Dai Fei Elmer Ker&#39;s research is supported by funding from the Food and Health Bureau, Hong Kong SAR (Health Medical and Research Fund: 08190466), Innovation and Technology Commission, Hong Kong SAR (Tier 3 Award: ITS/090/18; Health@InnoHK program), Research Grants Council of Hong Kong, Hong Kong SAR (Early Career Scheme Award: 24201720 and General Research Fund: 14213922), and The Chinese University of Hong Kong (Faculty Innovation Award: FIA2018/A/01). Dan Wang&#39;s research is supported by funding from the Food and Health Bureau, Hong Kong SAR (Health Medical and Research Fund, 07180686), Innovation and Technology Commission, Hong Kong SAR (Tier 3 Award: ITS/333/18; Health@InnoHK program), and Research Grants Council of Hong Kong, Hong Kong SAR (General Research Fund: 14118620 and 14121121).

All procedures must be performed using sterile surgical technique in an appropriately equipped room designated for animal surgeries according to a protocol approved by the institute&#39;s animal experimentation ethics committee. In the present study, rabbit surgeries were performed in accordance with a protocol approved by The Chinese University of Hong Kong Animal Experimentation Ethics Committee.
1. Surgical procedure
<ol>
	<li>To prepare the surgical area, prewarm a heating pad and overlay it with sterile surgical drapes to maintain the body temperature of the rabbit. Subsequently, lay out sterilized surgical tools and supplies (as specified in the Table of Materials) and organize them according to the surgeon&#39;s preference.</li>
	<li>Induce anesthesia via intramuscular administration of 35 mg/kg ketamine and 5 mg/kg xylazine to New Zealand white rabbits (weighing between 3.5 and 4.5 kg, about 5-6.5 months of age; two male and one female rabbits were used in this study). Subsequently, confirm anesthesia with a paw and/or tail pinch test.</li>
	<li>If additional anesthesia is required to sustain the surgical plane, administer 10 mg/kg ketamine and 3 mg/kg xylazine&#160;intravenously via the marginal ear vein28 and monitor the animal&#39;s breathing rate at regular intervals of 5-10 min.</li>
	<li>To prepare the surgical window, shave the intended incision site (the skin region superficial to the SSC muscle-tendon unit) and clean with three alternating applications of betadine and 70% alcohol. Use a cotton swab to apply betadine and 70% alcohol in circular motions (from inside to outside). Use eye ointment to keep the rabbit&#39;s eyes moist and lubricated. Administer 20 mg/kg cephalexin intramuscularly as an anti-infective agent.</li>
	<li>Make a 3-4 cm skin incision inferior to the clavicle, split the deltopectoral interval using a surgical No. 11&#160;scalpel, and retract to gain access to the shoulder (Figure 1A,B).</li>
	<li>To locate the SSC muscle-tendon unit, first, identify the coracobrachialis muscle (as tissue covering the SSC tendon attachment) and split it. Following this, identify the SSC tendon, and insert a right-angle clamp to expose the entire SSC tendon at its insertion on the lesser tuberosity of the humerus (Figure 1C).</li>
	<li>Prior to introducing the injury, isolate the SSC muscle-tendon (Figure 1D) and administer intraoperative anesthetic (0.2 mg/kg of 0.5% bupivacaine) locally near the transection site. Wrap the SSC muscle-tendon unit in silicone-based penrose tubing&#160;(Figure 1E) to prevent undesired attachment to the surrounding tissues and aid subsequent tissue retrieval.</li>
