Galantamine reversed early postoperative cognitive deficit via alleviating inflammation and enhancing synaptic transmission in mouse hippocampus

European Journal of Pharmacology 846 (2019) 63–72

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Galantamine reversed early postoperative cognitive deficit via alleviating inflammation and enhancing synaptic transmission in mouse hippocampus

T

Tianhai Wanga,1, Hongge Zhub,1, Yanshen Houa, Weixin Guc, Haichuan Wuc, Yiwen Luanc, ⁎⁎ ⁎ Cheng Xiaoc, , Chunyi Zhouc, a

Department of Anesthesiology, The third hospital, affiliated to the Xinjiang Medical University, Urumqi, Xinjiang, China Department of Second Pulmonary Medicine, The third hospital, affiliated to the Xinjiang Medical University, Urumqi, Xinjiang, China c Jiangsu Province Key laboratory in Anesthesiology, School of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Postoperative cognitive deficit Fear-conditioning Contextual memory AMPA receptors Galantamine Stabilized tibial fracture

Postoperative cognitive dysfunction (POCD) is commonly seen in patients undergoing major surgeries and may persist. Although neuroinflammation is one of the important contributors to the development of POCD, the mechanisms underlying POCD remain unclear. We performed stabilized tibial fracture operation in male mice. In comparison with sham mice (anesthesia only), the surgery mice exhibited cognitive deficits in a fear conditioning paradigm at postsurgery day 3-7, and increased numbers of microglia and elevated levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) without change of anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus. Electrophysiological recordings from CA1 hippocampal neurons revealed that POCD mice exhibited impairment in AMPA receptor-mediated evoked excitatory postsynaptic currents (eEPSCs) without alteration in the rectification property of AMPA receptors. Interestingly, daily intraperitoneal administration of galantamine, an inhibitor of acetylcholinesterase, reversed cognitive dysfunction in surgery mice and attenuated accumulation of microglia and protein levels of IL-1β, IL-6 and TNF-α in the hippocampus. Additionally, galantamine potentiated AMPA receptor-mediated eEPSCs in the hippocampus more prominent in surgery mice than in sham mice. Therefore, enhancement of cholinergic tone in the hippocampus might be a therapeutic strategy for early POCD in terms of suppression of inflammation and normalization of excitatory synaptic transmission.

1. Introduction Postoperative cognitive dysfunction (POCD) is one of the public health problems in patients undergoing major surgeries. Cognitive deficits often develop between several days and one year after operation (Brown and Deiner, 2016) in up to 60% of patients subjected to cardiac surgery and 10% of patients received non-cardiac major surgery (Steinmetz and Rasmussen, 2016). Among the surgery patients, either infant and child or senior (> 60 years old) ones have higher risks of POCD (Brown and Deiner, 2016; Evered et al., 2017; Terrando et al., 2011a). However, the exact mechanisms underlying POCD remain unclear. Numerous studies demonstrate that inflammation may mediate POCD following combinatory treatment of surgery and anesthetics

(Riedel et al., 2014). In surgery mice, folds of increase in proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, were found in both plasma (Eckenhoff and Planel, 2012) and the central nervous system (H. Tan et al., 2014; Zhu et al., 2016). But no significant inflammation is reported in rodents undergoing anesthesia alone (Cibelli et al., 2010). Moreover, TNF-α antibodies (Terrando et al., 2010), dexmedetomidine (Xiong et al., 2016; Zhu et al., 2016) and thalidomide (Guo and Hu, 2017) normalize the levels of inflammatory cytokines and prevent POCD. However, it is still unclear how the inflammation following surgery modifies synaptic transmission that confer cognitive dysfunction. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl-D-aspartate (NMDA) receptors are major ionotropic glutamate receptors and mediate fast excitatory synaptic

⁎ Correspondence to: Jiangsu Province Key laboratory in Anesthesiology, School of Anesthesiology, Xuzhou Medical University, 209 Tongshan Road, KJL-D423, Xuzhou, Jiangsu, China. ⁎⁎ Correspondence to: Jiangsu Province Key laboratory in Anesthesiology, Xuzhou Medical University, 209 Tongshan Road, KJL-D425, Xuzhou, Jiangsu, China. E-mail addresses: [email protected] (C. Xiao), [email protected] (C. Zhou). 1 These authors contributed equally in this study.

https://doi.org/10.1016/j.ejphar.2018.12.034 Received 12 August 2018; Received in revised form 20 December 2018; Accepted 20 December 2018 Available online 23 December 2018 0014-2999/ © 2018 Published by Elsevier B.V.

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once again. Two test sessions were performed at post operation day (POD) 3 and 7 and sham treatment. The mice were placed into the same fear-conditioning chamber (the contextual cue) and allowed to habituate for 2 min without any tone or shock. Before each training and test session, mice were brought to the experiment room and allowed to habituate for 60 min. The chamber was wiped with 70% alcohol between animals. Movement of the mice was monitored by an attached tracking system. Video Freeze software was used to calculate freezing time as a fraction of total time in the chamber. The percentage of freezing time spent in the presence of the contextual cue (testing chamber) is the index of the contextual memory.

