Neuroprotective Effects of Diets Containing Olive Oil and DHA/EPA in a Mouse Model of Cerebral Ischemia

Abstract

Stroke is one of the leading causes of death worldwide and while there is increasing evidence that a Mediterranean diet might decrease the risk of a stroke, the effects of dietary fat composition on stroke outcomes have not been fully explored. We hypothesize that the brain damage provoked by a stroke would be different depending on the source of dietary fat. To test this, male C57BL/6J mice were fed for 4 weeks with a standard low-fat diet (LFD), a high-fat diet (HFD) rich in saturated fatty acids (HFD-SFA), an HFD containing monounsaturated fatty acids (MUFAs) from olive oil (HFD-OO), or an HFD containing MUFAs from olive oil plus polyunsaturated fatty acids (PUFAs) docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) (HFD-OO-ω3). These mice were then subjected to transient middle cerebral artery occlusion (tMCAo). Behavioural tests and histological analyses were performed 24 and/or 48 h after tMCAo in order to elucidate the impact of these diets with different fatty acid profiles on the ischemic lesion and on neurological functions. Mice fed with HFD-OO-ω3 displayed better histological outcomes after cerebral ischemia than mice that received an HFD-SFA or LFD. Furthermore, PUFA- and MUFA-enriched diets improved the motor function and neurological performance of ischemic mice relative to those fed with an LFD or HFD-SFA. These findings support the use of DHA/EPA-omega-3-fatty acid supplementation and olive oil as dietary source of MUFAs in order to reduce the damage and protect the brain when a stroke occurs.

Keywords: DHA; EPA; MUFA; PUFA; SFA; behaviour; cerebral ischemia; middle cerebral artery occlusion (MCAo); neurological function; neuroprotection; olive oil; omega-3 fatty acids; stroke.

Figure 1

Representative scheme showing the procedure followed to study the effect of HFDs with distinct fatty acid compositions on mice subjected to transient middle cerebral artery occlusion (tMCAo).

Figure 2

(A) Body weight monitoring and (B) Body weight gain. Experimental diets: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). No significant differences between the groups were found after ANOVA. The results represent the mean ± standard error of the mean (SEM) of 9 to 12 animals. Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO) and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).

Figure 3

Representation of cerebral blood flow (CBF) traces recorded by laser Doppler flowmetry during the tMCAo procedure. CBF values were recorded continuously during the procedure, analysing the following time intervals: baseline (0 min), 0–20 min, 20–40 min, 40–60 min, and the onset of reperfusion at 60–65 min. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green) and HFD-OO-ω3 (Blue). No significant differences between the groups were found (2-way ANOVA), the results represent the mean ± SEM of 8 to 10 animals. Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO), and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).

Figure 4

HFD-OO-ω3 diet reduces the infarct area in an experimental model of focal cerebral ischemia. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). (A) Representative images of TTC staining. Seven coronal slices along the rostro-caudal axis are shown, from coordinates bregma AP: +2.80 to bregma AP: −2.46 (Franklin and Paxinos atlas [41]). (B) The HFD-OO-ω3 diet reduced the infarct area compared to HFD-SFA and LFD. (C,D) show the Infarct volume and edema analyses, respectively. These data reflect a slight decrease in both parameters, yet no significant differences were found. The results represent the mean ± SEM of 8 to 10 animals: * p ≤ 0.05, ** p ≤ 0.01 versus HFD-SFA and ⁺ p ≤ 0.05 versus LFD (ANOVA + Bonferroni post-hoc). Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO), and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).

Figure 5

HFD-OO-ω3 diet improves infarct severity in mice subjected to transient focal cerebral ischemia. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). (A) Representative grayscale images of TTC staining to determine infarct severity. Three coronal sections along the rostro-caudal axis are shown, as well as the ipsilateral and contralateral ROI probes used in the analysis (yellow circles). (B) HFD-OO-ω3 diet improved brain infarct severity compared to HFD-SFA and LFD. The results represent the mean ± SEM of 8 to 9 animals: * p ≤ 0.05 (ANOVA + Bonferroni post-hoc). Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO), and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).

Figure 6

Haemorrhagic transformation analysis. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). (A) Representative images of the classes of macroscopic haemorrhages found during the analysis: no haemorrhages (NO HT), haemorrhagic infarction type 1 (HI-1), haemorrhagic infarction type 2 (HI-2). (B) Haemorrhagic transformation score. No significant differences were found between the groups (Kruskal–Wallis + Dunn´s post-hoc). Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO), and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).

Figure 7

Differences in motor deficits and neurological function in mice fed with HFD-OO or HFD-OO-ω3. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). (A) HFD-OO and HFD-OO-ω3 diets improved grip strength 24 and 48 h after reperfusion relative to the LFD or HFD-SFA. Mice belonging to HFD-SFA group displayed less grip strength 48 h after the onset of tMCAo than animals on LFD. (B) HFD-OO-fed mice had better neurological scores 24 and 48 h after reperfusion compared to HFD-SFA-fed or the HFD-SFA and LFD-fed groups, respectively. The results represent the mean ± SEM in A and the median ± interquartile range in B of 6 to 11 animals: * p ≤ 0.05 and ** p ≤ 0.01 (ANOVA + Bonferroni post-hoc were performed in (A) and Kruskal–Wallis + Dunn´s tests were performed in (B)).

Figure 8

The effect of dietary interventions with a HFD containing a specific fatty acid composition on gait coordination and weight loss. Groups: LFD (Black), HFD-SFA (Red), HFD-OO (Green), and HFD-OO-ω3 (Blue). (A) Mice on a HFD-OO-ω3 and LFD diet had better coordination of the right hindlimb 48 h after reperfusion than those on a HFD-SFA diet. (B) Mice fed with HFD-OO-ω3 or LFD showed better steps/m values than mice fed a HFD-SFA diet. HFD-OO was not as beneficial as HFD-OO-ω3 48 h after the onset of reperfusion. (C) When the weight loss was analysed 48 h after tMCAo, no significant differences were found between the groups. The results represent the mean ± SEM of 6 to 10 mice: * p ≤ 0.05 and ** p ≤ 0.01 (ANOVA + Bonferroni post-hoc). Low-fat diet (LFD), high-fat diet rich in saturated fatty acids (HFD-SFA), high-fat diet rich in olive oil (HFD-OO), and high-fat diet rich in olive oil plus DHA and EPA (HFD-OO-ω3).