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 mice were raised in an environment that meets specific pathogen-free (SPF) standards.</li>
	<li>Acclimatize the mice to the new rearing environment (a light/dark cycle of 12 h/12 h, and a constant temperature and humidity of 18-22 &#176;C and 40%-70%, respectively) for 2 weeks 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 end without stopping, 1 week before the experiment.</li>
	<li>At the age of 8 weeks, anesthetize the mouse using isoflurane inhalation by wetting a cotton ball with 2 mL of isoflurane and placing it in an airtight container for full volatilization. Monitor the mouse&#39;s activity and continue inhalation until the activity of the mouse has significantly reduced. Determine the depth of anesthesia by pinching the toes of the mice to observe their reactions. Inhale vaporized isoflurane (2%) through a facemask to maintain anesthesia in mice during subsequent operations. Use veterinary eye ointment during anesthesia to prevent the eyes from drying.</li>
	<li>After anesthetizing, remove the hair on the ankle joint of the mouse&#39;s right hindlimbs with a shaver, and disinfect the exposed skin three times with alternating rounds of iodine cotton balls and alcohol cotton balls. Administer 5 mg/kg carprofen by subcutaneous injection. Transfer the mouse to the microsurgery animal operating room using a sterile surgical pad.</li>
	<li>For the transverse cervical ligament and anterior talofibular ligament (CL + ATFL) group, make a 7 mm oblique downward longitudinal incision with a scalpel on the skin above the right ankle joint, and probe with microscopic straight forceps in front of the ankle joint.</li>
	<li>Ensure the exposure of the ATFL at the lower border of the talus body and the fibula after probing and cut it gently with a scalpel. Separate the long and short fibular tendons and the extensor digitorum longus tendons, expose the CL, and cut it with a scalpel.</li>
	<li>For the transverse CL + deltoid ligament (DL) group, make an 8 mm vertical incision on the medial skin of the right ankle joint and bluntly separate the DL from the medial malleolus to finally cut it. Then, cut the CL as described in step 1.5.</li>
	<li>For the sham group (sham surgery group), perform sham surgery on the right ankle joint but do not cut any ligaments.</li>
	<li>Flush the incision with sterile normal saline and suture it with 5-0 surgical nylon thread. Finally, disinfect the sutured incision with an iodine cotton ball.</li>
	<li>Keep the mouse under observation until it can maintain sternal recumbency, and supervise until it is fully conscious. Disinfect the ankle incision twice per day with an iodine cotton ball and administer carprofen (5 mg/kg, subcutaneous injection), once a day for 1 week. Rear alone after the operation, and pay close attention to the postoperative condition of the mouse.</li>
	<li>Two weeks after surgery and when the ankle joint swelling has reduced, start exercising the mice in the mouse rotary fatigue machine for 1 h every day.</li>
</ol>
2. Balance beam test
<ol>
	<li>Clamp a circular wooden beam with a length of 1 m and a diameter of 20 mm, inclined at 15&#176; at one end, with a photography tripod clip, and place the other end on a work surface connected to a closed cassette.</li>
	<li>Perform balance beam training 1 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 s without pausing.</li>
	<li>Perform two consecutive trials for each mouse and spray the balance beam with 75% alcohol after each trial to prevent the residual odor of the previous mouse from affecting the next mouse.</li>
	<li>Perform the balance beam test preoperatively, 3 days, 1 week, 4 weeks, 8 weeks, and 12 weeks after surgery. Record the average time of each mouse passing the balance beam twice in a row and the number of times the right hindfoot slips off the beam as dependent variables.</li>
</ol>
3. Footprint analysis
<ol>
	<li>Place a U-shaped plastic channel with a length of 50 cm, a width of 10 cm, and a height of 10 cm on the experimental table and connect one end of the plastic channel to a closed cassette.</li>
	<li>Place a plain pigment paper flat in the channel, and then hold the mouse with both hands and paint their front and rear feet evenly with non-toxic red and green pigment.</li>
	<li>Place the painted mouse gently at one end of the channel and let them move to the other end of the cassette. Take the pigmented paper with the footprints out, mark it, and place it on a rack to dry in a ventilated and shaded place.</li>
	<li>After each mouse has passed, spray the U-shaped plastic channel with 75% alcohol so as to prevent the previous mouse&#39;s residual odor from affecting the next mouse.</li>
	<li>Select three consecutive clear mouse footprints on each paper, and use a ruler to measure the step length of the footprint of the mouse&#39;s right foot, as well as the step width between the left and right footprints.</li>
</ol>
4. Thermal nociception assessment
<ol>
	<li>During the experiment, use the plantar test to record the thermal nociception reaction time of the mouse foot and the reaction time of the mouse during activity and resting, respectively. Record the measurement data in seconds.</li>
