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Effects of chronic infusion of endotoxin on renal and cardiovascular function in rats Azzarolo Carvallo, Ana Maria 1982

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EFFECTS OF CHRONIC INFUSION OF ENDOTOXIN ON RENAL AND CARDIOVASCULAR FUNCTION IN RATS ANA MARIA {AZZAROLO CARVALLO Pharmaceutical Chemist, Universidad de Concepcion, C h i l e , 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1982 © Ana Maria A z z a r o l o C a r v a l l o , 1982 by MASTER IN SCIENCE i n In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of P h £ £ i O l a ft y The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A ^ e i L HO , Z DE-6 (3/81) ABSTRACT A study was made of the r e n a l and c a r d i o v a s c u l a r responses of r a t s to the c h r o n i c intravenous a d m i n i s t r a t i o n of b a c t e r i a l endotoxin. Endotoxin (E c o l i ; 026: B6) was i n f u s e d i n t r a v e n o u s l y a t the r a t e o f 10 ug/h f o r a p e r i o d of 4-6 days by means of subcutaneously implanted osmotic mini-pumps. The c h r o n i c endotoxemia r e s u l t e d i n a l a r g e f a l l i n the r e n a l output of sodium, a decrease i n u r i n e o s m o l a l i t y and an i n c r e a s e i n potassium output. Ald o s t e r o n e c o n c e n t r a t i o n i n plasma was not changed. The r e n a l r e t e n t i o n of sodium was a s s o c i a t e d with an i n c r e a s e d plasma volume, sodium space and t o t a l body exchangeable sodium. A study of r e n a l f u n c t i o n u s i n g c l e a r a n c e methods showed t h a t the above changes o c c u r r e d i n the absence of s t a t i s t i c a l l y s i g n i f i c a n t changes i n glomerular f i l t r a t i o n r a t e , r e n a l plasma flow or f i l t r a t i o n f r a c t i o n . Measurements of r e n a l medullary plasma flow by means of an is o t o p e accumulation method i n d i c a t e d t h a t no s i g n i f i c a n t r e d i s t r i b u t i o n o f i n t r a r e n a l b l o o d flow had o c c u r r e d which might account f o r e i t h e r the r e t e n t i o n of sodium or the impaired r e n a l c o n c e n t r a t i n g a b i l i t y . An i n v e s t i g a t i o n of c a r d i o v a s c u l a r f u n c t i o n showed t h a t r a t s w i t h c h r o n i c endotoxemia had a s i g n i f i c a n t l y i n c r e a s e d c a r d i a c output as measured by the r e f e r e n c e sample technique u s i n g r a d i o - l a b e l l e d p l a s t i c microspheres. These animals a l s o had an i n c r e a s e d s t r o k e volume, b r a d y c a r d i a , hypotension - i i i -and a decreased t o t a l p e r i p h e r a l r e s i s t a n c e . The r e n a l f r a c t i o n o f the c a r d i a c output was s i g n i f i c a n t l y reduced but t o t a l r e n a l blood flow was unchanged. The f r a c t i o n a l d i s t r i b u t i o n o f n u t r i t i o n a l b l o o d flow t o the kidney, as 8 6 measured with RbCl, was not s i g n i f i c a n t l y changed. Because of t h e o r e t i c a l , but p o s s i b l e , sources of e r r o r i n h e r e n t i n the use of microspheres to measure c a r d i a c out-put an attempt was made to c o n f i r m the above data u s i n g an a l t e r n a t i v e method. Measurement of c a r d i a c output u s i n g the d i r e c t F i c k method d i d not d i s c l o s e a s i g n i f i c a n t d i f f e r e n c e i n c a r d i a c output. Measurements were a l s o made of the c i r c u l a t i n g h a l f -l i f e of endotoxin i n plasma u s i n g " ^ C r - l a b e l l e d endotoxin. There was a very l a r g e i n c r e a s e i n the r a t e of plasma c l e a r a n c e of endotoxin i n r a t s s u b j e c t e d to c h r o n i c endo-toxemia but no change i n the f r a c t i o n a l uptake of endotoxin by s e l e c t e d organs. I t i s suggested t h a t the observed changes i n r e n a l f u n c t i o n were secondary to the hemodynamic e f f e c t s of endo-t o x i n r a t h e r than to d i r e c t nephrotoxic e f f e c t s . - i v -TABLE OF CONTENTS Abs t r a c t i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements x i I n t r o d u c t i o n 1 Methods and M a t e r i a l s 7 A. General p r e p a r a t i o n 7 B. Measurements 9 1. Water, e l e c t r o l y t e s and sodium outputs .. 9 2. Sodium space and plasma volume 9 3. Measurement of aldosterone i n plasma .... 11 4. Renal f u n c t i o n t e s t s 11 5. Cardiovascular measurements 15 a. Radioactive microsphere technique ... 16 b. Measurement of c a r d i a c output by a d i r e c t F i c k p r i n c i p l e 19 c. I n d i r e c t e s t i m a t i o n of c a r d i a c output 26 d. Measurement of the d i s t r i b u t i o n of c a r d i a c output to the kidneys using 8 6 R b C l 26 6. Renal medullary plasma flow measurement . 28 7. Determination of h a l f l i f e of endotoxin 51 i n plasma and the uptake of Cr-endo-t o x i n by d i f f e r e n t organs 31 8. Hexobarbital s l e e p i n g time measurements . 33 - v -C. S t a t i s t i c a l a n a l y s i s 34 Results 35 1. Water and s o l u t e output 35 2. Plasma a n a l y s i s 41 3. Renal clearance data 42 4. Cardiovascular measurements 55 a. Cardiac output values using the d i r e c t F i c k method 55 b. Cardiac output values using microspheres 57 c. D i s t r i b u t i o n of c a r d i a c output 6 2 5. Half l i f e of endotoxin i n plasma 63 6. Hexobarbital s l e e p i n g time 73 7. Renal medullary plasma flow 73 D i s c u s s i o n 77 A. Renal f u n c t i o n 77 B. Blood and plasma volume s t u d i e s 81 C. Development of t o l e r a n c e i n chronic endo-toxaemia 83 D. Cardiovascular f u n c t i o n 85 B i b l i o g r a p h y 96 - v i -LIST OF TABLES Oxygen content i n a r t e r i a l and mixed venous b l o o d and the d i f f e r e n c e i n oxygen content between a r t e r i a l and mixed venous blood, i n c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin 56 R e s i s t a n c e of the kidneys or h e a r t to b l o o d flow i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin < .68 H a l f l i f e , time c o n s t a n t and r a t e constant of disappearance of ^ C r - e n d o t o x i n from plasma f o r c o n t r o l r a t s and f o r endotoxin t r e a t e d r a t s 70 Uptake of C r - l a b e l l e d endotoxin in:.various organs, i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin 72 - v i i -LIST. OF FIGURES Diagrammatic r e p r e s e n t a t i o n of the l i p o p o -l y s a c c h a r i d e . C learance d e t e r m i n a t i o n and b l o o d p r e s s u r e measurement i n the unanaesthetized r a t . Diagrammatic r e p r e s e n t a t i o n of the c l o s e d c i r c u i t - microspirometer apparatus. Sodium output i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. Food consumption by c o n t r o l r a t s and r a t s t r e a t e d with endotoxin. Percentage of body weight l o s s i n c o n t r o l r a t s and i n r a t s t r e a t e d with endotoxin. Renal sodium output i n p a i r - f e d c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. Sodium output as a percentage of sodium i n -take i n p a i r - f e d c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. Sodium space and exchangeable sodium i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endo-t o x i n . - v i i i -F i g u r e 10: Renal potassium output i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 11: U r ine c o n c e n t r a t i o n i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 12: Plasma e l e c t r o l y t e s i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 13: Plasma o s m o l a l i t y i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 14: Plasma p r o t e i n c o n c e n t r a t i o n i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 15: Plasma volume i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. F i g u r e 16: Haematoerit. v a l u e s f o r c o n t r o l r a t s and e n d o t o x i n - t r e a t e d r a t s . F i g u r e 17: R e l a t i o n s h i p between haematoerit and plasma volume i n r a t s t r e a t e d with endotoxin. F i g u r e 18: Glomerular f i l t r a t i o n r a t e and r e n a l plasma flow i n u n anaesthetized or a n a e s t h e t i z e d c o n t r o l r a t s and i n u n a naesthetized or a n a e s t h e t i z e d r a t s t r e a t e d w i t h endotoxin. F i g u r e 19: Oxygen consumption i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. - i x -C a r d i a c output i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. Mean a r t e r i a l b l o o d p r e s s u r e i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. Heart r a t e measurement i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. T o t a l p e r i p h e r a l r e s i s t a n c e i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. Renal f r a c t i o n of c a r d i a c output i n a n a e s t h e t i z e d c o n t r o l r a t s and i n r a t s t r e a t e d with endotoxin measured u s i n g 86 e i t h e r microspheres or RbCl. Blood flow to the kidneys and h e a r t i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endo-t o x i n . 51 Rate of disappearance of C r - l a b e l l e d endotoxin from plasma i n c o n t r o l r a t s and i n r a t s t r e a t e d with endotoxin. Organ weights expressed as percentages of bOdy weight i n c o n t r o l r a t s and i n r a t s t r e a t e d with endotoxin. H e x o b a r b i t a l s l e e p i n g time measured i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endo-t o x i n . - x -Figure 29: Medullary plasma flow f o r c o n t r o l r a t s and endotoxin-treated r a t s . Figure 30: Urine c o n c e n t r a t i o n at the time of medullary plasma flow measurement, i n c o n t r o l r a t s and i n r a t s t r e a t e d w i t h endotoxin. - x i -ACKNOWLEDGEMENTS I would l i k e t o express my s i n c e r e g r a t i t u d e t o my s u p e r v i s o r , Dr. R. Ke e l e r f o r h i s encouragement and guidance throughout the d u r a t i o n o f t h i s p r o j e c t . I would a l s o l i k e t o thank Dr. W. Nowaczynski f o r under-t a k i n g the plasma a l d o s t e r o n e measurements and to Dr. D. Godin f o r the advice i n the h e x o b a r b i t a l s l e e p i n g time experiments. S p e c i a l thanks t o T e r r y Meadows f o r the use of h i s i n g e n i o u s l y designed c l o s e d c i r c u i t microspirometer, and J . Ngsee f o r h i s t e c h n i c a l a s s i s t a n c e . - 1 -INTRODUCTION Endotoxins, h i g h m o l e c u l a r weight p h o s p h o l i p i d p o l y s a c c h a r i d e - p r o t e i n complexes c o n t a i n e d i n the c e l l w a l l s of Gram-negative b a c t e r i a are substances of h i g h b i o l o g i c a c t i v i t y . ( 2 6 ) . The l i p o p o l y s a c c h a r i d e s (L.P.S.), are c o n s i d e r e d t o be r e s p o n s i b l e f o r many of the t o x i c e f f e c t s of the Gram-nega t i v e b a c t e r i a . ( 4 9 ) . Chemically, endotoxin c o n s i s t s of three d i f f e r e n t r e g i o n s , a h y d r o p h i l i c r e g i o n , a c e n t r a l a c i d i c core and a l i p i d r i c h p a r t . ( F i g . 1 ) . The h y d r o p h i l i c p a r t c o n s i s t s of a h e t e r o p o l y s a c c h a r i d e c h a i n which c o n t a i n s the r e p e a t i n g 0 - s p e c i f i c a n t i g e n u n i t o f the c e l l . The k i n d and sequences of sugars i n t h i s c h a i n permit a f i n e d i s t i n c t i o n between the v a r i o u s b a c t e r i a l s t r a i n s which are otherwise p h e n o t y p i c a l l y i d e n t i c a l . The a c i d i c h e t e r o - o l i g o s a c c h a r i d e core c o n t a i n s 5 sugars and t h i s p a r t l i n k s the 0 - a n t i g e n and the l i p i d p o r t i o n c a l l e d l i p i d A.(42). L i p i d A i s b e l i e v e d t o be the a c t i v e p a r t of the endo-t o x i n and c o n s i s t s of a c h a i n of gl u c o s a m i n e d i s a c c h a r i d e u n i t s connected by pyrophosphate b r i d g e s t o which are att a c h e d long c h a i n f a t t y a c i d s . L i p o p o l y s a c c h a r i d e has been b a l l e d the endotoxin of - 2 -Gram-negative b a c t e r i a because i t i s f i r m l y a t t a c h e d to the c e l l s u r f a c e and can be r e l e a s e d by the c e l l s o n l y d u r i n g a c t i v e growth or when the c e l l s are l y s e d . ( 2 6 ) . Because of i t s complex s t r u c t u r e , endotoxin can e x e r t a l a r g e v a r i e t y of b i o l o g i c a l e f f e c t s . ( 2 7 ) . The g e n e r a l e f f e c t s observed a f t e r a d m i n i s t r a t i o n of endotoxin i n c l u d e : pyrogenic a c t i v i t y , h y p e r - o r hypo-glycemia, the Schwartzman phenomenon, bone marrow n e c r o s i s , •hypotension and death. Small doses l e a d t o l e u k o c y t o s i s but h i g h doses cause leukopenia. In a d d i t i o n endotoxin enhances ph a g o c y t o s i s , produces macrophage a c t i v a t i o n , a c t i v a t e s the complement system and Hageman f a c t o r and provokes a humoral immune response.(33) (16) . The r e t i c u l o e n d o t h e l i a l system (R.E.S.) i s the main c l e a r a n c e organ f o r endotoxin.(33). Since most of the R.E.S. i e . 