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Anesth Analg 2000;91:6-10
© 2000 International Anesthesia Research Society

PEDIATRIC ANESTHESIA

Nitrous Oxide Lacks the Antinociceptive Effect on the Tail Flick Test in Newborn Rats

Masahiko Fujinaga, MD*,{dagger},{ddagger}, Ryan Doone*,{dagger}, M. Frances Davies, PhD*,{dagger}, and Mervyn Maze, MB, ChB, FRCP, FRCA*,{dagger},{ddagger}

*Department of Anesthesia, Stanford University School of Medicine, Stanford; {dagger}Anesthesiology Service, V.A. Palo Alto Health Care System, Palo Alto, California; and {ddagger}Magill Department of Anaesthetics, Chelsea and Westminster Campus, Imperial College of Science, Technology and Medicine, University of London, London, United Kingdom

Address correspondence and reprint requests to Masahiko Fujinaga, MD, Magill Department of Anaesthetics, Chelsea and Westminster Hospital, 369 Fulham Rd., London, SW10 9NH, UK. Address e-mail to m.fujinaga{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Nitrous oxide (N2O) is commonly used for pediatric anesthesia under the assumption that it produces a similar analgesic response to that seen in adults. We examined the antinociceptive effect of 75% N2O on tail flick latency response in newborn rats at postnatal Day 1 (PD 1), PD 8, PD 15, PD 22, and PD 29. Up to PD 15, rats showed no analgesic effect to N2O. By PD 29, rats exhibited a comparable analgesic effect to that seen in adult animals. These data are consistent with the fact that the descending noradrenergic neurons, which are required for the analgesic action of N2O, are not anatomically or functionally developed at birth and take more than three weeks to fully develop in rats.

Implications: The present study indicates that rats below 3 wk old lack an antinociceptive effect to nitrous oxide by using the tail flick test. Because a 3-wk-old rat is comparable in neurological development with the toddler stage in humans, we may anticipate that patients below this age may not experience the usual analgesic effect of nitrous oxide.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Without a clear understanding of its underlying mechanisms or its efficacy as an analgesic, nitrous oxide (N2O) is often used during pediatric anesthesia and analgesia with the assumption that it produces a similar analgesic response to that in adults. For the anesthetic response, several studies have shown the minimum alveolar anesthetic concentration-reducing potential of N2O in infants and children (1–5). However, the anesthetic and analgesic response may be mediated and/or modulated by different mechanisms. Recently, we and others (6–9) have shown that a descending noradrenergic pathway is pivotally required to transduce the analgesic response to N2O as reflected by the tail flick latency response. Yet, it is apparent that these neuronal circuits are incompletely developed in newborn rats (10,11); therefore, one may predict that N2O may not be effective and would be an unsuitable analgesic for this age group. As no information regarding this subject exists, we have tested the analgesic effect of N2O in newborn rats at various stages of development, using the tail flick latency (TFL) response.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study protocol was approved by the institutional animal investigation committee (Stanford University and V.A. Palo Alto Health Care System). Sprague-Dawley rats were used for the study (B&K Universal, Inc., Fremont, CA). A total of five timed-pregnant rats were obtained from the breeder 3 days before the expected delivery date. Delivery date and time were confirmed, and the day of delivery was defined as postnatal day 0 (PD 0). Newborns from a single litter were divided into treatment (N2O) and control groups, and experiments for treatment group were conducted on PD 1, PD 8, PD 15, PD 22, and PD 29 (Table 1). To conduct experiments at the same time of the day for each group (i.e., between 10 AM and 1 PM), experiments for the control group were conducted on the next days after the experiment for the N2O group at each developmental stage. In addition, adult male rats (300–400 g) were examined as comparison by using the same experimental protocol as described.


