Suberoylanilide hydroxamic acid (SAHA) alleviates the learning and memory impairment in rat offspring caused by maternal sevoflurane exposure during late gestation

Abstract

Recent studies have shown that sevoflurane can cause long-term neurotoxicity and learning and memory impairment in developing and progressively neurodegenerative brains. Sevoflurane is a widely used volatile anesthetic in clinical practice. Late gestation is a rapidly developing period in the fetal brain, but whether sevoflurane anesthesia during late gestation affects learning and memory of offspring is not fully elucidated. Histone deacetylase 2 (HDAC2) plays an important regulatory role in learning and memory. This study examined the effect of maternal sevoflurane exposure on learning and memory in offspring and the underlying role of HDAC2. The Morris water maze (MWM) test was used to evaluate learning and memory function. Q-PCR and immunofluorescence staining were used to measure the expression levels of genes related to learning and memory. The results showed that sevoflurane anesthesia during late gestation impaired learning and memory in offspring rats (e.g., showing increase of the escape latency and decrease of the platform-crossing times and target quadrant traveling time in behavior tests) and upregulated the expression of HDAC2, while downregulating the expression of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and the N-methyl-D-aspartate receptor 2 subunit B (NR2B) mRNA and protein in the hippocampus of offspring in a time-dependent manner. HDAC2 inhibitor suberoylanilide hydroxamic acid (SAHA) treatment alleviated all of these changes in offspring rats. Therefore, the present study indicates that sevoflurane exposure during late gestation impairs offspring rat’s learning and memory via upregulation of the expression of HDAC2 and downregulation of the expression of CREB and NR2B. SAHA can alleviate these impairments.

INTRODUCTION

Two decades ago, it was thought that volatile anesthetics could be rapidly eliminated and caused no long-term neurotoxicity to the brain. However, recent clinical evidence showed that volatile anesthetics may cause long-term cognitive impairment in young and elderly patients, thus seriously affecting the patient’s rehabilitation and long-term quality of life (Royse et al., 2011). Recent experimental results showed that volatile anesthetics cause neuronal degeneration (Loepke et al., 2009) or apoptosis (Zhu et al., 2010), change the expression of cognition-related proteins (Fütterer et al., 2004), cause the loss of neuronal dendritic spines or neuronal morphological changes (Sanchez et al., 2011), and result in permanent impairment of learning and memory (Culley et al., 2004).

Sevoflurane is a widely used volatile anesthetic in clinical practice. Recent evidence demonstrates that sevoflurane can cause cognitive dysfunction in the developing and neurodegenerative brain (Aguiar et al., 2011). Sevoflurane is lipophilic and can cross the placenta easily. Gestation is one of the most susceptible periods for brain development (McGowan et al., 2008), and the last trimester (third trimester) is the peak of brain development. Noxious exposure during this period will affect the development and differentiation of neurons, synapse formation and plasticity, and even lead to permanent central nervous system dysfunction (Xiang et al., 2008). A significant number of pregnant women have to undergo nonobstetric operations during gestation (Goodman, 2002), and most of these operations have to be done during late gestation. Our previous study showed that anesthesia with 1.8% sevoflurane for 4 and 8 hr could not induce learning and memory impairment in the first trimester (5 to 7 days) of gestation (Li et al., 2010). However, it has been confirmed that general anesthetics cause neuronal apoptosis in a time-dependent manner, and different timing of exposure causes different effects on neurons in the brain (Palanisamy, 2012). However, whether sevoflurane anesthesia during the third trimester impairs the learning and memory of offspring in a time-dependent manner remains unclear.

The N-methyl-D-aspartate receptor (NMDAR) in the hippocampus has an important role in the formation and maintenance of learning and memory. Among the subunits of NMDAR, the NR2B subunit serves as a positive regulatory factor of learning and memory (Miwa et al., 2008). NR2B knockout results in cognitive dysfunction, while transfection with the NR2B gene facilitates the formation of long-term potentiation (LTP) and enhances learning and memory in mice (Von Engelhardt et al., 2008). Sevoflurane inhibits the transmission of synaptic cholinergic neurons and LTP in hippocampal synapses by blocking NMDA receptors, and it also facilitates long-term depression (LTD). Our previous study showed that maternal enflurane anesthesia during early gestation significantly impaired learning and memory in rat offspring and downregulated the expression of NR2B in the hippocampus (Liu et al., 2010b; Luo et al., 2011; Xu et al., 2012).

