Neurokinin-1 receptor antagonism improves postoperative neurocognitive disorder in mice

Accepted Manuscript Title: Neurokinin-1 receptor antagonism improves postoperative neurocognitive disorder in mice Authors: Zhanjun Li, Ting Luo, Xinyu Ning, Chao Xiong, Anshi Wu PII: DOI: Reference:

S0304-3940(18)30661-X https://doi.org/10.1016/j.neulet.2018.09.057 NSL 33846

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

6-6-2018 1-9-2018 27-9-2018

Please cite this article as: Li Z, Luo T, Ning X, Xiong C, Wu A, Neurokinin-1 receptor antagonism improves postoperative neurocognitive disorder in mice, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.09.057 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neurokinin-1 receptor antagonism improves postoperative neurocognitive disorder in mice

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Zhanjun Li 1,2, Ting Luo 1, Xinyu Ning 2, Chao Xiong 1*, Anshi Wu 1*

Department of Anesthesiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing

100020, China

Department of Anesthesiology, General Hospital of Chinese People's Armed Police Forces, Beijing

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Corresponding

Chao Xiong, M.D

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*

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100039, China

Department of Anesthesiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing

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100020, China E-mail address: [email protected]

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Anshi Wu, M.D.

Department of Anesthesiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing

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100020, China E-mail address: [email protected]

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Highlights:

• Surgery induces neurokinin-1 receptor (NK-1R) activation in the hippocampus • NK-1R antagonist improves cognitive decline after surgery • NK-1R antagonist reduces surgery-induced neuroinflammation • NK-1R antagonist attenuates blood-brain barrier disruption after surgery

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Abstract

Postoperative neurocognitive disorder (PND) is a major complication in surgical patients, especially

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the elderly, leading to mild memory impairment after surgery. The underlying pathophysiology

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remains unknown, although neuroinflammation and blood-brain barrier (BBB) disruption have been

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increasingly implicated in PND. Emerging evidence suggests that neurokinin-1 receptor (NK-1R), the

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principal target of proinflammatory neuropeptide substance P (SP), plays a pivotal role in modulating neuroinflammation and BBB integrity. In this study, we used an established mouse

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model for PND to investigate the effects of a selective NK-1R antagonist L-733,060 on PND-like features after peripheral surgery. Hippocampal SP started to increase at 6 h, peaked at 1 day, and

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returned to baseline at 3 days after surgery. At 1 day after surgery, NK-1R expression was increased in the hippocampus. At this time point, NK-1R antagonist pretreatment attenuated microgliosis and

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prevented neutrophil infiltration after surgery. Similarly, proinflammatory cytokines interleukin-1 beta and interleukin-6 were reduced in the hippocampus in NK-1R antagonist-treated mice at 6 h

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after surgery. Furthermore, surgery-induced BBB disruption, assessed by albumin deposition and expression of tight junction protein claudin-5, was attenuated by NK-1R antagonism at postoperative day 1. Finally, trace fear conditioning test revealed NK-1R blockade antagonism reversed surgeryinduced cognitive impairment at 3 days after surgery. Our findings suggest that inhibition of NK-1R

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signaling protects hippocampus-dependent memory from surgical insult, probably through modulations of neuroinflammation and BBB integrity.

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Abbreviations:

PND: Postoperative neurocognitive disorder; BBB: blood-brain barrier; NK-1R: neurokinin-1 receptor;

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CNS: central nervous system; SP: substance P; NK-1Ra: neurokinin-1 receptor antagonist; PBS:

phosphate-buffered saline; ELISA: enzyme-linked immunosorbent assay; RT: room temperature; PFA: paraformaldehyde; IBA1: ionized calcium-binding adapter molecule 1; ICAM-1: intercellular

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adhesion molecule-1; MPO: myeloperoxidase; ANOVA: one-way analysis of variance

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Introduction

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Keywords: substance P; neurokinin-1; surgery; neuroinflammation; blood-brain barrier; cognition

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Postoperative neurocognitive disorder (PND) is a common complication after both cardiac and noncardiac major surgery, resulting in short-term or even long-term mild memory impairment [25]. PND

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is more frequently seen in aged population and associated with increased mortality and morbidity post-surgery [20]. However, the pathogenesis underlying PND is unknown and evidence most findings come from human animal studies is lacking due to the difficulty of directly studying human brain tissues in non-neurosurgical populations.

