Scopolamine-induced Delirium Promotes Neuroinammation and Neuropsychiatric Disorder in Mice

Background Postoperative delirium is a common neuropsychiatric syndrome resulting in a high postsurgical mortality rate and decline in postdischarge function. Extensive research has been performed on both human and animal delirium models due to their clinical signicance, focusing on systemic inammation and consequent neuroinammation playing a key in the pathogenesis of postoperative cognitive dysfunctions. Since animal models are widely utilized for pathophysiological study of neuropsychiatric disorders, this study aimed at examining the validity of the scopolamine-induced delirium mice model with respect to the neuroinammatory hypothesis of delirium. Methods Male C57BL/6 mice were treated with intraperitoneal scopolamine (2 mg/kg). Neurobehavioural tests were performed to evaluate the changes in cognitive functions, including learning and memory, and the level of anxiety after surgery or scopolamine treatment. The levels of pro-inammatory cytokines (IL-1 (cid:0) , IL-18, and TNF-α) and inammasome components (NLRP3, ASC, and caspase-1) in different brain regions were measured. Gene expression proles were also examined using whole-genome RNA sequencing analyses to compare gene expression patterns of different mice models.

the activation of in ammation and immune systems is strongly regarded as the key mechanism for cognitive deterioration postsurgery (6). Surgery and anaesthesia unleash a body-wide in ammation releasing peripheral in ammatory cytokines affecting the integrity of the blood-brain barrier, thus, allowing increased in ltration of in ammatory factors and macrophages into the brain (7).
Forebrain cholinergic neurons play a fundamental role in controlling the central nervous system with regards to attention, memory, and cognitive function, and are implicated in cognitive decline and several neurodegenerative diseases (8)(9)(10). The impact of cholinergic pathways on the immune system is also well-documented (8,11) showing that systemic in ammatory responses are under the control of the cholinergic anti-in ammatory pathway supplied by connections of the vagus nerve (8,12). In a similar context, experimental and clinical studies focusing on the pathogenesis of delirium show that it is accompanied by cholinergic pathways and agents (13,14).
Scopolamine is an anti-cholinergic drug that antagonises the muscarinic cholinergic receptors (mAChRs) and is capable of producing de cits in the processes of learning, acquisition, and consolidation (15). Scopolamine-treated animal models are widely used in neurocognitive studies because scopolamine administration induces both the behavioural and molecular features of Alzheimer's disease and other neurocognitive disorders, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission in the hippocampus and prefrontal cortex (16)(17)(18)(19). However, scopolamine treatment has also been reported to show opposite effects such as anti-depressant and anti-anxiety effects (20).
Despite the highly analogous clinical traits and alterations in cholinergic neurotransmission by sustained neuroin ammation in scopolamine-treated animal models (21,22), the reproducibility of the exact pathogenesis of POD still remains insu cient. The aim of this study was to validate the effectiveness of scopolamine-treated animal models as POD experimental models, by identifying the change in in ammation-related cytokines and candidate genes using RNA sequencing technology by comparing a scopolamine-induced delirium mice model and surgery mice.

Animals
All in vivo experimental procedures were certi ed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System. All experiments were conducted according to the speci ed guidelines. All mice were housed under controlled environment with 12-h light/dark cycles, and ad libitum access to chews and water in a pathogen-free facility at the Yonsei Biomedical Research Institute. Male C57BL/6 mice aged 9-12 weeks from Orient Bio (Seongnam, Gyeonggi-Do, South Korea) were used for the experiment.

Experimental Design and Procedure
The mice were randomly assigned into three groups: 1) a vehicle-treated control group (control), 2) a surgery group (surgery), 3) and a scopolamine-treated group (scopolamine). Mice in the control group were kept unaffected to the experimental conditions, while mice in the surgery group underwent abdominal surgery as a positive control for the scopolamine-treated group. For the scopolamine-treated group, scopolamine (2 mg/kg) was dissolved in sterile saline (0.9% NaCl w/v) with the volumes for the administration prepared according to the body weight. Mice in the surgery group performed behavioural tests at day 4 and 5 postsurgery. Mice in both the control and scopolamine-treated groups performed behavioural tests at day 1 before scopolamine or vehicle treatment. At day 2, the mice were injected with scopolamine intraperitoneally 30 min before the neurobehavioural tests. The same amount of sterile saline was administered into mice of the control group 30 min before the neurobehavioural tests ( Figure  1A and B). The surgical procedure was as follows. In the surgery group, mice were anaesthetised with 4% iso urane and were maintained with 1.5-2% iso urane in oxygen at a ow rate of 1 L/min. Mice were placed on a heating pad during anaesthesia to prevent hypothermia. Abdominal surgery was performed as mentioned previously, with some modi cations (23,24). After vertically incising along the midline following the linea alba, the mesenteric artery was clipped for 20 min, and intestines were rubbed for 30 sec. The exteriorised abdominal muscle and skin were placed back into the peritoneal cavity and closed using sutures. Mice were returned to the home cage. At day 5 postsurgery, mice brains, including the hippocampus, prefrontal cortex, and amygdala, were isolated after sacri ce.

