Anesthesiology 2000; 92:125–32 © 2000 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Neuronal Nitric Synthase Mediates - induced Cerebral Microvascular Dilation Michael Staunton, M.B., F.F.A.R.C.S.I.,* Cathy Drexler, M.D.,† Phillip G. Schmid III, M.D.,‡ Heather S. Havlik, B.S.,§ Antal G. Hudetz, B.M.D., Ph.D.,ʈ Neil E. Farber, M.D., Ph.D.# Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021

Background: The causes of volatile anesthetic-induced cere- tered and was calculated as a percentage of pre- bral vasodilation include direct effects on and constriction. indirect effects via changes in metabolic rate and release of Results: Halothane caused significant, dose-dependent dila- mediators from vascular and brain parenchyma. tion of hippocampal microvessels (halothane group). Inhibi- The role of and the relative importance of neuronal tion of nNOS by 7-NINA or nNOS ؉ eNOS by L-NAME similarly and endothelial (nNOS and eNOS, respec- attenuated halothane-induced dilation at 0.6, 1.6, and 2.6% halo- ؎ tively) are unclear. thane. The dilation (mean ؎ SEM) at 1.6% halothane was 104 -Methods: Rat brain slices were superfused with oxygenated 10%, 65 ؎ 6%, and 51 ؎ 9% in the halothane, 7-NINA ؉ halo ؉ artificial cerebrospinal fluid. Hippocampal arteriolar diameters thane and L-NAME halothane groups, respectively. The spec- were measured using computerized videomicrometry. Vessels ificity of 7-NINA was confirmed by showing that - induced dilation was not inhibited by 7-NINA but was converted were preconstricted with prostaglandin F2␣ (PGF2␣; halothane group) or pretreated with 7-nitroindazole (7-NINA, spe- to constriction by L-NAME. Conclusions: At clinically relevant concentrations, halothane -cific nNOS inhibitor, 7-NINA ؉ halothane group) or N-nitro-L potently dilates intracerebral . This dilation is medi- methylester (L-NAME; nonselective NOS inhibitor, ated, in part, by neuronally derived nitric oxide. Endothelial L-NAME ؉ halothane group) and subsequently given PGF ␣ to 2 NOS does not play a major role in halothane-induced dilation of achieve the same total preconstriction as in the halothane hippocampal microvessels. (Key words: Anesthesia; cerebral group. Increasing concentrations of halothane were adminis- blood flow; hippocampus; microcirculation.)

* Visiting Assistant Professor, Department of Anesthesiology, Medi- VOLATILE anesthetics cause cerebral vasodilation, cal College of Wisconsin. which may to increased cerebral blood flow (CBF), † Resident, Department of Anesthesiology, Medical College of Wis- cerebral blood volume, and intracranial pressure.1,2 consin. When there is decreased intracranial compliance or al- ‡ Instructor, Department of Anesthesiology; and Fellow, Neuroanes- thesiology Research, Medical College of Wisconsin. ready established intracranial hypertension, further in- creases in intracranial pressure may cause cerebral isch- § Medical Student, Medical College of Wisconsin. emia and herniation of brain tissue. The mechanisms of ʈ Professor, Departments of Anesthesiology and Physiology, Medical College of Wisconsin. volatile anesthetic-induced cerebral vasodilation are un- derstood incompletely and may include direct effects on # Associate Professor, Departments of Anesthesiology, Pharmacol- ogy and Toxicology, and Pediatrics; and Director, Pediatric Anesthesi- vascular smooth muscle and indirect effects via release ology Research, Medical College of Wisconsin and the Children’s of mediators from endothelial and parenchymal cells and Hospital of Wisconsin. changes in neuronal activity or cerebral metabolic Received from the Departments of Anesthesiology, Physiology, Phar- rate.1–3 The relative importance of these factors is un- macology and Toxicology, and Pediatrics, the Medical College of Wis- known. In particular, the role of nitric oxide (NO) and consin and the Children’s Hospital of Wisconsin, Milwaukee, Wiscon- the relative importance of NO derived from neuronal sin. Submitted for publication March 11, 1999. Accepted for publication June 29, 1999. Supported in part by RO1 grants and endothelial NO synthases (NOS; nNOS, type I NOS; no. GM56398 (to Drs. Hudetz and Farber) and 2T32GM08377 (to Dr. eNOS, type III NOS, respectively) are unclear. In vitro Schmid) from the National Institutes of Health, Bethesda, Maryland; studies using isolated large cerebral arterial rings have and by the Foundation for Anesthesia Education and Research, Roch- shown that vascular relaxation caused by volatile anes- ester, Minnesota; Ohmeda, Tewksbury, Massachusetts; and the Society thetics does not depend on the presence of an intact for Pediatric Anesthesia, Richmond, Virginia (Dr. Farber). endothelium.4,5 In contrast, in vivo studies of the pial Address reprint requests to Dr. Farber: Department of Anesthesiol- ogy, MEB, 462C, Medical College of Wisconsin, 8701 Watertown Plank circulation have shown that inhibition of NOS with the Road, Milwaukee, Wisconsin 53226. Address electronic mail to: nonselective NOS inhibitor, N-nitro-L-arginine methyl- [email protected] ester (L-NAME), decreases volatile anesthetic-induced di-

