Assessment of Inner Blood-Retinal Barrier: Animal Models and Methods.

Proper functioning of the neural retina relies on the unique retinal environment regulated by the blood-retinal barrier (BRB), which restricts the passage of solutes, fluids, and toxic substances. BRB impairment occurs in many retinal vascular diseases and the breakdown of BRB significantly contributes to disease pathology. Understanding the different molecular constituents and signaling pathways involved in BRB development and maintenance is therefore crucial in developing treatment modalities. This review summarizes the major molecular signaling pathways involved in inner BRB (iBRB) formation and maintenance, and representative animal models of eye diseases with retinal vascular leakage. Studies on Wnt/ß-catenin signaling are highlighted, which is critical for retinal and brain vascular angiogenesis and barriergenesis. Moreover, multiple in vivo and in vitro methods for the detection and analysis of vascular leakage are described, along with their advantages and limitations. These pre-clinical animal models and methods for assessing iBRB provide valuable experimental tools in delineating the molecular mechanisms of retinal vascular diseases and evaluating therapeutic drugs.

Assessment of histological and immunohistochemical features of retinal tissues using a novel tissue submission procedure.

We introduce a novel tissue submission procedure without additional equipment or storage facilities for assessing the histological and immunohistochemical features of retinal tissues. In total, 150 specimens were collected from patients who underwent vitrectomy or macular surgery from January to December 2020. Ninety-eight specimens were submitted using the new procedure, and 58 specimens were submitted as flat-mount slides to compare specimen adequacy. The tissues submitted using the new procedure were subjected to paraffin-embedding and sectioning for hematoxylin & eosin staining. Additional immunohistochemical analysis was performed to assess the cellular composition in retinal tissues with diverse etiologies. The new submission procedure had an adequacy ratio of 75.51%, which was comparable to that of the flat-mount method (p = 0.1397). The new method could produce high-quality images of histological features of tissues and facilitated immunohistochemical analysis to demonstrate cell origins. More glial cells (p = 0.000) and myofibroblasts (p = 0.012) were detected in the epiretinal membranes (ERMs) than in the internal limiting membranes (ILMs). Subgroup analysis revealed that secondary ERMs contained more macrophage-like cells (p = 0.001) and retinal pigment epithelial cells (p = 0.000) than did idiopathic ERMs. Our novel tissue submission procedure can be applied to routine clinical practice. Our study provides additional histological and immunohistochemical evidence of cellular components in retinal tissues based on a large number of human tissue samples. Moreover, tissues submitted using the new method can be permanently preserved, enabling future investigation for potential prognostic or therapeutic targets.

Assessment of the cellular integrity and expression of melatonin receptor (MTNR1A) in the retina assaulted by ethanol and acetaminophen.

Ethanol exposures have been reported to disrupt the development of the retina and optic nerve which can be considered as part of underlying mechanisms of visual pathway impairments. This study aims to investigate the cellular integrity of the retina and the expression of melatonin receptor (MTNR1A) in the retina when assaulted chronically and simultaneously by ethanol and acetaminophen. Animals were randomly grouped into five groups. Control (normal saline), Alcohol group (25% alcohol in 2% sucrose solution), Acetaminophen group, (100 mg/kg BW for 14 days), Acetaminophen + Alcohol group (25% alcohol in 2% sucrose solution + 100 mg/kg BW of paracetamol). Withdrawal group (25% alcohol in 2% sucrose solution + 100 mg/kg BW of paracetamol). The body weight and rectal temperature of the animals were taking every 2 days and a post mortem study was conducted by quantitatively assessing the markers of oxidative stress. Melatonin level was quantified in the retina tissue and Immunohistochemistry was done via MTNR1A to study the expression of melatonin receptor type 1A in the retina. These results demonstrate that alcohol and acetaminophen significantly reduced the activity of retina rat melatonin (MTNR1A) levels, lowers the SOD and MDA activity. Expression of MTNR1A was reduced in the ganglionic cell layer of Alcohol and acetaminophen group as compared to the control and withdrawal group. It can be inferred that chronic simultaneous intake/consumption of alcohol and acetaminophen altered the melatonin level in the retina and this may implicate the circadian clock and melatonin in Wistar rat visual system.

Phenotype Characterization of a Mice Genetic Model of Absolute Blindness.

