This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.
This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.
Cranial nerve injury in the head and neck region can be secondary to congenital, infectious, idiopathic, iatrogenic, traumatic, neurologic, oncologic, or systemic etiologies1. Cranial nerve VII, or the facial nerve, is commonly affected. The incidence of facial nerve dysfunction can be significant, as it affects 20 to 30 per 100,000 people each year2. The main motor branches of the facial nerve are the temporal, zygomatic, buccal, marginal mandibular, and cervical branches; depending on the branch involved, the consequences can include oral incompetence or drooling, corneal dryness, visual field obstruction secondary to ptosis, dysarthria, or facial asymmetry2,3. Long-term morbidity includes the phenomenon of synkinesis, or involuntary movement of one facial muscle group, with attempted voluntary contraction of a distinct facial muscle group. Ocular-oral synkinesis is the most common of the aberrant regeneration as a sequela of facial nerve injury and causes functional impairment, embarrassment, diminished self-esteem, and poor quality of life3. Injury to individual branches dictates the functions that are selectively compromised.
The clinical treatment of facial nerve injury is not well standardized and is in need of further research to improve outcomes. Steroids can alleviate acute facial nerve swelling, whereas Botox is useful for temporizing synkinetic movements; but, the primary reconstructive options in the practitioner's armamentarium involve surgical intervention through nerve repair, substitution, or reanimation3,4,5,6. Depending on the type of facial nerve injury sustained, the facial nerve surgeon may utilize a number of options. For simple transection, nerve reanastomosis is useful whereas cable-graft repair is better suited for a nerve defect; for a restoration of function, the surgeon may choose either static or dynamic facial reanimation procedures. In many cases of facial nerve injury and subsequent repair, even in the hands of experienced facial nerve surgeons, the best outcome still results in persistent facial asymmetry and functional compromise7.
These suboptimal outcomes have spurred extensive research on facial nerve regeneration. Broad topics of interest include perfecting and innovating nerve repair techniques, determining the effect of various nerve regeneration factors, and assessing the potential of specific neural inhibitors to help combat the long-term outcome of synkinesis8,9,10,11. While in vitro models can be used to assess some characteristics of pro-growth or inhibitory factors, true translational research on this subject matter is best accomplished via translatable animal models.
The decision of which animal model to utilize can be challenging, as researchers have utilized both large animals, such as sheep and small animal models, such as mice12,13. While large animal models offer ideal anatomic visualization, their use requires specialized equipment and personnel not readily or easily available. Furthermore, powering a study to demonstrate effect could be highly cost-prohibitive and potentially not within the feasible scope of many scientific centers. Thus, the small animal model is most frequently utilized. The mouse model can be utilized for assessing a number of outcomes related to facial nerve surgery; however, the limited length of the nerve can restrict the scientist's ability to model certain patterns, such as large-gap injury14.
Thus, the rat murine prototype has emerged as the workhorse model through which the scientist can perform innovative surgical procedures or utilize inhibitory or pro-growth factors and assess effect across a broad range of outcome parameters. The rat facial nerve anatomy is predictably and easily approached in a reproducible fashion. Its larger scale, in comparison to the mouse model, allows for modeling of a wide range of surgical defects, ranging from simple transection to 5 mm gaps15,16. This further allows for the application of complex interventions at the defect site, including the topical placement of factor, intraneural injections of factor, and the placement of isografts or bridges17,18,19,20,21,22,23.
The docile nature of the rat, its reliable anatomy, and its propensity for effective nerve regeneration allows for the collection of many outcome measures in response to the aforementioned surgical patterns of injury24. Via the rat model, the facial nerve scientist is able to assess electrophysiologic responses to injury, nerve and muscle histologic outcomes via immunohistochemistry, functional outcomes via tracking movement of the vibrissal pad and assessing eye closure, and micro- and macroscopic changes via fluorescent or confocal microscopy, among others11,22,23,25,26,27,28,29. Thus, the following protocol will outline a surgical approach to the rat facial nerve and the injury patterns that can be induced.
All interventions were performed in strict accordance with the National Institutes of Health (NIH) guidelines. The experimental protocol was approved by the University of Michigan's Institutional Animal Care & Use Committee (IACUC) prior to implementation. Ten-week-old adult female Sprague-Dawley rats were utilized.