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lation.3,6–8 A study that evaluated laser Doppler flow in achieving sufficient anesthesia, a midline thoracotomy the parietal cortex of nNOS gene-deficient mice and was performed and 20 ml NaCl, 0.9%, was infused into wild-type controls suggested that eNOS may be involved the left ventricle while simultaneously making a right in isoflurane-induced hyperemia at low anesthetic con- atrial incision for blood drainage. The animals were then centrations (1.2 and 1.8%), with nNOS involved at higher decapitated and the brains rapidly removed and rinsed concentrations (2.4%).9 These studies described changes with nutrient medium (artificial cerebrospinal fluid, in large cerebral arteries and superficial cerebral vessels, aCSF) of the following composition (mM): NaCl: 124; Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 but did not provide information about the mechanism of KCl: 5; CaCl2: 2.4; MgCl2: 1.3; glucose: 10; KH2PO4: anesthetic-induced effects on intraparenchymal arte- 1.24; NaHCO3: 26. Nutrient medium was prepared daily rioles specifically. and equilibrated with 95% and 5% diox- We previously used an in vitro rat brain slice prepara- ide (95% O –5% C ) to achieve a pH of 7.4. All mea- 2 O2 tion to show that halothane causes potent, dose-depen- surements of cerebral microvessel diameters were per- dent dilation of hippocampal arterioles10 and that halo- formed within5hofthetissue slice preparation. thane and isoflurane cause dilation that is region-specific Brains were cut freehand into blocks containing the and agent-specific.11 At equipotent doses, the two agents hippocampus. A vibratome mechanical tissue slicer cause similar dilation in hippocampal, but not in neocor- (OTS-3,000-03; FHC, Brunswick, ME) was used to imme- tical, vessels, in which halothane produces a greater diately section the block into coronal slices approxi- degree of dilation.11 mately 280-␮m thick. Throughout the slicing procedure, The objectives of this investigation were to assess the tissues were continuously bathed in the oxygenated contribution of NO to the resting tone of hippocampal aCSF at room temperature. Subsequently, the slices were intraparenchymal arterioles, to define the role of NO in transferred to a Plexiglas holding chamber (M & G Plastic the mechanism of halothane-induced microvascular dila- Specialists, West Allis, WI) and maintained at interface tion, and to determine the relative importance of NO with oxygenated aCSF at the same temperature. Individ- derived from eNOS and nNOS. An in vitro rat brain slice ual slices were then transferred for evaluation to a preparation was used to assess the effects of halothane recording chamber mounted on an inverted halogen on hippocampal arterioles in the presence and absence transillumination microscope (Nikon Diaphot 200; of NOS inhibition. The NOS inhibitors used were the Yokohama, Japan). nonselective NOS inhibitor L-NAME and the selective The recording chamber consisted of a central record- nNOS inhibitor 7-nitroindazole sodium (7-NINA). ing–superfusion compartment and a laterally placed ele- vated chamber to allow gentle vacuum suction. Nylon mesh beneath the brain slice allowed for circulation of Materials and Methods superfusate under and around the slice. Flow through the recording chamber was at a rate of 2.0 ml/min, All experimental procedures used in this investigation completely exchanging the volume in the chamber in were reviewed and approved by the Animal Use and less than 2 min. The chamber temperature was contin- Care Committee of the Medical College of Wisconsin, uously monitored and maintained at 34°C using a ther- with protocols completed in accordance with the Guid- moelectric Peltier device coupled to a sensing ther- ing Principles in the Care and Use of Laboratory Animals mistor. The slices were maintained in this chamber, of the American Physiologic Society and in accordance continuously superfused with the oxygenated aCSF for with National Institutes of Health (NIH) guidelines. All approximately 1 h before initiation of the experimental animals used in this investigation were housed within protocol. During this equilibration period an intracere- the animal facilities of the Medical College of Wisconsin, bral microvessel was located. The aCSF that superfused accredited by the American Association for the Accred- the brain slices was equilibrated with a mixture of oxy- itation of Laboratory Care. gen, , and air sufficient to maintain the bath at a pH of 7.35–7.45, a P of 35–45 mmHg, and CO2 General Preparation aP in the range 210 Ϯ 30 mmHg. Gas analysis (Radi- O2 Adult male Sprague-Dawley rats, weighing 200–300 g, ometer ABL3, Copenhagen, Denmark) of the superfused were placed in an animal holding chamber and under- fluid was obtained during the equilibration period and at went inhalational induction of anesthesia using 2% halo- least every 30 min during the experimental protocol. thane (Anaquest Inc., Madison, WI) in oxygen. After The P was also continuously measured with an in-line O2

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HALOTHANE AND CEREBRAL VASODILATION Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021

Fig. 1. Schematic representation of the experimental protocol. Vessel diameters were measured at the end of each time period.

