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BJA Advance Access originally published online on December 23, 2005
British Journal of Anaesthesia 2006 96(2):216-221; doi:10.1093/bja/aei309
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© The Board of Management and Trustees of the British Journal of Anaesthesia 2005. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

NEUROSCIENCES AND NEUROANAESTHESIA

Nitrous oxide depresses electroencephalographic responses to repetitive noxious stimulation in the rat

M. L. LeDuc1, R. Atherley1, S. L. Jinks1 and J. F. Antognini1,2,*

1 Department of Anesthesiology and Pain Medicine and 2 Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA, USA

* Corresponding author. E-mail: jfantognini{at}ucdavis.edu

Accepted for publication November 21, 2005.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Although N2O has been widely used as an anaesthetic adjuvant its effect on electroencephalographic (EEG) activity is poorly understood because it is usually studied in the presence of additional anaesthetics, including inhaled anaesthetics. We examined the EEG effects of N2O in rats using a hyperbaric chamber that permitted N2O to be the sole anaesthetic.

Methods. Rats (n=10) were anaesthetized with isoflurane and EEG activity was recorded from skull screws. The rats were placed into a hyperbaric chamber and mechanically ventilated. Isoflurane was eliminated while the chamber was pressurized with N2O. The minimum alveolar concentration (MAC) was determined in five rats by adjusting the chamber pressure and N2O concentration, and applying a tetanic noxious stimulus to the tail via an electrical pass-through. EEG responses to noxious stimulation (20 electrical pulses at 40 V applied to the tail at 0.1, 1 and 3 Hz, and 50 Hz tetanic stimulation at 60 mA applied for 30 s) were determined at 1.5 and 2 atm N2O.

Results. The N2O MAC was 1.7±0.1 atm. No consistent EEG activation occurred during electrical stimulation at either partial pressure of N2O, although spontaneous EEG activation often occurred. Blood pressure increased after the 3 and 50 Hz stimuli. Four other rats anaesthetized with isoflurane had EEG activation with the 3 and 50 Hz stimuli.

Conclusions. These data indicate that N2O at peri-MAC partial pressures prevents EEG activation resulting from noxious electrical stimulation. Unlike the situation with isoflurane, stimulus-evoked EEG activation did not occur at peri-MAC anaesthetic concentrations, suggesting that N2O potently blocked ascending nociceptive transmission.

Keywords: anaesthesia; anaesthetics gases, nitrous oxide; monitoring, electroencephalography


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Nitrous oxide (N2O) has been used as an anaesthetic adjuvant for more than 150 yr but its use as a sole anaesthetic is limited due to low potency. The minimum alveolar concentration (MAC) of N2O is ~1 atm in humans1 and 2 atm in rats.2–4 The electroencephalographic (EEG) effects of N2O are poorly understood. N2O is administered with other more potent anaesthetics, and most reports of the EEG effects of N2O are limited5–7 because the presence of these other drugs obscures interpretation. Despite this paucity of data, there is clear evidence that the EEG effect of N2O differs from that of more potent anaesthetics. For example, N2O has limited effects on the bispectral index (BIS),8 an EEG parameter that is a measure of anaesthetic depth, while potent anaesthetics such as isoflurane and propofol clearly produce EEG and BIS depression.9 Furthermore, little is known about how N2O, when administered as a sole anaesthetic, affects the EEG response to noxious stimulation. In the present study we determined the EEG effects of N2O, including the EEG response to noxious stimulation. We hypothesized that N2O would depress the EEG response to noxious stimulation in the approximate 0.9–1.2 MAC range, as we have previously observed with isoflurane and halothane.10


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The University of California, Davis Animal and Care Use Committee approved this study. Adult male Sprague–Dawley rats (retired breeders) were anaesthetized with isoflurane in a chamber. The rats were then removed and placed on mask anaesthesia with 2% isoflurane. A midline incision was made over the ventral surface of the neck and a tracheostomy tube placed using a 12 gauge catheter, after which the rat was mechanically ventilated. Catheters (PE-50) were placed into the internal jugular vein and carotid artery for administration of fluids and drugs, and measurement of mean arterial blood pressure (MAP), respectively. Four small stainless steel screws were inserted into the skull to record the frontal-occipital electroencephalogram bilaterally.

