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

Lower limb ischemia mainly reduces blood flow to the leg and can seriously affect tissue healings. Tibial cortex transverse transport of TTT is a surgical technique that help promotes new blood vessels growth and improve circulations. To better study how this works, we developed a rat model that closely replicate the clinical TTT procedure.

This model is straightforward, reliable, and easy to reproduce, allowing us to visualize blood flow recoveries and explore the mechanisms behind TTT. Begin with a fully anesthetized adult SD rat. In accordance with the guidelines for the use of laboratory animals, ophthalmic ointment was applied to protect the cornea and thermal support and physiological monitoring were provided throughout the procedure.

Before the operation, hair around the surgical site was completely removed and the area was disinfected with 10%povidone iodine. To facilitate the procedure, 0.5%lidocaine hydrochloride was locally injected into the surgical region. We then proceeded with the invasive portion of the protocol.

To establish the lower limb ischemia model, a 10 millimeters incision was made in the right groin using a number 11 scalpel blade to expose the superficial femoral artery vein and nerve. Using ophthalmic forceps, the superficial femoral artery was carefully dissected free between the femoral nerve and the superficial femoral vein. After isolating the artery, both the proximal and distal ends were ligated with absorbable sutures, followed by transection of the vessel and trimming of excess suture material.

Finally, the surgical area is re-sterilized using 10%iodophor solution, and then the surgical incision is closed with four to zero absorbable sutures. Use suture scissors to cut off excess threads. Next, to expose the tibia, a 20 millimeter skin incision was made along the medial aspect of the right lower limb using a number 11 scalpel blade, followed by careful layer by layer dissection of the subcutaneous tissue and muscle.

Throughout this process, particular attention was paid to avoid damaging the periosteum. An eight millimeters times four millimeters bone fragment was then outlined on the flat surface of the tibial cortex using the scalpel blade. Next, the holes are drilled along the pre-marked scribed lines using a 0.6 millimeters diameter drill bit in a continuous and tight pattern, ensuring that each hole is evenly spaced along the cut line.

After that, we will install a custom designed external fixator and fix the bone fragment. To place two fixation pins in the center of the bone fragment, positioning was first performed using a 0.6 mm drill bit with the two points spaced approximately 0.5 millimeters apart. A 0.8 mm Kirschner wires were then inserted perpendicularly through the marked points into the cortical bone to a depth of three millimeters, taking care to avoid penetrating the contralateral cortex.

Afterwards cut off the excessively long Kirschner wire, leaving about 10 millimeters, then drilled another Kirschner wire in the same manner. For ease of subsequent manipulation, three edges of the bone fragment were cut using ophthalmic scissors, leaving the medial edge intact. This preserved edge provides a stable plane for fixation and facilitates controlled mobilization of the fragment.

A custom designed external fixator was subsequently installed over this region. This device is a scaled down version of the tibial transverse transport external fixator, TTTF, and consists of a traction component and a fixation component, which enable controlled movement of the bone fragment through rotation of an internal threaded mechanism. The traction component of the external fixator was mounted along the two Kirschner wires, followed by attachment of the fixation component.

After fixation, the miniature lateral transfer frame was fixed to the tibia of the rat using a one millimeter diameter Kirschner wire. The Kirschner wire need to pass completely through the tibia in order to firmly and stably attach the mini lateral transfer device to the tibia, then use the Allen key to tighten the external screws. The other side has been fixed in the same way, and finally the excessively long Kirschner wire has been cut off.

Finally, the medial edge of the bone fragment was cut to completely free it. Rotation of the threaded mechanism allows upward or downward movement of the fragment. Finally, use 10%iodophor to completely sterilize the surgical area.

It then used absorbable sutures to close the muscle and skin layer by layer to ensure no continued bleeding, and then the finished suture site is sterilized again. To prevent postoperative pain, buprenorphine was administered subcutaneously. During the first three postoperative days, buprenorphine was given every 12 hours for continued analgesia thereafter as needed.

Subsequently, the rats were placed in a clean cage, and provided with an appropriate environment and temperature, then closely monitored for recovery. The therapeutic effect of this procedure is achieved through controlled movement of the bone fragment. We designed a specific transport protocol for fragment mobilization.

After a one day latency period, upward transport of the bone fragment was initiated on day one. This phase lasted for five days during which the fragment was advanced 0.5 millimeters every 12 hours. Upon completion of the upward transport, the fragment was kept stationary for one day, followed by the initiation of downward transport on day seven.

The downward phase similarly lasted five days, with a fragment moved 0.5 millimeters every 12 hours. After a total of 12 days of transport, the bone fragment returned to its original position. Finally, a two-day consolidation phase was applied to further stabilize the fragment.

To evaluate the therapeutic effect of the TTT procedure in the animal models, X-ray images and vascular perfusion images was performed to assess the positions of the bone fragment and microvascular perfusions in the lower limb. X-ray examination showed that the bone fragment was at its lowest position on day one and day 12, and reached its highest point on day seven. No fractures were observed around the corticotomy site throughout the transport process.

Additionally, vascular profusion imaging on day 19 demonstrated that although ligation of the superficial femoral artery reduced blood profusion in the lower limb, animals that underwent the TTT procedure exhibited markedly enhanced microvascular profusion. These findings indicate that TTT exerts a positive effect on improving blood circulation in ischemic limbs. Our study establishes a rat model for TTT-based treatments of lower limb ischemia.

TTT was shown to promote microcirculatory reconstructions and effectively eradicate ischemic conditions in the affecting limb. This model provides a valuable platforms for exploring the underlying mechanisms by which TTT is exerts therapeutic effect in ischemic disease.