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Caspase-1 inhibitor exerts brain-protective eﬀects against sepsis-associated encephalopathy and cognitive impairments in a mouse model of sepsis
Xi-e Xua, Lu Liub, Yu-chang Wanga, Chun-tao Wanga, Qiang Zhenga, Qin-xin Liua, Zhan-fei Lia,
Xiang-jun Baia, Xing-hua Liua,⁎
a Trauma Center/Department of Emergency and Trauma Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
b Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

A R T I C L E I N F O

Keywords:
Sepsis-associated encephalopathy Pyroptosis
Behavior Electrophysiology Inﬂammatory cytokine
A B S T R A C T

Sepsis-associated encephalopathy (SAE) manifested clinically in acute and long-term cognitive impairments and associated with increased morbidity and mortality worldwide. The potential pathological changes of SAE are complex and remain to be elucidated. Pyroptosis, a novel programmed cell death, is executed by caspase-1- cleaved GSDMD N-terminal (GSDMD-NT) and we investigated it in peripheral blood immunocytes of septic patients previously. Here, a caspase-1 inhibitor VX765 was treated with CLP-induced septic mice. Novel object recognition test indicated that VX765 treatment reversed cognitive dysfunction in septic mice. Elevated plus maze, tail suspension test and open ﬁeld test revealed that depressive-like behaviors of septic mice were relieved. Inhibited caspase-1 suppressed the expressions of GSDMD and its cleavage form GSDMD-NT, and reduced pyroptosis in brain at day 1 and day 7 after sepsis. Meantime, inhibited caspase-1 mitigated the expressions of IL-
1β, MCP-1 and TNF-α in serum and brain, diminished microglia activation in septic mice, and reduced sepsis-
induced brain-blood barrier disruption and ultrastructure damages in brain as well. Inhibited caspase-1 pro- tected the synapse plasticity and preserved long-term potential, which may be the possible mechanism of cog- nitive functions protective eﬀects of septic mice. In conclusion, caspase-1 inhibition exerts brain-protective ef- fects against SAE and cognitive impairments in a mouse model of sepsis.

⦁ Introduction

Sepsis often leads to diﬀuse brain dysfunction without direct central nervous system (CNS) infection. It is deﬁned as sepsis-associated en- cephalopathy (SAE), manifested clinically in acute and long-term cog- nitive impairment (Iwashyna et al., 2010; Widmann et al., 2014), and is associated with increased morbidity and mortality worldwide (Angus et al., 2001). Although neuroinﬂammation (Hernandes et al., 2014; Gao et al., 2017), neurotoxicity (Gao et al., 2016), and apoptosis (Sui et al., 2016; Zhang et al., 2014) have been considered as causative factors of SAE, the potential pathological changes of SAE are highly complex and multifactorial and remain to be elucidated.
Recently, pyroptosis of peripheral blood immunocytes in sepsis at- tracts much attention (Wang et al., 2018; Chen et al., 2018b). Pyr- optosis is taken as a novel programmed cell death, which is executed by caspase-1-cleaved GSDMD N-terminal (GSDMD-NT). The GSDMD-NT can bind to inner membrane lipids forming pores in the plasma mem- brane, which ultimately results in cellular lysis and released

inﬂammatory mediators IL-1β (Shi et al., 2015; Kayagaki et al., 2015). In our previous study, we found that pyroptosis of peripheral blood mononuclear cells predicted the development of sepsis in severe trauma
patients (Wang et al., 2018). Microglia, the tissue macrophages of the CNS, are considered to be the main cell type of the innate immune system in the CNS and participates in SAE during sepsis. Inﬂammatory mediators provoke a rapid change in phenotype of the microglia from surveilling state into active state, and triggered microglia producing inﬂammatory mediators. However, the precise mechanisms underlying pyroptosis in SAE and whether the inhibition of caspase-1 might protect the injured brain after sepsis remains unknown.
During systemic inﬂammation, releasing of cytokines and other immunoactive substances from circulating immune cells of peripheral blood and astrocytes, microglia and leukocytes modify a host of BBB functions, altering BBB integrity, BBB transporters, and the perme- ability of the BBB to pathogens and circulating immune cells (Banks, 2015). Speciﬁc caspase-1 inhibitor YVAD treatment completely pre- vented memory impairment in septic rodents (Bilbo et al., 2005; Zhu

⁎ Corresponding author at: Trauma Center/Department of Emergency and Trauma Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
E-mail address: [email protected] (X.-h. Liu).

https://doi.org/10.1016/j.bbi.2019.05.038

Received 11 January 2019; Received in revised form 21 May 2019; Accepted 26 May 2019
0889-1591/©2019ElsevierInc.Allrightsreserved.

X.-e. Xu, et al. Brain,Behavior,andImmunityxxx(xxxx)xxx–xxx

et al., 2017).
We hypothesized that sepsis-evoked inﬂammatory mediators in peripheral blood entered the brain via damaged BBB and triggered pyroptosis in brain, followed by microglia activation and neural dys- functions, leading cognitive impairments and depression ultimately.

