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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 R o b e r t M a u r i c e Bryan, J r . B.Sc,  U n i v e r s i t y o f Alabama,  1970  M.Sc,  U n i v e r s i t y o f Alabama, 1977  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE.DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES Department o f Z o o l o g y  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA November, I978  (c) R o b e r t M a u r i c e Bryan, J r . , 197,8  In presenting  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 o f the  r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . that permission f o r extensive  I f u r t h e r agree  copying of t h i s thesis f o r  s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood  that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada  i  ABSTRACT C e r e b r a l energy m e t a b o l i s m d u r i n g a p n e i c a s p h y x i a and s t e a d y s t a t e h y p o x i a was compared i n ducks and c h i c k e n s ; ducks t o l e r a t e apneic a s p h y x i a 3-8 times l o n g e r t h a n  chickens.  F l u c t u a t i o n s i n t h e reduced form o f r e s p i r a t o r y c h a i n n i c o t i n a mide adenine d i n u c l e o t i d e (NADH) were m o n i t o r e d  from t h e l e f t  c e r e b r a l hemisphere by a n o n i n v a s i v e f l u o r o m e t r i c t e c h n i q u e and used as an i n d i c a t o r o f m i t o c h o n d r i a l h y p o x i a .  Electro-  encephalogram (EEG) and s u r f a c e POg were r e c o r d e d from t h e r i g h t hemisphere.  F o r c e d d i v e s o f ^-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 b r a d y c a r d i a and an accumulation period.  o f NADH w h i c h i n c r e a s e d t h r o u g h o u t t h e d i v i n g  NADH r e t u r n e d t o t h e p r e a s p h y x i c l e v e l when b r e a t h i n g  was resumed.  I n l a t e r experiments a s p h y x i a was 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 p a r a l y z e d ducks.  Asphyxia  produced by t h i s means caused s i m i l a r changes i n t h e measured v a r i a b l e s ( h e a r t r a t e , b l o o d p r e s s u r e , NADH f l u o r e s c e n c e , and EEG) t o those o b t a i n e d i n f o r c e d submergence o f n o n p a r a l y z e d ducks. NADH f l u o r e s c e n c e was 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 100 AU was d e f i n e d as t h e f l u o r e s c e n c e change from normoxia to anoxia.  A f t e r 1 minute o f a s p h y x i a NADH f l u o r e s c e n c e i n -  c r e a s e d by 37 AU - 3.60 SEM ( n = 5^) in*-paralyzed c h i c k e n s and 8 AU - 1.^1 SEM ( n = 55) i n p a r a l y z e d ducks.  A f t e r 2 minutes  the f l u o r e s c e n c e i n c r e a s e d by o n l y 15 AU - 1.95 SEM i n ducks. Both s p e c i e s showed an i s o e l e c t r i c EEG when f l u o r e s c e n c e i n c r e a s e d "by a p p r o x i m a t e l y  35 AU i n d i c a t i n g t h a t a n a e r o b i c ATP p r o d u c t i o n  ii  i n ducks d i d n o t m a i n t a i n "brain f u n c t i o n (EEG) accumulation  o f r e s p i r a t o r y c h a i n NADH.  f o r a greater  A t a g i v e n decrease  i n t i s s u e P 0 ducks and c h i c k e n s showed t h e same l e v e l o f NADH 2  i n c r e a s e i n d i c a t i n g t h a t both s p e c i e s a r e e q u a l l y dependent on t i s s u e P 0 f o r the maintenance o f redox s t a t e . 2  Furthermore,  the i n h i b i t i o n o f c a r d i o v a s c u l a r adjustments by a t r o p i n e i n ducks caused NADH t o i n c r e a s e f a s t e r d u r i n g a p n e i c t h a n i n n o n a t r o p i n i z e d ducks.  asphyxia  I conclude t h a t t h e oxygen  c o n s e r v i n g c a r d i o v a s c u l a r adjustments a r e r e s p o n s i b l e f o r t h e i n c r e a s e d c e r e b r a l t o l e r a n c e t o apneic a s p h y x i a i n ducks w i t h o u t any i n v o l v e m e n t o f b i o c h e m i c a l mechanisms.  iii  TABLE OF CONTENTS General  Introduction  Chapter 1.  C h a p t e r 2.  1  G e n e r a l Methods - A n i m a l P r e p a r a t i o n  17  a.  Rats  17  D.  Ducks  17  c.  Chickens  20  S p e c i a l Techniques  21  The F l u o r o m e t r i c r e c o r d i n g o f NADH  21  a.  Descriptionr.of the fluorometer  22  b.  S t a b i l i t y of the fluorometer  c.  Fluorescence  d.  S t a b i l i t y of the b i o l o g i c a l  e.  B l o o d a r t i f a c t compensation  f.  NADH f l u o r e s c e n c e d u r i n g n i t r o g e n  "  emission spectra  26 29  preparation  35 36  ventila-  t i o n and a p n e i c a s p h y x i a  ^1  P o l a r o g r a p h i c measurement o f oxygen t e n s i o n of t h e c o r t i c a l s u r f a c e  ^8  a.  D e s c r i p t i o n o f t h e oxygen e l e c t r o d e  51  b.  Oxygen measurements from t h e c o r t i c a l s u r f a c e o f ducks d u r i n g h y p o x i a and hypercapnia  C h a p t e r 3.  5^  Changes i n t h e Redox S t a t e o f R e s p i r a t o r y C h a i n NADH D u r i n g A p n e i c A s p h y x i a  i n Ducks  60  Introduction  60  Methods  62  a.  Fluorescence ducks  r e c o r d i n g s from  nonparalyzed 62  iv  Chapter 3.  Methods b.  (cont'd)  Fluorescence  r e c o r d i n g s from p a r a l y z e d  ducks c.  62  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 a d j u s t ments d u r i n g apneic asphyxia  63  Results a.  65  Comparison of nonparalyzed  and  paralyzed  ducks b.  65  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 a d j u s t ments d u r i n g apneic asphyxia  i n ducks  Discussion Chapter k.  74 79  C e r e b r a l Energy Metabolism i n Ducks and -.. Chickens During Apneic  Asphyxia  and Hypoxia  82  Introduction  82  Methods  Qk  a.  Fluorescence ducks and  b.  r e c o r d i n g s from p a r a l y z e d  chickens  8^  Concurrent f l u o r o m e t r i c and r e c o r d i n g s d u r i n g hypoxia  Results  polarographic ^  '  86  a.  Comparison of ducks and chickens  b.  C r i t i c a l pyridine nucleotide reduction (CPNR) i n chickens  c.  8^4-  and ducks  86  90  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 PrpO^ i n ducks and  Discussion  chickens d u r i n g hypoxia  97 107  General D i s c u s s i o n  Bibliography  vi  LIST OF FIGURES F i g u r e 1.  The r e s p i r a t o r y c h a i n .  7  F i g u r e 2.  Diagram of the o p t i c a l d e s i g n o f the fluorometer.  F i g u r e 3.  S i m p l i f i e d diagram o f the f l u o r o m e t e r circuitry.  F i g u r e k.  23  Fluorescence  -  27 e m i s s i o n s p e c t r a from the  c e r e b r a l c o r t e x o f a duck d u r i n g normoc a p n i a and a n o x i a . F i g u r e 5•  Fluorescence  30  emission spectra,from  the  ..cerebral c o r t e x o f a duck d u r i n g a n o x i a and from a s o l u t i o n o f NADH.' " Figure 6.  O p t i c a l measurements r e c o r d e d  33 from the  c e r e b r a l c o r t e x o f 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  altered. F i g u r e 7«  37  The r e l a t i o n s h i p between r e f l e c t e d  excitation  l i g h t and e m i t t e d f l u o r e s c e n c e l i g h t  recorded  from the c e r e b r a l c o r t e x o f a duck when the hemoglobin i n t h e r e c o r d i n g f i e l d  was  altered. Figure 8 .  39  O p t i c a l measurements and b l o o d  pressure  of a duck d u r i n g n i t r o g e n v e n t i l a t i o n . F i g u r e 9.  ^-2  C o r r e c t e d f l u o r e s c e n c e r e c o r d e d from t h e c e r e b r a l c o r t e x o f ducks d u r i n g  apneic  a s p h y x i a when mean a r t e r i a l b l o o d fell.  ,  pressure ^5  vii  F i g u r e 10.  Diagram of an oxygen e l e c t r o d e .  49  F i g u r e 11.  Polarogram of an oxygen e l e c t r o d e .  52  F i g u r e 12.  P o l a r o g r a p h i c measurements of P^Og ^  r  o  m  the c o r t i c a l s u r f a c e of a duck d u r i n g hypoxia and hypercapnia. F i g u r e IJ.  Surface P r p 0  r  e  c  0  2  r  d  ed  55  froirr the r i g h t  cerebral  c o r t e x d u r i n g v a r i o u s l e v e l s of hypoxia. F i g u r e Ik-.  58  Comparison of h e a r t r a t e , mean a r t e r i a l blood pressure,  and c o r r e c t e d f l u o r e s c e n c e  ini.paralyzed and nonparalyzed apneic  ducks d u r i n g  asphyxia. i n a nonparalyzed  '  66  duck.  69  F i g u r e 15.  Submergence asphyxia  F i g u r e 16.  Apneic asphyxia  F i g u r e 17.  C o r r e c t e d f l u o r e s c e n c e and PipOg from the  i n a p a r a l y z e d duck.  c e r e b r a l c o r t e x o f a duck d u r i n g asphyxia  72  apneic  before and a f t e r 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. F i g u r e 18.  77  Comparison o f heart r a t e , mean a r t e r i a l pressure,  blood  and c o r r e c t e d f l u o r e s c e n c e i n  p a r a l y z e d ducks and chickens  during  apneic  asphyxia. F i g u r e 19.  Electroencephalogram  87 (EEG) and NADH  f l u o r e s c e n c e i n a c h i c k e n and duck d u r i n g apneic F i g u r e 20.  asphyxia.  95  A s p h y x i a t o l e r a n c e i n ducks p l o t t e d as a f u n c t i o n of b r a d y c a r d i a .  98  viii  F i g u r e 21.  Surface P^Og and NADH f l u o r e s c e n c e  of the  r i g h t and l e f t c e r e b r a l cortex r e s p e c t i v e l y d u r i n g v a r i o u s l e v e l s of hypoxia. F i g u r e 22.  NADH f l u o r e s c e n c e p l o t t e d as a f u n c t i o n of P r p 0  F i g u r e 23.  100  2  d u r i n g hypoxia i n chickens  102  and ducks.  NADH f l u o r e s c e n c e p l o t t e d as a f u n c t i o n o f P 0 T  ?  d u r i n g apneic  asphyxia  i n ducks.  ~  105  ix  LIST OF TABLES NADH f l u o r e s c e n c e i n p a r a l y z e d ducks d u r i n g apneic asphyxia before and a f t e r a t r o p i n e injections. Heart r a t e , c e s s a t i o n o f b r a i n  electrical  a c t i v i t y , and NADH f l u o r e s c e n c e i n p a r a l y z e d ducks d u r i n g apneic  asphyxia.  Heart r a t e , c e s s a t i o n o f b r a i n  electrical  a c t i v i t y , and NADH f l u o r e s c e n c e i n p a r a l y z e d chickens d u r i n g apneic  asphyxia.  X  A CKNOWLEGEMENTS  I thank Dr. David R. Jones f o r h i s guidance and support o f the r e s e a r c h presented i n t h i s t h e s i s .  His  p h i l o s o p h y o f and approach t o s c i e n c e has impressed me to the extent t h a t I s h a l l use these as g u i d e l i n e s as I persue a c a r e e r i n s c i e n c e . In a d d i t i o n I thank Dr. D.J. R a n d a l l , Dr. W.K. Milsom, Dr. M.S. Haswell, and Dr. O.S. Bamford f o r t h e i r helpful  comments p e r t a i n i n g t o t h i s r e s e a r c h .  I am g r a t e f u l  t o Dr. W.K. Milsom,  Chuck Daxboeck,  and Steve P e r r y f o r r e v i e w i n g t h i s manuscropt. r  T h i s r e s e a r c h was supported by a grant t o Dr. D.R. Jones from B r i t i s h Columbia Heart Foundation.  1  INTRODUCTION S i n c e t h e time o f A r i s t o t l e i t has been n o t e d t h a t t h e p o r p o i s e i s an a i r b r e a t h i n g i n water f o r p e r i o d s  a n i m a l which c a n remain submerged  o f time t h a t a r e f a t a l t o s t r i c t l y t e r r e s -  t r i a l mammals ( S t r a u s s , 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 t h e f o l l o w i n g 2 2 0 0 y e a r s ,  i t was  not u n t i l t h e 1 8 7 0 ' s t h a t P a u l B e r t attempted t o e x p l a i n t h e n a t u r e o f t h i s phenomenon (Andersen, I 9 6 6 ) . pioneering  Since  Bert's  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" h e l p i n e x p l a i n i n g the. t o l e r a n c e  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 r e v i e w s see S c h o l a n d e r , 1 9 ^ 0 ; Andersen, I 9 6 6 ; E i s n e r , I 9 6 9 ) .  The major problem d u r i n g a d i v e  i s t h e d e p l e t i o n o f oxygen and i t appears t h 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 , o r mammals, d e a l w i t h t h e problem i n a s i m i l a r manner.  D e s p i t e t h i s f a c t d i v e r s c a n 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 r e q u i r e m e n t .  F o r example l i z a r d s , snakes, and c r o c o d i l e s c a n s u r v i v e f o r 3 0 minutes ( B e l k i n , I 9 6 3 ) ,  anoxia  and t u r t l e s f o r over k hours  ( J a c k s o n , I 9 6 8 ) , whereas d i v i n g mammals and b i r d s c a n s u r v i v e a n o x i a f o r o n l y a m a t t e r o f seconds b e f o r e i r r e p a r a b l e damage occurs.  Because o f t h i s l a r g e d i f f e r e n c e i n t o l e r a n c e many o f  the g e n e r a l i z a t i o n s c o n c e r n i n g t h e p h y s i o l o g y  o f d i v i n g mammals  and b i r d s do n o t p e r t a i n i n any d e t a i l t o r e p t i l e s and amphibians.  F o r t h i s r e a s o n o n l y one o f the groups, b i r d s and  mammals, w i l l be c o n s i d e r e d  i n this thesis.  2  Since  e x t e r n a l r e s p i r a t i o n ceases d u r i n g the p e r i o d o f  the underwater e x c u r s i o n ,  animals such as mammals and b i r d s  t h a t are o b l i g a t e l y dependent on oxygen must make some prov i s i o n f o r i t s storage increasing  d u r i n g the d i v e .  (1) blood volume ( E i s n e r , I 9 6 9 ) ,  of the blood I966),  and c o n s e r v a t i o n  (Scholander, 1 9 ^ 0 ) ,  By  (2) oxygen c a p a c i t y  ( 3 ) l u n g volume (Andersen,  and ( 4 ) muscle myoglobin (Robinson, 1 9 3 9 ) d i v e r s can as  much as double the q u a n t i t y o f oxygen s t o r e d over t h a t o f a s i m i l a r l y s i z e d nondiver ( E i s n e r , I 9 6 9 ) . oxygen storage  However,  increased  does not appear t o be a b s o l u t e l y n e c e s s a r y s i n c e  some animals can endure extended p e r i o d s  o f breath  holding i n  the absence of many of these p h y s i o l o g i c a l a d a p t a t i o n s . example, the oxygen c a r r y i n g c a p a c i t y o f b l o o d (Scholander and I r v i n g , 19^-1), s e a l i o n  For  i n the manatee  ( F l o r k i n and R e d f i e l d ,  1931),  porpiose  (Green and R e d f i e l d , 1 9 3 3 ) 1  I966),  and penguin (Scholander, 19^-0) i s a c t u a l l y l e s s than  t h a t of man (Prosser, 1 9 7 3 ) •  duck (Andersen,  The b o t t l e nose whale and f i n  whale, although known t o be good d i v e r s , have a r e l a t i v e l y s m a l l l u n g volume (Andersen, I 9 6 6 ) has  and the F l o r i d a manatee  p r a c t i c a l l y no muscle myoglobin f o r the storage  o f oxygen  (Scholander and I r v i n g , 1 9 ^ 1 ) . More important t o underwater s u r v i v a l i s a s e r i e s o f c a r d i o v a s c u l a r adjustments d u r i n g the dive t h a t  increases  the v a s c u l a r r e s i s t a n c e i n most v a s c u l a r beds ( 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 ) and reduces the heart r a t e  (bradycardia)  ( I r v i n g , 1 9 3 ^ ; I r v i n g e t a l . , 1935; Scholander, 19^0; Scholander  e t a l . , I9^2b; Johansen, 1 9 6 ^ ; B u t l e r and Jones,  1971).  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 t o conserve "blood,  oxygen by r e d i s t r i b u t i n g b l o o d . and,  Tissues  such as the  heart  i n p a r t i c u l a r , the b r a i n w h i c h are most s e n s i t i v e t o oxygen  l a c k r e c e i v e p r i o r i t y f o r b l o o d f l o w over t i s s u e s and t h a t are l e s s e a s i l y damaged by a n o x i a .  The  organs  bradycardia  s e t s the i n c r e a s e d p e r i p h e r a l r e s i s t a n c e so t h a t b l o o d i s held constant  and,  offpressure  i n a d d i t i o n , i t a l s o reduces the work l o a d  on the h e a r t , thus f u r t h e r c o n s e r v i n g  oxygen.  A l t h o u g h the same  b a s i c response i s seen i n n o n d i v e r s d u r i n g apnea, i t ' h a s been c o n s i d e r a b l y r e f i n e d i n d i v e r s t o meet the needs of p r o l o n g e d apneic s u r v i v a l .  The  f u l l y u n d e r s t o o d and  c o n t r o l of the adjustments i s s t i l l i s p r e s e n t l y the o b j e c t o f i n t e n s e  t i g a t i o n ( f o r r e v i e w s of c o n t r o l see AngelaJames - and 1972;  Jones, 1976; The  not  inves-  Daly,  Jones and West, 1978).  n a t u r e of the c a r d i o v a s c u l a r adjustments d i v i d e s  t i s s u e s i n t o two  groups; those t h a t r e c e i v e b l o o d f l o w d u r i n g  d i v e , and those t h a t do n o t .  The  the r e s i d u a l t i s s u e oxygen i s d e p l e t e d . i n muscle and  a  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 a n a e r o b i c p r o d u c t i o n  produce ATP  the  The  of ATP* once c a p a c i t y to  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 o n l y make minor m o d i f i c a t i o n s t o 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 a r e a s :  * Abbreviations: ATP, adenosine t r i p h o s p h a t e ; ADP, d i p h o s p h a t e ; and AMP, adenosine monophosphate.  adenosine  k  (1) t i g h t e r c o n t r o l o f r e g u l a t o r y enzymes e n a b l i n g g l y c o l y t i c f l u x t o i n c r e a s e by s e v e r a l hundred f o l d and (2) maintenance of t h e c y t o p l a s m i c NAD/NADH* r a t i o , p r i m a r i l y t h r o u g h t h e a c t i o n of  l a c t a t e dehydrogenase, t o i n s u r e t h a t NAD i s a v a i l a b l e f o r  glycolysis  (Hochachka and S t o r e y , 1975)•  T i s s u e s r e m a i n i n g i n t h e 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 r e q u i r e m e n t s and t h e energy s u p p l y must n o t be i n t e r rupted during a dive.  