	<li>To induce injury, create a full-thickness transection at the muscle-tendon junction using a surgical No. 11&#160;scalpel (Figure 1F). Where necessary, stop bleeding by applying pressure with a piece of gauze and use saline to irrigate the wound as needed.</li>
	<li>To close the wound, use a 4-0 poly glycolic acid (PGA) suture to reapproximate the deltoid muscle tissue (Figure 1G) and a 4-0 nylon suture to close the skin wound (Figure 1H).</li>
	<li>Deliver post-surgery care through subcutaneous administration of 0.03 mg/kg buprenorphine as an analgesic (once immediately after surgery and twice daily for the next 48 h29).</li>
	<li>Allow the rabbits to recover on a covered heating pad and apply a soft collar to prevent undesired behavior, including self-mutilation, licking of surgical sites, and removal of sutures (Figure 1I).</li>
	<li>Monitor the animals for weight and behavioral changes. Report any decrease of more than 10% body weight and severe pain that cannot be controlled (assessed based on five behavioral actions: orbital tightening, cheek flattening, nostril shape changes, whisker position changes, and ear shape and position changes) to the vet to determine if intervention such as early euthanasia is required.</li>
</ol>
2. Specimen harvest
<ol>
	<li>Euthanize the rabbits at 4 weeks from the time of injury. Anesthetize the rabbits and give a lethal dose of sodium pentobarbital (more than 60 mg/kg). Confirm death by thoracotomy.</li>
	<li>Identify the humeral head and surgically excise it, while preserving the greater and lesser tubercles and all soft tissue attachments. Fix with 4% paraformaldehyde (PFA) for 72 h at 4 &#176;C before transferring to a solution of 10% ethylenediaminetetraacetic acid (EDTA) for 1 month at room temperature (with media change every 72 h) to decalcify bone.</li>
	<li>After decalcification, subject the samples to standard histological processing using graded ethanol dehydration, paraffin embedding, histological sectioning (8 &#181;m sections), and staining with hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome solutions30,31,32.</li>
	<li>Capture images with an upright microscope at 10x magnification.</li>
	<li>Perform semi-quantification of the H&#38;E and Masson&#39;s trichrome images by measuring the area and percentage of muscle, fibrous tissue, and fat within the muscle, as previously described33,34 using a graphic design software of choice. In this example, Adobe Photoshop software (https://www.adobe.com) is used.
	<ol>
		<li>Select a region of a particular color representing a certain tissue type using the magic wand tool (red is muscle tissue, blue is fibrosis, and white represents fat).</li>
		<li>Click the menu items Select | Inverse | Save selection | Name the section.</li>
		<li>Count the number of pixels within the marked region by clicking menu items Window | Measurement Log | Record Measurement to record these pixel values, and manually calculate the percentage of the selected tissue types.</li>
	</ol>
	</li>
</ol>
3. Statistical analysis
<ol>
	<li>For histological data, perform the statistical analysis using the analytical software of choice. Perform a Student&#39;s t-test for the comparison of two independent samples between the control and injured groups.</li>
	<li>Express the data as mean &#177; standard error of the mean. Consider a p value of &#60;0.05 as statistically significant.</li>
</ol>

