Original Article
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Vascularized versus Nonvascularized Island Median Nerve Grafts in the Facial Nerve Regeneration and Functional Recovery of Rats for Facial Nerve Reconstruction Study
1 Department of Plastic and Reconstructive Surgery, Tokyo Women’s
Medical University, School of Medicine, Tokyo, Japan 2 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan 3 Department of Oral and Maxillofacial Surgery, Global Center of Excellence (COE) Program, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan 4 Department of Physiology, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan 5 Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan
Address for correspondence Hajime Matsumine, MD, PhD, Department of Plastic and Reconstructive Surgery, Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan (e-mail: [email protected]).
J Reconstr Microsurg 2014;30:127–136.
Abstract
Keywords
► vascularized nerve transplantation ► facial nerve ► rat ► buccal branch ► median nerve
Histological and physiological basis of the therapeutic efficacy of the vascularized autologous nerve graft in facial nerve regeneration remains poorly understood because of no established rat model. The left median nerve and median artery/vein of Lewis rats were collectively ligated, and harvested as a vascularized island median nerve, which was transplanted to a 7-mm gap in the left buccal branch of facial nerve. Nerve regeneration was investigated. The numbers of myelinated fibers, axon diameter, and myelin thickness were significantly higher in the vascularized nerve graft group than in the nonvascularized nerve graft group. Compound muscle action potential measurement showed that the parameters of vascularized group were similar to those in the intact control group. A vascularized median nerve graft resulted in better facial nerve regeneration outcomes.
Nerve reconstruction with a nonvascularized autologous nerve graft has been traditionally performed in patients with a facial nerve deficit after trauma or tumor resection. However, a long and thick nerve graft may develop central necrosis after a few days of ischemia, resulting in an unsatisfactory outcome.1 A vascularized autologous nerve graft has been recently developed for compensating the shortcoming of nonvascularized grafts and providing a functional recovery to patients with a damaged nerve graft bed or longer nerve deficits that are unable to be treated adequately by conven-
tional nerve transplantation techniques.2,3 A vascularized nerve graft diminishes endoneurial scarring by maintaining the Schwann cell population and suppressing fibroblast infiltration, and provides an optimal nutritional environment that results in an increased rate of axonal regeneration.4 For example, Koshima et al have reported that facial nerve reconstruction using a vascularized auricular nerve graft in patients with a facial nerve defect gives good postoperative outcomes.3 Kashiwa et al have used a vascularized lateral femoral cutaneous nerve combined with a groin flap to
received July 2, 2013 accepted after revision August 13, 2013 published online October 25, 2013
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DOI http://dx.doi.org/ 10.1055/s-0033-1357500. ISSN 0743-684X.
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Hajime Matsumine, MD, PhD1,2 Ryo Sasaki, DDS, PhD2,3 Yuichi Takeuchi, PhD4 Mariko Miyata, MD, PhD4,5 Masayuki Yamato, PhD2 Teruo Okano, PhD2 Hiroyuki Sakurai, MD, PhD1
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
reconstruct a facial nerve defect and reported that satisfactory facial animation is obtained.5 However, the histological and physiological basis of the therapeutic efficacy of the vascularized autologous nerve graft in facial nerve regeneration remains poorly understood, because in part no established rat model with vascularized autologous nerve grafts for facial nerve regeneration is available. Although rat experimental models having sciatic and femoral nerve defects have been well used by many researchers for vascularized nerve studies,6–8 no facial nerve reconstruction model with a vascularized nerve is developed because of its complicated anatomy. This study established a rat model for facial nerve regeneration by transplanting a vascularized island median nerve, which had the brachial artery serving as a vascular pedicle, to a 7-mm deficit in the buccal branch of the facial nerve. The effectiveness of the facial nerve regeneration with a vascularized autologous nerve graft was investigated both histologically and physiologically.
aspect of the wrist (►Fig. 2B). After the skin incision was made, a subcutaneous tunnel was created to reach the preauricular incision to allow the facial nerve to be exposed. A cutaneous nerve and vein around the elbow were ligated from the medial to the short head of the biceps brachii muscle, and the median nerve and brachial artery/vein along the lower margin of the greater pectoral muscle were identified and exposed. An incision was then made on the antebrachial fascia from the elbow to wrist to expose the forearm flexor muscle group. After the palmaris longus tendon and flexor retinaculum were cut in the wrist, the median nerve and peripheral end of the median artery were exposed between the flexor carpi radialis and flexor digitorum superficialis in the forearm (►Fig. 2C). As the median artery divides into the radial and ulnar branches in the mid-forearm, the radial branch was ligated and cut, while the ulnar branch was preserved and used as a vascular pedicle. All branches of the median artery/vein distributed to the forearm muscle group were ligated and cut. The branches of the median nerve innervating the flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis muscle, and pronator teres muscle were also ligated. Another branch of median nerve located a few millimeters distal from the branch innervating the pronator teres muscle, equivalent to the interosseous nerve in humans, was also ligated. The superficial radial artery/vein, ulnar collateral artery/vein, and transverse cubital artery/vein arising from the brachial artery/vein were ligated around the elbow. Because the flexor carpi radialis muscle and pronator teres muscle were cut to expose the median nerve and median artery/vein coursing behind the muscles, they could be connected to the median nerve and brachial artery/vein that had been exposed proximal to the elbow. Then, the ulnar artery/vein coursing into the deep region was ligated. The superficial and deep pectoral muscles were cut up to the midclavicular line after the cephalic vein
Methods Surgical Procedure Lewis rats (8 weeks old, n ¼ 7) were anesthetized with 4% isoflurane using a nasal mask connected to a Univentor 400 Anesthesia Unit (Univentor, Zejtun, Malta) and placed in a right lateral decubitus position.9 The surgical procedure was performed as shown in ►Fig. 1. A preauricular incision with a marginal mandibular extension was made on the left side of the face, exposing the buccal and marginal mandibular branches, and parotid gland (►Fig. 2A). The rat was then repositioned into a supine position, and both forelimbs were stretched and fixed with tape. A slightly curved skin incision was made, starting from the midpoint of the left collar bone, passing over the left cubital fossa, and ending at the flexor
Fig. 1 Schematic illustration of the main branches of the brachial to median arteries and the brachial plexus to median nerve The present technique required the incisions of the greater pectoral muscle, pronator teres muscle, and flexor carpi radialis muscle for exposing the median nerve and brachial-median artery (the blue curved arrows indicate direction of movement). Areas of the median nerve and artery were used for a vascularized island nerve graft (blue ellipse). After being elevated, the vascularized median nerve was passed through the subclavian tunnel toward the head (green arrow in the upper left area of opened tissue). Journal of Reconstructive Microsurgery
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Fig. 2 Surgical procedure. (A) Left lateral facial view of a rat. A preauricular incision with a marginal mandibular extension was made on both sides of the face, exposing the buccal and marginal mandibular branches and parotid gland. (B) An incisional line on the left forelimb. A slightly curved line was drawn, starting from the midpoint of the left collar bone, passing over the left cubital fossa, and ending at the flexor aspect of the wrist. (C) Exposure of the median nerve and artery. After the palmaris longus tendon and flexor retinaculum were cut in the wrist, the median nerve (white arrow) and the peripheral end of the median artery (black arrow) were exposed between the flexor carpi radialis and flexor digitorum superficialis in the forearm. (D) Exposure of the brachial artery and brachial plexus. The flexor carpi radialis muscle and pronator teres muscles were cut to expose the median nerve and median artery/vein coursing behind the muscles, allowing them to be connected to the median nerve and brachial artery/vein that had been exposed proximal to the elbow. The sternocleidomastoid muscle, clavicular part of the trapezius and pectoral muscles, and clavicular part of the deltoid muscle were separated from the collar bone to expose the bone to create, behind the bone, a tunnel wide enough for a pedicled nerve to pass through. The cutaneous muscle of trunk was then cut to expose the brachial plexus. (E) Elevation of the vascularized median nerve. The median nerve and median artery/vein were collectively ligated, cut at the far distal end of the wrist, and elevated as a vascularized median nerve. (F) The pedicled median nerve graft was allowed to pass through the prepared subclavian tunnel toward the head.
