Maresin1 ameliorates postoperative cognitive dysfunction in aged rats by potentially regulating the NF-κB pathway to inhibit astrocyte activation

Postoperative cognitive dysfunction (POCD) is one of the most serious postoperative complications in the elderly population. Perioperative central neuroinflammation is considered to be an important pathological mechanism of POCD, with the activation of astrocytes playing a key role in central neuroinflammation. Maresin1 (MaR1) is a specific pro-resolving mediator synthesized by macrophages in the resolution stage of inflammation, and provides unique anti-inflammatory and pro-resolution effects by limiting excessive neuroinflammation and promoting postoperative recovery. However, the question remains whether MaR1 can have a positive effect on POCD. The objective of this study was to investigate the protective effect of MaR1 on POCD cognitive function in aged rats after splenectomy. Morris water maze test and IntelliCage test showed that splenectomy could cause transient cognitive dysfunction in aged rats; however, the cognitive impairment of rats was significantly mitigated when MaR1 pretreatment was administered. MaR1 significantly alleviated the fluorescence intensity and protein expression of glial fibrillary acidic protein and central nervous system specific protein in the cornu ammonis 1 region of the hippocampus. Simultaneously, the morphology of astrocytes was also severely altered. Further experiments showed that MaR1 inhibited the mRNA and protein expression of several key proinflammatory cytokines-interleukin-1β, interleukin-6, and tumor necrosis factor-α in the hippocampus of aged rats following splenectomy. The molecular mechanism underlying this process was explored by evaluating expression of components of the nuclear factor κB (NF-κB) signaling pathway. MaR1 substantially inhibited the mRNA and protein expression of NF-κB p65 and κB inhibitor kinase β. Collectively, these results suggest that MaR1 ameliorated splenectomy-induced transient cognitive impairment in elderly rats, and this neuroprotective mechanism may occur through regulating the NF-κB pathway to inhibit astrocyte activation.


