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ANESTHETIC PHARMACOLOGY

Thiopental Produces Immobility Primarily by Supraspinal Actions in Rats

Department of Anesthesia and Perioperative Care, University of California, San Francisco, California

Address correspondence and reprint requests to James M. Sonner, MD, Department of Anesthesia and Perioperative Care, S-455, University of California, San Francisco, CA 94143–0464. Address e-mail to sonnerj{at}anesthesia.ucsf.edu

	Abstract

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The spinal cord mediates most of the immobilizing action of inhaled anesthetics. In the present study we investigated whether spinal or supraspinal sites mediate the immobilizing action of thiopental in rats. Thiopental was administered IV, intrathecally (IT), intracerebroventricularly (ICV), or simultaneously IT and ICV. Only the IV infusion produced anesthesia, defined as immobility in response to application of a tail clamp (i.e., the equivalent of minimum alveolar concentration, MAC). Consequently, the MAC-sparing effect (for isoflurane) of thiopental was used to assess the immobilizing contribution of IT and ICV infusions of thiopental. Thiopental concentrations were determined in whole brain, spinal cord, and a slice of cerebral cortex distant from the infusion sites. These concentrations were correlated with the MAC-sparing effect of the thiopental infusions in a multiple regression model. To assess the rate at which thiopental penetrates the cord, rat spinal cords were equilibrated in a bath of thiopental ex vivo and the concentration of thiopental in the cord was measured as a function of equilibration time. This was repeated in vivo with IT infusions of thiopental spanning the time of the behavioral studies. We found that IT or ICV infusion of thiopental 25 µg/min decreased isoflurane MAC <25%. The associated thiopental concentrations in the spinal cord after IT infusion, and in the whole brain after ICV infusion of 25 µg/min thiopental, exceeded by 500% and 680%, respectively, the concentrations found in the spinal cord and in the whole brain after IV infusion of thiopental in a dose that produced anesthesia in the absence of isoflurane. The percentage decrease in the MAC of isoflurane correlated primarily with the concentration of thiopental found in cerebral tissue not in contact with the cerebral ventricles. The spinal cord infusion produced an approximately 20% decrease in MAC. Ex vivo IT thiopental readily diffused into the spinal cord, with a time constant of approximately 1 h. We conclude that, unlike inhaled anesthetics, the immobilizing action of thiopental is largely supraspinal. Centers in the brain other than those near the third and fourth ventricles produce the greatest effect.

IMPLICATIONS: Thiopental produces immobility in response to noxious stimuli predominantly by actions on supraspinal sites.

	Introduction

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The spinal cord mediates the immobility produced by volatile anesthetics in the face of noxious stimulation (i.e., mediates MAC, the minimum alveolar concentration required to eliminate movement in response to noxious stimulation in 50% of subjects.) In rats, cervical transection of the spinal cord does not change isoflurane MAC (1), demonstrating that supraspinal sites are not necessary to the immobilizing action of isoflurane. In goats, separate perfusion of the brain and spinal cord reveals that nearly three times the cerebral concentration of isoflurane is required to prevent pain-evoked movement when the brain alone is perfused with isoflurane compared with perfusion of both the brain and spinal cord (2). A still greater difference is seen for halothane (3). Thus, the supraspinal application of 1 MAC of isoflurane or halothane does not produce immobility in response to noxious stimulation.

Several studies have examined the actions of barbiturates on the spinal cord. Barbiturates enhance the response of spinal cord -aminobutyric acid A (GABA_A) receptors to GABA in vitro (4). Two case reports describe the accidental injection of thiopental into the epidural space of patients, finding that such injection leads to pain followed by sedation (5,6). Morrison et al. (7) injected ascending intrathecal (IT) doses of thiopental into patients. Smaller doses could induce analgesia, and motor blockade could be achieved with doses that induced sedation. Consistent with a capacity to produce analgesia, thiopental produces a direct but brief depression of dorsal horn neuron responses to noxious stimulation (8). Results of differential perfusion of thiopental to the spinal cord versus the brain suggest that both contribute to the immobility produced by thiopental (3). And, finally, thiopental minimally affects the F wave (9), a measure of motoneuron hyperexcitability that is depressed by inhaled anesthetics.

The present study assessed the importance of the spinal cord as a mediator of the immobility produced by thiopental. We examined the capacity of IT and/or intracerebroventricular (ICV) thiopental infusions to change isoflurane MAC and separately established the EC₅₀ of thiopental given as an IV infusion. The MAC-sparing effect of thiopental infusions was used as the measure of the capacity of IT and ICV to produce immobility because thiopental infused alone by these routes did not produce immobility in response to a noxious stimulus. In each of these studies we mea-sured the associated thiopental concentrations in the whole brain, a slice of cerebral cortex (one distant from ICV thiopental infusion), and the spinal cord. We correlated these concentrations with the MAC-sparing effect of thiopental in a multiple regression model to determine the relative importance of the brain and spinal cord to the immobilizing action of thiopental. We examined whether injury to the spinal cord or brain might explain any decrease in MAC produced by IT or ICV infusions. Finally, an ex vivo model of IT diffusion into the cord was used to demonstrate that equilibrium is reached in the spinal cord superfused with thiopental.

	Methods

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Animals and Cannula Placement
With approval of the Committee on Animal Research of the University of California, San Francisco, we studied male (Crl:CD(SD)BR) Sprague-Dawley rats weighing 270–357 g obtained from Charles River Laboratories (Hollister, CA). Cannulae were placed in the third cerebral ventricle, lumbar subarachnoid space, or internal jugular vein.

To place cannulae into the third ventricle, we anesthetized each rat with isoflurane, exposed the skull, and drilled a hole 1.8 mm posterior to the bregma in the midline. Through this hole we inserted a 24-gauge stainless steel guide cannula to a depth of 4.1 mm from the skull surface. The cannula was secured by wiring to two screws that were placed into the skull approximately 5 mm to either side of the cannula then cemented with dental acrylic.

To place catheters into the IT space, we anesthetized each rat with isoflurane, and placed a 32-gauge polyurethane catheter (Micor Inc., Allison Park, PA) through the atlantooccipital membrane according to methods described previously (10) and applied in our laboratory (11). The catheter was threaded caudally 6 to 8 cm towards the lumbar sac, the length depending on the size of the rat. Sutures fixed the catheter to adjacent muscle and skin at the neck.

IV catheters made of polyethylene 10 were placed under isoflurane anesthesia in the right internal jugular vein of the animals. The catheter was tunneled through the skin and brought out through the right ear. Rats were allowed to recover for at least 24 h from all cannulae insertions before subsequent study.

IT and/or ICV Studies
Determination of MAC.
MAC was determined concurrently in four rats. Each rat was placed in a 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 at the head end of the cylinder and exit of gases at the tail. A total flow rate of 4 L/min of oxygen containing isoflurane was delivered (average 1 L/min per cylinder), and the exiting gases were scavenged.

To determine MAC, isoflurane was introduced into the system via a conventional vaporizer, starting with a partial pressure of about 1.0% of an atmosphere. Anesthetic partial pressures were monitored using an infrared analyzer (Datascope, Helsinki, Finland). The isoflurane was applied for 30 min, after which time a tail clamp was applied for up to 1 min or until the animal moved. The isoflurane partial pressure was then measured by gas chromatography. If the animal moved, we increased the isoflurane partial pressure by 0.1% to 0.2% atmospheres. After equilibration for 30 min, a tail clamp was applied and isoflurane partial pressure was measured. This procedure was repeated to achieve a partial pressure at which each animal did not move.

Determination of the Effect of IT and/or ICV Infusion of Thiopental on MAC
Each study consisted of three MAC determinations. We first determined MAC (MAC0) during infusion of artificial cerebrospinal fluid (aCSF) at 1 µL/min through the intrathecal catheter and/or through a 32-gauge stainless steel blunt end needle inserted into the ICV guide cannula. The aCSF was made daily from stock solutions, with the pH adjusted to approximately 7.2 (measured with a pH meter) by bubbling CO₂ through it. The final composition of the aCSF was 154.7 mM Na⁺, 0.82 mM Mg²⁺, 2.9 mM K⁺, 132.49 mM Cl^–, 1.1 mM Ca²⁺, and 5.9 mM glucose, at a pH of 7.2.

We redetermined MAC (MAC1) while infusing thiopental dissolved in sterile purified water (concentrations up to 37.5 mg/mL) through the IT and ICV catheters. We used sterile water because the resulting osmolarity is similar to that of the aCSF (i.e., to have added the thiopental to aCSF would have produced a hyperosmolar solution, one with an excessive sodium concentration; such a concentration could affect MAC) (12). Methylene blue at a concentration of 0.01% was added to allow a determination of the extent of spread of the infused solutions. During the half-hour between the first and second parts of the study, the isoflurane concentration was decreased to a concentration at which each rat again responded to the tail clamp. The procedure used to determine MAC0 was used to determine MAC1, defined again 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 percentage decrease in the MAC1 relative to MAC0. We repeated this process one more time to give a second MAC value (MAC2). Comparison of the results of the MAC2 and MAC1 determinations allowed an estimate of the extent of equilibrium of the infusions with the cord and brain: a difference in the two MAC values might indicate incomplete equilibrium. We calculated the mean and SD for the change at each infusion rate of thiopental.

After MAC2 was determined, in dead animals (some rats were allowed to recover to permit a redetermination of MAC; i.e., whether injury might have caused a change in the MAC), isoflurane was given to assure immobility and a 1 mL blood sample was taken from the aorta and transferred to a 10 mL Vacutainer® filled with sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Subsequently, the animals were decapitated, and a necropsy was performed to determine the location of the methylene blue and, thereby, the correctness of the position of the cannulae and the minimum extent of infusion.

Determination of Thiopental Concentrations in Blood, Spinal Cord, Whole Brain, and a Cortical Site Distant from the Ventricles.
We determined thiopental concentrations in blood and various sites in the central nervous system (CNS) to allow an estimation of which sites best reflected the anesthetic effect of thiopental (i.e., where the concentration of thiopental best correlated with the anesthetic effect). Thiopental concentrations in plasma, spinal cord, whole brain, and a cortical slice distant from the infusion locations were determined in selected groups. The groups for which these determinations were made were IV infusion (see below for details); IT infusion of 12.5 and 25 µg/min; ICV infusion of 25 µg/min and simultaneous IT and ICV infusion of 0.625, 12.5, and 25 µg/min. After determining MAC or the EC₅₀ (i.e., for the IV studies) value, each rat was killed, and the brain and spinal cord removed. The spinal cord was rapidly and transiently immersed three times into saline 0.9% (pH, 4.6), excess water was removed with a dry sponge, the cord was weighed and placed into a 10-mL glass tube (Fisher Scientific, Pittsburgh, PA) filled with 50 µL of 5-ethyl-5-p-tolyl barbiturate (Sigma-Aldrich, St. Louis, MO) as an internal standard and 3 mL 0.1 M sodium phosphate buffer (pH, 5.59). The brain was cut sagittally in half and a slice of the cerebral cortex was taken. The brain and, separately, the cortical slice, were immersed three times into saline 0.9% (pH, 4.6), excess water was removed with a dry sponge, and the tissue was weighed and placed into a 10-mL glass tube filled as described below.

We conducted separate studies to detect gross neural injury after thiopental infusion. Ascending concentrations of thiopental were infused. Recovery of apparently normal motor coordination was examined at 24 h after the study. The MAC was remeasured after this recovery period and during infusion of aCSF to determine if the MAC differed significantly from MAC0.

IV Study
Determination of MAC and Measurement of Blood and Tissue Concentrations.
The thiopental EC₅₀ required to produce immobility was determined in four rats at a time. Each rat was placed in a clear plastic cylinder and breathed oxygen (1 L/min per cylinder). Thiopental, 2.5%, dissolved in sterile purified water was infused IV at 1.2 mg · kg^–1 · min^–1 until each animal did not respond to a tail clamp. Then a rectal temperature probe was inserted. An arterial catheter was placed into the femoral artery of the left lower limb and was secured by suturing it to adjacent tissue. Heparin, 140 U/kg, was given IV. Every 10 min the tail clamp was applied for 1 min or until the animal moved. When the animal moved to the noxious stimulus, the infusion of thiopental at the same infusion rate was reinstituted, and a 1-mL blood sample was taken. The rat was stimulated after an additional 10 min. If the rat moved, a new 1-mL blood sample was taken and the previous sample returned to the rat. This process continued until the rat failed to move in response to stimulation. We thus acquired blood samples at the largest concentration allowing movement and the smallest concentration suppressing movement. The two blood samples were added to separate 10-mL Vacutainers® filled with sodium heparin (Becton Dickinson). After taking the last blood sample, the animals were decapitated and tissue was harvested and treated as described for the IT and ICV studies.

