904

Workshop on B P D

The Journal of Pediatrics November 1979

number of primate centers in the country which should be able to provide this resource. DR. STAItLMAN: Keeping these animals alive will require support comparable to that given the human infant with respiratory distress, and this will require all the expertise of an intensive care nursery including oxygen and respirators. DR. TOOLEu But, apparently that is how we get BPD in the human infant! DR. BOAT: I am concerned that the bronchiolar system in the monkey is different than in the hmnan. Do they have the same number of generations of airways? I have the bias that changes in the bronchiolar system are important to the development of BPD. DR. HODSON: From the extensive modeling studies done by Dr. Boyden at our institution the monkey bronchiolar system appears to resemble the human quite closely. There are comparable numbers of airway generations. The major structural difference appears to be in the acinar unit, which is based on a single bronchiole rather than several bronchi01es as in the human. It is unclear whether thi,s would have any relevance to the development of BPD in the monkey, but I rather doubt it. DR. REID: It is important in discussing animal models that we not worry too much about how closely the animal lung resembles the human lung. The monkey is a remarkably good model of H M D and it should be used to study HMD and ge t that sorted out before moving on to BPD. It appears that we should be concerned with the !ung

damage occurring in the first hours of life and those factors contributing to early lesions. When you get around to more chronic models you have so many factors you will need to study other animals to examine different pathogenetic mechanisms of different agents such as oxygen toxicity in the premature animal. The important thing is the considerable body of knowledge about the monkey, so that if the normal development and pattern is understood, it is much easier to sort out deviations from normal. The most important thing is the degree of information obtained on the normal lung. All animal models will probably show structural differences from the human but I do not think that is critical. What is important is to assess damage, be it to bronchioles or alveoli, in contrast to the normal. I think the monkey provides an exciting model and that we should be able to state to the community that here is a critical model to work with. DR. HODSON: I agree. The monkey provides an excellent opportunity to study HMD. I am a bit uncertain at this stage whether it will be the ideal model for studying BPD. If H M D is a prerequisite to BPD, then there are precious few animal models which can fulfill that requirement. I also agree with Dr. Reid in that it will be important to study the reparative processes occurring in H M D and sort out the influences of oxygen and other variables which might adversely affect lung regeneration and repair. The monkey should prove an excellent animal to pursue these aspects.

Employment of pulmonary superoxide dismutase, catalase, and glutathione peroxidase activity as criteria for assessing suitable animal models for studies of bronchopulmonary dysptasia Robert J. Roberts, M.D., Ph.D., Iowa City, Iowa

A N UM n E R of investigations have been conducted in the past to identify a suitable animal model for studies of the pathogenesis of hyaline membrane disease? -~ Only a few animal species, the premature 6 and low-birth-weight 92~:. From the Divisions of Neonatology and Clinical Pharmacology, Department of Pediatf'ics, and The Toxicology Center, Department of Ph"hrmacology, University of Iowa College of Medicine. Reprint address: Department of Pediatrics, BSB, University of Iowa College of Medicine, Iowa City, IA 52242.

newborn rabbit, 7 premature lamb, 1 and piglet, ~ have actually been reported to spontaneously develop clinical and morph,ologic changes resembling HMD. The description of bronchopulmonary dysplasia by Abbreviations used HMD: hyaline membrane disease BPD: bronchopulmonary displasia SOD: superoxide dismutase Cat: catalase GP: glutathione peroxidase

0022-3476/79/110904+06500.60/0 9 1979 The C. V. Mosby Co.

Volume 95 Number 5, part 2

905

Models for BPD

B a s e l i n e L u n g A n t i o x i d a n t E n z y m e Levels in N e o n a t a l a n d A d u l t A n i m a l s of 5 S p e c i e s 7

6

14

oP o B o 9

R o

~

3

d

O M e o

9

C3

9 2

=

O Q.

9

~ .12 E .10

"~-

~. J.-.

R

o GP H

,~ rO

B

o

N :~ .08

E

6

9 Neonate

-~.

