Optimizing the Setup and Conditions for <em>Ex Vivo</em> 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 ...

University of Utah

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 Műller 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 Műller 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. 

Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of 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/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-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 pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

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.

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

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.

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

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 VEGF 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 VEGF, in Müller cells may have fewer deleterious effects than nonselective VEGFA inhibition to all cells in the retina.

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

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.

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

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.

Rod phototransduction and light signal transmission during type 2 diabetes.

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.

Diabetic photoreceptors: Mechanisms underlying changes in structure and function.

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 ceases, 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.

Silke Becker

John A. Moran Eye Center

University of Utah

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

Silke Becker

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University of Utah

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

John A. Moran Eye Center