We used a custom-made hyperbaric chamber that consisted of a cylindrical plexiglass tube 122 cm long and 33 cm in diameter, with wall thickness 1.3 cm. At both ends, 5 cm thick aluminium plates were placed that contained circular grooves and rubber gaskets, in which the ends of the cylinder would fit. On the outer limits of the plates beyond the circular grooves were six holes that permitted placement of 140 cm long rods that, when bolted, connected the two aluminium plates. Each plate had several electrical and gas pass-throughs. Attached to one of these was a pressure gauge that permitted measurement of the chamber pressure. A rodent ventilator (Harvard Apparatus, Holliston, MA, USA) was placed into the chamber, and the anaesthesia circuit included a small container of soda lime that removed exhaled carbon dioxide.

The rat was placed on a small supporting hammock and the head was lightly secured to a stereotaxic frame using ear bars, but the forelimbs and the hindlimbs were free to move. Rectal temperature was measured via a temperature probe that was attached to an electrical pass-through. Below the rat was a hot/cold pack that was wrapped around a metal coil through which warm water flowed from an external water bath. The rat's body temperature was maintained at 38.0±0.7°C by adjusting the temperature of the water bath. The tracheostomy tube was attached to the ventilator which received its fresh gas supply from an isoflurane vaporizer and oxygen tank, via a gas pass-through in one of the plates.

An Aspect 1050 BIS monitor (Aspect Medical, Newton, MA, USA) was used for EEG recording. The head stage was placed into the chamber and the leads attached to the screws in the skull to measure fronto-occipital EEG activity bilaterally. The head stage cable was attached to a custom-made electrical pass-through, and a cable exiting from the outside of the aluminium plate was attached to the BIS monitor. Two needle electrodes were placed subcutaneously at the base of the tail to permit application of electrical noxious stimuli. The carotid arterial catheter was attached to a transducer placed in the chamber, and the electrical cable of the transducer exited one of the aluminium plates via a pass-through. The jugular catheter was attached to a syringe pump that contained pancuronium 0.2 mg ml–1. The syringe pump was controlled via computer, and was not started until after MAC was determined.

The chamber was sealed and flushed with O2:N2O (40:60) until the nitrogen in the chamber was <1%, which usually required ~60 min. The gas flow to the ventilator was switched from the external oxygen source (and isoflurane vaporizer) to the ambient internal gas mixture. The chamber was pressurized with N2O reaching an approximate total pressure of 2–2.5 atm within 2–3 min. The rising N2O partial pressure maintained anaesthesia as the isoflurane concentration was decreasing. The pressure was maintained for 30–45 min while the residual isoflurane was exhaled by the rat into the chamber. A small PE-10 tubing sampled gas from the tracheostomy tube and this sample exited the chamber via a gas pass-through. Analysis with a calibrated anaesthetic agent analyser (Rascal II, Ohmeda, Salt Lake City, UT) demonstrated that residual isoflurane concentrations were <0.1%.

In five of the rats we first determined the N2O MAC as previously reported.2 In brief, we applied a tetanic stimulus (50 Hz, 60 mA, up to 1 min) to the tail electrodes using electrical pass-throughs. We looked for gross and purposeful movement, which consisted of a pawing motion of the legs; stiffening of the extremities was considered negative. Because the head was secured, we could not use head movement as a positive response. Depending on the initial response, the pressure in the chamber was either increased (by adding more N2O) or decreased (by releasing gas from the chamber) by ~15–20%. Fifteen minutes after each pressure change, the N2O partial pressure was determined by sampling the gas in the chamber (40–60 ml collected in a glass syringe over 30 s) and passing it through a calibrated agent analyser (Rascal II, Ohmeda, Salt Lake City, UT). The N2O concentration was then multiplied by the total chamber pressure. This process was repeated until two N2O partial pressures were found that just permitted and just prevented movement; N2O MAC was the average of these. Because the MAC was similar in these five rats ({approx}1.7 atm N2O) a MAC value of 1.7% was used for the remaining five rats.

After MAC determination (or after the initial N2O equilibration in the five rats that did not have MAC determined), the N2O pressure was adjusted to either {approx}1.5 or {approx}2 atm (order alternated), the syringe pump was started to infuse pancuronium {approx}0.3 mg followed by {approx}0.36 mg kg–1 h–1 and EEG recording commenced. The pancuronium eliminated any electromyographic artifact in the EEG recording. The BIS monitor recorded EEG activity at 256 Hz and the processed variables were downloaded to computer every 5 s. These included the spectral edge frequency at 95% power (SEF95) and the median edge frequency at 50% power (MEF). The filter settings were 1–2 Hz for the high pass and 70 Hz for the low pass, with a 60 Hz notch filter. The monitor used a 30 s ‘rolling’ average.