coursing across these muscles was ligated proximally. Sufficient care was taken to avoid possible damage to the external jugular vein coursing immediately medial to the midclavicular line (accidental tearing of this vein would result in death in a few seconds). The sternocleidomastoid muscle and the clavicular parts of trapezius muscle, pectoral muscle, and deltoid muscle were separated from the collar bone to expose the bone (only the regions lateral to the external jugular vein), and behind the bone, a tunnel wide enough for a pedicled nerve to pass through was created. The cutaneous muscle of the trunk was then cut to expose the brachial plexus, followed by ligation of the ulnar nerve (►Fig. 2D). The radial nerve was cut to expose the deep brachial artery/vein, which were then ligated. The medial anterior thoracic nerve, lateral thoracic artery/vein, circumflex scapular artery, and musculocutaneous nerve were ligated and cut in the axilla to facilitate the distal repositioning of the pedicled median nerve. Finally, the median nerve and median artery/vein were collectively ligated, cut at the far distal end of the wrist, and elevated as a vascularized median nerve (►Fig. 2E). Because the distal end of the median nerve divides into the medial and lateral branches at approximately one-third from the distal end of forearm, with maximum care, both branches were elevated along with the median artery. The vascularized median nerve was allowed to pass through the prepared subclavian tunnel toward the head (►Fig. 2F), and the tunnel route was expected to be the shortest route for the nerve graft to reach the head. After all ligations made on nerves and vascular
branches were examined and no twisting of the pedicled median nerve was confirmed, the vascularized median nerve was allowed to pass through the prepared subcutaneous pocket to the surgical field prepared in the face. The donor site in the left forelimb was closed with a 5–0 nylon suture (Nescosuture; Alfresa, Osaka, Japan), and the rat was repositioned back to a right lateral decubitus position. A gap of 7 mm was made in the buccal branches of the facial nerve on the left side of the head. At the far end of the vascularized median nerve, the nerve was separated from the artery/vein to secure a margin long enough for nerve anastomosis (1–2 mm). Epineural suture was made between the stumps of the buccal branch of the facial nerve and the median nerve with a 9–0 nylon suture (Nescosuture) under an operating microscope (M651; Leica Microsystems, Wetzlar, Germany). Then, approximately 2 mm of the median nerve was separated from the median artery/vein at 7 mm proximal from the anastomosed stump of the nerve graft, and only the median nerve was cut. The proximal end of the 7-mm island nerve prepared from the median nerve was connected to the proximal stump of the buccal branch of the facial nerve by epineural suture with a 9–0 nylon suture. The proximal stump of the median nerve was anchored to the masseter muscle or parotid gland with a 9–0 nylon suture for reducing possible mechanical force caused by body movement that pulls the anastomosed nerve site in the axillary direction (►Fig. 3A). In a nonvascularized nerve graft group (8 weeks old, n ¼ 7), a 7-mm graft of the median nerve was collected Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 3 Transplantation of the vascularized median nerve to facial nerve (A) The pedicled median nerve graft was allowed to pass through the prepared subcutaneous pocket to the head and was transplanted to a 7-mm gap in the buccal branch of facial nerve by epineural suture with a 9–0 nylon suture. (B, C) Protecting silicone tube. The transplanted median nerve graft was wrapped with a silicon tube (length: 10 mm, i.d.: 1.5 mm, o. d.: 2.5 mm) to prevent neovascularization from the graft bed. One scale mark: 1 mm.
from the left forelimb and transplanted by epineural suture with a 9–0 nylon suture. In both the vascularized and nonvascularized nerve graft groups, the transplanted median nerve graft was wrapped with a silicone tube (length: 10 mm, i.d.: 1.5 mm, o.d.: 2.5 mm) to prevent neovascularization from the graft bed (►Fig. 3B, C). Finally, the facial surgical wound was closed with a 5–0 nylon suture. All median nerve grafts (n ¼ 14) were removed at postoperative week (POW) 30 and subjected to physiological and histological examinations. An additional five rats (8 weeks old) were assigned to the intact control group. All animal care and handling procedures were performed in accordance with the “Principles of Laboratory Animal Care” of the Tokyo Women’s Medical University Animal Experimentation Committee.
Histological Analysis of Myelinated Fibers The transplanted rats were anaesthetized deeply with sodium pentobarbital, and the transplanted median nerves were extracted. The middle portion of each transplanted nerve was subsequently examined. Specimens were prefixed with 2% glutaraldehyde (Distilled EM Grade; EM Science, Gibbstown, NJ), 2% paraformaldehyde (Wako Pure Chemical, Osaka, Japan), and 0.1 mol/L cacodylate buffer (Wako Pure Chemical), and postfixed with 2% osmium tetroxide (Wako Pure Chemical) and 0.1 mol/L cacodylate buffer (Wako Pure Chemical) before being embedded in Quetol-812 resin (Nisshin EM, Tokyo, Japan) by polymerization at 60°C for 2 days. The embedded specimens were cut into approximately 1.5-μm sections with a glass knife and heat stained with 0.5% toluidine blue. Journal of Reconstructive Microsurgery
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The number of myelinated fibers in the middle portion (3.5 mm from the proximal end) of the transplanted median nerve specimens was counted using a NanoZoomer 2.0-HT scanner (Hamamatsu Photonics, Shizuoka, Japan) and its software (NDP view 1.2.25), and the average number of myelinated fibers was calculated.
Transmission Electron Microscopy of the Regenerated Nerves To precisely observe the sheath shape and the regeneration of axon and myelin inside the transplanted median nerve, the specimens were cut into 70-nm sections and mounted on grids (EM fine-grid F-200; Nisshin, Tokyo, Japan). The sections were then stained with 2% uranyl acetate and lead stain solution (Sigma-Aldrich, St. Louis, MO) and examined by a JEM1200EX transmission electron microscope (TEM) (JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV. Axon diameter and myelin thickness in five randomly selected fields were measured with Photoshop CS2 (Adobe Systems, San Jose, CA).
