- Email: [email protected]
Pathology study of rabbit calf muscles after repeated compression*
JY.-H. Orthop (1998) 3:209–215 BaiSci et al.: Muscle pathology after repeated compression
209
Pathology study of rabbit calf muscles after repeated compression* Yue-Hong Bai1,2, Masakazu Takemitsu1, Yuji Atsuta1, and Yoshiharu Takemitsu1 1 2
Department of Orthopaedic Surgery, Asahikawa Medical College, Nishikagura 4-5, 3-11, Asahikawa, Hokkaido 078-8510, Japan Department of Orthopaedic Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P. R. China
Abstract: To elucidate the pathogenesis of chronic compartment syndrome, we examined pathological changes in the soleus (red) and extensor digitorum longus (EDL; white) muscles in Japanese white rabbits after repeated compression with a pneumatic tourniquet. Repeated tourniquet compression via cuff inflation was carried out on the rabbits, calves daily, for 2 h, then stopped for 30 min, and then applied for another 2 h. The contralateral hindlimb, which was not compressed, served as a control. Animals were allocated to 15 groups, with pressures of 40, 80, and 120 mmHg for periods of 1 day, 3 days, 1 week, 2 weeks, and 4 weeks. Skeletal muscle specimens in each group were studied by histopathological and histochemical (ATPase) methods. After compression for 1 day, regardless of pressure, and compression for 3 days in the 40-mmHg pressure group, edematous changes in regions with mild inflammation and increases in fiber diameter were observed in the muscles. After compression for 3 days in the 80- and 120-mmHg pressure groups, and after 1, 2, or 4 weeks in the 40-mmHg pressure group, a few necrotic fibers and scattered fibers with some mononuclear cell infiltrates indicative of early-stage necrosis were detected. In the groups with 80 or 120 mmHg pressure for 1, 2, or 4 weeks, muscle fibers exhibited marked degenerative changes, which were more pronounced in the 120-mmHg group than in the 80-mmHg group. The pathological changes were more pronounced in the soleus than in the EDL muscles, indicating that these two muscles differed in sensitivity to repeated compression. Additionally, average muscle wet weight and average fiber diameter for both types of muscle were increased in the 1-day and 3-day compression groups and decreased in the 1-week, 2-
Offprint requests to: M. Takemitsu Received for publication on Oct. 22, 1997; accepted on Feb. 26, 1998 *No benefit in any form has been received or will be received from any commercial party related directly or indirectly to the subject of this article
209
week, and 4-week compression groups. These findings clearly differ from those of previously reported single-compression experiments. Our findings indicate that repeated compression may cause serious muscle degeneration, particularly in red muscles. Key words: skeletal muscle, pneumatic tourniquet, repeated compression, histopathology, histochemistry
Introduction Chronic compartment syndrome is a well known cause of muscle pain in humans, especially in the lower leg and lumbar muscles during and/or after physical exercise.1,3,5,14 The pathogenesis of this syndrome appears to be repeated elevation of intramuscular pressures, resulting in muscle damage, since repeatedly elevated intramuscular pressure of approximately 60–80 mmHg in the examined compartments has been demonstrated in physiological experiments.2,7,13 In many studies, pathological changes in damaged muscles have been demonstrated in acute compartment syndrome.6,9,12 Changes in muscles have also been experimentally investigated by several authors,8,10 most of whom used pneumatic tourniquet compression or an infusion of autologous plasma to increase intramuscular pressure. These experiments were based on a model of acute compartment syndrome in which the muscles were compressed only a single time. However, few studies of chronic compartment syndrome have been reported. The pathogenesis of chronic compartment syndrome may differ from that of acute compartment syndrome. In this study we examined histopathological changes in muscle after repeated pneumatic tourniquet compression mimicking chronic compartment syndrome in an animal model.
210
Y.-H. Bai et al.: Muscle pathology after repeated compression
Materials and methods Animal preparation Ninety-nine female Japanese white rabbits were used (age, 20 weeks; weight, 3–4 kg). They were kept under specific pathogen-free conditions and allowed free access to food and water throughout the experiments. Ninety animals were kept in cages, without anesthesia, during the muscle compression experiments and for histopathological evaluation. In 9 rabbits, a tipped catheter transducer (Camino, Neuro Care, San Diego, CA, USA) was used to measure the intramuscular pressure induced by the cuff of a pneumatic tourniquet (Nakamura Ika, Tokyo, Japan). These conditions had no obvious effect on the lives of any experimental animal (Ichikawaya, Tokyo, Japan). Compression testing Ninety rabbits were allocated to 15 groups, of 6 rabbits each. In each rabbit, the calf of one leg was compressed with the cuff of the pneumatic tourniquet (Fig. 1). The compression pressure and duration of pressure in each group are summarized in Table 1. The compression was applied twice a day as follows: cuff on for 2 h, off for 30 min, and on again for 2 h each day (i.e., the muscle was compressed for a total of 4 h each day). The contralateral hindlimb, which was not compressed, served as a control.
of fiber types in muscles in complete cross sections were observed microscopically. Photomicrographs were taken with a Microflex UFX-DX camera (Nikon, Tokyo, Japan) at a final image magnification of 1003. Muscle wet weight and fiber diameter The ratios of muscle wet weight to the animal’s body weight were compared for compressed and control muscles. In the stained cross sections, the diameters of 500 fibers in each muscle were measured with a TV camera (CCTV Camera HV-750, Hitachi, Tokyo, Japan) and digital picture analyzer (VM-1730; Hitachi, Tokyo, Japan). Statistical analysis Statistical analysis was performed with the paired Student’s t-test with P 5 0.05 being the minimum significant difference between the compressed and control muscles in muscle wet weight and fiber diameter. One-way analysis of variance (ANOVA) was also performed to compare differences between the compressed and control muscles in muscle wet weight and fiber diameter. All values are presented as means 6 SEM.
