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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical tools</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>Forceps with teeth</td>
			<td>Taobao, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors&#160;</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>11</td>
		</tr>
		<tr>
			<td>Suture (4-0 Silk)</td>
			<td>Taobao, China</td>
			<td>19054</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical accessories</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>Drugs</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>Tin Hang Tech</td>
			<td>b5274-5g</td>
			<td>1-2 mg/kg</td>
		</tr>
		<tr>
			<td>Cephalexin</td>
			<td>Santa Cruz Biotechnology (Genetimes)</td>
			<td>sc-487556</td>
			<td>20 mg/kg</td>
		</tr>
		<tr>
			<td>Ketamine&#160;</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>Equipment</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>Software</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>Other reagents&#160;</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>Ethylenediaminetetraacetic 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>Histological staining reagents</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&#160;(Masson)&#160;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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JoVE Journal

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1.0K Views.  The Chinese University of Hong Kong. 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...