The authors have declared that no competing interests exist.
Conceived and designed the experiments: ZT GQL YY. Performed the experiments: ZT SB HX. Analyzed the data: ZXY SZB ZT ZWY. Contributed reagents/materials/analysis tools: ZT SB. Wrote the paper: ZT YY.
Neonatal exposure to isoflurane may induce long-term memory impairment in mice. Histone acetylation is an important form of chromatin modification that regulates the transcription of genes required for memory formation. This study investigated whether neonatal isoflurane exposure-induced neurocognitive impairment is related to dysregulated histone acetylation in the hippocampus and whether it can be attenuated by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA).
C57BL/6 mice were exposed to 0.75% isoflurane three times (each for 4 h) at postnatal days 7, 8, and 9. Contextual fear conditioning (CFC) was tested at 3 months after anesthesia administration. TSA was intraperitoneally injected 2 h before CFC training. Hippocampal histone acetylation levels were analyzed following CFC training. Levels of the neuronal activation and synaptic plasticity marker c-Fos were investigated at the same time point.
Mice that were neonatally exposed to isoflurane showed significant memory impairment on CFC testing. These mice also exhibited dysregulated hippocampal H4K12 acetylation and decreased c-Fos expression following CFC training. TSA attenuated isoflurane-induced memory impairment and simultaneously increased histone acetylation and c-Fos levels in the hippocampal cornu ammonis (CA)1 area 1 h after CFC training.
Memory impairment induced by repeated neonatal exposure to isoflurane is associated with dysregulated histone H4K12 acetylation in the hippocampus, which probably affects downstream c-Fos gene expression following CFC training. The HDAC inhibitor TSA successfully rescued impaired contextual fear memory, presumably by promoting histone acetylation and histone acetylation-mediated gene expression.
The use of inhaled anesthetics has become widespread in the pediatric population, and its deleterious effects are causing increasing concern. Several recent studies showed that children with multiple exposures to anesthesia and surgery before 4 years of age could be at increased risk of developing learning disabilities[
The capabilities to form and retrieve long-term memories are regarded as major aspects of cognitive function. Generally, changes in gene expression immediately following learning are thought to be indispensable for long-term memory formation. A wide variety of mechanisms regulate gene expression, and among chromatin remodeling via histone acetylation plays a particularly important role. Recent studies have demonstrated that cognitive function is closely related to histone acetylation alterations in the central nervous system, and dysregulation of hippocampal histone acetylation has particular significance for neurocognitive impairment associated with mutations, brain aging, iron overload, and other precipitating factors[
Histone acetyltransferases (HATs) catalyze histone acetylation, whereas histone deacetylases (HDACs) have the opposite effect. In previous studies, HDAC inhibitors (HDACi) such as sodium butyrate or trichostatin A (TSA) were reported to rescue memory deficits in both aged and gene-mutant mice by elevating the level of hippocampal histone acetylation, and these compounds also showed therapeutic potential for depression and some neurodegenerative disorders such as Huntington’s disease (HD), Parkinson's disease (PD), and Alzheimer’s disease (AD) [
We therefore hypothesized that dysregulation of histone acetylation was involved in neurocognitive impairment caused by repeated neonatal exposure to isoflurane and that cognition impairment could therefore be ameliorated by the HDACi TSA. To test this hypothesis, we treated mice with 0.75% isoflurane for 4 h on postnatal days 7, 8, and 9 and assessed hippocampal histone acetylation and neurocognitive function using contextual fear conditioning (CFC) testing at 3 months after isoflurane exposure. Together with CFC, we carried out an open-field analysis to assess locomotor activity and anxiety levels in mice. In addition, we also determined whether TSA reversed changes in hippocampal histone acetylation and behavioral testing in isoflurane-treated mice.
