Myricetin preserves rat pial microcirculation from injury induced by cerebral hypoperfusion and reperfusion

Background/Objective: Myricetin, a flavonoid compound, is widely diffused in vegetables, fruits and beverages, well known for its antioxidant and anti-inflammatory properties. The present study was aimed to investigate the acute effects of myricetin on the pial microvascular alterations and oxygen-derived free radical formation, due to 30 min cerebral blood flow lowering (CBFL) and subsequent cerebral blood flow resumption (CBFR). Methods: Rat pial microvasculature was investigated using fluorescence microscopy through a closed cranial window. At first, arterioles were classified according to the Strahler’s ordering scheme. Then, arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (CPL) and red blood cell velocity (VRBC) were quantified by computerized methods. Finally, reactive oxygen species (ROS) production was investigated in vivo by 2 ′ -7 ′ -dichlorofluorescein- diacetate assay and infarct size by 2,3,5-triphenyltetrazolium chloride staining. Results: After 30 min CBFL and 60 min CBFR, a decrease of arteriolar diameter, CPL and VRBC was detected; furthermore, increases in microvascular leakage and leukocyte adhesion were observed in hypoperfused animals. Conversely, myricetin administration induced dose-related arteriolar dilation, reduction in microvascular permeability as well as leukocyte adhesion when compared to those detected in bilateral common carotid artery occlusion-submitted animals; moreover, CPL and VRBC were preserved. In animals treated with myricetin the ROS production was blunted and infarct size significantly reduced. Conclusion: In conclusion, myricetin acute administration showed dose-related protective effects on rat pial microcirculation during CBFL and subsequent CBFR, inducing arteriolar dilation and inhibiting ROS formation, consequently preserving the blood brain barrier integrity.


Introduction
Emerging research provides substantial evidence to classify food rich in flavonoids and polyphenols as functional food with several preventive and therapeutic health benefits. Flavonoids are natural compounds widely diffused in nature and characterized by several phenolic structures [1,2]. They are responsible for the colors in the skins of fruits and vegetables and are abundant also in tea and wine [3][4][5]. Many Authors focused the attention on the biological properties of these natural molecules, suggesting their protective role against coronary heart disease and mortality [6]. Flavonoids can be classified according to their molecular structure as flavones, flavanones, catechins and anthocyanins and are all characterized by a strong antioxidant capacity [7]. In particular, many studies suggest that flavones and catechins show stronger scavenger activity compared to others [8]. Among flavones, myricetin, a molecule diffused and abundant in berries, vegetables, teas and wines, manifests interesting antioxidant, anti-inflammatory and anticancer properties [9,10] and is well recognized for its nutraceuticals value. The strong scavenger activity of myricetin was previously documented; Rusak, et al. showed that this compound was able to counteract the 1,1′-diphenyl-2-picrilhydrazyl (DPPH) radicals [11], while Husain, et al. demonstrated it was able to scavenge hydroxyl (.OH) radicals generated by UV photolysis of hydrogen peroxide [12]. Moreover, myricetin inhibits liposomes peroxidation [13] and its antioxidant properties appeared higher than Vitamin E (d-α-tocopherol) [14]. Tzeng, et al. showed that this flavonoid inhibits platelet aggregation in rabbits and this ability may be due to the inhibition of thromboxane formation [15]. The mechanism of anti-aggregating activity appeared related to the inhibition of phosphodiesterase activity [16]. A recent work indicated that a myricetin pretreatment, 25 or 50 mg/kg b.w., reduced rat intestinal ischemia-reperfusion injury [17]. On the other hand, epidemiological evidence indicates that the polyphenols have a protective effect against some chronic diseases [18]. They play an important role in human nutrition and are endowed with numerous biological properties, such as antioxidant activity [19], anti-tumor, anti-atherosclerotic, antiinflammatory, hepatoprotective, anti-bacterial and anti-replicative for HIV [20]. Therefore, the aim of the present study was to in vivo assess the effects of myricetin on cerebral microvascular alterations caused by hypoperfusion and reperfusion in rat. We investigated the acute effects induced by myricetin on pial microcirculation in rats submitted to 30 min of cerebral blood flow lowering (CBFL) and 60 min of cerebral blood flow resumption (CBFR). values). The protocol of drug administration was previously described [23]. Appropriately mixing 2′-7′-dichlorofluorescein-diacetate (DCFH-DA) and artificial cerebrospinal fluid (aCSF) allowed us to superfuse the pial layer with 250 mM DCFH-DA solution for 30 min after CBFL [24,25]. Sigma Chemical, St. Louis, MO, USA supplied all drugs.

Rat preparation
All experiments conform to 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 [22]. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Pisa and Italian Health Ministry (Permit Number: 156/2017-PR).
Rats were anesthetized with intraperitoneal (i.p.) injection of α-chloralose, (60 mg/kg b.w. for induction; afterward 30 mg/kg b.w. for maintenance) and mechanically ventilated after tracheotomy, according to the protocol previously reported [22]. 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 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 6G, 1 mg/100 g b.w. in 0.3 mL, as a bolus with supplemental injection throughout CBFL 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 [23].
The visualization of pial microvasculature was carried out as previously reported [14,22,26]. A closed cranial window was positioned at the level of the left frontoparietal cortex through an incision in the skin to operate a craniotomy. Cerebral cortex was preserved by overheating caused by drilling with saline solution superfusion of the skull. The dura mater was gently cut and displayed on the corner; a quarz microscope coverglass was bound to the skull bone. Artificial cerebrospinal fluid 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% O2, 6.0% CO2 and 84.0% N2; pH 7.38 ± 0.02).
The lowering in cerebral blood flow (CBFL) was produced by clamping both common carotid arteries, previously prepared. The clamping was removed after 30 min; thereafter, the pial microvasculature was investigated during the resumption of cerebral blood flow (CBFR), lasting 60 min [27].

Experimental groups
Experiments were carried out utilizing male Wistar rats, 250-300g (Harlan, Italy), randomly assigned to three groups: (1) sham group (SH group), subjected to the same surgical procedure of the other experimental groups without changes in cerebral blood flow; (2)  Five animals for SH-Na subgroup, RF and M 2 group were investigated by in vivo fluorescence microscopy, to detect microcirculation damage; six rats were utilized to assess oxidative stress by Dichloro-dihydrofluorescein diacetate (DCFH-DA) assay after CBFL (n=3) and after CBFR (n=3); in three animals tissue damage was evaluated by TTC staining. The rats belonging to the SH-M2 and SH-L subgroups were utilized only for microcirculation investigations.

Drug administration
Each utilized drug (myricetin or L-NIO) was dissolved in 0.5 mL saline solution and, successively, i.v. injected to rats within 3 min, 10 min before CBFL and at the beginning of CBFR. We tested the effects of two different dosages of myricetin: 10 or 35 mg/kg b.w. The choice of the dosages derived from pilot experiment results indicating that myricetin dosages less than 10 mg / kg b.w. were ineffective on the pial microvasculature. On the other hand, dosages higher than 35 mg/kg b.w. did not improve microvascular protection detected in the animals administered with 35 mg/kg b.w myricetin before and after CBFL.
Finally, L-NIO, known to inhibit the NO release, was administered at the dosage of 10 mg/kg b.w., 10 min before i.v. infusion of the higher myricetin dosage (35 mg/kg b.w.) [21,22]. To test the right dosage of L-NIO, pilot experiments were performed where the intravenous administration L-NIO, at the dosage of 10 mg/kg b.w., was able to impede arteriolar dilation caused by i.v. injection of 10 mg/4 min L-arginine (diameter increase by 22.8 ± 2.0%, compared to basal values) or abolish the vasodilation due to topical administration of 100 μM acetylcholine (diameter increase by 5.0 ± 1.5%, compared to basal used for epiillumination with the corresponding filters for FITC and rhodamine 6G. A heat filter prevented overheating of the preparations (Leitz KG1). Pial microvascular networks were televised with a DAGE MTI 300 low-light level camera and stored through a computer-based frame grabber (Pinnacle DC 10 plus, Avid Technology, Burlington, MA, USA).

