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THE EFFECTS OF EXERCISE AND
HIGH PROTEIN DIET
ON THE RAT ACHILLES TENDON
b y
C h e r y l Ann T a u n t o n
B; Sc. ( K i n e s i o l o g y ) , S i m o n F r a s e r U n i v e r s i t y , 1969
A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF
THE REQUlRE34ENTS FOR THE DEGREE O F
MASTER OF SCIENCE (KINESIOLOGY)
i n t he D e p a r t m e n t
of
K i n e s i o l o g y
@ CHERYL ANN TAUNTON 1 9 7 6
SIMON FRASER UNIVERSITY
A p r i l I,
A l l r i g h t s reserved . T h i s thesis may n o t be reproduce(: i n w h o l e o r in p a r t , by p h o t o c o p y o r o t h e r means, without permission of t h 2 author .
APPROVAL
N a m e : C h e r y l A n n T a u n t o n
D e g r e e : M a s t e r of Science ( K i n e s i o l o g y )
T i t l e of Thesis : T h e E f f e c t s of Exercise and H i g h P ro te in
D i e t on t h e Rat A c h i l l e s T e n d o n
E x a m i n i n g C o m m i t t e e :
C h a i r m a n : D r . T.W. C a l v e r t
D r . N.M.G. B h a k t h a n Senior Supervisor
D r . A .E . ~ h a p r n g
D r . D.B. C l e m e n t
~arc&ret V. ~ a v a g e E x t e r n a l E x a m i n e r
PARTIAL COPYRIGHT L I C E N S E
I hereby g r a n t t o Simon F r a s e r U n i v e r s i t y the r i g h t t o lend
my t h e s i s o r d i s s e r t a t i o n ( t h e t i t l e of which i s shown below) t o u s e r s
of t h e Simon F r a s e r U n i v e r s i t y L i b r a r y , and t o make p a r t i a l or single
c o p i e s o n l y f o r . s u c h u s e r s o r i n r e sponse t o a r e q u e s t from t h e l i b r a r y
of any o t h e r u n i v e r s i t y , o r o t h e r e d u c a t i o n a l i n s t i t u t i o n , on i t s 'own
b e h a l f o r f o r one of i t s u s e r s . I f u r t h e r a g r e e t h a t pe rmiss ion f o r
m u l t i p l 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 g r a n t e d
by me o r t h e Dean of Graduate S t u d i e s . It is unders tood 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 n o t be a l lowed
w i t h o u t my w r i t t e n pe rmiss ion ,
T i t l e of ~ h e s i s / ~ i s s e r t a t i o n :
E F f k q s O F H F%GRC/SE A A U l4rC;rl
Author :
( s i g n a t u r e )
~ & T D N
(name )
k ~ P R / L . 14FL
( d a t e )
ABSTRACT
Achi l l e s t e n d o n i t i s is a common occurrence i n t h e endur-
ance runner. The endurance runners who experience A c h i l l e s
t e n d o n i t i s have been shown t o have an e l e v a t e d serum u r i c a c i d --
and t h e ques t ion has been r a i s e d a s t o whether t h e inflammation
of t h e tendon i s p r e c i p i t a t e d by t h e presence of u r i c a c i d
c r y s t a l s i n t h e tendon. This experiment was conducted t o inves-
t i g a t e whether u r i c a c i d c r y s t a l s could be a c a u s a t i v e f a c t o r i n
hyperuricemic t e n d o n i t i s . A high p r o t e i n d i e t was introduced
i n t o t h e experiment i n an at tempt t o p r e c i p i t a t e hyperuricemia
t o determine i f a r e s u l t a n t inflammation of t h e tendonous region
occurred.
A t o t a l of twenty male Sprague-Dawley r a t s , ranging i n
weight from 250 t o 325 grams were randomly ass igned t o four
groups of f i v e r a t s each; a c o n t r o l group which was n o t exer-
c i s e d and was f e d on r e g u l a r l a b chow, two e x e r c i s e groups, one
fed on high p r o t e i n d i e t and one f e d on r e g u l a r l a b chow and a
second sedentary group fed on high p r o t e i n d i e t , The e x e r c i s e
groups w e r e r u n on a motorized t r e a d m i l l f i v e days per week f o r
s i x weeks t o near exhaustion. Samples of Ach i l l e s tendon were
taken f o r determinat ion of t h e preseEze of u r i c a c i d c r y s t a l s
and f o r e l e c t r a n microscopic study.
The e l e c t r o n microscopic s tudier ; showed l i t t l e change i n
t h e f i n e s t r u c t u r e of t h e tendon due t o e x e r c i s e o r high
p r o t e i n d i e t . . /
The t i s s u e f i x e d f o r u r i c a c i d was observed under a Zeiss
Photomicroscope I1 with po la r i zed l i g h t attachments. I t w a s
found t h a t va r ious types of c r y s t a l s were p r e s e n t i n t h e t i s s u e .
The high p r o t e i n e x e r c i s e group e x h i b i t e d more c r y s t a l l i n e
m a t e r i a l than t h e o t h e r groups, followed by t h e r e g u l a r d i e t
e x e r c i s e group, t h e sedentary high p r o t e i n group and l a s t l y
t h e c o n t r o l group which exh ib i t ed only random c r y s t a l s . Fur the r
s p e c i f i c s t a i n i n g f o r u r i c ac id was c a r r i e d o u t t o e l imina te
t h e p o s s i b i l i t y of calcium and o t h e r c r y s t a l s being mistaken
f o r u r i c a c i d c r y s t a l s . This s t a i n i n g method showed more
u r i c c r y s t a l s i n t h e high p r o t e i n e x e r c i s e group than t h e o t h e r
experimental groups. None of t h e experimental animals showed
any symptoms of Ach i l l e s t e n d o n i t i s (inflammation and swel l ing
i n t h e tendon reg ion) . I t i s proposed t h a t trauma may be
necessary a s a precursor t o t h e inflammatory process of hyper-
uricemic t e n d o n i t i s . The increased u r i c a c i d d e p o s i t s i n t h e
tendon of t h e r a t s f ed on high p r o t e i n d i e t and exerc ised
i n d i c a t e d t h a t accumulation of u r i c a c i d may be an important
f a c t o r i n hyperuricemic t e n d o n i t i s i n endurance runners .
To Jack
ACKNOWLEDGMENT
I wish to thank Dr. Doug Clement and Jack Taunton coauthors
with myself on the study which led to this thesis. Their con-
tinued interest and support have been invaluable to me in the
completion of this thesis. I also wish to thank Dr. N.M.G.
Bhakthan, my senior supervisor, for his help in the final
preparation of this thesis and for his technical advice on
tissue preparation and also Dr. A.E. Chapman for his critical
evaluation of the thesis. Final thanks to Margaret Savage
for her encouragement and assistance and Brian Mist for his
assistance,
TABLE O F CONTENTS
Page no . Approval ......................................... ii
Abs t rac t ......................................... iii
Dedication . ....................................... v
Acknowledgment ................................... v i
In t roduc t ion ..................................... I
Exerc ise .................................... 1
High P r o t e i n Die t ........................... 2
Summary ..................................... 6
'Review of t h e L i t e r a t u r e ......................... Fine S t r u c t u r e of Tendon ..................... E f f e c t s of Exercise on Tendon ............... 10
E f f e c t s of High P r o t e i n D i e t on Tendon ...... 11
U r i c Acid And Tendonit is .................... 1 4
In t roduc t ion ........................... 1 4
Uric a c i d and acu te e x e r c i s e ........... 1 6
Mate r i a l and Methods ............................. 21
Treatment of t h e Animals .................... 2 1
High P r o t e i n D i e t ........................... 22
Exerc ise Procedure .......................... 22
Excision of t h e Tissue ...................... 2 3
Treatment of t h e Tissues .................... 24
Page no . .................... Electron microscopy 24
....................... Light microscopy 25
Results .......................................... 28
Light Microscopy ............................ 28
Electron Microscopy ......................... 37
Discussions ....................................... 49
Light Microscopy ............................ 49
Polarized light microscopy ............. 49
~xercise and uric acid ................. 49
Fine Structure of the Tendon ................ 52
Conclusions ...................................... 56
References ....................................... 58
Figure
LIST OF FIGURES
Page no.
1. The role of exercise in the alteration of uric acid levels...................-................ 3
2. Exercise, Hyperuricemia and Tendonitis ......-. 4a&4b 3. Theoretical Explanation of Hyperuricemic .................................... Tendonitis 15
4. Achilles tendon of a high protein exercise rat under compensated polarized light ............. 29a&29b
5. Achilles tendon of a regular diet exercise rat ............. under compensated polarized light 30a&30b
6. Achilles tendon of a high protein sedentary rat under compensated polarized light ............. 30a&30b
. 7. Achilles tendon of a control rat under compen- 1 : .......................... sated polarized light 30a&30b
8. Achilles tendon of a high protein exercise rat under compensated polarized light ............. 32ab32b
9. Achilles tendon of a high protein exercise rat under compensated polarized light ............. 32a&32b
10. Achilles tendon o$ a high protein exercise rat under compensated polarized light ............. 32a&32b
11. Achilles tendon of a high protein exercise rat /
under uncompensated polarized light ........... 34a&34b 12. Achilles tendon of a high protein exercise rat
under compensated polarized light ............. 34a&34b 13. Achilles tendon of a high protein exercise rat
under the light microscope .................... 34a&34b 14. Achilles tendon of hiqh protein exercise rat
under the light microscope .................... 35a&35b 15. Achilles tendon of high protein exercise rat
under the light microscope .................... 35a&35b
(ix)
Figure Page no.
16. A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e ...... r a t under uncompensated po la r i zed l i g h t 36a&36b
17. A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e ............... r a t under t h e l i g h t microscope 36a&36b
18. A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e ................ r a t under t h e l i g h t microscope 38a&38b
19. A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e ............... r a t under t h e l i g h t microscope 38a&38b
20. A c h i l l e s tendon of high p r o t e i n sedentary r a t .......... under uncompensated p o l a r i z e d l i g h t 39a&39b
21. A c h i l l e s tendon of high p r o t e i n sedentary r a t ................... under t h e l i g h t microscope 39a&39b
22. A c h i l l e s tendon of a high p r o t e i n sedentary r a t ................... under t h e l i g h t microscope 40a&405
23. A c h i l l e s tendon of a high p r o t e i n sedentary r a t ................... under t h e l i g h t microscope 40a&40b
24. A c h i l l e s tendon of c o n t r o l r a t under uncompen- ........................ s a t e d po la r i zed l i g h t 41a&41b
A c h i l l e s tendon of c o n t r o l r a t under t h e l i g h t ................................... microscope 41a&41b
A c h i l l e s tendon of a c o n t r o l r a t under t h e ............................. l i g h t microscope 42a&42b'
A c h i l l e s tendon of a c o n t r o l r a t under t h e l i g h t microscope ............................. 42a&42b
E lec t ron micrograph of t h e A c h i l l e s tendon of a high p r o t e i n sedentary r a t ................. 45a&45b
E lec t ron micrograph of t h e Acrhi l les tendon of a high p r o t e i n e x e r c i s e r a t .................. 46a&46b
The Ach i l l e s tendon of a high p r o t e i n e x e r c i s e r a t u:~der t h e e l e c t r o n inicrcscope. C r y s t a l s can be seen a s dark i r r e g u l a r shaped bodies ...... 4ia&47b
Figure Page no.
