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Department of Anesthesia and Perioperative Care, University of California, San Francisco, California
Address correspondence to James M. Sonner, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464.
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Abstract |
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-amino-n-butyric acid (GABAA) and glycine receptors to GABA and glycine. In this study, we antagonized GABAA and glycine responses by intrathecal administration of picrotoxin (a noncompetitive GABAA antagonist), strychnine (a competitive glycine antagonist), or combinations of these drugs. We measured the capacity of antagonist infusion to increase isoflurane MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). We found that these potent GABAA and glycine receptor antagonists had a ceiling effect, either alone or in combination increasing the MAC of isoflurane by at most 47%.
Implications: -amino-n-butyric acid and glycine receptors may in part be responsible for the immobilizing action of isoflurane. They are not, however, the only receptors that contribute to isoflurane-induced immobility (i.e., that determine the MAC of isoflurane).
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Introduction |
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-amino-n-butyric acid (GABAA) and glycine receptors may mediate the effects of inhaled anesthetics. Inhaled anesthetics, in doses of approximately 1 MAC (the minimum alveolar concentration of an inhaled anesthetic preventing response to a noxious stimulus in 50% of experimental subjects), enhance GABAA and glycine receptor responses to their respective agonists (1–3). IV anesthetics with substantial GABAergic actions, such as barbiturates, propofol, and anesthetic steroids, also potentiate GABAA responses (4). Nonimmobilizers (halogenated compounds related to anesthetics but that lack the immobilizing effect of anesthetics, and thus serve as a negative control) do not potentiate GABAA responses (5). Studies in rats and goats demonstrate that inhaled anesthetics produce immobility by an action on the spinal cord (6–9). In rats, cervical transection of the spinal cord does not change MAC (9). In goats, when the brain and spinal cord are separately perfused (one naturally and the other with extracorporeal circulation) the spinal cord is more sensitive to anesthetic than the brain. Nearly three times the cerebral concentration of isoflurane is required to prevent pain-evoked movement when the brain alone is perfused with anesthetic compared with perfusion of both the brain and spinal cord (6). Together, these studies demonstrate that MAC is spinally mediated.
It is not, however, known whether or to what extent GABAA and strychnine-sensitive glycine receptors contribute to the production of immobility by inhaled anesthetics. The spinal cord contains both GABAA and glycine receptors (10,11). Antagonism of the anesthetic effect of halothane can be produced by the intrathecal administration of GABAA and glycine receptor antagonists; however, the size of that effect was not measured (12,13). In the present investigation, we determined how much antagonism of GABAA and glycine receptors altered anesthetic potency (as measured by MAC) by blockade of spinal GABAA and/or glycine receptors with picrotoxin and strychnine. We did not use bicuculline in these studies because the free base is poorly soluble in aqueous solution, although the N-methyl salts of bicuculline are not specific for GABAA receptors (14). We conjectured that if GABAA and glycine receptors were solely responsible for the immobilizing effect of isoflurane, MAC would increase without limit with increasing doses of GABAA and strychnine-sensitive glycine receptor antagonists.
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Methods |
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Intrathecal catheters were placed in rats given intrathecal study drug. Rats were anesthetized with ketamine and xylazine, and a 32-gauge polyurethane catheter (Micor Inc., Allison Park, PA) was placed through the atlantooccipital membrane following the method of Yaksh and Rudy (15). The catheter was threaded caudally 6 to 8 cm toward the lumbar sac, the length depending on the size of the rat. At the neck, sutures were used to fix the catheter to adjacent muscle and skin. Rats were allowed to recover from anesthesia and surgery for at least 4 days before study.
Internal jugular catheters were placed in rats given IV study drugs. Rats were anesthetized with isoflurane, and an incision was made in the neck. A PE 10 catheter was placed approximately 2 cm into the internal jugular vein, tunnelled to the nape of the neck, and sutured closed. Rats were allowed to recover from anesthesia and surgery at least 2 days before study.
MAC was determined in four rats at a time. Each rat was placed in a gas-tight clear plastic cylinder. A rectal temperature probe was inserted, and the temperature probe and the tail of the rat were separately drawn through holes in the rubber stopper used to seal one end of the cylinder. Ports through the rubber stoppers in each end of the cylinder allowed delivery of gases. The gases entered at the head end of the cylinder and exited at the tail. Gas delivery was via a circuit that was connected to fresh gas containing oxygen and anesthetic, a carbon dioxide absorber, a fan that circulated gases, and ports for sampling and for scavenging waste anesthetic gases.
