Upsala J Med Sci 91: 233-238, 1986 The Technique of Whole Body Autoradiography-Some Examples of Applications Lars-Erik Appelgren Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, The Swedish University of Agricultural Sciences, Biomedicum, Uppsala, Sweden INTRODUCTION When Sven U l l b e r g i n t h e b e g i n n i n g o f t h e 1950’s s t a r t e d h i s Ph.D. t h e s i s work a l l d e t e r m i n a t i o n s o f t h e p e n i c i l l i n c o n c e n t r a t i o n i n organs o f t h e li- v i n g organism were done by m i c r o b i o l o g i c a l I n h i s a t t e m p t t o i n - v e s t i g a t e t h e f a t e o f p e n i c i l l i n i n v i v o U l l b e r g t r i e d d i f f e r e n t , by t h e n r e l a t i v e l y new methods. He produced r a d i o l a b e l l e d p e n i c i l l i n by growing t h e p e n i c i l l i n p r o d u c i n g f u n g i i n a r a d i o a c t i v e ( 3 5 S ) g r o w t h medium. A f t e r a h i - n i s t r a i o n o f t h i s l a b e l l e d p e n i c i l l i n t o e x p e r i m e n t a l animals measurements o f p e n i c i l l i n c o n c e n t r a t i o n s i n d i f f e r e n t organs were t h e n performed w i t h Geiger- M h l e r t e c h n i q u e and a u t o r a d i o g r a p h y o f s e c t i o n s from e x c i s e d organs. A prob- lem with which i s a water s o l u b l e substance, may be d i s s o l v e d when p r e p a r i n g t h e h i s t o l o g i c a l sec- t i o n s used. To be a b l e t o make a d i r e c t comparison o f t h e r a d i o a c t i v i t y i n d i f f e r e n t organs Sven U l l b e r g e l a b o r a t e d a method (31 which makes i t p o s s i b l e t o s i m u l t a n e o u s l y gans i n t h e body w i t h o u t r i s k i n g leakage o f water s o l u b l e substances. The d i f - f i c u l t p r e s e l e c t i o n o f organs which - f o r p r a c t i c a l reasons - u s u a l l y has t o be done i s t h u s needless. techniques. t r a d i t i o n a l a u t o r a d i o g r a p h y i s t h e f a c t t h a t p e n i c i l l i n , l o c a l i z e a r a d i o a c t i v e substance i n p r a c t i c a l l y a l l t h e o r - DESCRIPTION OF THE TECHNIQUE The technique can b r i e f l y be d e s c r i b e d as f o l l o w s (4): A f t e r a d m i n i s t r a t i - on o f t h e r a d i o a c t i v e substance t h e animals a r e s a c r i f i c e d a f t e r d i f f e r e n t s u r v i v a l t i m e s by an a n a e s t h e t i z i n g agent. T h e i r bodies a r e immediately f r o z e n i n a m i x t u r e o f hexane and s o l i d carbon d i o x i d e and t h e n s e c t i o n e d i n a s p e c i - a l microtome a t =: -2OOC. To be a b l e t o h a n d l e t h e l a r g e t i s s u e s e c t i o n s a p i e - ce o f Scotch t a p e i s pressed o n t o t h e b l o c k b e f o r e s e c t i o n i n g . It i s p o s s i b l e t o t a k e s e c t i o n s from animals up t o t h e s i z e o f e.g. a newborn p i g by t h i s technique. A f t e r f r e e z e - d r y i n g t h e s e c t i o n s they a r e p r e s s e d a g a i n s t a photo- g r a p h i c emulsion. A f t e r exposure t h e s e c t i o n s a r e separated from t h e f i l m s and t h e f i l m s a r e developed t o v i s u a l i z e t h e b l a c k e n i n g caused by t h e r a d i o a c t i v e 233 s u b s t a n c e i n t h e d i f f e r e n t o r g a n s . a u t o r a d i o g r a m s are shown i n F i g . 