A Mouse Model of Ankle-Subtalar Complex Joint Instability

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 annually^3,4,5. 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 reported^7,8,9. 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 reconstruction^11,12.

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 separately^14,15,16,17. 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 implemented^10,19. 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 model^7,8,9,19. 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.

Protokół

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

1. 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.
2. 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 °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.
3. 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'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.
4. After anesthetizing, remove the hair on the ankle joint of the mouse'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.
5. 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.
6. 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.
7. 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.
8. For the sham group (sham surgery group), perform sham surgery on the right ankle joint but do not cut any ligaments.
9. 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.
10. 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.
11. 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.

2. Balance beam test

1. Clamp a circular wooden beam with a length of 1 m and a diameter of 20 mm, inclined at 15° at one end, with a photography tripod clip, and place the other end on a work surface connected to a closed cassette.
2. 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.
3. 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.
4. 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.

3. Footprint analysis

1. 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.
2. 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.
3. 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.
4. After each mouse has passed, spray the U-shaped plastic channel with 75% alcohol so as to prevent the previous mouse's residual odor from affecting the next mouse.
5. Select three consecutive clear mouse footprints on each paper, and use a ruler to measure the step length of the footprint of the mouse's right foot, as well as the step width between the left and right footprints.

4. Thermal nociception assessment

1. 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.
2. 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'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.
3. 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.

5. Micro-CT scanning

1. 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.
2. 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 µm. Run the micro-CT scanner.
3. 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.
4. 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.
5. 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.

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.

1. 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.
2. 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.
3. 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.
4. Place the paraffin specimens in a 4 °C refrigerator for later use. Before sectioning, take the specimens from the 4 °C refrigerator and place them in a −20 °C freezer for about 10 min to facilitate the cutting of the complete plane.
5. Fix the specimens on the microtome and section them at a thickness of 6 µm. 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.
6. Adjust the water temperature of the tablet machine to 40 °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.

7. Hematoxylin and eosin (H&E) staining

1. Place the sections in a 60 °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.
2. 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.
3. 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.
4. 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.
5. 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.

8. Safranin O-fast green staining

1. Place the selected sections into a 60 °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.
2. 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.
3. 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.
4. 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.
5. 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.

9. Immunohistochemistry

1. Day 1: Place the selected sections in a 60 °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.
2. Antigen retrieval: Drop 20-50 µL of 0.25% trypsin into the circled specimen area, incubate in a 37 °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 °C overnight.
3. 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 °C, and then wash the sections 3x for 2 min each with PBS.
4. 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 µL of reagent to the sections, keep the samples protected from light for 5 min, and then wash the sections for 2 min with ddH₂O.
5. 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.
6. 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.

Wyniki

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 ± standard deviation (SD), and p < 0.05 was considered statistically significant.

Dyskusje

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 joint^23,24. In the right foot sliding test, we observed that the sliding times of the two groups of mice with severed...

Materiały

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