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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., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To investigate the biocompatibility of the new cyanine dye: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS) as a vital dye for intraocular application in an in vivo rat model and to evaluate the effects of this dye on retinal structure and function., Methods: DSS at a concentration of 0.5% was applied via intravitreal injections to adult Brown Norway rats with BSS serving as a control. Retinal toxicity was assessed 7 days later by means of retinal ganglion cell (RGC) counts, light microscopy, optical coherence tomography (OCT), and electroretinography (ERG)., Results: No significant decrease in RGC numbers was observed. No structural changes of the central retina were observed either in vivo (OCT) or under light microscopy. ERGs detected a temporary reduction of retinal function 7 days after injection; this was no longer evident 14 days after injection., Conclusions: DSS showed good biocompatibility in a well-established experimental in vivo setting and may be usable for intraocular surgery as an alternative to other cyanine dyes. In contrast to indocyanine green, it additionally offers fluorescence in the visual spectrum. Further studies with other animal models are needed before translation into clinical application.

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.

The kynurenine aminotransferases (KATs) KAT I and KAT II are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. KAT I and II have been found in avian, rodent, and human retina. Expression of KAT I in Müller cell endfeet and KAT II in retinal ganglion cells has been documented. Developmental changes in KAT expression and KYNA concentration in the avian and rodent retina have also been found. Studies of retinal neurodegeneration have shown alterations in KYNA synthesis in the retina in response to retinal ganglion cell loss. In DBA/2J mice, a model of ocular hypertension, an age-dependent decrease of retinal KYNA and KATs was found. In the corpora amylacea in the human retina intensive KAT I and II immunoreactivity was demonstrated. In summary, these findings point to the potential involvement of KYNA in the mechanisms of retinal aging and neurodegeneration.

Background: Corpora amylacea (CAm) are a hallmark of aging and neurodegeneration. The presence of kynurenine aminotransferases I and II (KAT I and II) in CAm in the human retina and optic nerve has been already shown. The present study aimed to examine kynurenine aminotransferase III (KAT III) immunoreactivity in CAm in the human retina and optic nerve., Material and Methods: Polyclonal antibody against KAT III was used on sections of human eyes enucleated due to malignant uveal melanoma. PAS-stained sections of CAm were compared with KAT III stained ones., Results: KAT III immunoreactivity was observed in CAm in the retina, prelaminar, laminar and retrolaminar region of the optic nerve with similar location to PAS-stained sections. The most intense staining was observed in the retrolaminar part of the optic nerve. KAT III immunoreactivity was also present in the cytoplasm of retinal ganglion cells., Conclusions: Expression of KAT III in CAm in the human retina and optic nerve indicates that this enzyme may be relevant in mechanisms of neurodegeneration leading to CAm formation.

Purpose: To investigate the biocompatibility of methyl blue and aniline blue as vital dyes for vitreoretinal surgery in an in vivo rat model and to evaluate the effect of these dyes on retinal structure and function., Methods: Adult Brown-Norway rats received intravitreal injections of 0.1%, 0.2%, and 2% methyl blue or aniline blue dissolved in balanced salt solution with balanced salt solution serving as a control. Retinal toxicity was assessed 7 days thereafter by means of retinal ganglion cell counts, light microscopy, and electroretinography., Results: No significant decrease in retinal ganglion cell counts at concentrations up to 0.2% was observed. At 2%, however, a significant retinal ganglion cell loss was detected with both dyes (more pronounced for aniline blue). Light microscopy showed no structural changes in the central retina for concentrations up to 0.2%. Electroretinographies detected no adverse effects of methyl blue or aniline blue on rod- or cone-driven responses at concentrations up to 0.2%., Conclusion: Methyl blue and aniline blue are very biocompatible and may, therefore, be usable for intraocular surgery. Further testing with other animal models will be necessary to confirm this. The safety margin of methyl blue is possibly higher than that of aniline blue.

