- Email: [email protected]
Ornithine decarboxylase: A biochemical marker of repair in damaged tissue
Life Sciences, Vol. 48, pp. 1501-1510 Printed in the U.S.A.
Pergamon Press
ORNITHINE DECARBOXYLASE: A B I O C H E M I C A L M A R K E R OF R E P A I R IN DAMAGED T I S S U E James H. G i l l e t t e I, John L.A. M i t c h e l l 2 IDepartment of A l l i e d H e a l t h P r o f e s s i o n s and 2Department of B i o l o g i c a l Sciences, N o r t h e r n Illinois U n i v e r s i t y DeKalb, Illinois 60115 (Received in final form February Ii, 1991) Summary The p u r p o s e of this study was to: (a) d e t e r m i n e the temporal p a t t e r n of e x p r e s s i o n of ornithine d e c a r b o x y l a s e (ODC), an established marker of cells engaged in proliferation and differentiation, during repair of t r a u m a t i z e d skeletal muscle, and (b) e v a l u a t e ODC as a b i o c h e m i c a l m a r k e r for indexing the extent and rate of repair of t r a u m a t i z e d skeletal m u s c l e in r e s p o n s e to t h e r a p e u t i c agents. Adult female W i s t a r rats w e i g h i n g 240 to 280 grams were a n e s t h e t i z e d and injected w i t h i00 ul of 2% lidocaine into the right or left a n t e r i o r t i b i a l i s m u s c l e to induce a localized injury. The animals were t r e a t e d 1 hour p o s t - i n j e c t i o n and every 12 hours for five m i n u t e s up to the time of sacrifice using u l t r a s o n i c i r r a d i a t i o n at 1.5 w a t t s / c m 2, sham-irradiation, or no treatment. An analysis of v a r i a n c e for r e p e a t e d m e a s u r e s and a Tukey's post hoc test were used to d e t e r m i n e the s i g n i f i c a n c e of t r e a t m e n t effects. Animals i r r a d i a t e d with u l t r a s o u n d d e m o n s t r a t e d an a c c e l e r a t e d p a t t e r n of change in ODC a c t i v i t y at 24 hours (P < 0.001), 30 hours (P < 0.003), and at 48 hours (P < 0.002) c o m p a r e d to control and sham-irradiated animals at these time intervals. I r r a d i a t e d animals also d e m o n s t r a t e d less edema at 48 hours compared to s h a m - i r r a d i a t e d and control animals (P < 0.003). These findings suggest that ODC is a useful b i o c h e m i c a l m a r k e r for d e t e r m i n i n g the extent and rate of tissue repair in t r a u m a t i z e d skeletal muscle, and it p r o v i d e s a temporal and q u a n t i f i a b l e p a r a m e t e r for e v a l u a t i n g the efficacy of t h e r a p e u t i c agents used to treat d a m a g e d skeletal muscle. T h e r a p e u t i c u l t r a s o u n d has been used for nearly five decades to restore function and p r o m o t e h e a l i n g of soft t i s s u e injuries. Clinical o b s e r v a t i o n s have s u p p o r t e d the use of u l t r a s o u n d for a wide v a r i e t y of c o n d i t i o n s including pain (i), p r e s s u r e sores (2), b u r s i t i s (3), and t r a u m a t i z e d skeletal m u s c l e (4). In spite of these observations, r e l a t i v e l y little research has been r e p o r t e d r e g a r d i n g t h e r a p e u t i c u l t r a s o u n d or other c o m m o n l y used agents, and e v i d e n c e of e f f i c a c y has been supported p r i m a r i l y by s u b j e c t i v e p a r a m e t e r s (5,6). In order to e s t a b l i s h the e f f e c t i v e n e s s of a s p e c i f i c t r e a t m e n t it is imperative that there be an o b j e c t i v e l y m e a s u r a b l e p a r a m e t e r that can be e m p l o y e d to d e t e r m i n e the e f f i c a c y 0024-3205/91 $3.C0 +.00 Copyright (c) 1991Pergmon Press plc
1502
Ornlthlne Decarboxylase During Repair
of the t h e r a p e u t i c
agent
Vol. 48, No. 16, 1991
(7).
Skeletal muscle can be damaged by a wide v a r i e t y of insults including the application of local anesthetics (8). Although b u p i v a c a i n e (Marcaine) is commonly used in the research laboratory to p r o d u c e muscle degeneration, Xylocaine (lidocaine) also has been shown to produce rapid necrosis of muscle fibers (9). The advantages of using local anesthetics such as lidocaine to e x p e r i m e n t a l l y induce injury are that the extent of damage to the muscle in each animal can be carefully controlled and the procedure is painless. R e g a r d l e s s of the method used to damage skeletal muscle, the muscle will undergo a repair process that appears to follow a common p a t h w a y (i0). Initially there is a period of muscle fiber d e g e n e r a t i o n c h a r a c t e r i z e d by the disruption and dissolution of sarcomeric units, mitochondria, sarcoplasmic reticula, and myonuclei. Satellite cells (myogenic stem cells), present beneath the basal lamina, survive the initial traumatic event and ensuing deleterious environment. Following removal of cellular components from the damaged muscle fiber, satellite cells are activated, giving rise to a p o p u l a t i o n of m y o b l a s t i c cells. Myoblasts, in turn, will fuse to form m u l t i n u c l e a t e d myotubes with centrally located nuclei, which then d i f f e r e n t i a t e into mature muscle fibers with p e r i p h e r a l l y located nuclei (8). Muscle damage and repair are often c h a r a c t e r i z e d by levels of Creatine P h o s p h o k i n a s e (CPK), Lactate Dehydrogenase (LDH), and other acute phase reactants (11,12). However, these analytes represent the acute inflammatory and degenerative stages of wound healing. Conversely, L-ornithine decarboxylase (EC 4.1.1.17, ODC) is an enzyme that demonstrates peak activity in all tissues stimulated to grow, proliferate and d i f f e r e n t i a t e (13-18). This enzyme catalyzes the initial, rate limiting step in the biosynthesis of the polyamines putrescine, spermidine, and spermine, which are essential for cell growth and differentiation. ODC activity has been recognized as a sensitive m a r k e r of protein synthesis, proliferation, and differentiation, d e m o n s t r a t i n g i0i00 fold elevations in activity following tissue stimulation (1921). Repair of damaged skeletal muscle is also accompanied by sharply elevated levels of ODC activity. Sadeh et al. (22) have shown that skeletal muscle injected with Marcaine results in muscle fiber necrosis followed by repair of the damaged tissue. More importantly, they used representative histological sections of muscle tissue as a function of time following injection to d e m o n s t r a t e that ODC levels are elevated during the period of repair and that ODC activity returns to control levels as the muscle returns to normal morphology. This suggests that levels of ODC activity are indicative of muscle cell p r o l i f e r a t i o n and d i f f e r e n t i a t i o n associated with repair, and not increased synthesis of excessive or unwanted connective tissue. This would infer that ODC activity may provide a useful biochemical m a r k e r for the time and extent of tissue repair in damaged skeletal muscle. In the present study we have determined the patterns of expression of ODC activity in damaged skeletal muscle, in the presence or absence of u l t r a s o n i c irradiation, in order to test the value of the enzyme as an objective index of v a r i a t i o n in the rate of tissue repair.
