Milicevic Nemanja, Sandu Cristina, Challet Etienne, Ihalainen Teemu O, Nymark Soile, Felder-Schmittbuhl Marie-Paule
Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
https://orcid.org/0000-0002-8062-7270.
Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):62. doi: 10.1167/iovs.66.6.62.
Our understanding of ocular clocks has been profoundly advanced by the development of real-time recording of bioluminescence of PER2-luciferase (PER2::LUC) knock-in mouse explants. However, the effect of sex, mouse strain, and culturing conditions on ocular clocks remains unknown. Here, we studied the role these variables play on PER2::LUC bioluminescence rhythms of ocular tissues: retinas, corneas, and posterior eye cups (PECs). We also tested the hypothesis that the sclera contains a circadian oscillator by using scraped PECs as a proxy.
Retinas, corneas, and intact and scraped PECs were obtained from male and female PER2::LUC knock-in mice maintained on either a pigmented C57BL/6J or albino RjOrl:SWISS background. PER2::LUC bioluminescence rhythms in ocular tissues were measured using a Lumicycle.
We compared PER2::LUC bioluminescence rhythms between ocular tissues and found that all ocular tissues oscillated, including the scraped PECs, which were previously not known to oscillate. The rhythms in scraped PECs had lower amplitudes, longer periods, and distinct acrophases compared with other ocular tissues. Immunolabeling revealed the presence of the protein product of the clock gene BMAL1 in the sclera. Ocular tissues of RjOrl:SWISS mice oscillated with higher amplitudes compared with the ones of C57BL/6J, with corneal rhythms being most affected by mouse strain. A 24-hour preincubation with Neurobasal A medium affected rhythms of ocular tissues, whereas sex differences were not detected for these rhythms.
We discovered a novel oscillator in the sclera. PER2::LUC bioluminescence rhythms in murine ocular tissues are affected by Neurobasal A medium preincubation mouse strain but not sex.
通过对PER2-荧光素酶（PER2::LUC）基因敲入小鼠外植体生物发光的实时记录，我们对眼内生物钟的理解有了极大的进展。然而，性别、小鼠品系和培养条件对眼内生物钟的影响仍然未知。在此，我们研究了这些变量对眼组织（视网膜、角膜和眼后杯（PEC））中PER2::LUC生物发光节律的作用。我们还通过使用刮取的PEC作为替代物来测试巩膜中存在昼夜节律振荡器的假设。
从维持在有色C57BL/6J或白化病RjOrl:SWISS背景下的雄性和雌性PER2::LUC基因敲入小鼠中获取视网膜、角膜以及完整和刮取的PEC。使用Lumicycle测量眼组织中的PER2::LUC生物发光节律。
我们比较了眼组织之间的PER2::LUC生物发光节律，发现所有眼组织都有振荡，包括刮取的PEC，而之前并不知道其会振荡。与其他眼组织相比，刮取的PEC中的节律振幅较低、周期较长且峰相位不同。免疫标记显示巩膜中存在生物钟基因BMAL1的蛋白质产物。与C57BL/6J小鼠相比，RjOrl:SWISS小鼠的眼组织振荡振幅更高，其中角膜节律受小鼠品系影响最大。用Neurobasal A培养基预孵育24小时会影响眼组织的节律，而未检测到这些节律存在性别差异。
我们在巩膜中发现了一种新的振荡器。小鼠眼组织中的PER2::LUC生物发光节律受Neurobasal A培养基预孵育、小鼠品系影响，但不受性别影响。
