Repeated Neonatal Sevo urane Induced Neurocognitive Impairment Through NF-κB- Mediated Pyroptosis

Jing Dai Jiangsu University A liated Jintan Hospital Xue Li Jiangsu Uinverisity A liated Jintan Hospital Cai Wang Jiangsu University A liated Jintan Hospital Shuxin Gu Jiangsu University A liated Jintan Hospital Lei Dai Jiangsu University A liated Jintan Hospital Jingyun Zhang Jiangsu University A liated Jintan Hospital Yunxia Fan (  fanyinjia@aliyun.com ) Jiangsu University https://orcid.org/0000-0003-4488-2511 Jing Wu Zhengzhou University First A liated Hospital


Introduction
Each year around the world, increasing numbers of children and infants receive surgical and diagnostic procedures under general anesthesia (GA), often requiring repeated exposures. Accumulating evidence from animal and preclinical studies has demonstrated that general anesthetics (GAs) cause neuroin ammation in the developing brain, leading to neurodevelopmental de cits later in life [1][2][3][4][5].
Sevo urane is the most common used inhalational GAs for pediatric patients, with excellent properties of respiratory tolerance, rapid onset, rapid offset, and hemodynamic stability [6]. It has been shown that sevo urane induces activation of nuclear factor (NF)-κB, a master regulator of in ammation, increases in ammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-(IL-) 1β and IL-6 in the developing brain, resulting in long-term cognitive dysfunction in adulthood [2]. Despite of role of NF-κB in neuroin ammation, a large gap remains in understanding of the consequences of NF-κB activation in the developing brain after neonatal GAs exposures.
Pyroptosis is a novel in ammatory form of programmed cell death. This type of cell death can be triggered through the canonical nod-like receptor pyrin domain-containing 3 (NLRP3) in ammasomecaspase-1 pathway and non-canonical caspase-4/5/11 pathway [7][8][9][10]. More speci cally, activated in ammatory caspases cleave Gasdermin D (GSDMD) protein, the executor of pyroptosis, into two fragments (the N domain and C domain). As a result, the N-terminal fragment of GSDMD (GSDMD-N) forms nanoscopic pores on the cell membrane, leading to cell swelling and the release of proin ammatory materials [7][8][9][10]. Our previous study has demonstrated that canonical NLRP3 in ammasome-caspase-1 pyroptotic pathway was involved in iso urane (a volatile anesthetic)-induced cognitive impairment in aged mice [11]. However, the link between GAs, pyroptosis and cognitive function remains largely unknown.
NF-κB is a nuclear transcription factor that participated in the control of a variety of cellular processes.
Recent studies have discovered that the activation of the NF-κB family of transcription factors is a key step in the regulation of pyroptosis, through promoting the transcription and translation of pyroptosisrelated proteins, including NLRP3, caspase-1 and caspase-11 [12][13][14]. Thus, the present study was set out to investigate whether GSDMD-induced pyroptosis mediated by in ammatory caspases is involved in the pathophysiology of neuroin ammation and cognitive de cits after repeated neonatal sevo urane exposures in developing rats. In addition, BAY 11-7082, a selective NF-κB inhibitor [15,16], was used to further investigate the link between sevo urane GA and pyroptosis.

Animals
Sprague-Dawley rat pups at postnatal day (PND) 6 were used in the present study. All experimental procedures and protocols were reviewed and approved by the Animal Investigation Ethics Committee of Jiangsu University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The pups were housed in a room maintained under constant environmental conditions (temperature 22-24 °C, a 12-h light/dark cycle, and 50 ± 10 % humidity) with their mothers till PND 20. At PND 21, the pups were weaned and housed 4-5 per cage in standard condition.

