Total ankle immobilization with cast or composite splint is a common practice after ankle sprain (Kerkhoffs et al., 2001) and metatarsal (Rammelt et al., 2004) as well as ankle fracture (Vandenborne et al., 1998a). The main inconveniences of such immobilization are the subsequent muscle atrophy and reduction in skeletal muscle functional capacity which may impair functional abilities (Young and Skelton, 1994), postural balance (Onambele and Degens, 2006; Onambele et al., 2006), or maximal physical capacities recovery in athletes (Wilkerson, 1992). Strategies that may minimize these effects are of considerable functional significance to the individual.
One of the most predictable consequences of joint immobilization is loss of lean muscle mass. The time course of changes in muscle cross-sectional area (CSA) is well documented showing a decrease following unilateral lower limb suspension (ULLS—based on Berg et al.'s model (Berg et al., 1991; Hather et al., 1992), immobilization (or a general decreased mobility) owing to a cast (Vandenborne et al., 1998b), bed rest (Akima et al., 2001), spaceflight (Narici et al., 2003), aging (Morse et al., 2005), and prolonged diseases including cancer (Evans, 2004). However, data on muscle volume modifications with cast or composite splint immobilization are poorly documented, with the only information coming from microgravity models: ULLS (Tesch et al., 2004), bed rest (Alkner and Tesch, 2004a), or spaceflight (LeBlanc et al., 2000). These authors reported a significant decrease in muscle volume following the hypoactivity period. To our knowledge, no data have been reported on the immobilization-induced changes in muscle morphology. As eluded earlier, the literature on the relative recovery of muscle volume/shape after immobilization is scarce. Indeed, while Akima et al. (2000) reported an estimated muscle volume recovery after short-term spaceflight, to our knowledge, no data has in fact been published on muscle shape modifications following a recovery period that precede an immobilization.
On the whole, despite a frequent practice of ankle joint immobilization after injury, the impact of said immobilization and the rate of recovery of both proximal and distal (to the immobilized joint) muscle groups is poorly characterized in terms of muscle morphology and volume changes. The aim of our current investigation was therefore to identify the changes associated with total ankle immobilization with cast, using the quadriceps, hamstring, and triceps surae (TS) muscles as models of atrophy.
The current is the case of a moderately active, 29-year old, Caucasian female, 171-cm tall, with a body mass of 58 kg. The subject was initially scanned by the authors who used an MRI scanner as part of a teaching program, designed to instruct students on how to carry out a full lower limb scan. The subject's right leg (from calcuneus to iliac crest) was used. Unfortunately for the subject, she broke the fifth metatarsal on her right foot as a result of a bad fall, a month after the “teaching scan” session. A cast was applied and remained in position for 28 days, during which time it served to minimize the movements of the right foot and ankle joint. Two days following cast removal, a second scan, replicating the protocol used during the first scan, was performed by the authors. Finally a third scan, similar to the previous two, was carried out after 2 months of recovery. Written informed consent was obtained for the current study, which was approved by the ethics committee of the Manchester Metropolitan University.
Muscle Volume Measurement
Cod-liver oil tablets were first positioned along the tibial and femur bone using a surgical tape to identify positions of the scan. These markers were spaced at 7 cm starting from the knee joint. The femur length, defined as the distance from the most distal border of the lateral femur condyle to the most proximal prominence of the greater trochanter, was determined carefully using ultrasonography (Mylab 25, Esaote Biomedica, Genova, Italy). From the ultrasound images, accurate positioning of the skin markers (cod-liver oil tablets) at 40% of the femur length, proximal from the lateral femur condyle, was feasible. The subject was then laid down in a 0.2 T MRI scanner (G-Scan, Esaote Biomedica) in the supine position, with the knee fully extended in a relaxed state. Subsequently, seven sets of 52 axial slides (i.e., a total of 364 slides per session) were obtained along the leg and the thigh using the appropriate coil for the anatomical site and the size of the study case (i.e., coil no. 2). Scans were run using T1-weighted three-dimensional isotrophic profiles (Turbo 3D T1 sequence) with the following scanning parameters: echo time = 16 ms; repetition time = 40 ms; field of view = 180 mm × 180 mm; matrix = 256 × 256; slice thickness = 2.8 mm; interslice gap = 0 mm.
