Daniels, Eskandari-Marandi, Nicholas - 1993 - The Role of Surfactant in the Static Lung Mechanics of...

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Respiration Physiology,94 (1993) 11-2 3 11Q 1993 E lsev ie r Sc ience Pub l i she rs B .V. Al l r igh ts r e se rved . 0034-5687 /93 / 06 .00

R E S P 0 20 61

he role of surfactant in the stat ic lung mechanics ofthe lizard ten ophor u s n u chal i s

C h r i s t o p h e r B . D a n i e l s, B o r i s D . E s k a n d a r i - M a r a n d i a n dTe r e n c e E . N i c h o l a s

Department of Hum an Physiology, School of Medicine, Th e Flinders University of South Australia, Adelaide,Australia

(Ac cep te d 17 M ay 1993)

A b s t r a c t W e prev ious ly sho we d tha t the lung o f the cen t ra l Aus t ra l i an l i za rd ,Ctenophorus n uchalis,c o n t a i n sa l arg e a m o u n t o f s u r f a c ta n t , th e c o m p o s i t i o n o f w h i c h v a r ie s w i t h b o d y t e m p e r a t u r e . W e n o w s h o w t h a tt h e s p e c i fi c c o m p l i a n c e o f t h e l u n gs o f t h e s e l iz a r d s r e m a i n s c o n s t a n t r e g a r d l e s s o f w h e t h e r t h e y w e r ema in ta ined a t 10 , 18, 27 , 37 o r 43 °C fo r 4 hours . In con t ras t , the open ing p ressure was con s tan t up to 27 °C ,b u t d e c r e a s e d a t 3 7 a n d 4 3 ° C . W h e n w e l a v a g e d th e l u n g sin situto r em ove the m ajor i ty o f su r fac tan t , spec i fi cc o m p l i a n c e d e c r e a s e d w h i le o p e n i n g p r e s s u r e i n c r e a s e d . T h e l u n g s o fC. nuchalisare essen t i a l ly two bubb les ,wi th the l e f t one l a rge r a t low an d in te rm edia te vo lum es . Af te r co l l aps ing bo th lungs , the l a rge r l e f t lung a lwaysin f la ted f ir s t. H ow ever, fo l lowing l avage the smal le r r igh t lung in f l a ted f ir s t. A s the l a rge r lung , whe n co l l apsed ,would ha ve a m uch g rea te r a rea o f ep i the l ia l con tac t , th i s r e su l t is cons i s t e n t wi th su r fac tan t ac t ing as an' an t ig lue ' . Dur ing de f la t ion the sm al le r lung co l l apsed f ir s t, cons i s t en t wi th the l aw of Lap lace . Com pl ianced id no t chan ge in the sa l ine - f il l ed lung sugges t ing tha t the gas - l iqu id in te r face does no t p lay a m ajor ro le .W e con c lude tha t in the lungs o f these l i za rds , su r fac tan t i s ac ting as an an t ig lue . Th i s m igh t be im por ta n tdur ing pe r iods o f apne a a t low body t em pera tu res , w hen res idua l vo lume i s smal l and ep i the l ia l su r faces ma y

c o m e i n to c o n t a c t .

Compl iance , su r fac tan t ; Mechan ics , compl iance , su r fac tan t ; Rep t i l e s , l i za rdCtenophorus nu chalis);Sur fac -tant , lung, ant iglue

The m ajor funct ion of pulm onary surfactant in mam ma ls i s thought to be in prom ot-ing alveolar stabil ity. Ac cordin g to the law of Y oun g and Lap lace, the pressu re in thebubble i s d i rec t ly propor t ional to the surface tens ion and inverse ly propor t ional to theradius . Each lung ofCtenophorus nuchal i sis unicameral with an extremely large airspace in the centre Danie lset al . 1989). U nicam eral lungs are extremely com pliantbec ause of both smal l absolute am ounts of t i ssue and the rela tively large propo r t ionof elastic tissue in the wa lls of the respiratory u nits faveoli). Th e co m pliance of iso-lated unica m eral lungs is determine d primarily by two force s. First , the overall rela-t ionship of the lung to i ts air sp ace, w hich is essentially balloon-l ike. Thus , lung press ure

Correspondence to:D r. C . B . D a n i e l s D e p t . o f H u m a n P h y s i o lo g y, F l i n d e r s M e d i c a l C e n t r e , B e d f o r d P a r k ,Sou th Aus t ra l i a 5042.



12

is r e la t ed t o t he fo rces desc r ibed by L ap lace . A secon d and sma l l e r i n f luence on lungcom pl i ance a r e fo rces i nvo lved in t he expan s ion and con t r ac t ion o f t he be l lows - l ikefaveo li . Such fo rces i nc lude the e l a st ic r eco i l o f t he f aveo la r wa l l and su r f ace t ens ion

of t he hy pop hase , t he l a tt e r e spec ia l l y i n r eg ions w he re a d j acen t ep i the li a l su r f aces maycom e in to con tac t . I n 1957, C lem en t s w ork ing wi th lung ex t r ac ts on the su r f ace ba l-ance , p rov ided an exp lana t ion a s t o how su r f ac t an t migh t s t ab i l i s e a lveo l i . Ex t r apo -l a ti ng to t he l ung: a s an a lveo lus con t r ac t s , t he m ono m olec u la r l aye r o f su r f ac t an t a tt he gas - li qu id i n t e r f ace is com pres s ed , p rog re s s ive ly squeez ing ou t d i f fe r en t com po -nen t s and f i na lly l eav ing a fi lm o f nea r ly pu re d ipa lm i toy lpho spha t idy l cho l ine (D PP C ) ,t h e p r in c i p a l c o m p o n e n t o f s u rf a c ta n t . A t 3 7 ° C , D P P C is in t h e ge l p h a s e , h e n c e t h em ono laye r p robab ly r es i st s any fu r the r com pres s ion , a t wh ich po in t the su r f ace ten -s i o n a p p r o a c h e s z e r o .

