Name: a mouse model of ankle-subtalar complex joint instability
Uploaded: 2022-10-28T14:40:00
Description: Watch this Scientific Journal Video about A Mouse Model of Ankle-Subtalar Complex Joint Instability at JoVE.com

This study was supported by the Jiangsu provincial government scholarship program and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

All animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Soochow University.
1. Surgical procedures
<ol>
	<li>Divide 21, 6-week-old C57BL/6 male mice into three groups: the transverse cervical ligament and anterior talofibular ligament group, the transverse cervical ligament and deltoid ligament group, and the sham surgery group. Ensure that all the m...

<ol>
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M., Garcia, J., Hoffmeyer, P., Fritschy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subtalar+sprain.+A+roentgenographic+study.">The subtalar sprain. A roentgenographic study.</a> Clinical Orthopaedics and Related Research. (226), 169-173 (1988).</li><li>Mittlmeier, T., Rammelt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+subtalar+joint+instability.">Update on subtalar joint instability.</a> Foot and Ankle Clinics. 23 (3), 397-413 (2018).</li><li>Chang, S. 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In this study, two mouse models of ASCJ instability were successfully constructed by transecting CL + ATFL or CL + DL. The time for the mice to pass through the balance beam increased significantly at 8 weeks and 12 weeks after surgery, which is similar to the results obtained by the Hubbard-Turner team by cutting the lateral ligament of the ankle joint23,24. In the right foot sliding test, we observed that the sliding times of the two groups of mice with severed...