flow-through P electrode (DO-166FT; Lazar Research prostaglandin F ␣ (PGF ␣; Sigma Chemical Co., St. Louis, O2 2 2 Laboratories, Los Angeles, CA). MO), given at a bath concentration of 1 ␮M, to produce a constriction of approximately 11–14% from base- Microvessel Analysis line.10,11 In the other two groups, vessels were pre- A vessel (range, 10–25 ␮m in diameter) was located treated with either 7-NINA (Tocris Cookson Inc., Ball- within the parenchyma of the hippocampus. The integ- win, MO), given at a bath concentration of 2 ␮M (7- rity and diameter were continuously monitored using NINA ϩ halothane group), or L-NAME (Sigma Chemical videomicroscopy. Arterioles were differentiated from Co., St. Louis, MO), given at a bath concentration of 50 venules by the presence and the characteristics of the ␮M (L-NAME ϩ halothane group). In initial dose-finding vascular smooth muscle. Equipment consisted of an in- experiments, we found that 7-NINA caused a maximal verted halogen transillumination microscope, a 40ϫ ob- constriction of 6–8% at a dose of 2 ␮M, whereas L-NAME jective (Olympus WPlanFL 160/0, Tokyo, Japan), and a caused a much greater degree of constriction at doses 2.25ϫ video projection lens (Nikon CCTV/Microscope more than 50 ␮M. Although other authors using the same Adapter, Yokohama, Japan). The image was transmitted preparation have given nonselective NOS inhibitors to to a video camera (CCD 72; Dage MTI, Michigan City, IN) produce a preconstriction of approximately 30%,12,13 and displayed on a video monitor (Sony HR Trinitron, we were reluctant to use higher doses of L-NAME be- Tokyo, Japan). Vessel diameter changes were recorded cause of the possibility of mechanisms unrelated to NOS 14 on videotape using a VHS video recorder (Magnavox, inhibition. After 7-NINA or L-NAME administration, ves-

Rebersburg, PA) and analyzed using a computerized im- sels were further constricted with PGF2␣, given at a bath aging analysis system (Metamorph Imaging System, Uni- concentration of 0.5–0.8 ␮M, to achieve the same total versal Imaging Corp., West Chester, PA) with an IBM- preconstriction as that in the halothane group. We chose compatible computer. doses of 7-NINA and L-NAME that caused a similar level

of constriction (6–8%) and used PGF2␣ to ensure the Experimental Protocol same total level of preconstriction so that the response There were three experimental groups: the halothane to halothane could be compared in vessels that have the group, the 7-NINA ϩ halothane group, and the same initial vascular tone. All drugs were diluted in aCSF L-NAME ϩ halothane group. The protocol is represented and infused directly into the recording chamber. schematically in figure 1. In all groups, vessel diameter After preconstriction, increasing doses of halothane was measured at the end of the equilibration period. In were given, dissolved in aCSF for 30 min at each dose the halothane group, vessels were preconstricted with (fig. 1). Halothane was volatilized into the aCSF by pass-

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ing the oxygen, carbon dioxide, and air mixture through Table 1. Baseline Data for the Experimental Groups a vaporizer (Model F100; OH Medical Products, Airco Group Inc., Madison, WI). The time necessary for equilibration of halothane concentrations in the recording chamber 7-NINA ϩ L-NAME ϩ Halothane Halothane Halothane was determined during previous experiments.10 The halothane vaporizer was set at dial settings of 0.4, 0.6, No. of slices 14 14 10 ␮ Ϯ Ϯ Ϯ 1.8, and 2.6%. At the end of each equilibration period, a Baseline diameter ( m) 15.4 1.4 16.5 1.1 17.9 0.9

