Nitric oxide (NO) has an important role as a neural messenger in the central and peripheral nervous system. It binds to guanyl cyclase, which catalyses the production of cyclic 3′,5′-guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) (the NO-cGMP pathway). However, the effect of NO synthase (NOS) inhibition on anaesthesia remains unclear. In previous reports, alterations in the minimum alveolar concentration (MAC) and/or the righting reflex ED50 of volatile agents were investigated after administration of non-selective NOS inhibitors [1-4]. MAC is used to measure the degree of analgesia produced by an anaesthetic and the righting reflex measures the effectiveness of an anaesthetic (degree of hypnosis produced). However, non-selective NOS inhibitors might also affect the systemic circulation, metabolism and the functions of many internal organs. Through these systemic effects, non-selective NOS inhibitors might further influence changes in MAC or the righting reflex. Recently, the effect of acute and chronic administration of the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI), on MAC has been described . However, comparisons have not been made between alterations in MAC and the righting reflex after acute and chronic neuronal NOS inhibition. Moreover, changes in the righting reflex have never before been measured after acute and chronic administration of a selective neuronal NOS inhibitor. Our hypothesis was that 7-NI might affect the righting reflex differently than it does MAC. To test the hypothesis, we determined the effect of the neuronal NOS inhibitor 7-NI on the righting reflex and MAC during sevoflurane anaesthesia in rats following acute and chronic administration and further to determine if there is a correlation between alterations in MAC and the righting reflex. We also measured cGMP concentrations within the brain, cerebellum and spinal cord of rats after acute treatment with 7-NI.
This study was approved by the Animal Care Committee of the Hamamatsu University School of Medicine. Thirty-two male Sprague-Dawley rats weighing 250-350 g (Japan SLC) were used. Twenty rats were randomly divided into two groups. Rats in Groups 1 (n = 10) and 2 (n = 10) were assigned to the acute and chronic protocol groups, respectively. MAC and the righting reflex were measured in both groups. The remaining 12 rats were used to measure cGMP concentrations in the brain, cerebellum and spinal cord before and after acute administration of 7-NI (100 mg kg−1). The studies were conducted in random order.
Ten rats were assigned to the acute protocol group. Each rat served as its own control. Three doses of 7-NI (0, 50 and 100 mg kg−1) were administered intraperitoneally to 10 rats. This study was conducted in random order. At least 1 week elapsed between successive studies in all rats. Each dose of 7-NI was suspended in peanut oil and 2 mL kg−1 of the suspension was administered intraperitoneally to the rats. Peanut oil (2 mL kg−1) without 7-NI was administered in the control study. After 60 min of administration, the righting reflex and MAC were determined.
We administered 0 and 75 mg kg−1 7-NI in 2 mL kg−1 peanut oil to 10 rats by gastric tube feeding every 12 h for 4 consecutive days. Each rat served as its own control. At least 1 week elapsed between successive studies in all rats. The righting reflex and MAC determinations were made 1 h after the last feeding on day 4.
Determination of righting reflex
Each rat was placed in an individual mesh cylinder cage that rotated at 4 revolutions min−1 in a 40 L plastic chamber. The chamber temperature was adjusted using a heater. Fresh gas containing a predetermined concentration of sevoflurane, 40% oxygen and balanced nitrogen was delivered to the chamber at 3 L min−1. Sevoflurane and oxygen concentrations were measured using a multigas analyser (Datex Capnomac Ultima®; Helsinki, Finland). We used the sevoflurane concentration data from the serial output port of the gas analyser, which has a resolution of 0.01 vol.%. For the detection of the righting reflex, rats that rolled over twice during five complete turns of the rotator failed the test and were considered anaesthetized . Initially, rats were examined for the righting reflex without sevoflurane; then they were anaesthetized with sevoflurane 0.15%. After a 30 min equilibration, the righting reflex was examined again. Starting from this point, the sevoflurane concentration was increased in steps of 0.15-0.20% and re-equilibration allowed for 30 min, after which rats were re-examined for their ability to right themselves. The concentration of sevoflurane was raised periodically in this manner until it was high enough for the rats to fail the righting reflex test. Likewise, it was also lowered in this manner until a sufficiently low anaesthetic concentration was attained for the rats to right themselves. Righting reflexes were calculated for each rat as the mean of these two anaesthetic concentrations (the concentrations required to pass and fail the righting reflex test).
