Hypoglossal-facial nerve “side”-to-side neurorrhaphy for persistent incomplete facial palsy

Laboratory investigation

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Object

Hypoglossal-facial nerve neurorrhaphy is a widely used method for treating complete facial palsy. However, the classic surgical procedure using a “side”-to-end neurorrhaphy is not suitable for incomplete facial palsy (IFP), because sectioning of the facial nerve for neurorrhaphy compromises remnant axons and potential spontaneous reinnervation. For the treatment of persistent IFP, the authors investigated in rats a modified method using hypoglossal-facial nerve “side”-to-side neurorrhaphy.

Methods

An IFP model was created by crushing the facial nerve and then ligating the injury site to limit axonal regeneration. After 9 weeks, rats with IFP were submitted to hypoglossal-facial nerve “side”-to-side neurorrhaphy: The gap between the 2 nerves was bridged with a predegenerated peroneal nerve graft, which was sutured to only one-half of the hypoglossal nerve and to the remnant facial nerve through a small window created by removing the epineurium, thus preserving regenerating facial axons.

Results

Four months after repair surgery, double innervation of the target whisker pad by hypoglossal and facial motor neurons was supported by the recording of muscle action potentials and their retrograde labeling. Regenerated hypoglossal and facial motor neurons effectively participated in the reinnervation of the whisker pad, significantly improving facial symmetry without evident synkinesis, compared with rats that underwent IFP without hypoglossal-facial nerve neurorrhaphy.

Conclusions

This study demonstrates that hypoglossal-facial nerve “side”-to-side neurorrhaphy with a predegenerated nerve graft can lead to rapid functional benefits for persistent IFP without compromising the remnants of facial axons, thus providing a proof-of-feasibility for further studies in humans.

Abbreviations used in this paper:CFP = complete facial palsy; CTB–Alexa 555 = cholera toxin subunit B conjugated with Alexa Fluor 555; DY = diamidino yellow; FN = facial nerve; HN = hypoglossal nerve; IFP = incomplete facial palsy; IFP-R = IFP treated with HN-FN “side”-to-side neurorrhaphy; MAP = muscle action potential; PNG = predegenerated nerve graft.

Abstract

Object

Hypoglossal-facial nerve neurorrhaphy is a widely used method for treating complete facial palsy. However, the classic surgical procedure using a “side”-to-end neurorrhaphy is not suitable for incomplete facial palsy (IFP), because sectioning of the facial nerve for neurorrhaphy compromises remnant axons and potential spontaneous reinnervation. For the treatment of persistent IFP, the authors investigated in rats a modified method using hypoglossal-facial nerve “side”-to-side neurorrhaphy.

Methods

An IFP model was created by crushing the facial nerve and then ligating the injury site to limit axonal regeneration. After 9 weeks, rats with IFP were submitted to hypoglossal-facial nerve “side”-to-side neurorrhaphy: The gap between the 2 nerves was bridged with a predegenerated peroneal nerve graft, which was sutured to only one-half of the hypoglossal nerve and to the remnant facial nerve through a small window created by removing the epineurium, thus preserving regenerating facial axons.

Results

Four months after repair surgery, double innervation of the target whisker pad by hypoglossal and facial motor neurons was supported by the recording of muscle action potentials and their retrograde labeling. Regenerated hypoglossal and facial motor neurons effectively participated in the reinnervation of the whisker pad, significantly improving facial symmetry without evident synkinesis, compared with rats that underwent IFP without hypoglossal-facial nerve neurorrhaphy.

Conclusions

This study demonstrates that hypoglossal-facial nerve “side”-to-side neurorrhaphy with a predegenerated nerve graft can lead to rapid functional benefits for persistent IFP without compromising the remnants of facial axons, thus providing a proof-of-feasibility for further studies in humans.

Facial palsy resulting from facial nerve (FN) injury affects up to 10% of patients undergoing removal of cerebellopontine angle tumors.15,31 The loss of facial symmetry and expression following facial palsy has a great impact on psychosocial conditions of the patients.21

Hypoglossal nerve–facial nerve neurorrhaphy is a conventional method for treating complete facial palsy (CFP) when the proximal FN stump is not available.3,5,16,25,26,32 Since complete transection of the hypoglossal nerve (HN) may result in paralysis and atrophy of the ipsilateral hemitongue,16,22 HN-FN “side”-to-end neurorrhaphy by using only one-half of the HN is preferable to their end-to-end neurorrhaphy.16,25,26

Incomplete facial palsy (IFP), which has a relatively high prevalence among patients who undergo cerebellopontine angle surgery, results from FN injury with remnant axons or insufficient spontaneous axonal regeneration. For the patients with persistent and important facial deficits, surgical repair of the injured FN is desirable. However, the classic HN-FN “side”-to-end neurorrhaphy is not suitable for these patients because complete sectioning of the FN for neurorrhaphy compromises its remnant axons and/or spontaneous reinnervation potential. We thus assessed a modified method for the treatment of persistent IFP by using a HN-FN “side”-to-side neurorrhaphy through a predegenerated nerve graft (PNG). The “side”-to-side neurorrhaphy was achieved through a window at the injured FN where only the epineurium was removed. This intervention does not interrupt the main structure of the FN at the neurorrhaphy site, thereby preserving the remnants of facial axons and their potential for spontaneous reinnervation. A rat model with persistent IFP was created by performing a crush injury of the FN and then ligating the injury site to limit axonal regeneration. Nine weeks after injury, functional evaluation was performed to select rats that developed IFP. In those rats, the HN-FN “side”-to-side neurorrhaphy was performed. The animals were then followed up for 4 months and functional, electrophysiological, and histological examinations were performed.

Methods

Experiments were approved by the local animal care committee and were performed by authorized investigators in accordance with French law.

