Investigating the Mediating Role of Pain in the Relationship between Ankle Joint Position Sense and Balance Assessed Using Computerized Posturography in Individuals with Unilateral Chronic Ankle Instability: A Cross-Sectional Study

: Chronic ankle instability (CAI) is characterized by impaired ankle joint position sense (JPS) and compromised balance. Despite previous studies investigating the correlation between ankle JPS and balance in individuals with CAI, the potential mediating effect of pain in this relationship remains unclear. Understanding the role of pain as a mediator between ankle JPS and balance could provide valuable insights into the complex interplay among these variables in individuals with CAI. Therefore, further research is needed to elucidate the mediating effect of pain and its implications in assessing and managing ankle JPS and balance deﬁcits in individuals with CAI. This cross-sectional study aimed to investigate the mediating role of pain in the association between ankle JPS and balance in individuals with unilateral CAI. Methods: Fifty-ﬁve individuals diagnosed with CAI participated in this study. Ankle JPS was assessed using a digital inclinometer, whereas the balance was measured using a computerized dynamic posturography device. Results: Participants with CAI showed impaired ankle JPS in the affected leg compared to that in the asymptomatic leg ( p < 0.001). Ankle JPS errors were greater in both dorsiﬂexion and plantarﬂexion directions in the CAI. Balance was compromised in the CAI leg ( p < 0.001). Moderate correlations ( p < 0.001, r = 0.31 to 0.48) were found between the balance variables. Pain signiﬁcantly mediated the ankle JPS-balance relationship in the CAI ( p < 0.05, Sobel test). The ﬁndings suggest that individuals with CAI exhibit impaired ankle JPS and compromised balance. Pain plays a mediating role in the association between ankle JPS and balance in individuals with CAI. These results highlight the importance of considering pain as a potential mediator when assessing and treating balance issues in individuals with CAI. Healthcare professionals should incorporate assessments of ankle JPS and pain into the management of interventions that address these factors and improve balance and functional ability.


Introduction
Maintaining equilibrium in static and dynamic movement pattern is crucial for minimizing the risk of injury and optimizing physical performance, especially in athletes [1]. Lateral ankle sprains resulting from rapid supination, leading to damage to the ankle's lateral ligaments and cartilaginous surfaces, are a common cause of injury [2]. These injuries can significantly affect the individuals' functional balance [2]. A more than fifty percent recurrence rate leads to a wide variety of long-term problems, including ankle

Design, Ethics, and Participants
A total of 56 participants who attended the physical therapy and health research center between August 2021 and January 2023 and met the inclusion criteria were enrolled in the study. The following inclusion criteria were used to select participants with CAI for the scientific study: (1) individuals who experienced multiple ankle sprains in the past, indicating a chronic instability of the ankle joint; (2) participants who reported persistent symptoms related to ankle instability, such as a feeling of giving way, instability during weight-bearing activities, or chronic pain/discomfort; (3) objective assessments, such as positive anterior drawer test, talar tilt test, or other clinical measures, indicating ligament laxity or mechanical instability of the ankle joint; (4) participants with a documented history of ankle instability symptoms for a minimum duration of, for example, six months or longer; (5) a specific age range for participants, such as adults between 18 and 40 years old; and (6) a score of 25 or less on the Cumberland Ankle Instability Tool [30]. Individuals with a history of lower extremity injury or surgery, neuromuscular or vascular diseases in the lower extremity, limb fractures, pregnancy, or those who were unable to understand or follow commands given by the examiner were excluded. Notably, the study did not involve any specific treatment outcomes but rather aimed to investigate the relationship between ankle JPS and balance in individuals with CAI. Ethical approval was obtained from the King Khalid University-approved scientific ethics committee (REC#24-09-2021). Before taking part in the trial, each subject provided their written consent after receiving appropriate information. The rights, well-being, and privacy of the participants were ensured throughout the research process.
The outcome assessments in this research were conducted by a physical therapist with extensive experience in musculoskeletal physical therapy following the completion of a Ph.D. To ensure impartiality, a separate evaluator, who had no prior knowledge of the study's protocol, documented the outcomes.

