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. 2015 Jan 22;21(5):398–409. doi: 10.1111/cns.12368

Surgical Stress Induces Brain‐Derived Neurotrophic Factor Reduction and Postoperative Cognitive Dysfunction Via Glucocorticoid Receptor Phosphorylation in Aged Mice

Xiao‐Sheng Tian 1,2, Ya‐Wei Tong 1, Zheng‐Qian Li 1,2, Lun‐Xu Li 1,2, Tao Zhang 1, Tian‐Yun Ren 1, Ting Zhou 1, He‐Cheng Wang 1, Rui Zhan 1, Yang Sun 1, Zhao Yan 2, Qiu‐Dian Wang 1, Dong‐Sheng Fan 3, Fan‐Jun Kong 4, Xiang‐Yang Guo 2, Wei‐Zhong Xiao 3, De‐Hua Chui 1,3,✉
PMCID: PMC6495659  PMID: 25611431

Summary

Aims

This study explored whether surgical stress‐induced glucocorticoid receptor (GR) phosphorylation is related to postoperative cognitive dysfunction (POCD) in aged individuals. Inhibition of GR activation could be an effective treatment for POCD.

Methods

A laparotomy was given to C57/BL6 mice in POCD group both 20 and 6 months old. Animals in control group were treated in identical manners except for laparotomy. Cognitive function was evaluated by Morris water maze and elevated plus maze. Western blot and Elisa assay were used to detect related molecules. Mifepristone and roscovitine were treated as inhibitions of GR phosphorylation.

Results

The cognitive function was impaired, and brain‐derived neurotrophic factor (BDNF) was found reduced in aged POCD group. GR translocation into nucleus and elevated GR phosphorylation were found in prefrontal cortex of aged POCD mice. Cyclin‐dependent Kinase 5 (CDK5), kinase for GR phosphorylation also elevated in aged POCD mice. With GR antagonist and CDK5 inhibitor, reduction of BDNF and cognitive dysfunction in aged mice were both rescued.

Conclusion

These results presented a mechanism that surgical stress‐induced GR phosphorylation contributes to POCD in aged individuals. Inhibition of GR activation and phosphorylation might be a potential treatment target of POCD.

Keywords: Aging, Brain‐derived neurotrophic factor (BDNF), GR phosphorylation, Postoperative cognitive dysfunction (POCD), Surgical stress

Introduction

The surgical stress response is intimately associated with adverse outcomes in surgeries of several areas 1. It has been recognized as one of the risk factors in the pathogenesis of postoperative cognitive dysfunction (POCD) especially in geriatric surgical population 2. POCD refers to a deficit in cognitive function subsequent to surgery, especially in elder patients 3, 4, 5. Surgery induces various kinds of stress such as surgical impairment, inflammation, ischemia, and psychological stress 6, 7, 8, 9. These stresses contribute to neuronal degeneration associated with hormones disturbance conditions such as aging 10. During acute or chronic stress, HPA axis (hypothalamic–pituitary–adrenal axis) is activated to release cortisol, which could activate glucocorticoid receptors (GR) leading to cognitive impairment 11, 12, 13. Activation of glucocorticoid receptors includes translocation into nucleus and phosphorylation (primary phosphorylation sites for activation in human: ser203, ser211, and ser226; mice: ser212, ser220, and ser234) following stress controls transcription of genes participating in stress and dementia 14, 15, 16, 17 such as BDNF and extracellular‐regulated protein kinases (ERK). Patients with POCD presented significantly higher cortisol levels and negatively correlated with Mini Mental State Examination (MMSE) scores 18, 19. However, whether GR activation participates in surgical stress‐induced POCD is not clearly explained.

In this research, we reported that surgical stress‐induced BDNF reduction and POCD via activating GR phosphorylation in aged but not in younger mice. GR translocation into nucleus and phosphorylation on ser220 were found in prefrontal cortex of aged mice. The cognitive impairment after surgical stress and following changes might be closely related to GR activation.

Materials and Methods

Animals

Aged (20 months old) and young (6 months old) male C57 mice were bred and kept under standardized housing conditions with food and water ad libitum. All work was approved by the Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch. Both aged and young animals were divided into four groups for different tests: control, POCD, POCD+RU486 (GR antagonist), and POCD+roscovitine (CDK5 inhibitor). As isoflurane inhalation was reported to increase CDK5 20, we administered an i.p. injection of 5% chloral hydrate for anesthesia and buprenorphine (0.1 mg/kg) for analgesia in all groups. No significant changes on CDK5 with or without chloral hydrate anesthesia (Fig. S2). All of the four groups received chloral hydrate anesthesia. Control group received sterile preparation without surgery as control for possible effects of handling, injection, and anesthesia. POCD group received a laparotomy surgery with saline injection. POCD+RU486 group received surgery with RU486 injection, and POCD+ roscovitine group received surgery with roscovitine.

Surgical Program

Mice were deeply anesthetized by i.p. injection of chloral hydrate (5%) and then received a minor surgery. After the surgical site was shaved and sterilized, a 1.5‐cm incision was made in the upper left quadrant through the skin and muscle wall. According to method of Rosczyk HA et al., with minor modification, a sterile probe was then inserted into the body cavity to gently manipulate the internal organs in turn of small intestine, liver, colon, stomach for 1 min. Three dissolvable sutures were used to close the muscle wall and four silk thread sutures were used to close the skin 21. Before collecting the samples, mice were anesthetized and transported to another room prior to sacrifice.

Drug Exposure

Mice were injected with RU486 (Sigma) dissolved in 30% (wt/vol) propylene glycol + PBS or vehicle acutely after the abdominal surgery as described with minor modification (40 mg/kg, i.p.) 22. As described with minor modifications 23, roscovitine (Santa Cruz, CA, USA) 10 mg/kg or equal volume vehicle (sterile oil with 0.2% dimethylsulfoxide) was injected through intraperitoneal route daily for 3 days postincision in interaction studies.

Physical Signs Analysis

Five extra mice in each treatment groups were prepared to measure the physical signs. 0.3 milliliter of blood was immediately drawn by cardiac puncture. Blood gas testing was carried out immediately using a handheld i‐STAT analyser (Abbott Point of Care Inc., Princeton, NJ, USA). The animals were then euthanized and not used for any other part of the study.

