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The Pomace Extract Taurisolo Protects Rat Brain From Ischemia-Reperfusion Injury

Taurisolo® is a pomace extract from Aglianico Grapes, a wine cultivar native to Campania (Southern Italy). It exhibits a very high polyphenolic content and, consumed as a nutraceutical, is effective in reducing the level of Trimethylamine N-oxide (TMAO), a cardiovascular disease risk factor marker. We here show the effects of Taurisolo® on rat brain microvascular alterations induced by a diminution in cerebral blood flow (CBFD) for 30 min, due to bilateral common carotid artery occlusion, and subsequent blood flow restoration (CBFR) for 60 min. The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed window implanted into the skull bone. The rat pial arterioles were classified according to Strahler’s ordering scheme, from smaller penetrating arterioles up to the larger ones. Western blotting analysis and mass spectrometry (MS)-based metabolomics were used to investigate the expression of endothelial nitric oxide synthase (eNOS) or the presence of peroxidized cardiolipin and several inflammatory mediators, respectively. Radical Oxygen Species (ROS) formation and neuronal loss were assessed. In rats CBFD and CBFR caused a decrease in arteriolar diameter, increase in fluorescent leakage and in adhesion of leukocytes to venular walls, reduction in the length of perfused capillaries and increment of ROS formation with large infarct size. Taurisolo®, intravenously or orally administered, induced pial arteriolar dilation (up to >30% of baseline), prevented fluorescent leakage, adhesion of leukocytes, ROS formation, while facilitated capillary perfusion and significantly reduced infarct size. These effects were accompanied by an increase in eNOS expression. Mass-spectrometry metabolomics analysis detected a marked decrease in the amount of peroxidized cardiolipin and pronounced reduction in pro-inflammatory prostaglandins and thromboxane Txb2. Altogether, these results extend the nutraceutical potential of Taurisolo® and suggest their eligibility for preventing brain damage due to ischemia-reperfusion injury.


INTRODUCTION
Endogenous antioxidants, like melatonin and glutathione, have been shown to exert preventive effects on an ischemiareperfusion injury, a complication resulting from the rapid restoration of the blood flow supply after an ischemic insult (Lapi et al., 2011;Zhou et al., 2018). Recently, plant extracts enriched in polyphenols (Brosková et al., 2013), epigallocatechin, epigallocatechin-3-gallate (ECCG; Aneja et al., 2004), oleuropein and malvidin (Lapi et al., 2015(Lapi et al., , 2016 have been included among the nutraceuticals endowed with a protective effect against ischemia-reperfusion injury. Their mechanisms of action have been mainly linked to their antioxidant potential and their ability to counterbalance oxidative stress (Zhou et al., 2018). Upon an IR event, indeed, a burst of reactive oxygen species (ROS) is produced by several cell types (Zorov et al., 2014). This massive ROS production induces oxidative damage, reduces ATP production and leads to necrotic or apoptotic cell death. ROS-mediated damage will also trigger the inflammatory response (Eltzschig and Eckle, 2011), i.e., a production of prostaglandins, contributing to the formation of interstitial edema, reduction of arteriolar diameter and venular wall alterations. Much evidence indicates that nutraceutical inhibits mitochondrial oxidative stress and ROS production resulting in a partial reduction of brain damage (Brosková et al., 2013).
The mitochondria are the primary intracellular source of ROS. Among phospholipid species, cardiolipin has interesting chemical and structural properties and has also a highly specialized physiological distribution, being almost exclusively located in the inner mitochondrial membrane where it is biosynthesized (Paradies et al., 2014). Several studies indicate that cardiolipin is involved in the regulation of main mitochondrial bioenergetic processes, optimizing the activity of key mitochondrial inner membrane proteins involved in oxidative phosphorylation (Houtkooper and Vaz, 2008). Furthermore, alterations to cardiolipin structure, content, and acyl chain composition are responsible for mitochondrial dysfunction in multiple tissues in a variety of physiopathological settings (Petrosillo et al., 2003;Paradies et al., 2009Paradies et al., , 2013. Our research group has recently identified the antioxidant property of the Pomace (called Taurisolo ), obtained from Aglianico Grapes, a wine cultivar native to Campania region (Southern Italy), listed as a Protected Geographical Indication (PGI) product [Commission Regulation (EC) No. 417/2006]. Taurisolo exhibits a very high polyphenolic content, specifically represented by resveratrol, catechins and their derivatives. On the basis of its anti-oxidant activity, we here challenged Taurisolo in preventing IR injury damage, by evaluating the effects of its acute (intravenous) or chronic (oral) treatment in rats subjected to cerebral hypoperfusion and reperfusion. We here show that Taurisolo reduced the main IR-dependent pial microvascular changes, such as microvascular permeability increase, leukocyte adhesion, reduction in perfused capillary length, ROS formation and neuronal loss.
Furthermore, employing high-resolution mass spectrometry (MS) techniques and measuring cardiolipin peroxidation and arginine catabolites, we further show that Taurisolo reduces cell membrane alterations and increases NO production.
