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Purpose: To examine corneal tissue for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) positivity regarding implications for tissue procurement, processing, corneal transplantation, and ocular surgery on healthy patients. We performed quantitative reverse transcription-polymerase chain reaction qRT-PCR-testing for SARS-CoV-2 RNA on corneal stroma and endothelium, bulbar conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium of coronavirus disease 2019 (COVID-19) postmortem donors., Methods: Included in this study were 10 bulbi of 5 COVID-19 patients who died because of respiratory insufficiency. Informed consent and institutional review board approval was obtained before this study (241/2020BO2). SARS-CoV-2 was detected by using a pharyngeal swab and bronchoalveolar lavage. Tissue procurement and tissue preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for each of the abovementioned tissues and intraocular fluids., Results: The qRT-PCRs yielded no viral RNA in the following ocular tissues and intraocular fluid: corneal stroma and endothelium, bulbar-limbal conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium., Conclusions: In this study, no SARS-CoV-2-RNA was detected in conjunctiva, anterior chamber fluid, and corneal tissues (endothelium, stroma, and epithelium) of COVID-19 donors. This implicates that the risk for SARS-CoV-2 infection using corneal or conjunctival tissue is very low. However, further studies on a higher number of COVID-19 patients are necessary to confirm these results. This might be of high importance for donor tissue procurement, processing, and corneal transplantation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

The appearance of the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV‑2) poses challenges in ophthalmology particularly for eye banks. A valid risk assessment for the removal and processing of donor corneas is difficult due to the lack of data. The risk to infect transplant recipients with SARS-CoV‑2 still appears very unlikely due to the experience with severe acute respiratory syndrome -coronavirus(‑1) (SARS-CoV(‑1)) and Middle East respiratory syndrome-coronavirus (MERS-CoV); however, due to the occurrence of angiotensin-converting enzyme 2 (ACE2) receptors in the cornea an infection of this tissue with SARS-CoV‑2 cannot be completely excluded. Therefore, routine testing of the organ culture medium used for donor corneas for SARS-CoV‑2 prior to transplantation during the coronavirus disease 2019 (COVID‑19) pandemic should be considered.

BACKGROUND This study investigated the effect of donor-dependent factors on contamination rates of conjunctival swabs of human donor eyes. MATERIAL AND METHODS From July 2015 to September 2017 a total of 1008 conjunctival swabs from 504 consecutive human donor eyes were analyzed. Cross-tabulation, chi-squared tests, and Fisher's exact tests were used to evaluate the effect of donor-dependent factors on contamination rates of conjunctival swabs. RESULTS The mean conjunctival swabs contamination rate was 28.4%. Donors with the diagnosis of carcinoma or metastases were associated with an increased conjunctival swab contamination rate [odds ratio (OR)=1.8, 95% confidence interval (CI)=1.2-2.6, p=0.007; OR=1.7, 95% CI=1.1-2.6, p=0.016; respectively]. However, the age, sex, diagnosis of diabetes mellitus, and donors who received chemotherapy did not significantly increase the conjunctival swab contamination risk. CONCLUSIONS Donors with the diagnosis of a carcinoma or metastases seemed to be predisposed to increased conjunctival swab contamination risk.

This study assessed the influence of donor, environmental, and logistical factors on the contamination rates of the conjunctival swabs and organ culture media of human donor eyes.In total, 1008 conjunctival swabs and 418 organ culture media samples from 504 consecutive human donor eyes were analyzed. Cross-tabulation, chi-squared tests, and Fisher's exact tests were used to assess the influence of the different factors on the contamination rates of the conjunctival swabs and organ culture media.The overall contamination rates were 28.4% for the conjunctival swabs and 1.0% for the organ culture media. A prolonged time between death and the conjunctival swab collection was associated with an increased conjunctival swab contamination rate [odds ratio (OR) = 1.9, 95% confidence interval (CI) = 1.2-3.0, P = .007]. The highest conjunctival swab contamination rate was found in the corneas procured in external institutions (outside the university hospital) (44.1%, OR = 3.6, 95%CI = 1.5-8.4, P = .003). Hospitalization times of 2 to 7 days prior to death were associated with an increased conjunctival swab contamination risk (OR = 2.6, 95%CI = 1.1-5.8, P = .021). However, the sex, age, cause of donor death, differentiation between septic and aseptic donors, differentiation between heart-beating brain-dead multiorgan donors and cadaveric donors, a warmer mean monthly temperature, and death to corneoscleral disc excision time did not significantly increase the conjunctival swab contamination risk. In addition, none of these factors affected the organ culture media contamination risk. Moreover, a positive conjunctival swab did not significantly increase the media contamination risk (P = .08). Surprisingly, the microorganisms causing media contamination were present at 50% of the amount detected on the conjunctival surface of the respective donor eye.A prolonged time between death and the conjunctival swab collection, a hospitalization time of 2 to 7 days prior to death, and corneal collection outside the university hospital seemed to be the main factors responsible for an increased conjunctival swab contamination risk. In addition, our investigation illustrated that a positive conjunctival swab is not a strong indicator for organ culture media contamination. Critical discussion is necessary regarding the validity of conjunctival swabs as prognostic parameters for organ culture media contamination.