1. s o l u t i o n t o t h e p r o b l e m i s a s i n g e n i o u s a s s i m p l e . The who: l e body a u t o r a d i o g r a p h y as i n t r o d u c e d by U l l b e r g h a s b e e n i n c r e a s i n g l y u s e d i n o u r d e p a r t m e n t s b u t a l s o i n many l a b o r a t o r i e s i n p r a c t i c a l l y a l l p a r t s o f t h e w o r l d . The p i o n e e r work i n c l u d e d s e c t i o n i n g t h e s p e c i m e n s i n a f r e e z e - room ( - 2 O O C ) w h i c h m e a n t t h a t f u r coats and g l o v e s were n e c e s s a r y e q u i p m e n t . Some i m p r o v e m e n t s o f t h e t e c h n i c a l d e t a i l s when h a n d l i n g t h e embedding o f t h e s p e c i m e n s and t h e s e c t i o n i n g h a s b e e n i n t r o d u c e d d u r i n g t h e y e a r s . The i n t r o - d u c t i o n o f a c r y o s t a t e w i t h a s p e c i a l l y d e s i g n e d , heavy m i c r o t o m e h a s f a c i - l i t a t e d t h e work c o n s i d e r a b l y . The d e v e l o p m e n t a l work f o r an a u t o m a t i c sec- t i o n i n g m a c h i n e h a s l a t e l y been s u c c e s s f u l and a p r o t o t y p e f o r t h i s p u r p o s e is now t e s t e d a t t h e d e p a r t m e n t o f T o x i c o l o g y , U p p s a l a U n i v e r s i t y . Examples o f w h o l e body S v e n U l l b e r g ’ s o t h e r F i . 1 Whole body a u t o r a d i o g r a m s from mice i n j e c t e d w i t h 3 s S - p r o m e t h a z i n e ( A ) +S-hydroxyethyl p r o m e t h a z i n e ( 5 ) . W h i t e areas c o r r e s p o n d t o h i g h a m o u n t s of r a d i o a c t i v i t y . Note t h e d i f f e r e n t u p t a k e of t h e compounds i n t h e b r a i n , w h i c h e x p l a i n s why s u b s t a n c e (B) d i d n o t c a u s e d r o w s i n e s s when u s e d f o r e . g . a l l e r g i c r h i n i t i s ( 2 ) . USE OF THE TECHNIQUE F o r r e f e r e n c e s e x e m p l i f y i n g t h e u s e o f t h e t e c h n i q u e t h e r e a d e r is r e f e r r e d t o (4), w h e r e an e x t e n s i v e b i b l i o g r a p h y of a p p l i c a t i o n s is p u b l i s h e d . The most a p p a r e n t a d v a n t a g e o f t h e w h o l e body a u t o r a d i o g r a p h y t e c h n i q u e as a s c i e n t i f i c t o o l is t h a t it o f f e r s a d e t a i l e d , b u t a l s o c o m p l e t e , view o f t h e d i s t r i b u t i o n o f a n i n j e c t e d compound i n a l l t h e t i s s u e s of t h e body. T h i s f a c t f a v o u r s t h e 234 p o s s i b i l i t y t o make unexpected d i s c o v e r i e s and a l s o c o n t r i b u t e s t o s u g g e s t i o n s f o r f u r t h e r research. The method is a l s o e x t e n s i v e l y used f o r r o u t i n e scree- n i n g work i n t h e pharmaceutical i n d u s t r y . The c o m b i n a t i o n of U l l b e r g ’ s method w i t h o t h e r t e c h n i q u e s has t o l a r g e e x t e n t c o n t r i b u t e d t o e l u c i d a t i n g t h e mech- a n i s m s o f a c t i o n s f o r many endogenous s u b s t a n c e s as well a s the p r i n c i p l e f o r c e r t a i n p h a r m a c o l o g i c a l and t o x i c o l o g i c a l e f f e c t s . As an example can be men- t i o n e d t h e s t u d i e s w h i c h g a v e t h e e x p l a n a t i o n why two a n t i - h i s t a m i n e s ( p r o m e t h a z i n e and h y d r o x y e t h y l p r o m e t h a z i n e ) i n f l u e n c e d t h e slee- p i n g p e r i o d a f t e r b a r b i t u r a t e a d m i n i s t r a t i o n i n d i f f e r e n t ways ( F i g . 