Intermediate-term impact on corneal endothelial cells and efficacy of Preserflo MicroShunt implantation in patients with open-angle glaucoma - a prospective study over two years.

INTRODUCTION: Preserflo MicroShunt is a novel microinvasive bleb forming device for the treatment of primary open-angle glaucoma. The intermediate- and long-term success and the impact of this procedure on corneal endothelial cell density remain to be investigated. METHODS: In this prospective observational study, 62 eyes of 55 glaucoma patients (mean age ± SD: 67.0 ± 15.0 years) receiving a Preserflo MicroShunt were included. Corneal endothelial cell density, intraocular pressure and best corrected visual acuity were assessed preoperatively and at 3, 6, 9, 12, 18 and 24 months postoperatively. Success rates, bleb revision rates and complications were analysed. Complete success was defined as an intraocular pressure reduction of ≥ 20% and achieving a target pressure of ≤ 18, ≤ 15 or ≤ 12 mmHg without antiglaucoma medication. Qualified success indicated that the criteria were reached with or without medication. RESULTS: Corneal endothelial cells showed no significant decline over 24 months (p > 0.05). Intraocular pressure showed a substantial reduction postoperatively (p < 0.001), decreasing from 29.6 ± 8,3 mmHg to 13.0 ± 4.3 mmHg after 24 months (p < 0.001). Complete and qualified success with a target pressure ≤ 15 mmHg was achieved in 52.9% and 54.6% of cases after 24 months, respectively. Best corrected visual acuity did not change after 24 months. CONCLUSION: Preserflo MicroShunt had no negative side effects on corneal endothelial cells and showed favourable success rates after 2 years in patients with open-angle glaucoma.

Early REperfusion Therapy with Intravenous Alteplase for Recovery of VISION in Acute Central Retinal Artery Occlusion (REVISION): Study Protocol of a Phase III Trial.

RATIONALE: Meta-analyses of case series of non-arteritic central retinal artery occlusion (CRAO) indicate beneficial effects of intravenous thrombolysis when initiated early after symptom onset. Randomized data is lacking to address this question. AIMS: REVISION investigates intravenous alteplase within 4.5 hours of monocular vision loss due to acute CRAO. METHODS: Randomized (1:1), double-blind, placebo-controlled, multicenter adaptive phase III trial. STUDY OUTCOMES: Primary outcome is functional recovery to normal or mildly impaired vision in the affected eye defined as best corrected visual acuity of the Logarithm of the Minimum An-gle of Resolution of 0.5 or less at 30 days (intention-to-treat analysis). Secondary efficacy out-comes include modified Rankin Score at 90 days and quality of life. Safety outcomes include symptomatic intracranial hemorrhage, major bleeding (International Society on Thrombosis and Haemostasis definition) and mortality. Exploratory analyses of optical coherence tomogra-phy/angiography, ultrasound and MRI biomarkers will be conducted. SAMPLE SIZE: Using an adaptive design with interim analysis at 120 patients, up to 422 participants (211 per arm) would be needed for 80% power (one-sided alpha 0.025) to detect a difference of 15%, assuming functional recovery rates of 10% in the placebo arm and 25% in the alteplase arm. DISCUSSION: By enrolling patients within 4.5 hours of CRAO onset, REVISION uses insights from meta-analyses of CRAO case series and randomized thrombolysis trials in acute ischemic stroke. Increased rates of early reperfusion and good neurological outcomes in stroke may trans-late to CRAO with its similar pathophysiology. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04965038; EU Trial Number: 2023-507388-21-00.

A Comparative Analysis of Traumatic Retinal Detachment After Open and Closed Globe Injuries in Children.