Hypo-reperfused group (H-group) induction of CBFD for 30 min and of CBFR for 60 min.
Taurisolo -treated group (T group) Tiv i.v. administration of Taurisolo , 10 mg/kg b.w. 10 min prior to CBFD and at the beginning of CBFR.
Tor oral administration of Taurisolo (20 mg/kg b.w./dye) for 1 month and induction of CBFD and of CBFR.
L-Tor oral administration of Taurisolo (20 mg/kg b.w./die) for 1 month and i.v administration of L-NIO 10 min prior to CBFD and at the beginning of CBFR.
the end of treatment animals were subjected to CBFD and CBFR.
Taurisolo dosages were determined by pilot experiments. We tried several dosages by intravenous administration: 3,5,8,10,12,15,18,20,22, 25 mg/kg b.w. dosages and we observed that a dosage lower of 5 mg was ineffective. In the range between 8 and 20 mg/kg b.w. Taurisolo exerted a protective effect on pial microcirculation. We observed as well that doses above 20 mg/kg b.w did not further improve the protective effects exerted by the lower dosages. Therefore, to avoid a high concentration of the substance, we chose to use 10 mg/kg b.w. a concentration similar to those of previously studied anti-oxidant molecules. Oral administration of Taurisolo at the dosages of 10, 15, 20, 25 mg/die shorter than 15 days did not have significant effects; therefore, we report the data obtained after 30 days of treatment at the dosage of 20 mg/kg b.w./die, effective in the protection.

Surgery Procedure
The experiments were performed following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and to institutional rules for the care and handling of experimental animals, as previously reported (Lapi et al., 2012a). The protocol was approved by the ''Federico II'' University Medical School of Naples, Ethical Committee (n • 2011/0059997, 24/05/2011). Animals were anesthetized with intraperitoneal (i.p.) injection of α-chloralose (60 mg/kg b.w. for induction; afterward 30 mg/kg b.w.) and mechanically ventilated after tracheotomy, according to the experimental protocol previously reported (Lapi et al., 2012b). Two catheters were placed, one in the right femoral artery and the other in the left femoral vein, respectively, for the measurement of arterial blood pressure and to inject the fluorescent tracers [fluorescein isothiocyanate, FITC bound to dextran, molecular weight 70 kDa (FD 70), 50 mg/100 g b.w., as 5% wt/vol solution in 3 min just once at the start of experiment after 30 min of the preparation stabilization; rhodamine 6 G, 1 mg/100 g b.w. in 0.3 ml, as a bolus with supplemental injection throughout CBFD and CBFR (final volume 0.3 ml·100 g −1 ·h −1 ) to label leukocytes for adhesion evaluation]. Both carotid arteries were prepared for clamping.
Blood gases were measured on arterial blood samples at 30 min intervals (ABL5; Radiometer, Copenhagen, Denmark). The parameters monitored in all animals were: heart rate, mean arterial blood pressure, respiratory CO 2 and blood gases values. They were stable within physiological ranges. Rectal temperature was recorded and maintained at 37.0 ± 0.5 • C, as previously reported (Lapi et al., 2016). The visualization of pial microvasculature was carried out as previously reported (Lapi et al., 2012a). Briefly, a closed cranial window was positioned at the level of the left frontoparietal cortex through an incision in the skin to operate a craniotomy. The cerebral cortex was preserved from overheating caused by drilling with saline solution superfusion of the skull. The dura mater was gently cut and displayed on the corner; a quartz microscope coverglass was bound to the skull bone. Artificial cerebrospinal fluid (aCSF) was superfused on the cerebral surface with a rate of 0.5 ml/min. The composition of the aCSF was 119.0 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 •7H 2 O, 1.0 mM NaH 2 PO 4 , 26.2 mM NaHCO 3 , 2.5 mM CaCl 2 and 11.0 mM glucose (equilibrated with 10.0% O 2 , 6.0% CO 2 and 84.0% N 2 ; pH 7.38 ± 0.02).
Hypoperfusion was induced by clamping both common carotid arteries, previously prepared (CBFD) for 30 min, and after removing the clamping the pial microcirculation was observed for 60 min (CBFR).

Determination of Microvascular Parameters
Microvascular parameters, index of microvascular damage, were measured off-line utilizing a computerized imaging technique (Lapi et al., 2012b(Lapi et al., , 2016. First, the arterioles were measured and classified in orders by Strahler's method (Lapi et al., 2008), utilizing a frame by frame computerized method (Microvascular Imaging Program, MIP).
The arteriolar diameter changes were evaluated in all orders of pial arterioles; however, we have shown the data collected in 30 arterioles of order 3 for each rat group or subgroup studied.
The increase in microvascular permeability was measured by evaluating fluorescent dextran extravasation from venules and expressed as normalized gray levels (NGLs): NGL = (I − Ir)/Ir, where, Ir was the baseline gray level at the microvasculature filling with fluorescence, and I was the value at the end of CBFD or CBFR. Gray levels were obtained using the MIP image program by averaging data derived from 5 windows, measuring 50 × 50 mM (10× objective) and located outside the venules. To localize the same regions of interest a computer-assisted device for XY movement of the microscope table was used.
