The French have a lower incidence of cardiovascular disease despite the relatively high proportion of saturated fat in their diet.1 This “French Paradox” has been explained by many factors, one of which is wine consumption. Polyphenolic compounds, which occur naturally in grape skin, may play an important role in the beneficial cardiovascular effects of wine. These compounds are particularly rich in red wine.2
Red wine polyphenols (RWP) have been shown to relax isolated, extracerebral vessels such as the femoral artery,3 the thoracic aorta,4 and the mesenteric artery.5 Relaxation was blocked by the nitric oxide synthase blocker, NG-nitro-L-arginine methyl ester (L-NAME). Moreover, RWP induced nitric oxide (NO) production and enhanced NO activity in endothelial cells in vitro.6,7 These studies strongly support the notion that RWP enhances endothelium-dependent vasodilation that involves an NO pathway. Despite the vast amount of literature addressing the effect of RWP on the cardiovascular system, there are a few studies on their effects on the cerebrovascular system. RWP reduce brain edema8 and infarct volume9 after cerebral ischemia in animals, but its effects on cerebral blood vessels are less known. In light of above, the goal of the present study was to investigate the effect of RWP on vasodilation in rat cerebral arterioles using an open cranial window technique in vivo. We studied the concentration-response relationship of RWP, an endothelium-dependent vasodilator adenosine diphosphate (ADP), an endothelium-dependent vasoconstrictor L-NAME, and an endothelium-independent vasodilator sodium nitroprusside (SNP) in cerebral arterioles. We then tested the hypothesis that RWP causes or improves vasodilation in the presence of ADP, L-NAME, and SNP.
The myogenic response, characterized by vasodilation after a reduction in intravascular pressure, is a mechanism that regulates vascular diameter that is particularly important in small, resistant vessels. Moreover, the myogenic response has been shown to play an important role in blood flow autoregulation in the cerebral circulation.10 Although the mechanism of this response is not completely understood, it is believed to be intrinsic to smooth muscle cell, and independent of endothelium.11 Considering the importance of the myogenic response on cerebral arterioles and the fact that the effect of RWP has not been studied, our second aim was to investigate the effect of RWP on the myogenic response induced by hemorrhagic hypotension.
MATERIALS AND METHODS
Male Wistar rats (396 ± 9 g, n = 24) were housed at 24°C, exposed to 12 hours of light (lights on at 06:00, off at 18:00), and allowed free access to standard rat chow (A04, SAPE, 89290 Angy, France) and water. Experiments were performed in accordance with the guidelines of the French Ministry of Agriculture, Paris, France (permits 54-4 and 03575).
Animals were anesthetized with sodium pentobarbital (60 mg/kg, IP) and were mechanically ventilated (50 strokes per minute, tidal volume 10 mL/kg) to maintain blood gases (pH, pCO2, pO2, blood gas analyzer 238, Ciba Corning, 95011 Cergy Pontoise, France) in the physiological range. A silicone catheter (Sigma Medical, 92000 Nanterre, France) was introduced into the right femoral vein for continuous infusion of sodium pentobarbital (0.25 mL/h; 20 mg/kg/h). The depth of anesthesia was periodically evaluated by testing for the absence of the corneal reflex, and the rate of infusion adjusted individually. A polyethylene cannula (Merck Biotrol, Chennevieres, France) was introduced into the right femoral artery; the cannula was connected to a low volume, strain-gauge transducer (Baxter, Bentley Laboratories, Europe) for measurement of systemic arterial mean blood pressure and heart rate. A second cannula was introduced into the left femoral artery to obtain blood samples for the measurement of arterial blood gases and withdrawal of blood to produce hypotension. Rectal temperature was maintained at 37 to 38°C.