It is known that sevoflurane may affect the ligand-gated ion channels and G-protein coupled receptors by disturbing the expression of NMDAR and/or γ-aminobutyric acid (GABA) receptors, then disrupting the phosphorylation of CREB, leading to learning and memory dysfunction after anesthesia (Wang et al., 2012). CREB is a nuclear factor in eukaryotes and has a critical role in neurogenesis, synaptogenesis and the formation and maintenance of learning and memory (Benito and Barco, 2010). Under physiological conditions, NMDA receptors exert neurotrophic effects mainly via the CREB signal transduction pathway, promoting the survival of nerve cells, regulating the development of the central nervous system, and mediating the release of neurotransmitters (Valera et al., 2008). A previous study found that activation of CREB promoted synaptic facilitation (Viosca et al., 2009), whereas dephosphorylation at the Ser-133 site of CREB in the hippocampus induced LTD in mice (Mauna et al., 2011).

Histone deacetylases (HDACs) play an important role in the modification of chromosomal structure and the regulation of the whole genome expression profile. HDACs are key enzymes that maintain the histone acetylation and histone deacetylation balance in the nucleosome (Nelson et al., 2004). Histone deacetylase 2 (HDAC2), one of the HDAC family members, plays a critical role in modulating LTP and learning and memory (Haettig et al., 2011; Reolon et al., 2011). HDAC2 overexpression can lead to cognitive dysfunction (Guan et al., 2009). Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor that is permeable to the blood-brain barrier, increases histone acetylation and facilitates gene transcription, resulting in restoration of memory performance in an AD model through inhibition of HDAC1, 2, 3, 6 and 8 (Kilgore et al., 2010) and improvement of pathological phenotypes in neurodegenerative animal models (Chuang et al., 2009). Two hours after SAHA administration, the acetylated histone level increased significantly in young rat hippocampus, and the phosphorylation of CREB (p-CREB) and the combination of p-CREB with the cAMP response element (CRE) in the NR2B promoter region were improved, there by increasing the expression of NR2B and enhancing learning and memory (Fujita et al., 2012). This finding indicates that HDAC2 regulates learning and memory via “HDAC2-CREB-NR2B” signaling pathway. Sevoflurane anesthesia can lead to cognitive dysfunction by reducing CREB phosphorylation (Culley et al., 2006; Dalle et al., 2011; Suzuki et al., 2011). However, whether this signaling pathway is involved in the cognitive dysfunction caused by sevoflurane anesthesia during late gestation is not known yet.

The MWM test is a reliable method of assessing spatial learning and memory in rodents (Bromley-Brits et al., 2011). In MWM trials, the time to find the platform reflects learning ability, while the time spent in the target quadrant and the platform crossing times reflect memory ability. Therefore, the pregnant rats were exposed to sevoflurane on G18 day, the learning and memory in rat offspring were tested with MWM from P30 and the expression levels of genes related to learning and memory were detected, then further verified with the HDAC2 inhibitor intervention in the present study. Thus, this study was performed to clarify whether sevoflurane exposure during late gestation (G18) can cause learning and memory impairment in rat offspring via the “HDAC2-CREB-NR2B” signaling pathway.

MATERIALS AND METHODS

Animals

The protocol was approved by the institutional review board of the First Affiliated Hospital of Nanchang University on the Animal Use in Research and Teaching and performed according to relevant guidelines and regulations. Ten-week old Sprague-Dawley (SD) female and male rats were provided by Nanchang University Animal Science Research Department (permission number JZDWNO: 2013-0095). Animals were kept in a temperature-controlled (22-25°C) room under a 12-hr light/dark period (light on at 7:00 AM) with ad libitum feeding. The Morris water maze (MWM) test was performed before group arrangement, and the rats with abnormal swimming posture and abnormal learning and memory were removed in order to minimize the effect of genetic factors on learning and memory in offspring. The female rats were then allowed to mate with male rats (1 male rat with 2 female rats per cage). After confirmed pregnancy, the pregnant rats were randomly assigned to sevoflurane exposure 2 hr group (Sev2h), 4 hr group (Sev4h) and control group, with 10 rats in each group.

