Although the effects of general anesthesia may be promptly and entirely reversible, we have previously demonstrated lasting impairment on a spatial memory task in rats after a single 2-h isoflurane (ISO)-nitrous oxide (N2O) anesthetic (1,2). In one study, where rats were trained on a radial arm maze for 2 mo before anesthesia and then tested for 8 wk beginning 24 h after anesthesia, the ability of aged rats to improve their maze performance was worse than that of identically treated, unanesthetized control rats (1). Similarly, in a study where young and aged rats were anesthetized with ISO-N2O and then tested for 3 wk beginning 48 h after anesthesia, the ability of both age groups to acquire the task was impaired after anesthesia (2). These results demonstrate that general anesthesia affects performance for longer than would be expected based on the pharmacology of the drugs and suggest that aged rats may be more susceptible to such disruption.
In both studies, ISO and N2O were used together, making it impossible to determine whether the effect is attributable to one or both anesthetics. N2O produces vacuoles in adult brain (3,4) and apoptosis, altered hippocampal synaptic function, and long-term learning impairment in neonatal rats (5). We speculated, therefore, that both N2O and ISO contributed to the persistent spatial learning deficits in aged rats and that the impairment is persistent enough to be evident even when testing begins 2 wk after anesthesia. Consequently, in this study, we examined the effect of ISO with and without 70% N2O on the ability of aged rats to acquire a spatial memory task beginning 2 wk after anesthesia.
This study was approved by the Standing Committee on the Use of Animals in Research and Teaching, Harvard University Faculty of Arts and Sciences. To evaluate the effect of general anesthesia on subsequent acquisition of a spatial memory task in aged rats, we acquired 18-mo-old Fischer 344 rats from the National Institutes of Health aged rat colony at Harlan. This age was chosen because Fischer 344 rats have a life expectancy of 26 mo and develop progressive age-related spatial memory impairment, but at 18 mo of age are not so impaired that floor (i.e., performance so poor that deterioration is difficult to detect) or ceiling (i.e., perfect performance, so impairment cannot be detected) effects are a problem (6). After a 2-wk acclimation period in the laboratory, rats were randomly assigned (n = 9 per group) to receive 1.2% ISO 70% N2O 30% oxygen (ISO+N2O group), 1.2% ISO 100% oxygen (ISO group), or an air-oxygen mixture with a Fio2 of 30% (control group). Anesthesia was induced by placing rats in a chamber flushed with 3% isoflurane and 100% oxygen, and the trachea was intubated with a 14-gauge catheter. Rats were then mechanically ventilated with the appropriate anesthetic for 2 h with a 2-mL tidal volume delivered at a rate of 45 breaths/min, which pilot studies demonstrated maintains the Paco2 at 43 ± 2 mm Hg (mean ± sem) in 18 mo old Fischer 344 rats. Rectal temperature was controlled to 37°C ± 0.5°C. Arterial oxygen saturation and mean arterial blood pressure (MAP) were measured noninvasively using a pulse oximeter and a rat tail cuff during anesthesia. After 2 h, the anesthetics were discontinued and 100% oxygen delivered. The rate of ventilation was reduced until spontaneous ventilation resumed and the trachea was extubated when the rat was responsive. Control rats were placed in a box flushed with 30% oxygen for 2 h and were not tracheally intubated. Arterial oxygen saturation and MAP were not measured in the control group used in the behavioral experiment to prevent the introduction of stress as a confounding variable. However, oxygen saturation and MAP were measured in a separate group of rats (n = 6) during exposure to 30% oxygen for 2 h. All animals were recovered for 20 min in a box flushed with 40% oxygen and then placed in their home cage.
