Copyright © 2015 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc. Unauthorized reproduction of this article is prohibited. Ophthal Plast Reconstr Surg, Vol. 31, No. 4, 2015 325 Purpose: To investigate and compare the histologic compositions of the pretarsal, preseptal, and orbital orbicularis oculi muscle (OOM) using nonpreserved, fresh-frozen, human cadavers. Methods: The OOM was exposed using sharp and blunt dissection. A metric ruler was used to measure and mark 0.5 cm × 1 cm samples from each portion of the right, superior OOM. Samples were excised, fixed in formalin, and completely embedded in paraffin. Five-micrometer-thick, hematoxylin- and eosin-stained sections were generated for each sample and analyzed by an anatomical pathologist. The relative percentages of the 4 main tissue types (skeletal muscle, fibrous tissue, adipose tissue, and neurovascular tissue) were quantified. Results: Forty-two samples were obtained from 14 Caucasian cadavers. On average, the pretarsal samples were composed of 83.5% skeletal muscle, 0.0% adipose, 5.0% neurovascular, and 11.5% fibrous tissue. Average preseptal OOM was 46.5% skeletal muscle, 12.7% adipose, 9.2% neurovascular, and 31.5% fibrous tissue. The orbital OOM was, on average, 42.7% skeletal muscle, 32.7% adipose tissue, 6.9% neurovascular, and 17.7% fibrous tissue. Conclusions: The OOM represents a histologically heterogeneous structure. (Ophthal Plast Reconstr Surg 2015;31:325–327) T he orbicularis oculi (orbicularis palpebrarum) muscle is critical to ocular health and an essential structure in oph- thalmic plastic surgery and neurotoxin therapy. The protractor is composed of pretarsal and preseptal portions (collectively, the pars palpebralis) and an orbital portion (pars orbitalis). 1,2 In general, the former controls involuntary narrowing of the palpebral fissure and the latter produces eyelid closure with increasing force and volition. The orbital orbicularis also acts as a major eyebrow depressor. 3,4 Along the eyelid margin, a very small component of the orbicularis called the muscle of Riolan likely plays a role in meibomian gland secretion, eyelid-globe apposition, and cilia position. 2,5,6 A PubMed search in September 2014 using the search parameter “orbicularis oculi” yielded 1,522 titles with only a few studies on orbicularis microstructure. Most of these stud- ies explored microscopic relationships to other structures, spe- cific muscle fiber types, or stained for neuromuscular junction locations. 7–15 Despite its importance, the authors are not aware of previous comparative histologic studies. They sought to com- pare the histologic compositions of the pretarsal, preseptal, and orbital orbicularis using light microscopic examination of fresh- frozen cadaveric human tissue. METHODS This study was performed using nonpreserved human cadavers registered in the Cleveland Clinic Body Donation Program. Specimens were excluded from this study if previous head and/or neck dissection had been performed or if there were signs of ocular and periocular trau- ma including cornea donation. The same surgeon (B.R.C.) performed all dissections and all measurements. Only the right upper eyelid was dissected, and this location was chosen at random. Data collection in- cluded age, gender, and race. Superficial skin incisions were created 1 cm lateral and parallel to a line connecting the supraorbital notch and the infraorbital foramen and from the lateral canthus to the superior border of the tragus. The orbicularis oculi muscle (OOM) was exposed using a combination of sharp and blunt dissection and 1.0 cm horizontal × 0.5 cm vertical rect- angles were excised using a no. 15 blade, 0.5 mm forceps, and Westcott scissors (Fig. 1). Each specimen was fixed in 10% buffered formalin, completely embedded in paraffin, cut in a singe 5-μm-thick section, and stained with hematoxylin and eosin. Each sample was evaluated by light microscopy at ×2 magnifica- tion for relative quantities of the 4 main histologic components, includ- ing skeletal muscle, fibrous tissue, adipose tissue, and neurovascular tissue by an anatomical pathologist (T.P.P.; Figs. 2–4). Percentages were determined by visual inspection and estimation using a technique simi- lar to that described by Markin et al. 16 for hepatic adiposity. Continuous DOI: 10.1097/IOP.0000000000000430 Accepted for publication January 14, 2015. The authors have no financial or conflicts of interest to disclose. Address correspondence and reprint requests to Bryan R. Costin, M.D., Cole Eye Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. E-mail: brc3@case.edu Regional Variations in Orbicularis Oculi Histology Bryan R. Costin, M.D.*, Thomas P. Plesec, M.D.†, Laura J. Kopplin, M.D., Ph.D.‡, Rao V. Chundury, M.D., M.B.A.*, Jennifer M. McBride, Ph.D.§, Mark R. Levine, M.D.║, and Julian D. Perry, M.D.* *Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio and †Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio; ‡Casey Eye Institute, Oregon Health and Science University, Portland, Oregon; §Department of Anatomy, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio; ║Lorain Institute, Cleveland Clinic, Lorain, Ohio. FIG. 1. External photograph of cadaver no. 10 demonstrates the dissected orbicularis with 0.5 cm × 1.0 cm sections marked for excision and analysis. ANATOMY & PHYSIOLOGY