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Quantitative studies of factors affecting bone blood flow based on bone clearance of radiostrontium (Sr85) Shim, Sun Shik 1965

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The Un i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of SUN SHIK SHIM ^ Severance Medical College, ^955 '*' Yonsei University, Seoul M..Sc.,.The University of B r i t i s h Columbia, 1963 MONDAY, MAY 3, 1965, AT 10:00 A.M.. IN PHYSIOLOGY SEMINAR ROOM #203 Block A Medical Sciences Building COMMITTEE IN CHARGE Chairman: I. McT. Cowan E. C. D. H. C. F i Black Copp Cramer L. Kraintz H. W. Mcintosh H. McLennan F. P. Patterson External Examiner: P. J . K e l l y Department of Orthopedic Surgery Mayo C l i n i c , Rochester, Minnesota QUANTITATIVE STUDIES ON FACTORS AFFECTING BONE BLOOD FLOW BASED ON BONE CLEARANCE OF RADIOSTRONTIUM ( S r 8 5 ) ABSTRACT Factors a f f e c t i n g bone blood flow were studied i n rabbits and dogs, A method for estimating bone blood flow was developed using the i n i t i a l bone clearance of radioactive Strontium (Sr 8-*) from' blood. The method i s based on Fick P r i n c i p l e and i s s i m i l a r to that, used i n estimating renal plasma flow from clearance of PAH or diodrast. The v a l i d i t y of the method depends on the e f f i c i e n c y of Sr8"* removal from blood by bone as indicated by the Extraction Ratio (ER). This represents the proportion of the Sr^5 which has been removed from the blood flow-ing through bone, It was determined i n 10 dogs by i n -j e c t i n g Sr&5 and plasma dye,T.-1824, into the nutrient artery of t i b i a . During the next 5 minutes, 87.3 i 2.9% of the plasma dye and 20.21+ 1.68% of the S r 8 5 were recovered from the corresponding femoral vein. The Extraction Ratio calculated from the data was 0.764._ 0.066 (SE) and i s comparable to the ER of 0.90 for extrac-t i o n of PAH by kidney. The high ER appears to j u s t i f y the use of i n i t i a l (0-5 min) bone clearance of S r 8 ^ as a measure of e f f e c t i v e bone blood flow. Divided by the Extraction Ratio, the clearance gives an indirect, measure of t o t a l bone blood flow. Using the above technique, the mean e f f e c t i v e bone blood flow for 270 bones from 80 rabbits was found to be 9.60 + 0.19 (SE) ml/min/100 g fresh weight, and for 46 bones from 10 dogs, the average value was 10.15 T, 0.61 (SE) ml/min/100 g fresh weight. Total s k e l e t a l blood flow was estimated to be 7.1 1" 0.25 (SE) % of the r e s t i n g cardiac output i n the rabbits and 7.3 1 0.95 (SE) % of the resting cardiac output i n the dogs. The nutrient artery of femur was found to supply 70% of the blood flow to the shaft and 1/3 of the blood flow to the ends. Blood flow to the ends of bone was s i g n i f i -cantly higher than that to the shaft. Various factors a f f e c t i n g bone blood flow were studied. Section of the s c i a t i c nerve increased blood flow to the bones of the leg and foot, presumably due to i n t e r r u p t i o n of vasomotor f i b e r s . In contrast, i n f u s i o n of epinephrine (2-4 /vg/kg/min) reduced blood flow to t i b i a and humerus by 78-81% and sharply reduced calcium exchange between blood and the l a b i l e calcium storage pool i n bone. Immobilization of the leg i n a p l a s t e r cast for 2 weeks resulted i n some decrease i n blood flow i n t i b i a and calcaneus but more prolonged immobilization (2 months) caused disuse osteoporosis i n these bones and a r e l a t i v e increase i n blood flow. The s u r g i c a l problem of fractures of the neck of femur was.studied, and it. was found that such fractures reduced blood flow to the femoral head by 52-83%. This interference with blood flow may account for high incidence of aseptic necrosis of the femoral head associated with such f r a c t u r e s . GRADUATE STUDIES Mammalian Physiology including laboratory D. H. Copp Human Anatomy Neuro-phys i ology S. M. Friedman H. McLennan Neur o-anat omy H. F„ Scherrer Surgery Seminar F. P. Patterson Physiology Seminar Research Supervisor E. C. Black D. H... Copp QUANTITATIVE STUDIES OF FACTORS AFFECTING BONE BLOOD FLOW BASED ON BONE CLEARANCE OF RADIOSTRONTIUM ( S r 8 5 ) by SUN SHIK SHIM M.D., Yonsei U n i v e r s i t y Severance M e d i c a l C o l l e g e , 1955 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY, i n the Department of PHYSIOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1965 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l -able f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permis-s i o n . i ACKNOWLEDGEMENTS The author would l i k e t o express h i s deep a p p r e c i a t i o n to Dr. D. H a r o l d Copp f o r h i s sponsorship o f the author's graduate study and r e s e a r c h work, and f o r h i s encouragement and guidance; to Dr. Edgar C. Black, Chairman of the Commit-tee f o r the author's graduate study, f o r h i s t i r e l e s s en-couragement, d i r e c t i o n and a d v i c e ; and to Dr. Hugh McLennan and Dr. C a r l F. Cramer f o r t h e i r counsel and v a l u a b l e sug-g e s t i o n s . The author i s g r a t e f u l t o Dr. Frank P. P a t t e r s o n f o r h i s co-sponsorship of the author's graduate study, and to Dr. A l l a n M. McKelvie of Washington, D.C. who i n t r o d u c e d the author to Dr. P a t t e r s o n i n January, 1961. The author owes many thanks to Mr. Kurt Henze who p r e -pared the graphs and photographs, to Miss Mary Hashimoto who a s s i s t e d i n p r e p a r a t i o n of the manuscript, and to other s t a f f members of the Departments of Physiology, Surgery and P e d i -a t r i c s f o r t h e i r h e l p and c o - o p e r a t i o n . The author would p a r t i c u l a r l y l i k e to express a p p r e c i a -t i o n to the M e d i c a l Research C o u n c i l of Canada which f o r the p a s t three years has p r o v i d e d support through a M e d i c a l Re-search F e l l o w s h i p . He would a l s o l i k e t o acknowledge i i a s s i s t a n c e from the Defence Research Board of Canada and the Trauma Research U n i t of the Department of Surgery a t the U n i v e r s i t y of B r i t i s h Columbia. ABSTRACT F a c t o r s a f f e c t i n g bone blo o d flow were s t u d i e d i n r a b b i t s and dogs. A method f o r e s t i m a t i n g bone blo o d flow was developed u s i n g the i n i t i a l bone c l e a r a n c e o f r a d i o a c t i v e s t r o n t i u m ( S r 8 5 ) from b l o o d . The method i s based on F i c k P r i n c i p l e and i s s i m i -l a r to t h a t used i n e s t i m a t i n g r e n a l plasma flow from c l e a r a n c e of PAH or d i o d r a s t . The v a l i d i t y of the method depends on the e f f i c i e n c y of or Sr removal from blood by bone as i n d i c a t e d by the E x t r a c t i o n R a t i o (ER). T h i s r e p r e s e n t s the p r o p o r t i o n of the S r 8 5 which has been removed from the bloo d f l o w i n g through bone. I t was determined i n 10 dogs by i n j e c t i n g S r 8 5 and plasma dye, T-1824, i n t o the n u t r i e n t a r t e r y of t i b i a . D u r i n g the next 5 minutes, 87.3 ± 2.9% of the plasma dye and 20.21 ± 1.68% of the S r 8 5 were recovered from the cor r e s p o n d i n g femoral v e i n . The Ex-t r a c t i o n R a t i o c a l c u l a t e d from the data was 0.764 ± 0.066 (SE) and i s comparable to the ER of 0.90 f o r e x t r a c t i o n of PAH by kid n e y . The h i g h ER appears to j u s t i f y the use of i n i t i a l 85 (0-5 min) bone c l e a r a n c e of Sr as a measure of e f f e c t i v e bone bl o o d flow. D i v i d e d by the E x t r a c t i o n R a t i o , the c l e a r a n c e g i v e s an i n d i r e c t measure of t o t a l bone blood flow. Using the above technique, the mean e f f e c t i v e bone b l o o d flow f o r 270 bones from 80 r a b b i t s was found to be 9.60 - 0.19 i v (SE) ml/min/100 g f r e s h weight, and f o r 46 bones from 10 dogs, the average value was 10.15 - 0.61 (SE) ml/min/100 g f r e s h weight. T o t a l s k e l e t a l b l o o d flow was estimated to be 7.1 - 0.25 (SE) % of the r e s t i n g c a r d i a c output i n the r a b b i t s and 7.3 - 0.95 (SE) % o f the r e s t i n g c a r d i a c output i n the dogs. The n u t r i e n t a r t e r y of femur was found to- supply 70% of the b l o o d flow to the s h a f t and 1/3 of the b l o o d flow to the ends. Blood f l o w to the ends of bone was s i g n i f i c a n t l y h i g h e r than t h a t to the s h a f t . V a r i o u s f a c t o r s a f f e c t i n g bone b l o o d flow were s t u d i e d . S e c t i o n of the s c i a t i c nerve i n c r e a s e d blood flow to the bones of the l e g and f o o t , presumably due to i n t e r r u p t i o n of vasomotor f i b e r s . In c o n t r a s t , i n f u s i o n of epinephrine (2-4 micro-gram/ kg/min) reduced b l o o d flow to t i b i a and humerus by 74-81% and s h a r p l y reduced c a l c i u m exchange between b l o o d and the l a b i l e c a l c i u m storage p o o l i n bone. I m m o b i l i z a t i o n of the l e g i n a p l a s t e r c a s t f o r 2 weeks r e s u l t e d i n some decrease i n b l o o d flow i n t i b i a and calcaneus but more prolonged i m m o b i l i z a t i o n (2 months) caused d i s u s e o s t e o p o r o s i s i n these bones and a r e l a t i v e i n c r e a s e i n b l o o d flow. The s u r g i c a l problem of f r a c t u r e s of the neck of femur was s t u d i e d , and i t was found t h a t such f r a c -t u r e s reduced b l o o d flow to the femoral head by 52-83%. T h i s i n t e r f e r e n c e w i t h b l o o d flow may account f o r h i g h i n c i d e n c e of V a s e p t i c n e c r o s i s of the femoral head a s s o c i a t e d w i t h such f r a c t u r e s . v i TABLE OF CONTENTS Chapter page I INTRODUCTION 1 I I REVIEW OF THE LITERATURE 4 1. V a s c u l a r anatomy of bone 4 2. Nerve supply o f bone 7 3. P h y s i o l o g y of bone b l o o d c i r c u l a t i o n . 8 A) Rate of bone b l o o d flow 8 B) Mechanisms of r e g u l a t i o n of bone bloo d flow 12 C) F a c t o r s a f f e c t i n g bone blo o d flow 14 D) F u n c t i o n a l aspects of bone b l o o d flow 15 I I I THE METHOD OF MEASUREMENT OF BONE BLOOD FLOW DEVELOPED AND USED IN THIS STUDY 18 1. Techniques and procedures 18 2. P r i n c i p l e 20 3. V a l i d i t y o f the method 25 4. P o s s i b l e sources of e r r o r 27 5.. Advantages and l i m i t a t i o n s 30 IV EXPERIMENTAL STUDIES 31 Purpose, s p e c i f i c method, r e s u l t s and d i s c u s s i o n , summary and c o n c l u s i o n i n each study 1. Dependence of bone uptake of S r 8 5 on bone bl o o d flow 32 v i i Chapter Page IV EXPERIMENTAL STUDIES (cont'd) 2. S r 8 ^ e x t r a c t i o n r a t i o f o r bone 35 3. Normal b l o o d flow t o v a r i o u s r e g i o n s i n femur... 44 4. Normal r a t e s of b l o o d flow through v a r i o u s bones and estimates of t o t a l s k e l e t a l b l o o d flow 48 5. . R e l a t i v e c o n t r i b u t i o n of the three a r t e r i a l systems i n long bone 51 6. Blood flow to bone with d i s u s e o s t e o p o r o s i s 58 7. E f f e c t o f s c i a t i c nerve s e c t i o n on bone blo o d flow 64 8. E f f e c t o f f r a c t u r e s of the femoral neck on b l o o d supply to femoral head 70 9. E f f e c t of e p i n e p h r i n e on bone .blood flow 79 10. R e l a t i o n s h i p of bone blo o d flow to blood-bone c a l c i u m t r a n s f e r 84 V GENERAL SUMMARY AND CONCLUSIONS 97 TABLES 100-115 BIBLIOGRAPHY 116-129 Chapter I INTRODUCTION Bone i s a s p e c i a l i z e d connective t i s s u e i n which c o l l a g e n p r o v i d e s r e s i l i e n c e w h i l e the c r y s t a l s of bone m i n e r a l impart r i g i d i t y . The bones make up the s t r u c t u r a l framework of the body and p r o t e c t i n t e r n a l organs. They a l s o p r o v i d e a v a s t m i n e r a l r e s e r v o i r , which p l a y s an important p a r t i n homeostatic c o n t r o l o f the c o n c e n t r a t i o n of a number of ions i n body f l u i d s -p a r t i c u l a r l y c a l c i u m . The blo o d flow through bone i s important i n making t h i s r e s e r v o i r a v a i l a b l e and a l s o i n the g e n e r a l n u t r i t i o n o f the t i s s u e . However, u n t i l r e c e n t l y , t h e r e was v e r y l i t t l e i n f o r m a t i o n a v a i l a b l e concerning q u a n t i t a t i v e aspects of bone b l o o d flow, and t h i s l a c k of knowledge has hampered progress i n bone p h y s i o l o g y and i n the management of bone d i s e a s e s and i n j u r i e s . The p h y s i o l o g i c a l study of bone blo o d c i r c u l a t i o n i s very d i f f i c u l t and has been l i m i t e d because of the i n v o l v e d vascu-l a r p a t t e r n , the r i g i d i t y o f the t i s s u e and deep l o c a t i o n o f bone under the s o f t t i s s u e s . The proper i n t e r p r e t a t i o n o f the r e s u l t s of s t u d i e s of bone b l o o d c i r c u l a t i o n i s a l s o d i f f i c u l t . The p h y s i o l o g y of bone c i r c u l a t i o n can be c o n v e n i e n t l y separated i n t o the f o l l o w i n g f o u r major a s p e c t s : 2. 1. the r a t e of bone b l o o d flow, 2. the mechanism of r e g u l a t i o n of bone b l o o d flow, 3. the f a c t o r s a f f e c t i n g bone b l o o d flow, and 4. the f u n c t i o n s o f bone blood flow. I t cannot be emphasized too s t r o n g l y t h a t a good knowledge of these q u a n t i t a t i v e and q u a l i t a t i v e a spects of the p h y s i o l o g y of bone b l o o d c i r c u l a t i o n and the c o r r e l a t i o n s w i t h bone meta-b o l i s m i s e s s e n t i a l f o r the proper understanding of normal and abnormal behaviour of bone. Accurate measurement of bone blo o d flow i s extremely d i f -f i c u l t . To date, o n l y a few methods of e s t i m a t i o n of bone blo o d flow i n animals have been proposed, and a l l have l i m i t a t i o n s . There i s a t p r e s e n t no s a t i s f a c t o r y method f o r the measurement of bone b l o o d flow i n man. Bi o c h e m i c a l s t u d i e s of the a r t e r i a l and venous b l o o d of bone have been a l s o l i m i t e d . The met a b o l i c r a t e of the o r g a n i c substances o f bone i s not known. In oth e r words, the amounts of oxygen consumption, carbon d i o x i d e p r o d u c t i o n , hence the r e s p i r a t o r y q u o t i e n t o f bone have not been s t u d i e d . C o r r e l a -t i v e s t u d i e s of bone blo o d flow and m i n e r a l metabolism of bone a l s o have been l i m i t e d . Thus l i t t l e i s known about the quan-t i t a t i v e r e l a t i o n s h i p o f bone blo o d flow to the metabolism and behaviour of bone as a l i v i n g t i s s u e and organ i n both h e a l t h and d i s e a s e . 3. T h i s t h e s i s i s concerned w i t h the q u a n t i t a t i v e s t u d i e s of c e r t a i n aspects of b l o o d flow to bone i n r a b b i t and dog, i n -c l u d i n g the f o l l o w i n g : 1. Development of an i n d i r e c t method f o r measurement of bone blood f l o w based on d e t e r m i n a t i o n of bone c l e a r a n c e of r a d i o s t r o n t i u m . 2. S t u d i e s of the normal r a t e s of bone blo o d flow to v a r i o u s bones i n the r a b b i t and dog. 3. Q u a n t i t a t i v e s t u d i e s of f a c t o r s a f f e c t i n g bone b l o o d flow i n the r a b b i t . 4. S t u d i e s of the r e l a t i o n s h i p between blo o d flow and the blood-bone m i n e r a l dynamics i n the dog. 4. Chapter I I REVIEW OF THE LITERATURE T h i s chapter w i l l review the c u r r e n t concepts o f the vas-c u l a r anatomy of bone, the nerve supply to bone, and the quan-t i t a t i v e and q u a l i t a t i v e aspects of the p h y s i o l o g y of b l o o d c i r c u l a t i o n i n bone. 1. . THE VASCULAR ANATOMY OF BONE Using many methods of gross and micro-angiography, the abundant v a s c u l a r i t y of bone, wi t h complex anastomosis of blood v e s s e l s i n the marrow, c o r t e x and periosteum, has been w e l l demonstrated. Ham (29) emphasized the f a c t t h a t c a l c i f i e d r i g i d s t r u c t u r e and abundant v a s c u l a r i t y are c h a r a c t e r i s t i c of bone. The c l a s s i c a l s t u d i e s and f i n d i n g s of Langer (41) i n 1876 and Lexer (44) i n 1903 have been m o d i f i e d l i t t l e by subse-quent workers, i n c l u d i n g Johnson (34) , T r u e t a e t a_l. (82, 84) , McNab (50), Morgan (54), H a l i b u r t o n e t a l . (28) and K e l l y e t a l . (39). In g e n e r a l , i t i s agreed t h a t there are three major a r -t e r i a l i n l e t s i n long b o n e s — t h e n u t r i e n t , the epi-metaphyseal and the p e r i o s t e a l a r t e r i e s . Most long bones have one n u t r i e n t a r t e r y which p e n e t r a t e s the bone i n the middle p a r t of the 5. d i a p h y s i s ( s h a f t ) , few epi-metaphyseal a r t e r i e s i n the regions of each end o f bone and m u l t i p l e p e r i o s t e a l a r t e r i e s e n t e r i n g the s u r f a c e of bone. The n u t r i e n t a r t e r y d i v i d e s i n t o two or more main branches toward each end of the bone which d i v i d e f u r t h e r i n t o m u l t i p l e s m a l l e r branches. Nelson et _ a l . (55) observed t h a t s m a l l a r -t e r i o l e s branch r a d i a l l y p a s s i n g outward toward the c o r t e x i n the zone of spongy bone and i n the medullary c a v i t y . These v e s s e l s f u r t h e r d i v i d e i n t o f i n e branches as they enter the h a v e r s i a n c a n a l s . Many h a v e r s i a n c a n a l s c o n t a i n more than one c a p i l l a r y type of v e s s e l . These v e s s e l s i n the h a v e r s i a n c a n a l s make f i n e anastomosis with the numerous p e r i o s t e a l a r -t e r i e s , many of which reach the i n t e r i o r of the c o r t e x through c r o s s channels which are known as volkmann's c a n a l s . As des-c r i b e d by Ham (29), the l o n g i t u d i n a l (haversian) and the t r a n s -verse (volkmann) canals p r o v i d e m u l t i p l e routes f o r v a s c u l a r supply and anastomosis i n the c o r t e x . The metaphyseal a r t e r i e s which d i s t r i b u t e t h e i r branches i n the end r e g i o n s o f bone anastomose w i t h the t e r m i n a l twigs of the n u t r i e n t a r t e r y a t about the boundary between s h a f t and end. The c a p i l l a r i e s of the metaphyseal v e s s e l s run to the e p i p h y s e a l p l a t e i n long l o o p s . The e p i p h y s e a l a r t e r i e s , which a r i s e from the j o i n t capsule, do not j o i n the metaphyseal a r -t e r i e s across the e p i p h y s e a l p l a t e u n t i l bony union i s complete. 