Histopathology and histochemistry The animals were killed by intravenous injection of an overdose of pentobarbital sodium 24 h after the last compression. The control and compressed soleus and EDL muscles were excised, weighed while wet, and fixed in isopentane chilled with liquid nitrogen. Two hundred slices of serially sectioned 10-µm cryosections of each muscle were stained with hematoxylin and eosin (H&E) and adenosine triphosphate (ATP)ase (routine and acid preincubation). Cryosections of each muscle several hundred microns away from the area compressed by the cuff were also stained with H&E. The pathological changes in each group and the distribution
Fig. 1. The right calf of a rabbit was compressed with the cuff of a pneumatic tourniquet
Table 1. Pressure and duration of compression I (n 5 6)
II (n 5 6)
III (n 5 6)
IV (n 5 6)
V (n 5 6)
VI (n 5 6)
VII (n 5 6)
VIII (n 5 6)
IX (n 5 6)
X (n 5 6)
XI (n 5 6)
XII (n 5 6)
XIII (n 5 6)
XIV (n 5 6)
XV (n 5 6)
Pressure (mmHg)
40
80
120
40
80
120
40
80
120
40
80
120
40
80
120
Duration (days)
1
1
1
3
3
3
7
7
7
14
14
14
28
28
28
Group
Y.-H. Bai et al.: Muscle pathology after repeated compression
Results Pathological changes Changes were more severe in specimens with a greater number of compressions and greater compression pressures. Changes characteristically included muscle fiber necrosis, interstitial fibrosis, and variation in fiber size, as well as hypertrophy, atrophy, or occasional small groups of basophilic regenerating fibers. Interstitial fibrosis and muscle fiber necrosis were more pronounced in the central zone than near the peripheral zone or adjacent to the bone. Regional necrosis was localized in the centers of fascicles. Regions near the necrotic fiber exhibited variation in fiber size and regeneration. Some fibers had some mononuclear cell infiltration, whereas others were of nearly normal appearance. These changes in each muscle were observed not only in sections from beneath the cuff but also in sections several hundred microns away from cuff areas. These changes were more pronounced in the soleus than in the EDL. In the 1-day compression groups (I, II, and III), the soleus and EDL muscles were almost normal in appearance except for mild edematous changes with inflammatory reaction and increase in fiber diameter. In the 3-day compression groups (IV, V, and VI), the severity of degenerative muscle changes depended on the pressure level. The soleus and EDL muscles in group IV were almost normal in appearance, as in the 1-day compression groups. Several fibers with some mononuclear cell infiltrates, indicating early-stage necrosis, were detected in the soleus in group V (Fig. 2a), but variation in fiber size and a few small foci of inflammation were noted in the EDL (Fig. 2b). In the soleus in group VI, scattered necrotic fibers with phagocytosis were observed, along with regenerative fibers and some hypertrophic fibers. A few necrotic fibers were seen in the EDL in group VI. In the 1-week compression groups (VII, VIII, and IX) soleus muscles had necrotic fibers with phagocytosis and regenerating fibers, the density of which increased with increases in pressure level. In the soleus in group VII, there were a few necrotic fibers and mild variation in fiber size. One-fifth of the cross-sectional area of the soleus in group VIII (Fig. 2c) was occupied by necrotic, atrophic, and regenerating fibers, and fibrosis. The pathological changes in the soleus in group IX were more pronounced than those in group VIII. In contrast, in the EDL in group VII, a few areas of focal fiber necrosis and local cellular infiltration were observed, but without central nuclei, and several necrotic and central nuclei fibers were seen in the EDL in groups VIII (Fig. 2d) and IX.
211
In the 2-week compression groups (X, XI, and XII), the densities of necrotic and regenerating fibers in the soleus muscles were increased compared with those in the 1-week compression group with the same pressures. Hypertrophic whorled fibers were present in the soleus in groups XI (Fig. 2e), but were rare in group X. Some fibers in group XI exhibited central nuclear aggregation, indicating partial necrosis and regeneration. There were no hypertrophic whorled fibers in group XII. EDL muscles in group X had findings similar to those in group VII. In the EDL in group XI (Fig. 2f), one-third of the muscle fibers were necrotic and many regenerating fibers were present. The density of the necrotic fibers in the EDL in group XII was higher than that in group XI. No hypertrophic whorled fibers were seen in any EDL muscle in groups X, XI, and XII. Even with 4 weeks of repeated compression, half of the cross-sectional area of the EDL muscles in groups XIV (Fig. 2g) and XV exhibited necrotic and regenerative fibers with mild interstital fibrosis, while in group XIII, several necrotic fibers were seen, but no hypertrophic whorled fibers. A few necrotic fibers were detected in the soleus muscles in group XIII, and no increased fibrosis was seen. Although numerous regenerative fibers were present in the soleus in groups XIV (Fig. 2h) and XV, almost all other fibers were necrotic, and the interstitium exhibited marked fibrosis. No hypertrophic whorled fibers were seen in the soleus in groups XIV and XV. Intrafusal fibers remained without necrosis in all groups, even though they were surrounded by marked fibrosis in groups VIII, IX, XI, XII, XIV, and XV. No identifiable abnormality was observed in vessel walls or nerve bundles in groups I, II, III, IV, V, VI, VII, X, and XIII. The intramuscular nerve bundles and blood vessels in groups VIII, IX, XI, XII, XIV, and XV were surrounded by fibrotic tissue. The densities of necrotic fibers in the soleus and EDL muscles in each group are summarized in Table 2. Histochemical analysis Histochemical analysis demonstrated fiber types according to ATPase staining intensity. In the control groups, the soleus consisted of approximately 71% type 1 fibers with scattered type 2 (A and B) fibers (29%), and the EDL consisted of 75% type 2 (A and B) fibers with scattered type 1 fibers (25%). Type 2C fibers accounted for less than 1% of the whole fiber population in the controls. In the 3-day, 1-week, 2-week and 4-week compression groups, almost all fibers without necrosis or regeneration in the soleus and EDL muscles were type 2B fibers.
