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Background:Primary hyperoxaluria type 1 (PH1) is a rare congenital metabolic disorder of the glyoxylate pathway, which manifests with nephrocalcinosis, urolithiasis, and end-stage renal failure (ESRD) as well as deposition of oxalate crystals within ocular tissues. This report demonstrates classical ocular features of PH1 of the posterior pole and furthermore highlights the ocular genotype–phenotype variability among siblings with identical compound heterozygous alanine-glyoxylate aminotransferase (AGXT) mutations. Materials and Methods: Two siblings, an 8-year-old boy and an 18-year-old girl, with genetically confirmedAGXTmutation (c.364C>T (p.R122X) and c.33dupC), but different renal phenotype underwent an ophthalmic examination, including slit-lamp examination and funduscopy as well as optical coherence tomography (OCT), near-infrared autofluorescence (NIA), and microperimetry examination. Results: The 8-year-old boy presented with a best-corrected visual acuity (BCVA) of 20/630. Fundus examination revealed bilateral, whitish oxalate deposits and prominent fibrotic macular scars. OCT imaging illustrated hyperdense deposits in all retinal layers and the choroid and the vitreous body along with a prominent dome-shaped macular fibrosis. NIA imaging outlined macular retinal pigment epithelium (RPE) atrophy with panretinal hyperreflective material. Bilateral symptomatic epiphora was putatively due to bilateral depositions of palpable nodular oxalate deposits at the level of the lacrimal sac. In contrary, the 18-year-old sister presented without any signs of ocular oxalate deposition and a BCVA of 20/20. Conclusions: PH1 is potentially accompanied with a considerable decline in visual acuity due to macular scaring and fibrosis, whereas a profound variability of ocular manifestations can be observed in PH1 patients with identical genotypes. [ABSTRACT FROM AUTHOR]

PurposeTo 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.MethodsDSS 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).ResultsNo 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.ConclusionsDSS 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. [ABSTRACT FROM AUTHOR]

Autosomal dominant optic atrophy (adOA) is a juvenile onset, progressive ocular disorder characterized by bilateral loss of vision, central visual field defects, colour vision disturbances, and optic disc pallor. adOA is most frequently associated with mutations in OPA1 encoding a dynamin-related large GTPase that localizes to mitochondria. Histopathological studies in adOA patients have shown a degeneration of retinal ganglion cells (RGCs) and a loss of axons in the optic nerve. However little is known about the molecular mechanism and pathophysiology of adOA due to the lack of appropriate in vivo models. Here we report a first mouse model carrying a splice site mutation (c.1065 + 5G --> A) in the Opa1 gene. The mutation induces a skipping of exon 10 during transcript processing and leads to an in-frame deletion of 27 amino acid residues in the GTPase domain. Western blot analysis showed no evidence of a shortened mutant protein but a approximately 50% reduced OPA1 protein level supporting haploinsufficiency as a major disease mechanism in adOA. Homozygous mutant mice die in utero during embryogenesis with first notable developmental delay at E8.5 as detected by magnetic resonance imaging (MRI). Heterozygous mutants are viable and of normal habitus but exhibit an age-dependent loss of RGCs that eventually progresses to a severe degeneration of the ganglion cell and nerve fibre layer. In addition optic nerves of mutant mice showed a reduced number of axons, and a swelling and abnormal shape of the remaining axons. Mitochondria in these axons showed disorganized cristae structures. All these defects recapitulate crucial features of adOA in humans and therefore document the validity and importance of this model for future research. [ABSTRACT FROM AUTHOR]