Sevoflurane exposure

On gestation day 18 (G18), the pregnant rats in the Sev2h and Sev4h groups received 1.8% sevoflurane in 100% oxygen for 2 hr or 4 hr respectively in an anesthetizing chamber. Saturation of pulse oximetry (SpO2), tail noninvasive blood pressure and electrocardiogram were continuously monitored during sevoflurane exposure. The rectal temperature was maintained at 37 ± 0.5°C with a thermal blanket. If the cumulative time of systolic blood pressure less than 80% and/or SpO2 less than 95% of the baseline was more than 5 min, the pregnant rat was removed. Another pregnant rat would be used to supply the sample. The gases were analyzed at the end of sevoflurane exposure. After that, the rats were kept in a chamber with 100% oxygen until 30 min after recovery of righting reflex. The rats in the control group received 100% oxygen for 4 hr instead.

HDAC2 inhibitor injection

Thirty days after birth (P30), the offspring rats born to each maternal rat were randomly divided into HDAC2 inhibitor subgroup (SAHA subgroup) and non-HDAC2 inhibitor subgroup (DMSO subgroup). Two hours before each MWM trial, 90 mg/kg HDAC2 inhibitor (SAHA, 50 mg/mL in DMSO solution) was intraperitoneally injected into the offspring in the SAHA subgroup (once a day for seven consecutive days). The same volume of DMSO solution was given to the offspring in the DMSO group.

Morris water maze test

The offspring rats’ learning and memory were determined by the MWM test as we previously reported (Zhong et al., 2016). Briefly, the training trial was performed once a day for six consecutive days (P30-P35). In this trial, the offspring rat was put into the water to look for the platform, and the time to find the platform was recorded as escape latency (reflecting learning ability). If the offspring found the platform within 120 sec, it was allowed to stay on the platform for 30 sec. Otherwise, the escape latency was recorded as 120 sec for the offspring, and then the offspring was guided to the platform and allowed to stay on the platform for 30 sec. On the seventh day (P36), the platform was removed, and the offspring was allowed to swim for 120 sec. The time spent in the original platform quadrant (target quadrant time) and the times crossing the original platform area (platform crossing times) were recorded. Both the target quadrant time and platform crossing times reflect memory function. The mean value of MWM test results of the offspring rats born to each maternal rat was calculated as one final result.

Hippocampal harvest

Twenty-four hours after the MWM test, the offspring rats were anesthetized and sacrificed by cervical dislocation. The left hippocampus was collected and placed in ribozyme-free Eppendorf tubes for total RNA extraction. The right hippocampus was immersed in 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4 at 4°C) and then embedded in paraffin for immunohistochemistry.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from hippocampus using the TRIzol kit (Takara, Shiga, Japan). cDNA was synthesized according to manufacturer’s instruction. qRT-PCR was performed using the SYBR Green PCR Kit (RR820Q, Takara). The primers were: HDAC2-forward primer (5’-AGC CCA TGG CGT ACA GTC AA-3’), reverse primer (5’-GGA TGA CCC TGG CCG TAA TAA TAA-3); CREB-forward primer (5’- ACA GTT CAA GCC CAG CCA CAG-3’), reverse primer (5’-GCA CTA AGG TTA CAG TGG GAG CAG A-3’); NR2B-forward primer (5’-TGG CTA TCC TGC AGC TGT TTG-3’), reverse primer (5’-TGG CTG CTC ATC ACC TCA TTC-3’); β-actin-forward primer (5’-GGA GAT TAC TGC CCT GGC TCC TA-3’), reverse primer (5’-GAC TCA TCG TAC TCC TGC TTG CTG-3’). The products of PCR were measured using the ABI 7500 system (Applied Biosystems, Waltham, MA, USA). The results were calculated using the 2^−ΔΔCt method (Ct is cycle thresholds). The relative expression levels of target genes were normalized to β-actin.

