European Journal of Pharmacology 670 (2011) 168–174

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European Journal of Pharmacology

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Behavioural Pharmacology

Effects of gestational isoflurane exposure on postnatal memory and learning in rats

Feijuan Kong a,1, Linhao Xu b,2, Daqiang He b,2, Xiaoming Zhang b,2, Huishun Lu a,⁎a Department of Anesthesiology, Women's Hospital, School of Medicine, Zhejiang University, Chinab Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China

⁎ Corresponding author at: Department of AnesthSchool of Medicine, Zhejiang University, Hangzhou,China. Tel.: +86 571 87061501 2410; fax: +86 571 87

E-mail address: lig08010915@163.com (H. Lu).1 Postal address: Department of Anesthesiology, Wom

cine, Zhejiang University, Hangzhou, Bachelor Road 1, 32 Postal address: Institute of Anatomy and Cell Biology

University, Hangzhou, Yuhangtang Road 388, 310058, P

0014-2999/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.ejphar.2011.08.050

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 July 2011Received in revised form 15 August 2011Accepted 27 August 2011Available online 14 September 2011

Keywords:IsofluraneMemory and learning impairmentHippocampusC/EBP homologous proteinCaspase-12Neuron apoptosis

A maternal fetal rat model was developed to study the effects of gestational isoflurane exposure on postnatalmemory and learning and investigate the potential mechanisms. Pregnant rats at gestational day 14 were ex-posed to 1.3% isoflurane for 4 h. Spatial learning and memory of the offspring were examined using the Mor-ris Water Maze. The expression levels of C/EBP homologous transcription factor protein (CHOP) and caspase-12 in the hippocampus of the pups were determined by immunohistochemistry and western blot analysis.Simultaneously, the ultrastructure changes of synapse in the hippocampal CA1 and dentate gyrus regionwere also observed by transmission electron microscopy (TEM). Prenatal exposure to isoflurane impairedpostnatal spatial memory and learning in the offspring rats as shown by the longer escape latency and thefewer times of original platform crossing in the Morris Water Maze test. The number of CHOP and caspase-12 positive neurons significantly increased by 138% and 147% respectively in the hippocampus of isoflur-ane-exposed pups, as well as the levels of CHOP and caspase-12 protein. Furthermore, TEM studies showedchanges of synaptic ultrastructure in isoflurane-exposed hippocampus characterized by the decreased synap-se number, the widened synaptic cleft and the thinned postsynaptic densities. These results demonstrate thatgestational exposure to a clinically relevant concentration of isoflurane could cause neuron apoptosis,changes of synaptic structure, and postnatal spatial memory and learning impairments in offspring. Ourstudy further showed that the up-regulation of CHOP and caspase-12 may contribute to isoflurane-inducedneuron apoptosis.

esiology, Women's Hospital,Bachelor Road 1, 310006, PR061878.

en's Hospital, School of Medi-10006, PR China., School of Medicine, ZhejiangR China.

rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Many pregnant women, fetuses, and infants are exposed to a varietyof anesthetic agents for surgical or diagnostic procedures each year.Pregnant women sometimes undergo general anesthesia during theirpregnancy for surgeries unrelated to the delivery, such as fetal andnon-obstetric surgeries, especially during midgestation (Goodman,2002; Tran, 2010). Since most general anesthetic agents are lipophilicand cross the placenta easily (Dwyer et al., 1995), the developing fetalbrains will be exposed to anesthetics as well. Inhalation anestheticssuch as isoflurane have been widely used in recent years in clinical andresearch practices. Preclinical studies demonstrate that early exposureto anesthetic agents causes neuroapoptosis and long-term cognitive im-pairments (Culley et al., 2004; Jevtovic-Todorovic et al., 2003; Ma et al.,2007), and recent clinical studies support the possibility (DiMaggio etal., 2008; Kalkman et al., 2009; Wilder et al., 2009). These observations

raise concerns about the potentially deleterious effects of general anes-thesia in the human fetus, neonate, and infant. Nevertheless, the major-ity of prior neurodevelopmental studies focused on postnatal subjectsrather than on the fetuses. In this study, we hypothesized that gestation-al exposure to isoflurane duringmaternal anesthesia may have deleteri-ous effects on the fetal brain that leads to postnatal spatial memory andlearning impairments in the offspring rats.

