PflLigersArchiv
Pflfigers Arch. 379, 209-214 (1979)
EuropeanJoomal of Physiology
9 by Springer-Verlag 1979
Effect of Ischemia on Contractile and Histochemical Properties of the Rat Soleus Muscle V~ra Hanzlikovfi and Ernest G u t m a n n ? Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia
Abstract. Ligature and section of the abdominal aorta
results in only minor and temporary functional and metabolic changes in the slow soleus muscle of the rat. A very small decrease in maximal tetanic tension corresponds to a few scattered areas of damaged and necrotic muscle fibres, in which decreased succinic dehydrogenase and loss of phosphorylase activity was observed. A new experimental approach, i.e. ligature and section of the abdominal aorta combined with terminal devascularisation, preserving intact tendons and innervation of the muscle causes maximal muscle ischemia, followed by an almost complete loss of tetanic tension output, marked shortening of contraction time and profound morphological and histochemical changes. The decrease in succinic dehydrogenase and ATPase activities and loss of phosphorylase activity occur in the majority of degenerating muscle fibres except for a thin rim of peripheral fibres during the first 4 days. Subsequently, the contractile properties recover gradually and enzyme activities reappear in the regenerating muscle fibres simultaneously with new revascularisation. Thirty days after the operation all the parameters observed returned to control values. Key words: Ischemia
- Slow skeletal muscle Contractile and histochemical properties - Recovery from ischemia.
Introduction
Different models for experimentally induced ischemia have been used for studying biochemical, physiological, ultrastructural and histochemical changes in skeletal muscles. However, the most common approach, i.e. ligature of the abdominal aorta or of the common illiac artery (Mendel et al., 1971 ; Janda et al., 1972; Karpati + Deceased August 6, 1977
et al., 1974) damaged only a small portion of the muscle mass. Models of ischemia, affecting in addition the nerves or tendons (Moore et al., 1956; Stenger et al., 1962; M/ikitie and Terfiv/iinen, 1977a) introduced further interfering factors, which enhanced the investigated changes to different extents. A controlled and reproducible experimental model was therefore necessary in order to study the effects of muscle ischemia and the recovery process. Our aim was to develop a model of arterial occlusion, producing not focal, but total ischemia, leaving the nerve and tendons of the muscle intact. Furthermore we studied the process of recovery from ischemia in the soleus muscle, which is known to be more affected by arterial occlusion than fast muscles (Karpati et al., 1974). Changes in contractile and histochemical properties were also investigated as these parameters of muscle function and metabolism are highly informative and could be obtained from the same muscle.
Material and Methods Two-month-old female Wistar rats, 160-i80 g body weight, were used. Ischemia of the soleus muscle was produced either by a (double ligature and section of the abdominal aorta above the bifurcation of the common illiac artery, after which the peritoneal cavity was closed and the skin sutured, or b) ligature and section of the abdominal aorta combined with "terminal devascularisation". The soleus muscle on the right side was freed from other muscles and the main vessels supplying it were carefully ligated, and the small vessels cut, thus achieving maximal devascularisation. Both the tendons and the nerve innervating the soleus muscle were left intact. In each experimental group 5 soleus muscles were removed 1,2, 4, 7,14 or 30 days after the operation. Unoperated animals of the same age and weight were used as controls.
Contractile Properties. The control and experimental soleus muscles
wereremovedand their contractileresponseswereelicitedin vitro in a chamber by direct mass stimulationwith rectangularpulses of 1.0 ms duration and supramaximal voltage (cca 12 V). Recordings were
0031-6768/79/0379/0209/$01.20
2~0
Pfl/igers Arch. 379 (1979)
made with an automatic analyzer previously described (Rohli6ek, 1968). The following contractile properties were measured: Twitch tension (TwT), maximal tetanic tension (TetT) using the frequency which elicited maximal force, latency period (LP), maximal rate of TwT development (TCC), expressed as a time parameter in ms (see RohH~ek and Gutmann, t972), contraction time (CT - time to peak) and half relaxation time (HRT). Histochemieal Procedures. Following the determination of contractile properties the muscles were frozen in liquid nitrogen, cut in a cryostat and sections were histochemically stained for myofibrillar ATPase activity at pH 9.4 (Padykula and Herman, 1955), using the modification of Guth and Samaha (1970), succinic dehydrogenase activity (SDH) according to Nachlas et al. (1957), phosphorylase (Ph) activity (Takeuchi and Kuriaki, 1968), phospholipid activity (Elleder and Lojda, 1973) and hematoxylin - eosin (HE).
~m 100-
II
II 75"
I .1.
50-
25-
contro[
iig. aorta abd
rig. aorta +devasc.
Fig. 1. Maximal isometric tetanic tension (g) of soleus muscles of control animals (1st bar), 48 h after ligature of the abdominal aorta (2nd bar) and after ligature of the abdominal aorta combined with terminal devascularisation (3rd bar). n = 5 (in each group), means +_ S.E.