Compound Muscle Action Potential Recordings of the Vibrissal Muscles Rats were anesthetized with urethane (1.2 g/kg body weight, i.p.) and placed on a stereotaxic apparatus. Rectal temperature was maintained around 35 to 36°C using a chemical thermo mat. Stages of anesthesia were maintained by confirming the lack of vibrissae movement and eyelid reflex.10 For recording compound muscle action potential (CMAP), a stainless-steel microelectrode (9–12 MΩ at 1 kHz) (UESMGCSELNNM-type; FHC, Bowdoin, ME) was first inserted into the
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vibrissal muscles between middle vibrissal rows C and D. A reference electrode (TN204–089B; Unique Medical, Tokyo, Japan) was placed in the caudal position of the skull. For stimulating the reconstructed facial nerve, a tandem hookshaped stimulation electrode (IMC-220224; InterMedical, Aichi, Japan) hooked up the exfoliated nerve, the vascularized nerve graft (n ¼ 7), nonvascularized nerve graft (n ¼ 5), or the normal buccal branch of the facial nerve (the intact control group, n ¼ 5), and stimulation pulses at a supramaximal level (2 mA, 100 μs monopolar pulses) were delivered at 0.2 Hz via an isolator (SS-202J; Nihon Kohden, Tokyo, Japan). Recorded signals were processed with a multichannel amplifier (MEG-6100; Nihon Kohden) at 15 to 10,000 Hz and then digitized at 40 kHz using a PowerLab4/ 30 and LabChart7 system (ADInstruments, Dunedin, New Zealand). Data were analyzed in an off-line manner using Igor Pro software (Wavemetrics, Lake Oswego, OR). The upward trace gave a negative deflection (depolarization), and 10 consecutive traces were averaged. Amplitude was measured as difference in voltage between the maximum and baseline CMAP amplitude. The duration of CMAP was calculated as the time between the two points where the baseline was crossed by the rising and declining CMAP curves. CMAP latency was estimated as the time between the stimulus artifact and the point where the baseline was crossed by the rising CMAP curve.
Matsumine et al.
Fig. 4 Macroscopic photographs of transplanted nerves. The photographs were taken at 30 weeks after transplantation. (A) Vascularized nerve graft and (B) nonvascularized nerve graft. One scale mark: 1 mm.
Statistical Analysis Results are expressed as mean SD. Probability less than 5% (p < 0.05) was considered statistically significant. Number of myelinated fibers, amplitude, duration, and latency in each group were compared using an unpaired t-test. Axon diameter and myelin thickness were compared by Mann– Whitney U tests. Statistical analyses were performed by using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA).
Results In all rats of experimental vascularized group (total n ¼ 7), the vascularized median nerve successfully reached the buccal branch of the facial nerve. A 100% survival rate was observed at POW 30, and only one of the rats developed the partial necrosis of the donor site in the left forelimb; the remaining seven rats showed no evidence of necrosis due to ischemia. Anastomosed nerve sites were confirmed to be sutured together even after POW 30, and the vascularized nerve grafts were observed to receive richer blood flow than the nonvascularized nerve graft (►Fig. 4A, B). Histologically, toluidine blue-stained transplanted nerves in the vascularized nerve graft group (►Fig. 5A) showed the median artery and vein (the feeding vessels to the median nerve) coursing along the transplanted median nerve and multiple large areas of dense axonal regeneration around the vessels. In contrast, in the nonvascularized nerve graft group (►Fig. 5B), blood vessels coursing along the regenerated nerves were very thin, with axonal regeneration observed in a comparatively smaller area. The vascularized group had a
longer diameter of the transplanted nerve than the nonvascularized group (►Fig. 5A, B). The number of myelinated fibers was significantly higher in the vascularized nerve graft group (7,986 2,794, n ¼ 7) than in the nonvascularized nerve graft group (5,264 774, n ¼ 7, p < 0.05, unpaired ttest) (►Fig. 5C). These results indicate that the vascularized nerve graft more effectively promoted the axonal regeneration than the nonvascularized nerve graft. On TEM, regenerated nerves in the vascularized nerve graft group had thick and beautifully laminated myelin sheathes (►Fig. 6A), while those in the nonvascularized nerve graft group had only small and thin myelin sheathes (►Fig. 6B). The vascularized nerve graft group showed a significantly larger axon diameter (7.69 2.75 μm, n ¼ 3 vs. 5.28 2.08 μm, n ¼ 3, p < 0.01, Mann–Whitney U tests) (►Fig. 6C) and a significantly higher myelin thickness (0.95 0.34 μm vs. 0.57 0.25 μm, p < 0.01) (►Fig. 6D). These results suggested the accelerated maturation of the regenerated nerves by vascularized nerve graft. For evaluating the effects of vascularization on functional recovery, this study applied electrical stimulation to the proximal part of the grafted nerve and then monitored CMAP from the extrinsic muscle of the whisker pads while observing whisker movements (►Figs. 7 and 8).11–13 Electrical stimulation on the vascularized nerve always elicited large and reliable movements of mystacial whiskers and pads, which can easily be recognized as in normal rats. In contrast, electrical stimulation on the nonvascularized nerve was unable to induce any obvious movements. Quite consistent with the movements, in the vascularized group, large and Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 5 Sectional microscopic photographs of transplanted nerves. Toluidine blue-stained specimen taken from the middle portion of the median nerve at postoperative week 30. (A) Vascularized nerve graft group and (B) nonvascularized nerve graft group. Dark brown dots in (A) are fat tissue. Scale bar: 1 mm. (C) Number of myelinated fibers in the cross sections of the middle portion of transplanted nerves. Mean number of myelinated fibers was significantly higher in the vascularized nerve graft group (mean SD: 7,986 2,794; n ¼ 7) than that in the nonvascularized nerve graft group (5,264 774; n ¼ 7). Unpaired t-test. p < 0.05.
sharp CMAPs were recorded shortly after stimulation (►Fig. 8A). By contrast, in the nonvascularized group, small and broad CMAPs were recorded with a relatively larger latency after nerve stimulation. Differences in amplitude, duration, and latency were all statistically significant for vascularized and nonvascularized groups (►Table 1) (►Fig. 8B–D). The results in the vascularized nerve graft group were similar to those in the intact control group (►Table 1). These results indicated that the vascularized nerve graft considerably promoted the recovery of physiological nerve functions, which were found to be comparable with those of intact animals.
Discussion Facial nerve deficits encountered in clinical practice are often caused by traumatic injury or facial nerve resection due to Journal of Reconstructive Microsurgery
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malignant parotid tumor. In these cases, autologous nerve grafting is performed with the graft bed with a poor blood supply due to scarring from the original trauma or radiotherapy following tumor resection. Graft beds in these cases are generally unable to revascularize. Although artificial nerve conduits widely used in clinical settings are associated with the segmental nature of the blood supply to nerves due to the semipermeable featrure,14 this study performed to insert both vascularized and nonvascularized nerve grafts in silicone tubes to simulate the actual clinical condition with poor revascularization in an animal model. In addition, for evaluating the direct reconstruction effect of vascularized nerve graft without any revascularization from the grafted bed, possible revascularization such as the segmental nature of the blood supply to nerves was completely blocked by using nonpermeable and non-bioabsorbable silicone tubes. Previous histological and physiological studies have reported
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Fig. 6 Transmission electron microphotographs of transplanted nerves. The cross sections of (A) vascularized nerve graft and (B) nonvascularized nerve graft were taken at postoperative week 30. Scale bar: 5 μm. (C) Axon diameter and (D) myelin thickness of the vascularized and nonvascularized nerve graft groups. Mean axon diameter was significantly higher in vascularized nerve graft groups (mean SD: 7.69 2.75 μm; n ¼ 3) than in nonvascularized nerve graft (5.28 2.08 μm; n ¼ 3). Mean myelin thickness was also significantly greater in the vascularized nerve graft group (0.95 0.34 μm vs. 0.57 0.25 μm; n ¼ 3). Mann–Whitney U tests. p < 0.01.