Geometric detection of microvascular network
In each animal, first we characterized the arteriolar network by stop-frame images and pial arterioles were assigned order according to Strahler's method, starting from capillaries to the largest arterioles (centripetal method), as previously reported [28,29]. In each experiment we studied one order 4 arteriole, two order 3 and two order 2 arterioles. Furthermore, we assessed the functional changes of each arteriolar order under the experimental conditions. We report, however, the results detected in order 3 arterioles.

Microvascular parameter assessment
Microvascular parameters were measured off-line utilizing a computerized imaging technique, previously described in details by Lapi, et al. [22,23]. Concisely, arteriolar diameters were measured with a computerized method, Microvascular Imaging Program (MIP), frame by frame. The increase in permeability was measured by evaluating fluorescent dextran extravasation from venules and expressed as normalized gray levels (NGL): NGL = (I -Ir)/Ir, where Ir is the baseline gray level at the microvasculature filling with fluorescence, and I is the value at the end of CBFL or CBFR. Gray levels were obtained using the MIP image program by average of 5 windows, measuring 50 × 50 mM (10x objective) and located outside the venules. During recordings the same regions of interest were localized by a computer-assisted device for XY movement of the microscope table.
Leukocytes sticking to the vessel walls (45 venules for every group) over a 30-s time-period were reported as number of adherent cells/100 μm of venular length (v.l.)/30 s, utilizing appropriate magnification (20 x and 32 x, objectives) [22]. Perfused capillaries were evaluated as the length of the capillaries showing blood flow (CPL), assessed by MIP image in an area of 150 × 150 μm [23,24].

Tissue damage estimation
At the end of CBFR, rats were sacrificed to evaluate tissue damage. The brains were isolated and rostro-caudally cut into coronal sections (1 mm) with a vibratome (Campden Instrument, 752 M; Lafayette, IN, USA). Slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (20 min) at 37 °C and in 10% formalin overnight, as previously reported [22]. TTC, a white salt, is reduced to red 1,3,5-triphenylformazan by dehydrogenases in living cells. The location and extent of necrotic areas were assessed by computerized image analysis (Image-Pro Plus; Rockville, MD, USA). Moreover, the infarct size was quantified by manual measurements, according to the following formula: [(area of nonhypoperfused, or area not subjected to cerebral blood flow lowering, cortex or striatum-area of remaining hypoperfused, or area subjected to cerebral blood flow lowering, cortex or striatum)/area of nonhypoperfused cortex or striatum] × 100 [32].

Western blotting
Bio-Rad protein assay (Bio-Rad, Berkeley, CA, USA) was utilized to evaluate the protein concentration. Equal amounts of proteins were separated by SDS-PAGE under reducing conditions and then transferred to PVDF (Invitrogen, Carlsbad, CA, USA). Successively, the immunoblot was blocked, incubated with specific antibodies at 4 °C overnight, washed and then incubated for one hour with horseradish peroxidase-conjugated secondary antibody (1:2000) (GE-Healthcare, Little Chalfont, UK). By enhanced chemiluminescence system (GE-Healthcare) was evaluated the peroxidase activity. The optical density of the bands was determined by the ChemiDoc Imaging System (Bio-Rad) and normalized to the optical density of α-Tubulin (1:5000). Finally, the protein of interest was detected by specific antibodies: rabbit polyclonal anti-eNOS (1:500) and rabbit polyclonal anti phosphorylated e NOS (Ser 1177) (1:200) (Santa Cruz, CA, USA).