	<li>Place the mouse in the measuring instrument, align the instrument with its right foot, and start to heat the instrument with the temperature increasing slowly. Observe the mouse&#39;s response. When the temperature increases above tolerance, the mouse will quickly withdraw or lick its right foot. Record the time using the instrument and define the time as the reaction time during the activity.</li>
	<li>To measure the reaction time at rest, allow the mouse to sit for 30 min on the rest table without heating, and then obtain the time data as explained in step 4.2. Use these acquired temporal data for subsequent analysis.</li>
</ol>
5. Micro-CT scanning
<ol>
	<li>Euthanize 12-week-old mice with carbon dioxide postoperatively, peel off the skin of their right ankles, and then cut their middle tibia and fibula with surgical scissors to obtain complete ankle specimens. Place the specimens in marked 15 mL centrifuge tubes containing 10% neutral formalin for 48 h.</li>
	<li>After fixation, place the specimens in batches (four specimens per batch) in a special sponge tank for micro-CT scanning. Set the machine parameters as follows: voltage = 50 kV, current = 200 mA, filter = 0.5 mmAl, and resolution = 9 &#181;m. Run the micro-CT scanner.</li>
	<li>After scanning, use the reconstruction software to delineate the range of the image, and select a specific angle position after adjusting the XYZ axis of the delineated image using commercial data analysis software, as described in Liu et al.10.</li>
	<li>Use micro-CT analysis software to select continuous 10-layer regions of interest in the restructured images adjusted by the XYZ axes, and quantitatively analyze the required joints to determine the bone volume fraction (BV/TV), as described in Liu et al.10.</li>
	<li>Finally, use three-dimensional medical image processing software to perform three-dimensional CT image processing of the mouse ankle joints, as described in Liu et al.10. After reconstruction, observe the wear and the formation of osteophytes in the ASCJ.</li>
</ol>
6. Section staining of articular cartilage
NOTE: All the staining steps are performed in a fume hood, and a mask is worn during the procedure.
<ol>
	<li>Use microscopic tweezers and scissors to remove excess soft tissue around the ankle specimens, and then place the specimens in a centrifuge tube containing 10% EDTA decalcification solution prepared with 44 g of NaOH, two packs of PBS, and 400 g of EDTA-2Na, with the pH adjusted to 7.35-7.45 with hydrochloric acid, and mark the different groups.</li>
	<li>Next, place the centrifuge tube on a shaking table (speed set to 20 rpm) for decalcification and change the decalcification solution once per day. Determine the decalcification of the specimens.</li>
	<li>After 1 month of decalcification, dehydrate the specimens with gradient alcohol and then use n-butanol for 8 h for clearing purposes. Finally, immerse the cleared specimens in paraffin in a coronal position for embedding.</li>
	<li>Place the paraffin specimens in a 4 &#176;C refrigerator for later use. Before sectioning, take the specimens from the 4 &#176;C refrigerator and place them in a &#8722;20 &#176;C freezer for about 10 min to facilitate the cutting of the complete plane.</li>
	<li>Fix the specimens on the microtome and section them at a thickness of 6 &#181;m. At the same time, use a microscope to observe whether the samples have been cut at the expected level-easy penetration of a syringe needle into the bone tissue.</li>
	<li>Adjust the water temperature of the tablet machine to 40 &#176;C in advance. Next, cut two to three complete paraffin sections in a row and transfer them to the tablet machine for full expansion. Then, remove the paraffin sections with a glass slide and drain the water. Finally, mark the slides with groups and numbers.</li>
</ol>
7. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Place the sections in a 60 &#176;C incubator in such a manner that the intact ASCJ joint structure can be observed under the microscope and bake the sections for 40-50 min. Then, dewax the sections with xylene 3x, numbered as I, II, and III for easy identification, for 15 min, 15 min, and 10 min, respectively.</li>
	<li>Place the dewaxed sections in 100% ethanol 2x, numbered as I and II for easy identification, 90% ethanol, and 80% ethanol for 3 min, 3 min, 5 min, and 5 min, respectively. Next, wash the sections with double-distilled water (ddH2O) for 5 min.</li>
	<li>After soaking with hematoxylin for 1 min, wash the sections with ddH2O until they become colorless. Soak the sections in 1% acid ethanol differentiation solution for 30 s and wash them 3x for 1 min with ddH2O. After that, stain the sections with 1% ammonia solution for 1 min, and then wash 3x for 1 min each with ddH2O.</li>
	<li>Next, stain the samples with eosin staining solution for 1 min, and then put them in 95% ethanol and 100% ethanol for 1 min each in succession. Finally, treat the sections with xylene IV for 1 min.</li>
	<li>Air-dry the sections, paste a drop of neutral resin onto the specimens on the slides, and cover them with a cover slip. Then, take images with an upright fluorescence microscope in brightfield at 5x and 20x.</li>
</ol>
8. Safranin O-fast green staining
<ol>
	<li>Place the selected sections into a 60 &#176;C incubator and bake the sections for 40-50 min. Then, dewax the sections with xylene I, II, and III for 15 min, 15 min, and 10 min, respectively.</li>
	<li>Place the dewaxed sections in 100% ethanol I, II, 90% ethanol, and 80% ethanol for 3 min, 3 min, 5 min, and 5 min, respectively. Next, wash the sections with ddH2O for 5 min.</li>