To determine MAC, isoflurane was introduced into the system via a conventional vaporizer, starting with a partial pressure of approximately 1.0% of an atmosphere. Anesthetic partial pressures were monitored by using an infrared analyzer (Datascope, Helsinki, Finland). Animals were exposed to isoflurane for 30 min. A tail clamp was then applied for 1 min or until the animal moved. The isoflurane partial pressure was measured by using gas chromatography. If the animal moved, the isoflurane partial pressure was increased by 0.1% to 0.2% atmospheres. After equilibration for 30 min, the tail clamp was applied again and isoflurane partial pressure measured. This procedure was repeated until a partial pressure at which the animals did not move was achieved.
Each study was composed of two parts. In the first, we infused artificial cerebrospinal fluid (aCSF) alone. The aCSF was made daily from stock solutions. Stock solution #1 was a monovalent stock solution made by adding NaCl 3.6963 g, NaHCO3 1.1551 g, KCl 0.0895 g, KH2PO4 0.0340 g, and Na2SO4 0.0355 g in deionized, distilled water to a volume of 500 mL. Stock solution #2 was a divalent solution made from CaCl2 · 2H2O 0.8086g and MgCl2 · 6H2O 0.8437 g in deionized, distilled water to a volume of 10 mL. To make aCSF, 25 mL stock solution #1 was added to 0.0266 g glucose, adjusted to pH 7.4 with bubbles of CO2 for approximately 10 min, and added to 50 µL divalent stock solution, giving a final composition of 154.7 mM Na+, 0.82 mM Mg2+, 2.9 mM K+, 132.49 mM Cl-, 1.1 mM Ca2+, 5.9 mM glucose, at a pH of 7.4. In the second study, we infused aCSF to which we added the GABAA blocker picrotoxin (Sigma Chemical, St. Louis, MO), the glycine blocker strychnine hydrochloride (Sigma Chemical), or a combination of picrotoxin and strychnine. All intrathecal infusions were at a rate of 1 µL/min to limit spread of the agent in the subarachnoid space. IV infusions at rates of up to 32 µL/min were used. Because larger concentrations of strychnine (>12 mg/mL) would not completely dissolve, we increased the partial pressure of carbon dioxide in the aCSF, producing pH values as low as 6.0, to promote solvation of strychnine.
The interval between the first and second parts of the study differed among the studies. For studies of intrathecal picrotoxin we allowed 2 days. For studies of intrathecal strychnine, or of IV picrotoxin or strychnine, we allowed only an hour, during which time the isoflurane concentration was decreased to the point that each rat again responded to the tail clamp.
Intrathecal picrotoxin was studied in four animals at each of eight doses: 0.02, 0.05, 0.1, 0.2, 0.3, 0.6, 1.2, and 2.4 mg/mL. Intrathecal strychnine was studied in four animals at each of seven doses: 0.5, 1, 2, 4, 8, 12, and 16 mg/mL. Because doses of strychnine greater than 16 mg/mL (the limit of solubility for this agent) could not be studied, three additional studies were performed to confirm the ceiling effects observed. First, a fixed dose 4.0 mg/mL of strychnine (producing a 24% increase in isoflurane MAC alone) was infused intrathecally, and increasing doses of picrotoxin were coinfused with it. Second, a fixed dose of picrotoxin (producing a 17% increase in isoflurane MAC by itself) 0.05 mg/mL was infused intrathecally, and increasing doses of strychnine were coinfused with it. Finally, we coinfused doses of strychnine (12 mg/mL) and picrotoxin (0.6 mg/mL) that individually produced maximum responses (respectively 40% and 43%).
To establish whether our results were confounded by toxic effects from either strychnine or picrotoxin, MAC to isoflurane was measured before and 2 days after infusion of either picrotoxin (1.2 mg/mL) or strychnine (at 12 mg/mL).
