Isoflurane Anesthesia Impairs Spatial Memory Recall And Its Related CREB And Egr1 Expression In Aged Mice

Isoflurane anesthesia impairs spatial memory recall and its related CREB and Egr1 expression in aged miceBackground and purposePostoperative cognitive dysfunction (POCD) is a decline in cognitive performance after surgery and anesthesia especially in elderly person. According to the result of a multicenter research conducted by ISPOCD (international study of POCD) group, the incidence of POCD in persons older than 65 years old receiving noncardiac surgery is 25.8% in 1 week after surgery, and 9.9% in 3 months after surgery. The inhaled anesthetic acts on central nervous system in a very delicate way whose actual mechanism is still far from being elucidated. However, it has been demonstrated that the inhaled anesthetics enhance GABA_A receptor while inhibiting NMDA receptor. The exact role of anesthesia in the development of POCD is still unclear. Some studies show that isoflurane has some residual memory effect after weaning from anesthesia. CREB and Egr1 are two important transcript modulator closely related to spatial memory, synaptic plasticity as well as the function of GABA_A and NMDA receptor. Our purpose is to clarify the effect of isoflurane on the recall of an already-learned spatial memory task and, to probe into its effect on CREB and Egr1 expression after recall of spatial memory.MethodsAged C57/BL6 mice were trained in Morris water maze (MWM) for 14 days. The criteria for memory formation is to make the distance percentage of swimming in the quadrant of platform exceed 30% in the probe test. After the acquisition of the memory of a hidden platform, the mice were allocated to isoflurane anesthetized group, oxygen control group and air control group. The mice of isoflurane anesthetized group were anaesthetized with isoflurane (1MAC, 4 hours). The probe tests were repeated at 1 day, 1 week and 1 month after anesthesia. Two way repeated measurement ANOVA combined with Bonferroni post test was employed to test the difference of target quadrant distance percentage of the three groups in different time points. Some other mice with a formed memory were allocated to WM group, WM+Ane group and naive group which received only watermaze probe test, watermaze probe test plus anesthesia and no treatment respectively. The protein level of Egr1 was detected by immunoflurescence and immunoblotting and p-CREB by immunoflurescence at 2 hours after probe test in the three time points mentioned above. Furthermore, the mRNA levels of Egr1 and CREB were detected with real time RT-PCR at 1hour after probe test. The inter-group difference of integral optimal densities (IOD) in immunoflurescent image, relative densities of blots in immunoblotting and normalized gene copies in RT-PCR were compared with one way ANOVA.ResultsThe mice did not show signs of anoxia all through the anesthesia. The results of arterial gas analysis after anesthesia were normal. We found that on 1 day and 1 week after anesthesia, the target quandrant distance percentage decreased dramatically which indicated the mice have difficulties in reading out the previously formed memory. Interesingly, the inhibition effect disappeared at 1 month post anestheia. The statistic results are as follows: on 1 day after anesthesia, Ane vs Actl: t=4.587, P<0.001, Ane vs Vctl: t=3.822, P<0.001; on 1 week after anesthesia, Ane vs Actl: t=2.733, P<0.05, Ane vs Vctl: t=2.621, P<0.05. The protein and mRNA level of Egr1 is profoundly inhibited by isoflurane anesthesia in 1 day and 1 week after anesthesia. However, the level of p-CREB was inhibited on 1 day postanesthesia and recovered on 1 week.Discussion and conclusionMWM is a well accepted tool for evaluating the learning and memory of rodent animal. There have been some researches about the memory effect of isoflurane. They concluded that spatial memory acquisition (12-arm radial maze) is impaired after isoflurane anesthesia in aged rats. Our research differs from those reports in that we proved that isoflurane anesthesia impairs the spatial memory retrieving, that is the read out of previously existed memory. Since the distance percentages of the target quadrant of the three groups were not different before anesthesia, and there were no promiscuous effect from hypoxia or hypotension, we conclude that the significant difference on 1 day and 1 week is the effect of isoflurane. Furthermore, we observed that the protein and mRNA level of Egr1 after place recall test was dramatically lower in anesthetized mice compared with those without anesthesia. The temporal evolution