]>EXG5379S0531-5565(00)00189-310.1016/S0531-5565(00)00189-3Elsevier Science Inc.Fig. 1Net percent gain in muscle weights of young and old rats after 4weeks of remobilisation. Data are expressed as means ±SD (n=5). ∗p<0.05 for muscles of old rats versus young rats.Fig. 2Net percent change of biochemical parameters in gastrocnemius muscles of young and old rats after 4weeks of remobilisation. CPK, creatine phosphokinase; ACP, acid phosphatase; LPX, lipid peroxidation. Data are expressed as means ±SD (n=5). ∗p<0.05 for biochemical data of old versus young muscles.Fig. 3Transverse sections of gastrocnemius muscle (frozen sections stained Hematoxylin and Eosin). (a) left control (contralateral) leg of young (6months old) rat; (b) left control (contralateral) leg of old (26months old) rat; (c) right immobilised leg of old rat (4weeks of external fixation); (d) right leg of old rat (4weeks of external fixation, 4weeks of remobilisation). The external fixation causes major atrophic changes in myofibres, which only manage partial recovery on remobilisation.Fig. 4Details of myofibres of gastrocnemius muscle of old rats after 4weeks of external fixation (a, b); and after 4weeks of external fixation followed by 4weeks of remobilisation (c, d). (a) Transverse section showing myofibre degeneration. Many myofibres develop small vacuoles. (b) Ultrastructural details of myofibre showing sarcomere disruption and loss of myofilaments. (c) Myopathic damage is still clearly evident and there is only limited recovery 4weeks after removal of the external fixation. (d) In some cases lysosome-like bodies are prominent in myofibers, which in many cases remain vacuolated. (a, c, d, 1μm epon sections stained alkaline toluidine blue; b, transmission electron micrograph)Table 1The mean (±SD) body-weight of old rats before and after 4weeks of external fixation and before and after an additional 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationBeforeAfterBeforeAfterBody-weight (g)398±69.8373.8±62.7428.3±63.3450±57Mean difference (g)−2521.795% confidence interval (CI) of difference−127.76 to 71.76−66.15 to 109.55Change (%)−6.1%5.14%p ValueNSNSTable 2The mean (±SD) change in the weight (mg) of the hindlimb muscles in old rats after 4weeks of external fixation and 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationMuscleContralateral legImmobilised legMean difference95% CI of differenceChange (%)Contralateral legImmobilised legMean difference95% CI of differenceChange (%)Gastrocnemius1556±204914±129−642−891 to −393−41.27*1510±1671073±118−437−648 to −226−28.9*Quadriceps2172±1001221±88−951−1088 to −814−43.77**2314±1261541±139−773−996 to −579−33.4*Plantaris231±11121±11−110−126 to −94−46.6**250±17144±9>−106−126 to −86−42.3*Soleus171±16103±20−6.8−94 to −42−39.5*166±15104±1−62−77 to −46−37.3**p<0.001.**p<0.01, contralateral control v immobilised legs.Table 3Mean (±SD) values for the activity of ACP, CPK and lipid peroxidation in the gastrocnemius muscle of old rats after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationMuscleContralateral legImmobilised legMean difference95% CI of difference (%)ChangeContralateral legImmobilised legMean difference95% CI of differenceChange (%)ACP activity (mU/mg)a28.6±1.9538.1±69.472.96 to 15.97+33.229.5±1.8732.6±1.763.10.45 to 5.75+10.7p valuep<0.02p<0.02CPK activity (U/mg)a4.5±0.793.73±0.78−0.77−1.91 to 0.37−17.24.17±0.413.63±0.31−0.54−1.07 to −0.01−12.6p valuep<0.01Lipid peroxidation (nmol MDA/mg protein)b4.13±16.61±0.352.481.38 to 3.57+60.04.04±1.75.26±1.71.22−1.26 to 3.70+30p valuep<0.02NSaActivity of enzymes is expressed per mg of soluble proteins in the supernatant.bMDA, malondialdehyde.Table 4Comparison between young and old rats in their capacities for recovery of hindlimb muscle weights after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationYounga % weight lossOld % weight lossYounga % weight lossOld % weight lossYoung net % gainOld net % gainYoung/Old relative % gainGastrocnemius−58−41.27−28.9−28.929.912.42.41Quadriceps−62−43.77−42.7−33.419.310.31.87Plantaris−46.5−46.6−35.7−42.310.94.32.53Soleus−46.6−39.5−41.0−37.35.62.22.54aThe data for the young animals are taken from a recent publication (Zarzhevsky et al., 1999).Table 5Comparison between young and old rats in their capacities