31. E lec t ron micrograph of Ach i l l e s tendon of a .................... r e g u l a r d i e t e x e r c i s e r a t 48a&48b
32. E lec t ron micrograph of A c h i l l e s tendon of a c o n t r o l r a t ................................... 49a&49b
CHAPTER 1
INTRODUCTION
A c h i l l e s t e n d o n i t i s is a major problem o f long d i s t a n c e
runners . The c u r r e n t view of i t s cause i s of a mechanical
n a t u r e , a s c r i b i n g it t o a s t r u c t u r a l weakening of t h e tendon
due t o t h e r e p e a t e d f o r c e s of take-off and l and ing i n running.
However, Clement -- e t a l . (1975) r e p o r t e d a s i g n i f i c a n t c o r r e l a -
t i o n between t e n d o n i t i s and hyperur icemia i n endurance a t h l e t e s .
They proposed t h a t t e n d o n i t i s i s a gou t - l i ke syndrome r e s u l t i n g
from t h e d e p o s i t i o n of u r i c a c i d c r y s t a l s i n t o t h e tendon
d u r i n g i n t e n s e and prolonged t r a i n i n g and p r e c i p i t a t e d by
trauma. The d e p o s i t i o n o f u r i c a c i d c r y s t a l s i n t h e tendons
of p a t i e n t s w i t h gout i s w e l l documented ( T a l b o t t , 1967) .
I. E x e r c i s e
There i s w e l l documented in fo rma t ion on t h e e f f e c t s of
e x e r c i s e on t h e f i n e s t r u c t u r e of s k e l e t a l muscle (Gol ln ick &
King, 1969; Gollni.ck -- e t a l . , 1969; Barnard -- e t a l . , 1970 and
Reitsma, 1970) . However, l i t t l e a t t e n t i o n i s pa id t o t h e
s t r u c t u r e of t h e tendon i n t h e s e animals. Mechanical ly , tendon
has been s t u d i e d e x t e n s i v e l y and t h e f o r c e s a p p l i e d t o t h e
A c h i l l e s tendon
of one thousand
i n running were found t o be of t h e magnitude
~ : o u n d s t o t h e squa re i n c h ( D e l l a , 1950) .
-1-
-2-
Endurance runners t r a i n from s i x t o seven days p e r week
a t a very h igh i n t e n s i t y and have been shown t o have an
inc reased serum u r i c a c i d l e v e l as a r e s u l t (Horvath, 1967).
Clement e t al. (1975) r epor ted t h a t e x e r c i s e appeared t o l ead -- t o an e l e v a t i o n of t h e serum u r i c a c i d (hyperuricemia) i n two
ways: by i n c r e a s i n g t h e t i s s u e n u c l e i c a c i d breakdown and
by decreas ing r e n a l exc re t ion . Tissue n u c l e i c breakdown was
e l e v a t e d by t h e i n c r e a s e i n muscle t i s s u e turnover , blood c e l l
breakdown and mitochondria1 breakdown. The decrease i n r e n a l
e x c r e t i o n was e f f e c t e d by dehydrat ion (causing decreased r e n a l
blood flow and decreased glomerular f i l t r a t i o n r a t e ) , increased
l a c t i c a c i d and ketone bodies (caus ing a decreased u r i n a r y
u r i c a c i d e x c r e t i o n ) and an inc rease i n catecholamines and
l o c a l hypoxia ( a l s o caus ing a decrease i n r e n a l blood flow and
decreased glomerular f i l t r a t i o n r a t e ) (Figure 1). The occur-
rence of hyperuricemia i n endurance a t h l e t e s was t h e basis f o r
t h e proposal of Clement -- e t a l . (1975) t h a t hyperuricemia and
t e n d o n i t i s were r e l a t e d (Figure 2 ) .
11. High P r o t e i n D i e t
High p r o t e i n d i e t s have been used by a t h l e t e s i n an e f f o r t
t o improve performance by i ~ c r e a s i n g endurance and/or s t r e n g t h
and a l s o f o r r ap id weight reduct ion. However, it does not
appeai- t o be u n i v e r s a l l y agceed t h a t e x t r a d i e t a r y p r o t e i n ,
PURINE SYNTHESIS
.. .- BODY PURINE
*
E x e r c i s e
I RENAL EXCRETION Exercise
E x e r c i s e : E l e v a t e s U r i c Acid ( H y p e r u r i c e m i a ) by:
1. f t i s s u e n u c l e i c a c i d breakdown - f m u s c l e t i s s u e t u r n o v e r , f RBC breakdown, f m i t o c h o n d r i a l breakdown
2 . + r e a a l e x c r e t i o n a ) d e h y d r a t i o n - + r e n a l b l o o d f l o w , +GFR b) f l a c t i c a c i d c ) f k e t o n e b o d i e s
+ u r i n a r y u r i c a c i d e x c r e t i o n
d ) f c a t e c h o l a m i n e s and l o c a l h y p o x i a - $ r e n a l b l o o d f l o w and CGFR
Figure 1. The role of exercise in the alteration of uric acid levels (from Clement et al., 1975, p. 42). --
@ 3 i . - . G Z x ~ r a 1 3 u u a , 3 ~ m~
I U ~ I m o > - > I m + m - ~ U N l c L C ) C % I s m O O v , C Q ) * - t C n O C
u%-- m C (DE m u m - - c.- mu- 6, 0- s E @ u ~ , - - Y m C -- E r m o s a, - -?C m r m ~ r u m a
- - C L Z . - O 3 E m r a -u r TI o m s
beyond t h e d a i l y neces sa ry requi rements improved performance.
yamaji (1951) , Yoshimura (1961, 1965) and Cvorkov e t a l e (1974) -- found an i n c r e a s e i n d i e t a r y p r o t e i n d u r i n g s t r enuous a c t i v i t y
t o be f avourab le f o r performance. However, Dar l ing e t a l . -- IA\ -
(1944) and Rasch and P i e r s o n (1962) found e x t r a d i e t a r y p r o t e i n
t o be of no v a l u e i n improving performance.
S ince t h e t i m e t h a t t h e m a j o r i t y of t h e s e s t u d i e s were
c a r r i e d o u t t h e r e has been an advancement i n endurance r a c e
p r e p a r a t i o n which r e q u i r e s d i e t manipulat ion. This p roces s ,
f i r s t e s t a b l i s h e d by Bergstrom e t a l . (1967) , i n Sweden, was -- c a l l e d ca rbohydra t e load ing . I t r e q u i r e d an over load ing of
p r o t e i n for t h r e e days fol lowed by an over load ing of carbohy-
d r a t e f o r t h r e e days i n t h e s i x days p r i o r t o t h e r a c e . The
purpose of t h i s d i e t w a s n o t t o i n c r e a s e performance th rough
high p r o t e i n i n t a k e b u t t o i n c r e a s e t h e glycogen s t o r e s s o a s
t o a l low t h e a t h l e t e t o perform f o r a g r e a t e r l e n g t h of t i m e
u s ing glycogen b e f o r e he must u t i l i z e f a t t y a c i d s .
A c l a s s i c a l p r e c i p i t a t o r of gout (deposi t ion 'of u r i c a c i d
c r y s t a l s i n j o i n t s ) was h igh p r o t e i n i n t a k e ( H a l l , 1971) .
Clement -- e t a l . (1975) p re sen ted d a t a i n d i c a t i n g t h a t A c h i l l e s
t e n d o n i t i s found i n endurance runne r s was a s s o c i a t e ~ w i t h hyper-
ur icemla which i s c h a r a c t e r i s ' . i c of gout . They pos::ulated that
h i g h p z o t e i n d i e t might p r e c i ~ i t a t e t e n d o n i t i s i n t ' le a l r e a d y
vu lne rab le tendon o f t h e enducance runner by f u r t h e r i n c r e a s i n g
t h e u r a t e c o n c e n t r a t i o n of t h e body f l u i d s . Egan (1972) h a s
r e p o r t e d t h a t many rats, e x e r c i s e d wh i l e on a h igh p r o t e i n
d i e t , have developed a c o n d i t i o n which he diagnosed as tendon-
i t i s . If t h i s a l s o occur red i n endurance a t h l e t e s t h e u s e of
h igh p r o t e i n d i e t by t h s e a t h l e t e s could be d e t r i m e n t a l .
The u s e of h igh p r o t e i n d i e t s by endurance a t h l e t e s may
be f u r t h e r i n c r e a s e d i n t h e f u t u r e as a r e s u l t o f two Japanese
s t u d i e s (Yamaji, 1951 and S h i r a h i e t a l . , 1974) - These a u t h o r s -7
r e p o r t e d t h a t a l a r g e p r o t e i n i n t a k e w a s neces sa ry t o c o r r e c t
t h e anemia found i n endurance a t h l e t e s as a r e s u l t of i n t e n s e
t r a i n i n g . The anemia occur red a t t h e beg inn ing o f t r a i n i n g
and t h e deg ree o f hemoglobin r e d u c t i o n and i t s rate o f r e t u r n
t o t h e p r e t r a i n i n g l e v e l w e r e i n f l u e n c e d by p r o t e i n i n t a k e .
Y a m a j i (1951) b e l i e v e d t h a t h i s r e s u l t s showed t h a t muscle w a s
formed a t t h e expense of blood p r o t e i n .
111. Summary
The purpose of t h i s s tudy w a s t o de te rmine t h e e f f e c t s ,
bo th s t r u c t u r a l ( e l e c t r o n microscopy) and h i s tokhemica l (po la r -
i z e d l i g h t microscopy and s e l e c t i v e s t a i n i n g ) , o f e x e r c i s e and
h igh p r o t e i n d i e t on r a t A c h i l l e s tendon. I t w a s hypothesized
t h a t e x e r c i s e and h igh p r o t e i n d i e t would l e a d t o t h e d e p o s i t i o n
of u r i c a c i d c r y s t a l s i n t h e tendon.