General anesthesia
Rat pups at PND 6 were randomly assigned to one of following four treatment protocols: control + vehicle (Con group), control + BAY 11-7082 (Con+BAY group), sevo urane + vehicle (Sev group), and sevo urane + BAY 11-7082 (Sev+BAY group). BAY 11-7082 (MilliporeSigma, USA) was rst dissolved in a small amount of DMSO and then diluted by PBS (phosphate buffered saline) according to the previously published method [17,18]. BAY 11-7082 (20 mg/kg) or PBS (vehicle) was intraperitoneally administered to the pups 30 min before gas inhalation [17,18]. Sevo urane anesthesia was induced by putting the rat pups in an anesthetizing chamber delivered with 3% sevo urane for 2 h daily for three consecutive days [19,20]. For control condition, 30% O 2 was delivered at the same ow rate. The composition of the chamber gas was continuously monitored using a DatexTM infrared analyzer (Capnomac, Finland). Rats were kept normothermic throughout the experiment. Six rat pups from each group were sacri ced immediately after 2 h gas inhalation at PND 8, and the brains were rapidly removed for histological and biochemical studies. Twelve rats from each group were used for behavioral studies at PND 40, 50 and 60.

Rat hippocampal neuronal culture and anesthetic exposure
Primary neuronal cultures were prepared from embryonic day 16-17 (E16-17) embryos of Sprague-Dawley rats as previously described [21]. Neurons were dissociated and seeded on poly-D-lysine-coated plates with neurobasal medium (Thermo Fisher Scienti c, Waltham, MA, USA) supplemented with B27 (Thermo Fisher Scienti c, USA), GlutaMAX-I (Thermo Fisher Scienti c, USA), 5% FBS (Invitrogen GIBCO Life Technologies, USA) and antibiotics. After 2 h incubation, primary cultures were maintained in neurobasal medium without FBS in 5% CO 2 incubator at 37 °C. Subsequently, half of the medium was replaced every 2 days. After 9 days in vitro (DIV), the neurons were treated with 3% sevo urane plus 5% CO 2 for 2 h daily for three consecutive days at 37 °C, whereas the control group was maintained in same amount of culture medium. BAY 11-7082 (5 μM) or equal volume of DMSO was added to the culture medium 30 min before GA exposure according to group assignment [22].

Cell viability assays
At DIV 11, neuronal cell viability was detected with the Cell Counting Kit-8 (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. Results were expressed as the percentage of reduction of absorbance at 450 nm by calibration with the absorbance of the control cells.

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β and IL-18 in the hippocampus were performed by ELISA kit following the manufacturer's instructions (Abcam, UK). Brie y, the supernatant of hippocampal tissue was added to 96well plates coated with the indicated antibodies. After the reaction between the enzyme and substrate, the absorbances of the sample were assessed at 450 nm using a microplate reader (Thermo Fisher Scienti c, USA).

Immunohistochemical (IHC) staining
IHC was used to detect the immunoreactivity of GSDMD. The brain tissues were immediately perfused with 4% paraformaldehyde in PBS after removal and embedded in para n for sectioning. Brain sections (4 μm thickness) were incubated overnight at 4 °C with primary antibody against GSDMD (1:200, Santa Cruz Biotechnology, USA). The sections were then incubated with a secondary antibody labeled with horseradish peroxidase for 30 min at room temperature. Cells with brownish-yellow cytoplasm were counted as positive cells. For quantitative immunostaining, GSDMD-positive cells were observed under an inverted microscope, and the CA1 region was counted for all groups in ImageJ software.

Open eld test
At PND 40, each rat (n = 12 for each group) was gently placed in the center of a black plastic chamber (100 cm × 100 cm × 40 cm) for 5 min. The exploratory behavior was automatically recorded by a video tracking system (XR-XZ301, Shanghai Soft Maze Information Technology Co., Ltd., China). The total distance and the amount of time traveled in the center area (50 cm × 50 cm) of the maze were measured.
After each test, the arena was cleaned with 75% alcohol to avoid the presence of olfactory cues.

Morris water maze (MWM) test
The MWM test (XR-XM101; Shanghai Xinruan Information Technology Co., Ltd., China) was performed at PND 50. In the training phase, the rat was allowed to face to the pool wall in four random places (N, S, E, W) in the pool to nd the xed platform. The trial was terminated once the rat reached the platform. If the rat failed to reach the platform within 60 s, it would be guided to the platform and allowed to stay for 10 s, and then the latency was recorded for 60 s. In the probe test, single-probe trial was conducted with the original platform removed 24 h after the last training session. The rat was released at the opposite position of the platform and allowed to swim for 60 s in the pool.

