This work was funded by the Frankel Foundation and the Northwestern Memorial Hospital McCormick Grant (Operation RESTORE). Research reported in this publication was supported by the National Institute of General Medicial Sciences of the National Institutes of Health under Award&#160;Number T32GM008152. This work was supported by the Northwestern University Microsurgery Core and Behavioral Phenotyping Core.

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 under protocol IS00001663.
NOTE: Two strains of rats were used, Lewis rats and August Copenhagen x Irish (ACI) rats. Animals were divided into three treatment groups: allotransplant without immune suppression (ACI to Lewis), allotransplant with conventional immune suppression (ACI to Lewis), and isotransplant (Lewis to Lewis or ACI to ACI). Lewis is an inbred strain, while ACI rats represent an out-bred wild-type, therefore this combination was chosen to model the worse-case rejection response. Conventional immunosuppression was administered subcutaneously either as rapamycin 1 mg/kg from post-operative day (POD) minus 1 to POD 28 or as FK506 3 mg/kg from POD 0 to POD 14, and then once weekly thereafter. Both male and female rats were eligible recipients from 8 to 16 weeks old, weighing between 250 and 400 grams at the time of surgery.
1. Donor right hind limb harvest
<ol>
	<li>Induce general anesthesia with 5% isoflurane in pure oxygen through a vaporizer with an appropriate scavenging system.</li>
	<li>Confirm adequate depth of anesthesia with toe pinch, and then use hair clippers to trim the fur off of the right hind limb and right groin surgical site</li>
	<li>Down-titrate the isoflurane through a rodent nose cone to 2-2.5%.</li>
	<li>Position the rat supine with spread limbs taped out to the sides on an operating board with a heating pad underneath. Disinfect the hairless skin with 70% rubbing alcohol and protect the surgical field with sterile gauze.</li>
	<li>Using an appropriate microsurgical microscope, microsurgical instruments, and with easy access to bipolar and monopolar electrocautery, begin the dissection.</li>
	<li>Use scissors to make a circumferential skin/subcutaneous tissue incision around the right hindlimb. Start in the inguinal crease medially at roughly the same level as the inguinal ligament and extend dorsal-laterally to complete the circumferential incision.</li>
	<li>Having exposed the muscular layer directly beneath the incision, dissect and cauterize the superficial epigastric vessels that lead from the muscular layer to the proximal skin/subcutaneous flap just created.</li>
	<li>Reflect the proximal flap superomedially to the inguinal ligament and the distal skin/subcutaneous flap inferolaterally to the knee.</li>
	<li>Use a wire retractor or rolled gauze to help expose the field.</li>
	<li>Observe that the inguinal anatomy of the rat is similar to humans; from lateral to medial lie the nerve, artery, and vein.</li>
	<li>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.</li>
	<li>Turning attention to the femoral artery and vein, use 4 cm 7-0 silk ties to atraumatically retract the vessels instead of handling them directly.</li>
	<li>Ligate all branches of the femoral vessels as they arise with 7-0 silk ties; divide the branches between the ties. For very small branches, bipolar cautery may be used instead of ties. 
	NOTE: 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 rat15. More distal branches of the femoral vessels such as the highest genicular and the saphenous branch do not usually require division.</li>
	<li>Systemically inject 500 international units of heparin through the penile vein in a male rat donor. Use the superficial epigastric vein if the donor rat is female.</li>
	<li>Allow the heparin to circulate systemically for 2 min before proceeding with the next steps.</li>
	<li>Ligate the femoral artery with 7-0 silk ties as proximal to the inguinal ligament as possible and divide between the ties.</li>
	<li>Similar to the artery, ligate and divide the femoral vein.</li>
	<li>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 here will minimize recipient blood loss after reperfusion.</li>
	<li>Deep to the ventral muscle groups, identify and sharply divide the sciatic nerve proximal to its branches. Three sciatic branches are usually visible: tibial, peroneal and sural. All three should all be preserved in the donor limb. A fourth cutaneous branch is not typically seen in this dissection15,16.</li>
	<li>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.</li>
	<li>Transect the femur bone at midshaft using a hand-held cordless rotary saw.</li>
	<li>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.</li>
	<li>Insert a 24-gauge angiocatheter into the graft artery stump and flush the graft with 250 international units of heparin diluted in 5 mL of ice-cold normal saline, watching it flow out clear through the opened vein.</li>
	<li>Slowly, gently flush the graft for around 3 min. Excess forceful flushing may damage the endothelium.</li>
	<li>Place the graft in a chilled saline dish nested in an ice bucket until transplantation.</li>
	<li>Euthanize the donor rat with bilateral thoracotomy.</li>
	<li>Clean all surgical instruments appropriately.</li>
</ol>
2. Recipient native right hind limb amputation
<ol>
	<li>Induce anesthesia with isoflurane at 5%, confirm depth, trim the fur, position the animal, and disinfect the skin with alcohol as described for the donor rat.</li>
	<li>Down-titrate isoflurane to 2-2.5% and inject subcutaneous preoperative analgesia with buprenorphine 1.2 mg/kg, and preoperative prophylaxis with enrofloxacin 7.5 mg/kg.</li>
	<li>Same as for the donor, make a circumferential incision in the inguinal crease, reflect skin flaps assuring hemostasis, and dissect out the femoral nerve, artery and vein, ligating the same branch vessels as above.</li>
	<li>Divide the femoral nerve more distally than for the donor, but proximally to the bifurcation if possible.</li>
	<li>Dissect out the femoral artery and vein with enough space to clamp each separately at the level of the inguinal ligament. Clamp the vein and artery with microsurgical bulldog clamps. Once clamped, divide each vessel sharply with scissors.</li>
	<li>Divide the ventral and dorsal muscles of the thigh at mid-thigh level with meticulous hemostasis, retracting the limb medially as necessary.</li>
	<li>Identify and divide the sciatic nerves proximal to their branch points as above.</li>
	<li>Transect the femur at midshaft using the saw.</li>
	<li>Remove the recipient native right hind limb and dispose appropriately.</li>
	<li>Down-titrate the isoflurane to 1-1.5% through the nose cone.</li>
</ol>
3. Donor to recipient limb implantation
<ol>
