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Original Article |

A New Model for Facial Nerve Research The Novel Transgenic Thy1-GFP Rat FREE

Christina K. Magill, MD; Amy M. Moore, MD; Gregory H. Borschel, MD; Susan E. Mackinnon, MD
[+] Author Affiliations

Author Affiliations: Department of Otolaryngology–Head and Neck Surgery (Drs Magill and Mackinnon) and Division of Plastic and Reconstructive Surgery (Drs Moore and Mackinnon), Washington University School of Medicine, St Louis, Missouri; and Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario, Canada (Dr Borschel).


Arch Facial Plast Surg. 2010;12(5):315-320. doi:10.1001/archfacial.2010.71.
Text Size: A A A
Published online

Objective  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.

Figures in this Article

The face is the epicenter of human emotion and expression. The inability to move the muscles of facial expression or to endure the contorted movements of synkinesis after facial nerve injury is devastating.1 In addition, patients with facial nerve injury may have oral incompetence or corneal exposure leading to ocular damage. The improvement of facial nerve function after injury is of the utmost importance to both patients and the physicians who treat them.2

Many strategies have been used to improve the recovery of facial function after facial nerve damage. These include the use of nerve guidance conduits at the site of injury,37 neurotrophic factors,6,8,9 nerve grafts and transfers,10,11 electrical stimulation,12 and stem cells.13,14 Despite the numerous strategies to improve nerve regeneration, interventions often result in suboptimal functional outcomes and aberrant reinnervation patterns.15,16

One of the main barriers to understanding aberrant regeneration and in improving functional recovery after injury is the complex anatomy of the facial nerve. The facial nerve has a tortuous path from the brainstem through the temporal bone and then subsequently exits the stylomastoid foramen into the periphery,17 where it receives and extends multiple communicating branches to other peripheral and cranial nerves. The nerve courses anteriorly to bisect the parotid gland into a superficial and deep lobe, and then terminates in a delicate network of innervating muscular branches distally.18,19

The complex anatomy of the facial nerve has been studied and surgically manipulated in several animal models to afford translation of findings to the clinical setting. One difficulty with this research has been the caliber of the nerve and its branches in small rodent models. In addition, the regeneration of the nerve after injury is difficult to quantify and has been previously done by analyzing nerve cross-section histomorphometry,20 in addition to retrograde labeling,2125 muscle force and power testing,26 stereologic testing,27 and a variety of other techniques.28 A further challenge in the study of facial nerve regeneration is the measurement of functional recovery.2935

Direct visualization of facial nerve regeneration is now possible because of a recent breakthrough in transgenic animal technology. The green fluorescent protein (GFP) gene, expressed intrinsically in the native Aequorea victoria jellyfish, has been successfully cloned, introduced, and rendered heritable in nonnative organisms.3638 Furthermore, by understanding the genetics of neural tissue development and expression, investigators have created transgenic mice that express GFP in targeted tissues, including axons and Schwann cells.3943 These transgenic animals allow nerve reinnervation to be directly visualized after injury. Fluorescence in the nerve may be lost and regained as seen under fluorescence microscopy, and confocal imaging reveals dynamics of motor end plate reinnervation in the target muscle.28,44

To study nerve regeneration in a larger animal model, we collaborated with investigators at genOway (Lyon, France) to engineer a Thy1-GFP rat. These animals express GFP under the regulation of the Thy1 promoter. The Thy1 regulatory element is found in neurons, thymocytes, and other supporting cells.43 By visualizing the neural fluorescing tissues in these animals, we were able to characterize their facial nerve anatomy. We performed a pilot study in which the proximal facial nerve trunk was crushed and then imaged at weekly intervals up to 8 weeks. In addition, we studied the denervation and reinnervation of the long, thin zygomaticus muscle with confocal microscopy to demonstrate regeneration dynamics at the motor end plate.