Immunofluorescence staining

Paraffin-embedded hippocampi (n = 6) were cut into 4 μm sections. After antigen retrieval and incubation in 3% H₂O₂, the sections were incubated with the primary antibodies: anti-HDAC2 (1:200, ab32117, Abcam, Cambridge, UK), anti-p-CREB (1:100, ab32096, Abcam) or anti-NR2B (1:250, ab65783, Abcam) dissolved in 1% bovine serum albumin (BSA) at 4°C overnight. Then, the sections were exposed to the green fluorescent-conjugated secondary antibody (1:250, TransGen Biotech, Beijing, China) for 1 hr and DAPI stained for 5 to 10 min. Finally, the sections were viewed immediately using an inverted fluorescence microscope (200X) (Olympus, Japan). The photos were taken and the fluorescent densities of HDAC2, p-CREB and NR2B were detected using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA). The images were converted to black and white. After intensity calibration, the pyramidal cell layer of hippocampal CA1 area was chosen, then the sum of area and the integrated optical density (IOD) were measured. The IOD /Area was calculated to determine the protein expression levels.

Statistical analysis

All of the data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The escape latency was analyzed by repeated measures two-way analysis of variance (RM two-way ANOVA), followed by least significant difference t (LSD-t) analysis when a significant difference for the factor was found (p < 0.05). The normality and homogeneity of the data were tested for weight, average litter size, blood gas, platform crossing times, target quadrant time, Q-PCR and immunofluorescence staining. If the data met the assumptions of normality and homogeneity, they were subjected to one-way ANOVA, followed by LSD-t when a significant difference for the factor was found. The Kruskal-Wallis test was performed if the assumptions of normality and homogeneity were unmet. Student’s t-test was used to analysis the effect of SAHA treatment when the assumption of normality was met, and the Mann-Whitney U test was performed if the assumption of normality was unmet. The survival rate and sex composition in offspring rats were analyzed using the Chi-square test. Statistical significance was declared at p < 0.05.

RESULTS

Physiological characteristics of offspring

The average litter size and the sex composition of offspring were calculated after the birth. The survival rate and the average body weight of the offspring were analyzed on P30. There were no significant differences in average litter size, sex composition, survival rate, or average body weight among three groups (p > 0.05) (Fig. 1a-d). These results indicate that the changes in offspring’s learning and memory observed in the present study were caused by sevoflurane exposure rather than physical differences.

Fig. 1

Physical characteristics of the offspring rats. (a) The average litter size of offspring rats, ANOVA, F(2, 27) = 0.309, p = 0.737. (b) Sex composition (the ratio of females to males), Chi-square test, X² = 0.401, p = 0.828. (c) Survival rate (survived to P30) of offspring, Chi-square test, X² = 0.007, p = 1.00. (d) The average weight (weight at P30) of the offspring rats, ANOVA, F(2, 27) = 0.292, p = 0.749. There were no significant differences. Sev2h = sevoflurane exposure 2 hr group, Sev4h = sevoflurane exposure 4 hr group. n = 10 in each group.

Blood gases analysis

Venous blood gas analysis showed that there were no significant differences in blood gases among the Sev2h, Sev4h and control groups (p > 0.05) (Table 1), suggesting that 1.8% sevoflurane exposure for 4 hr did not change the internal environment of pregnant rats. This indicated that the learning and memory changes observed in present study were caused by sevoflurane itself, rather than disturbance of the internal environment in pregnant rats.

Table 1. Blood gases in pregnant rats (mean ± SD).

