight-matched rats as controls for hypoxic rats so that growth of the lung could be assessed by ~omparing the parameters of lung growth, such as lung weight, lung DNA content and lung air volume, either in absolute terms or after correcting for body weight. Another point taken into consideration was the duration of hypoxia. Most previous studies (as quoted above; Burri and Weibel, 1971; Cunningham et al., 1974) have dealt with the effects of prolonged hypoxia on lung growth. Thus the information on the immediate effects of hypoxia on lung growth is lacking. Short episodes of hypoxia do not lead to a significant loss in body weight (Faridy et al., 1988) to have an influence on the interpretation of results. The results of the present study clearly show that hypoxia has a profound effect on lung growth in rats even when administered for only 1 or 2 days. All 3 parameters of lung growth measured in this study, as mentioned above, do alter, independently of each other and not simultaneously, in response to and in proportion to the duration of hypoxia. Analysis of figs. 1, 3 and 4 suggests that the effects of hypoxia on lung growth begins with an increase in lung weight, followed by stimulation of DNA synthesis and cell proliferation, and later on by an increase in lung distention. Similarities noted in the rate of DNA synthesis in lung explants from rats exposed to hypoxia for I day and 7 days suggest that lung DNA synthesis could be maximally stimulated by only 1-2 days continuous hypoxia (10~o 02); that the mechanism by which hypoxia stimulates lung DNA synthesis persists long after the hypoxia has been relieved; and that once the mechanism for DNA synthesis is triggered and maximally stimulated by low Po2, the continuation of stimulus (hypoxia) beyond that point, up to
LUNG GROWTH IN HYPOXIA
187
I week, would not generate additional effect. This conception may be justified since the total lung DNA content of Hpo-Air rats which are exposed to only 2 days hypoxia does not differ from that of Hpo rats (7 days hypoxia). In contrast to the response shown by lung cells, lung air volume is not affected by short episodes of hypoxia nor is it directly influenced by lung DNA content or cell number. This becomes apparent when the ratio of lung air volume to lung DNA is taken as a measure of lung distensibility for a given lung cell mass. For example we note that in Hpo-Air and Hpo rats the ratios are significantly below and above the control value, respectively, while their lung DNA contents are similar but larger than control. This implies that cell multiplication in lung does not necessarily make the lung or prepare the lung to become more distensible. Alterations in lung air volume and lung weight become more and more noticeable as hypoxia continues beyond 2 days. In searching for a cause for these changes one should note that in hypoxic environment the rats not only experience low Po2 but also hyperventilate. Olson and Dempsey (1978) found that rats during 14 days exposure to hypoxia (4,300 m) showed a time-dependent hyperventilation by increasing both the frequency of breathing and the tidal volume. This was associated with a 24~o fall in Vo2 after I h of hypoxia which returned to control by 4 days. A similar hyperventilation, but for a different reason might have occurred in our rats subjected to hypercapnia. Jennings and Chen (1976) exposed the dogs to 5~ CO2 for 14 days and found that ventilation tripled during the first hour and remained 25-100~o above control between days 4 and 14. Rezzonico et al. (1987) observed that ventilation in neonatal rats almost doubled with exposure to 7 % CO2 in air from postnatal day 1 to 7. Our data indicate that hypercapnia in the presence of normal Po, significantly increases lung air volume without affecting the rate of DNA synthesis in lungs, the total lung DNA content, or the lung weight; that the ratio of lung air volume to lung DNA content is similar whether the rat is subjected to hypoxia or to hypercapnia (Hpo, Hpo-CO2 and Air-CO2 rats); and that lung air volume may increase by hypercapnia in an animal which has no previous exposure to, and stimulation of lung tissue by, hypoxia (Air-CO2 rats). It is, therefore, conceivable that lung distensibility (MLV) increases as a consequence of hyperventilation and not of a direct effect of low Po~ on lung tissue. This statement contradicts that made by Bartlett and Remmers (1971). These investigators, in quoting the unpublished observations of Bartlett who had kept young rats in 5~o CO2 for 20 days, state that the rats showed no 'abnormali',ies of lung development' and thus negate the role of mechanical stimulation by hyperventilation on lung growth. Unfortunately they have not specified the parameters by which they assessed lung growth in the hypercapnic rats and in particular if lung air volume was measured. Wigglesworth et al. (1977) described pulmonary hypoplasia following interruption of breathing movements in fetal rabbits by transecting the cervical spinal cord. Their report stimulated an interest in the notion that fetal breathing movements are essential for normal fetal lung growth. Alcorn et al. (1980) performed bilateral phrenectomy in fetal lambs at gestational days of 103-113 and showed that in 10-28 days after surgery the wet weight of the lungs was significantly reduced compared to sham operated controls.
