Ir. J. Med. Sc, Sixth Series No, 485 pp. 195-201, ~ay, 1966

EXPERIMENTAL RESPIRATORY DISTRESS IN NEWBORN RABBITS

(FAILURE TO PRODUCE PULMONARY HYALINE MEMBRANES)

By BRIAN ~cNICHOLL and JOHN D. KENNEDY

From tl~e Departments of Paediatrics a?~d Pathology, University College, Galway.

M ANY of the attempts to reproduce the patho~ogic'al picture of pulmonary hyaline membrane were revie~ ed b)'T De and Anderson (1953); these included .such methods as intratracheal injection of

foreign substances including liquor amnii, cervic,~l vagotomy, poisoning with oxygen or carbon-dioxide, asphyxia in nitrogen and in the in,tact amniotic sac. ,Most o~ these studies were on adult animals, including ra'bbit~, guinea pigs, rats, mice and dogs. De and Anderson (1954) pro- duced hyaline membranes in asso,e.iation with ateleetasis in newborn guinea pigs by oxygen ,poisoning with and without cervical vagotomy, and stressed that. most previous reports of artefactual membranes d~d not describe associated a~electasis such as occurs in the human newborn. Snyder (1958) descri'bed some membrane for~nation and .atelecC~asis in new, born rabbits whose mothers had been heavily sedated during labour. C/aireaux produced some hyaline membrane in rats with hyalinised material from human liquor. Little mention is made by most of thcs'e authors of whether a respiratory distress syndrome (R.D.S.) similar to that in the human, was produced. Stahlman et al. (1963) described R.D.S. and hyaline membraaes in newborn lambs delivered by Caesare,a12 section, the duration of gestation not being stated; Davis (1964) also described R.D.S. in newborn rabbits whose mothers had been kept in I0 per cent oxygen for short periods, and in rabbits who were delivered into saline. Of the several methods described here, we found partiaI asphyx.ia in the amniatie sac .at Caesarean section and severe hypoxia in utero most suce,essful in producing R.D.S. and severe pulmonary patho- logy similar to th'at seen in the ne~born infant. No completely typical hyaline membranes were produced. Bilatera'] cervi.cal vagotomy, p,arti- cularly when combined with oxygen poisoning, produced the mos~ marl~ed pulmonary ,pathology. ~Sincc this work was completed, the produetior~ of some hyaline membrane in adult and newborn rabbits has bee~ described by Milley an,d C'asarett (1965) by bilateral vagotomy.

Methods: At autopsy, the trachea was tied before the thorax was opened; the lungs were fixed in formol saline, whole-lung sections being stained with hae~natoxylin and eosin, and with periodic-acid Schiff (P.A.S.). Autopsies were carried out within minutes of death in most cases, oth'crwise following some hours, usually with refrigeration. Caesarean section .was carried out under open drop ether anaesthesia. The newborn animals were observed periodically, respiratory (and occasionally cardiac) rates being recorded. The gestation period of the

195

196 IRISH JOURNAL OF MEDICAL SCIENCE

does varied from 29 to 33, average 31 days. Except in the intra-uterine hypoxia group, one animal in each litter was used as a control, being killed fr(~n 24 to 48 hours after birth.

Repeated Intra-tracheal Injection of Foreign Liq~dds Human liquor amnii was used in 7 animals, 4 born by section at 28

days, 3 born naturally at term: rabbit liquor, rabbit serum and rabbit (eitrated) blood were used in 2, 2 and 4 animals respectively, al.ternatcly born by section at 28 days or naturally at term.

Each animal had from 3 to 5 pereutaneous intratraeheal injections of the foreign liquid, amounts, varying from 0.2 to 0.5 ml; starting .within 24 hours of birt.h. Temporary distress, related to the amount of liquid, occurred after each injection, but the animals alapear(~ to be relatively little affected, surviving for from 20 to 70 hours. Where much liquid had been injected, large areas of lung showed partial alveolar collapse, the alveoli containing the injected liquid and the tissues being congest'ed and oedematous; where lesser amounts had been injected, smaller areas of liquid-filled alveoli were seen. The. injected liquid was lying mostly in the centres o~ the alveoli and alveolar ducts, only occasionally forming a membrane around the periphery of the air spaces.

