A high-resolution RNA expression atlas of Retinitis Pigmentosa genes in the human and mouse retinas

PURPOSE. Retinitis pigmentosa (RP) is one of the leading causes of visual handicap in the world population and is characterized by high genetic heterogeneity. The study of the disease mechanisms and the development of efficient therapeutic approaches have mostly relied on the availability of animal models for this condition, so far. Nevertheless, little information is available about the RNA expression profiles of RP genes in the human retina. An expression atlas of 34 known RP genes in human and murine retinas was generated to overcome this lack of information. METHODS. Appropriate templates were retrieved for 34 RP genes that were used to perform RNA in situ hybridization studies on human and murine adult eyes. RESULTS. Most of the genes displayed similar patterns between human and mouse retina. Different expression patterns were observed for the CNGB1, USH2A, and FSCN2 genes, compared with those in previously reported profiles. In addition, different expression profiles were detected for the RPGR, CA4, PAP1, RGR, and RLBP1 genes in human and mouse retinas. CONCLUSIONS. The first gene expression atlas has been generated of RP genes in human and murine retinas. Differences observed in the expression patterns of some genes in humans and mice, will open new perspectives on the function of these genes and their putative roles in disease pathogenesis

Human Corneal Endothelial Cell Assessment From Tissues Preserved in Serum-Based and Synthetic Storage Media

PURPOSE: To assess the difference between endothelial cells from tissues preserved in media supplemented with fetal bovine serum (FBS) and recombinant human serum albumin (rHSA). METHODS: In a donor-matched study, 48 tissues were preserved for 28 days at 31°C in Cornea Max and Cornea Syn supplemented with FBS and rHSA, respectively. Endothelial cells were visualized by 2 masked observers before and after preservation. Endothelial cell density (ECD) and the number of iatrogenic folds were counted manually. Alizarin red staining and tight junction protein (Zonula Occludens-1) were used to assess cell morphology (hexagonality and polymorphism). Intraobserver and interobserver cell counts were recorded and analyzed. Wilcoxon and one-way analysis of variance tests were used, where P < 0.05 was deemed statistically significantly different. RESULTS: Significant amount of iatrogenic folds were observed in the tissues supplemented with FBS compared with rHSA postpreservation (P = 0.0007). Approximately 69% and 71% hexagonal cells (P = 0.0303) and 29% and 26% polymorphic cells (P = 0.0234) were observed in the FBS and rHSA groups, respectively. Postpreservation, operator 1 counted 1766 cells/mm in FBS and 1864 cells/mm in rHSA. Operator 2 counted 1702 cells/mm in FBS and 1858 cells/mm in rHSA. ECD counts from FBS (interoperator) were statistically significant (P = 0.0429). However, significance was not observed in the ECD counts (interoperator) from the rHSA-preserved tissues (P = 0.8738). CONCLUSIONS: rHSA-supplemented media allow better visualization of the corneal endothelial cells. This reduces the rate of discard observed due to counting errors. Use of rHSA improves the current standard of care and reduces the use of animal-derived products

Corneal storage methods: considerations and impact on surgical outcomes

INTRODUCTION: With recent developments in the field of eye banking, human corneas are not only procured and preserved, but also processed and prepared for transplantation. However, one of the challenges that still persists is the long-term storage of tissues without damaging the corneal endothelial cells. Thus, the review aims at reporting the influence of tissue storage conditions on the clinical outcomes. AREAS COVERED: Endothelial cell loss (ECL), graft survival, and contamination from the tissues stored in hypothermic storage and organ culture and; other storage options such as cryopreservation and lyophilization. EXPERT OPINION: Hypothermic storage and organ culture have shown similar ECL. However, due to the relatively new techniques and limited long-term clinical studies, further evaluation is essential to assess the effect of storage time and conditions on the grafts deemed for endothelial keratoplasty

Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology

Impact of the COVID-19 Pandemic on Corneal Transplantation: A Report From the Italian Association of Eye Banks