All MRI images were then transferred to a Macintosh labtop (iBook G4) for measurement of muscle anatomical CSA (aCSA). The aCSA of every quadriceps and hamstring muscles were measured along the length of the femur up to the iliac crest. Muscle volumes were also measured along the length of the tibia for the TS muscle group. A segmental adipose volume index was evaluated for the leg (AVILeg) and the thigh (AVIThigh) from the most distal border of the lateral femur condyle, respectively, over 33.6 and 25.2 cm, and defined as the adipose tissue volume between the skin and the muscles. A DICOM file viewer and associated measurement software (OsiriX medical imaging software, OsiriX, Atlanta, USA) was used to analyze one in five slides. The same investigator completed the analysis in triplicate for all muscles of interest and the average was recorded. For lean skeletal muscle tissue measures, visible fat and connective tissue were not included within the measurement region. Cod-liver oil tablets, bone and muscle shape as well as veins locations were used to define the common slides between two consecutive scans.
Assessment of Muscle Volume
The aCSA data gaps between the analyzed image slides along each 7-cm segment were interpolated with the trapezoidal numerical integration method using Matlab (Matlab, The MathWorks, Natick, MA). Then, the muscle volume and segmental fat volume index were calculated as the sum of the measured and interpolated aCSA of each muscle of interest with the length of the segment.
The MRI scan (see Fig. 1) was taken at 40% of femur length. The use of cod-liver oil tablets, together with ultrasound imaging of the anatomical landmarks, allowed this positioning to be accurately replicated at the post and post+2 phases. In addition, within our sample MRI pictures shown in Fig. 1, as with all MRI slices, bone and muscle contours, as well as blood vessels locations, were also used to correctly match MRI slices between phases.
No external markers were used to identify a specific position along the leg. Instead, the position at 30% of total tibial length was defined using data analysis and, as with the thigh, this position was precisely characterized according to bone and muscle shape, as well as blood vessels location.
Timing of Measurements
As mentioned earlier, a first scan of the right thigh and leg was done a month before complete immobilization of the right foot and ankle, a second scan 2 days after the end of the 28-day immobilization period, and a third scan after 2 months of recovery.
Figure 1 shows typical MRI scans with the individual muscles identified in the thigh (Fig. 1A) and the leg (Fig. 1B) at different measurement points (Pre, Post, and Post+2). These MRI scans demonstrate muscle volume changes following immobilization and recovery periods. Thus, atrophy is shown after the 28 days of immobilization (Post) relative to Pre measurement in both the thigh and the leg muscles. In contrast, MRI scans of the thigh and leg after the 2-month recovery period show a degree of hypertrophy in comparison to immediately postimmobilization.
The aCSA in the muscle groups of interest (quadriceps, hamstring, and TS) is illustrated at the Pre phase in Fig. 2. As shown, all individual muscles constituting the quadriceps (see Fig. 2A), the hamstring (see Fig. 2B), and the TS (see Fig. 2C) muscles differs in length, origin, and insertion points, as well as shape and volume.