212
Fig. 2a–h. Photomicrographs. a The compressed soleus muscle with 80 mmHg pressure exerted for 3 days showed a few necrotic fibers (hollow arrow) and scattered fibers with some mononuclear cell infiltrations. b In the compressed extensor digitorum longus (EDL) muscle with 80 mmHg pressure in the 3-day group, no necrotic fibers were seen, but there was some variation in fibers size. The compressed soleus muscle with
Y.-H. Bai et al.: Muscle pathology after repeated compression
80 mmHg pressure in c 1-week, e 2-week, and g 4-week groups and the EDL muscles with 80 mmHg pressure in d 1-week, f 2-week, and h 4-week groups had necrotic, atrophic (hollow triangle; c), regenerating (black triangle; f, g), hypertrophic whorled fibers (black arrow; e), and interstitial fibrosis (hollow five-pointed star; h). These pathological changes were more servere in the soleus than in the EDL. H&E, 3100
Y.-H. Bai et al.: Muscle pathology after repeated compression
213
Table 2. Densities of necrotic muscle fibers (%) Groups SOL EDL
I (n 5 6)
II (n 5 6)
III (n 5 6)
IV (n 5 6)
V (n 5 6)
VI (n 5 6)
VII (n 5 6)
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
6.5 6 22 0.0 6 0.0
19 6 11 3.5 6 17
0.5 6 13 45 6 6.0 61 6 8.0 0.1 6 10 7.5 6 11 15 6 10
Values are expressed as percentages:
Necrotic muscle fiber number
Total muscle fiber number SOL, Soleus; EDL, extensor digitorum longus
VIII (n 5 6)
IX (n 5 6)
X (n 5 6)
XI (n 5 6)
XII (n 5 6)
XIII (n 5 6)
XIV (n 5 6)
XV (n 5 6)
0.6 6 15 0.2 6 9.0
80 6 8.0 34 6 8.0
86 6 9.0 42 6 8.0
1.0 6 15 0.4 6 13
89 6 9.0 46 6 6.0
99 6 11 55 6 7.0
3 100 (mean 6 SD)
Fig. 3a,b. Changes in a soleus (SOL) and b (EDL) muscle wet weight after repeated compression. Squares, 40 mmHg; dots, 80 mmHg; triangles, 120 mmHg
Fig. 4a,b. Changes in a soleus (SOL) and b EDL muscle fiber diameter after repeated compression. Squares, 40 mmHg; dots, 80 mmHg; triangles, 120 mmHg
Muscle wet weight and fiber diameter (Figs. 3 and 4) The compressed muscle wet weight changed in parallel with fiber diameter with changes in time and pressure. In the 80 mmHg compression groups (II, V, VIII, XI, XIV) and 120 mmHg compression groups (III, VI, IX,
XII, XV), muscle wet weight and fiber diameter increased day by day until the third day of compression, returned to control level by the seventh day, and then decreased further. The changes in wet weight and fiber diameter in the soleus were more pronounced than those in the EDL in all groups. Although changes in wet
214
Y.-H. Bai et al.: Muscle pathology after repeated compression
Table 3. Relationship between tourniquet compression pressure and intramuscular pressure Pressure (mmHg) Tourniquet Intramuscular
40 20.0 6 0.6
80 49.6 6 1.0
120 70.1 6 1.7
weight and fiber diameter in the 40 mmHg compression groups (I, IV, VII, X, XIII) exhibited a similar course to that in the other groups, there was no significant difference in either parameter between the 40 mmHg compression and control groups on any experimental day. In the second and fourth experimental weeks, the 80 (XI and XIV) and 120 mmHg compression groups (XII and XV) had significantly lighter wet weight and smaller fiber diameter than did the controls (P , 0.05), indicating muscle atrophy. The muscle wet weight and fiber diameter of the soleus muscle in group XV were the lightest and the smallest of either muscle in any group. Intramuscular pressure The compression-induced intramuscular pressure increased with increases in pneumatic tourniquet compression pressure (Table 3).
Discussion A repeatedly compressed muscle is thought to be a model of chronic compartment syndrome after exercise, since intramuscular pressure is repeatedly elevated in both conditions. Qvarfordt et al.11 reported that the average intramuscular pressure during excessive physical exercise was 80 mmHg. Hargens et al.4 showed that capillary blood flow was disturbed when the intramuscular pressure exceeded 30 mmHg. However, Pedowitz9 found no pathological changes in muscle that was compressed only once for 2 h at less than 125 mmHg pressure. Based on the above findings, we set the pneumatic tourniquet pressure levels to be tested at 40, 80 and 120 mmHg instead of using only one compression, and the periods of repeated compression were set at 1 day, 3 days, 1 week, 2 weeks, and 4 weeks. Clinically, chronic compartment syndrome is seen mostly in the anterior tibial compartment. One may ask why the EDL and soleus muscles were used in this study, and why the anterior tibial muscle was not. The anterior tibial muscle contains more type 1 fibers than the EDL does; it is not a “white” muscle model. It is difficult to evaluate the characteristic changes of “white” and “red” muscles with the anterior tibial muscle. We therefore used the EDL muscle in the same
anterior compartment as a “white” muscle model and the soleus in the superficial posterior compartment as a “red” muscle model. Although the thickness of the fascia and the muscle volume differ in each compartment, the cuff compression method can induce high intramuscular pressure in the compartments of interest. The behavior of muscles after repeated compression differs from that of muscles in acute compartment syndrome and muscles undergoing neurogenic atrophy.15 Although it is possible that pathological findings similar to those following repeated compression can be observed several weeks after acute compression, the threshold of intramuscular pressure for inducing myonecrosis with repeated compression is smaller than that required for acute compression. For example, with less than 125 mmHg compression for 2 h, no clear necrosis was detected in an acute compartment model;9 however, in the present study, repeated compression at 80 mmHg pressure for 2 h (intramuscular pressure level, 50 mmHg) resulted in marked degenerative changes. These findings suggest that excessive physical exercise in which there is an intramuscular pressure of 50 mmHg may induce muscle degenerative changes similar to those seen in the present study. There is a difference in the time course of degeneration between muscles compressed repeatedly and those compressed once. Usually, necrotic fibers with phagocytosis are seen within 1 day of acute compression. In the present study, in muscle which had undergone repeated 80-mmHg compression, obvious necrosis was detected after 1-week compression, although a few necrotic fibers were observed on the third day of compression. In contrast, in muscles which had undergone repeated 40-mmHg compression, few fibers were necrotic. In the latter condition, the average intramuscular pressure was below 30 mmHg, and muscle blood flow was apparently not reduced during compression.4 The pathological changes in muscles with 1-day compression (groups I, II, and III) were similar to those seen after 2–4 h of ischemia by Sanderson et al.,12 who reported the principal changes as acute inflammatory reaction with edema. Characteristic changes in repeatedly compressed muscles were necrotic, atrophic, regenerating, and hypertrophic (whorled) fibers, and interstitial fibrosis. The mechanism of formation of the hypertrophic whorled and centrally regenerating fibers remains unclear. These fibers may have been formed after partial necrosis induced by mild and repeated compression. However, no hypertrophic whorled fibers were detected in either the 4-week 80-mmHg compression group or the 2- or 4-week 120 mmHg compression group. There may be a threshold for the number of repetitions and/ or compression pressure above which hypertrophic whorled fibers are formed.