Fear conditioning test
Fear conditioning tests (XR-XC404; Shanghai Softmaze Information Technology Company Limited, China) was performed at PND 60. Each rat was placed into a conditioning chamber and allowed to explore freely for 3 min. Then one tone-foot-shock pairing (tone, 30 s, 85 dB, 2 kHz; foot-shock, 2 s, 0.8 mA) was delivered. The rat then stayed in the chamber for another 30 s and was then returned to the home cage. The contextual fear conditioning test (a hippocampus-dependent task) was performed 24 h later by placing each rat back in the same test chamber for 5 min without any stimulation. Two hours later, the tone fear conditioning test (a hippocampus-independent task) was performed by placing each rat in a novel chamber with a different shape, color, and smell from the previous chamber, and the same tone was presented for 3 min without foot shock. Freezing behavior, de ned as the absence of all visible movement except for respiration, was automatically recorded by the video tracking system.

Statistical analysis
Data are presented as the mean ± SEM and analyzed by the Graphpad Prism 8.0 software. The difference between the groups was determined by one-way analysis of variance followed by the Tukey's tests.
Comparisons for the spatial training sessions of MWM were performed by repeated two-way ANOVA followed by LSD test. A p value <0.05 was regarded as statistical signi cance.

Repeated sevo urane induces activation of the NF-κB signaling in the hippocampus of neonatal rats
Sevo urane has been shown to up-regulate the NF-κB signaling pathway in the hippocampus of neonatal rats [2,5]. Herein, our results showed that the protein level of phosphorylation IκBα (p-IκBα) was signi cantly increased and the total level of IκBα was signi cantly decreased in the hippocampus of the developing rats after repeated sevo urane exposure ( Figure 1A). Moreover, the protein level of nuclear NF-κB p65 increased while the level of cytosolic NF-κB p65 reduced in the sevo urane group compared with the control group ( Figure 1B). BAY 11-7082, the most popular NF-κB activation inhibitor, can freely cross the blood-brain barrier [23]. We further investigated the effect of BAY 11-7082 on the expression of NF-κB and IκB. Notably, changes of the protein levels of NF-κB p65 and IκBα induced by sevo urane GA were reversed by the administration of BAY 11-7082 ( Figure 1). Our results indicate that repeated sevo urane induces activation of NF-κB signaling in the hippocampus of neonatal rats and the activation is successfully inhibited by BAY 11-7082 administration.

Inhibiting NF-κB byBAY 11-7082 suppresses the activation of canonical and non-canonical in ammatory caspases in sevo urane anesthesia rats
Pyroptosis can be triggered in the canonical (caspase-1-mediated) and non-canonical (caspase-11mediated) in ammasome signaling pathways. Both processes are tightly controlled by the activation of NF-κB [12][13][14]. In the present study, we showed that the mRNA levels of NLRP3, caspase-1 and caspase-11 were obviously increased in the hippocampus of neonatal rats after repeated sevo urane exposures (Figure 2A). In addition, the protein levels of NLRP3, Pro-caspase-1, Cleaved-caspase-1, Pro-caspase-11 and Cleaved-caspase-11 were signi cantly elevated in the sevo urane group compared with the control group ( Figure 2B and 2C). These changes are molecular characteristics of canonical and non-canonical pyroptotic pathways. Remarkably, in the rats that were pre-treated with BAY 11-7082, sevo urane did not increase both the mRNA and protein levels of NLRP3, caspase-1 and caspase-11 when compared to those of control conditions (Figure 2), suggesting that inhibiting NF-κB activation by BAY 11-7082 suppressed both the canonical and non-canonical pyroptotic pathways after sevo urane anesthesia exposures.