Determination of MAC
A rat was placed in a 3 L plastic chamber. The chamber temperature was adjusted using a heater. The rectal temperature was maintained between 36.5 and 38.0°C. Fresh gas containing a predetermined concentration of sevoflurane, 40% oxygen, and balance nitrogen was delivered to the chamber at 0.6 L min−1. Sevoflurane and oxygen concentrations were measured as described for the determination of MAC. The initial concentration of sevoflurane was 0.80 and 1.80% in acute and chronic protocols, respectively, after a 30 min equilibration, a clamp was applied to the tail for 1 min and the rat observed for movement in response to stimulation. In every case, the tail was stimulated proximally to the previous test site. Only the middle third of the tail was used for clamping. Gross movements of the head, extremities and/or body were considered positive responses. The concentration of the sevoflurane was increased or decreased in increments of 0.15-0.20% from the initial concentration, after which the rats were re-examined by tail-clamping after a 30 min equilibration. The concentration of sevoflurane was continually adjusted in this manner until a sufficiently high anaesthetic concentration was attained whereby the rats gave a negative response and then until the concentration was sufficiently low for the rats to give a positive response. MAC was calculated for each rat as the mean of these two anaesthetic concentrations (the concentrations that permitted and prevented a positive response) .
To exclude the potential for hypoxia, hypercapnia and acidosis during the study, we sampled arterial blood from four rats during the acute protocol and at the conclusion of the MAC experiments by percutaneous left ventricular puncture. Blood samples were immediately analysed (Radiometer ABL® 50 and OSM® 3; Copenhagen, Denmark).
Assay of cGMP
Twelve rats were randomly divided into two groups (n = 6). In Group 1, the rats were given peanut oil (2 mL kg−1) without 7-NI to provide controls. Rats in Group 2 were given 7-NI (100 mg kg−1). The 7-NI was suspended in peanut oil and administered intraperitoneally to the rats at 2 mL kg−1. These studies were conducted in random order. One hour after drug administration, the rats were killed by dislocation of the cervical vertebrae. They were then perfused via the left ventricle with buffered solution for dissection. The solution used for dissection consisted of the following (mmol L−1): Na+ (144.44), K+ (4.10), Cl− (135.02), Ca2+ (1.01), Mg2+ (1.19), PO2−4 (1.54), SO2−4 (1.19), HEPES (24.90), glucose (10.0) and a phosphodiesterase inhibitor, isobutylmethyl-xanthine (IBMX) (0.10). The solution was aerated with 100% oxygen, and the pH of the solution was maintained at 7.40. The brain, cerebellum and spinal cord were immediately removed and rapidly frozen in liquid nitrogen. They were respectively minced in 6.0% trichloroacetic acid solution. After respective sonication of the tissue samples six times for 20 s, they were ultracentrifuged at 2000 g and 4°C for 15 min. The supernatant of each sample was extracted with water-saturated dimethyl ether four times and then lyophilized for 24 h. The concentration of cGMP in the brain, cerebellum and spinal cord was measured by an enzyme immunoassay system for cGMP (Amersham Laboratories, Amersham, UK). Each sample was dissolved in 2.0 mL of the assay buffer solution provided by the assay system. Of each sample, 1 mL was acetylated with 33.3% triethylamine. Aliquots (50 μL) of the standard cGMP sample provided in the assay system and 50 μL aliquots of the collected samples were incubated with 100 μL rabbit anti-cGMP serum in an anti-rabbit IgG-coated microtitre plate for 2 h at 4°C. Thereafter, 100 μL peroxidase conjugate was added to each well and the plate incubated for 1 h at 4°C. After each well was washed with washing solution, 200 μL tetramethylbenzidine was added to each well as the substrate. The microtitre plate was incubated for 30 min at room temperature and the reaction was then stopped by adding 100 μL 1.0 mol L−1 sulphuric acid. The absorbance of each well was read at 450 nm in a microtitre spectrophotometer. To compute the concentration of cGMP within each sample, a standard curve was used, which was obtained from the standard cGMP solution provided in this system. The concentration of protein in each sample was measured with a protein assay system (Bio-Rad, Hercules, CA, USA). The concentration of cGMP was expressed as pmol μg protein. The working range of the cGMP assay system was 2-512 fmol/well and that of the protein assay system was 1-1500 μg mL−1. The standard curve of the cGMP assay system (plotted on semilog area) and that of the protein assay system were both linear over the concentrations measured. The combination of the cGMP and the protein assay system had a lower limit of detection of 0.1 pmol mg−1 protein and a coefficient of variation of <5%.