1. Prior to the operative day
2. Preoperative setup
3. Anesthesia and preparation
4. Surgical approach and injury patterns
5. Wound closure
6. Postoperative recovery
Following the initial surgical procedure, there are two main types of outcome measures: serial measurements in the live animal and measurements that require sacrificing the animal. Examples of serial measurements include electrophysiological assays, such as a compound muscle action potential measurement30, assessments of facial muscle movement via laser-assisted or videography means9, or even repetitive live imaging of regrowth of the facial nerve in fluorescent transgenic animals31,32. Figure 1 illustrates live imaging of the main trunk of the facial nerve in an adult transgenic Thy1-GFP rat. A crush injury has been performed on the marginal mandibular branch, approximately 2-3 mm distal to the branch point of the first pes. Utilizing MetaMorph imaging software, we were able to quantify the fluorescent intensity at any point along the course of the facial nerve. In particular, it is possible to quantify fluorescence proximal and distal to an injury site, thereby serially assessing for the return of fluorescence as a marker for nerve regeneration. Figure 2 illustrates the gradual return of fluorescence (measured as a ratio of distal fluorescence to proximal fluorescence across a simple transection site) at the 1, 2, 3, and 4 week time-points in the Thy1-GFP rat.
Histomorphometric analysis of the nerve proper or muscle requires sacrificing the animal after a predetermined length of time to demonstrate the desired effect across groups. Figure 3 demonstrates cross-sectional images of the marginal mandibular division. This technique requires careful tissue handling, storage, preparation, sectioning, and staining to allow for a histomorphometric analysis across various groups. If appropriately performed, this technique allows for the quantification of the axonal diameter, amount of debris, nerve fiber, percentage of nerve, and density measurements.
Figure 1: Facial nerve anatomy demonstrated in the Thy1-GFP rat 1 week after a crush injury at the marginal mandibular nerve. The site of the crush injury is demonstrated by the white arrow. Sections of the nerve are labeled as MT (main trunk), B (buccal), and MM (marginal mandibular) branches. The scale bar represents 1.5 mm. Please click here to view a larger version of this figure.
Figure 2: Quantification of return of fluorescence as a ratio of the fluorescent intensity of the site immediately distal to the transection injury over the intensity of the site proximal to the transection injury. Four animals were studied, and a crush injury was modeled. The graph is plotted as mean proportion ± standard error of the mean. Please click here to view a larger version of this figure.
Figure 3: Axial cross-section of the marginal mandibular branch of the rat facial nerve following staining with toluidine blue. The scale bar represents 100 µm. Please click here to view a larger version of this figure.
The rat facial nerve injury model has emerged as the most versatile system for the evaluation of neurotrophic factors due to its surgical accessibility, branching pattern, and physiological significance27,29,33,34,35,36. The combination of video demonstration and application of transgenic animal data opens new possibilities for the scientific study of nerve regenerative phenomena. This model allows the systematic and detailed characterization of neural response to trauma, neurotrophic influences, immunomodulatory influences, and other aspects of the microenvironment. Although the primary objective of clinical facial nerve study relates to the recovery of motoneuron function, the model can also be used to better understand events at the neuromuscular junction, nuances of axonal transport, and the interplay of axonal-glial influences27,36,37,38,39,40,41,42,43,44. Mechanistic study using facial nerve has paradoxically led to important insights relating to central inflammation and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease45,46,47. There, the medical implications of work conducted in this model have notable implications for better understanding and eventually improving clinical care for patients afflicted with both peripheral and central nervous system disorders affecting both supporting cells and neuronal tissues.
There is an initial learning curve with performing facial nerve surgery in the rat model. While it is not necessary for the scientist to be surgically trained, they should be comfortable working underneath an operating microscope and utilizing binocular vision. The scientist should further be comfortable working with their nondominant hand, as adjusting the retraction with the Weitlaner retractor in the contralateral hand can significantly improve visualization. This is particularly true for identifying the main trunk of the facial nerve, as it is possible for the novice surgeon to lose landmarks and become disoriented in the three-dimensional space where the nerve exits the skull base. However, once experience is gained and the surgeon is consistently able to identify the tendon of the digastric muscle, then the procedures are quite straight-forward. Where the procedure-a simple transection of the nerve-could take as long as 30 min when the scientist is initially learning, in the hands of an experienced surgeon, it can be as expedient as 5 min from incision to wound closure. With an experienced assistant managing the preparation and the anesthesia of the animals-as well as restocking supplies as needed-it is feasible to operate on several animals in a single sitting. If complex intraoperative maneuvers are performed, such as a stereotaxic intraneural injection, then the time required will increase.