188
E.E. FARIDY AND W.Z. YANG
These investigators pointed out that their results did not differentiate the role of fetal breathing movements from that of tonic activity of the diaphragm in regulating lung growth. In 1979 Wigglesworth and Desai interrupted fetal breathing movements by transecting the cervical spinal cord in fetal rabbits about the level of the phrenic nucleus in order to distinguish between tonic and phasic components of diaphragmatic activity. They found that the lung wet weight of fetuses with high cord section was significantly less than that of sham operated controls and that of fetuses with low cord transection. They concluded that normal fetal lung growth depends on maintenance of a function involving integration of respiratory movements and lung liquid secretion. Recently Mansell et al. (1986) studied the effects of unilateral diaphragmatic paralysis on postnatal lung growth in cats a~-~dpiglets in an attempt to test the hypothesis that activity of respiratory muscles determines regional growth of lung parenchyma. They found that the growth of contralateral lungs relative to ipsilateral lungs was greater in the phrenectomized animals than in the controls. They concluded that regional growth of lung parenchyma by cell proliferation depends in part on regional distribution of respiratory muscle activity. The above observations indicate that lung size may be influenced by presence or absence of ventilation in some parts of the lung and that mechanical overdistension of alveoli is at the base of lung hyperplasia. This Was initially suggested by Gehrig in 1951 (Burri and Weibel, 1971) who found that lungs of rats trained by swimming show first an acute overdistension of the alveoli, followed by a thickening of the septa with cellular proliferation and later an increase of the number of alveoli. We know that lung distension and in a greater degree hyperventilation increase the metabolic rate 3f excised lungs (Faridy and Naimark, 1971), but no information is available in regard to the effects of distension and hyperventilation on lung DNA synthesis. The present study suggests that hyperventilation induced by hypercapnia and increase in lung distensibility as a result of hyperventilation do not affect lung DNA synthesis and cell proliferation. This may be a likely conclusion to arrive at since hypercapnia per se does not suppress lung DNA synthesis. The transient suppression of DNA synthesis which we observe immediately after transferring the rats to the environmental chamber is a phenomenon not unique to hypercapnia but common to hypercapnia, hypoxia, and air, and perhaps related to 'stress'. We can not speculate on the effects of prolonged hyperventilation on lungs since our studies did not extend beyond I week. However, the results of Pepelko (1970), showing that lung weight of rats subjected to hypercapnia for 1, 2, 4, 8, 16 and 32 days are not different from that of controls, may be taken as an indirect evidence against the possible impact the prolonged hyperventilation may have on lung cell proliferation. We cannot clearly define the underlying cause for increased lung distensibility in response to hyperventilation. Even though hyperventilation enhances both the rate of release and that of depletion ofpuimonary surfactant (Faridy et al., 1966; Wyszogrodski et ai., 1975; Oyarzun and Clements, 1977), it appears that in both hypoxic and hypercapnic animals the lung surface forces have not altered to influence lung distensibility. The evidence in favour of this notion is the similarity of air deflation pressure-volume curves of lungs of hyperventilated rats with those of controls.
LUNG GROWTH IN HYPOXIA
189
Since, in Hpo-CO 2 rats, hyperventilation did not increase lung weight it becomes evident that, in hypoxia, lung weight increases in direct response to low PO2"This increase in lung weight occurs prior to and exceeds that achieved by cell multiplication. In excised lung, blood accounts for 15-20% of the total weight of the lung (Faridy and Naimark, 1971). Lung weight in hypoxia may therefore increase as a result of increased intravascular volume (Burri and Weibel, 1971), increased hematocrit (from 38% in normoxia, to 55% in hypoxia within a week - Pepelko, 1970), or pulmonary edema (Bartlett and Returners, 1971). Since lung dry weight is also increased with hypoxia such that the dry weight to wet weight ratio does not significantly differ from control, pulmonary edema becomes an unlikely alternative. In summary it appears that adaptive or compensatory lung growth in hypoxia is brought about on one hand by the direct effect of low Po2 on lung cells, resulting in lung hyperplasia, and on the other hand by the mechanical stimulation of lung tissue by hyperventilation, causing lung distension.
Acknowledgement. This study was supported by a grant from the Medical Research Council of Canada.
References Alcorn, D., T.M. Adamson, J.E. Maloney and P.M. Robinson (1980). Morphological effects of chronic bilateral phrenectomy or vagotomy in fetal lamb lung. J. Anat. 130: 683-695. Bartlett, D., Jr. (1970). Postnatal growth of the mammalian lung: influence oflow and high oxygen tensions. Respir. Physiol. 9: 58-64. Bartlett, D., Jr., and J. E. Returners (! 97 I). Effects of high altitude exposure on the lungs ofyoung rats. Respir. Physiol. 13: 116-125. Burri, P.H. and E.R. Weibei (1971). Morphometric estimation of pulmonary diffusion capacity. !i. Effect of Po, on the growing lung. Adaptation of the growing rat lung to hypoxia and hyperoxia. Respir. Physiol. I 11: 247-264. Cunningham, E. L., J. S. Brody and B. P. Jain (1974). Lung growth induced by hypoxia. J. Appl. Physiol. 37: 362-366. Faridy, E. E., S. Permutt and R. L. Riley (1966). Effect of ventilation on surface forces in excised dogs' lungs. J. Appi. Physiol. 21: 1453-1462. Faridy, E. E. and A. Naimark (! 971 ). Effect ofdistension on metabolism of excised dog lung. J. Appl. Physiol. 31: 31-37. Faridy, E.E., M.R. Sanii and J.A. Thliveris (1988). Fetal lung growth: influence of maternal hypoxia and hyperoxia in rats. Respir. Physiol. 73: 225-242. Gribetz, I., N. R. Frank and M. E. Avery (1959). Static volume-pressure relations ofexcised lungs of infants with hyaline membrane disease, newborn and stillborn infants. J. Clin. Invest. 38: 2168-2175. Guleria, J. S., J. N. Pande, P.K. Sethi and S.B. Roy (1971). Pulmonary diffusing capacity at high altitude. J. Appi. Physiol. 31: 536-543. Hurtado, A. (1932). Respiratory adaptation in the Indian natives of the Peruvian Andes. Studies at high altitude. Am. J. Phys. Anthropol. 17: 137-165. Jennings, D.B. and C.C. Chen (1976). Ventilation in conscious dogs during acute and chronic hypercapnia. J. Appl. Physiol. 4 I: 839-847. Mansell, A. L., J. V. Rojas, E. M. Sillos, C.J. Stolar, M. H. Collins and S.J. Rozovski (1986). Diaphragmatic activity is a determinant of postnatal lung growth. J. Appl. Physiol. 61:1098-1103.
