THE TARGET SIGN: A Near Infrared Feature and Multimodal Imaging in a Pluri-Ethnic Cohort with RDH5-Related Fundus Albipunctatus.

PURPOSE: Retinol dehydrogenase 5 (RDH5)-related fundus albipunctatus can present with phenotypic variability. Our purpose was to investigate new clinical characteristics and multimodal imaging findings in patients from different ethnic origins, carrying different mutations. METHODS: Multicenter international retrospective case series of 18 patients with genetically confirmed RDH5-related fundus albipunctatus. Patients' files were reviewed for fundus images, visual acuity, macular optical coherence tomography scans, near-infrared images, fundus autofluorescence, electroretinogram, and genetic mutations. Imaging and electroretinogram findings. RESULTS: All eyes (n = 36, 100%) showed small circular findings seen on near-infrared images, termed as the "target sign," correlating to the yellowish dots seen clinically and to the distinct hyperreflective linear lesions on optical coherence tomography at the level between external limiting membrane and retinal pigment epithelium. Perifoveal atrophy with foveal sparing was seen in 4 eyes of 2 patients (both RDH5-c.160C>T, p.R54X mutation). Fundus autofluorescence revealed small hyperautofluorescent dots (n = 16, 44.4%). Scotopic electroretinograms were significantly reduced in all cases with an electronegative pattern, 66.7% displayed cone dysfunction. CONCLUSION: Our results show distinct imaging findings present in all patients with fundus albipunctatus independent of ethnicity or genetic mutation. Our results can facilitate the current algorithm to diagnose RDH5-related fundus albipunctatus and allow for targeted genetic testing.

PRCD is concentrated at the base of photoreceptor outer segments and is involved in outer segment disc formation.

Mutations of the photoreceptor disc component (PRCD) gene are associated with rod-cone degeneration in both dogs and humans. Prcd is expressed in the mouse eye as early as embryonic day 14. In the adult mouse retina, PRCD is expressed in the outer segments of both rod and cone photoreceptors. Immunoelectron microscopy revealed that PRCD is located at the outer segment rim and that it is highly concentrated at the base of the outer segment. Prcd-knockout mice present with progressive retinal degeneration, starting at 20 weeks of age and onwards. This process is reflected by a significant and progressive reduction of both scotopic and photopic electroretinographic responses and by thinning of the retina, and specifically of the outer nuclear layer, indicating photoreceptor loss. Electron microscopy revealed severe damage to photoreceptor outer segments, which is associated with immigration of microglia cells to the Prcd-knockout retina and accumulation of vesicles in the inter-photoreceptor space. Phagocytosis of photoreceptor outer segment discs by the retinal pigmented epithelium is severely reduced. Our data show that Prcd-knockout mice serve as a good model for retinal degeneration caused by PRCD mutations in humans. Our findings in these mice support the involvement of PRCD in outer segment disc formation of both rod and cone photoreceptors. Furthermore, they suggest a feedback mechanism which coordinates the rate of photoreceptor outer segment disc formation, shedding and phagocytosis. This study has important implications for understanding the function of PRCD in the retina, as well as for future development of treatment modalities for PRCD deficiency in humans.

A nationwide genetic analysis of inherited retinal diseases in Israel as assessed by the Israeli inherited retinal disease consortium (IIRDC).

Inherited retinal diseases (IRDs) cause visual loss due to dysfunction or progressive degeneration of photoreceptors. These diseases show marked phenotypic and genetic heterogeneity. The Israeli IRD consortium (IIRDC) was established in 2013 with the goal of performing clinical and genetic mapping of the majority of Israeli IRD patients. To date, we recruited 2,420 families including 3,413 individuals with IRDs. On the basis of our estimation, these patients represent approximately 40% of Israeli IRD patients. To the best of our knowledge, this is, by far, the largest reported IRD cohort, and one of the first studies addressing the genetic analysis of IRD patients on a nationwide scale. The most common inheritance pattern in our cohort is autosomal recessive (60% of families). The most common retinal phenotype is retinitis pigmentosa (43%), followed by Stargardt disease and cone/cone-rod dystrophy. We identified the cause of disease in 56% of the families. Overall, 605 distinct mutations were identified, of which 12% represent prevalent founder mutations. The most frequently mutated genes were ABCA4, USH2A, FAM161A, CNGA3, and EYS. The results of this study have important implications for molecular diagnosis, genetic screening, and counseling, as well as for the development of new therapeutic strategies for retinal diseases.