BACKGROUND This study aimed to investigate factors limiting corneal donation at the University Hospital Tübingen. MATERIAL AND METHODS We retrospectively studied all hospital deaths from January 2012 to December 2015, considering each deceased patient as a potential corneal donor. During this period an ophthalmic resident managed corneal donor procurement on a full-time basis. Various factors limiting corneal donation were examined. RESULTS Among the 3412 deaths, 2937 (86.1%) displayed nonfulfillment of corneal donation. Consent for corneal donation was obtained in 475 cases (13.9%). The mean annual corneal donation rate was 13.9 donors per 100 deaths (range: 11.2-17.8). The leading causes of nonfulfillment of corneal donations were refusal to donate (49.8%, 1698 cases) and medical contraindications (23.6%, 805 cases). After next-of-kin interview of 2173 potential donors (109 potential donors were excluded because of logistical problems), willingness to participate in corneal donation was present in 475 cases (21.9%), whereas in 1698 cases (78.1%) corneal donation was refused. CONCLUSIONS Our study showed refusal to donate is the most important factor limiting corneal donation. It seems that increasing the knowledge of people about corneal donation through public education and media are necessary to address the corneal shortage.

BACKGROUND This study aimed to report the rates and experiences of corneal donation at the University Hospital Tübingen. MATERIAL AND METHODS Data from all cornea donors from January 2002 to December 2015 were analyzed. The rates of corneal donation were examined. From 2002 to 2008, an ophthalmic resident handled the corneal donor procurement in addition to his ward work. From 2009 to 2015, an ophthalmic resident handled the corneal donor procurement on a full-time basis. RESULTS Among the 10 260 deaths, consent for corneal donation was obtained in 892 cases. The mean annual corneal donation rate was 8.7 donors per 100 deaths (range: 2.0-17.8). The mean corneal donation rate in the first period without a full-time ophthalmic resident in the eye bank team was 4.0 donors per 100 deaths (95 % confidence interval [CI]: 1.0-6.9) and in the second period 12.3 donors per 100 deaths (95 % CI: 3.2-21.5) (P<0.0001). Comparing the periods before and after the modification of the eye bank team, the corneal donation rate was increased by more than 3 times. CONCLUSIONS Our study showed the influence of the eye banking team on the corneal donation rate. Modification of the eye bank team, by recruiting a full-time resident at a University Hospital with 1500 beds, increased the corneal donation rate by more than 3 times. It appears necessary to strengthen this team with sufficient staff levels to improve donor rates.