Neurobehavioural Assessment
The neurobehavioural ndings of the mice were assessed using the open eld test (OFT), elevated plus maze (EPM), and novel object recognition test (NORT). The behavioural tests were performed 30 min after drug administration according to the treatment plan. All the neurobehavioural tests were recorded on video and analysed with an image analysing system (SMART v2.5.21 software and SMART video tracking system, Panlab Harvard Apparatus, Barcelona, Spain) by an assessor blinded to the treatment groups. The mice sequentially performed the OFT, EPM, and NORT.

Open Field Test
Mice were placed in a square open eld arena (40 × 40 × 40 cm), were allowed to explore for 5 min, and the behaviours were recorded simultaneously. Total distance moved was used as a measure of general activity and locomotor function. The animal's tendency to avoid the centre area re ects the anxiety related behavioural change (25).

Elevated Plus Maze
The EPM was performed to evaluate anxiety related behaviour, learning, and memory functions of the mice. The maze consisted of two open arms (31 × 6 × 1 cm) and two closed arms (31 × 6 × 15 cm) extended from a central platform (5 × 5 × 1 cm), and was elevated to a height of 50 cm (JEUNGDO Bio & Plant Co., Ltd.) from the oor. The mice were trained prior to surgery and tested at day 5 postsurgery.
Mice were individually placed at the end of the open arm facing the other open arm and were allowed to explore for 5 min. The total duration of time spent in the open and closed arms was recorded respectively. The apparatus was cleaned with 70% ethanol prior to all tests. Entry was de ned as the placement of all paws into the arm of the maze. The percentage of time spent in the open arms was also measured.
Learning index was calculated as follows: learning index = the rst latency time to enter the closed arm (training period) -the rst latency time to enter the closed arms (test period). The duration of time spent in the open arms also re ects anxiety related behaviour (26).

Novel Object Recognition Test
NORT was performed to evaluate cognition, especially recognition memory in the mice (27). During the habituation phase, each mouse was allowed to explore the square box (40 × 40 × 40 cm) freely for 5 min. During the familiarisation phase, the mice were placed into the box, which contained two identical objects (A + A), and were allowed to explore for 5 min. During the test phase, each mouse was returned to the box with the two objects, where one object was changed into a novel object (A + B), and mice were allowed to explore for 5 min. During both the familiarisation and the test phases, time spent in exploring each object was measured and recorded. At the end of each test, the apparatus and objects were cleaned with 70% ethanol. The habituation phase was performed immediately prior to surgery, the familiarisation phase was performed at day 4 postsurgery, and the test trial was performed at day 5 postsurgery. The discrimination index was evaluated as (time taken to explore novel object B) / (time taken to explore novel and familiar objects) × 100, which re ects cognitive ability.

Enzyme-linked Immunosorbent Assay (ELISA)
For the in vivo cytokine experiment, the hippocampus, prefrontal cortex, and amygdala of mice were obtained at day 5 postsurgery after the neurobehavioural tests and stored at −80°C until use. To measure the levels of TNF-α, IL-1 , and IL-18 in the three different regions, brains were lysed using tissue protein extraction reagent (T-PER® Tissue Protein Extraction Reagent, Thermo Scienti c™, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktail (100X Halt protease and phosphatase inhibitor cocktail, #1861281 Thermo Scienti c™). The tissues were then homogenised and centrifuged at 13,000 rpm for 10 min to obtain sample supernatants. Supernatant protein concentrations were measured using a BCA Protein Assay Kit (Thermo Scienti c™) according to the manufacturer's speci cations. Levels of TNF-α, IL-1 , and IL-18 in the lysates were assayed using high-sensitivity ELISA kits (Quantikine® ELISA, R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's speci cations. Brie y, samples were added to the assay plates at a volume of 50 μL/well and incubated for 2 h at room temperature. After washing plates with the wash buffer from the kit, TNF-α, IL-1 , and IL-18 conjugates were added to each well and incubated for 2 h. The reaction was stopped and the absorbance of each well was measured at 450 nm using a microplate reader. To measure the levels of NLR family pyrin domaincontaining protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and caspase-1 in lysates, ELISA kits from MyBioSource (San Diego, CA, USA) were used for this assay, and all procedures followed the manufacturer's instructions.

