The Dilator Naris Muscle as a Reporter of Facial Nerve Regeneration in a Rat Model.

OBJECTIVE: Many investigators study facial nerve regeneration using the rat whisker pad model, although widely standardized outcomes measures of facial nerve regeneration in the rodent have not yet been developed. The intrinsic whisker pad "sling" muscles producing whisker protraction, situated at the base of each individual whisker, are extremely small and difficult to study en bloc. Here, we compare the functional innervation of 2 potential reporter muscles for whisker pad innervation: the dilator naris (DN) and the levator labii superioris (LLS), to characterize facial nerve regeneration. METHODS: Motor supply of the DN and LLS was elucidated by measuring contraction force and compound muscle action potentials during stimulation of individual facial nerve branches, and by measuring whisking amplitude before and after DN distal tendon release. RESULTS: The pattern of DN innervation matched that of the intrinsic whisker pad musculature (ie, via the buccal and marginal mandibular branches of the facial nerve), whereas the LLS seemed to be innervated almost entirely by the zygomatic branch, whose primary target is the orbicularis oculi muscle. CONCLUSIONS: Although the LLS has been commonly used as a reporter muscle of whisker pad innervation, the present data show that its innervation pattern does not overlap substantially with the muscles producing whisker protraction. The DN muscle may serve as a more appropriate reporter for whisker pad innervation because it is innervated by the same facial nerve branches as the intrinsic whisker pad musculature, making structure/function correlations more accurate, and more relevant to investigators studying facial nerve regeneration.

The convergence of facial nerve branches providing whisker pad motor supply in rats: implications for facial reanimation study.

INTRODUCTION: Rodent whisking behavior is supported by the buccal and mandibular branches of the facial nerve, but a description of how these branches converge and contribute to whisker movement is lacking. METHODS: Eight rats underwent isolated transection of either the buccal or mandibular branch and subsequent opposite branch transection. Whisking function was analyzed after both transections. Anatomical measurements, and video recording of stimulation to individual branches, were taken from both facial nerves in 10 rats. RESULTS: Normal to near-normal whisking was demonstrated after isolated branch transection. Whisking was eliminated after transection of both branches. The buccal and mandibular branches form a convergence just proximal to the whisker pad, herein called "distal pes." Distal to this convergence, we identified consistent anatomy that demonstrated cross-innervation. CONCLUSION: The overlap of efferent supply to the whisker pad must be considered when studying facial nerve regeneration in the rat facial nerve model.

Toward the Bionic Face: A Novel Neuroprosthetic Device Paradigm for Facial Reanimation Consisting of Neural Blockade and Functional Electrical Stimulation.

BACKGROUND: Facial palsy is a devastating condition potentially amenable to rehabilitation by functional electrical stimulation. Herein, a novel paradigm for unilateral facial reanimation using an implantable neuroprosthetic device is proposed and its feasibility demonstrated in a live rodent model. The paradigm comprises use of healthy-side electromyographic activity as control inputs to a system whose outputs are neural stimuli to effect symmetric facial displacements. The vexing issue of suppressing undesirable activity resulting from aberrant neural regeneration (synkinesis) or nerve transfer procedures is addressed using proximal neural blockade. METHODS: Epimysial and nerve cuff electrode arrays were implanted in the faces of Wistar rats. Stimuli were delivered to evoke blinks and whisks of various durations and amplitudes. The dynamic relation between electromyographic signals and facial displacements was modeled, and model predictions were compared against measured displacements. Optimal parameters to achieve facial nerve blockade by means of high-frequency alternating current were determined, and the safety of continuous delivery was assessed. RESULTS: Electrode implantation was well tolerated. Blinks and whisks of tunable amplitudes and durations were evoked by controlled variation of neural stimuli parameters. Facial displacements predicted from electromyographic input modelling matched those observed with a variance-accounted-for exceeding 96 percent. Effective and reversible facial nerve blockade in awake behaving animals was achieved, without detrimental effect noted from long-term continual use. CONCLUSIONS: Proof-of-principle of rehabilitation of hemifacial palsy by means of a neuroprosthetic device has been demonstrated. The use of proximal neural blockade coupled with distal functional electrical stimulation may have relevance to rehabilitation of other peripheral motor nerve deficits.

The effects of venous ensheathment on facial nerve repair in the rat.

