Dataset Information

In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells.

ABSTRACT: A defining characteristic of optic neuropathies, such as glaucoma, is progressive loss of retinal ganglion cells (RGCs). Current clinical tests only provide weak surrogates of RGC loss, but the possibility of optically visualizing RGCs and quantifying their rate of loss could represent a radical advance in the management of optic neuropathies. In this study we injected two different adeno-associated viral (AAV) vector serotypes in the vitreous to enable green fluorescent protein (GFP) labelling of RGCs in wild-type mice for in vivo and non-invasive imaging. GFP-labelled cells were detected by confocal scanning laser ophthalmoscopy 1-week post-injection and plateaued in density at 4 weeks. Immunohistochemical analysis 5-weeks post-injection revealed labelling specificity to RGCs to be significantly higher with the AAV2-DCX-GFP vector compared to the AAV2-CAG-GFP vector. There were no adverse functional or structural effects of the labelling method as determined with electroretinography and optical coherence tomography, respectively. The RGC-specific positive and negative scotopic threshold responses had similar amplitudes between control and experimental eyes, while inner retinal thickness was also unchanged after injection. As a positive control experiment, optic nerve transection resulted in a progressive loss of labelled RGCs. AAV vectors provide strong and long-lasting GFP labelling of RGCs without detectable adverse effects.

SUBMITTER: Smith CA

PROVIDER: S-EPMC5784170 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells.

Smith Corey A CA Chauhan Balwantray C BC

Scientific reports 20180124 1

A defining characteristic of optic neuropathies, such as glaucoma, is progressive loss of retinal ganglion cells (RGCs). Current clinical tests only provide weak surrogates of RGC loss, but the possibility of optically visualizing RGCs and quantifying their rate of loss could represent a radical advance in the management of optic neuropathies. In this study we injected two different adeno-associated viral (AAV) vector serotypes in the vitreous to enable green fluorescent protein (GFP) labelling ...[more]

PMID: 29367685

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Project description:RATIONALE:Somatic overexpression in mice using an adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrial-specific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes. OBJECTIVE:To establish the platform of atrial specific in vivo gene delivery by AAV-ANF. METHODS AND RESULTS:We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP (green fluorescent protein) exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue. In contrast, similar vectors containing a cardiac troponin T promoter (AAV9-TNT4-GFP) showed GFP expression in all 4 chambers of the heart, while AAV9 with an enhanced chicken ?-actin promoter (AAV-enCB-GFP) caused ubiquitous GFP expression. Next, we used Rosa26mT/mG (membrane-targeted tandem dimer Tomato/membrane-targeted GFP), a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato before Cre-mediated excision, and membrane-targeted GFP after excision. AAV9-ANF-Cre led to highly efficient LoxP recombination in membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein mice with high specificity for the atria. We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26mT/mG allele. AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. Finally, we assessed whether the AAV9-ANF-Cre vector could be used to induce atrial-specific gene knockdown in proof-of-principle experiments using conditional JPH2 (junctophilin-2) knockdown mice. Four weeks after AAV9-ANF-Cre injection, a strong reduction in atrial expression of JPH2 protein was observed. Furthermore, there was evidence for abnormal Ca2+ handling in atrial myocytes isolated from mice with atrial-restricted JPH2 deficiency. CONCLUSIONS:AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. This vector is a novel reagent for both mechanistic and gene therapy studies on atrial diseases.

Dataset Information

In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells.

Publications

In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells.

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