All values are the mean ± SD. Data were analysed using the paired t-test and ANOVA followed by Bonferroni's multiple comparison tests. P < 0.05 was taken as being statistically significant.
The pre-experimental values of sevoflurane righting reflex and MAC were 1.09 ± 0.07 and 2.34 ± 0.19 (vol.%), respectively, in the acute protocol group, and 1.04 ± 0.15 and 2.31 ± 0.20 (vol.%), respectively, in the chronic protocol group. No significant differences were observed between the acute and chronic protocol groups with regard to the righting reflex and MAC.
There was no apparent hypoxia, hypercapnia or acidosis in the rats. Blood-gas analysis data were: pHa 7.44 ± 0.02, PaO2 15.2 ± 0.64 (kPa), PaCO2 5.5 ± 0.33 (kPa), HCO3 26.9 ± 0.94 (mmol L−1) and BE 2.65 ± 0.89 (mmol L−1).
Following acute and chronic peanut oil administration, the righting reflex was 1.03 ± 0.09 and 0.98 ± 0.09, respectively. Peanut oil had no effect on the righting reflex in either the acute or chronic protocol groups. We used the righting reflexes attained after acute and chronic peanut oil administration as our baseline values. Before sevoflurane anaesthesia, no rat acutely or chronically treated with 7-NI showed loss of the righting reflex. Acute administration of 7-NI was observed to reduce the righting reflex in a dose-dependent manner. The righting reflex fell from 1.03 ± 0.09 to 0.28 ± 0.14 vol.% (27.2 ± 14.1% of the baseline value, P < 0.01) and 0.14 ± 0.10 vol.% (14.3 ± 10.7% of the baseline value, P < 0.01) after administration of 50 and 100 mg kg−1, respectively. A significant difference was observed among the righting reflexes attained after administration of 7-NI in 50 and 100 mg kg−1 amounts (P < 0.05). Chronic administration of 7-NI also reduced the righting reflex from 0.98 ± 0.09 to 0.64 ± 0.15 vol.% (65.3 ± 17.3% of the baseline value, P < 0.01) (Fig. 1).
After acute and chronic administration of peanut oil, MAC was 2.40 ± 0.14 and 2.28 ± 0.18, respectively. Peanut oil had no effect on MAC in either the acute or chronic protocol groups. Therefore, we used the MAC attained after acute and chronic peanut oil administration as our baseline values.
Acute administration of 7-NI reduced MAC from 2.40 ± 0.14 to 1.41 ± 0.26 vol.% (58.8 ± 11.9% of the baseline value, P < 0.01) and 1.34 ± 0.29 vol.% (55.8 ± 12.4% of the baseline value, P < 0.01) after administration of 50 and 100 mg kg−1, respectively. No significant differences were observed among the MAC attained after administration of 50 and 100 mg kg−1(Fig. 2). Chronic administration of 7-NI did not reduce MAC (2.31 ± 0.18 vol.%; 101.3 ± 4.8% of the baseline value) (Fig. 2).
Relations between the magnitude of decline in MAC and that in the righting reflex after acute and chronic administration of 7-NI are shown in Figure 3. In the acute protocol, a decrease of MAC was not correlated with that of the righting reflex. In the chronic protocol, because the MAC did not change after the administration of 7-NI, a reduction of MAC was not correlated with that of righting reflex.
cGMP concentrations in the brain, cerebellum and spinal cord of the control rats were 7.2 ± 2.1, 44.9 ± 14.5 and 8.5 ± 3.8 pmol mg−1 protein, respectively. cGMP concentrations in the brain, cerebellum and spinal cord of rats treated with 7-NI 100 mg kg−1 were 6.8 ± 2.1, 20.0 ± 9.2 and 6.6 ± 4.4 pmol mg−1 protein, respectively. Acutely administrated 7-NI significantly reduced cGMP concentrations in the cerebellum (to 44.6% of the control group, P < 0.01) (Fig. 4).
Our results show that in rats: (1) acute intraperitoneal administration of 7-NI decreases the MAC and the righting reflex of sevoflurane, (2) 4-day-long gastric tube feeding with 7-NI decreases the righting reflex of sevoflurane, but does not alter the MAC of sevoflurane, (3) the reduction in MAC following acute and chronic administration of 7-NI is not correlated with that of righting reflex, and (4) acute intraperitoneal administration of 7-NI decreases the concentration of cGMP in the cerebellum; however, it does not alter cGMP concentrations in the brain and spinal cord. These findings demonstrate that this selective neuronal NOS inhibitor does not affect MAC and the righting reflex in a comparable manner.
