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International Journal of Developmental Neuroscience 75 (2019) 19–26 Contents lists available at ScienceDirect International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu Dexmedetomidine reduced sevoflurane-induced neurodegeneration and long-term memory deficits in neonatal rats T Toru Goyagi Department of Anesthesia and Intensive Care Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita, Akita 010-8543, Japan A R T I C L E I N F O A B S T R A C T Keywords: Sevoflurane Anesthesia Dexmedetomidine Cognitive function Neonate Neural toxicity Exposure to sevoflurane and other inhalational anesthetics can induce neurodegeneration in the developing brain. Although dexmedetomidine (DEX) has provided neuroprotection against hypoxic ischemic injury, rela- tively little is known about whether it has the neuroprotective effects against anesthetic-induced neurodegen- eration. This study examined whether DEX improves the long-term cognitive dysfunction observed after ex- posure of neonatal rats to 3% sevoflurane. Seven-day-old rats received intraperitoneal saline (DEX 0) or DEX (6.6, 12.5, 25 μg/kg) 30 min before exposure to 3% sevoflurane with 21% oxygen for 4 h (n = 10 per group). The pups in the control group received only DEX 25 μg/kg without anesthesia. The escape latency in the Morris water maze was significantly increased in the DEX 0 group compared with the sham and control group, and the escape latency, but not the swimming path length, was significantly shorter at post-natal day 47 in the DEX 25 than in the DEX 0 group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and control group, and the percent time spent in the quadrant was significantly in- creased in the DEX 25 group compared with the DEX 0 groups. The freezing times of the DEX 0 and 6.6 groups were significantly decreased compared with those in the sham, control and DEX 25 groups. The number of NeuN- positive cells in the CA1 region was significantly decreased in the DEX 0 and 6.6 groups compared with the sham, control and DEX 25 groups. These findings indicate pre-treatment with DEX may improve long-term cognitive function and ameliorate the neuronal degeneration induced by sevoflurane exposure in neonatal rats. 1. Introduction Exposure of the immature brain to general anesthetics causes structural and functional alterations, subsequently resulting in the neurodegenerations (Disma et al., 2016; Stratmann, 2011; Walters and Paule, 2017). Sevoflurane, an inhalational anesthetic, is widely used in pediatric anesthesia. Many experimental studies have shown exposure of the developing brain to sevoflurane causes neurological impairment in later adulthood (Amrock et al., 2015; Fang et al., 2012; Zheng et al., 2013). Furthermore, clinical reports have shown long-term behavioral impairments and memory dysfunction in infants and children after anesthesia exposure (Davidson et al., 2016; Sun et al., 2016; Warner et al., 2018). There is concern that infants and children could be af- fected if they receive general anesthesia for a prolonged period and multiple times during the neonatal and infant term (Andropoulos and Greene, 2017). As general anesthesia is necessary for surgery, the in- vestigation of method to ameliorate anesthesia-induced neurotoxicity is urgently needed. adjuvant of anesthesia to provide analgesia and sedation in the pre- operative and postoperative periods (Kamibayashi, 2000). These effects are useful for the prevention of postoperative delirium (Duan et al., 2018). Moreover, DEX protects organs in various injury models in- cluding ischemia (Dahmani et al., 2005; Engelhard et al., 2002; Eser et al., 2008; Goyagi et al., 2009; Zhu et al., 2013), inflammation (Vincent Degos et al., 2013), and traumatic injury (Schoeler et al., 2012), owing to its anti-apoptotic effects (Engelhard et al., 2002), its ability to decrease caspase-3 elevation (Dahmani et al., 2005; Eser et al., 2008), its effects on the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways (Zhu et al., 2013) via the α2A and imidazole 1 receptors (Ma et al., 2004). Pharmacological preventive strategies against neurodegeneration induced by general anesthesia have been introduced (Disma et al., 2016; Walters and Paule, 2017). DEX is likely to be a protective com- pound that could be used to prevent or ameliorate anesthesia-induced neurodegeneration (Walters and Paule, 2017). Based on previous stu- dies, DEX can ameliorated the neuroapoptosis and cognitive dysfunc- tion induced by exposure of the developing brain to ketamine (Duan et al., 