Early Human Development, 1980,414, 411-424 o Elsevier/North-Holland Biomedical Press
Oxygen-induced
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lung injury in the newborn piglet
JOHN YAM and ROBERT J. ROBERTS The Toxicology Center, Department of Pharmacology, Divisions of Clinical Pharmacology and Neonatology, Department of Pediatrics, The University of Iowa, Iowa City, Iowa 52242, U.S.A.
Accepted for publication 5 June 1980
SUMMARY
Oxygen-induced lung toxicity was studied in artificially fed newborn miniature piglets. Paired littermates of newborn piglets were exposed to either 96-98% oxygen or air for 2, 4, 7 or 10 days. Development of pulmonary edema, as monitored by both the lung wet weight to dry weight ratio and the lung wet weight to body weight ratio, was evident 4 days after the start of oxygen exposure. Examination of light and electron micrographs showed that pulmonary edema was located mainly in the perivascular and interstitial spaces. Endothelial and type I cells were normal in appearance throughout the oxygen exposure. After exposure to 10 days of oxygen, type II cells appeared to show a decrease in the size of lamellar bodies and an increase in the number and size of mitochondria. The activity of pulmonary antioxidant defenses, as measured by the activity of superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR), and the level of reduced glutathione (GSH), showed a. progressive increase in activity with duration of oxygen exposure, culminating in a significantly higher SOD, GP and GSH level in ‘I-day oxygen-exposed piglets. It is concluded that the newborn piglet is less susceptible to oxygen-induced lung injury compared to adults of other species, and the increase in the lung complement of SOD, GR, GP and GSH may contribute to the apparent resistance to oxygen toxicity. oxygen toxicity; newborn piglet
lung; bronchopulmonary
dysplasia; superoxide dismutase; glutathione;
INTRODUCTION
Northway et al. [19] first reported that prolonged oxygen exposure of infants with hyaline membrane disease (HMD) was associated with a
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syndrome of chronic lung disease which was referred to as bronchopulmonary dysplasia (BPD). Despite the risk, continued oxygen therapy for long periods of time is often required in infants with HMD to minimize infant mortality due to hypoxemia. Research directed at oxgen-induced lung toxicity in newborn animals may contribute significantly to the understanding of BPD in the human infant. Published reports on oxygen-induced lung toxicity in newborn animals are limited [ 81. Most of the available studies have been performed with newborn mice or rats [5, 15, 21, 24, 361. Systematic studies of pulmonary oxygen toxicity in neonatal rats or mice is complicated by ‘the fact that they are often destroyed by their mothers, their small lung size requires the pooling of tissue from a number of animals for biochemical analysis, and multiple studies on the same lung are not always possible. Their body size also precludes certain complex cardiovascular and/or pulmonary function measurements. The objective of this research was to study the miniature piglet as an alternate animal model to investigate oxygen-induced lung toxicity in newborns. The use of newborn piglets would not only circumvent the above disadvantages of using mice or rats, but the pig has been shown to be human-like in many organ systems including the respiratory system [3, 7, 101.
