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The effect of acute cigarette smoke exposure on regional pulmonary blood flow, volume, red cell transit… Lee, Sherman 1985

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THE EFFECT OF ACUTE CIGARETTE SMOKE EXPOSURE ON REGIONAL PULMONARY BLOOD FLOW, VOLUME, RED CELL TRANSIT AND POLYMORPHONUCLEAR LEUKOCYTE RETENTION IN THE RABBIT LUNG BY SHERMAN LEE B .Sc . ( H o n o u r s ) , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Depar tment o f P a t h o l o g y , F a c u l t y o f M e d i c i n e , U .B .C . ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UWiVERSITY OF/BRIT ISH COLUMBIA Augus t 1985 © S h e r m a n L e e , 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pathology  The University of B r i t i s h Columbia 1956 M a i n M a l l Vancouver, Canada V6T 1Y3 Date October 1st, 1985 ABSTRACT R e g i o n a l pu lmonary b l o o d f l o w and vo lume was measured i n ten r a b b i t s a n e s t h e t i z e d w i t h p e n t o b a r b i t a l (30 mg/kg ) . T racheos tomy was p e r f o r m e d and c a t h e t e r s were p l a c e d i n t o the j u g u l a r v e i n and c a r o t i d a r t e r y . The c a r d i a c 99m o u t p u t was measured u s i n g the i n d i c a t o r - d i l u t i o n t e c h n i q u e u s i n g Tc l a b e l l e d RBC f o l l o w e d by an i n j e c t i o n o f r a d i o l a b e l l e d macroagg rega te s (MAA) t o mark r e g i o n a l b l o o d f l o w . Measurements were made bo th b e f o r e and a f t e r e i t h e r e x p o s u r e t o c i g a r e t t e smoke (3 c i g a r e t t e s f o r ten m inu te s at 4 p u f f s / m i n u t e ) o r sham e x p o s u r e to a i r . The an ima l s were s a c r i f i c e d and the l u n g s were removed w i t h the v e s s e l s t i e d . The l ung s were then i n f l a t e d and r a p i d l y f r o z e n o v e r l i q u i d n i t r o g e n . The l u n g s were sampled i n t o s l i c e s by v e r t i c a l h e i g h t , each s l i c e was f u r t h e r sampled and then gamma c o u n t e d f o r t h e i n j e c t e d i s o t o p e s . R e g i o n a l pu lmonary b l o o d f l o w was c a l c u l a t e d by s e t t i n g the t o t a l l u n g MAA c o u n t s f o r each MAA e q u a l t o the c a r d i a c o u t p u t so t h a t the sample f l o w was c a l c u l a t e d as the f r a c t i o n o f sample coun t s t o t o t a l c o u n t s t imes t h e c a r d i a c o u t p u t . The b l o o d volume was marked by the l a b e l l e d RBC and RBC t r a n s i t was c a l c u l a t e d as b l o o d volume (ml) d i v i d e d by b l o o d f l o w ( m l / s e c ) . In a s e c o n d s e r i e s o f e x p e r i m e n t s (N=10) , "**Cr PMN were i n j e c t e d as a b o l u s a l o n g w i t h 9 9 m T C RBC i n an i n d i c a t o r - d i l u t i o n r u n . F o l l o w i n g the i n j e c t i o n o f the c e l l s , t he b l o o d f l o w was marked w i t h MAAs and then the same sham o r smoke t r e a t m e n t s were g i v e n as i n the p r e v i o u s e x p e r i m e n t s . A t t h e end o f t e n m i n u t e s , t he a n i m a l s were s a c r i f i c e d and the l ung s were - H i -p r o c e s s e d the same as b e f o r e . R e g i o n a l PMN r e t e n t i o n was c a l c u l a t e d as the (PMN r e t a i n e d ) PMN d e l i v e r e d The d a t a show t h a t smoke exposu re i n c r e a s e d pu lmonary b l o o d volume ( p ^ . 0 1 ) , pu lmonary t r a n s i t t ime (p<C.05) and the r a t i o o f l u n g b l o o d volume t o c e n t r a l b l o o d vo lume (p <C-05) w i t h o u t c h a n g i n g c e n t r a l b l o o d volume o r c a r d i a c o u t p u t . Smoke exposu re a l s o c a u s e d a r e d i s t r i b u t i o n o f b l o o d f l o w f r om upper to l o w e r l u n g r e g i o n s (p<^.05) . T h i s l e n g t h e n e d the r e g i o n a l RBC t r a n s i t t i m e s i n a l l r e g i o n s but p a r t i c u l a r l y i n t he upper z o n e s . These changes i n RBC t r a n s i t had no e f f e c t on PMN r e t e n t i o n . We c o n c l u d e t h a t a c u t e smoke e x p o s u r e l e n g t h e n s the RBC t r a n s i t th rough t h e pu lmonary c i r c u l a t i o n by i n c r e a s i n g b l o o d volume and r e d i s t r i b u t i n g b l o o d f l o w . T h i s change i n r e d c e l l b e h a v i o r was not a s s o c i a t e d w i t h a c o n s i s t e n t change i n PMN r e t e n t i o n i n t he l u n g s . - i i i i -TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS i i L I ST OF TABLES V L I ST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 METHODS A. E x p e r i m e n t a l De s i gn 18 1. Doub le F low Marker S t u d i e s 19 2. R a d i o l a b e l e d PMN S t u d i e s 19 B. C e l l L a b e l l i n g T e c h n i q u e s 1. Red B l o o d C e l l (RBC) L a b e l l i n g 22 2. L e u k o c y t e L a b e l l i n g 22 a . I s o l a t i o n o f L e u k o c y t e s 22 b. R a d i o l a b e l l i n g w i t h 51rjr 25 C. I n j e c t a t e P r e p a r a t i o n 25 D. S u r g i c a l P r e p a r a t i o n 27 E. I n d i c a t o r - D i l u t i o n T e c h n i q u e 29 F. M e a s u r i n g R e g i o n a l Pu lmonary B l o o d F low 29 Lung Removal and Samp l ing 34 DATA HANDLING AND CALCULATIONS A. C o r r e c t i n g Gamma Counts 39 B. I n d i c a t o r - D i l u t i o n Cu rve s 39 C. C a l c u l a t i n g B l o o d F l ow, Volume and T r a n s i t 39 D. PMN R e t a i n e d , D e l i v e r e d and % R e t e n t i o n 40 E. E s t i m a t i n g T o t a l B l o o d Volume and L a b e l l e d PMN D i s t r i b u t i o n 41 F. S t a t i s t i c a l A n a l y s e s 42 RESULTS A. BLOOD FLOW STUDY 1) S i n g l e b o l u s o f two mac roagg rega te s . 43 2) Two m a c r o a g g r e g a t e s i n j e c t e d 10 m inutes a p a r t 45 3) R e g i o n a l Lung Da ta a) Pu lmonary b l o o d f l o w 48 b) Pu lmonary b l o o d volume 54 c) RBC t r a n s i t t ime 54 B. RADIOLABELLED PMN STUDY 1) I n d i c a t o r - d i l u t i o n c u r v e d a t a 57 2) R e g i o n a l l u n g d a t a a) Pu lmonary b l o o d f l o w and RBC t r a n s i t t imes 57 b) Pu lmonary b l o o d volume 59 c) PMN r e t e n t i o n 59 d) D i s t r i b u t i o n o f ^ C r l a b e l l e d PMN i n the t i s s u e s . . . . 61 C. TRANSIT TIMES AND BLOOD VOLUME 63 D. ARTERIAL BLOOD GASES, WHITE CELL COUNTS AND ARTERIAL BLOOD PRESSURE 63 DISCUSSION 65 CONCLUSION 78 BIBLIOGRAPHY 80 - vi -L I ST OF TABLES T a b l e Page I. C a r d i a c o u t p u t : i n i t i a l vs f i n a l ; p r e d i c t e d and measured 46 I I . I n d i c a t o r - d i l u t i o n c u r v e d a t a - PMN s tudy 58 I I I . R e g i o n a l Lung B l o o d F l o w , B l o o d Volume and PMN R e t e n t i o n 60 IV. T i s s u e D i s t r i b u t i o n o f 5 1 C r l a b e l l e d PMN 62 V. RBC mean t r a n s i t t i m e , l u n g b l o o d vo lume, c e n t r a l b l o o d volume 64 - v i i -L IST OF FIGURES F i g u r e Page 1 P r o t o c o l I. B l o o d F low S t u d i e s 20 2 P r o t o c o l I I . L a b e l l e d PMN S t u d i e s 21 3 R a d i o l a b e l l i n g RBC 23 4 I s o l a t i n g P o l y m o r p h o n u c l e a r L e u k o c y t e s 24 5 L a b e l l i n g P o l y m o r p h o n u c l e a r L e u k o c y t e s 26 6 S u r g i c a l P r e p a r a t i o n 28 7 F r a c t i o n C o l l e c t o r 30 8 The Smoking A p p a r a t u s 31 9 The E x p e r i m e n t a l S e t - u p 32 10 I n d i c a t o r - d i l u t i o n run 33 11 F r o z e n I n f l a t e d Lung 35 12 S l i c e d F r o z e n Lung 36 13 Samp l i ng From Lung S l i c e s 37 14 Sampled Lung i n V i a l s 38 15 S i n g l e B o l u s Two M a c r o a g g r e g a t e s 44 16 Doub le B o l u s Two M a c r o a g g r e g a t e s 47 R e g i o n a l Lung B l o o d F l o w 17 Sham Group 49 18 Smoke Group 51 19 P r e - t r e a t m e n t Run 52 20 P o s t - t r e a t m e n t Run 53 21 R e g i o n a l Lung B l o o d Volume 55 22 R e g i o n a l Lung RBC T r a n s i t Time 56 - v i i i -ACKNOWLEDGEMENTS I wou ld l i k e to thank my s u p r e v i s o r Dr . J . C . Hogg f o r h i s g u i d a n c e , encouragment and p a t i e n c e t h r o u g h o u t the c o u r s e o f my r e s e a r c h I wou ld a l s o l i k e t o thank Ms. B r i d g e t M i l s o m f o r he r a s s i s t a n c e and f o r the many s u g g e s t i o n s o f f e r e d d u r i n g my work i n the l a b o r a t o r y , Mr. B a r r y Wiggs f o r the a s s i s t a n c e w i t h the s t a t i s t i c s . The c o n t r i b u t i o n s o f Joe Comeau w i t h the f i g u r e s and S t u a r t Greene and Dean E n g l i s h w i t h the p h o t o g r a p h y i s a c k n o w l e d g e d . The s e c r e t a r i a l s u p p o r t p r o v i d e d by Joan D ixon and Lee Kowk i s g r e a t l y a p p r e c i a t e d . - 1 -INTRODUCTION Emphysema i s a c h r o n i c l u n g d i s e a s e c h a r a c t e r i z e d by the permanent e n l a r g e m e n t o f t h e a i r w a y s d i s t a l t o the t e r m i n a l b r o n c h i o l e s accompan ied by the d e s t r u c t i o n o f i t s a s s o c i a t e d a l v e o l a r w a l l ( T h u r l b e c k , 1976) . Because t h e c r i t e r i a i s an anatomic one , emphysema i s i n c l u d e d i n a group o f c o n d i t i o n s r e c o g n i z e d c l i n i c a l l y as the c h r o n i c o b s t r u c t i v e pu lmonary d i s e a s e s (COPD). P a t i e n t s w i t h COPD u s u a l l y c o m p l a i n o f s h o r t n e s s o f b r e a t h , c h r o n i c c o u g h , and poor e x e r c i s e t o l e r a n c e . Lung f u n c t i o n t e s t s u s u a l l y r e v e a l i n c r e a s e d l u n g vo lumes ( i . e . t o t a l vo lume, r e s i d u a l vo lume, and f u n c t i o n a l r e s i d u a l c a p a c i t y ) , i n c r e a s e d c o m p l i a n c e , d e c r e a s e d v i t a l c a p a c i t y , d e c r e a s e d d i f f u s i n g c a p a c i t y , and i n c r e a s e d a i r f l o w r e s i s t a n c e (West, 1 9 8 2 ) . T h e r e a r e f o u r t y p e s o f pu lmonary emphysema: p a n l o b u l a r , c e n t r i l o b u l a r , p a r a s e p t a l , and p a r a c i c a t r i c i a l . A l t h o u g h the v a r i o u s t ypes o f emphysema may i n v o l v e d i f f e r e n t e t i o l o g i c a l and p a t h o g e n i c f a c t o r s , the f i n a l common pathway o f t i s s u e d e s t r u c t i o n i s b e l i e v e d t o the same f o r a l l t h e t y p e s . C e n t r i l o b u l a r emphysema i s the most common and i s c l o s e l y a s s o c i a t e d w i t h c i g a r e t t e smok ing . C i g a r e t t e smoking i s r e g a r d e d as the most i m p o r t a n t c au se o f emphysema ( T h u r l b e c k , 1976 ) . E p i d e m i o l o g i c a l e v i d e n c e compar ing the i n c i d e n c e o f emphysema i n non-smokers and smokers show t h a t the m a j o r i t y o f non-smokers do not have emphysema; emphysema i s o n l y f o u n d i n the o l d e r age g roups and i s l i m i t e d i n e x t e n t (Ryder §_t §_1. , 1 9 7 1 ) . On the o t h e r hand , the m a j o r i t y o f smokers do have emphysema wh ich i s o f t e n e x t e n s i v e . I t was uncommon f o r smokers under 60 y e a r s o f age to have l u n g s f r e e o f emphysema. The l e s i o n i n c e n t r i l o b u l a r emphysema o c c u r s a t the c e n t r e o f the l u n g l o b u l e ( s e c o n d a r y l o b u l e o f M i l l e r ) wh i ch u s u a l l y c o n t a i n s 6-8 t e r m i n a l b r o n c h i o l e s and t h e i r b r a n c h e s . These l e s i o n s a re more s e v e r e i n the upper zones t h a n the l o w e r zones o f the l u n g w i t h the most s e v e r e l y a f f e c t e d a r e a s i n the upper l o b e s ( T h u r l b e c k , 1970) . W i t h i n the l e s i o n s t h e r e i s expanded a i r s p a c e , b r o n c h i a l and b r o n c h i o l a r i n f l a m m a t i o n and ca rbonaceous p igment d e p o s i t i o n ( L e o p o l d and Gough, 1957 ) . A l t h o u g h the a s s o c i a t i o n between c i g a r e t t e smoking and emphysema i s w e l l e s t a b l i s h e d , t h e r o l e o f smoking i n t he p a t h o g e n e s i s o f emphysema i s p o o r l y u n d e r s t o o d . The most p o p u l a r e x p l a n a t i o n i s the p r o t e a s e - a n t i p r o t e a s e imba lance h y p o t h e s i s wh i ch a t t r i b u t e s t he damage t o l u n g c o n n e c t i v e t i s s u e to p r o t e a s e s r e l e a s e d f rom the i n f l a m m a t o r y c e l l s . The p o l y m o r p h o n u c l e a r l e u k o c y t e (PMN) o r n e u t r o p h i l i s the ma jo r c e l l u l a r component i n t he a c u t e i n f l a m m a t o r y r e s p o n s e . The i n f l a m m a t o r y p r o c e s s i s c a p a b l e o f i n d u c i n g p r o t e o l y s i s by r e l e a s i n g enzymes such as e l a s t a s e f rom t h e g r a n u l e s o f the p o l y m o r p h o n u c l e a r l e u k o c y t e . T h i s p r o t e o l y t i c f o r c e i s n o r m a l l y kep t i n c h e c k by n a t u r a l i n h i b i t o r s i n the p lasma such as a l p h a - l - p r o t e a s e i n h i b i t o r ( A - l - P I ) . D i s t u r b a n c e s i n t h i s b a l a n c e b e s t d e s c r i b e s the p r o p o s e d p a t h o g e n i c mechanism f o r pu lmonary emphysema where u n r e s t r a i n e d i n f l a m m a t o r y p r o c e s s e s a r e though t t o p roduce d e s t r u c t i o n o f l u n g t i s s u e . The c o n c e p t o f t h e r e b e i n g two m u t u a l l y a n t a g o n i s t i c f o r c e s i n the i n f l a m m a t o r y p r o c e s s i s no t a new one . E a r l y t h i s c e n t u r y i n 1905, Op ie examined the c o n t e n t s o f i n f l a m m a t o r y exuda te s and n o t e d the p r e s e n c e o f p r o t e a s e and a n t i p r o t e a s e components. The f o r m u l a t i o n o f the - 3 -p r o t e a s e - a n t i p r o t e a s e imba lance h y p o t h e s i s i n the p a t h o g e n e s i s o f emphysema r e s u l t e d f rom two c o i n c i d e n t a l o b s e r v a t i o n s by two i ndependen t i n v e s t i g a t o r s . L a u r e l l and E r i k s s o n ' s s t udy i n 1963 a s s o c i a t e d the d e v e l o p m e n t o f pu lmonary emphysema t o p a t i e n t s w i t h a r a r e i n h e r i t e d p la sma p r o t e i n a b n o r m a l i t y c a l l e d a l p h a - l - a n t i t r y p s i n d e f i c i e n c y . P a t i e n t s w i t h t h e homozygous serum e l e c t r o p h o r e t i c p a t t e r n o f the d e f i c i e n c y had an i n c r e a s e d t e n d e n c y t o d e v e l o p s e v e r e emphysema ( p a n l o b u l a r t y p e ) at an e a r l y age ( L a u r e l and E r i k s s o n , 1963; Kueppers et^ a l . , 1974 ) . A lmos t s i m u l t a n e o u s l y , G r o s s and h i s c o l l e a g u e s f ound they c o u l d p roduce l u n g l e s i o n s i n r a t s s i m i l a r t o those o f emphysema by a d m i n i s t e r i n g the p l a n t p r o t e a s e , p a p a i n i n t r a t r a c h e a l l y (Gros s et. a l . , 1964 ) . The p r o t e a s e - a n t i p r o t e a s e imba lance h y p o t h e s i s i s c u r r e n t l y the most p o p u l a r t h e o r y i n the p a t h o g e n e s i s o f emphysema and has g e n e r a t e d e x t e n s i v e r e s e a r c h i n r e c e n t y e a r s ( H o i d a l e t a l . , 1983; J a n o f f , 1983; S n i d e r , 1981 ) . The e n z y m e - i n d u c e d emphysema mode l o f G ro s s has s i n c e been r e p r o d u c e d by o t h e r i n v e s t i g a t o r s i n d i f f e r e n t s p e c i e s , u s i n g d i f f e r e n t enzyme p r e p a r a t i o n s and r o u t e s o f a d m i n i s t r a t i o n ( K a r l i n s k y and S n i d e r , 1978) . The m a j o r t i s s u e component a t t a c k e d i n e n z y m e - i n d u c e d emphysema i s e l a s t i n . The c o n t e n t s o f human PMN g r a n u l e s can d i g e s t human l u n g p r o t e i n s i n v i t r o ( J a n o f f e t a l , 1972) and f u r t h e r work has i d e n t i f i e d the p r o t e a s e as an e l a s t a s e ( K a p l a n e t al., 1973; S n i d e r e_t a l . , 1974 ) . E l a s t a s e has been p u r i f i e d as a c a t i o n i c p r o t e a s e i n t he g r a n u l e f r a c t i o n o f human l e u k o c y t e s and f o u n d to be s i m i l a r t o p a n c r e a t i c e l a s t a s e (K ruze e t a l , 1976 ) . I n t r a v e n o u s l y a d m i n i s t e r e d e l a s t a s e i n r a t s p roduce s the c h a r a c t e r i s t i c l e s i o n s as p r o d u c e d when e l a s t a s e was g i v e n i n t r a t r a c h e a l l y (B lackwood e t - 4 -a l • , 1974 ) . The emphysema- l i ke l e s i o n s were a l s o p r o d u c e d when human l e u k o c y t e e l a s t a s e was g