Facial Nerve Injury and Reconstruction

Thirty-four male Fisher-344 rats (Charles River, France) 6 weeks of age were used as experimental (n = 12), control (n = 12), intact (n = 4), and donor (n = 6) animals in this study. Rats underwent general anesthesia through an intraperitoneal injection of pentobarbital (72 mg/kg) for surgical intervention. For FN injury, the right FN was exposed under a surgical microscope (Leica M650). After the recording of muscle action potentials (MAPs) in the right whisker pad in response to FN electrostimulation using a Myto electromyography unit (EB-Neuro), we used microforceps to crush-injure the FN; this was done by sectioning all of the axons except the nerve's perineurium at the site close to the nerve's emergence from the stylomastoid foramen but distal to its posterior auricular branch, resulting in the total degeneration of the distal FN (Fig. 1A and B). The injury site was then ligated with 4-0 nylon sutures. In preliminary studies, this intervention resulted in spontaneous but limited axonal regeneration. The surgical wound was closed in layers with 4-0 nylon sutures, and amitriptyline (15 mg/kg/day) was added to the rats' drinking water for 2 weeks to reduce their neuropathic pain. Nine weeks later, MAPs were recorded again. MAP surface values that were reduced to approximately 10%–20% of their initial preinjury values qualified as persistent IFP. Rats with IFP underwent no repair surgery as controls (IFP rats, n = 6), representing spontaneous incomplete regeneration, or were subjected to HN-FN “side”-to-side neurorrhaphy using a 10-mm PNG (IFP-R rats, n = 12). Their right HN was exposed beneath the digastric muscle, and about 50% of its axons were cross-sectioned. One end of a PNG was anastomosed end-to-“side” to the HN at the partial injury site using 10-0 nylon microsutures. The other end of the PNG was anastomosed end-to-“side” to the distal stump of the injured FN through a window where only the epineurium was removed (Fig. 1C).

Fig. 1.
Fig. 1.

Schematic drawings showing surgical procedures for FN injury and repair in the rat. A: The right FN and HN were exposed. B: The FN was crushed by sectioning all of the its axons except the perineurium at the site close to the nerve's emergence from the stylomastoid foramen but distal to its posterior auricular branch; the nerve was then ligated using 4-0 sutures at the injury site, resulting in persistent IFP. C: Repair by HN-FN “side”-to-side neurorrhaphy was performed by placing a PNG. One end of the PNG was bridged end-to-“side” to the HN at the site where approximately 50% of axons were cross-sectioned, and the other end of the PNG was bridged end-to-“side” to the distal FN through a window where only the epineurium was removed. The site proximal to the bridged PNG is indicated at the FN.

Depending on the suitability of their diameter for the neurorrhaphy with HNs and FNs,36 PNGs were prepared from peroneal nerves of donor Fisher-344 rats (n = 6), Briefly, peroneal nerves were exposed and sectioned as close as possible to their origin from the sciatic nerve, leading to distal axonal degeneration and Schwann cell proliferation. One week later, the distal part of the lesioned peroneal nerve (10 mm long) was removed and used as a PNG, providing an environment supporting axonal regeneration.6,19 Because the Fisher-344 rats are syngeneic, the PNGs were considered as “autografts,” and no signs of immune responses or rejection were found in recipient rats.

For comparison with CFP, an additional control group of rats (n = 6) underwent sectioning of the FN followed by a double ligature, preventing spontaneous regeneration. CFP rats received no surgical repair.36

The progress of all animals was followed for a period of 4 months, from the time of 9 weeks after FN injury. At the end of the follow-up period, facial symmetry analysis and electrophysiological monitoring were performed before and after sectioning the partially regenerated right FN of IFP rats and either the right FN proximal to the neurorrhaphy site or the PNG of IFP-R rats. The delay between the 2 examinations was 1 week. Three days before the electrophysiological examination prior to sectioning of the FN or PNG, neuronal tracers were injected into the rats for subsequent retrograde labeling studies. The rats were euthanized with an overdose of pentobarbital (120 mg/kg) so that histological examination could be conducted after the last electrophysiological measure. IFP-R rats that underwent either FN or PNG cutting are presented as independent groups throughout the experiment: “IFP-R cut FN” and “IFP-R cut PNG” (n = 6 each). We also used 4 intact rats for histological examination to obtain normal data on retrograde labeling as well as optic and electron microscope studies.

Behavioral Testing

Each rat was photographed before and after surgery, and the Angle α between a line extending from the fold on the bridge of the nose and a line linking the outer corners of each of the eyes was measured (Fig. 2A).36 Measuring Angle α allows determination of changes in facial resting-tone symmetry. Before and after surgery video recording was performed to observe behaviors and synkinesis during eating and drinking. Body weight was recorded to assess the HN function related to feeding after surgery. All photographing, video recording, and analysis were performed in a blind manner. To assess which nerve pathways contributed to the recovery of facial symmetry at the end of the follow-up period, the PNG or the FN proximal to the bridged PNG was sectioned, and Angle α was measured again. The measurements of Angle α in the posttraumatic period were determined 1 week after injuring the FN at the initial surgery or sectioning the PNG or FN at the end of follow-up period to allow the rats recovery from surgery and anesthesia.

Fig. 2.
Fig. 2.

Facial symmetry was assessed by measuring the Angle α. A: Angle α is the angle created between the vertical line prolonging the fold on the bridge of the nose and the horizontal line linking the outer corners of the eyes. B: Measurements of Angle α prior to injury, 1 week after injury, 9 weeks after injury but prior to repair surgery, 4 months after repair surgery but before cutting the FN or the PNG, and 1 week after the cutting of either the FN or PNG. Results are presented as mean ± SEM and were analyzed by 2-way ANOVA: (time × treatment) [overall effect F(12, 70) = 17.8, p < 0.0001; effect of time F(4, 70) = 1598, p < 0.0001; and effect of treatment F(3, 70) = 52.6, p < 0.0001]. Post hoc multiple comparisons between groups (n = 4 to 5) were conducted using Newman-Keuls tests. **p < 0.01 compared to groups with mean values at or below the dotted line or as specifically indicated.

Electrophysiological Examination

We recorded MAPs using electromyography simultaneously at the right whisker pad and the hemitongue muscles during direct electrostimulation of either the FN or HN trunk or the PNG. Stimulation (0.1 msec, 0.3 mA) was delivered through 2 0.5-mm-diameter electrodes directly placed onto the injured FN, the PNG, or the HN proximal to the neurorrhaphy site. The MAPs were recorded by 2 other 0.5-mm-diameter electrodes inserted into the right whisker pad and the hemitongue. The amplitudes between the largest positive and negative peaks and the surface beneath the slope were measured. The latency was not measured because of its lack of reliability for such a short conduction distance. After 4 months of recovery following nerve repair, the PNG or the FN proximal to the bridged PNG was sectioned, and the electrostimulation was then immediately performed again to check which nerve pathway was responsible for active MAPs.