Background/aims: Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) is a neurodegenerative, blinding lysosomal storage disorder. The purpose of the current study was to characterise the progression of CLN2-associated retinal degeneration in patients under intraventricular enzyme replacement therapy (ERT) with cerliponase alfa., Methods: We analysed visual function, retinal morphology and neuropaediatric data using preferential looking test (PLT), Weill Cornell Batten Scale (WCBS), optical coherence tomography (OCT) imaging and the Hamburg Motor-Language late-infantile neuronal ceroid lipofuscinosis (LINCL) Scale (M-L scale)., Results: Fifty-six eyes of 28 patients had baseline PLT, WCBS and OCT. 15 patients underwent serial examinations, resulting in a total of 132 OCT scans and WCBS results, 66 Hamburg M-L scores and 49 PLT results during a mean follow-up time of 18.2 months (range 5-40). A negative correlation (r=-0.69, p<0.001) was found between central retinal thickness (CRT) values and age at examination with a maximal annual decrease of 23 µm between 56 and 80 months of age. A significant correlation was observed between PLT results and the age at examination (r=0.46, p=0.001), the WCBS scores (r=0.62; p<0.001) and CRT values (r=-0.64; p<0.001). The M-L score correlated with the ocular measurements (CRT: r=0.58, p<0.001; WCBS r=-0.64, p<0.001; PLT score: r=-0.57, p<0.001)., Conclusion: Despite intraventricular ERT, retinal degeneration progressed in patients with CLN2 and was particularly pronounced between 56 and 80 months of age. Retina-directed therapies should therefore be initiated before or as early as possible during the phase of rapid retinal degeneration. PLT and WCBS were identified as valuable outcome measures to monitor disease progression., Trial Registration Number: NCT04613089., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Myopia is a major cause of degenerative eye disease and increases the risk of secondary visual impairment. Mitigating its progression therefore has great potential of clinically relevant benefit as shown by using highly diluted atropine eye drops in children of Asian origin. However, limited evidence is available regarding the efficacy and safety of low-dose atropine therapy in non-Asian populations. Hence, the Low-dose AtropIne for Myopia Control in Children (AIM) study will test the efficacy and safety of 0.02% atropine vs placebo in a German population., Methods and Analysis: AIM is a national, multicentre, prospective, randomised, placebo-controlled, double-blind trial with two parallel arms. The primary objective is to assess the efficacy of atropine 0.02% eyedrops for myopia control in children of Caucasian origin. The primary outcome is the change in cycloplegic refraction after 1 year of treatment (D/year). Secondary and tertiary outcome measures comprise the change in axial length (mm/year) in children treated with 0.02% atropine compared with placebo, the myopic progression of participants treated with 0.01% compared with 0.02% atropine (D/year and mm/year), and the safety profile of both 0.02% and 0.01% atropine. Furthermore, the myopic progression 1 year after cessation of therapy with 0.02% atropine will be evaluated. Inclusion criteria are an age of 8-12 years and myopia of -1 D to -6 D with an estimated annual myopia progression of ≥0.5 D. After randomisation, patients will receive either atropine 0.02% (arm A) or placebo eye drops (arm B) in the first year of treatment. In the second year, they will continue to receive atropine 0.02% (arm A) or switch to atropine 0.01% (arm B). In the third year, they will switch to placebo (arm A) or continue with atropine 0.01% (arm B). To achieve a statistical power of 80%, the calculated sample size is 300. The trial has started in October 2021 with a planned recruitment period of 18 months., Ethics and Dissemination: AIM has been approved by the Central Ethics Committee of the University Medical Center Freiburg (21-1106), local ethics committees of each participating centre and the German Federal Institute for Drugs and Medical Devices (61-3910-4044659). It complies with the Declaration of Helsinki, local laws and ICH-GCP. Results and underlying data from this trial will be disseminated through peer-reviewed publications and conference presentations., Trial Registration Number: NCT03865160., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Kynurenines, products of tryptophan (TRP) metabolism, display neurotoxic (e.g., 3-hydroxykynurenine; 3-HK), or neuroprotective (e.g., kynurenic acid; KYNA) properties. Imbalance between the enzymes constituting the kynurenine pathway (KP) plays a role in several disease, including neurodegeneration. In this study, we track changes in concentrations of tryptophan and its selected metabolites after damage to retinal ganglion cells and link this data with expression of KP enzymes. Brown-Norway rats were subjected to intravitreal N -methyl-D-aspartate (NMDA) injection or partial optic nerve crush (PONC). Retinas were collected 2 and 7 days after the completion of PONC or NMDA injection. Concentrations of TRP, kynurenine (KYN), and KYNA were determined by high performance liquid chromatography (HPLC). Data on gene expression in the rat retina were extracted from GEO, public microarray experiments database. Two days after NMDA injection concentration of TRP decreased, while KYN and KYNA increased. At day 7 compared to day 2 decrease of KYN, KYNA and further reduction of TRP concentration were observed, but on day 7 KYN concentration was still elevated when compared to controls. At day 2 and 7 after NMDA injection no statistically significant alterations of 3-HK were observed. TRP and 3-HK concentration was higher in PONC group than in controls. However, both KYN and KYNA were lower. At day seven concentration of TRP, 3-HK, and KYN was higher, whereas concentration of KYNA declined. In vivo experiments showed that retinal damage or optic nerve lesion affect TRP metabolism via KP. However, the pattern of changes in metabolite concentrations was different depending on the model. In particular, in PONC KYNA and KYN levels were decreased and 3-HK elevated. These observations correspond with data on expression of genes encoding KP enzymes assessed after optic nerve crush or transection. After intraorbital optic nerve crush downregulation of KyatI and KyatIII between 24 h and 3 days after procedure was observed. Kmo expression was transiently upregulated (12 h after the procedures). After intraorbital optic nerve transsection (IONT) Kmo expression was upregulated after 48 h and 7 days, KyatI and KyatIII were downregulated after 12, 48 h, 7 days and upregulated after 15 days. Collected data point to the conclusion that development of therapeutic strategies targeting the KP could be beneficial in diseases involving retinal neurodegeneration., (Copyright © 2019 Fiedorowicz, Choragiewicz, Thaler, Schuettauf, Nowakowska, Wojtunik, Reibaldi, Avitabile, Kocki, Turski, Kaminska, Grieb, Zrenner, Rejdak and Toro.)