Introduction
Postoperative cognitive dysfunction (POCD) is one of the most serious postoperative complications in elderly patients (Deiner and Silverstein, 2009). The postoperative cognitive ability of such patients will be impaired, including learning, memory, orientation, attention, perception, awareness, and judgment (Kotekar et al., 2018;Lin et al., 2020). POCD is reported to occur days or weeks after surgery and can last for months or even years (Monk et al., 2008;Fodale et al., 2010;Evered et al., 2011). POCD can lead to severe cognitive deficits, which not only affect overall morbidity and mortality but also increase hospitalization costs, causing a heavy burden to individuals, families, and society (Olotu, 2020). However, the definition of POCD currently remains unclear, the evaluation methods are difficult to unify, and neither drug nor surgical methods have achieved satisfactory therapeutic efficacy.
The pathogenesis of POCD predominantly includes central neuroinflammation (Luo et al., 2019), oxidative stress (Netto et al., 2018), cholinergic system disorder (Heinrich et al., 2021), and amyloid deposition (Schaefer et al., 2019), etc., with perioperative central neuroinflammation being one of the most important mechanisms underlying the pathogenesis of POCD (Safavynia and Goldstein, 2018). Activation of glial cells, including microglia and astrocytes, is instrumental in the inflammatory response of the central nervous system (CNS) (Liu et al., 2022). When central nerve cells are damaged, astrocytes are activated, accompanied by increased secretion of glial fibrillary acid protein (GFAP), tumor necrosis factor (TNF-α), interleukin (IL-1β), interleukin-6 (IL-6) and other markers, which have toxic effects on neurons (Zhang et al., 2016). Another study found that surgical stimulation caused a blow similar to mild traumatic brain injury, which subsequently resulted in long-term cognitive impairment in mice, as shown by the influence of water maze test and olfactory test (Zhang et al., 2011). However, following administration of brain activator, which can inhibit astrocyte activation, the experimental mice exhibited improved performance on these tests (Zhang et al., 2011). These findings suggest that astrocytes may be involved in the occurrence of POCD, and also suggest that inhibition of astrocyte activation can improve the symptoms of POCD.
Nuclear factor-κB (NF-κB) is a transcription factor that plays an important role in cell survival and inflammation (Ridder and Schwaninger, 2009). NF-κB activation is triggered by phosphorylation and subsequent degradation of the inhibitor of NF-κB (IκB) (Hayden and Ghosh, 2011). When the signaling pathway is activated, IκB protein is degraded and NF-κB dimers enter the nucleus to regulate the expression of target genes (Hayden and Ghosh, 2011). The whole process is mediated by the IκB kinase (IKK) complex, which consists of two kinase subunits (IKKα and IKKβ) and a regulatory subunit (NEMO), with IKKβ regulating activation of the classical pathway through the phosphorylation of IκB (Lawrence, 2009). Inhibition of the NF-κB-IKKβ cascade is an attractive therapeutic method for the genesis of new model to combat certain chronic multifactorial diseases (Freitas and Fraga, 2018). NF-κB may also regulate the production of various proinflammatory factors in astrocytes, including IL-6, IL-1β, and TNF-α (Hwang et al., 2015;Phuagkhaopong et al., 2017). Zhu found that activation of the NF-κB pathway induced by lipopolysaccharide or sevoflurane exposure caused astrocyte activation and cognitive dysfunction using the POCD model . In addition, selective inhibition of NF-κB has been reported to inhibit the activation of astrocytes, leading to subsequent downregulation of the chemokines released by astrocytes, a reduction in macrophage and T cell infiltration, and thus a reduction in secondary inflammatory damage in CNS diseases (Yuan et al., 2017). These studies suggest that improving POCD symptoms by regulating NF-κB to inhibit astrocyte activation may be a potential therapeutic target. Maresin1 (MaR1) is a proinflammatory resolution lipid mediator derived from docosahexaenoic acid, which has anti-inflammatory and proinflammatory resolution activities (Serhan et al., 2009). In the mouse model of colitis, MaR1 reduced the release of inflammatory mediators such as TNF-α, IL-1β, and IL-6 by inhibiting the NF-κB pathway, thereby alleviating inflammation-related intestinal injury (Marcon et al., 2013). Furthermore, the use of MaR1 in the Alzheimer's disease mouse model reduced the activation of astrocytes in the hippocampus and cortex of the mice and thus improved cognitive function of the mice (Yin et al., 2020). However, there is currently limited research on whether MaR1 can alleviate POCD symptoms by inhibiting astrocyte activation through the NF-κB pathway.
We hypothesized that MaR1 could attenuate postoperative cognitive decline by inhibiting the degree of astrocyte activation through regulation of the NF-κB pathway. A series of experiments were performed in the current study to test this hypothesis, and the results suggest that pretreatment with MaR1 has a positive impact on POCD.

Animals
90 aged male Sprague-Dawley rats (18-20 months old, 450-600 g) were used for this study, that they were provided by the Laboratory Animal Center of Ningxia Medical University. All animals were reared under standard environmental conditions (specific pathogen-free, 12hour light-dark cycle, ambient temperature 22 ± 2 • C and relative humidity 60 ± 5 %). The experimental rats were provided with adequate food and water to satisfy free foraging. All protocols were approved by the Ethics Committee of Ningxia Medical University [Approval number SCXK(Ning) 2020-0001].

Anesthesia and surgery
A POCD model was established by sevoflurane anesthesia and splenectomy as previously reported (Lu et al., 2015;Feng et al., 2021). Rats were anesthetized for 2 min in an anesthesia induction box equipped with 5 % sevoflurane (Shanghai Hengrui Pharmaceutical Co., Ltd. China, cat: 22011051), and splenectomy was performed under 3 % sevoflurane maintenance for approximately half an hour. Briefly, the surgical area of the skin was prepared, followed by iodine disinfection, then a longitudinal incision of approximately 2 cm was made along the midline of the abdomen. The peritoneum was entered, the spleen was exposed, and the spleen was removed after adequate ligation of the splenic arteries and veins. The wound was closed in layers using sutures. Pulse oxygen and temperature were continuously monitored and maintained within the normal range during the splenectomy. After splenectomy, ropivacaine hydrochloride injection (Zhejiang Xianju Pharmaceutical Co. Ltd., China, cat: EE2202) (diluted to 1.5 mg/mL with normal saline, 2 mg/kg) was administered around the incision for local infiltration anesthesia and analgesia. Following completion of the splenectomy, the rats were resuscitated on a 40 • C thermostatic plate and were put back into their cage; the feeding was the same as before the splenectomy.

Animal grouping and drug treatment
The rats were randomized into five groups: i) Control group (C, n = 18); ii) anesthesia group (A, n = 18); iii) surgery group (S, n = 18); iv) surgery + normal saline group (S + NS, n = 18); and v) surgery + MaR1 group (S + MaR1, n = 18). The instruction of MaR1 was comparable to previous reports Wu et al., 2022). Rats in the C group underwent no treatment. Rats in the A group were only anesthetized (induced with 5 % sevoflurane for 2 min and maintained with 2 % sevoflurane for 30 min). Rats in S group underwent a splenectomy (as described above) following the same anesthesia as those in group A. In the S + NS group, normal saline (2 mL) was intraperitoneally injected (i. p.) 30 min before the induction of anesthesia, followed by splenectomy.

Behavioral test
Behavioral tests included the Morris Water Maze (MWM) and Intel-liCage. A series of behavioral experiments were performed from day 6 before splenectomy to day 10 after splenectomy (The day of splenectomy was defined as day 0, day 1 before splenectomy was defined as day − 1, day 1 after splenectomy as day 1, and so on). The experimental sequence was as Fig. 1.
MWM consists of two phases: training trails (days − 6 to − 2) and probe trails (days − 1, 1, 3, and 7). IntelliCage consists of four stages: free exploration (days − 6 to − 4), nosepoke learning (days − 3 to − 1), position learning (days 1 to 7), and reversal of position learning (days 8 to 10). At day 0, rats were treated with splenectomy or drug pretreatment. On the third day after splenectomy, the brain tissue was collected after the MWM test for subsequent experiments. The day of splenectomy was defined as day 0, day 1 before splenectomy was defined as day − 1, day 1 after splenectomy as day 1, and so on.

MWM
MWM was used to evaluate hippocampal-dependent memory and cognitive function for behavior in rats (Lissner et al., 2021). The hardware consisted of a cylindrical pool with a diameter of 120 cm and a height of 50 cm, and a cylindrical platform with a diameter of 10 cm where the rats could rest. The depth of the pool was 30 cm, the water temperature was 23 ± 2 • C, and opaque black dye was added to the water. Briefly, the pool was divided into four identical quadrants, and the circular platform was placed in the first quadrant (the target quadrant). Markers for each quadrant were placed on the wall of the bucket around the pool so that the rats could clearly see them, and these markers were fixed during all tests. MWM consists of two phases: training trails and probe trails. The training trails lasted for 5 days. The platform was placed 1.5 cm below the water surface. The experiment in this quadrant ended after the rat stayed on the circular platform for 3 s or if the rat swam in the water for 60 s. One to four quadrants were tested sequentially, with an interval of 15 min for each quadrant. After completing the training trails, the rats enter the probe trails. In this phase, the platform in the first quadrant was removed at day − 1, 1, 3, and 7 days. The rats were placed in the third quadrant (the opposite quadrant of the platform) and allowed to swim for 60 s. Latency to platform (time to find the hidden platform), latency for the first time to platform, percentage of time spent in the target quadrant, platformcrossing times, and swimming speed were recorded (Feng et al., 2021). Rat tracks and data were collected using Smart 3.0 Animal Behavior Acquisition and Analysis System (Panlab, Spain).