Vol. 48, No. 16, 1991
Ornlthine Decarboxylase During Repair
1503
Methods
Subjeats. A d u l t f e m a l e r a t s of t h e W i s t a r s t r a i n w e i g h i n g b e t w e e n 240 a n d 280 g r a m s w e r e u s e d in t h i s study. All animals w e r e h o u s e d o n e p e r s t a n d a r d cage, fed a s t a n d a r d p e l l e t d i e t a n d water, ab l i b i t u m , a n d k e p t in t e m p e r a t u r e - c o n t r o l l e d quarters (23°C) w i t h a 1 2 - h l i g h t - d a r k c y c l e (6:00 a.m. a n d 6:00 p.m.). The c a r e a n d u s e of t h e a n i m a l s w e r e in a c c o r d a n c e w i t h t h e A n i m a l s W e l f a r e Act. Instrumentation. Ultrasonic treatment was administered using a D y n a w a v e M o d e l 801 w i t h a o n e c e n t i m e t e r s o u n d - h e a d . The machine p r o v i d e s a f r e q u e n c y of 1.0 m e g a h e r t z . T h e m a x i m u m i n t e n s i t y is 2.0 w a t t s / c m 2 a n d t h e u l t r a s o u n d can be delivered pulsed or continuous. A UPM-DT-10 ultrasound power meter was used to c a l i b r a t e t h e u l t r a s o u n d m a c h i n e b e f o r e a n d d u r i n g t h e study. M u s c l e Injury. O n t h e d a y of a n y g i v e n e x p e r i m e n t t h e e n t i r e c a u d a l 1/3 of e a c h a n i m a l w a s c a r e f u l l y s h a v e d w i t h an e l e c t r i c hair clippers. Then each animal was randomly assigned t o an irradiated, sham-irradiated, or nonirradiated group. Following a n e s t h e s i a w i t h an i.p. i n j e c t i o n of I00 ul of k e t a m i n e a n d i00 ul of x y l a z i n e e a c h a n i m a l w a s r a n d o m l y i n j e c t e d w i t h i00 ul of 2% l i d o c a i n e i n t o e i t h e r t h e r i g h t or l e f t a n t e r i o r t i b i a l i s m u s c l e . 5/8 - i n c h 2 5 - g a u g e n e e d l e s w e r e m a r k e d w i t h an i n d e l i b l e m a r k e r at m e a s u r e d d i s t a n c e s of 2, 4, 6, 8, a n d i0 m m f r o m t h e t i p of t h e needle. The anterior tibialis muscle to be injected was gently squeezed from the medial and lateral margins. At a measured d i s t a n c e of 2 cm f r o m t h e l a t e r a l m a l l e o l u s t h e n e e d l e w a s i n s e r t e d l o n g i t u d i n a l l y a l o n g t h e l e n g t h of t h e m u s c l e to a d e p t h of I0 mm. As t h e n e e d l e w a s r e t r a c t e d a p p r o x i m a t e l y 25 ul of 2% l i d o c a i n e w a s d i s p e n s e d e v e r y 2 m m u n t i l t h e e n t i r e i00 ul h a d b e e n d i s t r i b u t e d . All i n j e c t i o n s w e r e a d m i n i s t e r e d b e t w e e n 2:00 a n d 4:00 p.m. Treatment. T r e a t m e n t s for t h e i r r a d i a t e d a n d s h a m - i r r a d i a t e d groups began approximately 1 hour post-injection of 2% l i d o c a i n e . P r i o r to e a c h t r e a t m e n t t h e a r e a o v e r t h e a n t e r i o r t i b i a l i s m u s c l e w a s w a s h e d a n d d r i e d to r e m o v e a n y d i r t or oil. For irradiated and sham-irradiated a n i m a l s u l t r a s o n i c c o n d u c t i n g gel w a s a p p l i e d to a t h i c k n e s s of a p p r o x i m a t e l y 1 m m w i t h a t o n g u e d e p r e s s o r . All a n i m a l s in t h e i r r a d i a t e d g r o u p r e c e i v e d c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for f i v e m i n u t e s . T h e s o u n d - h e a d w a s m o v e d in a circular, r h y t h m i c p a t t e r n o v e r t h e e n t i r e m u s c l e b e l l y of t h e anterior tibialis muscle. T h e s a m e p r o t o c o l w a s f o l l o w e d for all a n i m a l s in t h e s h a m - i r r a d i a t e d group except that the machine was t u r n e d off. A l l a n i m a l s in t h e i r r a d i a t e d and sham-irradiated g r o u p s w e r e t r e a t e d t w i c e p e r day. Treatments were administered between 4 : 0 0 p.m. a n d 6:00 p.m. a n d 8 : 0 0 a.m. a n d i 0 : 0 0 a.m. F o l l o w i n g i n j e c t i o n w i t h l i d o c a i n e , a n i m a l s in t h e n o n i r r a d i a t e d g r o u p s w e r e n o t h a n d l e d u n t i l t h e t i m e of s a c r i f i c e . Muscle Tissue S a m p l i n g . Animals from the irradiated, shamirradiated, and nonirradiated g r o u p s w e r e s a c r i f i c e d at 24 h o u r s ( e x p e r i m e n t 1), 30 h o u r s ( e x p e r i m e n t 2), o r 48 h o u r s ( e x p e r i m e n t 3) p o s t - i n j e c t i o n of 2% l i d o c a i n e . The injected and uninjected anterior tibialis muscles from each animal were rapidly excised, w e i g h e d , a n d p l a c e d in 5 ml of i c e - c o l d i s o l a t i o n b u f f e r (0.02 M E P P S p H 7.2, 0.5 m M EDTA, 50 u M p y r i d o x a l - 5 - p h o s p h a t e , 5 mM dithiothreitol). E a c h m u s c l e w a s h o m o g e n i z e d for 15 s e c o n d s w i t h
1504
Ornithine Decarboxylase During Repair
Vol. 48, No. 16, 1991
a B r i n k m a n P o l y t r o n h o m o g e n i z e r set at a speed of 5 followed by s o n i c a t i o n for 45 seconds using a Branson sonifier w i t h a microtip. H o m o g e n a t e s were c e n t r i f u g e d at 30,000 g for 15 m i n u t e s at 4°C. Five 175 ul r e p l i c a t e s from the r e s p e c t i v e s u p e r n a t a n t s were each added to 25 ul of isolation b u f f e r c o n t a i n i n g 0.08 uCi L-(I-14C) ornithine at a final concentration of 0.2 mM L-ornithine. R e a c t i o n s were stopped by the a d d i t i o n of 0.5 ml of 2 M citric acid (23). The r e l e a s e d Z4CO2 was c a p t u r e d by 1 cm squares of No. 3 W h a t m a n filter paper c o n t a i n i n g 50 ul of 30% p o t a s s i u m h y d r o x i d e located in center wells suspended from the rubber caps of the r e a c t i o n vials. The r a d i o a c t i v i t y was d e t e r m i n e d by s c i n t i l l a t i o n c o u n t i n g at an e f f i c i e n c y of 95%. Protein content was d e t e r m i n e d by the m e t h o d of B r a d f o r d (24).
Statistical Analysis. D i f f e r e n c e s in levels of ODC activity and in gross w e i g h t of a n t e r i o r tibialis m u s c l e s among the irradiated, s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups were tested by a o n e - w a y analysis of v a r i a n c e (ANOVA) for repeated measures. D i f f e r e n c e s at a level of 0.05 were c o n s i d e r e d significant. The sources of d i f f e r e n c e s identified by the one-way A N O V A were e x a m i n e d using a Tukey's m u l t i p l e - c o m p a r i s o n post hoc test. Results Levels of ODC a c t i v i t y found in a n t e r i o r tibialis muscles from animals r e c e i v i n g only an i.p. injection c o n t a i n i n g i00 ul of 2% lidocaine, i.p. injections c o n t a i n i n g i00 ul of K e t a m i n e and i00 ul of Xylazine, or no injection all d e m o n s t r a t e d levels of ODC a c t i v i t y less than 1 p m o l / h r / m g protein. However, repair of a n t e r i o r t i b i a l i s muscles d a m a g e d by an i.m. injection c o n t a i n i n g i00 ul of 2% lidocaine p r o d u c e d a c h a r a c t e r i s t i c p a t t e r n of change in ODC activity. As shown in Fig. 1 activity increased a p p r o x i m a t e l y 100-fold to peak levels at 30 hours post-injection, with a 50% d e c l i n e from peak a c t i v i t y by 48 hours post-injection. U n i n j e c t e d c o n t r a l a t e r a l anterior tibialis muscles of each animal in Fig. 1 (the u n i n j e c t e d side) also d e m o n s t r a t e d an increase in ODC activity, however, this was e x t r e m e l y small relative to the injected side. To a s c e r t a i n w h e t h e r m a s s a g i n g by the s o u n d h e a d or the general h a n d l i n g of the animals during t r e a t m e n t could, by themselves, affect the p a t t e r n of ODC activity during repair, some animals were s h a m - i r r a d i a t e d following injection with i00 ul of 2% lidocaine. There was no d i f f e r e n c e noted b e t w e e n the n o n i r r a d i a t e d and shami r r a d i a t e d groups in either the injected or u n i n j e c t e d anterior t i b i a l i s m u s c l e s (Fig. i). A n i m a l s injured by injection with I00 ul of 2% lidocaine and t r e a t e d w i t h c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five minutes d e m o n s t r a t e d an a c c e l e r a t e d p a t t e r n of change in ODC a c t i v i t y (Fig. 2). At 24 hours post-injection, ODC a c t i v i t y for irradiated animals reached peak levels and was nearly d o u b l e that of nonirradiated and sham-irradiated animals at the same time interval; P < 0.001. At 30 hours post-injection, animals treated w i t h u l t r a s o u n d d e m o n s t r a t e d a m a r k e d return toward b a c k g r o u n d levels at a time when n o n i r r a d i a t e d and s h a m - i r r a d i a t e d animals were a p p r o a c h i n g peak levels of ODC activity; P < 0.003. Even at 48 hours post-injection, ODC activity for irradiated animals c o n t i n u e d to d e m o n s t r a t e a return toward b a c k g r o u n d levels more
Vol. 48, No. 16, 1 9 9 1
Ornithine Decarboxylase During Repair
I00 4~
~
75
~
50
!
