Neuroprotection Afforded by an Enriched Mediterranean-like Diet Is Modified by Exercise in a Rat Male Model of Cerebral Ischemia

Abstract

Ischemic stroke is an important cause of mortality and disability worldwide. Given that current treatments do not allow a remarkably better outcome in patients after stroke, it is mandatory to seek new approaches to preventing stroke and/or complementing the current treatments or ameliorating the ischemic insult. Multiple preclinical and clinical studies highlighted the potential beneficial roles of exercise and a Mediterranean diet following a stroke. Here, we investigated the effects of a pre-stroke Mediterranean-like diet supplemented with hydroxytyrosol and with/without physical exercise on male rats undergoing transient middle cerebral artery occlusion (tMCAO). We also assessed a potential synergistic effect with physical exercise. Our findings indicated that the diet reduced infarct and edema volumes, modulated acute immune response by altering cytokine and chemokine levels, decreased oxidative stress, and improved acute functional recovery post-ischemic injury. Interestingly, while physical exercise alone improved certain outcomes compared to control animals, it did not enhance, and in some aspects even impaired, the positive effects of the Mediterranean-like diet in the short term. Overall, these data provide the first preclinical evidence that a preemptive enriched Mediterranean diet modulates cytokines/chemokines levels downwards which eventually has an important role during the acute phase following ischemic damage, likely mediating neuroprotection.

Keywords: EPCs; Milliplex; cerebral ischemia; exercise; hydroxytyrosol; inflammation; mediterranean diet; microglia; neuroprotection; tMCAO.

Figure 1

Experimental protocol. Schematic drawing of the protocols followed throughout the time course of the experiments.

Figure 2

Infarct size and edema development were assessed by means of magnetic resonance imaging (MRI) (A–E‴). T2-weighted images on each row correspond to one of the 2D slices of the same animals within each group at different time points: control rats (A–E), diet group (A′–E′), PA group (A″–E″), and PA/diet group (A‴–E‴). (F) Time course of infarct volumes. (G) Time course of the edema volumes. Bars show mean ± SEM. A total of 10 rats per group from basal to 14 days, and 7 rats per group at 28 days.

Figure 3

Time course of plasma concentrations of fourteen cytokines/chemokines: granulocyte colony-stimulating factor (G-CSF); eotaxin; macrophage inflammatory protein (MIP)-1α; MIP-2; interleukin (IL)-1α; IL-1β; IL-4; IL-5; IL-6; IL-12p70; IL-13; IL-17; IL-18; and interferon-gamma (IFNγ) Comparisons were made at each time point vs. control and represented in the figure if significant. * < 0.05, ** < 0.01, *** < 0.001, **** <  0.0001. p values are shown in Table S1. Bars show mean ± SEM. A total of 6 rats per group.

Figure 4

Time course of plasma 8-OHdG levels. * p = 0.0199. Bars show mean ± SEM. A total of 6 rats per group.

Figure 5

(A) Time course of circulating progenitor cell (CPC), hematopoietic progenitor cell (HPC), and endothelial progenitor cell (EPC) numbers from blood samples (250.000 cells counted in each one. Stats at each time point are vs. control. Bars show mean ± SEM. (B) Time course of normalized data from (A) against control values at the basal point. Stats at each time point vs. control. * < 0.05. p values are shown in Table S2. Bars show mean ± SEM. A total of 10 rats per group from basal to 14 days, and 7 rats per group at 28 days.

Figure 6

Assessment of motor and neurological behavior by the Bederson test (A), the Wahl test (B), and the cylinder test, both asymmetry (C) and laterality (D). Stats at each time point are vs. control. Bars show mean ± SEM. 10 rats per each group from basal to 14 days, and 7 rats per each group at 28 days.

Figure 7

Representative images of TUNEL (A–D), NeuN (A′–D′), and vGLUT1 (A‴–D‴) staining at the peri-infarct area in the sensorimotor cortex of the control group (A–A‴); diet group (B–B‴); PA group (C–C‴); and PA/diet group (D–D‴). (E) Quantification of the percentage (%) of NeuN+ cells with TUNEL labeling, * p = 0.0149. (F) Quantification of vGLUT1 immunoreactivity, * p = 0.0153 (diet group), * p = 0.0376 (PA group), ** p = 0.003 (PA/diet group). Scale bars: 100 μm. Bars show mean ± SEM. A total of 3 sham rats and 5 rats per remaining group were used in all experiments, with 250 cells quantified per animal.

Figure 8

Representative images of Ki67 (A–D), CD31 (A′–D′), and CD31+ area (A‴–D‴) staining at the subventricular zone (SVZ) of the control group (A–A‴); diet group (B–B‴); PA group (C–C‴); and PA/diet group (D–D‴). Representative images of IB4 (E–H) and Iba1 (E′–H′) staining at the peri-infarct area in the sensorimotor cortex of the control group (E–E″); diet group (F–F″); PA group (G–G″); and PA/diet group (H–H″). (I) Quantification of the percentage (%) of CD31+ cells with Ki67 labeling from the total number of CD31+ cells, ** p = 0.0071 (PA vs. sham), * p = 0.0446 (PA vs. control), ** p = 0.0018 (PA vs. diet). (J) Quantification of CD31+ area immunoreactivity. (K) Quantification of IB4 immunoreactivity. Scale bars: 400 μm. Bars show mean ± SEM. A total of 3 sham rats and 5 rats per remaining group were used in all experiments, with 250 cells quantified per animal.

Figure 9

Representative images of Ki67 (A–D), DCX (A′–D′), and DCX+ area (A‴–D‴) staining at the SVZ of the control group (A–A‴); diet group (B–B‴); PA group (C–C‴); and PA/diet group (D–D‴). (E) Quantification of the percentage (%) of DCX+ cells with Ki67 labeling from the total number of DCX+ cells, * p = 0.0468 (PA vs. sham), ** p = 0.0013 (PA vs. control). (F) Quantification of DCX+ area immunoreactivity, ** p = 0.0055 (control vs. sham), **** p < 0.0001 (PA vs. sham), * p = 0.0239 (PA vs. diet), ** p = 0.0083 (PA vs. PA/diet). Scale bars: 100 μm. Bars show mean ± SEM. A total of 3 sham rats and 5 rats per remaining group were used in all experiments, with 250 cells quantified per animal.

Figure 10

Late microglia activation in either exercise or Mediterranean-like diet or combined groups following injury. Representative images of Iba1 staining at 14 days (A–D) and 28 days (A′–D′) following ischemia in the peri-infarct area of control, diet, PA, and PA/diet groups. (E) Quantifications of Iba1 immunoreactivity and the number of Iba1+ cells at 14 days after injury, ** p = 0.0096. (F) Quantifications of Iba1 immunoreactivity and the number of Iba1+ cells at 28 days after injury, Iba1+ cells: * p = 0.0114 (control vs. sham), *** p = 0.0005 (diet vs. sham), **** p < 0.0001 (PA vs. sham), * p = 0.0404 (PA vs. PA/diet); Iba1 immunoreactivity: * p = 0.0359 (diet vs. sham), * p = 0.012 (PA vs. sham). (G) Positive correlation for Iba1 immunofluorescence (IF) signal and the number of Iba1+ cells. Scale bars: 100 μm. Bars show mean ± SEM. A total of 4 rats were used per group.