new_pdfs/10.1111_jcmm.13524.txt

Received: 22 September 2017 | Accepted: 12 December 2017 DOI: 10.1111/jcmm.13524

O R I G I N A L A R T I C L E

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

Jiamei Lin1,2 | Shengqiang Wang1 | Yunlin Feng1 | Weihong Zhao1 | Weilu Zhao1 | Foquan Luo1

| Namin Feng1

1Department of Anesthesiology, the First Affiliated Hospital, Nanchang University, Nanchang, China

Abstract

Propofol is widely used in clinical practice, including non-obstetric surgery in preg-

2Department of Anesthesiology, the Eastern Hospital of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

nant women. Previously, we found that propofol anaesthesia in maternal rats during

the third trimester (E18) caused learning and memory impairment to the offspring

Correspondence Foquan Luo Email: lfqjxmc@outlook.com

rats, but how about the exposure during early pregnancy and the underlying mecha-

nisms? Histone acetylation plays an important role in synaptic plasticity.

study, propofol was administered to the pregnant rats in the early pregnancy (E7).

In this

Funding information Natural Science Foundation of Jiangxi Province, Grant/Award Number: 20132BAB205022, 20171ACB20030; National Natural Science Foundation of China, Grant/Award Number: 81060093, 81460175

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, acety-

lated 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 off-

spring,

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 gesta- tion 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.

K E Y W O R D S

histone deacetylase, learning and memory, pregnancy, propofol

Jiamei Lin and Shengqiang Wang contributed equally to this work (co-first author).

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 2600 | wileyonlinelibrary.com/journal/jcmm J Cell Mol Med. 2018;22:2600–2611.

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1 |

I N T R O D U C T I O N

mainly targeting HDAC2, probably has therapeutic potentialities for the learning impairment caused by neurodegenerative diseases.22-24

Histone deacetylase inhibitors facilitated synaptic plasticity and

Accumulating evidence indicates general anaesthetics exposure dur-

memory by promoting the combination of CREB with CREB-binding

ing pregnancy may cause neurotoxic effects and induce persistent cognitive dysfunction of offspring rats.1-3 Propofol is commonly used in pregnancy for non-obstetric surgery. Xiong et al4 showed that

protein (CBP) domain, which subsequently activate CREB-mediated transcription.25-27 Our early researches showed that anaesthesia dur-

ing early gestation damaged the neurons and reduced the expression

anaesthesia with propofol on gestational day 18 (E18) associated

of NR2B in hippocampus, thus leading to learning and memory impairments in offspring rats.11,12 In this study, we aim to investigate

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

whether histone acetylation involves in the cognitive function

learning

impairment induced by propofol anaesthesia during early pregnancy.

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

2 | M A T E R I A L S A N D M E T H O D S

of brain-derived neurotrophic factor (BDNF) and synaptophysin in offspring hippocampus.5 Exposure to propofol for 5 hour caused

2.1 | Drugs

death of neurons and oligodendrocytes in foetal and neonatal NHP brain.6 However, little attention was paid to the early stage of gesta- tion, which is equivalent to the early pregnancy of human.7 It is

All drugs were prepared just before use: propofol (Diprivan; AstraZe-

Italy:

jc393, 20 mL: 200 mg); 20% intralipid

neca UK limited,

(2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC,

reported that 0.75% to 2% gestational women have to experience non-obstetric surgery due to various medical problems.8 This number

Houston, TX, USA). HGN antisense was synthesized by Sangon Bio- tech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was pur-

is increasing with the development of laparoscopic technique, and

chased from Nanjing SenBeiJia Biological Technology Co., Ltd.

the most common surgical procedure performed in the early preg- nancy is laparoscopy.9 It is reported that about 28% of the non- obstetric surgeries occurred in the first trimester.10 Our earlier stud-

(Jiangsu province, China).

