The study aims to validate mouse retinal explants for use as an efficient tool to investigate neuroprotective therapies for treatment in diseases such as glaucoma. Eyes from C57BL/6 mice were enucleated immediately post-mortem to make two retinal explants per eye. Explants were cultured at an air/medium interface on membrane inserts for 7 days ex-vivo. Explants were treated either with Z-VAD-FMK (a pan-caspase inhibitor; 100 μM) or vehicle. Retinal Ganglion cell (RGC) density was analysed by βIII tubulin and RNA-binding protein with multiple splicing (RBPMS) immunohistochemistry. Caspase activity was measured using Caspase 3/7 glo assay and western blot. Caspase-3 expression was quantified using RT-PCR and western blotting. Retinal explants treated with Z-VAD-FMK demonstrated a 1.5-fold (p = 0.027) increase in number of surviving RGCs on day 4 compared to the control treatment using βIII tubulin staining. RGC viability was 2-fold (p = 0.002) higher in RGC stained with RBPMS on day 1 compared to control. There was no RBPMS staining of RGCs beyond day 1 in either treatment. The caspase activity was 4.75 and 5.5-fold (p = 0.002 and 0.004 respectively) higher in control as compared to treatment with Z-VAD-FMK on day 1, 2 respectively. Increase in caspase activity in control group was also confirmed by western blot for day 1 protein lysates. Caspase-3 mRNA expression was 4.75-fold higher in Z-VAD-FMK treated explants compared to control on day 1 (p < 0.001). Culture conditions appropriate to retinal explant culture for investigation of RGC apoptosis was identified. Retinal cultures at day 4 were ideal for detecting neuroprotection using βIII tubulin staining. RBPMS acts as a viability marker as well as to define best time point for investigation of apoptosis related signalling pathways which is day 1. These findings suggest that mouse retinal explants are good model for studying ganglion cell specific apoptosis and are applicable to diseases such as glaucoma.
To investigate retinal ganglion cell (RGC) survival and activation of caspase 3 after optic nerve crush (ONC) or transection (ONT) and treatment with brain-derived neurotrophic factor (BDNF) or Z-DEVD_fmk.In albino Swiss mice, the left optic nerve was severed or crushed at 0.5 mm from the optic head and retinas were analyzed from 1 to 10 days. Additional groups were treated intravitreally with a single injection of BDNF (2.5 μg) or Z-DEVD_fmk (125 ng) right after injury, or with Z-DEVD_fmk at da...
To analyze the long-term effect of optic nerve injury on retinal ganglion cells (RGCs) and melanopsin+RGCs orthotopic and displaced, and on the rest of the ganglion cell layer (GCL) cells.In adult albino rats, the left optic nerve was crushed (ONC) or transected (ONT). Injured and contralateral retinas were analyzed at increasing survival intervals (up to 15 months). To study all GCL cells and RGCs, retinas were immunodetected with Brn3a and melanopsin to identify the general RGC population (Brn...
·AIM: To evaluate the reliability of β-III-Tubulin protein as a retinal ganglion cell(RGC) marker in the experimental glaucoma model.·METHODS: Glaucoma mouse models were established by injecting polystyrene microbeads into the anterior chamber of C57BL/6J mice, then their retinas were obtained 14 d and 28 d after the intraocular pressure(IOP)was elevated. Retinal flat mounts and sections were double-labeled by fluorogold(FG) and β-III-Tubulin antibody or single-labeled by β-III-Tubulin antibody,...
Last. Keith R Martin(University of Cambridge)H-Index: 54
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Purpose: An ex vivo organotypic retinal explant model was developed to examine retinal survival mechanisms relevant to glaucoma mediated by the renin angiotensin system in the rodent eye. Methods: Eyes from adult Sprague Dawley rats were enucleated immediately post-mortem and used to make four retinal explants per eye. Explants were treated either with irbesartan (10 µM), vehicle or angiotensin II (2 μM) for four days. Retinal ganglion cell density was estimated by βIII tubulin immunohistochemis...
Abstract In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m + RGC), Brn3a immunodetection to identify most RGCs b...
There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On Western blots these antibodies recognize a single band at ~24 kDa, corresponding to RBPMS, and they strongly ...
The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in ...
