Circadian Flux of Extravascular Polymorphonuclear Leukocytes in the Mouse Limbus

Purpose: In the uninjured mouse cornea, a recent study suggests extravascular PMNs are found at the limbus and their numbers rise and fall in a circadian rhythm, peaking in the evening. Additional studies in other tissues suggest metabolic syndrome resulting from the consumption of a HFD is associated with circadian disturbances. The studies outlined in this thesis were designed to test the hypothesis that the circadian accumulation of PMNs at the limbus is depressed by consumption of a HFD. Aim 1 will determine if there is a circadian flux of PMN extravasation at the limbus in the uninjured cornea, and Aim 2 will determine if consumption of a HFD affects PMN extravasation at the limbus. Methods: C57BL/6 male mice (7 weeks of age; n=65 total) were fed a normal diet (ND) or a HFD. Some mice were euthanized at 3h time intervals spanning a 24h period while other mice were euthanized at one of two time-points, evening (9 pm or 11 pm) or morning (11 am or 12 pm). Excised corneas were immunostained for PMNs and blood vessels. Using a DeltaVision fluorescence microscope, the limbal area for each cornea was measured and the total number of extravascular limbal PMNs was determined. Data are shown as mean ± SD and statistical significance was set at p≤0.05. Results: PMN infiltration at the limbus followed a circadian pattern and PMN numbers were ~30% higher in the evening (p≤0.05), but only after pooling the data to increase the sample size (n=20). Pooling was not possible for the HFD studies (n=5) and PMN counts were not significantly difference between mice fed a ND or a HFD. Conclusion: Collectively, the data show a large sample size is needed to evaluate PMN accumulation at the limbus. PMNs show a significant peak accumulation in the evening, but statistical significance is only evident after pooling the data to increase sample size (n=20). Pooling was not possible for the HFD study and no firm conclusions can be drawn because the study was underpowered by virtue of its smaller sample size (n=5).