Adhesion of leukocytes to the vessel walls (45 venules for every group or subgroup) over a 30-s time-period was reported as number of adherent cells/100 µm of venular length (v.l.)/30 s, utilizing appropriate magnification (20× and 32× objectives; Lapi et al., 2012a). Perfused capillaries were evaluated as the length of the capillaries showing blood flow (BFCL), assessed by MIP image in an area of 150 × 150 µm (Lapi et al., 2016).
During the whole experimental period, we monitored arterial blood pressure (mean) by a Gould Windograf recorder (model 13-6615-10S, Gould, OH, USA) and heart rate by Viggo-Spectramed P10E2 transducer; Oxnard, CA, USA, linked to the catheterized femoral artery (Lapi et al., 2012b). The arterial blood gases (ABL5; Radiometer, Copenhagen, Denmark) were measured at 30 min intervals, as previously reported (Lapi et al., 2012b), as well as the hematocrit in basal conditions, at the end of CBFD and CBFR.

ROS Production Assessment
The pial layer was superfused with aCSF, containing 250 mM 2 -7 -dichlorofluorescein-diacetate (DCFH-DA) at 37.0 ± 0.5 • . The consequent test was carried out after 30 min CBFD (n = 3) and 60 min CBFR (n = 3), as previously reported (Lapi et al., 2013). DCFH-DA is widely used in vivo as a marker for oxidative stress of the cells and tissues (Wang and Joseph, 1999). DCF fluorescence intensity, related to the intracellular ROS level, was assessed using an appropriate filter (522 nm) and measured by NGL (Watanabe, 1998). For this analysis, three rats from each experimental group were used.
A computerized image analysis (Image-Pro Plus; Rockville, MD, USA) was utilized to identify the location and extent of necrotic areas. The infarct size was quantified by manual measurements, according to the following formula: [(area of non-hypoperfused, or area not subjected to cerebral blood flow decrease, cortex or striatum − area of remaining hypoperfused, or area subjected to cerebral blood flow decrease, cortex or striatum)/area of non-hypoperfused cortex or striatum] × 100 (Bederson et al., 1986).

Western Blotting Protocol
Tissue specimens from cortex and striatum were treated as previously reported (Lapi et al., 2016). They were homogenized by Polytron (Brinkman Instruments, Westbury, NY, USA) in buffer with the following components: 50 mM HEPES, 150 mM NaCl, 5 mM EGTA, 150 mM MgCl 2 , 1% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM trypsin inhibitor. Bradford assay (BioRad, Berkeley, CA, USA) was used to quantify protein concentration. The stirred (1 h, 4 • C) homogenate was centrifuged at 14,000 rpm for 20 min. The Bradford procedure (BioRad, Berkeley, CA, USA) was utilized to determine the protein concentration in the supernatant. The same amounts of proteins were run on 7.5% Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) under reducing conditions, then transferred to polyvinylidene difluoride membranes (PVDF; Invitrogen, Carlsbad, CA, USA). 5% w/v BSA in Tris-buffered saline and 0.1% Tween 20 (TBST) were used to block the membrane for 1 h at room temperature. Filters were incubated with specific antibodies at 4 • C overnight, washed in TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000, GE-HealthCare, Little Chalfont, UK) for 1 h at room temperature. Peroxidase activity was detected by the ECL system (GE-HealthCare, Little Chalfont, UK) after washing. Normalized protein loading was obtained incubating the same filters with anti-tubulin antibody (Sigma-Aldrich, Milan, Italy) and the intensity of each band was quantified by densitometry (ChemiDoc, XRS, Bio-Rad). We assessed the protein concentration of endothelial NO synthase (eNOS) compared with tubulin concentration. To reveal the proteins of interest, specific antibodies were utilized: rabbit polyclonal anti-eNOS (1:1,000). We purchased anti-eNOS and anti-tubulin antibodies from Cell Signaling Technology Inc. (Danvers, MA, USA).

Grape Pomace Extract Supplement Preparation
Taurisolo was obtained from Aglianico cultivar grape, collected during the harvest in autumn 2016. The product was formulated by the Department of Pharmacy, University of Naples ''Federico II'' (Naples, Italy). Large scale production of Taurisolo has been accomplished by MBMed Company (Turin, Italy). The grape has been extracted with water at 50 • C. After centrifugation, the extract underwent a spray-drying process with maltodextrins as support, obtaining a fine powder, containing a pomace: maltodextrins ratio 1:1 (w/w). Taurisolo has been used to formulate the nutraceuticals.
The High Performance Liquid Chromatography-diodearray detector (HPLC-DAD) analysis indicates that the main polyphenols contained in Taurisolo

Mass Spectrometry-Based Metabolomics, Statistics and Analysis
Rat brains were homogenized using a potter in 1 ml of pre-chilled methanol/water 1:1 solution, containing 10 nmol of internal standard, and centrifuged at 10,000 g for 10 min at 4 • C (Ser et al., 2015). The resulting supernatants were collected and transferred into Eppendorf tubes and stored at −80 • C. Analyses were performed according to a previous protocol (Sommella et al., 2019). Data were acquired on a SolariX XR 7T (Bruker Daltonics, Bremen, Germany). The instrument was tuned with a standard solution of sodium trifluoracetate. Mass spectra were recorded in a broadband mode in the range 100-1,500 m/z, with an ion accumulation of 20 ms, with 32 scans using 2 million data points (2M). Nebulizing (N2) and drying gases (air) were set at 1 and 4 ml/min, respectively, with a drying temperature of 200 • C. Both positive and negative ESI ionization were employed. Five replicates of each injection were carried out. The instrument was controlled by Bruker FTMS Control, MS spectra were elaborated with Compass Data Analysis version 4.2 (Bruker); identification of compounds based on accurate MS measurements was performed by Compound Crawler ver. 3.0 and Metaboscape 3.0 (Bruker). Metabolites signals were normalized using internal standards.
We utilized three animals for microvascular studies, three for ROS quantification, three for TTC staining, three for Western blot analysis and three for mass-spectrometry metabolomics analysis for each experimental group or subgroup.

Statistical Analysis
Comparisons and differences were analyzed for statistical significance by two-way ANOVA and Bonferroni post hoc test. For comparison of diameter and length of vessels, due to distribution of data computed by the Kolmogorov-Smirnov test, nonparametric tests were used: Mann-Whitney and Kruskal-Wallis tests. All data (graphs, bars or lines) are reported as the mean and standard error of the mean (SEM). Statistical analysis was performed using Statistica software (StatSoft, Tulsa, OK, USA) and Minitab (Minitab Inc., State College, PA, USA).

Microvascular Changes
To examine whether Taurisolo alleviated brain hypoperfusionreperfusion damage we assessed the microvascular changes of rat pial microcirculation in sham-operated rats, in ischemic animals and in Taurisolo -treated rats by fluorescence microscopy. As reported in Figures 1A-C, in sham-operated rats there were no changes in arteriolar diameter at the end of observation time, while in ischemic animals we detected reduction in diameter of arterioles, increase in fluorescent dextran leakage on the venous side of microcirculation and in DCF fluorescence due to marked formation of ROS (Figures 1D-F). In Taurisolo -treated animals by i.v. or oral administration we detected an increase in arteriolar diameter accompanied by a significant decrease in fluorescent leakage on the venous side of microcirculation and reduction of DCF fluorescence, indicating a reduction in ROS formation (Figures 1G-I,J-L).
Moreover, TTC assays showed a significant decrease in infarct size induced by Taurisolo , as reported in Figure 2. In particular, the hypoperfusion and the subsequent reperfusion caused significant damage in cortex and striatum cerebral tissue of both hemispheres in H rats ( Figure 2B), compared to the SO-S subgroup (Figure 2A). Cortex infarct size was 9.2 ± 1.8% (p < 0.01 vs. non-hypoperfused cortex), while in the striatum the damage was more marked (striatum infarct size 32.5 ± 3.7%, p < 0.01 vs. non-hypoperfused cortex). Animals treated with Taurisolo , intravenously or orally administered, and subjected to CBFD and CBFR showed neuronal damage significantly diminished when compared to the H group (Figures 2C,D,  respectively). The damage appears to be localized to the striatum (infarct sizes were 7.0 ± 1.5% and 10.6 ± 1.0% in rats treated with i.v. Taurisolo or with oral Taurisolo , respectively, p < 0.01 vs. hypoperfused striatum).
These results prompted us to quantify the effects of Taurisolo on ROS formation, as indicated by DCF assays, and on the diameter changes of pial arterioles, classified as reported in Table 2. Figure 3 reports the DCF fluorescence intensity in sham-operated animals, in hypoperfused and in Taurisolo -treated rats. The ROS formation was really marked in hypoperfused animals, while Taurisolo was effective in reducing fluorescence intensity and consequently ROS formation. Moreover, we evaluated the diameter variations in all experimental groups (Figure 4). In the SO-S subgroup during the entire observation period, we did not detect any change in arteriolar diameter; furthermore, the rats treated with intravenous or oral administration of Taurisolo (SO-Tiv and SO-Tor subgroup) did not show any alteration in diameter. In addition, the intravenous administration of L-NIO, specific inhibitor of eNOS (SO-L subgroup), or of L-NIO plus Taurisolo (SO-LTiv subgroup) did not cause significant changes in arteriolar diameter. On the other hand, the animals FIGURE 2 | Triphenyl tetrazolium chloride (TTC) staining of coronal brain slices from a rat of Sham-operated group (A: sham-operated animal), from a rat of H group (B: hypoperfused after 60 min of CBFR), from a rat of Tiv subgroup (C: after 60 min of CBFR) and from a rat of Tor subgroup (D: after 60 min of CBFR). The lesion in the striatum is shown by the dashed black line.
subjected to hypoperfusion and reperfusion (H group) showed marked changes in diameter: CBFD caused a reduction in diameter (by 10.8 ± 1.5% of baseline in order 3 arterioles; p < 0.01 vs. baseline and SO-S subgroup; Table 2 and Figure 4).  After 60 min CBFR, pial arteriolar diameters significantly decreased (by 16.5 ± 1.8% of baseline, p < 0.01 vs. baseline and SO-S subgroup; Table 3 and Figure 4). Taurisolo , intravenously administered (Tiv subgroup), protected the pial microvasculature from damage induced by hypoperfusion and reperfusion. At the end of CBFD, pial arterioles slightly dilated by 5.0 ± 1.2% of baseline (p < 0.01 vs. baseline, SO-S subgroup and H group; Figure 4). At the end of CBFR, pial arteriolar diameters markedly increased (by 35.0 ± 1.8% of baseline, p < 0.01), indicating significant dilation of arterioles compared with H group and SO-S subgroup ( Table 3 and Figure 4). The animals fed with Taurisolo supplemented diet, 20 mg/kg b.w./die for 1 month (Tor subgroup), were highly protected from microvascular damage induced by CBFD and CBFR. After CBFD, pial arteriolar diameters did not significantly change compared to baseline conditions and the SO-S subgroup (diameter decreased by 2.0 ± 1.0% of baseline, p < 0.01 vs. H group; Figure 4). At the end of CBFR, the arteriolar diameters significantly increased by 12 ± 2% of baseline (p < 0.01 vs. baseline, SO-S subgroup and H group; Table 3 and Figure 4). To clarify the mechanism of pial arteriolar dilation induced by Taurisolo , we administered L-NIO, a specific inhibitor of eNOS, prior to Taurisolo . The animals treated with intravenous administration of L-NIO plus Taurisolo (L-Tiv subgroup) and subjected to CBFD and CBFR did not show any significant changes in arteriolar diameter compared with baseline conditions. Therefore, vasodilation induced by intravenous administration of Taurisolo was abolished by L-NIO administration. The same pattern was observed in rats belonging to the L-Tor subgroup; in these animals, the intravenous administration of L-NIO 10 min prior to CBFD and at the beginning of CBFR was able to abolish the vasodilation induced by the oral treatment of Taurisolo both at the end of CBFD and at the end of CBFR.
Then, we quantified the effects of Taurisolo on the changes in microvascular permeability, known to be markedly induced by CBFD and CBFR; therefore, we assessed FITC leakage on the venous side of microcirculation. In sham-operated animals (SO-S subgroup) we did not observe microvascular leakage (0.01 ± 0.01 NGL; Table 3, Figure 5) nor in the rats treated with intravenous or oral administration of Taurisolo (SO-Tiv and SO-Tor subgroup): microvascular permeability did not increase (0.02 ± 0.01 NGL and 0.01 ± 0.01 NGL, respectively). Finally, the intravenous administration of L-NIO (SO-L subgroup) or of L-NIO plus Taurisolo (SO-LTiv subgroup) did not cause significant changes in microvascular leakage. On the contrary, in H group rats CBFD caused an increase in microvascular permeability (0.18 ± 0.02 NGL; p < 0.01 vs. baseline and SO-S subgroup; Figure 5). The increase was further incremented at the end of 60 min CBFR when microvascular permeability was really marked TABLE 3 | Changes of the microvascular parameters evaluated at the end of the restoration of cerebral blood flow (CBFR) in SO-S, H, Tiv [10 mg/kg b.w. of Taurisolo administered i.v. prior to diminution of cerebral blood flow (CBFD) and at the beginning of CBFR], Tor (20 mg/kg b.w. of Taurisolo orally administered for 1 month and subjected to CBFD and CBFR), L-Tiv (L-NIO 10 mg/kg i.v. prior to i.v. Taurisolo i.v.) and L-Tor (L-NIO 10 mg/kg i.v. administered prior to CBFD and at the beginning of CBFR) subgroups.
The animals fed with Taurisolo supplemented diet, 20 mg/kg b.w./die for 1 month (Tor subgroup), were markedly protected from microvascular damage induced by CBFD and CBFR. After CBFD, microvascular leakage was prevented compared to the H group (0.07 ± 0.02 NGL, p < 0.01 vs. baseline, SO-S subgroup and H group; Figure 5).
The animals treated with intravenous administration of L-NIO plus Taurisolo (L-Tiv subgroup) and subjected to CBFD and CBFR did not show significant changes in fluorescent leakage compared with baseline conditions. L-NIO administration did not affect its protective effects on microvascular leakage that were observed also in the Tiv subgroup (Table 3 and Figure 5).
The same pattern was observed in rats belonging to the L-Tor subgroup; in these animals, the intravenous administration of L-NIO 10 min prior to CBFR and at the beginning of CBFD was unable to abolish the prevention of fluorescent leakage due to the oral treatment of Taurisolo both at the end of CBFD and at the end of CBFR.
An increase in leakage and arteriolar dilation have been indicated as key mechanisms related to leukocyte adhesion to vessel walls and to capillary perfusion. Therefore, we evaluated the number of leukocytes adhered to venular walls and the BFCL in the different experimental groups and subgroups. As reported in Figure 6, in SO-S animals there was no significant sticking of leukocytes to venular wall (1 ± 1/100 µm of v.l./30 s), while in H group there was a significant increase in the number of adhered leukocytes (7 ± 2/100 µm v.l./30 s; p < 0.01 vs. baseline and FIGURE 7 | Percent decrease in the length of perfused capillaries after 60 min of CBFR in the different experimental groups: SO-S, H, Tiv (10 mg/kg b.w. of Taurisolo administered i.v. prior to CBFD and at the beginning of CBFR), Tor (20 mg/kg b.w. of Taurisolo orally administered for 1 month and subjected to CBFD and CBFR), L-Tiv (L-NIO 10 mg/kg i.v. prior to i.v. Taurisolo i.v.) and L-Tor (L-NIO 10 mg/kg i.v. prior to oral Taurisolo ) subgroups, treated before CBFD and at the beginning of CBFR. • p < 0.01 vs. baseline, *p < 0.01 vs. SO-S subgroup and ∧ p < 0.01 vs. H group.
SO-S subgroup). In Taurisolo -treated animals by i.v. or oral administration, there were significant reductions in the number of adhered leukocytes (2 ± 1/100 µm v.l./30 s, p < 0.01 vs. H group; 3 ± 1/100 µm v.l./30 s, p < 0.01 vs. H group, respectively). Administration of L-NIO prior to Taurisolo was effective in partially reducing the protective effects of I.v. Taurisolo , while the oral route was less affected by L-NIO. The perfusion of capillaries was quantified by computer-assisted methods. We evaluated the overall BFCL: in SO-S animals all capillaries were perfused, while BFCL was markedly reduced in H animals (by 45.0 ± 2.2%, p < 0.01 vs. baseline and SO-S subgroup; Figure 7). In all Taurisolo -treated animals the capillaries were perfused, with percent reduction from 15.0 ± 1.2% of baseline in Tiv animals (p < 0.01 vs. baseline, SO-S subgroup and H group) to 6.0 ± 1.5% of baseline in Tor rats (p < 0.05 vs. baseline, SO-S subgroup; p < 0.01 vs. H group). The administration of L-NIO did not decrease the perfusion of capillaries preserved by Taurisolo .

Western Blotting
We tried to clarify the mechanism of arteriolar dilation induced by Taurisolo and we utilized a specific inhibitor of eNOS, known to play a crucial role in arteriolar dilation. The responses we obtained with L-NIO encouraged us to assess eNOS expression in the cortex and striatum of our experimental animals by Western Blotting. Diminution and subsequent restoration of cerebral blood flow did significantly decrease eNOS expression both in cortex and striatum of H group animals, compared to that detected in SO-S animals, as reported in Figure 8, where we normalized all data on SO-S rat results (assigned 1 value). There was a marked increase in the striatum eNOS expression of Tiv subgroup animals compared to the striatum values in H animals (p < 0.01); this FIGURE 8 | Western blotting of endothelial nitric oxide synthase (eNOS) expression in two regions: cortex (ctx) and striatum (str), at the end of CBFR in SO-S, SO-LTiv, H, Tiv (10 mg/kg b.w. of Taurisolo administered i.v. prior to CBFD and at the beginning of CBFR), Tor (20 mg/kg b.w. of Taurisolo orally administered for 1 month and subjected to CBFD and CBFR), L-Tiv (L-NIO 10 mg/kg i.v. prior to i.v. Taurisolo ) and L-Tor (L-NIO 10 mg/kg i.v. prior to oral Taurisolo ) subgroups, treated before CBFD and at the beginning of CBFR. We compared all data, normalizing the results on SO-S values. • p < 0.05 vs. H group; *p < 0.01 vs. SO-S subgroup and ∧ p < 0.01 vs. H group (same region). Each entry: nine evaluations.
trend was observed also in the cortex where the increase was slight compared to cortex values in H group (p < 0.05). In Tor subgroups rats, there was a significant increase in eNOS expression in the striatum compared to that observed in H animals, while in the cortex there was a decrease in expression. However, in L-Tiv rat subgroup there was no increase in eNOS expression compared to H animals, during inhibition by L-NIO; in L-Tor subgroup animals there was an increase in eNOS expression in the striatum when compared to H animals, notwithstanding eNOS inhibition, while a decrease was observed in eNOS expression in the cortex. Administration of L-NIO plus Taurisolo in sham-operated animals did not induce significant changes in the striatum eNOS expression, but a reduction in the cortex expression was observed when compared to SO-S data.

Metabolomic Profiles
To identify the mechanisms operative in the protection exerted by Taurisolo on hypoperfusion and reperfusion-induced microvascular damage, metabolites were extracted from brains of rats belonging to H group, Tiv or Tor subgroups, SO-S and SO-Tiv or Tor subgroups, respectively. Upon extraction, the sample was profiled by MS analysis and compared ( Table 4).
Brains from the H group presented a significant increase in peroxidized cardiolipins, indicating an increase in peroxidation of mitochondrial lipids (Horvath and Daum, 2013).
the release of free fatty acids, particularly arachidonic acid (AA), whose metabolites, in particular, COX products were investigated. Compared to SO-S animals, the H group showed significant increases in the pro-inflammatory prostaglandin PGF2 α, as well as in PGD2 and in 6-keto-PGFl α (a derivative of the prostacyclin PGI2; Tegtmeier et al., 1990;Bultel-Poncé et al., 2015). It is worth noting that thromboxane Txb2 levels were more than 10 times higher compared to the levels detected in SO-S animals. Tiv and Tor subgroups presented lower levels of peroxidized cardiolipins, almost half levels compared to H group values. All prostaglandins were significantly reduced by Taurisolo treatment, even though the response was different according to the properties of each prostaglandin. PGD2 values were more than 10 times higher in H animals compared to SO-S rats; the treatment with Taurisolo decreased the PGD2 levels to 2-3 times higher than those observed in SO-S animals. PGF2 α values increased more than five times in H group rats, while in Taurisolo treated animals the levels of this prostaglandin were not significantly different compared to those detected in SO-S animals. 6-keto-PGF1 α values increased more than three times in H rats compared to SO-S animals. Taurisolo , i.v. administered, was effective in decreasing the levels of this prostaglandin to the values observed in SO-S animals, while the oral administration of Taurisolo was able to halve the levels detected in H rats. The diminution in thromboxane Txb2 was marked: from 16 folds (on the average) to 3-2 folds after treatment compared to SO-S group. A decreased amount of prostaglandin PGD2 was detected also in SO-Tor subgroup, prior to hypoperfusion-induced damage, with the same trend observed in the SO-Tiv subgroup.
MS analysis of arginine and citrulline, two amino acids playing a fundamental role in the biosynthesis of nitric oxide, indicates that in H group animals, submitted to hypoperfusion and recovery of blood flow, the level of arginine did not change compared to SO-S group, while there was a modest increment in the levels of citrulline, indicating a slight increase in NO production. On the other hand, in animals treated with Taurisolo , the levels of citrulline, highly increased, while decreasing the levels of arginine, demonstrated that there was a significant increase in NO release ( Table 5). The levels of arginine in SO-S animals increased after Taurisolo treatment, pointing toward Taurisolo increasing intracellular arginine availability, useful for its conversion to citrulline and consequent NO release. Ornithine's levels, the third investigated amino acid, did not change among the different experimental groups, indicating substantial metabolic steady-state conditions in all animals.

DISCUSSION
The present data indicate that Taurisolo , a nutraceutical endowed with antioxidant activity, was effective in decreasing the damage to the brain microcirculation, induced by reduction and successive recovery of cerebral blood flow. Taurisolo administered to rats by intravenous infusion or by oral supplementation was effective in preventing the decrease in pial arteriolar diameter, the increase in microvascular permeability and adhesion of leukocytes, the reduction in capillary perfusion, when compared with the changes observed in ischemic animals (H group, submitted to bilateral occlusion of common carotid arteries). As detected by TTC staining of brain slice and survival of cerebral tissue, Taurisolo limited the infarct size.
The venous administration caused a fast response with higher increase in vessel diameter, at the end of cerebral blood flow decrease, and marked rise by 35.0 ± 1.8% of baseline at reperfusion, compared with the results by oral supplementation: no change at the end of CBFD and arteriolar dilation by 12.0 ± 2.0% of baseline at the end of reperfusion. Moreover, with the administration of i.v., there was a slight reduction in capillary perfusion (by 15.0 ± 1.2% of baseline compared to that observed in H group: by 45.0 ± 2.2% of baseline). It is important to note that the oral supplementation better preserved capillary perfusion with a reduction by 6.0 ± 1.5% of baseline, significantly different compared with i.v. Taurisolo -treated rats and H group animals. Both routes of administration did not differ on the ROS overall formation, because the amounts of fluorescence were quite in the same range, with a prevalence in prevention for the oral administration (0.05 ± 0.01 vs. 0.08 ± 0.02, respectively).
All these effects could be related to the antioxidant properties of Taurisolo able to decrease ROS formation, known to be a key factor in the mechanisms causing brain ischemia-reperfusion injury.
Usually, antioxidant molecules can have both protective as well as dangerous effects according to their dosages. However, we did not observe damage with higher dosages, because no one antioxidant was able to revert the prevention of increased leakage of macromolecules or decreased capillary perfusion. At least we did not observe any leakage with the highest dosages.
Moreover, it is worth noting that the dilation of arteriolar networks induced by Taurisolo appears to be crucial in preventing the decrease in capillary perfusion. Western blotting data, indeed, demonstrate the increase in expression of eNOS in both cortex and striatum in i.v.-Taurisolo -treated rats. Moreover, inhibition of eNOS by L-NIO blunted arteriolar dilation at the end of perfusion recovery in animals treated with Taurisolo , but L-NIO did not reduce the levels of eNOS expression induced by Taurisolo at least in the striatum of orally-treated animals.
Moreover, the present data confirm the microvascular changes in ischemic animals reported in previous studies, where the increase in ROS formation was one of the main factors in ischemia-reperfusion injury (Lapi et al., 2016).
The protective effects triggered by Taurisolo were confirmed by the Mass-Spectrometry analysis of metabolomic data. As reported in Tables 4, 5, cardiolipin oxidation, very high in H group animals, was reduced by Taurisolo administered by the intravenous or oral route, with the former more active. In previous studies, cardiolipin has been shown to play a pivotal role in the mitochondrial damage due to ischemia, with the increase in ROS production. Our data support the previous observations (Haines and Dencher, 2002;Petrosillo et al., 2003;Chicco and Sparagna, 2007;Claypool, 2009;Camara et al., 2011;Hausenloy and Yellon, 2013;Zhou et al., 2018).
Furthermore, Taurisolo effectively hampered the production of inflammation mediators such as all evaluated prostaglandins, especially PGD2. It is interesting to point out the dramatic reduction in thromboxane TxB2 values induced by Taurisolo , able to decrease the release of this vasoconstrictor factor produced by endothelial cells, effective in inducing intraluminal blood coagulation. Moreover, the MS analysis of arginine and citrulline levels demonstrates that the increase in NO release during hypoperfusion and recovery of blood flow in H group animals was modest, as indicated by the corresponding values of arginine and citrulline (1.1 vs. 1.3 on the average, respectively). On the other hand, in Taurisolo -treated animals the release of NO, taking into account the increase in citrulline levels and the decrease in arginine contents, was significantly higher compared to the values detected in H group animals. Consequently, these data indicate a very high release of NO-induced by Taurisolo treatment. Moreover, it is worth noting that Taurisolo in SO-S animals was able to increase the baseline levels of arginine, indicating that this could induce an increment in NO bioavailability and release. These effects on arginine and citrulline levels were accompanied by substantial metabolic steady-state conditions, represented by stable ornithine concentrations in all experimental group animals. Moreover, altogether these data suggest that Taurisolo may explicate a general anti-inflammatory effect. It is interesting to observe that metabolomic data corresponded to Western Blotting results, indicating an increase in eNOS expression in Taurisolo treated animals, observed mainly in i.v. injected animals compared with those orally administered. However, metabolomics data demonstrate that orally administered Taurisolo was effective in inducing a very high formation of citrulline and consequently a very high release of NO. The discrepancy between metabolomic and Western Blotting results may be due to the different protocols for the two assays because more tissue was used for metabolomic data compared to Western Blotting technique, focused on cortical and striatal regions, where different vascular structures and amounts could be investigated.
The effects of Taurisolo are likely the result of the synergistic activity of its components. Resveratrol and catechins have been extensively studied in the last decade revealing protective effects against ischemia-reperfusion injury in several organs in experimental models or in humans (Khurana et al., 2013;Menditto et al., 2015;Chen et al., 2016;Putignano et al., 2017;Koushki et al., 2018;Li et al., 2019). In our previous studies, we tried to assess the effects of quercetin, malvidin, cyanidin, catechin and other phenolic compounds in the same model of rat brain ischemia-reperfusion injury as in the present study. We observed that quercetin protects pial microvasculature against damage induce by hypoperfusion and reperfusion at the dosage of 5 and 10 mg/kg b.w. We used dosages of catechin and cyanidin in the same range as for quercetin and we found significant protection with all polyphenolic substances (Lapi et al., 2012a;Mastantuono et al., 2015Mastantuono et al., , 2018. The amounts of polyphenols present in the pomace extract are smaller than those previously used (Lapi et al., 2012a(Lapi et al., , 2015Mastantuono et al., 2015Mastantuono et al., , 2018; however, the protection exerted by Taurisolo was significant. Therefore, it is reasonable to suggest that the natural mixture of polyphenols, a component of the pomace extract, could potentiate the effects of each substance, facilitating protection of cerebral microcirculation during ischemia-reperfusion injury. In future experiments we will plan to assess the effects of gallic acid and syringic acid, two of the main components of Taurisolo , on hypoperfusion and reperfusion injury in rat pial microcirculation.
In conclusion, Taurisolo was effective in the preservation of vascular integrity and cellular functions, finally preventing brain damage due to cerebral hypoperfusion, one of the most important human pathological conditions in the World, the third cause of disability and death. Furthermore, the effects of Taurisolo , orally-administered, suggest that Taurisolo could be useful to prevent widespread oxidation in brain structure during aging-related changes in brain perfusion.