Fig 1.
Fecal samples collected after GPE treatment contain high levels of antioxidant activity.
Antioxidant activity (Trolox equivalents) in murine fecal samples (n = 6 mice) collected before (0 h) a single oral dose of GPE (32 mg total polyphenols/ kg) and A. every hour for 12 h after treatment while mice had ad libitum access to chow diet or B. every hour during a 12 h period of food restriction, after which chow diet was replaced and an additional fecal sample was collected at 24 h. C. Total polyphenols (as gallic acid equivalents, left) and total antioxidant capacity (as Trolox equivalents, right) in fecal samples collected before gavage (0 h, black bars) and in total feces collected over the 24 h period following oral GPE administration (crosshatched bars), as compared to the initial dose (white bars). N = 6 mice; Data are reported as mean ± SD.
Fig 2.
HFD-fed obese mice have higher levels of intestinal ROS than LFD-fed lean mice.
Mice fed HFD or LFD for 10 weeks (n = 6/ group) were imaged in stationary, ventral orientation (abdomen facing camera) 3 h after H-ICG administration (i.e. 4 h after GPE administration). A. Representative overlay of ROS-associated NIRF image and corresponding brightfield image of a HFD-fed obese mouse (left) and a LFD-fed lean mouse (right). NIRF intensity scale shown on the right, image was normalized accordingly using Carestream MI software. B. ROS-associated NIRF in healthy, lean mice (white bars) and in obese mice (black bars) at 1, 2, and 3 h after administration of H-ICG. N = 6 mice per group; Data are reported as mean ± SD. One-way ANOVA followed by the Tukey’s multiple comparison test was performed across both groups and all time points. Same letters indicate no difference between groups or time points while different letters indicate significant difference (p < 0.05).
Fig 3.
ROS-associated NIRF measured over a 360° rotational scan is higher in obese mice than in lean mice.
A. ROS-associated NIRF images in obese (HFD) and lean (LFD) mice superimposed onto brightfield images. Rotation started 45 min following H-ICG administration, which was 1 h 45 min after GPE administration. Images were taken at 30° rotational increments over a course of a 360° rotation completed in 24 min. Images were colorized using Carestream MI software according to the NIRF intensity scale shown on right. B. Intensity of NIRF measured at different orientational angles in HFD and LFD-fed mice. Zero angle represents ventral orientation (abdomen facing camera). C. Area under curve (AUC) calculated from panel B (left axis) and NIRF measured at 0° corresponding to the ventral orientation (right axis). N = 20 mice per group; Data are reported as mean ± SD. Significant difference between HFD and LFD groups was detected using unpaired Mann Whitney test, *** p < 0.0001.
Fig 4.
ROS-associated rotational NIRF of obese, HFD-fed mice treated with dietary antioxidants.
A. ROS-associated 360° rotational NIRF images of obese HFD-fed mice superimposed on brightfield images. Animals were gavaged (1 h 45 min before imaging) with GPE, B-type proanthocyanidins (PAC), β-carotene (β-car), or ATL (mixture of L-ascorbic acid, D-α-tocopherol succinate and α-lipoic acid) at 32 mg/kg dose, except for GPE, which was dosed to deliver 32 mg/kg dose of total polyphenols. Images were taken at 30° rotational increments over a course of a 360° rotation completed in 24 min. Images were colorized using Carestream MI software according to the NIRF intensity scale shown on right. B. Area under curve (AUC) calculated from 4A as a function of different antioxidant treatments. Numbers under x-axis are x107 (photons*deg)/s/mm2 AUC for the corresponding group. N = 5 mice per group. Data are reported as mean ± SD. Significant difference between groups was detected by one-way ANOVA (p = 0.002) followed by post hoc comparison to water-treated group using Dunnett’s test, * p< 0.05, ** p< 0.01.
Fig 5.
ROS-associated rotational NIRF of lean, LFD-fed mice treated with dietary antioxidants.
A. ROS-associated 360° rotational NIRF images of obese LFD-fed mice superimposed on brightfield images. Animals were gavaged (1 h 45 min before imaging) with GPE, B-type proanthocyanidins (PAC), β-carotene (β-car), or ATL (mixture of L-ascorbic acid, D-α-tocopherol succinate and α-lipoic acid) at 32 mg/kg dose, except for GPE which was dosed to deliver 32 mg/kg dose of total polyphenols. Images were taken at 30° rotational increments over a course of a 360° rotation completed in 24 min. Images were colorized using Carestream MI software according to the NIRF intensity scale shown on right. B. Area under curve (AUC) calculated from 5A as a function of different antioxidant treatments. Numbers under x-axis are x107 (photons*deg)/s/mm2 AUC for the corresponding group. N = 5 mice per group; Data are reported as mean ± SD. Significant difference between groups was detected by one-way ANOVA (p = 0.023) followed by post hoc comparison to water-treated group using Dunnett’s test, * p< 0.05.