effective vascularized nerve grafting over a period of 1 to 24 weeks using nerves such as sciatic nerve.6–8 For elucidating the efficacy of vascularized nerve grafting over an extended period, this study performed a histological and physiological evaluations over a period of 30 weeks, which was much longer than the periods investigated in previous studies. Using the vascularized median nerve model, this study successfully showed that vascularized autologous nerve grafts provided better outcome in several nerve-regeneration parameters than in nonvascularized grafts for repairing facial nerve deficits. The number of myelinated fibers in the sectional specimen stained with toluidine blue was significantly higher than that of the vascularized nerve graft group, suggesting that the presence of feeding vessels (i.e., the median artery and vein)
facilitated nerve regeneration more effectively than the conventional nerve transplantation method. On electron microscopy, the vascularized nerve graft group showed significantly better results in terms of axon diameter and myelin thickness than the nonvascularized nerve graft group, suggesting that the use of a vascularized nerve graft effectively facilitated the maturation of regenerated nerves. Finally, this study found that CMAP showed significantly better results in the vascularized nerve graft group for all examined electrophysiological parameters examined (i.e., amplitude, duration, and latency), which were also comparable with those of the intact control group. These physiological results indicated that the conduction and the neuromuscular transmission in the regenerated nerve in the vascularized nerve graft group was able to recover the almost normal functional level. Best et al Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 7 Recording of the compound muscle action potential (CMAP) of the vibrissal muscles. For recording CMAP, a stainless-steel microelectrode (white arrow) was inserted into the vibrissal muscles between middle vibrissal rows C and D. A reference electrode (black arrow) was placed in the caudal position of the skull. For stimulating the reconstructed facial nerve, a tandem hook-shaped stimulation electrode (blue arrow) hooked up the exfoliated nerve.
Fig. 8 Compound muscle action potential (CMAP) analysis. (A) Traces show CMAPs recorded from a whisker pad after the supramaximal stimulation of the intact buccal branch of the facial nerve (left), vascularized median nerve (center), and nonvascularized median nerve (right). Each trace was the average of 10 consecutive raw traces. The vascularized nerve graft group (n ¼ 7) showed significantly better results in terms of amplitude (5.06 4.44 mV vs. 0.57 0.32 mV) (graph B), duration (1.35 0.52 ms vs. 2.61 1.29 ms) (C), and latency (1.88 0.42 ms vs. 3.10 0.95 ms) (D) than the nonvascularized nerve graft group (n ¼ 5) ( ►Table 1). Results in the vascularized nerve graft group were similar to those of the intact control group (amplitude: 5.39 2.60 mV, duration: 1.44 0.34 ms, and latency: 1.25 0.48 ms; n ¼ 5), suggesting that the physiological functions of the vascularized nerve graft group were recovered. Unpaired t-test. p < 0.05, p < 0.01. Journal of Reconstructive Microsurgery
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135
Group
Amplitude (mV)
Duration (ms)
Latency (ms)
Intact control (n ¼ 5)
5.39 2.60
1.44 0.34
1.25 0.48
Vascularized (n ¼ 7)
5.06 4.44
1.35 0.52
1.88 0.42
Nonvascularized (n ¼ 5)
0.57 0.32
2.61 1.29
3.10 0.95
have reported that (1) small-diameter nerve grafts, such as rat sciatic nerve, spontaneously revascularize and (2) no revascularization with microvascular techniques is necessary.15 In the present study, however, revascularization using microvascular techniques greatly affected the long-term outcome of nerve regeneration in silicone tubes, suggesting that our microvascular technique is useful in the condition of poor blood supplied grafted bed. As a result, a facial nerve deficit of 7 mm created in this study was sufficient to reveal a statistically significant difference between vascularized and nonvascularized nerve grafts in rat. The route of the median nerve, which arises from the brachial plexus in rats, as well as the detailed anatomy of the arteries supplying the median nerve, including the brachial artery to median artery, has been described by Bertelli et al.16 The present study used the median nerve as a vascularized island nerve graft for facial nerve reconstruction. Although Ozcan et al have reported using the median nerve as a free vascularized nerve graft in a rabbit model,17 the application of their technique to a rat model by transplanting a vascularized nerve graft to the facial nerve in the present study was thought to be innovative. This technique eliminated necessary vascular anastomosis under a microscope and allowed the graft to reach the nerve. Our laboratory has previously reported a nerve suturing procedure allowing an appropriate-sized nerve conduit with an internal diameter of 1 mm to be inserted and transplanted into a 7-mm deficit created in the buccal branch of rat facial nerve.18 In the rat, the thickness of the median nerve in the forearm is reported to be only 0.6 mm.16 Therefore, the diameter of the median nerve is comparable with that of the buccal branch of the facial nerve serving as a graft bed, allowing nerve anastomosis to be performed without difficulty. In this study, except one rat with partial necrosis in the paw, all rats exhibited no clear evidence of disturbed blood flow in the limbs even at POW 30, because, even with the disruption of the brachial-median artery mainly supplying blood to the forelimb,16 sufficient blood flow could be maintained by collateral flow from the dorsal side of the forelimb. The treated rats were able to walk with the paralyzed forelimb and eat feed without any problem. Even under the same housing environment as the intact control rats, none of the seven treated rats died during the 30-week observation period. In conclusion, this study developed a rat model transplanted with vascularized median nerve transplantation to the buccal branch of the facial nerve. After being transplanted in a graft bed having a poor revascularization ability, the vascularized median nerve graft used in this study gave better
nerve regeneration outcomes histologically and physiologically than the nonvascularized median nerve graft. This study can also provide a useful animal model that allowed the efficacy of vascularized grafts used for treating old facial nerve injury to be evaluated. Furthermore, upon the transplantation of a nerve graft to a severely scarred graft bed with a poor graft-revascularization ability, the vascularized nerve grafting procedure have a potential to achieve a clinically significant outcome.
Acknowledgments This study was supported by the Formation of Innovation Center for Fusion of Advanced Technologies project sponsored by the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” and by the Global COE program at the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, as well as by the JST PRESTO program and Hiroto Yoshioka Memorial Fund for Medical Research.
Conflicts of Interest None. Funding Source None.
