Anesth Analg 2005;100:1075-1080
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000147709.52571.DC
PAIN MEDICINE
Intrathecal Neostigmine Prevents Intrathecal Clonidine from Attenuating Hypercapnic Cerebral Vasodilation in Rabbits
Motoyasu Takenaka, MD*,
Hiroki Iida, MD*,
Mami Iida, MD
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Kazuyuki Sumi, MD*,
Masahiko Kumazawa, MD*,
Shigeaki Tanahashi, MD*, and
Shuji Dohi, MD*
Departments of *Anesthesiology and Pain Medicine and Cardiology, Gifu University Graduate School of Medicine; and Department of Nutrition and Food Science, Gifu Women’s University, Gifu City, Japan
Address correspondence and reprint requests to Hiroki Iida, MD, Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan. Address e-mail to iida{at}cc.gifu-u.ac.jp.
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Abstract
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We previously demonstrated that lumbar intrathecal 
2 agonists attenuate hypercapnia-induced cerebral vasodilation. The combination of intrathecal clonidine and neostigmine is being investigated as pain therapy. The effects of their combination on cerebrovascular reactivity are unknown. We allocated rabbits anesthetized with pentobarbital to two groups: (a) clonidine (normal saline followed 30 min later by clonidine 2 µg/kg, both into the lumbar intrathecal space; n = 6), and (b) neostigmine-pretreatment (neostigmine 2 µg/kg followed 30 min later by clonidine 2 µg/kg, both into the lumbar intrathecal space; n = 6). We then evaluated the hypercapnia-induced changes in pial arteriolar diameter in these two groups using the closed cranial window preparation. The pial arteriolar dilator response to hypercapnia was significantly attenuated in the clonidine group (14% ± 4%, 4% ± 4%, 6% ± 6%, and 5% ± 7% for before and 30, 60, and 90 min, respectively). Neither normal saline nor neostigmine alone induced any change in the cerebral reactivity to hypercapnia. Pretreatment with neostigmine completely prevented the clonidine-induced attenuation of the hypercapnic cerebral vasodilation attenuated by intrathecal clonidine (16% ± 7%, 15% ± 6%, 12% ± 6%, and 16% ± 8%, respectively).
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Introduction
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Spinal administration of clonidine, an 2 adrenergic agonist, provides excellent analgesia without the risk of significant respiratory depression (1,2). Some experiments have suggested that activation of cholinergic spinal neurons and stimulation of muscarinic receptors by acetylcholine are involved in this analgesia (3–5). A cholinesterase inhibitor, neostigmine, enhances intrathecal clonidine-induced analgesia and counteracts clonidine-induced hypotension (6). Alpha2 agonists exert their effects by stimulating both presynaptic and postsynaptic 2 adrenergic receptors centrally and peripherally. We previously reported that intrathecal 2 agonists, such as clonidine, attenuate hypercapnic cerebral vasodilation in rabbits (7). This is likely to be mediated, at least in part, via a stimulation of 2 adrenergic receptors within the central nervous system (CNS) (7). The combination of spinal 2 agonists and neostigmine is under clinical investigation for potential use in the perioperative period. There is no report concerning the reactivity of cerebral vessels to hypercapnia after intrathecal clonidine with neostigmine pretreatment. We thought it important to examine the influences of these drugs on the changes in cerebral vessels induced by hypercapnia. The aim of the present study was to investigate the effects of intrathecal neostigmine on the clonidine-induced attenuation of hypercapnic cerebral vasodilation in anesthetized rabbits.