OBJECTIVE: To investigate the protective effect of autologous venous ensheathment on sutured rat facial nerve and to test whether the ensheathment could improve the functional recovery of repaired nerve and accuracy of axonal growth. STUDY DESIGN: In vivo study. METHODS: Forty-six rats were examined, with six rats serving as normal controls and 40 receiving facial nerve transection and suture repair (SR) or transection and suture repair with an additional venous ensheathment (VE). The rats were then subjected to functional testing, histological assessment of nerve specimens, or retrograde tracing, respectively. RESULTS: At the postoperative day (POD) 60, the venous ensheathment showed no adhesion at the surrounding tissues. No significant difference in neuroma formation was found between the two surgical manipulations (SR and VE groups) (P < 0.05). Retrogradely labeled motoneurons in facial nuclei were extremely disorganized after the facial nerve undertook surgical manipulation. In all manipulated groups, double retrogradely labeled neurons, indicative of aberrant axonal branching during regeneration, could be observed after peripheral manipulation across all time points. With the two facial surgical manipulations, the average count of double-labeled neurons at POD 60 was significantly less than at POD 21 (P < 0.05). CONCLUSION: Autologous venous ensheathment could not help with the functional recovery of facial nerve or improve the accuracy of axonal regeneration. Further studies are warranted to elucidate the effects of venous ensheathment in other motor and sensory nerve models. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1558-1564, 2017.

Bilateral Facial Paralysis: A 13-Year Experience.

BACKGROUND: Bilateral facial palsy is a rare clinical entity caused by myriad disparate conditions requiring different treatment paradigms. Lyme disease, Guillain-Barré syndrome, and leukemia are several examples. In this article, the authors describe the cause, the initial diagnostic approach, and the management of long-term sequelae of bilateral paralysis that has evolved in the authors' center over the past 13 years. METHODS: A chart review was performed to identify all patients diagnosed with bilateral paralysis at the authors' center between January of 2002 and January of 2015. Demographics, signs and symptoms, diagnosis, initial medical treatment, interventions for facial reanimation, and outcomes were reviewed. RESULTS: Of the 2471 patients seen at the authors' center, 68 patients (3 percent) with bilateral facial paralysis were identified. Ten patients (15 percent) presented with bilateral facial paralysis caused by Lyme disease, nine (13 percent) with Möbius syndrome, nine (13 percent) with neurofibromatosis type 2, five (7 percent) with bilateral facial palsy caused by brain tumor, four (6 percent) with Melkersson-Rosenthal syndrome, three (4 percent) with bilateral temporal bone fractures, two (3 percent) with Guillain-Barré syndrome, one (2 percent) with central nervous system lymphoma, one (2 percent) with human immunodeficiency virus infection, and 24 (35 percent) with presumed Bell palsy. Treatment included pharmacologic therapy, physical therapy, chemodenervation, and surgical interventions. CONCLUSIONS: Bilateral facial palsy is a rare medical condition, and treatment often requires a multidisciplinary approach. The authors outline diagnostic and therapeutic algorithms of a tertiary care center to provide clinicians with a systematic approach to managing these complicated patients.

Functional recovery after facial nerve cable grafting in a rodent model.

IMPORTANCE: Cable grafting is widely considered to be the preferred alternative to primary repair of the injured facial nerve; however, quantitative comparison of the 2 techniques has not been previously undertaken in a rodent model. OBJECTIVE: To establish functional recovery parameters after interposition autografting in a rodent facial nerve model. DESIGN, SETTING, AND PARTICIPANTS: Prospective randomized animal study at a tertiary care facial nerve center using 16 female Wistar Hannover rats. INTERVENTION: The experimental group received reversed autograft reconstruction of a 20-mm neural gap, and the control group received facial nerve transection and primary repair. MAIN OUTCOME AND MEASURE: Whisker excursion was measured weekly for 70 postoperative days using laser micrometers. RESULTS: The control group exhibited the most rapid recovery, with substantial return of whisker movement occurring during the third postoperative week. The experimental group demonstrated return of function beginning in the fourth postoperative week, eventually achieving a degree of function comparable to that of the control group by the sixth postoperative week (P = .68). CONCLUSIONS AND RELEVANCE: Recovery of facial function after cable grafting seems to be slower than, but eventually similar to, recovery after primary neurorrhaphy in a rodent model. In the present study we have established a benchmark for recovery of whisker movement across a 20-mm rodent facial nerve gap, which will be used for comparison of different facial nerve gap bridging materials in future studies. LEVEL OF EVIDENCE: NA.

Rat whisker movement after facial nerve lesion: evidence for autonomic contraction of skeletal muscle.

