Modeling complex age-related eye disease.

Modeling complex eye diseases like age-related macular degeneration (AMD) and glaucoma poses significant challenges, since these conditions depend highly on age-related changes that occur over several decades, with many contributing factors remaining unknown. Although both diseases exhibit a relatively high heritability of >50%, a large proportion of individuals carrying AMD- or glaucoma-associated genetic risk variants will never develop these diseases. Furthermore, several environmental and lifestyle factors contribute to and modulate the pathogenesis and progression of AMD and glaucoma. Several strategies replicate the impact of genetic risk variants, pathobiological pathways and environmental and lifestyle factors in AMD and glaucoma in mice and other species. In this review we will primarily discuss the most commonly available mouse models, which have and will likely continue to improve our understanding of the pathobiology of age-related eye diseases. Uncertainties persist whether small animal models can truly recapitulate disease progression and vision loss in patients, raising doubts regarding their usefulness when testing novel gene or drug therapies. We will elaborate on concerns that relate to shorter lifespan, body size and allometries, lack of macula and a true lamina cribrosa, as well as absence and sequence disparities of certain genes and differences in their chromosomal location in mice. Since biological, rather than chronological, age likely predisposes an organism for both glaucoma and AMD, more rapidly aging organisms like small rodents may open up possibilities that will make research of these diseases more timely and financially feasible. On the other hand, due to the above-mentioned anatomical and physiological features, as well as pharmacokinetic and -dynamic differences small animal models are not ideal to study the natural progression of vision loss or the efficacy and safety of novel therapies. In this context, we will also discuss the advantages and pitfalls of alternative models that include larger species, such as non-human primates and rabbits, patient-derived retinal organoids, and human organ donor eyes.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes.

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Revival of light signalling in the postmortem mouse and human retina.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Diabetic photoreceptors: Mechanisms underlying changes in structure and function.

Based on clinical findings, diabetic retinopathy (DR) has traditionally been defined as a retinal microvasculopathy. Retinal neuronal dysfunction is now recognized as an early event in the diabetic retina before development of overt DR. While detrimental effects of diabetes on the survival and function of inner retinal cells, such as retinal ganglion cells and amacrine cells, are widely recognized, evidence that photoreceptors in the outer retina undergo early alterations in diabetes has emerged more recently. We review data from preclinical and clinical studies demonstrating a conserved reduction of electrophysiological function in diabetic retinas, as well as evidence for photoreceptor loss. Complementing in vivo studies, we discuss the ex vivo electroretinography technique as a useful method to investigate photoreceptor function in isolated retinas from diabetic animal models. Finally, we consider the possibility that early photoreceptor pathology contributes to the progression of DR, and discuss possible mechanisms of photoreceptor damage in the diabetic retina, such as enhanced production of reactive oxygen species and other inflammatory factors whose detrimental effects may be augmented by phototransduction.

Rod phototransduction and light signal transmission during type 2 diabetes.

INTRODUCTION: Diabetic retinopathy is a major complication of diabetes recently associated with compromised photoreceptor function. Multiple stressors in diabetes, such as hyperglycemia, oxidative stress and inflammatory factors, have been identified, but systemic effects of diabetes on outer retina function are incompletely understood. We assessed photoreceptor physiology in vivo and in isolated retinas to better understand how alterations in the cellular environment compared with intrinsic cellular/molecular properties of the photoreceptors, affect light signal transduction and transmission in the retina in chronic type 2 diabetes. RESEARCH DESIGN AND METHODS: Photoreceptor function was assessed in BKS.Cs-Dock7m+/+Lepr db/J mice, using homozygotes for Leprdb as a model of type 2 diabetes and heterozygotes as non-diabetic controls. In vivo electroretinogram (ERG) was recorded in dark-adapted mice at both 3 and 6 months of age. For ex vivo ERG, isolated retinas were superfused with oxygenated Ames' media supplemented with 30 mM glucose or mannitol as iso-osmotic control and electrical responses to light stimuli were recorded. RESULTS: We found that both transduction and transmission of light signals by rod photoreceptors were compromised in 6-month-old (n=9-10 eyes from 5 animals, ***p<0.001) but not in 3-month-old diabetic mice in vivo (n=4-8 eyes from 2 to 4 animals). In contrast, rod signaling was similar in isolated retinas from 6-month-old control and diabetic mice under normoglycemic conditions (n=11). Acutely elevated glucose ex vivo increased light-evoked rod photoreceptor responses in control mice (n=11, ***p<0.001), but did not affect light responses in diabetic mice (n=11). CONCLUSIONS: Our data suggest that long-term diabetes does not irreversibly change the ability of rod photoreceptors to transduce and mediate light signals. However, type 2 diabetes appears to induce adaptational changes in the rods that render them less sensitive to increased availability of glucose.

