Circadian Rhythms in Murine Ocular Tissues Including Sclera Are Affected by Neurobasal A Medium Preincubation and Mouse Strain, but Not Sex

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1.

Sex does not affect circadian rhythms of PER2::LUC bioluminescence of ex vivo ocular tissues. Representative examples of bioluminescence rhythms in retina, PEC and cornea of (a–c) male and (d–f) female PER2::LUC mice bread on a C57BL/6J background. Animal sex did not significantly affect the following parameters of PER2::LUC bioluminescence rhythms: (g) period length, (h) amplitude, (i) acrophase, and (j) relative rhythmic power. The arrows indicate media change. Time in (i) is projected ZT with ZT12 = lights off. The gray rectangle represents subjective nighttime. Individual data points are plotted together with means ± SD. PEC, posterior eye cup. N = 4 to 18.

Figure 2.

Circadian rhythms of PER2::LUC bioluminescence in ex vivo ocular tissues. Representative examples of PER2::LUC rhythms in (a) the cornea, (b) the intact PECs, (c) the scraped PEC, and (d) the retinas of mice bred on a C57BL/6J background. The bioluminescence rhythms of scraped PEC show (e) longer periods and (f) lower amplitudes compared with other ocular tissues. (g) The first obvious peak of PER2::LUC bioluminescence occurred earlier within the 24-hour cycle in scraped PECs compared with other ocular tissues. (h) The bioluminescence rhythms of PECs oscillated with higher relative rhythmic power compared with the retinas. Time in (g) is projected ZT with ZT12 = lights off. The gray rectangle represents subjective nighttime. Individual data points are plotted together with means ± SD. Holm-Sidak's post hoc test comparison, *P < 0.05, **P < 0.01, ****P < 0.0001. PEC, posterior eye cup. N = 4 to 20.

Figure 3.

Immunohistochemistry of murine retina. Representative micrograph of BMAL1 and DAPI labeling in the murine (a) retina. Extraocular muscle fibers that show strong background labeling, are indicated by asterisks. (b) Zoomed-in view of BMAL1 and DAPI labeling in the sclera. Arrows indicate BMAL1 positive nuclei. The scale bar is 100 µm. INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Figure 4.

Mouse background influences circadian rhythms in ocular tissues. Representative examples of bioluminescence rhythms in the corneas, intact PECs, and scraped PECs obtained from PER2::LUC mice maintained on a (a–c) C57BL/6J background and, respectively, from a (d–f) RjOrl:SWISS background. Two-way ANOVA analysis was used to study the effects of background on (g) period length, (h) amplitude, (i) time of first peak, that is, acrophase, and (j) relative rhythmic power of PER2::LUC bioluminescence oscillations in the corneas, and intact and scraped PECs. Time in (i) is projected ZT with ZT12 = lights off. The gray rectangle represents subjective nighttime. Individual data points are plotted together with means ± SD. Holm-Sidak's post hoc test comparison, *P < 0.05, **P < 0.01, ****P < 0.0001. PEC, posterior eye cup. N = 3 to 28.

Figure 5.

Preincubation medium affects circadian rhythms of PER2::LUC bioluminescence of ex vivo ocular tissues. Ocular tissues were obtained from heterozygous C57BL/6J mPer2^Luc/+ mice. Representative examples of PER2::LUC bioluminescence rhythms in the retinas, PECs, and corneas that were preincubated for 24 hours with (a–c) M199 and (d–f) Neurobasal A medium. (g) Period length, (i) acrophase, and (j) relative rhythmic power in ocular tissues was affected by the choice of preincubation medium. Conversely, (h) amplitude of PER2::LUC bioluminescence rhythms in ocular tissues was not affected by the choice of preincubation medium. Time in (i) is projected ZT with ZT12 = lights off. The gray rectangle represents subjective nighttime. Individual data points are plotted together with means ± SD. Holm-Sidak's post hoc test comparison, *P < 0.05, ****P < 0.0001. PEC, posterior eye cup. N = 3.