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the Cardiovascular Research Center (D.Z., K.Y., C.Z., R.J.R., D.R.H.) Department of Physiology, Medical College of Wisconsin
Clement Zablocki VA Medical Center (D.R.H.), Milwaukee, Wis.
Abstract
Background and Purpose— Recent studies have suggested that autoregulation of cerebral blood flow (CBF) is impaired after traumatic and ischemic brain injury. Given that the levels of superoxide anion (O2·–) are increased in these conditions, we postulate that O2·– contributes to the impairment of CBF autoregulation.
Methods— CBF was monitored with laser Doppler flowmetry during increases in blood pressure.
Results— During the control period, CBF was well autoregulated after the increase in mean arterial pressure (MAP) from 98±3 to 140±6 mm Hg. The autoregulation index (AI; CBF/MAP) averaged 0.25±0.02 (n=6). O2·– in the brain was then increased by subdural perfusion of xanthine/xanthine oxidase (different concentrations) and catalase. Low concentrations of O2·– decreased basal CBF by 10±1.6% but had no effect on autoregulation (AI, 0.19±0.02; n=6). Higher concentrations of O2·– (0.2 mmol/L xanthine and either 3 or 20 mU xanthine oxidase) increased basal CBF by 30±2% and 42±4%, respectively, and impaired autoregulation of CBF (AI, 0.55±0.03 and 0.76±0.02; n=6). Inclusion of superoxide dismutase in the O2·–-generating system restored autoregulation (AI, 0.28±0.05; n=6). Neither inhibition of NO synthase nor the addition of deferioxamine had any effect on the ability of higher concentrations of O2·– to impair autoregulation of CBF (AI, 0.65±0.07 and 0.72±0.05 respectively; n=6). O2·– also increased the activity of KCa channels in cerebral vascular smooth muscle cells (VSMCs; n=8).
Conclusion— These results suggest that O2·– increases basal CBF and impairs autoregulation of CBF, likely through the activation of KCa channels in cerebral VSMCs.
Key Words: free radicals cerebral blood flow
Introduction
Considerable evidence suggests that superoxide anion (O2·–) has direct and indirect effects on vascular tone. O2·– increases the basal tone of vessels by reducing the availability of NO in a variety of tissues.1–3 However, recent studies suggest that O2·– can also dilate vessels by directly activating potassium (Kca) channels in vascular smooth muscle cells (VSMCs).4–6 As in other vascular beds, cerebral blood flow (CBF) is well autoregulated and remains constant despite fluctuations in arterial blood pressure. The importance of autoregulation of blood flow in cerebral circulation is greater than that in the peripheral vasculature because neuronal activity and viability is highly dependent on constant delivery of oxygen and glucose. In addition, increases in vascular resistance after elevations in blood pressure are required to protect the cerebral microcirculation from elevations of capillary hydrostatic pressure and overperfusion. Impaired autoregulation of CBF significantly contributes to disturbances of Frank-Starling forces and vasogenic brain edema, suggesting the importance of this mechanism in physiological and pathological conditions.7 The exact mechanism of CBF autoregulation remains elusive.8 Two major mechanisms, myogenic and metabolic, have been proposed, but it is unclear which plays a dominant role. Abnormalities in autoregulation of CBF have been reported after cerebral ischemia/reperfusion or traumatic brain injury, resulting in a shift of the autoregulatory curve toward higher pressures.9,10 Pretreatment of animals with antioxidants and scavengers of reactive oxygen species (ROS) have been reported to prevent the impairment in autoregulation of CBF and suggest that ROS may be involved.11,12 However, the influence of O2·– on autoregulation of CBF in the intact animal has not been studied directly. Thus, the present study examines the effect of various concentration of O2·– on autoregulation of CBF produced by superfusion of the brain of rats with artificial cerebrospinal fluid (aCSF) containing an O2·–-generating system.
Materials and Methods
Experiments were performed on 8- to 10-week-old male Sprague-Dawley rats purchased from Harlan Laboratories (Indianapolis, Ind). The rats were housed in the animal facility at the Medical College of Wisconsin, which has been approved by the American Association for Laboratory Animal Care. The institutional animal care committee approved all protocols involving animals.