CHAPTER 2
REVIEW O F THE LITERATURE
I. F ine S t r u c t u r e o f Tendon
There i s a d e a r t h of l i t e r a t u r e on t h e s t r u c t u r e of normal
tendon (see reviews by H a l l , 1965 and Holrnes, 1971) . The g r o s s
appearance o f t h e tendon i s t h a t of wate red s i l k (Scha fe r , 1912)
w i t h a l t e r n a t i n g dark and l i g h t bands ( ~ e r i n g a and Lohr, 1924) .
These bands were found t o be p r e s e n t i n r e l a x a t i o n b u t d i s ap -
peared under t e n s i o n ' ( ~ a u c h , 1931 and Rigby -- e t a l . , 1959) . .
e end on e x c i t e s a t t e n t i o n because it i s v i r t u a l l y synonymous
w i t h c o l l a g e n , a prime c o n s t i t u e n t p r o t e i n o f a l l connec t ive
t i s s u e , i n c l u d i n g sk in" (Cr i sp , 1972, p . 143 ) . The composi t ion
of tendon i s a package of c o l l a g e n f i b r e s and mucopolysaccharide
(Scha tzker and Branemark, 1969) . The c o l l a g e n f i b r e s w e r e l a i d
approximately p a r a l l e l t o t h e long a x i s of a tendon ( E l l i o t t ,
1965) and w e r e found t o be a s long a s t h e tendon i t s e l f , anas- C
tomosing w i t h each o t h e r a t a c u t e a n g l e s (Scha tzker and Brane-
mark, 1969) . An e x t r a c e l l u l a r t h r e a d o f p r o t e i n , t h e f i b r i l ,
w a s the s t r u c t u r a l u n i t of co l l agenous t i s s u e ( E l l i o t t , 1965) .
The f i b r i l s w e r e grouped i n t o bundles ( p r i m . ~ r y bundles o r f i b -
res) which were aggrsga ted i n t o secondary kundles (Schneider ,
195c') o r f a s c i c l e s (Edwards, 1946) . The s€c-ondary bundles i n
t u r ; ? formed t e r t i a r y bundles (Schne ider , 1 1 59) which t o g e t h e r
comprised t h e tendon. The col lagen f i b r e s were t h i c k wi th
f i b r i l l a r s t r u c t u r e and w e r e p a r a l l e l t o one another (DiFiore,
The i n d i v i d u a l f i b r i l s w e r e cha rac te r i zed by a d i s t i n c t
p a t t e r n of f i n e c r o s s - s t r i a t i o n s (Bear, 1942 and Schmit t , 1942) ,
Light microscopy revealed t h e s e s t r i a t i o n s t o be made up of a l -
t e r n a t i n g s e t s of bands and in terbands (Grover, 1965); There
were c o n f l i c t i n g r e p o r t s a s t o t h e i n n e r s t r u c t u r e of t h e c o l -
lagen f i b r i l . Kennedy (1955) (human) , Grassman (1955) ( r a b b i t )
and Potz and Nemetschek (1956) ( r a b b i t ) showed t h e col lagen f i b -
rils from human pe r iodon ta l membrane and r a b b i t s k i n t o have a
t u b u l a r s t r u c t u r e . However, Borysko (1963) r epor ted beef l e g
tendon f i b r i l s t o be s o l i d . He concluded t h a t t h e bands exten-
ded through t h e e n t i r e th ickness of t h e f i b r i l . Stromberg and
Wiederheilm (1969) found t h e f i b r i l s t o be d i s o r i e n t e d wi th re-
s p e c t t o t h e long a x i s of t h e tendon when a t r e s t i n g l eng th and
they hypothesized t h a t s t r e s s cond i t ions caused some of t h e f i b -
rils t o be l o n g i t u d i n a l l y al igned. This confirmed t h e e a r l i e r
s tudy of r a t t a i l tendon by E l l i o t t ( 1965 ) who found t h a t t h e
f i b r e s followed a wavy course through t h e tendon bundle and only
became s t r a i g h t and p a r a l l e l t o t h e a x i s when s t r e t c h e d , The
o t h e r e x t r a c e l ' l u l a r components of tendon included t h e amorphous
ground substance which was a g e l l ay ing between t h e f i b r i l l a r
elements and conta in ing mucopolysacch.~rides ( E l l i o t t , 1965).
The cel ls of tendons were f i l amen tous and l a y p a r a l l e l t o
t h e l ong a x i s of t h e tendon (Buck, 1953) . There w e r e two t y p e s
of c e l l s which predominated: t e n o b l a s t s ( f i b r o b l a s t s ) and teno-
c y t e s ( f i b r o c y t e s ) (Holmes, 1971) . The t e n o b l a s t s were des-
c r i b e d by Greenlee and Ross (1967) t o have prominent n u c l e a l i ,
rough endoplasmic r e t i cu lum and Golgi appa ra tus . Holmes (1971)
r e p o r t e d t h a t t e n o b l a s t s were more o f t e n found i n o l d rats,
which confirmed t h e s t u d i e s o f V i id ik (1966) and Gould (1968)
who d i s t i n g u i s h e d t e n o c y t e s a s t h e a c t i v e cel ls which produce
the c o l l a g e n and t e n o b l a s t s a s t h e major i n e r t c o n s t i t u e n t of
tendon. Many a u t h o r s d i d n o t d i s t i n g u i s h between t e n o b l a s t s an(
t e n o c y t e s , however, Holmes (1971) found t h a t t h e r e w e r e f i n e
s t r u c t u r e d i f f e r e n c e s between t e n o b l a s t s and t enocy te s . Teno-
b l a s t s appeared i n l o n g i t u d i n a l s e c t i o n a s rhomboid cells a r -
ranged i n rows w i t h ovoid n u c l e i . There w e r e one o r more nucle-
o l i p r e s e n t and a p a l e a r e a i n t h e cytoplasm o p p o s i t e t h e nucle-
us. Tenocytes appeared t o c o n s i s t s o l e l y of e longa ted n u c l e i
due t o poor ly s t a i n i n g cytoplasm.
The e p i t e n o n was a d e l i c a t e l a y e r of l o o s e connec t ive tis-
sue which covered t h e tendor: (Buck, 1953 and C r i s p , 1972) . I t
cont inued i n t o t h e i n t e r i o r of t h e tendon a s t h e endotenon o r
parakenon (Buck, 1953; E l l i p ) t t , 1965 and C r i s p , 1'371). This
cove r ing surrounded t h e t e n i o n bundles and he ld t h e m t o g e t h e r ,
a l lowing some movement of t h e bundles w i t h r e l a t i o n s h i p t o each
o the r . I t c a r r i e d a l l t h e blood v e s s e l s , lymphatics and nerves
(Schatzker and Branemark, 1969 and E l l i o t t , 1965). The endo-
tenon contained e l a s t i c f i b r e s ( H i r a i , 1959 and E l l i o t t , 1965).
I f l u b r i c a t i o n of t h e tendon was e s s e n t i a l due t o f r i c t i o n o r
d i r e c t i o n change, then t h e r e was a c o n s t r a i n i n g shea th , t h e
synovia l shea th , which ca te red f o r one o r s e v e r a l tendons
(Cr isp , 1972).
The A c h i l l e s tendon was t h e common tendon of t h e so leus
and gastrocnemius muscles and i n s e r t e d i n t o t h e calcaneus.
This tendon d i d n o t change d i r e c t i o n and s o had no need f o r a
synovia l shea th (Kiely, 1963 and Corr igan, 1967) . I t was,
however, surrounded by a r e o l a r f a t t y connect ive t i s s u e i n
which e l a s t i c f i b r e s w e r e i n t e rposed ( t h e epi tenon) (Arner and
Lindholm, 1959).
11. E f f e c t s of Exerc ise on Tendon
"Tendon i s a dynamic l i n k between bone and muscle, v i t a l
t o t h e e f f i c i e n t t r a n s l a t i o n of t h e body's energy i n t o e f f e c t i v e
ac t ion" (Welsh -- e t a l . , 1971) . Jewel1 and Wilkie (1958) con-
eluded t h a t t h e tendon played a r o l e i n damping t h e f o r c e pro-
duced by a muscle and thereby modified t h z e f f e c t of muscular
con t rac t ion . When e x t e r n a l f o r c e s w e r e z p p l i e d , g r e a t e r than
the accustomed load of a muscle, t h e tendon dampened the fo rce .
I n t h i s way i t played t h e r o l e of a safeky mechanism. The
tendon was two t i m e s s t r o n g e r t han i t s muscle ( E l l i o t t , 1967)
y e t Walker e t a1 (1964) found t h a t i n v ivo t h e stress on a -- -- tendon w a s never more t h a n one q u a r t e r of i t s u l t i m a t e t e n s i l e
s t r e n g t h .
Comparatively few i n v e s t i g a t i o n s w e r e r epo r t ed on t h e
e f f e c t s o f e x e r c i s e on t h e s t r u c t u r e o f tendon. E l l i o t t (1964,
1965) s t u d i e d t h e tendon of t h r e e week o l d r a b b i t s fo l lowing
e x e r c i s e . H e found t h a t t h e tendon hyper t rophied and t h a t t h i s
growth w a s r e l a t e d t o t h e degree o f t e n s i o n t o which t h e tendon
was s u b j e c t e d . The i n c r e a s e i n tendon t h i c k n e s s w a s proport ion- '
a1 t o ' t h e i n c r e a s e i n t h e muscle c r o s s s e c t i o n a l a r e a .
Inglemark (1948) r e p o r t e d an i n c r e a s e i n t h e concen t r a t i on o f
n u c l e i i n hype r t roph ied tendon of e x e r c i s e d growing m i c e and
r a b b i t s . Adul t m i c e and r a t s e x h i b i t e d on ly a smal l n u c l e a r
i n c r e a s e . A later s tudy by V i i d i k (1967) on tendons of exer-
c i s e d r a b b i t s found no change i n weight , wate r c o n t e n t and
c o l l a g e n c o n t e n t from t h e c o n t r o l t o t h e t r a i n e d . His conclu-
s i o n was t h a t t r a i n i n g induced q u a l i t a t i v e b u t n o t q u a n t i t a t i v e
changes i n tendon.
111. E f f e c t s o f High P r o t e i n D i e t on Tendon
The main s t r u c t u r a l c o n s t i t u e n t s of t i s s u e s were p r o t e i n s .
