Name: Orthotopic Hind-Limb Transplantation in Rats (Video) | JoVE Journal
Uploaded: 2010-07-12T17:40:00.000+00:00
Duration: 14 min 3 s
Description: Learn orthotopic hind-limb transplantation in rats using the cuff technique for revascularization. This method enables researchers to study ischemia, reperfusion injury, and immunological events in composite tissue allotransplantation.

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5 minutes

Donor Operation and Hind-Limb Harvest
<ol>
<li>	For the operative procedure a sterile set of microsurgical instruments is utilized. The surgeon should wear proper attire including sterile gloves, which have to be changed prior to the start of the surgical procedure.</li>
<li>	The rat is anesthetized with an intraperitoneal injection of pentobarbital (50mg/kg IP) and placed supine on a heated operation field, which facilitates a stable animal body temperature throughout the entire surgical procedure. </li>
<li>	Prior to skin incision the filed of operation is sanitized three times each with both, a disinfectant and alcohol. To prevent the animal&#8217;s corneas from drying out, eyes are lubricated with an ophthalmic ointment (Vidisic) prior to surgery. </li>
<li>	A skin incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels are dissected free with bipolar cautery and blunt dissection; deep branches are ligated with 8-0 silk. </li>
<li>	300&mu;l (50 IU) of heparin are injected via the penile vein for anti-coagulation.</li>
<li>	Femoral vessels are ligated at the level of the inguinal ligament and cut distal to the ligature. </li>
<li>	The hind limb is flushed with ice-cold (4&deg;C) HTK solution (5ml) via the femoral artery over 5 minutes. </li>
<li>	All muscle groups are sharply divided with scissors and osteotomy is performed with a rotating electrical saw at the level of the distal third of the femur.</li>
<li>	The harvested limb is then submerged in 20mL of ice-cold HTK solution for preservation and cold storage until transplantation.</li>
</ol>
Recipient Operation and Transplantation
<ol>
<li>	The rat is anesthetized with an intraperitoneal injection of pentobarbital and placed supine on the operation field. </li>
<li>	A skin incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels are dissected free with bipolar cautery and blunt dissection.</li>
<li>	Femoral vessels are ligated as distal as possible, but proximal to the epigastric vessel takeoff. Vascular clamps are placed at the level of the inguinal ligament on artery and vein.</li>
<li>	A polyethylene cuff is pulled over the artery (inside cuff diameter: 0.912 mm, cuff-wall thickness: 0.010 mm, cuff length: 2.0 mm) and vein (inside cuff diameter: 1.151 mm; cuff-wall thickness: 0.010 mm, cuff length: 2.0 mm). The edge of the vessel lumen is then pulled with a vessel dilator and the femoral artery and vein are everted over the whole body of the cuff (approximately 2mm) and fixed with a circumferential 8-0 silk ligature.</li>
<li>	To make room for the donor limb the recipient limb is removed with sharp dissection of muscle groups and osteotomy is performed at the mid-femur. </li>
<li>	The donor limb is removed from the HTK solution and transplantation begins with osteosynthesis of the femur using an 18-gauge needle (length: 1.5-2.0 cm) as an intramedullary rod.</li>
<li>	Ventral muscle groups are closely approximated with 4-0 Vicryl or Prolene interrupted sutures.</li>
<li>	Vascular anastomoses are performed by pulling the donor vein and artery over the recipient&#8217;s vein and cuff complex. A circumferential 8-0 silk ligature is again placed to secure the anastomosis. </li>
<li>	The venous clamp is released followed by the arterial clamp ,and reperfusion is visualized by return of colour to the distal limb and foot, as well as pulsatile flow through the artery.</li>
<li>	The remaining dorsal muscle groups are re-approximated with 4-0 Vicryl or Prolene interrupted sutures.</li>
<li>	The skin is closed with 4-0 vicryl or prolene interrupted sutures.</li>
<li>	The recipient rat is placed on a heating pad and monitored closely until recovery.</li>
<li>	Recipient animals receive prophylactic antibiotics (Cefazolin, 20 mg/day, IM) and Buprenex (0.1 mg/kg, SC) for three posttransplant days. </li>
</ol>

<ol><li>Shapiro, R. I., Cerra, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+model+for+reimplantation+and+transplantation+of+a+complex+organ%3A+the+rat+hind+limb%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">A model for reimplantation and transplantation of a complex organ: the rat hind limb.</a> J Surg Res. 24 (6), 501-506 (1978).</li>
<li>Petruzzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+international+registry+on+hand+and+composite+tissue+transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">The international registry on hand and composite tissue transplantation.</a> Transplantation. 86 (4), 487-492 (2008).</li>
<li>Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+forearm+transplantation%3A+outcome+at+3+years%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">First forearm transplantation: outcome at 3 years.</a> Am J Transplant. 7 (7), 1753-1762 (2007).</li>
</ol>

No conflicts of interest declared.

Decades of experimental research have preceded and enabled successful human hand transplantation in the recent past. In 1978, Shapiro et al. developed an orthotopic hind limb transplant model in rats, well suited to study various pathological, physiological, and immunological aspects as well as functional outcome of composite tissue grafts. We have modified the most challenging part of the procedure, namely the vascular anastomosis, by introducing a non-suture cuff technique. When compared to the conventional suture tec...