Background: Reports on autosomal recessive optic atrophy (arOA) are sparse and so far, only one gene has been specifically associated with non-syndromic arOA, namely TMEM126A. To date, all reports of pathogenic TMEM126A variants are from affected individuals of Maghrebian origin, who all carry an identical nonsense variant. Here we report two novel variants in the TMEM126A gene from non-Maghreb individuals, both found in affected individuals with an arOA phenotype., Case Presentation: We report three affected individuals from two families. The proband of family A, a 24-year-old Turkish woman, was diagnosed with visual loss in early childhood but a diagnosis of optic atrophy was only made at 14 years. A diagnostic gene panel revealed a splice donor variant (c.86 + 2 T > C) in homozygous state in the TMEM126A gene. Analysis of this variant based on RNA from whole blood revealed a single aberrant transcript lacking exon 2, presumably representing a functional null allele. Two siblings from family B, a 16-year old Iraqi girl and her 14-year old brother, were diagnosed with optic atrophy in early childhood. A missense variant p.(S36 L) in the TMEM126A gene was identified in homozygous state in a gene panel-based diagnostic setting in both siblings. This missense variant is ultra rare in the general population, affects a highly evolutionarily conserved amino acid and segregates with the disease within the family. The three probands reported in this study had a relatively mild clinical course without any evidence of a syndromic (e.g. neurological) comorbidity, which is in line with previous studies., Conclusions: We provide additional evidence for the implication of biallelic pathogenic TMEM126A variants in arOA. Our findings extend both the mutational spectrum and geographic presence of TMEM126A in arOA. Screening of the entire gene should be considered in affected individuals presenting with features resembling arOA and also from non-Maghrebian descent.