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Peripheral surgery induces rapid onset of systemic inflammation followed by hippocampal neuroinflammation in rodents [5, 28]. This immune response results in elevations of proinflammatory cytokines in the circulation and central nervous system (CNS) [13], migration of peripheral immune cells into the hippocampus through disrupted blood-brain barrier (BBB) [4, 13], and glia activation [33], all contributing to the subsequent memory deficits.

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Substance P (SP), a neuropeptide that is previously known to involve in pain perception and bowel motility, is now found to play a proinflammatory role in the development of many neurological

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disorders [6]. Neurokinin-1 receptor (NK-1R), the principal target of the SP, is expressed throughout the brain. Various cell types including microglia, astrocytes, neurons, endothelial cells, and

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circulating immune cells possess NK-1R [2, 10]. Recent studies show that activation of SP/NK-1R signaling contributes to activation of microglia as well as astrocytes, promoting proinflammatory

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cytokine production [2, 21]. This neuroinflammatory response could be ameliorated by genetic

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deletion or pharmacological inhibition of NK-1R in animal models of neurological disorders.[18, 32] Increased release of SP in the CNS has been shown to increase BBB permeability, leading to

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extravasation of plasma protein into the brain parenchyma [9, 31]. Furthermore, SP does not only alter BBB permeability but also involves in leukocyte infiltration into the brain [3]. NK-1R activation is implicated in facilitating neutrophil migration into the CNS via upregulation of adhesion molecules

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and chemokines [6, 15].

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In the present study, we aim to investigate the role of NK-1R signaling in PND, determining the effects of NK-1R blockade antagonism on PND-like features, including surgery-induced

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neuroinflammation, BBB disruption, and cognitive impairment.

Methods and materials Mice

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All experimental procedures were approved by Institutional Animal Care and Use Committee at Capital Medical University (Beijing, China). Adult young male C57BL/6 mice aged 12 to 14 weeks were used (Vital River Laboratory, Beijing, China). Mice were housed at the animal core facility of Beijing Chao-Yang Hospital with free access to food and water under a 12 h light/dark cycle.

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Surgery Peripheral surgery was performed as previously described to induce PND-like features [5, 33].

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Briefly, mice were subjected to open tibia fracture in the left hind paw under 2% isoflurane

anesthesia. The fracture was stabilized with intramedullary pin fixation. Analgesia was achieved by subcutaneous injection of buprenorphine (0.1 mg/kg) after anesthesia induction. Sham mice

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underwent no surgical trauma but received the same anesthesia and analgesia.

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Drug Administration

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NK-1R antagonist (NK-1Ra) L-733,060 (R&D systems, #1145) was dissolved in sterile phosphate-

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buffered saline (PBS). One hour before surgery, mice were intraperitoneally administered NK-1Ra

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(10mg/kg i.p.). Vehicle-treated mice received i.p. administration of the same volume of PBS. This antagonist has been extensively used for NK-1R blockade antagonism in treating CNS disorders in

Grouping

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rodents [8, 24].

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In the first experiment, mice were randomly assigned into 3 groups (n=5 per group): (1) Naïve; (2) Surgery; (3) Sham. In the second experiment, mice were randomly divided into 3 groups (n=5 per

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group): (1) Sham + Vehicle; (2) Surgery + Vehicle; (3) Surgery + NK-1Ra. Mice were sacrificed for harvesting brain tissues in these two experiments. A different cohort of mice were was used for behavioral test (n=10 per group): (1) Sham + Vehicle; (2) Surgery + Vehicle; (3) Surgery + NK-1Ra. Enzyme-linked Immunosorbent Assay

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At 6 h, 1 day, and 3 days after surgery, mouse brains were harvested after transcardiac perfusion with PBS. Hippocampal tissues were homogenized and stored at -80ºC before use. Protein quantification was performed using bicinchoninic acid (BCA) assay (ThermoScientific, #23225). Hippocampal levels of SP, proinflammatory cytokines interleukin-1β (IL-1β), and interleukin-6 (IL-6) were determined by Enzyme-linked immunosorbent assay (ELISA) (R&D Systems, #KGE007 and

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#MLB00C; ThermoScientific, # KMC0061).