6. Thus the three a r t e r i a l systems i n long bones anastomose w i t h each other abundantly and complexly i n the c o r t e x and medullary spaces. While the a r t e r i a l systems have been d e s c r i b e d w e l l , l i t t l e i s known about the venous system. In g e n e r a l , however, i t i s b e l i e v e d t h a t the c a p i l l a r i e s d r a i n through venules or s i n u s e s to the l a r g e v e i n s which are a s s o c i a t e d w i t h and p a r a l l e l to the a r t e r i e s . I t i s w e l l known t h a t the l a r g e n u t r i e n t a r t e r y and metaphyseal a r t e r i e s are accompanied by corresponding v e i n s . The v a s c u l a r anatomy of non-tubular c a n c e l l o u s bones has r e c e i v e d r e l a t i v e l y l i t t l e study. The head of the femur and the t a l u s are r e p r e s e n t a t i v e examples of such bones. Because of t h e i r c l i n i c a l s i g n i f i c a n c e , e s p e c i a l l y i n f r a c t u r e s , the v a s c u l a r anatomy of the head of the femur was s t u d i e d r e c e n t l y by T r u e t a and H a r r i s o n (82) and t h a t of t a l u s by H a l i b u r t o n e t a l . (28). The t a l u s i s the more a p p r o p r i a t e one f o r study of the g e n e r a l v a s c u l a r p a t t e r n o f c a n c e l l o u s bone. S e v e r a l a r t e r i e s e n t e r the bone and branch i n t o many a r t e r i o l e s and f u r t h e r into, t h i n w a l l e d c a p i l l a r i e s or s i n u s e s u n t i l they become o r i e n t e d to each i n d i v i d u a l marrow space formed by the t r a b e c u l a e . The main i n t r a o s s e o u s a r t e r i e s are a s s o c i a t e d w i t h v e i n s which pro-g r e s s i v e l y u n i t e to form l a r g e r v e i n s and e v e n t u a l l y leave the bone by ro u t e s which are almost i d e n t i c a l w i t h the a r t e r i a l i n l e t s . I t cannot be emphasized too s t r o n g l y t h a t bone of any type i s v e r y v a s c u l a r . . There i s a l s o an i n t i m a t e a s s o c i a t i o n betwee the c i r c u l a t i o n i n the marrow c a v i t y and i n bone, so t h a t the two cannot be c o n s i d e r e d s e p a r a t e l y . 2. NERVE SUPPLY OF BONE While i t i s g e n e r a l l y b e l i e v e d t h a t bone i s abundantly s u p p l i e d by both sensory and motor nerve f i b e r s , there are few anatomical s t u d i e s on t h i s s u b j e c t . O t t o l e n g h i (58) i n 1902 s t u d i e d the nerve supply o f bone marrow of man, sheep, dog, r a b b i t , guinea p i g and c h i c k e n . In 1964, Sherman (69) ob-served abundant d i s t r i b u t i o n o f nerve f i b e r s i n the marrow c a v i t y and space between t r a b e c u l a e i n man. Peterson e t a l . (59) observed medullated nerve f i b e r s which were a s s o c i a t e d w i t h the n u t r i e n t a r t e r y . O t t o l e n g h i (58) concluded from h i s f i n d i n g s t h a t : (a) the bone marrow i s r i c h l y s u p p l i e d w i t h medullated and non-medullated f i b e r s , (b) these nerves form f i n e plexuses i n the w a l l s o f the b l o o d v e s s e l s and many r a m i f i c a t i o n s reach the c a p i l l a r i e s , (c) i n the marrow pulp there are many medullated and non-medullated f i b e r s p a s s i n g e v e n t u a l l y t o d i s t a n t v e s s e l s 8. and (d) the e x i s t e n c e o f s p e c i a l nerve endings about independ-ent marrow elements cannot be d e f i n i t e l y determined. These f i n d i n g s i n d i c a t e t h a t s p e c i a l i z e d n e u r a l r e f l e x mechanisms may be mediated through these nerve f i b e r s . The p h y s i o l o g i c a l s t u d i e s of t h e i r p o s s i b l e r o l e s w i l l be reviewed l a t e r i n t h i s chapter. 3. THE PHYSIOLOGY OF BONE BLOOD CIRCULATION The scope o f t h i s s u b j e c t may be c o n v e n i e n t l y d i v i d e d i n t o the f o l l o w i n g a s p e c t s : A. The r a t e o f bone b l o o d flow B. The mechanism of r e g u l a t i o n C. The f a c t o r s a f f e c t i n g bone b l o o d flow D. The f u n c t i o n s o f bone blo o d flow G e n e r a l — M a i n t e n a n c e o f t i s s u e v i t a l i t y — N u t r i t i o n a l - -Defensive and r e p a r a t i v e — O t h e r s S p e c i f i c — M o b i l i z a t i o n o f b l o o d c e l l s — M i n e r a l homeostasis A. THE RATE OF BONE BLOOD FLOW Dr i n k e r e t a l . (23, 24) were perhaps the f i r s t group who attempted t o measure bone b l o o d flow by a d i r e c t method. In 1916 and 1922, they i s o l a t e d the t i b i a of the dog and pe r f u s e d 9. i t through the n u t r i e n t a r t e r y , u s i n g a pump. In t h e i r study, the venous o u t f l o w was c o l l e c t e d i n a pan and measured. Ob-v i o u s l y , the s t u d i e s were not made under o p t i m a l p h y s i o l o g i c a l c o n d i t i o n s . They found t h a t the r a t e of flow ranged from 2.6-15.2 ml/min when p e r f u s e d a t the dog's own c i r c u l a t o r y p r e s s u r e . In 1955, F r e d r i c k s o n e t a l . (26) proposed an i n d i r e c t and more g e n e r a l l y a p p l i c a b l e method, based on i n i t i a l bone c l e a r -ance of r a d i o a c t i v e c a l c i u m (Ca^->) , T h i s procedure used the same p r i n c i p l e as t h a t employed i n measuring : the e f f e c t i v e r e n a l plasma o r bloo d flow from d i o d r a s t or PAH c l e a r a n c e by kidne y . "Clearance" i s d e f i n e d as the volume o f blood o r plasma " c l e a r e d " of the substance per minute by uptake or removal by the organ. The v a l i d i t y o f the cl e a r a n c e method i n measuring of b l o o d flow through an organ depends on the c o n s i s t e n c y and completeness of removal o f the substance concerned by the organ d u r i n g a s i n g l e passage. T h i s e f f i c i e n c y o f removal o f the substance by the organ i s expressed by the E x t r a c t i o n R a t i o (ER), where ER = (A-V)/A, and A and V are the c o n c e n t r a t i o n s of the substance i n b l o o d of the a r t e r y and v e i n s u p p l y i n g the organ. The k i d n e y i s h i g h l y e f f i c i e n t i n removing d i o d r a s t and PAH from blood, w i t h an ER of about 0.90, so t h a t the r e n a l c l e a r -ance o f these substance g i v e s a u s e f u l q u a n t i t a t i v e measure of e f f e c t i v e b l o o d or plasma flow through the kid n e y . F r e d r i c k s o n 10. e t al. (26) made the assumption t h a t bone would remove Ca^ 5 w i t h g r e a t e f f i c i e n c y from the blood f l o w i n g through bone, by exchange w i t h the v e r y l a r g e r e s e r v o i r of n o n r a d i o a c t i v e c a l c i u m p r e s e n t i n bone. In r a t t i b i a , they o b t a i n e d C a 4 5 c l e a r a n c e r a t e s ( e f f e c t i v e bone blo o d flow) from 10-30 ml/min/100 g bone. From these data they estimated t h a t the t o t a l s k e l e t a l b l o o d flow was about 1/5 of the r e n a l blood flow i n the r a t . In 1962, Cumming (19, 20) made a study of b l o o d flow through bone marrow i n the r a b b i t by a d i r e c t method, c o l l e c t i n g venous dra i n a g e . He r e p o r t e d t h a t the r a t e of the femoral mar-row b l o o d f l o w was 0.41-0.51 ml/min/g. In the same year, Post and Shoemaker (61) a l s o measured the b l o o d flow through the femur i n dog by c o l l e c t i n g from the upper and lower venous e f -f l u x systems of the bone f o l l o w i n g l i g a t i o n of as many other v e i n s as p o s s i b l e . They o b t a i n e d an average r a t e of flow of 11 ml/min through t h i s bone wi t h a range of 4.3-25.1 ml. In 1963, Copp and Shim (13, 70) r e p o r t e d t h a t the r a t e s of b l o o d flow through v a r i o u s bones i n r a b b i t s and dogs were remarkably s i m i l a r w i t h an average r a t e of 10 ml/min/100 g. They estimated the s k e l e t a l b l o o d flow as about 5-8% of the r e s t i n g c a r d i a c output. In the same year, Ray e t al. (62) and Weinman e_t al. (85) a l s o measured the bone blo o d flow i n dogs. The r a t e o b t a i n e d by Ray est a l . was 4.9 ml/min/100 g 11. and the s k e l e t a l b l o o d flow was estimated to be 4-9% of the r e s t i n g c a r d i a c output. Weinman e t a l . o b t a i n e d average r a t e s of 5.6 ml and 7.7 ml/min/100 g i n mature and immature dogs r e -s p e c t i v e l y . T h e i r estimated s k e l e t a l b l o o d flow was about 5-10% of the r e s t i n g c a r d i a c output. A l l of these s t u d i e s used the method of i s o t o p e c l e a r a n c e . In 1964, White e t aJL. (88) proposed a d i f f e r e n t method of measurement of bone blo o d flow i n which the amount o f Cr^"*" tagged b l o o d d e l i v e r e d from the c e n t r a l c i r c u l a t i o n to bone was measured. T h i s i s e s s e n t i a l l y a measurement of bone b l o o d volume by a d i l u t i o n t e c h nique. In b r i e f , they shut o f f com-p l e t e l y the b l o o d c i r c u l a t i o n to a limb by a t o u r n i q u e t and i n j e c t e d C r ^ i n t o the systemic c i r c u l a t i o n . A p e r i o d of 11 minutes was allowed f o r e q u i l i b r a t i o n o f the c o n c e n t r a t i o n of the i s o t o p e i n the systemic c i r c u l a t i o n . Then, the i s o t o p e c o n c e n t r a t i o n per ml b l o o d of the h e a r t , o b t a i n e d by c a r d i a c puncture, was determined. F i n a l l y , the t o u r n i q u e t p l a c e d on the limb was r e l e a s e d to allow the c i r c u l a t i o n to the limb to r e -sume. The r a d i o a c t i v i t y i n the v a r i o u s t i s s u e s o f the limb, i n c l u d i n g bone, was analyzed a t i n t e r v a l s from 2 seconds t o 15 minutes a f t e r r e l e a s i n g the t o u r n i q u e t . By a r a t h e r com-p l i c a t e d a n a l y s i s of the curves obtained, they estimated the bloo d flow through the v a r i o u s t i s s u e s , i n c l u d i n g bone. 12. They r e p o r t e d an average r a t e o f bloo d flow through the t i b i a i n r a b b i t s of 0.16 ml/min/g. They a l s o r e p o r t e d the r a t e of b l o o d flow (ml/min/g) through other t i s s u e s measured a t the same time as f o l l o w s : s k i n , 0.54; s k e l e t a l muscle, 0.27; and tendon, 0.10. Since b l o o d flow was measured f o l l o w i n g a p e r i o d of a b s o l u t e ischemia o f more than 10 minutes, the hyperemic e f f e c t undoubtedly would have magn i f i e d t h e i r measured r a t e of f low. In summary, q u a n t i t a t i v e measurement of bone b l o o d flow i s ver y d i f f i c u l t and none of the p r e s e n t methods appears to be ap-p l i c a b l e i n man. However, the r a t e s of bone bloo d flow i n a n i -mals measured by i n d i r e c t methods by s e v e r a l groups o f workers are remarkably c o n s i s t e n t . The range of va l u e s r e p o r t e d are as f o l l o w s : i n the r a t , 10-30 ml/min/100 g; i n the r a b b i t , 10-16 ml/min/100 g; and i n the dog, 5-10 ml/min/100 g. In these s t u d i e s , the e n t i r e s k e l e t a l b l o o d flow was estimated to be ap-pr o x i m a t e l y 5-10% of the r e s t i n g c a r d i a c output. B. THE MECHANISMS OF REGULATION OF BONE BLOOD FLOW Although p o o r l y understood, there i s some evidence t h a t the bone blo o d c i r c u l a t i o n i s c o n t r o l l e d by both n e u r a l and  hormonal mechanisms. In 1916 and 1922, Dr i n k e r e t a l . (23,24), i n t h e i r experiments p e r f u s i n g the i s o l a t e d t i b i a of the dog through the n u t r i e n t a r t e r y , found t h a t the ou t f l o w of b l o o d 13. was decreased when they e l e c t r i c a l l y s t i m u l a t e d the nerve f i b e r s of the bone marrow. T h i s was a l s o the case when e p i -nephrine was added to the p e r f u s i n g b l o o d . In 1952, Bloomen-t h a l e t J L I . (5) observed t h a t the i n t r a m e d u l l a r y p r e s s u r e of long bone i n the dog f e l l . when epinephrine, n o r e p i n e p h r i n e or p i t r e s s i n were g i v e n . In 1958, S t e i n et; _ a l . (76) r e p o r t e d s i -m i l a r f i n d i n g s . In 1959, H e r z i g and Root (32) and Weiss and Root (87) found i n the c a t t h a t s t i m u l a t i o n o f p e r i p h e r a l ends of v a r i o u s p e r i p h e r a l nerves and a d m i n i s t r a t i o n o f ep i n e p h r i n e caused a f a l l i n the i n t r a m e d u l l a r y b l o o d p r e s s u r e . In 1962 and 1963, Copp and Shim (12, 70) confirmed the above f i n d i n g s i n dogs. They found a c o n s i s t e n t and p e r s i s t e n t f a l l of the i n t r a m e d u l l a r y b l o o d p r e s s u r e , and a decrease or c e s s a t i o n o f bone b l e e d i n g through the d r i l l h o l e s made through the c o r t e x to the medullary c a v i t y d u r i n g slow intravenous i n f u s i o n of p h y s i o l o g i c a l s a l i n e c o n t a i n i n g a minute amount of e p i n e p h r i n e . Q u a n t i t a t i v e evidence t h a t e p i n e p h r i n e reduces bone b l o o d flow w i l l be presented i n t h i s t h e s i s . In 1963, Shaw (67, 68) a l s o confirmed the above f i n d i n g s . In the same year, Trotman and K e l l y (80) r e p o r t e d a 27% i n -crease i n b l o o d flow to the t i b i a i n the a n a e s t h e t i z e d dog on the f o u r t h day a f t e r lumbar sympathectomy. T h e i r study was based on the r a t e o f bone uptake o f r a d i o a c t i v e rubidium (Rb 8 6) . 14. The anatomical f a c t that bone i s r i c h i n both medullated and non-medullated nerve f i b e r s w i l l be r e c a l l e d . Since e p i -nephrine, norepinephrine and p i t r e s s i n are a l l nat u r a l l y oc-curring substances i n the body, the above evidence strongly indicates that the bone blood c i r c u l a t i o n i s controlled by both sympathetic vasomotor nerves and by vasopressor hormones. C. FACTORS AFFECTING BONE BLOOD FLOW Since the bone blood c i r c u l a t i o n i s an important part of peripheral c i r c u l a t i o n and since bone i s an organ having many important functions i n the body, there must be many factors a f f e c t i n g bone blood c i r c u l a t i o n . They can be eith e r physio-l o g i c a l factors ( i . e . respiration) or pathological ( i . e . hemor-rhage), and either systemic factors ( i . e . shock) or l o c a l ( i . e . f r a c t u r e ) . The action therefore could be either d i r e c t or i n -d i r e c t , and the e f f e c t of such action could be either hyperemia or ischemia of bone. Active hyperemia i n bone following acute i n f e c t i o n (acute osteomyelitis) i s well known c l i n i c a l l y . Ischemia following fractures of bone, and hyperemia i n the stage of healing of fractures are also well known. Evidence was found i n the i s o l a t e d perfusion experiment of the t i b i a i n the dog by Drinker et aJL. (24) i n 1922, that anoxia would increase the bone blood flow. This e f f e c t could be of value since, in hypoxic or anoxic state, the body requires more c i r c u l a t i n g red 15. c e l l s which must be m o b i l i z e d from the bone marrow. L o c a l ischemia o f bone, whatever the cause, might r e s u l t i n accumu-l a t i o n o f C O 2 and a c i d m e t a b o l i t e s and i n c r e a s e l o c a l hydrogen i o n c o n c e n t r a t i o n , a l l o f which might lead e v e n t u a l l y t o a c t i v e hyperemia due to chemical v a s o d i l a t i o n (53). Experimental s t u d i e s on these f a c t o r s a f f e c t i n g bone b l o o d flow have been l i m i t e d and o b s e r v a t i o n s have been fragmentary. There i s need f o r more experimental s t u d i e s on both q u a l i t a t i v e and q u a n t i -t a t i v e a s p e c t s . D. THE FUNCTIONAL ASPECTS OF BONE BLOOD FLOW The prime f u n c t i o n o f bloo d c i r c u l a t i o n through any t i s s u e s or organs i s to mai n t a i n the l i f e o f c e l l s , t i s s u e and organ. T h i s i s c e r t a i n l y t r u e f o r bone. Ham (29) and H a r r i s and Ham (31) have s t r e s s e d the importance o f the v a s c u l a r supply to bone i n r e l a t i o n t o nourishment o f bone c e l l s . A v a s c u l a r or ischemic n e c r o s i s of bone by some known ( i . e . f r a c t u r e ) or unknown reason i s one of the s e r i o u s problems i n o r t h o p e d i c surgery (2, 66). Johnson (34) i n 1927 made an e x c e l l e n t study of. the r e -l a t i v e importance of each of the three a r t e r i a l systems i n long bone. F o l l o w i n g s e l e c t i v e l i g a t i o n or d e s t r u c t i o n o f two of the three a r t e r i a l systems o f the t i b i a i n the dog he ob-served s p e c i f i c areas o f dead bone. He concluded t h a t the 16. n u t r i e n t a r t e r y i s r e s p o n s i b l e f o r the nourishment of the bone marrow and the inn e r h a l f or 2/3 of the c o r t e x of the s h a f t , t h a t metaphyseal a r t e r i e s supply b l o o d to the metaphyseal areas and t h a t the p e r i o s t e a l a r t e r i e s supply the outer h a l f or 1/3 of the c o r t e x of the s h a f t . Subsequent workers have agreed w i t h Johnson although some workers questioned the r o l e o f the p e r i o s t e a l a r t e r i e s . However, T r u e t a and C a l a d i a s (84) i n 1964 s t u d i e d t h i s problem c a r e f u l l y and e x t e n s i v e l y i n the r a b b i t ' s r a d i u s and confirmed Johnson's f i n d i n g s . The f u n c t i o n o f bone blood flow i n f r a c t u r e h e a l i n g or r e p a i r o f other i n j u r i e s i s of s e r i o u s concern to the o r t h o -p e d i c surgeon. Although ischemia i s known to hamper the pro-cesses of r e p a i r of bone i n j u r y , the t h r e s h o l d l e v e l of quan-t i t a t i v e ischemia or harmful l e v e l o f hyperemia f o r r e p a i r of f r a c t u r e i s not known as has been emphasized by Ray (64). The f u n c t i o n o f the bone b l o o d c i r c u l a t i o n i n growth and development of bones i s w e l l known (81, 83). Hyperemia i n growing stage o f bone causes i n c r e a s e i n r a t e of bone growth. T h i s knowledge i s c l i n i c a l l y a p p l i e d to c o r r e c t l e g - l e n t h d i s -crepancy i n growing c h i l d r e n . T h i s has been accomplished by s u r g i c a l c r e a t i o n o f an a r t e r i o - v e n o u s f i s t u l a (33, 86), lum-bar sympathectomy (4, 30), and s u r g i c a l s t r i p p i n g o f periosteum. J u v e n i l e o s t e o c h o n d r o s i s and some c o n g e n i t a l anomalies ( i . e . 17. c o n g e n i t a l h i p d i s l o c a t i o n i n i n f a n t ) may be due to l o c a l de-f e c t o f v a s c u l a r development c a u s i n g l o c a l ischemia i n the developmental stage o f bone. L i t t l e i s known q u a n t i t a t i v e l y about the c o r r e l a t i o n of oxygen consumption of bone to bone metabolism (53, 65), but. the problem i s now b e i n g s t u d i e d i n t h i s l a b o r a t o r y . M o b i l i z a t i o n o f c i r c u l a t i n g blood c e l l s from the bone i n t o b l o o d and t r a n s p o r t o f hemopoietic m a t e r i a l s t o bone marrow are important s p e c i f i c f u n c t i o n s o f bone blood c i r c u l a t i o n . Another important s p e c i f i c f u n c t i o n o f bone bloo d flow i s the b l o o d -bone m i n e r a l t r a n s f e r which i s e s s e n t i a l f o r c a l c i f i c a t i o n o f bone and r e g u l a t i o n o f m i n e r a l metabolism. Bone i s a major m i n e r a l and e l e c t r o l y t e s r e s e r v o i r and p l a y s an important r o l e i n c a l c i u m homeostasis. These important aspects have been s t r e s s e d by McLean e t a i l . (48) , Neuman e t _ a l . (56) , Copp e t a l . (11, 15, 16), and many o t h e r s . 