Results
The effect of ischemia in the first experimental model (a) corresponds with the few scattered areas of ischemic changes previously observed (Karpati et al., 1974; Urbanovfi et al., 1974) and this damage causes a very small decrease in maximal tetanic tension (Fig. 1). After this procedure the majority of the muscle fibres are not altered, retain their enzyme activity, characteristic of the soleus muscle except for some affected regions, where swelling and necrosis of the muscle fibres, focal interstitial infiltration of lymphocytes and macrophages can be observed. Intramuscular oedema, loss of phosphorylase (Fig. 3a) and decrease of SDH activity are seen in these damaged areas. On the other hand our new experimental model (b), i.e. ligature and section of abdominal aorta combined with total devascularisation of the muscle results in a rapid decrease in tetanic tension almost to zero in the first 2 days after operation (Fig. 1 and Table 1). This experimental model of ischemia with intact tendons and well preserved nerve was therefore chosen for a study of the recovery processes in ischemic muscle. The changes in contraction time (CT) and maximal tetanic tension output (TetT) were investigated 1 30 days after the operation and compared with the control values tbr soleus muscles of normal 2- and 3month-old animals (Fig. 2). It can be seen that there is a shortening of CT in the first 2 days after operation, followed later by successive prolongation of CT. TetT is very low 1 day after the operation, however, its recovery is very rapid in the course of the following 30 days. Changes in latency period (LP) and half relaxation time (HRT) are small throughout the period of observation (Table 1). However, the time parameters of contraction reflecting changes in the maximal rate of twitch tension revealed rapid recovery.
Table 1. Changes in twitch tension (TwY-gm), latency period (LP), time constant of contraction (TCC), contraction time (CT) and half relaxation time (HRT) in ms, maximal tetanic tension output (TetT) and wet weight of the soleus muscle (WM-gm) 1 - 30 days after ligature of the abdominal aorta combined with muscle devascularisation, n = 5 (in each group), means _+ S.E. Soleus muscle Control 2monthsold Days after arterial occlusion-ld 2d 4d 7d 14d 30d
TwT
LP
TCC
CT
HRT
TetT
WM
20.98_+1.08
3.36+_0,10
23.24+_1.97
39.96+2.82
45.82+_4.18
9 3 . 6 2 ! 7.56
99.80+_ 3.58
0.12_+0.02 0.74+_0,45 3.44+_1.55 8.14_+1.77 11.50+_1.39 16.89_+1.03
4.18+_0.36 3.50_+0.21 4.t3+_0.14 3.86+_0.18 3.58-+0.12 3.64+-0.11
6.58_+0.66 I2.03_+2.04 20.10+_0.59 22.34+_0.71 21.26+_1.65 24.86+_0.68
23.48_+0.72 21.53+-0.72 36.20+_I.82 38.28+_1.19 34.52+_1.96 41.16+_1.72
37.20_+7.30 33.32+-4.68 48.85+_1.49 50.84_+3.12 40.36_+2.77 54.04+4.19
0.74_+ 0.12 5.52_+ 2.55 18.48_+ 6.90 34.56_+11.55 68.52+- 9.61 111.62+_13.39
147.50_+13.62 150.00+-10.80 86.00+_ 4.30 72.20+_ 6.10 82.80_+ 7.72 131.0 +_ 4.00
V. Hanzlikovfi and E. Gutmann: Ischemia of Slow Skeletal Muscle
gm ms 120
~
lOO-4oI
{. -...
80 -20
--~--
control
211
in SDH and Ph activity during this period (Figs. 3 g and 3h). Thirty days after operation the muscle fibre pattern with respect to phospholipids and ATPase activity is predominantly homogeneous and the fibre size distribution approaches normal values. The pattern of Ph activity is mixed, resembling that of the control muscle (Fig. 3h). An almost normal pattern of muscle fibre types is restored with respect to SDH activity.
L
Discussion 20-
012 4 7 14 30 days Fig. 2. Changes in isometric contraction time to peak (ms) (open symbols) and maximal tetanic tension (g) (filled symbols) of the soleus muscles 1 - 3 0 days after ligature of the abdominal aorta combined with terminal devascularisation. Control values for the soleus muscle for 2- and 3-month-old rats are indicated (2 m, 3 m)
Histochemically there is complete loss of phosphorylase activity in practically the whole soleus muscle 1 4 days after the operation with only a small rim of peripherally located fibres with intact enzyme activity (Fig. 3b). The muscle fibres are dispersed due to intramuscular oedema and small cell infiltration is found, especially perivascularly. Degeneration and advanced necrosis of muscle fibres occur. SDH activity decreases to a lesser extent. Four days after the operation peripheral fibres exhibit high SDH activity (Fig. 3 c). Seven days after the operation, when the ingrowth of new capillaries is observed (Fig. 3 d), phosphorylase, SDH and ATPase activities increase in centrally located groups of small, apparently regenerating muscle fibres (Figs. 3e and 3f). Stratification of the fibres is indicated, often producing 2 groups of fibres, i. e. a thin peripheral rim of fibres retaining normal diameter and enzyme activities and a central area of small fibres regenerating after previous degeneration. In the thin peripheral strip, the mixed enzyme pattern of muscle fibres with respect to ATPase activity can still be distinguished, whereas the mass of small, regenerating muscle fibres reveals a homogeneous high level of ATPase activity (Fig. 30. Many fibres are still in the myotubal stage and have central nuclei, some isolated fibres are swollen and/or hypertrophic. The same stratification of muscle fibres can be observed in sections stained for phospholipids. Fourteen to thirty days after the operation, perivascular infiltration decreases and the muscle fibres with central nuclei disappear. Marked recovery occurs
