ORIGINAL ARTICLE
Effects of sevoflurane on learning, memory, and expression of pERK1/2 in hippocampus in neonatal rats X. Yu1,2, Y. Liu1,2, S. Bo1,2 and L. Qinghua1,2 1 2
Department of Anesthesiology, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China Department of Anesthesiology, Yichang Central People’s Hospital, Yichang, China
Correspondence Xiangdi Yu, Department of Anesthesiology, The First College of Clinical Medical Science, China Three Gorges University, Yichang 443003, China E-mail: [email protected] Conflict of interest The authors declare no conflicts of interest. Funding This study was supported by YiChang Science and Technology Bureau (No. A14-30323). Submitted 13 June 2014; accepted 22 September 2014; submission 9 May 2014. Citation Yu Xiangdi, Liu Y, Bo S, Qinghua L. Effects of sevoflurane on learning, memory, and expression of pERK1/2 in hippocampus in neonatal rats. Acta Anaesthesiologica Scandinavica 2014 doi: 10.1111/aas.12433
Background: Sevoflurane may be associated with neural toxicity in the developing brain, but the mechanism is still unclear. Phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) are important for developing neurons. The aim of our study was to investigate the effects of sevoflurane on spatial learning and memory and on expression of pERK1/2 in hippocampus of neonatal rats. Methods: Sixty-three neonatal rats were randomly divided into three groups: control group, sevoflurane (sevo) group, and sham group. Rats in the control group were placed in a plastic chamber flushed continuously for 4 h with air alone, rats in the sevo group were exposed in 5% sevoflurane and air for 4 h, and rats in the sham group were exposed in 5% carbon dioxide and air for 4 h, with identical flow rates for all groups. All three groups were subjected to Morris water maze test 1 day after sevoflurane exposure. Moreover, expression of pERK1/2 was determined by immunochemistry and Western blot at 1, 3, and 6 weeks after exposure. Results: Compared with the control group, the escape latency was longer in sevo group and the expression of pERK1/2 was significantly inhibited in the sevo group (P < 0.01); no differences between control and sham groups were observed. Conclusion: Our study demonstrated that neonatal rats exposed to sevoflurane had impaired spatial learning and memory, and this may be attributed to decreased pERK1/2 in the hippocampus.
Several experimental studies have shown that sevoflurane induces neural toxicity in the developing brain and causes long-lasting neurocognitive dysfunction and learning disabilities.1–5 The study demonstrated that sevoflurane toxicity is related to activation of γ-aminobutyric acid and to inhibition of N-methyl-D-aspartate receptors.6–8 However, the mechanism of sevoflurane toxicity is still unclear. Hippocampal formation has been implicated in many learning and memory processes.9–13 Therefore, the learning and memory function is related to hippocampus, assuming that sevoflurane could
cause hippocampal impairments. Behavioral studies and hippocampal synaptic plasticity indicate that extracellular signal-regulated kinases 1/2 (ERK1/2) activation leads to induction of long-term potentiation (LTP), which contributes to memory formation.14 ERKs are widely expressed protein kinase intracellular signaling molecules that are involved in functions including the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. ERKs consist of two similar protein kinases originally called ERK1 and ERK2. phosphorylation of ERKs leads to the activation of their kinase activity.15 Acta Anaesthesiologica Scandinavica 59 (2015) 78–84
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The data of Straiko et al. indicate that phosphorylated ERK1/2 (pERK1/2) is the prosurvival signaling in developing neurons,16 so it is the key protein kinase for developing brain. The purpose of our study was to evaluate the effect of sevoflurane on spatial learning and memory and the expression of pERK1/2 in hippocampus in neonatal Sprague-Dawley (SD) rats.
Materials and methods Animals and anesthesia treatment This study was approved by the Committee for Animal Research at Three Gorges University (Hubei, China). Post-natal day 14 (p14) male Sprague-Dawley rats (n = 63) were raised by the Laboratory Animal Center of Three Gorges University. The animals were kept under a 12-h light–dark cycle, and room temperature was maintained at 22 ± 1°C. Experiments were conducted with the approval of the Animal Care Committee of Three Gorges University. Sixty-three rats were randomly divided into three groups, 21 rats per group: control group, sevoflurane (sevo) group, and sham group. The rats in the control group were placed in a chamber flushed continuously for 4 h with air alone; the rats in the sevo group were exposed in 5% sevoflurane and air for 4 h. To eliminate the influence of blood gas abnormalities caused by sevofluraneinduced respiratory suppression, the rats in the sham group were exposed in 5% carbon dioxide and air for 4 h. The flow rates were identical for all groups.
