BACKGROUND: Nitrous oxide is a commonly used anesthetic that inhibits the activity of methionine synthase, an enzyme involved in methylation reactions and DNA synthesis and repair. This inhibition triggers vacuole formation and degeneration of neurons in areas of the developing and mature brain that are important for spatial memory, raising the possibility that nitrous oxide might have sustained effects on learning.
METHODS: To test this possibility, we randomized 18-month-old Fischer 344 rats (n = 13 per group) to 4 h of 70% nitrous oxide + 30% oxygen or 70% nitrogen + 30% oxygen (control) and assessed memory using a 12-arm radial maze for 14 days beginning 2 days after nitrous oxide inhalation. In separate, identically treated groups of rats, we measured methionine synthase activity in the cortex and liver at the end of nitrous oxide exposure and 2 days later (n = 3 rats per group per time point) using a standard assay.
RESULTS: Liver and cortical methionine synthase was inhibited during nitrous oxide inhalation (6% and 23% of control in liver and cortex, respectively; P < 0.01). Liver enzyme activity remained depressed 2 days later, whereas cortical enzyme activity recovered. There was no difference in error rate between control and nitrous oxide treated rats. However, those exposed to nitrous oxide took more time to complete the maze and made fewer correct choices before first error (P < 0.05).
CONCLUSIONS: Sedation with 70% nitrous oxide profoundly, but transiently, reduces the activity of cortical methionine synthase but produces lasting impairment in spatial working memory in aged rats.
IMPLICATIONS: This study demonstrates that exposure of aged rats to 4 h of 70% nitrous oxide impairs acquisition of a spatial memory task when testing begins 2 days later, and suggests that subanesthetic concentrations of anesthetics may lead to persistent changes in the aged brain.
From the *Department of Anesthesiology Harvard Medical School, Brigham and Women’s Hospital, Boston; †Department of Psychology, Harvard University, Cambridge; ‡Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts; and §Department of Experimental Psychology, Oxford University, Oxford, United Kingdom.
Accepted for publication March 9, 2007.
Supported by an American Geriatrics Society Jahnigen Award (DJC), National Institutes of Health grants KO8 GM077057 (DJC), and RO1 AG20253 (GC).
Address correspondence and reprint requests to Gregory Crosby, MD, Department of Anesthesiology, Aging Neuroscience Laboratory, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Address e-mail to email@example.com.
Nitrous oxide is clinically used as an adjunct to general anesthesia and recreationally, in the form of “whippets,” as a drug of abuse (1,2). Although gross clinical recovery is rapid, there is concern that nitrous oxide might produce subtle neurotoxicity and cognitive deficits. Nitrous oxide inhibits methionine synthase, an enzyme important for methylation reactions and DNA synthesis and repair. Decreased methionine synthase activity produces a myelopathy and is implicated in dementing illness, possibly related to accumulation of homocysteine, a cytotoxic amino acid normally remethylated to methionine, an essential amino acid, by methionine synthase (3,4). In addition, high concentrations of nitrous oxide produce a reversible neurotoxic reaction in the mature brain characterized by mitochondrial swelling and vacuolization of neurons. With prolonged administration, the toxicity is irreversible and associated with massive apoptotic neurodegeneration and neuronal death (5,6). This neurotoxicity has been linked to nitrous oxide’s action as a N-methy-d-aspartate (NMDA) receptor antagonist. Nitrous oxide mimics other NMDA receptor antagonists in this regard, and the neurotoxicity of nitrous oxide is exacerbated when it is administered with other drugs (e.g., ketamine, MK801) that block glutaminergic neurotransmission (5–7). Moreover, in neonatal rodents exposed to nitrous oxide there is evidence of hippocampal dysfunction and learning impairment months later (8).