F o r example, -;the energy r e q u i r e m e n t s o f  the b r a i n may be as much as 20% o f t h e t o t a l energy consumed i n mammals (Dunn and Bondy, 197^) and c a n o n l y be met by p r o d u c i n g ATP a e r o b i c a l l y ( S e i s j f l , 1977). t h e p r o c e s s  depending  almost e x c l u s i v e l y on t h e s u b s t r a t e g l u c o s e ( S o k o l o f f , I976). Only i n i n f a n t s (Dunn and Bondy, 197^) o r d u r i n g s t a r v a t i o n (Himwich,  1976b) a r e o t h e r s u b s t r a t e s o x i d i z e d t o any s i g n i f i -  cant degree, b u t , even s o , g l u c o s e s t i l l accounts f o r t h e m a j o r i t y o f t h e s u b s t r a t e consumed (Dunn and Bondy, 197^0. S i n c e g l y c o g e n , t h e s t o r a g e form o f g l u c o s e , i s n o t p r e s e n t i n the b r a i n i n l a r g e enough q u a n t i t i e s t o s u p p o r t energy metab o l i s m (Himwich,  1976a)» t h e b l o o d i s r e s p o n s i b l e n o t o n l y f o r  a c o n t i n u o u s s u p p l y o f oxygen b u t a l s o a c o n t i n u o u s s u p p l y o f glucose. On e n t e r i n g t h e c e l l g l u c o s e i s o x i d i z e d by t h e c l a s s i c a l Embden-Meyerhoff pathway ( g l y c o l y s i s ) t o p y r u v a t e w i t h a n e t p r o d u c t i o n o f two moles o t ATP f o r each mole o f g l u c o s e * r>  NAD and NADH are t h e o x i d i z e d and reduced forms o f 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.  (1) describes the o v e r a l l  Equation  Glucose + 2NAD  +  + 2ADP + 2 P i  reaction. > (1)  2 p y r u v a t e + 2 NADH + 2 H + 2 A T P +•2H 0 2  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 p a t h w a y s . be a n a e r o b i c a l l y r e d u c e d  to lactate  i n the presence  I t can  of l a c t i c  d e h y d r o g e n a s e (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 The  NADH p r o d u c e d  L  D  H  )  2 l a c t a t e + 2 NAD  (2)  +  i n e q u a t i o n ( l ) i s now r e o x i d i z e d a n d r e a d y  to  be u s e d  of  oxygen t h e two m o l e c u l e s  c u l e s from  +  again i n the o x i d a t i o n of glucose.  I n the presence  o f NADH a n d t h e t w o p y r u v a t e  e q u a t i o n ( 1 ) may be o x i d i z e d v i a t h e c i t r i c  mole-  acid  c y c l e and t h e 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 m o l e s o f ATP p e r m o l e 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 +  2 NAD  ,  Under normoxic  + 6C0  2  >  ?  + kJm 0 z  +  (3)  36ATP  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 w h i l e 1 5 $ 2  is  oxidized only to lactate  t h e m o l e s o f ATP p r o d u c e d metabolism of ATP  ATP),  (Dunn a n d B o n d y , 1 9 7 * 0 •  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  (2 moles o f ATP) and a e r o b i c m e t a b o l i s m  aerobic metabolism  produced.  Considering  accounts  ( 3 6 moles  f o r a b o u t 95% o f t h e t o t a l  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 t h e a v a i l a b l e  o x y g e n ; a s o x y g e n becomes  p r o p o r t i o n a t e l y more g l u c o s e  limiting  i smetabolized to l a c t a t e .  6  I n r a t s when t h e b r a i n i s d e p l e t e d o f oxygen, g l y c o l y t i c  flux  can i n c r e a s e 5 f o l d ( S e i s j O , 1977)- However, even w i t h t h i s i n c r e a s e , a n a e r o b i c m e t a b o l i s m alone cannot s u p p l y t h e n e c e s s a r y 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% o f t h e oxygen used by t h e b r a i n i s h a n d l e d by the r e s p i r a t o r y c h a i n ( J f l b s i s , 197^) which c o n s i s t s o f 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  o r d e r o f t h e i r redox  p o t e n t i a l s ( F i g . 1) and a c t s as an energy g r a d i e n t t h a t t r a n s f e r s e l e c t r o n s from s u b s t r a t e t o oxygen.  On e n t e r i n g t h e  r e s p i r a t o r y c h a i n e l e c t r o n s f l o w from t h e members w i t h t h e more n e g a t i v e redox p o t e n t i a l s , NAD o r F P f ( f l a v o p r o t e i n ) , t o t h e more p o s i t i v e members and u l t i m a t e l y t o oxygen.  Each atom o f  oxygen r e c e i v e s two e l e c t r o n s from t h e r e s p i r a t o r y c h a i n p l u s two hydrogen i o n s from t h e media f o r m i n g one m o l e c u l e o f w a t e r . 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 c a p t u r e d and used i n o x i d a t i v e p h o s p h o r y l a t i o n ,  a  term g i v e n t o t h e 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 o f e l e c t r o n s passed down t h e r e s p i r a t o r y c h a i n from NAD t o oxygen a p p r o x i m a t e l y produced.  t h r e e m o l e c u l e s o f ATP a r e  E l e c t r o n o p a i r s e n t e r i n g t h e c h a i n v i a FP produce  o n l y two m o l e c u l e s o f ATP s i n c e t h e e l e c t r o n s bypass t h e f i r s t phosphorylation s i t e ( F i g . 1). Oxidative  phosphorylation  i s o b l i g a t e l y c o u p l e d t o t h e e l e c t r o n f l o w o f t h e c h a i n and t h e r e f o r e proceeds o n l y i n t h e presence o f oxygen.  When  oxygen becomes l i m i t i n g e l e c t r o n s e n t e r t h e r e s p i r a t o r y c h a i n  7  F i g u r e 1.  A diagram of the r e s p i r a t o r y c h a i n i n m i t o c h o n d r i a i l l u s t r a t i n g the f l o w of e l e c t r o n s and the  probable  s i t e s f o r ATP  NAD,  production.  Abbreviations:  n i c o t i n a m i d e adenine d i n u c l e o t i d e ; FP^ f l a v o p r o t e i n s ; CoQ, CYT,  cytochrome.  and  FPg,  u b i q u i n o n e or coenzyme Q;  ATP  ATP •CYT b  CYTc  -•CYTc  "CYTaa,  9  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 n e t r e d u c t i o n o f each e l e c t r o n c a r r i e r .  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 e l e c t r o n c a r r i e r can t h e r e f o r e 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 (Chance e t a l . , Changes i n the redox s t a t e o f the f i r s t  1 9 7 3 ) .  component o f the  r e s p i r a t o r y c h a i n , NAD, can be monitored i n i n t a c t t i s s u e s by a f l u o r o m e t r i c method which was f i r s t d e s c r i b e d by Chance e t a l . (1962).  The method takes  advantage of the f a c t t h a t the reduced  form o f NAD (NA-DH) i s a n a t u r a l fluorochrome which; can-be:• e x c i t e d by l i g h t wavelengths between 31° and 370 nm and g i v e s r i s e t o a f l u o r e s c e n c e emission ( J S b s i s e t a l . , I966).  i n the r e g i o n of ^-25-^75 nm  The o x i d i z e d form o f the coenzyme  does not f l u o r e s c e a t these wavelengths.  I n p r a c t i c e NADH i s  e x c i t e d by f o c u s s i n g 366 nm l i g h t , a n a t u r a l peak o f the mercury arc lamp, on the t i s s u e s u r f a c e and the f l u o r e s c e n c e o r i g i n a t i n g p r i m a r i l y from the t o p 1.5 mm of t i s s u e (Jtfbsis e t a l . , 1971) i s measured w i t h a p h o t o m u l t i p l i e r tube. The  l a b i l e f l u o r e s c e n c e s i g n a l from t i s s u e was  initially  l i n k e d t o NADH due t o s i m i l a r i t i e s i n t h e i r f l u o r e s c e n c e emiss i o n s p e c t r a (Chance e t a l . , 1962) and t o the f a c t t h a t NADH, determined by b i o c h e m i c a l of f l u o r e s c e n c e et  i nliver  a n a l y s i s , c o r r e l a t e s w i t h changes (Chance e t a l . , 1965a), h e a r t  a l . , 1965b) and b r a i n (Jflbsis e t a l . , 1971).  of anoxia  During  (Chance cycles  the k i n e t i c s of t i s s u e f l u o r e s c e n c e are i n synchrony  w i t h those o f the cytochromes i n d i c a t i n g a l a r g e c o n t r i b u t i o n of r e s p i r a t o r y chain, NADH t o the f l u o r e s c e n c e s i g n a l (Lubbers et  a l . 1 9 6 ^ ) . Furthermore, Chance e t a l . (I962) and Mayevsky  10  and Chance (197^) showed t h a t drugs which b l o c k t h e r e s p i r a t o r y c h a i n caused a l a r g e f l u o r e s c e n c e i n c r e a s e i n t h e k i d n e y and b r a i n of r a t s . However, t h e f l u o r e s c e n c e s i g n a l from t h e r e s p i r a t o r y c h a i n may  be contaminated w i t h f l u o r e s c e n c e from o t h e r p y r i d i n e  n u c l e o t i d e p o o l s i n the c e l l w h i c h are noti.at t h e same redox s t a t e as r e s p i r a t o r y c h a i n NAD and do n o t n e c e s s a r i l y show redox changes t h a t p a r a l l e l changes i n r e s p i r a t o r y c h a i n NADH. I n v e s t i g a t i o n s i n t o t h e o r i g i n o f t h e l a b i l e f l u o r e s c e n c e have, shown t h a t t h e s e n o n r e s p i r a t o r y c h a i n p o o l s c o n t r i b u t e i f any t o t h e f l u o r e s c e n c e s i g n a l .  Fluorescence  little  from r e s p i r a -  t o r y c h a i n NADH i s s u f f i c i e n t l y enhanced over t h e f l u o r e s c e n c e from c y t o p l a s m i c NADH, t h e p o o l which i s p r i m a r i l y i n v o l v e d w i t h t h e 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 c y t o p l a s m i c NADH c o n t r i b u t e l i t t l e  t o t h e f l u o r e s c e n c e s i g n a l ( J t f b s i s and  D u f f i e l d , 1967; O'Connor, 1977). is  The r e s p i r a t o r y c h a i n NADH  enzyme bound whereas much c y t o p l a s m i c NADH i s f r e e o r un-  bound.  As a g e n e r a l r u l e NADH t h a t i s enzyme bound has a g r e a t e r  quantum e f f i c i e n c y t h a n f r e e NADH (Boyer and T h e o r e l l , 1956). However, g l y c e r a l d e h y d e dant i n the cytoplasm  phosphate dehydrogenase w h i c h i s abun-  i s one o f t h e few enzymes t h a t a c t u a l l y  d i m i n i s h e s r a t h e r t h e n enhances f l u o r e s c e n c e when bound t o t h e coenzyme ( V e l i c k , I96I).  The o v e r a l l r e s u l t i s t h a t t h e  f l u o r e s c e n c e e f f i c i e n c y o f r e s p i r a t o r y c h a i n NADH i s 10-20 times g r e a t e r t h a n t h a t o f t h e c y t o p l a s m i c NADH p o o l ( J f l b s i s e t a l . , 1971). A n o t h e r NAD p o o l which i s thought t o s e r v e p r i m a r i l y i n  11  b i o s y n t h e s i s ( J O b s i s , 1964) i s l o c a t e d i n t h e m i t o c h o n d r i a but i s n o t d i r e c t l y i n v o l v e d w i t h t h e r e s p i r a t o r y c h a i n (Chance and H o l l u n g e r , I96I).  T h i s p o o l has n o t been as e x t e n -  s i v e l y i n v e s t i g a t e d as t h e c y t o p l a s m i c NAD p o o l b u t n e v e r t h e l e s s appears t o c o n t r i b u t e o n l y 1-2% o f t h e t o t a l f l u o r e s c e n c e s i g n a l in 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 t o those o f NADH a l s o occur i n t h e reduced form -of c y t o p l a s m i c and m i t o c h o n d r i a l n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate (NADPH) w h i c h may i n t e r f e r e w i t h t h e f l u o r e s c e n c e s i g n a l from t h e r e s p i r a t o r y c h a i n NADH.  However,  b i o c h e m i c a l a n a l y s e s have shown t h a t NADPH does n o t change i n the c o r t e x o f t h e s q u i r r e l monkey d u r i n g a n o x i a when f l u o r e s c e n c e i s maximal (Sundt e t a l . ,  1976).  Furthermore NADPH i s o n l y 3%  o f t h e t o t a l p y r i d i n e n u c l e o t i d e p o o l (Glock and McLean, 1955) and would t h e r e f o r e c o n t r i b u t e v e r y l i t t l e  t o the f l u o r e s c e n c e  signal. P0  2  o f t h e b r a i n , and thus t h e r e d o x s t a t e o f r e s p i r a t o r y  c h a i n NAD, i s dependent n o t o n l y on a r t e r i a l P 0 the r a t e a t which b l o o d p e r f u s e s t h e b r a i n . hypercapnia,  and a f a l l  2  (PaOg) b u t a l s o  Since  i n a r t e r i a l pH ( e i t h e r from  hypoxia, hypercapnia  or l a c t a t e ) , w h i c h occur d u r i n g b r e a t h h o l d i n g , i n c r e a s e b l o o d f l o w t o t h e b r a i n ( I n g v a r and L a s s e n , 1962; McDowall, I966; P u r v e s , 1972; Kogure e t a l . , 1975; G-rubb e t a l . , lar  1970; B e t z , 1972; Borgstrom e t a l . ,  1977 and 1978) by d e c r e a s i n g c e r e b r a l v a s c u -  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 m e d i a t e d  mechanisms (Purves, 1972; B e t z , 1972), b r a i n P 0 c a n be r e g u l a t e d 2  to  a degree d u r i n g d i v i n g .  However, d u r i n g a d i v e c e r e b r a l  12  b l o o d f l o w i n n a t u r a l d i v e r s has been r e p o r t e d t o r e m a i n unchanged ( G r i n n e l l e t a l . , 19^2;  Bron e t a l . , I 9 6 6 ) o r even  decrease s l i g h t l y from the p r e d i v e l e v e l (Van C r i t t e r s e t a l . , 1965;  E i s n e r e t a l . , I 9 6 6 ; B u t l e r and J o n e s , 1971,* Kerem and  Eisner, 1973)'  On the o t h e r hand, some s t u d i e s have shown  large increases i n cerebral'blood flow during a dive 196^;  —'  Dormer e t a l . , 1977;  c e r e b r a l b l o o d f l o w was  Jones e t a l . , 1 9 7 8 ) .  (Johansen,  Even when  r e p o r t e d to>have d e c r e a s e d i t was  nevertheless sustained at higher l e v e l s than that i n other tissues. S i n c e PaOg f a l l s d u r i n g a d i v e , the p r e d i v e POg b r a i n cannot be m a i n t a i n e d  even i f c e r e b r a l b l o o d f l o w i n c r e a s e s ,  but oxygen d e l i v e r y t o the t i s s u e can be m a i n t a i n e d o f f a l l i n g PaOg.  of the  i n the  I n f a c t , oxygen consumption of the b r a i n  (CMR0 ) and l e v e l s of the adenosine phosphates (ATP, ADP  and  2  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 ) ( D u f f y e t a l . , 1972; Seisjo*, 1971;  K e t y and Schmidt, 19^8;  MacMillan  1  i n c r e a s e once P a 0 capnic hypoxia  2  Although  the l a c t a t e / p y r u v a t e r a t i o begins  f a l l s below 6 . 6 5 k P a ( 5 0  1953;  Borgstrom et. a l . ,  p r o v i d i n g t h a t b l o o d p r e s s u r e does not f a l l .  CMROg i s m a i n t a i n e d ,  and  Lambertson e t a l . ,  Cohen e_t a l . , I 9 6 7 ; Johanssen and S e i s j o , 1975; 1975)  face  to  t o r r ) d u r i n g normo-  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, d u r i n g apneic a s p h y x i a i n dogs l a c t a t e p r o d u c t i o n the b r a i n does not i n c r e a s e even a f t e r P a 0  2  by  f a l l s w e l l below  2 . 6 6 k P a (Kerem and E i s n e r , 1 9 7 3 b ) 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 b l o o d f l o w i n c r e a s e s are f a r g r e a t e r  13  t h a n d u r i n g normocapnic The  hypoxia.  importance of the c a r d i o v a s c u l a r adjustments t o  c o n t i n u e d t r a i n f u n c t i o n d u r i n g d i v i n g was E i s n e r (1973a). 18.5  shown by Kerem and  Harbor s e a l s w h i c h t o l e r a t e d d i v e s  minutes b e f o r e the onset of h y p o x i c EEG  lasting  (electroencephalo-  gram) p a t t e r n s t o l e r a t e d d i v e s l a s t i n g o n l y 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 c o n s e r v i n g oxygen f o r the b r a i n and h e a r t alone,  then  r e s p i r a t o r y c h a i n NAD s h o u l d not be reduced as f a s t as i n the b r a i n of n o n d i v i n g animals  during forced dives.  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 t o a s p h y x i a i n d i v i n g animals may  be a b l e t o t o l e r a t e more s e v e r e h y p o x i a  t r i a l animals  since divers  .than t e r r e s -  (Ridgeway e t a l . , 1969; E i s n e r e t 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 a t l e a s t a i d e d , by some form of b i o c h e m i c a l r e g u l a t i o n i f ATP production i s to  continue.  S e v e r a l b i o c h e m i c a l changes t h a t enhance o x i d a t i v e phosp h o r y l a t i o n have been r e p o r t e d f o r animals Reynafarje  acclimated to  (1971/72) showed t h a t g u i n e a - p i g s  hypoxia.  native to high  a l t i t u d e s (4500 m) h a v e h e a r t m i t o c h o n d r i a w i t h a g r e a t e r 1  a f f i n i t y ( l o w e r K ) f o r ADP and concluded ADP  t h a n those n a t i v e t o s e a  t h a t the former can generate ATP  level  f a s t e r at  c o n c e n t r a t i o n s when oxygen t e n s i o n s are low.  lower  Heart mito-  c h o n d r i a from r a t s a c c l i m a t e d t o h y p o x i a 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  -1 mole cytochrome a a ^ animals  (moles Og consumed '  -1 " min  ) over m i t o c h o n d r i a from normoxic  (Mela e t a l . , 1976) and Park e t a l . (1973) have  t h a t t h e former may have l o w e r c r i t i c a l P ° 2 '  s  ^•'  lan  ^  e  suggested latter  presumably due t o an i n c r e a s e d cytochrome a ^ a f f i n i t y f o r oxygen. Isozymes o f cytochrome a ^ h a v i n g d i f f e r e n t oxygen a f f i n i t i e s have been r e p o r t e d i n t h e 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, t h e l o w e r a f f i n i t y cytochrome a ^ i s an oxygen s e n s o r and may n o t have a r o l e i n ATP p r o d u c t i o n . C l e a r l y , i n c r e a s e d a f f i n i t i e s f o r ADP, P i , and oxygen c o u l d enhance o r p r o l o n g ATP p r o d u c t i o n when t i s s u e h y p o x i a  occurs.  On t h e o t h e r hand, Simon e t 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 t h r e e s p e c i e s o f 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 d i v e times;)however, t h e s t u d y o n l y 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  relationship  between t h e enzyme and oxygen. Many d i v i n g animals appear t o have t h e c a p a c i t y f o r g e n e r a t i n g ATP f o r t h e b r a i n b y a n a e r o b i c g l y c o l y s i s . The b r a i n o f whales (Shoubridge  e t a l . , ,1976), beavers  (Messelt  and B l i x , 1974), s e a l s ( B l i x and Fromm, 1971). and e i d e r s ( B l i x and Fromm, 1971; B l i x e t a l . , 1973) a r e known t o have a g r e a t e r p r o p o r t i o n o f t h e muscle type isozyme o f l a c t a t e dehydrogenase (LDH) t h a n t e r r e s t r i a l mammals.  The muscle  isozyme f a v o r s t h e f o r m a t i o n o f l a c t a t e ( e q u a t i o n (2), page 5) and i s i n d i c a t i v e o f t i s s u e s more t o l e r a n t t o h y p o x i a ;  con-  v e r s e l y t h e h e a r t isozyme f a v o r s t h e f o r m a t i o n o f p y r u v a t e and  15  i s i n d i c a t i v e of h i g h l y a e r o b i c t i s s u e s ( H e l l u n g - L a r s e n Andersen, 1968;'Kaplan and E v e r s e , 1972).  