2014), isoflurane (Li et al., 2014; Sanders et al., 2010, 2009), Dexmedetomidine (DEX) is an α2-adrenoceptor agonist that has sedative and analgesic effects, and has been widely used clinically as an E-mail address: tgoyagi@doc.med.akita-u.ac.jp. https://doi.org/10.1016/j.ijdevneu.2019.04.002 Received 31 January 2019; Received in revised form 28 March 2019; Accepted 3 April 2019 Available online 05 April 2019 0736-5748/ © 2019 ISDN. Published by Elsevier Ltd. All rights reserved. T. Goyagi sevoflurane (Perez-Zoghbi et al., 2017) and propofol (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Moreover, DEX has neuropro- tective effects in the hypoxic-ischemic neonatal brain (Ren et al., 2016; Zhou et al., 2018) and in an acute hyperoxic neonatal rat model (Endesfelder et al., 2017; Sifringer et al., 2015). In contrast, DEX does not ameliorate the injury induced by sevoflurane, and induces neural apoptosis in neonatal rats (Lee et al., 2017). Although many reports have shown that DEX is protective against anesthesia-induced neuro- degeneration, few reports have shown high mortality rates under high- dose DEX with sevoflurane anesthesia in neonatal rats (Lee et al., 2017; Perez-Zoghbi et al., 2017). Thus, the efficiency of DEX against sevo- flurane-induced neurodegeneration is not fully understood. We hypothesized that DEX administration could ameliorate the neurodegeneration triggered by sevoflurane in the developing rat brain and improve cognitive function in the long-term. This study examined the effects of DEX on rat brain histological changes, of through the assessment of persistent normal cells, and cognitive function in later life following perinatal sevoflurane exposure. 2. Material and methods All animal protocols were approved by the animal research com- mittee of Akita University, Japan (Approval number: a-1-2625). Seven- day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15 g) were used in this study. Animals were housed under standard conditions (12 h light/12 h dark cycle at 22 °C) in the Animal Research Laboratory at Akita University. All efforts to reduce the number of animals and their suffering were made. The animals were randomly divided into 6 groups (n = 10 per group) as follows: no anesthesia and no injection (sham), no anesthesia and intraperitoneal 25 μg/kg DEX (control), intraperitoneal saline (DEX 0), intraperitoneal 6.6 μg/kg DEX intraperitoneal 12.5 μg/kg DEX (DEX 12.5), and in- (DEX 6.6), traperitoneal 25 μg/kg DEX (DEX 25). After 30 min intraperitoneal injection on P7, the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2 L/min of 21% oxygen for 4 h, and returned to their mother’s cage. The oxygen and sevoflurane concentration were measured using a gas analysis system (GE Healthcare BioSciences, Pittsburgh, PA). The chamber was main- tained at 30 ± 1 °C using an infrared heat lamp during the exposure. 2.1. Cognitive tests 2.1.1. Morris water maze Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018). At P27 – P29, acquisition trials were executed 4 times per day for 3 suc- cessive days. The latency and the swimming path length to reach the hidden platform were measured using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). If the rat could not reach the hidden platform within 90 s, it was placed on the platform for 30 s during an acquisition trial. At P47 – P49, retention trials were executed 4 times per day. If the rats failed to find the platform within 90 s, the latency was regarded as 90 s. In this study, a probe trial was not done during the acquisition trials. 2.1.2. Fear conditioning test Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018). The apparatus consisted of a clear rectangular Plexiglas box with a floor of for the delivery of electric currents. At P42, the rats were placed on the cleaned parallel metallic rods to be accustomed to new environment for 1 min, before they were presented with a 70-dB white noise for 30 s A mild foot shock (0.4-mA) was administered through the metallic rods during the last 1 s of the tone presentation. The tone-shock pairing was repeated once per minute for the next 2 min. The rats were left in 20 International Journal of Developmental Neuroscience 75 (2019) 19–26 Fig. 1. Morris water maze results 3 weeks after sevoflurane exposure. (A) Escape latency to reach the platform, (B) swimming path length to the platform, and (C) swimming speed were measured. Days 1, 2, and 3 of the acquisition trials were P27, P28, and P29, respectively. Although the escape latency on day 3 significantly decreased in all groups compared with the latency on day 1, no significant differences were found among the groups on days 1–3, whereas the swimming path length in the sham group was significantly shorter than that in the other groups. The data are expressed as means ± SDs (n = 10). DEX, dexmedetomidine. the cage for an additional 60 s before returned to their cage. At P49, cued fear memory was tested by placing rats into an unrelated en- vironment for 90 s without any tone and presenting the auditory cue for a further 60 s used for conditioning. Freezing time was measured by the percent of time during the tone presentation using a video image mo- tion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). 