Cerebral blood flow (CBF) autoregulation can be defined as the maintenance of constant cerebral perfusion during changes in cerebral perfusion pressure (CPP). The mechanism of CBF autoregulation is still not completely understood, but myogenic, metabolic, neurogenic, and endothelial factors seem to be involved . CBF autoregulation is mediated through vasodilation or vasoconstriction at the level of the resistance vessels, although changes in vascular tone of large cerebral arteries may shift the regulatory range . In rodents and humans, the autoregulatory range is within CPP values of approximately 60-150 mm Hg [3,4].
CBF autoregulation may be impaired in the presence of cerebral vasodilators. For example, volatile anesthetics seem to modulate CBF autoregulation in a dose-dependent fashion. Studies in dogs have shown that CBF autoregulation was maintained with 1 minimum alveolar anesthetic concentration (MAC) isoflurane but was pressure-passive with higher concentrations of the volatile anesthetic . Like isoflurane, sevoflurane produces cerebral metabolic suppression and electroencephalographic burst suppression without evidence of seizure activity. Although the favorable pharmacokinetic and pharmacodynamic char-acteristics of sevoflurane may be suitable in neurosurgical patients, it is unclear whether sevoflurane induces cerebrovascular dilation associated with changes in CBF autoregulation. In the present study, we therefore investigated the effects of sevoflurane on CBF as a function of decreases in mean arterial blood pressure (MAP) in rats.
This study was approved by our institutional animal care committee. Twenty-four adult nonfasted male Sprague-Dawley rats (300-540 g) were anesthetized in a bell jar with isoflurane. The trachea was intubated, and the lungs were mechanically ventilated. Anesthesia was maintained with 2 vol% isoflurane end-tidal in N2 O/O2 (fraction of inspired oxygen [FIO2] = 0.33) during surgical preparation. Catheters (inner diameter 0.58 mm) were inserted into the right femoral artery, both femoral veins, and the right jugular vein for continuous measurement of MAP, drug administration, and hemorrhage. A nonpenetrating burr hole 2 mm in diameter was drilled into the cranium, over the right parietal cerebral cortex 2 mm posterior to the bregma and 2 mm to the right of the midline to visualize the cortical vessels through the intact inner layer of the skull. The tip of the drill (5 mm) was continuously flushed with saline to avoid thermal injury. Red blood cell flow velocity (LDF) was measured continuously using a laser Doppler flowmeter (PeriFlux System 4001; Perimed, Stockholm, Sweden). The flow-probe (Probe 403; Perimed) was fixed to the skull using a custom frame to maintain the probe position constant over time. Care was taken to place the probe over a tissue area devoid of large blood vessels visible through the thinned bone. After confirmation of correct CBF measurement by transient hyperventilation, mechanical ventilation was adjusted to maintain PaCO2 at 38-42 mm Hg, and arterial pH was maintained at normal levels throughout the experiment by IV infusion of bicarbonate. Skull temperature was measured by insertion of a 22-gauge stainless steel needle thermistor beneath the temporalis muscle on the right side and was maintained at 38[degree sign]C by servomechanism using an overhead heating lamp. After surgical preparation, isoflurane was removed from the inspiratory gas mixture, and the animals were allowed an equilibration period of 45 min according to one of the following treatments: in Group 1 (n = 8, control) anesthesia was maintained using N2 O/O (2) (FIO2 0.33) and fentanyl (bolus 10 [micro sign]g/kg IV, followed by a fentanyl infusion of 25 [micro sign]g [center dot] kg-1 [center dot] h-1). In Group 2 (n = 8) and Group 3 (n = 8), anesthesia was maintained using 1 MAC sevoflurane (2 vol%)  or 2 MAC sevoflurane (4 vol%) in O2/air (FIO2 0.33), respectively. A norepinephrine infusion (0.2-0.5 [micro sign]g/min) was used to support baseline MAP when necessary. After baseline measurements at 100 mm Hg, MAP was reduced to the target pressures of 80, 60, 50, 40, and 30 mm Hg by graded hemorrhage. At each MAP target pressure with a range of 2 mm Hg, a stabilization was allowed for 5-8 min before the measurement. At the end of the experiment, the rat was killed with an IV bolus injection of 1% potassium chloride.
For statistical analysis, LDF in perfusion units was averaged over a 5-min period at each target MAP value and is expressed as percent change (CBF%) in relation to LDF at 100 mm Hg MAP.
Data are reported as mean +/- SD. Comparisons among different groups were made using unpaired t-tests or Welch's tests. Comparisons within the treatment groups were made using paired t-tests. The general Holm procedure  was applied to adjust for multiple tests. The significance level was set at 5%.