The EEG response to repetitive electrical stimulation was determined. We applied a pulse train of 20 stimuli (100 µs pulse duration, 40 V) at 0.1, 1, and 3 Hz (order varied), with 2–3 min elapsing between each pulse train.10 11 In addition, we determined the EEG response to a supramaximal tetanic stimulus (50 Hz, 60 mA) applied for 30 s. The N2O pressure was adjusted to 2 atm (or 1.5 atm, depending on the initial N2O pressure) and after 15–20 min the electrical stimulation paradigm was repeated. At the end of the experiment the animal was euthanized by increasing the N2O partial pressure and decreasing the oxygen in the chamber.

Because the initial N2O data indicated little EEG activation, we anaesthetized four rats with isoflurane 1% ({approx}0.9 MAC) and determined EEG responses to the repetitive stimuli, as used above in the N2O rats and as we have previously reported.10 11 These rats served as a positive control group.

EEG variables (SEF95, MEF) were analysed using repeated measures analysis of variance (ANOVA) for the 50 s period before and the 200 s period after initiation of stimulation.10 Likewise, the pre-stimulus MAP was compared with the peak MAP after initiation of the stimulus using ANOVA. Post hoc testing with the Student–Neuman–Kuels test or Student's t-test was performed as indicated. A P<0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
N2O MAC was 1.7±0.1 atm (n=5). During N2O anaesthesia EEG activity was noted to spontaneously fluctuate between a high amplitude, low frequency pattern to a low amplitude, high frequency pattern. Application of repetitive noxious stimuli did not consistently evoke an EEG response (Fig. 1). Summary data shown in Figure 2 demonstrate that the electrical stimuli did not elicit an EEG response.


Figure 1
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  		Fig 1 Electroencephalographic (EEG) tracings during N2O (pressure 2 atm) anaesthesia. Spontaneous EEG activation is shown in the upper tracing, with a change from a high amplitude, low frequency to a low amplitude, high frequency pattern. In the lower tracing there is no discernible EEG response to a tetanic electrical stimulus (30 s beginning at arrow).

 

Figure 2
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  		Fig 2 Summary data (mean and SD) of EEG responses to repetitive noxious stimulation during N2O anaesthesia at 1.5 and 2 atm; N=10. The spectral edge frequency at 95% power (SEF95) and the median edge frequency at 50% power (MEF) are plotted against time. The arrows indicate the onset of the stimulus and the black bars to the right of each arrow indicate the duration of the stimulus. The electrical stimuli consisted of 20 pulses at 0.1, 1, and 3 Hz and a tetanic stimulus (50 Hz) applied for 30 s. No significant changes were noted with stimulation.

 
The blood pressure tracing was severely dampened in three animals, possibly because of blood which backed up into the catheter and could not be flushed as the hyperbaric chamber prevented access to the animal. MAP in the seven other animals was noted to increase with the 3 and 50 Hz (tetanic) stimuli, indicating that the animals developed a cardiovascular response to these noxious stimuli (Table 1).


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  		Table 1 Mean arterial pressure (mm Hg) before and during stimulation. Mean and standard deviation, n=7; Pre-Stim=value prior to stimulation; Stim=peak value with stimulation. The 0.1, 1, and 3 Hz stimuli consisted of 20 pulses each, while the 50 Hz stimulus was a tetanic stimulus applied for 30 s.

 
The isoflurane anaesthetized rats developed an EEG activation response to the 3 and 50 Hz electrical stimuli (Fig. 3). This activation was observed primarily in the MEF.


Figure 3
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  		Fig 3 Summary data (mean and SD) of EEG responses to repetitive noxious stimulation during isoflurane anaesthesia (1%, 0.9 minimum alveolar concentration, MAC); N=4. The SEF95 and the MEF are plotted against time. The arrows indicate the onset of the stimulus and the black bars to the right of each arrow indicate the duration of the stimulus. The electrical stimuli consisted of 20 pulses at 0.1, 1, and 3 Hz and a tetanic stimulus (50 Hz) applied for 30 s. *P<0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The main finding of the present study was that N2O prevented EEG responses to noxious electrical stimulation. The cardiovascular response, as manifested as an increased MAP, was not abolished, indicating that N2O did not provide complete analgesia.