80 to 90% i s r e p r e s e n t e d by the Kuppfer c e l l s , i t i s l o g i c a l t o t h i n k t h a t the l i v e r would a c t as a f i l t e r of b a c t e r i a and t h e i r products between the i n t e s t i n a l t r a c t and the systemic c i r c u l a t i o n . Because endotoxins are commonly found i n the p o r t a l c i r c u l a t i o n i t might be expected t h a t the f a i l u r e of t h i s b a r r i e r f u n c t i o n , which might occur i n l i v e r d i s e a s e s , would l e a d t o e n t r y of these substances i n t o the g e n e r a l c i r c u l a t i o n . ( 2 5 ) ( 4 5 ) . - 3 -F i g u r e 1 Diagrammatic r e p r e s e n t a t i o n of the l i p o p o l y s a c c h a r i d e complex. The sequences and kinds of p o l y s a c c h a r i d e s (S^ - S^) g i v e type s p e c i f i c i t y t o the 0 a n t i g e n , p e r m i t t i n g the d i f f e r e n -t i a t i o n between b a c t e r i a which are p h e n o t y p i c a l l y i d e n t i c a l , (42) . L I P I D A . P O L Y S A C C H A R I D E " C O R E GLUCOSAMINE N-ACETYLGLUCOSAMINE GLUCOSE GALACTOSE FATTY ACIDS HEPTOSE PHOSPHATE PHOSPHATYDYLETHANOLAMINE 2-KET0-3 DEOXYTOCTONIC ACID •I T Y P E S P E C I F I C "0" A N T I G E N S 1 - S 2 - S 3 - S 4 - L - 4 -Thus, the use of the l i m u l u s g e l a t i o n t e s t , which d e t e c t s picogram/ml q u a n t i t i e s of endotoxin i n b i o l o g i c a l f l u i d s (62), has i n d i c a t e d the presence of endotoxin i n the systemic c i r c u l a t i o n i n the absence of i n f e c t i o n , i n p a t i e n t s with a s e v e r e l y damaged l i v e r o r w i t h marked p o r t a l h y p e r t e n s i o n . ( 5 0 ) . For t h i s reason i t has been suggested t h a t some h e p a t i c and e x t r a h e p a t i c m a n i f e s t a t i o n such as f u n c t i o n a l r e n a l f a i l u r e observed d u r i n g c e r t a i n l i v e r d i s e a s e s , f o r example, c i r r h o s i s or f u l m i n a n t h e p a t i c f a i l u r e c o u l d be due to the presence of endotoxins coming from i n t e s t i n a l b a c t e r i a . ( 3 3 ) (43) . On the o t h e r hand some of the r e n a l d i s t u r b a n c e s found i n f u l m i n a t i n g h e p a t i c f a i l u r e where endotoxaemia has developed are s i m i l a r to those found i n Gram ne g a t i v e s e p t i c e m i a . ( 5 0 ) . T h e r e f o r e , the c o r r e l a t i o n between impaired r e n a l f u n c t i o n and endotoxaemia suggests t h a t endotoxin might be an important f a c t o r i n the pathogenesis of t h i s type of r e n a l f a i l u r e . Renal f a i l u r e e v e n t u a l l y develops i n up t o 85% o f p a t i e n t s w i t h c i r r h o s i s and c o u l d be the immediate cause of death. The c l i n i c a l m a n i f e s t a t i o n of t h i s c o m p l i c a t i o n of l i v e r d i s e a s e s i s known as the h e p a t o - r e n a l syndrome. (10) . - 5 -The p a u c i t y of p a t h o l o g i c a l f i n d i n g s a t autopsy has suggested a f u n c t i o n a l b a s i s f o r the r e n a l f a i l u r e which i s c h a r a c t e r i z e d by a r e d u c t i o n of r e n a l plasma flow (R.P.F.), and glomerular f i l t r a t i o n r a t e (G.F.R.), a low u r i n a r y sodium c o n c e n t r a t i o n , an impaired r e n a l c o n c e n t r a t -i n g a b i l i t y , a normal u r i n e sediment and a poor p r o g n o s i s . (19) (8). The cause of t h i s syndrome s t i l l remains obscure, although the r a p i d i t y w i t h which the syndrome develops and the a s s o c i a t e d moderate decrease i n systemic b l o o d p r e s s u r e , have suggested a c i r c u l a t o r y mechanism i n , ' i t s pathogenesis. In s p i t e of many s t u d i e s c a r r i e d out upon the b i o l o g i c a l e f f e c t s of endotoxin, few i n v e s t i g a t i o n s have been made of the e f f e c t s of endotoxin on r e n a l f u n c t i o n . In a d d i t i o n , most o f the s t u d i e s performed have been c a r r i e d out u s i n g a l e t h a l dose of endotoxin i n j e c t e d as a s i n g l e b o l u s . T h i s c l e a r l y i s not what r e a l l y happens i n the c l i n i c a l s i t u a t i o n . I t has a l s o been demonstrated t h a t the e f f e c t s of endotoxin change from s p e c i e s t o s p e c i e s , and very few s t u d i e s have been performed on the r e n a l e f f e c t of endotoxin i n r a t s . (20) (24) (9) . Th e r e f o r e , the purpose o f these experiments was t o study the r e n a l and c a r d i o v a s c u l a r e f f e c t s of prolonged - 6 -a d m i n i s t r a t i o n of s m a l l q u a n t i t i e s of endotoxin i n r a t s . - 7 -METHODS AND MATERIALS A. General P r e p a r a t i o n Chronic endotoxaemia was induced i n r a t s by i n f u s i o n of endotoxin from E. C o l i (026:B6 D i f c o L a b o r a t o r i e s ) . T h i s i n f u s i o n was c a r r i e d out by means of implantable osmotic mini-pumps (M.P.) ( A l z e t Model 1701), primed with 170 u l of L.P.S. s o l u t i o n . The s o l u t i o n i n f u s e d was ob t a i n e d by d i s s o l v i n g the endotoxin (10 mg./ml.) i n 0.85% s a l i n e . 1 mg. of h e p a r i n was added t o the endotoxin s o l u t i o n t o prevent c l o t t i n g i n the intravenous cannula. Normal male Wistar r a t s weighing between 327 and 4 54 g. were a n a e s t h e t i z e d w i t h e t h e r . The r i g h t e x t e r n a l j u g u l a r v e i n was cannulated with approximately 10 cm. of s i l a s t i c t u b i n g (Dow Corning: 0.020 inches I.D., 0.037 inches O.D.), which had been f i l l e d w ith h e p a r i n - s a l i n e (1:1000). Because s i l a s t i c t u b i n g i s extremely p l i a b l e , a wire was i n s e r t e d to make i t s t i f f e r and thus a l l o w an e a s i e r c a n n u l a t i o n . A f t e r s e c u r i n g the cannula, the wire was withdrawn. The cannula was then brought out to the d o r s a l r e g i o n of the neck of the r a t through a s m a l l i n c i s i o n and connected to the M.P. which was implanted subcutaneously through the s k i n i n c i s i o n . The i n c i s i o n was c l o s e d with a u t o c l i p s . The M.P. was s l i g h t l y m o d i f i e d by s e a l i n g a 26g. needle i n t o the flow modulator, to permit attachment of the s i l a s t i c t u b i n g . - 8 -The r a t e of i n f u s i o n of endotoxin was 10 _ug./h. A f t e r the i m p l a n t a t i o n of the M.P., the r a t s were l e f t i n i n d i v i d u a l metabolism cages, with f r e e access to food and water. A group of c o n t r o l r a t s was a l s o s t u d i e d i n which the M.P.s were f i l l e d w ith o n l y 0.85% s a l i n e . The r a t s were monitored d a i l y f o r a p e r i o d of 6 days. The parameters measured were: body weight, food and water consumption, u r i n e volume, c o n c e n t r a t i o n of sodium and potassium i n u r i n e and u r i n e o s m o l a l i t y . Because i t was observed t h a t r a t s t r e a t e d with endotoxin had a reduced food i n t a k e , an a d d i t i o n a l group of c o n t r o l r a t s was s t u d i e d i n which each c o n t r o l r a t was p a i r - f e d with a r a t t r e a t e d w i t h endotoxin. The r e s u l t s of these experiments showed t h a t the maximum e f f e c t of endotoxin on r e n a l f u n c t i o n was between the f o u r t h and f i f t h day, t h e r e f o r e f u r t h e r experiments were c a r r i e d out i n which the r e n a l and c a r d i o v a s c u l a r e f f e c t s of endotoxin were s t u d i e d 4 or 5 days f o l l o w i n g i m p l a n t a t i o n of the M.P. In these experiments, i n a d d i t i o n to the parameters measured d a i l y , the c o n c e n t r a t i o n of sodium and potassium i n plasma, plasma o s m o l a l i t y , plasma p r o t e i n s , l e v e l s of a l d o s t e r o n e i n plasma, haematoerit ( H c r t ) , plasma volume, sodium space and t o t a l sodium i n the body were a l s o measured. Renal and c a r d i o v a s c u l a r f u n c t i o n t e s t s (see below) were a l s o c a r r i e d out a t t h i s time. - 9 -B. Measurements 1. Water, E l e c t r o l y t e s and s o l u t e outputs Sodium and potassium c o n c e n t r a t i o n i n u r i n e and plasma were determined by flame emission spectroscopy (Instrumentation L a b o r a t o r i e s , Model 143). Urine and plasma o s m o l a l i t y were determined by measuring the d e p r e s s i o n of the f r e e z i n g p o i n t (Osmette, P r e c i s i o n Osmometer). E l e c t r o l y t e output was expressed i n uEq/24h/100g.b.w. Osmolar output i n u0sm/24h/100g.b.w. 2. Sodium space and plasma volume On the f o u r t h day a f t e r the i m p l a n t a t i o n of the 22 M.P., 1 ml of NaCl (approximately 1 jiCi/ml) i n 0.85% s a l i n e was i n j e c t e d i n t r a p e r i t o n e a l l y i n t o the r a t s . The a c t i v i t y i n j e c t e d was determined as the 22 a c t i v i t y of NaCl i n the s y r i n g e b e f o r e the i n j e c t i o n 22 minus the a c t i v i t y of NaCl i n the s y r i n g e a f t e r the i n j e c t i o n . 22 NaCl a c t i v i t y was measured wi t h a P i c k e r S p e c t r o s c a l e r 4R and w e l l - c o u n t e r s e t a t a peak of 516 KV w i t h a 10% window. The r a t s were kept o v e r n i g h t without food or water. * Urine was c o l l e c t e d as u s u a l f o r 24h.;' f o l l o w i n g 22 the i n j e c t i o n of NaCl. To make c e r t a i n t h a t the animals emptied t h e i r b l a d d e r s , a few drops of ether - 10 -were p l a c e d on the backs of the r a t s a t the end of the 24h. u r i n e c o l l e c t i o n p e r i o d . Each metabolism cage was r i n s e d w i t h approximately 10 ml of d i s t i l l e d water. 22 The a c t i v i t y of NaCl i n the u r i n e and i n the wash was determined and s u b t r a c t e d from the a c t i v i t y 22 of NaCl i n j e c t e d i n t o the r a t s . The r a t s were then a n a e s t h e t i z e d with an i n t r a -p e r i t o n e a l i n j e c t i o n of I n a c t i n (100 mg/Kg.b.w.). The r i g h t c a r o t i d a r t e r y was cannulated with p o l y e t h y l e n e t u b i n g (P.E. 50) f i l l e d w i t h h e p a r i n -s a l i n e (1:1000). 0.1 ml/lOOg.b.w. of h e p a r i n - s a l i n e was a l s o i n j e c t e d i n t o the r a t . Mean A r t e r i a l Blood Pressure (M.A.B.P.) and Heart Rate (H.R.) were recorded by a p r e s s u r e transducer system. Plasma volume was next measured by the method of Wang and Hegsted.(55). A f t e r an abdominal i n c i s i o n was made, bloo d was c o l l e c t e d from the i n f e r i o r vena cava. A s m a l l sample of b l o o d was taken to measure the haematoerit. The remaining b l o o d was c e n t r i f u g e d and 22 the plasma used f o r the a n a l y s i s of Na, T-1824 and plasma e l e c t r o l y t e s and o s m o l a l i t y . Kidneys, l i v e r and lungs were removed and weighed. 22 The a c t i v i t y of NaCl was measured i n 1 ml of plasma. - 11 -C a l c u l a t i o n of Sodium Space and Exchangeable Sodium Sodium Space (ml/lOOg) = — 22 22 c.p.m. Na i n j e c t e d - c.p.m. Na exc r e t e d — c.p.m. Na i n 1 ml plasma mEq\ x 100 b.w.(g) Exchangeable Sodium \ Kg Sodium Space L ^x Plasma [ N a + ] m E q Kg \ L ; 3. Measurement of Al d o s t e r o n e i n plasma Plasma a l d o s t e r o n e l e v e l s were measured on the f o u r t h day a f t e r i m p l a n t a t i o n of the mini-pumps i n a group of r a t s t r e a t e d with endotoxin and i n a group of p a i r - f e d c o n t r o l s . The r a t s weighing between 209g and 419g were p l a c e d i n a j a r c o n t a i n i n g dry i c e u n t i l unconscious. Then, a f t e r an abdominal i n c i s i o n was made, blood was withdrawn from the vena cava i n t o a s y r i n g e c o n t a i n -i n g 0.07 ml of 15% E.D.T.A. used as a n t i c o a g u l a n t . The blood was c e n t r i f u g e d and the plasma was f r o z e n and sent f o r a l d o s t e r o n e d e t e r m i n a t i o n t o the l a b o r a t o r y f o r S t e r o i d Research a t S a i n t Pauls H o s p i t a l . 4. Renal f u n c t i o n t e s t s On the f o u r t h or f i f t h day a f t e r i m p l a n t a t i o n of the M.P.'s, the f o l l o w i n g r e n a l f u n c t i o n t e s t s were a l s o performed. Blood Urea N i t r o g e n (B.U.N.), 24h C r e a t i n i n e - 12 -e x c r e t i o n , I n u l i n c l e a r a n c e (G.F.R) and P.A.H. c l e a r -ance (R.P.F.). a) B.U.N was measured by the indophenol r e a c t i o n u s i n g a standard B.U.N, k i t . ( 1 1 ) . b) C r e a t i n i n e was measured as the a l k a l i n e p i c r a t e d e r i v a t i v e . c) Clearance Measurements G.F.R. and R.P.F. were estimated from the c l e a r -3 14 ances of ( H) I n u l i n and ( C) para amino h i p p u r a t e (P.A.H.) r e s p e c t i v e l y . These de t e r m i n a t i o n s were c a r r i e d out i n both i ) a n a e s t h e t i z e d and i i ) unanaesthetized r a t s . i ) Rats weighing between 216g and 441g were anaes-t h e t i z e d w i t h I n a c t i n (100 mg/Kg) i n t r a p e r i t o n e a l l y (i.p.) and p l a c e d on a thermoregulated t a b l e t o maintain t h e i r body temperature a t 37°C. The r i g h t c a r o t i d a r t e r y , l e f t e x t e r n a l j u g u l a r v e i n and bladder were cannulated with p o l y e t h y l e n e t u b i n g (P.E. 50). The a r t e r i a l and venous cannulas were f i l l e d w ith h e p a r i n - s a l i n e 1:1000 and 0.1 ml/lOOg body weight of h e p a r i n - s a l i n e was i n j e c t e d i n t r a v e n o u s l y . i i ) The b l o o d v e s s e l s and bl a d d e r were a l s o cannulated i n a second group of r a t s a n a e s t h e t i z e d w i t h e t h e r . F o l l o w i n g surgery, the r a t s were p l a c e d i n p l a s t i c h o l d e r s and allowed t o recover from the anaes-t h e t i c , (see F i g . 2). - 13 -3 A 0.85% s a l i n e s o l u t i o n c o n t a i n i n g ( H) I n u l i n 14 and ( C) P.A.H. (New England Nuclear Corp.) was i n f u s e d i n travenous ( i . v . ) i n both groups of r a t s . 3 The r a t e of i n f u s i o n was 0.829 uCi/min. of ( H) 14 I n u l i n and 0.414 uCi/min. of ( C) P.A.H. f o r the f i r s t seven minutes as a priming dose. The r a t e of i n f u s i o n 3 was then lowered to 0.083 uCi/min. of ( H) I n u l i n and 0.041 uCi/min. of ( 1 4C) P.A.H. f o r the r e s t of the c l e a r a n c e p e r i o d . The r a t e of s a l i n e i n f u s i o n was 1.2 ml/h (20 jil/min.) A f t e r 2h f o r e q u i l i b r a t i o n , two c o n s e c u t i v e u r i n e c o l l e c t i o n s were made f o r a p e r i o d of 20 minutes each. A sma l l sample of a r t e r i a l b l o o d (^ 50ul) was taken a t the mid-point of the u r i n e c o l l e c t i o n p e r i o d s 3 14 to determine the a c t i v i t y of ( H) and ( C) i n the plasma. At the end of the c l e a r a n c e p e r i o d s a second a r t e r i a l b l o o d sample was c o l l e c t e d . P a r t was used to measure the haematocrit. The r e s t of the bloo d was c e n t r i f u g e d and used to determine plasma e l e c t r o l y t e s (Na +, K +) and plasma p r o t e i n . The u r i n e samples c o l l e c t e d i n the 20 minute p e r i o d s were weighed and e l e c t r o l y t e s (Na +, K +) as 3 14 w e l l as the a c t i v i t y of ( H and ( C) were measured. 