Measurement of Cerebral Arteriolar Internal Diameter
We measured arteriolar internal diameter (ID), defined as the interface between circulating blood (red) and the arteriolar wall of first order pial arterioles through an open cranial window (Figure 1).12 Briefly, the head was placed in an adjustable head holder, and a 10-mm skin incision was made to expose the skull. A dam of dental acrylic cement was constructed on the exposed skull, and ports were placed for inflow and outflow of artificial cerebrospinal fluid (CSF). Craniotomy was performed over the left parietal cortex, and the dura was incised. The exposed surface was continuously superfused with artificial CSF (in mM: KCl 3.0; MgCl2 0.6; CaCl2 1.5; NaCl 131.9; NaHCO3 24.6; urea 6.7; glucose 3.7), warmed to 37 to 38°C, and equilibrated with a gas mixture of 5% CO2/95% N2.13 Arteriolar ID was monitored through a microscope (Stemi 200-C, Carl Zeiss Jena GMBH, Jena, Germany) connected to a video system using image analysis software (Saisam, Microvision Instruments, Evry, France; magnification, ×400). The precision of this system is 0.5 micron. After the cranial window procedure, animals were allowed to equilibrate for 30 minutes before exposure to drugs.
Blood samples were taken twice for blood gas analysis (see above) immediately after open window procedures and at the end of the experiment (2 hours later).
Effect of RWP on Cerebral Arterioles
Cerebral arterioles (n = 6 rats) were exposed to cumulative concentrations of RWP (0.003 to 1 μg/mL, dissolved in CSF), starting with the lowest concentration, for 5 minutes. A preliminary study showed that a plateau response was reached at 3 minutes after application of a vasoactive drug. Thus photographs were taken at +3 minutes after RWP application for measurement of ID. A higher concentration of RWP was then superfused, and the procedure was repeated. The percentage change of diameter was calculated as
Reactivity of Cerebral Arterioles to Vasoactive Agents
Cerebral arterioles (n = 5 rats) were exposed to cumulative concentrations of adenosine diphosphate (ADP, 3 × 10−6 to 10−3 M), NG-nitro-L-arginine methyl ester (L-NAME, 3 × 10−9 to 10−6 M), and sodium nitroprusside (SNP, 10−8 to 10−4 M) for 5 minutes. Changes in arteriolar ID were measured as described in the previous paragraph. Cerebral arterioles were washed with CSF until the ID returned to baseline after each drug exposure (5 minutes). The concentration-response relationship was analyzed for each drug by a sigmoid curve fit:
where Y is ID (μm), Emax is maximum effect, X is log (concentration), EC50 is the concentration that produces 50% of the maximum effect.
Effect of RWP on drug-induced changes in arteriolar diameter
Cerebral arterioles (n = 8 rats) were exposed to ADP (10−5, 10−4 M), L-NAME (10−8, 10−7 M), and SNP (10−5, 10−4 M). We chose sub-maximal concentrations based on results of the previous concentration-response study. The arterioles were washed with CSF until the ID returned to baseline after each drug exposure. RWP (10−2 mg/mL) was then superfused, and the procedures were repeated.
Effect of RWP on Hypotension-Induced Vasodilation
Hemorrhage-induced hypotension was performed (n = 5 rats) by withdrawing blood stepwise from the left femoral artery cannula in steps of 0.5 mL (8 steps, 4 mL total). Each step took about 2.5 minutes to complete. Blood was slowly withdrawn at the rate of 1 mL/min. After 2 minutes of stabilization, cerebral arteriolar ID and systemic mean arterial pressure [MAP: diastolic pressure + 1/3 (systolic pressure - diastolic pressure) measured at femoral artery] were recorded. Blood was reinjected at the end of this procedure, and the animals were allowed to recover for 30 minutes. RWP (10−2 mg/mL) were added into the CSF, and the blood withdrawal steps were repeated.
The preparation and characterization of the RWP extract from a French red wine (Corbieres, AOC, France) have been described previously.14 One liter of red wine produced 2.9 g of extract, which contained 471 mg/g total phenolic compounds expressed as gallic acid. The extract contained 8.6 mg/g catechin, 7.8 mg/g epicatechin, dimers (B1, 6.9 mg/g; B2, 8.0 mg/g; B3, 20.7 mg/g; B4, 0.7 mg/g), anthocyanins (malvidin-3-glucoside, 11.7 mg/g; peonidin-3-glucoside, 0.66 mg/g; cyanidin-3-glucoside, 0.06 mg/g), and phenolic acids (gallic acid, 5.0 mg/g; caffeic acid, 2.5 mg/g; caftaric acid, 12.5 mg/g).15
ADP, L-NAME, and SNP were purchased from Sigma Chemical (St Louis, MO). Sodium pentobarbital was purchased from Sanofi Sante Animale (Libourne, France). All superfused agents (including RWP) were prepared in CSF, and no alcohol was used.
Results were expressed as means ± SEM. Following analysis of variance, the significance of differences between means was analyzed by the Bonferroni test. The probability level for the rejection of the null hypothesis was P ≤ 0.05.
Effect of RWP on Cerebral Arterioles
RWP slightly dilated the cerebral arteriole, but the effect was not concentration-dependent. The lowest concentration that produced a significant increase in ID was 10−2 mg/mL (4 ± 1%; P < 0.05; unpaired t test versus no change, 0 ± 1%). This concentration was selected for subsequent experiments. The highest concentration of RWP (1 mg/mL) increased ID by 5 ± 1% (P > 0.05 versus 4 ± 1%).
Reactivity of Cerebral Arterioles to Vasoactive Agents
ADP dilated cerebral arterioles starting from a concentration of 3 × 10−6 M (6 ± 1%) with maximum dilation at 3 × 10−4 M (25 ± 6%) and an EC50 of 5.3 × 10−5 M (95% CI, 3.1 to 9.0 × 10−5 M) (Figure 1A). L-NAME modestly constricted cerebral arterioles. The maximum effect was at 10−6 M (-11 ± 1%) with an EC50 of 5.8 × 10−9 M (95% CI, 2.5 × 10−9 to 1.4 × 10−8 M) (Figure 1B). SNP substantially increased ID from 10−8 M (6 ± 1%) to 10−4 M (100 ± 18%). The EC50 was 1.0 × 10−6 M (95% CI, 9.2 × 10−7 to 1.1 × 10−6 M) (Figure 1C).
Effect of RWP on Drug-Induced Changes in Arteriolar Diameter
Figure 2 shows representative photographs of cerebral arterioles before (ID, 44 μm; Figure 2A) and after superfusion of ADP and SNP. Cerebral ID was measured at the same order of the arteriole as shown by arrows. ADP (10−4 M) or SNP (10−5 M) alone significantly increased ID by 14% and 70%, respectively (Figure 2, B and D). L-NAME slightly decreased ID by 5% (Figure 2C). RWP (10−2 mg/mL) significantly increased ADP-induced vasodilation at 10−5 M (from 12 ± 3% to 20 ± 4%; difference, 8%) and 10−4 M (from 17 ± 2% to 29 ± 4%; difference, 12%) (Figure 3). The effect was synergistic because RWP alone increased ID by only 4%. L-NAME alone slightly decreased ID, but it failed to inhibit the small vasodilatory action of RWP. RWP did not affect the dilation produced by the endothelium-independent vasodilator SNP.
Effect of RWP on Hypotension-Induced Vasodilation
Stepwise blood withdrawal decreased systemic blood pressure from 112 ± 3 to 56 ± 2 mm Hg, or by 50 ± 3% at 4 mL of blood withdrawn without RWP (Figure 4A). This was accompanied by an increase in ID from 42 ± 2 to 58 ± 3 μm (40 ± 9%), demonstrating an active myogenic response (Figure 4B). At the first blood withdrawal step (0.5 mL), RWP further reduced blood pressure from 118 ± 3 to 106 ± 4 mm Hg (11 ± 1% against 4 ± 2% without RWP). At the second step (1.0 mL), RWP further reduced blood pressure to 99 ± 4 mm Hg (16 ± 2%) and increased ID from 40 ± 1 to 43 ± 1 μm (8 ± 3%). There was no significant difference from the third blood step (1.5 mL; blood pressure, 89 ± 4 mm Hg) onwards.