Pain Intensity Assessment
A visual analogue scale (VAS) was used to measure the ankle pain observed by the subjects in the past 24 h using a 0-10 cm scale [31]. Each patient was instructed to represent their perceived pain intensity with a numerical rating scale ranging from '0' (no pain) to '10' cm (maximum imaginable pain) [31]. The VAS is a widely accepted and validated method for measuring pain in musculoskeletal conditions, exhibiting good test-retest reliability (ICC = 0.97) [31]. Notably, the VAS has also been shown to be a valid tool for identifying individuals with CAI [32], making it an appropriate assessment tool for the purposes of this study.

Ankle Joint Position Sense Assessment
Ankle proprioception was assessed using a digital inclinometer. The inter-rater reliability demonstrated excellent agreement, with ICC values ranging from 0.87 to 0.91. The standard error of measurement (SEM) for these tests was found to be between 0.06 and 0.07. The individuals were instructed to sit on a couch with their feet in a relaxed dangling posture, knees flexed to an angle of 90 degrees, and hips also flexed to the same degree. The primary inclinometer of the dual inclinometer was attached to the lateral aspect of the foot, while the secondary inclinometer was attached to the lateral aspect of the tibia at the shaft level and secured with a Velcro strap ( Figure 1).  The examiner performed the necessary calibration on the inclinometer in order to locate the neutral position (0 degrees of dorsiflexion and 0 degrees of plantar flexion). During the measurements of the ankle JPS, the subjects were told to close their eyes so that any potential influence from visual input could be eliminated. The examiner manipulated the participant's foot into a position of dorsiflexion or plantar flexion (at angles of 10 degrees and 15 degrees), starting from the neutral position. This position was then held for a period of five seconds. It was requested of the participants that they recall their place. After that, the examiner guided the foot back to its starting position (neutral position-0 degrees of dorsiflexion and 0 degrees of plantar flexion), and participants were given instructions to actively repeat the target angle based on their memories with as much precision as they could muster. The participants indicated when they reached the desired location by saying "YES", and the angular displacement was determined as the difference in degrees between the replicated angle and the target angle. The results of these trials were averaged for further examination, and the results of three consecutive trials were used for each of the directions that were investigated. The examiner provided instructions to the participants that were standardized and did not emphasize anything in particular to assist them in simply obtaining the target angle. The examiner performed the necessary calibration on the inclinometer in order to locate the neutral position (0 degrees of dorsiflexion and 0 degrees of plantar flexion). During the measurements of the ankle JPS, the subjects were told to close their eyes so that any potential influence from visual input could be eliminated. The examiner manipulated the participant's foot into a position of dorsiflexion or plantar flexion (at angles of 10 degrees and 15 degrees), starting from the neutral position. This position was then held for a period of five seconds. It was requested of the participants that they recall their place. After that, the examiner guided the foot back to its starting position (neutral position-0 degrees of dorsiflexion and 0 degrees of plantar flexion), and participants were given instructions to actively repeat the target angle based on their memories with as much precision as they could muster. The participants indicated when they reached the desired location by saying "YES", and the angular displacement was determined as the difference in degrees between the replicated angle and the target angle. The results of these trials were averaged for further examination, and the results of three consecutive trials were used for each of the directions that were investigated. The examiner provided instructions to the participants that were standardized and did not emphasize anything in particular to assist them in simply obtaining the target angle.

Balance Assessment
Techno-body, Iso-Free (Bergamo, Italy), reported that a computerized dynamic posturography device was used for the assessment of balance. Prior to the testing trials, participants underwent a familiarization period to acquaint themselves with the balance assessment procedure. This period allowed participants to become familiar with the singleleg stance task and the instructions provided. The participants stood on the force platform of the posturography device while wearing spandex shorts or another garment with a comparable feel. Balance assessment was conducted using the Modified Clinical Test of Sensory Interaction on Balance [33]. Participants were instructed to stand on a single leg (the testing leg), with the opposite leg flexed away from the force platform and their hands hanging freely by their sides (Figure 2).

