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 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.
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.
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
2. Recipient native right hind limb amputation
3. Donor to recipient limb implantation
4. Post-operative care
5. Post-operative sensation testing
6. Post-operative motor testing
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-operative days as seen in Figure 2. Once out of the acute post-operative window, treated allotransplanted animals could experience survival up to 58 post-operative days. Isografted rats lived indefinitely over the course of study while allograft transplanted rats had variable mortalities from rapamycin and FK506. Out of the treatments FK506 promoted the longest viability (day 57), while rapamycin was second best (day 20) over the untreated control (day 10).
Sensory and motor recovery can be shown in Figure 3. Animals were shown to have recovered sensory nerve function of the transplanted paw using the Hargreaves apparatus by day 30. Animals displayed significant recovery by four weeks after surgery (Aii). Animals shown marked improvements in motor function of the transplanted limb using a gait analysis treadmill and integrated software analysis platform. Example gait parameter based on specific limbs are shown (Bii) and a Sciatic Function Index (SFI) are also presented (Biii).
Figure 1: Operative design is depicted in cartoon format. (A) The rat is shown with (B) right hind leg cross-section depicting (i) the femoral bundle (nerve, artery, and vein) (ii) the sciatic nerve, and (iii) the bone. (C) Representative micrographs from the operating microscope (donor left and recipient right) were taken of the (i) sciatic nerve anastomosis (ii) the femoral nerve artery, and vein anastomoses (shown from top to bottom), and (iii) the 18-gauge needle intramedullary rod-femur bone coaptation. Note the donor structures appear to the left in each photo. Also note the femur is shown before full coaptation when both bones are opposed and the needle is concealed within. Please click here to view a larger version of this figure.
Figure 2: Percent survival of animals as presented days post-surgery (POD). Groups shown include isograft, allografts with no treatment, rapamycin, and FK506 immunosuppressant drugs. Please click here to view a larger version of this figure.
Figure 3: Sensory nerve recovery is demonstrated in (A) Hargreaves testing transplanted animals each at six post-operative time points and in (B) still shot of treadmill testing using DigiGait. (i) Representative pictures are shown with (ii) respective paw data. Respective color-coded images of paws are also in 0.025 ms frames. Digigate models (iii) are also shown. Significance was determined using a one-way ANOVA with a Bonferroni's multiple comparison test and SEM, where n=7 and p< 0.05. These particular DigiGait data were taken from an isogeneic animal tested at post-operative day 28. Please click here to view a larger version of this figure.
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-human primate models offer attractive translatability to human patients, but have been hampered by cost concerns and toxic levels of immunosuppression required4. Canine models have been seen as advantageous for specific similarities of muscular structure as humans as well as a more experienced immune system19,20. Porcine models offer the benefits of a large animal model where the immune system is increasingly well studied21,22. Mouse model systems present the most advanced techniques to study immunology, but despite important advances in cuffed vascular microsurgical anastomosis23, mouse limb transplant remains technically challenging and has some limitations in functional recovery assessment5,24,25,26.
Rat models in VCA have been utilized since 19783, providing a mature platform to investigate both immunological and neuromotor hypotheses6,9,13,14,17,27,28,38. The model here combines the advantages of hindlimb orthotopic approach, suture anastomosis, nerve re-approximation, and potential for gait analysis. Hindlimb orthotopic as opposed to forelimb transplant is less of an encumbrance to the rat during the recovery process and allows for continued normal grooming and feeding behaviors post operatively. Suture anastomosis although painstaking may potentially offer less technical confounding for long term studies. Nerve re-approximation allows for future investigation17,18 and gait analysis. This protocol relies on meticulous, time-tested microsurgical techniques well-described elsewhere29, requiring constant attention to avoid the immediate pitfalls of anesthetic overdose, anastomotic failure, anastomotic thrombosis, and excessive surgical blood loss. Although multiple microsurgeons can improve the workflow, we have described a method by which a single operating microsurgeon can achieve sufficient experimental output.
Autotomy or self-mutilation has been a phenomenon noted in several microsurgical models, and it has been hypothesized to inversely correlate with nerve healing30,31. Autotomy was overall controlled in this model, possibly related meticulous neural anastomotic technique. Autotomy also decreased farther into the learning curve. Bitter Safe Mist was a valuable adjunct in controlling this phenomenon.
Gait analysis in rats has been studied for multiple models of injury32,33,34, most relevantly for sciatic nerve injury35,36. Rats even when not limb transplant recipients are known to be heterogenous subjects for gait analysis, and investigators still debate which analysis parameters describe recovery37. In this model we have described several methods to obtaining the best data from transplanted recipients who are willing and able to walk. Preselection of adequate walkers was not predictive of post-operative cooperation. Although animals are able to move about their housing as soon as several hours after surgery, they are not ready for treadmill ambulation until at least four to six weeks after surgery.