Morris Water Maze (MWM)

On day 5 after surgery (3 days for recovery from surgery and 1 day for adaption of experimental environment), mice were used to complete Morris water maze test. As previously described 24 with minor modifications, all groups of 8 mice each were tested for memory using MWM test by investigators blinded to the group conditions. Briefly, the animals received four training trials daily for seven consecutive days. During each trial, the mice were placed gently in the water facing the wall of the maze at one of the four equally spaced start positions. The time to locate the submerged platform (defined as the latency cutoff time of 60 seconds) and swim velocity were recorded. On test day 8, a series of probe trials were conducted, whereby the platform was removed. The percentage of time spent in the previous target platform quadrant (IV) in 60‐s period was determined.

Elevated Plus Maze (EPM)

On day 5 after surgery, another groups of mice (n = 8 for each group) were used to complete the elevated plus maze. The protocol of elevated plus maze was as previously described 25 with minor modifications. Elevated plus maze test consists of four polycarbonate arms extending 11.5 inches in length, two of which have walls 6.5 inches in height. The plus maze was elevated from the floor at the height of 24 inches. Mice were placed in an open square dividing the four arms of the chamber with an overhead camera recording the time spent on the open arms of the plus maze during each 5‐min test session.

Preparation of Nuclear and Cytoplasmic Extracts

Tissue of mice in all groups was prepared after the elevated plus maze tests on day 7 after surgery. Preparation of nuclear and cytoplasmic extracts was carried out as previously described 26. To prepare cytoplasmic and nuclear extracts, tissue homogenates were allowed to swell by adding 0.4 μL/μg of cold lysis buffer (10 mM HEPES, 10 mM KCl,0.1 mM EDTA, 0.5% Nonidet P‐40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl ‐fluoride [PMS F, pH 7.9]) with protease and phosphatase inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 2 mM NaF, 2 mM Na4P2O7, 4 U/mL aprotonin, 2 mM DTT, 1 mM EGTA, 1 μM microcystin, and 1 mM benzamidine). The removed cytoplasmic fraction was stored at −20°C. The pellets containing crude nuclei were resuspended in 50‐μL extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM PMSF, pH 7.9), incubated on ice for 30 min, and centrifuged at 12,800 g for 10 min to obtain the supernatant containing nuclear extracts. Another two groups of mice were killed and took samples on day 1 and 13 after surgery.

Western Blot

Western blot analysis was used to determine the expression of related proteins as described 26 with the following modifications. Briefly, sixty micrograms of protein per lane was loaded on 10% SDS‐PAGE for protein in nuclear and forty micrograms for protein in cytosol. After transferred onto PVDF membrane, the following primary antibodies were used: sheep anti‐BDNF antibody (1:5000, millipore, Billerica, MA, USA); rabbit anti‐GR and anti‐p‐GRs220 antibody (1:12000, CST, Boston, MA, USA); rabbit anti‐CDK5 antibody (1:5000, BD, NJ, USA); rabbit anti‐p35 antibody (1:5000, BD); rabbit anti‐p‐GRs212 (1:2000, Anbo, San Francisco, CA, USA); and rabbit anti‐p‐GRs234 (1:12000, CST).

Immunohistochemistry and Confocal Microscopy

Tissue preparation and immunohistochemistry were performed as described (Xian et al., 2009) with minor modifications. Free‐floating sections (18 μm thick) were processed for free‐floating immunohistochemistry. The primary antibody was applied overnight at 4°C. Secondary antibody used was Alexa Fluor 488 labeled‐goat anti‐rabbit IgG (MicroProbe, Carlsbad, CA, USA 1:2000). Nuclei were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA, USA). Fluorescence images were acquired with a confocal laser scanning microscope (LSM510; Carl Zeiss Co., Oberkochen, Germany) using a 505‐nm‐long path filter for emission and an argon ion laser for excitation (488 nm). No fluorescence was detected with the primary antibody omitted.

Elisa Kit Assays

Plasma cortisol level was detected with ELISA kit (Enzo, Farmingdale, NY, USA ADI‐900‐071) according to the procedures provided by the manufacturer. Plasma supernatants were collected on day 1, 7, and 13 postoperation and stored at −80°C until analysis. IL‐1β, IL‐6, and TNFα was measured in the protein from prefrontal cortex of mice, using a commercially available ELISA kit according to the procedures provided by the manufacturer (IL‐1β and TNFα from Abcam; IL‐6 from Pierce). The supernatants were collected on day 7 after surgery and stored at −80°C until assays were performed.

Statistical Analysis

Statistics were calculated using functions provided in GraphPad Prism 5 for Windows (GraphPad Software Inc., La Jolla, CA, USA). All data in the text and figures were presented at least three independent experiments and were expressed as mean ± standard error (SEM). Data in escape latency trails of MWM were assessed with two‐way ANOVA (treatment condition × day) as described 27 with a minor modification. One‐way ANOVA followed by Tukey's post hoc tests was performed to compare data in probe trail and other data among multiple groups 27. Two‐tail unpaired t‐test was performed to compare the data between two groups. The correlation analysis was completed with function of GraphPad Prism 5. Mean values were considered significantly different at P < 0.05.

Results

Surgical Stress Induces Postoperative Cognitive Dysfunction in Aged but not in Younger Mice

We used Morris water maze and elevated plus maze to detect the effects of surgical stress on cognition and emotion in 20 (Figure 1A) or 6‐month‐old (Figure 1B) mice. There was a significant difference on escape latency in aged mouse (two‐way ANOVA, treatment condition × day, F(1,98) = 19.06, P < 0.001 for treatment condition) but not the younger mouse (F(1,98) = 0.2388, P = 0.6261 for treatment condition). We found the POCD group performed longer latency to reach the hidden platform on day 4, 5, 6, 7 (P < 0.05), and shorter time spent in target area (Q IV) with a removed platform (P = 0.0129) (Figure 1A) compared with 20‐month‐old control group in MWM tests. No significant difference was found in visible trail and crossing numbers (Fig. S1). The swimming speed had no difference between the two groups (P = 0.5595) (Fig. S6). We also found that 20‐month‐old POCD group spent shorter time in the open arm on elevated plus maze test (P = 0.0179) (Figure 1A). However, in 6‐month‐old group, no significant difference was recorded between POCD and control in both MWM (escape latency, F(1,98) = 0.2388, P = 0.6261 for treatment condition; probe time, P = 0.2208) and EPM tests (P = 0.2133) (Figure 1B). BDNF is a biomarker of neuron survival and cognition 28, 29, and it was involved in POCD 30. We detected BDNF level in prefrontal cortex of 20‐month‐old POCD and control mice on day 7 postoperation (Figure 1C). BDNF had an appreciable reduction in POCD group compared with control group (two‐tail unpaired t‐test, P < 0.001). We also compared BDNF among 4 groups with a two‐way ANOVA (treatment × age) analysis (Fig. S3). The result turns out to be age but not treatment factor had a significant effect on BDNF expression. The level of BDNF was increased in aged mice and was significantly increased while surgery along with aging (Fig. S3). There is no significant changes in 6‐month‐old mice (two‐tailed unpaired t‐test, P = 0.7660). These results indicated that surgical stress could induce the cognitive dysfunction in aged mice rather than in young.

