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Cerebral energy metabolism in mallard ducks during apneic asphyxia the role of oxygen conservation Bryan, Robert Maurice 1978

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CEREBRAL ENERGY METABOLISM IN MALLARD DUCKS DURING APNEIC ASPHYXIA THE ROLE OF OXYGEN CONSERVATION by Robert Maurice Bryan, J r . B. S c , U n i v e r s i t y of Alabama, 1970 M.Sc, U n i v e r s i t y of Alabama, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE.DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Zoology 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 November, I978 (c) Robert Maurice Bryan, J r . , 197,8 I n 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 l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th 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 reference and study. I f u r t h e r agree that permission f o r extensive 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 of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha 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 gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada i ABSTRACT Cerebr a l energy metabolism during apneic asphyxia and steady s t a t e hypoxia was compared i n ducks and chickens; ducks t o l e r a t e apneic asphyxia 3-8 times longer than chickens. F l u c t u a t i o n s i n the reduced form of r e s p i r a t o r y chain n i c o t i n a -mide adenine d i n u c l e o t i d e (NADH) were monitored from the l e f t c e r e b r a l hemisphere by a noninvasive f l u o r o m e t r i c technique and used as an i n d i c a t o r of m i t o c h o n d r i a l hypoxia. E l e c t r o -encephalogram (EEG) and surface POg were recorded from the r i g h t hemisphere. Forced dives of ^ -7 minutes d u r a t i o n on r e s t r a i n e d ducks were c h a r a c t e r i z e d by bradycardia and an accumulation of NADH which increased throughout the d i v i n g p e r i o d . NADH returned to the preasphyxic l e v e l when br e a t h i n g was resumed. I n l a t e r experiments asphyxia was produced by stopping a r t i f i c i a l v e n t i l a t i o n i n paralyzed ducks. Asphyxia produced by t h i s means caused s i m i l a r changes i n the measured v a r i a b l e s (heart r a t e , blood pressure, NADH fluorescence, and EEG) to those obtained i n f o r c e d submergence of nonparalyzed ducks. NADH fluorescence was expressed i n a r b i t r a r y u n i t s (AU) where 100 AU was defined as the fluorescence change from normoxia to anoxia. A f t e r 1 minute of asphyxia NADH fluorescence i n -creased by 37 AU - 3.60 SEM (n = 5^) in*-paralyzed chickens and 8 AU - 1.^1 SEM (n = 55) i n paralyzed ducks. A f t e r 2 minutes the fluorescence increased by only 15 AU - 1.95 SEM i n ducks. Both species showed an i s o e l e c t r i c EEG when fluorescence increased "by approximately 35 AU i n d i c a t i n g that anaerobic ATP production i i i n ducks d i d not maintain "brain f u n c t i o n (EEG) f o r a greater accumulation of r e s p i r a t o r y chain NADH. At a given decrease i n t i s s u e P 0 2 ducks and chickens showed the same l e v e l of NADH increase i n d i c a t i n g t h a t both species are e q u a l l y dependent on t i s s u e P 0 2 f o r the maintenance of redox s t a t e . Furthermore, the i n h i b i t i o n of c a r d i o v a s c u l a r adjustments by atr o p i n e i n ducks caused NADH to increase f a s t e r during apneic asphyxia than i n nonatropinized ducks. I conclude that the oxygen conserving c a r d i o v a s c u l a r adjustments are re s p o n s i b l e f o r the increased c e r e b r a l tolerance to apneic asphyxia i n ducks without any involvement of biochemical mechanisms. i i i TABLE OF CONTENTS General I n t r o d u c t i o n 1 Chapter 1. General Methods - Animal P r e p a r a t i o n 17 a. Rats 17 D. Ducks 17 c. Chickens 20 Chapter 2. S p e c i a l Techniques 21 The Fluorometric r e c o r d i n g of NADH 21 a. Descriptionr.of the fluorometer 22 b. S t a b i l i t y of the fluorometer " 26 c. Fluorescence emission s p e c t r a 29 d. S t a b i l i t y of the b i o l o g i c a l p r e p a r a t i o n 35 e. Blood a r t i f a c t compensation 36 f. NADH fluorescence during n i t r o g e n v e n t i l a -t i o n and apneic asphyxia 1^ Polarographic measurement of oxygen t e n s i o n of the c o r t i c a l surface ^ 8 a. D e s c r i p t i o n of the oxygen electrode 51 b. Oxygen measurements from the c o r t i c a l surface of ducks during hypoxia and hypercapnia 5^ Chapter 3. Changes i n the Redox State of R e s p i r a t o r y Chain NADH During Apneic Asphyxia i n Ducks 60 I n t r o d u c t i o n 60 Methods 62 a. Fluorescence recordings from nonparalyzed ducks 62 i v Chapter 3. Methods (cont'd) b. Fluorescence recordings from paralyzed ducks 62 c. I n h i b i t i o n of the cardiovascular adjust-ments during apneic asphyxia 63 Results 65 a. Comparison of nonparalyzed and paralyzed ducks 65 b. I n h i b i t i o n of the cardiovascular adjust-ments during apneic asphyxia i n ducks 74 Discussion 79 Chapter k. Cerebral Energy Metabolism i n Ducks and -. Chickens During Apneic Asphyxia and Hypoxia 82 Introduction 82 Methods Qk a. Fluorescence recordings from paralyzed ducks and chickens 8^ b. Concurrent fluorometric and polarographic recordings during hypoxia ^ 8^4-Results ' 86 a. Comparison of ducks and chickens 86 b. C r i t i c a l pyridine nucleotide reduction (CPNR) i n chickens and ducks 90 c. Relationship between fluorescence and PrpO^ i n ducks and chickens during hypoxia 97 Discussion 107 General Discussion Bibliography v i LIST OF FIGURES Figure 1. The r e s p i r a t o r y chain. 7 Figure 2. Diagram of the o p t i c a l design of the fluorometer. 23 Figure 3. S i m p l i f i e d diagram of the fluorometer c i r c u i t r y . - 27 Figure k. Fluorescence emission s p e c t r a from the c e r e b r a l cortex of a duck during normo-capnia and anoxia. 30 Figure 5• Fluorescence emission spectra,from the ..cerebral cortex of a duck duri n g anoxia and from a s o l u t i o n of NADH.' " 33 Figure 6 . O p t i c a l measurements recorded from the c e r e b r a l cortex of a duck when the amount of hemoglobin i n the r e c o r d i n g f i e l d was a l t e r e d . 37 Figure 7« The r e l a t i o n s h i p between r e f l e c t e d e x c i t a t i o n l i g h t and emitted fluorescence l i g h t recorded from the c e r e b r a l cortex of a duck when the hemoglobin i n the r e c o r d i n g f i e l d was a l t e r e d . 39 Figure 8 . O p t i c a l measurements and blood pressure of a duck during n i t r o g e n v e n t i l a t i o n . -^2 Figure 9. Corrected fluorescence recorded from the c e r e b r a l cortex of ducks during apneic asphyxia when mean a r t e r i a l blood pressure f e l l . , 5^ v i i Figure 10. Diagram of an oxygen electrode. 49 Figure 11. Polarogram of an oxygen electrode. 52 Figure 12. Polarographic measurements of P^Og ^ r o m the c o r t i c a l surface of a duck during hypoxia and hypercapnia. 55 Figure IJ. Surface P r p 0 2 r e c 0 r d e d froirr the r i g h t cerebral cortex during various levels of hypoxia. 58 Figure Ik-. Comparison of heart rate, mean a r t e r i a l blood pressure, and corrected fluorescence ini.paralyzed and nonparalyzed ducks during apneic asphyxia. ' 66 Figure 15. Submergence asphyxia i n a nonparalyzed duck. 69 Figure 16. Apneic asphyxia i n a paralyzed duck. 72 Figure 17. Corrected fluorescence and PipOg from the cerebral cortex of a duck during apneic asphyxia before and a f t e r i n h i b i t i o n of cardiovascular adjustments. 77 Figure 18. Comparison of heart rate, mean a r t e r i a l blood pressure, and corrected fluorescence i n paralyzed ducks and chickens during apneic asphyxia. 87 Figure 19. Electroencephalogram (EEG) and NADH fluorescence i n a chicken and duck during apneic asphyxia. 95 Figure 20. Asphyxia tolerance i n ducks plotted as a function of bradycardia. 98 v i i i Figure 21. Surface P^Og and NADH fluorescence of the ri g h t and l e f t cerebral cortex respectively during various le v e l s of hypoxia. 100 Figure 22. NADH fluorescence plotted as a function of P r p 0 2 during hypoxia i n chickens and ducks. 102 Figure 23. NADH fluorescence plotted as a function of P T0 ? during apneic asphyxia i n ducks. ~ 105 ix LIST OF TABLES NADH fluorescence i n paralyzed ducks during apneic asphyxia before and af t e r atropine i n j e c t i o n s . Heart rate, cessation of brain e l e c t r i c a l a c t i v i t y , and NADH fluorescence i n paralyzed ducks during apneic asphyxia. Heart rate, cessation of brain e l e c t r i c a l a c t i v i t y , and NADH fluorescence i n paralyzed chickens during apneic asphyxia. X A CKNOWLEGEMENTS I thank Dr. David R. Jones f o r his guidance and support of the research presented i n t h i s t h e s i s . His philosophy of and approach to science has impressed me to the extent that I s h a l l use these as guidelines as I persue a career i n science. In addition I thank Dr. D.J. Randall, Dr. W.K. Milsom, Dr. M.S. Haswell, and Dr. O.S. Bamford f o r t h e i r h e l p f u l comments pertaining to thi s research. I am gra t e f u l to Dr. W.K. Milsom, Chuck Daxboeck, and Steve Perry f o r reviewing rthis manuscropt. This research was supported by a grant to Dr. D.R. Jones from B r i t i s h Columbia Heart Foundation. 1 INTRODUCTION Since the time of A r i s t o t l e i t has been noted t h a t the porpoise i s an a i r breathing animal which can remain submerged i n water f o r periods of time t h a t are f a t a l to s t r i c t l y t e r r e s -t r i a l mammals (Strauss, 1 9 7 0 ) . Although the study of n a t u r a l d i v e r s has i n t r i g u e d man over the f o l l o w i n g 2 2 0 0 years, i t was not u n t i l the 1 8 7 0's t h a t P a u l Bert attempted to e x p l a i n the nature of t h i s phenomenon (Andersen, I 9 6 6 ) . Since Bert's p i o n e e r i n g i n v e s t i g a t i o n s p h y s i o l o g i c a l adjustments have been elucidated., which" help i n e x p l a i n i n g the. tolerance to-breath h o l d i n g i n n a t u r a l d i v e r s ( f o r reviews see Scholander, 1 9 ^ 0 ; Andersen, I 9 6 6 ; E i s n e r , I 9 6 9 ) . The major problem during a dive i s the d e p l e t i o n of oxygen and i t appears th a t n a t u r a l d i v e r s , whether amphibians, r e p t i l e s , b i r d s , or mammals, deal w i t h the problem i n a s i m i l a r manner. Despite t h i s f a c t d i v e r s can be d i v i d e d i n t o two major groups, ( 1 ) amphibians and r e p t i l e s , and ( 2 ) b i r d s and mammals, depending on t h e i r oxygen requirement. For example l i z a r d s , snakes, and c r o c o d i l e s can s u r v i v e anoxia f o r 30 minutes ( B e l k i n , I 9 6 3 ) , and t u r t l e s f o r over k hours (Jackson, I 9 6 8 ) , whereas d i v i n g mammals and b i r d s can su r v i v e anoxia f o r only a matter of seconds before i r r e p a r a b l e damage occurs. Because of t h i s l a r g e d i f f e r e n c e i n tolerance many of the g e n e r a l i z a t i o n s concerning the physiology of d i v i n g mammals and b i r d s do not p e r t a i n i n any d e t a i l to r e p t i l e s and amphi-bians. For t h i s reason only one of the groups, b i r d s and mammals, w i l l be considered i n t h i s t h e s i s . 2 Since external r e s p i r a t i o n ceases during the period of the underwater excursion, animals such as mammals and birds that are obligately dependent on oxygen must make some pro-v i s i o n f o r i t s storage and conservation during the dive. By increasing (1) blood volume (Eisner, I 9 6 9 ) , (2) oxygen capacity of the blood (Scholander, 1 9 ^ 0 ) , ( 3 ) lung volume (Andersen, I 9 6 6 ) , and ( 4 ) muscle myoglobin (Robinson, 1 9 3 9 ) divers can as much as double the quantity of oxygen stored over that of a s i m i l a r l y sized nondiver (Eisner, I 9 6 9 ) . However, increased oxygen storage does not appear to be absolutely necessary since some animals can endure extended periods of breath holding i n the absence of many of these ph y s i o l o g i c a l adaptations. For example, the oxygen carrying capacity of blood i n the manatee (Scholander and Irving, 19^-1), sea l i o n ( F l o r k i n and Redfield, 1 9 3 1 ) , porpiose (Green and Redfield, 1 9 3 3 ) 1 duck (Andersen, I 9 6 6 ) , and penguin (Scholander, 19^-0) i s a c t u a l l y less than that of man (Prosser, 1 9 7 3 )• The bottle nose whale and f i n whale, although known to be good divers, have a r e l a t i v e l y small lung volume (Andersen, I 9 6 6 ) and the F l o r i d a manatee has p r a c t i c a l l y no muscle myoglobin f o r the storage of oxygen (Scholander and Irving, 1 9^1). More important to underwater s u r v i v a l i s a series of cardiovascular adjustments during the dive that increases the vascular resistance i n most vascular beds (peripheral vasoconstriction) and reduces the heart rate (bradycardia) (Irving, 1 9 3 ^ ; Irving et a l . , 1935; Scholander, 19^0; Scho-lander et a l . , I9^2b; Johansen, 1 9 6 ^ ; Butler and Jones, 1 9 7 1 ) . 3 The p e r i p h e r a l v a s o c o n s t r i c t i o n i s thought to conserve "blood, oxygen by r e d i s t r i b u t i n g blood. Tissues such as the heart and, i n p a r t i c u l a r , the b r a i n which are most s e n s i t i v e to oxygen la c k r e c e i v e p r i o r i t y f o r blood flow over t i s s u e s and organs tha t are l e s s e a s i l y damaged by anoxia. The bradycardia o f f -sets the increased p e r i p h e r a l r e s i s t a n c e so that blood pressure i s h e ld constant and, i n a d d i t i o n , i t a l s o reduces the work load on the heart, thus f u r t h e r conserving oxygen. Although the same basi c response i s seen i n nondivers during apnea, i t ' h a s been considerably r e f i n e d i n d i v e r s to meet the needs of prolonged apneic s u r v i v a l . The c o n t r o l of the adjustments i s s t i l l not f u l l y understood and i s p r e s e n t l y the object of intense inves-t i g a t i o n ( f o r reviews of c o n t r o l see AngelaJames - and Daly, 1972; Jones, 1976; Jones and West, 1978). The nature of the c a r d i o v a s c u l a r adjustments d i v i d e s the t i s s u e s i n t o two groups; those that r e c e i v e blood flow during a d i v e , and those that do not. The t i s s u e s removed from the c i r c u l a t i o n must r e l y on anaerobic production of ATP* once the r e s i d u a l t i s s u e oxygen i s depleted. The c a p a c i t y to produce ATP i n muscle and other p e r i p h e r a l t i s s u e s i n t e r r e s -t r i a l animals through anaerobic g l y c o l y s i s i s s u f f i c i e n t that d i v e r s need only make minor m o d i f i c a t i o n s to t h i s b a s i c scheme. The o v e r a l l theme of the m o d i f i c a t i o n s i n v o l v e s two main areas: * A b b r e v i a t i o n s : ATP, adenosine triphosphate; ADP, adenosine diphosphate; and AMP, adenosine monophosphate. k (1) t i g h t e r c o n t r o l of r e g u l a t o r y enzymes enabling g l y c o l y t i c f l u x to increase by s e v e r a l hundred f o l d and (2) maintenance of the cytoplasmic NAD/NADH* r a t i o , p r i m a r i l y through the a c t i o n of l a c t a t e dehydrogenase, to insure t h a t NAD i s a v a i l a b l e f o r g l y c o l y s i s (Hochachka and Storey, 1975)• Tissues remaining i n the c e n t r a l c i r c u l a t i o n have l a r g e energy requirements and the energy supply must not be i n t e r -rupted during a di v e . For example, -;the energy requirements of the b r a i n may be as much as 20% of the t o t a l energy consumed i n mammals (Dunn and Bondy, 197^) and can only be met by pro-ducing ATP a e r o b i c a l l y ( S e i s j f l , 1977). the process depending almost e x c l u s i v e l y on the subs t r a t e glucose ( S o k o l o f f , I976). Only i n i n f a n t s (Dunn and Bondy, 197^) or durin g s t a r v a t i o n (Himwich, 1976b) are other substrates o x i d i z e d to any s i g n i f i -cant degree, but, even so, glucose s t i l l accounts f o r the m a j o r i t y of the substrate consumed (Dunn and Bondy, 197^0. Since glycogen, the storage form of glucose, i s not present i n the b r a i n i n la r g e enough q u a n t i t i e s to support energy meta-bolism (Himwich, 1976a)» the blood i s r e s p o n s i b l e not only f o r a continuous supply of oxygen but a l s o a continuous supply of glucose. On e n t e r i n g the c e l l glucose i s o x i d i z e d by the c l a s s i c a l Embden-Meyerhoff pathway ( g l y c o l y s i s ) to pyruvate w i t h a net production of two moles ot ATP f o r each mole of glucose * r> NAD and NADH are the o x i d i z e d and reduced forms of n i c o t i n a -mide adenine d i n u c l e o t i d e r e s p e c t i v e l y . 