Verifying complaints of difficulties in night vision using electroretinography and dark adaptation tests.

PURPOSE: To determine the electroretinographical and psychophysical parameters that can help to verify patients' complaints of reduced night vision. METHODS: We tested 275 consecutive patients with normal appearing fundi, complaining of visual difficulties at night, using flash electroretinography (ERG) and dark adaptation (DA) test. Two ERG parameters were used to assess a scotopic retinal function: the amplitude of the response to dim blue flash (the rod response) and the b-wave ratio (measured/expected). Dark adaptation was measured with green- and red-light stimuli after exposure to a bright, bleaching light. The psychophysical parameter of night vision was defined as the threshold for detection of the blue-green stimulus that was measured after 40-45 min in complete darkness. RESULTS: Fifty-five patients were excluded from the analysis because of a discrepancy between the two ERG parameters in assessment of scotopic retinal function. The remaining 220 patients were divided into 4 groups: (1) normal ERG and normal DA, (2) subnormal ERG and subnormal DA, (3) normal ERG and subnormal DA and (4) subnormal ERG and normal DA. The ERG and DA tests supported the complaint of visual difficulties at night in 67 patients (group 2), while 34 patients were characterized as having normal scotopic visual function (group 1). The other 119 patients (groups 3 and 4) presented a diagnostic dilemma because one test (ERG or dark adaptation) showed normal scotopic function, while the other indicated subnormal scotopic function. CONCLUSION: Our findings indicate that ERG is an essential, but not sufficient test for verifying patient's complaint on visual difficulties in the dark. We suggest using both electroretinography and psychophysical dark adaptation to test patients complaining of reduced night vision.

ISCEV extended protocol for the S-cone ERG.

The International Society for Clinical Electrophysiology of Vision (ISCEV) standard for full-field electroretinography (ERG) describes a minimum procedure for testing generalized retinal function but encourages more extensive testing. This extended protocol describes a method of assessing the function of the short-wavelength-sensitive cone (S-cone) retinal pathway, using a short-wavelength flash superimposed on a background that saturates the rods and adapts the L/M-cones to elicit a response, known as the S-cone ERG. Stimulus parameters such as the strength and luminance of the flash and background, respectively, and their spectral and temporal characteristics are specified. As a complement to the ISCEV standard, testing the S-cone ERG enables further characterization of light-adapted retinal function and may refine diagnosis of some retinal disorders. Typical applications are described including use in the diagnosis of rod monochromacy and S-cone monochromacy, identification and investigation of cone On-bipolar cell dysfunction and use of the technique to confirm the diagnosis of enhanced S-cone syndrome.

[THE ISRAELI INHERITED RETINAL DISEASES CONSORTIUM (IIRDC)- CLINICAL-GENETIC MAPPING AND FUTURE PERSPECTIVES].

INTRODUCTION: The sense of vision is highly important for humans and its loss markedly affects function and quality of life. Many inherited retinal diseases (IRDs) cause visual loss due to dysfunction or progressive degeneration of photoreceptor cells. These diseases show clinical and genetic heterogeneity. AIMS: The Israeli IRD consortium (IIRDC) was established with the goal of performing clinical and genetic mapping of IRDs in the Israeli population. METHODS: Clinical evaluation is carried out at electroretinography (ERG) centers and ophthalmology departments, where the patients undergo a comprehensive eye exam, including testing of visual acuity, refractive error, imaging techniques and ERG tests. Genetic analysis is performed using Sanger sequencing, analysis of founder mutations, and whole exome sequencing. RESULTS: We recruited over 2,000 families including more than 3,000 individuals with IRDs. The most common inheritance pattern is autosomal recessive (65% of families). The most common retinal phenotype is retinitis pigmentosa (RP- 45% of families), followed by cone/cone-rod dystrophy, Stargardt Disease and Usher syndrome. We identified the cause of disease in 51% of families, mainly due to mutations in ABCA4, USH2A, FAM161A, CNGA3, and EYS. IIRDC researchers were involved in the identification of 16 novel IRD genes. In parallel, IIRDC members are involved in the development of therapeutic modalities for these currently incurable diseases. CONCLUSIONS: IIRDC works in close collaborative efforts aiming to continue and recruit for the genotype - phenotype study from the vast majority of Israeli IRD families, to identify all disease-causing mutations, and to tailor therapeutic interventions to each IRD patient.

