Fig. 8.
High-fat diet fed mice show increased monocytic infiltration and greater neurological deficits in an experimental model of MS. (a) Female C57/bl6 mice were fed either a low-fat (LFD) or high-fat diet (HFD) for 5 weeks, and then immunized with 200 ng MOG35–55 using a protocol to induce EAE. (b) Ascending paralysis was graded on a scale of 0 to 5 with high scores indicating increased clinical disability (black = LFD-EAE mice, gray = HFD-EAE mice). Differences in EAE scores were evaluated between LFD-EAE and HFD-EAE mice at each time point using Mann-Whitney U test, respectively (*p < .05, **p < .01) (n = 15/group). (c) Weight change from baseline (i.e. day of immunization) was calculated for each time point and differences between LFD-EAE and HFD-EAE mice were evaluated by Sidak's multiple comparisons test (**p < .01). (d) Area under the curve of the EAE disease course and maximum EAE score were also used to evaluate differences in disease course and statistical analysis was performed using Mann-Whitney U test (*p < .05). (e-g) Mice were sacrificed at the peak of disease and spinal cord tissue sectioned for histological analysis. Spinal cord sections were stained with Fluoromyelin (FM)to stain myelin and lesion areas and Neurofilament H (NFH) to stain axons. Images were acquired using the Zeiss LSM 800 confocal microscope and the 20× objective (scale bar = 50um). Myelin and axonal content were quantified as the area of FM or NFH positivity, respectively, in relation to the whole spinal cord area. Spinal cord sections were also stained with Iba1 and CD45 to mark peripheral monocytes/activated microglia (Iba1+ CD45+) and leukocytes (CD45+) and counterstained with DAPI, to identify nuclei. Confocal images were acquired using the Zeiss LSM 800 and the 20× objective (scale bar = 25um). Iba1 + CD45+ area and CD45+ area was quantified within the lesion to enhance representation of peripheral monocytes and peripheral leukocytes, respectively, and was calculated as a percentage in relation to the total spinal cord area. Spinal cord sections were stained with 5mc and Iba1 to assess DNA methylation levels in monocytes. Images were acquired using the Zeiss LSM 800 at 63× (scale bar = 10um). 5mc intensity was normalized to DAPI intensity in Iba1+ cells within the lesion. Statistical differences between LFD-EAE (white) and HFD-EAE (gray) groups were assessed by Student's t-test (*p < .05) (n = 3 mice/group, n = 2 sections/mouse, n = 3 images/section).