Retrograde Labeling Study

At the end of the 4-month follow-up period, retrograde labeling with fluorescent tracers was performed in rats to detect regenerated motor neurons in the related hypoglossal and facial nuclei. Rats were reanesthetized and 20 μ l of 1% cholera toxin subunit B conjugated with Alexa Fluor 555 (CTB–Alexa 555) was injected into the right whisker pad at multiple points, while 10 μ l of 2% diamidino yellow (DY) was injected into the right hemitongue. Ten days later, rats were killed by intraperitoneal overdose injection of pentobarbital (120 mg/kg) and were perfused with 300 ml phosphate-buffered saline (0.1 M, pH 7) followed by 300 ml 4% paraformaldehyde. The brainstem was removed and postfixed in the paraformaldehyde fixative solution for 3 hours. The specimens were then immersed in 30% sucrose at 4° C for cryopreservation. Cross-sections (30 μ m) were cut with a freezing microtome. Using a Zeiss AxioPlan 2 imaging optic fluorescent microscope (200 M), labeled neurons were identified and counted in all sections covering the facial and hypoglossal nuclei.

Optic and Electron Microscope Analysis

Axonal elongation in the PNG and FN was assessed by optical and electron microscopy. The PNG and FN in each animal were removed at the end of the follow-up period and fixed in 3.6% glutaraldehyde for 3 hours. The specimens were postfixed with osmium tetroxide and then embedded in Epon. Semi-thin (0.35-μ m) and ultra-thin (0.07-μ m) cross-sections were acquired using an ultra-microtome (Reichert Ultracut S Wild M3z, Leica) and stained with thionin or uranyl acetate and lead citrate, respectively. Semi-thin sections were examined under an optic microscope, and ultra-thin sections were analyzed with a 1200ExII transmission electron microscope (JEOL).

Statistical Analysis

Group differences were analyzed using 2-way or 1-way ANOVA followed by Newman-Keuls post hoc tests. Data are presented as mean ± SEM. Statistica 64 software, version 10 (StatSoft) was used.

Results

Recovery of Facial Symmetry After HN-FN “Side”-to-Side Neurorrhaphy

Changes in facial symmetry were analyzed by measuring Angle α (Fig. 2A). This angle corresponded to an average of 90° in intact rats, whereas it decreased to about 71° 1 week after FN injury in rats with persistent IFP or CFP (Fig. 2B). It then remained unchanged in CFP rats until the end of the follow-up period; however, at 9 weeks postinjury, a slight recovery was observed in IFP rats (average angle 74°), indicating spontaneous regeneration and partial reinnervation by FN axons. In the absence of repair surgery (IFP rats), Angle α remained at an average of 74°. However, significantly higher values (p < 0.01), ranging from 76° to 80°, were observed 4 months after the HN-FN “side”-to-side neurorrhaphy using a PNG (IFP-R rats) (Fig. 2B).

When the PNG was sectioned in IFP-R rats at the end of the follow-up period (IFP-R cut PNG), the Angle α that was measured after 1 week decreased to the values observed before HN-FN neurorrhaphy was performed. When the FN was cut in IFP-R rats proximal to the neurorrhaphy site (IFP-R cut FN), the average Angle α decreased to 76°. In IFP rats that did not undergo repair surgery, Angle α decreased to about 71° after sectioning of the FN (Fig. 2B). Video recordings show normal eating and drinking in both IFP-R and IFP rats after surgery. The rats maintained a normal body weight, and there was no evident synkinesis while eating and drinking (Video 1).

Video 1. In an IFP-R rat 4 months after repair surgery, we can see that the rat was eating normally without evident synkinesis in the right side of the face. Copyright Song Liu. Published with permission. Click here to view with Media Player. Click here to view with Quicktime.

Nerve Conduction in the Reconstructed Pathways

To assess functional target innervation, MAPs were recorded in the whisker pad. In response to electrostimulation of the FN before injury, MAPs exhibited values of 6.97 ± 1.14 mV for the amplitude and 6.05 ± 0.96 mVmsec (millivolts times milliseconds) for the surface (Fig. 3). They disappeared completely after FN injury and were not recordable in CFP rats at any time after injury. However, 9 weeks after injury, MAPs could be again recorded (0.9 ± 0.54 mV for the amplitude and 1.11 ± 0.5 mVmsec for the surface) in IFP rats, indicating spontaneous reinnervation. The MAP values increased to 3.29 ± 0.8 mV for the amplitude and 3.45 ± 0.71 mVmsec for the surface in IFP-R rats 4 months after the HN-FN neurorrhaphy (p < 0.01), whereas they remained unchanged in IFP rats in the absence of repair surgery (Fig. 3).

Fig. 3.
Fig. 3.

Muscle action potentials were recorded from the right whisker pad during direct electrostimulation of the FN. Surface of MAPs are expressed in millivolts times milliseconds (mVmsec) and were measured prior to injury, 1 week after injury, 9 weeks after injury prior to repair surgery, 4 months after repair surgery before cutting the FN or the PNG, and 1 week after the cutting of either the FN or PNG. Results are presented as mean ± SEM and were analyzed by 2-way ANOVA: (time x treatment) [overall effect F(12, 74) = 6.67, p < 0.0001; effect of time F(4, 74) = 270, p < 0.0001; and effect of treatment F(3, 74) = 21.7, p < 0.0001]. Post hoc multiple comparisons between groups (n = 4–6) were conducted by Newman-Keuls tests. **p < 0.01 compared to groups with mean values at or below the dotted line.

Muscle action potentials could also be recorded in the whisker pad of IFP-R rats when the PNG was directly stimulated (amplitude 1.52 ± 0.47 mV, surface 1.63 ± 0.39 mVmsec). Importantly, evident muscle contraction was observed in the right whisker pad but not in the ipsilateral hemitongue when the PNG was intensely stimulated, indicating that the hypoglossal axons growing through the PNG selectively innervated facial muscles (Video 2).

Video 2. In an IFP-R rat 4 months after repair surgery, muscle contraction is evident in the right whisker pad while the PNG is intensely stimulated. At the same time, muscle contraction of the ipsilateral hemitongue is not observed, indicating that the hypoglossal axons growing through the PNG selectively innervated the facial muscles. Copyright Song Liu. Published with permission. Click here to view with Media Player. Click here to view with Quicktime.

Sectioning of the PNG at 4 months after repair surgery (IFP-R cut PNG rats) resulted after 1 week in a significant decrease in whisker pad MAPs in response to FN electrostimulation (amplitude 1.36 ± 0.56 mV, surface 1.38 ± 0.45 mVmsec) (Fig. 3). Muscle action potentials also decreased when the FN was sectioned proximal to the neurorrhaphy site in IFP-R rats (that is, the IFP-R cut FN; amplitude 1.69 ± 0.66 mV, surface 1.88 ± 0.61 mVmsec). In the absence of repair surgery, MAPs disappeared completely in IFP rats after sectioning of the FN, indicating that MAPs resulted from the partial regeneration of FN axons.