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Olson, E. B., Jr., and J. A. Dempsey (1978). Rat as a model for human like ventilatory adaptation to chronic hypoxia. J. Appl. Physiol. 44: 763-769. Oyarzun, M.J. and J.A. Clements (1977). Ventilatory and cholinergic control of pulmonary surfactant in the rabbit. J. Appl. Physiol. 43: 39-45. Pepelko, W.E. (1970). Effects of hypoxia and hypercapnia, singly and combined, on growing rats. J. Appl. Physiol. 28: 646-651. Rezzonico, R., M. Saetta, K. Sullivan and J.P. Mortola (1987). Respiratory adaptation to chronic hypercalmia in newborn rats..Phys~logist 30: 252. Schneider, W.C. (1957). Determination of nucleic acids in tissue by pentose analysis. Methods Enzymol. 3: 680-684. Thurlbeek, W.M. (1975). The state of the art: postnatal growth and development of the lung. Am. Rev. Respir. DIS. 111: 803-844. Timiras, P. S., A.A. Krum and N. Pace (1957). Body and organ weights of rats during acclimatization to an altitude of 12470 feet. Am. J. Physiol. 191: 598-604. Wigglesworth, J.S., R.M.L. Winston and K. Bartlett (1977). Influence of the central nervous system on fetal lung development. Arch. Dis. Child. 52: 965-967. Wigglesworth, J. S. and R. Desai (1979). Effects on lung growth ofcervical cord section in the rabbit fetus. Early Human Dev. 3: 51-65. Wyszogrodski, I., K. Kyer-Aboagye, H.W. Taeusch, Jr., and M.E. Avery (1975). Surfactant inactivation by hyperventilation: conservation by end-expiratory pressure. J. Appi. Physiol. 38: 461-466.
179
RSP 01530
Role of hyperventilation in hypoxia on lung growth in rats Edmund E. Faridy and Wen Z. Yang Department of Physiology. University of Manitoba, Winnipeg, Canada R3E OW3 (Accepted for publication 21 January 1989) Abstract. This study was conducted in an attempt to differentiate the contribution of hyperventilation, if any, from that of low Po2 on adaptive lung growth in response to hypoxia. Male albino rats were exposed to one ofthe following: (1) Room air for 7 days, as control; (2) 10% 02 in N 2 for 7 days; (3) 10% 02 for 6 h, 1 day or 2 days and air for the remaining of 7 days; (4) 10% 02 for 2 days and 7% CO2 in air for 5 days; (5) air for 2 days and 7% CO2 for 5 days; or (6) 7% CO2 in air for 7 days. Lung growth was assessed by measuring the lung weight, lung air volume, lung DNA content and rage of DNA synthesis in lung explants. Hypoxia stimulated lung DNA synthesis even when administered for only 6 h, and the effects persisted for a few days alter discontinuation of hypoxia. Hypercapnia did not stimulate DNA synthe~-~ in lung. In 2 clay hypoxic 5 day air rats the lung weight and lung DNA content increased, in 2 day air 5 day hypercapnic rats only the lung volume increased, and in 2 day hypoxic 5 day hypercapnic rats all parameters of lung growth, i.e., lung weight, DNA content and air volume increased as in 7 day hypoxic rats. The results suggest that adaptive or compensatory lung growth in hypoxia is brought about on one hand by the direct effect of low Po2 on lung cells, resulting in lung hyperplasia, and on the other hand by the mechanical stimulation of lung tissue by hyperventilation, causing lung distension.
Compensatory lung growth; Hypercapnia; Lung air volume; Lung DNA synthesis; Lung pressure-volume
Observations on humans native to high altitudes (Hurtado, 1932; Guleria et al., 1971) and on experimental animals exposed to hypoxic environment (Bartlett and Remmers, 1971; Burri and Weibel, 1971; Cunningham et ai., 1974) suggest that chronic hypoxia induces an adaptive growth response in the lung which is aimed to increase surface area for gas exchange. The mechanisms by which the lung grows in hypoxia are unclear. Furthermore, the role of hyperventilation as a consequence of low Po2 on adaptive lung growth in response to hypoxia has not been delineated. The present study, conducted in male albino rats, was undertaken in an attempt to differentiate the contribution of hyperventilation, if any, from that of low Po2 on lung growth.
