Propofol exposure during early gestation impairs learning and memory in rat offspring by inhibiting the acetylation of histone

Abstract Propofol is widely used in clinical practice, including non‐obstetric surgery in pregnant women. Previously, we found that propofol anaesthesia in maternal rats during the third trimester (E18) caused learning and memory impairment to the offspring rats, but how about the exposure during early pregnancy and the underlying mechanisms? Histone acetylation plays an important role in synaptic plasticity. In this study, propofol was administered to the pregnant rats in the early pregnancy (E7). The learning and memory function of the offspring were tested by Morris water maze (MWM) test on post‐natal day 30. Two hours before each MWM trial, histone deacetylase 2 (HDAC2) inhibitor, suberoylanilide hydroxamic acid (SAHA), Senegenin (SEN, traditional Chinese medicine), hippyragranin (HGN) antisense oligonucleotide (HGNA) or vehicle were given to the offspring. The protein levels of HDAC2, acetylated histone 3 (H3) and 4 (H4), cyclic adenosine monophosphate (cAMP) response element‐binding protein (CREB), N‐methyl‐D‐aspartate receptor (NMDAR) 2 subunit B (NR2B), HGN and synaptophysin in offspring's hippocampus were determined by Western blot or immunofluorescence test. It was discovered that infusion with propofol in maternal rats on E7 leads to impairment of learning and memory in offspring, increased the protein levels of HDAC2 and HGN, decreased the levels of acetylated H3 and H4 and phosphorylated CREB, NR2B and synaptophysin. HDAC2 inhibitor SAHA, Senegenin or HGN antisense oligonucleotide reversed all the changes. Thus, present results indicate exposure to propofol during the early gestation impairs offspring's learning and memory via inhibiting histone acetylation. SAHA, Senegenin and HGN antisense oligonucleotide might have therapeutic value for the adverse effect of propofol.


| INTRODUCTION
Accumulating evidence indicates general anaesthetics exposure during pregnancy may cause neurotoxic effects and induce persistent cognitive dysfunction of offspring rats. [1][2][3] Propofol is commonly used in pregnancy for non-obstetric surgery. Xiong et al 4 showed that anaesthesia with propofol on gestational day 18 (E18) associated with the up-regulation of caspase-3 and the loss of neurons, as well as associated with the down-regulation synaptophysin expression in offspring rats' hippocampus and caused persistent spatial learning impairment in offspring. Our previous study showed that propofol anaesthesia in the second trimester inhibits the cognitive function of the offspring that is related to down-regulation of the protein levels of brain-derived neurotrophic factor (BDNF) and synaptophysin in offspring hippocampus. 5 Exposure to propofol for 5 hour caused death of neurons and oligodendrocytes in foetal and neonatal NHP brain. 6 However, little attention was paid to the early stage of gestation, which is equivalent to the early pregnancy of human. 7 It is reported that 0.75% to 2% gestational women have to experience non-obstetric surgery due to various medical problems. 8 This number is increasing with the development of laparoscopic technique, and the most common surgical procedure performed in the early pregnancy is laparoscopy. 9 It is reported that about 28% of the nonobstetric surgeries occurred in the first trimester. 10 Our earlier studies demonstrated that propofol, ketamine, enflurane, isoflurane or sevoflurane anaesthesia in the early pregnancy inhibits the cognitive function, damages hippocampal neurons, reduces NR2B mRNA and increased HGN mRNA levels in offspring rats' hippocampus, [11][12][13][14] but the underlying pathogenesis needs to be clarified.
Long-term potentiation (LTP) is considered the cellular mechanism of memory formation and plays a role in synaptic plasticity. 15 NR2B is an important positive regulator of learning and memory by promoting synaptic plasticity and LTP. 16,17 The balance between positive and negative learning and memory-regulating genes and proteins is key to the formation, maintenance, as well as retrieval of memory. HGN is a negative regulating protein that highly expresses in hippocampus, acting suppression/clearance function in memory regulating. 18 Inhibiting HGN by antisense oligonucleotide induces an increase in performance of Morris water maze and LTP. This indicates that HGN negatively regulates synaptic plasticity and LTP and plays negative regulating role in the formation and maintenance of memory.
Persistent changes in synapses, which based on appropriate gene transcription and subsequent protein synthesis, are the structural basis of learning and memory processes. 19 Both compact chromatin structure and the accessibility of DNA to target genes can be modulated by chromatin remodelling, in particular, histone tail acetylation, thus to regulate their expression. 20,21 Histone acetylation regulates by acetyltransferases (HATs) and histone deacetylases (HDACs).
HATs serve as transcriptional activators, whereas HDACs serve as transcriptional repressors. Increased HDAC activity had been linked to neurodegeneration. Growing evidence indicated that SAHA, which mainly targeting HDAC2, probably has therapeutic potentialities for the learning impairment caused by neurodegenerative diseases. [22][23][24] Histone deacetylase inhibitors facilitated synaptic plasticity and memory by promoting the combination of CREB with CREB-binding protein (CBP) domain, which subsequently activate CREB-mediated transcription. [25][26][27] Our early researches showed that anaesthesia during early gestation damaged the neurons and reduced the expression of NR2B in hippocampus, thus leading to learning and memory impairments in offspring rats. 11,12 In this study, we aim to investigate whether histone acetylation involves in the cognitive function impairment induced by propofol anaesthesia during early pregnancy.