~,
0 ......
0
i ~-
t0
20
7--
I --QI
30
40
50
I
I
I
60
70
80
HOURS FIG.
1
ODC a c t i v i t y in m u s c l e s that have been injected with 2% lidocaine (~,[3) and in the u n i n j e c t e d c o n t r a l a t e r a l muscles (O,~)" F o l l o w i n g injection the d a m a g e d m u s c l e was e i t h e r u n t r e a t e d ( [ ~ , . ) or sham t r e a t e d ( Q , O ) by m a s s a g i n g w i t h the u l t r a s o u n d m a c h i n e turned off.
i1~
iO0 ,o Q.
~
.~".
75 I
. ~,
"..
I
0
/
50
-
// I
25
I
."
~-
/
U
o
dz-~" 0
J
I
I
I
I
I
I
iO
20
30
40
50
60
70
I 80
HOURS FIG.
2
O r n i t h i n e d e c a r b o x y l a s e a c t i v i t y of a n t e r i o r t i b i a l i s m u s c l e s injected with 2% lidocaine and r e c e i v i n g sham u l t r a s o u n d (machine turned off) for five m i n u t e s every t w e l v e hours (~), or c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five m i n u t e s every twelve hours ( A ) . R e s u l t s are means +/- S.E.M of 5 r e p l i c a t e samples. I r r a d i a t e d groups differ from s h a m - i r r a d i a t e d groups: P < 0.001 at 24 h; P < 0.003 at 30 h; P < 0.002 at 48 h.
1505
1506
Ornithine Decarboxylase During Repair
Vol. 48, No. 16, 1991
rapid than the n o n i r r a d i a t e d or s h a m - i r r a d i a t e d animals; P < 0.002. In contrast, the p a t t e r n of ODC a c t i v i t y in u n d a m a g e d m u s c l e was not a l t e r e d p e r c e p t i b l y by either direct u l t r a s o u n d i r r a d i a t i o n t r e a t m e n t or i r r a d i a t i o n of the d a m a g e d c o n t r a l a t e r a l m u s c l e (data not shown). To s t r e n g t h e n the p r e m i s e that ODC a c t i v i t y p r o v i d e s a b i o c h e m i c a l m a r k e r for skeletal m u s c l e u n d e r g o i n g repair, ODC a c t i v i t y was a s s a y e d at 24 h p o s t - i n j e c t i o n in r e s p o n s e to three d i f f e r e n t d o s a g e s of irradiation. As seen in Fig. 3 damaged skeletal m u s c l e irradiated at an intensity of 1.5 w a t t s / c m 2 d e m o n s t r a t e d the m o s t a c c e l e r a t e d p a t t e r n of ODC e x p r e s s i o n of the three d i f f e r e n t intensities of irradiation t e s t e d c o m p a r e d to noni r r a d i a t e d or s h a m - i r r a d i a t e d groups; P < .001. Damaged m u s c l e i r r a d i a t e d at 0.5 and 1.0 w a t t s / c m 2 was s u f f i c i e n t to elicit an a p p a r e n t a c c e l e r a t i o n in the p a t t e r n of ODC e x p r e s s i o n a l t h o u g h it 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 (.50 > P > .25 & .20 > P > .i0 respectively). C l i n i c a l l y h i g h e r intensities of i r r a d i a t i o n are not g e n e r a l l y used for t h e r a p e u t i c p u r p o s e s b e c a u s e of p o t e n t i a l l y harmful side effects such as c a v i t a t i o n (7). too
75
~
50
8 ~
25
o
FIG.
3
ODC a c t i v i t y in m u s c l e s that have been injected w i t h 2% l i d o c a i n e and r e c e i v e d no irradiation, sham-irradiation, i r r a d i a t i o n at 0.5, i. 0, or i. 5 w a t t s / c m 2. Irradiated g r o u p s d i f f e r from n o n - i r r a d i a t e d and s h a m - i r r a d i a t e d groups: P < 0.40 at 0.5; P < 0.20 at 1.0; P < 0.001 at 1.5. Clinical a s s e s s m e n t of soft tissue injuries often include m o n i t o r i n g of edema as a means of indexing p r o g r e s s toward recovery and it has been s u g g e s t e d that c o n t r o l l i n g edema d u r i n g early stages of i n f l a m m a t i o n may q u a l i t a t i v e l y affect later stages of repair. The course of edema was followed in all animals included in this i n v e s t i g a t i o n to assist in a s s e s s i n g the relative v a l u e of edema r e s o l u t i o n and the p a t t e r n of ODC e x p r e s s i o n as indices of the r e p a i r process. C o m p a r i s o n of gross w e i g h t s of a n t e r i o r tibialis m u s c l e s among normal uninjected, injected/irradiated,
Vol. 48, No. 16, 1991
Ornlthlne Decarboxylase During Repair
1507
injected/sham-irradiated, and injected/nonirradiated animals d e m o n s t r a t e d p r o n o u n c e d contrasts. Uninjected anterior tibialis m u s c l e s e x c i s e d from animals of e q u i v a l e n t size used in this study w e i g h e d a p p r o x i m a t e l y 600 milligrams. At 24 and 30 hours postinjection anterior tibialis muscles from irradiated, shamirradiated, and n o n i r r a d i a t e d animals d e m o n s t r a t e d a s i g n i f i c a n t increase in w e i g h t c o m p a r e d to m u s c l e s of u n i n j u r e d animals P < 0.001; (Fig. 3). However, no s i g n i f i c a n t w e i g h t d i f f e r e n c e s were found among the irradiated, sham-irradiated, or n o n i r r a d i a t e d groups at t h e s e time intervals (P > 0.25). At 48 hours posti n j e c t i o n a s i g n i f i c a n t w e i g h t increase r e m a i n e d in a n t e r i o r t i b i a l i s m u s c l e s from the s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups c o m p a r e d to m u s c l e s from irradiated or u n i n j u r e d animals (P < 0.001). Furthermore, w e i g h t s of m u s c l e s taken from i r r a d i a t e d animals had r e t u r n e d to levels observed in m u s c l e s of u n i n j u r e d animals (0.50 > P > 0.20).
1000
o~
CZ~ltn]ected ~x'mtn)ectsd,
Imtn]ected,
treated
shamtreated
control [ m u n i n J e c t e d control
-
750
I.--r LO W X
500
W ._1 r.J
¢n .,=.
250
24 h
30 h
48 h
TIME AFTER INJECTION
FIG.
4
Therapeutic ultrasound hastens resolution of edema. A n t e r i o r t i b i a l i s m u s c l e s were injected w i t h i00 ul of 2% lidocaine and received no irradiation, sham irradiation, or c o n t i n u o u s i r r a d i a t i o n at 1.5 watts/cm'. C o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 d e m o n s t r a t e d a s i g n i f i c a n t d e c r e a s e in w e i g h t of a n t e r i o r t i b i a l i s m u s c l e s c o m p a r e d to n o n - i r r a d i a t e d and s h a m - i r r a d i a t e d muscles. Injected, s h a m - t r e a t e d & injected, control c o m p a r e d to uninjected, control P < 0.001 at 24, 30, & 48 h; Injected, t r e a t e d c o m p a r e d to uninjected, control P < 0.001 at 24 & 30 h; P > 0.20 at 48 h. A n a l y s i s of body w e i g h t d e m o n s t r a t e d no s i g n i f i c a n t change from the time of i n j e c t i o n to the time of s a c r i f i c e for individual animals and no s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d among the groups of a n i m a l s (data not shown).