2.2. Histological analyses 2.2.1. Neuronal nuclei staining After finished the water maze task and fear conditioning test at P49, the rats’ brains were removed and embedded in paraffin following the perfusion of heparinized saline then 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4) to use further neuronal nuclei (NeuN) stain, as described previously (Goyagi, 2018). In brief, 3-μm-thick serial transverse sections were incubated with a mouse monoclonal antibody to NeuN antigen (NeuN; 1:100 diluted in blocking solution; Millipore Corporation, Temecula, CA) for 10 min at 37 °C. Immunodetection was performed using avidin-horse radish peroxidase complexes with T. Goyagi Fig. 3. Fear conditioning test results. The freezing time in response to the conditioned stimulus tone on P49 was significantly longer in the DEX 25 groups compared with the DEX 0 and 6.6 groups. The freezing time was significantly decreased in the DEX 0 group compared with the sham and the control group. * P < 0.05 vs sham, † P < 0.05 vs control, and ‡ P < 0.05 vs DEX 25. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine. biotinylated antibodies to rabbit and mouse IgG (MILLIPORE IHC Se- lectR Immunoperoxidase Secondary Detection System; Millipore Cor- poration), with diaminobenzidine. Then we counterstained those with hematoxylin. The NeuN-positive cells express as mature typical neurons after growth. We counted the number of NeuN-positive cells in bilateral 500 μm × 300 μm areas in the CA1 hippocampus, amygdala, and cer- ebral cortical layer 3, as described previously (Goyagi, 2018). 2.2.2. Positive cell density map (PCDM) The PCDM was made as described previously (Goyagi, 2018; Wada et al., 2006). In brief, the composite image was FFT- bandpass-filtered using the Image J program (National Institute of Health, Bethesda, MD) to eliminate low-frequency drifts (> 20 pixels [50 μm]) and high-fre- quency noises (< 1 pixel [2.5 μm]). The PDCM was made with a 21 International Journal of Developmental Neuroscience 75 (2019) 19–26 Fig. 2. Morris water maze results 6 weeks after sevoflurane exposure. (A) Escape latency to reach the platform, (B) swimming path length, (C) swimming speed, and (D) percent time to reach the quadrant were measured on day 1 (P47). The escape latency was significantly longer in the DEX 0 group than in the sham and the control group, and significantly shorter in the DEX 25 group than in the DEX 0 group. No significant differences were observed in swim- ming path length among the groups. The swimming speed in the DEX 0 and 12.5 groups was significantly decreased compared with that in the sham group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and the control group. However, it was significantly increased in the DEX 25 group compared with the DEX 0 group. * P < 0.05 vs sham, † P < 0.05 vs control, and ‡ P < 0.05 vs DEX 25. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine. custom-made program using MATLAB (MathWorks INC., Natick, MA) (Wada et al., 2006), then adjusted for each section automatically and enumeration of NeuN-positive cells in each 100 μm × 100 μm square section. Finally, the normalized PCDMs were seen as averaged for each group (Fig. 6 A). As mentioned our previous study (Goyagi, 2018), the PCDMs were analyzed whether the DEX-treated groups showed in- creased NeuN cell density compared with the DEX 0 group. The areas were mapped as colored to indicate significantly increased normal neurons in blocks where the P value was less than 0.05 (Fig. 6B). 2.3. Statistical analysis The escape latency, the swimming speed, the swimming path length, the freezing time, and the number of NeuN-positive cells are expressed as means ± standard deviation (SD). Comparisons of these variables among the groups were performed using a one-way or two- way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post hoc tests. Each PCDM using a Gaussian filter of the block size (SD = 100 μm) was analyzed using t-tests for each block. Differences with p-values less than 0.05 were considered statistically significant. We performed all analyses using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA). 