We have previously examined the effects of halothane, isoflurane, and propofol on EEG responses to repetitive noxious stimulation as has presently been used.10 11 At 0.8 MAC for halothane and isoflurane, noxious electrical stimulation shifted EEG activity to higher frequencies, as demonstrated by increased SEF and MEF.10 At 0.8 and 1.2 of the mean effective propofol dose to produce immobility (ED50) noxious stimulation did not evoke EEG changes.11 At 1.2 MAC isoflurane and halothane, less EEG activation occurred with noxious stimulation, as compared with the responses at 0.8 MAC. Thus, N2O is similar to propofol in that EEG activation was not consistently observed at doses that encompass those required to produce immobility. However, the SEF95 and MEF were generally lower during N2O anaesthesia in the present study as compared with those during halothane, isoflurane, and propofol anaesthesia.10 11

Nitrous oxide appears to act primarily at the N-methyl-D-aspartate (NMDA) receptor,12 although it also affects the gamma-amino butyric acid receptor.13 Nitrous oxide produces analgesia at a supraspinal site, including the noradrenergic nuclei of the brainstem, which release norepinephrine onto neurones of the dorsal horn of the spinal cord.14 15 Thus, it is possible that N2O acts on this supraspinal-spinal loop to block ascending transmission of impulses to the cortex. Neurones in the reticular formation are variably affected by N2O, with some neurones excited while others are depressed.16 17 The reticular formation is critical to consciousness and EEG generation,18 and the variable effect of N2O at these sites, and elsewhere in the CNS, might partially explain why N2O has been reported to have limited EEG effects.

We used repetitive noxious stimulation to elicit neuronal wind-up, which depends in part on the NMDA receptor system. Insofar as N2O acts primarily at the NMDA receptor,12 we speculated that the EEG response to repetitive noxious stimulation would be abolished by N2O. Because spontaneous EEG activation occurred, we believe that had the impulses been transmitted to the brain, EEG activation would have been observed. That no consistent EEG activation occurred suggests that N2O diminished transmission of nociceptive impulses to the cortex. The presence of a cardiovascular response indicates that some impulses were still transmitted to the CNS, but this likely involved spinal circuitry, as an intact brain is not needed to generate a cardiovascular response to noxious stimulation.19

Previous investigators have examined N2O effects on EEG activity and have reported mixed data. In rats, Russell and Graybeal reported that N2O produced progressive EEG depression, with a high amplitude, slow wave pattern observed at N2O pressure >1.5 atm.3 In addition, the somatosensory evoked potential was likewise depressed. In general, N2O does not appear to significantly alter EEG activity in humans. For example, Barr and colleagues8 reported no change in the BIS, which is derived from EEG activity, after administration of 0.7 atm N2O. EEG entropy was likewise unchanged by N2O.20 Some investigators, however, have reported EEG activation,5 while others have reported EEG depression.7 Collectively, these data suggest that N2O has variable EEG effects that might depend, in part, on the methods used to elicit and analyse EEG activity, and the presence of additional anaesthetics. The present data suggest that N2O and isoflurane differentially affect EEG responses to noxious stimulation and that, in the clinical setting, N2O would depress EEG activation responses to noxious surgical stimulation.

The MAC of N2O varies among species. In humans, N2O MAC is ~1 atm,1 while in rats it has been reported to be between 1.55 and 2.35 atm.3 4 In a prior study we determined that N2O MAC in rats was 1.9 atm.2 The lower MAC in the present study could be attributed to the use of older rats, which would be expected to have a lower MAC compared with younger rats used in prior studies. The results of the present study are limited because it is possible that, in younger rats, noxious stimulation might have caused EEG activation during N2O anaesthesia, even when accounting for the greater MAC.

In summary, we determined that repetitive noxious stimulation failed to significantly alter EEG activity in aged rats anaesthetized with N2O. The rats, however, had spontaneous EEG activation, suggesting that N2O was not likely depressing EEG activity directly in cortex, but might have done so at subcortical sites, including the brainstem and the spinal cord.