Arterial blood gas analysis In order to determine adequacy of ventilation and exclude the hypoxemia induced by anesthesia, nine rats were removed from three groups at random and arterial blood analysis was performed as the following: A single blood sample (100 μl) was obtained from the left cardiac ventricle immediately at the end of anesthesia. Bicarbonate concentration (in millimole per liter), oxygen saturation (%), pH, PaCO2 (partial pressure of carbon dioxide in mmHg), and PaO2 (partial pressure of oxygen in mmHg) were determined immediately after blood collection using an
i-STAT 200 blood gas analyzer (Abbott Labs Corporate, Abbott Park, IL, USA). Behavioral studies We followed the procedure established by Morris.17 The test was performed by recording the route and time of rats in Morris water maze (Three Gorges University). The circular pool (diameter, 180 cm; depth, 60 cm) was filled with warm (25 ± 1.0°C) opaque water and divided into four quadrants; at the center of the pool, a platform (diameter, 10.3 cm) was placed. Rats were subjected to three consecutive training days to familiarize them with finding and perching on the hidden platform that was maintained in a fixed location. At the start of each trial, rats were placed in the pool facing the wall and were allowed to swim for 60 s or until the platform was found. If the rat did not find the platform during the trial, it was guided to the platform and stayed there for 15 s. The tests were started at 8:00 in the morning after 1 week, 3 weeks, and 6 weeks for seven consecutive days after exposure into sevoflurane. The time to reach the platform (latency) and path lengths were recorded by software. Immunohistochemical staining On day 7, 21, and 42 after exposure, three rats that were picked up from each group were randomly selected and anesthetized with pentobarbital sodium (50 mg/k, i.p.) perfused through the ascending aorta with 0.9% NaCl, followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Hippocampi were dissected and embedded in paraffin. Cross-sections of paraffin-embedded tissue (5 μm) were cut and mounted on Vectabond adhesive-coated slides. Sections were deparaffinized in xylene and rehydrated in ethanol to water, then washed in PBS, and were incubated with the primary antibody (pERK1/2, 1 : 100; Cell Signaling, Danvers, MA, USA) at 4°C for 24 h. The sections were washed twice with PBS and incubated in goat anti-rabbit serum (1 : 200) (Chemicon Group, Danvers, MA, USA) for 1 h. This step was followed by three washes in PBS and staining using the ABC kit (Vector Laboratories, Burlingame, CA, USA). The 3,3’diaminobenzidine (DAB) chromogenic reaction
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was monitored for about 5 min and PBS was used to stop the reaction in time. Western blot analysis On day 7, 21, and 42 after exposure, three rats that were picked up from each group randomly anesthetized with pentobarbital sodium (50 mg/k, i.p.) were prepared for dissecting hippocampus out. The lysates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and the proteins were transferred onto cellulose acetate membranes. The membranes were incubated with primary antibody against pERK1/2 (pERK1/2, 1 : 1000, rabbit polyclonal; Cell Signaling) or β-tubulin (1 : 2000, mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA), then followed incubation with a secondary antibody. The membranes were developed using chemiluminescent agent (Millipore, Bedford, MA, USA), and the bands were analyzed by software Gel-Pro Analyzer (Media Cybernetic, Rockville, MD, USA). Statistical analyses Results were expressed as the mean ± standard error of at least three separate experiments. Results were analyzed by one-way analysis of variance followed by the Fisher’s Least Significant Difference (LSD) test. Differences with P values of < 0.05 were considered significant. Results The rats appeared pink throughout the 4-h sevoflurane exposure, suggesting no effect in metabolism or respiration. Animals recovered rapidly
after anesthesia. Blood gas analyses indicated no significant difference from the control group (P > 0.05; Table 1). The test in the Morris water maze revealed that basic value of escape latency and path length were not different among the three groups (P > 0.05). Rat spatial memory was impaired 1 week and 3 weeks after sevoflurane exposure. In terms of path length and escape latency, sevoflurane-treated rats required more time to find the platform (P < 0.05) (Table 2). The path length in sevoflurane group was significantly increased compared with the two other groups (P < 0.05) (Table 2). Until 6 weeks after exposure, the observed impairment still existed, as path length and escape latency were both increased compared with the two other experimental groups (P < 0.05) (Table 2). Immunohistochemical staining and Western blot analyses of pERK1/2 in the hippocampal CA1 areas were performed in all three groups at 1 week, 3 weeks, and 6 weeks after sevoflurane exposure. Compared with the control groups, sevoflurane-treated rats exhibited a timedependent decrease in pERK1/2 expression in hippocampal CA1 area. The expression of pERK1/2 was significantly different between the
Table 1 Arterial blood gas analyses. Arterial blood gas
Control
Sevoflurane
Sham
pH PaCO2 (mmHg) PaO2 (mmHg) SaO2 (%)
7.41 ± 0.03 27.9 ± 4.0 90.9 ± 7.0 94.0 ± 3.0
7.39 ± 0.05 30.2 ± 3.0 87.9 ± 6.0 92.9 ± 3.5
7.40 ± 0.06 29.9 ± 3.2 88.7 ± 5.6 93.5 ± 2.6
Exposure to sevoflurane does not induce significant metabolic or respiratory dysfunction. Arterial blood gas analyses revealed no significant difference in any measured parameters between the sevoflurane-exposure group and controls (P > 0.05).
Table 2 Comparison of escape latency and path length. Group
Basic value
1 week
3 weeks
6 weeks
Control group escape latency (s) Path length (cm) Sevoflurane group escape latency (s) Path length (cm) Sham group escape latency (s) Path length (cm)
120.07 ± 20.13 2551 ± 523 119.39 ± 31.07 2495 ± 497 123.57 ± 22.48 2631 ± 601
103.15 ± 16.7 2203 ± 412 110.75 ± 18.4* 2240 ± 400* 100.97 ± 17.4 2210 ± 390
90.78 ± 15.3 1950 ± 305 100.74 ± 16.6* 2219 ± 300* 89.79 ± 21.3 1890 ± 400
70.41 ± 12.7 1839 ± 480 96.71 ± 11.3* 2010 ± 360* 73.87 ± 17.4 1800 ± 370
*P < 0.05. Compared with control group.
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1 week
3 weeks
6 weeks
C group
S group
Fig. 1. Immunohistochemical staining of pERK1/2 with positive cells in control (C) group, sham (S) group, and sevoflurane (sevo) group, with similar numbers of positive cells in C group and S group and lower numbers of positive cells in sevo group (*P < 0.05 vs. S group, **P < 0.01 vs. S group). The expression of pERK1/2 was significantly different between the sevo group and the C group in the hippocampal CA1 area of rats after 3 weeks (*P < 0.05) and 6 weeks (**P < 0.01). pERK1/2, phosphorylated extracellular signal-regulated kinases.
sevoflurane and control groups in the hippocampal CA1 area of rats after 3 weeks (P < 0.05) and 6 weeks (P < 0.01), but not after 1 week (P > 0.05) (Figs 1 and 2). Discussion Neonatal rats exposed to sevoflurane have impaired spatial learning and memory ability 3 weeks later, and the impairment lasted until 6
weeks after exposure to sevoflurane in our experiments. By immunohistochemistry and Western blot analysis, we show that exposure of neonatal rats to sevoflurane inhibits the expression of pERK1/2 in the CA1 region of the hippocampus. The developing brain has several significant differences from the adult brain that provide a physiological basis for enhanced vulnerability to anesthetics. Early in development, the number of neurons formed is significantly greater than in
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1 week
3 weeks
6 weeks
Sevo group
Western blot 1 week after exposed to sevoflurane
Western blot 3 week after exposed to sevoflurane
Western blot 6 week after exposed to sevoflurane
Fig. 2. Western blot analysis showed that the pERK1/2 protein expression followed the same pattern, with the similar level of expression in control (C) group and sham (S) group and lower level expression in sevoflurane (sevo) group (*P < 0.05 vs. S group, **P < 0.01 vs. S group). The expression of pERK1/2 was significantly different between the sevo group and the C group in the hippocampal CA1 area of rats after 3 weeks (*P < 0.05) and 6 weeks (**P < 0.01). pERK1/2, phosphorylated extracellular signal-regulated kinases.
