Absence of detectable SARS-CoV-2 replication in ex vivo cultured cornea and cornea-derived epithelial cells.

PURPOSE: To study the possibility of SARS-CoV-2 to infect human corneal cells and tissues under standard corneal culture conditions using explants of COVID-19 donors and primary cornea-derived epithelial cells. METHODS: Cornea isolated from deceased COVID-19 donors was cultured for 4 weeks, and SARS-CoV-2 replication was monitored by qRT-PCR. Furthermore, primary corneal epithelial cells from healthy donors were cultured ex vivo and infected with SARS-CoV-2 and human cytomegalovirus (HCMV) as a control. Infection status was assessed by western blotting and reporter gene expression using green fluorescent protein-expressing viral strains. ACE2 and TMPRSS2 receptor expression levels in cornea and epithelial cells were assessed by qRT-PCR. RESULTS: We did not detect SARS-CoV-2 replication in 10 corneas isolated from deceased COVID-19 patients and cultured for 4 weeks, indicating absence of infection under natural conditions. Furthermore, high-titer SARS-CoV-2 infection of ex vivo cultured cornea-derived epithelial cells did not result in productive virus replication. In contrast, the same cells were highly permissive for HCMV. This phenotype could potentially be explained by low ACE2 and TMPRSS2 transcriptional activity in cornea and cornea-derived epithelial cells. CONCLUSIONS: Our data suggest that cornea and limbal epithelial cells are refractory to productive SARS-CoV-2 infection. This could be due to the absence of robust receptor expression levels necessary for viral entry. This study adds further evidence to support the very low possibility of transmission of SARS-CoV-2 from an infected corneal transplant donor to a recipient in corneal organ cultures.

Clinical results after single asymmetrical shark fin for graft orientation in DMEK.

PURPOSE: Evaluating the effect of a single peripheral triangular mark to ensure the correct anterior-posterior graft orientation in DMEK. METHODS: Retrospective study of patients scheduled for DMEK due to Fuchs endothelial dystrophy and divided into 2 study groups: Group -M (n = 184) had no mark of the EDM (Endothelial Descemet membrane) and group + M (n = 193) had a triangular peripheral mark. Follow-up time was 1 year after surgery. RESULTS: The postoperative graft turning and Re-DMEK rate could be significantly reduced by the use of a peripheral mark (p = 0.002, p = 0.001, respectively). Re-DMEK due to primary graft failure was significantly associated with prior graft turning (p < 0.001). Both groups showed comparable values for visual acuity, central corneal thickness and endothelial cell count after a follow-up of 1 year. CONCLUSION: Single peripheral triangular marking is a simple and cost-saving addition to EDM preparation to ensure the correct orientation of the graft intraoperatively and could lead to a significant reduction in graft turning and re-DMEK rate in this study.

Outcomes after Descemet membrane endothelial keratoplasty over a period of 7 years at a tertiary referral center: endothelial cell density, central corneal thickness, and visual acuity.

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.

Ketogenic diet attenuates NMDA-induced damage to rat's retinal ganglion cells in an age-dependent manner.

OBJECTIVE: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. METHODS: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed. RESULTS: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. CONCLUSION: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration.

Surgical treatment of open globe trauma complicated with the presence of an intraocular foreign body.

BACKGROUND: Open globe injuries complicated with the presence of an intraocular foreign body constitute a vision threatening condition. PURPOSE: To present the results of pars plana vitrectomy in patients with intraocular foreign body. MATERIAL AND METHODS: Medical records of 22 patients were analyzed. Retrospective analysis of data included visual acuity, age, gender and type of injury. RESULTS: All patients were men and the mean age was 37 years. All injuries occurred while working with a hammer. All patients were treated with pars plana vitrectomy combined with intraocular foreign body removal and internal limiting membrane peeling. The visual acuities improved in 9 cases (41%), in 13 cases (59%) the deterioration of visual acuity was observed, no eye was enucleated. In 14 eyes pars plana vitrectomy was combined with lens removal, in 14 eyes silicone oil was used as a tamponade. CONCLUSIONS: Surgical intervention with pars plana vitrectomy combined with intraocular foreign body removal and cataract extraction may preserve severely traumatized eyes and maintain or even improve vision. ocular trauma, vitrectomy, intraocular foreign body.