	<li>After soaking with hematoxylin for 1 min, wash the sections with ddH2O until they become colorless. Soak the sections in 1% acid ethanol differentiation solution for 30 s and wash them 3x for 1 min with ddH2O. Then, stain the sections with 1% ammonia solution for 1 min, and wash them 3x for 1 min each with ddH2O.</li>
	<li>Soak the sections in 0.05% fast green for 2 min, followed by soaking the sections in 1% acetic acid solution for 30 s and in 0.1% safranine for 5 min. Place the stained samples in 95% ethanol and 100% ethanol for 1 min, one after the other.</li>
	<li>Finally, treat the sections with xylene IV for 1 min. Air-dry the sections, paste a drop of neutral resin onto the specimens on the slides, and cover them with a cover slip. Then, take images with an upright fluorescence microscope in brightfield at 5x and 20x.</li>
</ol>
9. Immunohistochemistry
<ol>
	<li>Day 1: Place the selected sections in a 60 &#176;C incubator, bake the sections for 40-50 min, and then dewax them with xylene I, II, and III for 15 min, 15 min, and 10 min, respectively. Place the dewaxed sections in 100% ethanol I, II, 90% ethanol, and 80% ethanol for 3 min, 3 min, 5 min, and 5 min, respectively. Then, wash the sections with ddH2O for 5 min, use a histochemical pen to circle the area of the specimen, and place them in a dark box.</li>
	<li>Antigen retrieval: Drop 20-50 &#181;L of 0.25% trypsin into the circled specimen area, incubate in a 37 &#176;C incubator for 60 min, and then wash the specimens 3x for 2 min each with PBS. Block endogenous peroxidase by adding 3% H2O2, incubate the specimens for 10 min at room temperature in the dark, and then wash them 3x for 2 min each with PBS. Perform serum blocking by adding 10% goat serum at room temperature for 20 min, and then incubate with the primary antibody (mouse anti-mouse type II collagen, diluted 1,000 times) at 4 &#176;C overnight.</li>
	<li>Day 2: Rewarm the sections at room temperature for 30 min. Recover the primary antibody and wash the sections 3x for 2 min each with PBS. Incubate with the secondary antibody for 40 min in an incubator at 37 &#176;C, and then wash the sections 3x for 2 min each with PBS.</li>
	<li>DAB color development: Add 5 mL of ddH2O, two drops of buffer, four drops of DAB, and two drops of H2O2 to prepare the DAB reagent. Add 20-50 &#181;L of reagent to the sections, keep the samples protected from light for 5 min, and then wash the sections for 2 min with ddH2O.</li>
	<li>After soaking with hematoxylin for 1 min, wash the sections with ddH2O until they become colorless. Soak the sections in 1% acid ethanol differentiation solution for 30 s and wash them 3x for 1 min each with ddH2O. Stain the sections for 1 min with 1% ammonia solution, and then wash them 3x for 1 min each with ddH2O.</li>
	<li>Next, place the sections in 95% ethanol and 100% ethanol, respectively, for 1 min each. Finally, incubate the sections for 1 min with xylene IV. Air-dry the sections, paste a drop of neutral resin onto the specimens on the slides, and cover them with a cover slip. Then, take images with an upright fluorescence microscope in brightfield at 5x and 20x.</li>
</ol>

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None of the authors have any conflicting interests.

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">
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		<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>
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		<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>
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		<tr>
			<td>Aqueous acetic acid (1%)</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd.</td>
			<td>R20773</td>
			<td></td>
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		<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>
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			<td>Centrifuge tube 50ml</td>
			<td>Beijing Soleibo Technology Co., Ltd.</td>
			<td>YA0472</td>
			<td></td>
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			<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>
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		<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>
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		<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>
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		<tr>
			<td>Gait paper</td>
			<td>Baoding Huarong Paper Factory</td>
			<td></td>
			<td></td>
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		<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>
</table>

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.
<p class="jove_conten...

Watch this Scientific Journal Video about A Mouse Model of Ankle-Subtalar Complex Joint Instability at JoVE.com

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 ...

a-mouse-model-of-ankle-subtalar-complex-joint-instability

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

1.3K Views.  The First Affiliated Hospital of Soochow University. 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.

Video: A Mouse Model of Ankle-Subtalar Complex Joint Instability

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

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.

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