3 14 To measure the a c t i v i t y of the ( H) and ( C), 20 u l of e i t h e r u r i n e or plasma were p l a c e d i n i n d i v i d u a l v i a l s c o n t a i n i n g 3 mis of d i s t i l l e d water. - 14 -F i g u r e 2 Clearance d e t e r m i n a t i o n and b l o o d pressure measure-ment i n the u n a e s t h e t i z e d r a t . - 14a -- 15 -Then, 10 mis. of Aquasol.II was added and the mixture was shaken v i g o r o u s l y to make a g e l . The a c t i v i t i e s of both r a d i o i s o t o p e s were counted i n a L i q u i d S c i n t i l l a t i o n Counter (Beckman L.S. - 233) with a p p r o p r i a t e c o r r e c t i o n s f o r s p i l l - o v e r , The e x t r a c t i o n r a t i o of P.A.H. (E.P.A.H.) had been shown to be 85% f o r both c o n t r o l r a t s and r a t s t r e a t e d with endotoxin,(29) t h e r e f o r e the t r u e r e n a l plasma flow (R.P.F.) i s equal t o : R.P.F. = E - R - p - F - = CP.A.H. E.P.A.H. 0.85 T h i s v a l u e was used to c a l c u l a t e Renal Blood Flow (R.B.F.) and F i l t r a t i o n F r a c t i o n ( F . F . ) . R.B.F. = R - P ' F - F.F. = G.F.R. (1 - Hcrt) R.P.F. In these experiments, i t was assumed t h a t the whole kidney haematoerit was the same as the a r t e r i a l haematoerit. G.F.R., R.P.F. and R.B.F. were expressed as ml/min/lOOg b.w. 5. C a r d i o v a s c u l a r measurements Ca r d i a c Output (CO.) and i t s d i s t r i b u t i o n were measured on the f o u r t h day f o l l o w i n g i m p l a n t a t i o n of the M.P. C O . was measured by both the r e f e r e n c e sample technique u s i n g r a d i o a c t i v e microspheres(38) (23), and - 16 -by the d i r e c t F i c k method.(54). The d i s t r i b u t i o n of C O . to the kidneys was measured wi t h r a d i o a c t i v e microspheres and w i t h 8 6 R b C l . R.P.F. was determined a f t e r both C O . measure-14 ments by measuring the ( C) P.A.H. c l e a r a n c e as d e s c r i b e d above. R.B.F. was a l s o estimated from the r e n a l d i s t r i -b u t i o n of microspheres. a) R a d i o a c t i v e microsphere technique A simultaneous measurement of C O . and i t s 57 d i s t r i b u t i o n t o the kidneys and h e a r t u s i n g Co -l a b e l l e d microspheres was c a r r i e d out as f o l l o w s . Rats weighing between 29Og and 312g were a n a e s t h e t i z e d w i t h I n a c t i n , p l a c e d on a thermoregulated t a b l e and p o l y e t h y l e n e cannula (PE 50) was p l a c e d i n the l e f t v e n t r i c l e of the h e a r t v i a the r i g h t c a r o t i d a r t e r y . The c o r r e c t p o s i t i o n of the cannula i n the l e f t v e n t r i c l e was confirmed by r e c o r d i n g the c h a r a c t e r i s t i c v e n t r i c u l a r p r e s s u r e waves. The l e f t e x t e r n a l j u g u l a r v e i n and the b l a d d e r were a l s o cannulated u s i n g p o l y e t h y l e n e t u b i n g (PE 50). The femoral a r t e r y was cannulated u s i n g a s h o r t p i e c e of PE 10 t u b i n g (2cm) which was a t t a c h e d to a longer p i e c e of PE 50 t u b i n g (10cm). The femoral cannula was f i l l e d w i t h h e p a r i n -s a l i n e (1:1000) and connected to a 1 ml. s y r i n g e which - 17 -was attached to a withdrawl pump (Harward Apparatus, Model No 940). 0.1 ml/lOOg body weight of h e p a r i n - s a l i n e was i n j e c t e d i n t o the r a t s . A f t e r the r a t s had recovered from the s u r g i c a l procedures, a r t e r i a l blood pressure and heart r a t e were recorded from the femoral a r t e r y cannula. 57 Approximately 60,000 Co - l a b e l l e d micro-spheres (15± 3um, New England Nuclear) suspended i n 2% dextran and d i l u t e d w i t h 0.85% s a l i n e to a t o t a l volume of approximately 300 j i l , were i n j e c t e d i n t o the l e f t v e n t r i c l e of the r a t s through the r i g h t c a r o t i d a r t e r y cannula over a p e r i o d of about 20 seconds. Then, approximately 0.1 ml of blood was withdrawn t o wash out a l l the microspheres remaining i n the cannula. The syringe used f o r the i n j e c t i o n was modified i n such a way t h a t the dead=space was reduced to a minimum, (approximately 6.6ul). The a c t i v i t y of the microspheres i n j e c t e d i n t o 57 the r a t s was c a l c u l a t e d from the a c t i v i t y of Co -l a b e l l e d microspheres i n the syringe before the i n j e c -57 t i o n minus the a c t i v i t y of Co - l a b e l l e d microspheres i n the syringe a f t e r the i n j e c t i o n . Simultaneously w i t h the i n j e c t i o n of the micro-spheres, a blood sample was withdrawn from the femoral a r t e r y at the r a t e of 0.43 ml/min. 57 This blood sample was counted f o r Co a c t i v i t y . - 18 -To make sure t h a t the cannula was s t i l l i n the l e f t v e n t r i c l e d u r i n g the i n j e c t i o n of the microspheres, i t was atta c h e d again to the p r e s s u r e - t r a n s d u c e r system and the v e n t r i c u l a r p r essure waves were recorded a g a i n . A f t e r the de t e r m i n a t i o n of the CO., the blood withdrawn from the femoral a r t e r y was i n j e c t e d slowly back i n t o the r a t . The R.P.F. was then determined as d e s c r i b e d above f o r c l e a r a n c e measurements. A f t e r measuring the c l e a r a n c e of (14C) P.A.H., the r a t s were k i l l e d with an i . v . i n j e c t i o n of a s a t u r a t e d s o l u t i o n of KC'l. Kidneys and he a r t were removed, weighed and the 57 Co a c t i v i t y measured. These v a l u e s were used t o c a l c u l a t e the d i s t r i b u -t i o n of the C O . to these organs. C a l c u l a t i o n of the Ca r d i a c Output and i t s d i s t r i b u t i o n 57 u s i n g the Co - l a b e l l e d microsphere technique Microspheres, because of t h e i r s i z e , are trapped i n the c a p i l l a r i e s , b e i n g t h e r e f o r e c l e a r e d from the blood a f t e r one c i r c u l a t i o n . I f the microspheres are w e l l mixed with the blood, 57 the number of Co - l a b e l l e d microspheres i n an organ i s p r o p o r t i o n a l t o the bloo d flow through the organ. The percentage o f the C O . to an organ can be c a l c u l a t e d as: - 19 -o/-, ^ j_ c.TD.m. i n the organ %C.O. to the organ = " ^ x 10 0 T o t a l e.p.m. i n j e c t e d i n t o the r a t In the r e f e r e n c e sample method, a s y r i n g e connected to the femoral cannula f u n c t i o n s as an a r t i f i c i a l organ. Then, i f the r a t e of bloo d flow i n t o t h i s a r t i f i c i a l organ i s known, the C O . of the r a t can be found. C O . was t h e r e f o r e c a l c u l a t e d as f o l l o w s : C O . (ml/min) = T o t a l c.p.m. i n j e c t e d x S a m p l i n g r a t e ( 0 > 4 3 m l / m i n ) c.p.m. i n a r t i f i c i a l organ C O . was expressed i n ml/min/lOOg of body weight. b) Measurement of C a r d i a c Output by a d i r e c t F i c k P r i n c i p l e To determine C O . u s i n g the d i r e c t F i c k method, measurements of the oxygen consumption and the 0^ content i n a r t e r i a l and i n mixed venous b l o o d are needed. To measure oxygen consumption, a c l o s e d c i r c u i t m i crospirometer apparatus was used. (See F i g . 3 ) . Oxygen content of the bloo d samples was determined by an a d a p t a t i o n of the method of Linden, Ledsome and Norman.(35). Rats weighing between 251g and 319g were - 20 -Figure 3 Diagrammatic r e p r e s e n t a t i o n of the c l o s e d c i r c u i t micro-spirometer apparatus, used t o measure oxygen consumption (V n ) i n anaesthetized c o n t r o l and endotoxin t r e a t e d r a t s . " u2 Spirometers - I , lOOOml Venous Cannulae : Arter ia o C 0 2 Absorber - 21 -a n a e s t h e t i z e d w i t h I n a c t i n . The temperature of the body was kept a t 37°C by u s i n g a thermoregulated t a b l e . C a n n u l a t i o n of the r i g h t a t r i u m through the l e f t j u g u l a r v e i n , the l e f t v e n t r i c l e through the r i g h t c a r o t i d a r t e r y and of the bladder were done u s i n g p o l y e t h y l e n e t u b i n g (PE 50). H e p a r i n - s a l i n e (0.1 ml/lOOg of body weight) was i n j e c t e d i n t o the r a t . The l e n g t h of the bloo d v e s s e l cannulas was 28 cm, t h i s made i t p o s s i b l e t o o b t a i n blood samples w h i l e the r a t s were i n s i d e the p l a s t i c chamber of the micro-spirometer. Mean a r t e r i a l blood p r e s s u r e and h e a r t r a t e were measured through the c a r o t i d cannula by means of a pres s u r e t r a n s d u c e r system b e f o r e the cannula was advanced i n t o the l e f t v e n t r i c l e . Once the r a t s were i n s t a b l e c o n d i t i o n a f t e r the surgery, they were p l a c e d i n a p l a s t i c h o l d e r . A thermo-e l e c t r i c b l a n k e t was wrapped around the p l a s t i c h o l d e r to keep the body temperature of the r a t a t 37°C. The p l a s t i c h o l d e r was c l o s e d to the atmosphere wit h two rubber stoppers, one on e i t h e r end. A thermometer and a g l a s s "U" tube were f i x e d on these rubber stoppers. The g l a s s "U" tube was f i l l e d w ith heavy m i n e r a l o i l and used t o e x t e r i o r i z e the blood v e s s e l s cannulas from the c l o s e d system without l o s s of oxygen. - 22 -The r a t h o l d e r was connected through rubber tubes, a c t i n g as a c i r c u l a t i n g gas l i n e , to one s i d e with two spirometers of 1 L and 50 ml. c a p a c i t y r e s p e c t i v e l y , and to the other s i d e t o a tank c o n t a i n -i n g pure oxygen which served t o f i l l the sp i r o m e t e r s . The gas w i t h i n the c i r c u i t was r e c i r c u l a t e d u s i n g a Cole-Parmer pump whose speed was c o n t r o l l e d by a Cole-Parmer m a s t e r f l e x SCR c o n t r o l l e r . A C 0 2 absorber c o n t a i n i n g 20% NaOH was i n c l u d e d i n the gas l i n e s . Keeping the l a r g e spirometer open to the c i r c u i t and the sm a l l spirometer c l o s e d through a three way tap, the r a t s were allowed t o b r e a t h pure f o r a t l e a s t t e n minutes. At t h i s stage the e f f i c a c y of the C0 2 absorber was checked by t a k i n g a sample of gas from the c l o s e d c i r c u i t . The CC^ content of the gas c o l l e c t e d was determined by u s i n g a CC^ a n a l y z e r . To measure consumption, the sm a l l spirometer was f i l l e d w i t h t o a c e r t a i n l e v e l , keeping the l a r g e spirometer c l o s e d t o the c i r c u i t . The time r e q u i r e d f o r the r a t to b r e a t h 10 ml of C>2 from the smal l spirometer was measured u s i n g a stop watch. A f t e r t h i s , both spirometers were open and venous and a r t e r i a l b l o o d samples were c o l l e c t e d i n m o d i f i e d 1 ml s y r i n g e s . - 23 -The m o d i f i c a t i o n c o n s i s t e d of s e a l i n g a 23g needle i n t o the t i p of the s y r i n g e with epoxy r e s i n . Then, the needle was ground down i n such a way t h a t the needle s h a f t protruded approximately 3 mm from the sy r i n g e t i p . With t h i s m o d i f i c a t i o n the dead-space i n the s y r i n g e was l e s s than 1% of the volume of bloo d c o l l e c t e d . The amounts of a r t e r i a l and mixed venous bloo d withdrawn were 0.25 ml each. Venous samples were c o l l e c t e d f i r s t and the t i p of the s y r i n g e was c l o s e d w i t h a rubber cap to a v o i d any co n t a c t o f the blood with the atmosphere. Then the s y r i n g e was p l a c e d i n i c e water u n t i l the P n d e t e r m i n a t i o n was done. U 2 The a r t e r i a l blood sample was c o l l e c t e d next and a l s o a sample of blood t o measure the haematoerit. A 20 ml l u e r l o c k g l a s s s y r i n g e connected to a three way metal tap was f i l l e d w ith 10 ml of a s o l u t i o n c o n t a i n i n g saponin and potassium f e r r i c y a n i d e . T h i s s o l u t i o n was made by d i s s o l v i n g saponin (3g/L) and K f e r r i c y a n i d e (6g/L) i n d i s t i l l e d water. The water was p r e v i o u s l y b o i l e d t o reduce the q u a n t i t y of d i s s o l v e d C ^ i n i t . The reagent was kept i n an Erlenmeyer f l a s k under heavy m i n e r a l o i l to a v o i d c o n t a c t with the atmosphere. The reagent was withdrawn from the f l a s k u s i n g a 10 cm l e n g t h of p o l y e t h y l e n e t u b i n g (PE 100) atta c h e d - 24 -to a s y r i n g e through a three way tap. The three way tap a t t a c h e d to the 20 ml g l a s s s y r i n g e was l e f t open to the l a t e r a l c o n n e c t i o n where the 1 ml p l a s t i c s y r i n g e c o n t a i n i n g the b l o o d sample was a t t a c h e d . The b l o o d was mixed wi t h the reagent by emptying and f i l l i n g the 1 ml s y r i n g e and by r o t a t i o n of the 20 ml g l a s s s y r i n g e . Once the mixing was completed, the 20 ml s y r i n g e was shut o f f and the 1 ml s y r i n g e was removed. The p a r t i a l p r essure of 0 2 of the blood-reagent mixture was measured u s i n g a Corning/6 5/2 pH Blood Gas A n a l y z e r . The p a r t i a l p r essure of 0 2 of the K f e r r i c y a n i d e reagent was a l s o measured. A f t e r the c a l c u l a t i o n of CO., the r a t was p l a c e d on a thermoregulated t a b l e and i t s body tempera-t u r e kept a t 37°C. Approximately 0.5 ml of b l o o d was slowly i n j e c t e d i n t o the r a t s t o r e p l a c e the b l o o d t h a t had been w i t h -drawn . A f t e r a r e c o v e r y and e q u i l i b r a t i o n p e r i o d of 57 about 30 minutes, Co - l a b e l l e d microspheres were i n j e c t e d i n t o the l e f t v e n t r i c l e through the c a r o t i d a r t e r y cannula. 14 Then, an i . v . i n f u s i o n of ( C) P.A.H. was s t a r t e d i n order to determine the r e n a l plasma flow as - 25 -d e s c r i b e d under "clearance measurements" above. Once the c l e a r a n c e measurement was f i n i s h e d , the r a t was k i l l e d by an intravenous i n j e c t i o n of KC1. The kidneys and h e a r t were removed, weighed and 57 Co a c t i v i t y counted. These v a l u e s were used to c a l c u l a t e the f r a c t i o n a l d i s t r i b u t i o n of the C O . to the organs as d e s c r i b e d above. C a l c u l a t i o n of c a r d i a c output The content of oxygen i n 100 ml of whole blo o d was c a l c u l a t e d u s i n g the formula d e s c r i b e d by Linden, Ledsome and Norman.