adult mammals. At the same time, there is an exuberant burst of synapse formation (synaptogenesis) before synapses are eventually pruned to establish behaviorally relevant connections between neurons. Programmed cell death, or apoptosis, is responsible for the elimination of 50–70% of developing neurons under normal
circumstances.15,16 One significant difference between the immature and the mature mammalian brain with neuropharmacological implications is the developmentally regulated reversal of the transmembrane chloride gradient. This is relevant to anesthetic effects as many anesthetic agents enhance the activity of gammaActa Anaesthesiologica Scandinavica 59 (2015) 78–84
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aminobutyric acid type A (GABAA) receptors.17,18 Furthermore, GABAA receptor was demonstrated to play an important role in the process of learning and memory.19–22 Previous studies have suggested that the phosphorylation of ERK may be affected by the functional modulation of GABAA receptor under some stress conditions.23–25 A previous study has indicated that pERK1/2 is necessary for the induction of LTP in the hippocampus, which participates in learning and memory.26 pERK1/2 is abundantly expressed in neurons of the central nervous system and is associated with cell growth, proliferation, differentiation, and migration. Moreover, pERK1/2 coordinates neuronal responses to extracellular signals and may improve learning and memory by regulating synaptic remodeling, axonal growth, LTP formation, and neuronal excitability.26,27 In this study, we used 4% sevoflurane that did not inhibit respiration and circulation in rats. Arterial blood analyses confirmed that none of the rats experienced hypoxemia or hypercapnia during the 4-h sevoflurane exposure. In addition, no differences in the arterial blood gas analyses were shown in different groups. Thus, we hypothesize that duration of sevoflurane exposure is the key factor that inhibits the expression of pERK1/2 and impairs the spatial learning and memory ability of neonatal rats as they grow up. Our findings demonstrate that toxicity of sevoflurane is related to the activation of γ-aminobutyric acid by affecting the phosphorylation of ERK expression in the hippocampus. These findings are consistent with other recent reports that the administration of ketamine or propofol to postnatal day (PND) mouse pups decreased pERK1/2 expression and led to neuronal apoptosis. Lithium restored pERK1/2 levels and prevented ketamine and propofol-induced injury.28,29 It is important to underline that so far, there have been no convincing studies in humans showing neurotoxicity of sevoflurane resulting in cognitive and learning deficits. In summary, our findings indicate that exposure to sevoflurane impairs spatial learning and memory ability of neonatal rats when they grow up. Its mechanism may be related to inhibition of the expression of pERK1/2 in the hippocampus.
References 1. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009; 110: 628–37. 2. Bercker S, Bert B, Bittigau P, Felderhoff-Müser U, Bührer C, Ikonomidou C, Weise M, Kaisers UX, Kerner T. Neurodegeneration in newborn rats following propofol and sevoflurane anesthesia. Neurotox Res 2009; 16: 140–7. 3. Edwards DA, Shah HP, Cao W, Gravenstein N, Seubert CN, Martynyuk AE. Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology 2010; 112: 567–75. 4. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T. Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 2011; 115: 979–91. 5. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, Sall JW, Rau V, Bickler PE, Lalchandani GR, Yusupova M, Woodward E, Kang H, Wilk AJ, Carlston CM, Mendoza MV, Guggenheim JN, Schaefer M, Rowe AM, Stratmann G. Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 2012; 116: 586–602. 6. Piehl E, Foley L, Barron M, D’Ardenne C, Guillod P, Wise-Faberowski L. The effect of sevoflurane on neuronal degeneration and GABAA subunit composition in a developing rat model of organotypic hippocampal slice cultures. J Neurosurg Anesthesiol 2010; 22: 220–9. 7. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009; 110: 628–37. 8. Fang F, Xue Z, Cang J. Sevoflurane exposure in 7-dayold rats affects neurogenesis, neurodegeneration and neurocognitive function. Neurosci Bull 2012; 28: 499–508. 9. Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982; 297: 681–3. 10. Burgess N, Maguire EA, O’Keefe J. The human hippocampus and spatial and episodic memory. Neuron 2002; 35: 625–41.
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11. Bliss TV, Collingridge GL. A synaptic model of memory: longterm potentiation in the hippocampus. Nature 1993; 361: 31–9. 12. Gruart A, Muz MD, Delgado-Garca JM. Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci 2006; 26: 1077–87. 13. Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science 2006; 313: 1093–7. 14. Selcher JC, Weeber EJ, Christian J, Nekrasova T, Landreth GE, Sweatt JD. A role for ERK MAP kinase in physiologic temporal integration in hippocampalarea CA1. Learn Mem 2003; 10: 26–39. 15. Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases(ERKs) with antipeptide antibodies. Cell Regul 1991; 11: 569–74. 16. Straiko MMW, Young C, Cattano D, Creeley CE, Wang H, Smith DJ, Johnson SA, Li ES, Olney JW. Lithium protects against anesthesia-induced developmental neuroapoptosis. Anesthesiology 2009; 110: 862–8. 17. Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 1984; 11: 47–60. 18. Rakic S, Zecevic N. Programmed cell death in the developing human telencephalon. Eur J Neurosci 2000; 12: 2721–34. 19. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 2005; 135: 815–27. 20. Veyckemans F. Excitation phenomena during sevoflurane anaesthesia in children. Curr Opin Anaesthesiol 2001; 14: 339–43.
21. Sharma AC, Kulkarni SK. Evidence for GABA-BZ receptor modulation in short-term memory passive avoidance task paradigm in mice. Methods Find Exp Clin Pharmacol 1990; 12: 175–80. 22. Kulkarni SK, Sharma K. Alprazolam modifies animal behaviour on elevated plus-maze. Indian J Exp Biol 1993; 31: 908–11. 23. Zarrindast MR, Bakhsha A, Rostami P, Shafaghi B. Effects of intrahippocampal injection of GABAergic drugs on memory retention of passive avoidance learning in rats. J Psychopharmacol 2002; 16: 313–9. 24. Zarrindast MR, Jafari MR, Shafaghi B, Djahanguiri B. Influence of potassium channel modulators on morphine state-dependent memory of passive avoidance. Behav Pharmacol 2004; 15: 103–10. 25. Koya E, Uejima JL, Wihbey KA, Bossert JM, Hope BT, Shaham Y. Role of ventral medial prefrontal cortex in incubation of cocaine craving. Neuropharmacology 2009; 1: 177–85. 26. Kalluri HS, Ticku MK. Role of GABA (A) receptors in the ethanol-mediated inhibition of extracellular signal-regulated kinase. Eur J Pharmacol 2002; 451: 51–4. 27. Bachtell RK, Tsivkovskaia NO, Ryabinin AE. Alcohol-induced c-Fos expression in the Edinger-Westphal nucleus: pharmacological and signal transduction mechanisms. J Pharmacol Exp Ther 2002; 302: 516–24. 28. Selcher JC, Weeber EJ, Christian J, Nekrasova T, Landreth GE, Sweatt JD, et al. A role for ERK MAP kinase in physiologic temporal integration in hippocampalarea CA1. Learn Mem 2003; 10: 26–39. 29. Chin J, Angers A, Cleary LJ, Eskin A, Byrne JH. Transforming growth factor beta1alters synapsin distribution and modulates synaptic depression in Aplysia. J Neurosci 2002; 22: RC220.
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Effects of sevoflurane on learning, memory, and expression of pERK1/2 in hippocampus in neonatal rats X. Yu1,2, Y. Liu1,2, S. Bo1,2 and L. Qinghua1,2 1 2
Department of Anesthesiology, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China Department of Anesthesiology, Yichang Central People’s Hospital, Yichang, China
Correspondence Xiangdi Yu, Department of Anesthesiology, The First College of Clinical Medical Science, China Three Gorges University, Yichang 443003, China E-mail: [email protected] Conflict of interest The authors declare no conflicts of interest. Funding This study was supported by YiChang Science and Technology Bureau (No. A14-30323). Submitted 13 June 2014; accepted 22 September 2014; submission 9 May 2014. Citation Yu Xiangdi, Liu Y, Bo S, Qinghua L. Effects of sevoflurane on learning, memory, and expression of pERK1/2 in hippocampus in neonatal rats. Acta Anaesthesiologica Scandinavica 2014 doi: 10.1111/aas.12433
Background: Sevoflurane may be associated with neural toxicity in the developing brain, but the mechanism is still unclear. Phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) are important for developing neurons. The aim of our study was to investigate the effects of sevoflurane on spatial learning and memory and on expression of pERK1/2 in hippocampus of neonatal rats. Methods: Sixty-three neonatal rats were randomly divided into three groups: control group, sevoflurane (sevo) group, and sham group. Rats in the control group were placed in a plastic chamber flushed continuously for 4 h with air alone, rats in the sevo group were exposed in 5% sevoflurane and air for 4 h, and rats in the sham group were exposed in 5% carbon dioxide and air for 4 h, with identical flow rates for all groups. All three groups were subjected to Morris water maze test 1 day after sevoflurane exposure. Moreover, expression of pERK1/2 was determined by immunochemistry and Western blot at 1, 3, and 6 weeks after exposure. Results: Compared with the control group, the escape latency was longer in sevo group and the expression of pERK1/2 was significantly inhibited in the sevo group (P < 0.01); no differences between control and sham groups were observed. Conclusion: Our study demonstrated that neonatal rats exposed to sevoflurane had impaired spatial learning and memory, and this may be attributed to decreased pERK1/2 in the hippocampus.