Targeted Knockdown of Overexpressed VEGFA or VEGF164 in Müller cells maintains retinal function by triggering different signaling mechanisms.

Oxygen-induced retinopathy (OIR) upregulates Müller cell vascular endothelial growth factor A (VEGFA) that causes intravitreal neovascularization similar to severe retinopathy of prematurity (ROP). Safety concerns exist with anti-VEGF treatment for ROP. We evaluated long-term knockdown of Müller cell-VEGFA with short-hairpin RNAs to VEGFA or VEGF164 via subretinal lentivirus delivery (L-VEGFAshRNA, L-VEGF164shRNA) on retinal structure and function in a rat OIR model. Lectin-stained retinal flat mounts analyzed for areas of avascular/total retina (AVA) and intravitreal neovascular/total retina (IVNV) showed initial significantly reduced IVNV by L-VEGFAshRNA and L-VEGF164shRNA compared to control, luciferase-shRNA lentivirus, without late recurrence. Spectral-domain optical coherence tomography (OCT) and immunohistochemical sections (IHC) demonstrated changes in retinal layer thicknesses in L-VEGFAshRNA or L-VEGF164shRNA  compared to control. Ganzfeld electroretinograms were increased in L-VEGFAshRNA or L-VEGF164shRNA compared to control. Erythropoietin (EPO), brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3) mRNAs were increased in L-VEGFAshRNA, but not L-VEGF164shRNA retinas. In cultured rat Müller cells, knockdown of VEGF upregulated NT-3 and EPO, whereas treatment with EPO activated neuroprotective signaling. Methods to reduce IVNV by selective knockdown of VEGFA, and particularly VEGF164, in Müller cells may have fewer deleterious effects than nonselective VEGFA inhibition to all cells in the retina.

Automatic analysis of the retinal avascular area in the rat oxygen-induced retinopathy model.

Purpose: The aim of this study was to create an algorithm to automate, accelerate, and standardize the process of avascular area segmentation in images from a rat oxygen-induced retinopathy (OIR) model. Methods: Within 6 h of birth, full-term pups born to Sprague Dawley rat dams that had undergone partial bilateral uterine artery ligation at embryonic day 19.5 were placed into a controlled oxygen environment (Oxycycler, BioSpherix, Parish, NY) at 50% oxygen for 48 h, followed by cycling between 10% and 50% oxygen every 24 h until day 15. The pups were then moved into room air until day 18.5. Ten lectin-stained retinal flat mounts were imaged in montage fashion at 10x magnification. Three masked human reviewers measured two parameters, total retinal area and peripheral avascular area, for each image using the ImageJ freehand selection tool. The outputs of each read were measured as number of pixels. The gold standard value for each image was the mean of the three human reads. Interrater agreement for the measurement of total retinal area, avascular area, and percent avascular area was calculated using type A intraclass correlation coefficients (ICCs) with a two-way random effects model. Automated avascular area identification (A3ID) is a method written in ImageJ Macro that is intended for use in the Fiji (Fiji is Just ImageJ) image processing platform. The input for A3ID is a rat retinal image, and the output is the avascular area (in pixels). A3ID utilizes a random forest classifier with a connected-components algorithm and post-processing filters for size and shape. A separate algorithm calculates the total retinal area. We compared the output of both algorithms to gold standard measurements by calculating ICCs, performing linear regression, and determining the Dice coefficients for both algorithms. We also constructed a Bland-Altman plot for A3ID output. Results: The ICC for percent peripheral avascular/total area between human readers was 0.995 (CI: 0.974-0.999), with p<0.001. The ICC between A3ID and the gold standard was calculated for three image parameters-avascular area: 0.974 (CI: 0.899-0.993), with p<0.001; total retinal area: 0.465 (CI: 0.0-0.851), with p=0.001; and the percent peripheral avascular/total area: 0.94 (CI: 0.326-0.989), with p<0.001. In the linear regression analysis, the slope for prediction of the gold standard percent peripheral avascular/total area from A3ID was 0.98, with R2=0.975. A3ID and the total retinal area algorithm achieve an average Dice coefficient of 0.891 and 0.952, respectively. The Bland-Altman analysis revealed a trend for computer underestimation of the peripheral avascular area in images with low peripheral avascular area and overestimation of peripheral avascular area in images with large peripheral avascular areas. Conclusions: A3ID reliably predicts peripheral avascular area based on rat OIR retinal images. When the peripheral avascular area is particularly high or low, hand segmentation of images may be superior.