Vibrissal whisking is often employed to track facial nerve regeneration in rats; however, we have observed similar degrees of whisking recovery after facial nerve transection with or without repair. We hypothesized that the source of non-facial nerve-mediated whisker movement after chronic denervation was from autonomic, cholinergic axons traveling within the infraorbital branch of the trigeminal nerve (ION). Rats underwent unilateral facial nerve transection with repair (N=7) or resection without repair (N=11). Post-operative whisking amplitude was measured weekly across 10weeks, and during intraoperative stimulation of the ION and facial nerves at ⩾18weeks. Whisking was also measured after subsequent ION transection (N=6) or pharmacologic blocking of the autonomic ganglia using hexamethonium (N=3), and after snout cooling intended to elicit a vasodilation reflex (N=3). Whisking recovered more quickly and with greater amplitude in rats that underwent facial nerve repair compared to resection (P<0.05), but individual rats overlapped in whisking amplitude across both groups. In the resected rats, non-facial-nerve-mediated whisking was elicited by electrical stimulation of the ION, temporarily diminished following hexamethonium injection, abolished by transection of the ION, and rapidly and significantly (P<0.05) increased by snout cooling. Moreover, fibrillation-related whisker movements decreased in all rats during the initial recovery period (indicative of reinnervation), but re-appeared in the resected rats after undergoing ION transection (indicative of motor denervation). Cholinergic, parasympathetic axons traveling within the ION innervate whisker pad vasculature, and immunohistochemistry for vasoactive intestinal peptide revealed these axons branching extensively over whisker pad muscles and contacting neuromuscular junctions after facial nerve resection. This study provides the first behavioral and anatomical evidence of spontaneous autonomic innervation of skeletal muscle after motor nerve lesion, which not only has implications for interpreting facial nerve reinnervation results, but also calls into question whether autonomic-mediated innervation of striated muscle occurs naturally in other forms of neuropathy.

Whisking recovery after automated mechanical stimulation during facial nerve regeneration.

IMPORTANCE Recovery from facial nerve transection is typically poor, but daily mechanical stimulation of the face in rats has been reported to remarkably enhance functional recovery after facial nerve transection and suture repair. This phenomenon needs additional investigation because of its important clinical implications. OBJECTIVE To determine whether automated mechanical stimulation of the whisker pad improves whisking recovery after facial nerve transection and repair in a rat model. DESIGN AND SETTING Sixty-one rats underwent unilateral facial nerve transection and suture repair and were randomized into 8 groups. Six groups received daily automated whisker or whisker pad mechanical stimulation including 0.5-, 1.5-, and 8.0-Hz patterns. Two control groups received restraint without stimulation. Treatment started on postoperative day 8, occurred 5 days per week, and lasted throughout 15 weeks of recovery. Whisking amplitude, velocity, and acceleration were quantified weekly for 15 weeks. INTERVENTIONS Unilateral facial nerve transection, suture repair, and, for 6 groups, daily automated whisker or whisker pad mechanical stimulation. MAIN OUTCOMES AND MEASURES Quantification of whisking amplitude, velocity, and acceleration. RESULTS Rats receiving the low frequencies of stimulation of the whiskers or whisker pad did not demonstrate enhanced whisking recovery, and rats receiving stimulation at 8.0 Hz showed significantly worse whisking recovery compared with controls and previously published groups receiving lower dose manual stimulation. CONCLUSIONS AND RELEVANCE Although daily manual whisker pad stimulation has been shown to enhance whisking recovery, rats in this study did not demonstrate improved whisking recovery after automated mechanical stimulation across a wide range of driving frequencies. Moreover, faster stimulation (8.0 Hz) was actually detrimental to recovery. Further work is needed to understand the relationship between stimulation patterns and the physiologic mechanisms underlying improved or worsened functional outcomes after facial nerve transection and repair.

A system for delivering mechanical stimulation and robot-assisted therapy to the rat whisker pad during facial nerve regeneration.

Functional recovery is typically poor after facial nerve transection and surgical repair. In rats, whisking amplitude remains greatly diminished after facial nerve regeneration, but can recover more completely if the whiskers are periodically mechanically stimulated during recovery. Here we present a robotic "whisk assist" system for mechanically driving whisker movement after facial nerve injury. Movement patterns were either preprogrammed to reflect natural amplitudes and frequencies, or movements of the contralateral (healthy) side of the face were detected and used to control real-time mirror-like motion on the denervated side. In a pilot study, 20 rats were divided into nine groups and administered one of eight different whisk assist driving patterns (or control) for 5-20 minutes, five days per week, across eight weeks of recovery after unilateral facial nerve cut and suture repair. All rats tolerated the mechanical stimulation well. Seven of the eight treatment groups recovered average whisking amplitudes that exceeded controls, although small group sizes precluded statistical confirmation of group differences. The potential to substantially improve facial nerve recovery through mechanical stimulation has important clinical implications, and we have developed a system to control the pattern and dose of stimulation in the rat facial nerve model.