Y.-H. Bai et al.: Muscle pathology after repeated compression
The soleus muscle was more severely affected by repeated compression than the EDL; this may have been a consequence of the different metabolic styles and blood supplies of these two muscles. The soleus is a “red” muscle (consisting mainly of type 1 fibers), while the EDL is a “white” muscle (consisting almost entirely of type 2 fibers). Red muscle has a more extensive blood supply, higher oxidative enzyme activity, and lower glycolytic activity than white muscle, suggesting that red muscle may not be able to withstand the ischemia induced by compression for a long period. Muscle wet weight and fiber diameter exhibited parallel changes over time at each compression pressure. In the 80- and 120-mmHg compression groups, muscle wet weight and fiber diameter increased day by day until the third day of compression, returned to control level by the seventh day, and then decreased further. This pattern of change until the third day of compression may have reflected the edematous swelling of muscle fibers, increasing muscle wet weight and fiber diameter. Moore et al.6 reported that the application of a pneumatic tourniquet for 2 h was followed by an approximately 75% increase in water in ischemic tissue. They showed, in electron micrographs, that muscle fibers in advanced stages of degeneration had increased fluid content. This finding appears to be supported by findings of visible damage to the capillary endothelium and sarcolemma after tourniquet.6 Therefore, the main factor influencing muscle wet weight and fiber diameter in our 1- and 3day compression groups (I, II, III, IV, V, and VI) was probably edematous change. Although there were some hypertrophic fibers in groups with more than 1-week compression (VII, VIII, IX, X, XI, XII, XIII, XIV, and XV), many small regenerating, atrophic, and partial necrotic fibers, and interstitial fibrosis were observed in these groups as well. Muscle wet weight and fiber diameter may therefore have decreased as a result of the fiber necrosis and fibrotic changes. The question arises whether muscle that has degenerated following repeated compression can recover. It appears that it may be able to, since even the muscle with the longest and strongest compression (group XV) had numerous regenerating fibers. However, complete recovery usually does not occur, because of fibrosis.
215
For adequate planning of physical exercise, we need to clarify the threshold of intramuscular pressure and density below which complete recovery is possible in humans. In conclusion, our findings show that degenerative changes occur in muscle after repeated compression, and that these changes are particularly pronounced in red muscle.
References 1. Beckham SG, Grana WA, Buckley P, et al. A comparison of anterior compartment pressures in competitive runners and cyclists. Am J Sports Med 1993;21:36–40. 2. Binkhorst FMP, Slaaf DW, Kuipers H, et al. Exercise-induced swelling of rat soleus muscle: Its relationship with intramuscular pressure. J Appl Physiol 1990;69:67–73. 3. Davey JR, Rorabeck CH, Fowler PJ. The tibialis posterior muscle compartment. An unrecognized cause of exertional compartment. Am J Sports Med 1984;12:391–7. 4. Hargens AR, Akeson WH, Mubarak SJ, et al. Fluid balance within the canine anterolateral compartment and its relationship to compartment syndromes. J Bone Joint Surg Am 1978;60:499–505. 5. Lawson SK, Reid DC, Wiley JP. Anterior compartment pressures in cross-country skiers. A comparison of classic and skating skis. Am J Sports Med 1992;20:750–3. 6. Moore DH, Ruska H, Copenhaver WM. Electron microscopic and histochemical observations of muscle degeneration after tourniquet. J Biophys Biochem Cytol 1956;2:755–63. 7. Ogata K, Whiteside LA. Effects of external compression on blood flow to muscle and skin. Clin Orthop 1982;168:105–7. 8. Patterson S, Klienerman L. The effect of pneumatic tourniquets on the ultrastructure of skeletal muscle. J Bone Joint Surg Br 1979;61;178–83. 9. Pedowitz RA. Tourniquet-induced neuromuscular injury. A recent review of rabbit and clinical experiments. Acta Orthop Scand Suppl 1991;245:1–33. 10. Pedowitz RA, Rydevik BL, Gershuni DH, et al. An animal model for the study of neuromuscular injury induced beneath and distal to a pneumatic tourniquet. J Orthop Res 1990;8:899–908. 11. Qvarfordt P, Christenson JT, Eklöf B. Intramuscular pressure, muscle blood flow, and skeletal muscle metabolism in chronic anterior tibial compartment syndrome. Clin Orthop 1983;179:284–90. 12. Sanderson RA, Foley RK, McIvor GWD. Histological response of skeletal muscle to ischemia. Clin Orthop 1975;113:27–35. 13. Styf J, Körner L, Suurkula M. Intramuscular pressure and muscle blood flow during exercise in chronic compartment syndrome. J Bone Joint Surg Br 1987;69:301–5. 14. Styf J, Lysell E. Chronic compartment syndrome in the erector spinae muscle. Spine 1987;12:680–2. 15. Uchino M, Uyama E, Hirano T, et al. A histochemical and electron microscopic study of skeletal muscle in an adult case of Chédiak-Higashi syndrome. Acta Neuropathol 1993;86:521–4.