They combi.ned w i t h l i p i d t.o form t h e i n t e r n a l anci e x t e r n a l
c e l l u l a r nembranes and i n k e r f aces . Free amino a c i d s were
7
l i b e r a t e d by d i g e s t i o n of d i e t a r y p r o t e i n and w e r e taken up
by t h e t i s s u e s , e s p e c i a l l y t h e l i v e r , i n t e s t i n e and kidney.
Amino a c i d s were metabolized by incorpora t ion i n t o p r o t e i n and
rese rves of p r o t e i n were found i n t h e l i v e r , kidney, plasma i
and muscle. The amino ac ids which were n o t incorpora ted i n t o I I I
p r o t e i n were deaminated. The n i t rogen was exc re ted i n t h e u r i n e
a s urea and carbon was metabolized through a v a r i e t y of path-
ways t o t r i c a r b o x y l i c a c i d cyc le in te rmedia tes (Harper, 1969) -
F igure 2 (p. 4 b ) shows t h e p o i n t a t which high p r o t e i n
d i e t s may i n f l u e n c e t h e purine pool. This type of d i e t has (
been common f o r some a t h l e t e s who were a t tempt ing quick weight
l o s s and f o r some who were looking t o i n c r e a s e performance. i i Egan (1972) has repor ted t h a t high p r o t e i n d i e t was seen t o
induce what he diagnosed a s t e n d o n i t i s i n e x e r c i s i n g r a t s . This
r e s u l t e d i n inflammation about t h e Ach i l l e s tendon and a /!I cessa t ion of running.
1 I
High p r o t e i n d i e t s have been thought t o i n c r e a s e t h e per-
fornance of a t h l e t e s due t o increased endurance and s t r e n g t h 1
(Cvorkov e t al . , 1974). Some s t u d e n t s of n u t r i t i o n do no t -- agree w i t h t h i s and f e e l t h a t v a r i a t i o n s i n p r o t e i n in take
a f f e c t n e i t h e r phys ica l f i t n e s s nor muscle mass (Rasch, 1 9 6 0 ) . I
T k t i s was i n agreement w i t h an e a r l i e r study by ~ a r l i n g -- e t a l .
( s - 9 4 4 ) . Rasch and Pierson ( 1 9 6 2 ) confirmed these r e s u l t s i n
a s tudy on male c o l l e g e s tuden t s . They d iv ided t h e s tuden t s
into two groups, one given a daily dietary protein supplement
and the other given a placebo. The subjects were given three
weight training sessions per week for six weeks. The body
weight, arm volumes, arm girths and total weight lifted were
measured before and after training. ~t the end of six weeks
training there was no significant differences (P < .01) between
the pre and post training body weights, arm volumes or upper
arm girths, for either group. The total weight lifted was
significantly increased for both groups but there was no dif-
ference between the groups. Although these authors concluded
that-the protein supplement had no effect on muscular strength
or hypertrophy they also reported a need for further studies
in this area to cover a longer period of time. Yamaji (1951),
Kraut et al. (1953) and Yoshimura (1961 and 1965) found -- protein supplementation, during strenuous exercise, favourable
for performance. Cvorkov (1972) agreed with these findings.
He studied the endurance capabilities of rats on high protein
diet as compared to rats on a normal lab chow diet and found
a significantly higher endurance capacity in the high protein
diet group.
Dietary protein level has been reported to influence the
serum urate concentration, but to a lesser degree than the
protein content (Bien -- et al., 1953). The products of protein
metabolism contributed precursors for arate biosynthesis-
s e e g m i l l e r -- e t a l . (1961) s u b j e c t e d twenty-two normal and s i x t y
gouty i n d i v i d u a l s t o a pu r ine f r e e d i e t and no ted average f a l l s
in serum u r a t e c o n c e n t r a t i o n o f 0.63 and 1 .01 mg%, r e s p e c t i v e l y .
A l a t e r s t u d y (Seegmi l le r e t a l . , 1963) showed t h a t t h e inges- -- t i o n of a d i e t r i c h i n p u r i n e s enhanced serum u r a t e p roduc t ion .
Seven nongouty s u b j e c t s w e r e g iven a h i g h p u r i n e d i e t ( f o u r
grams of r i b o n u c l e i c a c i d p e r day) w i t h a r e s u l t a n t i n c r e a s e
i n serum u r a t e from an average o f 4.6 mg% t o 8.3 mg%.
UnfortunateLy, t h e r e i s no in fo rma t ion r e p o r t e d i n t h e
l i t e r a t u r e about t h e e f f e c t s o f h igh p r o t e i n d i e t on tendons.
IV. U r i c Acid and Tendon i t i s
A. I n t r o d u c t i o n
~ y p e r u r i c e m i a i s t h e presence o f sodium u r a t e ( u r i c
acid) i n t h e plasma a t a g r e a t e r t h a n normal c o n c e n t r a t i o n
(>7.0 mg%), r e s u l t i n g i n a s u p e r s a t u r a t e d s o l u t i o n . Under
a p p r o p r i a t e c o n d i t i o n s , t h e exces s u r a t e w i l l p r e c i p i t a t e and
r e s u l t i n sodium u r a t e c r y s t a l d e p o s i t i o n i n a v a r i e t y of
t i s s u e s i n c l u d i n g synovium and tendon . (Goldf inger , 1971) .
Hyperuricemia has been shown to be p r e s e n t i n gout and i n gouty
a r t h r i t i s ( H a l l , 1971) . I t has been hypothes ized t h a t hyper-
u r icemia , w i t h a r e s u l t a n t d e p o s i t i o n of c r y s t a l s , w a s t h e
cause o f t h e a l l t o o common A ( * h i l l e s t e n d o n i t i s found i n
enduran,r.e runne r s (Clement -- e t a l . , 1975) (F igu re 3 ) . As t h e r e
I ENDURANCE EXERCISE I b e t w e e n 30 m i l e s and
120 m i l e s p e r week
\L HYPER URICENIA
URIC ACID i s a m e t a b o l i c e x c r e t o r y p r o d u c t o f
p u r i n e d e g r a d a t i o n . H y p e r u r i c e m i a i s p r e s e n t
when serum uric a c i d e x c e e d s , 7.0 mg%.
I MONOSODIUM URaTE CRYSTAL DEPOSIT~ON
gsuty arthritic s t a t e
I
I TENDONITIS
I m e c h a n i c a l and b i o c h e m i c a l
FIGURE 3. Theoretical Explanation of Hyperuricemic Tendonitis (from Clerneilt - et -- al., 1975)
was no synovium assoc ia ted wi th t h e Ach i l l e s tendon, t h e
c r y s t a l s would have been depos i ted i n t h e tendon i t s e l f ,
s e t t i n g up an inflammatory r e a c t i o n which i n t u r n r e s u l t e d i n
pain and swell ing.
B. Uric a c i d and acute e x e r c i s e
Elevated serum u r i c a c i d has been repor ted t o be
as soc ia ted wi th e x e r c i s e s i n c e 1921 when ~ a k e s t r a w repor ted
e l e v a t i o n s i n serum u r i c a c i d i n women a f t e r acu te exe rc i se .
H i s r e s u l t s showed inc reases i n serum u r i c a c i d of 0.9 mg% i n
b r i e f and 1.5 mg% i n prolonged pe r iods of exe rc i se . I n a s tudy
by Levine -- e t a l . (1924) of runners i n t h e Boston marathon, p r e
r ace blood samples were taken on f i v e runners wi th a r e s u l t a n t
mean serum u r i c a c i d of 3.94 mg% while p o s t r ace samples on
eleven s u b j e c t s y ie lded a mean of 5.70 mg%. One runner was
re-examined fo r ty -e igh t hours l a t e r . H i s serum u r i c a c i d had
re tu rned t o t h e pre-race l e v e l .
These r e s u l t s have been supported by Nichols .- e t - a l .
(1951) who repor ted a c l o s e c o r r e l a t i o n between serum u r i c a c i d
and i n h i b i t i o n of u r i c ac id exc re t ion . These au thors observed
an i n c r e a s e i n serum u r i c a c i d l e v e l s a f t e r s t renuous ghys ica l
a c t i v i t y (mean inc rease of 0.5 mg% fo l lowing t h i r t y minutes of
running) whereas moderate e x e r t i o n l i k e walking d i d n c t
in f luence uricemia. Even ligh.2 e x e r c i s e ( l i g h t gymnastics with
running, walking and appa ra tus work f o r t h i r t y minutes) has
been r e p o r t e d t o r e s u l t i n a s m a l l rise i n serum u r i c a c i d
(0.16 -1- 0.7 mg%) i n twenty young men (Zachau-Chris t iansen,
1959). Severe s t r a i n such a s marathon running r e s u l t e d i n a
much g r e a t e r i n c r e a s e i n serum u r i c a c i d ( 1 . 4 mg%) (Horvath,
1967).
A number of reasons have been p u t forward t o e x p l a i n
t h e i n c r e a s e i n serum u r i c a c i d w i t h muscular e x e r t i o n , t h e
ear l ies t be ing t h a t t h e r e was a r e d u c t i o n i n u r i n a r y e x c r e t i o n
of u r i c a c i d due t o an i n c r e a s e d e x c r e t i o n o f f l u i d s as s w e a t - .
However, Horvath (1967) r e p o r t e d t h a t o t h e r f a c t o r s must be
involved because t h e dec rease i n u r i c a c i d e x c r e t i o n w a s a l s o
p r e s e n t i n s u b j e c t s whose d i u r e s i s remained a t a h igh l e v e l
d u r i n g stress. Rakestraw (1921) and Levine -- e t a l . ( 1 9 2 4 ) found
t h a t e x c e s s i v e s t r a i n was accompanied by a d e c r e a s e i n t h e
c l e a r a n c e o f u r i c a c i d which was a s s o c i a t e d w i t h a r e d u c t i o n o f
g lomeru la r f i l t r a t i o n and t u b u l a r dys func t ion . The t u b u l a r
dys func t ion w a s s een t o be caused by hyposec re t ion o f u r i c a c i d
(Nicho1.s -- e t a l . , 1951) . A f u r t h e r f a c t o r was in t roduced by
Horva t l~ (1967) who s t u d i e d a group o f ~ n t e r n a t i o n a l sportsmen
( l i g h t a t h l e t e s , heavy athle::es, hockey p l a y e r s , f o o t b a l l e r s ,
c y c l i s t s and gymnasts) d u r i ~ q r e s t and d u r i n g i n t e n s i v e t r a i n -
ing . Twenty-three of t h e a t h l e t e s were s t u d i e d d u r i n g a per iod
of no t r a i n i n g , and had a mcan serum u r i c a c i d level of 3.05
mg% while the remaining twenty-seven were studied during
intense training for their particular sporting event and showed
a mean serum uric acid of 4.90 mg%- Horvath (1967) concluded
that the increased serum uric acid in the intensive training
group was due to an increased destruction and new formation of
protein. The author presented the question as to whether the
increased uricenia of muscular exertion would eventually lead
to hyperuricemic syndrome or gout. Clement et al- (1975) -- postulated that this increase in uricemia lead to tendonitis
when the conditions were right. Figure 2 (p. 4b) outlines -
the exercise factors which could lead to an increased serum uric
acid level. Banister -- et a1 (1971) reported mitochondria1
breakdown in animals following strenuous exercise which also
suggested an increased turnover of tissue protein.
