Ana G. Duran - 30 April 2024
Zebrafish: A key asset in ophthalmology drug discovery
Readily assess visual function in a live organism
Advancements And Limitations in Ophthalmology Drug Discovery
A new era in ophthalmic research aiming at finding fundamental cures for inherited retinal diseases was initiated by the FDA's approval of Luxturna in December 2017. There are currently several ongoing clinical trials investigating various gene therapies for other ocular diseases, including neovascular age-related macular degeneration, retinitis pigmentosa, Usher syndrome and glaucoma [1].
Although such advancement has unlocked a long-standing lack of innovation in ocular drug development, there are still several limitations during early-drug discovery processes and how specific drugs are selected to continue into clinical development [2]. In particular, disease modelling and target identification are key initial steps in drug discovery that influence the probability of success at every step of drug development [3].
When it comes to disease modelling, there are important considerations around anatomical and pathophysiological differences between disease processes among species. Anatomical differences between human and rodent eyes, such as the rodent rod-dominated vision, as well as differences in the lens and vitreous cavity, are a significant limitation to the use and interpretation of rat and mouse models. Non-human primate models have a number of pathophysiological advantages but they are expensive and have limited availability. Therefore, newly developed models of eye disease may provide important insights into pathogenesis of disease, as well as, efficacy of potential therapeutic approaches.
Leveraging Zebrafish Eye Power
Benefits of Zebrafish in Ophthalmology Research
Zebrafish models, although phylogenetically more distant from humans than rodents, share 82% genetic homology in human disease-associated genes. Zebrafish represent an attractive model for ophthalmology research due to their cone-rich retina and the structural similarity to the human macula, which results in good colour vision and high-acuity vision [4]. In fact, zebrafish models of a variety of ocular diseases are currently available, including cone dystrophies, which are difficult to reproduce in nocturnal mammals [4]. Zebrafish have already shown their use in providing pre-clinical data prior to testing genetic therapies in clinical trials, such as antisense oligonucleotide therapy for Usher syndrome [5].
Understanding Zebrafish's Visual System
Similar to humans, the zebrafish retina consists of an outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). It also possesses the same broad classes of human retinal neurons, such as retinal ganglion cells (RGC), bipolar cells, horizontal cells, and amacrine cells, and the same glial elements including Müller cells, astrocytes, and microglia (Figure 1) [6]. In terms of visual function, zebrafish show visual responsiveness and adult human-like retinal morphology and function as early as 72 hours post fertilisation [7].
Figure 1. Cross-sectional histology of the human and zebrafish retina demonstrates similarities in the arrangement of cells and structural features that define the distinct retinal layers. Adapted from [6].
ZeClinics, your partner in ophthalmology research
Using zebrafish well-described behavioural features, ZeClinics scientists have established phenotypic-based tests to evaluate colour-vision, vision acuity, and perception of light with medium to high throughput.
Vision acuity | Visually guided hunting ability
A golden test is the prey consumption assay, which is designed to evaluate vision acuity. Visual acuity is a measure of the ability of the eye to distinguish shapes and the details of objects at a given distance. This test leverages the ability of zebrafish to hunt small prey such as paramecia or rotifers, a behaviour that predominantly relies on vision to be initiated and successfully completed [8].
In the prey capture assay visual information about prey location flows from the retina to two contralateral visual areas, and this detection of prey-like objects triggers the prey capture sequence. The prey capture behaviour is a highly stereotyped sensory-motor behaviour that consists of the following steps (Figure 2) [8,9]:
- Visual observation of target
- Eye converge and J-bend tail flicks to reorient body toward the prey
- Slow swim towards the prey
- Capture swim to suction the prey
Figure 2: Prey capture sequence. Adapted from [8].
The quantification of the consumption of rotifers is an indirect measure of this ability to visualise/detect the prey. In the dark, hunting events are rare, and appear to be initiated by physical contact with the prey rather than visual cues [10,11]. In addition, disruptions of visual signalling impair the larva's ability to hunt [12,13,14], further highlighting the central role played by vision.
In our genetic model of cone-dystrophy, the prey consumption test allowed us to functionally prove vision impairment. When incubated with rotifers, mutant larvae were unable to successfully perform hunting behaviour while wildtype siblings could efficiently detect, track and eat their prey. The quantification of the number of remaining rotifers confirmed that, in the same time period, mutants consumed a significantly lower number of prey. (Figure 3).
Figure 3. Prey consumption assay scheme. The remaining rotifers percentage measures larval hunting proficiency, thus indicating their visual acuity in prey detection. Impaired vision larvae exhibit diminished hunting capability, resulting in a higher percentage of remaining rotifers within the same timeframe.
In summary, the biological and physiological characteristics of the zebrafish, coupled with the availability of visually guided behavioural tests and ease of genetic manipulation, make the zebrafish an extremely attractive model for ophthalmic research. The number of applications range from ocular disease modelling, target identification to phenotypic-based safety and efficacy evaluation. Gene therapy and optogenetics constructs, antiangiogenic compounds, neuroprotective drugs, and potential ocular toxic compounds can be evaluated using this model.
REFERENCES
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By Ana G. Duran
Ana is a biotechnologist with expertise in gene and cell therapy. She holds a Master’s degree in Biomedical Sciences and a PhD in Regenerative Therapies from the Charité University in Berlin. Her PhD research was focused on the development of human induced pluripotent stem cell (hiPSC)-based strategies to regenerate human heart muscle after myocardial infarction. In 2022, she joined ZeClinics as a Sales and Account manager, acting as a liaison between customers and ZeClinics to help advance drug discovery and development using the zebrafish model.