~ 10 s EL ~ 8 ~

o Adult

~ .14

M o

12

5 O ck 4

9

o 4

O

R O

~ 32.06

M

~ C~

9

'~ o

~

9 .0,4

H O

o

1

2

:~.02

;

9

9

i

0

0

ck

Fig. 1. Base-line lung antioxidant enzyme activity for SOD, CAT, and GP in adult and neonatal animals of five species (GP-guinea pig; H = hamster; M = mouse; B = rabbit; R = rat). Figure indicates maturational changes for individual enzymes in separate species. (From Frank L, Bucher JR, and Roberts R J: J Appl Physiol 45:699, 1979.) Northway et al 9 in infants with H M D who had been treated with high concentrations of oxygen and ventilatory support, together with the recognition that m a n y of the morphologic features o f oxygen-induced lung injury resemble those of BPD, ~~ 1, precipitated several investigations utilizing an oxygen-induced lung injury animal model to study the pathogenesis of BPD. 12-1" The major objective of these studies has been to characterize the morphologic a n d / o r functional lesions produced in the newborn mouse, guinea pig, or lamb lung by prolonged exposur e to oxygen. The results of the morphologic and functional studies provide substantial evidence favoring a major contributing role for oxygen in the development of BPD. Recently, we began studies to investigate the role of several protective enzyme systems, superoxide dismutase, catalase; and glutathione peroxidase, in the pathogenesis of H M D and BPD. These investigations have included an exploration of a biochemical approach to the evaluation of suitable animal models for BPD. Briefly, the protective enzyme systems SOD, Cat, and GP are thought to regulate or control within their cellular environment certain reactive molecular species. The complex reactions involved have been the subject of m a n y investigations and reviews. 17 Actual or relative deficiency of any one or all of these protective devices could

Table I. SOD activity in h u m a n lung and RBC*t

Group

Fetal (18-20 wk) (6) Premature (28-32 wk) Normal (5) HMD (5) Term (8) Infant 1-5 mo (4) BPD (3) Adult (5)

Lung units~gin hmg

Blood units/l~rnole heine

109 _+ 11

-

152 _+ 11 -152 + 32 192 _+ 70 182 + 19

56 53 60 79 78 93

_+ 1 _+ 8 + 5 _+ 1 _+ 5 _+ 3

*From Autor AP, Frank L, and Roberts R J: Pediatr Res 10:154, 1976. tValues represent means _+ SE derived from 3 to 8 samples (parentheses).

conceivably render critical cellular constituents vulnerable to injury, resulting in loss a n d / o r compromise of cell function or death. Two basic approaches have been employed in exploring suitable animal models for BPD. Both approaches are based on the assumptions that ( 1 ) o x y g e n is intimately involved in the critical events leading to the development of BPD and (2) that the protective enzyme systems (SOD, Cat, GP) are major determinants of the outcome of the disease and treatment processes. The initial studies were

906

Workshop on B P D

The Journal of Pediatrics November 1979

Relationship Between Lung Antioxidant Enzyme Changes And Survival Time in Hyperoxia N SOD --] CAT

to ,< E LU

...I

130%

Adult3d

120% 110%'

e.,-~.~ 1Adult 4-5d

Adult5-6d ~

-~ 1000 (,.1

.~

~

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~

80O/ol

I

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Adult5d Adult4-5d

N

el Neonate=,=,7d 99 m ~] Neonate>=.7d 99

140% to 130%

r E ~, IN

Neonate>=,7dI* 120% Neonate

110% Neonate

O r

4-5d

lO0% 90% 80%

IGu'neaP'g]

5d

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Fig. 2. Results represent percent change in activity compared to controls of the three protective enzymes (SOD, Cat, GP) in adults (upper) and neonates (lower) of five species exposed to >95% oxygen for 24 hc~'rs. The time (days) to 50% survival in hyperoxia (separate study) for adults and neonates of the five species is shown above the enzyme activity data. The asterisk indicates significant difference from control enzyme activity data or adult survival data (P < 0.05). (Adapted from Frank L, Bucher JR, and Roberts RJ: J Appl Physiol 45:699, 1979.)

designed to explore changes in enzyme activities as related to maturation-analogous to previous studies of the lung surfactant system. The results obtained in studies of neonatal and adult guinea pigs, hamsters, mice, rabbits, and rats are shown in Fig. 1. SOD and GP; activity was consistently less in newborn animals versus,that in adults. The same was true for Cat activity except f,or the guinea pig and rat, in which adult values were less than those for the neonate. Analysis of SOD activity in human lung tissue was done for purposes of comparison with the various animal species; the results are shown in Table I.