Anti-b-actin and anti-rabbit

IgG secondary antibody were

obtained from Cell Signaling Technology (Cell Signaling Tech, MA,

ies demonstrated that propofol, ketamine, enflurane,

isoflurane or

USA). Anti-CREB (Phospho S133), anti-NMDAR2B, anti-HDAC2,

sevoflurane anaesthesia in the early pregnancy inhibits the cognitive

antisynaptophysin, anti-Ac-H4K12 and anti-Ac-H3K14 antibodies

function, damages hippocampal neurons, reduces NR2B mRNA and increased HGN mRNA levels in offspring rats’ hippocampus,11-14 but

were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti-

HGN antibody was synthesized by Kitgen Bio-tech Co., Ltd.(Zhejiang

the underlying pathogenesis needs to be clarified.

province, China).

is considered the cellular mecha- nism of memory formation and plays a role in synaptic plasticity.15

Long-term potentiation (LTP)

NR2B is an important positive regulator of learning and memory by promoting synaptic plasticity and LTP.16,17 The balance between

2.2 | Animals

The protocol in this study was approved by the institutional review

positive and negative learning and memory-regulating genes and pro-

board of the First Affiliated Hospital of Nanchang University on the

teins is key to the formation, maintenance, as well as retrieval of

Use of Animals in Research and Teaching. All the methods in this

memory. HGN is a negative regulating protein that highly expresses

study were performed in co-ordination with relevant guidelines and

in hippocampus, acting suppression/clearance function in memory regulating.18 Inhibiting HGN by antisense oligonucleotide induces an

regulations. Sprague Dawley (SD) rats were purchased from the ani-

mal science research department of the Jiangxi Traditional Chinese

increase in performance of Morris water maze and LTP. This indi-

Medicine College (JZDWNO: 2011-0030; Nanchang, Jiangxi,China).

cates that HGN negatively regulates synaptic plasticity and LTP and

The learning and memory functions of

the parental

rats were

plays negative regulating role in the formation and maintenance of

assessed using the Morris water maze (MWM) system before mating,

memory.

so that to minimize the hereditary difference. Animals were housed

Persistent changes in synapses, which based on appropriate gene

separately under standard laboratory conditions with 12:12 light/ dark cycle, 24 (cid:1) 1°C and had free access to tap water. Two female

transcription and subsequent protein synthesis, are the structural basis of learning and memory processes.19 Both compact chromatin

rats in cages with one male rat per cage for mating. Pregnancy was

structure and the accessibility of DNA to target genes can be modu-

diagnosed by the sign of vaginal plug.

lated 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).

2.3 | Drug treatment

HATs serve as transcriptional activators, whereas HDACs serve as

rats received intravenous infusion of propo- (n = 10 dams) with the rate of 20 mg kg(cid:3)1 h(cid:3)1 for 4 hours,

On E7, pregnant

transcriptional repressors. Increased HDAC activity had been linked

fol

to neurodegeneration. Growing evidence indicated that SAHA, which

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LIN ET AL.

equal volume of saline (n = 10 dams) or intralipid (n = 5 dams),

previously described,18,29 once daily for seven consecutive days

before MWM trial.

respectively.

Electrocardiograms, saturation of pulse oximetry (SpO2) and tail

non-invasive blood pressure were continuously monitored during

2.4 | Morris water maze test

maternal propofol exposure. Using heating lamp and temperature

the rectal

to monitor

Spatial learning and memory were assessed by the MWM test from post-natal day 30 (P30) to P36 according to previously described5,30

temperature and keep it at controller 37 (cid:1) 0.5°C. Arterial blood sampling from lateral caudal artery for

with SLY-WMS Morris water maze test system (Beijing Sunny Instru- ments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o’clock in

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

the morning in the MWM system with the pool was filled with

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

water to a height of 1.0 cm above the top of a 15-cm-diameter plat-

size, so as to exclude the interfering effect of maternal hypotension

form, in the second quadrant (target quadrant), and the water main- tained at 24 (cid:1) 1°C. The training trial was performed once a day for

or hypoxia on cognitive function in the pup rats.

six consecutive days. In each training trial, offspring rats were placed

After delivery, the offspring rats born to the same pregnant rat

were randomly subdivided into the SAHA, SEN, HGNA group and

in the water facing the wall of the pool in the third quadrant, the

their relative control groups (DMSO, NS(1) and NS(2) group, respec-

farthest one from the target quadrant. The animals were allowed to

tively; Figure 1). It has been proved that the acetylation level of his-

search for the hidden platform or for 120 seconds. They were

after

increased 2 hour

the tone in hippocampus obviously administration of HDAC inhibitor.27 Therefore, 90 mg kg(cid:3)1 SAHA (HDAC inhibitor), at a concentration of 0.6 lmol L(cid:3)1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA

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

group by the intraperitoneal route at 2 hours before each MWM

gently guided to the platform and allowed to stay there for 30 sec-

onds, and their escape latency was recorded as 120 seconds. At the

trial. The same volume of DMSO was given to the DMSO group.

