Diagnostic Algorithm for Surgical Management of Limbal Stem Cell Deficiency.

BACKGROUND: Limbal stem cell deficiency (LCSD) presents several challenges. Currently, there is no clearly defined systematic approach to LSCD diagnosis that may guide surgical tactics. METHODS: The medical records of 34 patients with LSCD were analyzed. Diagnostic modalities included standard (visometry, tonometry, visual field testing, slit-lamp biomicroscopy with corneal fluorescein staining, Schirmer test 1, ultrasonography) and advanced ophthalmic examination methods such as anterior segment optical coherence tomography, in vivo confocal microscopy, impression cytology, and enzyme-linked immunoassay. RESULTS: Standard ophthalmological examination was sufficient to establish the diagnosis of LSCD in 20 (58.8%) cases, whereas advanced evaluation was needed in 14 (41.2%) cases. Depending on the results, patients with unilateral LSCD were scheduled to undergo glueless simple limbal epithelial transplantation (G-SLET) or simultaneous G-SLET and lamellar keratoplasty. Patients with bilateral LSCD with normal or increased corneal thickness were enrolled in the paralimbal oral mucosa epithelium transplantation (pLOMET) clinical trial. CONCLUSIONS: Based on the diagnostic and surgical data analyzed, the key points in LSCD diagnosis were identified, helping to guide the surgeon in selecting the appropriate surgical procedure. Finally, we proposed a novel step-by-step diagnostic algorithm and original surgical guidelines for the treatment of patients with LSCD.

Lipofuscin-Mediated Photic Stress Induces a Dark Toxic Effect on ARPE-19 Cells.

Lipofuscin granules from retinal pigment epithelium (RPE) cells contain bisretinoid fluorophores, which are photosensitizers and are phototoxic to cells. In the presence of oxygen, bisretinoids are oxidized to form various products, containing aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that bisretinoid oxidation and degradation products have both hydrophilic and amphiphilic properties, allowing their diffusion through the lipofuscin granule membrane into the RPE cell cytoplasm, and are thiobarbituric acid (TBA)-active. The purpose of the present study was to determine if these products exhibit a toxic effect to the RPE cell also in the absence of light. The experiments were performed using the lipofuscin-fed ARPE-19 cell culture. The RPE cell viability analysis was performed with the use of flow cytofluorimetry and laser scanning confocal microscopy. The results obtained indicated that the cell viability of the lipofuscin-fed ARPE-19 sample was clearly reduced not immediately after visible light irradiation for 18 h, but after 4 days maintaining in the dark. Consequently, we could conclude that bisretinoid oxidation products have a damaging effect on the RPE cell in the dark and can be considered as an aggravating factor in age-related macular degeneration progression.

Labial mucosal epithelium grafting in an ex vivo human donor cornea model.

The purpose of the study was to establish a simple ex vivo corneal re-epithelization model and study the labial mucosal epithelium grafting as a potential approach for ocular surface reconstruction. Four human donor corneal buttons were overstored in a corneal cold storage solution at 4 °C for 32-52 days. Four labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed informed consent. The substantia propria was trimmed off, and the resulting graft was sutured near the corneal limbus with running sutures (thus forming the tissue construct). Constructs were cultured under the standard conditions with the anterior corneal side outwards. After 3 weeks of culture, constructs were removed, washed, and fixed. Sections were stained with hematoxylin and eosin (HE), anti-keratins 4, 13, 19, and p63. Nuclei were counterstained with Hoechst. After the cultivation, all constructs were integral with the attached graft and non-loosened sutures. The native cells were absent in all donor corneas. Histological evaluation demonstrated that the labial mucosal grafts were attached to the Bowman's membrane (BM), and its cellular outgrowths were found to be transit from the graft to the BM over the anterior surface in all constructs. Cells expressed mucosal epithelial keratins 4, 13, and 19, and several were p63-positive in nuclei. In the study, a simple ex vivo corneal re-epithelization model was successfully established. The model was potent in studying the labial mucosal epithelium grafting as an option for autologous ocular surface reconstruction in patients with bilateral limbal stem cell deficiency.