Correspondence address: Edmund E. Faridy, Department of Physiology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. 0034-5687/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
180
E.E. FARIDY AND W.Z. YANG
Methods Male Sprague-Dawley rats weighing between 135 and 150 g were used for this study. The rats were kept in a room with controlled temperature and humidity and supplied with standard rat chow and water ad libitum. For studies of DNA synthesis two groups of rats were used as controls: (1)normal controls - these rats were kept in wire cages (one rat in each cage) and were sacrificed at various days from 0 to 7; and (2) experimental controls - on day 0, these rats (in groups of 12) were transferred into an environmental chamber, and exposed to air for up to 7 days. The experimental rats, on day 0, were also transferred into the environmental chamber but exposed to either: 1) hypoxia (10% 02 in N2) for 6 h or 1 day and air for the remaining of 7 days; 2)hypoxia for up to 7 days; or 3)hypercapnia (7 % CO2 in air) for 7 days. The chamber was made of plexiglass and had a volume of 98 L. A wire cage within the chamber had sufficient space to accommodate 12 rats. The chamber gas was maintained at predetermined concentrations of 02, CO 2 and N z. A continuous flow of gas from air, 02, CO2 and N2 cylinders washed out the excess CO2 and humidity from the chamber. The gas flow ranged between 5-7 L/min depending on the number of rats in the chamber. The chamber gas was continuously mixed by a noiseless fan, and passed over a temperature controlled copper coil. The inflow and the outflow gases were monitored by 02 and CO2 analyzers (Beckman). A temperature of 23 °C + 0.5 and a CO2 concentration of less than 0.2% (in air and in hypoxia experiments) were maintained over the duration of the experiment. The rats were then sacrificed on days 1, 2, 3, 4, 5 and 7 of exposure to above gases. On the day of sacrifice, the rats were exsanguinated under sodium pentobarbital anesthesia (5 mg/100 g BW) by cutting the abdominal aorta. The lungs were excised, under sterile conditions, dissected free of extrapulmonary airways and tissues, washed with chilled culture medium and chopped into explants of approximately I mm cubes with a pair of sharp surgical blades. All procedures were carried out in a laminar flow hood and completed within 10 min. The explants were transferred into multi-wall plastic culture plates (Flow Lab., Inc., McLean, Virginia) containing Waymouth MB 752/1 culture medium (Gibco Lab., Grand Island, New York) with 0.1 #Ci/ml of [3H]thymidine (specific radioactivity 6.7 Ci/mM. New England Nuclear, Inc., Boston, MA), and were incubated at 37 °C in a humidified atmosphere of 5 ~ C02 in air. At the end of 6 h of incubation, the explants were removed from the medium. The tissues from four wells were pooled as one sample and washed in 10% thrichloroacetic acid (TCA) and then in 95~ ethyl alcohol. The explants were digested by incubating in 2 N sodium hydroxide in 37 °C water bath overnight. The nucleus fraction was precipitated in 2N perchloric acid by centrifugation. DNA was dissolved from the pellets in 5~o TCA by incubating in 90 °C water bath for 15 rain. One millilitre of DNA extract was mixed with 10 ml of Biofluor liquid scintillation cocktail (New England Nuclear, Inc.) and counted in a liquid scintillation spectrephotometer. Another millilitre of DNA extract was used to measure DNA content by the method of Schneider (1957). The results are expressed as DPM/#g DNA x 10- 3
LUNG GROWTH IN HYPOXIA
181
For studies of lung growth the rats were divided into 5 groups. Each group was placed in the environmental chamber and subjected to one of the following gas mixtures for 7 days: (1)room air (Air) as control. This group consisted of two subgroups: (a)agematched rats (C~) as control for all experimental groups; and (b) body weight-matched (3 days younger) rats (C2) as control for hypoxic rats which do not grow as rapidly as normoxic rats; (2) Hypoxia (10% 0 2 in N2) (Hpo); (3) Hypoxia for 6 h, 1 day or 2 days and air for the remaining of 7 days (Hpo-Air); (4) Hypoxia for 2 days and hypercapnia (7% CO2 in air) for 5 days (Hpo-CO2); and (5)air for 2 days and hypercapnia for 5 days (Air-CO2). With the exception of some rats in C2 (body weight-matched controls) and Hpo (10~o 02 in N2) groups which were sacrificed on days 1, 2, 3, and 5, the rest were all sacrificed at the end of 7 days exposure to various gases. On the day of sacrifice, the rats were weighed, exsanguinated, as mentioned above, and a pneumothorax was produced by cutting the diaphragm adjacent to the xyphoid. The opening in the diaphragm was enlarged and the lungs were inspected under a magnifying glass. If the lungs were not uniformly pink in color and showed signs of possible infection (such as even a single red or grey spot) the rat was excluded from the study. The trachea immediately below the larynx was cannulated and the lungs, left intact in the chest, were degassed by placing the animal in a vacuum jar. The chest was then opened by bisecting the sternum, and the large vessels ligated at the base of the heart. The ca~mu|ated degassed lungs were then attached to a pressure-volume apparatus similar to that previously described (Gribetz etaL, 1959). The lungs were inflated with air to 30 cm H20 pressure. This inflating pressure was maintained until the lung air volume remained constant for 15 sec. The air volume observed at this transpulmonary pressure, considered as maximal lung air volume (MLV), was designated as 100~ and each volume subsequently observed after deflation to a predetermined transpulmonary pressure (20, 15, 10, 5 and 0 cm H20) was expressed as a percentage of MLV. These pressures were maintained for 20 sec before the volumes were read at each pressure. If, during the procedure, the lung air volume did not remain constant at high pressure, air leaks were assumed to be present, and the data were excluded from the study. All pressure-volume measurements were performed at room temperature. At the end ofak PV measurements the lungs were separated from the extrapulmonary airways, weighed and used to measure lung DNA and dry weight. The lung deoxyribonucleic acid (DNA) content was determined by the method of Schneider (1957) using 0.5 g samples of lung tissue. The remaining lung tissues were dried in an oven at 60 °C for one week. Statistical analysis of the data was carded out using a t-test of unpaired variates and a multiple range test for analysis of variance by Duncan's method, where applicable. Results
Only rats subjected to hypoxia for 7 days (Hpo) had body weights significantly lower than control (table 1).