209
Pathology study of rabbit calf muscles after repeated compression* Yue-Hong Bai1,2, Masakazu Takemitsu1, Yuji Atsuta1, and Yoshiharu Takemitsu1 1 2
Department of Orthopaedic Surgery, Asahikawa Medical College, Nishikagura 4-5, 3-11, Asahikawa, Hokkaido 078-8510, Japan Department of Orthopaedic Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P. R. China
Abstract: To elucidate the pathogenesis of chronic compartment syndrome, we examined pathological changes in the soleus (red) and extensor digitorum longus (EDL; white) muscles in Japanese white rabbits after repeated compression with a pneumatic tourniquet. Repeated tourniquet compression via cuff inflation was carried out on the rabbits, calves daily, for 2 h, then stopped for 30 min, and then applied for another 2 h. The contralateral hindlimb, which was not compressed, served as a control. Animals were allocated to 15 groups, with pressures of 40, 80, and 120 mmHg for periods of 1 day, 3 days, 1 week, 2 weeks, and 4 weeks. Skeletal muscle specimens in each group were studied by histopathological and histochemical (ATPase) methods. After compression for 1 day, regardless of pressure, and compression for 3 days in the 40-mmHg pressure group, edematous changes in regions with mild inflammation and increases in fiber diameter were observed in the muscles. After compression for 3 days in the 80- and 120-mmHg pressure groups, and after 1, 2, or 4 weeks in the 40-mmHg pressure group, a few necrotic fibers and scattered fibers with some mononuclear cell infiltrates indicative of early-stage necrosis were detected. In the groups with 80 or 120 mmHg pressure for 1, 2, or 4 weeks, muscle fibers exhibited marked degenerative changes, which were more pronounced in the 120-mmHg group than in the 80-mmHg group. The pathological changes were more pronounced in the soleus than in the EDL muscles, indicating that these two muscles differed in sensitivity to repeated compression. Additionally, average muscle wet weight and average fiber diameter for both types of muscle were increased in the 1-day and 3-day compression groups and decreased in the 1-week, 2-
Offprint requests to: M. Takemitsu Received for publication on Oct. 22, 1997; accepted on Feb. 26, 1998 *No benefit in any form has been received or will be received from any commercial party related directly or indirectly to the subject of this article
209
week, and 4-week compression groups. These findings clearly differ from those of previously reported single-compression experiments. Our findings indicate that repeated compression may cause serious muscle degeneration, particularly in red muscles. Key words: skeletal muscle, pneumatic tourniquet, repeated compression, histopathology, histochemistry
Introduction Chronic compartment syndrome is a well known cause of muscle pain in humans, especially in the lower leg and lumbar muscles during and/or after physical exercise.1,3,5,14 The pathogenesis of this syndrome appears to be repeated elevation of intramuscular pressures, resulting in muscle damage, since repeatedly elevated intramuscular pressure of approximately 60–80 mmHg in the examined compartments has been demonstrated in physiological experiments.2,7,13 In many studies, pathological changes in damaged muscles have been demonstrated in acute compartment syndrome.6,9,12 Changes in muscles have also been experimentally investigated by several authors,8,10 most of whom used pneumatic tourniquet compression or an infusion of autologous plasma to increase intramuscular pressure. These experiments were based on a model of acute compartment syndrome in which the muscles were compressed only a single time. However, few studies of chronic compartment syndrome have been reported. The pathogenesis of chronic compartment syndrome may differ from that of acute compartment syndrome. In this study we examined histopathological changes in muscle after repeated pneumatic tourniquet compression mimicking chronic compartment syndrome in an animal model.
210
Y.-H. Bai et al.: Muscle pathology after repeated compression
Materials and methods Animal preparation Ninety-nine female Japanese white rabbits were used (age, 20 weeks; weight, 3–4 kg). They were kept under specific pathogen-free conditions and allowed free access to food and water throughout the experiments. Ninety animals were kept in cages, without anesthesia, during the muscle compression experiments and for histopathological evaluation. In 9 rabbits, a tipped catheter transducer (Camino, Neuro Care, San Diego, CA, USA) was used to measure the intramuscular pressure induced by the cuff of a pneumatic tourniquet (Nakamura Ika, Tokyo, Japan). These conditions had no obvious effect on the lives of any experimental animal (Ichikawaya, Tokyo, Japan). Compression testing Ninety rabbits were allocated to 15 groups, of 6 rabbits each. In each rabbit, the calf of one leg was compressed with the cuff of the pneumatic tourniquet (Fig. 1). The compression pressure and duration of pressure in each group are summarized in Table 1. The compression was applied twice a day as follows: cuff on for 2 h, off for 30 min, and on again for 2 h each day (i.e., the muscle was compressed for a total of 4 h each day). The contralateral hindlimb, which was not compressed, served as a control.
of fiber types in muscles in complete cross sections were observed microscopically. Photomicrographs were taken with a Microflex UFX-DX camera (Nikon, Tokyo, Japan) at a final image magnification of 1003. Muscle wet weight and fiber diameter The ratios of muscle wet weight to the animal’s body weight were compared for compressed and control muscles. In the stained cross sections, the diameters of 500 fibers in each muscle were measured with a TV camera (CCTV Camera HV-750, Hitachi, Tokyo, Japan) and digital picture analyzer (VM-1730; Hitachi, Tokyo, Japan). Statistical analysis Statistical analysis was performed with the paired Student’s t-test with P 5 0.05 being the minimum significant difference between the compressed and control muscles in muscle wet weight and fiber diameter. One-way analysis of variance (ANOVA) was also performed to compare differences between the compressed and control muscles in muscle wet weight and fiber diameter. All values are presented as means 6 SEM.