⦁ Materials and methods

⦁ Reagents

Antibodies for GSDMD (mouse, sc-393656) and β-actin (mouse, sc- 47778) were obtained from Santa Cruz Biotechnology. Iba-1 (rabbit, ab178847), Caspase-1 (rabbit, ab179515), Synapsin 1 (SYN1, rabbit,
ab64581), NMDAR2B (NR2B, rabbit, ab65783) and PSD95 (rabbit, ab76115) were purchased from Abcam. 594-conjugated donkey poly- clonal secondary antibody to Rabbit IgG-H&L (711-585-152) were ob- tained from Jackson Immuno Research. Evans blue dye was purchased from Sigma. LEGENDplexTM Mouse Inﬂammation Panel was purchased from BioLegend (B254913). VX765 was bought from ApexBio Technology (A8238).

⦁ Animals

Male BALB/c mice aged 8–12 weeks and weighing 20–25 g were enrolled in this study. The mice were obtained from Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology and placed in Experimental Animal Center of Tongji Hospital, Huazhong University of Science and Technology. The mice were housed at 22 °C with relative humidity of 50–60% and al- ternate 12 h of light/dark cycle. Standard food and water were provided ad libitum. All animal experiments were approved by the Experimental Animal Ethical Committee of Tongji Hospital aﬃliated to Huazhong University of Science and Technology (IACUC N umber: S813). All animals had acclimated to the environment for at least 7 days prior to experiments.

⦁ Preparation of cecal ligation and puncture (CLP) model

Mice were subjected to cecal ligation and puncture to induce polymicrobial abdominal sepsis according to a previously used protocol (Rittirsch et al., 2009; Ayala et al., 1995). Brieﬂy, after in- traperitoneally anaesthetized with pentobarbital sodium (50 mg/kg), a midline incision (1 cm) was made in the lower abdomen. The cecum was ligated using 3.0 silk and punctured with a 21-gauge needle. Thereafter, feces were extruded through the puncture wound by gently squeezing the cecum. The intestinal tract was then returned to the peritoneal cavity, and the abdomen was sutured. Mice received ﬂuid resuscitation immediately after surgery by sterile saline solution (0.9℅, 24 ml/kg) subcutaneous injection. In the sham group, mice underwent the same operation as described above but they were neither ligated nor punctured.

⦁ Treatment protocol

Animal treatment followed previous studies with some modiﬁca- tions (Chen et al., 2018a; Wannamaker et al., 2007). Animals were randomly divided into 3 groups: a sham with saline group (sham group), a cecal ligation and puncture with corn oil treatment group (CLP group), a cecal ligation and puncture with VX765 (VX765 group). As there was no statistical diﬀerence in behavioral analysis between the 1st and 7th day of the sham groups (the data did not show), here we chose the 7th day after sham operation as sham group. As low dosage of VX765 (0.2 mg per mouse) did not inﬂuence the behaviors of mice (Zhang et al., 2015), we omitted the control group of sham + VX765. VX765 powder was dissolved in corn oil with a ﬁnal concentration of 1 mg/ml. These three groups of animals received intragastric
administration of corn oil (0.2 ml per mouse), corn oil (0.2 ml per mouse), or VX765 (0.2 ml per mouse) respectively 30 min before sur- gery and an equal dose administrated twice a day (10 a.m. and 4 p.m.) until mice being sacriﬁced.

⦁ Novel object recognition test (NORT)

Mice subjected to NORT underwent three phases: habituation phase, familiarization phase, and discrimination phase. The habituation phase was carried out before the surgery. Each mouse was placed in a square open ﬁeld (40 × 40 × 40 cm) individually for 10 min per day for three consecutive days. Then the animals entered into the familiarization phase. In this phase, each mouse was allowed to explore in the open ﬁeld with two identical objects (A1 and A2) located in opposite and equidistant positions for 10 min. After a three-hour retention interval, the mouse returned to the open ﬁeld, with one of the familiar objects (A1) replaced by a novel object (A3). For the discrimination phase, each mouse was allowed to explore for 10 min and the time for exploring each object was recorded. Mice touching an object or facing an object within 2 cm around the object were taken as measure of object ex- ploration behavior. To eliminate olfactory cues, the objects and ﬁeld were cleaned with 75% ethanol between each trial. The preference index was determined as [time spent in exploring the novel object (A3)/ time spent in exploring the two objects (A2 and A3)] × 100%.

⦁ Open ﬁeld test (OFT)

The open ﬁeld test was conducted in an arena consisting of a plat- form (50 × 50 cm) surrounded by a wall with height of 50 cm. The platform was split into 25 grids with black lines and the centric 9 grids were deﬁned as central area. Each mouse was allowed to explore in the arena for 5 min and between each trail the arena was cleaned with 75% ethanol. Time spent in exploring central area was recorded. Center duration was calculated as (time spent exploring central area/ 300 s) × 100%.

⦁ Tail suspension test (TST)

The test was conducted following previous studies with some modiﬁcations (Anderson et al., 2015). The tail was attached to a holder with a height of 50 cm using adhesive tape located 1 cm from the tip of the tail. The experiment was videotaped for 5 min. The ﬁrst time when mice appeared to be standstill and the total duration of immobility throughout the entire 5 min were scored.