Senegenin, a kind of Chinese medicine, was proved to up-regulate

end of the reference training (P37), the platform was removed. The

offspring rats were allowed to perform spatial probe test (memory

the expression of NR2B mRNA and protein, thus to mitigate cogni- tive dysfunction.28 So, 15 mg kg(cid:3)1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM

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

trial to SEN or NS(1) groups, respectively. HGN antisense oligonu- cleotide (0.25 nmol lL(cid:3)1, 1.5 lL) or normal saline (1.5 lL) was injected to offspring’s hippocampus in HGNA or NS(2) group as

of the platform crossing times, escape latency and speed of the off-

spring 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

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green light, while the DAPI was performed by UV blue light. All images were recorded at 10 9 209 (Exp Acq-700mmm, Offset Acq-

2.5 | Brain hippocampus harvest

1, Gain Acq-1, Gamma Acq-300). The density of HDCA2 and p-

The day after the MWM test, rats were anaesthetized with isoflu-

rane and killed by cervical dislocation. Hippocampus tissues were

CREB staining was conducted on the images using Image-Pro Plus

harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at (cid:3)80°C (for Western blot analyses) or

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

immersed in 4% paraformaldehyde (for immunofluorescence assay).

(IOD) was measured. IOD/Area was calculated as the protein expres-

sion level.

2.6 | Western blot analysis

The hippocampus (n = 6, with three male and three female offspring

2.8 | Statistical analysis

rats from each group) were homogenized on ice in lysis buffer con-

All analyses were performed with SPSS 17.0 software (SPSS, Inc.,

taining a protease inhibitors cocktail. Protein concentration was

Chicago, IL, USA). Data from escape latency in the MWM test were

determined by the bicinchoninic acid protein assay kit. Protein sam- ples (20 lg) were separated by sodium dodecyl sulphate polyacry-

subjected to a repeated measures two-way analysis of variance (RM

lamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF

two-way ANOVA) and were followed by least significant difference t

membrane. The membranes were blocked by non-fat dry milk buffer for 1.5 h and then incubated overnight at 4°C with antihistone H3

(LSD-t) analysis when a significant overall between-subject factor was found (P < 0.05). Data from Western blot and immunofluores-

cence staining results were subjected to one-way ANOVA analysis.

(acetyl K14)

(1:10000), anti-

(1:2000), antihistone H4 (acetyl K12)

NMDAR2B antisynaptophysin (1:10000) and anti-b-actin (1:2000), respectively. Thereafter, the

All data well provided for any of the variables. The LSD t test was

anti-HGN (1:1000),

(1:1000),

used to determine the difference between groups. Statistical signifi- cance was declared at P < .05.

membranes were washed three times with TBS-T buffer for 15 min-

utes and incubated with the horseradish peroxidase (HRP)-conju-

gated secondary antibody for 2 hours at room temperature. The

3 | R E S U L T S

immune complexes were washed three times with TBS-T buffer and

detected using the ECL system (Millipore Corporation, MA, USA).

3.1 | Physiological parameters of maternal propofol anaesthesia

The images of Western blot products were collected and analysed

(Wayne Rasband, National

Institutes of Health,

by ImageJ 1.50i

During propofol infusion, the maternal body temperature, respiratory

USA). The density of observed protein band was normalized to that of b-actin in the same sample. The results of offspring from all the

rate, arterial oxygen saturation, heart rate and non-invasive blood

other group were then normalized to the average values of normal saline control offspring (control+NS group) in the same Western blot.

pressure were continuously monitored and recorded every five min-

utes. No significant change in these physiological parameters had

The mean expression level of all of the offspring born to the same

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

mother rat in the same group was calculated as the final expression

level of the observed proteins.