	<li>Using the hand-held power saw, shave off any irregularities from both donor and recipient femur cut ends.</li>
	<li>Using the saw, cut off the hub end of an 18-gauge needle, which will become the femur intramedullary rod.</li>
	<li>Before manipulating the bone, apply a small amount of bone wax to the recipient cut end of femur bone to reduce marrow bleeding during the reaming process.</li>
	<li>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.</li>
	<li>As needed, remove the needle and trim it to an appropriate length so that both bones fit smoothly over the needle with no needle showing in between the bone.</li>
	<li>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.</li>
	<li>Reapproximate the ventral muscle groups with eight to ten 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.</li>
	<li>Periodically irrigate the graft and surgical field with ice-cold saline for better visualization and to reduce warm ischemic reperfusion injury.</li>
	<li>Align the donor and recipient femoral arteries and anastomose them 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.</li>
	<li>Similar to the artery, anastomose the donor and recipient femoral veins in end to end fashion. The vein requires six to eight sutures. 
	NOTE: 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.</li>
	<li>Place a small amount of hemostatic cellulose powder around both anastomoses, and then remove the proximal microsurgical bulldog clamps on the vein and the artery.</li>
	<li>Inspect both anastomoses for good patency and flow. Use cotton swab sticks to gently prod the vein and assure good hemostasis of both anastomoses. Hold pressure over bleeding sites and place more hemostatic cellulose powder if needed. Another suture may be placed through a bleeding hole at the risk of &#8220;back-walling&#34; the needle only as a last resort.</li>
	<li>When both anastomoses are confirmed satisfactory, trim any remaining long stay suture tails short to match the others.</li>
	<li>Reposition the rat to the left lateral decubitus position, use liberal electrocautery to attain meticulous hemostasis of any reperfusion muscle bleeding.</li>
	<li>Turn attention to the nerve anastomoses once muscle hemostasis is assured. Trim back any nerve cut ends that appear ragged.</li>
	<li>Reapproximate the dorsal muscle groups under sciatic nerve with simple interrupted 5-0 polyglactin sutures.</li>
	<li>Reapproximate the sciatic nerve. Eight to ten 10-0 nylon neural simple interrupted sutures will usually suffice.</li>
	<li>Reapproximate the reminding dorsal muscle groups and then close the dorsal skin with 4-0 polyglactin continuous suture.</li>
	<li>Reposition the rat back to supine position and reapproximate the femoral nerve. Two to three 10-0 nylon neural simple interrupted sutures will usually suffice.</li>
	<li>Close the ventral skin with 4-0 polyglactin continuous suture. Avoid excess suture tail, which can be irritating to the rat once awake.</li>
</ol>
4. Post-operative care
<ol>
	<li>Recover animals in their cages with a heating pad under the cage and ready access to food and water, monitoring for early complications daily for the first week.</li>
	<li>Provide post-operative analgesia with subcutaneous meloxicam 1 mg/kg daily injection through POD 2. Provide post-operative antibiotic prophylaxis dilute enrofloxacin spray. Provide disincentive for autotomy (self-mutilation) with Bitter Safe Mist sprayed twice daily to the graft through POD 7.</li>
	<li>Maintain transplanted rats in cages with other rats, to stimulate return to daily activities and rehabilitate the transplanted limb.</li>
</ol>
5. Post-operative sensation testing
<ol>
	<li>Apply the Hargreaves testing of thermal sensation protocol, also described elsewhere17,18.</li>
	<li>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 confirmed to be working with the investigator&#8217;s finger.</li>
	<li>Before testing, confirm that the rat is awake and the tested paw is positioned over the infrared motion detector.</li>
	<li>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.</li>
	<li>Obtain five trials per tested limb, excluding the highest and lowest value before calculating the mean withdrawal latency time for each animal.</li>
</ol>
6. Post-operative motor testing
<ol>
	<li>Using a gait analysis treadmill and integrated software analysis platform, select candidates for treadmill testing at four to six weeks post-surgery.</li>
	<li>Trim all rat toenails one or two days before testing.</li>
	<li>Acclimatize animals to the testing room for one hour before testing, and allow for one minute of pre-test petting to calm anxiety.</li>
	<li>Placing the rat inside the treadmill, run the treadmill at trials of increasing speed, from 10 cm/s, to 14 cm/s, to the goal 18 cm/s. If the rat is reticent and cannot be coaxed to walk, abort the testing that day to avoid negative conditioning. Allow high performers to walk up to 24 cm/s.</li>
	<li>Rinse the treadmill apparatus with 70% ethanol in between tested animals.</li>
	<li>Gait parameters are output from the analysis platform&#8217;s proprietary software.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+quantitative+gait+analysis+during+overground+locomotion+in+the+rat:+its+application+to+spinal+cord+contusion+and+transection+injuries.">Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries.</a> Journal of Neurotrauma. 18 (2), 187-201 (2001).</li><li>Deumens, R., Marinangeli, C., Bozkurt, A., Brook, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+motor+outcome+and+functional+recovery+following+nerve+injury.">Assessing motor outcome and functional recovery following nerve injury.</a> Methods in Molecular Biology. 11622, 179-188 (2014).</li><li>Bozkurt, A., 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=Aspects+of+static+and+dynamic+motor+function+in+peripheral+nerve+regeneration:+SSI+and+CatWalk+gait+analysis.">Aspects of static and dynamic motor function in peripheral nerve regeneration: SSI and CatWalk gait analysis.</a> Behavioural Brain Research. 219 (1), 55-62 (2011).</li><li>Neckel, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+quantify+the+velocity+dependence+of+common+gait+measurements+from+automated+rodent+gait+analysis+devices.">Methods to quantify the velocity dependence of common gait measurements from automated rodent gait analysis devices.</a> Journal of Neuroscience Methods. 253, 244-253 (2015).</li><li>Yeh, L. S., Gregory, C. R., Theriault, B. R., Hou, S. M., Lecouter, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+model+for+whole+limb+transplantation+in+the+rat.">A functional model for whole limb transplantation in the rat.</a> Plastic and Reconstructive Surgery. 105 (5), 1704-1711 (2000).</li></ol>