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orthotopic hind-limb transplantation in rats

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

I am Robert Suha from the Daniels Roski Research Laboratory of the Department of Visceral Transplant and Thoracic Surgery from Innsbrook Medical University. Today we are going to show you the procedure of he limb transplantation in the red, introducing a cuff technique for revascularization. This he limb transplantation surgery is performed by our surgical residents, including myself.We use this animal model to study ischemia and reperfusion associated injuries in a composite tissue allograft. Furthermore, we use this procedure to study immunological events of composite tissue allograft rejection, as well as strategies to prevent rejection from occurring. These strategies include the minimization or even avoidance of maintenance immunosuppression through the use of targeted immunomodulatory regimens.The red hind limb transplantation procedure begins with the harvest of the donor composite tissue graft in which the right hind limb is removed from the donor rat and stored on ice. Next, the right hind limp is removed from the recipient rat to make room for the donor limp. The donor limp is then removed from ice and transplanted to the recipient rat.In this model, vascular anastomosis are performed using a cuff technique. For our experiments, male Louis Reds weighing 250 grams were used. Anesthesia was induced using 50 milligrams per kilogram intraperitoneal injection of pentobarbital.The anesthetized red is shaved and sanitized over the groin and limp for the procedure. A complete set of microsurgical instruments is required including straight microsurgical, scissors, vessel dilators, and two curved forceps. Furthermore, the most important instruments are the microsurgical vessel clamps shown here.To test depth of anesthesia, the toe pinch reflex is utilized. The rat is placed on a sterile platform and secured with all four Limbs in full extension prior to incision. The limp is whipped free of Any axis here to prevent contamination.An incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels appear immediately under the skin and they're dissected free with bipolar tery and blunt dissection. The epigastric vessels are then dissected free and cauterized.A Single loop as holding suture is placed around the vein and artery to allow traction without directly handling the vessels with instruments after separation. The deep branches of the artery and vein are ligated with eight oh silk suture proximally and cauterized distally Before harvest of the donor limp. 50 units of heparin are administered via the penal Vein.After one minute, Attention is returned to the femoral vessels. The femoral artery is ligated as approximately as possible using eight o silk, and the same procedure is repeated for the Fal. Both vessels are then cut distally from the ligature and the hind limb is flashed through the artery with heparinized ice cold HDK solution over five Minutes.Next, all muscle groups are carefully divided with scissors. The osteotomy is performed using a rotating electrical saw at the level of the distal third of the femur. The harvested limp is then submerged in 20 milliliters of ice cold HDK solution for preservation and cold storage until transplantation.The recipient operation begins similarly to the donor operation with isolation and dissection of the femoral vessels. Following careful dissection, the vessels are ligated using eight o silk as distal as possible, but proximal to the epigastric vessels Takeoff. We then proceed with the cuff mounting of the recipient vein by pulling the polyline cuff over the vessel.A single vein Clamp is applied proximal to the cuff over the vein and cuff handle to ensure complete hemostasis. The ligature at the distal end of the vein is cut and the vein is then averted Over the cuff. An eight O Ligature is tied circumferentially around the vessel and the cuff securing the recipient vein in Place.The exact same procedure is performed for the artery with the smaller diameter cuff. The recipient's limp Is dissected similarly to the donor limp with sharp dissection of muscle groups and osteotomy performed in the mid femur. We are now ready to Transplant the hind limp.The donor limp is removed from the HDK solution. Transplantation begins with osteo synesis of the femur with an 18 gauge needle as an intramedullary rod. The tip is cut off and the remaining needle is cut to one and a half to two centimeters.Ventral Muscle groups are closely approximated with four oh Vicryl or proline interrupted sutures to stabilize the limb and prepare for vascular Anastomosis. For Cuff R anastomosis, the donor vein is dilated, pulled over the recipient's vein and cuff and secured with another circumferential eight o ligature. The same procedure is performed for the donor artery.Finally, Once both vessels are estamos, the venous clamp is released, followed by the arterial Clamp. Reperfusion Is immediate and easily visualized by return of color of the distal limb and foot, as well as pulsitile flow through the artery. The remaining dorsal muscle groups are re approximated.Skin is closed with four oh Vicryl or proline interrupted sutures, and transplantation Is complete Postoperatively. The red is warmed on a heating pad and analgesics and antibiotics are administered. The red is monitored closely for 48 to 72 hours for signs of distress or surgical complications.The cuff technique of re anastomosis has been widely performed in different surgical research areas over the last decades from marine kidney transplant models to heterotopic heart transplantation models. It allows for studying various aspects of his chemo, reperfusion associated injuries, as well as immunological phenomena. Following our transplantation.Here we show the first application of this unique cuff technique to he limb composite tissue transplantation. In a small animal model, the cuff technique significantly reduces operative time, microsurgical expertise required by the operator and intraoperative blood loss when compared to conventional suture techniques for anastomosis.

orthotopic-hind-limb-transplantation-in-rats

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Learn orthotopic hind-limb transplantation in rats using the cuff technique for revascularization. This method enables researchers to study ischemia, reperfusion injury, and immunological events in composite tissue allotransplantation.

Orthotopic Hind-Limb Transplantation in Rats

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