⦁ Elevated plus maze test (EPMT)

The plus maze height of 40 cm consisted of a central square, two open arms, and two closed arms. All arms extended from the central square of diameter 10 cm. The two closed arms were 40 cm in length and 10 cm in width and enclosed by walls with height of 20 cm. In contrast, open arms had no walls. Mice were placed in the central square facing one of the open arms and allowed to explore individually for 5 min. At the end of each trail, the ﬁeld was cleaned with 75% ethanol. The total time spent in the open arms was calculated. The deﬁnition of entries was that all paws of a mouse were included in an arm.

⦁ Immunoﬂuorescence

Following anaesthetized, animals were perfused transcardially by phosphate-buﬀer saline (PBS) and 4℅ paraformaldehyde (PFA) suc- cessively. Brains were removed and ﬁxed in PFA at 4 °C for more than 24 h. Thereafter, brains were immersed in 30% sucrose for dehydration
and then cut into 30-μm-thick slices on a cryostat. Tissue sections were incubated with 0.5℅ Triton X-100 for 20 min for permeation, and

subsequently immersed in 3℅ bovine serum albumin for 1 h at room temperature for blocking. The sections were then incubated with pri- mary antibodies (1:200) overnight at 4 °C. After washed with PBS three times, the sections were incubated with secondary antibodies (1:1000) for 1 h at room temperature. Following rinses in PBS, the sections were stained with DAPI. Images were ultimately acquired with the aid of laser-scanning confocal microscope (Olympus) and analyzed using ImageJ software.

⦁ Western blotting

Brain tissues were homogenized with RIPA lysis buﬀer including protease inhibitor and centrifuged at 12000 rpm for 15 min at 4 °C. The supernatants were then collected as total protein and determined for ﬁnal concentrations using bicinchoninic acid protein assay kit. After
boiled for 10 min, equal amounts (5 μg for β-actin detecting, 30 μg for GSDMD detecting, 20 μg for Caspase-1 detecting, and 10 μg for other proteins detecting) of denatured protein were separated by 12% SDS-
PAGE and transferred to polyvinylidene diﬂuoride (PVDF) membrane. Membranes were blocked with 3% bovine serum albumin for 1 h at room temperature, incubated with primary antibodies (1:1000) over- night at 4 °C, and subsequently washed by TBST three times. Membranes were then incubated with horseradish peroxidase con- jugated secondary antibodies (1:2000) for 1 h at room temperature and washed in TBST three times before detected using electro- chemiluminescence (ECL) and X-ray ﬁlms. The densities of blots were examined using ImageJ software.

⦁ Transmission electron microscopy

After anaesthetized deeply, the mice were perfused intracardially by PBS. Prefrontal cortex were taken out quickly and immersed in 2℅ glutaraldehyde for more than 24 h. Following rinsed in PBS, brain tis- sues were postﬁxed with 2% osmium tetroxide for 1 h. Tissues were then dehydrated through a graded series of ethanol, embedded in resin, and cut into 700-nm-thick slices. The slices were stained respectively with 4℅ uranyl acetate for 20 min and 5℅ lead citrate for 5 min. Images were ﬁnally captured under a transmission electron microscope. The numbers of mitochondria and synapses were counted manually in an unbiased manner. The characters of mitochondria are bilayer mem- branes with intima protruding inward into ﬂat ridges. Electron micro- scopy of synapses is characterized by electron dense deposits, and presynaptic vesicles.

⦁ Assessment of BBB integrity

A modiﬁed Evans blue extravasation method was used to evaluate BBB integrity (Chu et al., 2004). Brieﬂy, Evans blue dye (4% in saline) was given at 2 ml/kg via tail vein 2 h before sacriﬁce. The mice were perfused with normal saline to wash residual dye from the blood ves- sels. Brain samples were then collected, weighed, and homogenized in PBS. Evans blue was extracted from tissue homogenates by incubating samples in formamide at 60 °C for 14–18 h. The inﬁltrated Evans blue dye was quantiﬁed using a spectrophotometer at 610 nm. The dye in the cerebellum was used as an internal control. The content of the dye was calibrated with a standard curve of known dyes. Data are expressed as micrograms per milligram brain weight.

⦁ Evaluation of inﬂammatory factors

Inﬂammatory-related factors, including IL-1β, MCP-1 and TNF-α, were evaluated using LEGENDplex TM Mouse Inﬂammation Panel with
V-bottom plate according to the manufacturer’s guideline. BDTM FACS Calibur ﬂow cytometer was used to collect data, and the results were analyzed using LEGENDplex TM data analysis software. The assay sen- sitivities of IL-1β, MCP-1 and TNF-α are 3.3–10000 pg/ml,
1.8–10000 pg/ml and 2.1–10000 pg/mg (serum) respectively according to instruction.

