The primary purpose of this study was to develop and validate a new rat model of severe SCI combined with cast immobilization, to better reproduce the condition observed in humans with SCI. Patients with SCI experience further muscle inactivity following injury, 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 contusion SCI in rats produced a greater loss in muscle size/mass and force production than SCI alone, and prevented spontaneous recovery. Our data suggest that SCI+IMM can be used to better identify the respective influences of the neural input and mechanical loading on skeletal muscle adaptations after SCI, providing a clinically relevant rodent model of SCI.
Spontaneous reversal of the muscle atrophy process after moderate contusion SCI has been previously observed by our group,20
as well as in several other laboratories.14,30
Recently, Caudle et al.18
used hindlimb wheelchair immobilization (15–18
h/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 24
h/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 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%).31
The present work proposes a reproducible 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.14
using the same injury protocol. One unique aspect of our study is that muscle size was characterized longitudinally, using noninvasive MRI. We demonstrated that CSAmax
of the TS muscles was highly correlated to muscle wet weight measured at 21 days post-injury, regardless of the group. A maximum decline of 25% in TS CSAmax
was 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.30
after moderate SCI. Although the amount of atrophy was similar between severe and moderate SCI after 1 week, several major differences 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 significantly affected in moderate SCI.30
Second, the time course of muscle mass recovery was significantly slower after severe SCI. Muscle mass was still 27% lower in severe SCI compared with controls after 3 weeks in the soleus, a major postural muscle. This is in contrast with the value reported in moderate contusion SCI, in which soleus wet weight recovered within 3 weeks.30
Overall, severe SCI produced the same maximal amount of atrophy as the moderate models, and showed a significant delay in spontaneous recovery.
Unlike human SCI patients, ambulation and movement are unrestricted in rats following experimental SCI once they are returned to the cage. This results in a considerable amount of mechanical loading.14,20
To 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.32–34
For example, in humans with incomplete and complete SCI, partial lower limb loading significantly increased the amplitude of gastrocnemius EMG activity.35
It 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%.36
In addition, loading activity modulates the stepping and stance in locomotor control as the main source of afferent input to the spinal central pattern generator.37,38
Our data showed that cast immobilization 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,39–42
in 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–traumatic complications.32
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 atrophy. 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 immobilization is also highly dependent on the length of the immobilized muscle.43
Muscles immobilized in a shortened position experience a greater reduction in the normalized peak tetanic force.44,45
In 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.44
Interestingly, 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 normalized tetanic force in the muscle can increase,46
or be maintained47,48
after SCI. A potential confounding factor in determining normalized peak tetanic force might be the inaccurate estimation of the contractile muscle mass or area. Muscle degeneration is often paralleled by an increase in intramuscular fat, and eventually by fibrosis as observed in aging49
and clinical conditions such as muscular dystrophy.50
In human chronic SCI, intramuscular fat infiltration and tissue scaring have been observed,51
and correction for muscle fat content is necessary when measuring muscle size, in order to avoid inaccurate estimation.52,53
Another 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.54,55
Several studies suggested that fast-twitch muscle fibers generate a higher specific force than do slower fibers.56,57
Assessment of myosin heavy chain composition may be warranted in future studies.
To further characterize this new model, we uniquely investigated the changes in muscle size across different disuse models in the soleus muscle, which is the primary loading muscle in the hindlimb in rodents. The present results showed that the fiber size distribution 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 about the degree of atrophy in the individual muscle fibers. The size distribution of muscle fibers has important implications for the quality of the muscle (cellularity), and is correlated with the body size.58
The maximum force is proportional to the cross-sectional area of the individual muscle fibers. It is likely that the size distribution 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 muscle volume.
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 asymmetry of the animals. It was interesting to observe that the cast immobilization affected both limbs in a symmetrical way. On the other hand, SCI led to an increased asymmetry within 7 days. This most likely reflects a slight asymmetry in the way the contusion was produced on the spinal cord. Surprisingly, the asymmetry in muscle 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 reloading 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,59
which have been documented to produce further damage up to several weeks post-injury.60
Changes in neural activation and/or limb loading contribute to skeletal muscle atrophy and hypertrophy, especially in the hindlimb muscles of rodents. Therefore, it is important to determine the differences and similarities across different models of muscle atrophy. From a rehabilitation standpoint, it is also essential to address 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 human SCI, extended bed rest after injury further decreases muscle loading in addition to the reduced neural activation, which causes a greater degree of muscle atrophy to be reached before rehabilitation interventions are initiated. The new model of SCI-IMM implemented in this study will allow for an accurate assessment of the efficacy of therapeutic interventions and facilitate the translation to the clinical setting.