Exploring the Healthy Eye Microbiota Niche in a Multicenter Study.

PURPOSE: This study aims to explore and characterize healthy eye microbiota. METHODS: Healthy subjects older than 18 years were selected for this descriptive cross-sectional study. Samples were collected with an eSwab with 1 mL of Liquid Amies Medium (Copan Brescia, Italy). Following DNA extraction, libraries preparation, and amplification, PCR products were purified and end-repaired for barcode ligation. Libraries were pooled to a final concentration of 26 pM. Template preparation was performed with Ion Chef according to Ion 510, Ion 520, and Ion 530 Kit-Chef protocol. Sequencing of the amplicon libraries was carried out on a 520 or 530 chip using the Ion Torrent S5 system (Thermo Fisher; Waltham, MA, USA). Raw reads were analyzed with GAIA (v 2.02). RESULTS: Healthy eye microbiota is a low-diversity microbiome. The vast majority of the 137 analyzed samples were highly enriched with Staphylococcus, whereas only in a few of them, other genera such as Bacillus, Pseudomonas, and Corynebacterium predominate. We found an average of 88 genera with an average Shannon index of 0.65. CONCLUSION: We identified nine different ECSTs. A better understanding of healthy eye microbiota has the potential to improve disease diagnosis and personalized regimens to promote health.

The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane.

The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-ß1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

Glasses-Assisted 3D Display System-Guided Descemet Membrane Endothelial Keratoplasty Tissue Preparation.

PURPOSE: The aim of this study was to evaluate the feasibility of Descemet membrane endothelial keratoplasty (DMEK) tissue preparation using a glasses-assisted 3-dimensional (3D) display system and to compare it with a conventional surgical microscope. METHODS: Healthy pairs of human corneas suitable for penetrating keratoplasty surgery were selected for this study. The tissues were randomly divided into 2 groups. Each pair of corneas had 1 cornea (group 1) prepared with NGENUITY (Alcon) with a 5-second staining time with vision blue, and the fellow cornea (group 2) was prepared using a OPMI Lumera 700 surgical microscope (Carl Zeiss Meditec, Jena, Germany) with a 30-second staining time. DMEK graft preparation time, speed of stripping, graft width, and endothelial cell loss were evaluated. RESULTS: Twenty-eight pairs of corneas were included in this study. The graft preparation time was significantly higher in the 3D group than in the conventional group (498 ± 147 vs. 418 ± 85 seconds, P value = 0.031). The mean speed of stripping was 0.59 ± 0.081 mm/s in group 1 and 0.089 ± 0.005 mm/s in group 2 ( P value = 0.024). The mean endothelial cell density in group 1 and group 2 before tissue preparation was 2162 ± 115.21 and 2153 ± 122.45, respectively ( P value > 0.1). After tissue preparation, the endothelial cell density reduced to 1911 ± 150.72 in group 1 and 1998 ± 90.72 in group 2 ( P value = P value > 0.05). The graft width was 5.05 ± 0.71 mm in group 1 and 4.92 ± 0.23 mm in group 2 ( P value > 0.05). CONCLUSIONS: DMEK tissue preparation with 3D display system NGENUITY is feasible with a slightly increased preparation time. The improved visualization allows a reduced staining time that could be beneficial for eye banks because it may reduce the toxic effect of staining colorants.

Long-Term Dehydrated Donor Lamella Survival in Anterior Keratoplasty: Keratocyte Migration and Repopulation of Corneal Stroma.

PURPOSE: The aim of this study was to investigate the ability of host keratocytes to colonize the donor lamella transplanted without viable cells (dehydrated) in Descemetic (deep anterior lamellar keratoplasty) and in pre-Descemetic keratoplasty (excimer laser-assisted lamellar keratoplasty). METHOD: A total of 17 eyes (8 deep anterior lamellar keratoplasties and 9 excimer laser-assisted lamellar keratoplasties) were included in this observational retrospective study; patients underwent ophthalmic examinations, and histological staining was performed ex vivo on the graft in cases of failure. RESULTS: In Descemetic keratoplasty, the long-term survival of the graft is compromised with the central corneal thickness decreasing; corneal pachymetry and in vivo and ex vivo keratocyte densities are significantly reduced (pachymetric reduction of -86 µm in the apex and -87 µm in the thinnest point; density cell reduction of 72% at a depth of 100 µm, 62% at a depth of 250 µm, and -66% at a depth of 400 µm). In pre-Descemetic keratoplasty, clinical complications, reduction of central thickness, or alterations of keratocyte density were not observed. CONCLUSIONS: In Descemetic keratoplasty, the migration of the host peripheral keratocytes does not seem enough to repopulate the donor graft, whereas in pre-Descemetic keratoplasty, long-term survival of the graft is good. Keratocyte repopulation was observed only by extensive contact between the donor and host parenchyma.

Deep anterior lamellar keratoplasty with dehydrated, 4 °C-stored, and rehydrated lenticules.

PURPOSE: To retrospectively evaluate the outcomes of 124 consecutive deep anterior lamellar keratoplasties (DALK) performed by using dehydrated, 4 °C stored, and rehydrated lenticules. METHODS: A total of 124 eyes of 109 patients, which had undergone DALK from August 11, 2000, to December 13, 2007, were included in our retrospective study; 88 cases were male and 36 female, with a mean age of 39.45±15.52 years (range 6-85). A total of 109 (87.9%) had keratoconus, 7 (5.6%) had deep or superficial corneal scar, 3 (2.4%) had epikeratophakia, 3 (2.4%) had pellucid degeneration, 1 (0.8%) a malignant pterygium, and 1 (0.8%) had a severe alkali burn. All the patients underwent ophthalmic examinations, including best-corrected visual acuity (BCVA), central ultrasound pachymetry, computerized topography, specular microscope endothelial cell count, and confocal microscopy. RESULTS: The mean follow-up was 23.9±3.4 months. The mean preoperative BCVA was 0.16±0.18; the postoperative BCVA was 0.68±0.20 after 2 years. The mean preoperative central corneal thickness (CCT) was 439.4±10.5 µm; after 2 years, it was 532.1±9.7 µm. The mean preoperative endothelial cell count (ECC) was 2301.3±180.2 cells/mm2; after 2 years it was 1986.0±169.5 with a decrease of 13.7%. Baring of Descemet membrane (DM) during DALK was achieved in 112 (90.32%) eyes; 4 eyes (3.26%) had a large perforation of the DM with consequent PK. Small intraoperative perforation of DM during DALK occurred in 18 cases (14.52%). CONCLUSIONS: The results showed that the method of preservation and distribution of dehydrated, 4 °C stored, and rehydrated lenticules is safe, cheap, and simple. Furthermore, it does not appear to influence the outcomes of the DALK surgery.

Enzymatic descemetic lamellar keratoplasty: pilot study.

PURPOSE: This was a pilot study to evaluate the efficacy of a new surgical technique of descemetic anterior lamellar keratoplasty (DALK) with the help of enzymatic hyaluronidase solution. METHODS: We selected 10 patients, 6 male and 4 female, with surgical keratoconus, with a mean age of 45+/-16 years (range 29-61), with best-corrected visual acuity (BCVA)