| Animals
The protocol in this study was approved by the institutional review The learning and memory functions of the parental rats were assessed using the Morris water maze (MWM) system before mating, so that to minimize the hereditary difference. Animals were housed separately under standard laboratory conditions with 12:12 light/ dark cycle, 24 AE 1°C and had free access to tap water. Two female rats in cages with one male rat per cage for mating. Pregnancy was diagnosed by the sign of vaginal plug.
Electrocardiograms, saturation of pulse oximetry (SpO2) and tail non-invasive blood pressure were continuously monitored during maternal propofol exposure. Using heating lamp and temperature controller to monitor the rectal temperature and keep it at 37 AE 0.5°C. Arterial blood sampling from lateral caudal artery for blood gas analysis at the end of propofol anaesthesia. If the total time of SpO2 <95% and/or the systolic blood pressure <80% of the baseline in excess of 5 minutes, the pregnant rat was got rid of the study, and other pregnant rats were chosen to supply the sample size, so as to exclude the interfering effect of maternal hypotension or hypoxia on cognitive function in the pup rats.
After delivery, the offspring rats born to the same pregnant rat were randomly subdivided into the SAHA, SEN, HGNA group and their relative control groups (DMSO, NS(1) and NS(2) group, respectively; Figure 1). It has been proved that the acetylation level of histone in hippocampus obviously increased 2 hour after the administration of HDAC inhibitor. 27 Therefore, 90 mg kg À1 SAHA (HDAC inhibitor), at a concentration of 0.6 lmol L À1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA group by the intraperitoneal route at 2 hours before each MWM trial. The same volume of DMSO was given to the DMSO group.
Senegenin, a kind of Chinese medicine, was proved to up-regulate the expression of NR2B mRNA and protein, thus to mitigate cognitive dysfunction. 28 So, 15 mg kg À1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM trial to SEN or NS(1) groups, respectively. HGN antisense oligonucleotide (0.25 nmol lL À1 , 1.5 lL) or normal saline (1.5 lL) was injected to offspring's hippocampus in HGNA or NS(2) group as previously described, 18,29 once daily for seven consecutive days before MWM trial.

| Morris water maze test
Spatial learning and memory were assessed by the MWM test from post-natal day 30 (P30) to P36 according to previously described 5,30 with SLY-WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o'clock in the morning in the MWM system with the pool was filled with water to a height of 1.0 cm above the top of a 15-cm-diameter platform, in the second quadrant (target quadrant), and the water maintained at 24 AE 1°C. The training trial was performed once a day for six consecutive days. In each training trial, offspring rats were placed in the water facing the wall of the pool in the third quadrant, the farthest one from the target quadrant. The animals were allowed to search for the hidden platform or for 120 seconds. They were allowed to remain on the platform for 30 seconds when they found the platform and the time for the animal to find the platform was recorded as escape latency (indicating learning ability). For those who did not find the platform within 120 seconds, the animals were gently guided to the platform and allowed to stay there for 30 seconds, and their escape latency was recorded as 120 seconds. At the end of the reference training (P37), the platform was removed. The offspring rats were allowed to perform spatial probe test (memory function test) for 120 seconds. Times across the platform (platform crossing times, indicate memory function), the swimming trail and speed were automatically recorded by the system. The mean value of the platform crossing times, escape latency and speed of the offspring born to the same pregnant rats was taken as the final results.
F I G U R E 1 Experimental design. Pregnant dams were exposed to Propofol, 20% Intralipid or normal saline on E7, and the offspring were treated with SAHA, Senegenin, HGNA or vehicles two hours before behavioural testing. The number in parentheses represents the number of animals: F, female; M, male; SAHA, suberoylanilide hydroxamic acid, also known as vorinostat; DMSO, dimethyl sulphoxide; SEN, Senegenin; NS(1), Normal saline intraperitoneal injection; NS(2), Normal saline intrahippocampus injection; HGNA, HGN antisense oligonucleotide

| Brain hippocampus harvest
The day after the MWM test, rats were anaesthetized with isoflurane and killed by cervical dislocation. Hippocampus tissues were harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at À80°C (for Western blot analyses) or immersed in 4% paraformaldehyde (for immunofluorescence assay).

