Hindlimb Muscle Morphology and Function
in a New Atrophy Model Combining Spinal Cord Injury
and Cast Immobilization
Fan Ye,1,* Celine Baligand,2,* Jonathon E. Keener,3Ravneet Vohra,1Wootaek Lim,1Arjun Ruhella,1
Prodip Bose,3–5Michael Daniels,6Glenn, A. Walter,2Floyd Thompson,3,4,7and Krista Vandenborne1
Contusion spinal cord injury (SCI) animal models are used to study loss of muscle function and mass. However, parallels
to the human condition typically have been confounded by spontaneous recovery observed within the first few post-injury
weeks, partly because of free cage activity. We implemented a new rat model combining SCI with cast immobilization
(IMM) to more closely reproduce the unloading conditions experienced by SCI patients. Magnetic resonance imaging was
used to monitor hindlimb muscles’ cross-sectional area (CSA) after SCI, IMM alone, SCI combined with IMM (SCI+
IMM), and in controls (CTR) over a period of 21 days. Soleus muscle tetanic force was measured in situ on day 21, and
hindlimb muscles were harvested for histology. IMM alone produced a decrease in triceps surae CSA to 63.9–4.9% of
baseline values within 14 days. In SCI, CSA decreased to 75.0–10.5% after 7 days, and recovered to 77.9–10.7% by day
21. SCI+IMM showed the greatest amount of atrophy (56.9–9.9% on day 21). In all groups, muscle mass and soleus
tetanic force decreased in parallel, such that specific force was maintained. Extensor digitorum longus (EDL) and soleus
fiber size decreased in all groups, particularly in SCI+IMM. We observed a significant degree of asymmetry in muscle
CSA in SCI but not IMM. This effect increased between day 7 and 21 in SCI, but also in SCI+IMM, suggesting a minor
dependence on muscle activity. SCI+IMM offers a clinically relevant model of SCI to investigate the mechanistic basis
for skeletal muscle adaptations after SCI and develop therapeutic approaches.
Key words: atrophy; immobilization; magnetic resonance imaging; SCI; skeletal muscle
ations after spinal cord injury (SCI), including profound atro-
phy and loss of muscle strength.1Impairments in skeletal muscle
greatly increase the risk for secondary health complications, such
as type 2 diabetes,2cardiovascular disease,3,4osteoporosis,5and
fractures.6Strength of the lower extremity muscles has also proven
to be an important predictor of locomotor recovery in persons with
SCI.7,8Accordingly, the development of effective therapeutic
strategies that promote the recovery of muscle mass and function
after SCI is critically important.
Animal models of SCI have proven to be invaluable for the
development of novel experimental therapies. Current animal
models of SCI include complete transection, hemisection, com-
pression, and contusion.9–11Models in which the spinal cord is
keletal muscle experiences extensive detrimental alter-
sharply transected have been widely used to study the anatomic
regeneration of axons, because they allow for a reproducible
complete SCI. However, most human SCIs do not involve tran-
section of the cord, and are caused by transient compression or
contusion of the spinal cord.12Experimental contusion models of
SCI have been developed that more closely reproduce important
features of the pathology observed in clinical injuries.11,13,14These
models have been widely used to characterize neuromuscular,
pathophysiological, and functional changes after SCI.14–17and to
assess the efficacy of rehabilitation strategies such as locomotor
inamoderatecontusionSCI ratmodelaccelerates muscle massand
force recovery.20However, the untrained animals also showed a
progressive increase in muscle mass, suggestive of a considerable
amount of spontaneous recovery. This recovery is likely the result
of limb movement and loading (self-training) in the unrestricted
Departments of1Physical Therapy,2Physiology and Functional Genomic,3Physiological Sciences,5Neurology, and7Neuroscience, University of
Florida, Gainesville, Florida.
4North Florida/ South Georgia Veterans Heath System, Brain Rehabilitation Research Center of Excellence, Gainesville, Florida.
6Section of Integrative Biology, University of Texas, Austin, Texas.
*The first two authors contributed equally.