Ankle sprains are one of the most common sports injuries worldwide. It is estimated that 10,000 people are injured daily in the United States1, of which sports-related injuries account for 15%-45%2. The medical costs associated with treating ankle sprains in the United States amount to $4.2 billion annually3,4,5. Chronic foot instability is a common problem following ankle sprains and occurs in approximately 74% of ankle sprains6, including ankle or subtalar instability. However, due to the similar clinical symptoms and signs, it is difficult for medical staff to distinguish whether chronic ankle instability is also accompanied by chronic subtalar joint instability in the clinic, and as a result, chronic subtalar instability can be easily missed. Therefore, the true incidence of chronic ankle-subtalar complex joint (ASCJ) instability (a specific type of chronic foot instability that includes both chronic ankle instability and chronic subtalar instability) may be higher than reported7,8,9. If left untreated, chronic ankle-subtalar complex joint instability can cause repeated ankle sprains, leading to a vicious circle of ankle sprains and chronic ankle-subtalar complex instability. Long-term chronic ankle-subtalar complex instability can lead to degeneration of the ASCJ and post-traumatic osteoarthritis, which can affect the adjacent joints in severe cases10. For these diseases, the current clinical treatment is mainly conservative, in addition to surgical treatment methods such as ligament repair and ligament reconstruction11,12.
ASCJ is the core structure of the foot and maintains the balance of the body during movement13. Extensive research has been conducted on the structure of the ankle joint and the subtalar joint separately14,15,16,17. However, research on the whole ankle-subtalar joint is rare. About one-quarter of the cases of ankle injury are associated with subtalar joint injury18. Due to the complex injury mechanism of ASCJ instability, there is no consensus on diagnosing and treating it in the clinical setting. Considering the current situation of ankle injuries in the clinic, a more scientific method is needed to study the ankle and subtalar joint as a whole, thereby providing a new understanding for studying foot diseases.
Since the anatomical structure of the mouse hindfoot at the musculoskeletal level is comparable to that of the human foot19, in several studies, mouse models for foot/ankle research have already been implemented10,19. Chang et al.19 successfully developed three different mouse models of ankle osteoarthritis. Inspired by the successful establishment of ankle instability in the mouse model, we established a mouse model for ankle-subtalar complex instability, hypothesizing that the transection of the partial ligaments in the mouse hindfoot would result in mechanical instability of the ASCJ, which would lead to post-traumatic osteoarthritis (PTOA) of the ASCJ. The ASCJ instability animal model could be used for the treatment of both ankle instability and subtalar instability, which is more in line with the actual clinical situation than the currently used simple ankle instability model7,8,9,19. To test this hypothesis, two mouse models of ligament transection-induced instability of the ASCJ were designed. The results for the sensory-motor function-the balance beam test, footprint analysis, and thermal nociception assessment-were used to evaluate the feasibility of the model, and micro-computed tomography (CT) and histological staining were used to evaluate the damage and degeneration of the mouse articular cartilage. The successful establishment of a mouse model of ASCJ instability not only provides a new understanding for studying foot diseases but also provides a valuable reference for clinical research on the injury-related mechanisms, provides better treatment options for ankle sprains, and is helpful for further studies on the disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-0 Surgical Nylon Suture</td>
			<td>Ningbo Medical Needle Co., Ltd.</td>
			<td>191104</td>
			<td></td>
		</tr>
		<tr>
			<td>Acidic ethanol differentiation solution (1%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R20778</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive slides</td>
			<td>Jiangsu Shitai Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution (1%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R20788</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Shanghai Sinopharm Group Chemical Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous acetic acid (1%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R20773</td>
			<td></td>
		</tr>
		<tr>
			<td>Black cube cassette</td>
			<td>Shanghai Yizhe Instrument Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 15ml</td>
			<td>Beijing Soleibo Technology Co., Ltd.</td>
			<td>YA0476</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 50ml</td>
			<td>Beijing Soleibo Technology Co., Ltd.</td>
			<td>YA0472</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Jiangsu Shitai Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CTAn software</td>
			<td>Blue scientific</td>
			<td></td>
			<td>micro-CT analysis software</td>
		</tr>
		<tr>
			<td>Dataview software</td>
			<td>AEMC instruments</td>
			<td></td>
			<td>commercial data analysing software</td>
		</tr>
		<tr>
			<td>Disodium ethylenediaminetetraacetate (EDTA-2Na)</td>
			<td>Beijing Soleibo Technology Co., Ltd.</td>
			<td>E8490</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric incubator</td>
			<td>Suzhou Huamei Equipment Factory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding paraffin</td>
			<td>Leica, Germany</td>
			<td>39001006</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin staining solution (alcohol soluble, 1%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R30117</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast green staining solution</td>
			<td>Sigma-Aldrich, USA</td>
			<td>F7275</td>
			<td></td>
		</tr>
		<tr>
			<td>Gait paper</td>
			<td>Baoding Huarong Paper Factory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0</td>
			<td>Graphpad software</td>
			<td></td>
			<td>online statistical analysis tools</td>
		</tr>
		<tr>
			<td>Iodophor cotton balls</td>
			<td>Qingdao Hainuo Bioengineering Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica 818 blade</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Skyscan, Belgium</td>
			<td>SkyScan 1176</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulation microscope</td>
			<td>Suzhou Omet Optoelectronics Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mimics software</td>
			<td>Materialise&#160;</td>
			<td></td>
			<td>3D medical image processing software&#160;</td>
		</tr>
		<tr>
			<td>Modified Harris Hematoxylin Stain</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R20566</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse type II collagen</td>
			<td>American Abcam Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shanghai Sinopharm Group Chemical Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-butanol</td>
			<td>Shanghai Sinopharm Group Chemical Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral formalin fixative (10%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral resin</td>
			<td>Sigma-Aldrich, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nrecon reconstrcution software&#160;</td>
			<td>Micro Photonics Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oaks hair clipper</td>
			<td>Oaks Group Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Embedding Machine</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Shanghai Leitz Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>American Biosharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline (for mammals, sterile)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R22172</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O-staining solution</td>
			<td>Sigma-Aldrich, USA</td>
			<td>HT90432</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (0.9%)</td>
			<td>Shanghai Baxter Medical Drug Co., Ltd.</td>
			<td>309107</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Haimen Qilin Bell Instrument Manufacturing Co., Ltd.</td>
			<td>2008779</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 23</td>
			<td>IBM</td>
			<td></td>
			<td>online statistical analysis tools</td>
		</tr>
		<tr>
			<td>Tablet machine</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ugo Basile</td>
			<td>Ugo Basile Biological Research Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright fluorescence microscope</td>
			<td>Zeiss Axiovert, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shaped plastic channel</td>
			<td>Shanghai Yizhe Instrument Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary eye ointment</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Shanghai Sinopharm Group Chemical Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>YLS-10B Wheel Fatigue Tester</td>
			<td>Jinan Yiyan Technology Development Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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a mouse model of ankle-subtalar complex joint instability