| Western blot analysis
The hippocampus (n = 6, with three male and three female offspring rats from each group) were homogenized on ice in lysis buffer containing a protease inhibitors cocktail. The mean expression level of all of the offspring born to the same mother rat in the same group was calculated as the final expression level of the observed proteins.

| Immunofluorescence staining
Immunofluorescence staining was used to assess HDAC2 and phospho-CREB in the hippocampus of offspring rats after the MWM test.
Hippocampus from offspring rats (n = 6, with three male and three female offspring rats from each group) were fixated in paraformaldehyde. Five-lm frozen sections of the hippocampus were used for the immunofluorescence staining. The sections were incubated with anti-HDAC2 (1:300) and anti-CREB (1:100) dissolved in 1% bovine serum albumin in phosphate-buffered saline at 4°C overnight. Then, the sections were incubated with fluorescent-conjugated anti-rabbit secondary antibody (1:300) for 1 hour in the dark at room temperature. Negative control sections were incubated with PBS as a substitute for primary antibody. Finally, the sections were wet mounted and viewed immediately using a inverted fluorescence microscope (2009) (Olympus, Japan). The target protein was red, and nuclei were blue. The proteins of HDAC2 and p-CREB were excited by the green light, while the DAPI was performed by UV blue light. All images were recorded at 10 9 209 (Exp Acq-700mmm, Offset Acq-1, Gain Acq-1, Gamma Acq-300). The density of HDCA2 and p-CREB staining was conducted on the images using Image-Pro Plus 6.0 (Media Cybernetics Inc., USA). The images were converted it into black and white pictures. After intensity calibration, hippocampal CA1 area was chosen to analyse and the integrated optical density (IOD) was measured. IOD/Area was calculated as the protein expression level.

| Statistical analysis
All analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data from escape latency in the MWM test were subjected to a repeated measures two-way analysis of variance (RM two-way ANOVA) and were followed by least significant difference t (LSD-t) analysis when a significant overall between-subject factor was found (P < 0.05). Data from Western blot and immunofluorescence staining results were subjected to one-way ANOVA analysis.
All data well provided for any of the variables. The LSD t test was used to determine the difference between groups. Statistical significance was declared at P < .05.

| Physiological parameters of maternal propofol anaesthesia
During propofol infusion, the maternal body temperature, respiratory rate, arterial oxygen saturation, heart rate and non-invasive blood pressure were continuously monitored and recorded every five minutes. No significant change in these physiological parameters had been seen during propofol exposure (4 hours). Tail artery blood was collected from pregnant rats for blood gas analysis after propofol perfusion, and no significant difference (P > .05) was observed (Table 1). These results suggested that propofol has no side effect on the physiological parameters in pregnant rats, indicating the T A B L E 1 Maternal arterial blood gas at the end of propofol exposure or normal saline (n = 10, mean AE SD) results of offspring rats in this study are likely caused directly by propofol rather than secondary effects of maternal propofol infusion.