Last. John Danias(SUNY Downstate Medical Center)H-Index: 27
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Purpose: To determine whether short-term pressure elevation affects complement gene expression in the retina in vitro and in vivo. Methods: Muller cell (TR-MUL5) cultures and organotypic retinal cultures from adult mice and monkeys were subjected to either 24-h or 72-h of pressure at 0, 15, 30, and 45 mmHg above ambient. C57BL/6 mice were subjected to microbead-induced intraocular pressure (IOP) elevation for 7 days. RNA and protein were extracted and used for analysis of expression levels of co...
Visual fields measured with standard automated perimetry are a benchmark test for determining retinal function in ocular pathologies such as glaucoma. Their monitoring over time is crucial in detecting change in disease course and, therefore, in prompting clinical intervention and defining endpoints in clinical trials of new therapies. However, conventional change detection methods do not take into account non-stationary measurement variability or spatial correlation present in these measures. A...
Abstract A recently patented one-step methodology was used for the formulation of chitosan (CS) coated polylactic- co -glycolic acid (PLGA) nanoparticles containing dexamethasone (DXM) as a model drug. SEM investigations showed that nanoparticles (NPs) were spherical in shape with smooth surface. CS coating switched NPs ζ -potential from negative to positive, without modifying particle size distribution. Moreover, CS coating allowed a significant modulation of in vitro drug release, providing a ...
Abstract null null Retinal degeneration is a leading cause of visual impairment and blindness worldwide. Microglia reactivity is a hallmark of neurodegenerative diseases and a driving force for retinal cell death and disease progression. Thus, immunomodulation emerges as a potential therapeutic option. AhR deficiency is known to trigger inflammation and previous studies revealed important roles for AhR ligands in neuroprotection without focusing on microglia. Here, we investigate the anti-inflam...
Last. Alexa Klettner(CAU: University of Kiel)H-Index: 22
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Abstract For many retinal diseases, including age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR), the exact pathogenesis is still unclear. Moreover, the currently available therapeutic options are often unsatisfactory. Research designed to remedy this situation heavily relies on experimental animals. However, animal models often do not faithfully reproduce human disease and, currently, there is strong pressure from society to reduce animal research. Overall, this cre...
Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and...
Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury or disease have permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules. We achieved this by expressing the adaptor molecule Protrudin which enabled robust central nervous system regeneration both in vitro and in vivo in the injured adult optic nerve. Protrudin expression facilitated the accumulation of endoplasmic reticulum and ...
Last. Miriam Kolko(UCPH: University of Copenhagen)H-Index: 19
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Abstract Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l -lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released ...
Last. Ulrike Grünert(USYD: University of Sydney)H-Index: 49
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Purpose: Particle-mediated gene transfer has been used in animal models to study the morphology and connectivity of retinal ganglion cells. The aim of the present study was to apply this method to transfect ganglion cells in postmortem human retina. Methods: Postmortem human eyes from male and female donors aged 40 to 76 years old were obtained within 15 hours after death. In addition, two marmoset retinas were obtained immediately after death. Ganglion cells were transfected with an expression ...
Abstract Discovery ophthalmic research is centered around delineating the molecular and cellular basis of ocular diseases and finding and exploiting molecular and genetic pathways associated with them. From such studies it is possible to determine suitable intervention points to address the disease process and hopefully to discover therapeutics to treat them. An investigational new drug (IND) filing for a new small-molecule drug, peptide, antibody, genetic treatment, or a device with global heal...
Preclinical toxicity screening of the new retinal compounds is an absolute requirement in the pathway of further drug development. Since retinal neuron cultivation and in vivo studies are relatively expensive and time consuming, we aimed to create a fast and reproducible ex vivo system for retinal toxicity screening. For this purpose, we used rat retinal explant culture that was retrogradely labeled with the FluoroGold before the isolation. Explants were exposed to a toxic concentration of genta...
Last. Andrew White(USYD: University of Sydney)H-Index: 23
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For many years, oxidative stress arising from the ubiquitous production of reactive oxygen species (ROS) has been implicated in the pathogenesis of various eye diseases. While emerging research has provided some evidence of the important physiological role of ROS in normal cell function, disease may arise where the concentration of ROS exceeds and overwhelms the body’s natural defence against them. Additionally, ROS may induce genomic aberrations which affect cellular homoeostasis and may result...
ABSTRACTDiabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1−/− mouse retinal explants from...