for recovery of biochemical parameters (% change) after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationYoungaOldYoungaOldYoung net % gainOld net % gainYoung/Old relative % gainACP activity+82.4+33.2−7.3−10.789.745.91.95CPK−36.5−17.2+2.9−12.639.44.68.56Lipid peroxidation+132.5+60.0+32.4+30.0100.130.03.33aThe data for the young animals are taken from a recent publication (Zarzhevsky et al., 1999).Recovery of muscles of old rats after hindlimb immobilisation by external fixation is impaired compared with those of young ratsNZarzhevskyaECarmelibDFuchscRColemanaHSteindA.ZReznicka*reznick@tx.technion.ac.ilaDepartment of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, IsraelbPhysical Therapy Program, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, IsraelcDepartment of Orthopaedics, Bnai Zion Medical Center, Haifa 31408, IsraeldDeparment of Orthopaedics A, Rambam Medical Center, Haifa 31096, Israel*Corresponding author. Tel.: +972-4-829-5388; fax: +972-4-829-5392AbstractThe right hindlimbs of 24-month-old female Wistar rats were immobilised for 4weeks using external fixation of the knee joint. In a further group, after the external fixation was removed, the rats were allowed to remobilise for an additional 4weeks. Hindlimb immobilisation for 4weeks caused a 32–42% reduction in wet weights of the hindlimb muscles of the rats as compared to those of the contralateral non-immobilised legs. After 4weeks of remobilisation the hindlimb muscles had not returned to the “control” weights. Biochemical changes in the gastrocnemius muscle resulting from the external fixation showed greatly elevated acid phosphatase activities (33.2%) and markedly reduced creatine phosphokinase activities (17.2%), which did not recover to preimmobilisation values after 4weeks of remobilisation. Light and transmission electron microscopy showed that remobilisation for 4weeks (after external fixation) resulted in only partial morphological restoration of the damage to the muscles in these aged rats. A comparison of similar hindlimb external fixation and remobilisation in young (6months old) rats showed that remobilisation caused a substantial recovery in biochemical parameters in both age groups, with the muscles of the young group (but not the old group) often reaching almost complete recovery accompanied by morphological restoration. We conclude that the net gain in the recovery period of biochemical and morphological parameters is significantly greater in the young rats compared to the old rats indicating that muscle metabolism and capacity for recovery from disuse atrophy is impaired with ageing.KeywordsDisuse atrophySkeletal muscleExternal fixationRemobilisationAgeingRats1IntroductionAs early as 1944, the cause-and-effect relationship of limb immobilisation and disuse atrophy was recognised (Eccles, 1944; Thomsen and Luco, 1944). Inactivity, bed rest, non-weight bearing, external casts or external fixation all result in loss of muscle mass or muscular atrophy. Atrophy is associated with a net loss of muscle protein synthesis initiated within hours of immobilisation (Goldspink, 1977; Booth and Seider, 1979). The initial atrophy is very rapid, especially within the first week of immobilisation (Booth, 1977; Maxwell et al., 1992; Appell, 1990). Recently a comparison of two different modes of hindlimb immobilisation (Plaster of Paris casts and external fixation) was reported (Reznick et al., 1995). In these studies several biochemical and morphological parameters were investigated as a measure of damage to muscles of immobilised limbs. It was shown that external fixation, with its more rigid immobilisation, resulted in more drastic changes and damage to the hindlimb muscles (Reznick et al., 1995).The capacity for recovery after immobilisation has been little investigated, though it is of the utmost clinical importance as the orthopaedic surgeon needs to decide at what stage the cast or external fixation should be removed in order to achieve the best outcome of the operation. Common clinical experience reveals that muscle recovery following limb immobilisation is more rapid and complete in young individuals rather than in the elderly. Relatively little is known about the effects of muscle hypoactivity in old age (Ansved, 