| Physical features of the offspring
The birth rate (total number of neonates born to each mother rat), survival rate (survived more than 30 days), gender ratio (the ratio of females to males) and the average weight of the offspring on day P30 in propofol exposure group were not significantly different from nor- F I G U R E 2 Maternal propofol exposure had no effect on the physical features of the offspring rats. The physical features of the offspring rats between propofol exposure and normal saline control group had no significant difference (P > .05). A, The birth rate (average litter size, total number of neonates born to each mother rat). B, Survival rate of offspring (survived more than 30 days). C, Gender ratio (the ratio of females to males, gender composition). D, The average weight of the offspring on day P30 3.4 | Reduced histone acetylation levels and the mitigating effect of SAHA,Senegenin and HGN antisense oligonucleotide Histone deacetylation was implicated in memory impairments. 31,32 The acetylation of histone is regulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs). 33,34 HATs acetylate multiple lysine residues on histones, and different acetylated sites result in different downstream biological effects. H3K14 and H4K12 acetylation have been shown to play a crucial part in learning, memory and synaptic plasticity. 35 The results showed that propofol exposure during pregnancy up-regulated HDAC2 protein expression in F I G U R E 3 Maternal Propofol exposure impaired learning and memory in offspring. Post-natal thirty days (P30), the learning and memory were assessed using the Morris water maze (mean AE SD). A, Escape latency (indicating learning ability) among control groups. B, Platform crossing times (indicating memory ability) among control groups. C, Propofol exposure increased escape latency in offspring compared to the saline control condition (*P < .05). No statistically significant difference was observed between the saline control and intralipid group. D, Propofol exposure decreased platform crossing times in offspring compared to the saline control condition (*P < .05). No significant difference was observed between the saline control and intralipid group F I G U R E 4 SAHA, SEN and HGNA treatment mitigated the learning and memory impairment (mean AE SD). A, Propofol exposure increased the escape latency in offspring compared to the control condition (*P < .05), and SAHA treatment significantly reversed the effect ( # P < .05). B, SEN treatment significantly reversed the effect ( # P < .05). C, HGNA treatment significantly reversed the effect ( # P < .05). D, Propofol exposure decreased the platform crossing times in offspring compared with the control condition (*P<.05), and SAHA treatment reversed the effect ( # P < .05). E, SEN treatment reversed the effect caused by propofol exposure ( # P < .05). F, HGNA treatment reversed the effect caused by propofol exposure ( # P < .05), and SAHA, SEN and HGNA treatment had no significant effect on learning and memory in offspring that were not exposed to propofol during pregnancy. Error bar = SD LIN ET AL. Maternal exposure to propofol decreased the expression of phospho-CREB protein in offspring compared to the control condition (P < .001). SAHA, SEN and HGNA treatment significantly increased phospho-CREB protein level (P = .006, P = .006, P = .016, respectively). The protein levels of phospho-CREB in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05) Maternal body temperature, respiratory rate, saturation of pulse oximetry, heart rate and non-invasive blood pressure were continuously monitored during propofol exposure, and no obvious abnormality was observed. Furthermore, maternal artery blood gases were analysed after the 4 hours propofol infusion and showed no significant change (Table 1). Moreover, there was no significant difference in birth rate, offspring survival rate, the ratio of sex or basic physical development of offspring between propofol and saline group. These results suggested that the impaired learning and memory of the rats' offspring may be not caused by pathological disorders but caused by the pregnant rats propofol anaesthesia in the current study.
Several animal studies showed that anaesthetics exposure during gestation induced apoptosis in foetal brain. 1  neurodegeneration-associated memory impairments. Abolished the memory impairments in connection with neurodegeneration. 35 Our earlier study suggested that maternal isoflurane anaesthesia during third trimester impairs the spatial learning and memory of the offspring rats, and its mechanism in connection with the up-regulation of HDAC2 mRNA and subsequent inhibits the expression of CREB mRNA and NR2B, while HDAC2 inhibition reversed these changes. 30 Consistent to our previous study, our results suggest that maternal propofol anaesthesia on E7 impairs learning and memory in offspring rats, causes the overexpression of HDAC2 and inhibits the acetylation of H3K14 and H4K12, and these effects were reversed by SAHA. Senegenin and HGN antisense oligonucleotide treatment also showed similar effects.
NMDA receptors play a crucial role in neuronal development and circuit formation. Subunit NR2B is critical to learning and memory. 45 It is reported that the enhancement of pre-frontal cortical long-term potentiation (LTP) and working memory via the up-regulate expression of NR2B specifically in the forebrain region. 46 While decreased expression of NR2B subunit suppressed NMDA-dependent longterm potentiation (LTP) and impaired spatial learning. 47 Therefore, NR2B acts as a positive regulator in memory process by promoting synaptic plasticity and long-term potential (LTP). Not only the activation of positive regulatory mechanisms that favour memory storage but also the removal of inhibitory constraints that prevent memory storage are required for long-lasting of synaptic plasticity. 37 Negative regulators play an important role in the formation and maintenance of memory. Hippyragranin (HGN) is a protein which expresses in rat hippocampus and involves in negative memory regulation. 18  which induced robust activation of gene transcription afterwards.
The activity of CREB is essential to the gene transcription of NR2B, and expression of NR2B relies on the binding of p-CREB to its binding site at the promoter of the NR2B gene. 51  Present study has several limitations. First, we had not accessed the pathological changes of neurons in the foetal brains immediately after maternal propofol exposure and during various period of brain development (e.g., post-natal day 1 to 10). Second, in the present study, we only used MWM to evaluate learning and memory.
Although MWM is recognized as an appropriate way to evaluate the spatial learning and memory in rodents, to provide a more compre- propofol anaesthesia in pregnant rats induced caspase-3 activation and microglial response in foetal rats. They found that the activated caspase-3-positive cells were abundant and heavily concentrated in the cortex, thalamus and hypothalamus regions. 55 Whether maternal propofol anaesthesia will affect the histone acetylation in other brain regions should be studied. We had only evaluated the short-term therapeutic effects of SAHA, Senegenin and HGNA on behaviour performance and proteins. The long-term or long-lasting therapeutic effects of these drugs on learning and memory deficits and protein expression changes caused by propofol exposure on E7 should be evaluated in future study.
Taken together, the results of the present study suggest that propofol anaesthesia during first trimester causes learning and memory deficit in offspring rats by inhibiting histone acetylation. SAHA, Senegenin and HGN antisense oligonucleotide can ameliorate these impairments.