and  The presence of a  l a r 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 i n c r e a s e d a n a e r o b i c animals.  c a p a c i t y i n the b r a i n of the above  On the o t h e r hand, LDH isozyme p a t t e r n s of the b r a i n  of the W e d d e l l s e a l (Murphy and Hochachka, 1978) n a r w h a l ( V o g e l , 1978)  and  the  f a v o r the h e a r t type s u b u n i t 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 W e d d e l l  s e a l (Murphy and Hochachka, 1978)  are s i m i l a r t o t e r r e s t r i a l  a n i m a l s , the s u b s t r a t e f o r a n a e r o b i c metabolism,  glycogen,  appears t o be a t l e a s t 2 t o 3 times h i g h e r i n W e d d e l l s e a l b r a i n t h a n f o r o t h e r n o n d i v i n g mammals (Kerem et a l . , 1973).P y r u v a t e k i n a s e , 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 w h i c h 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 g r e a t e r a c t i v i t y w i t h i n c r e a s i n g maximal d i v e times i n the sea l i o n , h a r b o r  seal,  and W e d d e l l s e a l (Simon e t a l . , 197*0. Although  the a n a e r o b i c machinery i s p r e s e n t , can  d i v e r produce s i g n i f i c a n t amounts of ATP bically?  the  f o r the b r a i n anaero-  D u r i n g maximal d i v e s i n h a r b o r s e a l s the  CMROg  decreases and b r a i n l a c t a t e p r o d u c t i o n i n c r e a s e s f o r the 5 minutes b e f o r e the onset of h y p o x i c EEG E i s n e r , 1973a).  last  p a t t e r n s (Kerem and  D u r i n g submaximal d i v e s (20 m i n u t e s ) i n W e d d e l l  s e a l s , Murphy and Hochachka (1978) found no a p p r e c i a b l e i n brain lactate production.  S i n c e a n a e r o b i c ATP  increase  production  i s much l e s s e f f i c i e n t t h a n a e r o b i c p r o d u c t i o n i t i s q u e s t i o n a b l e  16  that s u f f i c i e n t tain cerebral The metabolism can  energy  integrity  purpose  (Seisjo  of this  1  glycolytically  t h e s i s was t o s t u d y  asphyxia.  above was u s e d  of r e s p i r a t o r y  c h a i n NAD  w h i c h have d i f f e r e n t  cerebral  (Andersen,  1959  The f l u o r o m e t r i c t e c h n i q u e  t o compare changes i n t h e r e d o x i n the c e r e b r a l  I966).  f u n c t i o n . i s maintained apneic  asphyxia,  times  After  d u c k s and c h i c k e n s ,  chemical adjustments  longer than  Ducks a r e chickens  establishing that  brain  l o n g e r i n ducks t h a n c h i c k e n s  I. examined t h e r o l e  state  c o r t e x o f two s p e c i e s  tolerances to asphyxia,  a s p h y x i a 3-8  and  energy  i n animals'that  d u r i n g a p n e i c a s p h y x i a and s t e a d y s t a t e h y p o x i a . known t o t o l e r a t e  t o main-  e t a l . , 197*0.  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  endure p r o l o n g e d  described  c a n he p r o d u c e d  physiological  p l a y i n the maintenance  during  and b i o -  of brain  function.  17  CHAPTER 1 G e n e r a l Methods - A n i m a l 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 e x p e r i m e n t a l  s i n c e they have been e x t e n s i v e l y used i n experiments  animals where  c o r t i c a l NADH was measured by t h e f l u o r o m e t r i c t e c h n i q u e (Chance e t a l . , 1 9 6 2 ) .  F i f t e e n W i s t a r r a t s (3OO-5OO g) o f  both sexes were a n e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l o f urethane  ( 1 . 3 g/kg, BDH Chemicals  L t d . , Poole,  injection  England).  A p o l y e t h y l e n e c a t h e t e r ( P E 5 0 ) was i n s e r t e d i n t o t h e 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 . rat  A f t e r a tracheostomy  each  was p a r a l y z e d 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 t u r b o c u r a r i n e  c h l o r i d e ( 1 mg/kg, Burroughs Wellcome and Co., M o n t r e a l , Canada) and v e n t i l a t e d w i t h a Narco type V5KG c o n s t a n t p r e s s u r e r a t o r (Narco Bio-Systems,  I n c . , Houston, T e x a s ) .  respi-  For fluoro-  m e t r i c r e c o r d i n g s t h e s c a l p over t h e l e f t p a r i e 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 r e t r a c t e d .  A window  approximately  1 cm x 0.5 cm was c u t i n the u n d e r l y i n g c a l v a r i a and t h e l e f t c e r e b r a l hemisphere was exposed.  The dura was l e f t  intact.  Only b l o o d - f r e e p r e p a r a t i o n s were used f o r f l u o r o m e t r i c recordings. b.  Ducks.  Experiments  were done on a t o t a l o f 75 m a l l a r d  ducks, Anas p l a t y r h y n c h o s , o f both sexes and w e i g h i n g 1 . 0 - 2 . 2 k g . A l l o p e r a t i o n s except c r a n i e c t o m i e s were performed a f t e r a l o c a l i n j e c t i o n , o f 2% w/v x y l o c a i n e ( A s t r a P h a r m a c e u t i c a l , Mississauga>, O n t a r i o ) .  T h i s procedure  produced l o c a l a n e s t h e s i a  which was s u s t a i n e d f o r s e v e r a l hours and t h e ducks showed  18  no s i g n s of s t r e s s e i t h e r d u r i n g o r a f t e r s u r g e r y .  Craniec-  tomies were performed u s i n g g e n e r a l a n e s t h e s i a 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% h a l o t h a n e venous i n j e c t i o n of urethane  i n a i r o r by an  ( 1 . 0 g/kg).  Ducks t h a t were  a n e s t h e t i z e d w i t h urethane were used f o r d e v e l o p i n g the (Chapter 2)  metric technique  out the experiment.  intra-  fluoro-  and remained a n e s t h e t i z e d through-  Halothane was  used f o r c r a n i e c t o m i e s i n  a l l o t h e r ducks and a k$ minute r e c o v e r y p e r i o d from a n e s t h e s i a preceeded a l l e x p e r i m e n t s . A polyethylene cannula  (PE 9 0 ) was  l e f t s c i a t i c a r t e r y and connected  i n s e r t e d i n t o the  t o a Statham P23GB p r e s s u r e  transducer f o r monitoring blood pressure. cock connected  A three-way s t o p -  the t r a n s d u c e r t o the c a n n u l a and a l l o w e d w i t h -  d r a w a l of a r t e r i a l b l o o d samples v i a the s i d e arm f o r b l o o d gas a n a l y s i s ( P a 0 (PE 9 0 ) was  2 >  PaCOg, pHa).  A second p o l y e t h y l e n e  cannula  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 t o c a l i b r a t e the f l u o r o m e t e r and f o r drug i n j e c t i o n s . e l e c t r o c a r d i o g r a m (ECG) e l e c t r o d e s , one  Heart r a t e was which was  determined  from the  o b t a i n e d from 2 copper w i r e  i n s e r t e d subcutaneously  i n the l e f t s i d e of  the c h e s t and the o t h e r i n s e r t e d i n the r i g h t t h i g h . was  a m p l i f i e d and f e d i n t o a r a t e m e t e r .  was m a i n t a i n e d  a t kl"—  Cloacal  c o n t r o l l e d feedback  r e g u l a t e d a h e a t i n g pad p l a c e d over the duck. 9 ducks a c a n n u l a was  signal  temperature  0 . 5 ° C by a t h e r m i s t o r i n s e r t e d  the c l o a c a and a temperature  The  into  unit that  I n a l l but  i n s e r t e d i n the t r a c h e a towards the  l u n g and the c l a v i c u l a r a i r sac was  punctured.  After paralysis  19  w i t h an intravenous  i n j e c t i o n of gallamine  F l a x e d i l , Poulenc L t d . , Montreal,  triethiodile  (1 mg/kg,  Quebec) t h e d u c k s 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 through  the t r a c h e a l cannula  a t r a t e s up t o 1 l / m i n .  b l o o d s a m p l e s were w i t h d r a w n f r o m t h e s c i a t i c  Arterial  a r t e r y and a n a l y z e d  u s i n g a R a d i o m e t e r PHM 71 g a s m o n i t o r w i t h o x y g e n a n d c a 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 m e t e r , Copenhagen, Denmark). a r t e r i a l blood values PaC0 The  2  f o rPa0  (Radio-  The a i r f l o w was a d j u s t e d t o g i v e 2  o f 11.3-14.0 k P a ( 8 5 - 1 0 5  o f 3 . 3 - 4 . 4 k P a (25-33 t o r r ) ;  remaining  u n i t t y p e E5021  torr);  a n d pHa o f 7 . 4 5 - 7 - 5 0 .  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 u s e d i n e x p e r i m e n t s i n v o l v i n g n o n p a r a l y z e d ducks (Chapter  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 f r o m 2 s t a i n l e s s s t e e l screws cemented i n t h e 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  b e t w e e n t h e s c r e w s was a m p l i f i e d 1000 t i m e s  with a Frederick  H a e r A m p l i f i e r ( F r e d e r i c k H a e r a n d Co., B r u n s w i c k , M a i n e ) a n d and  d i s p l a y e d on a 6 c h a n n e l  Watanabe WTR 281 p e n 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 , T o k y o , J a p a n ) a l o n g  with  o t h e r measured v a r i a b l e s . For f l u o r o m e t r i c recordings  animals  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 t h e h e a d 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 and  earbars.  clamp  The s c a l p o v e r t h e l e f t f r o n t a l r e g i o n o f t h e  s k u l l was i n c i s e d a n d a h o l e 1.0 underlying calvaria.  cm i n d i a m e t e r  was c u t i n t h e  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 o f t h e l e f t hemisphere, P a r s o r a l i s , "below the F i s s u r a d o r s a l i s . was  I n a l l b u t 14 ducks t h e exposed c o r t e x  covered w i t h p l a s t i c f i l m t o p r e v e n t  drying.  I n the remaining  14 ducks t h e exposed c o r t e x was covered w i t h a g l a s s window 1 cm i n d i a m e t e r and cemented t o t h e s k u l l w i t h d e n t a l cement ( H y g i e n i c M f g . Co., A k r o n ,  Ohio).  T i s s u e oxygen t e n s i o n (P^Og) was r e c o r d e d c a l l y from t h e c o r t i c a l s u r f a c e . on t h e r i g h t was  A craniectomy  polarographiwas performed  f r o n t a l r e g i o n o f t h e s k u l l and an oxygen e l e c t r o d e  placed i n contact w i t h the surface of the c o r t i c a l  corresponding  area  t o t h a t from w h i c h t h e f l u o r o m e t r i c r e c o r d i n g s  were made on t h e o p p o s i t e s i d e . i n diameter) supported  C o i l e d copper w i r e  (0.25  mm  t h e e l e c t r o d e and a l l o w e d i t t o move  w i 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 o f 43 hens, G a l l u s d o m e s t i c u s, w e i g h i n g  between 1.2 and 2.2 k g were used.  Roosters  s i n 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 procedures f o r c h i c k e n s were i d e n t i c a l  were n o t used surgery.  Operative  t o those done on ducks.  21  CHAPTER 2 S p e c i a l Techniques The The  Fluorometric Recording  1  of NADH  r e s p i r a t o r y chain i s the f o c a l point f o r bioener-  g e t i c s i n t h e b r a i n s i n c e i t l i n k s oxygen uptake w i t h ATP production  ( J O b s i s , 1972).  The r e s p i r a t o r y c h a i n a c t s as an  energy g r a d i e n t t h a t t r a n s f e r s r e d u c i n g e q u i v a l e n t s from subs t r a t e t o oxygen f o r m i n g ATP w i t h t h e 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  c h a i n f a s t e r t h a n t h e y c a n be removed b y oxygen r e s u l t i n g i n a net r e d u c t i o n o f t h e r e s p i r a t o r y c h a i n 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 o f any r e s p i r a t o r y c h a i n component c a n t h e r e f o r e s e r v e as an i n d i c a t o r o f m i t o c h o n d r i a l h y p o x i a .  The  f l u o r o m e t r i c method d e s c r i b e d by Chance e t a l . (1962) p r o v i d e s a n o n d e s t r u c t i v e , d i r e c t , and c o n t i n u o u s  (Chance e t a l . , 1970)  r e a d o u t o f changes i n t h e o x i d a t i o n - r e d u c t i o n s t a t e o f t h e f i r s t member o f t h e r e s p i r a t o r y c h a i n , NAD. S i n c e hemoglobin absorbs b o t h e x c i t a t i o n and f l u o r o e s c e n c e light,,changes o f b l o o d 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 f l u o r e s c e n c e  ( S e n n i t g e r e t a l . , 1965;  Chance and Schoener, 1965; Granholm e t a l . , 1969; K o b a y a s h i e t a l . , 1971a and b; J f l b s i s e t a l . , 1971).' J O b s i s and S t a i n s b y (I968) and J O b s i s e t a l . (1971) i n t r o d u c e d a second p h o t o m u l t i p l i e r system t o measure t h e 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 t o compensate f o r t h e hemoglobin a r t i f a c t . outputs  I f the  from t h e two p h o t o m u l t i p l i e r s a r e a d j u s t e d t o g i v e an  22  e q u a l response f o r a g i v e n , hemoglobin change i n the absence of a concomitant  NADH change t h e n t h e d i f f e r e n c e between t h e  two outputs must be due s o l e l y t o NADH.  However, when t h e  a r t i f a c t i s l a r g e , f u l l compensation i s q u e s t i o n a b l e et a l . , 1971). average,  (JObsis  S i n c e b l o o d f l o w t o t h e b r a i n i n c r e a s e s , on  8.5 times i n ducks d u r i n g p r o l o n g e d a p n e i c  asphyxia  (Jones e t a l . , 1978), compensation by u s i n g r e f l e c t e d l i g h t c o u l d be inadequate  excitation  during diving conditions.  The purpose o f t h i s s e c t i o n was t o determine  i f the blue  f l u o r e s c e n c e o r i g i n a t i n g from t h e c e r e b r a l c o r t e x o f ducks when e x c i t e d w i t h UV l i g h t was due t o NADH and, f l u o r o m e t e r t o m o n i t o r t h e NADH change.  i f so, develop a  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 e v a l u a t e d t o determine i f t h e r e was adequate compensation f o r t h e hemodynamic a r t i f a c t over t h e range o f b l o o d f l o w changes expected  d u r i n g apneic  asphyxia. a.  D e s c r i p t i o n of the fluorometer The f l u o r o m e t e r was a m o d i f i e d v e r s i o n o f t h a t d e s c r i b e d  by J O b s i s e t 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 o f a 1000 W water c o o l e d mercury a r c 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 I n c . , Sunnyvale, i n a s t a i n l e s s s t e e l housing  C a l i f o r n i a ) encased  (made b y Ron Overaker, Department  of P h y s i o l o g y and Pharmacology, Duke U n i v e r s i t y M e d i c a l S c h o o l , Durham, N o r t h C a r o l i n a ) . Power was s u p p l i e d b y a model T507 step-up t r a n s f o r m e r ( I l l u m i n a t i o n I n d u s t r i e s ) and t h e v o l t a g e t o t h e lamp (700 VAC) was c o n t r o l l e d b y a Powerestat  I36 v a r i a b l e  23  F i g u r e 2.  Diagram of the o p t i c a l d e s i g n of the used to monitor f l u c t u a t i o n s NADH i n i n t a c t t i s s u e .  fluorometer  i n respiratory  chain  2k  FLUORESCENCE PMT  450nm  SECONDARY FILTERS  BEAM SPLITTER  REFLECTANCE PMT  450nm LIGHT-  -360nm LIGHT  360nm LIGHT UV SOURCE ULTROPAK ASSEMBLY  EXCITATION FILTER 360 nm  .SPECIMEN  25  transformer (Superior E l e c t r i c  Co., B r i s t o l ,  p l a c e d on t h e p r i m a r y s i d e o f t h e step-up  Connecticut)  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 p r i m a r y  filter  ( L e i t z , W e t z l a r , Germany) h a v i n g a h a l f power band w i d t h (HPBW) of 10 nm and r e f l e c t e d onto t h e s u r f a c e o f t h e t i s s u e a t an angle o f 45-60° u s i n g a L e i t z U l t r o p a k assembly as a n i n c i d e n t light illuminator.  The l i g h t i n t e n s i t y was measured by an  E p p l e y t h e r m o p i l e No. 106-57 (The E p p l y L a b o r a t o r y , I n c . , Newport, Rhode I s l a n d ) and a t t e n u a t e d b y p l a c i n g round m i c r o scope c o v e r s l i p s (1.8 cm i n d i a m e t e r ) between t h e l i g h t  source  and t h e specimen, s i n c e 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 ( R o s e n t h a l , 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 f l u o r e s c e n c e 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 o r 2.3 mm i n diameter by a low (3.8x) o r h i g h (6.5x) power o b j e c t i v e and d i v i d e d b y an 80:20 beam s p l i t t e r ( L e i t z ) .  Changes i n  f l u o r e s c e n c e i n t e n s i t y were m o n i t o r e d w i t h a n EMI 9 5 2 4 B photom u l t i p l i e r tube (EMI Gencom I n c . , P l a i n v i e w , New Y o r k ) from the Q0% p o r t i o n o f l i g h t a f t e r t h e 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  filter  t a t i o n l i g h t was m o n i t o r e d  (Leitz).  The r e f l e c t e d  exci-  from t h e 20% p o r t i o n o f l i g h t  after  the f l u o r e s c e n c e 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, I n c . , F l u s h i n g , New Y o r k ) and a K n o t t h i g h s t a b i l i t y power s u p p l y type NSHM ( K n o t t E l e k t r o n i k , Munich, West Germany). I n l a t e r experiments  v o l t a g e s f o r b o t h p h o t o m u l t i p l i e r s were  d e r i v e d from t h e Kepco power s u p p l y by s p l i t t i n g t h e v o l t a g e  26 and v a r y i n g i t s e p a r a t e l y t o each p h o t o m u l t i p l i e r . The  f l u o r e s c e n c e s i g n a l ( F ) and t h e r e f l e c t a n c e s i g n a l  (R) were o b t a i n e d 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).  I n a d d i t i o n t h e e l e c t r o n i c s u b t r a c t i o n o f F-R  (mixer a m p l i f i e r i n F i g . 3) p r o v i d e d a t h i r d s i g n a l termed corrected fluorescence recorded  (CF).  A l l t h r e e o p t i c a l s i g n a l s were  on the 6 c h a n n e l Watanabe pen r e c o r d e r .  E q u a l i z a t i o n o f p h o t o m u l t i p l i e r outputs  t o a g i v e n hemo-  g l o b i n change was s i m i l a r t o t h e method used by JtJbsis e t a l . (1971).  