THE ANATOMICAL RECORD 233:376-386 (1992)
Muscle Atrophy Continues After Early Cast Removal Following Tendon Repair L.C. MAXWELL, M.R. MOODY, AND C.S. ENWEMEKA Department of Physiology (L.C.M.,, M.R.M.) and Division of Physical Therapy (C.S.E.), University of Texas HeaEth Scaence Center at S a n Antonio, Sun Antonio, Texas 78284-7756;Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine, and Research Service, Veterans Affairs Medical Center, Miami, Florida 33146 (C.S.E.)
ABSTRACT We studied soleus (SOL), plantaris (PLN), and gastrocnemius (GST) muscles to determine whether early cast removal minimizes muscle atrophy or permits recovery from atrophy after tendon repair. After right tendocalcaneus (Achilles tendon) was transected and repaired, rabbit right hindlimbs were immobilized with the ankle plantar flexed and the knee flexed to 90". Rabbits were maintained in the cast and sacrificed at 5,15, or 21 days postoperatively or the cast was removed on day 5 and the animals sacrificed at day 15 or 21. SOL, PLN, and GST muscles of both limbs were removed and weighed, and then histochemical analyses were performed on SOL and PLN muscles. Immobilization decreased SOL muscle wet weights, mean fiber cross-sectional area, and percentage of Type I fibers and increased the percentage of Type IIc fibers. Ten days after cast removal (i.e., postoperative day 151, SOL muscle atrophy and fiber composition did not differ significantly from continuously immobilized controls. However, 16 days after cast removal (i.e., postoperative day 211, SOL muscle fiber cross-sectional area and fiber composition were near normal, differing significantly from continuously casted controls. At each of the time intervals studied, PLN (containing many glycolytic fibers) did not atrophy as much as SOL (containing mainly oxidative fibers). Our results indicate that 1)early cast removal prevents atrophy of PLN glycolytic fibers, but not oxidative fibers in either PLN or SOL, and 2) early cast removal promotes recovery from atrophy of both oxidative and glycolytic fibers. In spite of the many differences between rabbits and humans, these findings suggest that, although early cast removal may not prevent oxidative muscle fiber atrophy after postoperative immobilization, i t may facilitate recovery from atrophy. 0 1992 Wiley-Liss, Inc.
It is well documented that chronic limb immobilization results in severe muscle atrophy, especially in those muscles immobilized in a shortened position (Summers and Hines, 1951; Ferguson et al., 1957; Booth and Kelso, 1973a,b; Edgerton e t al., 1975; Booth, 1977; Goldspink, 1977; Herbison e t al., 1978, 1979; Sjostrom, 1979; Mayer et al., 1981; Spector et al., 1982; Appell, 1986; Gossman et al., 1986). Atrophy is associated with a net loss of muscle protein initiated by a decreased rate of protein synthesis within hours of immobilization (Goldspink, 1977; Booth and Seider, 1979a). Initial muscle atrophy is rapid, with up to one-half of the final extent of atrophy occurring within the first week of immobilization, but the severity of muscle atrophy increases for several weeks after application of the cast (Booth, 1977; Sjostrom et al., 1979; Appell, 1986). Much of the loss of muscle mass can be accounted for by reduction in muscle fiber cross-sectional area. However, fibers of muscles immobilized in a shortened position also lose sarcomeres from the end of the fibers (Tabary et al., 1972; Williams and Goldspink, 1978; Baker and Matsumoto, 1988). In rats, speed of contraction of the slow twitch soleus 0 1992 WILEY-LISS, INC
(SOL) muscle increases following immobilization. This correlates with a decrease in the percentage of Type I fibers and a n increase in the percentage of Type I1 fibers after 4 weeks of immobilization (Booth and Kelso, 197313). These authors interpreted their data as a preferential loss of a number of Type I fibers. However, Cardenas et al. (19771, using a different technique, found no significant difference in the number of fibers in rat SOL after 4 weeks of immobilization. The decreasing percentage of Type I fibers during immobilization likely results from transformation of fiber types rather than a loss of Type I fibers. The rate of recovery from atrophy induced by immobilization appears related to the duration of immobilization. In SOL muscles of rats that had hindlimbs immobilized for 42 days, 4 and 7 days are required for recovery of contraction time and half-relaxation time, respectively, and 28 days are needed for recovery of
Received December 18, 1990; accepted October 4, 1991.
