full-field-optical-coherence-microscopy-for-histology-like-analysis

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Sorbonne University, UM 80 / INSERM, UMR_S 968 / CNRS, UMR_7210

Quinze Vingts National Ophthalmology Hospital

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts. 

Three-dimensional structure of the mammalian limbal stem cell niche.

We uncover the significance of a previously unappreciated structural feature in corneal stroma, important to its biomechanics. Vogt striae are a known clinical indicator of keratoconus, and consist of dark, vertical lines crossing the corneal depth. However we detected stromal striae in most corneas, not only keratoconus. We observed striae with multiple imaging modalities in 82% of 118 human corneas, with pathology-specific differences. Striae generally depart from anchor points at Descemet's membrane in the posterior stroma obliquely in a V-shape, whereas in keratoconus, striae depart vertically from posterior toward anterior stroma. Optical coherence tomography shear wave elastography showed discontinuity of rigidity, and second harmonic generation and scanning electron microscopies showed undulation of lamellae at striae locations. Striae visibility decreased beyond physiological pressure and increased beyond physiological hydration. Immunohistology revealed striae to predominantly contain collagen VI, lumican and keratocan. The role of these regions of collagen VI linking sets of lamellae may be to absorb increases in intraocular pressure and external shocks.

Stromal striae: a new insight into corneal physiology and mechanics.

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer. To fill this gap, we propose dynamic full-field OCT (D-FFOCT) as a candidate for live observation of in vitro RPE phenotype. In this way, we monitored primary porcine and human stem cell-derived RPE cells in stress model conditions by performing scratch assays. In this study, we quantified wound healing parameters on the stressed RPE, and observed different cell phenotypes, displayed by the D-FFOCT signal. In order to decipher the subcellular contributions to these dynamic profiles, we performed immunohistochemistry to identify which organelles generate the signal and found mitochondria to be the main contributor to D-FFOCT contrast. Altogether, D-FFOCT appears to be an innovative method to follow degenerative disease evolution and could be an appreciated method in the future for live patient diagnostics and to direct treatment choice.

Article	Year
Three-dimensional structure of the mammalian limbal stem cell niche. Experimental eye research\| PubMed ID: 26297801	2015
Stromal striae: a new insight into corneal physiology and mechanics. Scientific reports\| PubMed ID: 29051516	2017
Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model. Communications biology\| PubMed ID: 35688936	2022

Kate Grieve

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