PURPOSE: Pediatric traumatic retinal detachment (RD) resulting from open globe (OGI) or closed globe injuries (CGI) presents unique challenges due to complexity often resulting in lifelong sequelae. This study compares pediatric traumatic RD outcomes and prognostic factors following OGI and CGI. METHODS: A retrospective analysis reviewed 47 cases of pediatric traumatic RD cases (age <18 years) undergoing retinal detachment surgery between 2002 and 2021. Among them, 25 cases were caused by CGI and 22 cases by OGI. Demographics, retinal detachment characteristics, surgical procedures, anatomical and functional results were assessed. Predictive factors for visual outcomes were investigated. RESULTS: In the CGI group, mean (± standard deviation) age was 11 ± 4 years, and 10 ± 5 years in the OGI group. CGI traumatic RD had significantly better preoperative (CGI: logMAR 1.39 ± 0.19 (mean ± standard error); OGI: logMAR 2.12 ± 0.20) and follow-up (CGI: logMAR 0.94 ± 0.19; OGI: logMAR 1.85 ± 0.20) best corrected visual acuity (BCVA) (p<0.05). Initial BCVA improvement was observed in CGI only. In multivariable analysis, prognostic factors for favorable BCVA outcomes included higher preoperative BCVA, older age and absence of proliferative vitreoretinopathy (p<0.05). CONCLUSION: Visual prognosis for pediatric traumatic RD remains limited, favoring CGI cases compared to OGI. Baseline BCVA emerged as major determinant of final visual acuity. Tailored management approaches can optimize treatment results.

Incidence of central retinal artery occlusion peaks in winter season.

Introduction: Stroke incidence exhibits seasonal trends, with the highest occurrences observed during winter. This study investigates the incidence of central retinal artery occlusion (CRAO), a stroke equivalent of the retina, and explores its monthly and seasonal variations, as well as potential associations with weather and ambient air pollutants. Methods: A retrospective search of medical records spanning 15 years (January 2008-December 2022) was conducted at the University Eye Hospital Tübingen, Germany, focusing on diagnosed cases of CRAO. Incidences were evaluated on a monthly and seasonal basis (winter, spring, summer, fall). Weather data (temperature, precipitation, atmospheric pressure) and concentrations of ambient air pollutants [fine particulate matter (PM2.5), coarse particulate matter (PM10), nitrogen dioxide (NO2), and ozone (O3)], were analyzed for a potential association with CRAO incidence. Results: Out of 432 patients diagnosed with CRAO between 2008 and 2022, significantly varying incidences were observed monthly (p = 0.025) and seasonally (p = 0.008). The highest rates were recorded in February and winter, with the lowest rates in June and summer. Concentrations of NO2, PM2.5 and lower ambient air temperature (average, minimum, maximum) showed significant correlations with CRAO incidence. Discussion: This comprehensive 15-year analysis reveals a pronounced winter peak in CRAO incidence, with the lowest occurrences in summer. Potential associations between CRAO incidence and ambient air pollutants and temperature underscore the importance of considering seasonal trends and call for further investigations to elucidate contributing factors, potentially leading to targeted preventive strategies and public health interventions.

Comparing the Quality of ChatGPT- and Physician-Generated Responses to Patients' Dermatologic Questions in the Electronic Medical Record.

BACKGROUND: Chat Generated Pre-trained Transformer ('ChatGPT,' Open AI, San Francisco, USA) is a free artificial intelligence (AI)-based natural language processing tool that generates complex responses to inputs from users. OBJECTIVE: To determine whether ChatGPT is able to generate high-quality responses to patient-submitted questions in the patient portal. METHODS: Patient-submitted questions and their corresponding responses from their dermatology physician were extracted from the electronic medical record for analysis. The questions were input into ChatGPT (version 3.5), and the outputs were extracted for analysis, with manual removal of verbiage pertaining to ChatGPT's inability to provide medical advice. Ten blinded reviewers (n=7 physicians, n=3 non-physicians) rated and selected their preference in terms of 'overall quality,' 'readability,' 'accuracy,' 'thoroughness,' and 'level of empathy,' of the physician- and ChatGPT-generated responses. RESULTS: Thirty-one messages and responses were analyzed. The physician-generated response was vastly preferred over the ChatGPT response by both physician and non-physician reviewers and received significantly higher ratings for 'readability' and 'level of empathy.' CONCLUSIONS: The results of this study suggest that physician-generated responses to patients' portal messages are still preferred over ChatGPT, but generative AI tools may still be helpful in generating first drafts of responses and education resources for patients.

