Diurnal changes and topographical distribution of ocular surface epithelial dendritic cells in humans, and repeatability of density and morphology assessment

Abstract

Purpose Dendritic cells (DC) play a crucial role in ocular surface defence. DC can be visualised in vivo by confocal microscopy but have not yet been fully characterised in humans. This study investigated the diurnal variation, topographical distribution and repeatability of DC density and morphology measurements. Methods In vivo confocal microscopy (IVCM) was conducted on 20 healthy participants (mean age 32.7 ± 6.4 years, 50% female) at baseline and repeated after 30 minutes, 2, 6 and 24 h. Images were captured at the corneal centre, inferior whorl, corneal periphery, limbus and bulbar conjunctiva. DC were counted manually, and their morphology was assessed for cell body size, presence of dendrites, and presence of long and thick dendrites. Mixed‐model analysis, non‐parametric analyses, Bland and Altman plots, coefficient of repeatability (CoR) and kappa were used. Results There were no significant changes in DC density (p ≥ 0.74) or morphology (p > 0.07) at any location over the 24‐h period. The highest DC density was observed at the corneal limbus followed by the peripheral cornea (p < 0.001), with the lowest density at the corneal centre, inferior whorl and bulbar conjunctiva. Most DC at the corneal periphery, limbus and bulbar conjunctiva had larger cell bodies compared with the corneal centre (p ≤ 0.01), and the presence of long dendrites was observed mostly at non‐central locations. Day‐to‐day CoR for DC density ranged from ±28.1 cells/mm2 at the corneal centre to ±56.4 cells/mm2 at the limbus. Day‐to‐day agreement of DC morphology determined by kappa ranged from 0.5 to 0.95 for cell body size, 0.60 to 0.95 for presence of dendrites, and 0.55 to 0.80 for the presence of long dendrites at various locations. Conclusions No diurnal changes are apparent in corneal or conjunctival DC. Substantial topographical differences exist in DC density and morphology. IVCM provides good repeatability of DC density and acceptable agreement of DC morphology.

Increased dendritic cell density and altered morphology in allergic conjunctivitis

“…The bulbar conjunctiva had the lowest DC density compared to corneal locations in both groups. This aligns with previous reports [11,22,42] and animal work [45].…”

“…A gradient density from high DC density at the limbus to low density at the corneal centre and inferior whorl was observed. This aligns with previously reported in vivo findings in normal and inflamed corneas and ex vivo studies [11,12,22,36,[41][42][43][44]. DC have been observed to move the limbus to the corneal centre in porcine eyes in response to contact lens wear, and in mice following bone marrow cell transplantation [16,17].…”

“…Sample size was calculated using GPower 3.1. If the controls show a mean DC density of 30 cells/mm 2 at the corneal centre (SD = 35 cells/ mm 2 ) and the allergy group shows a mean density of 58 cells/mm 2 and similar SD, there would be greater than 80% power to detect the mean difference with 27 participants per group (54 total), assuming alpha of 0.05 [22]. A 15% adjustment allowed for possible non-normal distribution in outcome variable, for a total 33 per group.…”

“…The reliability and reproducibility of DC measurements using IVCM must be established. We have reported inter and intraobserver repeatability of DC morphology and intra-observer of DC density [21,22]. However, the inter-observer repeatability of DC density at various locations has not been reported.…”

“…The HRT III confocal microscope with Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany) was used to assess corneal and conjunctival DC [22]. Five locations of the corneal subbasal epithelium and conjunctival epithelium including the corneal anatomical centre, inferior whorl, peripheral cornea (temporal, 1 mm inside limbus), limbal cornea (temporal), and bulbar conjunctiva (temporal, 2-3 mm from the limbus) of the right eye were scanned, in that order.…”