In IFP-R rats (n = 6) 4 months after partial sectioning of the HN for HN-FN neurorrhaphy, amplitude and surface MAPs recorded in the right hemitongue during electrostimulation of the HN were 2.86 ± 1.56 mV and 3.49 ± 1.61 mVmsec, respectively. These values were decreased to approximately one-half of their original values (amplitude 8.7 ± 2.93 mV and surface 8.42 ± 2.75 mVmsec) before injury. Thus, hypoglossal functions were partially conserved, confirming the results of a previous study.36

Anatomical Connections Between the Regenerated Motor Neurons and the Target Muscles

Retrograde labeling was performed by injecting CTB–Alexa 555 into the right whisker pad and DY into the right hemitongue. In intact rats (n = 4), 927 ± 115 CTB–Alexa 555–labeled neurons were counted in the facial nucleus and 523 ± 44 DY-labeled neurons in the hypoglossal nucleus. In CFP rats (n = 4), no labeled cells were found in the right facial nucleus at the end of the follow-up period, confirming the lack of regeneration in these rats. In IFP rats (n = 5), 303 ± 91 CTB–Alexa 555–labeled neurons were observed in the right facial nucleus, confirming spontaneous partial reinnervation of the whisker pad by regenerated FN axons (Fig. 4). As expected, no labeling of hypoglossal neurons was observed. In contrast, in IFP-R rats (n = 5), 83 ± 48 and 284 ± 80 CTB–Alexa 555–labeled neurons were found, respectively, in the right hypoglossal and facial nuclei 4 months after HN-FN neurorrhaphy, demonstrating successful double innervation of the whisker pad by both facial and hypoglossal axons (Fig. 4). Consistent with a previous study36 and with a partial preservation of hypoglossal functions, 177 ± 30 DY-stained neurons were observed in the hypoglossal nucleus (n = 5). Interestingly, we also found 14 ± 5 DY-labeled neurons in the facial nucleus, suggesting that a small number of regenerated facial axons may have innervated the ipsilateral hemitongue.

Fig. 4.
Fig. 4.

A: Representative photomicrographs of retrogradely labeled motor neurons in the hypoglossal nucleus and/or facial nucleus after injection of CTB–Alexa 555 into the right whisker pad and DY into the right hemitongue. Upper row: CTB–Alexa 555–labeled (red) and DY-labeled (green) neurons in the hypoglossal nucleus of an IFP-R rat 4 months after neurorrhaphy. Center row: CTB–Alexa 555–labeled labeled neurons in the facial nucleus of the same IFP-R rat. Few DY-labeled neurons were observed in the ipsilateral facial nucleus. Lower row: CTB–Alexa 555–labeled neurons in the facial nucleus of an IFP rat in the absence of repair surgery, indicating spontaneous reinnervation of the ipsilateral whisker pad by FN axons. Bar = 50 μ m. B: Retrograde CTB–Alexa 555–labeled motor neurons were scored in the sections covering the whole facial or hypoglossal nucleus 4 months after nerve reconstruction. The mean ± SEM of CTB–Alexa 555–labeled neurons of intact (n = 4), CFP (n = 4), IFP (n = 5), and IFP-R (n = 5) rats are shown.

Axonal Regrowth in PNGs and FNs

The presence of myelinated nerve fibers was further analyzed on semi-thin thionin-stained sections of the FN distal to the neurorrhaphy site and of the PNG. In intact rats (n = 4), we observed 3026 ± 141 myelinated axons within the FN (Fig. 5A). In contrast, at the end of the follow-up period, only aspects of degeneration were observed in the FN of CFP rats (n = 4, Fig. 5B). In IFP rats (n = 4), 856 ± 172 myelinated axons were counted in the FN, consistent with the aforementioned observation of partial spontaneous recovery of facial symmetry, nerve conduction, and target muscle reinnervation (Fig. 5C). Importantly, as many as 1472 ± 316 myelinated axons were observed in the FN of IFP-R rats 4 months after HN-FN “side”-to-side neurorrhaphy (n = 4, p < 0.001 [Fig. 5D]). In these IFR-R rats, about 677 ± 84 myelinated nerve fibers crossing the PNG were also observed (Fig. 5E), suggesting that they contributed to nearly one-half of the fibers reinnervating the right whisker pad after repair surgery (Fig. 5F). The presence of both myelinated and nonmyelinated axons in the FN and PNG of IFP-R rats 4 months after repair surgery and their absence in CFP rats was confirmed by electron microscopy (Fig. 6).

Fig. 5.
Fig. 5.

A–E: Representative photomicrographs of myelinated axons on thionin-stained sections of the FN, at a level distal to the neurorrhaphy site, and of the PNG. A: Myelinated axons in the FN of an intact rat. B: Only degenerated fibers were observed in the injured FN of CFP rats. C: Spontaneous regeneration of myelinated axons in the FN of an IFP rat. D: Myelinated axons in the FN of an IFP-R rat 4 months after repair surgery. E: Myelinated axons in the PNG of the same IFP-R rat. Bar = 100 μ m. F: Comparison between the number of myelinated axons in the right FN of intact, CFP, IFP, and IFP-R rats, and in the PNG graft of IFP-R rats (n = 4). Results are presented as mean ± SEM and were analyzed by 1-way ANOVA: [F(4, 15) = 167, p < 0.001] followed by Newman-Keuls post hoc tests. ***p < 0.001.

Fig. 6.
Fig. 6.

Representative electron microscopic images of ultra-thin sections of the FN distal to the neurorrhaphy site or the PNG. A: Absence of axons in the FN of a CFP rat. B: Myelinated and nonmyelinated axons in the FN of an IFP rat. C: Myelinated and nonmyelinated axons in the FN of an IFP-R rat. D: Numerous myelinated and nonmyelinated axons were observed in the PNG at 4 months after repair surgery. Bar = 2 μ m.