(35). c o 2 =tX 100 A v (V+v) Prb©2 - Pr°2 V ( ) v+V where: C C , = C X -v V Prob, Pro, Content of 0^ i n 100 ml of b l o o d Oxygen s o l u b i l i t y expressed as ml 0 2 a t normal temperature and p r e s s u r e (NTP)/ml solution/mmHg. At 37°C,cx has a v a l u e of 3.5 10~ 5 ml 0 2/ml blood/mmHg Volume of b l o o d used Volume of reagent used + volume of the dead space of the s y r i n g e Oxygen t e n s i o n of the b l o o d - saponin K f e r r i c y a n i d e mixture Oxygen t e n s i o n of the saponin K f e r r i -cyanide C a r d i a c output = V ° 2 x 100 i -i / • \ Cao 0 - Cvo„ (ml/mm) 2 2 Where: Vc>2 : Oxygen consumption (ml/min) Cao 2 : Oxygen content i n a r t e r i a l blood Cvo 2 : Oxygen content i n the mixed venous blo o d C a r d i a c output was expressed i n ml/min/lOOg of body weight, c) I n d i r e c t e s t i m a t i o n of c a r d i a c output Using the va l u e s o b t a i n e d from R.P.F. and the percent of d i s t r i b u t i o n of the C O . to the kidneys, C O . was a l s o c a l c u l a t e d as f o l l o w s : _, ,. _ . . Renal b l o o d flow C a r d i a c Output = . n , . . % of d i s t r i b u t i o n of C O . (ml/mm) t o t h e k i d n e y s d) Measurement of the d i s t r i b u t i o n of C a r d i a c Output 86 to the kidneys u s i n g RbCl As a means of c o n f i r m i n g the va l u e s found f o r the d i s t r i b u t i o n of C O . to the kidneys measured 57 p r e v i o u s l y w i t h C o - l a b e l l e d microspheres, a s a l i n e 8 6 s o l u t i o n c o n t a i n i n g both RbCl and r a d i o a c t i v e micro-spheres was i n j e c t e d i n t o the r a t s . Rats whose weight ranged from 220g and 440g were a n a e s t h e t i z e d with an i . p . i n j e c t i o n of I n a c t i n , and pl a c e d on a thermoregulated t a b l e to keep the body temperature a t 3 7 ° C - 27 -The l e f t v e n t r i c l e of the heart was cannulated through the r i g h t c a r o t i d a r t e r y using polyethylene tubing (PE 50). Before p l a c i n g the cannula i n the v e n t r i c l e , the M.A.B.P. and the H.R. were monitored through the c a r o t i d a r t e r y cannula using a pressure transducer system. A small a r t e r i a l blood sample was withdrawn to measure the haematoerit and plasma p r o t e i n c o n c e n t r a t i o n 6 then, a mixture c o n t a i n i n g approximately 10 c.p.m. of 86 6 57 RbCl and 10 c.p.m. of C o - l a b e l l e d microsphere i n a t o t a l volume of 250 u l of 0.85% s a l i n e s o l u t i o n was i n j e c t e d i n t o the l e f t v e n t r i c l e v i a the c a r o t i d a r t e r y . The i n j e c t i o n was completed i n approximately 10 seconds, a f t e r which 0.1 ml of blood was withdrawn to wash out the r a d i o a c t i v i t y l e f t i n the cannula. The r a t s were then k i l l e d w i t h an i n t r a - a r t e r i a l i n j e c t i o n of saturated KC1 50 seconds a f t e r completing the i n j e c t i o n . A m i d l i n e abdominal i n c i s i o n was made, both 57 kidneys were removed, weighed and counted f o r Co and ^ R b C l i n a gamma counter. The t o t a l r a d i o a c t i v i t y i n j e c t e d i n t o the r a t was c a l c u l a t e d as the d i f f e r e n c e between the r a d i o -a c t i v i t y of the isotopes i n the syringe before and a f t e r the i n j e c t i o n . When ^Rb and ^ C o are counted together i n a 86 mixture, the spectrum of Rb appears to i n t e r f e r e w i t h 57 the counting of Co. - 28 -T h e r e f o r e , t o check i f the counts o b t a i n e d 57 f o r the C o - l a b e l l e d microspheres were c o r r e c t , kidneys removed from r a t s which were i n j e c t e d w i t h a mixture of both r a d i o i s o t o p e s were kept i n formaldehyde 86 u n t i l the a c t i v i t y of Rb decayed t o an i n s i g n i f i c a n t l e v e l (4 months), ( t A 8 6 R b C l = 18.7 d ) . 2 57 The a c t i v i t y of Co i n the kidneys was then re-measured and c o r r e c t e d f o r r a d i o a c t i v e decay. 86 In an a d d i t i o n a l group of r a t s , Rb only was i n j e c t e d . 6) Renal Medullary plasma flow measurement The method used i n these experiments t o measure medullary plasma flow (M.P.F.), i s a m o d i f i c a t i o n of the L i l i e n f i e l d method.(34). I t i s based on the f a c t t h a t the t r a n s i t time f o r c i r c u l a t i n g albumin i n the r e n a l p a p i l l a of the normal r a t i s 4 0 sec. Then, i f r a d i o a c t i v e albumin i s allowed t o c i r c u l a t e through the kidneys f o r 30 s e c , the accumu-l a t i o n r a t e of p a p i l l a r y r a d i o a c t i v i t y d u r i n g t h i s time w i l l be p r o p o r t i o n a l t o the plasma flow r a t e . A f t e r the second day of i m p l a n t a t i o n of the M.P.'s when the u r i n e c o n c e n t r a t i o n was very low, r a t s weighing between 239g and 324g were a n a e s t h e t i z e d with I n a c t i n i . p . and p l a c e d on a thermoregulated t a b l e t o ma i n t a i n the body temperature a t 37°C. The t r a c h e a and l e f t j u g u l a r v e i n were cannulated u s i n g p o l y e t h y l e n e t u b i n g . - 29 -To check at the same time i f the u r i n e concen-t r a t i n g defect was caused by a d e f i c i e n c y i n endogenous a n t i d i u r e t i c hormone (A.D.H.), the r a t s were i n j e c t e d w i t h 0.1 ml of A.D.H. tannate (2U/ml) i n t r a m u s c u l a r l y (41), or inf u s e d w i t h a r g i n i n e vasopressin (2uU/min/100g). C o n t r o l r a t s were i n f u s e d i . v . f o r approximately 4 hours wi t h 0.85% s a l i n e and r a t s t r e a t e d w i t h endo-t o x i n were in f u s e d w i t h a s o l u t i o n c o n t a i n i n g endotoxin (0.013 mg/ml) i n 0.85% s a l i n e , i n both cases the s o l u t i o n was i n f u s e d at the r a t e of 13 ul/min. Urine was c o l l e c t e d during t h i s p e r i o d of time and the volume, sodium and potassium conce n t r a t i o n and o s m o l a l i t y were measured each h a l f hour. Then, a mid-l i n e abdominal i n c i s i o n was made, both kidneys were exposed and loose non-occlusive s i l k threads (3-0) were placed around the r e n a l p e d i c l e s . The threads were e x t e r i o r i z e d from the r a t abdominal c a v i t y through polyethylene tubing (PE 100), p e r m i t t i n g o c c l u s i o n of the r e n a l p e d i c l e while keeping the abdominal c a v i t y c l o s e d . Care was taken to not i n t e r r u p t the r e n a l c i r c u l a t i o n a t t h i s stage. The c a r o t i d a r t e r y was then cannulated using polyethylene tubing (PE 50) and 0.1 ml/lOOg of body weight of h e p a r i n - s a l i n e was i n j e c t e d i n t o the r a t . The c a r o t i d cannula was connected to a 1 ml syringe which was attached to a pump (Harvard). - 30 -During the surgery a s o l u t i o n c o n t a i n i n g 5% of albumin i n 0.85% s a l i n e s o l u t i o n was i n f u s e d i n t o c o n t r o l r a t s to avoid l o s s of plasma volume and con-sequent increase i n haematocrit.(37). A s o l u t i o n c o n t a i n i n g 5% albumin plus endo-t o x i n (0.0034 mg/ml) i n 0.85% s a l i n e was i n f u s e d i n t o experimental r a t s . In both groups, the i n f u s i o n r a t e was 50 ^ul/min. A f t e r surgery the r a t s were allowed to reach steady s t a t e f o r a p e r i o d of about h a l f an hour. No i n f u s i o n was given during t h i s time. Then, a s i n g l e bolus of a concentrated s o l u t i o n of Evans blue i n 0.85% s a l i n e was i n j e c t e d through the j u g u l a r v e i n to determine the c i r c u l a t i o n time between the j u g u l a r v e i n and the kidney. Once the t r a n s i t time was determined (^3 sec) 125 a s o l u t i o n of I - l a b e l l e d albumin (1 uCi/ml) i n 0.85% , s a l i n e was i n f u s e d through the j u g u l a r v e i n at the r a t e of 0.37 ml/min f o r a p e r i o d of 30 seconds a f t e r the t r a n s i t time p e r i o d . Simultaneously, an a r t e r i a l blood sample was c o l l e c t e d through a c a r o t i d cannula at the r a t e of 1.08 ml/min f o r the l a s t 30 seconds of the i n f u s i o n p e r i o d . At the end of the i n f u s i o n p e r i o d the snares around the kidneys were p u l l e d t i g h t to stop the c i r c u l a -t i o n to the kidneys. - 31 -The r a t was k i l l e d by opening the c h e s t c a v i t y . The kidneys were removed, keeping the snares i n p l a c e , and c u t i n h a l f from pole to p o l e i n such a way t h a t a l l the p a p i l l a remained i n j u s t one h a l f of the kidney. The p a p i l l a was removed from the r e s t of the kidney, wiped with non-absorbent paper t o remove adher-i n g b lood, and p l a c e d i n t o a capped, preweighed p l a s t i c tube. A s m a l l sample of a r t e r i a l b l o o d was used to determine the haematoerit. 125 A f t e r weighing the p a p i l l a , the T a c t i v i t y , 125 together w i t h the I a c t i v i t y of an a l i q u o t of the a r t e r i a l b l o o d sample were measured i n a gamma counter. The medullary plasma flow was c a l c u l a t e d by d i v i d i n g p a p i l l a r y a c t i v i t y by plasma r a d i o a c t i v i t y and was expressed i n u n i t s of ml plasma/min/lOOg of p a p i l l a . 7) Determination of H a l f L i f e of Endotoxin i n plasma 51 and the uptake of Cr-endotoxin by d i f f e r e n t  organs A f t e r the f o u r t h day of i m p l a n t a t i o n of the M.P., the c i r c u l a t i n g h a l f l i f e ( t x ) of endotoxin i n the plasma was measured i n a n a e s t h e t i z e d and i n unanaesthetized r a t s by an i n t r a - a r t e r i a l i n j e c t i o n of a s i n g l e b olus of 51 endotoxin l a b e l l e d with Cr. 51 Endotoxin was l a b e l l e d w i t h hexavalent Cr as d e s c r i b e d by Braude e t a l . ( 3 ) . - 32 -51 The hexavalent Cr-endotoxin has been r e p o r t e d to be a s p e c i f i c tagged product which maintains the l a b e l and t o x i c i t y i n plasma f o r s e v e r a l hours a f t e r the i n j e c t i o n . ( 6 ) . Rats weighing between 233g and 383g were anaes-t h e t i z e d with e t h e r , the r i g h t c a r o t i d a r t e r y was cannulated u s i n g p o l y e t h y l e n e t u b i n g (PE 50) and 0.1 ml/lOOg of body weight of h e p a r i n - s a l i n e (1:1000) was i n j e c t e d i n t o the r a t s . The r a t s were p l a c e d i n a p l a s t i c h o l d e r and they' were allowed to recover from the a n a e s t h e s i a . The c a r o t i d cannula was connected to a s y r i n g e v i a a three way tap. When the r a t s were awake, a s o l u t i o n of 0.125 mg 51 of C r - l a b e l l e d endotoxin i n 250 u l of 0.85% s a l i n e , was i n j e c t e d i n t o the r a t s through the c a r o t i d cannula. The s o l u t i o n was washed i n with s a l i n e . Two t u b e r c u l i n e s y r i n g e s were then a t t a c h e d to the t h r e e way tap and blood samples (approximately 0.3ml) were taken a t i n t e r v a l s . The three way tap was cleaned a f t e r each sampling. Then, a c c u r a t e l y measured samples of 0.2ml were used to measure the "^*"Cr a c t i v i t y i n a gamma counter. The same procedure was c a r r i e d out with o t h e r groups of r a t s which were a n a e s t h e t i z e d w i t h I n a c t i n . 51 Counts per minute (c.p.m.) of Cr i n the blood vs time were graphed on . s e m i l o g a r i t h m i c paper. - 33 -From t h i s graph, the t A of endotoxin i n plasma, 2 was es t i m a t e d . The r a t e c onstant (K) and the time c o n s t a n t (TC) were c a l c u l a t e d from the f o l l o w i n g formula: TC = 1.4 4 x t x K = _ i TC A f t e r the measurement o f the tk o f endotoxin i n 2 plasma, the r a t s were k i l l e d and the kidneys, l i v e r , 51 lungs and s p l e e n removed, weighed and the Cr a c t i v i t y i n these organs was measured. 8) H e x o b a r b i t a l s l e e p i n g time measurements To determine i f the s h o r t t ^ of endotoxin i n plasma observed i n r a t s t r e a t e d w i t h endotoxin was due i n p a r t t o g e n e r a l i z e d enzymatic i n d u c t i o n , measurement of h e x o b a r b i t a l s l e e p i n g time was c a r r i e d out. The measurement of h e x o b a r b i t a l s l e e p i n g time i s used as an index of h e x o b a r b i t a l metabolism. Four days a f t e r i m p l a n t a t i o n of the M.P., a 0.85% s a l i n e s o l u t i o n c o n t a i n i n g hexobarbitone sodium i n a dose of 70 mg/kg of the body weight (22)(59) was i n j e c t e d i . p . i n t o male r a t s weighing between 226g and 279g. The s l e e p i n g time was measured as the time between the l o s s and the recove r y of the r i g h t i n g r e f l e x . - 34 -C) S t a t i s t i c a l A n a l y s i s R e s u l t s as shown as mean ± SEM. S t u d e n t ' s t w o - t a i l e d t t e s t f o r p a i r e d o r u n p a i r e d d a t a a s a p p r o p r i a t e , was u s e d t o d e t e r m i n e t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e between c o n t r o l r a t s and r a t s t r e a t e d w i t h e n d o t o x i n . When t h e number o f a n i m a l s p e r g r o u p was l e s s t h a n 6, a Mann-Whitney U t e s t was u s e d t o e v a l u a t e d i f f e r e n c e s between t h e two g r o u p s . - 35 -RESULTS 1. Water and s o l u t e output Chronic i n f u s i o n of endotoxin (10 ug/h) i n t o r a t s f o r a p e r i o d of s i x days r e s u l t e d i n a s i g n i f i c a n t decrease i n r e n a l sodium output. Sodium output was minimum at the f o u r t h day a f t e r the i m p l a n t a t i o n of the M.P.s 21 ± 8 uEq/ 24h/100g of the b.w., f o r r a t s t r e a t e d w i t h endotoxin com-pared wi t h 138 ± 17 pEq/24h/100g of the b.w. f o r c o n t r o l . A f t e r the f o u r t h day, sodium output returned t o normal v a l u e s . ( F i g . 4 ) . Food consumption by r a t s t r e a t e d w i t h endotoxin was 54% of the amount consumed by c o n t r o l r a t s . ( F i g . 5 ), and the r e f o r e a s i g n i f i c a n t d i f f e r e n c e was found i n the percent of weight l o s s between the two groups, 3.00 ± 1.7% i n c o n t o r l r a t s and 9.67 ±3.9% i n r a t s t r e a t e d w i t h endotoxin. ( F i g . 