Effect of elevated irrigation bottle height during cataract surgery on corneal endothelial cells in porcine eyes.

BACKGROUND: Cataract surgery induces corneal endothelial cell loss (ECL). This study investigates the relationship between bottle height (BH) and ECL induced due to irrigation and aspiration (I/A) in cataract surgery and quantifies protective effects of intraoperatively used ophthalmic viscoelastic substances. METHODS: Intermittent I/A without phacoemulsification was performed in porcine eyes for 10 min with varying BHs of 100 cm (BH100), 125 cm (BH125), 150 cm (BH150) or no treatment (control, no I/A). Additionally, in one group a dispersive ophthalmic viscoelastic substance was injected into the anterior eye chamber before treatment with I/A at a BH of 150 cm (BH150 + V). After exposure of the corneal endothelium to I/A, the corneas were prepared to split corneal buttons on day 0 and cultivated for 15 days. Endothelial cell density (ECD) was analyzed blinded on days 1, 8 and 15. RESULTS: Relative ECL significantly correlated with irrigation BH (control (n = 13): -9.69 ± 6.03% (average ± standard deviation); BH100 (n = 12): -9.69 ± 4.81%-p = 1.000; BH125 (n = 14): -19.44 ± 7.30% - p < 0.001; BH150 (n = 13): -21.99 ± 6.70%-p < 0.001). I/A-induced ECL was significantly decreased by the injection of ophthalmic viscoelastic, as BH150 + V (n = 14; -10.92 ± 4.09%-p = 1.000) showed a cell loss comparable to the control group. CONCLUSIONS: ECL is altered by I/A BH and reduced when viscoelastic substances are used.

[Optical coherence tomography in the differential diagnostics of important neuro-ophthalmological disease patterns]. / Die optische Kohärenztomographie in der Differenzialdiagnostik wichtiger neuroophthalmologischer Krankheitsbilder.

There are many disease patterns that are treated jointly by neurologists and ophthalmologists, for which optical coherence tomography (OCT) is of important differential diagnostic significance. In this context neurologists are mainly confronted by two patient collectives: patients with an acute ischemic event, who present with an acute but painless monocular visual deterioration (for central retinal artery occlusion) or with a monocular visual field defect (for arterial branch occlusion or anterior ischemic optic neuropathy). The second collective is patients without ophthalmological symptoms but with conspicuous optic nerve findings (papilledema or optic disc drusen). In this overview article both patient collectives are considered separately. In addition, the most important OCT findings for optic neuritis are presented. Before the disease patterns are described in detail, the normal OCT findings and the diagnostic possibilities of OCT are explained.

Inner Retinal Layer Hyperreflectivity Is an Early Biomarker for Acute Central Retinal Artery Occlusion.

Purpose: To investigate inner retinal hyperreflectivity on optical coherence tomography (OCT) as a potential biomarker indicating acute central retinal artery occlusion (CRAO). Methods: A total of 56 patients at two university hospitals with acute CRAO (symptom onset ≤48 h) were included in this retrospective study. The optical intensity of the inner retinal layers was determined in both eyes and the relationship between symptom onset and inner retinal layer optical intensity in OCT scans compared to the unaffected fellow eye was analyzed. Several differential diagnoses [central retinal vein occlusion, anterior ischemic optic neuropathy, diabetic macular edema, and subretinal fibrosis/disciform scar (Junius-Kuhnt)] served as controls to validate optical intensity-based diagnosis of CRAO. Results: CRAO strongly correlated with an increased inner retinal layer hyperreflectivity in this cohort with acute CRAO with a time since symptom onset ranging from 1.1 to 48.0 h. Receiver operating characteristic (ROC) analysis showed an area under the curve of 0.99 to confirm CRAO with a true positive rate of 0.93 and a false positive rate of 0.02. No correlation between optical intensity and time since symptom onset was noticeable. None of the differential diagnoses did show an elevated optical intensity of the inner retinal layers as it was detectable in CRAO. Conclusion: OCT-based determination of inner retinal layer hyperreflectivity is a very promising biomarker for a prompt diagnosis of CRAO in an emergency setting. This may be of major interest to speed up the administration of a possible thrombolytic treatment.