TRANSGENIC RATS

The construct of the Thy1-XFP mouse lines43 (gift from Jeffrey Lichtman, PhD, and Joshua Sanes, PhD, and colleagues) was used to create transgenic Sprague Dawley rat founder lines in collaboration with genOway (Lyon, France) using pronuclear injection. Further characterization of F2 and subsequent generations was performed.45 The presence of the GFP transgene was confirmed by polymerase chain reaction (PCR) using genomic DNA extracted from rat tail specimens, and expression was verified by examination of the retina under fluorescence microscopy. Primer sequences (Integrated DNA Technologies) were provided by genOway (PCR size, 422 base pairs [bp]; BOR1-C1: 5′-CTGAGGTATTCATCATGTGCTCCGTGG-3′; BOR1D1: 5′GCG-GACTTGAAGAAGTCGT-GCTGC-3′). A total of 8 transgenic animals were used in this study, with 4 used as uninjured control animals for facial anatomy characterization (Figure 1). The relatively recent development and characterization of the Thy1-GFP founder line mandated a low total number of animals for use in this study. The rats are maintained and bred in a central animal housing facility, and all described procedures were performed according to protocols approved by the Division of Comparative Medicine at Washington University School of Medicine.

Place holder to copy figure label and caption
Figure 1.

Control animals were used to characterize facial nerve anatomy. A, Bright field microscopic imaging of the right hemiface of the Thy1-GFP rat. B, The right hemiface of the Thy1-GFP rat after depilation and followed by removal of skin to reveal the underlying facial musculature, including the zygomaticus muscle. C, Under the dissecting fluorescence microscope, green fluorescence is visualized in nerve branches.

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SURGICAL PROCEDURE

Transgenic animals were anesthetized for surgery with subcutaneous injections of ketamine (75 mg/kg) and medetomidine (100 mg/kg). The right side of the face was carefully shaved and then depilated with Nair hair removal cream (Church and Dwight Co, Inc, Princeton, New Jersey) prior to skin preparation with alcohol swabs. Great care was taken to avoid caustic or exposure injury to the eye. The skin was incised 1 mm anterior and inferior to the tragus and carried in an arch posteriorly. Following the tragal pointer medially, the facial nerve trunk was identified at an original magnification of ×16 as it exits the stylomastoid foramen. The main nerve trunk was then crushed with a No. 5 Jeweler's forceps for 30 seconds,46 and the crush site was marked with a 10-0 nylon stitch. The wound was then irrigated, and skin was reapproximated with interrupted 6-0 nylon sutures. Animals recovered on a heated surface following anesthesia reversal with atipamezole hydrochloride (1 mg/kg). Animals were inspected and weighed weekly to ensure that nerve damage did not impair the ability to eat or cause ocular damage. At the time of imaging evaluation, animals were reanesthetized, and the surgical site was prepared. Following this, they were perfused transcardially with heparinized paraformaldehyde, 4%, in 0.1M phosphate-buffered saline (pH 7.4). The superficial subcutaneous tissue of the face was then dissected completely off of the underlying superficial musculoaponeurotic system, and the vibrissae were transected under the skin. Images were then taken under an Olympus MVX10 dissecting microscope equipped with fluorescent cube filters (Olympus Corporation, Center Valley, Pennsylvania), followed by removal of the superficial musculoaponeurotic system and imaging of the nerve trunk and its branches. The zygomaticus muscle was removed with careful dissection, placed on a Sylgard resin-coated dish (Dow Corning Corporation, Midland, Michigan) and rinsed with phosphate-buffered saline. It was then stained with α-bungarotoxin Alexa Fluor-594 (10 μg/mL; Invitrogen, Carlsbad, California) for 30 minutes at room temperature. The muscle was then rinsed again with phosphate-buffered saline and mounted under a coverslip in Vectashield (Vector Laboratories, Burlingame, California) for confocal microscopic imaging.