Balance Assessment
Techno-body, Iso-Free (Bergamo, Italy), reported that a computerized dynamic posturography device was used for the assessment of balance. Prior to the testing trials, participants underwent a familiarization period to acquaint themselves with the balance assessment procedure. This period allowed participants to become familiar with the singleleg stance task and the instructions provided. The participants stood on the force platform of the posturography device while wearing spandex shorts or another garment with a comparable feel. Balance assessment was conducted using the Modified Clinical Test of Sensory Interaction on Balance [33]. Participants were instructed to stand on a single leg (the testing leg), with the opposite leg flexed away from the force platform and their hands hanging freely by their sides (Figure 2). The participants were given instructions to maintain their balance while standing on one leg for a period of thirty seconds while looking directly at the target mark "X" displayed on the computer monitor. The posturography device measured balance variables, including anterior to posterior (A-P) sway in millimeters (mm), medial to lateral (M-L) sway in millimeters (mm), and center of pressure (COP) ellipse area in square millimeters (mm 2 ) over the course of a duration of thirty seconds. A resting time of 30 s was provided The participants were given instructions to maintain their balance while standing on one leg for a period of thirty seconds while looking directly at the target mark "X" displayed on the computer monitor. The posturography device measured balance variables, including anterior to posterior (A-P) sway in millimeters (mm), medial to lateral (M-L) sway in millimeters (mm), and center of pressure (COP) ellipse area in square millimeters (mm 2 ) over the course of a duration of thirty seconds. A resting time of 30 s was provided between each trial. During this resting period, participants were allowed to stand. The tests were conducted in both the CAI leg and the asymptomatic leg. Every test was conducted thrice, and the results of the most successful attempt were used in the subsequent analysis.
All ankle proprioception and posturography assessments were conducted in a quiet, well-ventilated environment to minimize any distractions that could interfere with accurate measurements. The measurements were recorded by an independent evaluator who was blinded to the study protocol to ensure objectivity.

Sample Size Calculation
In order to determine the appropriate sample size for this study, we referred to the sample size estimate made by Alshahrani et al. for joint position sense in patients with CAI [10]. Using this estimate as a guide, we ultimately arrived at a final participant count of 55. Additionally, we employed the G*Power software (Version 4) to calculate the sample size needed with an 80% statistical power, a threshold of 0.05, and an effect size of f = 0.32 in mind. Based on these calculations, a total sample size of 55 was determined to be appropriate for our study. By following established guidelines and employing appropriate software tools, we ensured that our sample size was sufficient to achieve our research objectives and obtain meaningful results.

Statistical Analysis
To compare the ankle JPS and balance measures between the CAI leg and the asymptomatic leg, paired t-tests were conducted. Pearson's correlation coefficient (r) was calculated to determine the correlation between ankle JPS and balance. The strength and direction of the correlation were interpreted based on the magnitude of the correlation coefficient (r) and its statistical significance (p-value). To evaluate the mediation effect of pain on the relationship between ankle JPS and balance parameters, a mediation model with X (independent variable: ankle JPS)-M (mediator variable: pain intensity)-Y (dependent variable: balance variables) was created ( Figure 3). between each trial. During this resting period, participants were allowed to stand. The tests were conducted in both the CAI leg and the asymptomatic leg. Every test was conducted thrice, and the results of the most successful attempt were used in the subsequent analysis.
All ankle proprioception and posturography assessments were conducted in a quiet, well-ventilated environment to minimize any distractions that could interfere with accurate measurements. The measurements were recorded by an independent evaluator who was blinded to the study protocol to ensure objectivity.

Sample Size Calculation
In order to determine the appropriate sample size for this study, we referred to the sample size estimate made by Alshahrani et al. for joint position sense in patients with CAI [10]. Using this estimate as a guide, we ultimately arrived at a final participant count of 55. Additionally, we employed the G*Power software (Version 4) to calculate the sample size needed with an 80% statistical power, a threshold of 0.05, and an effect size of f = 0.32 in mind. Based on these calculations, a total sample size of 55 was determined to be appropriate for our study. By following established guidelines and employing appropriate software tools, we ensured that our sample size was sufficient to achieve our research objectives and obtain meaningful results.