METHODS
Newborn Pitman Moore miniature piglets were obtained from the University of Iowa Pediatric Research Center at the Oakdale Campus. The newborn piglets were kept with the mother sow and maintained on colostrum for 1 day after birth before transporting to the Toxicology Center for use in these studies. It has been shown that maintenance of the piglet on mother’s colostrum reduces the possibility of the piglet acquiring infection [7]. Littermates of newborn piglets were paired according to body weight and sex and were randomly assigned to either the oxygen-exposed or air control groups. Oxygen-exposed and control piglets were placed in infant Isolettes (Air-Shields, Hatboro; PA) in clean, open-top boxes. Oxygen was supplied by a pressurized liquid oxygen tank at a flow rate of 5-6 l/min. The oxygen concentration in the Isolettes was monitored frequently by a Beckman model OMll oxygen analyzer (Beckman Instruments, Schiller Park, IL) and was maintained at 96-98% throughout the exposure period. Carbon dioxide concentration in the Isolettes, as analyzed by a Beckman model LB-2 gas analyzer, was maintained at less than 0.4% during the exposure period. The temperature of the Isolettes was maintained between 24 and 26°C. The cardboard boxes in which the piglets were housed were changed daily, which required less than 20 set to accomplish. After the change, the oxygen in the Isolette was quickly (within 30 set) brought back to 96-98% by flushing with excess oxygen. The piglets.were fed 4-6 times daily (220 Cal/kg/day) liquid infant formula (Ross with concentrated Similac plus iron
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Laboratories, Columbus, OH) using a no. 4 French feeding tube (Pharmaseal, Toa Alta, Puerto Rico). To prevent a decrease in oxygen concentration in the Isolette during feeding, the oxygen flow was increased during feeding. Monitoring of oxygen concentration during feeding showed less than a 2% drop in oxygen concentration. At a specified time, litter-mates of air- and oxygen-exposed newborn piglets were sacrificed by pentobarbital euthanasia and the lungs removed, trimmed of extraparenchymal tissue, and weighed. The left middle lobe was isolated and removed for determination of lung wet weight to dry weight ratio. Preweighed wet lung tissue blocks were dried in an oven until constant weight was achieved (4-7 days). Histological studies were performed on the left caudal lobe of the piglet lung. The left bronchus was cannulated and Kamovsky’s formaldehyde-glutaraldehyde fixative [ll] was instilled via the cannula at a perfusion pressure of 20-25 cm of water [14] . After overnight fixation at the above pressure, several tissue blocks were obtained from the lobe. The tissue samples were postfixed in 2% phosphate-buffered osmium tetroxide (pH 7.2), washed briefly in saline, dehydrated in graduated methanols, placed in a 1:l mixture of propylene oxide and Epon 812 overnight and embedded in Epon 812. Tissue samples were then placed in a 60°C oven to harden. The specimens were sectioned on a LKB III ultramicrotome and l-2-pm sections for photomicrotomy were stained with toludine blue 0, while the 80-nm sections for electron microscopy were stained with uranyl acetate followed by lead citrate. Specimens were examined on a Siemens 101 electron microscope equipped with an image intensifier (Siemens, Rosemont, IL). For biochemical assays, the right lung was perfused with ice-cold isotonic buffer through the right pulmonary artery. The lungs were then blotted dry and homogenized with 0.005 M phosphate buffer as previously described [ 351. Pulmonary superoxide dismutase activity [ 171, reduced glutathione level [9] and DNA content [ 271 were determined in the lung homogenate. Lung glutathione reductase [25] and glutathione peroxidase activity [23] were analyzed in the 15,000 X g lung supematant. Enzyme activities and GSH levels were expressed as units per mg DNA. Lung DNA content was calculated as mg per lung and expressed as % control mean. The paired t-test was .employed for statistical comparison of data [31]. A significance level of P < 0.05 was selected for rejection of the null hypothesis.
RESULTS
Both oxygen- and air-exposed newborn piglets were fed equal calories per body weight of concentrated Similac infant formula with iron during these studies. Paired littermates in both groups had a similar gain in body weight (average about 8% increase over the original body weight at day 2, 15% at day 4, and 13% at day 7 of exposure). A total of 28 piglets were used in
