Abstract
Purpose.:
The rat facial nerve (CN VII) controls the orbicularis oculi (OO) muscle, which contracts to close the palpebral fissure during blinking. It was recently observed that rats are able to achieve nearly complete eye closure shortly after CN VII lesion, and hypothesized that the retractor bulbi (RB) muscle assumes an important compensatory role after CN VII lesion. This study was undertaken to determine the maintenance of rat corneal health and eye closure capability after lesion of the OO, RB, or both.
Methods.:
Twenty-two rats underwent RB transection; 12 of them had undergone complete unilateral CN VII transection (OO denervation) 15 weeks earlier. Corneal appearance and ability to blink in response to a corneal air puff was monitored weekly for 9 weeks. An additional 13 rats received CN VII transection and were video recorded (1000 frames/s) during elicited blinks at days 1, 3, 5/6, and 11 after surgery.
Results.:
Rats achieved nearly full or full eye closure after OO paralysis or RB myotomy, respectively. Ninety-two percent of rats maintained good corneal health after OO denervation over 9 weeks, consistent with compensatory eyelid movement served by the RB muscles. In contrast, only 40% of rats with loss of RB function alone and only 17% of rats with concurrent OO and RB paralysis were able to maintain corneal health by week 3.
Conclusions.:
Like other small mammals, the rat RB musculature can support nearly complete eye closure when CN VII is lesioned, and must be carefully considered when using blink as a functional recovery parameter of facial nerve lesion.
The rat facial nerve (CN VII) controls the orbicularis oculi (OO) muscle, which is responsible for rapid eye closure during blinking. Division of the nerve results in an initial loss of complete eye closure during blinking, both spontaneously and in response to corneal stimulation.
1 We recently observed that rats regain their ability to close their eyes nearly completely within the first week of CN VII lesion,
1 well before regenerated axons would be expected to arrive to repopulate the neuromuscular junctions of the OO.
2 This observation, coupled with our prior findings that rats are able to maintain their corneal health throughout periods of facial paralysis,
3 indicates that corneal protection can be achieved in the absence of CN VII function. Although retractor bulbi (RB) muscles have been described in the rat,
4,5 their role in eyelid closure has not yet been described. Based on rat ocular anatomy and the time course of compensatory eyelid closure after facial nerve lesion, we hypothesized that rat eyelid closure may be under dual OO and RB control, as has been shown in some other small mammals.
6–8 In the present study, we report our observations of corneal health and elicited ocular closures after OO paralysis, RB myotomy, or both, and demonstrate multiple eye closure mechanisms in the rat. Such dual input to ocular closure behavior must be carefully considered by investigators who employ rodent models of facial nerve injury and recovery.
9–12
Thirty-eight adult female Wistar rats weighing 200 to 300 g underwent surgical manipulations, in which anesthesia was induced by intramuscular injection of ketamine HCl and medetomidine HCl (60 mg/kg and 0.5 mg/kg, respectively). First they underwent implantation of a previously described titanium head fixation device
13 for facial nerve testing and behavioral conditioning to our previously described rodent facial nerve-testing apparatus.
1 In the first group (
n = 12), rats underwent complete unilateral facial nerve transection to paralyze the OO, where the proximal nerve stump was buried in the sternomastoid muscle belly to prevent axonal resprouting to facial muscle targets. This OO paralysis group underwent weekly monitoring of corneal health as well as global facial nerve testing across postoperative weeks 3 to 12. Eyelid movement was measured by determining changes in infrared (IR) light reflection from the eye surface with dual IR emitter/detector units located in front of each eye.
1,14,15 Animal experimentation adhered to protocols approved by the Massachusetts Eye and Ear Infirmary IRB and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Fifteen weeks after CN VII transection, 11 of these rats underwent a second surgical manipulation, in which resection of the ipsilateral RB muscle was performed via a superior orbitotomy in the retrobulbar region, transitioning them to an OO paralysis and RB myotomy state. Eye surgery was performed by a team of two microsurgeons, under microsurgical magnification (40×) with a double-headed microscope (Wild M651; Leitz, Rockleigh, NJ), being careful not to damage the long ciliary nerves responsible for corneal sensation. The RB was lysed with a microsurgical dissecting needle as it inserted circumferentially onto the deep surface of the globe. Rats underwent weekly testing sessions and visual inspections of corneal health for an additional 9 weeks. Testing of particular rats was stopped if the IR tracing became uninterpretable because of corneal opacification or ulceration.