All animal experiments were approved by the Animal Ethics Committee of Xiangya Hospital, Central South University, China (Approval number: 2011–11028). A total of 234 male C57BL/6 mice purchased from the Experimental Animal Center of Central South University were used for this study. Mice were housed in group cages(5–6 animals per cage) with free access to food and water. The environment was controlled on a 12/12-h light/dark cycle at a temperature of 25±2°C.
The neonatal mice were exposed to 0.75% isoflurane three times (postnatal days 7, 8, and 9) in groups of 12–20 using a gas-delivery chamber. Each isoflurane exposure lasted 4 h. The gas was carried by 30% O2, and the total flow was controlled at 2 L/min. The concentration of isoflurane was measured in the gas-delivery chamber outlet using a Capnomac Ultima anesthesia monitor (Daetex-Ohmeda of GE Healthcare, Wauwatosa, WI, USA). The control group was exposed to 30% O2-enriched air. The environmental temperature of gas-delivery chamber was controlled at 36±1°C. Arterial blood specimens were obtained with an interval of 2 h during the first isoflurane exposure and immediately following the second and third exposures; mice were sacrificed by cervical dislocation and the hearts were quickly exposed, then the samples of blood in left cardiac ventricle were drew into syringe for blood-gas and blood glucose analyses.
To assess the effect of TSA on CFC memory in isoflurane-treated mice, TSA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 4% dimethyl sulfoxide (DMSO), and the concentration was controlled at 0.5 μg/μl. TSA (2 mg kg-1) or vehicle was intraperitoneally injected 2 h before CFC training. An equal number of air-exposed mice were used as controls and also received TSA or vehicle.
months after gas exposure, the mice underwent Open-field test and CFC trial. The mice were handled for 5 d, and on the day of behavioral testing they were transported to the laboratory at least 2 h before behavioral testing.
Open-field test was performed in a white plastic box(75×75×45 cm).The mice were placed in the center of the box and allowed to explore it for 5 min under a weak light condition(about 5 lux), the travel trace was captured by a camera using software Smart JUNIOR(Panlab Harvard Apparatus, Barcelona, Spain). Locomotor activity of mice was measured by the total distance (centimeters) traveled in 5 min and anxiety level was assessed by the exploration time in the center of the open field.
For CFC, the mice were placed in a pellucid Perspex chamber (40×30×26 cm) in a soundproof cabinet (75×60×45 cm). The training procedure of each mouse was recorded by a high-resolution camera located on the ceiling of the soundproof cabinet equipped with ANY-maze software (Stoelting Co, Wood Dale, IL, USA). The floor of the training chamber consisted of 28 iron bars that delivered electric footshocks. The CFC training was conducted for 5 min. At the beginning of the fifth minute, mice received a 0.75-mA footshock for 2 s. ANY-maze software was used to analyze the video files to determine whether or not the mice were in freezing behavior. The observation time-window for freezing behavior during CFC training was from the second minute until footshock administration. The CFC testing took place 24 h later, and we measured the freezing time in 3 consecutive min when the mice were placed into the same chamber.
