A Low-Fat/Sucrose Diet Rich in Complex Carbohydrates Reverses High-Fat/Sucrose Diet-Induced Corneal Dysregulation

Abstract

High-fat/sucrose diet feeding in mice causes loss of corneal nerve function and impairs corneal wound healing. While changing to a diet with a low fat/sugar composition and enrichments in complex carbohydrates mitigates the reduction in nerve function, it remains to be determined if it has an effect on corneal wound healing. In this study, 6-week-old C57BL/6 male mice were fed either a normal diet or a high-fat/sucrose diet for 20 weeks. A third group (diet reversal) was placed on a high-fat/sucrose diet for 10 weeks followed by a normal diet for an additional 10 weeks. A central corneal epithelial abrasion wound was created, and wound closure was monitored. Neutrophil and platelet recruitment was assessed by immunofluorescence microscopy. Mice fed the high-fat/sucrose diet-only had greater adiposity (p < 0.005) than normal diet-only fed mice; diet reversal markedly reduced adiposity. Following corneal abrasion, wound closure was delayed by ~6 h (p ≤ 0.01) and, at 30 h post-wounding, fewer neutrophils reached the wound center and fewer extravascular platelets were present at the limbus (p < 0.05). Diet restored normal wound closure and neutrophil and platelet influx in the injured cornea. These data suggest compositional changes to the diet may be an effective diet-based therapeutic strategy for maintaining or restoring corneal health.

Keywords: cornea; high-fat/sucrose diet; macronutrient; neutrophils; obesity; platelets.

Figure 1

High-fat/sucrose diet feeding for 10 weeks causes significant adiposity and liver weight gain. (A) Cumulative weight change (B) Epididymal adipose tissue weight, (C) body adiposity index, and (D) liver weight in mice fed a normal diet or high-fat/sucrose diet (n ≥ 6 per group). Data expressed as means ± SD. ND₁₀—10-week normal diet; HFSD₁₀—10-week high-fat/sucrose diet; *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2

High-fat/sucrose diet feeding for 10 weeks causes dysregulated insulin metabolism. (A) Fasting blood glucose, (B) fasting plasma insulin, (C) insulin resistance, (D) insulin sensitivity, and (E) beta cell function in mice fed a normal diet or high-fat/sucrose diet for 10 weeks (n ≥ 6 per group). Data expressed as means ± SD. ND₁₀—10-week normal diet; HFSD₁₀—10-week high-fat/sucrose diet; * p ≤ 0.05.

Figure 3

High-fat/sucrose diet feeding for 10 weeks impairs corneal wound healing. (A) Corneal wound closure in mice fed a normal or high-fat/sucrose diet. Top: Wound closure kinetics. Bottom: Representative images of the epithelial wound immediately after wounding, 12 h, 18 h, 24 h, and 30 h after wounding. (B) 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 periphery to the center of the cornea & (C) Basal epithelial cell density in each region of the cornea at 30 h after wounding. Data expressed as mean ± S.D. ND₁₀—10-week normal diet; HFSD₁₀—10-week high-fat/sucrose diet; L—limbus; PL—paralimbus/periphery; PW—parawound; PC—paracentral; C—center; ** p ≤ 0.01.

Figure 4

High-fat/sucrose diet feeding for 10 weeks dysregulates the inflammatory response of the cornea to abrasion when measured 30 h after wounding. (A) Left: The distribution of neutrophils over the limbal and central regions of the corneal stroma. Right: Sum of neutrophils in four fields of view from the periphery to the center of the cornea (n ≥ 6), (B) Platelet counts at the limbus (n ≥ 6). (C) Venule diameter at the corneal limbus of mice fed the normal or high-fat/sucrose diet. Data expressed as means ± SD. ND₁₀—10-week normal diet; HFSD₁₀—10-week high fat diet; n.s.—not significant; * p ≤ 0.05, *** p ≤ 0.001.

Figure 5

Diet reversal attenuates adiposity and liver weight gain. Mice were fed for a total of 20 weeks. (A) Cumulative weight change (B) Epididymal adipose tissue weight, (C) body adiposity index, and (D) liver weight in mice fed only the normal diet, only the high-fat/sucrose diet, and the diet reversal mice (n ≥ 7 per group). Data expressed as mean ± S.D. ND₂₀—20-week normal diet; HFSD₂₀—20-week high fat diet; DiR—diet reversal; *** p ≤ 0.001.