There was contradictory evidence that showed that physical
exertion in high school sports lead to a decrease in serum
uric acid (Montoye -- et al., 1967). However, the high school
athletes studied had resting serum uric acid levels signifi-
cantly higher than non-athletes. The serum uric acid levels
of twenty male college athletes were measured twice, seven
months apart. These athletes had a substantially elevated
serum uric acid when compared to o~her non-athletic grouFs
(Greenleaf -- et al. , 1969). A study by ~osco et al. (1970) on the - --
effect of eight weeks of chronic physical exercise on thirty
ten - control group - bowling class - sedentary) showed that chronic exercise lowered uric acid in eighty per cent of the
athletic and training groups' subjects. The exercise training
period lasted for eight weeks and consisted of two hour daily
physical conditioning sessions of sprint swimming, resistive
exercise, water polo drills and weekly school competition for
the athletic group. The moderate activity group ran one mile
per day, played one hour of soccer per week and did one-half
hour of circuit training twice a week for muscular endurance.
The control group had two one-half hour bowling sessions per
week, participated in little or no organized physical activity
and led relatively sedentary lives. The athletes' activity
was estimated, in terms of caloric expenditure, to be six times
more than the soccer class and twenty-five times more than the
bowling class. However, the intensity of the exercise in this
study did not closely approximate that of endurance athletes,
many of whom train twice a day resulting in an increase in
metabolic demands and cellular turnover. This increased the
quantity of purines and proteins added to the total purine
Pool (Ficure 2, p. 4b) which inzreased uric acid concentration.
Seer miller and Howell (1963) proposed a self-propogating
inflammaiory reaction theory ilxvolving urate crystals.
The initiating event of the cycle could be stress, trauma,
adrenocorticotropic hormone withdrawl or exercise. All these I I
factors, especially exercise, involve the development of a pH I
gradient between tissue and blood, favouring the precipitation I
I
of urate (Clement -- et al., 1975). Seegmiller, Laster and Howell I
(1963) stated that "strenuous exercise increased the serum ~ urate level through the urate-retaining action of the elevated
lactic acid levels".
CHAPTER 3
MATERIAL AND METHODS
Twenty male a l b i n o r a t s of t h e Sprague-Dawley s t r a i n ,
averaging 280 grams i n weight, were used i n t h e study. The
r a t s were randomly divided i n t o four groups of f i v e r a t s each.
I. Treatment of t h e Animals
Group I (n = 5) - Exercise High P r o t e i n Diet
This group of r a t s was exe rc i sed by running t o nea r
exhaust ion f i v e days pe r week f o r a s i x week period. They were -
maintained on a high p r o t e i n d i e t (powder) given ad l ib i tum.
Group I1 (n = 5) - Sedentary High P r o t e i n D i e t
Group I1 were maintained i n t h e i r cages f o r a per iod
of s i x weeks. They were fed on a d a i l y d i e t of high p r o t e i n
powder given - ad l ib i tum.
Group I11 (n = 5) - Exerc ise Regulak D i e t Group
These r a t s were exe rc i sed by running t o near exhaus-
t i o n f i v e days p e r week f o r s i x weeks. They were fed on a r a t
l a b chow (mixed) d i e t - . ad l ib i tum.
Group I V ( n = 5) - Control Group
This group of r a t s were maintained i n t h e i r cages
f o r a s i x week period. Thei r d i e t w a s ra t l a b chow (mixed) and
was given ad l ib i tum. - - .-
11. High P r o t e i n Die t
This powdered d i e t was obta ined from N u t r i t i o n a l Biochemi-
c a l Corporat ion, Cleveland, Ohio. The high p r o t e i n d i e t con-
t a ined :
Mo . was :
Vitamin Free Casein . . . 6 4 %
Sucrose . . . . . . . . . . 2 2 %
Vegetable O i l . . . 89
Brewers Yeast U. S. P. . 2%
. S a l t Mixture U.S.P. . . . 4%
The l a b chow was obtained from Purina Company, S t . Louis,
The con ten t , according t o t h e manufacturers s p e c i f i c a t i o n s
Crude p r o t e i n . . . . . . . . . 22%
Crude f a t . . . . . . . . . . 4 %
Crude f i b e r . . . . . . . . . . 5%
Ash . . . . . . . . . . . . . 9%
Exerc ise Procedure
Groups I and I11 were exe rc i sed f i v e days pe r week, f o r
s i x weeks, t o near exhaust ion, on motor d r i v m small animal
t r e a d m i l l (Quinton Instrument Co. , S e a t t l e , \Tashington) , The
p o i n t of c e s s a t i o n of e x e r c i s e was determine:' by t h e r a t s '
behaviour. They were removed from t h e t r e a d n i l l a t near
exhaus t ion , when they l a y on t h e t r e a d m i l l a-id would al low
themselves to be carried into the shock electrodes (Holloszy,
1970). The grade of the treadmill was maintained at zero per
cent while the speed was increased progressively to maintain
the time of exercise at approximately sixty minutes per
exercise period (Cvorkov -- et al., 1974). Initially, the speed
of the treadmill varied between 0.5 and 1.0 km/hr and at the
end of the training period the rats were running at varied
speeds between 1.9 and 3.5 km/hr.
IV. Excision of the Tissue
The five rats from each gorup were sacrificed under a light
anaesthesia induced with ether and tissue was taken for light
and/or electron microscopic study. When the animals were light-
ly sedated (determined by a lack of eyelid reflex when the
eye was gently touched) they were placed ventral surface down
on the operating board and their limbs were secured in an out-
stretched manner. An incision was made at the mLdpoint of one
lower leg and this was extended in both directions to include
the entire lower leg. The skin and fascia were removed to
reveal the Achilles tendon.
Four per cent glutaraldehyee in phosphate buffer was
pipetted into the area immediately following exposure and the
tissue was periodically bathed with this solution to p-eserve
a good p r o t e i n f i x a t i v e (Pease, 1964).
Small p i e c e s of t h e Ach i l l e s tendon w e r e
i n v i a l s con ta in ing buffered g lu tara ldehyde ,
p ieces of tendon were taken.
exc ised and placed
Three s e p a r a t e
The second l e g of-each rat was prepared f o r t i s s u e exc i s ion
a s above. The muscle and tendon of t h e lower l e g were exposed
and t h e tendon was immediately excised and placed i n one hun-
dred p e r c e n t a lcohol . This t i s s u e was prepared f o r u r i c
a c i d demonstration. Again, t h r e e s e p a r a t e p ieces of each
tendon were used f o r t h e study. One hundred per c e n t a l coho l
has been shown t o be t h e f i x a t i v e of choice a s u r i c a c i d i s
s o l u b l e i n water (Merck, 1972).
V. Treatment of t h e Tissue
A. E lec t ron microscopy
The excised tendon was p o s t f i x e d i n two p e r c e n t
buffered osmium t e t r o x i d e i n phosphate b u f f e r f o r two hours
(Mil lonig, 1962). Following p o s t f i x a t i o n t h e t i s s u e was
processed through grades of e t h y l a l coho l (dehydra t ion) ,
propylene oxide ( c l e a r i n g ) and was f l a t embedded i n Epon 812
(Weakley, 1972). Thc t i s s u e was o r i e n t e d ort blank blocks
f o r Longitudinal s e c t i o n s and t h i n s e c t i o n s were c u t on a
Por t sr-Blum
witbL uranyl
MT-1 ultxamicroto~ne. The s e c t i m s were s t a i n e d
a c e t a t e and lead c i t r a t e (Reynclds, 1963) and were
examined under an RCA EMU-3~/Zeiss 9A electron microscope.
The three specimens from each of three animals of all four
groups were scanned for fine structure changes.
B. Light microscopy I
The excised tissue was cut into pieces and was fixed
in cold absolute alcohol in order to preserve uric acid
crystals if they were present. Fixation was for forty-eight
hours. This tissue was processed through toluene as the
clearing agent. Washing and dehydration were not carried out
as washing would have dissolved any uric acid crystals present-
and the tissue was fixed in absolute alcohol so dehydration
was not necessary. Embedding was in paraffin. Sections,
five mp thick, were cut and passed through xylene and alcohol
and were mounted on glass slides with Canada balsam.
Uric acid crystals have been easily identified in syno-
vial fluid by the use of compensated polarized microscopy
(Phelps, 1968). Polarized light was attained by use of a
polarizing microscope which had two polarizing filters, one
positioned below the condenser (polarizer) and one inserted
at some point above the objective (analyzer). A removable
first-order red compensator was placed between the objective
and the analyzer. In practice, a crystal was centred in the
field of view and the stage was rotated so that the 1o:lg axis
of t h e c r y s t a l was p a r a l l e l t o t h e d i r e c t i o n of v i b r a t i o n of t h e
slower component of l i g h t i n t h e compensator.
The p o l a r i z i n g f i l t e r passed l i g h t i n only one plane
and t h e p o l a r i z i n g f i l t e r was s e t s o t h a t no l i g h t passed
through it and t h e r e was a black background. I f a c r y s t a l
( o p t i c a l l y a n i s o t r o p i c ) was placed between t h e p o l a r i z e r and t h e
analyzer t h e l i g h t passed by t h e p o l a r i z e r w a s r o t a t e d by t h e
c r y s t a l a l lowing l i g h t t o pass through t h e analyzer . Only t h e 1 8 ,
8 v l 13 li I i
light which passed through t h e c r y s t a l was v i s i b l e and t h e r,,,,".. 1
c r y s t a l appeared white. 1 I
( 1 1 - ! ' I
- I n o r d e r t o i d e n t i f y one c r y s t a l from another ( i .e. sodium I1 4 1
ura te from calcium pyrophosphate) it was necessary t o add a
f i r s t o r d e r red compensator (Phelps, 1971) . This device
separa ted l i g h t according t o components of f a s t and slow I ;:;,ll~l
v ib ra t ion . I t was inser ted-between t h e o b j e c t i v e and t h e analy- .
zer and r e t a r d e d red l i g h t s o t h a t t h e background became red
ins tead of b lack (Phelps, 1968). The u r a t e c r y s t a l s were s h o r t
and rod-shaped wi th rounded ends and p a r a l l e l s i d e s i n t h e j o i n t
f l u i d b u t were long and needle-shaped i n topl-.aceous m a t e r i a l
(Phelps, 1971) . - Urate c r y s t a l s were s t r o n g l y nega t ive ly
b i re f c ingent such tha:: under compensated p ~ l ~ r r i z e d l i g h t they
were yellow when t h e long a x i s of t h e crysta.: was p a r a l l e l t o
. t he a x i s of slow v i b r a t i o n of t h e compensatc~r; a t t h e perpendic-
i- u l a r they were b lue . Calcium pyrophosphate, which was weakly
p o s i t i v e l y b i r e f r i n g e n t , exhibi ted the oppos i t e colours .