Histopathology and histochemistry The animals were killed by intravenous injection of an overdose of pentobarbital sodium 24 h after the last compression. The control and compressed soleus and EDL muscles were excised, weighed while wet, and fixed in isopentane chilled with liquid nitrogen. Two hundred slices of serially sectioned 10-µm cryosections of each muscle were stained with hematoxylin and eosin (H&E) and adenosine triphosphate (ATP)ase (routine and acid preincubation). Cryosections of each muscle several hundred microns away from the area compressed by the cuff were also stained with H&E. The pathological changes in each group and the distribution
Fig. 1. The right calf of a rabbit was compressed with the cuff of a pneumatic tourniquet
Table 1. Pressure and duration of compression I (n 5 6)
II (n 5 6)
III (n 5 6)
IV (n 5 6)
V (n 5 6)
VI (n 5 6)
VII (n 5 6)
VIII (n 5 6)
IX (n 5 6)
X (n 5 6)
XI (n 5 6)
XII (n 5 6)
XIII (n 5 6)
XIV (n 5 6)
XV (n 5 6)
Pressure (mmHg)
40
80
120
40
80
120
40
80
120
40
80
120
40
80
120
Duration (days)
1
1
1
3
3
3
7
7
7
14
14
14
28
28
28
Group
Y.-H. Bai et al.: Muscle pathology after repeated compression
Results Pathological changes Changes were more severe in specimens with a greater number of compressions and greater compression pressures. Changes characteristically included muscle fiber necrosis, interstitial fibrosis, and variation in fiber size, as well as hypertrophy, atrophy, or occasional small groups of basophilic regenerating fibers. Interstitial fibrosis and muscle fiber necrosis were more pronounced in the central zone than near the peripheral zone or adjacent to the bone. Regional necrosis was localized in the centers of fascicles. Regions near the necrotic fiber exhibited variation in fiber size and regeneration. Some fibers had some mononuclear cell infiltration, whereas others were of nearly normal appearance. These changes in each muscle were observed not only in sections from beneath the cuff but also in sections several hundred microns away from cuff areas. These changes were more pronounced in the soleus than in the EDL. In the 1-day compression groups (I, II, and III), the soleus and EDL muscles were almost normal in appearance except for mild edematous changes with inflammatory reaction and increase in fiber diameter. In the 3-day compression groups (IV, V, and VI), the severity of degenerative muscle changes depended on the pressure level. The soleus and EDL muscles in group IV were almost normal in appearance, as in the 1-day compression groups. Several fibers with some mononuclear cell infiltrates, indicating early-stage necrosis, were detected in the soleus in group V (Fig. 2a), but variation in fiber size and a few small foci of inflammation were noted in the EDL (Fig. 2b). In the soleus in group VI, scattered necrotic fibers with phagocytosis were observed, along with regenerative fibers and some hypertrophic fibers. A few necrotic fibers were seen in the EDL in group VI. In the 1-week compression groups (VII, VIII, and IX) soleus muscles had necrotic fibers with phagocytosis and regenerating fibers, the density of which increased with increases in pressure level. In the soleus in group VII, there were a few necrotic fibers and mild variation in fiber size. One-fifth of the cross-sectional area of the soleus in group VIII (Fig. 2c) was occupied by necrotic, atrophic, and regenerating fibers, and fibrosis. The pathological changes in the soleus in group IX were more pronounced than those in group VIII. In contrast, in the EDL in group VII, a few areas of focal fiber necrosis and local cellular infiltration were observed, but without central nuclei, and several necrotic and central nuclei fibers were seen in the EDL in groups VIII (Fig. 2d) and IX.
211
In the 2-week compression groups (X, XI, and XII), the densities of necrotic and regenerating fibers in the soleus muscles were increased compared with those in the 1-week compression group with the same pressures. Hypertrophic whorled fibers were present in the soleus in groups XI (Fig. 2e), but were rare in group X. Some fibers in group XI exhibited central nuclear aggregation, indicating partial necrosis and regeneration. There were no hypertrophic whorled fibers in group XII. EDL muscles in group X had findings similar to those in group VII. In the EDL in group XI (Fig. 2f), one-third of the muscle fibers were necrotic and many regenerating fibers were present. The density of the necrotic fibers in the EDL in group XII was higher than that in group XI. No hypertrophic whorled fibers were seen in any EDL muscle in groups X, XI, and XII. Even with 4 weeks of repeated compression, half of the cross-sectional area of the EDL muscles in groups XIV (Fig. 2g) and XV exhibited necrotic and regenerative fibers with mild interstital fibrosis, while in group XIII, several necrotic fibers were seen, but no hypertrophic whorled fibers. A few necrotic fibers were detected in the soleus muscles in group XIII, and no increased fibrosis was seen. Although numerous regenerative fibers were present in the soleus in groups XIV (Fig. 2h) and XV, almost all other fibers were necrotic, and the interstitium exhibited marked fibrosis. No hypertrophic whorled fibers were seen in the soleus in groups XIV and XV. Intrafusal fibers remained without necrosis in all groups, even though they were surrounded by marked fibrosis in groups VIII, IX, XI, XII, XIV, and XV. No identifiable abnormality was observed in vessel walls or nerve bundles in groups I, II, III, IV, V, VI, VII, X, and XIII. The intramuscular nerve bundles and blood vessels in groups VIII, IX, XI, XII, XIV, and XV were surrounded by fibrotic tissue. The densities of necrotic fibers in the soleus and EDL muscles in each group are summarized in Table 2. Histochemical analysis Histochemical analysis demonstrated fiber types according to ATPase staining intensity. In the control groups, the soleus consisted of approximately 71% type 1 fibers with scattered type 2 (A and B) fibers (29%), and the EDL consisted of 75% type 2 (A and B) fibers with scattered type 1 fibers (25%). Type 2C fibers accounted for less than 1% of the whole fiber population in the controls. In the 3-day, 1-week, 2-week and 4-week compression groups, almost all fibers without necrosis or regeneration in the soleus and EDL muscles were type 2B fibers.