Recent technological development requires new approaches to address the problem of blindness. Such approaches need to be able to ensure that no cells with photosensitive capability remain in the retina. The presented model, Opn4-/- × Pde6brd10/rd10 (O×Rd) double mutant murine, is a combination of a mutation in the Pde6b gene (photoreceptor degeneration) together with a deletion of the Opn4 gene (responsible for the expression of melanopsin in the intrinsically photosensitive retinal ganglion cells). This model has been characterized and compared with those of WT mice and murine animal models displaying both mutations separately. A total loss of pupillary reflex was observed. Likewise, behavioral tests demonstrated loss of rejection to illuminated spaces and a complete decrease in visual acuity (optomotor test). Functional recordings showed an absolute disappearance of various wave components of the full-field and pattern electroretinogram (fERG, pERG). Likewise, visual evoked potential (VEP) could not be recorded. Immunohistochemical staining showed marked degeneration of the outer retinal layers and the absence of melanopsin staining. The combination of both mutations has generated an animal model that does not show any photosensitive element in its retina. This model is a potential tool for the study of new ophthalmological approaches such as optosensitive agents.

Quantitative Assessment of the Apical and Basolateral Membrane Expression of VEGFR2 and NRP2 in VEGF-A-stimulated Cultured Human Umbilical Vein Endothelial Cells.

Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood-retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.

A deep-learning system for the assessment of cardiovascular disease risk via the measurement of retinal-vessel calibre.

Retinal blood vessels provide information on the risk of cardiovascular disease (CVD). Here, we report the development and validation of deep-learning models for the automated measurement of retinal-vessel calibre in retinal photographs, using diverse multiethnic multicountry datasets that comprise more than 70,000 images. Retinal-vessel calibre measured by the models and by expert human graders showed high agreement, with overall intraclass correlation coefficients of between 0.82 and 0.95. The models performed comparably to or better than expert graders in associations between measurements of retinal-vessel calibre and CVD risk factors, including blood pressure, body-mass index, total cholesterol and glycated-haemoglobin levels. In retrospectively measured prospective datasets from a population-based study, baseline measurements performed by the deep-learning system were associated with incident CVD. Our findings motivate the development of clinically applicable explainable end-to-end deep-learning systems for the prediction of CVD on the basis of the features of retinal vessels in retinal photographs.

Assessment of necroptosis in the retina in a repeated primary ocular blast injury mouse model.

Primary blast injury (caused by the initial rapid increase in pressure following an explosive blast) to the retina and optic nerve (ON) causes progressive visual loss and neurodegeneration. Military personnel are exposed to multiple low-overpressure blast waves, which may be in quick succession, such as during breacher training or in combat. We investigated the necroptotic cell death pathway in the retina in a mouse repeated primary ocular blast injury (rPBI) model using immunohistochemistry. We further evaluated whether intravitreal injections of a potent necroptosis inhibitor, Necrostatin-1s (Nec-1s), protects the retina and ON axons by retinal ganglion cells (RGC) counts, ON axonal counting and optical coherence tomography (OCT) analysis of vitreous haze. Receptor interacting protein kinase (RIPK) 3, increased in the inner plexiform layer 2 days post injury (dpi) and persisted until 14 dpi, whilst RIPK1 protein expression did not change after injury. The number of degenerating ON axons was increased at 28 dpi but there was no evidence of a reduction in the number of intact ON axons or RNA-binding protein with multiple splicing (RBPMS)+ RGC in the retina by 28 dpi in animals not receiving any intravitreal injections. But, when intravitreal injections (vehicle or Nec-1s) were given there was a significant reduction in RBPMS+ RGC numbers, suggesting that rPBI with intraocular injections is damaging to RGC. There were fewer RGC lost after Nec-1s than vehicle injection, but there was no effect of Nec-1s or vehicle treatment on the number of degenerating axons. OCT analysis demonstrated no effect of rPBI on vitreous haze, but intravitreal injection combined with rPBI increased vitreous haze (P = 0.004). Whilst necroptosis may be an active cell death signalling pathway after rPBI, its inhibition did not prevent cell death, and intravitreal injections in combination with rPBI increased vitreous inflammation and reduced RBPMS+ RGC numbers, implying intravitreal injection is not an ideal method for drug delivery after rPBI.

Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases.

Purpose: Synucleinopathies such as multiple system atrophy (MSA) and Parkinson's disease are associated with a variety of visual symptoms. Functional and morphological retinal aberrations are therefore supposed to be valuable biomarkers for these neurodegenerative diseases. This study examined the retinal morphology and functionality resulting from human α-synuclein (α-Syn) overexpression in the transgenic Plp-α-Syn mouse model. Methods: Immunohistochemistry on retinal sections and whole-mounts was performed on 8- to 11-week-old and 12-month-old Plp-α-Syn mice and C57BL/6N controls. Quantitative RT-PCR experiments were performed to study the expression of endogenous and human α-Syn and tyrosine hydroxylase (TH). We confirmed the presence of human α-Syn in the retina in western blot analyses. Multi-electrode array (MEA) analyses from light-stimulated whole-mounted retinas were used to investigate their functionality. Results: Biochemical and immunohistochemical analyses showed human α-Syn in the retina of Plp-α-Syn mice. We found distinct staining in different retinal cell layers, most abundantly in rod bipolar cells of the peripheral retina. In the periphery, we also observed a trend toward a decline in the number of retinal ganglion cells. The number of TH+ neurons was unaffected in this human α-Syn overexpression model. MEA recordings showed that Plp-α-Syn retinas were functional but exhibited mild alterations in dim light conditions. Conclusions: Together, these findings implicate an impairment of retinal neurons in the Plp-α-Syn mouse. The phenotype partly relates to retinal deficits reported in MSA patients. We further propose the suitability of the Plp-α-Syn retina as a biological model to study synuclein-mediated mechanisms.

In Vivo Assessment of Retinal Biomarkers by Hyperspectral Imaging: Early Detection of Alzheimer's Disease.

A noninvasive and cost-effective means to detect preclinical Alzheimer's disease (AD) and monitor disease progression would be invaluable. The retina is a developmental extension of the brain and has been viewed as a window to evaluate AD-related pathology. Cross-sectional studies have shown structural changes in the retina of AD patients that include thinning of the retinal nerve-fiber layer and changes in retinal vasculature. However, such changes do not manifest in early stages of the disease nor are they specific biomarkers for AD. Described herein is the utilization of our retinal hyperspectral imaging (rHSI) technique as a biomarker for identification of AD-related early pathological changes in the retina. Specifically, this account concerns the translation of our rHSI technique from animal models to human AD subjects. The underlying principle is Rayleigh light scattering, which is expected from low-order Aß aggregates present in early pathology. Recruitment was restricted to AD subjects (N = 19) and age-matched controls, with no family history of AD (N = 16). To limit the influence of skin pigmentation, subjects were restricted to those with skin pigmentation values of 2-3 on the Fitzpatrick scale. The largest spectral deviation from control subjects, rHSI signature, was obtained at the MCI stage with MMSE scores ⩾22, suggesting higher sensitivity of this technique in early disease stages. The rHSI signature observed is unaffected by eye pathologies such as glaucoma and cataract. Age of the subjects minimally influenced the spectral signatures. The rHSI technique shows promise for detection of preclinical AD; it is conducted in a truly noninvasive manner, without application of an exogenous label, and is thus potentially suitable for population screening.

Targeted next generation sequencing reveals genetic defects underlying inherited retinal disease in Iranian families.

Purpose: Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous showing progressive retinal cell death which results in vision loss. IRDs include a wide spectrum of disorders, such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and Stargardt disease (STGD1). Methods: In this study, we performed targeted next-generation sequencing based on molecular inversion probes (MIPs) that allowed the sequence analysis of 108 IRD-associated genes in 50 Iranian IRD probands. Results: The sequencing and variant filtering led to the identification of putative pathogenic variants in 36 out of 50 (72%) probands. Among 36 unique variants, we identified 20 novel variants in 15 genes. Four out of 36 probands carry compound heterozygous variants, and 32 probands carry homozygous variants. Conclusions: Employing a cost-effective targeted next-generation sequencing procedure, we identified the genetic causes of different retinal disorders in the majority of Iranian families in this study.

In vivo MRI assessment of bioactive magnetic iron oxide/human serum albumin nanoparticle delivery into the posterior segment of the eye in a rat model of retinal degeneration.

BACKGROUND: Retinal degeneration diseases affect millions of patients worldwide and lead to incurable vision loss. These diseases are caused by pathologies in the retina and underlying choroid, located in the back of the eye. One of the major challenges in the development of treatments for these blinding diseases is the safe and efficient delivery of therapeutics into the back of the eye. Previous studies demonstrated that narrow size distribution core-shell near infra-red fluorescent iron oxide (IO) nanoparticles (NPs) coated with human serum albumin (HSA, IO/HSA NPs) increase the half-life of conjugated therapeutic factors, suggesting they may be used for sustained release of therapeutics. In the present study, the in vivo tracking by MRI and the long term safety of IO/HSA NPs delivery into the suprachoroid of a rat model of retinal degeneration were assessed. RESULTS: Twenty-five Royal College of Surgeons (RCS) pigmented rats received suprachoroidal injection of 20-nm IO/HSA NPs into the right eye. The left eye was not injected and used as control. Animals were examined by magnetic resonance imaging (MRI), electroretinogram (ERG) and histology up to 30 weeks following injection. IO/HSA NPs were detected in the back part of the rats' eyes up to 30 weeks following injection by MRI, and up to 6 weeks by histology. No significant differences in retinal structure and function were observed between injected and non-injected eyes. There was no significant difference in the weight of IO/HSA NP-injected animals compared to non-injected rats. CONCLUSIONS: MRI could track the nanoparticles in the posterior segment of the injected eyes demonstrating their long-term persistence, and highlighting the possible use of MRI for translational studies in animals and in future clinical studies. Suprachoroidal injection of IO/HSA NPs showed no sign of adverse effects on retinal structure and function in a rat model of retinal degeneration, suggesting that suprachoroidal delivery of IO/HSA NPs is safe and that these NPs may be used in future translational and clinical studies for extended release drug delivery at the back of the eye.

Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model.

PURPOSE: Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time. METHODS: Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point. RESULTS: The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants. CONCLUSIONS: This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease.

A machine learning approach for automated assessment of retinal vasculature in the oxygen induced retinopathy model.

Preclinical studies of vascular retinal diseases rely on the assessment of developmental dystrophies in the oxygen induced retinopathy rodent model. The quantification of vessel tufts and avascular regions is typically computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network. Such manual measurements are time-consuming and hampered by user variability and bias, thus a rapid and objective method is needed. Here, we introduce a machine learning approach to segment and characterize vascular tufts, delineate the whole vasculature network, and identify and analyze avascular regions. Our quantitative retinal vascular assessment (QuRVA) technique uses a simple machine learning method and morphological analysis to provide reliable computations of vascular density and pathological vascular tuft regions, devoid of user intervention within seconds. We demonstrate the high degree of error and variability of manual segmentations, and designed, coded, and implemented a set of algorithms to perform this task in a fully automated manner. We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools. The source code of our implementation is released under version 3 of the GNU General Public License ( https://www.mathworks.com/matlabcentral/fileexchange/65699-javimazzaf-qurva ).

Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2-photon imaging.

Two-photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2-photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom-built 2-photon microscope for 5-dimensional microscopy with a near infrared broadband sub-15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.

Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants.

Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

Assessment of trans-scleral iontophoresis delivery of lutein to the human retina.

The efficacy of novel scleral iontophoresis device for in situ delivery of lutein to the human retina was assessed by Resonance Raman spectroscopy (RRS) technique. Eight human donor eye globes were used for experiments, 6 of which underwent trans-scleral iontophoresis delivery of lutein and the other 2 were used as controls. The scleral iontophoresis applicator was filled with liposome-enriched 0.1% lutein solution and the generator's current was set at 2.5 mA and delivered for 4 min. A custom RRS setup was used for detecting lutein in the inner sclera, choroid, retinal periphery and macula of treated samples and controls. Forty minutes after iontophoresis, the inner sclera, choroid and retinal periphery were greatly enriched with lutein (P < .05); no lutein was found in the same ocular regions of non-treated samples. In the same period, the average concentration of lutein in the macula (4.8 ± 1.7 ng/mm2 ) of treated samples was 1.3 times greater than controls (3.7 ± 1.0 ng/mm2 ; P = .4). Scleral iontophoresis was shown to be effective in delivering lutein to the human retina. Future studies will aim at assessing if this therapeutic strategy is valuable to enrich the macular pigment in human subjects.

Assessment of PAXgene Fixation on Preservation of Morphology and Nucleic Acids in Microdissected Retina Tissue.

AIM: To compare the effect of the two fixatives on tissue morphology and utility to obtain good quality nucleic acids for molecular analysis from micro-dissected retinal samples. METHODS: Enucleated specimens from New Zealand white rabbits were fixed in formalin or PAXgene fixative according to standard protocols, and then processed and embedded in paraffin for sectioning. Sections were stained with hematoxylin and eosin to assess the structural integrity of retina. Retinal tissue on slides was micro-dissected. DNA/RNA were extracted and assessed for preservation of the quality and quantity of the retinal tissue. RESULTS: The retinal morphology was well preserved with both PAXgene and formalin fixation. The RNA yield obtained using both fixation methods was similar, but RNA from PAXgene fixed paraffin embedded (PFPE) samples had better purity than that from formalin fixed paraffin embedded (FFPE) samples. There was a twofold greater yield of DNA in PFPE compared to FFPE samples but with similar purity. Quantitative reverse transcription PCR analyses showed that the mean cycle threshold values for beta-actin, beta-microglobin, Opsin 1-sw, Rhodopsin, and 18S RNA of the PFPE group were significantly lower than those of the FFPE group (p < 0.01). Greater than 10-fold greater levels of gene expression were detected in PFPE relative to FFPE for the above genes. CONCLUSION: PAXgene fixed tissue retinal morphology is comparable to FFPE tissue. PAXgene may be a good alternative to formalin, providing good tissue morphology and ability to isolate high quality nucleic acids from micro-dissected paraffin embedded retinal samples.

Assessment of P-Glycoprotein Transport Activity at the Human Blood-Retina Barrier with (R)-11C-Verapamil PET.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity. Methods: We performed PET scans with the ABCB1 substrate (R)-11C-verapamil on 5 healthy male volunteers without and with concurrent infusion of the ABCB1 inhibitor tariquidar. We estimated the rate constants for radiotracer transfer across the BRB (K1, k2) and total retinal distribution volume VTResults: During ABCB1 inhibition, retinal VT and influx rate constant K1 were significantly, by 1.4 ± 0.5-fold and 1.5 ± 0.3-fold, increased compared with baseline. Retinal efflux rate constant k2 was significantly decreased by 2.8 ± 1.0-fold. Conclusion: We found a significant increase in (R)-11C-verapamil distribution to the retina during ABCB1 inhibition, which provides first in vivo evidence for ABCB1 transport activity at the human BRB. The increase in retinal distribution was approximately 2.5-fold less pronounced than previously reported for the blood-brain barrier.

Noninvasive Electroretinography Assessment of Intravitreal Sustained-Release Methotrexate Microimplants in Rabbit Eyes.

PURPOSE: The purpose of this study is to noninvasively evaluate the safety and toxicity of a chitosan (CS) and polylactic acid (PLA)-based sustained-release methotrexate (MTX) intravitreal microimplant in normal rabbit eyes using electroretinography (ERG). METHODS: PLA-coated CS-based microimplants containing 400 µg of MTX and placebo microimplants (without drug) were surgically implanted in the vitreous of the right and the left eyes, respectively, in each of the 8 New Zealand rabbits using minimally invasive technique. At each predetermined time points (days 5, 12, 19, and 33), ERG was conducted on 2 rabbits to evaluate the safety of the microimplants administered in each eye. ERG was carried out using 2 protocols, scotopic and photopic, on each eye prior to surgery (PS) and prior to euthanasia (PE) conditions. The safety of the microimplants was assessed using statistical analysis of the ERG data (B/A ratio analysis, oscillatory potential analysis, and Naka-Rushton analysis) and subsequently quantifying and comparing functional integrity of the retina between the PS and PE conditions of each eye. RESULTS: Statistical analysis of the ERG data showed no change in retinal functional integrity because of the PLA-coated CS-based MTX microimplant and the placebo microimplant. ERG analysis also revealed absence of any evident bioelectrical dysfunction caused by the microimplants. CONCLUSION: ERGs were performed to determine whether the microimplants containing MTX and the placebo microimplants were associated with any profound retinal bioelectrical dysfunction that might be attributable to toxicity not apparent on histological studies of such eyes. The results shown in this report indicate that there were no such evident adverse effects of the microimplants or contained drug.

Assessment of visual function and retinal structure following acute light exposure in the light sensitive T4R rhodopsin mutant dog.

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.