Figure 1.

Figure 1

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Surgical stress induces postoperative cognitive dysfunction in aged but not younger mice. (A), a seven‐day escape latency trail for two groups of 20‐month‐old mice in Morris water maze test. POCD group spent longer latency to reach the hidden platform, compared with control mice (two‐way ANOVA, treatment condition × day, F(1,98) = 19.06, P < 0.001 for treatment condition, POCD compared with control). The POCD group performed longer latency to reach the hidden platform on day 4, 5, 6, 7 (two‐tailed unpaired t‐test for comparisons in each day, P < 0.05). A probe trail with a removed platform was detected. POCD group displayed decreased time in target quadrant (Q IV) (two‐tailed unpaired t‐test, P = 0.0129). Elevated plus maze was detected in 20‐month‐old and 6‐month‐old mice. The time spent in open arm was shorter in POCD group at 20 months old (two‐tailed unpaired t‐test, P = 0.0179). (B), no significant difference on behavioral tests between POCD and control group at 6 months old. (Escape Latency:two‐way ANOVA, treatment condition × day, F(1,98) = 0.3392, P = 0.6261 for treatment condition, POCD compared with control; two‐tailed unpaired t‐test for probe trail, P = 0.2208; elevated plus maze, two‐tailed unpaired t‐test, P = 0.2133). (C), the expression of BDNF level in prefrontal cortex was tested with Western blot in POCD and control mice. BDNF was downregulated in 20‐month POCD group compared with control (two‐tailed unpaired t‐test, P < 0.001). No significant changes on BDNF level in 6‐month groups (two‐tailed unpaired t‐test, P = 0.7660). (*P < 0.05, ***P < 0.001, POCD compared with control) (n = 8 mice/group). Error bar represent SEM.

Surgical Stress Elevated Glucocorticoid Receptor Expression in Nucleus in Prefrontal Cortex of POCD Mice

To demonstrate whether surgical stress could induce GR activation, translocation into nucleus and phosphorylation, we prepared nuclear and cytoplasmic extracts of prefrontal cortex of POCD and control mice on day 7 postoperation. The level of GR in nucleus was found elevated in 20‐month‐old POCD group compared with control group (P = 0.0253) (Figure 2A & B). Oppositely, GR level had a slight decrease in cytosol (P = 0.0331) (Figure 2A & B). The rate of GR level in nucleus/cytosol had a significant difference (P = 0.0257) (Figure 2A & B). No significant change was found on GR translocation in 6‐month‐old group (P > 0.05) (Figure 2C & D). Results are similar in immunofluorescence assay (Fig. S4). Increased GR translocation into nucleus was found in POCD group rather than control. These data suggested that surgical stress induced GR translocation into nucleus in elder but not in younger mice.

Figure 2.

Figure 2

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Surgical stress elevated glucocorticoid receptor expression in nucleus in prefrontal cortex of POCD mice. (A) Glucocorticoid receptor level in nucleus and cytosol of prefrontal cortex were tested with Western blot in POCD and control mice. Glucocorticoid receptor in nucleus was upregulated in POCD group compared with control (two‐tailed unpaired t‐test, P = 0.0253). Oppositely, GR level in cytosol was decreased in POCD group (two‐tailed unpaired t‐test, P = 0.0331). The ratio of nucleus/cytosol GR significantly increased in POCD group compared with control (two‐tailed unpaired t‐test, P = 0.0257). Total GR in prefrontal cortex had no significant difference between two groups (two‐tailed unpaired t‐test, P = 0.2371). (B), quantification of Western blot in 20‐month groups. (*P < 0.05, POCD compared with control) (n = 8 mice/group). Error bar represents SEM. (C), no significant changes on GR translocation in 6‐month groups (two‐tailed unpaired t‐test, P = 0.7874 for GR in nucleus; P = 0.3628 for GR in cytosol; P = 0.5937 for ratio of nucleus/cytosol GR; P = 0.7915 for total GR). (D), quantification of Western blot in 6‐month groups. (*P < 0.05, POCD compared with control) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Surgical Stress Elevated GR Phosphorylation at ser220 by CDK5 and its Regulator in Prefrontal Cortex of POCD Mice

While activated by ligands, GR transferred into nucleus and then phosphorylated. The phosphorylation of GR was detected in POCD and control mice on day 7 postoperation. Ser220, one of the most principal phosphorylation sites of mice in nucleus for GR activation, was found elevated in 20‐month‐old POCD mice (P = 0.0136) (Figure 3A). Other sites were also considered such as ser212 (P = 0.2303) and ser234 (P = 0.3860), without significant changes, however (Figure 3A). We also compared pGRs220 among four groups with a two‐way ANOVA (treatment x age) analysis (Fig. S3). Age but not treatment factor had a significant effect on pGRs220 expression. The level of pGRs220 was increased in aged mice and was significantly increased while surgery along with aging (Fig. S3). CDK5 is an important kinase for GR phosphorylation. We use Western blot to detect whether CDK5 participates in surgical stress‐induced GR phosphorylation. The level of CDK5 was found elevated in 20‐month‐old POCD mice compared with control group (P = 0.0178) (Figure 3B). We also detected the level of CDK5 regulator p35 and p25 in POCD and control mice. Both p35 (P = 0.0140) and p25 (P = 0.0139) had an appreciable increase in POCD group compared with control group (Figure 3B). These data indicated that the increase of CDK5 and its regulator might be one of the reasons for the elevation of GR phosphorylation. The relationship between GR phosphorylation and cognitive dysfunction was also examined by correlation analysis conducted with Prism 5. CDK5 and pGRs220 both have negative correlation with behavioral index that represents cognitive function (CDK5 with MWM, r2 = 0.75, P < 0.001; CDK5 with EPM, r2 = 0.78, P < 0.001; pGRs220 with MWM, r2 = 0.63, P < 0.001; pGRs220 with EPM, r2 = 0.73, P < 0.001) (Figure 3C). The level of pGRs220 and CDK5 had no significant changes in 6‐month‐old mice (P > 0.05) (Figure 3D). These results indicated that GR phosphorylation might be a key difference between aged and young individuals in POCD.

