This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Congressionally Directed Medical Research Program under Award No. W81XWH-17-1-0680. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

All animals received humane care in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC-protocol 2017N000184) and Animal Care and Use Review Office (ACURO) approved all animal protocols. Inbred male Lewis rats (250-400 g) were used for all experiments.
1. Surgery
<ol>
	<li>Anesthetize the Lewis rats using isoflurane inhalation. Induce anesthesia with 5% isoflurane in the induction chamber, and maintain anesthesia with 1.5-3% isoflurane inhalation through a breathing cone.</li>
	<li>Apply eye lubricant before surgery in survival procedures. Shave the surgical site, treat with depilatory cream, scrub, and drape with sterile drapes.</li>
	<li>Confirm total anesthesia with a toe pinch test before incision and regularly during the procedure. Monitor heart and respiratory rates throughout the entire procedure. For all surgeries, maintain sterile conditions by using sterile instruments, supplies, drapes, and gloves. See the Table of Materials for the list of instruments used for the procedures.</li>
</ol>
2. Donor right partial hindlimb procurement
<ol>
	<li>Make a circumferential incision of the skin above the ankle at the distal third of the leg.</li>
	<li>Skeletonize and cauterize the saphenous artery and the terminal branch of the popliteal artery using bipolar forceps. Cauterize and cut off the gastrocnemius, soleus, tibialis anterior, and biceps femoris muscles until the tibial bone is exposed.</li>
	<li>Make a 2.5 cm incision at the right inguinal crease. Dissect out the inguinal fat pad and retract it distally to expose the femoral vessels. Use a fishhook retractor to grasp the inguinal ligament and clamping forceps to hold the inguinal fat pad distally. 
	NOTE: The inguinal fat pad is included in the harvest of the partial limb.</li>
	<li>Dissect the femoral vessels, individualize Murphy branches (deep muscular collateral branches usually located halfway between the inguinal ligament and the epigastric branch), and ligate with 8-0 nylon ties.</li>
	<li>Heparinize the donor rat with 100 IU/kg heparin, injected in the penile dorsal vein using a 27.5 G needle.</li>
	<li>Complete the skin incision around the hip.</li>
	<li>Cauterize the biceps femoris and gluteus superficialis muscles using bipolar forceps. Cauterize and cut the sciatic nerve at mid femur length. Expose the femur proximally at the level of the posterior femoral crest. 
	NOTE: Adductor and quadriceps muscles are left out of the procurement. The innominate pedicle is preserved.</li>
	<li>Ligate femoral vessels with 8/0 nylon ties at the level of the inguinal ligament. Perform an arteriotomy on the femoral artery just below the ligature and dilate to allow for the insertion of a 24 G angio-catheter.</li>
	<li>Cauterize and cut remaining muscle underneath the pedicle, exposing the anterior side of the femur.</li>
	<li>Cut the tibia and femur using a bone cutter as proximally and distally as possible, respectively (mid-length).</li>
	<li>Flush the partial hindlimb with 2 mL of heparin saline (100 IU/mL) to obtain a clear venous outflow. Store on ice in a sterile gauze until microvascular transfer (Figure 1).</li>
	<li>While the animal is under general anesthesia, perform euthanasia by exsanguination.&#160;Confirm death by absence of respiratory movement and heartbeat.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62586/62586fig01.jpg" /> 
Figure 1: Rat partial hindlimb harvested. A 24 G angiocath is inserted in the femoral artery, ready for heterotopic microvascular transfer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Recipient surgery
<ol>
	<li>Before the incision, shave the back of the neck, and administer buprenorphine 0.01-0.05 mg/kg subcutaneously. Place the rat in a supine position on a heating pad.</li>
	<li>Make a 2.5 cm incision in the right inguinal crease. Dissect the inguinal fat pad and recline it distally to expose the femoral vessels. Use a hook to retract the inguinal ligament and clamping forceps to hold the inguinal fat pad distally.</li>
	<li>Dissect the femoral vessels, individualize the Murphy branches, and ligate with 8/0 nylon ties.</li>
	<li>Ligate both vessels above the epigastric vessels using 8/0 nylon ties. Place approximator clamps proximally and dilate vessel ends; rinse with heparin saline.</li>
	<li>Make an incision on the left flank above the hip, and create a subcutaneous pocket with a subcutaneous tunnel to the inguinal crease. 
	NOTE: The inset incision is made above the range of motion of the hip to ensure that the animal maintains a normal hindlimb motion. Additionally, keeping a cutaneous bridge between the graft inset and the microvascular transfer site allows for better fixation of the graft (Figure 2).</li>
	<li>Place the proximal part of the partial limb and the inguinal fat pad through the subcutaneous tunnel for microvascular transfer. Perform venous and arterial anastomoses using 10/0 nylon sutures. Remove both approximator clamps, and observe revascularization of the limb. Perform a &#34;milking test&#34; on both vessels to assess the patency of each anastomosis. 
	NOTE: Eight to nine sutures are usually necessary for venous anastomosis, 6 sutures on average for arterial anastomosis.</li>
	<li>Make a longitudinal skin incision on the medial side of the transplanted limb, and insert the graft. Remove excess skin of the graft, and close the wound with separate sutures and a running suture using absorbable 4/0 sutures.</li>
	<li>Suture together the inguinal fat pads of the transplanted limb and the recipient using two separate absorbable sutures, and close the inguinal crease at the very end after a last checkup of the microvascular anastomoses. 
	&#8203;NOTE: Inguinal fat pads are sutured tightly to add a protective layer of fat above the anastomoses and ensure a secured position of the graft and its pedicle. A meticulous closure is better for wound healing; it also prevents residual bleeding from the wound and decreases the risk of self-mutilation.</li>
	<li>Compensate fluid loss subcutaneously with 1-3 mL of saline according to the amount of perioperative bleeding.</li>
	<li>Place an Elizabethan collar around the neck of the animal, and apply 2 loose sutures to the skin to maintain it in the correct position.</li>
	<li>Stop isoflurane inhalation, and monitor the animal continuously on a warming pad until fully conscious and ambulatory.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62586/62586fig02.jpg" /> 
Figure 2:&#160;Perioperative image before inset of the osteomyocutaneous limb. A cutaneous bridge of approximately 1 cm is preserved between the inguinal crease incision and the inset of the graft above the hip. The graft is placed under the bridge, maintaining it steady for microvascular transfer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Postoperative care
<ol>
	<li>Monitor the animal twice daily for 72 hours, then once daily until postoperative day (POD) 7, and then twice per week. 
	NOTE: Monitoring must be adjusted to the animal and graft condition (pale eyes might require supplementary fluids, porphyrin staining as an indicator of animal pain, abnormal graft color/temperature), and further care should be discussed with the veterinarian. Single housing is required for the recipient rats during the entire study period to avoid any damage to the graft.</li>
	<li>Perform analgesia with subcutaneous injection of buprenorphine and/or non-steroid anti-inflammatory drug according to IACUC guidelines.</li>
	<li>Evaluate the graft, and perform physical examination daily with pictures using the same device. 
	NOTE: Using hair removal cream on the graft&#39;s skin is helpful to better assess the skin color of the transplant.</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penis+transplantation:+first+US+experience.">Penis transplantation: first US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Grajek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+complex+allotransplantation+of+neck+organs:+larynx,+trachea,+pharynx,+esophagus,+thyroid,+parathyroid+glands,+and+anterior+cervical+wall:+a+case+report.">First complex allotransplantation of neck organs: larynx, trachea, pharynx, esophagus, thyroid, parathyroid glands, and anterior cervical wall: a case report.</a> Annals of Surgery. 266 (2), 19-24 (2017).</li><li>Pribaz, J. J., Caterson, E. 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The authors have no disclosures.