18. Chapter I I I THE METHOD OF MEASUREMENT OF BONE BLOOD FLOW DEVELOPED AND USED IN THE STUDY The i n d i r e c t method of measurement of bone b l o o d flow which was developed and used i n t h i s study w i l l be d e s c r i b e d i n t h i s c h a p ter. The method i s based on measurement of bone c l e a r a n c e of r a d i o a c t i v e s t r o n t i u m ( S r 8 5 ) i n the i n i t i a l 5 minutes f o l l o w i n g intravenous i n j e c t i o n s . Clearance i s d e f i n e d as the volume of bl o o d c l e a r e d o f the i s o t o p e by bone uptake expressed as ml per minute. The advantages and l i m i t a t i o n s o f the method w i l l be d i s -cussed l a t e r . 1. THE TECHNIQUES AND PROCEDURES 1. In the a n a e s t h e t i z e d animal, the c a r o t i d a r t e r y i s cannulated w i t h a p o l y e t h y l e n e tube (PE 60) d i r e c t e d towards the h e a r t . 2. The cannula i s connected t o a slow constant r a t e s y r i n g e pump as shown i n F i g u r e 2. (The s y r i n g e pump used was No. 5-8292, American Instrument Co., S i l v e r Spring, Mary-land .) 3. A known amount of r a d i o s t r o n t i u m ( S r 8 5 d 2 / 5-10 micro-c u r i e s / k g o f animal weight) i s i n j e c t e d i n t o an ear v e i n 19. or j u g u l a r v e i n . T h i s i n j e c t e d dose i s same as t h a t i n the standard s o l u t i o n prepared. 4. The pump i s turned on immediately f o l l o w i n g the i n -j e c t i o n and the c a r o t i d a r t e r i a l b l o o d i s withdrawn c o n t i n u -o u s l y a t the r a t e o f approximately 1.5-2.0 ml/min. 5. At the end of the f i f t h minute, acute c a r d i a c a r r e s t i s induced i n the animal t o secure instantaneous c i r c u l a t o r y a r r e s t . T h i s i s done by i n j e c t i o n of about 10 ml of 6-10% EDTA s o l u t i o n i n t o the h e a r t through the j u g u l a r v e i n which pro-duces immediate c a r d i a c a r r e s t . 6. The withdrawn b l o o d i s g e n t l y shaken t o mix thoroughly and 1 ml a l i q u o t s are p i p e t t e d i n t o two or three p l a n c h e t s . 7. The bone(s) f o r study, are removed q u i c k l y , s o f t t i s s u e i s removed and the f r e s h weight of the bone i s d e t e r -mined. 8. The bones are then ashed and d i s s o l v e d i n IN HNO^. 9. The average r a d i o a c t i v i t y i n the 1 ml bloo d a l i q u o t s i s determined. The i s o t o p e uptake by the bones i s a l s o de-termined by measuring the r a d i o a c t i v i t y i n a l i q u o t s of bone ash d i s s o l v e d i n HNO^. 10. The i n d i v i d u a l bone uptake of r a d i o a c t i v i t y i s d i v i d e d by the r a d i o a c t i v i t y per ml bloo d to o b t a i n the 5 minute c l e a r a n c e of i s o t o p e by t h a t bone. 20. 11. T h i s v a l u e i s d i v i d e d by 5 to o b t a i n the c l e a r a n c e i n ml/min. 2. PRINCIPLE The method i s based on F i c k P r i n c i p l e , and i s e s s e n t i a l l y the same as t h a t f o r de t e r m i n i n g b l o o d flow through kidney by measuring c l e a r a n c e of para-aminohippuric a c i d (PAH) (9) or d i o d r a s t (18). The F i c k P r i n c i p l e s t a t e s t h a t the b l o o d flow through an organ i s e q u a l t o the amount o f the substance taken up from the b l o o d p a s s i n g through i t d i v i d e d by the a r t e r i o v e n o u s con-c e n t r a t i o n d i f f e r e n c e of the substance. I t i s formulated i n the f o l l o w i n g e q u a t i o n : F = Q/A-V, where F i s the volume of bl o o d flow, Q i s the amount o f the r e f e r e n c e substance taken up (or added) by the organ, and A and V are, r e s p e c t i v e l y , the c o n c e n t r a t i o n s o f the substance i n 1 ml of b l o o d from the a r t e r y and v e i n . I t i s easy to determine the S r 8 5 c o n c e n t r a t i o n i n blo o d of the n u t r i e n t a r t e r y because t h i s w i l l be the same as t h a t ob-t a i n e d by sampling any convenient a r t e r y . However, c o l l e c t i o n of venous b l o o d from bone i s extremely d i f f i c u l t because of the v e r y s m a l l s i z e o f these v e s s e l s . However, as F r e d r i c k s o n e t a l . (26) p o i n t e d out, i f the Sr i s exchanged almost completely f o r n o n - r a d i o a c t i v e c a l c i u m from the v a s t m i n e r a l s t o r e i n bone d u r i n g the p e r i o d t h a t the b l o o d i s f l o w i n g through the bone v e s s e l s , the venous c o n c e n t r a t i o n w i l l be ve r y low (V-yO), and the formula becomes F = Q/A. T h i s i s the same as the c l e a r a n c e of Sr 8-^ by bone uptake, given by the formula: c _ Bone uptake of S r 8 5 S r 8 ^ S r 8 5 cone, i n 1 ml systemic a r t e r i a l b l o o d Here, we s h a l l c o n s i d e r some dynamic aspects o f the method. Since both the a r t e r i a l c o n c e n t r a t i o n o f S r 8 ^ and bone uptake change c o n t i n u o u s l y , the S r O J c l e a r a n c e must be measured on a dynamic b a s i s . F i g u r e 1 i l l u s t r a t e s the t i m e - c o n c e n t r a t i o n curve f o r S r 8 5 disappearance from the s y s t e m i c . a r t e r y c o n s t r u c t e d w i t h the averaged data from 10 r a b b i t s . In the f i r s t f i v e minutes f o l l o w i n g i n j e c t i o n , the S r 8 ^ c o n c e n t r a t i o n changes r a p i d l y , but changes s l o w l y a f t e r t h a t time. The bone uptake of Sr8-> (dU) , d u r i n g an i n f i n i t e l y s h o r t p e r i o d o f time (dt),. under the s p e c i f i c a c t i v i t y or c o n c e n t r a t i o n of the i s o t o p e i n 1 ml b l o o d ( S d t ) , can be expressed as f o l l o w s : dU = C x Sdt Sr where C i s S r 8 ^ c l e a r a n c e and presumably i s constant d u r i n g Sr the c l e a r a n c e p e r i o d . I n t e g r a t i o n f o r 5 minutes can be ex-pressed as f o l l o w s : 22. F i g u r e 1 T i m e - c o n c e n t r a t i o n curve f o r Sr disappearance from b l o o d . F i g u r e 2 i t Method of Continuous Withdrawal of Systemic Arterial Blood for Integration of Average Isotope Concentration per Ml. Blood. Inject Radioisotope into Jugular Vein. Method of continuous withdrawal of systemic a r t e r i a l b l o o d f o r i n t e g r a t i o n o f average i s o t o p e c o n c e n t r a t i o n per ml b l o o d . 24. F i g u r e 3 Sr c o n c e n t r a t i o n i n 1 ml c a r o t i d a r t e r i a l b l o o d (average f o r 10 dogs) a t 10 seconds and each minute. 25. Th e r e f o r e , the 5 minute bone c l e a r a n c e of the i s o t o p e would be C. expressed as: (5 4 d u A s d t The average S r 8 ^ c l e a r a n c e by bone, C, i n ml/min can be ob-t a i n e d by d i v i d i n g the 5 minute c l e a r a n c e by 5. That i s : c = 5 ftdt 4* The above formula means, e s s e n t i a l l y , t h a t the average  Sr8-> c l e a r a n c e o f bone (ml blood/min) can be o b t a i n e d by d i v i d -i n g the average minute bone uptake of i s o t o p e per minute by the  average c o n c e n t r a t i o n o f the i s o t o p e i n 1 ml of systemic a r -t e r i a l b l o o d c o l l e c t e d d u r i n g the 5 minute c l e a r a n c e p e r i o d . T h i s may be determined by drawing the a r t e r i a l blood c o n t i n u o u s l y from the c a r o t i d d u r i n g the c l e a r a n c e p e r i o d u s i n g a co n s t a n t r a t e s y r i n g e pump. T h i s b l o o d i s then mixed w e l l and the aver-age c o n c e n t r a t i o n o f the i s o t o p e i n 1 ml of the b l o o d i s d e t e r -mined. 3. THE VALIDITY OF THE METHOD In e v a l u a t i n g the v a l i d i t y o f the method, the f o l l o w i n g q u e s t i o n s are p e r t i n e n t : 1. Is bone uptake o f the i s o t o p e dependent upon bone bl o o d flow? 2. How e f f i c i e n t i s bone i n removing the i s o t o p e from the b l o o d p a s s i n g through bone? The answers to these q u e s t i o n s are given i n d e t a i l i n the chapter on Experiments I and I I r e s p e c t i v e l y . I t was proven t h a t the bone uptake of S r 8 ^ i s e n t i r e l y dependent on bone blood flow. The ischemic heads of the femur had o n l y 1.5% of the is o t o p e uptake of the normal head. Secondly, the v a l i d i t y of S r 8 ^ c l e a r a n c e as a measure of bone b l o o d flow depends on the c o n s i s t e n c y and completeness of removal o f the i s o t o p e by bone d u r i n g a s i n g l e passage. T h i s e f f i c i e n c y of bone i s expressed by the e x t r a c t i o n r a t i o (ER), where ER = (A-V)/A, and A and V are the c o n c e n t r a t i o n s of the i s o t o p e i n a r t e r i a l and venous b l o o d of bone. Here, i t w i l l be r e c a l l e d t h a t the venous c o n c e n t r a t i o n (V) was taken as zero i n the course of d e r i v a t i o n o f the c l e a r a n c e formula. I t i s important to know whether i t i s , i n f a c t , zero or f o r example, 50% of the a r t e r i a l c o n c e n t r a t i o n , i n order to know the e f f i c i e n c y o f removal of the i s o t o p e by bone and to know how much of a c t u a l bone blo o d flow i s measured by c l e a r a n c e . I f bone removes the i s o t o p e i n the bone a r t e r y completely, the venous c o n c e n t r a t i o n w i l l be zero, and t h i s c l e a r a n c e w i l l e q u a l the a c t u a l bone b l o o d flow. In such cases, the e x t r a c -t i o n r a t i o would be 1.0. 27. The average e x t r a c t i o n r a t i o determined f o r t i b i a i n 10 dogs was 0.75 and was f a i r l y c o n s i s t e n t f o r the f i r s t 5 one minute c l e a r a n c e periods-and from dog to dog (vide i n f r a , Experiment I I ) . These f i n d i n g s j u s t i f y the use o f i n i t i a l bone c l e a r a n c e o f S r 8 5 as a measure of bone b l o o d flow. The cl e a r a n c e value (ml/min) i s r e f e r r e d to as "the e f f e c t i v e bone bl o o d f l o w " . A c t u a l bone b l o o d flow can be c a l c u l a t e d r e a d i l y by d i v i d i n g t h i s v a l u e by the e x t r a c t i o n r a t i o , 0.75. 4. POSSIBLE SOURCES OF ERROR The f o l l o w i n g p o s s i b l e sources of e r r o r must be c o n s i d e r e d . a. E r r o r s r e l a t e d to i n d u c t i o n of instantaneous c i r c u l a -t o r y ( c a r d i a c ) a r r e s t : As we co n s i d e r e d the dynamic aspect of the method w i t h r e g a r d to r a p i d and continuous changes i n con-c e n t r a t i o n of the is o t o p e i n c i r c u l a t i n g b l o o d and i n bone up-take, any t i m i n g e r r o r i s c r i t i c a l . T h e r e f o r e , a t the end of 5 minute c l e a r a n c e time, the c a r d i a c a r r e s t must be induced a c c u r a t e l y and i n s t a n t a n e o u s l y . I t was proven t h a t i n j e c t i o n o f EDTA s o l u t i o n (10-15 ml o f 6-10%) i n t o the j u g u l a r v e i n caused immediate d i a s t o l i c c a r d i a c a r r e s t due to c a r d i a c hypo-c a l c e m i a . b. E r r o r s r e l a t e d to withdrawal o f c a r o t i d a r t e r i a l b l o o d : The continuous withdrawal of the c a r o t i d a r t e r i a l b l o o d through a p o l y e t h y l e n e tube u s i n g a constant r a t e s y r i n g e pump i s an 2 8 . important p a r t of the technique to determine the i n t e g r a t e d average c o n c e n t r a t i o n of the i s o t o p e i n 1 ml b l o o d . Any t e c h -n i c a l : f a i l u r e i n t h i s p r o c e s s would r e s u l t i n e r r o r i n the c l e a r a n c e d e t e r m i n a t i o n . Timing e r r o r , dead space i n the tube and mechanical d e f e c t of the pump i n withdrawing c o n s t a n t l y would produce h i g h e r or lower average c o n c e n t r a t i o n of the i s o -tope i n the blood, hence, lower or h i g h e r r a t e of c l e a r a n c e r e s p e c t i v e l y . C a r e f u l checking p r i o r to and d u r i n g the e x p e r i -ments should minimize p o s s i b l e e r r o r s a r i s i n g from t h i s mechan-i c a l a s pect. c. Sources of e r r o r r e l a t e d to the l e n g t h of c l e a r a n c e time: I t was found t h a t the bone e f f i c i e n c y o f i s o t o p e uptake, t h a t i s , Sr8-> e x t r a c t i o n r a t i o of bone, g r a d u a l l y decreases as 85 time passes by. Obviously, the longer the p e r i o d of Sr ac-cumulation i n the s k e l e t o n , the h i g h e r w i l l be the i s o t o p e con-c e n t r a t i o n i n the l a b i l e exchangeable m i n e r a l p o o l of bone, hence, i n the venous b l o o d . In order to minimize the e r r o r , a s h o r t e r p e r i o d of c l e a r a n c e ( i . e . l e s s than 5 minutes) would be more i d e a l . The i n i t i a l 5 minute c l e a r a n c e time was chosen i n t h i s study a f t e r a n a l y z i n g the t i m e - c o n c e n t r a t i o n curve (Figure 1) f o r the i s o t o p e disappearance from the systemic a r -t e r i a l b l o o d . At the end of 5 minutes f o l l o w i n g i s o t o p e i n j e c -t i o n , the curve i n d i c a t e s t h a t e q u i l i b r i u m between b l o o d and t i s s u e c o n c e n t r a t i o n s of i s o t o p e may be approached. 2 9 . d. Errors related to determining bone weight: Muscles, tendons and ligaments attached to bone must be c a r e f u l l y cleaned o f f p r i o r to weighing the bone. Since the clearance i s computed on the basis of bone weight, t h i s must be determined accurately. In general, the weights of bones on both sides ( i . e . l e f t and r i g h t tibiae) are s u r p r i s i n g l y similar i f the soft tissues are completely removed. Any discrepancy generally indicates incomplete removal of the soft tissues on either side, unless there i s u n i l a t e r a l pathology of bone ( i . e . osteoporo-sis) . e. Error related to radioisotope counting: Since the radioisotope i s continuously decaying (half l i f e of S r 8 5 i s 64-65 days), the bone and blood samples should be counted on the same day. A l l counting times including that of background must be standardized. Because of the random nature of radioactive decay, i f N disintegrations are counted, the standard error of t h i s determination i s f[N . For example, for 100 counts stand-ard deviation i s 100 or I0„(i.e. 10% of the count). For a count of 10,000, the standard deviation i s 100 ( i . e . 1% of the count). For t h i s reason, greater precision i s obtained with higher counts. However, at very high counting rates, a second error i s introduced i f two counts are so close together that the detector records them as a single count. This coincidence error i s a function of the lag time of detector and the sca l i n g c i r c u i t . 30. P o s s i b l e e r r o r s due to d i f f e r e n c e s i n geometry of the c o u n t i n g chamber and samples must be a l s o c o n s i d e r e d . A l l must be s t a n d a r d i z e d i n c l u d i n g the p h y s i c a l c o n d i t i o n , of the samples ( s i z e , shape and d e n s i t y , e t c . ) . C a r e f u l p r e p a r a t i o n of the standard s o l u t i o n of the i s o t o p e i s of g r e a t importance. 5. ADVANTAGES AND LIMITATIONS Th i s i s an i n d i r e c t method of measurement of bone b l o o d flow based on sound p h y s i o l o g i c a l p r i n c i p l e s . Two important advantages over the d i r e c t method must be noted. F i r s t , i t i s g e n e r a l l y a p p l i c a b l e to any bone and any animal. Second, there i s no s u r g i c a l i n t e r v e n t i o n so t h a t the p h y s i o l o g i c a l c o n d i t i o n of the c i r c u l a t i o n i s not a l t e r e d b e f o r e and d u r i n g the study. The method developed and used i n t h i s study appears to be simple, r e l i a b l e and g e n e r a l l y a p p l i c a b l e to any animal or bone. At present, i t i s not a p p l i c a b l e to man s i n c e i t r e q u i r e s removal of bones f o r S r 8 5 assay at the end of the experiment. However, i t might be p o s s i b l e to take a s u i t a b l e bone b i o p s y f o r study, or perhaps o b t a i n a bone specimen a t amputation. Chapter IV EXPERIMENTAL STUDIES I n * t h i s chapter, the f o l l o w i n g s e r i e s o f ex p e r i m e n t a l s t u d i e s w i l l be r e p o r t e d . In each study, the purpose, the s p e c i f i c method, r e s u l t s and d i s c u s s i o n , and the summary of the study w i l l be g i v e n . 1. Dependence of bone uptake of S r 8 5 on bone blo o d flow. 85 2. Sr e x t r a c t i o n r a t i o f o r bone. 3. Normal b l o o d flow t o v a r i o u s r e g i o n s i n femur. 4. Normal r a t e s of b l o o d flow through v a r i o u s bones and esti m a t e s of t o t a l s k e l e t a l b l o o d flow. 5. R e l a t i v e c o n t r i b u t i o n of the three a r t e r i a l systems i n long bone. 6. Blood flow to bone wi t h d i s u s e o s t e o p o r o s i s . 7. E f f e c t o f s c i a t i c nerve s e c t i o n on bone blo o d flow. 8. E f f e c t of f r a c t u r e s of femoral neck on blo o d supply to femoral head. 9. E f f e c t of ep i n e p h r i n e on bone b l o o d flow. 10. R e l a t i o n s h i p of bone b l o o d flow to blood-bone c a l c i u m t r a n s f e r . 