Bovine Retina Research Articles

Sitagliptin and the Blood-Retina Barrier: Effects on Retinal Endothelial Cells Manifested Only after Prolonged Exposure.

Inhibitors of dipeptidyl peptidase-4 (DPP-4) are widely used to treat diabetes mellitus, but data concerning their effects on the barrier stability of retinal endothelial cells (REC) in vivo and in vitro are inconsistent. Therefore, we studied whether the barrier properties of immortalized endothelial cells of the bovine retina (iBREC) were affected by the inhibitors of DPP-4 sitagliptin (10-1000 nM) and diprotin A (1-25 μM). Their effects were also investigated in the presence of VEGF-A165 because diabetic patients often develop macular edema caused by VEGF-A-induced permeability of REC. To detect even transient or subtle changes of paracellular and transcellular flow as well as adhesion of the cells to the extracellular matrix, we continuously monitored the cell index (CI) of confluent iBREC grown on gold electrodes. Initially, the CI remained stable but started to decline significantly and persistently at 40 h or 55 h after addition of sitagliptin or diprotin A, respectively. Both inhibitors did not modulate, prevent, or revert the persistent VEGF-A165-induced reduction of the CI. Interestingly, sitagliptin and diprotin A increased the expression of the tight-junction protein claudin-1 which is an important component of a functional barrier formed by iBREC. In contrast, expressions of CD29—a subunit of the fibronectin receptor—or of the tetraspanin CD9 were lower after extended treatment with the DPP-4 inhibitors; less of the CD9 was seen at the plasma membrane after prolonged exposure to sitagliptin. Because both associated proteins are important for adhesion of iBREC to the extracellular matrix, the observed low CI might be caused by weakened attachment of the cells. From our results, we conclude that extended inhibition of DPP-4 destabilizes the barrier formed by microvascular REC and that DPP-4 inhibitors like sitagliptin do not counteract or enhance a VEGF-A165-induced barrier dysfunction as frequently observed in DME.

Submicromolar copper (II) ions stimulate transretinal signaling in the isolated retina from wild type but not from Cav2.3-deficient mice

BackgroundSo far, only indirect evidence exists for the pharmacoresistant R-type voltage-gated Ca2+ channel (VGCC) to be involved in transretinal signaling by triggering GABA-release onto ON-bipolar neurons. This release of inhibitory neurotransmitters was deduced from the sensitivity of the b-wave to stimulation by Ni2+, Zn2+ and Cu2+. To further confirm the interpretation of these findings, we compared the effects of Cu2+ application and chelation (using kainic acid, KA) on the neural retina from wildtype and Cav2.3-deficient mice. Furthermore, the immediately effect of KA on the ERG b-wave modulation was assessed.MethodsTransretinal signaling was recorded as an ERG from the superfused murine retina isolated from wildtype and Cav2.3-deficient mice.ResultsIn mice, the stimulating effect of 100 nM CuCl2 is absent in the retinae from Cav2.3-deficient mice, but prominent in Cav2.3-competent mice. Application of up to 3 mM tricine does not affect the murine b-wave in both genotypes, most likely because of chelating amino acids present in the murine nutrient solution. Application of 27 μM KA significantly increased the b-wave amplitude in wild type and Cav2.3 (−|-) mice. This effect can most likely be explained by the stimulation of endogenous KA-receptors described in horizontal, OFF-bipolar, amacrine or ganglion cells, which could not be fully blocked in the present study.ConclusionCu2+-dependent modulation of transretinal signaling only occurs in the murine retina from Cav2.3 competent mice, supporting the ideas derived from previous work in the bovine retina that R-type Ca2+ channels are involved in shaping transretinal responses during light perception.

Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality.