377
ATROPHY CONTINUES AFTER EARLY CAST REMOVAL
peak tetanic tension (Witzmann et al., 1982). In rats that had hindlimbs immobilized for 90 days in plantar flexion, muscle protein concentration and wet weight require 60 days, and maximum isometric tension requires 120 days to return to control values following cast removal (Booth and Seider, 197913). Upon remobilization, muscle fibers return to normal length within 6 weeks by adding sarcomeres (Tabary et al., 1972). Despite detrimental effects on muscle, it is common clinical practice to immobilize the lower leg with muscles in a shortened position for 6 to 8 weeks after surgical repair of the tendocalcaneus (Christensen, 1954; Arner and Lindholm, 1959; Lea and Smith, 1972; Quigley and Sheller, 1980; Nistor, 1981; Carden et al., 1987). This procedure produces atrophy and weakening of the calf muscles, thereby necessitating extensive postoperative rehabilitation. The treatment of tendon ruptures without pronounced muscular atrophy would have major clinical benefit by expediting rehabilitation and restoration of function. Baker and Matsumoto (1988) suggested casting ankles in neutral position to reduce atrophy, and Kellam et al. (1985) advocate a s much dorsiflexion as repair of Achilles tendon will allow. Recent data on rabbits indicate that the tendocalcaneus is capable of bearing weight as soon as 5 days following repair (Enwemeka et al., 1988). The purposes of this investigation were to test the hypotheses that 1) early cast removal will minimize atrophy of th e SOL, plantaris (PLN), and gastrocnemius (GST) muscles following tenotomy and repair of the rabbit tendocalcaneus and 2) early cast removal will permit recovery from atrophy induced during immobilization. Little information is available on the effects of immobilization of one limb upon the muscles in the contralateral limb. Therefore, 3) we also determined the influence of unilateral hind limb immobilization on the muscles of the contralateral uncasted leg. MATERIALS AND METHODS Animal Care
Twenty-three adult female New Zealand rabbits were randomly assigned to control or experimental groups. Animals were housed in 30.5 x 71 x 51 cm rabbit cages and were kept on a 12 hrl12 h r lightldark cycle. The room temperature was maintained a t 2123°C. Rabbits were fed rabbit chow and water ad libitum and were examined regularly by a veterinarian.
TABLE 1. Experimental desim ~
Group'
Control CT 05 CT15 CT 21 WB15 WB21
N 3
4 3 SOL 5PLN 5 GST 3 3 3
Immobili- Days in Days withSurgery zation cast out cast None None None Yes
Yes
5
None
Yes
Yes
15
None
Yes Yes Yes
Yes Yes Yes
21 5 5
None 10 16
'CT, continuously casted, numbers indicate postoperative days; WB, early cast removal, numbers indicate postoperative days.