transmission in the central nervous system. In acute hippocampal slices, Lipopolysaccharide (LPS) induced inflammation enhances excitatory postsynaptic currents (EPSCs) through presynaptic mechanisms (Pascual et al., 2012), while in striatal slices, LPS potentiates postsynaptic AMPA receptors on D2 receptor-containing medium spiny neurons (Winland et al., 2017). The enhancement of AMPA and NMDA receptor function in rat hippocampus is also observed 4 days after the induction of colonic inflammation (Riazi et al., 2015). In Tau transgenic mice, microglia activation appears before the formation of neurofilament tangle, and is accompanied by attenuation of excitatory synaptic transmission in the hippocampus (Yoshiyama et al., 2007). In summary, acute, intermediate, and chronic neuroinflammation potentiates excitatory synaptic transmission. As the inflammation following surgery may not be the same as that in above-mentioned studies, the postoperative alteration of excitatory synaptic transmission could differ, but needs further investigations. In the present study, we employed a POCD mouse model with unilateral stabilized tibial fracture operation and found that the surgery induced neuroinflammation in the hippocampus and impairment of AMPARs. Consistent with previous studies showing that cholinergic system is implicated in synaptic plasticity and learning and memory (Al-Onaizi et al., 2017) and counteracts the detrimental effects of LPS on neuroinflammation and spatial memory in mice (Liu et al., 2018), we found that enhancing cholinergic system with galantamine, an inhibitor of acetylcholinesterase and potentiator of nicotinic acetylcholine receptors, could reverse these pathophysiological alterations and improve cognitive function in the mice. Therefore, enhancement of cholinergic system might be a therapeutic strategy for the treatment of early POCD.

2.4. Motor behaviors

2. Materials and methods

The open field test was used to measure general locomotion 3 and 7 days after surgery and sham treatment. The open field apparatus is a Plexiglas round cylinder with a diameter of 30 cm and a height of 50 cm. Before tests, the mice at POD 3 and POD 7 were first acclimated to the experimental test room for at least 60 min. They then were allowed to move freely in the cylinder for 25 min, while their movement was acquired with an animal behavioral tracking system (Noldus Ethovision 11.5 software, Noldus Information Technology, Netherland) through an infrared camera. The percentage of time in mobility and moving velocity during the last 20 min were analyzed. The apparatus was cleaned with 70% ethanol between two mice. The Pole climbing test involves a finely textured rod that is 1 cm thick and 60 cm long. The rod is vertically fixed on a metal platform, which is placed in a mouse cage and covered with beddings. On the top of the rod, a hemisphere with a diameter of 1.5 cm is attached for a mouse to stand. The time that the mouse spent climbing from the top of the rod down into the cage is recorded.

2.1. Animals

2.5. ELISA assay of pro-inflammatory and anti-inflammatory cytokines

Male C57/BL6 mice, aged 10–12 months, were housed in a standard 12 h light / dark cycle with free access to food and water. All animal care and experimental protocols comply with Guide for the care and use of laboratory animals (National Institute of Health, USA), and were approved by the Institution of Animal Care and Use Committee and the Office of Laboratory Animal Resources in the Xinjiang Medical University and the Xuzhou Medical University.

Pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) and anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus were measured using mouse ELISA kits (Invitrogen, Thermo Fisher Sci, Shanghai, China; MultiSciences Biotechnology, Hangzhou, China), according to instruction provided by manufacturer. Briefly, the mice at POD 3 were euthanized with CO2, and the hippocampus tissue was dissected and harvested from each group, then, was immediately frozen in liquid nitrogen and stored at −80 °C until use. Total protein from each sample was extracted using RIPA lysis and extraction buffer (Thermo Fisher Sci, Shanghai, China). Protein concentration was determined with BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). 100 μl (200 μg protein) sample / standard diluent was tested in duplication, and the absorbance of samples was measured with a plate reader at 450 nm minus 55 nm. Protein level was expressed as pg/ml of total proteins determined over a standard curve.

2.2. Surgery A unilateral stabilized tibial fracture operation with intramedullary pinning was performed as described previously (Terrando et al., 2011b, 2013). Mice were anesthetized with sevoflurane (4% for induction and 3% for maintenance). A longitudinal 0.5 cm incision was made below the left knee under a strict aseptic condition. Muscles were detached to expose the tibia and the periosteum was stripped. Osteotomy was performed and then a 0.38 mm thick 5 mm long stainless-steel pin was inserted into the intramedullary canal of the tibia. The wound was irrigated and sutured. The mice were allowed to recover on a heating pad until woke up before returning to the home cage. The surgery was performed 4–5 h after the training session of fear conditioning assay.

2.6. Immunohistochemistry The mice were euthanized with CO2 and subjected to cardiac perfusion with 7 ml PBS with heparin, and then with 35 ml 4% paraformaldehyde in PBS. The mouse brain was removed and sectioned into 50 µm slices with a vibratome (VT-1200S, Leica). The slices were mounted onto glass slides, dried at room temperature, and frozen at −20 °C. The frozen slices were thawed at room temperature (15 min), washed twice (10 min each) with cold PBS (4 °C), permeabilized for 1 h at room temperature in PBS / 0.1% triton X-100, blocked for 45 min in PBS / 10% donkey serum, incubated in primary antibodies (1:500, rabbit anti-Iba1- IgG, Wako, Japan) in PBS / 4% donkey serum at 4 °C overnight (18 h), washed 3 times (15 min each) in PBS, incubated in secondary antibody (1:500, Cy3-conjugated donkey anti-mouse IgG, Jackson Immunology Inc.) in PBS / 4% donkey serum at room