Several experimental studies have shown that sevoflurane induces neural toxicity in the developing brain and causes long-lasting neurocognitive dysfunction and learning disabilities.1–5 The study demonstrated that sevoflurane toxicity is related to activation of γ-aminobutyric acid and to inhibition of N-methyl-D-aspartate receptors.6–8 However, the mechanism of sevoflurane toxicity is still unclear. Hippocampal formation has been implicated in many learning and memory processes.9–13 Therefore, the learning and memory function is related to hippocampus, assuming that sevoflurane could
cause hippocampal impairments. Behavioral studies and hippocampal synaptic plasticity indicate that extracellular signal-regulated kinases 1/2 (ERK1/2) activation leads to induction of long-term potentiation (LTP), which contributes to memory formation.14 ERKs are widely expressed protein kinase intracellular signaling molecules that are involved in functions including the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. ERKs consist of two similar protein kinases originally called ERK1 and ERK2. phosphorylation of ERKs leads to the activation of their kinase activity.15 Acta Anaesthesiologica Scandinavica 59 (2015) 78–84
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The data of Straiko et al. indicate that phosphorylated ERK1/2 (pERK1/2) is the prosurvival signaling in developing neurons,16 so it is the key protein kinase for developing brain. The purpose of our study was to evaluate the effect of sevoflurane on spatial learning and memory and the expression of pERK1/2 in hippocampus in neonatal Sprague-Dawley (SD) rats.
Materials and methods Animals and anesthesia treatment This study was approved by the Committee for Animal Research at Three Gorges University (Hubei, China). Post-natal day 14 (p14) male Sprague-Dawley rats (n = 63) were raised by the Laboratory Animal Center of Three Gorges University. The animals were kept under a 12-h light–dark cycle, and room temperature was maintained at 22 ± 1°C. Experiments were conducted with the approval of the Animal Care Committee of Three Gorges University. Sixty-three rats were randomly divided into three groups, 21 rats per group: control group, sevoflurane (sevo) group, and sham group. The rats in the control group were placed in a chamber flushed continuously for 4 h with air alone; the rats in the sevo group were exposed in 5% sevoflurane and air for 4 h. To eliminate the influence of blood gas abnormalities caused by sevofluraneinduced respiratory suppression, the rats in the sham group were exposed in 5% carbon dioxide and air for 4 h. The flow rates were identical for all groups.
Arterial blood gas analysis In order to determine adequacy of ventilation and exclude the hypoxemia induced by anesthesia, nine rats were removed from three groups at random and arterial blood analysis was performed as the following: A single blood sample (100 μl) was obtained from the left cardiac ventricle immediately at the end of anesthesia. Bicarbonate concentration (in millimole per liter), oxygen saturation (%), pH, PaCO2 (partial pressure of carbon dioxide in mmHg), and PaO2 (partial pressure of oxygen in mmHg) were determined immediately after blood collection using an
i-STAT 200 blood gas analyzer (Abbott Labs Corporate, Abbott Park, IL, USA). Behavioral studies We followed the procedure established by Morris.17 The test was performed by recording the route and time of rats in Morris water maze (Three Gorges University). The circular pool (diameter, 180 cm; depth, 60 cm) was filled with warm (25 ± 1.0°C) opaque water and divided into four quadrants; at the center of the pool, a platform (diameter, 10.3 cm) was placed. Rats were subjected to three consecutive training days to familiarize them with finding and perching on the hidden platform that was maintained in a fixed location. At the start of each trial, rats were placed in the pool facing the wall and were allowed to swim for 60 s or until the platform was found. If the rat did not find the platform during the trial, it was guided to the platform and stayed there for 15 s. The tests were started at 8:00 in the morning after 1 week, 3 weeks, and 6 weeks for seven consecutive days after exposure into sevoflurane. The time to reach the platform (latency) and path lengths were recorded by software. Immunohistochemical staining On day 7, 21, and 42 after exposure, three rats that were picked up from each group were randomly