The authors have nothing to disclose.

Limb transplant, under the broader category of vascularized component allotransplantation (VCA), has widely applicable therapeutic promise as yet unfulfilled. The main roadblocks lie in unsolved immunological issues unique to VCA and neuromotor recovery techniques used currently. Development of new techniques will depend on animal modeling that is flexible, robust, and reproducible.
Many animal models have been established in VCA, each with specific advantages4. Non-hum...

Limb transplant under the broader category of vascularized-composite allotransplant (VCA) or composite tissue allotransplant (CTA) has yet to fulfill its therapeutic promise. Since the first successful human hand transplants in Lyon, France and Louisville, Kentucky in 1998 and 1999, over 100 upper extremity transplants have been performed worldwide in carefully selected patients1. Wider applicability has been stymied by substantial immunosuppression and limited functional neuromotor recovery. Current immunosuppression strategies result in 85% incidence of acute rejection in the face of 77% incidence of opportunistic infection2. On the other hand, functional recovery after hand transplant occurs; mean Disability of Arm Shoulder and Hand (DASH) scores improve from 71 to 43, but that level of function may still qualify as a disability2. Given the nonlife saving nature of limb transplant, current techniques must be refined in animal models to take the next step in VCA.
Since the first rat model of limb transplant in 19783, many innovative animal models have been developed to advance the field of VCA4, incorporating vascular cuffed anastomoses to minimize operative time5,6, heterotopic osteomyocutaneous transplants to minimize physiologic insult to the recipient animal7,8,9,10,11, and novel immunologic approaches7,12,13,14. The rat model of orthotopic right hind limb mid-thigh transplant presented here emphasizes meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and neural coaptation as an upfront investment in a robust, reproducible model platform to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery.