Figure 3.

Figure 3

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Surgical stress elevated GR phosphorylation at ser220 by CDK5 and its regulator in prefrontal cortex of POCD mice. (A), GR phosphorylation was tested with Western blot. The phosphorylation at ser220 site was significantly elevated in POCD group (two‐tailed unpaired t‐test, P = 0.0136 for pGRs220; P = 0.2303 for pGRs212; P = 0.3860 for pGRs234), compared with control group. (B) The level of CDK5 and its regulators p35, p25 in prefrontal cortex was tested with Western blot in POCD and control mice. The level of CDK5 was upregulated in POCD group compared with control (two‐tailed unpaired t‐test, P = 0.0178). CDK5 regulators p35 (P = 0.0140), p25 (P = 0.0139) also elevated in prefrontal cortex compared with control. (C), nonparametric correlation analysis of GR phosphorylation and behavioral index in 20‐month groups (CDK5 with MWM, r2 = 0.75, P < 0.001; CDK5 with EPM, r2 = 0.78, P < 0.001; pGRs220 with MWM, r2 = 0.63, P < 0.001; pGRs220 with EPM, r2 = 0.73, P < 0.001) (C). (*P < 0.05, POCD compared with control) (n = 8 mice/group). Error bar represents SEM. (D), no significant changes on pGRs220 (P = 0.7576), CDK5 (P = 0.8640) and its regulators (P = 0.8182 for p35 and P = 0.8844 for p25) in 6‐month groups. (*P < 0.05, POCD compared with control) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Surgical Stress‐Induced BDNF Reduction and Cognitive Dysfunction are Rescued by GR Antagonist

To demonstrate whether surgical stress‐induced cognitive dysfunction was mediated by GR activation, we use a specific GR antagonist RU486 on behavior tests in aged mouse. There was a significant effect of Ru486 on escape latency (two‐way ANOVA, treatment condition × day, F(1,98) = 20.15, P < 0.001; POCD+Ru486 compared with POCD group by followed Tukey's post hoc test, P < 0.001). One‐way ANOVA following with Tukey's post hoc test was used to detect other data for multiple groups. We found the POCD+RU486 group performed lower latency to reach the hidden platform (P < 0.05 on day 5, 6; P < 0.01 on day 7) (Figure 4A) and spent longer time in target area (Q IV) of removed platform (P = 0.0189) (Figure 4A) compared with POCD group in MWM tests. The swimming speed had no difference between all groups (P = 0.8762, POCD+RU486 compared with POCD) (Figure 4A). We also found POCD+RU486 group spent longer time in the open arm in elevated plus maze test (P = 0.0388) (Figure 4A) compared with POCD group. We also used RU486 to detect whether the BDNF reduction is induced by GR activation. We found BDNF level was rescued in POCD+RU486 group compared with POCD group (P = 0.0354) (Figure 4B). These data suggested that surgical stress‐induced cognitive dysfunction and BDNF reduction were mediated by GR activation.

Figure 4.

Figure 4

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Surgical stress‐induced BDNF reduction and cognitive dysfunction are rescued by GR antagonist. (A), Mifepristone, a specific GR antagonist was treated just after the surgery. The performance on Morris water maze and elevated plus maze were rescued with treatment of RU486. POCD+RU486 group performed shorter latency to find the hidden platform (two‐way ANOVA, treatment condition × day, F(1,98) = 20.15, P < 0.001; POCD+Ru486 compared with POCD group followed by Tukey's post hoc test, P < 0.001; one‐way ANOVA following with Tukey's post hoc test was used to compare latency on each day, P < 0.05 on day 5, 6 and P < 0.01 on day 7, POCD+Ru486 compared with POCD) and spent longer time in target area (Q IV) with a removed platform (one‐way ANOVA following with Tukey's post hoc test, P = 0.0189), compared with POCD group. No significant difference on swimming speed among groups (P = 0.8762, POCD+Ru486 compared with POCD) and the swimming paths were also presented. In elevated plus maze test, time spent in open arm was also rescued with RU486 treatment (one‐way ANOVA following with Tukey's post hoc test, P = 0.0388, POCD+Ru486 compared with POCD), compared with POCD group. (B), the expression of BDNF level in prefrontal cortex was also rescued in POCD+RU486 group compared with POCD group (one‐way ANOVA following with Tukey's post hoc test, P = 0.0354). (*P < 0.05, POCD compared with control; # P < 0.05, POCD+RU486 compared with POCD) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Surgical Stress‐Induced GR Phosphorylation, BDNF Reduction, and Cognitive Dysfunction are Rescued by CDK5 Inhibitor

To demonstrate whether surgical stress‐induced cognitive dysfunction was mediated by CDK5‐induced GR phosphorylation, we use a specific CDK5 inhibitor roscovitine in behavior tests. There was a significant effect of roscovitine on escape latency (two‐way ANOVA, treatment condition × day, F(1,98) = 11.21, P < 0.001; POCD+roscovitine compared with POCD group followed by Tukey's post hoc test, P < 0.001). One‐way ANOVA followed by Tukey's post hoc test was used to detect other data for multiple groups. We found that POCD+roscovitine group performed lower latency to reach the hidden platform (P < 0.05 on day 6, 7) (Figure 5A) and spent longer time in target area (Q IV) with removed platform (P = 0.0446) (Figure 5A) compared with POCD group in MWM tests. The swimming speed had no difference between all groups (POCD+roscovitine compared with POCD, P = 0.8075) (Figure 5A). We also found POCD+roscovitine group spent longer time in the open arm in elevated plus maze test (P = 0.0199) (Figure 5A) compared with POCD group. We also used roscovitine to detect whether the BDNF reduction is mediated by CDK5 induced GR phosphorylation. The elevation of GR phosphorylation at ser220 was rescued by roscovitine (P = 0.0277) (Figure 5B). The level of BDNF was also rescued in POCD+roscovitine group compared with POCD group (P = 0.0162) (Figure 5B). These results indicated that the BDNF reduction in POCD was mediated by CDK5‐induced GR phosphorylation. These data also suggested that surgical stress‐induced cognitive dysfunction and BDNF reduction were mediated by CDK5‐induced GR phosphorylation.