(Table 1). These results suggested that propofol has no side effect

indicating the

on the physiological parameters in pregnant rats,

2.7 |

Immunofluorescence staining

Immunofluorescence staining was used to assess HDAC2 and phos-

pho-CREB in the hippocampus of offspring rats after the MWM test. Hippocampus from offspring rats (n = 6, with three male and three

T A B L E 1 Maternal arterial blood gas at the end of propofol exposure or normal saline (n = 10, mean (cid:1) SD)

female offspring rats from each group) were fixated in paraformalde- hyde. Five-lm frozen sections of the hippocampus were used for

Normal Saline exposure pregnant rats

Propofol exposure pregnant rats

Indexes

the immunofluorescence staining. The sections were incubated with

7.39 (cid:1) 0.04

7.38 (cid:1) 0.05

pH

anti-HDAC2 (1:300) and anti-CREB (1:100) dissolved in 1% bovine serum albumin in phosphate-buffered saline at 4°C overnight. Then,

94.00 (cid:1) 3.52

97.17 (cid:1) 3.49

PO2 (mm Hg)

45.33 (cid:1) 2.88

the sections were incubated with fluorescent-conjugated anti-rabbit

44.83 (cid:1) 5.78

PCO2 (mm Hg) HCO(cid:3) K+ (mmol L(cid:3)1) Na+ (mmol L(cid:3)1) Ca2+ (mmol L(cid:3)1)

3 (mmol L(cid:3)1)

secondary antibody (1:300) for 1 hour in the dark at room tempera-

27.95 (cid:1) 3.21

26.68 (cid:1) 2.32

ture. Negative control sections were incubated with PBS as a substi-

3.48 (cid:1) 0.29

3.47 (cid:1) 0.39

tute for primary antibody. Finally, the sections were wet mounted

141.67 (cid:1) 1.03

140.83 (cid:1) 1.47

and viewed immediately using a inverted fluorescence microscope (2009)

1.38 (cid:1) 0.05

1.34 (cid:1) 0.03

(Olympus, Japan). The target protein was red, and nuclei

9.18 (cid:1) 0.99

9.57 (cid:1) 0.55

Glu

were blue. The proteins of HDAC2 and p-CREB were excited by the

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LIN ET AL.

results of offspring rats in this study are likely caused directly by

the data of offspring from normal saline and intralipid infusion group

into one control group in the following data analysis. Propofol expo-

propofol rather than secondary effects of maternal propofol infusion.

sure increased escape latency, while decreased platform crossing

times in offspring compared to the saline control condition (Fig- ure 3C,D, P < .05),

3.2 | Physical features of the offspring

indicating propofol anaesthesia on E7 impairs

spatial learning and memory in offspring.

The birth rate (total number of neonates born to each mother rat), sur-

vival rate (survived more than 30 days), gender ratio (the ratio of

SAHA, Senegenin and HGN antisense oligonucleotide have been

shown to improve learning and memory by facilitating histone acety-

females to males) and the average weight of the offspring on day P30

lation, increasing NR2B expression and inhibiting HGN expression, respectively.18,27,28 Therefore, we assessed whether they can amelio-

in propofol exposure group were not significantly different from nor-

mal saline control group (Figure 2). Dyskinesia was not observed in

either of the two groups. These results indicate that maternal propofol

rate the learning and memory impairment caused by propofol expo-

anaesthesia at the early pregnant stage (E7) has no significant effects

sure during pregnancy. Based on the previous discovery on the pharmacodynamics,18,27,28 SAHA or Senegenin was intraperitoneally

on physical development of offspring rats, indicating the differences in

injected into the offspring 2 hours before each MWM test, while

learning and memory observed in this study are caused by propofol

HGN antisense oligonucleotide was injected into hippocampus

exposure during pregnancy rather than physical differences.

2 hours before each MWM test. The results showed that SAHA,

Senegenin or HGN antisense oligonucleotide treatment ameliorated

3.3 | and the ameliorating effect of SAHA, Senegenin and HGN antisense oligonucleotide

Impaired learning and memory in offspring

the cognitive function deficit caused by propofol exposure during pregnancy (Figure 4A-F, P < .05). SAHA, Senegenin or HGN anti-

sense oligonucleotide had no obvious effect on the learning and

There was no obvious difference in offspring between normal saline

memory in offspring that had not exposed to propofol during preg-

and intralipid infusion group (Figure 3C,D). Therefore, we merged

nancy (Figure 4A-F).