Constriction (%) Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 1-ml sample of aCSF was taken from the recording cham- 7-NINA/L-NAME 7.2 Ϯ 0.7 6.7 Ϯ 0.6 Ϯ Ϯ Ϯ ber for measurement of halothane concentrations by gas PGF2␣ 13.2 0.7 12.9 0.6 12.7 0.8 chromatography (Sigma 3B; Perkin-Elmer, Norwalk, CT). Preconstricted diameter ␮ Ϯ Ϯ Ϯ A glass coverslip was placed over the chamber to ensure ( m) 13.3 1.2 14.3 0.9 15.6 0.8 Percent of baseline 86.8 Ϯ 0.7 87.1 Ϯ 0.6 87.3 Ϯ 0.8 a consistent relation between the vaporizer dial settings Gas analysis and the measured halothane concentrations. Measured pH 7.37 Ϯ 0.02 7.38 Ϯ 0.01 7.39 Ϯ 0.01 P (mmHg) 38.2 Ϯ 1.5 36.5 Ϯ 0.4 36.4 Ϯ 0.4 aqueous concentrations (mM) were converted to partial CO2 P (mmHg) 232 Ϯ 19 194 Ϯ 5 221 Ϯ 5 pressures (percent of one atmosphere, %), yielding mean O2 15 values of 0.4, 0.6, 1.6, and 2.6%. In the rat, these partial Data are expressed as mean Ϯ SEM. There are no significant differences pressures are equivalent to minimum alveolar concentra- between groups. PGF ϭ prostaglandin F ;P ϭ carbon dioxide partial pressure; P ϭ tions (MAC) of 0.3, 0.5, 1.5, and 2.3 (MAC value for the 2␣ 2␣ CO2 O2 rat ϭ 1.1%).16 oxygen partial pressure. To evaluate the specificity of 7-NINA and L-NAME at the doses used in this investigation, the dilator effect of where DN is the new diameter and DP is the diameter acetylcholine was assessed during control conditions after preconstriction (fig. 1). (acetylcholine group, n ϭ 6) and in the presence of 7-NINA (7-NINA ϩ acetylcholine group, n ϭ 6) and Statistical Analysis L-NAME (L-NAME ϩ acetylcholine group, n ϭ 7). In the Statistical analysis was performed using one-way anal- acetylcholine group, vessels were preconstricted with 1 ysis of variance (between groups) and repeated-mea- ␮ ϩ sures one-way analysis of variance (within groups). In M PGF2␣. In the 7-NINA acetylcholine and the L-NAME ϩ acetylcholine groups, vessels were pretreated both cases, follow-up multiple comparisons were made with 2 ␮M 7-NINA or 50 ␮ML-NAME and further con- using the Duncan multiple range test. Differences were stricted with 0.5–0.8 ␮M PGF . After preconstriction, considered statistically significant when the P value was 2␣ Ϯ acetylcholine was given at bath concentrations of 10 ␮M less than 0.05. All data are expressed as the mean SEM. and 100 ␮M in sequence for 20 min at each dose. Results Data Analysis A total of 50 hippocampal slices were obtained from Because changes may be nonuniform, mi- 24 animals. Brain slices were excluded from the analyses crovessel diameters were derived as an average of 10–13 if microvessels could not be adequately visualized or if measurements taken every 6–10 ␮m along approxi- the luminal diameters of the microvessels were not mately 80 ␮m of vessel length. The percentage constric- clearly discernible during the experiment. The number tion of the cerebral from the baseline diameter of slices that completed the experimental protocol and was calculated using the following equation: baseline data for the experimental groups are shown in % constriction ϭ ͑D Ϫ D ͒/D ϫ 100 table 1. The arteriolar diameters before preconstriction BL N BL did not differ significantly between groups. 7-NINA and L-NAME caused similar microvascular constriction in the where DBL is the baseline diameter at the start of the 7-NINA ϩ halothane and L-NAME ϩ halothane groups. experiment and DN is the new diameter. The percentage dilation of the cerebral arteriole was calculated as a The total preconstriction after administration of PGF2␣ percentage of the total amount of preconstriction using was also similar in all groups. the following equation:10,11 Halothane alone caused significant and dose-depen- dent microvascular dilation (halothane group). How- ϭ ͑ Ϫ ͒ ͑ Ϫ ͒ ϫ % dilation DN DP / DBL DP 100 ever, this was significantly less in the presence of 7-NINA

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7-NINA and L-NAME caused similar constriction in the 7-NINA ϩ acetylcholine and L-NAME ϩ acetylcholine groups (7.8 Ϯ 1% and 6.6 Ϯ 0.9%, respectively). The

total preconstriction after administration of PGF2␣ was also similar in all groups (total preconstriction of 11.1 Ϯ 1.9%, acetylcholine group; 13.1 Ϯ 1.3%, 7-NINA ϩ ace- tylcholine group; 11.8 Ϯ 1.4%, L-NAME ϩ acetylcholine group). Vessel diameters were significantly greater than Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 the control diameters at both concentrations of acetyl- choline in the acetylcholine and acetylcholine ϩ 7-NINA groups (percentage change from the preconstricted di- ameter 9.4 Ϯ 2.1% and 9.8 Ϯ 1.5%, respectively; 100 ␮M acetylcholine; fig. 3). There was no significant difference between the effect of acetylcholine in the presence and absence of 7-NINA. In contrast, the dilator response to acetylcholine was converted to constriction in the Fig. 2. Microvessel dilation caused by halothane alone and in L-NAME ϩ acetylcholine group. In this group, vessel the presence of 7-NINA and L-NAME. Dilation was calculated as diameters were significantly different from the control a percentage of the amount of preconstriction (see text). Halo- thane partial pressures (%) were calculated from measured bath diameters and from those in the acetylcholine and concentrations (mM). Error bars represent SEM. *P < 0.05 ver- 7-NINA groups at both concentrations of acetylcholine sus halothane group. †P < 0.05 versus 7-NINA ؉ halothane (percentage change from the preconstricted diameter group. There was similar attenuation of halothane-induced di- 15.3 Ϯ 1.8%; 100 ␮M acetylcholine; fig. 3). lation by 7-NINA and L-NAME at 0.6, 1.6, and 2.6% halothane. and in the presence of L-NAME (fig. 2). Halothane, 0.6% Discussion and 2.6%, caused dilation of 63 Ϯ 7% and 143 Ϯ 18%, respectively. Halothane, 0.6%, caused dilation of 37 Ϯ Nitric oxide is recognized as an ubiquitous 7% and 36 Ϯ 6% in the presence of 7-NINA and L-NAME, that has an important role in many physiologic and respectively. Halothane, 2.6%, caused dilation of 85 Ϯ 11% and 65 Ϯ 8% in the presence of 7-NINA and L-NAME, respectively. 7-NINA and L-NAME caused similar attenu- ation of halothane-induced dilation at 0.6, 1.6, and 2.6% halothane. At 0.4% halothane, halothane-induced dila- tion was significantly less in the presence of L-NAME than in the presence of halothane alone or in the presence of 7-NINA (35 Ϯ 4%, 30 Ϯ 7%, and 14 Ϯ 3% dilation at 0.4% halothane in the halothane, 7-NINA ϩ halothane, and L-NAME ϩ halothane groups, respectively). The mean bath concentration of halothane 1 h after the anesthetic was discontinued was 0.07–0.08 mM in all groups. These aqueous concentrations are equivalent to mean partial pressures of 0.24–0.27%. Vessel diameters had decreased toward their preconstricted control val- Ϯ Ϯ Ϯ Fig. 3. Dilation of hippocampal arterioles in response to acetyl- ues (mean dilation 15 5%, 3 4%, and 10 11% in choline during control conditions (acetylcholine group [ACh]) -the halothane, halothane ϩ 7-NINA, and halothane ϩ and after pretreatment with either 7-NINA (7-NINA ؉ acetylcho ؉ L-NAME groups, respectively), and significant microvas- line group) or L-NAME (L-NAME acetylcholine group). The doses of 7-NINA and L-NAME were the same as those that were cular dilation (compared with baseline) was not ob- given in the presence of halothane. Dilation or constriction was served in any group. calculated as a percentage change from the preconstricted di- In experiments to evaluate the specificity of 7-NINA ameter. Acetylcholine-induced dilation was similar in the pres- ence and absence of 7-NINA. Error bars represent SEM. *P < 0.05 -and L-NAME, baseline diameters did not differ signifi- versus acetylcholine group. †P < 0.05 versus 7-NINA ؉ acetyl cantly between groups (mean diameters 15.8–16.8 ␮m). choline group. §P < 0.05 versus 10 ␮M acetylcholine.

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pathologic processes. In the brain, NO is continuously increases in cGMP content in the isolated aorta.27 In in produced by eNOS and nNOS.17 Once formed, NO dif- vitro preparations of brain tissue and vascular endothe- fuses into vascular smooth muscle in which it activates lial and smooth muscle cells, volatile anesthetics have soluble (GC). This increases the syn- minimal effect on NOS activity,28 GC activity,29 and thesis of cyclic guanosine 3Ј,5Ј-monophosphate (cGMP) cGMP content.30,31 and to vasodilation.17 In this investigation, we In contrast to in vitro studies, administration of non- assessed the effects of halothane on hippocampal arte- selective NOS inhibitors in vivo, either topically or sys- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 rioles in the presence and absence of