Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

This video will provide detailed description, how to perform an orthotopic right hind limb, mid-thigh transplanted rat. The donor rat right hind limb is amputated at mid-thigh level, then stored on ice. The recipient rat right hind limb is also amputated at mid-thigh level.

The donor rat limb is then attached to the recipient rat. We will describe how to reattach the bone, artery, vein, nerve, muscle, and skin systems. Limb transplant, more specifically, vascularized composite allotransplant, VCA, has wide therapeutic promise.

But it's currently limited by techniques, immunosuppression, and poor functional neuromotor recovery. Our model emphasizes meticulous time-tested microsurgical techniques, such as hand-sewn vascular anastomosis and neural coaptation. We've performed over 200 of these surgeries in over a year.

Although there's an initial learning curve to the beginning microsurgeon, by using this video to learn our method and their committing to dedicated practice, any microsurgeon will be able to master this model. All experiments were conducted in accordance with the guide for the care and use of laboratory animals of the National Institutes of Health, NIH, and were approved by the Northwestern University Animal Care and Use Committee. The specific procedures were performed on a protocol IS 00001663.

Select a rat aged eight to 16 weeks old weighing between 250 and 400 grams at the time of surgery. Induce general anesthesia with 5%isoflurane through a vaporizer before trimming the rat's hind limb fur. Using a nose cone to maintain anesthesia between two to 2.5%position the rat on the operating board with a heater underneath.

Disinfect the hairless skin with 70%rubbing alcohol and protect the surgical field with sterile gauze. Using an appropriate microsurgical microscope and the microsurgical instruments shown here, along with easy-to-access bipolar and monopolar electrocautery, you will be ready to begin the dissection. Use a scissors to make a circumferential and skin incision around the right hind limb.

Start in the inguinal crease immediately at roughly the same level as the inguinal ligament and extend dorsal laterally to complete the circumferential incision. And then expose the muscular layer directly beneath the incision, dissect and cauterized the superficial epigastric vessels, which lead from the muscular layer to the proximal skin flap just created. Reflect the proximal flap superomedially to the inguinal ligament and the distal skin flap inferolaterally to the knee.

Use a wire retractor or rolled gauze to help expose the field. Observed that the inguinal anatomy of the rat is similar to humans;from lateral to medial lie the nerve, artery, and vein. Dissect out the femoral nerve, divide it sharply at the inguinal ligament proximal to the bifurcation, if possible.

Retract the divided nerve inferiorly, keeping it safely out of the way, covered beneath moist gauze. Turning attention to the femoral artery and vein use four centimeter long 7-0 silk ties to atraumatically retract the vessels instead of handling them directly. Paying close attention now will improve your final anastomotic result.

Ligate all branches of the femoral vessels with 7-0 silk ties. Divide the branches between the ties. Arterial and venous branches which require division include the superficial circumflex iliac and the muscular vessels.

The superficial circumflex iliac is usually largest and appears to dive deep as would the profunda femoral in humans, but the profunda is absent in the rat. More distal branches of the femoral vessels, such as highest genicular and the saphenous branch do not usually require division. Now ligate the femoral artery with 7-0 silk ties as proximal to the inguinal ligament as possible and divide between the ties.

Similar to the artery, ligate and divide the femoral vein. Reflect both artery and vein inferiorly, safely out of the way, covered beneath moist gauze together with the femoral nerve covered previously. Dissect the ventral muscle groups, taking care to cauterize any visible vessel that arises.

Attention to hemostasis now will minimize recipient blood loss during reprofusion later. Deep to the ventral muscle groups, identify and sharply divide the sciatic nerve proximal to its branches. All three sciatic branches are usually visible, tibial, peroneal and sural.

All three should be preserved in the donor limb. A fourth cutaneous branch is not typically seen in this dissection. Finish dividing the remaining ventral and dorsal muscle groups at mid-thigh level with meticulous hemostasis.

It may be necessary to retract the limb medially to complete dividing the muscles. Transect the femur bone at mid-shaft using a handheld cordless rotary saw. The limb is now free from the donor.

Having removed the limb graft from the donor, cut the silk tied ends from the graft side femoral artery and vein stumps, thereby re-opening the vessels. Insert a 24-gauge angiocatheter into the graft artery stump and flush the graft with 250 international units of heparin diluted in 5 milliliters of ice-cold normal saline, being careful to watch it flow out of the open vein. Slowly, gently flush the graft for around three minutes.

Excess forceful flushing may damage the endothelium. Place the graft in a chilled saline dish nested in an ice bucket until transplantation. Euthanize the donor rat with bilateral thoracotomy.

The recipient surgery begins similar to the donor surgery, anesthetizing the rat, trimming the fur, making a circumferential incision in the inguinal crease, reflecting the skin flaps, dissecting out the femoral nerve, artery, and vein. And ligating the same branch vessels as described previously. However, during this operation, divide the femoral nerve more distally than for the donor, but proximally to the bifurcation if possible.