The authors have no competing interests to declare.

A reproducible and physiologically-relevant animal model provides the ability to advance the understanding of disease pathogenesis, evaluate the outcomes of clinical therapies, and improve and further develop surgical treatments35. In this study, a reliable and accurate rabbit SSC model that mimics aspects of human RC anatomy and pathophysiology was established. RC tears are related to progressive and likely irreversible muscular degenerative changes, resulting in a reduced healing potential. For example, Ko et al. showed that the reattachment of rabbit SSP at 6 weeks did not reverse muscle atrophy or FD in the following 6 weeks. Such FD-mediated muscle atrophy influences several important clinical parameters, including tendon-muscle strength and joint range of motion, which may affect the surgical outcomes36,37.
The protocol established here showed significant chronic-like attributes after the transection of SSC muscle-tendon units. Specifically, these changes include visibly decreased muscle mass and increased adipose content and fibrotic tissue (Figure 2, Figure 3, and Figure&#160;4). These findings are consistent with degenerative changes reported in human RC tears38. In recent years, the rat has emerged as one of the most intensively studied animal models for RC disease and injury due to its high anatomical similarities with both human and rat SSPs traveling under the acromion38,39,40. However, it should be noted that the portion of rat SSP which passes under the acromial arch is muscular as opposed to tendinous, which is the case in humans41. Most importantly, Barton et al. recognized a lack of significant fat accumulation after SSP tendon detachment in rats23, which stands in contrast to the human condition42. As such, it is believed that the rabbit SSC complex may provide an appropriate model to mimic the chronic RC tear of humans.
To ensure the reproducibility of this model, two points are worth noting when performing this protocol. First, after the transection of muscle-tendon units, the free-end of the transected tendon may be at risk of forming adhesions, which can make tendon retrieval challenging for subsequent manipulations. To avoid this issue, a non-resorbable silicone tubing was used to wrap the free end of the muscle-tendon junction following transection to avoid spontaneous adhesion to surrounding tissues as well as spontaneous healing (Figure 1E). Further, the transected muscle-tendon unit during a second procedure for intervention (i.e., to perform a secure repair; data not shown) can be clearly identified by wrapping the end of injured tissues at the time of initial surgery. This technique is economical, effective, and can be easily implemented in surgery43. Second, rabbits are a highly sensitive species that may exhibit detrimental behavior following surgery. To avoid such issues, it is highly recommended that a soft collar is also applied to prevent undesired behavior, including self-mutilation, licking of surgical sites, and removal of sutures (Figure 1I). Compared to commercially conventional E-collars that are made of rigid plastic, the self-made soft collar did not cause any skin injury or other side effects that affected animal welfare or the quality of scientific inquiry. Together, such steps are critical to create an accurately reproducible rabbit RC injury model and provide the possibility for studying the regenerative repair strategies.
To study tendon pathophysiology and healing in an animal model, a distinct and reproducible injury must be created, and the study time points must be carefully selected. The vast majority of studies on tendon injury and healing have been performed on fully transected animal tendons44, as transection is a simple procedure that is highly reproducible and can adequately simulate the clinical scenario45,46. Huegel et al. showed that the injury of a partially transected tendon was less severe than that of a fully transected tendon, and immobilization had a detrimental effect on tendon mechanics, including increased joint stiffness47. To evaluate the atrophy and FD that is seen in the setting of massive RC tear, it is essential to define the experimentally observed characteristic time points. Gupta et al. have validated a RC injury model in the male rabbit and observed muscle atrophy at 2 and 6 week time points, with increased fat content at later time points (less than 5% fat content at 2 weeks vs. more than 10% fat content at 6 weeks), consistent with the pathological process observed in human RC tears11. In this study, a massive RC tear was created by transection of the SSC muscle-tendon unit in male and female rabbits for 4 weeks, which resulted in SSC muscle FD (36.5% fat content). Thus, a 4 week time point is appropriate for generating SSC muscle FD in male and female New Zealand white rabbits.
Several limitations to this study exist. These include: (i) steps associated with animal model generation, such as a relatively short time point and potentially inflammatory materials (silicone-based penrose tubing) for chronic-like injury generation; (ii) animal model characterization and analysis, such as lack of gait analysis and electromyography to assess joint kinematics and muscle contractile force generation; and (iii) animal model comparison, such as lack of comparison with other RC injury sites.
In terms of model generation, human RC injuries typically involve progressive atrophy and FD that may occur over the span of several years, which is relatively longer than the 4 week time point reported here. This is deemed to be acceptable, since an animal model that generates around 36.5% intramuscular fat in a relatively short time span will be logistically convenient and can be prolonged if deemed necessary. Moreover, the biocompatibility of silicone-based implants, such as penrose tubing, has been a source of long-standing controversy due to reports of cellular immune response and inflammation47; therefore, an alternative inert material, such as polyethylene glycol (PEG), may be substituted for wrapping the resected tendon if pursuing inflammation-associated RC studies.
In terms of animal model characterization and analysis, the lack of gait analysis49 and electromyograph studies50 may limit the study&#39;s findings to qualitative histological data. These aspects may be addressed in future studies by using video motion analysis51 and surface electromyography50 to generate quantitative data on shoulder kinematics and RC muscle performance.
In terms of model comparison, since the SSP and infraspinatus tendons in the rabbits have also been widely used for RC studies, comparing injury severity, including FD among these different injury sites in the future, will identify additional sites for model optimization.
In summary, this study has developed a protocol for modeling chronic-like RC injuries in male and female rabbits. This model is convenient for investigators owing to its simplicity (transection) and relatively short period to induce chronicity (4 weeks) while generating a large degree (36.5%) of intramuscular FD. As such, this protocol is expected to aid investigators in the study of RC pathophysiology, as well as facilitate the development of novel therapeutics for muscle-tendon repair and regeneration.

