Beneficial Effects of Melatonin on Apolipoprotein-E Knockout Mice by Morphological and 18F-FDG PET/CT Assessments

Figure 1. Morphometrical evaluation. Photomicrographs of the aorta from the control (a,c) and MLT-treated (b,d) mice. Each panel shows a full-field image at 400× (a,b) (scale bars: 20 µm) and at 1000× (c,d) (scale bars: 10 µm). Control group aorta showed larger areas of WAT, characterized by white lipid drop, with a minimum presence of multilocular brown adipocytes. In addition, ApoE-/- mice aorta is also characterized by a disarrangement of normal vascular structure (a). On the contrary, MLT-treated mice showed a higher presence of BAT, with a significant reduction of white adipocyte infiltration and are also characterized by relatively unremarkable changes in vascular cytoarchitecture and organization (b). Photomicrographs of perivascular adipose tissue from the control (c) and from treated (d) mice. Graphs summarize the morphometrical analyses of the percentage per area of periaortic BAT (e) and WAT (f) obtained evaluating, for each experimental animal, fifteen non-overlapping fields with the same area. Statistical analyses comparing multiple continuous outcomes were performed using one-way analyses of variance test corrected by Bonferroni for morphometrical evaluations. Continuous…

Figure 2. Assessment of the inflammatory state. Labelling of the aorta’s wall with VCAM-1 (identified in red staining, panels (a) and (d) and ICAM-1 (identified in green staining, panels (b) and (e) considering them as general markers of the inflammatory state. Each panel shows a full-field image at 400× (scale bars: 20 µm). Double immunofluorescence photomicrograph of the control mice shows a moderate expression of both adhesion proteins (merged expression reported in panel (c). In contrast, the double immunofluorescence photomicrograph of ApoE-/- mice treated with melatonin highlights, for the same inflammatory markers, an absent/very weak positivity at the tunica intima level (merged expression reported in panel (f). Graphs summarize, as arbitrary units, the positivity, respectively, of VCAM-1 (g) and ICAM-1 (h) obtained, for both adhesion molecules, evaluating fifteen non-overlapping fields with the same area for each experimental animal. The negative control of the double immunofluorescence staining without primary antibody and in the presence of isotype matched immunoglobulins G is reported in (i). Statistical analyses comparing multiple continuous outcomes were performed using a…

Figure 3. Immunohistochemical characterization of macrophage population. Labelling of the aorta’s wall with CD68 (a,b) and CD163 (d,e) antibodies. Each panel shows a full-field image at 1000× (scale bars: 20 µm). Immunohistochemical photomicrograph shows the positivity for CD68, mainly localized in the subendothelial space and in the tunica adventitia of control mice (a). In contrast, the immunohistochemical photomicrograph of ApoE-/- mice treated with melatonin highlights that this immunopositivity slightly decreased in the subendothelial space, even if not significantly, after melatonin supplementation (b). CD163 expression was negative or really weak in control mice (d). In contrast, the photomicrograph of MLT-treated mice showed that CD163 was moderately/strongly expressed both in the subendothelial space and tunica adventitia, increasing its positivity after melatonin administration (e). Graphs summarize the number of CD68 (c) and CD163 (f) positive cells, obtained for both macrophage markers, evaluating five non-overlapping fields with the same area from three non-consecutive aorta sections. For both CD68 (g) and CD163 (h), sections without primary antibody and in the presence of…

Figure 4. Immunofluorescence characterization of macrophage cytokine production. Labelling of the aorta’s wall with TNF-α (a,b) and TGF-β (d,e) antibodies. Each panel shows a full-field image at 400× (scale bars: 20 µm). Immunofluorescence photomicrograph of control mice showed a moderate TNF-α expression at the subendothelial and adventitia levels, confirming that these mice were characterized by an infiltrate of macrophages predominantly polarized in the M1 direction (a). In contrast, the immunofluorescence photomicrograph of ApoE-/- mice treated with melatonin highlights, for the same M1 marker, an absent/very weak positivity (b). Immunofluorescence photomicrograph also showed an absent expression of TGF-β in the aorta of the control mice (d). Differently, the immunofluorescence photomicrograph of MLT-treated mice showed a moderate TGF-β expression in the tunica adventitia (e). Graphs summarize, as arbitrary units, the immunomorphometrical analyses of TNF-α (c) and TGF-β (f) obtained, for both pro-inflammatory markers, evaluating fifteen non-overlapping fields with the same area for each experimental animal, respectively. For both TNF-α (g) and TGF-β (h), sections without primary…

Figure 5. Aortic 18F-FDG uptake and biodistribution. Results were obtained from scans that were performed on a dedicated small animal PET/CT scanner and are expressed as mean %ID/mL (%injected dose per mL of tissue) from 18F-FDG PET/CT (a,b) and activity concentration (mean pCi/gm) from biodistribution data (c). The latter was divided by 105 for the purpose of visualization. Error bars represent the 95% confidence interval around the mean; * indicates the level of significance, p ≤ 0.05; CTR control group; MLT, mice treated with melatonin; %ID/mL, % injected dose per mL of tissue; pCi/g, picocurie per gram.

Figure 6. 18F-FDG PET/CT evaluation. Sagittal views of MLT-treated mouse (c,d) and comparable views of a control mouse (a,b). Each panel shows, from left to right, CT only, PET only, and fused PET/CT images. Liver is recognized as a large solid structure in CT image and vertebral column as a hyperdense (white) posterior structure. In PET and fused PET/CT images, the sites with the most intense activity are the heart and the urinary bladder due to the normal excretion of 18F-FDG by the kidneys (not shown in this section). The brain may demonstrate variable levels of 18F-FDG uptake. The structure lying anterior to the vertebral column represents the abdominal aorta. Mild 18F-FDG uptake is seen in its proximal part at the baseline PET/CT images taken from the control (a) and MLT-treated (c) mice. After 14 weeks of the atherogenic diet, a second PET/CT scan showed nearly unchanged FDG activity in the control mice (b) while, the MLT-treated group presented a significantly higher 18F-FDG uptake in the same region of the aorta (d) compared to the baseline (respectively (a) for the control group and c for the MLT-treated mice). %ID/cc %injected dose per mL of tissue; white arrow indicates the…