JOURNAL OF NEUROTRAUMA 30:227–235 (February 1, 2013)
ª Mary Ann Liebert, Inc.
setting of their home cage. Accordingly, the recovery induced by
self-training in rodents can represent a limitation in the interpre-
tation of pre-clinical rehabilitation studies and the transferability of
findings in this model to patients.18,19
of muscle afferent input during recovery. Even though unloading was
periodic in this approach (up to 18 hours per day, 5 days per week), it
demonstrated the considerable impact of free cage ambulation on
around-the-clock disuse condition experienced by patients more clo-
of the hindlimbs using cast immobilization. Cast immobilization
The implementation of a modified rodent cast design model, allowing
for bladder expression and appropriate animal care in contusion SCI
animals, may offer a more clinically relevant approach to study ther-
apeutic strategies targeting skeletal muscle in SCI.
The primary purpose of the present study was to develop and
validate a new contusion SCI rat model, which minimizes loading
of the hindlimbs by using cast immobilization. We compared this
new model to two other models of disuse: cast immobilization
alone and severe SCI alone. Using magnetic resonance imaging
(MRI) we performed a noninvasive longitudinal assessment of
changes in muscle size. In addition, in situ force mechanics and
muscle histology were performed to further evaluate the effect of
each of the models on muscle function and fiber morphology.
Adult female Sprague–Dawley rats (16 weeks, weighing
294–16g at the start of the study) were obtained from Charles
River Laboratories and housed in a temperature (22–1?C) and
humidity (50–10%) controlled room with a 12:12h light:dark
cycle. Animals were provided rodent chow and water ad libitum
and were given 1 week to acclimatize to the environment. Fifty-six
rats were randomly separated into four groups: severe SCI (SCI,
n=19), bilateral cast immobilization (IMM, n=12) for 2 weeks,
severe SCI plus 2 weeks of bilateral cast immobilization (SCI+
presented in detail in Figure 1. All procedures were performed in
accordance with the United States Government Principle for the
Utilization and Care of Vertebrate Animals and were approved by
Florida. Experimental animals were given access to a soft high-
protein content transgenic dough (Bio-Serv,NJ, product #S3472,
21.2% protein, 3.83 kcal/g), placed on the bottom of the cage.
All surgical procedures were performed under aseptic condi-
tions. Severe SCI was produced using a NYU-MASCIS injury
device as previously described.14Briefly, the animals were deeply
anesthetized with a combination of ketamine (90mg/kg body
weight) and xylazine (8mg/kg body weight) and a dorsal lami-
nectomy was performed at the thoracic vertebral level T7-T9 to
expose the spinal cord.28Clamps attached to the spinous processes
of T7 and T9 stabilized the vertebral column. Contusion was pro-
duced by dropping a 10g cylinder from the height of 50mm onto
the T8/T9 segment of the spinal cord. To further ensure the re-
producibility of the injuries, the MASCIS device was connected to
a computer to record force/velocity data from each drop (height:
2.55–0.44mm). The impactor was left on the spinal cord for 7sec
after the drop. Analgesia was given in the form of Buprinex
(0.025mg/kg) and ketoprofen (22mg/kg) once daily over the first
48h after SCI. Subcutaneous lactated Ringers’ solution and am-
picillin (200mg/kg) were administrated after completion of the
surgery. The animals were kept under vigilant postoperative care,
including daily examination for signs of distress, weight loss, de-
hydration, and bladder dysfunction. Manual expression of bladders
was performed two to three times daily, as required, and animals
were monitored for the possibility of urinary tract infection. Ani-
mals were housed individually. At postoperative day 7, open field
locomotion was assessed during 4min by two different scorers
using the Basso, Beattie, Bresnahan (BBB) scoring scale13and two
animals that did not fall within a preset score range (0–4) were
of 49 surgical procedures were performed to account for potential
attrition of the groups caused by possible surgical complication.
Mortality rates are reported in the Results section.
Bilateral cast immobilization procedures
At day 8 after SCI, bilateral cast immobilization was performed
were anesthetized with gaseous isoflurane (3% for induction, 0.5–
2.5% for maintenance). The casting tape (Patterson Medical, Bo-
lingbrook, IL) was applied on both hindlimbs using the following
procedure. Joints were fixed at the following angles: ankle, 125
degrees, knee 180 degrees, and hip 160 degrees, except for a slight
abduction. The casting tape encompassed the caudal fourth of the
body (superior to tail); however, care was taken not to cover the
abdominal region, to allow easy access for bladder expression in
SCI+IMM. A thin layer of padding was placed underneath the
casting tape in order to prevent skin abrasions. Slight pressure was
applied when wrapping the casting tape on the body, so that there
was minimal room for movement.
Daily preventive maintenance consisted of adjusting and repla-
cing chewed-off casting tape. In addition, the rats were checked
twice a day for skin lesions, hygiene, and fecal clearance. Animals
maintained good mobility, healthy skin, normal grooming behav-
iors, and adequate food intake.
MR images of each of the hindlimbs were acquired separately at
baseline and on days 7, 14, and 21, respectively, to noninvasively
assess changes in muscle cross-sectional area (CSA) (Fig. 1). Ex-
periments were performed in horizontal magnets at 4.7T using
VnmrJ (Agilent, Inc., Palo Alto, CA) and at 11.1T using Paravision
3.0.2 (Bruker, Ettlingen, Germany). Animals were anesthetized
with isoflurane (3% for induction, 1–2% for maintenance) and
placed in a prone position on a plastic cradle. One foot was firmly
secured with tape on a 5mm thick support placed underneath the
animal to ensure that the hindlimb was kept horizontal, centered
and aligned with the z-axis of the magnet. For casted animals, the
ments in the four different groups. Black triangle indicates the day
of spinal cord injury (SCI).
Experimental time line of interventions and measure-
228 YE ET AL.
foot support was 1cm thick to match the cast thickness, and the
animal body slightly angled for appropriate alignment of the hin-
dlimb. A concave1H quadrature surface coil (2cm·4cm) was
placed on top of the lower hindlimb, centered on the belly of the
muscle. Three-dimensional gradient echo images were acquired at
4.7T (TR/TE=100/7.6ms) or 11.1T (TR/TE=120/5.4ms) with a
spatial resolution of 97·97·384lm3.
Images were converted to Digital Imaging and Communications
in Medicine (DICOM) format using either Paravision for Bruker
data, or a custom-made IDL code for Varian data (IDL, ITT Visual
Information Systems, Boulder, CO), and analyzed with Osirix
(www.osirix-viewer.com). Hypointense signal arising from the
muscle facia provided sufficient contrast to allow for the delinea-
tion of the triceps surae (TS) muscle group (soleus and gastroc-
nemius) and the tibialis anterior (TA), as illustrated in Figure 2A.
The maximal CSA (CSAmax) was calculated as the mean of the
consecutive three greatest CSAs. CSAmaxmeasurements on day 21
were compared with muscle wet weight (see next section) for
methodological validation. To study the potential asymmetry in
longitudinal changes of muscle size, an asymmetry index was de-
fined as a relative difference between left and right hindlimb sizes
(CSAleft-CSAright)/ average (CSAleft,CSAright)·100.
Muscle force measurements and tissue harvest
At the end of the 3 week experiment, in situ soleus force
measurements were performed on both hindlimbs as described
previously.29Briefly, the rats were anesthetized with isoflurane
(3% for induction, 1–2% for maintenance), and a small dorsal,
midline incision was made to expose the gastrocnemius-soleus
complex. The soleus and gastrocnemius muscles were carefully
separated and the distal tendon of the soleus was connected to a
variable range force transducer (Biopac Systems, Goleta, CA
TSD105A) using a steel wire suture. The tibial nerve was sur-
rounded by a bipolar electrode cuff proximal to its innervation to
the soleus muscle. The animal was then placed in supine posi-
tion, such that the soleus muscle was oriented in a horizontal
plane, and the hindlimb was secured in placed by a pair of screw-
driven pins at the condyles of the femur. The body temperature
was maintained at 37?C, and a mineral oil drip (30?C) was used
to maintain muscle temperature and prevent the muscle from
drying during testing.
The soleus muscle was first stimulated using supramaximal
(*7V, 0.2 msec) unidirectional square-wave pulses applied to the
tibial nerve (Grass S88 stimulator; West Warwick, RI). The
physiological tests were performed on the soleus adjusted to the
isometric optimum length, determined by measuring maximal
isometric forces generated at graded muscle lengths. Subsequently,
three maximal isometric tetanic force measurements were per-
formed with a 5min interval between stimulations (20–30 V,
80Hz, 1500 msec duration). The absolute peak tetanic force (P0in
N) was recorded and normalized to muscle weight (MW in N.g-1).
Immediately after completion of the force mechanics the hindlimb
[EDL]) were harvested and weighed. The muscles were pinned at
optimal length with optimal cutting temperature (OCT) compound,
rapidly frozen in isopentane pre-cooled in liquid nitrogen, and
subsequently stored at -80?C.
Immunohistochemistry was used to determine experimental
changes in soleus and EDL muscle fiber size. Transversal cryostat
sections (10lm) were taken from the midbelly region and mounted
serially on gelatin-coated glass slides. Rabbit anti-laminin (Neo-
marker, Labvision, Fremont, CA) and rhodamine-conjugated goat
anti-rabbit IgG (Nordic Immunological Laboratories, Langendijk,
The Netherlands) were used to outline the muscle fibers. Stained
sections were mounted in mounting medium for fluorescence
(Vector Laboratories, Burlingame, CA). Image acquisition and
analysis was performed on a Leitz DMR microscope (Leica
Camera Inc, Allendale, NJ). The average muscle fiber size (EDL
and soleus) was determined in both legs of each animal based on
analysis of 250–300 fibers at the mid-belly section, and the mean
value and standard deviation of all animals is reported for each
were also examined. The fullwidth at half maximum (FWHM) was
establish a discriminative threshold among the different groups.
Statistical analyses were performed on final sample sizes of
n=12 for CTR, IMM, and SCI+IMM, and n=19 for SCI. All data
FIG. 2. (A)
outlined in white. Data were acquired with a slice thickness of 385lm and a field of view of 2.5·2.5cm2. (B) Correlation between wet
weight and CSAmaxmeasured in the TS muscle in all groups on experimental day 21.
Example of MRI transverse image of the rat hindlimb. The tibialis anterior (TA) and triceps surae (TS) muscles are
MUSCLE ATROPHY AFTER SCI AND CAST IMMOBILIZATION229
were analyzed using a mixed model with random subject effect,
varying residual variance, and estimation of auto correlation pa-
rameters for repeated measurements. A Tukey’s adjusted mean
separation test was used to estimate which model-estimated least
squares means were significantly different.
differently in the text. Statistical significance was accepted for
Body weight and animal health
SCI+IMM animals showed normal grooming behaviors
able to ambulate using their forelimbs, and maintained adequate
food intake. We did not observe any sign of stress after cast im-
mobilization. Body weight for each of the animal groups at the end
of the 3 week experiment is provided in Table 1. Whereas baseline
animal body weights were similar among groups, the body weights
at 3 weeks of the CTR, SCI, IMM and SCI+IMM animals were
309–18g, 292–18g, 267–10g, and 281–17g, respectively
(Table 1). The weight of the SCI+IMM animals was on average
10% lower than that of the control group, and not different in
comparison with SCI. We observed a mortality rate of 18% 1 week
after surgery. By the end of the 3 week experimental period, ad-
ditional attritions of 14% and 25% were observed in the SCI and
observation period was 29.5% in SCI and 38.5% in SCI+IMM.
Muscle wet weight
Muscle wet weight measurements acquired at the 3 week time
point showed significant atrophy in all three experimental groups
studied, compared with controls (Table 1). As expected, both SCI
and IMM alone produced a marked loss in soleus absolute muscle
weight (115–20g and 86–8g, respectively) compared with the
control group (158–17g, p<0.0001). After normalization to body
weight measured on day 21, the relative soleus muscle mass was
decreased by 20% in the SCI group and by 25% in the IMM group
compared with controls (p<0.05). An additional 20% relative
weight loss was observed in the soleus muscle of SCI+IMM ani-
mals compared with SCI animals (p<0.05). The combination of
SCI and bilateral cast immobilization also induced a greater rela-
tive muscle mass loss compared with SCI in the other muscles
studied: TS, TA, and EDL (Table 1).
Muscle cross-sectional area
MRI was implemented to quantify in vivo longitudinal changes
in muscle size in the three experimental groups. As an internal
validation of our MRI measures, the relationship between CSAmax
obtained on day 21 and wet weight of the TS, (sum of gastrocne-
mius and soleus), was examined. A strong correlation (Pearson
coefficient of 0.94) was found between the two measures (Fig. 2B),
which demonstrates that the in vivo MRI CSA measures reliably
reflect measures of muscle mass in the posterior compartment.
The initial baseline CSAmax of the TS muscle group was
113.0–7mm2. Figure 3 graphically illustrates the evolution of the
average TS CSAmaxin each of the groups relative to baseline val-
ues. The control group presented a non-significant relative increase
day 21, p=0.76). On postoperative day 7, the TS CSAmaxof SCI
animals significantly decreased to 75.1–10.5% of baseline values
(p<0.0001). In animals with SCI only, the CSAmaxremained rel-
atively stable over time and was 77.9–10.7% of pre-injury
levels by day 21 post-surgery. In the cast immobilization only
(IMM) group, TS CSAmaxdecreased to 79.2–5.4% on day 14
(p<0.0001), after 1 week of immobilization, and showed a further
decline in the second week, reaching 63.9–4.9 % on day 21
(p<0.0001). The largest amount of atrophy was observed in the
SCI+IMM model (Fig. 3A), in which the TS CSAmaxwas de-
creased to 56.9–9.9 % of baseline by day 21 (p<0.0001).
The TA muscle CSAmaxare presented in Figure 3B. IMM alone
did not show a significant decrease in TA CSAmax. On the other
hand, SCI alone induced a significant decrease in TA CSAmaxon
postoperative day 7 (79.4–6.8%, p<0.0001). A significant re-
covery was observed by postoperative day 21 (p<0.001). This was
prevented in the SCI+IMM group, in which atrophy was main-
tained throughout the experimental period.
Muscle force measurements
significant decrease, compared with controls, in all the experi-
mental conditions tested (p=0.0001). At the 3 week time point,
absolute peak P0was decreased by 25% in the SCI group, 42% in
Table 1. Muscle Wet Weight and Muscle Wet Weight/Body Weight for Hindlimb Muscles on Day 21.
Data Are Presented as Mean– SD
Muscle wet weight (mg)
Extensor digitorum longus
Muscle wet weight/body weight on day 21 (mg.g-1)
Extensor digitorum longus
281–17* Body weight on day 21 (g)
*Significantly different from control.
#Significantly different from SCI (p<0.05).
230YE ET AL.
the IMM group, and 46% in the SCI+IMM group compared with
controls. The addition of the cast in the SCI+IMM group led to a
significant additional loss of 22% compared with SCI alone
muscles (normalized to muscle wet mass) did not significantly
differ among the groups (p=0.85) (Fig. 4).
Muscle fiber size
decrease in average muscle fiber size compared with controls (Fig.
5, p<0.05). The decrease was more dramatic in the SCI+IMM
group, with the soleus fibers being on average 66% smaller than in
controls (p<0.05). In the EDL muscle, a larger amount of atrophy
was also observed in SCI+IMM compared with SCI alone
Frequency distributions of the soleus fiber size are displayed for
each experimental condition in Figure 6. In control animals, fiber
sizes were broadly distributed, ranging from 500lm2to 6000lm2
approximately, which can be described by an FWHM of 2400lm2.
shift at the terminal time point, resulting in a large decrease in the
FWHM (600lm2in SCI and in SCI+IMM, 800lm2in IMM), and
a positive skewness of the histogram. Interestingly, the SCI+IMM
showed the largest shift toward small size fibers. Only 5 % of the
muscle fibers were<1000lm2in CTR animals, a threshold defined
as the fifth percentile of the distribution. In contrast, 26% of soleus
fiber sizes were below this threshold in CTR animals, 42% in IMM
and up to 71 % in SCI+IMM.
Asymmetry in muscle size
The degree of asymmetry in muscle size between left and right
hindlimbs for each group of animals was assessed and compared
over time. At baseline, the asymmetry coefficient for the TS
CSAmaxranged from 2.6 to 3.4% in all groups (Table 2). The cast
immobilization procedure induced a rather symmetrical amount of
atrophy, as shown by the 4.4–1.2% coefficient of asymmetry on
(IMM), and both spinal cord injured and immobilized (SCI+IMM). (A) Example of transverse image of the hindlimb in a SCI+IMM
rat on experimental days 0, 7, 14, and 21. (B) TS CSAmaxand (C) tibialis anterior (TA) CSAmax, expressed as a percentage of baseline
measurements as a function of time in all groups. *Significantly different from CTR.#Significantly different from SCI (p<0.05)
Time course of the relative changes in triceps surae (TS) CSAmaxin controls (CTR) spinal cord injured (SCI), immobilized
MUSCLE ATROPHY AFTER SCI AND CAST IMMOBILIZATION231
postoperative days 14 and 21, which was not significantly dif-
ferent from baseline levels of asymmetry (p=0.16). Interest-
ingly, whereas 1 week after SCI animals displayed a limited
amount of asymmetry (6.9–1.0 in SCI and 4.7–0.8 in SCI+
IMM), this progressively increased (Table 2). The asymmetry
coefficient was 11.8–2.4 on day 14 (p=0.002) and 9.6–2.4 on
day 21 (Table 2). This elevation in the degree of muscle size
asymmetry was not observed to be reduced by cast immobili-
zation in the SCI+IMM group, and progressively reached sig-
nificantly higher values at days 14 and 21 compared with day 7:
11.7–2.3 on day 14, and 14.9–3.4 on day 21, p=0.009,
The primary purpose of this study was to develop and validate a
new ratmodel of severeSCI combinedwith castimmobilization, to
better reproduce the condition observed in humans with SCI. Pa-
tients with SCI experience further muscle inactivity following in-
jury, because of being on bed rest during the acute phase of
recovery, and having limited mobility or using a wheelchair during
the chronic phase. Overall, cast immobilization following contu-
sion SCI in rats produced a greater loss in muscle size/mass and
force production than SCI alone, and prevented spontaneous re-
covery. Our data suggest that SCI+IMM can be used to better
identify the respective influences of the neural input and mechan-
ical loading on skeletal muscle adaptations after SCI, providing a
clinically relevant rodent model of SCI.
Spontaneous reversal of the muscle atrophy process after mod-
erate contusion SCI has been previously observed by our group,20
as well as in several other laboratories.14,30Recently, Caudle
et al.18used hindlimb wheelchair immobilization (15–18h/day and
5 days/week) to reduce the in-cage activity, which dramatically
delayed spontaneous functional recovery after moderate SCI. Here,
we extended this approach by restricting muscle activity for 24h/
day and 7 days/week. The model described here: 1) significantly
reduced the reloading input; 2) was easily performed, with no
evidence of infection at the surgical site or casted skin and no
different models. (A) Representative experimental records of the
force trace from the soleus muscle in controls (CTR) spinal cord
injured (SCI), immobilized (IMM), and both spinal cord injured
and immobilized (SCI+IMM). (B) Soleus absolute peak tetanic
force (P0), (C) Soleus specific peak tetanic force (P0/MW).
*Significantly different from CTR.#Significantly different from
In situ contractile properties of soleus muscle in the
sectional area (lm2). *Significantly different from controls (CTR).
#Significantly different from spinal cord injured (SCI) (p<0.05).
Soleus and extensor digitorum longus (EDL) fiber cross-
muscle of control (CTR) spinal cord injured (SCI), immobilized
(IMM), and both spinal cord injured and immobilized (SCI+
IMM) animals. The dashed line represents the fifth percentile
threshold for the CTR group.
Averaged muscle fiber size distributions in soleus
232 YE ET AL.
significant loss in body weight compared with SCI alone; 3)
maintained mortality rates of 29.5% in SCI and 38.5% in SCI+
IMM, which was in the range of the values reported in the literature
for moderate SCI (33.3%).31The present work proposes a repro-
ducible experimental model of severe spinal contusion combined
with bilateral limb cast immobilization.
Our model produced a dramatic drop of the BBB scores after 7
days, consistent with the values reported by Basso et al.14using the
same injury protocol. One unique aspect of our study is that muscle
size was characterized longitudinally, using noninvasive MRI. We
demonstrated that CSAmaxof the TS muscles was highly correlated
the group. A maximum decline of 25% in TS CSAmaxwas reached
after 1 week in SCI. This was similar to our previous observation20
in a model of moderate SCI after 2 weeks, and was consistent with
the changes in muscle mass reported by Hutchinson et al.30after
moderate SCI. Although the amount of atrophy was similar be-
tween severe and moderate SCI after 1 week, several major dif-
ferences were observed. First, our model induced significant
atrophy in all of the tested muscles – including 10% decrease in
EDL muscle wet weight after 3 weeks – whereas the EDL was not
muscle mass recovery was significantly slower after severe SCI.
Muscle mass was still 27% lower in severe SCI compared with
in contrast with the value reported in moderate contusion SCI, in
which soleus wet weight recovered within 3 weeks.30Overall, se-
vere SCI produced the same maximal amount of atrophy as the
moderate models, and showed a significant delay in spontaneous
Unlike human SCI patients, ambulation and movement are un-
restricted in ratsfollowing experimental SCI once they are returned
to the cage. This results in a considerable amount of mechanical
loading.14,20To overcome this limitation, we applied bilateral cast
immobilization in the SCI rats to reduce muscle loading. Muscle
loading during locomotion has been shown to play an important
role in shaping muscle activity after SCI.32–34For example, in
humans with incomplete and complete SCI, partial lower limb
loading significantly increased the amplitude of gastrocnemius
EMG activity.35It has also been demonstrated that unloading one
of the hindlimbs during treadmill training in de-cerebrated cats
reduces the magnitude of ankle extensor EMG by 70%.36In ad-
dition, loading activity modulates the stepping and stance in lo-
comotor control as the main source of afferent input to the spinal
central pattern generator.37,38Our data showed that cast immo-
bilization alone led to a similar or even larger percentage of loss
in muscle mass and size in the posterior compartment muscles in
comparison with severe SCI, which further demonstrates the
importance of loading input in muscle plasticity. In the SCI+
IMM group, the addition of cast immobilization minimized the
loading on the muscle, allowing for examination of the role of
neuromuscular activity and/or loading in the muscle adaptations
after SCI. This model is more consistent with what is generally
observed in human complete or chronic incomplete SCI,39–42in
which patients usually experience an extended period of bed rest
and reduced activity during the acute phase of the injury, up to
several weeks. The primary muscles of ambulation continue to
experience reduced loading for several weeks to months after
SCI as activity-based rehabilitation is delayed by other post–
Whereas muscle peak tetanic force production was significantly
reduced in all of the disuse conditions implemented in this study,
the change in force was proportional to the extent of muscle atro-
phy. The normalized peak tetanic force of the soleus muscle among
the different disuse models was similar to control values. From a
functional perspective, this finding is of interest, as a greater loss of
muscle mass induced by cast immobilization in the SCI model was
not accompanied by loss in the normalized peak tetanic force or
muscle quality. The degree of muscle atrophy from cast immobi-
lization is also highly dependent on the length of the immobilized
muscle.43Muscles immobilized in a shortened position experience
a greater reduction in the normalized peak tetanic force.44,45In our
study, muscles were immobilized in a neutral (resting) position and
we did not observe a decrease in the normalized peak tetanic force,
which is consistent with the literature.44Interestingly, we did not
see a significant change in the normalized peak tetanic force of the
soleus muscle in SCI+IMM either. It has been reported that nor-
maintained47,48after SCI. A potential confounding factor in de-
termining normalized peak tetanic force might be the inaccurate
estimation of the contractile muscle mass or area. Muscle degen-
eration is often paralleled by an increase in intramuscular fat, and
such as muscular dystrophy.50In human chronic SCI, intramus-
cular fat infiltration and tissue scaring have been observed,51and
correction for muscle fat content is necessary when measuring
muscle size, in order to avoid inaccurate estimation.52,53Another
reason for the discrepancies in the findings of normalized tetanic
force may be the initial loss of slow rather than fast myofibril
proteins in the early stages of muscle disuse.54,55Several studies
suggested that fast-twitch muscle fibers generate a higher specific
force than do slower fibers.56,57Assessment of myosin heavy chain
composition may be warranted in future studies.