1508
Ornlthine Decarboxylase During Repair
Vol. 48, No. 16, 1991
Discussion Because polyamines are essential for p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of all cells the c h r o n o l o g i c a l e x p r e s s i o n of ODC, the rate l i m i t i n g enzyme in the b i o s y n t h e s i s of the polyamines, p r o v i d e s a good index of the respective stages of repair of d a m a g e d skeletal muscle. Normal adult skeletal m u s c l e in a quiescent, nonp r o l i f e r a t i n g state has very low levels of c o n s t i t u t i v e ODC. During repair, when there is extensive growth and p r o l i f e r a t i o n of myoblasts, ODC a c t i v i t y is rapidly and m a r k e d l y elevated, followed by a return to b a c k g r o u n d levels as the m y o b l a s t s fuse and d i f f e r e n t i a t e into m a t u r e m u s c l e fibers. R e p a i r of rat anterior t i b i a l i s m u s c l e s t r a u m a t i z e d w i t h 2% lidocaine is m a r k e d by a peak in ODC a c t i v i t y at 30 hours post-injection, followed by an a p p r o x i m a t e l y 50% r e d u c t i o n from peak levels at 48 hours postinjection. This p a t t e r n of ODC activity d u r i n g repair of skeletal m u s c l e c o m p a r e s c l o s e l y to that shown by others (22, 25). A n i m a l s irradiated with c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five m i n u t e s not only d e m o n s t r a t e d an a c c e l e r a t e d rise in ODC activity, w i t h p e a k levels occurring at 24 hours p o s t - i n j e c t i o n rather than at 30 hours post-injection, but also an a c c e l e r a t e d return t o w a r d b a c k g r o u n d levels at 30 and 48 hours p o s t - i n j e c t i o n c o m p a r e d to the n o n i r r a d i a t e d and s h a m - i r r a d i a t e d groups at these time intervals. This a c c e l e r a t i o n in the ODC a c t i v i t y v a r i a t i o n suggests that the u l t r a s o u n d t r e a t m e n t has p e r m i t t e d a more rapid onset and t e r m i n a t i o n of the p r o l i f e r a t i o n phase of the muscle repair. This c o n c l u s i o n is c o n s i s t e n t with the results of other i n v e s t i g a t o r s w h i c h suggest that u l t r a s o u n d may be e f f e c t i v e in a c c e l e r a t i n g the repair of damaged skeletal muscle. S t r a t t o n et al. (26) found that u l t r a s o u n d at an intensity of 1.5 w a t t s / c m 2 was h i g h l y s i g n i f i c a n t compared to 0.5 w a t t s / c m 2 for improving repair of h e m a t o m a s in skeletal muscle. Hogan et al. (27) d e m o n s t r a t e d that i r r a d i a t i o n of skeletal m u s c l e with u l t r a s o u n d improves blood flow in ischemic tissue and can enhance p e r f u s i o n in v a s c u l a r l y impaired muscle compared to that o b s e r v e d in noni r r a d i a t e d muscle. S h a m - i r r a d i a t e d animals reveal nearly identical levels of ODC a c t i v i t y c o m p a r e d to n o n i r r a d i a t e d animals at each time point assayed. This w o u l d suggest that the h a n d l i n g of the animals and m a s s a g i n g effect of the soundhead for each sham t r e a t m e n t do not influence levels of ODC activity. Similarly, lack of a m a s s a g i n g effect from the s o u n d h e a d also was observed in a study involving a n o t h e r tissue (7). Interestingly, the u n i n j e c t e d / u n t r e a t e d c o n t r a l a t e r a l limbs of irradiated, sham irradiated, and nonirradiated animals d e m o n s t r a t e d an increase in ODC activity, a l t h o u g h r e l a t i v e l y small (0.50 > P > 0.20). P r e s u m a b l y this may result from a modest h y p e r t r o p h y of the u n i n j e c t e d limb m u s c u l a t u r e r e s p o n d i n g to the added d e m a n d s imposed on it s e c o n d a r y to g u a r d i n g of the injected limb. This p r o v i d e s evidence for the relative importance of ODC d u r i n g c o m p e n s a t o r y growth as well as m u s c l e repair. Edema is an early and frequent clinical m a n i f e s t a t i o n of injury that may p r o l o n g the inflammatory process if left unchecked. Previous studies e v a l u a t i n g the effects of u l t r a s o u n d on edema have yielded contradictory results. Goddard et al. (28) found
Vol. 48, No. 16, 1991
0rnithine Decarboxylase During Repair
1509
u l t r a s o u n d to b e ~ t o t a l l y ineffective in s u p p r e s s i n g or r e d u c i n g i n f l a m m a t o r y edema. Conversely, Fyfe and Chahl (29) have shown that a l t h o u g h u l t r a s o u n d may increase edema d u r i n g the first 24 hours, by 48 hours the edema is s i g n i f i c a n t l y reduced. Findings p r e s e n t e d here show that the w e i g h t s of a n t e r i o r t i b i a l i s m u s c l e s t r e a t e d w i t h u l t r a s o u n d are s i g n i f i c a n t l y less than a n t e r i o r t i b i a l i s m u s c l e s from s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups at 48 hours, even though the groups are not s i g n i f i c a n t l y d i f f e r e n t at 24 and 30 hours. Moreover, the w e i g h t s of the irradiated anterior tibialis muscles at 48 hours return to levels c h a r a c t e r i s t i c a l l y seen in u n i n j e c t e d muscles. R e s o l u t i o n of edema is an important o b j e c t i v e in the t r e a t m e n t of soft tissue injuries and is often used as an index of repair. However, findings from this study d e m o n s t r a t e d peak levels of ODC a c t i v i t y in t r a u m a t i z e d skeletal m u s c l e that is m a r k e d l y edematous. These results suggest that ODC a c t i v i t y p r o v i d e s a more s e n s i t i v e and s p e c i f i c m a r k e r of repair d u r i n g the early stages of w o u n d healing. Acknowledgements This w o r k s was s u p p o r t e d in part by NIH R e s e a r c h Grant GM 33841. The authors w o u l d like to g r a t e f u l l y a c k n o w l e d g e Mary Jane Harris for her t e c h n i c a l assistance. References I. 2. 3. 4. 5. 6.
E. MUNTING, Physiotherapy 6__44 (6) 180-181 (1978) T. MCDIARMID, P.N. BURNS, G.T. LEWITH, D. M A C H I N 71 (2) 6670 (1985) D.S. D O W N I N G and A. WEINSTEIN, Phys Ther 66194-199 (1986) E.M. OAKLEY, Br J Cancer 45 (Suppl V) 233-237 (1982) C.J. PARTRIDGE, Physiotherapy 7_/3 (4) 166-168 (1987) G.H. CLARKE and L. STENNER, Physiotherapy 62 (6) 185-190
(1976) 7. 8. 9.
M. DYSON and J. SUCKLING, Physiotherapy 6__44 (4) 105-108 (1978) B.M. C A R L S O N and J.A. FAULKNER, Med Sci Sports Exer 1_55 187198 (1983) A.H. FOSTER and B.M. CARLSON, Anesth Analg 5_99 275-282
(1980) i0. II. 12. 13. 14.
D. ALBROOKE, M u s c l e Nerve 4 234-254 (1981) F.S. A P P L E and M. RHODES, J Appl Physiol 65 (6) 2598-2600 (1988) C.M. COLLEY, A. FLECK, B.R. MULLER, and M.A. MYERS J Clin Pathol 3-6 203-207 (1983) D.H. R U S S E L L and S.H. SYNDER, Proc Natl Acad Sci U S A 60 1420-1427 (1968) C.A. M A N E N and D.H. RUSSELL, Biochem Pharmocol 2-5 2379-2384
(1977) 15. 16. 17. 18.
D.H. R U S S E L L and B.G.M. DURIE, Polyamines as M a r k e r s of Normal and M a l i q n a n t Growth, Raven Press, New Y o r k (1978) D.H. RUSSELL, Med Biol 5-5 286-295 (1981) M.K. H A D D O X and D.H. RUSSELL, Proc Natl Acad Sci U S A 78 1712-1716 (1981) J.F.F. SCOTT and D.H. RUSSELL, J Cell Physiol iii 111-116
(1982) 19. 20.
S.L. ZAHNER, K.V. PRAHALAD, and J.L.A. MITCHELL, Cytobios 4-5 25-34 (1986) S.J. FRIEDMAN, R.A. BELLANTONE, and E.S. CANELLAKIS, Biochim
1510
21. 22. 23. 24. 25.
0rnlthlne Decarboxylase During Repair
Biophys Acta 261 188-193 (1972) O. HEBY, Differentiation 19 1-20 (1981) M. SADEH, L.Z. STERN, K. CZYZEWSKI, P.R. FINLEY, and D.H. RUSSELL, Life sci 34 483-488 (1984) J.L.A. M I T C H E L L and G.K. MITCHELL, B i o c h e m Biophys Res Comm 105 1189-1197 (1982) M.M. BRADFORD, Anal B i o c h e m 7_/2 248-254 (1976) C.A. CONOVER, S.J. ROZOVSKI, and N.B. RUDERMAN, Diabetes 300
678-684 26. 27.
29.
(1981)
S.A. STRATTON, R. HECKMANN, and R.S. FRANCIS, J Ortho Sports Phys Ther 5 278-281 (1984) R.D. HOGAN, K.M. BURKE, and T.D. FRANKLIN, M i c r o v a s c Res 2__33
370-379 28.