1). An i m p o r t a n t q u e s t i o n when s t u d y i n g the d i s t r i b u t i o n o f a n t i b i o t i c s is the con- c e n t r a t i o n i n t h e s i t e o f t h e i n f e c t i o n , e.g. t h e a b i l i t y t o p e n e t r a t e the membrane o f an e n c a p s u l a t e d a b s c e s s . The c o n d i t i o n f o r 3 5 S - p e n i c i l l i n i n t h i s c o n t e x t is shown i n F i g . 2. a u t o r a d i o g r a p h i c Abscess membrane Abscess c o n t e n t F i g . 2 Autoradiogram showing the d i s t r i b u - t i o n of r a d i o a c t i v i t y i n two 30 day o l d ab- scesses a f t e r i n t r a v e n o u s i n j e c t i o n o f 3 5 ~ - p e n i c i l l i n i n t o a mouse. White areas re- p r e s e n t h i g h amounts of r a d i o a c t i v i t y . P e n i - c i l l i n p e n e t r a t e s the abscess membrane and a r e l a t i v e l y h i g h c o n c e n t r a t i o n is s e e n immediately i n s i d e t h e membrane. The con- c e n t r a t i o n is t h e n d i m i n i s h e d towards t h e c e n t e r of t h e a b s c e s s ( 3 ) . From t h e t o x i c o l o g i c a l p o i n t o f view r e t e n t i o n of a s u b s t a n c e i n a c e r t a i n tissue may be o f i n t e r e s t . The p r e v i o u s l y n o t known, s t r o n g a c c u m u l a t i o n o f t e t r a c y c l i n e s i n bone and t e e t h , w h i c h was o b s e r v e d by whole body a u t o r a d i - o g r a p h i c s t u d i e s i n 1956 h a s c o n t r i b u t e d t o t h e u n d e r s t a n d i n g of t h e s i d e e f - f e c t s r e p o r t e d r e g a r d i n g f o e t a l growth and m a l f o r m a t i o n s of the enamel i n the d e c i d u o u s t e e t h when u s i n g these a n t i b i o t i c s i n p r e g n a n t i n d i v i d u a l s . To be a b l e t o e v a l u a t e the a u t o r a d i o g r a p h i c image i n a q u a n t i t a t i v e way d i f f e r e n t methods have been t r i e d . Simple s y s t e m s u s i n g ’ i s o t o p e staircases’, t i n y p h o t o c e l l s and advanced systems w i t h c o m p u t e r i z e d image a n a l y s i s have 235 been used. Whole body s e c t i o n s have a l s o been used f o r p a r a l l e l i n v e s t i g a t i o n s such as q u a n t i t a t i o n and r a d i o c h e m i c a l a n a l y s i s o f p i e c e s punched o u t from t h e d i f f e r e n t t i s s u e s i n t h e s e c t i o n s . H i s t o c h e m i c a l methods have been a d j u s t e d t o whole body s e c t i o n s i n a t t e m p t s t o f u r t h e r e x p l a i n t h e mechanism o f a c t i o n i n r e l a t i o n t o an a u t o r a d i o g r a p h i c a l l y found l o c a l i z a t i o n , e.g. enzyme i n h i b i - t i o n . Substances with f l u o r e s c e n c e o f t h e i r own (e.9. t e t r a c y c l i n e s and c e r - t a i n m y c o t o x i n s ) have been i d e n t i f i e d d i r e c t l y i n whole body s e c t i o n s by u l t r a v i o l e t r a d i a t i o n . By u s i n g a s p e c i a l d r y mounting technique whole body sec- t i o n s and p h o t o g r a p h i c emulsions can be permanently mounted t o enable a more p r e c i s e l o c a l i z a t i o n o f t h e r a d i o a c t i v i t y t o s p e c i a l t y p e s o f c e l l s (’semi m i c r o t e c h n i q u e ’ ) (Fig. 3 ) . When i n t e r p r e t i n g t h e autoradiograms i t i s impor- t a n t r e p r e s e n t t h e o r i g i n a l substance as w e l l as r a d i o l a b e l l e d m e t a b o l i t e s . As an example can be mentioned t h a t t h e r a d i o a c t i v i t y i n F i g . 