skin was incised, and the tendocalcaneus of the right hindlimb was exposed by blunt dissection, separated from surrounding tissue, and severed transversely 3 cm proximal to the calcaneal attachment. The tendon was sutured with No. 3 nonreabsorbable silk so that the tendon components were aligned end to end (Enwemeka, 1989). Following surgery, a rigid plaster cast was applied to the limb with the ankle in full plantar flexion and the knee flexed 90". Casts were inspected frequently and repaired or replaced as needed. No animal required more than one replacement during the experimental period. Muscle Samples
Rabbits were weighed and anesthetized a s described above. The SOL, a slow twitch muscle, and the PLN and GST, predominantly fast twitch muscles, of the experimental and contralateral limb were removed. The muscles were weighed, mounted on tongue depressors, precooled in ice-cold saline, and quick frozen in isopentane cooled in liquid nitrogen for histochemical analysis. Precooling of the excised muscles was necessary because atrophied experimental muscles were so fragile that cutting a sample from the midpoint of the muscle belly was impossible without causing major disruption of muscle organization. Precooling allowed the relatively large intact muscles to be frozen without serious freezing artifact. Histochemical Analysis
The frozen muscles were equilibrated to the cryostat temperature (-2O"C), and a 3-kmm-thick slice was cut from the thickest portion of the belly perpendicular to Experimental Design the long axis of the muscle. The remainder of each The right tendocalcaneus of anesthetized experimen- frozen muscle was weighed and then oven dried to tal animals was severed, and repaired, and the hind- constant weight for determination of water content. The limb was placed in a cast with the knee angle at 90" frozen slice was mounted on a cryostat chuck, and 10 pm and the ankle in full plantar flexion (Enwemeka, cross sections were cut. Following room temperature air 1989). Groups of animals were maintained either with drying of the cross sections for 30-60 min, enzymatic continuous hindlimb immobilization until the time of analyses were performed. Enzymatic reactions for mysacrifice or with immobilization terminated by cast re- ofibrillar adenosine triphosphatase (ATPase), performed after alkaline or acid preincubation, were used moval a t 5 days (Table 1). as correlates of contractile velocity (Dubowitz and Operative Procedures Brooke, 1973). Fibers were classified as Type I or Type The rabbits were weighed and anesthetized with in- I1 based on low or high ATPase activity, respectively, tramuscular injection of 1 mU1.5 kg body weight of a after alkaline preincubation. Type I1 muscle fibers that mixture containing 100 mg/ml ketamine, 20 mg/ml xy- did not demonstrate a reversal in ATPase activity follazene, and 10 mg/ml acepromazine. Operative proce- lowing acid preincubation were labeled Type IIc. The dures were performed under sterile conditions. The remaining Type I1 fibers were classified Type 110 or
378
L.C. MAXWELL ET AL.
TABLE 2. Muscle weight
Experimental Group' Control CT 06 CT 15 CT 21 WB 15 WB 21
SOL 1.32 t 0.04 0.96 2 0.12* 0.75 t 0.21" 0.55 t 0.02* 0.48 t 0.02* 0.68 t 0.03*
PLN 3.59 f 0.35 2.86 f 0.20 3.14 f 0.34 2.32 f 0.12* 2.35 f 0.43* 2.73 f 0.14
Contralateral GST
SOL
8.11 f 0.86 8.99 f 0.78 7.89 f 0.70 8.38 k 0.26 5.54 t 0.47' 7.38 +- 1.12
1.32 f 0.04 1.36 f 0.04 1.82 t 0.22 1.89 f 0.24 1.08 f 0.10+ 1.08 f 0.17'
PLN 3.59 f 0.35 3.41 f 0.15 4.00 f 0.47 4.31 f 0.23 3.04 f 0.08 3.58 f 0.16+
GST 8.11 f 0.86 9.88 f 0.87 9.90 f 0.71 10.87 k 0.53 7.36 f 0.48' 8.55 1.06'
*
'CT,continuously casted, numbers indicate postoperative days; WB, early cast removal, numbers indicate postoperative days. *Experimental significantly different from control ( P < 0.05). 'WB 15 significantly different from CT 15 or WB 21 significantly different from CT 21 ( P < 0.05).
