A total of 56 animals were studied in six groups, as shown in Table 40.1. The “graft” term was assigned to those non-vascularized composite tissue transplants, while the “flap” term was used when vascular repairs were performed. In group I (anatomic study group, n = 8), Lewis rats were subject to harvesting technique trialing and the mystacial vascular network was drawn. In group II (flap viability group, n = 8), the mystacial pad elevated and reset in Lewis rats to check flap survival based on the facial vessels. In group III (isograft group, n = 8), viability without vascular pedicles was explored in Lewis to Lewis transplantation. In this group, a mystacial defect was created in the recipient, where the mystacial pad was set as a composite graft. In group IV (isoflap group, n = 8), vascularized mystacial pad flaps were transplanted from Lewis to Lewis rats and nerves were repaired, to serve as controls of the alloflaps. In group V (allograft group, n = 8), graft viability without vascular repairs was explored in Lewis-Brown-Norway to Lewis transplantation. Group VI was the study group, in which transplantations from Lewis-Brown-Norway to Lewis were done. Group VIa (non-neurotized alloflap transplant group, n = 8) represented the vascularized mystacial pad flap transplantation in the absence of nerve repairs, while in group VIb (neurotized alloflap transplant group, n = 8), the mystacial pad flaps were transplanted and the BL, uMM and ZyO branches of the facial nerve, and the Io nerve were repaired.

The mystacial pad flap was elevated under microscope magnification (Leyca Microsystems, M300, magnification × 10 × 25 × 40). The skin was incised deep to the platysma in the anterior neck, and superficial to the facial muscles in the mystacial pad and frontal region. In the neck, the external jugular vein (EJV) and anterior facial vein were preserved, but the posterior facial vein was ligated. The submandibular gland was excised after ligation of the glandular branches of the facial artery and vein. The sternocleidomastoid muscle and facial vein were retracted to expose the CCA and its main branches, the external (ECA) and internal carotid arteries. The posterior belly of the digastricus muscle was excised, the omohyoid muscle was transected and the greater horn of the hyoid bone was excised. The hypoglossal nerve crossing anteriorly to the ECA needs to be transected. The facial artery is included in the flap as the main vessel to the mystacial pad. The internal carotid artery, superior thyroid artery, ascending pharyngeal artery, lingual artery, ascending palatine artery and the superficial temporal artery were ligated and transected. The facial artery and vein were undermined from the masseter muscle to the labial commissure, and protected creating a cuff of muscle to avoid skeletonization of the vessels. At this point uMM and BL branches were transected. The animal was placed laterally and the incision runs peri-oro-nasally. The posterior incision in the mystacial pad reached the frontal area to harvest the ZyO branch. From the frontal incision dissection was carried out downwards subperiostically and then just over the oral mucosa to include the facial vessels, reaching the insertion of the masseter muscle tendon. The fan IoN was transected proximally. The flap was elevated based on the anterior facial vein draining to EJV and on the facial artery off the ECA [35] (Fig. 40.2). The vascular pedicle was only transected at the end of the recipient preparation to minimize the time of warm ischemia.

The midline skin of the recipient’s neck was incised to approach the EJV and CCA. The sternocleidomastoid was freed from the sternoclavicular insertion and removed. The vagus and frenicus nerves were preserved. The incision ran to the labial commissure, where the facial artery was ligated, and then surrounded the mystacial area preserving the eyelids, nose and lips. The BL, uMM, ZyO branches were transected and the mystacial pad was removed leaving the oral mucosa intact. The IoN was transected as distal as possible for ease of repair. Then the mystacial pad flap was transferred to the recipient. End-to-end intimo-intimal EJV anastomosis was performed using 10-0 nylon sutures. Next, end-to-side CCA anastomosis was performed using 10-0 nylon. Then BL, uMM, and ZyO were sutured by epineural repair using 10-0, while the fan IoN was repaired with 8-0 (Figs. 40.3 and 40.4). Finally the skin was closed using 6-0 running absorbable suture. After the transplant the recipients received 2 mL of subcutaneous lactated Ringer’s solution to compensate for perioperative fluid loss.

Tapered subcutaneous tacrolimus (Astellas Pharma, Munich, Germany) immunosuppression monotherapy was initiated after surgery in groups V and VI. The tapering scheme consisted of 6 mg/kg/day during the first week, 4 mg/kg/day in the second week, and 2 mg/kg/day thereafter. The animals were weighted weekly for dosage preparation.

In group I, the vascular territory of the mystacial pad was drawn using radiographic studies by injecting a mixture of gelatin (market available) and barium (Barigraf, Juste, Madrid, Spain) into the common carotid artery (CCA) of two animals.

The general condition of the recipients in the other groups was observed daily for 8 weeks, and weight was measured every week. A clinical sign of graft rejection was defined as post-transplant hair loss, while hard touch was assumed to be a clinical sign of necrosis. Electrodiagnostic (EDX) evaluation was performed after 6 weeks under ketamine sedation (10 mg/kg) by a neurophysiologist unaware of the nerve repair procedures. Two percutaneous electrodes (one active, one reference) were placed at the retroauricular region to stimulate the facial nerve. A bipolar receptor electrode was placed percutaneously in the mystacial pad. The stimulation voltage was 2.8 mA, the electrical pulses were of 1 ms duration, and the frequency was 1 Hz. The presence, duration and amplitude of the conduction potentials were recorded if present in the conduction study. The presence of denervation activity or electrical silence during rest was noted. Motor unit potentials were also registered during whisking, and the records were classified into normal activity, moderate activity, or absence of activity (denervation activity) according to the neurophysiologist subjective criteria. After 8 weeks the animals were euthanized and biopsies of the mystacial region were taken. They were formalin-fixed and paraffin embedded; 5-mm thick sections were examined microscopically (Olympus Vanox AH-3, Japan) at ×300 and ×600 magnification after hematoxylin-eosin staining for the presence of the neurokeratin artifact. It traduces the presence of spirally rolled Schwann cells plasmatic membrane around the axons, providing the myelin to the nerve fibers. In the absence of axons, Schwann cells do not roll and the neurokeratin artifact is not seen. The presence of neurokeratin was noted for each sample as positive or negative by the first author, who was aware of the nerve repair procedure.

Quantitative variables are presented as mean ± standard deviation and qualitative variables as percentages. Pairwise comparisons in the duration and amplitude of conduction studies in groups IV and VIb based on the explored side were performed using the Wilcoxon test. Mann- Whitney U test was used for the comparison of these variables in the transplanted side between IV and VIb and also between VIa and VIb groups. The differences in resting and whisking EDX, sensitivity test, and histology in the transplanted side based on these groups were analyzed using the Fisher exact test. Statistical significance was taken if P ≤ 0.05. Statistical analysis was performed using the SPSS Statistical Package, version 15 for Windows (SPSS Inc, Chicago, IL).