Muscle Volume Changes
In the TS, the soleus muscle (Sol) is the largest of the three individual muscles, with the second largest being the gastrocnemius medialis (GM) and the smallest being the gastrocnemius lateralis (GL) (Table 1). Neither the immobilization nor the recovery periods modify that hierarchy. After 4 weeks of immobilization, gastrocnemii muscles volumes are the most affected compared to Pre injury data. Between Post and Post+2 measurement points, the results indicates that the gastrocnemii muscles, the most affected by immobilization, in fact showed the greatest volume improvement (Table 1). Even so, after 2 months of recovery, the total TS muscle volume was still 9.5% diminished, compared to Pre immobilization data (Table 1)
Table 1. Quadriceps, hamstring, and triceps surae muscle volume as well as subcutaneous thigh and leg adipose tissue volume index before and after immobilization and after 2 months of recovery
Among the quadriceps muscles, vastus lateralis (VL) is the largest muscle, followed by the vastus intermedius (VI), the vastus medialis (VM), and finally the rectus femoris (RF; Table 1). As with the TS, this order was not influenced by immobilization nor changed after recovery. The foot and ankle immobilization leads to an important decrease in the muscle volume of all the individual muscles constituting the quadriceps, and importantly, even though this muscle group is not directly immobilized. Between Pre and Post measurement points, muscle volumes are decreased, and after 2 months of recovery, a significant increase in all the individual quadriceps muscle volumes is evident. It is notable that the greatest increments here were exhibited by those muscles that had the greatest decrease in muscle volume after immobilization (Table 1). Despite the volume increments at Post+2 relative to Post and between Pre and Post+2, the whole quadriceps muscle volume was still 5.2% smaller
As for the hamstring muscle group, the semitendinosus (ST) is the largest of the four muscles constituting this muscle group, followed in size by the biceps femoris long head (BFLH), the semimembranosus (SM), and the biceps femoris short head (BFSH; Table 1). As with the two previous muscle groups, this order is not affected by immobilization or recovery period. Immobilization leads to a decrease in the total hamstring muscle volume of 6.5%, with variations in individual muscle values. Interestingly, between Pre and Post+2 the total hamstring muscle volume in fact shows a small increase (2.7%), though with a large disparity between individual muscles (Table 1).
Muscle Morphology Changes
Owing to the fact that MRI scanners are not readily available, coupled with the fact that analysis of a full MRI data set is time consuming, in the view of facilitating future comparative work, we also determined the precise location of aCSA modifications along the length of individual muscles within the muscle groups of interest (Fig. 3). Thus, we describe here the changes seen at discreet quartiles along the length of the limbs these muscles are associated with.
Overall, the TS aCSA was decreased along its length after immobilization (Fig. 3D). At the Post+2 phase, the TS aCSA was completely recovered over the first 40% of its length, was increased (though not back to baseline values) between 40% and 60% of its length, and did not show any modifications relative to the Postphase over the last 40% of its length. More details on individual TS muscles are shown on Fig. 3A–C
By and large, the quadriceps muscle group showed a reduction in aCSA along its length after 1 month of immobilization (Fig. 4E). After 2 months of recovery, the quadriceps aCSA had recovered its baseline values in the first two quartiles but was still slightly reduced in the last two quartiles, though values came close to Pre data. More details on individual quadriceps muscles are shown on Fig. 4A–D.
All in all, hamstring aCSA was slightly decreased after immobilization in the first quartile, was unchanged in the middle two quartiles, and decreased in the last quartile (Fig. 5E). After 2 months of recovery, whilst in the first quartile hamstring aCSA is still the same as Post values, it is increased over and above Pre values in the second and third quartiles, and in the last quartile, hamstring aCSA has recovered to Pre values. More details on individual hamstring muscles are shown in Fig. 5A–D.
Subcutaneous Adipose Tissue Content Changes
The subcutaneous adipose tissue volume in both the thigh and the leg shows conspicuous decreases of 9.0% and 10.2%, respectively, between Pre and Post measurements (Table 1). After 2 months of recovery, both values slightly increased by 2.3% and 1.2%, respectively, for the thigh and the leg. At the Post+2 relative to the Pre phase, the subcutaneous adipose tissue volumes were still smaller by 6.9% and 9.2% for the thigh and the leg, respectively (Table 1). As shown in Fig. 6, the decrease in the subcutaneous adipose tissue volume after immobilization does not show any localization as it is evenly distributed along the two limbs of interest. The recovery period did not modify to any noticeable degree, the distribution of subcutaneous fat either in the thigh or in the leg when comparing the Post+2 with the Post phase.
The subject's right femur and tibia lengths were, respectively, 41.0 and 40.3 cm. The quadriceps length was 42.8 cm with individual muscles measuring 32.2 cm (RF), 33.9 cm (VI), 35.3 cm (VL) and 36.7 cm (VM). The total hamstring length was 41.4 cm with individual muscle lengths of 23.2, 28.8, 34.4 and 26.0 cm, respectively, for BFSH, BFLH, ST, and SM. Finally, the TS muscle length was 39.5 cm, made up of one long [the Sol muscle (35.3 cm)], and two shorter [the GL and GM muscles (23.8 and 24.6 cm, respectively)] components.