<table>
	<tbody>
		<tr>
			<td>Anesthesia machine</td>
			<td>Vet Equip</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5cc syringe</td>
			<td>Exel</td>
			<td>26018</td>
			<td></td>
		</tr>
		<tr>
			<td>18-gauge needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>22-gauge needle</td>
			<td>BD</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>24-gauge angiocatheter</td>
			<td>Sur-Vet</td>
			<td>SROX2419V</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>Exel</td>
			<td>26403</td>
			<td></td>
		</tr>
		<tr>
			<td>3 cc syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>5cc syringe</td>
			<td>Exel</td>
			<td>26230</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Fisher Scientific</td>
			<td>HC-600-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction chamber</td>
			<td>Vet Equip</td>
			<td>941443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas scavenger system</td>
			<td>Vet Equip</td>
			<td>931401</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar electrocautery</td>
			<td>Aura</td>
			<td>26-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bitter Spray Mist</td>
			<td>Henry Schein</td>
			<td>5553</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>CP Medical</td>
			<td>CPB31A</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathing circuit</td>
			<td>Vet Equip</td>
			<td>921413</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenophine</td>
			<td>Reckitt Benckiser</td>
			<td>12496075705</td>
			<td></td>
		</tr>
		<tr>
			<td>Castro-Viejos needle drivers</td>
			<td>Roboz</td>
			<td>RS-6416</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless rotary saw</td>
			<td>Dremel</td>
			<td>8050-N/18</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab stick</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
			<td>For hemostasis</td>
		</tr>
		<tr>
			<td>DigiGait Appparatus and Software</td>
			<td>Mouse Specifics</td>
			<td>MSI-DIG, DIG-SOFT</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps (#4)</td>
			<td>Roboz</td>
			<td>RS-4972</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps (#5)</td>
			<td>Roboz</td>
			<td>RS-5035</td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Norbrook</td>
			<td>ANADA 200-495</td>
			<td></td>
		</tr>
		<tr>
			<td>FK-506</td>
			<td>Astellas</td>
			<td>301601</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Kendall</td>
			<td>1903</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Microflex</td>
			<td>DGP-350-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>Oster</td>
			<td>078005-010-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld monopolar electrocautery</td>
			<td>Bovie</td>
			<td>AA00</td>
			<td></td>
		</tr>
		<tr>
			<td>Hargreaves Apparatus</td>
			<td>Ugo Basile S.R.L. Gemonio, Italy</td>
			<td>37370</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Walgreens</td>
			<td>126987</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresenius Kabi</td>
			<td>42592K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Corning</td>
			<td>PC-351</td>
			<td>For warming resusscitation fluid</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated ringers</td>
			<td>Baxter</td>
			<td>2B2074</td>
			<td></td>
		</tr>
		<tr>
			<td>Large petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>For donor graft while in chilled saline</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Henry Schein</td>
			<td>49755</td>
			<td></td>
		</tr>
		<tr>
			<td>micro Collin Hartmann retractor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5841</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfibrillar collagen powder</td>
			<td>BD</td>
			<td>1010590</td>
			<td>For hemostasis</td>
		</tr>
		<tr>
			<td>Microvascular clips</td>
			<td>Roboz</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Baxter</td>
			<td>2F7124</td>
			<td></td>
		</tr>
		<tr>
			<td>Opthalmic lube</td>
			<td>Dechra</td>
			<td>IS4398</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapmycin</td>
			<td>MedChem Express</td>
			<td>HY-10219</td>
			<td></td>
		</tr>
		<tr>
			<td>Small petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td>For warmed resusscitation fluid</td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td>ProAdvantage</td>
			<td>N207100</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gown</td>
			<td>Cardinal Health</td>
			<td>9511</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical mask</td>
			<td>3M</td>
			<td>1805</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope, optic model OPMIMD</td>
			<td>Zeiss</td>
			<td>169756</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope, Universal S3</td>
			<td>Zeiss</td>
			<td>243188</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 10-0 nylon</td>
			<td>Covidien</td>
			<td>N2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 5-0 vicryl</td>
			<td>Ethicon</td>
			<td>J213H</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture 7-0 silk tie</td>
			<td>Teleflex</td>
			<td>103-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>3M</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic instrument cleaner</td>
			<td>Roboz</td>
			<td>RS-9911</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel dilation forceps</td>
			<td>Roboz</td>
			<td>RS-5047</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Survival and recovery depend on meticulous surgical technique. Attention to the vascular anastomoses and the neural anastomoses, as well as the bone coaptation as described above is crucial maximizing the success of this model. Operative design and representative anastomotic results are shown in Figure 1.
Overall mortality was dependent on immunosuppression strategy, with the majority of isotransplanted animals attaining the study endpoint of 100-200 post-operativ...

Watch this Scientific Journal Video about Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat at JoVE.com

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

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 ...

taking-next-step-neural-coaptation-orthotopic-hind-limb-transplant

Research

JoVE Journal

Medicine

7.3K Views.  Northwestern University. 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.

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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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 ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

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 ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...