A l though th i s hypo thes i s has been t e s t ed i n t he mod i f i ed Wi lhe lmy ba l ance and theb u b b l e s u r f a c t o m e t e r, a n d m o r e r e c e n tl y b y a c a p t iv e b u b b l e m e t h o d ( S c h i ir c he t a l .

1989), i t has been diff icul t to testin v ivo .H o w e v e r , B a c h o f e ne t a l . (1987) successfu l lyused a m ic rod ro p le t me th od to mo n i to r d i r ec t l y t he su r f ace t ens ion in a lveo l i o f i so -l a te d p e r f u s e d r a b b it l u n gs . T h e y f o u n d t h a t t h e s u r f a c e te n s i o n v a r i ed f r o m 3 0 m N / ma t t o t a l l u n g c a p a c i t y ( T L C ) to a b o u t 1 m N / m a t 4 0 ° ; T L C .

W e have r ecen t ly fou nd tha t t he cen t r a l Aus t r a l i an l i z a rd ,C t e n o p h o r u s n u c h a l i sh a sfaveo li wh ich a r e 10 to 100 t imes l a rge r t han m am ma l i an a lveo li , wh i l e p re sen t ing on ly1 °/o of the to ta l sur face a rea (D anie lse t a l . 1989). Th is l i zard has tw o ba l loo n- l ike lungs

o f unequa l s ize . The m a jo r i t y o f t he r e sp i r a to ry po r t i on o f t he l ung is l oca t ed in t hean te r io r t h i rd (Dan ie l se t a l . 1989) , which , in v ivo a p p e a r s h e l d o p e n b y c o n n e c t i v et i ssue be tw een the p l eu ra l su r f ace an d the i n s ide o f t he ches t w a ll . Dur ing the non -ven t i la tory per iods the lungs hav e a la rge res idua l vo lum e, wi th the le f t lung con ta in-i n g m o r e a i r. F u n c t i o n a l r e s i d u a l v o l u m e d e c r e a s e s w i th d e c r e a s i n g b o d y t e m p e r a t u r e(F rapp e l l and D an ie l s , 1991b) . Th e lungs o fC . n u c h a l i scon ta in s even ty t imes mores u r f a c t a n t- t y p e p h o s p h o l i p i d s p e r r e s p i r a t o r y s u rf a c e a r ea t h a n d o t h o s e o f a c o m p a -r a b l e s i z e d m a m m a l ( D a n i e l se t a l . 1 9 8 9 ) . F u r t h e r m o r e , t h e c h o l e s t e r o l / D P P C r a t i oo f t he a lveo la r su r f ac t an t i nc reases ve ry s ign i fi can t ly a s bo dy t em pera tu re is l owered(Dan ie l s e t a l . 1990) . How th i s a f f ec t s l ung compl i ance o r open ing p re s su re i s un -k n o w n .

Th e d i spa ra t e s i ze s o f t he l ungC . n u c h a l i s t he i r ba l loon - l ike s t ruc tu re , t he p re senc eo f la rge amo un t s o f ea si ly l avageab le su fac t an t and the f ac t t ha t r e s idua l vo lume i s sos m a ll a t l o w b o d y t e m p e r a t u r e s , a p p e a r e d t o p r o v i d e a r e a d y m o d e l w i t h w h i c h t o t e s tt he e ff ec t o f su r f ac t an t , bo th on the fo rces desc r ibed by Youn g and Lap lace and a s aposs ib l e an t ig lue , p r even t ing adhe s ion o f ad j acen t ep i the l ia l li n ings . The re fo re , wehave f i r s t examined the i n f l a t i on /de f l a t i on cha rac t e r i s t i c s and open ing p re s su re s o flungs bo th i n s i t u and i so l a t ed , bo th be fo re and a f t e r l avage . Second we have inves t i -ga t ed the ro l e o f a lveo la r su r f ac t an t i n the spec if ic com pl i ance and open ing p re s su re so f l ungs o f l i z a rds ma in t a ined a t d i f fe r en t bod y t em pera tu re s .



13

a t e r i a l s a n d m e t h o d s

C. nuchal is 10 to 25 g ) were co l l ec t ed f rom cen t r a l Au s t r a l ia a nd hou sed a s p rev ious ly

d e s c r i b e d D a n i e l set a l . 1989 , 1990) . L i za rds were anaes the t i zed wi th me thohex i tonesod iu m 20 mg /kg in t r ape r i tonea l ly, E l i L i ll y, Sydney) , a f t e r f ir s t be ing m a in t a ined a tv a r i o u s t e m p e r a t u r e s f o r 4 h . T h e m e a n p r e f e r re d b o d y t e m p e r a t u r e o fC. nuchal is is3 7 ° C , w h e r e a s 2 7 ° C is th e a p p r o x i m a t e s u m m e r m o r n i n g e m e rg e n c e t e m p e r a t u r ereco rde d in the fi eld He a tw o le , 1970).