Figure 5.

Figure 5

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Surgical stress‐induced GR phosphorylation, BDNF reduction and cognitive dysfunction are rescued by CDK5 inhibitor. (A) Roscovitine, a specific CDK5 inhibitor was treated after the surgery. The performance on Morris water maze and elevated plus maze was rescued with treatment of roscovitine. POCD+roscovitine group performed shorter latency to find the hidden platform (two‐way ANOVA, treatment condition × day, F(1,98) = 11.21, P < 0.001; POCD+roscovitine compared with POCD group followed by Tukey's post hoc test, P < 0.001; one‐way ANOVA following with Tukey's post hoc test was used to compare latency on each day, P < 0.05 on day 6 and 7, POCD+roscovitine compared with POCD) and spent longer time in target area with a removed platform (one‐way ANOVA following with Tukey's post hoc test, P = 0.0446), compared with POCD group. No significant difference on swimming speed among groups (P = 0.8075, POCD+roscovitine compared with POCD) and swimming paths were also presented. In elevated plus maze test, time spent in open arm was also rescued with roscovitine treatment (one‐way ANOVA following with Tukey's post hoc test, P = 0.0199, POCD+roscovitine compared with POCD). (B), the level of GR phosphorylation at ser220 site in nucleus was rescued in POCD+roscovitine group compared with POCD (P = 0.0277). The expression of BDNF level in prefrontal cortex was also rescued in POCD+roscovitine group compared with POCD (P = 0.0162). (*P < 0.05, POCD compared with control; # P < 0.05, POCD+roscovitine compared with POCD) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Surgical Stress Induces Sustaining GR Phosphorylation in Aged but not in Young Mouse

We also collected samples on day 1 and 13 postoperation (postop) to detect GR phosphorylation in different time points. The level of pGRs220 in control groups was set as 100% to compare the elevation of pGRs220 in POCD groups. On day 1 postop, GR phosphorylation at ser220 was found elevated in both aged (P = 0.009) and younger POCD mice (P = 0.0228) compared with each control groups (Figure 6A & B). On day 13 postop, pGRs220 level in younger group was recovered (P = 0.6822) (Figure 6B). However, GR phosphorylation at ser220 was still at a high level in aged POCD mouse on day 13 postoperation (P = 0.0187) compared with control group (Figure 6A). Plasma cortisol level was also detected in both ages on day 1, 7, and 13 postop (Figure 6C). The cortisol level elevated in early stage and recovered gradually (P < 0.01 on day 1 postop). These data indicated that cortisol might be involved in the elevation of GR phosphorylation in early stage. However, the sustaining GR phosphorylation is cortisol‐independent.

Figure 6.

Figure 6

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Surgical stress induces sustaining GR phosphorylation in aged but not in young mouse. (A & B), we used Western blot to detect the pGRs220 level in prefrontal cortex of 20‐month‐old and 6‐month‐old mouse. The samples were collected on day 1 and 13 after surgery. The level of pGRs220 was compared to the control group on each time point. The percentage of pGRs220 elevation was presented. On day 1 postop, pGRs220 was elevated in POCD groups compared with each control, in both 20 (two‐tail unpaired t‐test, P = 0.0090, elevated by 51.9%) and 6 (two‐tail unpaired t‐test, P = 0.0228, elevated by 48.2%) months of mouse. On day 13 postop, pGRs220 was still at a high level in 20‐month‐old group, compared with control (P = 0.0187). In 6‐month‐old groups, no significant difference on pGRs220 level between POCD and control mouse (P = 0.6822). (C), plasma cortisol level compared to control groups in both aged and young mouse.(*P < 0.05, POCD group compared with control group; **P < 0.01, POCD group compared with control group; C, control; P, POCD; postop, postoperation) (n = 8 mice/group). Data are Mean ± SEM (Error bars).

Discussion

We first found GR translocation into nucleus and elevated pGRs220 in prefrontal cortex of aged but not younger POCD groups. Then, we demonstrated the reduction of BDNF expression and cognitive dysfunction after surgical stress. The reduction of BDNF and cognitive dysfunction was rescued by GR antagonist and CDK5 inhibitor. These data indicated that stress‐induced GR phosphorylation might be a significant neuropathology and treatment target of POCD.

GR Activation is Mediated by both Ligand and Kinase

A recent study has reported that the level of cortisol, a primary glucocorticoid, was increased in plasma of POCD patients 18. As binding to cortisol, GR will be phosphorylated by CDK5 and translocated into nucleus from cytosol to regulate transcription 31. Therefore, ligand (cortisol) and kinase (CDK5 as a representative) could be a double control to the phosphorylation of GR. In our study, we use two kinds of inhibitor to rescue the phosphorylation of GR, RU486, and roscovitine. RU486 is a kind of GR antagonist which could competitively bind to GR with cortisol 32. Roscovitine is a kind of CDK5 inhibitor. Consistent with our hypothesis, both of the two inhibitors rescued BDNF reduction and postoperative cognitive dysfunction. Moreover, Ru486 and roscovitine have multiple substrates including GR and CDK5. These results presented a phenomenon that GR signaling was involved in POCD. We also found that plasma cortisol elevated in early stage after surgery and recovered gradually (Figure 6C). However, sustaining GR phosphorylation lasted 1–2 weeks in aged groups (Figure 6A & B). These results indicated that surgical stress‐induced cortisol elevation might be involved in GR activation in early stage. Yet, it may have little effects on sustaining of GR phosphorylation in late period after surgery which probably is controlled by other mechanisms.

GR Activation and Inflammation

Lots of researches report that GR was closely related to inflammation 32, 33, 34, 35, 36, and surgery‐induced inflammation probably leads to dementia and POCD 37, 38. As for surgical stress, activation of inflammation‐associated NF‐kB pathway and elevation of the downstream pro‐inflammatory cytokines are closely linked to the incidence of cognitive impairment 37, 39. We also detect IL‐1β, IL‐6 and TNFɑ on day 7 after surgical stress. These inflammatory factors were elevated in aged POCD group (Fig. S5), and the results were similar to the work mentioned above 38, 39. The elevation of inflammatory factors was probably a direct effect of surgical stress or an indirect effect by surgical stress–GR–inflammation circle. There is no doubt that GR and inflammation have a closely interaction, and the relationship between GR and inflammation in POCD should be studied further.