6) To c o n t r o l the e f f e c t s t h a t d i f f e r e n c e s i n food intake may have had on r e n a l f u n c t i o n , experiments were done i n which each c o n t r o l r a t was p a i r fed with"a r a t t r e a t e d w i t h endotoxin. The r e s u l t s of these experiments showed tha t even when both groups ingested the same amount of sodium, 277.5 ± 16.8 ^iEq/24h/100g of the b.w. f o r c o n t r o l (n=6) and 294.8 ± 86.1 uEq/24h/100g of the b.w. f o r r a t s t r e a t e d w i t h endotoxin (n=6) there was s t i l l a s i g n i f i c a n t decrease i n - 36 -F i g u r e 4 Sodium output i n c o n t r o l r a t s (n=6) and i n r a t s t r e a t e d with endotoxin (n=6), f o r a p e r i o d of s i x days. * v a l u e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). Mean sodium output i n unoperated c o n t r o l r a t s : 528 ± 11 uEq/24h/100g - 36a -6001 Control Endotoxin o g N c <«• CVJ N '5 5 450 300 3 o E 3 •5 o 150 6 - 37 -F i g u r e 5 Food consumption by c o n t r o l r a t s (n=6) and r a t s t r e a t e d with endotoxin (n=6) i n 24 hours. * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). - 37a -| | Control Endotoxin cvi \ o> c o E co c o o o o 0 - 38 -F i g u r e 6 Percentage of body weight l o s s i n c o n t r o l r a t s (n=6) and r a t s t r e a t e d w i t h endotoxin (n=6), on the f o u r t h day f o l l o w i n g i m p l a n t a t i o n of the mini-pumps. * value s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). - 38a -10 i -8 | | Control Endotoxin w 6 £ 4 0 - 39 -F i g u r e 7 Renal sodium output i n p a i r - f e d c o n t r o l r a t s (n=6) and r a t s t r e a t e d w i t h endotoxin (n=6) a t the f o u r t h day of i n f u s i o n with endotoxin. * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). - 39a -150 [ | Control Endotoxin O O \ OJ \ =3 cr LU 100 3 CL o E TD O C O 50 0 - 40 -r e n a l sodium output i n r a t s t r e a t e d w i t h endotoxin.(Fig. 7) Thus, the r a t i o of sodium output to sodium input i n r a t s t r e a t e d w i t h endotoxin showed a s i g n i f i c a n t decrease from a c o n t r o l value of 50.2 ± 7.2% to 12.0 ± 6.0%. (F i g . 8) A s i g n i f i c a n t decrease i n the values of the r a t i o of sodium-output to c r e a t i n i n e output was a l s o found i n r a t s t r e a t e d w i t h endotoxin (129.3 ± 28.9) compared w i t h c o n t r o l values (671.67 ± 56.8) (n = 6). In a d d i t i o n to a decreased r e n a l output of sodium, a s i g n i f i c a n t increase i n sodium space and t o t a l exchangeable sodium i n the body was found i n the r a t s t r e a t e d w i t h endo-t o x i n compared w i t h c o n t r o l values. Sodium space was 279.1 ±7.0 ml/Kg i n the c o n t r o l group and 313.0 ± 7.0 ml/Kg i n the endotoxin t r e a t e d r a t s (p<0.01). T o t a l sodium i n the body was found to be 38.52 ± 0.8 mEq/Kg i n the c o n t r o l r a t s and 43.98 ± 0.9 mEq/Kg i n the r a t s t r e a t e d w i t h endotoxin (p <0.01).(Fig. 9) Urine a n a l y s i s showed a l s o a s i g n i f i c a n t increase i n potassium output i n r a t s t r e a t e d w i t h endotoxin during the t h i r d , f o u r t h and f i f t h days a f t e r the im p l a n t a t i o n of the M.P.s. ( F i g . 10) The r e n a l potassium output at the f o u r t h day of impl a n t a t i o n of the M.P. was 286.5 ± 25 ^ iEq/24h/100g i n the - 41 -c o n t r o l group and 602.4 ± 78 uEq/24h/100g i n the endotoxin t r e a t e d r a t s . Urine volume was s i g n i f i c a n t l y increased i n r a t s t r e a t e d w i t h endotoxin ( F i g . 11a), and u r i n e c o n c e n t r a t i o n was s i g n i f i c a n t l y lower ( F i g . l i b ) . The lowest value f o r u r i n e c o n c e n t r a t i o n was found at the second day a f t e r the im p l a n t a t i o n of the M.P.s 596 ± 112 mOs/Kg, compared w i t h 1400 ± 104 mOs/Kg i n the c o n t r o l group. 2. Plasma a n a l y s i s Plasma a n a l y s i s showed t h a t there was no s i g n i f i c a n t d i f f e r e n c e s i n plasma sodium and potassium l e v e l s between c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. Sodium concen t r a t i o n i n plasma was 139.2 ± 0.8 mEq/L f o r c o n t r o l r a t s and 14 0.5 ± 0.9 mEq/L f o r r a t s t r e a t e d w i t h endotoxin. The conce n t r a t i o n of potassium i n the plasma was 3.90 ± 0.20 mEq/L i n the c o n t r o l group and 3.74 ± 0.25 mEq/L i n r a t s t r e a t e d w i t h e n d o t o x i n . ( F i g . 12). There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n plasma o s m o l a l i t y between c o n t r o l r a t s and r a t s t r e a t e d w i t h e n d o t o x i n . ( F i g . 13). There was a l s o no s i g n i f i c a n t d i f f e r e n c e i n plasma p r o t e i n c o n c e n t r a t i o n between the two groups.(Fig. 14). - 42 -Measurement of a l d o s t e r o n e l e v e l s i n the plasma showed no s i g n i f i c a n t d i f f e r e n c e between c o n t r o l r a t s (n=6), 2.5 ± 1.1 n g / d l and r a t s t r e a t e d w i t h endotoxin (n=6), 2.0 ± 0.6 n g / d l . Measurement of plasma volume showed a s i g n i f i c a n t i n c r e a s e i n r a t s t r e a t e d with endotoxin (46.8 ± 0.3 ml/Kg) compared w i t h c o n t r o l r a t s (35.9 ± 0.3 m l / K g ) . ( F i g 15) The i n f u s i o n of endotoxin a l s o caused a s i g n i f i c a n t decrease of 27.3% i n haematoerit v a l u e s , being 47.7 ± 0.85% i n the c o n t r o l group and 34.7 ± 1.6% i n r a t s t r e a t e d with e n d o t o x i n . ( F i g . 16) The r e l a t i o n s h i p between haematoerit and plasma volume i n r a t s t r e a t e d w i t h endotoxin i s shown i n F i g . 17. These va l u e s are compared wi t h a t h e o r e t i c a l curve which shows how haematoerit v a l u e s vary when plasma volume changes i n c o n t r o l r a t s . 3. Renal c l e a r a n c e data Renal f u n c t i o n t e s t s showed t h a t b l o o d urea n i t r o g e n (B.U.N.) 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 two groups. B.U.N, f o r c o n t r o l was 15.5 ± 1.1 mg/dl and 17.0' ± 1.0 f o r r a t s t r e a t e d w i t h endotoxin. A l s o , no s i g n i f i c a n t d i f f e r e n c e was found f o r c r e a t e . i n i n e - o u t p u t between the two groups, showing t h e r e f o r e a complete u r i n e c o l l e c t i o n i n the r a t s t r e a t e d w i t h endotoxin. - 43 -F i g u r e 8 Sodium output as a percentage of sodium i n t a k e i n p a i r -f e d c o n t r o l r a t s (n=6) and r a t s t r e a t e d with endotoxin (n=6), fo u r days a f t e r i m p l a n t a t i o n of the mini-pumps. * value s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). 6 0 5 0 4 0 3 0 2 0 10 -0 -| | Control Endotoxin - 44 -F i g u r e 9 Sodium space and exchangeable sodium i n c o n t r o l r a t s (n=6) and r a t s t r e a t e d with endotoxin (n=7). * v a l u e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). - 44a -i 4 0 0 : ™ 3 0 0 g 2 0 0 a. (0 E W 1 0 0 I I Control Endotoxin -i 5 0 0 L -4 0 ~ 3 0 2 0 cr o (0 o i o g LU - 45 -F i g u r e 10 Renal potassium output i n c o n t r o l r a t s (n=6) and endotoxin t r e a t e d r a t s (n=6), f o r a p e r i o d of s i x days. * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s (p<0.05). Mean potassium output i n unoperated c o n t r o l r a t s : 1162 ± 25 uEq/24h/100g - 45a -10001 | | Control Endotoxin 800 8 c <\i 600| > 3 CT LU =5. 3 CL. 400 E 3 W o 200| o 0. rr nr if 2 3 4 5 6 Days - 46 -F i g u r e 11a Urine volume i n c o n t r o l r a t s (n=6) and i n endotoxin-t r e a t e d r a t s (n=8), f o r a p e r i o d of s i x days. * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s , (p<0.05). Mean u r i n e volume i n unoperated c o n t r o l r a t s : 4.81 ± 0.14 ml/24h/100g F i g u r e l i b Urine c o n c e n t r a t i o n i n c o n t r o l r a t s (n=6) and i n endotoxiri-t r e a t e d r a t s (n=6), f o r a p e r i o d o f s i x days. * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s , (p<0.05). Mean u r i n e c o n c e n t r a t i o n i n unoperated c o n t r o l r a t s : 1636 ± 53 mOs/Kg - 46a -- 46b -I 1 1 1 1 1 0 1 2 3 4 5 Days - 47 -F i g u r e 12 Plasma e l e c t r o l y t e s i n c o n t r o l r a t s (n=6) and i n r a t s t r e a t e d with endotoxin (n=6). Plasma Sodium(mEquiv./L) 01 o o o T ai o m o 73 O a. 3 ° =r o o X _L — ro O J Plasma Potassium (mEquiv./L) - 48 -F i g u r e 13 Plasma o s m o l a l i t y i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). - 48a -| | Control Endotoxin 3 0 0 \ C O O E 2 0 0 o o E co O o E co o Q_ I 0 0 0 - 49 -F i g u r e 14 Plasma p r o t e i n c o n c e n t r a t i o n i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). - 49a -| | Control fg| Endotoxin 6 0 i -5 0 h 3 4 0 3 0 2 0 0 0 - 50 -F i g u r e 15 Plasma volume i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). - 50a -| | Control 50 r-EndotoxTn « 4 0 XL \ E 2 3 0 s o E (0 o 2 0 0 0 - 51 -F i g u r e 16 Haematoerit v a l u e s f o r c o n t r o l r a t s (n=6) and endotoxin t r e a t e d r a t s (n=6). * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , ( p ^O.05). - 51a -50 i - • Control Endotoxin R e l a t i o n s h i p between haematoerit and plasma volume i n r a t s t r e a t e d with endotoxin. The s o l i d l i n e shows how the mean value of haematoerit of c o n t r o l r a t v a r i e s with d i f f e r e n t degrees of expansion of plasma volume.. Mean c o n t r o l v a l u e s f o r blood volume and haematoerit are 69.0 ml/Kg and 48% r e s p e c t i v e l y . - 53 -No s i g n i f i c a n t d i f f e r e n c e between the two groups was found i n G.F.R. values estimated as i n u l i n clearance or i n R.P.F. estimated as P.A.H. clearance. G.F.R. was 0.914 ± 0.06 and 0.892 ± 0.05 ml/min/lOOg i n the c o n t r o l group and 0.966 ± 0.03 and 0.873 +0.06 ml/min/lOOg of the b.w. i n r a t s t r e a t e d w i t h endotoxin f o r unanaesthetized and anaesthetized r a t s r e s p e c t i v e l y . ( F i g . 18). The values f o r R.P.F. found i n unanaesthetized and anaesthetized r a t s were: 3.178 ± 0.3 ml/min/lOOg and 2.722 ± 0.3 ml/min/lOOg i n the c o n t r o l group and 3.800 ± 0.3 ml/min/lOOg and 2.748 ± 0.1 ml/min/lOOg i n r a t s t r e a t e d w i t h e n d o t o x i n . ( F i g . 18) No s i g n i f i c a n t d i f f e r e n c e s were found i n the values c a l c u l a t e d f o r R.B.F. and F.F. between c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin i n both unanaesthetized and anaesthetized r a t s . R.B.F. was 5.95 ±'0.8 ml/min/lOOg i n the c o n t r o l ' rats~ and 6.25 ± 0.5 ml/min/lOOg i n the endotoxin t r e a t e d group f o r unanaesthetized r a t s . In the case of anaesthetized r a t s , R.B.F. was 5.31 ± 0.5 ml/min/lOOg f o r c o n t r o l r a t s (n=6), and 4.97 ± 0.3 ml/min/lOOg i n r a t s t r e a t e d w i t h endotoxin (n=6). F i l t r a t i o n f r a c t i o n was found to be 0.291 ± 0.017 i n the unanaesthetized c o n t r o l r a t s (n=6), and 0.264 ± 0.020 - 54 -F i g u r e 18 Glomerular f i l t r a t i o n r a t e and r e n a l plasma flow i n unanaesthetized (UA) and a n a e s t h e t i z e d (A) c o n t r o l r a t s (n=6) and i n unanaesthetized :(UA) and a n a e s t h e t i z e d (A) endotoxin t r e a t e d r a t s (n=6) I I Control • O r o> 0 .8 h O g c 0.6 h E 1 0.4 |-or hi 0 . 2 h d 0.0 Endotoxin t 1X1 4 _ o> O 3 2 c 2 "e \ E 0_ -i 0 of UA UA - 55 -i n the unanaesthetized endotoxin t r e a t e d r a t s (n=6). In anaesthetized c o n t r o l r a t s (n=6), F.F. was 0.335 ± 0.020 and 0.366 ± 0.017 i n r a t s t r e a t e d w i t h endotoxin (n=6) . 4. Card i o v a s c u l a r measurements a) Cardiac output values using the Direct F i c k Method No s i g n i f i c a n t d i f f e r e n c e was found between anaesthetized c o n t r o l r a t s and r a t s t r e a t e d w i t h endo-t o x i n i n the values f o r CO., measured using the F i c k method. CO. was 27.35 ± 0.81 ml/min/lOOg of the b.w. i n the c o n t r o l group (n=4) and 29.36 ± 1.24 ml/min/lOOg of the b.w. i n the endotoxin t r e a t e d r a t s (n=6). A s i g n i f i c a n t decrease i n the content of oxygen i n a r t e r i a l blood was found i n the r a t s t r e a t e d w i t h endotoxin (13.85 ± 0.69 ml O2/100 ml blood,) compared w i t h the c o n t r o l group (18.76 ± 1.80. ml O2/100 ml bloo d ) . However no s i g n i f i c a n t d i f f e r e n c e e i t h e r i n the content of oxygen i n the mixed venous blood or i n the a r t e r i a l - v e n o u s d i f f e r e n c e i n blood oxygen content, was found between the two groups.(Table 1) - 56 -Table 1 Oxygen content i n a r t e r i a l ( C a 0 2 ) and mixed venous blood (CyC^) and the d i f f e r e n c e i n oxygen content between a r t e r i a l and mixed venous b l o o d , ( C a 0 2 - C V O 2 ) i n c o n t r o l r a t s (n=4) and i n r a t s t r e a t e d with endotoxin (n=6). * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p < 0.02). CONTROL C a 0 2 (ml/dl) 18.76 ± C v 0 2 (ml/dl) 13.67 ± C a 0 2 - C v 0 2 5.08 ± (ml/dl) ENDOTOXIN TREATED 1.80 * 13.85 ± 0.69 1.81 9.21 ± 0.72 0.09 4.50 ± 0.42 - 57 -There was no s i g n i f i c a n t d i f f e r e n c e i n the oxygen consumption v a l u e s i n r a t s t r e a t e d w i t h endotoxin compared with c o n t r o l r a t s . ( F i g . 19) b) C a r d i a c output v a l u e s u s i n g microspheres In c o n t r a s t t o the absence of a s i g n i f i c a n t d i f f e r e n c e i n the C O . value o b t a i n e d u s i n g the d i r e c t F i c k method, a s i g n i f i c a n t i n c r e a s e i n C O . was observed i n r a t s t r e a t e d w i t h endotoxin when C O . was measured with microspheres u s i n g the r e f e r e n c e sample method and a l s o when C O . was c a l c u l a t e d from the bloo d flow to the kidneys and the percentage of the C O . going to the k i d n e y s . ( F i g . 20) The C O . v a l u e s o b t a i n e d by the microsphere technique were 23.1 ± 0.8 ml/min/lOOg i n the c o n t r o l group (n=6), and 28.3 ± 0.5 ml/min/lOOg i n r a t s t r e a t e d w i t h endotoxin (n=6). Using the valu e s f o r R.B.F. and the percentage d i s t r i b u t i o n of C O . to the kidneys, C O . was found t o be 26.6 ± 0.5 ml/min/lOOg i n the c o n t r o l group (n=6) and 30.9 ± 0.8 ml/min/lOOg i n the r a t s i n f u s e d w i t h endotoxin (n=6). The mean a r t e r i a l blood p r e s s u r e was s i g n i f i c a n t l y decreased by 17.8% i n r a t s t r e a t e d w i t h endotoxin from a mean c o n t r o l value of 140.8 ± 1.5 mmHg.(Fig. 21) A s i g n i f i c a n t decrease i n h e a r t r a t e was found i n r a t s t r e a t e d with endotoxin (353 ± 13 beats/min) compared wit h c o n t r o l v a l u e s (397 ± 14 b e a t s / m i n ) . ( F i g . 