Corresponding Author: Jeong-Min Hwang, MD. Department of Ophthalmology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea. Functional electrical stimulation (FES) has been proposed as a potential therapeutic approach for restoring motor function [1-5]. FES may help prevent muscle atrophy, facilitate muscle reinnervation from motor neurons, support the reconnection of the muscle to its original endplate and nerve fibers, and promote the potential for spontaneous recovery [6-8]. However, any histologic study to investigate whether FES is safe for the ocular tissues when applied to the extraocular muscles has not been performed. Additionally, for FES to be finally applied in humans, both safety and minimal discomfort of foreign body sensation should be ensured.
Ocular motor nerve palsies are clinically important because they are most commonly associated with microvascular ischemia [9-11], and could be a significant risk factor of stroke [12]. Furthermore, ocular motor nerve palsies can cause diplopia and loss of stereopsis, which could be debilitating and severely impact the quality of life [9].
To investigate whether FES would be helpful for ocular motor nerve palsy, we need an electrode to stimulate the extraocular muscle. Needle electrodes can cause ocular morbidity including hematoma, edema, and scleral laceration [13]. The purpose of our study was to develop an effective, noninvasive and safe electrode for the electric stimulation of extraocular muscles, and to explore the safety and feasibility of the electrode to stimulate the extraocular muscles using rabbits.
Materials and Methods
Ethics statement
The study protocol was approved and monitored by the Institutional Animal Care and Utilization Committee of Seoul National University Bundang Hospital (No. BA-2202-338-002-10) and adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All methods were conducted in strict accordance with the relevant guidelines and regulations. All experiments of this study comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Development of a flexible electrode for electrical stimulation of extraocular muscles
The electrodes were fabricated using a standard photolithography process (Fig. 1, 2A-2C) [14]. Initially, a 20-μm-thick layer of photosensitive polyimide was deposited onto a titanium/silicon substrate and developed in a cyclopenta-none solution. The developed polyimide layer was then thermally treated in an oven at 300 °C for 1 hour. Next, a photoresist (AZ 5214E, Merck) was patterned onto the polyimide layer, followed by the deposition of a 30 nm titanium layer and a 300-nm platinum layer. After the patterning process, the metal-photoresist was removed and cleaned using AZ 300 MIF solution (Merck). An additional 10-μm-thick polyimide layer was introduced as a capping layer. The titanium bottom layer was etched in a buffered oxide etchant solution to release the electrodes from the substrate. The area of the exposed electrode circles is 0.16π mm2. Finally, the fabricated electrodes were connected to commercially available wires (TE/S43-638, Technomed).
Electric stimulation of rabbit extraocular muscles
Five New Zealand white rabbits, weighing 3 to 4 kg, underwent general anesthesia with intramuscular 30 to 45 mg/kg of alfaxalone (Alfaxan, Jurox) and 5 mg/kg of xylazine hydrochloride. Topical anesthesia using proparacaine hydrochloride (Paracaine, Hanmi Pharmaceutical) was also done. A total of 12 times of electric stimulation composed of 1 pulse per second and 2.0 mA for 0.1 milliseconds on the right superior rectus muscle for 5 minutes (Fig. 2), then on the inferior rectus muscle for 5 minutes using an electromyograph (Medelec Synergy PIU, Natus Neurology) three times a week for 4 weeks in the right eye was performed.
Histologic examination
Following a 4-week electric stimulation period, the rabbits were anesthetized via intravenous injection of alfaxalone 6 mg/kg and xylazine 5 mg/kg. After the administration of anesthesia, a double dose of alfaxalone and xylazine was utilized to induce deeper anesthesia, after which the animals were sacrificed via intravenous injection of potassium chloride. Following sacrifice, the eyes were enucleated. The enucleated eyes were fixed in 10% buffered formalin for 48 hours, and the part of the superior rectus muscle and the inferior rectus muscle in contact with the electrode were resected from both eyeballs. Each eyeball was divided into superior and inferior halves. Serial cross sections of each muscle and eyeball were separately fixed in each cassette. Eight specimens were obtained from each rabbit, consisting of right and left, superior rectus muscle, inferior rectus muscle, superior and inferior eyeball including retinal pigment epithelium, sclera, and ciliary body. Total 40 specimens were formalin-fixed and paraffin-embedded by routine tissue processing methods, and 4-μm-thick sections were cut and stained by hematoxylin-eosin stain and Masson trichrome stain. To investigate the impact of electric stimulation on extraocular muscles and ocular structure, light microscopic evaluation was performed by a pathologist.
Results
Direct electrical stimulation of extraocular muscles using a flexible electrode could elicit eye movement successfully in all the rabbits without any event. By histologic examination, electrically stimulated right side was compared with nonstimulated left side in extraocular skeletal muscles and ocular structures. In both sides, there was no histological features of electrical injury (electric burn) such as coagulation necrosis, rhabdomyolysis, degenerated myofiber, degeneration of collagen, acute or chronic inflammation, hemorrhage, endomysial fibrosis, and scar tissue formation. Ocular structures including retinal pigment epithelium, full thickness of sclera and ciliary body revealed no abnormal histologic changes in the right side as well as the left side. Both superior and inferior rectus muscles showed smaller diameter of myofibers in the peripheral area near fascia compared to the central area myofibers. Endomysial fibrosis was noted in the subfascial peripheral area with smaller myofibers. This perifascicular atrophy-like histologic feature was noted in the nonstimulated left side as well as the electrically stimulated right side (Fig. 3A-3F). In addition, the diameter of myofibers showed no difference between the right side and the left side when central area and peripheral area were compared, respectively. Therefore, electrically stimulated right superior rectus muscle and inferior rectus muscle showed neither hypertrophy nor atrophy, compared with nonstimulated left extraocular muscles.