Effect of subretinal injection on retinal structure and function in a rat oxygen-induced retinopathy model.

Purpose: Subretinal injections are used to deliver agents in experimental studies of retinal diseases, often through viral vectors. However, few studies have investigated the effects of subretinal injections alone on the structure and function of the healthy or diseased retina, particularly in models of oxygen-induced retinopathy (OIR). We report on the effects of subretinal injections in a rat OIR model, which is used to study mechanisms of retinopathy of prematurity. Methods: Within 6 h of birth, neonatal rat pups were exposed to repeated cycles of oxygen between 50% and 10% O2 every 24 h for 14 days and subsequently moved to room air. On postnatal day 8 (P8), animals were treated in both eyes with advancement of the injection needle into the vitreous (pilot-treated) or with a subretinal PBS injection (sPBS-treated) or were left untreated (untreated). Additional control animals were exposed to microscope light after eyelid opening only (light-treated). Retinal fundus images were recorded on P26. Areas of the avascular retina and intravitreal neovascularization were determined in flat mounted retinas stained with isolectin B4 on P32. Retinal function of the respective eyes was assessed with the Ganzfeld electroretinogram (ERG) on P31 or P32 and with focal ERG in the central retina on P28 or P29. The thickness of the retinal layers was measured with spectral domain optical coherence tomography (OCT) on P30 and in opsin- and TO-PRO 3-stained retinal cryosections from pups euthanized on P32. Two sections were analyzed in each pup. For each section, three images of three different locations were analyzed accounting for 18 thickness measurements per pup. Results: Compared to untreated animals, the avascular area of the retina was greater in the pilot-treated (p<0.05) and sPBS-treated eyes (p<0.01), and the sPBS-treated eyes had a greater avascular retinal area compared to the pilot-treated eyes (p<0.01). The intravitreal neovascular area was larger in the sPBS-treated eyes compared to the untreated eyes (p<0.01). The outer nuclear and outer segment layers were thinner in the pilot- (p<0.01) and sPBS-treated eyes (p<0.05) compared to the untreated eyes as measured with OCT and immunohistochemical staining of the retinal cryosections. Compared to the untreated eyes, the amplitudes of the scotopic a- and b-waves in the Ganzfeld ERG were reduced in the pilot-treated eyes (p<0.001 and p<0.01, respectively), but only the a-wave was reduced in the sPBS-treated eyes (p<0.001). The a-wave amplitude in the focal ERG was reduced in the pilot- and sPBS-treated eyes, and no difference was seen in the b-wave amplitude between any of the groups. There was no difference between the light-treated and untreated eyes in the areas of the avascular retina or intravitreal neovascularization or Ganzfeld or focal ERG. Conclusions: Pilot injections alone without injection into the subretinal space resulted in an increased avascular retinal area, reduced thickness of the photoreceptors, and reduced ERG function compared to the untreated animals. Although subretinal PBS injections further increased the areas of avascular retina and intravitreal neovascularization and resulted in similar retinal thinning compared to the pilot treatment, inner retinal function was improved, as evidenced by higher Ganzfeld b-wave amplitudes. Differences in the Ganzfeld and focal ERGs may indicate that the peripheral retina is more susceptible to remote beneficial effects from potential protective mechanisms induced by subretinal injection. This study stresses the importance of appropriate controls in experiments with subretinal delivery of agents.

Protective effect of maternal uteroplacental insufficiency on oxygen-induced retinopathy in offspring: removing bias of premature birth.