Transcription Regulation by GR Phosphorylation Participates in Cognitive Dysfunction and Stress

Glucocorticoid and its receptor are closely related to stress and cognition 10, 40, 41. Activation of GR by phosphorylation following behavioral or cellular stress 17 controls transcription of genes participates in stress and cognitive dysfunction. In this work, we detected GR phosphorylation in three common sites as representative. The ser220 site is one of the most important sites for GR activation and its downstream functions. However, GR has lots of modifiable sites, and GR activation is a complex progress 42. Much more work need to be carried out to discover its beyond mechanism. Contribute to the survival of neuron and synapse, BDNF is often correlated with memory, stress, emotion, and dementia 28, 29. A large number of researches demonstrate that GR has close relationship to BDNF 39, 42, 43. Dexamethasone (DEX), a potent GR agonist could inhibit the expression of BDNF in vivo and in vitro 44, 45. A recent study found BDNF decreased in POCD animal models 30. This result suggested that BDNF was involved in POCD. Another study found that BDNF level was not changed in peripheral samples of patients with POCD 46. However, in parenchyma, the level of BDNF is found decreased after surgery 30. Much more work is to be carried out to discover the exactly mechanism. In our study, the level of BDNF was reduced in POCD group and was rescued by GR phosphorylation inhibition. These data probably suggested that surgical stress‐induced GR activation could impaired the postoperative cognitive function via BDNF. Recent studies reported that formaldehyde was related to POCD and phosphorylation 47, 48. Formaldehyde elevation is one of the changes after surgical stress. Another study reported that formaldehyde is related to CDK5, which participated in GR phosphorylation 49. This does not contradict with our finding. GR activation probably is induced by several changes after surgical stress such as formaldehyde, cortisol, and inflammatory factors. The relationship between formaldehyde stress and GR activation is an interesting topic that needs to be studied in future.

Sustaining GR Phosphorylation in Elder but not in Younger Patients

The morbidity of POCD is closely related to the age of patients who had clinical surgery 3, 4. So, what is the difference between elder and younger patients in pathological process of POCD? In this work, we found elevated GR phosphorylation in both ages on early stage after surgery. However, sustaining GR phosphorylation in late period after surgery only existed in aged groups and was cortisol‐independent (Figure 6). Large numbers of clinical researches demonstrated that POCD could last several weeks or even months in elder patients 50. According to these results, we hypothesized that the ability of self‐regulation on HPA axis might be the difference between elder and younger individuals.

Surgical Stress‐Induced POCD in a Laparotomy Animal Model

Different models have been used in POCD studies, inhalation anesthesia and various kinds of surgical style included 23, 51, 52, 53, 54. In this research, we chose laparotomy to exclude the effects of surgical style and viscera injury and the general incidence of POCD was reported to have little difference between the types of surgeries 55. A few studies also attach great importance of the effects of surgical incision itself 23, 56. Moreover, the kinase of GR phosphorylation, CDK5, was reported increased in the model of isoflurane inhalation 19. Therefore, we used a general anesthesia with chloral hydrate intraperitoneal injection instead for excluding the effects of isoflurane.

Conclusion

Our data indicated that surgical stress‐induced BDNF reduction and cognitive dysfunction were mediated by GR phosphorylation in aged mice. These results suggested that surgical stress‐induced GR activation and sustaining phosphorylation might be a potential mechanism and treatment target of POCD.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Figure S1. We used a visible platform to detect the visual effect in Morris water maze. No significant difference was found with a one‐way ANOVA analysis (p > 0.05).

Click here for additional data file. (2.6MB, tif)

Figure S2. Western blot was used to measure the effect of cholral hydrate anesthesia. The level of pGRs220, cdk5 and BDNF were detected.

Click here for additional data file. (2.6MB, tif)

Figure S3. Western blot was used to detect the level of pGRs220 and BDNF among 4 groups(sham control and POCD mice on 20 and 6 months old).

Click here for additional data file. (2.6MB, tif)

Figure S4. Immunofluorescence was used to verify GR translocation and CDK5 expression.

Click here for additional data file. (2.6MB, tif)

Figure S5. (A)&(B), we used Elisa kit assays to detect the level of IL1β, IL6 and TNFα level in prefrontal cortex of 20 and 6 months mice on day 6 after surgery.

Click here for additional data file. (2.6MB, tif)

Figure S6. The swimming speed was recorded during Morris water maze.

Click here for additional data file. (2.6MB, tif)

 

Click here for additional data file. (12.2KB, docx)

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (973 Program No. 2012CB911004) and the National Natural Science Foundation of China (Grants No. 81171015 and No. 81371205).