MOTOR END PLATE EVALUATION

The staining of the zygomaticus muscle with α-bungarotoxin conjugated to Alexa 594 (Molecular Probes, Eugene, Oregon)-labeled motor end plates with red fluorescence and allowed the axon and the motor end plate to be viewed together using different lasers specific to separate excitation wavelengths. The entire muscle was placed on a glass slide with a coverslip to facilitate whole-mount imaging, allowing the course of nerves and their branches to be viewed throughout the entire muscle specimen. The muscle was surveyed and imaged under low magnification before higher power imaging of nerve terminals and motor end plates. The confocal microscope (Olympus FV 1000; Olympus America Inc, Center Valley, Pennsylvania) captures GFP-labeled axons with a spectral detection window at 488 nm and motor end plates at 568 nm. Final confocal images were taken with a ×20 objective (numerical aperture, 0.75) and were merged from pictures taken at 5-μm intervals through the depth of the muscle. These were used to create a motor end plate reinnervation map to visualize the branching system of the facial nerve to the zygomaticus muscle. Image data was obtained in uninjured control animals (n = 4) and after injury (n = 4) at 1, 2, 4, and 8 weeks following nerve crush. At each time point, regions of interest within the image were calibrated to examine the number of denervated and reinnervated motor end plates.

ANATOMY OF THE Thy1-GFP FACIAL NERVE

The rodent facial nerve anatomy has been previously described and studied.12,47 Notable features of the nerve include the lateral location of the lacrimal gland, which is located outside of the bony orbit, and branching patterns of the nerve distally that innervate the rodent vibrissae. To facilitate study of reinnervation patterns after injury, a thin, reliable muscular branch was required. The anatomical study of several control animals (n = 4) under a dissecting fluorescent microscope revealed that the zygomaticus muscle had a persistent innervating branch that was reliably imaged under low and high power (Figure 1 and Figure 2). The long, thin anatomy of this muscle was conducive to fluorescence microscopy as well as confocal imaging of the muscle whole mount.

Place holder to copy figure label and caption
Figure 2.

Facial nerve branch to the zygomaticus muscle. The facial nerve branches of a Thy1-GFP rat are visualized under fluorescent light (A), with the branch to the zygomaticus muscle imaged grossly (A1), and then under high power (A2).

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FLUORESCENCE MICROSCOPY

The facial nerve in uninjured control animals reliably expressed GFP in axons (Figure 1C and Figure 2). After nerve crush and marking of the crush site, a clear loss and recovery of a fluorescent regenerative front was not clearly seen at any of the time points chosen for the study. However, fluorescence in the frontal, buccal, and marginal branches was decreased at 1 and 2 weeks after nerve crush and increasingly robust at 4 and 8 weeks (Figure 3). Interestingly, high-powered imaging showed a definitive return of fluorescence in the zygomaticus muscular branch at 4 weeks after injury, which corresponds to reinnervation of motor end plates seen with confocal microscopy at this same time point (Figure 4D and F).

Place holder to copy figure label and caption
Figure 3.

Fluorescence microscopy after proximal facial nerve crush injury. The exposed rat hemiface is shown over time (t) after injury under low (A-D) and high (A1-D1) power. Proximal frontal (+), buccal (ˆ), and mandibular (*) branches are shown on the right-hand side with decreased fluorescence at 2 weeks (B1). The triangle represents the crush site.

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Place holder to copy figure label and caption
Figure 4.

Confocal imaging of the zygomaticus muscle. Acetylcholine receptors (AChRs) appear red after staining with α-bungarotoxin. A control animal is shown (A), followed by denervation up to 2 weeks (B and C), and then reinnervation, which is also visualized under fluorescence microscopy (D and F). By 8 weeks, images approximate the appearance of controls (E).

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CONFOCAL IMAGING

In studies of uninjured control animals, adult Thy1-GFP rats were noted to have a 1:1 relationship between terminal axons and motor end plates in the zygomaticus muscle (Figure 4A). After nerve crush, this relationship changed as Wallerian degeneration occurred and motor end plates were denervated and then reinnervated over time. Imaged nerve terminals were completely denervated at 1 week following nerve crush injury with only “ovoids” of fluorescing debris visible (Figure 4B). This persisted at 2 weeks after injury. Reinnervation of end plates is visualized at 4 weeks (Figure 4D). The axonal relationship with motor end plates at 4 weeks did not show any hyperinnervation after nerve crush injury, as has been seen in other animal models.28 However, there was disorganization of the axonal branches, and unmatched axons were seen in addition to fluorescing debris, which may represent phagocytosis of GFP from degenerated axons, or fluorescing lymphoid cells.43 By 8 weeks after nerve crush injury, the 1:1 axon–to–end plate relationship was reestablished, and images approximated uninjured controls (Figure 4A and E).