Vol. 48, No. 16, 1991
(1982)
D.H. GODDARD, P.A. REVELL, J. CASON, S. G A L L A G H E R and H.L.F. CURREY, Ann R h e u m Dis 4_/2 582-584 (1983) M.C. FYFE and L.A. CHAHL, U l t r a s o u n d in Med & Biol 1_!1 (2) 273-283 (1985)
Pergamon Press
ORNITHINE DECARBOXYLASE: A B I O C H E M I C A L M A R K E R OF R E P A I R IN DAMAGED T I S S U E James H. G i l l e t t e I, John L.A. M i t c h e l l 2 IDepartment of A l l i e d H e a l t h P r o f e s s i o n s and 2Department of B i o l o g i c a l Sciences, N o r t h e r n Illinois U n i v e r s i t y DeKalb, Illinois 60115 (Received in final form February Ii, 1991) Summary The p u r p o s e of this study was to: (a) d e t e r m i n e the temporal p a t t e r n of e x p r e s s i o n of ornithine d e c a r b o x y l a s e (ODC), an established marker of cells engaged in proliferation and differentiation, during repair of t r a u m a t i z e d skeletal muscle, and (b) e v a l u a t e ODC as a b i o c h e m i c a l m a r k e r for indexing the extent and rate of repair of t r a u m a t i z e d skeletal m u s c l e in r e s p o n s e to t h e r a p e u t i c agents. Adult female W i s t a r rats w e i g h i n g 240 to 280 grams were a n e s t h e t i z e d and injected w i t h i00 ul of 2% lidocaine into the right or left a n t e r i o r t i b i a l i s m u s c l e to induce a localized injury. The animals were t r e a t e d 1 hour p o s t - i n j e c t i o n and every 12 hours for five m i n u t e s up to the time of sacrifice using u l t r a s o n i c i r r a d i a t i o n at 1.5 w a t t s / c m 2, sham-irradiation, or no treatment. An analysis of v a r i a n c e for r e p e a t e d m e a s u r e s and a Tukey's post hoc test were used to d e t e r m i n e the s i g n i f i c a n c e of t r e a t m e n t effects. Animals i r r a d i a t e d with u l t r a s o u n d d e m o n s t r a t e d an a c c e l e r a t e d p a t t e r n of change in ODC a c t i v i t y at 24 hours (P < 0.001), 30 hours (P < 0.003), and at 48 hours (P < 0.002) c o m p a r e d to control and sham-irradiated animals at these time intervals. I r r a d i a t e d animals also d e m o n s t r a t e d less edema at 48 hours compared to s h a m - i r r a d i a t e d and control animals (P < 0.003). These findings suggest that ODC is a useful b i o c h e m i c a l m a r k e r for d e t e r m i n i n g the extent and rate of tissue repair in t r a u m a t i z e d skeletal muscle, and it p r o v i d e s a temporal and q u a n t i f i a b l e p a r a m e t e r for e v a l u a t i n g the efficacy of t h e r a p e u t i c agents used to treat d a m a g e d skeletal muscle. T h e r a p e u t i c u l t r a s o u n d has been used for nearly five decades to restore function and p r o m o t e h e a l i n g of soft t i s s u e injuries. Clinical o b s e r v a t i o n s have s u p p o r t e d the use of u l t r a s o u n d for a wide v a r i e t y of c o n d i t i o n s including pain (i), p r e s s u r e sores (2), b u r s i t i s (3), and t r a u m a t i z e d skeletal m u s c l e (4). In spite of these observations, r e l a t i v e l y little research has been r e p o r t e d r e g a r d i n g t h e r a p e u t i c u l t r a s o u n d or other c o m m o n l y used agents, and e v i d e n c e of e f f i c a c y has been supported p r i m a r i l y by s u b j e c t i v e p a r a m e t e r s (5,6). In order to e s t a b l i s h the e f f e c t i v e n e s s of a s p e c i f i c t r e a t m e n t it is imperative that there be an o b j e c t i v e l y m e a s u r a b l e p a r a m e t e r that can be e m p l o y e d to d e t e r m i n e the e f f i c a c y 0024-3205/91 $3.C0 +.00 Copyright (c) 1991Pergmon Press plc
1502
Ornlthlne Decarboxylase During Repair
of the t h e r a p e u t i c
agent
Vol. 48, No. 16, 1991
(7).
Skeletal muscle can be damaged by a wide v a r i e t y of insults including the application of local anesthetics (8). Although b u p i v a c a i n e (Marcaine) is commonly used in the research laboratory to p r o d u c e muscle degeneration, Xylocaine (lidocaine) also has been shown to produce rapid necrosis of muscle fibers (9). The advantages of using local anesthetics such as lidocaine to e x p e r i m e n t a l l y induce injury are that the extent of damage to the muscle in each animal can be carefully controlled and the procedure is painless. R e g a r d l e s s of the method used to damage skeletal muscle, the muscle will undergo a repair process that appears to follow a common p a t h w a y (i0). Initially there is a period of muscle fiber d e g e n e r a t i o n c h a r a c t e r i z e d by the disruption and dissolution of sarcomeric units, mitochondria, sarcoplasmic reticula, and myonuclei. Satellite cells (myogenic stem cells), present beneath the basal lamina, survive the initial traumatic event and ensuing deleterious environment. Following removal of cellular components from the damaged muscle fiber, satellite cells are activated, giving rise to a p o p u l a t i o n of m y o b l a s t i c cells. Myoblasts, in turn, will fuse to form m u l t i n u c l e a t e d myotubes with centrally located nuclei, which then d i f f e r e n t i a t e into mature muscle fibers with p e r i p h e r a l l y located nuclei (8). Muscle damage and repair are often c h a r a c t e r i z e d by levels of Creatine P h o s p h o k i n a s e (CPK), Lactate Dehydrogenase (LDH), and other acute phase reactants (11,12). However, these analytes represent the acute inflammatory and degenerative stages of wound healing. Conversely, L-ornithine decarboxylase (EC 4.1.1.17, ODC) is an enzyme that demonstrates peak activity in all tissues stimulated to grow, proliferate and d i f f e r e n t i a t e (13-18). This enzyme catalyzes the initial, rate limiting step in the biosynthesis of the polyamines putrescine, spermidine, and spermine, which are essential for cell growth and differentiation. ODC activity has been recognized as a sensitive m a r k e r of protein synthesis, proliferation, and differentiation, d e m o n s t r a t i n g i0i00 fold elevations in activity following tissue stimulation (1921). Repair of damaged skeletal muscle is also accompanied by sharply elevated levels of ODC activity. Sadeh et al. (22) have shown that skeletal muscle injected with Marcaine results in muscle fiber necrosis followed by repair of the damaged tissue. More importantly, they used representative histological sections of muscle tissue as a function of time following injection to d e m o n s t r a t e that ODC levels are elevated during the period of repair and that ODC activity returns to control levels as the muscle returns to normal morphology. This suggests that levels of ODC activity are indicative of muscle cell p r o l i f e r a t i o n and d i f f e r e n t i a t i o n associated with repair, and not increased synthesis of excessive or unwanted connective tissue. This would infer that ODC activity may provide a useful biochemical m a r k e r for the time and extent of tissue repair in damaged skeletal muscle. In the present study we have determined the patterns of expression of ODC activity in damaged skeletal muscle, in the presence or absence of u l t r a s o n i c irradiation, in order to test the value of the enzyme as an objective index of v a r i a t i o n in the rate of tissue repair.