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Table 1 Compound muscle action potential recordings of the vibrissal muscles
Vascularized Nerve for Facial Nerve Reconstruction in Rat 7 Vargel I. Impact of vascularization type on peripheral nerve
Matsumine et al. 13 Byrne PJ, Stuart RM, Fakhry C, Lehar M, Flint PW. An electrophysi-
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vascularized nerve graft: evaluation of nerve regeneration using choline acetyltransferase activity. Microsurgery 2001;21(2): 43–51 9 Sasaki R, Matsumine H, Watanabe Y, Yamato M, Ando T. Anesthesia
for research on reconstructive facial surgery in rats. J Reconstr Microsurg 2013;29(3):209–210 10 Friedberg MH, Lee SM, Ebner FF. Modulation of receptive field
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properties of thalamic somatosensory neurons by the depth of anesthesia. J Neurophysiol 1999;81(5):2243–2252 11 Nguyen QT, Kleinfeld D. Positive feedback in a brainstem tactile
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sensorimotor loop. Neuron 2005;45(3):447–457 12 Veronesi C, Maggiolini E, Franchi G. Postnatal development of
vibrissae motor output following neonatal infraorbital nerve manipulation. Exp Neurol 2006;200(2):332–342
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ologic model for functional assessment of effects of neurotrophic factors on facial nerve reinnervation. Arch Facial Plast Surg 2005; 7(2):114–118 Konofaos P, Ver Halen JP. Nerve repair by means of tubulization: past, present, future. J Reconstr Microsurg 2013;29(3):149–164 Best TJ, Mackinnon SE, Evans PJ, Hunter D, Midha R. Peripheral nerve revascularization: histomorphometric study of small- and large-caliber grafts. J Reconstr Microsurg 1999;15(3):183–190 Bertelli JA, Taleb M, Saadi A, Mira JC, Pecot-Dechavassine M. The rat brachial plexus and its terminal branches: an experimental model for the study of peripheral nerve regeneration. Microsurgery 1995;16(2):77–85 Ozcan G, Shenaq S, Spira M. A new vascularized nerve graft model in the rabbit. J Reconstr Microsurg 1992;8(1):35–40 Matsumine H, Sasaki R, Takeuchi M, Yamato M, Sakurai H. Surgical procedure for transplanting artificial nerve conduits for peripheral nerve regeneration. Plast Reconstr Surg 2011;128(2):95e–97e
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Vascularized versus Nonvascularized Island Median Nerve Grafts in the Facial Nerve Regeneration and Functional Recovery of Rats for Facial Nerve Reconstruction Study
1 Department of Plastic and Reconstructive Surgery, Tokyo Women’s
Medical University, School of Medicine, Tokyo, Japan 2 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan 3 Department of Oral and Maxillofacial Surgery, Global Center of Excellence (COE) Program, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan 4 Department of Physiology, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan 5 Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan
Address for correspondence Hajime Matsumine, MD, PhD, Department of Plastic and Reconstructive Surgery, Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan (e-mail: [email protected]).
J Reconstr Microsurg 2014;30:127–136.
Abstract
Keywords
► vascularized nerve transplantation ► facial nerve ► rat ► buccal branch ► median nerve
Histological and physiological basis of the therapeutic efficacy of the vascularized autologous nerve graft in facial nerve regeneration remains poorly understood because of no established rat model. The left median nerve and median artery/vein of Lewis rats were collectively ligated, and harvested as a vascularized island median nerve, which was transplanted to a 7-mm gap in the left buccal branch of facial nerve. Nerve regeneration was investigated. The numbers of myelinated fibers, axon diameter, and myelin thickness were significantly higher in the vascularized nerve graft group than in the nonvascularized nerve graft group. Compound muscle action potential measurement showed that the parameters of vascularized group were similar to those in the intact control group. A vascularized median nerve graft resulted in better facial nerve regeneration outcomes.
Nerve reconstruction with a nonvascularized autologous nerve graft has been traditionally performed in patients with a facial nerve deficit after trauma or tumor resection. However, a long and thick nerve graft may develop central necrosis after a few days of ischemia, resulting in an unsatisfactory outcome.1 A vascularized autologous nerve graft has been recently developed for compensating the shortcoming of nonvascularized grafts and providing a functional recovery to patients with a damaged nerve graft bed or longer nerve deficits that are unable to be treated adequately by conven-
tional nerve transplantation techniques.2,3 A vascularized nerve graft diminishes endoneurial scarring by maintaining the Schwann cell population and suppressing fibroblast infiltration, and provides an optimal nutritional environment that results in an increased rate of axonal regeneration.4 For example, Koshima et al have reported that facial nerve reconstruction using a vascularized auricular nerve graft in patients with a facial nerve defect gives good postoperative outcomes.3 Kashiwa et al have used a vascularized lateral femoral cutaneous nerve combined with a groin flap to
received July 2, 2013 accepted after revision August 13, 2013 published online October 25, 2013
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DOI http://dx.doi.org/ 10.1055/s-0033-1357500. ISSN 0743-684X.
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Hajime Matsumine, MD, PhD1,2 Ryo Sasaki, DDS, PhD2,3 Yuichi Takeuchi, PhD4 Mariko Miyata, MD, PhD4,5 Masayuki Yamato, PhD2 Teruo Okano, PhD2 Hiroyuki Sakurai, MD, PhD1
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
reconstruct a facial nerve defect and reported that satisfactory facial animation is obtained.5 However, the histological and physiological basis of the therapeutic efficacy of the vascularized autologous nerve graft in facial nerve regeneration remains poorly understood, because in part no established rat model with vascularized autologous nerve grafts for facial nerve regeneration is available. Although rat experimental models having sciatic and femoral nerve defects have been well used by many researchers for vascularized nerve studies,6–8 no facial nerve reconstruction model with a vascularized nerve is developed because of its complicated anatomy. This study established a rat model for facial nerve regeneration by transplanting a vascularized island median nerve, which had the brachial artery serving as a vascular pedicle, to a 7-mm deficit in the buccal branch of the facial nerve. The effectiveness of the facial nerve regeneration with a vascularized autologous nerve graft was investigated both histologically and physiologically.
aspect of the wrist (►Fig. 2B). After the skin incision was made, a subcutaneous tunnel was created to reach the preauricular incision to allow the facial nerve to be exposed. A cutaneous nerve and vein around the elbow were ligated from the medial to the short head of the biceps brachii muscle, and the median nerve and brachial artery/vein along the lower margin of the greater pectoral muscle were identified and exposed. An incision was then made on the antebrachial fascia from the elbow to wrist to expose the forearm flexor muscle group. After the palmaris longus tendon and flexor retinaculum were cut in the wrist, the median nerve and peripheral end of the median artery were exposed between the flexor carpi radialis and flexor digitorum superficialis in the forearm (►Fig. 2C). As the median artery divides into the radial and ulnar branches in the mid-forearm, the radial branch was ligated and cut, while the ulnar branch was preserved and used as a vascular pedicle. All branches of the median artery/vein distributed to the forearm muscle group were ligated and cut. The branches of the median nerve innervating the flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis muscle, and pronator teres muscle were also ligated. Another branch of median nerve located a few millimeters distal from the branch innervating the pronator teres muscle, equivalent to the interosseous nerve in humans, was also ligated. The superficial radial artery/vein, ulnar collateral artery/vein, and transverse cubital artery/vein arising from the brachial artery/vein were ligated around the elbow. Because the flexor carpi radialis muscle and pronator teres muscle were cut to expose the median nerve and median artery/vein coursing behind the muscles, they could be connected to the median nerve and brachial artery/vein that had been exposed proximal to the elbow. Then, the ulnar artery/vein coursing into the deep region was ligated. The superficial and deep pectoral muscles were cut up to the midclavicular line after the cephalic vein
Methods Surgical Procedure Lewis rats (8 weeks old, n ¼ 7) were anesthetized with 4% isoflurane using a nasal mask connected to a Univentor 400 Anesthesia Unit (Univentor, Zejtun, Malta) and placed in a right lateral decubitus position.9 The surgical procedure was performed as shown in ►Fig. 1. A preauricular incision with a marginal mandibular extension was made on the left side of the face, exposing the buccal and marginal mandibular branches, and parotid gland (►Fig. 2A). The rat was then repositioned into a supine position, and both forelimbs were stretched and fixed with tape. A slightly curved skin incision was made, starting from the midpoint of the left collar bone, passing over the left cubital fossa, and ending at the flexor
Fig. 1 Schematic illustration of the main branches of the brachial to median arteries and the brachial plexus to median nerve The present technique required the incisions of the greater pectoral muscle, pronator teres muscle, and flexor carpi radialis muscle for exposing the median nerve and brachial-median artery (the blue curved arrows indicate direction of movement). Areas of the median nerve and artery were used for a vascularized island nerve graft (blue ellipse). After being elevated, the vascularized median nerve was passed through the subclavian tunnel toward the head (green arrow in the upper left area of opened tissue). Journal of Reconstructive Microsurgery
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Fig. 2 Surgical procedure. (A) Left lateral facial view of a rat. A preauricular incision with a marginal mandibular extension was made on both sides of the face, exposing the buccal and marginal mandibular branches and parotid gland. (B) An incisional line on the left forelimb. A slightly curved line was drawn, starting from the midpoint of the left collar bone, passing over the left cubital fossa, and ending at the flexor aspect of the wrist. (C) Exposure of the median nerve and artery. After the palmaris longus tendon and flexor retinaculum were cut in the wrist, the median nerve (white arrow) and the peripheral end of the median artery (black arrow) were exposed between the flexor carpi radialis and flexor digitorum superficialis in the forearm. (D) Exposure of the brachial artery and brachial plexus. The flexor carpi radialis muscle and pronator teres muscles were cut to expose the median nerve and median artery/vein coursing behind the muscles, allowing them to be connected to the median nerve and brachial artery/vein that had been exposed proximal to the elbow. The sternocleidomastoid muscle, clavicular part of the trapezius and pectoral muscles, and clavicular part of the deltoid muscle were separated from the collar bone to expose the bone to create, behind the bone, a tunnel wide enough for a pedicled nerve to pass through. The cutaneous muscle of trunk was then cut to expose the brachial plexus. (E) Elevation of the vascularized median nerve. The median nerve and median artery/vein were collectively ligated, cut at the far distal end of the wrist, and elevated as a vascularized median nerve. (F) The pedicled median nerve graft was allowed to pass through the prepared subclavian tunnel toward the head.