2.3. Contextual fear conditioning Fear conditioning is a useful behavioral paradigm for assessing learning and memory (Shi et al., 2016). Contextual fear-conditioning was conducted as reported previously (Johansen et al., 2011; Shi et al., 2016). The test included three sessions：one training session and two test sessions. In the training session, mice were first placed in the fearconditioning chamber and allowed to explore for 120 s; a tone (70 dB) was then presented for 20 s and a constant mild foot shock (0.8 mA) was given at the last 2 s; 120 s later, the tone and foot shock were presented 64

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the stimulation- and voltage-dependence of eEPSCs, five consecutive eEPSCs were averaged to represent the eEPSCs recorded in each condition. To examine galantamine effect on eEPSCs, eEPSCs were recorded for at least 5 min to establish a stable baseline, and the effect of bath-applied galantamine on eEPSCs was quantified with % increase of eEPSCs from baseline. SigmaPlot 11.0 (SPSS Inc.) was used to plot graphs and to perform statistical analysis. Data were presented as mean ± S.E.M. For comparison between two groups of data, student's two-tailed t-test was used, while comparison among multiple groups, one-way ANOVA with pairwise multiple comparison tests (Holm-Sidak method) and two-way ANOVA repeated measures were applied. P < 0.05 is considered statistically significant.

temperature for 1 h. Samples were washed 3 times (10 min each) in PBS, dried at room temperature, immersed in mounting medium (Vector laboratories Inc.), and then cover-slipped. We imaged the slices with a confocal microscope (Fv-1000, Olympus), equipped with a 10 × Plan Apo objective (numerical aperture: 0.45) and a 20 × Plan Apo objective (numerical aperture: 0.75). Cy3 was excited with a 561 nm laser and the emission light was detected between 580 and 640 nm. Iba1-positive cells were counted with Image J (Schneider et al., 2012). 2.7. Electrophysiological recordings Patch-clamp recordings were performed on brain slices, which were prepared on POD 3 from sham or surgery mice, using the protocol described with minor modifications (Xiao et al., 2016, 2006, 2015). In brief, the mice were deeply euthanized with CO2, and then were decapitated. The brain was removed and 300 µm thick coronal slices were prepared with a vibratome (VT-1200S, Leica), while immersed in icecold modified sucrose-based artificial cerebral spinal fluid (sACSF) saturated with 95% O2 / 5% CO2 (carbogen) containing (mM): 85 NaCl, 75 sucrose, 2.5 KCl, 1.25 NaH2PO4, 4.0 MgCl2, 0.5 CaCl2, 24 NaHCO3 and 25 glucose (Cho et al., 2017). Hippocampal slices were allowed to recover at 32 ± 1 °C in a holding chamber, filled with carbogenated sACSF. One hour later, the slices were transferred into normal ACSF, containing (mM): 125 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 26 NaHCO3, and 11 glucose, and were kept at room temperature. One of the slices was transferred into the recording chamber and superfused (1.5–2.0 ml/min) with carbogenated ACSF at 32 ± 0.5 °C. Three to four slices per mouse were used for recordings in each day. The neurons in brain slices were visualized with an upright microscope (FN-1, Nikon) equipped with a CMOS CCD-camera (Flash 4.0 LTE, Hamamatsu) and near-infrared illumination. Whole-cell patchclamp techniques were used to record electrophysiological signals from hippocampal neurons in CA1 region with MultiClamp 700B amplifiers (Molecular Devices, CA), Digidata 1550B analog-to-digital converters (Molecular Devices), and pClamp 10.7 software (Molecular Devices). A patch electrode had a resistance of 4–6 MΩ when filled with intrapipette solution (in mM): 135 potassium gluconate, 5 KCl, 5 EGTA, 0.5 CaCl2, 10 HEPES, 2 Mg-ATP, and 0.1 GTP. The pH of these solutions was adjusted to 7.2 with Tris-base, and the osmolarity was adjusted to 300 mOsm with sucrose. The junction potential between patch pipette and bath solutions was nulled just before gigaseal formation. Series resistance was monitored without compensation throughout the experiment using Multiclamp 700B. The data were discarded if the series resistance (15–30 MΩ) changed by more than 20% during whole-cell recordings. Data were sampled at 10 kHz and filtered at 2 kHz. Postsynaptic currents were evoked by electrical stimuli (100 μs, 0.05 Hz, 0.1–0.2 mA) through a glass pipette placed at Schaffer Collaterals 400–500 µm away from the recording pipette.