Chronic rotator cuff (RC) tears are characterized by degenerative changes in musculature and tendons, including the atrophy of muscles, accumulation of adipose tissue, and fibrosis, which can compromise the outcome of RC repair and ultimately cause shoulder pain and dysfunction1,2,3,4,5. To better understand the RC tear pathogenesis and improve surgical results, it is crucial to develop appropriate animal models that can mimic aspects of human RC anatomy and pathophysiology. Specifically, RC injury models should meet the following criteria: (i) lack spontaneous healing after injury; (ii) contain a significant presence of fibrosis, muscular atrophy, and accumulation of adipose tissue; and (iii) be of adequate size to allow for the approximation of surgical techniques used in humans6.
Within this context, the rabbit subscapularis (SSC) muscle can be used as an accurate and reliable animal model for the study of RC pathophysiology, given its unique anatomy, pathophysiological response, and biomechanical properties7. Indeed, rabbit SSC RC anatomy is similar to human supraspinatus (SSP) RC, which is the muscle-tendon unit most often associated with injury stemming from overuse8,9. Specifically, the rabbit SSC tendon complex passes through a bony tunnel and under the coracobrachialis muscle, which is analogous to the situation in humans where the SSP tendon complex passes through the subacromial bony tunnel and under the coracoacromial ligament7. This anatomical similarity results in rabbit SSC undergoing similar musculoskeletal motions as human SSP, in which the tendon travels under the acromion during elevation and abduction of the humerus7,10.
Furthermore, pathohistological changes, similar to human RC tears11, have been observed in the rabbit after SSC tear. Specifically, the muscle belly undergoes severe FD, with a significant loss of muscle mass, decreased muscle-fiber cross-sectional area, and increased adiposity. In addition, Otarodifard et al. assessed the biomechanical characteristics of the rabbit SSC after (1) single-row, (2) double-row, and (3) transosseous-equivalent RC repair techniques, and found that the initial biomechanical characteristics of these repairs were similar to human SSP RC repairs performed in cadaveric specimens12. As such, the anatomical, physiological, and biomechanical similarity of rabbit SSC with human SSP makes it useful for modeling RC injuries.
Although many species of animals&#160;including rats, mice, dogs, and sheep, have been used in the study of RC disease and repair6,13,14,15, the degree of injury chronicity is a key consideration. This is because RC tears can be asymptomatic and may often be diagnosed much later when the tear has enlarged and become chronic in nature, with both the tendon and muscle exhibiting severe degeneration16,17,18. However, most RC repair models employ acute injury models, in which the healthy tendon is transected and then immediately repaired19,20,21,22. This largely occurs for reasons of logistical expediency and technical ease, resulting in few studies that examine the RC pathophysiology within a chronic-like setting. Moreover, several animal models may possess attributes that hinder their use for chronic RC studies.
For example, although the rat has been extensively used to model RC tear and intervention, the lack of significant adipose accumulation following injury contrasts with the human condition, and its small size makes repeated surgical procedures challenging23. Further, although Gerber et al. used the infraspinatus of sheep to study muscle atrophy and FD after chronic RC tear24, there exists some anatomical dissimilarity between sheep infraspinatus and human SSP, as well as numerous logistical challenges for studying and housing such a large animal model. In addition, Gerber et al. developed a delayed RC injury model in sheep by releasing the superficial head of the infraspinatus muscle and tendon to mimic the features of a chronic RC tear, and then evaluated the efficacy of different repair techniques on the tendon at 4 to 6 weeks. Unfortunately, this chronic-like sheep model possessed a limitation, in that the end of the released tendon became indistinguishable from scar tissue during the second surgical procedure25.
Coleman et al. also developed a chronic RC tear model in sheep by covering the transected tendon end with a synthetic membrane at the time of the initial surgery, which allowed for nutrient diffusion and efficiently minimized scar tissue formation around the injured tissue, while improving discrimination between the tendon and scar tissue26. Meanwhile, Turner&#160;et al. suggested that a delayed repair should be conducted within 4 weeks, since direct reattachment rarely happens in a massive tendon retraction27. Together, these studies have contributed toward reproducible and reliable protocols for the successful establishment of a chronic-like rabbit SSC RC injury model.
In this protocol, a chronic-like rabbit RC injury model is established at 4 weeks, in which pathologic changes related to fibrosis and FD-mediated muscle atrophy can be studied via histologic assessments. In particular, wrapping the free end of the muscle-tendon junction using a silicone-based penrose tubing at the time of the initial surgery enables clear identification of the RC tissues during the second surgical procedure and, consequently, facilitates a secure repair to study RC healing with and without scaffold augmentation. Altogether, a chronic-like rabbit SSC model may better mimic RC pathophysiology and pose minimal technical and logistical requirements.