The cellular and molecular mechanisms of anesthetics-mediatedneurotoxicity remain unclear. Previous studies indicate that endo-plasmic reticulum (ER) stress is associated with a range of diseases,including ischemia/reperfusion injury, neurodegeneration, and dia-betes (Oyadomari and Mori, 2004), making ER stress a probable in-stigator of pathological cell death and dysfunction. At least threepathways contribute to ER stress-mediated cell death: transcriptionactivation of the C/EBP homologous transcription factor (CHOP)(Oyadomari and Mori, 2004), activation of the IRE1-tumor necrosisfactor receptor-associated factor (TRAF2) pathway (Matsukawa etal., 2004) and activation of ER-resident caspase-12 (Nakagawa andYuan, 2000; Nakagawa et al., 2000). CHOP, a member of the C/EBP transcription factor family, is induced by ER stress and thuscauses apoptosis. Caspase-12, an ER-specific caspase, participatesin apoptosis under ER stress. In the current study, we hypothesizedthat CHOP and caspase-12 play a role in the mechanisms of isoflur-ane-induced neuron apoptosis.

Fig. 1. Effects of rats exposed to isoflurane on postnatal memory and learning ability.(A) Place trial demonstrating the latency for offspring rats to reach platform measuringspatial information acquisition. (B) Probe trial demonstrating the number of originalplatform crossing measuring memory retention capabilities. *Pb0.05 compared withcontrol.

169F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174

In the present study, using a maternal fetal rat model, we testedthe capacity for learning and memory in pups of fetal exposure to iso-flurane with the Morris Water Maze. Then we used transmission elec-tron microscopy (TEM) to investigate synaptic ultrastructure changesin the hippocampal area. We also measured the levels of CHOP andcaspase-12 protein in the hippocampal area, and analyzed their rela-tionship with isoflurane-induced neuron apoptosis.

2. Materials and methods

2.1. Animals

All of the animals were treated according to the guidelines of theGuide for the Care and Use of Laboratory Animals (China Ministry ofHealth). The Laboratory Animal Care Committee of Zhejiang Universi-ty approved all experimental procedures and protocols. All effortswere made to minimize the number of animals used and their suffer-ing. The dams were housed in polypropylene cages, and the roomtemperature was maintained at 22 °C, with a 12-hour light–darkcycle. The dams at gestational day 14 were used for all experiments,because this time corresponds approximately to midgestation inhumans (Clancy et al., 2001, 2007), the period when most nonobste-tric surgeries and fetal interventions are performed (Goodman, 2002;Tran, 2010).

2.2. Anesthesia exposure

Ten dams were randomly divided into a control and an isofluranegroup (n=5). The dams were placed in plastic containers resting inwater baths with a constant temperature of 38 °C. In these boxes,the dams were either exposed to 1.3% isoflurane (Lot 826005U, AB-BOTT, USA) in a humidified 30% oxygen carrier gas or simply humid-ified 30% oxygen without any inhalational anesthetic for 4 h. Wechose 1.3% as the anesthetic concentration because it represents 1minimum alveolar concentration (MAC) in the pregnant rats(Mazze et al., 1985). The determination of anesthetic duration basedon our preliminary study which indicated that maternal physiologicalstates remained stable throughout a 4-hour isoflurane exposure. Theisoflurane concentration in the box was monitored with an agent gasmonitor (Vamos, Drager Medical AG & Co. KgaA). Otherwise, controland experimental animals were under the same treatment and envi-ronment. During isoflurane anesthesia, arterial blood gases and bloodglucose were measured at the end of the 4-hour anesthetic exposure.The rectal temperature was maintained at 37±0.5 °C. After exposure,the dams were returned to their cages and allowed to deliver natural-ly. The postnatal body weights of the rat pups were monitored.

2.3. Memory and learning studies

Four rat pups (2 females and 2 males) from each damwere select-ed to determine cognitive function at postnatal day 28 with a MorrisWater Maze test with minor modifications (Jevtovic-Todorovic et al.,2003). A round pool (diameter, 150 cm; depth, 50 cm) was filled with

Table 1Maternal physiological parameters during isoflurane anesthesia.