Parameter	Control (n = 4)			Sev2h (n = 3)			Sev4h (n = 3)			P value
pH	7.41	±	0.03	7.42	±	0.01	7.43	±	0.01	0.24
PaO2 (mmHg)	48.75	±	6.18	54.00	±	4.58	52.33	±	2.52	0.40
PaCO2 (mmHg)	50.25	±	1.71	48.67	±	3.21	51.33	±	2.08	0.42
HCO3- (mmol/L)	31.18	±	0.35	29.73	±	2.31	30.17	±	1.61	0.47
Na+ (mmol/L)	135.25	±	1.26	136.67	±	3.06	136.67	±	1.15	0.55
BE (mmol/L)	3.85	±	1.22	3.93	±	1.76	3.77	±	0.38	0.98
K+ (mmol/L)	4.68	±	0.74	4.90	±	0.30	4.63	±	0.15	0.80
Ca2+ (mmol/L)	1.23	±	0.23	1.44	±	0.04	1.45	±	0.02	0.17

Learning and memory impairments in offspring and the therapeutic effect of SAHA

The MWM test showed that the offspring in the sevoflurane exposure group had to spend more time to find the platform than offspring in the control group (Fig. 2a). Meanwhile, the platform-crossing times in sevoflurane exposure groups were less than in the control group, though the difference was not significant (p > 0.05) (Fig. 2b). The travel time to the target quadrant was decreased in the sevoflurane anesthesia group, especially in the Sev4h+DMSO group (p < 0.05) (Fig. 2c). There was no significant difference in swimming speed among the three groups. These results indicate that maternal sevoflurane anesthesia in the third trimester impaired rat offspring’s learning and memory in a time-dependent effect.

Fig. 2

Sevoflurane anesthesia in late gestation impaired learning and memory in offspring. (a) Escape latency (reflecting learning ability). Compared with the control group, the escape latency of offspring was significantly prolonged in the sevoflurane anesthesia group, and the escape latency in the Sev4h+DMSO group was significantly longer than in the Sev2h+DMSO group, RM two-way ANOVA, F(2, 27) = 24.708, p < 0.001. (b) Platform crossing times (reflecting memory ability), ANOVA, F(2, 27) = 2.444, p = 0.106. (c) The target quadrant traveling time (reflecting memory ability). Target quadrant traveling time in the Sev4h+DMSO group was significantly shorter than in the Control+DMSO group, Kruskal-Wallis, H = 6.452, p = 0.04. (d) Swimming speed in the three subgroups, F(2, 27) = 0.377, p = 0.69. There was no obvious difference. ^&p < 0.05, Sev2h+DMSO vs. Control+DMSO; *p < 0.05, Sev4h+DMSO vs. Control+DMSO; ^ap < 0.05, Sev4h+DMSO vs. Sev2h+DMSO. DMSO = Dimethyl sulfoxide. n = 10 in each group.

The acetylation and deacetylation of histones, which are modulated by the interaction of HDACs and histone acetyltransferases (HATs), play an important role in learning and memory function (Benoit et al., 2016). To verify the potential role of HDAC2 in the learning and memory impairment caused by sevoflurane anesthesia during late gestation, the HDAC2 inhibitor SAHA was used to interfere with the expression of HDAC2. The results showed that SAHA significantly improved learning and memory in sevoflurane anesthesia groups, as the escape latency decreased significantly (p < 0.05, Sev2h+SAHA vs. Sev2h+DMSO, Sev4h+SAHA vs. Sev4h+DMSO) (Fig. 3b-c), while the target quadrant traveling time increased significantly (p < 0.05) when compared to their relative DMSO control subgroups (Sev2h+SAHA vs. Sev2h+DMSO, Sev4h+SAHA vs. Sev4h+DMSO) (Fig. 3e). After treatment with SAHA, the platform-crossing times also increased compared to the DMSO control subgroups, though the differences were not significant (p > 0.05, Fig. 3d). There were no significant differences in swimming speed in SAHA subgroups compared to their relative DMSO control subgroups (Fig. 3f). These findings suggest that HDAC2 mediated the learning and memory impairment caused by sevoflurane exposure.