Orthotopic limb transplantation models in rodents have been described in the literature16,17,18; however, they require a nerve repair, muscle reattachment, and a perfect osteosynthesis of the femur, which can be a very difficult step. These models are also associated with a higher morbidity and mortality rate in rodents14, especially in the short-term follow-up as the recovery of a normal function of a transplanted hindlimb can take several months19. However, they allow for a longer-term assessment of graft function in case of success. The main limitation in heterotopic limb transplantation is the muscle atrophy induced by the lack of reinnervation of the graft. As previously published in the literature, muscle fiber injuries can occur as soon as five days post-denervation20. This causes significant muscle atrophy and fatty degeneration after one month, preventing the use of this model for in vivo studies lasting more than three weeks. From this standpoint, partial heterotopic osteomyocutaneous flap models are best for short-term studies on tissue preservation10, tissue bioengineering, and immunosuppression strategies21.
In the proposed model, both surgical and postoperative complications are limited and can be easily and rapidly addressed by a microsurgical researcher. We estimate that success can be achieved in this model after 3-6 transplantations for a young surgeon, given that they have received basic microsurgery courses. It is also a procedure that can be performed by a single operator in less than 3 hours with an ischemia time below 2 hours. This model can also be performed by two operators, shortening the ischemia time to the time of microvascular anastomoses only. The critical step in this model is the graft inset and ensuring good placement of the pedicle to avoid any kinking or tension that would cause microvascular thrombosis. For this reason, dissection of the femoral vessels should proceed as proximally and distally as possible in the donor and the recipient, respectively. Cutaneous vascularization should be thoroughly preserved by keeping the skin wrapped around the limb until graft revascularization to avoid any shearing effect on the arterial skin perforators, which can cause low skin perfusion and subsequent skin necrosis.
In the literature, complications in rodent VCA models are not well-reported. The knowledge of every possible outcome is essential to be able to prevent and anticipate complications. This study stresses the need for good communication with the staff veterinarian. Acquiring skills in rat handling and physical examination is crucial for the well-being of the animal and the proper conduct of the study. Once this technique is mastered, this model can be used for VCA research with a success rate close to 100%. Although non-functional models are not suited for long-term evaluations, they are of great interest for early graft assessment in immunological research on full-mismatch VCA transplantation or evaluation of ischemia-reperfusion injuries. The described model offers a large skin component and muscle volume that can be repeatedly sampled using punch biopsies, generating several histological and immunological assays as well as imaging evaluation techniques13. This precise protocol offers a reproducible and reliable VCA model in rats with reduced morbidity once the possible complications are foreseen and actively prevented.

