Neuroprotection afforded by an enriched Mediterranean-like diet is modified by exercise in a rat model of cerebral ischemia

doi:10.21203/rs.3.rs-3454550/v1

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Neuroprotection afforded by an enriched Mediterranean-like diet is modified by exercise in a rat model of cerebral ischemia

https://doi.org/10.21203/rs.3.rs-3454550/v1

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Ischemic stroke is an important cause of mortality and disability worldwide. Given that current treatments do not allow a remarkable better outcome in patients after stroke, it is mandatory to seek new approaches 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 stroke. Here, we examined whether a Mediterranean-like diet supplemented with hydroxytyrosol with/without physical exercise enhances the good outcome of rats submitted to a transient middle cerebral artery occlusion (tMCAO). We also assessed a potential synergistic effect with physical exercise. We found that an enriched Mediterranean-like diet decreased infarct/edema volumes, delayed acute immune response (modulates cytokines/chemokines levels) and increased acute functional recovery after ischemic injury. Strikingly, although physical exercise did improve cellular and some functional outcomes compared to control animals, it did not synergize with the Mediterranean-like diet but even impaired the positive short-term outcomes. Overall, these data provide the first preclinical evidence that an enriched Mediterranean diet mediates neuroprotection probably by the modulation of cytokines/chemokines levels downwards that eventually has an important role during the acute phase following ischemic damage.

Cerebral ischemia

EPCs

exercise

hydroxytyrosol

inflammation

Mediterranean diet

microglia

Milliplex

neuroprotection

tMCAO

Ischemic stroke remains one of the leading causes of mortality and disability worldwide [1]. Following the occlusion of a blood vessel, the pathological paths of the ischemic cascade are activated, which eventually leads to neuronal death by necrosis or apoptosis [2]. Unfortunately, although the current reperfusion therapies (mechanical thrombectomy and thrombolysis) are currently extended to larger therapeutic windows and larger infarcts, still less than one third of patients receive those treatments [3–5], and the probability of a long-term good functional outcome is still lower than 50% even after a successful reperfusion treatment. Therefore, new approaches such as stroke-preventing alternatives and/or complementary treatments to reperfusion therapies are needed to improve the recovery of stroke patients. Along these lines, healthy lifestyle habits such as physical exercise and the Mediterranean diet have been constantly associated with a lower incidence and mortality in cardio- and cerebrovascular diseases [6–8]. Importantly, beyond the impact on suffering from stroke, both exercise and diet may have positive effects on neuroprotection and neurorepair mechanisms following ischemia [9, 10].

Although the exact molecular mechanisms underlying the beneficial outcome of exercise following stroke are not fully understood, its impact on vasculogenesis, angiogenesis, arteriogenesis and neurogenesis processes was reported [11]. Several studies showed that pre-stroke physical activity was associated with a lower initial severity and infarct volume as well as a better short-term functional outcome [12–14]. Furthermore, clinical studies from our group revealed that a higher rate of pre-stroke exercise reduced the severity and the infarct volume and increased the rate of recanalization following recombinant tissue plasminogen activator (rtPA) treatment and the long-term functional recovery, and this beneficial effect might be mediated by an increase in the vascular endothelial growth factor (VEGF) levels [15, 16].

The Mediterranean diet is based on the abundant consumption of food such as fruits, vegetables, legumes, nuts, and olive oil as the principal fat source, among others. It provides several beneficial nutrients (e.g., monounsaturated fatty acids), a well-balanced proportion of essential fatty acids omega-3/omega-6, high amounts of fiber and antioxidants [17, 18]. So far, numerous in vivo and in vitro studies highlighted the importance of polyphenols as neurorepair substances following ischemia through anti-apoptotic, antioxidant and anti-inflammatory mechanisms due to their ability to cross the BBB [19–25]. Specifically, hydroxytyrosol (HT), an extra-virgin olive oil-derived polyphenol, has been suggested as a promising therapeutic compound following stroke due to its biological effects such as antioxidant, anti-inflammation, and neuroprotection [26–31]. Preclinical studies have shown that the pre-stroke consumption of either olive oil or olive leaf extract positively impacts cellular and behavioral outcomes following ischemia [28, 29]. Similarly, post-stroke administration of HT also revealed beneficial effects in the recovery of animals undergoing stroke [30, 31]. Results from clinical studies assessing the relationship Mediterranean diet and stroke have observed associations with less atherosclerotic etiology and better outcomes, specifically with the consumption of olive oil [32–36].

Physical activity and a balanced Mediterranean diet may exert synergistic effects on ischemic tolerance. Both, the Mediterranean diet and exercise have been also postulated as endothelial progenitor cells (EPCs)-stimulating factors, which mediate vasculogenesis and the optimization of endothelial function following stroke [37, 38]. EPCs are a subpopulation of CD34⁺ bone marrow-derived progenitor cells (BMPCs) that exhibit characteristics of both endothelial and stem cells, participating in angiogenesis and/or the maintenance of the endothelium [39]. Importantly, our group has demonstrated the positive role of EPCs within the first week following stroke [40–42]. Moreover, some previous studies evaluated the synergy of exercise and diet in cognition [43–46]; nevertheless, to our knowledge, no studies have assessed the potential synergistic effect of combining both exercise and diet previously to stroke.

These considerations prompted us to test the possibility that the combination of physical exercise and a Mediterranean-like diet before stroke could act synergistically reducing the infarct volume as well as increasing the functional recovery following injury.

Animal handling

Male adult Sprague-Dawley (SD) rats (300–350 g) were used. Rats were kept in day/night cycles of 12/12 h at a mean temperature of 22 ± 1°C and humidity of 60 ± 5%, and they had water and food ad libitum. For surgical procedures and magnetic resonance imaging (MRI) acquisitions, anesthesia was induced by inhalation of 6% sevoflurane in a N₂O/O₂ mixture (70/30) and maintained at 3–4% sevoflurane during procedures. Body temperature was maintained at 37°C with a feedback-controlled heating pad (Neos Biotec, Pamplona, Spain) until animals completely recovered from anesthesia.

Forty-five SD rats were randomly divided for each condition as follows: 5 rats for the sham group, 10 rats for the control group (normal diet, no physical activity), 10 rats for the diet group (Mediterranean-like rich in polyphenols diet, no physical activity), 10 rats for the PA group (normal diet, physical activity) and 10 rats for the diet/PA group (Mediterranean-like rich in polyphenols diet, physical activity) (Fig. 1).