⦁ Electrophysiology

The brain of anesthetized mouse was rapidly removed within 1 min and transferred to ice cold, oxygenated (95% O2, 5% CO2) artiﬁcial cerebrospinal ﬂuid (ACSF, containing in mM: 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgSO4, 1 NaH2PO4, 25 NaHCO3, and 10 Glucose; pH 7.4).
Brain was horizontally sliced with a precooled vibratome to produce 350 µm slices. The slices were incubated in oxygenated ACSF at 29 °C for at least 90 min to recover. After recovery, a single slice was trans- ferred to a MED probe (MED-P515A, 8 × 8 array, interpolar distance 150 μm, Panasonic) and positioned in such a way that the perforant
path ﬁbers (PP, stimulating region) and DG region (recording region)
covered most of electrodes, immersed with 29 °C oxygenated ACSF, stabilized carefully with a meshed anchor, and allowed to equilibrate for at least 30 min. Oxygenated fresh ACSF was continuously perfused at the rate of 2 ml/min with the aid of a peristaltic pump during the entire experiment. Field excitatory postsynaptic potentials (fEPSPs) were recorded at PP-DG synapses. Electrical stimulation was delivered to one channel located within the PP, and evoked fEPSPs were mon- itored and recorded from the other 63 channels. The stimulation in- tensity was approximately 40% of intensity that induced the maximal fEPSPs. For LTP recording, baseline responses were evoked for at least 30 min until stabilized, followed by a high frequency stimulation (HFS) protocol (consisted of 10 bursts, each containing 4 pulses at 100 Hz with an inter-burst interval of 200 ms) was given at a stimulation in- tensity that was adjusted to elicit 40% of the maximal response. After HFS, the test stimulus was repeatedly delivered once every minute for at least 1 h to record LTP.

⦁ Golgi staining

Brains were placed directly into Golgi solution (1 g potassium chromate, 1 g mercuric chloride, 0.8 g potassium chloride and 100 ml double-distilled water) where they remained in a bottle kept in dark for 6 weeks. Thereafter, the solution was exchanged sequentially in 10, 20 and 30% sucrose solutions in light-protected jars to aid in maintaining
histological structure. The brains were sectioned at 100 μm thickness with a vibratome and placed onto gelatin-coated glass slides. After
rinsing with double-distilled water, slides were incubated in ammonium hydroxide for 30 min. Following a water wash, the slides were in- cubated for 30 min in a black and white ﬁlm developer diluted 1:9 with water and then rinsed with ﬁnal double-distilled water. Slides were mounted with resin and cover-slipped. All subsequent quantitative analyses were examined using ImageJ software and conducted in an unbiased manner to group designation with repeated measurements.

⦁ Statistical analysis

The study was conducted by researchers following the principle of randomization and blinding. All data were analyzed using the GraphPad Prism version 5.04 statistical package. Data were expressed mean ± SEM. For LTP, the last ﬁve minutes of fEPSPs values in 3 groups were analyzed by ANOVA followed by Newman-Keuls test. Other results were all analyzed by ANOVA followed by Newman-Keuls test. P < 0.05 was considered statistically signiﬁcant.

⦁ Results

⦁ Inhibited caspase-1 improved survival rate and ameliorated cognitive and emotional dysfunction of CLP-induced sepsis in mice

All of the sham animals were normal with 100% survival throughout the 7-day study period. However, post-septic mice showed

Fig. 1. Caspase-1 inhibition improved survival rate and ameliorated cognitive and emotional dysfunction of CLP-induced sepsis in mice. (A) Survival curve. 12 of 25 mice of CLP group survived by day 7. 13 of 17 mice of VX765 survived. (B–D) Time spend with novel object, counteracts with novel object and preference index in the NORT respectively. (E, F) In the EPMT, entries of the open arms and time consumed in the open arms respectively. (G) In the TST, total immobility times during 5 min. (H) In the OFT, percentage of center duration in the 5 min test. Data were shown as mean ± SEM (n = 6 per group). *p < 0.05 and **p < 0.01 versus sham, #p < 0.05 versus the CLP.

48% survival (12 of 25 mice survived) by day 7. In the VX765 groups, the survival curve revealed survival rate improvement (13 of 17 mice survived) when compared to the septic mice (Fig. 1A).
Behavioral tests were carried out before sacriﬁce at 1 day and 7 days after CLP surgery. The hippocampal mediated working memory in all surviving mice of each experimental group was assessed by the NORT.
During the familiarization phase, the exploratory preference for the objects was not inﬂuenced by the treatments (Data did not show). In the discrimination phase of NORT, septic mice exhibited a signiﬁcantly decreased exploratory preference and less counteracts with the novel object at 1 day and 7 days after sepsis onset compared with the sham group. In contrast, inhibited caspase-1 remarkably mitigated sepsis-

induced recognition memory impairments when compared to the CLP group (Fig. 1B–D). To analyze the eﬀects of caspase-1 inhibitor treat- ment on septic mice emotional behaviors, EPMT, OFT and TST were carried out at diﬀerent time points after sepsis onset. In EPMT, the entry and time consuming in the open arms of septic mice were decreased signiﬁcantly compared to sham, while the entry and duration time of VX765-treated mice were increased without statistic diﬀerence when compared with CLP (Fig. 1E and F). In TST, the mean immobility time of septic mice at 1 day increased signiﬁcantly but did not diﬀer from that of caspase-1 inhibitor treated mice. However, 7 days after surgery, the immobility time of caspase-1 inhibitor treated mice was shorter than that of septic mice, which showed no diﬀerence to the sham (Fig. 1G). Septic mice spent less time in the central area of open ﬁeld than that of sham during the course, which indicated caspase-1 in- hibitor treatment could remarkably improved eﬀect on exploring cen- tral ﬁeld (Fig. 1H).
In general, these results suggest that CLP-induced septic mice ex- hibit cognitive dysfunction and emotional disorder, while caspase-1 inhibitor treatment prevents these behavioral deﬁcits.

