Ocular surface neutrophils are morphologically distinct in keratoconus

Abstract

Purpose : Increasing evidence suggests inflammation plays a role in keratoconus (Kc), yet the contribution of leukocytes is not known. This study used ultra-high resolution transmission electron microscopy (TEM) to evaluate morphological characteristics of ocular surface neutrophils in Kc and compare them to normal controls.

Methods : A total of 5 Kc and 5 age and gender-matched controls were recruited. Subjects washed their ocular surface with 3mL saline to collect an overnight tear wash (TW). Samples were centrifuged (1400 rpm for 10 min) and the cells isolated, resuspended, and fixed in 0.1M sodium cacodylate buffer containing 2% glutaraldehyde, embedded in agarose followed by processing for routine TEM. Ultrathin sections from each subject were collected and 15 neutrophil profiles were randomly imaged at 9900X using the Tecnai G2 12 TEM (FEI Company, Hillsboro, OR). Image analysis was done using Celleste Image Analysis Software (Invitrogen, Waltham, MA) and cell morphological parameters were assessed and compared between normal and Kc using independent t-test.

Results : Cell circularity was significantly lower in Kc subjects (0.58 ± 0.06) compared to controls (0.71 ± 0.01) (P = .01), and there was a greater number of phagosomes per cytoplasmic area in Kc (0.053 ± 0.003 counts/um² cytoplasm) compared to controls (0.038 ± 0.005 counts/um² cytoplasm) (P = .001). A significantly greater percentage of light vesicle area in total cytoplasmic area in Kc (4.9 ± 1.5%) was observed compared to controls (2.6 ± 0.8%) (P < .05). However, no significant difference in percentage of dark vesicle area in total cytoplasmic area in Kc (7.5 ± 1.9%) was observed compared to controls (5.4 ± 0.9%) (P = 0.06). No significant differences in total cell area were observed in Kc (44.6 ± 8.8 µm²) compared to controls (42.1 ± 6.7 µm²) (P = .62).

Conclusions : Ocular surface neutrophils are morphologically distinct in Kc. The reduced circularity (increased irregularity) is consistent with a more active phenotype. Increased numbers of phagosomes per cytoplasmic area and increased percentage of light vesicle area supports this conclusion and raises the possibility of increased neutrophil activity in Kc and warrants further investigation.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.