To address the hypothesis that maternal uteroplacental insufficiency (UPI) increases severity of retinopathy of prematurity, we developed a composite rat model of UPI and oxygen-fluctuations and removed premature birth as a confounding factor. Timed-pregnant Sprague-Dawley dams underwent bilateral uterine artery ligation or anesthesia (control) at e19.5. Full-term pups developed in room air (RA) or an oxygen-induced retinopathy (OIR) model. Isolectin-stained retinal flat-mounts were analyzed for percent of areas of avascular/total retina (AVA) and of intravitreal neovascular/total retina (IVNV). Pup weights and serum and mRNA of liver and kidney VEGF, IGF-1, and erythropoietin (EPO) were determined. Multivariable mixed effects linear regressions and Pearson correlations were performed using STATA14. Postnatal growth restriction occurred in pups in UPI/RA, but not in UPI/OIR. Weight gain was similar between UPI/OIR and control/OIR pups. AVA was reduced and a trend toward reduced IVNV was seen in UPI/OIR compared to control/OIR. No difference in birth weights of UPI/OIR vs. control/OIR pups occurred. Serum and renal IGF-1 and EPO were significantly increased in UPI/OIR compared to control/OIR pups. In the absence of prematurity, UPI increased angiogenic factors in association with reduced OIR severity, suggesting that ischemia from UPI could yield protective angiogenic effects by offspring.

Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/ß-catenin complexes, increased transepithelial electrical resistance through an interaction of ß-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of Choroidal Neovascularization.

Inhibition of chemokine C-C motif receptor 3 (CCR3) signaling has been considered as treatment for neovascular age-related macular degeneration (AMD). However, CCR3 is expressed in neural retina from aged human donor eyes. Therefore, broad CCR3 inhibition may be harmful to the retina. We assessed the effects of CCR3 inhibition on retina and choroidal endothelial cells (CECs) that develop into choroidal neovascularization (CNV). In adult murine eyes, CCR3 colocalized with glutamine-synthetase labeled Muller cells. In a murine laser-induced CNV model, CCR3 immunolocalized not only to lectin-stained cells in CNV lesions but also to the retina. Compared to non-lasered controls, CCR3 mRNA was significantly increased in laser-treated retina. An intravitreal injection of a CCR3 inhibitor (CCR3i) significantly reduced CNV compared to DMSO or PBS controls. Both CCR3i and a neutralizing antibody to CCR3 increased TUNEL+ retinal cells overlying CNV, compared to controls. There was no difference in cleaved caspase-3 in laser-induced CNV lesions or in overlying retina between CCR3i- or control-treated eyes. Following CCR3i, apoptotic inducible factor (AIF) was significantly increased and anti-apoptotic factor BCL2 decreased in the retina; there were no differences in retinal vascular endothelial growth factor (VEGF). In cultured human Muller cells exposed to eotaxin (CCL11) and VEGF, CCR3i significantly increased TUNEL+ cells and AIF but decreased BCL2 and brain derived neurotrophic factor, without affecting caspase-3 activity or VEGF. CCR3i significantly decreased AIF in RPE/choroids and immunostaining of phosphorylated VEGF receptor 2 (p-VEGFR2) in CNV with a trend toward reduced VEGF. In cultured CECs treated with CCL11 and/or VEGF, CCR3i decreased p-VEGFR2 and increased BCL2 without increasing TUNEL+ cells and AIF. These findings suggest that inhibition of retinal CCR3 causes retinal cell death and that targeted inhibition of CCR3 in CECs may be a safer if CCR3 inhibition is considered as a therapy for neovascular AMD.

Allogeneic Transplantation of Müller-Derived Retinal Ganglion Cells Improves Retinal Function in a Feline Model of Ganglion Cell Depletion.