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

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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 (cid:1) 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 (cid:1) 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

(HDACs) and histone acetyltransferases (HATs).33,34 HATs acetylate

3.4 | Reduced histone acetylation levels and the mitigating effect of SAHA,Senegenin and HGN antisense oligonucleotide

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, mem- ory and synaptic plasticity.35 The results showed that propofol expo-

Histone deacetylation was implicated in memory impairments.31,32

The acetylation of histone is regulated by histone deacetylases

sure during pregnancy up-regulated HDAC2 protein expression in

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offspring rat’s hippocampus (Figure 6, P < .05), whereas decreased

and maintenance.37 NR2B is critical positive regulating factor,38 while HGN is considered as an important

recognized as

the acetylation levels of H3K14 and H4K12 significantly (Figure 5, P < .05). SAHA, Senegenin and HGN antisense oligonucleotide alle- viated these changes (Figures 5 and 6, P < .05). These results indi-

factor.18 The

negative

results

in this

study

showed that

propofol anaesthesia during pregnancy resulted in decrease in NR2B protein (Figure 8A, P < .05), while increased the level of HGN protein (Figure 8B, P < .05), resulted in decreased ratio of NR2B/HGN in offspring rats’ hippocampus (Figure 8C, P < 0.05).

cate that propofol anaesthesia during pregnancy inhibits histone acetylation in offspring rats’ hippocampus, which could be alleviated

by SAHA, Senegenin or HGN antisense oligonucleotide.

The ratio of NR2B/HGN was reversed significantly by SAHA,

or HGN antisense

oligonucleotide

Senegenin P < .05).

(Figure 8A-C,

3.5 | Decreased phosphorylated CREB levels in hippocampus and the mitigating effect of SAHA, Senegenin and HGN antisense oligonucleotide

3.7 | Down-regulated expression of synaptophysin in the hippocampus of offspring rats and the improving effect of SAHA, Senegenin and HGN antisense oligonucleotide

Phosphorylation of CREB is recognized as a molecular marker of mem- ory processing in the hippocampus for spatial learning.36 Therefore, we

investigated the phosphorylation of CREB in this study. The results

showed that propofol anaesthesia during pregnancy resulted in decrease in phospho-CREB protein in offspring rats’ hippocampus.

Synaptophysin plays an important

role in the exocytosis of

SAHA, Senegenin or HGN antisense oligonucleotide treatment allevi- ated the effects (Figure 7, P < .05). These results suggest that propofol

synaptic vesicles and acknowledged as a marker of synaptic density.39 Synapse loss is closely associated with cognitive dys- function and learning impairment.40,41 The results showed that the

anaesthesia during pregnancy on E7 can down-regulate the phosphory-

lation of CREB in hippocampus of the offspring, whereas SAHA, Sene-

protein level of synaptophysin in maternal propofol exposure

lower

than control condition (Figure 9),

group was

indicating

genin or HGN antisense oligonucleotide ameliorates this effect.

maternal propofol exposure on E7 impairs the synaptic plasticity in offspring rats’ hippocampus, whereas the level of synaptophysin

3.6 | Decreased the ratio of NR2B/HGN in offspring rat’s hippocampus and the reversing effect of SAHA, Senegenin or HGN antisense oligonucleotide

in SAHA, Senegenin or HGN antisense oligonucleotide-treated

group was higher than propofol exposure group (Figure 9), sug-

gesting that SAHA, Senegenin and HGN antisense oligonucleotide

The balance between positive and negative regulating factors

can reverse the down-regulated expression of

synaptophysin

of learning and memory plays a key role in the memory obtain

caused by propofol.

F I G U R E 5 Maternal propofol exposure reduced the level of histone acetylation and the reversed effect of SAHA, SEN and HGNA treatment. Acetylation level of H3K14 and H4K12 was detected by Western blot (mean (cid:1) SD). Maternal exposure to propofol decreased the acetylation level of H3K14 and H4K12 in offspring compared to the control condition (P < .001), and SAHA treatment significantly increased acetylated H3K14 (P = .016) and H4K12 (P = .003) levels; SEN treatment significantly increased acetylated H3K14 (P = .012) and H4K12 (P = .002) levels; HGNA treatment significantly increased acetylated H3K14 (P = .042) and H4K12 (P = .029) levels. The protein levels of acetylated H3K14 and H4K12 in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)

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LIN ET AL.