The observed MAC of sevoflurane in rats in the present experiments are in accord with previous reports . Although the righting reflex of sevoflurane in rats has not been reported, Ichinose and colleagues showed that the righting reflex of isoflurane in mice is 0.45 MAC . The present study found the righting reflex of sevoflurane in rats as 0.46 MAC, which is close to that reported for isoflurane.
In the present study, acute administration of 7-NI reduced the MAC of sevoflurane. This finding corresponds to those of previous reports in which mice were anaesthetized with sevoflurane  and rats with isoflurane . However, the reduction rates of 41.2 and 44.2% after the administration of 7-NI 50 and 100 mg kg−1, respectively, from baseline values were larger than those of previous reports of about 17 and 24% after 7-NI 60 and 120 mg kg−1, respectively ; and of 18.7 and 39.0% after 7-NI 60 and 120 mg kg−1, respectively . Differences in the design of the studies might explain this discrepancy. The precise mechanism by which general anaesthesia is induced remains largely unknown; however, several mechanisms have been proposed for a reduction in MAC. It has been suggested that 7-NI might reduce the concentration of cGMP through the NO-cGMP pathway. In the present study, the concentration of cGMP within the cerebellum was significantly reduced after 7-NI administration. Volatile anaesthetics have demonstrated an inhibitory effect toward cGMP concentrations [9-11]. A number of studies have suggested that central nervous system (CNS) depression is at least partially related to a decrease in cGMP [12,13]. This suggests that 7-NI may be a CNS depressant and, as such, might potentiate the anaesthesia induced by volatile agents. Another possibility for the reduction in MAC is the analgesic effect of NO reduction. It has been reported that administration of the non-selective NOS inhibitor, nitroG-L-arginine methylester (L-NAME), produces antinociception through a direct effect within the CNS . In addition, spinal NOS inhibition has been observed following antinociception . Thus, the antinociceptive effect of NOS inhibition might also cause a decrease in the MAC of sevoflurane. However, it is also possible that the reduction in MAC caused by 7-NI is not mediated by a mechanism involving guanylate cyclase-cGMP. Adachi and colleagues demonstrated that acute and chronic administration of L-NAME does not reduce the MAC of halothane [2,3] and they did not observe a relationship between the NO-cGMP pathway and the MAC of halothane. NO interacts with several kinds of neurotransmitters [16,17]. Therefore, a slight possibility exists that 7-NI modifies the sensitivity to general anaesthesia via a different cGMP-independent mechanism.
The current study reveals that acute administration of 7-NI causes a dose-dependent reduction in the righting reflex of sevoflurane; moreover, chronic administration also decreases the righting reflex of sevoflurane. The mechanism by which the righting reflex is reduced is, at least partially, due to the potentiating effect of neuronal NOS inhibition on the anaesthetic action of this volatile agent. In a previous study, acute administration of the non-specific NOS inhibitor L-NAME caused a 62.5% maximal reduction in the righting reflex of isoflurane from its baseline in mouse . This is consistent with the results of the present study. However, a much greater reduction in the righting reflex was observed in the current study than in this previous report. It is possible that the decline in the righting reflex produced by 7-NI does not level out. Our observations, therefore, suggest that the righting reflex is strongly dependent on a mechanism related to neuronal NOS.
In the present study, chronic administration of 7-NI did not alter MAC. This result is consistent with a previous investigation in mice in which sevoflurane MAC was reduced only for the first 2 days and returned to its baseline after 3 days of 7-NI feeding . The exact mechanism for this lack of effect is unknown. However, alternative neural mechanisms may compensate for a lack of NO in the CNS when neural NOS inhibitor is chronically administered. It is interesting that chronic administration of 7-NI did not decrease MAC, yet it reduced righting reflex. This result is not supported by the findings of a previous report . In Ichinose and colleagues' study , acute intraperitoneal L-NAME decreased MAC and the righting reflex in the wild-type mouse but did not alter the values in the knockout mouse. The wild-type mouse, when given L-NAME for 1 week, showed an MAC and the righting reflex identical to that of untreated wild-type animals. Methodological differences in experimental design such as the neuronal selectivity of the NOS inhibitors used, the species of animal used and the volatile anaesthetics administered might explain this discrepancy. Furthermore, in our acute protocol, MAC reduction by 7-NI had a ceiling effect; however, the righting reflex reduction did not. The NO-cGMP pathway might play a less important role in MAC reduction than it does in alterating the righting reflex.