W e co nd uc te d 2 t ypes o f expe r imen t s . F i r s t we exam ined in t he i so l a ted l ung thee f fe c t o f re m o v i n g s u r f a c ta n t o n t h e v o l u m e o f a ir a n d c o r r e s p o n d i n g p r e s s u r e r e q u i re dto open each lung . Second we examined the i n f l a t i on and de f l a t i on cha rac t e r i s t i c s o fthe two lungs bo th be fo re and a f t e r l avage , bo thin situ a n d in vitro. The lungs in vitro

we re r emov ed f rom the an ima l and suspe nded ve r t ica l ly i n 100~o sa tu ra t ed a ir.

Opening pressure . I n o r d e r t o m e a s u r e o p e n i n g p r e ss u r e s d e f in e d a s t h e p re s s u r erequ i red to in i t ia lly inf la te a fu l ly co l lap sed lung dur ing con t inua l in fus ion of a i r a t1 ml /min ) , and in fus ion vo lume s , a tr achea l ca the t e r was i n se r t ed and con nec t ed to aS t a t h a m p r e s s u r e tr a n s d u c e r m o d e l P 2 3 D J c , H a t o R e y, P R ) a n d t h e n t o a p r e a m -p li fi er m o d e l 7 P I E G r a s s I n s t ru m e n t s , Q u i n c y M A ) a n d t o a O m n i s c r ib e c h a r t re -c o r d e r m o d e l D 5 0 0 0 ; H o u s t o n I n s t ru m e n t s , A u s t in T X ) . I s o l a t e d l u ng s w e r e m a i n -t a ined in a humid if i ed cham ber. T he lungs were gen t ly eva cua t e d to a back p re s su re

o f - 4 c m H 2 0 w h i c h r e m o v e d a l l a ir b u t d i d n o t d a m a g e t h e i n te r n a l a rc h i te c t u re .P re s su re was t hen equ i l i b r a t ed wi th a tmosphe r i c and a i r was i n fused in to t he t r achea lca the t e r v i a a sy ringe pum p mo de l 341B, Sage , Cam br idge , U .K . ) a t 1 ml /min . Th epres su re sp ike i nd i ca t ed the open ing o f t he lungs F ig . 1 ). Op en ing p re s su re s w eremeasu red in t r i p l i ca t e and the mean t aken .

Compliance. F o l l o w i n g th e m e a s u r e m e n t o f o p e n i n g p r e s s u r e , t h e l un g s w e r e a g a ine v a c u a t e d t o - 4 c m H 2 0 a n d s in g le s te p in f u s io n s o f 0 .4 m l w e r e c o m m e n c e d a n dcon t inued un t i l T L C was r eached . Ea ch in fus ion was fo l lowed by a 30 sec equ i li b ra -

t io n p e r i o d b e f o r e t h e p r e s s u r e re a d i n g s w e r e ta k e n . T L C w a s t a k e n a s th e v o l u m er e a c h e d w h e n t h e r e w a s a g r e at e r th a n 1 c m H 2 0 i n c re a s e in p r e s s u r e p e r 0 .4 m lin fus ion o f a ir. Th i s avo ided da mag ing the ve ry compl i an t , t h in -wa l l ed l ungs . Th e lungwas then d e f l a ted i n t he s am e de c rem en t s a nd the r ead ings t a ken a f t e r a 30 sec equ il i-b ra t i on . De f l a t i on con t inued un t i l e i t he r ze ro p re s su re was r eached o r t he en t i r e vo l -u m e o f a ir in f u s e d w a s w i t h d r a w n . L u n g c o m p l i a n c e w a s m e a s u r e d t w i c e a n d c o m -pa red a t 40 and 60~o TLC.

Th e vo lum e in t he r i gh t and l e ft l ung was de t e rm ined in a s ing le ana es thes i zed l iz -a r d b y c o m p u t e r i s e d a x ia l t o m o g r a p h y C T - s c a n , G E 9 8 0 0 , G e n e r a l E l e c tr ic , U S A ) .X - r a y p h o t o g r a p h s w e r e t a k e n a t re a l b o d y s i z e a t 3 m m d i s ta n c e s f r o m t h e m o s tan t e r io r t o the m os t d i s t a l end o f t he lungs . On each X - ray the t r ansve r se a r ea o f eachlung i s de l inea t ed and the t r ace i n t eg ra t ed fo r a r ea u s ing a d ig i t i ze r. To ta l vo lume i st h e n c a l c u l a t e d b y m u l t ip l yi n g t h e a r e a o f e a c h l u n g o n e a c h X - r a y b y t h e n u m b e r o f



14

4

I-

2

o~

4 -

2 21 22 23

T i m e ( s e c )

Fig . I . The r aw t r ace o f t he in f l a tion o f un lavaged lungsin situ L u n g s w e re d e f l a te d t o - 4 c m H 2 0 a n d t h e ne q u i l ib r a t e d t o a t m o s p h e r i c p r e s s u r e b e f o r e a n i n fu s i o n o f a ir w a s c o m m e n c e d a t 1 m l / m i n . T h e t i m e isr e c o r d e d o n t h e a b s c i s s a a n d t h e p r e s s u r e i s r e c o r d e d o n t h e o r d i n a t e . T h e s u d d e n f a ll i n p r e s s u r e i n d i c a t e s

t h a t a l u n g h a s o p e n e d . T h e a r r o w i n d i c a t e s t h e o p e n i n g o f t h e s e c o n d l u ng .