Statistical Analysis
To compare the ankle JPS and balance measures between the CAI leg and the asymptomatic leg, paired t-tests were conducted. Pearson's correlation coefficient (r) was calculated to determine the correlation between ankle JPS and balance. The strength and direction of the correlation were interpreted based on the magnitude of the correlation coefficient (r) and its statistical significance (p-value). To evaluate the mediation effect of pain on the relationship between ankle JPS and balance parameters, a mediation model with X (independent variable: ankle JPS)-M (mediator variable: pain intensity)-Y (dependent variable: balance variables) was created ( Figure 3).  All statistical analyses were performed using statistical software (SPSS) at a predetermined level of significance (p < 0.05). The specific statistical tests, software version, and any relevant parameters used in the analyses were recorded for transparency and reproducibility.

Results
For this cross-sectional study, we enrolled 55 individuals diagnosed with CAI, and Table 1 provides an overview of their demographic and physical characteristics. Our findings demonstrated a significant decrease in ankle JPS in the affected leg (CAI) in comparison to the asymptomatic leg (p < 0.001). The degree of ankle JPS error was greater in both dorsiflexion and plantarflexion directions in the CAI ankle than in the asymptomatic leg (p < 0.001). Furthermore, balance was compromised in the CAI leg compared to the asymptomatic leg. Specifically, the COP ellipse area (p < 0.001), A-P sway (p < 0.001), and M-L sway (p < 0.001) were all significantly larger in the CAI leg compared to the asymptomatic leg, as outlined in Table 1 and Figure 4.  The correlation coefficients between ankle JPS and balance variables were examined in this study, and the results are presented in Table 2   All correlations were significant at p < 0.001. The JPS at 10 degrees and 15 degrees of dorsiflexion and plantarflexion was positively correlated with balance variables, including COP ellipse area, anterior-posterior sway, and medial-lateral sway. The strongest correlations were found between JPS at 15 degrees of plantarflexion and anterior-posterior sway (r = 0.49) and between JPS at 10 degrees of plantarflexion and medial-lateral sway (r = 0.48). These findings suggest that ankle JPS is associated with balance in individuals with chronic ankle instability. The correlation coefficients between ankle JPS and balance variables were examined in this study, and the results are presented in Table 2    All correlations were significant at p < 0.001. The JPS at 10 degrees and 15 degrees of dorsiflexion and plantarflexion was positively correlated with balance variables, including COP ellipse area, anterior-posterior sway, and medial-lateral sway. The strongest correlations were found between JPS at 15 degrees of plantarflexion and anterior-posterior sway (r = 0.49) and between JPS at 10 degrees of plantarflexion and medial-lateral sway (r = 0.48). These findings suggest that ankle JPS is associated with balance in individuals with chronic ankle instability. Table 3 summarizes the mediation analysis using pain as mediation on the relationship between ankle joint position sense and balance variables. Table 3. Mediation analysis using pain as mediation between ankle joint position sense and balance variables.  The mediation analysis using pain as mediation between ankle joint position sense and balance variables showed that all test variables had a significant total effect (c + a × b) with p-values ranging from 0.001 to 0.012. All variables had a significant direct effect (c-Path) with p-values < 0.001, except for pain x JPS −10 • of PF × A-P sway with p = 0.011. All variables also had a significant indirect effect (b-Path), with p-values ranging from 0.002 to 0.004. The unstandardized coefficients for the total effect ranged from 0.42 to 0.58; for the direct effect, it ranged from 0.13 to 0.31; and for the indirect effect, it ranged from 0.04 to 0.11. These results suggest that pain mediates the relationship between ankle joint position sense and balance variables in individuals with chronic ankle instability.

Test Variables Total Effect-Direct and Indirect (c + a × b) Direct Effect (c-Path) Indirect Effect (b-Path
The Sobel test for the moderation effect examined whether the magnitude of the overall interaction effect of ankle JPS and the moderator variable (pain) on balance remained significant once the mediator variable was accounted for in the model. The results presented in Table 4 show the Sobel test for the indirect effect of statistical significance.
All the variables showed significant p-values, indicating that pain had a significant mediating effect on the relationship between ankle JPS and balance variables. The indirect effect was significant for all variables tested, with Sobel test values ranging from 0.26 to 0.53 and p-values ranging from 0.010 to 0.040. These findings suggest that pain intensity played a significant role in mediating the relationship between ankle JPS and balance variables in individuals with CAI.