References

  • 1. Orci LA, Toso C, Mentha G, Morel P, Majno PE. Systematic review and meta‐analysis of the effect of perioperative steroids on ischaemia‐reperfusion injury and surgical stress response in patients undergoing liver resection. Br J Surg 2013;100:600–609. [DOI] [PubMed] [Google Scholar]
  • 2. Monk TG, Price CC. Postoperative cognitive disorders. Curr Opin Crit Care 2011;17:376–381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Monk TG, Weldon BC, Garvan CW, et al. Predictors of cognitive dysfunction after major noncardiac surgery. Anesthesiology 2008;108:18–30. [DOI] [PubMed] [Google Scholar]
  • 4. Steinmetz J, Christensen KB, Lund T, Lohse N, Rasmussen LS. Long‐term consequences of postoperative cognitive dysfunction. Anesthesiology 2009;110:548–555. [DOI] [PubMed] [Google Scholar]
  • 5. Vanderweyde T, Bednar MM, Forman SA, Wolozin B. Iatrogenic risk factors for Alzheimer's disease: Surgery and anesthesia. J Alzheimers Dis 2010;22(Suppl 3):91–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Hartholt KA, van der Cammen TJ, Klimek M. Postoperative cognitive dysfunction in geriatric patients. Z Gerontol Geriatr 2012;45:411–416. [DOI] [PubMed] [Google Scholar]
  • 7. Perouansky M. Liaisons dangereuses? General anaesthetics and long‐term toxicity in the CNS. Eur J Anaesthesiol 2007;24:107–115. [DOI] [PubMed] [Google Scholar]
  • 8. Perouansky M. General anesthetics and long‐term neurotoxicity. Handb Exp Pharmacol 2008;182:143–157. [DOI] [PubMed] [Google Scholar]
  • 9. He HJ, Wang Y, Le Y, et al. Surgery upregulates high mobility group box‐1 and disrupts the blood‐brain barrier causing cognitive dysfunction in aged rats. CNS Neurosci Ther 2012;18:994–1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Mizoguchi K, Kunishita T, Chui DH, Tabira T. Stress induces neuronal death in the hippocampus of castrated rats. Neurosci Lett 1992;138:157–160. [DOI] [PubMed] [Google Scholar]
  • 11. Butts KA, Weinberg J, Young AH, Phillips AG. Glucocorticoid receptors in the prefrontal cortex regulate stress‐evoked dopamine efflux and aspects of executive function. Proc Natl Acad Sci U S A 2011;108:18459–18464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Brureau A, Zussy C, Delair B, et al. Deregulation of hypothalamic‐pituitary‐adrenal axis functions in an Alzheimer's disease rat model. Neurobiol Aging 2013;34:1426–1439. [DOI] [PubMed] [Google Scholar]
  • 13. Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui DH, Tabira T. Chronic stress differentially regulates glucocorticoid negative feedback response in rats. Psychoneuroendocrinology 2001;26:443–459. [DOI] [PubMed] [Google Scholar]
  • 14. Espinosa‐Oliva AM, dePablos RM , Villaran RF, et al. Stress is critical for LPS‐induced activation of microglia and damage in the rat hippocampus. Neurobiol Aging 2011;32:85–102. [DOI] [PubMed] [Google Scholar]
  • 15. Graff J, Rei D, Guan JS, et al. An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature 2012;483:222–226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Ismaili N, Garabedian MJ. Modulation of glucocorticoid receptor function via phosphorylation. Ann N Y Acad Sci 2004;1024:86–101. [DOI] [PubMed] [Google Scholar]
  • 17. Martinez‐Finley EJ, Goggin SL, Labrecque MT, Allan AM. Reduced expression of MAPK/ERK genes in perinatal arsenic‐exposed offspring induced by glucocorticoid receptor deficits. Neurotoxicol Teratol 2011;33:530–537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Ji MH, Shen JC, Gao R, et al. Early postoperative cognitive dysfunction is associated with higher cortisol levels in aged patients following hip fracture surgery. J Anesth 2013;27:942–944. [DOI] [PubMed] [Google Scholar]
  • 19. Rasmussen LS, O'Brien JT, Silverstein JH, et al. Is peri‐operative cortisol secretion related to postoperative cognitive dysfunction? Acta Anaesthesiol Scand 2005;49:1225–1231. [DOI] [PubMed] [Google Scholar]
  • 20. Wang WY, Luo Y, Jia LJ, et al. Inhibition of aberrant cyclin‐dependent kinase 5 activity attenuates isoflurane neurotoxicity in the developing brain. Neuropharmacology 2014;77:90–99. [DOI] [PubMed] [Google Scholar]
  • 21. Rosczyk HA, Sparkman NL, Johnson RW. Neuroinflammation and cognitive function in aged mice following minor surgery. Exp Gerontol 2008;43:840–846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Gourley SL, Swanson AM, Jacobs AM, et al. Action control is mediated by prefrontal BDNF and glucocorticoid receptor binding. Proc Natl Acad Sci USA 2012;109:20714–20719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Zhang X, Xin X, Dong Y, et al. Surgical incision‐induced nociception causes cognitive impairment and reduction in synaptic NMDA receptor 2B in mice. J Neurosci 2013;33:17737–17748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Li ZQ, Rong XY, Liu YJ, et al. Activation of the canonical nuclear factor‐kappaB pathway is involved in isoflurane‐induced hippocampal interleukin‐1beta elevation and the resultant cognitive deficits in aged rats. Biochem Biophys Res Commun 2013;438:628–634. [DOI] [PubMed] [Google Scholar]
  • 25. Lotfipour S, Byun JS, Leach P, et al. Targeted deletion of the mouse alpha2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine‐modulated behaviors. J Neurosci 2013;33:7728–7741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Yu Y, Zhou L, Sun M, et al. Xylocoside G reduces amyloid‐beta induced neurotoxicity by inhibiting NF‐kappaB signaling pathway in neuronal cells. J Alzheimers Dis 2012;30:263–275. [DOI] [PubMed] [Google Scholar]
  • 27. Fitz NF, Cronican A, Pham T, et al. Liver X receptor agonist treatment ameliorates amyloid pathology and memory deficits caused by high‐fat diet in APP23 mice. J Neurosci 2010;30:6862–6872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Acheson A, Conover JC, Fandl JP, et al. A BDNF autocrine loop in adult sensory neurons prevents cell death. Nature 1995;374:450–453. [DOI] [PubMed] [Google Scholar]
  • 29. Huang EJ, Reichardt LF. Neurotrophins: Roles in neuronal development and function. Annu Rev Neurosci 2001;24:677–736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Hovens IB, Schoemaker RG, van der Zee EA, Absalom AR, Heineman E, van Leeuwen BL. Postoperative cognitive dysfunction: Involvement of neuroinflammation and neuronal functioning. Brain Behav Immun 2014;38:202–210. [DOI] [PubMed] [Google Scholar]
  • 31. Kino T, Ichijo T, Amin ND, et al. Cyclin‐dependent kinase 5 differentially regulates the transcriptional activity of the glucocorticoid receptor through phosphorylation: Clinical implications for the nervous system response to glucocorticoids and stress. Mol Endocrinol 2007;21:1552–1568. [DOI] [PubMed] [Google Scholar]
  • 32. Fiala C, Gemzel‐Danielsson K. Review of medical abortion using mifepristone in combination with a prostaglandin analogue. Contraception 2006;74:66–86. [DOI] [PubMed] [Google Scholar]