To evaluate CBF autoregulation, CBF% values were plotted against the corresponding MAP values. Linear regression modeling was applied to visualize the autoregulation process, as well as to distinguish among the three treatment groups. Autocorrelation due to repeated measurements in the individual animal was taken into account by assuming an autoregressive error structure. This implies that each response depends directly on the immediately preceding response. Fitted likelihood ratio tests were used to establish the best model.
The appropriate position of the flowprobe to measure cortical CBF within 1 mm3 of cortex tissue was confirmed by testing the response of CBF to transient hyperventilation in each animal. Hyperventilation (Delta PaCO2 10 mm Hg) reduced cortical CBF in all groups to a similar extent (fentanyl/N2 O 4.8% +/- 0.8%/mm Hg, 1 MAC sevoflurane 4.2% +/- 0.8%/mm Hg, and 2 MAC sevoflurane 4.1% +/- 2.0%/mm Hg). Norepinephrine (26.0 +/- 7.9 [micro sign]g) was only used in Group 3 (2 MAC sevoflurane).
(Table 1) shows physiological variables during graded hemorrhage. There were no differences in arterial blood gases and arterial pH over time because they were maintained constant according to the protocol. Hemoglobin was decreased by 3-4 g/dL in all groups to a similar extent.
(Table 2) shows CBF% during hemorrhagic hypotension. The individual CBF profiles are displayed in Figure 1, Figure 2, and Figure 3. In Groups 1 (fentanyl/N2 O) and 2 (1 MAC sevoflurane), cortical CBF was constant within the MAP range of 100-40 mm Hg. In Group 3 (2 MAC sevoflurane), cortical CBF decreased during hemorrhage (P < 0.05). In these animals, the decline at each target MAP value was greater compared with that in Group 2 (P < 0.05) within the MAP range of 100-50 mm Hg.
With linear regression, the functional relationship between CBF% and MAP is best described in terms of a second-degree polynomial. Thus, CBF% is modeled as a function of MAP and MAP2 in Groups 1 and 2 (Figure 4). In Group 3, the regression coefficient for MAP2 vanished with 2 MAC sevoflurane. This indicates that CBF% decreased in a linear fashion within the MAP range of 100-30 mm Hg in these animals.
The present study shows that CBF autoregulation in the rat cerebral cortex is intact during hemorrhagic hypotension with 1 MAC sevoflurane. In contrast, autoregulation is impaired under 2 MAC sevoflurane. The dose-dependent impairment of CBF autoregulation is consistent with previous experimental data showing substantial changes in the regulation of CBF with larger doses of volatile anesthetics [5,8,9].
Volatile anesthetics produce direct cerebral vasodilation and increases in CBF in a dose-dependent fashion. In rats, CBF is increased with higher concentrations (1.7-2.0 MAC) of halothane, isoflurane, and sevoflurane [8,10,11]. This is consistent with data in humans, in which sevoflurane increased CBF as a result of a dose-dependent reduction in cerebrovascular resistance (CVR) . Consequently, pharmacological cerebrovascular dilation (i.e., low baseline cerebrovascular tone) induced by higher concentrations of volatile anesthetics may further impair autoregulatory decreases in CVR.
Autoregulation of CBF is a function of changes in CVR in response to changes in CPP. This mechanism is sensitive to the status of the individual baseline cerebrovascular tone. If CBF autoregulation is intact, CBF remains constant as CPP changes. Several studies have shown dose-dependent changes in CBF autoregulation with volatile anesthetics. In goats, CBF autoregulation was impaired with 1 MAC but not 0.5 MAC halothane or enflurane . Similarly, CBF autoregulation was intact during 1 MAC isoflurane in animals and humans, whereas higher concentrations of isoflurane impaired CBF autoregulation [6,12]. In these studies, the CVR continuously decreased as CPP or MAP was decreased by hemorrhage with 1 MAC isoflurane, whereas CVR did not change during 2 MAC isoflurane. These experimental and clinical data are consistent with the present results, in which CBF autoregulation was maintained with 1 MAC sevoflurane, whereas 2 MAC sevoflurane impaired the autoregulatory response to graded hypotension. The data support the view that the extent of autoregulatory vasodilation is an immediate function of the preexisting baseline cerebrovascular tone that determines the vasodilatory capacity. However, the decrease in CBF with hemorrhage does not necessarily induce cerebral hypoperfusion, as the baseline CBF should be significantly higher with 2 MAC sevoflurane compared with 1 MAC sevoflurane or fentanyl/N2 O.
