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ANESTHETIC PHARMACOLOGY

The Role of Adrenergic and Cholinergic Transmission in Volatile Anesthetic-Induced Pain Enhancement

Department of Anesthesiology, Columbia University, New York, New York

Address correspondence and reprint requests to Pamela Flood, Department of Anesthesiology, Columbia University, 630 West 168th Street, NY, NY 10032. Address e-mail to pdf3{at}columbia.edu.

	Abstract

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Volatile anesthetic drugs have a biphasic effect on pain transmission. At very small concentrations they enhance pain sensitivity whereas at larger subanesthetic concentrations they have an analgesic effect. Previous work has suggested that nicotinic inhibition could mediate the pronociceptive action of isoflurane. Furthermore, activation of nicotinic receptors facilitates the release of norepinephrine in the spinal cord. We hypothesize that nicotinic modulation of norepinephrine release in the spinal cord mediates isoflurane's pronociceptive action. We used hindpaw withdrawal latency as a measure of pain sensitivity after inhibition of adrenergic activity or treatment with nicotine in mice. Isoflurane's effect on pain is separable by concentration. The 50% effective concentration for pain enhancement is 0.16% isoflurane whereas the 50% effective concentration for the antinociceptive action of isoflurane is 0.8%. Depletion of systemic norepinephrine with the neurotoxin DSP-4 caused a reduction in baseline withdrawal latencies and prevented isoflurane pronociception. Baseline latency was also reduced by intrathecal yohimbine. After treatment with yohimbine, isoflurane had no additional pronociceptive effect. Nicotine administered through intracerebroventricular injection increased baseline latency but did not prevent isoflurane pronociception. Conversely, intrathecal applications of nicotine caused a slight reduction in baseline latency and prevented isoflurane's pronociceptive effect. We conclude that spinal noradrenergic transmission seems to be necessary for isoflurane pronociception to occur. Isoflurane may act by inhibiting tonically active nicotinic receptors that modulate the release of norepinephrine in the spinal cord.

	Introduction

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Isoflurane and other volatile anesthetics have a biphasic effect on pain transmission. They enhance pain sensitivity at very small concentrations, whereas they have analgesic effects at larger subanesthetic concentrations (1–3). The pain-enhancing actions of volatile anesthetics require connections from brain to spinal cord, as this paradoxical effect is absent after spinal cord transaction. A potential role for modulatory noradrenergic input has been suggested (1). However, there is no evidence for direct effects of volatile anesthetics on either 1-adrenergic or 2-adrenergic receptors (4,5). In fact, the volatile anesthetic concentrations that cause the pain-enhancing response (as small as 0.1% isoflurane) are far smaller than those that act on most known targets. One potential direct target of these small concentrations of volatile anesthetics is the neuronal nicotinic receptors (6,7). Inhibition of nicotinic receptors by nicotinic antagonists also enhances pain sensitivity whereas treatment with the classical agonist nicotine prevents the pain enhancing action of isoflurane (2).

Activation of nicotinic receptors facilitates the release of norepinephrine in the spinal cord (8) and the pain-relieving effect of nicotinic agonists is thought to involve norepinephrine release (9–12). Thus we hypothesize that small concentrations of isoflurane enhance pain sensitivity by inhibiting tonically active nicotinic receptors that control the release of norepinephrine in the spinal cord.

	Methods

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With the approval of the animal use and care committee at Columbia University, female 129J mice (Jackson Laboratories, Bar Harbor, ME) at 6 to 10 wk of age were used. They were housed in groups of five and had free access to food and water. Animals were housed in an American Association of Laboratory Animal Care-approved facility. All mice had undergone surgical oophorectomy, and inhibition of cycling was verified with sequential vaginal smears on days before experimentation. We used ovaryectomized females because we have previously shown that both testosterone and estrogen modulate the pronociceptive action of isoflurane (13). Pain sensitivity was tested as hindpaw withdrawal latency to a standard heat source. Mice were unrestrained in separate small Plexiglas enclosures on a warmed glass surface. They were allowed 30 min for exploration before testing. Latencies were tested at baseline and in the presence of isoflurane between 0.13% and 1.20%, as described in our previous studies (2,13). Isoflurane was administered to spontaneously breathing mice in a closed hood that covered the testing apparatus. Concentrations were verified continually with a respiratory gas monitor calibrated with gas chromatography. Mice were equilibrated at each concentration for at least 15 min before testing.

