Impact of hydroxytyrosol on stroke: tracking therapy response on neuroinflammation and cerebrovascular parameters using PET-MR imaging and on functional outcomes

Abstract

Immune cells have been implicated in influencing stroke outcomes depending on their temporal dynamics, number, and spatial distribution after ischemia. Depending on their activation status, immune cells can have detrimental and beneficial properties on tissue outcome after stroke, highlighting the need to modulate inflammation towards beneficial and restorative immune responses. Novel dietary therapies may promote modulation of pro- and anti-inflammatory immune cell functions. Among the dietary interventions inspired by the Mediterranean diet, hydroxytyrosol (HT), the main phenolic component of the extra virgin olive oil (EVOO), has been suggested to have antioxidant and anti-inflammatory properties in vitro. However, immunomodulatory effects of HT have not yet been studied in vivo after stroke. The aim of this project is therefore to monitor the therapeutic effect of a HT-enriched diet in an experimental stroke model using non-invasive in vivo multimodal imaging, behavioural phenotyping and cross-correlation with ex vivo parameters. Methods: A total of N = 22 male C57BL/6 mice were fed with either a standard chow (n = 11) or a HT enriched diet (n = 11) for 35 days, following a 30 min transient middle cerebral artery occlusion (tMCAo). T₂-weighted (lesion) and perfusion (cerebral blood flow)-/diffusion (cellular density)-weighted MR images were acquired at days 1, 3, 7, 14, 21 and 30 post ischemia. [¹⁸F]DPA-714 (TSPO, neuroinflammation marker) PET-CT scans were acquired at days 7, 14, 21 and 30 post ischemia. Infarct volume (mm³), cerebral blood flow (mL/100g/min), apparent diffusion coefficient (10^-4·mm²/s) and percentage of injected tracer dose (%ID/mL) were assessed. Behavioural tests (grip test, rotarod, open field, pole test) were performed prior and after ischemia to access therapy effects on sensorimotor functions. Ex vivo analyses (IHC, IF, WB) were performed to quantify TSPO expression, immune cells including microglia/macrophages (Iba-1, F4/80), astrocytes (GFAP) and peripheral markers in serum such as thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) 35 days post ischemia. Additionally, gene expression of pro- and anti-inflammatory markers were assessed by rt-qPCR, including tspo, cd163, arg1, tnf and Il-1β. Results: No treatment effect was observed on temporal [¹⁸F]DPA-714 uptake within the ischemic and contralateral region (two-way RM ANOVA, p = 0.71). Quantification of the percentage of TSPO⁺ area by immunoreactivity indicated a slight 2-fold increase in TSPO expression within the infarct region in HT-fed mice at day 35 post ischemia (p = 0.011) correlating with a 2-3 fold increase in Iba-1⁺ cell population expressing CD163 as anti-inflammatory marker (R² = 0.80). Most of the GFAP⁺ cells were TSPO^-. Only few F4/80⁺ cells were observed at day 35 post ischemia in both groups. No significant treatment effect was observed on global ADC and CBF within the infarct and the contralateral region over time. Behavioural tests indicated improved strength of the forepaws at day 14 post ischemia (p = 0.031). Conclusion: An HT-enriched diet significantly increased the number of Iba-1⁺ microglia/macrophages in the post-ischemic area, inducing higher expression of anti-inflammatory markers while no clear-cut effect was observed. Also, HT did not affect recovery of the cerebrovascular parameters, including ADC and CBF. Altogether, our data indicated that a prolonged dietary intervention with HT, as a single component of the Mediterranean diet, induces molecular changes that may improve stroke outcomes. Therefore, we support the use of the Mediterranean diet as a multicomponent therapy approach after stroke.

Keywords: Hydroxytyrosol; TSPO; [18]DPA-714; multimodal imaging; neuroinflammation; transient middle artery occlusion.

Figure 1

Study design. After a 30 minutes tMCAo, N = 22 mice were randomly divided into a control (green) or HT (red) diet group. MR studies were conducted at days 1, 3, 7, 14, 21 and 30 post ischemia while [¹⁸F]DPA-714 PET scans were acquired at days 7, 14, 21 and 30 post ischemia. Mice were also tested for motor and cognitive impairments using four behavioural tests: open field, grip strength test, pole test, and rotarod prior and after a 30 min tMCAo. After the last behavioural test (at day 35), all animals were killed and brains were collected for ex vivo analysis.

Figure 2

Longitudinal monitoring of the edema volume using T₂w-MR imaging after ischemic stroke. (A) Representative T₂w-MR images from control and HT-fed mice acquired at days 1, 3 and 7 post ischemia. The edema volume (white) was delineated by an atlas-based thresholding approach. (B) Edema volume decreased from day 3 post ischemia for either control (green, n = 8) and HT (red, n = 8) fed mice. No treatment effect was observed on edema volume. Individual data are plotted in mm³. Statistical analysis was carried out with two-way RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.005; * vs. day 3, # vs. day 7).

Figure 3

Longitudinal monitoring of the mean cerebral blood flow by FAIR-MRI 1, 3, 7, 14, 21 and 30 days after stroke. CBF was assessed within the infarct defined on the T₂w-MR images from day 1 post ischemia and this mirrored ROI in the contralateral side in control (green) and HT-fed (red) mice. (A) Representative CBF images at days 1, 3, 7, 14, 21 and 30 post ischemia for both control (upper row) and HT-fed mice (lower row) and the corresponding T₂w-MR image at day 1 post ischemia. CBF maps showed hypoperfused infarct regions within the first week for both groups. At later time points, CBF was partially recovered in both groups. (B) Quantification of the mean CBF within the infarct and contralateral striatum showed no treatment effect on tissue reperfusion. Values represent mean ± sd (n = 8 per group). Statistical analysis was carried out with RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (# p < 0.05, ## p < 0.01, ### p < 0.005, # vs. contralateral).

