Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

Summary

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

Abstract

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and Müller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

Introduction

Being the only optically accessible part of the central nervous system, the retina serves as a valuable model for neuroscience research¹. Retinal ganglion cells (RGCs), the output neurons of the retina that transmit visual information to the brain, play a crucial role in visual function. Their loss or dysfunction leads to vision impairment and irreversible blindness, as seen in glaucoma and other optic neuropathies². Müller glia, the principal glial cells in the retina, are essential for maintaining retinal homeostasis, providing structural and metabolic support to neurons, regulating neurotransmitter levels, and co....

Protocol

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing. Four-week-old adult male C57BL/6J mice (weighing between 15-20 g) were used for all experiments and housed in temperature-controlled rooms with a 12/12-h light/dark cycle. Standard rodent chow and water were available ad libitum. The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. AAV-mediated retinal cell transfection

Results

Following the presented protocol, different retinal cells were successfully visualized and tracked in vivo using a combination of AAV-mediated gene delivery and CSLO. AAV2-hSyn-eGFP effectively transduced RGCs, resulting in robust eGFP expression throughout the retina, as confirmed by CSLO and colocalization with the RGC-specific marker, RNA binding protein with multiple splicing (RBPMS), specifically found in the ganglion cell layer (Figure 2 and Figure 3

Discussion

The presented protocol details a robust and accessible method for in vivo surveillance of specific retinal cell populations, harnessing the power of both AAV-mediated gene delivery and CSLO imaging. This approach offers several advantages over traditional methods, facilitating longitudinal studies of retinal cell dynamics and their responses to injury or disease under physiological or pathological conditions.

The success of this method hinges on several critical steps. Firstly, achiev.......

Materials

Name	Company	Catalog Number	Comments
33 Gauge Needle	Hamilton Corp., Reno, NV, USA	7803-05	For intravitreal injection
0.5% proparacaine	Santen Pharmaceutical Co., Ltd.		Topical Aneasthetics
AAV2-hSyn-eGFP	OBiO Technology Corp., China		Virus titer: 2.7 x 1012 viral genomes (vg)/mL
AAV9-GfaABC1D-eGFP	WZ Biosciences Inc., China		Virus titer: 4.5 x 1012 viral genomes (vg)/mL
Betadine	Healthy medical company	001651	Topical Antiseptics
Corneal scelar forceps (toothed)	Mingren Eye Instruments, China	MR-F301A	For eyelid secure during intravitreal injection
Dumont 05# forceps	FST	51-AGT5385	For optic nerve crush
Graphpad prism	GraphPad Prism, USA		Graph drawing and statistical analysis
HRA Spectralis	Heidelberg Engineering, GmbH, Dossenheim, Germany		"IR" and "FA" mode for CSLO imaging
Image J/Fiji	National Institutes of Health, USA		Image processing
Maxitrol antibiotic ointment	Alcon Laboratories, INC. USA	0065-0631	Topical antibiotics
Microliter Syringe	Hamilton Corp., Reno, NV, USA	7633-01	For intravitreal injection
Mydrin-P Ophthalmic solution	Santen Pharmaceutical Co.,Ltd, Japan		Pupil dilation
Ophthalmic surgical microscope	Leica AG, Heerbrugg, Switzerland	M220	For surgical operations
Pentorbarbitol Sodium	Sigma Aldrich, USA	57-33-0	Genereal Aneasthetics
Powerpoint	Microsoft Corporation, USA		Image alignment and cropping
VISCOTEARS Liquid Gel (Carbomer)	Dr. Gerhard Mann, Chem.-Pharm. Fabrik, Germany		Topical lubricant