5 consumed. E q u a t i o n ( 1 ) d e s c r i b e s the o v e r a l l r e a c t i o n . G l u c o s e + 2 N A D + + 2ADP + 2 P i > ( 1 ) 2 p y r u v a t e + 2 NADH + 2H + 2 A T P +•2H20 A t t h i s p o i n t p y r u v a t e may e n t e r one o f two pathways. I t can be a n a e r o b i c a l l y r e d u c e d t o l a c t a t e i n the presence o f l a c t i c dehydrogenase (LDH) w i t h o u t f u r t h e r ATP p r o d u c t i o n : 2 p y r u v a t e + 2NADH + 2 H + L D H ) 2 l a c t a t e + 2 NAD + ( 2 ) The NADH produced i n e q u a t i o n ( l ) i s now r e o x i d i z e d and r e a d y t o be used a g a i n i n the o x i d a t i o n o f g l u c o s e . I n the presence of oxygen the two m o l e c u l e s o f NADH and the two p y r u v a t e mole-c u l e s from e q u a t i o n ( 1 ) may be o x i d i z e d v i a the c i t r i c a c i d c y c l e and the r e s p i r a t o r y c h a i n t o produce an a d d i t i o n a l 36 moles o f ATP per mole o f g l u c o s e i n i t i a l l y consumed. 2NADH + 2 H + + 2 p y r u v a t e + 6 0 ? + 36ADP + 3 ^ P i > , ( 3 ) 2 NAD + 6C0 2 + kJmz0 + 3 6 A T P Under normoxic c o n d i t i o n s Q5% o f t h e g l u c o s e e n t e r i n g t h e b r a i n i n mammals i s c o m p l e t e l y o x i d i z e d t o C 0 2 w h i l e 1 5 $ i s o x i d i z e d o n l y t o l a c t a t e (Dunn and Bondy, 1 9 7 *0• C o n s i d e r i n g the moles o f ATP produced p e r mole o f g l u c o s e f o r a n a e r o b i c m e t a b o l i s m (2 moles o f ATP) and a e r o b i c m e t a b o l i s m ( 3 6 moles of ATP), a e r o b i c m e t a b o l i s m a c c o u n t s f o r about 95% o f the t o t a l ATP produced. The p r o p o r t i o n o f g l u c o s e o x i d i z e d t o l a c t a t e depends on the a v a i l a b l e oxygen; as oxygen becomes l i m i t i n g p r o p o r t i o n a t e l y more g l u c o s e i s m e t a b o l i z e d t o l a c t a t e . 6 In r a t s when the b r a i n i s depleted of oxygen, g l y c o l y t i c f l u x can increase 5 f o l d ( S e i s j O, 1977)- However, even w i t h t h i s increase, anaerobic metabolism alone cannot supply the necessary ATP r e q u i r e d f o r b r a i n f u n c t i o n . Over $0% of the oxygen used by the b r a i n i s handled by the r e s p i r a t o r y chain ( J f l b s i s , 197^) which c o n s i s t s of a s e r i e s of e l e c t r o n c a r r i e r s arranged i n ascending order of t h e i r redox p o t e n t i a l s ( F i g . 1) and acts as an energy gradient t h a t t r a n s -f e r s e l e c t r o n s from substrate to oxygen. On e n t e r i n g the r e s p i r a t o r y chain e l e c t r o n s flow from the members w i t h the more negative redox p o t e n t i a l s , NAD or F P f ( f l a v o p r o t e i n ) , to the more p o s i t i v e members and u l t i m a t e l y to oxygen. Each atom of oxygen r e c e i v e s two e l e c t r o n s from the r e s p i r a t o r y chain plus two hydrogen ions from the media forming one molecule of water. At s e v e r a l steps along the chain the f r e e energy of e l e c t r o n t r a n s f e r i s captured and used i n o x i d a t i v e phosphorylation, a term given to the energy r e q u i r i n g r e a c t i o n : ADP + P i -> ATP + H £ 0 (k) For each p a i r of e l e c t r o n s passed down the r e s p i r a t o r y chain from NAD to oxygen approximately three molecules of ATP are produced. E l e c t r o n o p a i r s e n t e r i n g the chain v i a FP produce only two molecules of ATP sin c e the e l e c t r o n s bypass the f i r s t p hosphorylation s i t e ( F i g . 1). Oxidative phosphorylation i s o b l i g a t e l y coupled to the e l e c t r o n flow of the chain and there f o r e proceeds only i n the presence of oxygen. When oxygen becomes l i m i t i n g e l e c t r o n s enter the r e s p i r a t o r y chain 7 Figure 1. A diagram of the r e s p i r a t o r y chain i n mitochondria i l l u s t r a t i n g the flow of e l e c t r o n s and the probable s i t e s f o r ATP production. A b b r e v i a t i o n s : NAD, nicotinamide adenine d i n u c l e o t i d e ; FP^ and FPg, f l a v o p r o t e i n s ; CoQ, ubiquinone or coenzyme Q; CYT, cytochrome. ATP ATP •CYT b CYTc -•CYTc "CYTaa, 9 faster than they can be removed by oxygen r e s u l t i n g i n a net reduction of each electron c a r r i e r . The oxidation-reduction state of any electron c a r r i e r can therefore serve as an i n d i -cator of mitochondrial hypoxia (Chance et a l . , 1 9 7 3 ) . Changes i n the redox state of the f i r s t component of the respiratory chain, NAD, can be monitored i n inta c t tissues by a fluorometric method which was f i r s t described by Chance et a l . (1962). The method takes advantage of the fact that the reduced form of NAD (NA-DH) i s a natural fluorochrome which; can-be:• excited by l i g h t wavelengths between 31° and 370 nm and gives r i s e to a fluorescence emission i n the region of ^ -25-^ 75 nm (JSbsis et a l . , I966). The oxidized form of the coenzyme does not fluoresce at these wavelengths. In practice NADH i s excited by focussing 366 nm l i g h t , a natural peak of the mercury arc lamp, on the tissue surface and the fluorescence o r i g i n a t i n g primarily from the top 1.5 mm of tissue (Jtfbsis et a l . , 1971) i s measured with a photomultiplier tube. The l a b i l e fluorescence s i g n a l from tissue was i n i t i a l l y l inked to NADH due to s i m i l a r i t i e s i n t h e i r fluorescence emis-sion spectra (Chance et a l . , 1962) and to the fac t that NADH, determined by biochemical analysis, correlates with changes of fluorescence i n l i v e r (Chance et a l . , 1965a), heart (Chance et a l . , 1965b) and brain (Jflbsis et a l . , 1971). During cycles of anoxia the ki n e t i c s of tissue fluorescence are i n synchrony with those of the cytochromes i n d i c a t i n g a large contribution of respiratory chain, NADH to the fluorescence s i g n a l (Lubbers et a l . 1 9 6 ^ ) . Furthermore, Chance et a l . (I962) and Mayevsky 10 and Chance (197^) showed th a t drugs which block the r e s p i r a t o r y chain caused a larg e fluorescence increase i n the kidney and b r a i n of r a t s . However, the fluorescence s i g n a l from the r e s p i r a t o r y chain may be contaminated w i t h fluorescence from other p y r i d i n e n u c l e o t i d e pools i n the c e l l which are noti.at the same redox s t a t e as r e s p i r a t o r y chain NAD and do not n e c e s s a r i l y show redox changes that p a r a l l e l changes i n r e s p i r a t o r y chain NADH. I n v e s t i g a t i o n s i n t o the o r i g i n of the l a b i l e fluorescence have, shown th a t these n o n r e s p i r a t o r y chain pools c o n t r i b u t e l i t t l e i f any to the fluorescence s i g n a l . Fluorescence from r e s p i r a -t o r y chain NADH i s s u f f i c i e n t l y enhanced over the fluorescence from cytoplasmic NADH, the pool which i s p r i m a r i l y i n v o l v e d w i t h the g l y c o l y t i c pathway, t h a t f l u c t u a t i o n s i n cytoplasmic NADH co n t r i b u t e l i t t l e to the fluorescence s i g n a l (Jtfbsis and D u f f i e l d , 1967; O'Connor, 1977). The r e s p i r a t o r y chain NADH i s enzyme bound whereas much cytoplasmic NADH i s f r e e or un-bound. As a general r u l e NADH tha t i s enzyme bound has a gre a t e r quantum e f f i c i e n c y than f r e e NADH (Boyer and T h e o r e l l , 1956). However, glyceraldehyde phosphate dehydrogenase which i s abun-dant i n the cytoplasm i s one of the few enzymes t h a t a c t u a l l y diminishes r a t h e r then enhances fluorescence when bound to the coenzyme ( V e l i c k , I96I). The o v e r a l l r e s u l t i s that the fluorescence e f f i c i e n c y of r e s p i r a t o r y chain NADH i s 10-20 times greater than that of the cytoplasmic NADH pool (J f l b s i s et a l . , 1971). Another NAD pool which i s thought to serve p r i m a r i l y i n 11 b i o s y n t h e s i s (JObsis, 1964) i s l o c a t e d i n the mitochondria but i s not d i r e c t l y i n v o l v e d w i t h the r e s p i r a t o r y chain (Chance and Hollunger, I96I). This pool has not been as exten-s i v e l y i n v e s t i g a t e d as the cytoplasmic NAD pool but nevertheless appears to c o n t r i b u t e only 1-2% of the t o t a l fluorescence s i g n a l i n r e s t i n g mitochondria (J f l b s i s and D u f f i e l d , 1967). Fluorescence p r o p e r t i e s , i d e n t i c a l to those of NADH a l s o occur i n the reduced form -of cytoplasmic and m i t o c h o n d r i a l nicotinamide adenine d i n u c l e o t i d e phosphate (NADPH) which may i n t e r f e r e w i t h the fluorescence s i g n a l from the r e s p i r a t o r y chain NADH. However, biochemical analyses have shown th a t NADPH does not change i n the cortex of the s q u i r r e l monkey during anoxia when fluorescence i s maximal (Sundt et a l . , 1976). Furthermore NADPH i s only 3% of the t o t a l p y r i d i n e n u c l e o t i d e pool (Glock and McLean, 1955) and would th e r e f o r e c o n t r i b u t e very l i t t l e to the fluorescence s i g n a l . P 0 2 of the b r a i n , and thus the redox s t a t e of r e s p i r a t o r y chain NAD, i s dependent not only on a r t e r i a l P 0 2 (PaOg) but a l s o the r a t e at which blood perfuses the b r a i n . Since hypoxia, hypercapnia, and a f a l l i n a r t e r i a l pH ( e i t h e r from hypercapnia or l a c t a t e ) , which occur during breath h o l d i n g , increase blood flow to the b r a i n (Ingvar and Lassen, 1962; McDowall, I966; Purves, 1972; Kogure et a l . , 1970; Betz, 1972; Borgstrom et a l . , 1975; G-rubb et a l . , 1977 and 1978) by decreasing c e r e b r a l vascu-l a r r e s i s t a n c e through l o c a l and p o s s i b l y c e n t r a l l y mediated mechanisms (Purves, 1972; Betz, 1972), b r a i n P 0 2 can be re g u l a t e d to a degree during d i v i n g . However, during a dive c e r e b r a l 12 blood flow i n n a t u r a l d i v e r s has been reported to remain un-changed ( G r i n n e l l et a l . , 19^2; Bron et a l . , I 9 6 6 ) or even decrease s l i g h t l y from the predive l e v e l (Van C r i t t e r s et a l . , —' 1 9 6 5 ; E i s n e r et a l . , I 9 6 6 ; B u t l e r and Jones, 1971,* Kerem and E i s n e r , 1 9 7 3 ) ' On the other hand, some s t u d i e s have shown la r g e increases i n c e r e b r a l ' b l o o d flow during a dive (Johansen, 196^; Dormer et a l . , 1 9 7 7 ; Jones et a l . , 1 9 7 8 ) . Even when c e r e b r a l blood flow was reported to>have decreased i t was nevertheless sustained at higher l e v e l s than t h a t i n other t i s s u e s . Since PaOg f a l l s d uring a d i v e , the predive POg of the b r a i n cannot be maintained even i f c e r e b r a l blood flow increases, but oxygen d e l i v e r y to the t i s s u e can be maintained i n the face of f a l l i n g PaOg. I n f a c t , oxygen consumption of the b r a i n (CMR02) and l e v e l s of the adenosine phosphates (ATP, ADP and AMP) do not change i n t e r r e s t r i a l mammals u n t i l PaOg f a l l s below 2 . 6 6 k P a (20 t o r r ) (Duffy et a l . , 1 9 7 2 ; MacMillan and Seisjo*, 1971; Kety and Schmidt, 19^8; Lambertson et a l . , 1 9 5 3 ; Cohen e_t a l . , I 9 6 7 ; Johanssen and Seisjo 1, 1 9 7 5 ; Borgstrom et. a l . , 1975) p r o v i d i n g that blood pressure does not f a l l . Although CMROg i s maintained, the l a c t a t e / p y r u v a t e r a t i o begins to increase once Pa0 2 f a l l s below 6 . 6 5 k P a ( 5 0 t o r r ) during normo-capnic hypoxia i n d i c a t i n g t h a t g l y c o l y t i c f l u x has increased. However, during apneic asphyxia i n dogs l a c t a t e production by the b r a i n does not increase even a f t e r Pa0 2 f a l l s w e l l below 2 . 6 6 k P a (Kerem and E i s n e r , 1973b) i n d i c a t i n g t h a t perhaps under these c o n d i t i o n s blood flow increases are f a r greater 13 than during normocapnic hypoxia. The importance of the c a r d i o v a s c u l a r adjustments to continued t r a i n f u n c t i o n during d i v i n g was shown by Kerem and E i s n e r (1973a). Harbor s e a l s which t o l e r a t e d dives l a s t i n g 18.5 minutes before the onset of hypoxic EEG (electroencephalo-gram) patterns t o l e r a t e d dives l a s t i n g only 5.5 minutes a f t e r the c a r d i o v a s c u l a r adjustments were i n h i b i t e d w i t h a t r o p i n e . I f enhanced c a r d i o v a s c u l a r adjustments i n d i v e r s p r o t e c t the b r a i n by conserving oxygen f o r the b r a i n and heart alone, then r e s p i r a t o r y chain NAD should not be reduced as f a s t as i n the b r a i n of nondiving animals during f o r c e d d i v e s . P h y s i o l o g i c a l mechanisms cannot t o t a l l y account f o r the c e r e b r a l t o l e r a n c e to asphyxia i n d i v i n g animals s i n c e d i v e r s may be able t o t o l e r a t e more severe hypoxia .than t e r r e s -t r i a l animals (Ridgeway et a l . , 1969; E i s n e r et a l . , 1970; Kerem and E i s n e r , 1973a and b ) . When oxygen d e p l e t i o n i s f a r advanced p h y s i o l o g i c a l adjustments must be superceded, .or at l e a s t aided, by some form of biochemical r e g u l a t i o n i f ATP production i s t o continue. S e v e r a l biochemical changes that enhance o x i d a t i v e phos-p h o r y l a t i o n have been reported f o r animals acclimated to hypoxia. Reynafarje (1971/72) showed th a t guinea-pigs n a t i v e to high a l t i t u d e s (4500 m) hav e 1 h e a r t mitochondria w i t h a grea t e r a f f i n i t y (lower K ) f o r ADP than those n a t i v e to sea l e v e l and concluded t h a t the former can generate ATP f a s t e r at lower ADP concentrations when oxygen tensions are low. Heart mito-chondria from r a t s acclimated to hypoxia show a t h r e e - f o l d 14 increase i n the r e s p i r a t o r y chain a c t i v i t y (moles Og consumed ' -1 -1 mole cytochrome aa^ " min ) over mitochondria from normoxic animals (Mela et a l . , 1976) and Park et a l . (1973) have suggested t h a t the former may have lower c r i t i c a l P ° 2 ' s ^•'lan ^ e l a t t e r presumably due to an increased cytochrome a^ a f f i n i t y f o r oxygen. Isozymes of cytochrome a^ having d i f f e r e n t oxygen a f f i n i t i e s have been reported i n the c a r o t i d body ( M i l l s , 1972; M i l l s and J f l b s i s , 1972); however, the lower a f f i n i t y cytochrome a^ i s an oxygen sensor and may not have a r o l e i n ATP production. Clearly, increased a f f i n i t i e s f o r ADP, P i , and oxygen could enhance or prolong ATP production when t i s s u e hypoxia occurs. On the other hand, Simon et a l . (1974) found no s i g n i f i c a n t d i f f e r e n c e s inccytochrome oxidase (cytochrome a, a^) a c t i v i t y i n three species of d i v i n g mammals w i t h w i d e l y v a r y i n g maximal dive times;)however, the study only q u a n t i f i e d cytochrome oxidase a c t i v i t y without i n v e s t i g a t i n g the k i n e t i c r e l a t i o n s h i p between the enzyme and oxygen. Many d i v i n g animals appear to have the c a p a c i t y f o r generating ATP f o r the b r a i n by anaerobic g l y c o l y s i s . The b r a i n of whales (Shoubridge et a l . , ,1976), beavers (Messelt and B l i x , 1974), seals ( B l i x and Fromm, 1971). and eid e r s ( B l i x and Fromm, 1971; B l i x et a l . , 1973) are known to have a greater p r o p o r t i o n of the muscle type isozyme of l a c t a t e dehydrogenase (LDH) than t e r r e s t r i a l mammals. The muscle isozyme favors the formation of l a c t a t e (equation (2), page 5) and i s i n d i c a t i v e of t i s s u e s more t o l e r a n t to hypoxia; con-v e r s e l y the heart isozyme favors the formation of pyruvate and 15 i s i n d i c a t i v e of h i g h l y aerobic t i s s u e s (Hellung-Larsen and Andersen, 1968;'Kaplan and Everse, 1972). The presence of a lar g e p r o p o r t i o n of the muscle isozyme t h e r e f o r e i n d i c a t e s an increased anaerobic c a p a c i t y i n the b r a i n of the above animals. On the other hand, LDH isozyme patterns of the b r a i n of the Weddell s e a l (Murphy and Hochachka, 1978) and the narwhal (Vogel, 1978) f a v o r the heart type subunit and are not s t r i k i n g l y d i f f e r e n t from the LDH p a t t e r n observed i n t e r r e s t r i a l mammals. Although the LDH isozymes of the b r a i n of the Weddell s e a l (Murphy and Hochachka, 1978) are s i m i l a r to t e r r e s t r i a l animals, the substrate f o r anaerobic metabolism, glycogen, appears to be at l e a s t 2 to 3 times higher i n Weddell s e a l b r a i n than f o r other nondiving mammals (Kerem et a l . , 1973).