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and can eventually lead to post-traumatic osteoarthritis (PTOA) in the long term. However, due to the complexity of the injury mechanism and the clinical manifestations, such as ecchymosis, hematoma, or tenderness in the lateral foot, there is no clinical consensus on diagnosing and treating ASCJ instability. Since the musculoskeletal structure of the bones and ligaments of the mouse hindfoot is comparable to that of humans, an animal model of ASCJ instability in mice was established by the transection of ligaments around the ASCJ. The model was well-validated through a series of behavioral tests and histological analyses, including a balance beam test, a footprint analysis (an assessment of exercise level and balance ability in mice), a thermal nociception assessment (an assessment of foot sensory function in mice), micro-computed tomography (CT) scanning, and section staining of the articular cartilage (an assessment of articular cartilage damage and degeneration in mice). The successful establishment of a mouse model of ASCJ instability will provide a valuable reference for clinical research on the injury mechanism and result in better treatment options for ankle sprain.

The statistical analysis of the correlation data was performed using online statistical analysis tools. The data that met the two tests of normal distribution and homogeneity of variance were used for further statistical analysis by one-way analysis of variance. If the data did not meet the two tests, the Kruskal-Wallis test was used for the statistical analysis. The data are expressed as the mean &#177; standard deviation (SD), and p &#60; 0.05 was considered statistically significant.
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A Mouse Model of Ankle-Subtalar Complex Joint Instability

The ankle-subtalar complex joint (ASCJ) is the core of the foot and plays a key role in balance control in daily activities. Sports injuries often lead to instability in this joint. Here, we describe a mouse model of ligament transection-induced instability of the ASCJ.

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and ...