1995). Our working hypothesis was that muscles of old animals will be affected more severely by immobilisation and will be slower to recover compared with muscles of young animals. We have recently reported experimental studies on the capacity for recovery of hindlimb muscles of young (6month old) rats after external fixation (Zarzhevsky et al., 1999). In these studies, 4weeks of remobilisation resulted in the restoration of biochemical and morphological parameters to preimmobilisation values, whereas muscle mass was still lower than preimmobilisation values. In the present study we report similar experiments conducted in aged rats (24months old) and evaluate various biochemical and morphological parameters as indicators of the capacity for recovery from disuse atrophy in these old animals.2Materials and methods2.1Animals15 female Wistar rats (24months old) were housed in standard plastic breeding cages, in groups of three, at 20°C with a 12-h light–dark cycle. They were fed a standard diet and water ad libitum. The animals were maintained in conformity with the Guiding Principles in the Care and Use of Animals of the American Physiological Society and the experimental protocol received approval by the Animal Welfare and Ethics Committee of the Technion Faculty of Medicine.The animals were divided into three groups each of five rats. Group 1 consisted of rats in which the right knee was immobilised by external fixation in a position of 40–50° flexion for 4weeks (Reznick et al., 1995). Group 2 had similar hindlimb immobilisation for 4weeks after which the external fixation was removed and the limb remobilised for a further 4weeks. Group 3 consisted of untreated age-matched animals.2.2Immobilisation by external fixation (Reznick et al., 1995)Rigid immobilisation was achieved by the insertion of two Kirschner wires (0.8mm diameter) driven in pairs through the lateral plane of the femur and tibia. Then, two threaded brass rods fitted with nuts, connected them in order to construct a rigid frame. The brass rods were 4.8mm diameter and 33mm long. Each rod was cut longitudinally from both ends to an equal length of 13mm. These cuts were 1.0–1.2mm in width in order to contain the Kirschner wires. The overall weight of the above device was 12g.2.3Biochemical studiesAnimals were sacrificed by ether anesthesia at 4weeks for Group 1 and 8weeks for Group 2. Group 3 (untreated controls) were sacrificed at 24months of age prior to the start of the experimental hindlimb immobilisation. Muscle specimens for biochemical studies (200mg each) were taken from the belly of the gastrocnemius and quadriceps muscles of the right (immobilised) and left (contralateral) hindlimbs. (The soleus and plantaris muscles weigh less than 200mg and are insufficient for biochemical analysis). The muscles were immersed in 1.4ml of 50mM tris(hydroxymethyl) aminomethane buffer, pH 7.4. The mixture was homogenised in a Polytron homogeniser (Kinematica GmbH, Lucerne, Switzerland) three times for 15s. The homogenate was centrifuged for 30min at 14,000g. The supernatant was separated and utilised for enzymatic assays. Acid phosphatase activities were determined as previously described (Reznick et al., 1995). Creatine phosphokinase assay was performed according to the method of Tanzar and Gilvard (1959) using 0.05M glycine buffer, pH 7.4. Lipid peroxidation measurements were performed using thiobarbituric acid (TBA) according to the procedure of Ohkawa et al. (1979). Protein concentration in muscle extracts was determined according to the method of Lowry et al. (1951).2.4Light and electron microscopyAt sacrifice, the gastrocnemius muscle was rapidly removed. The belly of the gastrocnemius muscle was rapidly frozen in isobutane cooled by liquid nitrogen. 