Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy

Laboratory investigation

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Facial nerve injury results in facial palsy that has great impact on the psychosocial conditions of affected patients. Reconstruction of the facial nerve to restore facial symmetry and expression is still a significant surgical challenge. In this study, the authors assessed a hypoglossal-facial nerve anastomosis method combined with neurotrophic factor gene therapy to treat facial palsy in adult rats after facial nerve injury.


Surgery consisted of the interposition of a predegenerated nerve graft (PNG) that was anastomosed with the hypoglossal and facial nerves at each of its extremities. The hypoglossal nerve was cut approximately 50% for this anastomosis to conserve partial hypoglossal function. Before their transplantation, the PNGs were genetically engineered using lentiviral vectors to induce overexpression of the neurotrophic factor neurotrophin-3 (NT-3) to improve axonal regrowth in the reconstructed nerve pathway. Reconstruction was performed after facial nerve injury, either immediately or after a delay of 9 weeks. The rats were followed up for 4 months postoperatively, and treatment outcomes were then assessed.


Compared with the functional innervation in control rats that underwent facial nerve injury without subsequent treatment, functional innervation of the paralyzed whisker pad by hypoglossal motoneurons in rats treated 4 months after nerve reconstruction was evidenced by the retrograde transport of neuronal tracers, the recording of muscle action potentials conducted by the PNG, and the recovery of facial symmetry. Although a better outcome was observed when reconstruction was performed immediately after facial nerve injury, reconstruction with NT3-treated PNGs significantly improved functional reinnervation of the paralyzed whisker pad even when implantation occurred 9 weeks posttrauma.


Results demonstrated that hypoglossal-facial nerve anastomosis facilitates innervation of paralyzed facial muscle via hypoglossal motoneurons without sacrificing ipsilateral hemitongue function. Neurotrophin-3 treatment through gene therapy could effectively improve such innervation, even after delayed reconstruction. These findings suggest that the combination of surgical reconstruction and NT-3 gene therapy is promising for its potential application in treating facial palsy in humans.

Abbreviations used in this paper:CTB-Alexa555 = cholera toxin subunit B Alexa fluor 555 conjugate; DY = diamidino yellow; FG = Fluoro gold; GFP = green fluorescent protein; LV = lentiviral vector; MAP = muscle action potential; NT = neurotrophin; PFA = paraformaldehyde; PNG = predegenerated nerve graft; RT-PCR = reverse transcription–polymerase chain reaction.

Article Information

Drs. Wan and Zhang contributed equally to this work.

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

Please include this information when citing this paper: published online April 12, 2013; DOI: 10.3171/2013.1.JNS121176.

© AANS, except where prohibited by US copyright law.



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    Drawings and photographs showing surgical procedures of rat facial nerve injury and reconstruction. The right facial (VII n) and hypoglossal (XII n) nerves were exposed under a surgical microscope (a and b). The facial nerve was transected close to its emergence from the stylomastoid foramen but distal to its posterior auricular branch. The sectioned facial nerve was doubly ligated with 6-0 sutures to prevent spontaneous regeneration (c and d). Approximately 50% of axons in the right hypoglossal nerve were cross-sectioned, and hypoglossal-facial anastomosis was performed using a PNG. One end of the PNG was anastomosed end-to-side to the hypoglossal nerve at the site of the partial section. The other end of the PNG was anastomosed to the distal segment of the injured facial nerve (e and f). Adapted with permission from Dörfl: J Anat 142:173–184, 1985.8

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    Efficiency of transduction with the LVs and NT-3 expression in PNGs. a: A GFP signal was observed in the cells of PNG cross-sections. The intensity of the GFP signal was greater in the PNGs 1 week after transduction than 4 months after transplantation. Bars = 100 μm. b: The amounts of NT-3 mRNA were measured by quantitative RT-PCR in tissue extracts prepared from GFP or NT-3 PNGs removed 1 week after transduction or 4 months after transplantation (4 rats per subgroup, or 16 rats total). Values represent the means ± SEM. *p < 0.05, 1-way ANOVA followed by Newman-Keuls post hoc tests.