Discussion

Progress in microsurgical techniques now allows anatomical preservation of the FN in most cases during cerebellopontine angle surgery. For treating facial palsy after the FN injury, repair surgery is usually only performed in the absence of spontaneous recovery, requiring an important delay, often more than 1.5 years after the trauma.8,32 Unfortunately, such a long waiting period may result in an important and sometimes irreversible atrophy of the paralyzed facial muscles.8,13 Considering the clinical relevance, we developed a rat model of persistent IFP, and we explored a new “side”-to-side neurorrhaphy procedure for preserving the remnant facial axons and/or potential spontaneous regeneration. With such a surgical procedure, there would also be no more need for delaying repair surgery. On the other hand, the early innervation by the HN can prevent the occurrence of irreversible atrophy of the paralyzed facial muscles before spontaneous reinnervation, which has been referred to as the “baby-sitter” effect.27

Experimental studies have shown that axonal regrowth occurs across a supercharged end-to-side4,30 or side-toside23,37 nerve neurorrhaphy. Although this type of neurorrhaphy remains an area of intense scrutiny,10,17,20,35 many studies stress that such a nerve repair relies on injury to the donor nerve.17,24,28,33 Therefore, it is likely that injury to the donor nerve not only determines axonal regeneration from the donor to recipient nerve but also the absolute number of regenerated axons. Because the number of axons that effectively regenerate and the speed of elongation toward their targets constitutes the main factors that influence functional reinnervation,12,18 the cutting of one-half of the HN is probably necessary to provide an adequate source of motor axons. It has indeed been shown that in humans the cross-sectioned area and the number of myelinated axons of the normal FN account for about 61.5% of the area and 73.2% of the axon number in the normal HN.2 As expected, bridging one-half of the HN and the remnant axons of the injured FN with a PNG resulted in the double innervation of the paralyzed whisker pad by hypoglossal and facial motor neurons, leading to improved functional recovery. Successful double innervation was evidenced by the MAPs, the retrograde labeling of muscle innervation, and the analysis of regenerated facial axons and HN fibers growing through the PNG. Importantly, “side”-to-side neurorrhaphy with a PNG did not impair the regenerative capacity of spared facial axons, as approximately the same number of retrogradely labeled neurons was observed in the facial nuclei of IFP and IFP-R rats after injection of CTB–Alexa 555 into the whisker pad. However, the additional innervation of whisker pad muscles by hypoglossal axons regenerating through the PNG significantly improved functional outcomes. It is difficult to correlate the improvement of facial symmetry in rats by measuring the Angle α to grades of the House-Brackmann system in humans. In the present study, we measured the Angle α between a line extending from the fold on the bridge of the nose and a line linking the outer corners of each of the eyes, and found that α was about 90° in intact rats and 71° in controls with complete facial paralysis. Although it is possible to create a scale system in rats based on Angle α measurements, it may not reflect outcome evaluation in humans.

By sectioning either the PNG or the FN in IFP-R and IFP rats at the end of follow-up period, we also investigated which nerve pathway contributed to the recovery of facial symmetry. In IFP rats, sectioning of the FN resulted in the alteration of Angle α, which became comparable to that of CFP rats, and the loss of MAPs, indicating spontaneous functional reinnervation of whisker pad muscles by facial axons. In IFP-R rats, cutting either the PNG or the FN proximal to the neurorrhaphy site led to a deterioration of facial symmetry and a decrease in MAPs, demonstrating that both axons deviated from the hypoglossal and spared facial axons contributed to successful functional reinnervation. We assume that even more functional benefits may be obtained if the regeneration of hypoglossal axons through the PNG into the FN is further promoted, such as could occur using a PNG expressing NT-3 cDNA through lentiviral vector transduction.36

In the present study, we performed nerve crush injury by sectioning all of the FN axons except the nerve's perineurium and then ligated the injury site with 4-0 nylon sutures, which resulted in IFP after the spontaneous regeneration of part of the injured FN fibers. This method was assessed in our preliminary and present studies, in which the FN injury was standardized and nearly 20%–30% of the facial motor neurons spontaneously regenerated and reinnervated the FN, as evidenced by CTB–Alexa 555 retrograde labeling and MAP recording. However, in a clinical situation, it is difficult to precisely evaluate the FN damage, particularly for those patients whose FN is anatomically preserved. Progress in microsurgical techniques now allows us to anatomically preserve the FN in most cases during cerebellopontine angle surgery. Therefore, one of our objectives was to also test HN-FN “side”-to-side neurorrhaphy. Because the “side”-to-side neurorrhaphy at the injured FN is achieved through a window where only the epineurium is removed, this intervention does not interrupt the main structure of the FN at the neurorrhaphy site, thereby retaining the remnants of facial axons and the potential for spontaneous reinnervation. We hope this method will also be of interest for the treatment of other FN injuries, such as ischemia, stretch, thermal, and so on.

Surgical creation of new neural networks using end-to-side neurorrhaphy between a donor and a recipient nerve can give rise to synkinesis. However, we did not find any evidence of synkinesis in our IFP-R rats. This is consistent with the observation that the incidence of synkinesis is reduced after facial rehabilitation surgery if facial muscles are not only innervated by transposed hypoglossal axons but also by remnant facial axons.14 Hypoglossal neurons project axons to the facial nucleus at the level of the brainstem, and neurons of the parvocellular reticular nucleus innervate both facial and hypoglossal nuclei. These connections may explain coordinated movements of the tongue and facial muscles while swallowing and vocalizing.9,34 The shared innervation of facial and hypoglossal nuclei within the brainstem may prevent synkinesis of the doubly innervated facial muscles.1,7 Efficient facial network reconstruction should thus take into account existing connections within the brain. We assume that a conscious use of facial muscles may be regained after appropriate training during rehabilitation.11,29

Conclusions

This study shows that HN-FN “side”-to-side neurorrhaphy through a PNG can effectively treat persistent IFP in adult rats. The double innervation of the paralyzed whisker pad by such a neurorrhaphy could lead to rapid functional benefits without compromising the remnants of the facial axons, suggesting its potential application to treat persistent IFP in humans.

Disclosure

This work was supported by grants from Beijing Tiantan Hospital Affiliated to Capital Medical University and Beijing Neurosurgical Institute (Beijing, China); the Fondation de l'Avenir (Paris, France); and the Institut pour la Recherche sur la Moelle épinière et l'Encéphale (IRME) (Paris, France).

Author contributions to the study and manuscript preparation include the following. Conception and design: Liu, Wan, Zhang. Acquisition of data: Liu, Wan, Zhang, Li, Hao, Feng. Analysis and interpretation of data: Liu, Wan, Zhang. Drafting the article: Liu. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Liu. Statistical analysis: Schumacher. Administrative/technical/material support: Liu, Wan, Zhang, Li, Hao, Feng, Oudinet. Study supervision: Liu, Wan, Zhang, Schumacher.