The f l u o r o m e t e r was f o c u s s e d  on the b r a i n s u r f a c e , and  the v o l t a g e t o t h e p h o t o m u l t i p l i e r s was a d j u s t e d u n t i l t h e a m p l i f i e d outputs were each o f 4 v o l t s . a c c o m p l i s h e d by e q u a l i z i n g t h e outputs  F i n e adjustment was o f t h e F and R photo-  m u l t i p l i e r s when the amount o f hemoglobin i n t h e f i e l d was reduced i n the absence o f 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 w h i c h was i n t r o d u c e d t h r o u g h t h e venous c a n n u l a and t h e s e n s i t i v i t y o f t h e 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 a d j u s t e d by a l t e r i n g the  input  v o l t a g e u n t i l t h e response t o t h e f l u s h e q u a l l e d t h a t o f t h e fluorescence photomultiplier.  Generally, c a l i b r a t i o n of the  fluorometer required 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 o f t h e f l u o r o m e t e r was determined by f o c u s s i n g  the f l u o r o m e t e r  on a p i e c e o f paper and r e c o r d i n g the a m p l i f i e d  v o l t a g e outputs  o f t h e F and R p h o t o m u l t i p l i e r tubes over  3 hours.  S i n c e no NADH was p r e s e n t  the 450 nm f i l t e r -normally  27  Figure  3-  Simplified  diagram  of the  PMT's, p h o t o m u l t i p l i e r  fluorometer  tubes.  circuitry.  FLUORESCENCE FLUORESCENCE CHANNEL CORRECTED FLUORESCENCE REFLECTANCE CHANNEL REFLECTANCE  PMT'S  VARIABLE G A I N  MIXER AMPLIFIER  BUFFERS  INPUT A M P L I F I E R S  OD  29 i n s e r t e d i n f r o n t of the f l u o r e s c e n c e p h o t o m u l t i p l i e r tube was  r e p l a c e d with a 5% t r a n s m i s s i o n f i l t e r  the l i g h t .  The angle  of i l l u m i n a t i o n and r e f l e c t i o n of the  paper was a d j u s t e d t o simulate n e a r l y as p o s s i b l e . for  c.  a b i o l o g i c a l p r e p a r a t i o n as  Fluorescence  and r e f l e c t a n c e were f o l l o w e d  3 hours with l e s s than 1% d r i f t  output  ( L e i t z ) t o attenuate  i n the a m p l i f i e d v o l t a g e  i neither.  Fluorescence  emission  Fluorescence  spectra  emission s p e c t r a were made from the l e f t  c e r e b r a l cortex of p a r a l y z e d a n e s t h e t i z e d ducks d u r i n g normoxia and  anoxia  ( F i g . 4).  To make the f l u o r e s c e n c e emission  spectra  the 450 nm secondary f i l t e r was r e p l a c e d by a continuous  inter-  ference f i l t e r  over  ( V e r i l B-60),  a range of 428-507 nm.  which was manually operated  Since the p h o t o m u l t i p l i e r s e n s i t i v i t y  v a r i e d with wavelength and the i n t e r f e r e n c e f i l t e r  d i d not  t r a n s m i t l i g h t e q u a l l y over the s p e c t r a l range of t h i s measurement, the f l u o r e s c e n c e s p e c t r a i n F i g s . 4 and 5 were c o r r e c t e d for  these  inequalities.  The normoxic spectrum was taken when  the duck was v e n t i l a t e d with a i r and the anoxic spectrum was taken a f t e r death ( d e f i n e d as a blood pressure  of 0 kPa)  produced by n i t r o g e n v e n t i l a t i o n . An  i n c r e a s e i n c o r t i c a l f l u o r e s c e n c e accompanied the  t r a n s i t i o n from normoxia t o anoxia without f l u o r e s c e n c e peak (467 s h i f t s or f a l l s of blood  nm).  any s h i f t  A f t e r death the c o r t e x  i n the often  away from the f l u o r o m e t e r when i t i s d r a i n e d  (Jflbsis e t a l . , 1971).  To ensure t h a t the d i f f e r e n c e  30  Figure 4 .  Fluorescence  emission  s p e c t r a recorded  from, the  c e r e b r a l cortex of a duck d u r i n g normoxia anoxia  (death).  both s p e c t r a was  The  f l u o r e s c e n c e maximum f o r  4 6 7 nm.  a n d ' l y i n g between the two  The  square at 4 6 7  s p e c t r a was  d u r i n g extreme hypoxia before blood fell  significantly.  and  taken pressure  nm  ANOXIA  /  \  /  I  FLUORESCENCE  /  ARBITRARY UNITS  /  /  \  NORMOXIA  I  \ \ V \  \ +—4 420  440  •\  460  480  nm WAVELENGTH  1  1 500  1  I 520  32 between t h e normoxic and a n o x i c to  (death) s p e c t r a was n o t due  an a r t i f a c t caused by a movement o f t h e c o r t e x , t h e  f l u o r e s c e n c e was a l s o measured d u r i n g extreme h y p o x i a blood pressure f e l l s i g n i f i c a n t l y .  before  The square i n F i g . 4 a t  467 nm and l y i n g between the two s p e c t r a r e p r e s e n t s t h e h y p o x i c measurement.  I f a s h i f t o f t h e c o r t e x caused an a r t i f a c t i t  c o u l d be no l a r g e r t h a n t h e d i f f e r e n c e between t h e square and the a n o x i c spectrum a t 467 nm.  C l e a r l y , a t l e a s t 90^ o f t h e  f l u o r e s c e n c e i n c r e a s e a t 467 nm i s n o t a r t i f a c t u a l .  The time  r e q u i r e d f o r t h e f l u o r e s c e n c e measurements d i d n o t p e r m i t a f u l l spectrum d u r i n g extreme h y p o x i a .  Fluorescence  spectra  b e f o r e and a f t e r a n o r m o x i c - a n o x i c t r a n s i t i o n i n t h e r a t were s i m i l a r t o those  o f t h e duck, b u t t h e f l u o r e s c e n c e maximum  was between 4 6 4 and 4 6 7 nm. I n o r d e r t o c o n f i r m t h a t NADH was r e s p o n s i b l e f o r t h e change i n f l u o r e s c e n c e i n t e n s i t y d u r i n g t h e n o r m o x i c - a n o x i c t r a n s i t i o n , t h e f l u o r e s c e n c e e m i s s i o n spectrum i n a duck d u r i n g  -4 anoxia  was compared t o t h a t o f a s o l u t i o n o f NADH (7.0 x 10  Sigma, S t . L o u i s , M i s s o u r i ) ( F i g . 5 ) . was expressed  as a p e r c e n t  Fluorescence  intensity  o f t h e peak i n t e n s i t y w i t h  i n t e n s i t y a r b i t r a r i l y s e t a t 4 2 8 nm.  M,  zero  Peak f l u o r e s c e n c e  occurred  a t 467 nm and 470-473 nm f o r t h e a n o x i c b r a i n and NADH s o l u t i o n respectively.  S i n c e f l u o r e s c e n c e increased!.'during a n o x i a , a  c o n d i t i o n when NADH i s known t o i n c r e a s e , and f l u o r e s c e n c e s p e c t r a from t h e a n o x i c c o r t e x and pure NADH a r e s i m i l a r , I conclude t h a t t h e l a b i l e f l u o r e s c e n c e s i g n a l from t h e c e r e b r a l  33  F i g u r e $.  F l u o r e s c e n c e e m i s s i o n s p e c t r a from a n o x i c c e r e b r a l c o r t e x o f a duck and a s o l u t i o n o f NADH (7.0 x 1 0 " ^ M ) .  Fluorescence i n t e n s i t y  i s e x p r e s s e d as a p e r c e n t o f t h e peak i n t e n s i t y with 0 intensity a r b i t r a r i l y set at 428 nm.  F l u o r e s c e n c e maxima were 467 nm >and  470-473 nm f o r t h e a n o x i c c o r t e x and NADH respectively.  ANOXIC  NADH  BRAIN 100 T-  80  PERCENT OF  6  +  0  MAXIMUM FLUORESCENCE 40  , +  20  I—I—I—I—I—I—I 420  440  480  460  nm WAVELENGTH  500  35  c o r t e x i n ducks o r i g i n a t e s from NADH.  B i n d i n g o f the NADH t o  c e l l u l a r c o n s t i t u e n t s , m o s t l y enzymes, i s p r o b a b l y  responsible  f o r the s h i f t o f the f l u o r e s c e n c e maximum t o 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 e t a l . (1962) r e p o r t e d a s i m i l a r s h i f t i n t h e  f l u o r e s c e n c e maximum i n a n o x i c k i d n e y when compared t o pure NADH.  On t h e o t h e r hand H a r b i g e t a l . (1976) and Sundt and  Andersen (1975a) d i d n o t f i n d a s h i f t i n t h e f l u o r e s c e n c e spectrum o f t h e c e r e b r a l c o r t e x when compared t o 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 t o f i l t e r  character-  i s t i c s which a t t e n u a t e d t h e s h o r t e r wavelengths o f t h e ' s p e c t r a i n t i s s u e and s h i f t e d the apparent f l u o r e s c e n c e maximum towards t h a t o f 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 o f t h e 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  m o n i t o r i n g f l u c t u a t i o n s of-'the f l u o r e s c e n c e s i g n a l from the c e r e b r a l c o r t e x o f a r a t and a duck d u r i n g s t e a d y s t a t e c o n d i tions.  Fluorescence  was m o n i t o r e d d u r i n g normoxia f o r 3 hours  i n both s p e c i e s and a f t e r death f o r 1 hour i n a duck. t i o n i n c o r r e c t e d f l u o r e s c e n c e was l e s s than  —j>fo  Fluctua-  of the a m p l i f i e d  v o l t a g e output p e r hour when t h e l i g h t i n t e n s i t y was 0.5 mW/cm . I f p h o t o d e c o m p o s i t i o n o f NADH o r o t h e r background m a t e r i a l o c c u r r e d t h e f l u o r e s c e n c e i n t e n s i t y would have shown a n e t decrease. decrease,  S i n c e f l u o r e s c e n c e i n t e n s i t y d i d n o t show a n e t changes of. fluorochrome  decomposition  was n e g l i g a b l e .  c o n c e n t r a t i o n s due t o photo-  36  e.  Blood a r t i f a c t  compensation  Ducks were p r e p a r e d 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 as p r e v i o u s l y d e s c r i b e d , and a p o l y e t h y l e n e c a n n u l a (PE 9 0 ) was i n s e r t e d i n t h e c a r o t i d a r t e r y towards t h e b r a i n .  Bolus i n -  j e c t i o n s o f oxygenated s a l i n e i n t o t h e 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 t h e 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 i n c r e a s e 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 p e r c e n t i n c r e a s e ( p o s i t i v e ) o r p e r c e n t decrease  (negative) i n l i g h t  intensity.  E l e c t r o n i c s u b t r a c t i o n o f F-R (CF) compensated f o r changes i n hemoglobin i n t h e f i e l d when t h e change was s m a l l ; however, when t h e change was l a r g e CF i n c r e a s e d s l i g h t l y .  The same  e x p e r i m e n t a l p r o t o c o l was r e p e a t e d w i t h b o l u s i n j e c t i o n s o f concentrated r e d blood c e l l s  (oxygenated) ( F i g . 6 , B ) . When  the b o l u s reached t h e r e c o r d i n g a r e a o f t h e c e r e b r a l c o r t e x b o t h F and R decreased.  However, as b e f o r e , CE was o n l y a f f e c t e d  when t h e a r t i f a c t was l a r g e . I n F i g . ? t h e 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 r e f l e c t a n c e was examined i n 4' ducks d u r i n g f l u s h e s w i t h s a l i n e and c o n c e n t r a t e d b l o o d .  The s o l i d l i n e through t h e i n t e r c e p t  of t h e a x i s r e p r e s e n t s a 1 t o 1 correspondence  between F and  R and t h e r e f o r e f u l l compensation f o r t h e hemodynamic a r t i f a c t . P o i n t s t o t h e r i g h t o f t h e o r d i n a t e a r e from b o l u s  injections  of s a l i n e , and p o i n t s t o t h e l e f t a r e from b o l u s i n j e c t i o n s o f concentrated blood.  W i t h i n t h e range from a p p r o x i m a t e l y -15%>  to +15% change i n F, t h e R c h a n n e l f u l l y compensated f o r t h e hemodynamic a r t i f a c t , however, o u t s i d e o f t h i s range  full  37  F i g u r e 6.  Changes i n f l u o r e s c e n c e corrected fluorescence  ( F ) , r e f l e c t a n c e (R) (CF) r e c o r d e d  from the  c e r e b r a l c o r t e x of a duck when the b r a i n f l u s h e d w i t h oxygenated s a l i n e (A) and blood (B).  Each s p i k e r e p r e s e n t s one  F, R, and CF are expressed  was  concentrated flush.  as a p e r c e n t  increase  ( p o s i t i v e ) or decrease ( n e g a t i v e ) i n the i n t e n s i t y from the normoxic b a s e l i n e . s c a l e (bottom) a p p l i e s t o b o t h A and  and  The B.  light time  A  B  10 % CHANGE  0  -10  10 1 CHANGE  CO  0  -10  10 7o C H A N G E  0  AJU.  -10  1 MIN.  CF  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  ( o r d i n a t e ) and recorded b r a i n was  emitted f l u o r e s c e n c l i g h t  (abscissa)  from the c e r e b r a l cortex of ducks when the f l u s h e d with oxygenated s a l i n e  concentrated  blood  (oxygenated).  and f l u o r e s c e n c e are expressed  and  Both r e f l e c t a n c e  as a percent  i n c r e a s e ( p o s i t i v e ) or decrease (negative) i n light  i n t e n s i t y from the normoxic b a s e l i n e .  P o i n t s to the r i g h t of the o r d i n a t e are from s a l i n e f l u s h e s and p o i n t s to the l e f t are from f l u s h e s of concentrated  blood.  The  solid  line  through the i n t e r c e p t of the a x i s r e p r e s e n t s 1 to 1 correspondence between r e f l e c t a n c e and fluorescence.  a  41  compensation was q u e s t i o n a b l e .  However, t h e n a t u r e  of the  d e v i a t i o n s u g g e s t s t h a t t h e r e were accompanying a l t e r a t i o n s i n t h e NADH f l u o r e s c e n c e .  When l a 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 t h e f l u o r e s c e n c e i n c r e a s e was g r e a t e r t h a n the r e f l e c t a n c e i n c r e a s e , s u g g e s t i n g a r e d u c t i o n o f NAD which would be expected i f s a l i n e caused s l i g h t h y p o x i a . hand, when l a r g e a r t i f a c t s were produced by b o l u s of c o n c e n t r a t e d  On t h e o t h e r injections  oxygenated b l o o d , f l u o r e s c e n c e decrease was  not as l a r g e as t h e r e f l e c t a n c e decrease,  suggesting oxidation  of NADH w h i c h would be e x p e c t e d i f t h e oxygenated b l o o d s l i g h t hyperoxia.  C o n s e q u e n t l y i t appears t h a t " t r u e " b l o o d  a r t i f a c t compensation cannot be a s s e s s e d  by t h i s method i n any  a b s o l u t e sense a l t h o u g h 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 f.  Fluorescence  caused  acceptable.  during a c y c l e of hypoxia  F i g . 8 shows a r e c o r d i n g o f a r t e r i a l b l o o d and changes i n F, R, and CF r e c o r d e d  pressure  from t h e c e r e b r a l c o r t e x  of a duck d u r i n g h y p o x i a produced by Ng v e n t i l a t i o n . changes a r e expressed  Optical  i n a r b i t r a r y u n i t s (AU) where t h e CF  change from normoxia t o a n o x i a  (death) was d e f i n e d as 100 AU.  Approximately  11 seconds a f t e r t h e t r a n s i t i o n from a i r t o N »  F increased.  Hemodynamic a r t i f a c t s i d e n t i c a l t o t h e r e f l e c t a n c e  2  (R) change were superimposed on t h e f l u o r e s c e n c e  (F) trace.  E l e c t r o n i c s u b t r a c t i o n o f F-R produced t h e CF t r a c e w h i c h was due  s o l e l y t o NADH change.  As h y p o x i a p r o g r e s s e d  corrected  f l u o r e s c e n c e i n c r e a s e d t o a maximum o f 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 t o a i r , CF r e c o v e r e d and  42 I  Figure 8 .  Blood pressure  (BP) and changes i n f l u o r e s c e n c e  ( F ) , r e f l e c t a n c e (R), 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 c e r e b r a l c o r t e x of a duck  d u r i n g 3 5 seconds of n i t r o g e n v e n t i l a t i o n i n d i c a t e d by a r r o w s ) .  (as  In 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 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 a l l o p t i c a l t r a c e s are e x p r e s s e d  i n arbitrary units  where the CF change from normoxia t o (death) was  d e f i n e d as 1 0 0 AU.  as k i l o p a s c a l s ( k P a ) .  BP i s  (AU),  anoxia expressed  30 kPa  BP 15  1  100 A U  1  100AU  R  ]  CF  N  AIR  2  1 1 MIN.  100AU  44  s t a b i l i z e d a t the prehypoxic b a s e l i n e while increased blood i n t h e f i e l d caused F and R t o overshoot t h e b a s e l i n e .  After  s e v e r a l minutes C and F g e n e r a l l y r e t u r n e d t o t h e i r c o r r e s ponding prehypoxia b a s e l i n e s . E i g h t e e n p e r i o d s o f a p n e i c a s p h y x i a i n 6 ducks produced s i m i l a r responses  t o Ng v e n t i l a t i o n , w i t h t h e d i f f e r e n c e l y i n g  m a i n l y i n t h e time course o f t h e o p t i c a l change. t r i a l s t h e r e f l e c t a n c e decreased,  I n a l l 18  b u t d i d n o t exceed -15%>  change from t h e b a s e l i n e i n any i n s t a n c e except one, w h i c h showed a -17%  change.  I n t h e s e cases f u l l compensation f o r  the b l o o d a r t i f a c t was assumed t o have been a c h i e v e d . On o c c a s i o n s d u r i n g a s p h y x i a a f a l l i n b l o o d p r e s s u r e was m i r r o r e d by a CF i n c r e a s e and o c c u r r e d whether b l o o d  pressure  f e l l g r a d u a l l y ( F i g . 9 , A) o r r a p i d l y ( F i g . 9 , B ) . S i n c e t h e r e l a t i o n s h i p between b l o o d p r e s s u r e and CF was s t r i k i n g i n these i n s t a n c e s I s u s p e c t e d t h a t t h e CF change was an a r t i f a c t . The  e f f e c t s o f b l o o d p r e s s u r e change on NADH f l u o r e s c e n c e were  i n v e s t i g a t e d d u r i n g normoxia by d e c r e a s i n g t h e b l o o d p r e s s u r e 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 L t d . ) o r s l o w l y w i t h an a n t i h y p e r t e n s i v e agent ( D i a z o x i d e  (Hyperstat)  10 mg/kg, S c h e r i n g C o r p o r a t i o n L t d . , P o i n t e C l a i r e , Quebec) and t h e n r a p i d l y i n c r e a s i n g t h e b l o o d p r e s s u r e w i t h e p i n e p h r i n e (10 ug/kg, P a r k e - D a v i s  Co. L t d . , B r o o c k v i l l e , M a r y l a n d ) .  During  normoxia a r a p i d f a l l i n b l o o d p r e s s u r e produced a t r a n s i e n t CF i n c r e a s e and t r a n s i e n t decrease  i n b r a i n POg ( r e c o r d e d w i t h  an oxygen e l e c t r o d e as d e s c r i b e d l a t e r i n t h i s c h a p t e r ) w h i l e a slow decrease  i n b l o o d p r e s s u r e over s e v e r a l minutes d i d n o t  ^5  F i g u r e 9-  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 t h e c e r e b r a l c o r t e x i n ducks d u r i n g a p n e i c a s p h y x i a when mean a r t e r i a l b l o o d p r e s s u r e (MABP) f e l l . I n A a g r a d u a l f a l l i n MABP was m i r r o r e d by a CF increase. pronounced.  