coursing across these muscles was ligated proximally. Sufficient care was taken to avoid possible damage to the external jugular vein coursing immediately medial to the midclavicular line (accidental tearing of this vein would result in death in a few seconds). The sternocleidomastoid muscle and the clavicular parts of trapezius muscle, pectoral muscle, and deltoid muscle were separated from the collar bone to expose the bone (only the regions lateral to the external jugular vein), and behind the bone, a tunnel wide enough for a pedicled nerve to pass through was created. The cutaneous muscle of the trunk was then cut to expose the brachial plexus, followed by ligation of the ulnar nerve (►Fig. 2D). The radial nerve was cut to expose the deep brachial artery/vein, which were then ligated. The medial anterior thoracic nerve, lateral thoracic artery/vein, circumflex scapular artery, and musculocutaneous nerve were ligated and cut in the axilla to facilitate the distal repositioning of the pedicled median nerve. Finally, the median nerve and median artery/vein were collectively ligated, cut at the far distal end of the wrist, and elevated as a vascularized median nerve (►Fig. 2E). Because the distal end of the median nerve divides into the medial and lateral branches at approximately one-third from the distal end of forearm, with maximum care, both branches were elevated along with the median artery. The vascularized median nerve was allowed to pass through the prepared subclavian tunnel toward the head (►Fig. 2F), and the tunnel route was expected to be the shortest route for the nerve graft to reach the head. After all ligations made on nerves and vascular
branches were examined and no twisting of the pedicled median nerve was confirmed, the vascularized median nerve was allowed to pass through the prepared subcutaneous pocket to the surgical field prepared in the face. The donor site in the left forelimb was closed with a 5–0 nylon suture (Nescosuture; Alfresa, Osaka, Japan), and the rat was repositioned back to a right lateral decubitus position. A gap of 7 mm was made in the buccal branches of the facial nerve on the left side of the head. At the far end of the vascularized median nerve, the nerve was separated from the artery/vein to secure a margin long enough for nerve anastomosis (1–2 mm). Epineural suture was made between the stumps of the buccal branch of the facial nerve and the median nerve with a 9–0 nylon suture (Nescosuture) under an operating microscope (M651; Leica Microsystems, Wetzlar, Germany). Then, approximately 2 mm of the median nerve was separated from the median artery/vein at 7 mm proximal from the anastomosed stump of the nerve graft, and only the median nerve was cut. The proximal end of the 7-mm island nerve prepared from the median nerve was connected to the proximal stump of the buccal branch of the facial nerve by epineural suture with a 9–0 nylon suture. The proximal stump of the median nerve was anchored to the masseter muscle or parotid gland with a 9–0 nylon suture for reducing possible mechanical force caused by body movement that pulls the anastomosed nerve site in the axillary direction (►Fig. 3A). In a nonvascularized nerve graft group (8 weeks old, n ¼ 7), a 7-mm graft of the median nerve was collected Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 3 Transplantation of the vascularized median nerve to facial nerve (A) The pedicled median nerve graft was allowed to pass through the prepared subcutaneous pocket to the head and was transplanted to a 7-mm gap in the buccal branch of facial nerve by epineural suture with a 9–0 nylon suture. (B, C) Protecting silicone tube. The transplanted median nerve graft was wrapped with a silicon tube (length: 10 mm, i.d.: 1.5 mm, o. d.: 2.5 mm) to prevent neovascularization from the graft bed. One scale mark: 1 mm.
from the left forelimb and transplanted by epineural suture with a 9–0 nylon suture. In both the vascularized and nonvascularized nerve graft groups, the transplanted median nerve graft was wrapped with a silicone tube (length: 10 mm, i.d.: 1.5 mm, o.d.: 2.5 mm) to prevent neovascularization from the graft bed (►Fig. 3B, C). Finally, the facial surgical wound was closed with a 5–0 nylon suture. All median nerve grafts (n ¼ 14) were removed at postoperative week (POW) 30 and subjected to physiological and histological examinations. An additional five rats (8 weeks old) were assigned to the intact control group. All animal care and handling procedures were performed in accordance with the “Principles of Laboratory Animal Care” of the Tokyo Women’s Medical University Animal Experimentation Committee.
Histological Analysis of Myelinated Fibers The transplanted rats were anaesthetized deeply with sodium pentobarbital, and the transplanted median nerves were extracted. The middle portion of each transplanted nerve was subsequently examined. Specimens were prefixed with 2% glutaraldehyde (Distilled EM Grade; EM Science, Gibbstown, NJ), 2% paraformaldehyde (Wako Pure Chemical, Osaka, Japan), and 0.1 mol/L cacodylate buffer (Wako Pure Chemical), and postfixed with 2% osmium tetroxide (Wako Pure Chemical) and 0.1 mol/L cacodylate buffer (Wako Pure Chemical) before being embedded in Quetol-812 resin (Nisshin EM, Tokyo, Japan) by polymerization at 60°C for 2 days. The embedded specimens were cut into approximately 1.5-μm sections with a glass knife and heat stained with 0.5% toluidine blue. Journal of Reconstructive Microsurgery
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The number of myelinated fibers in the middle portion (3.5 mm from the proximal end) of the transplanted median nerve specimens was counted using a NanoZoomer 2.0-HT scanner (Hamamatsu Photonics, Shizuoka, Japan) and its software (NDP view 1.2.25), and the average number of myelinated fibers was calculated.