-Pyruvate kinase, a r e g u l a t o r y enzyme of the g l y c o l y t i c pathway which has been used as a q u a n t i t a t i v e index of g l y c o l y t i c c a p a c i t y (Simon and Robin, I972), shows a greater a c t i v i t y w i t h i n c r e a s i n g maximal dive times i n the sea l i o n , harbor s e a l , and Weddell s e a l (Simon et a l . , 197*0. Although the anaerobic machinery i s present, can the d i v e r produce s i g n i f i c a n t amounts of ATP f o r the b r a i n anaero-b i c a l l y ? During maximal dives i n harbor s e a l s the CMROg decreases and b r a i n l a c t a t e production increases f o r the l a s t 5 minutes before the onset of hypoxic EEG patterns (Kerem and E i s n e r , 1973a). During submaximal dives (20 minutes) i n Weddell s e a l s , Murphy and Hochachka (1978) found no appreciable increase i n b r a i n l a c t a t e production. Since anaerobic ATP production i s much l e s s e f f i c i e n t than aerobic production i t i s questionable 16 t h a t s u f f i c i e n t energy can he produced g l y c o l y t i c a l l y to main-t a i n c e r e b r a l i n t e g r i t y (Seisjo 1 et a l . , 197*0. The purpose of t h i s t h e s i s was to study c e r e b r a l energy metabolism and b r a i n f u n c t i o n d u r i n g d i v i n g i n an i m a l s ' t h a t can endure prolonged asphyxia. The f l u o r o m e t r i c technique d e s c r i b e d above was used to compare changes i n the redox s t a t e of r e s p i r a t o r y c h a i n NAD i n the c e r e b r a l c o r t e x of two s p e c i e s which have d i f f e r e n t t o l e r a n c e s to asphyxia, ducks and chickens, d u r i n g apneic asphyxia and steady s t a t e hypoxia. Ducks are known to t o l e r a t e asphyxia 3-8 times l o n g e r than chickens (Andersen, 1959 and I966). A f t e r e s t a b l i s h i n g t h a t b r a i n f u n c t i o n . i s maintained longer i n ducks than chickens d u r i n g apneic asphyxia, I. examined the r o l e p h y s i o l o g i c a l and b i o -chemical adjustments p l a y i n the maintenance of b r a i n f u n c t i o n . 17 CHAPTER 1 General Methods - Animal P r e p a r a t i o n a. Rats. Rats were used i n i t i a l l y as experimental animals since they have been e x t e n s i v e l y used i n experiments where c o r t i c a l NADH was measured by the f l u o r o m e t r i c technique (Chance et a l . , 1 9 6 2 ) . F i f t e e n Wistar r a t s (3OO-5OO g) of both sexes were anesthetized w i t h 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 urethane ( 1 . 3 g/kg, BDH Chemicals L t d . , Poole, England). A polyethylene catheter ( P E 5 0 ) was i n s e r t e d i n t o the r i g h t j u g u l a r v e i n f o r drug i n j e c t i o n s . A f t e r a tracheostomy each r a t was paralyzed w i t h an intravenous i n j e c t i o n of turbocurarine c h l o r i d e (1 mg/kg, Burroughs Wellcome and Co., Montreal, Canada) and v e n t i l a t e d w i t h a Narco type V5KG constant pressure r e s p i -r a t o r (Narco Bio-Systems, Inc., Houston, Texas). For f l u o r o -m e tric recordings the s c a l p over the l e f t p a r i e t a l r e g i o n of the s k u l l was i n c i s e d and r e t r a c t e d . A window approximately 1 cm x 0.5 cm was cut i n the un d e r l y i n g c a l v a r i a and the l e f t c e r e b r a l hemisphere was exposed. The dura was l e f t i n t a c t . Only blood-free preparations were used f o r f l u o r o m e t r i c r e c o r d i n g s . b. Ducks. Experiments were done on a t o t a l of 75 m a l l a r d ducks, Anas platyrhynchos, of both sexes and weighing 1 . 0 - 2 . 2 kg. A l l operations except craniectomies were performed a f t e r a l o c a l i n j e c t i o n , of 2% w/v xy l o c a i n e ( A s t r a Pharmaceutical, Mississauga>, O n t a r i o ) . This procedure produced l o c a l anesthesia which was sustained f o r s e v e r a l hours and the ducks showed 18 no signs of s t r e s s e i t h e r during or a f t e r surgery. Craniec-tomies were performed using general anesthesia e i t h e r "by v e n t i l a t i n g the ducks w i t h 1% halothane i n a i r or by an i n t r a -venous i n j e c t i o n of urethane ( 1 . 0 g/kg). Ducks th a t were anesthetized w i t h urethane were used f o r developing the f l u o r o -metric technique (Chapter 2) and remained anesthetized through-out the experiment. Halothane was used f o r craniectomies i n a l l other ducks and a k$ minute recovery p e r i o d from anesthesia preceeded a l l experiments. A polyethylene cannula (PE 9 0 ) was i n s e r t e d i n t o the l e f t s c i a t i c a r t e r y and connected to a Statham P23GB pressure transducer f o r monitoring blood pressure. A three-way stop-cock connected the transducer to the cannula and allowed w i t h -drawal of a r t e r i a l blood samples v i a the s i d e arm f o r blood gas a n a l y s i s ( P a 0 2 > PaCOg, pHa). A second polyethylene cannula (PE 9 0 ) was i n s e r t e d i n t o the vena cava v i a the b r a c h i a l v e i n and used f o r s a l i n e i n j e c t i o n s to c a l i b r a t e the fluorometer and f o r drug i n j e c t i o n s . Heart r a t e was determined from the electrocardiogram (ECG) which was obtained from 2 copper wire e l e c t r o d e s , one i n s e r t e d subcutaneously i n the l e f t s i d e of the chest and the other i n s e r t e d i n the r i g h t t h i g h . The s i g n a l was a m p l i f i e d and fed i n t o a ratemeter. C l o a c a l temperature was maintained at kl"— 0 .5°C by a t h e r m i s t o r i n s e r t e d i n t o the c l o a c a and a temperature c o n t r o l l e d feedback u n i t t h a t regulated a heating pad placed over the duck. I n a l l but 9 ducks a cannula was i n s e r t e d i n the trachea towards the lung and the c l a v i c u l a r a i r sac was punctured. A f t e r p a r a l y s i s 19 w i t h an i n t r a v e n o u s i n j e c t i o n o f g a l l a m i n e t r i e t h i o d i l e (1 mg/kg, F l a x e d i l , P o u l e n c L t d . , M o n t r e a l , Quebec) the ducks were u n i -d i r e c t i o n a l l y v e n t i l a t e d by p a s s i n g a stream o f h u m i d i f i e d a i r t h r o u g h the t r a c h e a l c a n n u l a a t r a t e s up t o 1 l / m i n . A r t e r i a l b l o o d samples were withdrawn from the s c i a t i c a r t e r y and a n a l y z e d u s i n g a Radiometer PHM 71 gas m o n i t o r w i t h oxygen and ca r b o n d i o x i d e e l e c t r o d e s and a m i c r o e l e c t r o d e u n i t type E5021 ( R a d i o -meter, Copenhagen, Denmark). The a i r f l o w was a d j u s t e d t o g i v e a r t e r i a l b l o o d v a l u e s f o r P a 0 2 o f 11 .3-14 .0 k P a ( 8 5 - 1 0 5 t o r r ) ; P a C 0 2 o f 3 . 3 - 4 . 4 kPa (25-33 t o r r ) ; and pHa o f 7 . 4 5 - 7 - 5 0 . The r e m a i n i n g 9 ducks w h i c h were n o t p a r a l y z e d o r u n i d i r e c -t i o n a l l y v e n t i l a t e d were used i n experiments i n v o l v i n g nonpara-l y z e d ducks ( C h a p t e r 3 ) . B r a i n e l e c t r i c a l a c t i v i t y was m o n i t o r e d from 2 s t a i n l e s s s t e e l screws cemented i n the a n t e r i o r and p o s t e r i o r a r e a s o f the r i g h t f r o n t a l r e g i o n . o f t h e s k u l l . The p o t e n t i a l d i f f e r e n c e between t h e screws was a m p l i f i e d 1000 times w i t h a F r e d e r i c k Haer A m p l i f i e r ( F r e d e r i c k Haer and Co., Brunswic k , Maine) and and d i s p l a y e d on a 6 c h a n n e l Watanabe WTR 281 pen r e c o r d e r (Watanabe I n s t r u m e n t C o r p o r a t i o n , Tokyo, Japan) a l o n g w i t h o t h e r measured v a r i a b l e s . F o r f l u o r o m e t r i c r e c o r d i n g s a n i m a l s were p l a c e d v e n t r a l s i d e down on a m e t a l o p e r a t i n g t a b l e w i t h the head s e c u r e d i n a s t a i n l e s s s t e e l head h o l d e r w h i c h c o n s i s t e d o f a b i l l clamp and e ar b a r s . The s c a l p over the l e f t f r o n t a l r e g i o n o f the s k u l l was i n c i s e d and a h o l e 1.0 cm i n di a m e t e r was c u t i n t h e u n d e r l y i n g c a l v a r i a . The d u r a was r e t r a c t e d e x p o s i n g t h e 20 a n t e r i o r p o r t i o n of the l e f t hemisphere, Pars o r a l i s , "below the F i s s u r a d o r s a l i s . I n a l l but 14 ducks the exposed cortex was covered w i t h p l a s t i c f i l m to prevent d r y i n g . In the remaining 14 ducks the exposed cortex was covered w i t h a glass window 1 cm i n diameter and cemented to the s k u l l w i t h d e n t a l cement (Hygienic Mfg. Co., Akron, Ohio). Tissue oxygen t e n s i o n (P^Og) was recorded p o l a r o g r a p h i -c a l l y from the c o r t i c a l s u r f a c e . A craniectomy was performed on the r i g h t f r o n t a l r e g i o n of the s k u l l and an oxygen electrode was placed i n contact w i t h the surface of the c o r t i c a l area corresponding to tha t from which the f l u o r o m e t r i c recordings were made on the opposite s i d e . C o i l e d copper wire (0.25 mm i n diameter) supported the electro d e and allowed i t to move wi t h p u l s a t i o n s of the b r a i n . c. Chickens. A t o t a l of 43 hens, Ga l l u s domesticus, weighing between 1.2 and 2.2 kg were used. Roosters were not used sin c e a l a r g e comb i n t e r f e r e d w i t h c r a n i a l surgery. Operative procedures f o r chickens were i d e n t i c a l to those done on ducks. 21 CHAPTER 2 S p e c i a l Techniques The Fluorometric Recording 1 of NADH The r e s p i r a t o r y chain i s the f o c a l p o i n t f o r bioener-g e t i c s i n the b r a i n since i t l i n k s oxygen uptake w i t h ATP production (JObsis, 1972). The r e s p i r a t o r y chain acts as an energy gradient t h a t t r a n s f e r s reducing equivalents from sub-s t r a t e t o oxygen forming ATP wit h the l i b e r a t e d f r e e energy. During hypoxia, the reducing equivalents enter the r e s p i r a t o r y chain f a s t e r than they can be removed by oxygen r e s u l t i n g i n a net r e d u c t i o n of the r e s p i r a t o r y chain c a r r i e r s . The o x i d a t i o n -r e d u c t i o n s t a t e of any r e s p i r a t o r y chain component can there-fore serve as an i n d i c a t o r of m i t o c h o n d r i a l hypoxia. The fl u o r o m e t r i c method described by Chance et a l . (1962) provides a nondestructive, d i r e c t , and continuous (Chance et a l . , 1970) readout of changes i n the o x i d a t i o n - r e d u c t i o n s t a t e of the f i r s t member of the r e s p i r a t o r y chain, NAD. Since hemoglobin absorbs both e x c i t a t i o n and fluoroescence light,,changes of blood i n the r e c o r d i n g f i e l d produce a r t i f a c t s i n the apparent NADH fluorescence (Sennitger et a l . , 1965; Chance and Schoener, 1965; Granholm et a l . , 1969; Kobayashi et a l . , 1971a and b; J f l b s i s et a l . , 1971).' JObsis and Stainsby (I968) and JObsis et a l . (1971) introduced a second photomulti-p l i e r system to measure the r e f l e c t e d e x c i t a t i o n l i g h t and used i t to compensate f o r the hemoglobin a r t i f a c t . I f the outputs from the two p h o t o m u l t i p l i e r s are adjusted to give an 22 equal response f o r a given, hemoglobin change i n the absence of a concomitant NADH change then the d i f f e r e n c e between the two outputs must be due s o l e l y to NADH. However, when the a r t i f a c t i s l a r g e , f u l l compensation i s questionable (JObsis et a l . , 1971). Since blood flow to the b r a i n i n c r e a s e s , on average, 8.5 times i n ducks duri n g prolonged apneic asphyxia (Jones et a l . , 1978), compensation by using r e f l e c t e d e x c i t a t i o n l i g h t could be inadequate d u r i n g d i v i n g c o n d i t i o n s . The purpose of t h i s s e c t i o n was to determine i f the blue fluorescence o r i g i n a t i n g from the c e r e b r a l cortex of ducks when ex c i t e d w i t h UV l i g h t was due to NADH and, i f so, develop a fluorometer to monitor the NADH change. I n p a r t i c u l a r , the r e f l e c t e d e x c i t a t i o n l i g h t has been evaluated to determine i f there was adequate compensation f o r the hemodynamic a r t i f a c t over the range of blood flow changes expected during apneic asphyxia. a. D e s c r i p t i o n of the fluorometer The fluorometer was a modified v e r s i o n of that described by JObsis et a l . (1971) ( F i g . 2 ) . The u l t r a v i o l e t (UV) source c o n s i s t e d of a 1000 W water cooled mercury arc lamp AH6-1-B ( I l l u m i n a t i o n I n d u s t r i e s Inc., Sunnyvale, C a l i f o r n i a ) encased i n a s t a i n l e s s s t e e l housing (made by Ron Overaker, Department of Physiology and Pharmacology, Duke U n i v e r s i t y M e d i c a l School, Durham, North C a r o l i n a ) . Power was s u p p l i e d by a model T507 step-up transformer ( I l l u m i n a t i o n I n d u s t r i e s ) and the voltage to the lamp (700 VAC) was c o n t r o l l e d by a Powerestat I36 v a r i a b l e 23 Figure 2. Diagram of the o p t i c a l design of the fluorometer used to monitor fluctuations i n respiratory chain NADH i n intact t i s s u e . 2k FLUORESCENCE PMT BEAM SPLITTER 450nm LIGHT-360nm LIGHT UV SOURCE EXCITATION FILTER 360 nm SECONDARY 450nm FILTERS REFLECTANCE PMT -360nm LIGHT ULTROPAK ASSEMBLY .SPECIMEN 25 transformer (Superior E l e c t r i c Co., B r i s t o l , Connecticut) placed on the primary side of the step-up transformer. E x c i t a t i o n l i g h t (360 nm) was s e l e c t e d by a primary f i l t e r ( L e i t z , Wetzlar, Germany) having a h a l f power band width (HPBW) of 10 nm and r e f l e c t e d onto the surface of the t i s s u e at an angle of 45-60° using a L e i t z Ultropak assembly as an i n c i d e n t -l i g h t i l l u m i n a t o r . The l i g h t i n t e n s i t y was measured by an Eppley thermopile No. 106-57 (The Epply Laboratory, Inc., Newport, Rhode Island) and attenuated by p l a c i n g round micro-scope c o v e r s l i p s (1.8 cm i n diameter) between the l i g h t source and the specimen, since l i g h t i n t e n s i t y above 0.8 mW/cm may cause t i s s u e damage (Rosenthal, 1976). R e f l e c t e d e x c i t a t i o n l i g h t and NADH fluorescence were c o l l e c t e d from c o r t i c a l f i e l d s e i t h e r 3.5 mm or 2.3 mm i n diameter by a low (3.8x) or high (6.5x) power o b j e c t i v e and d i v i d e d by an 80:20 beam s p l i t t e r ( L e i t z ) . Changes i n fluorescence i n t e n s i t y were monitored w i t h an EMI 9 5 2 4 B photo-m u l t i p l i e r tube (EMI Gencom Inc., P l a i n v i e w , New York) from the Q0% p o r t i o n of l i g h t a f t e r the e x c i t a t i o n l i g h t was removed w i t h a 450 nm secondary f i l t e r ( L e i t z ) . The r e f l e c t e d e x c i -t a t i o n l i g h t was monitored from the 20% p o r t i o n of l i g h t a f t e r the fluorescence l i g h t was removed w i t h a L e i t z UV UG1 f i l t e r . I n i t i a l l y the p h o t o m u l t i p l i e r s were powered by a Kepco OPS 2000 (Kepok, Inc., F l u s h i n g , New York) and a Knott high s t a b i l i t y power supply type NSHM (Knott E l e k t r o n i k , Munich, West Germany). In l a t e r experiments voltages f o r both p h o t o m u l t i p l i e r s were derived from the Kepco power supply by s p l i t t i n g the voltage 26 and v a r y i n g i t s e p a r a t e l y to each p h o t o m u l t i p l i e r . The fluorescence s i g n a l (F) and the r e f l e c t a n c e s i g n a l (R) were obtained by a m p l i f y i n g the p h o t o m u l t i p l i e r outputs ( F i g . 