PurposeThe retinal rod outer segment (OS) disk membranes, devoid of mitochondria, conducts oxidative phosphorylation (OxPhos). This study aimed at identifying which proteins expressed in the retinal rod OS disks determined the considerable adenosine‐5'‐triphosphate production and oxygen consumption observed in comparison with retinal mitochondria.ProceduresCharacterization was conducted by immunogold transmission electron microscopy on retinal sections. OxPhos was studied by oximetry and luminometry. The proteomes of OS disks and mitochondria purified from bovine retinas were studied by mass spectrometry. Statistical and bioinformatic analyses were conducted by univariate, multivariate, and machine learning methods.ResultsWeighted gene coexpression network analysis identified two protein expression profile modules functionally associated with either retinal mitochondria or disk samples, in function of a strikingly different ability of each sample to utilized diverse substrate for F1Fo‐ATP synthase. The OS disk proteins correlated better than mitochondria with the tricarboxylic acids cycle and OxPhos proteins.ConclusionsThe differential enrichment of the expression profile of the OxPhos proteins in the disks versus mitochondria suggests that these proteins may represent a true proteome component of the former, with different functionality. These findings may shed new light on the pathogenesis of rod‐driven retinal degenerative diseases.

Selective retina therapy monitoring by speckle variance optical coherence tomography for dosimetry control.

.Significance: Selective retina therapy (SRT) selectively targets the retinal pigment epithelium (RPE) and reduces negative side effects by avoiding thermal damages of the adjacent photoreceptors, the neural retina, and the choroid. However, the selection of proper laser energy for the SRT is challenging because of ophthalmoscopically invisible lesions in the RPE and different melanin concentrations among patients or even regions within an eye.Aim: We propose and demonstrate SRT monitoring based on speckle variance optical coherence tomography (svOCT) for dosimetry control.Approach: M-scans, time-resolved sequence of A-scans, of ex vivo bovine retina irradiated by duration laser pulses were obtained by a swept-source OCT. SvOCT images were calculated as interframe intensity variance of the sequence. Spatial and temporal temperature distributions in the retina were numerically calculated in a 2-D retinal model using COMSOL Multiphysics. Microscopic images of treated spots were obtained before and after removing the upper neural retinal layer to assess the damage in both RPE and neural layers.Results: SvOCT images show abrupt speckle variance changes when the retina is irradiated by laser pulses. The svOCT intensities averaged in RPE and photoreceptor layers along the axial direction show sharp peaks corresponding to each laser pulse, and the peak values were proportional to the laser pulse energy. The calculated temperatures in the neural retina layer and RPE were linearly fitted to the svOCT peak values, and the temperature of each lesion was estimated based on the fitting. The estimated temperatures matched well with previously reported results.Conclusion: We found a reliable correlation between the svOCT peak values and the degree of retinal lesion formation, which can be used for selecting proper laser energy during SRT.

Determination of clenbuterol, salbutamol, and cimaterol in bovine retina by electrospray ionization-liquid chromatography-tandem mass spectrometry.

An electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI/LC/MS/MS) method was developed for the simultaneous determination of the beta-agonists clenbuterol, salbutamol, and cimaterol in bovine retina. The tissue was homogenized in alkaline buffer and spiked to give 10, 15, and 20 ng/g each of the 3 analytes together with the internal standards d6-salbutamol and d6-clenbuterol. The mixture was incubated with protease enzyme to release any protein-bound analytes and then made alkaline before extraction with isobutanol. The extract was dissolved in water and transferred to a clenbuterol immunoaffinity column. After washing, the analytes were eluted and analyzed by ESI/LC/MS/MS using a C18 column with acetic acid-methanol as mobile phase. No interferences were observed from the spiked retina extract at the various single-reaction monitoring modes. Average recoveries for clenbuterol, salbutamol, and cimaterol were 94, 85, and 87% with coefficients of variation (CVs) of 9.4, 9.9, and 8.6%, respectively. A correlation coefficient of r2 = 0.9999 was obtained for all analytes. The limits of detection for clenbuterol, salbutamol, and cimaterol, determined from 3 times the standard deviation of 7 replicates of the lowest spike, were 2.5, 3.5, and 2.0 ng/g with CVs of 8.9, 11.6, and 7.2%, respectively.

Oxidized Lipids Decrease Phagocytic Activity of ARPE‐19 Cells In Vitro

Phospholipids, representing 80% of retinal lipids, contain a high level of polyunsaturated fatty acids, which are susceptible to oxidation. It has recently been shown that Folch’ extract of bovine neural retinas reveals moderate photoreactivity by generating reactive oxygen species upon irradiation with blue light. Importantly, the photoreactivity markedly increases upon oxidation of the exctracted lipids. In this work, the phototoxic potential of the Folch’ extract of bovine neural retinas (BRex), subjected to controlled oxidation, is studied in vitro employing a retinal pigment epithelium cell line (ARPE‐19). Multilamellar liposomes prepared from BRex, equilibrated with air, are oxidized in the dark at 37 °C for up to 400 h. The liposomes are then introduced into ARPE‐19 cells by phagocytosis. The impact of BRex at different stages of oxidation on viability and specific phagocytic activity of ARPE‐19 cells exposed to visible light is analyzed. While the oxidized BRex, at the light doses employed, does not induce any measurable killing of the cells, the treatment noticeably decreases their specific phagocytic activity.Practical Applications: Lipid oxidation products are commonly considered as an efficient protein modification factor occurring in the retina. Although possible consequences of oxidation of retinal lipids for the onset and development of age‐related macular degeneration are widely discussed, their potential photoreactivity and possible phototoxicity have not been considered. This is an important issue; taking into account that reactive products of lipid oxidation present in the outer retina, it may be exposed to short‐wavelength visible radiation. If photoreactive, peroxidized retinal lipids may act as acquired endogenous sensitizers increasing the risk of retina photodamage. In this work, the phototoxic potential of the Folch’ extract of bovine neural retinas (BRex), subjected to controlled oxidation, is studied in vitro employing a retinal pigment epithelium cell line (ARPE‐19).Lipids present in the retina are characterized by high level of unsaturation that makes them susceptible to oxidation. When oxidized, retinal lipids become photoreactive under visible light. Treatment of retinal pigment epithelium cells (ARPE‐19) with liposomes prepared from oxidized Folch’ extract of bovine neural retinas followed by irradiation with visible light, reduces phagocytic activity of ARPE‐19 cells.

The Endocannabinoid System Is Present in Rod Outer Segments from Retina and Is Modulated by Light.

The aim of the present research was to evaluate if the endocannabinoid system (enzymes and receptors) could be modulated by light in rod outer segment (ROS) from bovine retina. First, we analyzed endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in purified ROS obtained from dark-adapted (DROS) or light-adapted (LROS) retinas. To this end, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), and lysophosphatidate phosphohydrolase (LPAP) enzymatic activities were analyzed using radioactive substrates. The protein content of these enzymes and of the receptors to which cannabinoids bind was determined by immunoblotting under light stimulus. Our results indicate that whereas DAGL and MAGL activities were stimulated in retinas exposed to light, no changes were observed in LPAP activity. Interestingly, the protein content of the main enzymes involved in 2-AG metabolism, phospholipase C β1 (PLCβ1), and DAGLα (synthesis), and MAGL (hydrolysis), was also modified by light. PLCβ1 content was increased, while that of lipases was decreased. On the other hand, light produced an increase in the cannabinoid receptors CB1 and CB2 and a decrease in GPR55 protein levels. Taken together, our results indicate that the endocannabinoid system (enzymes and receptors) depends on the illumination state of the retina, suggesting that proteins related to phototransduction phenomena could be involved in the effects observed.

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.

Investigating retinal toxicity of a lutein-based dye in a model of isolated and perfused bovine retina

Retidyne™ is a new lutein-based dye for internal limiting membrane staining. It uses the intrinsic staining characteristics of lutein which is already known to act as an antioxidant and blue-light filter in the human retina. We investigated retinal tolerance to different staining times measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount. For functionality, testing bovine retinas were prepared and perfused with an oxygen saturated standard solution and the ERG was recorded until stable b-wave amplitudes were reached. Then the perfusion was stopped and Retidyne™ was applied directly onto the retinal surface for exposure times of 60 or 120s. After restarting the perfusion with standard solution, the ERG amplitudes were monitored for 75min. To investigate the effects on photoreceptor function alone, 1mM asparate was added to block b-waves. For an exposure time of 60s amplitudes of a- and b-waves remained stable throughout the experiment. Exposure times of 120s caused an initial drop of amplitudes that reached statistical significance only for a-waves (a, - 21%, p = 0.047; b, - 14%, p = 0.052). This effect was only seen during the first minutes of the washout and the ERG recovered completely. In the model of isolated and perfused bovine retina, Retidyne™ showed a good safety profile for common intraoperatively used staining times. An initial toxic effect regarding the transient drop of amplitudes cannot be ruled out but the effect might also be explained by the partial blockage of the flashlight due to a more intense staining effect at the beginning of the washout.

Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute.

PurposeTo characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS’s influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing.MethodsPig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR (Bax/Bcl–2-ratio, GFAP- and PGP9.5-levels) or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and β-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant.ResultsThe IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes (b-wave reduction 14.2%, p>0.05; a-wave reduction 23.9%, p>0.05). qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h.ConclusionsThe manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility.

Interaction between hydrogen sulfide, nitric oxide, and carbon monoxide pathways in the bovine isolated retina.

PurposeNitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are physiologically relevant gaseous neurotransmitters that are endogenously produced in mammalian tissues. In the present study, we investigated the possibility that NO and CO can regulate the endogenous levels of H2S in bovine isolated neural retina.MethodsIsolated bovine neural retina were homogenized and tissue homogenates were treated with a NO synthase inhibitor, NO donor, heme oxygenase-1 inhibitor, and/donor. H2S concentrations in bovine retinal homogenates were measured using a well-established colorimetric assay.ResultsL-NAME (300 nM–500 µM) caused a concentration-dependent decrease in basal endogenous levels of H2S by 86.2%. On the other hand, SNP (10–300 µM) elicited a concentration-related increase in H2S levels from 18.3 nM/mg of protein to 65.7 nM/mg of protein. ZnPP-IX (300 nM–10 µM) caused a concentration-dependent increase in the endogenous production of H2S whereas hemin (300 nM–20 µM) attenuated the basal levels of H2S.ConclusionWe conclude that changes in the biosynthesis and availability of both NO and CO can interfere with the pathway/s involved in the production of H2S in the retina. The demonstrated ability of NO, CO and H2S to interact in the mammalian retina affirms a physiological/pharmacological role for these gaseous mediators in the eye.

Prevention of Oxidative Stress-induced Metabolic Aberrations in the Neural Retina by Caffeine

Oxyradical-induced damage to the retina has been implicated as one of the contributing factors in the pathogenesis of vision-impairing diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). It is hypothesized that caffeine, a nutraceutical antioxidant, will be effective in preventing metabolic aberrations in the neural retina exposed to oxygen radicals. This hypothesis is based on our previous studies demonstrating its effectiveness in preventing oxidative damage to the lens, and in protecting the neural retina against UV-A- and peroxide-induced biochemical damage. Bovine neural retinas were incubated in medium 199 at 37°C for 6 hours. Xanthine (XA)-xanthine oxidase (XO) were used to generate reactive oxygen species (ROS). Incubations were conducted in 3 groups- control, experimental (with XA+XO), and caffeine group (XA + XO+ 5mM caffeine). Retinas were then processed for determining protein, lactate and pyruvate concentrations. Lactate concentration in the controls was 2.62±0.43mM/mg protein, decreasing to 1.04±0.3 mM/mg protein in the presence of XA+XO. Its level in the caffeine group was significantly higher, 2.44±0.65 mM/mg protein, close to the controls. Pyruvate concentration in the controls was 0.16±0.05mM/mg protein, which declined significantly with XA+XO to 0.066±0.02 mM/mg protein. Such decrease was substantially prevented in the caffeine group, wherein its concentration was 0.156±0.03mM/mg protein. Caffeine was thus found to be highly effective in preventing metabolic aberrations, due to its ability to scavenge oxyradicals and thereby possibly prevent inactivation of key enzymes. Such effect of caffeine in maintaining metabolism of the neural retina exposed to ROS has been shown for the first time.

Reciprocal modulation of Cav2.3 voltage‐gated calcium channels by copper(II) ions and kainic acid

Kainic acid (KA) is a potent agonist at non-N-methyl-D-aspartate (non-NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav 2.3 voltage-gated calcium channels have been implicated in the pathogenesis of KA-induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu2+ , Zn2+ ) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage-dependent gating. Using whole-cell patch-clamp recordings in human embryonic kidney (HEK-293) cells stably transfected with human Cav 2.3d and β3 -subunits, we identified a novel, glutamate receptor-independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu2+ and produces a hyperpolarizing shift in channel voltage-dependence by about 10 mV, thereby reconciling the effects of Cu2+ chelation with tricine. When tricine was used as a surrogate to study the receptor-independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b-wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav 2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu2+ -induced allosteric suppression, presumably via formation of stable kainate-Cu2+ complexes, could contribute to the receptor-mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA invitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.