To our knowledge, the current case study is the first systematic documentation of muscle volume and shape changes following a complete ankle immobilization and recovery period for all the individual muscles that constitute the ankle flexors, the knee extensors, and the knee flexors.
In the present case study based on a cast-induced ankle immobilization, we have found a disparity between the individual muscle volume changes within the TS as well as within the muscles indirectly affected, including the quadriceps and hamstrings. The rate of muscle loss was in fact −0.78%/day for the TS. Within the quadriceps and hamstring muscle groups, the rate of muscle loss were −0.86%/day and −0.23%/day, respectively. Tesch et al. (2004), using ULLS for 5 weeks, found an 8.8% and a 10.5% decrease in quadriceps and TS muscles volumes, respectively, corresponding to rates of muscle losses of −0.25 and −0.30%/day, respectively. Similarly, Alkner and Tesch (2004a) reported decreases in both quadriceps and TS volumes of 10% and 16%, respectively, after 29 days of bed rest. They report also that the vasti muscles decreased by 10%, whilst the RF did not change. This was unlike the events that Vanderborne et al. (1998b) report for the maximal CSA of the TS, with a recorded −20% to −32% decrease after 8 weeks of cast immobilization (i.e., −0.36 to −0.57%/day). Similarly, following a 17 days spaceflight, Le Blanc et al. (2000) found a decrease in muscle volume of 10% in the TS (−0.58%/day), 6% in the quadriceps (−0.35%/day), and 3% in the hamstrings (−0.17%/day). The data in the current case study would suggest that complete ankle immobilization may lead to a greater lost of muscle volume than the other hypoactivity models, where whilst movements are still allowed, degree of loading is substantially decreased.
Furthermore, compared to the previously cited studies we have shown that the antigravity knee and ankle extensor muscles are the most affected by immobilization. Surprisingly, we have found that ankle immobilization leads to a relatively greater atrophy in the quadriceps than in the TS, the latter being directly immobilized with the ankle cast. More specifically, the present case study showed that, in the TS, a greater influence of immobilization can be seen on the gastrocnemii muscles compared to the soleus muscle. These results would tend to contrast with previous studies reporting that muscles with predominantly slow muscle fibers (such as the soleus with ∼75% slow oxidative fibers) are more affected than those with relatively fewer slow fibers (e.g., the gastrocnemii, where only ∼55% of fibers are slow oxidative), in response to unloading (Fitts et al., 2000). For the quadriceps muscle, as in previous studies (Akima et al., 2001; Alkner and Tesch, 2004b; Tesch et al., 2004), we have found that the three vastii muscles are more affected by immobilization than the RF. The RF, being a multijoint muscle, was less susceptible to immobilization-induced changes compared with the three vasti muscles that only act as knee extensors. In the current case study, similar to the events in the quadriceps muscle, in the hamstring muscle, atrophy following immobilization is mainly confined to the only monoarticular muscle (BFSH) while the three biarticular muscles (BFLH, ST, and SM) are less affected by unloading.
To our knowledge, the current case study is the first systematic description of all individual muscle shape changes for the three muscle groups involved in the ankle immobilization (TS, quadriceps, and hamstring muscles). As illustrated in Figs. 3–5, the regional modifications due to unloading are muscle dependent. However, the main loss of muscle volume occurs in the greatest extent around the region of peak CSA (the muscle “belly”). Nonetheless, for a number of individual muscles (including soleus, GL, GM, VL, VM, and BFLH) hypoactivity seems to also lead to marked changes in proximal or distal muscle shape. The mechanisms for this lack of uniformity in CSA losses remain to be investigated.
The causes of muscle atrophy resulting from extended and rigid immobilization are likely to stem from a fall in muscle protein synthesis associated with an increase in muscle protein breakdown as previously evidenced through (i) decreased muscle fiber CSA (Edgerton et al., 1995), (ii) reduced fiber diameter (Widrick et al., 1999), (iii) sarcomere dissolution and endothelial degradation (Oki et al., 1995), (iv) reduced overall number of muscle fibers (Kasper et al., 2002), and (v) decreased proportion of slow-twitch fibers (Edgerton et al., 1975). However, the inconveniences of such immobilization treatment may not only be focused on the decrease in the contractile element but may also result in (vi) an increase in the intramuscular connective tissue (Oki et al., 1995), (vii) a reduction in the number of mitochondrias (Rifenberick et al., 1973), and (viii) a decrease in capillary density within the muscle tissue (Jozsa et al., 1990). Hence, a combination of some or all of the earlier changes would lead to a loss of muscle extensibility, strength, and endurance (Kannus et al., 1992a, b) thus contributing to a characteristically slow recovery of the skeletal muscle fibers (Booth and Seider, 1979; Fitts and Brimmer, 1985; Kannus et al., 1992b, 1998a, b; Kvist et al., 1995).
Recovery of Skeletal Muscle Volume and Shape
The current case study has shown substantial though localized muscle volume recovery in the three muscle groups of interest, following 2 months of recovery. The most affected muscle groups by the immobilization were both the TS and the quadriceps, as they both still showed a degree of muscle volume deficit after the 2-month recovery period. The rate of muscle growth over the recovery period was 0.26%/day for the TS and 0.41%/day for the quadriceps surae. Different from the aforementioned muscle groups, the hamstring muscle group was much less affected by immobilization, was seen to fully recover, and in fact surpassed preinjury muscle volume at the postrecovery phase. The rate of muscle growth was 0.16%/day for the hamstring. To our knowledge, Akima et al. (2000) are the only authors reporting data on muscle volume recovery using a 9- to 16-day spaceflight model. On the one hand, these authors observed a complete recovery in knee extensor and flexor muscles volumes after 1 month in two astronauts, and after 4 months in a third. On the other hand, in the TS, one astronaut recovered after 1 month and the other 2 after 4 months. Unfortunately, these authors do report numerical data associated with the recovery. This, however, is unsurprising as few studies to date in fact report rates of change in CSA over a reloading period in human models. A rare comparable example to the current case study is a case report of a cast immobilization (4 weeks nonweight bearing; 4 weeks weight bearing) after bimalleolar fracture. The authors showed that, after 1 month of recovery, the lateral gastrocnemius exhibited ∼5% greater CSA, whereas the medial gastrocnemius and soleus muscles still showed ∼10% CSA deficit, compared to preimmobilization (Vandenborne et al., 1998a). Other comparable studies have been based on 7 weeks of cast immobilization after unilateral ankle malleolar fractures. These studies have reported an incomplete recovery after 10 weeks of reloading, in the CSA of the TS muscle (Stevens et al., 2004, 2006). Lastly, Kawashima et al (2004) reported a complete recovery of knee extensors and flexors CSA after a 1-month reloading period, following a 20-day bed rest period. The earlier studies are thus evidence that data on volume changes, during a recovery period, after cast immobilization, is in short supply.
The present case study is, to our knowledge, also the first to report data on muscle shape changes during the ambulatory period following a 4-week ankle immobilization. Similar to events during the immobilization period, the recovery period led to a great variability in the muscle shape changes. Indeed, although all individual muscles showed a trend toward returning to baseline values after 2 months of recovery, it is notable that recovery was not uniform along the length of individual muscles. We have found that for all individual muscles, either the distal or the proximal region depending on the muscle under consideration, did not in fact recover by the end of the recovery period. The few studies concerned with muscle shape changes were conducted alongside resistance training effects and have in fact led to contradictory results. Indeed, whilst a greater increase at the distal end of the VL has been reported by Housh et al. (1992), other authors have noted greater changes in the proximal region of this muscle (Narici et al., 1989, 1996). Changes in muscle shape along the length of the VI with training (Housh et al., 1992) are reported as being minimal.