3). In a d d i t i o n the e l e c t r o n i c s u b t r a c t i o n of F-R (mixer a m p l i f i e r i n F i g . 3) provided a t h i r d s i g n a l termed co r r e c t e d fluorescence (CF). A l l three o p t i c a l s i g n a l s were recorded on the 6 channel Watanabe pen recorder. E q u a l i z a t i o n of p h o t o m u l t i p l i e r outputs to a given hemo-g l o b i n change was s i m i l a r to the method used by JtJbsis et a l . (1971). The fluorometer was focussed on the b r a i n s u r f a c e , and the voltage to the p h o t o m u l t i p l i e r s was adjusted u n t i l the a m p l i f i e d outputs were each of 4 v o l t s . Fine adjustment was accomplished by e q u a l i z i n g the outputs of the F and R photo-m u l t i p l i e r s when the amount of hemoglobin i n the f i e l d was reduced i n the absence of a concomitant NADH change. The b r a i n was f l u s h e d w i t h oxygenated s a l i n e which was introduced through the venous cannula and the s e n s i t i v i t y of the r e f l e c t a n c e p h o t o m u l t i p l i e r was f i n e l y adjusted by a l t e r i n g the input voltage u n t i l the response to the f l u s h equalled that of the fluorescence p h o t o m u l t i p l i e r . Generally, c a l i b r a t i o n of the fluorometer r e q u i r e d 2 or 3 f l u s h e s . b. S t a b i l i t y of the fluorometer S t a b i l i t y of the fluorometer was determined by f o c u s s i n g the fluorometer on a piece of paper and r e c o r d i n g the a m p l i f i e d voltage outputs of the F and R p h o t o m u l t i p l i e r tubes over 3 hours. Since no NADH was present the 450 nm f i l t e r -normally 2 7 F i g u r e 3- S i m p l i f i e d diagram of the f l u o r o m e t e r c i r c u i t r y . PMT's, p h o t o m u l t i p l i e r tubes. F L U O R E S C E N C E C H A N N E L R E F L E C T A N C E C H A N N E L PMT'S V A R I A B L E G A I N MIXER AMPLIFIER INPUT AMPLIFIERS F L U O R E S C E N C E C O R R E C T E D F L U O R E S C E N C E R E F L E C T A N C E BUFFERS OD 29 inserted i n front of the fluorescence photomultiplier tube was replaced with a 5% transmission f i l t e r (Leitz) to attenuate the l i g h t . The angle of ill u m i n a t i o n and r e f l e c t i o n of the paper was adjusted to simulate a b i o l o g i c a l preparation as nearly as possible. Fluorescence and reflectance were followed for 3 hours with less than 1% d r i f t i n the amplified voltage output i n either. c. Fluorescence emission spectra Fluorescence emission spectra were made from the l e f t cerebral cortex of paralyzed anesthetized ducks during normoxia and anoxia (Fig. 4). To make the fluorescence emission spectra the 450 nm secondary f i l t e r was replaced by a continuous i n t e r -ference f i l t e r ( V e r i l B-60), which was manually operated over a range of 428-507 nm. Since the photomultiplier s e n s i t i v i t y varied with wavelength and the interference f i l t e r did not transmit l i g h t equally over the spectral range of t h i s measure-ment, the fluorescence spectra i n Figs. 4 and 5 were corrected fo r these i n e q u a l i t i e s . The normoxic spectrum was taken when the duck was ven t i l a t e d with a i r and the anoxic spectrum was taken a f t e r death (defined as a blood pressure of 0 kPa) produced by nitrogen v e n t i l a t i o n . An increase i n c o r t i c a l fluorescence accompanied the t r a n s i t i o n from normoxia to anoxia without any s h i f t i n the fluorescence peak (467 nm). Af t e r death the cortex often s h i f t s or f a l l s away from the fluorometer when i t i s drained of blood (Jflbsis et a l . , 1971). To ensure that the difference 30 Figure 4 . Fluorescence emission spectra recorded from, the cerebral cortex of a duck during normoxia and anoxia (death). The fluorescence maximum f o r both spectra was 467 nm. The square at 4 6 7 nm and'lying between the two spectra was taken during extreme hypoxia before blood pressure f e l l s i g n i f i c a n t l y . FLUORESCENCE ARBITRARY UNITS ANOXIA / / / \ \ I / NORMOXIA / I + — 4 \ \ V \ \ 420 440 460 480 nm W A V E L E N G T H •\ 1 1 1 I 500 520 32 between the normoxic and anoxic (death) s p e c t r a was not due to an a r t i f a c t caused by a movement of the cortex, the fluorescence was a l s o measured during extreme hypoxia before blood pressure f e l l s i g n i f i c a n t l y . The square i n F i g . 4 at 467 nm and l y i n g between the two s p e c t r a represents the hypoxic measurement. I f a s h i f t of the cortex caused an a r t i f a c t i t could be no l a r g e r than the d i f f e r e n c e between the square and the anoxic spectrum at 467 nm. C l e a r l y , at l e a s t 90^ of the fluorescence increase at 467 nm i s not a r t i f a c t u a l . The time r e q u i r e d f o r the fluorescence measurements d i d not permit a f u l l spectrum during extreme hypoxia. Fluorescence s p e c t r a before and a f t e r a normoxic-anoxic t r a n s i t i o n i n the r a t were s i m i l a r to those of the duck, but the fluorescence maximum was between 4 6 4 and 4 6 7 nm. In order to confirm that NADH was res p o n s i b l e f o r the change i n fluorescence i n t e n s i t y during the normoxic-anoxic t r a n s i t i o n , the fluorescence emission spectrum i n a duck during -4 a n o x i a was compared to that of a s o l u t i o n of NADH (7.0 x 10 M, Sigma, S t . L o u i s , M i s s o u r i ) ( F i g . 5 ) . Fluorescence i n t e n s i t y was expressed as a percent of the peak i n t e n s i t y w i t h zero i n t e n s i t y a r b i t r a r i l y s e t at 4 2 8 nm. Peak fluorescence occurred at 467 nm and 470-473 nm f o r the anoxic b r a i n and NADH s o l u t i o n r e s p e c t i v e l y . Since fluorescence increased!.'during anoxia, a c o n d i t i o n when NADH i s known to increase, and fluorescence s p e c t r a from the anoxic cortex and pure NADH are s i m i l a r , I conclude t h a t the l a b i l e fluorescence s i g n a l from the c e r e b r a l 33 Figure $. Fluorescence emission s p e c t r a from anoxic c e r e b r a l cortex of a duck and a s o l u t i o n of NADH (7.0 x 10"^ M ) . Fluorescence i n t e n s i t y i s expressed as a percent of the peak i n t e n -s i t y w i t h 0 i n t e n s i t y a r b i t r a r i l y s e t at 428 nm. Fluorescence maxima were 467 nm >and 470-473 nm f o r the anoxic cortex and NADH r e s p e c t i v e l y . 1 0 0 T-8 0 + P E R C E N T O F 6 0 M A X I M U M FLUORESCENCE , 4 0 + 2 0 4 2 0 A N O X I C N A D H BRAIN I—I—I—I—I—I—I 4 4 0 4 6 0 4 8 0 5 0 0 nm W A V E L E N G T H 35 cortex i n ducks o r i g i n a t e s from NADH. Binding of the NADH to c e l l u l a r c o n s t i t u e n t s , mostly enzymes, i s probably r e s p o n s i b l e f o r the s h i f t of the fluorescence maximum to s h o r t e r wavelengths i n the b r a i n (Boyer and T h e o r e l l , 1956; Duysens and Amesz, 1957)• Chance et a l . (1962) reported a s i m i l a r s h i f t i n the fluorescence maximum i n anoxic kidney when compared to pure NADH. On the other hand Harbig et a l . (1976) and Sundt and Andersen (1975a) d i d not f i n d a s h i f t i n the fluorescence spectrum of the c e r e b r a l cortex when compared to NADH i n s o l u t i o n . The former authors a t t r i b u t e d t h e i r r e s u l t s to f i l t e r c haracter-i s t i c s which attenuated the s h o r t e r wavelengths of the'spectra i n t i s s u e and s h i f t e d the apparent fluorescence maximum towards t h a t of pure NADH.. d. S t a b i l i t y of the b i o l o g i c a l p r e p a r a t i o n S t a b i l i t y of the b i o l o g i c a l p r e p a r a t i o n was t e s t e d by monitoring f l u c t u a t i o n s of-'the fluorescence s i g n a l from the c e r e b r a l cortex of a r a t and a duck during steady s t a t e condi-t i o n s . Fluorescence was monitored during normoxia f o r 3 hours i n both species and a f t e r death f o r 1 hour i n a duck. F l u c t u a -t i o n i n c o r r e c t e d fluorescence was l e s s than —j>fo of the a m p l i f i e d voltage output per hour when the l i g h t i n t e n s i t y was 0.5 mW/cm . I f photodecomposition of NADH or other background m a t e r i a l occurred the fluorescence i n t e n s i t y would have shown a net decrease. Since fluorescence i n t e n s i t y d i d not show a net decrease, changes of. fluorochrome concentrations due to photo-decomposition was n e g l i g a b l e . 3 6 e. Blood a r t i f a c t compensation Ducks were prepared f o r f l u o r o m e t r i c recordings as p r e v i o u s l y described, and a polyethylene cannula (PE 9 0 ) was i n s e r t e d i n the c a r o t i d a r t e r y towards the b r a i n . Bolus i n -j e c t i o n s of oxygenated s a l i n e i n t o the c a r o t i d a r t e r y produced t r a n s i e n t d i l u t i o n s i n hemoglobin i n the r e c o r d i n g f i e l d and r e s u l t e d i n a t r a n s i e n t increase i n F and R ( F i g . 6 , .A).'. F l u c t u a t i o n s i n F and R were expressed as a percent increase ( p o s i t i v e ) or percent decrease (negative) i n l i g h t i n t e n s i t y . E l e c t r o n i c s u b t r a c t i o n of F-R (CF) compensated f o r changes i n hemoglobin i n the f i e l d when the change was s m a l l ; however, when the change was l a r g e CF increased s l i g h t l y . The same experimental p r o t o c o l was repeated w i t h bolus i n j e c t i o n s of concentrated red blood c e l l s (oxygenated) ( F i g . 6 , B). When the bolus reached the r e c o r d i n g area of the c e r e b r a l cortex both F and R decreased. However, as before, CE was only a f f e c t e d when the a r t i f a c t was l a r g e . In F i g . ? the r e l a t i o n s h i p between fluorescence and r e f l e c t a n c e was examined i n 4' ducks during f l u s h e s w i t h s a l i n e and concentrated blood. The s o l i d l i n e through the i n t e r c e p t of the a x i s represents a 1 to 1 correspondence between F and R and therefore f u l l compensation f o r the hemodynamic a r t i f a c t . P o i n t s to the r i g h t of the ordinate are from bolus i n j e c t i o n s of s a l i n e , and points to the l e f t are from bolus i n j e c t i o n s of concentrated blood. W i t h i n the range from approximately -15%> to +15% change i n F, the R channel f u l l y compensated f o r the hemodynamic a r t i f a c t , however, outside of t h i s range f u l l 37 Figure 6 . Changes i n fluorescence ( F ) , r e f l e c t a n c e (R) and c o r r e c t e d fluorescence (CF) recorded from the c e r e b r a l cortex of a duck when the b r a i n was f l u s h e d w i t h oxygenated s a l i n e (A) and concentrated blood (B). Each sp i k e represents one f l u s h . F, R, and CF are expressed as a percent increase ( p o s i t i v e ) or decrease (negative) i n the l i g h t i n t e n s i t y from the normoxic b a s e l i n e . The time s c a l e (bottom) a p p l i e s to both A and B. A B 10 % C H A N G E 0 -10 10 1 CHANGE 0 -10 CO 10 7o C H A N G E 0 -10 A J U . CF 1 MIN. 39 Figure 7 « The r e l a t i o n between r e f l e c t e d e x c i t a t i o n l i g h t (ordinate) and emitted fluorescenc l i g h t (abscissa) recorded from the cerebral cortex of ducks when the brain was flushed with oxygenated saline and concentrated blood (oxygenated). Both reflectance and fluorescence are expressed as a percent increase (positive) or decrease (negative) i n l i g h t i n t e n s i t y from the normoxic baseline. Points to the r i g h t of the ordinate are from saline flushes and points to the l e f t are from flushes of concentrated blood. The s o l i d l i n e through the intercept of the axis represents a 1 to 1 correspondence between reflectance and fluorescence. 41 compensation was questionable. However, the nature of the d e v i a t i o n suggests t h a t there were accompanying a l t e r a t i o n s i n the NADH flu o r e s c e n c e . When la r g e a r t i f a c t s were produced by s a l i n e f l u s h e s the fluorescence increase was greater than the r e f l e c t a n c e increase, suggesting a r e d u c t i o n of NAD which would be expected i f s a l i n e caused s l i g h t hypoxia. On the other hand, when l a r g e a r t i f a c t s were produced by bolus i n j e c t i o n s of concentrated oxygenated blood, fluorescence decrease was not as la r g e as the r e f l e c t a n c e decrease, suggesting o x i d a t i o n of NADH which would be expected i f the oxygenated blood caused s l i g h t hyperoxia. Consequently i t appears t h a t " t r u e " blood a r t i f a c t compensation cannot be assessed by t h i s method i n any absolute sense although w i t h i n l i m i t s i t i s c e r t a i n l y acceptable. f . Fluorescence during a c y c l e of hypoxia F i g . 8 shows a r e c o r d i n g of a r t e r i a l blood pressure and changes i n F, R, and CF recorded from the c e r e b r a l cortex of a duck during hypoxia produced by Ng v e n t i l a t i o n . O p t i c a l changes are expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was defined as 100 AU. Approximately 11 seconds a f t e r the t r a n s i t i o n from a i r to N 2» F increased. Hemodynamic a r t i f a c t s i d e n t i c a l to the r e f l e c t a n c e (R) change were superimposed on the fluorescence (F) t r a c e . E l e c t r o n i c s u b t r a c t i o n of F-R produced the CF tra c e which was due s o l e l y to NADH change. As hypoxia progressed c o r r e c t e d fluorescence increased to a maximum of 60 AU a f t e r 45 seconds. When the v e n t i l a t o r y gas was changed to a i r , CF recovered and I 4 2 Figure 8 . Blood pressure (BP) and changes i n fluorescence ( F ) , r e f l e c t a n c e (R), and c o r r e c t e d fluorescence (CF) recorded from the c e r e b r a l cortex of a duck during 3 5 seconds of n i t r o g e n v e n t i l a t i o n (as i n d i c a t e d by arrows). I n a l l o p t i c a l traces an upward d e f l e c t i o n of the t r a c e i n d i c a t e d an increase i n l i g h t i n t e n s i t y and a l l o p t i c a l t r a c e s are expressed i n a r b i t r a r y u n i t s (AU), where the CF change from normoxia to anoxia (death) was defined as 1 0 0 AU. BP i s expressed as k i l o p a s c a l s (kPa). BP 3 0 15 k P a 1 100 A U R 1 1 0 0 A U CF N 2 AIR 1 1 M I N . ] 1 0 0 A U 44 s t a b i l i z e d at the prehypoxic b a s e l i n e while increased blood i n the f i e l d caused F and R to overshoot the b a s e l i n e . A f t e r s e v e r a l minutes C and F g e n e r a l l y returned to t h e i r c o r r e s -ponding prehypoxia b a s e l i n e s . Eighteen periods of apneic asphyxia i n 6 ducks produced s i m i l a r responses to Ng v e n t i l a t i o n , w i t h the d i f f e r e n c e l y i n g mainly i n the time course of the o p t i c a l change. I n a l l 18 t r i a l s the r e f l e c t a n c e decreased, but d i d not exceed -15%> change from the ba s e l i n e i n any instance except one, which showed a -17% change. I n these cases f u l l compensation f o r the blood a r t i f a c t was assumed t o have been achieved. On occasions during asphyxia a f a l l i n blood pressure was mirrored by a CF increase and occurred whether blood pressure f e l l g r a d u a l l y ( F i g . 9 , A) or r a p i d l y ( F i g . 