Inhibiting NF-κB by BAY 11-7082 attenuatessevo urane-induced pyroptosis and neuroin ammation
The GSDMD is the substrate of active caspase-1 and caspase-11 and the executor of proptosis. Activated caspase-1 or caspase-11 cleaves the N-and C-terminals of GSDMD and triggers pyroptosis [7][8][9][10]. In vivo study, we showed that repeated sevo urane exposures induced upregulations of GSDMD, GSDMD-N and in ammatory cytokines IL-1β and IL-18 ( Figure 3A and 3B) in the hippocampus of developing rats. These ndings were further con rmed with increased number of GSDMD-positive cells in CA1 area of brain sections in sevo urane-treatment group ( Figure 3C and 3D). In vitro study, western blot analysis from primary neuronal cultures showed that the protein levels of GSDMD and GSDMD-N were signi cantly increased in hippocampal neurons after repeated sevo urane exposures ( Figure 4A). Moreover, ICC staining showed that the intensity of GSDMD-immuno uorescence was elevated in the sevo urane group ( Figure 4B and 4C). Notably, BAY 11-7082 attenuated the cleavage of GSDMD and the release of in ammatory cytokines (Figure 3 and 4), suggesting that inhibiting NF-κB by BAY 11-7082 successfully attenuates pyroptosis and neuroin ammation induced by sevo urane anesthesia.

BAY 11-7082 rescues sevo urane-induced neuronal damage and synaptic dysfunction
Pyroptosis plays important roles in regulating neuronal cell death and in maintaining synaptic integrity and thereby modulating neural networks. To tested whether attenuating NF-κB-mediated pyroptosis could rescue sevo urane-induced neuronal damage. Primary neurons were treated with BAY 11-7082, and MAP2 immunostaining was performed to examine neuronal morphology and neuronal outgrowth. We observed that neurons exposed to sevo urane displayed impaired morphology and dystrophic neurites ( Figure 5A). Remarkably, neurons pre-treated with BAY 11-7082 showed signi cantly improved morphology compared with Sevo urane group. The treatment did not alter neuron morphology in Con+BAY group, indicating that NF-κB inhibitors do not generally improve neuronal outgrowth and branching, but rather protect sevo urane-mediated neurotoxicity. In addition, the cell viability assay showed that BAY 11-7082 treatment increased neuronal viability in the neurons exposed to sevo urane compared to that of vehicle treatment ( Figure 5B).
We further measured the protein levels of synapsin 1 and PSD-95, two important indicators of synaptic structure [24], in the hippocampus of developing rat. Our results showed that repeated sevo urane exposures induced downregulation of synapsin 1 and PSD-95 and BAY 11-7082 pretreatment signi cantly attenuated the downregulation ( Figure 5C), thus suggesting a protective effect of BAY 11-7082 toward synaptic integrity. Collectively, our results provide important evidence that BAY 11-7082 rescues sevo urane-induced neuronal damage and synaptic dysfunction.

BAY 11-7082 ameliorates sevo urane-induced neurocognitive de cits in adolescent rats
To further verify the role of NF-κB-mediated pyroptosis in sevo urane-induced cognitive de cits, open eld test, MWM test and fear conditioning test were performed at PND 40, 50 or 60, respectively. Open eld test showed no difference among the four groups in the spontaneous locomotor activity as re ected by the total distance ( Figure 6A) and the time spent in the center ( Figure 6B), excluding the possibility that locomotor activity per se affected the results in MWM test and fear conditioning test.
The MWM test is a hippocampus-dependent memory test for assessing spatial learning and memory [25,26]. We showed that BAY 11-7082 pretreatment successfully shortened the escape latency in training test ( Figure 6C) and increased the target quadrant time ( Figure 6D) and crossing platform times (Figures 6E) in probe trial in developing rats exposed to sevo urane. The contextual fear conditioning test was used to evaluate the ability of hippocampus-dependent memory [27,28]. We showed that BAY 11-7082 pretreatment ameliorated sevo urane-induced reduction in percentage of freezing time ( Figure 6F). There was no difference in the cued fear conditioning test results among the four groups ( Figure 6G). Our results suggest that NF-κB-mediated pyroptosis may involve in pathogenesis of sevo urane-induced neurocognitive de cits and BAY 11-7082 has a cognitive protective effect in adolescent rats after early exposure of sevo urane.

