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SYNCING SUCCESS: Elevating Data Quality in Preclinical Retinal Research with the Circadian Advantage

January 2024

Background

Circadian rhythms are commonly associated with our daily wake and sleep patterns, but their influence extends far beyond just sleep-wake cycles. These rhythms pervade almost every tissue and organ system, orchestrating various physiological functions, including sleep-wake cycles, hormone secretion, metabolism, and core body temperature. The circadian system, often referred to as the body's internal clock, is controlled by a cluster of cells in the brain called the suprachiasmatic nucleus (SCN) and is synchronised with the daily cycle through external cues like light. Somewhere between 10 and 15% of all gene transcription is rhythmic with as many as 50% of mammalian genes predicted to be rhythmically expressed in at least one tissue 1. The eye is no exception to this, with as many as 9% of genes expressed rhythmically in the retina 2. Circadian rhythms exert a profound impact on visual function and eye health, influencing genes associated with angiogenesis 3, inflammation 4, and responses to hypoxia 5, among others. These genetic changes from day to night have implications for clinical assessments of the eye, affecting vessel permeability, retinal thickness 6, and light responses 7.

Recognising the significance of circadian rhythms in vision research is crucial in unveiling the critical role they play not only in maintaining health but also in influencing various biological processes in disease. By considering circadian rhythms in experimental design, we enhance the rigor and reproducibility of research. In this article, we delve into the profound implications of circadian rhythms on ocular health, explore some of their impact on reproducible science, and reveal how we at MediNect Ophtho considers these insights for better and more reliable research solutions. 

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Many ocular outputs cycle each day due to changes driven either by light or circadian rhythms. Where a process does display circadian rhythmicity, these rhythms can often be changed in disease. Considering this in research might unveil changes between experimental groups that would otherwise be missed (a & b). Conversely, ignoring these changes might result in large variation within experimental groups. 

Visual performance throughout the day

Have you ever wondered why your vision seems sharper and clearer during certain times of the day while appearing blurred and strained at others?

Our vision from day to night is governed by two main players: cone-driven signalling which prioritises high visual acuity, and rod-driven signalling that amplifies scarce photons to boost sensitivity. The circadian clock orchestrates this by adjusting electrical photoreceptor coupling to anticipate daily light changes 8.

Circadian control of visual processing in the retina is reflected in electroretinograms (ERG) which measure the retinal response to light. Rhythms in ERG response were first shown only in light adapted ERG 7 and so considered to be driven by cone photoreceptors. Since then, rhythms have also been demonstrated in dark adapted ERG responses as well 9, and so the mechanisms involved have been reconsidered. More recently, the changes between day and night have been isolated to the mesopic range of light intensity only, when both rods and cones are at play 10. These rhythms likely come from circadian changes in gap junction coupling between rod and cone cells. Understanding these fluctuations is crucial for designing experiments that yield consistent and repeatable results, where factors such as lighting conditions and timing can significantly impact outcomes.

Intraocular pressure

Did you know that your eye pressure experiences daily rhythms, peaking during the early morning hours?

 

This phenomenon of circadian regulation influences intraocular pressure (IOP), a critical aspect of eye health. Several studies have revealed rhythmic fluctuations in IOP, with peak levels often observed during the early hours of the day 11 ,12This circadian regulation is through direct neural control via sympathetic innervation and also via hormonal control, specifically by melatonin 13. Glaucoma, a leading cause of irreversible blindness worldwide, is characterised by progressive damage to the optic nerve, often associated with increased IOP. Acknowledging IOP rhythms is invaluable in the development of glaucoma therapies, for example in considering the importance of timing when administering medications to target the peak IOP and optimise treatment efficacy.

 

As well as rhythms in visual processing and pressure, the response of the eye to injury also changes from day to night. The time of day plays a significant role in determining the extent of damage, with intense light exposure at night resulting in greater loss of photoreceptors 14. Studies in phototoxicity in rats suggest that retinoids are involved in retinal damage and the extent of light damage fluctuates alongside rhythms in RPE mediated phagocytosis, the process by which photoreceptor outer segments are internalised and digested by RPE cells. The mechanisms of changing susceptibility to light damage are complex and not yet fully understood. 

 

Also relevant to injury is the inflammatory response. It stands to reason that the very well documented circadian control of inflammatory responses around the body extend to the eye as well. Microglia exhibit rhythmic expression of circadian genes and associated changes in morphology and function 15 as well as daily changes in the amplitude of their response to immune stimuli 16. For example, microglia are more active and phagocytic during the day than at night.

 

Finally, the role of angiogenesis, the formation of new blood vessels, has emerged as a crucial player in many ocular diseases. It is now widely appreciated that the molecular clock, orchestrating our circadian rhythms, has a profound impact on angiogenesis, both in development and disease. Studies have shown that the expression of genes involved in angiogenesis, such as vascular endothelial growth factor (VEGF), exhibits a circadian rhythm in the retina and RPE 3,17. In addition, circadian disruption has been shown to impair angiogenesis in the retina and rhythmic environmental light is needed for normal vessel density 18. These findings suggest that circadian rhythms are important for regulating angiogenesis in the retina. Disruption of circadian rhythms may contribute to retinal diseases that involve angiogenesis, such as diabetic retinopathy and age-related macular degeneration.