3. Results 3.1. DEX improves memory retention in the Morris water maze The escape latencies and swimming path lengths to the hidden platform in the acquisition trials during postnatal day 27–29 (P27–29) are shown in Fig. 1. No statistically significant differences among the groups were found for the DEX treated, sham and control groups, in- dicating that all rats acquired the task equally well. As shown in Fig. 2, the escape latency was significantly increased in the DEX 0 group compared with the sham, the control and the DEX 25 group, and the escape latency, but not the swimming path length, was significantly shorter in the DEX 25 group than in the DEX 0 group at P47. In addi- the swimming speed in the DEX 0 and 12.5 groups was tion, T. Goyagi International Journal of Developmental Neuroscience 75 (2019) 19–26 Fig. 4. NeuN staining of brain sections from sevoflurane-exposed rats. Representative NeuN staining at -3 ± 0.2 mm from bregma is shown. NeuN-positive cells in the entire image at lower magnification (A), CA1 hippocampus at higher magnification (B), amygdala at higher magnification (C), and cortex at higher magnification (D) are shown for each group. Squares indicate the measuring field in the CA1, amygdala, and cortex. Scale bar: 3 mm (A) or 100 μm (B, C, and D). DEX, dexmedetomidine. 22 T. Goyagi Fig. 5. Numbers of NeuN-positive cells in the hippocampus, amygdala, and cortex of sevoflurane-exposed rats. The number of NeuN-positive cells in the CA1 region of the hippocampus was decreased in the DEX 0 group compared with the sham, control, DEX 12.5 and 25 groups. The number of NeuN-positive cells was increased in the DEX 12.5 and 25 groups compared with the DEX 0 group (A). The number of NeuN- positive cells in the amygdala was decreased in the DEX 0 group compared with the sham and the control group, and increased in the DEX 12.5 and 25 groups compared with the DEX 0 group (B). The number of NeuN-positive cells in the cortex was decreased in the DEX 0 group compared with the sham and the control group (C). * P < 0.05 vs Sham, † P < 0.05 vs control, ‡ P < 0.05 vs DEX 25, § P < 0.05 vs DEX 12.5. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine. significantly decreased compared with that in the sham group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and the control group, and the percent time spent in the quadrant was significantly increased in the DEX 25 group compared with the DEX 0 group. This indicates that the DEX 25 improved the escape latency, the swimming speed, and he percent time spent in the quadrant. 3.2. Fear conditioning test The freezing times in response to the conditioned stimulus tone were significantly decreased in the DEX 0 and 6.6 groups compared with the sham, the control and DEX 25 groups. The data indicate that the DEX 25 group had a freezing time that was similar to that in the sham group. (Fig. 3). 23 International Journal of Developmental Neuroscience 75 (2019) 19–26 3.3. DEX attenuates the reduction in neuronal numbers in anesthesia- exposed brains Fig. 4 shows NeuN staining of brain sections from the 5 groups at -3 mm caudally to bregma. The number of NeuN-positive cells per 0.15 mm2 in the hippocampal CA1 region was significantly decreased in the DEX 0 and 6.6 groups compared with the sham, the control and the DEX 25 groups. In addition, the DEX 12.5 and 25 groups had an increased number of positive cells compared with those in the DEX 0 group (Fig. 5A). The number of NeuN-positive cells per 0.15 mm2 in the amygdala was significantly decreased in the DEX 0 group compared with the sham and the control group, and the number of cells in the DEX 12.5 and 25 groups were significantly increased compared with those in the DEX 0 group, suggesting that DEX 12.5 and 25 increased the number of intact cells in the amygdala (Fig. 5B). The number of NeuN-positive cells per 0.15 mm2 in the cortex was significantly de- creased in the DEX 0 group compared with the sham and the control group (Fig. 5C). 3.4. PCDM and statistical parameter mapping of positive cell density Fig. 6A shows the NeuN PCDM for each group. The positive cell density tended to increase in the cortex. DEX-treated rats showed an increase in the hippocampal and cortical PCDM compared with the DEX 0 group. The differences among the groups are shown in detail in Fig. 6B. DEX-treated rats had significantly increased NeuN-positive cell densities when compared with those in the DEX 0 group. Increased NeuN expression was observed in almost all cortical and hippocampal areas, indicating that the numbers of normal cells in the DEX 12.5 and 25 groups were profoundly higher than those in the DEX 0 group. 