The origin of the skeletal muscle hypertrophy exhibited during the recovery period is likely to come from the opposite effects to those associated with immobilization. Indeed, the main factor regulating the synthesis/degradation of proteins in skeletal muscles has been shown to be the increase/decrease in the mechanical load applied to the muscular system (for a review, see Boonyarom and Inui, 2006). Thus, the increase in load on skeletal muscle during the recovery period after long-term complete immobilization can be assimilated to either/both exercise training and/or mechanical stretching activities, which are known to lead to an upregulation of protein synthesis (Hornberger and Esser, 2004) and thus to result in muscle fiber hypertrophy (Widrick et al., 2002). However, few studies have focused on the effects of reloading on skeletal muscles. Animal studies have shown that muscle reloading after spaceflight (12.5 days) or hind limb suspension (2 weeks) leads to myofiber lesions (Krippendorf and Riley, 1993, 1994) and, particularly, sarcomere damage (Krippendorf and Riley, 1994; Vijayan et al., 1998, 2001). The reloading period has also been shown to result in an increase in hydroperoxide levels in rat soleus muscle, indicating an oxidative stress elevation (Lawler et al., 2006) as well as inflammation, and muscle membrane damage (Nguyen and Tidball, 2003a, b). In human subjects, it is inferred that the aforementioned microdamage is responsible of the soreness express by subjects within the first few reloading days. Dix and Eisenberg (1990) reported that stretching muscle to a new rest length leads to the accumulation of polysomes end mitochondria at the myotendinous junctions, the appearance of nascent sarcomeres, and elevated concentration of mRNA for myosin heavy chain at the myotendinous junctions. These effects would tend to explain the rapid recovery of the CSA and muscle mass after immobilization or unloaded situation (Bajotto and Shimomura, 2006).
Changes in Subcutaneous Adipose Tissue Levels
We report here a decrease in the segmental AVILeg as well as AVIThigh after 1-month total ankle immobilization. The opposite effect was found after the 2-month recovery period, though values still differed from preimmobilization data. The loss of adipose volume was evenly distributed along the leg and the thigh. These results are in contrast with the only study to our knowledge concerned with changes in fat content in the thigh following plaster immobilization. Indeed, Ingemann-Hansen and Halkjaer-Kristensen (1977) reported unchanged calculated fat thigh volume after 4–5 weeks of one-leg immobilization in soccer players. However, these authors were focusing on thigh fat and not on subcutaneous adiposity. This would explain the difference with the results in the current study. Further investigations are therefore needed to better understand the changes in leg and thigh adiposity after ankle immobilization.
Recommendations for Future Work
With such striking atrophy in the limbs directly in series with the cast, we would propose that, to limit immobilization effects, the recovery of the subject needs to not only follow the standard rehabilitation protocol, but also, immobilization protocols ought to be adapted so as to enable a degree of exercise training. For instance, if practitioners were encouraged to use a removable splint, this would enable the patient to detach the contraption for a short period every day, thus allowing them to exercise, and thus limit the process of atrophy. Indeed, it has been shown that application of moderate stretching (30 min/day) helps prevent fiber shortening during disuse (Williams, 1990). Furthermore, studies show that adding small amounts of stretching/mobilization during detraining, arrests muscle atrophy in the soleus (Yamashita-Goto et al., 2001; Gomes et al., 2007). In a recommendable intervention programme, exercise would initially entail slowly and carefully moving the ankle joint in order to provide a contractile/stretching stimulus to the muscles. Another solution might be to use electrostimulation, during the recovery period, directly onto the muscles of interest, with the cast/splint in situ. In either case, exercise intensity would then be gradually increased as recovery progresses, hence preventing/limiting atrophy.
We have systematically investigated the manner in which both the volume and the shape of the quadriceps, hamstring, and TS muscles change following a month-long ankle immobilization and a 2-month recovery period. Muscle volumes changes varied extensively within and between muscle groups. Interestingly, even though the knee was not immobilized, the quadriceps muscle volume was more affected by ankle immobilization than the TS. The results of the current case study need to be confirmed on a larger population; yet they do suggest that knee exercises during ankle immobilization could be used to prevent the likely marked quadriceps atrophy. Moreover, the present study suggests the importance of awareness of the localized aspect of the degree of CSA changes. Indeed, based on our current data, we propose that a conclusion that hyper/hypoactivity has resulted in a seemingly nonexistent modification in CSA, may in fact only be owing to where, along the length of the muscle, the CSA snapshot has been taken and is therefore not necessarily representative of what the muscle as a whole will have experienced.
The authors are indebted to Albane, without whom this study would not have been possible.