F I G U R E 6 Maternal propofol exposure increased the level of HDAC2 and the reversed effect of SAHA, SEN and HGNA treatment. HDAC2 protein level was determined by immunofluorescence (mean (cid:1) SD). Maternal exposure to propofol up-regulated the expression of HDAC2 protein in offspring compared to the control condition (P = .001). SAHA treatment significantly inhibited the expression of HDAC2 protein (P = .029); SEN treatment significantly decreased HDAC2 protein level (P = .032); HGNA treatment significantly decreased HDAC2 protein level (P = .006). The protein levels of acetylated HDAC2 in propofol+SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)

F I G U R E 7 Maternal propofol exposure decreased the level of phospho-CREB and the reversed effect of SAHA, SEN and HGNA treatment. Phospho-CREB protein level was determined by immunofluorescence (mean (cid:1) SD). 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)

4 | D I S C U S S I O N

H3K14 and H4K12 and the phosphorylation of CREB, down-regu-

lates the expression of NR2B, up-regulates the expression of HGN

The current study findings suggest that pregnant rats propofol

and decreases the ration of NR2B/HGN and the expression of

anaesthesia on E7 impairs the learning and memory in offspring rats,

synaptophysin. SAHA, Senegenin and HGN antisense oligonucleotide

increases the expression of HDAC2,

inhibits the acetylation of

ameliorate all these changes.

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2608 |

LIN ET AL.

F I G U R E 8 Maternal propofol exposure broken the balance between NR2B and HGN and the mitigating effect of SAHA, SEN and HGNA treatment. Expression of NR2B and HGN protein was detected by Western blot (mean (cid:1) SD). A, Maternal exposure to propofol decreased the NR2B protein level (P < .001). B, Maternal exposure to propofol increased the HGN protein level (P < .001). C, The ratio of NR2B/HGN was significantly reduced in offspring compared with the control condition (P < .001). SAHA, SEN and HGNA treatment significantly increased NR2B protein level and decreased HGN protein level and reversed the ratio of NR2B/HGN (P < .05). The protein levels of NR2B and HGN and the ratio of NR2B/ HGN 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

the 2nd trimester of pregnancy) may cause learning deficits in the rat offspring.42 Our previous study showed that ketamine, propofol

oximetry, heart rate and non-invasive blood pressure were continu-

ously monitored during propofol exposure, and no obvious abnor-

and enflurane anaesthesia during early gestation (on gestation day 7) induced learning and memory impairment in offspring rats,11-14 asso-

mality was observed. Furthermore, maternal artery blood gases were

analysed after the 4 hours propofol infusion and showed no signifi-

ciated with hippocampal neuron injury, NR2B receptor subunit

cant change (Table 1). Moreover, there was no significant difference

reduction and increased level of HGN mRNA.

in birth rate, offspring survival rate, the ratio of sex or basic physical

How does propofol anaesthesia during pregnancy impair the

development of offspring between propofol and saline group. These results suggested that the impaired learning and memory of the rats’

learning and memory in offspring? The consolidation and mainte-

nance of memory require specific genes expression, and histone

acetylation promotes the expression of these genes, while histone deacetylation represses their expression.21,43 Histone deacetylases

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.1,2,40 Xiong et al and our

(HDACs) inhibit the expression of these genes, while histone acetyl- transferases (HATs) promote their expression.44 Among the HDACs,

HDAC2 was implicated in learning and memory, it negatively regu-

previous study showed that prenatal propofol exposure resulted in learning and memory deficit in offspring.4,5 While these studies

lates synaptic plasticity and memory process by suppressing memory specific genes’ expression, and loss function of HDAC2 facilitates synaptic plasticity and learning and memory.32 Graff et al showed

mainly focused on the second and third trimester, there is little

information in relation to the effect of propofol anaesthesia during

early pregnancy on the cognitive function in offspring. Because some

that HDAC2 overexpression reduced the histone acetylation of his-

tone and inhibited the expression of memory specific genes. HDAC2

of non-obstetric surgeries during pregnancy occurred in the first tri- mester,10 our current study mainly focus on gestation day 7, which distinct with the exposure time-point in previous studies,4-6 the dif-

is significantly enriched near the histones of genes shown to play a

key role in learning, memory and synaptic plasticity, such as H2B

lysine (K) 5, H3K14, H4K5, and H4K12. Reversing the build- up of

ferent exposure time-point may alter the vulnerability to general

anaesthetics for

the developing brain. Halothane and enflurane

HDAC2 by short-hairpin-RNA -mediated knockdown activated these

exposure on gestation day 6 and 10 (amount to the early and early

genes, reinstated structural and synaptic plasticity and abolished the

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| 2609

LIN ET AL.