Fig. 3

SAHA alleviated the learning and memory impairment induced by sevoflurane anesthesia. (a-c) The escape latency. After treatment with SAHA, the escape latency was significantly reduced, RM two-way ANOVA, Control+DMSO vs. Control+SAHA: F(1, 18) = 152.673, p < 0.001; Sev2h+DMSO vs. Sev2h+SAHA: F(1, 18) = 36.834, p < 0.001; Sev4h+DMSO vs. Sev4h+SAHA: F(1, 18) = 140.655, p < 0.001. (d) Platform-crossing times. After treatment with SAHA, the platform-crossing times increased, but there was no significant difference, Mann-Whitney U test, DMSO vs. SAHA: Control, U = 34.5; Sev2h, U = 29; Student’s t-test, DMSO vs. SAHA, Sev4h, t₁₈ = -2.038. (e) Target quadrant traveling time. After treatment with SAHA, the target quadrant traveling time increased significantly, Student’s t-test, DMSO vs. SAHA: Control, t_14.4 = -8.251; Sev2h, t₁₈ = -2.283; Sev4h, t₁₈ = -3.815. (f) Swimming speed in six subgroups. There was no difference between DMSO and SAHA subgroups. *p < 0.05, SAHA subgroup vs. DMSO subgroup. DMSO = Dimethyl sulfoxide, and SAHA is the HDAC2 inhibitor subgroup. n = 10 in each group.

Changes of HDAC2, CREB and NR2B mRNA expression and the effect of SAHA

The results showed that the levels of HDAC2 mRNA in the hippocampus of sevoflurane-exposed offspring were upregulated (Fig. 4a), whereas the levels of both CREB mRNA (Fig. 4b) and NR2B mRNA were downregulated (Fig. 4c). HDAC2 inhibitor SAHA significantly alleviated these changes (Fig. 4d-f).

Fig. 4

Sevoflurane anesthesia upregulated the expression of HDAC2 mRNA while downregulating that of CREB and NR2B mRNA. (a, d) HDAC2 mRNA levels in offspring hippocampus. Compared to the control group, the levels of HDAC2 mRNA were increased in sevoflurane anesthesia groups, ANOVA, F(2, 27) = 13.972, p < 0.001. SAHA decreased the levels of HDAC2 mRNA, Student’s t-test, DMSO vs. SAHA: Control, t₁₈ = 7.025; Sev2h, t₁₈ = 2.242; Sev4h, t₁₈ = 5.273. n = 10 in each group. (b, e) CREB mRNA level in offspring’s hippocampus. Compared to the control group, the levels of CREB mRNA in sevoflurane anesthesia groups were reduced, Kruskal–Wallis, H = 13.727, p < 0.001. SAHA mitigated the reduction of CREB mRNA expression, Mann-Whitney U test, DMSO vs. SAHA: Control, U = 20; Student’s t-test, DMSO vs. SAHA: Sev2h, t₁₄ = -8.352; Sev4h, t₁₄ = -2.887. n = 8 in each group. (c, f) NR2B mRNA levels in offspring’s hippocampus. The expression of NR2B mRNA was downregulated by sevoflurane anesthesia, ANOVA, F(2, 24) = 17.733, p < 0.001. SAHA alleviated this changes, Student’s t-test, DMSO vs. SAHA: Control, t₁₆ = -8.813; Sev2h, t₁₆ = -4.637; Sev4h, t₁₆ = -3.305. n = 9 in each group. DMSO = Dimethyl sulfoxide, and SAHA is the HDAC2 inhibitor subgroup.

Changes in HDAC2, p-CREB and NR2B protein expression and the effect of SAHA

The CA1 region of the hippocampus is important for the retrieval of spatial and contextual memories (Tanaka et al., 2014; Goshen et al., 2011). Further evidence has shown that sevoflurane can impair the development of neurons in the hippocampus and the plasticity of pyramidal cells in area CA1 in the rapidly developing brain, which are important for the formation and consolidation of learning and memory (Yang et al., 2014). Therefore, present experiments mainly studied the changes in HDAC2, p-CREB and NR2B protein levels in hippocampal CA1 region of offspring rats. The results showed there were detectable levels of HDAC2, p-CREB and NR2B protein in all the offspring’s hippocampal neurons (Fig. 5a-c). Sevoflurane anesthesia significantly increased the levels of HDAC2 protein in offspring’s hippocampus (Fig. 5g), while decreased the levels of p-CREB and NR2B proteins (Fig. 5h, i). SAHA significantly alleviated the changes in these proteins (Fig. 5d-f).