Bilateral Cervical Vogotomy: 18 animals, born naturally, at term, from 5 litters, had bilateral vagotomy at the level of the lower border of the larynx wilhin 24 hours of birth, under local anaesthesia with xylo-

:FIG. 1.--Bilateral cervical vago tomy with oxygen poisoning; resorbtion ate |ectasis with dilated alveolar ducts .

E X P E R I M E N T A L R E S P I R A T O R Y D I S T R E S S I N N E W B O R N RABBITS 197

caine. A'll showed considerable initial distress with inspi ra tory retrac- tion and laryngeal str idor; all the animals except 2 died 1 to 20 hours a f te r vagotomy, mean 8 hours: 2 animals we.re quite vigorous when killed 13 and 18 hours af ter vagotomy. All lungs except 2 showed consider- able pathology, hypervaseular i ty and oedema being widespread, with some alveolar haemorrhage in the most affected areas. Resorbtion atelectasis occurred to a considerab]'e but variable extent. An interesting finding was the localisation of the most severe changes to the upper �88 to �89 of the lungs, which were often mostly " hepatic " on macroscopic and microscopic examina.tion. Traces of eosinophilic membranes were present in the affected areas but were thin and less opaque than the human variety.

Bilateral Cervical Vagotomy with Oxygen Poisoning: 7 animals, born

Fro. 2.--Oxygen poisoning; death in room air ; moderate congestion, occasional resorbtio~ atelectasis, ruptured alveolar ducts.

na tu ra l ly at term, f rom 2 litters, were vagotomised as above but then kept in oxygen (over 90 per cent at atmo~.pheric pre~sure) until death or killing. Two died 16 and 30 hours af ter vagotomy, the remainder being killed a f t e r 22 to 41 hours, their survival thus being prolonged in a high oxygen environment as compared to the group left in air a f te r vagotomy. All lungs showed more severc changes than those of the " vagotomy without oxygen " grou, p, ttre uppe r .~ to �89 of the lungs being plum eoloured and solid. Sections showed marked congestion with f requent haemorrhages, extensive resorbtion atelectasis with di!ated alveolar duets, the uppe r port ions of the lungs being " hepatic "

198 IRISH JOURNAL OF MEDICAL SCIENCE

(Fig. 1). All lungs showed traces of hyaline membranes, these being nearest to those seen in the human but not typical. Two of the animals dying in oxygen, showed almost completely solid lungs (v. i.).

Oxygen Paisoning: 13 animals born naturally at term to 5 does, were kept in oxygen over 90 per cent at ~tmospheric pressure, 3 dying 13 to 62 hours 1.ater, the re~min,der being killed after 23 to 50 hours. The animals showed few or no signs of distress. Seven were kept in room air for between 5 and 10 minutes before being killed; their lungs showed moderate congestion and occasion'a] resorbtion atelectasis (Fig. 2). Six killed and left in oxygen showed almost completely solid lungs that part ly sank in formol saline; sections showed almost no patent alveoli or alveolar ducts (Fig. 3), this being the complete resorbtion atelectasis as ,described by Ahvenainen (1948). No hyaline membranes were seen. When an animal dies with respiratory arrest in 90 per cent to 100 per cent oxygen and the heart continues to beat for som'e minutes longer, all or most of the oxygen is removed from the air spaces, t~he ~bsenee of any significant amounts of nitrogen or carbon-dioxide allowing almost complete collapse. Although the term " resorbtion ateleetasis " pre- sumably refers to absorption of air from .previously expanded air- spaces by a related mechanism, one wonders if the oxygen level in the environmen,t of infants .dying of respiratory failure is always taken into account when assessing the degree of atelectasis at autopsy, since high alveolar oxygen tensions may be produced in an incubator or by posi- tive pressure resuscitation with oxygen via an endotracheal tube.

FIr . 3.--Oxygen poisoning ; death in oxygen "hepatic" lung, almost no "'a~r spaces".