⦁ Inhibited caspase-1 suppressed pyroptosis and ameliorated sepsis- induced BBB disruption in brain of CLP-induced sepsis in mice

The expressions of cleaved-caspase-1 (C-caspase-1, 10 kD), GSDMD and GSDMD-NT in brain were remarkably increased in the CLP group comparing with the sham group, but were dampened in VX765-treated mice comparing with septic mice at 1 day and 7 days after surgery. The expressions of Pro-caspase-1 (45 and 42kD) showed no diﬀerence at 7 days after surgery around three groups (Fig. 2A and B).
These results indicate that VX765 inhibited pyroptosis in brain ef- fectively, therefore we investigated the details of brain ultrastructure by transmission electron microscope. 1 day after sepsis onset, the integrity of BBB was breaching, the endothelial cell nuclear chromatin was coacervate and assembled around the edge of karyolemma. In the VX765-treated mice, the BBB was intact and the damage of endothelial cell was clearly attenuated (Fig. 2C–E). In line with the altered BBB ultrastructure, we observed that inhibiting caspase-1 resulted in a sig- niﬁcant improvement in the sepsis-induced loss of BBB integrity 1 day after sepsis, which evidenced by an improvement in Evans blue extra- vasation (Fig. 2F).
In the septic mice, the synapse density (Fig. 3A and D) and the number of mitochondria were decreased signiﬁcantly (Fig. 3A–C), the continuity of double neural nuclear and mitochondria membrane was interrupted (Figs. 3B and 4), and the neural contents were decreased remarkably (Fig. 4). In the caspase-1 inhibitor administrated mice, the synapse density and the number of mitochondria were preserved (Fig. 3A–C), the double nuclear membrane was clear and intact, and the cell content was abundant (Fig. 4). These results demonstrated that VX765 treatment diminished pyroptosis in brain and ameliorated ul- trastructure damage in septic mice.

⦁ Caspase-1 inhibitor treatment mitigated the inﬂammatory cytokine response and diminished microglia activation in septic mice

Sepsis is a systemic inﬂammatory response by the immune system to injury, and induced multiple organs dysfunction. We analyzed the le- vels of inﬂammatory cytokines in serum and brain. In serum, the levels
of IL-1β, MCP-1 and TNF-α were elevated at 1 day and 7 days after surgery, while the levels of these inﬂammatory cytokines were de-
creased dramatically when treated with VX765 at both time points (Fig. 5A, C and E). These brain tissue homogenate inﬂammatory cyto- kines were also measured in order to assess the presence of neuroin- ﬂammation in the cortex and hippocampus, and the results showed that sepsis notably up-regulated the IL-1β levels in the cortex and hippo-
campus at 1 day and 7 days after surgery, while caspase-1 inhibitor
administration signiﬁcantly reduced the increases of IL-1β. VX765
treatment decreased MCP-1 and TNF-α in hippocampus but not cortex at 7 days after sepsis onset (Fig. 5B, D and F). The microglia im- munoreactivity was evaluated by Iba-1 in the hippocampus and cortex.
In the hippocampus and cortex, immunohistochemical images showed more Iba-1 positive cells at 1 day and 7 days after septic onset. A magniﬁed image of Iba1-positive cells in septic mice showed an en- larged cell body with thick, shrunk processes and were consistent with the ameboid morphology of activated microglia. On the other hand, an Iba-1 positive cell in sham group showed a thin cell body with ﬁne and long processes, consistent with the ramiﬁed morphology of resting microglia. Measuring Iba-1 expression showed that the Iba-1 positive cells were increased signiﬁcantly in septic mice compared to sham in the whole brain, including hippocampus and cortex (Fig. 6B–D). In VX765 treated mice, though the number of activated microglia in the hippocampus and cortex were dramatically decreased, higher than that of sham group (Fig. 6B–D). Similarly, the area of Iba-1 positive cell body was increased in septic mice, while shrunk in VX765-treated mice (Fig. 6E).

