It has been confirmed that repeated application of propofol, as an intravenous and short-fast-acting anesthetic, in neonatal animals or humans may produce long-term deficits in cognitive functions. With the aim of explaining the neurotoxic effects of repeated administration of propofol on neonatal rat pups from P7 to P9 especially from an epigenetic perspective, the present study used the Morris water maze to detect cognitive deficits in spatial learning and memory, Sequenom methylation on the CpG island located in the promoter region of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to assess the methylation level of this region, and Western blot to measure the expression of EFEMP1, TIMP-3, and MMP-9. As the results have shown, repeated propofol administration on neonatal rats caused significant systemic growth retardation, impairment of spatial learning and memory, and hypermethylation of the CpG sites in the promoter region of EFEMP1 accompanied by lower expression of EFEMP1 and TIMP-3 and enhanced expression of MMP-9. These data suggest that repeated propofol administration in neonatal rats may generate hypermethylation in the promoter region of EFEMP1 which results in downregulation of the expression of EFEMP1 and tissue inhibitor of metalloproteinase-3 (TIMP-3) but upregulation of the expression of matrix metalloproteinase-9 (MMP-9), which together may affect the stability of ECM to hamper the development of the central nervous system and therefore lead to deficits in cognitive functions.
Though general anesthesia has its undoubted role in facilitating critical surgeries, still people started to consider its double-bladed actions on CNS since its principal mechanism is to blockade nervous traffic. Based on the current research, multiple or long-lasting anesthesia may cause some persistent neurotoxicity including deficits in memory and learning, at extreme ages [
As one type of often-used intravenous general anesthetics, propofol has been demonstrated to cause significant associated cognitive dysfunction at clinically relevant concentrations and durations [
Twenty-two P3 (postnatal day 3) Sprague-Dawley male rat pups and their lactating dams (
The 22 pups were randomly divided into three groups: control group (
The Morris water maze test was performed for 5 days as previously described [
Rats were sacrificed at P29. Then, the hippocampus was first homogenized with an ice-cold lysis buffer and then centrifuged at 12,000 rpm for 20 min at 4°C. Lastly, the supernatant was collected for protein content analysis. After quantitation of protein concentration via a BCA protein assay reagent, samples were separated by 10% SDS-PAGE gel, transferred to PVDF membranes under electrophoresis, and blocked with 5% bovine serum albumin for 2 hours. Subsequently, the membranes were incubated overnight at 4°C with the primary antibody for EFEMP1 (1 : 1000, ab106429, Abcam), TIMP-3 (1 : 1000, 5673, Cell Signaling Technology), and GAPDH (1 : 1000, 2118s, Cell Signaling Technology), then again incubated for another 2 hours with the HRP-conjugated secondary antibody. Immunoreactive proteins were detected using an enhanced chemiluminescence method, and the signal was normalized to GAPDH.
Genomic DNA was extracted from the hippocampus with the QIAamp DNA Mini Kit (Qiagen 51304, Germany) according to the manufacturer’s instructions. The concentration and purity of the DNA were determined by absorbance at 260 and 280 nm. A total of 1.5
All data were expressed as
The change in body weight was obtained by subtraction of the weight of the day from the weight of the day immediately prior to the day. As shown in Figure
Alteration in weight from P4 to P29 after exposure to propofol (P7-P9). The body weight of animals at different measure time and related statistical data are shown in Table
The Morris water maze test was performed on P29 to reflect the effects of propofol on long-term cognitive functions. As shown in Figure
Effects of propofol on cognitive functions by the Morris water maze test. (a) Representative traces of rat movement in the open field test. The red circle in the left upper quadrant represented the position of the platform underwater, and the red dot represented the point from where rats were released into the water. (b) The percentage of time the rats remained in the target quadrant during the probe trial of the MWM test. (c) Escape latency for the rats to locate the platform during the probe trial of the MWM test. Related statistical data are shown in Table
The EFEMP1 gene sequence was confirmed via GenBank, and a 112-base pair-long CpG island was found in the promoter region of EFEMP1. As shown in Figure
Schematic diagram of long interspersed nucleotide EFEMP1. EFEMP1 transcript consists of a 5
Comparison in the methylation levels of the CpG sites in the EFEMP1 promoter region between the vehicle group and the propofol group. (a) Was the heat map representing the methylation level of CpG sites in the EFEMP1 promoter region. Every row represented the methylation level of a CpG site while every line represented the methylation level of a sample. The discrepancy in the methylation level was displayed in red or white color. The more reddish the color, the higher the methylation level. (b–f) Were used to show the comparison in the methylation level of each CpG site in the EFEMP1 promoter region between the vehicle group and the propofol group. Due to missing data in the third CpG site, no result was obtained from it. #
As displayed in Figures
Alterations in the expression of EFEMP1 and TIMP-3 in the rat hippocampus. (a) The Western blotting results of EFEMP1, TIMP-3, and GAPDH. (b–d) Comparison in the levels of EFEMP1, TIMP-3, and MMP-9 by densitometry analysis among different groups. Related statistical data and parameters are shown in Table
Previous research on animals and human beings has confirmed that repeated/long-lasting/multiple application of general anesthesia (GA) may trigger neurotoxic changes in the developing, immature brain, which ultimately leads to a persistent deleterious impact on CNS, such as neuroapoptosis, disturbance in synaptogenesis, and cognitive deficits in spatial learning and memory [
Exposure to propofol at BGS may affect glial cells such as oligodendrocytes, the myelin sheath origin for the neurons in CNS, leading to the lack of myelination of neuronal axons and induction of further apoptosis of these unmyelinated neurons [
In normal tissues, EFEMP1 is usually highly expressed by epithelial and endothelial cells, interacting with several other proteins of the extracellular matrix (ECM), contributing not only to the integrity of the basement membrane but also to the assembly of elastic fibers during embryonic development [
MMP-9, as one pericellularly acting endopeptidase, regulates numerous cell processes and physiological functions via its role in remodeling the extracellular matrix [
Hence, to explore the mechanism by which the expression of MMP-9 was elevated in the hippocampus on repeated exposure of propofol became the ultimate objective for our research. As displayed by previous studies, MMP activity is strictly regulated via various mechanisms including transcription, activation of the precursor zymogens, and inhibition by tissue inhibitor of metalloproteinase (TIMP) [
On the other hand, as for the functions of the pair of TIMP-3 and EFEMP1, there are several studies that can be cited below. Han et al. [
In conclusion, it may be hypothesized from our results that propofol administration in neonatal rats may generate hypermethylation in the promoter region of EFEMP1 which results in downregulation of the expression of EFEMP1 and TIMP-3 but upregulation of the expression of MMP-9 and subsequent neurotoxicity.
Data included in this paper are available on request.
The authors declare that there are no conflicts of interest.
G.P.C. and H.F. conceptualized and designed the experiments. N.Z. and P.W.W. performed most of the experiments and analyzed the data. Z.Y.L. carried out bioinformatic analysis and behavioral data analysis. N.Z. and Z.Y.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Nu Zhang, Zhiyi Liao, and Pinwen Wu contributed equally to this work and shared the first authorship.
This work was supported by the National Natural Science Foundation of China (81971863), the Committee of Science and Technology of Jinshan (2017-3-04), and the Funding of Minhang Hospital (2019MHJC07).
All the statistical data and parameters related to the statistical analysis in the current study are shown in the following four tables. Table 1: body weight of rats in different groups at different measure time. Table 2: data measured in the Morris water maze test. Table 3: methylation level of every CpG site. Table 4: expression of EFEMP1, TIMP-3, and MMP-9 in the hippocampus of rats.