Type IIg (Maxwell et al., 1989) based on high or low oxidative capacity indicated by the activity of reduced nicotinamide adenine dinucleotide tetrazoleum reductase (NADH-TR). Following completion of enzymatic reactions, sections were dehydrated in a n ascending series of ethanol (70%, 95%, and loo%), cleared in xylene, and mounted with Permount. Muscle sections were observed with a Zeiss microscope equipped with a drawing tube that allows tracing of a magnified image of individual muscle fibers. Three sample sites of each muscle were chosen using a random stratified sampling scheme. Each muscle cross section was grossly divided into three regions, and one sample site was randomly selected from each of these regions. This ensured that all regions of the muscle had equal opportunity to be sampled. Drawings were made of fiber profiles within a standardized rectangular sample area. Magnification was selected that included approximately 50 fibers in each sample site. Fibers crossing the top and right edges of the rectangle including the top left corner were included in the drawings, while those crossing the left and bottom edges and bottom right corner were excluded. The fiber type composition of each muscle was determined by counting the number of fibers of each type in the sample area. The crosssectional area of each fiber was measured from the drawings using a Bioquant digitizer. The mean fiber area for each fiber type of each muscle sample was calculated.
Muscle Wet Weight
Soleus
SOL wet weight was progressively reduced throughout 21 days of continuous casting (Table 2; Figure 1A). In WB animals, this decline in muscle weight (MW) through postoperative day 15 (10 days of recovery) was not prevented by removal of the cast at day 5. However, between postoperative days 15 and 21, the weight of SOL muscles in WB animals became greater than that in CT animals. Expressed a s muscle weight to body weight ratio (MW/BW), the recovery between days 15 and 21 was statistically significant (Table 3; P < 0.05). Contralateral SOL muscles of CT animals became heavier during continuous casting. Expressed as muscle weight per body weight, the hypertrophy of contralateral SOL in CT relative to control SOL was significant a t postoperative days 15 and 21 (P < 0.05). Although contralateral SOL of WB animals did not differ from control SOL in MW or MW/BW, they weighed significantly less t h a n their counterparts in CT animals (Tables 2, 3; Fig. 1A; P < 0.05). Plantaris In CT animals, PLN MW was significantly less than control only a t 21 days (Table 2; Fig. 1B; P < 0.051, but MWlBW declined significantly throughout continuous immobilization (Table 3; P < 0.05). In WB animals, PLN MW was less than control a t 10 but not 16 days after cast removal. MW/BW was significantly less than control a t both times. Differences from control in PLN MW or MW/BW of contralateral limbs in CT and WB Statistical Analysis animals were not significant. However, contralateral Data was pooled for each group, and the mean, stan- PLN weight in CT animals was significantly greater dard deviation, and standard error of the mean were than in WB animals (Table 2; P < 0.05). calculated. Data are presented as mean ? standard error. Analysis of variance and Student Neuman Gastrocnemius Kuehl tests were used to compare the fiber area and There were no significant differences of MW or MWI percentages of fiber types in muscles of experimental BW from control for experimental or contralateral GST groups and controls. An unpaired Student's t test was of CT (Tables 2,3; Fig. 1C). Experimental GST muscles performed to compare continuously immobilized to cast of WB were smaller than control at 10, but not at 16, removal groups a t common time points. All statistical procedures were performed a s described by Zar (1974). RESULTS Body Weight
There was no significant difference from control body weight in any experimental group nor between experimental groups a t any postoperative time.
CT GST PLN SOL uc WB
Ab breuiations continuously casted following tendon repair gastrocnemius muscle plantaris muscle soleus muscle muscle from contralateral uncasted limb cast removal at post-operative day 5.
ATROPHY CONTINUES AFTER EARLY CAST REMOVAL
379
days after cast removal. However, MW was significantly less for WB than CT experimental and contralateral GST at 10 days after cast removal, and for WB than CT contralateral GST after 16 days after cast removal. MW/BW was significantly less than control in experimental GST of WB 15, and MW/BW of WB 15 GST was significantly less than of CT 15 GST for experimental and contralateral limbs (P < 0.05). Muscle Dry Weight/Wet Weight
Compared with controls, no significant differences were found in the ratio of dry to wet muscle weights for any experimental group. Fiber Type Composition
Soleus
The SOL of control animals is a predominantly slow, oxidative muscle, containing -97% Type I fibers,
Muscle Atrophy Continues After Early Cast Removal Following Tendon Repair L.C. MAXWELL, M.R. MOODY, AND C.S. ENWEMEKA Department of Physiology (L.C.M.,, M.R.M.) and Division of Physical Therapy (C.S.E.), University of Texas HeaEth Scaence Center at S a n Antonio, Sun Antonio, Texas 78284-7756;Department of Orthopaedics and Rehabilitation, Division of Physical Therapy, University of Miami School of Medicine, and Research Service, Veterans Affairs Medical Center, Miami, Florida 33146 (C.S.E.)