3 i n t h e a d r e n a l c o r t e x r e p r e s e n t s o n l y 10 % o f un- changed c h o l e s t e r o l a c c o r d i n g t o p a r a l l e l radiochromatographic s t u d i e s . The m a j o r i t y o f 1 4 C r e p r e s e n t s d i f f e r e n t c h o l e s t e r o l e s t e r s which a r e t h e s t o r a g e forms f o r t h e s t a r t i n g m a t e r i a l f o r s y n t h e s i s o f c o r t i c a l s t e r o i d hormones. t o remember t h a t t h e r a d i o a c t i v i t y can G l o m e r u l a r zone F a s c i c u l a r zone R e t i c u l a r zone M e d u I la F i g . 3 D F t a i l o f ’semi microauto- radiogram (whole body s e c t i o n and p h o t o g r a p h i c emulsion permanently mounted t o g e t h e r ) from a mouse in- j e c t e d i n t r a v e n o u s l y with “C-cho- l e s t e r o l and s a c r i f i c e d a f t e r f o u r days. B l a c k areas correspond t o h i g h amounts o f r a d i o a c t i v i t y . The r a d i o - a c t i v i v t y i s c o n f i n e d t o o n l y one c e l l t y p e l a y e r o f t h e c o r t e x (Zona f a s c i c u l a t a ) (1). Whole body autoradiography i m p l i e s t h a t r a d i o l a b e l l e d substances a r e a t hand. I n t h e l a t e 1950’ t h e r e were n o t t o o many l a b e l l e d p h a r m a c o l g i c a l or t o x i c o l o g i c a l substances a v a i l a b l e . Beside t h e b i o s y n t h e s i s o f l a b e l l e d sub- 236 s t a n c e s U l l b e r g and h i s coworkers s p e n t much time f o r ' c h e m i c a l ' l a b e l l i n g of i n t e r e s t i n g compounds b y e x c h a n g e r e a c t i o n s ( t r i t i u m , i o d i n e exchange etc.). To p o s s i b l y improve the a u t o r a d i o g r a p h i c r e s o l u t i o n , an i n v e n t o r y o f r a d i o - n u c l i d e s w i t h s u i t a b l e low e n e r g y r a d i a t i o n was made i n c o l l a b o r a t i o n w i t h t h e Royal T e c h n i c a l C o l l e g e i n Stockholm. S e v e r a l o f these r a d i o n u c l i d e s were s u c - c e s s f u l l y a p p l i e d i n b i o l o g i c a l e x p e r i m e n t s . A l s o v e r y s h o r t - l i v e d r a d i o n u c l i - d e s s u c h a s "F ( h a l f - l i f e 110 min.) h a v e been used f o r b o t h whole body a u t o - r a d i o g r a p h y a n d t h e a b o v e mentioned 'semi m i c r o t e c h n i q u e ' . I n t e r e s t i n g a r e a l s o t h e t o use t h e v e r y same s e c t i o n f o r l o c a l i z a t i o n of ''Ca as well as "F by u s i n g t h e i r d i f f e r e n c e s i n h a l f - l i v e s . S e v e r a l v o l a t i l e s u b s t a n c e s have been s t u d i e d by a s o f i s t i c a t e d method. Af- ter f r e e z i n g t h e whole a n i m a l , t h e s p e c i m e n s are k e p t a t - 8 O O C and after saw- i n g t h e b l o c k i n t o two p a r t s a t d r y ice t e m p e r a t u r e the ' h a l v e s ' are p l a c e d a g a i n s t X-ray films and exposed w h i l e k e p t a t the same low t e m p e r a t u r e . By t h i s method it is p o s s i b l e t o l o c a t e q u i t e many v o l a t i l e s u b s t a n c e s as well a s t h e i r l a b e l l e d m e t a b o l i t e s . When s e c t i o n s p r e p a r e d i n t h e t r a d i t i o n a l way are