(8)
(8)
1.20 + 0.06
(8)
(8)
1.26 + 0.05
10.10 + 0.45
(8)
(8)
10.39 + 0.36
4.12 + 0.15
(8)
7.80 + 0.29
(8)
4.06 + 0.10
(8)
6.56 + 0.21
(8)
4.65 + 0.11
(8)
8.48 + 0.28
(8)
3.73 + 0.15
(8)
6.78 + 0.24
(8)
96.4 + 1.77
(8)
(8)
90.4 + 1.78
0.156 + 0.004
(8)
(8)
0.164 + 0.002
118.6 + 2.04
(8)
121.4 _+ 3.04
0.776 + 0.017
(8)
0.818 + 0.015
161.4 + 1.83
(8)
181.9 + 2.12
(8)
Air (C2)
(7)
1.44 + 0.05"*
(7)
10.31 + 0.34
(7)
6.64 + 0.26**
(7)
10.70 + 0.30**
(8)
4.66 + 0.08**
(8)
7.53 + 0.20**
(8)
133.4 + 4.14"*
(8)
0.216 + 0.008**
(8)
139.2 + 2.61"*
(8)
1.048 + 0.036**
(8)
161.5 _+2.28*
Hpo
(6)
1.09 + 0.06"*
(6)
9.52 + 0.43
(6)
4.61 + 0.10'
(6)
8.52 + 0.13'
(8)
4.17 + 0.13"*
(8)
7.71 _+0.27*
(8)
97.3 + 2.28**
(8)
0.180 + 0.005**
(S)
117.6 + 3.60*
(8)
0.903 + 0.023**
(8)
185.1 _+ 1.88'
Hpo-Air
(7)
1.34 + 0.04 s
(7)
! !.23 + 0.39 !
(7)
5.41 + 0.10 **0
(7)
9.75 + 0.20**o
(8)
4.10 + 0.11'
(8)
7.33 + 0.25
(8)
98.2 + 2.08**
(8)
0.176 + 0.005*
(8)
118.8 + 3.68*
(8)
0.864 + 0.028*
(8)
178.6 + 2.93*
Hpo-CO 2
(8)
1.44 + 0.05*
(8)
12.14 + 0.37*
(8)
5.35 + 0.14"
(8)
9.59 + 0.28*
(8)
3.74 + 0.10
(8)
6.69 + 0.16 a
(8)
90.8 + 2.43 a
(8)
0.162 +_0.003 a
(8)
119.4 + 4.51
(8)
0.794 + 0.016 a
(8)
179.1 + 1.66
Air-CO 2
Hpo = hypoxia (10~o 02 in N2) for 7 days; Hpo-Air = 2 days hypoxia, 5 days air; H p o - C O 2 = 2 days hypoxia, 5 days 7~o CO2 in air; A i r - C O 2 -- 2 days air, 5 days 7~o CO2 in air. BW = body weight at the day of sacrifice. MLV -- lung air volume at 30 cm H20 transpulmonary pressure. Data are expressed as mean + 1 SE; numbers in parentheses indicate the number of rats studied. Significantly different: *from CI (P<0.001 - P < 0 . 0 5 ) ; t H p o from C2 (P < 0.001-P < 0.007); *Hpo-Air and H p o - C O 2 from Hpo ( P < 0.001 - P < 0.02); §Hpo-CO2 from Hpo-Air ( P < 0.001 - P < 0.02); a Air-CO2 from H p o - C O 2 (P < 0.05). Comparisons are not made between C1 and C2, and between A i r - C O 2 and Hpo groups.
MLV (ml/mg DNA)
MLV (ml/g lung)
MLV (ml/100 g BW)
MLV (ml)
Lung DNA (rag/100 g BW)
Lung DNA (mg)
Lung dry wt (mg/100 g.BW)
Lung dry wt (g)
Lung wet wt (mg/mg DNA)
Lung wet wt (g)
Body weight (g)
Air (C,)
Lung measurements in control (Ci and C2) and experimental rats at the end of 7 day exposure to various gases.