This group has experience working with the adult Lewis and Sprague-Dawley rats and the adult Thy1-GFP rat. These models have demonstrated impressive resilience, both intraoperatively and postoperatively. It would be prudent to allow time for the acclimation of the animal (typically 1 week) if ordered from a separate facility as mandated by the NIH Guide for Care and Use of Laboratory Animals, as operating too soon after a transfer could result in excessive stress and poor postoperative health. With unilateral facial nerve injury, the rat does not demonstrate untoward signs of malnourishment or corneal irritation postoperatively. Furthermore, they tolerate repetitive periods of anesthesia quite well48, as previous protocols have demanded serial electrophysiological analyses of the return of whisker function until eventual sacrifice. Although the operators should attempt to maintain intraoperative sterility to the best of their abilities, we have not noted postoperative infections with any of the operated animals. On occasion, the animal will attempt to scratch at their incision site; however, it typically returns to baseline status with regrowth of hair within 1-2 weeks postoperatively. If the parotid gland is injured or inadvertently removed, then sialocele will result, which may necessitate repeat anesthesia for drainage.
Modifications can be made to the location of injury, type of injury, or intervention performed. Injury can be induced at any point along the course of the facial nerve, from intracranial facial nerve transection to injury to the main trunk or any of its peripheral branches49. The broad patterns of injury include crush injury, simple transection with or without repair, and gap-defect with or without repair or bridge11. The range of possible interventions is exhaustive. Briefly, intervention can be undertaken at the level of the nerve proper17, at the site of the injury23, or at the muscular end-organ50. The list of possible outcome parameters is equally lengthy. Workhorse histomorphometric parameters include axial-based quantification of nerve count, nerve density, and percentage of nerve, among others. Additional measures include histologic analyses of longitudinal sections to illustrate the extent of regeneration and neuromuscular junction quantification to demonstrate the reinnervation of target facial musculature51. Novel methods of assessing outcomes continue to be developed22. For example, Hadlock et al. have demonstrated a complex method for assessing the contracture of independent facial zones via scent- or puff-induced reactions; this has potential for the assessment and eventual treatment of troublesome synkinetic regeneration11.
As with any animal model, there are limitations in translating the results to human patients. The mouse and rat models both exhibit the superior regenerative potential inherent in the rodent nervous system; this property allows the rodent to demonstrate regenerative results that could not possibly be achieved in humans and higher animals24. Therefore, the facial nerve scientist must carefully select appropriate time-points for the evaluation of nerve regeneration and recovery; if a prolonged time-point is selected, the narrow window of opportunity where experimental groups may reliable demonstrate the effect from their unique intervention may be missed24.
The authors have nothing to disclose.
S.A.A. is funded by the American Academy of Facial Plastic and Reconstructive Surgery Leslie Bernstein Grants Program.
1.8% isoflurane | VetOne | 13985-030-40 | |
11-0 nylon microsutures | AROSuture | TK-117038 | |
4-0 monocryl suture | VWR | 75982-084 | |
Buprenorphine SR | ZooPharm | MIF 900-006 | |
Carprofen | Sigma-Aldrich | MFCD00079028 | |
Chlorhexidine | VWR | IC19135805 | |
Jeweler forceps | VWR | 21909-458 | |
Micro Weitlaner retractor | VWR | 82030-146 | |
Micro-scissors | VWR | 100492-348 | |
Mini tenotomy scissors | VWR | 89023-522 | |
Number 15 scalpel blade | VWR | 102097-834 | |
Operating microscope | Leica | ||
Petrolatum eye gel | Pharmaderm | B002LUWBEK | |
Sterile water | VWR | 89125-834 | |
Tissue adhesive | Vetbond, 3M | NC9259532 | |
Water conductor pad | Aqua Relief System | ARS2000B |