X - r a y p h o t o g r a p h s a n d t h e n b y t h r e e . T h e p r e s s u r e w a s m e a s u r e d a s p r e v i o u s l y d e -

s c r i b e d . T h e e n t i r e o p e n i n g p r e s s u r e , l u n g c o m p l i a n c e m e a s u r i n g s y s t e m w a s p e r f o r m e d

i n a v e r y l a r g e c o n s t a n t t e m p e r a t u r e c a b i n e t p r e s e t t o t h e b o d y t e m p e r a t u r e o f t h e

a n i m a l .

TA B L E 1

S i z e a n d m a s s o f l i z a rd s u s e d i n t h e d i f fe r e n t e x p e r i m e n t s .

Te m p e r a t u r e N u m b e r M a s s S n o u t - v en t l en g th Ta b l e s( ° C ) ( g ) ( m m )

27 5 19.0_+2.83 82 .8+ 1.11 2,337 5 14.7 + 3.15 82.2 + 4.48 2,3

10 5 15.9_+ 1,22 82 .2 + 1.32 618 5 14 .5+0 .73 81 .2 1 .32 627 10 18.7 + 2,70 85.5 _+ 3.81 637 16 14.9 + 2.41 82.1 +_3.53 643 5 11.8 + 0.7 2 80.2_+ 1.24 6

37 5 26 .9+ 4.1 3" 103.0_+ 10.54 4

A l l r e s u lt s a r e e x p r e s s e d a s m e a n i S E M .* P < 0 .05 : s ign i fi can t d i f f e rence be tw een th i s g roup and o the r g roups by ana lys i s o f va r iance .



15

In all cases the lungs were lavaged with three volume s of ice-cold 0.15 M NaC1

(0.2 mg/g body weight), each volume being instilled and withdrawn three times. Ice-

cold saline inactivates lavaged enzymes and prevents com posit io nal changes after

extraction of the surfactant. This proce dure re moved m ore th an 90 ~o of the surfac tantthat could be removed by using 9 such volumes (Daniels e t a l . 1989).

Lungs were also evacuated to a pressure of -4 cmH20 immersed in a large wide-

mou the d beak er filled with 0.15 M (20 o C) NaC1 and then infused with 0.9~o saline and

P/V curves constructed in the manner described above (see the method of Perry and

Duncker, 1978).The dat a was analyzed by a one way analysis of variance, Student 's t- tests and paired

t-tests. All dat a are expressed as me an + SEM .

e s u l t s

There were no differences in mass or snou t-ve nt length betwe en an y of the respective

test an d contr ol groups (Table 1). However , there were some mi nor differences in body

size between the groups used in the different experiments.

TABLE 2

Opening pressure and specific compliance of C. nuchal i slungs measured sequentially nitially n si tu thenisolated before and after lavage.

Opening Pressure Pressurepressure at 60% TLC at 40% TLC

Inflation Deflation Deflation

Tempera tu re : 27 ° CLung in s i tu

Air-filled

Iso la ted lung

Air-filledLavaged

Saline-filled

4.21 ± 0.54*

5.00±0.145.76 + 1.00

2.04 ± 0.36* 1.30 ± 0.14 0.78 ± 0.31

0.95 ± 0.20* 0.67 ± 0.13 0.46 ± 0.07*1.32 ± 0.69 0.57 ± 0.29 0.33 + 0.381.70 ± 0.40 1.17 ± 0.74* 0.74 ± 0.28*

Tempera tu re : 37 ° CLung in s i tu

Air-filled 2.78 ± 0.52* 2.19 + 0.34 1.20 + 0.27* 0.72 ± 0.08

Iso la ted lungAir-filled 5.18 + 0.65 1.61 + 0.27 0.75 +_ 0.20 0.92 ± 0.47Lavaged 5.10 + 0.30 1.08 ± 0.27* 0.59 + 0.22* 0.58 ± 0.31

Results are expressed in cmH20 as mean ± SEM, number of lizards at each temperature = 5. Lizards weremaintained at temperature for 4h prior to the experiment. * Significant difference (P<0.05) between thalgroup and one or more of the other groups by analysis of variance and paired t-test. TLC is total lung ca-pacity.



16

Opening pressure. W h e n a ir w a s i n f u s e d a t 1 m l / m i n i n t o a n e v a c u a t e d p a i r o f l u n g s,e i the r in situ o r in vitro t h e p r e s s u r e i n c r e a s e d u n t i l i t w a s s u f f ic i e n t t o i n fl a te t h ec o n d u c t i n g a i r w a y s a n d o n e lu n g. T h e p r e s s u re t h e n d e c r e a s e d . T h e s e c o n d l u n g

o p e n e d a v e r y s h o r t t i m e l a t e r ( F i g . 1 ) .I n c r e a s i n g t h e b o d y t e m p e r a t u r e f r o m 2 7 to 3 7 ° C f o r 4 h d e c r e a s e d t h e o p e n i n g