I n B t h e f a l l i n MABP was\more MABP i s expressed i n k i l o p a s c a l s  (kPa) and CF i s 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 t h e CF change from normoxia t o a n o x i a (death) was d e f i n e d as 1 0 0 AU. r e f e r s t o b o t h A and B.  The time b a r  ^7  produce a change i n e i t h e r CF o r t i s s u e P 0 . 2  A rapid increase  i n b l o o d p r e s s u r e , e i t h e r from- the normal l e v e l or h y p o t e n s i v e l e v e l ( a f t e r D i a z o x i d e ) a l s o produced POg.  no change i n CF o r t i s s u e  The CF change d u r i n g apnea does not appear t o be  an  a r t i f a c t but r a t h e r a t r u e NADH change due t o changes i n the t i s s u e POg.  A slow f a l l i n b l o o d p r e s s u r e p r o b a b l y a l l o w e d  time f o r i n c r e a s e s i n c e r e b r a l b l o o d f l o w t o m a i n t a i n b r a i n POg  whereas a r a p i d f a l l i n b l o o d p r e s s u r e was  by a t r a n s i e n t drop i n P 0 to  2  accompanied  c o r r e s p o n d i n g t o t h e time i t took  a l t e r c e r e b r a l blood flow. D u r i n g a p n e i c a s p h y x i a c e r e b r a l b l o o d f l o w i n ducks  i n c r e a s e s (Jones e t a l . , falls tion.  1975)  a t a time when c a r d i a c output  (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 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  b l o o d p r e s s u r e c o u l d not be accompanied by f u r t h e r r e d u c t i o n s i n vasomotor t o n e .  Consequently,  a drop i n b l o o d p r e s s u r e  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 PO . 2  J  48  P o l a r o g r a p h i c measurements o f oxygen t e n s i o n of t h e c o r t i c a l s u r f a c e The importance o f d i s s o l v e d oxygen i n l i f e p r o c e s s e s has l e d t o t h e development  of a polarographic electrode t o  m o n i t o r oxygen t e n s i o n ( P 0  2  Brink, 1942; Clark, 1 9 5 3 ) .  ) i n l i v i n g t i s s u e s (Davies and The e l e c t r o d e c o n s i s t s o f a p l a t i n u m  cathode w h i c h i s p o l a r i z e d a t -O.65 v o l t s w i t h r e s p e c t t o a s i l v e r / s i l v e r c h l o r i d e anode.  When t h e p o l a r i z e d e l e c t r o d e  i s i n c o n t a c t w i t h l i v i n g t i s s u e oxygen m o l e c u l e s from t h e t i s s u e a r e reduced a t t h e cathode: 0  2  + 2H 0 + 2e" 2  > . 'H 0 2  2  + 2 0 H " + 2e~  >  40H~  (5)  The r e d u c t i o n s t a r t s a c u r r e n t f l o w from the cathode i n t o t h e t i s s u e w h i c h i s d i r e c t l y p r o p o r t i o n a l t o t h e oxygen t e n s i o n . A t t h e anode C l ~ from t h e t i s s u e f l u i d i s a t t r a c t e d by t h e p o s i t i v e p o l a r i z i n g v o l t a g e and used i n t h e o x i d a t i o n o f silver: 4Ag  + 4C1~ — >  4AgCl + 4e"  (6)  E l e c t r o n s r e l e a s e d a t t h e anode f l o w t h r o u g h t h e e x t e r n a l c i r c u i t o f t h e oxygen e l e c t r o d e ( F i g ; 1 0 ) and equal' f l o w from t h e cathode i n t o t h e t i s s u e s .  the current  The c u r r e n t which  i s measured w i t h an ammeter i s t h e r e f o r e p r o p o r t i o n a l . t o t h e oxygen t e n s i o n o f t h e t i s s u e .  49  Figure 1 0 .  Diagram of an oxygen e l e c t r o d e 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  Ag-AgCI  Anode( + )  cr  4Ag+4Cr—*4AgCI  51  a.  D e s c r i p t i o n of the  electrode  e l e c t r o d e c o n s i s t e d of a 5 mm p i e c e of  The  i n l e a d g l a s s t u b i n g , 1.5 cm i n  wire^25 urn i n d i a m e t e r ^ f u s e d l e n g t h and 5 mm i n d i a m e t e r . e l e c t r o d e was AC  platinum  The  a c t i v e end of the  platinum  p o l i s h e d w i t h an o i l s t o n e , and d i p p e d i n Rhoplex  35 (Rohm and Hass, West H i l l ,  t o reduce p r o t e i n p o i s o n i n g .  Ontario) to provide a covering  A s i l v e r / s i l v e r chloride reference  e l e c t r o d e , 250 pa i n d i a m e t e r , was surface adjacent  t o the cathode.  ments were made, each e l e c t r o d e was  p l a c e d d i r e c t l y on the b r a i n Before p h y s i o l o g i c a l measureconditioned'by  placing i t  i n 0.9% s a l i n e and a p p l y i n g - 0 . 8 V t o the p l a t i n u m u n t i l the c u r r e n t s t a b i l i z e d ( a p p r o x i m a t e l y e l e c t r o d e was  —  cathode  3° m i n u t e s ) .  t e s t e d by m e a s u r i n g the c u r r e n t - . i n the  Each  electrode  c i r c u i t when the v o l t a g e 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 t o oxygen c o n c e n t r a t i o n show a p l a t e a u of the v o l t a g e p l o t between 0.5 and 0.7 v o l t s . acceptable  F i g . 11 shows an  c u r r e n t - v o l t a g e p l o t (polarogram) f o r l i n e a r i t y o f  oxygen t e n s i o n between 0 and 2 0 . 0  kPa  (150 t o r r ) .  output showed a r e s t i n g c u r r e n t of a p p r o x i m a t e l y for  n i t r o g e n s a t u r a t e d s a l i n e and a p p r o x i m a t e l y  for  aP0  was  3 t o 6 seconds f o r 90%  2  current-  of 2 0 kPa  ( a i r saturated s a l i n e ) .  The  of a f u l l r e s p o n s e .  Electrode 0.1-0.2 nA  5•0-6.0 nA response time Only e l e c t r o d e s  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.  52  Figure 11.  Polarogram of an a c c e p t a b l e Og Abbreviation:  nA,  nanoamps.  electrode.  19-p 18-17-16-15-14-13 -12 -11 10 CURRENT nA •  9 -8 -7 -6 -5 -4 3  +  2 1 0  +  54  b.  Oxygen measurements from the c o r t i c a l s u r f a c e of ducks d u r i n g h y p o x i a and The  hypercapnia  e l e c t r o d e was  t e s t e d by m e a s u r i n g oxygen t e n s i o n  from the c e r e b r a l c o r t e x  of a duck d u r i n g  B ) and d i f f e r e n t l e v e l s of h y p o x i a  (Fig. 1 2 , The  (P^Og)  e l e c t r o d e was  hypercapnia A).  (Fig. 1 2 ,  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 b e f o r e and a f t e r the p h y s i o l o g i c a l measurements. the v e n t i l a t o r y gas was  changed from a i r t o  9%  oxygen,  When P  T  0  decreased s h a r p l y and formed a p l a t e a u ; when the duck was t i l a t e d w i t h $% oxygen the pronounced. PrpOg  PrpOg  change was  2  ven-  s i m i l a r o n l y more  H y p e r c a p n i a ( F i g . 1 2 , B ) caused an i n c r e a s e i n  due presumably.to v a s o d i l a t i o n . F o r the purposes of t h i s s t u d y i t was  CF i n c r e a s e as a f u n c t i o n of  necessary  during hypoxia;  PrpOg  to  express  however,  the h e t e r o g e n e i t y of oxygen t e n s i o n i n the b r a i n p r e c l u d e s use  of a b s o l u t e  P r p 0  2  f o r t h i s measurement.  t e n s i o n s may v a r y from 0 kPa t o over 9 . 0 kPa  the  F o r example, oxygen ( 6 8 t o r r ) depending  on the g e o m e t r i c 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 , Lttbbers, 1 9 7 1 ;  Smith, 1 9 7 7 )  much as 9 . 0 kPa (Ltibbers, 3.5  I 9 7 I ) .  I 9 6 6 ;  and oxygen t e n s i o n can change as  ( 6 8 t o r r ) i n a d i s t a n c e of l e s s t h a n 0 , 5 I M The  r e c o r d i n g f i e l d f o r the f l u o r o m e t e r  was  mm or 2 . 3 mm i n diameter, depending on the o b j e c t i v e used,  and f l u o r e s c e n c e 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 1 9 7 1 ) .  P  T  0  2  something l e s s t h a n 1 . 5 mm ( J O b s i s e t a l . ,  In order f o r  P  T  0  2  to adequately  r e f l e c t CF,  f o r the volume of t i s s u e i n the f l u o r e s c e n c e  would have t o be determined.  the average recording  I f , however, f o r a g i v e n 1  level  55  Figure 12.  P o l a r o g r a p h i c measurements cortical gas was oxygen  surface  o f P^Og  o f a duck when t h e  ^  r  o  m  "t  n e  ventilatory  c h a n g e d f r o m a i r t o 9% o x y g e n and 5% (A).  I n B,  7%  v e n t i l a t o r y mixture. b o t h A and  B.  COg  was  added t o t h e  The t i m e b a r r e f e r s  to  56  <  57 of h y p o x i a t h e decrease i n P^Og i s p r o p o r t i o n a l t h r o u g h o u t t h e brain,then P 0 T  2  can be r e p o r t e d as a p e r c e n t decrease from t h e  normoxic c o n d i t i o n s , e x c e p t , o f c o u r s e , where P^Og i s 0.  A  s e r i e s o f experiments showed t h a t t h e above assumption was v a l i d . P^0  2  was r e c o r d e d from t h e c e r e b r a l c o r t e x 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 c h i c k e n when t h e oxygen i n t h e 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  e x p r e s s e d as a p e r c e n t decrease o f t h e e l e c t r o d e c u r r e n t where the decrease from normoxia t o a n o x i a (death) was c o n s i d e r e d 100%.  The r e s u l t s a r e 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  t o 1.0  c h i c k e n w h i c h had an r  except f o r 1 h y p o x i c regime i n t h e o f 0.65.  An a n a l y s i s o f c o v a r i a n c e  showed t h a t t h e 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 s l o p e and y - i n t e r c e p t w i t h a common e q u a t i o n o f y = 99 - 3»9x.  Not  o n l y was t h e assumption t h a t a r t e r i a l h y p o x i a produced a u n i f o r m decrease i n PrpOg throughout an i n d i v i d u a l v a l i d b u t i t a l s o h e l d for different individuals.  These r e s u l t s a r e c o n s i s t e n t w i t h  the work o f L e n i n g e r - F o l l e r t e t a l .  (1976) who showed t h a t  a r t e r i a l h y p o x i a produced a u n i f o r m decrease i n oxygen t e n s i o n r e c o r d e d s i m u l t a n e o u s l y from 8 d i f f e r e n t l o c a t i o n s i n t h e c e r e b r a l cortex.  5 8  F i g u r e 1J.  S u r f a c e POg (PrpOg)  r e c o r d e d from' the r i g h t  c e r e b r a l c o r t e x when oxygen i n the v e n t i l a t o r y gas was v a r i e d from 1 5 $  to 5% i n 4 ducks  (solid  l i n e s ) 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 (dotted l i n e s ) .  Each  line  r e p r e s e n t s a l i n e a r r e g r e s s i o n from a minimum of 1 2 p o i n t s .  PrpO-g was 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 t o anoxia (death) was c o n s i d e r e d 100%.  DUCKS CHICKEN  °/o 0  2  IN VENTILATORY  GAS  6 0  CHAPTER 3 Changes i n t h e Redox S t a t e o f R e s p i r a t o r y C h a i n NAD During Apneic Asphyxia  i n Ducks  Introduction D i v i n g mammals and "birds, a r e known t o t o l e r a t e p e r i o d s of apneic a s p h y x i a t h a t a r e d e t r i m e n t a l t o t e r r e s t r i a l (Andersen,  1 9 6 6 ) .  t o the r e f i n e m e n t ments (Scholander,  The i n c r e a s e d t o l e r a n c e has been r e l a t e d o f oxygen c o n s e r v i n g c a r d i o v a s c u l a r a d j u s t 1 9 ^ 0 ) w h i c h a r e thought t o p r o t e c t t h e  h e a r t and t h e b r a i n (Andersen,  1 9 6 6 ;  Blix,  1 9 7 6 ) .  oxygen c o n s e r v a t i o n i s w e l l documented (Scholander, B u t l e r and J o n e s ,  animals  1 9 7 l ) »  Although 19^0;  i"t has n o t been d i r e c t l y r e l a t e d t o  the b i o c h e m i c a l events i n v o l v e d w i t h ATP p r o d u c t i o n o f the b r a i n . S i n c e oxygen i s l i n k e d t o ATP p r o d u c t i o n v i a t h e r e s p i r a t o r y chain, the e f f e c t s o f i t s c o n s e r v a t i o n s h o u l d be r e f l e c t e d i n the redox s t a t e o f NAD. The  purpose o f t h i s c h a p t e r was t o examine t h e redox  change o f r e s p i r a t o r y c h a i n NAD from the c e r e b r a l c o r t e x o f ducks d u r i n g apneic a s p h y x i a and determine t h e e f f e c t s o f t h e c a r d i o v a s c u l a r adjustments on the redox s t a t e . ments were performed on n o n p a r a l y z e d on a m e t a l o p e r a t i n g t a b l e .  Although  I n i t i a l experi-  ducks w h i c h were r e s t r a i n e d the head was h e l d r i g i d ,  s t r u g g l e s which produced s l i g h t movements o f 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 t o s u b s t i -  t u t e p a r a l y z e d ducks f o r n o n p a r a l y z e d  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 t h e movement a r t i f a c t s produced by the s t r u g g l e s  61  could be e l i m i n a t e d .  I compared c a r d i o v a s c u l a r responses and  change i n c o r t i c a l NADH i n p a r a l y z e d determine i f indeed  and nonparalyzed ducks to  t h i s s u b s t i t u t i o n was p o s s i b l e .  62  Methods a.  Fluorescence  r e c o r d i n g s from n o n p a r a l y z e d  NADH f l u o r e s c e n c e was m o n i t o r e d  ducks  from t h e l e f t c e r e b r a l  c o r t e x o f 9 ducks u s i n g g l a s s windows as d e s c r i b e d i n Chapter 1. The  b i r d s were s e c u r e d v e n t r a l s i d e down on a m e t a l  operating  t a b l e and t h e head was h e l d m o t i o n l e s s w i t h t h e b i l l a t 4-5° below h o r i z o n t a l by 3 m e t a l screws ( 3 cm l o n g ) , s e c u r e d t o t h e s k u l l w i t h d e n t a l cement, and b o l t e d t o rods f i x e d t o the table.  metal  I n 5 o f t h e 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 t h e 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 .  Ducks were s u b j e c t e d t o p e r i o d s o f a p n e i c a s p h y x i a ( 4 t o 7 m i n u t e s ) produced by submerging the b i l l , water.  n a r e s , and eyes i n  Each duck was exposed t o 2 p e r i o d s o f submergence  w i t h a 4 5 - 6 0 minute r e c o v e r y p e r i o d between them. b.  Fluorescence  r e c o r d i n g s from p a r a l y z e d ducks  NADH f l u o r e s c e n c e was m o n i t o r e d  from t h e l e f t c e r e b r a l  c o r t e x o f 28 p a r a l y z e d ducks u s i n g p l a s t i c f i l m t o c o v e r t h e exposed c o r t e x t o p r e v e n t d r y i n g .  Ducks were s u b j e c t e d t o 2  or 3 p e r i o d s o f apneic a s p h y x i a l a s t i n g from 2 t o 9 minutes w i t h a 3 ° minute r e c o v e r y p e r i o d between a s p h y x i c p e r i o d s . A b l o o d sample was withdrawn f o r b l o o d gas a n a l y s i s b e f o r e each a s p h y x i c p e r i o d and v e n t i l a t i o n was a d j u s t e d t o g i v e s u i t a b l e b l o o d gas v a l u e s  (Chapter 1 ) .  A d d i t i o n a l b l o o d samples  were t a k e n f o r b l o o d gas a n a l y s i s a t v a r i o u s times v e n t i l a t i o n was stopped.  after  63  c.  I n h i b i t i o n o f t h e c a r d i o v a s c u l a r adjustments d u r i n g  apneic  a s p h y x i a i n ducks E i g h t m a l l a r d ducks o f e i t h e r sex, w e i g h i n g 1 . 2 - 2 . 0 kg> were used i n t h i s s e r i e s o f experiments and were p r e p a r e d as d e s c r i b e d i n s e c t i o n b, except i n 2 ducks t h e window method was used f o r o p t i c a l r e c o r d i n g s , 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 a p p r o p r i a t e ,  and CF were measured d u r i n g  2 minute p e r i o d s o f a p n e i c a s p h y x i a b e f o r e and a f t e r t h e 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 s u l f a t e ( 2 . 5 mg/kg, BDH C h e m i c a l s L t d . , P o o l e , E n g l a n d ) .  The e f f e c t s  of a t r o p i n e were t e s t e d b e f o r e and a f t e r a p n e i c a s p h y x i a by i n j e c t i o n s o f 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). hypotension  The  and b r a d y c a r d i a n o r m a l l y produced by a c e t y l c h o l i n e  i s a b o l i s h e d by a t r o p i n e and the absence o f h y p o t e n s i o n  was  c o n s i d e r e d as t h e i n h i b i t i o n o f c a r d i o v a s c u l a r a d j u s t m e n t s . Each duck was exposed t o 4 - 5 p e r i o d s o f apneic 2-3  asphyxia,  p r e - a t r o p i n e 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. F o r t h e purpose o f e x p r e s s i n g 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 f l u o r e s c e n c e i n t e n s i t y d u r i n g normoxia ( b a s e l i n e ) was d e f i n e d as zero and t h e f l u o r e s c e n c e  intensity  f o l l o w i n g death by a n o x i a was d e f i n e d 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 t h a t c o r r e s p o n d  to i n t e n s i t i e s  greater  t h a n the b a s e l i n e (NAD r e d u c t i o n ) a r e d e s i g n a t e d w i t h a p o s i t i v e s i g n ; c o n v e r s e l y , o p t i c a l changes t h a t have i n t e n s i t i e s l e s s t h a n the b a s e l i n e (NADH o x i d a t i o n ) a r e d e s i g n a t e d w i t h a • ~-_  64  negative s i g n .  Numerical  values i n the f i g u r e s are expressed  as means —1 standard e r r o r 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 (P<0.05)  analysis  of the data w i t h 5 $  c o n s i d e r e d the a c c e p t a b l e l e v e l of s i g n i f i c a n c e .  65  Results a.  Comparison o f n o n p a r a l y z e d  and p a r a l y z e d ducks  H e a r t r a t e , mean a r t e r i a l b l o o d p r e s s u r e  (MABP), and  c o r r e c t e d f l u o r e s c e n c e were compared i n p a r a l y z e d and nonp a r a l y z e d ducks d u r i n g t h e f i r s t 2 minutes o f a s p h y x i a and 4 0 seconds o f r e c o v e r y ( F i g . 1 4 ) . was  A s p h y x i a i n " n o n p a r a l y z e d ducks  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 .  Since the  l e n g t h o f the a s p h y x i c p e r i o d s i n both p a r a l y z e d and n o n p a r a l y z e d ducks v a r i e d , t h e r e c o v e r y phase i n F i g . 14 s t a r t e d from' t e r m i n a t i o n of asphyxia.  Mean p r e - a s p h y x i c h e a r t r a t e f o r p a r a l y z e d  and nonparalyzedcducks  was 2 5 5 ~ 3 2 » 0 4 beats/minute  o f a s p h y x i a i n 20 ducks) and 247 ~ 1 3 ' ° 5 beats/minute of a s p h y x i a i n 6 ducks) r e s p e c t i v e l y .  (55 periods (11  periods  D u r i n g t h e f i r s t minute  of a s p h y x i a mean h e a r t r a t e f e l l t o 4 4 $ and 2 3 $ o f t h e c o n t r o l r a t e i n p a r a l y z e d and n o n p a r a l y z e d  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 o f a s p h y x i a . A l t h o u g h mean h e a r t r a t e i n n o n p a r a l y z e d  ducks was l o w e r d u r i n g  a s p h y x i a , o n l y a t 60 seconds was t h e d i f f e r e n c e between p a r a l y z e d and n o n p a r a l y z e d  ducks s i g n i f i c a n t .  Nonparalyzed  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 a f t e r emersion;  ducks  immediately  h e a r t r a t e i n c r e a s e d from l e s s t h a n 5 0 b e a t s /  minute t o 3 7 6 — 2 4 . 0 0 beats/minute  (11 periods of asphyxia  i n 6 ducks) a f t e r 10 seconds and g r a d u a l l y r e t u r n e d t o p r e a s p h y x i c l e v e l s d u r i n g t h e n e x t 40 seconds. was  When v e n t i l a t i o n  resumed i n p a r a l y z e d ducks, h e a r t r a t e r e t u r n e d t o t h e p r e -  a s p h y x i c l e v e l a f t e r 90-120 seconds w i t h o u t a t a c h y c a r d i a .  66  F i g u r e Ik.  Comparison between h e a r t r a t e (HR), mean a r t e r i a l "blood p r e s s u r e  (MABP), and c o r r e c t e d f l u o r e s c e n c e  (CF) d u r i n g 11 p e r i o d s o f a p n e i c a s p h y x i a i n 6 nonparalyzed  ducks ( d o t t e d l i n e ) and 55 p e r i o d s  of a p n e i c a s p h y x i a i n 20 p a r a l y z e d ducks line).  (solid  A s p h y x i a was produced "by submerging t h e  f a c e i n water and by s t o p p i n g t h e 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 p a r a l y z e d ducks  respectively.  (abscissa) during  E l a p s e d time  a s p h y x i a and t h e r e c o v e r y i s g i v e n 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 t o a n o x i a was  d e f i n e d as 100 AU.  (death)  Each p o i n t r e p r e s e n t s  the mean — SEM and t h e SEM i s c o n t a i n e d w i t h i n the p o i n t when absent.  NONPARALYZED PARALYZED-  400 350 300 250 HEART RATE BEATS/MIN.  200 150  +  100 50 0  J-  25 20 MABP  15  kPa  10  ri»m  5 0 25 20 CF  15  AU  10 5 0 -5  1  10  1  20  1  30  1  40  h  50  60  H  70  1  80  1  90  1  100  ASPHYXIA  h  110  120  10  20  RECOVERY TIME (SECONDS)  68  Before a s p h y x i a MABP was 2 0 . 1 - 1 . 0 4 0 k P a ( 1 5 1 and  17.8 - 0 . 6 6 0 k P a ( 1 3 4 -  7 . 8 mm Hg)  4 . 5 mm Hg) i n n o n p a r a l y z e d and  p a r a l y z e d ducks r e s p e c t i v e l y , and showed o n l y minor changes d u r i n g and a f t e r  asphyxia.  An i n c r e a s e i n CF o c c u r r e d i n b o t h p a r a l y z e d and nonp a r a l y z e d ducks d u r i n g a s p h y x i a .  Moreover t h e r a t e s o f i n c r e a s e  were s i m i l a r and n e a r l y c o n s t a n t between s a m p l i n g  intervals.  A f t e r 1 2 0 seconds o f a s p h y x i a , CF i n c r e a s e d by 1 5 - 1 . 9 4 AU (n = 5 5 p e r i o d s o f a s p h y x i a i n 2 0 ducks) and 18.5 -  6 . 3 5 AU  (n = 1 1 p e r i o d s o f a s p h y x i a i n 6 ducks) i n p a r a l y z e d and nonp a r a l y z e d ducks r e s p e c t i v e l y . When a s p h y x i a was t e r m i n a t e d mean CF d e c r e a s e d and was below t h e b a s e l i n e 4 0 seconds a f t e r a s p h y x i a i n b o t h groups.  I n b o t h p a r a l y z e d and n o n p a r a l y z e d  ducks, r e c o v e r y o f CF a f t e r a s p h y x i a 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 o v e r s h o o t o f t h e b a s e l i n e which g e n e r a l l y r e t u r n e d to  t h e >preasphyxic l e v e l a f t e r 5 - 1 ° m i n u t e s . A 4 . 7 minute p e r i o d o f submergence a s p h y x i a and t h e  subsequent r e c o v e r y i n a s i n g l e n o n p a r a l y z e d Fig.  15.  duck i s snown i n  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 t h e d u r a t i o n o f submergence. MABP was m a i n t a i n e d  n e a r t h e p r e d i v e 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 t r a c e s from t h e t o p d e s c r i b e t h e o p t i c a l s i g n a l s . I n a l l cases an i n c r e a s e i n l i g h t d e t e c t e d by t h e 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 o f t h e pen. Note t h e change i n s c a l e i n t h e CF t r a c e ( f i f t h t r a c e ) from t h e F and R t r a c e s ( t h i r d and f o u r t h t r a c e s r e s p e c t i v e l y ) . The a b r u p t  69  Figure  15.  A 4 . 7 minute p e r i o d o f submergence a s p h y x i a and recovery  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 r r o w a t t h e b o t t o m i n d i c a t e s s u b m e r s i o n and t h e upward p o i n t i n g arrow cates  emersion.  indi-  An upward d e f l e c t i o n o f t h e  o p t i c a l traces represents  an i n c r e a s e  i n light  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 t h e CF change f r o m normoxia t o anoxia  ( d e a t h ) was d e f i n e d a s 1 0 0 AU.  Time bars, 1 s e c o n d a n d 1 m i n u t e r e s p e c t i v e l y , denote t h e changes i n c h a r t Abbreviations« a r t e r i a l blood fluorescence;  HR,  heart  pressure;  speed.  r a t e ; MABP, mean k P a , k i l o p a s c a l s ; F,  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, microvolts.  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 t h e large, slower, n e g a t i v e d e f l e c t i o n a f t e r emersion a r e artifacts  caused most p r o b a b l y by s l i g h t movement o f t h e c o r t e x .  Note t h a t these a r t i f a c t s  a r e p a r a l l e l e d i n t h e R t r a c e and  t h a t e l e c t r o n i c s u b t r a c t i o n o f F-R g i v e s t h e CF t r a c e which i s free of a r t i f a c t s  and due s o l e l y t o NADH.  About 80 seconds  a f t e r submersion,CF began t o i n c r e a s e and g r a d u a l l y i n c r e a s e d to 5 AU a f t e r 2 m i n u t e s .  The t r e n d c o n t i n u e d u n t i l about  4.5 minutes a f t e r submersion markedly; occurred.  when b l o o d p r e s s u r e dropped  a t t h i s p o i n t a s l i g h t l y s h a r p e r i n c r e a s e i n CF EEG a c t i v i t y d i m i n i s h e d (bottom t r a c e ) when CF was  a p p r o x i m a t e l y 32 AU.  I n ducks t h a t d i d n o t show a p r e c i p i t o u s  drop i n b l o o d p r e s s u r e t h e EEG d i m i n i s h e d 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  o c c u r r e d when f l u o r e s c e n c e had  i n c r e a s e d from between 28-40 AU i n a l l n o n p a r a l y z e d  ducks.  On emersion h e a r t r a t e r o s e above t h e p r e d i v e 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 r e t u r n e d t o t h e p r e d i v e 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 f l u o r e s c e n c e decreased s h a r p l y and was f o l l o w e d by a g r a d u a l decrease over 3 minutes and even b r i e f l y d i p p e d below the b a s e l i n e b e f o r e r e t u r n i n g t o t h e p r e d i v e l e v e l .  Some o t h e r  ducks showed an immediate b a s e l i n e overshoot o m i t t i n g t h e 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 o f a p n e i c a s p h y x i a p r o duced 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 a p a r a l y z e d duck, and t h e v a r i a b l e s measured appear t o show t h e same t r e n d s as i n nonparalyzed^ a n i m a l s ^  72  F i g u r e 16.  Response i n a p a r a l y z e d duck t o a 2 minute p e r i o d of a p n e i c a s p h y x i a produced by s t o p p i n g ventilation.  Fluorescence  artificial  (F), reflectance (R),  and c o r r e c t e d f l u o r e s c e n c e (CF) were 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 and 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 t h e CF change from normoxia t o a n o x i a (death) was d e f i n e d as 100 AU. The p e r i o d o f a s p h y x i a i s i n d i c a t e d by t h e 2 arrows.  Other a b b r e v i a t i o n s :  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 ; k P a , k i l o pascals.  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  d u r i n g apneic  a s p h y x i a i n ducks E i g h t ducks were exposed t o 2 minute p e r i o d s o f a p n e i c a s p h y x i a b e f o r e and a f t e r the c a r d i o v a s c u l a r adjustments a p n e i c a s p h y x i a were a b o l i s h e d . and b r a d y c a r d i a produced was  Over  to  of the h y p e r t e n s i o n  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  a b o l i s h e d by a t r o p i n e .  T a b l e I shows the CF i n c r e a s e i n  8 ducks a f t e r 1 and 2 minutes of a p n e i c a s p h y x i a b e f o r e a f t e r atropine treatment.  I n e v e r y duck CF was  and  greater after  1 2 0 seconds of a s p h y x i a f o l l o w i n g t r e a t m e n t w i t h a t r o p i n e t h a n i n a s p h y x i a b e f o r e a t r o p i n e t r e a t m e n t and the d i f f e r e n c e was  significant.  (8 -  Note t h a t CF was  s i m i l a r i n the normal  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 a s p h y x i a and o n l y 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 a more o x i d i z e d r e s p i r a t o r y c h a i n a p p a r e n t . ;  P 0 T  2  i n maintaining  F i g . 17  shows  and CF t r a c e s d u r i n g a p n e i c a s p h y x i a b e f o r e (C) and  (A) an a t r o p i n e i n j e c t i o n .  After  2  minutes o f a s p h y x i a  after PrpOg  decreased by 6 1 $ and CF i n c r e a s e d 25 AU b e f o r e 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 72$  i n c r e a s e d 4 4 AU i n the same time p e r i o d .  and  CF  75  Table I .  I n c r e a s e i n c o r r e c t e d f l u o r e s c e n c e (CF) i n p a r a l y z e d ducks a f t e r 60 and 120 seconds o f a p n e i c a s p h y x i a b e f o r e and a f t e r a t r o p i n e t r e a t ment.  The numbers i n parentheses  of a s p h y x i c p e r i o d s . bottom o f t h e t a b l e .  a r e the numbers  Mean — SEM AU a r e " a t the  76  Apneic Asphyxia Before A t r o p i n e Treatment  Apneic Asphyxia A f t e r A t r o p i n e Treatment  Corrected  Corrected  Fluorescence  Fluorescence  60 s e c  120 s e c  60 s e c  120 s e c  1  8(2)*  39(2)  8(2)  47(2)  2  3(1)  6(1)  1(2)  16(2)  3  5(3)  23(3)  11(2)  44(2)  4  4(2)  19(2)  10(2)  36(2)  5  8(3)  21(3)  8(2)  43(2)  6  18(3)  23(3)  23(2)  50(2)  7  19(2)  3K2)  25(2)  45(2)  3(2)  29(2)  7(2)  32(2)  Duck Number  Mean - SEM  1  2.29  The numbers i n p a r e n t h e s e s asphyxia.  23 ~ 3-39  12 - 2 . 9 0  39 - 3 . 8 9  a r e t h e number o f p e r i o d s o f a p n e i c  77  Figure 17.  POg  o f t h e r i g h t c o r t i c a l s u r f a c e (Pr^Og) and  corrected fluorescence  (CF) from t h e l e f t  s u r f a c e d u r i n g 2 minutes o f a p n e i c (indicated  cortical  asphyxia  by a r r o w s ) i n a p a r a l y z e d duck b e f o r e  (C) and a f t e r an a t r o p i n e i n j e c t i o n ( A ) . i s expressed  Prp £ 0  as a p e r c e n t decrease o f t h e e l e c -  t r o d e c u r r e n t when t h e decrease from normoxia to a n o x i a  (death) was d e f i n e d as a 1 0 0 $  CF was expressed  i n arbitrary units  (AU) when  the CF change from n o r m o x i a t o a n o x i a was d e f i n e d as 1 0 0 A U .  decrease.  (death)  79  Discussion The  a p n e i c a s p h y x i a produced i n p a r a l y z e d ducks "by  s t o p p i n g 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 s i m u l a t e s the onset o f c a 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 c h a i n NADH i n n o n p a r a l y z e d  ducks d u r i n g f o r c e d submersion.  Although  b r a d y c a r d i a d u r i n g a s p h y x i a i n p a r a l y z e d ducks was n o t as severe as i n n o n p a r a l y z e d  ducks, t h e r e were no s i g n i f i c a n t  d i f f e r e n c e s between t h e 2 groups except a t t h e 6 0 second sampling  interval.  The major d i f f e r e n c e was found i n t h e  r e c o v e r y p e r i o d where t h e p a r a l y z e d group l a c k e d t h e t y p i c a l postasph'yxic.tachycardia; . instead heart rate g r a d u a l l y returned t o t h e p r e - a s p h y x i c r a t e over a p e r i o d o f 9 0 - 1 2 0 seconds. Bamford and Jones ( 1 9 7 4 ) r e p o r t e d s i m i l a r r e s u l t s f o r p a r a l y z e d and n o n p a r a l y z e d  ducks and l a t e r c l a i m e d i t i s caused by t h e  absence o f n e u r a l i n p u t from t h e 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 p a r a l y z e d animals does n o t cause an i n c r e a s e i n lung pressure during postasphyxic v e n t i l a t i o n Jones, 1 9 7 6 ) .  (Bamford and  NADH f l u o r e s c e n c e i n b o t h groups g r a d u a l l y  i n c r e a s e d d u r i n g t h e f i r s t 2 minutes o f a s p h y x i a and r e t u r n e d t o t h e b a 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 f l u o r e s c e n c e was almost i d e n t i c a l i n  both groups d u r i n g t h e e n t i r e c y c l e . " Only 1 0 seconds a f t e r t h e t e r m i n a t i o n o f a s p h y x i a d i d NADH f l u o r e s c e n c e d i f f e r  signifi-  c a n t l y due t o a f a s t e r r e c o v e r y r a t e i n t h e n o n p a r a l y z e d The  redox s t a t e o f r e s p i r a t o r y c h a i n components .has  animals'. been  s t u d i e d i n t h e b r a i n d u r i n g h y p o x i a and a n o x i a which were produced by v a r y i n g oxygen i n t h e v e n t i l a t o r y gas from 0 - 2 0 $  80  (Chance  e t a l . , 1962,  1964,  1973; Chance and Schoener,  1962),  and by r e d u c i n g o r s t o p p i n g b l o o d f l o w ( i s c h e m i a ) (Sundt and Andersen, 1975h; Sundt e t a l . , 1976; R o s e n t h a l e t a l . ,  1976b;  LaManna e t a l . , 1977; G i n s b e r g e t a l . , 1976); b u t s t u d i e s d u r i n g a p n e i c a s p h y x i a have n e v e r been r e p o r t e d b e f o r e t h i s study.  A l l t h r e e means o f p r o d u c i n g a f a l l i n t i s s u e oxygen  t e n s i o n s h a r e t h e same t y p e o f r e s p o n s e , namely a r e d u c t i o n of t h e r e s p i r a t o r y c h a i n components; however, t h e k i n e t i c s of t h e r e d u c t i o n a r e d i f f e r e n t due t o d i f f e r e n t r a t e s o f oxygen depletion.  A t t h e onset o f h y p o x i a , r e d u c i n g e q u i v a l e n t s from  the s u b s t r a t e a r e passed i n t o t h e r e s p i r a t o r y c h a i n f a s t e r t h a n t h e y a r e removed by oxygen.  The reduced form o f t h e  c a r r i e r s i n c r e a s e s a t t h e expense'-o"f-the o x i d i z e d - f o r m ' (NADH and NAD i n t h e case o f t h e p r e s e n t s t u d y ) and s e e k . a more reduced redox s t a t e .  I n a p n e i c a s p h y x i a oxygen i s c o n t i n u a l l y  d e p l e t e d and r e d u c t i o n approaches  t h e maximum o b t a i n a b l e a t  d e a t h u n l e s s v e n t i l a t i o n i s resumed. The importance o f t h e c a r d i o v a s c u l a r a d j u s t m e n t s f o r p r o t e c t i n g t h e b r a i n d u r i n g a p n e i c a s p h y x i a was demonstrated by m o n i t o r i n g NADH f l u o r e s c e n c e and b r a i n PrpOg d u r i n g a p n e i c a s p h y x i a i n ducks b e f o r e and a f t e r t h e 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 o f t h e c a r d i o v a s c u l a r adjustments produced a g r e a t e r 1  r e d u c t i o n o f NAD and a g r e a t e r decrease i n b r a i n PjpOg t h a n before the i n h i b i t i o n .  However, t h e e f f e c t s o f t h e c a r d i o -  v a s c u l a r adjustments on CF and P 0 T  d u r i n g t h e f i r s t minute  2  were n o t r e a d i l y  apparent  o f a s p h y x i a ( T a b l e I and F i g . 17).  81  For example, a f t e r 1 minute o f asphyxia, CF was 12 AU and 8 AU i n a t r o p i n i z e d and n o n - a t r o p i n i z e d ducks r e s p e c t i v e l y . I t was e x p e c t e d t h a t e f f e c t s o f t h e c a r d i o v a s c u l a r adjustments s h o u l d a l s o be apparent d u r i n g t h e f i r s t minute o f a s p h y x i a . 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 n o n - a t r o p i n i z e d ducks (as i n d i c a t e d from h e a r t r a t e ) , and thus oxygen c o n s e r v a t i o n , were n o t complete u n t i l 4 0 - 6 0 seconds a f t e r v e n t i l a t i o n was stopped ( F i g . 1 4 ) .  artificial  Only i n t h e second m i n u t e ,  when oxygen c o n s e r v a t i o n i s maximal, a r e l a r g e d i f f e r e n c e s i n PrpOg between t h e p e r i o d s  o f a s p h y x i a b e f o r e and a f t e r a t r o p i n e  i n j e c t i o n s r e a d i l y apparent.  Second, t h e r e l a t i o n s h i p between  NADH f l u o r e s c e n c e and Prp0 was s u c h t h a t v e r y l i t t l e 2  i n NADH f l u o r e s c e n c e o c c u r r e d u n t i l PrpOg (Fig.  22, Chapter 4 ) .  After P Q T  changes i n t i s s u e o x y g e n a t i o n  2  h  a  s  change  d e c r e a s e d by 5 ° $  has d e c r e a s e d by 5 0 $ s m a l l  caused l a r g e i n c r e a s e s i n CF.  S i n c e P T 0 2 d i d n o t f a l l below 5 0 $ i n e i t h e r t h e a t r o p i n i z e d or n o n - a t r o p i n i z e d animals  d u r i n g t h e f i r s t minute o f a s p h y x i a ,  l a r g e d i f f e r e n c e i n CF would n o t be expected.  82 CHAPTER 4C e r e b r a l Energy M e t a b o l i s m  i n Ducks and Chickens  D u r i n g A p n e i c A s p h y x i a and Hypoxia Introduction The p r i m a r y means o f ATP p r o d u c t i o n i n t h e b r a i n ( o x i d a t i v e p h o s p h o r y l a t i o n ) r e q u i r e s oxygen t o a c c e p t  reducing  e q u i v a l e n t s from t h e r e s p i r a t o r y c h a i n and b r a i n f u n c t i o n c a n c o n t i n u e o n l y as l o n g 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 c a n m a i n t a i n b r a i n f u n c t i o n over 4- times l o n g e r d u r i n g 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).  