<table><tbody><tr><td>&lt;strong&gt;Surgical tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4-0 Poly glycolic acid (PGA)</td><td>e-Sutures</td><td>GBK884</td><td></td></tr><tr><td>Toothed Adson forceps</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Hemostatic forceps</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Needle holders</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Surgical scalpel with handle</td><td>Taobao, China</td><td></td><td>No. 11 blade</td></tr><tr><td>Suture (4-0 Nylon)</td><td>Taobao, China</td><td>19054</td><td>Either nylon or silk sutures are acceptable for skin closure. Each suture has its own advantages and disadvantages and users are advised to choose one according to their preference.</td></tr><tr><td>&lt;strong&gt;Surgical accessories&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cotton balls</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Gauze</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Razor</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Surgical heating pad</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>Surgical lamp</td><td></td><td></td><td></td></tr><tr><td>Syringe with needles</td><td>Taobao, China</td><td></td><td>1 mL, 5 mL, 10 mL</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Buprenorphine</td><td>LASEC, CUHK</td><td></td><td>0.12 mg/kg</td></tr><tr><td>Bupivacaine</td><td>Sigma-Aldrich</td><td>b5274-5g</td><td>1-2 mg/kg</td></tr><tr><td>Cephalexin</td><td>Santa Cruz Biotechnology</td><td>sc-487556</td><td>20 mg/kg</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>LASEC, CUHK</td><td></td><td>35 mg/kg</td></tr><tr><td>Sodium pentobarbital</td><td>LASEC, CUHK</td><td></td><td>more than 60 mg/kg</td></tr><tr><td>Xylazine</td><td>LASEC, CUHK</td><td></td><td>5 mg/kg</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nikon Ni-U Eclipse Upright Microscope</td><td>Nikon Instruments Inc, USA</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe Photoshop 20.01</td><td>Adobe Inc, USA</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Other reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Betadine</td><td>Taobao, China</td><td></td><td>5%</td></tr><tr><td>Ethanol</td><td>Taobao, China</td><td></td><td>70%</td></tr><tr><td>Ethylene diamine tetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>EDS-1KG</td><td>10%</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15713</td><td>4%</td></tr><tr><td>Silicone tubing</td><td>Easy Thru, China</td><td>ISO13485</td><td></td></tr><tr><td>Saline</td><td>Taobao, China</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Histological staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eosin Stain Solution</td><td>Sigma-Aldrich</td><td>R03040</td><td>5% Aqueous</td></tr><tr><td>Hematoxylin Solution</td><td>Sigma-Aldrich</td><td>HHS32</td><td></td></tr><tr><td>Trichrome Stain&amp;nbsp;(Masson)&amp;nbsp;Kit</td><td>Sigma-Aldrich</td><td>HT15</td><td></td></tr></tbody></table>

development of a rabbit chronic-like rotator cuff injury model for study of fibrosis and muscular fatty degeneration

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which negatively influences clinically relevant parameters, such as strength and retraction of the muscle-tendon/myotendinous unit, ultimately causing loss of shoulder function and negatively affecting RC repair outcomes. Animal models that mimic aspects of human RC anatomy and pathophysiology are crucial for advancing the conceptual understanding of injury progression and developing effective tissue engineering and regenerative medicine-based therapeutics.
Within this context, a rabbit subscapularis (SSC) model is suitable due to (i) its anatomical similarity to the human supraspinatus (SSP) bone-tendon-muscle unit, which is the most frequently injured RC site; (ii) its pathophysiological similarity to humans in terms of fibrosis and muscle fatty degeneration (FD); and (iii) its amenability to surgical procedures. Therefore, the goal of this study is to describe the surgical techniques for inducing SSC RC injury. Briefly, the procedure involves the isolation of the SSC by identifying the coracobrachialis muscle followed by a full-thickness transection at the muscle-tendon junction and wrapping the free end of the muscle-tendon junction with a silicone-based penrose tubing to prevent spontaneous reattachment. Histologic evaluations are performed to monitor the progression of muscle FD at 4 weeks post-surgery using hematoxylin and eosin (H&#38;E) as well as Masson&#39;s trichrome staining.
Loss of muscle and FD were evident 4 weeks after transection of the SSC muscle-tendon junction, similar to human RC pathophysiological conditions. This protocol demonstrates the steps for successfully establishing a chronic-like rabbit SSC RC injury model, which can serve as a powerful tool to study skeletal muscle changes associated with RC pathophysiology and aid the development of novel therapeutic strategies for chronic-like RC tears.

