Transretinal electroretinography for preclinical drug and nanostructure testing
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School of Science |
G5 Artikkeliväitöskirja
| Defence date: 2025-04-17
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Authors
Date
2025
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Mcode
Degree programme
Language
en
Pages
109 + app. 87
Series
Aalto University publication series Doctoral Theses, 45/2025
Abstract
The retina, a complex neural tissue, converts light into electrical signals through phototransduction, making it a valuable model for studying neural mechanisms, drug effects, and retinal diseases. Its high sensitivity to physical and chemical disturbances allows photoreceptor signaling to serve as an effective tool for biocompatibility testing of potential drug molecules and nanostructures. Transretinal electroretinography (tERG), an ex vivo technique for recording extracellular field potentials across isolated retinae, provides a controlled platform for evaluating photoreceptor and bipolar cell functions. This method enables rapid assessment of drug effects by analyzing changes in response amplitude, kinetics, and light sensitivity. In the initial studies of this thesis, tERG was employed to evaluate and quantify the inhibition constants of CB-5083, a valosin-containing protein (VCP) inhibitor, and avanafil, a phosphodiesterase-5 (PDE5) inhibitor, on light-activated PDE6 in murine retinae. PDE6 is a critical enzyme in the G-protein signaling cascade in phototransduction, regulating cyclic GMP levels and controlling photoreceptor activity. G-protein-coupled pathways are significant targets for pharmacological intervention. CB-5083 clinical trials for cancer treatment were halted because of adverse visual effects, including photophobia and dyschromatopsia, which were believed to stem from the inhibition of PDE6. Our tERG analysis confirmed this hypothesis, revealing that CB-5083 inhibits PDE6 with an inhibition constant of 80 nM. Avanafil was also found to cause dose-dependent PDE6 inhibition, primarily affecting rod and cone photoreceptors by slowing their response kinetics. The inhibition constants for light-activated PDE6 were determined as 1.74 μM in rods and 6.3 μM in cones, indicating a weaker inhibitory effect compared to other PDE5 inhibitors such as sildenafil and zaprinast. No significant impact was observed on rod bipolar cell signaling, suggesting that avanafil's effects were primarily confined to photoreceptor function. The second phase of this research contained development of a closed-circulation tERG system for murine retinae to facilitate testing with minimal amounts of drug molecules or nanostructures. This system maintained retinal viability and functionality during extended experiments, with rod responses remaining stable for at least 42 hours and rod and cone bipolar cell responses lasting at least 12 hour closed-perfusion period. The setup was validated using IBMX, a non-specific PDE inhibitor, and demonstrated its utility in biocompatibility testing with zwitterionic polymers such as poly(sulfobetaine methacrylate) (PSMBA) and its monomer SBMA, both of which showed no adverse effects on rod and bipolar cell responses. This method proved effective for long-term assessments using small volumes of perfusate, providing a scalable and cost-efficient platform for preclinical studies. In the final stage, the small-volume closed-circulation tERG system was adapted for postmortem human retinal tissue. Donor eyecups, which had enucleation delays of one to four hours, successfully maintained steady rod photoreceptor responses throughout a 12-hour perfusion period. This method bridges preclinical research and clinical applications, reducing reliance on animal models and enhancing translational relevance. As a summary, this work establishes tERG as a powerful tool for preclinical research, offering detailed insights into drug-induced retinal effects and supporting the development of effective therapeutic strategies.Description
Supervising professor
Koskelainen, Ari, Prof., Aalto University, Department of Neuroscience and Biomedical Engineering, FinlandThesis advisor
Koskelainen, Ari, Prof., Aalto University, Department of Neuroscience and Biomedical Engineering, FinlandKeywords
electroretinography, closed-perfusion, phosphodiesterase, phototransduction, photoreceptor cell, rod, cone, preclinical testing, retina
Other note
Parts
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[Publication 1]: Leinonen Henri, Cheng Cheng, Pitkänen Marja, L. Sander Christopher, Zhang Jianye, Saeid Sama, Turunen Teemu, Shmara Alyaa, Weiss Lan, Ta Lac, Ton Timothy, Koskelainen Ari, D. Vargas Jesse, Kimonis Virginia, and Palczewski Krysztof. 2021. A p97/Valosin-Containing Protein Inhibitor Drug CB-5083 Has a Potent but Reversible Off-Target Effect on Phosphodiesterase-6. The Journal of Pharmacology and Experimental Therapeutics, volume 378, issue1, pages 31-41.
DOI: 10.1124/jpet.120.000486 View at publisher
- [Publication 2]: Saeid Sama, Vinberg Frans, and Koskelainen Ari. 2024. Impact of 2nd generation PDE 5 inhibitor, avanafil, on retinal function: studies from ex vivo ERG. Submitted.12/2024
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[Publication 3]: Saeid Sama, Pitkänen Marja, Ilonen Emma, Niskanen Jukka, Tenhu Heikki, Vinberg Frans, and Koskelainen Ari. 2024. Closed-perfusion transretinal ERG setup for preclinical drug and nanostructure testing. IEEE Transactions on Biomedica Engineering.
DOI: 10.1109/TBME.2024.3493616 View at publisher
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[Publication 4]: Becker Silke, Allen Jordan, L’Ecuyer Zia, Saeid Sama, Koskelainen Ari, and Vinberg Frans. 2024. Restoring and sustaining human postmortem retinal light responses with scalable methods for testing degenerative disease therapies. Submitted. 11/2024.
DOI: 10.1101/2024.11.04.621932 View at publisher