p r e s s u r e o f t h e l u n gin situ ( Ta b l e 2 ). L a v a g e i n c r e a s e d t h e p r e s s u r e r e q u i r e d t o o p e nt h e l u n g in situ ( Ta b l e 3 ). I s o l a t i n g t h e l u n g i t s e lf a l s o i n c r e a s e d t h e o p e n i n g p r e s s u r e( Ta b l e 2 ); l a v a g i n g t h e n h a d n o f u r t h e r e f f ec t. H o w e v e r , w h e r e a s i t w a s t h e l a rg e le ftl u n g w h i c h o p e n e d f ir st in t h e i s o l a t e d l u n g, f o l l o w i n g l av a g e , it w a s t h e r i g h t lu n g w h i c ho p e n e d f i r st (F i g . 2 , Ta b l e 4 ). T h i s o r d e r o f o p e n i n g a n d t h e s u b s e q u e n t r e v e r s a l a f te rl a v a g e w a s a p p a r e n t a t b o t h 2 7 a n d 3 7 ° C ( Ta b l e 4) . T h e o p e n i n g p r e s s u r e r e s u l t s a r es u m m a r i z e d i n F i g . 3 .

A l t h o u g h m a i n t a i n in g t h e li z a r d s' b o d y t e m p e r a t u r e s a b o v e 2 7 ° C d e c r e a s e d t h eo p e n i n g p r e s s u r e o f t h e l u n gin situ ( Ta b l e s 2 a n d 5 ), o p e n i n g p r e s s u r e w a s u n a l t e r e da t t e m p e r a t u r e s b e t w e e n 1 0 a n d 2 7 ° C ( Ta b l e 5).

Lun g compliance. T h e r e w a s n o c h a n g e i n t h e c o m p l i a n c e o f th e l u ngin situ a t e i the r4 0 o r 6 0 ~ , o f to t a l l u n g c a p a c i t y r e g a r d l e s s o f w h e t h e r t h e l i z a r d h a d b e e n m a i n t a i n e da t 1 0, 1 8 , 2 7 , 3 7 o r 4 3 ° C f o r 4 h ( Ta b l e 5 ) . H o w e v e r, f o l l o w i n g l u n g l a v a g e o f l i z a r d sw h i c h h a d b e e n m a i n t a i n e d a t 3 7 ° C f o r 4 h o u r s , t h e r e w a s a s i gn i f ic a n t d e c r e a s e i ns p e c i f i c c o m p l i a n c e u p o n d e f l a t i o nin situ a t b o t h 4 0 a n d 6 0 ~ o o f T L C ( Ta b l e 3 ). T h i sw a s a l s o c l e a r ly r e f le c t e d in t h e o n e l i z a r d w e s u b j e c t e d t o C T- s c a n s b e f o r e a n d a f te rl a v a g e ( F i g . 4 ; Ta b l e 6 ). L a v a g i n g t h e i s o l a t e d l u n g h a d n o e f fe c t o n c o m p l i a n c e( Ta b l e 2 ). T h e s p e c if i c c o m p l i a n c e w a s n o t a l t e r e d b y b o d y t e m p e r a t u r e , w h e t h e r t h el u n g w a s # zsitu o r in vitro ( Ta b l e s 2 a n d 5 ) ( s u m m a r i z e d i n F i g. 5).

R e g a r d l e s s o f w h e t h e r t h e l un g w a sin situ o r i s o la t e d , o r w h e t h e r i t w a s l a v a g e d o rn o t , c o m p l i a n c e w a s a l w a y s g r e a t e r d u r i n g d e f l a t i o n t h a n d e f l a ti o n . F i l li n g t h e l u n g w i t hs a li n e ( 2 0 ° C ) g r e a t ly r e d u c e d t h is h y s t e r e s is , b u t i t a ls o s l ig h tl y d e c r e a s e d t h e c o m -p l i a n c e o f t h e l u n g ( T a b l e 2 , F i g . 5) .

TABLE 3

Effect of lavage on the initial opening pressure and specific com pliance of lungsin situ at 37°C.

Opening Pressure at 60% TLC Pressure at 40' 0 TLCpressure

Inflation Deflation Deflation

Before lavage 3.04 + 0.67 2.39 + 0.24 1.03 + 0.07 0.60 + 0.12

Afte r lavag e 4.06 + 0.69* 2.38 + 0.12 1.42 + 0.12* 1.05 + 0.10"

Results expressed in cm H2 0 as m ean + SEM. Num ber of lizards = 5. Lizards were maintained at 37°C for4h p rior to the experiment. * Significant difference (P < 0.05) using a paired t-test. TLC is total lung capacity.



7

B E F O R EL V G E

F T E RL V G E

I N F L AT I O Nm l

4 m l

7 m l

Fig. 2. Isolated lungs were deflated to -4 cmH /O and t hen equilibrated with air before they were inflatedin step s up to a volum e of 7 ml and then deflated. On ly 1, 4 and 7 ml volum es during inflation are show n,

i s c u s s i o n

T h e p r in c i p a l t e c hn i c a l p r o b l e m i n m e a s u r i n g c o m p l i a n c e o f C .n u c h a l i s l ungs i s the ve rylow in f la t i on p r e s su re s i nvo lved ; t he se p r e s su re s a r e le s s t han 5~o o f t hos e i n a co m -p a r a b l e s i ze d m a m m a l . T h e r e a r e a l s o m a r k e d d i ff e re n c e s i n t h e i n fl a ti o n c h a r a c t e ri s t ic so f t h e f r a gi le li z a r d l un g . W h e r e a s t h e a l v e o li o f t h e m a m m a l i a n l u n g c o m p r i s e s b y f a rt h e m a j o r i t y o f t h e l un g v o l u m e , t h e f a v e o l a r v o l u m e o fC . n u c h a l i s c o m p r i s e s o n l y av e r y s m a l l p e r c e n t a g e D a n i e l se t a l . 1989) o f t o t a l l ung vo lum e . Th i s r e l a t i onsh ip i sc h a r a c t e r i s ti c o f u n i c a m e r a l l u n g s P e r r ye t a l . 1 98 9). H e n c e th e m e a s u r e d c o m p l i a n c e



18

TABLE 4

Effect of lavage on the opening pre ssu rc of the right and left isolated lungs at 27 and 37 : C.