0 h

Control 1.3% isofl

pH 7.43±0.02 7.43±0PaCO2 (mm Hg) 35.8±2.47 36.7±1PaO2 (mm Hg) 169.5±4.32 168.2±6SaO2 (%) 95.4±1.1 94.2±1Glucose (mg/dl) 113±21 115±1

1.3% isoflurane did not affect arterial blood gas values and blood glucose levels significantlyPaCO2 = arterial carbon dioxide tension; PaO2 = arterial oxygen tension; SaO2 = arterial o

warm (24 °C) opaque water to a height of 1.5 cm above the top of themovable clear 15-cm-diameter platform in the third quadrant. Avideo tracking system recorded the swimming motions of animals,and the data were analyzed using motion-detection software for theMorris Water Maze (Actimetrics Software, Evanston, IL, USA). Afterevery trial, each rat was wiped before returning to its regular cage,keeping warm and free diet.

2.3.1. Place trialsThe place trials were performed at postnatal day 29 for 4 days to

determine the rats' ability to obtain spatial information. At postnatalday 28, the rats were made to know the existence of the platformthrough a 30-second swimming training. A dark black curtain sur-rounded the pool to prevent confounding visual cues. All rats received4 trials per day in each of the four quadrants of the swimming pool.On each trial, rats were placed in a fixed position into the swimmingpool facing the wall. They were allotted 120 s to find the platformupon which they sat for 20 s before being removed from the pool. Ifa rat did not find the platform within 120 s, the rat was gently guidedto the platform and allowed to remain there for 20 s. For all trainingtrials, swim speed and the time to reach the platform (escape latency)were recorded. The less time it took a rat to reach the platform, the

4 h

urane Control 1.3% isoflurane

.01 7.41±0.02 7.39±0.01

.34 36.6±2.39 37.9±3.25

.19 166.2±6.41 165.3±5.32

.2 95.1±0.9 94.6±1.16 116±22 115±12

.xygen saturation.

170 F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174

better the learning ability. We took the average of four trials as the es-cape latency each day.

2.3.2. Probe trialsProbe trials were conducted immediately after the four-day period

to evaluate memory retention capabilities. The probe trials involvedremoving the submerged platform from the pool and allowing therats to swim for 120 s in any of the four quadrants of the swimmingpool. Time spent in the third quadrant and the number of originalplatform crossing in the third quadrant was recorded.

2.4. Transmission electron microscopy

After theMorrisWaterMaze test, three pups per groupwere anesthe-tized with a lethal dose of Nembutal. The thoracic cavities were openedandperfused intracardiallywith 100 mLof normal saline. Then thehippo-campus, including CA1 and dentate gyrus area, of each rat was taken out

Fig. 2. The expression of CHOP and caspase-12 increased significantly in the hippocampus ofical staining in control pups ×400. (Ab) CHOP immunohistochemical staining in isoflurane×400. (Ad) Caspase-12 immunohistochemical staining in isoflurane-exposed pups ×400. Twere compared between the control and 1.3% isoflurane treatment groups. **Pb0.01 comp

immediately. Immersion fixation was completed on tissues about 1 mm3

from the hippocampus. Samples were rinsed in cold phosphate-bufferedsaline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissuewas rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h.Then, the tissuewas rinsedwith distilledwater before undergoing a grad-ed ethanol dehydration series and was infiltrated using a mixture of halfpropylene oxide andhalf resin overnight. Twenty-four hours later, the tis-sue was embedded in resin. 120 nm sections were cut and stained with4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastruc-ture changes of synapse in the hippocampus were observed under atransmission electron microscope (Phliphs Tecnai 10, Holland).

2.5. Tissue section preparation

After the Morris Water Maze test, two pups from each dam wereanesthetized by intraperitoneal injection of a lethal dose of Nembutal.The aorta was cannulated and the animal was firstly perfused with

isoflurane-exposed pups showed by immuno-reaction. (Aa) CHOP immunohistochem--exposed pups ×400. (Ac) Caspase-12 immunohistochemical staining in control pupshe number and optical density of the CHOP (B) and caspase-12 (C) positive neuronsared to control. Scale bar=50 μm.