RNA Extraction and Gene Expression Pro ling
Total RNA from mouse brain tissue was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and quantity were assessed using Agilent 2100 bioanalyser (Agilent Technologies, USA) and ND-1000 spectrophotometer (NanoDrop Technologies, USA), respectively. RNA samples were used as input into the Affymetrix procedure (Affymetrix, Santa Clara, CA, USA) as recommended by protocol (http://www.affymetrix.com), of which total RNA from each sample was converted to double-stranded cDNA. Ampli ed RNA (cRNA) was generated from the double-stranded cDNA template through an IVT (in vitro transcription) reaction using a random hexamer incorporating a T7 promoter and puri ed with the Affymetrix sample cleanup module. cDNA was regenerated from a random-primed reverse transcription using a dNTP mix containing dUTP. UDG and APE 1 restriction endonucleases were used for fragmenting cDNA, which was then end-labelled by terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labelled cDNA was hybridised to the Affymetrix arrays for 16 hours (45℃ and 60 rpm) as described in the Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). The chips were stained using SAPE (Streptavidin Phycoerythrin), washed in a Genechip Fluidics Station 450 (Affymetrix) and scanned using Affymetrix Model 3000 7G scanner. The scanned image data were extracted through Affymetrix Command Console 1.1 software to generate raw CEL les, which show expression intensity data. Expression data were generated by Transcriptome Analysis Console 4.0.1. For the normalisation, RMA (Robust Multi-Average) algorithm implemented in Transcriptome Analysis Console software was used.

RNA Sequencing Analysis of Differentially Expressed Genes
Genes with a more than two-fold difference in the normalised signals as compared to those in control group were selected as differentially expressed genes (DEG). Gene ontology analysis of the DEGs was performed by exDEGA (Excel based Differentially Expressed Gene Analysis, eBIOGEN, Inc., Seoul, Korea) tool. Categorisation of the genes was based on a search performed using DAVID v6.8 (http://david.abcc.ncifcrf.gov). In each group, gene expression level was converted to a log2 value, and the relative level with respect to the control group was presented. The clustering heatmap pro les of DEGs were compared across the experimental groups using the Multiple Experiment Viewer software program v4.9 (MeV). The average fold change (FC) for each gene was expressed as a standardised zscore.

Statistical Analysis
Statistical analyses were performed using SPSS v25.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7.00 software (GraphPad Software, San Diego, CA, USA). Values are presented as mean ± standard error of the mean (SEM). Unpaired t-tests were performed to determine statistical signi cance. p-values < 0.05 were considered signi cant.

Scopolamine Treatment Induces Delirium-like Cognitive Dysfunction in Mice
Delirium is related to accelerated cognitive dysfunction (2). Therefore, we examined the role and validity of scopolamine treatment as the POD model by causing cognitive impairment, similar to POCD caused by the abdominal surgery model. Behavioural tests of EPM and NORT were carried out to assess the changes in learning and memory of mice before/after surgery or scopolamine treatment ( Figure 2). During the EPM test, surgery mice exhibited signi cant decrease in learning index as compared to control mice, which indicated POCD. Similarly, scopolamine-treated mice displayed a signi cant drop in learning index with no difference between the surgery and scopolamine groups (Figure 2A), as compared to mice from the control group. The mice were made to perform the NORT to examine hippocampal-dependent learning and memory. The duration of time spent exploring the novel object (calculated in percentage using the formula) signi cantly decreased in the surgery and scopolamine-treated groups, emphasising the effect of surgery and scopolamine treatment on impairment of cognitive ability ( Figure 2B). The SMART video tracking system showed real-time tracking of each group in NORT ( Figure 2C). Conclusively, neurobehavioural tests indicated that scopolamine treatment signi cantly caused memory impairments and decrease in cognition in mice, similar to the results seen in mice of the surgery group.