TABLE 1
Z
Z
ql
.re .m
oo
LUNG GROWTH IN HYPOXIA
183
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600
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, o
300
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D~ation of Expos~e to Various~ e s
6 (Day)
Fig. l. The tritiated thymidine incorporation in lung tissues, in organ culture, obtained from rats previously exposed to hypoxia. Closed triangles represent lungs from rats exposed to 6 h hypoxia and 3/4 to 63/4 days air; open triangles: 1 day hypoxia and I to 6 days air; and closed circles: 1 to 7 days hypoxia. Each point and bar represents the mean + 1 SE (n -- 8). The horizontal broken lines are the mean + 1 SE (n = 60) for normal controls, i.e. rats kept singly in wire cages and exposed to room air. *Significantly different (P < 0.001-P < 0.05) from normal controls.
Figures 1 and 2 show that lung DNA synthesis as measured by tritiated thymidine incorporation was initially suppressed when rats were transferred into the environmental chamber, regardless of whether the gas within the chamber was normoxic, hypoxic or hypercapnic. Thereafter, neither air nor hypercapnia, but only hypoxia stimulated lung DNA synthesis. This phenomenon was observed even with as little as 6 h exposure to hypoxia. The stimulatory effect of hypoxia was more pronounced as exposure to hypoxia prolonged. The effects of hypoxia on lung DNA synthesis, however, persisted for a few days after discontinuation of hypoxia (6 h and 1 day hypoxic rats). g ~,
'o
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o c Z --
O
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1o0
~8 0
0 Duration of Exposure to Various Gases(Day)
Fig. 2. The tritiated thymidine incorporation in lung tissues, in organ culture, obtained from rats previously exposed to room air (experimental control - see text; closed circles) and hypercapnia (7 % COs in air; open circles). Each point and bar represents the mean + ! SE (n = 8). The horizontal broken lines are the mean + 1 SE (n = 60) for normal controls (see text). * Significantly different (P < 0.001) from normal controls.
184
E.E. FARIDY A N D W.Z. Y A N G ,~ 1.1 '~ 1.0 .? O.g
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Fig. 3. Lung weight (upper panel), lung DNA content (middle panel) and lung air volume at 30 cm H20 pressure (lower panel) of rats after 1, 2, 3, 5 and 7 days of exposure to hypoxia (10~o 02 in N2; broken line) or room air (C,, i.e. body weight-matched experimental controls; solid line). Each point and bar represents the mean ± 1 SE (n = 5 to 9). * Significantly different (P < 0.001-P < 0.05) from experimental controls.
Daily alterations in lung weight, lung DNA content and lung air volume in rats exposed to hypoxia compared to controls (body weight=matched rats, C2) are shown in fig. 3. While lung weight progressively increased from the second day of exposure to hypoxia, lung DNA content and lung air volume remained below or near the control values for about 5 days and exceeded those of controls on day 7. As shown in table 1, with 7 day continuous hypoxia, lung weight, lung DNA content and lung air volume increased not only above those of body weight=matched controls (C2) but also of age-matched controls (C~). Figure 4 shows that these parameters ,of lung growth, measured at day 7, changed in proportion to the duration of exposure to hypoxia but did not change simultaneously. For example, lung DNA content increased with prolongation of hypoxia from 6 h to 2 days (air for the remaining of 7 days) but thereafter remained unchanged, while lung weight continued to increase and lung air volume enlarged only with more than 2 days hypoxia. It is obvious from figs. 3-5 that increase in lung weight is not mainly brought about by lung cell multiplicatioll. It is interesting to note (table 1) that the differences between Hpo-Air (2 days hypoxia and 5 days air) and Hpo rats (7 days hypoxia) are in lung weight and lung air volume,
LUNG GROWTH IN HYPOXIA
185
~ 1.1
-J~0.9 y 0,8 &O
~=
•<~
1o
s
//
J
9
i
p
s S
e.
i 7 6 Duration
i
i
i
5
4
3 2 1 0 Air to Hypoxia & Air (Day)
i
6
7 Hypoxia
ofExposure
Fig. 4. Lung weight (upper panel), lung DNA content (middle panel) and lung air volume at 30 cm H20 pressure (lower panel) of rats at the end of 7 day exposure to either air (C1: see text) (point at far leA), or hypoxia of short duration (6 h, I day, 2 days) followed by air for the remaining of 7 days (Hpo-Air), or hypoxia for 7 days (Hpo; point at far right). Each point and bar represents the mean + 1 SE (n = 6-8). The lines drawn through the corresponding points are hand-drawn approximations.
8.0
~
Hpo-AIr
7.6
Hpo(I d)l/-~t
7.2
Hpo(6MI~/ Air~C021~l
_~6.8
c2
6.4 I_
0
o'.e
o19
I'.O
Lung Wet Weight (g)
1:1
.~11
o.'16 o118 o;2o o122 Lung Dry Weight (g)
Fig. 5. Relationship between lung wet weight and DNA content (let~ panel) and lung dry weight and DNA (right panel). Each point and bar represents mean +_1 SE for a group of rats, namely from bottom to top (closed circles), C2, Air-CO2, CI, Hpo(6 h)-Air, Hpo(1 day)-Air, Hpo-CO2, and Hpo-Air (see text for explanation). Line drawn through these points is hand-drawn approximation. Open circle is for rats subjected to hypoxia for 7 days. (For statistical significance of lung wet weight/DNA see table 1).