Fig. 3. The levels of CHOP and caspase-12 protein remarkably increased in the hippo-campus of isoflurane-exposed pups. (A) Representative changes of CHOP and cas-pase-12 by western blot analysis. (B, C) The quantified CHOP (B) and caspase-12(C) bands were normalized to the loading control β-actin. **Pb0.01, ***Pb0.001 com-pared to control.

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200 mL of normal saline, then with 250 mL of 4% formaldehyde(freshly made from paraformaldehyde) for 20–30 min. The fixedbrain was then removed from the cranial cavity and post-fixed over-night in the same fixative at 4 °C. The tissues were embedded in par-affin, and transverse paraffin sections containing the hippocampalarea (5 mm thick) were mounted on silanecoated slides. Sectionswere deparaffinaged and rehydrated. Then the sections were treatedfor antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1,for 20 min at 95 °C for immunohistochemistry.

2.6. Immunohistochemistry analysis

The sections mentioned above were washed in 0.01 M PBS con-taining 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in5% normal goat serum in 0.01 M PBS. The sections were then incubat-ed in the primary antibodies rabbit polyclonal against anti-CHOP orcaspase-12 (1:100, Santa Cruz Biotechnology, USA) overnight at4 °C. After a thorough wash in PBS, sections were incubated with bio-tinylated goat anti-rabbit IgG antibody (1:200, Boster, China) for 2 hat room temperature, followed by avidin–biotin–peroxidase complexsolution (ABC, 1:100, Boster) for 2 h at room temperature. Immunola-beling was visualized with 0.05% diaminobenzdine (DAB) plus 0.3%H2O2 in PBS and the reaction was stopped by rinsing the slides with0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-L-lysine-coated slides and allowed to dry at room temperature. Then the sec-tions were dehydrated through a graded series of alcohols, clearedin xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200,Biomeda Corporation, USA) was used instead of primary antibody asa negative control. Three sections from each animal were selected atrandom and images were photographed under 400× magnificationin 3 visual fields/per section, the CHOP and caspase-12 positive neu-rons were counted in the same area. The optical densities of CHOPand caspase-12 positive neurons were measured quantitativelyusing NIH image software (ImageJ, National Institutes of Health, Be-thesda, MD).

2.7. Western blot analysis

After Morris Water Maze test, two pups from each pregnant moth-er were anesthetized with a lethal dose of Nembutal. Then their tho-racic cavities were opened and perfused intracardially with 100 mL ofnormal saline. Hippocampus, including CA1 and dentate gyrus field,of each rat was taken out immediately to obtain fresh tissue speci-mens. Protein concentration was determined by the BCA methodusing bovine serum albumin as the standard. Protein samples(50 μg) were separated by 12% sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellu-lose membrane. The membranes were blocked by nonfat dry milkbuffer for 2 h and then incubated overnight at 4 °C with primary an-tibody against CHOP or caspase-12 (1:500, Santa Cruz Biotechnology,USA). The membranes were subsequently incubated with horseradishperoxidase-conjugated secondary antibodies and developed with ECLkit. The optical densities of bands were quantitatively analyzed usingBio-Rad Quantity One 4.6.2 (Bio-Rad Laboratories, USA). The resultswere expressed as a relative density. Equal protein loading in eachlane was confirmed by hybridization with a 1:2000 dilution of β-actin antibody (Santa Cruz Biotechnology, USA).

2.8. Statistical analysis

All data were presented as mean±S.E.M. Results of weight ofpostnatal rat pups and place trials of postnatal rats were analyzedusing 2-way ANOVA for repeated measurements. Other data were an-alyzed using Student's t-test for comparison of two groups. A P valueof b0.05 was considered statistically significant. All statistical tests

and graphs were performed or generated, respectively, using Graph-Pad Prism Version 4.0 (GraphPad Prism Software, Inc. CA, USA).

3. Results

3.1. Physiologic parameters

As shown in Table 1, ABG values and blood glucose levels werewithin the normal physiologic range. There were no significant differ-ences in ABG values and blood glucose levels before and after expo-sure in both the control group and the 1.3% isoflurane treatmentgroup. All pups were viable and there were no significant differencesin growth rate of the rat pups between the two groups (data notshown).