Temperature

~c)Before lavage After lavage

Infusion Opening Order of Infusion Opcningvolume pressure opening volume pressureat lung at lungopening opening

Order ofopening

Left Lung (larger)27 0.3 ± 0.02 5.0 ± 0.14 1st 2.5 _+ 0.44 - 2nd37 0. 3+ 0. 05 5.2+_0.64 1st 2.2+_0.74 - 2nd

Right Lung (smaller)

27 1.4 _+ 0.28 - 2nd 0.4 _+ 0.03 5.7 ± 0.64 1st37 1.9+0.69 - 2nd 0.320 .03 5.1+0 .30 1st

Results expressed as mean ± SEM, Number of lizards at each temperature = 5. Infusion volume exprcssedin ml. Pressure expressed in cmH20. Lizards were maintained at the body temperature for 4h prior to theexperiment.

p r o b a b l y r e fl ec t s p r e d o m i n a n t l y t h e d i s t en s i b il it y o f t h e o u t e r s u r f a ce o f t h e l u n g s a n dt r a b e c u l a e r a t h er t h a n t h a t o f t h e w a l ls o f t h e f a v e o li . F u r t h e r m o r e , t h e s u r fa c e t e n s i o na t t h e r e l a t i v e l y s m a l l f a v e o l a r g a s - l i q u i d i n t e r f a c e w o u l d p r o b a b l y p l a y a v e r y m i n o r

r o l e i n d e t e r m i n i n g o v e r a l l c o m p l i a n c e . C e r t a i n l y t h i s w o u l d b e c o n s i s t e n t w i t h o u rp r e s e n t f i n d i n g t h a t c o m p l i a n c e i s n o t i n c r e a s e d b y f i l l i n g t h e i s o l a t e d l u n g w i t h s a l i n e .I n fa c t , t h i s fi ll in g w i t h s a l i n e t e n d s t o d e c r e a s e t h e c o m p l i a n c e , w h i c h i s s o r a d i c a l lyd i ff e re n t f r o m t h e c l a s s ic f in d i n g s o f v o n N e e rg a a r d 1 9 2 9 ) i n c a t lu n g s , t h a t w e w e r ei n i ti a ll y c o n v i n c e d t h a t w e w e r e d e a l i n g w i t h a n ar t if a c t. H o w e v e r , n o a r t i fa c t w a sa p p a r e n t a n d t h e s a m e f i n d in g w a s r e p o r t e d b y P e r ry a n d D u n c k e r 1 9 7 8 ) .

TABLE 5

Relationship between body temperature and the opening pressure and specific compliance of the lungs ins i t u

Temper ature N Opening Pressure at 60 TLC Pressure at 40°o TLC(° C) pressure

Inflation Deflation Inflation

10 5 4.42 _+ 0.37 2.13 _+ 0.27 0.88 _+ 0.14 0.50 + 0.0718 5 4.27 + 0.32 2.12 + 0.12 1.05 + 0.09 0.58 + 0.1227 10 4.55+0 .41 2.07_+0.11 1.31_+0.09 0.80_+0.0737 15 3.02 ± 0.25* 2.23 ± 0.09 1.19 ± 0.03 0.67 ± 0.0543 5 2.81 ± 0.05* 1.87 _+ 0.16 0.96 ± 0.11 0.54 _+ 0.05

Results expresse d in cm H2 0 as mean _+ SEM. N, number of lizards. TLC, total lung capacity. Lizards weremaint ained at the temperatu re for 4h prior to the experiment. *, significantly different P< 0.05, using n lysisof variance and a Stu dent's t-test.



9

¢N

E

.o

ww

1.

6 -

solated Lung

Lavaged Lungin Thorax solated

Lavagod Lung

Lung in Thorax

III

I

I

0 i I0 1

Vo l u m e m l )

nfused a t 1 m l / m i n

F i g . 3 . S u m m a r y o f th e m e a n o p e n i n g p r e s s u r e s o f l u n g sn s t u or i so l a t ed be fo re and a f t e r l avage , a f t e rd e g a s s i n g u n d e r b a c k p r e s s u r e o f - 4 c m H 2 0 , a n d s u b s e q u e n t i n fu s i o n o f a i r a t 1 m l / m i n .

