Learning and memory deficits have been reported in adult patients following surgery and anaesthesia.1,2 This persistent decline in cognitive function, referred to as postoperative cognitive dysfunction (POCD), increases in incidence and severity in elderly patients, but also occurs in young and middle-aged patients. Monk et al.2 reported an incidence of POCD of 36.6% in young (18–39 years), 30.4% in middle-aged (40–59 years) and 41.4% in elderly (≥60 years) patients at the time of discharge from hospital. Three months after surgery, POCD was found to persist only in elderly patients.
Clinical studies have lacked power to identify anaesthesia as a cause of POCD and in humans it has not been possible to separate the effects of anaesthesia from those of surgery. However, experimental animal studies have suggested that anaesthesia alone may induce cognitive decline and, consistent with human data showing a greater risk of cognitive deficits in elderly patients, aged rodents appear to be more susceptible, demonstrating impaired cognition up to 3 weeks after exposure to the inhaled anaesthetic isoflurane.3,4 Reports of the effects of anaesthetics in young adult rats and mice have yielded inconsistent findings and have not been well investigated in middle-aged animals. Researchers report impaired,3–5 improved4,6,7 or unaltered cognition8 following exposure of young adult rodents to isoflurane. Many differences exist among these studies in drug, dosage, duration and time points examined after anaesthesia, making comparisons difficult. In addition, the large variability in the reported incidence of clinical POCD has been associated with differences in the number and type of neuropsychological tests employed,9 and so the inconsistent findings in animal studies may also be related to differences in cognitive testing methods among studies.
To emphasise this point, subsequent studies performed by the same research group have reported both improved and impaired cognitive performance under nearly identical experimental conditions.3,4,10 Culley et al.4 reported a significant improvement in cognitive performance following isoflurane [1 minimum alveolar concentration (MAC), 2 h] at 1, 3 and 8 weeks after exposure but, in a subsequent study, found persistent deficits in both young and aged rats exposed to the same dosage regimen of isoflurane. Culley et al.3–5,10,11 employed N2O/O2 as the carrier gas in all of their experiments, but control groups variably received 30% O2 or 70% N2O/30%O2 which could potentially account for the different findings.
In an attempt to differentiate the effects of anaesthesia in young and aged rats, we have compared a moderate duration of exposure to isoflurane at 1 MAC in 100% oxygen in young adult (3 month old) and middle-aged (12 month old) rats on cognitive function. The study aimed to investigate the effects of isoflurane (1 MAC, 4 h) on task acquisition and memory retention in the Morris water maze, with a hypothesis that there would be a greater effect of the anaesthetic in middle-aged rats. Our findings indicate specific deficits in memory retention in both young adult and middle-aged rats after exposure to isoflurane.
Ethics approval for this study (0811071) was granted by the Pharmacology, Physiology, Biochemistry and Molecular Biology and Bio21 Institute Animal Ethics Committee of the University of Melbourne (Chairperson Dr C. Wright) on the use of animals in a research project on 23 June 2009. Young adult Sprague Dawley rats (8–10 weeks of age, n = 25) and ex-breeders (12 months of age, n = 20) were obtained from the Animal Resources Centre, Canning Vale, Western Australia. These aged rats are referred to as ‘middle-aged’ throughout. Rats were housed one to two per cage in a climate and humidity controlled room on a 12-h light–dark cycle with free access to food and water in the animal facilities of the Department of Medicine, Royal Melbourne Hospital, University of Melbourne.
Rats were pseudorandomly assigned to receive anaesthetic exposure or sham exposure. Rats randomised to the anaesthesia group (n = 12 young, n = 10 middle-aged) were placed in groups of three to four in an anaesthetic induction chamber filled with isoflurane (5%) for approximately 1–2 min until unconscious. Rats were then removed and attached to one of the nose cones of our custom-designed anaesthetic apparatus, where they received 1-MAC isoflurane (1.2%) in 100% oxygen for 4 h. The apparatus allowed up to 10 rats to be anaesthetised simultaneously and ensured that rats were exposed to the same amount of anaesthetic because each nose cone was connected to the chamber into which the anaesthetic was pumped at a flow rate of approximately 3 l min−1 and adjusted constantly to maintain MAC, O2 and CO2 concentrations within the chamber. Gases within the anaesthetic chamber were monitored continuously (Ohmeda Excel 210 SE anaesthetic machine Datex Instrumentarium Corp., Helsinki, Finland). Sham exposure rats were placed in the induction chamber containing 100% oxygen only for 10 min. Because the bodies of the rats were outside the anaesthetic chamber, it was possible to monitor blood pressure and maintain normothermia in the anaesthetised animals. Mean arterial blood pressure was monitored noninvasively by tail cuff (Coda, Kent Scientific, Connecticut, USA) at intervals during anaesthetic exposure. Normothermia (37 ± 1°C) was maintained using warming mats and rectal temperature was measured at 30-min intervals.
The behavioural tests were performed in a closed, quiet, light-controlled room in the Behavioural Testing Facility at the Department of Medicine, Royal Melbourne Hospital, University of Melbourne. Rats were acclimatised to the conditions of the facility for at least 30 min before testing on each day.
Each rat was placed in a 160-cm diameter black plastic pool filled to a depth of 30 cm with clear water maintained at 24 ± 1°C and was required to locate a submerged platform using visual cues around the edges of the pool and within the room as described previously,12 with modifications.13 On each trial, a rat was placed gently into the pool at one of four different locations and allowed 90 s to locate a 10-cm diameter black platform submerged 2 cm below the surface of the water, located in one of four randomly assigned positions. The position of the platform was kept constant for each individual rat. If unsuccessful in the 90-s trial, the animal was gently guided to the platform. Once on the platform, animals were allowed to remain there for 30 s. At the end of each trial, the rats were removed from the pool, towel dried and returned to their home cage. Four trials were conducted during each session with an intertrial interval of 30 min and an intersession interval of 24 h. The experiment extended for 4 consecutive days, beginning 1 week after anaesthetic exposure. Each trial was videotaped and swim paths were tracked using Ethovision Video-Tracking Software (v3.1.16 Noldus Information Technology, Wageningen, Netherlands. Statsoft Inc. Oklahoma, USA). The latency to reach the platform, distance travelled and swim velocity were calculated for each trial, and then averaged over each daily session of four trials. Additionally, distance travelled in the periphery (within a 20-cm annulus from the edge) of the pool, or thigmotaxis, was monitored on each trial. On the fifth day, a probe trial was conducted in which rats were placed in the pool for 90 s without the platform and the duration of time spent in each quadrant was determined. In middle-aged rats, a second probe trial was conducted 4 weeks after isoflurane or sham exposure to assess long-term memory retention.
Mean arterial pressure (MAP) was analysed using unpaired Student's t-tests. Three-way analysis of variance (ANOVA) with factors treatment, age and session, the last being a repeated measures factor, was used for analysis of acquisition in the Morris water maze, and thigmotactic behaviour. Probe trials, comparing time spent in each quadrant, were analysed comparing treatments within an age group using two-way ANOVA and Bonferroni's post-hoc test. Data were analysed using Statistica (StatSoft Inc. USA) and Prism GraphPad (GraphPad Software Inc.) and in all cases, statistical significance was defined as P value less than 0.05.
MAP was higher in middle-aged rats compared with young rats during isoflurane exposure, but the difference did not reach statistical significance (Student's t-test, P > 0.05 at 2 h and 4 h; MAP was 78.5 ± 4.0 and 98.5 ± 3.4 mmHg after 2 h and 75.8 ± 4.8 and 96.4 ± 7.1 mmHg after 4 h in young adult and middle-aged isoflurane-exposed rats, respectively). Normothermia (37 ± 1°C) was maintained throughout isoflurane exposure in young and middle-aged rats.
In the Morris water maze test, all rats were able to learn the task successfully, as evidenced by reduced latency to locate the hidden platform over the four daily sessions [F(3.123) = 110.35, P < 0.0001; Fig. 1a]. Exposure to isoflurane had no significant effect on the latency to find the platform in either young adult rats or in middle-aged rats compared with their corresponding shams [Fig. 1a; F(1.41) = 0.06, P > 0.05]. Irrespective of treatment, middle-aged rats took significantly longer to locate the hidden platform compared with young rats [Fig. 1a; F(1.41) = 7.94, P < 0.01]. There was also a significant overall effect of age on the distance travelled while searching for the hidden platform, with older rats travelling significantly further than young adult rats [F(1.41) = 17.49, P < 0.001, data not shown]. However, within each age group, distance travelled was unaffected by prior exposure to isoflurane [F(1.41) = 0.12, P > 0.05]. The increased latency and distance travelled in middle-aged rats were not due to slower swim speeds; the middle-aged rats swam faster than the young adult rats [F(1.41) = 7.70, P < 0.01; Fig. 1b). To investigate the reason for increased latency and distance in the absence of a slower swim speed in the middle-aged rats, the swim paths were examined. It was observed that middle-aged rats persisted swimming around the edges of the pool well into the second daily session, whereas young adult rats had already begun searching for the escape platform (Fig. 2a). When distance travelled in the periphery was measured, this observation was confirmed, with middle-aged rats spending significantly more time swimming around the edges of the pool [thigmotaxis, Fig. 2b; F(1.41) = 9.97, P < 0.01]. Exposure to isoflurane did not affect thigmotactic swimming in either young or middle-aged rats [Fig. 2B; F(1.41) = 0.44, P > 0.05].