3.2. Morris Water Maze test

As shown in Fig. 1A, pups in both groups showed a rapid decreasein latency, while the pups of the isoflurane group spent more time tofind the platform than those of control group in the place trial(Pb0.05). Swimming speeds were also analyzed during place trials,and no differences were observed between the two groups (datanot shown). In the probe test, the number of crossing over the formerplatform location in isoflurane-treated pups was fewer than the cor-responding control animals (Fig. 1B, Pb0.05), but the time spent inthe third quadrant where the platform located has no difference(data not shown).

172 F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174

3.3. Immunoreactivity assay

In the isoflurane-exposed pups, the expression of CHOP in the hip-pocampal CA1 and dentate gyrus area increased compared with thecontrol (Fig. 2A a and b). The caspase-12 expression displayed thesame tendency of increase in the hippocampal area in the isoflur-ane-treated pups (Fig. 2Ac and d). Quantitative analysis of the num-ber and optical densities of CHOP and caspase-12 positive neuronsfor the whole hippocampal CA1 and dentate gyrus area showed thattheir immunoreactivity was increased compared with the control(Fig. 2B and C, Pb0.01).

3.4. CHOP and caspase-12 protein levels

Consistent with the findings of immunohistochemistry studies,western blot analysis showed that the levels of CHOP and caspase-12 protein markedly increased in the hippocampal region of isoflur-ane-exposed pups when compared with the control (Fig. 3, Pb0.01).

3.5. Ultrastructure changes in synapse of hippocampus

Synapses with postsynaptic densities, an inerratic synaptic cleftand a presynaptic vas were clearly visible in the control pups(Fig. 4A and C). In contrast, in the isoflurane-treated pups, the num-ber of synapses decreased in the dentate gyrus and CA1 area, whilea widened synaptic cleft, thinned postsynaptic densities and loss ofa presynaptic vas were observed (Fig. 4).

4. Discussion

In the present study, we employed a new model, a maternal fetalrat model, to study the behavioral and neurotoxic effects of exposureto anesthetics and investigate the potential mechanisms. Our results

Fig. 4. Ultrastructural changes of synapse in the CA1 and dentate gyrus area of hi

demonstrate that gestational exposure to a clinically relevant concen-tration of isoflurane causes postnatal spatial memory and learningimpairments in the offspring rats. Moreover, neuron apoptosis andchanges of synaptic structure were also observed at the hippocampallevel in pups subject to isoflurane.

Our present work confirmed that the levels of CHOP and caspase-12 increased at hippocampal level in isoflurane-exposed rats, as indi-cated by the significant increase in the amount and densities of CHOPand caspase-12-positive cells, as well as the levels of CHOP and cas-pase-12 protein. Neuronal cell death after general anesthesia has re-cently been documented in several immature animal models. Somestudies proposed that inhalational anesthetics, such as isoflurane, in-duced cell death processes through activation of γ-aminobutyric acidand inhibition of N-methyl-D-aspartate receptors (Ikonomidou et al.,1999; Olney et al., 2004). However, the mechanisms of the effectare not clear or fully understood. Recent advances indicate that ER re-sponses play a pivotal role in cellular apoptosis after exposure to var-ious stresses, such as hypoxia, calcium dysregulation and oxidativestress (Larner et al., 2005; Schroder and Kaufman, 2005). C/EBP ho-mologous protein (CHOP), also known as GADD153 (growth arrest-and DNA damage-inducible gene 153), is a member of the C/EBP fam-ily of bZIP transcription factors, and its low expression under normalconditions is induced to high levels by ER stress. The role of CHOP inER stress-induced apoptosis has been illustrated in Chop−/− mice(Oyadomari et al., 2001; Zinszner et al., 1998). Caspase-12 has beenproposed as a key mediator of ER stress-induced apoptosis (Szegezdiet al., 2003). CHOP activation occurs concomitantly with the activa-tion of caspase-12, and activated caspase-12 in turn produces activa-tion of the caspase cascade (Rao et al., 2002). Caspase-12 activation ismediated mainly by calpain, which is released from the ER membraneby tumor necrosis factor receptor-associated factor. Subsequently,caspase-12 interacts with caspase-9, which forms part of the ‘intrinsic’apoptotic pathway, leading to activation of the executer caspase-3.

ppocampus under TEM. (A, C) control pups, (B, D) isoflurane-exposed pups.