The purpose of this study was to demonstrate the utility of a new transgenic model in facial nerve research. The Thy1-GFP rat, which constitutively expresses GFP in axons, allows the intricate facial nerve anatomy to be studied. The increased nerve caliber of this animal model, compared with other currently available models, allows for more reliable manipulation and use in a variety of surgical paradigms that are translationally powerful.43 In this study, we performed a proximal facial nerve crush and, for the first time to our knowledge, directly visualized facial nerve regeneration and muscle reinnervation using fluorescence and confocal microscopy in a rat model.

The use of fluorescence microscopy allowed for the direct visualization of nerve regeneration. After nerve injury, axons underwent Wallerian degeneration and fluorescence was lost. As axons expressing GFP regenerated, fluorescence was again visualized and could be used as a marker for regeneration. In the present study, the uninjured Thy-GFP rats revealed expression of GFP in the facial nerve. Following injury, fluorescence was not markedly diminished in the main nerve trunk or immediately distal to the marked crush site at the time points captured. However, evaluation of the nerve branches demonstrated diminished fluorescence at weeks 1 and 2, followed by an increase at 4 and 8 weeks after injury (Figure 3). Specifically, high-power imaging revealed return of fluorescence in the zygomatic muscle branch at 4 weeks (Figure 4F). The persistence of fluorescent protein at earlier time points after nerve injury has been previously documented in other fluorescence-expressing animal models.28,44,48 This persistence of fluorescence has been overcome by using a “double-crush” technique, in which an initial crush is followed by a second nerve injury to allow fluorescent debris to be cleared.49 The double-crush method was considered in the study design but was decided against in order to establish baseline data in a single nerve crush model using this new transgenic rat line. In future studies, a double-crush technique could be used and coupled with line scanning to quantify the degree of fluorescence intensity over time.50 In addition, live in vivo imaging techniques with fluorescence microscopy are also possible in these transgenic animals to follow the same nerve branch and its specific morphologic characteristics at serial time points.49,51

The long, thin zygomaticus muscle is well suited for confocal imaging after facial nerve injury. We visualized the return of innervation at 4 weeks after the crush injury. The thin nature of the superficial musculoaponeurotic system affords other facial muscles to be used in reinnervation studies as well. By correlating the morphologic characteristics of axonal regrowth in different injury paradigms, the Thy1-GFP model will assist in understanding aberrant reinnervation patterns and the development of synkinesis.

There are a variety of assays that can be used to evaluate facial nerve regeneration after injury; however, few techniques confer the direct visualization of nerve reinnervation or the possibility of a live in vivo study. The outcome measures used to assess nerve regeneration in this study were chosen based on the novel fluorescing properties of this new transgenic tool. The direct visualization of axonal behavior in the Thy1-GFP line may allow questions regarding aberrant nerve regeneration to be answered and improve treatment modalities available for patients with facial nerve injury.

Correspondence: Susan E. Mackinnon, MD, Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, 660 S Euclid Ave, Campus Box 8238, St Louis, MO 63110 (mackinnon@wudosis.wustl.edu).

Accepted for Publication: April 29, 2010.

Author Contributions:Study concept and design: Magill, Moore, Borschel, and Mackinnon. Acquisition of data: Magill and Mackinnon. Analysis and interpretation of data: Magill, Moore, Borschel, and Mackinnon. Drafting of the manuscript: Magill, Moore, and Mackinnon. Critical revision of the manuscript for important intellectual content: Magill, Moore, Borschel, and Mackinnon. Obtained funding: Moore and Borschel. Administrative, technical, and material support: Magill, Moore, Borschel, and Mackinnon. Study supervision: Magill, Borschel, and Mackinnon.

Financial Disclosure: None reported.

Funding/Support: This study was made possible by funding awarded to Dr Mackinnon by the National Institutes of Health (grant RO1 NS051706) and to Dr Borschel by the American Society for Peripheral Nerve. The Barnes-Jewish Foundation provided confocal microscopy equipment and support.

Additional Contributions: Alice Tong, MS, taught confocal techniques used in this work, and Andrew R. Magill, BS, assisted with image calibration and the figures.  

