new_pdfs/10.1016_j.ejphar.2011.08.050.txt

European Journal of Pharmacology 670 (2011) 168–174

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e j p h a r

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, China b Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China

a r t i c l e

i n f o

a b s t r a c t

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

Keywords: Isoflurane Memory and learning impairment Hippocampus C/EBP homologous protein Caspase-12 Neuron apoptosis

A maternal fetal rat model was developed to study the effects of gestational isoflurane exposure on postnatal memory 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 region were also observed by transmission electron microscopy (TEM). Prenatal exposure to isoflurane impaired postnatal spatial memory and learning in the offspring rats as shown by the longer escape latency and the fewer 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 showed changes 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 that gestational 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. Our study further showed that the up-regulation of CHOP and caspase-12 may contribute to isoflurane-induced neuron apoptosis.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Many pregnant women, fetuses, and infants are exposed to a variety of anesthetic agents for surgical or diagnostic procedures each year. Pregnant women sometimes undergo general anesthesia during their pregnancy for surgeries unrelated to the delivery, such as fetal and non-obstetric surgeries, especially during midgestation (Goodman, 2002; Tran, 2010). Since most general anesthetic agents are lipophilic and cross the placenta easily (Dwyer et al., 1995), the developing fetal brains will be exposed to anesthetics as well. Inhalation anesthetics such as isoflurane have been widely used in recent years in clinical and research practices. Preclinical studies demonstrate that early exposure to 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 et al., 2008; Kalkman et al., 2009; Wilder et al., 2009). These observations

⁎ Corresponding author at: Department of Anesthesiology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Bachelor Road 1, 310006, PR China. Tel.: + 86 571 87061501 2410; fax: + 86 571 87061878.

E-mail address: lig08010915@163.com (H. Lu).

1 Postal address: Department of Anesthesiology, Women's Hospital, School of Medi-

cine, Zhejiang University, Hangzhou, Bachelor Road 1, 310006, PR China.

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 subjects rather than on the fetuses. In this study, we hypothesized that gestation- al exposure to isoflurane during maternal anesthesia may have deleteri- ous effects on the fetal brain that leads to postnatal spatial memory and learning impairments in the offspring rats.

The cellular and molecular mechanisms of anesthetics-mediated neurotoxicity 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 three pathways contribute to ER stress-mediated cell death: transcription activation of the C/EBP homologous transcription factor (CHOP) (Oyadomari and Mori, 2004), activation of the IRE1-tumor necrosis factor receptor-associated factor (TRAF2) pathway (Matsukawa et al., 2004) and activation of ER-resident caspase-12 (Nakagawa and Yuan, 2000; Nakagawa et al., 2000). CHOP, a member of the C/ EBP transcription factor family, is induced by ER stress and thus causes apoptosis. Caspase-12, an ER-specific caspase, participates in apoptosis under ER stress. In the current study, we hypothesized that CHOP and caspase-12 play a role in the mechanisms of isoflur- ane-induced neuron apoptosis.

2 Postal address: Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang

University, Hangzhou, Yuhangtang Road 388, 310058, PR China.

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

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

169

In the present study, using a maternal fetal rat model, we tested the 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 changes in the hippocampal area. We also measured the levels of CHOP and caspase-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 the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang Universi- ty approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffer- ing. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-hour light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to midgestation in humans (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 isoflurane group (n = 5). The dams were placed in plastic containers resting in water 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. We chose 1.3% as the anesthetic concentration because it represents 1 minimum alveolar concentration (MAC) in the pregnant rats (Mazze et al., 1985). The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 4-hour isoflurane exposure. The isoflurane concentration in the box was monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA). Otherwise, control and experimental animals were under the same treatment and envi- ronment. During isoflurane anesthesia, arterial blood gases and blood glucose 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 dam were select- ed to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications (Jevtovic-Todorovic et al., 2003). A round pool (diameter, 150 cm; depth, 50 cm) was filled with

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 measuring spatial information acquisition. (B) Probe trial demonstrating the number of original platform crossing measuring memory retention capabilities. *P b 0.05 compared with control.

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

2.3.1. Place trials

The place trials were performed at postnatal day 29 for 4 days to determine the rats' ability to obtain spatial information. At postnatal day 28, the rats were made to know the existence of the platform through a 30-second swimming training. A dark black curtain sur- rounded the pool to prevent confounding visual cues. All rats received 4 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 swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, 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

Table 1 Maternal physiological parameters during isoflurane anesthesia.

0 h

4 h

Control

1.3% isoflurane

Control

1.3% isoflurane

pH PaCO2 (mm Hg) PaO2 (mm Hg) SaO2 (%) Glucose (mg/dl)

7.43 ± 0.02 35.8 ± 2.47 169.5 ± 4.32 95.4 ± 1.1 113 ± 21

7.43 ± 0.01 36.7 ± 1.34 168.2 ± 6.19 94.2 ± 1.2 115 ± 16

7.41 ± 0.02 36.6 ± 2.39 166.2 ± 6.41 95.1 ± 0.9 116 ± 22

7.39 ± 0.01 37.9 ± 3.25 165.3 ± 5.32 94.6 ± 1.1 115 ± 12

1.3% isoflurane did not affect arterial blood gas values and blood glucose levels significantly. PaCO2 = arterial carbon dioxide tension; PaO2 = arterial oxygen tension; SaO2 = arterial oxygen saturation.

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

2.3.2. Probe trials

Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved removing the submerged platform from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.

immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a grad- ed ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty-four hours later, the tis- sue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastruc- ture changes of synapse in the hippocampus were observed under a transmission electron microscope (Phliphs Tecnai 10, Holland).

2.4. Transmission electron microscopy

2.5. Tissue section preparation

After the Morris Water Maze test, three pups per group were anesthe- tized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippo- campus, including CA1 and dentate gyrus area, of each rat was taken out

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

Fig. 2. The expression of CHOP and caspase-12 increased significantly in the hippocampus of isoflurane-exposed pups showed by immuno-reaction. (Aa) CHOP immunohistochem- ical staining in control pups × 400. (Ab) CHOP immunohistochemical staining in isoflurane-exposed pups × 400. (Ac) Caspase-12 immunohistochemical staining in control pups × 400. (Ad) Caspase-12 immunohistochemical staining in isoflurane-exposed pups × 400. The number and optical density of the CHOP (B) and caspase-12 (C) positive neurons were compared between the control and 1.3% isoflurane treatment groups. **P b 0.01 compared to control. Scale bar = 50 μm.

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

200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20–30 min. The fixed brain 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 hippocampal area (5 mm thick) were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for 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 in 5% normal goat serum in 0.01 M PBS. The sections were then incubat- ed in the primary antibodies rabbit polyclonal against anti-CHOP or caspase-12 (1:100, Santa Cruz Biotechnology, USA) overnight at 4 °C. After a thorough wash in PBS, sections were incubated with bio- tinylated goat anti-rabbit IgG antibody (1:200, Boster, China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (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 with 0.2 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, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Three sections from each animal were selected at random and images were photographed under 400× magnification in 3 visual fields/per section, the CHOP and caspase-12 positive neu- rons were counted in the same area. The optical densities of CHOP and caspase-12 positive neurons were measured quantitatively using NIH image software (ImageJ, National Institutes of Health, Be- thesda, MD).

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 were within 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 treatment group. All pups were viable and there were no significant differences in growth rate of the rat pups between the two groups (data not shown).

3.2. Morris Water Maze test

As shown in Fig. 1A, pups in both groups showed a rapid decrease in latency, while the pups of the isoflurane group spent more time to find the platform than those of control group in the place trial (P b 0.05). Swimming speeds were also analyzed during place trials, and no differences were observed between the two groups (data not shown). In the probe test, the number of crossing over the former platform location in isoflurane-treated pups was fewer than the cor- responding control animals (Fig. 1B, P b 0.05), but the time spent in the third quadrant where the platform located has no difference (data not shown).

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 of normal 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 method using 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 milk buffer 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 horseradish peroxidase-conjugated secondary antibodies and developed with ECL kit. The optical densities of bands were quantitatively analyzed using Bio-Rad Quantity One 4.6.2 (Bio-Rad Laboratories, USA). The results were expressed as a relative density. Equal protein loading in each lane 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 of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were an- alyzed using Student's t-test for comparison of two groups. A P value of b0.05 was considered statistically significant. All statistical tests

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. **P b 0.01, ***P b 0.001 com- pared to control.

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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 the control (Fig. 2A a and b). The caspase-12 expression displayed the same 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 neurons for the whole hippocampal CA1 and dentate gyrus area showed that their immunoreactivity was increased compared with the control (Fig. 2B and C, P b 0.01).

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, P b 0.01).

3.5. Ultrastructure changes in synapse of hippocampus

Synapses with postsynaptic densities, an inerratic synaptic cleft and 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, while a widened synaptic cleft, thinned postsynaptic densities and loss of a presynaptic vas were observed (Fig. 4).

4. Discussion

demonstrate that gestational exposure to a clinically relevant concen- tration of isoflurane causes postnatal spatial memory and learning impairments in the offspring rats. Moreover, neuron apoptosis and changes of synaptic structure were also observed at the hippocampal level 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 CHOP and 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. Some studies proposed that inhalational anesthetics, such as isoflurane, in- duced cell death processes through activation of γ-aminobutyric acid and inhibition of N-methyl-D-aspartate receptors (Ikonomidou et al., 1999; Olney et al., 2004). However, the mechanisms of the effect are 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 oxidative stress (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 normal conditions is induced to high levels by ER stress. The role of CHOP in ER stress-induced apoptosis has been illustrated in Chop−/− mice (Oyadomari et al., 2001; Zinszner et al., 1998). Caspase-12 has been proposed as a key mediator of ER stress-induced apoptosis (Szegezdi et 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 is mediated mainly by calpain, which is released from the ER membrane by 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.

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

Fig. 4. Ultrastructural changes of synapse in the CA1 and dentate gyrus area of hippocampus under TEM. (A, C) control pups, (B, D) isoflurane-exposed pups.

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

Therefore, CHOP and caspase-12-mediated ER stress-induced cell death appear to be the major mediators 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 and memory capabilities in pups as manifested by the longer escape latency and the fewer times of original platform crossing in the Morris Water 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 sensorimotor disturbances. Consistent with previous studies in maternal fetal rat models, these findings indicate that rats exposed to anesthetics in 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 used during the development of fetal brains on postnatal memory and learning ability are controversial, with transient improvement (Li et al., 2007), no effects (McClaine et al., 2005) and permanent im- pairment (Chalon et al., 1981; Palanisamy et al., 2011) all being reported. These discrepancies could be due to methodological dif- ferences, species differences (rats vs. mice), pharmacological differ- ences in anesthetic concentrations (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 their timing relative to gestational age, it is expected that the vulnerabil- ity of the brain to the adverse effects of the anesthetic agents would 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 of isoflurane exposure in the current study corresponds approximately to midgestation in human, and studies in several animal species suggest that susceptibility is limited to a brain developmental state corresponding to the human second trimester of pregnancy.

in postnatal

spatial

learning

(isoflurane vs.

sevoflurane), differences

Acknowledgments

We thank Shu Han, M.D., Ph.D. (Associate Professor, Institute of Anatomy 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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