Over the past two decades, VCA has evolved as a revolutionary treatment for patients who suffer severe disfigurement including face1, upper limb amputations2, penile3, and other complex tissue defects4,5. However, the consequences of life-long immunosuppression still hinder a broader application of these complex reconstructive surgeries. Basic research is crucial to improve anti-rejection strategies. Increasing VCA preservation time is also essential to improve transplantation logistics and increase the donor pool (as VCA donors must fulfill more criteria than solid organ donors, including skin tone, anatomic size, gender). In this context, rat limb transplantations are widely used in studies on the immune rejection of allografts6,7, novel tolerance induction protocols8, and preservation studies9,10,11. Hence, these VCA models are a key element to master for VCA translational research.
Osteomyocutaneous flaps have been described in the literature as reliable models to study VCA in rats8,12,13,14. Although orthotopic whole-limb transplantations allow for long-term evaluation of graft function, it is a time-consuming procedure associated with higher postoperative morbidity and mortality rates14. In contrast, heterotopic limb transplantation models are non-functional, but enable reproducible studies on VCA. Postoperative outcomes can be reliably anticipated before the start of a rat VCA transplantation study. This study reports a partial heterotopic osteomyocutaneous flap transplantation model in the rat that includes frequent possible outcomes and complications that can arise intra-operatively and postoperatively during a follow-up period of three weeks.