Investigating retinal toxicity of tempol in a model of isolated and perfused bovine retina.

PURPOSE: Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl) is a membrane-permeable superoxide dismutase and potentially neuroprotective substance. This study evaluates the retinal tolerance of 0.5 mM, 1 mM, 2 mM, and 5 mM tempol measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. METHODS: For functionality testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution, and the ERG was recorded until stable b-wave amplitudes were reached. Tempol concentrations of 0.5 mM, 1 mM, 2 mM, and 5 mM were tested for 45 minutes. To investigate the effects on photoreceptor function, 1 mM aspartate was added to suppress the b-wave and obtain isolated a-waves. ERG amplitudes were monitored for 100 minutes. RESULTS: While no toxic effects for concentrations of 0.5 mM and 1 mM tempol could be detected, concentrations of 2 mM tempol and higher caused statistically significant negative effects on the b-wave amplitude (-38 %, p = 0.02 for 2 mM; -54 %, p = 0.02 for 5 mM). The a-wave amplitude remained stable even at higher concentrations. CONCLUSIONS: Although the photoreceptors seem to have a tolerance to high concentrations of tempol, higher intravitreal concentrations than 1 mM should be considered critical.

DMEK outcome after one year - Results from a large multicenter study in Germany.

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) accounts for >50% of all corneal transplants in Germany. So far, no data from such a large multicenter study have been published. METHODS: This retrospective study included 3200 DMEKs at seven departments performed for Fuchs endothelial corneal dystrophy (FECD) or bullous keratopathy (BK). We evaluated best corrected visual acuity (BCVA, logMAR), endothelial cell density (ECD, cells/mm2 ), minimal corneal thickness (CT, µm), rebubbling-, primary transplant failure- and immune reaction-rate. Changes over time were evaluated by linear mixed models for repeated measures and correlation with case number by center by weighted linear regression. RESULTS: For patients without vision-limiting comorbidities (74% of all analysed eyes, n = 2270), mean BCVA improved from 0.6 ± 0.4 logMAR to 0.2 ± 0.2 logMAR 6 months (p < 0.001, n = 1441) and 0.1 ± 0.2 logMAR 12 months (p = 0.001, n = 1402) postoperatively. BK- had a worse BCVA compared to FECD-patients (0.3 ± 0.5 vs. 0.1 ± 0.2 logMAR [p < 0.001] at 1 year). ECD declined from 2465 ± 259 cells/mm2 (n = 2876 preoperatively) to 1587 ± 433 cells/mm2 after 12 months (p < 0.001, n = 1237). Mean rebubbling rate was 0.4 ± 0.7/eye. 784 eyes (25%) received at least one rebubbling. More rebubblings correlated with a lower ECD, a worse BCVA, a higher CT, and higher transplant failure and rejection rates (p < 0.001, p = 0.013 for BCVA at 12 months). A single rebubbling did not influence the BCVA (p = 0.785). Graft failure rate was 3% (n = 67), rejection rate 1.5% (n = 48). CONCLUSION: Descemet membrane endothelial keratoplasty increases visual acuity with low transplant failure- and rejection-rates. FECD has a better outcome than BK. Since a quarter of all patients need a rebubbling, this should be included in the informed consent. Remarkably, one rebubbling has no influence on the outcome.

Ketogenic diet increases concentrations of kynurenic acid in discrete brain structures of young and adult rats.

Targeting mechanisms that result in increased concentrations of kynurenic acid (KYNA) in the brain has been considered as a therapeutic approach for the treatment of epilepsy and certain neurodegenerative disorders. Recently, KYNA has been implicated in the effects produced by the high-fat and low-protein/carbohydrate ketogenic diet (KD) in a report demonstrating an increased production of KYNA in vitro by one of the ketone bodies, ß-hydroxybutyrate, elevated by the KD. To further explore this association, brain concentrations of KYNA were compared in young (3 weeks old) and adult (8-10 weeks old) rats that were chronically exposed to the KD and regular diet. Exposure to the KD resulted in the anticipated elevations of ß-hydroxybutyrate with accompanying decreases in glucose concentrations. In comparison to rats fed the regular diet, KYNA concentrations were significantly (p < 0.05) increased in the hippocampus (256 and 363% increase in young and adult rats, respectively) and in the striatum (381 and 191% increase in young and adult rats, respectively) in KD-fed rats. KD-induced increases in KYNA concentrations in young versus adult rats in the hippocampus and striatum were comparable (p > 0.05). Exposure to the KD had no effect on KYNA concentrations in the cortex of young and adult rats (p > 0.05). In summary, chronic exposure to the KD resulted in several-fold increases in KYNA concentrations in discrete brain structures in the rats. Thus, the relevant clinical question for further exploration is whether KD-induced increases in KYNA concentrations can translate into clinically significant improvements in neuropsychiatric diseases associated with KYNA hypofunction.