Transcriptome Analysis Shows Scopolamine Treatment Alters Gene Expression Pattern in the Hippocampus
To investigate whether surgery or scopolamine treatment affects the gene expression pattern in animal models, we used RNA sequencing analysis to compare the gene expression of hippocampal samples from control, surgery, and scopolamine-treated mice ( Figure 3 and Table 1). The heat map showed the two-way hierarchical clustering comparing the gene expression levels among different groups: surgery or scopolame ( Figure 3A). Dock8, Myo1f, Treml2, Gas5, Lpar2, and Ralbp1, were upregulated or downregulated after surgery or scopolamine treatment. The normalised repeatability coe cient (RC) of Dhx58, which is involved in processes of the immune system, was upregulated in both the surgery and scopolamine-treated groups, as compared to the control group. The gene expression levels of Dock8, Myo1f, and Treml2 were downregulated in both the surgery and scopolamine-treated groups. In the case of Gas5, only the surgery group showed downregulated levels, as compared to the control group. Only the scopolamine-treated group exhibited upregulated levels of Lpar2. Compared to the surgery group, the level of Ralbp1 was downregulated in both the control and scopolamine-treated groups ( Figure 3B). In addition, genes involving hippocampal function and the nervous system, such as Lrrn4, S100a10, Slc5a7, and Wnt6 were altered by surgery or scopolamine treatment. Gene expression of Lrrn4 and S100a10 was upregulated, while that of Slc5a7 was downregulated in both the surgery and scopolamine-treated groups, as compared to the control group. However, only the scopolamine-treated group showed e ciently downregulated expression of Wnt6, as compared to the control and surgery groups ( Figure 3C).

Hippocampus
Based on the changes of in ammatory genes in RNA sequencing analysis, the levels of pro-in ammatory cytokines were measured from the hippocampal samples using ELISA to compare the effects of surgery and scopolamine on the in ammatory reaction (Figure 4). Both surgery and scopolamine-treated mice showed signi cantly increased levels of pro-in ammatory cytokines (TNF-α, IL-1β, and IL-18) ( Figure 4A, B, and C). The levels of pro-in ammatory cytokines IL-1β and IL-18 are regulated by in ammasomes. Therefore, we measured protein levels of in ammasome components in the hippocampus. NLRP3 in ammasome, an intracellular sensor that detects a broad range of microbial motifs, has been demonstrated to increase the level of pro-in ammatory cytokines such as IL-1β and IL-18 in the brain by activating caspase-1. NLRP3 in ammasome further promotes the aggregation of innate immune cells and initiates the downstream in ammatory cascade that ultimately accelerates the pathological progression of neurocognitive disease (28). The expression level of NLRP3 in ammasome components such as NLRP3, ASC, and caspase-1 in the hippocampus were signi cantly upregulated in both the surgery and scopolamine-treated groups, as compared to the control group ( Figure 4D, E, and F).

Scopolamine Treatment Promotes Delirium-like Anxiety and Hyper-activation in Mice
We measured the level of anxiety to investigate whether scopolamine administration could lead to psychological disturbance, a characteristic trait of delirium. Mice behaviour tests were conducted using EPM and OFT ( Figure 5). Mice exhibiting the state of anxiety are known to display a tendency of staying in the closed arms of the EPM, thus, scopolamine-treated mice spent signi cantly less amount of time in the open arms as compared to mice from the control and surgery groups, while surgery mice also showed less time in open arms, as compared to those in the control group ( Figure 5A). In OFT, there was no signi cant difference between the total distance travelled by the control and surgery group mice. However, mice in the scopolamine-treated group exhibited hyperactive motor activity resulting in signi cantly greater total distance travelled, as compared to mice in the surgery group ( Figure 5B). High activity is used as an index of low emotionality (29). Additionally, scopolamine-treated mice showed lesser distance travelled in the central zone of the OFT ( Figure 5C), suggesting increased level of anxiety. The SMART video tracking system showed visual tracking of each group in EPM and OFT ( Figure 5D and E).