synaptic plasticity and long-term potential (LTP). Not only the activa-

tion 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 Down-

regulation of HGN by antisense oligonucleotide in the hippocampal

CA1 region caused enhanced learning and memory as well as ele-

vated LTP. Therefore, we hypothesize that the balance between the

positive regulator NR2B and negative regulator HGN plays a pivotal

role in the learning and memory process. Present study showed that

Senegenin treatment reversed the protein levels of NR2B, HGN and

the ratio of NR2B/HGN, as well as enhanced learning and memory,

which in accordance with the previous research that Senegenin

attenuates cognitive impairment by up-regulating expression of hip- pocampal NR2B expression in rats.28 While treatment with HGN

antisense oligonucleotide inhibited the expression of HGN protein,

reversed the ratio of NR2B/HGN and the learning and memory impairment as previous report.18

Transcription factor CREB (cAMP response element-binding pro-

tein) shows an important role in synaptic plasticity underlying learn- ing memory.48,49 CREB is a critical mediator of cAMP- and calcium-

F I G U R E 9 Maternal propofol exposure decreased the expression of synaptophysin and the reversed effect of SAHA, SEN and HGNA treatment. Synaptophysin level was determined by Western blot (mean (cid:1) SD). Maternal exposure to propofol decreased the expression of synaptophysin in offspring compared to the control condition (P = .002). SAHA, SEN and HGNA treatment significantly increased synaptophysin protein level (P = .026, P = .007, P = .027, respectively). The protein levels of synaptophysin in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)

inducible transcription, whereas the phosphorylation of serine 133

is its main

in its kinase-inducible domain (KID)

(phospho-Ser133)

transactivating form. Phospho-Ser133 plays a role in CREB to bind the KIX domain of the coactivators CBP and p300 (CBP/p300).50 Vecsey et al25 demonstrated that enhancement of hippocampus-

dependent memory and synaptic plasticity by HDAC inhibitors was

relied on the binding of CREB and CREB-binding protein (CBP),

which induced robust activation of gene transcription afterwards.

The activity of CREB is essential to the gene transcription of NR2B,

neurodegeneration-associated memory impairments. Abolished the memory impairments in connection with neurodegeneration.35 Our

and expression of NR2B relies on the binding of p-CREB to its bind- ing site at the promoter of the NR2B gene.51 Fujita et al27 have

demonstrated that HDAC inhibitor up-regulated the expression of

isoflurane anaesthesia during

earlier study suggested that maternal

acetylated histones and NR2B mRNA in the hippocampus, and up-

third trimester impairs the spatial

learning and memory of the off-

spring rats, and its mechanism in connection with the up-regulation

regulated expression of acetylated histones was accompanied by

enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene.27 These findings indicated that HDAC inhibitor pro-

of HDAC2 mRNA and subsequent inhibits the expression of CREB mRNA and NR2B, while HDAC2 inhibition reversed these changes.30

motes learning and memory by increasing the acetylation of histone

Consistent to our previous study, our results suggest that maternal

propofol anaesthesia on E7 impairs learning and memory in offspring

and the phosphorylation of CREB, and subsequent increase of NR2B

rats, causes the overexpression of HDAC2 and inhibits the acetyla-

expression. Our previous study has demonstrated that isoflurane

tion of H3K14 and H4K12, and these effects were reversed by

anaesthesia during the third trimester impaired learning and memory in offspring rats via “HDAC2-CREB-NR2B” pathway.30

SAHA. Senegenin and HGN antisense oligonucleotide treatment also

showed similar effects.

Synaptophysin is a synaptic protein marker and provides a struc- tural basis for synaptic plasticity.52 Decrease in synaptophysin is implicated in learning and memory impairment.1,4,5 Graff et al35

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

demonstrated that HDAC2 overexpression reduced synaptophysin

potentiation (LTP) and working memory via the up-regulate expres- sion of NR2B specifically in the forebrain region.46 While decreased

protein level and caused memory impairments, HDAC2 inhibition

reversed the effects. As synaptophysin is one of the CREB target genes,53 we detected the expression of synaptophysin in the present

expression of NR2B subunit suppressed NMDA-dependent long- learning.47 Therefore,

term potentiation (LTP) and impaired spatial

study. The results showed that propofol anaesthesia during preg- nancy reduced the protein level of synaptophysin in offspring’s

NR2B acts as a positive regulator in memory process by promoting

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2610 |

LIN ET AL.