All intrathecal and intracerebroventricular injections were in a volume of 5 µL. Intrathecal injections were performed free hand between the L5 and L6 lumbar spaces in unanesthetized mice according to Hylden and Wilcox's method (14). The injections were administered using a glass microsyringe with a 30-gauge needle attached. The accurate placement of the needle was confirmed by a brief twitch of the tail. Intraventricular injections were performed according to the method of Pedigo et al. (15). Mice were lightly anesthetized with isoflurane, and an incision was made in the scalp such that the bregma was exposed. A guide hole was made in the skull at a site 2 mm lateral and 2 mm caudal to the bregma. Injections were performed awake using a 26-gauge needle with a sleeve of polyethylene 20 tubing to control the depth of the injection to 2 mm. After experimentation and death, 5 µL methylene blue dye was injected through the guide hole as above to confirm intraventricular injection.

Mice were treated with an intraperitoneal injection of DSP-4 (50 mg/kg) or saline in an equal volume 7 days before latency testing. After testing, all mice were killed with CO₂, decapitated, and their spinal cords were harvested. Neurotransmitter levels in the spinal cord tissue were measured using high-performance liquid chromatography (16).

In a separate study, yohimbine (30 µg/5 µL) was injected intrathecally with a free hand injection according to the method described by Damaj et al. (17). After 10 min to allow the drug to take effect, the mice were tested for hindpaw withdrawal latency in the presence of isoflurane from 0% to 0.75%. Intrathecal yohimbine at this dose completely reverses the action of adrenergic agonists (18–20).

In a third experiment, the effect of intracerebroventricular and intrathecal nicotine (5 µg) was compared to an equal volume injection of saline to determine whether either would modulate isoflurane hyperalgesia. Five minutes after injection, the mice were tested for hindpaw withdrawal latency in the presence of isoflurane from 0% to 0.75% by an investigator blinded as to treatment. The nicotine dose chosen was the minimal effective dose in a concentration response curve for nicotine nociceptive effects and was intended to mimic the small systemic dose that prevented isoflurane hyperalgesia in our previous work (2,9,17)

The concentration response relationship in Figure 1 is a scatter graph of all latency responses in the presence of isoflurane at concentrations from 0% to 1.2%. These data were evaluated in two different ways. First, two Hill equations were fitted to the data with an Excel macro (Non Mem) as follows:

View larger version (19K): [in this window] [in a new window]   Figure 1. Hindpaw withdrawal latency response to isoflurane. Scatter graph of withdrawal latency responses at isoflurane concentrations from 0% to 1.2%. Each dot represents the response of an individual animal (n = 5–29). When two Hill equations were used to fit the data simultaneously (solid line) the concentration of isoflurane that produced the half-maximal pronociceptive effect for the pronociceptive action of isoflurane was 0.16% isoflurane and the Hill coefficient is 1.5. Withdrawal latency was reduced from 14.5 ± 0.8 s to a minimum of 8.5 ± 0.3 s by 0.5% isoflurane. The concentration that caused the half-maximal inhibitory effectfor the analgesic phase is 0.8% isoflurane and the Hill coefficient is 10.8. Our maximum cut off value for latency (30 s.) is reached at 1.2% isoflurane. When a single Hill equation is used to fit the antinociceptive portion of the curve (0.05%–1.2% isoflurane, dotted line) the resulting values are not significantly different.