3. Results 3.1. Contextual memory was impaired in surgery mice To understand the mechanisms underlying postoperative cognitive dysfunction (POCD), we adopted a bone fracture mouse model. In this set of experiment, we performed fear conditioning in mice 4–5 h before surgery (Fig. 1A) so that we can examine whether surgery could damage the retrieval of contextual memory. Before the training session, we tested baseline levels of fear behavior (such as freezing) in the apparatus and observed no significant difference in freezing time between sham and surgery mice. Both 3 and 7 days after training, sham and surgery mice showed increased freezing time in the context, in comparison with baseline levels (Fig. 1B, C, D). As expected, 3 and 7 days after training, the freezing time in surgery mice was significantly less than that in sham mice, suggesting an impairment in contextual memory. These results indicate that we successfully established POCD mouse models with stabilized tibia fracture operation. 3.2. Microglia were activated in the hippocampus of surgery mice Inflammation in the hippocampus, including accumulation of microglia, is a common feature of pathophysiology in POCD (Riedel et al., 2014; H. Tan et al., 2014; Zhu et al., 2016). We performed immunohistochemistry in the hippocampus of both sham and surgery mice (3 days after sham and surgery treatment) to label microglia using Iba-1 antibody staining (Fig. 2A, B). We observed that the numbers of microglia were dramatically increased (Fig. 2B) by about 2-fold in CA1 (Fig. 2C), CA3 and DG (Fig. 2D) without regional specificity. The activation of microglia in the hippocampus of surgery mice was further supported by the elevation of pro-inflammatory cytokines in the hippocampus (Fig. 2E, F, G), including IL-1β, TNF-α and IL-6. In our observation, the levels of these cytokines in surgery mice were about twofold of those in sham mice. The protein levels of anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus were not changed by surgery (Fig. 2H, I). These results suggest that POCD mice exhibited neuroinflammation in the hippocampus.

2.8. Chemicals and applications 3.3. Excitatory synaptic transmission in the hippocampus was impaired in surgery mice

Tocris furnished most of the chemicals: 1(S), 9(R)-(−)-Bicuculline methiodide, 6-Cyano-7-nitro quinoxaline-2,3-dione disodium salt hydrate (CNQX), DL-2-Amino-5-phosphonovaleric acid lithium salt (AP-5) and galantamine. Stock solutions (> 1000x) were made, aliquoted and stored at −20 °C. The final dilutions were freshly made before experiments and the drugs were applied with bath-perfusion. For in vivo tests, galantamine was dissolved in 0.9% normal saline and injected intraperitoneally (i.p.) at dose of 4 mg/kg once per day.

It is demonstrated that excitatory synaptic transmission in the hippocampus is implicated in learning and memory (Isaac et al., 2007; Meng et al., 2003; Shepherd, 2012; Wiltgen et al., 2010), and is one of the target of cytokines (Riazi et al., 2015; Winland et al., 2017). To address whether neuroinflammation in the hippocampus of the POCD mice is strong enough to affect excitatory synaptic transmission, we recorded EPSCs from voltage-clamped CA1 pyramidal neurons (VH = −75 mV) by stimulating Schaffer-Collateral pathway in both sham and surgery mice 3 days after sham and surgery treatment. As illustrated in Fig. 3A, in our experimental condition, 100, 120, 140, 160 and 200 μA electrical stimuli evoked EPSCs with amplitudes

2.9. Data analysis Electrically evoked excitatory postsynaptic currents (eEPSCs) were analyzed with Clampfit 10.7 software (Molecular Devices). To illustrate 65

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Fig. 1. Surgery impaired contextual learning in mice. Sevoflurane-anesthetized mice were subjected to unilateral stabilized tibial fracture operation (surgery) and anesthesia only (sham) for the same period of time. (A) 4–5 h before the operation, both surgery and sham mice singly placed in an apparatus for fear-conditioning. After 2 min habituation, the mice received 18 s sound stimulation (70 dB), followed by 2 s sound + electric foot shock (0.8 mA). 3 and 7 days later, the mice were placed in the same apparatus for 2 min without any stimulation. Freezing time of mice in both sham and surgery groups was tested before surgery (t = 0.61, P = 0.55, two-tailed student's t-test) (B), and 3 days (F3,23 = 46.5, P ≤ 0.001, one-way ANOVA) (C) and 7 days (F3,23 = 60.4, P < 0.001, one-way ANOVA) (D) after surgery. The numbers in parentheses represent the numbers of mice tested. * *P < 0.01, compared with baseline of sham; ## P < 0.01, compared with sham on POD 3 or POD 7.

evoked-EPSCs recorded in CA1 pyramidal neurons in both sham and surgery mice (Fig. 3C). Our summarized data in Fig. 3D show that the paired-pulse ratios did not differ between sham and surgery mice, suggesting that the impairment in excitatory synaptic transmission in surgery mice may result from a postsynaptic mechanism. We observed that the EPSCs in CA1 pyramidal neurons, recorded at −75 mV, were blocked by 20 μM CNQX, indicating that the currents were mostly mediated by AMPA receptors. But at more depolarized

gradually increasing with stimulation intensity. The analysis of stimulation-response curve showed that the amplitudes of evoked EPSCs at every stimulation intensity were significantly smaller in surgery mice than in sham mice (Fig. 3B). It is known that the ratio of two EPSCs evoked by paired electrical stimuli (paired-pulse ratio) has an inverse correlation with the release probability of glutamate from presynaptic terminals (Xiao et al., 2006, 2009). To clarify whether this impairment involves presynaptic transmission, we measured paired-pulse ratio in

Fig. 2. Microglia in the hippocampus were activated in surgery mice. (A) Diagram of the hippocampus, modified from brain atlas. (B) Microglia were labelled with antibody of its marker protein, Iba-1 (red). Typical images showing that microglia are more abundant in CA1 region of the hippocampus in surgery mice than in sham mice. Microglia were counted in CA1 (C) (t = -3.6, P = 0.02) and CA3 (t = -6.18, P = 0.003) and DG (t = -8.6, P = 0.001) regions (D) in the hippocampus. Data of each group come from 4 counting areas (one from each mouse). Scale bars: 10 µm. The hippocampus from both sham and surgery mice was collected and IL-1β (E), TNF-α (F), IL-6 (G), IL-4 (H) and IL-10 (I) were measured with ELISA assay (IL-1β: t = -3.17, P = 0.01; TNF-α: t = -3.68, P = 0.006; IL-6: t = -2.69, P = 0.03; IL-4: t = -2.1, P = 0.08; IL-10: t = 0.35, P = 0.74). The numbers in parentheses represent the numbers of mice tested. * P < 0.05, * * P < 0.01, compared with sham group.