9 , B). Since the r e l a t i o n s h i p between blood pressure and CF was s t r i k i n g i n these instances I suspected t h a t the CF change was an a r t i f a c t . The e f f e c t s of blood pressure change on NADH fluorescence were i n v e s t i g a t e d during normoxia by decreasing the blood pressure r a p i d l y w i t h a c e t y l c h o l i n e (5-1° mg/kg, BDH Chemicals Ltd.) or s l o w l y w i t h an an t i h y p e r t e n s i v e agent (Diazoxide (Hyperstat) 10 mg/kg, Schering Corporation L t d . , Pointe C l a i r e , Quebec) and then r a p i d l y i n c r e a s i n g the blood pressure w i t h epinephrine (10 ug/kg, Parke-Davis Co. L t d . , B r o o c k v i l l e , Maryland). During normoxia a r a p i d f a l l i n blood pressure produced a t r a n s i e n t CF increase and t r a n s i e n t decrease i n b r a i n POg (recorded w i t h an oxygen electrode as described l a t e r i n t h i s chapter) while a slow decrease i n blood pressure over s e v e r a l minutes d i d not ^5 Figure 9- Corrected fluorescence (CF) recorded from the c e r e b r a l cortex i n ducks during apneic asphyxia when mean a r t e r i a l blood pressure (MABP) f e l l . I n A a gradual f a l l i n MABP was mirrored by a CF inc r e a s e . I n B the f a l l i n MABP was\more pronounced. MABP i s expressed i n k i l o p a s c a l s (kPa) and CF i s expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was defined as 100 AU. The time bar r e f e r s to both A and B. ^7 produce a change i n e i t h e r CF or t i s s u e P0 2. A r a p i d increase i n blood pressure, e i t h e r from- the normal l e v e l or hypotensive l e v e l ( a f t e r Diazoxide) a l s o produced no change i n CF or t i s s u e POg. The CF change during apnea does not appear to be an a r t i f a c t but r a t h e r a true NADH change due to changes i n the t i s s u e POg. A slow f a l l i n blood pressure probably allowed time f o r increases i n c e r e b r a l blood flow to maintain b r a i n POg whereas a r a p i d f a l l i n blood pressure was accompanied by a t r a n s i e n t drop i n P0 2 corresponding to the time i t took to a l t e r c e r e b r a l blood flow. During apneic asphyxia c e r e b r a l blood flow i n ducks increases (Jones et a l . , 1 9 7 5 ) at a time when ca r d i a c output f a l l s (Jones and Holeton, 4 9 7 2 ) , i n d i c a t i n g c e r e b r a l v a s o d i l a -t i o n . I f v a s o d i l a t i o n i s maximal or near maximal a f a l l i n blood pressure could not be accompanied by f u r t h e r reductions i n vasomotor tone. Consequently, a drop i n blood pressure would d i r e c t l y r e s u l t i n a drop i n CBF and thus a drop i n t i s s u e -P O 2 . J 48 Polarographic measurements of oxygen t e n s i o n  of the c o r t i c a l surface The importance of d i s s o l v e d oxygen i n l i f e processes has l e d to the development of a polarographic electrode to monitor oxygen t e n s i o n ( P 0 2 ) i n l i v i n g t i s s u e s (Davies and Brink , 1 9 4 2 ; C l a r k , 1 9 5 3 ) . The electrode c o n s i s t s of a platinum cathode which i s p o l a r i z e d at -O.65 v o l t s w i t h respect to a s i l v e r / s i l v e r c h l o r i d e anode. When the p o l a r i z e d electrode i s i n contact w i t h l i v i n g t i s s u e oxygen molecules from the t i s s u e are reduced at the cathode: 0 2 + 2 H 2 0 + 2 e " > . 'H 2 0 2 + 2 0 H " + 2e~ > 4 0 H ~ ( 5 ) The r e d u c t i o n s t a r t s a current flow from the cathode i n t o the t i s s u e which i s d i r e c t l y p r o p o r t i o n a l to the oxygen t e n s i o n . At the anode C l ~ from the t i s s u e f l u i d i s a t t r a c t e d by the p o s i t i v e p o l a r i z i n g voltage and used i n the o x i d a t i o n of s i l v e r : 4 A g + 4 C 1 ~ — > 4 A g C l + 4 e " ( 6 ) E l e c t r o n s r e l e a s e d at the anode flow through the e x t e r n a l c i r c u i t of the oxygen electrode ( F i g ; 1 0 ) and equal' the current flow from the cathode i n t o the t i s s u e s . The current which i s measured w i t h an ammeter i s ther e f o r e p r o p o r t i o n a l . t o the oxygen t e n s i o n of the t i s s u e . 49 Figure 1 0 . Diagram of an oxygen electrode and the e l e c t r o l y t i c d i s s o c i a t i o n at the anode and cathode. external c ircuit A g - A g C I Anode( + ) cr 4 A g+4Cr— * 4 A g C I 51 a. D e s c r i p t i o n of the electrode The electrode c o n s i s t e d of a 5 mm piece of platinum wire^25 urn i n diameter^fused i n l e a d glass t u b i n g , 1.5 cm i n — length and 5 mm i n diameter. The a c t i v e end of the platinum electrode was p o l i s h e d w i t h an o i l stone, and dipped i n Rhoplex AC 35 (Rohm and Hass, West H i l l , Ontario) to provide a covering to reduce p r o t e i n poisoning. A s i l v e r / s i l v e r c h l o r i d e reference e l e c t r o d e , 250 pa i n diameter, was placed d i r e c t l y on the b r a i n surface adjacent to the cathode. Before p h y s i o l o g i c a l measure-ments were made, each electr o d e was conditioned'by p l a c i n g i t i n 0.9% s a l i n e and a p p l y i n g - 0 . 8 V to the platinum cathode u n t i l the current s t a b i l i z e d (approximately 3° minutes). Each electrode was t e s t e d by measuring the current-.in the e l e c t r o d e c i r c u i t when the voltage between the anode and cathode was v a r i e d between 0 and 1 v o l t . E l e c t r o d e s t h a t have a l i n e a r response to oxygen co n c e n t r a t i o n show a pla t e a u of the current-voltage p l o t between 0.5 and 0.7 v o l t s . F i g . 11 shows an acceptable current-voltage p l o t (polarogram) f o r l i n e a r i t y of oxygen t e n s i o n between 0 and 2 0 . 0 kPa (150 t o r r ) . E l e c t r o d e output showed a r e s t i n g current of approximately 0.1-0.2 nA fo r n i t r o g e n saturated s a l i n e and approximately 5•0-6.0 nA f o r a P 0 2 of 2 0 kPa ( a i r s a t u r a t e d s a l i n e ) . The response time was 3 to 6 seconds f o r 90% of a f u l l response. Only electrodes t h a t f i t t e d the above c r i t e r i a were used f o r c o r t i c a l P 0 g measurements. 5 2 F i g u r e 1 1 . Polarogram of an acceptable Og e l e c t r o d e . A b b r e v i a t i o n : nA, nanoamps. CURRENT nA • 19-p 18--17--16--15--14--13 --12 --11 10 9 --8 --7 --6 --5 --4 3 + 2 1 + 0 5 4 b. Oxygen measurements from the c o r t i c a l surface of ducks during hypoxia and hypercapnia The e l e c t r o d e was t e s t e d by measuring oxygen t e n s i o n from the c e r e b r a l cortex (P ^ O g ) of a duck during hypercapnia ( F i g . 1 2 , B ) and d i f f e r e n t l e v e l s of hypoxia ( F i g . 1 2 , A ) . The electrode was c a l i b r a t e d i n a i r and n i t r o g e n s a t u r a t e d s a l i n e before and a f t e r the p h y s i o l o g i c a l measurements. When the v e n t i l a t o r y gas was changed from a i r to 9% oxygen, P T 0 2 decreased s h a r p l y and formed a pla t e a u ; when the duck was ven-t i l a t e d w i t h $% oxygen the PrpOg change was s i m i l a r only more pronounced. Hypercapnia ( F i g . 1 2 , B ) caused an increase i n PrpOg due presumably.to v a s o d i l a t i o n . For the purposes of t h i s study i t was necessary to express CF increase as a f u n c t i o n of PrpOg during hypoxia; however, the heterogeneity of oxygen t e n s i o n i n the b r a i n precludes the use of absolute P r p 0 2 f o r t h i s measurement. For example, oxygen tensions may vary from 0 kPa to over 9 . 0 kPa ( 6 8 t o r r ) depending on the geometric p o s i t i o n i n the c a p i l l a r y network ( S i l v e r , I 9 6 6 ; Lttbbers, 1 9 7 1 ; Smith, 1 9 7 7 ) and oxygen t e n s i o n can change as much as 9 . 0 kPa ( 6 8 t o r r ) i n a distance of l e s s than 0 , 5 I M (Ltibbers, I 9 7 I ) . The r e c o r d i n g f i e l d f o r the fluorometer was 3 . 5 mm or 2 . 3 mm i n diameter, depending on the o b j e c t i v e used, and fluorescence o r i g i n a t e d from an i n d e f i n i t e depth i n the t i s s u e which was something l e s s than 1 . 5 mm (JObsis et a l . , 1 9 7 1 ) . In order f o r P T 0 2 to adequately r e f l e c t CF, the average P T 0 2 f o r the volume of t i s s u e i n the fluorescence r e c o r d i n g would have to be determined. I f , 1 however, f o r a given l e v e l 55 F i g u r e 1 2 . P o l a r o g r a p h i c measurements of P^Og ^ r o m " t n e c o r t i c a l s u r f a c e of a duck when the v e n t i l a t o r y gas was changed from a i r to 9% oxygen and 5% oxygen (A). I n B, 7% COg was added to the v e n t i l a t o r y m i x t u re. The time bar r e f e r s t o both A and B. 5 6 < 57 of hypoxia the decrease i n P^Og i s p r o p o r t i o n a l throughout the brain,then P T 0 2 can be reported as a percent decrease from the normoxic c o n d i t i o n s , except, of course, where P^Og i s 0. A s e r i e s of experiments showed th a t the above assumption was v a l i d . P^02 was recorded from the c e r e b r a l cortex i n k ducks and from k- d i f f e r e n t r e c o r d i n g s i t e s i n a s i n g l e chicken when the oxygen i n the v e n t i l a t o r y gas was v a r i e d from 5% to 15%. PrpOg was expressed as a percent decrease of the electrode current where the decrease from normoxia to anoxia (death) was considered 100%. The r e s u l t s are d i s p l a y e d g r a p h i c a l l y i n F i g . 13. The 2 c o e f f i c i e n t s of c o r r e l a t i o n ( r ) f o r the l i n e a r regressions ranged from 0.87 to 1.0 except f o r 1 hypoxic regime i n the chicken which had an r of 0.65. An a n a l y s i s of covariance showed that the 8 l i n e a r r e g r e s s i o n l i n e s had a common slope and y - i n t e r c e p t w i t h a common equation of y = 99 - 3»9x. Not only was the assumption t h a t a r t e r i a l hypoxia produced a uniform decrease i n PrpOg throughout an i n d i v i d u a l v a l i d but i t al s o h e l d f o r d i f f e r e n t i n d i v i d u a l s . These r e s u l t s are c o n s i s t e n t w i t h the work of L e n i n g e r - F o l l e r t et a l . (1976) who showed that a r t e r i a l hypoxia produced a uniform decrease i n oxygen t e n s i o n recorded simultaneously from 8 d i f f e r e n t l o c a t i o n s i n the cere-b r a l cortex. 5 8 Figure 1J. Surface POg (PrpOg) recorded from' the r i g h t cerebral cortex when oxygen i n the v e n t i l a t o r y gas was varied from 1 5 $ to 5% i n 4 ducks ( s o l i d l i n e s ) and from k d i f f e r e n t recording s i t e s i n a single chicken (dotted l i n e s ) . Each l i n e represents a l i n e a r regression from a minimum of 1 2 points. PrpO-g was expressed as a'percent decrease of the electrode current when the decrease from normoxia to anoxia (death) was considered 100%. DUCKS CHICKEN °/o 0 2 IN VENTILATORY GAS 6 0 CHAPTER 3 Changes i n the Redox State of R e s p i r a t o r y Chain NAD  During Apneic Asphyxia i n Ducks I n t r o d u c t i o n D i v i n g mammals and "birds, are known to t o l e r a t e periods of apneic asphyxia that are de t r i m e n t a l to t e r r e s t r i a l animals (Andersen, 1 9 6 6 ) . The increased tolerance has been r e l a t e d to the refinement of oxygen conserving c a r d i o v a s c u l a r a d j u s t -ments (Scholander, 1 9 ^ 0 ) which are thought to p r o t e c t the heart and the b r a i n (Andersen, 1 9 6 6 ; B l i x , 1 9 7 6 ) . Although oxygen conservation i s w e l l documented (Scholander, 1 9 ^ 0 ; B u t l e r and Jones, 1 9 7 l ) » i"t has not been d i r e c t l y r e l a t e d to the biochemical events i n v o l v e d w i t h ATP production of the b r a i n . Since oxygen i s l i n k e d to ATP production v i a the r e s p i r a t o r y chain, the e f f e c t s of i t s conservation should be r e f l e c t e d i n the redox s t a t e of NAD. The purpose of t h i s chapter was to examine the redox change of r e s p i r a t o r y chain NAD from the c e r e b r a l cortex of ducks during apneic asphyxia and determine the e f f e c t s of the ca r d i o v a s c u l a r adjustments on the redox s t a t e . I n i t i a l e x p e r i -ments were performed on nonparalyzed ducks which were r e s t r a i n e d on a metal operating t a b l e . Although the head was held r i g i d , s t r u g g l e s which produced s l i g h t movements of the b r a i n caused a r t i f a c t s i n the o p t i c a l t r a c e s . I f i t was p o s s i b l e to s u b s t i -tute paralyzed ducks f o r nonparalyzed ducks i n these f l u o r o m e t r i c s t u d i e s , t h e n the movement a r t i f a c t s produced by the st r u g g l e s 61 could be eliminated. I compared cardiovascular responses and change i n c o r t i c a l NADH in paralyzed and nonparalyzed ducks to determine i f indeed this s u b s t i t u t i o n was possible. 62 Methods a. Fluorescence recordings from nonparalyzed ducks NADH fluorescence was monitored from the l e f t c e r e b r a l cortex of 9 ducks using glass windows as described i n Chapter 1. The b i r d s were secured v e n t r a l side down on a metal operating t a b l e and the head was held motionless w i t h the b i l l a t 4-5° below h o r i z o n t a l by 3 metal screws ( 3 cm lo n g ) , secured to the s k u l l w i t h d e n t a l cement, and b o l t e d to rods f i x e d to the metal t a b l e . I n 5 of the 9 ducks b r a i n e l e c t r i c a l a c t i v i t y was monitored b i p o l a r l y from the r i g h t f r o n t a l r e g i o n of the s k u l l . Ducks were subjected to periods of apneic asphyxia ( 4 to 7 minutes) produced by submerging the b i l l , nares, and eyes i n water. Each duck was exposed to 2 periods of submergence w i t h a 4 5 - 6 0 minute recovery p e r i o d between them. b. Fluorescence recordings from paralyzed ducks NADH fluorescence was monitored from the l e f t c e r e b r a l cortex of 28 paralyzed ducks us i n g p l a s t i c f i l m to cover the exposed cortex to prevent d r y i n g . Ducks were subjected to 2 or 3 periods of apneic asphyxia l a s t i n g from 2 to 9 minutes w i t h a 3 ° minute recovery p e r i o d between asphyxic p e r i o d s . A blood sample was withdrawn f o r blood gas a n a l y s i s before each asphyxic p e r i o d and v e n t i l a t i o n was adjusted to give s u i t a b l e blood gas values (Chapter 1 ) . A d d i t i o n a l blood samples were taken f o r blood gas a n a l y s i s at various times a f t e r v e n t i l a t i o n was stopped. 6 3 c. I n h i b i t i o n of the c a r d i o v a s c u l a r adjustments during apneic  asphyxia i n ducks E i g h t m a l l a r d ducks of e i t h e r sex, weighing 1 . 2 - 2 . 0 kg> were used i n t h i s s e r i e s of experiments and were prepared as described i n s e c t i o n b, except i n 2 ducks the window method was used f o r o p t i c a l recordings, and c o r t i c a l oxygen t e n s i o n was measured i n 4 ducks. PrpOg, where appropriate, and CF were measured during 2 minute periods of apneic asphyxia before and a f t e r the c a r d i o v a s c u l a r adjustments were i n h i b i t e d w i t h atropine s u l f a t e ( 2 . 5 mg/kg, BDH Chemicals L t d . , Poole, England). The e f f e c t s of atropine were t e s t e d before and a f t e r apneic asphyxia by i n j e c t i o n s of a c e t y l c h o l i n e c h l o r i d e ( 5 - 1 0 mg/kg, BDH). The hypotension and bradycardia normally produced by a c e t y l c h o l i n e i s abolished by atropine and the absence of hypotension was considered as the i n h i b i t i o n of c a r d i o v a s c u l a r adjustments. Each duck was exposed to 4 - 5 periods of apneic asphyxia, 2 - 3 pre-atropine and 2 p o s t - a t r o p i n e , w i t h a 3 0 minute recovery between each. For the purpose of expressing the o p t i c a l changes i n a q u a n t i t a t i v e manner, the fluorescence i n t e n s i t y during normoxia (baseline) was defined as zero and the fluorescence i n t e n s i t y f o l l o w i n g death by anoxia was defined as 1 0 0 a r b i t r a r y u n i t s (AU). O p t i c a l changes that correspond to i n t e n s i t i e s g r eater than the ba s e l i n e (NAD reduction) are designated w i t h a p o s i t i v e s i g n ; conversely, o p t i c a l changes that have i n t e n s i t i e s l e s s than the ba s e l i n e (NADH o x i d a t i o n ) are designated w i t h a • ~-_ 64 negative sign. Numerical values i n the figures are expressed as means —1 standard error of the mean (SEM) and the t- t e s t was used i n the s t a t i s t i c a l analysis of the data with 5 $ (P< 0 . 0 5 ) considered the acceptable l e v e l of s i g n i f i c a n c e . 65 Results a. Comparison of nonparalyzed and paralyzed ducks Heart r a t e , mean a r t e r i a l blood pressure (MABP), and corr e c t e d fluorescence were compared i n paralyzed and non-paralyzed ducks during the f i r s t 2 minutes of asphyxia and 4 0 seconds of recovery ( F i g . 1 4 ) . Asphyxia in"nonparalyzed ducks was produced by stopping a r t i f i c i a l v e n t i l a t i o n . Since the length of the asphyxic periods i n both paralyzed and nonparalyzed ducks v a r i e d , the recovery phase i n F i g . 14 s t a r t e d from' termina-t i o n of asphyxia. Mean pre-asphyxic heart r a t e f o r paralyzed and nonparalyzedcducks was 2 5 5 ~ 3 2 » 0 4 beats/minute ( 5 5 periods of asphyxia i n 20 ducks) and 247 ~ 1 3 ' ° 5 beats/minute ( 1 1 periods of asphyxia i n 6 ducks) r e s p e c t i v e l y . During the f i r s t minute of asphyxia mean heart r a t e f e l l to 4 4 $ and 2 3 $ of the c o n t r o l r a t e i n para l y z e d and nonparalyzed ducks r e s p e c t i v e l y and remained r e l a t i v e l y s t a b l e u n t i l t e r m i n a t i o n of asphyxia. Although mean heart r a t e i n nonparalyzed ducks was lower during asphyxia, only at 60 seconds was the d i f f e r e n c e between para-l y z e d and nonparalyzed ducks s i g n i f i c a n t . Nonparalyzed ducks c h a r a c t e r i s t i c a l l y showed a t a c h y c a r d i a almost immediately a f t e r emersion; heart r a t e increased from l e s s than 5 0 beats/ minute to 3 7 6 — 2 4 . 0 0 beats/minute ( 1 1 periods of asphyxia i n 6 ducks) a f t e r 10 seconds and g r a d u a l l y returned to pre-asphyxic l e v e l s during the next 40 seconds. When v e n t i l a t i o n was resumed i n paralyzed ducks, heart r a t e returned to the pre-asphyxic l e v e l a f t e r 90-120 seconds without a t a c h y c a r d i a . 66 Figure Ik. Comparison between heart r a t e (HR), mean a r t e r i a l "blood pressure (MABP), and co r r e c t e d fluorescence (CF) during 11 periods of apneic asphyxia i n 6 nonparalyzed ducks (dotted l i n e ) and 55 periods of apneic asphyxia i n 20 paralyzed ducks ( s o l i d l i n e ) . Asphyxia was produced "by submerging the face i n water and by stopping the a r t i f i c i a l v e n t i l a t i o n i n nonparalyzed and paralyzed ducks r e s p e c t i v e l y . Elapsed time (abscissa) d u r i n g asphyxia and the recovery i s given i n seconds. CF i s expressed i n a r b i t r a r y u n i t s (AU) where the CF changes from normoxia to anoxia (death) was defined as 100 AU. Each p o i n t represents the mean — SEM and the SEM i s contained w i t h i n the p o i n t when absent. HEART RATE BEATS/MIN. 400 350 300 250 200 150 + 100 50 0 J-NONPARALYZED PARALYZED-MABP kPa 25 20 15 10 5 0 r i » m CF AU 25 20 15 10 5 0 -5 1 1 1 1 h 10 20 30 40 50 60 70 ASPHYXIA H 1 1 1 h 80 90 100 110 120 10 20 RECOVERY TIME (SECONDS) 6 8 Before asphyxia MABP was 2 0 . 1 - 1 . 0 4 0 kPa ( 1 5 1 - 7 . 8 mm Hg) and 17.8 - 0 . 6 6 0 kPa ( 1 3 4 - 4 . 5 mm Hg) i n nonparalyzed and paralyzed ducks r e s p e c t i v e l y , and showed only minor changes during and a f t e r asphyxia. An increase i n CF occurred i n both paralyzed and non-paralyzed ducks during asphyxia. Moreover the rat e s of increase were s i m i l a r and n e a r l y constant between sampling i n t e r v a l s . A f t e r 1 2 0 seconds of asphyxia, CF increased by 1 5 - 1 . 9 4 AU (n = 5 5 periods of asphyxia i n 2 0 ducks) and 18.5 - 6 . 3 5 AU (n = 1 1 periods of asphyxia i n 6 ducks) i n paralyzed and non-paralyzed ducks r e s p e c t i v e l y . When asphyxia was terminated mean CF decreased and was below the base l i n e 4 0 seconds a f t e r asphyxia i n both groups. I n both paralyzed and nonparalyzed ducks, recovery of CF a f t e r asphyxia was always a s s o c i a t e d w i t h a t r a n s i t o r y overshoot of the ba s e l i n e which g e n e r a l l y returned to the >preasphyxic l e v e l a f t e r 5 - 1 ° minutes. A 4 . 7 minute p e r i o d of submergence asphyxia and the subsequent recovery i n a s i n g l e nonparalyzed duck i s snown i n F i g . 1 5 . Heart r a t e decreased r a p i d l y during the f i r s t 3 ° seconds and remained low f o r the d u r a t i o n of submergence. MABP was maintained near the predive l e v e l u n t i l 4 . 5 minutes a f t e r submersion when i t f e l l p r e c i p i t o u s l y . The t h i r d , f o u r t h and f i f t h traces from the top describe the o p t i c a l s i g n a l s . I n a l l cases an increase i n l i g h t detected by the p h o t o m u l t i p l i e r i s i n d i c a t e d by an upward d e f l e c t i o n of the pen. Note the change i n s c a l e i n the CF tra c e ( f i f t h t r a c e ) from the F and R trac e s ( t h i r d and f o u r t h traces r e s p e c t i v e l y ) . The abrupt 69 F i g u r e 1 5 . A 4 . 7 minute p e r i o d o f submergence a s p h y x i a and r e c o v e r y i n a n o n p a r a l y z e d , r e s t r a i n e d duck. The downward p o i n t i n g a rrow a t the bottom i n d i c a t e s s ubmersion and t h e upward p o i n t i n g arrow i n d i -c a t e s emersion. An upward d e f l e c t i o n o f the o p t i c a l t r a c e s r e p r e s e n t s an i n c r e a s e i n l i g h t i n t e n s i t y and o p t i c a l t r a c e s a r e e x p r e s s e d i n a r b i t r a r y u n i t s (AU) where the CF change from n o r m o x i a t o a n o x i a ( d e a t h ) was d e f i n e d as 1 0 0 AU. Time bars, 1 second and 1 minute r e s p e c t i v e l y , denote the changes i n c h a r t speed. Abbreviations« HR, h e a r t r a t e ; MABP, mean a r t e r i a l b l o o d p r e s s u r e ; kPa, k i l o p a s c a l s ; F, f l u o r e s c e n c e ; R, r e f l e c t a n c e ; CF, c o r r e c t e d f l u o r e s c e n c e ; EEG, e l e c t r o e n c e p h a l o g r a m ; uV, m i c r o v o l t s . 71 p o s i t i v e d e f l e c t i o n i n the P trace s h o r t l y a f t e r submersion and the large, slower, negative d e f l e c t i o n a f t e r emersion are a r t i f a c t s caused most probably by s l i g h t movement of the cortex. Note that these a r t i f a c t s are p a r a l l e l e d i n the R trace and that e l e c t r o n i c s u b t r a c t i o n of F-R gives the CF tra c e which i s f r e e of a r t i f a c t s and due s o l e l y to NADH. About 80 seconds a f t e r submersion,CF began to increase and g r a d u a l l y increased to 5 AU a f t e r 2 minutes. The trend continued u n t i l about 4.5 minutes a f t e r submersion when blood pressure dropped markedly; at t h i s p o i n t a s l i g h t l y sharper increase i n CF occurred. EEG a c t i v i t y diminished (bottom t r a c e ) when CF was approximately 32 AU. In ducks t h a t d i d not show a p r e c i p i t o u s drop i n blood pressure the EEG diminished more g r a d u a l l y ; however, i s o e l e c t r i c i t y s t i l l occurred when fluorescence had increased from between 28-40 AU i n a l l nonparalyzed ducks. On emersion heart r a t e rose above the predive r a t e w i t h i n s e v e r a l seconds and g r a d u a l l y returned to the predive l e v e l . Blood pressure a l s o increased. A few seconds a f t e r emersion c o r r e c t e d fluorescence decreased s h a r p l y and was f o l l o w e d by a gradual decrease over 3 minutes and even b r i e f l y dipped below the b a s e l i n e before r e t u r n i n g to the predive l e v e l . Some other ducks showed an immediate b a s e l i n e overshoot o m i t t i n g the slower o x i d a t i o n described here. F i g . 16 shows a 2 minute p e r i o d of apneic asphyxia pro-duced by stopping a r t i f i c i a l v e n t i l a t i o n i n a paralyzed duck, and the v a r i a b l e s measured appear to show the same trends as i n nonparalyzed^ animals^ 72 Figure 16. Response i n a paralyzed duck to a 2 minute p e r i o d of apneic asphyxia produced by stopping a r t i f i c i a l v e n t i l a t i o n . Fluorescence ( F ) , r e f l e c t a n c e (R), and c o r r e c t e d fluorescence (CF) were recorded from the l e f t c e r e b r a l cortex and are expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was defined as 100 AU. The p e r i o d of asphyxia i s i n d i c a t e d by the 2 arrows. Other a b b r e v i a t i o n s : HR, heart r a t e ; MABP, mean a r t e r i a l blood pressure; kPa, k i l o -p a s c a l s . 7^ b. I n h i b i t i o n of the c a r d i o v a s c u l a r adjustments during apneic  asphyxia i n ducks Ei g h t ducks were exposed to 2 minute periods of apneic asphyxia before and a f t e r the c a r d i o v a s c u l a r adjustments to apneic asphyxia were a b o l i s h e d . Over of the hypertension and bradycardia produced by the i n j e c t i o n of a c e t y l c h o l i n e was abolished by a t r o p i n e . Table I shows the CF increase i n 8 ducks a f t e r 1 and 2 minutes of apneic asphyxia before and a f t e r atropine treatment. In every duck CF was greater a f t e r 1 2 0 seconds of asphyxia f o l l o w i n g treatment w i t h atropine than i n asphyxia before atropine treatment and the d i f f e r e n c e was s i g n i f i c a n t . Note t h a t CF was s i m i l a r i n the normal ( 8 - 2 . 2 9 AU, n = 8 ) and a t r o p i n i z e d ( 1 2 - 2 . 9 0 AU, n = 8 ) ducks a f t e r 1 minute of asphyxia and only i n the second minute were the e f f e c t s of c a r d i o v a s c u l a r adjustments i n m a i n t a i n i n g a more o x i d i z e d r e s p i r a t o r y chain ;apparent. F i g . 1 7 shows P T 0 2 and CF traces during apneic asphyxia before (C) and a f t e r (A) an atropine i n j e c t i o n . A f t e r 2 minutes of asphyxia PrpOg decreased by 6 1 $ and CF increased 2 5 AU before an a t r o p i n e i n j e c t i o n while a f t e r atropine, PrpOg decreased by 7 2 $ and CF increased 4 4 AU i n the same time p e r i o d . 75 Table I . Increase i n co r r e c t e d fluorescence (CF) i n paral y z e d ducks a f t e r 60 and 120 seconds of apneic asphyxia before and a f t e r atropine t r e a t -ment. The numbers i n parentheses are the numbers of asphyxic p e r i o d s . Mean — SEM AU are"at the bottom of the t a b l e . 76 Apneic Asphyxia Before Atropine Treatment Corrected Fluorescence Duck Number 1 2 3 4 5 6 7 60 sec 8(2)* 3(1) 5(3) 4(2) 8(3) 18(3) 19(2) 3(2) 120 sec 39(2) 6(1) 23(3) 19(2) 21(3) 23(3) 3 K 2 ) 29(2) Apneic Asphyxia A f t e r Atropine Treatment Corrected Fluorescence 60 sec 8(2) 1(2) 11(2) 10(2) 8(2) 23(2) 25(2) 7(2) 120 sec 47(2) 16(2) 44(2) 36(2) 43(2) 50(2) 45(2) 32(2) Mean - SEM 1 2 .29 23 ~ 3-39 12 - 2 .90 39 - 3 .89 The numbers i n parentheses are the number of periods of apneic asphyxia. 77 Figure 1 7 . POg of the r i g h t c o r t i c a l surface (Pr^Og) and corr e c t e d fluorescence (CF) from the l e f t c o r t i c a l surface during 2 minutes of apneic asphyxia ( i n d i c a t e d by arrows) i n a paralyzed duck before (C) and a f t e r an atropine i n j e c t i o n ( A ) . Prp 0£ i s expressed as a percent decrease of the e l e c -trode current when the decrease from normoxia to anoxia (death) was defined as a 1 0 0 $ decrease. CF was expressed i n a r b i t r a r y u n i t s (AU) when the CF change from normoxia to anoxia (death) was defined as 1 0 0 A U . 79 D i s c u s s i o n The apneic asphyxia produced i n paralyzed ducks "by stopping u n i d i r e c t i o n a l v e n t i l a t i o n simulates the onset of ca r d i o v a s c u l a r adjustments and changes i n r e s p i r a t o r y chain NADH i n nonparalyzed ducks duri n g f o r c e d submersion. Although bradycardia during asphyxia i n paralyzed ducks was not as severe as i n nonparalyzed ducks, there were no s i g n i f i c a n t d i f f e r e n c e s between the 2 groups except at the 6 0 second sampling i n t e r v a l . The major d i f f e r e n c e was found i n the recovery p e r i o d where the paralyzed group lacked the t y p i c a l postasph'yxic.tachycardia; . i n s t e a d heart r a t e g r a d u a l l y returned to the pre-asphyxic r a t e over a p e r i o d of 9 0 - 1 2 0 seconds. Bamford and Jones ( 1 9 7 4 ) reported s i m i l a r r e s u l t s f o r paralyzed and nonparalyzed ducks and l a t e r claimed i t i s caused by the absence of n e u r a l input from the lungs s i n c e u n i d i r e c t i o n a l v e n t i l a t i o n i n paralyzed animals does not cause an increase i n lung pressure during postasphyxic v e n t i l a t i o n (Bamford and Jones, 1 9 7 6 ) . NADH fluorescence i n both groups g r a d u a l l y increased d u r i n g the f i r s t 2 minutes of asphyxia and returned to the ba s e l i n e a f t e r a t r a n s i t o r y overshoot when v e n t i l a t i o n was resumed. The NADH fluorescence was almost i d e n t i c a l i n both groups during the e n t i r e cycle." Only 1 0 seconds a f t e r the ter m i n a t i o n of asphyxia d i d NADH fluorescence d i f f e r s i g n i f i -c a n t l y due to a f a s t e r recovery r a t e i n the nonparalyzed animals'. The redox s t a t e of r e s p i r a t o r y chain components .has been s t u d i e d i n the b r a i n during hypoxia and anoxia which were produced by v a r y i n g oxygen i n the v e n t i l a t o r y gas from 0 - 2 0 $ 80 (Chance et a l . , 1962, 1964, 1973; Chance and Schoener, 1962), and by reducing or stopping blood flow (ischemia) (Sundt and Andersen, 1975h; Sundt et a l . , 1976; Rosenthal et a l . , 1976b; LaManna et a l . , 1977; Ginsberg et a l . , 1976); but s t u d i e s d u r i n g apneic asphyxia have never been reported before t h i s study. A l l three means of producing a f a l l i n t i s s u e oxygen t e n s i o n share the same type of response, namely a r e d u c t i o n of the r e s p i r a t o r y chain components; however, the k i n e t i c s of the r e d u c t i o n are d i f f e r e n t due to d i f f e r e n t r a t e s of oxygen d e p l e t i o n . At the onset of hypoxia, reducing equivalents from the substrate are passed i n t o the r e s p i r a t o r y chain f a s t e r than they are removed by oxygen. The reduced form of the c a r r i e r s increases at the expense'-o"f-the oxidized-form ' (NADH and NAD i n the case of the present study) and seek.a more reduced redox s t a t e . I n apneic asphyxia oxygen i s c o n t i n u a l l y depleted and r e d u c t i o n approaches the maximum obtainable at death unless v e n t i l a t i o n i s resumed. The importance of the c a r d i o v a s c u l a r adjustments f o r p r o t e c t i n g the b r a i n during apneic asphyxia was demonstrated by monitoring NADH fluorescence and b r a i n PrpOg during apneic asphyxia i n ducks before and a f t e r the c a r d i o v a s c u l a r a d j u s t -ments were i n h i b i t e d w i t h a t r o p i n e . Apneic asphyxia f o l l o w i n g i n h i b i t i o n 1 o f the c a r d i o v a s c u l a r adjustments produced a gr e a t e r r e d u c t i o n of NAD and a greater decrease i n b r a i n PjpOg than before the i n h i b i t i o n . However, the e f f e c t s of the c a r d i o -v a s c u l a r adjustments on CF and P T 0 2 were not r e a d i l y apparent during the f i r s t minute of asphyxia (Table I and F i g . 17). 81 For example, a f t e r 1 minute of asphyxia, CF was 12 AU and 8 AU i n a t r o p i n i z e d and non-atropinized ducks r e s p e c t i v e l y . I t was expected t h a t e f f e c t s of the c a r d i o v a s c u l a r adjustments should a l s o be apparent during the f i r s t minute of asphyxia. S e v e r a l f a c t o r s w i l l shed some l i g h t on t h i s observation. F i r s t , maximum c a r d i o v a s c u l a r adjustments i n non- a t r o p i n i z e d ducks (as i n d i c a t e d from heart r a t e ) , and thus oxygen conser-v a t i o n , were not complete u n t i l 4 0 - 6 0 seconds a f t e r a r t i f i c i a l v e n t i l a t i o n was stopped ( F i g . 1 4 ) . Only i n the second minute, when oxygen conservation i s maximal, are l a r g e d i f f e r e n c e s i n PrpOg between the periods of asphyxia before and a f t e r atropine i n j e c t i o n s r e a d i l y apparent. Second, the r e l a t i o n s h i p between NADH fluorescence and Prp02 was such t h a t very l i t t l e change i n NADH fluorescence occurred u n t i l PrpOg h a s decreased by 5 ° $ ( F i g . 22, Chapter 4 ) . A f t e r P T Q 2 has decreased by 5 0 $ s m a l l changes i n t i s s u e oxygenation caused l a r g e increases i n CF. Since P T 0 2 d i d not f a l l below 5 0 $ i n e i t h e r the a t r o p i n i z e d or non-atropinized animals during the f i r s t minute of asphyxia, l a r g e d i f f e r e n c e i n CF would not be expected. 82 CHAPTER 4-C e r e b r a l Energy Metabolism i n Ducks and Chickens  During Apneic Asphyxia and Hypoxia I n t r o d u c t i o n The primary means of ATP production i n the b r a i n ( o x i -d a t i v e phosphorylation) r e q u i r e s oxygen to accept reducing equivalents from the r e s p i r a t o r y c h ain and b r a i n f u n c t i o n can continue only as long as oxygen i s a v a i l a b l e . However, n a t u r a l d i v e r s can maintain b r a i n f u n c t i o n over 4- times longer during dives than t h e i r t e r r e s t r i a l counterparts before b r a i n f u n c t i o n ceases (Kerem and E i s n e r , 1973a). I n Chapter 3 I showed that p h y s i o l o g i c a l adjustments which conserved oxygen during asphyxia were r e s p o n s i b l e f o r prolonging both t i s s u e oxygenation and re d u c t i o n of r e s p i r a t o r y chain NAD. Since the b r a i n i s o b l i -g a t e l y dependent on oxygen, i t f o l l o w s that b r a i n f u n c t i o n i s prolonged d i r e c t l y as a r e s u l t of these p h y s i o l o g i c a l a d j u s t -ments. However, n a t u r a l d i v e r s may t o l e r a t e more severe hypoxia than t e r r e s t r i a l animals ( E i s n e r et a l . , 1970; Kerem and E i s n e r , 1973 a and b; Ridgeway et a l , , 1969) i n d i c a t i n g t h a t i n addi-t i o n to p h y s i o l o g i c a l adjustments some form of biochemical adaptation i s present i n d i v e r s . I n theory, these could be adaptations which would (1) a l l o w o x i d a t i v e phosphorylation to continue at l e v e l s of hypoxia t h a t cannot be t o l e r a t e d by nondivers and/or (2) enhance anaerobic ATP production to supplement o x i d a t i v e phosphorylation i n the face of decreasing oxygen. 83 The purpose of t h i s chapter was t o , f i r s t , t e s t the hypo-t h e s i s t h a t the r e d u c t i o n of r e s p i r a t o r y chain NAD proceeds at a slower r a t e during apneic asphyxia i n ducks, Anas P l a t y - rhynchos, than chickens, G a l l u s domesticus. Ducks are known to t o l e r a t e apneic asphyxia 3-8 times longer than chickens (Andersen, 1959 and I 9 6 6 ; Scholander, 1964) . Second, I i n v e s t i -gated the r e l a t i o n between the redox s t a t e of r e s p i r a t o r y chain NAD and EEG i n ducks and chickens d u r i n g apneic asphyxia to e l u c i d a t e any major c o n t r i b u t i o n to ATP production by anaero-b i c metabolism i n ducks. I n v i v o f l u o r o m e t r i c recordings i n the anesthetized r a t have i d e n t i f i e d the c r i t i c a l p y r i d i n e n u c l e o t i d e r e d u c t i o n (CPNR) above which b r a i n e l e c t r i c a l a c t i -v i t y ceases (Chance and Schoener, 1962; Mayevsky and Chance, 1973)- During Ng v e n t i l a t i o n the CPNR occurs when the fluorescence increase from the normoxic l e v e l i s approximately 3/4 of the l e v e l recorded at death. I f ducks r e l y on l a r g e anaerobic c o n t r i b u t i o n s of ATP f o r the c o n t i n u a t i o n of b r a i n function, they should have a .more reduced r e s p i r a t o r y c h ain than the chicken before the appearance of gross a l t e r a t i o n s i n EEG. T h i r d , to t e s t f o r biochemical adaptations i n v o l v i n g o x i d a t i v e phosphorylation the redox s t a t e of r e s p i r a t o r y chain NAD of ducks was compared to chickens at various l e v e l s of hypoxia. I f biochemical adaptations are present i n ducks, then the accumulation of r e s p i r a t o r y chain NADH f o r a given l e v e l of hypoxia should be l e s s i n ducks than chickens. 84 Methods a. Fluorescence recordings from'.paralyzed ducks and chickens NADH fluorescence was monitored from the l e f t c e r e b r a l cortex of 2 0 paralyzed chickens and 3 ° paralyzed ducks. P l a s t i c f i l m was: placed over the exposed cortex to prevent d r y i n g . Chickens and ducks were subjected to 2 or 3 periods of apneic asphyxia l a s t i n g 1 minute and from 2 to 9 minutes r e s p e c t i v e l y . Asphyxic periods were separated by a 2 0 - 6 0 minute recovery p e r i o d ; the longest recovery periods f o l l o w e d the longest periods of asphyxia. I n 1 3 ducks and 1 1 chickens EEG was recorded and the asphyxic p e r i o d was continued' u n t i l b r a i n e l e c t r i c a l a c t i v i t y ceased ( r e f e r r e d to i n t h i s paper as EEG endpoint). I n these experiments each animal was exposed to only one asphyxic t r i a l . A blood sample was withdrawn f o r blood gas a n a l y s i s before each asphyxic p e r i o d and v e n t i l a t i o n was adjusted to give normal blood gas values (Chapter 1 ) . b. Concurrent f l u o r o m e t r i c and polarographic recordings  during hypoxia NADH fluorescence was recorded from the l e f t c e r e b r a l cortex of 1 3 ducks and 9 chickens as described i n s e c t i o n a. PrpOg was recorded from the area of the r i g h t c e r e b r a l cortex t h a t corresponded to the area used f o r o p t i c a l recordings on the opposite hemisphere. Both species were exposed to s e v e r a l l e v e l s of normocapnic hypoxia ( 1 minute i n duration) by v a r y i n g the oxygen i n the v e n t i l a t o r y gas from 2 - 2 0 $ i n a balance of ni t r o g e n . The gases were mixed w i t h flowmeters complete w i t h 85 screw valves and analyzed f o r percent oxygen using a Beckman Model F3 oxygen analyzer (Beckman Instruments, Inc., F u l l e r t o n , C a l i f o r n i a ) . I n a d d i t i o n , progressive hypercapnic hypoxia was induced i n ducks "by stopping a r t i f i c i a l v e n t i l a t i o n f o r 2 - 4 minutes. 8 6 r Results a. Comparison of ducks and chickens Heart r a t e , MABP, and CF recorded from ducks ( s o l i d l i n e ) and chickens (dotted l i n e ) during apneic asphyxia are shown i n F i g . 18. Mean preasphyxic' heart r a t e f o r 1 9 chickens and 2 0 ducks was 1 5 4 - 9 . 2 9 and 2 5 5 - 3 2 . 0 4 beats'minute - 1 r e s p e c t i v e l y . A f t e r a 1 5 second l a t e n t p e r i o d from the time a r t i f i c i a l v e n t i l a t i o n was stopped mean heart r a t e i n chickens f e l l s t e a d i l y > u n t i l apnea was terminated a t 6 0 seconds. In c o n t r a s t , ducks showed a decrease i n mean heart r a t e during the f i r s t 1 5 seconds which corresponded to 42$ of the t o t a l b r adycardia. Although t h i s r a t e of decrease was not p a r a l l e l e d between any other subsequent sampling periods, the heart r a t e , n e v e r t h e l e s s , s t e a d i l y decreased to 3 5 $ at 9 0 seconds w i t h 88$ of the bradycardia o c c u r r i n g d u r i n g the f i r s t minute. At 6 0 seconds heart r a t e i n chickens and ducks was 5 5 $ and 4 4 $ of the preasphyxic; r a t e r e s p e c t i v e l y . When a r t i f i c i a l v e n t i l a t i o n was resumed, mean heart r a t e i n both species returned to the preasphyxic. r a t e a f t e r 6 0 - 9 0 seconds without the post-asphyxic t a c h y c a r d i a - c h a r a c t e r i s t i c of nonparalyzed birds/. MABP d i d not change s i g n i f i c a n t l y i n e i t h e r species during asphyxia or the subsequent recovery. Asphyxia i n both species was c h a r a c t e r i z e d by an increase i n NADH which continued at a n e a r l y l i n e a r r a t e u n t i l a r t i f i -c i a l v e n t i l a t i o n was resumed. Although r e d u c t i o n was i n e v i t a b l e d u r i n g apnea s i n c e oxygen was d i m i n i s h i n g , ducks showed b e t t e r c o n t r o l at r e g u l a t i n g redox balance by decreasing the r a t e of 87 Figure 18. Heart r a t e (HR), mean a r t e r i a l blood pressure (MABP), and co r r e c t e d fluorescence (CF) during 1 and 2 minute periods of apneic asphyxia i n paralyzed chickens ( 5 4 periods of asphyxia i n 1 9 chickens) and ducks ( 5 5 periods of asphyxia i n 2 0 ducks) r e s p e c t i v e l y . The dotted l i n e s represent chickens and the s o l i d l i n e s represent ducks. The arrows i n d i c a t e the asphyxic p e r i o d . CF i s expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was defined as 1 0 0 AU. Each p o i n t represents the mean - SEM and the SEM i s contained w i t h i n the p o i n t when absent. -15 -L | | 1 | | 1 | | | I I 1 I I I 1 1 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 SECONDS TIME 8 9 r e d u c t i o n to 1 / 5 of that i n chickens. For example, CF was s i g n i f i c a n t l y lower a f t e r 1 minute of apnea i n ducks ( 8 — 1.41 AU) than chickens ( 3 7 - 3-60 AU). Furthermore, i f the r a t e of r e d u c t i o n continued.unchanged i n ducks from the f i r s t 2 minutes, then i t would take over 4 . 5 minutes of apnea before CF equalled the CF i n chickens at 60 seconds. When v e n t i l a t i o n was resumed CF returned to the b a s e l i n e i n both species a f t e r a t r a n s i e n t overshoot, u s u a l l y of 5-1° minutes d u r a t i o n . A f t e r 6 0 seconds of apnea i n chickens Pa0 2 was 5 . 8 — O . 3 6 6 kPa ( 4 4 - 2 . 5 7 t o r r ) and a f t e r 120 seconds of apnea i n ducks Pa0 2 was 5.6 - G . 5 5 I kPa ( 3 8 - 4.14 t o r r ) . I t i s i n t e r e s t i n g to note that when mean Pa0 2's were very s i m i l a r mean CF increase i n chickens was over twice that i n ducks ( 3 7 AU versus 1 5 AU). To c l a r i f y the enigma 2-4 blood samples were withdrawn from the femoral a r t e r y at random i n t e r v a l s a f t e r a r t i f i c i a l v e n t i l a t i o n was stopped i n 8 ducks and 6 chickens, and the r e l a t i o n s h i p between CF increase and Pa0 2 was examined f o r the best f i t to e i t h e r a s t r a i g h t l i n e , an exponential curve, a power curve, or a l o g a r i t h m i c curve. C o r r e l a t i o n was poor i n a l l cases w i t h a l i n e a r r e g r e s s i o n having the l a r g e s t p c o e f f i c i e n t s of c o r r e l a t i o n ( r ), 0 . 2 4 and O . 3 7 i n chickens and ducks r e s p e c t i v e l y . Therefore, Pa0 2 d i d not a c c u r a t e l y r e f l e c t redox s t a t e of the r e s p i r a t o r y chain. PaC0 2 of the various blood samples ranged from 4 . 6 - 7 . 3 kPa ( 3 5 - 5 5 mm Hg). 9 0 b. C r i t i c a l p y r i d i n e n u c l e o t i d e r e d u c t i o n (CPNR) i n chickens and ducks EEG and CF were recorded simultaneously during prolonged apneic asphyxia i n chickens and ducks. I n 1 1 chickens and 1 0 ducks CF was 3 4 - 2 . 1 9 AU and 3 8 - I . 9 0 AU r e s p e c t i v e l y when the EEG endpoint was reached (Tables I I and I I I ) . CF was not s i g n i f i c a n t l y d i f f e r e n t at the EEG endpoint; however, the time from the beginning of apnea to the endpoint was over 5 - f o l d longer i n ducks than chickens. Chickens maintained b r a i n e l e c t r i c a l a c t i v i t y d u r i n g apnea f o r 6 3 - 4 . 3 8 seconds (range 4 2 - 9 3 seconds) and ducks f o r 3 3 8 - 3 2 . 3 6 seconds (range 2 3 2 -5 4 9 seconds). F i g . 1 9 shows r e s u l t s from 2 i n d i v i d u a l s ; the top 2 traces (EEG and CF) are from a duck and the bottom 2 traces (EEG and CF) are from a'chicken. I n both cases CF increased when the a r t i f i c i a l v e n t i l a t i o n was stopped ( f i r s t arrow) and continued u n t i l the EEG endpoint was reached at which time the v e n t i l a t i o n was resumed (second arrow) and CF returned to the b a s e l i n e . Although the EEG endpoint was reached sooner i n chickens, CF at the endpoints was s i m i l a r , 3 8 AU and 3 2 AU i n the chicken and duck r e s p e c t i v e l y . I n ducks i t appeared that those which showed the gr e a t e s t bradycardia maintained EEG f o r longer periods of apnea than those i n which bradycardia was not so pronounced ( T a b l e ' I l ) . Although bradycardia i s only a crude measure of c a r d i o v a s c u l a r adjustments, s i n c e i t does not account f o r p e r i p h e r a l v a s o c o n s t r i c t i o n , i t nevertheless c o r r e l a t e s w i t h d i v i n g performance. Considering the data from Table I I ,the time t o l e r a t e d during asphyxia before the EEG — 91 Table I I . Heart r a t e , time to EEG endpoint, and CF at EEG endpoint i n paralyzed ducks during apneic asphyxia. Mean - SEM AU at bottom of t a b l e . Time (seconds) during apneic asphyxia Time at EEG 0 15 30 60 120 180 240 300 36O 420 480 540 endpoint CF at EEG endpoint Ducks Heart rate (beats •minute' - 1 ) 1 305 208 171 73 49 37 39 2 293 273 220 208 104 85 81 3 141 102 90 115 119 4 240 200 150 125 112 109 121 5 281 203 172 142 125 108 107 7 175 145 122 119 121 125 136 8 270 175 140 130 91 84 95 10 301 209 185 156 130 121 135 11 270 161 135 112 92 81 79 12 261 149 92 42 37 42 35 Mean - SEM 254 ± 182 i - 149 1 119 +~ 98 ± 91 1 92 17.26 14.83 12.39 14.49 10.00 9.97 12 (n=10) (n=10) (n=10) (n=10) (n=10) (n=10) (n= 42 35 49 431 263 232 253 35 28 32 31 142 311 247 41 38 93 87 367 31 129 313 38 85 77 415 34 39 41 47 49 65 549 45 88 -17.46 (n=6) 338 i 32.26 (n=10) 38 i 1.90 (n=10) 93 Table I I I . Heart r a t e , time to EEG endpoint, and CF at EEG endpoint i n paralyzed chickens during apneic asphyxia. Mean - SEM AU at the bottom of t a b l e . Time (seconds) during apneic asphyxia 0 1 5 3 0 6 0 9 0 Time at CF at E E G E E G endpoint endpoint Chickens Heart rate (beats'minute" ) 1 114 9 6 114 48 62 2 7 2 163 158 118 42 3 2 3 202 1 9 6 184 147 9 8 93 3 6 4 204 186 I 6 3 120 77 37 5 1 9 5 1 9 5 108 94 • 75 41 6 108 1 0 7 95 56 5 0 7 1 4 0 140 1 2 5 5 1 2 5 8 1 9 6 1 9 6 1 7 2 4 9 4 0 9 201 1 9 5 142 126 61 3 0 1 0 138 1 5 1 88 57 31 1 1 147 147 1 2 6 124 64 3 0 - S E M 164 - 160 - 1 3 0 - 110 - 62 ± 34 ± 11.11 1 0 . 9 8 9.45 14 . 1 7 4 . 3 8 2 . 1 9 (n=ll) (n=ll) (n=ll) (n=6) (n=ll) (n=l lK 9 5 Figure 19• Electroencephalogram (EEG) and corrected fluorescence (CF) recorded from a duck (upper 2 traces) and a chicken (bottom 2 traces) during apneic asphyxia. The downward pointing arrow f o r each p a i r of traces indicates the beginning of asphyxia and the upward pointing arrow i n d i -cates the end of asphyxia. The time bar applies to a l l four traces. EEG i s expressed i n micro-vol t s (uV) and CF i s expressed i n a r b i t r a r y units (AU) where the CF change from normoxia to anoxia (death) represents 100 AU. DUCKS CHICKENS E E G C F E E G C F J lOOuV 50 A U J 100 pV J 50 A U 97 endpoint shows a c o r r e l a t i o n w i t h the maximum decrease i n he a r t r a t e (expressed as a perc e n t of the pre-asphyxic r a t e ) ( F i g . 20). A r e l a t i o n between degree of b r a d y c a r d i a and time to the EEG endpoint was not observed i n chickens (Table I I I ) . c. R e l a t i o n s h i p between f l u o r e s c e n c e and PrpOo i n ducks and  chickens d u r i n g hypoxia NADH f l u o r e s c e n c e and PrpOg were measured s i m u l t a n e o u s l y from opposite c e r e b r a l hemispheres i n ducks and chickens d u r i n g steady s t a t e hypoxia produced by v a r y i n g the oxygen i n the v e n t i l a t o r y gas. As t i s s u e PrnOg f e l l , CF i n c r e a s e d , and the more severe the hypoxia the g r e a t e r the CF i n c r e a s e . When the duck was r e t u r n e d to 20$ oxygen, PrpOg and CF even-t u a l l y r e t u r n e d to normal. F i g u r e 21 shows the r e s u l t s from a s i n g l e duck. The are a between the arrows r e p r e s e n t s the pe r i o d s when v a r i o u s l e v e l s of hypoxia were a p p l i e d . P I J°2 i s expressed as a percent decrease of the e l e c t r o d e c u r r e n t when ;the decrease from normoxia to anoxia (death) was d e f i n e d as a 100$ decrease; CF was expressed i n AU's as p r e v i o u s l y d e s c r i b e d . Proceeding from a to d, hypoxia became more severe as i n d i c a t e d by the decrease i n Prn 0 ^* Experiments performed on chickens (not shown) showed a s i m i l a r r e l a t i o n s h i p between P T 0 2 and CF. When NADH f l u o r e s c e n c e was p l o t t e d as a f u n c t i o n of the decrease i n PjpOg (Fig« 22) an e x p o n e n t i a l curve b e s t d e s c r i b e d the r e l a t i o n s h i p i n 8 chickens ( l i g h t c i r c l e s ) and 9 ducks (dark c i r c l e s ) . S ince the EEG endpoint was reached before 98 Figure 2 0 . Length of time during apneic asphyxia i n paralyzed ducks before the cessation of brain e l e c t r i c a l a c t i v i t y i n r e l a t i o n to the decrease i n heart rate (expressed as a percent of the preasphyxic heart r a t e ) . Each point represents a single period of apneic asphyxia. Values are taken from Table I I . 100 F i g u r e 21. Surface POg from the r i g h t c e r e b r a l c o r t e x (PrpOg) and c o r r e c t e d f l u o r e s c e n c e (CF) r e c o r d e d from the l e f t c e r e b r a l c o r t e x i n p a r a l y z e d ducks d u r i n g steady s t a t e hypoxia ( i n d i c a t e d by arrows) produced by v a r y i n g oxygen i n the v e n t i l a t o r y gas. Proceeding from a to d hypoxia became more se v e r e . PfpOg i s expressed as a percent decrease of the e l e c t r o d e c u r r e n t when the decrease .from normoxia to anoxia (death) was d e f i n e d as a 100$ decrease. CF was expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was d e f i n e d as 100 AU. 102 Figure 2 2 . Corrected fluorescence (CF) i n r e l a t i o n to P 0 2 recorded from c o r t i c a l surface (P^Og) i n 8 chickens ( l i g h t c i r c l e s ) and 9 ducks (dark c i r c l e s ) . PrpOg i s expressed as a percent decrease of the ele c t r o d e c u r r e n t where the decrease from normoxia to anoxia (death) was defined as 100$ decrease. CF was expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was defined as 100 AU. 70 + 60 + 5 0 + 4 0 + 20 + DUCKS• •• o CHICKENS o o o • • • • • o • • o • ° o o o 30+ o g o o o 8 °o 8 ° o • i • o o* ° ° ° ° * 8 ° • • * o • o —I 1 1 1 1 1 1 1 1 1 10 20 30 40 50 60 70 80 90 100 P TO 2 PERCENT DECREASE 104 CF i n c r e a s e d by 5 ° AU, even i n the most extreme case (Tables II and I I I ) , only the p o i n t s t h a t f e l l below 5 ° AU were c o n s i d e r e d here. Equations t h a t b e s t d e s c r i b e the data f o r chickens and ducks are y = 3 . 8 2 e ° ' ° 3 x ( r 2 = 0 . 7 5 ) and y = 3 . 7 4 e ° * ° 3 x ( r 2 = 0.8?) r e s p e c t i v e l y . For purposes of comparing ducks and chickens the e x p o n e n t i a l equations were transformed i n t o a l i n e a r r e g r e s s i o n by p l o t t i n g l n CF versus P T 0 2 . A f t e r determining t h a t the r e s i d u a l v a r i a n c e s between chickens and ducks were homogeneous, s l o p e s and y - i n t e r c e p t s were compared by the a n a l y s i s of covariance (Snedecor and Cochran, 1 9 7 4 ) . The groups were not s i g n i f i c a n t l y d i f f e r e n t at the 9 5 $ confidence 0 03x l e v e l and can be r e p r e s e n t e d by a common equ a t i o n y = 3 ' 7 l e " • . F i g . 2 3 shows a continuous p l o t of CF versus P r ^ O g i n 16 p e r i o d s of p r o g r e s s i v e hypercapnic hypoxia produced by s t o p p i n g a r t i f i c i a l v e n t i l a t i o n i n 8 ducks and the combined data can be described', by the equation y = . 3 . 0 0 e ° ' ° ^ x ( r 2 = 0 . 8 7 ) . P r o g r e s s i v e hypercapnic hypoxia d i d not d i f f e r s i g n i f i c a n t l y from t h a t obtained d u r i n g steady s t a t e normocapnic hypoxia i n d i c a t i n g t h a t COg had no d i r e c t e f f e c t on the redox s t a t e of NAD i n ducks. 1 0 5 F i g u r e 2 3 . C o r r e c t e d f l u o r e s c e n c e (CF) r e c o r d e d from the l e f t c e r e b r a l c o r t e x as a f u n c t i o n o f P^Og r e c o r d e d from the r i g h t c o r t i c a l s u r f a c e (P^Og) d u r i n g apneic asphyxia i n ducks (16 p e r i o d s of apneic asphyxia i n 8 ducks). Each t r a c e i s the r e s u l t from a s i n g l e asphyxic p e r i o d . PrnOg i s expressed as a percent decrease of the e l e c t r o d e c u r r e n t where the decrease from normoxia to anoxia (death) was d e f i n e d as a 100$ decrease. CF was expressed i n a r b i t r a r y u n i t s (AU) where the CF change from normoxia to anoxia (death) was d e f i n e d as 100 AU. 106 107 D i s c u s s i o n S t u d i e s have been performed-on the t o l e r a n c e s of chickens and ducks to d i v i n g and have d e f i n e d death i n s e v e r a l d i f f e r e n t ways. Most were based on l a s t h e a r t beat or l a s t s t r u g g l e a f t e r apnea (Andersen, I 9 6 6 ) . Probably a b e t t e r measure f o r t o l e r a n c e i s the p o i n t a t which b r a i n f u n c t i o n ceases s i n c e i n the absence' of b r a i n f u n c t i o n an animal can no l o n g e r c o o r d i -nate the muscular movements f o r escape i f i t i s indeed aware of the need to escape. E i s n e r et a l . (1970) and Kerem and E i s n e r ( 1973a and b) used the time to the onset of slow wave pa t t e r n s i n the EEG, which are c h a r a c t e r i s t i c of unconsciousness d u r i n g hypoxia, f o r t h e i r endurance s t u d i e s . In t h i s study the l o s s of EEG ( i s o e l e c t r i c i t y ) was d e f i n e d as the "EEG end-p o i n t " and the time from v e n t i l a t o r y a r r e s t to the EEG endpoint was d e f i n e d as maximum t o l e r a n c e of the i n d i v i d u a l to apneic asphyxia. The time to the EEG endpoint i n ducks and chickens d u r i n g apneic asphyxia was 338 — 3 2 . 3 6 seconds (n = 10) and 62 — 4 . 3 8 seconds (n = 11) r e s p e c t i v e l y ; over 5 - f o l d l o n g e r i n ducks. Chickens have been p r e v i o u s l y r e p o r t e d to endure 3 minutes of apneic asphyxia (Andersen, I 9 6 6 ) and ducks have been r e p o r t e d to endure 1 0 - 2 3 minutes of apneic asphyxia (Andersen, 1959 and 1 9 6 6 ; Scholander, 1961/1962, 1 9 6 4 ) . Although the endurance times i n the p r e s e n t experiments are l e s s than those p r e v i o u s l y r e p o r t e d , p r o b a b l y because a l e s s s o p h i s t i c a t e d measure of t o l e r a n c e was used i n p r e v i o u s experiments, the r a t i o of en-durance times i n chickens to ducks i s compatible, 3 -8 f o r the 108 previous r e p o r t s and 5 *5 f o r the presen t study. During apneic asphyxia the r e s p i r a t o r y c h a i n NADH charac-t e r i s t i c a l l y i n c r e a s e d i n the c e r e b r a l c o r t e x of chickens and ducks and continued to i n c r e a s e almost l i n e a r l y f o r the d u r a t i o n of asphyxia ( 1 and 2 minutes r e s p e c t i v e l y ) . The d i f f e r e n c e between the 2 s p e c i e s l a y i n the r e l a t i v e r a t e a t which t h a t NADH f l u o r e s c e n c e i n c r e a s e d . For example, NADH f l u o r e s c e n c e i n -creased a t a r a t e of 0 . 6 2 AU/second i n chickens over a 1 minute p e r i o d and 0 . 1 3 AU/second i n ducks over a 2 minute p e r i o d , r e p r e s e n t i n g over a 4 - f o l d d i f f e r e n c e i n r a t e . As apneic asphyxia progressed the c r i t i c a l r e d u c t i o n l e v e l ( 3 5 AU) of the r e s p i r a t o r y c h a i n NAD was approached f a s t e r i n chic k e n s . These r e s u l t s are compatible w i t h the known t o l e r a n c e s of the 2 s p e c i e s to apneic asphyxia. Berger ( 1 9 3 8 ) suggested t h a t EEG frequency i s c l o s e l y r e l a t e d to the degree of o x i d a t i v e metabolism i n nervous t i s s u e (Ingvar et a l . , I 9 7 6 ) . Although there have been r e p o r t s to the c o n t r a r y (Mangold et a l . , I955i Kennedy and S o k o l o f f , 1 9 5 7 ) the m a j o r i t y of evidence supports Berger's theory (Him-wick et a l . , 19^7; Ingvar et a l . , 1 9 7 6 ; Brodersen et a l . , 1 9 7 3 ; Sundt et a l . , 1 9 7 6 ; Meyer et a l . , 1 9 6 7 ; Gleichmann et a l . , I 9 6 2 ) . Furthermore, LaManna et a l . ( l 9 7 5 ) have shown t h a t EEG frequency and redox s t a t e of the r e s p i r a t o r y c h a i n (cytochrome a^) are s t r i c t l y r e l a t e d to oxygen a v a i l a b i l i t y . I t f o l l o w s t h a t , i f anaerobic metabolism prolongs b r a i n f u n c t i o n by s i g n i f i c a n t ATP production, then the r e l a t i o n s h i p d e s c r i b e d by LaManna et a l . ( 1 9 7 5 ) should be l o s t . In other words, d u r i n g apneic asphyxia 109 an animal r e l y i n g on a l a r g e anaerobic ATP c o n t r i b u t i o n f o r s u r v i v a l should have a more reduced r e s p i r a t o r y c h a i n before gross a l t e r a t i o n s i n EEG than one without l a r g e anaerobic c o n t r i b u t i o n s . R e s u l t s from t h i s study show t h a t the EEG endpoint i n both s p e c i e s d u r i n g apneic asphyxia o c c u r r e d a f t e r p r e c i s e l y the same i n c r e a s e i n NAD r e d u c t i o n (approximately 35 AU). F o r these reasons I conclude t h a t anaerobic ATP pro-d u c t i o n , i f i t occurs, gives the duck no advantage over the ch i c k e n i n p r o l o n g i n g b r a i n f u n c t i o n and s u r v i v a l d u r i n g apneic asphyxia. I n these experiments n e i t h e r the absolute p o o l s i z e of NAD nor the absolute r a t e of i n c r e a s e i n NADH has been q u a n t i -t a t e d . I f p o o l s i z e i s l a r g e r i n ducks than chickens and i f the a b s o l u t e r a t e t h a t NADH accumulates i s the same i n both species, vthen the time to the c r i t i c a l p y r i d i n e n u c l e o t i d e r e d u c t i o n (Approximately 35 AU i n both s p e c i e s ) would be*longer i n ducks. In both chickens and ducks d u r i n g steady s t a t e hypoxia a s i m i l a r r e l a t i o n s h i p h e l d between the f a l l i n c o r t i c a l PrpOg and accumulation i n c o r t i c a l NADH which i s independent of p o o l s i z e or ab s o l u t e r a t e s - of accumulation of NADH. But d u r i n g p r o g r e s s i v e hypercapnic hypoxia the r e l a t i o n s h i p between the decrease i n PjpOg and i n c r e a s e i n NADH s t i l l holds, s t r o n g l y s u g g e s t i n g t h a t any d i f f e r e n c e i n p o o l s i z e or ab s o l u t e r a t e of NADH accumulation i s of no s i g n i f i c a n c e i n promoting t o l e r a n c e to apneic asphyxia i n ducks. R e s u l t s from t h i s study p r o v i d e no evidence to support the s u p p o s i t i o n t h a t b i o c h e m i c a l adaptations enhance o x i d a t i v e 1 1 0 p h o s p h o r y l a t i o n i n ducks d u r i n g hypoxia. When chickens and ducks were exposed to a g i v e n l e v e l of t i s s u e hypoxia, 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 redox change of NADH between the 2 s p e c i e s . That m i t o c h o n d r i a l K m f o r oxygen i n the b r a i n i s 1 / 1 0 t h a t of other t i s s u e s ( C l a r k e t a l . , 1 9 7 6 ) may be p r o t e c t i o n enough to ensure maximum use of a v a i l a b l e oxygen at low oxygen t e n s i o n s . P r o g r e s s i v e hypercapnic hypoxia had the same e f f e c t ,on the redox s t a t e of the r e s p i r a t o r y c h a i n as steady s t a t e normocapnic hypoxia. In view of the f a c t t h a t the CF response to p r o g r e s s i v e hypercapnic hypoxia and steady s t a t e normocapnic hypoxia can be d e s c r i b e d by a common equation, I conclude t h a t hypercapnia does not d i r e c t l y a f f e c t the redox s t a t e of the r e s p i r a t o r y c h a i n or e l e c t r o n flow per se. Rosenthal et a l . ( 1 9 7 6 a ) showed t h a t , when 5 $ COg was i n t r o d u c e d i n the v e n t i l a t o r y gas i n r a b b i t s and c a t s , a s l i g h t o x i d a t i o n of cytochrome aa^ always occurred r e g a r d l e s s of the t i s s u e oxygenation s t a t e , but the authors d i d not d i s t i n g u i s h between d i r e c t e f f e c t s of COg on the r e s p i r a t o r y c h a i n and i n d i r e c t e f f e c t s caused by an i n c r e a s e d c e r e b r a l blood f l o w and thus an i n c r e a s e d P j O g . Both the i n c r e a s e i n PrpOg shown when 7f0 COg was added to the v e n t i l a t o r y gas (Chapter 2 , F i g . 1 2 ) and the l a c k of any e f f e c t of hypercapnia on changes i n redox s t a t e d u r i n g hypoxia suggest t h a t the e f f e c t must have been due to i n c r e a s e d c e r e b r a l b l o o d flow and P^Og. Rosenthal et a l . ( 1 9 7 6 a ) r e p o r t e d t h a t changes i n v e n t i l a -t o r y oxygen c o n c e n t r a t i o n produced a c o r r e s p o n d i n g change i n the redox s t a t e of cytochrome aa^ t h a t was continuous from I l l 1 0 0 $ oxygen to n i t r o g e n . On the other hand, Chance et a l . ( 1 9 6 4 ) r e p o r t e d t h a t NADH f l u o r e s c e n c e d i d not i n c r e a s e u n t i l oxygen i n the v e n t i l a t o r y gas was reduced to 5 $ . R e s u l t s from t h i s study confirm the c o n c l u s i o n s of Rosenthal et a l . ( 1 9 7 6 ) showing t h a t f i r s t l y , no abrupt changes occurred d u r i n g the hypoxic regime, only a continuum of change, and secondly, CF in c r e a s e d when PrpOg had decreased by only 2 0 $ . A 2 0 $ decrease i n PipO.g corresponds to oxygen c o n c e n t r a t i o n s i n the v e n t i l a t o r y gas of g r e a t e r than 15$> w e l l above the c r i t i c a l 5 $ l e v e l r e p o r t e d by Chance et a l . ( 1 9 6 4 ) . 1 1 2 GENERAL DISCUSSION The problem of continued ATP p r o d u c t i o n d u r i n g d i v i n g l i e s i n the i n a b i l i t y to m a i n t a i n the nece s s a r y homeostasis necessary f o r ATP p r o d u c t i o n when e x t e r n a l r e s p i r a t i o n ceases. N e v e r t h e l e s s , the c a r d i o v a s c u l a r adjustments enable n a t u r a l d i v e r s to t o l e r a t e p e r i o d s of apneic asphyxia t h a t would be d e t r i m e n t a l t o man and other t e r r e s t r i a l v e r t e b r a t e s . These adjustments do not all o w the d i v e r to u n c o n d i t i o n a l l y m a i n t a i n homeostasis but r a t h e r p r o l o n g i t . The s t r a t e g i e s f o r t i s s u e s v a r y and are r e f l e c t e d by t h e i r b l o o d s u p p l y d u r i n g the d i v e . Those t h a t are p o o r l y p e r f u s e d depend on enhanced g l y c o l y t i c c a p a c i t y f o r the p r o d u c t i o n of ATP once the r e s i d u a l oxygen i n t h a t p a r t i c u l a r t i s s u e i s de p l e t e d . On the other hand the he a r t and b r a i n are e a s i l y and i r r e p a r a b l y damaged by severe and p r o t r a c t e d hypoxia (Andersen, I 9 6 6 ) and must be w e l l per-f u s e d throughout the d i v e . Although harbor s e a l s appear t o t o l e r a t e more severe hypoxia than dogs before the onset o f gross EEG a l t e r a t i o n s (Kerem.,and E i s n e r , 1 9 7 3 a and b ^ i n t h i s study ducks c o u l d t o l e r a t e hypoxia no b e t t e r than c h i c k e n s . The s e m i q u a n t i t a t i v e method f o r m o n i t o r i n g c o r t i c a l NADH i n t h i s study allowed comparisons between i n d i v i d u a l s and s p e c i e s r e g a r d l e s s of NAD p o o l s i z e . Once NADH had i n c r e a s e d a p p r o x i -mately 3 5 $ above the normoxic l e v e l i n chickens and duc k s , b r a i n e l e c t r i c a l a c t i v i t y ceased. Furthermore, the 3 5 $ i n c r e a s e i n NADH occurred w i t h the same decrease i n b r a i n POg i n both s p e c i e s . Although there i s a s t r o n g c o r r e l a t i o n between NADH f l u o r e s c e n c e i n c r e a s e and EEG endpoint, EEG may not be d i r e c t l y r e l a t e d to 1 1 3 the redox s t a t e . I t may be t h a t EEG and NADH f l u o r e s c e n c e both r e f l e c t the l e v e l of t i s s u e oxygenation. I n t e r r e s t r i a l mammals the f u n c t i o n a l s t a t e of the b r a i n changes (s l o w i n g of EEG) before the t i s s u e shows any d e t e c t a b l e changes i n the adenosine phosphates (ATP, ADP, AMP) d u r i n g severe hypoxia (Seisjo" et a l . , 1 9 7 3 and 1 9 7 4 ) and i n d i c a t e s t h a t energy f a i l u r e (ATP p r o d u c t i o n cannot match the demand) may not be r e s p o n s i b l e f o r the l o s s of b r a i n f u n c t i o n . However, the t i s s u e measurement,;is an average value f o r the adenosine phosphates, and may not r e f l e c t the energy s t a t e at c r i t i c a l s i t e s or i s o l a t e d areas of the b r a i n ( S e i s j O , 1 9 7 7 ) ' A l t e r n a t i v e l y , other oxygen r e q u i r i n g r e a c t i o n s i n the b r a i n which are used i n the b i o s y n t h e s i s of neurotrans-m i t t e r s ( t y r o s i n e and tryptophane hydroxylase) and t h e i r d e g r a d a t i o n (monoamine oxidase) may be a l t e r e d d u r i n g hypoxia c a u s i n g a l o s s of b r a i n f u n c t i o n . Although these r e a c t i o n s account f o r l e s s than 1 5 $ of the t o t a l oxygen consumed i n the b r a i n (JObsis, 197*+), they are n e v e r t h e l e s s s e n s i t i v e to s m a l l changes i n oxygen. For example, s e r o t o n i n , a product of the tryptophane hydroxylase r e a c t i o n , decreases l i n e a r l y w i t h a f a l l i n P T 0 2 between 5 0 and 2 0 t o r r (JObsis, 197*+). On the other hand, most of these r e a c t i o n s can be s p e c i f i c a l l y i n h i b i t e d w i t h chemical b l o c k e r s or b r a i n l e s i o n s without the l o s s of b r a i n f u n c t i o n (Snyder, 1 9 7 6 ; Sourkes, 1 9 7 6 ) . The nervous system of a l l b i r d s and mammals, whether d i v e r s or nondivers, appears to be dependent on o x i d a t i v e metabolism f o r i t s s u r v i v a l . . Therefore d i v e r s must ensure t h a t oxygen s u p p l y to the b r a i n i s maintained throughout a d i v e . 114 While other tissues appear capable of r e l y i n g on anaerobic metabolism during the dive, the brain cannot. It may be that "the complexity of the brain i s not compatable with anoxic tolerance. 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