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these studies and all survived. Four oxygen-exposed piglets and their littermate air controls were sacrificed at 2, 4, or 7 days after starting exposure. The remaining 4 piglets were maintained in oxygen or air for a total of 10 days. Development of pulmonary edema in newborn piglets exposed to oxygen was monitored by both the lung wet weight to dry weight ratio and the lung wet weight to body weight ratio (Fig. 1). The lung wet weight to dry weight ratio (Fig. 1A) was found to be a more sensitive indicator of pulmonary edema, as edematous changes were detected using this method as early as 4 days after the start of oxygen exposure. Both lung wet weight to lung dry weight ratio and percent lung wet weight to body weight were significantly higher in the 7-day oxygen-exposed piglets as compared to their air controls. At autopsy, no pleural effusion was detected in any of the oxygen-exposed piglets. However, lungs from 4-, 7- or lo-day oxygen-exposed piglets were edematous, and showed patchy hemorrhagic areas. Lungs of all airexposed animals were found to be free of such abnormalities. Light and electron microscopy studies were performed on 4-, 7- and lo-day air- and oxygen-exposed piglets. Light micrographs of piglets exposed to air or oxygen are shown in Figure 2. The alveoli space of 7- and IO-day oxygenexposed piglets contained debris and a few mononuclear cells and macrophages, and the alveolar septa showed focal areas of thickening. Compared to controls, the lo-day oxygen-exposed animals showed evidence of an increased number of blood vessels with marked congestion and interstitial edema. The severity of these pathologic features was much less than that commonly reported in lungs of oxygen-exposed adult animals and newborn guinea pigs and mice [ 5, 8, 20, 241. Figures 3, 4 and 5 are electron micrographs of piglets exposed to air or oxygen. The normal appearance of lung cells in control piglets is shown in 4
024
B
710 Days
of Oxygen
Exposure
Fig. 1. Effect of oxygen on lung wet weight to dry weight ratio and % lung wet weight to body weight ratio in newborn piglets, Paired piglet littermates (l-day-old) were exposed to either air or 96-9896 oxygen for 2, 4, 7 or 10 days. Shown are lung wet weight to lung dry weight ratios (A) and % lung wet weight to body weight (B) or air (e) and oxygen-exposed (0) newborn piglets at various times after starting exposure. Values shown are mean f SE. Numbers in parentheses indicate the number of animals in each experimental group. * indicates significant difference from the air-exposed group (P < 0.05).
Fig. 2. Light micrographs of air- and oxygenexposed piglet lungs. Paired l-day-old piglet litter-mates were exposed to either air or 96-9896 oxygen. At different intervals after exposure, the animals were sacrificed and their lungs were fixed and stained as described in the Methods. Shown are light microscopic appearances of lung obtained at different intervals after exposure (176X ): 4 days after air (A) or oxygen (D) exposure; 7 days after air (B) or oxygen (E) exposure; 10 days after air (C) or oxygen (F) exposure.
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Fig. 3. Electron micrographs of lungs of control air-exposed piglets. Paired l-day-old piglets were exposed to either air or 96-98% oxygen. At different intervals after exposure, control air-exposed piglets were sacrificed and their lungs were fixed and stained for electron microscopic examination as described in Methods. A: 4-day airexposed piglet (6240~ ); B: 7-day air-exposed piglet (7800x ): C: lo-day air-exposed piglet (13,500x ). T, : ‘type I epithelial cell; T z : type II epithelial cell; lum: capillary lumen; as: alveolar space; rbc: red blood cell; lb: lamellar body.
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Fig. 4. Electron micrographs of lungs of 4- and 7-day oxygen-exposed piglets. One-dayold newborn piglets were exposed to 96-98% oxygen for 4 or 7 days. At the end of exposure, the animals were sacrificed and their lungs were fixed and stained for electron microscopic examination as described in Methods. A: C-day oxygenexpoeed piglet (10,000~ ). Edema is oherved in interstitium (*). Tubular myelin appears about to be released from a type II cell (arrow). B: ‘I-day oxygenexposed piglet (10,000~ ). Pulmonary edema is limited to perivascular and interstitial space (*). T, : type I epithelial cell; T, : type II epithelial cell; en: endothelial cell; lum: capillary lumen; as: alveolar space; rbc: red blood cell; lb: lamellar body.
Fig. 6. Electron micrographs of lungs of lo-day oxygenexposed piglets. One-day-old newborn piglets were exposed to 96-982 oxygen for a total of 10 days. At the end of exposure, the animals were sacrificed and their lungs were fixed and stained for electron microscopic examination as described in Methods. A: perivascular and interstitial edema (*) are extensive after 10 days of oxygen exposure. Increased numbers of mitochondria are seen in type I (T,) and type II (T,) epithelial cells (7000X ); B: type II cells appear to have smaller lamellar bodies (arrow) and an apparent increase in the number and size of mitochondria (m) after 10 days of oxygen exposure (19,200X). en: endothelial cell; lum: capillary lumen; as: alveolar space.