References

  • 1

    Asaoka KSawamura Y: Hypoglossal-facial nerve side-to-end anastomosis. J Neurosurg 91:1631641999. (Letter)

  • 2

    Asaoka KSawamura YNagashima MFukushima T: Surgical anatomy for direct hypoglossal-facial nerve side-to-end “anastomosis”. J Neurosurg 91:2682751999

  • 3

    Atlas MDLowinger DSG: A new technique for hypoglossal-facial nerve repair. Laryngoscope 107:9849911997

  • 4

    Barbour JYee AKahn LCMackinnon SE: Supercharged end-to-side anterior interosseous to ulnar motor nerve transfer for intrinsic musculature reinnervation. J Hand Surg Am 37:215021592012

  • 5

    Conley JBaker DC: Hypoglossal-facial nerve anastomosis for reinnervation of the paralyzed face. Plast Reconstr Surg 63:63721979

  • 6

    Danielsen NKerns JMHolmquist BZhao QLundborg GKanje M: Pre-degenerated nerve grafts enhance regeneration by shortening the initial delay period. Brain Res 666:2502541994

  • 7

    Darrouzet VGuerin JBébéar JP: New technique of side-to-end hypoglossal-facial nerve attachment with translocation of the infratemporal facial nerve. J Neurosurg 90:27341999

  • 8

    Fenton JEChin RYKShirazi AFagan PA: Prediction of postoperative facial nerve function in acoustic neuroma surgery. Clin Otolaryngol Allied Sci 24:4834861999

  • 9

    Fernandez ELauretti LDenaro LMontano NDoglietto FNovegno F: Motoneurons innervating facial muscles after hypoglossal and hemihypoglossal-facial nerve anastomosis in rats. Neurol Res 26:3954002004

  • 10

    Fernandez ELauretti LTufo TD'Ercole MCiampini ADoglietto F: End-to-side nerve neurorrhaphy: critical appraisal of experimental and clinical data. Acta Neurochir Suppl 100:77842007

  • 11

    Fernandez EPallini RPalma PLauretti L: Hypoglossal-facial nerve anastomosis. J Neurosurg 87:6496521997. (Letter)

  • 12

    Fu SYGordon T: The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol 14:671161997

  • 13

    Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation. J Neurosci 15:388638951995

  • 14

    Furukawa HSaito AMol WSekido MSasaki SYamamoto Y: Double innervation occurs in the facial mimetic muscles after facial-hypoglossal end-to-side neural repair: rat model for neural supercharge concept. J Plast Reconstr Aesthet Surg 61:2572642008

  • 15

    Gharabaghi AHeckl SKaminsky JTorka WNägele TTatagiba M: [Cranial nerve deficits caused by uncommon skull base lesions of the cavernous sinus.]. HNO 55:2782802007. (Ger)

  • 16

    Hammerschlag PE: Facial reanimation with jump interpositional graft hypoglossal facial anastomosis and hypoglossal facial anastomosis: evolution in management of facial paralysis. Laryngoscope 109:2 Pt 2 Suppl 901231999

  • 17

    Hayashi APannucci CMoradzadeh AKawamura DMagill CHunter DA: Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy. Exp Neurol 211:5395502008

  • 18

    Höke A: Mechanisms of disease: what factors limit the success of peripheral nerve regeneration in humans?. Nat Clin Pract Neurol 2:4484542006

  • 19

    Kerns JMDanielsen NHolmquist BKanje MLundborg G: The influence of predegeneration on regeneration through peripheral nerve grafts in the rat. Exp Neurol 122:28361993

  • 20

    Kettle SJStarritt NEGlasby MAHems TE: End-to-side nerve repair in a large animal model: how does it compare with conventional methods of nerve repair?. J Hand Surg Eur Vol 38:1922022013

  • 21

    Kiese-Himmel CLaskawi RWrede S: [Psychosocial problems and coping with illness by patients with defective healing after facial paralysis.]. HNO 41:2612671993. (Ger)

  • 22

    Kunihiro THigashino KKanzaki J: Classic hypoglossal-facial nerve anastomosis after acoustic neuroma resection. A review of 46 cases ORL. J Otorhinolaryngol Relat Spec 65:162003

  • 23

    Ladak ASchembri POlson JUdina ETyreman NGordon T: Side-to-side nerve grafts sustain chronically denervated peripheral nerve pathways during axon regeneration and result in improved functional reinnervation. Neurosurgery 68:165416662011

  • 24

    Lykissas MG: Current concepts in end-to-side neurorrhaphy. World J Orthop 2:1021062011

  • 25

    Manni JJBeurskens CHvan de Velde CStokroos RJ: Reanimation of the paralyzed face by indirect hypoglossal-facial nerve anastomosis. Am J Surg 182:2682732001

  • 26

    May MSobol SMMester SJ: Hypoglossal-facial nerve interpositional-jump graft for facial reanimation without tongue atrophy. Otolaryngol Head Neck Surg 104:8188251991

  • 27

    Mersa BTiangco DATerzis JK: Efficacy of the “baby-sitter” procedure after prolonged denervation. J Reconstr Microsurg 16:27352000

  • 28

    Pannucci CMyckatyn TMMackinnon SEHayashi A: End-to-side nerve repair: review of the literature. Restor Neurol Neurosci 25:45632007

  • 29

    Pitty LFTator CH: Hypoglossal-facial nerve anastomosis for facial nerve palsy following surgery for cerebellopontine angle tumors. J Neurosurg 77:7247311992

  • 30

    Sherif MMAmr AH: Intrinsic hand muscle reinnervation by median-ulnar end-to-side bridge nerve graft: case report. J Hand Surg Am 35:4464502010

  • 31

    Sood SAnthony RHomer JJVan Hille PFenwick JD: Hypoglossal-facial nerve anastomosis: assessment of clinical results and patient benefit for facial nerve palsy following acoustic neuroma excision. Clin Otolaryngol Allied Sci 25:2192262000

  • 32

    Stennert E: I. Hypoglossal facial anastomosis: its significance for modern facial surgery. II. Combined approach in extra-temporal facial nerve reconstruction. Clin Plast Surg 6:4714861979

  • 33

    Stipp-Brambilla EJViterbo FLabbé DGarbino JABernardelli MM: Double muscle innervation using end-to-side neurorrhaphy in rats. Sao Paulo Med J 130:3733792012

