Onkar B. Sawant

Purpose: We propose supplementing a novel hypothermic corneal storage solution (CSS), Eversol, with the Rho kinase inhibitor HA-1077 (EversolPlus) to better nurture corneal endothelial cells (CEC) during storage, ultimately, to improve transplantation success. The goal of this study is to evaluate the ex vivo stability, safety, and efficacy of HA-1077–supplemented hypothermic CSS over a 14-day period. Methods: Corneal endothelium was injured by brushing, then corneas were cultured, and wound healing was assessed (n = 7). Two independent studies compared corneas stored at 2 to 8°C for 14 days recovered into EversolPlus or Eversol (n = 8), and into Optisol-GS or EversolPlus (n = 11). Central corneal thickness (CCT), CEC density, and morphology were assessed on days 1, 3, 7, 10, and 14. Results: After endothelial brushing, corneas stored in HA-1077–supplemented media demonstrated markedly improved healing compared with the control group ( P = 0.0402), confirming the compound's beneficial effect for endothelial health under physiological conditions. During hypothermic storage, corneas stored in EversolPlus demonstrated reduced CCT on day 1 ( P = 0.0028) and day 3 ( P = 0.0090) compared with the Eversol group. Corneas stored in EversolPlus displayed reduced CCT on day 1 ( P = 0.0024) and day 3 ( P = 0.0048) compared with the Optisol-GS group. CEC density and morphometric parameters were within acceptable ranges for all study groups over 14 days. Conclusions: Supplementing CSS with HA-1077 results in overall thinner corneas during the first 7 days of hypothermic storage, likely because of improved nurturing of CECs. We postulate that ROCKi containing CSS, such as EversolPlus, could improve clinical outcomes after endothelial keratoplasty procedures.

Purpose: Limbal stem cell deficiency is a blinding corneal disease that requires transplantation to replenish the limbal epithelial stem cells (LESCs) and restore vision. Keratolimbal allografts from cadaveric donors are commonly used as a source for LESCs. The goal of this study was to verify the efficacy of Food and Drug Administration-approved hypothermic corneal storage solutions in preserving LESCs within human donor corneas over time. Methods: Limbal segments from deceased donor corneas were stored in Food and Drug Administration-approved corneal storage solutions for 0, 4, or 7 days. Tissues were then processed for immunohistochemistry, and number of p63α+ cells within the limbus were calculated. LESC density was calculated as the number of p63α+ cells per total number of cells. LESCs were isolated from donor corneas after 7 days of hypothermic storage and subjected to a colony formation assay, and the numbers of holoclones were calculated. Results: The average density of p63α+ cells was 37.4% on day 0, 27.3% on day 4, and 10.9% on day 7 (P < 0.05) in limbal segments. Holoclones (15.33 ± 3.5) were formed by cells isolated from the limbal region of donor corneas after 7 days in the hypothermic storage conditions, indicating that viable LESCs are still present in human donor corneas after 7 days in storage. Conclusions: Although the density of p63α+ LESCs significantly decreases over time, viable LESCs are present in the donor corneas after 7 days in hypothermic storage conditions. Thus, failure of detection or absence of expression of standard marker(s) does not necessarily indicate the loss of functional LESCs.

Purpose: The goal of this study was to evaluate the effect of various Food and Drug Administration–approved storage solutions on endothelial cell density (ECD) and central corneal thickness (CCT). Method: We analyzed 6220 tissues used for endothelial keratoplasty procedures between January 2022 and June 2023 that were stored in either Life4°C, Optisol-GS, Kerasave, or Eusol-C under hypothermic conditions. We analyzed preprocessing CCT, success rate of meeting surgeon's Descemet stripping automated endothelial keratoplasty (DSAEK) thickness preferences, and preprocessing and postprocessing ECD. Results were analyzed using one-way analysis of variance, followed by multiple pairwise comparisons using the Tukey test. Results: Mean preprocessing CCT was significantly lower in the Life4°C group (532 μm) than in Optisol-GS (549 μm), Kerasave (582 μm), and Eusol-C (589 μm) groups (all P < 0.0001). Preprocessing CCT in the Optisol-GS group was significantly lower than in Kerasave and Eusol-C groups (F (3, 3273) = 153.1, all P < 0.0001). Success rate of meeting surgeon DSAEK preferences was statistically similar among all 4 groups. Preprocessing ECD of the Kerasave group (2821 cells/mm²) was numerically higher than of the Eusol-C (2791 cells/mm²), Life4°C (2759 cells/mm²), and Optisol-GS (2768 cells/mm²) groups (P = 0.3232, 0.0004, and 0.0015, respectively). Conclusions: Corneal tissues stored in Kerasave and Eusol-C are significantly thicker than those in Life4°C and Optisol-GS. However, the success rate of meeting surgeon DSAEK preferences is similar among all 4 storage solutions.