How then can we explain our finding that lavage significantly depressed lung com-pliance in situ but not in vitro? Possibly the lung in vitro tends to collapse primarilybecause of the retractile nature of the lung tissue. This tendency in situ may be coun-teracted by the extensive connective tissue between its outer surface and the inside ofthe chest wail McGregor et al. 1993). Hence lavaging in situ lungs appears to reveal

the small contribution to compliance of surface tension at the gas-liquid interface. Thesmaller lung deflates faster than the larger as predicted by the law of Young andLaplace, but lavage did not exacerbate this differential deflation. Higher pressures wererequired to inflate initially in situ lungs after lavage and static pressures were also higheron deflation which suggests that surfactant is reducing surface tension. Hence whilesurfactant may not be varying surface tension with volume in the same manner asmammalian surfactant, its contribution to the overall lung mechanics in partially in-flated in situ lungs is small, and negligible in isolated lungs.

The degree of hysteresis exhibited by the pressure-volume curves was greater withthe lung in situ than in vitro suggesting that forces involving the chest wall must beovercome before there are changes in lung volume Perry and Duncker, 1978). The factthat saline filling greatly reduced this hysteresis is consistent with its being primarilya function of the gas-liquid interface and possibly influenced by surfactant. However,



2

B E F O R EL V G E

F T E RL V G E

I N F L T I O N

D E F L T I O N

Fig. 4. A tracheal catheter was inserted and different volumesof air injected while the lung was viewed undera CT -scan. Only the effects of instilling and withdrawing 0.5 m l is illustrated, both pre - and post-lavage.

aga in , the fac t tha t sa l ine fil ling ha d l it tl e e ffec t on c om pl ia nc e sugg es t s tha t the gas -l i qu id i n t e r f ace mus t p l ay on ly a m ino r r o l e . Th i s obse rva t i on con f i rms t he ea r l i e rf in d i ng s o f P e r r y a n d D u n c k e r 1 9 7 8) .

W e h a v e f o u n d t h a t l o w b o d y t e m p e r a t u r e s i n d u c e d p e r i o d s o f a p n e a i nC . n u c h a l i s

d u r i n g w h i c h th e l u n g s c o l l a p s e a n d t h e e p it h e li a l su r f a c e s m a y c o m e in c o n t a c t F r a p -p e ll a n d D a n i e l s , 1 9 9 1 a ,b ; M c G r e g o re t a l . 1 99 3; C . B . D a n i e l s , L . K . M c G r e g o r a n dT. E . N i c h o l a s , u n p u b l i s h e d ) . W e h a v e h y p o t h e s i z e d t h a t s u r f a c t a n t in li z a r d lu n g s m a yac t a s an an ti g lue t o p r ev en t ep i the l i a l su r f ace s f r om a dhe r ing i n t h is s i t ua t i on Da n i e l se t a l . 1 99 0; M c G r e g o re t a l . 1993). I f th i s hyp oth es i s i s t rue i t should be m or e d i ff i-cu l t t o op en t he l a rge r l ung t han t he sm a l l e r l ung a f t e r l avage becau se o f t he l a rg e re p it h el ia l s u r f a c e s in c o n t a c t . O u r m e t h o d f o r m e a s u r i n g o p e n i n g p r e s s u r e i n v o l v e sco l l ap s ing bo th l ungs t o a cons t an t nega t i ve back p r e s su re . I n fu s ing a i r un t i l one l u ngp o p s o p e n , r e s u lt s i n a p r e s s u r e e q u a l t o t h e s u m o f t h e p r e s s u r e s r e q u i re d t o o p e n a n y



TA B L E 6

Co m pliance of individual lungs of an anaesthe tized l izard determined by CT -scan .

2

Volum e infused Pressure Volum e of lungm l ) c m H 2 0 )

Left large) Right small)

BeforelavageIn f la t ion

1.0 1,2 0.53 4 0.3632.0 1 .5 1 .119 0 .9485 .0 4 .2 2 .385 2 .214

eflation5 .0 4 .2 2 .385 2 .214

2.0 0 .55 1 .101 0 .9631.0 0 .22 0 .474 0 .405

f ter avageInflation

1.0 1.65 0.58 2 0.69 32.0 2 .42 0 .984 1 .0625 .0 7 .50 2 .535 2 .889

Def la t ion5 .0 7 .50 2 .535 2 .8892.0 1.20 0.97 5 1.1221.0 0 .80 0 .589 0 .597

This experiment wa s performed on one l izard of body m ass23 g.

collapsed airways and then op en the easiest lung. The seco nd lung opened a short t imelater. H enc e the order o f lung opening reflects the lung wh ich has the lowe st openingpressure. We found that the lowest opening pressure existed in the large lung beforelavage and the small lung after lavage. This is consistent with an antiglue function forpulmonary surfactant. The order of opening of the isolated lungs and the subsequent

reversal after lavage wer e apparent at both 27 and 37 °C .Th e increase in bod y temperature decre ased the op ening pressure of the lungi n s i t u .

Th is probably reflects an increase in fluidity of the surfactant Da nielse t a l . 1990).Alternatively the higher temperatures may result in an increase in blood perfusionkeeping the faveoli op en by an erectile effect. There were no chan ges in o penin g pressurebe low 27 ° C, suggesting that the surfactant has a significant insensitivity to co ld. W ehave p reviously sho wn that within 2 h o f reducing the bo dy temperature o f initiallywarm 37 °C) C . n u c h a l i s to 19 ° C there wa s an increase in the cholesterol /phosph olipidratio in lavage material Da niels e t a l . 1990). This increased cholesterol content mayprovide th e thermal insensitivity b y lowering th e ph ase transition temperature o f theph osph olipid m ixtures. Alternatively the lung tissue itself ma y be insen sitive to tem-perature. Clements 197 6) reported that the elasticity o f rat lungs filled wit h saline isonly slightly temperature dependent.