L-NAME and temically, decreases volatile anesthetic-induced cerebral 6,7 7-NINA. L-NAME is an L-arginine analog that causes a vasodilation. Similarly, studies of regional CBF found 3,8 prolonged, competitive inhibition of eNOS and that L-NAME attenuates anesthetic-induced hyperemia. nNOS.18,19 In contrast, 7-nitroindazole20 (7-NI) and its Okamoto et al. found that isoflurane-induced increases more -soluble sodium salt, 7-NINA,21 cause a selec- in laser Doppler flow were decreased by L-NA at all tive inhibition of nNOS. concentrations in wild-type mice, but only at lower con- The unchanged acetylcholine-induced dilation in the centrations in nNOS gene-deficient (knockout) mice.9 presence of 7-NINA suggests that when 7-NINA is given The authors concluded that eNOS (with or without in our preparation and at the dosage used in our study, nNOS) may be involved in alterations of CBF at lower it does not inhibit eNOS. In contrast, the dilator response concentrations of isoflurane, with nNOS only involved in to acetylcholine was converted to constriction in the high-dose isoflurane-induced hyperemia.9 presence of L-NAME, suggesting a direct smooth muscle A role for NO in volatile anesthetic-induced cerebral effect of acetylcholine. Fergus and Lee12 also found that vasodilation cannot be excluded by in vitro studies that the dilator response to acetylcholine was inhibited by use isolated arteries. These vessels are removed from ␻ N -nitro-L-arginine (L-NA) (nonselective NOS inhibitor) their neuronal and glial framework and therefore cannot 22–24 but not by 7-NI (10 ␮M). In contrast, the dilator response be under the influence of NO derived from nNOS. to NMDA was inhibited by L-NA and 7-NI, showing that In addition, the role of NO may be different in vessels of NMDA-induced microvascular dilation is mediated, in different size.32 Although volatile anesthetic-induced pial part, by NO derived from nNOS.12 Taken together, these vessel dilation may depend, in part, on NO derived from findings suggest that 7-NINA selectively inhibited nNOS. eNOS,7,9 it may also, similar to the response to hyper- Traditionally, eNOS is considered to be present in vas- capnia,33 be influenced by NO derived from nNOS. cular endothelial cells and nNOS is considered to be We previously used the brain slice microvessel prepa- present in neurons, perivascular nerves, and astro- ration to demonstrate that vasomotor responses to alter- cytes.22–24 This delineation may not be entirely correct ations in oxygen34 and carbon dioxide35 levels are well- because nNOS may also be present in the endothelium.25 preserved. The model used in this investigation is the However, endothelium-derived nNOS does not appear to first to evaluate the effects of anesthetic agents on intra- mediate endothelium-dependent dilation.12,17 parenchymal arterioles and on the relation between The role of NO in volatile anesthetic-induced cerebral these vessels and their surrounding neurons and glial vasodilation is controversial. In isolated cerebral arteries, cells. The findings of our study suggest that NO is in- relaxation caused by halothane and isoflurane is not volved intimately in halothane-induced dilation of these affected by removal of the endothelium4,5 or, in the case vessels. The lack of a difference between the effects of G of isoflurane, by the nonselective NOS inhibitor, N - L-NAME and 7-NINA at halothane concentrations of 0.6, 5 monomethyl-L-arginine. The results of studies to deter- 1.6, and 2.6% suggests that, at these concentrations, NO mine the effects of halothane on GC and cGMP content derived from nNOS but not eNOS is involved. In addi- depend on whether the direct actions of halothane or its tion, the greater inhibitory effect of L-NAME at 0.4% effects on agonist-induced increases in these variables halothane suggests a role for eNOS at this concentration. were evaluated. Although halothane increases basal These findings agree with those of Okamoto et al.,9 who cGMP levels in isolated canine middle cerebral arteries, it suggested that cerebral hyperemia at low concentrations is the particulate (non–NO-dependent), rather than the of isoflurane is modulated by eNOS. However, our re- soluble (NO-dependent), GC activity that increases.26 sults contrast with those of Koenig et al.,7 who sug- Halothane decreases endothelium-dependent relaxation gested a more important role for eNOS in isoflurane- of isolated carotid arteries and the aorta.4,27 Halothane induced pial arteriolar dilation. The cause of this also decreases NO-induced relaxation and NO-induced discrepancy is unclear, but may be related to regional