186
E.E. FARIDY AND W.Z. YANG
both significantly lower in Hpo-Air, but not m lung DNA content. Because of this the ratio of MLV/DNA is significantly lower in Hpo-Air rats than that in control and Hpo rats. When 7 ~o CO2 in air instead of air was given to 2 days hypoxic rats (Hpo-CO2) the lung air volume significantly increased above that of Hpo-Air rats but the lung DNA content and lung weight remained the same as in Hpo-Air rats. The ratio of MLV/DNA, therefore, approached that of Hpo rats. In Air-CO2 rats neither the lung weight nor the lung DNA content changed but the lung air volume increased as it did in Hpo-CO2 rats. The ratio of MLV/DNA in this case resembled that of Hpo rats. In summary, 2 days hypoxic rats had larger lung weight and DNA content than control, and hypercapnic rats had larger lung air volume. Finally no significant differences in the deflation pressure-volume curves of lungs were noted among five groups of rats studied. Discussion
It is well known that rats raised in hypoxic environment have smaller body weight than their counterpart in normoxia (Timiras et al., 1957; Bartlett, 1970; and Pepelko, 1970). Since in most studies the effects of hypoxia on lung growth were assessed by correcting for body weight this has raised a doubt that perhaps hypoxia does not stimulate lung growth but suppresses body growth instead (Thurlbeck, 1975). For this reason in our study we included age-matched and body weight-matched rats as controls for hypoxic rats so that growth of the lung could be assessed by ~omparing the parameters of lung growth, such as lung weight, lung DNA content and lung air volume, either in absolute terms or after correcting for body weight. Another point taken into consideration was the duration of hypoxia. Most previous studies (as quoted above; Burri and Weibel, 1971; Cunningham et al., 1974) have dealt with the effects of prolonged hypoxia on lung growth. Thus the information on the immediate effects of hypoxia on lung growth is lacking. Short episodes of hypoxia do not lead to a significant loss in body weight (Faridy et al., 1988) to have an influence on the interpretation of results. The results of the present study clearly show that hypoxia has a profound effect on lung growth in rats even when administered for only 1 or 2 days. All 3 parameters of lung growth measured in this study, as mentioned above, do alter, independently of each other and not simultaneously, in response to and in proportion to the duration of hypoxia. Analysis of figs. 1, 3 and 4 suggests that the effects of hypoxia on lung growth begins with an increase in lung weight, followed by stimulation of DNA synthesis and cell proliferation, and later on by an increase in lung distention. Similarities noted in the rate of DNA synthesis in lung explants from rats exposed to hypoxia for I day and 7 days suggest that lung DNA synthesis could be maximally stimulated by only 1-2 days continuous hypoxia (10~o 02); that the mechanism by which hypoxia stimulates lung DNA synthesis persists long after the hypoxia has been relieved; and that once the mechanism for DNA synthesis is triggered and maximally stimulated by low Po2, the continuation of stimulus (hypoxia) beyond that point, up to
LUNG GROWTH IN HYPOXIA
187
I week, would not generate additional effect. This conception may be justified since the total lung DNA content of Hpo-Air rats which are exposed to only 2 days hypoxia does not differ from that of Hpo rats (7 days hypoxia). In contrast to the response shown by lung cells, lung air volume is not affected by short episodes of hypoxia nor is it directly influenced by lung DNA content or cell number. This becomes apparent when the ratio of lung air volume to lung DNA is taken as a measure of lung distensibility for a given lung cell mass. For example we note that in Hpo-Air and Hpo rats the ratios are significantly below and above the control value, respectively, while their lung DNA contents are similar but larger than control. This implies that cell multiplication in lung does not necessarily make the lung or prepare the lung to become more distensible. Alterations in lung air volume and lung weight become more and more noticeable as hypoxia continues beyond 2 days. In searching for a cause for these changes one should note that in hypoxic environment the rats not only experience low Po2 but also hyperventilate. Olson and Dempsey (1978) found that rats during 14 days exposure to hypoxia (4,300 m) showed a time-dependent hyperventilation by increasing both the frequency of breathing and the tidal volume. This was associated with a 24~o fall in Vo2 after I h of hypoxia which returned to control by 4 days. A similar hyperventilation, but for a different reason might have occurred in our rats subjected to hypercapnia. Jennings and Chen (1976) exposed the dogs to 5~ CO2 for 14 days and found that ventilation tripled during the first hour and remained 25-100~o above control between days 4 and 14. Rezzonico et al. (1987) observed that ventilation in neonatal rats almost doubled with exposure to 7 % CO2 in air from postnatal day 1 to 7. Our data indicate that hypercapnia in the presence of normal Po, significantly increases lung air volume without affecting the rate of DNA synthesis in lungs, the total lung DNA content, or the lung weight; that the ratio of lung air volume to lung DNA content is similar whether the rat is subjected to hypoxia or to hypercapnia (Hpo, Hpo-CO2 and Air-CO2 rats); and that lung air volume may increase by hypercapnia in an animal which has no previous exposure to, and stimulation of lung tissue by, hypoxia (Air-CO2 rats). It is, therefore, conceivable that lung distensibility (MLV) increases as a consequence of hyperventilation and not of a direct effect of low Po~ on lung tissue. This statement contradicts that made by Bartlett and Remmers (1971). These investigators, in quoting the unpublished observations of Bartlett who had kept young rats in 5~o CO2 for 20 days, state that the rats showed no 'abnormali',ies of lung development' and thus negate the role of mechanical stimulation by hyperventilation on lung growth. Unfortunately they have not specified the parameters by which they assessed lung growth in the hypercapnic rats and in particular if lung air volume was measured. Wigglesworth et al. (1977) described pulmonary hypoplasia following interruption of breathing movements in fetal rabbits by transecting the cervical spinal cord. Their report stimulated an interest in the notion that fetal breathing movements are essential for normal fetal lung growth. Alcorn et al. (1980) performed bilateral phrenectomy in fetal lambs at gestational days of 103-113 and showed that in 10-28 days after surgery the wet weight of the lungs was significantly reduced compared to sham operated controls.