Purpose: The purpose of this study was to establish a validated method, consistent with Eye Bank Association of America medical standards, for evaluating endothelial cell loss (ECL) from an entire Descemet membrane endothelial keratoplasty (DMEK) graft using trypan blue dye as an alternative to specular microscopy. Method: Twenty-nine corneas were prepared for preloaded DMEK by a single technician, and the endothelium was stained with trypan blue dye for 30 seconds. The technician estimated total cell loss as a percentage of the graft and captured an image. Images were evaluated by a blinded technician using ImageJ software to determine ECL and compared with endothelial cell density from specular microscopy. Tissue processing intervals were analyzed for 4 months before and after implementation of this method. Results: For the 29 grafts, there was no statistically significant difference (t test, P = 0.285) between ECL estimated by a processor (mean = 5.8%) and ECL calculated using an ImageJ software (mean = 5.1%). The processor tended to estimate greater ECL than the actual ECL determined by ImageJ (paired t test, P = 0.022). Comparatively, postprocessing endothelial cell density measured by specular microscopy were higher compared with the preprocessing endothelial cell density (mean = 4.5% P = 0.0006). After implementation of this evaluation method, DMEK graft processing time intervals were reduced by 47.9% compared with specular microscopy evaluation (P < 0.001). Conclusions: Our results show that visual ECL estimation using trypan blue staining by a DMEK graft processor is a reliable and efficient method for endothelial assessment. Unlike specular microscopy, this method achieves comprehensive visualization of the entire endothelium, reduces total time out of cold storage, and decreases total time required to prepare and evaluate DMEK grafts.

Purpose: The purpose of this study was to assess the effect of a second povidone-iodine (PVP-I) application at the time of donor tissue recovery on overall tissue quality and to analyze the rate of positive fungal and bacterial rim cultures before and after implementing increased PVP-I exposure. Methods: The left cornea was recovered after a single application of PVP-I, while the right cornea was recovered after double PVP-I application in research-consented donors. The epithelial cell death rate was estimated using viability assay in corneal whole mounts under 10× objective (n = 5). Clinical characteristics of epithelium, stroma, and endothelium; positive rim culture rate; and incidences of infectious postoperative adverse reactions were compared for a period of 14 months before and after implementation of increased PVP-I protocol. Results: The average epithelial cell death rate was unaltered between single and double PVP-I exposure groups. We observed a modest 10% increase in the number of tissues with mild edema after implementation of increased PVP-I exposure. Nonetheless, the percentage of tissues with moderate or severe edema was unaltered. The average positive rim culture rate decreased from 1.17% to 0.88% (P = 0.075) after implementation of the double PVP-I soak procedure. There has been only one report of infectious postoperative adverse reactions since this procedure change. By contrast, there were 5 reports for a period of 14 months before implementation of this protocol. Conclusions: These results indicate that new donor preparation methods with an additional 5 minutes of PVP-I exposure do not affect tissue quality, reduce positive rim cultures, and lead to lower incidence of postoperative infection.

Purpose: The purpose of this study was to assess the impact of COVID-19 guidelines for corneal donor tissue screening and the utility of routine postmortem COVID-19 testing of donors intended for surgical use at a single eye bank. Methods: A retrospective analysis of referrals to and eligible donors from an eye bank between March 1, 2020, and June 30, 2020, was performed, with the same time period in 2019 as a control. Referrals who were not procured because of Eye Bank Association of America COVID-19 guidelines and eye bank–specific restrictions were noted. The results of 1 month of routine postmortem testing performed by the eye bank were examined. Analysis of variance tests were performed to assess the change between donors from 2019 to 2020. Results: There was a significant reduction in both the number of total referrals to the eye bank (P = 0.044) and donors eligible for surgical transplantation (P = 0.031). Eye Bank Association of America COVID-19 guidelines reduced the number of referrals over this period by 4% to 14%. Of the 266 surgically eligible donors who received postmortem COVID-19 testing in June by the eye bank, 13 resulted positive (4.9%). Conclusions: Despite a reduction in referrals and eligible corneal transplant donors at a single eye bank, there was a surplus of surgically suitable corneal tissue during the first wave of the COVID-19 pandemic. Eye banks should consider routine postmortem COVID-19 testing to identify asymptomatic infected donors although the risk of transmission of COVID-19 from infected donors is unknown.

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1β) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

Background: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. Methods: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors. Findings: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ∼13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection. Interpretations: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation. Funding: Research grant from EBAA and National Institutes of Health.