⦁ Caspase-1 inhibitor treatment protected synapse plasticity in CLP- induced sepsis model

Caspase-1 inhibitor treatment prominently improved CLP-induced hippocampus-dependent cognitive dysfunction (Fig. 1B–D) and there- fore we recorded hippocampal LTP at 1 day after sepsis onset which underlay the mechanism of learning and memory (Nicoll, 2017). Our data showed that septic mice manifested a suppression of PP-DG LTP, which might be the cause of the dysfunction of memory retrieval. VX765 administration apparently improved the inhibited LTP of septic mice (Fig. 7A). In consistent with the results of electrophysiology, the levels of synaptic associated proteins including presynaptic protein synapsin 1 (SYN1), postsynaptic protein NMDA receptors (NR2B) and PSD95 were decreased remarkably in the cortex and hippocampus of septic mice at 1 day, while the levels of these proteins recovered in VX765 treated mice. 7 days after surgery, the expressions of SYN1 in septic and VX765 treated mice were recovered. On the other hand, the levels of NR2B and PSD95 in septic mice were still lower but partly recovered in the VX765 treated mice. Similarly, Golgi staining results revealed that the dendritic spine density in DG of septic mice was de- creased compared to sham, while VX765 treatment alleviated the da- mages in dendritic spine of SAE (Fig. 7B and C). These results revealed that VX765 treatment rescued the impairments of synaptic plasticity in septic mice.

⦁ Discussion

Sepsis is often as a result in SAE and associated with signiﬁcant mortality and morbidity. Some studies have demonstrated an associa- tion between brain lesions and long-term psychological or cognitive disorders in SAE (Angus et al., 2001; Barichello et al., 2005b; Mina et al., 2014; Calsavara et al., 2013). Consistent with the results, our CLP induced septic animals exhibited serious cognitive impairments at 1 day and 7 days after CLP accessed by NORT. Shimizu et al showed that memory retention performance was disturbed 48 h after the CLP pro- cedure in rats (Shimizu et al., 1999). Studies with sepsis surviving rats were extended up to 30 days from surgery and also showed decrease in both aversive learning and memory (Barichello et al., 2005a; Tuon et al., 2008). Caspase-1 inhibitor administration alleviated the cogni- tive dysfunctions at 1 day and 7 days after CLP surgery, suggesting that hippocampus-dependent memory was recovered. In previous studies, the septic rats demonstrated depressive-like behavior after 10 and 30 days (Tuon et al., 2008; Barichello et al., 2007), but it did not persist up to 60 days (Tuon et al., 2008). In the present study, the anxiety behaviors of caspase-1 treated mice were attenuated at 1 day and 7 days after sepsis onset. In addition to increasing anxiety-like behaviors, post- septic animals also exhibited serious depression in the TST and OFT;

Fig. 2. Caspase-1 inhibition treatment suppressed pyroptosis and ameliorated sepsis-induced BBB disruption in the brain. (A) Expressions of Pro-caspase-1 and its activated form cleaved-caspase-1 (C-caspase-1), GSDMD and GSDMD-NT in cortex were analyzed by western blots. (B) Statistical analysis of western blots of A (n = 3 per group). (C) One day after sepsis onset, representative transmission electron micrographs of the BBB ultrastructure of CLP. (D) Higher magniﬁcation of BBB among three groups showed details of tight junction (red arrow). (E) The endothelial cell nuclear chromatin was remarkably coacervated and assembled around the edge of karyolemma in the CLP group, while VX765 signiﬁcantly attenuated those kinds of impairment. (F) The BBB permeability was evaluated by Evans blue extravasation.
**p < 0.01 versus sham, #p < 0.05 and ##p < 0.01 versus the CLP group. nu: nuclear; peri: pericyte; er: erythrocyte; lu: lumen. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

caspase-1 inhibitor treatment migrated the depressive-like behaviors at 1 day and 7 days after surgery.
In previous studies, caspase inhibitory treatment oﬀers protective eﬀects on the outcome of sepsis. The broad-spectrum caspase inhibitor, Z-VAD, provided signiﬁcant neuroprotections by decreasing
hippocampal neuronal death in a rabbit model of pneumococcal me- ningitis (Braun et al., 1999), and also improved survival rate in the CLP model (Hotchkiss et al., 2000). Inhibition of caspase activity by Z-VAD signiﬁcantly reduced lipopolysaccharide (LPS) and Staphylococcus aureus (SAC) induced release of mature IL-1beta in septic patients and

Fig. 3. Caspase-1 inhibition alleviated ultrastructural damage of sepsis. (A) Representative transmission electron micrographs of mitochondria and synapses in cortex among three groups 1 day after surgery. Red stars: synapses; Blue arrows: mitochondria. (B) Representative image of mitochondria double membrane disruptions. Stars showed the double membrane continuity interruptions. (C and D) Statistical analysis of the densities of mitochondria and synapses. **p < 0.01 versus sham, ##p < 0.01 versus the CLP mice, #p < 0.05 versus the CLP mice. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