Human Müller glia with stem cell characteristics (hMGSCs) have been shown to improve retinal function upon transplantation into rat models of retinal ganglion cell (RGC) depletion. However, their translational potential may depend upon successful engraftment and improvement of retinal function in experimental models with anatomical and functional features resembling those of the human eye. We investigated the effect of allogeneic transplantation of feline Müller glia with the ability to differentiate into cells expressing RGC markers, following ablation of RGCs by N-methyl-d-aspartate (NMDA). Unlike previous observations in the rat, transplantation of hMGSC-derived RGCs into the feline vitreous formed aggregates and elicited a severe inflammatory response without improving visual function. In contrast, allogeneic transplantation of feline MGSC (fMGSC)-derived RGCs into the vitrectomized eye improved the scotopic threshold response (STR) of the electroretinogram (ERG). Despite causing functional improvement, the cells did not attach onto the retina and formed aggregates on peripheral vitreous remnants, suggesting that vitreous may constitute a barrier for cell attachment onto the retina. This was confirmed by observations that cellular scaffolds of compressed collagen and enriched preparations of fMGSC-derived RGCs facilitated cell attachment. Although cells did not migrate into the RGC layer or the optic nerve, they significantly improved the STR and the photopic negative response of the ERG, indicative of increased RGC function. These results suggest that MGSCs have a neuroprotective ability that promotes partial recovery of impaired RGC function and indicate that cell attachment onto the retina may be necessary for transplanted cells to confer neuroprotection to the retina. Significance: Müller glia with stem cell characteristics are present in the adult human retina, but they do not have regenerative ability. These cells, however, have potential for development of cell therapies to treat retinal disease. Using a feline model of retinal ganglion cell (RGC) depletion, cell grafting methods to improve RGC function have been developed. Using cellular scaffolds, allogeneic transplantation of Müller glia-derived RGC promoted cell attachment onto the retina and enhanced retinal function, as judged by improvement of the photopic negative and scotopic threshold responses of the electroretinogram. The results suggest that the improvement of RGC function observed may be ascribed to the neuroprotective ability of these cells and indicate that attachment of the transplanted cells onto the retina is required to promote effective neuroprotection.

Successful re-evaluation of broth medium T for growth of Francisella tularensis ssp. and other highly pathogenic bacteria.

Pavlovich's medium T was compared with other broadly used media and extensively checked by growth of various subspecies of Francisellatularensis as well as other risk group 3 bacteria. The medium was successfully re-evaluated as an optimal liquid medium suitable for enrichment of fastidious and/or highly pathogenic bacteria.

Short Communication: Contribution of Voltage-Gated Sodium Channels to the Rabbit Cone Electroretinograms.

PURPOSE: Although the rabbit eye is of a similar size to the human eye, our limited understanding of the differences in retinal physiology to other species hinders its use in retinal research. The role of voltage-gated sodium channels (Nav) in the propagation of excitatory potentials along bipolar cells remains unclear, as conflicting data have been reported in the rabbit. The present study assesses the relative contributions of Nav to the scotopic and photopic flash ERGs as well as the wavelength-dependence of Nav blockade on the rabbit flicker ERG. MATERIALS AND METHODS: Tetrodotoxin (TTX, 1 µM) was injected into the vitreous cavity of Chinchilla bastard rabbits. Scotopic ERGs were evoked by white flashes ranging from 10(-5) to 10 cds m(-2), photopic ERGs on a background of 25 cdm(-2) using flash intensities of 0.032-25 cds m(-2). Flicker ERGs (3-50 Hz) were elicited by blue, green and yellow stimuli at 2.34 cds m(-2) on a white background of 30 cdm(-2). RESULTS: The a- and b-waves of the scotopic ERG were unaffected by intravitreal injection of the Nav blocker TTX. In contrast, the b-wave, but not the a-wave, of the photopic ERG was selectively blocked by TTX. The reduction by TTX of the flicker ERG was greater for blue than for green and yellow stimuli. DISCUSSION: The data suggest that Nav selectively contribute to the generation of the photopic b-wave in the rabbit, indicating that they play an important role in the propagation of excitatory signals on bipolar cells in the cone, but not rod pathways. Importantly, the present study resolves conflicting previous reports into the role of Nav in the retinal function of the rabbit.

Genetic background and light-dependent progression of photoreceptor cell degeneration in Prominin-1 knockout mice.

PURPOSE: Mutations in the Prominin-1 (Prom1) gene are known to cause retinitis pigmentosa and Stargardt disease, both of which are associated with progressive photoreceptor cell death. There are no effective therapies for either disorder. The aim of this study was to investigate the mechanism of the retinal degeneration in Prom1-deficient mouse models. METHODS: We constructed Prom1 knockout mice with two distinct genetic backgrounds of C57BL/6 and C57BL/6xCBA/NSlc, and investigated the photoreceptor degeneration by means of histology and functional tests.. In addition, we examined the effect of light on the Prom1(-/-) retina by rearing the mice in the normal light/dark cycle and completely dark conditions. Finally, we investigated if the retinoic-acid derivative Fenretinide slowed the pace of retinal degeneration in these mouse models. RESULTS: The Prom1(-/-)-knockout mice with both backgrounds developed photoreceptor degeneration after eye opening, but the CB57/BL6-background mice developed photoreceptor cell degeneration much faster than the C57BL/6xCBA/NSlc mice, demonstrating genetic background dependency.. Interestingly, our histologic and functional examination showed that the photoreceptor cell degeneration of Prom1-knockout mice was light-dependent, and was almost completely inhibited when the mutant mice were kept in the dark. The Prom1-knockout retina showed strong downregulation of expression of the visual cycle components, Rdh12 and Abca4. Furthermore, administration of Fenretinide, which lowers the level of the toxic lipofuscin, slowed the degeneration of photoreceptor cells. CONCLUSIONS: These findings improve our understanding of the mechanism of cell death in Prominin-1-related disease and provide evidence that fenretinide may be worth studying in human disease.

Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects.

AIMS: Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. METHODS: Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2 , and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. RESULTS: In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. CONCLUSIONS: In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker.

Optimized feline vitrectomy technique for therapeutic stem cell delivery to the inner retina.

OBJECTIVE: To describe an optimized surgical technique for feline vitrectomy which reduces bleeding and aids posterior gel clearance in order to facilitate stem cell delivery to the inner retina using cellular scaffolds. PROCEDURES: Three-port pars plana vitrectomies were performed in six-specific pathogen-free domestic cats using an optimized surgical technique to improve access and minimize severe intraoperative bleeding. RESULTS: The surgical procedure was successfully completed in all six animals. Lens sparing vitrectomy resulted in peripheral lens touch in one of three animals but without cataract formation. Transient bleeding from sclerotomies, which was readily controlled, was seen in two of the six animals. No cases of vitreous hemorrhage, severe postoperative inflammation, retinal detachment, or endophthalmitis were observed during postoperative follow-up. CONCLUSIONS: Three-port pars plana vitrectomy can be performed successfully in the cat in a safe and controlled manner when the appropriate precautions are taken to minimize the risk of developing intraoperative hemorrhage. This technique may facilitate the use of feline models of inner retinal degeneration for the development of stem cell transplantation techniques using cellular scaffolds.

Transplantation of photoreceptors derived from human Muller glia restore rod function in the P23H rat.

Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia-derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC-derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a-wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell-replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.

Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors.

PURPOSE: Human Müller glia with stem cell characteristics (hMGSCs) can be induced to express genes and proteins of retinal ganglion cells (RGCs) upon in vitro inhibition of Notch-1 activity. However, it is not known whether expression of these markers is accompanied by acquisition of RGC function. This study investigated whether hMGSCs that express RGC markers also display neural functionality, as measured by their intracellular calcium concentration ([Ca(2+)]i) responsiveness following neurotransmitter stimulation in vitro. METHODS: Changes in mRNA expression of RGC markers and neurotransmitter receptors were assessed either by conventional or quantitative reverse transcription PCR (RT-PCR), while changes in protein levels were confirmed by immunocytochemistry. The [Ca(2+)]i levels were estimated by fluorescence microscopy. RESULTS: We showed that while undifferentiated hMGSCs displayed a profound elevation of [Ca(2+)]i after stimulation with N-methyl-D-aspartate (NMDA), this was lost following Notch-1 inhibition. Conversely, untreated hMGSCs did not respond to muscarinic receptor stimulation, whereas [Ca(2+)]i was increased in differentiated hMGSCs that expressed RGC precursor markers. Differentiated hMGSC-derived RGCs, but not undifferentiated hMGSCs, responded to stimulation by nicotine with a substantial rise in [Ca(2+)]i, which was inhibited by the α4ß2 and α6ß2 nicotinic receptor antagonist methyllycaconitine. Notch-1 attenuation not only caused a decrease in the gene expression of the Notch effector HES1 and increased expression of RGC markers, but also an increase in the gene and protein expression of α4 and α6 nicotinic receptor subunits. CONCLUSIONS: These observations suggest that in response to Notch-1 inhibition, hMGSCs differentiate into a population of RGCs that exhibit some of the functionality observed in differentiated RGCs.

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release.

Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 µm) or nifedipine (3 µm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 µm) or SKF96365 (50 µm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 µm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 µm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 µm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 µm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 µm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 µm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 µm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 µm) nor FK506 (10 µm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 µm) and attenuated by the protein kinase C inhibitor Gö6983 (3 µm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 µm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 µm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.