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ANESTHETIC PHARMACOLOGY

Alterations in Spinal, but Not Cerebral, Cerebrospinal Fluid Na⁺ Concentrations Affect the Isoflurane Minimum Alveolar Concentration in Rats

Michael J. Laster, DVM^*, Yi Zhang, MD, Edmond I. Eger, II, MD^*, Dimitry Shnayderman, BS^*, and James M. Sonner, MD^*

From the *Department of Anesthesia and Perioperative Care, University of California, San Francisco, California; and Department of Anesthesiology, Fuwai Hospital and Cardiovascular Institute, Beijing, China.

Address correspondence and reprint requests to Edmond I Eger, II, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia.ucsf.edu.

	Abstract

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BACKGROUND: Previous studies demonstrated that MAC (the minimum alveolar concentration of an inhaled anesthetic that produces immobility in 50% of subjects exposed to noxious stimulation) for halothane directly correlates with the central nervous system concentration of Na⁺. However, those studies globally altered Na⁺ concentrations, and thus did not distinguish effects on the spinal cord from cerebral effects. This is an important distinction because the cord appears to be the primary site for mediation of the immobility produced by inhaled anesthetics. Accordingly, in the present study, we examined the effect of altering intrathecal versus intracerebroventricular concentrations of Na⁺ on MAC.

METHODS: In rats prepared with chronic indwelling catheters or stylets, we infused solutions deficient in Na⁺ and with an excess of Na⁺ into the lumbar subarachnoid and intracerebroventricular spaces and measured MAC for isoflurane before, during, and after infusion.

RESULTS: MAC of isoflurane correlated directly with concentrations of Na⁺ infused intrathecally but did not correlate with concentrations infused intracerebroventricularly.

CONCLUSION: The results are consistent with a mediation or modulation of MAC by Na⁺ channels. These might include voltage-gated or ligand-gated channels or other Na-sensitive targets (e.g., pumps, transporters, exchangers).

	Introduction

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Halothane (1), isoflurane (2), and propofol (2) can inhibit sodium (Na⁺) channel activity, and inhibition of such activity may mediate halothane's capacity to decrease the release of the excitatory neurotransmitter glutamate (3). Enflurane and isoflurane also can decrease glutamate release (4). F3(1-chloro-1,2,2-trifluorocyclobutane), a cyclobutane anesthetic, inhibits glutamate release from synaptosomes derived from cerebral cortical cells and inhibits Na⁺ currents in lumbar dorsal root ganglion neurons (5), whereas the nonimmobilizer cyclobutane F6(1,2-dichlorohexafluorocyclobutane) neither inhibits glutamate release nor Na⁺ currents (5). Such actions of inhaled anesthetics would be consistent with a role of Na⁺ channels as mediators of inhaled anesthetic action, particularly on immobility and MAC (the minimum alveolar concentration of an inhaled anesthetic that produces immobility in 50% of subjects exposed to noxious stimulation).

We previously demonstrated in dogs that MAC for halothane correlates directly with the concentration of Na⁺ in cerebrospinal fluid (6). MAC changed from 0.79% ± 0.05% halothane at 133 mEq/L Na⁺ to 1.48% ± 0.10% at 175 mEq/L, an 87% increase. Such results suggest that central nervous system Na⁺ may markedly influence anesthetic requirement. This experiment did not distinguish between spinal and cerebral effects of Na⁺, and, particularly, did not show that the spinal cord mediated at least a portion of the effect of the change in Na⁺. Such a demonstration would be important to the interpretation of the potential importance of Na⁺ channels as mediators of MAC because of the central role of the spinal cord in the production of immobility by inhaled anesthetics (7,8).

Accordingly, the present study sought to demonstrate whether MAC correlated directly with changes in the Na⁺ concentration surrounding the lumbar cord versus cerebral portions of the rat central nervous system. We predicted that changes in the region of the cord, but not the brain, would affect MAC.

	METHODS

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Materials
Isoflurane was obtained from Baxter Healthcare Corp. (New Providence, NJ).