22) - 58 -F i g u r e 19 Oxygen consumption i n c o n t r o l r a t s (n=4) and endotox t r e a t e d r a t s (n=6) - 58a -14 12 ~ 10 \ £ 8 E \ S 6 -4 -2 -0 -CVJ O > | | Control Endotoxin - 59 -F i g u r e 20 Car d i a c outputs measured u s i n g the d i r e c t F i c k method and the microsphere r e f e r e n c e sample, (M.S.) and c a r d i a c outputs c a l c u l a t e d from r e n a l b l o o d flow (R.B.F.), and the percentage of the c a r d i a c output ( C O . ) , t o the kidneys, i n c o n t r o l r a t s and endotoxin t r e a t e d r a t s . * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s a t p<0.05. - 59a -| | Control [5] Endotoxin - 60 -F i g u r e 21 Mean a r t e r i a l b l o o d p r e s s u r e (M.A.B.P.) i n c o n t r o l r a t s (n=6) and i n r a t s t r e a t e d with endotoxin (n=6). - 60a -• Control 160 :3 Endotoxin _ 120 cn I e E CO < 8 0 4 0 0 L . - 61 -F i g u r e 22 Heart r a t e measurements i n c o n t r o l r a t s (n=6) and i n endo-t o x i n t r e a t e d r a t s (n=6). - 61a -| [ Control Endotoxin 4 0 0 c E \ w o CD n Q) o o r o cu I 3 0 0 2 0 0 100 0 L-From CO. and H.R. values, stroke volume (S.V.) was c a l c u l a t e d . A s i g n i f i c a n t increase i n S.V. was found i n r a t s t r e a t e d w i t h endotoxin (n=6) (81.3 ± 4 pi/ lOOg of b.w.), compared w i t h c o n t r o l r a t s (n=6) (58.6 ± 3 ul/lOOg of b.w.). The t o t a l p e r i p h e r a l r e s i s t a n c e (T.P.R.) was s i g n i f i c a n t l y decreased by 34.1% i n r a t s t r e a t e d w i t h endotoxin from a mean value of 6.23 ± 0.33 mmHg/ml ^ min i n the c o n t r o l group.(Fig. 23) In these experiments, the haematocrit values f o r c o n t r o l r a t s (n=6) (43.3 ± 1.3%) and endotoxin t r e a t e d r a t s (n=6) (42.0 ± 0.1%) were not s t a t i s t i c a l l y s i g n i f i -c a n t l y d i f f e r e n t . c) D i s t r i b u t i o n of Cardiac Output F i g . 24 shows the d i s t r i b u t i o n of CO. to the kidneys i n anaesthetized c o n t r o l and i n endotoxin 57 t r e a t e d r a t s , measured w i t h Co - l a b e l l e d microspheres or w i t h 8 6 R b C l . The value obtained f o r the r e n a l d i s t r i b u t i o n of CO. using microspheres showed a s i g n i f i c a n t decrease i n r a t s t r e a t e d w i t h endotoxin (0.171 ± 0.008) compared wit h c o n t r o l values (0.200 ± 0.005). However, no s i g n i f i c a n t d i f f e r e n c e was found i n the r e n a l f r a c t i o n of CO. between the c o n t r o l group 8 6 and experimental r a t s when RbCl was used. The r e n a l f r a c t i o n of the CO. i n the c o n t r o l group was 0.172 ± 0.006, and 0.173 ± 0.007 i n the endotoxin t r e a t e d r a t s . - 63 -There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the c a l c u l a t e d b l o o d flow t o the kidneys and he a r t between c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. ( F i g . 25) The r e s u l t s are expressed i n ml/min/g of kidney or h e a r t r e s p e c t i v e l y . A l s o , no s i g n i f i c a n t d i f f e r e n c e s were found between the two groups i n the r e s i s t a n c e s of the kidneys and h e a r t t o b l o o d flow. The v a l u e s are shown i n Table 2. 5. H a l f l i f e of endotoxin i n plasma The h a l f l i f e o f endotoxin i n plasma was measured f o u r 51 days a f t e r the i m p l a n t a t i o n of the M.P.s, u s i n g C r - l a b e l l e d endotoxin. A l a r g e and h i g h l y s i g n i f i c a n t d i f f e r e n c e i n the r a t e of the disappearance of the endotoxin from plasma was obser-ved between r a t s t r e a t e d w i t h endotoxin and c o n t r o l r a t s . ( F i g . 26) The t ^ of endotoxin i n plasma being very much s h o r t e r i n r a t s t r e a t e d w i t h endotoxin (0.49 ± 0.07 min) compared wi t h c o n t r o l r a t s (5.42 ± 1.06 min).(Table 3) From the above measurements of t x i t was p o s s i b l e to estimate the plasma c o n c e n t r a t i o n of endotoxin i n the experimental group of r a t s and a l s o the volume of d i s t r i b u -t i o n o f i n f u s e d endotoxin. The c o n c e n t r a t i o n of endotoxin i n plasma was c a l c u l a t e d u s i n g the f o l l o w i n g formula: K where: C = e q u i l i b r i u m c o n c e n t r a t i o n of endotoxin i n plasma (ng/ml) A = i n p u t of endotoxin i n t o the r a t (ng/h/ml of volume of d i s t r i b u t i o n ) K = r a t e c o n s t a n t The i n i t i a l plasma c o n c e n t r a t i o n of endotoxin i n r a t s t r e a t e d w i t h endotoxin was estimated to be approximately 103 hg/ml d e c r e a s i n g to approximately 6.6 hg/ml a f t e r i n f u s i n g endotoxin f o r f o u r to f i v e days. The volume of d i s t r i b u t i o n of endotoxin was equal to the plasma, volume. The weights of v a r i o u s organs and the uptake of ~*^Cr-l a b e l l e d endotoxin by these organs was measured. The weight of the kidneys, lungs and h e a r t expressed as a percentage of b.w. were not found to be 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 groups. However, a s i g n i f i c a n t i n c r e a s e i n the weight of the spleen and l i v e r i n r a t s t r e a t e d w i t h endotoxin was o b s e r v e d . ( F i g . 27) There was no s i g n i f i c a n t d i f f e r e n c e i n the amount of ^ C r - l a b e l l e d endotoxin taken up by the kidneys, lungs, spleen and l i v e r between c o n t r o l r a t s and endotoxin t r e a t e d r a t s . ( T a b l e 4) 51 The uptake of C r - l a b e l l e d endotoxin by each organ was c a l c u l a t e d as a percentage of the t o t a l amount i n j e c t e d . - 65 -F i g u r e 23 T o t a l P e r i p h e r a l R e s i s t a n c e i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). - 65a -7 r-T | | Control Endotoxin - 66 -F i g u r e 24 Renal f r a c t i o n of C a r d i a c Output i n a n a e s t h e t i z e d c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=7) measured 8 6 u s i n g microspheres or RbCl. * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s a t p<£0.05. - 66a -| | Control - 67 -F i g u r e 25 Blood flow to the kidneys and h e a r t i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). I I Control i Endotoxin 1 Kidneys Heart - 68 -Table 2 Res i s t a n c e of the kidneys or h e a r t to blood flow (mmHg/ ml '''min g t i s s u e i n c o n t r o l r a t s (n=6) and i n endotoxin t r e a t e d r a t s (n=6). CONTROL ENDOTOXIN TREATED Kidneys Heart 21.53 ± 1.5 39.33 ± 3.0 18.25 ± 2.0 33.30 ± 6 . 0 - 69 -F i g u r e 26 Rate of disappearance of ^ C r - l a b e l l e d endotoxin from plasma i n c o n t r o l r a t s (n=6) and i n r a t s t r e a t e d with endotoxin (n=5) . The a c t i v i t i e s of a l l samples have been normalized t o 100% at zero time. - 69a -Time (min) - 70 -Table 3 H a l f l i f e ( t j , Time constant (T.C.) and Rate c o n s t a n t (K) i 51 of disappearance of Cr-endotoxin from plasma f o r c o n t r o l r a t s (n=7) and f o r endotoxin t r e a t e d r a t s (n=6). * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s from c o n t r o l v a l u e s , at (p<0.001). t x (min) T.C. (min) K CONTROL 5.42 ± 1.06' 7.8 0 ± 1.53 0.15 ± 0.03 ENDOTOXIN TREATED * 0.49 ± 0.07 * 0.70 ± 0.10 * 1.52 ± 0.16 - 71 -F i g u r e 27 Organ weights expressed as percentages of body weight, i n c o n t r o l r a t s (n=7) and i n endotoxin t r e a t e d r a t s (n=6). * v a l u e s s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , (p<0.05). Organ Weight (% Body Weight) O — N> CM ^ Oi 0> I 1 1 1 1 1 1 OL 3 <D c 3 CO m • m a . o o >< 5" o o CO •a CD a> 3 < CD CD O Hi - BIZ. -- 72 -Table 4 Uptake of ^ C r - l a b e l l e d endotoxin (percentage of the i n j e c t e d dose) i n v a r i o u s organs, i n c o n t r o l r a t s (n=6) and i n endo-t o x i n t r e a t e d r a t s (n=5). 51 C r - l a b e l l e d endotoxin uptake Kidneys Lungs Spleen L i v e r CONTROL 0.85 ± 0.19 4.37 ± 0.98 5.80 ± 0.8 62.18 ± 4.41 ENDOTOXIN TREATED 0.60 ± 0.10 4.86 ± 0.46 4.18 ± 0.83 65.74 ± 2.59 - 73 -6. H e x o b a r b i t a l S l e e p i n g Time There was no s i g n i f i c a n t d i f f e r e n c e i n the h e x o b a r b i t a l s l e e p i n g time between c o n t r o l r a t s (28 ± 2.5 min) and r a t s t r e a t e d with endotoxin (33.5 ± 3.7 m i n ) . ( F i g . 28) 7. Renal Medullary plasma flow Measurement of r e n a l medullary plasma flow showed t h a t there was no s i g n i f i c a n t d i f f e r e n c e between c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. Medullary plasma flow was found to be 24.4 ± 1.0 ml/min/ lOOg p a p i l l a f o r c o n t r o l r a t s and 26.0 ± 0.9 ml/min/lOOg p a p i l l a i n r a t s t r e a t e d with e n d o t o x i n . ( F i g . 29) In s p i t e of the i n j e c t i o n of v a s o p r e s s i n tannate, or i n f u s i o n of a r g i n i n e v a s o p r e s s i n there was a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n u r i n e c o n c e n t r a t i o n a t the time of the medullary plasma flow measurement between the two groups. The u r i n e c o n c e n t r a t i o n was 1757 ± 103 mOs/Kg i n the c o n t r o l group and 703 ± 75 mOs/Kg i n r a t s t r e a t e d with endotoxin. ( F i g . 30) No s i g n i f i c a n t d i f f e r e n c e i n haematocrit value was found between c o n t r o l r a t s (45.9 ± 0.9%) (n=7) and endotoxin t r e a t e d r a t s (44.5 ± 1.6%) (n=7), i n these experiments. - 74 -F i g u r e 28 H e x o b a r b i t a l s l e e p i n g time measured i n c o n t r o l r a t s (n=6) and i n r a t s t r e a t e d with endotoxin (n=6). - 74a -• Control I r"~^ Endotoxin 2 30 CD E | 2 0 <D (D CO O . Q O X <D 0 - 75 -F i g u r e 29 Medullary plasma flow f o r c o n t r o l r a t s (n=7) and endotoxin t r e a t e d r a t s (n=7). - 75a -6 £ GL O Q_ cn O O X c I I % i a e CO O Q_ 30 | | Control Endotoxin L L . O 20 10 0 « -Urine c o n c e n t r a t i o n a t the time of medullary plasma flow measurement, i n c o n t r o l r a t s (n=5) and i n endotoxin t r e a t e d r a t s (n=5). * v a l u e s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (p<0.05). - 76a -2000 r | | Control SI Endotoxin 1500 CT \ (0 O E | 1000 o t_ c CD O c o o CD .E 500 DISCUSSION Almost a l l s t u d i e s of experimental endotoxaemia have been performed u t i l i z i n g b olus i n j e c t i o n s of endotoxin, however i n most r e a l cases, endotoxin e n t e r s the c i r c u l a -t i o n i n s m a l l amounts over a prolonged p e r i o d of time. The p r e s e n t experiments were t h e r e f o r e designed to achieve low plasma c o n c e n t r a t i o n s of endotoxin which c o u l d be maintained f o r s e v e r a l days. During t h i s time r e n a l and c a r d i o v a s c u l a r f u n c t i o n s were s t u d i e d . A. Renal F u n c t i o n Endotoxin has been r e p o r t e d to a l t e r r e n a l f u n c t i o n i n humans and experimental animals.(32). Very l i t t l e work has been done i n r a t s . ( 2 0 ) . In the p r e s e n t experiments a s i g n i f i c a n t decrease i n r e n a l sodium output was observed i n r a t s t r e a t e d w i t h endotoxin. T h i s r e s u l t was not secondary to weight l o s s or decreased sodium i n t a k e , s i n c e a s i m i l a r e f f e c t was observed when endotoxin t r e a t e d r a t s were compared wi t h p a i r - f e d c o n t r o l r a t s . Nor was the decrease i n sodium output due to incomplete u r i n e c o l l e c t i o n as shown by the f a c t that:mo s i g n i f i c a n t d i f f e r e n c e was found i n c r e a t i n i n e output v a l u e s between c o n t r o l r a t s and r a t s t r e a t e d w i t h endotoxin. In a d d i t i o n , the lower r a t i o of sodium o u t p u t / t o t a l osmolar output found i n r a t s t r e a t e d with endotoxin showed t h a t sodium r e t e n t i o n was s e l e c t i v e . - 78 -The f a c t t h a t no s i g n i f i c a n t d i f f e r e n c e was observed i n G.F.R. between c o n t r o l r a t s and endotoxin t r e a t e d r a t s , i n d i c a t e s t h a t sodium r e t e n t i o n was not caused by a decrease i n G.F.R. but by an i n c r e a s e i n t u b u l a r r e a b s o r p t i o n . Because of the s i g n i f i c a n t i n c r e a s e i n K + output observed i n r a t s t r e a t e d w i t h endotoxin a t the time when sodium output was minimum, i t seemed p o s s i b l e t h a t a l d o s -terone might have pla y e d a r o l e i n the r e n a l sodium r e t e n -t i o n and t h e r e f o r e the i n c r e a s e i n sodium r e a b s o r p t i o n would have o c c u r r e d i n the c o l l e c t i n g d u c t s . Measurement of plasma l e v e l s of a l d o s t e r o n e however, showed no s i g n i f i c a n t d i f f e r e n c e s between c o n t r o l r a t s and r a t s t r e a t e d with endo-t o x i n . On the other hand, we can not d i s c a r d the p o s s i b i l i t y t h a t t h i s i n c r e a s e i n sodium r e a b s o r p t i o n occurs i n proximal segments i n the nephron. Even though F.F. was found normal i n r a t s t r e a t e d w i t h endotoxin, which would i n d i c a t e no d i f f e r e n c e s i n o n c o t i c p r e s s u r e i n the p e r i t u b u l a r c a p i l l a r i e s , the f a c t t h a t a low mean a r t e r i a l b l o o d p r e s s u r e was found i n these r a t s , r a i s e s the p o s s i b i l i t y t h a t the decreased M.A.B.P. c o u l d r e s u l t i n a decrease i n h y d r o s t a t i c p r e s s u r e i n the p e r i t u b u l a r c a p i l l a r i e s and t h e r e f o r e t h i s c o u l d i n c r e a s e the sodium r e a b s o r p t i o n i n the proximal t u b u l e . I t was not p o s s i b l e , however, from these experiments t o determine which segment ,(s) o f the nephron are i n v o l v e d i n the i n c r e a s e i n sodium r e a b s o r p t i o n and t h e r e f o r e more s t u d i e s u s i n g micropuncture techniques need t o be done. - 79 -The second impairment of r e n a l f u n c t i o n found i n r a t s t r e a t e d w i t h endotoxin was a reduced a b i l i t y of t h e i r kidneys t o conc e n t r a t e the u r i n e . T h i s impairment precedes sodium r e t e n t i o n which suggests t h a t i t i s caused by an u n r e l a t e d change i n r e n a l f u n c t i o n . T h i s d e f e c t i n u r i n e c o n c e n t r a t i o n c o u l d be caused by a decrease i n the plasma l e v e l of v a s o p r e s s i n (A.D.H.), or to a r e d u c t i o n i n the c o n c e n t r a t i o n g r a d i e n t i n the medulla. E l e v a t e d c o n c e n t r a t i o n s of A.D.H. have been r e p o r t e d i n dogs and baboons d u r i n g endotoxaemia(60), however th e r e are no data about l e v e l s of A.D.H. i n r a t s i n t h i s c o n d i t i o n . Although the c o n c e n t r a t i o n of A.D.H. i n plasma was not measured, the presen t experiments showed t h a t v a s o p r e s s i n tannate (200 mU/rat) or i n f u s i o n or argenine v a s o p r e s s i n (2 uU/min/lOOg), which i n c r e a s e s the c o n c e n t r a t i o n of u r i n e i n normal r a t s , d i d not improve the a b i l i t y of the kidneys of the r a t s t r e a t e d with endotoxin t o co n c e n t r a t e the u r i n e . T h i s suggests t h a t a low l e v e l of endogenous A.D.H. i s not the cause of the r e n a l impairment, but to be c e r t a i n measurement of t h i s hormone would be necessary. A l t e r n a t i v e l y , a reduced c o n c e n t r a t i o n g r a d i e n t c o u l d be due to a "wash out" of the medulla by i n c r e a s i n g the medullary plasma flow, or by i n h i b i t i o n of the a c t i v e NaCl t r a n s p o r t i n the t h i c k loop of Henle or by d e c r e a s i n g the d i s t a l d e l i v e r y of NaCl. - 80 -Measurement of the plasma medullary flow u s i n g the 125 I-albumin uptake technique i n r a t s t r e a t e d w i t h endo-t o x i n , showed t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between these r a t s and c o n t r o l s , suggesting t h a t a "wash out" of the medullary i n t e r s t i t i u m was not the cause of the l o s s of r e n a l c o n c e n t r a t i n g a b i l i t y . On the other hand, the f a c t t h a t a normal or a decreased sodium e x c r e t i o n was observed i n r a t s t r e a t e d with endotoxin when the r e n a l c o n c e n t r a t i n g d e f e c t was p r e s e n t i s opposed to the i d e a t h a t i n h i b i t i o n of the a c t i v e t r a n s p o r t of NaCl i n the t h i c k limb of the loop of Henle had o c c u r r e d , u n l e s s a decrease i n d i s t a l sodium d e l i v e r y i s a l r e a d y p r e s e n t due to i n c r e a s e i n sodium r e a b s o r p t i o n i n the proximal t u b u l e . These however, are o n l y c o n j e c t u r e s and more d e t a i l e d s t u d i e s need to be done. The mechanisms of the r e n a l d y s f u n c t i o n due to endo-t o x i n i s s t i l l not c l e a r , however some authors c o n s i d e r t h a t t h i s r e n a l impairment i s secondary to haemodynamic changes r a t h e r than to a nephrotoxic e f f e c t of the endotoxin.(18). In a d d i t i o n , i n the case of the h e p a t o r e n a l syndrome, because the syndrome develops r a p i d l y w i t h c i r c u l a t o r y hypotension and few p a t h o l o g i c a l s i g n s , t h i s suggests a c i r c u l a t o r y mechanism as a cause of the r e n a l f a i l u r e . T h i s i s confirmed by s t u d i e s i n which kidneys from c i r r h o t i c p a t i e n t s d y i n g from the h e p a t o r e n a l syndrome w i l l s t a r t t o f u n c t i o n immediately when they are t r a n s p l a n t e d . ( 1 0 ) . - 81 -A l s o , intravenous i n j e c t i o n of endotoxin i n t o baboons caused a pronounced decrease i n R.B.F. due to i n c r e a s e i n r e n a l v a s c u l a r r e s i s t a n c e and these changes were seen to develop b e f o r e any a l t e r a t i o n i n C O . or M.A.B.P. was observed, showing t h a t r e n a l impairment i n t h i s case may be r e l a t e d t o a primary a c t i o n on r e n a l c i r c u l a t i o n . ( 5 8 ) . I t has been shown i n most of the acute experiments t h a t the kidneys m a n i f e s t an i n i t i a l v a s o c o n s t r i c t i o n which i s f o l l o w e d by v a s o d i l a t i o n a f t e r endotoxin i n j e c t i o n . ( 1 1 ) . Perhaps t h i s a l s o o c c u r r e d i n the p r e s e n t experiments, but because of the longer p e r i o d of time used to a d m i n i s t e r : the endotoxin, any change o c c u r r i n g i n r e n a l f u n c t i o n a t the beginning of the a d m i n i s t r a t i o n p e r i o d were undetected. On the other hand, whether endotoxin has a v a s o d i l a t o r or v a s o c o n s t r i c t o r a c t i o n i s a l s o dose and s p e c i e s dependent. (57) . B. Blood and plasma volume s t u d i e s The f a c t t h a t the haematocrit v a l u e s i n r a t s t r e a t e d with endotoxin were s i g n i f i c a n t l y lower than c o n t r o l v a l u e s , i n d i c a t e s t h a t an i n c r e a s e i n plasma volume or a decrease i n the number of red b l o o d c e l l s or both had o c c u r r e d . Because the r e d u c t i o n of the haematocrit o c c u r r e d i n a very s h o r t time and no haemolysis was observed i n the plasma samples t e s t e d , i t seemed t h a t the low haematocrit v a l u e s observed i n the r a t s t r e a t e d w i t h endotoxin were caused by a s i g n i f i c a n t i n c r e a s e i n plasma volume r a t h e r than by - 82 -a l t e r a t i o n s i n the formation or d e s t r u c t i o n of the e r y t h r o c y t e s . However, the r e l a t i o n s h i p between the haematoerit and the plasma volume i n the r a t s t r e a t e d with endotoxin when compared wi t h the t h e o r e t i c a l v a r i a t i o n i n haematoerit with plasma volume, showed t h a t the low haema-t o e r i t i n these r a t s i s not o n l y due t o haemodilution f a c t o r s but to a decrease i n the number of the r e d b l o o d c e l l s . In the p r e s e n t experiments i t was not p o s s i b l e t o measure the t ^ of the e r y t h r o c y t e s because i t r e q u i r e s approximately 60 days while the e f f e c t s of the endotoxin i n our experiments o n l y l a s t e d f o r a few days. In more r e c e n t experiments, although sodium r e t e n t i o n was observed i n r a t s t r e a t e d with endotoxin, the haematoerit v a l u e s were found to be normal. In these experiments however, plasma volumes were not measured. T h i s d i f f e r e n c e i n haematoerit might have been due to the f a c t t h a t the endotoxin which was used was a d i f f e r e n t b a tch even though i t was the same serotype. S l i g h t but f u n c t i o n a l l y important d i f f e r e n c e s might have been pres e n t between the two batches. I t has a l s o been p o s t u l a t e d t h a t the i n c r e a s e i n plasma volume c o u l d be caused by a s h i f t of water from the i n t e r -s t i t i a l space as a consequence of the decrease i n b l o o d p r e s s u r e caused by endotoxin(47), however, t h i s seems improbable because i n our experiments sodium space was a l s o i n c r e a s e d . - 83 -C. Development of t o l e r a n c e i n c h r o n i c endotoxaemia In most s t u d i e s of acute experimental endotoxaemia or of the he p a t o - r e n a l syndrome, the degree of r e n a l impairment becomes worse wi t h time and the s u b j e c t s u s u a l l y d i e . In the pre s e n t experiments however, we observed improvement i n r e n a l f u n c t i o n a f t e r f i v e or s i x days w i t h almost 100% re c o v e r y . T h i s d i f f e r e n c e might be due to the f a c t t h a t endotoxin enhances the R.E.S. a c t i v i t y ( 4 8 ) . I t has been r e p o r t e d t h a t exposure of animals t o s m a l l doses of endotoxin w i l l i n c r e a s e the c a p a c i t y of the R.E.S. to i n a c t i v a t e endotoxin and t h e r e -f o r e these animals become t o l e r a n t t o endotoxin.(56). Thus, animals which r e c e i v e s u b l e t h a l doses of endotoxin every day can s u r v i v e s u p r a l e t h a l doses of e n d o t o x i n . ( 1 ) . I t has been r e p o r t e d t h a t t o l e r a n t animals removed c i r c u l a t i n g endotoxin more r a p i d l y than normal animals with a g r e a t e r l o c a l i z a t i o n of the L.P.S. i n the l i v e r . ( 1 5 ) ( 5 3 ) . An attempt t o v e r i f y t h i s was made by measuring the t ^ of endotoxin i n plasma a t the f o u r t h day a f t e r the implanta-t i o n of the M.P.s.. A l a r g e and h i g h l y s i g n i f i c a n t r e d u c t i o n i n the t ^ of endotoxin i n plasma was observed i n r a t s t r e a t e d w i t h endo-t o x i n when compared with c o n t r o l v a l u e s , demonstrating t h a t t h e r e was a s i g n i f i c a n t i n c r e a s e i n the r a t e o f c l e a r a n c e of endotoxin. The d i f f e r e n c e i n plasma c o n c e n t r a t i o n of endotoxin found between the begi n n i n g of the i n f u s i o n of the L.P.S. and a f t e r f o u r days of the i n f u s i o n c o u l d be t h e r e f o r e one of the - 84 -reasons f o r the recovery of the r a t s . No s i g n i f i c a n t d i f f e r e n c e was found i n the f r a c t i o n a l 51 uptake of Cr conjugated with endotoxin by the l i v e r s of c o n t r o l r a t s or r a t s t r e a t e d with endotoxin. However, there was presumably an i n c r e a s e i n the r a t e of uptake of the endo-t o x i n by the l i v e r i n the experimental r a t s . I t has been r e p o r t e d t h a t t o l e r a n c e i s t r a n s f e r a b l e , and t h a t i n man t o l e r a n c e can occur i n the absence of any i n c r e a s e i n the R.E.S. a c t i v i t y . ( 5 3 ) . T h i s suggests t h a t some humoral f a c t o r c o u l d p l a y a r o l e i n the r e s i s t a n c e of these animals to endotoxin. In a d d i t i o n , i t has been r e p o r t e d t h a t a f t e r 72 hours of i n j e c t i o n of endotoxin, a n t i b o d i e s a g a i n s t endotoxin have been found i n plasma of the r a t s . ( 1 5 ) . We d i d not look f o r a n t i b o d i e s a g a i n s t endotoxin i n our experiments, and t h i s i s one of the s t u d i e s t h a t should be done to i n v e s t i g a t e the mechanism r e s p o n s i b l e f o r the i n c r e a s e d c l e a r a n c e of endotoxin from plasma. The p o s s i b i l i t y t h a t the i n c r e a s e d r a t e o f c l e a r a n c e of endotoxin was caused by g e n e r a l i z e d enzymatic i n d u c t i o n i n the l i v e r was a l s o i n v e s t i g a t e d . To study t h i s , a b a r b i t u r a t e drug, hexobarbitone sodium, which i s metabolized o n l y by the l i v e r was t e s t e d . ( 4 ) . As i s known, the metabolism of t h i s drug i n v i v o can be estimated by measuring the s l e e p i n g time. Thus, any i n c r e a s e i n r a t e of metabolism of the drug t h a t occurs i f g e n e r a l i z e d enzymatic i n d u c t i o n i s present, w i l l decrease the tx of the drug i n plasma and a s h o r t e r than - 85 -normal s l e e p i n g time w i l l be observed. In the presen t experiments there was no s i g n i f i c a n t d i f f e r e n c e i n the value s f o r s l e e p i n g time i n endotoxin-t r e a t e d r a t s compared wi t h c o n t r o l v a l u e s , which i n d i c a t e s t h a t g e n e r a l i z e d enzymatic i n d u c t i o n was not produced by endotoxin i n these r a t s . On the other hand, there have been r e p o r t s t h a t the a d m i n i s t r a t i o n of endotoxin t o mice prolongs the d u r a t i o n of b a r b i t u r a t e - i n d u c e d s l e e p w i t h i n a few hours by s t i m u l a t i o n of the degra d a t i o n of cytochrome P-4 50.(2). The d i f f e r e n c e between our r e s u l t s and those p u b l i s h e d c o u l d be due t o s p e c i e s d i f f e r e n c e and a l s o due to the d i f f e r e n t dose and k i n d of endotoxin used. D. C a r d i o v a s c u l a r f u n c t i o n The c a r d i o v a s c u l a r e f f e c t s o f endotoxin vary from one sp e c i e s to another,(17) however one of the common c h a r a c t e r -i s t i c s observed i n almost a l l the s p e c i e s s t u d i e d i s a decrease i n M.A.B.P. a f t e r the i n j e c t i o n of the L.P.S. The time a t which t h i s o c c u r s , the degree of response and a l s o the mechanisms by which t h i s o c c u r s , seem t o vary a l s o from s p e c i e s t o s p e c i e s . ( 1 6 ) . I t has a l s o been demonstrated i n dogs t h a t the i n i t i a l hypotension observed a f t e r the bolus i n j e c t i o n of a l e t h a l dose of endotoxin i s due to h e p a t i c venous c o n s t r i c t i o n which w i l l p o o l a l a r g e amount of bl o o d i n the s p l a n c h n i c bed producing a decrease i n venous r e t u r n and t h e r e f o r e a decrease - 86 -i n C O . wit h consequent decrease i n bloo d p r e s s u r e . (1) (16) . When the l i v e r was removed, no immediate decrease i n bloo d p r e s s u r e or venous r e t u r n was observed.(16). An i n c r e a s e i n the weight of the l i v e r and an i n c r e a s e i n v a s c u l a r r e s i s t a n c e has a l s o been observed i n pe r f u s e d l i v e r s t r e a t e d w i t h endotoxin which would i n d i c a t e h e p a t i c venous c o n s t r i c t i o n and p o o l i n g of the b l o o d . ( 3 6 ) . I t was proposed t h e r e f o r e t h a t the f i r s t organ i n which endotoxin acted was the l i v e r and t h a t the v a s c u l a r changes produced by the L.P.S. i n t h i s organ were r e s p o n s i b l e f o r the observed systemic hypotension. On the other hand, i n the case of the primate, no h e p a t i c p o o l i n g was observed and the hypotension observed i n t h i s s p e c i e s was caused by p e r i p h e r a l v a s o d i l a t i o n which produced p e r i p h e r a l p o o l i n g of the b l o o d . ( 5 3 ) . Acute i n j e c t i o n of endotoxin i n r a t s has been r e p o r t e d to cause a f a l l i n bloo d p r e s s u r e which o c c u r r e d e a r l y i n the second hour a f t e r the i n j e c t i o n . ( 1 6 ) . A s m a l l drop i n pre s s u r e a l s o o c c u r r e d immediately a f t e r the i n j e c t i o n . An e a r l i e r study from t h i s l a b o r a t o r y showed however t h a t i n unan a e s t h e t i z e d r a t s the acute a d m i n i s t r a t i o n of endotoxin f a i l e d t o decrease M.A.B.P. from c o n t r o l v a l u e s . The l a c k of a hypotensive e f f e c t has a l s o been r e p o r t e d by Ross(46), who has shown t h a t a l e t h a l dose of endotoxin i n u n a naesthetized r a t s produced o n l y a moderate decrease i n blo o d p r e s s u r e , f o l l o w e d by a s u s t a i n e d and grad u a l r i s e i n pr e s s u r e . - 87 -In the p r e s e n t study the c h r o n i c a d m i n i s t r a t i o n of endotoxin r e s u l t e d i n a s i g n i f i c a n t decrease i n M.A.B.P. measured when r e n a l sodium output was minimum. There was a l s o a s i g n i f i c a n t i n c r e a s e i n the weight of the l i v e r and spleen which might i n d i c a t e p o o l i n g of b l o o d i n these areas, however measurement of C O . w i t h the micro-sphere r e f e r e n c e sample method, showed t h a t r a t s t r e a t e d w i t h endotoxin had h i g h e r C O . s than the c o n t r o l r a t s , t h e r e f o r e i t seems t h a t the decrease i n b l o o d p r e s s u r e was not a r e s u l t of a decrease i n venous r e t u r n due to p o o l i n g of b l o o d i n the s p l a n c h n i c area as occurs i n the dog, but due to the s i g n i f i c a n t decrease i n t o t a l p e r i p h e r a l r e s i s -tance, with a consequent r i s e i n C O . as an attempt to maintain B.P. I t has a l s o been p o s t u l a t e d t h a t hypotension f o l l o w i n g the a d m i n i s t r a t i o n of endotoxin c o u l d be a r e s u l t of a r e l e a s e of some v a s o a c t i v e substance. Thus, the k a l l i k r e i n -b r a d y k i n i n system may be an important mediator of the de-creased B.P. d u r i n g endotoxaemia.