p h y s i o l o g i c a l adjustments  I n Chapter  w h i c h conserved  3 I showed t h a t  oxygen d u r i n g a s p h y x i a  were r e s p o n s i b l e f o r p r o l o n g i n g both t i s s u e o x y g e n a t i o n and r e d u c t i o n o f r e s p i r a t o r y c h a i n 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 t h a t b r a i n f u n c t i o n i s p r o l o n g e d d i r e c t l y as a r e s u l t o f 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 s e v e r e  t h a n t e r r e s t r i a l animals 1973  ( E i s n e r e t a l . , 1970;  a and b; Ridgeway e t a l , , 1969)  hypoxia  Kerem and E i s n e r ,  indicating that i n addi-  t i o n t o p h y s i o l o g i c a l adjustments  some form o f b i o c h e m i c a l  adaptation i s present i n d i v e r s .  I n t h e o r y , these c o u l d be  a d a p t a t i o n s which would (1)  allow oxidative phosphorylation  t o c o n t i n u e a t l e v e l s o f h y p o x i a t h a t cannot be t o l e r a t e d by n o n d i v e r s and/or (2) enhance a n a e r o b i c ATP p r o d u c t i o n t o supplement o x i d a t i v e p h o s p h o r y l a t i o n i n t h e f a c e o f d e c r e a s i n g oxygen.  83  The purpose o f t h i s c h a p t e r was t o , f i r s t , t e s t t h e hypot h e s i s t h a t t h e r e d u c t i o n of r e s p i r a t o r y c h a i n NAD proceeds a t a s l o w e r r a t e d u r i n g a p n e i c a s p h y x i a i n ducks, Anas P l a t y rhynchos,  t h a n c h i c k e n s , G a l l u s domesticus.  Ducks a r e known  t o t o l e r a t e a p n e i c a s p h y x i a 3-8 times l o n g e r t h a n 1959 and I 9 6 6 ; S c h o l a n d e r ,  (Andersen,  1964).  chickens  Second, I i n v e s t i -  gated t h e r e l a t i o n between t h e redox s t a t e o f r e s p i r a t o r y c h a i n NAD and EEG i n ducks and c h i c k e n s d u r i n g a p n e i c  asphyxia  t o e l u c i d a t e any major c o n t r i b u t i o n t o ATP p r o d u c t i o n by anaerob i c metabolism  i n ducks.  In vivo fluorometric recordings i n  the a n e s t h e t i z e d r a t have i d e n t i f i e d t h e 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 w h i c h b r a i n e l e c t r i c a l v i t y ceases 1973)-  acti-  (Chance and Schoener, 1962; Mayevsky and Chance,  D u r i n g Ng v e n t i l a t i o n t h e CPNR occurs when t h e  f l u o r e s c e n c e i n c r e a s e from t h e normoxic l e v e l i s a p p r o x i m a t e l y 3/4 of the l e v e l r e c o r d e d a t death.  I f ducks r e l y on l a r g e  a n a e r o b i c c o n t r i b u t i o n s o f ATP f o r the c o n t i n u a t i o n o f b r a i n f u n c t i o n , t h e y s h o u l d have a .more reduced r e s p i r a t o r y c h a i n t h a n the c h i c k e n b e f o r e t h e appearance o f gross a l t e r a t i o n s i n EEG.  Third, to t e s t f o r biochemical adaptations  involving  o x i d a t i v e p h o s p h o r y l a t i o n the redox s t a t e o f r e s p i r a t o r y c h a i n NAD o f ducks was compared t o c h i c k e n s a t v a r i o u s l e v e l s o f hypoxia.  I f b i o c h e m i c a l a d a p t a t i o n s a r e p r e s e n t i n ducks, then  the a c c u m u l a t i o n o f r e s p i r a t o r y c h a i n NADH f o r a g i v e n l e v e l of h y p o x i a s h o u l d be l e s s i n ducks t h a n c h i c k e n s .  84  Methods a.  F l u o r e s c e n c e r e c o r d i n g s from'.paralyzed ducks and c h i c k e n s NADH f l u o r e s c e n c e was m o n i t o r e d  from t h e l e f t c e r e b r a l  c o r t e x o f 2 0 p a r a l y z e d c h i c k e n s and 3 ° p a r a l y z e d ducks.  Plastic  f i l m was: p l a c e d over t h e exposed c o r t e x t o p r e v e n t d r y i n g . Chickens  and ducks were s u b j e c t e d t o 2 o r 3 p e r i o d s o f a p n e i c  a s p h y x i a l a s t i n g 1 minute  and from 2 t o 9 minutes r e s p e c t i v e l y .  A s p h y x i c p e r i o d s were s e p a r a t e d by a 2 0 - 6 0 minute r e c o v e r y 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 c h i c k e n s EEG was  r e c o r d e d and t h e a s p h y x i c 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 endpoint).  ( r e f e r r e d t o i n t h i s paper as EEG  I n these experiments  o n l y one a s p h y x i c t r i a l .  each a n i m a l was exposed t o  A b l o o d sample was w i t h d r a w n f o r  b l o o d gas a n a l y s i s b e f o r e each a s p h y x i c p e r i o d and v e n t i l a t i o n was  a d j u s t e d t o g i v e normal b l o o d gas v a l u e s (Chapter  b.  Concurrent  1).  f l u o r o m e t r i c and p o l a r o g r a p h i c r e c o r d i n g s  during hypoxia NADH f l u o r e s c e n c e was r e c o r d e d from t h e l e f t c e r e b r a l c o r t e x o f 1 3 ducks and 9 c h i c k e n s as d e s c r i b e d i n s e c t i o n a. PrpOg was r e c o r d e d  from t h e a r e a o f t h e r i g h t c e r e b r a l c o r t e x  t h a t corresponded  t o t h e a r e a used f o r o p t i c a l r e c o r d i n g s on  the o p p o s i t e hemisphere.  Both s p e c i e s were exposed t o s e v e r a l  l e v e l s o f normocapnic h y p o x i a (1 minute i n d u r a t i o n ) by v a r y i n g the oxygen i n t h e v e n t i l a t o r y gas from 2 - 2 0 $ i n a b a l a n c e o f nitrogen.  The gases were mixed w i t h flowmeters  complete w i t h  85 screw v a l v e s and a n a l y z e d f o r p e r c e n t oxygen u s i n g a Beckman Model F3 oxygen a n a l y z e r (Beckman I n s t r u m e n t s , I n c . , F u l l e r t o n , California).  In addition,  progressive hypercapnic hypoxia  was i n d u c e d i n ducks "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 f o r 2-4  minutes.  86 r  Results a.  Comparison o f ducks and c h i c k e n s Heart r a t e , MABP, and CF r e c o r d e d from ducks  (solid  l i n e ) and c h i c k e n s ( d o t t e d l i n e ) d u r i n g a p n e i c a s p h y x i a a r e shown i n F i g . 18. Mean p r e a s p h y x i c ' h e a r t r a t e f o r 1 9 c h i c k e n s and 2 0 ducks was 1 5 4 - 9 . 2 9 and 2 5 5 - 3 2 . 0 4 respectively.  beats'minute  -1  A f t e r a 1 5 second l a t e n t p e r i o d from t h e 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 h e a r t r a t e i n c h i c k e n s f e l l s t e a d i l y > u n t i l apnea was t e r m i n a t e d a t 6 0 seconds. In  c o n t r a s t , ducks showed a decrease  i n mean h e a r t r a t e d u r i n g  the f i r s t 1 5 seconds w h i c h corresponded bradycardia.  t o 42$ o f t h e t o t a l  A l t h o u g h t h i s r a t e o f decrease was n o t p a r a l l e l e d  between any o t h e r subsequent s a m p l i n g p e r i o d s , the h e a r t 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  t o 3 5 $ a t 9 0 seconds w i t h  88$ o f t h e b r a d y c a r d i a o c c u r r i n g d u r i n g t h e f i r s t m i n u t e . A t 6 0 seconds h e a r t r a t e i n c h i c k e n s and ducks was 5 5 $ and 44$  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 h e a r t r a t e i n b o t h s p e c i e s r e t u r n e d to  t h e p r e a s p h y x i c . r a t e a f t e r 6 0 - 9 0 seconds w i t h o u t t h e p o s t -  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 n o t change s i g n i f i c a n t l y i n e i t h e r s p e c i e s d u r i n g a s p h y x i a o r t h e subsequent r e c o v e r y . A s p h y x i a i n both s p e c i e s was c h a r a c t e r i z e d by an i n c r e a s e i n NADH w h i c h c o n t i n u e d a t a n e a r l y l i n e a r r a t e u n t i l c i a l v e n t i l a t i o n was resumed.  artifi-  A l t h o u g h 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 a t r e g u l a t i n g redox b a l a n c e by d e c r e a s i n g t h e r a t e o f  87  F i g u r e 18. H e a r t r a t e (HR), mean a r t e r i a l b l o o d p r e s s u r e (MABP), and c o r r e c t e d f l u o r e s c e n c e (CF) d u r i n g 1 and 2 minute p e r i o d s o f a p n e i c a s p h y x i a i n paralyzed chickens ( 5 4 periods of asphyxia i n 19  c h i c k e n s ) and ducks ( 5 5 p e r i o d s o f a s p h y x i a  i n 2 0 ducks) r e s p e c t i v e l y .  The d o t t e d l i n e s  r e p r e s e n t c h i c k e n s and t h e s o l i d l i n e s ducks.  The arrows i n d i c a t e t h e a s p h y x i c 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 t o a n o x i a was  represent  d e f i n e d as 1 0 0 AU.  (death)  Each p o i n t r e p r e s e n t s  the mean - SEM and t h e SEM i s c o n t a i n e d w i t h i n the p o i n t when absent.  -15  -L  |  |  0  10  1 20  |  |  30  40  1 50  |  60  |  70  |  80  SECONDS TIME  I  90  I  100  1 110  I  120  I  130  I  140  1 1 150  160  8 9  reduction to 1 / 5 of that i n chickens.  F o r example, CF was  s i g n i f i c a n t l y l o w e r a f t e r 1 minute o f apnea i n ducks ( 8 — 1.41  AU) t h a n c h i c k e n s ( 3 7 - 3-60 AU). Furthermore,  r a t e o f r e d u c t i o n continued.unchanged 2 minutes,  i n ducks from t h e f i r s t  t h e n i t would take over 4 . 5 minutes o f apnea b e f o r e  CF e q u a l l e d t h e CF i n c h i c k e n s a t 6 0 seconds. was  i f the  When v e n t i l a t i o n  resumed CF r e t u r n e d t o t h e b a s e l i n e i n b o t h s p e c i e s a f t e r  a t r a n s i e n t o v e r s h o o t , u s u a l l y o f 5 - 1 ° minutes d u r a t i o n . A f t e r 6 0 seconds o f apnea i n c h i c k e n s P a 0 O.366  kPa  ducks P a 0  2  ( 4 4  -  2.57  was 5.6 -  was 5 . 8 —  2  t o r r ) and a f t e r 120 seconds o f apnea i n G.55I  kPa  ( 3 8  - 4.14 t o r r ) .  It is  i n t e r e s t i n g t o note t h a t when mean P a 0 ' s were v e r y s i m i l a r 2  mean CF i n c r e a s e i n c h i c k e n s was over t w i c e t h a t i n ducks ( 3 7 AU v e r s u s 1 5 AU). To c l a r i f y t h e enigma 2-4 b l o o d samples were withdrawn from t h e f e m o r a l a r t e r y a t random i n t e r v a l s  after  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 c h i c k e n s , and t h e r e l a t i o n s h i p between CF i n c r e a s e and P a 0 for  2  was examined  t h e b e s t f i t t o e i t h e r a s t r a i g h t l i n e , an e x p o n e n t i a l  c u r v e , a power c u r v e , o r a l o g a r i t h m i c c u r v e .  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 h a v i n g t h e l a r g e s t p  c o e f f i c i e n t s o f c o r r e l a t i o n ( r ), 0 . 2 4 and O . 3 7 i n c h i c k e n s 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 o f t h e r e s p i r a t o r y c h a i n . v a r i o u s b l o o d samples ranged from  4.6-7.3  kPa  PaC0  2  ( 3 5 - 5 5  of the 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 c h i c k e n s and  ducks  EEG and CF were r e c o r d e d s i m u l t a n e o u s l y d u r i n g p r o l o n g e d a p n e i c a s p h y x i a i n c h i c k e n s and ducks.  I n 1 1 c h i c k e n s 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 e n d p o i n t was reached ( T a b l e s I I and I I I ) .  CF was n o t  s i g n i f i c a n t l y d i f f e r e n t a t t h e EEG endpoint; however, t h e time from the b e g i n n i n g o f apnea t o t h e e n d p o i n t was over 5 - f o l d l o n g e r i n ducks t h a n c h i c k e n s .  Chickens m a i n t a i n e d 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 4 2 - 9 3  seconds) and ducks f o r  3 3 8  -  3 2 . 3 6  seconds  (range  (range  2 3 2 -  549  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 ; t h e  top  2 t r a c e s (EEG and CF) a r e from a duck and t h e bottom 2  t r a c e s (EEG and CF) a r e from a ' c h i c k e n .  I n b o t h cases CF  i n c r e a s e d when t h e a r t i f i c i a l v e n t i l a t i o n was stopped  (first  arrow) and c o n t i n u e d u n t i l t h e EEG e n d p o i n t was reached a t which time t h e v e n t i l a t i o n was resumed (second arrow) and CF returned t o the baseline.  A l t h o u g h t h e EEG e n d p o i n t was reached  sooner i n c h i c k e n s , CF a t t h e e n d p o i n t s was s i m i l a r , 3 8 AU and 3 2 AU i n t h e c h i c k e n and duck r e s p e c t i v e l y .  I n ducks i t appeared  t h a t those which showed t h e g r e a t e s t b r a d y c a r d i a m a i n t a i n e d EEG f o r l o n g e r p e r i o d s o f apnea t h a n those i n which b r a d y c a r d i a was n o t so pronounced  (Table'Il).  Although bradycardia i s only  a crude measure o f 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 n e v e r t h e l e s s c o r r e l a t e s w i t h d i v i n g performance.  C o n s i d e r i n g t h e d a t a from  Table I I ,the time t o l e r a t e d d u r i n g a s p h y x i a b e f o r e t h e EEG  —  91  Table I I .  H e a r t r a t e , time t o EEG  e n d p o i n t , and CF a t  EEG  e n d p o i n t i n p a r a l y z e d ducks d u r i n g a p n e i c a s p h y x i a . Mean - SEM AU a t bottom o f t a b l e .  Time (seconds) d u r i n g apneic asphyxia Ducks  15  0  Heart r a t e  1  305  208  2  293 141  273 102  240  200  5  281  7 8  175 270  3 4  60  120  49 104  90 150  115 125  119 112  203  172  142  145  122 140  119 130  185 135 92  301  11  270  161  12  261  149  300  36O  420  37  39 81  42  35  49  121  125 121  108  107  125  136  84  95  156  91 130  121  135  112  92  81  79  37  42  42 1  85 109  182 i - 149 254 119 ~ 17.26 14.83 12.39 14.49 (n=10) (n=10) (n=10) (n=10) ±  240  480  540  1  73 208  10  180  CF a t EEG endpoint  (beats •minute'- ) 171 220  175 209  Mean - SEM  30  Time a t EEG endpoint  +  35  98 ± 92 91 10.00 9.97 12 (n=10) (n=10) (n= 1  431  142 93 85 39 88 -  17.46 (n=6)  28  232  32  253 311  41  247 87  367  77 41  415 549  129  313  47  49  65  35  263  338 i 32.26  (n=10)  31 38 31 38 34  45 38  i  1.90 (n=10)  93  T a b l e I I I . Heart r a t e , time t o EEG  e n d p o i n t , and CF a t  EEG  endpoint i n p a r a l y z e d chickens d u r i n g apneic asphyxia.  Mean - SEM AU a t the bottom of t a b l e .  Time (seconds) during apneic asphyxia Chickens  0  15  Heart rate  30  60  90  Time at  CF at  EEG  EEG  endpoint  endpoint  62  27  42  32  93  36  77 • 75 56  37 41  (beats'minute" ) 48  158  114 118  196  184  204  186  I63  147 120  5 6  195  195  108  94  108  107  95  7 8  140  140  125  51  25  196  196  172  49  40  9  201  195  142  126  61  30  10  138  151  88  31  11  147  147  126  124  57 64  164 11.11  160 -  130  (n=ll)  (n=ll) (n=ll) (n=6)  1  114  96  2  163 202  3 4  -  SEM  10.98  9.45  -  110 14.17  98  62 ±  50  30  34 ±  4.38  2.19  (n=ll)  (n=llK  95  F i g u r e 19•  Electroencephalogram fluorescence  (EEG) and c o r r e c t e d  (CF) r e c o r d e d from a duck  (upper  2 t r a c e s ) and a c h i c k e n (bottom 2 t r a c e s ) d u r i n g apneic asphyxia.  The downward p o i n t i n g arrow  f o r each p a i r of t r a c e s i n d i c a t e s the b e g i n n i n g of asphyxia and the upward p o i n t i n g arrow cates the end of a s p h y x i a . to a l l f o u r t r a c e s .  EEG  The  indi-  time bar a p p l i e s  i s expressed i n micro-  volts  (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) r e p r e s e n t s 100  AU.  EEG  J  lOOuV  DUCKS 50 A U CF  EEG CHICKENS CF  J  J  100 pV  50 A U  97  endpoint  shows a c o r r e l a t i o n w i t h  heart rate (expressed (Fig. to  as a p e r c e n t  t h e maximum d e c r e a s e i n of the pre-asphyxic  2 0 ) . A r e l a t i o n between degree  t h e EEG e n d p o i n t  rate)  o f b r a d y c a r d i a and time  was n o t o b s e r v e d  i n chickens  (Table I I I ) .  R e l a t i o n s h i p b e t w e e n f l u o r e s c e n c e a n d PrpOo i n d u c k s a n d  c.  chickens  during  hypoxia  NADH f l u o r e s c e n c e a n d PrpOg were m e a s u r e d from  opposite c e r e b r a l hemispheres  during steady  state hypoxia  the v e n t i l a t o r y  gas.  t h e more s e v e r e  the hypoxia  i n d u c k s and c h i c k e n s  produced  As t i s s u e  simultaneously  by v a r y i n g t h e oxygen i n  PrnOg f e l l ,  CF i n c r e a s e d , a n d  t h e g r e a t e r t h e CF i n c r e a s e .  When t h e duck was r e t u r n e d t o 20$ oxygen, PrpOg a n d CF e v e n tually  returned t o normal.  a single  duck.  F i g u r e 21 shows t h e r e s u l t s  The a r e a b e t w e e n t h e a r r o w s r e p r e s e n t s t h e  p e r i o d s when v a r i o u s l e v e l s is  expressed  as a p e r c e n t  when the decrease ;  from  as a 100$ d e c r e a s e ; described. as  P 0 T  decrease  i n d i c a t e d by the decrease  2  of the electrode current  normoxia to anoxia  from  PIJ°2  o f h y p o x i a were a p p l i e d .  CF was e x p r e s s e d  Proceeding  on c h i c k e n s  from  ( d e a t h ) was d e f i n e d  i n AU's a s p r e v i o u s l y  a t o d, h y p o x i a i n Prn ^*  became more  Experiments  0  severe  performed  ( n o t shown) showed a s i m i l a r r e l a t i o n s h i p  between  and CF. When NADH f l u o r e s c e n c e was p l o t t e d  decrease  i n PjpOg (Fig«  22) a n e x p o n e n t i a l c u r v e  the r e l a t i o n s h i p  i n 8 chickens  (dark  Since  circles).  as a f u n c t i o n o f t h e  (light  best described  c i r c l e s ) and 9 ducks  t h e EEG e n d p o i n t  was r e a c h e d  before  98  F i g u r e 2 0 . Length of time d u r i n g apneic asphyxia i n p a r a l y z e d ducks b e f o r e the c e s s a t i o n of b r a i n  electrical  a c t i v i t y i n r e l a t i o n to the decrease  i n heart  r a t e (expressed as a p e r c e n t of the p r e a s p h y x i c heart r a t e ) .  Each p o i n t r e p r e s e n t s a s i n g l e  p e r i o d of apneic a s p h y x i a . from Table I I .  Values are  taken  100  Figure  21.  S u r f a c e POg f r o m  the r i g h t  (PrpOg) and c o r r e c t e d from  the l e f t  cortex  f l u o r e s c e n c e (CF) r e c o r d e d  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  during steady state produced  cerebral  hypoxia  ( i n d i c a t e d by arrows)  by v a r y i n g oxygen i n t h e v e n t i l a t o r y gas.  