Scopolamine-treated Mice Display Pro-in ammatory Cytokines and In ammasome Components in the Prefrontal Cortex and Amygdala
The prefrontal cortex and amygdala are engaged in emotional response and mood regulation, such as anxiety and depression (30). We further checked the levels of pro-in ammatory cytokines and NLRP3 in ammasome components in the prefrontal cortex and amygdala ( Figure 6). Compared to the control group, levels of pro-in ammatory cytokines, such as TNF-α, IL-1 , and IL-18, were remarkably higher in the surgery group mice. Additionally, mice of the scopolamine-treated group showed increased levels of TNFα, IL-1 , and IL-18; although, IL-1 and IL-18 protein levels in scopolamine-treated mice were signi cantly reduced as compared to those in the surgery group ( Figure 6A, B, and C). The levels of NLRP3 in ammasome components, such as NLRP3, ASC, and caspase-1, were highly expressed in the prefrontal cortex and amygdala postsurgery. Similar to the surgery group, the scopolamine-treated group showed increased levels of NLRP3 in ammasome components in the prefrontal cortex. Furthermore, the levels of NLRP3 in the amygdala were different between mice in the surgery and scopolamine-treated groups with no incremental changes in the latter. However, ASC and caspase-1 expressions were higher in the scopolamine-treated group ( Figure 6D, E, and F).