Crumley  RL Mechanisms of synkinesis. Laryngoscope 1979;89 (11) 1847- 1854
PubMed Link to Article
May  MSchaitkin  B The Facial Nerve.  New York, NY Thieme2000;
Spector  JGLee  PDerby  A  et al.  Facial nerve regeneration through semipermeable chambers in the rabbit. Laryngoscope 1992;102 (7) 784- 796
PubMed Link to Article
Spector  JGLee  P 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
PubMed
Hadlock  TACheney  ML Update on facial nerve repair. Facial Plast Surg 1998;14 (3) 179- 184
PubMed Link to Article
Barras  FMKuntzer  TZurn  ADPasche  P Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair. Laryngoscope 2009;119 (5) 846- 855
PubMed Link to Article
Kitahara  AKNishimura  YShimizu  YEndo  K Facial nerve repair accomplished by the interposition of a collagen nerve guide. J Neurosurg 2000;93 (1) 113- 120
PubMed Link to Article
Barras  FMPasche  PBouche  NAebischer  PZurn  AD Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat. J Neurosci Res 2002;70 (6) 746- 755
PubMed Link to Article
Shi  YZhou  LTian  JWang  Y Transplantation of neural stem cells overexpressing glia-derived neurotrophic factor promotes facial nerve regeneration. Acta Otolaryngol 2008;129 (8) 1- 9
PubMed
Hadlock  TSheahan  THeaton  JSundback  CMackinnon  SCheney  M Baiting the cross-face nerve graft with temporary hypoglossal hookup. Arch Facial Plast Surg 2004;6 (4) 228- 233
PubMed Link to Article
Hwang  KKim  SGKim  DJ Hypoglossal-facial nerve anastomosis in the rabbits using laser welding. Ann Plast Surg 2008;61 (4) 452- 456
PubMed Link to Article
Sinis  NHorn  FGenchev  B  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
PubMed Link to Article
Guo  BFDong  MM Application of neural stem cells in tissue-engineered artificial nerve. Otolaryngol Head Neck Surg 2009;140 (2) 159- 164
PubMed Link to Article
Zhang  HWei  YTTsang  KS  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;667
PubMed Link to Article
Moran  CJNeely  JG Patterns of facial nerve synkinesis. Laryngoscope 1996;106 (12, pt 1) 1491- 1496
PubMed Link to Article
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 1994;44 (11) 2165- 2173
PubMed Link to Article
Baxter  A Dehiscence of the Fallopian canal: an anatomical study. J Laryngol Otol 1971;85 (6) 587- 594
PubMed Link to Article
Larrabee  WFJMakielski  KH Surgical Anatomy of the Face.  New York, NY Raven Press1993;
Davis  RAAnson  BJBudinger  JMKurth  LR 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
PubMed
Hunter  DAMoradzadeh  AWhitlock  EL  et al.  Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J Neurosci Methods 2007;166 (1) 116- 124
PubMed Link to Article
Brushart  TM Preferential reinnervation of motor nerves by regenerating motor axons. J Neurosci 1988;8 (3) 1026- 1031
PubMed
Brushart  TM Preferential motor reinnervation: a sequential double-labeling study. Restor Neurol Neurosci 1990;1281- 287
Brushart  TM Motor axons preferentially reinnervate motor pathways. J Neurosci 1993;13 (6) 2730- 2738
PubMed
Hayashi  AMoradzadeh  AHunter  DA  et al.  Retrograde labeling in peripheral nerve research: it is not all black and white. J Reconstr Microsurg 2007;23 (7) 381- 389
PubMed Link to Article
Dohm  SStreppel  MGuntinas-Lichius  O  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
PubMed
Yoshimura  KAsato  HCederna  PSUrbanchek  MGKuzon  WM The effect of reinnervation on force production and power output in skeletal muscle. J Surg Res 1999;81 (2) 201- 208
PubMed Link to Article
Larsen  JO Stereology of nerve cross sections. J Neurosci Methods 1998;85 (1) 107- 118
PubMed Link to Article
Magill  CKTong  AKawamura  D  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
PubMed Link to Article
Hadlock  TAHeaton  JCheney  MMackinnon  SE Functional recovery after facial and sciatic nerve crush injury in the rat. Arch Facial Plast Surg 2005;7 (1) 17- 20
PubMed Link to Article
Heaton  JTKowaleski  JMBermejo  RZeigler  HPAhlgren  DJHadlock  TA 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
PubMed Link to Article
Hadlock  TKowaleski  JLo  D  et al.  Functional assessments of the rodent facial nerve: a synkinesis model. Laryngoscope 2008;118 (10) 1744- 1749
PubMed Link to Article
Hadlock  TKowaleski  JMackinnon  SHeaton  JT A novel method of head fixation for the study of rodent facial function. Exp Neurol 2007;205 (1) 279- 282