In the probe trial conducted 24 h after the last water maze acquisition session, young rats previously exposed to isoflurane spent significantly less time in the target quadrant compared with shams [Fig. 3a; F(3.99) = 3.76, P < 0.05; Bonferroni post-hoc test, P < 0.05]. No such statistically significant difference was observed between the middle-aged sham and isoflurane treated rats in their probe trial [Fig. 3b; F(3.72) = 5.92, P < 0.05; Bonferroni post-hoc test, P > 0.05].
We decided to investigate whether there could be a delayed effect of isoflurane in the middle-aged rats. The middle-aged rats underwent a second probe test 4 weeks after exposure (2 weeks after completion of task acquisition). Under these conditions, sham, but not isoflurane-treated rats, showed a significant preference for the quadrant in which the platform had been located previously (Fig. 3c; one-way ANOVA and Bonferroni test, P < 0.05). Rats which had been exposed to isoflurane explored all quadrants equally, suggesting that isoflurane exposure reduced the ability of rats to form a stable long-term memory of the position of the platform. However, this is not a robust effect because there was no statistically significant difference when comparing preference for target quadrants between sham and isoflurane-exposed rats [Fig. 3C; F(3.72) = 1.68, P > 0.05].
Our findings are not in agreement with the majority of previous studies, which showed that young adult rats do not develop cognitive deficits after exposure to isoflurane, and may even show improved performance in memory tasks.5,6,11 Different methodologies, including dosage, rat strain, duration of exposure, outcome measurements or anaesthetic carrier gas may have contributed to these different findings. With respect to the last, Culley et al.3 reported improved working memory performance in the radial arm maze in adult rats 2 weeks after exposure to 1.2% isoflurane for 2 h, but only in animals which also received nitrous oxide. In contrast, these authors reported impaired memory in the same task in adult rats tested 2 days after exposure to isoflurane and nitrous oxide, but different outcome measures were affected (time to complete the maze and decreased correct choices before the first error). This research group also reported a similar finding in aged rats exposed to nitrous oxide alone.14 These studies show that the addition of nitrous oxide can confound interpretation of the effects of isoflurane alone. In our studies, 100% oxygen was used as the carrier gas.
The finding in the present study that young adult rats were more vulnerable to isoflurane-induced memory impairments than the older rats was unexpected and is difficult to explain. The blood pressure was lower, but not significantly, in young adult rats than in the middle-aged rats during exposure to isoflurane. The MAPs were within physiological limits throughout the experiment. Greater vulnerability of very young animals to anaesthesia has been reported, albeit in neonates or juveniles,15–18 but these studies are also not without their opposing reports. Fetal (day 21 of gestation) or postnatal day 7 mice exposed to isoflurane for 6 h showed no cognitive deficits in the Morris water maze when tested as adults despite immediate increased brain cell degeneration.19,20In vitro, isoflurane inhibited synaptic plasticity in the hippocampus of both juvenile and adult mice, with greater effects seen in juveniles.18 The effect of isoflurane on synaptic plasticity in the aged brain under these circumstances is unknown. These studies illustrate that a clear age-dependent effect of isoflurane on memory processes is unpredictable.
In the current study, the memory deficit found in the probe trial in young adult isoflurane-treated rats 1 week after exposure was small but statistically significant and indicative of a detrimental effect on memory. Working memory in this task is defined by the ability of the rat to recall the location of the platform during each daily session once the platform has been found on that day; reference memory is the ability to recall the platform position over longer periods between days.21 In the Morris water maze protocol used in the present study, the platform location is fixed for the entire experiment for each rat and so working and reference memory are not distinguishable during the acquisition of the task. However, deficits in the probe trial in the present study do suggest isoflurane-induced defects in reference memory. The two other studies employing the Morris water maze to assess cognition following anaesthesia have also used a fixed platform protocol. An isoflurane-induced deficit in Morris water maze performance was reported by Zhang et al.22 in both young adult and aged rats (20 months old) after the same dose and duration of isoflurane used in the present study. However, the deficit was in latency to locate the platform which we did not find. Although a probe trial was conducted, no specific results were described by the authors. The anaesthetic carrier gas was not reported. Stratmann et al.6 reported a long-term improvement in spatial reference memory 4 months after exposure to 1-MAC isoflurane for 4 h, evidenced by a greater proportion of time spent in the target quadrant compared with controls. Again, the carrier gas was not reported. The great variation in effects of isoflurane exposure suggests a complex interaction of this agent with memory and with age which warrants further investigation. Further studies are needed to try and determine what factors are responsible for differences in vulnerability to developing specific cognitive deficits after isoflurane exposure.