Few studies have investigated cognitive performance in adult or aged animals after nitrous oxide inhalation. This is a potentially important issue, because many elderly patients suffer long-lasting cognitive impairment after surgery, and because nitrous oxide is commonly used in anesthesia (9). We previously demonstrated that general anesthesia with a combination of nitrous oxide and isoflurane impairs acquisition of a spatial memory task for weeks in aged rodents (10–12). The role of nitrous oxide alone in this impairment has not been explored, but it is noteworthy that nitrous oxide-induced vacuolization of neurons occurs in pyramidal and multipolar neurons in layers III–V of the posterior cingulate/retrosplenial cortex, a region important for spatial memory acquisition, and that the aged brain is especially sensitive to the neurodegenerative effects of NMDA receptor antagonists (13–15). Accordingly, we hypothesized that inhalation of nitrous oxide may produce a persistent learning/memory deficit in aged animals. To test this hypothesis, we examined the influence of nitrous oxide inhalation on methionine synthase activity and radial arm maze (RAM) performance.
The study was approved by the Standing Committee on the Use of Animals in Research and Teaching, Harvard University/Faculty of Arts and Sciences. Thirty, 18-month-old, male Fischer 344 rats were acquired from the National Institute of Health aged rat colony at Harlan (Harlan Sprague-Dawley, Inc., Indianapolis, IN) and housed individually in a climate-and humidity-controlled room on a 12-h light–dark cycle with continuous access to food and water. After a 1-wk acclimation period in the laboratory, rats were placed on a restricted feeding schedule to reduce their body weight to 85% of free-feeding levels and habituated to a 12-arm RAM for 10 min daily over 3 days. During acclimation, the rats were able to freely explore the maze in which food rewards [quarter pieces of Froot Loops cereal (Kellogg’s, Battle Creek, MI)] were scattered randomly. The purpose of habituation is to acclimatize animals to the unfamiliar environment of the maze; because food is distributed randomly, habituation does not directly assist subsequent maze learning. Four rats were killed before anesthesia. Two of the rats had suspected eye disease, one fell off the maze during habituation, and one was losing weight before food restriction. Thereafter, rats were randomly assigned to an anesthesia or control group. Rats randomized to the anesthesia group (n = 13 rats) received 70% nitrous oxide:30% oxygen for 4 h in a anesthetizing chamber whereas the control group (n = 13 rats) received air:oxygen (Fio2 0.3) at identical flow rates for 4 h in identical chambers. Oxygen concentrations were measured (Ohmeda, Madison, WI) and temperature of the anesthetizing chamber controlled to maintain rats body temperature at 37 ± 0.5°C. The experiment was terminated by removing the animal from the anesthetizing chamber and placing it for 20 min in a chamber flushed with 40% oxygen. Rats recovered for 48 h so as to avoid the confounding influence of residual anesthetic before beginning 14 days of RAM testing.
The RAM tests spatial working and reference memory and can detect subtle differences caused by aging, sedative medications, and anesthetics (11,12, 16,17). The maze consists of a central platform that communicates with 12 arms, each of which is baited with a hidden food reward. The walls of the room display simple geometric designs providing fixed, extra-maze cues to assist spatial navigation. The food restriction schedule ensured motivated performance; rats had free access to water in the home cage. Testing consisted of a daily 15-min session in which the rat was placed on the central platform of the maze with all arms baited. The rat was allowed to choose arms in any order until all 12 arms were visited or 15 min elapsed. A correct choice was defined as one in which the rat entered and proceeded more than 80% down a baited arm not previously explored, whereas an error was scored when the rat entered and proceeded more than 80% down an arm it had previously visited or failed to enter the arm in 15 min. The number of correct choices before first error, number of errors, and time to complete the maze was measured for each trial.
