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Anesth Analg 1999;89:1035
© 1999 International Anesthesia Research Society

GENERAL ARTICLES

Halothane Minimum Alveolar Anesthetic Concentration and Neuronal Nitric Oxide Synthase Activity of the Dorsal Horn and the Locus Ceruleus in Rats

Taeko Fukuda, MD, PhD*, Shigeyuki Saito, MD, PhD*, Shigehito Sato, MD, PhD{dagger}, Izumi Harukuni, MD*, and Hidenori Toyooka, MD, PhD*

*Department of Anesthesiology, Institute of Clinical Medicine, Tsukuba University, Tsukuba-city, Ibaraki; and {dagger}Department of Anesthesiology and Intensive Care, Hamamatsu University School of Medicine, Hamamatsu-city, Sizuoka, Japan

Address correspondence and reprint requests to Taeko Fukuda, MD, PhD, Department of Anesthesiology, Institute of Clinical Medicine, Tsukuba University, Tsukuba-city, Ibaraki, 305-8575, Japan. Address e-mail to taekof{at}md.tsukuba.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
There is some evidence of a relationship between nitric oxide and pain control pathways. However, it is still controversial whether nitric oxide synthase (NOS) inhibitors affect minimum alveolar anesthetic concentration (MAC). We examined the effects of 7-nitro indazole (7-NI), a selective neuronal NOS (nNOS) inhibitor, on halothane MAC. With nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, we also investigated the nNOS activity of the dorsal horn and the locus ceruleus in 26 Sprague-Dawley rats. 7-NI (100, 500, 1000 mg/kg intraperitoneally) reduced halothane MAC to 0.34% ± 0.12%, 0.1% ± 0.03%, and 0.05% ± 0.12%, dose dependently (P < 0.01). 7-NI also reduced the number of nicotinamide adenine dinucleotide phosphate diaphorase-positive cells by 20% to 65% (P < 0.05 or 0.01) and the staining intensity of the axons in the locus ceruleus and lumbar and thoracic spinal cord as compared with the control group. 7-NI reduced the MAC observed with halothane anesthesia, which was accompanied by nNOS activity suppression in the spinal cord and the locus ceruleus. Our results support the hypothesis that the nitric oxide signaling pathway is related to MAC.

Implications: We examined the effects of a selective neuronal nitric oxide synthase inhibitor, 7-nitro indazole, on halothane minimum alveolar anesthetic concentration and measured the nitric oxide synthase activity in the spinal cord and the locus ceruleus of Sprague-Dawley rats using nicotinamide adenine dinucleotide phosphate diaphorase staining method. 7-Nitro indazole decreased both the minimum alveolar anesthetic concentration and neuronal nitric oxide synthase activity.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) apparently is involved in nociception and the maintenance of wakefulness (1–3). However, there continues to be disagreement as to whether the NO signaling pathway is related to minimum alveolar anesthetic concentrations (MAC) in the use of inhaled anesthetics. Nitric oxide synthase (NOS) inhibitors, nitroG-L-arginine methyl ester (L-NAME) and 7-nitro indazole (7-NI), were reported to reduce the threshold for halothane or isoflurane anesthesia (4,5). These two structurally distinct NOS inhibitors dose-dependently reduced MAC; MAC reduction was reversed by L-arginine. These results suggest that the NO signaling pathway exerts an influence on MAC. On the other hand, L-NAME reduced cerebellar NOS activity and cyclic guanosine monophosphate, but it did not modify MAC (6,7). Furthermore, the targeted disruption of the neuronal NOS (nNOS) gene did not modify isoflurane MAC (8). A dissociation between sevoflurane MAC and cyclic guanosine monophosphate was observed during chronic treatment with 7-NI (9). These results suggest either the existence of unknown compensatory mechanisms or that the relationship between MAC and NO activity is complicated, and therefore warrants further investigation.

Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining has contributed to many studies of NOS, especially as an alternative to the use of antibodies and antisense in in situ probes. This staining technique depends on the ability of the C-terminal portion of NOS to transfer electrons from NADPH to nitroblue tetrazolium (NBT), thus reducing the substrate NBT to an insoluble purple formazan product that produces the characteristic diaphorase reaction (10). Therefore, this technique reflects NOS activity quite easily. In the central nervous system, NADPH diaphorase and nNOS are identical after tissue is exposed to 4% paraformaldehyde fixative (11). For example, after salt treating the rats to stimulate vasopressin secretion, there is a substantial upregulation of nNOS immunoreactivity, NADPH diaphorase staining, and there is a parallel increase in nNOS mRNA (12). Thus, NADPH diaphorase histochemistry permits analysis of the nNOS activity in a small area or in nuclei (13,14).