Surgical Preparation
Anesthesia was induced by using 4.5% isoflurane and maintained at 2.0% using a gas anesthesia mask for small rodents (model 51610; Stoelting). The femoral vessels were cannulated with polyethylene (PE, 50) tubing (Intramedic; Clay Adams) for intravenous infusion and measurement of arterial pressure. Body temperature was maintained at 37°C using a thermostatically regulated heated surgical table. The head was fixed in a stereotaxic apparatus (model 900; David Kopf), and the closed cranial window was made in the right parietal bone using a low-speed drill as described previously. Two small holes were drilled on the opposite sides of the cranial window, and PE-10 tubing (pulled to a diameter of 100 to 200 μm) was inserted under the dura and advanced to the border of the cranial window for subdural perfusion. The subdural space was perfused continuously with aCSF at a rate of 3 μL/min and inflow pressure averaged between 5 and 8 mm Hg. Composition of the aCSF was (in mmol/L): 2.9 KCl, 38 MgCl2, 1.99 CaCl2, 131.9 NaCl, 19 NaHCO3, 6.63 urea, and 3.69 glucose, with pH adjusted daily to 7.4. After surgery, the isoflurane anesthesia was withdrawn, and anesthesia was maintained by continuous intravenous infusion of -chloralose (40 mg/kg; Sigma). Cortical microvascular blood flow was monitored with a laser Doppler flowmeter (PF3; Perimed) and a flow probe (PF316; Perimed).
Experimental Protocol
After surgery and a 30-minute equilibration period, CBF was monitored as arterial pressure was increased by increasing the rate of intravenous infusion of phenylephrine from 10 μg/mL per minute to 30 μg/mL per minute. Steady-state cortical blood flow was recorded over a 3-minute period after reaching a stable level of mean arterial pressure (MAP). After a control autoregulation curve was obtained, the infusion of phenylephrine was terminated and the subdural perfusion was switched to aCSF containing the O2·–-generating system. After a 15-minute equilibration period, the experimental relationship between CBF and MAP was re-evaluated.
Autoregulation of CBF was also tested under the condition in which basal CBF was increased. In these experiments, rats were intubated, ventilated, and normobaric normocapnic hypoxia was used to increase CBF. Hypoxic conditions were achieved by decreasing oxygen concentration in the inspired mixture from 21% to 10% and confirmed by blood gas analysis in the arterial blood samples (0.07 mL; Stat Profile; Nova Biomedical). After 15 minutes of hypoxia, during which the increase of basal CBF stabilized, autoregulation of CBF was determined by increasing blood pressure with phenylephrine infusion.
Additional experiments were performed to determine the role of NO in mediating the effects of O2·–. In these experiments, animals were pretreated by subdural perfusion of aCSF containing 50 μmol/L of N (omega)-nitro-L-arginine methyl ester (L-NAME) at a rate 3 μL/min for 45 minutes before perfusion with an O2·–-generating system.
Generation of O2·–
O2·– was produced using a xanthine (0.2 mmol/L)/xanthine oxidase (XO; 0.2, 2, 3, and 20 mU/L) reaction. Components of generating system were dissolved in aCSF, and catalase (250 U/L) was added to prevent accumulation of hydrogen peroxide. The steady-state concentration of O2·– generated by this system was measured, in vitro, by the reduction of ferricytochrome C (25 μmol/L horse heart type III; Sigma) in the presence and absence of superoxide dismutase (SOD; 150 U/mL; Sigma). Reduction of ferricytochrome C was determined spectrophotometrically at 550 nm (Beckman DU-640), and the concentration of O2·– was calculated using a molar extinction coefficient () of 21 000.13
Dihydroethidine Staining
Dihydroethidine (DHE) was used to determine the distribution of O2·– in the brain after superfusion with the generating system. In response to O2·–, DHE is oxidized intracellularly to the fluorescent product ethidium bromide (EB+), which is membrane impermeable and trapped within the cell by intercalation with DNA.14,15 In these experiments, the brains of rats were superfused with aCSF containing the O2·–-generating system for 15 minutes followed by superfusion of the brain with aCSF containing DHE (3 μmol/L) at a rate of 3 μL/min for 10 minutes. The brain was then quickly removed and frozen in liquid nitrogen. Frozen brains were sectioned on a cryostat, mounted, cover slipped, and examined using a Nikon E-600 microscope equipped with epifluorescence.
Measurement of NO Synthesis
NO synthase (NOS) activity in the brain was determined by measuring the conversion of L-[14C]-arginine to L-citrulline using reverse-phase high-performance liquid chromatography equipped with a radioactive flow detector as described previously.16,17 Comparisons of NOS activity were made between rats treated with L-NAME (50 μmol/L) and vehicle (aCSF).