173F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174

Therefore, CHOP and caspase-12-mediated ER stress-induced cell deathappear to be themajormediators of anesthesia-mediated apoptotic cellu-lar death.

Learning and memory are important aspects of cognitive func-tion. Our results showed that prenatal exposure to isoflurane dis-played deficits in postnatal spatial learning and memorycapabilities in pups as manifested by the longer escape latencyand the fewer times of original platform crossing in the MorrisWater Maze test. The lack of differences in swimming speeds be-tween the two groups suggested that the learning and memory def-icits observed in our study were not due to sensorimotordisturbances. Consistent with previous studies in maternal fetalrat models, these findings indicate that rats exposed to anestheticsin utero during fetal neurodevelopment is capable of causing be-havioral abnormalities in adolescent animals (Chalon et al., 1981;Palanisamy et al., 2011). However, the effects of anesthesia usedduring the development of fetal brains on postnatal memory andlearning ability are controversial, with transient improvement (Liet al., 2007), no effects (McClaine et al., 2005) and permanent im-pairment (Chalon et al., 1981; Palanisamy et al., 2011) all beingreported. These discrepancies could be due to methodological dif-ferences, species differences (rats vs. mice), pharmacological differ-ences (isoflurane vs. sevoflurane), differences in anestheticconcentrations (0.5–2 MAC), or differences in anesthetic durations(1–6 h). Last but not the least is the time of isoflurane exposure.Since different neurodevelopmental events are performed in theirtiming relative to gestational age, it is expected that the vulnerabil-ity of the brain to the adverse effects of the anesthetic agentswould be different depending on the time of exposure. Correspond-ingly, behavioral outcome varies as a function of the neurodevelop-mental events occurring at the time of exposure. The time ofisoflurane exposure in the current study corresponds approximatelyto midgestation in human, and studies in several animal speciessuggest that susceptibility is limited to a brain developmentalstate corresponding to the human second trimester of pregnancy.

In the present study, our results demonstrated that the number ofsynapses decreased significantly in the dentate gyrus and CA1 areawith widened synaptic clefts, thinned postsynaptic densities and aloss of the presynaptic vas in the isoflurane-exposed rats. It is widelyrecognized that there is a relationship between hippocampal synapticplasticity and memory (Gruart et al., 2006; Sametsky et al., 2010;Thompson et al., 2008). The synaptic cleft is a region of informationtransmission among neurons and plays an important role in the dy-namics of synaptic activity. The thickness of postsynaptic densitiesand the ability of learning and memory training and memory reten-tion go hand in hand. These changes impaired synapse normal struc-ture and resulted in interruption of synaptic connections. Our resultssuggest that the interruption of synaptic connections may be a mech-anismwhich further leads to changes of synaptic plasticity and conse-quent memory and learning impairments.

In summary, the current findings demonstrate that prenatal expo-sure to anesthetic drugs during a critical period of neural develop-ment causes neuron apoptosis, changes of synaptic structure, andpostnatal spatial memory and learning impairments in the offspringrats. We speculate that the up-regulation of CHOP and caspase-12may contribute to neural cell apoptosis, leading to damage in synapsenumber and function and consequent impairments in synaptic plas-ticity, all of which would contribute to the long-term neurocognitivedecline. With the gradual rise in the occurrence of fetal and non-ob-stetric surgery during pregnancy under general anesthesia, it is im-perative that further animal studies into the mechanism as well asclinical studies defining human susceptibility are both urgently need-ed. A better understanding of the inhalational anesthetics mecha-nisms will help us to guide clinical trials aiming to define the scopeof the problem in humans and may lead to preventive and therapeu-tic strategies.

Acknowledgments

We thank Shu Han, M.D., Ph.D. (Associate Professor, Institute ofAnatomy and Cell Biology, School of Medicine, Zhejiang University,China) for the technical support and thought-provoking discus-sions. Our work was supported by the Medical and Health Re-search Fund of Health Department of Zhejiang Provincial, China(no. 2010KYA129).

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