of Egr1 and CREB inhibition coincided with that of behavioral parameters. Taken together, we concluded that isoflurane anesthesia impairs the retrieving of an existed spatial memory in aged C57/BL6 mice, probably through inhibiting the Egr1 and CREB expression and CREB activation closely related to the spatial memory recall. Subclinical concentration of sevoflurane potentiates neuronal apoptosis in the developing C57BL/6 mouse brainObjectiveAdvances in the pediatric surgery have increased the duration of neonatal exposure to anesthetic agents. To minimize the associated risks, it is necessary to investigate the influence of these agents on the developing brain. Although the acting mechanism of general anesthetics remains unclear, mounting evidence supports the theory that some anesthetics act on postsynaptic receptors. Considering the GABAmimetic or NMDA antagonistic properties of general anesthetics and their wide use in pediatric anesthesia as well as occasional recreational abuse in pregnant woman (in the case of ketamine), the effect of these agents on infant brain has received growing attention. Sevoflurane, with its lowpungency, non-irritating odor, and low blood/gas partition coefficient, is widely used in pediatric anesthesia. There is still a lack of studies concerning sevoflurane's effect on the developing brain. Therefore, we sought to investigate the influence of sevoflurane on cell apoptosis in the developing brains of C57BL/6 mice through the detection of cleaved caspase-3 and single-strand DNA (ssDNA), as well as a morphological study of apoptotic cells using a transmission electron microscopy.Materials and MethodsSeven-day-old (P7) mice from litters that had a mean body weight in the range of 2.5 to 3.5 g were used in all experiments. Pups from the same litter were randomly allocated to sevoflurane, ethanol, vehicle, air or blank control group. The left pups were used for detection of blood glucose after sevoflurane anesthesia (n=11). The duration of exposure was 2 hours. Anesthetics was inhaled through a transparent chamber in which 0.75MAC sevoflurane in oxygen was introduced through a portal on one side and the other side was connected to a sensing device gas analyzer to measure the concentration of anesthetics, oxygen and carbon dioxide. The flow rate of oxygen was regulated to keep the concentration of O₂ around 30% and CO₂ less than 1%. Volatile anesthetics were provided by vaporizer. Pups in vehicle and air group inhaled 30% O₂ and room air respectively. Pups in the blank control group were kept with their mother. For ethanol positive control, pups were injected subcutaneously with 2.5g/kg ethanol twice at 0h and 2h. Ethanol was prepared as 20% solution with normal saline. Twelve hours after exposure, the pups were euthanized. For the electon microscopy, the mice were sacrificed at 24 hours after anesthesia. Apoptotic detection methods included immunofluorescence stain of activated caspase-3 and ssDNA, DAPI stain and immunoblotting of activated caspase-3. The morphology of apoptotic neuron was confirmed by electromicroscopy.ResultsThere were no obvious clinical signs of hypoxia in the treated mice throughout the experimental period. No hypoglycemia happened in the sevoflurane inhaled pups. Both activated caspase-3 and ssDNA positive cell percentages in sevoflurane group were significantly higher than that of vehicle, air or blank control group (P<0.001). DAPI stain displayed many cells with condensed chromatin in sevoflurane group as well as in ethanol group. We did not find difference among the three control groups, so only blank control pups were used in immunoblotting assay. The band densities of sevofurane group were significantly greater than those of control group but less than ethanol group. We observed the typical ultrastructural features of ongoing apoptosis, including cytoplasmic shrinkage and nuclear condensation in the sevoflurane treated mouse brains.Discussion and ConclusionCaspase-3 is activated in apoptotic cascade and ultimately leads to breakdown of cellular components. Antibody for ssDNA stains only the condensed chromatin from apoptotic cells, more specifically than traditional TUNEL technique. Using these two methods combined with electron microscopy confirmed apoptotic morphology, we have proved sevoflurane (0.75MAC, 2 hours) could induce significant neuroapoptosis in brains of postnatal C57BL/6. Although it is inappropriate to discredit the safety of sevoflurane in pediatric anesthesia through our results, it suggests that it is necessary to perform clinical investigation about sevoflurane's effect on developing brain.