The Authors would like to acknowledge Mr. and Mrs. Tom and Phyllis Froeschle for providing financial support towards this project.

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
<ol>
	<li>Use 10 Sprague-Dawley rats that are 13 weeks old at the time of surgery and that range between 250&#8211;300 g of body weight.</li>
</ol>
2. Surgical Procedur...

<ol>
	<li>Bunker, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+for+a+new+name+for+'frozen+shoulder'.">Time for a new name for 'frozen shoulder'.</a> British medical journal. 290 (6477), 1233-1234 (1985).</li><li>Bunker, T. D., Anthony, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+frozen+shoulder.+A+Dupuytren-like+disease.">The pathology of frozen shoulder. A Dupuytren-like disease.</a> The Journal of bone and joint surgery. British volume. 77 (5), 677-683 (1995).</li><li>Kilian, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frozen+shoulder.+Arthroscopy,+histological+findings+and+transmission+electron+microscopy+imaging.">The frozen shoulder. Arthroscopy, histological findings and transmission electron microscopy imaging.</a> Chirurg. 72 (11), 1303-1308 (2001).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+in+idiopathic+adhesive+capsulitis:+a+case+control+study.">Risk factors in idiopathic adhesive capsulitis: a case control study.</a> Journal of Shoulder and Elbow Surgery. 22 (7), e24-e29 (2013).</li><li>Milgrom, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+idiopathic+frozen+shoulder.">Risk factors for idiopathic frozen shoulder.</a> The Israel Medical Association Journal. 10 (5), 361-364 (2008).</li><li>Huang, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthyroidism+is+a+risk+factor+for+developing+adhesive+capsulitis+of+the+shoulder:+a+nationwide+longitudinal+population-based+study.">Hyperthyroidism is a risk factor for developing adhesive capsulitis of the shoulder: a nationwide longitudinal population-based study.</a> Scientific Reports. 4, 4183 (2014).</li><li>Struyf, F., Meeus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+evidence+on+physical+therapy+in+patients+with+adhesive+capsulitis:+what+are+we+missing.">Current evidence on physical therapy in patients with adhesive capsulitis: what are we missing.</a> Clinical Rheumatology. 33 (5), 593-600 (2014).</li><li>Song, A., Higgins, L. D., Newman, J., Jain, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glenohumeral+corticosteroid+injections+in+adhesive+capsulitis:+a+systematic+search+and+review.">Glenohumeral corticosteroid injections in adhesive capsulitis: a systematic search and review.</a> Journal Of Physical Medicine And Rehabilitation. 6 (12), 1143-1156 (2014).</li><li>Schollmeier, G., Sarkar, K., Fukuhara, K., Uhthoff, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+changes+in+the+canine+shoulder+after+cessation+of+immobilization.">Structural and functional changes in the canine shoulder after cessation of immobilization.</a> Clinical Orthopaedics and Related Research. 323 (323), 310-315 (1996).</li><li>Kanno, A., Sano, H., Itoi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+shoulder+contracture+model+in+rats.">Development of a shoulder contracture model in rats.</a> Journal of Shoulder and Elbow Surgery. 19 (5), 700-708 (2010).</li><li>Villa-Camacho, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+kinetic+evaluation+of+an+adhesive+capsulitis+model+in+rats.">In vivo kinetic evaluation of an adhesive capsulitis model in rats.</a> Journal of Shoulder and Elbow Surgery. 24 (11), 1809-1816 (2015).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Decalcification+Techniques+for+Rat+Femurs+Using+HE+and+Immunohistochemical+Staining.">Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining.</a> BioMed Research International. 2017, 9050754 (2017).</li><li>Gonzalez-Chavez, S. A., Pacheco-Tena, C., Macias-Vazquez, C. E., Luevano-Flores, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+different+decalcifying+protocols+on+Osteopontin+and+Osteocalcin+immunostaining+in+whole+bone+specimens+of+arthritis+rat+model+by+confocal+immunofluorescence.">Assessment of different decalcifying protocols on Osteopontin and Osteocalcin immunostaining in whole bone specimens of arthritis rat model by confocal immunofluorescence.</a> International Journal of Clinical and Experimental Pathology. 6 (10), 1972-1983 (2013).</li><li>Sanjai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+comparison+of+decalcification+agents+on+the+human+teeth.">Evaluation and comparison of decalcification agents on the human teeth.</a> Journal of Oral and Maxillofacial Pathology. 16 (2), 222-227 (2012).</li><li>Rolls, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Decalcification. , (2013).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> Journal of Histochemistry &amp; Cytochemistry. 59 (1), 6-12 (2011).</li><li>Sarver, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+rotator+cuff+repair,+stiffness--but+not+the+loss+in+range+of+motion--increased+transiently+for+immobilized+shoulders+in+a+rat+model.">After rotator cuff repair, stiffness--but not the loss in range of motion--increased transiently for immobilized shoulders in a rat model.</a> Journal of Shoulder and Elbow Surgery. 17 (1 Suppl), 108S-113S (2008).</li><li>. Variations within Outbred Strains: Know Your Strains and Stocks | Taconic Biosciences Available from: <a target="_blank" href="https://www.taconic.com/taconic-insights/quality/variations-within-outbred-strains-know-your-strains-and-stocks.html">https://www.taconic.com/taconic-insights/quality/variations-within-outbred-strains-know-your-strains-and-stocks.html</a> (2016)</li><li>Liu, Y. L., Ao, Y. F., Cui, G. Q., Zhu, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+histology+and+capsular+collagen+in+a+rat+shoulder+immobilization+model.">Changes of histology and capsular collagen in a rat shoulder immobilization model.</a> Chinese Medical Journal. 124 (23), 3939-3944 (2011).</li></ol>