ABSTRACT We studied soleus (SOL), plantaris (PLN), and gastrocnemius (GST) muscles to determine whether early cast removal minimizes muscle atrophy or permits recovery from atrophy after tendon repair. After right tendocalcaneus (Achilles tendon) was transected and repaired, rabbit right hindlimbs were immobilized with the ankle plantar flexed and the knee flexed to 90". Rabbits were maintained in the cast and sacrificed at 5,15, or 21 days postoperatively or the cast was removed on day 5 and the animals sacrificed at day 15 or 21. SOL, PLN, and GST muscles of both limbs were removed and weighed, and then histochemical analyses were performed on SOL and PLN muscles. Immobilization decreased SOL muscle wet weights, mean fiber cross-sectional area, and percentage of Type I fibers and increased the percentage of Type IIc fibers. Ten days after cast removal (i.e., postoperative day 151, SOL muscle atrophy and fiber composition did not differ significantly from continuously immobilized controls. However, 16 days after cast removal (i.e., postoperative day 211, SOL muscle fiber cross-sectional area and fiber composition were near normal, differing significantly from continuously casted controls. At each of the time intervals studied, PLN (containing many glycolytic fibers) did not atrophy as much as SOL (containing mainly oxidative fibers). Our results indicate that 1)early cast removal prevents atrophy of PLN glycolytic fibers, but not oxidative fibers in either PLN or SOL, and 2) early cast removal promotes recovery from atrophy of both oxidative and glycolytic fibers. In spite of the many differences between rabbits and humans, these findings suggest that, although early cast removal may not prevent oxidative muscle fiber atrophy after postoperative immobilization, i t may facilitate recovery from atrophy. 0 1992 Wiley-Liss, Inc.
It is well documented that chronic limb immobilization results in severe muscle atrophy, especially in those muscles immobilized in a shortened position (Summers and Hines, 1951; Ferguson et al., 1957; Booth and Kelso, 1973a,b; Edgerton e t al., 1975; Booth, 1977; Goldspink, 1977; Herbison e t al., 1978, 1979; Sjostrom, 1979; Mayer et al., 1981; Spector et al., 1982; Appell, 1986; Gossman et al., 1986). Atrophy is associated with a net loss of muscle protein initiated by a decreased rate of protein synthesis within hours of immobilization (Goldspink, 1977; Booth and Seider, 1979a). Initial muscle atrophy is rapid, with up to one-half of the final extent of atrophy occurring within the first week of immobilization, but the severity of muscle atrophy increases for several weeks after application of the cast (Booth, 1977; Sjostrom et al., 1979; Appell, 1986). Much of the loss of muscle mass can be accounted for by reduction in muscle fiber cross-sectional area. However, fibers of muscles immobilized in a shortened position also lose sarcomeres from the end of the fibers (Tabary et al., 1972; Williams and Goldspink, 1978; Baker and Matsumoto, 1988). In rats, speed of contraction of the slow twitch soleus 0 1992 WILEY-LISS, INC
(SOL) muscle increases following immobilization. This correlates with a decrease in the percentage of Type I fibers and a n increase in the percentage of Type I1 fibers after 4 weeks of immobilization (Booth and Kelso, 197313). These authors interpreted their data as a preferential loss of a number of Type I fibers. However, Cardenas et al. (19771, using a different technique, found no significant difference in the number of fibers in rat SOL after 4 weeks of immobilization. The decreasing percentage of Type I fibers during immobilization likely results from transformation of fiber types rather than a loss of Type I fibers. The rate of recovery from atrophy induced by immobilization appears related to the duration of immobilization. In SOL muscles of rats that had hindlimbs immobilized for 42 days, 4 and 7 days are required for recovery of contraction time and half-relaxation time, respectively, and 28 days are needed for recovery of
Received December 18, 1990; accepted October 4, 1991.