i v e n to hamsters ( S e n i o r e t a l . , 1977) and when dog l u n g s were exposed bo th i n v i t r o and i n v i v o t o p u r i f i e d e l a s t a s e s f rom homologous l e u k o c y t e s ( S l o a n e t a l . , 1981 ) . These s t u d i e s d e m o n s t r a t e d t h a t e x p e r i m e n t a l emphysema can be g e n e r a t e d by i n c r e a s i n g the p r o t e a s e burden i n the l u n g o f a n i m a l s . I t i s not d i f f i c u l t t o see how l u n g i n j u r y r e s u l t s i n a l p h a - l - a n t i p r o t e a s e d e f i c i e n t p e r s o n s i n t h a t the p r o t e a s e s a r e f r e e t o a c t on l u n g c o n n e c t i v e t i s s u e unopposed by the a n t i p r o t e a s e s . C o n v e r s e l y , by i n c r e a s i n g the p r o t e a s e l o a d i n l u n g as i n the e n z y m e - i n d u c e d emphysema, the a n t i p r o t e a s e s a re overwhelmed and the b a l a n c e a g a i n f a v o r s t he p r o t e a s e . How p r o t e a s e - m e d i a t e d l u n g i n j u r y o c c u r s i n p e r s o n s w i t h g e n e t i c a l l y s u f f i c i e n t l e v e l s o f a n t i p r o t e a s e i s l e s s c l e a r l y u n d e r s t o o d . A h y p o t h e s i s p r o p o s e d by J a n o f f c o n s i d e r s t h r e e d i f f e r e n t ways c i g a r e t t e smoking i s i n v o l v e d i n the p a t h o g e n e s i s o f emphysema ( J a n o f f , 1983 ) . F i r s t l y , c i g a r e t t e smoking d e p r e s s e s the e l a s t a s e i n h i b i t o r f u n c t i o n i n the l u n g ( J a n o f f e t a l . , 1 9 7 9 ) . S e c o n d l y , t h e r e i s e v i d e n c e t o sugges t t h a t agents i n c i g a r e t t e smoke i n h i b i t the e l a s t i n r e p a i r mechanisms i n the l u n g ( L a u r e n t e_t a l . , 1983 ) . T h i r d l y , c i g a r e t t e smoking appears t o i n c r e a s e the pu lmonary r e c r u i t m e n t o f " e l a s t a s e - v e c t o r c e l l s " by c h e m o t a c t i c components i n c i g a r e t t e smoke ( T o t t i et_ a l . , 1979) o r by the a c t i o n o f smoke on the a l v e o l a r macrophage wh ich then r e l e a s e s c h e m o t a c t i c f a c t o r s f o r the PMN (Hunninghake e t a l . , 1978) . The " e l a s t a s e - v e c t o r c e l l s " i n c l u d e the p o l y m o r p h o n u c l e a r l e u k o c y t e and the a l v e o l a r macrophage. Some i n v e s t i g a t o r s b e l i e v e the r o l e o f the a l v e o l a r macrophage may be c e n t r a l s i n c e i t s - 5 -e l a s t a s e i s l e s s e f f e c t i v e l y i n h i b i t e d by a l p h a - l - a n t i p r o t e a s e (Kuhn e t a l . , 1978) and the a l v e o l a r macrophage r e l e a s e s c h e m o t a c t i c f a c t o r s wh ich a t t r a c t PHN t o the l u n g (Hunninghake e t a l . , 1980; K imbe l e t a l . , 1980) . The r e c r u i t m e n t o f " e l a s t a s e - v e c t o r c e l l s " was d e m o n s t r a t e d i n a p a t i e n t s t udy where i n c r e a s e d numbers o f n e u t r o p h i l s were found i n the l u n g s o f smokers as compared to non-smokers by b r o n c h o a l v e o l a r l a v a g e and by d i r e c t i s o l a t i o n f r om l u n g b i o p s y m a t e r i a l (Hunninghake and C r y s t a l , 1983 ) . I t appear s t h a t c i g a r e t t e smoking can i n c r e a s e the p r o t e a s e burden i n the l u n g by i n c r e a s i n g t h e r e c r u i t m e n t o f pu lmonary p h a g o c y t e s . T h e r e i s e v i d e n c e t o sugges t t h a t the deve lopment o f the p r o t e a s e i m b a l a n c e i n emphysema o c c u r s as a r e s u l t o f c h r o n i c i n t r a v a s c u l a r l e u k o c y t e s e q u e s t r a t i o n . E x p e r i m e n t a l l y , t h i s was i n d u c e d by g i v i n g r e p e t i t i v e doses o f i n t r a v e n o u s e n d o t o x i n t o a n i m a l s ( W i t t e l s e t 1974; P i n g l e t o n e t a l . , 1975 ; Guen te r e t a l . , 1 9 8 1 ) . In a s t u d y where e n d o t o x i n was g i v e n r e p e a t e d l y t o monkeys, modera te damage t o the a l v e o l i was d e t e c t e d at the end o f n i n e weeks. The a u t h o r s i n t e r p r e t e d t h e s e l e s i o n s t o be e q u i v a l e n t t o t h e changes seen e a r l y i n emphysema and they s p e c u l a t e d t h a t the d i s e a s e d e v e l o p s a f t e r r e p e t i t i v e pu lmonary i n s u l t s i n the v a s c u l a r bed ove r a l i f e t i m e ( W i t t e l s e t a l . , 1974 ) . The same t e c h n i q u e i n dogs p roduced m i l d emphysematous l e s i o n s i n the l u n g wh i ch when compared to p a p a i n - i n d u c e d emphysema, d i d no t d e m o n s t r a t e as g r e a t a l o s s i n l u n g e l a s t i c i t y (Guenter e t a l . , 1981) . The r e c r u i t m e n t o f " e l a s t a s e - v e c t o r c e l l s " i n the l u n g by i n t r a v a s c u l a r s e q u e s t r a t i o n appears t o be a p l a u s i b l e mechanism o f p r o t e a s e imba lance s i n c e the l u n g i s known t o be the ma jo r s i t e o f the b o d y ' s m a r g i n a t i n g p o o l - 6 -o f l e u k o c y t e s ( B i e r m a n , 1955 ) . W i th a l a r g e r e s e r v e o f s e q u e s t e r e d l e u k o c y t e s m a r g i n a t i n g w i t h i n the pu lmonary c i r c u l a t i o n i t i s c o n c e i v a b l e t h a t c i g a r e t t e smoking c o u l d a c t i v a t e the c e l l s a n d / o r a l t e r the s i z e o f the p o o l w i t h i n the l u n g . The m i g r a t i o n o f l e u k o c y t e s towards the v e s s e l w a l l s i n the m i c r o c i r c u l a t i o n i s r e f e r r e d t o as m a r g i n a t i o n and i s u s u a l l y but no t a lways f o l l o w e d by w a l l a d h e s i o n and e m i g r a t i o n o f the w h i t e c e l l i n t o the e x t r a v a s c u l a r s p a c e . A l t h o u g h the f i r s t d e s c r i p t i o n s o f l e u k o c y t e m a r g i n a t i o n a re a t t r i b u t e d to D u t r o c h e t i n 1824, i t was s t u d i e s by Cohnheim i n the l a t e 1 8 0 0 ' s t h a t g e n e r a t e d i n t e r e s t i n the s u b j e c t . Cohnheim s t u d i e d t h e m i c r o c i r c u l a t i o n i n the mesen te ry and tongue o f the l i v e f r o g under the l i g h t m i c r o s c o p e . By o b s e r v i n g the v e s s e l s i n v i v o he n o t i c e d t h a t as the b l o o d f l o w s l o w e d , t he w h i t e c e l l s t ended t o move towards the p e r i p h e r y o f t h e f l o w i n g s t r e a m and l i n e up a l o n g the w a l l s o f t he v e n u l e s w h i l e t he r e d b l o o d c e l l s s t a y e d w i t h i n the c e n t r a l s t r e a m (Cohnheim, 1889 ) . E v e n t u a l l y t h e s e c e l l s e i t h e r a d h e r e d t o the v e s s e l w a l l and e m i g r a t e d f rom the v a s c u l a r space o r t h e y r e - e n t e r e d the s t r e a m o f f l o w i n g b l o o d w i t h o r w i t h o u t exchange w i t h c i r c u l a t i n g c e l l s . Cohnhe im ' s work ex tended to the s t u d y o f the hemodynamic e v e n t s f o l l o w i n g i n j u r y and i n p a r t i c u l a r d e s c r i p t i o n s o f t h e e v e n t s i n the i n f l a m m a t o r y r e s p o n s e . S t u d i e s by V e j l e n s i n 1938 showed t h a t l e u k o c y t e m a r g i n a t i o n can be a s s o c i a t e d w i t h r e d b l o o d c e l l a g g r e g a t i o n . By i n j e c t i n g s u b s t a n c e s such as g e l a t i n i n t o the c i r c u l a t i o n he was a b l e t o i n d u c e l e u k o c y t e m a r g i n a t i o n by a g g r e g a t i n g the r e d c e l l s . S i m i l a r l y , r e d b l o o d c e l l a g g r e g a t i o n c a u s e d by r e d u c i n g b l o o d f l o w was a l s o shown to be a s s o c i a t e d w i t h l e u k o c y t e - 7 -m a r g i n a t i o n ( P h i b b s , 1967 ) . Recent s t u d i e s show t h a t the w h i t e b l o o d c e l l i n t h e c a p i l l a r y i s n o r m a l l y f ound i n the m a r g i n a l s t ream and t r a v e l s at a l o w e r v e l o c i t y t han the r e d b l o o d c e l l i n the a x i a l s t r e a m ( S c h m i d - S c h o e n b e i n , 1980 ) . M a r g i n a t i o n has been a t t r i b u t e d t o the i n t e r a c t i o n s between the s m a l l e r , d e f o r m a b l e , d i s c o i d a l r e d c e l l s and the more r i g i d , s p h e r i c a l l e u k o c y t e s as they l e a v e the c a p i l l a r i e s . S c h m i d - S c h o e n b e i n m o d e l l e d t h e s e c o n d i t i o n s an i n v i t r o model c o n t a i n i n g a s u s p e n s i o n o f r i g i d s phe re s and f l e x i b l e d i s c s . In the m o d e l , t he l e u k o c y t e t e n d e d t o move towards the p e r i p h e r y o f the p o s t - c a p i l l a r y v e n u l e upon l e a v i n g the c a p i l l a r y b e d . The deve lopment o f r e l i a b l e t e c h n i q u e s t o r a d i o l a b e l t he g r a n u l o c y t e b o t h i n v i v o and i n v i t r o (Athens e t a l . , 1959; Maurer e t a l . , 1960) w i t h 32 d i i s o p r o p y l f l u o r o p h o s p h a t e (DF P) has a l l o w e d i n v e s t i g a t o r s t o measure the s i z e o f the c i r c u l a t i n g and m a r g i n a t i n g g r a n u l o c y t e p o o l s . A thens e t a l . , i n 1961 e s t i m a t e d the t o t a l body g r a n u l o c y t e p o o l (TBGP) to be a p p r o x i m a t e l y t w i c e t h a t o f the c i r c u l a t i n g g r a n u l o c y t e p o o l (CGP). When l a b e l l e d g r a n u l o c y t e s were r e - i n f u s e d i n t o s u b j e c t s o v e r a 15 to 20 m inute p e r i o d , by the end o f i n f u s i o n o n l y about f i f t y p e r c e n t o f the l a b e l l e d c e l l s were s t i l l f o u n d w i t h i n the c i r c u l a t i o n . The d i s a p p e a r a n c e o f t h e s e c e l l s was p o s t u l a t e d to be due to t h e i r d i s t r i b u t i o n i n t o a l a r g e r p o o l o f a p p r o x i m a t e l y e q u a l s i z e and not due t o c e l l damage o r l a b e l e l u t i o n . The TBGP i s c o n c e p t u a l i z e d as h a v i n g two d y n a m i c a l l y i n t e r a c t i v e compar tments , t h e c i r c u l a t i n g g r a n u l o c y t e p o o l (CGP) and the m a r g i n a t i n g g r a n u l o c y t e p o o l (MGP) wh i ch e x i s t i n r a p i d e q u i l i b r i u m w i t h each o t h e r . T h i s dynamic e q u i l i b r i u m was d e m o n s t r a t e d by the f a c t t h a t l a b e l l e d c e l l s c o u l d be - 8 -b r o u g h t back i n t o c i r c u l a t i o n by e x e r c i s e o r by g i v i n g e p i n e p h r i n e to the s u b j e c t s (Athens e t a l . , 1961, M u i r e t a l . , 1984 ) . The l u n g has l o n g been known as a major s i t e o f l e u k o c y t e s e q u e s t r a t i o n . The i n i t i a l i n t e r e s t was i n the mechanism o f l e u k o c y t e r e g u l a t i o n ba sed on o b s e r v a t i o n s o f l e u k o c y t e l e v e l s f o l l o w i n g the t r a n s f u s i o n o f l a r g e numbers o f l e u k o c y t e s ( W e i s b e r g e r e_t a l . , 1950, W e i s b e r g e r §_t a l . , 1951, B ierman e t a l . , 1 9 5 1 ) . In W e i s b e r g e r ' s s t u d i e s , r a d i o l a b e l l e d l e u k o c y t e s were t r a n s f u s e d i n t o r a b b i t s . The c o n s i s t e n t f i n d i n g was t h a t the m a j o r i t y o f the r a d i o a c t i v i t y was l a t e r found i n t he l u n g s . T h i s f i n d i n g was c o r r e l a t e d w i t h the h i s t o l o g i c appearance o f the. l u n g wh i ch showed a marked engorgement o f t he i n t r a - a l v e o l a r s e p t a w i t h l e u k o c y t e s . The s p e c i f i c i t y o f the l e u k o c y t e f o r the pu lmonary c a p i l l a r y bed was d e m o n s t r a t e d by i n j e c t i n g the l a b e l l e d c e l l s i n t o the a o r t a so t h a t t h e y wou ld f i r s t pass t h rough the s y s t e m i c c i r c u l a t i o n . The l ung s a g a i n d e m o n s t r a t e d the m a j o r i t y o f the r a d i o a c t i v i t y . The l u n g , l i v e r and s p l e e n were f o u n d t o be the most a c t i v e i n the remova l o f t r a n s f u s e d l e u c k o y t e s f r o m t h e p e r i p h e r a l c i r c u l a t i o n . S t u d i e s i n dog (Ambrus e t a l . , 1954; Thomassen e t a l . , 1984) and i n man ( B i e r m a n , 1955 ; B ie rman e_t a l . , 1955) show the pu lmonary c i r c u l a t i o n c a p a b l e o f r emov ing and d e l i v e r i n g l a r g e numbers o f l e u c k o y t e s f rom and i n t o the c i r c u l a t i o n . B ie rman d e s c r i b e d the p r o c e s s as an " e b b and f l o w t i d e " r e f l e c t i n g the r a p i d f l u c t u a t i o n s i n the p e r i p h e r a l l e u k o c y t e l e v e l and the changes o c c u r r i n g w i t h the r e s p i r a t o r y movements. A c c o r d i n g to B i e r m a n , t he l e u k o c y t e s a r e t r a n s i e n t l y t r a p p e d i n the pu lmonary c i r c u l a t i o n by m a r g i n a t i o n and n o r m a l l y n e a r l y e q u a l numbers l e a v e the l u n g to t ho se t h a t - 9 -e n t e r . T h i s h y p o t h e s i s i s s u p p o r t e d by a r e c e n t s t udy by Thomassen (1984) where l e u k o c y t e s removed by the l u n g i n low f l o w were r e l e a s e d back i n t o the c i r c u l a t i o n i n s i m i l a r numbers once the f l o w was r e s t o r e d . The s i z e o f the m a r g i n a t i n g p o o l i n t he l u n g has been e s t i m a t e d to be 3.0 t imes t h a t o f the c i r c u l a t i n g p o o l i n sheep (S taub e t &1., 1982 ) . The l o c a t i o n o f the m a r g i n a t i n g p o o l was thought to be i n the p r e - c a p i l l a r y a r t e r i o l a r v e s s e l s . O t h e r s t u d i e s o f t he s y s t e m i c c i r c u l a t i o n have found the s i t e o f m a r g i n a t i o n t o be i n the p o s t - c a p i l l a r y v e n u l e s ( S c h m i d - S c h o e n b e i n , 1980) o r e v e n l y d i s t r i b u t e d between the a r t e r i o l e s and v e n u l e s ( V e j l e n s , 1938 ) . The c a t h e t e r i z a t i o n o f the c e n t r a l c i r c u l a t i o n has a l l o w e d the uptake and r e l e a s e o f l e u k o c y t e s by the l u n g t o be d e t e c t e d as d i f f e r e n c e s i n l e u k o c y t e c o u n t s a c r o s s the l u n g (B ie rman e t a l . , 1952; Thomassen e t a l . , 1 9 8 4 ) . B ie rman showed t h a t s m a l l f l u c t u a t i o n s i n t he p e r i p h e r a l l e u k o c y t e c o u n t a r e dependent on the dep th and phase o f the r e s p i r a t o r y c y c l e . The e x a g g e r a t e d i n s p i r a t i o n o f the M u e l l e r manoeuver c au sed the w h i t e c e l l l e v e l s t o r i s e on e i t h e r s i d e o f the l u n g w i t h the g r e a t e r r i s e o c c u r r i n g on t h e a r t e r i a l s i d e . The M u e l l e r manoeuver r e s u l t e d i n the r e l e a s e o f l e u k o y t e s f r om the l u n g w h i l e c o n v e r s e l y , the V a l s a l v a maneuver p e r f o r m e d by e x a g g e r a t i n g e x p i r a t o r y e f f o r t c a u s e d l e u k o c y t e l e v e l s t o f a l l on the a r t e r i a l s i d e o f t he l u n g . In b o t h i n s t a n c e s when t h e s e maneuvers were s t o p p e d the l e v e l s q u i c k l y r e t u r n e d back t o n o r m a l . The l u n g s o f no rma l s u b j e c t s r e c e i v i n g b l o o d t r a n s f u s i o n s f rom l e u k e m i c d o n o r s d e m o n s t r a t e the a b i l i t y t o " f i l t e r " l a r g e numbers o f w h i t e c e l l s b e c a u s e d e s p i t e the l o a d i n g o f w h i t e c e l l s f rom the donor b l o o d , the p e r i p h e r a l w h i t e c e l l coun t s o f the r e c i p i e n t were m a i n t a i n e d c l o s e to - 10 -n o r m a l (B ierman e t a l . , 1951 ) . The a d m i n i s t r a t i o n o f i n t r a v e n o u s e p i n e p h r i n e e l i c i t s a marked a r t e r i a l l e u k o c y t o s i s w i t h no change i n venous l e u k o c y t e l e v e l s f o r a t l e a s t one to two m inu te s a f t e r t he d rug (B ierman e t a l . , 1 9 5 2 ) . The l e u k o c y t o s i s was accompan ied by a marked t h r o m b o c y t o s i s s u g g e s t i n g t h a t p l a t e l e t s a r e a l s o s e q u e s t e r e d i n the l u n g . R e s u l t s s i m i l a r t o t h o s e i n man were f ound i n c r o s s - t r a n s f u s i o n a l and S t a r l i n g h e a r t - l u n g s t u d i e s i n t he dog (Ambrus e t a l . , 1954 ) . In the h e a r t - l u n g p r e p a r a t i o n s , t h e w h i t e b l o o d c e l l s were r a p i d l y removed f rom the h e p a r i n i z e d b l o o d u n t i l a c e r t a i n l e v e l was r e a c h e d . These w h i t e c e l l l e v e l s were m a i n t a i n e d even though more w h i t e c e l l s were added t o the s y s tem. The c r o s s - t r a n s f u s i o n a l s t u d i e s i n the i n t a c t dog showed the f a l l i n l e u k o c y t e c o u n t s t o be m a i n l y due t o the l o s s o f g r a n u l o c y t e s . Recen t s t u d i e s by M a r t i n e t a l . (1982) w i t h 5 1 C r l a b e l l e d PMN i n the dog d e m o n s t r a t e d an 80 t o 90% e x t r a c t i o n o f t h e s e c e l l s f rom the c i r c u l a t i o n i n a s i n g l e pass t h rough the l u n g and the r e t e n t i o n o f PMN was found t o be i n v e r s e l y r e l a t e d to b l o o d f l o w . T h i s i n v e r s e r e l a t i o n s h i p was d e m o n s t r a t e d i n two s t u d i e s . The f i r s t s tudy examined w h i t e c e l l c oun t s a c r o s s the l u n g i n t h e dog as b l o o d f l o w was d e c r e a s e d by wedg ing an i n f l a t e d b a l l o o n - t i p p e d c a t h e t e r i n the i n f e r i o r vena cava j u s t be low the d i aphragm. A r t e r i o - v e n o u s d i f f e r e n c e s r e s u l t e d as the f l o w was d e c r e a s e d s u g g e s t i n g a ne t uptake o f c e l l s by the l u n g . These r e s u l t s a re s u p p o r t e d by f u r t h e r s t u d i e s where the same number o f c e l l s removed by the l u n g i n low b l o o d f l o w were r e l e a s e d back i n t o the c i r c u l a t i o n once the f l o w t o the l u n g was r e s t o r e d (Thomassen e t a l . 