~Xlt'ERIMENTAL RESPIRATORY DISTRESS IN NEW-BORN RABBITS 199

Asphyxia in the Intact Amniotic Sac: Caesarean section was performed at 28 days in ,two does and at 30 days in two others the uterus being opened under saline at 37 ~ C; the animals were left in the amniotic sac from 5 to 20 minutes a f te r each p'l~acenta had been separated from the uterus, most of the animals making some respira tory movement in the sacs. ~Most of the 14 animals showed initial mild R.D.S. deaths occur- r ing ~t from 2 to 48 hours af ter birth, other animals being killed at intervening periods. Sections of animals dying soon af ter bir th showed mostly " intra-uterine " lung as seen in the stillborn animal, but with some areas of as,phyxial dilatation of alveolar duct~ and of resorbtion atel'ectasis. Animals living for longer .periods showed much resorbtion ateleetasis, most of the air spaces being distended alveolar ducts; occasional areas of " h e p a t i c " lung were also present. )5oderate amounts of amniotic debris were seen in some lungs, part icularly of those dying within a few hours of birth.

Hypoxia iu utero: 7 does were kep~t in a low oxygen environment for periods of up to 28 hours, between the 27th and 28th days of gestation, Caesarea,~ ~ct ion being performed on the 28th day under open drop ether anasthesia ,(~McNicholl, 1965). The original aim was to keep the doe in 9 per cent to 11 per cent oxygen for 12 hours or more before bii~h, but it was found that if the oxygen level fell below 9 per cent for more than a short period, survival of the l i t ter was unlikely; 4 l i t ters did not survive birth. 'Three does gave live litters, the average oxygen level for t~vo of them for 12 hours before bir th being 11 per cent and 12 per cent approximately; in the third doe (No. 25), the oxygen level was between 9 per cent and 10.5 per cent for 12 hours and the l i t ter was removed post-mortem, owing to the death of the doe under anaesthesia. The 29 animals in these three li t ters were somewhat chilled af ter bir th and breathed poorly at fir'.~t, many needing resuscitation .with positive airway oxygen; 7 of the an4mals did not respond to resuscitation. All animals sho.wed some initial R.D.S. ,with retr~c.tion and were kept in 45 per cent oxygen for about 2 hours af ter birth, then being kept in warm room air until deas or killing, with the exeeption of 5 w~hieh were kept in 45 per cent oxygen throughout. Animals were killed from 6 to 47 hours af ter birth, little R.D.S. being evident a f t e r about 9 hours of age. Four animals died between 1 and 10 hours af ter birth, and it is of interest that they were all f rom li t ter No. 25 deli~e.red post-mortem; 3 had been kept in room air af ter the initial 2 hours in 45 per cent oxygen, the four th dying at 1 hour in oxygen; 4 of their l i t ter mates, kept in 45 per cent oxygen throughout, appeared almost normal when killed 27 to 47 hours af ter birth, suggesting a beneficial affect of oxygen in prolonging survival.

S t r ik ing ,pulmonary pathology was present in all the animals, very fe.w alveoli being expanded; the major i ty of the air spaces were distended alveo'lar ducts, some of which were over-distended or rup tured ; " hepat'ic " segments of lung were seen f requent ly (Fig. 4). Traces of thin eosinophilic membranes were occasionally seen but these were not typical of those seen in the infant. Very. little amniotic debris was seen; patchy infective changes were found in the lungs of 2 animals. The general pulmonary histology was very like that o f " severe ateleetasis without hyaline membrane " as seen in the human newborn. Foetal

200 IRISH JOURNAL OF MEDICAL SCIENCE

:FIG. 4 . - - H y p o x i a in utero; " H e p a t i c " s e g m e n t s a l t e rna t i ng wi th d i la ted alveolar duc t s , a l m o s t no r~ormal a lveolar d i l a t a t ion .

hypoxia followed by Caesarean section as described seems to be a con- sistent way of producing this pulmonary picture in the ra'bbit.