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Figure 3 and has been previously described [4, 61. Pulmonary edema in oxygen-exposed piglets was present as wide electron-lucent spaces and was limited to perivascular and interstitial spaces (Figs. 4 and 5). Pulmonary edema was evident at 4 days after starting oxygen exposure. The only observable alteration in endothelial cells was the swelling of mitochondria after 10 days of oxygen exposure. No changes were detected in type I cells except in lo-day oxygen-exposed piglets, where the type I cells appear to have greater numbers of mitochondria. No significant changes were detected in type II cells before 10 days of oxygen exposure. After exposure to 10 days of oxygen, type II cells appeared to show a decrease in the size of lamellar bodies and an increase in the number and size of mitochondria. Decreased lamellar body size and hypertrophy of mitochondria in type II cells have also been observed in adult rats exposed to sublethal concentrations of oxygen [16, 281. The effect of oxygen on lung DNA content in newborn piglets is shown in Figure 6. Pulmonary DNA content of oxygen-exposed piglets was 92 + 12% of controls at day 2, and significantly lower than air-exposed animals on day 4 of oxygen exposure (68 + 4% of control mean). However, 7-day oxygenexposed piglets consistently showed higher DNA content in their lungs as compared to the controls. Pulmonary DNA content in lo-day oxygenexposed piglets remained higher than that of the controls (124 + 6% control mean). Pulmonary antioxidant enzymes (SOD, GP and GR) showed a progressive increase in activity with duration of oxygen exposure, culminating in significantly higher SOD and GP activities in 7-day oxygen-exposed piglets (Fig. 7). Pulmonary activity of SOD (139 f 12% control mean), GP (241 f 50%) and GR (305 + 60%) in lo-day oxygen-exposed piglets remained above control values. Reduced glutathione levels were significantly increased throughout the oxygen exposure. 150 DNA
Days
.
of oxygen
Exposure
Fig. 6. Effect of oxygen on total lung DNA content in newborn piglets. Paired l-day-old piglet littermates were exposed to either air or 96-98% oxygen for 2, 4, 7 or 10 days. Pulmonary DNA content was assayed as described in Methods. Each bar respresents the % of control mean (k SE) of n = 3-4 for 2-, 4- or 7-day air- vs. oxygenexposed group and n = 2 for lo-day air- vs. oxygen-exposed group. * indicates significant difference from the air-exposed group (P < 0.05).
420
*
t
GSH O-OGR
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7 :,
‘\ ‘.
t4GP
T
7
10
300-
? z ,Q
5 f
200-
E :, 0 S loo-
1
0
I 2
4 Daysof
Oxygen Exposure
Fig. 7. Effect of oxygen on selected lung biochemical parameters in newborn piglets. Paired l-day-old piglet litter-mates were exposed to either air or 96-98% oxygen for 2,4, 7 or 10 days. Pulmonary superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GP) activities and reduced glutathione (GSH) levels were assayed as described in Methods. The various enzyme activities and GSH levels were expressed on the basis of per mg DNA. Each value represents the % control mean (* SE) of n = 3-4 for 2-, 4- or ‘I-day air- vs. oxygen-exposed group and n = 2 for lo-day air- vs. oxygenexposed group. * indicates significant difference from the air-exposed group (P < 0.06). (From R.J. Roberts, 1979, Journalof Pediatrics,Vol. 96, pp. 904-909.)