377
ATROPHY CONTINUES AFTER EARLY CAST REMOVAL
peak tetanic tension (Witzmann et al., 1982). In rats that had hindlimbs immobilized for 90 days in plantar flexion, muscle protein concentration and wet weight require 60 days, and maximum isometric tension requires 120 days to return to control values following cast removal (Booth and Seider, 197913). Upon remobilization, muscle fibers return to normal length within 6 weeks by adding sarcomeres (Tabary et al., 1972). Despite detrimental effects on muscle, it is common clinical practice to immobilize the lower leg with muscles in a shortened position for 6 to 8 weeks after surgical repair of the tendocalcaneus (Christensen, 1954; Arner and Lindholm, 1959; Lea and Smith, 1972; Quigley and Sheller, 1980; Nistor, 1981; Carden et al., 1987). This procedure produces atrophy and weakening of the calf muscles, thereby necessitating extensive postoperative rehabilitation. The treatment of tendon ruptures without pronounced muscular atrophy would have major clinical benefit by expediting rehabilitation and restoration of function. Baker and Matsumoto (1988) suggested casting ankles in neutral position to reduce atrophy, and Kellam et al. (1985) advocate a s much dorsiflexion as repair of Achilles tendon will allow. Recent data on rabbits indicate that the tendocalcaneus is capable of bearing weight as soon as 5 days following repair (Enwemeka et al., 1988). The purposes of this investigation were to test the hypotheses that 1) early cast removal will minimize atrophy of th e SOL, plantaris (PLN), and gastrocnemius (GST) muscles following tenotomy and repair of the rabbit tendocalcaneus and 2) early cast removal will permit recovery from atrophy induced during immobilization. Little information is available on the effects of immobilization of one limb upon the muscles in the contralateral limb. Therefore, 3) we also determined the influence of unilateral hind limb immobilization on the muscles of the contralateral uncasted leg. MATERIALS AND METHODS Animal Care
Twenty-three adult female New Zealand rabbits were randomly assigned to control or experimental groups. Animals were housed in 30.5 x 71 x 51 cm rabbit cages and were kept on a 12 hrl12 h r lightldark cycle. The room temperature was maintained a t 2123°C. Rabbits were fed rabbit chow and water ad libitum and were examined regularly by a veterinarian.
TABLE 1. Experimental desim ~
Group'
Control CT 05 CT15 CT 21 WB15 WB21
N 3
4 3 SOL 5PLN 5 GST 3 3 3
Immobili- Days in Days withSurgery zation cast out cast None None None Yes
Yes
5
None
Yes
Yes
15
None
Yes Yes Yes
Yes Yes Yes
21 5 5
None 10 16
'CT, continuously casted, numbers indicate postoperative days; WB, early cast removal, numbers indicate postoperative days.