C O N F L I C T O F I N T E R E S T S

hippocampus, whereas SAHA, Senegenin and HGN antisense

oligonucleotide mitigated the reduced synaptophysin levels; mean-

The authors declare that they have no conflict of interest.

while, the increased expression of synaptophysin was companied

with decreased HDAC2 protein level, increased histone acetylation

A U T H O R C O N T R I B U T I O N S

and CREB phosphorylation level. The BDNF-TrkB signalling pathway

is one of the downstream regulating targets of histone acetylation,

F.Q.L. and J.M.L. conceived and designed the experiments. J.M.L.,

so BDNF-TrkB signalling pathway may be one of the underlying

Y.L.F. and S.Q.W. performed the experiments. J.M.L and F.Q.L. anal-

downstream mechanisms of learning and memory deficits induced by

ysed the data. J.M.L. contributed reagents/materials/analysis tools.

propofol exposure during early gestation.

It

is confirmed that

J.M.L and F.Q.L. wrote the article. All the authors read and approved

HDAC2 up-regulation will impair BDNF-TrkB signalling pathway and results in cognitive impairments induced by isoflurane.54 Our previ-

the final manuscript.

ous study also verified the role of BDNF-TrkB signalling pathway in

O R C I D

the cognitive deficits induced by propofol during late pregnant stage.5 Whether BDNF-TrkB signalling pathway involves in the

Foquan Luo

http://orcid.org/0000-0003-0106-0710

learning and memory impairments induced by maternal propofol

anaesthesia needs to be explored in future study.

Present study has several limitations. First, we had not accessed

R E F E R E N C E S

the pathological changes of neurons in the foetal brains immediately

1. Zheng H, Dong Y, Xu Z, et al. Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. Anesthesiol- ogy. 2013;118:516-526.

after maternal propofol exposure and during various period of brain

in the present

development (e.g., post-natal day 1 to 10). Second,

study, we only used MWM to evaluate learning and memory.

2. Zhao T, Li Y, Wei W, et al. Ketamine administered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiol Dis. 2014;68:145-155.

Although MWM is recognized as an appropriate way to evaluate the

spatial learning and memory in rodents, to provide a more compre-

3. Palanisamy A, Baxter MG, Keel PK, et al. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011;114:521-528.

hensive assessment of learning and memory in rat offspring, multiple

behavioural test such as open field test, step-through test and the

fear conditioning test should be used in future study. Third, we have

4. Xiong M, Li J, Alhashem HM, et al. Propofol exposure in pregnant rats induces neurotoxicity and persistent learning deficit in the off- spring. Brain Sci. 2014;4:356-375.

explored the underlying mechanisms only from hippocampus. Mater-

nal propofol exposure may also affect other brain regions, such as cortex, thalamus and hypothalamus regions. Li et al55 found that

5. Liang Z, Luo F, Zhao W, et al. Propofol exposure during late stages of pregnancy impairs learning and memory in rat offspring via J Cell Mol Med. 2016;20:1920-1931. the BDNF-TrkB signalling pathway.

propofol anaesthesia in pregnant rats induced caspase-3 activation

and microglial response in foetal rats. They found that the activated

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effects of these drugs on learning and memory deficits and protein

expression changes caused by propofol exposure on E7 should be

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evaluated in future study.

Taken together, the results of the present study suggest that

propofol anaesthesia during first trimester causes learning and mem-

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ory deficit in offspring rats by inhibiting histone acetylation. SAHA,

Senegenin and HGN antisense oligonucleotide can ameliorate these

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A C K N O W L E D G E M E N T S

13. Li Gang Z, Wei-lu L. Fo-quan. Effect of ketamine anesthesia in early pregnancy on the c-fos mRNA and c-jun mRNA expression in off- spring of rats. Chin J Anesthesiol. 2010;30:1333-1335.

We thank other members of the laboratory for valuable discussion

and technical help. This research was supported by National Natural

14. Bing-da L, Fuo-quan L, Wei-lu Z, et al. Effect of ketamine anesthesia in early pregnancy on expression of hippyragranin mRNA in hip- pocampus in offspring of rats. Chin J Anesthesiol. 2012;32:1334- 1336.

Science Foundation of China (NO. 81460175, 81060093) and Natu-

Jiangxi Province of China

ral Science Foundation of

(NO.

20171ACB20030, 20132BAB205022).

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https://doi.org/10.1111/jcmm.13524