(58). P r o s t a g l a n d i n s , on the other hand, which have been shown to be i n c r e a s e d i n a v a r i e t y of s p e c i e s d u r i n g endotoxaemia c o u l d a l s o p l a y a r o l e i n hypotension.(5). Thus, p r o s t a -c y c l i n which a c t s as a v a s o d i l a t o r , has been i m p l i c a t e d as one of the f a c t o r s r e s p o n s i b l e f o r the decrease i n the a r t e r i a l b l o o d p r e s s u r e d u r i n g endotoxic shock.(13). C a r d i a c output was found to be s i g n i f i c a n t l y g r e a t e r i n r a t s t r e a t e d w i t h endotoxin compared with c o n t r o l v a l u e s - 88 -when C O . was measured by the microsphere r e f e r e n c e sample method or when c a l c u l a t e d from R.B.F. v a l u e s o b t a i n e d from c l e a r a n c e measurements and from the r e n a l f r a c t i o n of the C O . as measured wi t h r a d i o a c t i v e microspheres. When the d i r e c t F i c k method was used, C O . was found t o be i n c r e a s e d but t h i s i n c r e a s e was not s t a t i s t i c a l l y s i g n i f i -c ant. S e p s i s i n man has a l s o been c h a r a c t e r i z e d by a hyper-dynamic s t a t e with a decrease i n t o t a l p e r i p h e r a l r e s i s t a n c e and an i n c r e a s e i n C O . ( 3 0 ) . However, the g e n e r a l observa-t i o n i n most s p e c i e s has been a decrease i n C O . f o l l o w i n g acute a d m i n i s t r a t i o n of endotoxin.(16). E a r l i e r measurement of C O . i n t h i s l a b o r a t o r y u s i n g the microspheres r e f e r e n c e sample method showed t h a t c h r o n i c i n f u s i o n of endotoxin i n r a t s decrease C O . by 14% from c o n t r o l v a l u e s . (28). T h i s d i s c r e p a n c y i n the e f f e c t s of endotoxin was not on l y seen i n C O . v a l u e s but a l s o i n other parameters such as haematocrit, u r i n a r y sodium and potassium e x c r e t i o n and r e n a l c o n c e n t r a t i n g a b i l i t y . The d i f f e r e n c e s are perhaps due to the f a c t t h a t the b i o l o g i c a l e f f e c t s of endotoxin are v a r i a b l e depending on the dose, p e r i o d of a d m i n i s t r a t i o n , s p e c i e s and a l s o the e f f e c t w i l l depend on the batch of endotoxin used f o r the s t u d i e s . In our case the d i f f e r e n c e appeared t o be a r e s u l t of the f a c t t h a t the endotoxin came from d i f f e r e n t batches, one of the batches being much more potent i n i t s b i o l o g i c a l e f f e c t than the o t h e r . - 89 -I t has been r e p o r t e d t h a t the microsphere technique and the F i c k method g i v e s i m i l a r v a l u e s f o r C O . i n r a t s , (52) (39), however i n our experiments the c o n t r o l v a l u e s o b t a i n e d f o r C O . d i f f e r e d s i g n i f i c a n t l y u s i n g these two methods. The r e s u l t s show t h a t both methods g i v e reproducable data t h e r e f o r e the d i f f e r e n c e observed might be due to d i f f e r e n c e s i n technique or to a x i a l streaming of the micro-spheres . In the case of the microsphere technique, the p r e c i s i o n of t h i s method depends mainly on adequate mixing and on the numbers of microspheres found i n the organs and i n the r e f e r e n c e sample. Thus, i t has been found i n dogs, sheep and r a t s t h a t 400 microspheres r e p r e s e n t s a minimum number t h a t must be trapped i n each sample to g i v e a c c u r a t e measure-ments of C O . or estimated v a l u e s f o r b l o o d flow, a v o i d i n g t h e r e f o r e e r r o r s due to non-random d i s t r i b u t i o n . ( 5 2 ) . I t has a l s o been r e p o r t e d t h a t t o t a l cummulative i n j e c t i o n s of more than 100,000 microspheres, have a l t e r e d c a r d i o v a s c u l a r haemodynamics i n r a t s . (51). These e x p e r i -ments however were c a r r i e d out w i t h microspheres suspended i n 10% dextran which, by i t s e l f , has a severe hypotensive and a n a p h y l a c t i c - l i k e e f f e c t i n r a t s . More r e c e n t r e p o r t s have shown t h a t i n j e c t i o n s of up to 850,000 microspheres i n i s o t o n i c s a l i n e s o l u t i o n d i d not decrease b l o o d p r e s s u r e or h e a r t r a t e i n r a t s . ( 1 2 ) . - 90 -In the present experiments, approximately 60,000 microspheres were i n j e c t e d i n 2% dextran. This amount of dextran has no s i g n i f i c a n t c a r d i o v a s c u l a r e f f e c t s and even la r g e amounts of dextran (10%) do not i n f l u e n c e the d i s t r i b u -t i o n of microspheres during the time they are' c l e a r e d from the c i r c u l a t i o n (a few seconds) when a s i n g l e i n j e c t i o n i s used. One of the major l i m i t a t i o n s i n the a p p l i c a t i o n of t h i s method i s ensuring homogenous mixing and d i s t r i b u t i o n of the microspheres w i t h blood. Thus, to o b t a i n a v a l i d r e l a t i o n s h i p between the a c t i v i t y of l a b e l l e d microspheres i n one organ and the blood flow through t h a t organ, the d i s t r i b u t i o n of the microspheres i n blood should be homogenous, i n such a way t h a t at any b i f u r c a t i o n p o i n t of an a r t e r y the blood which enters i n any of the daughter branches should have the same concen t r a t i o n of microspheres as i n the main v e s s e l . I t has been shown however th a t changing the diameter of the spheres used to measure blood flow (from 9u to 64u), showed a considerable v a r i a t i o n i n the measurement depending upon the s i z e of the microspheres.(44). This e f f e c t has been explained by an uneven r a d i a l d i s t r i b u t i o n of the microspheres i n the blood v e s s e l s the c o n c e n t r a t i o n being increased c e n t r i p e t a l l y w i t h a progressive decrease i n the number of microspheres i n the periphery. This r a d i a l d i s t r i b u t i o n seems to be propor-t i o n a l t o both, absolute sphere diameter and s i z e r e l a t i v e to a r t e r y diameter. - 91 -I t was found a l s o t h a t microspheres of 7.5 to 10 )i diameter are the ones which more c l o s e l y approach red blood c e l l d i s t r i b u t i o n i n blood v e s s e l s . ( 4 4 ) . On the other hand, i t has been shown th a t red blood c e l l d i s t r i b u t i o n i n c a p i l l a r y blood v e s s e l s depends on the v e l o c i t y of flow, t h e r e f o r e i f at a b i f u r c a t i o n p o i n t of a blood v e s s e l , the daughter branches have d i f f e r e n t blood flow, the one i n which the v e l o c i t y of blood flow i s f a s t e r w i l l get a bigger number of e r y t h r o c y t e s . This i s because the f o r c e s t h a t determine i n which branch the red blood c e l l s must go, are the r e s u l t a n t of pressure and shear s t r e s s i n the f l u i d , and both forces tend to p u l l the c e l l s i n t o the f a s t e r channel. (61) . Microspheres, due to t h e i r s i m i l a r i t y to red blood c e l l s a l s o must be subjected to the same kinds of mechanical f o r c e s , t h e r e f o r e i f the v e l o c i t y i n the d i f f e r e n t branches i s not the same, t h i s w i l l r e s u l t i n d i f f e r e n t concentrations of microspheres i n the d i f f e r e n t branches, y i e l d i n g erroneous values i n the determination of CO. and organ blood flow values. In our experiments the f a c t t h a t equal numbers of microspheres were observed i n both kidneys, would i n d i c a t e t h a t mixing of microspheres w i t h blood i n the l e f t v e n t r i c l e was adequate, but we can not be sure that no change i n the d i s t r i b u t i o n of microspheres w i t h i n the blood v e s s e l s occurred. When the d i r e c t F i c k method was used to measure CO., the values found f o r r a t s t r e a t e d w i t h endotoxin were s i m i l a r - 92 -to those found u s i n g microsphere technique, however no s i g n i f i c a n t d i f f e r e n c e was found between c o n t r o l v a l u e s and r a t s t r e a t e d with endotoxin, because c o n t r o l v a l u e s o b t a i n e d with the F i c k method were somewhat h i g h e r than those o b t a i n e d u s i n g the microsphere technique. I t has been suggested t h a t the h i g h e r v a l u e s of C O . measured by the d i r e c t F i c k method ( i f we compare t h i s method with the e l e c t r o m a g n e t i c probe method) (54) might be e x p l a i n e d i f s u f f i c i e n t b r o n c h i a l b l o o d f l o w e n t e r e d the l e f t s i d e o f the h e a r t from the b r o n c h i a l v e i n s to lower the Pa0 2 content and g i v e e r r o n e o u s l y s m a l l a 0 2 - v 0 2 d i f f e r e n c e s . ( 5 4 ) The v a l u e s f o r the a r t e r i a l oxygen content i n our c o n t r o l r a t s however were s i m i l a r to those found elsewhere (54), t h e r e f o r e i t seems t h a t t h i s f a c t o r does not account f o r the d i f f e r e n c e observed between the two methods. The a r t e r i a l oxygen content was s i g n i f i c a n t l y lower i n r a t s t r e a t e d with endotoxin, which agrees with p r e v i o u s r e p o r t s which have shown t h a t endotoxin produces a decrease i n Pa0 2, P v 0 2 and an i n c r e a s e i n PvC0 2 i n r a t s . ( 2 1 ) . In our r a t s t r e a t e d w i t h endotoxin the mean Pv0 2 was a l s o lower than c o n t r o l v a l u e s , but t h i s d i f f e r e n c e was not s i g n i f i c a n t . There was no s i g n i f i c a n t d i f f e r e n c e i n the. v a l u e of a 0 2 - v 0 2 between the two groups. T h i s i n d i c a t e s t h a t the amount of oxygen taken up by the t i s s u e s i n the r a t s t r e a t e d w i t h endotoxin was the same as the c o n t r o l r a t s . Mean oxygen consumption was s m a l l e r i n r a t s t r e a t e d - 93 -with endotoxin but the d i f f e r e n c e was not s i g n i f i c a n t . The b l o o d samples taken to measure p a r t i a l p r e s s u r e of oxygen (PG^), were kept i n i c e water u n t i l they were ready to be measured, t h i s procedure would decrease the r a t e of metabolism and oxygen u t i l i z a t i o n from l e u c o c y t e s , t h e r e -f o r e , as has been p o s t u l a t e d , the decrease i n a r t e r i a l oxygen content i n r a t s might be due to a - v shunts i n lungs produced by the a c t i o n of endotoxin(21) s i n c e i t was u n l i k e l y t h a t l o s s of oxygen o c c u r r e d d u r i n g h a n d l i n g of the samples. In agreement w i t h the f i n d i n g s of other workers(31), the p r e s e n t experiments a l s o showed t h a t endotoxin produced a decrease i n h e a r t r a t e . The mechanism(s) of t h i s b r a d y c a r d i a i s not known, but i t has been r e p o r t e d t h a t the a d d i t i o n of endotoxin to an i s o l a t e d h e a r t p r e p a r a t i o n does not a l t e r h e a r t r a t e . ( 3 1 ) . In i n t a c t animals, endotoxin has no e f f e c t on h e a r t r a t e a f t e r c u t t i n g both v a g i and blockade of the sympathetic system(31). I t seems t h e r e f o r e , t h a t the e f f e c t of endo-t o x i n on h e a r t r a t e i s mediated through t h e . n e u r a l pathway r a t h e r than by a d i r e c t a c t i o n on the h e a r t . A d d i t i o n a l r e p o r t s have a l s o shown t h a t endotoxin causes b r a d y c a r d i a by d e c r e a s i n g sympathetic d i s c h a r g e and i n c r e a s -i n g v a g a l d i s c h a r g e . (7). I t has been p o s t u l a t e d a l s o t h a t the r e l e a s e of vaso-a c t i v e substances such as histamine, s e r o t o n i n e or brady-k i n i n might be a d d i t i o n a l f a c t o r s producing b r a d y c a r d i a through the B e z o l d - J a r i s c h r e f l e x . ( 2 1 ) . - 94 -Renal f r a c t i o n of c a r d i a c output Because of p o s s i b l e causes of uneven d i s t r i b u t i o n of \ the microspheres d i s c u s s e d b e f o r e , the r e n a l f r a c t i o n of 57 the C O . was measured u s i n g both C o - l a b e l l e d microspheres ftfi and RbCl. Even though the d i s t r i b u t i o n of C O . t o the kidneys u s i n g these two methods has been r e p o r t e d to be i d e n t i c a l i n normal r a t s (40). (14) , the pr e s e n t experiments showed a s i g n i f i c a n t d i f f e r e n c e between c o n t r o l v a l u e s . A l s o , a s i g n i f i c a n t r e d u c t i o n i n the f r a c t i o n of the : C O . going to the kidneys was found i n r a t s t r e a t e d w i t h 57 endotoxin when C o - l a b e l l e d microspheres was used, while no s i g n i f i c a n t d i f f e r e n c e was found when r e n a l f r a c t i o n of C O . was measured w i t h 8 ^ R b C l . The d i f f e r e n t r e s u l t s o b t a i n e d by the two methods might be due to the f a c t t h a t the two methods have important d i f f -erences, i e . Rb + measures n u t r i t i o n a l b l o o d flow, t h i s means the b l o o d flow through v e s s e l s where there i s exchange of the i s o t o p e with the t i s s u e , w h i l e l a b e l l e d microspheres measure i n t r a v a s c u l a r or anatomical b l o o d flow. Our r e s u l t s suggest t h a t endotoxin does not i n t e r f e r e w i t h the exchange of Rb + w i t h the t i s s u e . I t w i l l be noted t h a t measurement of C O . with micro-spheres showed t h a t C O . was s i g n i f i c a n t l y i n c r e a s e d i n r a t s t r e a t e d w i t h endotoxin while measurement of C O . u s i n g the d i r e c t F i c k method d i d not show a s t a t i s t i c a l l y s i g n i f i -c ant d i f f e r e n c e because of the hig h v a l u e s f o r C O . in-the c o n t r o l r a t s . - 95 -The reason f o r t h i s d i f f e r e n c e i s not known. An a l t e r n a t i v e method of e s t i m a t i n g C O . i s from the simultaneous measurement of r e n a l b l o o d flow u s i n g standard c l e a r a n c e techniques and the f r a c t i o n a l d i s t r i b u t i o n of microspheres t o the kidneys. C a l c u l a t i o n of C O . by t h i s method gave v a l u e s i n agreement wi t h the microsphere determina-t i o n of C O . i e . t h a t C O . was s i g n i f i c a n t l y i n c r e a s e d a f t e r the i n f u s i o n of endotoxin. 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