The successful establishment of the ASCJ inability mouse model extends a simple ankle inability animal model, and provides a new concepts for the study of the clinical foot diseases. We have established an animal model that's more in line with the actual clinical situation, which can be used for further research on the diagnosis and the treatment of the foot's diseases. One week before the experiment, subject the mice to balance beam and gait training, going from one end of a balance beam or U-shaped pipe to the other, without stopping.After anesthetizing and removing the hair on the ankle joint of the mouse's right hind limbs, disinfect the exposed skin with an iodine swab. Transfer the mouse to the microsurgery animal operating room using a sterile surgical pad. For the transverse cervical and anterior talofibular ligament groups, make a seven millimeter oblique downward longitudinal incision with a scalpel on the skin above the right ankle joint.And probe with microscopic straight forceps in the front of the ankle joint. Ensuring the exposure of the anterior talofibular ligament at the lower border of the talus body and the fibula, cut it gently with a scalpel. Separate the long and short fibular tendons, and the extensor digitorum longus tendons.Expose the cervical ligament, and cut it with a scalpel. For the transverse cervical ligament plus deltoid ligament group, make an eight-millimeter vertical incision on the medial skin of the right ankle joint, and bluntly separate the DL from the medial malleolus to cut it. Then cut the cervical ligament as demonstrated earlier.For the sham group, perform sham surgery on the right ankle joint. After flushing the incision with sterile normal saline, suture it with 5-0 surgical nylon thread, followed by disinfecting the sutured incision with an iodine swab. Once the angle joint swelling has reduced at two weeks after surgery, exercise the mice in the mouse rotary fatigue machine for one hour every day.For the balance beam test, clamp a circular wooden beam inclined at 15 degrees at one end with a photography tripod clip, and place the other end on a work surface connected to a closed cassette. Perform balance beam training one week before surgery to ensure that the mice smoothly move from one end of the beam to the other. Consider a mouse to have passed the test when it passes through the beam twice in 60 seconds without pausing.After each trial, spray the balance beam with 75%alcohol to present the residual odor of the previous mouse from affecting the next mouse. Perform the balance beam test and record the average time of each mouse passing the balance beam twice in a row. And the number of times the right hind foot slips off the beam, as dependent variables.For footprint analysis, place a U-shaped plastic channel on the experimental table, and connect one end of the plastic channel to a closed cassette. Place a plain pigment paper flat in the channel. Then hold the mouse with both hands, and paint the front and rear feet evenly with non-toxic red and green pigment.Now, place the painted mouse at one end of the channel, and allow it to move to the other end of the cassette. Take the pigmented paper with the footprints, mark it, and allow it to dry in a ventilated and shaded space. Spray the U-shaped plastic channel with 75%alcohol after each mouse had passed, to prevent the previous mouse's residual odor from affecting the next mouse.Select three consecutive clear mouse footprints on each paper. Using a ruler, measure the step length of the footprint of the mouse's right foot, with the step width between the left and right footprints. Use microscopic tweezers and scissors to remove excess soft tissue around the ankle specimens.And then, place the specimens in a centrifuge tube containing a 10%EDTA decalcification solution, and mark the different groups. Next, place the centrifuge tube on a shaking table for decalcification. Change the decalcification solution once per day, and determine the decalcification of the specimens.After one month of decalcification, dehydrate the specimens with gradient alcohol, and then use N-butanol for eight hours for clearing purposes. Finally, immerse the cleared specimens in paraffin in a coronal position for embedding. Before sectioning, transfer the specimens from the four degree Celsius refrigerator to a minus 20 degree Celsius freezer for about 10 minutes, to facilitate the complete planes cutting.Fix the specimens on the microtome and section them in a thickness of six micrometers. At the same time, use a microscope to observe whether the samples have been cut at the expected level for easy penetration of a syringe needle into the bone tissue. After cutting two or three complete paraffin sections in a row, transfer them to the tablet machine, set at 40 degrees Celsius, for full expansion.Then remove the paraffin sections with a glass slide. Finally, mark the slides with groups and numbers. To observe the intact ASCJ joint structure under a microscope.Place the sections in an incubator set at 60 degrees Celsius for 40 to 50 minutes. Then de-wax the sections with xylene thrice, as described in the manuscript. Place the de-waxed sections in 100%ethanol twice for three minutes, followed by 90%and 80%ethanol for five minutes each.Then wash the sections with double distilled water for five minutes. After soaking in Hematoxylin for one minute, wash the sections with double distilled water until they become colorless. Soak the sections in 1%acid ethanol differentiation solution for 30 seconds, and wash them with double-distilled water.Once the sections are stained with 1%ammonia solution, and washed with double-distilled water, stain the samples with eosin staining solution for one minute. And then put them in 95%and 100%ethanol in succession for one minute each. Finally, treat the sections with xylene four for one minute.Air dry the sections and cover them with a cover slip, after pasting a drop of neutral resin onto the specimens on the slides. Then take images with an upright fluorescence microscope in bright field at five and 20X. Soak the hematoxylin and ammonia-stained sections in 0.05%fast green for two minutes.Followed by soaking the sections in 1%acetic acid solution for 30 seconds, and 0.1%safranin for five minutes. Then process the slides with ethanol and xylene as demonstrated earlier. Take images with an upright fluorescence microscope in bright field at 5X and 20X.After three days, eight weeks, and 12 weeks of surgery, the severed ligament groups took significantly longer to pass through the balance beam than the sham group. Before surgery, the right hind foot step length was comparable among the three mice groups. However, 12 weeks after surgery, the step length was shorter in the ligament cut group than in the sham group.Before surgery, the thermal nociception response time of the mice's feet during activity is similar among the three groups of mice. After surgery, the mice in the ligament cut group displayed longer response times compared to the sham group. The ASCJ of the right hind foot in severed ligament groups showed rough surfaces, osteophyte accumulation, degenerative changes.And significantly higher bone volume fraction than the sham group. The cartilage layer of the ASCJ in the ligament cut group showed discontinuity, and decreased number of chondrocytes. Also, the Malkin and Osteoarthritis Research Society International scores of the mice with ligament amputation were significantly higher than the sham group.The type two collagen in the sham group's ASCJ articular cartilage layer of the right hind foot was more uniform, and higher than that of the two groups of mice with severed ligaments. This study can be used for the diagnosis and the treatments for ankle instability, and provide an experimental reference for the clinical treatments of this disease.

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