Discussion
In the present study, scopolamine-treated animals and postsurgery animals both showed characteristic delirium-like behavioural patterns, and the biochemical ndings were coherent with neuroin ammatory changes. RNA sequencing pro les also showed consistent changes in gene expression patterns relevant to the immune/in ammatory reaction and nervous system development in both the surgery and scopolamine-treated groups, though there are some discrepancies in the expression patterns of in ammation-related genes. These results suggest the compatibility of scopolamine-treated delirium animal models in re-enacting the clinical features of POD, although there could still be undisclosed pathophysiology and insu cient explanation for neuroin ammatory-based POD. The cholinergic neurotransmitter system is responsible for the control of cognitive processes, acquisition, and retention of information as well as task performance. Numerous studies have reported the role of cholinergic function in age-related memory dysfunction and other neurocognitive diseases (31,32). Scopolamine is known to reproduce delirium-like state in both human and experimental animals (33)(34)(35)(36) by inducing dysregulation of cholinergic signals in the brain; such cognitive declining properties of scopolamine have contributed to its wide application in neurocognitive research. Oxidative stress has also been proposed as the possible cause of neurocognitive disease along with the cholinergic hypothesis. Regarding the use of scopolamine, previous studies have reported the association of systemic administration of scopolamine with increased oxidative stress in the brain, especially the areas associated with memory and learning, such as the hippocampus and prefrontal cortex, some of which are reported to be involved in mitochondrial dysfunction (16,37,38). Furthermore, scopolamine-induced memory impairment is successfully attenuated by several anti-oxidant compounds, emphasising the crucial role of oxidative stress in scopolamine-induced amnesia (39,40). Although dysregulation of the cholinergic system and oxidative stress have been a promising hypothesis explaining pathogenesis for POCD and delirium, increased in ammation still plays an important part in explaining the development of neurocognitive disorders, of which increased level of pro-in ammatory cytokines, such as TNF-α IL-1β, IL-18, and IL-6, can be observed in the brain and blood samples of patients with dementia, delirium, and POCD (41)(42)(43)(44). Scopolamine administration has also been shown to cause increase in pro-in ammatory cytokines, and in ammation has been proposed as the theoretical basis for scopolamine-induced memory impairment (16,45). The signi cance of neuroin ammation in delirium is consistent with our results showing the increase of pro-in ammatory cytokines in both the surgery and scopolamine-treated groups.
NLRP3 in ammasome is an essential mediator of host immune responses through the activation of caspase-1 and leads to the maturation of pro-in ammatory cytokines (46). Its activation had been the marker for host immune defence mechanism in various in ammatory diseases with no exceptions to the development of neurodegenerative disorders (46)(47)(48). In this study, the activation of NLRP3 in ammasome was observed in three different brain regions, of which the hippocampal area of scopolamine-treated mice showed increased activation. Increased activation of NLRP3 in ammasome was also seen in the hippocampus of surgery mice implying POCD. Therefore, NLRP3 in ammasome may be the main cause of cognitive impairment after surgery as well as scopolamine treatment.
Amygdala is the brain region crucial for executive function, memory, attention, and especially for the pathogenesis of delirium, along with the frontal lobes, diencephalon, and hippocampus (35,49). In addition, the dysregulated prefrontal cortex-to-amygdala pathway is associated with anxiety (30). Previous studies have shown the signi cant diminution of neurotransmitters, including dopamine, 3,4dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), and acetylcholine in the amygdala of scopolamine-treated animals as compared to control group animals and also compared to the hippocampus area within the same scopolamine-treated groups (35,49). In addition, evidence has also suggested altered neurotransmission especially in the regions related to memory, such as the hippocampus and amygdala, in relation to outcomes seen in POD. The signi cance of in ammasomes is very important in the neuroin ammatory-based hypothesis for POD. In the present study, levels of NLRP3 in ammasome components were all higher in the prefrontal cortical area of scopolamine-treated mice; however, the expression level was different in the amygdala, showing increase in in ammasome component levels after scopolamine treatment, but not in NLRP3 levels. Detailed explanation for transition aspects of NLRP3 in ammasome in the amygdala could be limited due to restricted information focusing on the direct changes of in ammasome in the amygdala, of which reduced expression of NLRP3 in ammasome can only be indirectly inferred from altered or abnormal expression of neurotransmitters in the amygdala or other kinds of in ammasome complexes (e.g. NLRP2, NLRC4, and AIM2) might be involved in the pathogenesis of delirium.
RNA sequencing analysis technique has been widely used for various human diseases, and a large amount of genetic data has been produced over the past decades from various diseases. With the help of RNA sequencing analyses from different treatment groups, this study successfully identi ed the altered gene expression patterns from each group to further investigate the reliability of scopolamine-treated animal models as compatible delirium models from the neuroin ammatory point of view. This study also identi ed the genes with signi cant expression pro les related to in ammation and the development of the nervous system that presented with a more distinct expression pattern in surgery mice as compared with scopolamine-treated mice (Table 1). For example, gene expression of Ralbp1, which was signi cantly upregulated in surgery mice compared to control mice and downregulated in scopolaminetreated mice compared to surgery mice, plays a role in receptor-mediated endocytosis and is a downstream effector of the small GTP-binding protein RAL (50). Ralbp1 is known for its oncogenic role and necessary role in cell proliferation and invasion in carcinogenesis (51), and it is also known for regulating obesity-promoting pro-in ammatory cytokines (52). Lpar2 encodes a member of family I of the G protein-coupled receptors, as well as the endothelial cell differentiation gene (EDG) family of proteins. Lysophosphatidic acid signalling via receptors regulates diverse malignant cellular functions such as cell proliferation, motility, invasion, and metastasis (53). Furthermore, Lpar2 antagonism prevents ageing-related cognitive impairment in mice (54). From the in ammation and immune perspective, Lpar2 functions as the negative regulator for innate immune response to prevent excess in ammation, tissue injury, and to promote homeostasis, with increased expression of Lpar2 to suppress T-helper type 2 (Th2)driven in ammation in mice models (55). Lpar2 gene expression was upregulated 1.5 times in scopolamine-treated mice, while no dramatic changes were seen in surgery mice in this study. This could imply the possible suppression of Th2-driven in ammation followed by increased expression of Lpar2, despite the decreased acetylcholine (Ach) level seen in scopolamine-treated group, since Ach is known to modulate key processes of the allergic in ammatory response via muscarinic and nicotinic receptors (11). Increased levels of Ach are known to activate Th2 differentiation and polarisation via native T cells and dendritic cells, respectively (11). Such changes in the Ach level from scopolamine treatment could also affect the in ammatory and immune response via Lpar2 expression levels in the scopolaminetreated mice group.
However, gene expression patterns from both the surgery and scopolamine-treated groups showed that a number of notable genes were associated with the analogous cellular functions, mostly focusing on in ammatory reactions and the development of the neuronal system, although expression levels and signi cance vary to some extent. For example, Dhx58 coding LGP2, is related to the regulation of innate immune responses, the immune system, and apoptosis during viral infection (56). Myo1f encodes myosin-1F protein and is expressed mainly in the immune system (57). It is involved in the regulation of M1-polarisation during the in ammatory process, whereas Myo1f de ciency is known to attenuate the commitment of macrophages into a pro-in ammatory phenotype. Myo1f de ciency model strongly reduces the secretion of pro-in ammatory cytokines, decreases epithelial damage, ameliorates disease activity, and enhances tissue repair (58,59). Treml2 is a transmembrane protein, which expresses in various immune cells, such as monocytes, macrophage, and microglia. Treml2 is involved in innate and adaptive immunity and causes increase in number of macrophages under in ammatory conditions (60).
Another gene, Lrrn4, which was upregulated in both the surgery and scopolamine-treated groups, is a protein coding gene playing a crucial role in hippocampus-dependent long lasting memory (61). S100a10 encodes a member of the S100 family of protein containing 2 EF-hand calcium-binding motifs called S100A10 or p11, which are involved in the regulation of various cellular processes such as cell cycle progression and differentiation. Upregulated levels of S100a10 in the hippocampus engage in processing emotional memory and altered hippocampal functionality (62), which is due to the interaction of p11 with serotonin-signalling proteins. Moreover, as cognitive impairments are common in delirium due to dysfunctional serotonin neurotransmission, p11 protein is known to interact with serotonin-signalling proteins and correlate with symptoms of mood disorders (62,63). Scl5a7 is implicated in the delivery of the precursor choline from the synaptic space into the presynaptic terminal, which is important in cholinergic neuronal communication (64). To summarise, scopolamine administration can successfully reproduce characteristic neuropsychiatric behavioural changes of delirium in accordance with the neuroin ammatory hypothesis, although less consistency was noted in descriptive RNA sequencing analysis studies. In this study, we have only suggested candidate genes involved in scopolamine-treatment delirium. Therefore, further studies focusing on delirium and the involvement of candidate genes for pathophysiological studies are needed.
Delirium represents the large spectrum of cognitive and behavioural abnormalities from hypoactive form, with negative symptoms of inattention and at affect, to hyperactive form, with characteristic agitation and anxiousness (65). In this study, neurobehavioural tests showed signi cant increase in the level of anxiety and hyper motor activity, and impairment in memory and cognitive function in mice from the scopolamine-treated group; this seems to be associated with increased level of in ammatory response. Scopolamine is particularly known to damage learning and short-term memory functions in rodents and humans by disrupting cholinergic transmission (16), which makes it the most widely used drug for reproducing delirium in animals. However, disagreements regarding altered cognitive function in scopolamine-treated delirium animal models, perhaps due to lack of credibility of neurobehavioural tests, especially those focusing on cognition, were also witnessed. Different neurobehavioural changes could possibly be due to a large spectrum of behavioural delirium, or potentiality of other distinct mechanisms underlying the development of cognitive dysfunction, apart from the disruption of cholinergic transmission by scopolamine. For one reason or another, inconsistent ndings of neurobehavioural patterns obtained from the tests raise doubts on the credibility of scopolamine-treated delirium animal models in re-enacting characteristic ndings of delirium in humans. Thereby, the reliability of scopolamine treatment as a standard protocol for reproducing delirium via neuroin ammation-based pathophysiology still needs to be studied further.