selected and anesthetized with pentobarbital sodium (50 mg/k, i.p.) perfused through the ascending aorta with 0.9% NaCl, followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Hippocampi were dissected and embedded in paraffin. Cross-sections of paraffin-embedded tissue (5 μm) were cut and mounted on Vectabond adhesive-coated slides. Sections were deparaffinized in xylene and rehydrated in ethanol to water, then washed in PBS, and were incubated with the primary antibody (pERK1/2, 1 : 100; Cell Signaling, Danvers, MA, USA) at 4°C for 24 h. The sections were washed twice with PBS and incubated in goat anti-rabbit serum (1 : 200) (Chemicon Group, Danvers, MA, USA) for 1 h. This step was followed by three washes in PBS and staining using the ABC kit (Vector Laboratories, Burlingame, CA, USA). The 3,3’diaminobenzidine (DAB) chromogenic reaction
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was monitored for about 5 min and PBS was used to stop the reaction in time. Western blot analysis On day 7, 21, and 42 after exposure, three rats that were picked up from each group randomly anesthetized with pentobarbital sodium (50 mg/k, i.p.) were prepared for dissecting hippocampus out. The lysates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and the proteins were transferred onto cellulose acetate membranes. The membranes were incubated with primary antibody against pERK1/2 (pERK1/2, 1 : 1000, rabbit polyclonal; Cell Signaling) or β-tubulin (1 : 2000, mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA), then followed incubation with a secondary antibody. The membranes were developed using chemiluminescent agent (Millipore, Bedford, MA, USA), and the bands were analyzed by software Gel-Pro Analyzer (Media Cybernetic, Rockville, MD, USA). Statistical analyses Results were expressed as the mean ± standard error of at least three separate experiments. Results were analyzed by one-way analysis of variance followed by the Fisher’s Least Significant Difference (LSD) test. Differences with P values of < 0.05 were considered significant. Results The rats appeared pink throughout the 4-h sevoflurane exposure, suggesting no effect in metabolism or respiration. Animals recovered rapidly
after anesthesia. Blood gas analyses indicated no significant difference from the control group (P > 0.05; Table 1). The test in the Morris water maze revealed that basic value of escape latency and path length were not different among the three groups (P > 0.05). Rat spatial memory was impaired 1 week and 3 weeks after sevoflurane exposure. In terms of path length and escape latency, sevoflurane-treated rats required more time to find the platform (P < 0.05) (Table 2). The path length in sevoflurane group was significantly increased compared with the two other groups (P < 0.05) (Table 2). Until 6 weeks after exposure, the observed impairment still existed, as path length and escape latency were both increased compared with the two other experimental groups (P < 0.05) (Table 2). Immunohistochemical staining and Western blot analyses of pERK1/2 in the hippocampal CA1 areas were performed in all three groups at 1 week, 3 weeks, and 6 weeks after sevoflurane exposure. Compared with the control groups, sevoflurane-treated rats exhibited a timedependent decrease in pERK1/2 expression in hippocampal CA1 area. The expression of pERK1/2 was significantly different between the
Table 1 Arterial blood gas analyses. Arterial blood gas
Control
Sevoflurane
Sham
pH PaCO2 (mmHg) PaO2 (mmHg) SaO2 (%)
7.41 ± 0.03 27.9 ± 4.0 90.9 ± 7.0 94.0 ± 3.0
7.39 ± 0.05 30.2 ± 3.0 87.9 ± 6.0 92.9 ± 3.5
7.40 ± 0.06 29.9 ± 3.2 88.7 ± 5.6 93.5 ± 2.6
Exposure to sevoflurane does not induce significant metabolic or respiratory dysfunction. Arterial blood gas analyses revealed no significant difference in any measured parameters between the sevoflurane-exposure group and controls (P > 0.05).
Table 2 Comparison of escape latency and path length. Group
Basic value
1 week
3 weeks
6 weeks
Control group escape latency (s) Path length (cm) Sevoflurane group escape latency (s) Path length (cm) Sham group escape latency (s) Path length (cm)
120.07 ± 20.13 2551 ± 523 119.39 ± 31.07 2495 ± 497 123.57 ± 22.48 2631 ± 601
103.15 ± 16.7 2203 ± 412 110.75 ± 18.4* 2240 ± 400* 100.97 ± 17.4 2210 ± 390
90.78 ± 15.3 1950 ± 305 100.74 ± 16.6* 2219 ± 300* 89.79 ± 21.3 1890 ± 400
70.41 ± 12.7 1839 ± 480 96.71 ± 11.3* 2010 ± 360* 73.87 ± 17.4 1800 ± 370
*P < 0.05. Compared with control group.