Cognitive function was tested in a 12-arm radial arm maze (RAM) (7). This RAM task tests spatial working memory, assesses the integrity of the frontal cortex, entorhinal cortex, and hippocampus (8,9), and can detect subtle differences in learning caused by aging, sedatives and anesthetics (1,2,8,10,11). The maze consists of a central platform that communicates with 12 arms, each of which is baited with a hidden food reward (quarter Froot Loops cereal). The walls of the maze display simple geometric designs providing fixed, extra-maze cues to assist spatial navigation. To ensure motivated performance, rats were food-restricted to 85% of free-feeding body weight starting 11 days after anesthesia but had free access to water in the home cage. Rats were adapted to the maze for 10 min/day during days 11–13 after anesthesia during which the maze was randomly scattered with food rewards and the rat was allowed to freely explore the maze. Formal testing began 14 days after anesthesia and consisted of a daily 15-min session in which the rat was placed on the central platform of the maze and all arms were baited. The rat was allowed to choose arms in any order until all 12 arms were visited or until 15 min had 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. An error was scored when the rat entered and proceeded more than 80% down an arm it has previously visited or failed to enter the arm in 15 min. Number of correct choices before first error, error rate, and time to complete the maze were recorded.
Trial results were grouped and analyzed in 2-day blocks. Measures of performance (error rate, number of correct choices to first error, and time to complete the maze) were analyzed with repeated-measures analysis of variance, with treatment group as a between-subjects factor and day of testing as the within-subjects factor. All analyses were performed in SYSTAT 7.0 for Windows (Systat, Richmond, CA). Statistical analysis of MAP and oxygen saturation was performed using a one-way analysis of variance followed by Dunnett’s test for multiple comparisons.
Anesthesia and mechanical ventilation were physiologically well tolerated in both groups of anesthetized rats. Relative to the control group, MAP was 8% lower with ISO+N2O and ISO anesthesia (121 ± 2 versus 111 ± 3 and 112 ± 2 mm Hg, respectively; P ≤ 0.05) but similar in both anesthetized groups. Measurement of Sao2 proved to be technically impossible in control rats because of movement artifact. Sao2 remained within the physiologically acceptable range in the anesthetized rats but was slightly less in the ISO+N2O group compared with the ISO group (97% ± 0% versus 99% ± 2%, respectively; P < 0.01).
One rat in the ISO+N2O group was euthanized before RAM testing owing to the development of a skin lesion and was not included in the analysis. Aged previously anesthetized rats demonstrated impaired RAM acquisition compared with controls. In terms of number of correct choices before first error, there was a main effect of day (P < 0.0005) and a main effect of group (P ≤ 0.05) indicating, respectively, learning across trials and an anesthetic-specific effect, but there was no interaction between group and day (P = 0.79) (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 the main effect of group (P = 0.09) and interaction between day and group (P = 0.6) did not achieve significance. With regard to time to complete the maze, repeated-measures analysis of variance was not possible because of an absence of variability on some test days (i.e., all rats required the maximum time, 900 s, to complete the maze). Therefore, a one-way analysis of variance was computed on the average time to complete the maze across the 14 days of testing (Fig. 3), which showed an effect of anesthesia group that did not achieve significance (P = 0.09). A post hoc analysis comparing control rats (n = 9) to all anesthetized rats (n = 17) reveals anesthetized rats performed worse than controls on number of correct choices to first error (P ≤ 0.05) and time to complete the maze (P ≤ 0.05) but not error rate (P ≤ 0.06).
Our study demonstrates that aged rats subjected to general anesthesia without surgery are less able than control rats that were not anesthetized to learn and perform a spatial memory task, even when the first trial is 2 weeks after anesthesia. This shows that in aged rats performance on a spatial memory task remains impaired for at least 2 weeks after general anesthesia. Consistent with our previous work, this performance impairment lasted for at least 1 month after anesthesia. These effects are not easily explained either by incomplete drug clearance or by physiologic perturbations because, respectively, both ISO and N2O should be fully eliminated by the time testing began (12,13) and measured physiologic variables were well within the normal range.