A protocol’s ability to measure nerve recovery in VCA is dependent on its strategy for rehabilitation. This protocol explicitly promotes transplant recipients interacting with other rats as inducement to function. This strategy is cognizant of the importance of modeling rehabilitation, yet is simple, economical, and is largely standard. Future strategies may include more active rehabilitation such as treadmill training.
The immunologic techniques applicable to this model are beyond the scope of this discussion, but in particular, comparing isotransplant versus allotransplanted animals provides a useful control to differentiate allograft immunologic phenomena and rejection from the ischemic reperfusion injury, inflammation, revascularization, and post-surgical infection processes inherent in the transplant surgery itself. Isotransplants provide a similar control for nerve function studies for the same reason.
Using this platform, investigators may be able to advance both VCA immunology and neuromotor recovery.
The authors have nothing to disclose.
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 Number T32GM008152. This work was supported by the Northwestern University Microsurgery Core and Behavioral Phenotyping Core.
Anesthesia machine | Vet Equip | 911103 | |
0.5cc syringe | Exel | 26018 | |
18-gauge needle | BD | 305196 | |
1cc syringe | BD | 309659 | |
22-gauge needle | BD | 305156 | |
24-gauge angiocatheter | Sur-Vet | SROX2419V | |
25-gauge needle | Exel | 26403 | |
3 cc syringe | BD | 309657 | |
5cc syringe | Exel | 26230 | |
Alcohol | Fisher Scientific | HC-600-1GAL | |
Anesthesia induction chamber | Vet Equip | 941443 | |
Anesthetic gas scavenger system | Vet Equip | 931401 | |
Bipolar electrocautery | Aura | 26-500 | |
Bitter Spray Mist | Henry Schein | 5553 | |
Bone wax | CP Medical | CPB31A | |
Breathing circuit | Vet Equip | 921413 | |
Buprenophine | Reckitt Benckiser | 12496075705 | |
Castro-Viejos needle drivers | Roboz | RS-6416 | |
Cordless rotary saw | Dremel | 8050-N/18 | |
Cotton swab stick | Fisher Scientific | 23-400-101 | For hemostasis |
DigiGait Appparatus and Software | Mouse Specifics | MSI-DIG, DIG-SOFT | |
Dumont forceps (#4) | Roboz | RS-4972 | |
Dumont forceps (#5) | Roboz | RS-5035 | |
Enrofloxacin | Norbrook | ANADA 200-495 | |
FK-506 | Astellas | 301601 | |
Gauze | Kendall | 1903 | |
Gauze | Covidien | 8044 | |
Gloves | Microflex | DGP-350-M | |
Hair clippers | Oster | 078005-010-003 | |
Handheld monopolar electrocautery | Bovie | AA00 | |
Hargreaves Apparatus | Ugo Basile S.R.L. Gemonio, Italy | 37370 | |
Heating pad | Walgreens | 126987 | |
Heparin | Fresenius Kabi | 42592K | |
Hot plate | Corning | PC-351 | For warming resusscitation fluid |
Isoflurane | Henry Schein | 29405 | |
Lactated ringers | Baxter | 2B2074 | |
Large petri dish | Fisher Scientific | FB0875713 | For donor graft while in chilled saline |
Meloxicam | Henry Schein | 49755 | |
micro Collin Hartmann retractor | |||
Micro dissecting scissors | Roboz | RS-5841 | |
Microfibrillar collagen powder | BD | 1010590 | For hemostasis |
Microvascular clips | Roboz | RS-5420 | |
Normal saline | Baxter | 2F7124 | |
Opthalmic lube | Dechra | IS4398 | |
Rapmycin | MedChem Express | HY-10219 | |
Small petri dish | Fisher Scientific | FB0875713A | For warmed resusscitation fluid |
Sterile drapes | ProAdvantage | N207100 | |
Surgical gown | Cardinal Health | 9511 | |
Surgical mask | 3M | 1805 | |
Surgical microscope, optic model OPMIMD | Zeiss | 169756 | |
Surgical microscope, Universal S3 | Zeiss | 243188 | |
Suture 10-0 nylon | Covidien | N2512 | |
Suture 5-0 vicryl | Ethicon | J213H | |
Suture 7-0 silk tie | Teleflex | 103-S | |
Tape | 3M | 1530-1 | |
Ultrasonic instrument cleaner | Roboz | RS-9911 | |
Vessel dilation forceps | Roboz | RS-5047 |