212
Fig. 2a–h. Photomicrographs. a The compressed soleus muscle with 80 mmHg pressure exerted for 3 days showed a few necrotic fibers (hollow arrow) and scattered fibers with some mononuclear cell infiltrations. b In the compressed extensor digitorum longus (EDL) muscle with 80 mmHg pressure in the 3-day group, no necrotic fibers were seen, but there was some variation in fibers size. The compressed soleus muscle with
Y.-H. Bai et al.: Muscle pathology after repeated compression
80 mmHg pressure in c 1-week, e 2-week, and g 4-week groups and the EDL muscles with 80 mmHg pressure in d 1-week, f 2-week, and h 4-week groups had necrotic, atrophic (hollow triangle; c), regenerating (black triangle; f, g), hypertrophic whorled fibers (black arrow; e), and interstitial fibrosis (hollow five-pointed star; h). These pathological changes were more servere in the soleus than in the EDL. H&E, 3100
Y.-H. Bai et al.: Muscle pathology after repeated compression
213
Table 2. Densities of necrotic muscle fibers (%) Groups SOL EDL
I (n 5 6)
II (n 5 6)
III (n 5 6)
IV (n 5 6)
V (n 5 6)
VI (n 5 6)
VII (n 5 6)
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
0.0 6 0.0 0.0 6 0.0
6.5 6 22 0.0 6 0.0
19 6 11 3.5 6 17
0.5 6 13 45 6 6.0 61 6 8.0 0.1 6 10 7.5 6 11 15 6 10
Values are expressed as percentages:
Necrotic muscle fiber number
Total muscle fiber number SOL, Soleus; EDL, extensor digitorum longus
VIII (n 5 6)
IX (n 5 6)
X (n 5 6)
XI (n 5 6)
XII (n 5 6)
XIII (n 5 6)
XIV (n 5 6)
XV (n 5 6)
0.6 6 15 0.2 6 9.0
80 6 8.0 34 6 8.0
86 6 9.0 42 6 8.0
1.0 6 15 0.4 6 13
89 6 9.0 46 6 6.0
99 6 11 55 6 7.0
3 100 (mean 6 SD)
Fig. 3a,b. Changes in a soleus (SOL) and b (EDL) muscle wet weight after repeated compression. Squares, 40 mmHg; dots, 80 mmHg; triangles, 120 mmHg
Fig. 4a,b. Changes in a soleus (SOL) and b EDL muscle fiber diameter after repeated compression. Squares, 40 mmHg; dots, 80 mmHg; triangles, 120 mmHg
Muscle wet weight and fiber diameter (Figs. 3 and 4) The compressed muscle wet weight changed in parallel with fiber diameter with changes in time and pressure. In the 80 mmHg compression groups (II, V, VIII, XI, XIV) and 120 mmHg compression groups (III, VI, IX,
XII, XV), muscle wet weight and fiber diameter increased day by day until the third day of compression, returned to control level by the seventh day, and then decreased further. The changes in wet weight and fiber diameter in the soleus were more pronounced than those in the EDL in all groups. Although changes in wet
214
Y.-H. Bai et al.: Muscle pathology after repeated compression
Table 3. Relationship between tourniquet compression pressure and intramuscular pressure Pressure (mmHg) Tourniquet Intramuscular
40 20.0 6 0.6
80 49.6 6 1.0
120 70.1 6 1.7
weight and fiber diameter in the 40 mmHg compression groups (I, IV, VII, X, XIII) exhibited a similar course to that in the other groups, there was no significant difference in either parameter between the 40 mmHg compression and control groups on any experimental day. In the second and fourth experimental weeks, the 80 (XI and XIV) and 120 mmHg compression groups (XII and XV) had significantly lighter wet weight and smaller fiber diameter than did the controls (P , 0.05), indicating muscle atrophy. The muscle wet weight and fiber diameter of the soleus muscle in group XV were the lightest and the smallest of either muscle in any group. Intramuscular pressure The compression-induced intramuscular pressure increased with increases in pneumatic tourniquet compression pressure (Table 3).
Discussion A repeatedly compressed muscle is thought to be a model of chronic compartment syndrome after exercise, since intramuscular pressure is repeatedly elevated in both conditions. Qvarfordt et al.11 reported that the average intramuscular pressure during excessive physical exercise was 80 mmHg. Hargens et al.4 showed that capillary blood flow was disturbed when the intramuscular pressure exceeded 30 mmHg. However, Pedowitz9 found no pathological changes in muscle that was compressed only once for 2 h at less than 125 mmHg pressure. Based on the above findings, we set the pneumatic tourniquet pressure levels to be tested at 40, 80 and 120 mmHg instead of using only one compression, and the periods of repeated compression were set at 1 day, 3 days, 1 week, 2 weeks, and 4 weeks. Clinically, chronic compartment syndrome is seen mostly in the anterior tibial compartment. One may ask why the EDL and soleus muscles were used in this study, and why the anterior tibial muscle was not. The anterior tibial muscle contains more type 1 fibers than the EDL does; it is not a “white” muscle model. It is difficult to evaluate the characteristic changes of “white” and “red” muscles with the anterior tibial muscle. We therefore used the EDL muscle in the same
anterior compartment as a “white” muscle model and the soleus in the superficial posterior compartment as a “red” muscle model. Although the thickness of the fascia and the muscle volume differ in each compartment, the cuff compression method can induce high intramuscular pressure in the compartments of interest. The behavior of muscles after repeated compression differs from that of muscles in acute compartment syndrome and muscles undergoing neurogenic atrophy.15 Although it is possible that pathological findings similar to those following repeated compression can be observed several weeks after acute compression, the threshold of intramuscular pressure for inducing myonecrosis with repeated compression is smaller than that required for acute compression. For example, with less than 125 mmHg compression for 2 h, no clear necrosis was detected in an acute compartment model;9 however, in the present study, repeated compression at 80 mmHg pressure for 2 h (intramuscular pressure level, 50 mmHg) resulted in marked degenerative changes. These findings suggest that excessive physical exercise in which there is an intramuscular pressure of 50 mmHg may induce muscle degenerative changes similar to those seen in the present study. There is a difference in the time course of degeneration between muscles compressed repeatedly and those compressed once. Usually, necrotic fibers with phagocytosis are seen within 1 day of acute compression. In the present study, in muscle which had undergone repeated 80-mmHg compression, obvious necrosis was detected after 1-week compression, although a few necrotic fibers were observed on the third day of compression. In contrast, in muscles which had undergone repeated 40-mmHg compression, few fibers were necrotic. In the latter condition, the average intramuscular pressure was below 30 mmHg, and muscle blood flow was apparently not reduced during compression.4 The pathological changes in muscles with 1-day compression (groups I, II, and III) were similar to those seen after 2–4 h of ischemia by Sanderson et al.,12 who reported the principal changes as acute inflammatory reaction with edema. Characteristic changes in repeatedly compressed muscles were necrotic, atrophic, regenerating, and hypertrophic (whorled) fibers, and interstitial fibrosis. The mechanism of formation of the hypertrophic whorled and centrally regenerating fibers remains unclear. These fibers may have been formed after partial necrosis induced by mild and repeated compression. However, no hypertrophic whorled fibers were detected in either the 4-week 80-mmHg compression group or the 2- or 4-week 120 mmHg compression group. There may be a threshold for the number of repetitions and/ or compression pressure above which hypertrophic whorled fibers are formed.