Transmission Electron Microscopy of the Regenerated Nerves To precisely observe the sheath shape and the regeneration of axon and myelin inside the transplanted median nerve, the specimens were cut into 70-nm sections and mounted on grids (EM fine-grid F-200; Nisshin, Tokyo, Japan). The sections were then stained with 2% uranyl acetate and lead stain solution (Sigma-Aldrich, St. Louis, MO) and examined by a JEM1200EX transmission electron microscope (TEM) (JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV. Axon diameter and myelin thickness in five randomly selected fields were measured with Photoshop CS2 (Adobe Systems, San Jose, CA).
Compound Muscle Action Potential Recordings of the Vibrissal Muscles Rats were anesthetized with urethane (1.2 g/kg body weight, i.p.) and placed on a stereotaxic apparatus. Rectal temperature was maintained around 35 to 36°C using a chemical thermo mat. Stages of anesthesia were maintained by confirming the lack of vibrissae movement and eyelid reflex.10 For recording compound muscle action potential (CMAP), a stainless-steel microelectrode (9–12 MΩ at 1 kHz) (UESMGCSELNNM-type; FHC, Bowdoin, ME) was first inserted into the
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vibrissal muscles between middle vibrissal rows C and D. A reference electrode (TN204–089B; Unique Medical, Tokyo, Japan) was placed in the caudal position of the skull. For stimulating the reconstructed facial nerve, a tandem hookshaped stimulation electrode (IMC-220224; InterMedical, Aichi, Japan) hooked up the exfoliated nerve, the vascularized nerve graft (n ¼ 7), nonvascularized nerve graft (n ¼ 5), or the normal buccal branch of the facial nerve (the intact control group, n ¼ 5), and stimulation pulses at a supramaximal level (2 mA, 100 μs monopolar pulses) were delivered at 0.2 Hz via an isolator (SS-202J; Nihon Kohden, Tokyo, Japan). Recorded signals were processed with a multichannel amplifier (MEG-6100; Nihon Kohden) at 15 to 10,000 Hz and then digitized at 40 kHz using a PowerLab4/ 30 and LabChart7 system (ADInstruments, Dunedin, New Zealand). Data were analyzed in an off-line manner using Igor Pro software (Wavemetrics, Lake Oswego, OR). The upward trace gave a negative deflection (depolarization), and 10 consecutive traces were averaged. Amplitude was measured as difference in voltage between the maximum and baseline CMAP amplitude. The duration of CMAP was calculated as the time between the two points where the baseline was crossed by the rising and declining CMAP curves. CMAP latency was estimated as the time between the stimulus artifact and the point where the baseline was crossed by the rising CMAP curve.
Matsumine et al.
Fig. 4 Macroscopic photographs of transplanted nerves. The photographs were taken at 30 weeks after transplantation. (A) Vascularized nerve graft and (B) nonvascularized nerve graft. One scale mark: 1 mm.
Statistical Analysis Results are expressed as mean SD. Probability less than 5% (p < 0.05) was considered statistically significant. Number of myelinated fibers, amplitude, duration, and latency in each group were compared using an unpaired t-test. Axon diameter and myelin thickness were compared by Mann– Whitney U tests. Statistical analyses were performed by using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA).
Results In all rats of experimental vascularized group (total n ¼ 7), the vascularized median nerve successfully reached the buccal branch of the facial nerve. A 100% survival rate was observed at POW 30, and only one of the rats developed the partial necrosis of the donor site in the left forelimb; the remaining seven rats showed no evidence of necrosis due to ischemia. Anastomosed nerve sites were confirmed to be sutured together even after POW 30, and the vascularized nerve grafts were observed to receive richer blood flow than the nonvascularized nerve graft (►Fig. 4A, B). Histologically, toluidine blue-stained transplanted nerves in the vascularized nerve graft group (►Fig. 5A) showed the median artery and vein (the feeding vessels to the median nerve) coursing along the transplanted median nerve and multiple large areas of dense axonal regeneration around the vessels. In contrast, in the nonvascularized nerve graft group (►Fig. 5B), blood vessels coursing along the regenerated nerves were very thin, with axonal regeneration observed in a comparatively smaller area. The vascularized group had a
longer diameter of the transplanted nerve than the nonvascularized group (►Fig. 5A, B). The number of myelinated fibers was significantly higher in the vascularized nerve graft group (7,986 2,794, n ¼ 7) than in the nonvascularized nerve graft group (5,264 774, n ¼ 7, p < 0.05, unpaired ttest) (►Fig. 5C). These results indicate that the vascularized nerve graft more effectively promoted the axonal regeneration than the nonvascularized nerve graft. On TEM, regenerated nerves in the vascularized nerve graft group had thick and beautifully laminated myelin sheathes (►Fig. 6A), while those in the nonvascularized nerve graft group had only small and thin myelin sheathes (►Fig. 6B). The vascularized nerve graft group showed a significantly larger axon diameter (7.69 2.75 μm, n ¼ 3 vs. 5.28 2.08 μm, n ¼ 3, p < 0.01, Mann–Whitney U tests) (►Fig. 6C) and a significantly higher myelin thickness (0.95 0.34 μm vs. 0.57 0.25 μm, p < 0.01) (►Fig. 6D). These results suggested the accelerated maturation of the regenerated nerves by vascularized nerve graft. For evaluating the effects of vascularization on functional recovery, this study applied electrical stimulation to the proximal part of the grafted nerve and then monitored CMAP from the extrinsic muscle of the whisker pads while observing whisker movements (►Figs. 7 and 8).11–13 Electrical stimulation on the vascularized nerve always elicited large and reliable movements of mystacial whiskers and pads, which can easily be recognized as in normal rats. In contrast, electrical stimulation on the nonvascularized nerve was unable to induce any obvious movements. Quite consistent with the movements, in the vascularized group, large and Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 5 Sectional microscopic photographs of transplanted nerves. Toluidine blue-stained specimen taken from the middle portion of the median nerve at postoperative week 30. (A) Vascularized nerve graft group and (B) nonvascularized nerve graft group. Dark brown dots in (A) are fat tissue. Scale bar: 1 mm. (C) Number of myelinated fibers in the cross sections of the middle portion of transplanted nerves. Mean number of myelinated fibers was significantly higher in the vascularized nerve graft group (mean SD: 7,986 2,794; n ¼ 7) than that in the nonvascularized nerve graft group (5,264 774; n ¼ 7). Unpaired t-test. p < 0.05.
sharp CMAPs were recorded shortly after stimulation (►Fig. 8A). By contrast, in the nonvascularized group, small and broad CMAPs were recorded with a relatively larger latency after nerve stimulation. Differences in amplitude, duration, and latency were all statistically significant for vascularized and nonvascularized groups (►Table 1) (►Fig. 8B–D). The results in the vascularized nerve graft group were similar to those in the intact control group (►Table 1). These results indicated that the vascularized nerve graft considerably promoted the recovery of physiological nerve functions, which were found to be comparable with those of intact animals.