Purpose: To analyze the impact of COVID-19 on Italian corneal transplantation from March-2020 to February 2021 compared to the same timeframe of the 2 previous years, in order to identify potential consequences of a global pandemic on corneal procurement and transplantation services during this time. Methods: This national, multicentric, retrospective cohort study evaluated data collected from 12 (100%) Italian eye banks from March 2020 to February 2021 (Group A). The number of tissues collected, distributed and discarded were compared with the same time-frame of the 2 previous years: 2019 and 2018 (group B and C, respectively). The different type of transplants performed were reported. Data were analyzed using a non-parametric Friedman test. Results: Corneal procurement and the percentage of distributed tissues reduced in 2020 by more than 30 and 15%, respectively, compared to the 2 previous years. During the pandemic corneal transplant surgery showed only a modest drop: the number of the penetrating keratoplasties (PKs) and the anterior lamellar keratoplasties (ALKs) decreased by about 30 and 20% in comparison with groups B and C, respectively; between the Endothelial Keratoplasties (EKs), the Descemet membrane endothelial keratoplasty (DMEK) increased slightly from March 2020 to February 2021. Conclusions: Italy was one of the first countries most affected by the outbreak of COVID-19, and the Italian government adopted severe measures to limit viral transmission. The pandemic generated several implications in corneal transplant activity during the first lockdown. Then an efficacious reaction with constant, vigorous work led to a resumption of transplant surgery to a near-normal standard. The increase of EKs, despite the pandemic, is a sign that the advance in corneal transplantation has gone ahead and it continues to evolve

Cross-Country Transportation Efficacy and Clinical Outcomes of Preloaded Large-Diameter Ultra-Thin Descemet Stripping Automated Endothelial Keratoplasty Grafts

PURPOSE: To evaluate the clinical outcomes of preloaded large-diameter ultra-thin grafts for Descemet stripping automated endothelial keratoplasty (UT-DSAEK) after cross-country shipment. METHODS: A laboratory study in an eye bank and a clinical cohort study in an academic tertiary care center were performed. UT-DSAEK (9.5 mm diameter) grafts (n = 7) were prepared, loaded into a commercial device (iGlide; Eurobio, Les Ulis, France), preserved for 4 days at room temperature in transport medium, and analyzed. In a retrospective study, preloaded tissues (n = 39) for clinical use were prepared, transported from Italy to the United Kingdom, and surgically delivered into the eyes of patients undergoing UT-DSAEK. Central and peripheral endothelial cell density (ECD) and viability were measured before and after loading and storage of the grafts in the laboratory study. Clinically, best-corrected visual acuity, ECD before and at final follow-up, dislocation rate, primary graft failure, and surgical time were recorded. RESULTS: In the laboratory study, postcut central graft thickness was 93.3 ± 17.2 μm. ECD and cell mortality did not change significantly before and after preservation (P = 0.8). Cell loss after 4 days of preservation was 1.7% ± 1.6%. Clinically, 39 eyes of 39 patients at final follow-up showed a mean central graft thickness of 88 ± 22 μm and a best-corrected visual acuity of 0.34 ± 0.24 logMAR. Nine of 39 cases (23%) needed rebubbling, and 28% cell loss was observed at final follow-up. CONCLUSIONS: Large-diameter UT-DSAEK grafts can be prepared and preloaded in the eye bank using the iGlide and transported to the surgical center facilitating surgery for patients undergoing UT-DSAEK, potentially reducing tissue wastage, surgical time, and costs related to surgery

Effect of Covid-19 on Eye Banks and Corneal Transplantations: Current Perspectives

The coronavirus disease 2019 (COVID-19) pandemic exerted a great impact on medical practice, which was reframed according to the actual needs. Ophthalmological services and procedures including corneal transplantation did not represent an exception. The adoption and implementation of new standard operating procedures as well as of new technologies for remote consultation and smart-working reshaped daily activities of both eye bankers, physicians, researchers, and patients. Regulatory restrictions were issued redefining corneal donor eligibility criteria, as well as handling and harvesting procedures of donor ocular tissues. Surgical schedules underwent an abrupt contraction with prioritization of urgent procedures. Local lockdowns and confinement strategies resulted in both a reduction and redirection of research activities. The evaluation of SARS-CoV-2 colonization of ocular tissues, long-term corneal storage techniques, new disinfection strategies, split corneal transplants and cell-based therapies for the treatment of corneal disease peaked in the pipeline. Aim of this article is to summarizes the overall impact of the pandemic on the corneal transplantation machinery, and the current and future perspectives for the corneal transplant community

Effects of serine phospholipids on human and murine lymphocytes.

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