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  		Table 1. Summary of Data from Experimental Groups at Different Developmental Stages
 
Gas exposure was performed in a plexiglas chamber (20 inches long, 35 inches wide, and 15 inches high) with a sliding door on one side (for insertion of animals). A mixture of N2O and oxygen (O2) gas was continuously delivered from an anesthetic machine into the chamber via an inflow port and circulated throughout the chamber by two small fans. Gas flow rate was 10 L/min. Gas concentrations—including those for N2O, O2, and carbon dioxide (CO2)—in the chamber were measured continuously by infrared gas spectrometry (Datex 254 airway monitor; Datex Medical Instrument, Tewksbury, MA) and recorded on a strip chart recorder. N2O concentration was maintained between 70% and 75%. We selected this concentration range because we wanted to administer the maximum possible concentration of N2O under ambient pressure to detect a change in efficacy, if it were present. O2 concentration was maintained between 20% and 25%. (CO2 concentration was always <0.1% during the experiments.) Newborn rats at different stages of development were exposed to either 75% N2O/25% O2 (N2O group) or room air (control group), and each rat was tested in triplicate for their TFL every 30 min for 3 h; the mean of three measurements was used as a result. Adult male rats were examined in the same fashion with three animals per each experiment because of the limitation of chamber size.

We used the tail flick test because we wanted to unambiguously measure nociception and not have it confounded by any sedative action of N2O, which is a centrally active anesthetic. Tail-Flick Analgesia Meter (Columbus Instruments, Columbus, OH) was used for the tail flick tests within the chamber. A beam of radiant heat (a single-fixed aperture) was focused on the underside of the tail on the middle third of the rat’s tail. The latency between the exposure to the radiant heat source and the movement of the tail away from the focused beam was referred to as TFL. Intensity of heat was determined by preliminary experiment so that basal TFL fits between 3 and 4 s for each developmental stage, and the time of maximal exposure to the radiant heat stimulus was determined to be 10 s ("cut-off time"). Data were expressed as the percentage of maximal possible effect (%MPE) as follows (12): Go


Newborns were tested without restrainers, whereas adult animals were kept within the restrainers during the gas exposure and during the tail flick test.

Data within each group were analyzed on raw data by using one-way analysis of variance with repeated measurements, and post hoc significance was assessed by using the paired t-test. Data between control and N2O groups at each age group were analyzed on %MPE data by using one-way analysis of variance. A P value <0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The unprocessed data from tail flick tests are shown in Table 2. TFL was significantly increased when compared with baseline TFL after N2O exposure only in PD 22 (t = 3.95, df = 4; P < 0.0169), PD 29 (t = 9.83, df = 5; P < 0.0002), and adult (t = 22.28, df = 4; P < 0.0001) cohorts. Figure 1 shows %MPE data after 30 min of N2O exposure. Again, the processed data reveal that N2O showed no effect on the TFL test before PD 22. (The result on PD 22 was not statistically significant on this analysis.) At PD 29, rats exhibited a similar response to N2O as was seen in adult rats. At this age, animals also developed tolerance to N2O after 60 min (Fig. 2A) as was seen in adult animals (Fig. 2B).


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  		Table 2. Raw Data from Tail Flick Tests at Different Developmental Stages
 


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  		Figure 1. Effects of 75% nitrous oxide (N2O) (after 30 min of exposure) on tail flick test at different ages indicated by maximal possible effect (%MPE) (mean ± SD, n = 3–7). *P < 0.05 versus control.

 


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  		Figure 2. Time course of the effects of 75% N2O on tail flick test at 29-day-old newborn and adult rats indicated by maximal possible effect (%MPE) (mean ± SD, n = 5–6).

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Nociceptive stimulation is recognized as pain when a signal received at sensory receptors (e.g., in the skin) reaches the sensory area of the cerebral cortex. The initial signal is mediated by nociceptive afferent nerves that synapse on spinothalamic tract neurons located in the dorsal horn of the spinal cord. Processing of nociceptive information is modulated by various descending inhibitory neurons. For example, activation of descending noradrenergic inhibitory neurons (13,14) results in suppression of activity in the spinothalamic tract neurons through stimulation of {alpha}2-adrenoceptors in the dorsal horn of the spinal cord (15), thereby decreasing nociception. The noradrenergic descending inhibitory neurons are also modulated by stimulation of opiate receptors in the periaqueductal gray area and {alpha}2-adrenoceptors in the locus coeruleus of the brain stem (16,17).