Divide and free the femoral artery and vein and proximately clamp them respectively at the level of the inguinal ligament with microsurgical bulldog clamps. Continue the surgery as described previously, dividing the ventral endorsing muscles of the thigh at mid-thigh level with meticulous hemostasis, while retracting the limb medially as necessary. Identify and divide the sciatic nerve proximal to the branch point and transect the femur at mid-shaft using the rotary saw.

Remove the recipient native right hind limb and dispose appropriately in accordance with local Animal Care and Use Committee guidelines. It is very important at this stage to turn down the isoflurane to one to 1.5%through the nose cone. Too much anesthesia during this less painful part of the procedure will lead to the rat's death.

It is time to coapt the bones together. Using the handheld rotary saw, shave off any irregularities from both the donor and recipient femur cut ends. Cut off the hub end of an 18-gauge needle, which will become the femur intramedullary rod.

Before manipulating the bone, apply a small amount of a bone wax to the recipient cut end of the femur bone to reduce marrow bleeding during the reaming process. Coapt the donor and recipient femoral bones using the 18-gauge needle as an intramedullary rod. Some force is necessary, but do not ream either bone so far as to fracture the cortex.

As needed, remove the needle and trim it to inappropriate length so that both bones fit smoothly over the needle with no needle showing in between the bone. Place a small support such as a pad of gauze or a small rock or modelling clay underneath the donor limb to keep it off tension. Reapproximate the ventral muscle groups with eight to 10 simple interrupted 5-0 polyglactin sutures so that the graft does not rotate around the femur needle.

This gives the limb stability for the anastomoses. Now begin the anastomoses. Periodically irrigate the graft and surgical field with ice-cold saline for better visualization and to reduce ischemic reperfusion injury.

Align the donor and recipient femoral arteries and anastomose in end-to-end fashion using simple interrupted 10-0 nylon suture, avoiding both tension and looping. The artery requires an average of six sutures. Similar to the artery, anastomose the donor and recipient femoral veins in end-to-end fashion.

The vein requires six to eight sutures. Generous cold saline irrigation, atraumatic vessel handling technique, and leaving long tails to serve as stay sutures for vessel retraction are important tools for effective microsurgical anastomoses. Place a moderate amount of hemostatic cellulose powder on both anastomoses, and subsequently remove the proximal microsurgical bulldog clamps on the vein and the artery.

Inspect both anastomoses for good patency and flow. Use cotton swab sticks to gently prod the vein and inspect for good hemostasis on both anastomoses. When both anastomoses are confirmed satisfactory, trim any remaining long stay suture tails to match the others.

Repositioning the rat to the left lateral decubitus position, use liberal electrocautery to attain meticulous hemostasis of any reperfusion muscle bleeding. Attention is turned to the nerve anastomoses once muscle hemostasis is assured. Reapproximate the dorsal muscle groups which lie directly under the sciatic nerve with simple interrupted 5-0 polyglactin sutures.

Trim back any nerve cut ends that appear ragged. Reapproximate the sciatic nerve with 10-0 nylon sutures striving for epineural bites. Eight to 10 simple interrupted sutures will usually suffice.

Reapproximate the remaining dorsal muscle groups with 5-0 polyglactin sutures. Then close the dorsal skin with 4-0 polyglactin continuous running suture. Repositioning the rat to supine position, reapproximate the femoral nerve with 10-0 nylon suture, again, striving for epineural bites.

Two to three simple interrupted sutures will usually suffice. Close the remaining ventral skin in the same fashion as the dorsal skin. Avoid excess suture tail, which can be irritating to the rat once awake.

Once the animals are out of the immediate postoperative window, they can be tested for neuromotor recovery. The Hargreaves test is used to test thermal sensation. Place the rat in the testing container and allowed it to acclimatize for 20 minutes.

The apparatus glass is confirmed clean, and the heat source is confirmed to be working with the investigator's finger. Before testing, confirm the rat is awake and the tested paw is positioned over the infrared motion detector. Transmit thermal energy at intensity level 90.

Time delay in the animal moving its paw away from the heat source is recorded. If no movement occurs within 20 seconds, the test is aborted to prevent injury. Obtain five trials per tested limb, excluding the highest and lowest value before calculating the mean withdrawal latency time for each animal.

Here's depicted operative design in cartoon format. The right hind limb of both donor and recipient rats are amputated at mid-thigh level as sciatic cross-section. Femoral artery, vein, and nerve, as well as the sciatic nerve and femur bone must then be reconnected in the recipient surgery.

The model can be used to show differential survival effects of immune suppression and transplant strategy. With this technique, long-term survival is achievable. The model can be used to investigate sensory nerve recovery, which may be evident by four weeks postoperatively.

Motor function can also be interrogated using a gait analysis platform. Here, we have presented a rat model platform with favorable survival characteristics, which we use to probe sensory and motor recovery as seen in the proceeding figures. Limb transplant under the broader category of VCA, vascularized component allotransplantation has widely applicable therapeutic promise is yet unfulfilled.

The main roadblocks line current immunosuppression and neuromotor recovery techniques. Development of new techniques will depend on flexible, robust, and reproducible animal modeling. Using this platform, it is our hope that investigators will be able to advance multiple facets of limb transplant.

Thank you for watching and good luck in your next steps.