  • 34

    Streppel MPopratiloff AGruart AAngelov DNGuntinas-Lichius ODelgado-Garcia JM: [Morphological connections between the Hypoglassal and facial nerve in the brain stem of the rat.]. HNO 48:9119162000. (Ger)

  • 35

    Tos PArtiaco SPapalia IMarcoccio IGeuna SBattiston B: Chapter 14: End-to-side nerve regeneration: from the laboratory bench to clinical applications. Int Rev Neurobiol 87:2812942009

  • 36

    Wan HZhang LBlanchard SBigou SBohl DWang C: Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy. Laboratory investigation. J Neurosurg 119:7397502013

  • 37

    Yüksel FKaracaoğlu EGüler MM: Nerve regeneration through side-to-side neurorrhaphy sites in a rat model: a new concept in peripheral nerve surgery. Plast Reconstr Surg 104:209220991999

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Article Information

Address correspondence to: Song Liu, M.D., Ph.D., UMR 788, INSERM et Université Paris-Sud, 80 rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France. email: song.liu@inserm.fr.

Drs. Wan and Zhang contributed equally to this work.

Please include this information when citing this paper: published online November 8, 2013; DOI: 10.3171/2013.9.JNS13664.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Schematic drawings showing surgical procedures for FN injury and repair in the rat. A: The right FN and HN were exposed. B: The FN was crushed by sectioning all of the its axons except the perineurium at the site close to the nerve's emergence from the stylomastoid foramen but distal to its posterior auricular branch; the nerve was then ligated using 4-0 sutures at the injury site, resulting in persistent IFP. C: Repair by HN-FN “side”-to-side neurorrhaphy was performed by placing a PNG. One end of the PNG was bridged end-to-“side” to the HN at the site where approximately 50% of axons were cross-sectioned, and the other end of the PNG was bridged end-to-“side” to the distal FN through a window where only the epineurium was removed. The site proximal to the bridged PNG is indicated at the FN.

  • View in gallery

    Facial symmetry was assessed by measuring the Angle α. A: Angle α is the angle created between the vertical line prolonging the fold on the bridge of the nose and the horizontal line linking the outer corners of the eyes. B: Measurements of Angle α prior to injury, 1 week after injury, 9 weeks after injury but prior to repair surgery, 4 months after repair surgery but before cutting the FN or the PNG, and 1 week after the cutting of either the FN or PNG. Results are presented as mean ± SEM and were analyzed by 2-way ANOVA: (time × treatment) [overall effect F(12, 70) = 17.8, p < 0.0001; effect of time F(4, 70) = 1598, p < 0.0001; and effect of treatment F(3, 70) = 52.6, p < 0.0001]. Post hoc multiple comparisons between groups (n = 4 to 5) were conducted using Newman-Keuls tests. **p < 0.01 compared to groups with mean values at or below the dotted line or as specifically indicated.

  • View in gallery

    Muscle action potentials were recorded from the right whisker pad during direct electrostimulation of the FN. Surface of MAPs are expressed in millivolts times milliseconds (mVmsec) and were measured prior to injury, 1 week after injury, 9 weeks after injury prior to repair surgery, 4 months after repair surgery before cutting the FN or the PNG, and 1 week after the cutting of either the FN or PNG. Results are presented as mean ± SEM and were analyzed by 2-way ANOVA: (time x treatment) [overall effect F(12, 74) = 6.67, p < 0.0001; effect of time F(4, 74) = 270, p < 0.0001; and effect of treatment F(3, 74) = 21.7, p < 0.0001]. Post hoc multiple comparisons between groups (n = 4–6) were conducted by Newman-Keuls tests. **p < 0.01 compared to groups with mean values at or below the dotted line.

  • View in gallery

    A: Representative photomicrographs of retrogradely labeled motor neurons in the hypoglossal nucleus and/or facial nucleus after injection of CTB–Alexa 555 into the right whisker pad and DY into the right hemitongue. Upper row: CTB–Alexa 555–labeled (red) and DY-labeled (green) neurons in the hypoglossal nucleus of an IFP-R rat 4 months after neurorrhaphy. Center row: CTB–Alexa 555–labeled labeled neurons in the facial nucleus of the same IFP-R rat. Few DY-labeled neurons were observed in the ipsilateral facial nucleus. Lower row: CTB–Alexa 555–labeled neurons in the facial nucleus of an IFP rat in the absence of repair surgery, indicating spontaneous reinnervation of the ipsilateral whisker pad by FN axons. Bar = 50 μ m. B: Retrograde CTB–Alexa 555–labeled motor neurons were scored in the sections covering the whole facial or hypoglossal nucleus 4 months after nerve reconstruction. The mean ± SEM of CTB–Alexa 555–labeled neurons of intact (n = 4), CFP (n = 4), IFP (n = 5), and IFP-R (n = 5) rats are shown.

  • View in gallery

    A–E: Representative photomicrographs of myelinated axons on thionin-stained sections of the FN, at a level distal to the neurorrhaphy site, and of the PNG. A: Myelinated axons in the FN of an intact rat. B: Only degenerated fibers were observed in the injured FN of CFP rats. C: Spontaneous regeneration of myelinated axons in the FN of an IFP rat. D: Myelinated axons in the FN of an IFP-R rat 4 months after repair surgery. E: Myelinated axons in the PNG of the same IFP-R rat. Bar = 100 μ m. F: Comparison between the number of myelinated axons in the right FN of intact, CFP, IFP, and IFP-R rats, and in the PNG graft of IFP-R rats (n = 4). Results are presented as mean ± SEM and were analyzed by 1-way ANOVA: [F(4, 15) = 167, p < 0.001] followed by Newman-Keuls post hoc tests. ***p < 0.001.

  • View in gallery

    Representative electron microscopic images of ultra-thin sections of the FN distal to the neurorrhaphy site or the PNG. A: Absence of axons in the FN of a CFP rat. B: Myelinated and nonmyelinated axons in the FN of an IFP rat. C: Myelinated and nonmyelinated axons in the FN of an IFP-R rat. D: Numerous myelinated and nonmyelinated axons were observed in the PNG at 4 months after repair surgery. Bar = 2 μ m.