Preparation of Samples
The blood samples were centrifuged (TJ-6; Beckman, Fullerton, CA) at 2000 rpm for 20 min, and 300 µL of the plasma phase was transferred to 15-mL glass tubes (Fisher Scientific) filled with 5 µg internal standard and 500 µL 0.1 molar sodium phosphate buffer (pH, 5.59). The brain and spinal cord samples together with 5 µg of 5-ethyl-5-p-tolyl barbiturate as internal standard solution and 3 mL 0.1 molar sodium phosphate buffer (pH, 5.59) were homogenized (Kinematica CO, Kriens, Luzern, Switzerland). The homogenate was transferred to 15 mL glass tubes. Pentane 8 mL (Fisher Scientific) was added to the tissue and to the plasma samples in the glass tubes. All samples were vortexed for 1 min and centrifuged at 2000 rpm for 20 min. We transferred 5.5 mL of the organic phase to 10 mL glass tubes and dried this phase with a flow of nitrogen. The dried samples were frozen until analyzed.

Calibration samples were prepared using various amounts of stock thiopental (1.125 µg, 2.25 µg, 4.5 µg, and 9 µg), a constant amount of 5-ethyl-5-p-tolyl barbiturate as an internal standard (5 µg) and of methanol (100 µL) (Fisher Scientific) and 300 µL rat plasma. The calibration sample material was mixed in 15-mL glass tubes and then treated as were the samples described above.

Chromatographic Analyses
A model 1100 high-performance-liquid chromatograph (Hewlett-Packard 1100 series, Agilent Technologies Inc., Mountain View, CA), equipped with a 20 µL volume injector and a detector allowing changes of wavelength settings, was used. Analyses were performed on a 5 µm C8 econosil column (25 cm x 4.6 mm inner diameter) (Alltech Associates, Inc., Deerfield, IL) operating at ambient room temperature (20°C to 25°C). The analytical column was protected by a small pre-column (7.5 mm x 3.2 mm inner diameter) packed with 5 µm C8 bonded silica (Alltech Associates, Inc.).

Following the technique for analysis of thiopental described by Houdret et al. (13), methanol and 0.01 M potassium phosphate (1:1) adjusted to pH 4.40 ± 0.05 with 0.15 M phosphoric acid were used as the mobile phase. The flow rate was 1.9 mL/min. The frozen plasma and tissue samples were dissolved in 100 µL of mobile phase and a 40-µL aliquot was injected. Quantitation was performed using the peak area ratio method with 5-ethyl-p-tolylbarbituric acid as the internal standard. Calibration graphs were obtained from unweighted least-squares linear regression analysis of the data after injection of two separately prepared samples for each of the four calibration concentrations. Based on studies by Houdret et al. (13) and Avram and Krejcie (14), 220 nm was chosen as the detector wavelength for the 5-ethyl-5-p-tolyl barbiturate and 260 nm was chosen as the detector wavelength for thiopental.

Gas Chromatographic Analysis
We used a Gow-Mac 580 flame ionization detector gas chromatograph (Gow-Mac Instrument Corp., Bridgewater, NJ) to analyze the isoflurane concentrations. The 4.6-meter-long, 0.22 cm (inner diameter) column was packed with SF-96. The column temperature was 152°C. The detector was maintained at a temperature approximately 30°C greater. The carrier gas flow was nitrogen at a flow of 16 mL/min. The detector received 35 mL/min hydrogen and 320 mL/min air. Primary standards (obtained by injection of an aliquot of liquid isoflurane into a flask of known volume) were prepared for isoflurane and the linearity of the response of the chromatograph determined. We commonly used secondary (cylinder) standards referenced to the primary standards.

Equilibration of Thiopental into Spinal Cords Ex Vivo
To demonstrate that thiopental could penetrate the spinal cord rapidly enough to reach any target sites of action during in vivo infusions, fresh spinal cords were harvested, weighed, and placed into 50 mL syringes filled with 0.025% thiopental solution at a pH of approximately 7.2. The syringes were placed into an incubator and rotated at 4 rpm. The spinal cords were removed from the syringes after equilibration times of 15, 30, 60, 120, 240, 480, and 1440 min. Subsequently, the cords were dried on gauze, then twice dipped rapidly and transiently into a saline solution, and dried again with gauze. Then they were placed into 10 mL glass tubes filled with 5 µg of 5-ethyl-5-p-tolyl barbiturate as an internal standard and 3 mL 0.1 molar sodium phosphate buffer (pH, 5.59). The samples were prepared for high performance liquid chromatographic analysis as described above.

Equilibration of Thiopental into Spinal Cords In Vivo
This portion of the research tested whether the duration of IT infusion of thiopental was sufficient to produce a steady-state in the spinal cord. It also allowed a test of the extent to which the infused thiopental was restricted to the lower portion of the spinal cord. IT catheters were inserted into rats as described above. Eight rats were studied at a time. Each rat was placed in a 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 at the head end of the cylinder and exit of gases at the tail. A total flow rate of 4 L/min of oxygen and isoflurane at a partial pressure of approximately 1.5% of an atmosphere was delivered (average 1 L/min per cylinder), and the exiting gases were scavenged. Thiopental was infused at 25 µg/min IT. Four rats were killed after 2 h and 4 rats after 4 h, the spinal cords were removed, dipped twice into saline, dried with gauze, divided into upper (thoracocervical) and lower (lumbosacral) portions, and each portion was weighed and placed into separate 10-mL glass tubes filled with 5 µg of 5-ethyl-5-p-tolyl barbiturate as an internal standard and 3 mL 0.1 molar sodium phosphate buffer (pH, 5.59). The samples were prepared for high performance liquid chromatographic analysis as described above.

Statistical Analysis
Mean and standard deviation values were calculated, and Student’s t-test was performed as appropriate. Regression analysis was performed using SPSS 10.0. P < 0.05 was taken as statistically significant.

	Results

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The largest IT and ICV concentrations of thiopental that did not produce apparent neurological deficit in the animals decreased the MAC of isoflurane <22% (Fig. 1 and Table 1). Doses exceeding 12.5 µg/min and up to 37.5 µg/min thiopental IT decreased the MAC of isoflurane <25% despite the development of neurologic deficits in the lower limbs of some rats at the larger doses. Despite the presence of neurologic deficits in some rats, the MAC of isoflurane on the day subsequent to study did not differ from control (MAC0) in these animals. Therefore, we did not exclude their data from the study. Thiopental was infused for 254 ± 2 min IT, for 240 ± 16 min ICV, and for 260 ± 25 min simultaneously IT and ICV. When thiopental was given IT at 25 µg/min (the slowest rate used that caused some injury), the thiopental concentration in the spinal cord (113 ± 16 µg/g) exceeded the concentration found in the spinal cord after IV infusion (24 ± 4 µg/g) by approximately fivefold (Table 2). The IV infusion produced immobility whereas the IT infusion did not despite the latter’s larger concentration in the spinal cord. Thiopental infused at 25 µg/min into the third cerebral ventricle produced a whole brain thiopental concentration (430 ± 39 µg/g) that exceeded the whole brain concentration after IV infusion of thiopental (63 ± 6 µg/g) by nearly sevenfold (Table 2). Again, the IV infusion produced immobility while the ICV infusion did not. The largest concentrations of thiopental in cortical slices (63 ± 6 µg/g) were found in association with IV infusions that produced anesthesia (Table 2).

View larger version (14K): [in this window] [in a new window]   Figure 1. Intrathecal (IT) and intracerebroventricular (ICV) infusion of thiopental produce a dose-related decrease in isoflurane MAC (the minimum alveolar concentration of anesthetic producing immobility in 50% of animals). Separate IT and ICV infusion of thiopental decrease MAC for isoflurane <25%. A ceiling effect was found with both routes of infusion. The first determination of MAC during the IT infusion (MAC1) did not differ from the second determination of MAC during infusion (MAC2), suggesting that a steady-state had been achieved by the time of determination of the first MAC. Values are presented as mean, SD.

IT thiopental diffused into isolated ex vivo spinal cords with a time constant (i.e., a 63% approach to equilibrium) of approximately 1 h (Fig. 2). The identity of the in vivo concentrations at 2 and 4 h are consistent with this rate of equilibration (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Figure 2. Ex vivo spinal cords were equilibrated in a 0.025% thiopental solution for different durations. Sixty-one percent and 79% of thiopental’s steady-state concentrations in the spinal cords are achieved after approximately 1 h and 2 h, respectively. The largest thiopental concentration in cords (the largest concentrations were found at 480 or 1440 min) is assumed to equal the steady-state concentration.

View larger version (38K): [in this window] [in a new window]   Figure 3. Thiopental concentrations in the lower part of spinal cords after intrathecal infusion of 2.5% of thiopental for 2 h did not differ from those found after infusion for 4 h (four rats per study, comparison by Student’s t-test.) Similarly, the concentrations in the upper part of the spinal cords did not differ after 2 versus 4 h, but the concentrations in the upper part of the cord were much less than those in the lower part.

Neither the intercept nor the brain concentration of thiopental differed significantly from zero (a₀ = 4.94 ± 7.0, P = 0.48; a₂ = –0.0024 ± 0.016, P = 0.89, with all coefficients reported as mean ± SE). Both the effect of the spinal cord and cortical concentrations of thiopental were significant (a₁ = –0.21 ± 0.07, P = 0.007; a₃ = –1.38 ± 0.16, P < 0.001) with the effect of the cortical concentration on the MAC-sparing effect of thiopental being 6.5 times as large as the cord concentration. In this model, the brain concentration reflects the effect of cerebral sites near the cerebral ventricles, whereas the cortical concentration reflects the impact of cerebral centers distant from the ventricles, on the immobilizing effect of thiopental. The correlation coefficient for the correlation of change in MAC as a function of the cortical concentration in individual rats (Fig. 4) was 0.82 (r² = 0.67).

View larger version (23K): [in this window] [in a new window]   Figure 4. Using regression analysis, we found that the percentage change in minimum alveolar concentration (MAC) correlated significantly with thiopental concentrations in cortical samples for individual rats (P < 0.001). Each point in the graph represents the concentration found in an individual rat, and the legend indicates the mode of administration of thiopental for that rat. No other correlation of MAC change with thiopental concentrations (either whole brain or spinal cord) was significant.

Necropsy examination after IT and ICV infusion of thiopental with 0.01% methylene blue with a rate of 1 to 2 µL/min confirmed that the infusions were confined to the lumbar and lower thoracic portions of the IT space after IT infusion and to the third and fourth ventricle after ICV infusion. Studies of thiopental infusion, with and without application of methylene blue 0.01%, did not differ in the decrease in isoflurane MAC that the infusion produced.

	Discussion

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Our goal was to determine whether, like isoflurane (1,2,15) and halothane (3), thiopental produced immobility predominantly by an action on the spinal cord. This was accomplished by correlating the MAC-sparing effect of thiopental, administered by different routes, with the concentrations achieved in three parts of the CNS. We found that thiopental produced immobility by an action on cortical sites not proximate to the cerebral ventricles. Our estimates suggest that approximately 20% of the capacity to produce immobility results from an action on the spinal cord. Qualitatively, this agrees with Antognini et al.’s (3) finding that both cord and higher centers mediate the capacity of thiopental to produce immobility but they would assign approximately equal responsibility to cord and higher centers. The difference between our findings and theirs may lie in the different selection of techniques and test animals.

The lack of a larger effect of thiopental on the cord was not attributable to insufficient penetration of the cord by thiopental. As shown in Figure 2, thiopental achieves approximately 61% of its steady-state concentration in the cord in 1 hour. The in vivo concentration achieved in the cord with an IT infusion rate of 25 µg/min thiopental was 5 times that achieved by an immobilizing dose of thiopental when administered IV, yet the effect of the IT infusion was only one fifth of the effect of the IV infusion. Thus, on average, the IT infusion produced a cord concentration 25 times that required for immobility if the cord were the site of the immobilizing action of thiopental. Reports that the IT injection of thiopental in humans also has an apparently direct effect on the much larger human cord also suggest that cord penetration should not have provided a confounding factor in the present studies.

One mechanism thought to underlie the anesthetic effect of barbiturates is their potentiation of the effect of GABA at GABA_A receptors. Barbiturates enhance the action on GABA_A receptors more than volatile anesthetics (16), and this difference increases further at concentrations exceeding the physiologic range (personal communication, RA Harris, University of Texas, Austin, TX). We produced exceedingly large average concentrations with IT and ICV infusions of thiopental. The 20%–25% maximum decrease in MAC produced by IT thiopental may therefore define an upper limit to GABA_A-mediated capacity of the spinal cord to prevent pain-evoked movement. Because inhaled anesthetics produce immobility by acting on the spinal cord and they have smaller enhancing effects on GABA_A receptors than barbiturates, the finding for thiopental is consistent with (but, given the involvement of GABA_A receptors in complex feedback loops, does not prove) the notion that the effect of inhaled anesthetics on spinal GABA_A receptors is of minimal importance to their action. This is also consistent with our observation that GABA_A antagonists equally affect the MAC of inhaled anesthetics with widely different in vitro capacities to enhance the action of GABA on GABA_A receptors (17).

The thiopental concentration in the cerebral cortical tissue predicted approximately 80% of the immobilizing action of thiopental. The limited effect of ICV infusion (Fig. 1) suggests that cerebral centers other than those proximate to the third and fourth cerebral ventricles mediate the immobilizing effect of thiopental. Our data do not distinguish cortical versus subcortical structures as mediators of the effect of thiopental. Measurement of the anesthetizing concentrations of thiopental in decorticated animals compared with controls might provide this information.

We do not know what actions of thiopental on higher centers result in immobility, although these are likely actions on GABA_A receptors. Because GABA_A receptors are distributed throughout the CNS, including the spinal cord, actions on GABA_A receptors per se do not explain why the brain is the more crucial site. We might add here that these studies do not reveal where in the higher centers thiopental acts to produce immobility, nor do we know the descending pathways involved in the production of immobility.

The ratio of the thiopental concentrations found in the spinal cord versus brain in our study (1:3) differed from the ratio found in the study of Mather et al. (2:1) (18). Studies in cats indicate that blood flow to the spinal cord is substantially less than in the brain (19). Because we used a 2.5 times shorter equilibration time than did Mather et al. (18), thiopental might have had insufficient time to accumulate in the more lipid-rich white matter of the spinal cord compared with the cortical regions of the brain.

Two case reports of accidental injection of thiopental into the epidural space of humans observed sedation and one also described severe pain after injection of 10 to 15 mL of 2%–2.5% thiopental into the epidural space (5,6). Those reports found no evidence of neural injury after several days. Morrison et al. (7) described the result of intentional injection of thiopental 5% into the IT space of 22 patients. A dose of at least 150 mg produced sensory anesthesia, a result consistent with the finding of Sudo et al. (8) that thiopental can directly depress dorsal horn neuronal responses to noxious stimulation. The finding of sensory analgesia also suggests that thiopental can penetrate into cord tissues in humans. Given the greater depths needed in humans versus rats, this indicates that penetration in the present study should have been adequate to produce whatever spinal cord effect might be obtained with thiopental. Morrison et al. also found that thiopental doses larger than 450 mg could induce complete blockade of the motor response, concurrently causing deep sedation (7). Such results appear to be similar to those of the present study; both studies suggest that any immobilizing effect of thiopental results in part from the transfer of thiopental to supraspinal centers.

In summary, our results suggest that the major site that mediates the immobility produced by thiopental resides in the cerebral cortex and/or in subcortical nuclei deep within the brain but not proximate to the third or fourth ventricles. The spinal cord mediates a small portion of the immobility produced by thiopental.

	Acknowledgments

 
Supported, in part, by National Institutes of Health grant 1P01GM47818–10 and the Adolf-Messer-Stiftung, Koenigstein, Germany

We appreciate the several suggestions made by Dr. Joseph F. Antognini.

	Footnotes

 
Presented, in part, at the International Anesthesia Research Society (IARS) meeting, March 19, 2001, Fort Lauderdale, Florida.

Dr. Eger is a paid consultant to Baxter Healthcare Corp. who donated the isoflurane used in these studies.

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