6μm cryostat transverse sections were stained with Hematoxylin and Eosin (H and E). For higher resolution sections, a 2mm-thick transverse slice of the fresh gastrocnemius muscle was cut with a razor blade from a site approximately 3mm distant from the tendon of origin and immersion fixed in 3% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 containing 0.01% calcium chloride at room temperature. Blocks of tissue approximately 1mm3 were cut and left in the fixative for 3h prior to rinsing and storage overnight in 0.1M sodium cacodylate buffer containing 7.5% (wt/vol.) sucrose. This was followed by post-fixation in 1% unbuffered aqueous osmium tetroxide for 2h, dehydration in ascending ethanols, treatment with propylene oxide and epoxy embedding in Pelco Eponate 12 (Pelco International, Redding, CA, USA). After heat polymerisation (60°C for 18h), 1μm thick sections were cut on an ultramicrotome and stained with 0.1% toluidine blue in 1% borax for light microscopy. Sections (60–90nm thick) were cut with a diamond knife on the ultramicrotome, collected on uncoated copper grids, and contrast-stained with 1% uranyl acetate in 70% ethanol followed by 1% lead citrate prior to examination in a JEOL 100SX transmission electron microscope at 80kV.2.5Statistical analysisStatistical analysis was performed using either paired or independent Student's t-test. Statistical significance was set at p<0.05. Results are reported as ±SD.3Results3.1Body weightsThe body weights of animals with hindlimb immobilisation by external fixation for 4weeks and remobilisation are shown in Table 1. After 4weeks of immobilisation (Group 1), the old animals lost 6.1%, which was not significant. On remobilisation for 4weeks (Group 2) the rats had made a complete recovery of body weights and even showed a slight increase (5.14%). The differences between body weights at the start of the experiment and following external fixation and remobilisation were not significant.3.2Muscle weightsTable 2 shows the weights of the four muscles studied in the old animals after 4weeks of external fixation and after further 4weeks of remobilisation. The weights of the gastrocnemius, quadriceps, plantaris and soleus muscles were reduced by 41.2, 43.7, 46.6 and 39.5%, respectively. Following remobilisation the weights of these muscles were still much reduced (28.0, 33.4, 42.3 and 37.3%, respectively) compared with those of the contralateral legs, which was still statistically significant (p<0.001).3.3Biochemical studiesThe changes in enzymatic activities of acid phosphatase (ACP), creatine phosphokinase (CPK) and lipid peroxidation of gastrocnemius muscle in old rats after external fixation and remobilisation are shown in Table 3. Immobilisation for 4weeks caused a 33.2% increase in acid phosphatase activities, whereas there was a decrease of 17.2% in CPK activities. Lipid peroxidation increased by 60% as measured by the TBA assay. These changes were significant for acid phosphatase (p<0.02), lipid peroxidation (p<0.02) and CPK activities (p<0.01). On remobilisation for 4weeks, activities of acid phosphatase and CPK remained significantly lower than the control values indicating an incomplete recovery of enzymatic activities after 4weeks of remobilisation. Lipid peroxidation values recovered on remobilisation and were no longer significantly different from the contralateral control values.3.4Comparative age-associated studiesTable 4 presents a comparison of changes in hindlimb muscle weights of young and aged rats after external fixation and remobilisation. As a result of hindlimb immobilisation in the young rats the larger muscles, quadriceps and gastrocnemius, lose about 60% of their muscle weight, but in the aged rats these muscles lose only 41–43%. The weight loss in the smaller muscles, plantaris and soleus, was similar in both age groups. After 4weeks of remobilisation, none of the muscles of both age groups recovered to preimmobilisation weights. If values are expressed as net % gain (the difference between maximum loss in % after 4weeks of EF and the percentage weight loss after 4weeks of remobilisation) the values for the young animals are much higher than those of the aged animals. The relative % gain as calculated from the ratio young: old in the net % gain for the gastrocnemius, quadriceps, plantaris and soleus muscles was 2.41, 1.87, 2.53 and 2.54, respectively. The net % gain of both young and old animals is illustrated graphically in Fig. 1.Table 5 presents a comparison of the capacity of enzyme activities (acid phosphatase, creatine phosphokinase) and lipid peroxidation in the gastrocnemius muscle of young and old rats to recover after hindlimb immobilisation and remobilisation. After 4weeks of remobilisation in young rats the % change of acid phosphatase, CPK