To introduce a Thy1-GFP transgenic rat model, whose axons constitutively express green fluorescent protein (GFP), in order to study facial nerve regeneration. Facial nerve injury can cause devastating physical and social sequelae. The functional recovery of the facial nerve can result in synkinesis and permanent axonal misrouting. Facial nerve research has been hindered by the lack of available animal models and reliable outcome measures.
Methods
Transgenic Thy1-GFP rats underwent a proximal facial nerve crush injury and were imaged at 0, 1, 2, 4, and 8 weeks after injury. Nerve regeneration was assessed via confocal imaging and fluorescence microscopy.
Results
Uninjured animals reliably demonstrated facial nerve fluorescence and had predictable anatomical landmarks. Fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. This was confirmed with the visualization of denervation and reinnervation of zygomaticus muscle motor end plates using confocal microscopy.
Conclusions
The Thy1-GFP rat is a novel transgenic tool that enables direct visualization of facial nerve regeneration after injury. The utility of this model extends to a variety of clinical facial nerve injury paradigms.
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References
1.
CrumleyRL. Mechanisms of synkinesis.. Laryngoscope. 1979; 89(11):1847–1854 502707
2.
MayM, SchaitkinB. The Facial Nerve.. 2nd ed. New York, NY: Thieme; 2000
3.
SpectorJG, LeeP, DerbyA, et al.. Facial nerve regeneration through semipermeable chambers in the rabbit.. Laryngoscope. 1992; 102(7):784–796 1319531
4.
SpectorJG, LeeP. Axonal regeneration in severed peripheral facial nerve of the rabbit: relation of the number of axonal regenerates to behavioral and evoked muscle activity.. Ann Otol Rhinol Laryngol. 1998; 107(2):141–148 9486909
HwangK, KimSG, KimDJ. Hypoglossal-facial nerve anastomosis in the rabbits using laser welding.. Ann Plast Surg. 2008; 61(4):452–456 18812720
12.
SinisN, HornF, GenchevB, et al.. Electrical stimulation of paralyzed vibrissal muscles reduces endplate reinnervation and does not promote motor recovery after facial nerve repair in rats.. Ann Anat. 2009; 191(4):356–370 19481914
13.
GuoBF, DongMM. Application of neural stem cells in tissue-engineered artificial nerve.. Otolaryngol Head Neck Surg. 2009; 140(2):159–164 19201281
14.
ZhangH, WeiYT, TsangKS, et al.. Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model.. J Transl Med. 2008; 6:67 18986538
BakerRS, StavaMW, NelsonKR, MayPJ, HuffmanMD, PorterJD. Aberrant reinnervation of facial musculature in a subhuman primate: a correlative analysis of eyelid kinematics, muscle synkinesis, and motoneuron localization.. Neurology. 1994; 44(11):2165–2173 7969978
17.
BaxterA. Dehiscence of the Fallopian canal: an anatomical study.. J Laryngol Otol. 1971; 85(6):587–594 5581361
18.
LarrabeeWFJ, MakielskiKH. Surgical Anatomy of the Face.. New York, NY: Raven Press; 1993
19.
DavisRA, AnsonBJ, BudingerJM, KurthLR. Surgical anatomy of the facial nerve and parotid gland based upon a study of 350 cervicofacial halves.. Surg Gynecol Obstet. 1956; 102(4):385–412 13311719
20.
HunterDA, MoradzadehA, WhitlockEL, et al.. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve.. J Neurosci Methods. 2007; 166(1):116–124 17675163
21.
BrushartTM. Preferential reinnervation of motor nerves by regenerating motor axons.. J Neurosci. 1988; 8(3):1026–1031 3346713
22.
BrushartTM. Preferential motor reinnervation: a sequential double-labeling study.. Restor Neurol Neurosci. 1990; 1:281–287
23.
BrushartTM. Motor axons preferentially reinnervate motor pathways.. J Neurosci. 1993; 13(6):2730–2738 8501535
24.
HayashiA, MoradzadehA, HunterDA, et al.. Retrograde labeling in peripheral nerve research: it is not all black and white.. J Reconstr Microsurg. 2007; 23(7):381–389 17979067
25.
DohmS, StreppelM, Guntinas-LichiusO, et al.. Local application of extracellular matrix proteins fails to reduce the number of axonal branches after varying reconstructive surgery on rat facial nerve.. Restor Neurol Neurosci. 2000; 16(2):117–126 12671214
26.