Y.-H. Bai et al.: Muscle pathology after repeated compression
The soleus muscle was more severely affected by repeated compression than the EDL; this may have been a consequence of the different metabolic styles and blood supplies of these two muscles. The soleus is a “red” muscle (consisting mainly of type 1 fibers), while the EDL is a “white” muscle (consisting almost entirely of type 2 fibers). Red muscle has a more extensive blood supply, higher oxidative enzyme activity, and lower glycolytic activity than white muscle, suggesting that red muscle may not be able to withstand the ischemia induced by compression for a long period. Muscle wet weight and fiber diameter exhibited parallel changes over time at each compression pressure. In the 80- and 120-mmHg compression groups, muscle wet weight and fiber diameter increased day by day until the third day of compression, returned to control level by the seventh day, and then decreased further. This pattern of change until the third day of compression may have reflected the edematous swelling of muscle fibers, increasing muscle wet weight and fiber diameter. Moore et al.6 reported that the application of a pneumatic tourniquet for 2 h was followed by an approximately 75% increase in water in ischemic tissue. They showed, in electron micrographs, that muscle fibers in advanced stages of degeneration had increased fluid content. This finding appears to be supported by findings of visible damage to the capillary endothelium and sarcolemma after tourniquet.6 Therefore, the main factor influencing muscle wet weight and fiber diameter in our 1- and 3day compression groups (I, II, III, IV, V, and VI) was probably edematous change. Although there were some hypertrophic fibers in groups with more than 1-week compression (VII, VIII, IX, X, XI, XII, XIII, XIV, and XV), many small regenerating, atrophic, and partial necrotic fibers, and interstitial fibrosis were observed in these groups as well. Muscle wet weight and fiber diameter may therefore have decreased as a result of the fiber necrosis and fibrotic changes. The question arises whether muscle that has degenerated following repeated compression can recover. It appears that it may be able to, since even the muscle with the longest and strongest compression (group XV) had numerous regenerating fibers. However, complete recovery usually does not occur, because of fibrosis.
215
For adequate planning of physical exercise, we need to clarify the threshold of intramuscular pressure and density below which complete recovery is possible in humans. In conclusion, our findings show that degenerative changes occur in muscle after repeated compression, and that these changes are particularly pronounced in red muscle.
References 1. Beckham SG, Grana WA, Buckley P, et al. A comparison of anterior compartment pressures in competitive runners and cyclists. Am J Sports Med 1993;21:36–40. 2. Binkhorst FMP, Slaaf DW, Kuipers H, et al. Exercise-induced swelling of rat soleus muscle: Its relationship with intramuscular pressure. J Appl Physiol 1990;69:67–73. 3. Davey JR, Rorabeck CH, Fowler PJ. The tibialis posterior muscle compartment. An unrecognized cause of exertional compartment. Am J Sports Med 1984;12:391–7. 4. Hargens AR, Akeson WH, Mubarak SJ, et al. Fluid balance within the canine anterolateral compartment and its relationship to compartment syndromes. J Bone Joint Surg Am 1978;60:499–505. 5. Lawson SK, Reid DC, Wiley JP. Anterior compartment pressures in cross-country skiers. A comparison of classic and skating skis. Am J Sports Med 1992;20:750–3. 6. Moore DH, Ruska H, Copenhaver WM. Electron microscopic and histochemical observations of muscle degeneration after tourniquet. J Biophys Biochem Cytol 1956;2:755–63. 7. Ogata K, Whiteside LA. Effects of external compression on blood flow to muscle and skin. Clin Orthop 1982;168:105–7. 8. Patterson S, Klienerman L. The effect of pneumatic tourniquets on the ultrastructure of skeletal muscle. J Bone Joint Surg Br 1979;61;178–83. 9. Pedowitz RA. Tourniquet-induced neuromuscular injury. A recent review of rabbit and clinical experiments. Acta Orthop Scand Suppl 1991;245:1–33. 10. Pedowitz RA, Rydevik BL, Gershuni DH, et al. An animal model for the study of neuromuscular injury induced beneath and distal to a pneumatic tourniquet. J Orthop Res 1990;8:899–908. 11. Qvarfordt P, Christenson JT, Eklöf B. Intramuscular pressure, muscle blood flow, and skeletal muscle metabolism in chronic anterior tibial compartment syndrome. Clin Orthop 1983;179:284–90. 12. Sanderson RA, Foley RK, McIvor GWD. Histological response of skeletal muscle to ischemia. Clin Orthop 1975;113:27–35. 13. Styf J, Körner L, Suurkula M. Intramuscular pressure and muscle blood flow during exercise in chronic compartment syndrome. J Bone Joint Surg Br 1987;69:301–5. 14. Styf J, Lysell E. Chronic compartment syndrome in the erector spinae muscle. Spine 1987;12:680–2. 15. Uchino M, Uyama E, Hirano T, et al. A histochemical and electron microscopic study of skeletal muscle in an adult case of Chédiak-Higashi syndrome. Acta Neuropathol 1993;86:521–4.