Automated IntelliCage testing
IntelliCage was used to evaluate behaviors such as spontaneous activity, behavioral flexibility, spatial learning, and memory in social rats (Kiryk et al., 2020). The equipment includes the computer that runs the analysis software (Analyzer, TSE, Germany) and a behavioral detection device (WMT-100S, TSE, Germany). After induction of sevoflurane anesthesia, the sensor was implanted into the subcutaneous tissue of the posterior neck of the rats (Wu et al., 2017). After successful implantation, the information in the sensor of each rat was entered into the system. The experiment included four stages. Stage i) Free exploration (3 days): the rats could move freely in the cage, the valves in each corner of the cage were open, and after entering the valve, the rats could drink water by themselves. Stage ii) Nosepoke adaptation (3 days): the program defaults that the valves at each corner are closed. When the rats reach the sensing area, the sensing device opens the valve, and the rats can drink water. There is a nose-touch recognition signal point near the sensing door. After the nosepoke learning, the rats leave, the valve closes, and the visit ends. Stage iii) Position learning (behavioral sequencing task; 7 days): the position with the least number of visits in the nose-touch learning experiment was defined as the "target position", and the rest of the positions were defined as "non-target positions". Each valve was still closed by default, and the sensor device could not open the valve until the rat reached the designated position. However, only the valve in the "target position" had a storage tank behind it, thus the rats could only drink at the "target position". Stage iv) Reversal of position learning (3 days): the position opposite to the "target position" in the position learning stage is redefined as the "target position", and the rest of the positions are redefined as "non-target positions". All rats could still only drink at the "target position" behind the valve. In this experiment, the system detected that the rat entered the peripheral switch through the sensor in the rat's neck, which was defined as a "Visit". The system detected if the rat touched the sensor device near the switch, which was recorded as a "Nosepoke". A "Lick" was recorded after the rat opened the valve and licked the water bottle to drink. In addition to measuring the total number of visits, nosepokes, and licks, the following metrics were calculated: % correct = (number of correct visits with nosepoke and lick) / (total visits), and % incorrect = (number of correct awareness without a nosepoke and lick) / (total visits) (Winslow et al., 2021).

Slice preparation
After sevoflurane anesthesia induction, 0.9 % normal saline and 4 % paraformaldehyde were injected into the heart of the rats. The brain tissue was excised and fixed with 4 % paraformaldehyde for 16 h at room temperature (RT). The fixed tissues were dehydrated in gradient alcohol, made transparent with xylene, and embedded in wax. The tissue was sliced into 5-μm sections using a paraffin microtome.

Transmission electron microscopy (TEM)
After sevoflurane-induced anesthesia, the brain was quickly excised on ice and the hippocampus was dissected. The cornu ammonis 1 (CA1) region was cut under a microscope and fixed in 2.5 % glutaraldehyde solution at 4 • C for 48 h, and then rinsed with 1 % PBS (15 min, three times). The specimens were fixed in 1 % osmic acid solution at 4 • C for 4 h and then rinsed with 1%PBS (15 min, three times). After embedding, polymerization and block repair, tissue sections (70 nm thick) were stained with uranium (10 min) and rinsed with deionized water (15 min, three times). Finally, the samples were stained with lead (10 min) and rinsed with deionized water (5 min, three times). The samples were observed under TEM (H-7650, Hitachi) and images were captured.

Real-time quantitative polymerase chain reaction (RT-qPCR)
The hippocampal region of rats was collected. Total RNA was extracted by TRNzol Universal Reagent (DP424, Tiangen) at RT. After detection and quantification of the isolated RNA, the primers for different target fragments were designed and synthesized (Table 1). The transcriptional levels of NF-κB p65, IKKβ, IL-6, IL-1β, and TNF-α were detected by RT-qPCR using the SYBR Green chimeric fluorescence method with SYBR Premix Ex Taq (Perfect Real Time) Kit reagent. The comparative 2− ΔΔCt value quantitative method was used for relative quantification. Specificity of RT-qPCR amplification was analyzed by solubilization curve.

Statistical analysis
All data in this experiment were statistically analyzed using PRISM 9.0 software (GraphPad). Multivariate differences were compared by one-way or repeated measures two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Data are expressed as the mean ± standard deviation (mean ± SD). P < 0.05 was considered to indicate a statistically significant difference.

Cognitive decline in aged rats after splenectomy
To assess the effects of anesthesia and surgical trauma on cognitive function in rats, their spatial learning and memory abilities were examined using MWM and IntelliCage tests.
In the MWM training test, rats in groups C, A, and S showed similar spatial learning and memory abilities during five consecutive days of training before splenectomy ( Fig. 2A, B). During the probe trials, group S spent less time in the target quadrant and had lower platform-crossing times and longer first time to the platform on postoperative day 3 compared with group C. The performance of rats in this group returned to normal on postoperative day 7 (Fig. 2C-F). However, there was no major variation in the performance on postoperative days 1, 3, and 7 between group C and group A (Fig. 2C-F). No difference in swimming speed was observed among the three groups (Fig. 2G), indicating that poor performance of the groups is not caused by a decrease of motor ability or by postoperative nociception of the rats.
Another group of rats were used for IntelliCage testing. There was no significant difference in spatial learning and memory ability between the three groups during free exploration and nosepoke adaptation before splenectomy (Fig. 2H-K). In the position-learning experiment, compared with group C, the correct access rate of rats in group S was significantly reduced 1-4 days after splenectomy, and there was no obvious abnormality in the correct access rate from the fifth day (Fig. 2L). However, there was no significant difference in performance between groups C and A after splenectomy (Fig. 2L). In the reversal position-learning experiment, the rats were observed to learn and remember new target locations quickly. There was no difference in Table 1 Primer sequences for RT-qPCR.