Studies of MAC in Rats
With approval of the Committee on Animal Research of the University of California, San Francisco, we studied 65 male, 12–15-wk-old, Long-Evans rats weighing 300–450 g obtained from Charles River Laboratories (Hollister, CA). Each animal was caged with up to as many as two additional rats before insertion of catheters or cannulae, and was housed singly thereafter. All had continuous access to standard rat chow and tap water before study.

Effect of Lumbar Intrathecal Infusions of Solutions Containing Lower and Higher Concentrations of Na⁺
Rats were anesthetized with isoflurane, and a 32-gauge polyurethane catheter (Micor Inc., Allison Park, PA) was placed through the atlantooccipital membrane according to the method described by Yaksh and Rudy (9). The catheter was threaded caudally 6–8 cm towards 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, and the wound was infiltrated with 0.25% bupivacaine. Rats recovered from anesthesia and surgery for at least 24 h before study.

Rats were studied in groups of eight placed in individual clear plastic cylinders. Each cylinder received approximately 1 L/min oxygen containing approximately 2% isoflurane. An infusion of artificial cerebrospinal fluid (aCSF) was begun immediately after induction of anesthesia at 4 µL/min via the previously placed intrathecal catheters. The aCSF contained 155 mEq/L of Na⁺ and was buffered with 37 mEq/L of bicarbonate. (The complete composition was as follows: 126.6 mM NaCl; 27.4 mM NaHCO₃; 2.4 mM KCl; 0.50 mM KH₂PO₄; 0.49 mM NaSO₄; 1.10 mM CaCl₂; 0.83 mM MgCl₂; and 5.49 mM glucose. pH was adjusted to 7.3 by addition of a small amount of NaOH. Total osmolarity approximately 330 mOsm.) A rectal temperature probe was inserted. The isoflurane concentration was decreased to 1.0%–1.2% and sustained at this concentration for 50 min, after which the tail was clamped and moved by rolling the clamp at 1–2 Hz for up to 1 min (less if the rat moved). After certifying that movement had occurred, the isoflurane concentration was increased by approximately 0.15%–0.2%, and after a 30-min period of equilibration, the tail clamp was again applied and movement or lack of movement determined. Isoflurane partial pressures were monitored using an infrared analyzer (Datascope, Helsinki, Finland), and immediately after determination of the response to tail clamp, a sample of gas was obtained from one of the chambers and analyzed for isoflurane by gas chromatography. This process continued until all rats failed to move in response to application of the tail clamp. MAC was calculated as the average of the largest concentration that permitted movement and the smallest concentration that suppressed movement. This value was designated MAC1. Anesthetic administration then was discontinued, and the rats recovered.

The next day, the rats again were anesthetized with isoflurane and MAC redetermined (MAC2). However, on this day, half the rats received an infusion of aCSF (the control group), and half received an infusion of a solution containing a decreased (28.5 mEq/L) or an increased (192.5 mEq/L) concentration of Na⁺ (the experimental group). A decreased concentration was produced by substituting mannitol for the NaCl component of aCSF, supplying sufficient mannitol to maintain a constant osmolarity. An increased concentration was produced by adding NaCl. When the concentration of Na⁺ was increased, the aCSF given the control group was altered by addition of sufficient mannitol to produce an osmolarity equal to that produced by the addition of NaCl in the experimental group. Anesthetic administration then was discontinued, and the rats recovered. The investigator making the determination of MAC was blinded to the contents of the infusions.

On the third day, the rats again were anesthetized with isoflurane and the process of MAC determination repeated (MAC3). On this day, all rats received only an infusion of aCSF. The rats again were allowed to recover and were examined for gross abnormalities in hindlimb function.

Thus, these measurements supplied two control assessments. The change in MAC with treatment could be compared with the MAC in the same rat when given aCSF. And the change in MAC with treatment could be compared with the MAC in a comparable group of rats given aCSF. Injury from treatment could be assessed by noting whether a gross abnormality in motor function, particularly of the hindlimbs, was evident after the third anesthetic, and whether MAC3 differed from MAC1 in the experimental group more than in the control group.

Effect of Cerebral Ventricular Infusions of Solutions Containing Lower and Higher Concentrations of Na⁺
To place cannulae into a lateral ventricle, each rat was anesthetized with isoflurane, the skull was exposed, and a hole was drilled 0.5 mm posterior to the bregma and 1.5 mm lateral to the midline. Through this hole, we inserted a 24-gauge stainless steel guide cannula to a depth of 3.2 mm from the brain (dural) surface. The wound edges were infiltrated with 0.25% bupivacaine. The cannula was secured by wiring to two screws placed into the skull approximately 5 mm to either side of the cannula. Infusions of solutions containing various concentrations of Na⁺ as described above were made through a needle inserted into this guide cannula. Infusion rates equaled those for intrathecal studies (4 µL/min). The studies of MAC during intracerebroventricular infusions were made as described above for such studies using intrathecal infusions.

Gas Chromatographic Analysis
We used a Gow-Mac 580 flame ionization detector gas chromatograph (Gow-Mac Instrument Corp., Bridgewater, NJ) to analyze isoflurane concentrations. The 4.6-m long, 0.22-cm (ID) column was packed with SF-96. The column temperature was 107°C with the detector maintained at a temperature approximately 50°C greater. The carrier gas flow was nitrogen at a flow of 16 mL/min. The detector received 35 mL/min hydrogen and 250 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 the primary standards.

Statistical Analyses
Mean values and standard deviations were determined for the MAC determinations. The first and third determinations of MAC were averaged for each rat, and the percent that the determination on the second day deviated from this average was calculated. An analysis of variance was done to determine whether infusion of altered Na⁺ concentrations significantly affected MAC for either intrathecal or intracerebroventricular infusions. Deviations were analyzed using a paired Student's t-test. Because we made multiple comparisons, we accepted a value of P < 0.01 as significant.

	RESULTS

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Two rats were lost from study. One died before completion of study and the other pulled out his catheter between study days. A new rat was substituted for the rat that died. Thus, results were available for 63 rats.

Analysis of variance revealed a significant effect of infusion of altered Na⁺ concentrations into the intrathecal space (P < 0.001), but not infusion into the cerebral ventricles (P > 0.05). Subsequent paired t-tests revealed that administration of a decreased Na⁺ concentration to the lumbar intrathecal space significantly decreased isoflurane MAC (P < 0.001), and administration of an increased concentration did the converse (Table 1, Fig. 1, P < 0.01). In contrast, intracerebroventricular delivery of the same volume of decreased or increased Na⁺ concentration did not significantly affect MAC (P > 0.01).

View larger version (11K): [in this window] [in a new window]   Figure 1. Minimum alveolar concentration directly correlates with the concentration of Na+ infused into the intrathecal space of rats, but does not correlate with the concentration of Na+ infused into the right lateral cerebral ventricle. Infusion in all rats equaled 4 µL/min.

We also found that repeated determination of MAC in control rats did not change MAC (Fig. 2).

View larger version (8K): [in this window] [in a new window]   Figure 2. In rats receiving only normal Na+ artificial cerebrospinal fluid into either the intrathecal space or the right lateral cerebral ventricle, minimum alveolar concentration did not change over the course of 3 days of repeated determination (one determination per day).

	DISCUSSION

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Our results indicate that MAC for isoflurane in rats correlates directly and rectilinearly with the concentration of Na⁺ infused intrathecally, but not with the concentration infused intracerebroventricularly (Fig. 1). The finding for the effect of intrathecal infusion confirms earlier work showing that MAC correlates with Na⁺ concentration in cerebrospinal fluid (6). However, the earlier work modified the Na⁺ concentration in cerebrospinal fluid by IV infusions of solutions with Na⁺ concentrations less and more than normal (i.e., 5% glucose solutions in water and hypertonic saline), or by administration of concentrated solutions of mannitol to draw water from the central nervous system. Thus, these earlier studies produced global changes in central nervous system Na⁺ rather than changes limited to one portion of the central nervous system. The present findings suggest that the effect of Na⁺ results from an action on the spinal cord rather than a cerebral action. Such an observation is consistent with the present perception that the spinal cord is the primary mediator of the immobility produced by inhaled anesthetics (7,8). It supports the notion that blockade of Na⁺ channels might mediate a portion of the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation.