Discussion
In the present study, we assessed whether NF-κB-mediated pyroptosis is involved in the pathophysiology of neuroin ammation and cognitive de cits after repeated neonatal sevo urane exposures in developing rats. We found that repeated neonatal sevo urane exposures upregulated the expression of NF-κB, NLRP3, caspase-1 and caspase-11, and induced neuroin ammation and pyroptosis in the hippocampus of developing mice. Remarkably, pretreatment with BAY 11-7082, a selective NF-κB inhibitor, inhibited the activation of NF-κB signaling and canonical and non-canonical pyroptotic pathways. Consequently, BAY 11-7082 rescued the hippocampal neuronal damage and synaptic dysfunction and improved the longterm cognitive function in adolescent rats after early exposure of sevo urane. Collectively, our ndings suggested that NF-κB-mediated pyroptosis may play an important role in sevo urane-induced cognitive de cits in developing brain, and NF-κB inhibition might be a potential target for ameliorating neuroin ammation and GAs-induced, developing-related neurocognitive dysfunction.
Neuroin ammation is an essential process in the pathophysiology of GAs-induced neuronal damage and cognitive de cits in developing brain [1][2][3][4][5]. Pyroptosis is a novel and unique type of in ammatory form of programmed cell death and GSDMD has recently been identi ed as the key effector in pyroptosis. Cleavage of GSDMD frees the GSDMD-N domain, which oligomerizes to form pores on the cell membrane. These pores lead to the increase of membrane permeability and the release of in ammatory cytokines, which trigger a cascade of in ammatory response [7][8][9][10]. Pyroptosis has been implicated in the pathogenesis of many in ammatory and non-in ammatory diseases [7][8][9][10]. Our previous study and reports from others have recently demonstrated that the GSDMD-induced pyroptosis is involved in iso urane (a volatile anesthetic)-induced cognitive impairment in aged mice [11] and in ketamine (an intravenous anesthetic)-induced hippocampal neurotoxicity in mouse primary hippocampal neurons [29].
Here, our in vitro study showed that repeated neonatal sevo urane exposures upregulated the expression of GSDMD and GSDMD-N in primary hippocampal neurons, induced impairment of neuronal morphology and network, and caused the decrease of neuronal viability. In addition, our in vivo study showed that sevo urane upregulated the expression of GSDMD, GSDMD-N, IL-1β, IL-18 and synaptic synapsin 1 and PSD-95 in the hippocampus of developing rat. More importantly, sevo urane caused long-term cognitive de cits in adolescent rats. Thus, we speculate that GSDMD-induced pyroptosis is involved in neuroin ammation and neurocognitive impairment after repeated neonatal sevo urane exposures in developing brain.
GSDMD is the substrate for active caspase-1 and caspase-11. The canonical caspase-1 pathway can be triggered by the NLRP3 in ammasome that functions through an interaction with apoptosis-associated speck-like protein (ASC) and the subsequent recruitment of the precursor form of caspase-1 (Pro-caspase-1), leading to the cleavage of caspase-1 and the maturation of IL-1β and IL-18. The non-canonical caspase-11 in ammasome pathway is activated by lipopolysaccharide molecules in the cytoplasm of infected cells. Upon activation, cleaved caspase1/11 can directly mediate GSDMD cleavage and thus serve as important checkpoints in GSDMD-mediated pyroptosis [7][8][9][10]. It has been demonstrated that the levels of pyroptosis-related proteins, including NLRP3, Pro-caspase-1, Pro-caspase-11, Cleaved-caspase-1, and Cleaved-caspase-11 signi cantly increased after multiple doses of ketamine administration in mouse primary hippocampal neurons [29]. Our previous study also observed that the protein levels of NLRP3 and Cleaved-caspase-1 increased in the hippocampus of aged mice after iso urane anesthesia [11]. In line with these studies, the present study data showed that the protein levels of NLRP3, Pro-caspase-1, Cleaved-caspase-1, Pro-caspase-11 and Cleaved-caspase-11 were signi cantly elevated in the hippocampus of neonatal rats after repeated sevo urane exposures. Consistent results were also found in RT-PCR assay with increased mRNA levels. These results indicated that both the canonical and noncanonical pyroptotic pathways were activated in the developing brain after sevo urane anesthesia.
The mechanisms underlying in ammatory caspases activation in pyroptosis are multifaceted. Recent studies showed that NF-κB is an essential transcription factor in pyroptosis. NF-κB is normally sequestered in the cytoplasm, bound to the regulatory protein IκB. Upon activation, IκB gets phosphorylated by the enzyme IκB kinase, which results in the release of the NF-κB. The liberated NF-κB then translocates to the nucleus and induces expression of target genes [30,31], including NLRP3,caspase-1 and caspase-11, for the canonical and non-canonical in ammatory responses [12][13][14]. NF-κB signaling is considered a key factor in the regulation of neuroin ammation and is reported to be activated by inhalation anesthetics, such as iso urane and sevo urane [2,5,32,33]. In this study, repeated sevo urane exposures decreased IκBα and cytosolic NF-κB p65 and increased p-IκBα and nuclear NF-κB p65 in the hippocampus, suggesting the activation of NF-κB signaling after sevo urane anesthesia. Furthermore, after NF-κB activation, NF-κB translocates into the nucleus and subsequently initiate the transcription and translation of NLRP3, caspase-1 and caspase-11 proteins. Therefore, our study suggested a correlation between NF-κB activation and pyroptosis in the developing brain after neonatal GAs exposures.
In order to further demonstrate the role of NF-κB-mediated pyroptosis in cognitive impairment induced by sevo urane anesthesia, Bay 11-7082, a selective inhibitor of NF-κB, was selected for this study. Bay 11-7082 has been shown to irreversibly inhibit IκBa phosphorylation and NF-κB activation [15,16]. It has been proven that Bay11-7802 could attenuate in ammation-induced memory injury in neurodegenerative diseases [34,35]. Our results showed that Bay 11-7082 pretreatment attenuates sevo urane-induced pyroptosis and neuroin ammation by inhibiting activation of canonical and non-canonical in ammatory caspases. Moreover, Bay 11-7082 protected against neuronal damage and synaptic dysfunction and attenuated cognitive impairment in developing rats after sevo urane anesthesia. GSDMD is the executioner of pyroptosis and recent efforts are focusing on the development of inhibitors to interfere with the pore-forming function of GSDMD. In addition to inhibit NF-κB activation, Bay 11-7082 has been shown to potently inhibit GSDMD pore formation in liposomes and in ammasome-mediated pyroptosis and IL-1β secretion in human and mouse cells [36,37]. Thus, inhibition of GSDMD pore formation is supposed to be another protective effect of Bay 11-7082 in GA-induced cognitive de cits. Future studies are needed to con rm this hypothesis.