In the human lung tissue (and RBCs) there is a significant increase in SOD activity (per gram of lung or per mg D~A) "from fetal to adult lung. Data regarding lung Cat and GP activity have been very difficult to obtain because of problems with enzyme stabili!y in postmortem tissue and with blood contamination. An additional motivation for these studies was the exploration of the hypothesis that deficiencies in the protective enzyme systems may contribute to the development of HMD and/or BPD.I,,. 20 No gross differences were identified in SOD activity in lung tissue obtained from infants dying from

Volume 95 Number 5, part 2

Models for BPD

//:t

o---e GSH

3O0

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~: "~

A- -..A GP ~ SOD

~,

T

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I

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10

Days of O x y g e n Exposure Fig. 3. Effect of oxygen on selected lung protective systems in newborn piglets. Paired one-day-old piglet litter mates were exposed to either air or >95% oxygen for 2, 4, 7, or 10 days. Each value represents the % control mean (_+ SE) of N = 3-4 for 2, 4, or 7 days and N = 2 for ten-day groups. Asterisk (*) indicates significant difference from the air-exposed group (P < 0.05). GSH = Glutathione; GR = glutathione reductase; GP = glutathione peroxidase; SOD = superoxide dismutase. Table

II.

Lung

SOD

activity in p r e m a t u r e

animals

e x p o s e d to h y p e r o x i a *

Degree ofprematurity Species

(d~vs)

Survival of Control and E n d o t o x i n - T r e a t e d Adult Rats in H y p e r o x i a (95 + % 02 - 72 Hours) 80/80

Units~gin lung~( Control

24-hr h vpero.via

265/274

100 %

change

\\\xxxx k\\\%\\ ,X.NNNN ~\ \\\\\\\\\\\\x) ~xxx\\\ \x\\\\\

80' Rabbit Rat

28 20-2l

90 _+ 4 667 ~+ 42

128 _+ 19 929 + 62

+42 +39

*From Autor AP, Frank L, and Roberts RJ: Pediatr Res 10:154, 1976. ]'Values shown are means ( _+ SE) obtained from 4 to 8 animals which were randomly chosen from different litters and exposed to air or >95% O~ for 24 hours. Values in the hyperoxia groups are significantly different from the respective controls (P < 0.05). H M D or B P D versus values o f infants 1 to 5 m o n t h s o f age d y i n g f r o m n o n p u l m o n a r y

~x...

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20

causes. O b v i o u s l y , the

ideal c o n t r o l values are lacking. O n the o t h e r h a n d , c o n s i d e r i n g t h e fact that the i n f a n t s with H M D a n d B P D were e x p o s e d to o x y g e n , a p p r a i s a l o f the effect o f o x y g e n in n e w b o r n

,\.\\\\",~

a n i m a l s s e e m e d w a r r a n t e d . T h e effect o f

o x y g e n o n l u n g p r o t e c t i v e e n z y m e activity was, in fact, o u r s e c o n d a p p r o a c h to e x p l o r a t i o n o f a suitable a n i m a l m o d e l o f B P D . Fig. 2 s h o w s the results o f p r o l o n g e d c o n t i n o u s o x y g e n e x p o s u r e ( > 9 5 % ) on lung S O D , Cat, a n d G P activity in n e o n a t a l a n d a d u l t a n i m a l s o f five species. N e o n a t a l mice, rabbits, a n d rats all s h o w e d

Air Control

02 Control

02 + Endotoxin

Fig. 4. Alteration of oxygen-induced lethality in adult rats. Figure represents the percent survival after 72 hours of continuous exposure to >95% oxygen in the three treatment groups. Numbers indicate number of survivors/total number in group. Survival in the O~-control group was significantIy different from the other treatment groups (P < 0.0l). (Adapted from Frank L, Yam J, and Roberts RJ: J Clin Invest 61:269, 1977; Frank L, and Roberts RJ: J Appl Physiol [in press]; and Frank L, and Roberts RJ: Toxicol Appl Pharmacol [in press].)

908

Workshop on B P D

The Journal of Pediatrics November 1979

Table IlL Lung protective enzyme activities in control and endotoxin-treated adult rats exposed to > 9 5 % oxygen for 72 hours*t

Treatment

Air-control O._,-control O._, + endotoxin

SOD (u/lung)

Ghttathione peroxidase (Ixmol NA DPH oxidized ~rain~lung)

Catalase (IU/hmg)

842 +_ 48 8.89 • 0.45 1,917 • 79 884 _+ 50 10.32 +_ 0.78 2,277 • 65w 1.190 • 38:~ 11.32 • 0.44w 2,768 _+ 132~

*From Frank L, Yam J, and Roberts RJ: J Clin Invest 61:269, 1977 tEnzyme activities expressed on per lung basis (mean _+ SE) represent values determined from 6 to l I experimental animals for each group. IU for catalase activityis defined as the amount of enzymewhich reacts with 1 /mlol of H~O~per minute. ~Significantly different from both air-control and O~-control groups (e < 0,01). w different from air-control group (P < 0.0l).