Synthesis, staining properties, and biocompatibility of a new cyanine dye for ILM peeling.

BACKGROUND: The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS). METHODS: Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295 mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300 s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750 mW/cm(2)) for 10 min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes. RESULTS: DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300 s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591 nm; the even more bathochromic fluorescence proceeds with a common Stokes' shift where maxima at 620 and 660 nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. CONCLUSIONS: Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.

Tempol protects against intravitreous indocyanine green-induced retinal damage in rats.

PURPOSE: Indocyanine green (ICG) has been widely used as a vital dye for macular surgery. However, ICG can be toxic to retinal cells. Here we evaluate whether tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a free radical scavenger, can protect against ICG-induced retinal damage in rats. METHODS: Brown Norway rats received intravitreal injections of ICG 0.5 % or BSS as controls. Tempol (20 mg/kg BW) or PBS as a control was administered intraperitoneally 24 h and 30 min before ICG and once daily for 7 consecutive days. Tempol was detected in the retina using electron paramagnetic resonance (EPR) spectroscopy. One week after ICG injections, the effects of tempol on retinal toxicity were assessed by retinal ganglion cell (RGC) back-labeling and by light microscopy. Electroretinography (ERG) was performed after 1 and 2 weeks. RESULTS: ICG administration reduced RGC numbers by 17 % (1,943 ± 45 vs. 2,342 ± 31 RGCs/mm(2)). Tempol treatment rescued RGCs in a significant manner (2,258 ± 36, p < 0.01) and diminished morphological changes detected by light microscopy. ICG-injected eyes showed a significant reduction of ERG potentials only in PBS-treated animals (V(max) 530 ± 145 µV vs. 779 ± 179 µV, p = 0.0052), but not in the tempol-treated group. CONCLUSIONS: Tempol significantly attenuates ICG-induced toxicity in rat retinas and may therefore be considered for further evaluation as accompanying treatment in ICG-assisted chromovitrectomy.

Caspase inhibitors protect against NMDA-mediated retinal ganglion cell death.

BACKGROUND: Apoptosis is a major mechanism of cell death in glutamate-induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N-methyl-D-aspartate (NMDA). METHODS: Animals were injected intravitreally with one of six caspase inhibitors (against caspases 1, 3, 4, 6, 8 and 9). Seven hours later, NMDA or phosphate-buffered saline as a control was injected intravitreally into the respective eyes. The neuroprotective potential against NMDA toxicity was assessed by retinal ganglion cell quantification. Additionally, wholemount TUNEL was performed. RESULTS: Statistical analysis revealed significant neuroprotective effects for the inhibitors of caspases 3, 6, 8 and 9, but not for those of caspases 1 and 4. The inhibitors of caspases 6 and 9 showed greater neuroprotective potential than those of caspases 3 and 8, although cell death was not entirely averted in any case. Results of ganglion cell counts were confirmed for the most pronounced treatment groups using wholemount TUNEL. CONCLUSION: Excitotoxic retinal ganglion cell death after NMDA injection is mediated mainly through apoptosis, whereby extrinsic as well as intrinsic pathways of caspase activation play a role.

Can SARS-CoV-2 infect the eye? An overview of the receptor status in ocular tissue.