The many discrepancies in studies on metabolic and functional changes in the ischemic muscle are due to the different degree of arterial occlusion brought about by different experimental models. Unless occlusion of the terminal arterial branches is performed together with complete occlusion of the main artery, maximal reduction of blood flow cannot be expected. After ligature and section of the common iliac artery in rats, blood flow in the tibialis anterior muscle decreases to 20 % (Janda et al., 1974) 1 - 14 days after the operation, and a decrease to 54% of control values was demonstrated (Kohout et al., 1976) 3 weeks after the operation. Apparently the reduced blood supply is still sufficient to ensure a n oxygen supply to large parts of the muscle in the early stages of ischemia and this corresponds with the findings that only a small portion of the muscle showed morphological changes (Karpati et al., 1974). Almost maximal experimental ischemia was produced by section of the neurovascular pedicle to the sartorius muscle (Stenger et al., 1962). However, this model involves section and resuture of the tendons and therefore not allow representative studies on the recovery from ischemia. In the latter case loss of contraction was observed as early as 61/2 h after operation. In our experiments (ligature and section of abdominal aorta and terminal devascularisation) a very rapid decrease of tetanic tension, but not complete loss of contractility was observed when the muscle was stimulated directly. The vulnerability of neuromuscular transmission is known to be higher than that of the muscle fibres themselves (Holubfi~ et al., 1952). The initial shortening of contraction time in the ischemic muscle is most likely due to the decrease in pH in muscle and venous blood (Billings and Magraith, 1937), to the altered energy metabolism and to the changed depolarisation of cellular membrane systems (Scully et al., 1971). After brief temporarily induced ischemia, degeneration of nerve terminals without degeneration of the postischemic zone was found and this was assumed to be due to an increased susceptibility of the nerve structure to ischemia or to compression and lesion of the nerve by the
Fig. 3. a Soleus muscle 48 h after ligatm:e and cuting of the abdominal aorta (experiment a). Only focal degeneration of some muscle fibres (arrows) and loss of phosphorylase (Ph) activity staining in the affected areas are to be seen. b - - h Experiment b in which ligature of abdominal aorta was combined with muscle devascularisation, b Complete loss of Ph-activity in the soleus muscle, except for a rim of peripheral fibres with high activity, 24 h after the operation, c Peripherally located fibres retain high SDH activity even 4 days after the operation, d Irregular ingrowth of new capillaries detected by alkaline phosphatase activity 7 days after the operation, e 7 days after the operation. Increased SDH activity appears in small regenerating muscle fibres, f At the same time high ATPase activity occurs in large groups of regenerating muscle fibres. g 14 days after the operation. Stratification of muscle fibres is still clearly seen, the size of regenerating fibres becomes greater (SDH activity). h Fibre size and phosphorylase activity practically normal 30 days after the operation. Magn. 120 x in all figures
V. Hanzlikov~ and E. Gutmann: lschemia of Slow Skeletal Muscle
213
t o u r n i q u e t m e t h o d ; a x o n a l r e c o n n e c t i o n with the newly regenerating muscle fibres was r a p i d a n d is first seen on the 18th p o s t i s c h e m i c d a y (MS.kitie a n d Terfiv/iinen, 1977b). In o u r e x p e r i m e n t a l m o d e l o f c o m p l e t e ischemia b u t with i n t a c t i n n e r v a t i o n , the recovery process is shortened, a n d 30 d a y s after o p e r a t i o n the m a x i m a l tetanic tension a n d c o n t r a c t i o n time r e t u r n to their n o r m a l values. It s h o u l d be recalled t h a t c o n t r a c t i o n time o f the soleus muscle shows a b i p h a s i c d e v e l o p m e n t a l c h a n g e f r o m slow to fast a n d subsequently f r o m fast to slow c o n t r a c t i o n (Close, 1964), i.e. a progressive slowing o f c o n t r a c t i o n time between the second a n d third m o n t h s after birth. A similar course can be seen in the p o s t i s c h e m i c recovery p e r i o d (Fig. 2). D e g e n e r a t i o n o f capillaries after t e m p o r a r y ischemia a n d r a p i d restitution o f new capillaries a n d b l o o d flow has been d e s c r i b e d ( D r u r y , 1959; M/ikitie, 1977). These findings are in a g o o d a g r e e m e n t with o u r histochemical o b s e r v a t i o n s as to alkaline p h o s p h a t a s e activity. It is difficult however to asses a n d c o m p a r e results on m e t a b o l i c changes o b t a i n e d in different models. I n a d d i t i o n , the histochemical changes can n o t be expected to be the same for all enzymes studied. In o u r e x p e r i m e n t s p h o s p h o r y l a s e (Ph) activity a p p e a r s to be the m o s t vulnerable. Persistence o f the Ph-activity in p e r i p h e r a l l y l o c a t e d muscle fibres can m o s t easily be e x p l a i n e d to be caused by diffusion o f substrates f r o m the periphery. A f t e r t e m p o r a r y ischemia i n d u c e d by a t o u r n i q u e t (M/ikitie a n d Terfivfiinen, 1977a) Phactivity in skeletal muscle fibres has also been f o u n d to be the m o s t sensitive o f the histochemically o b s e r v e d enzyme activities. C o m p l e t e loss o f a m y l o p h o s p h o rylase activity a n d o f P A S - s t a i n a b l e glycogen were f o u n d in muscle fibres at early stages o f e x p e r i m e n t a l l y i n d u c e d ischemia ( K a r p a t i et al., 1974). Since hist o c h e m i c a l p h o s p h o r y l a s e staining was d e m o n s t r a t e d to be d e p e n d e n t on cellular glycogen (Schulze et al., 1971; M a r t i n a n d Engel, ~972) lack o f a p p a r e n t p h o s p h o r y l a s e activity m i g h t be due to glycogen depletion in the first few h o u r s after inducing ischemia. W e f o u n d A T P a s e activity to be less affected by ischemia a n d this c o r r e s p o n d s with the relatively late f r a g m e n t a t i o n o f filaments (Stenger et al., 1962). T h e earliest lesions affect m i t o c h o n d r i a l structures ( K a r p a t i et al., 1974; Hanzlikovfi a n d Schiaffino, 1977), reflecting oxygen a n d s u b s t r a t e d e p r i v a t i o n (Ingwall et al., 1975). O u r findings also indicate a decrease o f S D H activity in the early stages o f ischemia. T h e recovery f r o m ischemia, b r o u g h t a b o u t b y collateral circulation a n d i n g r o w t h o f new capillaries, was r a p i d a n d c o m plete as early as 30 d a y s after o p e r a t i o n . I n general a g o o d c o r r e l a t i o n was o b s e r v e d between the recovery o f histochemical p a t t e r n o f the muscle fibres a n d the contractile, p r o p e r t i e s o f the muscle.