YoshimuraK, AsatoH, CedernaPS, UrbanchekMG, KuzonWM. The effect of reinnervation on force production and power output in skeletal muscle.. J Surg Res. 1999; 81(2):201–208 9927541
MagillCK, TongA, KawamuraD, et al.. Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice.. Exp Neurol. 2007; 207(1):64–74 17628540
29.
HadlockTA, HeatonJ, CheneyM, MackinnonSE. Functional recovery after facial and sciatic nerve crush injury in the rat.. Arch Facial Plast Surg. 2005; 7(1):17–20 15655169
30.
HeatonJT, KowaleskiJM, BermejoR, ZeiglerHP, AhlgrenDJ, HadlockTA. A system for studying facial nerve function in rats through simultaneous bilateral monitoring of eyelid and whisker movements.. J Neurosci Methods. 2008; 171(2):197–206 18442856
31.
HadlockT, KowaleskiJ, LoD, et al.. Functional assessments of the rodent facial nerve: a synkinesis model.. Laryngoscope. 2008; 118(10):1744–1749 18641527
32.
HadlockT, KowaleskiJ, MackinnonS, HeatonJT. A novel method of head fixation for the study of rodent facial function.. Exp Neurol. 2007; 205(1):279–282 17397835
33.
MehtaRP, ZhangS, HadlockTA. Novel 3-D video for quantification of facial movement.. Otolaryngol Head Neck Surg. 2008; 138(4):468–472 18359356
34.
TomovTL, Guntinas-LichiusO, GroshevaM, et al.. An example of neural plasticity evoked by putative behavioral demand and early use of vibrissal hairs after facial nerve transection.. Exp Neurol. 2002; 178(2):207–218 12504880
35.
SerpeCJ, TetzlaffJE, CoersS, SandersVM, JonesKJ. Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice.. Brain Behav Immun. 2002; 16(6):808–812 12480509
36.
TsienRY. The green fluorescent protein.. Annu Rev Biochem. 1998; 67:509–544 9759496
37.
ShimomuraO, JohnsonFH, SaigaY. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.. J Cell Comp Physiol. 1962; 59:223–239 13911999
38.
ChalfieM, TuY, EuskirchenG, WardWW, PrasherDC. Green fluorescent protein as a marker for gene expression.. Science. 1994; 263(5148):802–805 8303295
39.
ZuoY, LubischerJL, KangH, et al.. Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.. J Neurosci. 2004; 24(49):10999–11009 15590915
40.
MagillC, WhitlockE, SolowskiN, MyckatynT. Transgenic models of nerve repair and nerve regeneration.. Neurol Res. 2008; 30(10):1023–1029 19079976
41.
MignoneJL, KukekovV, ChiangAS, SteindlerD, EnikolopovG. Neural stem and progenitor cells in nestin-GFP transgenic mice.. J Comp Neurol. 2004; 469(3):311–324 14730584
42.
VivesV, AlonsoG, SolalAC, JoubertD, LegraverendC. Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice.. J Comp Neurol. 2003; 457(4):404–419 12561079
43.
FengG, MellorRH, BernsteinM, et al.. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.. Neuron. 2000; 28(1):41–51 11086982
44.
HayashiA, PannucciC, MoradzadehA, et al.. Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy.. Exp Neurol. 2008; 211(2):539–550 18433746
45.
MooreAM, TongAY, YanY, et al.. A novel model transgenic rat expressing green fluorescent protein (GFP) in peripheral nerve: Abstracts of the Plastic Surgery Research Council 54th Annual Meeting: May 27-30, 2009: Pittsburgh, Pennsylvania [abstract].. Plast Reconstr Surg. 2009; 123(6)(suppl):41
46.
BridgePM, BallDJ, MackinnonSE, et al.. Nerve crush injuries—a model for axonotmesis.. Exp Neurol. 1994; 127(2):284–290 8033968
47.
DörflJ. The innervation of the mystacial region of the white mouse: a topographical study.. J Anat. 1985; 142:173–184 17103584
48.
KoobJW, MoradzadehA, TongA, et al.. Induction of regional collateral sprouting following muscle denervation.. Laryngoscope. 2007; 117(10):1735–1740 17713446
49.
PanYA, MisgeldT, LichtmanJW, SanesJR. Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.. J Neurosci. 2003; 23(36):11479–11488 14673013
50.
HayashiA, MoradzadehA, TongA, et al.. Treatment modality affects allograft-derived Schwann cell phenotype and myelinating capacity.. Exp Neurol. 2008; 212(2):324–336 18514192