Recent studies have suggested that N2O exerts its analgesic properties by activating descending noradrenergic inhibitory pathways. N2O induces release of endogenous opioid peptides in the periaqueductal gray area of the midbrain (6), which in turn leads to stimulation of noradrenergic descending inhibitory neurons (7). We reported that systemic administration of opiate receptor antagonists and {alpha}2-adrenoceptor antagonist both blocked the analgesic effect of N2O on the tail (8). Whereas intrathecal injection of {alpha}2-adrenoceptor antagonist blocked the analgesia, intrathecal opiate receptor antagonists were without effect. Subsequently, we showed that bilateral microinjection of opiate receptor antagonist but not {alpha}2-adrenoceptor antagonist into the ventrolateral periaquedactal gray blocked the analgesic effect of N2O (9). These data firmly support the conclusion that N2O produces supraspinal opiate receptor stimulation, which activates a descending noradrenergic pathway, which modulates nociceptive processing via {alpha}2-adrenoceptors in the dorsal horn of the spinal cord.

Descending noradrenergic inhibitory neurons are not anatomically (10,18) or functionally (11,19,20) developed at birth and take at least three weeks to fully develop in rats. Thus, our finding that N2O is without analgesic effect appears to be due to the lack of the appropriate modulatory pathways required for nociceptive processing. Although the sequence of events that take place in neurological development in rat and human central nervous systems are not totally comparable, the rat is the most accepted animal model for development of the central nervous system at the present time. Investigators who are familiar with both rat and human development estimate that the central nervous system of the newborn rat at birth is equivalent to that of a human fetus at 24 weeks of gestation (21), whereas at PD 7 in rats, it becomes equivalent to the full-term neonate in humans. At three weeks after birth in rats, it becomes equivalent to that of toddler stage. Thus, our results suggest that N2O may not be an effective analgesic at least until toddler stage in humans.

Attractive as our hypothesis may be, we are not able to discount the possibility that a technical artefact may have been introduced inadvertently in the testing of younger animals. Because a fixed aperture device was used, a proportionally larger area of the tail was exposed to the noxious heat stimulus than was evident in the older animals. To compensate for this problem, the intensity of the heat was reduced in younger rats, which may have resulted in similar basal TFL values. Also, there may be local differences in the tail at the various stages of development which may have contributed to the lack of an antinociceptive response to N2O. Conway et al. (22) reported differences in the radiant heat absorption properties of the tail during development. Again, because the intensity of the heat was reduced in younger rats, such differences should have been compensated in our study. Another concern is a possible gender difference. Although only male animals were tested for adults, both genders were used for newborn animals. We used mixed genders because we wanted to use newborns from the same litter for control and N2O cohorts. Conway et al. (22) also demonstrated no gender differences in response to thermal stimulation at any age groups from newborns to adults. Since no gender difference is reported for the antinociceptive effect of N2O, a concern of gender differences in our study is minimized.

As previously reported in animals (23,24) and in humans (25,26), the analgesic response to N2O diminishes during continuous administration, a phenomenon referred to as "tolerance." Similar development of "tolerance" is also reported on other variables [e.g., electroencephalogram activity in cats (27) and anticonvulsant action in cats (28)]. Although the underlying mechanism for development of tolerance is not well understood, it is suggested that acute depletion of opiate peptides in the central nervous system may be the cause (29). The results from our study clearly indicate that, at the time when N2O begins to show an analgesic effect, the underlying mechanism for development of tolerance to N2O has already been established.

If the analgesic properties of N2O are lacking in newborns and infants, then the administration of this drug for its analgesic properties should be avoided at this juncture in the patient’s life because of the potential for adverse events. For example, N2O may cause gas-filled cavities (such as pneumothorax or abdominal obstruction) to expand further, thus compromising ventilation. Also, N2O is associated with diffusion hypoxia on emergence from anesthesia. Furthermore, in many settings, N2O is coadministered with a potent volatile anesthetic since the dose of the latter can be safely reduced while maintaining a satisfactory depth of anesthesia. The analgesic components of the "anesthetic–sparing" action of N2O may be absent in newborns and infants.

In summary, we have demonstrated that N2O lacks an antinociceptive effect on tail flick test in newborn rats. Our results suggest that N2O may not be an effective analgesic at least until toddler stage in humans.


   Acknowledgments
 
This study was supported by the Veterans Administration and National Institute of Health (Grant GM 30232).


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication November 4, 1999.





This Article
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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press