Acknowledgement. The authors wish to acknowledge the expert technical assistance of Mrs. J. Stichovfi and A. Herbrychovfi and the helpful criticism of Dr. P. Hnlk during preparation of the manuscript.
References Billings, F. T., Magraith, B. C. : Chemical changes in tissues following obstruction of the blood supply. Qt. J. Exp. Physiol. 27, 249270 (1937) Close, R. : Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. (Lond.) 173, 24-95 (1964~ Drury, D. R. : Circulatory adjustments to aortic constriction. XXI. Int. Congress IUPS, Buenos Aires, p. 79 (1959) Elleder, M., Lojda, Z.: Studies in lipid histochemistry. XI. New, rapid, simple and selective method for the domonstration of phospholipids. Histochemie 36, 149-166 (1973) Guth, L., Samaha, F. J.: Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol. 28, 365-367 (1970) Hanzlikov/t, V., Schiaffino, S. : Mitochondrial changes in ischemic skeletal muscle. J. Ultrastruct. Res. 60, 121 - 133 (1977) HolubS.}, J., Schiick, M., Saravec, C. : Effect of low temperature on recovery of the neuro-muscular apparatus after ischemia. (In Czech) Cas L~k. (2es. 91,755-760 (1952) Ingwal, J. S., DeLuca, M., Sybers, H. D., Wildenthal, K.: Fetal mouse hearts: A model for studying ischemia. Proc. Nat. Acad. Sci. USA 72, 2809-2813 (1975) Janda, J., Urbanovfi, D., MrhovA, O., Linhart, J.: The effect of muscle training on the activity of some enzymes of skeletal muscle in chronic ischemia. Cor Vasa 14, 312-320 (1972) Janda, J., Linhart, J., Kasalick), J. : Experimental chronic ischemia of the skeletal muscle in the rat. Physiol. Bohemoslov. 23, 521 - 526 (1974) Karpati, G., Carpenter, S., Melmed, C., Eisen, A. A. : Experimental ischemic myopathy. J. Neuroi. Sci. 23, 129-161 (1974) Kohout, M., Poledne, R., Janda, J., Linhart, J.: Blood flow and transport of free acids in striated muscle under chronic ischemia. Pflfigers Arch. 367, 49-53 (1976) Martin, D. L., Engel, W. K. : Dependency of histochemical phosphorylase staining on amount of cellular glycogen. J. Histochem. Cytochem. 20, 476 479 (1972) MS.kitie, J. : Microvasculature of rat striated muscle after temporary ischemia. Acta Neuropath. (Berl.)37, 247-253 (1977) M/ikitie, J., Terfiv/iinen, H. : Histochemical studies of striated muscle after temporary ischemia in the rat. Acta Neuropath. (Berl.) 37, 10i - 109 (1977a) Mfikitie, J., Ter~iv~iinen,H. : Ultrastructure of striated muscle of the rat after temporary ischemia. Acta Neuropath. (Berl.) 37, 237245 (1977b) Mendell, J. R., Engel, W. K., Derrer, E. C.: Duchenne dystrophy: Functional ischemia reproduces its characteristic lesions. Science 172, 1143-1145 (1971) Moore, D. H., Ruska, H., Copenhaven, W. H. : Electron microscopic and histochemical observations of muscle degeneration after tourniquet. J. Biophys. Biochem. Cytol. 2, 755 763 (1956) Nachlas, M. K., Tsou, K. C., Souza, E., Cheng, C. S., Seligman, A. M. : Cytocbemical demonstration of succinic dehydrogenase by the use of a new P-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5, 420-436 (1957) Padykula, H. A., Herman, E.: The specifity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3, 170--195 (1955) Rohli~ek, V. : An automatic analyzer of muscle contraction. SNTL Tech. Dig. 61, 383-387 (1968)
214 Rohli~ek, V., Gutmann, E.: Time constant of contraction, a new expression of maximal rate of tension development. Physiol. Bohemoslov. 21, 430--431 (1972) Schulze, W., Krause, E. G., Wollenberger, A. : On the fate of glycogen phosphorylase in the ischemic and infarcting myocardium. J. Mol. Cell. Cardiol. 2, 241-251 (1971) Scully, R. E., Shannon, F. M., Dickersin, G. R. : Factors involved in recovery from experimental skeletal muscle ischemia produced in dogs. Part I. (Histologic and histochemical pattern of ischemic muscle) Am. J. Path. 39, 721-738 (1961) Stenger, R. J., Spiro, D., Scully, R. E., Shannon, J. M.: Ultrastructural and physiological alterations in ischemic skeletal muscle. Am. J. Pathol. 60, 1 - 1 9 (1962)
Pfliigers Arch. 379 (1979) Takeuchi, T., Kuriaki, M. : Histochemical and electron microscopic differences between native glycogen and polyglucose synthetized by phosphorylase in tissue cells. Acta Histochem. Cytochem. 1, 6 3 - 7 8 (1968) Urbanovfi, D., Janda, J., Mrhovfi, O., Linhart, J. : Enzyme changes in the ischaemia of skeletal muscle and the effect of physical conditioning. A histochemical study. Histochem. J. 6, 147-155 (1974)
Received December 2, 1977
Pflfigers Arch. 379, 209-214 (1979)
EuropeanJoomal of Physiology
9 by Springer-Verlag 1979
Effect of Ischemia on Contractile and Histochemical Properties of the Rat Soleus Muscle V~ra Hanzlikovfi and Ernest G u t m a n n ? Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia
Abstract. Ligature and section of the abdominal aorta
results in only minor and temporary functional and metabolic changes in the slow soleus muscle of the rat. A very small decrease in maximal tetanic tension corresponds to a few scattered areas of damaged and necrotic muscle fibres, in which decreased succinic dehydrogenase and loss of phosphorylase activity was observed. A new experimental approach, i.e. ligature and section of the abdominal aorta combined with terminal devascularisation, preserving intact tendons and innervation of the muscle causes maximal muscle ischemia, followed by an almost complete loss of tetanic tension output, marked shortening of contraction time and profound morphological and histochemical changes. The decrease in succinic dehydrogenase and ATPase activities and loss of phosphorylase activity occur in the majority of degenerating muscle fibres except for a thin rim of peripheral fibres during the first 4 days. Subsequently, the contractile properties recover gradually and enzyme activities reappear in the regenerating muscle fibres simultaneously with new revascularisation. Thirty days after the operation all the parameters observed returned to control values. Key words: Ischemia
- Slow skeletal muscle Contractile and histochemical properties - Recovery from ischemia.