4. Discussion Our study shows that the pre-treatment of neonatal rats with DEX before exposure to sevoflurane anesthesia improved cognitive function, and increased the number of intact neurons in the cortex, hippocampus, and amygdala in the adulthood, indicating that DEX attenuates the neural degeneration induced by exposure of the developing rat brain to sevoflurane. Although the doses of DEX used in this study were 6.6, 12.5, and 25 μg/kg, we did not find a dose-response relationship. The rats in the DEX 25 group obtained the maximum effects compared with those in the other groups, as seen in the water maze, fear conditioning test, and histological results. These results were consistent with previous similar studies (Li et al., 2014; Sanders et al., 2010, 2009), though inconsistent with others (Lee et al., 2017; Perez-Zoghbi et al., 2017). In previous studies, DEX ameliorated the neuroapoptosis and cognitive dysfunction induced by exposure of the developing brain to isoflurane (Li et al., 2014; Sanders et al., 2010, 2009), sevoflurane (Perez-Zoghbi et al., 2017), and propofol (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Sanders et. al. showed that DEX 25, 50, and 75 μg/kg prevents cortical apoptosis in vitro and in vivo, whereas higher doses of DEX do not further increase the protection against isoflurane-induced injury in the cortex (Sanders et al., 2010). In contrast, the study from Perez- Zoghbi et al. showed that co-administration of DEX (1 μg/kg) during 2.5% sevoflurane exposure for 6 h provided significant neuroprotection, as measured by a reduction in the number of caspase-3 positive cells in several brain regions, whereas DEX at doses of 10, and 25 μg/kg in combination with sevoflurane increased mortality (Perez-Zoghbi et al., 2017). In contrast to previous studies, the aim of this study was to measure cognitive function, using the fear conditioning test, and the number of intact neurons in the brain during adulthood, as in our previous study (Goyagi, 2018). We only measured the freezing time to tone but did not use contextual stimuli. The amygdala contributes to the acquisition of conditioned fear responses to a cue, whereas the amygdala and the T. Goyagi International Journal of Developmental Neuroscience 75 (2019) 19–26 Fig. 6. Positive cell density map (PCDM) and statistical parameter mapping of positive cell densities in the brains of sevoflurane-exposed rats. (A) The NeuN PCDM for each group is shown. (B) Statistical parametric mapping showing the areas (red) where the DEX-treated groups yielded significantly higher NeuN-positive cell densities than the DEX 0 group, indicating the numbers of normal cells in the DEX-treated group were higher than in the DEX 0 group. DEX, dexmedetomidine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). hippocampus contribute to the acquisition of contextual conditioned fear responses (Phillips and LeDoux, 1992; Selden et al., 1991; Stanton, 2000). In this study, we measured the number of NueN-positive cells in both the amygdala and CA1 hippocampus. The cells in both regions increased in the DEX 12.5 and 25 groups, which was consistent with the behavioral results. The behavioral and histological results from this study suggested that administration of 12.5 and 25 μg/kg DEX improved learning and memory following sevoflurane-induced neuro- logical impairment in the developing brain. Neural toxicity induced by exposure to general anesthesia in neo- natal rodents has been shown to lead to neurological impairments in adulthood (Disma et al., 2016; Stratmann, 2011; Walters and Paule, 2017). Numerous animal studies have reported that anesthetic neuro- toxicity occurs via several pathways (Acharya et al., 2015; Jevtovic- 24 T. Goyagi Todorovic, 2018; Shen et al., 2013; Sinner et al., 2014). We have pre- viously reported that the use of oxygen as a carrier gas at concentra- tions higher than 21% ameliorates sevoflurane-induced neurodegen- eration. (Goyagi, 2018). Therefore, we used air as a carrier for sevoflurane anesthesia to minimize the neuroprotective effects of oxygen. According to the results of a previous study, co-administration of high dose DEX (5, 25 and 50 μg/kg) and 2.5% sevoflurane in rat pups causes high mortality rates (Perez-Zoghbi et al., 2017), and DEX pro- duces significant cellular degeneration and apoptosis