[Optical coherence tomography in the differential diagnostics of important neuro-ophthalmological disease patterns]. / Die optische Kohärenztomographie in der Differenzialdiagnostik wichtiger neuroophthalmologischer Krankheitsbilder.

There are many disease patterns that are treated jointly by neurologists and ophthalmologists, for which optical coherence tomography (OCT) is of important differential diagnostic significance. In this context neurologists are mainly confronted by two patient collectives: patients with an acute ischemic event, who present with an acute but painless monocular visual deterioration (for central retinal artery occlusion) or with a monocular visual field defect (for arterial branch occlusion or anterior ischemic optic neuropathy). The second collective is patients without ophthalmological symptoms but with conspicuous optic nerve findings (papilledema or optic disc drusen). In this overview article both patient collectives are considered separately. In addition, the most important OCT findings for optic neuritis are presented. Before the disease patterns are described in detail, the normal OCT findings and the diagnostic possibilities of OCT are explained.

[Acute diagnostics of central retinal artery occlusion and biomarkers of ischemia : Diagnostics with consequences?] / Akutdiagnostik des retinalen Zentralarterienverschlusses und Biomarker der Ischämie : Diagnostik mit Konsequenzen?

BACKGROUND: Acute central retinal artery occlusion (CRAO) represents an ophthalmological emergency with neurological implications, which must be immediately investigated and treated. Intravenous thrombolysis could improve the prognosis only within the first 4.5 h due to limited retinal tolerance to ischemia. Accordingly, ophthalmological acute diagnostics should be reduced to the minimum necessary followed by immediate referral to a clinic with neurovascular expertise. The typical anamnesis is well-suited for triaging and should be carried out ín a standardized way in order to determine the onset of symptoms as exactly as possible. In addition to pathognomic findings in fundoscopy, there are characteristic ischemia-related changes in optical coherence tomography (OCT), which document ischemia in the inner retina and under some circumstances can provide inferences for the onset of ischemia. OBJECTIVE: This review article summarizes the acute ophthalmological diagnostic management of acute CRAO with a focus on the typical OCT and transorbital ultrasound (TOUS) findings and discusses their potential use as ischemic biomarkers. CONCLUSION: Characteristic biomarkers make OCT an important diagnostic tool in the management of acute CRAO. Additional information can be obtained by TOUS. With an evidence-based treatment established in the future both tools could be used for indications for treatment and for estimating the prognosis.

Determination of Ischemia Onset Based on Automatically Generated Spectralis SD-OCT Values in Acute Central Retinal Artery Occlusion.

Acute central retinal artery occlusion (CRAO) induces a time-dependent increase in retinal thickness. By manually measuring the relative retinal thickness increase (RRTI) in comparison to the contralateral eye based on optical coherence tomography (OCT), ischemia onset within the past 4.5 hours could be determined with 100% sensitivity and 94.3% specificity. To enable examiner-independent and quicker diagnostics, we analyzed the RRTI using the automatic retinal thickness measurement. In this retrospective study, 28 eyes were evaluated with an acute CRAO (<46 hours). All patients received a Spectralis SD-OCT image of both eyes. The RRTI was calculated for the ETDRS sectors using the Segmentation Module for Single Retinal Layer Analysis. Receiver operating characteristic (ROC) analysis was performed to determine patients ≤4.5 hours by RRTI. In all sectors, time to OCT (TTO) and RRTI correlated positively. The optimal cutoff point to detect CRAOs ≤4.5 hours was between 18.7% nasally and 22.9% RRTI temporally. Sensitivity and specificity varied between the sectors with 90-95% sensitivity and 89-100% specificity. In conclusion, the automatic measurement of RRTI also allows the differentiation of CRAOs within a possible therapeutic time window ≤4.5 hours and CRAOs ≥4.5 hours with a high sensitivity and specificity. Additionally, it offers quicker, easier, and a user-independent assessment of ischemia onset, helping to set a base for establishing automatic indices generated by the OCT machines.