Figure 4

Hydroxytyrosol does not affect the microstructure after stroke. Apparent diffusion coefficient was assessed within the infarct defined on the T₂w-MR images from day 1 post ischemia and a mirrored reference ROI (contralateral) in control (green) and HT-fed (red) mice. (A) Representative diffusion-weighted images (with the highest b value of 2400 s/mm²) and the respective ADC map at days 1, 3, 7, 14, 21 and 30 days post ischemia for each experimental group. (B) Time course of the ADC values determined in the infarct and the contralateral area in control (green) and HT-fed (red) mice. RM ANOVA indicated main effect of time (p < 0.001) and time * region (p < 0.001) but not of treatment (p = 0.34). For both groups, the mean ADC value within the infarct hemisphere significantly recovered from day 14 post ischemia. Values represent mean ± sd. Control: n = 8, HT: n = 8. Statistical analysis was carried out with RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.005; * vs. contralateral, # vs. day 1, & vs. day 3 and + vs. day 7).

Figure 5

A HT diet does not affect TSPO expression as detected by [¹⁸F]DPA-714 PET imaging after ischemic injury. The mice were scanned for [¹⁸F]DPA-714 PET at days 7, 14, 21 and 30 post ischemia to assess TSPO expression within the infarct (white circle) and the contralateral striatum. The percentage of injected dose per millimetre (%ID/mL) was quantified within both regions. (A) Representative axial co-registered [¹⁸F]DPA-714-PET-CT images of a mouse fed with either control (upper row) or HT (lower row) diet after a 30 min tMCAo with comparable T₂w-infarct on day 1 post ischemia (white delineation). (B) Similar uptake was measured between both groups either within the infarct or contralateral striatum over time. (C) Infarct-to-contralateral ratios did not differ over time, indicative of constant chronic neuroinflammatory reaction in the infarcted hemisphere compared to the contralateral side (p > 0.05). Values represent mean ± sd. Control: n = 8, HT: n = 8. Statistical analysis was carried out with two-way RM ANOVA.

Figure 6

A HT diet induces a slight increase in TSPO expression at day 35 post ischemia. (A) Representative TSPO staining within the infarct and contralateral side obtained by immunohistochemistry at day 35 post ischemia from both control and HT-fed mice (white scale bar: 30 µm, black scale bar: 8 µm). For both experimental groups, immune cells and endothelial cells showed TSPO immunoreactivity. A HT diet slightly increased TSPO-related inflammatory level (n = 3/group, with 3 fields of view per animal). (B) Representative western blot for TSPO from brain protein extracts obtained at day 35 post ischemia (n = 6/group). A HT diet did not change TSPO protein levels in both the infarct (I) and contralateral (C) hemispheres at day 35 post ischemia. Values are normalized relative to GAPDH band. Statistical analysis was carried out with two-way ANOVA for multiple comparisons followed by Holm Sidak's post hoc test for multiple comparisons. Values represent mean ± sd. (*p < 0.05, ** p < 0.01, ***p < 0.005; * vs. contralateral, # vs. diet).

Figure 7

TSPO is mainly expressed in microglia/macrophages within the infarct at day 35 post ischemia. Co-localization of TSPO with immune cells markers such as Iba-1, GFAP and F4/80 at day 35 post ischemia highlighted strong microgliosis independent of TSPO expression. No or minor colocalization of GFAP or F4/80 positive cells with TSPO was found. Nuclei are counterstained with DAPI (blue). White scale bar: 40 µm.

Figure 8

HT increases Iba-1 positive cells number within the infarct at day 35 post ischemia. (A) Representative immunohistochemistry for Iba-1 in brain of control (upper row) and HT-fed (lower row) mice within the infarct and at the contralateral side 35 days after tMCAo. Iba-1 positive cells within the ischemic lesion exhibited an amoeboid morphology in brain of HT-fed mice compared to control mice brain (ramified shape) (black scale bar: 50 µm, white bar: 9 µm). (B) Quantification of the percentage of Iba-1 stained area showed increased Iba-1 positive area within the infarct in HT-fed mice (red) compared to controls (control), indicative of HT's immunomodulatory effect on Iba-1 positive cells (n = 4/group, with 3 fields of view per region). (C) Most of the Iba-1 positive cells co-expressed CD163, an anti-inflammatory marker (red scale bar: 10 µm). (D) Gene expression confirmed immunofluorescence data and indicated a trend toward an increased expression of anti-inflammatory markers after HT treatment at day 35 post ischemia. Statistical analysis was carried out with two-way RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.005, * vs. contralateral, # vs. diet).

Figure 9

Hydroxytyrosol does not improve locomotion. Distance moved [cm], velocity [cm/s], frequency and duration [s] were assessed like index of locomotion and anxiety. Overall, there was no dietary effect observed on locomotor activity and anxiety level assessed by open field. The time spent in the central zone significantly decreased at day 30 post ischemia in control mice compared to baseline while no difference was observed in HT-fed mice. Values represent mean ± sd. Control: n = 8, HT: n = 8. Statistical analysis was carried out with two-way RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.005).

Figure 10

Hydroxytyrosol improves strength recovery of the forelimbs after ischemia. (A) Grip test revealed a HT diet improved forelimbs strength and thus recovery at day 14 post ischemia (p = 0.031). In control mice, the grip strength was decreased 14 days after stroke while no difference was observed in HT-fed mice (p = 0.031). (B) However, no difference was observed in total limbs strength over time. Values represent mean ± sd. Control: n = 8, HT: n = 8. Statistical analysis was carried out with two-way RM ANOVA followed by Holm Sidak's post hoc test for multiple comparisons (*p < 0.05).