Is the new coronavirus SARS-CoV­2 able to infect ocular tissue and thus poses a risk of infection through the tissue in addition to the risk of contact? This is the question that has occupied ophthalmologists since the beginning of the outbreak. In order to infect a certain type of tissue specific receptors for each virus and sometimes also coreceptors or other proteins must be present. The aim of this review was to summarize and reflect the current state of research with the help of the currently available literature as of 28 May 2020. At the time of the research, angiotensin-converting enzyme 2 (ACE2) was clearly identified as the receptor and transmembrane serine protease 2 (TMPRSS2) as the necessary protease to enable the infection of human cells with SARS-CoV­2. In the eye both ACE2 and TMPRSS2 are expressed, although sometimes very weakly and with varying degrees in different tissues. It is noteworthy that very different results were obtained with different methods. Several reasons can account for this effect: Firstly, the method of detection or preservation of the tissue, secondly, the possibly different expression of the tested tissue samples and thirdly, a possibly rapid loss of receptor expression post-mortem. Therefore, an infection of the eye seems possible, which has already been reported in various publications. The amount of virus or receptor expression necessary to cause an infection is not known. According to current state of knowledge the eye is not considered to be a high-risk tissue due to the low ACE2 and TMPRSS2 expression. Nevertheless, appropriate protective measures are necessary for both medical personnel and patients. In cases of corneal transplantation an infection of the donor tissue with SARS-CoV­2 must be excluded.

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in Human Corneal Tissues.

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.

First results of investigations of SARS-CoV-2 RNA in human corneal tissue.

Preliminary investigations of human corneal tissues from coronavirus disease 2019 (COVID-19) cadaveric donors indicated that no severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is present. Current eye banking guidelines do not recommend any type of routine testing for SARS-CoV­2 RNA in post-mortem donor tissue. This is partly based on factors that can influence the test results of the reverse transcription polymerase chain reaction (RT-PCR).

Severe acute respiratory Syndrome-Coronavirus-2: Can it be detected in the retina?

BACKGROUND/OBJECTIVES: The systemic organ involvement of SARS-CoV-2 needs to be thoroughly investigated including the possibility of an ocular reservoir in humans. To examine retinal tissues and vitreous for histopathology and SARS-CoV-2 presence with regard to possible effects on the human retina and/ or vitreous. We performed histopathological analyses and quantitative (q)RT-PCR-testing for SARS-CoV-2 RNA on retinal tissues and vitreous of COVID-19 postmortem donors. SUBJECTS/METHODS: Included in this study were 10 eyes of 5 deceased COVID-19 patients. The diagnosis of SARS-CoV-2 infection was confirmed via pharyngeal swabs and broncho-alveolar fluids. The highest level of personal protective equipment (PPE) and measures was employed during fluid-tissue procurement and preparation. Histopathological examinations and qRT-PCR-testing were carried out for all retinal tissues and vitreous fluids. RESULTS: The histopathological examinations revealed no signs of morphologically identifiable retinal inflammation or vessel occlusions based on hematoxylin and eosin stains. By qRT-PCRs, we detected no significant level of viral RNA in human retina and vitreous. CONCLUSIONS: In this study, no significant level of SARS-CoV-2-RNA was detected in the human retinal and vitreous fluid samples of deceased COVID-19 patients. Histopathological examinations confirmed no morphological sign of damage to retinal vasculature or tissues. Further studies are needed to confirm or refute the results.

SARS-CoV-2: Impact on, Risk Assessment and Countermeasures in German Eye Banks.

INTRODUCTION: Since the beginning of the COVID-19 pandemic there has been some debate regarding the risk of transmission through tissue transplantation and tissue banking processes. AIM OF THE STUDY: To analyze the changes that SARS-CoV-2 has caused regarding the harvesting of corneal donor tissue and eye bank activities in Germany. METHODS: A questionnaire was provided to 26 eye banks in Germany, consisting of questions about adaptations made in the screening of potential donors and the harvesting of corneal tissue following the pandemic spread of SARS-CoV-2. RESULTS: Eighteen eye banks actively reduced recruitment of donors and two banks ceased all activity. Additional diagnostic screening was performed in eight banks, using conjunctival swabs and/or nasopharyngeal swabs. In six eye banks, additional protective measures, such as FFP2 masks and/or facial shields, were implemented. Overall, a mean reduction in the number of obtained donor tissues of 17% was observed. DISCUSSION: Conjunctival and/or nasopharyngeal swabs of donors have been implemented by a minority. Reasons for not performing additional tests may be moderate sensitivity and lack of validation for postmortem use of RT-PCR testing. Also, the hazard of SARS-CoV-2 entering the corneal donor pool with subsequent transmission might be perceived as theoretical. Face shields provide a sufficient barrier against splash and splatter contamination but may be insufficient against aerosols. Additional face masks would provide support against aerosols, but it remains debatable if corneal harvesting can be considered an aerosol-producing procedure. In the future we expect to see changes in current guidelines because of a surge in scientific activities to improve our understanding of the risks involved with cornea donation in the COVID-19 pandemic, and because current practice may reduce the availability of donor corneas due to new exclusion criteria while the demand remains unchanged.