To confirm that drugs infused at 1 µL/min via intrathecal catheter remained confined to the spinal subarachnoid space, 0.1% methylene blue in aCSF was infused at this rate in three rats, and MAC determined in each rat. The extent of spread of the methylene blue was determined visually on necropsy of the animals. Methylene blue 0.1% was then combined with 1.2 mg/mL picrotoxin in aCSF, MAC to isoflurane determined while infusing this solution at 1 µL/min intrathecally in four rats, and necropsies performed to determine the spread of the methylene blue in the CSF. MAC in animals receiving 0.1% methylene blue with or without picrotoxin was measured to establish that methylene blue had no effect on MAC and could be coinfused as a marker of the extent of spread of intrathecal drug.
The effect of IV administration of picrotoxin or strychnine was determined after MAC to isoflurane was first measured infusing vehicle alone. Picrotoxin was administered IV at rates of 1.2 (2 rats), 2.4 (6 rats), 4.8 (4 rats), 9.6 (4 rats), 19.2 (4 rats), or 38.4 (4 rats) µg/min, and strychnine at 16 (4 rats), 32 (8 rats), 64 (4 rats), or 128 (4 rats) µg/min. These experiments were performed to eliminate the possibility that picrotoxin or strychnine exerted their effects, when administered intrathecally, simply by absorption into the systemic circulation.
MAC was defined as the average of the partial pressures that just prevented and permitted movement in response to clamping the tail. The change in MAC was calculated as the ratio of the MAC for the second part of each study to the first part. We calculated the mean and SD for the change at each dose of each antagonist. To determine whether a ceiling effect in MAC had been reached, for each antagonist or combination of antagonists a one-way analysis of variance was performed. A Student-Newman-Keuls test was then used to determine differences in MAC for different doses of antagonist. A ceiling effect was deemed to exist if three successively larger doses of the antagonist producing the greatest change in MAC were not determined to be significantly different from each other on the Student-Newman-Keuls test. P < 0.05 was taken as statistically significant.
We used a Gow-Mac 750TM gas chromatograph (Gow-Mac Instrument Corp., Bridgewater, NJ) equipped with a flame ionization detector to measure isoflurane partial pressures. The 4.6 meter-long, 0.22 cm (inner diameter) column was packed with SF-96. The column temperature was 138°C. The detector was maintained at temperatures approximately 50°C warmer than the column. The carrier gas flow was nitrogen at a flow of 15 mL/min. The detector received 38 mL/min hydrogen and 2400 mL/min air. Primary standards were prepared for isoflurane and the linearity of the response of the chromatograph determined. We commonly used secondary (cylinder) standards referenced to primary standards.
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Results |
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Picrotoxin, with or without the addition of 4 mg/mL strychnine, produced a ceiling effect, with a maximum increase in isoflurane MAC of 43% (Fig. 1). For picrotoxin alone the one-way analysis of variance was highly significant for all seven doses considered together (P < 0.001), with no significant difference in the change in isoflurane MAC for the four largest doses. For the addition of 4 mg/mL strychnine to the picrotoxin, there was no significant difference among picrotoxin doses tested (P = 0.11).
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Picrotoxin 1.2 mg/mL did not have a toxic effect as assessed by a change in MAC after infusion of the drug (isoflurane MAC before infusion 1.28 ± 0.08, MAC after infusion 1.34 ± 0.11, with four animals in each group; P = 0.41). Strychnine at 12 mg/mL also did not injure the spinal cord as assessed by measurement of MAC (isoflurane MAC before infusion 1.34 ± 0.09 for three animals, MAC after infusion 1.41 ± 0.00, P = 0.25)
Intrathecal infusion of 0.1% methylene blue had no effect on isoflurane MAC (change in MAC ± SD, n = 3: 0.26% ± 0.65; P = 0.53 compared with no change in MAC). Methylene blue was confined to the thoracolumbar subarachnoid space in all three animals.
The addition of 0.1% methylene blue to 1.2 mg/mL picrotoxin had no effect on the MAC isoflurane compared with intrathecal infusion of 1.2 mg/mL picrotoxin alone (P = 0.65). Methylene blue stained only the thoracolumbar spinal cord in all four animals.
Isoflurane MAC increased with infusion of picrotoxin IV, but only at doses exceeding those producing a maximal effect when administered spinally. The effect of spinal application of picrotoxin is therefore not explained by absorption of the drug into the systemic circulation, with delivery to supraspinal sites. No ceiling effect was obtained with IV administration of picrotoxin; however, larger doses could not be given because the animals did not survive this treatment.
IV strychnine also increased isoflurane MAC, but at doses larger than those used intrathecally, eliminating systemic absorption of intrathecal strychnine as the cause of its effect on MAC. There was no ceiling effect seen with IV strychnine. The animals died when given 128 µg/min strychnine.
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Discussion |
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We introduced antagonists into the lumbar subarachnoid space to assess the effect of these drugs on a spinally mediated effect of anesthetics (immobility). This approach has been widely used in other fields to assess the spinal action of other drugs. It has been used much less frequently in the study of anesthetic mechanisms. Mason et al. (13) administered strychnine, bicuculline, and picrotoxin intrathecally in bolus doses to rats to antagonize the immobilizing effect of halothane. Antagonism was evidenced by a prolongation in the latency to nocifensive movement in response to a tail clamp. Because of the careful selection of doses of antagonists, using only those doses that did not alter baseline responses to painful stimuli, the authors could conclude that anesthetics acted on GABAA and glycine receptors in the spinal to suppress nocifensive responses. In contrast to the current study, the size of the effect from antagonism of each receptor was not quantified.
There have been few other pharmacologic investigations in intact animals of other ion channels that may be targets of anesthetic action. N-methyl-D-aspartate (NMDA) and 
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are thought to be important to inhaled anesthetic action, and antagonists to these receptors have been given to rats, both intrathecally and IV, but with considerable differences in the size of the effect seen. Ishizaki et al. (16) found a 30% maximum reduction in isoflurane MAC in Wistar rats administered intrathecal bolus doses of the NMDA antagonists APV and MK801. In a subsequent study they reported that intrathecally administered CPP (a competitive antagonist at the NMDA receptor) or 7CKA (a selective antagonist at the glycine site of the NMDA receptor) produced at most a 15% decrease in isoflurane MAC (17). These results contrast with those of McFarlane et al. (18) who administered NMDA or AMPA antagonists IV to rats and found much larger effects, ranging from 58% to 85%. However, as noted by these investigators, the supraspinal or sedating actions of these drugs may have contributed to the MAC-sparing effect of the antagonists, completely aside from their antagonism of the immobilizing effect of halothane, which is largely spinal in origin.
It is possible that the potentiating effect of inhaled anesthetics on GABAA and glycine receptors is not a major determinant of anesthetic-induced immobility; currently, there are no data in animals that address this question. The present study places an upper limit on the effect of blockade of GABAA and glycine receptors on MAC, but not on the portion of the GABAA effect that results from the potentiating action of isoflurane on the receptor. By using recent information about the action of anesthetics on ion channels and the techniques we have described, it should be possible to measure the potentiating effect of isoflurane on GABAA receptors in animals with MAC as the anesthetic endpoint. For example, xenon has no effect on GABAA receptors (19). Thus, if picrotoxin were administered spinally to animals anesthetized with xenon, the difference in the ceiling effect on MAC between xenon and isoflurane should be because of the potentiating effect of isoflurane on GABAA receptors. If the potentiating effect of isoflurane on GABAA receptors is important to the immobilizing effect of isoflurane, then picrotoxin should produce a smaller increase on the MAC of xenon than isoflurane.
The current study raises an intriguing question regarding the mechanism of action of IV anesthetics thought to act largely via GABAA receptors, such as barbiturates and propofol. We found that picrotoxin, a potent antagonist of GABAA receptors, had a limited effect, increasing the MAC of isoflurane by approximately 40%. One might expect, accordingly, that IV anesthetics that act by potentiating the action of GABA at GABAA receptors should have a limited effect on the spinal cord and therefore a limited effect in reducing MAC. That is, they should not be able to immobilize an animal by a spinal action on GABAA receptors alone. If so, then either these anesthetics produce immobility by a supraspinal action (in contrast to inhaled anesthetics, which produce immobility predominantly by an action on the spinal cord), or they have a major, unappreciated molecular mechanism of action.
In summary, we report glycine and GABAA receptors both may partially contribute to isoflurane-induced immobility. Further studies using intrathecal neurotransmitter antagonists should help dissect the pharmacologic basis by which anesthetics produce immobility.
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Acknowledgments |
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The isoflurane used in these studies was generously donated by Baxter, Pharmaceutical Products Inc.
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Footnotes |
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