To assess the chronicity of RC pathology following the transection of SSC muscle-tendon units, overall tissue morphology and cellular changes were characterized via gross evaluation and histological analysis (H&#38;E and Masson&#39;s trichrome staining, respectively), at 4 weeks post-injury (Figure 2, Figure 3,&#160;and Figure 4). Representative images of gross tissue morphology showed the appearance of white adipose-like tissue in injured SSC muscles, which was absent in the control group (Figure 2). H&#38;E staining confirmed loss of muscle cellularity and organization, which was replaced with large numbers of adipocytes (empty spaces surrounded by thin rims of cytoplasm that contained compressed nuclei) in injured SSC muscles relative to the control group (Figure 3A).
Semi-quantitative assessment of H&#38;E images showed a high degree of intramuscular adipocytes present in injured SSC muscles (36.5% &#177; 8.5%) relative to the control group (0.69% &#177; 0.18%) (Figure 3B). Masson&#39;s trichrome staining also confirmed muscle atrophy and disorganized collagen fiber arrangements in injured SSC muscles relative to the control group (Figure 4A). Semi-quantitative assessment of Masson&#39;s trichrome images showed a reduction in muscle cellularity for injured SSC muscles (41.3% &#177; 2.6%) relative to the control group (99.2% &#177; 0.16%) (Figure 4B). Although further semi-quantitative assessment did not show any significant difference for fibrotic tissue formation between injured SSC muscles (22.3% &#177; 13.1%) and the control group (0.07% &#177; 0.05%), a high degree of fibrosis was observed in injured SSC muscles (Figure 4C). Together, gross tissue morphology and histological analysis showed that injured rabbit SSC muscle-tendon exhibited severe muscle atrophy, fatty accumulation, and fibrosis, which are known hallmarks of chronic RC pathophysiology.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64828/64828fig01.jpg" /> 
Figure 1: Surgical procedure for chronic-like SSC muscle-tendon injury model. (A)&#160;A surgical window was created and anatomical landmarks such as the humerus, humeral head, and clavicle were identified by palpation. (B) A 3.0 cm skin incision was made inferior to the clavicle. (C) The coracobrachialis muscle was split to expose the SSC muscle. (D)&#160;The SSC muscle-tendon unit was isolated. (E) A silicone-based penrose drain was used to wrap the SSC muscle-tendon tissue. (F) The SSC muscle-tendon was transected. (G) The coracobrachialis muscle was reapproximated using PGA sutures. (H) The&#160;skin incision was closed using nylon sutures. (I) Post-surgery, the rabbits were given a soft collar to wear. Abbreviations: SSC = subscapularis; PGA = poly glycolic acid. <a href="https://www.jove.com/files/ftp_upload/64828/64828fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64828/64828fig02.jpg" /> 
Figure 2: Gross morphology of representative SSC muscles. Black arrows represent white adipose tissues. Abbreviation: SSC = subscapularis. <a href="https://www.jove.com/files/ftp_upload/64828/64828fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64828/64828fig03.jpg" /> 
Figure 3: Histological analysis of chronic-like RC injury model at 4 weeks. (A) Representative H&#38;E-stained histology images showed atrophic muscle fibers and accumulation of adipocytes. (B)&#160;Quantification of injured muscle fat accumulation percentage. n = 3 rabbits. Error bars indicate SEM. *, statistically significant (p&#8804; 0.05). Scale bars = 5,000 &#181;m (A, left column), 600 &#181;m (A, right column). Abbreviations: SSC = subscapularis; RC = rotator cuff; H&#38;E = hematoxylin and eosin. <a href="https://www.jove.com/files/ftp_upload/64828/64828fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64828/64828fig04.jpg" /> 
Figure 4: Histological analysis of chronic-like RC injury model at 4 weeks. (A)&#160;Masson&#39;s trichrome-stained images showed substantial fibrosis. Fibrous connective tissue are stained blue. (B)&#160;Quantification of the proportion of muscle and (C)&#160;fibrotic tissue. n = 3 rabbits. Error bars indicate SEM. *, statistically significant (p&#8804; 0.05). Scale bars = 5,000 &#181;m (A, left column), 200 &#181;m (A, right column). Abbreviations: SSC = subscapularis; RC = rotator cuff. <a href="https://www.jove.com/files/ftp_upload/64828/64828fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration at JoVE.com

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

This study details procedures for establishing a chronic-like rabbit rotator cuff (RC) injury. Specifically, the injury is created in the subscapularis (SSC) muscle-tendon/myotendinous unit to mimic human RC anatomy and pathophysiology, including severe muscle fatty degeneration (FD). This protocol can be applied to study RC injuries and assess regenerative therapies.