22

T L C - -

G

Em

0>

- - I s o l a t e d L u n g - -

. ui i i t

I I I Ia l •

1 / /I/ ?t t

t•

- - I so la t ed Lavaged Lung m

~; ; ; , ,~ , ,~* - - Sahne

/ - - L u n g in T h o r a x - -

T .

/ II1

II

h S J

F i l l e d L u n g - -

t L a v a g e d L u n g ~in T h o r a x

1 I6

P r e s s u r e cm H20~

Fig. 5. A sum mary of the changes in the pressure-volum e curves both before and after lavage of the lungin the thorax and then isolated. A c urv e is a lso included of the saline-filled isolated lung.

In conclusion w e s u g g e s t t h a t t h e m a i n r o l e f o r s u r f a c t a n t i n t h e l u n g s o fC. nuchalis

i s a s a n a n t i gl u e . T h i s f u n c t i o n m i g h t b e v e r y i m p o r t a n t d u r i n g p e r i o d s o f a p n e a a t l o wb o d y t e m p e r a t u r e s w h e n r e s i d u a l v o l u m e i s g r e a t l y r e d u c e d a n d e p i th e li a l s u r f a c e sw i t h i n t h e l u n g c o m e i n t o c o n t a c t . T h i s a n t i g lu e f u n c t i o n m i g h t a l s o b e i m p o r t a n t i nt h e a ir w a y s . C e r t a i n ly Vo n N e e r g a a r d 1 9 2 9 ) w o u l d h a v e c o m e t o a v e r y d i ff e re n tc o n c l u s i o n a s t o th e r o l e o f s u r f a c e t e n s i o n i n lu n g s t a b il it y i f h e h a d p e r f o r m e d h i se x p e r i m e n t s o nC. nuchalis r a t h e r t h a n m a m m a l s . O u r f in d in g s re v e al m a r k e d d if fe r-e n c e s i n t h e r o l e o f s u r f a c t a n t - t y p e l i p i d s in t h e l u n g s o fC. nuchalis f r o m t h a t i nm a m m a l i a n l u n g s .



3

AcknowledgementsWe are g rate fu l to M r. Ed mu nd A rozoo and the D epar tm ent o f Rad io logy for pe rfo rmingthe CT-scanning . L iza rds were co l lec ted under the Nor thern Ter r i to ry Conserva t ion Commiss ion Permi t

A/89 /19 gran ted to C .B. Danie l s . The p ro jec t was funded by the Aus t ra l i an Research Counc i l and theNat iona l H ea l th and Medica l Research C ounc i l o f Aus t ra l ia .

e f e r e n c e s

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Clem ents , J .A . (1957). Surface tension of lung extracts .Proc. Soc. E xp. Biol. Med.95: 170-172.Clem ents , J .A. (1976). Fu nct io ns of the alveolar lining.Am. Rev. Resp. Dis.115: 67-71 .Danie l s , C .B. , H .A. Bar r and T.E . Nicho las (1989). A com par i son of the sur fac tan t assoc ia ted l ip ids de -

r ived from rept i l ian and mammalian lungs.Respir. Physiol.75: 335-348.

Danie l s , C .B. , H .A. Bar r, J .H .T. Power and T.E . Nicho las (1990) . Body tempera tu re a l t e r s the l ip idcompo s i t ion o f pu lmonary sur fac tan t in the l i za rdCtenophorus nuchalis. Exp. Lung Res.16: 435-449.

Frappel l , P.B. and C.B. Dan iels (1991a). V ent i la t ion and oxygen con sum ption in agamid l izards .Physiol.Zool. 64: 985-1001.

Frappel l , P.B. and C.B. Daniels (1991b) . Temperature effects on vent i la t ion and metabol ism in the l izardCtenophorus nuchalis. Respir. Physiol.86: 257-270.

Heatwo le, H. (1970). T herm al ecology o f the deser t dragonArnphibolurus inermis Ecol. M onogr.40: 425-453,McG regor, L .K . , C .B. Danie l s and T. E . N icho las (1993). Lung u l t ras t ruc tu re and sur factan t -l ike sys tem

of the cen t ra l ne t t ed d ragon ,Ctenophorus Nuchalis. Copeia.1993: 320-333.Perry, S.F. and H.R. Duncker (1978) . Lung archi tecture , volume and s ta t ic mechanics in f ive species of

lizards. Respir. Physiol.34: 61-81.

Per ry, S .F. , A .M . Bauer, A .P. Russe l l, J .T. A ls ton and J .E . Maloney (1989). Lu ngs o f the geckoRhacodactylus leachianus(Rept i l ia : Gekk onidae) : a correlative gross ana tom ical and l ight and electron micro-scopic study.J. Morph. 199: 23-40 .

Scht i rch, S., H. Bachofen, J . Go erke and F . Poss ma yer (1989). A cap t ive bubble metho d reprod uces thein situbehav iour o f lung su r fac tan t monolayers .J. Appl. Physiol.67: 2389-2396.

Von Neergaard , K . (1929) . N eue A uffassungen t iber e inen Grundb egr i ff de r Atem mechanik . Die R e t rak t ions -Kraf t de r Lunge, abhangig v o n d er Ober f l~chenspannun g in den Alveo len .Z. Ges . Exp. M ed.66:373-394 .

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