PubMed Link to Article
Mehta  RPZhang  SHadlock  TA Novel 3-D video for quantification of facial movement. Otolaryngol Head Neck Surg 2008;138 (4) 468- 472
PubMed Link to Article
Tomov  TLGuntinas-Lichius  OGrosheva  M  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
PubMed Link to Article
Serpe  CJTetzlaff  JECoers  SSanders  VMJones  KJ Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice. Brain Behav Immun 2002;16 (6) 808- 812
PubMed Link to Article
Tsien  RY The green fluorescent protein. Annu Rev Biochem 1998;67509- 544
PubMed Link to Article
Shimomura  OJohnson  FHSaiga  Y Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962;59223- 239
PubMed Link to Article
Chalfie  MTu  YEuskirchen  GWard  WWPrasher  DC Green fluorescent protein as a marker for gene expression. Science 1994;263 (5148) 802- 805
PubMed Link to Article
Zuo  YLubischer  JLKang  H  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
PubMed Link to Article
Magill  CWhitlock  ESolowski  NMyckatyn  T Transgenic models of nerve repair and nerve regeneration. Neurol Res 2008;30 (10) 1023- 1029
PubMed Link to Article
Mignone  JLKukekov  VChiang  ASSteindler  DEnikolopov  G Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 2004;469 (3) 311- 324
PubMed Link to Article
Vives  VAlonso  GSolal  ACJoubert  DLegraverend  C Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice. J Comp Neurol 2003;457 (4) 404- 419
PubMed Link to Article
Feng  GMellor  RHBernstein  M  et al.  Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 2000;28 (1) 41- 51
PubMed Link to Article
Hayashi  APannucci  CMoradzadeh  A  et al.  Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy. Exp Neurol 2008;211 (2) 539- 550
PubMed Link to Article
Moore  AMTong  AYYan  Y  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
Bridge  PMBall  DJMackinnon  SE  et al.  Nerve crush injuries—a model for axonotmesis. Exp Neurol 1994;127 (2) 284- 290
PubMed Link to Article
Dörfl  J The innervation of the mystacial region of the white mouse: a topographical study. J Anat 1985;142173- 184
PubMed
Koob  JWMoradzadeh  ATong  A  et al.  Induction of regional collateral sprouting following muscle denervation. Laryngoscope 2007;117 (10) 1735- 1740
PubMed Link to Article
Pan  YAMisgeld  TLichtman  JWSanes  JR Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J Neurosci 2003;23 (36) 11479- 11488
PubMed
Hayashi  AMoradzadeh  ATong  A  et al.  Treatment modality affects allograft-derived Schwann cell phenotype and myelinating capacity. Exp Neurol 2008;212 (2) 324- 336
PubMed Link to Article
Nguyen  QTSanes  JRLichtman  JW Pre-existing pathways promote precise projection patterns. Nat Neurosci 2002;5 (9) 861- 867
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Control animals were used to characterize facial nerve anatomy. A, Bright field microscopic imaging of the right hemiface of the Thy1-GFP rat. B, The right hemiface of the Thy1-GFP rat after depilation and followed by removal of skin to reveal the underlying facial musculature, including the zygomaticus muscle. C, Under the dissecting fluorescence microscope, green fluorescence is visualized in nerve branches.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Facial nerve branch to the zygomaticus muscle. The facial nerve branches of a Thy1-GFP rat are visualized under fluorescent light (A), with the branch to the zygomaticus muscle imaged grossly (A1), and then under high power (A2).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Fluorescence microscopy after proximal facial nerve crush injury. The exposed rat hemiface is shown over time (t) after injury under low (A-D) and high (A1-D1) power. Proximal frontal (+), buccal (ˆ), and mandibular (*) branches are shown on the right-hand side with decreased fluorescence at 2 weeks (B1). The triangle represents the crush site.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Confocal imaging of the zygomaticus muscle. Acetylcholine receptors (AChRs) appear red after staining with α-bungarotoxin. A control animal is shown (A), followed by denervation up to 2 weeks (B and C), and then reinnervation, which is also visualized under fluorescence microscopy (D and F). By 8 weeks, images approximate the appearance of controls (E).