Patch-Clamp Experiments
Vascular muscle cells were freshly isolated from middle cerebral arteries of rats as described previously.18 Membrane currents were recorded at room temperature using a GeneClamp 500 amplifier (Axon Instruments) and a Digidata 1200A analog-to-digital converter (Axon Instruments). Currents were recorded at 10-kHz bandwidth with a low-pass Bessel filter at 1 kHz. Data were analyzed using pCLAMP 6.0 software (Axon Instruments). Patch pipettes were pulled from borosilicate glass (No. 7052; Garner Glass) by use of a vertical puller (Narashige) and gently heat polished using a microforge (model MF-83, Narashige). The indifferent electrode was an Ag-AgCl plug connected to a bath chamber via a 140-mmol/L agar bridge. The effect of O2·– on KCa channel current activity was examined using the cell-attached configuration and symmetrical K+ solutions (145 mmol/L). KCa channel current activity was compared in the presence and absence of O2·– generated by adding xanthine and XO to the bath solution (final concentration 0.2 mmol/L xanthine and 20 mU XO).
Statistics
Mean values±SE are presented. Changes in baseline CBF are expressed as the percent change from control values. The autoregulatory capacity of cerebral vessels was expressed as an autoregulation index (AI, CBF/BP). Differences between groups were analyzed using ANOVA with Holm-Sidak post hoc test. A P value <0.05 was considered to be statistically significant.
Results
During the control period, CBF was well autoregulated and increased by 11±4% when MAP was increased from 98±3 to 140±6 mm Hg. The AI averaged 0.25±0.02 (Figure 1A and 1B). Subdural perfusion of the brain with the lowest concentration of O2·– (0.2 mmol/L xanthine and 0.2 mU XO) studied had no significant effect on basal CBF (Figure 2) or autoregulation of CBF (data not shown). A slightly higher concentration of O2·– decreased basal CBF by 10±1.6% (Figure 2) but had no significant effect on autoregulation of CBF (AI averaged 0.19±0.02). Concentration of O2·–, produced by superfusion of the brain with 0.2 mmol/L xanthine and 3.0 or 20 mU XO, increased basal CBF by 30±2 and 42±4%, respectively (Figure 2; n=6), and significantly impaired autoregulation of CBF. The AI averaged 0.55±0.03 and 0.76±0.02, respectively (Figure 1B). Addition of SOD (150 U) to the superfusate prevented the effect of O2·– on baseline flow and autoregulation of CBF (AI, 0.28±0.05; n=6; Figure 1B).
Because O2·– significantly increased basal CBF, we determined autoregulation of CBF, in which a similar increase in basal CBF was induced by hypoxia. Hypoxic conditions were confirmed by blood gas analysis (control pH 7.41±0.01, PO2 98.5±4.4, PCO2 33±0.03; hypoxia pH 7.44±0.01, PO2 40.4±4, PCO2 29±0.02). Hypoxia increased basal CBF by 38±7%; however, increasing blood pressure under hypoxic conditions resulted in no further increase in CBF (control AI, 0.11±0.01; hypoxia AI, 0.08±0.03; n=6; Figure 3A and 3B).
Other experiments were performed to determine whether blocking the synthesis of NO affects the ability of O2·– to impair autoregulation of CBF. NOS activity in the brain was inhibited by perfusion of the subdural space with aCSF containing the nonselective inhibitor L-NAME (50 μmol/L). The ability of L-NAME to inhibit NOS activity was confirmed by an 88±7% reduction of the conversion of L-[14C]-arginine to L-citrulline in the homogenized brain tissue (data not shown). Superfusion of the brain with L-NAME decreased basal CBF by 18±2% (n=6; data not shown) and blocked the effect of the low concentration of O2·– (0.2 mmol/L xanthine and 0.2 mU XO) on basal CBF. In addition, L-NAME significantly enhanced autoregulation of CBF compared with control conditions (AI, 0.13±0.01; Figure 4B). In contrast, L-NAME did not alter the ability of a higher concentration of O2·– to increase basal CBF and impair autoregulation. In animals pretreated with L-NAME, higher concentrations of O2·– (0.2 mmol/L xanthine and 20 mU XO) still raised basal CBF by 63±6% and impaired autoregulation of CBF (AI, 0.65±0.07; n=6; Figure 4A and 4B).
To determine the role of hydroxy (OH · ) radical in mediating the effects of O2·– on basal CBF and autoregulation, we examined the effect of adding the iron chelator deferioxamine (DFX) to the O2·–-generating system. The effects of O2·– on basal CBF and autoregulation were not altered by addition of 1 mmol/L of DFX to perfusate solution. Under these conditions, basal CBF increased by 39±9%, and the AI averaged 0.72±0.05 (n=4; data not shown).