Introduction
Different models for experimentally induced ischemia have been used for studying biochemical, physiological, ultrastructural and histochemical changes in skeletal muscles. However, the most common approach, i.e. ligature of the abdominal aorta or of the common illiac artery (Mendel et al., 1971 ; Janda et al., 1972; Karpati + Deceased August 6, 1977
et al., 1974) damaged only a small portion of the muscle mass. Models of ischemia, affecting in addition the nerves or tendons (Moore et al., 1956; Stenger et al., 1962; M/ikitie and Terfiv/iinen, 1977a) introduced further interfering factors, which enhanced the investigated changes to different extents. A controlled and reproducible experimental model was therefore necessary in order to study the effects of muscle ischemia and the recovery process. Our aim was to develop a model of arterial occlusion, producing not focal, but total ischemia, leaving the nerve and tendons of the muscle intact. Furthermore we studied the process of recovery from ischemia in the soleus muscle, which is known to be more affected by arterial occlusion than fast muscles (Karpati et al., 1974). Changes in contractile and histochemical properties were also investigated as these parameters of muscle function and metabolism are highly informative and could be obtained from the same muscle.
Material and Methods Two-month-old female Wistar rats, 160-i80 g body weight, were used. Ischemia of the soleus muscle was produced either by a (double ligature and section of the abdominal aorta above the bifurcation of the common illiac artery, after which the peritoneal cavity was closed and the skin sutured, or b) ligature and section of the abdominal aorta combined with "terminal devascularisation". The soleus muscle on the right side was freed from other muscles and the main vessels supplying it were carefully ligated, and the small vessels cut, thus achieving maximal devascularisation. Both the tendons and the nerve innervating the soleus muscle were left intact. In each experimental group 5 soleus muscles were removed 1,2, 4, 7,14 or 30 days after the operation. Unoperated animals of the same age and weight were used as controls.
Contractile Properties. The control and experimental soleus muscles
wereremovedand their contractileresponseswereelicitedin vitro in a chamber by direct mass stimulationwith rectangularpulses of 1.0 ms duration and supramaximal voltage (cca 12 V). Recordings were
0031-6768/79/0379/0209/$01.20
2~0
Pfl/igers Arch. 379 (1979)
made with an automatic analyzer previously described (Rohli6ek, 1968). The following contractile properties were measured: Twitch tension (TwT), maximal tetanic tension (TetT) using the frequency which elicited maximal force, latency period (LP), maximal rate of TwT development (TCC), expressed as a time parameter in ms (see RohH~ek and Gutmann, t972), contraction time (CT - time to peak) and half relaxation time (HRT). Histochemieal Procedures. Following the determination of contractile properties the muscles were frozen in liquid nitrogen, cut in a cryostat and sections were histochemically stained for myofibrillar ATPase activity at pH 9.4 (Padykula and Herman, 1955), using the modification of Guth and Samaha (1970), succinic dehydrogenase activity (SDH) according to Nachlas et al. (1957), phosphorylase (Ph) activity (Takeuchi and Kuriaki, 1968), phospholipid activity (Elleder and Lojda, 1973) and hematoxylin - eosin (HE).
~m 100-
II
II 75"
I .1.
50-
25-
contro[
iig. aorta abd
rig. aorta +devasc.
Fig. 1. Maximal isometric tetanic tension (g) of soleus muscles of control animals (1st bar), 48 h after ligature of the abdominal aorta (2nd bar) and after ligature of the abdominal aorta combined with terminal devascularisation (3rd bar). n = 5 (in each group), means +_ S.E.
