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

Laboratory investigation

Restricted access

Object

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.

Methods

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.

Results

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.

Conclusions

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: song.liu@inserm.fr.

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.

Headings

Figures

  • View in gallery

    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

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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).

  • View in gallery

    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.

References

1

Arai HSato KYanai A: Hemihypoglossal-facial nerve anastomosis in treating unilateral facial palsy after acoustic neurinoma resection. J Neurosurg 82:51541995

2

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

3

Atlas MDLowinger DS: A new technique for hypoglossalfacial nerve repair. Laryngoscope 107:9849911997

4

Baker RSStava MWNelson KRMay PJHuffman MDPorter JD: Aberrant reinnervation of facial musculature in a subhuman primate: a correlative analysis of eyelid kinematics, muscle synkinesis, and motoneuron localization. Neurology 44:216521731994

5

Bascom DASchaitkin BMMay MKlein S: Facial nerve repair: a retrospective review. Facial Plast Surg 16:3093132000

6

Boyd JGGordon T: Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury. Mol Neurobiol 27:2773242003

7

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

8

Dörfl J: The innervation of the mystacial region of the white mouse: a topographical study. J Anat 142:1731841985

9

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

10

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

11

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

12

Geuna SRaimondo SRonchi GDi Scipio FTos PCzaja K: Chapter 3: Histology of the peripheral nerve and changes occurring during nerve regeneration. Int Rev Neurobiol 87:27462009

13

Gordon THegedus JTam SL: Adaptive and maladaptive motor axonal sprouting in aging and motoneuron disease. Neurol Res 26:1741852004

14

Grafstein B: Role of slow axonal transport in nerve regeneration. Acta Neuropathol 5:Suppl 51441521971

15

Guntinas-Lichius O: The facial nerve in the presence of a head and neck neoplasm: assessment and outcome after surgical management. Curr Opin Otolaryngol Head Neck Surg 12:1331412004

16

Guntinas-Lichius OEffenberger KAngelov DNKlein JStreppel MStennert E: Delayed rat facial nerve repair leads to accelerated and enhanced muscle reinnervation with reduced collateral axonal sprouting during a definite denervation period using a cross-anastomosis paradigm. Exp Neurol 162:981112000

17

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

18

Hendriks WTEggers RCarlstedt TPZaldumbide ATannemaat MRFallaux FJ: Lentiviral vector-mediated reporter gene expression in avulsed spinal ventral root is short-term, but is prolonged using an immune “stealth” transgene. Restor Neurol Neurosci 25:5855992007

19

Hendriks WTRuitenberg MJBlits BBoer GJVerhaagen J: Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord. Prog Brain Res 146:4514762004

20

Hoffman PNLasek RJ: Axonal transport of the cytoskeleton in regenerating motor neurons: constancy and change. Brain Res 202:3173331980

21

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

22

Höke AGordon TZochodne DWSulaiman OA: A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation. Exp Neurol 173:77852002

23

Jubran MWidenfalk J: Repair of peripheral nerve transections with fibrin sealant containing neurotrophic factors. Exp Neurol 181:2042122003

24

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

25

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

26

Kunihiro TKanzaki JYoshihara SSatoh YSatoh A: Hypoglossal-facial nerve anastomosis after acoustic neuroma resection: influence of the time anastomosis on recovery of facial movement. ORL J Otorhinolaryngol Relat Spec 58:32351996

27

Liu SBlanchard SBigou SVitry SBohl DHeard JM: Neurotrophin 3 improves delayed reconstruction of sensory pathways after cervical dorsal root injury. Neurosurgery 68:4504612011

28

Lykissas MGBatistatou AKCharalabopoulos KABeris AE: The role of neurotrophins in axonal growth, guidance, and regeneration. Curr Neurovasc Res 4:1431512007

29

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

30

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

31

Pellat JLBonnefille EZanaret MCannoni M: [Hypoglossalfacial anastomosis. A report of 60 cases.]. Ann Chir Plast Esthet 42:37431997. (Fr)

32

Pradat PFKennel PNaimi-Sadaoui SFiniels FOrsini CRevah F: Continuous delivery of neurotrophin 3 by gene therapy has a neuroprotective effect in experimental models of diabetic and acrylamide neuropathies. Hum Gene Ther 12:223722492001

33

Salonen VLehto MVaheri AAro HPeltonen J: Endoneurial fibrosis following nerve transection. An immunohistological study of collagen types and fibronectin in the rat. Acta Neuropathol 67:3153211985

34

Son YJTrachtenberg JTThompson WJ: Schwann cells induce and guide sprouting and reinnervation of neuromuscular junctions. Trends Neurosci 19:2802851996

35

Spector JG: Mimetic surgery for the paralyzed face. Laryngoscope 95:149415221985

36

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

37

Sulaiman OAGordon T: Role of chronic Schwann cell denervation in poor functional recovery after nerve injuries and experimental strategies to combat it. Neurosurgery 65:4 SupplA105A1142009

38

Tannemaat MRBoer GJEggers RMalessy MJVerhaagen J: From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve. Prog Brain Res 175:1731862009

39

Tannemaat MRBoer GJVerhaagen JMalessy MJA: Genetic modification of human sural nerve segments by a lentiviral vector encoding nerve growth factor. Neurosurgery 61:128612962007

40

Tannemaat MREggers RHendriks WTde Ruiter GCWvan Heerikhuize JJPool CW: Differential effects of lentiviral vector-mediated overexpression of nerve growth factor and glial cell line-derived neurotrophic factor on regenerating sensory and motor axons in the transected peripheral nerve. Eur J Neurosci 28:146714792008

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 46 46 18
Full Text Views 120 120 20
PDF Downloads 62 62 8
EPUB Downloads 0 0 0

PubMed

Google Scholar