1984 ) . The second g roup o f s t u d i e s by M a r t i n e t a l . t ook advantage o f t h e wide range o f b l o o d f l o w s i n the l u n g to show t h a t PMN r e t e n t i o n a l s o - 11 -v a r i e d w i t h the no rma l v a r i a t i o n i n r e g i o n a l pu lmonary b l o o d f l o w i n the l u n g . PMN r e t e n t i o n i s c a l c u l a t e d as the number o f PMN r e t a i n e d by b l o o d f l o w d i v i d e d by the number o f PMN d e l i v e r e d i n a sample o f l u n g . By s a m p l i n g the e n t i r e l u n g , t h e y showed t h a t r e g i o n s w i t h low f l o w had the h i g h e s t r e t e n t i o n o f PMN w h i l e h i g h f l o w r e g i o n s have l ower r e t e n t i o n . The g r e a t e s t PMN r e t e n t i o n o c c u r r e d i n r e g i o n s w i t h b l o o d f l o w l e s s than 7 m l / m i n / g wet l u n g and the l e a s t i n r e g i o n s w i t h b l o o d f l o w s g r e a t e r than 15 m l / m i n / g wet l u n g . R e g i o n a l pu lmonary b l o o d f l o w has been s t u d i e d i n the e r e c t human l u n g o f l i v i n g s u b j e c t s w i t h r a d i o a c t i v e gases such as xenon ( B a l l e_t a l . , 1962) and c a r b o n d i o x i d e (West e t a l . , 1960 ) . These s t u d i e s s u p p o r t the f i n d i n g t h a t pu lmonary b l o o d f l o w d e c r e a s e s a lmos t l i n e a r l y f rom the l u n g bases t o the a p i c e s ( D o l l e r y and West , 1960 ) . The r e g i o n a l d i s t r i b u t i o n o f pu lmonary b l o o d f l o w i s b e s t e x p l a i n e d by the r e l a t i o n s h i p s between the pu lmonary a l v e o l a r ( P . ) , a r t e r i a l (P ), and venous (P ) p r e s s u r e s m o d e l l e d i n A a v t h e S t a r l i n g r e s i s t o r . The S t a r l i n g r e s i s t o r can be thought o f as a c o l l a p s i b l e tube i n a c l o s e d box w i t h t h r e e communica t ions t o the o u t s i d e , two o f wh i ch a r e t h e i n l e t and the o u t l e t f o r the t u b e . The t h i r d p o r t r e g u l a t e s the p r e s s u r e w i t h i n the box o r the p r e s s u r e s u r r o u n d i n g the c o l l a p s i b l e tube i n t he box . In t h i s m o d e l , the pu lmonary a r t e r i a l p r e s s u r e i s r e p r e s e n t e d by t h e i n f l o w p r e s s u r e , the mean l e f t a t r i a l p r e s s u r e by the o u t f l o w p r e s s u r e , and the a l v e o l a r p r e s s u r e i s r e p r e s e n t e d by the p r e s s u r e s u r r o u n d i n g the c o l l a p s i b l e tube (West e t a l . , 1964 ) . B l o o d f l o w b e g i n s a t a l e v e l where the P exceeds the P and t h e r e f o r e the d r i v i n g f o r c e i s a A t h e d i f f e r e n c e between P and P. . Above t h i s l e v e l t he P. exceeds the a A A P and no b l o o d f l o w o c c u r s . West has d e s i g n a t e d t h r e e main zones i n the a l u n g a c c o r d i n g to t h e s e p r e s s u r e r e l a t i o n s h i p s where Zone I: P a ^ P , Zone I I : P P. > P , and Zone I I I : P > P > P . . An v a ' k s v a ' v ' A a d d i t i o n a l f o u r t h zone was l a t e r added to e x p l a i n the r e d u c t i o n i n b l o o d f l o w a t the ext reme l u n g bases due t o i n c r e a s e d i n t e r s t i t i a l p r e s s u r e s f e l t by t h e e x t r a a l v e o l a r v e s s e l s i n t h i s zone (Hughes e_t a l . , 1968 ) . R e g i o n a l pu lmonary b l o o d f l o w can be a l s o measured u s i n g r a d i o l a b e l l e d m i c r o s p h e r e s and m a c r o a g g r e g a t e s o f human serum a lbumin (Tow et^ a l . . 1962; Ueda and K a i h a r a , 1965; Rudo lph and Heymann, 1967; K a i h a r a e t a l . , 1968 ) . The m i c r o s p h e r e s and mac roagg rega te s d i s t r i b u t e i n the pu lmonary m i c r o v a s c u l a t u r e i n p r o p o r t i o n to the amount o f b l o o d f l o w to an a r e a b e c a u s e most o f the p a r t i c l e s a re o f s u f f i c i e n t s i z e t o be removed by the m i c r o v a s c u l a r bed i n a f i r s t pass but too s m a l l i n numbers t o e f f e c t the b l o o d f l o w . Comp le te m i x i n g o f t he p a r t i c l e s w i t h the b l o o d i n the r i g h t v e n t r i c l e f o l l o w i n g i n j e c t i o n i s assumed i n o r d e r f o r the measurement to be v a l i d . The ave rage i n j e c t i o n o f MAA c o n t a i n s about 1 to 2 m i l l i o n p a r t i c l e s and t h e ave rage number o f c a p i l l a r i e s i n the l u n g has been e s t i m a t e d t o be 280 b i l l i o n ( W e i b e l , 1962) o f wh ich p r o b a b l y o n l y 70 b i l l i o n v e s s e l s a re s m a l l enough to t r a p the p a r t i c l e s . The i n j e c t i o n o f a c o u p l e o f m i l l i o n p a r t i c l e s t h e r e f o r e wou ld b l o c k o n l y a v e r y s m a l l f r a c t i o n o f the t o t a l number o f v e s s e l s p r e s e n t i n the l u n g . T h i s was d e m o n s t r a t e d e x p e r i m e n t a l l y by g i v i n g m u l t i p l e i n j e c t i o n s o f m i c r o s p h e r e s to dogs (Reed e t a l . , 1970) . The pu lmonary a r t e r i a l (P ) and l e f t a t r i a l p r e s s u r e s ( P T . ) were a LA m o n i t o r e d so t h a t pu lmonary v a s c u l a r r e s i s t a n c e (P ) was d e t e r m i n e d . p v r i . e . P = (P - P T . ) / C 0 . No changes were n o t i c e d a f t e r m u l t i p l e p v r a LA - 13 -i n j e c t i o n s o f m i c r o s p h e r e s . They f ound t h a t p r e v i o u s i n j e c t i o n s w i t h m i c r o s p h e r e s d i d no t a l t e r the d i s t r i b u t i o n o f subsequent i n j e c t i o n s . The ma jo r f i n d i n g o f R e e d ' s s t u d i e s was t h a t the v e r t i c a l d i s t r i b u t i o n o f pu lmonary b l o o d f l o w i s h i g h l y dependent on g r a v i t y but not f u l l y e x p l a i n e d by t h e S t a r l i n g r e s i s t o r m o d e l . M i c r o s p h e r e i n j e c t i o n s were g i v e n t o a n i m a l s p l a c e d i n s i x d i f f e r e n t body p o s i t i o n s and the same r e g i o n a l f l o w r e l a t i o n s h i p s t o g r a v i t y o b s e r v e d . However, t he g r a v i t y e f f e c t was not f o u n d t o be the s o l e d e t e r m i n a n t s i n c e p l e u r a l g r a d i e n t s and r e g i o n a l d i f f e r e n c e s i n v a s c u l a r tone were sugge s ted as o t h e r f a c t o r s . One o f the n e c e s s a r y r e q u i r e m e n t s o f u s i n g m i c r o s p h e r e s o r m a c r o a g g r e g a t e s i s the p r i o r measurement o f c a r d i a c o u t p u t . In the c a l c u l a t i o n o f r e g i o n a l b l o o d f l o w s i n s m a l l samples o f l u n g , the t o t a l r a d i o a c t i v e coun t s f r om a l l samples a re s e t e q u a l t o the t o t a l f l o w o r the c a r d i a c o u t p u t . The f l o w t o the sample i s c a l c u l a t e d as a f r a c t i o n o f the t o t a l f l o w based on the f r a c t i o n o f the t o t a l c o u n t s i n t h a t sample . A commonly used method f o r m e a s u r i n g b l o o d f l o w i s the d y e - d i l u t i o n o r i n d i c a t o r d i l u t i o n t e c h n i q u e . The t e c h n i q u e i s a l s o known as the S t e w a r t - H a m i l t o n i n d i c a t o r d i l u t i o n t e c h n i q u e a f t e r the two i n v e s t i g a t o r s t h a t f i r s t d e v e l o p e d i t s u s e . S tewar t i n v e s t i g a t e d the v a r i o u s c i r c u l a t i o n t i m e s t h r o u g h the o rgans by i n j e c t i n g s a l t s o l u t i o n s i n t o the c i r c u l a t i o n and m e a s u r i n g the d e l a y t i m e s f o r the s o l u t i o n . Changes i n e l e c t r i c a l r e s i s t a n c e were measured a t the v e s s e l s by e l e c t r o d e s c o n n e c t e d to a g a l v a n o m e t e r ( S t e w a r t , 1 8 9 3 ) . T h i s t e c h n i q u e was l a t e r m o d i f i e d f o r the i n j e c t i o n o f dye and the t h e o r y o f i t s use f o r c a r d i a c o u t p u t d e t e r m i n a t i o n was p r o p o s e d ( S t e w a r t , 1898 ) . By a p p l y i n g the same t e c h n i q u e as used f o r - 14 -m e a s u r i n g c i r c u l a t i o n t i m e s , b l o o d volume i n the l u n g was d e t e r m i n e d by the e q u a t i o n : Q = V / T x 60 V = volume o f b l o o d i n the l u n g s Q = b l o o d f l o w (ml/min) T = mean pu lmonary c i r c u l a t i o n t ime o r s i m p l y , t ime ( s e c ) = b l o o d volume (ml) b l o o d f l o w ( m l / s e c ) R e f i n e m e n t s t o t he i n d i c a t o r d i l u t i o n t e c h n i q u e have r e s u l t e d f r om H a m i l t o n ' s c o n t r i b u t i o n s wh ich i n c l u d e the v e r i f i c a t i o n o f i t s a c c u r a c y by compar i ng c a r d i a c o u t p u t measurements t o t ho se d e t e r m i n e d by F i c k p r o c e d u r e ( H a m i l t o n et_ a l . , 1928; H a m i l t o n e_t a l . , 1919 ) . The d e r i v a t i o n o f the S t e w a r t - H a m i l t o n e q u a t i o n f o r the d e t e r m i n a t i o n o f b l o o d f l o w s , c i r c u l a t i o n t imes and b l o o d vo lumes a re f u l l y d e s c r i b e d i n l i t e r a t u r e ( M e i r e r and Z i e r l e r , 1954; Z i e r l e r , 1962 ) . The v a s c u l a r marker f o r the i n d i c a t o r - d i l u t i o n measurement i n t h i s s t udy i s the r a d i o l a b e l e d r e d b l o o d c e l l (RBC). The r a d i o l a b e l e d RBC has been used p r e v i o u s l y as an i n t r a v a s c u l a r marker i n the i n d i c a t o r - d i l u t i o n t e c h n i q u e ( C h i n a r d and Enns , 1954; Go re sky e_t a l . , 1969; M a r t i n §_t a l . , 1984 ) . The f i r s t i s o t o p e u sed to l a b e l the RBC was "^Chromium (^*Cr) by Gray and S t e r l i n g i n 1951. "^Chromium has a h a l f - l i f e o f 26.5 days and t h e RBC has an a f f i n i t y f o r the l a b e l wh i ch b i n d s to the g l o b i n f r a c t i o n o f h e m o g l o b i n . " ^ C r a l s o b i n d s to p lasma p r o t e i n s and as d e s c r i b e d l a t e r can be u sed to l a b e l l e u k o c y t e s . Wi th t h i s t e c h n i q u e the RBC can be l a b e l l e d b o t h i n v i v o and i n v i t r o and has been used f o r i n t r a v a s c u l a r s u r v i v a l s t u d i e s and f o r imag ing o rgans such as the s p l e e n . The i s o t o p e c u r r e n t l y 99m 99m used i s T e c h n e t i u m ( Tc ) because o f i t s s h o r t h a l f - l i f e o f 6 hour s - 15 -and h i g h e n e r g y . The l a b e l l i n g can be p e r f o r m e d f rom a k i t wh ich i s as q u i c k and s i m p l e as l a b e l l i n g w i t h "**Cr. The r a d i o l a b e l l e d RBC e n a b l e s t h e c a r d i a c o u t p u t and RBC mean t r a n s i t t imes to be d e t e r m i n e d f rom the i n d i c a t o r - d i l u t i o n c u r v e . The l a b e l l e d RBC i s a l s o used as a volume marker f o r e s t i m a t i n g the t o t a l b l o o d volume and r e g i o n a l pu lmonary b l o o d vo lumes ( B a i l e e_t a l . , 1982 ) . R e g i o n a l r e d c e l l t r a n s i t t imes can be d e t e r m i n e d f r om the e q u a t i o n : Time ( sec ) = [ B l ood volume ( m l ) / B l o o d f l o w ( m l / s e c ) ] . The r e g i o n a l b l o o d f l o w s a re measured w i t h f l o w m a r k e r s . T h i s method has been used i n the dog to s tudy the r e d c e l l t r a n s i t t imes i n the norma l l u n g (Hogg e t a l . , 1985 ) . The p o l y m o r p h o n u c l e a r l e u k o c y t e (PMN) can be r a d i o l a b e l l e d l i k e the RBC f o r s t u d y i n g i t s k i n e t i c s i n the body. The most commonly u sed i s o t o p e f o r l a b e l l i n g l e u k o c y t e s i s "^Chromium a l t h o u g h d i i s o p r o p y l f l u o r o p h o s p h a t e 32 111 (DFP ) (A thens e_t a l . , 1959) and more r e c e n t l y , Ind ium ( T h a k u r , 1981) have been u s e d . The t e c h n i q u e f o r l a b e l l i n g l e u k o c y t e s w i t h "*^Cr has been d e s c r i b e d ( M c C a l l §_t a l . , 1955 ) . The l a b e l does not e f f e c t the v i a b i l i t y o f the c e l l as d e m o n s t r a t e d by m o b i l i t y , T r y p a n b l u e e x c l u s i o n , and p h a g o c y t o s i s t e s t s and l a b e l e l u t i o n o r r e u t i l i z a t i o n was no t d e m o n s t r a t e d ( M c M i l l a n and S c o t t , 1955 ) . The r e l a t i v e l a b e l l i n g e f f i c i e n c y w i t h "*^Cr was h i g h e r f o r the l e u k o c y t e than the e r y t h r o c y t e . The r a d i o a c t i v i t y o f one l e u k o c y t e was e q u i v a l e n t to t h a t o f 10-20 e r y t h r o c y t e s ( E y r e e t a l . , 1970 ) . C e l l l a b e l l i n g has a l l o w e d i n v e s t i g a t o r s t o s t udy the k i n e t i c s o f the l e u k o c y t e i n v i v o . F o r example , l a b e l l e d c e l l s were used to e s t i m a t e the - 16 -s i z e o f the c i r c u l a t i n g and m a r g i n a t i n g p o o l s i n the body (A thens e_t a l . , 1961a) and the r e t e n t i o n o f PMNs i n the l u n g have been measured w i t h "**Cr l a b e l l e d PMN i n the dog ( M a r t i n e t a l . , 1984) . S t u d i e s i n the r a b b i t have d e m o n s t r a t e d t h a t i n t r a v e n o u s l y i n j e c t e d c e l l s l a b e l l e d w i t h "**Cr a re f u n c t i o n a l l y norma l i n t h e i r r e s p o n s e to c h e m o t a c t i c s t i m u l i s i t u a t e d o u t s i d e the c i r c u l a t i o n i n the t i s s u e s ( I s s e k u t z , 1981a; I s s e k u t z , 1981b; I s s e k u t z and Movat , 1980; I s s e k u t z and Movat , 1982 ) . The r a b b i t i s a good a n i m a l t o use i n l e u k o c y t e s t u d i e s s i n c e I s s e k u t z ' s work p r o v i d e s e v i d e n c e f o r t h e v i a b i l i t y o f the l a b e l l e d c e l l s and p r e v i o u s s t u d i e s have e s t a b l i s h e d the l i f e span o f the l e u k o c y t e ( W e i s k o t t e n , 1 9 3 0 ) , i t s d i s t r i b u t i o n i n the t i s s u e s (Nye e t &L. , 1932) and the phenomenon o f l e u k o c y t o s i s and l e u k o p e n i a ( W e l l s , 1917 ) . The r a b b i t was s e l e c t e d f o r use i n t h i s s t u d y on the b a s i s o f the p r e v i o u s work done u s i n g l a b e l l e d c e l l s and the numerous advantages o v e r the dog i n terms o f expense and a v a i l a b i l i t y . The same t e c h n i q u e s o f mea su r i n g PMN r e t e n t i o n i n the dog l u n g w i l l be a p p l i e d t o the c u r r e n t i n v e s t i g a t i o n i n the r a b b i t . The o b j e c t i v e o f t h i s s tudy a r e t o i n v e s t i g a t e the e f f e c t o f a c u t e c i g a r e t t e smoke e x p o s u r e on : 1) r e g i o n a l pu lmonary b l o o d f l o w , vo lume, and RBC t r a n s i t t ime 2) r e t e n t i o n o f PMN i n the l u n g . The e f f e c t o f c i g a r e t t e smoke on r e g i o n a l pu lmonary b l o o d f l o w i s o f p a