Evidence of autophagic vesicles in a patient with Lisch corneal dystrophy

Abstract

Lisch corneal dystrophy is a rare corneal disease characterized by the distinctive feature of highly vacuolated cells. Although this feature is important, the nature of these vacuoles within corneal cells remains unknown. Here, we sought to analyze corneal cells from a patient diagnosed with Lisch dystrophy to characterize the vacuoles within these cells. Analyses using histopathology examination, confocal microscopy, and transmission electron microscopy were all consistent with previous descriptions of Lisch cells. Importantly, the vacuoles within these cells appeared to be autophagosomes and autolysosomes, and could be stained with an anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody. Taken together, these findings indicate that the vacuoles we observed within superficial corneal cells of a patient with Lisch corneal dystrophy constituted autophagosomes and autolysosomes; this finding has not been previously reported and suggests a need for further analyses to define the role of autophagy in this ocular disease.

Figure 1

(A-C) Slit-lamp examination (SL) in the right eye showing a gray, banded shape, sharply demarcated corneal opacity with an intraepithelial dense microcystic aspect. (B) SL, retroillumination. (D) Anterior segment optical coherence tomography showing a hyperreflective epithelium in the affected region. (E) In vivo confocal microscopy showing abnormal epithelial cells with intracytoplasmic vacuoles, with highly hyperreflective cytoplasm and hyporeflective nuclei.

Figure 2

(A) Microscopy analysis of toluidine blue staining revealing scat- tered areas of vacuoles within basal and suprabasal cells. The vacuoles appear optically empty (400×). (B) Electron microscopy (EM) panoramic view showing vacuolated squamous epithelium. Vacuoles are larger and more abundant in superficial cells than in those of the basal layer. Uranyl acetate and lead citrate, original magnification 1,700×. C) EM, lead citrate and uranyl acetate staining, showing supranuclear vacuoles of different sizes along the entire thickness of the epithelium. Myelinoid bodies and debris are also present, especially at the deeper layers. These findings are consistent with autophagosome phenotype (6,000×). (D) EM, vacuoles of different sizes showing myelinoid structures. Uranyl acetate and lead citrate, original magnification 6,000×. (E) EM, details of vacuoles with multilayered bodies of thin electron-dense membranous and amorphous material, representing autophagic vacuoles. Uranyl acetate and lead citrate, original magnification 16,500×. (F) Immunoelectron microscopy showing gold particles, evidenced as a highly electron-dense band (yellow asterisks) concentrated in autophagic vacuole walls. Original magnification 43,000×.