Objective The objective of this article is to evaluate advanced techniques of diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) measurements of the optic nerve in patients with optic neuritis. Methods In this prospective and institutional review board-approved trial, we examined 15 patients with acute visual loss and clinical signs of optic neuritis including thin-slice multi-shot segmented readout of long variable echo trains (rs-EPI, RESOLVE) DWI and reduced field-of view DWI using a parallel transmit system (rFOV-EPI). Conventional single-shot echo-planar DWI (ss-EPI) of the whole brain was available in 13 patients. Subjective image quality was compared using a four-point scale and objective ADC measurements were performed in comparison with the non-affected side. Results In the intraorbital segment, subjective image quality was significantly higher in rFOV-EPI (score 3.3 ± 0.8) compared with rs-EPI (score 2.1 ± 0.8) and ss-EPI (score 0.9 ± 0.8). Diagnosis was hampered in the canalicular segment ( n = 3) and the intracranial segment ( n = 1) in all applied DWI techniques. ADC measurements of the affected side differed significantly in all DWI sequences ss-EPI (sensitivity 54%, accuracy 77%), rs-EPI (sensitivity 71%, accuracy 86%), and rFOV-EPI (sensitivity 73%, accuracy 87%). Conclusion Optic neuritis in the intraorbital segment can be detected with high sensitivity without the need for contrast application. Using rFOV-EPI improves subjective image quality compared with rs-EPI and ss-EPI. Due to its higher spatial resolution, rFOV-EPI was the preferred technique in our study and can ensure the diagnosis in the intraorbital segment. However, artefacts occur in the canalicular and intracranial segment of the optic nerve, therefore contrast-enhanced T1-weighted images must still be considered as the gold standard.

Purpose: To prospectively compare the image quality and diagnostic performance of orbital MR images obtained by using a dual-source parallel transmission (pTX) 3D sequence (Sampling Perfection with Application optimized Contrasts using different flip angle Evolution, SPACE) with the image quality of conventional high-resolution standard protocol for clinical use in patients at 3T., Materials and Methods: After obtaining institutional review board approval and patient consent, 32 patients with clinical indication for orbital MRI were examined using a high-resolution conventional sequences and 3D pTX SPACE sequences. Quantitative measurements, image quality of the healthy orbit, incidence of artifacts, and the subjective diagnostic performance to establish diagnosis was rated. Statistical significance was calculated by using a Student's t-test and nonparametric Wilcoxon signed rank test., Results: Length measurements were comparable in the two techniques, 3D pTX SPACE resulted in significant faster image acquisition with higher spatial resolution and less motion artifacts as well as better delineation of the optic nerve sheath. However, estimated contrast-to-noise and signal-to-noise and overall image quality as well as subjective scores of the conventional TSE imaging were rated significantly higher. The conventional MR sequences were the preferred techniques by the readers., Conclusion: This study demonstrates the feasibility of 3D pTX SPACE of the orbit resulting in a rapid acquisition of isotropic high-resolution images. Although no pathology was missed in 3D pTX SPACE, conventional MRI techniques showed the higher diagnostic confidence in our study, presumably due to the higher signal-to-noise and contrast-to-noise ratios. We observed high-resolution TSE imaging to be the preferred technique, 3D pTX SPACE cannot replace conventional MRI so far., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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., (© 2015 S. Karger AG, Basel.)

Background: The efficacy of tempol and its acyl derivative tempol-C8 as retinoprotective agents was compared in a rat model of NMDA-induced retinal ganglion cell (RGC) damage., Material and Methods: Tempol or tempol-C8 in different doses was administered intraperitoneally to 6 weeks old (pre-adolescent) and 9-10 weeks old (young adult) rats before and after an intravitreous NMDA injection. Retinal ganglion cell were retrogradely labeled with the fluorescent tracer hydroxystilbamidine and RGC counting was performed on retinal flatmounts., Results: Intravitreal NMDA reduced RGC counts by about 90%, independently of age < 0.001). In pre-adolescent animals tempol-C8, but not tempol unmodified, showed a significant, dose-dependent RGC rescue effect, with peak activity at 5.8 µmol/kg (p < 0.001). In young adult animals, however, no neuroprotective effect was found for either tempol or tempol-C8., Conclusions: In contrast to tempol itself, tempol-C8 acyl ester was neuroprotective in pre-adolescent rats in the NMDA- induced RGC damage model. Therefore, neuroprotection by tempol acyl esters seems to be superior to that of tempol under certain conditions.