Physical activity

Animals from PA and diet/PA groups performed physical activity sessions (rotarod) five days per week during the 4 weeks prior to surgery consisting of 5 minutes accelerating from 0.5 to 3 m/min plus 20 min at 3 m/min. All animals were previously adapted to rotarod during the first week by performing 5 minutes at 0.5 m/min. Following surgery, animals were allowed to recover for one week and then they performed physical activity for 3 weeks more until the final follow-up. Sham, control and diet rats performed control training sessions consisting of the placement of the animal in stopped rotarod.

Diet

Within the first week in the animal facility and during the resting week following surgery, all rats were fed on the same food (Teklad Global 19% Protein Extruded Rodent Diet, ref: 2018, ENVIGO, Spain). Then, rats from sham, control and PA groups were fed on a Western-like diet (control diet with dairy butter as the fat source, ref: U8978-version-0177, AIN93, SAFE, France) during the 4 weeks prior to surgery. Animals from diet and diet/PA groups were fed on a Mediterranean-like diet containing 85% olive oil/15% anchovy sardine oil as fat source (ref: U8978 Version 01 AIN93, SAFE, France) during the 4 weeks prior to surgery and for 3 weeks more until the final follow-up. Moreover, rats from diet and diet/PA groups were supplied with a supplemental of HT (ref.: 21001, Nutexa) diluted in the water (200 mg/kg/day).

Surgical procedures

Transient focal ischemia was induced in rats by using the transient middle cerebral artery occlusion (tMCAO) model, which is considered one of the best models to mimic human ischemic stroke and has been used in numerous studies [47–49]. tMCAO surgery was performed as previously described [47] with slight modifications. Briefly, the left common, external, and internal carotid arteries were dissected from connective tissue through a midline neck incision under a surgical microscope (Leica MZ6, Leica Microsystems, Germany). Then, the left external carotid artery and pterygopalatine artery of the internal carotid artery were separated and ligated by 6 − 0 silk sutures. A silicon rubber-coated monofilament (403512PK5Re; Doccol Corporation, USA) was inserted through the external carotid into the left common carotid artery and advanced into the internal carotid artery to 20 mm from the bifurcation to occlude the origin of the MCA. A laser-Doppler flow probe (tip diameter 1 mm) attached to a flowmeter (PeriFlux 5000, Perimed AB, Sweden) was located over the thinned skull in the MCAO territory (4 mm lateral and 1 mm posterior to bregma) to obtain a continuous measure of relative cerebral brain flow during the arterial occlusion. Once the artery occlusion was achieved, as indicated by Doppler signal reduction, each animal was carefully moved from the surgical bench to the magnetic resonance imaging (MRI) system for ischemic lesion assessment using apparent diffusion coefficient maps (defined as T0 ischemic lesion). Moreover, a magnetic resonance angiography (MRA) was also performed to ensure that the artery remained occluded throughout the procedure. After magnetic resonance (MR) analysis, animals were returned to the surgical bench and the Doppler probe was repositioned. The suture was removed after 75 min of occlusion and the left pterygopalatine artery was reperfused while the left external carotid (used to introduce the suture) remained tied to avoid bleeding. As previously described [50], this surgical protocol represents a reliable method to reduce variability intergroup and to guarantee the reproducibility of the infarct volumes during the tMCAO surgery.

Animal experimental procedures

The following exclusion criteria were used: 1) less than 70% reduction in relative cerebral blood flow; 2) arterial malformations, as determined by MRA; 3) baseline lesion volume of less than 25% or greater than 45% with respect to the ipsilateral hemisphere, as measured using ADC maps and 4) absence of reperfusion or prolonged reperfusion (more than 10 min until the achievement of at least 50% of the baseline cerebral blood flow) after monofilament removal.

Experimental procedures were performed following five criteria derived from the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines for preclinical evaluation of stroke therapeutics [51, 52] which are: 1) cerebral blood flow was measured to confirm the vascular occlusion as an index of the reliability of the ischemic model; 2) animals were randomly assigned to treatment groups of the study; 3) researchers were blinded to treatment administration; 4) researchers were blinded to treatments during outcome assessment; and 5) temperature was monitored during the surgery.

Magnetic resonance image protocol

MRI studies were conducted on a 9.4-T horizontal bore magnet MRI system, Biospec 94/20USR, (Bruker BioSpin, Germany) with a 20 cm wide actively shielded gradient coils (440 mT/m). Radiofrequency transmission was achieved with a birdcage volume resonator; the signal was detected using a four-element surface coil, positioned over the head of the animal, which was fixed with a tooth bar, earplugs, and adhesive tape. Transmission and reception coils were actively decoupled from each other.

Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore. During occlusion, MRA was carried out to identify the circle of Willis and evaluate the successful occlusion of the MCA. Axial MRA images of the rat brain were a stack of 58 slices mapping the whole brain. The parameters for the TOF-MRA 3D Flash sequence were field-of-view (FOV) 30.72 x 30.72 x 14 mm3, image matrix 256 x 256 x 58, repetition time 15 ms, 2 averages and echo time 2.5 ms. Immediately after MRA, diffusion coefficient maps ADC maps were acquired using a spin-echo echo-planar imaging sequences with the following acquisition parameters: FOV 24 x 16 mm2, image matrix 96 x 64, 14 consecutive slices of 1 mm thickness, repetition time 4 s, 4 averages, echo time 26.91 ms, spectral bandwidth 200.000 Hz and 7 diffusion b values: 0; 300; 600; 900; 1200; 1600 and 2000. T2-weighted images were acquired at 3, 7, 14, and 28 days after tMCAO using a RARE (factor n = 4) sequence with the following acquisition parameters: FOV 19.2 x 19.2 mm2, image matrix 192 x 192, 14 consecutive slices of 1 mm thickness, repetition time = 3 s, effective echo time = 45 ms.

All images were processed, and maps were constructed with ImageJ software. Diffusion volumes were determined from DWI maps whereas infarct volumes were determined from T2 maps, both by manual selection. Edema was estimated by measuring the volumes of the affected (V_Les) and contralateral (V_c) hemispheres and using the formula: edema (%) = 100 x [(V_Les - V_c)/V_c]. The V_c/V_Les ratio was also used to correct lesion volumes for edema formation.