Purpose: To investigate the membrane physiology of Müller glial cells from retinae of DBA/2J mice (which develop ocular hypertension) and of C57BL/6 control mice of different ages., Methods: Retinae were obtained at the ages of 3, 6, and 12 months from DBA/2J mice and from C57BL/6 controls. Immunohistochemistry was performed using antibodies against glial fibrillary acidic protein (GFAP). Whole-cell membrane currents, membrane potentials and capacitances were recorded from freshly isolated Müller cells., Results: Strong immunostaining for GFAP was found in Müller cells of 12-month-old DBA/2J mice, whereas only astrocytes were immunopositive in C57BL/6 retinae. No significant alterations of membrane currents or potentials of Müller cells from DBA/2J mice as compared to controls were observed at any age; however, the membrane capacitance was increased in Müller cells from DBA/2J mice at the age of 6 months., Conclusions: Although Müller cells of DBA/2J mice display some symptoms of reactive gliosis, the lack of significant alterations of the membrane physiology confirm previous data demonstrating that these cells undergo a nonproliferative gliosis.

Background/aims: To evaluate the retinal toxicity of Brilliant Blue G (BBG) following intravitreal injection in rat eyes and examine the biocompatibility and the staining properties in humans., Methods: BBG was injected into the 11 rat eyes to evaluate toxic effects with balanced salt solution (BSS) serving as control. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts and by light microscopy 7 days later. In addition, BBG was applied during vitrectomy for macular hole (MH) (n = 15) or epiretinal membranes (ERM) (n = 3) in a prospective, non-comparative consecutive series of patients. Before and after surgery, all patients underwent a complete clinical examination including measurement of best corrected visual acuity (VA) and intraocular pressure, perimetry, fundus photography and optical coherence tomography. Patients were seen 1 day before surgery and then in approximately four weeks intervals., Results: No significant reduction in RGC numbers and no morphological alterations were noted. A sufficient staining of the internal limiting membrane (ILM) was seen in patients with MH, while the staining pattern in ERM cases was patchy, indicating that parts of the ILM were peeled off along with the ERM in a variable extent. All MHs could be closed successfully. VA improved in 10 eyes (56%; 8/15 MH patients, 2/3 ERM patients), was unchanged in four eyes (22%; all MH patients) and was reduced in four eyes (22%; 3/15 MH, 1/3 ERM). No toxic effects attributable to the dye were noted during patient follow-up. The ultrastructure of tissue harvested during surgery was unremarkable., Conclusion: Brilliant Blue provides a sufficient and selective staining of the ILM. No retinal toxicity or adverse effects related to the dye were observed in animal and human studies. The long-term safety of this novel dye will have to be evaluated in larger patient series and a longer follow-up.

Purpose: To investigate the intraocular effect of rhodamine 6G (R6G) on retinal structures and function in an in vivo rat model and to develop an in vivo method for accurate evaluation of new dyes for intraocular surgery., Methods: R6G in physiologic saline solution (PSS) was injected into the vitreous of adult Brown Norway rats at concentrations of 0.0002%, 0.002%, 0.02%, 0.2%, and 0.5%. Control animals received only PSS. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts, light microscopy 7 days later, photopic electroretinography (ERG), and measurement of scotopic sensitivity and recovery of dark adaptation 48 hours and 7 days after intravitreous injection., Results: R6G at concentrations of 0.2% and 0.5% led to a dose-dependent loss of RGC. The most significant loss occurred at 0.5%. Lower concentrations (0.0002%, 0.002%, and 0.02%) produced no statistically significant retinal ganglion cell loss. Analysis of the eyes by light microscopy showed no structural changes in the central retina, although injections of 0.5% R6G were followed by impressive degenerative changes adjacent to the injection sites. ERGs showed no effects of the highest R6G concentration on rods, kinetics of rhodopsin recovery after bleaching, or cone-driven responses., Conclusions: R6G can be safely injected in doses of up to 0.02% in rats, but has a toxic effect on retinal ganglion cells at higher concentrations. Accumulation of R6G may be a problem at higher concentrations, particularly at the injection site.