Characterization of the retina-induced relaxation in mice.

The retinal relaxing factor (RRF) is a continuously released factor from the retina that causes vasorelaxation, the identity and potential role in physiology of which remain largely unknown. Experiments were performed to find out whether the RRF-induced relaxation is influenced by serotonin, glutamate, L-cysteine, the cytochrome P450 pathway, the cyclooxygenase pathway, or oxidative stress. In addition, the sensitivity of retinal and non-retinal arteries towards the RRF was compared. In vitro tension measurements were performed on isolated mouse femoral or bovine retinal arteries to study the vasorelaxing effect of the RRF, induced by mouse or bovine retinas. The presence of serotonin, glutamate, or L-cysteine did not alter the RRF-induced relaxation. Increasing oxidative stress by hydroquinone and diethyldithiocarbamic acid sodium salt enhanced the RRF response. Inhibition of the cytochrome P450 or the cyclooxygenase pathway did not cause any alteration. Surprisingly, the RRF-induced relaxation was enhanced by the presence of flufenamic acid or carbenoxolone. Furthermore, bringing retinal tissue in close contact with retinal or non-retinal arteries induced comparable relaxations. Serotonin, glutamate, L-cysteine, the cytochrome P450, and the cyclooxygenase pathway do not influence the RRF-induced relaxation and the RRF-induced relaxation seems to be resistant to oxidative stress. The mechanism responsible for the enhanced RRF-induced relaxation in the presence of flufenamic acid or carbenoxolone remains elusive and the RRF does not show more effectivity on retinal arteries.

Regulation of Hydrogen Sulfide Levels by Cannabinoids in the Bovine Isolated Retina

Both endocannabinoids and hydrogen sulfide (H2S) have been reported to exert neuroprotective actions in neural tissues (Biermann et al., Mol Vis. 2011;17:1275–86; Nucci et al., Invest Ophthalmol Vis Sci. 2007;48(7):2997–3004). It remains to be determined if these modulators can interact with each other and/or share a common pathway in their neuroprotective actions.PurposeThe aim of the present study was, therefore, to investigate the pharmacological actions of endocannabinoids on the biosynthesis of H2S in isolated bovine neural retina.MethodsIsolated bovine retinae were homogenized and exposed to different concentrations of methanandamide (1 nM – 10 uM) and 2‐arachidonyl glycerol (2‐AG) (1 – 3 uM) for 90 minutes at 37ºC. Retinal homogenates were then incubated with ethylenediaminetetraacetic acid (EDTA) (1%), Zinc acetate (1%), and Borate buffer (pH 11) for 30 mins followed by 10‐minute incubation with N, N dimethyl p‐phenylenediamine (20 mM) and FeCl3 (300 mM). Samples were then centrifuged at 5000 g for three minutes and supernatant was assessed for H2S content using the well‐established Methylene Blue method.ResultsBoth methanandamide and 2‐AG elicited a concentration‐dependent decrease in basal H2S production in the isolated retina. For instance, methanandamide (10 μM) significantly (p <0.05) reduced basal production of H2S by 30% when compared to controls. Likewise, 2‐AG (10 μM) also elicited a 30% decrease in basal retinal H2S concentration when compared to controls.ConclusionsWe conclude that exogenously applied endocannabinoids can alter the basal production of H2S in the isolated neural retina. The observed effect of endocannabinoids on the pathway leading to H2S biosynthesis suggests that a cross‐talk exists between both neuromodulators in the retina.Support or Funding InformationSupported by NIH/NEI Research Grant 1R15EY022215‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isolation of Phagosomes Containing Bovine Photoreceptor Outer Segments from Retinal Pigment Epithelial Cells by Magnetic Selection