P r o c e e d i n g from  a t o d h y p o x i a became more  PfpOg i s e x p r e s s e d  as a p e r c e n t d e c r e a s e  of the  electrode  c u r r e n t when t h e d e c r e a s e .from  to anoxia  ( d e a t h ) was  CF was e x p r e s s e d  severe.  normoxia  d e f i n e d as a 100$ d e c r e a s e .  i n arbitrary units  (AU) where  t h e CF change f r o m n o r m o x i a t o a n o x i a was d e f i n e d as 100 AU.  (death)  102  Figure 2 2 . Corrected fluorescence  (CF) i n r e l a t i o n t o P 0  recorded  from c o r t i c a l s u r f a c e ( P ^ O g ) i n 8  chickens  ( l i g h t c i r c l e s ) and 9 ducks (dark  PrpOg i s expressed  2  circles).  as a p e r c e n t decrease of t h e  e l e c t r o d e c u r r e n t where t h e decrease from normoxia t o a n o x i a (death) was d e f i n e d as 1 0 0 $ decrease.  CF was e x p r e s s e d  i n arbitrary units  (AU) where t h e CF change from normoxia t o a n o x i a (death) was d e f i n e d as 1 0 0 AU.  DUCKS• CHICKENS o  ••  o  70 + o o  60 +  • 50  +  40  +  •  ° o  o  •  o  o  30+  •••  • o • o  g  o 20 +  o °o  8  i  ° °*  8  10  20  —I • •  1  o  •  •  o o*  ° *  o  1  30  o  8 °  ° •  1  40  o  ° 1  1  50 P O T  60  2  PERCENT DECREASE  1  70  1  80  1  90  1  100  104  CF  i n c r e a s e d by 5 ° AU,  and  e v e n i n t h e most extreme c a s e  I I I ) , o n l y the p o i n t s t h a t f e l l  here.  Equations  ducks are y =  t h a t b e s t d e s c r i b e the  3.82e°'°  respectively.  3 x  (r  that  the  plotting  0 . 7 5 ) and  y =  3.74e°*°  were t r a n s f o r m e d versus  P  T  0  2  into  .  g r o u p s were n o t  a  After and  3 x  significantly  and  Cochran,  different  =  2  0.8?)  linear determining d u c k s were the The  1974).  at the  (r  and  chickens  y - i n t e r c e p t s were compared b y (Snedecor  II  considered  data f o r chickens  r e s i d u a l v a r i a n c e s between c h i c k e n s  of covariance  were  o f c o m p a r i n g d u c k s and  l n CF  homogeneous, s l o p e s and analysis  =  2  For purposes  the e x p o n e n t i a l equations r e g r e s s i o n by  b e l o w 5 ° AU  (Tables  9 5 $ confidence 0  level  and  . 16  c a n be  r e p r e s e n t e d by  F i g . 2 3 shows a c o n t i n u o u s  periods  c a n be  Progressive from  ventilation  d e s c r i b e d ' , by hypercapnic  the  NAD  t h a t COg  i n ducks.  had  hypoxia  equation  hypoxia  no  o f CF v e r s u s  y  d i d not  direct  =  Pr^Og  the  "  •  in by  combined  .3.00e°'°^ (r =0.87).  differ  x  2  significantly  normocapnic  effect  3'7le  produced  i n 8 d u c k s and  that obtained during steady state  indicating of  plot  of p r o g r e s s i v e hypercapnic  stopping a r t i f i c i a l data  a common e q u a t i o n y =  03x  hypoxia  on t h e r e d o x  state  105  Figure 23.  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 left  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  r e c o r d e d from  the r i g h t  during apneic  asphyxia  apneic  asphyxia  result  from  is  electrode  as  (AU)  CF was  where t h e CF  anoxia  ( d e a t h ) was  surface  period. of  decrease  ( d e a t h ) was expressed  (P^Og)  (16 p e r i o d s  decrease  c u r r e n t where t h e  P^Og  of  Each t r a c e i s the  asphyxic  a percent  normoxia to anoxia decrease.  i n ducks  i n 8 ducks).  a single  expressed  cortical  the  PrnOg the from  d e f i n e d as  in arbitrary  change f r o m  normoxia to  d e f i n e d as 100  AU.  a  100$  units  106  107  Discussion Studies and  ducks  ways.  have b e e n p e r f o r m e d - o n  the t o l e r a n c e s  t o d i v i n g and have d e f i n e d d e a t h  M o s t were b a s e d  of chickens  i n several  different  on l a s t  h e a r t beat or l a s t  a f t e r apnea (Andersen,  I966).  P r o b a b l y a b e t t e r measure f o r  tolerance  a t which  i s the p o i n t  b r a i n f u n c t i o n ceases  i n t h e a b s e n c e ' o f b r a i n f u n c t i o n an a n i m a l c a n no nate  t h e m u s c u l a r movements f o r e s c a p e  of the need  to escape.  Eisner  ( 1 9 7 3 a and b) u s e d  Eisner  patterns  i n t h e EEG,  which  during hypoxia, f o r t h e i r the l o s s  o f EEG  p o i n t " and was  et a l . (1970)  the time  longer coordi-  and Kerem  and  t o t h e o n s e t o f s l o w wave  are c h a r a c t e r i s t i c endurance  studies.  ( i s o e l e c t r i c i t y ) was  as maximum t o l e r a n c e  since  i f i t i s i n d e e d aware  of  unconsciousness  In t h i s  d e f i n e d as t h e  the time from v e n t i l a t o r y a r r e s t  defined  struggle  study  "EEG  t o t h e EEG  end-  endpoint  o f the i n d i v i d u a l to a p n e i c  asphyxia. The  time  t o t h e EEG  a p n e i c a s p h y x i a was seconds  ( n = 11)  338  e n d p o i n t i n ducks  — 3 2 . 3 6 seconds  respectively;  and  chickens during  ( n = 10)  t o endure  10-23  minutes  I966)  Scholander, 1961/1962,  times  i n the p r e s e n t experiments  r e p o r t e d , p r o b a b l y because  4.38  and  ducks  3 minutes  1964).  a less  A l t h o u g h the  are l e s s  1959  used  than those  i n previous experiments,  durance  i n c h i c k e n s t o ducks  and  endurance previously  s o p h i s t i c a t e d measure  t o l e r a n c e was  of  have b e e n r e p o r t e d  of apneic a s p h y x i a (Andersen,  1966;  times  62 —  over 5 - f o l d l o n g e r i n ducks.  C h i c k e n s have b e e n p r e v i o u s l y r e p o r t e d t o endure apneic a s p h y x i a (Andersen,  and  the r a t i o  i s c o m p a t i b l e , 3-8  of of  en-  f o r the  108  r e p o r t s and 5 * 5 f o r t h e p r e s e n t  previous  study.  During apneic asphyxia the r e s p i r a t o r y teristically  increased i n the cerebral  asphyxia  linearly  ( 1 and 2 minutes r e s p e c t i v e l y ) .  between t h e 2 s p e c i e s l a y i n t h e r e l a t i v e NADH f l u o r e s c e n c e i n c r e a s e d . creased a t a rate period  representing  over a 4 - f o l d  f o r the duration  The d i f f e r e n c e  rate  a t which  i n ducks  i n chickens over a 1 minute over a 2 minute p e r i o d ,  difference  i nrate.  As apneic  asphyxia progressed the c r i t i c a l reduction l e v e l the r e s p i r a t o r y These r e s u l t s 2  c h a i n NAD was a p p r o a c h e d  a r e compatible  species t o apneic Berger  related tissue to  wick  I 9 7 6 ) .  the m a j o r i t y of evidence  redox  strictly  Ingvar  1 9 7 6 ;  Although  state related  supports  i n nervous  t h e r e have b e e n r e p o r t s  Berger's  e t a l . , 1 9 7 6 ; Brodersen  a l . ( l 9 7 5 )  of the r e s p i r a t o r y  1 9 6 7 ;  t h e o r y (Himet a l . ,  1 9 7 3 ;  Gleichmann e t a l . ,  I 9 6 2 ) .  have shown t h a t EEG f r e q u e n c y c h a i n (cytochrome  t o oxygen a v a i l a b i l i t y .  I t follows  a^) a r e that,if  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  production, then the r e l a t i o n s h i p  ( 1 9 7 5 )  i s closely  I 9 5 5 i K e n n e d y and S o k o l o f f ,  Meyer e t a l . ,  LaManna e t  anaerobic metabolism ATP  t h a t EEG f r e q u e n c y  of o x i d a t i v e metabolism  (Ingvar e t a l . ,  Furthermore, and  suggested  t o t h e degree  Sundt e t a l . ,  i n chickens.  asphyxia.  ( 1 9 3 8 )  e t a l . , 19^7;  faster  ( 3 5 AU) o f  w i t h t h e known t o l e r a n c e s o f t h e  t h e c o n t r a r y (Mangold e t a l . ,  1957)  that  F o r example, NADH f l u o r e s c e n c e i n -  o f 0 . 6 2 AU/second  and 0 . 1 3 AU/second  charac-  c o r t e x o f c h i c k e n s and  d u c k s and c o n t i n u e d t o i n c r e a s e a l m o s t of  c h a i n NADH  s h o u l d be l o s t .  d e s c r i b e d b y LaManna e t a l .  I n o t h e r words, d u r i n g a p n e i c  asphyxia  109  an a n i m a l  relying  on a l a r g e  a n a e r o b i c ATP c o n t r i b u t i o n f o r  s u r v i v a l s h o u l d have a more r e d u c e d gross a l t e r a t i o n s contributions. endpoint  i n EEG t h a n  R e s u l t s from  respiratory  one w i t h o u t this  large  chain before anaerobic  s t u d y show t h a t  i n both s p e c i e s during apneic  t h e EEG  asphyxia occurred  after  p r e c i s e l y t h e same i n c r e a s e i n NAD r e d u c t i o n ( a p p r o x i m a t e l y 35 A U ) .  F o r these reasons  duction,  i f i t o c c u r s , g i v e s t h e duck no a d v a n t a g e  I conclude  t h a t a n a e r o b i c ATP p r o -  c h 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  over the  during apneic  asphyxia. In these  experiments  NAD n o r t h e a b s o l u t e r a t e tated.  I f pool size  the absolute r a t e  a similar and size  i n ducks t h a n c h i c k e n s and i f  t h a t NADH a c c u m u l a t e s  v  t o the c r i t i c a l  (Approximately  i n ducks.  o f i n c r e a s e i n NADH h a s b e e n q u a n t i -  i s larger  s p e c i e s , t h e n t h e time reduction  n e i t h e r the absolute pool size of  In both  pyridine nucleotide  35 AU i n b o t h s p e c i e s ) w o u l d  hypercapnic hypoxia  o f NADH.  any d i f f e r e n c e  NADH a c c u m u l a t i o n  of pool  But d u r i n g  t h e r e l a t i o n s h i p between t h e  i n PjpOg a n d i n c r e a s e i n NADH s t i l l  suggesting that  hypoxia  i n c o r t i c a l PrpOg  i n c o r t i c a l NADH w h i c h i s i n d e p e n d e n t  or absolute rates - of accumulation  progressive  be*longer  c h i c k e n s and ducks d u r i n g s t e a d y s t a t e  r e l a t i o n s h i p h e l d between t h e f a l l  accumulation  decrease  i s t h e same i n b o t h  i n pool size  i s o f no s i g n i f i c a n c e  holds, s t r o n g l y or absolute rate of  i n promoting  tolerance  to apneic asphyxia i n ducks. Results  from  this  s t u d y p r o v i d e no e v i d e n c e  t o support  t h e 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 a d a p t a t i o n s enhance  oxidative  110  phosphorylation  i n ducks d u r i n g h y p o x i a .  ducks were e x p o s e d t o a g i v e n was  no  significant  between the brain be  level  i s 1 / 1 0 that  r e d o x change  That m i t o c h o n d r i a l K  of other  oxygen t e n s i o n s .  same e f f e c t ,on steady CF  the  Progressive  to p r o g r e s s i v e  normocapnic h y p o x i a can conclude state  that hypercapnia  of the  introduced  et a l .  does n o t  i n the  tissue  oxygenation s t a t e , but  between d i r e c t indirect t h u s an 7f  0  the  state due  the  the  o f COg  caused by  increased PjOg.  directly  occurred authors  on t h e  an  lack  o f any  to increased  cerebral blood et a l .  (1976a)  oxygen c o n c e n t r a t i o n  flow  se.  5$  COg  regardless  slight  of  the  and flow  and  i n PrpOg shown when  (Chapter on  2, F i g . 1 2 )  changes  i n redox been  P^Og.  and  that  changes i n v e n t i l a -  produced a corresponding  redox s t a t e of cytochrome aa^  I  was  cats, a  e f f e c t must have  reported  state  redox  per  c e r e b r a l blood  increase  t h a t the  the  the  d i d not d i s t i n g u i s h  of hypercapnia  during hypoxia suggest  steady  respiratory chain  increased  Both the  effect  affect  i n r a b b i t s and  added t o t h e v e n t i l a t o r y gas  Rosenthal tory  effects  effects  COg was  and  always  that  a common e q u a t i o n ,  showed t h a t , when  v e n t i l a t o r y gas  o x i d a t i o n of cytochrome aa^  by  the  as  fact  h y p o x i a and  or e l e c t r o n f l o w  (1976a)  h y p o x i a had  In view of the  described  respiratory chain  Rosenthal  the  o f a v a i l a b l e oxygen  r e s p i r a t o r y chain  hypercapnic  be  o f NADH  f o r oxygen i n  hypercapnic  redox s t a t e of the  s t a t e normocapnic hypoxia.  response  m  there  t i s s u e s ( C l a r k e t a l . , 1 9 7 6 ) may  p r o t e c t i o n enough t o e n s u r e maximum use  a t low  and  of t i s s u e hypoxia,  d i f f e r e n c e i n the  2 species.  When c h i c k e n s  t h a t was  change  continuous  from  in  Ill  100$  oxygen t o n i t r o g e n .  (1964)  reported  On t h e o t h e r hand,  t h a t NADH f l u o r e s c e n c e  Chance  d i dnot increase  oxygen i n t h e v e n t i l a t o r y gas was r e d u c e d t o 5 $ . this  study  confirm  the conclusions  showing t h a t f i r s t l y , hypoxic  no a b r u p t  et a l .  of Rosenthal  changes o c c u r r e d  until  Results  e t a l . (1976) during the  r e g i m e , o n l y a c o n t i n u u m o f change, a n d s e c o n d l y ,  i n c r e a s e d when PrpOg h a d d e c r e a s e d  by only 2 0 $ .  i n PipO.g c o r r e s p o n d s  t o oxygen c o n c e n t r a t i o n s  gas o f g r e a t e r t h a n  15$> w e l l above t h e c r i t i c a l  r e p o r t e d b y Chance  et a l . (1964).  from  A 20$  CF  decrease  i n the v e n t i l a t o r y 5$ level  112  GENERAL DISCUSSION The lies  problem  o f c o n t i n u e d ATP p r o d u c t i o n d u r i n g  i n the i n a b i l i t y  necessary  the necessary  homeostasis  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  Nevertheless, divers  to maintain  diving  the c a r d i o v a s c u l a r adjustments  to tolerate  periods  of apneic  enable  asphyxia  natural  t h a t w o u l d be  d e t r i m e n t a l t o man a n d o t h e r t e r r e s t r i a l v e r t e b r a t e s . a d j u s t m e n t s do n o t a l l o w  and a r e r e f l e c t e d  f o r the production  in that particular  it.  The s t r a t e g i e s  tissue  depend on e n h a n c e d  p r o t r a c t e d hypoxia  fused  throughout  i s depleted.  (Andersen,  the dive.  t o l e r a t e more s e v e r e EEG a l t e r a t i o n s  study  ducks c o u l d t o l e r a t e  The this  I966)  Although  hypoxia  gross  than  regardless  allowed  a n d must be w e l l  harbor  (Kerem.,and E i s n e r ,  o f NAD p o o l s i z e .  activity  NADH o c c u r r e d w i t h  the onset o f  1 9 7 3 a  and b ^ i n  no b e t t e r t h a n  ceased.  c o r t i c a l NADH i n  t h e same d e c r e a s e  Although  there  increase  a n d EEG e n d p o i n t ,  i n chickens  Furthermore,  this  chickens.  and s p e c i e s  Once NADH h a d i n c r e a s e d level  per-  s e a l s appear t o  comparisons between i n d i v i d u a l s  m a t e l y 3 5 $ above t h e n o r m o x i c electrical  damaged b y s e v e r e  dogs b e f o r e  hypoxia  oxygen  On t h e o t h e r hand t h e  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 study  glycolytic  o f ATP once t h e r e s i d u a l  h e a r t and b r a i n a r e e a s i l y and i r r e p a r a b l y and  for tissues  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 a r e p o o r l y p e r f u s e d capacity  These  the d i v e r t o u n c o n d i t i o n a l l y maintain  homeostasis but r a t h e r prolong vary  ceases.  approxi-  and d u c k s , b r a i n  the 3 5 $ increase i n  i n b r a i n POg i n b o t h  species.  i s a s t r o n g c o r r e l a t i o n b e t w e e n NADH f l u o r e s c e n c e EEG may n o t be d i r e c t l y  related to  113  the  redox s t a t e .  reflect the  I t may be t h a t  the l e v e l  of tissue  functional state  EEG a n d NADH f l u o r e s c e n c e  oxygenation.  of thebrain  phosphates 1973  (ATP,  ADP, AMP) d u r i n g  and 1 9 7 4 ) and i n d i c a t e s  that  c a n n o t m a t c h t h e demand) may n o t of b r a i n f u n c t i o n . v a l u e f o r the energy s t a t e  1977)'  (SeisjO, i n the  (tyrosine  degradation  (JObsis,  energy f a i l u r e  (ATP  used i n the  a n d may n o t  biosynthesis  of brain  function.  2  197*+), t h e y a r e  total  of the b r a i n reactions  of neurotrans-  nevertheless  or brain  a product  decreases l i n e a r l y  with a On t h e inhibited  lesions without the loss of  nervous system o f a l l b i r d s  metabolism f o r i t s  197*+).  of the  c a n be s p e c i f i c a l l y  (Snyder, 1 9 7 6 ; Sourkes,  or nondivers,  reactions  sensitive to small  F o r example, s e r o t o n i n ,  with chemical blockers  hypoxia  o x y g e n consumed i n t h e  between 5 0 and 2 0 t o r r ( J O b s i s ,  brain function  loss  r e f l e c t the  Although these  o t h e r hand, most o f t h e s e r e a c t i o n s  divers  f o r the  or i s o l a t e d areas  tryptophane hydroxylase r e a c t i o n ,  The  production  and tryptophane h y d r o x y l a s e ) and t h e i r  c h a n g e s i n oxygen.  in PT0  (Seisjo" e t a l . ,  t i s s u e measurement,;is a n a v e r a g e  account f o r l e s s t h a n 1 5 $ o f the  fall  adenosine  (monoamine o x i d a s e ) may be a l t e r e d d u r i n g  causing a loss  brain  changes i n the  A l t e r n a t i v e l y , o t h e r oxygen r e q u i r i n g  b r a i n which are  mitters  o f EEG)  severe hypoxia  adenosine phosphates, sites  (slowing  be r e s p o n s i b l e  However, t h e  at c r i t i c a l  I n t e r r e s t r i a l mammals  changes  b e f o r e t h e t i s s u e shows a n y d e t e c t a b l e  both  1976). a n d mammals, w h e t h e r  a p p e a r s t o be d e p e n d e n t on o x i d a t i v e survival.. Therefore  t h a t oxygen s u p p l y t o t h e b r a i n  d i v e r s must  i s maintained  ensure  throughout a dive.  114  While  other t i s s u e s appear capable of r e l y i n g on anaerobic  metabolism d u r i n g the dive, the b r a i n cannot.  I t may  complexity of the b r a i n i s not compatable w i t h anoxic I n any event i t appears to modify  that the b a s i c s t r a t e g y  be t h a t "the tolerance.  of d i v e r s i s  the defense mechanisms f o r apneic asphyxia found i n  terrestrial  animals  to meet the needs of d i v i n g .  Biochemical  and p h y s i o l o g i c a l s t r a t e g i e s f o r t o l e r a t i n g d i v i n g c e r t a i n l y cannot be c l a s s i f i e d as t r u e l y  unique.  115  BIBLIOGRAPHY A n d e r s e n , H.T. a i r i n the  240-243.  1959. A n o t e on t h e c o m p o s i t i o n o f a l v e o l a r d i v i n g duck* A c t a P h y s i o l . S c a n d . 46:  A n d e r s e n , H.T. I966. 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