Summary

Partial Heterotopic Hindlimb Transplantation Model in Rats

Published: June 09, 2021
doi:

Summary

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

Abstract

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.

Introduction

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.

Protocol

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

  1. 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.
  2. Apply eye lubricant before surgery in survival procedures. Shave the surgical site, treat with depilatory cream, scrub, and drape with sterile drapes.
  3. 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.

2. Donor right partial hindlimb procurement

  1. Make a circumferential incision of the skin above the ankle at the distal third of the leg.
  2. 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.
  3. 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.
  4. 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.
  5. Heparinize the donor rat with 100 IU/kg heparin, injected in the penile dorsal vein using a 27.5 G needle.
  6. Complete the skin incision around the hip.
  7. 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.
  8. 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.
  9. Cauterize and cut remaining muscle underneath the pedicle, exposing the anterior side of the femur.
  10. Cut the tibia and femur using a bone cutter as proximally and distally as possible, respectively (mid-length).
  11. 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).
  12. While the animal is under general anesthesia, perform euthanasia by exsanguination. Confirm death by absence of respiratory movement and heartbeat.

Figure 1
Figure 1: Rat partial hindlimb harvested. A 24 G angiocath is inserted in the femoral artery, ready for heterotopic microvascular transfer. Please click here to view a larger version of this figure.

3. Recipient surgery

  1. 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.
  2. 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.
  3. Dissect the femoral vessels, individualize the Murphy branches, and ligate with 8/0 nylon ties.
  4. Ligate both vessels above the epigastric vessels using 8/0 nylon ties. Place approximator clamps proximally and dilate vessel ends; rinse with heparin saline.
  5. 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).
  6. 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 "milking test" 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.
  7. 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.
  8. 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.
    ​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.
  9. Compensate fluid loss subcutaneously with 1-3 mL of saline according to the amount of perioperative bleeding.
  10. 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.
  11. Stop isoflurane inhalation, and monitor the animal continuously on a warming pad until fully conscious and ambulatory.

Figure 2
Figure 2: 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. Please click here to view a larger version of this figure.

4. Postoperative care

  1. 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.
  2. Perform analgesia with subcutaneous injection of buprenorphine and/or non-steroid anti-inflammatory drug according to IACUC guidelines.
  3. Evaluate the graft, and perform physical examination daily with pictures using the same device.
    NOTE: Using hair removal cream on the graft's skin is helpful to better assess the skin color of the transplant.

Representative Results

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' experience and advice for beginners in rodent microsurgery (Supplementary Table 1).

Figure 3
Figure 3: 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. Please click here to view a larger version of this figure.

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'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 (<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's hindlimb to mimic the movement'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).

Figure 4
Figure 4: Postoperative venous thrombosis. The skin appears blue and becomes darker each day. Abbreviation: POD = postoperative day. Please click here to view a larger version of this figure.

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 5AC). 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's surgical wounds. Repeated autophagia or deep mutilation requires euthanasia (Figure 5D).

Figure 5
Figure 5: Postoperative self-mutilation of the non-sensate graft. (AC) 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. Please click here to view a larger version of this figure.

Late complications (>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's or the graft'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.

Figure 6
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. Please click here to view a larger version of this figure.

Complication Solution Prevention
Peri-operative Microvascular thrombosis Re-do the anastomosis rapidly after rinsing the vessel ends with heparin saline and attesting good flow. Heparinize the donor, flush the flap, master the anastomosis technique, and use adequate instruments and suture.
Bleeding Cauterize if from muscles, reclamp if from the anastomosis and not stopping spontaneously within 3 min. Thorough cauterization during graft harvest
Anesthesia-related death Discuss the encountered problem with vet staff. Training in use of anesthesia machine and perioperative rat monitoring
Early (<POD7) Microvascular thrombosis Early euthanasia and necropsy to reveal the cause of thrombosis Depends on the cause
Auto-mutilation Discuss surgical repair with the vet if first event and superficial damage to the graft. E-collar from POD-2 to POD7
Late (>POD7) Bone exposure Discuss surgical revision with the vet. Cut the bone with bone cutter, make sure the edge is smooth, recover with surrounding muscles, account for later muscle atrophy (cut short).
Dermal cyst Discuss surgical drainage and antibiotic treatment with the vet Wash the inset site with water; avoid leaving any hair in surgical site.

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.

Five reasons for consulting the staff veterinarian
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
2) Special diet or supplementary nutrients that can help improve the animal condition during the study
3) Unexpected anesthesia-related death
4) Unexplained worsening of the animal condition
5) Surgical complication to assess salvaging possibilities or indications for euthanasia

Table 2: When to call the vet. Communication with the staff is essential for the conduct of an in vivo study.

Supplementary Table 1: "Dos & Don'ts." Advice for young microsurgical trainees. Please click here to download this Table.

Discussion

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.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Riferimenti

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Citazione di questo articolo
Goutard, M., Randolph, M. A., Taveau, C. B., Lupon, E., Lantieri, L., Uygun, K., Cetrulo Jr., C. L., Lellouch, A. G. Partial Heterotopic Hindlimb Transplantation Model in Rats. J. Vis. Exp. (172), e62586, doi:10.3791/62586 (2021).

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