The present results suggest other possibilities for testing the importance of particular channels as mediators of inhaled anesthetic actions. We previously attempted to assess the effect of changes in potassium ion concentration on MAC for halothane in dogs (6). No effect was found, but the changes in cerebrospinal fluid potassium were only 0.2–0.4 mEq/L, and thus did not provide a sufficient range to adequately test the importance of potassium. This limitation could be circumvented using the present approach. Such an experiment might shed light on the proposal that potassium channels are important mediators of the capacity of inhaled anesthetics to produce immobility (10). Other ion effects (e.g., calcium) might similarly be tested.

However, the present approach has limitations. The infusion bathes both the cord and the nerves issuing there from, not distinguishing between the two. Barriers to diffusion plus the removal of ions by blood coursing through the cord must produce ionic gradients with some parts of the cord more affected than others. Differential perfusion of gray versus white matter (11) further influences these gradients. Thus, this must remain a qualitative rather than quantitative technique.

The observation that changing Na⁺ changes MAC suggests an effect on processes that are sensitive to Na⁺, and specifically on the proteins that mediate these processes. Cation channels that conduct Na⁺ could be one such protein target, because changes in the Na⁺ gradient across the channel will change channel currents. Beyond voltage-gated sodium channels, such channels might include N-methyl-d-aspartate receptors, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, kainate receptors, neuronal nicotinic acetylcholine receptors, and transient receptor potential channels [e.g., see Ref. (12)]. Transporters that exchange Na⁺ for other ions, such as Na/K transporters, might similarly be affected. Proteins that are sensitive to Na⁺ or ionic strength but which do not conduct Na⁺ might also contribute to the observed effect on immobility. Further studies will be required to identify which targets underlie the observed effect on immobility.

Although the present results are consistent with Na⁺ channels as mediators of the immobility produced by inhaled anesthetics, other interpretations are possible. For example, an increased or decreased intrathecal Na⁺ may simply increase and decrease excitation in the spinal cord. An increased Na⁺ concentration will increase depolarization, and neurotransmitter release is exponentially connected to the extent of depolarization (13). Thus, an increased extracellular Na⁺ concentration might increase excitatory transmitter release and excitation of the spinal cord. Although such an effect might antagonize the depression produced by an inhaled anesthetic (i.e., increase MAC), it would represent a modulation, rather than be a mediation, of the capacity of inhaled anesthetics to produce immobility.

However, such an effect likely would be counterbalanced by a parallel effect, an enhancement at inhibitory synapses. That is, an increase in extracellular Na⁺ concentration might increase inhibitory as well as excitatory transmitter release. Inhibitory neurons are more numerous in the central nervous system, and, moreover, their effects are sustained over a longer period of time (14). Thus, an increase in extracellular Na⁺ concentration might be predicted to cause overall depression of the spinal cord and decrease MAC—the opposite to what was found. Conversely, a decrease in extracellular Na⁺ concentration might excite the cord and increase MAC. Consistent with this interpretation of excitation/inhibition, pathological hyponatremia can produce convulsions (15).

These speculations are not consistent with an indirect, modulatory effect of changes in intrathecal Na⁺. They are not consistent with what we found for MAC. Instead, they might be consistent with a blockade of Na⁺ channels by inhaled anesthetics that is reversed in part by larger Na⁺ concentrations and augmented by decreased Na⁺ concentrations. They might be more consistent with a mediation rather than a modulation of anesthetic effect by Na⁺ channels.

Such reasoning might also apply to the effect of extracellular Na⁺ concentration changes on the function of specific receptors. Regardless of the correctness of these speculations, the present results further support the notion that the spinal cord is crucial to the production of immobility; if excitation or depression is important, it is excitation or depression of the cord and not the brain. One caveat to generalization of our findings is that we tested the effect of changes in intrathecal Na⁺ on the MAC of isoflurane; we assumed that isoflurane is a prototypic anesthetic representative of all inhaled anesthetics. That is, we assume a unitary theory of narcosis, at least as concerns MAC.

Finally, we confirm previous observations in limited numbers of animals (16) that repeated measurements of MAC over days does not affect MAC (Fig. 2).

	Footnotes

 
Accepted for publication May 25, 2007.

Supported by NIH grant 1P01GM47818.

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

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