Modeling Diabetic Retinopathy in Zebrafish: High-Throughput Screening Solutions

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus caused by prolonged hyperglycaemia, leading to progressive damage of the retinal vasculature and surrounding neural tissue. The disease is characterized by increased vascular permeability, capillary degeneration, inflammation, and, in advanced stages, pathological neovascularization that can result in irreversible vision loss. 

The anatomical organization of the zebrafish eye enables direct in vivo analysis of retinal vascular and neuronal alterations associated with diabetic retinopathy. The retina displays a conserved layered structure and a functional neurovascular unit comparable to that of humans (Figure 1), allowing investigation of blood-retinal barrier disruption, vascular permeability, and inflammatory responses under hyperglycaemic conditions. In addition, optical transparency during early development permits high-resolution visualization of retinal vessels, supporting quantitative phenotypic assessment and high-throughput drug screening in preclinical diabetic retinopathy studies.

Figure 1. Anatomy of the zebrafish retina: Left: 4X magnification of a paraffin cut of the zebrafish retina, periodic-acid Schiff’s (PAS) stain. Right: 20X magnification of the zebrafish retina with a schematic overview of the different cell types. Scale bar = 100µm. Source: Middel CS, Hammes H-P, Kroll J. Advancing Diabetic Retinopathy Research: Analysis of the Neurovascular Unit in Zebrafish. Cells. 2021; 10(6):1313. 

Diabetic Retinopathy in Zebrafish: Induction Protocols and Pathway Targets

Several experimental strategies have been established to induce diabetic retinopathy-like phenotypes in zebrafish:

Hyperglycaemia by glucose immersion
One of the most widely used approaches consists of exposing zebrafish embryos or larvae to high-glucose solutions. Immersion in glucose concentrations from early developmental stages induces hyperglycaemic conditions that lead to retinal vascular alterations, including increased hyaloid and retinal vessel diameter (Figure 2), blood-retinal barrier disruption, and inflammatory responses. This method reproducibly activates key diabetic retinopathy-associated pathways such as VEGF upregulation and pro-inflammatory cytokine signaling.

Figure 2. (A) Average diameter and fluorescence images of retinal vessel morphologies in zebrafish Tg (flk: EGFP) at 6 dpf seen by fluorescence microscope (Leica DM 2500). Scale bar = 100 μm. (B) Retinal vessel diameters according to glucose concentrations. The mean ± SD (n = 40 in each group). **, *** and ****: statistically significant compared with normal group (p < 0.01, p < 0.001, and p < 0.0001). Adapted from: Lee Y, Yang J. Development of a zebrafish screening model for diabetic retinopathy induced by hyperglycemia: Reproducibility verification in animal model. Biomed Pharmacother. 2021 Mar;135:111201.

One of the main advantages of glucose-soaked zebrafish models is their rapid establishment. Following glucose exposure starting at 3 days post-fertilization (dpf), retinal vascular and inflammatory alterations characteristic of early diabetic retinopathy can be detected within a few days, typically by 5-6 dpf, enabling fast experimental turnaround for early-stage studies.

Hypoxia-induced retinal angiogenesis
Experimental hypoxia has been used to model proliferative aspects of diabetic retinopathy in zebrafish. Controlled reduction of oxygen levels induces retinal neovascularization through VEGF-dependent mechanisms. Importantly, this angiogenic response can be pharmacologically inhibited by anti-VEGF compounds, demonstrating that the pathway is VEGF-dependent, as it is in mammals, and is suitable for therapeutic testing.

Pharmacological induction of diabetes
In adult zebrafish, systemic hyperglycaemia has been induced using compounds such as streptozotocin (STZ), which disrupts pancreatic β-cell function. STZ-induced diabetic retinopathy in zebrafish results in sustained elevated glucose levels and retinal alterations, including thinning of retinal layers and vascular dysfunction, although increased mortality and variability have been reported.

Genetic models affecting glucose homeostasis
Targeted genetic manipulation of key metabolic regulators, including genes involved in pancreatic development and glucose metabolism, has generated zebrafish lines that exhibit chronic hyperglycaemia and retinal vascular phenotypes. Genetic models of DR enable mechanistic investigation of molecular pathways, particularly those linked to VEGF signaling, oxidative stress, and neurovascular unit dysfunction.

At the molecular level, hyperglycaemia activates pathways closely associated with human DR pathogenesis. Increased expression of vascular endothelial growth factor (VEGF), pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, and activation of STAT3 signaling have all been documented in zebrafish diabetic retinopathy models. These changes are accompanied by disruption of tight junction proteins, including ZO-1, contributing to blood-retinal barrier dysfunction and increased vascular permeability. 

