A Rat Model of Tibial Cortex Transverse Transport for the Treatment of Lower Limb Ischemia

Summary

This procedure demonstrates tibial cortex transverse transport in rats, enabling visualization and analysis of revascularization during limb ischemia.

Abstract

Tibial cortex transverse transport (TTT) is an emerging surgical technique used to promote revascularization in ischemic lower limbs. This video demonstrates a reproducible rat model of TTT, detailing the surgical creation of a cortical bone fragment, the transport protocol, and postoperative assessments. The procedure enables controlled upward and downward transport of a bone fragment to stimulate angiogenic responses. Representative X-ray images acquired on postoperative days 1, 7, and 12, together with microvascular perfusion imaging performed on Day 19 after euthanasia, show clear bone fragment movement and improved limb perfusion following TTT.

This model provides a practical and reproducible platform for investigating the molecular mechanisms underlying TTT-induced angiogenesis and evaluating potential therapeutic strategies for ischemic diseases. To construct this model, we simulated the clinical TTT procedure in Sprague-Dawley rats, involving three main surgical steps: ligation of the superficial femoral artery, creation and mobilization of a cortical bone fragment, and application of a custom external fixator. Over 12 days, the fragment was gradually transported forward and then backward. Perfusion imaging confirmed enhanced vascularization in the ischemic limb after the completion of the transport cycle. Overall, this standardized rat model closely mimics clinical TTT procedures and offers a valuable experimental system for studying mechanotransduction, angiogenesis, and vascular regeneration in ischemic limb conditions.

Introduction

Lower limb ischemic diseases, such as arteriosclerosis obliterans (ASO), thromboangiitis obliterans (TAO), and diabetic foot (DF), are characterized by restricted arterial blood flow to the lower extremities^1,2. In the early stages, patients typically experience intermittent claudication and resting pain. As the disease progresses, prolonged ischemia can result in tissue necrosis, infection, and, in severe cases, limb amputation¹. Current standard treatments-including pharmacological therapy, endovascular interventions, and open surgical procedures-often yield limited efficacy, particularly in advanced stages of the disease³.

Tibial cortex transverse transport (TTT) is an innovative therapeutic strategy for the treatment of various lower limb ischemic diseases⁴. Rooted in the Ilizarov method and the "tension-stress rule"^5,6, this technique involves creating a mobile cortical bone flap fragment in the tibia through osteotomy. A continuous, slow, and controlled distraction force is then applied to stimulate the regeneration of bone, blood vessels, and surrounding soft tissues. This regenerative process facilitates the reestablishment of microcirculation and helps restore the balance of oxygen supply in the affected limb. Ultimately, TTT leads to improved blood perfusion and functional recovery in ischemic tissues⁷.

Conventional vascular reconstruction techniques for lower limb ischemia can restore blood flow in large arteries of the calf but are often ineffective in revascularizing smaller arteries in the foot or promoting the repair and regeneration of damaged microvessels, limiting their ability to fully resolve ischemia⁸. TTT continuously applies traction to the bone and surrounding soft tissues, thereby inducing the robust release of chemotactic factors and cytokines⁹. These chemotactic factors regulate various functions of stem cells and, under ischemic conditions, are abundantly secreted into circulation. They actively recruit endogenous stem cells to sites of injury, thereby facilitating vascular repair and regeneration without requiring direct stem cell transplantation. Owing to these advantages, TTT has emerged as a promising therapeutic strategy for lower limb ischemia and is increasingly employed in the treatment of diverse ischemic conditions affecting the lower extremities^7,9,10.

Although rabbits and dogs are commonly used in fundamental TTT research, the lack of suitable analytical tools for these species limits mechanistic investigations. In contrast, the rat TTT model offers greater compatibility with available molecular and imaging techniques while maintaining practicality and lower cost, making it a more accessible and reproducible platform for mechanistic studies. However, to date, no study has systematically described or evaluated the efficacy of TTT in a rat model of lower limb ischemia.

The rat TTT model requires refined microsurgical skills due to the small tibial size and thin cortex, and its distraction range is limited by cortical thickness. These practical constraints should be taken into account when evaluating the model's suitability for specific research environments.

In this study, we developed a rat-specific TTT model by replicating the core structure and operative principles of the clinical tibial transverse transport apparatus. The procedure was refined from clinically validated protocols to ensure feasibility and consistency in small animals. Our objective is to establish and validate a standardized and reproducible rat TTT model suitable for investigating angiogenesis and mechanotransduction in ischemic limb disease.