increases in the protective enzymes and survived prolonged exposures to > 9 5 % oxygen. In the neonatal guinea pig and hamster no increase was observed in SOD activity, and lethality was similar to that in all the adult species, in which no increase in lung SOD activity occurred subsequent to hyperoxic exposure. These results support the impression that survival from hyperoxic exposure is consistently associated with a significant increase in SOD activity. Similar findings have recently been obtained in the neonatal piglet, which survives prolonged exposure to > 9 5 % oxygen exposure (Fig. 3). Studies in premature rabbits and rats exposed to > 9 5 % oxygen for 24 hours also resulted in increases in lung SOD activity (Table ll) similar to the studies in the term animals (Fig. 2). The effect of oxygen exposure on the protective enzyme systems in the h u m a n premature lung remains to be determined, but the unavailability of optimal controls (i.e., normal premature lung exposed to oxygen) may make the task difficult. In related investigations we found that very small doses of endotoxin resulted in protection of adult rats from oxygen-induced lung injury? ..... A summary of these studies is shown in Fig. 4. Histologic examination revealed that endotoxin pretreatment provided essentially complete protection of the adult rat from the effects of three or seven days of hyperoxic exposure (.>25%) including the chronic lung changes manifest follgwing recovery in room air for 31 days? ~ 23 This protective effect of endotoxin was associated with a significant increase in the activity of lung protective enzyme systems (Table III). These observations have significant implications in the

premature infant, in w h o m gram-negative infections are known to occur concurrently with H M D . Studies are in progress to examine the relationship between the development of BPD and evidence for preceding culture positive infections in infants with H M D . In summary, assessment of the lung protective enzyme systems (SOD, Cat, GP) may be a valuable tool to explore the appropriateness of various animal species as models for studies of BPD. Studies of the protective pulmonary enzyme systems may also provide insights into the pathogenesis of BPD. Critical data remain to be gathered regarding the response of the human infant lung protective enzyme system to oxygen exposure. The data presented represent the dedicated elIbrts of Dr. John Yam, Dr. Lee Frank, and Mr. John Bucher in the fulfillment of the requirements for the Ph.D. in Pharmacology accomplished in the author's laboratory. REFERENCES 1. Stahlman M, LeQuire VS, Young WC, Merrill RE, Birmingham RT, Payne GA, and Gray J: Pathophysiology of respiratory distress in newborn lambs, Am J Dis Child 108:375, 1964. 2. McAdams AJ, Coen R, Kleinman LI, Tsang R, and Sutherland J: Experirnental production of hyaline membranes in premature Rhesus monkeys, Am J Pathol 70:277, 1973. 3. Kikkawa Y, Motoyama EK, and Gluck L: Study of the lungs of fetal and newborn rabbits, Am J Pathol 52:177, 1968. 4 Berezin A: Histochemical study of the hyaline membrane of newborn infants and of that produced in guinea pigs. Biol Neonat 14:90, 1969. 5. Reynolds EOR, Jacobson HN, Motoyama EK, Kikkawa Y, Craig JM, Orzalesi MM, and Cook CD: The effect of immaturity and prenatal asphyxia on the lungs and pulmonary function of newborn lambs. The experimental production of respiratory distress, Pediatrics 35:382, 1965. 6. Ogawa J, and Yamaguchi H: Hyaline membrane in the lungs of premature newborn mammals, Nagoya Med J 7:17, 1961. 7. Shanklin DR, and Beerman PA: An experimental model for hyaline membrane disease, Biol Neonate 6:340, 1964. 8. Bradley R, and Wrathall AE: Barker (neonatal respiratory distress) syndrome inthe pig: The ultrastructural pathology of the lung, J Pathol 122:145, 1977. 9. Northway WH Jr, Rosan RC, and Porter DY: Pulmonary disease following respiratory therapy of hyaline membrane disease: Bronchopulmgnary dysplasia, N Engl J Med 276:357, 19fi7. 10. Hellstrom B, and Nergardh A: The effect of high oxygen concentrations and hypotherrnia on the lung of the newborn mouse, Acta Paediatr Scand 54:457, 1965. 11. Clark JM, and Lambertsen C J: Pulmonary oxygen toxicity: A review, PharmacolRev 23:37, 1971. 12. Northway WH, Rosan RC, Shahinian L Jr, Castellino RA, Gyepes MT, and Durbridge T: Radiological and histological investigation of pulmonary oxygen toxicity in newborn guinea pigs, Invest Radiol 4:148, 1969.