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load.

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5-8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

Neuroprotection by acetoacetate and ß-hydroxybutyrate against NMDA-induced RGC damage in rat--possible involvement of kynurenic acid.

PURPOSE: This study investigated the effects of systemically administered lithium acetoacetate (ACA) and sodium ß-hydroxybutyrate (BHB) in a rat model of N-methyl-D-aspartate (NMDA)-induced damage of retinal ganglion cells (RGC). Additionally, the influence of ACA and BHB on kynurenic acid (KYNA) production was assessed in vitro in bovine retinal slices. METHODS: Female adult Brown-Norway rats in groups of 5-8 animals were used. ACA and BHB were administered intraperitoneally once a day for 21 consecutive days, and phosphate buffered saline (PBS) was administered to control animals. After 2 weeks, the animals received intraocular NMDA (2 µl of a 10 mM solution in PBS) or intraocular PBS as a control. On day 19, retinal ganglion cells were labeled retrogradely with hydroxystilbamidine. Two days later, RGC density (cells per mm(2)) was assessed on retinal flatmounts. Additionaly, bovine retinal slices were incubated with NMDA and ACA or BHB at concentrations of 1.0 mM and 3.0 mM, and de novo KYNA production was measured using HPLC. RESULTS: Intraperitoneal ACA (250 mg/kg) or BHB (291.2 mg/kg) significantly protected RGC against NMDA-induced neurodegeneration. De novo KYNA production in bovine retinal slices was lowered by NMDA. Both ACA and BHB at a concentration of 3.0 mM significantly reduced the effects of NMDA. CONCLUSIONS: ACA and BHB had a significant dose-dependent neuroprotective effect on RGC in a rat model of NMDA-induced RGC damage. Both ketone bodies also significantly attenuated NMDA-induced reduction of retinal KYNA production in vitro, suggesting that this mechanism may be essential for the neuroprotective effects of ACA and BHB in vivo. Our results imply that ketone bodies may represent an additional treatment option in chronic neurodegenerative disorders of the eye.

Effects of bevacizumab on apoptosis, Na+ -K+ -adenosine triphosphatase and zonula occludens 1 expression on cultured corneal endothelial cells.

BACKGROUND: This laboratory study was undertaken to investigate the influence of bevacizumab on apoptosis, Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) and zonula occludens 1 (ZO-1) expression on cultured human corneal endothelial cells (HCECs). METHODS: Annexin V binding combined with propidium iodide (PI) costaining was used to distinguish viable, early and late apoptotic cells. Immunolocalization of ZO-1 and Na(+)-K(+)-ATPase was performed to analyze intercellular cell integrity after exposure to 5.0 mg/ml bevacizumab for 24 h. RESULTS: No significant induction of apoptosis or necrosis was seen in HCECs after exposure to 5.0 mg/ml bevacizumab (p = 0.689, p = 0.516, respectively). The mean number of annexin-V-FITC- and PI-positive cells did not change significantly. Additionally, no significant changes in expression were detectable, neither for ZO-1 nor for Na(+)-K(+)-ATPase in comparison with the control. For ZO-1, 70.0% of the cells stained intensely, 24.7% stained moderately, and 5.3% stained weakly in the control group. After exposure to 5.0 mg bevacizumab, only minor changes were observable: 68.8% stained intensely, 25.4% moderately and 5.8% weakly (p = 0.524). For Na(+)-K(+)-ATPase, 19.3% of the cells stained intensely, 59.4% moderately, and 21.3% weakly in the control group. After exposure to 5.0 mg bevacizumab, again only minor changes were observable in the expression pattern: 18.2% stained intensely, 60.3% moderately and 21.5% weakly. The changes were not significant compared with the control (p = 0.492). CONCLUSIONS: Bevacizumab, at concentrations used clinically, did not induce apoptosis or necrosis in HCECs in vitro. Additionally, no alteration of ZO-1 or Na(+)/K(+)-ATPase expression was detected after exposure to 5.0 mg/ml bevacizumab for 24 h.