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which ...

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1.1K Views.  The Chinese University of Hong Kong. Dit protocol demonstreert een complexe chirurgische techniek voor het induceren van een chronisch letsel aan de rotator cuff bij konijnen, en is nuttig bij het bestuderen van de pathogenese van de rotator cuff en het ontwikkelen van regeneratieve therapieën. Konijnen zijn middelgrote tot grote diermodellen die logistiek gemakkelijk te huisvesten zijn, chirurgisch te manipuleren zijn en hoge niveaus van rotator cuff spiervetdegeneratie vertonen wanneer ze gewond raken, vergelijkbaar met mense...

Video: Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Medicine

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.

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 ...

Bioengineering

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Targeted Tuina Stimulation Protocol in Lumbar Region of IDD Model Rabbits

Operational and Intervention Effects of Targeted Tuina in Lumbar Intervertebral Disc Degeneration Model Rabbits

Tui Na or massage therapy alleviates symptoms related to intervertebral disc degeneration (IDD). However, precise, repeatable, standardized instructions for Tuina manipulation are lacking. This study establishes IDD model rabbits induced by fibrous ring puncture, creates targeted Tuina stimulation protocols at the acupuncture points in the lumbar region, and describes in detail the operation methods and requirements of kneading, pointing, and flicking. New Zealand male white rabbits (n = 15) were selected and randomly divided into a blank group, a model group, and a Tuina group. The rabbits in the model group and the Tuina group were molded by fibrous ring puncture; the rabbits in the model group were only immobilized on the operating table without treatment. In contrast, the Tuina group used the "8N/10N, 30 cycles/min" prescription for kneading, pointing, and flicking to perform the intervention, using tactile sensory aids to monitor and regulate the intensity of the Tuina operation. Imaging diagnosis and pathological tests were used to assess the effect of Tuina in rabbits, and the results showed improved imaging features and significantly lowered pathology scores of lumbar disc degeneration in the Tuina group compared to the model group (P &lt; 0.01). Targeted Tuina in the lumbar region may be beneficial in the alleviation of lumbar disc degeneration, but further verification is needed. By regularly performing Tuina and recording the mechanical information involved enables reproducible manipulation prescriptions and helps to observe the basic features of the underlying mechanism of Tuina for IDD.

The finger tactile measurement system quantifies tactile pressure on the finger using a highly sensitive electrosurgical pressure sensor, with software displaying and accurately recording the pressure data and video in real time. Two analysis modules unify the data processing for thumb tracking and conditioning during Tuina.

Tui Na or massage therapy alleviates symptoms related to intervertebral disc degeneration (IDD). However, precise, repeatable, standardized instructions ...

Tuina Therapy for Frozen Shoulders in a Rat Model

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Author Spotlight: Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder 

This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

Induction of Lumbar Disc Degeneration in Rabbits Through a Transabdominal Approach

Lower back pain is an extremely common medical issue in populations worldwide. One of the main contributors to lower back pain is intervertebral disc (IVD) degeneration. An ideal animal model of IVD degeneration is essential to study the pathophysiology of lower back pain and investigate potential therapeutic strategies. Rabbit models are reliable, economical, and easily established animal models. The retroperitoneal approach has been widely used to induce IVD degeneration in rabbit models. However, there are reported complications associated with this technique, such as the avulsion of segmental arteries and nerve root injury. In this paper, we aim to show a surgical protocol using needle puncture to establish rabbit lumbar disc degeneration via a transabdominal approach. Consequently, radiological checks and histological analyses indicated that lumbar disc degeneration was successfully established in rabbits. This surgical protocol presents the precise location of target discs and high reproducibility of IVD degeneration models with fewer complications.

Here, we describe a surgical protocol using needle puncture to establish rabbit lumbar disc degeneration via a transabdominal approach. Radiological checks and histological analyses were used to confirm the successful establishment of lumbar disc degeneration.

Lower back pain is an extremely common medical issue in populations worldwide. One of the main contributors to lower back pain is intervertebral disc ...

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

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