Retinal Distribution Research Articles

Establishment and comprehensive characterization of a novel dark-reared zebrafish model for myopia studies

Myopia is predicted to impact approximately 5 billion people by 2050, necessitating mechanistic understanding of its development. Myopia results from dysregulated genetic mechanisms of emmetropization, caused by over-exposure to aberrant visual environments; however, these genetic mechanisms remain unclear. Recent human genome-wide association studies have identified a range of novel myopia-risk genes. To facilitate large-scale in vivo mechanistic examination of gene-environment interactions, this study aims to establish a myopia model platform that allows efficient environmental and genetic manipulations. We established an environmental zebrafish myopia model by dark-rearing. Ocular biometrics including relative ocular refraction were quantified using optical coherence tomography images. Spatial vision was assessed using optomotor response (OMR). Retinal function was analyzed via electroretinography (ERG). Myopia-associated molecular contents or distributions were examined using RT-qPCR or immunohistochemistry. Our model produces robust phenotypic changes, showing myopia after 2 weeks of dark-rearing, which were recoverable within 2 weeks after returning animals to normal lighting. 2-week dark-reared zebrafish have reduced spatial-frequency tuning function. ERG showed reduced photoreceptor and bipolar cell function (a- and b-waves) after only 2 days of dark-rearing, which worsened after 2 weeks of dark-rearing. We also found dark-rearing-induced changes to expression of myopia-risk genes, including egr1, vegfaa, vegfab, rbp3, gjd2a and gjd2b, inner retinal distribution of EFEMP1, TIMP2 and MMP2, as well as transiently reduced PSD95 density in the inner plexiform layer. Coupled with the gene editing tools available for zebrafish, our environmental myopia model provides an excellent platform for large-scale investigation of gene-environment interactions in myopia development.

Barcode lipids for absolute quantitation of liposomes in ocular tissues

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

PEDF-derived peptide protects against Amyloid-β toxicity in vitro and prevents retinal dysfunction in rats

Amyloid-beta (Aβ), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aβ42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aβ42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aβ-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aβ42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aβ, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aβ42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aβ42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aβ42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aβ42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aβ42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aβ42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aβ42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aβ42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aβ42 in the retina and suggest concepts on the molecular mechanism of Aβ retinal pathogenicity.

Shotgun lipidomics of human subretinal fluids under rod-dominant retina reveals cone-dominated lipids

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases’ mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina.

Characterization of the whole Advillin expressing retinal ganglion cell population in the rodent retina

Aims/Purpose: We have quantified and examined the topography of the total population of retinal ganglion cells (RGC) expressing Advillin (Advillin+RGCs), an actin‐binding protein expressed in somatic sensory neurons, in the retinas, as well as their projections to the brain, in control adult Advillin‐Cretg/+ mice (Zurborg et al. Mol Pain 2011, 7:66).Methods: Retinas from adult Advillin‐Cretg/+ Ai14fl/fl C57BL/6J mice (Frutos‐Rincón et al., Int J Mol Sci 2022, 23:2997) expressing tdTomato were prepared as whole‐mounts, labelled with antibodies recognizing Brn3a and melanopsin. The entire Advillin+RGC population was quantified and its retinal distribution analysed by nearest neighbour maps. The brains were serial coronal crysectioned and the axonal Advillin+RGC projections in the brain were anatomically analysed.Results: Advillin protein was mainly located in the somas and axons of a subset of RGCs. Total number of Advillin+RGCs was 1947 ± 157 (mean ± SD; n = 7), which constitutes 5% of the total RGC population. Approximately 17.5% (340 ± 76) and 0% (3 ± 1) of the Advillin+RGC population co‐expressed Brn3a or melanopsin proteins, respectively. The Advillin+RGC population was distributed across the entire retina, with a higher concentration in the temporal region. The axons of this RGC subpopulation project to the main retinal recipient regions in the brain, such as the lateral geniculate nucleus (LGN) and superior colliculus (SC). However, none of these axons project to the suprachiasmatic nucleus. These data indicates that Advillin+RGCs is not a subpopulation of ipRGCs.Conclusions: The Advillin protein, which is characteristic of sensory neurons, is also expressed in a specific RGC subpopulation of the adult mouse retina. A small percentage of Advillin+RGCs expressed the Brn3a transcription factor, while none expressed melanopsin. The axons of Advillin+RGCs project to the typical retino‐recipient areas in the brain, excluding the suprachiasmatic nucleus.