32. EXPERIMENT I DEPENDENCE OF BONE UPTAKE OF S r 8 5 ON BONE BLOOD FLOW Ray e t _ a l . (63) observed uptake of Sr^O by dead bone which had been reimplanted, and on t h i s b a s i s , questioned whether the is o t o p e uptake was dependent on bone bloo d flow. In t h e i r ex-periments, the i s o t o p e was admin i s t e r e d 3 days be f o r e bone up-take was measured. Since the method of measuring bone b l o o d flow i n t h i s study i s based on the uptake and c l e a r a n c e of S r 8 ^ by bone, as d e s c r i b e d i n the p r e c e d i n g chapter, the f o l l o w i n g experiment was c a r r i e d out to determine whether immediate up-take of Sr 8-' by bone (over a 5 minute period) was dependent on an i n t a c t c i r c u l a t i o n . METHOD Ten a d u l t white r a b b i t s weighing 2.1-2.6 kg (average 2.4 kg) were used. They were f a s t e d o v e r n i g h t and a n a e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n o f 1 g urethane and 250 mg bar-bi t o n e / k g body weight. Through a p o s t e r i o r approach, one h i p j o i n t i n each animal was exposed and the neck of the femur was f r a c t u r e d a t the s u b c a p i t a l l e v e l ( j u s t below the head). Then the ligamentum t e r e s was a l s o cut and the head of the femur was removed completely from the h i p j o i n t . Thus the e n t i r e b l o o d supply t o the femoral head was cut o f f . The femoral head then was p l a c e d back i n t o the acetabulum, b l e e d i n g p o i n t s were c a u t e r i z e d and the wound was c l o s e d . W i t h i n a few hours a f t e r the surgery, 5-8 m i c r o - c u r i e s of c a r r i e r f r e e S r 8 5 C l 2 i n 1 ml of normal s a l i n e were i n j e c t e d i n t o the systemic c i r c u l a t i o n through an ear v e i n . F i v e minutes a f t e r the i n j e c t i o n , the a n i -mals were s a c r i f i c e d and the S r 8 5 uptake of the experimental (ischemic) femoral head was compared wi t h t h a t o f the c o n t r o l femoral head from the o p p o s i t e s i d e . RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table I . The s i g n i f i c a n c e of the r e s u l t s i s q u i t e apparent. The r a d i o i s o t o p e uptake i n the i n i t i a l 5 minutes f o l l o w i n g systemic i n j e c t i o n was 28 and 1850 count per minute i n the completely ischemic and normal femoral heads r e s p e c t i v e l y . The r e s u l t s c l e a r l y i n d i c a t e t h a t the S r 8 5 uptake by bone i n the i n i t i a l 5 minutes f o l l o w i n g the i s o t o p e i n j e c t i o n i s e n t i r e l y dependent on i n t a c t b l o o d flow. Over long p e r i o d s , i t i s p o s s i b l e t h a t a v a s c u l a r bone can take up r a d i o i s o t o p e t o some ext e n t by d i f f u s i o n . Ray et. a l . (63) removed the r a t r a d i u s , k i l l e d the c e l l s by b o i l i n g and reimplanted the dead bone i n t o h e a l t h y muscles. A dose of S r 9 0 was i n j e c t e d , and the uptake of i s o t o p e by dead bone 3 days l a t e r was found to be the same as the uptake by the normal bone on the o p p o s i t e s i d e . Boyd e t _ a l . (7) a l s o observed uptake of 34. p32 k v a d e a G : femoral head i n man s e v e r a l hours a f t e r the i s o -tope had been i n j e c t e d . I t seems probable t h a t t h i s r e p r e s e n t s i s o t o p e which has been c a r r i e d to the bone by d i f f u s i o n and ex-changed w i t h c a l c i u m i n the c r y s t a l s o f bone m i n e r a l . I t i s s i g n i f i c a n t t h a t Ray e t j a l . (63) observed more removal of the de-p o s i t e d i s o t o p e i n normal bone a s s o c i a t e d w i t h bone r e m o d e l l i n g . SUMMARY AND CONCLUSION The r a d i o s t r o n t i u m uptake by the normal and completely ischemic femoral heads i n 10 r a b b i t s i n the i n i t i a l 5 minutes f o l l o w i n g intravenous i n j e c t i o n was s t u d i e d . The uptake by the completely ischemic femoral heads was 1.5 ± 0.5% of the normal. I t i s concluded t h a t the S r 8 5 uptake  and c l e a r a n c e of bone i n the i n i t i a l 5 minutes f o l l o w i n g i n t r a -venous i n j e c t i o n i s e n t i r e l y dependent on bone blo o d flow. 35. EXPERIMENT I I S r 8 5 EXTRACTION RATIO FOR BONE The v a l i d i t y of the method of e s t i m a t i n g b l o o d flow through c e r t a i n organs such as k i d n e y and bone by measuring the c l e a r -ance of c e r t a i n substances depends on the organ's e f f i c i e n c y of removal of the substance from the c i r c u l a t i n g b l o o d . T h i s e f -f i c i e n c y i s expressed by the e x t r a c t i o n r a t i o (ER), ER = (A-V)/A, where, A and V are the c o n c e n t r a t i o n s o f the substance concerned i n i t s a r t e r y and v e i n r e s p e c t i v e l y . The k i d n e y i s h i g h l y e f -f i c i e n t i n removing PAH and d i o d r a s t w i t h an e x t r a c t i o n r a t i o about 0.9 f o r these substances. To assess the v a l i d i t y of bone c l e a r a n c e of S r 8 5 as a measure o f bone b l o o d flow, the S r 8 5 e x t r a c t i o n r a t i o of bone was d e t e r -mined i n the f o l l o w i n g experiments. METHOD The study was c a r r i e d out on 10 a d u l t mongrel dogs (18-25 kg) which were a n a e s t h e t i z e d w i t h p e n t o b a r b i t a l (nembutal, 30 mg/ k g ) . The S r 8 5 e x t r a c t i o n r a t i o of a r e p r e s e n t a t i v e bone, the t i b i a , was s t u d i e d by i n j e c t i n g 1 ml of s o l u t i o n c o n t a i n i n g about 5 m i c r o - c u r i e s of S r 8 5 and 3.75 mg of Evans blue (T-1824) i n t o the n u t r i e n t a r t e r y and a n a l y z i n g the blood c o l l e c t e d from the i p s i l a t e r a l femoral v e i n i n the 5 minutes f o l l o w i n g the i n j e c -t i o n . The n u t r i e n t a r t e r y was exposed as f o l l o w s . In c o n t r a s t to man, the n u t r i e n t a r t e r y i n dog normally a r i s e s from the a n t e r i o r t i b i a l a r t e r y r a t h e r than the p o s t e r i o r t i b i a l . A l o n g i t u d i n a l i n c i s i o n was made along the l a t e r a l border of the t i b i a from a p o i n t two inches p r o x i m a l to the head of the f i b u l a to the j u n c t i o n o f the middle and lower one t h i r d o f the l e g . The s u p e r f i c i a l f a s c i a was i n c i s e d , the p e r o n e a l muscles were r e t r a c t e d l a t e r a l l y and the extensors of the f o o t m e d i a l l y . The a r t e r y was d i s s e c t e d f r e e , and the muscular branches i n the upper p o r t i o n were l i g a t e d . In most dogs, the n u t r i e n t a r t e r y comes o f f the a n t e r i o r t i b i a l a r t e r y w i t h i n the f i r s t 2-3 inches d i s t a l to the p o i n t where i t passes through the i n t e r -osseous membrane. I t i s then d i r e c t e d downward and p o s t e r i o r l y to e n t e r the n u t r i e n t foramen a t the p o s t e r o l a t e r a l aspect of the t i b i a . Great care was taken i n h a n d l i n g the v e s s e l s t o avoid i n j u r y and spasm. The femoral v e i n on the same s i d e was exposed i n the femoral t r i a n g l e and prepared f o r c a n n u l a t i o n . . The animal was then given 1 mg heparin/kg i . v . and the v e i n was cannulated. The c o n t r o l b l o o d was c o l l e c t e d f o r few minutes. The a n t e r i o r t i b i a l a r t e r y was then l i g a t e d immediately d i s t a l to the o r i g i n o f the n u t r i -ent a r t e r y . The prepared s o l u t i o n , as mentioned above, was i n -j e c t e d i n t o the n u t r i e n t a r t e r y s l o w l y (over a one minute period) u s i n g a 23 gauge needle. Beginning immediately a f t e r the s t a r t of i n j e c t i o n , the femoral v e i n b l o o d was c o l l e c t e d f o r 5 minutes i n the f i r s t group of 5 dogs and i n each o f 5 minutes i n the second group of 5 dogs. The per cent of the i n j e c t e d dose of S r 8 5 a n c j 0 f Evans b l u e i n t h i s b l o o d was determined. The former was done by measuring the r a d i o a c t i v i t y i n s u i t a b l e a l i q u o t s u s i n g a s c i n t i l l a t i o n d e t e c t o r . The l a t t e r was determined on plasma u s i n g a Klett-Summerson p h o t o e l e c t r i c c o l o r i m e t e r . The he m a t o c r i t r e a d i n g on t h i s b l o o d was a l s o determined. The per cent dose of Evans bl u e c o l l e c t e d gave a measure of the f r a c t i o n of the tagged b l o o d e n t e r i n g the n u t r i e n t a r t e r y which was u l t i -mately recovered i n the femoral v e i n . At the end of the 5 minute c o l l e c t i o n p e r i o d , acute c a r d i a c a r r e s t was induced i n each animal as d e s c r i b e d p r e v i o u s l y and the t i b i a was s p e e d i l y removed f o r Sr a n a l y s i s . The bone was cleaned, d r i e d and ashed a t 600° C (1110° F) f o r 24 hours and then d i s s o l v e d i n lN HNO3. CALCULATIONS Symbols: % dose Eb = percentage of the dose of Evans bl u e i n -j e c t e d i n t o the n u t r i e n t a r t e r y . % dose S r 8 5 = percentage of the dose of S r 8 5 i n j e c t e d i n t o the n u t r i e n t a r t e r y . (% dose E b ) € = % Eb c o l l e c t e d from femoral v e i n . 38. (% dose S r 8 5 ) = % S r 8 5 c o l l e c t e d from femoral v e i n , f v U t = % S r 8 ^ taken up by the t i b i a i n t minutes. Q C S = c o n c e n t r a t i o n o f Sr 0 -^ i n b l o o d expressed as % S r 8 5 / m l . ER = E x t r a c t i o n R a t i o f o r S r 8 5 = (A-V)/A = (Q -Q )/Q where A A V A and V are the a r -t e r i a l and venous c o n c e n t r a t i o n s of S r 8 ^ i n b l o o d e n t e r i n g and D C l e a v i n g bone, and and r e p r e s e n t the q u a n t i t y of S r O J as 85 % dose Sr i n the blood e n t e r i n g and l e a v i n g bone d u r i n g the c l e a r a n c e p e r i o d . E x t r a c t i o n R a t i o I f a l l the b l o o d c o n t a i n i n g Evans bl u e and S r 8 ^ which en-t e r e d the n u t r i e n t a r t e r y was subsequently c o l l e c t e d from the femoral v e i n , (% dose Eb) should equal 100%; Q would be 100% f v A and Qy would equal (% dose S r 8 - " ) ^ . The e x t r a c t i o n r a t i o would be g i v e n by the formula: , 100% - (% dose S r 8 5 ) f v (1) ER = However, l e s s than 100% of the dose of Evans bl u e was i n f a c t c o l l e c t e d from the femoral v e i n . The s m a l l d i f f e r e n c e (13%) p r o b a b l y r e p r e s e n t s b l o o d which had by-passed the femoral v e i n and has r e t u r n e d by o t h e r venous channels. The l a b e l l e d b l o o d e n t e r i n g the n u t r i e n t a r t e r y which was u l t i m a t e l y 39, c o l l e c t e d from the femoral v e i n should be reduced by t h i s amount, and the p r o p o r t i o n would be r e p r e s e n t e d by (% dose Eb) . In t h i s case, Q_A = (% dose E b ) f and the formula f o r e x t r a c t i o n r a t i o becomes: (% dose Eb) f._ - (% dose S r 8 5 ) . (2) ER = — — — v (% dose E b ) f v T h i s formula has been used i n c a l c u l a t i n g the data presented i n t h i s t h e s i s . 85 Recovery of Sr The "recovery" o f the i n j e c t e d dose of S r 8 5 was c a l c u l a t e d on the b a s i s of the 5 minute uptake by the t i b i a p l u s the Sr c o l l e c t e d from the femoral v e i n c o r r e c t e d f o r the a d d i t i o n a l S r 8 5 which by-passed t h i s v e i n . The formula used was: (% dose S r 8 5 ) f v (3) Recovery of S r B b = U 5 + ER RESULTS AND DISCUSSION The r e s u l t s are summarized i n F i g u r e 4 and Tables I I and I I I . N e a r l y 90% of the i n j e c t e d dose of Evans bl u e was r e -covered i n the femoral v e i n whereas o n l y about 20% of the i s o -tope was recovered i n the same bl o o d . The average e x t r a c t i o n r a t i o was 0.764 - 0.066 (S.E.) w i t h the range 0.63 - 0.80. T h i s compares f a v o r a b l y w i t h v a l u e s (0.34 - 0.12) r e p o r t e d f o r removal of b r o m s u l f o n p h t h a l e i n (BSP) by l i v e r (73), but i s not as s a t i s f a c t o r y as those o b t a i n e d f o r e x t r a c t i o n r a t i o of 40. F i g u r e 4 Diagrammatic i l l u s t r a t i o n of d e t e r m i n a t i o n of S r 8 5 e x t r a c t i o n r a t i o o f t i b i a d i o d r a s t (18), 0.79-0.96, and PAH (9), 0.81-0.96 by the kidney. T h i s i s not s u r p r i s i n g i n view of the p o s s i b l e b l o o d flow through shunts and i n bone marrow. However, the data f o r bone are remarkably c o n s i s t e n t , and i n view of t h i s , the i n i t i a l 5 minute S r 8 ^ c l e a r a n c e should give a v a l i d and u s e f u l measure of the e f f e c t i v e bone b l o o d flow through those r e g i o n s i n bone where a c t i v e m i n e r a l exchange i s . t a k i n g p l a c e . The c l e a r a n c e value would be the minimal v a l u e . I t would give a measure of 75% average o f the a c t u a l bone b l o o d flow and the l a t t e r c o u l d be r e a d i l y e s t imated by d i v i d i n g the .clearance value by the e x t r a c -t i o n r a t i o . S i m i l a r s t u d i e s on the bone e f f i c i e n c y of Ca^ and C a ^ uptake were c a r r i e d out by Ray e t al. (62) , and Weinman e_t a l . (85). Both groups s t u d i e d the femur i n dogs. Ray e t a_l. found the C a ^ e x t r a c t i o n r a t i o was 0.53 f o r the f i r s t 5 minutes, and 47 0.48 f o r the f i r s t 10 minutes. Weinman e t al. u s i n g Ca ob-t a i n e d an e x t r a c t i o n r a t i o of 0.55 f o r the f i r s t 10 minutes. Although these data are somewhat lower than the data o b t a i n e d i n the p r e s e n t study, they are q u i t e comparable. Ray e t a i l . (62) found t h a t the bone e f f i c i e n c y of removal of C a ^ 5 decreased w i t h time. The data shown i n Table I I I a l s o i n d i c a t e d e c r e a s i n g e x t r a c t i o n e f f i c i e n c y , from f i r s t minute to t h i r d , 0.80, 0.74 and 0.63 r e s p e c t i v e l y . T h i s might w e l l be due 42. to g r a d u a l accumulation of the i s o t o p e i n t r o d u c e d i n the l a b i l e m i n e r a l p o o l . The r a p i d l y exchangeable or l a b i l e c a l c i u m p o o l o f bone i n man has been estimated as approximately 40-80 mg Ca/kg body weight. O b v i o u s l y , the longer the p e r i o d of i s o t o p e accumulation i n the bone, the h i g h e r w i l l be the i s o t o p e con-c e n t r a t i o n i n t h i s p o o l , and i n the venous blo o d i n e q u i l i b r i u m 8 5 w i t h i t . From data on Sr accumulation i n bone, i t i s e s t i -mated t h a t the e r r o r i n t r o d u c e d by t h i s would be l e s s than 5% a t 5 minutes. S h o r t e r c l e a r a n c e p e r i o d s would minimize t h i s e r r o r . The f a c t t h a t 87% of Evans blue i n j e c t e d i n t o the n u t r i e n t a r t e r y was recovered i n the i p s i l a t e r a l femoral venous b l o o d at the l e v e l of the femoral t r i a n g l e i n d i c a t e s t h a t most of the venous drainage from the t i b i a takes t h i s r o u t e . The remaining 13% of the dye c o u l d have by-passed the femoral v e i n , or pos-s i b l y be r e t a i n e d i n the bone. In any case, the v a l u e s are i n good agreement w i t h the data o b t a i n e d by Post and Shoemaker (61). Although e x t e n s i v e s u r g i c a l procedures were i n v o l v e d i n t h e i r method, they c o l l e c t e d 73 - 12% of the b l o o d tagged w i t h C r 5 1 and Evans bl u e i n the upper and lower venous e f f l u x systems of the femur f o l l o w i n g i n j e c t i o n i n t o the n u t r i e n t a r t e r y of the femur i n dogs. These f i n d i n g s i n d i c a t e t h a t Evans blue i s a v e r y u s e f u l n o n - d i f f u s i b l e dye i n such s t u d i e s . SUMMARY AND CONCLUSION The e x t r a c t i o n r a t i o f o r removal of Sr 8-* by a r e p r e s e n t a -t i v e bone, the t i b i a , was measured i n 10 dogs. I t was d e t e r -mined by i n j e c t i n g a n o n - d i f f u s i b l e plasma dye, Evans blue, and S r 8 ^ i n a mixed s o l u t i o n i n t o the n u t r i e n t a r t e r y of the t i b i a and a n a l y z i n g the r e c o v e r y of these i n the femoral venous bl o o d . E i g h t y - s e v e n per cent of the dye was recovered i n t h i s b lood i n the next 5 minutes, whereas o n l y 21% of i n j e c t e d S r 8 5 was so re c o v e r e d . By d i v i d i n g the d i f f e r e n c e by 87, the ex-t r a c t i o n r a t i o was o b t a i n e d . The average value f o r the e x t r a c t i o n r a t i o i n the 10 dogs was 0.764 * 0.066 (S . E . ) . T h i s h i g h value (comparable to the ER of 0.90 f o r PAH e x t r a c t i o n by kidney) would appear to j u s t i f y use o f i n i t i a l S r 8 ^ c l e a r a n c e as a measure of e f f e c t i v e bone bl o o d flow. 44. EXPERIMENT I I I NORMAL BLOOD FLOW TO VARIOUS REGIONS IN FEMUR Anatomical s t u d i e s and c l i n i c a l experience i n d i c a t e t h a t c e r t a i n r e g i o n s ( i . e . metaphyses) are more v a s c u l a r i z e d than o t h e r areas i n the same long bone. However, no q u a n t i t a t i v e study of the r e l a t i v e r e g i o n a l b l o o d flow i n long bones have been made to date. I t was s t u d i e d i n the f o l l o w i n g experiments. METHOD The s t u d i e s were c a r r i e d out on 50 a d u l t white r a b b i t s weighing 2.1-3.6 kg (average weight 2.7 k g ) . They were f a s t e d o v e r n i g h t and a n a e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n of 1 g urethane and 250 mg b a r b i t o n e . C a r r i e r - f r e e S r 8 5 was i n -j e c t e d as d e s c r i b e d above f o r d e t e r m i n a t i o n of bone c l e a r a n c e . In each case, the animal was k i l l e d 5 minutes a f t e r the i n j e c -t i o n and the femur was removed from one s i d e . The bone was then d i v i d e d i n t o 4 segments by c u t t i n g w i t h a f i n e e l e c t r i c saw j u s t below the head; j u s t below the l e s s e r t r o c h a n t e r and j u s t above the condyle. These 4 segments (head, t r o c h a n t e r , s h a f t and con-dyle) were cleaned, weighed and analyzed f o r S r 8 5 . The e f f e c -t i v e bone b l o o d flow through each was determined on the b a s i s of i n i t i a l S r 8 5 c l e a r a n c e , and i s expressed as ml/min/100 g f r e s h weight. 45. RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table IV. There were s t a t i s -t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the r e l a t i v e b l o o d flow through these v a r i o u s r e g i o n s . The g r e a t e s t flow was observed i n the head (18.67 - 0.52 ml/min/100 g) and the lowest flow was through the s h a f t (7.50 ± 0.16 ml/min/100 g ) . These data i n d i c a t e t h a t the areas c o n t a i n i n g a l a r g e pro-p o r t i o n of c a n c e l l o u s spongeybone have a h i g h e r r a t e of blood flow than those which are predominantly composed of compact bone. These o b s e r v a t i o n s agree with the anatomical f i n d i n g and the c l i n i c a l experience t h a t the metaphyseal r e g i o n s are more v a s c u l a r i z e d than the s h a f t . The data a l s o support the c l i n i c a l e xperience t h a t f r a c t u r e s i n the metaphyseal r e g i o n s , i n g e n e r a l , h e a l f a s t e r than those i n the s h a f t . An i n t e r e s t i n g q u e s t i o n a r i s e s as to why the femoral head has such a h i g h r a t e of b l o o d flow compared w i t h other r e g i o n s i n the same bone. Is i t because the femoral head i s a s m a l l bulbous bone which has to w i t h s t a n d tremendous weight b e a r i n g s t r e s s and hence r e q u i r e s h i g h e r r a t e o f metabolism? I f t h i s i s t r u e , the head of the humerus should have a s i m i l a r h i g h r a t e of b l o o d flow s i n c e i n quadrupeds l i k e the r a b b i t , the humeral head i s a l s o s u b j e c t e d to weight b e a r i n g s t r e s s . I t was found, i n f a c t , t h a t the b l o o d flow to the head of the 46. humerus i n 22 r a b b i t s s t u d i e d was as h i g h as t h a t to the fem-o r a l head, w i t h an average flow of 17 ml/min/100 g bone. C a l a n d r u c c i o and h i s a s s o c i a t e s (8) made c o r r e l a t i v e s t u d i e s between the "trochanter-head r a t i o " , (T/H r a t i o ) , o f p-^ 2 up-take and the p r o g n o s i s of h e a l i n g of the femoral neck f r a c t u r e and v i a b i l i t y of the femoral head. The T/H r a t i o would be 1 i f the r a t e s of i s o t o p e uptake by the t r o c h a n t e r and head of the •femur were the same. I f the uptake of the femoral head i s h i g h e r than t h a t o f the t r o c h a n t e r , the T/H r a t i o would be l e s s than 1. T h i s i s the normal case i n man as w e l l as i n the rab-b i t . The lowest T/H r a t i o which was found by C a l a n d r u c c i o e_t a l . (8) i n f r a c t u r e of the femoral neck w i t h good p r o g n o s i s was 0.4-0.5, which means t h a t the i s o t o p e uptake of the femoral head was twice as h i g h as t h a t of the t r o c h a n t e r . The data ob-t a i n e d i n t h i s study i n the r a b b i t agree wi t h the data f o r man. o n As Table IV i n d i c a t e s , the S r O J c l e a r a n c e by the t r o c h a n t e r was 10.38 and by the head, 18.78, which gi v e s a T/H r a t i o o f 0.55. A h i g h r a t i o i n d i c a t e s r e l a t i v e r e d u c t i o n i n S r 8 5 uptake and b l o o d flow to the femoral head, and i s i n g e n e r a l a s s o c i a t e d w i t h poor p r o g n o s i s f o r h e a l i n g of a f r a c t u r e of the femoral neck. SUMMARY AND CONCLUSION The r e l a t i v e b l o o d flow through the head, t r o c h a n t e r , s h a f t and c o n d y l a r r e g i o n s was s t u d i e d i n femurs from 50 r a b b i t s . The b l o o d flow through these r e g i o n s were s i g n i f i c a n t l y d i f f e r e n t w i t h h i g h e s t flow to the femoral head and the l e a s t to the s h a f t . I t i s suggested t h a t t h i s may be due to a hig h e r p r o p o r t i o n of c a n c e l l o u s bone i n the head. The b l o o d flow t o the femoral head was twice as h i g h as t h a t to the t r o c h a n t e r g i v i n g a normal T/H r a t i o of 0.55, comparable to t h a t observed i n man. 48. EXPERIMENT IV NORMAL RATES OF BLOOD FLOW THROUGH VARIOUS BONES AND ESTIMATES OF TOTAL SKELETAL BLOOD FLOW St u d i e s o f bl o o d flow through v a r i o u s bones have been d i f -f i c u l t and l i m i t e d . D r i n k e r est a l . (23, 24), Post and Shoemaker (61), and Cumming (19, 20) attempted to measure the blood flow through the t i b i a or femur i n dog or r a b b i t by c o l l e c t i n g the venous o u t f l o w from these bones. . F r e d r i c k s o n e t a l . (26) e s t i -mated bone bloo d flow i n the r a t by an i n d i r e c t method based on measurement of the i n i t i a l bone c l e a r a n c e o f a r a d i o a c t i v e c a l -cium ( C a ^ ) . By the same method, but u s i n g d i f f e r e n t i s o t o p e s , Copp and Shim (13, 14, 70), Weinman e t a l . (85, 86), and Ray e t a l . (62) measured b l o o d flow i n r a b b i t s and dogs. White et. a l . (88) used a d i l u t i o n technique employing C r ^ tagged red c e l l s . An e x t e n s i v e study o f bloo d flow to femur, t i b i a , humerus, t a l u s , calcaneus and the v e r t e b r a i n the r a b b i t and the dog was- made i n the f o l l o w i n g experiments. METHOD The animals used were white a d u l t r a b b i t s weighing 1.8-3.5 kg (average weight 2.5 kg), and a d u l t mongrel dogs weighing 9-25 kg (average weight 18 k g ) . They were a n a e s t h e t i z e d as 49. p r e v i o u s l y d e s c r i b e d . The blood flow through v a r i o u s bones were measured by the method d e s c r i b e d i n Chapter I I I . RESULTS AND DISCUSSION As shown i n Table V, the r a t e s of b l o o d flow were remark-ab l y s i m i l a r f o r the v a r i o u s bones i n r a b b i t s and dogs. The average r a t e s were 10.15 - 4.12 (S.D.) ml/min/100 g wet weight i n 10 dogs f o r 46 bones, and 9.60 - 3.28 (S.D.) ml/min/100 g wet weight i n 80 r a b b i t s f o r 270 bones. These were the average r a t e s f o r i n i t i a l S r 8 5 c l e a r a n c e which has been d e f i n e d as " e f -f e c t i v e bone b l o o d flow". The average r a t e s of t o t a l b l o o d flow, c o r r e c t e d f o r the e x t r a c t i o n r a t i o (0.75), were 13.20 - 5.35 (S. D.) and 12.48 ^ 4.26 (S.D.) ml/min/100 g wet weight, i n dog and  r a b b i t r e s p e c t i v e l y . From these data, and by t a k i n g the s k e l e -t a l weight as 10% of the body weight, and the r e s t i n g c a r d i a c output as 182 ml/min/kg i n the dog (72), and 175 ml/min/kg i n the r a b b i t (25), the r a t e of the s k e l e t a l b l o o d flow was e s t i -mated as 2 38 - 96 ml/min or 7.3 - 3.0% of the r e s t i n g c a r d i a c output i n the dog, and 31 - 10 ml/min or 7.1 - 2.3% of the r e s t -i n g c a r d i a c output i n the r a b b i t . These v a l u e s are i n g e n e r a l agreement w i t h those of Weinman e t a l . (85) who r e p o r t e d a s k e l e t a l b l o o d flow e q u i v a l e n t to 5-7% of the r e s t i n g c a r d i a c output i n dogs, and Ray e t _a l . (62) who ob t a i n e d v a l u e s of 3.5-9.0%. 50. Based on e f f e c t i v e bone blood flow per 100 g f r e s h weight, the v a l u e s r e p o r t e d here are somewhat h i g h e r than those obtained by Weinman e t a l . (5.6-7.7 ml/min/100 g) and by Ray e t al. (ave-rage value 4.9 ml/min/100 g) and are somewhat lower than the f i g u r e o f 16 ml/min/100 g given by White e t _ a l . (88) . However, these l a t t e r s t u d i e s i n v o l v e d longer c l e a r a n c e p e r i o d s , and ac-cumulation of S r 8 ^ i n exchangeable bone p o o l would tend t o reduce the S r 8 ^ c l e a r a n c e . Bone blo o d flow appears h i g h r e l a t i v e t o the me t a b o l i c needs o f bone, which c o n t a i n s so much i n e r t m a t e r i a l . However, t h i s may be r e l a t e d to i t s important homeostatic f u n c t i o n i n m i n e r a l metabolism. Whatever the reason, the data o b t a i n e d i n t h i s study i n d i c a t e q u a n t i t a t i v e l y t h a t the blo o d c i r c u l a t i o n through the s k e l e t o n i s an important p a r t of the p e r i p h e r a l c i r c u l a t i o n . SUMMARY AND CONCLUSIONS The r a t e s of b l o o d flow through v a r i o u s bones i n the r a b b i t and the dog were s t u d i e d . The average r a t e f o r 270 bones from 80 r a b b i t s was 12.48 - 4.26 (S.D.), and f o r 46 bones from 10 dogs was 13.20 * 5.35 (S.D.) ml/min/100 g wet weight. S k e l e t a l b l o o d flow was estimated to be 7.3 ± 3.0 (S.D.) % of the r e s t i n g c a r d i a c output i n the dog and 7.1 - 2.3 (S.D.) % of the r e s t i n g c a r d i a c output i n the r a b b i t . EXPERIMENT V RELATIVE CONTRIBUTION OF THE THREE ARTERIAL SYSTEMS IN LONG BONE A t y p i c a l long bone r e c e i v e s i t s b l o o d supply from t h r e e a r t e r i a l systems w i t h abundant anastomoses: the n u t r i e n t , e p i -metaphyseal and p e r i o s t e a l v e s s e l s . T h i s has been f i r m l y es-t a b l i s h e d by many workers s i n c e the c l a s s i c a l study.made i n 1903 by Lexer (44). The r e l a t i v e p h y s i o l o g i c a l importance o f each of the three a r t e r i a l systems was s t u d i e d by o b s e r v i n g the e f f e c t s of s e l e c -t i v e s u p p r e s s i o n of one or more sources of b l o o d supply. John-son (34) i n 1927 was the f i r s t t o study t h i s problem and h i s f i n d i n g s p r o v i d e a b a s i s o f our understanding of t h i s s u b j e c t . By i n t e r f e r i n g w i t h two of the three sources of b l o o d supply and o b s e r v i n g the e x t e n t of the n e c r o t i c bone produced, he concluded t h a t the n u t r i e n t a r t e r y of the t i b i a i n the dog i s r e s p o n s i b l e f o r the nourishment of the bone marrow and the i n n e r h a l f of the c o r t e x of the s h a f t ; the metaphyseal v e s s e l s supply the meta-physes and are able to supply the area of the n u t r i e n t a r t e r y , w h i l e the p e r i o s t e a l a r t e r i e s supply the o u t e r h a l f of the c o r t e x . Thus, he found t h a t the n u t r i e n t a r t e r y system i s the most im-p o r t a n t , the metaphyseal i s the i n t e r m e d i a t e , and the p e r i o s t e a l i s the l e a s t important. S e v e r a l workers have subsequently con-f i r m e d the f i n d i n g s of Johnson (34), although McNab (50) and McAuley (46) both f e l t t h a t the p e r i o s t e a l system p l a y e d o n l y a minor r o l e . T r u e t a and C a l a d i a s (84) i n 1964 confirmed Johnson's f i n d i n g s i n a s e r i e s of c r i t i c a l s t u d i e s on the r a d i u s of the r a b b i t u s i n g s i m i l a r methods. Cuthbertson ejt _ a l . (21) r e c e n t l y r e p o r t e d t h a t l i g a t i o n of the n u t r i e n t a r t e r y of the t i b i a and humerus i n _ t h e dog caused an immediate and profound f a l l of the i n t r a m e d u l l a r y p ressure i n these bones. However, to date, there has been no q u a n t i t a t i v e study of the r e l a t i v e c o n t r i b u t i o n of each of the three a r t e r i a l systems to the b l o o d supply to long bone. T h i s was i n v e s t i g a t e d i n the f o l l o w i n g experiments. METHOD The study was c a r r i e d out on 13 a d u l t white r a b b i t s weighing 2.4-3.2 kg (average weight 2.8 k g ) . They were a n a e s t h e t i z e d as d e s c r i b e d p r e v i o u s l y and the areas of the t h i g h and g r o i n were shaved. The n u t r i e n t a r t e r y of one femur i n each animal was ex-posed through an a n t e r i o r approach. T h i s v e s s e l o r i g i n a t e s from the deep femoral a r t e r y and passes o b l i q u e l y downward and i n -t e r i o r l y along the i l i o p s o a s muscle to reach the n u t r i e n t foramen at the area a n t e r o - i n f e r i o r to the l e s s e r t r o c h a n t e r . The p r o x i -mal end of the n u t r i e n t a r t e r y was c a r e f u l l y d i s s e c t e d f r e e from the n u t r i e n t v e i n and the other adjacent t i s s u e s . A l i g a t u r e loop was p l a c e d around i t so t h a t i t could be l i g a t e d r e a d i l y . The n u t r i e n t a r t e r y on the o p p o s i t e s i d e ( c o n t r o l ) was merely exposed i n a sham o p e r a t i o n . Then, the c a r o t i d a r t e r y was can-n u l a t e d p r e p a r a t o r y to measurement of S r 8 5 c l e a r a n c e of bone as d e s c r i b e d i n Chapter I I I . The n u t r i e n t a r t e r y was l i g a t e d and o c the s o l u t i o n of Sr was immediately i n j e c t e d i n t o an ear v e i n . At the end of 5 minutes, the animal was s a c r i f i c e d and both femurs were q u i c k l y removed. The bones were cleaned and weighed. They were then d i v i d e d i n t o three segments by t r a n s v e r s e cuts a t the l e v e l of i n f e r i o r margin o f the l e s s e r t r o c h a n t e r and a t the supracondylar l e v e l u s i n g a f i n e e l e c t r i c saw. Each of these segments was weighed and the S r 8 5 c l e a r a n c e of these segments was determined as d e s c r i b e d above. RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table V I I I and F i g u r e 5. L i g a t i o n of the n u t r i e n t a r t e r y reduced the r a t e of b l o o d flow to the s h a f t by 71%, to the upper end by 37%, and to the lower end by 33%. The normal r a t e s o f b l o o d flow through the upper segment, s h a f t and lower segment were 10.8, 8.5 and 10.8 ml/ min/100 g wet weight, r e s p e c t i v e l y . L i g a t i o n of the n u t r i e n t a r t e r y reduced these r a t e s to 6.8, 2.5 and 7.2 ml/min/100 g wet weight, r e s p e c t i v e l y . F i g u r e 5 Effect of Ligation of Nutrient Artery of Femur on S r 8 5 Clearance in 13 Rabbits. ( ml / min./ 100 gm. ) 10.8 ± 0.8 8.5 ± 0.9 10.8 ± 1.0 Ligation 6.8 ±0 .4 (-37%) 2.5 ±0 .4 (-7196) 7.2 ± 0.8 (-3396) Intact Ligated E f f e c t o f l i g a t i o n of n u t r i e n t a r t e r y o f femur on S r 8 5 c l e a r a n c e i n 13 r a b b i t s . (ml/min/100 g) •The observed r e d u c t i o n of b l o o d flow f o l l o w i n g l i g a t i o n of the n u t r i e n t a r t e r y should p r o v i d e a d i r e c t i n d i c a t i o n of the r e l a t i v e c o n t r i b u t i o n of t h i s v e s s e l to the supply of these bone segments, p r o v i d e d t h a t there has been no compensatory flow through other anastomotic channels. Since such anastomotic flow, i f i t o c c u r r e d , would i n c r e a s e the apparent blood flow, the f i g u r e s presented must be c o n s i d e r e d minimal r e d u c t i o n s , and i t i s p o s s i b l e t h a t the c o n t r i b u t i o n of the n u t r i e n t a r t e r y to b l o o d supply to the s h a f t may be c o n s i d e r a b l y h i g h e r than 71%. The remaining b l o o d supply (29% of c o n t r o l flow) to the s h a f t a f t e r l i g a t i o n of the n u t r i e n t a r t e r y must be from p e r i o s t e a l v e s s e l s and the epi-metaphyseal system s u p p l y i n g the bone ends. The same a p p l i e s to the r e s i d u a l b l o o d flow to the upper (63%) and lower (67%) ends. I t i s , u n f o r t u n a t e l y , n o t - p o s s i b l e to d i f -f e r e n t i a t e the c o n t r i b u t i o n of the epi-metaphyseal system from t h a t o f the p e r i o s t e a l v e s s e l s i n these experiments, although from the work of o t h e r s , i t appears probable t h a t p e r i o s t e a l c o n t r i b u t i o n i s not g r e a t . The profound r e d u c t i o n of b l o o d flow through the s h a f t and through each end of the bone f o l l o w i n g the l i g a t i o n of the n u t r i -ent a r t e r y confirms q u a n t i t a t i v e l y the f i n d i n g s of Johnson (34), T r u e t a and C a l a d i a s (84) , Cuthbertson e_t _ a l . (21) and many o t h e r s who s t u d i e d q u a l i t a t i v e l y the r e l a t i v e importance of the F i g u r e 6 Regional Blood Supply of Normal Femur. 63 96 by upper Epi - metaphyseal Artery 37% by Nutrient Artery 71 % by Nutrient Artery 2 9 % by Periosteal Arteries ( + epi-metaphyseal artery) 6 7 % by Lower Epi-metaphyseal Artery 3 3 % by Nutrient Artery R e l a t i v e c o n t r i b u t i o n of the v a r i o u s v a s c u l a r systems to b l o o d supply to femur. r o u t e s of bloo d supply i n long bone. The 71% r e d u c t i o n i n bloo d flow t o the s h a f t a f t e r l i g a t i o n o f the n u t r i e n t a r t e r y agrees with Johnson's f i n d i n g t h a t the n u t r i e n t a r t e r y i s r e s p o n s i b l e f o r the bloo d supply to the marrow space and much of the s h a f t c o r t e x . I t a l s o supports the f i n d i n g of Cuthbertson et a_l. (21) t h a t l i g a t i o n of the n u t r i e n t a r t e r y caused an immediate and profound f a l l i n the i n t r a m e d u l l a r y p ressure o f the dog femur. SUMMARY AND CONCLUSIONS The r e l a t i v e c o n t r i b u t i o n of the three a r t e r i a l systems to bl o o d supply of femur was s t u d i e d i n 13 a d u l t r a b b i t s . The nu-t r i e n t a r t e r y was l i g a t e d and the r e s u l t i n g changes of the r a t e s of b l o o d flow through the upper epi-metaphyseal area, the s h a f t and the lower metaphyseal areas were determined 5 minutes a f t e r l i g a t i o n . The data i n d i c a t e d t h a t the n u t r i e n t a r t e r y was r e -s p o n s i b l e f o r at l e a s t 71% of the bloo d supply to the s h a f t , 37% of the flow to the upper end and 33% of the flow to the lower end. The remaining b l o o d supply to each area c o u l d be a t t r i b u t e d to the r e s p e c t i v e r e g i o n a l a r t e r i a l systems. 58. EXPERIMENT VI BLOOD FLOW TO BONE WITH DISUSE OSTEOPOROSIS Ost e o p o r o s i s i s a common me t a b o l i c bone d i s e a s e c h a r a c t e r -i z e d by decrease i n the mass of bone without s i g n i f i c a n t change i n the r a t i o o f the o r g a n i c (matrix) and i n o r g a n i c (mineral) components. I t can be produced i n l a b o r a t o r y animals by v a r i o u s methods. O s t e o p o r o s i s may be l o c a l i z e d or g e n e r a l i z e d . I t i s o f t e n found i n v a r i o u s c l i n i c a l c o n d i t i o n s such as Cushing's d i s e a s e and syndrome, rheumatoid a r t h r i t i s , scurvy, hyperpara-t h y r o i d i s m , s e n i l i t y and f o l l o w i n g i m m o b i l i z a t i o n , i . e . , t y p i -c a l l y i n a p l a s t e r c a s t . However, the pathogenesis o f o s t e o p o r o s i s i s p o o r l y under-stood. The c l a s s i c a l t h e o r y of A l b r i g h t (1) a t t r i b u t e s the con-d i t i o n t o a lack o f fo r m a t i o n o f o r g a n i c m a t r i x . Many subsequent s t u d i e s d i d not e n t i r e l y support the A l b r i g h t h y p o t h e s i s . I t i s known t h a t the ost e o p o r o s i s , which occurs i n o s t e i t i s f i b r o s a g e n e r a l i s a t a r e s u l t i n g from h y p e r p a r a t h y r o i d i s m i s i n f a c t due to i n c r e a s e d bone r e s o r p t i o n r a t h e r than reduced bone f o r m a t i o n . More r e c e n t l y some workers, p a r t i c u l a r l y Nordin (57) and Jowsey e t a l . (36), have expressed the o p i n i o n t h a t o s t e o p o r o s i s i s due to prolonged negative c a l c i u m balance. Jowsey and Gershon-Cohen (36) r e p o r t e d i n 1964 t h a t o s t e o p o r o s i s c o u l d be produced by a prolonged f e e d i n g of c a l c i u m d e f i c i e n t d i e t t o a d u l t c a t s and t h a t i t c o u l d be cured by a normal or h i g h c a l c i u m d i e t . McLean and U r i s t (48) s t a t e t h a t o s t e o p o r o s i s i s a type o f a t r o -phy o c c u r r i n g i n bone. At present, there are two main t h e o r i e s as to the cause o f o s t e o p o r o s i s . The f i r s t suggests t h a t i t i s due to reduced bone formation, the second proposes t h a t i t i s due to i n c r e a s e d bone r e s o r p t i o n , o f t e n secondary t o c a l c i u m de-f i c i e n c y . I t i s p o s s i b l e t h a t both f a c t o r s c o n t r i b u t e t o s e n i l e o s t e o p o r o s i s . I t has a l s o been suggested t h a t reduced b l o o d supply may be r e s p o n s i b l e f o r the bone atrophy. There i s l i t t l e q u a n t i t a t i v e data on t h i s p o i n t , so i t was decided to use the methods f o r measurement of bone b l o o d flow d e s c r i b e d i n Chapter I I I to determine whether the d i s u s e o s t e o p o r o s i s as a r e s u l t of prolonged i m m o b i l i z a t i o n i n a c a s t , might be a s s o c i a t e d w i t h changes i n blood flow. METHOD The study was made u s i n g 22 a d u l t r a b b i t s weighing 2.0-3.3 kg (average weight 2.5 k g ) . A long l e g c a s t o f p l a s t e r was ap-p l i e d to one h i n d l e g i n each animal with the knee i n p a r t i a l f l e x i o n . Care was taken to av o i d undue pressure and c i r c u l a t o r y embarrassment from the p l a s t e r c a s t . The animals were then d i v i -ded i n t o two groups of equal number. One group of 11 animals was used to study the e f f e c t s o f a s h o r t term (1-2 weeks) immo-b i l i z a t i o n and the second group of 11 animals was used f o r