To investigate the effect of nitrous oxide administration on methionine synthase activity, we measured enzyme activity during and 48 h after nitrous oxide administration using a standard assay (18). Aged Fischer 344 rats were food-restricted as described above and randomly assigned to anesthetized or control groups. Each group was placed in identical anesthetizing chambers for 4 h as described above and killed either at the end of anesthesia (n = 3 control rats, n = 3 anesthesia rats) or 48 h later (n = 3 control, n = 3 anesthesia). The cortex and liver of the animals were removed and rapidly homogenized and frozen at −80°C for subsequent assay for methionine synthase activity as previously described (18). Briefly, 100 μL of tissue homogenate was added to lysis buffer, sonicated and then centrifuged at 10,000g for 10 min. The supernatant was collected and used for the assay. The reaction mixture contained the following: 100 mM dibasic potassium phosphate, pH 7.2, 500 μM homocysteine, 152 μM S-adenosylmethionine, 2 mM titanium citrate, 250 μCi (6R,S)-5-[14C]-methyltetrahydrofolate (methylTHF) and enzyme in a final volume of 500 μL and run in the presence of hydroxocobalamin (10 μM). The reaction was initiated by addition of the radioactive methylTHF, incubated for 60 min at 37°C, and terminated by heating at 98°C for 2 min. At the end of the reaction, vials were uncapped and cooled on ice. Radiolabeled methionine was separated from un-reacted radiolabeled methylTHF by passing the reaction mixture through an anion exchange column of Dowex 1-× 8 (chloride form). The column was washed with 1 mL of water, and the aqueous samples were collected in scintillation vials, to which 8 mL of scintillation fluid (ScintiSafe™ Econo1) was added and the radioactivity counted. All reported values are corrected for the counts observed in control assays, in which sample enzyme was omitted. Protein concentrations were determined by the Lowry method for protein quantification and bovine serum albumin was used as the standard (19).
Measures of performance from the RAM (time to complete the maze, number of correct choices to first error and error rate) were grouped into 2-day blocks and analyzed with repeated measures ANOVA, with anesthesia group as a between-subjects factor and day of testing as the within-subjects factor. Methionine synthase activity was compared at each time point using a one-way ANOVA.
Nitrous oxide-treated rats appeared sedated but occasionally moved around the anesthesia chamber. Upon discontinuing nitrous oxide, gross recovery was rapid, with rats ambulating and exploring the cage within minutes. One rat in the nitrous oxide group died on day 11 of RAM testing of unknown causes; data from that animal were excluded (post hoc analysis revealed that excluding it does not change the results). Aged rats anesthetized with nitrous oxide had impaired RAM performance compared to controls, as judged by choice accuracy and time to complete the maze but not error rate. Choice accuracy, assessed by number of correct choices before first error, revealed a main effect of day (P < 0.001) and a main effect of group (P < 0.001) indicating, respectively, learning across trials and an anesthetic-specific effect, but there was no interaction between group and day (Fig. 1). In contrast, for total number of errors (Fig. 2), there was a main effect of day (P < 0.05), indicating learning across days, but no main effect of group (P ≥ 0.05) and no interactions between day and group (P ≥ 0.05). With regard to time to complete the maze, repeated-measures ANOVA was not possible because of absence of variability on some test days (i.e., all rats required the maximum time, 900 s). Therefore, a one-way ANOVA was computed on the average time to complete the maze across the 14 days of testing and demonstrated that rats exposed to nitrous oxide took longer than control rats to complete the maze (P < 0.05; Fig. 3).
Methionine synthase activity was reduced to 6% and 23% of control in liver and cortex, respectively, of aged rats during administration of nitrous oxide (P < 0.01; Fig. 4). However, activity returned to control values within 2 days in the brain (96% of control) but not the liver (56% of control; P < 0.05), indicating that there are differences in the effect of nitrous oxide on liver and brain methionine synthase.
The primary finding of this study is that in aged rats spatial memory is impaired when maze testing begins 2 days after inhaling 70% nitrous oxide. This is remarkable because 70% nitrous oxide is a subanesthetic dose, corresponding to less than half of the minimum alveolar concentration required to prevent movement in response to a surgical stimulus (20). This indicates that several hours of exposure to a sedating concentration of nitrous oxide can have persistent cognitive consequences, and that the state of general anesthesia is not a necessary condition for development of such a persistent deficit. Neither incomplete drug clearance nor physiologic abnormalities during administration of nitrous oxide are likely confounders. Nitrous oxide has low lipid solubility and is eliminated from the brain in a matter of minutes; its effects on arterial blood pressure and ventilation are minor, even when combined with isoflurane (21,22). Therefore, these data support the concept that nitrous oxide, or the sedation it produces, causes persistent memory/ learning deficits in aged rats.