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in mouse and pig photoreceptors

Inherited retinal dystrophies caused by dominant mutations in photoreceptor cell expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor cell expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor cell expressed genes with 1-2 intravitreal injections per year.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

The Degree of Adeno-Associated Virus-Induced Retinal Inflammation Varies Based on Serotype and Route of Delivery: Intravitreal, Subretinal, or Suprachoroidal.

Adeno-associated virus (AAV)-mediated gene therapy has great potential for treating a wide range of retinal degenerative diseases. However, some initial enthusiasm for gene therapy has been tempered by emerging evidence of AAV-associated inflammation, which in several instances has contributed to clinical trial discontinuation. Currently, there is a paucity of data describing the variable immune responses to different AAV serotypes, and similarly, little is known regarding how these responses differ depending on route of ocular delivery, including in animal models of disease. In this study, we characterize the severity and retinal distribution of AAV-associated inflammation in rats triggered by delivery of five different AAV vectors (AAV1, AAV2, AAV6, AAV8, and AAV9), each of which contained enhanced green fluorescent protein (eGFP) driven under control of the constitutively active cytomegalovirus promoter. We further compare the inflammation across three different potential routes (intravitreal, subretinal, and suprachoroidal) of ocular delivery. Compared to buffer-injected controls for each route of delivery, AAV2 and AAV6 induced the most inflammation across all routes of delivery of vectors tested, with AAV6 inducing the highest levels of inflammation when delivered suprachoroidally. AAV1-induced inflammation was highest when delivered suprachoroidally, whereas minimal inflammation was seen with intravitreal delivery. In addition, AAV1, AAV2, and AAV6 each induce infiltration of adaptive immune cells like T cells and B cells into the neural retina, suggesting an innate adaptive response to a single dose of virus. AAV8 and AAV9 induced minimal inflammation across all routes of delivery. Importantly, the degree of inflammation was not correlated with vector-mediated transduction and expression of eGFP. These data emphasize the importance of considering ocular inflammation when selecting AAV serotypes and ocular delivery routes for the development of gene therapy strategies.

Newly-established in vitro inner BRB spheroids to elucidate retinal Ang2-linked substance transfer.

Conjugation of angiopep-2 (Ang2) with drugs/compounds is known to increase plasma membrane permeability across endothelial barriers. The inner blood-retinal barrier (BRB) regulates retinal drug distribution and is formed by retinal capillary endothelial cells, supported by Müller cells and retinal pericytes. To elucidate the potential of Ang2 conjugation in promoting retinal drug distribution after peripheral administration across the inner BRB, an in vivo administration study and in vitro transport experiments using newly developed multicellular inner BRB spheroids were performed. After intravenous administration of Ang2-linked green fluorescence protein (GFP-Ang2) in mice, GFP-derived signals were observed in the neural retina. In contrast, GFP-derived signals were not observed after intravenous GFP administration, suggesting the promotion of the retinal distribution of substances by Ang2 conjugation. To overcome the limitations of in vitro studies using cells cultured on dishes, inner BRB spheroids were established using conditionally immortalized rat retinal capillary endothelial cells, Müller cells, and retinal pericytes. Immunocytochemistry of marker molecules suggests that the central part of the spheroids is occupied by Müller cells, and encapsulated by retinal pericytes and capillary endothelial cells. Studies on the expression and functions of tight junctions suggest that tight junctions are formed on the surface of the inner BRB spheroids by retinal capillary endothelial cells. The functional expression of drug transporters, such as P-glycoprotein, was observed in the spheroids, implying that the inner side of the spheroids reflects the retinal side of the inner BRB. In the inner BRB spheroids, energy-dependent accumulation of GFP-Ang2 and Ang2-linked 5(6)-carboxyfluorescein (FAM-Ang2) was observed. Moreover, an endocytic inhibition study revealed that clathrin-dependent endocytosis/transcytosis was involved in the transcellular transport of Ang2-conjugated drugs/compounds across the inner BRB. Consequently, it is suggested that the Ang2 linkage is useful for promoting retinal drug distribution via clathrin-dependent transcytosis at the inner BRB.

Magnetoreceptory Function of European Robin Retina: Electrophysiological and Morphological Non-Homogeneity.

The avian magnetic compass allows orientation during migration and is shown to function properly under short-wavelength but not long-wavelength visible light. Therefore, the magnetoreceptive system is assumed to be light- and wavelength-dependent and localized in the retina of the eye. Putative candidates for the role of primary magnetosensory molecules are the cryptochromes that are known to be expressed in the avian retina and must be able to interact with phototransduction proteins. Previously, we reported that in migratory birds change in magnetic field direction induces significant effects on electroretinogram amplitude in response to blue flashes, and such an effect was observed only in the nasal quadrant of the retina. Here, we report new electroretinographic, microscopic and microspectrophotometric data on European robins, confirming the magnetosensitivity of the retinal nasal quadrant after applying the background illumination. We hypothesized that magnetoreceptive distinction of this region may be related to its morphology and analyzed the retinal distribution and optical properties of oil droplets, the filtering structures within cones. We found that the nasal quadrant contains double cones with the most intensely colorized oil droplets compared to the rest of the retina, which may be related to its magnetosensory function.

Bile Duct Ligation Impairs Function and Expression of Mrp1 at Rat Blood-Retinal Barrier via Bilirubin-Induced P38 MAPK Pathway Activations.

Liver injury is often associated with hepatic retinopathy, resulting from accumulation of retinal toxins due to blood–retinal barrier (BRB) dysfunction. Retinal pigment epithelium highly expresses MRP1/Mrp1. We aimed to investigate whether liver injury affects the function and expression of retinal Mrp1 using bile duct ligation (BDL) rats. Retinal distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione were used for assessing Mrp1 function. BDL significantly increased distributions of the two substrates and bilirubin, downregulated Mrp1 protein, and upregulated phosphorylation of p38 and MK2 in the retina. BDL neither affected the retinal distribution of FITC-dextran nor expressions of ZO-1 and claudin-5, demonstrating intact BRB integrity. In ARPE-19 cells, BDL rat serum or bilirubin decreased MRP1 expression and enhanced p38 and MK2 phosphorylation. Both inhibiting and silencing p38 significantly reversed the bilirubin- and anisomycin-induced decreases in MRP1 protein. Apparent permeability coefficients of fluorescein in the A-to-B direction (Papp, A-to-B) across the ARPE-19 monolayer were greater than Papp, B-to-A. MK571 or bilirubin significantly decreased Papp, A-to-B of fluorescein. Bilirubin treatment significantly downregulated Mrp1 function and expression without affecting integrity of BRB and increased bilirubin levels and phosphorylation of p38 and MK2 in rat retina. In conclusion, BDL downregulates the expression and function of retina Mrp1 by activating the p38 MAPK pathway due to increased bilirubin levels.

Characterization of West African Crystalline Macular Dystrophy in the Ghanaian Population

West African crystalline maculopathy (WACM) is characterized by the presence of macular hyperrefractile crystal-like deposits. Although the underlying pathophysiology has not been elucidated, a few biologic drivers have been proposed. We analyzed a large WACM case series to gain a more robust understanding of its features and etiology. Prospective, cross-sectional cohort study. Participants with WACM were selected from the large cohort recruited in the Ghana Age-Related Macular Degeneration Study. Demographic and detailed medical histories, full ophthalmic examinations, digital color fundus photographs, and OCT images were obtained. All cases with WACM were evaluated by 3 retina experts. Crystal numbers, location, and distribution were determined. Associations between WACM and White age-related macular degeneration (AMD) risk variants were assessed using Firth's bias-reduced logistic regression, including age and sex as covariates. Phenotypic features of, and genetic associations with, WACM. West African crystalline maculopathy was identified in 106 eyes of 53 participants: 22 were bilateral and 24 were unilateral. Grading for AMD was not possible in 1 eye in 7 participants with WACM; therefore, laterality was not assessed in these subjects. Thirty-eight participants were women and were 14 men; sex was unrecorded for 1 participant. The mean age was 68.4 years (range, 45-101 years). Typical WACM crystals were demonstrated on OCT, which were more easily identified at high contrast and predominantly located at the inner limiting membrane. In eyes with copathology, crystals localized deeper in the inner retina, with wider retinal distribution over copathology lesions. There was no association with age or sex. A significant association was observed between the complement factor H (CFH) 402H risk variant and WACM. This study confirms the localization of crystals adjacent to the inner limiting membrane and distribution over lesions in eyes with copathology. The evaluation of OCT images under high contrast allows improved identification. West African crystalline maculopathy may be associated with the CFH-CFHR5 AMD risk locus identified among Whites; however, it is also possible that the combination of crystals and the CFH 402H allele increases the risk for developing late AMD. Further analyses using larger sample sizes are warranted to identify causalities between genotype and WACM phenotype.

Role of the Blood-Retinal Barrier Transporters: Antiaging in Retina

Since the retina continuously receives light to enable vision, reactive oxygen species (ROS) are easily generated in neural retina. The oxidative stress induced by ROS may be involved in the onset and progression of blinding aging diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Although supply of antioxidants to the retina is important to maintain the redox homeostasis in neural retina, the blood-retinal barrier (BRB) is created by complex tight-junctions of retinal capillary endothelial cells and retinal pigment epithelial cells to prevent the free diffusion of substances. The BRB is equipped with several membrane transporters to supply nutrients and essential molecules including antioxidants and drugs which exhibit antiaging effect to the retina from the circulating blood. In this review, the transporter-mediated retinal distribution of key endogenous compounds and drugs, such as vitamin C, l-cystine and gabapentin, is introduced for antiaging of the retina.

Quantitative modeling of regular retinal microglia distribution

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia’s morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell–cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

Resveratrol inhibits neural apoptosis and regulates RAX/P-PKR expression in retina of diabetic rats

ABSTRACT Purpose This study was to investigate the effect of resveratrol (RSV) administration on diabetes-induced neural apoptosis and on RNA-dependent-protein-kinase (PKR)-associated protein X (RAX), PKR and phosphorylated PKR (P-PKR) expression and distribution in retina of diabetic rats. Methods Retina was obtained from normal and diabetic Sprague–Dawley rats with or without RSV (5 and 10 mg/kg/d) treatment at 30-, 32-, 34- and 36-weeks. Apoptosis of retinal neural cells and distribution of RAX/P-PKR was assessed by TUNEL and immunofluorescence methods. Expression of RAX, PKR and P-PKR was evaluated by qRT-PCR and western-blotting methods. Results Our study showed that the TUNEL-positive cells were mainly localized in ganglion cells layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) of the diabetic rat's retina at 30-, 32-, 34- and 36-weeks. RSV administration effectively suppressed the neural apoptosis in GCL, INL and ONL. Almost no TUNEL-positive cells were observed in retina of normal control and RSV-treated normal control rats. Our study also showed that the expression level of RAX, P-PKR in diabetic rats retina at 30-, 32-, 34-, and 36-weeks was elevated. With supplementation of 5 and 10 mg/kg/d RSV, the expression level of RAX and P-PKR was decreased (P < 0.05). The expression level of RAX and P-PKR in the retina of normal control rats was not altered by RSV. The expression level of PKR was not altered by streptozotocin injection and RSV treatment. Conclusions Our results suggested that RSV attenuates retinal neural apoptosis in diabetic rats retina may be via regulation RAX/P-PKR expression.

Comprehensive Evidence of Carrier-Mediated Distribution of Amantadine to the Retina across the Blood-Retinal Barrier in Rats.

Amantadine, a drug used for the blockage of NMDA receptors, is well-known to exhibit neuroprotective effects. Accordingly, assessment of amantadine transport at retinal barriers could result in the application of amantadine for retinal diseases such as glaucoma. The objective of this study was to elucidate the retinal distribution of amantadine across the inner and outer blood–retinal barrier (BRB). In vivo blood-to-retina [3H]amantadine transport was investigated by using the rat retinal uptake index method, which was significantly reduced by unlabeled amantadine. This result indicated the involvement of carrier-mediated processes in the retinal distribution of amantadine. In addition, in vitro model cells of the inner and outer BRB (TR-iBRB2 and RPE-J cells) exhibited saturable kinetics (Km in TR-iBRB2 cells, 79.4 µM; Km in RPE-J cells, 90.5 and 9830 µM). The inhibition of [3H]amantadine uptake by cationic drugs/compounds indicated a minor contribution of transport systems that accept cationic drugs (e.g., verapamil), as well as solute carrier (SLC) organic cation transporters. Collectively, these outcomes suggest that carrier-mediated transport systems, which differ from reported transporters and mechanisms, play a crucial role in the retinal distribution of amantadine across the inner/outer BRB.

Effect of early exogenous supplementation of rhIGF-1 on oxygen-induced retinopathy in a mice model of prematurity, and on expressions of IGF-1 and VEGF

Purpose: To investigate the effect of early exogenous supplementation of recombinant human insulinlike growth factor (rhIGF-1) on oxygen-induced mouse model of retinopathy of prematurity (ROP).&#x0D; Methods: Three groups of healthy SPF grade C57BL/6 mice were used in this study, with 20 mice in each group. Hyperoxia saline (HS) and hyperoxia rhIGF-1 (HrGF) groups were placed in a closed oxygen chamber for one week and returned to the normal environment on the 15th day. The hyperoxia rhIGF-1 (HrGF) group was intraperitoneally injected with rhIGF-1 (1.5 mg/kg), while mice in high-oxygen saline (HS) group received normal saline. The air group (AG) was untreated. Changes in retinal blood vessel distributions, expression levels of serum IGF-1 and VEGF, and retinal IGF-1 and VEGF were determined.&#x0D; Results: On day 20, pronounced neo-vascularization was observed, but the distribution was disordered. Serum IGF-1 levels in AG and HrGF were significantly higher than that in HS group, but VEGF level was lower in HS mice (p &lt; 0.05). VEGF level in hyperoxia rhIGF-1 group on days 11 and 15 decreased, relative to control value, while retinal IGF-1 and VEGF in AG and hyperoxia rhIGF-1 mice were elevated, relative to corresponding values in HS mice (p &lt; 0.05).&#x0D; Conclusion: Early exogenous supplementation of rhIGF-1 exerts a therapeutic effect on ROP. Thus, rhIGF-1 may be a potential drug regimen for ROP in clinics.

ABCB1 and ABCG2 Together Limit the Distribution of ABCB1/ABCG2 Substrates to the Human Retina and the ABCG2 Single Nucleotide Polymorphism Q141K (c.421C> A) May Lead to Increased Drug Exposure.

The widely expressed and poly-specific ABC transporters breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1) are co-localized at the blood-brain barrier (BBB) and have shown to limit the brain distribution of several clinically used ABCB1/ABCG2 substrate drugs. It is currently not known to which extent these transporters, which are also expressed at the blood-retinal barrier (BRB), may limit drug distribution to the human eye and whether the ABCG2 reduced-function single-nucleotide polymorphism (SNP) Q141K (c.421C > A) has an impact on retinal drug distribution. Ten healthy male volunteers (five subjects with the c.421CC and c.421CA genotype, respectively) underwent two consecutive positron emission tomography (PET) scans after intravenous injection of the model ABCB1/ABCG2 substrate [11C]tariquidar. The second PET scan was performed with concurrent intravenous infusion of unlabelled tariquidar to inhibit ABCB1 in order to specifically reveal ABCG2 function.In response to ABCB1 inhibition with unlabelled tariquidar, ABCG2 c.421C > A genotype carriers showed significant increases (as compared to the baseline scan) in retinal radiotracer influx K 1 (+62 ± 57%, p = 0.043) and volume of distribution V T (+86 ± 131%, p = 0.043), but no significant changes were observed in subjects with the c.421C > C genotype. Our results provide the first evidence that ABCB1 and ABCG2 may together limit the distribution of systemically administered ABCB1/ABCG2 substrate drugs to the human retina. Functional redundancy between ABCB1 and ABCG2 appears to be compromised in carriers of the c.421C > A SNP who may therefore be more susceptible to transporter-mediated drug-drug interactions at the BRB than non-carriers.

Glutathione Metabolism and the Novel Role of Mitochondrial GSH in Retinal Degeneration.

Glutathione (GSH) is present ubiquitously, and its role as a crucial cellular antioxidant in tissues, including the retina, is well established. GSH’s antioxidant function arises from its ability to scavenge reactive oxygen species or to serve as an essential cofactor for GSH S-transferases and peroxidases. This review summarizes the general functions, retinal distribution, disorders linked to GSH deficiency, and the emerging role for mitochondrial GSH (mGSH) in retinal function. Though synthesized only in the cytosol, the presence of GSH in multiple cell organelles suggests the requirement for its active transport across organellar membranes. The localization and distribution of 2-oxoglutarate carrier (OGC) and dicarboxylate carrier (DIC), two recently characterized mitochondrial carrier proteins in RPE and retina, show that these transporters are highly expressed in human retinal pigment epithelium (RPE) cells and retinal layers, and their expression increases with RPE polarity in cultured cells. Depletion of mGSH levels via inhibition of the two transporters resulted in reduced mitochondrial bioenergetic parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) and increased RPE cell death. These results begin to reveal a critical role for mGSH in maintaining RPE bioenergetics and cell health. Thus, augmentation of mGSH pool under GSH-deficient conditions may be a valuable tool in treating retinal disorders, such as age-related macular degeneration and optic neuropathies, whose pathologies have been associated with mitochondrial dysfunction.

Visual function in guinea pigs: behavior and electrophysiology

ABSTRACT Clinical relevance Guinea pig visual function is characterised based on behavioural and electrophysiological measures and retinal ganglion cell density is examined to further develop the guinea pig as a model of human ocular conditions. Background Guinea pigs are an important model of human ocular conditions. Here, guinea pig spatial frequency discrimination, pattern and full-field photopic electroretinography (ERG), and retinal ganglion cell distribution were investigated. Methods Adult guinea pigs (n = 6) were included. Optomotor responses to square-wave gratings from 0.3 to 2.4 cycles per degree (cpd) were assessed. Pattern ERG responses were recorded using square-wave gratings from 0.025 to 0.25 cpd at 100% contrast, alternating at a temporal frequency of 1.05 Hz. Full-field ERG responses were recorded using a 10.0 cd.s/m2 flash. Ganglion cell density was determined histologically from retinal whole mounts. Results Maximum spatial frequency discrimination was 1.65 ± 0.49 cpd for stimuli rotating temporally to nasally and 0.75 ± 0.16 cpd for stimuli rotating nasally to temporally. For pattern ERG, a maximum amplitude of 3.50 ± 1.16 µV for the first negative to positive peak (N1P1) was elicited with a 0.025 cpd grating, and 2.5 ± 0.1 µV for the positive to second negative peak (P1N2) was elicited with a 0.05 cpd grating. For full-field ERG, a-wave amplitude was 19.2 ± 4.24 µV, b-wave amplitude was 33.6 ± 8.22 µV, and the PhNR was 24.0 ± 5.72 µV. Peak retinal ganglion cell density was 1621 ± 129 cells/mm2, located 1–2 mm superior to the optic nerve head. Conclusion Guinea pigs show directional selectivity for stimuli moving in the temporal to the nasal visual field. Guinea pigs demonstrate a quantifiable PhNR in the full-field ERG and negative and positive waveforms in the pattern ERG. The visual streak is located in the superior retina.