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Western Blotting

At postoperative day 1, mice were perfused with PBS for brain harvesting. Hippocampal supernatant was obtained and stored at -80ºC before use. Protein concentration of hippocampal supernatant

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was determined by BCA assay. Proteins were denatured and separated by 10% SDS-PAGE and then

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transferred onto polyvinylidene fluoride membranes. After wet transfer, membranes were rinsed

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and then blocked with 5% non-fat milk for 1 h at room temperature (RT). After blocking, membranes

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were incubated overnight at 4°C with primary antibodies against NK-1R (1:1000, Novus Biologicals, # NB300-101), claudin-5 (1:1000, ThermoScientific, #34-1600), albumin (1:2000, Abcam, #ab207327),

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or beta-actin (1:2000, ThermoScientific, # MA5-15739). After primary antibody incubation, membranes were washed and then incubated with horseradish peroxidase conjugated secondary antibodies (1:5000, ThermoScientific) for 30 min at RT. Band visualization was achieved with

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ChemiDoc XRS+ system (Bio-Rad). The relative intensities of each band were quantified by Image Lab

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software (Bio-Rad).

Immunohistochemistry

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At 1 day after surgery, each mouse was perfused with 30 mL cold PBS, followed by 20 mL PBS containing 4% paraformaldehyde (PFA). Mouse brains were harvested and post-fixed in 4% PFA overnight at 4°C. Brains were rinsed with PBS and then immersed in 30% sucrose at 4°C for 2 to 3 days. Frozen brain tissues were cut into free-floating coronal sections of 30 μm thickness. Sections were blocked with 1% bovine serum albumin at RT for 1h and then incubated with primary

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antibodies against ionized calcium-binding adapter molecule 1(IBA1) (1:500, Wako, #019-19741), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), or Myeloperoxidase (MPO (1:100, Abcam, #ab9535) for 24h at 4°C. Ater After primary antibody incubation, sections were washed with PBS for 3 times and then incubated with fluorophore-conjugated secondary antibodies (1:200, Invitrogen) for 90 min at dark at RT. After washing with PBS for 3 times, sections were

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mounted on pre-coated slides and sealed with ProLong Gold mounting medium (ThermoScientific) and coverslips (ThermoScientific). A Leica SP8 confocal microscope was used to capture all the

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images under the same settings. Adobe Photoshop software (version CS6) was used to make proper

adjustment under identical conditions. Quantification was performed using ImageJ software (version

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2.00).

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Trace Fear Conditioning

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Trace fear conditioning has been extensively used for determining the effects of orthopedic surgery

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on hippocampal-dependent memory [5, 13]. Thirty minutes after NK-1Ra or vehicle administration, mice were scheduled for a training session to build the association between a conditional stimulus (a

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conditioning chamber) and an unconditional stimulus (two periods of 2-s foot-shocks of 0.75 mA). Thirty minutes after training, either orthopedic surgery or sham surgery was performed. At 3 days after the training session, mice underwent a context test without any unconditional stimulus. Mouse

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movement was recorded and analyzed automatically by a camera-based monitoring system (Xeye

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Fcs system, Beijing MacroAmbition S&T Development Co., Ltd., Beijing, China). Reduction of freezing behavior in the context test indicates loss of memory.

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Statistics

Statistical analyses were performed using GraphPad Prism V6 (GraphPad Software, La Jolla, CA, USA). One-way analysis of variance (ANOVA), accompanied with Tukey’s or Student-Newman-Keuls test, was used to make comparisons between different groups. P < 0.05 indicates statistical significance. Data are presented as means ± standard error of the mean.

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Results Peripheral surgery activates NK-1R signaling in the hippocampus To determine whether SP/NK-1R pathway is involved in PND in mice, we assessed the expression of SP and NK-1R in the hippocampus after surgery by ELISA and western blotting, respectively.

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Compared to naïve, hippocampal SP was elevated at 6 h (Surgery 6h vs Naïve: p < 0.05; Fig. 1A), peaked at 1 day (Surgery 1d vs Naïve: p < 0.05; Fig. 1A), and returned to baseline at 3 days after

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surgery (Surgery 3d vs Naïve: p > 0.05; Fig. 1A). Of note, sham surgery did not alter SP expression

within the same postoperative period (p > 0.05; Fig. 1A). Orthopedic surgery significantly elevated hippocampal level of NK-1R compared with the naïve group (Surgery vs Naïve: p < 0.0001; Fig. 1B, C)

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at 1 day postoperatively, the peak of surgery-induced SP elevation in this model. In contrast, NK-1R

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expression was not increased in the sham group (Sham vs Naïve: p > 0.05; Fig. 1B, C). These findings

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indicate peripheral surgery but not anesthesia alone activates NK-1R signaling in the hippocampus.