Protocol

All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (approved No. 2025-E0642). Male Sprague-Dawley rats (300-350g) were housed in the Guangxi Medical University Animal Research Center under standardized conditions. Rats were provided with normal chow and clean drinking water ad libitum and maintained on a 12 h artificial light/dark cycle. The animal facility was kept at a controlled temperature of 20-26 °C with a relative humidity of 40%-70%. All surgical instruments were thoroughly sterilized prior to use. Detailed information on all reagents and materials used in this study is provided in Table 1.

Euthanasia was performed by intraperitoneal injection of an overdose of sodium pentobarbital. Death was confirmed by the permanent cessation of heartbeat and respiration, pupillary dilation, and the absence of response to noxious stimuli. All procedures were conducted by trained personnel to minimize animal distress, and the euthanasia protocol had been reviewed and approved in advance by the Institutional Animal Care and Use Committee.

1. External fixator for TTT

1. Use a scaled-down, custom-designed version of the clinical tibial cortex transverse transport external fixator (TTT-EF), consisting of a bone fragment traction unit and an external fixation frame.
2. Enable vertical mobilization of both components by incorporating an internal screw-thread mechanism (Figure 1).
3. Drill two through-holes on each side of the cross-sections of both the traction unit and the fixation frame to allow insertion of Kirschner wires.
4. Insert the Kirschner wires through the designated holes and secure them laterally using hexagonal screws to ensure stabilization.
5. Connect the traction component of the TTT-EF to the bone fragment using 0.8 mm Kirschner wires.
6. Anchor the fixation component to the proximal and distal ends of the tibia using 1.0 mm Kirschner wires.

2. Surgical procedure

1. Perform the TTT procedure on 8-week-old male Sprague-Dawley (SD) rats. Anesthetize the rats by intraperitoneal injection of 0.35 mg/kg sodium pentobarbital and 0.3 mg/kg chloral hydrate.
2. Assess the anesthetic effect by testing the pedal withdrawal reflex and corneal reflex. Continuously monitor vital signs and the depth of anesthesia throughout the procedure. Apply ophthalmic ointment to the rat's eyes prior to surgery to protect the cornea, following the Guidelines for the Use of Laboratory Animals.
3. Shave the surgical area and administer 0.5% lidocaine to minimize postoperative pain.
4. Make a 10 mm skin incision in the right inguinal region using a No. 11 scalpel.
5. Bluntly dissect the fascia to expose the superficial femoral artery.
6. Isolate the artery between the femoral nerve and the superficial femoral vein using ophthalmic forceps. Ligate and transect both ends of the artery.
7. Suture the incision using 4-0 absorbable suture.
8. Make a vertical 15 mm incision on the medial upper tibia using a No. 11 scalpel.
9. Separate the subcutaneous tissue and underlying muscle carefully to avoid periosteal injury. Gently dissect the soft tissue to fully expose the medial tibial surface.
10. Outline the bone fragment area (8 mm × 4 mm) on the medial tibial surface using a surgical blade. Drill closely spaced perforations along the marked boundary using a 0.6 mm drill bit. Create a bone window fragment (8 mm × 4 mm) on the upper medial tibia.
11. Implant two 0.8 mm threaded Kirschner wires into the bone fragment, spaced 0.5 mm apart, to facilitate transport. Connect the Kirschner wires to the TTT-EF and secure them.
12. Insert two 1.0 mm Kirschner wires on either side of the fragment, penetrating the posterior cortex of the tibia.
13. Assemble and install the TTT-EF.
14. Perform osteotomy along the marked lines using ophthalmic scissors.
15. Gently elevate the bone fragment to enable vertical mobility, avoiding marrow injury.
16. Irrigate the surgical site with 10% povidone-iodine followed by sterile saline.
17. Suture the incision in layers using 4-0 absorbable suture.
18. Administer buprenorphine (0.05 mg/kg) subcutaneously immediately post surgery. Continue dosing every 12 h for up to 3 days to ensure effective analgesia.

3. Postoperative monitoring

1. Closely monitor animals after surgery to ensure proper recovery and detect potential complications early. Examine animals every 6-8 h during the first 48 h postoperatively. Assess general activity, posture, grooming behavior, food and water intake, and surgical incision condition.
2. Perform monitoring at least once daily from postoperative Day 3 through the distraction and consolidation phases. Confirm adequate recovery by verifying normal ambulation, stable body weight, consistent food and water intake, and absence of wound discharge, excessive swelling, or behavioral signs of pain.
3. Continue analgesia as needed in accordance with institutional guidelines.
4. Treat wound infections (e.g., erythema, swelling, purulent discharge) with local wound care and systemic antibiotics, following veterinary recommendations.
5. Correct any loosening or displacement of the external fixator immediately under brief anesthesia to maintain alignment and enable accurate distraction.
6. Promptly assess and manage any potential complications. Evaluate animals with severe complications affecting mobility, feeding, or wound healing in consultation with veterinary staff.
7. Euthanize animals humanely if they meet predefined humane endpoint criteria.