   Acknowledgments
 
Supported in part by NIH GM57970, 61283 and 47818. The authors thank Edmond Eger, M.D. and Michael Laster D.V.M. who assisted with the construction of the hyperbaric chamber.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Hornbein TF, Eger EI, Winter PM, et al. The minimum alveolar concentration of nitrous oxide in man. Anesth Analg 1982; 61: 553–6[Abstract/Free Full Text]

2 Antognini JF, Lewis BK, Reitan JA. Hypothermia minimally decreases nitrous oxide anesthetic requirements. Anesth Analg 1994; 79: 980–2[Abstract/Free Full Text]

3 Russell GB, Graybeal JM. Direct measurement of nitrous oxide MAC and neurologic monitoring in rats during anesthesia under hyperbaric conditions. Anesth Analg 1992; 75: 995–9[Abstract/Free Full Text]

4 Gonsowski CT, Eger EI. Nitrous oxide minimum alveolar anesthetic concentration in rats is greater than previously reported. Anesth Analg 1994; 79: 710–2[Abstract/Free Full Text]

5 Yli-Hankala A, Lindgren L, Porkkala T, et al. Nitrous oxide-mediated activation of the EEG during isoflurane anaesthesia in patients. Br J Anaesth 1993; 70: 54–7[Abstract/Free Full Text]

6 Inada T, Shingu K, Nakao S, et al. Effects of nitrous oxide on haemodynamic and electroencephalographic responses induced by tetanic electrical stimulation during propofol anaesthesia. Anaesthesia 1999; 54: 423–6[CrossRef][Web of Science][Medline]

7 Hans P, Bonhomme V, Benmansour H, et al. Effect of nitrous oxide on the bispectral index and the 95% spectral edge frequency of the electroencephalogram during surgery. Anaesthesia 2001; 56: 999–1002[CrossRef][Web of Science][Medline]

8 Barr G, Jakobsson JG, Owall A, et al. Nitrous oxide does not alter bispectral index: study with nitrous oxide as sole agent and as an adjunct to i.v. anaesthesia. Br J Anaesth 1999; 82: 827–30[Abstract/Free Full Text]

9 Sebel PS, Lang E, Rampil IJ, et al. A multicenter study of bispectral electroencephalogram analysis for monitoring anesthetic effect. Anesth Analg 1997; 84: 891–9[Abstract]

10 Barter L, Dominguez CL, Carstens E, et al. The effect of isoflurane and halothane on electroencephalographic activation elicited by repetitive noxious c-fiber stimulation. Neurosci Lett 2005; 382: 242–7[CrossRef][Web of Science][Medline]

11 Orth M, Barter L, Dominguez C, et al. Halothane and propofol differentially affect electroencephalographic responses to noxious stimulation. Br J Anaesth 2005; 95: 477–84[Abstract/Free Full Text]

12 Jevtovic-Todorovic V, Todorovic SM, Mennerick S, et al. Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat Med 1998; 4: 460–3[CrossRef][Web of Science][Medline]

13 Yamakura T, Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol. Anesthesiology 2000; 93: 1095–101[CrossRef][Web of Science][Medline]

14 Sawamura S, Kingery WS, Davies MF, et al. Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors. J Neurosci 2000; 20: 9242–51[Abstract/Free Full Text]

15 Zhang C, Davies MF, Guo TZ, et al. The analgesic action of nitrous oxide is dependent on the release of norepinephrine in the dorsal horn of the spinal cord. Anesthesiology 1999; 91: 1401–7[CrossRef][Web of Science][Medline]

16 Antognini JF, Chen XG, Sudo M, et al. Variable effects of nitrous oxide at multiple levels of the central nervous system in goats. Vet Res Commun 2001; 25: 523–38[CrossRef][Web of Science][Medline]

17 Shimoji K, Matsuki M, Shimizu H, et al. Dishabituation of mesencephalic reticular neurons by anesthetics. Anesthesiology 1977; 47: 349–52[Web of Science][Medline]

18 Steriade M. Arousal: revisiting the reticular activating system. Science 1996; 272: 225–6[CrossRef][Web of Science][Medline]

19 Gramm HJ, Zimmermann J, Meinhold H, et al. Hemodynamic responses to noxious stimuli in brain-dead organ donors. Intensive Care Med 1992; 18: 493–5[CrossRef][Web of Science][Medline]

20 Anderson RE, Jakobsson JG. Entropy of EEG during anaesthetic induction: a comparative study with propofol or nitrous oxide as sole agent. Br J Anaesth 2004; 92: 167–70[Abstract/Free Full Text]


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