ISCEV extended protocol for the dark-adapted red flash ERG.

The International Society for Clinical Electrophysiology of Vision (ISCEV) standard for full-field electroretinography (ERG) describes a minimum procedure, but encourages more extensive testing. This ISCEV extended protocol describes an extension to the ERG standard, namely the dark-adapted (DA) red flash ERG. The DA red flash ERG can be incorporated conveniently within the ISCEV standard ERG protocol after a minimum of 20-min DA and recorded after the DA 0.01 ERG to a flash strength of 0.3 phot cd s m-2, eliciting a waveform with two positive peaks in healthy individuals. The first positive component is the cone-mediated x-wave with a peak at 30-50 ms; the second is a rod-mediated b-wave with a peak time of approximately 100 ms. Shorter DA times may be desirable to shorten the recording time or to alter the prominence of the early cone-mediated x-wave relative to the rod-mediated b-wave. The DA red flash ERG is used to aid the diagnosis of achromatopsia (rod monochromacy), cone dystrophy and other forms of cone system dysfunction, including "Bradyopsia" (RGS9/R9AP-retinopathy), when the DA red flash ERG x-wave is preserved in the absence of ISCEV standard LA ERGs. The DA red flash ERG can also help determine the origin of residual DA ERGs in cases of severe rod dysfunction, for example in disorders such as vitamin A deficiency, fundus albipunctatus (RDH5-retinopathy), Oguchi disease (SAG- or GRK1-retinopathy) and some rod-cone dystrophies. To shorter DA periods, the x-wave may be elicited without the following rod b-wave, shown to be helpful in abbreviated protocols for children.

Safety of intravitreal clindamycin in albino rabbit eyes.

PURPOSE: To study the potential toxic effects of intravitreal clindamycin on the retina of albino rabbits, by assessing functional and morphological retinal changes. METHODS: Eight albino rabbits were included in the study. In each rabbit, 1 mg/0.1 ml clindamycin was injected into the vitreous of the right (experimental) eye, and 0.1 ml saline was injected into the vitreous of the left (control) eye. The electroretinogram (ERG) was recorded before injection, 3 days, 1, 2, and 4 weeks post-injection. The visual evoked potential (VEP) was recorded 4 weeks post-injection. Clinical examination was conducted at all time points. The eyes were enucleated at the termination of the follow-up period in order to prepare the retinas for histology in order to assess retinal structure. RESULTS: ERG and VEP responses that were recorded from the experimental eye at different times following intravitreal clindamycin injection were very similar to the corresponding responses that were recorded from the control eyes. Clinical examination was normal in all eyes, and no histological damage was observed. CONCLUSIONS: Intravitreal injection of 1 mg clindamycin does not cause functional or morphological signs of retinal toxicity in albino rabbits, during a period of 4 weeks post-injection. These findings support the clinical use of 1 mg intravitreal clindamycin.

Infliximab exerts a dose-dependent effect on retinal safety in the albino rabbit.