We found in this study that ADP and L-NAME concentration-dependently induced vasodilation and vasoconstriction, respectively, indicating the existence of a functional endothelium in our model. Although RWP alone only slightly (and concentration-independently) dilated cerebral arterioles, it significantly enhanced endothelium-dependent vasodilation induced by ADP but not the endothelium-independent agent SNP. After hypotensive hemorrhage, RWP further increased vasodilation and decreased blood pressure at higher systemic blood pressure values (early blood withdrawal steps), but had no effect at lower pressure.
Our observation that RWP alone did not substantially dilate cerebral arterioles contrasts with results from other extracerebral vessels such as the aorta4 and the mesenteric artery5 in vitro. To our knowledge, this is the first study of local administration of RWP on the cerebral arteriole in vivo; as a result, direct comparison with in vitro studies is not appropriate because the in vivo situation is a complex balance of agonistic and antagonistic process. Secondly, the cerebral vascular system may respond differently compared to extracerebral arteries. Furthermore, the amplitude of the dilatory effect of RWP varies according to the constituents.16 Anthocyanins are strongly vasodilatory,16 whereas catechins and simple phenolic acids like gallic acid are not.4,17 As the content of anthocyanins in our RWP is not particularly high (12.96 mg/g, 15% of total polyphenols) and mixed with “ineffective” catechins (8.6 mg/g, 10%) and phenolic acids (total of 19.5 mg/g, 23%), it is not surprising that the dilatory effect is not pronounced. Although the RWP we used did not markedly alter arteriolar ID alone, endothelium-dependent vasodilation induced by ADP was significantly enhanced. Previous studies showed that RWP increased NO activity or bioavailability in endothelial cells.6,7 Thus RWP enhance vasodilation after NO release induced by ADP. Moreover, RWP did not modify the vasodilation induced by the NO donor SNP, supporting the idea that RWP acts on an endothelium-dependent NO pathway. However, it is unlikely that RWP acts solely on endothelium. With the application of L-NAME, cerebral arterioles constricted in response to reduced availability of NO. This constriction was reversed by RWP, indicating a possible NO-independent pathway, although we cannot rule out the possibility of NO from an L-NAME-insensitive source.
This report represents the first study of RWP on the effect of myogenic tone. Myogenic response is another mechanism that regulates vascular diameter. It is particularly important in the control of local blood flow in response to changes of blood pressure in small resistant vessels.11 RWP further increased arteriolar diameter after 1 mL of blood was withdrawn. This enhanced myogenic response was accompanied by a further decrease in blood pressure. As the myogenic response is independent of endothelium, it is possible that RWP increase the sensitivity of smooth muscle in response to changes in blood pressure, although further study is needed to support this claim. However, it is important to note that the effect of RWP on the myogenic response was limited to higher systemic blood pressure. As the myogenic tone produced substantial vasodilation (increased by 40%) at lower pressures, one might expect any effect of RWP to be masked.
Several previous studies have shown that RWP such as epicatechin18 and anthocyanins19 reach the brain quickly after oral administration. This study gives insight into the acute beneficial effect of RWP after drinking red wine in humans. Results from the present study support the idea that RWP causes vasodilation via endothelium. In a pressure-induced vasodilation study, we found that RWP also improves myogenic response by mechanisms other than endothelium. Whether these effects would be beneficial in cerebral disease states such as ischemic stroke deserves further investigation.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
cerebral arterioles; endothelium; hypotension; red wine polyphenols