Figure 6

Diet reversal ameliorates dysregulated insulin sensitivity (A) Fasting blood glucose levels (n ≥ 7 per group). (B) Plasma insulin levels, (C) Insulin resistance, (D) Insulin sensitivity, and (E) Beta cell function in mice fed only the normal diet, only the high-fat/sucrose diet, and the diet reversal mice (n ≥ 7 per group). Data expressed as mean ± S.D. ND₂₀—20-week normal diet; HFSD₂₀—20-week high fat diet; DiR—diet reversal; ** p ≤ 0.01, *** p ≤ 0.001.

Figure 7

Diet reversal ameliorated impaired corneal wound healing and halted the progression of corneal nerve sensitivity loss observed with high-fat/sucrose consumption. (A) Corneal nerve sensitivity in uninjured mice and (B) Corneal wound closure Top: Wound closure kinetics. Bottom: Representative images of the epithelial wound at different time points following a 2 mm diameter corneal epithelial abrasion wound. (C) Left: Dividing basal epithelial cells in each cornea region. Right: Sum of dividing basal epithelial cells in four fields of view of the cornea (periphery, parawound, paracentral and center) and (D) Basal epithelial cell density in each cornea region in mice fed only the normal diet, only the high-fat/sucrose diet, and the diet reversal mice at 30 h post wounding. Data expressed as means ± SD; ND₂₀—20-week normal diet; HFSD₂₀—20-week high-fat/sucrose diet; DiR—diet reversal; L—limbus; PL—paralimbus/periphery; PW—parawound; PC—paracentral; C—center * p ≤ 0.05, ** p ≤ 0.01 (normal diet vs. high-fat/sucrose diet); † p ≤ 0.05 and †† p ≤ 0.01 (high-fat/sucrose diet vs. diet reversal); # p ≤ 0.05 (comparing corneal sensitivity in normal diet fed mice at different time points).

Figure 7

Diet reversal ameliorated impaired corneal wound healing and halted the progression of corneal nerve sensitivity loss observed with high-fat/sucrose consumption. (A) Corneal nerve sensitivity in uninjured mice and (B) Corneal wound closure Top: Wound closure kinetics. Bottom: Representative images of the epithelial wound at different time points following a 2 mm diameter corneal epithelial abrasion wound. (C) Left: Dividing basal epithelial cells in each cornea region. Right: Sum of dividing basal epithelial cells in four fields of view of the cornea (periphery, parawound, paracentral and center) and (D) Basal epithelial cell density in each cornea region in mice fed only the normal diet, only the high-fat/sucrose diet, and the diet reversal mice at 30 h post wounding. Data expressed as means ± SD; ND₂₀—20-week normal diet; HFSD₂₀—20-week high-fat/sucrose diet; DiR—diet reversal; L—limbus; PL—paralimbus/periphery; PW—parawound; PC—paracentral; C—center * p ≤ 0.05, ** p ≤ 0.01 (normal diet vs. high-fat/sucrose diet); † p ≤ 0.05 and †† p ≤ 0.01 (high-fat/sucrose diet vs. diet reversal); # p ≤ 0.05 (comparing corneal sensitivity in normal diet fed mice at different time points).

Figure 8

Diet reversal restored normal abrasion-induced inflammation. (A) Left: The distribution of neutrophils over the limbus and center of the cornea 30 h after wounding. Right: Sum of neutrophils in four fields of view of the cornea (periphery, parawound, paracentral and center) at 30 h after wounding. (B) Platelets count at the limbus 30 h after wounding. (C) Diameter of venules at the corneal limbus. (D) Representative images of platelets at the limbus; images captured with a 60X oil objective lens (scale bar = 15 µm). Data expressed as means ± SD; ND₂₀—20-week normal diet; HFSD₂₀—20-week high-fat/sucrose diet; DiR—diet reversal; L—limbus; C—center * p ≤ 0.05, ** p ≤ 0.01. Scale bars: D = 15 µm.