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SEVOFLURANE AND MEMORY IN RATS
1 week
3 weeks
6 weeks
C group
S group
Fig. 1. Immunohistochemical staining of pERK1/2 with positive cells in control (C) group, sham (S) group, and sevoflurane (sevo) group, with similar numbers of positive cells in C group and S group and lower numbers of positive cells in sevo group (*P < 0.05 vs. S group, **P < 0.01 vs. S group). The expression of pERK1/2 was significantly different between the sevo group and the C group in the hippocampal CA1 area of rats after 3 weeks (*P < 0.05) and 6 weeks (**P < 0.01). pERK1/2, phosphorylated extracellular signal-regulated kinases.
sevoflurane and control groups in the hippocampal CA1 area of rats after 3 weeks (P < 0.05) and 6 weeks (P < 0.01), but not after 1 week (P > 0.05) (Figs 1 and 2). Discussion Neonatal rats exposed to sevoflurane have impaired spatial learning and memory ability 3 weeks later, and the impairment lasted until 6
weeks after exposure to sevoflurane in our experiments. By immunohistochemistry and Western blot analysis, we show that exposure of neonatal rats to sevoflurane inhibits the expression of pERK1/2 in the CA1 region of the hippocampus. The developing brain has several significant differences from the adult brain that provide a physiological basis for enhanced vulnerability to anesthetics. Early in development, the number of neurons formed is significantly greater than in
Acta Anaesthesiologica Scandinavica 59 (2015) 78–84 © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
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1 week
3 weeks
6 weeks
Sevo group
Western blot 1 week after exposed to sevoflurane
Western blot 3 week after exposed to sevoflurane
Western blot 6 week after exposed to sevoflurane
Fig. 2. Western blot analysis showed that the pERK1/2 protein expression followed the same pattern, with the similar level of expression in control (C) group and sham (S) group and lower level expression in sevoflurane (sevo) group (*P < 0.05 vs. S group, **P < 0.01 vs. S group). The expression of pERK1/2 was significantly different between the sevo group and the C group in the hippocampal CA1 area of rats after 3 weeks (*P < 0.05) and 6 weeks (**P < 0.01). pERK1/2, phosphorylated extracellular signal-regulated kinases.
adult mammals. At the same time, there is an exuberant burst of synapse formation (synaptogenesis) before synapses are eventually pruned to establish behaviorally relevant connections between neurons. Programmed cell death, or apoptosis, is responsible for the elimination of 50–70% of developing neurons under normal
circumstances.15,16 One significant difference between the immature and the mature mammalian brain with neuropharmacological implications is the developmentally regulated reversal of the transmembrane chloride gradient. This is relevant to anesthetic effects as many anesthetic agents enhance the activity of gammaActa Anaesthesiologica Scandinavica 59 (2015) 78–84
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aminobutyric acid type A (GABAA) receptors.17,18 Furthermore, GABAA receptor was demonstrated to play an important role in the process of learning and memory.19–22 Previous studies have suggested that the phosphorylation of ERK may be affected by the functional modulation of GABAA receptor under some stress conditions.23–25 A previous study has indicated that pERK1/2 is necessary for the induction of LTP in the hippocampus, which participates in learning and memory.26 pERK1/2 is abundantly expressed in neurons of the central nervous system and is associated with cell growth, proliferation, differentiation, and migration. Moreover, pERK1/2 coordinates neuronal responses to extracellular signals and may improve learning and memory by regulating synaptic remodeling, axonal growth, LTP formation, and neuronal excitability.26,27 In this study, we used 4% sevoflurane that did not inhibit respiration and circulation in rats. Arterial blood analyses confirmed that none of the rats experienced hypoxemia or hypercapnia during the 4-h sevoflurane exposure. In addition, no differences in the arterial blood gas analyses were shown in different groups. Thus, we hypothesize that duration of sevoflurane exposure is the key factor that inhibits the expression of pERK1/2 and impairs the spatial learning and memory ability of neonatal rats as they grow up. Our findings demonstrate that toxicity of sevoflurane is related to the activation of γ-aminobutyric acid by affecting the phosphorylation of ERK expression in the hippocampus. These findings are consistent with other recent reports that the administration of ketamine or propofol to postnatal day (PND) mouse pups decreased pERK1/2 expression and led to neuronal apoptosis. Lithium restored pERK1/2 levels and prevented ketamine and propofol-induced injury.28,29 It is important to underline that so far, there have been no convincing studies in humans showing neurotoxicity of sevoflurane resulting in cognitive and learning deficits. In summary, our findings indicate that exposure to sevoflurane impairs spatial learning and memory ability of neonatal rats when they grow up. Its mechanism may be related to inhibition of the expression of pERK1/2 in the hippocampus.
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