r t i c u l a r i n t e r e s t s i n c e r e c e n t s t u d i e s have shown the r e t e n t i o n o f the PMN i n the l u n g to be i n v e r s e l y r e l a t e d t o b l o o d f l o w ( M a r t i n e t 1982 ) . T h e r e i s a l s o p r e l i m i n a r y e v i d e n c e s u g g e s t i n g t h a t PMN r e t e n t i o n i s d i r e c t l y r e l a t e d t o RBC t r a n s i t t ime (Hogg e t a l . , a b s t r a c t F e d . P r o c e e d i n g s , 1 9 8 5 ) . Changes i n b l o o d f l o w o r RBC t r a n s i t due to smoking c o u l d i n c r e a s e the t ime spent by t h e PMN i n the l u n g and t h e r e f o r e i n c r e a s e the p r o b a b i l i t y f o r t h a t c e l l t o become a c t i v e and r e l e a s e i t s enzymes. I f t h e s e changes i n PMN r e t e n t i o n a r e o n l y s p e c i f i c t o c e r t a i n r e g i o n s i n the l u n g t h i s wou ld p r e d i s p o s e tho se a r e a s t o t he i n c r e a s e d r i s k o f i n j u r y . The pu rpo se o f t h i s s t u d y i s t o i n v e s t i g a t e t h e s e p o s s i b l e mechanisms i n the p a t h o g e n e s i s o f emphysema and t o d e t e r m i n e i f t h e r e a re c o n s i s t e n c i e s w i t h the p a t h o l o g i c a l f i n d i n g s o f t h e d i s e a s e . - 18 -METHODS E x p e r i m e n t s were p e r f o r m e d on New Z e a l a n d a l b i n o w h i t e r a b b i t s r a n g i n g i n w e i g h t f r om 5.0 t o 7.0 k g . B l o o d was c o l l e c t e d f rom the c e n t r a l e a r a r t e r y i n t o 50 ml s t e r i l e p l a s t i c tubes ( C o r n i n g #25320, C o r n i n g , New Y o r k ) u s i n g a 20 gauge n e e d l e . X y l e n e was a p p l i e d t o the ea r t o d i l a t e the a r t e r y and f a c i l i t a t e b l e e d i n g . The a n t i c o a g u l a n t s C i t r a t e Phosphate D e x t r o s e w i t h A d e n o s i n e (CPD-A, C u t t e r L a b o r a t o r i e s , C a l g a r y , A l t a . ) and A c i d C i t r a t e D e x t r o s e (ACD, F r o s s t , M o n t r e a l , P.Q.) were used a c c o r d i n g to the p r o t o c o l s o u t l i n e d be low. A l l s o l u t i o n s were p r e p a r e d f rom s t e r i l e p y r o g e n - f r e e water o r s a l i n e ( B a x t e r - T r a v e n o l L a b o r a t o r y , M a l t o n , O n t a r i o ) and s t e r i l e p l a s t i c w a r e was u s e d . A . EXPERIMENTAL DESIGN Two s t u d i e s were d e s i g n e d t o examine the e f f e c t o f a c u t e c i g a r e t t e smoke e x p o s u r e o n : 1) R e g i o n a l pu lmonary b l o o d f l o w and r e d b l o o d c e l l (RBC) t r a n s i t t imes 2) R e g i o n a l p o l y m o r p h o n u l c e a r l e u k o c y t e (PMN) r e t e n t i o n i n the l u n g . Each s t u d y g roup c o n s i s t s o f a c o n t r o l and a smoke-exposed g roup . The method and d u r a t i o n o f smoke a d m i n i s t r a t i o n were the same i n b o t h o f the s t u d i e s . The r a b b i t s were a n e s t h e t i z e d and s u r g i c a l l y p r e p a r e d as d e s c r i b e d b e l o w . The a n i m a l s were s p o n t a n e o u s l y b r e a t h i n g t h r o u g h o u t the e x p e r i m e n t . C i g a r e t t e smoke was a d m i n i s t e r e d f rom an automated smoking machine ( L a b t r o n i c s , P.Q.) o v e r a t r a c h e o s t o m y tube a t a r a t e o f A p u f f s / m i n . a t a - 19 -volume o f 20 ml ( F i g u r e 8 ) . C i g a r e t t e s were r e f e r e n c e s t a n d a r d s s u p p l i e d by the C a n a d i a n T o b a c c o M a n u f a c t u r e r ' s C o u n c i l w i t h f i l t e r s removed . A p l a s t i c mask was l o o s e l y p l a c e d o v e r the neck a r e a to h e l p c o n c e n t r a t e the smoke. B l o o d s a m p l i n g d i f f e r e d i n the two s t u d i e s and t h e r e f o r e a r e d e s c r i b e d s e p a r a t e l y i n more d e t a i l . 1. Doub le F l o w Marker S t u d i e s The p r o t o c o l i s o u t l i n e d i n F i g u r e 1. The e x p e r i m e n t a l run b e g i n s w i t h the s a m p l i n g o f b l o o d f o r a r t e r i a l b l o o d gases (ABG) and w h i t e b l o o d c e l l (WBC) c o u n t s . The f i r s t i n d i c a t o r - d i l u t i o n run was p e r f o r m e d and f o l l o w e d i m m e d i a t e l y by the i n j e c t i o n o f r a d i o l a b e l l e d mac roagg rega te s (MAA) to mark b l o o d f l o w i n the l u n g . C i g a r e t t e smoke was g i v e n as d e s c r i b e d to the smoke exposed g r o u p . ABG and WBC b l o o d samples were t aken a t 5 and 10 m inu te s a f t e r t he s t a r t o f t r e a t m e n t . A second i n d i c a t o r - d i l u t i o n run was p e r f o r m e d a t 10 m i n u t e s and was f o l l o w e d by the i n j e c t i o n o f a d i f f e r e n t MAA. A p p r o x i m a t e l y 1 m i n u t e a f t e r the MAA i n j e c t i o n , e n d - b l o o d and a WBC b l o o d was t a k e n b e f o r e the a n i m a l was s a c r i f i c e d w i t h p e n t o b a r b i t a l and KC1. 2. R a d i o l a b e l l e d PMN S t u d i e s The p r o t o c o l i s o u t l i n e d i n F i g u r e 2. C o n t r o l ABG and WBC b l o o d samples were t a k e n and then the i n d i c a t o r d i l u t i o n run was p e r f o r m e d . The r a d i o l a b e l l e d PMN was i n j e c t e d as a b o l u s a l o n g w i t h the l a b e l l e d RBC i n the i n d i c a t o r r u n . B l o o d f l o w was marked i m m e d i a t e l y a f t e r the run w i t h the MAA. The t r e a t m e n t g roup then r e c e i v e d c i g a r e t t e smoke f o r 10 m i n u t e s . S e t s o f ABG and WBC samples were taken a t 5 and 10 m inu te s a f t e r the s t a r t o f the smoke e x p o s u r e . The a n i m a l was s a c r i f i c e d a f t e r t he l a s t s e t o f b l o o d samples were t a k e n . - 20 -PROTOCOL I / 2 3 4 5 6 7 8 9 10 jble Blood Flow Marker Studies Time (min) 0 ABG, WBC 0 to 12.5 sec. Indicator-dilution #1 bolus 9 9 m T c RBC 1 MAA #1 Treatment Shorn Controls (Room Air) Smoke ( 4 pufts/min tor 10 min.) 6 ABG, WBC 11 Stop Treatment 12.5 sec. Indicator-dilution #2 bolus 9 9 m T c RBC 12 MAA #2 13+ End-blood. ABG, WBC Pentobarbital, KCI 4 6 TREATMENT 1 2 3 5 7 9 8 10 M A A = M A A a n d 12SI MAA, randomized F i R . 1: PROTOCOL FOR FLOW MARKER STUDIES - 21 -PROTOCOL II PMN Studies 1 2 3 4 Time (min) 0 ABG, WBC 0 to 12.5 sec. Indicator-dilution bolus 9 9 m T c RBC, 5 1 C r 1 1 2 5 l MAA Treatment Sham Controls (Room Air) Smoke ( 4 puffs/min for 10 min.) 5 6 ABG, WBC 6 11 Stop Treatment 7 12+ End-blood, ABG, WBC 8 Pentobarbital, KCI 1 2 3 TREATMENT 7 8 F ie ; . 2: PROTOCOL FOR LABELLED PMN STUDY - 22 -B. CELL LABELLING TECHNIQUES 1. Red B l o o d C e l l (RBC) L a b e l l i n g ; ( F i g u r e 3) A 10 ml sample o f b l o o d was c o l l e c t e d i n t o a 50 ml s t e r i l e p l a s t i c tube c o n t a i n i n g 2.0 ml ACD. A v i a l o f G l u c o s c a n (New E n g l a n d N u c l e a r , NRP-180, L a c h i n e , P .Q. ) was r e c o n s t i t u t e d w i t h 1.0 ml s t e r i l e s a l i n e , 0.3 ml o f the s o l u t i o n was added t o the b l o o d and i n c u b a t e d at room t e m p e r a t u r e (RT) f o r 10 m i n u t e s . The c e l l s were washed t w i c e w i t h s t e r i l e s a l i n e to remove e x c e s s G l u c o s c a n . The packed c e l l s were then r e s u s p e n d e d i n 5 ml s a l i n e 99m c o n t a i n i n g 180 m i c r o C u n e s o f Tc and then i n c u b a t e d f o r 10 m inutes a t RT. The c e l l s were washed o f exce s s l a b e l w i t h s t e r i l e s a l i n e . 2. L e u k o c y t e L a b e l l i n g a . I s o l a t i o n o f L e u k o c y t e s ( F i g u r e 4) A 30 ml vo lume o f b l o o d was c o l l e c t e d i n t o a 50 ml s t e r i l e p l a s t i c tube c o n t a i n i n g 4.2 ml o f CPD a n t i c o a g u l a n t . The b l o o d was added t o 7.5 ml o f 1% h y d r o x y e t h y l c e l l u l o s e (HEC, P o l y s c i e n c e s I n c . , W a r r i n g t o n , P e n n s y l v a n i a ) and g e n t l y m ixed by i n v e r s i o n and a l l o w e d to s t a n d a t 3 7 ° C . (The 1% HEC was p r e v i o u s l y p r e p a r e d by d i s s o l v i n g 0.5 grams o f HEC powder i n 50 ml o f s t e r i l e s a l i n e . The HEC was then d i s s o l v e d by g e n t l e h e a t i n g at 60°C i n a w a t e r b a t h . The s o l u t i o n was c e n t r i f u g e d a t 1000 g t o remove s e d i m e n t , a l i q u o t t e d i n 4.5 and 5.0 ml p o r t i o n s and s t o r e d f r o z e n a t 0°C ) . L e u k o c y t e - r i c h p l a sma (LRP) was h a r v e s t e d f r om the m i x t u r e and p l a c e d i n t o a n o t h e r tube on i c e . Over 30 m i n u t e s , a p p r o x i m a t e l y 20 ml o f LRP was c o l l e c t e d . The LRP was d i l u t e d w i t h an e q u a l volume o f s t e r i l e s a l i n e and l a y e r e d i n two 20 ml p o r t i o n s onto 2 x 10 ml P e r c o l l ( d e n s i t y 1.084 g/ml) i n 50 ml s t e r i l e p l a s t i c t u b e s . T h i s P e r c o l l was p r e p a r e d by add ing 9 vo lumes o f s t o c k P e r c o l l ( P h a r m a c i a , D o r v a l , P.Q.) t o one volume o f 10 t imes - 23 -Radiolabelling Red Blood Cells with 9 9 m Tc 1) Collect 10ml blood, add 2.0ml Acid Citrate Dextrose. 2) Add 0.3ml reconstituted Glucoscan (1.0ml saline). 3) Incubate 10 minutes at room temperature (RT) 4 ) Wash twice in sterile saline 5) Add 180yu & 9 9 m Tc in 5ml saline, incubate 10 min. at RT 6) Wash away excess label F i R . 3: PROCEDURE FOR RADIOLABELLING RBC - 24 -Isolation of Polymorphonuclear Leukocytes 30ml blood + 4.2ml Citrate Phosphate Dextrose 1 l R P Add to 7.5ml 1% Hydroxyethyl Cellulose (HEC). 60°C 11 Remove Leukocyte-rich plasma (LRP), put on ice K r — Add equal volume of saline, total volume 40ml | Layer in two portions of 20ml onto two 10ml Percoll \ ) centrifuge, 600g, 20 minutes R B C + H E C Wash PMN, RBC pellet Hemacytometer count - 25 -( l O x ) i s o t o n i c C a ^ + , M g ^ + - f r e e T y r o d e ' s s o l u t i o n . T h i s s o l u t i o n was d i l u t e d t o 67% (%w/v) w i t h i s o t o n i c ( l x ) C a ^ + , M g ^ + - f r e e T y r o d e ' s s o l u t i o n . The c e l l s were c e n t r i f u g e d a t 600 x g i n a Beckman T J - 6 r e f r i g e r a t e d c e n t r i f u g e f o r 20 m inu te s a t 4 ° C . The p l a sma , monocyte l a y e r and P e r c o l l were c a r e f u l l y removed and d i s c a r d e d . The p e l l e t c o n t a i n i n g the g r a n u l o c y t e s were r e s u s p e n d e d and washed once i n i s o t o n i c ( l x ) C a ^ + , M g ^ + - f r e e T y r o d e ' s s o l u t i o n w i t h 10% (%v/v) p l a t e l e t - p o o r p lasma (PPP ) . A sample o f the c e l l s u s p e n s i o n was c o u n t e d i n a hemacytometer . b. R a d i o l a b e l l i n g w i t h 51Cr ( F i g u r e 5) 8 9 The c e l l s u s p e n s i o n c o n t a i n i n g 10 t o 10 c e l l s was i n c u b a t e d a t 3 7 ° C f o r 30 m inu te s i n 100 m i c r o C u r i e s o f N a ^ C r O ^ i n 1 ml s a l i n e . The c e l l s were washed once i n b u f f e r and than r e s u s p e n d e d to a s m a l l volume ( a p p r o x i m a t e l y 1 m l ) . The c e l l s were then mixed w i t h 21 ml RBC + PPP ( o b t a i n e d f rom the same an ima l ) wh ich was then added t o 5 ml o f 1% HEC. LRP was a g a i n h a r v e s t e d f rom the m i x t u r e o v e r 30 m i n u t e s . The LRP was washed i n b u f f e r t o remove e x c e s s l a b e l . A f i n a l c e l l count was done w i t h a hemacytometer b e f o r e the i n j e c t a t e was p r e p a r e d . C. INJECTATE PREPARATION The i n j e c t a t e p r e p a r e d i n bo th s t u d i e s were kep t a t a volume o f a p p r o x i m a t e l y 0.5 m l . The d o u b l e f l o w marker s t u d i e s r e q u i r e d two i n j e c t a t e s t o be p r e p a r e d w i t h each c o n t a i n i n g a p p r o x i m a t e l y 500,000 coun t s p e r m i n u t e (cpm) o f l a b e l l e d RBC. The r a d i o l a b e l l e d PMN s t u d i e s r e q u i r e d a s i n g l e i n j e c t a t e c o n t a i n i n g the two l a b e l l e d c e l l s (RBC and PMN). The dose o f each i s o t o p e was kep t e q u a l a t a round 300,000 cpm. - 26 -Rodiolobelling Polymorphonuclear Leukocytes with Cr 10 8 to 10 9 cells + 100p 6 5 l C r , incubate 30 min., 37 CC Wash in buffer Add to 21ml RBC + PPP Add to 5ml 1% HEC Collect LRP, put on ice Wash cells in buffer Hemacytometer count Cells into injectate F ie ; . 5: RADIOLABELLING THE PMNS - 27 -A l l i n j e c t a t e was p r e p a r e d i n 3 ml s t e r i l e p o l y p r o p y l e n e tubes ( F a l c o n 2053, O x h a r d , C a l i f o r n i a ) and was drawn up i n t o 1 ml t u b e r c u l i n - t y p e s y r i n g e s . The s y r i n g e s were c o u n t e d and w e i g h t e d b e f o r e and a f t e r i n j e c t i o n t o d e t e r m i n e the c o u n t s and volume i n j e c t e d . D. SURGICAL PREPARATION ( F i g u r e 6) The r a b b i t s were a n e s t h e t i z e d w i t h i n t r a v e n o u s p e n t o b a r b i t a l (30 mg/kg body wt) v i a the m a r g i n a l e a r v e i n . Ma in tenance doses were g i v e n p e r i o d i c a l l y when r e q u i r e d . The v e n t r a l a s p e c t o f the neck was shaven and i n f i l t r a t e d w i t h X y l o c a i n e ( A s t r a P h a r m a c e u t i c a l s L t d . , M i s s i s s a u g a ) . The r a b b i t s were t r a c h e o s t o m i z e d w i t h a t e f l o n c a n n u l a . The c a r o t i d a r t e r y and t h e e x t e r n a l j u g u l a r v e i n were c a n n u l a t e d w i t h p o l y e t h y l e n e c a t h e t e r s ( c a r o t i d : P E - 2 0 0 , j u g u l a r : P E - 2 4 0 , I n t r a m e d i c C l ay -Adams , P e r i s p a n n y , N . J . ) and h e p a r i n was a d m i n i s t e r e d (100 U / k g ) . A l l l i n e s were c o n t i n u o u s l y f l u s h e d w i t h h e p a r i n i z e d s a l i n e (1 U/ml) v i a the C . F . S . I n t r a f l o c o n t i n u o u s f l u s h sy s tem ( S o r e n s o n R e s e a r c h C o . , S a l t Lake C i t y , U t a h ) . A r t e r i a l b l o o d p r e s s u r e was m o n i t o r e d w i t h a p r e s s u r e t r a n s d u c e r ( H e w l e t t - P a c k a r d 1280C) and a permanent t r a c i n g was o b t a i n e d on a c h a r t r e c o r d e r ( H e w l e t t - P a c k a r d mode l 9800, P a l o A l t o , C a l i f o r n i a ) . The s e t - u p i s shown i n F i g u r e 9. - 28 -F I R . 6: SURGICAL PREPARATION - 29 -E. INDICATOR-DILUTION A b o l u s o f l a b e l l e d c e l l s were i n j e c t e d i n t o the c a t h e t e r l e a d i n g t o the RA and q u i c k l y f l u s h e d i n t o the c i r c u l a t i o n w i t h 2 ml o f h e p a r i n i z e d s a l i n e . B l o o d was sampled f rom the c a r o t i d a r t e r y i n t o p r e w e i g h e d tubes and c o l l e c t e d f o r 12.5 s e c o n d s . A r o t a t i n g t u r n t a b l e on wh ich the tubes were p l a c e d e n a b l e d the b l o o d t o be sampled e v e r y 0.5 seconds p e r tube ( F i g u r e s 7 and 1 0 ) . F r a c t i o n c o l l e c t o r c o n s t r u c t i o n was ba sed on p l a n s by K i r k e t a l . ( 1 9 6 9 ) . The b l o o d was c o u n t e d i n a gamma c o u n t e r (Beckman model 7000, P a l o A l t o , C a l i f o r n i a ) and we i ghed . An i n d i c a t o r - d i l u t i o n c u r v e was c o n s t r u c t e d and t h e c a r d i a c o u t p u t was c a l c u l a t e d f rom the c u r v e . F. MEASURING REGIONAL PULMONARY BLOOD FLOW R e g i o n a l pu lmonary b l o o d f l o w was marked u s i n g r a d i o l a b e l l e d m a c r o a g g r e g a t e s o f human serum a lbumin (MAA, F r o s s t , P o i n t e C l a i r e , P . Q . ) . 125 131 MAA l a b e l l e d w i t h I and I were used i n the f l o w s t u d i e s . A s e p a r a t e s e r i e s o f e x p e r i m e n t s were p e r f o r m e d where a s i n g l e b o l u s o f bo th MAAs were i n j e c t e d . These e x p e r i m e n t s showed t h a t the MAA l a b e l l e d w i t h d i f f e r e n t i s o t o p e s d i s t r i b u t e i n the l u n g i d e n t i c a l l y when i n j e c t e d i n the l u n g a t the same t i m e . The o r d e r o f i n j e c t i o n o f the two MAA was r andomized i n t h e s e s t u d i e s . The MAA were i n j e c t e d i m m e d i a t e l y a f t e r the i n d i c a t o r - d i l u t i o n runs as o u t l i n e d i n the E x p e r i m e n t a l D e s i g n s e c t i o n above. A p p r o x i m a t e l y 1.5 to 2.5 125 131 m i c r o C u r i e s o f I and I MAA c o n t a i n i n g up to 75,000 p a r t i c l e s were i n j e c t e d pe r r u n . P a r t i c l e s i z e s r anged f r om 5 and 20 m i c r o n s . On ly one 125 f l o w marker was r e q u i r e d f o r the l a b e l l e d PMN s t u d i e s and I MAA was u s e d . - 30 -F ie, . 