PURPOSE: To assess the retinal toxicity of an intravitreal injection of infliximab, a monoclonal antibody to tumor necrosis factor α, in a rabbit model. MATERIALS AND METHODS: Two groups of adult albino rabbits (n = 5) received intravitreal injections of infliximab (0.1 ml) in the study eye and balanced salt solution (BSS, 0.1 ml) in the control eye at baseline. Group 1 was administered with 1.5 mg/0.1 ml, and group 2 was injected with 7.5 mg/0.1 ml of infliximab solution. Electroretinography (ERG) was performed at baseline and at 1, 7, 30, and 45 days after the injection. Visual evoked potentials (VEPs) were recorded at 7 and 45 days after the injection. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology and immunhistochemistry for glial fibrillary acidic protein (GFAP) were performed. RESULTS: ERG responses demonstrated no significant deficit in retinal function in eyes injected with infliximab. Mean dark-adapted a-wave and b-wave maximal amplitude and semi-saturation constant values at baseline and throughout the 45 days of follow-up after the injection indicated no remarkable difference in outer retinal function between the control and experimental eyes. VEP responses were similar at each time point (7 and 45 days). No difference was seen in retinal histopathology and immunocytochemistry sections in eyes receiving the 1.5 mg/0.1 ml dose compared to the control eyes. However, increased GFAP labeling in retinal Müller cells was detected in rabbit eyes treated with the 7.5 mg/0.1 ml dose. CONCLUSIONS: Intravitreal injection of 1.5 mg/0.1 ml infliximab dose has no toxic effect on the integrity (functional or structural) of the retina in rabbits. A higher dose of 7.5 mg/0.1 ml may be slightly toxic as suggested by positive Müller cell GFAP expression. Additional studies of retinal toxicity at higher doses and after multiple injections are needed to establish the retinal safety of intravitreal infliximab therapy in humans.

An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy.

PURPOSE: To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family. METHODS: Patients underwent a detailed ophthalmic evaluation, including eye examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potential (VEP). Genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing. RESULTS: The affected family members are three siblings who have various degrees of progressive visual deterioration, glare, color vision abnormalities, and night vision difficulties. Visual field tests revealed central scotomas of different extension. Cone and rod ERG responses were reduced, with cones more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice sites. However, in intron 21, proximate to the intron-exon junction, we observed a homozygous 10 bp deletion between positions -26 and -17 (c.2281-26_-17del). The deletion was linked to a known SNP, c.2281-6C>G. The deletion cosegregated with the disease in the family, and was not detected in public databases or in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a recognition site for the SRSF2 splicing factor is located within the deleted sequence. The in vitro splicing assay demonstrated that c.2281-26_-17del leads to complete exon 22 skipping. CONCLUSIONS: A novel and unique intronic mutation of PROM1, underlying autosomal recessive CRD in a consanguineous Israeli family, was found. This report expands the spectrum of pathogenic mutations of PROM1 and further demonstrates the importance of intronic mutations.

Best Disease: Global Mutations Review, Genotype-Phenotype Correlation, and Prevalence Analysis in the Israeli Population.

Purpose: To review all reported disease-causing mutations in BEST1, perform genotype-phenotype correlation, and estimate disease prevalence in the Israeli population. Methods: Medical records of patients diagnosed with Best disease and allied diseases from nine Israeli medical centers over the past 20 years were collected, as were clinical data including ocular findings, electrophysiology results, and retina imaging. Mutation detection involved mainly whole exome sequencing and candidate gene analysis. Demographic data were obtained from the Israeli Bureau of Statistics (January 2023). A bibliometric study was also conducted to gather mutation data from online sources. Results: A total of 134 patients were clinically diagnosed with Best disease and related conditions. The estimated prevalence of Best disease was calculated to be 1 in 127,000, with higher rates among Arab Muslims (1 in 76,000) than Jews (1 in 145,000). Genetic causes were identified in 76 individuals (57%), primarily showing autosomal-dominant inheritance due to BEST1 mutations (58 patients). Critical conserved domains were identified consisting of a high percentage of dominant missense mutations, primarily in transmembrane domains and the intracellular region (Ca2+ binding domain) of the BEST1 protein. Conclusions: This study represents the largest cohort of patients with Best disease reported in Israel and globally. The prevalence in Israel is akin to that in Denmark but is lower than that in the United States. Critical conserved domains within the BEST1 protein are pivotal for normal functioning, and even minor missense alterations in these areas lead to a dominant disease manifestation. Genetic testing is indispensable as the gold standard for Best disease diagnosis due to the variable clinical presentation of the disease.