4. Bone fragment transport protocol

1. Use the tibial cortex transverse transport external fixator (TTT-EF) to perform postoperative bone fragment transport.
2. Designate Day 1 after surgery as the latency period.
   NOTE: Do not manipulate the bone fragment during this period.
3. Initiate the upward transport phase on Day 2. Advance the bone fragment at a rate of 0.5 mm every 12 h for 5 consecutive days (Day 2-Day 6). Pause the transport process on Day 7 to allow for stabilization.
4. Initiate the downward transport phase on Day 8.
5. Retract the bone fragment at the same rate of 0.5 mm every 12 h for 5 days (Day 8-Day 12). Ensure that the bone fragment returns to its original position by the end of Day 12.
6. Continue with a 2-day consolidation phase (Days 13-14), with no further transport.
7. Remove the external fixator at the end of the consolidation phase.
   NOTE: Refer to Figure 2 for a visual timeline of the TTT transport procedure and evaluation schedule.

5. Evaluation

NOTE: Use imaging techniques to evaluate both the bone fragment position and blood flow restoration.

1. Perform X-ray imaging of the lower limbs on Days 1, 7, and 12 to monitor the progression and final position of the transported bone fragment.
   1. Anesthetize rats at designated imaging time points using 2.0% isoflurane in oxygen.
   2. Place each rat on a temperature-maintained imaging platform equipped with a warming pad to prevent hypothermia during the procedure.
   3. Monitor anesthetic depth by assessing respiratory rate and pedal reflex to ensure stable immobilization.
   4. Position the limbs and external fixator in a standardized orientation across all animals to minimize variability in X-ray acquisition.
   5. Acquire X-ray images to confirm bone fragment displacement and repositioning.
2. Conduct vascular perfusion imaging on Day 19 postoperatively to assess microvascular perfusion in the lower limb.
   1. Euthanize the rats and perfuse contrast agent via the femoral artery to visualize vasculature.
   2. Place the euthanized rats in a standardized supine position for consistent imaging setup.
   3. Allow complete vascular filling by perfusing the contrast agent through the femoral artery under steady room-temperature conditions.
   4. Conduct vascular imaging to assess microvascular architecture and perfusion. Ensure all specimens are aligned consistently with identical limb orientation and fixator alignment to support reproducibility across imaging sessions.
   5. Use the resulting images to evaluate the restoration of blood flow and vascular architecture in the lower limbs.

Results

Figure 3 illustrates the procedure for constructing a TTT-treated lower limb ischemia animal model. In our hands, the procedure demonstrated a surgical success rate of approximately 90%. The limited number of failures was largely attributable to technical issues such as external fixator loosening or intraoperative bone fragment fracture, which rendered subsequent distraction infeasible. The model was established by exposing and ligating the superficial femora...

Discussion

All procedures involving anesthesia and surgery were performed with careful attention to animal safety and institutional animal care guidelines. The depth of anesthesia was confirmed prior to incision using pedal withdrawal or corneal reflex testing and was monitored regularly throughout the procedure. Adequate thermal support (such as a heating pad) was provided to prevent hypothermia, and ophthalmic ointment was applied to protect the corneas during prolonged anesthesia.

Potential risks asso...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micro Transverse Retractor	Tianjin Xinzhong Medical Equipment Co., Ltd	A61B17/66	Perform bone fragment traction
Small Orthopedic Drill	Yancheng Ruiao Technology Co. LTD	RA-II	For tibial drilling osteotomy
0.6 mm Stainless Steel Drill Bit	Changzhou Borche Medical Equipment Co., Ltd	-	For tibial drilling osteotomy
0.8 mm Thread type Kirschner wire	Changzhou Borche Medical Equipment Co., Ltd	-	For attachment of bone flaps and retractor
1.0 mm Thread type Kirschner wire	Changzhou Borche Medical Equipment Co., Ltd	-	For fixation of retractor
Pentobarbital sodium	Shanghai Pharmaceuticals Holding Co., Ltd	-	For anesthetizing animals
Allen key	Changzhou Borche Medical Equipment Co., Ltd	-	For fixing screws
Buprenorphine	Shanghai Pharmaceuticals Holding Co., Ltd		For analgesia animal
MICROFIL	Flow Tech,Inc Co., Ltd		Angiography of animals
Isoflurane	Hebei Yipin Pharmaceutical Co., Ltd.		For induction and maintenance of inhalation anesthesia
Lidocaine Hydrochloride (0.5%)	Hebei Yipin Pharmaceutical Co., Ltd.		Used for local infiltration to reduce postoperative pain and minimize tissue irritation during surgical procedures