After head fixation device implantation and conditioning, a second set of rats (n = 10) underwent unilateral RB resection without facial nerve lesion, followed by the same 9-week monitoring of postoperative corneal health and induced blink. As in the other lesioned rats, testing was stopped if the IR tracing became uninterpretable due to deterioration of corneal health.
In a third set of rats (
n = 13), elicited blinks were video recorded with high speed equipment after complete unilateral CN VII transection on days 1, 5, or 6, and 11 after surgery. Video centered on the eye at close range was captured at 1000 frames/s (240 × 256 pixels), and a single frame representing baseline (open) and maximal eyelid closure was analyzed using ImageJ software (available at
http://rsb.info.nih.gov/ij/ developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Eyelid closure was elicited by several instances of periocular stimulation with a blunt probe, while being careful to exclude instances in which this instrument may have influenced eyelid position. Analyzed still-frame images were thresholded to identify the exposed corneal surface, and the automated ellipse tool in ImageJ was used to objectively define and measure the shape of the exposed cornea within the palpebral fissure. Complete closure was not achieved by the OO paralyzed rats, and so there was measurable corneal surface in each trial. The major-over-minor axis length was measured and averaged among rats for each day and condition to represent average exposed corneal shape during attempted closure by postsurgical day. Averages were statistically compared in six two-tailed
t-tests (α level of
P < 0.05).
The ability of mammals to retract the globe into the orbit during blinking has been described in many species.
6 When OO function is lost in some small mammals such as rabbits and cats, eyelid closure is achieved through compensatory contraction of the RB muscle system.
6–8 The muscle inserts circumferentially on the deep surface of the globe and retracts the globe into the orbit causing the eyelids to passively slide across the cornea to achieve eyelid closure.
In this study, the rat RB was shown to play a compensatory role when the facial nerve was lesioned, enabling substantial (yet incomplete) eyelid movements in response to periocular stimulation within the first week of OO paralysis. Moreover, the RB may normally contribute to reflexive and spontaneous blinking, since RB myotomy slightly attenuated eyelid movement in response to corneal air puff, and corneal health was degraded in most of the RB-lesioned rats as soon as 3 weeks after myotomy. Both of these observations, however, could be attributable to ocular bulging after RB myotomy and the possible reduction of corneal sensation caused by sensory nerve injury to the eye (despite concerted attempts to protect the ciliary nerve), particularly in light of the fact that rats receiving RB myotomy typically achieved complete reflexive eyelid closure in our testing apparatus, yet usually failed to maintain ocular health. Nevertheless, the fact that elicited eyelid movements persisted after complete facial nerve lesion (OO paralysis) is consistent with the presence of RB musculature in rats
4,5 and the RB-mediated eye closure demonstrated in some other small mammals.
6–8
Our identification of dual eyelid closure mechanisms with differing neural inputs in the rat has significant implications for those studying rodent facial nerve regeneration. Many investigators have depended on the rat vibrissal system alone in gauging functional recovery after nerve manipulation, although this represents only a single function of the multibranched facial nerve. Current research includes examining functional outcomes in multiple facial zones,
1,16 to address aberrant regeneration and synkinetic facial movements. The present findings show how it is quite possible to mistake RB-mediated eyelid movements for those controlled by the facial nerve. For example, some investigators have recently claimed that the appearance of a “semieyeblink” response in rats is the hallmark of early facial nerve recovery,
17 drawing conclusions about facial nerve manipulations based on this eyelid response without acknowledging the potential role of RB contraction.
Unfortunately, mitigating the RB contribution to eyelid movement after facial nerve lesion is not a simple process. With the current findings, we have established that rats cannot tolerate complete loss of blink reflex, as in our dual OO/RB lesion condition, because corneal exposure leads almost invariably to ulceration. Botulinum toxin injection of the orbital muscles can effectively paralyze the RB (Hadlock TA, unpublished observation, 2008), but leads to massive proptosis and globe herniation in the absence of OO function. It may be possible to identify and selectively lesion the brain stem motoneurons supplying the RB, but placement of chronic electromyographic electrodes in the OO muscles for investigating facial nerve-mediated eyelid movement is likely to be an easier approach. Future studies will be conducted to explore these possibilities, along with signal analysis techniques that differentiate OO from RB eyelid IR sensor responses characteristics with the goal of “subtracting” the RB contribution from the eyeblink measurements after facial nerve manipulation.
Supported by National Institute of Dental and Craniofacial Research (NIDCR) Grant K-08 DE015665-01A2.
Disclosure:
J.T. Heaton, None;
J. Kowaleski, None;
C. Edwards, None;
C. Smitson, None;
T.A. Hadlock, None