At each time point after CFC training, mice were sacrificed by cervical dislocation. Each brain was quickly dissected and cut into coronal slices, and the cornu ammonis (CA)1 regions of the hippocampi were separated from transverse hippocampal slices under a dissecting microscope and were stored in liquid nitrogen. The details of micro-dissection and subsequent histone extraction and protein sample preparation were described in our previous study [
Tissue preparation and immunohistochemistry procedures were performed as we previously described [
All statistical tests were performed with SPSS 13.0 software (SPSS, Chicago, IL, USA). Data are expressed as mean ± standard deviation (SD). Differences in blood gas analysis and blood glucose value were analyzed by Student’s
Prolonged isoflurane anesthesia may result in respiratory depression and pathoglycemia. A previous study demonstrated that 1.5% isoflurane exposure for 6 h induced hypercapnia, hypoglycemia, and increased mortality in neonatal mice[
Group | Time point | n | pH | PaO2, mmHg | PaCO2,mmHg | Hct, % | Glucose,mg/dl |
---|---|---|---|---|---|---|---|
30% O2-enriched air | 2 h in 1st exposure | 8 | 7.34±0.05 | 154.0±10.2 | 30.2±5.6 | 34.2±3.8 | 75.6±10.3 |
4 h in 1st exposure | 8 | 7.32±0.08 | 142.5±11.1 | 31.5±5.9 | 34.0±3.5 | 70.4±12.3 | |
2nd exposure | 8 | 7.31±0.08 | 159.7±9.7 | 33.4±6.1 | 34.6±4.1 | 69.8±10.9 | |
3rd exposure | 8 | 7.33±0.09 | 164.7±13.9 | 29.6±6.9 | 34.4±4.2 | 71.6±11.5 | |
0.75% isoflurane | 2 h in 1st exposure | 8 | 7.37±0.07 | 164.2±9.6 | 33.4±5.8 | 34.1±4.1 | 70.4±12.7 |
4 h in 1st exposure | 8 | 7.38±0.06 | 155.7±13.6 | 35.6±6.8 | 34.7±4.0 | 55.5±13.2 | |
2nd exposure | 8 | 7.40±0.07 | 153.8±15.6 | 34.8±5.4 | 34.3±3.7 | 54.6±13.7 | |
3rd exposure | 8 | 7.37±0.06 | 160.1±12.3 | 33.6±6.8 | 34.5±3.5 | 60.5±11.5 |
Hct: hematocrit; PaCO2: arterial carbon dioxide tension; PaO2: arterial oxygen tension.
There were no significant differences in blood gases or blood glucose between mice exposed to isoflurane and those exposed to O2-enriched air. Mean (SD) values are shown.
In the open-field test, there was no significant difference between O2-enriched air group and isoflurane group in the total distance and the percentage of time spent in the center of the open-field(t = 0.143, df = 22,
(A) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed normal traveled total distance in Open-field tests. (B) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed normal affective state(anxiety level), which was measured by the percentage of time spent in the center of open-field. (C) Mice that received repeated neonatal exposure to 0.75% isoflurane displayed decreased freezing time (** p<0.01 versus control) during contexual fear conditioning (CFC) test. The control group inhaled 30% O2-enriched air during the neonatal period.
In the present study, we assessed the neurocognitive function of mice using CFC trials. During CFC training, all mice exhibited few freezing behavior before the footshock was given; there was no significant difference between O2-enriched air group and isoflurane group (t = 0.197, df = 22,
We assessed the levels of histone acetylation in the CA1 hippocampal area at different time points after CFC training. The results showed that acetylation of H3K9, H3K14, H4K5, and H4K12 at 1 h after CFC training were significantly increased relative to baseline level at naive condition in the mice exposed to 30% O2-enriched air (H3K9 F(3,20) = 10.358,
(A) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region at 15 min,1 h, and 24 h after CFC training. Control mice were not subjected to CFC. (B) Quantification of the immunoblots from mice that received repeated exposure to 30% O2-enriched air. * p<0.05 and ** p<0.01 versus control. (C) Quantification of the immunoblots in mice that received repeated neonatal exposure to isoflurane. * p<0.05 and ** p<0.01 versus control. Mean (SD) values are shown.
To examine whether the HDACi TSA was capable of attenuating isoflurane-induced neurocognitive impairment, we divided the mice into four groups: an air+vehicle group subjected to three neonatal exposures to O2-enriched air and injected with DMSO (vehicle) 2 h before CFC training, an air+TSA group with neonatal exposure to O2-enriched air injected with TSA, an ISO+vehicle group that received repeated neonatal exposures to 0.75% isoflurane and vehicle injection 2 h before CFC training, and an ISO+TSA group that received isoflurane exposures and TSA injection. During the CFC training phase, there were no differences in freezing times among the four groups (F(3,44) = 0.606,
(A) TSA injection improved CFC performance in mice that received repeated neonatal exposures to isoflurane. The freezing times of each group (n = 12 mice per group) during CFC training and testing are shown. * p<0.05. (B) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region for each group 1 h after CFC training. (C) Quantification of (B). * p<0.05 versus the Air+vehicle group. Mean (SD) values are shown.