Sudden onset diplopia may occur secondary to something as simple as uncorrected refractive error or as complicated as brainstem disorders in conjunction with other symptoms. Therefore, all complaints of diplopia must be a cause for concern. Ophthalmologists have to determine if diplopia is the first sign of a systemic or neurological disorder, which needs to be referred to a specialist or can be managed by the practitioner. In this paper the importance of the case history, primary diagnostic options, the indications for supplementary testing with computed tomography (CT) or magnetic resonance imaging (MRI) as well as treatment options when a patient complains of sudden onset diplopia are discussed.

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.

Purpose: The aim of this study is to search for more effective derivatives of the superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). Although tempol is neuroprotective in a rat partial optic nerve crush (PONC) model, relatively high doses are required to exert this effect., Methods: Tempol acyl esters with different-length fatty acids (tempol-C4, tempol-C8, tempol-C12 and tempol-C16) were synthesized and the following properties were evaluated: water-octanol partition coefficient, liposome-liposome energy transfer, and electron paramagnetic resonance (EPR). Brown Norway rats underwent PONC and received tempol or acyl esters intraperitoneally once daily for 7 consecutive days. We then compared the effects of tempol and its four esters on retinal ganglion cell (RGC) damage using a retrograde labelling method., Results: The water-octanol partition coefficient increased with increasing length of attached acyl chain. However, the energy of the liposome-liposome transfer seemed to be optimal for tempol-C8 and tempol-C12. The EPR signal was very similar for all tested compounds, suggesting similar efficiency of superoxide scavenging. Partial optic nerve crush in vehicle-treated animals reduced RGC numbers by approx. 59% when compared with sham-operated eyes. Tempol did not affect RGC loss at a dose of 1 mg/kg. In contrast, at molar doses equivalent to 1 mg/kg of tempol, tempol-C8 showed a significant neuroprotective effect, whereas tempol-C4, tempol-C12 and tempol-C16 did not act neuroprotectively., Conclusion: Manipulating the hydrophobicity of tempol seems to be a promising tool for developing more potent neuroprotectants in the PONC degeneration model. However, the resulting compounds need further pharmacological evaluation., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

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., (© 2011 The Authors. Clinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: To evaluate capsule-staining properties and biocompatibility of the triarylmethane dyes methyl blue and aniline blue compared with patent blue and trypan blue on cultured human corneal endothelial cells., Setting: Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany., Design: Experimental study., Methods: Human corneal endothelial cell cultures were harvested from human donor cells and exposed to various concentrations (0.025 to 5.0 mg/mL) of methyl blue, aniline blue, patent blue, and trypan blue. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test after 24 hours of incubation. Calcein live cell staining was performed at the same time point. The dyes were also used to stain pig lens capsules in vitro by incubating the lenses for 1 minute with 3 concentrations (0.5, 1.5, and 2.5 mg/mL) of dye, after which the staining properties were evaluated., Results: No significant cytotoxicity was detected for patent blue and methyl blue at any tested concentration. However, aniline blue exerted significant cytotoxicity at concentrations of 1.5 mg/mL or higher and trypan blue at 2.5 mg/mL or higher. Capsule staining of the tested triarylmethane dyes was suitable for performing capsulorhexis, but only at higher concentrations than with trypan blue., Conclusions: High concentrations and long incubation times of trypan blue and aniline blue showed significant cytotoxicity to human cultured endothelial cells in contrast to patent blue and methyl blue. All tested dyes were able to stain lens capsules sufficiently for capsulorhexis creation., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

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.