The mechanism and site at which general anesthetics modulate somatic unresponsiveness remain unknown. However, two studies have demonstrated that it is not the forebrain but rather the brainstem and spinal cord that represent the important sites of the action of inhaled anesthetics in blocking motor responses (15,16). We examined the effects of 7-NI, a selective nNOS inhibitor, on halothane MAC, and measured the nNOS activity in the spinal cord and the locus ceruleus of Sprague-Dawley rats using NADPH diaphorase staining.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This experiment was reviewed and approved by the Animal Care and Use Committee of Tsukuba University. Twenty-eight male Sprague-Dawley rats (300–350 g) were divided into a control group and three 7-NI groups (100, 500, and 1000 mg/kg). 7-NI and a vehicle (arachis oil) were obtained from the Sigma Chemical Company (Sigma-Aldrich Japan, Tokyo, Japan).

Rats were kept in a plexiglass chamber, spontaneously breathing a mixture of halothane in air. The halothane concentration was monitored continuously by a Capnomac Ultima (Datex, Helsinki, Finland). The control MAC was established according to methods described by Quasha et al. (17). A pair of 15-cm hemostat forceps was clamped to the first ratchet lock on the tail for 1 min. The tail was always stimulated proximal to a previous test site. Gross movements of the head, extremities, and/or body were taken as a positive sign, whereas grimacing, swallowing, chewing, or tail flicking were considered negative. Halothane concentrations were reduced in decrements of 0.2% until the negative responses became positive; 12- to 15-min of equilibration were allowed after changes were made in concentrations. The MAC was considered to be midway between the highest concentration that permitted movement in response to stimulation and the lowest concentration that prevented movement. After measurement, the rats were allowed more than 4 h to recover in open air and were then administered 7-NI (100, 500, 1000 mg/kg) or the vehicle (arachis oil, 0.1–0.15 mL/10 g) intraperitoneally. Thirty minutes later, the MAC was determined once again with the same method described above.

After the second MAC measurement, the rats were anesthetized with pentobarbital (100 mg/kg) and perfused transcardially through the ascending aorta with phosphate buffered saline, followed by 500 mL of 4% paraformaldehyde in 0.1 M of phosphate buffer (pH 7.4). After 2 h postfixation, the brain and spinal cord (thoracic 6–7 and lumbar 5–6 regions) were cut in 40-µm slices with a microtome. All sections of the locus ceruleus nuclei and five sections of the lumbar or thoracic spinal cord were used, and these sections were cut according to an atlas (18). The free-floating sections were incubated in 0.1 M of phosphate buffer (pH 7.4) containing 0.2% Triton X-100, 0.1 mg/mL NBT, and 1.0 mg/mL ß-NADPH at 37°C for exactly 30 min (19). After the reaction, sections were rinsed in phosphate buffer (pH 7.4) and mounted onto slides.

We numbered all the slides in random order, and the number of NADPH diaphorase positive cells was counted by one researcher who did not know the ordering. Positive neurons were scored when they contained cytoplasmic and dendritic staining and a nucleus. The data were analyzed with a one-way analysis of variance. All data are presented as mean ± SD. A P value of <0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Two rats were excluded from the 500 and 1000 mg/kg groups because of an intrabowel injection of 7-NI.

There was no significant difference in the preinjection value for halothane MAC among the four groups. Arachis oil had no effect on halothane MAC in the control group. The different dosages (100 and 500 mg/kg) of 7-NI elicited a dose-dependent decrease of the halothane MAC from 0.76 ± 0.08 to 0.1 ± 0.03 (P < 0.01). However, no further decrease was seen in the largest dose (1000 mg/kg) of the 7-NI group (Table 1).


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  		Table 1. The Effect of 7-Nitro Indazole on Halothane MAC
 
The 7-NI reduced the number of stained cells in the locus ceruleus and in the spinal cord (P < 0.05 or 0.01), with the exception of the thoracic I-III laminae area in the 100 mg/kg of the 7-NI group. The magnitude of the decrements was 20%–65%; the deeper laminae (IV-V) of the spinal cord was especially prominent. Although the halothane MAC decreased dose dependently, there was no significant difference in the number of NADPH diaphorase histochemistry stained cells among the 7-NI groups (Table 2).


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  		Table 2. The Effect of 7-Nitro Indazole on NADPH Diaphorase Histochemistry Stained Cell Numbers in the LC and the Dorsal Horn
 
In the 7-NI groups, decreases in nNOS activity were demonstrated by reductions in the number of stained cells and also by the lower intensity of stains observed in the axons surrounding blood vessels (Fig. 1, arrows).