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NK-1R antagonism rescues surgery-induced cognitive impairment

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For assessing the role of NK-1R activation in memory deficits after surgery, we examined the effects of NK-1Ra pretreatment on cognition using trace fear conditioning. At 3 days after surgery, there was a reduction of freezing time compared with sham (Surgery + Vehicle vs Sham + Vehicle: p <

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0.0001; Fig. 2). Of note, NK-1Ra treatment increased freezing time to baseline level after surgery

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(Surgery + Vehicle vs Surgery + NK-1Ra: p < 0.01; Fig. 2) (Surgery + NK-1Ra vs Sham + Vehicle: p > 0.05; Fig. 2). Thus, our findings showed that NK-1R blockade antagonism rescued PND at

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postoperative day 3. NK-1R blockade antagonism attenuates hippocampal neuroinflammation after surgery Surgery-induced neuroinflammation is featured by elevations of proinflammatory cytokines and glial activation [5, 13]. To examine the role of NK-1R activation in CNS inflammation after surgery, we assessed whether NK-1R antagonist could mitigate proinflammatory cytokine elevations and

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microgliosis in the hippocampus. At 6 h after surgery, we found that surgical trauma significantly elevated IL-1β and IL-6 in the hippocampus (Surgery + Vehicle vs Sham + Vehicle: p < 0.05 for IL-1β, Fig. 3A; p < 0.001 for IL-6, Fig. 3B). At the same time point, NK-1R antagonism reduced IL-1β and IL-6 to baseline levels after surgery (Surgery + NK-1Ra vs Surgery + Vehicle: p < 0.05 for IL-1β, Fig. 3A; p < 0.01 for IL-6, Fig. 3B; Surgery + NK-1Ra vs Sham + Vehicle: p > 0.05 for both IL-1β and IL-6, Fig. 3A, B).

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Microgliosis occurred in the hippocampus at 1 day after surgery (Surgery + Vehicle vs Sham + Vehicle: p < 0.01; Fig. 3C, D). This surgery-induced microglial activation was prevented by

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pretreatment with NK-1Ra (Surgery + NK-1Ra vs Surgery + Vehicle: p < 0.05; Fig. 3C, D). These findings showed that NK-1Ra curbed hippocampal neuroinflammation after surgery.

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Blood-brain barrier disruption after surgery is prevented by NK-1R antagonist treatment

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BBB disruption is implicated in the pathogenesis of PND. To evaluate whether NK-1R signaling is

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associated with surgery-induced BBB damage in the hippocampus, we assessed the effects of NK-

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1Ra on BBB integrity after surgery. At postoperative day 1, peripheral surgery reduced the expression of tight junction protein claudin-5 and increased albumin deposition in the hippocampus

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(Surgery + Vehicle vs Sham + Vehicle: p < 0.001 for claudin-5, Fig. 4A, C; p < 0.0001 for albumin, Fig. 4B, D), suggesting that surgery disrupted tight junction integrity and increased BBB permeability. Of note, this BBB disruption after surgery could be mitigated by pretreatment with NK-1Ra (Surgery +

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NK-1Ra vs Surgery + Vehicle: p < 0.01 for claudin-5, Fig. 4A, C; p < 0.01for albumin, Fig. 4B, D).

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Surgery-induced neutrophil infiltration is prevented by NK-1R blockade Brain endothelial cells mediates leukocyte recruitment into the inflamed CNS upon activation.

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Surgical insult has been reported to be associated with neutrophil infiltration in the hippocampus. Here, we examined whether NK-1R signaling is involved in endothelial cell activation and neutrophil recruitment after surgery. At 1 day post-surgery, peripheral surgery significantly elevated ICAM-1 expression in the hippocampus compared with the sham control (Surgery + Vehicle vs Sham + Vehicle: p < 0.01; Fig.5 A, B). In contrast, surgery-induced ICAM-1 upregulation were prevented by

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NK-1Ra (Surgery + NK-1Ra vs Surgery + Vehicle: p < 0.05; Fig.5 A, B). Furthermore, MPO+ neutrophils were detected in the hippocampus at 1 day postoperatively while none of these cells were seen in the Sham + Vehicle or Surgery + NK-1Ra group (Fig. 5C). Our findings indicate that NK-1Ra treatment prevented surgery-induced neutrophil recruitment in the hippocampus, probably through downregulation of ICAM-1 expression.