the 60. long term (2 months) experiments. At the end o f the p e r i o d of i m m o b i l i z a t i o n , the b l o o d flow through the calcaneus, t i b i a and femur on both limbs was measured by the method d e s c r i b e d i n Chapter I I I . Roentgenographs were made of the t i b i a from the long term group, and ash/dry weight r a t i o s were measured. RESULTS AND DISCUSSION The r a t e s of b l o o d flow are summarized i n Table IX, and the v a l u e s f o r f r e s h , dry and ash weight of the bones are summarized i n Table X. There were no s i g n i f i c a n t changes i n bone weight a f t e r 1-2 weeks of i m m o b i l i z a t i o n , but the b l o o d flow to the calcaneus and t i b i a was s i g n i f i c a n t l y reduced. In the second group, i n which the limb was immobilized f o r 2 months, there was d e f i n i t e evidence o f l o c a l o s t e o p o r o s i s , p a r t i c u l a r l y i n the c a l -caneus and t a l u s . The b l o o d flow to the e n t i r e bone was not changed a p p r e c i a b l y , but because of the reduced mass of the bones from the immobilized s i d e , b l o o d flow per 100 g f r e s h weight was a c t u a l l y i n c r e a s e d s i g n i f i c a n t l y i n the case of t i b i a and c a l -caneus . Although the i n i t i a l e f f e c t of i m m o b i l i z a t i o n appeared to be a r e d u c t i o n i n b l o o d flow, t h i s was not t r u e a f t e r the pro-longed i m m o b i l i z a t i o n when a c t u a l o s t e o p o r o s i s had developed. For t h i s reason, i t i s d i f f i c u l t to b e l i e v e t h a t the osteoporo-s i s i s due p r i m a r i l y t o reduced b l o o d flow. I t i s i n t e r e s t i n g t h a t the most marked e f f e c t was observed i n the calcaneus, while t h e r e was l i t t l e e f f e c t on femur. As shown i n F i g u r e 7, t h e r e was d e f i n i t e r o e n t g e n o g r a p h ^ evidence of o s t e o p o r o s i s i n the t i b i a , w i t h the most s t r i k i n g e f f e c t on the c a n c e l l o u s bone of the metaphysis. T h i s i s i n agreement w i t h the c l i n i c a l observa-t i o n t h a t c a n c e l l o u s bone i s more a f f e c t e d by t h i s c o n d i t i o n . In the t i b i a , such changes were apparent, even though the aver-age l o s s of bone m i n e r a l was o n l y 16%. SUMMARY AND CONCLUSIONS A p r e l i m i n a r y study was made of q u a n t i t a t i v e changes i n bone b l o o d flow i n d i s u s e o s t e o p o r o s i s i n the r a b b i t . The bones of the limb which had been immobilized i n a long l e g p l a s t e r c a s t f o r 1-2 weeks had a c o n s i d e r a b l e decrease i n the r a t e of bone bloo d flow without s i g n i f i c a n t change i n the bone weight. The bones i n the limbs which were immobilized f o r 2 months became o s t e o p o r o t i c as demonstrated by roentgenographs and ash weight a n a l y s e s . These o s t e o p o r o t i c bones, however, d i d not show any change i n b l o o d flow on the b a s i s of each i n d i v i d u a l bone com-pared to the c o r r e s p o n d i n g bone i n the c o n t r o l limb. However, when the r a t e of b l o o d flow to t i b i a and calcaneus was computed on the b a s i s of bone weight, i t was i n c r e a s e d s i g n i f i c a n t l y be-cause of the marked decrease i n bone weight. Roentgenograph o f t i b i a (E) from limb immobilized i n p l a s t e r c a s t f o r 2 months and c o n t r o l bone (C) from the op p o s i t e s i d e . 6 3 . The r e s u l t s suggest t h a t a decrease i n the bone b l o o d flow occurs i n the e a r l y stages of i m m o b i l i z a t i o n . However, a f t e r o s t e o p o r o s i s has developed, the b l o o d flow c a l c u l a t e d on a weight b a s i s was normal or even i n c r e a s e d , so t h a t i t i s d i f f i -c u l t to a t t r i b u t e t h i s c o n d i t i o n t o reduced b l o o d flow. 64. EXPERIMENT VII EFFECT OF SCIATIC NERVE SECTION . ON BONE BLOOD FLOW There i s c o n s i d e r a b l e evidence f o r the c o n t r o l o f bone bl o o d c i r c u l a t i o n by both sympathetic vasomotor nerve and vaso-p r e s s o r hormones. Anatomical s t u d i e s o f nerve supply t o bone by O t t o l e n g h i (58) and Sherman (69) d i s c l o s e d t h a t the bone i s abundantly s u p p l i e d by both myelinated and unmyelinated nerve f i b e r s . Trotman and K e l l y (80) r e p o r t e d a 27% i n c r e a s e i n bone bl o o d flow f o l l o w i n g lumbar sympathectomy i n the dog. Weiss and Root (87) observed i n the c a t t h a t e l e c t r i c a l s t i m u l a t i o n of the d i s t a l cut ends of c e r t a i n p e r i p h e r a l nerves i n the limb caused a f a l l i n the i n t r a m e d u l l a r y b l o o d p r e s s u r e . Bloomenthal e t a l . (5), S t e i n e t aL. (76), Copp and Shim (12, 70) and Shaw (67, 68) a l l observed f a l l s i n i n t r a m e d u l l a r y b l o o d pressure f o l l o w -i n g a d m i n i s t r a t i o n o f e p i n e p h r i n e , norepinephrine or p i t r e s s i n . In o r d e r to study the e f f e c t of complete s c i a t i c nerve i n -j u r y ( s e c t i o n ) on bone blo o d flow i n the limb, the f o l l o w i n g experiments were c a r r i e d out i n the r a b b i t . METHOD The s t u d i e s were made i n 10 a d u l t r a b b i t s weighing 1.9-2.7 kg (average weight 2.4 k g ) . The animals were l i g h t l y anaesthe-t i z e d w i t h e t h e r . The g l u t e a l r e g i o n was shaved. The s c i a t i c 65. nerve on one s i d e was exposed behind the h i p j o i n t through a p o s t e r i o r approach by s p l i t t i n g the gluteus maximus. The nerve was then i d e n t i f i e d a t the i n f e r i o r border of the p y r i f o r m i s muscle and was c u t completely w i t h a k n i f e or s c i s s o r s under d i r e c t v i s i o n . The wound was c l o s e d w i t h b l a c k s i l k . Post-o p e r a t i v e l y , the animals r e c e i v e d 30,000 u n i t s of p r o c a i n e p e n i c i l l i n . The wound he a l e d by the 7th day. The r a t e s of b l o o d flow through the l e g bones ( t i b i a - f i b u l a ) and the r e p r e s e n t a t i v e f o o t bones ( t a l u s - c a l c a n e u s ) i n both normal and experimental s i d e s i n a l l animals were s t u d i e d by the method d e s c r i b e d i n Chapter I I I between 8th and 14th day a f t e r the o p e r a t i o n . RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table XI. There was no s i g -n i f i c a n t change i n the weights o f the bones i n the p a r a l y z e d limb. The average r a t e of b l o o d flow i n the normal t i b i a - f i b u l a was 9.89 - 1.70 ml/min/100 g wet weight w h i l e , on the p a r a l y z e d s i d e , i t was 12.87 - 1.81 ml/min/100 g wet weight. The i n c r e a s e i n the p a r a l y z e d s i d e was 2.98 - 2.42 ml which was not s t a t i s t i -c a l l y s i g n i f i c a n t . The r a t e o f blood flow to the f o o t bones was 7.57 - 1.52 ml/min/100 wet weight i n the c o n t r o l limb as com-pared to 13.52 ± 1.86 ml/min/100 g wet weight on the s i d e of s c i a t i c nerve s e c t i o n . The r a t e of i n c r e a s e i n t h i s case was 5.95 - 2.48 ml/min/100 g which was s t a t i s t i c a l l y s i g n i f i c a n t (0.05> P > 0.025) . 6 6 . The r e s u l t s i n d i c a t e t h a t s c i a t i c nerve s e c t i o n may cause a c o n s i d e r a b l e i n c r e a s e i n bone bloo d flow i n the l e g and f o o t . The e f f e c t i s more marked i n the f o o t bones than i n the l e g bones. The s c i a t i c nerve, the l o n g e s t somatic nerve i n the body, c o n t a i n s three elements: the somatic motor, sensory and sympa-t h e t i c vasomotor. The i n c r e a s e i n the bone b l o o d flow c o u l d be e x p l a i n e d by the f a c t t h a t the s e c t i o n of the s c i a t i c nerve i n -t e r r u p t s the sympathetic o u t f l o w to the b l o o d v e s s e l s i n the l e g and f o o t i n c l u d i n g the v e s s e l s i n bones. The sympathetic vaso-motor nerve supply below the knee i s almost e n t i r e l y conveyed by the s c i a t i c nerve (40, 92). A knowledge of the b a s i c p a t t e r n of the sympathetic nerve supply to the b l o o d v e s s e l s i n the limbs i s e s s e n t i a l to under-stand the r e s u l t s of t h i s study. T h i s s u b j e c t was s t u d i e d by Kramer and Todd (40) and Woolard (92) . I t was a l s o i l l u s t r a t e d w e l l by Lockhart eit _ a l . (45). There are two d i s t i n c t p a t t e r n s : the p r o x i m a l and the d i s t a l . The p a t t e r n of the p r o x i m a l v a s c u l a r i n n e r v a t i o n does not extend beyond the l a r g e r v e s s e l s o f the limbs and i s l i m i t e d to the p r o x i m a l p o r t i o n s of the femoral and b r a c h i a l a r t e r i e s . In the case o f the upper limb, the v a s c u l a r nerve supply o r i g i n a t e s from the middle and lower c e r v i c a l g a n g l i a of the c e r v i c a l sympa-t h e t i c c h ain, and i s then conveyed to the s u b c l a v i a n and a x i l l a r y 67. a r t e r i e s . The p r o x i m a l v a s c u l a r nerve supply to the lower limb i s d e r i v e d from the a o r t i c p l e x e s i n the abdomen and conveyed i n a p l e x i f o r m manner along the common and e x t e r n a l i l i a c a r -t e r i e s . The d i s t a l p a t t e r n o f v a s c u l a r nerve supply o r i g i n a t e s from the s p i n a l nerve trunks and i s c a r r i e d to the p e r i p h e r a l v e s s e l s by the somatic p e r i p h e r a l nerves. The upper l i m i t s are the p r o x i m a l p o r t i o n s of the b r a c h i a l and femoral a r t e r i e s , and the p e r i p h e r a l l i m i t s are the l e v e l s of the a r t e r i o l e s and c a p i l -l a r i e s i n the hand and f o o t . The p e r i p h e r a l sympathetic f i b e r s i n the limbs, t h e r e f o r e , t r a v e l e x c l u s i v e l y w i t h the somatic nerve trunks and thus the v a s o c o n s t r i c t o r impulses are conveyed to the minute v e s s e l s of the limbs. Hence the s e c t i o n of a  p e r i p h e r a l nerve, such as the s c i a t i c nerve, causes complete  d e n e r v a t i o n o f the sympathetic v a s o c o n s t r i c t o r s i n the area of  i t s d i s t r i b u t i o n . The femoral a r t e r y i s i n n e r v a t e d by the sympathetic vaso-c o n s t r i c t o r f i b e r s conveyed w i t h i n the femoral and o b t u r a t o r nerves and the p o p l i t e a l a r t e r y by the vasomotor f i b e r s c a r r i e d by the femoral and saphenous nerves. However, the sympathetic  supply below the knee i s conveyed e n t i r e l y by the s c i a t i c nerve. I t then d i v i d e s along the medial and l a t e r a l p o p l i t e a l nerves, the a n t e r i o r and p o s t e r i o r t i b i a l nerves, and the p l a n t a r nerves 68. to supply t h e i r c o r r e s p o n d i n g a r t e r i e s a t v a r i o u s l e v e l s i n -c l u d i n g the t e r m i n a l a r t e r i o l e s and p r e c a p i l l a r y s p h i n c t e r s . The tendency f o r an i n c r e a s e i n bone b l o o d flow i n the limb f o l l o w i n g s e c t i o n of the s c i a t i c nerve noted i n t h i s study sup-p o r t s the f i n d i n g of Trotman and K e l l y (80), who observed t h a t lumbar sympathectomy i n dog caused a 27% i n c r e a s e i n bone b l o o d flow i n the l e g . The f a c t t h a t e l e c t r i c a l s t i m u l a t i o n of the p e r i p h e r a l cut ends of c e r t a i n nerves i n the limb caused a f a l l i n the i n t r a m e d u l l a r y pressure of bone, as observed by Weiss and Root (87), c o u l d w e l l be due to the v a s o c o n s t r i c t i o n and decrease i n bone b l o o d flow due to i n c r e a s e d sympathetic tone l o c a l l y . T h i s knowledge c o u l d be a p p l i e d to c l i n i c a l c o n d i t i o n s i n which i n c r e a s e d bone blo o d flow i s d e s i r a b l e . Such i n c r e a s e d flow might promote f r a c t u r e h e a l i n g and prevent non-union i n r e g i o n s of low v a s c u l a r i t y , such as the j u n c t i o n of the middle and lower t h i r d of t i b i a . B l o c k i n g the p e r o n e a l or s c i a t i c nerve should i n c r e a s e bone b l o o d flow i n t h i s area and i n the f o o t . SUMMARY AND CONCLUSION The e f f e c t of the s e c t i o n of the s c i a t i c nerve on the b l o o d flow of bones i n the limb was s t u d i e d i n the r a b b i t . The e f f e c t was e v a l u a t e d between 1-2 weeks a f t e r complete s u r g i c a l s e c t i o n of the s c i a t i c nerve. T h i s procedure r e s u l t e d i n a tendency 6 9 . towards i n c r e a s e d b l o o d flow to the l e g bones and a s i g n i f i c a n t i n c r e a s e i n flow to the f o o t bones. I t i s suggested t h a t t h i s i n c r e a s e i n bone blo o d flow i s due to i n t e r r u p t i o n of the sym-p a t h e t i c vasomotor o u t f l o w to the l e g and f o o t as a r e s u l t of c u t t i n g the s c i a t i c nerve. The r e s u l t s of the study support the view t h a t sympathectomy should i n c r e a s e bone b l o o d flow i n the limb, and agrees w i t h the f i n d i n g s of Trotman and K e l l y (80). 70. EXPERIMENT V I I I EFFECT OF FRACTURES OF FEMORAL NECK ON BLOOD SUPPLY TO FEMORAL HEAD No matter how t r e a t e d , the f r a c t u r e s o f the neck of the femur are f r e q u e n t l y complicated by nonunion, degenerative a r -t h r i t i s or a s e p t i c n e c r o s i s o f the femoral head ( 2 ) . The im-pairment of the b l o o d supply to the femoral head i s g e n e r a l l y known to be an important cause of these c o m p l i c a t i o n s . Because of the unique anatomy of the v a s c u l a r supply to the neck and head o f the femur, the f r a c t u r e of the neck alone may w e l l be s e r i o u s enough a t the time the f r a c t u r e occurs t o i n t e r r u p t b l o o d supply to the head. In a d d i t i o n , however, there are many othe r f a c t o r s which may aggravate the degree of impairment of b l o o d supply to the head. F r a c t u r e h i g h i n the neck, d i s p l a c e -ment of the fragments, r o t a t i o n o f the p r o x i m a l fragment (71) and i n c r e a s e d i n t r a a r t i c u l a r p r e s s u r e (74) are a few examples. The normal anatomy o f the v a s c u l a r supply to the femoral head i n man was s t u d i e d so we 11 by T r u e t a and H a r r i s o n (82) t h a t t h e i r d e s c r i p t i o n has been a standard r e f e r e n c e on t h i s s u b j e c t . There are a t l e a s t f o u r sources of b l o o d supply: the l a t e r a l and medial ( f o v e a l ) e p i p h y s e a l a r t e r i e s , and the s u p e r i o r and i n f e r i o r metaphyseal a r t e r i e s . However, the r e l a t i v e importance of these v e s s e l s has not been c r i t i c a l l y s t u d i e d to date. The i n j u r i e s o f these v e s s e l s i n femoral neck f r a c t u r e s and t h e i r s i g n i f i c a n c e i n b l o o d supply to the femoral head i s a matter of s e r i o u s concern to the o r t h o p e d i c surgeon i n connec t i o n w i t h p r e v e n t i o n and p r e d i c t i o n of a v a s c u l a r n e c r o s i s and v i a b i -l i t y o f the femoral head. Boyd e t al. (6, 7) measured P 3 2 up-take by the femoral head f o l l o w i n g f r a c t u r e of the femoral neck. M i l e s (51) observed the i n t r a m e d u l l a r y p r e s s u r e of the femoral head; Woodhouse (89) s t u d i e d the presence or absence of d i s s o l v e d oxygen i n the medullary space i n the femoral head u s i n g an oxy-meter, and M i l c h (52) determined the d i s t r i b u t i o n o f t e t r a c y c l i n e i n the femoral head f o l l o w i n g i t s a d m i n i s t r a t i o n . Smith (71) i n v e s t i g a t e d the e f f e c t o f r o t a t i o n and valgus m a l p o s i t i o n of the pro x i m a l fragment on the b l o o d supply to the femoral head. He a l s o made important o b s e r v a t i o n s on the r o l e of the medial ( f o v e a l ) e p i p h y s e a l a r t e r y f o l l o w i n g the t r a n s c e r v i c a l f r a c t u r e i n the bloo d supply to the femoral head. He observed decrease or a r r e s t of b l e e d i n g from the p r o x i m a l s u r f a c e of the f r a c t u r e when the pr o x i m a l fragment was r o t a t e d . To date, however, the normal volume or the normal r a t e of blood flow to the femoral head and i t s q u a n t i t a t i v e changes f o l l o w i n g t r a n s c e r v i c a l f r a c t u r e s have not been, s t u d i e d . T h i s was i n v e s t i g a t e d i n the f o l l o w i n g experiments. In a number of d i s s e c t i o n s i n both l i v i n g and dead r a b b i t s , w i t h the a i d of a d i s s e c t i n g microscope, i t was found t h a t the v a s c u l a r anatomy of the femoral head and neck i n the r a b b i t i s v e r y s i m i l a r to t h a t i n man. T h e r e f o r e , i t was f e l t t h a t measurement of normal b l o o d supply to the femoral head and a study of the changes pro-duced i n the experimental t r a n s c e r v i c a l f r a c t u r e s of the femoral neck i n t h i s animal might p r o v i d e v a l u a b l e i n f o r m a t i o n on t h i s s e r i o u s c o n d i t i o n i n man. METHOD The study was made i n 80 a d u l t r a b b i t s weighing 2.2-3.5 kg (average weight 2.7 k g ) . They were d i v i d e d i n t o 4 groups. The f i r s t and second groups (10 r a b b i t s i n each) served as c o n t r o l s . In each animal, the femur on the o p p o s i t e (unoperated) s i d e served as a c o n t r o l . The animals were a n a e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n o f urethane (1 g/kg) and b a r b i t o n e (250 mg/kg). The g l u t e a l r e g i o n s were shaved. The h i p j o i n t was exposed through a poste-r i o r approach by s p l i t t i n g the gluteus maximus. Then, the j o i n t c apsule was opened a t the p o s t e r i o r a s p e c t . During the opera-t i o n , the b l e e d i n g p o i n t s were c a u t e r i z e d . In the f i r s t group of 10 animals, the e f f e c t of the p o s t e r i o r capsulotomy on the b l o o d supply to the femoral head was s t u d i e d 1-2 hours a f t e r the o p e r a t i o n . In the second group of 10 animals, the e f f e c t of a combination of s u b c a p i t a l f r a c t u r e and s e c t i o n of the ligamentum t e r e s was s t u d i e d . In the t h i r d group of 20 animals, the e f f e c t o f s u b c a p i t a l f r a c t u r e on the b l o o d supply to the femoral head was determined. In the f o u r t h group of 40 r a b b i t s , the e f f e c t o f n o n - s u b c a p i t a l f r a c t u r e s was i n v e s t i g a t e d . F r a c t u r e s were induced under d i r e c t v i s i o n u s i n g a bone c u t t i n g f o r c e p s or sharp p o i n t e d s c i s s o r s . The p r o x i m a l fragment was not r o t a t e d . The wound was c l o s e d without c l o s i n g the j o i n t c a p s u l e . One or two hours a f t e r the o p e r a t i o n , the r a t e s of b l o o d flow through the femoral head were measured by the method d e s c r i b e d i n Chap-t e r I I I . RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table X I I and F i g u r e s 8 and 9 Simple p o s t e r i o r capsulotomy of the h i p j o i n t d i d not a f f e c t the b l o o d supply to the femoral head. On the other hand, the b l o o d supply to the head was completely cut o f f when two procedures of s u b c a p i t a l f r a c t u r e and s e c t i o n of the t e r e s ligament were combined. S u b c a p i t a l f r a c t u r e alone reduced the b l o o d supply to the femoral head by an average of 83% (range 72-90%) , l e a v i n g o n l y 17% of the normal b l o o d supply. T h i s presumably came through the ligamentum t e r e s . On the other hand, n o n - s u b c a p i t a l f r a c t u r e s ( o b l i q u e f r a c t u r e w i t h a c a l c a r beak, and f r a c t u r e s through the base of the neck) reduced the blood supply to the femoral head by o n l y 52% (range 23-70%) . F i g u r e 8 Effect of Subcapital Fracture on Blood Supply to Femoral Head. ( 2 0 Rabbits) Control Fractured i 15.6 ± 0 . 