At 1 h after CFC training, western blotting analysis showed that TSA increased H3K9, H3K14, H4K5, and H4K12 acetylation in the hippocampal CA1 area in all groups (all
c-Fos belongs to the activator protein-1 family of transcription factors and is an immediate early gene (IEG). Many previous studies have demonstrated that c-Fos expression is rapidly elevated by various experiential stimuli including conditioned and unconditioned aversive stimuli [
(A) Representative immunohistochemistry images of c-Fos in the CA1 hippocampal region 1 h after CFC training. Hippocampal neuronal nuclei were stained purple with hematoxylin, and nuclear c-Fos protein is stained brown by DAB. (B) c-Fos-positive cell counts. Mice that received repeated neonatal exposures to isoflurane showed reduced numbers of c-Fos-positive neurons in the CA1 hippocampal region 1 h after CFC training, but this decrease was rescued by systemic TSA administration. * p<0.05. Mean (SD) values are shown.
The mechanism underlying neurocognitive impairment induced by inhalational anesthetic in the developing brain is thought to involve neurodegeneration, although the involved pathways are not entirely clear. The anesthetic properties of inhalational anesthetic are ascribed to a compound effect on the potentiation of γ-aminobutyric acid (GABAA)-gated receptors and inhibition of N-methyl-D-aspartate (NMDA)-gated receptors [
We used CFC testing to assess mouse neurocognitive function; this paradigm is commonly used to assess hippocampus-dependent associative learning and memory, and previous findings have demonstrated that the acetylation of several specific histone lysine residues (H3K9\14 and H4K5\8\12\16) in the hippocampus might be related to CFC memory formation[
In previous studies, administration of global HDACis such as TSA or sodium butyrate was found to increase long-term potentiation (LTP) at Schaffer collaterals in CA1 of the hippocampus and rescue memory deficits in both aged and gene-mutant mice [
There is a wide range of mechanisms that contribute to the protective effect of HDACis on neurocognitive function, including epigenetic and non-epigenetic pathways [
There are several noteworthy limitations of this study: First, it should be noted that the behavioral testing paradigm used in this study to assess learning and memory did not distinguish the different memory processes, including acquisition, consolidation and retrieval. Therefore, there is a possibility that the memory defect induced by neonatal isoflurane exposure may be just derived from the impairment of a specific memory process, such as acquisition impairment. Second, our observations just focused on the hippocampal CA1 subregion after mice received repeated neonatal exposure and intraperitoneal injection. Although histone acetylation changes in CA1 region were likely to play an important role in contributing to the observed effects on behavioral testing, it cannot be excluded that the similar changes in other regions of brain were implicated as well. Third, because we did not assess acetylation in other lysine sites or perform a genome-wide analysis of transcription after CFC, it is possible that the dysregulation of histone acetylation for other specific sites or expression deficits in other memory-related genes were overlooked by us. At last, we also did not assess the interaction between histone acetylation and target gene promoters using chromatin immunoprecipitation, and therefore further studies are required to identify specific effect of H4K12 acetylation on transcription regulation of genes in mice with memory impairment induced by neonatal isoflurane exposure together with the assessment of other memory-related genes.
In conclusion, our findings suggest that memory impairment induced by repeated neonatal exposures to 0.75% isoflurane is associated with dysregulated acetylation of histone H4K12 in the hippocampal CA1 region, which probably affects the downstream expression of memory-related genes such as c-Fos. TSA mitigated the isoflurane-induced memory impairment, most likely by enhancing histone acetylation levels and increasing c-Fos gene expression in the hippocampus.