Apoptosis Occurs in the Anterior Talofibular Ligament of Patients With Chronic Lateral Ankle Instability: An In Vitro Study
Authors: Choi Youn-Ho , Cho Sung-Hyun , Seo JeongYong , Ahn Jae Hoon , Kim Yoon-Chung 
Published: August 2022

Abstract

Background
Chronic lateral ankle instability is treated operatively, whereas most acute ankle sprains associated with acute anterior talofibular ligament injury are usually treated nonoperatively. This treatment strategy is widely accepted and has been validated using a variety of clinical or radiological methods. We suspected that there may be biological differences between chronic and acutely injured ligaments, particularly with respect to apoptosis. Apoptosis is known to cause ligament degeneration. If it could be demonstrated that apoptosis occurs more in the anterior talofibular ligament tissues of patients with chronic lateral ankle instability compared with patients with acute anterior talofibular ligament injury, biological evidence could be supported.

Questions/purposes
We sought to (1) elucidate the difference in the extent of apoptosis between patients with chronic lateral ankle instability and those with acute anterior talofibular ligament injury. In addition, we asked: (2) What is the expression level of apoptotic enzymes such as caspases 3, 7, 8, and 9 and cytochrome c in each patient group? (3) Is there a correlation between apoptotic activities and the symptom duration period of chronic lateral ankle instability?

Methods
Between March 2019 and February 2021, 50 patients were prospectively enrolled in this study. Anterior talofibular ligament tissues were harvested from patients who were divided into two groups: the chronic lateral ankle instability group and the acute anterior talofibular ligament injury group. Patients with insufficient remaining ligaments were excluded from the chronic lateral ankle instability group, and cases in which the tissue was severely damaged or the quality of collected tissue was insufficient because of severe impingement into the fracture site were excluded from the acute anterior talofibular ligament injury group. Tissues were collected from 21 patients (11 males and 10 females) in the chronic lateral ankle instability group with a mean age of 37 ± 14 years and from 17 patients (6 males and 11 females) in the acute anterior talofibular ligament injury group with a mean age of 49 ± 17 years. To investigate our first purpose, apoptotic cells were counted using a TUNEL assay. To answer our second question, Western blotting for apoptotic enzymes such as caspases 3, 7, 8, and 9 and cytochrome c was performed to investigate apoptotic activity. Immunohistochemistry was also used to detect apoptotic enzymes. To answer our third question, the time elapsed after the first symptom related to chronic lateral ankle instability occurred and the expression level of each enzyme was investigated.

Results
More apoptotic cells were observed in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group in the TUNEL assay. Western blotting revealed that the apoptotic activities of the chronic lateral ankle instability group were higher than those of the acute anterior talofibular ligament injury group: caspase 3 was 117 in the chronic lateral ankle instability group and 59 in the acute anterior talofibular ligament injury group (mean difference 58 [95% confidence interval (CI) 31 to 86]; p < 0.001), caspase 7 was 138 in the chronic lateral ankle instability group and 45 in the acute anterior talofibular ligament injury group (mean difference 93 [95% CI 58 to 128]; p < 0.001), caspase 8 was 126 in the chronic lateral ankle instability group and 68 in the acute anterior talofibular ligament injury group (mean difference 58 [95% CI 29 to 89]; p < 0.001), caspase 9 was 128 in the chronic lateral ankle instability group and 54 in the acute anterior talofibular ligament injury group (mean difference 74 [95% CI 44 to 104]; p < 0.001), and cytochrome c was 139 in the chronic lateral ankle instability group and 51 in the acute anterior talofibular ligament injury group (mean difference 88 [95% CI 46 to 129]; p < 0.001). Immunohistochemistry revealed higher expression of caspases 3, 7, 8, and 9 and cytochrome c in the chronic lateral ankle instability group compared with those in the acute anterior talofibular ligament injury group. Caspases 3, 7, and 9 showed no correlation with duration of chronic lateral ankle instability symptoms: the Pearson correlation coefficient was 0.22 [95% CI -0.25 to 0.69] for caspase 3 (p = 0.36), 0.29 [95% CI -0.16 to 0.74] for caspase 7 (p = 0.23), and 0.29 [95% CI -0.16 to 0.74] for caspase 9 (p = 0.23).

Conclusion
In chronic lateral ankle instability, apoptotic activity in the anterior talofibular ligament was higher than in acute anterior talofibular ligament injury.

Clinical Relevance
Apoptosis occurs more in chronic injured ligaments than in acutely injured ligaments. Although urgent surgical repair is not required for acute anterior talofibular ligament injury, chronic lateral ankle instability may progress if the nonoperative treatment is not successful. Further research should focus not only on timing of apoptotic progression, but also on biological augmentation to reverse or prevent apoptosis within the anterior talofibular ligament.