Conclusion
This study demonstrated, for the rst time, that the NF-κB-mediated pyroptosis may be involved in neuroin ammation and cognitive impairment after repeated neonatal sevo urane. When inhibiting the activation of NF-κB by Bay 11-7082, the pyroptosis mediated by canonical and non-canonical in ammatory caspases was signi cantly alleviated. Our study provides a promising strategy for the treatment of cognitive de cits in the developing brain involving pyroptosis.

Declarations
Availability of data and materials All data generated in this study are included in this manuscript. Figure 1 BAY 11-7082 inhibits sevo urane-induced NF-κB activation. Rat pups at PND 6 were randomly assigned to one of following four treatment protocols: control + vehicle (Con group), control + BAY 11-7082 (Con+BAY group), sevo urane + vehicle (Sev group), and sevo urane + BAY 11-7082 (Sev+BAY group).
BAY 11-7082 (20 mg/kg) or PBS (vehicle) was intraperitoneally administered to the pups 30 min before gas inhalation. Sevo urane anesthesia was induced by putting the rat pups in an anesthetizing chamber delivered with 3% sevo urane for 2 h daily for three consecutive days. For control condition, 30% O2 was delivered at the same ow rate. (A) and (B) Representative western blotting and quantitative analysis of protein levels of IκBα, p-IκBα, cytoplasmic NF-κB and nuclear NF-κB from fresh hippocampal tissue homogenates obtained on PND 8. Values are presented as mean ± SEM (n = 6 rats/group). *p < 0.05 versus the Con group; #p < 0.05 versus the Sev group.  (C) Representative western blotting and quantitative analysis of protein levels of Pro-caspase-11 and cleaved caspase-11 in the hippocampus in developing rats. Values are presented as mean ± SEM (n = 6 rats/group). *p < 0.05 versus the Con group; #p < 0.05 versus the Sev group.