Purpose: This study investigated the effects of systemically administered lithium acetoacetate (ACA) and sodium β-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.

Purpose: To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage., Methods: Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors., Results: RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations., Conclusions: Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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: The purpose of this study was to investigate the biocompatibility of aniline and methyl blue in a well-established cell culture model and assess the staining properties of these dyes at the level of the internal limiting membrane (ILM) in human donor eyes., Methods: Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (ARPE-19 and primary human retinal pigment epithelium) cell proliferation. Cell viability was also quantified based on a two-color fluorescence assay (Life-Dead Assay). Aniline blue and methyl blue at a concentration of 0.2% was applied over the macula during vitrectomy in human donor eyes to evaluate the staining properties at the level of the ILM., Results: Both dyes and dye concentrations of 0.1% and 0.2% showed no toxic effect on ARPE-19 and primary human retinal pigment epithelium cell proliferation for exposure times of 1 and 10 minutes, respectively. Cell viability was also not affected at all. Both dyes provided a good contrast at the level of the ILM and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted., Conclusion: Our results indicate that aniline blue and methyl blue might be applicable for intraocular surgery, providing a very good biocompatibility and required selective staining characteristics at the level of the ILM.

Purpose: To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions., Methods: Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days., Results: Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC., Conclusions: These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.

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.

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: This study set out to document the early electrophysiological and immunohistochemical changes that occur in the retina of experimentally induced diabetic rats., Methods: Diabetes was induced in rats by intraperitoneal injection of 60 mg/kg of streptozotocin (STZ). Electroretinogram readings were taken monthly under either short-duration or long-duration stimuli for up to 3 months after STZ. Oscillatory potentials (OP) and the amplitudes and implicit times of a- and b-waves were analysed, and b-wave amplitudes were analysed using a Naka-Rushton fit. Scotopic a-waves were analysed with photoreceptor models, and Rmp3 (the maximum a-wave amplitude) and S (sensitivity) were calculated. Three months after STZ injection, immunohistochemistry for glial fibrillary acidic protein was performed on the retinas of the STZ-treated rats and age-matched controls., Results: The implicit OP times were significantly longer in the diabetic rats as compared with the controls, and this difference was noted as early as 1 month following STZ treatment. Other electrophysiological parameters, such as OP amplitudes, a- and b-wave amplitude as well as the implicit times, did not differ from controls at this stage. The sacrificed STZ-treated rats also demonstrated marked enhancement of glial fibrillary acidic protein immunoreactivity, suggesting that at least in experimentally induced diabetic retinopathy there is increased Müller cell reactivity., Conclusion: The results of this study indicated that functional alterations in the retina develop rapidly after the onset of diabetes. Analysis of each electroretinogram component may be useful in further investigating the development mechanisms of diabetic retinopathy.

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.

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression.

Purpose: To determine the prevalence of FOXC1 and PITX2 mutations and to assess clinical phenotypes in a cohort of German patients with Axenfeld-Rieger malformations., Methods: All coding exons of the FOXC1 and PITX2 genes were amplified by PCR from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by restriction fragment length polymorphism (RFLP) analysis., Results: Sequence variants were identified by DNA sequencing in 15 of 19 cases. Mutation screening identified four potentially pathogenic FOXC1 mutations causing amino acid substitutions (P79R, Y115S, G149D, and M161V) that were not present in 100 control subjects. In addition, two different 1-bp deletions causing a frameshift and subsequent premature stop codon were identified in two subjects. One patient harbored a FOXC1 nonsense mutation (S48X). Mutation screening also identified two potentially pathogenic PITX2 mutations (P64L and P64R) in two index patients that were excluded in 100 healthy control subjects., Conclusions: The findings in the present study clearly demonstrate that FOXC1 and PITX2 mutations are responsible for a significant proportion of Axenfeld-Rieger malformations in Germany.

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: Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN., Methods: Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC)., Results: After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration., Conclusions: This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers.