Inflammation and the Nervous System: The Connection in the Cornea in Patients with Infectious Keratitis The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Cruzat, Andrea, Deborah Witkin, Neda Baniasadi, Lixin Zheng, Joseph B. Ciolino, Ula V. Jurkunas, James Chodosh, Deborah Pavan-Langston, Reza Dana, and Pedram Hamrah. 2011. “Inflammation and the Nervous System: The Connection in the Cornea in Patients with Infectious Keratitis.” Investigative Opthalmology & Visual Science 52 (8) (July 11): 5136. doi:10.1167/ iovs.10-7048. Published Version 10.1167/iovs.10-7048 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:34387112 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Cornea Inflammation and the Nervous System: The Connection in the Cornea in Patients with Infectious Keratitis Andrea Cruzat,1,2 Deborah Witkin,1 Neda Baniasadi,1,2 Lixin Zheng,1 Joseph B. Ciolino,2 Ula V. Jurkunas,2 James Chodosh,2 Deborah Pavan-Langston,2 Reza Dana,2 and Pedram Hamrah1,2 PURPOSE. To study the density and morphologic characteristics diminishment of corneal nerves and the increase of DC density of epithelial dendritic cells, as correlated to subbasal corneal was observed (r ϭϪ0.44; P Ͻ 0.0005). nerve alterations in acute infectious keratitis (IK) by in vivo CONCLUSIONS. IVCM reveals an increased density and morpho- confocal microscopy (IVCM). logic changes of central epithelial DCs in infectious keratitis. METHODS. IVCM of the central cornea was performed prospec- There is a strong and significant correlation between the in- tively in 53 eyes with acute bacterial (n ϭ 23), fungal (n ϭ 13), crease in DC numbers and the decreased subbasal corneal and Acanthamoeba (n ϭ 17) keratitis, and in 20 normal eyes, nerves, suggesting a potential interaction between the immune by using laser in vivo confocal microscopy. Density and mor- and nervous system in the cornea. (Invest Ophthalmol Vis Sci. phology of dendritic-shaped cells (DCs) of the central cornea, 2011;52:5136–5143) DOI:10.1167/iovs.10-7048 corneal nerve density, nerve numbers, branching, and tortuos- ity were assessed and correlated. It should be noted that due to orneal nerves are of great interest to clinicians and scien- the “in vivo” nature of the study, the exact identity of these Ctists, because of their important roles in regulating corneal DCs cannot be specified, as they could be monocytes or tissue sensation, epithelial integrity, proliferation, wound healing, macrophages, but most likely dendritic cells. and for their protective functions.1,2 The cornea is a densely RESULTS. IVCM revealed the presence of central corneal DCs in innervated tissue supplied by the terminal branches of the 3 all patients and controls. The mean DC density was signifi- ophthalmic division of the trigeminal nerve as ciliary nerves. cantly higher in patients with bacterial (441.1 Ϯ 320.5 cells/ Corneal nerves penetrate the corneal periphery in a radial mm2; P Ͻ 0.0001), fungal (608.9 Ϯ 812.5 cells/mm2; P Ͻ distribution, parallel to the superficial corneal surface, be- 0.0001), and Acanthamoeba keratitis (1000.2 Ϯ 1090.3 cells/ tween the Bowman’s layer and the basal epithelium, configur- 2 Ͻ Ϯ ing the subbasal nerve plexus that supplies the overlying cor- mm ; P 0.0001) compared with controls (49.3 39.6 cells/ 3 mm2). DCs had an increased size and dendrites in patients with neal epithelium. In vivo confocal microscopy (IVCM) is a noninvasive pro- IK. Corneal nerves were significantly reduced in eyes with IK cedure that allows imaging the living cornea at the cellular compared with controls across all subgroups, including nerve level, providing images comparable with histochemical meth- density (674.2 Ϯ 976.1 vs. 3913.9 Ϯ 507.4 ␮m/frame), total ods. IVCM enables the study of corneal cells, nerves, and the nerve numbers (2.7 Ϯ 3.9 vs. 20.2 Ϯ 3.3), main trunks Ϯ Ϯ Ϯ immune cells in different ocular and systemic diseases, and (1.5 2.2 vs. 6.9 1.1), and branching (1.2 2.0 vs. after corneal surgery.4,5 The complex stromal and epithelial Ϯ Ͻ 13.5 3.1; P 0.0001). A strong association between the branching of corneal nerves is not visible by conventional slit-lamp biomicroscopy, but can be visualized by IVCM. Our group has recently demonstrated the loss of corneal From the 1Ocular Surface Imaging Center and 2Cornea and Refrac- sensation in patients with herpes simplex keratitis (HSK), tive Surgery Service, Massachusetts Eye & Ear Infirmary, Department of which is strongly correlated with the profound diminishment Ophthalmology, Harvard Medical School, Boston, Massachusetts. of the subbasal nerve plexus in these patients by IVCM with a Presented in part at the Biennial Cornea Research Conference, slit scanning confocal microscope (Confoscan 4; Nidek Inc., Boston, Massachusetts, October 2009, and at the annual meeting of the Gamagori, Japan).6 Furthermore, IVCM revealed that the loss of Association of Research in Vision and Ophthalmology, Fort Lauderdale, the subbasal nerve plexus started within days of acute HSK Florida, May 2010. onset. More recently, we also observed a profound diminish- Supported by NIH K12-EY016335 (PH), NIH K08-EY020575 (PH), ment of the subbasal corneal nerve plexus in a pilot study of NIH K24-EY19098 (RD), New England Corneal Transplant Research Fund (PH), Falk Medical Research Foundation (PH), and an unre- patients with fungal and Acanthamoeba keratitis by laser stricted grant to the Department of Ophthalmology, Harvard Medical IVCM, where we observed the close proximity and apposition School, from Research to Prevent Blindness, New York, New York. of subbasal nerves with epithelial dendritic cells in the cornea The funding organizations had no role in the design or conduct of this (Kurbanyan K, et al. IOVS 2009;50:ARVO E-Abstract 2402). research. Dendritic cells, the most potent antigen-presenting cells Submitted for publication December 13, 2010; revised February 8, (APCs) of the body, are strategically positioned as immune and March 19, 2011; accepted March 26, 2011. sentinels ready to respond to invading pathogens in peripheral Disclosure: A. Cruzat, None; D. Witkin, None; N. Baniasadi, tissues.7 Dendritic cells have been shown to be critical for the None; L. Zheng, None; J.B. Ciolino, None; U.V. Jurkunas, None; initiation of adaptive immune responses and for maintenance J. Chodosh, None; D. Pavan-Langston, None; R. Dana, None; P. Hamrah, None of peripheral tolerance. Dendritic cells also represent the prin- Corresponding author: Pedram Hamrah, Ocular Surface Imaging cipal immune sentinels to the foreign world in the cornea and Center, Cornea and Refractive Surgery Service, Massachusetts Eye & ocular surface (reviewed in Refs. 8 and 9). While central cor- 10–12 Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA neal dendritic cells were initially described in mice, their 02114; [email protected]. presence has now been confirmed in humans, both by ex vivo Investigative Ophthalmology & Visual Science, July 2011, Vol. 52, No. 8 5136 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. IOVS, July 2011, Vol. 52, No. 8 Dendritic Cells and Corneal Nerves in Infectious Keratitis 5137 studies13–15 and more recently by IVCM at the level of the basal section of the cornea of 400 ϫ 400 ␮m, which is 160,000 ␮m2,ata epithelium and Bowman’s layer in the central cornea.16–18 selectable corneal depth and is separated from adjacent images by Previous studies in the skin and gut literature have demon- approximately 1 to 4 ␮m, with a lateral resolution of 1 ␮m/pixel. strated the bidirectional interplay of the immune and nervous Digital images were stored on a computer workstation at 30 frames per system.19–21 Further, experimental studies have shown the second. A disposable sterile polymethylmethacrylate cap (Tomo-Cap; immunomodulatory function of nerves via neuropeptides in Heidelberg Engineering GmbH, Dossenheim, Germany), filled with a mice.20–22 In the present study, our aim was to evaluate the layer of hydroxypropyl methylcellulose 2.5% (GenTeal gel; Novartis density and morphologic characteristics of epithelial dendritic- Ophthalmics, East Hanover, NJ) in the bottom, was mounted in front of shaped cells (DCs) and to correlate them to the subbasal the cornea module optics for each examination. One drop of topical corneal nerve alterations by laser IVCM in a prospective fash- anesthesia 0.5% proparacaine hydrochloride (Alcaine; Alcon, Fort ion in patients with infectious keratitis (IK). Although tissue Worth, TX) was instilled in both eyes, followed by a drop of hydroxy- macrophages or monocytes can have this morphology, we propyl methylcellulose 2.5% (GenTeal gel, Novartis Ophthalmics) in attributed the dendritic-shaped cells to dendritic cells. Our data both eyes. One drop of hydroxypropyl methylcellulose 2.5% was also demonstrates that diminishment of the subbasal nerve plexus placed on the outside tip of the cap to improve optical coupling, and in IK significantly correlates with the increase of epithelial DC manually advanced until the gel contacted the central surface of the density in these patients. cornea. A total of six to eight volume and sequence scans were obtained from the center of each cornea, at least three of which were sequence PATIENTS AND METHODS scans with particular focus on the subepithelial area, the subbasal We conducted a prospective, cross-sectional study, in a controlled, nerve plexus, and epithelial dendritic cells, typically at a depth of 50 to single-blinded fashion. Fifty-three eyes of 53 patients with diagnosis of 80 ␮m (Fig.