Vol. 48, No. 16, 1991
Ornlthine Decarboxylase During Repair
1503
Methods
Subjeats. A d u l t f e m a l e r a t s of t h e W i s t a r s t r a i n w e i g h i n g b e t w e e n 240 a n d 280 g r a m s w e r e u s e d in t h i s study. All animals w e r e h o u s e d o n e p e r s t a n d a r d cage, fed a s t a n d a r d p e l l e t d i e t a n d water, ab l i b i t u m , a n d k e p t in t e m p e r a t u r e - c o n t r o l l e d quarters (23°C) w i t h a 1 2 - h l i g h t - d a r k c y c l e (6:00 a.m. a n d 6:00 p.m.). The c a r e a n d u s e of t h e a n i m a l s w e r e in a c c o r d a n c e w i t h t h e A n i m a l s W e l f a r e Act. Instrumentation. Ultrasonic treatment was administered using a D y n a w a v e M o d e l 801 w i t h a o n e c e n t i m e t e r s o u n d - h e a d . The machine p r o v i d e s a f r e q u e n c y of 1.0 m e g a h e r t z . T h e m a x i m u m i n t e n s i t y is 2.0 w a t t s / c m 2 a n d t h e u l t r a s o u n d can be delivered pulsed or continuous. A UPM-DT-10 ultrasound power meter was used to c a l i b r a t e t h e u l t r a s o u n d m a c h i n e b e f o r e a n d d u r i n g t h e study. M u s c l e Injury. O n t h e d a y of a n y g i v e n e x p e r i m e n t t h e e n t i r e c a u d a l 1/3 of e a c h a n i m a l w a s c a r e f u l l y s h a v e d w i t h an e l e c t r i c hair clippers. Then each animal was randomly assigned t o an irradiated, sham-irradiated, or nonirradiated group. Following a n e s t h e s i a w i t h an i.p. i n j e c t i o n of I00 ul of k e t a m i n e a n d i00 ul of x y l a z i n e e a c h a n i m a l w a s r a n d o m l y i n j e c t e d w i t h i00 ul of 2% l i d o c a i n e i n t o e i t h e r t h e r i g h t or l e f t a n t e r i o r t i b i a l i s m u s c l e . 5/8 - i n c h 2 5 - g a u g e n e e d l e s w e r e m a r k e d w i t h an i n d e l i b l e m a r k e r at m e a s u r e d d i s t a n c e s of 2, 4, 6, 8, a n d i0 m m f r o m t h e t i p of t h e needle. The anterior tibialis muscle to be injected was gently squeezed from the medial and lateral margins. At a measured d i s t a n c e of 2 cm f r o m t h e l a t e r a l m a l l e o l u s t h e n e e d l e w a s i n s e r t e d l o n g i t u d i n a l l y a l o n g t h e l e n g t h of t h e m u s c l e to a d e p t h of I0 mm. As t h e n e e d l e w a s r e t r a c t e d a p p r o x i m a t e l y 25 ul of 2% l i d o c a i n e w a s d i s p e n s e d e v e r y 2 m m u n t i l t h e e n t i r e i00 ul h a d b e e n d i s t r i b u t e d . All i n j e c t i o n s w e r e a d m i n i s t e r e d b e t w e e n 2:00 a n d 4:00 p.m. Treatment. T r e a t m e n t s for t h e i r r a d i a t e d a n d s h a m - i r r a d i a t e d groups began approximately 1 hour post-injection of 2% l i d o c a i n e . P r i o r to e a c h t r e a t m e n t t h e a r e a o v e r t h e a n t e r i o r t i b i a l i s m u s c l e w a s w a s h e d a n d d r i e d to r e m o v e a n y d i r t or oil. For irradiated and sham-irradiated a n i m a l s u l t r a s o n i c c o n d u c t i n g gel w a s a p p l i e d to a t h i c k n e s s of a p p r o x i m a t e l y 1 m m w i t h a t o n g u e d e p r e s s o r . All a n i m a l s in t h e i r r a d i a t e d g r o u p r e c e i v e d c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for f i v e m i n u t e s . T h e s o u n d - h e a d w a s m o v e d in a circular, r h y t h m i c p a t t e r n o v e r t h e e n t i r e m u s c l e b e l l y of t h e anterior tibialis muscle. T h e s a m e p r o t o c o l w a s f o l l o w e d for all a n i m a l s in t h e s h a m - i r r a d i a t e d group except that the machine was t u r n e d off. A l l a n i m a l s in t h e i r r a d i a t e d and sham-irradiated g r o u p s w e r e t r e a t e d t w i c e p e r day. Treatments were administered between 4 : 0 0 p.m. a n d 6:00 p.m. a n d 8 : 0 0 a.m. a n d i 0 : 0 0 a.m. F o l l o w i n g i n j e c t i o n w i t h l i d o c a i n e , a n i m a l s in t h e n o n i r r a d i a t e d g r o u p s w e r e n o t h a n d l e d u n t i l t h e t i m e of s a c r i f i c e . Muscle Tissue S a m p l i n g . Animals from the irradiated, shamirradiated, and nonirradiated g r o u p s w e r e s a c r i f i c e d at 24 h o u r s ( e x p e r i m e n t 1), 30 h o u r s ( e x p e r i m e n t 2), o r 48 h o u r s ( e x p e r i m e n t 3) p o s t - i n j e c t i o n of 2% l i d o c a i n e . The injected and uninjected anterior tibialis muscles from each animal were rapidly excised, w e i g h e d , a n d p l a c e d in 5 ml of i c e - c o l d i s o l a t i o n b u f f e r (0.02 M E P P S p H 7.2, 0.5 m M EDTA, 50 u M p y r i d o x a l - 5 - p h o s p h a t e , 5 mM dithiothreitol). E a c h m u s c l e w a s h o m o g e n i z e d for 15 s e c o n d s w i t h
1504
Ornithine Decarboxylase During Repair
Vol. 48, No. 16, 1991
a B r i n k m a n P o l y t r o n h o m o g e n i z e r set at a speed of 5 followed by s o n i c a t i o n for 45 seconds using a Branson sonifier w i t h a microtip. H o m o g e n a t e s were c e n t r i f u g e d at 30,000 g for 15 m i n u t e s at 4°C. Five 175 ul r e p l i c a t e s from the r e s p e c t i v e s u p e r n a t a n t s were each added to 25 ul of isolation b u f f e r c o n t a i n i n g 0.08 uCi L-(I-14C) ornithine at a final concentration of 0.2 mM L-ornithine. R e a c t i o n s were stopped by the a d d i t i o n of 0.5 ml of 2 M citric acid (23). The r e l e a s e d Z4CO2 was c a p t u r e d by 1 cm squares of No. 3 W h a t m a n filter paper c o n t a i n i n g 50 ul of 30% p o t a s s i u m h y d r o x i d e located in center wells suspended from the rubber caps of the r e a c t i o n vials. The r a d i o a c t i v i t y was d e t e r m i n e d by s c i n t i l l a t i o n c o u n t i n g at an e f f i c i e n c y of 95%. Protein content was d e t e r m i n e d by the m e t h o d of B r a d f o r d (24).
Statistical Analysis. D i f f e r e n c e s in levels of ODC activity and in gross w e i g h t of a n t e r i o r tibialis m u s c l e s among the irradiated, s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups were tested by a o n e - w a y analysis of v a r i a n c e (ANOVA) for repeated measures. D i f f e r e n c e s at a level of 0.05 were c o n s i d e r e d significant. The sources of d i f f e r e n c e s identified by the one-way A N O V A were e x a m i n e d using a Tukey's m u l t i p l e - c o m p a r i s o n post hoc test. Results Levels of ODC a c t i v i t y found in a n t e r i o r tibialis muscles from animals r e c e i v i n g only an i.p. injection c o n t a i n i n g i00 ul of 2% lidocaine, i.p. injections c o n t a i n i n g i00 ul of K e t a m i n e and i00 ul of Xylazine, or no injection all d e m o n s t r a t e d levels of ODC a c t i v i t y less than 1 p m o l / h r / m g protein. However, repair of a n t e r i o r t i b i a l i s muscles d a m a g e d by an i.m. injection c o n t a i n i n g i00 ul of 2% lidocaine p r o d u c e d a c h a r a c t e r i s t i c p a t t e r n of change in ODC activity. As shown in Fig. 1 activity increased a p p r o x i m a t e l y 100-fold to peak levels at 30 hours post-injection, with a 50% d e c l i n e from peak a c t i v i t y by 48 hours post-injection. U n i n j e c t e d c o n t r a l a t e r a l anterior tibialis muscles of each animal in Fig. 1 (the u n i n j e c t e d side) also d e m o n s t r a t e d an increase in ODC activity, however, this was e x t r e m e l y small relative to the injected side. To a s c e r t a i n w h e t h e r m a s s a g i n g by the s o u n d h e a d or the general h a n d l i n g of the animals during t r e a t m e n t could, by themselves, affect the p a t t e r n of ODC activity during repair, some animals were s h a m - i r r a d i a t e d following injection with i00 ul of 2% lidocaine. There was no d i f f e r e n c e noted b e t w e e n the n o n i r r a d i a t e d and shami r r a d i a t e d groups in either the injected or u n i n j e c t e d anterior t i b i a l i s m u s c l e s (Fig. i). A n i m a l s injured by injection with I00 ul of 2% lidocaine and t r e a t e d w i t h c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five minutes d e m o n s t r a t e d an a c c e l e r a t e d p a t t e r n of change in ODC a c t i v i t y (Fig. 2). At 24 hours post-injection, ODC a c t i v i t y for irradiated animals reached peak levels and was nearly d o u b l e that of nonirradiated and sham-irradiated animals at the same time interval; P < 0.001. At 30 hours post-injection, animals treated w i t h u l t r a s o u n d d e m o n s t r a t e d a m a r k e d return toward b a c k g r o u n d levels at a time when n o n i r r a d i a t e d and s h a m - i r r a d i a t e d animals were a p p r o a c h i n g peak levels of ODC activity; P < 0.003. Even at 48 hours post-injection, ODC activity for irradiated animals c o n t i n u e d to d e m o n s t r a t e a return toward b a c k g r o u n d levels more
Vol. 48, No. 16, 1 9 9 1
Ornithine Decarboxylase During Repair
I00 4~
~
75
~
50
!
~,
0 ......