Nationwide Prevalence of Inherited Retinal Diseases in the Israeli Population.

Importance: Data regarding the prevalence of various inherited retinal diseases (IRDs) are limited and vary across populations; moreover, nationwide prevalence studies may be limited to a specific IRD phenotype, potentially leading to inaccurate prevalence estimations. Therefore, nationwide prevalence data are needed. Objective: To determine the prevalence of 67 IRD phenotypes in the Israeli population. Design, Setting, and Participants: This cohort study collected nationwide data regarding the number of individuals affected with IRD phenotypes assessed in 10 clinical and academic centers in Israel as part of the research activity of the Israeli inherited retinal disease consortium. Data were collected in May 2023 on 9396 individuals residing in Israel who were diagnosed by an ophthalmologist with an IRD using either electroretinography or retinal imaging where included. Individuals with retinal diseases known to have a nonmendelian basis or without a clear genetic basis and those who were reported as deceased at the time of data collection were excluded from this study. Main Outcomes and Measures: Prevalence of 67 IRD phenotypes. Results: Among the 9396 participants in our cohort, the most common IRD in Israel was retinitis pigmentosa with a disease prevalence of approximately 1:2400 individuals, followed by cone-rod dystrophy (approximately 1:14 000), Stargardt disease (approximately 1:16 000), Usher syndrome (approximately 1:16,000), and congenital stationary night blindness (approximately 1:18 000). The prevalence of all IRDs combined was 1:1043 individuals. Conclusions and Relevance: The current study provides large prevalence dataset of 67 IRD phenotypes, some of which are extremely rare, with only a single identified case. This analysis highlights the potential importance of performing additional nationwide prevalence studies to potentially assist with determining the prevalence of IRDs worldwide.

Retinal toxicity of intravitreal rituximab in albino rabbits.

PURPOSE: To evaluate retinal toxicity of intravitreal rituximab. METHODS: Twelve albino rabbits were included in the study. 1 mg/0.1 mL of rituximab was injected to the right (experimental) eye of each rabbit, and 0.1 mL of saline was injected into the left (control) eye. Electroretinogram was recorded before injection and 3 hours, 4 days, 1 week, 2 weeks and 4 weeks after injection (12 rabbits), and visual evoked potential was recorded before injection and 4 weeks after injection (10 rabbits). Histology and glial fibrillary acidic protein immunocytochemistry (12 rabbits) were performed at 4 weeks after injection. Clinical examination was conducted at all time points in all rabbits. RESULTS: The average dark-adapted electroretinogram b-wave Vmax ratios, and the average light-adapted b-wave amplitude ratios were approximately 1, and the average log σ difference was around zero throughout the follow-up period. The average visual evoked potential amplitude ratio and the average visual evoked potential implicit time difference were approximately 1 and 0, respectively. No histologic damage was seen, but glial fibrillary acidic protein was mildly expressed in 6 of 12 experimental eyes. Results of clinical examination were normal in all the eyes. CONCLUSION: Intravitreal injection of 1 mg rituximab does not cause functional or histologic signs of retinal toxicity in albino rabbits. Mild glial fibrillary acidic protein expression in Müller cells probably indicates a mild degree of retinal stress.

Reduced Electroretinogram Responses in Morphologically Normal Retina in Patients with Primary Hyperoxaluria Type 1.