Statistical analysis
All data were reported as mean ± SEM. Normal distribution of data was assessed with the Kolmogorov-Smirnov test. Parametric (Student's t-tests, ANOVA and Bonferroni post hoc test) or nonparametric tests (Wilcoxon, Mann-Whitney and Kruskal-Wallis tests) were utilized, according to data distribution; diameter and length data among experimental groups were compared with nonparametric tests, as previously reported [23]. Data derived from DCFH-DA treated rats were analyzed with non-parametric tests. SPSS 14.0 statistical package (IBM Italia, Segrate, MI, Italy) was used. Statistical significance was set at p < 0.05.

Results
Pial microvascular networks were classified, under baseline conditions, according to the Strahler's method, as previously described [29]. Arterioles were classified in five orders; order five was assigned to the largest vessels (average diameter 60.8 ± 4.5 μm; average length: 1157 ± 212 μm, n= 88), the following were assigned decreasing orders up to the smaller arterioles of order 1 with an average diameter of 17.0 ± 2.3 μm and average length of 146 ± 58 μm (n=163); finally, order 0 was assigned to the capillaries that sprout from order 1 arterioles. The diameters and length were significantly different among the different orders (p<0.01).

SH group
At the end of observations, the animals belonging to the SH-Na subgroup did not show significant differences compared with the baseline conditions in microvascular parameters; moreover, we did not detect changes in fluorescence intensity after the DCFH-DA superfusion ( Table 1).
The same behavior was observed in the rats treated with myricetin (at the dose 10 or 35 mg/kg b.w.) (SH-M 1 and SH-M 2 subgroup, respectively) as well as in the animals treated with L-NIO (SH-L subgrouo) (Table 1).

M group
Rats treated with 10 mg/kg b.w. myricetin (M 1 subgroup) did not show significant changes in arteriolar diameter at the end of CBFL, compared to baseline conditions ( Figure 1). Moreover, microvascular permeability was significantly reduced compared to RF group (NGL: 0.17 ± 0.02; p < 0.01 vs. baseline, SH-Na subgroup and RF group) (Figure 2).

2,3,5-triphenyltetrazolium chloride (TTC) staining
Thirty minutes of CBFL and 60 min CBFR caused a significant damage in cerebral cortex and striatum of animals belonging to RF doi: 10.15761/VDT.1000169 Volume 4: 6-9 °p < 0.01 vs. baseline, ^p < 0.01 vs. SH-Na subgroup, *p < 0.01 vs. RF group group (p < 0.01 vs. SO-Na subgroup, Figure 7A). The cortex infarct size was 6.5 ± 1.3% (p < 0.01 vs. nonhypoperfused cortex), while the extension of damage appeared particularly marked in the striatum (33.2 ± 2.5 %, p < 0.01 vs. nonhypoperfused cortex). Myricetin i.v. infused at the lower dosage slightly preserved the neuronal tissues from the damage induced by CBFL and CBFR ( Figure 7B). The infarct size in the cortex was 3.5 ± 1.0% (p < 0.01 vs. nonhypoperfused cortex and RF group) while in the striatum was 20.5 ± 1.8% (p < 0.01 vs. nonhypoperfused cortex and RF group). On the other hand, myricetin administered at the higher dosage, significantly protected neuronal tissues from the hypoperfusion and reperfusion injury ( Figure 7C). The infarct size, indeed, was 12.4 ± 1.5% in the striatum (p < 0.01 vs. hypoperfused striatum, M 1 subgroup and RF group), while cerebral cortex appeared entirely protected ( Figure 7C).  Figure 7. Images of the TTC staining of coronal brain slice. In A coronal brain slice of a rat submitted to CBFL and CBFR, the lesion in the cortex and striatum is delineated by the dashed black line. In B coronal brain slice of a rat treated with low dose myricetin (10 mg/ kg b.w.) where it is possible to observe a modest reduction of the lesion area.
In C coronal brain slice of a rat treated with high dose myricetin (35 mg/kg b.w.) where it is possible to observe a marked reduction of the lesion area that has been reduced to striatum only

eNOS protein expression
At the end of CBFR, in animals treated with myricetin at higher dosage we observed a significant increased of eNOS protein concentration by western blot analysis compared to RF group and SO-Na subgroup. In particular, the eNOS expression was higher in the cortex than in the striatum; the total and phosphorylated eNOS proteins increased to the same extent ( Figure 8).