Daily phagocytosis of the distal tips of photoreceptor cells by the retinal pigment epithelium (RPE) is known to be an essential mechanism in the maintenance of the retina. The outermost portions of the photoreceptor cells, termed outer segments, are ingested and subsequently processed by the RPE. Previous studies involving the isolation of phagosomes have utilized latex beads to induce phagocytosis in macrophages, which lacks specificity due to the large foreign body being ingested. Therefore, the purpose of this study was to develop a standard protocol for the isolation of outer segment enriched phagosomes from RPE cells. To generate phagosomes with the selected cargo, outer segments isolated from bovine retinas were covalently labelled with magnetite particles and then fed to RPE‐J cells. After feeding, the cells were washed and gently lysed with a Dounce homogenizer. The phagosomes containing the magnetite bound outer segments were collected via magnetic rack isolation and stored in a homogenization solution. Immunoblots using antibodies against Mertk, N and C‐terminal rhodopsin, and cathepsin D confirmed the presence of outer segment‐containing phagosomes of varying maturity in the isolated fraction. The protocol we have developed has successfully isolated outer segment enriched phagosomes from RPE‐J cells by magnetic selection. This study differentiates itself from other studies that employ latex beads to isolate phagosomes through density gradient centrifugation. The isolation of these phagosomes will potentially play an important role in investigating protein interactions that impact RPE membrane trafficking and maturation.Support or Funding InformationFlorida Southern College Faculty‐Student Collaborative Research GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [3H]D-Aspartate Release from Bovine Isolated Retinae.

We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, L-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10nM-10µM), L-cysteine (100nM-10µM) and N-acetylcysteine (10µM-1mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10µM, the rank order of activity was as follows: L-cysteine > GYY 4137 > N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3mM) reversed the inhibitory responses caused by GYY 4137, L-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and L-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and L-cysteine onK+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), L-NAME (300µM). Furthermore, a specific inhibitor of inducible NOS(iNOS), aminoguanidine (10µM) blocked the inhibitory action of L-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.

Comparison of Different Cell Culture Media in the Model of the Isolated and Superfused Bovine Retina: Investigating the Limits of More Physiological Perfusion Solutions

ABSTRACTPurpose: The isolated superfused retina is a standardized tool in ophthalmological research. However, stable electroretinogram (ERG) responses can only be obtained for around eight hours; therefore, limiting its use. The aim of this study was to evaluate the short-term potential of different cell culture media and to promote long-term testing based on the results obtained.Materials and Methods: For the experimental procedure bovine retinae were prepared and perfused with the standard Sickel solution and an ERG was performed. After recording stable a- or b-waves, different media (Dulbecco’s Modified Eagle’s Medium (DMEM), MACS, and Neurobasal) were superfused for 45 minutes. ERG recovery was monitored overall for 75 minutes. Analysis of the mRNA expression of Thy-1, GFAP, Bax/Bcl-2-ratio, Rhodopsin, and Opsin via qRT-PCR was performed directly after ERG recording on the same retina.Results: None of the tested media had a negative effect on a-wave amplitudes, although b-wave amplitudes decreased (DMEM) or increased (MACS and Neurobasal) compared to the standard solution (Sickel) after 45 minutes of exposure. However, after 75 minutes of wash-out, no difference to the standard solution alone could be observed. Exposure to different media either had no effect or decreased the Opsin and Rhodopsin mRNA levels. Thy-1 expression was strongly diminished in DMEM and MACS (by 2–3-fold), whereas incubation in Neurobasal medium led to a slight increase compared to incubation with the standard solution. Furthermore, the Bax/Bcl-2 ratio indicated an anti-apoptotic effect (Bax/Bcl-2 = 0.16; p < 0.05) for Neurobasal.Conclusion: Neurobasal medium displayed the best electrophysiological properties in the short-term and may be applicable for stable long-term escalation testing.

Oxidation-Induced Increase In Photoreactivity of Bovine Retinal Lipid Extract

The mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation—carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch’ extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 °C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (1O2, 1Δg) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch’ extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch’s extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as α-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch’ extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation.

Pericellular interphotoreceptor matrix dictates outer retina critical surface tension

Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal-pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina–RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR-IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR-IR depth penetration was established through ellipsometric measurement of barium-stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin-6-sulfate and IRBP, and imaging by IR-microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium-stearate calibration studies established that the MAIR-IR spectroscopy penetration depth was 0.2 μm. Ultrastructural observations and MAIR-IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips.