Simple RGC: ImageJ Plugins for Counting Retinal Ganglion Cells and
                        Determining the Transduction Efficiency of Viral Vectors in Retinal
                        Wholemounts

Cross, Tiger; Navarange, Rasika; Son, Joon-Ho; Burr, William W.; Singh, Arjun; Zhang, Kelvin; Rusu, Miruna; Gkoutzis, Konstantinos; Osborne, Andrew; Nieuwenhuis, Bart

doi:10.5334/jors.342

Cited by 10 publications

(6 citation statements)

References 16 publications

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“…The quantification of the expression efficiency of viral vectors and the transgene expression levels in retinal wholemounts was analysed using the ImageJ plugins “RGC Transduction” and “RGC batch” that are part of “Simple RGC” (version 1.1.0) [ 51 ]. This software identified co-localisation between Brn3a-postive cells (RGCs) and eGFP-positive cells (transduced cells) to specifically identify transduced RGCs.…”

Section: Methodsmentioning

confidence: 99%

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

et al. 2023

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Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

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Section: Methodsmentioning

confidence: 99%

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

et al. 2023

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“…Briefly, retinas were excised from the eye cup, flattened and stained with the RGC specific nuclei marker BRN3A (Santa Cruz, sc-31984, RRID: AB_2167511 , 1:200). Images were captured, blindly, from eight regions per retina using a 20 X objective, and automatically quantified with the Fiji plugin ‘Simple RGC’ ( Cross et al, 2021 ). The representative images shown were acquired on a confocal microscope (Leica Microsystems) using a 40 X objective.…”

Section: Methodsmentioning

confidence: 99%

Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Loreto

Angeletti

et al. 2021

eLife

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Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the nicotinamide adenine dinucleotide (NAD)-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet know is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.

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“…Automated RGC counting was then performed using SimpleRGC, a FIJI plugin. 76 Finally, RGC counts at the central, midperipheral, and peripheral retina were obtained by averaging that respectively counts from each quadrant.…”

Section: Methodsmentioning

confidence: 99%

Long-Term Effects on Retinal Structure and Function in a Mouse Endothelin-1 Model of Retinal Ganglion Cell Degeneration

Lakshmanan¹,

Wong²,

Chan

2023

Invest. Ophthalmol. Vis. Sci.

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Purpose To study the long-term effects of endothelin-1 (ET-1)–induced retinal pathologies in mouse, using clinically relevant tools. Methods Adult C57BL/6 mice (7–9 weeks old) were intravitreally injected with PBS ( n = 10) or 0.25 ( n = 8), 0.5 ( n = 8), or 1 nmol ET-1 ( n = 9) and examined using electroretinogram, optical coherence tomography (OCT), and Doppler OCT at baseline and postinjection days 10, 28, and 56. Retinal ganglion cell (RGC) survival in retinal whole mount was quantified at days 28 and 56. Results ET-1 induced immediate retinal arterial constriction. The significantly reduced total blood flow and positive scotopic threshold response in the 0.5- and 1-nmol ET-1 groups at day 10 were recovered at day 28. A-wave magnitude was also significantly reduced at days 10 and 28. While a comparable and significant reduction in retinal nerve fiber layer thickness was detected in all ET-1 groups at day 56, the 1-nmol group was the earliest to develop such change at day 28. All ET-1 groups showed a transient inner retinal layer thinning at days 10 and 28 and a plateaued outer layer thickness at days 10 to 56. The 1-nmol group showed a significant RGC loss over all retinal locations examined at day 28 as compared with PBS control. As for the lower-dosage groups, significant RGC density loss at central and midperipheral retina was detected at day 56 when compared with day 28. Conclusions ET-1 injection in mice resulted in a transient vascular constriction and reduction in retinal functions, as well as a gradual loss of retinal nerve fiber layer and RGC in a dose-dependent manner.

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Simple RGC: ImageJ Plugins for Counting Retinal Ganglion Cells and Determining the Transduction Efficiency of Viral Vectors in Retinal Wholemounts

Cited by 10 publications

References 16 publications

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Long-Term Effects on Retinal Structure and Function in a Mouse Endothelin-1 Model of Retinal Ganglion Cell Degeneration

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