This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.
This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60° and initial internal rotation of 80°.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 ± 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.
Adhesive capsulitis of the shoulder is frequently referred to as frozen shoulder or shoulder contracture. It is characterized by restricted glenohumeral motion and pain, presumably as a result of advanced fibrosis and joint contracture1,2,3. The condition involves fibroblast and myofibroblast cell recruitment with a resultant dense collagen matrix (types I and III) in the joint capsule2,3. There are many possible risk factors for developing a joint contracture, including gender, diabetes mellitus, hyperthyroidism, traumatic injury, and prolonged immobilization4,5,6.
Effective treatment options are lacking and mostly include physical therapy, with intervention in the form of surgical release in extreme cases that have not improved with conservative care. The best treatment method remains undetermined and has been a subject of great interest for years in the medical field7,8. Development of novel therapeutic options will require a reproducible animal model for the condition that does not rely on intra-articular induced trauma. The optimal adhesive capsulitis model should involve the two main characteristics of the disease: contracture of the shoulder capsule and a prolonged reduction in range of motion (ROM). Schollmeier et al.9 described one of the first joint contracture models by using a cast to develop shoulder contracture in canines. They also reported that changes in ROM and intra-articular pressure returned to normal levels after cessation of immobilization9. However, an important limitation mentioned in the study is the variation in limb position between animals because of the use of a cast technique.In order to obtain a more reproducible model, Kanno et al.10 later presented an adhesive capsulitis rat model using rigid internal fixation of the shoulder. However, although they achieved a significant reduction in ROM with their model, they did not state whether these changes were temporary or long lasting. The aim of our study was to create a suitable in vivo shoulder contracture rat model by investigating the effect of prolonged extra-articular glenohumeral joint immobilization on ROM and joint stiffness.
The study was approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center. Care was taken to avoid unnecessary prolonged anesthesia and also to avoid hypothermia. Animals were weighted at each ROM measurement session and monitored for weight loss.
1. Study Subjects
2. Surgical Procedure
3. Closing the Incision
4. Suture Removal 8 Weeks After Immobilization
5. Range of Motion and Joint Stiffness Measurements
6. Post-mortem Immunohistologic Analysis
Range of motion
On follow-up day 0, we found a 63% decrease in total ROM compared to baseline (P < .001). We observed a gradual improvement of ROM until week 5 of follow-up, when progression stopped at 19% restriction (P <0.001). The remaining restriction, 18% of total ROM, was still apparent at 8 weeks of follow-up (P <0.001).
Stiffness
On follow-up day 0, we found an increase of 13.3 Nmm in total torque compared with baseline (P <0.001); 8.9 Nmm externally (P = .002) and 4.4 Nmm internally (P <0.001), resulting in a 138.8% increase in external rotation torque, 159.6% increase in internal rotation torque, and a total of 149.2% increased torque overall. On week 8 of follow-up, we found the total measured torque to be 1.4 ± 0.2 Nmm higher than baseline (P = 0.115), with a 0.6 ± 0.1 Nmm increase of external torque (P = 0.369) and 0.7 ± 0.2 Nmm increase of internal torque (P = 0.036). This indicates torque increases 10% externally and 25.7% internally, for an overall 17.9% increase. At the beginning of post-operative week 3, the improvement of stiffness plateaued.
Histologic Results
As seen in Figure 2A, the intact group displayed proper separation between the capsule and articular surface of the femoral head and normal cellular organization. In addition, normal cellular organization was also observed in the synovial tissue and articular cartilage. However, the surgically immobilized group shows evidence of capsular adhesions in the inferior aspect of the glenohumeral joint. Moreover, the surrounding tissue appears to be denser when compared to the intact shoulders, leading to a tighter capsule with a decreased joint space (Figure 2B). Slices stained for fibronectin show an increased capsular thickness in the contracted surgical group (Figure 2B). when compared to healthy controls (Figure 2A). These findings are in agreement with previously reported literature on animal models of joint immobilization10 and support the creation of a proper contracture model.
Figure 1: Testing Apparatus. (A) Immobilization of the glenohumeral joint with 2 braided polyester sutures, passed firmly between the lateral edge of scapular and the humerus; (B) Customized device for measurement of ROM and passive shoulder mechanics; a) A stepper monitor. A sensor assembly consisting of b) a reaction torque sensor, and c) an orientation sensor. d) an arm clamp. (C) Internal or (D) external rotation of the glenohumeral joint11. Please click here to view a larger version of this figure.