<table><tbody><tr><td>24 GA angiocatheter</td><td>BD Insyte Autoguard</td><td>381412</td><td></td></tr><tr><td>4-0 suture Black monofilament non absorbable suture</td><td>Ethicon</td><td>1667</td><td>Used to suture the E-collar to the back of the neck</td></tr><tr><td>4-0 suture Coated Vicryl Plus Antibacterial</td><td>Ethicon</td><td>VCP496</td><td></td></tr><tr><td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td><td>ASSI</td><td>ASSI.ATK26426</td><td></td></tr><tr><td>Bipolar cords</td><td>ASSI</td><td>228000C</td><td></td></tr><tr><td>Black Polyamide Monofilament USP 10-0, 4 mm 3/8c</td><td>AROSurgical</td><td>T04A10N07-13</td><td>Used to perform the microvascular anastomoses</td></tr><tr><td>Buprenorphine HCl</td><td>Pharmaceutical, Inc</td><td>42023-179-01</td><td></td></tr><tr><td>Dilating Forceps</td><td>Fine science tools (FST)</td><td>18131-12</td><td></td></tr><tr><td>Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern</td><td>ASSI</td><td>ASSI.SAS15RVL</td><td></td></tr><tr><td>Double Micro Clamps 5.5 x 1.5 mm</td><td>Fine science tools (FST)</td><td>18040-22</td><td></td></tr><tr><td>Elizabethan collar</td><td>Braintree Scientific</td><td>EC-R1</td><td></td></tr><tr><td>Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2)</td><td>ASSI</td><td>ASSI.JFL31</td><td></td></tr><tr><td>Halsey Micro Needle Holder</td><td>Fine science tools (FST)</td><td>12500-12</td><td></td></tr><tr><td>Heparin Lock Flush Solution, USP, 100 units/mL</td><td>BD PosiFlush</td><td>306424</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>14043-704-06</td><td></td></tr><tr><td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td><td>ASSI</td><td>ASSI.BPNS11223</td><td></td></tr><tr><td>Lone Star Elastic Stays</td><td>CooperSurgical</td><td>3314-8G</td><td>Used to retract the inguinal ligament for femoral vessels dissection</td></tr><tr><td>Lone Star Self-Retaining Retractors</td><td>CooperSurgical</td><td>3301G</td><td></td></tr><tr><td>Micro-Mosquito Hemostats</td><td>Fine science tools (FST)</td><td>13010-12</td><td>Used to retract the inguinal fat pad distally</td></tr><tr><td>Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock</td><td>ASSI</td><td>ASSI.B1582</td><td></td></tr><tr><td>Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c</td><td>Ethilon</td><td>2808G</td><td>Used to ligate collateral branches on the femoral vessels</td></tr><tr><td>Offset Bone Nippers</td><td>Fine science tools (FST)</td><td>16101-10</td><td></td></tr><tr><td>S&amp;amp;T Vascular Clamps 5.5 x 1.5 mm</td><td>Fine science tools (FST)</td><td>00398-02</td><td></td></tr><tr><td>Walton scissors</td><td>Fine science tools (FST)</td><td>14077-09</td><td></td></tr></tbody></table>

partial heterotopic hindlimb transplantation model in rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

In this single-operator study, 30 syngeneic heterotopic partial limb transplants were performed. Success was defined at postoperative day 21 as the absence of VCA failure or complications requiring euthanasia. The normal evolution of the graft is represented in Figure 3. The mean duration for partial limb procurement and graft inset in the recipient were 35 and 105 min, respectively; the mean ischemia time was 105 min. During follow-up, two types of complications occurred (Table 1)-early or late. Some required euthanasia, others were salvaged, all were discussed with staff veterinarians (Table 2). This study reports the authors&#39; experience and advice for beginners in rodent microsurgery (Supplementary Table 1).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62586/62586fig03.jpg" /> 
Figure 3:&#160;Normal evolution of the heterotopic hindlimb model until the end of the study. Hair regrowth is observed during the first postoperative week; cutaneous retraction appears after 2 weeks. Abbreviation: POD = postoperative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Management of perioperative complications 
Arterial or venous thrombosis is the most common perioperative complication. The most important tip to overcome this and turn the surgery into a success is to make an early diagnosis, i.e., continuous monitoring of the flap color/bleeding and recurrent patency test are fundamental before closing the anastomoses surgical site. The prevention of this complication should be in the surgeon&#39;s mind as soon as an incision is made. The donor should be heparinized with IV injection of heparin 100 IU/kg 5 min prior to graft arterial ischemia. Once harvested, the graft should be flushed with heparin saline (100 IU/mL) until venous outflow is clear. In this study, perioperative arterial thrombosis happened in 10% of cases.
Bleeding is a less frequent occurrence and is easily mitigated with a cautious cauterization of the graft muscles and a thorough dissection of the recipient site. The primary cause of bleeding is the anastomotic leak in the artery. This should resolve by itself within 3 min, or if not, re-clamping and revision of the leaking anastomosis are necessary. Anesthesia-related complications, which occurred in 6.7% of cases, are more frequent with untrained microsurgeons. Using isoflurane is a reliable way to anesthetize rats, and the length of anesthesia can be adjusted in real time. The surgeon must be trained to use the machine correctly, follow the guidelines for induction and maintenance regimen, and closely monitor the heart and respiratory rates throughout the operation. Depending on the species, age, or weight of the animal, the amount of isoflurane needed may vary. Unpredictable animal loss is a rare event and is usually not explained by an anesthetic or surgical mistakes.
Early complications (&#60;POD7) 
VCA failure can occur during the first postoperative week due to microvascular thrombosis (venous thrombosis more frequent than arterial). Perioperative pedicle inset at the groin level is a very important step. Moving the rat&#39;s hindlimb to mimic the movement&#39;s effect on the pedicle is crucial; the pedicle should never be too loose nor too tight. Intensive monitoring is a fundamental requirement, as necropsy must be performed as soon as a VCA failure diagnosis is made. During necropsy, an analysis of the position of the pedicle (kinking or tension) and quality of the thrombosed anastomosis (back wall suture, intraluminal flap) provides much information on what can be improved during the next procedure and thus, should be performed by the operating surgeon. Venous thrombosis was a cause of early euthanasia in 20% of cases, all of which occurred before POD5 (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62586/62586fig04.jpg" /> 
Figure 4: Postoperative venous thrombosis. The skin appears blue and becomes darker each day. Abbreviation: POD = postoperative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Self-mutilation (or autophagia) is a serious concern in non-sensate grafts; it often occurs between POD2 and POD7. If limited to less than a third of the graft surface and concerning only skin, surgical debridement and suture using non-absorbable sutures can be discussed with the staff veterinarian (Figure 5A-C). In such case, non-absorbable sutures should be removed under sedation after 10-14 days. Prevention relies on the use of an E-collar stitched to the neck15 until POD7 and the cleaning of any blood or crust on the animal&#39;s surgical wounds. Repeated autophagia or deep mutilation requires euthanasia (Figure 5D).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62586/62586fig05.jpg" /> 
Figure 5: Postoperative self-mutilation of the non-sensate graft. (A-C) The limited surface of auto-mutilation at (A) POD2. (B) Surgically debrided and re-sutured; (C) aspect at POD21. (D) Severe autophagia of multiple layers of the graft leading to euthanasia of the animal. Abbreviation: POD = postoperative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Late complications (&#62;POD7) 
Less frequent and less lethal, these complications demand veterinarian consultation to provide adequate treatment. First, bone exposure can be observed in this model, usually after initial healing on the third week post-operation. Prevention is based on careful bone-cutting (use of bone cutter creating smooth edges); covering the bone edges with surrounding muscles while accounting for later muscle atrophy is helpful. If detected early and the animal is in good condition, surgical revision can be discussed with the veterinarian. Second, dermal cysts can occur around the surgical site after two weeks (Figure 6). They usually do not interfere with the rat&#39;s or the graft&#39;s condition, but can fistulize to the skin and get infected. Washing the surgical site of the graft inset to avoid any residual hair in the wound prevents the creation of the cyst. The indication for surgical drainage can be evaluated with the veterinarian.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62586/62586fig06.jpg" /> 
Figure 6: Dermal cysts. Dermal cysts appearing(A) after POD14, (B) sometimes with a cutaneous necrotic center prior to the fistula. Abbreviation: POD = postoperative day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/62586fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Complication</td>
			<td>Solution</td>
			<td>Prevention</td>
		</tr>
		<tr>
			<td rowspan="3">Peri-operative</td>
			<td>Microvascular thrombosis</td>
			<td>Re-do the anastomosis rapidly after rinsing the vessel ends with heparin saline and attesting good flow.</td>
			<td>Heparinize the donor, flush the flap, master the anastomosis technique, and use adequate instruments and suture.</td>
		</tr>
		<tr>
			<td>Bleeding</td>
			<td>Cauterize if from muscles, reclamp if from the anastomosis and not stopping spontaneously within 3 min.</td>
			<td>Thorough cauterization during graft harvest</td>
		</tr>
		<tr>
			<td>Anesthesia-related death</td>
			<td>Discuss the encountered problem with vet staff.</td>
			<td>Training in use of anesthesia machine and perioperative rat monitoring</td>
		</tr>
		<tr>
			<td rowspan="2">Early (&#60;POD7)</td>
			<td>Microvascular thrombosis</td>
			<td>Early euthanasia and necropsy to reveal the cause of thrombosis</td>
			<td>Depends on the cause</td>
		</tr>
		<tr>
			<td>Auto-mutilation</td>
			<td>Discuss surgical repair with the vet if first event and superficial damage to the graft.</td>
			<td>E-collar from POD-2 to POD7</td>
		</tr>
		<tr>
			<td rowspan="2">Late (&#62;POD7)</td>
			<td>Bone exposure</td>
			<td>Discuss surgical revision with the vet.</td>
			<td>Cut the bone with bone cutter, make sure the edge is smooth, recover with surrounding muscles, account for later muscle atrophy (cut short).</td>
		</tr>
		<tr>
			<td>Dermal cyst</td>
			<td>Discuss surgical drainage and antibiotic treatment with the vet</td>
			<td>Wash the inset site with water; avoid leaving any hair in surgical site.</td>
		</tr>
	</tbody>
</table>
Table 1: Potential outcomes. Prevention and solutions.
End of study 
The end of the study in this model was set at POD21; animals were euthanized using CO2 asphyxiation or by exsanguination. As a non-functional graft, muscle atrophy and fatty degeneration were observed as consequences of the lack of reinnervation. Biopsies of the skin and muscle are saved for histological analysis. The inguinal crease is reopened for the evaluation of the vascular pedicle (placement, patency) if euthanasia is performed before the end of the study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Five reasons for consulting the staff veterinarian</td>
		</tr>
		<tr>
			<td>1) Before the start of the study to inform her/him of the nature of your study, planned analgesia, and follow-up strategies and expected outcomes</td>
		</tr>
		<tr>
			<td>2) Special diet or supplementary nutrients that can help improve the animal condition during the study</td>
		</tr>
		<tr>
			<td>3) Unexpected anesthesia-related death</td>
		</tr>
		<tr>
			<td>4) Unexplained worsening of the animal condition</td>
		</tr>
		<tr>
			<td>5) Surgical complication to assess salvaging possibilities or indications for euthanasia</td>
		</tr>
	</tbody>
</table>
Table 2: When to call the vet. Communication with the staff is essential for the conduct of an in vivo study.
Supplementary Table 1: &#34;Dos &#38; Don&#39;ts.&#34; Advice for young microsurgical trainees. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62586/Supplementary Table. Dos &amp; Dont.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Partial Heterotopic Hindlimb Transplantation Model in Rats

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

partial-heterotopic-hindlimb-transplantation-model-in-rats

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.7K Views.  Massachusetts General Hospital, Harvard Medical School. We presenteren een betrouwbaar en reproduceerbaar chirurgisch model van heterotope achterpoottransplantatie bij ratten. Deze techniek is ideaal voor kortlopende studies naar conservering of immuunafstoting bij VCA. De stapsgewijze beschrijving stelt cursisten in staat om de operatie snel onder de knie te krijgen.Maak om te beginnen een omtrekkende incisie op de huid boven de enkel bij het distale derde deel van het been. Skeletoniseer en cauteriseer de sapheneuze slagader en de termina...

Video: Partial Heterotopic Hindlimb Transplantation Model 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 ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Medicine

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

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

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...

Partial Heterotopic Hindlimb Transplantation Model in Rats

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