0
i ~-
t0
20
7--
I --QI
30
40
50
I
I
I
60
70
80
HOURS FIG.
1
ODC a c t i v i t y in m u s c l e s that have been injected with 2% lidocaine (~,[3) and in the u n i n j e c t e d c o n t r a l a t e r a l muscles (O,~)" F o l l o w i n g injection the d a m a g e d m u s c l e was e i t h e r u n t r e a t e d ( [ ~ , . ) or sham t r e a t e d ( Q , O ) by m a s s a g i n g w i t h the u l t r a s o u n d m a c h i n e turned off.
i1~
iO0 ,o Q.
~
.~".
75 I
. ~,
"..
I
0
/
50
-
// I
25
I
."
~-
/
U
o
dz-~" 0
J
I
I
I
I
I
I
iO
20
30
40
50
60
70
I 80
HOURS FIG.
2
O r n i t h i n e d e c a r b o x y l a s e a c t i v i t y of a n t e r i o r t i b i a l i s m u s c l e s injected with 2% lidocaine and r e c e i v i n g sham u l t r a s o u n d (machine turned off) for five m i n u t e s every t w e l v e hours (~), or c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five m i n u t e s every twelve hours ( A ) . R e s u l t s are means +/- S.E.M of 5 r e p l i c a t e samples. I r r a d i a t e d groups differ from s h a m - i r r a d i a t e d groups: P < 0.001 at 24 h; P < 0.003 at 30 h; P < 0.002 at 48 h.
1505
1506
Ornithine Decarboxylase During Repair
Vol. 48, No. 16, 1991
rapid than the n o n i r r a d i a t e d or s h a m - i r r a d i a t e d animals; P < 0.002. In contrast, the p a t t e r n of ODC a c t i v i t y in u n d a m a g e d m u s c l e was not a l t e r e d p e r c e p t i b l y by either direct u l t r a s o u n d i r r a d i a t i o n t r e a t m e n t or i r r a d i a t i o n of the d a m a g e d c o n t r a l a t e r a l m u s c l e (data not shown). To s t r e n g t h e n the p r e m i s e that ODC a c t i v i t y p r o v i d e s a b i o c h e m i c a l m a r k e r for skeletal m u s c l e u n d e r g o i n g repair, ODC a c t i v i t y was a s s a y e d at 24 h p o s t - i n j e c t i o n in r e s p o n s e to three d i f f e r e n t d o s a g e s of irradiation. As seen in Fig. 3 damaged skeletal m u s c l e irradiated at an intensity of 1.5 w a t t s / c m 2 d e m o n s t r a t e d the m o s t a c c e l e r a t e d p a t t e r n of ODC e x p r e s s i o n of the three d i f f e r e n t intensities of irradiation t e s t e d c o m p a r e d to noni r r a d i a t e d or s h a m - i r r a d i a t e d groups; P < .001. Damaged m u s c l e i r r a d i a t e d at 0.5 and 1.0 w a t t s / c m 2 was s u f f i c i e n t to elicit an a p p a r e n t a c c e l e r a t i o n in the p a t t e r n of ODC e x p r e s s i o n a l t h o u g h it 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 (.50 > P > .25 & .20 > P > .i0 respectively). C l i n i c a l l y h i g h e r intensities of i r r a d i a t i o n are not g e n e r a l l y used for t h e r a p e u t i c p u r p o s e s b e c a u s e of p o t e n t i a l l y harmful side effects such as c a v i t a t i o n (7). too
75
~
50
8 ~
25
o
FIG.
3
ODC a c t i v i t y in m u s c l e s that have been injected w i t h 2% l i d o c a i n e and r e c e i v e d no irradiation, sham-irradiation, i r r a d i a t i o n at 0.5, i. 0, or i. 5 w a t t s / c m 2. Irradiated g r o u p s d i f f e r from n o n - i r r a d i a t e d and s h a m - i r r a d i a t e d groups: P < 0.40 at 0.5; P < 0.20 at 1.0; P < 0.001 at 1.5. Clinical a s s e s s m e n t of soft tissue injuries often include m o n i t o r i n g of edema as a means of indexing p r o g r e s s toward recovery and it has been s u g g e s t e d that c o n t r o l l i n g edema d u r i n g early stages of i n f l a m m a t i o n may q u a l i t a t i v e l y affect later stages of repair. The course of edema was followed in all animals included in this i n v e s t i g a t i o n to assist in a s s e s s i n g the relative v a l u e of edema r e s o l u t i o n and the p a t t e r n of ODC e x p r e s s i o n as indices of the r e p a i r process. C o m p a r i s o n of gross w e i g h t s of a n t e r i o r tibialis m u s c l e s among normal uninjected, injected/irradiated,
Vol. 48, No. 16, 1991
Ornlthlne Decarboxylase During Repair
1507
injected/sham-irradiated, and injected/nonirradiated animals d e m o n s t r a t e d p r o n o u n c e d contrasts. Uninjected anterior tibialis m u s c l e s e x c i s e d from animals of e q u i v a l e n t size used in this study w e i g h e d a p p r o x i m a t e l y 600 milligrams. At 24 and 30 hours postinjection anterior tibialis muscles from irradiated, shamirradiated, and n o n i r r a d i a t e d animals d e m o n s t r a t e d a s i g n i f i c a n t increase in w e i g h t c o m p a r e d to m u s c l e s of u n i n j u r e d animals P < 0.001; (Fig. 3). However, no s i g n i f i c a n t w e i g h t d i f f e r e n c e s were found among the irradiated, sham-irradiated, or n o n i r r a d i a t e d groups at t h e s e time intervals (P > 0.25). At 48 hours posti n j e c t i o n a s i g n i f i c a n t w e i g h t increase r e m a i n e d in a n t e r i o r t i b i a l i s m u s c l e s from the s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups c o m p a r e d to m u s c l e s from irradiated or u n i n j u r e d animals (P < 0.001). Furthermore, w e i g h t s of m u s c l e s taken from i r r a d i a t e d animals had r e t u r n e d to levels observed in m u s c l e s of u n i n j u r e d animals (0.50 > P > 0.20).
1000
o~
CZ~ltn]ected ~x'mtn)ectsd,
Imtn]ected,
treated
shamtreated
control [ m u n i n J e c t e d control
-
750
I.--r LO W X
500
W ._1 r.J
¢n .,=.
250
24 h
30 h
48 h
TIME AFTER INJECTION
FIG.
4
Therapeutic ultrasound hastens resolution of edema. A n t e r i o r t i b i a l i s m u s c l e s were injected w i t h i00 ul of 2% lidocaine and received no irradiation, sham irradiation, or c o n t i n u o u s i r r a d i a t i o n at 1.5 watts/cm'. C o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 d e m o n s t r a t e d a s i g n i f i c a n t d e c r e a s e in w e i g h t of a n t e r i o r t i b i a l i s m u s c l e s c o m p a r e d to n o n - i r r a d i a t e d and s h a m - i r r a d i a t e d muscles. Injected, s h a m - t r e a t e d & injected, control c o m p a r e d to uninjected, control P < 0.001 at 24, 30, & 48 h; Injected, t r e a t e d c o m p a r e d to uninjected, control P < 0.001 at 24 & 30 h; P > 0.20 at 48 h. A n a l y s i s of body w e i g h t d e m o n s t r a t e d no s i g n i f i c a n t change from the time of i n j e c t i o n to the time of s a c r i f i c e for individual animals and no s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d among the groups of a n i m a l s (data not shown).