7: THE FRACTION COLLECTOR - 31 -F i g . 8: THE SMOKING APPARATUS - 32 -F i R . 9: THE EXPERIMENTAL SET-UP - 33 -F i g . 10: INDICATOR-DILUTION RUN - 34 -Lung, Removal and Sampling; J u s t b e f o r e s a c r i f i c e , a r e f e r e n c e b l o o d sample and samples f o r b l o o d gas and WBC d e t e r m i n a t i o n s were t a k e n . Deep a n a e s t h e s i a was i n d u c e d w i t h sod ium p e n t o b a r b i t a l and the h e a r t was s t o p p e d by g i v i n g an i n j e c t i o n o f h y p e r t o n i c KC1 i n t o the c a r o t i d a r t e r y . The t h o r a x was q u i c k l y opened and the s t e r n u m was s p l i t . The r i b s were s p r e a d and the p e r i c a r d i a l sac was p i c k e d up and d i s s e c t e d t o expose the h e a r t . A f i r m s i l k l i g a t u r e was t i e d a round the base o f the h e a r t t o p r e v e n t b l o o d f rom l e a v i n g o r e n t e r i n g the ma jo r v e s s e l s a t the h i l u m . The l u n g was c a r e f u l l y l i f t e d out o f the t h o r a x towards the head as t he t r a c h e a was d i s s e c t e d f r e e . The h e a r t was c u t away, a c a n n u l a p l a c e d i n t he t r a c h e a and the l u n g s were i n f l a t e d w i t h a i r to a p o s i t i v e p r e s s u r e o f 25 cm H O (P ). The t r a c h e a was then c lamped and t h e l u n g s were r a p i d l y f r o z e n o v e r l i q u i d n i t r o g e n ( F i g u r e 1 1 ) . The l u n g was s l i c e d c o r o n a l l y , f rom p o s t e r i o r t o a n t e r i o r s u r f a c e s , on b l o c k s o f d r y i c e ( F i g u r e 1 2 ) . Each s l i c e was then c u t i n t o s m a l l e r samples and p l a c e d i n t o p r e w e i g h e d g l a s s s c i n t i l l a t i o n v i a l s ( F i g u r e 13 and 1 4 ) . These v i a l s were t h e n gamma c o u n t e d and r e w e i g h e d . - 35 -Fie,. 11: FROZEN INFLATED LUNG Fie.• 12: FROZEN SLICED LUNG FJR. 13: SAMPLING LUNG SLICES - 38 -F i K . 14: SAMPLED LUNG IN VIALS - 39 -DATA HANDLING AND CALCULATIONS A . CORRECTING GAMMA COUNTS The raw gamma c o u n t s were c o r r e c t e d f o r backg round and o v e r l a p . These c a l c u l a t i o n s were done w i t h an A p p l e computer s o f t w a r e program w r i t t e n f o r t h e Beckman model 7000 gamma c o u n t e r . T h i s program a l l o w s the c o u n t e r to work a t s p e c i f i e d window s e t t i n g s wh i ch can be changed to s u i t the i s o t o p e u s e d . B. INDICATOR-DILUTION CURVES Count s f rom the i n d i c a t o r d i l u t i o n d a t a was c o r r e c t e d f o r background and o v e r l a p and t h e n was n o r m a l i z e d to the f r a c t i o n o f i n j e c t a t e v a l u e . U n - n o r m a l i z e d c o u n t s = F r a c t i o n o f i n j e c t a t e x 10^. ( i n j . c t s . / i n j . w t . ) x sample wt. The n o r m a l i z e d d a t a was then p l o t t e d a g a i n s t t ime (o r tube number) t o g i v e an i n d i c a t o r - d i l u t i o n c u r v e . A r e a s o f each c u r v e were d e t e r m i n e d to the p o i n t o f RBC r e c i r c u l a t i o n f o r the % R e c o v e r y . The c a r d i a c o u t p u t and mean t r a n s i t t i m e s were d e t e r m i n e d f rom the c u r v e w i t h the a i d o f a computer p r o g r a m . C. CALCULATING BLOOD FLOW. BLOOD VOLUME AND TRANSIT E n d - b l o o d sample gamma coun t s ( c t s ) f o r each i s o t o p e used were e x p r e s s e d as a r a t i o o f t he RBC l a b e l c o u n t s i n the e n d - b l o o d sample ( e . g . 99m 125 T c / I ) . Each r a t i o was d i v i d e d i n t o the l u n g sample RBC c t s t o o b t a i n the c i r c u l a t i n g o r f r e e l a b e l c o u n t s a s s o c i a t e d w i t h the MAA or PMN. - 40 -These f r e e c o u n t s were s u b t r a c t e d away f rom the sample MAA and PMN coun t s t o y i e l d t he n o n - c i r c u l a t i n g MAA and PMN c o u n t s . The n o n - c i r c u l a t i n g coun t s were t h e n used t o c a l c u l a t e the b l o o d f l o w or PMN r e t e n t i o n i n each sample . B l o o d f l o w i n each sample was c a l c u l a t e d as f o l l o w s . A l l the 125 131 n o n - c i r c u l a t i n g MAA coun t s i n the l u n g were summed ( I and I t o t a l l e d s e p a r a t e l y i n the f l o w s t u d y ) . These t o t a l coun t s were d i v i d e d by t h e i r c o r r e s p o n d i n g c a r d i a c o u t p u t measurement f o r the run as d e t e r m i n e d by i n d i c a t o r - d i l u t i o n . T h i s v a l u e d e t e r m i n e d f o r each MAA i s then used to c a l c u l a t e the f l o w i n e v e r y sample o f l u n g by d i v i d i n g the MAA count i n the sample by the v a l u e . The f l o w to each sample was then n o r m a l i z e d to b l o o d - f r e e wet l u n g we i gh t and ave rages were c a l c u l a t e d f o r each s l i c e o f l u n g . B l o o d vo lumes i n each sample were c a l c u l a t e d by d i v i d i n g the RBC gamma c o u n t s by the e n d - b l o o d s p e c i f i c a c t i v i t y f o r the RBC ( i . e . RBC c t s / g b l o o d ) . B l o o d w e i g h t i n grams were then c o n v e r t e d t o volume i n m l . Red c e l l t r a n s i t t imes were c a l c u l a t e d f o r each sample by d i v i d i n g the b l o o d vo lume by the b l o o d f l o w . B l o o d volume (ml) = T r a n s i t t ime ( s e c ) B l o o d f l o w ( m l / s e c ) D. PMN RETAINED. PMN DELIVERED AND % RETENTION F o r t he l a b e l l e d PMN s t u d i e s , t he number o f PMN r e t a i n e d i n each l u n g s l i c e was c a l c u l a t e d as f o l l o w s . The s p e c i f i c a c t i v i t y o f the l a b e l l e d PMN was c a l c u l a t e d . A . t o t a l PMN gamma c t s i n j e c t e d = PMN gamma c t s C a r d i a c o u t p u t (ml /min) (ml /min) - 41 -B. t o t a l PMN gamma c t s i n j e c t e d = PMN gamma c t s I n j e c t i o n volume (ml) (ml) B was d i v i d e d by the hemacytometer coun t s (#PMN/ml) t o g i v e the number o f gamma c o u n t s pe r PMN r e t a i n e d = C. The c e l l l a b e l coun t s i n each sample were d i v i d e d by C t o o b t a i n the number o f l a b e l l e d c e l l s r e t a i n e d i n the s amp le . PMN r e t a i n e d v a l u e s were n o r m a l i z e d f o r b l o o d - f r e e wet l u n g we i gh t and a v e r a g e by s l i c e . The d e l i v e r y was c a l c u l a t e d f rom each sample b l o o d f l o w . The d e l i v e r y c o n s t a n t D was c a l c u l a t e d f rom p r e v i o u s l y d e t e r m i n e d v a l u e s A and C. D=A/C=PMN d e l i v e r e d per m l /m in b l o o d f l o w By m u l t i p l y i n g each sample b l o o d f l o w by D the number o f PMN d e l i v e r e d to t h e sample i s c a l c u l a t e d . P e r c e n t PMN r e t e n t i o n i s c a l c u l a t e d a s : % R e t e n t i o n = (PMN r e t a i n e d / P M N d e l i v e r e d ) x 100%. E. ESTIMATING TOTAL BLOOD VOLUME AND LABELLED PMN DISTRIBUTION The e n d - b l o o d RBC gamma c o u n t s i n c t s / g b l o o d was c o n v e r t e d t o g/ml b l o o d by m u l t i p l y i n g by 1.06 ( a p p r o x i m a t e d e n s i t y o f b l o o d ) . The t o t a l RBC c o u n t s i n t he i n j e c t a t e was n o r m a l i z e d t o c t s / m l by d i v i d i n g the t o t a l c o u n t s by the i n j e c t a t e vo lume. The volume d i s t r i b u t i o n e q u a t i o n was used where c-^^ = C 2 V 2 ' T ^ e C l a n c * C 2 v a ^ u e s a r e *-he i n j e c t a t e and e n d - b l o o d RBC c t s / m l , r e s p e c t i v e l y . V^ i s the i n j e c t a t e volume and the e q u a t i o n i s s o l v e d f o r V^. T h i s v a l u e i s the e s t i m a t e f o r the t o t a l RBC volume o f d i s t r i b u t i o n o r t o t a l b l o o d vo lume. The body d i s t r i b u t i o n o f l a b e l l e d PMN was c a l c u l a t e d f o r each organ f rom the t o t a l c oun t s r e c o v e r e d f rom each o f the f o l l o w i n g t i s s u e s : l u n g , l i v e r , s p l e e n , and b l o o d . The d i s t r i b u t i o n i n the b l o o d i s c a l c u l a t e d f rom the PMN gamma c o u n t s per gram e n d - b l o o d m u l t i p l i e d by the RBC volume o f d i s t r i b u t i o n as d e t e r m i n e d p r e v i o u s l y . The PMN count s r e c o v e r e d i n each organ i s e x p r e s s e d as a p e r c e n t a g e o f t he amount i n j e c t e d . F. STATIST ICAL ANALYSES P a i r e d s t u d e n t s t - t e s t s were used i n t he compar i s on o f d a t a i n v o l v i n g the two measurements ( e . g . i n i t i a l and f i n a l b l o o d f l o w s ) i n the same an ima l o r sample o f l u n g . T h i s t e s t was chosen s i n c e each i n i t i a l b l o o d f l o w s e r v e s as t he c o n t r o l f o r the f i n a l measurement. The s i g n i f i c a n c e i n each s l i c e was s e t a t P-<Co.01 and a s s i g n e d a s i g n i f i c a n c e o f p<^0.05 i f i t met t h e c r i t e r i a o f p ^ O . O l . T h i s was because m u l t i p l e t e s t s were pe r f o rmed and the B o n f e r r o n i c o r r e c t i o n was r e q u i r e d . The u n p a i r e d t - t e s t was used i n a l l t he g rouped d a t a ( e . g . LBV, C O . e t c . ) f o r c o m p a r i s o n between the c o n t r o l s and t r e a t m e n t s . Examples o f t he se a r e d a t a i n T a b l e s I I I , IV, and V. S i g n i f i c a n c e was s e t a t p ^ 0 . 0 5 . The s l i c e d a t a ( i . e . b l o o d f l o w , b l o o d vo lume, RBC t r a n s i t t ime and PMN r e t e n t i o n ) c o m p a r i s o n a c r o s s g roups was p e r f o r m e d u s i n g two-way a n a l y s i s o f v a r i a n c e (AN0VA) w i t h one f a c t o r b e i n g t r e a t m e n t , the o t h e r l u n g s l i c e . A B o n f e r r o n i c o r r e c t i o n was p e r f o r m e d where m u l t i p l e t e s t s were made. - 43 -RESULTS A. BLOOD FLOW STUDY 1) S i n g l e B o l u s o f Two D i f f e r e n t Mac roagR reRa te s . The r e g i o n a l pu lmonary b l o o d f l o w was measured s i m u l t a n e o u s l y w i t h two 125 131 d i f f e r e n t r a d i o l a b e l l e d macroagg rega te s ( I and I MAA) by i n j e c t i n g t h e two t o g e t h e r as a s i n g l e b o l u s . The b l o o d f l o w s f rom one MAA was then compared t o the o t h e r by p l o t t i n g the f l o w f o r e v e r y p i e c e o f l u n g so t h a t each MAA d e t e r m i n e d f l o w i s on a d i f f e r e n t a x i s ( F i g . 1 5 ) . Each p o i n t on the g raph r e p r e s e n t s a s i n g l e l u n g sample . A s l o p e o f 1.00 wou ld be e x p e c t e d i f each MAA marks the b l o o d f l o w i d e n t i c a l l y . The r e g r e s s i o n l i n e 2 f r om the p l o t o f t he l u n g p i e c e s has a s l o p e o f 0.99 and an R v a l u e o f 0 . 9 7 . The d i s t r i b u t i o n o f the d a t a p o i n t s c l o s e l y a round the l i n e o f i d e n t i t y shows t h a t the two MAA used i n t h i s s t udy mark the same r e g i o n a l b l o o d f l o w s i d e n t i c a l l y . - 44 -SINGLE BOLUS INJECTION OF TWO MACROAGGREGATES I MAA BLOOD FLOW (ml/min) FIGURE 15 _ 45 -2) Two Mac roagRreRa te s I n j e c t e d Ten M i n u t e s A p a r t The change i n b l o o d f l ows to each sample a c r o s s ten m inu te s o f sham smoking was measured by g i v i n g an i n i t i a l and a f i n a l MAA i n j e c t i o n . The o r d e r o f MAA i n j e c t i o n on e i t h e r s i d e o f t e n m inu te s was r a n d o m i z e d . The b l o o d f l o w s to each sample a t the i n i t i a l and f i n a l measurements were p l o t t e d a g a i n s t each o t h e r ( F i g . 16) and a r e g r e s s i o n l i n e was c a l c u l a t e d . The change i n c a r d i a c o u t p u t ( C 0 ( 2 ) / C 0 ( l ) ) o v e r the ten m inu te s was 0.74 i n t he examp le . The s l o p e d e t e r m i n e d by l i n e a r r e g r e s s i o n was 0.80 w i t h an 2 R o f 0 . 9 2 . T h i s d a t a shows t h a t each p i e c e o f l u n g i n the c o n t r o l s i t u a t i o n t e n d e d t o s h a r e e q u a l l y i n the change i n b l o o d f l o w as p r e d i c t e d by the change i n c a r d i a c o u t p u t . The f r a c t i o n a l change i n c a r d i a c o u t p u t (CO (2 ) /CO(1 ) ) f o r a l l t he f l o w e x p e r i m e n t s were compared to the s l o p e s o f t he i n i t i a l v s . f i n a l b l o o d f l o w 2 p l o t s ( T a b l e 1 ) . The s l o p e s and R v a l u e s were d e t e r m i n e d by l i n e a r r e g r e s s i o n . T a b l e 1 shows the c a r d i a c o u t p u t s (ml /min) f o r the sham smoke (N=5) and the smoke exposed (N=5) groups as d e t e r m i n e d by i n d i c a t o r - d i l u t i o n . The i n i t i a l ( C O ( D ) , f i n a l (C0 (2 ) ) c a r d i a c o u t p u t and the d i f f e r e n c e (ACO = CO(2) - C O ( l ) ) a r e shown. The h e a r t r a t e a t t e n m inu te s i s a l s o shown. T h e r e were no s i g n i f i c a n t d i f f e r e n c e s between the c o n t r o l and smoke groups i n any o f C 0 ( 1 ) , C O ( 2 ) , ACO, and HR by u n p a i r e d t - t e s t s . - 46 -SHAM-SMOKE CONTROLS (N=5) n C0(1) C0(2) ACO HR C 0 ( 2 ) / C 0 ( l ) S l o p e R 2 1 581.8 430.8 -151 294 0.74 0.80 0.92 2 851.4 706.9 -145 192 0.83 0.79 0.92 3 617 .8 580.8 -37 246 0.94 0.84 0.76 4 713.9 755.2 +42 276 1.06 1.21 0.82 5 633.2 646.0 +13 240 1.02 1.16 0 .79 mean 679.6 623.9 -53 250 0.92 0.96 0.84 SE 48.1 56.5 40 18 0.06 0.09 0.03 SMOKE EXPOSED (N=5) n C0(1) C0(2) ACO HR C 0 ( 1 ) / C 0 ( 1 ) S l o p e R 2 1 546.8 450.9 -97 270 0.82 0.87 0.89 2 841.4 721.4 -120 260 0.86 0.78 0.55 3 671.7 622.7 -49 240 0.93 0.88 0.43 4 939.3 1019.1 +80 318 1.08 1.08 0.66 5 906.8 922.4 +16 320 1.02 1.00 0.78 mean 781.2 747.3 -34 281 0.94 0.92 0.66 SE 74.6 102.1 37 16 0.05 0.05 0.08 T a b l e 1: DATA FROM DOUBLE FLOW MARKER STUDIES I n i t i a l and f i n a l c a r d i a c o u t p u t i n m l /m in ( C 0 ( 1 ) , C 0 ( 2 ) ) and the change i n c a r d i a c o u t p u t , ACO = C 0 ( 2 ) - C 0 ( l ) . H e a r t r a t e at 10 m inu te s = HR. C 0 ( 2 ) / C 0 ( l ) i s t he f r a c t i o n a l change i n c a r d i a c o u t p u t f rom C0(1) t o CO (2 ) . The s l o p e and the R 2 a re the r e g r e s s i o n v a l u e s f rom i n i t i a l v e r s u s f i n a l l u n g sample b l o o d f l o w p l o t s ( F i g . 16 i s e x a m p l e ) . - 47 -F ie , . 16 : Two Macroag,g,ree,ates I n j e c t e d Ten M i n u t e s A p a r t - 48 -3) R e g i o n a l Lung, Da ta F i g u r e s 17 to 22 a re p l o t t e d to show each o f the f o l l o w i n g p a r a m e t e r s : i n i t i a l and f i n a l b l o o d f l o w , b l o o d vo lume, and RBC t r a n s i t t ime as a f u n c t i o n o f the l u n g h e i g h t o r s l i c e number. F i v e l u n g s l i c e s a re p l o t t e d a l o n g the y - a x i s f rom the p o s t e r i o r ( S l i c e 1) t o t he a n t e r i o r ( S l i c e 5) s u r f a c e s o f the l u n g i n a s u p i n e r a b b i t . Each p o i n t r e p r e s e n t s the mean v a l u e f o r the s l i c e w i t h s t a n d a r d e r r o r s , a) Pu lmonary B l o o d F low The changes i n r e g i o n a l pu lmonary b l o o d f l o w a f t e r t e n m inu te s o f sham ( F i g . 17) and smoke e x p o s u r e ( F i g . 18) a re not t he same. S l i c e f l o w s a c r o s s runs ( i n i t i a l vs f i n a l ) w i t h i n a group were compared u s i n g the p a i r e d t - t e s t s i n c e each s l i c e had an i n i t i a l f l o w to s e r v e as i t s c o n t r o l . The sham c o n t r o l g roup shows a u n i f o r m f a l l i n b l o o d f l o w t o most s l i c e s but o n l y s l i c e s 3 and 4 ( a n t e r i o r ) had s i g n i f i c a n t ( p ^ 0 . 0 5 ) d e c r e a s e s . The s l i c e s 1, 2, and 5 d i d no t show any s i g n i f i c a n t changes . The c a r d i a c o u t p u t changed to 92 + 6% o f b a s e l i n e o v e r the t e n m i n u t e s . - 49 -- 50 -The b l o o d f l o w t o s l i c e s 4 and 5 a n t e r i o r l y d e c r e a s e d i n the smoke expo sed g roup (p<^0.05) w h i l e s l i c e s 2 and 3 i n the m i d d l e r e g i o n d i d not show s i g n i f i c a n t change ( F i g . 