S l i d e s prepared from the th ree p ieces of tendon from each
of t h e r a t s were examined under compensated po la r i zed l i g h t .
A s t h e t i s s u e had been sect ioned the c r y s t a l s were not o r i e n t e d
such t h a t t h e long a x i s could be determined. More s e c t i o n s
w e r e prepared. The s l i d e s were then placed i n twenty per
c e n t s i l v e r n i t r a t e solukion and were exposed t o s t rong sun-
l i g h t f o r one t o four hours (u ra tes become b r i g h t rose c o l o u r ) .
A s o l u t i o n of t e n m l . of t h r e e per c e n t g e l a t i n i n hot water , 11 I
t h r e e m l . of twenty pe r cent s i l v e r n i t r a t e and two m l . of two I
per c e n t hydroquinone was poured over t h e s l i d e s u n t i l t h e I
I
u r a t e s turned black and t h e connective t i s s u e was yellow. The I(
I1
s l i d e s w e r e washed i n hot water (50•‹c), dehydrated and mounted 1
with Canada Balsam. They were observed under t h e l i g h t and I po la r i zed l i g h t microscope.
CHAPTER 4
RESULTS
I. Light Microscopy
The results of the light microscopic study are presented
as colour photomicrographs. Initially, all of the slides
prepared for light microscopy were scanned under the polarizing
microscope for the presence of crystals. Figures 4 to 7 are
photomicrographs taken under the compensated polarized micro-
scope. Figure 4 shows the presence of crystals in the tendon
of a high protein exercise rat. The crystals, which are 1
scattered through the tissue, are seen as blue and yellow while
the tissue is red. These colours are due to the presence of
the first-order red compensator plate as well as the polarizing
filter and analyzer in the path of the beam. It is not possible
from this micrograph alone to differentiate the various crys-.
tals. Figures 5, 6 and 7 are photomicrographs of tendon from
regular diet exercise, high protein sedentary and control rats,
respectively. These micrographs also demonstrate the presence
of crystals which cannot be differentiated.
As uric acid crystals are strongly negatively birefringent
and cdcium crystals are weakly positively birefringent it was
necessary to rotate the slides and observe wP.ether the crystals
chang-:d colour from yc>llow to blue or from b..ue to yellow.
FIGURE 4.' Achilles tendon of a high protein exercise rat under compensated polarized light. Crystals are seen as yellow and blue (X 1250).
Figure 5 . A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e r a t under compensated l i g h t . C r y s t a l s are seen as ye l low and b l u e ( X 2 5 0 ) .
F igu re 6. A c h i l l e s tendon of a h igh p r o t e i n s eden ta ry r a t under compensated p o l a r i z e d l i g h t . C r y s t a l s are seen as ye l low and b l u e ( X 5 0 0 ) .
F igu re 7. A c h i l l e s tendon of a c o n t r o l r a t un2er compensated p o l a r i z e d l i g h t . C r y s t a l s a r e seen a s yel low and b l u e (X5OO).
Figures 8, 9, and 10 are of one slide, that of a high protein
exercise rat. The first micrograph (Fig. 8) has only the
polarizing analyzer and filter in place, causing the black out
of light. The crystals are illuminated and appear as a bright
cluster in the upper right hand corner of the micrograph.
The next two figures (9 and 10) were taken with the first-
order red compensator in place and were taken at right angles
to one another (i.e. the stage was rotated ninety degrees after
the first micrograph was taken). A close observation of the
crystals present in these two micrographs shows that the yellow
crystals have turned blue and the blue crystals yellow. The
possibility was considered that the yellow crystals present in
the first micrograph were uric acid and the blue calcium.
However, this was not valid as it was not possible to identify t
the long axis of the crystals in the sectioned tissue which is H
a requirement for positive identification (uric acid crystals
are yellow when their long axis is parallel to the axis of slow
vibration of the compensator plate). A further step in the
identification process followed.
Tissue was sectioned for selective staining for uric acid,
a procedure which turns thc crystals coral and thcn black in
colour. The remaining colour photomicrographs are of the
selective~y stained tendor. (Figures 11 to 27). P ; a quantita-
tive analysis of the relaAive amounts oE uric acj 1 in the
Figure 8. Achilles tendon of a high protein exercise rat under compensated polarized light. Crystals are white ( X 5 0 0 ) .
Figure 9. Achilles tendon of a high protein exercise rat under compensated polarized light. Crystals are yellow and blue. This micrograph is at right angles to Figure 7 ( X 5 0 0 ) .
Figure 10- Achilles tendon of a high protein exercise rat under compensated polarized light. Crystals are yellow and blue. The micrograph is at right angles to Figure 6 ( X 5 0 0 ) .
t i s s u e of t h e four groups was n o t p o s s i b l e , photomicrographs
from t h r e e animals of each group a r e presented.
The tendon of t h e high p r o t e i n e x e r c i s e r a t s exh ib i t ed
t h e most u r i c a c i d c r y s t a l s . F igures 11 and 12 show c r y s t a l s
wi th t h e analyzer and f i l t e r i n p l a c e and wi th t h e ana lyze r ,
f i l t e r and compensator i n p lace , r e spec t ive ly . The t h i r d
micrograph (Fig. 13) was taken wi thout t h e ana lyze r , f i l t e r
o r compensator. This micrograph shows a number of black
d e p o s i t s t o be p resen t a s we l l a s a number of c o r a l coloured
d e p o s i t s which a r e more evident i n F igure 11. It would appear
t h a t t h e black and c o r a l coloused d e p o s i t s a r e both u r i c acid,
t h e c o r a l ones having not been washed wi th t h e s i l v e r n i t r a t e ,
g e l a t i n and hydroquinone mix f o r a long enough per iod . The
presence of o t h e r c r y s t a l l i n e m a t e r i a l is evident from Figures
11 and 1 2 . F igures 1 4 and 15 (p. 35b) a r e of high p r o t e i n
e x e r c i s e r a t tendon from two o t h e r r a t s . They were taken
without t h e p o l a r i z i n g equipment i n p l a c e and show t h e u r i c
a c i d a s b lack depos i t s .
The r e g u l a r d i e t e x e r c i s e t i s s u e (Figs . 16 and 17, p. 36b)
a l s o showed t h e presence .af u r i c a c i d , bu t t o a l e s s e r degree
than t h e high p r o t e i n ex~ : rckse group.. There a r c no c o r a l
coloured d e p o s i t s visiblcl; a l l t h e u r i c ac id i s b lack . Figure
1 6 with t h e ana lyzer anc: p l a t e i n p l a c e shows the presence
of o t h e r c r y s t a l l i n e mat, :r ial . Photomicrograpl-,s of tendon frori
Figure 11. A c h i l l e s tendon o f a h igh p r o t e i n e x e r c i s e r a t under uncompensated p o l a r i z e d l i g h t . S t a i n e d f o r u r i c a c i d . C r y s t a l s a r e wh i t e , u r i c a c i d i s b l ack and c o r a l (X500) .
Figure 12. A c h i l l e s tendon of a h igh p r o t e i n e x e r c i s e r a t under compensated p o l a r i z e d l i g h t . S t a i n e d f o r u r i c a c i d . C r y s t a l s a r e ye l low and b lue . U r i c a c i d i s b l a c k (X500).
Figure 13. A c h i l l e s tendon of h igh prot :? in e x e r c i s e . r a t under t h e l i g h t microscope. S t a i n e d f o r u r i c a c i d . U r i c a c i d i s b lack and c o r a l (X500).
Figure 14. A c h i l l e s tendon of high p r o t e i n e x e r c i s e r a t under t h e l i g h t microscope. S ta ined f o r u r i c a c i d . U r i c a c i d i s b lack (X5OO).
Figure 15. A c h i l l e s tendon of high p r o t e i n e x e r c i s e r a t under t h e l i g h t microscope. S ta ined f o r u r i c a c i d . U r i c a c i d i s black ( X 5 0 0 ) .
FIGURE 16. A c h i l l e s tendon of reguLar d i e t e x e r c i s e r a t under uncompensated p o l a r i z e d l i g h t . S t a i n e d f o r u r i c ac id . C r y s t a l s a r e whi te . U r i c a c i d i s b l ack (X3l) .
FIGURE 17. A c h i l l e s tendon of r e g u l a r d i e t e x e r c i s e r a t under t h e l i g h t microscope. S t a i n e d f o r u r i c a c i d . Ur ic a c i d i s b lack (X31).
two o t h e r r e g u l a r d i e t exe rc i se r a t s a r e presented i n Figures
18 and 19. Uric a c i d i s s t a i n e d black.
Fewer d e p o s i t s of u r i c a c i d w e r e p resen t i n t h e high
p r o t e i n sedentary rats a s observed i n F'igure 21 (P- 39b) I
taken without t h e p o l a r i z i n g equipment. Again, t h e r e i s some
o t h e r c r y s t a l l i n e m a t e r i a l p resen t (p. 39b, Fig. 20) and t h e
u r i c a c i d has been changed from c o r a l t o black. F igures 22
and 2 3 (p. 40b) a r e photomicrographs from two d i f f e r e n t high I(,
p r o t e i n sedentary rats. Uric a c i d c r y s t a l s are domonstrated. I I'
The f i n a l four co lour micrographs (Figs . 2 4 , 25, 26 and
27; pps. 41b and 42b) a r e of tendon from t h r e e c o n t r o l group
rats. This group exh ib i t ed t h e fewest d e p o s i t s of u r i c ac id
and t h e s m a l l e s t amount of o t h e r c r y s t a l l i n e ma te r i a l . I
I '
P o s i t i v e i d e n t i f i c a t i o n of t h e presence of u r i c a c i d , I/
d e p o s i t s i n t h e tendons of a l l t h e r a t s w a s made. Other, non-
i d e n t i f i a b l e c r y s t a l s were a l s o p resen t . The t i s s u e was n o t
u s e f u l f o r t h e s tudy of f i n e s t r u c t u r e changes.