In summary, histologic examination demonstrated no evidence of detrimental effects of electrical stimulation.
Discussion
At present, electrical stimulation devices for human extraocular muscles remain unavailable. This study tried to lay the foundation for electric stimulation as a new treatment modality for ocular motor nerve palsy, with the ultimate goal of developing an electrical stimulation device. In this study, we developed a very thin and flexible electrode for electrical stimulation of extraocular muscle and evaluated its safety of applying direct electrical stimulation to extraocular muscles as well as its potential effects on ocular tissues in rabbits [14]. We found that direct electrical stimulation of extraocular muscles using a flexible electrode could safely elicit eye movement without ocular damage in rabbits.
Electrical stimulation has been used as a treatment for nervous system disorders for a long time, despite poor understanding of the mechanism [15]. Functional improvements from electrical stimulation could achieve locomotion after spinal cord injury [16-20]. Furthermore, electrical stimulation of facial muscles using surface electrodes produced better movement even in complete or long-standing facial nerve palsies. More recently, synchronized FES with the unaffected side in the paralyzed orbicularisoculi muscle was tried to achieve a natural-looking eye blinking [21]. If we could develop such system for unilateral long-standing ocular motor nerve palsy, it could be helpful for these patients. However, electrical stimulation has not been applied to ocular motor palsy in humans. Therefore, it would be necessary to find out whether electrical stimulation also has a similar effect on extraocular muscles. As a first step in investigating the therapeutic effect of electrical stimulation of extraocular muscles, we tried to develop an electrode in this study.
The most critical part of developing an electrode to stimulate extraocular muscles is the safety of the eye. Another vital consideration for the development of an electrode may be its feasibility. The electrode should not be invasive nor cause any pain. We designed such an electrode that is biocompatible, thin, small, and flexible. In addition, the surface should be smooth so there is no damage to the surrounding tissue. Compared to previous electrodes for extraocular muscles [22,23], our platinum electrode could provide high biocompatibility and ensure greater stability [14]. With a thickness of less than 30 μm, it exhibits high flexibility and can be easily applied to the confined ocular space. The use of semiconductor microelectromechanical system processing allows for the fabrication of small and fine structures. Due to the thin electrode thickness, the impedance could be higher than those reported [22,23]. With these characteristics of smooth, thin, flexible, higher biocompatibility, and greater stability of platinum electrodes, our electrode could be promising as a future electric stimulation system for extraocular muscles in humans.
Regarding the dose of electric stimulation, there have been many studies on skeletal muscles with a spinal cord injury. A long and low-force electrically induced exercise with stimulation frequencies <5 Hz might be safe for chronic spinal cord injury [16,24]. Petrie et al. [16,24,25] reported that the 3 Hz protocol is more acceptable than the 1 Hz protocol for chronic spinal cord injury. In terms of the adequate dose for an extraocular muscle, any treatment that causes extraocular muscle contraction through electrical stimulation has not yet been established, so there is still much to be researched. Further studies of electrically induced exercise on extraocular muscles may provide valuable insight to determine the specific dose required to stress paralyzed extraocular muscles adequately.
There are some limitations in this study. First, there are many differences between extraocular muscles and other skeletal muscles including an inner global layer and an outer orbital layer, nontwitch muscle fibers with multiple endplate zones, and palisade endings in addition to twitch muscle fibers with a single endplate zone [26]. Extraocular muscles have less time to peak tension, one-half relaxation time, higher twitch to tetanic tension ratio than skeletal muscles, and much higher maximum firing frequencies. These characteristics could contribute to making the extraocular muscles more energy-demanding than the skeletal muscles. Therefore, the response of extraocular muscles to electrical stimulation and the treatment effect may be different from that of other skeletal muscles. Second, extraocular muscles differently respond to denervation with less atrophy and without postparetic fiber-type grouping as in other skeletal muscles [27]. In addition, most of the response to denervation in extraocular muscles is in orbital singly innervated muscles, sparing multiply innervated fibers. Therefore, the efficacy of electric stimulation of denervated extraocular muscles may be different from that of other denervated skeletal muscles. Third, human extraocular muscles differ from those of other species in many respects [28]. Therefore, we could not extend the conclusions from our study in rabbits to humans. Fourth, only five rabbits were included, which may limit the generalizability of the findings. However, the electrode’s characteristics—extremely thin (less than 30 μm), highly flexible, and with a very smooth surface—are unlikely to cause significant differences in tissue response between individuals. Lastly, our study assessed histological changes after 4 weeks of electric stimulation. Additional studies evaluating longer-term stimulation effects are necessary before considering human applications.
In conclusion, our preliminary results are promising as an effective electrical stimulation of extraocular muscles without significant damage to the ocular structure. Further research is necessary to establish safety standards, and to determine optimal treatment settings.
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