Theranostics 2021; 11(9):4030-4049. doi:10.7150/thno.48110

Research Paper

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

Cristina Barca1,2✉, Maximilian Wiesmann3, Jesús Calahorra4, Lydia Wachsmuth5, Christian Döring1, Claudia Foray1,2, Ali Heiradi6, Sven Hermann1,7, Maria Ángeles Peinado4, Eva Siles4, Cornelius Faber5, Michael Schäfers1,8, Amanda J Kiliaan3, Andreas H. Jacobs1,2,7,9✉*, Bastian Zinnhardt1,2,7,8,10*

1. European Institute for Molecular Imaging (EIMI), University of Münster, Münster, Germany.
2. PET Imaging in Drug Design and Development (PET3D).
3. Department of Medical Imaging, Anatomy, Radboud University Medical Center, Nijmegen, The Netherlands.
4. Department of Experimental Biology, University of Jaén, Campus las Lagunillas, Jaén, Spain.
5. Department of Radiology, Translational Research Imaging Center (TRIC), University Hospital Münster, Germany.
6. Institute of Physiological Chemistry and Pathobiochemistry, University Hospital Münster, Germany.
7. Immune Image, Innovative Medicines Initiative (IMI).
8. Department of Nuclear Medicine, University Hospital Münster, Germany.
9. Department of Geriatrics and Neurology, Johanniter Hospital, Bonn, Germany.
10. Biomarkers and Translational Technologies, Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
*These authors contributed equally to this work.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Barca C, Wiesmann M, Calahorra J, Wachsmuth L, Döring C, Foray C, Heiradi A, Hermann S, Peinado MÁ, Siles E, Faber C, Schäfers M, Kiliaan AJ, Jacobs AH, Zinnhardt B. Impact of hydroxytyrosol on stroke: tracking therapy response on neuroinflammation and cerebrovascular parameters using PET-MR imaging and on functional outcomes. Theranostics 2021; 11(9):4030-4049. doi:10.7150/thno.48110. Available from

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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, transient middle artery occlusion, neuroinflammation, [18]DPA-714, multimodal imaging, TSPO