Dissociation between Corneal and Cardiometabolic Changes in Response to a Time-Restricted Feeding of a High Fat Diet

Abstract

Mice fed a high fat diet (HFD) ab libitum show corneal dysregulation, as evidenced by decreased sensitivity and impaired wound healing. Time-restricted (TR) feeding can effectively mitigate the cardiometabolic effects of an HFD. To determine if TR feeding attenuates HFD-induced corneal dysregulation, this study evaluated 6-week-old C57BL/6 mice fed an ad libitum normal diet (ND), an ad libitum HFD, or a time-restricted (TR) HFD for 10 days. Corneal sensitivity was measured using a Cochet-Bonnet aesthesiometer. A corneal epithelial abrasion wound was created, and wound closure was monitored for 30 h. Neutrophil and platelet recruitment were assessed by immunofluorescence microscopy. TR HFD fed mice gained less weight (p < 0.0001), had less visceral fat (p = 0.015), and had reduced numbers of adipose tissue macrophages and T cells (p < 0.05) compared to ad libitum HFD fed mice. Corneal sensitivity was reduced in ad libitum HFD and TR HFD fed mice compared to ad libitum ND fed mice (p < 0.0001). Following epithelial abrasion, corneal wound closure was delayed (~6 h), and neutrophil and platelet recruitment was dysregulated similarly in ad libitum and TR HFD fed mice. TR HFD feeding appears to mitigate adipose tissue inflammation and adiposity, while the cornea remains sensitive to the pathologic effects of HFD feeding.

Keywords: corneal dysregulation; high fat diet; neutrophils; obesity; platelets; time-restricted feeding.

Figure 1

Time-restricted feeding attenuated body weight gain and adiposity. (A) Schematic representation of the different feeding regimens. TR HFD fed mice were allowed access to food from 8 p.m. (ZT13) through 4 a.m. (ZT21). (B) Caloric intake in ND, ad libitum HFD, and TR HFD fed mice. Left: average daily caloric intake. Right: Cumulative average caloric intake over the 10-day experimental feeding period. (C) Body weight gain and (D) visceral adiposity (eAT) in ND, ad libitum HFD, and TR HFD fed mice. ND—normal diet; TR—time-restricted; HFD—high fat diet; n.s.—not significant; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 2

Time-restricted feeding did not attenuate reduced corneal sensitivity and impaired corneal wound healing. (A) Corneal sensitivity in 10 day ND fed mice compared to ad libitum and TR HFD fed mice (n ≥ 6 per group). (B) Corneal wound closure in mice fed an ad libitum ND, an ad libitum HFD and a TR HFD. Left: Wound closure kinetics indicating complete wound closure by 24 h in ad libitum ND fed mice, while the wound remained open up to 30 h in the ad libitum HFD and TR HFD fed mice. Right: Representative images of the epithelial wound immediately after wounding, 12 h, 18 h, 24 h and 30 h after wounding. (C) Left: Dividing basal epithelial cells in each region of the cornea. Right: Sum of dividing basal epithelial cells in four fields of view from the paralimbus to the center of the cornea at 30 h after wounding (n ≥ 6 per group). Data expressed as means ± SD. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (ad libitum HFD compared to ad libitum ND); †† p ≤ 0.01, ††† p ≤ 0.001, †††† p ≤ 0.0001 (TR HFD compared to ND).

Figure 3

Inflammatory response of the cornea to abrasion. (A) The distribution of neutrophils over the limbal (L), paralimbal (PL), parawound (PW), paracentral (PC) and central (C) regions of the corneal stroma at 30 h after wounding (n ≥ 6). (B) Data showing platelet counts at the limbus 30 h after wounding (n ≥ 6). (C) Representative images of the abraded cornea of ad libitum ND, ad libitum HFD, and TR HFD 30 h after abrasion, showing extravasated neutrophils (green, anti-Ly-6G) and vasculature (red, anti-CD31) at the limbus. (D) Representative images of the abraded cornea of ad libitum ND, ad libitum HFD and TR HFD 30 h after abrasion, showing extravasated platelets (green, anti-CD41) and vasculature at the limbus. Data expressed as mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 (ad libitum HFD compared to ad libitum ND); †† p ≤ 0.01 and †††† p ≤ 0.0001 (TR HFD compared to ad libitum ND). Scale bars: C = 34 µm; D = 35 µm.