Purpose: To describe ocular findings in individuals with primary hyperoxaluria type 1 (PH1), focusing on the correlations between retinal anatomy and retinal function. To characterize the retinal alterations that occur at different disease stages by evaluating individuals with diverse degrees of renal impairment associated with PH1. Design: A cross-sectional study. Participants: Patients diagnosed with PH1 based on clinical criteria and genetic testing, treated in the Pediatric Nephrology Unit of the Ruth Children's Hospital, Rambam Health Care Campus, Haifa, Israel between 2013 and 2021. Methods: The ophthalmological assessment included a slit-lamp biomicroscopy of the anterior and posterior segment or indirect ophthalmoscopy. Electroretinography was employed for assessment of the retinal function, and retinal imaging included spectral-domain OCT and fundus autofluorescence. A systematic evaluation of the disease stage was based on clinical criteria including physical examination, purposeful imaging (X-ray, echocardiography, and US abdomen), and laboratory tests as needed. Main Outcome Measures: Anatomical and functional assessment of the retina in patients with PH1, and the relationship between retinal dysfunction and kidney impairment. Results: A total of 16 eyes were examined in the study of 8 children ranging in age from 4 to 19 years. Four eyes (25%) showed normal structural and functional retinal findings, 8 eyes (50%) presented functional impairment in the absence of pathological structural findings, and 4 eyes (25%) had advanced retinal damage that manifested as significant morphological and functional impairment. There was no direct relationship between the severity of the renal disease and the severity of the retinal phenotype. Conclusions: Subjects with PH1 present varying severity levels of the retinal phenotype, with possible discrepancy between the clinical retinal morphology and the retinal function noted on electroretinography. These findings raise questions about the molecular basis of the retinal manifestations in PH1. The presence of functional impairment in the absence of evident crystal deposition in the retina suggests that, in addition to oxalate crystal accumulation, other biomolecular processes may play a role in the development of retinopathy.

Safety of intravitreal bevacizumab in the developing rabbit retina.

PURPOSE: The purpose of this was to study the long-term effects of a single intravitreal dose of bevacizumab injected at different postnatal age on retinal function and development of retinal blood vessels in the adult albino rabbit. METHODS: Bevacizumab was injected into the right eye of newborn rabbits, aged 11 days to 25 days, whereas the left eye of each rabbit was injected with identical volume of saline and served as control. The electroretinogram was recorded 1 week and 10 weeks after injection. Visual evoked potentials were recorded 10 weeks after injection. At termination of the follow-up period, the rabbits were killed and the retinas were prepared for histopathologic studies at the light microscopic level, for glial fibrillary acidic protein immunoreactivity, and for reduced form of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry to assess the integrity of the retinal vascular system. RESULTS: The electroretinogram responses demonstrated normal retinal function in adult rabbits injected at postnatal age of 11 days to 25 days. Mean Vmax and σ values calculated for each group of rabbits, 10 weeks after bevacizumab injection, indicated similar retinal function of the experimental and control eyes. Visual evoked potentials recorded by stimulating each eye separately were also similar. The histopathologic studies yielded similar results; no structural retinal damage was observed in the experimental eyes compared with the control eyes of rabbits from all age groups, and no increased glial fibrillary acidic protein immunoreactivity in Müller cells was observed in the experimental eyes. Staining of blood vessels with reduced form of nicotinamide adenine dinucleotide phosphate diaphorase revealed decreased branching of the capillary network in the experimental eyes compared with the control eyes in all age groups. CONCLUSION: The electroretinographic and morphologic data showed no deleterious effects of a single intravitreal dose of bevacizumab, injected during the first 30 days postnatally, on the structural and functional integrity of the sensory retina in the adult rabbit. Even partial blockage of vascular endothelial growth factor with bevacizumab applied during retinal development seems to interfere with the development of the retinal vascular system in the albino rabbit. However, extrapolation from rabbits to humans should be made with caution.

Safety evaluation of repeated intravitreal injections of bevacizumab and ranibizumab in rabbit eyes.