Results
The effect of ischemia in the first experimental model (a) corresponds with the few scattered areas of ischemic changes previously observed (Karpati et al., 1974; Urbanovfi et al., 1974) and this damage causes a very small decrease in maximal tetanic tension (Fig. 1). After this procedure the majority of the muscle fibres are not altered, retain their enzyme activity, characteristic of the soleus muscle except for some affected regions, where swelling and necrosis of the muscle fibres, focal interstitial infiltration of lymphocytes and macrophages can be observed. Intramuscular oedema, loss of phosphorylase (Fig. 3a) and decrease of SDH activity are seen in these damaged areas. On the other hand our new experimental model (b), i.e. ligature and section of abdominal aorta combined with total devascularisation of the muscle results in a rapid decrease in tetanic tension almost to zero in the first 2 days after operation (Fig. 1 and Table 1). This experimental model of ischemia with intact tendons and well preserved nerve was therefore chosen for a study of the recovery processes in ischemic muscle. The changes in contraction time (CT) and maximal tetanic tension output (TetT) were investigated 1 30 days after the operation and compared with the control values tbr soleus muscles of normal 2- and 3month-old animals (Fig. 2). It can be seen that there is a shortening of CT in the first 2 days after operation, followed later by successive prolongation of CT. TetT is very low 1 day after the operation, however, its recovery is very rapid in the course of the following 30 days. Changes in latency period (LP) and half relaxation time (HRT) are small throughout the period of observation (Table 1). However, the time parameters of contraction reflecting changes in the maximal rate of twitch tension revealed rapid recovery.
Table 1. Changes in twitch tension (TwY-gm), latency period (LP), time constant of contraction (TCC), contraction time (CT) and half relaxation time (HRT) in ms, maximal tetanic tension output (TetT) and wet weight of the soleus muscle (WM-gm) 1 - 30 days after ligature of the abdominal aorta combined with muscle devascularisation, n = 5 (in each group), means _+ S.E. Soleus muscle Control 2monthsold Days after arterial occlusion-ld 2d 4d 7d 14d 30d
TwT
LP
TCC
CT
HRT
TetT
WM
20.98_+1.08
3.36+_0,10
23.24+_1.97
39.96+2.82
45.82+_4.18
9 3 . 6 2 ! 7.56
99.80+_ 3.58
0.12_+0.02 0.74+_0,45 3.44+_1.55 8.14_+1.77 11.50+_1.39 16.89_+1.03
4.18+_0.36 3.50_+0.21 4.t3+_0.14 3.86+_0.18 3.58-+0.12 3.64+-0.11
6.58_+0.66 I2.03_+2.04 20.10+_0.59 22.34+_0.71 21.26+_1.65 24.86+_0.68
23.48_+0.72 21.53+-0.72 36.20+_I.82 38.28+_1.19 34.52+_1.96 41.16+_1.72
37.20_+7.30 33.32+-4.68 48.85+_1.49 50.84_+3.12 40.36_+2.77 54.04+4.19
0.74_+ 0.12 5.52_+ 2.55 18.48_+ 6.90 34.56_+11.55 68.52+- 9.61 111.62+_13.39
147.50_+13.62 150.00+-10.80 86.00+_ 4.30 72.20+_ 6.10 82.80_+ 7.72 131.0 +_ 4.00
V. Hanzlikovfi and E. Gutmann: Ischemia of Slow Skeletal Muscle
gm ms 120
~
lOO-4oI
{. -...
80 -20
--~--
control
211
in SDH and Ph activity during this period (Figs. 3 g and 3h). Thirty days after operation the muscle fibre pattern with respect to phospholipids and ATPase activity is predominantly homogeneous and the fibre size distribution approaches normal values. The pattern of Ph activity is mixed, resembling that of the control muscle (Fig. 3h). An almost normal pattern of muscle fibre types is restored with respect to SDH activity.
L
Discussion 20-
012 4 7 14 30 days Fig. 2. Changes in isometric contraction time to peak (ms) (open symbols) and maximal tetanic tension (g) (filled symbols) of the soleus muscles 1 - 3 0 days after ligature of the abdominal aorta combined with terminal devascularisation. Control values for the soleus muscle for 2- and 3-month-old rats are indicated (2 m, 3 m)
Histochemically there is complete loss of phosphorylase activity in practically the whole soleus muscle 1 4 days after the operation with only a small rim of peripherally located fibres with intact enzyme activity (Fig. 3b). The muscle fibres are dispersed due to intramuscular oedema and small cell infiltration is found, especially perivascularly. Degeneration and advanced necrosis of muscle fibres occur. SDH activity decreases to a lesser extent. Four days after the operation peripheral fibres exhibit high SDH activity (Fig. 3 c). Seven days after the operation, when the ingrowth of new capillaries is observed (Fig. 3 d), phosphorylase, SDH and ATPase activities increase in centrally located groups of small, apparently regenerating muscle fibres (Figs. 3e and 3f). Stratification of the fibres is indicated, often producing 2 groups of fibres, i. e. a thin peripheral rim of fibres retaining normal diameter and enzyme activities and a central area of small fibres regenerating after previous degeneration. In the thin peripheral strip, the mixed enzyme pattern of muscle fibres with respect to ATPase activity can still be distinguished, whereas the mass of small, regenerating muscle fibres reveals a homogeneous high level of ATPase activity (Fig. 30. Many fibres are still in the myotubal stage and have central nuclei, some isolated fibres are swollen and/or hypertrophic. The same stratification of muscle fibres can be observed in sections stained for phospholipids. Fourteen to thirty days after the operation, perivascular infiltration decreases and the muscle fibres with central nuclei disappear. Marked recovery occurs
The many discrepancies in studies on metabolic and functional changes in the ischemic muscle are due to the different degree of arterial occlusion brought about by different experimental models. Unless occlusion of the terminal arterial branches is performed together with complete occlusion of the main artery, maximal reduction of blood flow cannot be expected. After ligature and section of the common iliac artery in rats, blood flow in the tibialis anterior muscle decreases to 20 % (Janda et al., 1974) 1 - 14 days after the operation, and a decrease to 54% of control values was demonstrated (Kohout et al., 1976) 3 weeks after the operation. Apparently the reduced blood supply is still sufficient to ensure a n oxygen supply to large parts of the muscle in the early stages of ischemia and this corresponds with the findings that only a small portion of the muscle showed morphological changes (Karpati et al., 1974). Almost maximal experimental ischemia was produced by section of the neurovascular pedicle to the sartorius muscle (Stenger et al., 1962). However, this model involves section and resuture of the tendons and therefore not allow representative studies on the recovery from ischemia. In the latter case loss of contraction was observed as early as 61/2 h after operation. In our experiments (ligature and section of abdominal aorta and terminal devascularisation) a very rapid decrease of tetanic tension, but not complete loss of contractility was observed when the muscle was stimulated directly. The vulnerability of neuromuscular transmission is known to be higher than that of the muscle fibres themselves (Holubfi~ et al., 1952). The initial shortening of contraction time in the ischemic muscle is most likely due to the decrease in pH in muscle and venous blood (Billings and Magraith, 1937), to the altered energy metabolism and to the changed depolarisation of cellular membrane systems (Scully et al., 1971). After brief temporarily induced ischemia, degeneration of nerve terminals without degeneration of the postischemic zone was found and this was assumed to be due to an increased susceptibility of the nerve structure to ischemia or to compression and lesion of the nerve by the
Fig. 3. a Soleus muscle 48 h after ligatm:e and cuting of the abdominal aorta (experiment a). Only focal degeneration of some muscle fibres (arrows) and loss of phosphorylase (Ph) activity staining in the affected areas are to be seen. b - - h Experiment b in which ligature of abdominal aorta was combined with muscle devascularisation, b Complete loss of Ph-activity in the soleus muscle, except for a rim of peripheral fibres with high activity, 24 h after the operation, c Peripherally located fibres retain high SDH activity even 4 days after the operation, d Irregular ingrowth of new capillaries detected by alkaline phosphatase activity 7 days after the operation, e 7 days after the operation. Increased SDH activity appears in small regenerating muscle fibres, f At the same time high ATPase activity occurs in large groups of regenerating muscle fibres. g 14 days after the operation. Stratification of muscle fibres is still clearly seen, the size of regenerating fibres becomes greater (SDH activity). h Fibre size and phosphorylase activity practically normal 30 days after the operation. Magn. 120 x in all figures
V. Hanzlikov~ and E. Gutmann: lschemia of Slow Skeletal Muscle
213
t o u r n i q u e t m e t h o d ; a x o n a l r e c o n n e c t i o n with the newly regenerating muscle fibres was r a p i d a n d is first seen on the 18th p o s t i s c h e m i c d a y (MS.kitie a n d Terfiv/iinen, 1977b). In o u r e x p e r i m e n t a l m o d e l o f c o m p l e t e ischemia b u t with i n t a c t i n n e r v a t i o n , the recovery process is shortened, a n d 30 d a y s after o p e r a t i o n the m a x i m a l tetanic tension a n d c o n t r a c t i o n time r e t u r n to their n o r m a l values. It s h o u l d be recalled t h a t c o n t r a c t i o n time o f the soleus muscle shows a b i p h a s i c d e v e l o p m e n t a l c h a n g e f r o m slow to fast a n d subsequently f r o m fast to slow c o n t r a c t i o n (Close, 1964), i.e. a progressive slowing o f c o n t r a c t i o n time between the second a n d third m o n t h s after birth. A similar course can be seen in the p o s t i s c h e m i c recovery p e r i o d (Fig. 2). D e g e n e r a t i o n o f capillaries after t e m p o r a r y ischemia a n d r a p i d restitution o f new capillaries a n d b l o o d flow has been d e s c r i b e d ( D r u r y , 1959; M/ikitie, 1977). These findings are in a g o o d a g r e e m e n t with o u r histochemical o b s e r v a t i o n s as to alkaline p h o s p h a t a s e activity. It is difficult however to asses a n d c o m p a r e results on m e t a b o l i c changes o b t a i n e d in different models. I n a d d i t i o n , the histochemical changes can n o t be expected to be the same for all enzymes studied. In o u r e x p e r i m e n t s p h o s p h o r y l a s e (Ph) activity a p p e a r s to be the m o s t vulnerable. Persistence o f the Ph-activity in p e r i p h e r a l l y l o c a t e d muscle fibres can m o s t easily be e x p l a i n e d to be caused by diffusion o f substrates f r o m the periphery. A f t e r t e m p o r a r y ischemia i n d u c e d by a t o u r n i q u e t (M/ikitie a n d Terfivfiinen, 1977a) Phactivity in skeletal muscle fibres has also been f o u n d to be the m o s t sensitive o f the histochemically o b s e r v e d enzyme activities. C o m p l e t e loss o f a m y l o p h o s p h o rylase activity a n d o f P A S - s t a i n a b l e glycogen were f o u n d in muscle fibres at early stages o f e x p e r i m e n t a l l y i n d u c e d ischemia ( K a r p a t i et al., 1974). Since hist o c h e m i c a l p h o s p h o r y l a s e staining was d e m o n s t r a t e d to be d e p e n d e n t on cellular glycogen (Schulze et al., 1971; M a r t i n a n d Engel, ~972) lack o f a p p a r e n t p h o s p h o r y l a s e activity m i g h t be due to glycogen depletion in the first few h o u r s after inducing ischemia. W e f o u n d A T P a s e activity to be less affected by ischemia a n d this c o r r e s p o n d s with the relatively late f r a g m e n t a t i o n o f filaments (Stenger et al., 1962). T h e earliest lesions affect m i t o c h o n d r i a l structures ( K a r p a t i et al., 1974; Hanzlikovfi a n d Schiaffino, 1977), reflecting oxygen a n d s u b s t r a t e d e p r i v a t i o n (Ingwall et al., 1975). O u r findings also indicate a decrease o f S D H activity in the early stages o f ischemia. T h e recovery f r o m ischemia, b r o u g h t a b o u t b y collateral circulation a n d i n g r o w t h o f new capillaries, was r a p i d a n d c o m plete as early as 30 d a y s after o p e r a t i o n . I n general a g o o d c o r r e l a t i o n was o b s e r v e d between the recovery o f histochemical p a t t e r n o f the muscle fibres a n d the contractile, p r o p e r t i e s o f the muscle.
Acknowledgement. The authors wish to acknowledge the expert technical assistance of Mrs. J. Stichovfi and A. Herbrychovfi and the helpful criticism of Dr. P. Hnlk during preparation of the manuscript.
References Billings, F. T., Magraith, B. C. : Chemical changes in tissues following obstruction of the blood supply. Qt. J. Exp. Physiol. 27, 249270 (1937) Close, R. : Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. (Lond.) 173, 24-95 (1964~ Drury, D. R. : Circulatory adjustments to aortic constriction. XXI. Int. Congress IUPS, Buenos Aires, p. 79 (1959) Elleder, M., Lojda, Z.: Studies in lipid histochemistry. XI. New, rapid, simple and selective method for the domonstration of phospholipids. Histochemie 36, 149-166 (1973) Guth, L., Samaha, F. J.: Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol. 28, 365-367 (1970) Hanzlikov/t, V., Schiaffino, S. : Mitochondrial changes in ischemic skeletal muscle. J. Ultrastruct. Res. 60, 121 - 133 (1977) HolubS.}, J., Schiick, M., Saravec, C. : Effect of low temperature on recovery of the neuro-muscular apparatus after ischemia. (In Czech) Cas L~k. (2es. 91,755-760 (1952) Ingwal, J. S., DeLuca, M., Sybers, H. D., Wildenthal, K.: Fetal mouse hearts: A model for studying ischemia. Proc. Nat. Acad. Sci. USA 72, 2809-2813 (1975) Janda, J., Urbanovfi, D., MrhovA, O., Linhart, J.: The effect of muscle training on the activity of some enzymes of skeletal muscle in chronic ischemia. Cor Vasa 14, 312-320 (1972) Janda, J., Linhart, J., Kasalick), J. : Experimental chronic ischemia of the skeletal muscle in the rat. Physiol. Bohemoslov. 23, 521 - 526 (1974) Karpati, G., Carpenter, S., Melmed, C., Eisen, A. A. : Experimental ischemic myopathy. J. Neuroi. Sci. 23, 129-161 (1974) Kohout, M., Poledne, R., Janda, J., Linhart, J.: Blood flow and transport of free acids in striated muscle under chronic ischemia. Pflfigers Arch. 367, 49-53 (1976) Martin, D. L., Engel, W. K. : Dependency of histochemical phosphorylase staining on amount of cellular glycogen. J. Histochem. Cytochem. 20, 476 479 (1972) MS.kitie, J. : Microvasculature of rat striated muscle after temporary ischemia. Acta Neuropath. (Berl.)37, 247-253 (1977) M/ikitie, J., Terfiv/iinen, H. : Histochemical studies of striated muscle after temporary ischemia in the rat. Acta Neuropath. (Berl.) 37, 10i - 109 (1977a) Mfikitie, J., Ter~iv~iinen,H. : Ultrastructure of striated muscle of the rat after temporary ischemia. Acta Neuropath. (Berl.) 37, 237245 (1977b) Mendell, J. R., Engel, W. K., Derrer, E. C.: Duchenne dystrophy: Functional ischemia reproduces its characteristic lesions. Science 172, 1143-1145 (1971) Moore, D. H., Ruska, H., Copenhaven, W. H. : Electron microscopic and histochemical observations of muscle degeneration after tourniquet. J. Biophys. Biochem. Cytol. 2, 755 763 (1956) Nachlas, M. K., Tsou, K. C., Souza, E., Cheng, C. S., Seligman, A. M. : Cytocbemical demonstration of succinic dehydrogenase by the use of a new P-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5, 420-436 (1957) Padykula, H. A., Herman, E.: The specifity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3, 170--195 (1955) Rohli~ek, V. : An automatic analyzer of muscle contraction. SNTL Tech. Dig. 61, 383-387 (1968)
214 Rohli~ek, V., Gutmann, E.: Time constant of contraction, a new expression of maximal rate of tension development. Physiol. Bohemoslov. 21, 430--431 (1972) Schulze, W., Krause, E. G., Wollenberger, A. : On the fate of glycogen phosphorylase in the ischemic and infarcting myocardium. J. Mol. Cell. Cardiol. 2, 241-251 (1971) Scully, R. E., Shannon, F. M., Dickersin, G. R. : Factors involved in recovery from experimental skeletal muscle ischemia produced in dogs. Part I. (Histologic and histochemical pattern of ischemic muscle) Am. J. Path. 39, 721-738 (1961) Stenger, R. J., Spiro, D., Scully, R. E., Shannon, J. M.: Ultrastructural and physiological alterations in ischemic skeletal muscle. Am. J. Pathol. 60, 1 - 1 9 (1962)
Pfliigers Arch. 379 (1979) Takeuchi, T., Kuriaki, M. : Histochemical and electron microscopic differences between native glycogen and polyglucose synthetized by phosphorylase in tissue cells. Acta Histochem. Cytochem. 1, 6 3 - 7 8 (1968) Urbanovfi, D., Janda, J., Mrhovfi, O., Linhart, J. : Enzyme changes in the ischaemia of skeletal muscle and the effect of physical conditioning. A histochemical study. Histochem. J. 6, 147-155 (1974)
Received December 2, 1977