skin was incised, and the tendocalcaneus of the right hindlimb was exposed by blunt dissection, separated from surrounding tissue, and severed transversely 3 cm proximal to the calcaneal attachment. The tendon was sutured with No. 3 nonreabsorbable silk so that the tendon components were aligned end to end (Enwemeka, 1989). Following surgery, a rigid plaster cast was applied to the limb with the ankle in full plantar flexion and the knee flexed 90". Casts were inspected frequently and repaired or replaced as needed. No animal required more than one replacement during the experimental period. Muscle Samples
Rabbits were weighed and anesthetized a s described above. The SOL, a slow twitch muscle, and the PLN and GST, predominantly fast twitch muscles, of the experimental and contralateral limb were removed. The muscles were weighed, mounted on tongue depressors, precooled in ice-cold saline, and quick frozen in isopentane cooled in liquid nitrogen for histochemical analysis. Precooling of the excised muscles was necessary because atrophied experimental muscles were so fragile that cutting a sample from the midpoint of the muscle belly was impossible without causing major disruption of muscle organization. Precooling allowed the relatively large intact muscles to be frozen without serious freezing artifact. Histochemical Analysis
The frozen muscles were equilibrated to the cryostat temperature (-2O"C), and a 3-kmm-thick slice was cut from the thickest portion of the belly perpendicular to Experimental Design the long axis of the muscle. The remainder of each The right tendocalcaneus of anesthetized experimen- frozen muscle was weighed and then oven dried to tal animals was severed, and repaired, and the hind- constant weight for determination of water content. The limb was placed in a cast with the knee angle at 90" frozen slice was mounted on a cryostat chuck, and 10 pm and the ankle in full plantar flexion (Enwemeka, cross sections were cut. Following room temperature air 1989). Groups of animals were maintained either with drying of the cross sections for 30-60 min, enzymatic continuous hindlimb immobilization until the time of analyses were performed. Enzymatic reactions for mysacrifice or with immobilization terminated by cast re- ofibrillar adenosine triphosphatase (ATPase), performed after alkaline or acid preincubation, were used moval a t 5 days (Table 1). as correlates of contractile velocity (Dubowitz and Operative Procedures Brooke, 1973). Fibers were classified as Type I or Type The rabbits were weighed and anesthetized with in- I1 based on low or high ATPase activity, respectively, tramuscular injection of 1 mU1.5 kg body weight of a after alkaline preincubation. Type I1 muscle fibers that mixture containing 100 mg/ml ketamine, 20 mg/ml xy- did not demonstrate a reversal in ATPase activity follazene, and 10 mg/ml acepromazine. Operative proce- lowing acid preincubation were labeled Type IIc. The dures were performed under sterile conditions. The remaining Type I1 fibers were classified Type 110 or
378
L.C. MAXWELL ET AL.
TABLE 2. Muscle weight
Experimental Group' Control CT 06 CT 15 CT 21 WB 15 WB 21
SOL 1.32 t 0.04 0.96 2 0.12* 0.75 t 0.21" 0.55 t 0.02* 0.48 t 0.02* 0.68 t 0.03*
PLN 3.59 f 0.35 2.86 f 0.20 3.14 f 0.34 2.32 f 0.12* 2.35 f 0.43* 2.73 f 0.14
Contralateral GST
SOL
8.11 f 0.86 8.99 f 0.78 7.89 f 0.70 8.38 k 0.26 5.54 t 0.47' 7.38 +- 1.12
1.32 f 0.04 1.36 f 0.04 1.82 t 0.22 1.89 f 0.24 1.08 f 0.10+ 1.08 f 0.17'
PLN 3.59 f 0.35 3.41 f 0.15 4.00 f 0.47 4.31 f 0.23 3.04 f 0.08 3.58 f 0.16+
GST 8.11 f 0.86 9.88 f 0.87 9.90 f 0.71 10.87 k 0.53 7.36 f 0.48' 8.55 1.06'
*
'CT,continuously casted, numbers indicate postoperative days; WB, early cast removal, numbers indicate postoperative days. *Experimental significantly different from control ( P < 0.05). 'WB 15 significantly different from CT 15 or WB 21 significantly different from CT 21 ( P < 0.05).