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differences in vascular reactivity (intraparenchymal . are involved in volatile anesthetic-induced cerebral vaso- pial arterioles), agent-specific differences (halothane vs. dilation.1–3 The residual anesthetic-induced dilation that isoflurane), differences in the experimental model used was not inhibited by NOS inhibitors may be caused by (in vitro vs. in vivo microvessel diameter), and differ- incomplete acute NOS inhibition, but may also reflect ences in the technique used to inhibit endothelium- direct smooth muscle effects, effects on local neuronal dependent dilation (NOS inhibitor vs. endothelial injury). activity or neuronal-vascular coupling, stimulation of par- It is important to interpret these results with caution. ticulate (non–NO-dependent) GC,26 or involvement of Although microvascular constriction by NOS inhibitors other endothelial or parenchymal mediators, such as Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 strongly suggests basal production of NO in our prepa- prostaglandins.3 Just as anesthetic agents may differ with ration, the relative baseline activities of eNOS and nNOS respect to regional effects on CBF and ,3,11 so and the relative degree of inhibition by nonselective too the mechanism of cerebrovascular effects may vary NOS inhibitors are unknown. An important stimulus for depending on the agent and region evaluated and the the synthesis of NO by eNOS in vivo is intraluminal dosage of anesthetic used.9 Endothelium-dependent re- blood flow and pressure,36 which are absent in our sponses may exhibit wide regional heterogeneity,32,39 preparation. Nevertheless, agonist-induced release of reflecting variations in the distribution of NOS,22 neural NO occurs, as shown by acetylcholine-induced dilation control of NO release,40 and sensitivity of vascular endo- 12 39 inhibited by L-NA, L-NAME, or a period of hypoxia– thelial and smooth muscle cells. reoxygenation.37 In conclusion, halothane-induced dilation of intrapa- Administration of NOS inhibitors in vivo causes cere- renchymal cerebral arterioles appears to depend, in part, bral vasoconstriction6 and decreased CBF.3,8,9,38 This on the synthesis of NO. These results suggest that the may inhibit the response to anesthetics and other vaso- NO that contributes to this dilation is derived mainly dilators because of a nonspecific vasoconstrictor effect, from nNOS. The source of nNOS for regulating cerebro- rather than because of a deficiency of NO. We addressed vascular tone in the presence of halothane may be this difficulty in our study by ensuring the same total perivascular or astrocytic, rather than brain parenchy- preconstriction in all three experimental groups. Smith mal, because the latter may involve a greater diffusion et al.8 evaluated the role of NO in the cerebrocortical distance. In this in vitro brain slice preparation, NO laser Doppler flow response to halothane in rats. They derived from eNOS does not appear to play an important found that L-NAME significantly inhibited this response; role. Future studies using this model will investigate the however, the use of vasoactive agents to restore baseline effects of volatile anesthetic agents on intracerebral mi- cerebrocortical flow and mean arterial pressure in a crovessels in nNOS gene-deficient mice. subset of the L-NAME–treated rats resulted in hyperemia to halothane, which was not different from that ob- The authors thank David A. Schwabe, Dale C. Ekbom, and Nicole served during control conditions.8 The discrepancy be- Beauvais for expert technical assistance. tween our findings and those of Smith et al.8 may reflect regional differences in vascular reactivity or methodolog- ical differences (vessel diameter vs. erythrocyte flow, References absent vs. present intraluminal blood flow, no baseline 1. Todd MM, Drummond JC: A comparison of the cerebrovascular anesthesia vs. baseline anesthesia, use of va- and metabolic effects of halothane and isoflurane in the cat. ANESTHE- soconstrictors vs. vasodilators to equalize vascular tone). SIOLOGY 1984; 60:276–82 It has been suggested that NO may act, not as a classic 2. Drummond JC, Todd MM, Scheller MS, Shapiro HM: A compari- mediator, but as a “permissive modulator8” or provide a son of the direct cerebral vasodilating potencies of halothane and tonic, background effect on the cerebral vasculature in isoflurane in the New Zealand white rabbit. ANESTHESIOLOGY 1986; 38 65:462–7 the presence of anesthetic agents. This means that, 3. Moore LE, Kirsch JR, Helfaer MA, Tobin JR, McPherson RW, although volatile anesthetics do not directly stimulate Traystman RJ: Nitric oxide and prostanoids contribute to isoflurane- NOS or cause an increased production or release of induced cerebral hyperemia in pigs. ANESTHESIOLOGY 1994; 80:1328–37 NO,28,29 the presence of a certain critical level of NO or 4. Muldoon SM, Hart JL, Bowen KA, Freas W: Attenuation of endo- cGMP may be necessary for maximal dilation. The find- thelium-mediated vasodilation by halothane. ANESTHESIOLOGY 1988; 68: 31–7 ing that halothane continued to cause some vasodilation 5. Flynn NM, Buljubasic N, Bosnjak ZJ, Kampine JP: Isoflurane pro- despite NOS inhibition supports the hypothesis that NO duces endothelium-independent relaxation in canine middle cerebral represents one of several mediators or modulators that arteries. ANESTHESIOLOGY 1992; 76:461–7