the changes in muscle size across different disuse models in the
soleus muscle, which is the primary loading muscle in the hindlimb
of soleus was identical in the SCI and casted rats. However, a
greater shift towards smaller muscle fiber sizes was observed in the
SCI+IMM group. It has been reported that SCI results in reduced
average muscle fiber size after the injury. However, little is known
distribution of muscle fibers has important implications for the
quality of the muscle (cellularity), and is correlated with the body
size.58The maximum force is proportional to the cross-sectional
area of the individual muscle fibers. It is likely that the size dis-
tribution of fiber diameters will determine the time course for the
progression of force production as well as the structural changes,
such as amounts of collagen and cytoskeletal proteins per unit
Table 2. Degree of Asymmetry in the TS Muscles Size
between Left and Right Limbs for All Groups
Asymmetry coefficient (%)
day CTR IMM SCISCI+IMM
*Significantly different from control values.
#Significantly different from baseline values.
MUSCLE ATROPHY AFTER SCI AND CAST IMMOBILIZATION233
An original observation of the present study is the changes in
asymmetry between limb sizes over time. The levels of asymmetry
measured in control animals were low and stable over time and
might reflect the small variability in animal positioning in the
magnet, in manual outlining of the muscles, as well as a natural
asymmetryofthe animals. Itwas interestingtoobserve thatthe cast
immobilization affected both limbs in a symmetrical way. On the
other hand, SCI led to an increased asymmetry within 7 days. This
producedonthe spinalcord.Surprisingly,the asymmetryinmuscle
size further increased at days 14 and 21, not only in SCI alone, but
also in SCI+IMM, suggesting that despite the cast, the modulation
in muscle size was still partially driven by the consequences of the
injury. Whereas in SCI only the possibility of an asymmetric re-
loading pattern with free activity preferentially enhancing muscle
size in one hindlimb cannot be excluded, SCI+IMM may offer a
more unbiased model to specifically observe the neural aspect of
muscle changes post-injury. In this case, the increased asymmetry
in muscle size is likely to be related either to asymmetry in residual
white matter pathways and subsequent ability to contract motor
neuron and motor units, or to the progression of secondary injury
mechanisms,59which have been documented to produce further
damage up to several weeks post-injury.60
Changes in neural activation and/or limb loading contribute to
muscles of rodents. Therefore, it is important to determine the
differences and similarities across different models of muscle at-
rophy. From a rehabilitation standpoint, it is also essential to ad-
dress the possible changes in muscle quality and function
associated with SCI and cast immobilization. Our results show that
SCI combined with cast immobilization adequately represents the
disuse conditions inherent to the clinical condition of SCI. In hu-
man SCI, extended bed rest after injury further decreases muscle
loading in addition to the reduced neural activation, which causes a
interventions are initiated. The new model of SCI-IMM im-
plemented in this study will allow for an accurate assessment of the
efficacy of therapeutic interventions and facilitate the translation to
the clinical setting.
Theauthorsthank JamesColee forstatisticalanalysis.Thiswork
was supported by grants from the National Institutes of Health
(PO1 HD059751-01A1), the United States Department of Veterans
Affairs (VA Rehabilitation R&D Merit Review B5037R), and the
National High Magnetic Field Laboratory.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Celine Baligand, PhD
Department of Physiology and Functional Genomic
University of Florida
PO Box 100274
Gainesville, FL 32610
MUSCLE ATROPHY AFTER SCI AND CAST IMMOBILIZATION235