Discussi(~n Measures which have bccn stated ~o produce pulmonary hyaline

membranes in adult rabbits, and in other animals both adul:t and newborn, do not appear to do so in the newborn rabbit, in our hands. This is rather surprising since severe pu].monary pathology, similar to that seen as~)ciated with hyaline membranes in premature infants, was produced in these rabbits. The two methods nearest to the clinical situations that usually prevail in the human disease, aypoxia in utero and in the amniotic sac, with birth by section at 28 days, produced some R.D.S. and severe ateleetatic changes in the lungs but did not produce typical hyaline membrane. It may be argued that 28 days, corresponding roughly to 36 weeks in the human, was not sufficiently premature, but our impression was that not many animals would have survived birth by section had they been more premature. Bilateral cervical vagotomy at laryngeal level includes section of the recurrent laryngeal nerves, probably accounting for the inspiratory stridor and retraction and probably contributing to the severe pulmonary change~. The fact that severe rosorbtion atelectasis can be pl~duced with some consistency by hypoxia in utero may be useful as an experimental model.

EXPERIMENTAL RESPIRATORY DISTRESS IN NEWBORN RABBITS 201

S ll ~D~lctry

AttempLs at production of pulmonary hyaline membranes in full term and prema:turely born rabbits were unsuece~uful; methods in- eluded intratraeheal injection of human and rabbit liquor amnii, rabbit blood and serum; oxygen poisoning; bilateral cervical vagotomy ~,ith and without subsc~luent oxygen poisoning; hypoxia in the intact amniotic sac during Caesarean section, and hypoxia in utei~ by means of maternal hypoxia.

Moderate respirator)" distress and severe resorbtion atelectasis fol- lowed hypoxia in the amniotic sac and in utero and could serve as an experimental model. Severe pulmonary pathology was a l~ produced by other methods.

The level of oxygen in an infant's environment at the time of death should be taken into account by the pathologist in assessing the degree of "ateleetasis " at autopsy since respiratory failure in high oxygen mixtures may result in marked alveolar collapse.

Acknowledgements

This work was largely suppor ted by a grant to one of us (B. MEN.) by the Medical Research Council of Ireland. We wish to t h a n k Professor W. J . E. Jes sop for his suppor t , Professor J . Donegan for his advice a t various s tages arid successive hospi ta l res iden ts and s t ude n t s for the i r assis tance.

References

Ahvenainen , E. K., On changes ir~ di la ta t ion and signs of asp i la t ion ir~ foetal and neonata l lur.gs, Acta Paediatrica, Supp.3, Vot. 35, 1948.

Claireaux, A. E., Hyal ine m e m b r a n e in t he neonatal lurLg, Lancet, 2 : 749, 1953. Davis J . , Abs t rac t s of 10~h In te rna t iona l Paediat r ic Congress, Lisbon, 1962. De, Tran-Dinh, and Anderson, G.W., Hyaline-like m e m b r a n e s associa ted wi th disease

of t he newborn lungs : a review of the l i terature, Obstct. and Gyn. Survey, 8 : 1, 1953.

De, Tran-Dinh, and Anderson, G. W., The exper imenta l p roduc t ion of pu lmonary hyaline-like membranes wi th atelec~asis, Am. J. Obstet. and Gyn. 68. : 1557, 1954.

Milley, 1 ). S., and Casarett , L. J . , Bilateral vago tomy and hyalit~e membrane formation, Pediatrics, 36, 173, 1965.

Snyder , F , F., Pu lmonary hyal ine m e m b r a n e ; eontamiimtiorx o f the lungs by fluid of t he b i r th canal ; s tudies in rabbi ts , Obstetrics and Gynaecology. 2 : 599, 1958.

S tah lman, M. T., le Quire, V. S., Young, W. C., Marrill, R. E. and Payne , G. A., The pa thophys io logy of resp i ra tory dis t ress it1 newbort t lambs, Jour. Pcdiat. (Abstracts) 63 : (Par t 2), 757, 1963.

McNicholl B., Survival of rabbi t foetus i~x mate rna l hypoxia , Irish J. Med. Sc. March, 147, 1965.

Rc~.eived for publ icat ion 4 J anua ry , 1966.