t h e n e x p o s e d , o n l y t h e n o n - v o l a t i l e l a b e l l e d material is v i s i b l e i n the a u t o - r a d i o g r a m s . After e x t r a c t i o n w i t h d i f f e r e n t s o l v e n t s r e - e x p o s u r e o f t h e secti- o n s can r e v e a l i f f i r m l y ( c o v a l e n t l y ? ) bound m e t a b o l i t e s a r e p r e s e n t . d u r i n g pregnancy and t o x i c o l o g y e v a l u a t i o n of compounds p o s s i b l y e f f e c t i n g p r e g n a n t women many c o n t r i b u t i o n s have been a c h i e v e d t h r o u g h t h e good r e s o l u t i o n of whole body a u t o r a d i o g r a p h y making it p o s s i b l e t o trace s u b s t a n c e s i n o r g a n s o f the growing f o e t u s . The whole body a u t o r a d i o g r a p h y t e c h n i q u e h a s a l s o been v e r y u s e f u l f o r f i n - d i n g s u i t a b l e s u b s t a n c e s w i t h s e l e c t i v e l o c a l i z a t i o n f o r d i a g n o s t i c and/or t h e r a p e u t i c u s e s i n c o n n e c t i o n w i t h n e o p l a s t i c diseases. S u b s t a n c e s w i t h s p e - c i f i c l o c a l i z a t i o n t o c e r t a i n may be l a b e l l e d w i t h y - e m i t t i n g i s o t o - pes f o r s u c h p u r p o s e s . Lately t h e u s e of s h o r t - l i v e d p o s i t r o n e m i t t i n g n u c l i - des i n c o n n e c t i o n w i t h p o s i t r o n e m i s s i o n tomography (PET) h a s a l s o i n c r e a s e d t h e need f o r f i n d i n g s u b s t a n c e s w i t h s e l e c t i v e l o c a l i z a t i o n s . p o s s i b i l i t i e s I n c o n n e c t i o n w i t h d r u g use t i s s u e s CONCLUSION T h a t t h e whole body a u t o r a d i o g r a p h y t e c h n i q u e is s t i l l r e l e v a n t and up t o date w i t h i n b i o m e d i c a l research is shown by its e x t e n s i v e use ( a l o n e and i n c o m b i n a t i o n w i t h o t h e r methods) i n f i e l d of pharmacology and t o x i c o l o g y . T h e i n t r o d u c t i o n o f PET h a s a l s o g i v e n c u r r e n t i n t e r e s t t o the p o s s i b i l i t i e s t o f i n d d i s t i n c t , s p e c i f i c l o c a l i z a t i o n s f o r which t h e whole body a u t o r a d i o - graphy i s s t i l l t h e u n r i v a l l e d method. t h e 231 REFERENCES 1. Appelgren, L.-E.: S i t e s o f s t e r o i d hormone formation. A u t o r a d i o g r a p h i c s t u d i e s u s i n g l a b e l l e d p r e c u r s o r s . Acta P h y s i o l Scand (SupEl 301) 1-108, 1967. 2. Hansson, E. Schmiterlow, C.G.: A comparison o f t h e d i s t r i b u t i o n , e x c r e t i o n and metabolism o f a t e r - t i a r y (promethazine) and a q u a r t e n a r y ( A p r o b i t ) p h e n o t h i a z i n e com- pound l a b e l l e d w i t h S 3 ' . Archs I n t Pharmacodyn ThCr 131:309-324, 1961. 3. U l l b e r g , S.: S t u d i e s on t h e d i s t r i b u t i o n and f a t e o f S3 ' - l a b e l l e d b e n z y l p e n i c i l l i n i n t h e body. Acta Radio1 (Suppl 118) 1-110, 1954. 4. U l l b e r g , S.: The technique o f whole body autoradiography. C r y o s e c t i o n i n g o f l a r g e specimens. Science Tools. The LKB I n s t r u m e n t J o u r n a l (Sweden). S p e c i a l I s s u e on Whole Body Autoradiography 1-29, 1977. Address f o r r e p r i n t s : L a r s - E r i k Appelgren Dept o f Pharmacology Toxicology Biomedicum, Box 573 S-751 23 Uppsala, Sweden 238