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6. Koenig HM, Pelligrino DA, Albrecht RF: Halothane vasodilation 25. Rosenblum WI, Murata S: Antisense evidence for two function- and nitric oxide in rat pial vessels. J Neurosurg Anesthesiol 1993; ally active forms of nitric oxide synthase in brain microvascular endo- 5:264–71 thelium. Biochem Biophys Res Commun 1996; 224:535–43 7. Koenig HM, Pelligrino DA, Wang Q, Albrecht RF: Role of nitric 26. Eskinder H, Hillard CJ, Flynn N, Bosnjak ZJ, Kampine JP: Role of oxide and endothelium in rat pial vessel dilation response to isoflurane. guanylate cyclase-cGMP systems in halothane-induced vasodilation in Anesth Analg 1994; 79:886–91 canine cerebral arteries. ANESTHESIOLOGY 1992; 77:482–7 8. Smith JJ, Hudetz AG, Bosnjak ZJ, Kampine JP: The role of nitric 27. Jing M, Hart JL, Masaki E, Van Dyke RA, Bina S, Muldoon SM: oxide in cerebrocortical laser Doppler flow response to halothane in Vascular effects of halothane and isoflurane: cGMP dependent and the rat. J Neurosurg Anesthesiol 1995; 7:187–95 independent actions. Sci 1995; 56:19–29 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/92/1/125/399507/0000542-200001000-00023.pdf by guest on 03 October 2021 9. Okamoto H, Meng W, Ma J, Ayata C, Roman RJ, Bosnjak ZJ, 28. Tobin JR, Martin LD, Breslow MJ, Traystman RJ: Selective anes- Kampine JP, Huang PL, Moskowitz MA, Hudetz AG: Isoflurane-induced thetic inhibition of brain nitric oxide synthase. ANESTHESIOLOGY 1994; cerebral hyperemia in neuronal nitric oxide synthase gene deficient 81:1264–9 mice. ANESTHESIOLOGY 1997; 86:875–84 29. Zuo Z, Johns RA: Halothane, enflurane, and isoflurane do not 10. Harkin CP, Hudetz AG, Schmeling WT, Kampine JP, Farber NE: affect the basal or agonist-stimulated activity of partially isolated solu- Halothane-induced dilatation of intraparenchymal arterioles in rat brain ble and particulate guanylyl cyclases of rat brain. ANESTHESIOLOGY 1995; slices: A comparison to . ANESTHESIOLOGY 1997; 83:395–404 86:885–94 30. Johns RA, Tichotsky A, Muro M, Spaeth JP, Le Cras TD, Ren- 11. Farber NE, Harkin CP, Niedfeldt J, Hudetz AG, Kampine JP, Schmeling WT: Region-specific and agent-specific dilation of intrace- gasamy A: Halothane and isoflurane inhibit endothelium-derived relax- ing factor-dependent cyclic guanosine monophosphate accumulation rebral microvessels by volatile anesthetics in rat brain slices. ANESTHE- SIOLOGY 1997; 87:1191–8 in endothelial cell-vascular smooth muscle co-cultures independent of 12. Fergus A, Lee KS: Regulation of cerebral microvessels by gluta- an effect on guanylyl cyclase activation. ANESTHESIOLOGY 1995; 83: matergic mechanisms. Brain Res 1997; 754:35–45 823–34 13. Fergus A, Lee KS: GABAergic regulation of cerebral microvascu- 31. Rengasamy A, Pajewski TN, Johns RA: lar tone in the rat. J Cer Blood Flow Metab 1997; 17:992–1003 effects on rat cerebellar nitric oxide and cyclic guanosine monophos- 14. Buxton IL, Cheek DJ, Eckman D, Westfall DP, Sanders KM, Keef phate production. ANESTHESIOLOGY 1997; 86:689–98 KD: NG-nitro L-arginine methyl ester and other alkyl esters of arginine 32. Faraci FM: Role of endothelium-derived relaxing factor in cere- are muscarinic receptor antagonists. Circ Res 1993; 72:387–95 bral circulation: Large arteries vs. microcirculation. Am J Physiol 1991; 15. Franks NP, Lieb WR: Selective actions of volatile general anaes- 261:H1038–42 thetics at molecular and cellular levels. Br J Anaesth 1993; 71:65–76 33. Wang Q, Pelligrino DA, Baughman VL, Koenig HM, Albrecht RF: 16. White PF, Johnston RR, Eger EI: Determination of anesthetic The role of neuronal nitric oxide synthase in regulation of cerebral requirement in rats. ANESTHESIOLOGY 1974; 40:52–7 blood flow in normocapnia and hypercapnia in rats. J Cereb Blood 17. Faraci FM, Heistad DD: Regulation of the cerebral circulation: Flow Metab 1995; 15:774–8 Role of endothelium and channels. Physiol Rev 1998; 78: 34. Staunton M, Dulitz MG, Fang C, Schmeling WT, Kampine JP, 53–97 Farber NE: The effects of graded hypoxia on intraparenchymal arte- 18. Moore PK, Handy RL: Selective inhibitors of neuronal nitric rioles in rat brain slices. Neuroreport 1998; 9:1419–23 oxide synthase—Is no NOS really good NOS for the nervous system? 35. Harkin CP, Schmeling WT, Kampine JP, Farber NE: The effects Trends Pharmacol Sci 1997; 18:204–11 of hyper- and hypocarbia on intraparenchymal arterioles in rat brain 19. Dwyer MA, Bredt DS, Snyder SH: Nitric oxide synthase: Irrevers- slices. Neuroreport 1997; 8:1841–4 ible inhibition by L-NG- in brain in vitro and in vivo. 36. Rubanyi GM, Freay AD, Kauser K, Johns A, Harder DR: Mech- Biochem Biophys Res Commun 1991; 176:1136–41 anoreception by the endothelium: Mediators and mechanisms of pres- 20. Moore PK, Wallace P, Gaffen Z, Hart SL, Babbedge RC: Charac- sure- and flow-induced vascular responses. Blood Vessels 1990; 27: terization of the novel nitric oxide synthase inhibitor 7-nitro 246–57 and related : Antinociceptive and cardiovascular effects. Br J 37. Staunton M, Drexler C, Dulitz MG, Ekbom DC, Schmeling WT, Pharmacol 1993; 110:219–24 Farber NE: Effects of hypoxia–reoxygenation on microvascular endo- 21. Silva MT, Rose S, Hindmarsh JG, Aislaitner G, Gorrod JW, Moore PK, Jenner P, Marsden CD: Increased striatal efflux in vivo thelial function in the rat hippocampal slice. ANESTHESIOLOGY 1999; following inhibition of cerebral nitric oxide synthase by the novel 91:1462–69 monosodium salt of 7-nitro indazole. Br J Pharmacol 1995; 114:257–8 38. Todd MM, Wu B, Warner DS, Maktabi M: The dose-related 22. Bredt DS, Hwang PM, Snyder SH: Localization of nitric oxide effects of nitric oxide synthase inhibition on cerebral blood flow synthase indicating a neural role for nitric oxide. Nature 1990; 347: during isoflurane and anesthesia. ANESTHESIOLOGY 1994; 768–70 80:1128–36 23. Murphy S, Minor RL Jr, Welk G, Harrison DG: Evidence for an 39. Vanhoutte PM, Miller VM: Heterogeneity of endothelium-depen- -derived vasorelaxing factor with properties similar to nitric dent responses in mammalian blood vessels. J Cardiovasc Pharmacol oxide. J Neurochem 1990; 55:349–51 1985; 7:S12–23 24. Iadecola C, Beitz AJ, Renno W, Xu X, Mayer B, Zhang F: Nitric 40. Faraci FM, Breese KR: Nitric oxide mediates vasodilatation in oxide synthase-containing neural processes on large cerebral arteries response to activation of N-methyl-D-aspartate receptors in brain. Circ and cerebral microvessels. Brain Res 1993; 606:148–55 Res 1993; 72:476–80

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