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Discussion

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Substance P/neurokinin-1 receptor signaling is increasingly implicated in the pathophysiology of

neurological disorders, playing a pivotal role in modulating neuroinflammatory response [7, 31]. However, whether NK-1R activation contributes to the development of PND is unknown. In the

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current study with the use of a clinically relevant mouse model of PND and a selective NK-1R

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antagonist, we showed that peripheral surgery induced NK-1R activation in the hippocampus and

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NK-1Ra pretreatment attenuated CNS inflammation, BBB disruption, and memory decline after

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surgery, suggesting the potentially therapeutic effects of NK-1R blockade antagonism for PND.

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SP/NK-1R signaling has been shown to be activated in animal models of neurological diseases including traumatic brain injury, sepsis septic encephalopathy, stroke, and lipopolysaccharideinduced neuroinflammation [6, 12, 19, 30]. Similarly, we found that orthopedic surgery elevated the

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expressions of SP and it receptor NK-1R expression in the hippocampus at 1 day after surgery, suggesting that surgical insult induced activation of NK-1R signaling in the CNS. However, NK-1R is

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expressed on various types of cells in the brain, including microglia, astrocytes, neurons, and

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endothelial cells [2, 10]. The cell-type-specific expression changes of NK-1R by surgical trauma still need to be determined. In addition, the role of downstream signaling of NK-1R in PND warrants investigation. The pathogenesis of PND remains unclear, although surgery-induced neuroinflammatory response has been suggested to contribute to its development [25]. Microglial activation is a hallmark of PND

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[33], which occurs in the hippocampus after surgery. Interventions aiming at reducing microgliosis have shown promising effects on preventing improving PND via ameliorating surgery-induced neuroinflammation [5, 35]. Here, we showed that microglial activation in the hippocampus after surgery was attenuated by NK-1Ra pretreatment. Thus, our finding suggests activation of NK-1R on microglia might be involved in surgery-induced microgliosis.

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Orthopedic surgery induces rapid elevations of proinflammatory cytokines in both the peripheral circulation and hippocampus [5, 11, 13]. Previous studies showed that pharmacological blockade

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antagonism of either IL-1 receptor or IL-6 receptor ameliorates surgery-induced neuroinflammation and memory decline, identifying the essential role of IL-1β and IL-6 in mediating PND [5, 13]. In the

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current study, we found that NK-1R blockade antagonism reduced IL-1β and IL-6 levels in the hippocampus at 6h after orthopedic surgery. In previous studies, activation of SP/NK-1R signaling

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has been shown to initiate glial activation and promote production of proinflammatory mediators

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from glial cells via nuclear factor kappa pathway, exacerbating neuroinflammation [2, 21]. Thus, our

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findings indicate that surgery-induced NK-1R activation might be involved in the increase of

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proinflammatory cytokines in the hippocampus, probably through promoting IL-1β and IL-6 production from glial cells.

Blood-brain barrier is the interface between the circulation system and CNS. Maintenance of BBB is

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essential for the homeostasis of CNS, preventing noxious compounds from entering the brain [34].

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Tight junction (TJ) is the structure that seal the gaps between brain endothelial cells [16]. The disruption of TJ increases BBB permeability, allowing more molecules to pass through BBB [16].

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Surgical trauma has been shown to result in leakage of hippocampal BBB, which might facilitate the infiltrate of blood-borne proinflammatory cytokines and immune cells into the vulnerable brain [4, 27]. Similarly, we found that orthopedic surgery reduced TJ marker claudin-5 and increased albumin deposition in the hippocampus, suggesting the disruption of BBB after surgery. Interestingly, this surgery-induced BBB breakdown could be ameliorated by pretreatment with NK-1Ra. In line with our

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findings, previous studies showed that SP increases the permeability of BBB, which results in increased plasma protein extravasation [14]. In addition, SP treatment has been reported to reduce TJ proteins claudin-5 and ZO-1 in brain endothelial cells in vitro [17]. Together with our results, these findings implicate that NK-1R signaling is involved in the alteration of BBB integrity after surgery. Leukocyte infiltration into the inflamed CNS exacerbates neuroinflammation and BBB disruption

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[26]. This process is mediated by activated brain endothelial cells which express elevated adhesion molecules [23]. In vivo, SP/NK-1R signaling is implicated in mediating leukocyte trafficking into

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different tissues outside the brain [29] [22]. In vitro, SP treatment upregulates the expression of adhesion molecule ICAM-1 in brain endothelium [1]. In the current study, we showed that

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orthopedic surgery elevated ICAM-1 expression in endothelium and recruited MPO+ neutrophils into the hippocampus, which were both prevented by pretreatment with NK-1Ra. Thus, our findings

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probably through upregulation of ICAM-1.