Quantifying Retinal Damage: Readouts and Phenotypic Assays in Zebrafish

Zebrafish retain a highly conserved neurovascular unit, composed of endothelial cells, pericytes, glial cells, and neurons, mirroring key aspects of mammalian retinal organization. This enables integrated assessment of vascular, inflammatory, and neuronal phenotypes within a single in vivo system.

A key advantage of zebrafish models lies in their transparent bodies in early developmental stages. It allows to obtain robust, quantifiable retinal readouts. Vascular phenotyping is commonly performed by measuring hyaloid and retinal vessel diameter using fluorescence microscopy, particularly in transgenic lines expressing endothelial reporters (Figure 2). Hyperglycaemia induces a dose-dependent increase in vessel thickness, providing a sensitive and reproducible morphological endpoint for disease modeling and therapeutic evaluation. 

Histological and molecular analyses further enable characterization of retinal damage. Hematoxylin and eosin staining reveals structural alterations across retinal layers, while immunofluorescence assays detect changes in VEGF, GFAP, and tight junction proteins. Apoptotic activity within retinal tissues can be quantified using TUNEL assays, linking vascular dysfunction to neuronal injury. At the transcriptional level, RT-PCR analysis of whole-eye samples allows monitoring of inflammatory and angiogenic gene expression in response to hyperglycaemia or pharmacological intervention.

Zebrafish in High-Throughput Drug Discovery for Diabetic Retinopathy

Zebrafish models are particularly well-suited for high-throughput drug discovery in diabetic retinopathy. Their small size and compatibility with multi-well plate formats enable parallel testing of multiple compounds at different concentrations, while early-stage embryos allow rapid assessment of toxicity, efficacy, and mechanism of action.

Hyperglycaemia-induced zebrafish DR models have been successfully used to evaluate clinically relevant therapies such as aflibercept, which reduces retinal vessel diameter, suppresses inflammatory marker expression, and restores vascular integrity at non-toxic doses (Li and Yang, 2021). These responses closely parallel therapeutic effects observed in mammalian systems and clinical practice, validating zebrafish as a translational screening platform. 

More recent studies conducted have expanded screening applications to novel therapeutic candidates, including peptides with vascular-protective properties. In zebrafish DR models, selected peptide candidates demonstrated favorable safety profiles and efficacy in reducing retinal vessel thickness, accompanied by downregulation of VEGF and modulation of Angiopoietin/Tie2 signaling pathways (Lee, Cha and Yang, 2025). These findings highlight the capacity of zebrafish to support early-stage identification and prioritization of drug candidates targeting non-proliferative diabetic retinopathy before moving to costly mammalian models. 

Limitations and Considerations When Modeling Diabetic Retinopathy in Zebrafish

Despite their advantages, zebrafish models present specific limitations that must be considered when interpreting results. Notably, zebrafish lack intraretinal vasculature, with retinal vessels confined to the surface of the inner limiting membrane. As a result, certain aspects of proliferative diabetic retinopathy, such as intraretinal neovascular invasion, cannot be fully recapitulated.

Additionally, some hyperglycaemia-induced phenotypes may be influenced by osmotic stress, underscoring the importance of appropriate controls and experimental validation. Differences in disease chronicity between zebrafish and human DR also necessitate cautious extrapolation to late-stage pathology.

Nevertheless, when used as part of an integrated preclinical strategy, zebrafish provide a powerful, fast, and efficient platform for mechanistic studies and high-throughput drug screening. Their ability to model early vascular and inflammatory events makes them particularly valuable for accelerating therapeutic discovery in diabetic retinopathy.

At ZeClinics, we boost the potential of zebrafish models. We provide a preclinical CRO service to evaluate drugs targeting ocular disorders such as diabetic retinopathy. Our ophthalmic diseases experts are ready to partner with you at the early stages of drug and target discovery, providing innovative zebrafish in vivo models to shorten your product’s time to market.

We have experience in genetic manipulation, disease model generation, and phenotype-based drug screening. 

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Sources

Lee Y, Cha Y, & Yang J. Screening and evaluation of therapeutic candidates with vascular protective effects in zebrafish models of diabetic retinopathy. Sci Rep. 2025; 15:35946. doi: 10.1038/s41598-025-20272-7.

Lee Y, Yang J. Development of a zebrafish screening model for diabetic retinopathy induced by hyperglycemia: Reproducibility verification in animal model. Biomed Pharmacother. 2021 Mar;135:111201. doi: 10.1016/j.biopha.2020.111201. 

Middel CS, Hammes H-P, Kroll J. Advancing Diabetic Retinopathy Research: Analysis of the Neurovascular Unit in Zebrafish. Cells. 2021; 10(6):1313. doi:10.3390/cells10061313

By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.