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Methods
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The procedures used in the present study conformed to the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiologic Society. The experimental protocols were approved by the Institutional Committee for Animal Care at Gifu University Graduate School of Medicine, Gifu City, Japan. The experiments were performed on 18 anesthetized rabbits weighing 2.3–2.6 kg. Anesthesia was induced with pentobarbital sodium (20 mg/kg, IV) and maintained with a continuous infusion of the same drug (4 mg · kg–1 · h–1). Each rabbit was mechanically ventilated through a tracheotomy tube with oxygen-enriched room air (arterial oxygen content, 14–15 vol%), with the tidal volume and respiratory rate adjusted to maintain a Paco2 between 35 and 40 mm Hg. A catheter was placed in the femoral vein for the administration of fluids and drugs. Another catheter was placed in the femoral artery for continuous monitoring of mean arterial blood pressure (MAP) and heart rate (HR) and to provide blood samples for the determination of arterial blood gas tensions and pH values. Rectal temperature was maintained between 38.0°C and 39.0°C by a heating blanket.
A closed cranial window was used to observe the cerebral pial microcirculation. Each rabbit was placed in the sphinx posture, the scalp was retracted, and a 5-mm-diameter hole was made in the parietal bone. The dura and arachnoid membranes were opened carefully, and a ring with a cover glass was placed over the hole and secured with dental acrylic. The space under the window was filled with artificial cerebrospinal fluid, the composition of which was Na+ 151 mEq/L, K+ 4 mEq/L, Ca2+ 3 mEq/L, Mg2+ 1.3 mEq/L, Cl– 134 mEq/L, HCO3– 25 mEq/L, urea 40 mg/dL, and glucose 67 mg/dL (pH value adjusted to 7.48). The solution was freshly prepared each day and bubbled with 5% CO2 in air at 38.5°C for 15 min just before use. Two polyethylene catheters were inserted through the ring; one was attached to a reservoir bottle containing artificial cerebrospinal fluid to maintain the desired level of intra-window pressure (5 mm Hg), and the other was used for continuous monitoring of the pressure within the window. The volume below the window was between 0.5 and 1 mL. The temperature within the window, which was monitored using a thermistor (Model 6510; Mallinckrodt Medical, St Louis, MO), placed before the use of dental acrylic, was maintained at 38.0°C–39.0°C. The lumbar paraspinal muscles were exposed by a longitudinal midline skin incision from the lower lumbar level to the upper sacral level. After the spinous process had been removed using a rongeur, a laminectomy was performed using an electric drill at the sixth lumbar vertebra. A polyethylene catheter was inserted 1 cm rostral into the intrathecal space.
The diameters of three pial arterioles were measured in each cranial window using a videomicrometer (Olympus Flovel Videomicrometer, Model VM-20; Flovel, Tokyo, Japan) on a television monitor. This received pictures from a microscope (Model SZH-10; Olympus, Tokyo, Japan). The data from the pial views were stored on videotape for later playback and analysis. The percentage changes recorded for individual arteriolar segments were averaged in each rabbit. This average value was used in the statistical analysis. Pial arterioles under normocapnic conditions ranged from 60 to 110 µm in diameter.
All experiments were conducted after at least 30 min of recovery from the surgical preparation. In the first set of experiments, intrathecal normal saline (0.1 mL/kg; control) was given as pretreatment, and 30 min later, clonidine was injected intrathecally (2 µg/kg in 0.1 mL/kg of normal saline; n = 6). Hypercapnic challenges (addition of carbon dioxide gas to the inspiratory gases) were delivered at baseline (before pretreatment) and then (after pretreatment) before and at 30, 60, and 90 min after the intrathecal administration of clonidine. Each time, pial arteriolar diameters were measured after 5 min at a stable level (arterial carbon dioxide partial pressure, approximately 60 mm Hg). The hypercapnia-induced changes in pial arteriolar diameter were calculated, and physiologic variables (MAP, HR, rectal temperature, arterial blood gas tensions, and pH value) were evaluated (STAT profile-5; NOVA Biomedicals, Waltham, MA) whenever we measured pial arteriolar diameters. In the second set of experiments, intrathecal neostigmine (2 µg/kg in 0.1 mL/kg of normal saline) was given as pretreatment, and 30 min later, clonidine was injected intrathecally (2 µg/kg in 0.1 mL/kg of normal saline; n = 6). Hypercapnia-induced changes in pial arteriolar diameters and physiologic values were measured exactly as in the first set of experiments. In an additional set of experiments, we investigated the time-control hypercapnic challenges after the sole administration of intrathecal neostigmine (2 µg/kg in 0.1 mL/kg of normal saline; n = 6).