References

1

Asaoka KSawamura Y: Hypoglossal-facial nerve side-to-end anastomosis. J Neurosurg 91:1631641999. (Letter)

2

Asaoka KSawamura YNagashima MFukushima T: Surgical anatomy for direct hypoglossal-facial nerve side-to-end “anastomosis”. J Neurosurg 91:2682751999

3

Atlas MDLowinger DSG: A new technique for hypoglossal-facial nerve repair. Laryngoscope 107:9849911997

4

Barbour JYee AKahn LCMackinnon SE: Supercharged end-to-side anterior interosseous to ulnar motor nerve transfer for intrinsic musculature reinnervation. J Hand Surg Am 37:215021592012

5

Conley JBaker DC: Hypoglossal-facial nerve anastomosis for reinnervation of the paralyzed face. Plast Reconstr Surg 63:63721979

6

Danielsen NKerns JMHolmquist BZhao QLundborg GKanje M: Pre-degenerated nerve grafts enhance regeneration by shortening the initial delay period. Brain Res 666:2502541994

7

Darrouzet VGuerin JBébéar JP: New technique of side-to-end hypoglossal-facial nerve attachment with translocation of the infratemporal facial nerve. J Neurosurg 90:27341999

8

Fenton JEChin RYKShirazi AFagan PA: Prediction of postoperative facial nerve function in acoustic neuroma surgery. Clin Otolaryngol Allied Sci 24:4834861999

9

Fernandez ELauretti LDenaro LMontano NDoglietto FNovegno F: Motoneurons innervating facial muscles after hypoglossal and hemihypoglossal-facial nerve anastomosis in rats. Neurol Res 26:3954002004

10

Fernandez ELauretti LTufo TD'Ercole MCiampini ADoglietto F: End-to-side nerve neurorrhaphy: critical appraisal of experimental and clinical data. Acta Neurochir Suppl 100:77842007

11

Fernandez EPallini RPalma PLauretti L: Hypoglossal-facial nerve anastomosis. J Neurosurg 87:6496521997. (Letter)

12

Fu SYGordon T: The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol 14:671161997

13

Fu SYGordon T: Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation. J Neurosci 15:388638951995

14

Furukawa HSaito AMol WSekido MSasaki SYamamoto Y: Double innervation occurs in the facial mimetic muscles after facial-hypoglossal end-to-side neural repair: rat model for neural supercharge concept. J Plast Reconstr Aesthet Surg 61:2572642008

15

Gharabaghi AHeckl SKaminsky JTorka WNägele TTatagiba M: [Cranial nerve deficits caused by uncommon skull base lesions of the cavernous sinus.]. HNO 55:2782802007. (Ger)

16

Hammerschlag PE: Facial reanimation with jump interpositional graft hypoglossal facial anastomosis and hypoglossal facial anastomosis: evolution in management of facial paralysis. Laryngoscope 109:2 Pt 2 Suppl 901231999

17

Hayashi APannucci CMoradzadeh AKawamura DMagill CHunter DA: Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy. Exp Neurol 211:5395502008

18

Höke A: Mechanisms of disease: what factors limit the success of peripheral nerve regeneration in humans?. Nat Clin Pract Neurol 2:4484542006

19

Kerns JMDanielsen NHolmquist BKanje MLundborg G: The influence of predegeneration on regeneration through peripheral nerve grafts in the rat. Exp Neurol 122:28361993

20

Kettle SJStarritt NEGlasby MAHems TE: End-to-side nerve repair in a large animal model: how does it compare with conventional methods of nerve repair?. J Hand Surg Eur Vol 38:1922022013

21

Kiese-Himmel CLaskawi RWrede S: [Psychosocial problems and coping with illness by patients with defective healing after facial paralysis.]. HNO 41:2612671993. (Ger)

22

Kunihiro THigashino KKanzaki J: Classic hypoglossal-facial nerve anastomosis after acoustic neuroma resection. A review of 46 cases ORL. J Otorhinolaryngol Relat Spec 65:162003

23

Ladak ASchembri POlson JUdina ETyreman NGordon T: Side-to-side nerve grafts sustain chronically denervated peripheral nerve pathways during axon regeneration and result in improved functional reinnervation. Neurosurgery 68:165416662011

24

Lykissas MG: Current concepts in end-to-side neurorrhaphy. World J Orthop 2:1021062011

25

Manni JJBeurskens CHvan de Velde CStokroos RJ: Reanimation of the paralyzed face by indirect hypoglossal-facial nerve anastomosis. Am J Surg 182:2682732001

26

May MSobol SMMester SJ: Hypoglossal-facial nerve interpositional-jump graft for facial reanimation without tongue atrophy. Otolaryngol Head Neck Surg 104:8188251991

27

Mersa BTiangco DATerzis JK: Efficacy of the “baby-sitter” procedure after prolonged denervation. J Reconstr Microsurg 16:27352000

28

Pannucci CMyckatyn TMMackinnon SEHayashi A: End-to-side nerve repair: review of the literature. Restor Neurol Neurosci 25:45632007

29

Pitty LFTator CH: Hypoglossal-facial nerve anastomosis for facial nerve palsy following surgery for cerebellopontine angle tumors. J Neurosurg 77:7247311992

30

Sherif MMAmr AH: Intrinsic hand muscle reinnervation by median-ulnar end-to-side bridge nerve graft: case report. J Hand Surg Am 35:4464502010

31

Sood SAnthony RHomer JJVan Hille PFenwick JD: Hypoglossal-facial nerve anastomosis: assessment of clinical results and patient benefit for facial nerve palsy following acoustic neuroma excision. Clin Otolaryngol Allied Sci 25:2192262000

32

Stennert E: I. Hypoglossal facial anastomosis: its significance for modern facial surgery. II. Combined approach in extra-temporal facial nerve reconstruction. Clin Plast Surg 6:4714861979

33

Stipp-Brambilla EJViterbo FLabbé DGarbino JABernardelli MM: Double muscle innervation using end-to-side neurorrhaphy in rats. Sao Paulo Med J 130:3733792012

34

Streppel MPopratiloff AGruart AAngelov DNGuntinas-Lichius ODelgado-Garcia JM: [Morphological connections between the Hypoglassal and facial nerve in the brain stem of the rat.]. HNO 48:9119162000. (Ger)

35

Tos PArtiaco SPapalia IMarcoccio IGeuna SBattiston B: Chapter 14: End-to-side nerve regeneration: from the laboratory bench to clinical applications. Int Rev Neurobiol 87:2812942009

36

Wan HZhang LBlanchard SBigou SBohl DWang C: Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy. Laboratory investigation. J Neurosurg 119:7397502013

37

Yüksel FKaracaoğlu EGüler MM: Nerve regeneration through side-to-side neurorrhaphy sites in a rat model: a new concept in peripheral nerve surgery. Plast Reconstr Surg 104:209220991999

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