Retinal Degeneration Model
Dry Age-related Macular Disease (Dry-AMD) is a common eye disorder leading to a gradual reduction of vision, characterized by progressive loss of the photoreceptor cell population in the retina.
Given the high anatomic and functional conservation between human and zebrafish retina, adult zebrafish are very suitable to model human eye disorders.
We offer a comprehensive and biologically translatable method to study human retinal degeneration pathogenesis and evaluate the efficacy of Dry-AMD therapies.
Applications
- Study retinal degeneration and regeneration processes.
- High throughput screening of compounds with potential capacity to protect from degeneration or enhance regeneration of photoreceptors in the retina.
- Study the molecular and physiological mechanisms of underlying response to compounds in Dry-AMD model.
Advantages
Zebrafish and human retina are highly conserved both anatomically and functionally.
The fast development of the eye.
Zebrafish retina has the long-life ability to regenerate upon injury, allowing screening of compounds potentially accelerating tissue recovery.
Transparency of the model.
Method description
The assay is divided into different phases. The order of the phases depends on the aim of the experiment.
Neural retina protection assay
For testing the compound’s capacity to protect from degeneration, the order of phases is the following:
- Adaptation of adult fish in complete darkness
- Intravitreal injection of the compound of interest
- Constant light exposure (60 hours) for inducing retinal damage (Light-Induced Degeneration, LID)
Neural retina regeneration assay
For testing pro-regenerative compounds, the order is the following:
- Adaptation of adult fish in complete darkness
- Constant light exposure (60 hours) for inducing retinal damage (Light-Induced Degeneration, LID)
- Intravitreal injection of the compound of interest
In both cases, treated and control eyes are enucleated and fixed. Cryosections of the eyes are immuno-stained against a photoreceptor marker to evaluate this cell population. Imaging is done by confocal microscopy, and analysis is performed through a custom image analysis software that allows process automation and unbiased analysis between samples.
Figure 1. Neural retina protection assay: retinal cryosections of adult zebrafish eyes immunostained for a photoreceptor marker (red) and nucleus marker (blue). A) NO LID: fish not exposed to constant light (without retinal damage); B) DAMAGED: fish exposed to 60 h LID; C) NEGATIVE CONTROL: fish exposed to 60 h LID upon injection with the vehicle; D) RESCUED: fish exposed to 60 h LID upon injection with an active compound that protects from regeneration. The white bar shows the thickness of the outer nuclear layer (ONL). INL: inner nuclear layer.
Readouts
- The thickness and cell number of the Outer Nuclear Layer (ONL), the retinal layer populated by rod and cone photoreceptors.
Figure 2. Neural retina protection assay: ONL cell thickness and cell number evaluation on four different conditions. NO LID, fish not exposed to constant light (without retinal damage); DAMAGED, fish exposed to 60 h LID; NEGATIVE CONTROL, fish exposed to 60 h LID upon injection with the vehicle; TEST COMPOUND, fish exposed to 60 h LID upon injection with an active compound that protects from regeneration.
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References
- Cocchiaro P, Di Donato V, Rubbini D, Mastropasqua R, Allegretti M, Mantelli F, Aramini A, Brandolini L. Intravitreal Administration of rhNGF Enhances Regenerative Processes in a Zebrafish Model of Retinal Degeneration. Front Pharmacol. 2022 Mar 7;13:822359.
- Di Donato V, De Santis F, Albadri S, Auer TO, Duroure K, Charpentier M, Concordet JP, Gebhardt C, Del Bene F. An Attractive Reelin Gradient Establishes Synaptic Lamination in the Vertebrate Visual System. Neuron. 2018 Mar 7;97(5):1049-1062.e6.