Purpose: To investigate in a retrospective review the histological and ultrastructural findings after failed primary and early Descemet membrane endothelial keratoplasty (DMEK), propose possible pathomechanisms of graft failure and give clinical implications., Methods: The explanted grafts underwent light- and electromicroscopical investigations in eight failed DMEK cases. Haematoxylin - Eosin, periodic acid Schiff and Alcian blue stainings were performed. Special note was given to any residual stromal remnants, absence of endothelial cells, lamellar structure and 'activation' of keratocytes., Results: Of the eight cases, six were re-DMEKs and two penetrating keratoplasties. Partial graft separation was seen in six and no graft separation in two of the cases. The average time-interval to the re-DMEK or penetrating keratoplasty was 4.6 months. Light and electron microscopy of the two explanted stromal specimens showed varying degrees of keratocyte activation. Endothelial cell loss was observed in essentially all explants with varying degrees and positive correlation with intraoperative difficulty. Assumed upside-down situations showed large areas of intact endothelial cells. In addition, a new layer, situated between the endothelial cell layer and the posterior nonbanded layer, was observed with loose intercellular structure., Conclusion: A loss of the endothelial cell layer of varying degrees and positive correlation with intraoperative difficulty are the prominent feature of primary and early DMEK graft failure. Of note is the upside-down situation, in which in some cases, the endothelial cell layer not only remains intact but also demonstrates metabolical activity in forming a novel cellular layer., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: Congenital anterior staphyloma is a rare, complex malformation syndrome of the anterior segment. Only a few reports on associated systemic malformations have been published. We herein present a rare manifestation of congenital anterior staphyloma (CAS) combined with amniotic band disruption syndrome (ABS)., Patient and Methods: Shortly after birth, a massive enlargement of the left eye was observed in a female child. Furthermore, an extensive bilateral congenital cleft lip and cleft alveolar ridge with oblique facial cleft extending into the left medial canthal region, coloboma(s) of the left eyelids, extensive adhesions between lids and eye bulb, as well as circumferential grooves, clubfeet, and terminal transverse defects in both hands and feet were present. Due to severe progression of eye bulb protrusion with thinning of the sclera, enucleation of the left eye was performed at the age of 3 years in order to prevent complications including perforation of the globe and with the aim of improving cosmetic aspects., Results: Histopathological examination of the enucleated eye disclosed findings typical of congenital anterior staphyloma, including massive corneal staphylomatic deformation with superficial vascularization and elapsed corneoscleral margin, destruction of Bowman's layer, absence of Descemet's layer, corneal endothelium, and angle structures. The lens was only partially formed, and had mainly dissolved. The neural retina appeared normal. The optic nerve disc revealed a pronounced excavation. Facial clefts, lid colobomas, congenital constriction bands, and amputation of distal limbs match ABS. This malformation complex develops in early pregnancy, probably prior to 35 days post conception., Conclusion: This is the first report on an association of these two rare complex congenital malformations, congenital anterior staphyloma and amniotic band syndrome. The anterior staphyloma was unilateral, and related to facial clefts and lid coloboma in the area adjacent to the anterior staphyloma. Furthermore, the systemic deformities are clearly due to the amniotic bands, and the timing of the development of both complex malformations seems to be similar. All findings suggest that the presence of amniotic bands is a causative factor for all observed abnormalities including anterior staphyloma.

Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs)., Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), natrium hydrogen carboanhydrase (Na(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry., Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na(+)/HCO(3)(-), and Na(+)/K(+)-ATPase., Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.

In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4–5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2–3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks. [ABSTRACT FROM AUTHOR]

Background: This study aimed to analyze if the outcome after corneal crosslinking (CXL) in progressive keratoconus patients depends on the stage at which the procedure is performed. This knowledge would help to improve success of CXL and to define surgery indications in those patients.Methods: In this retrospective study, 124 consecutive eyes of 100 patients with progressive keratoconus undergoing corneal CXL at the University Eye Hospital Tübingen were included. The eyes were graded according to modified Krumeich stages depending on induced myopia or astigmatism, corneal radii, minimum corneal thickness, and morphological changes. The observation period covered November 2008 to September 2018. Preoperatively, 12 and 24 months after CXL, the best corrected visual acuity (BCVA) was determined and astigmatism as well as tomographic parameters (Kmax, Kmin, central corneal thickness (CCT), minimum corneal thickness (MCT)) were measured by means of a Scheimpflug camera system.Results: BCVA results showed significant differences between the modified Krumeich stages at 12 months (p = 0.014) and at 24 months postoperatively (p = 0.032). Also, astigmatism differed significantly among the stages at 24 months after CXL (p = 0.023). However, no significant differences regarding astigmatism were detectable after 12 months. In terms of Kmax, Kmin, CCT, and MCT, no significant differences between the Krumeich stages were observed.Conclusions: BCVA showed a significantly higher improvement after CXL in the early stage of keratoconus compared to a higher stage. However, the postinterventional tomographic values did not differ significantly between the different modified Krumeich stages. The significantly higher improvement in BCVA after CXL in the early stage might indicate that earlier intervention provides a higher subjective benefit to the individual. Further studies with larger sample sizes are needed to confirm these findings. [ABSTRACT FROM AUTHOR]