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&#160;used a standardized torque for all of the animals in order to determine ex vivo ROM changes...

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.

<table>
	<tbody>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Charles River Laboratories, Wilmington, MA, USA</td>
			<td></td>
			<td>250-300 g</td>
		</tr>
		<tr>
			<td>Surgical tool:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td></td>
			<td></td>
			<td>BD 1&#39; 30 guage</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2% isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nose cone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skalpel and skalpel holder</td>
			<td></td>
			<td></td>
			<td>No. 11 scalpel</td>
		</tr>
		<tr>
			<td>Curved hemostat forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Staright hemostat forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue retractor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tissue forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plain tissue forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skin clip applicator</td>
			<td></td>
			<td></td>
			<td>Any standard staples for wound closure</td>
		</tr>
		<tr>
			<td>Immobilization material</td>
			<td>Ethicon</td>
			<td></td>
			<td>No. 2-0 braided polyester ethibond suture was used for immobilization</td>
		</tr>
		<tr>
			<td>Other materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Costumized device for ROM: 1)Sensor assembly, 2)pivoting axle, 3)arm clamp</td>
			<td></td>
			<td></td>
			<td>Assembly that is described in relaxin paper and adhesive capsulitis paper</td>
		</tr>
		<tr>
			<td>Orientation sensor (part of sensor assembly)</td>
			<td>MicroStrain Inc., Williston, VT, USA</td>
			<td></td>
			<td>3DM-GX3-15</td>
		</tr>
		<tr>
			<td>Reaction torque sensor (part of sensor assembly)</td>
			<td>Futek Inc., Irvine, CA, USA</td>
			<td></td>
			<td>TFF400</td>
		</tr>
		<tr>
			<td>Stepper Motor</td>
			<td>SparkFun Electronics, Niwot, CO 80503</td>
			<td></td>
			<td>https://www.sparkfun.com/products/13656</td>
		</tr>
		<tr>
			<td>Microcontroller</td>
			<td>Torino, Italy).</td>
			<td></td>
			<td>Arduino UNO, R3</td>
		</tr>
		<tr>
			<td>MATLAB code</td>
			<td>MATLAB 7.13.0.564, Natick, Ma, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weight Scale</td>
			<td></td>
			<td></td>
			<td>Ohaus</td>
		</tr>
	</tbody>
</table>

rat model of adhesive capsulitis of the shoulder

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&#176; and initial internal rotation of 80&#176;.
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 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

Range of motion
On follow-up day 0, we found a 63% decrease in total ROM compared to baseline (P &#60; .001). We observed a gradual improvement of ROM until week 5 of follow-up, when progression stopped at 19% restriction (P &#60;0.001). The remaining restriction, 18% of total ROM, was still apparent at 8 weeks of follow-up (P &#60;0.001).
Stiffness
<p class="jove_conten...