These results are consistent with previous studies in which we demonstrated that ISO+N2O anesthesia adversely affects the ability of aged rats to both master a maze task they had already partially learned and to acquire a new maze task (1,2). In the former study, rats were trained on the maze for 2 months before 1.2% ISO-70% N2O general anesthesia and then were tested for 8 weeks beginning 24 hours after anesthesia. That study showed that aged previously anesthetized rats failed to improve their performance to the same extent as nonanesthetized controls (1). In a subsequent study, we anesthetized rats with ISO+N2O and initiated maze testing 48 h later. Here, too, aged previously anesthetized rats lagged behind age-matched nonanesthetized controls in their ability to acquire and perform the maze task (2). The present study extends these observations and shows that there is learning impairment even 2 weeks after general anesthesia and that ISO+N2O may be worse in this regard than ISO alone. Moreover, because animals in our previous studies breathed spontaneously during anesthesia whereas these were mechanically ventilated, the mode of ventilation does not appear to have an influence.
Even though the anesthetics are rapidly eliminated, the brain may be altered both morphologically and functionally for some time after general anesthesia. In the neonatal brain, for example, ISO+N2O produce massive neurodegeneration and persistent learning impairment (5). Furthermore, at clinically relevant concentrations, anesthetics such as N2O and ketamine that block excitatory neurotransmission at N-methyl-d-aspartate (NMDA) receptors produce a distinctive neurotoxic reaction in the cerebral cortex of adult rats (4). Whether the aged brain is similarly susceptible is unknown, but a preliminary report suggests that this neurotoxic reaction is increased in the cerebral cortex of aged rats (3).
In our model, there were no statistically significant performance differences between rats anesthetized with ISO alone and those anesthetized with ISO+N2O despite differences in receptor mechanisms of the anesthetics and a larger MAC equivalent of anesthesia in the latter group. At clinically relevant concentrations, isoflurane enhances gamma-aminobutyric acid (GABA) receptor function by potentiating the response to submaximal concentrations of endogenous GABA and prolonging inhibitory postsynaptic currents (14–16). Isoflurane also inhibits NMDA- and non-NMDA-mediated excitation, producing significant NMDA receptor blockade at 1 MAC (17,18). N2O, in contrast, blocks NMDA glutaminergic and, to a lesser degree, non-NMDA receptors, at clinical concentrations and produces only weak and variable potentiation of GABA receptor-mediated currents (16,19). Moreover, both isoflurane and, to a lesser extent, N2O inhibit neuronal nicotinic acetylcholine (nACh) receptors at clinically relevant concentrations (20). Thus, the multiplicity of receptor actions of these anesthetics precludes conclusions about the mechanisms involved other than that our results implicate the GABA, glutaminergic, and/or ACh systems. To further elucidate receptor mechanisms of anesthesia-related cognitive impairment, therefore, it will be necessary to use anesthetics with more selective receptor effects.
There are a few important limitations of this study. First, our conclusion that the performance deficit reflects cognitive impairment is an inference from the behavior and not a direct measure of learning. The RAM is a standard and well-validated test of spatial learning and memory, but noncognitive variables, such as desire to eat and ability to walk, can influence it. Insufficient desire to eat is unlikely to have been a problem because during the period of food restriction with a fixed daily food allotment rats maintain a stable weight, and body weight is stable after ISO anesthesia in Fisher 344 rats (21). There is also no persistent postanesthetic locomotor impairment to account for poor performance on the RAM (2). Stress, such as might occur from the new surroundings of the anesthetic chamber or smelling the anesthetic vapor, is also unlikely to have influenced the results because if the anesthetic chamber is stressful, the controls should be most affected because they spent the most time in it awake. Moreover, for stress to affect learning behavior adversely and persistently it must be chronic and sustained (22). Finally, impaired smell or poor health can be excluded because rats use extramaze cues, not smell, to navigate the maze (23), and even aged rats suffer no apparent ill effects in the first month after general anesthesia (1). Second, for obvious reasons, control rats were not tracheally intubated or mechanically ventilated. However, we have seen similar performance deficits in two previous studies using spontaneously breathing ISO+N2O anesthetized rats, making it unlikely that intubation and/or mechanical ventilation explain our results (1,2).
We conclude, therefore, that general anesthesia produces long-lasting impairment in the ability of rats to acquire and perform a spatial memory task. Because this impairment was evident even 2 weeks after anesthesia, it cannot be explained by the pharmacokinetics of the drugs involved. We infer that the performance impairments observed potentially represent persistent anesthesia-induced changes in neural structures and/or biochemical cascades mediating learning and memory. If this is true, not only do the results provide a basis for examining the neurobiological bases of anesthesia-related impairments in cognitive function, they may have implications for understanding prolonged postoperative cognitive dysfunction in humans.