Figure 2: Coronal slices of the humeral head. Image stained for fibronectin (IHC) obtained at 40X magnification. Scale bar = 200 µm. (A) Healthy control. (B) Surgical control. Red arrows outline joint capsular thickness. Please click here to view a larger version of this figure.
Figure 3: Range of motion versus normalized torque for both healthy and surgically restricted rat glenohumeral joints. Internal Rotation is denoted as positive, external rotation is negative. The shaded region shows the 95% Confidence Interval (CI). Please click here to view a larger version of this figure.
This study presents a rat model of adhesive capsulitis of the shoulder through internal fixation of the glenohumeral joint. Furthermore, it shows an extended reduction of total ROM for at least 8 weeks after removal of the fixation. In order to calculate the alterations in ROM at different time points, measurements were compared to animal specific baselines. Conversely, Kanno et al.10 used a standardized torque for all of the animals in order to determine ex vivo ROM changes.
In 2008, Sarver et al.17 reported on joint stiffness of the shoulder resulting from non-surgical external fixation. Their study showed a transient increase in joint stiffness after immobilization of injured and treated shoulders, which was resolved by week 8 of follow-up. Yet, in the present study, we did not find a linear relationship between torque and angle, rather a polynomial fit (Figure 3). Furthermore, we only found a statistically significant difference in joint stiffness compared to baseline during assessment of internal rotation, where a 25.7% increase in stiffness persisted after 8 weeks of immobilization.
We used baseline ROM and stiffness measurements for each animal as their own internal control. Given the possible variation between the animals18, using the contralateral shoulder of the same animal as internal control increases internal validity and helps reduce the number of animals required.
One of the limitations of our study is that the apparatus used to measure ROM does not stabilize the scapula. However, a rat's scapula is more obliquely oriented with even more upward rotation than in humans. Having the rats in supine position should theoretically control for scapular tilting as the scapula rests against the firm plate of the testing jig. A further limitation of the study is that we only assess internal-rotation and external-rotation of the glenohumeral joint. This is partly due to the fact that abduction, flexion, and overhead activities require thorough external fixation or restriction of the scapulothoracic joint during ROM testing, which requires a different system than ours.
The pathogenesis and treatment of adhesive capsulitis continues to be inadequately understood. Regardless of the etiology, it has been shown that it is the contracture of the capsule that causes pain and limits glenohumeral movement1,3,11. Furthermore, it has been suggested that there are inflammatory triggers that result in joint fibrosis, thereby causing contracture10. Although our rat model cannot imitate the initial inflammatory insult of a primary contracture, nevertheless, it adequately replicates the characteristic kinetics of adhesive capsulitis and its pathologic changes10,19. This model invokes a lasting reduction in ROM and increased joint stiffness, allowing for a comprehensive assessment of current and potential therapeutic treatments for shoulder contracture.
The authors have nothing to disclose.
The Authors would like to acknowledge Mr. and Mrs. Tom and Phyllis Froeschle for providing financial support towards this project.
Sprague-Dawley rats | Charles River Laboratories, Wilmington, MA, USA | 250-300 g | |
Surgical tool: | |||
Injection needle | BD 1' 30 guage | ||
Needle holder | |||
5% isoflurane | |||
2% isoflurane | |||
Nose cone | |||
Skalpel and skalpel holder | No. 11 scalpel | ||
Curved hemostat forceps | |||
Staright hemostat forceps | |||
Tissue retractor | |||
Toothed tissue forceps | |||
Plain tissue forceps | |||
Dissecting scissors | |||
Suture scissors | |||
Skin clip applicator | Any standard staples for wound closure | ||
Immobilization material | Ethicon | No. 2-0 braided polyester ethibond suture was used for immobilization | |
Other materials: | |||
Costumized device for ROM: 1)Sensor assembly, 2)pivoting axle, 3)arm clamp | Assembly that is described in relaxin paper and adhesive capsulitis paper | ||
Orientation sensor (part of sensor assembly) | MicroStrain Inc., Williston, VT, USA | 3DM-GX3-15 | |
Reaction torque sensor (part of sensor assembly) | Futek Inc., Irvine, CA, USA | TFF400 | |
Stepper Motor | SparkFun Electronics, Niwot, CO 80503 | https://www.sparkfun.com/products/13656 | |
Microcontroller | Torino, Italy). | Arduino UNO, R3 | |
MATLAB code | MATLAB 7.13.0.564, Natick, Ma, USA | ||
Weight Scale | Ohaus |