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Fig. 3. The excitatory synaptic transmission in the hippocampus was impaired in surgery mice. (A) Excitatory postsynaptic currents (eEPSCs) were recorded from voltage-clamped pyramidal neurons (VH = −80 mV) by placing a stimulating electrode in CA1 region proximate to CA3 region to stimulate Schaffer collateral pathway. The stimulation intensity was adjusted from 100 to 200 μA. The amplitude of eEPSCs increased with stimulating intensity, which is summarized in (B), n = 5 at each point, P = 0.023. (C) Two consecutive stimuli separated 100 ms apart were used to evoke paired-EPSCs in hippocampal neurons. (D) The ratio of paired-EPSCs evoked by paired stimuli with intervals ranging from 100 to 1200 ms were measured in hippocampal neurons from both sham (neurons: n = 8) and surgery (neurons: n = 11) mice, P = 0.45. (E) AMPA receptor mediated EPSCs were isolated by adding 50 μM APV and 20 μM bicuculline, and voltage-dependence of AMPAR-EPSCs was analyzed. The number of neurons is 6 in each group, P = 0.42. (F) The rectification index of AMPAR-EPSCs was calculated by dividing EPSC amplitude at −60 mV with that at + 40 mV (t = -0.71, P = 0.48). The numbers in parentheses represent the numbers of neurons recorded. * P < 0.05, * * P < 0.01, compared with sham. Student's ttest in (F), two-way ANOVA repeated measures in (B), (D) and (E).

holding potentials (higher than 0 mV), both GABAA receptors and NMDA receptors may become important components to mediate the outward currents. To isolate AMPA receptor-mediated EPSCs, we included 50 μM APV and 20 μM bicuculline to block NMDA and GABAA receptors, respectively. In this condition, we evoked EPSCs with 140 μA stimulation and varied holding potentials from −60 mV to + 40 mV (Fig. 3E). The ratio between the EPSCs at −60 mV to EPSCs at + 40 mV (rectification index) represents the current rectification property of AMPA receptors (Fig. 3F). We observed that the rectification indices were at the similar levels between sham and surgery mice. As the percentage increase of Ca2+ permeable-components (lacking GluA2 subunits) in AMPA receptors (Hell, 2016) enhances the rectification, our data suggest that the components of AMPA receptors may not be altered in these POCD mice. We next examined whether EPSCs mediated by NMDA receptors are altered in surgery mice. In this set of experiments, we firstly recorded AMPA receptor-mediated EPSCs at −70 mV (Fig. 4A, lower traces showing inward currents). We then switched the hold potential to + 40 mV and bath applied 20 μM CNQX and 20 μM bicuculline to block the outward currents mediated by AMPA receptors and GABAA receptors. In this condition, the NMDA receptor-mediated currents were isolated (Fig. 4A, upper traces showing outward currents). The results showed that AMPA receptor-mediated EPSCs (Fig. 4B) but not NMDA receptor-mediated EPSCs (Fig. 4C) were reduced in surgery mice, in comparison with those in sham mice. We further calculated the ratio of AMPA receptor currents at −70 mV to NMDA receptor currents at + 40 mV and found that the ratio was significantly smaller in surgery mice than in sham mice (Fig. 4D). The data suggest that the detrimental effect of surgery on AMPARs may be stronger than that on NMDA receptors. Taken together, the results suggest that excitatory synaptic transmission, especially the AMPA receptor-mediated component, was impaired in surgery mice.

Fig. 4. AMPA receptor function in the hippocampus was impaired in surgery mice. Electrical stimulation-evoked EPSCs were recorded in CA1 pyramidal neurons in the presence of 20 μM bicuculline. The AMPA receptor mediated EPSCs were recorded at −80 mV, while NMDA receptor mediated EPSCs were recorded at + 40 mV in the presence of both 20 μM bicuculline and 20 μM CNQX. (A) Typical traces from sham (left) and surgery (right) mice. The amplitude of AMPA receptor EPSCs, NMDA receptor EPSCs, and their ratio in individual neurons were summarized in (B) (t = 2.4, P = 0.02), (C) (t = 1.5, P = 0.15), and (D) (t = 2.4, P = 0.02). The numbers in parentheses represent the numbers of neurons recorded. * P < 0.05, compared with sham.