Measurement of O2·– Production
The steady-state concentration of O2·– produced by the generating system was measured in vitro by the reduction of cytochrome C. The results indicate that the production of O2·– from the generating system was well correlated with the concentration of XO (data not shown).
DHE Staining
The results of these experiments are presented in Figure 5. Under control conditions (aCSF perfusion), there is some EB+ fluorescence, which most likely represents endogenously produced O2·– (Figure 5A). Superfusion of the brain with low concentration of O2·– had no effect on the intensity of EB+ staining (Figure 5B), suggesting that at this concentration, O2·– most likely remains in the extracellular space. In contrast, superfusion of the brain with a higher concentration of O2·– markedly increased the fluorescence intensity (Figure 5C). This response was abolished by the addition of cell-impermeable SOD to the perfusate solution (Figure 5D), suggesting that the observed changes were attributable to diffusion of extracellularly generated O2·–. Figure 5E and 5F depict the cerebral arterioles stained with DHE after exposure to low (Figure 5E) and high (Figure 5F) concentration of O2·–.
Electrophysiology
The effect of O2·– on KCa channel current activity in cerebral smooth muscle cells was examined using a cell-attached configuration of the patch-clamp technique. Comparisons of the current activity were made before and after addition of the O2·–-generating system to the bath. The addition of the O2·–-generating system to the bath solution significantly increased KCa channel current activity (Figure 6A). The mean open state probability of the channel increased up to 5-fold (Figure 6B). The opening frequency of the channel was not affected by an inactive generating system (boiled XO).
Discussion
Considerable evidence supports a vasoactive role for O2·–. Previous studies suggest that elevated levels of O2·– dilate cerebral arterioles.19,20 In the present study, we confirmed that superfusion of the rat brain with an O2·–-generating system that raised intracellular levels of O2·– increased baseline cortical blood flow and impaired autoregulation of CBF. In contrast, lower concentrations of O2·– that did not change intracellular levels of O2·– decreased baseline CBF and had no significant effect on autoregulation of CBF. The ability of low concentrations of O2·– to reduce CBF appears to be related to the scavenging of extracellular NO because this response was completely blocked by the L-NAME pretreatment. The ability of higher O2·– concentrations to increase baseline CBF and impair autoregulation of CBF is independent of NO because these responses were not affected by the inhibition of NOS activity. The increase in baseline CBF and impairment of autoregulation of CBF were prevented by the addition of SOD, indicating that O2·– likely mediates these effects. However, these effects may be mediated by secondary reactive species such as hydrogen peroxide or hydroxy radicals.21 The inclusion of catalase in the perfusion solution prevented the formation of hydrogen peroxide in the present experiment and therefore excludes hydrogen peroxide as the mediator of the effects. In addition, responses of baseline CBF and autoregulation to higher concentration of O2·– were also not altered by the addition of DFX to the perfusate solution, suggesting that these effects are not likely to be mediated by OH · radicals.
The opposite effects of different concentrations of O2·– on CBF in our experiments are very likely attributable to the presence of O2·– in different tissue compartments. By crossing the cell membrane, O2·– is capable of reacting and modifying a number of transmembrane and intracellular proteins that affect cellular responses. In this regard, recent studies have indicated that KCa channels play an important role in the regulation of basal CBF and autoregulation of CBF.22–24 Furthermore, the -regulatory subunit of this channel is highly redox sensitive, making it an attractive target for modulation by ROS.25 This led us to examine the effects of O2·– on the activity of KCa channels in cerebral VSMCs. Our results indicate that O2·– markedly increases the open probability of KCa channels. This would be expected to hyperpolarize VSMCs and reduce the activity of voltage-gated Ca2+ channels, which are crucial for influx of Ca2+ and the maintenance of vascular tone.26 The effects of O2·– on KCa channels activity in vitro are also consistent with our observation that superfusion of the brain with the higher concentration of O2·– increases basal CBF and impaired autoregulation of CBF.
In conclusion, the results of the present study indicate that the effects of O2·– on basal CBF and autoregulation are concentration dependent. Increased amounts of O2·– in the extracellular space decreased basal CBF, likely by scavenging NO. In contrast, superfusion of the brain with higher concentrations of O2·– that also increased intracellular levels of O2·– in the brain dilated cerebral arteries and impaired autoregulation of CBF by the mechanism that might involve activation of KCa channels in VSMCs.
Acknowledgments
This study was supported in part by grants HL33833-21, 2PO1 HL059996-06A1, and 5PO1 HL068769-04 from the National Institutes of Health, and by grant 3440-02P from the Department of Veterans Affairs Administration.
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