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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.
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.
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 contributing to retinal repair and regeneration3. Their dysfunction is implicated in various retinal diseases, including diabetic retinopathy4, age-related macular degeneration5, and ocular ischemic syndrome6. RGCs and Müller glia exhibit close interactions and interdependence; Müller glia provide essential support to RGCs, while RGC activity can influence Müller glia function3,7. Studying both RGCs and Müller glia is crucial for understanding retinal function and developing effective treatments for multiple retinal diseases.
Current assessments in retinal research primarily utilize techniques like optical coherence tomography (OCT) to measure the thickness of the retinal nerve fiber layer or the trajectories of axon bundles8,9. While these methods are invaluable for detecting RGC loss, they do not provide a detailed view of RGC morphology and glial cells due to limited resolution. Similarly, although advanced techniques like adaptive optics scanning laser ophthalmoscopy (AO-SLO) enable cellular-level imaging of RGCs, photoreceptors, and glial cells in the living human retina10, their technical complexity and limited accessibility confine their use primarily to specialized research settings. Given these constraints, there is an ongoing need for developing more accessible and reliable methods for the in-depth study of specific retinal cell populations in vivo.
Accordingly, this protocol aims to introduce an alternative imaging approach suited for research applications in retinal cells. It combines the power of AAV-mediated cell-type-specific labeling with the non-invasive nature of CSLO imaging. Adeno-associated viruses (AAVs) are versatile gene delivery vectors known for their low immunogenicity and ability to transduce a broad range of cell types, including both dividing and non-dividing cells11. This makes them ideal tools for targeting specific cell populations within the complex retinal environment. By utilizing AAV vectors with carefully selected serotypes and promoters, selective expression of fluorescent proteins can be achieved in multiple cell types of interest, such as RGCs and Müller glia. For example, AAV2 is known for its higher transduction efficiency in RGCs12,13, while AAV8 is markedly effective at targeting photoreceptors14, and AAV9 demonstrates strong transfection capabilities in Müller glia15, showing broad efficiency across various retinal cell layers. It is important to note that the effectiveness of AAV relies not only on the choice of serotype but also on the promoters, which dictate the intensity and cell specificity of transgene expression, underscoring the importance of careful selection to achieve optimal transduction.
For RGC labeling, this protocol employs AAV2 with the human synapsin (hSyn) promoter. AAV2 exhibits efficient transduction of RGCs following intravitreal injection13, and the hSyn promoter, a ubiquitous neuronal promoter, drives strong and specific transgene expression within these cells16. For Müller glia, the protocol utilizes AAV9 vectors driven by the GfaABC1D promoter17, which demonstrates strong transgene expression in these cells15. This targeted labeling approach enables researchers to distinguish these cells from the surrounding retinal tissue and track them over time, providing a basis for in vivo surveillance of retinal cells and their responses to bio-environmental changes.
Confocal scanning laser ophthalmoscopy (CSLO) is a non-invasive imaging technique that provides high-resolution images of the living retina, enabling real-time visualization of fluorescently labeled retinal cell populations18,19,20. A focused laser beam scans across the retina, capturing emitted light that passes through a pinhole to eliminate out-of-focus signals, resulting in sharper images with enhanced contrast. This protocol utilizes a Heidelberg Spectralis CSLO system, which has been widely used for retinal cell imaging in live animals, including studies visualizing transgenic-labeled RGCs21,22and microglia23. By employing the HRA CSLO unit with a 488 nm laser and appropriate filters, researchers can image fluorescently labeled RGCs or Müller glia in live animals following intravitreal injection of AAV vectors carrying fluorescent reporter genes. The longitudinal imaging protocol, with weekly sessions covering both central and peripheral retina, tracks changes over time. To prioritize animal welfare, the protocol utilizes the automatic eye-tracking system (ART) of the HRA CSLO unit, enabling precise image acquisition without the need for general anesthesia or contact lenses.
This protocol harnesses the combined power of AAV and CSLO to enable the longitudinal monitoring of specific retinal cell types in vivo. By pairing the cell-type specificity of AAV-mediated labeling with the non-invasive, high-resolution imaging capabilities of CSLO, this method allows researchers to study the dynamic changes in RGCs and Müller glia in response to various stimuli or interventions. These insights hold significant potential for informing the development of new diagnostic and therapeutic strategies for retinal diseases.
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
NOTE: For targeted transduction of RGCs, this protocol uses AAV2-hSyn-eGFP, which incorporates the hSyn promoter to drive robust expression of enhanced GFP. Müller glia is targeted using AAV9-GfaABC1D-eGFP. To achieve optimal transduction efficiency and robust transgene expression, a minimum AAV titer of 1 x 1012 viral genomes (vg)/mL is recommended for intravitreal injections in mice13.
2. Preparation of viral vectors and animals
3. Handling and intravitreal injection of viral vectors
4. In vivo imaging with CSLO
5. Image processing and analysis
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
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...
The authors have nothing to disclose.
This work was supported by a grant from the National Natural Science Foundation of China (82130029). Figure 2A and Figure 4A were created with BioRender.com.
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 |
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