Discussion Facial nerve deficits encountered in clinical practice are often caused by traumatic injury or facial nerve resection due to Journal of Reconstructive Microsurgery
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malignant parotid tumor. In these cases, autologous nerve grafting is performed with the graft bed with a poor blood supply due to scarring from the original trauma or radiotherapy following tumor resection. Graft beds in these cases are generally unable to revascularize. Although artificial nerve conduits widely used in clinical settings are associated with the segmental nature of the blood supply to nerves due to the semipermeable featrure,14 this study performed to insert both vascularized and nonvascularized nerve grafts in silicone tubes to simulate the actual clinical condition with poor revascularization in an animal model. In addition, for evaluating the direct reconstruction effect of vascularized nerve graft without any revascularization from the grafted bed, possible revascularization such as the segmental nature of the blood supply to nerves was completely blocked by using nonpermeable and non-bioabsorbable silicone tubes. Previous histological and physiological studies have reported
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Fig. 6 Transmission electron microphotographs of transplanted nerves. The cross sections of (A) vascularized nerve graft and (B) nonvascularized nerve graft were taken at postoperative week 30. Scale bar: 5 μm. (C) Axon diameter and (D) myelin thickness of the vascularized and nonvascularized nerve graft groups. Mean axon diameter was significantly higher in vascularized nerve graft groups (mean SD: 7.69 2.75 μm; n ¼ 3) than in nonvascularized nerve graft (5.28 2.08 μm; n ¼ 3). Mean myelin thickness was also significantly greater in the vascularized nerve graft group (0.95 0.34 μm vs. 0.57 0.25 μm; n ¼ 3). Mann–Whitney U tests. p < 0.01.
effective vascularized nerve grafting over a period of 1 to 24 weeks using nerves such as sciatic nerve.6–8 For elucidating the efficacy of vascularized nerve grafting over an extended period, this study performed a histological and physiological evaluations over a period of 30 weeks, which was much longer than the periods investigated in previous studies. Using the vascularized median nerve model, this study successfully showed that vascularized autologous nerve grafts provided better outcome in several nerve-regeneration parameters than in nonvascularized grafts for repairing facial nerve deficits. The number of myelinated fibers in the sectional specimen stained with toluidine blue was significantly higher than that of the vascularized nerve graft group, suggesting that the presence of feeding vessels (i.e., the median artery and vein)
facilitated nerve regeneration more effectively than the conventional nerve transplantation method. On electron microscopy, the vascularized nerve graft group showed significantly better results in terms of axon diameter and myelin thickness than the nonvascularized nerve graft group, suggesting that the use of a vascularized nerve graft effectively facilitated the maturation of regenerated nerves. Finally, this study found that CMAP showed significantly better results in the vascularized nerve graft group for all examined electrophysiological parameters examined (i.e., amplitude, duration, and latency), which were also comparable with those of the intact control group. These physiological results indicated that the conduction and the neuromuscular transmission in the regenerated nerve in the vascularized nerve graft group was able to recover the almost normal functional level. Best et al Journal of Reconstructive Microsurgery
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Vascularized Nerve for Facial Nerve Reconstruction in Rat
Vascularized Nerve for Facial Nerve Reconstruction in Rat
Matsumine et al.
Fig. 7 Recording of the compound muscle action potential (CMAP) of the vibrissal muscles. For recording CMAP, a stainless-steel microelectrode (white arrow) was inserted into the vibrissal muscles between middle vibrissal rows C and D. A reference electrode (black arrow) was placed in the caudal position of the skull. For stimulating the reconstructed facial nerve, a tandem hook-shaped stimulation electrode (blue arrow) hooked up the exfoliated nerve.
Fig. 8 Compound muscle action potential (CMAP) analysis. (A) Traces show CMAPs recorded from a whisker pad after the supramaximal stimulation of the intact buccal branch of the facial nerve (left), vascularized median nerve (center), and nonvascularized median nerve (right). Each trace was the average of 10 consecutive raw traces. The vascularized nerve graft group (n ¼ 7) showed significantly better results in terms of amplitude (5.06 4.44 mV vs. 0.57 0.32 mV) (graph B), duration (1.35 0.52 ms vs. 2.61 1.29 ms) (C), and latency (1.88 0.42 ms vs. 3.10 0.95 ms) (D) than the nonvascularized nerve graft group (n ¼ 5) ( ►Table 1). Results in the vascularized nerve graft group were similar to those of the intact control group (amplitude: 5.39 2.60 mV, duration: 1.44 0.34 ms, and latency: 1.25 0.48 ms; n ¼ 5), suggesting that the physiological functions of the vascularized nerve graft group were recovered. Unpaired t-test. p < 0.05, p < 0.01. Journal of Reconstructive Microsurgery
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Group
Amplitude (mV)
Duration (ms)
Latency (ms)
Intact control (n ¼ 5)
5.39 2.60
1.44 0.34
1.25 0.48
Vascularized (n ¼ 7)
5.06 4.44
1.35 0.52
1.88 0.42
Nonvascularized (n ¼ 5)
0.57 0.32
2.61 1.29
3.10 0.95
have reported that (1) small-diameter nerve grafts, such as rat sciatic nerve, spontaneously revascularize and (2) no revascularization with microvascular techniques is necessary.15 In the present study, however, revascularization using microvascular techniques greatly affected the long-term outcome of nerve regeneration in silicone tubes, suggesting that our microvascular technique is useful in the condition of poor blood supplied grafted bed. As a result, a facial nerve deficit of 7 mm created in this study was sufficient to reveal a statistically significant difference between vascularized and nonvascularized nerve grafts in rat. The route of the median nerve, which arises from the brachial plexus in rats, as well as the detailed anatomy of the arteries supplying the median nerve, including the brachial artery to median artery, has been described by Bertelli et al.16 The present study used the median nerve as a vascularized island nerve graft for facial nerve reconstruction. Although Ozcan et al have reported using the median nerve as a free vascularized nerve graft in a rabbit model,17 the application of their technique to a rat model by transplanting a vascularized nerve graft to the facial nerve in the present study was thought to be innovative. This technique eliminated necessary vascular anastomosis under a microscope and allowed the graft to reach the nerve. Our laboratory has previously reported a nerve suturing procedure allowing an appropriate-sized nerve conduit with an internal diameter of 1 mm to be inserted and transplanted into a 7-mm deficit created in the buccal branch of rat facial nerve.18 In the rat, the thickness of the median nerve in the forearm is reported to be only 0.6 mm.16 Therefore, the diameter of the median nerve is comparable with that of the buccal branch of the facial nerve serving as a graft bed, allowing nerve anastomosis to be performed without difficulty. In this study, except one rat with partial necrosis in the paw, all rats exhibited no clear evidence of disturbed blood flow in the limbs even at POW 30, because, even with the disruption of the brachial-median artery mainly supplying blood to the forelimb,16 sufficient blood flow could be maintained by collateral flow from the dorsal side of the forelimb. The treated rats were able to walk with the paralyzed forelimb and eat feed without any problem. Even under the same housing environment as the intact control rats, none of the seven treated rats died during the 30-week observation period. In conclusion, this study developed a rat model transplanted with vascularized median nerve transplantation to the buccal branch of the facial nerve. After being transplanted in a graft bed having a poor revascularization ability, the vascularized median nerve graft used in this study gave better
nerve regeneration outcomes histologically and physiologically than the nonvascularized median nerve graft. This study can also provide a useful animal model that allowed the efficacy of vascularized grafts used for treating old facial nerve injury to be evaluated. Furthermore, upon the transplantation of a nerve graft to a severely scarred graft bed with a poor graft-revascularization ability, the vascularized nerve grafting procedure have a potential to achieve a clinically significant outcome.
Acknowledgments This study was supported by the Formation of Innovation Center for Fusion of Advanced Technologies project sponsored by the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” and by the Global COE program at the Multidisciplinary Education and Research Center for Regenerative Medicine (MERCREM), funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, as well as by the JST PRESTO program and Hiroto Yoshioka Memorial Fund for Medical Research.
Conflicts of Interest None. Funding Source None.
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