Therefore, we hypothesized that galantamine may improve memory deficits in surgery mice. To test this hypothesis, we divided sham and surgery mice into two subgroups, respectively received intraperitoneal injection of galantamine (4 mg/kg) and the same volume of saline once daily after sham and surgery treatment. We performed fear conditioning in the mice with the same paradigm shown in Fig. 1A. Before

3.4. Galantamine partially restored deficits in contextual memory of surgery mice Cholinergic drugs are commonly used as memory enhancers, and galantamine is a representative one among them (C.C. Tan et al., 2014). 67

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Fig. 5. Galantamine partially restored contextual memory deficit in surgery mice at POD 3. (A) After receiving either saline or galantamine (Gal) treatment, sham and surgery mice were subjected to fear conditioning tests. (B) Before training, the freezing time of mice in all groups observed in apparatus was at the same level (F3,23 = 0.11, P = 0.95). After training, the freezing time was counted and analyzed 3 days (C) (F3,23 = 19.7, P < 0.001; P < 0.001, surgery + saline vs sham + saline; P = 0.016, surgery + saline vs surgery + gal) and 7 days (D) (F2,20 = 8.54, P = 0.001; P < 0.001, surgery + saline vs sham + saline; P = 0.02, surgery + saline vs surgery + Gal) after surgery. Percentage of time in mobility was analyzed in the open field test on POD 3 (E) (F3,25 = 0.73, P = 0.55) and POD 7 (F) (F3,19 = 0.96, P = 0.44). The moving velocity was analyzed in the open field test on POD 3 (G) (F3,25 = 0.35, P = 0.79) and POD 7 (H) (F3,19 = 0.12, P = 0.95). The total time (latency) that mice spent climbing down the pole was recorded on POD 3 (I) (F3,27 = 1.56, P = 0.23) and POD 7 (J) (F3,19 = 0.71, P = 0.56). The numbers in parentheses represent the numbers of mice tested. * P < 0.05, * * P < 0.01, compared with sham; # P < 0.05, ## P < 0.01, compared with surgery + saline.

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4. Discussion

surgery and sham treatment, we tested the freezing time of the mice in the apparatus (Fig. 5A) and confirmed that the mice in different groups were similarly active (Fig. 5B). As illustrated in Fig. 5C and D, 3 and 7 days’ treatment of galantamine improved contextual memory in surgery mice, while left the memory intact in sham mice. The data suggest that galantamine as a memory enhancer for presenile dementia may also have therapeutic potential for early POCD. To exclude the possibility that freezing behaviors observed in fear conditioning test may be due to impairment of motor activity by tibial fracture, we further measured locomotor activities and coordination in sham and surgery mice subjected to galantamine or saline treatment with open field and pole climbing tests, respectively. The total mobility time and average velocity in the open field test did not differ among all experimental groups at POD 3 and POD 7 (Fig. 5E, F, G and H). In the pole test, the latency for the mice to climb down the pole was similar between sham and surgery mice (Fig. 5I, J). Additionally, 3 or 7 days’ treatment of galantamine did not affect their performance in both open field arena and pole tests (Fig. 5E, F, G, H, I and J). These results indicate that surgery or galantamine treatment affected neither locomotion nor coordination in mice.

In this study, we established POCD mouse models with unilateral stabilized tibial fracture surgery under sevoflurane (3%) anesthesia. Consistent with previous studies (Cibelli et al., 2010; Terrando et al., 2011b; Vacas et al., 2017; Xin et al., 2017), we observed contextual memory deficit in surgery mice. In our experiment, we used mature adult mice and sevoflurane anesthesia to eliminate the confounding factors that confer vulnerability of a subject to POCD, such as, young or elderly ages (Brown and Deiner, 2016; Evered et al., 2017; JevtovicTodorovic, 2011; Jevtovic-Todorovic et al., 2003; Terrando et al., 2011a) and non-volatile general anesthetics (Royse et al., 2011; Schoen et al., 2011). We kept anesthesia depth similar between sham and surgery mice by applying the same duration of anesthesia with the same concentration of sevoflurane. Therefore, our study supports that surgery may result in the pathophysiological outcomes we observed even when the aforementioned vulnerability does not exist. Fear-conditioning was commonly used to test contextual memory for POCD (Shi et al., 2016; Vacas et al., 2017; Xin et al., 2017). Performing surgery before and after fear-conditioning respectively represents the effect of surgery on the formation and retrieval of contextual memory. To test the effect of surgery on the memory formation requires fear-conditioning after surgery. However, after the surgery, the tibial becomes fragile and is vulnerable to fracture when the mouse jumps upon foot shock. Thus, the fracture may lead to inability to move and confound the freezing behavior. Therefore, we did not examine the effect of surgery on contextual memory formation. Instead, we started fear-conditioning 4–5 h before surgery. After 3 and 7 days recovery from surgery, we analyzed freezing behavior of the mice in the conditioned chamber, but without any stimulation. At these time periods, the pain symptoms following the surgery might have minor effects on freezing behavior in mice. We observed that surgery mice showed a shortened freezing time in the chamber (Fig. 1), indicating that the mice may somehow forget the distress caused by foot shock. Therefore, surgery impaired contextual memory and the deficit can last for at least 7 days. Rodent models suggest that surgery trauma leads to infiltration of blood borne immune cells such as macrophages into the brain through transiently permeable blood brain barrier (BBB) (Degos et al., 2013). The close crosstalk between infiltrated immune cells with microglia may leads to neuroinflammation in the hippocampus. The other mechanism underling neuroinflammation may include that peripheral cytokines cross the BBB and stimulate microglia in the hippocampus (Terrando et al., 2011b). Here, we examined microglia activation and also provided insight in which key cytokines were changed. Accompanied with memory deficit observed 3 days after surgery, we found neuroinflammation in the hippocampus. On the one hand, microglia accumulated to similar extent in CA1, CA3 and DG regions of the hippocampus (Fig. 2A-D). On the other hand, our ELISA data showed that surgery elevated IL-1β, TNF-α and IL-6 without changing anti-inflammatory cytokines (IL-4 and IL-10) in the hippocampus. These results are consistent with previous studies, showing that stabilized tibial fracture operation increases the levels of proinflammatory cytokines in the hippocampus (Vacas et al., 2017; Zhu et al., 2016). The neuroinflammation in the hippocampus following surgery might be an important contributor to POCD because 1) IL-1β has been reported to maintain synaptic plasticity in the hippocampus, while TNF-α and IL-6 negatively regulate synaptic plasticity in the hippocampus (Arisi, 2014); 2) blocking or knocking out IL-1β receptors or neutralizing TNFα with antibody mitigates neuroinflammation in the hippocampus and memory deficit in surgical rodents (Cibelli et al., 2010; Terrando et al., 2010); 3) anti-inflammatory drugs improve memory deficit in surgery rodents (Guo and Hu, 2017; Xiong et al., 2016; Zhu et al., 2016). We propose that targeting pro-inflammatory cytokines could be an appropriate strategy to alleviate POCD. Glutamatergic synaptic transmission is an essential component that