Discussion
Several experimental results indicate that the hypoperfusion and reperfusion injury is a common feature of ischemic stroke, which occurs when blood supply is restored after a period of ischemia. The spontaneous reperfusion that occurs after hypoperfusion, despite the beneficial effect of restored oxygen supply, also causes deleterious effects that include oxidative stress, leukocyte infiltration, platelet adhesion and aggregation, complement activation, mitochondrial mediated mechanisms and blood-brain barrier disruption, ultimately leading to edema or hemorrhagic transformation in the brain [33][34][35]. Recently, against ischemia and reperfusion several studies have been focused on the fundamental role of antioxidant substances [24,36]. We have studied for the first time the effects of myricetin on damages induced by hypoperfusion and reperfusion on rat pial microcirculation, utilizing as experimental model the transient bilateral common carotid artery occlusion [37]. Although their numerous protective effects widely reported, myricetin (or flavonoids) have been poorly studied and few authors have analyzed the effects of hypoperfusion and reperfusion, showing often conflicting results [33]. The effects of myricetin have been detected in rat models of intestinal ischemia and reperfusion, demonstrating that the pretreatment with myricetin induced selective protection without affecting corresponding normal controls; moreover, p-MKK7 has been shown to be the key target in the protective actions [16]. Using the experimental model of permanent occlusion of the rat middle cerebral artery, myricetin exhibited marked effects by reducing ischemic cerebral injury; this protection may be associated to the reduction in the expression levels of IL-1β, IL-6, TNF-α, decrease in MDA (malondialdehyde) test and to the increase in GSH/GSSG ratio and SOD activity [38]. However, no data were reported on the direct effects of myricetin on smaller cerebral arterioles, representing the main site of the blood flow regulation in the capillary networks. We demonstrate, for the first time, that myricetin was able to counteract the microvascular changes induced by hypoperfusion and reperfusion in a dose-dependent manner. The administration of myricetin not only preserved the arteriolar diameter at the end of hypoperfusion, but also induced a dose-dependent vasodilatation, involving the small arterioles responsible for regulation of the capillary blood flow after 60 minutes of reperfusion. Moreover, it was observed a significant reduction in microvascular permeability and ROS formation that represent the key mechanisms of damage, induced by reperfusion. These results underline the protective effect of this antioxidant drug on the blood brain-barrier. It is possible to suggest that the role of this drug is likely linked to the scavenger action against reactive oxygen species. Moreover, myricetin was able to prevent the adhesion of leukocyte to the venular walls and to preserve the capillary perfusion. In order to clarify the mechanism of action triggered by myricetin to induce vasodilation, L-NIO, a specific inhibitor of the eNOS, has been infused before the injection of higher-dose myricetin. In these rats, the vasodilation was completely abolished. Therefore, it is possible to hypothesize that myricetin stimulates eNOS expression and NO release. This hypothesis was confirmed by Western blotting analysis, carried out on samples of brain tissue. It has been shown, indeed, that the administration of myricetin induced a dose-dependent increase of eNOS expression. In contrast, a dramatic reduction of eNOS expression was observed in animals treated with L-NIO. These data are in agreement with a previous study demonstrating that myricetin is effective in the regulation of NO bioavailability, through the stimulation of the biosynthetic pathway of eNOS / NO by Akt phosphorylation [39].

Conclusion
In conclusion, myricetin protects the cerebral microcirculation in a condition of acute injury due to transient hypoperfusion and reperfusion; this effect appear to be related to its antioxidant properties and to modulation of NO release. However, further studies of these pathways are required to clarify all the mechanisms operative in the microvascular damages due to cerebral blood flow reduction.