We also confirmed the inhibitory effect of nitrous oxide on methionine synthase activity and, in addition, found tissue-specific differences. Methionine synthase is a cobalamin (vitamin B12)-dependent enzyme that catalyzes the re-methylation of homocysteine to methionine and the concurrent de-methylation of 5-methylTHF to tetrahydrofolate (3,4). Methionine is an essential amino acid that plays a pivotal role in numerous methylation reactions and biosynthetic pathways crucial for DNA synthesis and repair. Methionine synthase can be inhibited via both reversible and irreversible mechanisms; the former occurs by oxidation of cobalamin, a required cofactor in the methyl transfer reaction, whereas the latter reflects damage to the enzyme by nitric oxide produced upon cobalamin-mediated reduction of nitrous oxide (23–25). Nitrous oxide profoundly reduced methionine synthase activity in both the brain and liver of our rats, results that confirm the findings of others (26,27). Catalytic activity of the liver enzyme remained impaired 2 days later, as described previously (26,27), whereas the brain enzyme recovered completely (Fig. 4). Although further studies are required, this suggests the liver and brain enzymes are structurally different, and that the brain enzyme is reversibly inhibited whereas the liver enzyme is presumably irreversibly damaged.
The fact that brain methionine synthase activity was normal when behavioral testing began 2 days after nitrous oxide administration indicates that altered catalytic activity of the enzyme does not directly explain the deficits in learning and memory described herein. However, the possibility remains that inactivation of methionine synthase during nitrous oxide inhalation contributes to subsequent and prolonged alterations in the capacity to learn. This could occur by hypomethylation of key substrates and/or by accumulation of toxic byproducts of hypomethylation. For example, methionine synthase is required for dopamine-stimulated phospholipid methylation, a membrane signaling mechanism unique to the D4 dopamine receptor and implicated in cognition and attention (28,29). Reduced methionine synthase activity also leads to accumulation of homocysteine, a cytotoxic, sulfur-containing nonessential amino acid. Epidemiologic studies have shown that mild to moderate chronic hyperhomocysteinemia is a risk factor for vascular and neurodegenerative disease, including dementia (3,4,30), and human studies indicate that plasma homocysteine remains elevated for 24 h or more after anesthesia using nitrous oxide (25). Such transient hyperhomocysteinemia may be deleterious, because homocysteine has been implicated in excitotoxic neuronal damage (31,32). In vitro, an acute increase in homocysteine induces DNA breakage, oxidative stress, apoptosis, accelerates processing of amyloid precursor protein to amyloid β, a protein implicated in the pathogenesis of Alzheimer’s disease, and increases amyloid β neurotoxicity (32–34).
In addition to its effects on methionine synthase, nitrous oxide may persistently impair spatial memory through neurotransmitter receptor mechanisms or neurotoxicity. Nitrous oxide is a neuronal nicotinic cholinergic receptor antagonist and an NMDA glutamate receptor antagonist (35,36). Both neurotransmitter systems participate in memory, but NMDA glutamate receptor-mediated calcium influx is crucial to encode information (37). Not surprisingly, therefore, nitrous oxide is an amnesic, a property it shares with other NMDA receptor antagonists. Nitrous oxide interferes with acquisition and retrieval, but not retention, of memory (1); in humans, it deactivates both the posterior cingulate cortex and hippocampus, regions which mediate learning and memory (38). There is an assumption that these effects on learning resolve completely with discontinuation of the drug but the capacity to learn days later has not been assessed. One study that examined memory processing minutes after discontinuation of nitrous oxide found rapid recovery, but the memory test (object recognition), age of the animals (young adult), duration (5–15 min.) and concentration (15%–50%) of nitrous oxide used differed substantially from those we used, making comparison with our results difficult (1). However, prolonged behavioral effects of other NMDA receptor antagonists have been reported. For example, a single dose of MK-810, a potent noncompetitive NMDA antagonist, produces impairment in rats and mice lasting for days to weeks on acquisition of water escape and food-motivated spatial learning tasks (39–42).