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Tables

References

Crumley  RL Mechanisms of synkinesis. Laryngoscope 1979;89 (11) 1847- 1854
PubMed Link to Article
May  MSchaitkin  B The Facial Nerve.  New York, NY Thieme2000;
Spector  JGLee  PDerby  A  et al.  Facial nerve regeneration through semipermeable chambers in the rabbit. Laryngoscope 1992;102 (7) 784- 796
PubMed Link to Article
Spector  JGLee  P 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
PubMed
Hadlock  TACheney  ML Update on facial nerve repair. Facial Plast Surg 1998;14 (3) 179- 184
PubMed Link to Article
Barras  FMKuntzer  TZurn  ADPasche  P Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair. Laryngoscope 2009;119 (5) 846- 855
PubMed Link to Article
Kitahara  AKNishimura  YShimizu  YEndo  K Facial nerve repair accomplished by the interposition of a collagen nerve guide. J Neurosurg 2000;93 (1) 113- 120
PubMed Link to Article
Barras  FMPasche  PBouche  NAebischer  PZurn  AD Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat. J Neurosci Res 2002;70 (6) 746- 755
PubMed Link to Article
Shi  YZhou  LTian  JWang  Y Transplantation of neural stem cells overexpressing glia-derived neurotrophic factor promotes facial nerve regeneration. Acta Otolaryngol 2008;129 (8) 1- 9
PubMed
Hadlock  TSheahan  THeaton  JSundback  CMackinnon  SCheney  M Baiting the cross-face nerve graft with temporary hypoglossal hookup. Arch Facial Plast Surg 2004;6 (4) 228- 233
PubMed Link to Article
Hwang  KKim  SGKim  DJ Hypoglossal-facial nerve anastomosis in the rabbits using laser welding. Ann Plast Surg 2008;61 (4) 452- 456
PubMed Link to Article
Sinis  NHorn  FGenchev  B  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
PubMed Link to Article
Guo  BFDong  MM Application of neural stem cells in tissue-engineered artificial nerve. Otolaryngol Head Neck Surg 2009;140 (2) 159- 164
PubMed Link to Article
Zhang  HWei  YTTsang  KS  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;667
PubMed Link to Article
Moran  CJNeely  JG Patterns of facial nerve synkinesis. Laryngoscope 1996;106 (12, pt 1) 1491- 1496
PubMed Link to Article
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 1994;44 (11) 2165- 2173
PubMed Link to Article
Baxter  A Dehiscence of the Fallopian canal: an anatomical study. J Laryngol Otol 1971;85 (6) 587- 594
PubMed Link to Article
Larrabee  WFJMakielski  KH Surgical Anatomy of the Face.  New York, NY Raven Press1993;
Davis  RAAnson  BJBudinger  JMKurth  LR 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
PubMed
Hunter  DAMoradzadeh  AWhitlock  EL  et al.  Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J Neurosci Methods 2007;166 (1) 116- 124
PubMed Link to Article
Brushart  TM Preferential reinnervation of motor nerves by regenerating motor axons. J Neurosci 1988;8 (3) 1026- 1031
PubMed
Brushart  TM Preferential motor reinnervation: a sequential double-labeling study. Restor Neurol Neurosci 1990;1281- 287
Brushart  TM Motor axons preferentially reinnervate motor pathways. J Neurosci 1993;13 (6) 2730- 2738
PubMed
Hayashi  AMoradzadeh  AHunter  DA  et al.  Retrograde labeling in peripheral nerve research: it is not all black and white. J Reconstr Microsurg 2007;23 (7) 381- 389
PubMed Link to Article
Dohm  SStreppel  MGuntinas-Lichius  O  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
PubMed
Yoshimura  KAsato  HCederna  PSUrbanchek  MGKuzon  WM The effect of reinnervation on force production and power output in skeletal muscle. J Surg Res 1999;81 (2) 201- 208
PubMed Link to Article
Larsen  JO Stereology of nerve cross sections. J Neurosci Methods 1998;85 (1) 107- 118