3.5. Galantamine attenuated microglia activation in the hippocampus of surgery mice As we observed massive activation of microglia in the hippocampus of surgery mice, we tested whether galantamine is capable of alleviating the inflammatory reaction. After surgery and sham treatment, we intraperitoneally administered galantamine and saline once daily for 3 days in separate groups of mice. We found that galantamine administration dramatically attenuated the numbers of microglia in the hippocampus of surgery mice (Fig. 6A, lower panels), in contrast to non-effect in sham mice (Fig. 6A, upper panels). In surgery mice, microglia accumulated in CA1 region 150% more than those in sham mice, while after galantamine treatment, the numbers of microglia in CA1 region increased by 50% in surgery mice, in comparison with that in sham mice (Fig. 6B). Accordingly, we examined the hippocampal protein levels of proinflammatory (IL-1β, TNFα and IL-6) and anti-inflammatory cytokines (IL-4 and IL-10) of sham and surgery mice subjected to saline or galantamine treatment. Galantamine treatment significantly attenuated the production of all tested anti-inflammatory cytokines in the hippocampus (Fig. 6C, D and E), while did not change anti-inflammatory IL-4 and IL-10 in surgery mice (Fig. 6F, G). The results support that galantamine attenuated microglia activation and inflammation in the hippocampus of surgery mice.

3.6. Galantamine improved excitatory synaptic transmission in the hippocampus of surgery mice Our preceding electrophysiological data indicate that AMPA receptor-mediated component in the EPSCs was impaired in surgery mice (Figs. 3 and 4), showing neuroinflammation in the hippocampus and cognitive deficit. We were wondering whether this impairment can be rescued by galantamine. To achieve this goal, we recorded EPSCs from hippocampal CA1 pyramidal neurons at a holding potential of −70 mV when stimulated Schafer collaterals (0.1 ms, 140 μA). After the recordings began, we collected 6 min baseline EPSCs, then bath-applied 10 μM galantamine for 5 min, and washed out galantamine for more than 10 min. After initiation of galantamine application, the EPSCs were gradually increased, and reached peaks within 5 min (Fig. 7A, B). Interestingly, the enhancement of EPSCs by galantamine was significantly stronger in surgery mice than in sham mice (Fig. 7B, C). The data suggest that galantamine may restore the impaired AMPA receptor-mediated synaptic transmission in surgery mice. 69

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Fig. 6. Galantamine attenuated microglia activation in the hippocampus of Surgery mice. Mice in groups: sham, sham + Gal, surgery, and surgery + Gal were sacrificed 3 days after training and surgery. Microglia in the hippocampus were labelled with Iba-1 antibody staining (A). Microglia were counted in CA1 (B) region of the hippocampus (F3,11 = 22.7, P < 0.001; P < 0.001, surgery + saline vs sham + saline; P = 0.006, surgery + Gal vs surgery + saline). Each group contained data from 3 mice. Scale bar 50 µm. The hippocampal protein levels of IL-1β (C), TNFα (D), IL-6 (E), IL-4 (F) and IL-10 (G) were measured at POD 3. (IL-1β: F3,19 = 13.4, P < 0.001; P < 0.001, surgery + saline vs sham + saline, P = 0.005, surgery + Gal vs surgery + saline. TNFα: F3,19 = 28.4, P < 0.001; P < 0.001, surgery + saline vs sham + saline, P < 0.001, surgery + Gal vs surgery + saline. IL-6: F3,19 = 7.3, P = 0.003; P = 0.003, surgery + saline vs sham + saline, P = 0.02, surgery + Gal vs surgery + saline. IL-4: F3,19 = 1.3, P = 0.3. IL-10: n = 5, F3,19 = 2.02, P = 0.15). The numbers in parentheses represent the numbers of mice tested. * P < 0.05, * * P < 0.01, compared with sham; # P < 0.05, ## P < 0.01, compared with surgery + saline.