1. Culley DJ, Yukhananov RY, Baxter MG, Crosby G. Memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 2003;96:1004–9.
2. Culley DJ, Baxter MG, Yukhananov RY, Crosby G. Long term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats. Anesthesiology 2004;100:309–14.
3. Beals JK, Carter LB, Jevtovic-Todorovic V. Neurotoxicity of nitrous oxide and ketamine is more severe in aged than in young rat brain. Ann N Y Acad Sci 2003;993:115.
4. Jevtovic-Todorovic V, Benshoff N, Olney JW. Ketamine potentiates cerebrocortical damage induced by the common anaesthetic agent nitrous oxide in adult rats. Br J Pharmacol 2000;130:1692–8.
5. Jevtovic-Todorovic V, Hartman RE, Izumi Y, et al. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003;23:876–82.
6. Frick KM, Baxter MG, Markowska AL, et al. Age-related spatial reference and working memory deficits assessed in the water maze. Neurobiol Aging 1995;16:149–60.
7. McMahan RW, Sobel TJ, Baxter MG. Selective immunolesions of hippocampal cholinergic input fail to impair spatial working memory. Hippocampus 1997;7:130–6.
8. Decker MW, Gallagher M. Scopolamine-disruption of radial arm maze performance: modification by noradrenergic depletion. Brain Res 1987;417:59–69.
9. Baxter MG, Holland PC, Gallagher M. Disruption of decrements in conditioned stimulus processing by selective removal of hippocampal cholinergic input. J Neurosci 1997;17:5230–6.
10. Borde N, Jaffard R, Beracochea D. Effects of chronic alcohol consumption or Diazepam administration on item recognition and temporal ordering in a spatial working memory task in mice. Eur J Neurosci 1998;10:2380–7.
11. Luine V, Rodriguez M. Effects of estradiol on radial arm maze performance of young and aged rats. Behav Neural Biol 1994;62:230–6.
12. Chen M, Olsen JI, Stolk JA, et al. An in vivo
19F NMR study of isoflurane elimination as a function of age in rat brain. NMR Biomed 1992;5:121–6.
13. Bailey JM. Context-sensitive half-times and other decrement times of inhaled anesthetics. Anesth Analg 1997;85:681–6.
14. Simon W, Hapfelmeier G, Kochs E, et al. Isoflurane blocks synaptic plasticity in the mouse hippocampus. Anesthesiology 2001;94:1058–65.
15. de Sousa SL, Dickinson R, Lieb WR, Franks NP. Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. Anesthesiology 2000;92:1055–66.
16. Krasowski MD, Harrison NL. General anaesthetic actions on ligand-gated ion channels. Cell Mol Life Sci 1999;55:1278–303.
17. Nishikawa K, MacIver MB. Excitatory synaptic transmission mediated by NMDA receptors is more sensitive to isoflurane than are non-NMDA receptor-mediated responses. Anesthesiology 2000;92:228–36.
18. Bickler PE, Buck LT, Hansen BM. Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices. Anesthesiology 1994;81:1461–9.
19. Mennerick S, Jevtovic-Todorovic V, Todorovic SM, et al. Effect of nitrous oxide on excitatory and inhibitory synaptic transmission in hippocampal cultures. J Neurosci 1998;18:9716–26.
20. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994;367:607–14.
21. Rice SA, Fish KJ. Anesthetic metabolism and renal function in obese and nonobese Fischer 344 rats following enflurane or isoflurane anesthesia. Anesthesiology 1986;65:28–34.
22. Mercier S, Canini F, Buguet A, et al. Behavioural changes after an acute stress: stressor and test types influences. Behav Brain Res 2003;139:167–75.
23. Olton DS, Samuelson RJ. Remembrance of places passed: spatial memory in rats. J Exp Psychol Anim Behav Process 1976;2:97–116.