In the present study, we determined the effect of 7-NI on cGMP concentrations in the brain, cerebellum and spinal cord. We observed a much higher cGMP concentration in the cerebellum than in either the spinal cord or the brain before 7-NI administration. Furthermore, the concentration of cGMP fell significantly only in the cerebellum after administration of 7-NI. Some reports support these results [5,11,18]. Ichinose and colleagues reported a reduction in the cGMP concentration of the cerebellum after acute and chronic 7-NI administration . They speculate that cGMP should decrease by the same amount in the spinal cord and cerebellum after 7-NI administration because the enzyme activity of NOS in the cerebellum and spinal cord shows very similar changes after acute administration of 7-NI . However, the present study reveals that the concentration of cGMP within the spinal cord does not decrease following acute 7-NI administration. Although alterations in cGMP within the spinal cord have not described in many reports, Kawamata and colleagues reported that acute administration of the non-selective NOS inhibitor, NG-monomethyl-L-arginine acetate (L-NMMA), alone does not reduce the concentration of cGMP in the spinal cord . This result is consistent with the results of the present study. It is possible that the concentration of cGMP does not change in accordance with NOS activity, especially in the spinal cord. Nitric oxide interacts with various kinds of neurotransmitters. 7-NI may modify other cGMP-independent mechanisms in the spinal cord. Previous studies have shown that MAC relies more heavily on the workings of the spinal cord than the supraspinal structures [21-24]. On the other hand, the righting reflex may be more reliant on activity within the supraspinal structures. The righting reflex depends on activity within the cerebellum, statolith, vestibular nucleus and mesoencephalon, as well as the sense of vision (mediated by the cerebral cortex). Therefore, an agent that reduces MAC might not necessary reduce righting reflexes in the same manner . Although the NO-cGMP pathway exists within supraspinal structures as well as in the spinal cord [18,26,27], in the present study we observed dissociation between MAC and the righting reflex reduction following acute and chronic administration of 7-NI. Thus, it is possible that MAC is less affected by the NO-cGMP pathway than the righting reflex. The NO-cGMP pathway might contribute more heavily to the righting reflex (mediated by supraspinal structures, especially the cerebellum) than it does to MAC (mediated by the spinal cord) because the concentration of cGMP within the cerebellum is greater than in the spinal cord. Therefore, following chronic administration of 7-NI, the mechanism that determined MAC might be easily compensated by other mechanisms. On the other hand, the mechanism that determined the righting reflex might not be compensated because the righting reflex depends more heavily on the NO-cGMP system. Furthermore, the effect of 7-NI on cGMP concentrations in the cerebellum and the spinal cord was observed to differ in magnitude. Acute administration of 7-NI did not decrease the concentration of cGMP in the spinal cord but it did reduce significantly the concentration of cGMP in the cerebellum. In a previous report, although the concentration of cGMP within the spinal cord was not measured, chronic administration of 7-NI reduced cGMP concentrations in the cerebellum; however, MAC did not decrease . These factors might explain the dissociation between a reduction of MAC and the righting reflex following acute and chronic administration of 7-NI. The reduction in MAC did not correlate with that of righting reflex after acute and chronic administration of 7-NI. The lack of relationship between the reduction in MAC and the righting reflex following acute and chronic administration of 7-NI can be at least partially explained by differences in their dependence on the NO-cGMP system.
In conclusion, alterations in the MAC and the righting reflex of sevoflurane after administration of the neuronal NOS inhibitor, 7-NI, were dissociated. Acute administration of 7-NI decreased both MAC and the righting reflex; however, a ceiling effect was observed for the reduction in MAC which was not seen for the righting reflex. Chronic 7-NI treatment decreased the righting reflex, but did not alter MAC. The cGMP concentration in the cerebellum was much higher than that of the spinal cord or the brain before 7-NI administration and only the cGMP concentration of the cerebellum was significantly reduced after 7-NI administration. The dissociation between MAC and the righting reflex reduction by 7-NI suggests that NO modulates the anaesthetic action of sevoflurane via a different mechanism. The NO-cGMP pathway might play a less important role in the determination of MAC than righting reflex.
The authors thank S. Nishizawa, MD, PhD, Assistant Professor of the Department of Neurosurgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan, for technical assistance in the measurement of cGMP.
Supported by Grant-in-Aid for Scientific Research No. C2-12671461 from the Ministry of Education Culture, Sports, Science and Technology, Tokyo, Japan.
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