1508
Ornlthine Decarboxylase During Repair
Vol. 48, No. 16, 1991
Discussion Because polyamines are essential for p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of all cells the c h r o n o l o g i c a l e x p r e s s i o n of ODC, the rate l i m i t i n g enzyme in the b i o s y n t h e s i s of the polyamines, p r o v i d e s a good index of the respective stages of repair of d a m a g e d skeletal muscle. Normal adult skeletal m u s c l e in a quiescent, nonp r o l i f e r a t i n g state has very low levels of c o n s t i t u t i v e ODC. During repair, when there is extensive growth and p r o l i f e r a t i o n of myoblasts, ODC a c t i v i t y is rapidly and m a r k e d l y elevated, followed by a return to b a c k g r o u n d levels as the m y o b l a s t s fuse and d i f f e r e n t i a t e into m a t u r e m u s c l e fibers. R e p a i r of rat anterior t i b i a l i s m u s c l e s t r a u m a t i z e d w i t h 2% lidocaine is m a r k e d by a peak in ODC a c t i v i t y at 30 hours post-injection, followed by an a p p r o x i m a t e l y 50% r e d u c t i o n from peak levels at 48 hours postinjection. This p a t t e r n of ODC activity d u r i n g repair of skeletal m u s c l e c o m p a r e s c l o s e l y to that shown by others (22, 25). A n i m a l s irradiated with c o n t i n u o u s u l t r a s o u n d at 1.5 w a t t s / c m 2 for five m i n u t e s not only d e m o n s t r a t e d an a c c e l e r a t e d rise in ODC activity, w i t h p e a k levels occurring at 24 hours p o s t - i n j e c t i o n rather than at 30 hours post-injection, but also an a c c e l e r a t e d return t o w a r d b a c k g r o u n d levels at 30 and 48 hours p o s t - i n j e c t i o n c o m p a r e d to the n o n i r r a d i a t e d and s h a m - i r r a d i a t e d groups at these time intervals. This a c c e l e r a t i o n in the ODC a c t i v i t y v a r i a t i o n suggests that the u l t r a s o u n d t r e a t m e n t has p e r m i t t e d a more rapid onset and t e r m i n a t i o n of the p r o l i f e r a t i o n phase of the muscle repair. This c o n c l u s i o n is c o n s i s t e n t with the results of other i n v e s t i g a t o r s w h i c h suggest that u l t r a s o u n d may be e f f e c t i v e in a c c e l e r a t i n g the repair of damaged skeletal muscle. S t r a t t o n et al. (26) found that u l t r a s o u n d at an intensity of 1.5 w a t t s / c m 2 was h i g h l y s i g n i f i c a n t compared to 0.5 w a t t s / c m 2 for improving repair of h e m a t o m a s in skeletal muscle. Hogan et al. (27) d e m o n s t r a t e d that i r r a d i a t i o n of skeletal m u s c l e with u l t r a s o u n d improves blood flow in ischemic tissue and can enhance p e r f u s i o n in v a s c u l a r l y impaired muscle compared to that o b s e r v e d in noni r r a d i a t e d muscle. S h a m - i r r a d i a t e d animals reveal nearly identical levels of ODC a c t i v i t y c o m p a r e d to n o n i r r a d i a t e d animals at each time point assayed. This w o u l d suggest that the h a n d l i n g of the animals and m a s s a g i n g effect of the soundhead for each sham t r e a t m e n t do not influence levels of ODC activity. Similarly, lack of a m a s s a g i n g effect from the s o u n d h e a d also was observed in a study involving a n o t h e r tissue (7). Interestingly, the u n i n j e c t e d / u n t r e a t e d c o n t r a l a t e r a l limbs of irradiated, sham irradiated, and nonirradiated animals d e m o n s t r a t e d an increase in ODC activity, a l t h o u g h r e l a t i v e l y small (0.50 > P > 0.20). P r e s u m a b l y this may result from a modest h y p e r t r o p h y of the u n i n j e c t e d limb m u s c u l a t u r e r e s p o n d i n g to the added d e m a n d s imposed on it s e c o n d a r y to g u a r d i n g of the injected limb. This p r o v i d e s evidence for the relative importance of ODC d u r i n g c o m p e n s a t o r y growth as well as m u s c l e repair. Edema is an early and frequent clinical m a n i f e s t a t i o n of injury that may p r o l o n g the inflammatory process if left unchecked. Previous studies e v a l u a t i n g the effects of u l t r a s o u n d on edema have yielded contradictory results. Goddard et al. (28) found
Vol. 48, No. 16, 1991
0rnithine Decarboxylase During Repair
1509
u l t r a s o u n d to b e ~ t o t a l l y ineffective in s u p p r e s s i n g or r e d u c i n g i n f l a m m a t o r y edema. Conversely, Fyfe and Chahl (29) have shown that a l t h o u g h u l t r a s o u n d may increase edema d u r i n g the first 24 hours, by 48 hours the edema is s i g n i f i c a n t l y reduced. Findings p r e s e n t e d here show that the w e i g h t s of a n t e r i o r t i b i a l i s m u s c l e s t r e a t e d w i t h u l t r a s o u n d are s i g n i f i c a n t l y less than a n t e r i o r t i b i a l i s m u s c l e s from s h a m - i r r a d i a t e d and n o n i r r a d i a t e d groups at 48 hours, even though the groups are not s i g n i f i c a n t l y d i f f e r e n t at 24 and 30 hours. Moreover, the w e i g h t s of the irradiated anterior tibialis muscles at 48 hours return to levels c h a r a c t e r i s t i c a l l y seen in u n i n j e c t e d muscles. R e s o l u t i o n of edema is an important o b j e c t i v e in the t r e a t m e n t of soft tissue injuries and is often used as an index of repair. However, findings from this study d e m o n s t r a t e d peak levels of ODC a c t i v i t y in t r a u m a t i z e d skeletal m u s c l e that is m a r k e d l y edematous. These results suggest that ODC a c t i v i t y p r o v i d e s a more s e n s i t i v e and s p e c i f i c m a r k e r of repair d u r i n g the early stages of w o u n d healing. Acknowledgements This w o r k s was s u p p o r t e d in part by NIH R e s e a r c h Grant GM 33841. The authors w o u l d like to g r a t e f u l l y a c k n o w l e d g e Mary Jane Harris for her t e c h n i c a l assistance. References I. 2. 3. 4. 5. 6.
E. MUNTING, Physiotherapy 6__44 (6) 180-181 (1978) T. MCDIARMID, P.N. BURNS, G.T. LEWITH, D. M A C H I N 71 (2) 6670 (1985) D.S. D O W N I N G and A. WEINSTEIN, Phys Ther 66194-199 (1986) E.M. OAKLEY, Br J Cancer 45 (Suppl V) 233-237 (1982) C.J. PARTRIDGE, Physiotherapy 7_/3 (4) 166-168 (1987) G.H. CLARKE and L. STENNER, Physiotherapy 62 (6) 185-190
(1976) 7. 8. 9.
M. DYSON and J. SUCKLING, Physiotherapy 6__44 (4) 105-108 (1978) B.M. C A R L S O N and J.A. FAULKNER, Med Sci Sports Exer 1_55 187198 (1983) A.H. FOSTER and B.M. CARLSON, Anesth Analg 5_99 275-282
(1980) i0. II. 12. 13. 14.
D. ALBROOKE, M u s c l e Nerve 4 234-254 (1981) F.S. A P P L E and M. RHODES, J Appl Physiol 65 (6) 2598-2600 (1988) C.M. COLLEY, A. FLECK, B.R. MULLER, and M.A. MYERS J Clin Pathol 3-6 203-207 (1983) D.H. R U S S E L L and S.H. SYNDER, Proc Natl Acad Sci U S A 60 1420-1427 (1968) C.A. M A N E N and D.H. RUSSELL, Biochem Pharmocol 2-5 2379-2384
(1977) 15. 16. 17. 18.
D.H. R U S S E L L and B.G.M. DURIE, Polyamines as M a r k e r s of Normal and M a l i q n a n t Growth, Raven Press, New Y o r k (1978) D.H. RUSSELL, Med Biol 5-5 286-295 (1981) M.K. H A D D O X and D.H. RUSSELL, Proc Natl Acad Sci U S A 78 1712-1716 (1981) J.F.F. SCOTT and D.H. RUSSELL, J Cell Physiol iii 111-116
(1982) 19. 20.
S.L. ZAHNER, K.V. PRAHALAD, and J.L.A. MITCHELL, Cytobios 4-5 25-34 (1986) S.J. FRIEDMAN, R.A. BELLANTONE, and E.S. CANELLAKIS, Biochim
1510
21. 22. 23. 24. 25.
0rnlthlne Decarboxylase During Repair
Biophys Acta 261 188-193 (1972) O. HEBY, Differentiation 19 1-20 (1981) M. SADEH, L.Z. STERN, K. CZYZEWSKI, P.R. FINLEY, and D.H. RUSSELL, Life sci 34 483-488 (1984) J.L.A. M I T C H E L L and G.K. MITCHELL, B i o c h e m Biophys Res Comm 105 1189-1197 (1982) M.M. BRADFORD, Anal B i o c h e m 7_/2 248-254 (1976) C.A. CONOVER, S.J. ROZOVSKI, and N.B. RUDERMAN, Diabetes 300
678-684 26. 27.
29.
(1981)
S.A. STRATTON, R. HECKMANN, and R.S. FRANCIS, J Ortho Sports Phys Ther 5 278-281 (1984) R.D. HOGAN, K.M. BURKE, and T.D. FRANKLIN, M i c r o v a s c Res 2__33
370-379 28.
Vol. 48, No. 16, 1991
(1982)
D.H. GODDARD, P.A. REVELL, J. CASON, S. G A L L A G H E R and H.L.F. CURREY, Ann R h e u m Dis 4_/2 582-584 (1983) M.C. FYFE and L.A. CHAHL, U l t r a s o u n d in Med & Biol 1_!1 (2) 273-283 (1985)