Type IIg (Maxwell et al., 1989) based on high or low oxidative capacity indicated by the activity of reduced nicotinamide adenine dinucleotide tetrazoleum reductase (NADH-TR). Following completion of enzymatic reactions, sections were dehydrated in a n ascending series of ethanol (70%, 95%, and loo%), cleared in xylene, and mounted with Permount. Muscle sections were observed with a Zeiss microscope equipped with a drawing tube that allows tracing of a magnified image of individual muscle fibers. Three sample sites of each muscle were chosen using a random stratified sampling scheme. Each muscle cross section was grossly divided into three regions, and one sample site was randomly selected from each of these regions. This ensured that all regions of the muscle had equal opportunity to be sampled. Drawings were made of fiber profiles within a standardized rectangular sample area. Magnification was selected that included approximately 50 fibers in each sample site. Fibers crossing the top and right edges of the rectangle including the top left corner were included in the drawings, while those crossing the left and bottom edges and bottom right corner were excluded. The fiber type composition of each muscle was determined by counting the number of fibers of each type in the sample area. The crosssectional area of each fiber was measured from the drawings using a Bioquant digitizer. The mean fiber area for each fiber type of each muscle sample was calculated.
Muscle Wet Weight
Soleus
SOL wet weight was progressively reduced throughout 21 days of continuous casting (Table 2; Figure 1A). In WB animals, this decline in muscle weight (MW) through postoperative day 15 (10 days of recovery) was not prevented by removal of the cast at day 5. However, between postoperative days 15 and 21, the weight of SOL muscles in WB animals became greater than that in CT animals. Expressed a s muscle weight to body weight ratio (MW/BW), the recovery between days 15 and 21 was statistically significant (Table 3; P < 0.05). Contralateral SOL muscles of CT animals became heavier during continuous casting. Expressed as muscle weight per body weight, the hypertrophy of contralateral SOL in CT relative to control SOL was significant a t postoperative days 15 and 21 (P < 0.05). Although contralateral SOL of WB animals did not differ from control SOL in MW or MW/BW, they weighed significantly less t h a n their counterparts in CT animals (Tables 2, 3; Fig. 1A; P < 0.05). Plantaris In CT animals, PLN MW was significantly less than control only a t 21 days (Table 2; Fig. 1B; P < 0.051, but MWlBW declined significantly throughout continuous immobilization (Table 3; P < 0.05). In WB animals, PLN MW was less than control a t 10 but not 16 days after cast removal. MW/BW was significantly less than control a t both times. Differences from control in PLN MW or MW/BW of contralateral limbs in CT and WB Statistical Analysis animals were not significant. However, contralateral Data was pooled for each group, and the mean, stan- PLN weight in CT animals was significantly greater dard deviation, and standard error of the mean were than in WB animals (Table 2; P < 0.05). calculated. Data are presented as mean ? standard error. Analysis of variance and Student Neuman Gastrocnemius Kuehl tests were used to compare the fiber area and There were no significant differences of MW or MWI percentages of fiber types in muscles of experimental BW from control for experimental or contralateral GST groups and controls. An unpaired Student's t test was of CT (Tables 2,3; Fig. 1C). Experimental GST muscles performed to compare continuously immobilized to cast of WB were smaller than control at 10, but not at 16, removal groups a t common time points. All statistical procedures were performed a s described by Zar (1974). RESULTS Body Weight
There was no significant difference from control body weight in any experimental group nor between experimental groups a t any postoperative time.
CT GST PLN SOL uc WB
Ab breuiations continuously casted following tendon repair gastrocnemius muscle plantaris muscle soleus muscle muscle from contralateral uncasted limb cast removal at post-operative day 5.
ATROPHY CONTINUES AFTER EARLY CAST REMOVAL
379
days after cast removal. However, MW was significantly less for WB than CT experimental and contralateral GST at 10 days after cast removal, and for WB than CT contralateral GST after 16 days after cast removal. MW/BW was significantly less than control in experimental GST of WB 15, and MW/BW of WB 15 GST was significantly less than of CT 15 GST for experimental and contralateral limbs (P < 0.05). Muscle Dry Weight/Wet Weight
Compared with controls, no significant differences were found in the ratio of dry to wet muscle weights for any experimental group. Fiber Type Composition
Soleus
The SOL of control animals is a predominantly slow, oxidative muscle, containing -97% Type I fibers,