9 2.9 ± 0.8 I-i 1 (ml Blood / Min. / l O O g m . fresh Bone) E f f e c t of s u b c a p i t a l f r a c t u r e on bl o o d supply-to femoral head. (20 r a b b i t s ) 75. F i g u r e 9 Effect of Non - Subcapital Fractures of Femoral Neck on Blood Supply to Head. (40Rabb i fs ) Control Fractured 20.6 ± 0.7 9.7 ± 0.5 ( ml Blood / Min. / lOO gm. fresh Bone ) r E f f e c t of n o n - s u b c a p i t a l f r a c t u r e s of femoral neck on b l o o d supply t o head. (40 r a b b i t s ) 76. The r e s u l t s i n d i c a t e t h a t any type o f complete t r a n s c e r -v i c a l f r a c t u r e s i n the h i p j o i n t causes a marked impairment of b l o o d supply to the femoral head. S u b c a p i t a l f r a c t u r e reduces the b l o o d supply to the femoral head f a r more than those a t other s i t e s . The data i n d i c a t e t h a t the femoral head w i t h the s u b c a p i t a l f r a c t u r e r e c e i v e s o n l y 17% of the normal b l o o d supply presumably v i a the ligamentum t e r e s , which i n d i c a t e s a c r i t i c a l r e d u c t i o n of the b l o o d supply to the head i n t h i s case. The r a t e of r e d u c t i o n ranged from 72 to 90% which i n d i c a t e s a f a i r l y con-s i s t e n t p a t t e r n . On the other hand, an o b l i q u e f r a c t u r e w i t h a c a l c a r beak and b a s a l neck f r a c t u r e impaired the b l o o d supply to the femoral head to an e x t e n t which v a r i e d from 23-70% w i t h the average r e d u c t i o n of 52%. I t suggests t h a t these types of f r a c t u r e cause v a s c u l a r i n j u r y to a v a r y i n g e x t e n t . I t must be noted t h a t the neck of the femur i n the r a b b i t i s r e l a t i v e l y s h o r t and i t was d i f f i c u l t t o produce s i m i l a r type of f r a c t u r e even when made under d i r e c t v i s i o n . I t was o n l y p o s s i b l e to make two d i s t i n c t types of f r a c t u r e s , s u b c a p i t a l and non-s u b c a p i t a l . The c o n s i s t e n t and g r e a t r e d u c t i o n of b l o o d supply to the femoral head i n the case of s u b c a p i t a l f r a c t u r e c o u l d be due to i n j u r i e s of a l l b l o o d v e s s e l s except the f o v e a l or medial e p i -p h y s e a l a r t e r y which i s a s s o c i a t e d w i t h the ligamentum t e r e s . T h i s i s s u b s t a n t i a t e d by the f a c t t h a t there was no b l o o d supply to the femoral head a t a l l when the s u b c a p i t a l f r a c t u r e and s e c t i o n of the ligamentum t e r e s were combined, as shown i n Table X I I . I t should be noted, however, t h a t b l e e d i n g from the l i g a -mentum t e r e s was not observed when i t was c u t . T h i s was probably due to v a s c u l a r spasm caused by c u t t i n g ligament. The r e s u l t s i n t h i s study support the c l i n i c a l o b s e r v a t i o n t h a t s u b c a p i t a l type of femoral neck f r a c t u r e causes nonunion of the f r a c t u r e and a s e p t i c n e c r o s i s o f the femoral head more o f t e n than n o n - s u b c a p i t a l types do. The f a c t t h a t the n o n - s u b c a p i t a l types reduce bl o o d supply to the femoral head l e s s s e v e r e l y than the s u b c a p i t a l type, c o u l d w e l l be due to l e s s e r i n j u r y to the b l o o d v e s s e l s i n the neck and a t the base of the femoral head which were contained i n the e l a s t i c r e t i n a c u l u m (or i n t e r n a l f o l d i n g of the capsule) on the s u r f a c e of the femoral neck. Although many b l o o d v e s s e l s were observed i n the a n t e r o - s u p e r i o r p a r t of the r e t i n a c u l u m which was s t i l l connected w i t h the upper fragment, i t was not p o s s i b l e to determine whether or not they were f u n c t i o n i n g . A c t i v e b l e e d i n g from the d i s t a l s u r f a c e o f the f r a c t u r e was e a s i l y observed. But i t was the r u l e , r a t h e r than the e x c e p t i o n , t h a t there was no a c t i v e b l e e d i n g from the p r o x i m a l s u r f a c e of the f r a c t u r e . I t was a l s o d i f f i c u l t to observe b l e e d i n g from the p r o x i m a l s u r f a c e under the microscope. 78. These r e s u l t s have important i m p l i c a t i o n s w i t h r e g a r d t o the s e r i o u s o r t h o p e d i c problem of f r a c t u r e s i n t h i s r e g i o n . SUMMARY AND CONCLUSION 1. The e f f e c t s of the experimental f r a c t u r e s of the femoral neck on the b l o o d supply to the femoral head were s t u d i e d i n a d u l t r a b b i t s . 2. The average r a t e of normal b l o o d supply to the femoral head i n 80 r a b b i t s was about 18 ml/min/100 g wet weight. 3. S u b c a p i t a l f r a c t u r e i n 20 r a b b i t s reduced b l o o d supply t o the femoral head by 83% (range 72-90%) . 4. The n o n - s u b c a p i t a l types o f f r a c t u r e i n 40 r a b b i t s reduced the b l o o d supply to the femoral head by an average of 52% (range 2 3-70%). 5. There was e s s e n t i a l l y no bloo d supply t o the femoral head when s u b c a p i t a l f r a c t u r e and s e c t i o n of the ligamentum t e r e s were combined. 6. P o s t e r i o r capsulotomy of the h i p j o i n t had no e f f e c t on the blood supply to the femoral head. 7. The medial or f o v e a l e p i p h y s e a l a r t e r y i n the ligamentum t e r e s appears to be r e s p o n s i b l e f o r about 17% of the b l o o d supply of the femoral head i n the a d u l t r a b b i t . 79. EXPERIMENT IX EFFECT OF EPINEPHRINE ON BONE BLOOD FLOW Evidence t h a t e p i n e p h r i n e reduces bone b l o o d flow has been accumulated through v a r i o u s s t u d i e s . D r i n k e r et al. (23, 24) i n t h e i r experiments on p e r f u s i o n of i s o l a t e d t i b i a through the n u t r i e n t a r t e r y i n the dog found t h a t o u t f l o w of the b l o o d from the bone was reduced when epi n e p h r i n e was added to the i n f l o w -i n g b l o o d . Bloomenthal et a l . (5) observed a f a l l i n the i n t r a -medullary b l o o d p r e s s u r e of long bone when ep i n e p h r i n e was given to the dog. S i m i l a r o b s e r v a t i o n s were made by S t e i n e t _ a l . (76), H e r z i g and Root (32), Copp and Shim (12, 70) and Shaw (67, 68). In a d d i t i o n , S t e i n jet _ a l . and Copp and Shim a l s o observed t h a t e p i n e p h r i n e reduced and/or stopped bone b l e e d i n g through d r i l l h o l e s made i n the c o r t e x of bone. Thus v a r i o u s q u a l i t a t i v e s t u d i e s p r o v i d e evidence t h a t e p i -nephrine reduces bone bloo d c i r c u l a t i o n presumably through i t s v a s o c o n s t r i c t o r a c t i o n on bone b l o o d v e s s e l s . However, to date, t h i s e f f e c t has not been demonstrated by q u a n t i t a t i v e measure-ment of changes i n bone b l o o d flow. T h i s was i n v e s t i g a t e d i n the f o l l o w i n g experiment. METHOD The animals used were 20 white r a b b i t s weighing 1.4-2.6 kg (average weight 1.90 k g ) . They were d i v i d e d i n t o two groups. 80. The animals were f a s t e d o v e r n i g h t and a n a e s t h e t i z e d by i n t r a -p e r i t o n e a l i n j e c t i o n of 1 g of urethane and 250 mg b a r b i t o n e per kg body weight. The f i r s t group of 10 animals served as c o n t r o l and the second 10 r a b b i t s f o r study of the e f f e c t s of epineph-r i n e . The b l o o d flow was measured i n two r e p r e s e n t a t i v e bones (the humerus and t i b i a - f i b u l a ) u s i n g the method d e s c r i b e d i n Chapter I I I . E p i n e p h r i n e , about 2-4 micro-gram/kg/min i n 1:100,000 or 1:150,000 d i l u t i o n of e p i n e p h r i n e h y d r o c h l o r i d e , was s l o w l y i n f u s e d i n t o an ear v e i n w i t h a constant r a t e s y r i n g e pump. I t was given f o r the 5 minutes of the S r 8 5 c l e a r a n c e p e r i o d . RESULTS AND DISCUSSION The r e s u l t s are summarized i n Table X I I I and F i g u r e 10. E p i n e p h r i n e reduced the r a t e of b l o o d flow to the humerus and t i b i a - f i b u l a by 74 and 81%, r e s p e c t i v e l y . The average e f f e c t i v e bone b l o o d flow w i t h standard e r r o r i n the normal ( c o n t r o l ) group was 16.43 - 1.02 ml/min/100 g f r e s h weight and was 3.69 -0.35 ml/min/100 g f r e s h weight i n the group which r e c e i v e d e p i -nephrine. The r e d u c t i o n i n b l o o d flow (12.74 - 1.04) was h i g h l y s i g n i f i c a n t (p , 0.0001). The data demonstrate t h a t e p i n e p h r i n e g r e a t l y reduces bone b l o o d flow. They demonstrate the e f f e c t of e p i n e p h r i n e on bone b l o o d c i r c u l a t i o n q u a n t i t a t i v e l y and c o n f i r m the evidence o b t a i n e d by v a r i o u s q u a l i t a t i v e methods of study. The r e s u l t s support the o b s e r v a t i o n s of D r i n k e r e t oi l . (24) who 81. F i g u r e 10 85 Initial 5 Minutes Bone Clearance of Sr in Rabbits ( ml. blood /min./ IOOgm. bone ) ml./min./lOO gm. 30 i Control 16.43 ± 1.02 (S.E.) 4.59 (S.D.) Epinephrine 3.69 ± 0.35 (S.E.) 1.57 (S.D.) 20 • 10 • 0 J 1 1 2 3 4 5 6 7 8 9 10 I 2 3 4 5 6 7 8 9 10 WW 10 Rabbits I n i t i a l 5 minutes bone c l e a r a n c e of Sr 0-* i n r a b b i t s (ml blood/min/100 g bone) 82. found t h a t outflow o f bloo d from p e r f u s e d bone i n the dog was decreased when epinephrine was added to the i n f l o w i n g b l o o d . I t a l s o e x p l a i n s the reasons f o r the f a l l of i n t r a m e d u l l a r y p r e s -sure o f bone f o l l o w i n g a d m i n i s t r a t i o n of ep i n e p h r i n e i n the dog observed by Bloomenthal e t a l . (5) and many o t h e r s . In a sense, the medullary c a v i t y o f bone i s a space i n s i d e a r i g i d c l o s e d box. The bloo d p r e s s u r e i n the medullary space should f a l l i f bl o o d flow i n t o the space i s decreased, and v i c e v e r s a . The r e -s u l t s a l s o e x p l a i n the f a c t t h a t b l e e d i n g from bone was reduced or even stopped when epi n e p h r i n e was g i v e n to the experimental animals. T h i s was observed by S t e i n e t _ a l . (76) and Copp and Shim (12, 70). The reduced bone b l o o d flow c o u l d w e l l be due to c o n s t r i c t i o n of i n t r a o s s e o u s v e s s e l s . SUMMARY AND CONCLUSION The e f f e c t o f ep i n e p h r i n e on the r a t e of bone bloo d flow i n the r a b b i t was s t u d i e d . The b l o o d flow through r e p r e s e n t a t i v e bones i n the f r o n t and h i n d limbs (humerus and the t i b i a - f i b u l a ) , was reduced to 1/4 of the r a t e o f flow i n the normal animals when 2-4 micro-gram/kg/min of epi n e p h r i n e h y d r o c h l o r i d e was given i n t r a v e n o u s l y . The average r a t e o f bone blood' flow w i t h the standard e r r o r i n the c o n t r o l group was 16.43 - 1.02 ml/min/100 g wet weight as compared t o 3.69 - 0.35 ml/min/100 g wet weight i n the group which r e c e i v e d e p i n e p h r i n e . The r e s u l t s i n d i c a t e t h a t v e s s e l s of the s k e l e t o n , l i k e those of the s p l a n c h n i c area and s k i n , are c o n s t r i c t e d by e p i -nephrine, and may be c o n s i d e r e d expendable i n emergency s i t u a -t i o n s . 84. EXPERIMENT X RELATIONSHIP OF BONE BLOOD FLOW TO BLOOD-BONE CALCIUM TRANSFER I t i s w e l l known t h a t bone f u n c t i o n s as an important m i n e r a l r e s e r v o i r (11, 16, 48, 56). The movement or t r a n s f e r o f m i n e r a l between the two compartments, b l o o d and bone, must depend on the c i r c u l a t i o n through bone. I t i s reasonable to assume t h a t there i s a q u a n t i t a t i v e c o r r e l a t i o n between bone b l o o d flow and b l o o d -bone m i n e r a l t r a n s f e r . In the immediately p r e c e d i n g chapter, i t was shown t h a t e p i n e p h r i n e g r e a t l y reduced bone b l o o d flow. I t was f e l t t h a t i t would be i n t e r e s t i n g t o determine whether ep i n e p h r i n e a l s o reduced c a l c i u m movement between the bl o o d and bone compartments. T h i s was s t u d i e d i n the f o l l o w i n g experiments. METHOD The experiments were c a r r i e d out on 20 a d u l t mongrel dogs weighing 12-29 kg (average weight 19 kg) which had been f a s t e d o v e r n i g h t . In 10 dogs, e p i n e p h r i n e was i n f u s e d f o r one hour at the r a t e of 2-4 micro-gram/kg/min and the e f f e c t on plasma c a l c i u m and i n o r g a n i c phosphate was determined. In two o f these animals, b l o o d sugar was a l s o determined by the method of F o l i n and Wu, through the c o u r t e s y of the C l i n i c a l I n v e s t i g a t i o n U n i t a t Shaughnessy H o s p i t a l . In a second group of 5 dogs, c a l c i u m 85. (as C a C l 2 i n i s o t o n i c s o l u t i o n ) was i n f u s e d i n t r a v e n o u s l y f o r 1 hour, u s i n g a constant r a t e s y r i n g e pump. The dose adminis-t e r e d was e q u i v a l e n t to 9.0 mg Ca/kg per one hour p e r i o d . The plasma c a l c i u m r e t u r n e d to the c o n t r o l l e v e l w i t h i n a few hours, and the c a l c i u m i n f u s i o n was then repeated, but with the addi-. t i o n a l i n f u s i o n o f 2-4 micro-gram epinephrine/kg/min. Plasma c a l c i u m and i n o r g a n i c phosphate was determined on samples drawn from the j u g u l a r v e i n a t 15-30 minute i n t e r v a l s . In the t h i r d group o f 5 dogs, plasma c a l c i u m was lowered by i . v . i n f u s i o n of a c h e l a t i n g agent, e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA), f o r one hour i n a dose s u f f i c i e n t to c h e l a t e 8.5 mg Ca/kg. Plasma c a l -cium r e t u r n e d to the c o n t r o l l e v e l w i t h i n a few hours, and the i n f u s i o n was then repeated with simultaneous i n f u s i o n of 2-4 micro-gram epinephrine/kg/min f o r the one hour p e r i o d . Plasma c a l c i u m was determined by a semiautomatic micro-t i t r a t i o n w i t h EDTA, u s i n g the method of Copp (17). I n o r g a n i c phosphate was determined by the Taussky and Shorr (79) m o d i f i -c a t i o n o f the method of F i s k e and Subbarow. RESULTS AND DISCUSSION The r e s u l t s are summarized i n Tables XIV-XVT, and are i l -l u s t r a t e d i n F i g u r e s 11-14. There was no s i g n i f i c a n t e f f e c t of e p i n e p h r i n e on plasma calcium, but the i n o r g a n i c phosphate l e v e l i n plasma f e l l from 5.72 to 3.10 mg-%. In the two dogs F i g u r e 11 Effects of Epinephrine on Co and P in Plasma. Ca 10 • E a O I—*—H-i—* I I Averages in 10 Dogs - 5 O ) E L 3 - I Averages in 6 Dogs Epinephrine 4/ugm/kg/min. IV. Infusion + I Time in Hours. E f f e c t o f ep i n e p h r i n e i n f u s i o n on c a l c i u m and i n o r g a n i c phosphate l e v e l s i n plasma. 87. F i g u r e 12 Ef fec t of Epinephrine on Blood C a , P, and Sugar. Averages in 2 Dogs. Epi -Infusion 4ugm./kgVmi. ' i i i i 0 1 2 3 4 Time in Hours. E f f e c t of epi n e p h r i n e i n f u s i o n on l e v e l s of plasma calcium, i n o r g a n i c phosphate and glu c o s e . 88. i n which blo o d glucose was determined, the i n f u s i o n r e s u l t e d i n a 2-3 f o l d i n c r e a s e from the f a s t i n g l e v e l . I t seems prob-able t h a t the f a l l i n plasma i n o r g a n i c phosphate i s the r e s u l t of i n c r e a s e d u t i l i z a t i o n i n the p h o s p h o r y l a t i o n processes i n -vo l v e d i n g l y c o g e n o l y s i s and enhanced m e t a b o l i c a c t i v i t y due to e p i n e p h r i n e . I t i s s i g n i f i c a n t t h a t the f a l l i n i n o r g a n i c phosphate i s not a s s o c i a t e d w i t h any change i n plasma calcium. A l b r i g h t and R e i f e n s t e i n (1) have suggested t h a t there i s a r e c i p r o c a l r e l a t i o n s h i p between the c o n c e n t r a t i o n s of these two ions i n blood, and t h a t the r i s e i n plasma c a l c i u m f o l l o w i n g parathormone a d m i n i s t r a t i o n i s secondary to the f a l l i n i n o r -ganic phosphate. The above r e s u l t s r e f u t e t h i s c l a i m , f o r the f a l l i n i n o r g a n i c phosphate induced by ep i n e p h r i n e had no e f f e c t on the plasma c a l c i u m l e v e l . Calcium I n f u s i o n s As shown i n Table XV and F i g u r e 13, i n f u s i o n o f c a l c i u m f o r one hour r a i s e d the l e v e l o f plasma c a l c i u m 2.12 mg-%. The l a t t e r r e t u r n e d to the o r i g i n a l c o n t r o l l e v e l 2 1/2 hours l a t e r . When the c a l c i u m i n f u s i o n was repeated w i t h simultaneous a d m i n i s t r a t i o n of e p i n e p h r i n e , the r i s e i n plasma c a l c i u m a t the end of the hour was almost twice as g r e a t (+3.94 mg-%). The average t o t a l u r i n a r y e x c r e t i o n d u r i n g the f i r s t i n f u s i o n was 1.85 mg, and 8 9 . F i g u r e 13 Effects of Epinephrine on Hypercalcemia by Ca Infusion. T - 1 1 1 1 1 I 1 l 0 + 1 2 3 4 5 6 7 8 Time in Hours. E f f e c t of e p i n e p h r i n e on the hypercalcemia produced by c a l c i u m i n f u s i o n . 