1 8 ) . The p o s t e r i o r s l i c e ( S l i c e 1) t ended t o have g r e a t e r f l o w bu t t h e s e were no t s i g n i f i c a n t . The c a r d i a c o u t p u t f o l l o w i n g t e n m i n u t e s o f smoking changed to 94 + 5% o f b a s e l i n e f o r the smoke expo sed g r o u p . The b l o o d f l o w s t o each s l i c e were a l s o compared by run ( i . e . p r e - t r e a t m e n t vs p o s t - t r e a t m e n t ) between the c o n t r o l and smoke groups by 2-way ANOVA. F o r t he p r e - t r e a t m e n t r u n , c o n t r o l and smoke groups d i d not show s i g n i f i c a n t d i f f e r e n c e s a c r o s s groups e x c e p t i n S l i c e 1 where the smoke g roup had h i g h e r b l o o d f l o w (p<^0.05) than the c o n t r o l g roup ( F i g . 1 9 ) . The p o s t - t r e a t m e n t b l o o d f l o w s were l ower a n t e r i o r l y i n S l i c e 5 f o r the smoke g roup compared t o the c o n t r o l g roup ( F i g . 20) w i t h p < £ 0 . 0 1 . The b l o o d f l o w i n the p o s t e r i o r s l i c e s ( S l i c e 1 and 2) were s i g n i f i c a n t l y i n c r e a s e d ( p ^ O . 0 1 ) w h i l e t h e m i d d l e s l i c e s ( S l i c e s 3 and 4) were not s i g n i f i c a n t l y d i f f e r e n t . - 51 -- 52 -BLOOD FLOW (ml/min/gm) - 53 -- 54 -b) Pu lmonary b l o o d volume S l i c e b l o o d vo lumes (m l / g b l o o d - f r e e l u n g ) between the sham and smoke g r o u p s were compared by 2-way ANOVA ( F i g . 2 0 ) . The b l o o d volumes were s i g n i f i c a n t l y g r e a t e r i n t he smoke group f o r S l i c e s 2 ( p - ^ . 0 0 1 ) , 3 (p<C 0 . 0 0 1 ) , and 4 (p<C.0.001). The b l o o d vo lumes i n s l i c e s 1 and 5 were not d i f f e r e n t a c r o s s t he g r o u p s . c ) RBC T r a n s i t t ime The RBC t r a n s i t t ime was g r e a t e r i n t he smoke exposed an ima l s i n a l l s l i c e s e x c e p t s l i c e 1 ( F i g . 2 1 ) . S l i c e s 2 , 3 , 4 , had l o n g e r t r a n s i t t imes f o r t h e smokers ( p < £ . 0 . 0 0 1 ) . In b o t h g r o u p s , t he l o n g e s t t r a n s i t t imes were i n the a n t e r i o r - m o s t r e g i o n ( S l i c e 5) where the RBC t r a n s i t t ime was 1.58 + 0 .34 s e c . i n the c o n t r o l g roup and 5.25 + 1.11 sec i n the smoke g roup . The t r a n s i t t ime i n the smoke group was s i g n i f i c a n t l y g r e a t e r than the c o n t r o l t r a n s i t t ime (p<C .0.05). - 55 -POSTERIOR t 0 - I I J L. 0.3 0.6 0.9 1.2 BLOOD VOLUME (ml/gm) J 1.5 POSTERIOR 1 , 1 1 1 0 1.0 2.0 3.0 4.0 RBC TRANSIT TIME (sec) J 5.0 - 57 -B. RADIOLABELLED PMN STUDY 1) I n d i c a t o r - d i l u t i o n c u r v e d a t a The i n d i c a t o r - d i l u t i o n c u r v e d a t a i s shown i n t a b l e I I . The mean t r a n s i t t ime f o r t he l a b e l l e d RBC and PMN th rough the c e n t r a l c i r c u l a t i o n and t h e c a r d i a c o u t p u t s were o b t a i n e d f r om the i n d i c a t o r c u r v e s . These d a t a were compared a c r o s s the two groups by u n p a i r e d t - t e s t s and no s i g n i f i c a n t d i f f e r e n c e s were f o u n d . 2) R e g i o n a l Lung Data a) R e g i o n a l Pu lmonary B l o o d F low The r e g i o n a l pu lmonary b l o o d f l o w i n t h i s s tudy was measured o n l y once ( a t the t ime o f the l a b e l l e d PMN i n j e c t i o n ) so t h a t the PMN d e l i v e r y c o u l d be c a l c u l a t e d f rom the b l o o d f l o w . S i n c e the measurements were done b e f o r e the t r e a t m e n t p e r i o d , the b l o o d f l o w s by s l i c e f o r the two g roups were no t d i f f e r e n t by AN0VA. T h i s r e s u l t i s s i m i l a r t o t he compar i s on o f t h e p r e - t r e a t m e n t runs i n P a r t A ( B l o o d F low S t u d y ) . The r e g i o n a l b l o o d f l o w d a t a i s t a b u l a t e d i n T a b l e I I I . U n f o r t u n a t e l y , a second f l o w marker was no t u sed t o measure the changes t o f l o w f o l l o w i n g the t r e a t m e n t as i n P a r t A because we were t e c h n i c a l l y l i m i t e d t o t h r e e i s o t o p e s . - 58 -CONTROL (N=5) SMOKE (N=5) Mean t r a n s i t t i m e : RBC ( s e c ) 5 . 2 + 0 . 3 5 . 9 + 0 . 2 PMN ( s e c ) 9 . 7 + 0 . 9 8 . 5 + 1 . 2 C O . (m l /min ) 674.4 + 51.1 684.1 + 81 .9 TABLE I I : I n d i c a t o r - d i l u t i o n c u r v e d a t a (mean + SE) - 59 -The r e g i o n a l RBC t r a n s i t t imes were not o b t a i n e d i n t h i s s tudy because t h e b l o o d f l o w measurement was made t e n m inu te s b e f o r e s a c r i f i c e , t he p o i n t when t h e b l o o d volume was measured ( F i g . 2 ) . S i n c e the p r e v i o u s s t udy showed t h a t r e g i o n a l b l o o d f l o w changes o v e r the t e n m inu te p e r i o d , the b l o o d f l o w c o u l d not be used w i t h the b l o o d volume to c a l c u l a t e the r e g i o n a l RBC t r a n s i t t i m e . b) R e g i o n a l Pu lmonary B l o o d Volume As i n the b l o o d f l o w s t u d y , the pu lmonary b l o o d vo lumes were measured u s i n g the l a b e l l e d RBC a t the t ime o f s a c r i f i c e . The smoke exposed a n i m a l s had s i n g i f i c a n t l y g r e a t e r b l o o d vo lumes i n the p o s t e r i o r and m i d d l e s l i c e s ( T a b l e I I I ) . S l i c e s 1,3 and 4 were s i g n i f i c a n t l y d i f f e r e n t (p<^0.001) by ANOVA ( S l i c e s 2 ,3 ,4 were g r e a t e r i n the smoke group i n P a r t A ) . These two s t u d i e s have c o n s i s t e n t r e s u l t s wh i ch show t h a t f o l l o w i n g smoke e x p o s u r e , the r e g i o n a l pu lmonary b l o o d volume i n c r e a s e s o v e r a l l e s p e c i a l l y i n the m i d d l e and p o s t e r i o r r e g i o n s . c ) R e g i o n a l P o l y m o r p h o n u c l e a r L e u k o c y t e R e t e n t i o n PMN r e t e n t i o n v a r i e d r e g i o n a l l y i n the l u n g such t h a t the r e t e n t i o n i n c r e a s e d w i t h the v e r t i c a l h e i g h t o f t he l u n g s l i c e ( T a b l e I I I ) . The p o s t e r i o r and m i d d l e l u n g s l i c e s had PMN r e t e n t i o n s o f a p p r o x i m a t e l y 30%. Towards the a n t e r i o r s u r f a c e the r e t e n t i o n i n c r e a s e s t o about 70% i n t h e sham c o n t r o l g roup . A compar i son o f PMN r e t e n t i o n a c r o s s the c o n t r o l and smoke g roups by s l i c e u s i n g ANOVA showed no s i g n i f i c a n t d i f f e r e n c e s . - 60 -SHAM SMOKE CONTROLS (N=5) SMOKE EXPOSED (N=5) S l i c e B l o o d F low ( m l / m i n / g b l o o d - f r e e l u n g ) 1 2 3 4 5 68 .0 + 7.0 73.4 + 5.9 73.7 + 5.9 52.5 + 7.2, 30.3 + 5.6 72,6 + 6.8 75.6 + 4 .9 79.3 + 6.0 60.6 + 5.7 39 .9 + 6.7 S l i c e B l o o d Volume (m l / g g l o o d - f r e e l u n g ) 1 2 3 4 5 0.551 0.733 0.554 0.496 0.526 + 0.033 + 0.047 + 0.034 + 0.044 + 0.063 0.731 + 0.042 * 0.767 + 0.044 0.748 + 0.048 * 0.655 + 0.077 * 0.553 + 0.085 S l i c e % R e t e n t i o n 32.2 + 3.9 29.7 + 2.7 32.5 + 4 .0 46.4 + 4 .9 72.1 + 7.5 36.2 + 3.4 32.4 + 3.3 39.6 + 4.4 53.7 + 6.1 64.1 + 6.6 T a b l e I I I : R e g i o n a l Lung B l o o d F l o w , B l o o d Vo lume, and PMN R e t e n t i o n The r e g i o n a l d a t a f o r b l o o d f l o w , b l o o d volume and PMN r e t e n t i o n i n f i v e s l i c e s . S l i c e 1 i s p o s t e r i o r and S l i c e 5 i s a n t e r i o r . The mean + SE a r e shown. The a s t e r i s k i n d i c a t e s p<^0.05 f o r compar i s on s a c r o s s g roups by ANOVA. - 61 -b) D i s t r i b u t i o n o f L a b e l l e d PMN i n the T i s s u e s T a b l e IV shows the t i s s u e d i s t r i b u t i o n o f the l a b e l l e d PMN i n the sham c o n t r o l and smoke exposed groups a t ten m inu te s a f t e r i n j e c t i o n . The p e r c e n t a g e o f t he i n j e c t a t e r e c o v e r e d i n each t i s s u e and the t o t a l r e c o v e r y a r e shown. F o r b o t h g r o u p s , the amount r e c o v e r e d f rom each t i s s u e i n o r d e r f r om l o w e s t t o h i g h e s t was s p l e e n , b l o o d , l u n g , and l i v e r . The smoke group had t h e l a r g e r p r o p o r t i o n o f l a b e l l e d c e l l s i n the b l o o d ( C o n t r o l : 13.7 + 1.3%, Smoke: 22 .2 + 4.8%) w h i l e the r e v e r s e was t r u e i n the l i v e r ( C o n t r o l : 35 .8 + 2.0%, Smoke: 24.7 + 2 .7%) . Compar i sons were made w i t h a u n p a i r e d t - t e s t f o r p/^.0.05. The d e c r e a s e i n c e l l s i n the l i v e r o f the smokers was s i g n i f i c a n t t o p<C.0.01 w h i l e the b l o o d i n c r e a s e was not s i g n i f i c a n t . I t a p p e a r s t he i n c r e a s e i n l a b e l l e d c e l l s i n t he b l o o d o f smokers comes f rom t h e l i v e r s i n c e the amounts i n the o t h e r o rgans d i d no t change s i g n i f i c a n t l y . - 62 -C o n t r o l s (N=5) SMOKE (N=5) p_ Lung 29.0 + 1.3 30 .0 + 4.5 N.S. B l o o d ' 13.7 + 1.3 22.2 + 4.8 N.S. L i v e r 35.8 + 2.0 24.7 + 2.7 p < 0 . 0 1 S p l e e n 3.3 + 0.7 2.1 + 0 .6 N.S. T o t a l R e c o v e r y 8 1 . 9 + 1 . 4 7 8 . 7 + 3 . 1 N.S. T a b l e IV: D i s t r i b u t i o n o f L a b e l l e d PMN i n the T i s s u e s Compar i s on o f t he d i s t r i b u t i o n o f l a b e l l e d PMN r e c o v e r e d i n the t i s s u e s . V a l u e s a re e x p r e s s e d as the % I n j e c t a t e (mean + S E ) . - 63 -C. THE EFFECT SMOKING ON BLOOD VOLUME AND TRANSIT TIME THROUGH THE LUNG The c e n t r a l b l o o d volume (CBV) and t r a n s i t t ime t h r o u g h the c e n t r a l b l o o d vo lume ( < ^ t ^ C B V ) was c a l c u l a t e d by the i n d i c a t o r d i l u t i o n t e c h n i q u e . The pu lmonary b l o o d volume was measured by add ing v a l u e s f o r each l u n g p i e c e and t h e t r a n s i t t ime t h r o u g h the l u n g was c a l c u l a t e d as f o l l o w s : < t > L B V = /£ t">CBV x (LBV/CBV) The e q u a t i o n e s t i m a t e s the f r a c t i o n o f the i n d i c a t o r d i l u t i o n RBC t r a n s i t t ime s p e n t i n the l u n g b l o o d volume measured i n v i t r o w i t h the l a b e l l e d b l o o d c e l l s . The d a t a i s shown i n T a b l e V. D. ARTERIAL BLOOD GASES. WHITE BLOOD CELL COUNTS, AND ARTERIAL BLOOD  PRESSURE A r t e r i a l b l o o d gases (ABG) and w h i t e c e l l c oun t s (WBC) were measured a t t he b e g i n n i n g , m i d d l e , and end o f the t e n m inu te t r e a t m e n t p e r i o d s o f e v e r y e x p e r i m e n t . The c r i t e r i a f o r the e x p e r i m e n t t o be i n c l u d e d i n the s tudy was p ° 2 75 mmMg i n a l l t h r e e measurements . A n i m a l s w i t h PO^^SmmMg were c o n s i d e r e d h y p o x i c and were not u s e d . The WBC coun t s were t aken and no t r e n d s were n o t i c e d nor were t h e r e any d i f f e r e n c e s i n w h i t e c e l l c oun t s between the g r o u p s . The ABP were m o n i t o r e d and any major changes were n o t e d . The smoke-exposed r a b b i t s t ended t o have a h i g h e r b l o o d p r e s s u r e than the c o n t r o l g roup at the end o f the t e n minute t r e a t m e n t p e r i o d , ( c o n t r o l : 90+4% b a s e l i n e , smoke: 104+4% b a s e l i n e ) ( p < £ 0 . 0 5 , u n p a i r e d t - t e s t ) . - 64 -CONTROL (N=8) SMOKE (N=9) P ^ t > C B V ( s e c ) 5.2 + 0.2 5.6 + 0. ,2 N.S. C O . (m l /min ) 650.9 + 45.2 682.0 + 59. .0 N.S. CBV (ml) 56.1 + 3.1 61.2 + 5. .3 N.S. LBV (ml) 5.6 + 0.5 7.9 + 0. ,5 p < 0 . 0 1 < t > L B V ( s e c ) 0.52 + 0.05 0.75 + 0, .07 p < 0 . 0 5 % LBV/CBV 10 .0 + 0.9 13.5 + 1. .2 p < 0 . 0 5 T a b l e V : T r a n s i t T ime and B l o o d Volume (mean + SE) Da ta d e t e r m i n e d by i n d i c a t o r - d i l u t i o n a re c e n t r a l c i r c u l a t i o n t r a n s i t t ime ( < C t ^ _ ), c a r d i a c o u t p u t ( C O . ) , and c e n t r a l b l o o d volume (CBV) . LBV i s CBV t h e sum o f a l l b l o o d vo lumes i n the l u n g samples i n v i t r o . The f r a c t i o n o f t h e c e n t r a l t r a n s i t t ime spent i n the sampled p a r t o f the l u n g i s the ^C.t^? . The p e r c e n t a g e o f the c e n t r a l volume i n the samples i s % LBV/CBV. - 65 -DISCUSSION The e f f e c t o f a c u t e c i g a r e t t e e x p o s u r e on r e g i o n a l pu lmonary b l o o d f l o w , v o l u m e , RBC t r a n s i t and PMN r e t e n t i o n was i n v e s t i g a t e d i n a t o t a l o f 20 a n e s t h e t i z e d , s u p i n e , s p o n t a n e o u s l y b r e a t h i n g r a b b i t s . The e x p e r i m e n t s were p e r f o r m e d as two s e p a r a t e s t u d i e s each c o n s i s t i n g o f 10 an ima l s e a c h , i n two g roups o f f i v e . The f i r s t s tudy measured r e g i o n a l pu lmonary b l o o d f l o w , vo lume and RBC t r a n s i t and the second s tudy measured r e g i o n a l PMN r e t e n t i o n . In each s t u d y , the two groups o f f i v e e i t h e r r e c e i v e d t e n m i n u t e s o f sham smoking w i t h room a i r as a c o n t r o l o r t e n m inu te s o f smoke e x p o s u r e f rom 3 c i g a r e t t e s at 4 p u f f s p e r m i n u t e . A r t e r i a l b l o o d p r e s s u r e s , p e r i p h e r a l w h i t e c e l l c o u n t s and a r t e r i a l b l o o d gases were m o n i t o r e d i n a l l e x p e r i m e n t s . 1) P r e l i m i n a r y E x p e r i m e n t s T e s t i n g Two M a c r o a g g r e g a t e s A s e t o f p r e l i m i n a r y e x p e r i m e n t s (N=3) were p e r f o r m e d t o t e s t the d i s t r i b u t i o n o f two d i f f e r e n t r a d i o l a b e l l e d mac roagg rega te (MAA) f l o w 125 m a r k e r s i n t he l u n g . Mac roagg rega te s l a b e l l e d w i t h I o d i n e and 131 125 131 I o d i n e ( I and I MAA) were i n j e c t e d t o g e t h e r as a s i n g l e b o l u s i n t o the r i g h t a t r i u m . The d i s t r i b u t i o n o f each f l o w marker was l a t e r measured by gamma c o u n t i n g f o r r a d i o a c t i v i t y i n the sampled l u n g and c a l c u l a t i n g the b l o o d f l o w s f rom each MAA. F i g . 15 shows the sample b l o o d f l o w s c a l c u l a t e d f rom one f l o w marker c o r r e l a t e d w e l l w i t h the second and t h e r e g r e s s i o n l i n e w i t h a s l o p e c l o s e t o 1.00 i s p r e d i c t e d i f bo th MAA mark t h e same f l o w . - 66 -The a p p l i c a t i o n o f the d o u b l e f l o w marker t e c h n i q u e i n the r a b b i t has no t been p r e v i o u s l y r e p o r t e d . In p r e v i o u s s t u d i e s , r e g i o n a l b l o o d f l o w s were measured w i t h m i c r o s p h e r e s i n the dog ( K a i h a r a e_t a l . , 1968) and sheep (Rudo lph and Heymann, 1967) and w i t h MAAs i n man (Rhodes e t a l . , 1969; Tow e t a l . , 1966; Ueda e t a l . , 1965) and i n dog (Hogg e t a l . , 1971, B a i l e e t a l . , 1982; Hogg e_t a l , 1985 ) . These p r e l i m i n a r y e x p e r i m e n t s have 125 d e m o n s t r a t e d the s u c c e s s f u l use o f r a d i o l a b e l l e d MAA f l o w markers ( I 131 and I MAA) f o r m e a s u r i n g r e g i o n a l pu lmonary b l o o d f l o w i n the r a b b i t . S i n c e t h e s e two MAAs were f o u n d to behave s i m i l a r l y , t h e y can t h e r e f o r e be u s e d i n tandem t o measure changes i n r e g i o n a l b l o o d f l o w a c r o s s t he sham smoke and smoke e x p o s u r e p e r i o d s . F i g . 16 i s an example o f a sample b l o o d f l o w p l o t o f i n i t i a l and f i n a l b l o o d f l o w s measured u s i n g two MAAs a c r o s s a t e n m i n u t e sham smoke p e r i o d . The pu lmonary b l o o d f l o w s were marked w i t h MAAs b o t h b e f o r e and a f t e r t e n m i n u t e s o f sham smoke and smoke e x p o s u r e . The i n i t i a l and f i n a l c a r d i a c o u t p u t s were no t s i g n i f i c a n t l y d i f f e r e n t among the two groups ( T a b l e I ) . The s l o p e o f t he r e g r e s s i o n l i n e s f rom the i n i t i a l v s f i n a l sample b l o o d f l o w p l o t s were compared t o the change i n c a r d i a c o u t p u t f rom the i n i t i a l t o f i n a l measurement ( C 0 ( 2 ) / C 0 ( 1 ) ) . The s l o p e o f the r e g r e s s i o n l i n e s were c o m p a r a b l e t o the changes p r e d i c t e d by the change i n o v e r a l l f l o w r e p r e s e n t e d as C 0 ( 2 ) / C 0 ( l ) ( T a b l e I ) . The c o n t r o l g roup p o i n t s had l e s s s c a t t e r a round the p r e d i c t e d l i n e than the smoke g roup i n d i c a t i n g t h a t each p i e c e o f l u n g i n t he c o n t r o l s t ended to sha re e q u a l l y i n the change i n o v e r a l l f l o w ( c a r d i a c o u t p u t ) . The d e v i a t i o n o f c e r t a i n p o i n t s f r om the p r e d i c t e d l i n e ( C 0 ( 2 ) / C 0 ( l ) ) i n the smoke exposed group sugge s t s t h a t t h e r e - 67 -a r e n o n - u n i f o r m changes i n b l o o d f l o w t o c e r t a i n l u n g r e g i o n s . The b l o o d f l o w d i s t r i b u t i o n can be f u l l y a p p r e c i a t e d when the l u n g i s s e p a r a t e d i n t o d i f f e r e n t r e g i o n s a c c o r d i n g t o v e r t i c a l h e i g h t . 