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indicate NK-1R signaling might mediate neutrophil infiltration into the hippocampus after surgery,

Surgical insult impairs hippocampal-dependent memory function in animal models of PND [25]. BBB

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leakage, increased proinflammatory cytokines, glial activation, and peripheral immune cells infiltration early after surgery all contribute to the following cognitive decline [13, 27, 28]. In this study, cognitive dysfunction at 3 days after surgery was attenuated by pretreatment with NK-1Ra.

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The benefit of NK-1R antagonism for memory function may be due to its effects on alleviating

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neuroinflammation and BBB disruption that occurred before the development of PND.

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Conclusions Using an established mouse model for PND, our results show that peripheral surgery induces NK-1R signaling activation in the hippocampus, which might involve in surgery-induced proinflammatory cytokine elevations, microgliosis, BBB disruption, and neutrophil recruitment into the hippocampus, likely leading to the subsequent cognitive impairment. In clinical practice, NK-1R antagonists have

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been widely used for prophylaxis of postoperative nausea and vomiting; our study indicates that they might also have the therapeutic potential for preventing alleviating cognitive impairment after surgery. Competing interests

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The authors declare that they have no competing interests.

Funding

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This work was supported by the National Natural Science Foundation of China, Beijing, China [grant No. 81371199] (to Dr. Wu).

Authors' contributions

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CX and AW designed the experiments. JL, TL, XN, and CX performed experiments and statistical analyses. JL, CX, and AW prepared the manuscript. All authors read and approved the final manuscript.

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Acknowledgements

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Not applicable.

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Figure legends

Figure 1. NK-1R expression activation in the hippocampus after surgery. (A) Hippocampal SP was assayed by ELISA (n=5). (B) Representative images of western blotting of

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NK-1R. (C) Quantification of NK-1R expression (n = 5). One-way analysis of variance was used for data analyses, followed by Student-Newman-Keuls test (A) or Tukey post hoc test (C). *P < 0.05, ***

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Figure 2. The effects of NK-1R blockade antagonism on behavioral test at postoperative day 3.

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Quantification of the percentage of freezing time in the trace fear conditioning test (n=10). One-way

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analysis of variance was used for data analyses, followed by Tukey post hoc test. ** P < 0.01, **** P

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Figure 3. The effects of NK-1R blockade antagonism on proinflammatory cytokines and microglial

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Quantification of IL-1β (A) and IL-6 (B) in hippocampus homogenates (n=5; one-way analysis of variance followed by Student-Newman-Keuls test). (C) Representative images of IBA1 (red)

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immunostaining in the hippocampus; nuclear counterstaining with DAPI (blue); scale bar = 100 μm. (D) Quantification of the percentage of IBA1+ area (n=5; one-way analysis of variance followed by

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Tukey post hoc test). *P < 0.05, ** P < 0.01, *** P < 0.001.

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Figure 4. The effects of NK-1R blockade antagonism on blood-brain barrier integrity. Representative images of western blotting of claudin-5 (A) and albumin (B). Quantification of claudin-5 (C) and albumin (D) expressions (n = 5). One-way analysis of variance was used for data analyses, followed by Tukey post hoc test. **P < 0.01, *** P < 0.001.

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Figure 5. The effects of NK-1R blockade antagonism on neutrophil infiltration. (A)Representative images of ICAM-1 (green) immunostaining in the hippocampus; scale bar = 500 μm. (B) Quantification of the mean gray value of ICAM-1 immunostaining (n=5; one-way analysis of variance followed by Tukey post hoc test). (C) Representative images of MPO (green) immunostaining in the hippocampus; white arrows indicate MPO+ cells; scale bar = 100 μm; nuclear

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counterstaining with DAPI (blue) (A and C). *P < 0.05, ** P < 0.01.

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