controls (Oberholzer et al., 2000). Using a selective caspase-3 inhibitor (Hotchkiss et al., 2000) or a siRNA directed against caspase-8 gene decreased apoptosis and improved survival in the CLP model of sepsis (Wesche-Soldato et al., 2005). Caspase-1 inhibitory treatment also provided protected eﬀects for cognitive functions on the sepsis. Speciﬁc caspase-1 inhibitor YVAD treatment completely prevented LPS-induced memory impairment in neonatally infected rats (Bilbo et al., 2005). Bilbo et al. showed that neonatal infection-induced memory impair- ment after lipopolysaccharide in adulthood is prevented via caspase-1 inhibition (Bilbo et al., 2005). In adult LPS-exposed mice, YVAD blocked long-term behavioral dysfunctions (Zhu et al., 2017). Rats that intracerebroventricularly received the YVAD exhibited reduced spon- taneous nonrapid eye movement sleep and nonrapid eye movement sleep enhancement induced by lipopolysaccharide (Imeri et al., 2006). VX765 is an orally absorbed and BBB permeable inhibitor of caspases belonging to the caspase-1 subfamily (Alexander et al., 2007) and shown to reduce the production of IL-1β and IL-18 both in vitro and in
vivo in correlation with tissue-protective eﬀects in animal models of
inﬂammatory disease (Wannamaker et al., 2007; Zhang et al., 2015; Flores and Noel, 2018). For these, VX765 may be considered as a sepsis potential therapeutic agent.
In agreement with the key role of caspase-1 in the pyroptosis, in our study, VX765 administration reduced pyroptosis remarkably in cortex and suppressed inﬂammatory cytokines release in brain and serum. Consistence with our previous study (Wang et al., 2018), we found that caspase-1 inhibitor treatment reduced the levels of IL-1β, TNF-a and
MCP-1 in serum and brain of post-septic mice. IL-1β and TNF-a are the
pro-inﬂammatory cytokines that mediate the initial response of the innate immune system to injury or infection. Mature IL-1β is cleaved by activated caspase-1 and released via the pore in the cell membrane and induces pyroptosis (Shi et al., 2015). Using IL-1β receptor antagonist
was able to prevent the BBB disruption and decreased the levels of IL-1β and TNF-α in the pre-frontal cortex and striatum at 24 h post sepsis (Mina et al., 2014). In our study, caspase-1 inhibitor treatment in septic
mice remarkably dampened the levels of caspase-1 and its activated form in brain, mitigated pyroptosis and reduced mature IL-1β produc- tion and inhibited inﬂammation ultimately. TNF-a is increased in cir-
culation following LPS administration (Tsao et al., 2001; Merrill and Benveniste, 1996). Serum TNF-a is a mediator of the acute phase re- sponse and does not correlate with the alterations in brain during stroke (Intiso et al., 2004). TNF-a is also produced by activated microglia and astrocytes in sepsis. TNF-a participates in many pathophysiological conditions in the central nervous system (Pan et al., 1997), including neurodegenerative diseases (Alexander et al., 2007). The signiﬁcance of TNF-a in these neuropathological conditions is not totally understood, since it has potentially both detrimental and beneﬁcial roles in the CNS.
TNF-α and IL-1β activate endothelial cells and attract circulating polymorphonuclear leukocytes (PMNs) to the site (Lakshmikanth et al.,
2016). Caspase-1 inhibitor administration dramatically down-regulated the levels of TNF-a in serum and brain in our research.
MCP-1, monocyte chemoattractant protein 1, is primarily secreted by monocytes, macrophages and dendritic cells (Semple et al., 2010) and recruits monocytes, memory T cells, and dendritic cells to the sites of inﬂammation (Carr et al., 1994; Xu et al., 1996). In CNS, MCP-1 predominantly produced by astrocytes (Berman et al., 1996; Glabinski et al., 1996) and rapidly elevated in response to stab-wound, aspiration, and diﬀuse axonal injury (Glabinski et al., 1996; Hausmann et al., 1998; Muessel et al., 2000; Rancan et al., 2001). MCP-1 is predominantly involved in the neuroinﬂammatory processes that takes place in the neuronal degeneration (Gerard and Rollins, 2001), epilepsy (Foresti et al., 2009; Fabene et al., 2010), brain ischemia (Kim et al., 2016), Alzheimer’s disease (Hickman and El Khoury, 2010) and traumatic

Fig. 4. Caspase-1 inhibition protected the ultrastructure of neurons one day after sepsis onset. Right panels were magnify images of left panels, showing details of double nuclear membrane. Blue arrows showed the interruptions of continuity of double neural nuclear membrane in CLP group. The neural contents were decreased in CLP group. Red arrows showed nuclear autophagy. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

brain injury (Semple et al., 2010). In the present study, the levels of MCP-1 in serum and brain were elevated in 1 day and 7 days after CLP surgery, and VX765 dampened the expressions of MCP-1 in both serum and brain, suggesting an anti-inﬂammation of VX765.
The serum pro-inﬂammatory cytokines may enter brain from the disrupted BBB. The BBB is primarily formed by cerebrovascular en- dothelial cells that are sealed with tight junctions (Tran et al., 2016) and is highly restrictive of the transport of substances between blood
and the CNS. In agreement with previous researches (Wang et al., 2017; Zhao et al., 2014; Avtan et al., 2011), ultrastructural analysis reveals that disruption of the BBB integrity occurring as a consequence of sepsis. A recent work suggested that endothelial pyroptosis worsens sepsis in CLP-induced septic mice (Chen et al., 2019). The nuclear chromatin of endothelial cells in septic mice was coacervate and as- sembled around the edge of karyolemma but endothelial cells nuclear toxicity was not observed in caspase-1 inhibitor treated mice. In septic