2) The E f f e c t o f C i g a r e t t e Smoking on B l o o d F l o w , Volume and RBC T r a n s i t In t h i s s t u d y , i n d i c a t o r d i l u t i o n measurements were made b o t h b e f o r e and a f t e r a t e n m i n u t e t r e a t m e n t p e r i o d where the a n i m a l s were e i t h e r sham smoked (N=5) o r smoke exposed (N=5). D i f f e r e n t f l o w marker s were i n j e c t e d i m m e d i a t e l y a f t e r each i n d i c a t o r run to mark r e g i o n a l pu lmonary b l o o d f l o w i n o r d e r t o d e t e r m i n e i f any changes o c c u r r e d a c r o s s the t r e a t m e n t s . The c a r d i a c o u t p u t , c e n t r a l b l o o d volume (CBV) and the r e d c e l l t r a n s i t t ime t h r o u g h the c e n t r a l c i r c u l a t i o n ( <ft~> ) was d e t e r m i n e d f rom each / CBV i n d i c a t o r r u n . The c e n t r a l b l o o d volume wh ich c o n s i s t s o f the b l o o d volume between the r i g h t a t r i u m and the a o r t i c r o o t were not s i g n i f i c a n t l y d i f f e r e n t between the two g roups ( T a b l e V) however , t he b l o o d volume i n the l u n g parenchyma (LBV) was s i g n i f i c a n t l y g r e a t e r ( p ^ 0 . 0 5 ) i n the smokers . These d a t a sugges t an i n c r e a s e i n l u n g b l o o d volume t h a t c o u l d be due t o an i n c r e a s e i n d r i v i n g p r e s s u r e a c r o s s the pu lmonary c i r c u l a t i o n (P -P ). The d r i v i n g p r e s s u r e has been p r e v i o u s l y r e p o r t e d to i n c r e a s e by 15% f o l l o w i n g c i g a r e t t e smoke e x p o s u r e i n t he i n t a c t a n i m a l ( L a r s o n e t al_. , 1 9 6 5 ) . T h i s i s c o n s i s t e n t w i t h the f i n d i n g t h a t an i n c r e a s e d d r i v i n g p r e s s u r e i n the pu lmonary c i r c u l a t i o n i n c r e a s e s c a p i l l a r y r e c r u i t m e n t and v e s s e l d i l a t a t i o n ( G r o v e r et. f Q . , 1984) and t h e r e f o r e b l o o d vo lume. N i c o t i n e has a l s o been shown to cause v a s o d i l a t a t i o n i n the pu lmonary c i r c u l a t i o n (Comroe, 1960 ) . - 68 -The RBC t r a n s i t t ime th rough the c e n t r a l c i r c u l a t i o n ( ) as CBV d e t e r m i n e d by the i n d i c a t o r d i l u t i o n t e c h n i q u e was not s i g n i f i c a n t l y d i f f e r e n t between the g roups ( T a b l e V) but when t h e s e t r a n s i t t imes were m u l t i p l i e d by the f r a c t i o n o f the l u n g b l o o d volume to c e n t r a l b l o o d volume ( L B V / C B V ) , an a p p r o x i m a t i o n o f the t ime spent by the RBC i n the c a p i l l a r i e s and s m a l l v e s s e l s was o b t a i n e d . < f c > L B V = < f c > C B V X L B W C B V The <^t*> i n t h e smoke exposed group was found to be s i g n i f i c a n t l y LBV g r e a t e r ( p . ^ 0 . 0 5 ) t h a n i n the sham smoked g roup . T h i s d a t a shows t h a t the o v e r a l l RBC t r a n s i t t ime t h r o u g h the LBV i s l o n g e r i n smoke exposed a n i m a l s t h a n i n sham smoke c o n t r o l s due to g r e a t e r LBV. No d i f f e r e n c e s i n CBV were d e t e c t e d . The p r e v i o u s d a t a showed t h a t the o v e r a l l l u n g b l o o d volume and the o v e r a l l RBC t r a n s i t t ime th rough the LBV i n c r e a s e d f o l l o w i n g a c u t e smoke e x p o s u r e . R e g i o n a l l y , pu lmonary b l o o d volume was g r e a t e r i n most l u n g r e g i o n s f o l l o w i n g smoking e x c e p t a t the t o p o f the l u n g ( F i g . 2 1 ) . The g r e a t e r b l o o d vo lume i n t h e s e r e g i o n s c o u l d e x p l a i n why the RBC t r a n s i t t i m e s were l o n g e r i n t h e s e same r e g i o n s ( F i g . 2 2 ) . The r e d c e l l t r a n s i t t i m e s were l o n g e s t i n t he upper l u n g r e g i o n s f o r bo th the c o n t r o l and smoke g r o u p . The range i n RBC t r a n s i t t imes was 0.3 to 1.0 second f o r the c o n t r o l s and 0.75 t o 5.0 seconds i n the smoke exposed g r o u p . L i k e the b l o o d v o l u m e s , t he RBC t r a n s i t t imes were g r e a t e r i n the smokers than the c o n t r o l s i n most r e g i o n s ( F i g . 2 2 ) . The g r e a t e s t l e n g t h e n i n g i n t r a n s i t o c c u r r e d a t the t o p o f the l u n g i n t he smokers . A t t he t o p o f t he l u n g , t he b l o o d vo lumes were the same i n bo th groups - 69 -( F i g . 21) but t he RBC t r a n s i t t ime was g r e a t e r f o r the smokers ( F i g . 2 2 ) . The r e l a t i o n s h i p : T r a n s i t = B l o o d v o l u m e / B l o o d f l o w p r e d i c t s t h a t i n o r d e r f o r t r a n s i t t o i n c r e a s e a t a c o n s t a n t b l o o d vo lume, the b l o o d f l o w must d e c r e a s e . The measurements o f the e f f e c t o f a c u t e c i g a r e t t e smoke exposure on r e g i o n a l pu lmonary b l o o d f l o w a re shown i n F i g s . 1 7 - 2 0 . The r e g i o n a l pu lmonary b l o o d f l o w measured i n both g roups at the p r e - t r e a t m e n t p e r i o d have t h e f a m i l i a r v e r t i c a l p a t t e r n s o f b l o o d f l o w d i s t r i b u t i o n as d e s c r i b e d by West ( F i g . 1 9 ) . The b l o o d f l o w i s g r e a t e s t i n the m i d d l e r e g i o n s and b l o o d f l o w i s s l i g h t l y r e d u c e d a t the b o t t o m . T h i s f i n d i n g i s s i m i l a r t o t h o s e i n t he dog where the v e r t i c a l d i s t r i b u t i o n o f pu lmonary b l o o d f l o w was f o u n d t o be g r a v i t y dependent and r e g a r d l e s s o f the body p o s i t i o n , the b l o o d f l o w was a lways l o w e s t i n the most s u p e r i o r p a r t o f the l u n g (Reed and Wood, 1 9 7 0 ) . The r a b b i t s i n t h i s s t udy were i n t he s u p i n e p o s i t i o n t h e r e f o r e the most s u p e r i o r p a r t o f t he l u n g was on the a n t e r i o r s u r f a c e . The b l o o d f l o w p a t t e r n s i n the two groups were not d i f f e r e n t b e f o r e t r e a t m e n t ( F i g . 19) bu t f o l l o w i n g ten m i n u t e s o f t r e a t m e n t w i t h e i t h e r sham o r smoke e x p o s u r e , t he r e g i o n a l l u n g b l o o d f l o w p a t t e r n s d i v e r g e ( F i g . 2 0 ) . C i g a r e t t e smoke e x p o s u r e r e d u c e d the b l o o d f l o w to the s u p e r i o r l u n g r e g i o n s and i n c r e a s e d t h e f l o w t o the dependent r e g i o n s . The r e a s o n f o r t h i s r e d i s t r i b u t i o n c a n n o t be d e t e r m i n e d f rom measurements made i n t h i s s t u d y . Bo th g roups e x p e r i e n c e d the same n e g l i g i b l e d e c r e a s e i n c a r d i a c o u t p u t f o l l o w i n g t r e a t m e n t ( T a b l e I ) . A r e d u c t i o n i n f l o w a t t he t o p o f the l u n g c o u l d be due t o a f a l l i n pu lmonary a r t e r i a l p r e s s u r e D u t . t he se measurements were no t made. P r e v i o u s s t u d i e s have f o u n d t h a t P e i t h e r a does no t change ( T a k a s a k i , 1 9 6 3 ) ; Nakamura e t a l . , 1985) o r i n c r e a s e s - 70 -(Samenek and A v i a d o , 1966; L a r s o n e t &1., 1968) f o l l o w i n g c i g a r e t t e smoke e x p o s u r e o r n i c o t i n e a d m i n i s t r a t i o n . Under t h e s e c o n d i t i o n s , i f o t h e r v a r i a b l e s do not c h a n g e , t he b l o o d f l o w p a t t e r n s h o u l d rema in unchanged o r f l o w s h o u l d i n c r e a s e a t the t o p wh i ch was c o n t r a r y t o ou r f i n d i n g . However, a v a r i a b l e l i k e a l v e o l a r p r e s s u r e (P ) most l i k e l y changed due t o A a l t e r a t i o n s i n a i r w a y r e s i s t a n c e . A r e c e n t s t u d y i n the i s o l a t e d dog l u n g showed t h a t e x p o s u r e to o n l y t w o - t h i r d s o f a c i g a r e t t e r e s u l t e d i n a 239% i n c r e a s e i n a i r w a y s r e s i s t a n c e (Nakamura e_t a l . , 1985) w i t h most o f the r e s i s t a n c e o c c u r r i n g i n the p e r i p h e r a l a i r w a y s ( <2mm d i a m e t e r ) . They a l s o f ound t h a t e x p o s u r e o f the upper a i rw ay s ( t r a c h e a , l a r y n x , na sopharynx ) t o smoke was a d d i t i v e t o the a i r w a y r e s i s t a n c e . I n h a l a t i o n o f f i n e c h a r c o a l d u s t i n man and i n the c a t a l s o p r o d u c e d an i n c r e a s e i n a i rw a y s r e s i s t a n c e t h a t was f o u n d t o be dependen t on i n t a c t p a r a s y m p a t h e t i c r e f l e x e s (Nade l §_t a l . , 1962 ) . The e x p o s u r e o f the na sopha rynx and l a r y n x t o smoke was no t n e c e s s a r y t o e l i c i t a r e s p o n s e . In t h i s c u r r e n t s t u d y , the r a b b i t s had t h e i r upper a i rways b y p a s s e d by t r a c h e o s t o m y so t h a t most o f the smoke e n t e r e d the t r a c h e a d i r e c t l y a l t h o u g h some s i d e s t r e a m smoke might e n t e r the no se . C i g a r e t t e smoke i s a v e r y complex s u b s t a n c e c o n t a i n i n g o v e r s e v e r a l h u n d r e d d i f f e r e n t compounds e i t h e r i n the p a r t i c u l a t e o r gaseous phases o f c i g a r e t t e smoke (Smoking and H e a l t h , Dept . o f N a t i o n a l H e a l t h and W e l f a r e , Canada , 196A) . Of t h e s e c o n s t i t u e n t s n i c o t i n e has known c a r d i o p u l m o n a r y e f f e c t s . The amount o f smoke o r e s t i m a t e d dose o f n i c o t i n e g i v e n t o the r a b b i t s i n t h i s s t u d y was s i m i l a r t o t h o s e p r o d u c i n g an i n c r e a s e a i rways r e s i s t a n c e i n p r e v i o u s s t u d i e s . A dosage o f 5-20 micrograms n i c o t i n e / k g was - 71 -s u f f i c i e n t t o i n c r e a s e P , pu lmonary v a s c u l a r r e s i s t a n c e (PVR) , and cause a b r o n c h o c o n s t r i c t i o n i n the i s o l a t e d dog l u n g (Samenek and A v i a d o , 1966 ) . Nakamura ' s s t udy wh i ch i n c r e a s e d the a i rway r e s i s t a n c e by 239%, i n v o l v e d a dose o f 1.44 mg o f n i c o t i n e f rom a c i g a r e t t e o v e r 1 m inute i n 14 kg dogs o r a p p r o x i m a t e l y 0.1 mg/kg /min . The r a b b i t s i n t h i s s tudy were g i v e n smoke f r o m t h r e e c i g a r e t t e s a t about 3 mg o f n i c o t i n e pe r c i g a r e t t e (no f i l t e r s ) o r 9 mg o v e r 10 m i n u t e s . The r a b b i t s we i ghed no more than 10 kg so the dosage was 0 .9 mg/kg o r 0.09 mg/kg/min. The dose o f n i c o t i n e i n the smoke g i v e n t o the r a b b i t s i n t h i s s t udy p r o b a b l y i n c r e a s e d the a i rway r e s i s t a n c e . T h i s c o u l d o n l y be c o n f i r m e d by l u n g f u n c t i o n t e s t s wh ich were no t p e r f o r m e d . By assuming an i n c r e a s e i n a i r w a y s r e s i s t a n c e (R) f o l l o w i n g smoking, i t f o l l o w s t h a t i f r e s i s t a n c e i n c r e a s e s and a i r f l o w (V ) rema ins c o n s t a n t o r A i n c r e a s e s , the a l v e o l a r p r e s s u r e must a l s o i n c r e a s e (P = V x R) . The A A common f i n d i n g f o l l o w i n g c i g a r e t t e smoke e x p o s u r e o r n i c o t i n e a d m i n i s t r a t i o n i s an i n c r e a s e i n t he r a t e and d e p t h o f b r e a t h i n g (Comroe, I960; Z a p a t a e t a l . , 1976) t h e r e f o r e smoking wou ld t e n d t o i n c r e a s e a i r f l o w . The b a s i c p r i n c i p l e s g o v e r n i n g pu lmonary b l o o d f l o w by W e s t ' s zones i n d i c a t e s t h a t an i n c r e a s e i n P beyond P r e s u l t s i n a s h i f t f rom Zone I I t o Zone I A a c o n d i t i o n s , i n o t h e r words f l o w d e c r e a s e s i n t h a t r e g i o n . The i n c r e a s e i n P i s not o f t he same magn i tude as the i n c r e a s e i n a i rway r e s i s t a n c e and a has been r e p o r t e d as 120% o f c o n t r o l ( L a r s o n e t a l . , 1968) o r no i n c r e a s e ( T a k a s a k i , 1963; Nakamura §_t a l . , 1985 ) . R e c a l l t h a t smoking i n c r e a s e d bo th a i r w a y r e s i s t a n c e and a i r f l o w . T h e r e f o r e , i t i s h i g h l y l i k e l y t h a t the t o t a l e f f e c t o f t he smoke i s t o i n c r e a s e P. g r e a t e r than P r e s u l t i n g i n A a a zone change and a d e c r e a s e i n f l o w to the s u p e r i o r r e g i o n s . The i n c r e a s e i n b l o o d f l o w a t t he bo t tom o f the l u n g (Zone I I I ) i s p r o b a b l y due to the i n c r e a s e i n t he d r i v i n g p r e s s u r e (P -P ) wh i ch has been r e p o r t e d t o a v i n c r e a s e by about 15% f o l l o w i n g smoking ( L a r s o n e_t a l . , 1968) . The r e s u l t s o f t h i s s tudy show t h a t a c u t e c i g a r e t t e smoke exposu re r e s u l t s i n an o v e r a l l i n c r e a s e i n the pu lmonary b l o o d volume (LBV) wh ich c a u s e s t he o v e r a l l RBC t r a n s i t t ime ( < t > ) t o be l o n g e r . The i n c r e a s e LBV i n r e g i o n a l RBC t r a n s i t s c o i n c i d e d w i t h tho se r e g i o n s where the r e g i o n a l b l o o d vo lumes were i n c r e a s e d . The e x c e p t i o n was at the upper l u n g r e g i o n where b l o o d vo lumes were no t d i f f e r e n t but where the g r e a t e s t l e n g t h e n i n g o f RBC t r a n s i t o c c u r r e d i n smokers . The r e l a t i o n s h i p : t r a n s i t t ime = b l o o d v o l u m e / b l o o d f l o w p r e d i c t s t h a t an i n c r e a s e i n t r a n s i t at c o n s t a n t b l o o d vo lume must be a s s o c i a t e d w i t h a d e c r e a s e i n b l o o d f l o w . A r e d u c t i o n i n b l o o d f l o w a t the t o p o f the l ung s was i n f a c t measured w i t h the f l o w m a r k e r s . The i n c r e a s e i n RBC t r a n s i t t ime and d e c r e a s e i n b l o o d f l o w t o the t o p o f t he l u n g m i gh t be o f impor t ance i n the mechanism wh i ch d e t e r m i n e s the p r e d i l e c t i o n o f t he upper l o b e s o f the l u n g to c e n t r i l o b u l a r emphysema. 3) The E f f e c t o f C i g a r e t t e Smokins on PMN R e t e n t i o n In t h i s s t u d y , r a b b i t s were i n j e c t e d w i t h "**Cr PMN b e f o r e they were g i v e n t h e same sham and smoke t r e a t m e n t s as i n the p r e v i o u s s t u d y . The f i n d i n g o f t h i s s t u d y was t h a t t h e r e were no d i f f e r e n c e s between the sham or smoked g roups i n e i t h e r r e g i o n a l ( T a b l e I I I ) o r o v e r a l l ( T a b l e IV) PMN r e t e n t i o n i n t he l u n g . The top o f the l u n g s had the g r e a t e s t r e t e n t i o n s when compared t o o t h e r l u n g r e g i o n s . T h i s r e g i o n a l v a r i a t i o n o f PMN - 73 -r e t e n t i o n i s a f u n c t i o n o f the i n v e r s e r e l a t i o n s h i p between b l o o d f l o w and PMN r e t e n t i o n . F o l l o w i n g smok ing , t he PMN r e t e n t i o n might be e x p e c t e d to i n c r e a s e i n a l l r e g i o n s because o f l o n g e r RBC t r a n s i t t imes ( F i g . 2 2 ) . The f a c t t h a t no such i n c r e a s e was o b s e r v e d sugge s t s t h a t some o t h e r mechanisms m i gh t be i n o p e r a t i o n . E p i n e p h r i n e i s known to m o b i l i z e w h i t e c e l l s f rom t h e m a r g i n a t i n g p o o l i n the l u n g (A thens e t a l . , 1961b; B ierman e t a l . , 1952b; M u i r e t a l . , 1984 ) . T h i s e f f e c t wou ld t end to c o u n t e r a c t any i n c r e a s e i n r e t e n t i o n due to a l e n g t h e n i n g o f the t r a n s i t t i m e . E p i n e p h r i n e r e l e a s e o c c u r s f o l l o w i n g c i g a r e t t e smoking (Wat t s , 1960) and the m i n i m a l dose o f n i c o t i n e r e q u i r e d t o cause r e l e a s e f rom the a d r e n a l m e d u l l a i s 0 . 0 1 - 0 . 