and lipid peroxidation was −7.3, +2.9 and +32.4%, respectively, which was not significantly different from that of the contralateral control leg. The % change for these same parameters in the old rats was −10.7, −12.6 and +30.0, respectively, which was significant for both acid phosphatase and CPK.Hence, it was possible to conclude that the net % change for young animals was much more pronounced for all the biochemical parameters studied compared with the values for the old animals. The net % change in biochemical parameters is illustrated graphically in Fig. 2. It is clear that in all categories of muscles, net % weight gain and biochemical differences, the changes of muscles of the young animals were much more pronounced than in the muscle of old animals.3.5Morphological studiesThe morphological studies were performed on gastrocnemius muscle of the aged rats. Frozen sections (Fig. 3a and b) show that the morphological appearance of myofibres is fairly similar in gastrocnemius muscles of control legs in both the young (6months old) and old (26months old) rats. Fig. 3c shows that 4weeks of external fixation in the old rats causes marked myopathic changes with the myofibres becoming markedly shrunken and distorted. On remobilisation after external fixation (Fig. 3d) there is only a partial structural recovery of the fibres. Fig. 4a shows greater detail of the myopathic changes after external fixation. Many myofibres show marked degeneration and numerous small vacuoles are seen in myofibres. At the ultrastructural level (Fig. 4b) the myopathic changes are clearly visible including sarcomere disruption and myofilament loss. After remobilisation (Fig. 4c and d), myopathic damage is still clearly evident and there is only limited recovery of myofibre structure. Many myofibres remain swollen and distorted. Sarcomeres in many myofibres remain irregular and distorted. Many myofibres still are highly vacuolated and others contain large numbers of lipid droplets. Some myofibres develop large numbers of lysosome-like bodies (Fig. 4d). Rounded cells with large regular nuclei and pronounced nucleoli (possibly activated satellite cells), as well as inflammatory cells and adipose cells are common in the connective tissue between the myofibres.4DiscussionExternal fixation is a highly effective and widely used method for bone and joint immobilisation that requires the invasive use of pins through bone and muscle. In the present study we showed that the body weights of aged rats subjected to unilateral external fixation of a hindlimb were not changed significantly as a result of the limb immobilisation or after subsequent remobilisation. We also demonstrated that none of the muscles studied from the immobilised limb were capable of returning to preimmobilisation weights or to the weights of the non-immobilised contralateral muscles after 4weeks of remobilisation. This interesting observation confirms that of Maeda et al. (1993), who wrote: “Both muscle and bone require a time period longer than the period of immobilisation in order to make a complete recovery from temporary deterioration”. Similar observations were reached by Booth (1978), who showed that gastrocnemius muscle of rats immobilised in plaster-of-Paris casts for 28days returned to preimmobilisation weight only 50days following the cast removal. However, in another study of 90 day immobilisation of rat hindlimbs, the soleus muscle weight was shown to return to control values 14days after the start of the recovery period (Booth and Seider, 1979). It should be noted that the loss of muscle weight due to immobilisation does not occur as a result of changes in muscle water content, as it was previously shown that the ratio of muscle dry weight to muscle wet weight remains the same for immobilised and control legs (Reznick et al., 1995).Alkaline phosphatase, acid phosphatase and CPK activities have been shown to change considerably after limb immobilisation (Witzmann et al., 1982). Using acid phosphatase and CPK activities as criteria for normal levels of muscle function and metabolism, we have shown that hindlimb immobilisation in old rats altered the level of activities of these enzymes in gastrocnemius muscles, however, after 4weeks of remobilisation, the levels