  • 33. Finsterwald C, Alberini CM. Stress and glucocorticoid receptor‐dependent mechanisms in long‐term memory: From adaptive responses to psychopathologies. Neurobiol Learn Mem 2013;112:17–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Lim CS, Hwang YK, Kim D, et al. Increased interactions between PKA and NF‐kappaB signaling in the hippocampus following loss of cholinergic input. Neuroscience 2011;192:485–493. [DOI] [PubMed] [Google Scholar]
  • 35. Lim W, Park C, Shim MK, Lee YH, Lee YM, Lee Y. Glucocorticoids suppress hypoxia‐induced COX‐2 and hypoxia inducible factor‐1alpha expression through the induction of glucocorticoid‐induced leucine zipper. Br J Pharmacol 2014;171:735–745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF‐kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA 1994;91:752–756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Li Z, Cao Y, Li L, et al. Prophylactic angiotensin type 1 receptor antagonism confers neuroprotection in an aged rat model of postoperative cognitive dysfunction. Biochem Biophys Res Commun 2014;449:74–80. [DOI] [PubMed] [Google Scholar]
  • 38. Felger JC, Lotrich FE. Inflammatory cytokines in depression: Neurobiological mechanisms and therapeutic implications. Neuroscience 2013;246:199–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Kapila AK, Watts HR, Wang T, Ma D. The impact of surgery and anesthesia on postoperative cognitive decline and Alzheimer's disease development: Biomarkers and preventive strategies. J Alzheimers Dis 2014;41:1–13. [DOI] [PubMed] [Google Scholar]
  • 40. Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui DH, Tabira T. Chronic stress induces impairment of spatial working memory because of prefrontal dopaminergic dysfunction. J Neurosci 2000;20:1568–1574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Lambert WM, Xu CF, Neubert TA, Chao MV, Garabedian MJ, Jeanneteau FD. Brain‐derived neurotrophic factor signaling rewrites the glucocorticoid transcriptome via glucocorticoid receptor phosphorylation. Mol Cell Biol 2013;33:3700–3714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Barik J, Marti F, Morel C, et al. Chronic stress triggers social aversion via glucocorticoid receptor in dopaminoceptive neurons. Science 2013;339:332–335. [DOI] [PubMed] [Google Scholar]
  • 43. Revest JM, Le Roux A, Roullot‐Lacarriere V, et al. BDNF‐TrkB signaling through Erk1/2 phosphorylation mediates the enhancement of fear memory induced by glucocorticoids. Mol Psychiatry 2013;19:1001–1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Brossaud J, Roumes H, Moisan MP, Pallet V, Redonnet A, Corcuff JB. Retinoids and glucocorticoids target common genes in hippocampal HT22 cells. J Neurochem 2013;125:518–531. [DOI] [PubMed] [Google Scholar]
  • 45. Tongjaroenbuangam W, Ruksee N, Mahanam T, Govitrapong P. Melatonin attenuates dexamethasone‐induced spatial memory impairment and dexamethasone‐induced reduction of synaptic protein expressions in the mouse brain. Neurochem Int 2013;63:482–491. [DOI] [PubMed] [Google Scholar]
  • 46. Ji MH, Yuan HM, Zhang GF, et al. Changes in plasma and cerebrospinal fluid biomarkers in aged patients with early postoperative cognitive dysfunction following total hip‐replacement surgery. J Anesth 2013;27:236–242. [DOI] [PubMed] [Google Scholar]
  • 47. Wang J, Su T, Liu Y, Yue Y, He R. Postoperative cognitive dysfunction is correlated with urine formaldehyde in elderly noncardiac surgical patients. Neurochem Res 2012;37:2125–2134. [DOI] [PubMed] [Google Scholar]
  • 48. Lu J, Miao Y, Su T, Liu Y, He R. Formaldehyde induces hyperphosphorylation and aggregation of Tau protein both in vitro and in vivo. Biochim Biophys Acta 2013;1830:4102–4116. [DOI] [PubMed] [Google Scholar]
  • 49. Wang C, Chou W, Hung K, et al. Intrathecal administration of roscovitine inhibits Cdk5 activity and attenuates formalin‐induced nociceptive response in rats. Acta Pharmacol Sin 2005;26:46–50. [DOI] [PubMed] [Google Scholar]
  • 50. Newman S, Stygall J, Hirani S, Shaefi S, Maze M. Postoperative cognitive dysfunction after noncardiac surgery: A systematic review. Anesthesiology 2007;106:572–590. [DOI] [PubMed] [Google Scholar]
  • 51. Degos V, Vacas S, Han Z, et al. Depletion of bone marrow‐derived macrophages perturbs the innate immune response to surgery and reduces postoperative memory dysfunction. Anesthesiology 2013;118:527–536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Li RL, Zhang ZZ, Peng M, et al. Postoperative impairment of cognitive function in old mice: A possible role for neuroinflammation mediated by HMGB1, S100B, and RAGE. J Surg Res 2013;185:815–824. [DOI] [PubMed] [Google Scholar]
  • 53. Tang JX, Mardini F, Janik LS, et al. Modulation of murine Alzheimer pathogenesis and behavior by surgery. Ann Surg 2013;257:439–448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Vacas S, Degos V, Tracey KJ, Maze M. High‐mobility group box 1 protein initiates postoperative cognitive decline by engaging bone marrow‐derived macrophages. Anesthesiology 2014;120:1160–1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55. Xu T, Bo L, Wang J, et al. Risk factors for early postoperative cognitive dysfunction after non‐coronary bypass surgery in Chinese population. J Cardiothorac Surg. 2013;8:204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56. Chi H, Kawano T, Tamura T, et al. Postoperative pain impairs subsequent performance on a spatial memory task via effects on N‐methyl‐D‐aspartate receptor in aged rats. Life Sci 2013;93:986–993. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. We used a visible platform to detect the visual effect in Morris water maze. No significant difference was found with a one‐way ANOVA analysis (p > 0.05).

Click here for additional data file. (2.6MB, tif)

Figure S2. Western blot was used to measure the effect of cholral hydrate anesthesia. The level of pGRs220, cdk5 and BDNF were detected.

Click here for additional data file. (2.6MB, tif)

Figure S3. Western blot was used to detect the level of pGRs220 and BDNF among 4 groups(sham control and POCD mice on 20 and 6 months old).

Click here for additional data file. (2.6MB, tif)

Figure S4. Immunofluorescence was used to verify GR translocation and CDK5 expression.

Click here for additional data file. (2.6MB, tif)

Figure S5. (A)&(B), we used Elisa kit assays to detect the level of IL1β, IL6 and TNFα level in prefrontal cortex of 20 and 6 months mice on day 6 after surgery.

Click here for additional data file. (2.6MB, tif)

Figure S6. The swimming speed was recorded during Morris water maze.

Click here for additional data file. (2.6MB, tif)

 

Click here for additional data file. (12.2KB, docx)

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