0 2 mg/kg/min i n the i n t a c t a n i m a l ( S i l v e t t e e t a l . , 1961 ) . R e c a l l , t h e e s t i m a t e d dose r e c e i v e d by the r a b b i t s i n t h i s s tudy was 0.09 mg /kg /m in . The amount o f c i g a r e t t e smoke g i v e n to the r a b b i t s would be above t h e m i n i m a l amount r e q u i r e d to r e l e a s e c a t e c h o l a m i n e s f rom the a d r e n a l s . The a r t e r i a l b l o o d p r e s s u r e changes i n the smoke-exposed r a b b i t s were 108 + 4% (mean + SE) o f base l i n e a t 5 m inu te s and 104 + 4% a t 10 m i n u t e s . T h e s e a r e s i m i l a r t o p r e v i o u s s t u d i e s i n the dog showing b l o o d p r e s s u r e s o f 110% o f b a s e l i n e a t 5 m inu te s and 104% at 10 m inu te s f o l l o w i n g a dose o f 0 .02 mg/kg/min o f n i c o t i n e ( L a r s o n e t a l . , 1968 ) . The a r t e r i a l b l o o d p r e s s u r e changes f r om b a s e l i n e f o r the r a b b i t s a t t e n m inu te s were 90 + 4% f o r c o n t r o l s and 104 + 4% i n the smoke exposed g roup . These changes were s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . The a r t e r i a l b l o o d p r e s s u r e changes i n t h e smoke exposed r a b b i t s were s i m i l a r t o t ho se changes p r e v i o u s l y r e p o r t e d i n t he dog g i v e n doses o f n i c o t i n e s u f f i c i e n t t o r e l e a s e e p i n e p h r i n e . A l s o - 74 -t he b l o o d p r e s s u r e a t t e n m inu te s was s i g n i f i c a n t l y g r e a t e r i n the smokers compared w i t h the sham c o n t r o l s . Both t h e s e f i n d i n g s sugges t t h a t c a t e c h o l a m i n e s were r e l e a s e d d u r i n g the smoke t r e a t m e n t . D i r e c t measurements o f c a t e c h o l a m i n e s were not p e r f o r m e d t h e r e f o r e t h i s f i n d i n g c a n n o t be c o n f i r m e d . The d i s t r i b u t i o n o f l a b e l l e d PMN r e c o v e r e d (as % o f i n j e c t a t e ) f rom the l u n g , l i v e r , s p l e e n and b l o o d a re shown i n T a b l e IV. Between t r e a t m e n t g r o u p s , o n l y the amounts r e c o v e r e d f rom the l i v e r were s i g n i f i c a n t l y d i f f e r e n t (p < 0 . 0 1 ) . The amounts i n the l u n g and s p l e e n were no t d i f f e r e n t . The amount i n b l o o d tended to be g r e a t e r i n t he smokers but t h i s was no t s i g n i f i c a n t . T h i s f i n d i n g was a l s o seen i n the a r t e r i a l w h i t e b l o o d c e l l c o u n t s ( C o u l t e r ) wh i ch showed no t r e n d s o r d i f f e r e n c e s among the g r o u p s . T h i s r e s u l t i s a c o n t r a d i c t i o n to p r e v i o u s d a t a i n human smokers f i n d i n g t h a t p e r i p h e r a l w h i t e c e l l c oun t s were i n c r e a s e d i n c i g a r e t t e smokers ( C o r r e e t a^ . , 1971 ) . A more c u r r e n t s tudy examined the v a r i a t i o n i n mean l e u k o c y t e c o u n t i n a l a r g e p o p u l a t i o n o f p u l p and paper and s m e l t e r w o r k e r s and f o u n d t h a t c u r r e n t smokers had s i g n i f i c a n t l y h i g h e r l e u k o c y t e c o u n t s t h a n non - smoker s and ex - smokers (Yeung e t a l , 1984) . T h i s d i s c r e p a n c y c o u l d be due to many rea sons such as s p e c i e s d i f f e r e n c e s , a n e s t h e s i a , s u r g i c a l s t r e s s and smoking t h r o u g h a t r a c h e o s t o m y . The r e s u l t s can be compared to those d e t e r m i n e d by Nye §_t a l . (1932) wh i ch were l u n g : 39%, b l o o d : 31%, l i v e r : 23%, and s p l e e n : 7%. The v a l u e s on T a b l e IV a r e c o n v e r t e d f rom % on i n j e c t a t e t o % o f t o t a l r e c o v e r e d . These new v a l u e s a re l i v e r (44% o f t o t a l r e c o v e r e d ) , l u n g (35%) , b l o o d (17%) , and s p l e e n (4%) i n t he c o n t r o l a n i m a l s . In smoke exposed a n i m a l s , the g r e a t e s t amount was r e c o v e r e d f rom the l u n g s (38%) f o l l o w e d by the l i v e r (28%) , b l o o d ( 2 8 % ) , and s p l e e n (7%) . Nye d e t e r m i n e d the d i s t r i b u t i o n o f n e u t r o p h i l s i n t h e o r g a n s by u s i n g m o r p h o l o g i c a l t e c h n i q u e s . The amount i n the b l o o d was e s t i m a t e d by assuming t h a t the b l o o d volume i n the r a b b i t s was 4 .7% o f the body w e i g h t . The c o n t r o l g roup v a l u e s o f t h i s s t udy a re d i f f e r e n t than the normal v a l u e s d e t e r m i n e d by Nye. More PMN were f o u n d i n the l i v e r and l e s s i n the b l o o d i n the c o n t r o l r a b b i t s o f t h i s s t udy compared t o the p r e v i o u s s t u d y . The b e s t e x p l a n a t i o n f o r t h i s i s t h a t the c e l l l a b e l l i n g t e c h n i q u e used i n t h e c u r r e n t s t udy i n v o l v e s a p r e p a r a t i o n t ime o f 3 t o 4 h o u r s . The h a l f - l i f e o f t he ^ * C r l a b e l l e d r a b b i t l e u k o c y t e was f o u n d to be 3.2 t o 3.8 h o u r s ( I s s e k u t z §_t a l . , 1980) so t h a t by the t ime the c e l l s a re i n j e c t e d back i n t o t o a n i m a l , many o f the c e l l s w i l l be s e n e s c e n t c e l l s wh ich a re i m m e d i a t e l y removed f r om the b l o o d by the l i v e r . The a s s o c i a t i o n between c i g a r e t t e smoking and the deve lopment o f emphysema i s w e l l e s t a b l i s h e d (Auerbach e t a l . , 1972; T h u r l b e c k e_t a l . . 1970; N iewoehner e t a l . , 1974 ) . However, the p a t h o g e n e s i s o f the d i s e a s e and t h e r o l e o f c i g a r e t t e smoking i n t he mechanism i s p o o r l y u n d e r s t o o d . C e n t r i l o b u l a r emphysema i s the most common t ype and i s the form a s s o c i a t e d w i t h c i g a r e t t e smok ing . The l e s i o n s have the g r e a t e s t p r e d i l e c t i o n to the upper l o b e s o f the l u n g and the a p i c a l r e g i o n s o f the l ower l o b e ( T h u r l b e c k , 1 9 7 6 ) . By c o n s i d e r i n g the p r o t e a s e - a n t i p r o t e a s e imba lance h y p o t h e s i s as a b a s i c mechan i sm, i t i s t e m p t i n g to s p e c u l a t e on how the s p e c i f i c p a t t e r n o f d i s e a s e r e s u l t s as a consequence o f the i n t e r a c t i o n between c i g a r e t t e smoke, - 76 -t h e PMN, and serum a n t i p r o t e a s e . An i n t e r e s t i n g h y p o t h e s i s has been p r o p o s e d wh ich a t tempt s t o e x p l a i n t h i s p a t t e r n on the b a s i s o f the norma l v e n t i l a t i o n / p e r f u s i o n r a t i o s i n the l u n g ( C o c k c r o f t and H o m e , 1982 ) . The i n h a l e d i r r i t a n t s o f c i g a r e t t e smoke a r e p r o p o s e d t o d i s t r i b u t e i n the manner o f the pu lmonary v e n t i l a t i o n w h i l e t h e a l p h a - l - a n t i t r y p s i n d i s t r i b u t e s i n the manner o f the pu lmonary p e r f u s i o n . The b l o o d f l o w i n the l u n g d e c r e a s e s i n an a lmost l i n e a r f a s h i o n f r o m the base t o the apex ( D o l l e r y and West , 1960) and l i k e w i s e , the a l v e o l i a t t he ba se s see l a r g e r changes i n volume ( i . e . g r e a t e r v e n t i l a t i o n ) than t h e a l v e o l i a t t he a p i c e s ( M i l i c - E m i l i e_t a l . , 1966) . S i n c e the p e r f u s i o n g r a d i e n t f r om t o p t o bo t tom i s g r e a t e r than the v e n t i l a t i o n g r a d i e n t , the v e n t i l a t i o n / p e r f u s i o n r a t i o g r a d i e n t v a r i e s f rom 3:1 a t the apex to 0 .6 :1 a t t he base (West, 1979 ) . The h y p o t h e s i s i s t h a t emphysema has the t endency to d e v e l o p i n a r e a s o f h i g h v e n t i l a t i o n - . p e r f u s i o n r a t i o s s i n c e the s m o k e : a n t i p r o t e a s e r a t i o i s h i g h . The a r e a c o r r e s p o n d i n g to t h i s i s at the l u n g apex. T h i s p r o p o s a l i s q u a l i f i e d by the c a u t i o n t h a t n e i t h e r has t he d i s t r i b u t i o n o f smoke been c o r r e l a t e d to v e n t i l a t i o n , nor a l p h a - l - a n t i t r y p s i n t o p e r f u s i o n . I f t he above h y p o t h e s i s i s m o d i f i e d to c o n s i d e r the r e g i o n a l d i s t r i b u t i o n o f PMN i n the l u n g and the changes t h a t o c c u r to r e g i o n a l pu lmonary b l o o d f l o w and RBC t r a n s i t f o l l o w i n g smok ing , some i n t e r e s t i n g s p e c u l a t i o n s can be made. T h i s s t udy and o t h e r s have shown PMN r e t e n t i o n t o v a r y r e g i o n a l l y w i t h the g r e a t e s t r e t e n t i o n a t the t o p o f the l u n g ( T a b l e I I I ) . C i g a r e t t e smoke has been shown to go p r e f e r e n t i a l l y t o the upper l o b e s o f the l u n g (Ander son e_t a l . , 1966) . Both c i g a r e t t e smoke and PMN a r e - 77 -o p t i m a l l y p l a c e d i n t o the upper l o b e s , where c e n t r i l o b u l a r emphysema i s u s u a l l y f o u n d . I f we assume t h a t the t r a n s i t o f p l a sma (and a l p h a - l - p r o t e a s e i n h i b i t o r ) i s c l o s e l y a p p r o x i m a t e d by the t r a n s i t o f t he RBC, t h e l o n g e r RBC t r a n s i t t imes i n the upper l u n g r e g i o n s would t h e r e f o r e mean t h a t t h e r e i s a g r e a t e r e x p o s u r e t o smoke and more i n h i b i t i o n o f A - l - P I ( J a n o f f et. a l . , 1 9 7 9 ) . The i n h i b i t i o n o f A - l - P I o c c u r s by an o x i d a t i v e p r o c e s s and r e d u c e s t he a c t i v i t y o f the a n t i p r o t e a s e by a f a c t o r o f 2000 ( J a n o f f , 1983 ) . As t h i s o c c u r s v e r y r a p i d l y , t he A - l - P I i s i n h i b i t e d at the b e g i n n i n g o f i t s j o u r n e y th rough the pu lmonary c i r c u l a t i o n , and rema ins i n h i b i t e d u n t i l t he e n d . T h e r e f o r e e l a s t a s e r e l e a s e d d u r i n g the j o u r n e y i s expo sed t o l e s s a c t i v e i n h i b i t o r and i s more c a p a b l e o f i n j u r i n g the l u n g . T h i s i s compounded by the f a c t t h a t PMNs have g r e a t e s t r e t e n t i o n s a t the t o p o f t he l u n g where t h e smoke goes p r e f e r e n t i a l l y . A l t h o u g h c i g a r e t t e smoking does no t appear t o i n c r e a s e PMN r e t e n t i o n i n the l u n g , t h e f a c t t h a t RBC t r a n s i t i s i n c r e a s e d and b l o o d f l o w i s d e c r e a s e d p r o v i d e s a p o s s i b l e w o r k i n g h y p o t h e s i s f o r t he o b s e r v e d l o c a l i z a t i o n o f c e n t r i l o b u l a r emphysema i n the upper l o b e s . The d e c r e a s e i n b l o o d f l o w a t the top o f t he l u n g wou ld mean t h a t l e s s a n t i p r o t e a s e i s p r e s e n t as p r o p o s e d by C o c k c r o f t e t a l . ( 1 9 8 2 ) . The f a c t t h a t RBC t r a n s i t s a re l o n g e r means t h a t the a n t i p r o t e a s e p r e s e n t a t t h e t o p o f the l u n g has more t ime to be i n a c t i v a t e d by smoke. PMN r e t e n t i o n , a l t h o u g h no t i n c r e a s e d by smok ing , i s a lways g r e a t e s t at the upper l u n g r e g i o n s . T h e r e f o r e the same p r o t e a s e f o r c e i s p r e s e n t i n t h i s r e g i o n even though t h e r e may be l e s s t o t a l i n h i b i t o r and l e s s a c t i v e i n h i b i t o r p r e s e n t . L o c a l imba l ance s o v e r ex tended p e r i o d s o f t ime wou ld l e a d t o p r o t e o l y t i c damage o f the l u n g t i s s u e s . T h i s s t udy shows t h a t a c u t e - 78 -c i g a r e t t e smoking by i n c r e a s i n g RBC t r a n s i t t ime and d e c r e a s i n g pu lmonary b l o o d f l o w t o the t o p o f the l ung s can c r e a t e l o c a l i z e d p r o t e a s e - a n t i p r o t e a s e imba l ance i n t h e s e r e g i o n s . T h i s e f f e c t may be the b a s i s f o r a w o r k i n g h y p o t h e s i s f o r why c e n t r i l o b u l a r emphysema has t he t e n d e n c y t o d e v e l o p a t the l u n g a p i c e s . - 79 -CONCLUSION A t e c h n i q u e u s i n g two d i f f e r e n t r a d i o l a b e l l e d m a c r o a g g r e g a t e s (MAA) was d e v e l o p e d f o r m e a s u r i n g changes i n r e g i o n a l pu lmonary b l o o d f l o w i n the r a b b i t . In a s e r i e s o f p r e l i m i n a r y e x p e r i m e n t s , two MAAs l a b e l l e d w i t h 125 131 I and I were i n j e c t e d t o g e t h e r i n t o the r a b b i t as a s i n g l e b o l u s . The two MAAs were shown to d i s t r i b u t e i n the l u n g i n the same manner w i t h r e s p e c t t o the f l o w . T h i s d o u b l e f l o w marker t e c h n i q u e was then used to d e t e r m i n e the e f f e c t o f a c u t e c i g a r e t t e smoke e x p o s u r e on r e g i o n a l pu lmonary b l o o d f l o w i n the r a b b i t . The r e d c e l l t r a n s i t t ime th rough the l u n g b l o o d vo lume ( < C t / ' ) LBV was d e t e r m i n e d w i t h t he f o l l o w i n g c a l c u l a t i o n : < f c > L B V = < t > C B V X L B V / C B V « ^ < t > C B V » W h e r e < t > C B V i s t he RBC t r a n s i t t ime t h r o u g h the c e n t r a l c i r c u l a t i o n as d e t e r m i n e d by the i n d i c a t o r - d i l u t i o n t e c h n i q u e , and LBV/CBV as the r a t i o o f l u n g b l o o d volume t o c e n t r a l b l o o d vo lume. The <^ t^ > was g r e a t e r (p<^0.05) i n the smoke LBV e x p o s e d r a b b i t s (0 .75 + 0.07 sec ) than the sham smoked r a b b i t s (0 .52 + 0.05 sec .) . The < t > _ o l f was the same i n bo th g r o u p s . The l o n g e r t r a n s i t t ime CBV i n t he smokers was f o u n d to be due to an i n c r e a s e i n the l u n g b l o o d volume f r om 5.6 + 0.05 ml i n the c o n t r o l s to 7.9 + 0.05 ml i n t he smokers (p - C o .O l ) . R e g i o n a l l y , the RBC t r a n s i t t imes were l o n g e r i n the smokers i n most l u n g r e g i o n s . The l o n g e r t r a n s i t t imes were a s s o c i a t e d w i t h b l o o d volume i n c r e a s e s i n t h e s e r e g i o n s w i t h the e x c e p t i o n o f the top o f t he l u n g where t h e l o n g e r t r a n s i t t ime was due t o a change i n b l o o d f l o w . - 80 -A c u t e c i g a r e t t e smoke e x p o s u r e r e d i s t r i b u t e s r e g i o n a l pu lmonary b l o o d f l o w f r o m the upper l u n g r e g i o n s to the l ower r e g i o n s . The b l o o d f l o w i n the upper r e g i o n s was r e d u c e d by 65% i n the smokers compared to 25% i n the c o n t r o l g r o u p . These changes were s i g n i f i c a n t l y d i f f e r e n t ( p ^ O . 0 5 ) . T h e r e were no changes i n r e g i o n a l PMN r e t e n t i o n a s s o c i a t e d w i t h the i n c r e a s e i n RBC t r a n s i t t ime i n the l u n g f o l l o w i n g a c u t e c i g a r e t t e smoke e x p o s u r e . The t i s s u e d i s t r i b u t i o n o f PMN d i d no t d e m o n s t r a t e any d i f f e r e n c e s i n the number o f PMN r e c o v e r e d i n the l u n g s o f the smokers and c o n t r o l s . L e s s PMNs were r e c o v e r e d f rom the l i v e r o f smoke exposed a n i m a l s t han sham smoked an ima l s ( p < 0 . 0 1 ) . The f a c t t h a t RBC t r a n s i t i s i n c r e a s e d and b l o o d f l o w i s d e c r e a s e d p r o v i d e s a p o s s i b l e w o r k i n g h y p o t h e s i s f o r the l o c a l i z a t i o n o f c e n t r i l o b u l a r emphysema i n the upper l o b e s . The d e c r e a s e i n b l o o d f l o w at the t o p o f the l u n g wou ld mean t h a t l e s s a n t i p r o t e a s e i s d e l i v e r e d t o t h o s e r e g i o n s i f a l p h a - l - p r o t e a s e i n h i b i t o r has the same d i s t r i b u t i o n as p e r f u s i o n as p r o p o s e d by C o c k c r o f t §_t a l . A l o n g e r RBC t r a n s i t means t h a t the a n t i p r o t e a s e p r e s e n t a t the top o f the l u n g has more t ime t o be i n a c t i v a t e d by smoke. A l t h o u g h c i g a r e t t e smoking does not appear t o i n c r e a s e PMN r e t e n t i o n i n the l u n g , the o b s e r v a t i o n t h a t PMN r e t e n t i o n s a re a lways g r e a t e s t a t the upper l u n g r e g i o n s sugge s t s t h a t a c o n s t a n t heavy p r o t e a s e bu rden e x i s t s i n t he r e g i o n and t h a t an imba l ance between the p r o t e a s e s and a n t i p r o t e a s e s may be more dependent on the l a c k o f i n h i b i t o r y c a p a c i t y o f t he a n t i p r o t e a s e s than an i n c r e a s e i n p r o t e a s e f o r c e s . - 81 -BIBLIOGRAPHY Ambrus , C M . , J . L . Ambrus, G . C J o h n s o n , E.W. Packman, W.S. C h e r n i c k , N. Back , and J . W . E . H a r r i s s o n . 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