PURPOSE: Repeated intravitreal injections of ranibizumab or bevacizumab are a common treatment for several retinal diseases. The aim of the present study was to evaluate the long-term retinal toxicity of repeated injections of ranibizumab and bevacizumab in rabbits. METHODS: Albino rabbits were injected intravitreally with ranibizumab (1 mg/0.1 mL) or bevacizumab (2.5 mg/0.1 mL) into the right eye, whereas the left eye of each rabbit was injected with saline. Nine consecutive injections were administered at 14-day intervals. Electroretinographic responses and flash visual-evoked potentials were recorded periodically. After 18 weeks of follow-up, the rabbits were killed, and the retinas were prepared for morphologic examination and for immunocytochemistry for glial fibrillary acidic protein. RESULTS: Electroretinographic and visual-evoked potential responses of the experimental and control eyes were similar in amplitude and pattern throughout the follow-up period. The histopathologic studies yielded similar results. No retinal damage was observed in the experimental and control eyes of all rabbits. Glial fibrillary acidic protein immunoreactivity showed staining only in retinal astrocytes but not in Müller cells in all rabbits. CONCLUSION: The electrophysiological tests and the morphologic data indicate that the repeated intravitreal injections of ranibizumab or bevacizumab have no cumulative long-term toxic effect on the retina in rabbits.

Induction of retinopathy by fibrillar oxalate assemblies.

The formation of metabolite fibrillar assemblies represents a paradigm shift in the study of human metabolic disorders. Yet, direct clinical relevance has been attributed only to metabolite crystals. A notable example for metabolite crystallization is calcium oxalate crystals observed in various diseases, including primary hyperoxaluria. We unexpectedly observed retinal damage among young hyperoxaluria patients in the absence of crystals. Exploring the possible formation of alternative supramolecular organizations and their biological role, here we show that oxalate can form ordered fibrils with no associated calcium. These fibrils inflict intense retinal cytotoxicity in cultured cells. A rat model injected with oxalate fibrils recaptures patterns of retinal dysfunction observed in patients. Antibodies purified from hyperoxaluria patient sera recognize oxalate fibrils regardless of the presence of calcium. These findings highlight a new molecular basis for oxalate-associated disease, and to our knowledge provide the first direct clinical indication for the pathogenic role of metabolite fibrillar assemblies.

The developing mammalian retina is partially protected from gentamicin toxicity.

Gentamicin retinal toxicity was tested in numerous studies on adult animal models, but not in the developing retina. Since differentiating cells and developing tissues may exhibit different degrees of sensitivity to toxic drugs, we aimed here to test the susceptibility of the developing mammalian retina to the toxic action of gentamicin. Gentamicin was injected in the right eye of newborn rabbits, aged 11-38 days. The left eye of each rabbit was injected with saline. Eight weeks after injection, electroretinographic (ERG) responses were recorded in the dark- and light-adapted states. The rabbits were then sacrificed, and the retinas were prepared for morphological examination and immunostaining for Glial Fibrillary Acidic Protein (GFAP) and Protein Kinase C (PKC). The ERG responses demonstrated practically no functional damage to the retina of rabbits injected at postnatal age of 11 and 13 days, and a gradually increasing severity of functional damage with increasing postnatal age of intravitreal gentamicin injection. The histopathological studies yielded similar results. GFAP immunoreactivity showed staining in Müller cells only in retinas that exhibited functional and structural damage. PKC immunoreactivity indicated lesser damage to the rod-bipolar cells compared to the photoreceptors. The ERG data and morphological observations suggest that processes involved in development of the rabbit retina, such as outer segment elongation may provide partial or even complete protection to the retina from toxic insults such as a single dose of gentamicin.

Testing retinal toxicity of drugs in animal models using electrophysiological and morphological techniques.

Drugs are frequently tested for retinal toxicity in animal models in order to address applied and basic research questions. When a retinal toxicity study is designed, the researcher needs to consider several factors depending on his/her research questions. Among the factors that need to be addressed before a toxicity study is conducted are: the animal species to be used, choice of experimental (functional and/or morphological) techniques, procedure of testing, period of follow-up, and modes of data analysis. This review is a summary of 20 years' experience of studying retinal toxicity of different drugs in rabbits and rats. The use of the electroretinogram and the visual evoked potential for assessment of outer and inner retinal function, respectively, is described as well as the use of morphological techniques (histology, histochemistry, and immunocytochemistry). The advantages and limitations of functional and morphological techniques are discussed with specific examples from my experience. Recommendations for future drug toxicity studies are summarized.