Discussion
This study aimed to investigate the relationship between ankle JPS and balance in individuals with unilateral CAI, as well as the potential mediating effect of pain. The findings revealed significant impairment in ankle JPS on the affected leg compared to that on the asymptomatic side. Additionally, moderate correlations were observed between ankle JPS and all balance variables, indicating that reduced ankle JPS is associated with decreased balance. Notably, the study identified pain as a significant mediator in the relationship between ankle JPS and balance. This suggests that pain intensity may influence the association between ankle JPS and balance. These results emphasize the importance of considering pain as a potential mediator when examining the relationship between ankle JPS and balance in individuals with CAI. This study provides valuable insights for clinicians working with patients with CAI, highlighting the intricate interplay between ankle JPS, pain, and balance.
Based on the findings of our study, several previous studies have also demonstrated significant correlations between ankle joint position sense and balance [34,35]. In line with these studies, Alshahrani et al. [10] conducted a study investigating the relationship between ankle joint position sense and balance in 55 individuals with CAI. The results indicated a moderate positive correlation between ankle joint position sense and postural sway. These findings highlight the importance of assessing ankle proprioception and balance in the management of individuals with FAI. According to previous research, Gribble et al. [36] demonstrated that individuals with better ankle joint position sense exhibited improved balance in both static and dynamic balance tasks. Similarly, de Oliveira et al. [37] observed that individuals with a better ankle joint position sense displayed enhanced balance during a single-leg stance task. These findings can be explained by the notion that accurate ankle joint position sense enables individuals to make precise adjustments to their ankle joint position, resulting in better control over their body's spatial position [38]. Another possible explanation is the interconnectedness of the neural pathways involved in ankle joint position sense and balance, suggesting that improvements in one domain may lead to improvements in the other [39]. However, it is important to acknowledge that not all studies have reported significant correlations between ankle joint position sense and balance. For instance, Basta et al. [40] found that while ankle joint position sense correlated with balance in healthy individuals, this relationship did not hold significance in individuals with ankle instability. Overall, although there is some evidence supporting a potential correlation between ankle joint position sense and balance, further research is necessary to comprehensively understand the nature of this relationship and its implications for assessing and treating balance and proprioceptive deficits.
In this study, patients with CAI had increased anterior-posterior and medial-lateral postural sway in the affected leg compared to the normal side. Moreover, patients with CAI showed a larger ellipse area, indicating greater oscillations in their postural sway. The impaired proprioception in patients with CAI may result from damage to the ligaments and sensory receptors in the ankle joint, leading to reduced sensitivity to the joint position and movement [8]. Furthermore, patients with CAI may exhibit altered muscle activation patterns and reduced ankle joint range of motion, further contributing to impaired proprioception and balance [8]. Interventions aimed at improving proprioception and balance in patients with CAI have been proposed as potential strategies to reduce the risk of future ankle injuries and improve functional outcomes [41]. For instance, proprioceptive training exercises, such as balance and stability exercises, have been shown to improve balance and reduce the risk of recurrent ankle sprains in patients with CAI [41][42][43]. Interventions aimed at improving proprioception and balance may be effective strategies to reduce the risk of recurrent ankle injuries and improve functional outcomes in this population.
This study aimed to investigate the mediating role of pain in the association between ankle joint position sense (JPS) and balance in individuals with unilateral chronic ankle instability (CAI). Previous studies by ALMohiza et al. [44] and Alshahrani et al. [45] have demonstrated the mediating effects of pain on the relationship between kinesiophobia and joint position sense in chronic low back pain and fibromyalgia syndrome, respectively. In line with these findings, a study by Abdullah Raizah [26] revealed impaired cervical proprioception in elderly individuals with chronic neck pain. Building upon this knowledge, our study aimed to examine the mediating effect of pain on the relationship between ankle JPS and balance impairments in individuals with CAI. Understanding this mediation effect may provide insights into targeted treatments and interventions to improve functional outcomes and reduce the risk of recurrent ankle injuries in this population.
Pain is a complex phenomenon that can have a significant impact on an individual's ability to maintain balance [46]. Pain can affect the sensory and motor systems, leading to changes in proprioception, muscle activation patterns, and movement strategies [47]. Pain can also affect an individual's motivation to maintain balance, as individuals may adopt compensatory strategies to avoid exacerbating their pain [47]. Given the potential impact of pain on balance, it is plausible that pain may mediate the relationship between ankle joint position sense and balance [48]. In other words, pain may influence the strength and direction of the relationship between ankle joint position sense and balance. To examine the potential mediating effect of pain, researchers can employ statistical techniques such as mediation analysis. This approach involves assessing the association between two variables (ankle joint position sense and balance) and the potential mediating variable (pain). If pain is found to significantly mediate the relationship between ankle joint position sense and balance, it indicates that pain plays a crucial role in the connection between these variables. The potential mediating effect of pain on the relationship between ankle joint position sense and balance underscores the importance of accounting for pain as a potential confounding or moderating factor in studies investigating this relationship. Future research should delve deeper into the role of pain to gain a more comprehensive understanding of the intricate interplay among ankle joint position sense, balance, and pain.