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Fig. 7. Galantamine enhanced AMPAR-mediated EPSCs in surgery mice at POD3. (A) Typical traces showing that bath-application of galantamine (5 μM, right panels) enhanced AMPAR-mediated EPSCs in the hippocampal CA1 pyramidal neurons of both sham (upper panels) and surgery (lower panels) mice. Time course (B) and summary (C) of the effect of Gal on AMPAR-mediated EPSCs in the hippocampal CA1 pyramidal neurons of sham and surgery mice (t = -3.1, P = 0.01). The numbers in parentheses represent the numbers of neurons recorded. * P < 0.05, compared with sham + Gal.

innervation. As cholinergic system is an endogenous inflammation-resolving pathway, which involves α7 subunit-containing nicotinic ACh receptors (Terrando et al., 2011b; Zhu et al., 2016), enhancement of this system could reduce neuroinflammation. Indeed, in the present study, galantamine did alleviate the accumulation of microglia in the hippocampus (Fig. 6). Additionally, acute bath-application of galantamine restored surgery-impaired AMPAR-mediated EPSCs in CA1 pyramidal neurons in hippocampal slices (Fig. 7). The data are consistent with previous findings that ACh enhances neurotransmitter release in the hippocampus (Cheng and Yakel, 2015). We observed that the enhancement of AMPAR-mediated EPSCs by galantamine was significantly greater in hippocampal slices from surgery mice than in those from sham mice. There could be at least two interpretations for this phenomenon. First, surgery reduces cholinergic tone in modulating glutamate release, and in this situation, inhibition of acetylcholinesterase could lead to a greater percentage of enhancement in cholinergic tone. Second, galantamine may alleviate neuroinflammation and disinhibit glutamate release. Further investigations are needed to elucidate how ACh receptors in glutamatergic terminals and microglia contribute to the enhancement of AMPAR-mediated EPSCs by galantamine in surgery mice. In summary, early POCD may be related to the accumulation of microglia and preferential impairment of AMPARs vs NMDA receptors in the hippocampus. Galantamine, a cholinesterase inhibitor, reversed these pathophysiological alterations, and improved contextual memory in POCD mice. This study provides evidence that enhancement of cholinergic tone could be a therapeutic strategy for early POCD in terms of restoration of synaptic transmission and attenuation of neuroinflammation.

mediates synaptic plasticity in the hippocampus and hippocampus-dependent learning and memory. Numerous evidence shows that it is subjected to regulation by neuroinflammation (Riazi et al., 2015; H. Tan et al., 2014; Winland et al., 2017). In surgery mice, in addition to neuroinflammation in the hippocampus, we observed impaired excitatory synaptic transmission in CA1 pyramidal neurons (Figs. 3 and 4). Comparing the effect of surgery on AMPARs and NMDA receptors, we found that surgery exerted much stronger effect on AMPARs than on NMDA receptors, which is represented by the decreased ratio of AMPA receptor-mediated currents to NMDA receptor-mediated currents (Fig. 4D). We observed that AMPAR-mediated EPSCs evoked by electrical stimulation ranging from 0.1 to 0.2 mA were significantly smaller in surgical mice than in sham mice (Fig. 3B). The fact that surgery did not alter paired-pulse ratios with various inter-pulse intervals (0.1–1.2 s) (Fig. 3D) supports the involvement of a postsynaptic mechanism (such as, receptor trafficking and phosphorylation) in the impairment of AMPAR-mediated synaptic transmission by surgery. Our findings are consistent with a previous study showing that surgery increases the levels of IL-1β and IL-6 and inhibits trafficking and phosphorylation of GluA1-AMPARs, following persistent membrane depolarization (H. Tan et al., 2014). There are also studies showing enhanced AMPAR currents in CA1 stratum radiatum of the hippocampus in mice subjected to colonic inflammation (Riazi et al., 2015) and in the striatum exposed to lipopolysaccharide (Winland et al., 2017). Additionally, IL-1β, IL-6, TNF-α, and chemokine display negative or positive regulation of synaptic transmission in the hippocampus (Arisi, 2014). The discrepancy could come from the severity of inflammation, neuronal cell types, and localization of neurons. As GluA2-containing and -lacking AMPARs are respectively impermeable and permeable to Ca2+, and display no and an apparent inward rectification, correspondingly (Hell, 2016). These types of AMPARs play distinct roles in synaptic plasticity. We observed that surgery did not alter the rectification index of AMPARs in CA1 pyramidal neurons, indicating that no change occurs in the proportion of GluA2-lacking AMPARs. But we could not exclude the possibility that surgery could increase GluA2-lacking AMPARs in other neurons, such as, neurons in CA1 stratum radiatum of the hippocampus, as shown in another study (Riazi et al., 2015). Interestingly, we found that daily administration of galantamine significantly improved contextual memory deficit in surgical mice (Fig. 5). It was reported that surgery causes downregulation of choline acetyl transferase, the synthase of acetylcholine (ACh) (Zhang et al., 2018). Galantamine elevates extracellular levels of ACh by inhibiting hydrolysis of ACh, thereafter, counteracts deficiency of cholinergic

CRediT authorship contribution statement Tianhai Wang: Methodology, Writing - original draft, Writing review & editing, Investigation, Formal analysis. Hongge Zhu: Investigation, Formal analysis. Yanshen Hou: Investigation, Formal analysis. Weixin Gu: Investigation, Formal analysis. Haichuan Wu: Investigation, Formal analysis. Yiwen Luan: Investigation, Formal analysis. Cheng Xiao: Methodology, Writing - original draft, Writing - review & editing, Supervision. Chunyi Zhou: Methodology, Writing original draft, Writing - review & editing, Supervision. Acknowledgements This work was supported by a grant to Tianhai Wang from the Natural Science Foundation of Xinjiang, China (No. 2016D01C359) 71

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Conflict of interest

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