11. Elec t ron Microscopy
Exerc ise has been repor ted t o have l i t z l e e f f e c t on tendon
f in f : s t r u c t u r e . However, a s t h e l i t e r a t u r e i n t h i s f i e l d of
i n v s s t i g a t i o n i s l i m i t e d , it was decided t o examine t h e ~ c h i l l e s
tendon under t h e e l e c t r o n microscope.
Figure 18. A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e r a t under t h e l i g h t microscope. S t a ined f o r u r i c ac id . U r i c a c i d i s b lack (X5OO).
Figure 1 9 . A c h i l l e s tendon of a r e g u l a r d i e t e x e r c i s e r a t under t he l i g h t microscope. s t a i n e d f o r u r i c a c i d . Ur ic a c i d i s b lack (X500).
. I . . . Y:.;a . - .!<
FIGURF, 20. Ach i l l e s tendon of high p r o t e i n sedentary r a t under uncompensated po la r i zed l i g h t . S ta ined f o r u r i c ac id . Crys ta l s a r e white . U r i c a c i d i s black (X125).
FIGURE 21 . A c h i l l e s tendon of high p r o t e i n sedentary r a t under t h e l i g h t m i s c r o s c o p ~ . S ta ined f o r u r i c ac id . Uric a c i d i s black (Xl25).
Figure 22. A c h i l l e s tendon of a high p r o t e i n sedentary ra t under t h e l i g h t microscope. S ta ined f o r u r i c ac id . Uric a c i d i s black (X5OO).
Figure 23. A c h i l l e s tendon of a high p r o t e i n sedentary ra t under t h e 1 ig l . t microscope. Stained f o r u r i c ac id . Uric al:id i s black (X500).
FIGURE 24. Achilles tendon of control rat under uncompensated polarized light. Stained for uric acid. Crystals are white. uric acid is black (Xl25).
FIGURE 25. Achilles tendon of control rat under the light microscope. Stained for uric acid. Uric acid is black (Xl25).
Figure 26. A c h i l l e s tendon of a c o n t r o l r a t under t h e l i g h t microscope. S ta ined f o r u r i c a c i d . U r i c a c i d i s b lack (X5OO).
Figure 27. A c h i l l e s tendon of a c o n t r o l r a t under t h e l i g h t microscope. S ta ined f o r u r i c a c i d . U r i c a c i d i s b lack (X5OO).
The u l t r a t h i n s e c t i o n s of t h e Ach i l l e s tendon were
scanned under t h e e l e c t r o n microscope. ~t was noted t h a t t h e r e
were no d i f f e r e n c e s i n t h e tendon of one group a s compared t o
any o t h e r group. The e x e r c i s e and high p r o t e i n d i e t had l i t t l e
e f f e c t on t h e f i n e s t r u c t u r e of t h e tendon. The col lagen
f i b r e s were s t i l l r e g u l a r l y arranged and f i b r o b l a s t s were seen
between t h e f i b r e bundles. I t i s q u i t e p o s s i b l e t h a t t h e
number of co l lagen bundles may have changed as an e f f e c t of
e x e r c i s e , however no q u a n t i t a t i v e a n a l y s i s was made on t h e s i z e I
I I1
U 1
and number of t h e s e f i b r e s because it was beyond t h e scope ( 1 1
of t h i s study. I
1 1 1 1
Collagen m i c r o f i b r i l s were seen , wi th t h e c h a r a c t e r i s t i c I I
p a t t e r n of c r o s s s t r i a t i o n s of a x i a l p e r i o d i c i t y (Ham, 1969).
They demonstrated l i t t l e u n i t s of s t r u c t u r e along t h e i r l e n g t h s 0 1 1 1
which repeated themselves every 640 A. These u n i t s of s t r u c t u r e
were made up of t ropocol lagen molecules. Hodge and Petruska
(1964) proposed a hypothesis t o e x p l a i n t h e p e r i o d i c i t y . Accor-
ding t o t h e s e authors each m i c r o f i b r i l had l i g h t and dark seg-
ments which repeated themselves, wi th each l i g h t segment accoun-
t i n g f o r s l i g h t l y more than h a l f a per iod and each dark segment
s l i g h t l y less than ha l f a per iod. The t ropocol lagen macromole-
c u l e s were arranged s i d e by s i d e i n a s taggered manner i n such a
way t h a t gaps between t h e i r ends f e l l i n t h e di-rk region .
E lec t ron micrograghs of t h e Ach i l l e s tendon t i s s u e a r e
presented a s F igures 28 t o 32. Figures 28 and 29 a r e longi-
t u d i n a l s e c t i o n s showing t h e col lagen f i b r e s . The wavy
d i s t r i b u t i o n of l i g h t and dark bands may be seen i n Figure 28.
This t i s s u e i s from a high p r o t e i n sedentary r a t . The h igher
magnif icat ion of Figure 29 (p. 46b) enables one t o see t h e
a x i a l p e r i o d i c i t y of t h e col lagen f i b r e s of a high p r o t e i n
e x e r c i s e r a t . The t i s s u e of one high p r o t e i n r a t showed
c r y s t a l s t o be p r e s e n t (Figure 30, p. 47b), however it i s
n o t p o s s i b l e t o i d e n t i f y t h e c r y s t a l s .
1 1 A s e c t i o n of tendon from a r e g u l a r d i e t e x e r c i s e r a t is
I
I presented i n Figure 31 (p. 48b). This micrograph shows normal ,
t r a n s v e r s e and l o n g i t u d i n a l s e c t i o n s of co l lagen f i b r e s . I
Sec t ions from a c o n t r o l r a t showed p o r t i o n s of f i b r o b l a s t s
between t h e col lagen f i b r e s .
The experimental cond i t ions d i d n o t appear t o induce any
conspicuous change i n t h e f i n e s t r u c t u r e of t h e r a t A c h i l l e s
tendon. Therefore, only a few r e p r e s e n t a t i v e micrographs
which show t h e tendon f i n e s t r u c t u r e a r e presented.
FIGURE 28. E lec t ron micrograph of t h e a c h i l l e s tendon of a high p r o t e i n sedentary r a t (X20,900)
I
I I
I FIGURE 29, Elec t ron micrograph of t h e A c h i l l e s tendon of Ill I a high p r o t e i n exe rc i se r a t (X20,900). 1 , I 0 1
Figure 30. The A c h i l l e s tendon of a h igh p r o t e i n e x e r c i s e r a t under t h e e l e c t r o n microscope. C r y s t a l s can be s een as dark i r r e g u l a r shaped bod ie s (X20,900) .
FIGURE 31. E lec t ron micrograph of Ach i l l e s tendon of a r e g u l a r d i e t e x e r c i s e r a t (X7300).
FIGURE 3 2 , Elec t ron micrograph of Ach i l l e s tendon of a c o n t r o l r a t ( X 7 3 O O ) .
CHAPTER 5
DISCUSSIONS
I. Light Microscopy
A. Polarized light microscopy
The identification of uric acid crystals in the
tendon itself was initially attempted using polarized microscopy.
The presence of crystals within the tissue was confirmed,
however, their identification was not possible as it was /
impossible to identify their long axis.
A suitable stain which was specific for uric acid
was chosen (De Galantha, 1935). The slides obtained showed
the presence of both uric acid and other crystals when studied
under uncompensated polarized light.
B. Exercise and uric acid
Although the presence of uric acid was confirmed in
all of the rat tissue it was most prevalent in the high protein
exercise group, and less so in the regular diet exercise group.
Th;-s indicates a relationship between exercise and uric acid
de,)osition in the Achilles tendon of rats. Uric acid crystals
h a ~ e been seen in the te~tdon of a patient who had a long history
oi tendonitis leading to tendon rupture (personal communication
w. th Clement, 1975).
-50-
The presence of e l e v a t e d serum u r i c a c i d l e v e l s i n a t h l e t e s
has been r e p o r t e d by a number of a u t h o r s (Rakestraw, 1921;
Levine -- e t a l . , 1924; Nichols e t a l . , 1951 and Horvath, 1967) . -- Evidence, c o n t r a d i c t o r y t o t h i s f i n d i n g , has a l s o been p re sen ted
(Montoye -- e t a l . , 1967 and ~ o s c o e t a l . , 19701, however, t h e -- e x t e n t oE e x e r t i o n i n t h e l a s t two s t u d i e s d i d n o t c l o s e l y
approximate t h e s u s t a i n e d p h y s i c a l stress of endurance a t h l e t e s .
Many o f t h e s e a t h l e t e s t r a i n e d t w i c e a day w i t h t h e r e s u l t t h a t
t h e r e w a s less chance f o r t h e serum u r i c a c i d , e l e v a t e d du r ing
t h e t r a i n i n g p e r i o d t o r e t u r n t o a lower l e v e l . The t r a i n i n g
s e y s i o n s i n c r e a s e d t h e me tabo l i c demand and c e l l u l a r t u r n o v e r
which i n t u r n i n c r e a s e d t h e amount o f p u r i n e s and p r o t e i n added
t o t h e t o t a l poo l w i t h a r e s u l t a n t i n c r e a s e i n serum u r i c a c i d I1
I
c o n c e n t r a t i o n (Clement -- e t a l . , 1975) .
Clement -- e t a l . (1975) proposed t h a t t h e i n c r e a s e d u r i c
a c i d c o n c e n t r a t i o n s could l e a d t o A c h i l l e s t e n d o n i t i s i n t h e
endurance a t h l e t e . They viewed t e n d o n i t i s as a gou t - l i ke
syndrome, w i t h hyperur icemia a r ~ d a r e s u l t a n t c r y s t a l d e p o s i t i o n
i n t h e tendon i t s e l f . Horvath (1967) and Greenleaf e t a l . --
(1969) had proposed i n v e s t i g a t i o n on t h e r e l a t i o n s h i p of t h e
e l e v a t 3 d serum u r i c a c i d 1 e v e : s seen i n sportsmen t o t h e
occur rence of gout i n l a t e r l i . f e .
:;erum u r i c a c i d l e v e l s w . r e n o t measured i n t h t ra t s of
t h i s ; tudy and it was t h e r e f o r e n o t p o s s i b l e t o c o r - e l a t e t h e
presence of the uric acid crystals in the rat Achilles tendon
with serum uric acid levels.
There were no gross signs of tendonitis in the rats. It
would appear that tendonitis may be precipitated by trauma.