Several definitions and criteria that analyze the presence or absence of CBF autoregulation have been published [8,10,14-20]. Most of these previous autoregulation studies have defined arbitrary CBF thresholds (i.e., 10% or 20% change of flow from baseline) [16,18] or investigated significant changes in CBF from baseline [15,19,20] as MAP or CPP were manipulated. One of the major disadvantages of these definitions is that such rigid criteria do not meet the physiological reality, because autoregulation is a dynamic process, not an on/off phenomenon. Nevertheless, even in our present experiment, some rats showed exactly this behavior, which is related to the fact that CBF measurements were performed at predefined MAP levels. Despite this, we applied linear regression analysis to simulate the functional relationship between CBF% and MAP smoothly, rather than specifying end points or using piecewise straight regression lines . Furthermore, the repeated measurement design was adequately taken into account by allowing for autocorrelation between the CBF% measurements in a single animal. Autocorrelation was found to be a significant effect in the applied regression model and therefore endorses the hypothesis of a dynamic behavior. Because of the substantial positive correlation between two measurements, it is unlikely that there will be a sudden steep decrease in CBF%.
Volatile anesthetics decrease MAP in a dose-dependent fashion [21,22]. During the present study, a norepinephrine infusion (0.2-0.5 [micro sign]g/min) was required to maintain baseline MAP at 100 mm Hg in rats anesthetized with 2 MAC sevoflurane (Group 3), and the use of this vasoconstrictor may represent a confounding factor. However, small doses of norepinephrine (approximately 1.0 [micro sign]g/min) have little to no effect on cerebral vascular resistance as long as the blood-brain barrier is intact [23,24]. In contrast, doses of 8-10 [micro sign]g/min produce vasodilation when the blood-brain barrier is defective or when the upper limit of autoregulation is exceeded . During the present study, only animals in Group 3 received small doses of norepinephrine (<or=to1.0 [micro sign]g/min) to maintain baseline MAP. Therefore, it is unlikely that these norepinephrine concentrations changed CVR.
Laser Doppler flowmetry produces blood flow readings by multiplying the number of moving red blood cells with their mean velocities within a certain tissue sample. During the present study, the plasma hemoglobin concentration was gradually decreased during hemorrhagic hypotension, which may confound CBF measurements using laser Doppler flowmetry. Studies in rats subjected to isovolemic hemodilution have shown that decreases in cerebral hematocrit exceed systemic hematocrit . Other animal experiments suggest increases in capillary red blood cell velocity during isovolemic hemodilution or during graded hemorrhage  in response to the decreases in hematocrit and arterial oxygen content. However, if systemic hematocrit (i.e., macrohematocrit) is >30%, this increase in capillary red blood cell velocity is counteracted by either vasoconstriction of resistance vessels, enhanced shunting of flow, or redistribution of capillary flow to slow-perfusion territories . This suggests that changes in systemic hematocrit do not affect microcirculatory hematocrit. This is supported by CBF measurements using radioactive microspheres in fentanyl/N2 O-anesthetized rats , in which an autoregulatory CBF pattern after reduction of systemic hematocrit during hemorrhage was nearly identical to the present LDF data. These data indicate that laser Doppler flowmetry represents a valid technique for the investigation of the CBF autoregulation.
In conclusion, CBF autoregulation was maintained with 1 MAC sevoflurane anesthesia. In contrast, CBF autoregulation was impaired with 2 MAC sevoflurane. This is likely related to a reduction of baseline cerebrovascular tone with higher concentrations of sevoflurane, which results in a decreased capacity of autoregulatory cerebrovascular dilation during hemorrhage.
The authors thank Ms. Doris Droese for the excellent technical assistance.
1. Iadecola C, Pelligrino DA, Moskowitz MA, Lassen NA. Nitric oxide synthase inhibition and cerebrovascular regulation (state of the art review). J Cereb Blood Flow Metab 1994;14:175-92.
2. Naveri L. The role of angiotensin receptor subtypes in cerebrovascular regulation in the rat. Acta Physiol Scand 1995;155:1-48.
3. Hernandez MJ, Brennan RW, Bowman GS. Cerebral blood flow autoregulation in the rat. Stroke 1978;9:150-5.
4. Drummond JC. The lower limit of autoregulation: time to revise our thinking? Anesthesiology 1997;86:1431-3.
5. McPherson RW, Traystman RJ. Effects of isoflurane on cerebral autoregulation in dogs. Anesthesiology 1988;69:493-9.
6. Warner DS, McFarlane C, Todd MM, et al. Sevoflurane and halothane reduce focal ischemic brain damage in the rat: possible influence on thermoregulation. Anesthesiology 1993;79:985-92.
7. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat 1979;6:65-70.
8. Hoffman WE, Edelman G, Kochs E, et al. Cerebral autoregulation in awake versus isoflurane-anesthetized rats. Anesth Analg 1991;73:753-7.
9. Manohar M, Parks CM. Porcine systemic and regional organ blood flow during 1.0 and 1.5 minimum alveolar concentrations of sevoflurane anesthesia without and with 50% nitrous oxide. J Pharmacol Exp Ther 1984;231:640-8.
10. Lee JG, Hudetz AG, Smith JJ, et al. The effects of halothane and isoflurane on cerebrocortical microcirculation and autoregulation as assessed by laser-Doppler flowmetry. Anesth Analg 1994;79:58-65.
11. Conzen PF, Vollmar B, Habazettl H, et al. Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992;74:79-88.
12. Kuroda Y, Murakami M, Tsuruta J, et al. Preservation of the ratio of cerebral blood flow/metabolic rate for oxygen during prolonged anesthesia with isoflurane, sevoflurane, and halothane in humans. Anesthesiology 1996;84:555-61.
13. Miletich DJ, Ivankovich AD, Albrecht RF, et al. Absence of autoregulation of cerebral blood flow during halothane and enflurane anesthesia. Anesth Analg 1976;55:100-9.
14. Kontos HA, Wei EP, Navari RM, et al. Responses of cerebral arteries and arterioles to acute hypotension and hypertension. Am J Physiol 1978;234:H371-83.
15. Barry DI, Strandgaard S, Graham DI, et al. Cerebral blood flow in rats with renal and spontaneous hypertension: resetting of the lower limit of autoregulation. J Cereb Blood Flow Metab 1982;2:347-53.
16. Dirnagl U, Pulsineli W. Autoregulation of cerebral blood flow in experimental focal brain ischemia. J Cereb Blood Flow Metab 1990;10:327-36.
17. Hauerberg J, Juhler M. Cerebral blood flow autoregulation in acute intracranial hypertension. J Cereb Blood Flow Metab 1994;14:519-25.
18. Olsen KS, Henriksen L, Owen-Falkenberg A, et al. Effect of 1 or 2 MAC isoflurane with or without ketanserin on cerebral blood flow autoregulation in man. Br J Anaesth 1994;72:66-71.
19. Kitaguchi K, Ohsumi H, Kuro M, et al. Effect of sevoflurane on cerebral circulation and metabolism in patients with ischemic cerebrovascular disease. Anesthesiology 1993;79:704-9.
20. Cho S, Fujigaki T, Uchiyama Y, et al. Effects of sevoflurane with and without nitrous oxide on human cerebral circulation: transcranial Doppler study. Anesthesiology 1996;85:755-60.
21. Scheller MS, Nakakimura K, Fleischer JE, et al. Cerebral effects of sevoflurane in the dog: comparison with isoflurane and enflurane. Br J Anaesth 1990;65:388-92.
22. Fujibajyshi T, Yanagimoto M, Harada J, Goto Y. Brain energy metabolism and blood flow during sevoflurane and halothane anesthesia: effects of hypocapnia and blood pressure fluctuations. Acta Anaesthesiol Scand 1994;38:413-8.
23. Olesen J. The effect of intracarotid epinephrine, norepinephrine, and angiotensin on the regional cerebral blood flow in man. Neurology 1972;22:978-87.
24. MacKenzie ET, McCulloch J, O'Keane M, et al. Cerebral circulation and norepinephrine: relevance of the blood-brain barrier. Am J Physiol 1976;231:483-8.
25. Todd MM, Weeks JB, Warner DS, et al. Cerebral blood flow, blood volume, and brain tissue hematocrit during isovolemic hemodilution with hetastarch in rats. Am J Physiol 1992;263:H75-82.
26. Waschke KF, Riedel M, Albrecht DM, et al. Regional heterogeneity of cerebral blood flow response to graded volume-controlled hemorrhage. Intensive Care Med 1996;22:1026-33.
27. Kummer R, Scharf J, Back T, et al. Autoregulatory capacity and the effect of isovolemic hemodilution on local cerebral blood flow. Stroke 1988;19:594-7.
28. Hoffman WE, Werner C, Kochs E, et al. Cerebral and spinal cord blood flow in awake and fentanyl-N2
O anesthetized rats: evidence for preservation of blood flow autoregulation during anesthesia. J Neurosurg Anesthesiol 1992;4:31-5.