Functional tests

All animals underwent a battery of functional tests in order to assess the motor function (cylinder test) and evaluate the sensorimotor deficit (Bederson and Wahl’s tests) at different time points during the darkness cycle: baseline (before tMCAO) as well as 3, 7, 14, and 28 days following surgery.

A cylinder test was performed to evaluate limb asymmetry during the exploratory activity, and so, the spontaneous use of forelimb, as previously described.⁵³ Briefly, each animal was put in a cylinder with a 20-cm-diameter transparent base with a video camera underneath recording the vertical exploratory movements of animals. Then, the laterality index was calculated as follows: the number of times that the animal touched the cylinder with the right leg during the ascendant movement by the number of times that the animals touched with each leg. This index is close to 0.5 for healthy animals and tends to 0 or 1 for animals that have preferential use of the left or the right paw, respectively [54].

On the other hand, sensorimotor deficits were evaluated with both Bederson and Wahl scales that mainly assess the presence or absence of reflex and spontaneous activity of the animal [55, 56]. The Bederson scale included the following items: spontaneous movement, spontaneous rotation, spontaneous flexion of the contralateral forelimb, edge detection, turn after tail suspension, reflection of protection. Likewise, the Wahl scale included the following items: edge detection, visual placing, spontaneous flexion of contralateral forelimb and hindlimb, circular spontaneous movement, and both extensor reflex and thoracic torsion when suspended.

Flow cytometry analysis of hematopoietic lineages

Blood samples were drawn from the tail vein before tMCAO (basal sample), and at 1 h, and days 3, 7, 14, and 28 days after tMCAO. The samples were collected into K2EDTA tubes (BD Microtainer, USA). Immunofluorescence cell staining was performed with fluorescent conjugated antibodies anti-CD34 (ref.: sc-7324, Santa Cruz Biotechnology), anti-ckit (ref.: sc-19619, Santa Cruz Biotechnology), anti-KDR (ref.: bs-10412R, Bioss) and anti-CD43 (ref.: 202814, Biolegend). Cell fluorescence was measured 15 minutes after staining by flow cytometry with BD FACS Aria II (BD, Bioscience, Franklin Lakes, NJ, USA). Numbers of each cellular lineage were calculated using the FACSDiva software (BD Biosciences, USA).

Immunomodulator effect and oxidative DNA damage

Blood samples were drawn from the tail vein before tMCAO (basal sample), and at 1 h, and days 3, 7, 14, and 28 days after tMCAO. The samples were collected into K2EDTA tubes (BD Microtainer, USA) and centrifugated at 1000 g for 7 min, storing serum at -80 ºC until analysis. Serum levels of cytokines/chemokines were determined using Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel kit (Cat. RECYTMAG-65K, EMD Millipore, Darmstadt, Germany). Similarly, the measurement of oxidative DNA damage in serum was performed by an ELISA kit (8-hydroxydeoxyguanosine assay, 8-OHdG) (Cat. STA-320; Cell Biolabs, San Diego, CA, USA).

Tissue processing

Three animals per group at 14 days and the rest of them at 28 days were sacrificed by an overdose of anesthesia, and then transcardially perfused with an ice-cold solution of PBS followed by 4% paraformaldehyde (PFA). Finally, brains were dissected out, and the tissues were post-fixed overnight at 4°C in the same fixative solution.

Tissue processing was performed as described previously [57]. Briefly, tissues were incubated in 30% sucrose for cryo-protection. Brains were embedded in OCT compound and frozen. Tissues were transversely sectioned with a cryostat at a thickness of 20 µm. For immunohistochemistry, sections were first washed with 0.2% triton-PBS and antigen retrieval protocol was carried out with sodium citrate (pH 6.0) at 99 ºC for 20 min. Then, sections were blocked and permeabilized (0.4% triton, 5% sera matching the species of the secondary antibodies, 1× PBS) for 1 h at room temperature. Incubation in the primary antibody solution (see concentrations below) was carried out at 4°C overnight. Sections were washed, followed by secondary antibody staining for 2 h at room temperature (antibody solutions at 1:500). Next, sections were washed again, incubated with Hoechst 33342 for 10 min, and mounted with Aqua-Poly/Mount (Polysciences).

The following antibodies were used: mouse anti-Ki67 (ref.: M724029-2, 1:50, Agilent Technologies), rabbit anti-DCX (ref.: ab18723, 1:500, Abcam), guinea pig anti-NeuN (ref.: ABN90, 1:200, Sigma-Aldrich), goat anti-CD31 (ref.: AF3628, 1:100, Novus Biologicals), rabbit anti-VGLUT1 (ref.: 12331, 1:100, Cell Signaling Technology), rabbit anti-Iba1 (ref.: MA5-36257, 1:100, Thermo Scientific), and anti-IB4 (ref.: L2140, 1:50, Sigma-Aldrich).

TUNEL labeling

The Tdt-mediated dUTP Nick End Labelling (TUNEL) Kit (Roche, Mannheim, Germany) was used to detect apoptotic nuclei as previously described [58, 59]. Briefly, sections were pre-treated with MetOH at − 20°C for 15 min and then with 0.01 M citrate buffer pH 6.0 for 30 min at 90°C. Following washes with PBS, each slide was incubated with a mixture of 5 µL of enzyme solution (terminal deoxynucleotidyl transferase) and 45 µL of labelling solution (TMR red labeled nucleotides) for 90 min at 37°C. Then, sections were rinsed in PBS, allowed to dry for 30 min at 37°C, and mounted with Aqua-Poly/Mount.

Image acquisition

Stained tissue sections were photographed using an inverted microscope (Leica CTR6000). Three sections from coronal brain sections were photographed for each animal. Photomicrographs were taken at 5× (areas DCX⁺ or CD31⁺) and 20× (TUNEL, neurogenesis, angiogenesis, synapsis and microglia studies) magnification without changing the amplifier gain or the offset to avoid the introduction of experimental variability. All images were processed with ImageJ software.

Following quantifications, contrast and brightness were minimally adjusted in figures, uniformly across panels for each experiment, with Adobe Photoshop CS4 (Adobe Systems).

Immunofluorescence quantifications

To quantify the intensity of each immunofluorescence (IF) signal in the cortical area surrounding the ischemic area (Iba1, IB4 and vGLUT1 signals), mean fluorescent intensity (mean grey value) was determined using ImageJ software. All values underwent internal normalization to the same contralateral areas and then they were normalized against control values. The number of TUNEL⁺ neurons was calculated by quantifying the NeuN⁺/TUNEL⁺ cells in the cortical area surrounding the ischemic area, in the same way for the numbers of Iba1⁺ cells. The experimenter was blinded during quantifications.

For neurogenesis analysis, Ki67⁺ cells were measured within the DCX⁺ area at the subventricular zone (SVZ). Similarly, angiogenesis was calculated by measuring the number of Ki67⁺ cells within CD31⁺ vessels present at the SVZ.

Statistical analysis

The sample size was calculated using EPIDAT software (http://www.sergas.es/Saude-publica/EPIDAT-4-2), based upon α = 0.05 and power of β = 0.8. Statistical analysis was carried out using Prism 8 (GraphPad software, La Jolla, CA). Data were presented as mean ± S.E.M. Normality of the data was determined by two different tests depending on the n numbers: the D’Agostino-Pearson omnibus test when n numbers were equal or higher than 10, and the Shapiro-Wilk normality test when n numbers were below 10. Data with multiple comparisons were analyzed by either one-way ANOVA or Kruskal–Wallis test where appropriate, and post-hoc Dunn’s multiple comparisons tests. Weight data were analyzed via two-way repeated measures ANOVA with Dunnett post-hoc test. Correlation analysis was assessed with the Spearman correlation coefficient test. The significance level was set at 0.05. In the figures, significance values were represented by different number of asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Mediterranean-like diet reduces the infarct volume following tMCAO

Given that physical activity and polyphenol-rich diets have been associated with a neuroprotective effect following cerebral ischemia [12–14, 28–31], we examined the effect of each condition per separate and together on edema and the infarct volume following stroke (Fig. 2). The analysis and quantification of MR images showed that rats from all conditions had a non-significant reduction of infarct volume at the basal time after tMCAO in comparison with the control group, although the diet group exhibited the highest decreased trend throughout all post-stroke times, specially at 14 days post-ischemia (p = 0.1717) (Fig. 2A-E’’’, F). We also observed similar trends when assessing edema development (Fig. 2G). Animals from the PA group exhibited similar infarct volumes to the diet group at basal, then this volume increased up to control values at 3 days and went down from 7 to 28 days after tMCAO, always higher than the diet group values (Fig. 2A-D’’’, E). Unexpectedly, no effects on infarct volume were observed for the PA/diet group, and if anything, may have slightly increased measurements compared to diet rats (Fig. 2A-D’’’, E). Together, these results revealed that only the Mediterranean-like diet had an impact on the evolution of the ischemic damage, and this positive impact was diminished when combining diet and PA.

Mediterranean-like diet attenuates the systemic inflammatory response and reduces oxidative DNA damage following tMCAO

To determine the effects of either exercise or diet on the inflammatory response pre- and post-stroke plasma samples collected at different time points were analyzed using a magnetic beads-based panel, which allows the measurement of 27 cytokines/chemokines in a single sample (Suppl. Table 1). Remarkably, the diet group exhibited the lowest cytokine/chemokine values in 21 out 27 metabolites at basal (pre-stroke) (Suppl. Table 1). From these, levels of 14 were significantly lower in the diet group compared to controls: granulocyte colony-stimulating factor (G-CSF) (Kruskal-Wallis, p = 0.0075), eotaxin (Kruskal-Wallis, p = 0.0043), macrophage inflammatory protein (MIP)-1α (Kruskal-Wallis, p = 0.006), MIP-2 (one-way ANOVA, p = 0.0314), interleukin (IL)-1α (Kruskal-Wallis, p = 0.0055), IL-1β (one-way ANOVA, p = 0.0115), IL-4 (one-way ANOVA, p = 0.0064), IL-5 (one-way ANOVA, p < 0.0001), IL-6 (Kruskal-Wallis, p = 0.0075), IL-12p70 (one-way ANOVA, p = 0.0002), IL-13 (one-way ANOVA, p = 0.0158), IL-17 (one-way ANOVA, p = 0.0023), IL-18 (Kruskal-Wallis, p = 0.0086) and interferon gamma (IFNγ) (one-way ANOVA, p = 0.0091) (Fig. 3A, Suppl. Table 1). Importantly, the Mediterranean-like diet appeared to attenuate the inflammatory response until 7 days post-stroke by showing the lowest levels of cytokines/chemokines compared to controls (Suppl. Table 1). Indeed, data normalization against basal values revealed that the Mediterranean-like group strikingly increased the levels of most of the inflammatory markers from 7 days post-stroke (Suppl. Figure 1). In contrast, the PA group only showed 6 metabolites, all of them included in the diet group, with values significantly lower than control ones at basal: IL-1α (Kruskal-Wallis, p = 0.0156), IL-1β (one-way ANOVA, p = 0.0494), IL-5 (one-way ANOVA, p = 0.0085), IL-12p70 (one-way ANOVA, p = 0.0098), IL-17 (one-way ANOVA, p = 0.0362) and IFNγ (one-way ANOVA, p = 0.0457) (Fig. 3A, Suppl. Table 1,). Moreover, there was an acute increase of these basal values at 1h, and then at 14 days post-injury in the PA group (Fig. 3A, Suppl. Figure 1, Suppl. Table 1). Finally, the PA/diet group had 3 values significantly lower than controls at basal: MIP-1α (Kruskal-Wallis, p = 0.0035), IL-5 (one-way ANOVA, p = 0.0002) and IL-12p70 (one-way ANOVA, p = 0.0022) (Fig. 3A, Suppl. Table 1). Similar to the PA group, there was an overall increase of basal values at 14 days post-injury which decreased at 28 days in the PA/diet group (Fig. 3A, Suppl. Figure 1, Suppl. Table 1).

We then measured plasma levels of 8-OHdG as a marker of oxidative stress and found that the control group exhibited the highest basal levels of 8-OHdG, although they were not statistically significant (Fig. 3B). Intriguingly, post-ischemia timepoints revealed that the PA/diet group had higher levels of 8-OHdG at 7 and 28 days, and this increase was statistically significant when compared with diet group at 7 days post-injury (Fig. 3B; one-way ANOVA, p = 0.0199), and it was close to be significant compared with the PA group (Fig. 3B; one-way ANOVA, p = 0.0619).

In summary, rats fed on a Mediterranean-like diet exhibited the lowest pre-stroke levels of cytokines/chemokines and contained the acute inflammatory response up to 7 days pos-stroke, whereas the combination of diet and exercise seemed to block this basal anti-inflammatory response and the PA group showed an acute increase following injury.

Mediterranean-like diet triggers acute EPCs mobilization

Several works from our group revealed the positive impact of higher numbers of CD34⁺ BMPCs, including EPCs, within the first week following stroke [40–42, 60]. Furthermore, both physical activity and a Mediterranean diet have been postulated as EPCs-stimulating factors [37, 38]. Then, we analyzed the levels of circulating progenitor cells (CPCs), identified as CD34 positive (CD34⁺); hematopoietic progenitor cells (HPCs, labeled as CD34 and CD43 positive (CD34⁺/ CD43⁺); and EPCs, positive to CD34, ckit and KDR, by flow cytometry.

Measurements of circulating levels of CPCs and HCs showed that the diet group have the highest levels of CPCs from basal to 3 days post-stroke and HCs from basal to 1h post-injury although they were not statistically significant (Fig. 4A, Suppl. Table 2). The PA group had a non-significant peak at 7 days post-injury, whereas the PA/diet group exhibited the highest numbers in CPCs and HCs at 3 days post-stroke without statistical significance (Fig. 4A, Suppl. Table 2). Regarding EPCs, the diet group showed significantly lower numbers of EPCs than controls at basal (Kruskal-Wallis, p = 0.0291) (Fig. 4A, Suppl. Table 2), which remarkably increased at 1h post-stroke and continued higher up to 28 days post-injury as confirmed by normalized analyses against basal values (one-way ANOVA, p = 0.0122; Fig. 4B). The exercise group did not show significant changes in EPCs levels following stroke and the PA/diet group did have the highest level of EPCs at 3 days post-lesion although without statistical significance (Fig. 4A, Suppl. Table 2). Therefore, the diet group showed high and constant numbers of both CPCs and HPCs but had the lowest value of EPCs at basal, that then exhibited a remarkable acute increase that was maintained.

Mediterranean-like diet improves functional recovery following an ischemic insult

Afterward, we analyzed the recovery of animals undergoing tMCAO by using different functional tests. Rats from the diet group showed the lowest values in the Bederson scale throughout all the post-injury times, especially at 3 days post-stroke, where the difference is close to be statistically significant (one-way ANOVA, p = 0.0544) (Fig. 5A). However, both PA and PA/diet groups exhibited a similar trend as controls (Fig. 4A). Likewise, the diet group showed the lowest reduction in the Wahl scale at 3 days post-injury (Kruskal-Wallis, p = 0.1503) (Fig. 5B), but eventually the evolution up to 28 days following injury was similar between groups (Fig. 5B). Strikingly, the cylinder test revealed that PA and PA/diet groups had the worst scores in both the use of the impaired forelimb and laterality index from 3 to 14 days following injury (Fig. 5C-D). The diet group mimicked control scores until 28 days post-stroke, where it showed better scores as well as the PA group (Fig. 5C-D). Importantly, rats undergoing diet and exercise had higher limb asymmetry than rats fed on diet, and so, the exercise seems to damper the effect of the diet following injury (Fig. 5C-D). Overall, the diet group exhibited a better functional recovery following stroke, which was negatively influenced when combined with exercise.

Each condition acts differently on molecular mechanisms underlying functional improvement following tMCAO

Given that exercise, the Mediterranean diet and/or the polyphenol HT have been related to either neuroprotection, vasculogenesis, neurogenesis, or anti-inflammatory processes [11, 19–23], we carried out immunohistochemical analyses to assess the impact of each condition on these mechanisms.

Cortical pyramidal neurons at layers V/VI are the main players not only for motor functions but also integrating sensory inputs [61, 62]. As glutamatergic neurons, vesicular glutamate transporter 1 (vGLUT1) plays a vital role in glutamatergic transmission, and so, this has been suggested as beneficial at the chronic phase following stroke [63, 64]. We observed higher vGLUT1 immunoreactivity in diet, PA and PA/diet groups than both control and sham groups in the peri-infarct tissue at cortical layers V/VI after 28 days post-injury, although this was statistically significant only in those against the sham group (Kruskal-Walllis, diet: p = 0.0153; PA: p = 0.0376; PA/diet: p = 0.0030) (Fig. 6D-D’’’, F). Moreover, this agrees with the analysis of TUNEL labeling in NeuN⁺ cells at cortical layers V/VI, where less apoptotic neuronal death has been detected in all experimental conditions than controls, although this difference was only significant in the PA/diet group (one-way ANOVA, p = 0.0149) (Fig. 6A-C’’’, E). Altogether, all experimental conditions were able to increase the numbers of cortical glutamatergic pyramidal neurons near the lesion, and this was especially remarkable in the PA/diet group.

Angiogenesis and neurogenesis are two important mechanisms to improve functional recovery following stroke [65, 66]. Our results showed that the PA group remarkably increased the numbers of CD31⁺/Ki67⁺ vessels at the subventricular zone (SVZ) not only compared to sham and control groups (one-way ANOVA, p = 0.0071 and p = 0.0446, respectively) but also compared to the diet group at 28 days post-stroke (one-way ANOVA, p = 0.0018) (Fig. 7A-E’’, J). Furthermore, these results agreed with the higher CD31⁺ areas seen in diet, PA and PA/diet groups compared to controls (Fig. 7A’’’-E’’’, K). We also observed the pattern of expression of the IB4 staining in the peri-infarct area 28 days following stroke. The diet group exhibited the highest increase in IB4 staining but it was not statistically significant (Fig. 7F-I’’, L). Our results may suggest the existence of more vascular repair in the diet group than the others after tMCAO.

Regarding neurogenesis, only the PA group showed statistically higher levels of neurogenesis (DCX⁺/Ki67⁺ cells) than sham and control groups at the SVZ (Kruskal-Wallis, p = 0.0468 and p = 0.0013, respectively) (Fig. 8A-E’’, F). Regarding the DCX⁺ area, the PA group exhibited remarkable higher labeling than the rest of conditions (one-way ANOVA, vs sham: p < 0.0001, vs diet: p = 0.0239, vs PA/diet: p = 0.0083) (Fig. 8A’’’-E’’’, G). The control group also had a significant higher DCX⁺ area than sham animals at the SVZ (one-way ANOVA, p = 0.0055) (Fig. 8B’’’, G).

Summarizing, rats performing physical exercise showed more angiogenesis and neurogenesis than the other conditions at 28 days post-injury.

The role of microglial cells following stroke and its relationship with inflammation processes, although controversial, seems to depend on the time post-injury [67–70]. We used the Iba1 antibody, the microglia-specific marker, in the cortical peri-infarct area at two different time-points after tMCAO, 14 and 28 days. After 14 days post-stroke, the control group showed higher non-significant levels of Iba1⁺ compared to the sham group (Kruskal-Wallis, p = 0.0608) and the other conditions (Fig. 9A-D, F). Interestingly, this was accompanied by a significant increase in the number of Iba1⁺ cells from the control group compared to sham animals (Kruskal-Wallis, p = 0.0096) (Fig. 9A-D, F). At 28 days post-injury, the immunohistochemical signal in the control group was reduced, the diet group showed almost the same value as at 14 days, but it was statistically significant vs the sham group (one-way ANOVA, p = 0.0359) (Fig. 9A’-D’, G). Likewise, the PA group exhibited the highest increase which was significant vs sham animals (one-way ANOVA, p = 0.012) (Fig. 9C’, G). Moreover, the presence of Iba1⁺ cells was increased in both diet and PA groups compared to sham animals (Kruskal-Wallis, p = 0.0005 and p < 0.0001, respectively), whereas the control group showed a slightly reduction but still significant vs the sham group (Kruskal-Wallis, p = 0.0114), and the PA/diet group remain unchanged (Fig. 9G). Importantly, there was a positive correlation for Iba1⁺ immunofluorescence signal with the numbers of Iba1⁺ cells surrounding the peri-infarct cortical area (Spearman test, r = 0.6444, p < 0.0001) (Fig. 9H). Together, these results suggested that increases in the Iba1 signal are due to a higher number of Iba1⁺ cells, rather than an enlargement of the cell body and/or increased expression of Iba1.⁶⁷

In this study, we have provided new data showing that a Mediterranean-like diet supplemented with HT may act as a pre-conditioning factor that protects against the acute damage following a tMCAO injury, and this beneficial effect is attenuated when these animals also perform physical activity. This represents the first in vivo demonstration that an enriched Mediterranean diet can attenuate the acute systemic inflammatory response and this leads to a reduction in the edema volume as well as it improves the functional recovery at the acute phase following ischemic injury. Our data suggest that this occurs by reducing the basal levels (pre-stroke) of cytokines/chemokines which slows down the acute and harmful stroke-induced immune response up to 7 days post-injury, and the post-stroke consumption of an enriched Mediterranean diet has no apparent effects. Here we discuss the implications and possible mechanisms underlying this enriched diet-mediated protection after tMCAO.

Our study has found important data showing basal low levels of circulating cytokines/chemokines and a delayed immune response in rats fed on enriched Mediterranean-like diet following ischemic damage. It is well-known that the spleen is the largest secondary lymphoid organ with a major role in the induction of adaptive immune responses following damage, including ischemia [71, 72] and, interestingly, some studies have suggested that either a Mediterranean diet [73] or polyphenols [24, 74] has a positive immunomodulatory effect on the spleen. Interestingly, previous studies showed that either the pre-tMCAO removal of the spleen or its post-tMCAO irradiation results in lower infarct volumes and white blood cell counts [75–77]. Therefore, we hypothesize that such low basal levels of circulating proinflammatory cytokines/chemokines in animals fed on a Mediterranean-like diet plus HT are due to a direct impact on spleen function. Likewise, this gradual and delayed activation of the immune cascade is maintained during the acute phase (from 1h to 3 days) which likely reduces the edema. At first glance, the apparent dual role of physical exercise could be striking: whereas exercise decreased the levels of circulating cytokines/chemokines when comparing animals fed on a Western-like diet (control and PA groups), exercise increased the levels of inflammation markers when comparing animals fed on a Mediterranean-like diet and HT (diet and PA/diet groups). However, it is well-known that a Western-like diet leads to chronic inflammation [78] and that exercise has a positive effect by counteracting many mechanisms triggered by this diet [79], which agrees with our present results. It is important to note that the crosstalk between oxidative stress and inflammation has a major role after stroke [80]. Here, we found a surprising result when the PA/diet group showed the highest levels of 8-OHdG in plasma following tMCAO. However, a recent work showed that a dose of HT of 300 mg/kg/day in animals undergoing physical exercise appears to trigger harmful effects by switching the role of HT from anti- to pro-oxidant [81], as previously suggested in vitro [82]. Therefore, our results support previous ones and point at a pro-oxidant effect of the 200 mg/kg/day dose of HT when combined with exercise, that may negatively impact the immune response following ischemic damage. Further studies will be needed to fully elucidate the mechanisms of action for each process.

Most of these circulating cytokines/chemokines target microglia and astrocytes that are pivotal players in the immune response following ischemia as well, being both activated within minutes after damage and having a great impact on neurotoxicity and edema development. Although the harmful impact of astrocytes is generally accepted, there is controversy about the exact role of microglia following ischemic injury [67–69]. Remarkably, it has been shown that microglia and newly recruited macrophages keep an anti-inflammatory state M2 at initial steps following damage that it is gradually changed to a pro-inflammatory state M1 [83]. Based on our data, we propose that the diet-mediated low levels of pro-inflammatory cytokines/chemokines may extend this M2 state in microglia during the acute phase after ischemia which would attenuate the cerebral inflammation and, hence, reduce infarct volume. Furthermore, the number of microglia is rapidly increased during the first 10 days after the insult [84]. Although we did not perform analysis at 10 days post-injury, we did observe that the number of Iba1⁺ cells decreased in the control group from 14 to 28 days post-injury, and this was contrary to the diet group where we found more Iba1⁺ cells at 28 days than at 14 days post-injury with no effect of re-starting the diet at 7 days post-injury, which is consistent with previous work at 35 days post-ischemia [31]. This agrees with the increasing levels of cytokines/chemokines seen in the diet group from 7 days post-injury and may explain this late increase in Iba1⁺ cells. Furthermore, the PA group exhibited a temporal increase in cytokines/chemokines at 14 days compared to 7 days post-stroke, which could explain the increased numbers of Iba1⁺ cells at 28 days as acute bouts of exercise transiently increase inflammation [85]. Therefore, it is plausible that basal/acute low levels of pro-inflammatory cytokines/chemokines promoted by diet/HT could slow down the activation and recruitment of microglia until the sub-chronic/chronic phase of ischemia, and that the restoration of physical exercise following injury may cause an acute increase in circulating cytokines/chemokines that eventually activates microglia.

Vasogenic edema is the main component of brain edema, and it is caused by the disruption of endothelial tight junctions in the BBB due to inflammatory molecules and oxidative stress following brain injuries [86]. Specifically, glial and peripheral immune cells as well as their released cytokines/chemokines have a key role in the pathophysiology of vasogenic edema [86]. Our results from flow cytometry analysis revealed an acute increase in EPCs levels only in the diet group following injury, and this is a remarkable result given that these animals had the lowest level of EPCs at the basal time. Similarly, both CD34⁺ and HPCs numbers were the highest in the diet group from basal to 3 days post-injury. Interestingly, several works assessed the effect of a Mediterranean diet on CD34⁺ and EPCs numbers following different injuries but not ischemic damage [87–89], overall seeing an increase in EPC numbers due to the diet. Recently, an in vitro study reported that low concentrations of HT can stimulate the migration of endothelial cells [90]. Moreover, previous clinical studies have shown that CD34⁺ BMPCs, including EPCs, are related to a good functional recovery and outcome following stroke [57–60], being consistent with the results of our study. Hence, it is conceivable that either diet or HT or both acts on CD34⁺ progenitor cells, primarily EPCs, allowing them to reach the site of injury, due to the high concentration of angiogenic factors and inflammatory cytokines, from which they paracrinally release different factors promoting angiogenesis and recruiting EPCs, which either restore the endothelium or form new vessels guided [91]. Furthermore, immunohistochemical analysis showed a higher CD31⁺ area but slightly lower numbers of proliferating CD31⁺ cells in the SVZ of the diet group, besides an increase in IB4 labeling in the peri-infarct area, compared to controls at 28 days post-injury. Overall, these results may suggest that either low neuroinflammation or increased acute levels of CD34⁺ BMPCs/EPCs or both protects BBB-forming endothelial cells, and this reduces cerebral edema. Given that animals fed on an enriched Mediterranean-like diet displayed larger CD31⁺ area and IB4 staining but few proliferating CD31⁺ cells at 28 days post-injury, it could be possible that EPCs exert a protective role rather than promoting angiogenesis. The exercise group did not show important changes in CD34⁺ BMPCs numbers, however, it did show a remarkable increase in proliferating CD31⁺ cells at 28 days post-injury. Previous studies addressed the role of exercise in angiogenesis with positive results [11, 92], and so, we speculate that animals fed on a Western-like diet that underwent physical activity showed higher angiogenesis by incrementing CD31⁺ cells proliferation. Future studies will be needed to decipher the exact mechanism on cerebral vasculature underlying both Mediterranean diet/HT and exercise-positive outcomes.

Several studies highlighted the ability of polyphenols to trigger anti-apoptotic and antioxidant mechanisms [19–25]. Importantly, HT is a well-known minor component found in the extra-virgin olive oil that has an important physiological effect following ischemic damage such as neuroprotection [26–31, 93]. Likewise, physical exercise has been related to higher neuronal survival following injury [94–97]. Here, we reported that either diet or PA or PA/diet leads to a reduced neuronal death in cortex that agrees with a higher vGLUT1 intensity at 28 days post-injury. Based on the above previous works, we speculate that such reduction in TUNEL labeling can be related to either HT supplement present in the Mediterranean-like diet or exercise or both. Interestingly, the area occupied by DCX, a marker for migrating immature neurons, is smaller in both diet and PA/diet groups than in control animals at 28 days post-injury, which could suggest that those animals fed on the enriched diet did not need to replace death neurons as much as control rats. Interestingly, a Mediterranean-like diet supplied with HT slightly maintained a high rate of proliferating neurons at 28 days post-injury compared to controls, and this trend appeared to increase in the PA/diet, that maybe indicate a more remarkable influence of exercise in promoting neuron proliferation. Indeed, the PA group exhibited a striking rate of neuron proliferation almost 10 times higher than the control group at 28 days post-injury, which agrees with the proliferating rate seen in CD31⁺ cells and may explain the highest DCX⁺ area in the PA group.

Regarding functional outcomes, the consumption of a Mediterranean-like diet and HT led to a clearer improvement in functional recovery at short times (3 and 7 days post-injury). The pattern of effects on neuroinflammation, edema and cellular mechanisms may support the hypothesis that an enriched Mediterranean diet allows faster behavioral recovery through a combination of these three aspects rather than one alone. In this regard, our study agrees with previous studies in that either pre-injury olive oil compounds^28,29 or post-injury HT administration [30, 31] shows less infarct volume and better functional recovery in rodent models of ischemic damage. The other conditions PA and PA/diet also showed a better trend than controls in behavioral tests at acute times post-injury (3 days), likely by increasing cellular proliferation. However, it is also important to note that all conditions eventually achieved similar levels of functional recovery at 28 days post-injury.

Together, our findings indicate that a HT-enriched Mediterranean-like diet downregulates basal cytokines/chemokines pre-ischemia and attenuates the acute immune response post-injury, resulting in lower infarct volume, delayed microglia activation and faster functional improvement. Our study provides the first in vivo demonstration that the regular consumption of a Mediterranean diet supplemented with HT can positively modulate the immune system, supporting the hypothesis that inflammation has a key role in the pathophysiology of ischemic damage. Unexpectedly, physical exercise exerts a dual role: it is beneficial in animals fed on a Western-like diet, while it reduces some of the beneficial outcomes of a Mediterranean-like diet. Future studies will be required to determine the exact cellular mechanisms underlying the beneficial effect of either HT or the Mediterranean-diet or both following ischemic damage.

BMPCs: Bone marrow-derived progenitor cells

CPCs: Circulating progenitor cells

EPCs: Endothelial progenitor cells

HT: Hydroxytyrosol

HPCs : Hematopoietic progenitor cells

IFNγ: Interferon gamma

IL: Interleukin

MIP-1α: Macrophage inflammatory protein-1α

MIP-2: Macrophage inflammatory protein-2

MRA: Magnetic resonance angiography

MRI: Magnetic resonance imaging

rtPA: Recombinant tissue plasminogen activator

SVZ: Subventricular zone

tMCAO: Transient middle cerebral artery occlusion

vGLUT1: vesicular glutamate transporter 1

Ethics approval and consent to participate

Experimental protocols were approved by the University Clinical Hospital of Santiago de Compostela Animal Care Committee (15010/2019/004), according to the European Union (EU) rules (86/609/CEE, 2003/65/CE and 2010/63/EU) and with the ARRIVE guidelines.

Funding

This study was partially supported by grants from the Xunta de Galicia (TS: IN607D 2020/09 & IN607A2022/07, AC: IN606A-2021/015), Ministry of Science and Innovation (TS: RTI2018-102165-B-I00, RTC2019-007373-1, and PDC2022-134000-I00) and Instituto de Salud Carlos III (ISCIII) (MC: CM19/00235; EL-C: PI18/01096 and INT19/00075; TS: PI22/00938) and CIBERNED (CB22/05/00067). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No. 101066444). Furthermore, this study was also supported by grants from the INTERREG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC project), INTER-REG V A España Portugal (POCTEP) (0624_2IQBIONEURO_6_E), and the European Regional Development Fund (ERDF).

The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The data that support the findings of this study are available from corresponding authors (AO and EL-C), upon reasonable request.

Author contributions

DR-S, MC, EL-C, and TS designed and conceptualized the research framework, interpreted results of experiments, and drafted the manuscript; DR-S, MC, EL-A, AC, CM and AO performed experiments; DR-S, MC, EL-C and TS analyzed data; AO, EL-C and TS performed supervision and critical review; and EL-C and TS got funding. DR-S and MC have equally contributed as first authors, and EL-C and TS have equally contributed as Senior authors.

All authors read and approved the final version of the manuscript.

Consent for publication

All authors declared to consent for publication.

Conflict of interests

The authors declare no conflict of interest.

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No competing interests reported.

SupplementaryTable1.xlsx
SupplementaryTable2.xlsx
Supplementaryfigure1.tif
Supplementary Figure 1. Rats fed on an enriched Mediterranean-like diet increases circulating cytokines/chemokines later. Time course of normalized data, against control values at basal, from concentrations of twenty-seven cytokines/chemokines. (1) Granulocyte colony-stimulating factor (G-CSF): *p=0.0247 (diet 14 days). (2) Eotaxin: **p=0.0074 (diet 28 days). (3) Macrophage inflammatory protein (MIP)-1α: *p=0.0444 (PA/diet 3 days), *p=0.0114 (diet 7 days), *p=0.024 (diet 14 days). (4) MIP-2. (5) Interleukin (IL)-1α. (6) IL-1β: **p=0.0058 (PA 1h). (7) IL-4. (8) IL-5: *p=0.0252 (PA/diet 3 days), **p=0.0035 (diet 14 days). (9) IL-6. (10) IL-12p70: *p=0.0453 (PA/diet 3 days), *p=0.011 (diet 14 days), *p=0.0169 (diet 28 days). (11) IL-13: *p=0.0116 (diet 28 days). (12) IL-17: *p=0.0125 (diet 7 days), *p=0.0121 (diet 14 days), **p=0.0018 (diet 28 days). (13) IL-18: *p=0.0283 (diet 3 days), *p=0.0421 (diet 7 days). (14) Interferon gamma (IFNγ): *p=0.0303 (PA 1h), **p=0.0067 (diet 7 days), **p=0.0097 (diet 28 days). (15) Regulated on activation, normal T cell expressed and secreted (RANTES). (16) Tumor necrosis factor alpha (TNFα). (17) Vascular endothelial growth factor (VEGF): *p=0.0229 (diet 3 days). (18) Epidermal growth factor (EGF). (19) Fractalkine: *p=0.046 (diet 3 days). (20) Granulocyte-macrophage colony-stimulating factor (GM-CSF): p=0.0363 (diet 14 days). (21) GRO/KC. (22) IL-2: **p=0.0034 (PA 3 days), **p=0.0034 (PA/diet 3 days), **p=0.0092 (diet 14 days), *p=0.0272 (PA 14 days), **p=0.0046 (PA/diet 14 days), **p=0.0099 (PA 28 days), **p=0.0027 (PA/diet 28 days). (23) IL-10: *p=0.0103 (diet 1h), *p=0.0375 (diet 7 days). (24) Interferon gamma-induced protein 10 (IP-10): *p=0.0429 (diet 3 days),*p=0.0281 (PA 7 days), *p=0.0341 (diet 14 days). (25) Leptin. (26) LPS-induced CXC (LIX): ***p=0.0005 (PA 3 days), *p=0.0239 (PA 28 days). (27) Monocyte chemoattractant protein-1 (MCP-1). 6 rats per each group. Stats: One-way ANOVA at each time point vs control. Bars show mean ± SEM.

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Neuroprotection afforded by an enriched Mediterranean-like diet is modified by exercise in a rat model of cerebral ischemia

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Animal handling

Physical activity

Diet

Surgical procedures

Animal experimental procedures

Magnetic resonance image protocol

Functional tests

Flow cytometry analysis of hematopoietic lineages

Immunomodulator effect and oxidative DNA damage

Tissue processing

TUNEL labeling

Image acquisition

Immunofluorescence quantifications

Statistical analysis

RESULTS

Mediterranean-like diet reduces the infarct volume following tMCAO

Mediterranean-like diet attenuates the systemic inflammatory response and reduces oxidative DNA damage following tMCAO

Mediterranean-like diet triggers acute EPCs mobilization

Mediterranean-like diet improves functional recovery following an ischemic insult

Each condition acts differently on molecular mechanisms underlying functional improvement following tMCAO

DISCUSSION

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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