Watch this Scientific Journal Video about Rat Model of Adhesive Capsulitis of the Shoulder at JoVE.com

Rat Model of Adhesive Capsulitis of the Shoulder

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 ...

This method can help answer key questions in the orthopedic surgery field, such as, Would relaxing to improve the range of motion in patients suffering from arthrofibrosis? The main advantage of this technique is that it avoid the section of shoulder girdle muscles and any intra-articular trauma, thus preserving capsular and articular continuity and anatomic integrity. Before beginning the procedure, connect the microcontroller of a Stepper Motor to a computer and set up an in-house-developed Matlab code to control the microcontroller.Next, position a 13-week-old, 250 to 300 gram Sprague Dawley rat in a customized range of motion device with the aid of a laser guide. Then, position the left forelimb of the rat on the arm clamp in 90 degrees forward flexion with the sensing axis aligned with the long axis of the humerus and secure the forelimb by the wrist and elbow. Then, use the microcontroller to control the passive forelimb rotation and cycle the sensor assembly three times between 60 degrees of external rotation and 80 degrees of internal rotation to obtain baseline torque measurements using inputs from the inclinometer in conjunction with those from a torque sensor to indicate the start and end of the measurements.To perform the surgical immobilization of the shoulder joint, after confirming a lack of response to toe pinch, make a posterior longitudinal incision parallel to the humeral shaft starting just below the shoulder joint and extending for about three centimeters. Using two braided polyester sutures, pierce through the medial edge of the scapula and around the distal two-thirds of the humeral shaft. And subsequently tighten the sutures to immobilize the shoulder joint.Use wound clips to close the incisions and monitor the incision sites for infection daily during the first post-operative week. Eight weeks after the immobilization, make an incision on top of the scar from the previous procedure and remove the sutures from humerus and the scapula. Then close the incision with fresh wound clips and measure the passive shoulder mechanics as demonstrated.To measure the range of motion, use the torque values at baseline as preset input variables in the program to detect changes in the rotation range of motion with a 0.2 degree resolution. For stiffness measurements, use the original rotation angles of 60 degrees external rotation and 80 degrees internal rotation as the preset inputs in the program to detect the changes in torque with a resolution of 0.01 newton millimeters. Weigh the animal on the same day as each range of motion assessment as one of the tools for evaluating animal health status during the study.Then return the animal to its cage with daily monitoring during the first week for signs of infection, pain, or distress, and removing the wound clips on the 10th day after the second surgery. The intact group displays a proper separation between the capsule and the articular surface of the humeral head as well as a normal cellular organization in the synovial tissue and articular cartilage. The surgically immobilized group, however, exhibits evidence of capsular adhesions in the inferior aspect of the shoulder joint.Moreover, the surrounding tissue appears to be denser as compared to the intact shoulders, leading to a tighter capsule with a decreased joint space. While attempting this procedure, it's important to remember to position the animals correctly. Following this procedure, other methods like intraperitoneal injection of Relaxin-2 can be performed to answer additional questions about the systemic effects of Relaxin-2.After its development, this technique paved the way for researchers in the field of orthopedic surgery to explore the use of Relaxin-2 in reversing fibrosis in different joints due to various underlying pathologies. Don't forget that working with animals can be dangerous and that precautions such as proper animal handling and wearing personal protective equipment should always be taken while performing this procedure.

rat-model-of-adhesive-capsulitis-of-the-shoulder

Research

JoVE Journal

Bioengineering

7.3K Görüntüleme.  Beth Israel Deaconess Medical Center and Harvard Medical School. Bu yöntem gibi ortopedik cerrahi alanında anahtar sorulara cevap yardımcı olabilir, gibi, Artrofibrozis muzdarip hastalarda hareket aralığını artırmak için rahatlatıcı mı? Bu tekniğin en büyük avantajı omuz korse kasları ve eklem içi travma kesitini önlemek, böylece kapsül ve eklem sürekliliği ve anatomik bütünlüğü koruyarak. İşleme başlamadan önce, Stepper Motor'un mikrodenetleyicisini bir bilgisayara bağlayın ve mikrodenetleyiciyi kontrol etmek için şirk...