Fig. 5

Immunofluorescence staining of HDAC2, p-CREB and NR2B proteins in hippocampus. (a-c) Immunofluorescence staining for HDAC2, p-CREB, and NR2B in hippocampal tissues of rat offspring. Scale bar = 100 μm. SO = Strata Oriens, SP = Stratum Pyramidale, SR = Strata Radiatum. (d, g) HDAC2 protein: the expression levels of HDAC2 protein in sevoflurane anesthesia groups were significantly higher than in the control group, ANOVA, F(2, 15) = 34.43, p < 0.001. SAHA decreased the levels of HDAC2 protein, Student’s t-test, DMSO vs. SAHA: Control, t₁₀ = 2.314; Sev2h, t₁₀ = 4.167; Sev4h, t₁₀ = 2.1. (e, h) p-CREB protein: the expression levels of p-CREB protein in sevoflurane groups were significantly lower than in the control group, ANOVA, F(2, 15) = 26.868, p < 0.001. SAHA mitigated this change, Student’s t-test, DMSO vs. SAHA: Control, t₁₀ = -2.266; Sev2h, t₁₀ = -1.264; Sev4h, t₁₀ = -3.386. (f, i) NR2B protein: the expression levels of NR2B protein in sevoflurane anesthesia groups were significantly lower than in the control group, ANOVA, F(2,15) = 9.513, p = 0.002. SAHA mitigated this change, Student’s t-test, DMSO vs. SAHA: Control, t₁₀ = -2.322; Sev2h, t₁₀ = -1.219; Sev4h, t₁₀ = -2.222. DMSO = Dimethyl sulfoxide, and SAHA is the HDAC2 inhibitor subgroup. n = 6 in each group.

DISCUSSION

The present study showed that maternal exposure to sevoflurane on G18 impaired learning and memory in offspring rats, upregulated the expression of HDAC2, and downregulated the expression of p-CREB and NR2B in the hippocampus of offspring. An HDAC2 inhibitor alleviated all these changes.

There was no significant difference in offspring’s physiological characteristics among the three groups. The body temperature, respiratory rate, heart rate, and blood gases analysis of maternal rats were monitored during sevoflurane inhalation. No significant differences were found in these indexes. Therefore, the learning and memory impairments in rat offspring were not caused by the offspring’s physiological characteristics or maternal pathophysiological disturbance but were caused by sevoflurane itself.

The MWM test is a reliable method of assessing spatial learning and memory in rodents, especially for learning and memory directly related to hippocampal function (Bromley-Brits et al., 2011; Abdul et al., 2008). During MWM trials, the time for offspring rats to find the platform was defined as escape latency (reflects learning ability). The time spending in the original platform-quadrant was defined as target quadrant time and the times crossing the original platform area was defined as platform crossing times. Both the target quadrant time and the platform crossing times reflect the memory ability. While the offspring learn how to find the platform during MWM trials depends on memory in some degree. Therefore, it is very difficult to separate the memory components from learning ability, though the escape latency mainly reflects learning ability. In the present study, MWM results showed that 1.8% sevoflurane anesthesia during the third trimester impaired the learning and memory in offspring rats in a time-dependent manner. Hui Zheng et al. found that 2.5% sevoflurane anesthesia on G18 had adverse effects on rat fetuses’ brains (Zheng et al., 2013). Multiple exposures to 2.5% sevoflurane during late gestation caused learning and memory impairment in offspring (Fang et al., 2017). One animal study showed that maternal exposure to 1.5% sevoflurane for 6 hr on E17 did not cause short-term learning and memory impairment in offspring (Suehara et al., 2016). This indicates that sevoflurane concentration is an important factor in the effect on learning and memory in offspring. It has been confirmed that learning and memory disorder caused by anesthesia is related to anesthetic type, physical and chemical properties of the anesthetics, anesthetic dosage and anesthetic exposure time (Suehara et al., 2016; Shang and Yao, 2006).