Neurotoxicity could also be a factor in the working memory impairment we identified. Nitrous oxide, and other NMDA receptor antagonists, produce mitochondrial swelling and vacuolization of neurons in the cingulate and retrosplenial cortex, but the changes are reversible if the duration of exposure is short. With longer exposure and supraclinical concentrations (i.e., 120%–150% nitrous oxide for 3 h or more), it produces irreversible damage consisting of apoptosis and neuronal degeneration in the developing and mature brain, with changes most prominent in the retrosplenial and entorhinal cortex, hippocampal subiculum, and thalamus (5). These neurotoxic reactions do not require inhibition of methionine synthase, because they also occur with NMDA receptor antagonists such MK-801 and ketamine that have no effect on the enzyme and are relevant here because lesions in the retrosplenial cortex are associated with a deficit in RAM performance (43,44) and activation of the retrosplenial cortex occurs during RAM learning (45). Moreover, NMDA receptor antagonist-induced vacuole formation and apoptotic neurodegeneration are age-dependent, with animals at the extremes of age being most vulnerable (6,8). NMDA antagonist-related neurotoxicity appears to depend upon the balance between excitatory and inhibitory neurotransmission. Nitrous oxide-induced vacuolization and neurodegeneration are exacerbated by concurrent administration of another NDMA receptor antagonist and reduced by agents that function as γ-aminobutyric acid (GABA) receptor agonists or modulators (36). This is potentially important, because nitrous oxide has low anesthetic potency and therefore, in clinical practice, is usually combined with more potent anesthetics, most of which act as GABA receptor agonists or modulators. However, comparing the present results with those from a previous experiment in our laboratory shows that impairment in spatial working memory acquisition after anesthesia for 2 h with nitrous oxide + isoflurane, a GABA modulator, is similar to that observed here with nitrous oxide alone (12).
Perhaps the main limitation of this study is that our conclusion that memory is persistently altered after nitrous oxide is an inference from the behavior and not a direct measure of learning. Such an inference seems reasonable, however, inasmuch as potential confounders, such as stress, poor appetite, and impaired mobility, can be excluded with some confidence. If the anesthetic chamber is stressful, the controls should be most affected because they spent the most time in it unsedated. Moreover, for stress to affect learning behavior adversely and persistently it must be chronic and sustained, which is certainly not the case here (46). Insufficient motivation to eat is unlikely because each rat received the same daily food allotment, consumed it avidly within minutes, and maintained stable weight throughout the experiment. Another limitation is that we examined a single dose and duration of exposure to nitrous oxide. Because the dose and duration of exposure affect the development of irreversible neurotoxicity with nitrous oxide, we cannot exclude the possibility that no learning deficit would be detected at lower dosages or after shorter periods of inhalation. Lastly, because we have no control for the effects of prolonged sedation, we cannot exclude the possibility that the behavioral impairment observed is a consequence of nitrous oxide’s sedative effects rather than its unique pharmacologic or neurotoxic properties.
In summary, inhalation of a subanesthetic concentration of nitrous oxide for 4 h impairs acquisition of a memory task for at least 2 days in aged rats. This indicates that, in aged animals, the state of general anesthesia is not a necessary condition for development of persistent memory impairment with this drug and suggests that sedation itself could be associated with prolonged learning dysfunction. Given that the RAM task depends upon hippocampal-cortical memory circuits, these data imply that dysfunction in these circuits after nitrous oxide exposure lasts longer than gross clinical recovery would indicate. The fact that nitrous oxide-induced memory impairment was preceded by, if not coincident with, reduced activity of cortical methionine synthase raises the possibility that altered methylation reactions or accumulation of homocysteine may contribute to the learning deficit.
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