Conclusion
In conclusion, our results show that scopolamine-induced POD animal models had succeeded in showing the analogous neurobehavioural patterns, and thus, present ndings coherent with neuroin ammatory reactions in POD. However, genetic analyses showed the limitations as an indispensable explanation between candidate genes and delirium. Undeniably, further studies addressing the possible interactions between delirium and candidate genes after scopolamine treatment for pathophysiological studies are necessary.

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Availability of data and materials
All data supporting the conclusions of this manuscript are provided in the text and gures.

Competing interests
The authors declare no con icts of interest. The funders had no role in the design of this study, the writing of the manuscript, or the decision of publishing this paper.  Figure 1 Experimental design and procedure. (A) Scopolamine (2 mg/kg) was injected intraperitoneally. Mice were subjected to abdominal surgery by clipping the mesenteric artery and rubbing the intestines. All mice brains were isolated, and brain sub-regions, such as the hippocampus, prefrontal cortex, and amygdala, were analysed after surgery or scopolamine treatment. (B) Mice from the surgery group performed behavioural tests at day 4 and 5 postsurgery. Mice from both the control and scopolamine-treated group performed behavioural tests at day 1 before scopolamine or vehicle treatment.   The levels of pro-in ammatory cytokines and NLRP3 in ammasome components in the hippocampus.