Within groups, all physiologic variables were examined using a one-way analysis of variance for repeated measurements followed by Scheffe F-test for post hoc comparisons. Differences between groups were examined using a two-way, with an unpaired t-test being used to assess any differences found. A paired t-test was used to assess differences between variables before and after hypercapnia. Significance was considered to be P < 0.05. All results are expressed as mean ± sd.
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Results
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In the presence of clonidine but without neostigmine pretreatment, the pial arteriolar dilator response to hypercapnia was significantly attenuated at 30, 60, and 90 min after the intrathecal administration of clonidine (14% ± 4%, 4% ± 4%, 6% ± 6%, and 5% ± 7% for before and at 30, 60, and 90 min, respectively) versus before the clonidine administration (P < 0.05; Fig. 1). Although intrathecal neostigmine did not induce any change in the reactivity of pial arterioles to hypercapnia during the experimental period (time-control study), neostigmine pretreatment completely prevented the above effect of intrathecal clonidine (16% ± 7%, 15% ± 6%, 12% ± 6%, and 16% ± 8%, respectively) (P < 0.05; Fig. 1). Intrathecal injection of clonidine in the lumbar region did not itself affect cerebral pial arteriolar diameters (Table 1). MAP did not change after the intrathecal administration of either pretreatment solution (saline or neostigmine) or clonidine in any group, nor did it change in response to hypercapnia (Table 2). HR did not change after the intrathecal administration of either pretreatment solution (saline or neostigmine) or clonidine in any group, but it decreased significantly in response to hypercapnia (P < 0.05; Table 2). Arterial pH decreased significantly in response to hypercapnia at all time points in both groups (without changes in arterial oxygen tension) (Table 2).
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Figure 1. The effects of intrathecal clonidine with or without neostigmine pretreatment (n = 6 each) and neostigmine pretreatment only (n = 6) on reactivity of cerebral pial arterioles to hypercapnia. Data are expressed as percentage change in diameter induced by hypercapnia at baseline (before pretreatment), before (before the administration of clonidine but after pretreatment), and at 30, 60, and 90 min after the intrathecal administration of clonidine. Clonidine attenuated the arteriolar dilation induced by hypercapnia. Pretreatment with neostigmine completely prevented the clonidine-induced decrease in the reactivity to hypercapnia. Values are mean ± sd. *P < 0.05 compared with before clonidine administration; #P < 0.05 between the indicated values.
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Table 1. Values Obtained for Pial Arteriolar Diameter (µm) during Normocapnia and Hypercapnia With or Without Intrathecal Administration of Test Drugs
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Table 2. Levels of Physiologic Variables During Normocapnia and Hypercapnia With or Without Intrathecal Administration of Test Drugs
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Discussion
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The major findings of the present study were that the intrathecal administration of clonidine attenuates hypercapnia-induced dilation of cerebral pial arterioles and that this effect of clonidine is completely prevented by pretreatment with neostigmine, a cholinesterase inhibitor. Intrathecal neostigmine did not induce any change in the cerebral vasodilation induced by hypercapnia.
Hypercapnia induces cerebrovascular dilation. Authors who have studied the effects of systemically administered 2-adrenergic agonists on hypercapnic cerebrovascular dilation have variously concluded that they attenuate (8), do not alter (9), or enhance (10) it. We previously demonstrated (7), and confirmed here, that spinally administered clonidine did not change cerebral vascular diameter during normocapnia, but rather it attenuated the cerebral vascular dilation induced by hypercapnia. This effect of clonidine is likely to be mediated via 2 adrenergic stimulation within the CNS (7,11).