Volume 95 Number 5, part 2 13. deLemos R, Walfsdorf J, Nachman R, Block AJ, Leiby G, Wilkinson HA, Allen T, Haller JA, Morgan W, and Avery ME: Lung injury from oxygen in lambs: The role of artificial ventilation, Anesthesiology 30:609, 1969. 14. Ludwin SK, Northway WH Jr, and Bensch KG: Oxygen toxicity in the newborn. Necrotizing bronchiolitis in mice exposed to 100 percent oxygen, Lab Invest 31:425, 1974. 15. Bieber MM, Cogan MG, Durbridge TC, and Rosan RC: Oxygen toxicity in the newborn guinea pig lung, Biol Neonatl 17:35, 1971. 16. Bonikos DS, Bensch KG, and Northway WH Jr.: Oxygen toxicity in the newborn. The effect of chronic continuous 100 percent oxygen exposure in the lungs of newborn mice, Am J Pathol 85:623, 1976. 17. Pryor WA, editor: Free radicals in biology, vol I, New York, 1976, Academic Press, Inc. 18. Frank L, Bucher JR, and Roberts RJ: Oxygen toxicity in neonatal and adult animals of various species, J Appl Physiol 45:699, 1979.

Mode&Jbr BPD

909

19. Autor AP, Frank L, and Roberts RJ: Developmental characteristics of pulmonary superoxide dismutase: Relationship to idiopathic respiratory distress syndrome, Pediatr Res 10:154, 1976. 20. Frank L, Autor AP, and Roberts RJ: Oxygen therapy and hyaline membrane disease. The effect of hyperoxia on pulmonary superoxide dismutase activity and the mediating role of plasma on serum, J PEDIATR90:105, 1977. 21. Frank L, Yam J, and Roberts R J: The role of endotoxin in protection of adult rats from oxygen-induced lung toxicity, J Clin Invest 61:269, 1977. 22. Frank L, and Roberts RJ: Endotoxin protection against oxygen-induced acute and chronic lung injury, J Appl Physiol (in press). 23. Frank L, and Roberts RJ: Oxygen Toxicity: Protection of the lung by bacterial lipopolysaccharide (Endotoxin), Toxicol Appl Pharmacol (in press).

Models for BPD." Discussion Dr. Alan Hodson, Moderator

DR. STERN: Do the effects in the premature animals result from 95% oxygen? DR. ROBERTS: We believe that oxygen is the stimulus. In more recent work the responses (biochemical and morphologic) appear to vary quantitatively and qualitatively depending upon the concentration of oxygen (20 to 100%) and the duration of exposure (one to 12 days). DR. STERN: It is a linear response? DR. ROBERTS: The SOD appears to be linear during the first 24 hours of exposure to 95% oxygen. DR. STERN: How does endotoxin exert its effects? DR, ROBERTS: We have not identified the mechanism but investigations are continuing. DR. STAHLMAN: What endotoxin did you use? DR. ROBERTS: We have used several different endotoxin preparations (Escherichia coli 011/:84 and 26:86, Salmonella typhimurium, Shigella,flexneri) and all appear effective against oxygen-induced lung injury in the rat. DR. NELSON: Was the endotoxin given before the oxygen exposure? DR. ROBERTS: It can be given as late as 24 hours after the onset of oxygen exposure and still show some protective effect, but it is much more effective if given at the time the animals is put in oxygen. We have done a 24-hour pre-exposure and this also has a protective effect.

DR. STERN: Does endotoxin stimulate SOD in the lung as well as in the white cells? DR. ROBERTS: Yes. There is a significant increase in SOD activity with endotoxin alone either in vivo or in vitro. Oxygen exposure is required in addition to endotoxin treatment. It poses an interesting question as to whether endotoxin producing infections in premature infants with H M D can influence the lung response to oxygen (i.e., development of BPD). DR. THURLBECr~: What do you know about the lung structure of the newborn hamster? The mouse, the rat, and the rabbit have completely nonalveolated lungs at birth. The guinea pig, on the other hand, has relatively mature lungs at birth. W h a t is known about the hamster lung? DR. ROBERTS: We have not done any developmental morphology work in the hamster. DR. NELSON: Was it classic serendipity that led you to use endotoxin? DR. ROBERIS: Yes. We were in the process of following up our observation that a serum factor is necessary for the increase in SOD enzyme activity in vitro with oxygen exposure. (Serum from infants with hyaline m e m b r a n e disease and bronchopulmonary dysplasia appears to lack the factor that is required for an increase in SOD activity in vitro.) We were experimenting with plasmanate as a

0022-3476/79/110909+02500.20/0 9 1979 The C. V. Mosby Co.