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These investigators pointed out that their results did not differentiate the role of fetal breathing movements from that of tonic activity of the diaphragm in regulating lung growth. In 1979 Wigglesworth and Desai interrupted fetal breathing movements by transecting the cervical spinal cord in fetal rabbits about the level of the phrenic nucleus in order to distinguish between tonic and phasic components of diaphragmatic activity. They found that the lung wet weight of fetuses with high cord section was significantly less than that of sham operated controls and that of fetuses with low cord transection. They concluded that normal fetal lung growth depends on maintenance of a function involving integration of respiratory movements and lung liquid secretion. Recently Mansell et al. (1986) studied the effects of unilateral diaphragmatic paralysis on postnatal lung growth in cats a~-~dpiglets in an attempt to test the hypothesis that activity of respiratory muscles determines regional growth of lung parenchyma. They found that the growth of contralateral lungs relative to ipsilateral lungs was greater in the phrenectomized animals than in the controls. They concluded that regional growth of lung parenchyma by cell proliferation depends in part on regional distribution of respiratory muscle activity. The above observations indicate that lung size may be influenced by presence or absence of ventilation in some parts of the lung and that mechanical overdistension of alveoli is at the base of lung hyperplasia. This Was initially suggested by Gehrig in 1951 (Burri and Weibel, 1971) who found that lungs of rats trained by swimming show first an acute overdistension of the alveoli, followed by a thickening of the septa with cellular proliferation and later an increase of the number of alveoli. We know that lung distension and in a greater degree hyperventilation increase the metabolic rate 3f excised lungs (Faridy and Naimark, 1971), but no information is available in regard to the effects of distension and hyperventilation on lung DNA synthesis. The present study suggests that hyperventilation induced by hypercapnia and increase in lung distensibility as a result of hyperventilation do not affect lung DNA synthesis and cell proliferation. This may be a likely conclusion to arrive at since hypercapnia per se does not suppress lung DNA synthesis. The transient suppression of DNA synthesis which we observe immediately after transferring the rats to the environmental chamber is a phenomenon not unique to hypercapnia but common to hypercapnia, hypoxia, and air, and perhaps related to 'stress'. We can not speculate on the effects of prolonged hyperventilation on lungs since our studies did not extend beyond I week. However, the results of Pepelko (1970), showing that lung weight of rats subjected to hypercapnia for 1, 2, 4, 8, 16 and 32 days are not different from that of controls, may be taken as an indirect evidence against the possible impact the prolonged hyperventilation may have on lung cell proliferation. We cannot clearly define the underlying cause for increased lung distensibility in response to hyperventilation. Even though hyperventilation enhances both the rate of release and that of depletion ofpuimonary surfactant (Faridy et al., 1966; Wyszogrodski et ai., 1975; Oyarzun and Clements, 1977), it appears that in both hypoxic and hypercapnic animals the lung surface forces have not altered to influence lung distensibility. The evidence in favour of this notion is the similarity of air deflation pressure-volume curves of lungs of hyperventilated rats with those of controls.
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Since, in Hpo-CO 2 rats, hyperventilation did not increase lung weight it becomes evident that, in hypoxia, lung weight increases in direct response to low PO2"This increase in lung weight occurs prior to and exceeds that achieved by cell multiplication. In excised lung, blood accounts for 15-20% of the total weight of the lung (Faridy and Naimark, 1971). Lung weight in hypoxia may therefore increase as a result of increased intravascular volume (Burri and Weibel, 1971), increased hematocrit (from 38% in normoxia, to 55% in hypoxia within a week - Pepelko, 1970), or pulmonary edema (Bartlett and Returners, 1971). Since lung dry weight is also increased with hypoxia such that the dry weight to wet weight ratio does not significantly differ from control, pulmonary edema becomes an unlikely alternative. In summary it appears that adaptive or compensatory lung growth in hypoxia is brought about on one hand by the direct effect of low Po2 on lung cells, resulting in lung hyperplasia, and on the other hand by the mechanical stimulation of lung tissue by hyperventilation, causing lung distension.
Acknowledgement. This study was supported by a grant from the Medical Research Council of Canada.
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