It has been reported that hypercapnic cerebrovascular dilation is mediated by the decrease in perivascular pH that occurs secondary to the increase in arterial CO2 (12,13). The increase in H+ ion concentration reportedly enhances nitric oxide synthase (NOS) activity (14). Nitric oxide (NO) participates in the mechanism by which extracellular acidosis induces smooth muscle relaxation, and therefore vasodilation, by its activation of guanylyl cyclase in vascular smooth muscle and the subsequent increase in cyclic guanosine monophosphate (15). NO is a mediator of, and a necessary factor for, hypercapnic vasodilation. However, it is not the only factor involved (16) because NOS inhibitors preserve 10%–70% of hypercapnia-mediated cerebral vasodilator and attenuate the vasodilation only within a certain range of Paco2 (e.g., 50
60 mm Hg, but not Paco2 > 100 mm Hg) (17,18). It is known that 2 adrenergic stimulation causes contraction of vascular smooth muscle and a simultaneous release of NO by vascular endothelium, the latter possibly serving to attenuate vasoconstriction (19). In hypercapnic cerebral vasodilation of neonatal animals, NO does not play a role (20), whereas prostanoids play an important role (21). In other words, an 2-adrenergic agonist has two opposing effects: a direct vasoconstriction and vasodilation (in which NO may play an important role). This being so, the NO released by 2-adrenergic agonists is unlikely to participate in the mechanism by which spinally administered 2-adrenergic agonists attenuate the cerebrovascular dilation induced by hypercapnia.
Furthermore, activation of the sympathetic nervous system attenuates cerebrovascular dilation induced by hypercapnia (22). Intrathecally administered 2-adrenergic agonists cause cardiovascular depression by inhibiting sympathetic preganglionic activity. Intrathecally administered neostigmine counteracts clonidine-induced hypotension by amplifying the action by which acetylcholine activates sympathetic preganglionic neurons. Clearly this mechanism cannot be invoked to explain the results obtained in the present study (in which intrathecal neostigmine had no effect alone but prevented the intrathecal clonidine-induced attenuation of the dilation response to hypercapnia).
Cholinergic mechanisms play a role in the cerebrovascular reactivity to hypercapnia. The cholinesterase inhibitor physostigmine, which can cross the blood-brain barrier, enhances cerebral vasodilation associated with cortical activation and hypercapnia (23,24). Intrathecal neostigmine increases sympathetic outflow (2) and releases acetylcholine and NO and thus increases spinal cord blood flow. This intrathecal neostigmine-induced increase in spinal cord blood flow occurs via an amplification of the action of acetylcholine on sympathetic preganglionic neurons and also via an increase in the acetylcholine concentration in the cerebrospinal fluid (25), which induces NOS activity. Although the precise mechanism is not yet clear, an enhancement of NOS activity by intrathecal neostigmine is one of the candidates to explain the present result (the preventive effect of neostigmine on the intrathecal clonidine-induced attenuation of the pial vasodilator response to hypercapnia).
In conclusion, the present study shows that intrathecal clonidine attenuates the dilation of cerebral pial arterioles that occurs in response to hypercapnia, even though it does not itself have a vasoconstrictor effect on cerebral vessels. Because pretreatment with neostigmine completely prevented the decreased vasoreactivity induced by clonidine during hypercapnia, it is possible that enhancement of the action of acetylcholine or NOS activity might participate in the mechanism underlying this preventive action of neostigmine.
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Footnotes
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Presented, in part, at the annual meeting of the American Society of Anesthesiologists, Orlando, FL, October 12–16, 2002.
Supported, in part, by Grant-in Aid for Scientific Research No. 13671570 (Ministry of Education, Science, and Culture, Japan).
Accepted for publication September 23, 2004.
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Abstract
Introduction