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  		Figure 1. Nicotinamide adenine dinucleotide phosphate diaphorase histochemical staining in the locus ceruleus (A,B) and the dorsal horn Lumbar (C,D) and thoracic (E,F) spinal cord. A, C, and E sections are the control group; B, D, and E sections are the 7-nitro indazole group (100 mg/kg). Note stained cells and axons decreased in the 7-nitro indazole group. The arrows show stained axons surrounding blood vessels. LC = locus ceruleus, LDTg = laterodorsal tegmental nuclei. Scale bar = 400 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The present study showed that 7-NI reduced halothane MAC dose dependently in rats. Our observations are in accordance with those made by other investigators (4,5). However, there is no universal agreement yet regarding the effects of 7-NI on MAC (6,7). The discrepancies may be attributed to technical differences; for example, there may have been differences in the specificity of the NOS inhibitors, doses, routes of administration, or the condition of MAC measurements. It might also be relevant that NO plays various roles at several sites of pain and pain-control pathways—peripheral, spinal, and supraspinal. NO evokes pain on intracutaneous application; apparently, it does so by exciting the cutaneous nociceptors (20). Depending on the tissue level, peripheral NO also plays a dual role in pain modulation, inducing either nociceptive or antinociceptive responses (21). NO facilitates the perception of nociceptive inputs at the spinal level and enhances the descending inhibition on the spinal nociception (22). The supraspinal application of NO exerts a dual action on thermal nociception in mice (23). In summary, NO apparently either evokes or facilitates pain; however, it induces antinociceptive responses under some conditions.

We selected the dorsal horn of the spinal cord and the locus ceruleus as regions of staining study. In the spinal cord, nNOS is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but it appears to be absent in motoneurons (24,25). Thus, we did not study the ventral horn of the spinal cord. On the other hand, the locus ceruleus forms a part of the descending inhibitory system, and includes nNOS positive cells (26). Recently, Xu et al. (27) reported that NOS inhibitors enhanced the excitatory postsynaptic potential evoked by focal electrical stimulation in the locus ceruleus of brain slices. It is possible that NOS inhibitors increase activity of the locus ceruleus and decrease MAC.

In the present study, the reduction of MAC by 7-NI was striking (maximal effect: 90%–95%). Previous studies showed about a 40% reduction of MAC at 1000 mg/kg of 7-NI (5,9); therefore, we did not anticipate this result. One reason for this result may be the volume of arachis oil used for solving 7-NI. Because we used two to four times the volume of arachis oil, the absorption rate of 7-NI might be higher than in other studies. However, that fact alone may not be the complete explanation. There is, as yet, no satisfactory explanation for this large reduction of MAC. In the present study, 7-NI reduced nNOS activity in the locus ceruleus and spinal cord, but the dose dependency was not clear. Our method of counting cells may not be appropriate for a quantitative analysis, because slight differences of intensity are hard to distinguish. However, cell counting is a popular technique in histochemical studies, and the staining method permits us to evaluate nNOS activity in a small area or in nuclei without destroying the structure of the brain. Therefore, we believe our method is useful. This result may also be affected by that fact that nNOS activity is influenced by many factors, i.e., the density of nNOS positive cells, their basic activity, nearby neurons and synapses, and the existence of scavengers. In a nNOS rich area, some NO may play a role in blocking the scavenger activity and accelerating the rest of the NO activity. If this is the case, a sigmoid curve, not a linear relationship, between nNOS activity and its effects might be observed in those areas. Even if nNOS activity decreases slightly, its effect on MAC may be greater than that suggested by the figures.

An additional effect of 7-NI that we observed was a decrease in the staining intensity of the axons surrounding blood vessels. Because we fixed the tissue with 4% paraform-aldehyde, endothelial NOS did not exhibit NADPH diaphorase reactivity (11). NOS neurons lie near branching points of the arteriole, and these neurons are thought to control regional blood flow on anatomical grounds (28). Cerebral blood flow (CBF) is regulated predominantly by endothelial NOS activity. However, nNOS also contributes partially to CBF regulation. For example, administration of 7-NI (80 mg/kg) reduced cortical brain NOS activity by 57%, the resting CBF by 19% to 27%, and the CBF response to hypercapnia by 60% (29). Another investigator reported that 7-NI (25 and 50 mg/kg) resulted in a decrease in CBF of between 20% and 60% (30). We speculate that these changes of CBF or CBF control ability may be related to a reduction in MAC. In this regard, much remains to be investigated.

In conclusion, the nNOS selective inhibitor 7-NI decreased halothane MAC and nNOS activity in the locus ceruleus and dorsal horn regions of Sprague-Dawley rats. It is likely that the NO signaling pathway is related to MAC observed with inhaled anesthetics.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication June 17, 1999.




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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