Fig. 5. Caspase-1 inhibition reduced inﬂammatory cytokines levels in serum and brain. (A and B) Levels of IL-1β in serum and brain. (C and D) Levels of MCP-1 in serum and brain. (E and F) Levels of TNF-a in serum and brain. n = 6 per group. **p < 0.01 and *p < 0.05 versus sham, #p < 0.05 and ##p < 0.01 versus the CLP group.

mice, the continuity of double nuclear membrane of neuron was in- terrupted, and the neural contents were decreased remarkably. The synapse density decreased signiﬁcantly in septic mice. These would be the pathological basis for the cognitive dysfunctions. Moreover, our
results indicated that synapse plasticity was impaired 1 day after sepsis. Caspase-1 inhibitor treatment was able to reverse the abnormal struc- tures of neurons and synapses and protected the synapse plasticity after sepsis. Evidence from the literature supports the hypothesis that

Fig. 6. Caspase-1 inhibition diminished microglia activation. (A) Representative IBA-1 and DAPI immunoﬂuorescence staining in the hippocampus and cortex (scale bar: 100 μm). Representative magnify Iba-1 positive cells were showed in upper right corner of each group (scale bar: 20 μm insets). (B–D) Statistical analysis of IBA-1 positive cell counts in whole brain, hippocampus and cortex. (E) Statistical analysis of IBA-1 positive cell body area (n = 3 per group, 3 slices per animal).
**p < 0.01 versus sham, ##p < 0.01 versus the CLP mice.

mitochondrial dysfunction occurred in sepsis (Kim et al., 2016; Zhang et al., 2017; Rademann et al., 2017).
Energy metabolism is disturbed in sepsis. Sepsis will initially induce an increase in metabolic activity that seems to be fueled by an increase in mitochondrial respiration (Singer, 2007). However, with prolonged sepsis, there is mitochondrial dysfunction and damage, plus down- regulation of genes expressing mitochondrial protein (Singer, 2007). Oxidative stress and mitochondrial dysfunction may occur because of the increased metabolism and energy needed by brain cells secondary to neuroinﬂammation during sepsis (van Gool et al., 2010). Mi- tochondrial dysfunction further causes production of reactive oxygen or nitrogen species, which may result in apoptosis of glial cells and neu- rons, causes SAE (van Gool et al., 2010; Cunningham, 2011). Our re- sults showed that the density of mitochondria was increased in the
brain of septic mice but the continuity of mitochondrial bilayer mem- branes was disrupted, which suggests high energy demands in the early stage of sepsis but the functions of the mitochondria were disordered too seriously to provide energy. Caspase-1 inhibitor treatment pro- tected the structure of mitochondria and maintained the density of mitochondria in a normal level.
Previous in vitro and in vivo models of sepsis demonstrated that autophagy was activated initially in sepsis, followed by a subsequent phase of impairment (Lin et al., 2014a,b; Lee et al., 2014; Piquereau et al., 2013; Su et al., 2015; Takahashi et al., 2013; Tang et al., 2013). During sepsis, autophagy counteracts microbial invasion by actively eliminating intracellular microbes, promoting antigen presentation, modulating inﬂammatory responses and removing damaged host cell organelles, such as mitochondria, in an attempt to maintain

Fig. 7. Caspase-1 inhibitor treatment protected synapse plasticity in CLP-induced sepsis model. (A) The LTP of PP-DG. (HFS, high frequency stimulation. n = 5 per group, 4–5 slices per animal). (B) Representative dendritic spines in DG of three groups (scale bar, 5 μm). (C) Quantiﬁcation of spine density in the three groups (n = 3 per group, 6 slices per animal). (D) Expressions of SYN1, NR2B and PSD95 in cortex and hippocampus were analyzed by western blots. (E, F) Statistical analysis of western blots of D (n = 3 per group). **p < 0.01 versus sham, #p < 0.05 and ##p < 0.01 versus the CLP group.

homeostasis (Schmid and Munz, 2007). The observation of autophagy induction in sepsis has largely relied on protein analysis of homo- genized organs/tissues harvested from animal models of sepsis. Liver showed the highest level of autophagy induction, followed by heart and spleen (Takahashi et al., 2013). CLP-induced sepsis showed increased autophagosome formation and lysosome activation in the hippo- campus. These changes are accompanied by increased LC3-II and de- creased BECN1, LAMP1 and RAB7. Inhibition of NFKB by pyrrolidine dithiocarbamate increases the level of LC3-II, BECN1, LAMP1 and RAB7 (Su et al., 2015). However, the mechanisms underlying sepsis-induced neural nuclear autophagy have to be elucidated.
In conclusion, in the current study we demonstrated that caspase-1 inhibition dramatically down regulated pyroptosis and reduced in- ﬂammatory cytokines release, protected ultrastructure of brain and preserved cognitive functions in CLP induced experimental sepsis.

Acknowledgment

This study was supported by grants from Natural Science Foundation of China (No. 81571891, No. 81772129, No. 81801072).

Declaration of Competing Interest

None.

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