where Y₀ is the baseline latency, Ymax is the maximal latency, and Ymin is the minimal latency. IC₅₀ is the concentration of isoflurane that produced the half-maximal pronociceptive effect and EC₅₀ is the concentration that caused the half-maximal inhibitory effect. The Hill coefficient for the pronociceptive effect is n1 and that for the antinociceptive effect is n2. The equation Y = Ymin + (30 – Ymin)(X/(EC₅₀ⁿ² + Xⁿ²) was separately fitted to the data for isoflurane from 0.5% to 1.2%. Data comparing latency in the presence and absence of drug were compared with a paired Student's t-test because the same animals were tested in both conditions. The presence or absence of a pronociceptive response to isoflurane was tested with analysis of variance with repeated measures. All statistical calculations were performed with Analyze-it in Excel (Analyse-it Software, ltd., Leeds, England).

	Results

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Isoflurane elicited a biphasic response with increased pain sensitivity to 0.5%. At baseline there is a wide range of latencies; however, when isoflurane is introduced the variability is diminished. Only lower latencies remain at maximally pronociceptive concentrations (Fig. 1). The IC₅₀ for the pronociceptive action of isoflurane is 0.16% and the Hill coefficient is –1.5. Isoflurane caused a maximal reduction in withdrawal latency of 41%. The variability in latency was larger at baseline than at the maximal pronociceptive concentration tested. There was no correlation between the baseline latency value and that at the maximal pronociceptive concentration (correlation coefficient 0:0.5% = 0.48).

At concentrations larger than 0.5%, latency progressively increased to an imposed maximum of 30 s at 1.2% isoflurane. The EC₅₀ for the antinociceptive action is 0.8% isoflurane and the Hill coefficient is 10.8. When a Hill equation was fitted to the data obtained at isoflurane concentrations from 0.5% to 1%–2%, the resulting curve was not significantly different from that obtained from fitting to the full range of data.

To determine whether the noradrenergic nervous system was involved in the pronociceptive action of isoflurane we depleted norepinephrine stores with the adrenergic neurotoxin DSP-4. DSP-4 treatment caused a reduction in spinal norepinephrine whereas spinal serotonin and dopamine were not changed (Table 1). Seven days after DSP-4 treatment, baseline withdrawal latency was reduced (Fig. 2; P < 0.01, Student's t-test). Isoflurane had no significant pronociceptive action after DSP-4 treatment (analysis of variance with repeated measures).

View larger version (15K): [in this window] [in a new window]   Figure 2. Isoflurane hyperalgesia requires spinal 2-noradrenergic transmission. a) Norepinephrine depletion with the adrenergic neurotoxin DSP-4 (closed circles) decreases baseline withdrawal latency (*P < 0.05, Student's t-test; n = 15). From this lower baseline, isoflurane does not further reduce the withdrawal latency (analysis of variance with repeated measures). b) Blocking 2- adrenergic receptors with intrathecal yohimbine (open circles) had a similar effect, lowering baseline withdrawal latency (**P < 0.01, Student's t-test; n = 15). After intrathecal yohimbine, isoflurane caused no further reduction in withdrawal latency. Each point represents the mean ± se.

In a separate experiment to determine the site of the receptors for norepinephrine that are important in isoflurane hyperalgesia, we administered the ₂-noradrenergic antagonist, yohimbine, intrathecally (Fig. 2). Once again, baseline withdrawal latency was reduced (P < 0.05, Student's t-test). After intrathecal yohimbine injection, there was no further decrease in latency in the presence of isoflurane (analysis of variance with repeated measures).

To determine the anatomical location for the action of nicotine on isoflurane's pronociceptive action, intrathecal or intracerebroventricular injections of nicotine, 5 µg, were given to 2 groups of mice who were then tested with isoflurane. When administered intrathecally, nicotine (5 µg) decreased the baseline withdrawal latency (Fig. 3) compared with the control that was injected with intrathecal saline. Isoflurane caused no further reduction in withdrawal latency. In contrast, when nicotine (5 µg) was injected intracerebroventricularly, it caused an increase in baseline withdrawal latency (Fig. 3). However, isoflurane's pronociceptive effect was unchanged.