Background: The DBA/2J mouse spontaneously develops ocular hypertension and time-dependent progressive retinal ganglion cell (RGC) loss. This study examines changes in amino acid levels in the vitreous, and changes in the expression of retinal glutamate transporters and receptors that occur during the progression of this pathology., Methods: Retinas were obtained from DBA/2J mice at ages 3, 6 and 11 months. C57BL/6 mice were used as age-matched controls. Vitreal amino acid content was measured with HPLC. Western blotting and immunohistochemistry were performed using specific antibodies against the glutamate transporters (GLAST, GLT-1v, EAAC-1) and glutamate receptors, particularly NMDA (NR1, NR2A, NR2B) and AMPA (GluR1, GluR2/3, GluR4) receptors., Results: HPLC showed retinal concentrations of glutamate, glutamine, glycine, alanine, lysine, serine, and arginine to be significantly higher in DBA/2J mice at 11 months of age compared to age-matched controls. Western Blots revealed a moderate decrease of GLAST and GLT-1v expression in DBA/2J mice at 6 and 11 months as compared to age-matched controls while there was no change in EAAC1. Immunohistochemically, no changes in expression of NMDA and AMPA receptors were seen., Conclusion: Alterations of amino acid content and enhanced glutamate neurotransmission might be involved in the pathogenesis of retinal neurodegeneration in the DBA/ 2J mouse model of ocular hypertension. Moreover, these mice provide an animal model for studying excitotoxic retinal damage.

Purpose: To investigate the effect of intravitreal injections of new vital dyes on the retina, the retinal pigment epithelium (RPE) and the choroid in an in vivo rat model., Methods: Rats were injected intravitreally with four dyes: light-green SF yellowish (LGSF), copper(II)phthalocyanine-tetrasulfonic acid (E68), bromphenol blue (BPB), and Chicago blue (CB) dissolved in physiologic saline solution (PSS) at concentrations of 0.5% and 0.02%. PSS served as the control. Additional animals were treated with single injections of 0.5%, 0.02%, 0.002%, and 0.0002% ICG or 0.002% E68 into one eye. Adverse effects on anterior and posterior segments were evaluated by slit lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by histology and retinal ganglion cell (RGC) quantification 7 days after dye administration., Results: Eyes treated with 0.5% E68, 0.5% ICG, or 0.5% CB showed discrete staining of both cornea and lens not seen at lower concentrations or with other dyes. Histology revealed dose-dependent reactions after E68 administration. ICG 0.5% induced significant thinning of inner retinal layers compared with PSS. ICG 0.02% caused focal degenerative changes of the outer retina in three of seven eyes, whereas 0.002% and 0.0002% ICG did not. CB led to heterogeneous morphologic alterations. BPB- or LGSF-treated eyes showed normal retinal morphology. ICG at all tested concentrations induced significant RGC loss, as did E68 at 0.5% but not at lower concentrations., Conclusions: BPB or LGSF produced no significantly detectable toxic effects on the retina in vivo. The safety of these new dyes must be established in other models and/or in preclinical studies before the clinical use of any of these dyes.

Purpose: To investigate the effect of intraocular erucylphosphocholine (ErPC) on the retina, the retinal pigment epithelium (RPE), and the choroid in an in vivo rat model., Methods: Adult male Brown Norway rats were injected intravitreally with ErPC dissolved in balanced salt solution (BSS) at a final concentration of 10 or 100 microM with BSS serving as control. Adverse effects on the anterior and posterior segment were assessed by slit-lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by electroretinography (ERG), retinal ganglion cell (RGC) quantification, and histology 7 days after intravitreal administration of ErPC., Results: There was neither a statistically significant difference in the clinical examination nor in the ERG waves of treated versus control rats 7 days after intravitreal administration of ErPC. Correspondingly, the number of RGC after BSS injection did not differ significantly from ErPC-injected animals. Histologic sections of the posterior segment of 10 and 100 microM ErPC-injected rats did not show any signs of retinal toxicity. Electron microscopy did not display a difference between the 10 microM and the control group. Only the 100 microM-injected animals showed a discrete irregularity of the Müller cell and the retinal ganglion cell cytoplasm at the ultrastructural level., Conclusions: ErPC can safely be injected into the vitreous of adult rats at a concentration of 10 microM without any retinal toxicity. Even a 10-fold increase in ErPC concentration leads only to a discrete cytoplasmic irregularity of the innermost retinal layers.