Adult male New Zealand White rabbits were obtained from Bakkom Rabbitry (Viroqua, WI) and housed in the AALAC-approved animal facility at the University of Minnesota. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Minnesota, as well as complied with the guidelines for the Use of Animals in Research published by the Association for Research in Vision and Ophthalmology as well as the guidelines of the National Institutes of Health.
Rabbits were anesthetized via an intramuscular injection of ketamine/xylazine (1:1) at a dose of 10 mg/kg:2 mg/kg, respectively. The cornea was anesthetized by placement of a drop of proparacaine HCl in the conjunctival cul-de-sac. Three groups of animals were prepared. Group one received a single injection of 5 units of botulinum toxin A (Botox; Allergan, Los Angeles, CA) in 1 cc of sterile isotonic saline in one randomly selected upper eyelid and examined after one, two, or four weeks post-botulinum toxin treatment. All injections were made by inserting the needle into the central region of the eyelid with the needle tip pointing toward the medial canthus, with half the volume being dispensed and the needle slowly pulled toward the needle entry point. With the needle in place, the syringe was rotated and the needle directed toward the lateral canthus. The same slow injection procedure was performed with slow withdrawal of the needle. After dispensing the full volume, the needle was left in place for 30 seconds to prevent leakage. This method minimizes leakage, as only one needle stick is performed. Previous studies demonstrated that single injections of this volume spread into all regions of the treated eyelids.
22 Group two received a single injection of 5 units of botulinum toxin, followed on post-treatment days three and five with injection of corticotropin releasing factor (CRF; Peninsula Laboratories, Belmont, CA) (150 μg in 1 mL sterile saline).
18,23 Eyelids were examined two weeks after the final treatment. Group 3 received botulinum toxin injections similar to groups 1 and 2, followed on post-treatment days three and five with injections of an antibody to insulin growth factor-I receptor (anti-IGFIR; R&D Systems, Minneapolis, MN) at a dose of 30 μg/mL sterile saline.
23 Again, eyelids were examined two weeks after the final treatment. The contralateral upper eyelids were injected with sterile saline only in comparable volumes.
At the appropriate post-injection intervals, the rabbits were anesthetized deeply with ketamine and xylazine, followed by an overdose of barbiturate anesthesia. Both eyelids were trimmed to remove the fur and dissected completely to include the muscle at both the medial and lateral canthi. They were pinned to their in situ length in embedding molds, surrounded by tragacanth gum, frozen in methylbutane chilled to a slurry on liquid nitrogen, and stored at −80°C until sectioned and processed. The muscles were sectioned completely in the longitudinal plane at 12 μm, and the sections were mounted on gelatin-subbed microslides. Every tenth section was immunostained for the presence of neuromuscular junctions using α-bungarotoxin conjugated to Alexa Fluor 488 (Molecular Probes) at a concentration of 1:100 overnight at 4°C. The slides were coverslipped with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and analyzed the same day they were immunostained.
The muscle sections were examined for neuromuscular junction position and number using a microscope (Leica DMR; Leica, Wetzlar, Germany). Using image analysis software (Topographer program of NovaPrime; Bioquant, Nashville, TN), the area of the entire orbicularis oculi muscle in longitudinal section was measured at 1.6×. Every neuromuscular junction was located at 20× and marked with X and Y coordinates recorded in the Topographer program. This analysis was repeated for every tenth section through the entire eyelid. The image analysis program (Bioquant Topographer) was used to reconstruct the entire muscle, including the area outlines and locations of each neuromuscular junction. This allowed for a three-dimensional reconstruction of all the neuromuscular junctions in their actual X, Y, and Z planes within the entire muscle. Four orbicularis oculi muscles were examined for each of the experimental paradigms.
Density of neuromuscular junctions was calculated as the number of neuromuscular junctions per mm2. Statistical significance was determined between the densities of neuromuscular junctions in the saline-treated control muscles, the botulinum toxin A–only treated orbicularis oculi, and the co-treated eyelid muscles. Statistics were performed using an unpaired t-test aided by statistical software (Prism and Statmate software; Graphpad, San Diego, CA). An F-test was used to verify that the variances were not significantly different. Data were considered significantly different if P ≤ 0.05.