A Rat Model of Lateral Ankle Sprain Induced by Manual Manipulation, with Controlled Force and Angle: An Experimental Study

Closed modeling methods for lateral ankle sprain (ALAS) avoid surgical drawbacks but lack standardization due to reliance on the operator's subjective force, leading to variable outcomes. This study aimed to refine a closed ALAS rat model by quantifying manipulation parameters and establishing optimal ranges for different injury grades. Ninety rats were randomly assigned to groups receiving manual ankle inversion under different combinations of force (0-8 N or 8-16 N) and plantar flexion angle (100°-130° or 130°-160°), with control groups. A flexible thin-film pressure sensor and a goniometer were used to standardize the applied force and angle. Multimodal assessments were conducted, including ankle thickness and calcaneofibular ligament (CFL) length measurement, micro-CT, MRI, histopathology (HE and Masson's staining), pain threshold testing, and CatWalk gait analysis at multiple time points up to 28 days postmodeling. The severity of the injury was directly correlated with the applied force and angle. Group D (8-16 N, 130°-160°) exhibited the most severe damage, including avulsion fractures, significant CFL elongation and partial tearing, diffuse MRI signal alterations, and prolonged pain and gait instability (>28 days). Groups A and B (0-8 N, both angles) induced mild injuries (slight edema, minor fiber loosening) with rapid functional recovery by day 7. Group C (8-16 N, 100°-130°) resulted in moderate, partial-thickness ligament injuries with recovery by day 10. Behavioral and imaging findings consistently demonstrated a dose-dependent response to the modeling parameters. This study successfully established a modified and quantifiable closed ALAS rat model. The optimal parameters for a grade I ALAS model are 0-8 N of force with plantar flexion of 100°-130° or 130°-160°. For a grade II ALAS model, the parameters are 8-15 N of force with plantar flexion of 100°-130°. This standardized model enhances reproducibility and provides a reliable foundation for future research into ALAS mechanisms and therapies.