Dry eye is a multifactorial disease that affects the ocular surface and tear fluid. Current treatment options include lubricant eye drop application several times a day. However, these eye drops often cause local side effects like ocular allergies or blurred vision after the application. To test new treatment options, a robust dry eye model is needed. Here, a porcine ex vivo model was established by means of incubation of porcine corneas in low humidity (LH) and characterized by histological damage evaluation, epithelial thickness and by relevant dry eye markers, such as interleukin 1 beta (IL-1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), occludin and galectin-3. In the dry eye model proposed, an increased secretion of IL-1β was observed, as well as an upregulation of NF-κB, occludin and galectin-3 mRNA expression. Moreover, the model presented a higher rate of cell death in comparison to the controls. These effects could be reversed with successful treatment of dexamethasone (dexa) and partially reversed with hyaluronic acid (HA) containing eye drops. Furthermore, medium-molecular-weight HA stimulated an increase in IL-1β in the model proposed. In conclusion, this dry eye model mimics the in vivo condition and hence allows for animal-free testing of novel dry eye treatments. [ABSTRACT FROM AUTHOR]

Purpose: To better assess clinical trajectories of patients with or without ocular comorbidity after Descemet membrane endothelial keratoplasty. Background: To report on the outcomes of eyes with differing starting conditions following surgery. Design: Retrospective study at a University Eye Hospital. Participants: 361 eyes separated into group 1 (n=229; eyes with endothelial disease only) and group 2 (n=132; eyes with additional ocular comorbid conditions, such as herpetic eye disease 18/132 (13.6%), glaucoma 16/132 (12.1%), dry age-related macular degeneration 14/132 (10.6%), epiretinal membranes 10/132 (7.6%), and wet age-related macular degeneration 9/132 (6.8%)). Methods: Consecutive eyes that underwent Descemet membrane endothelial keratoplasty over a follow-up period of up to 7 years at a tertiary referral center were reviewed. Main outcome measures were best-corrected visual acuity, postoperative complications, graft survival, central corneal thickness, and endothelial cell density. Results: Postoperative best-corrected visual acuity at year 1 improved in both groups significantly (Wilcoxon signed rank test: group 1, p =.002;.63 to.23 logMAR; group 2, p <.001; 1.15 to.87 logMAR) with a group difference in favor of group 1 (p =.009, Mann-Whitney-Wilcoxon). A decrease of the endothelial cell density and central corneal thickness was noted at postoperative year 1 for both groups (paired t-tests (group 1, p <.001; group 2, p =.045) and paired t-tests (group 1, p <.001; group 2, p =.003). Complications were less common, and graft longevity was superior in group 1. Conclusion: Eyes with different starting conditions might experience a visual improvement and benefit from surgery. Descemet membrane endothelial keratoplasty is a valid treatment for endothelial disorders in manifold of eyes. Further long-term studies are required. [ABSTRACT FROM AUTHOR]

Background: To assess the cytotoxic properties of voriconazole and sulfobutylether-β-cyclodextrin (SBECD) on cultured primary human corneal epithelial cells. Methods: Human corneal epithelial cells were cultured and exposed to various concentrations of SBECD (0.016-32 mg/ml) and voriconazole (0.001-2 mg/ml). Cellular cytotoxicity of SBECD and voriconazole on human corneal epithelial cells was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test and the LIVE/DEAD Viability/Cytotoxicity Assay with fluorescence microscopy analysis. Cell damage was assessed with phase-contrast microscopy after 24 h of exposure to SBECD and voriconazole. Results: The cytotoxicity tests and the morphological characteristic demonstrated the dose-dependent toxic effect of SBECD and voriconazole on human corneal epithelial cells. No corneal epithelial cytotoxicity was observed below the concentration of 0.08 and 0.025 mg/ml after 24-hour exposure to SBECD and voriconazole, respectively. Conclusions: The results of the study reveal the dose-dependent cytotoxic effect of SBECD and voriconazole on cultured human corneal epithelial cells. Therefore, voriconazole eye drops should be used cautiously in the treatment of fungal corneal ulcers. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Purpose: Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent. Methods: Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium. Results: Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium. Conclusions: Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis. [ABSTRACT FROM AUTHOR]