View larger version (15K): [in this window] [in a new window]   Figure 3. Isoflurane hyperalgesia is prevented by intrathecal but not intracerebroventricular nicotine. a) Intrathecal nicotine (5 µg), open squares, caused a reduction in baseline withdrawal latency (*P < 0.05, Student's t-test; n = 5). From this lower baseline, isoflurane caused no further reduction in withdrawal latency. b) In contrast, when administered to the cerebral ventricles, open circles, nicotine (5 µg) increased baseline withdrawal latency (**P < 0.01, Student's t-test; n = 10). The hyperalgesic response to isoflurane after intracerebroventricular nicotine was not different from control. All values are mean ± se.

	Discussion

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Although the biphasic nature of isoflurane's nociceptive actions has been documented, this is the first work to carefully identify the relevant concentration range for the two effects (1–3). Furthermore, our findings suggest that the two opposing actions of isoflurane can be separated by concentration. The reduction in experimental variance at the maximal pronociceptive isoflurane concentrations may represent the blockade of variable inhibitory input by isoflurane. The lack or correlation between baseline latency and latency at maximal pronociceptive concentration of isoflurane would be expected if our hypothesis is correct. If inhibitory tone (at least in part noradrenergic) is variable at baseline and removed by isoflurane, we would expect the latency to nadir near the same point, irrespective of its baseline value.

It has been reported that both systemic and intrathecal yohimbine has a pronociceptive effect in intact rats (1,21). Yet other studies by the same authors have found a pronociceptive effect of yohimbine in the tail flick but not hindpaw withdrawal assays (22) It may be that the activity of the 2-adrenergic system is variable. We noted a large degree of variability in baseline latency that was reduced by isoflurane (Fig. 1). The differing responses to yohimbine could reflect that variability.

Kingery et al. (1) concluded that spinal 2-adrenergic receptors were not involved in the pronociceptive phase because isoflurane did not have a pronociceptive effect 6–7 days after spinal cord transection. However, after spinal cord injury transected axons retract and thus these data do not exclude the involvement of spinal 2-adrenergic receptors but rather underscore the importance of descending inhibitory connections in this behavior. If the axons had retracted, the postsynaptic -2 receptors would still be intact, but a presynaptic effect on nicotinic receptors would be expected to disappear.

Nicotine, when given intrathecally (Fig. 3), reduced baseline latency. This response is thought to be attributable to potentiation of glutamate release (23). From this lower baseline, isoflurane had no further pronociceptive action. These data raise the possibility that the nicotinic modulation of norepinephrine release is not monosynaptic. Rather the adrenergic axon could be modulated by an excitatory interneuron with presynaptic nicotinic receptors. This possibility remains to be tested with direct experiments on nicotinic control of norepinephrine release in the spinal cord. Although nicotine clearly evokes the release of norepinephrine in the spinal cord, it is not known if the connection is monosynaptic. Intraventricular administration of nicotine caused antinociception as previously described (9,24,25). However, the pronociceptive effect of isoflurane was not changed (Fig. 3). Nicotinic receptors are modulatory on both excitatory and inhibitory nerve terminals in the central nervous system. The subtype of nicotinic receptor expressed at the various terminals appears to be different, however, raising the possibility of differential pharmacological modulation (26).

In summary, the data obtained from this investigation support our hypothesis that small concentrations of isoflurane intensify pain sensitivity by inhibiting tonically active nicotinic receptors in the spinal cord and thereby hindering the release of spinal norepinephrine. This study also confirms that the administration of nicotine in conjunction with small concentrations of isoflurane produces a means to overcome isoflurane pronociception.

	Footnotes

 
Supported, in part, by award GM00695 from NIGMS (to PF).

Accepted for publication September 23, 2004.

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