While the effect of time of day on laser induced lesions commonly used for the study of neovascularisation and retinal fibrosis has not yet been investigated, both the inflammation and angiogenesis that characterise these lesions are under known circadian influence. It therefore stands to reason that circadian rhythms might influence retinal injury, and should be considered in the design of experimental trials that make use of injury to model disease at the very least to minimise variability.

Response to injury

Did you know that depending on the time of day, the response to eye injury changes?

Learn more about our retinal disease models

Are you integrating timing of drug delivery or retinal analysis into your research?

At MediNect Ophtho, we understand the critical role circadian rhythms play in vision and eye-related research. We diligently integrate this knowledge to optimise the timing of your therapeutic's delivery, ensuring maximum effectiveness. Furthermore, to minimise data variability, we consistently conduct ERG measurements, and other bioimaging techniques, at the same time each day and for every time point. This meticulous approach decreases variability and increases the reliability and accuracy of our results.

References

  1. Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E. & Hogenesch, J. B. A circadian gene expression atlas in mammals: implications for biology and medicine. Proceedings of the National Academy of Sciences of the United States of America 111 (2014). https://doi.org:10.1073/pnas.1408886111

  2. Silk, R. P. et al. Mapping the daily rhythmic transcriptome in the diabetic retina. BioRxiv (2023). https://doi.org:10.1101/2023.05.27.542572

  3. Jensen, L. D. et al. Opposing effects of circadian clock genes bmal1 and period2 in regulation of VEGF-dependent angiogenesis in developing zebrafish. Cell Reports 2, 231-241 (2012). https://doi.org:10.1016/j.celrep.2012.07.005

  4. Wang, Q. et al. Regulation of retinal inflammation by rhythmic expression of MiR-146a in diabetic retina. Investigative Ophthalmology & Visual Science 55, 3986-3994 (2014). https://doi.org:10.1167/iovs.13-13076

  5. Peek, C. B. et al. Circadian Clock Interaction with HIF1α Mediates Oxygenic Metabolism and Anaerobic Glycolysis in Skeletal Muscle. Cell Metabolism 25, 86-92 (2017). https://doi.org:10.1016/j.cmet.2016.09.010

  6. Kotsidis, S. T., Lake, S. S., Alexandridis, A. D., Ziakas, N. G. & Ekonomidis, P. K. 24-Hour variation of optical coherence tomography-measured retinal thickness in diabetic macular edema. European Journal of Ophthalmology 22 (2012). https://doi.org:10.5301/ejo.5000119

  7. Storch, K. F. et al. Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information. Cell 130, 730-741 (2007). https://doi.org:10.1016/j.cell.2007.06.045

  8. Cao, J., Ribelayga, C. P. & Mangel, S. C. A circadian clock in the retina regulates rod-cone gap junction coupling and neuronal light responses via activation of adenosine A 2A receptors. Frontiers in Cellular Neuroscience 14, 605067 (2021). https://doi.org:10.3389/fncel.2020.605067

  9. Di, R., Luo, Q., Mathew, D. & Bhatwadekar, A. D. Diabetes alters diurnal rhythm of electroretinogram in db/db mice. The Yale Journal of Biology and Medicine 92, 155-167 (2019).

  10. Allen, A.  Vol. 42   8795–8806 (Journal of Neuroscience, 2022).

  11. Aihara, M., Lindsey, J. D. & Weinreb, R. N. Twenty-four-hour pattern of mouse intraocular pressure. Experimental Eye Research 77 (2003). https://doi.org:10.1016/j.exer.2003.08.011

  12. Moore, C. G., Johnson, E. C. & Morrison, J. C. Circadian rhythm of intraocular pressure in the rat. Current Eye Research 15 (1996). https://doi.org:10.3109/02713689608997412

  13. Alcantara-Contreras, S., Baba, K. & Tosini, G. Removal of melatonin receptor type 1 increases intraocular pressure and retinal ganglion cells death in the mouse. Neuroscience Letters 494 (2011). https://doi.org:10.1016/j.neulet.2011.02.056

  14. Organisciak, D. T., Darrow, R. M., Barsalou, L., Kutty, R. K. & Wiggert, B. Circadian-dependent retinal light damage in rats. Investigative Ophthalmology & Visual Science 41 (2000).

  15. Wang, Z., Zeng, Y., Cui, Z., Chen, J. & Tang, S. The involvement of circadian rhythm gene REV-ERBα in regulation of ocular inflammation. Investigative Ophthalmology & Visual Science 61, 707-707 (2020).

  16. Fonken, L. K. et al. Microglia inflammatory responses are controlled by an intrinsic circadian clock. Brain, Behavior, and Immunity 45 (2015). https://doi.org:10.1016/j.bbi.2014.11.009

  17. Klettner, A., Kampers, M., Töbelmann, D., Roider, J. & Dittmar, M. The influence of melatonin and light on VEGF secretion in primary RPE cells. Biomolecules 11 (2021). https://doi.org:10.3390/biom11010114

  18. Jidigam, V. K. et al. Neuronal Bmal1 regulates retinal angiogenesis and neovascularization in mice. Communications Biology 5 (2022). https://doi.org:10.1038/s42003-022-03774-2

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