Twenty-four hours after maze acquisition, middle-aged rats were able to recall the platform location and spent more time in the target quadrant compared with other quadrants. However, when tested 4 weeks after exposure to isoflurane, middle-aged rats showed no preference for any quadrant in the pool, in contrast to sham-exposed middle-aged rats, which retained a preference for the target quadrant. It is unlikely that the lack of memory for platform location in isoflurane-exposed rats in the 4-week probe trial is due to the middle-aged rats not acquiring memory in the acquisition phase because the middle-aged rats exposed to isoflurane showed a clear preference for the target quadrant in the 1-week probe trial. A statistically significant difference between treatments in preference for the target quadrant was not found but within-treatment differences in quadrant preferences may suggest an isoflurane-induced deficit in long-term reference memory, although this effect is subtle.
In the present study, exposure to isoflurane had no significant effect on acquisition of the water maze task, but middle-aged rats performed worse than young adult rats both in the latency to locate the platform and in the distance travelled. These differences could not be explained by differences in swim speed because middle-aged rats swam faster than the young rats, suggesting possible differences in strategies used to locate the platform between the two age groups. Examination of swim paths showed that middle-aged rats spent more time swimming around the edges of the pool (thigmotaxis). All rats tend to swim around the edges looking for an escape during the first trials in the water maze but after being placed on the platform at the end of each trial, rats gradually learn that there is an escape platform and will venture into the centre of the pool to locate it. In the present study, by the second daily session, young adult rats had learnt the task and rapidly located the platform. However, middle-aged rats were still looking for an escape and spent significantly more time in thigmotaxis, resulting in increased latency to locate the platform. This most probably represents slower learning in the middle-aged rats compared with young adult rats, similar to previous findings that older rats learn the task but require more trials to reach the same level of performance.23,24 Reduced spatial memory with ageing may play a role in these observations.25,26
The mechanism through which isoflurane exposure leads to memory impairment is unknown. Hallmarks of neurodegenerative diseases such as Alzheimer's disease, which is characterised by memory deficits, have been identified in mouse brain after exposure to isoflurane.27–30 Zhang et al.22 showed that the N-methyl-D-aspartate (NMDA) partial antagonist memantine attenuated isoflurane-induced elevation in cytosolic calcium, caspase-3 activation and apoptosis. They postulated that, although normal NMDA receptor function is necessary for learning and memory, isoflurane may cause overactivation of NMDA receptors, leading to excess calcium influx and apoptosis which in turn could lead to memory deficits. This research group recently reported that isoflurane may promote Alzheimer's neuropathogenesis by causing neuroinflammation.31
Recent studies have reported isoflurane-induced alterations in brain metabolism in mice.32 Marked dose-dependent increases in extracellular lactate and pyruvate concentrations were detected in the striatum and hippocampus during exposure to isoflurane for 90 min, increasing rapidly at the onset of anaesthesia and declining gradually after cessation. Although metabolic alterations could potentially influence cognitive outcomes,33 unpublished studies in our laboratory investigating sevoflurane have shown no cognitive deficits in young adult rats, whereas Horn and Klein32 found similar metabolic changes with both isoflurane and sevoflurane, arguing against anaesthesia-induced transient increases in lactate concentration as a cause of delayed cognitive deficits.
In murine acute hippocampal slice preparations, isoflurane blocks synaptic plasticity (long-term potentiation, LTP – a presumed cellular correlate of memory) in a dose-dependent manner but only in a relatively narrow dose range34; only concentrations higher than 1-MAC blocked LTP. Dose-dependent effects of isoflurane on memory have been reported,35 but have not yet been explored in a study of POCD. Variations in responses to 1-MAC isoflurane between young adult and aged rats used in the present study could conceivably have influenced cognitive outcome but further work is needed to explore this possibility.
There were some limitations to the present study. Young adult rats were not retained for further testing at 4 weeks and, hence, comparisons between young and middle-aged rats in the 4-week memory test were not possible. Further studies investigating long-term outcomes are required to determine persistence of anaesthetic effects. An additional limitation of this study is that histological and neuropathological analyses were not carried out after treatment to determine differential effects of isoflurane between groups.
In conclusion, the present study has found a small but significant deficit in memory in young adult rats following exposure to isoflurane at 1 MAC in 100% oxygen for 4 h and a subtle delayed detrimental effect on memory in middle-aged rats.
This work was supported by the National Heart Foundation, Australia (grant number 809274).
None for J.K.C. or N.C.J. C.F.R. has received funding for clinical studies, travel and consultancy from Baxter Healthcare which is an organisation that could have an interest in the drug being studied.
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