Quantitative analysis of muscle histologic method in rodent facial nerve injury.

OBJECTIVE: To describe denervation features of facial musculature following facial nerve injury in a rodent model. METHODS: Six Wistar-Hannover rats underwent unilateral transection and immediate repair of the facial nerve. After 8 weeks, muscular bundles consisting of dilator naris and levator labii superioris from both sides were analyzed for mean muscle cell diameter and the percentage of muscle cell cross-sectional area using image processing software. The atrophic features of facial muscles were quantified and compared with the contralateral, healthy side of the face. RESULTS: Weekly postoperative whisking assessment demonstrated the anticipated course of recovery. We observed significant differences between the normal side and the manipulated side, respectively, in the percentage of muscle specimen cross-sectional area attributable to muscle cell profiles (57% vs 29%; P = .006) and total fiber counts (1346 vs 794; P = .02). The mean cross-sectional area of individual muscle fibers was higher on the normal side (1129 vs 928 µm2; P = .39); however, this difference was statistically nonsignificant. CONCLUSION: The objective, quantitative measures of muscle microstructure used in this report provide a valuable point of comparison for whisking function and electrophysiologic measures and can be used in future studies to assess muscle atrophic features associated with facial nerve injury and repair techniques.

Facial nerve repair: fibrin adhesive coaptation versus epineurial suture repair in a rodent model.

OBJECTIVES/HYPOTHESIS: Repair of the transected facial nerve has traditionally been accomplished with microsurgical neurorrhaphy; however, fibrin adhesive coaptation (FAC) of peripheral nerves has become increasingly popular over the past decade. We compared functional recovery following suture neurorrhaphy to FAC in a rodent facial nerve model. STUDY DESIGN: Prospective, randomized animal study. METHODS: Sixteen rats underwent transection and repair of the facial nerve proximal to the pes anserinus. Eight animals underwent epineurial suture (ES) neurorrhaphy, and eight underwent repair with fibrin adhesive (FA). Surgical times were documented for all procedures. Whisking function was analyzed on a weekly basis for both groups across 15 weeks of recovery. RESULTS: Rats experienced whisking recovery consistent in time course and degree with prior studies of rodent facial nerve transection and repair. There were no significant differences in whisking amplitude, velocity, or acceleration between suture and FA groups. However, the neurorrhaphy time with FA was 70% shorter than for ES (P < 0.05). CONCLUSION: Although we found no difference in whisking recovery between suture and FA repair of the main trunk of the rat facial nerve, the significantly shorter operative time for FA repair makes this technique an attractive option. The relative advantages of both techniques are discussed.

Enhanced photolysis in aerosols: evidence for important surface effects.

While there is increasing evidence for unique chemical reactions at interfaces, there are fewer data on photochemistry at liquid-vapor junctions. This paper reports a comparison of the photolysis of molybdenum hexacarbonyl, Mo(CO)(6), in 1-decene either as liquid droplets or in bulk-liquid solutions. Mo(CO)(6) photolysis is faster by at least three orders of magnitude in the aerosols than in bulk-liquids. Two possible sources of this enhancement are considered: (1) increased light intensity due to either Morphology-Dependent Resonances (MDRs) in the spherical aerosol particles and/or to increased pathlengths for light inside the droplet due to refraction, which are termed physical effects in this paper; and (2) interface effects such as an incomplete solvent-cage at the gas-liquid boundary and/or enhanced interfacial concentrations of Mo(CO)(6), which are termed chemical effects. Quantitative calculations of the first possibility were carried out in which the light intensity distribution in the droplets averaged over 215-360 nm was obtained for 1-decene droplets. Calculations show that the average increase in light intensity over the entire droplet is 106%, with an average increase of 51% at the interface. These increases are much smaller than the observed increase in the apparent photolysis rate of droplets compared to the bulk. Thus, chemical effects, i.e., a decreased solvent-cage effect at the interface and/or enhancement in the surface concentration of Mo(CO)(6), are most likely responsible for the dramatic increase in the photolysis rate. Similar calculations were also carried out for broadband (290-600 nm) solar irradiation of water droplets, relevant to atmospheric conditions. These calculations show that, in agreement with previous calculations by Mayer and Madronich [B. Mayer and S. Madronich, Atmos. Chem. Phys., 2004, 4, 2241] MDRs produce only a moderate average intensity enhancement relative to the corresponding bulk-liquid slabs when averaged over a range of wavelengths characteristic of solar radiation at the Earth's surface. However, as in the case of Mo(CO)(6) in 1-decene, chemical effects may play a role in enhanced photochemistry at the aerosol-air interface for airborne particles.