Consequences of ankle joint immobilisation: insights from a morphometric analysis about fibre typification, intramuscular connective tissue, and muscle spindle in rats

Orthosis immobilisations are routinely used in orthopaedic procedures. This intervention is applicable in bone fractures, ligament injuries, and tendonitis, among other disorders of the musculoskeletal system. We aimed to evaluate the effects of ankle joint functional immobilisation on muscle fibre morphology, connective tissue, muscle spindle and fibre typification triggered by a novel metallic orthosis. We developed a rodent-proof experimental orthosis able to hold the tibiotalar joint in a functional position for short and long terms. The tibialis anterior muscles of free and immobilised legs were collected and stained by histology and histochemistry techniques to investigate general muscle morphology, connective tissue and muscle fibre typification. Morphometric analysis of muscle cross-section area, fibre type cross-section area, fibre type density, percentage of intramuscular connective tissue, and thickness of the muscle spindle capsule were obtained to gain insights into the experimental protocol. We found that short- and long-term immobilisation decreased the cross-section area of the muscles and induced centralisation of myonuclei. The connective tissue of immobilised muscle increased after 2 and 4 weeks mainly by deposition of type III and type I collagen fibres in the perimysium and endomysium, respectively, in addition to muscle spindle capsule thickening. Type IIB muscle fibre was severely affected in our study; the profile assumed odd shapes, and our data suggest interconversion of these fibre types within long-term immobilisation. In conclusion, our protocol has produced structural and histochemical changes in muscle biology. This method might be applied to various rodent models that enable genetic manipulation for the investigation of muscle degeneration/regeneration processes.