DISCUSSION
Based on the histological findings in this study, the newborn piglet appears to be less susceptible to oxygen-induced lung toxicity than most adult animals and newborn guinea pigs and mice. Marked histological derangement including atelectasis, pulmonary edema, inflammatory cellular exudation and hyaline membrane formation are observed in alveoli of adult animals [ 81, newborn guinea pigs [ 201 and newborn mice [ 5,241 after only 3-4 days of oxygen exposure. Newborn piglets showed a more mild degree of alveolar space debris and perivascular and interstitial edema but marked vascular congestion of the lung after 10 days of oxygen exposure (Figs. 2-5). Neonatal rats, which can survive longer than 5 days of oxygen exposure with minimal alveolar damage [36], are probably equally as sensitive to pulmonary oxygen toxicity as newborn piglets. All previous studies of prolonged (> 6 days) oxygen-induced lung injury in newborn animals were accomplished employing some form of maternal nourishment (suckling). It has therefore not been possible to exclude maternal-derived factor(s) as contributing to the relative tolerance of newborn animal species to oxygen-induced lung injury. The studies described herein were accomplished under conditions of artificial feeding. Although adult pigs were not examined with respect to their vulnerability to oxygen toxicity, it seems reasonable to conclude that the comparative resistance of neonatal pig lung to oxygen-induced injury relates to an intrinsic phenomena
421
rather than factor(s) acquired from maternal sources during suckling. Neonatal rats have repeatedly been shown to increase their pulmonary activities of SOD, CR and GP and levels of GSH in response to oxygen [2, 13, 32, 33, 361. Adult rats, most of whom succumbed within 3 days of oxygen exposure, do not show any significant increase in the activities of these enzymes and GSH levels in the lung as compared to the air-exposed adult animals [36] . In the present study, newborn piglets, who survived at least 10 days of oxygen exposure, were also observed to show an increase in the lung complement of SOD, GR, GP and GSH (Fig. 7). These results further support the antioxidant protective role of these enzymes and GSH in the lung [2,13,32,33,36]. Because little is known regarding the qualitative and quantitative aspects of the reactive molecular species which are substrates for the protective enzyme systems or their functional proximity to each other, the relative importance of the increases in enzyme activities cannot be determined at this time [34]. Additional endogenous protective systems have recently been reported which should also be considered in future studies [l] . The results showing an initial decrease of DNA levels at 4 days followed by increased levels at 7 days are consistent with the observations of Northway et al. [21] in experiments in newborn mice. The mechanism responsible for the apparent reversal of oxygen inhibition of DNA synthesis, which may be critically important to the mechanism of tolerance, remains to be identified. Although hyaline membranes were not observed in piglets exposed to prolonged oxygen, the use of piglets as an animal model for studying BPD has several potential advantages over newborns of other species. The present study shows that both air- and oxygen-exposed piglets can be successfully pair-fed on artificial diets by intragastric feeding. They gained equally in body weight and were apparently free of disease. The applicability of pairfeeding in newborn piglets not only eliminates the requirement of foster mothers, which sometimes complicates the design of experiments, but it also allows control of the effect of nutrition on the experimental outcome. The small size of newborn rats and mice also compromises their usefulness in complex multiparameter studies. In addition, measurement of the quantitative lung morphology and oxygen consumption in newborn animals shows that the lung of the piglet is very similar to that of the human [3]. Rendas et al. recently found that there was a common pattern of lung development in the pig and human, particularly regarding the vascular bed [26] . They concluded that the piglet was a suitable animal for studies of the pulmonary circulation. Although the lung of the newborn lamb, goat and monkey also resemble human, their small litter size complicates studies that require large numbers of animals. Finally, an inherited disease or ‘Barker’s’ syndrome which closely resembles HMD has been described in piglets [6] , which adds additional encouragement for the use of the piglet as an animal model to study BPD, It remains to be established whether or not HMD can be produced
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routinely in piglets by artificially induced premature delivery. Prematurely born rabbits [22, 291, lambs [30 ] and monkeys [18] spontaneously developed hyaline membranes without any experimental interventions, which is similar to the situation observed in premature human infants; Prematurity-associated deficiency in surfactant is generally regarded as the major predisposing factor for the development of hyaline membrane in infants with HMD [12]. Since prenatal development of surfactant in pig has been documented histologically by Baskerville [4], hyaline membranes can conceivably be induced in piglets which are ’delivered before the complete development of the surfactant system in their lung. Studies of the effect of oxygen on these prematurely born piglets may provide important clues to management of human BPD. The data obtained in the present study provide important background information for these studies. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Thomas K. Shires, Ph.D., for his thoughtful discussion and helpful comments on the histology and Mr. Jon P. Burke, B.S., for his skillful assistance in the preparation of the light and electron micrographs. John Yam was supported by a predoctoral fellowship from Procter and Gamble Company, Cincinnati, OH, This study was supported by USPHS Grants GM00141, GM07013, GM07069 and GM12675.
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