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    Retrograde labeling of hypoglossal motoneurons with CTB-Alexa555, FG, and DY through the reconstructed nerve pathway. a: Drawing showing that CTB-Alexa555 was injected into the right whisker pad muscle and DY into the ipsilateral hemitongue 4 months after reconstruction. Fluoro-gold was injected into the distal stump of the injured and repaired facial nerve 1 week later. Adapted with permission from Dörfl: J Anat 142:173–184, 1985. b: Retrograde-labeled hypoglossal motoneurons were counted in the sections covering the whole hypoglossal nucleus 4 months after nerve reconstruction. The mean number (± SEM) of neurons labeled with CTB-Alexa555/FG, FG, and DY in the immediate- and delayed-treatment rats with either GFP or NT-3 PNGs are shown (5 rats per subgroup, or 20 rats total). *p < 0.05 and **p < 0.01, 1-way ANOVA followed by Newman-Keuls post hoc tests. c: Representative section of the right hypoglossal nucleus of a delayed-treatment rat with an NT-3 PNG showing motoneurons labeled with CTB-Alexa555 (red), FG (blue), and DY (green). Bars = 50 μm.

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    Axonal regrowth within the PNGs under different conditions. a: There were 627 ± 171 and 938 ± 244 myelinated axons in semithin GFP and NT-3 PNGs, respectively, when transplantation was performed immediately after facial nerve injury and 449 ± 42 and 912 ± 219 when transplantation was performed 9 weeks posttrauma (3 rats per subgroup, or 12 rats total). Data represent the means ± SEM. *p < 0.05 (2-way ANOVA followed by Newman-Keuls post hoc tests). b: Optical microscope images (left column) showing numerous myelinated axons in semithin sections of GFP or NT-3 PNGs of both immediate- and delayed-treatment rats 4 months after reconstruction. Bars = 100 μm. Electron microscopy images (center column) of ultrathin sections from 1 rat from each subgroup demonstrating the presence of myelinated axons in the GFP or NT-3 PNGs as well as their distal anastomosed facial nerves. Bars = 5 μm. Histograms (right column) showing the representative distribution of myelinated axon diameters in the GFP or NT-3 PNGs of both transplantation conditions.

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    Evidence of electrophysiological conduction within the PNG-reconstructed hypoglossal-facial nerve pathway. Muscle action potentials were recorded from the right whisker pad muscle of both immediate- and delayed-treatment rats during electrostimulation of the GFP or NT-3 PNGs 4 months after nerve reconstruction (6 rats per subgroup, or 24 rats total). Amplitude (left) and surface (right) of the MAPs were measured. Data represent the means ± SEM. *p < 0.05, **p < 0.01 (unpaired Student t-test).

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    Improvement in facial symmetry in immediate- and delayed-treatment rats with GFP or NT-3 PNGs. a: Facial symmetry was investigated by comparing the α angle between a line extending from the fold on the bridge of the nose and a line linking the outer corners of the eyes. Measurements were performed in intact rats (8 rats), control rats (6 rats), and rats treated with PNG transplantation either immediately (immediate GFP or NT-3 PNGs; 8 rats for each subgroup, 16 rats total) or 9 weeks after facial nerve injury (delayed GFP or NT-3 PNGs; 8 rats for each subgroup, 16 rats total). The α angle was measured 1 week or 4 months postsurgery. Data represent the means ± SEM. **p < 0.01, ***p < 0.001 (1-way ANOVA followed by Newman-Keuls post hoc tests). b: Representative photographs of a delayed-treatment rat with an NT-3 PNG showing α assessment: α is equal to 88.9° before facial nerve injury (intact) and 71.5° (1 week) or 83.2° (4 months) after injury and reconstruction.



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