Previous studies have shown that abnormal expression of PSD-95, synaptophysin, interleukin-6 (IL-6) (Zheng et al., 2013) and caspase-3 (in the thalamus and parietal cortex) as well as abnormal neuronal precursor cell proliferation in the hippocampus (Lei et al., 2013) were involved in the learning and memory impairments caused by sevoflurane. The expression of PSD-95 and synaptophysin were regulated by NMDA receptors (Liu et al., 2010a), which play an important role in neuronal survival, synaptic transmission, synaptic plasticity, learning and memory and other cognition. The functional properties of the entire NMDA receptor are modified by its subunit NR2B (Collingridge et al., 2013). Sevoflurane can cause hippocampal neuronal damage in the rapidly developing brain and impair the plasticity of pyramidal cells in CA1 area, which are closely related to the formation and consolidation of learning and memory (Yang et al., 2014). Therefore, the present experiment mainly studied the changes in NR2B expression in the hippocampal CA1 region of offspring rats. The present results show that sevoflurane anesthesia in the third trimester decreased the expression levels of NR2B mRNA and protein.

Epigenetic regulation plays an essential role in the formation and maintenance of learning and memory (Bintu et al., 2016). Epigenetic modifications, such as histone acetylation, are extremely important regulatory processes in the formation of memory. HDAC2, one of the members of the HDAC family, plays an important role in the synaptic transmission of mature neurons (Montgomery et al., 2009; Akhtar et al., 2009). Overexpression of HDAC2 decreases the histone acetylation in the promoter regions of genes involved in cognition, causes downregulation of corresponding protein expression and synaptic plasticity disorder, and leads to learning and memory impairment (Wang et al., 2010; Shibasaki et al., 2011). In the present study, we found that sevoflurane anesthesia on G18 increased the levels of HDAC2 mRNA and protein, decreased the levels of p-CREB and NR2B mRNA and proteins in the hippocampus of offspring and caused learning and memory impairments in offspring. To further confirm the role of HDAC2 in the learning and memory impairments caused by sevoflurane, HDAC2 inhibitor SAHA was given to the offspring at 2 h before each MWM test. It is difficult to know whether the behavioral improvements caused by SAHA in offspring exposed to sevoflurane are due to SAHA’s compensatory effects or its specific reversal of sevoflurane’s effects. However, the effects of SAHA on the offspring exposed to sevoflurane, along with the effects of sevoflurane on the offspring’s behavior and the expression levels of HDAC2, CREB and NR2B, suggest that sevoflurane may act on HDAC2, CREB and NR2B to impair learning and memory in rat offspring.

Our previous study found that propofol exposure in late gestation caused learning and memory impairments in rat offspring by interfering with the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling pathway in the hippocampus (Zhong et al., 2016). Histone acetylation is one of the upstream regulatory mechanisms of the “BDNF-TrkB” signaling pathway (Ji et al., 2014). Juhasz G et al. showed that CREB can affect the expression of BDNF (Réus et al., 2011; Juhasz et al., 2011). Blocking NR2B receptors decreases the expression of BDNF (Crozier et al., 1999). Thus, BDNF-TrkB may be one of the downstream targets of the “HDAC2-CREB-NR2B” pathway. However, the role of the “BDNF-TrkB” signaling pathway in the impairment of learning and memory caused by maternal sevoflurane anesthesia during the third trimester is still unknown and needs further study.

The present study first revealed that maternal sevoflurane anesthesia during late gestation impairs learning and memory in rat offspring via interference with the expression of HDAC2, p-CREB and NR2B in the hippocampus of offspring. SAHA can alleviate the learning and memory impairment caused by maternal sevoflurane exposure during late gestation.

ACKNOWLEDGMENTS

We thank other member of the laboratory for valuable discussion and technical helps. This research was supported by National Natural Science Foundation of China (81460175, 81060093), Natural Science Foundation of Jiangxi Province of China (20171ACB20030) and Traditional Chinese Medicine Research Project Foundation of Jiangxi health and family planning commission (2017A292).

Conflict of interest

The authors declare that there is no conflict of interest.

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