of acid phosphatase and CPK have still not returned to pre-immobilisation levels. Lipid peroxidation was shown to increase considerably after 4weeks of limb immobilisation but on remobilisation recovered by 30% to values that were not statistically significantly different from the contralateral controls.In comparing the recovery responses of young rats to aged rats after external fixation of hindlimbs we have shown that the relative % change of muscle weights and of biochemical parameters is by far greater in the young animals. This may indicate that the capacity for active metabolism and turnover of muscle protein is indeed far greater in young animals than in the aged animals. It would appear that the older animals need much longer times to recover from the stress and disuse atrophy of limb immobilisation than the younger animals and this reflects common clinical experience on traumatic healing of muscle and bone in the elderly.These conclusions are confirmed by the morphological studies. We recently showed that the gastrocnemius muscle of young rats subjected to hindlimb immobilisation by external fixation shows an excellent structural recovery after 4weeks of remobilisation (Zarzhevsky et al., 1999). In contrast, the present study has shown that under similar experimental circumstances, the gastrocnemius muscle of old rats shows only a very limited structural recovery in this time period. We are led to conclude that skeletal muscles of hindlimbs of aged rats show very limited recovery processes after disuse atrophy compared with their younger counterparts.As can be seen from Table 4, % weight loss of gastrocnemius and quadriceps muscles of immobilised hindlimbs was higher in young animals compared to old ones. However, the % weight loss was similar for plantaris and soleus muscles for both age groups (Table 4). Indeed it appears that the larger muscles, such as the gastrocnemius and quadriceps, are undergoing a faster rate of catabolism in young animals compared to old ones. This phenomenon of reduction in the rate of protein breakdown in tissues of old animals compared to young ones has been observed in the past. A recent paper has shown using 3-methylhistidine excretion, that whole body muscle protein catabolism is slower in elderly humans compared to younger ones, by as much as 30–40% (Morais et al., 1997). Similarly, various subcellular fractions of proteins, as well as total proteins, are degraded more slowly in livers of old mice compared to livers of young mice (Lavie et al., 1982). A very recent study has shown that chaperone-mediated autophagy of lysosomes is reduced in livers of ageing rats as compared with young rats (Cuervo and Dice, 2000). Thus it is not unique that muscles of old animals in our experimental model were degraded to a lesser extent that those of the younger animals.The capacity of muscle recovery from muscle degeneration following bupivacaine injection in aging rats was studied by Marsh et al (1997a,b). The mass of tibialis anterior muscle was restored to control values within 21days in young rats (3months old), whereas in adult (18months old) and old rats (31months old) it remained 40% less than that of the contralateral controls at 21 and 28days of recovery. Furthermore, expression of myogenin and myoD mRNA levels increased in regenerated muscles of young, adult and old rats at 5 and 14days, respectively. Whereas in the young (3months old) rats mRNA levels returned to preinjection control values by 21days, in old animals they remained elevated even at 28days of recovery. It appears that either old animals have diminished capacities to down-regulate myogenin and MyoD mRNAs, or possibly their reduced ability for myogenic effect includes elevated levels of these mRNAs (Marsh et al., 1997a). Similar results were also obtained with IGF-1 mRNA, where impaired regeneration of the tibialis anterior was associated with a prolonged elevation of IGF-1 mRNA expression in the old muscle (Marsh et al., 1997b). Since the turnover of muscle proteins (synthesis and degradation) appears to slow down with age, it will be of great interest in future studies to investigate the molecular events behind the differences between young and old animals. 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