Abstract
Purpose :
In-vivo confocal microscopy (IVCM) allows non-invasive examination of corneal immune cells, but controversy exists due to morphology-based identification using this technique. This observational study aimed to (i) confirm dendritic-like cell identity in human donor corneas using immunohistochemistry (IHC) on precisely matched tissue locations assessed with IVCM and (ii) perform morphological comparisons
Methods :
Four post-mortem human corneas were fixed in 4% PFA and imaged with IVCM. Subsequently, 15 biopsies from central and peripheral corneal regions were immunostained (anti-HLA-DR or anti-CD11c). Confocal microscopy captured images of immune cells (w/wo large dendrites (LDs)), and cell density was determined from 3 randomly selected images per biopsy (~400x400µm FOV). ImageJ was used to calculate cell parameters
Results :
Out of the 15 biopsies, 40 cells were imaged using IVCM—20 from each of the central and peripheral regions. Post IHC, 38 cells were HLA-DR+—18 from central and 20 from the periphery. In total, 39 cells were CD11c+—20 from each of the regions. CD11c+ total cell density (cell/mm2) in the central cornea (35±7) was significantly higher (p<0.05) than donor IVCM (10±3) and HLA-DR+ (11±5). HLA-DR+ cells (31±24) were more prevalent in the periphery but did not reach significance (p=0.25). In the central cornea, cells woLD area (µm) was higher in CD11c+ (99±28) than donor IVCM (75±38, p=0.02), and donor IVCM's cells area was lower than HLA-DR+ (91±20, p=0.02). HLA-DR+ cell perimeter (µm) was higher (90±43) than donor IVCM (46±8; p=0.01), and CD11c+ cell length (µm) was longer (14±1; p= 0.02) than donor IVCM. Donor IVCM cell roundness index (0.57±0.21) was higher than HLA-DR+ (0.41±0.16, p<0.05). In the central cornea, CD11c+ cell width (µm) and circularity index were higher than donor IVCM and HLA-DR (p<0.01). In the peripheral cornea, CD11c+ cell perimeter (µm) was significantly higher (207±125) than donor IVCM and HLA-DR+ (p=0.02). However, CD11c+ cell circularity index (0.08±0.06) was lower than donor IVCM and HLA-DR+ (p< 0.01). Notably, cells woLD did not show differences between acquisition modes in the peripheral cornea
Conclusions :
IHC on precisely matched IVCM-assessed tissue confirmed dendritic-like cells in donor corneas. Morphological comparisons showed higher CD11c+ density, area, and length in central cornea than donor IVCM and HLA-DR+
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.