in primary sen- sory brain regions (Pancaro et al., 2016), though we did not find the degeneration induced by DEX administration alone (control group) in this study. Although we also used 3% sevoflurane in this study, 25 μg/ kg DEX did not cause a high rate of mortality. This difference may be because of the different methodology used and the environment. Fur- ther studies are needed to clarify the neurotoxic effects of DEX in neonatal brain, with or without sevoflurane anesthesia. This study has several limitations. First, the rat pups were main- tained under spontaneous ventilation during sevoflurane anesthesia. Since sevoflurane depresses ventilation in a dose-dependent manner, hypercapnia may be seen during the anesthesia exposure. Although we did not measure the blood gas analysis in this study, there was no significant difference in the rate of hypercapnia among the groups in our previous study (Goyagi, 2018). Therefore, hypercapnia was not examined here. Second, the rat pups received sevoflurane with 21% oxygen in this study. According to our previous study, rat pups that receive sevoflurane with 21% oxygen exhibit a profound increase in neurodegeneration compared with those that receive 30% oxygen (Goyagi, 2018). DEX has neuroprotective effects against hypoxic brain and spinal insult (Dahmani et al., 2005; Engelhard et al., 2002; Eser et al., 2008; Goyagi et al., 2009; Goyagi and Tobe, 2014; Ma et al., 2004; Zhu et al., 2013), and in animal neonatal models, particularly in the hypoxic-ischemic neonatal brain (Ren et al., 2016; Zhou et al., 2018). It is possible that DEX might be protective against the hypoxic effects of 21% oxygen rather than the neurotoxicity induced by sevo- flurane here. Although a carrier gas with 21% oxygen might increase the neurodegeneration induced by sevoflurane anesthesia in the neo- natal brain compared with higher oxygen concentrations, DEX ame- liorated the neurodegeneration induced by sevoflurane exposure with 21% oxygen in this study, as previously described (Perez-Zoghbi et al., 2017). Third, we used the Morris water maze and fear conditioning test to measure neurocognitive memory function in later adulthood in this study. As there are many tests for cognitive function in rats, further studies are warranted to measure the effects on other type of cognitive function. Fourth, the mechanisms underlying the protective effects of DEX were not clear from this study. Based on a previous study, DEX may attenuate the reduction in the expression of anti-apoptotic sig- naling pathways mediated by Bcl-2 and phosphor-ERK1/2 induced by isoflurane anesthesia, in a similar manner to that of neonatal brain injury (Li et al., 2014). Moreover, DEX has anti-apoptotic effects (Engelhard et al., 2002), decreases caspase-3 elevation (Dahmani et al., 2005; Eser et al., 2008) and attenuates the activation of the PI3k/Akt/ glycogen synthase kinase (GSK)3beta pathway in propofol-induced neuroapoptosis (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Further research is warranted in order to ascertain the precise neuro- protective mechanisms of DEX against neural toxicity induced by an- esthesia exposure in the neonatal brain. 5. Conclusion Four-hour administration of sevoflurane anesthesia in neonatal rats caused significant cognitive impairment in adulthood. Administration of a single dose of 25 μg/kg DEX before sevoflurane exposure improved cognitive functions, and maximum effects were observed at this dose compared with the other doses. Also, DEX attenuates the reduction in neuronal numbers in sevoflurane-exposed brains. DEX is likely to be useful as a neuroprotective agent in pediatric anesthesia. 25 International Journal of Developmental Neuroscience 75 (2019) 19–26 Ethics approval The animal experiments were performed according to international ethical standards and approved by the research ethics committee of Akita University (a-1-2625). Funding This study was supported in part by a Grant-in-Aid for Scientific Research (C), JSPS KAKENHI Grant Number JP 24592291. Conflict of interest The authors declare no conflicts of interest. Acknowledgements I would like to thank Mr. Yoshitsugu Tobe and Dr. Yoko Masaki, Ph.D. from the Department of Anesthesia and Intensive Care Medicine, Akita Graduate School of Medicine, for technical assistance. I am also grateful to Dr. Makoto Wada, M.D., Ph.D. from the Department of Rehabilitation for Brain Functions, National Rehabilitation Center for Persons with Disabilities, for technical advice and the use of his soft- ware. 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