Clement et al. (1975) suggested trauma induced the uric acid -- deposition. However, it appears that in this study the crystals
may already be present in the tendon, with no resultant inflam-
mation. It seems logical therefore to suggest that the inflam-
matory process may be initiated by trauma in the tendon. A /
second possibility is that the trauma may cause a further
precipitation of uric acid crystals and other crystalline mater-
ial into the tendon of the hyperuricemic athlete and then the
inflammatory process is initiated.
The positive identification of the presence of uric acid
crystals in the tendon puts the problem of treatment of choice
in tendonitis closer to resolution. At present, tendonitis
is treated with or21 anti-inflammatory drugs (i.e. phenylbuta-
zone) and corticos?.eroid injections which may be responsible
for weakening the tendon. Clement -- et al. (1975) treated seven
of their subjects who had hyperuricemic tendonitis with the
uricosuric agent probenecid in combination with the gout
specific anti-inf'ammatory drug colchicine. Abatement of
symptoms was seen in fotlr to twenty wee! s and all subjects
returned to full training and competition without relapse
whi l e remaining on t h e drug therapy .
11. Fine S t r u c t u r e o f Tendon
The l a c k of f i n e s t r u c t u r e changes i n t h e A c h i l l e s tendon
due t o e x e r c i s e and h igh p r o t e i n d i e t ag reed w i t h e a r l i e r
s t u d i e s ( Inglemark, 1948 and v i i d i k , 1967) . The tendon was
composed a lmos t e n t i r e l y of dense co l l agenous connec t ive
t i s s u e . The c o l l a g e n f i b r e s are ga the red i n t o c o a r s e bundles
i n an o r d e r l y p a r a l l e l arrangement. The tendon i s s u b j e c t e d /
t o stress and i t s bundles are bound i n t o ' s t r o n g f a s c i c l e s
w i t h l i t t l e i n t e r v e n i n g ground subs t ance and r e l a t i v e l y few
cel ls o r f i b r o b l a s t s . A s m a l l number o f e l a s t i c f i b r e s a r e
p r e s e n t i n tendon.
The tendon seen i n t h e micrographs ( F i g s , 28 and 29; pps.
45b and 46b) i s i n an u n s t r a i n e d state. Abrahams (1967)
s t u d i e d t h e e f f e c t s o f s t r a i n i n g tendon on i t s f i n e s t r u c t u r e .
H e c u t a h o r s e e x t e n s o r tendon i n t o t h r e e p a r t s , two t e n s i l e
t e s t specimens and one c o n t r o l specim.?n. The two t e n s i l e t e s t
specimens were s t r a i n e d t o a predetermined l e v e l . One was
f i x e d i n fo rma l in whi le s t i l l under t h e stress whi le t h e o t h e r
was a l lowed t o r e l a x be fo re f i x a t i o n . The c o n t r o l was a l s o
f i x e d i n fo rn t a l i n . The c o n t r o l tissue, under t h e l i g h t mit ro-
scope , showed c o l l a g e n f i b r e s which have a c l e a r l y de f ined wavy
p a t t e r n . Thc s t r a i n e d f i b r e s , f i x e d whi le under s t r a i n , a t t h e
-54-
3.5 p e r c e n t s t r a i n l e v e l , exh ib i t ed p a r a l l e l rows of co l lagen
f i b r e s wi th t h e f i b r e s o r i e n t e d i n t h e d i r e c t i o n of t h e appl ied
load. The second s t r a i n e d t i s s u e was a l s o s t r a i n e d a t t h e 3.5
per c e n t l e v e l b u t was then allowed t o r e l a x before f i x a t i o n .
This specimen showed some wavy col lagen f i b r e s and some p a r a l l e l
co l lagen f i b r e s which had not re turned t o t h e i r o r i g i n a l
uns t ra ined o r i e n t a t i o n . The author concluded t h a t some perman-
e n t s t r u c t u r a l change may have occurred e i t h e r i n t h e col lagen
network o r i n t h e bonding of t h e mucopolysaccharides i n t h e I
col lagen network due t o pass ing t h e e l a s t i c l i m i t of t h e tendon.
The e x e r c i s e s i t u a t i o n i n t h i s p resen t s tudy d i d not s t r e s s t h e
tendon t o t h e p o i n t of causing a permanent deformation. Also,
f i x a t i o n of t h e t i s s u e occurred, p r imar i ly , wi th t h e tendon
excised .
A number of f a c t o r s have repor ted t o lower t h e s t r e n g t h
of t h e tendon: s y p h i l i s , gonorrhea, t u b e r c u l o s i s , u r a t i c
a r t h r i t i s and pyogenic i n f e c t i o n s (Ljungqvist , 1968). Also
impl ica ted a r e twnours and c y s t s (Chr is t iansen , 1954) and
t e n d o n i t i s ( S i l f v e r s h i o l d , 1 9 4 1 ) . The p resen t study was
designed with t h e admin i s t r a t ion of high p r o t e i n d i e t t o h a l f
of t h e r a t s i n an at tempt t o v- t r i fy Egan's (1972) - f i n d i n g s of
t e n d o n i t i s i n e x e r c i s i n g r a t s Eed on high p ro te in d i ~ t . How-
e v e r , t h e r e were no gross s i g r s of t e n d o n i t i s o r edematous
swe l l ing observed i n any of t1.e r a t s .
Although e x e r c i s e p e r se has n o t been shown t o cause f i n e
s t r u c t u r a l changes i n ~ c h i l l e s t endons , it may i n d i r e c t l y
cause a p a t h o l o g i c a l p roces s which might l e a d t o r u p t u r e of I
t h e A c h i l l e s tendon. Trauma, i n t h e form of d i r e c t and i n d i r e c t
v io l ence , can cause tendon r u p t u r e . The more common cause is I
i n d i r e c t v io l ence . Arner and Lindholn (1959) proposed t h r e e I
main t y p e s of i n d i r e c t v io l ence which occu r s i n t h e A c h i l l e s , I
tendon :
(1) pushing o f f w i t h t h e weight b e a r i n g f o o t f l e x e d i n
a p l a n t a r d i r e c t i o n whi le ex tending t h e l e g a t t h e knee,
( 2 ) v i o l e n t d o r s i f l e x i o n
i n t e r m e d i a t e p o s i t i o n , and
( 3 ) v i o l e n t d o r s i f l e x i o n
d i r e c t i o n .
I t has been no ted t h a t tendon
o f t h e ank le when t h e f o o t i s i n
of t h e f o o t f l e x e d i n a p l a n t a r
Ill
degene ra t ion i s common i n p a t i e n t s I I
w i t h r u p t u r e d tendon (Arner -- e t a l . , 1959) . The h i s t o l o g i c a l
changes observed were a t t r i b u t e d t o impai red blood supply and
t h i s was suppor ted by angiographic s t u d i e s which showed A c h i l l e s
tendon r u p t u r e t o occur f r e q u e n t l y i n t h e most a v a s c u l a r p a r t i o n
of t h e tendon (Lacjergren and Lindholm, 1959) . A l l o f Arner
and Lindholm's ( 1 3 5 9 ) A c h i l l e s tendon r b p t u r e s t u d i e s were done
on former a th l e t e ; . They concluded t h a k t h e i n c r e a s e d stress
impl ied by s t r e n ~ ~ u s e x e r c i s e may r e s u l t i n degene ra t ive
changes i n t h e t i ndon t i s s u e w i t h a d e c r e a s e i n it re s i e ; t ance .
-56-
Ricklin (1962) maintained that the exhaustion of physical
training played an important role in causing structural changes
in the ~chilles tendon leading to a greater risk of rupture,
This might be associated with the deposition of uric acid
crystals in the tendon of endurance athletes. One case of
ruptured quadriceps tendons as a result of gouty affectation
of the tendon has been reported (Levy et al., 1971). --
The ruptured tendons studied by Ljungqvist (1968) showed
histological degeneration in the form,of partially devitalized I
I
tendon tissues with obliterated fibrin structure and some I
I fraying of the collagen bundles. There was loose granulation !
tissue, rich in collagen and of a wavy structure unlike normal I tendon collagen attached to the devitalized tissue.
Although an attempt was made to induce Achilles tendonitis
in rats through the administration of high protein diet, no
inflamed or swollen tendons were observed. The stre.ss on the
tendon was induced 12y exercising on the motorized treadmill,
however, no elevatian of the treadmill was used. The stress of
running may not be sufficient to cause strain changes or
degenerative changes in the rat Achilles tendon.
CHAPTER 6
CONCLUSIONS
u r i c a c i d c r y s t a l s were found t o be p r e s e n t i n t h e tendon
of a l l t h e r a t s s t u d i e d i n t h i s exper iment . These c r y s t a l s
can be p o s i t i v e l y i d e n t i f i e d by s e l e c t i v e s t a i n i n g . The exer -
c i s e c o n d i t i o n l e d t o a g r e a t e r u r i c a c i d d e p o s i t i o n i n t h e
tendon when compared t o t h e non e x e r c i s e groups. High p r o t e i n
d i e t a l s o i n c r e a s e d t h e d e p o s i t i o n of u r i c a c i d c r y s t a l s over \
r e g u l a r d i e t . These f i n d i n g s have impor t an t i m p l i c a t i o n s i n
t h e s tudy o f t h e hyperur icemic t e n d o n i t i s r e p o r t e d by Clement
e t a l . (1975) i n endurance runners . They p o s t u l a t e d t h a t trauma --
p r e c i p i t a t e d t h e d e p o s i t i o n of u r i c a c i d c r y s t a l s i n t h e tendon
w i t h t h e r e s u l t a n t inflammatory r e a c t i o n . From t h e f i n d i n g s of
t h e p r e s e n t s tudy it would appear t h a t t h e u r i c a c i d c r y s t a l s
may be p r e s e n t i n a l l tendon and t o a g r e a t e r degree i n t h e
e x e r c i s e d tendon, l e a d i n g t h e a u t h o r t o p o s t u l a t e t h a t trauma
causes an inflammatory r e a c t i o r and p o s s i b l y t h e p r e c i p i t a t i o n
o f f u r t h e r u r i c a c i d c r y s t a l s . I t is recommended t h a t s t u d i e s
be c a r r i e d o u t on normal tendon from seden ta ry humans and on
rup tu rcd tendon from enduranctx a t h l e t e s t o conf i rm t h e above
f i n d i n p f o r humans.
I i n e s t r u c t u r e s t u d i e s sllowed e x e r c i s e t o have l i t t l e
ef fect on t h e tendon. Some c z y s t a l s were observed under t h e
e l e c t r o n microscope b u t it was n o t p o s s i b l e t o i d e n t i f y them.
These c r y s t a l s w e r e p r e s e n t i n t h e A c h i l l e s tendon of one o f
t h e h i g h . p r o t e i n e x e r c i s e rats.
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!
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