90. d u r i n g the second one-hour i n f u s i o n w i t h e p i n e phrine, i t was 1.07 mg. The reduced e x c r e t i o n i n the l a t t e r , d e s p i t e h i g h e r plasma c a l c i u m l e v e l s , may be due to v a s o c o n s t r i c t i o n and r e -duced b l o o d flow to k i d n e y . However, the t o t a l e x c r e t i o n was i n both cases l e s s than 1% of the i n f u s e d calcium, so t h a t t h i s cannot account f o r the d i f f e r e n c e observed. EDTA I n f u s i o n As shown i n Table XVI and F i g u r e 14, i n f u s i o n o f EDTA f o r an hour r e s u l t e d i n a f a l l o f 1.76 mg-% i n the plasma c a l c i u m l e v e l . W i t h i n a few hours, plasma c a l c i u m had r e t u r n e d t o the o r i g i n a l l e v e l , presumably as a r e s u l t of c a l c i u m m o b i l i z a t i o n from bone. When the EDTA i n f u s i o n was repeated w i t h simultane-ous i n f u s i o n o f e p i n e p h r i n e , the f a l l i n plasma c a l c i u m was a l -most twice as g r e a t (-3.30 mg-%). The d o t t e d l i n e i n F i g u r e 14 i n d i c a t e s the h y p o t h e t i c a l f a l l i n plasma c a l c i u m which would have o c c u r r e d i f the same q u a n t i t y o f ca l c i u m had been removed from the e x t r a c e l l u l a r f l u i d c a l c i u m p o o l , w i t h no c o n t r i b u t i o n from bone. The d i f f e r e n c e between t h i s and the observed curve may be a t t r i b u t e d to c a l c i u m m o b i l i z a t i o n from the s k e l e t o n . I t i s s i g n i f i c a n t t h a t the curve o b t a i n e d when epi n e p h r i n e was i n -fused along w i t h the EDTA approached the h y p o t h e t i c a l f a l l ( dotted l i n e ) which would have been expected i f the c a l c i u m had been removed o n l y from the e x t r a c e l l u l a r c a l c i u m p o o l w i t h 91. F i g u r e 14 E f f e c t of e p i n e p h r i n e on the hypocalcemia pro-duced by i . v . i n f u s i o n of EDTA. The s l o p i n g d o t t e d l i n e s i n d i c a t e the f a l l i n plasma c a l c i u m t h a t might have been expected i f the c a l c i u m had been removed from the e x t r a c e l l u l a r p o o l alone, without any con-t r i b u t i o n from bone, and i s the curve t h a t would be expected i f bone b l o o d flow was zero. 9 2 . no contribution from bone. This i s consistent with the view that profound vasoconstriction and reduction i n bone blood flow as a r e s u l t of the epinephrine infusion also sharply reduces the a v a i l a b i l i t y of bone calcium for homeostatic control of hypocalcemia. It has been postulated that there i s an equilibrium between plasma calcium and the calcium situated on the surface of the c r y s t a l s of bone s a l t accessible to the c i r c u l a t i o n (11, 16). Such an equilibrium i s i l l u s t r a t e d diagrammatically i n Figure 15. Experiments with radioactive calcium and strontium have i n -dicated that 0.3-0.6% of the calcium i n bone i s r e a d i l y exchange-able. This would correspond to the calcium i n equilibrium with blood calcium mentioned above, and would constitute a l a b i l e calcium storage pool in which calcium might be stored, or from which i t might be withdrawn.• The possible manner in which th i s might occur i s i l l u s t r a t e d i n Figures 16 and 17. When calcium i s infused, r a i s i n g the plasma calcium l e v e l , there w i l l be a concentration difference and calcium w i l l flow into the l a b i l e bone pool. If the plasma calcium l e v e l i s lowered with EDTA, the opposite w i l l occur. I t w i l l be noted i n the model that the rate of transfer w i l l be dependent on the capacity of the pipe connecting the two pools (blood and bone) which w i l l cor-respond to the bone blood flow. As i l l u s t r a t e d i n Figure 17, 93. F i g u r e 15 Labile Calcium Storage in Bone. Crystal of Bone Salt. Bone Blood Flow Exchangeable Ca * Blood C a + + Bone - Blood Calcium Equilibrium Model i n d i c a t i n g h y p o t h e t i c a l bone-blood c a l c i u m e q u i l i b r i u m . 94. F i g u r e 16 Model i l l u s t r a t i n g the p o s s i b l e r o l e of bone b l o o d flow i n the exchange of c a l c i u m between bloo d and the l a b i l e c a l c i u m storage p o o l i n bone. F i g u r e 17 [ C a + + ] + C a T i s s u e -Fluid 350 mg Labile Calc ium in Bone Storage Pool _ 2000 mg_ Epinephrine g ives reduced - Bone Blood Flow C a ( E D T A ) Model i l l u s t r a t i n g the p o s s i b l e e f f e c t of e p i n e p h r i n e induced v a s o c o n s t r i c t i o n on m i n e r a l t r a n s f e r between b l o o d and the' l a b i l e c a l c i u m storage p o o l i n bone. c o n s t r i c t i o n o f the bone v e s s e l s by epi n e p h r i n e should s h a r p l y reduce the communication between the two po o l s and impair the " b u f f e r i n g " e f f e c t of the l a b i l e bone p o o l . T h i s e f f e c t was c l e a r l y demonstrated i n the experiments d e s c r i b e d above. SUMMARY AND CONCLUSIONS Intravenous i n f u s i o n o f 9.0 mg Ca/kg over a 1 hour p e r i o d r a i s e d the plasma Ca 2.12 mg-%. When the i n f u s i o n was repeated i n the same animals w i t h simultaneous i n f u s i o n o f 2-4 micro-gram epinephrine/kg/min, the c a l c i u m rose 3.94 mg-%. The d i f -f erence was a t t r i b u t e d to ep i n e p h r i n e - i n d u c e d v a s o c o n s t r i c t i o n and reduced b l o o d flow, which i n t u r n would reduce the r a t e a t which c a l c i u m c o u l d be s t o r e d i n bone. S i m i l a r l y , i . v . i n f u -s i o n o f EDTA s u f f i c i e n t t o c h e l a t e and remove 8.5 mg Ca/kg over a 1 hour p e r i o d reduced the plasma c a l c i u m 1.79 mg-%; s i m i l a r i n f u s i o n with simultaneous e p i n e p h r i n e i n f u s i o n r e s u l t e d i n a f a l l o f 3.30 mg-%. The d i f f e r e n c e i s a l s o a t t r i b u t e d to vaso-c o n s t r i c t i o n and reduced b l o o d flow, which would i n t e r f e r e w i t h m o b i l i z a t i o n o f c a l c i u m from the s k e l e t o n . These experiments emphasize the importance o f bone blood flow i n the homeostatic r o l e of the s k e l e t o n i n r e g u l a t i n g the plasma c a l c i u m l e v e l . 97. Chapter V GENERAL SUMMARY AND CONCLUSIONS An i n d i r e c t method f o r measuring bone blo o d flow has been developed. I t i s based on the i n i t i a l (0-5 min) bone c l e a r a n c e o f r a d i o a c t i v e s t r o n t i u m ( S r 8 5 ) , a bone-seeking r a d i o i s o t o p e . The method appears t o be v a l i d , g e n e r a l l y a p p l i c a b l e and u s e f u l f o r s t u d y i n g q u a n t i t a t i v e l y some of the f a c t o r s a f f e c t i n g bone b l o o d flow. Bone uptake of r a d i o s t r o n t i u m i n the f i r s t 5 minutes f o l -lowing intravenous i n j e c t i o n i s e n t i r e l y dependent on i n t a c t bone b l o o d flow. 85 The e f f i c i e n c y o f removal of Sr i n bloo d p a s s i n g through bone i s determined by the e x t r a c t i o n r a t i o (ER). A method was developed f o r determining t h i s v a l u e . In determina-t i o n s on t i b i a s from 10 dogs, the mean value f o r the e x t r a c -t i o n r a t i o was 0.764 ± 0.066 (SE). T h i s h i g h v a l u e would appear to j u s t i f y use of i n i t i a l bone c l e a r a n c e o f S r 8 5 as a measure of e f f e c t i v e bone b l o o d flow. T o t a l bone b l o o d flow can be estimated by d i v i d i n g t h i s v a lue by the e x t r a c t i o n r a t i o . For 2 70 bones from 80 r a b b i t s , the mean e f f e c t i v e bone b l o o d flow was 9.60 ± 0.19 (SE) ml/min/100 g f r e s h weight. For 46 98. bones from 10 dogs, the e f f e c t i v e bone blo o d flow was 10.15 ± 0.61 (SE) ml/min/100 g f r e s h weight. Values f o r i n d i v i d u a l bones were remarkably c o n s i s t e n t . 6. T o t a l s k e l e t a l b l o o d flow was estimated to be 7.1 - 0.25 (SE) % of the r e s t i n g c a r d i a c output i n the r a b b i t s s t u d i e d , and 7.30 ± 0.95 (SE) % of the r e s t i n g c a r d i a c out-put i n the dogs. 7. Blood flow i n ml/min/100 g f r e s h weight was s i g n i f i c a n t l y h i g h e r i n the head of femur (18.67 ± 0.52) than i n the s h a f t (7.50 ± 0.16). 8. In r a b b i t femur, the n u t r i e n t a r t e r y appears to supply 70% of the b l o o d flow to the s h a f t and 1/3 of the flow to the two ends. 9. I m m o b i l i z a t i o n of one l e g i n a long p l a s t e r c a s t f o r 2 weeks r e s u l t e d i n a s m a l l decrease i n b l o o d flow to the f o o t bones; i m m o b i l i z a t i o n f o r 2 months r e s u l t e d i n d i s u s e o s t e o p o r o s i s of t i b i a and calcaneus, and a s i g n i f i c a n t i n -crease i n r e l a t i v e bone b l o o d flow per u n i t weight. 10. Complete s e c t i o n of the s c i a t i c nerve i n c r e a s e d b l o o d flow to t i b i a and f o o t bones, presumably due to i n t e r r u p t i o n of sympathetic v a s o c o n s t r i c t o r f i b e r s . 11. F r a c t u r e s of the neck of femur reduced b l o o d flow to the femoral head by 52-83%, the g r e a t e s t r e d u c t i o n o c c u r r i n g 9 9 . w i t h s u b c a p i t a l f r a c t u r e s . When the l a t t e r was combined wit h s e c t i o n of the ligamentum t e r e s , there was e s s e n t i a l l y no b l o o d flow to the head of femur. 12. Continuous i n f u s i o n of e p i n e p h r i n e (2-4 micro-gram/kg/ min) reduced bone b l o o d flow to 20-25% of normal, presum-a b l y due to c o n s t r i c t i o n of the v e s s e l s s u p p l y i n g bone. 13. Continuous i . v . i n f u s i o n of e p i n e p h r i n e (2-4 micro-gram/kg/ min) i n the dog r a i s e d the b l o o d glucose l e v e l and lowered the plasma i n o r g a n i c phosphate, but had no e f f e c t on plasma c a l c i u m . However, epi n e p h r i n e i n f u s i o n d i d s e r i o u s l y i n -t e r f e r e w i t h c a l c i u m storage i n or m o b i l i z a t i o n from bone, i n d i c a t i n g the importance of bone b l o o d flow i n homeostatic c o n t r o l of plasma c a l c i u m . 100. Table I .85 Sr uptake by normal and ischemic femoral heads (5 minutes a f t e r i . v . i n j e c t i o n ) R abbit Normal CPM Ischemic CPM % of normal 1 2 3 4 5 6 7 8 9 10 Average - SE 2432 2315 2656 2040 1365 1402 2206 1160 1608 1305 50 38 67 28 8 14 12 28 22 13 2.0 1.6 2.5 1.4 0.6 1.0 0.6 2.4 1.4 1.0 1850 ± 13.4 28 ± 6 1.5 ± 0.16 CPM: Counts per minute c o r r e c t e d f o r background. (Note t h a t there i s e s s e n t i a l l y no uptake by ischemic bone.) 101. Table I I E x t r a c t i o n r a t i o f o r uptake of S r 8 ^ by t i b i a ( i n i t i a l 5 min)* Number of dogs 10 Evans bl u e c o l l e c t e d (a), % 87.3 - 2.9 S r 8 5 c o l l e c t e d (b), % 20.6 ± 1.6 S r 8 5 e x t r a c t i o n r a t i o , a-b/a 0.754± 0.066 A c t u a l bone uptake of S r 8 5 , % 70.9 ± 1.1 T o t a l S r 8 5 recovery, % 94.6 ± 1 . 6 * Averages ± standard e r r o r Table I I I Q C Sr 0-" e x t r a c t i o n r a t i o a t 1 minute i n t e r v a l s * I n t e r v a l % Evans dye % S r 8 5 E x t r a c t i o n ( i n minutes) c o l l e c t e d (a) c o l l e c t e d (b) r a t i o , a-b/a 0 - 1 63.2 12.8 0.80 1 - 2 18.4 5.5 0.74 2 - 3 4.9 2.0 0.63 3 - 4 4.1 1.1 0.76 4 - 5 2.0 0.6 0.65 0 - 5 92.6 22.0 * Average f o r 5 dogs. 0.75 103. Table IV E f f e c t i v e bone b l o o d flow ( S r 8 ^ clearance) i n v a r i o u s r e g i o n s o f r a b b i t femur Region Number of Average - SE animals ml/min/100 g f r e s h weight Head Trochanter S h a f t Condyle 50 50 50 50 18.67 - 0.52 10.38 - 0.46 7.50 - 0.16 12.06 - 0.54 Table V 85 E f f e c t i v e b l o o d flow (Sr clearance) i n v a r i o u s bones expressed as ml/min/100 g f r e s h weight DOGS (10) RABBITS (80) + + Bone n Average - SE n Average - SE Femur 8 9.28 ± 1.28 80 9.70 ± 0.27 T i b i a 10 10.48 - 2.20 70 10.33 - 0.41 Humerus 10 10.14 - 1.46 50 10.40 - 0.53 Tal u s 9 10.30 - 2.27 20 10.45 - 1.00 Calcaneus 9 11.00 - 2.66 30 8.80 - 0.42 V e r t e b r a - 20 11.30 - 0.50 Average * 46 10.15 ± 0.61 270 9.60 - 0.19 SE Blood flow c o r - 13.20 - 0.79 12.48 - 0.26 r e c t e d f o r ex-t r a c t i o n r a t i o Table VI Estimate of t o t a l s k e l e t a l b l o o d flow i n dogs and r a b b i t s * Dogs Rabbits Number of animals s t u d i e d 10 80 Number of bones 46 270 E f f e c t i v e bone blo o d flow ( S r 8 5 clearance) ml/min/100 g f r e s h bone 10.15 ± 4.12 9.60 ± 3.28 Bone b l o o d flow c o r r e c t e d f o r e x t r a c t i o n r a t i o o f 0.75 13.20 - 5.35 12.48 - 4.26 T o t a l s k e l e t a l b l o o d flow ml/min ( s k e l e t o n estimated as 10% of body weight) 238 ± 1 4 31 ± 10 T o t a l s k e l e t a l b l o o d flow as % of r e s t i n g c a r d i a c output 7.3 ± 3.0 7.1 ± 2.3 * The v a l u e s are average * standard d e v i a t i o n . Table VII Estimated bone blo o d flow i n dog femur Flow per 100 g Bone Weight Whole Femur f r e s h weight g ml/min ml/min 37 4.2 11.3 62 4.9 7.8 63 6.3 10.1 105 9.7 9.1 110 14.7 13.3 110 21.8 19.6 120 13.6 11.2 120 25.2 21.0 Average 91 - 11 12 . 55 - 2 . 76 12 . 92 ± 1. ± SE 107. Table V I I I E f f e c t o f l i g a t i o n o f n u t r i e n t a r t e r y on e f f e c t i v e b l o o d flow to r a b b i t femur Regions i n femur n Upper metaphysis 13 D i a p h y s i s (shaft) 13 Lower metaphysis 13 ml blood/min/100 g*  C o n t r o l E x p e r i m e n t a l 10.8 i 0.8 6.8 ± 0.4 8.5 - 0.9 2.5 - 0.4 10.8 - 1.0 7.2 - 0.8 Reduc-t i o n % -37% -71% -33% * Averages - SE Table IX E f f e c t of i m m o b i l i z a t i o n of limb on e f f e c t i v e bone b l o o d flow i n r a b b i t Bone n C o n t r o l Side Immobilized Side D i f f e r e n c e Group I, Calcaneus T i b i a Femur 1-2 weeks i m m o b i l i z a t i o n i n long l e g c a s t . E f f e c t i v e blood flow i n ml/min/100 g f r e s h weight* 11 9.36 ± 0.15 11 9.33 ± 0.11 11 9.71 ± 0.10 7.43 ± 0.15 8.19 ± 0.14 9.27 ± 0.13 -1.93 ± 0.21 9.2 -1.14 ± 0.18 6.3 -0.44 ± 0.16 2.7 0.0001 0.001 0.05 Group I I . 2 months i m m o b i l i z a t i o n i n long l e g c a s t . E f f e c t i v e blood flow i n ml/min/100 g f r e s h weight* Calcaneus T i b i a Femur 8 8.40 ± 0.13 8 9.65 ± 0.13 8 10.31 ± 0.23 11.79 ± 0.14 11.29 ± 0.19 10.70 ± 0.21 +3.39 ± 0.19 17.9 +1.64 ± 0.22 7.4 +0.39 ± 0.31 1.2 0.001 0.001 NS E f f e c t i v e blood flow i n ml/min/whole bone* Calcaneus 8 0.096 ± 0.010 0.099 ± 0.012 +0.003 ± 0.016 0.20 NS T i b i a 8 0.743 ± 0.101 0.769 ± 0.135 +0.026 ± 0.169 0.15 NS Femur 8 0.963 ± 0.113 0.979 ± 0.109 +0.016 ± 0.156 0.10 NS * Averages - SE t " F i s h e r " t e s t r a t i o = d i f f e r e n c e / S E p p r o b a b i l i t y t h a t d i f f e r e n c e i s due to chance NS not s t a t i s t i c a l l y s i g n i f i c a n t 2 109. Table X Bone weight changes i n d i s u s e o s t e o p o r o s i s * (8 r a b b i t s ) Bones Wet wt. Dry wt. Ash wt. Ash wt. _2_ Dry wt. x 100 CALCANEUS C o n t r o l 1.16 0.83 0.45 E x p ' n t l 0.83 0.52 0.28 -% -28% -38% -38% 56.4% 53.8% -2.6% TIBIA C o n t r o l 9.81 5.44 3.03 E x p ' n t l 6.88 4.65 2.58 -% -12% -14% -16% 56.6% 55.3% -1.3% FEMUR C o n t r o l 9.17 5.66 3.16 E x p ' n t l 8.73 5.08 2.75 -% -9% -10% -13% 56.2% 54.5% -1.7% Disuse o s t e o p o r o s i s due to i m m o b i l i z a t i o n f o r 2 months i n a long l e g c a s t . 110. Table XI E f f e c t o f s c i a t i c nerve s e c t i o n on bone b l o o d flow i n the limb Bones ml/min/100 q wet weight C o n t r o l Experimental D i f . - SE Leg bones* 9.89 ± 1.70 12.87 ± 1.81 2.98 ± 2.42 Foot bones** 7.91 ± 1.52 13.52 ± 1.86 5.95 ± 2.48 * T i b i a - f i b u l a ** T a l u s - c a l c a n e u s The d i f f e r e n c e i n the f o o t bones, w i t h standard e r r o r , i s s i g n i f i c a n t with 0.05> P >0.025. The v a l u e s are average f o r 20 bones i n 10 r a b b i t s . Table XII E f f e c t of v a r i o u s procedures on e f f e c t i v e b l o o d flow to femoral head Procedure No. of ml blood/min/100 g f r e s h bone* Rabbits C o n t r o l E x p ' n t l D i f f . ± SE t P o s t e r i o r 10 21.1 ± 1.48 21.7 ± 1.71 +0.6 ± 2.26 capsulotomy S u b c a p i t a l f r a c t u r e 10 17.5 ± 1.00 0.3 ± 0.1 -17.2 ± 1.01 17.0 + s e c t i o n of (-98.5%) ligamentum t e r e s S u b c a p i t a l f r a c t u r e 20 15.6 ± 0.99 2.9 ± 0.87 -12.7 ± 1.31 9.7 of heck (-83%) No n - s u b c a p i t a l 40 2 0 . 6 * 0 . 7 1 9.7 ± 0.51 -10.9 ± 0.87 12.5 f r a c t u r e of neck (-52%) t = t e s t r a t i o ( F i sher) p = p r o b a b i l i t y t h a t d i f f e r e n c e occurred by chance. * = average - standard e r r o r . Table X I I I E f f e c t o f epi n e p h r i n e on e f f e c t i v e bone b l o o d flow ( S r 8 5 c l e a r a n c e i n ml/min/100 g f r e s h weight) Humerus T i b i a + F i b u l a Both Bones Number of bones C o n t r o l group 10 16.95±1.43 (mean - SE) Ep i n e p h r i n e i n f u s e d 4.42+-0.49 (mean - SE) D i f f e r e n c e % decrease " F i s h e r " t e s t r a t i o , t P r o b a b i l i t y t h a t d i f f e r e n c e i s due to chance, p -12.53±1.52 74% 8.3 0.0001 10 15.91±1.41 81% 8.6 0.0001 20 16.43±1.02 2.96±0.51 3.69±0.35 •12.95±1.50 -12.74±1.04 78% 12.1 0.0001 Table XIV E f f e c t of i . v . i n f u s i o n o f e p i n e p h r i n e on plasma c a l c i u m and i n o r g a n i c phosphate mg/100 ml plasma Average - SE Time i n hours Ca (10 dogs) P (6 dogs) C o n t r o l 0 9.85 + 0.29 5.12 + 0.48 1/2 9.84 ± 0.27 5.43 + 0.51 1 9.82 + 0.27 5.72 + 0.43 E p i n e p h r i n e i n f u s i o n 1/2 9.83 + 0.30 3.90 + 0.40 3/4 9.89 + 0.38 3.54 + 0.55 1 9.73 + 0.15 3.10 + 0.56 A f t e r i n f u s i o n 1/2 9.79 + 0.18 4.74 + 0.80 1 9.79 + 0.25 5.00 + 0.80 1 1/2 9.92 + 0.28 4.74 + 0.93 2 10.00 + 0.30 5.02 + 0.90 114. Table XV E f f e c t of i . v . c a l c i u m i n f u s i o n w i t h and without simultaneous epinephrine i n f u s i o n on plasma c a l c i u m and phosphate i n 5 dogs Time i n hours mg/100 ml plasma* Ca C o n t r o l p e r i o d 0 10.23 ± 0. 14 4 .66 + 0. 17 1/4 10.25 + 0. 03 5 .21 + 0. 24 1/2 10.31 + 0. 01 5 .38 + 0. 18 Calcium i n f u s i o n , 9.0 m g/kg/hour 1/2 11.82 + 0. 27 5 .81 + 0. 41 3/4 12.35 + 0. 29 5 .75 + 0. 10 1 12.43 + 0. 31 5 .94 + 0. 19 I n f u s i o n stopped 1/2 11.94 + 0. 15 5 .45 + 0. 17 1 1/2 11.12 + 0. 10 5 .31 + 0. 22 2 1/2 10.49 + 0. 20 5 .33 + 0. 15 Calcium i n f u s i o n , 9.0 m g/kg/hour p l u s e p i n e p h r i n e i n f u s i o n , 2 -4 micro-g/kg/min. 1/2 12.69 + 0. 12 3 .73 + 0. 52 3/4 13.77 + 0. 13 3 .16 + 0. 50 1 14.43 + 0. 13 3 .07 + 0. 61 I n f u s i o n stopped + + 1/2 13.03 0. 23 4 .49 0. 53 1 1/2 12.12 + 0. 10 4 .00 + 0. 15 3 1/2 10.48 ± 0. 02 - -* Average v a l u e s - standard e r r o r Table XVI E f f e c t o f EDTA i n f u s i o n ( i . v . ) without and wit h simultaneous i n f u s i o n of epi n e p h r i n e on plasma c a l c i u m and i n o r g a n i c phosphate i n 5 dogs Txme i n hours mg/100 ml plasma* Ca C o n t r o l p e r i o d 0 10.04 ± 0. 15 5.25 ± 0. 10 1/4 10.09 + 0. 16 5.40 + 0. 11 1/2 10.05 + 0. 15 5.51 + 0. 10 EDTA i n f u s i o n 1/2 9.01 + 0. 23 5.35 + 0. 12 3/4 8.60 + 0. 17 5.30 + 0. 23 1 8.26 ± 0. 14 5.03 + 0. 11 I n f u s i o n stopped + + 1/2 9.32 0. 10 5.16 0. 14 1 1/2 10.06 + 0. 12 5.39 + 0. 13 2 1/2 10.17 + 0. 10 5.72 + 0. 12 EDTA i n f u s i o n w i t h e p i n e p h r i n e i n f u s i o n 1/2 8.04 + 0. 14 3.58 + 0. 14 3/4 7.41 + 0. 11 3.03 + 0. 15 1 6.87 + 0. 17 2.35 + 0. 15 I n f u s i o n stopped 1/2 8.42 + 0. 12 2.91 + 0. 10 1 1/2 9.80 + 0. 11 4.17 + 0. 20 2 1/2 10.14 + 0. 12 4.59 + 0. 25 * Average v a l u e s - standard e r r o r 116. 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