Localized bullous pemphigoid after knee replacement surgery.

Bullous pemphigoid (BP) most commonly presents as widespread, itchy, tense blisters in older patients. Localized bullous pemphigoid is a less common form of BP that can be more difficult to diagnose because of its similarity to more common conditions such as allergic contact dermatitis or bullous cellulitis. Prompt recognition of localized BP is important so that appropriate treatment can be started. We present a 57-year-old woman who presented with pruritic tense bullae overlying the surgical scar from a knee replacement 6 months prior on her anterior right knee. This case illustrates the potential for localized BP to be triggered by surgical procedures.

Optical coherence tomography-based determination of ischaemia onset - the temporal dynamics of retinal thickness increase in acute central retinal artery occlusion.

PURPOSE: Acute central retinal artery occlusion (CRAO) induces ischaemic retinal oedema. The purpose of this study was to define sensitivity and specificity of optical coherence tomography (OCT) based retinal thickness analysis in determining ischaemia onset in CRAO. METHODS: The relative retinal thickness increase (RRTI) in comparison with the fellow eye was analysed retrospectively in OCT scans of 66 patients diagnosed with CRAO between January 2010 and December 2019 within 48 hr of ischaemia onset. The natural course of RRTI and the sensitivity and specificity of OCT-based determination of ischaemia onset in identifying CRAO within 4.5 hr using the RRTI were evaluated. RESULTS: Relative retinal thickness increase (RRTI) in acute CRAO follows a hyperbolic curve with a steep incline within the early phase after which it reaches a plateau. Optical coherence tomography (OCT)-based retinal thickness analysis in CRAO allows to differentiate patients with ischaemia onset within the past 4.5 hr or thereafter with a sensitivity of 100% and a specificity of 94.3%. CONCLUSION: Relative retinal thickness increase (RRTI) allows to identify CRAO patients that are eligible for a potentially beneficial reperfusion therapy within a therapeutic window of 4.5 hr with a high accuracy. Especially in patients with unknown ischaemia onset, this diagnostic tool could be of major importance in the future clinical management.

Retinal oedema in central retinal artery occlusion develops as a function of time.

PURPOSE: Time is the key criterion in the management of non-arteritic central retinal artery occlusion (NA-CRAO). However, the precise onset of vision loss is often difficult to determine. This study aimed to evaluate the temporal changes of retinal thickness in acute NA-CRAO and the potential of this parameter to be used as a surrogate marker to estimate the onset of retinal ischaemia. METHODS: Optical coherence tomography was used to continuously assess retinal thickness and oedema progression rate in six porcine eyes. Additionally, a retrospective analysis of 12 patients with acute NA-CRAO was performed to determine association strength and progression rate between retinal thickness and onset of ischaemia. All Optical coherence tomography (OCT) scans (pigs and NA-CRAO patients) were performed within an ischaemic time frame of up to 9 hr. RESULTS: Retinal oedema progression rate in pigs was 25.32 µm/hr [CI 95%: 24.24-26.40 µm/hr]. Retrospective analysis of the patients revealed a strong correlation between retinal oedema and duration of ischaemia (Spearman's rho = 0.77, p = 0.004) with an estimated progression rate of 10.02 µm/hr [CI 95%: 3.30-16.74 µm/hr]. CONCLUSION: Retinal thickness increases with oedema formation, and ischaemia onset is strongly correlated with this structural biomarker in both, pigs and NA-CRAO patients. Prospective clinical trials will have to determine the clinical feasibility of retinal thickness measurements as a biomarker to support clinical management of NA-CRAO.

Effects of ultrasound energy on the porcine corneal endothelium - Establishment of a phacoemulsification damage model.

PURPOSE: The purpose of this study was to establish a standardized in vitro phacoemulsification damage model for future investigations of the effects of phacoemulsification, surgical devices, protective ophthalmic viscoelastic devices (OVDs), irrigation solutions and other aspects related to cataract phacoemulsification surgery on the corneal endothelium using porcine eyes. METHODS: Thirty-four porcine eyes were randomly assigned to three groups (phacoemulsification (n = 13), irrigation (n = 9), control (n = 12)). A total of 5 min of ultrasound energy with intermittent irrigation/aspiration was applied in the eyes of the phacoemulsification group. The eyes of the irrigation group received the identical treatment, but without the application of ultrasound energy. The control group was left untreated. All eyes were then prepared to split corneal buttons followed by 15 days of cultivation. Endothelial cell density (ECD) was assessed blinded on day 15. RESULTS: Endothelial cell density declined significantly more until day 15 in the phacoemulsification group (2567 ± 317/267 cells/mm² (median ± 25%/75%-quartiles), -32.5 ± 7.0/6.4%) compared to the irrigation (3450 ± 350/383 cells/mm², -11.8 ± 5.3/2.6%; p < 0.001) and the control group (3650 ± 288/258 cells/mm², -10.2 ± 3.2/4.6%; p < 0.001). CONCLUSION: The phacoemulsification damage model presented in this study is sensitive to phacoemulsification energy and may reliably be used to investigate various factors involved in phacoemulsification with regard to their influence on corneal endothelial cells. This method is able to replace animal experiments or in vitro cell culture experiments that often do not translate well to the in vivo situation in humans.

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet's membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Effects of Perfluorobutylpentane (F4H5) on Corneal Endothelial Cells.

Purpose: To evaluate the effects of perfluorobutylpentane (F4H5) on corneal endothelial cell density (ECD) and morphology using a porcine corneal endothelial organ culture model. Materials and methods: "Split corneal buttons" were cultivated for 15 days (d) after incubation in F4H5 (15, 30, 60, and 120 min) or BSS (controls). ECD was assessed manually on d1, d8, and d15. After histological staining (trypan blue, alizarin red S) on d15 morphological changes (reformation figures, rosette formations, and alizarin red cells) were evaluated. Results: ECD was significantly reduced after incubation in F4H5 for 120 min (median ± 25%/75%-quartile; 3281 ± 43/222 cells/mm2; p = 0.046) on d15 compared to controls (3658 ± 129/296 cells/mm2), but not after shorter incubation times (15, 30, and 60 min). Morphological assessment supports these findings as reformation figures (F4H5 120 min: 10.5 ± 9.3/13.9/mm2 vs. controls: 5.2 ± 2.8/7.2/mm2; p = 0.010), rosette formations (F4H5 120 min 25.566 ± 17.044/36.219/mm2 vs. controls: 8.333 ± 0.000/15.667/mm2; p = 0.002), and alizarin red cells (F4H5 120 min: 38.350 ± 29.827/51.333/mm2 vs. controls: 20.833 ± 10.417/25.000/mm2; p = 0.049) were significantly more prevalent after incubation in F4H5 for 120 min compared to controls. Also, F4H5 60 min showed significantly more rosette formations (25.452 ± 16.968/36.057/mm2; p = 0.006) and alizarin red cells (46.662 ± 42.420/50.903/mm2; p = 0.007), but not reformation figures (7.0 ± 2.2/1.6 %; p = 0.953). Conclusion: Short exposure (≤30 min) of porcine corneal endothelial cells to F4H5 does not have significant effects on ECD or morphological characteristics. Longer exposure times (≥60-120 min) may cause ECD decline and/or induce morphological changes.

Staining of endothelial cells does not change the result of cell density.

To determine the corneal endothelial cell density human donor corneas are usually placed in hypotonic balanced salt solution for unstained cell counting. Experimental studies often stain the corneal endothelium to highlight cells and cell borders and simplify counting. However, staining normally terminates the experiments. Up to date, there is no publication comparing endothelial cell counts before and after staining. This study compared the counting results of unstained (hypotonic balanced salt solution) and stained (trypan blue & alizarin red S) assessment of the corneal endothelial cell density. No evidence for significantly differing cell counts was found between unstained and stained analysis of the corneal endothelial cell density. Therefore, we consider unstained counting non-inferior to stained counting.

Establishment of a porcine corneal endothelial organ culture model for research purposes.

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.