Clinical Implications
The findings of our study have important clinical implications for the assessment and rehabilitation of individuals with CAI. By understanding the relationship between ankle JPS, balance, and pain, clinicians can develop targeted interventions to improve functional outcomes and reduce the risk of recurrent ankle injuries in this population. First, the study highlights the importance of assessing ankle proprioception and balance in individuals with CAI. Impaired ankle JPS was found to be associated with decreased balance, indicating the significance of addressing proprioceptive deficits in the assessment process. Clinicians can incorporate objective measures of ankle JPS, such as digital inclinometers, to identify proprioceptive impairments and tailor interventions accordingly. Furthermore, this study identified pain as a significant mediator in the relationship between ankle JPS and balance in individuals with CAI. This emphasizes the need to consider pain management strategies and address fear-avoidance behaviors when designing rehabilitation protocols. Interventions that combine proprioceptive training exercises, pain management techniques, and balance exercises may yield better outcomes in terms of improving balance and functional stability. Proprioceptive training exercises, such as balance and stability exercises, have shown promise in enhancing balance and reducing the risk of recurrent ankle sprains in individuals with CAI. Clinicians can incorporate these exercises into rehabilitation programs to improve proprioceptive acuity and promote better balance control. Additionally, the study underscores the importance of a comprehensive approach that considers the interplay between ankle JPS, pain, and balance. By integrating the assessments of ankle JPS, pain evaluation, and balance measures, clinicians can develop individualized treatment plans that address the underlying factors contributing to balance impairments in individuals with CAI. It is essential for clinicians to recognize that pain can have a significant impact on balance and proprioception. By addressing pain as a potential confounding or moderating factor, clinicians can better understand the complex relationship between ankle JPS and balance and tailor interventions accordingly. Future research should further investigate the role of pain in this relationship to gain a more comprehensive understanding of the intricate interplay between ankle JPS, balance, and pain.

Limitations of the Study
This study has a few limitations. First, the cross-sectional design limits the ability to establish causality and determine the temporal relationship between the ankle JPS and balance deficits. Future longitudinal studies are warranted to explore the dynamic nature of these relationships. Second, the sample size was relatively small, which may have affected the generalizability of the findings. Further research with larger sample sizes is needed to confirm the present findings. Additionally, the study focused on individuals with CAI attending a specific physical therapy and health research center, which may have introduced selection bias. Future studies should aim to include participants from diverse settings and populations to enhance the external validity of the findings.

Conclusions
In conclusion, this study suggests that individuals with CAI exhibit impaired ankle joint position sense (JPS), and this impairment is significantly correlated with reduced balance. Furthermore, this study indicates that pain mediates the relationship between ankle JPS and balance. These findings have practical implications for the management of patients with CAI, emphasizing the need to consider both ankle JPS and pain in designing interventions aimed at improving balance and overall functional ability. Therefore, healthcare professionals, such as physiotherapists or sports medicine physicians, should include assessments of ankle JPS and pain in the evaluation and treatment of individuals with CAI to develop targeted interventions that address these factors to improve their balance and functional ability.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The corresponding authors (RSR) have access to the data and can provide it upon request.