PubMed Link to Article
Magill  CKTong  AKawamura  D  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
PubMed Link to Article
Hadlock  TAHeaton  JCheney  MMackinnon  SE Functional recovery after facial and sciatic nerve crush injury in the rat. Arch Facial Plast Surg 2005;7 (1) 17- 20
PubMed Link to Article
Heaton  JTKowaleski  JMBermejo  RZeigler  HPAhlgren  DJHadlock  TA 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
PubMed Link to Article
Hadlock  TKowaleski  JLo  D  et al.  Functional assessments of the rodent facial nerve: a synkinesis model. Laryngoscope 2008;118 (10) 1744- 1749
PubMed Link to Article
Hadlock  TKowaleski  JMackinnon  SHeaton  JT A novel method of head fixation for the study of rodent facial function. Exp Neurol 2007;205 (1) 279- 282
PubMed Link to Article
Mehta  RPZhang  SHadlock  TA Novel 3-D video for quantification of facial movement. Otolaryngol Head Neck Surg 2008;138 (4) 468- 472
PubMed Link to Article
Tomov  TLGuntinas-Lichius  OGrosheva  M  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
PubMed Link to Article
Serpe  CJTetzlaff  JECoers  SSanders  VMJones  KJ Functional recovery after facial nerve crush is delayed in severe combined immunodeficient mice. Brain Behav Immun 2002;16 (6) 808- 812
PubMed Link to Article
Tsien  RY The green fluorescent protein. Annu Rev Biochem 1998;67509- 544
PubMed Link to Article
Shimomura  OJohnson  FHSaiga  Y Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962;59223- 239
PubMed Link to Article
Chalfie  MTu  YEuskirchen  GWard  WWPrasher  DC Green fluorescent protein as a marker for gene expression. Science 1994;263 (5148) 802- 805
PubMed Link to Article
Zuo  YLubischer  JLKang  H  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
PubMed Link to Article
Magill  CWhitlock  ESolowski  NMyckatyn  T Transgenic models of nerve repair and nerve regeneration. Neurol Res 2008;30 (10) 1023- 1029
PubMed Link to Article
Mignone  JLKukekov  VChiang  ASSteindler  DEnikolopov  G Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 2004;469 (3) 311- 324
PubMed Link to Article
Vives  VAlonso  GSolal  ACJoubert  DLegraverend  C Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice. J Comp Neurol 2003;457 (4) 404- 419
PubMed Link to Article
Feng  GMellor  RHBernstein  M  et al.  Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 2000;28 (1) 41- 51
PubMed Link to Article
Hayashi  APannucci  CMoradzadeh  A  et al.  Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy. Exp Neurol 2008;211 (2) 539- 550
PubMed Link to Article
Moore  AMTong  AYYan  Y  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
Bridge  PMBall  DJMackinnon  SE  et al.  Nerve crush injuries—a model for axonotmesis. Exp Neurol 1994;127 (2) 284- 290
PubMed Link to Article
Dörfl  J The innervation of the mystacial region of the white mouse: a topographical study. J Anat 1985;142173- 184
PubMed
Koob  JWMoradzadeh  ATong  A  et al.  Induction of regional collateral sprouting following muscle denervation. Laryngoscope 2007;117 (10) 1735- 1740
PubMed Link to Article
Pan  YAMisgeld  TLichtman  JWSanes  JR Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J Neurosci 2003;23 (36) 11479- 11488
PubMed
Hayashi  AMoradzadeh  ATong  A  et al.  Treatment modality affects allograft-derived Schwann cell phenotype and myelinating capacity. Exp Neurol 2008;212 (2) 324- 336
PubMed Link to Article
Nguyen  QTSanes  JRLichtman  JW Pre-existing pathways promote precise projection patterns. Nat Neurosci 2002;5 (9) 861- 867
PubMed Link to Article

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