Target
Sequences ( cognitive ability among the three groups of rats (Fig. 2M).
Collectively, these data suggest that anesthesia and surgery lead to impaired spatial learning and memory, with the most severe cognitive impairment on postoperative day 3, which is followed by recovery until near normal cognitive performance on day 7. Anesthesia alone had no significant effect on cognitive function.

MaR1 pretreatment ameliorates postoperative cognitive function in aged rats
The effect of MaR1 pretreatment on postoperative cognitive function of aged rats was analyzed by comparing the spatial learning and memory ability of C, S, S + NS, and S + MaR1 groups by MWM and IntelliCage tests.
In the MWM training test, the four groups of rats also showed similar spatial learning and memory abilities during five consecutive days of training before splenectomy (Fig. 3A-B). In the probe trial, both the time in the target area and platform-crossing times in the S + NS group were less than those in the S + MaR1 group on postoperative days 1 and 3 ( Fig. 3C-F). The latency for the first time to platform in group S + NS was longer compared with that of group S + MaR1 on days 1 and 3 postoperatively (Fig. 3C-F). The greatest difference between the two groups was on the third day after splenectomy, while there was no difference between the four groups on day 7 after splenectomy ( Fig. 3C-F). There was no significant difference in performance between the S and S + NS groups before splenectomy and on days 1, 3, and 7 after splenectomy ( Fig. 3C-F). In addition, there was no difference in swimming speed among the four groups of rats (Fig. 3G), indicating that MaR1 did Data are expressed as mean ± SD. n = 6 rats per experiment. Significance was assessed by two-way ANOVA and Tukey's post-hoc test. *P < 0.05, **P < 0.01. not alter the swimming speed of rats.
The IntelliCage test results showed that the four groups of rats exhibited similar spatial learning and memory abilities during free exploration and nosepoke adaptation before splenectomy (Fig. 3H-K). In the position-learning test, compared with the S + NS group, the correct access rate of rats in the S + MaR1 group was significantly reduced 1-4 days after splenectomy, and there was no obvious abnormality in the correct access rate from the fifth day (Fig. 3L). However, there was no significant difference in cognitive function between the S and S + NS groups throughout the process (Fig. 3L). In the reversal position-learning test, there was no difference in spatial learning and memory of rats in each group (Fig. 3M).
The above data indicated that the postoperative cognitive function of rats was improved after MaR1 pretreatment, and the drug remission Data are expressed as mean ± SD. n = 6 rats per experiment. Significance was assessed by two-way ANOVA and Tukey's post-hoc test. *P < 0.05**. effect was the best on the third day after splenectomy. In addition, normal saline had no effect on cognitive function in rats.

MaR1 pretreatment inhibited postoperative hippocampal astrocyte activation in aged rats
In the previous experiments, the cognitive impairment of elderly rats was the most severe on the third day after splenectomy, and the drug effect was the best on the third day after MaR1 pretreatment. Consequently, in the subsequent experiment, only the third day after splenectomy was selected as the observation time.
To determine the involvement of MaR1 in the activation of hippocampal astrocytes, the activation of astrocytes in the hippocampal CA1 region of the four groups of rats on day 3 after splenectomy was examined by WB ( Fig. 4A-C), IF (Fig. 4D-F), and TEM (Fig. 4G). The fluorescence intensity and protein expression of GFAP and S100β were significantly increased in group S compared with group C (Fig. 4B-C, E-F). There was no significant change between the S group and the S + SN group (Fig. 4B-C, E-F). Compared with the S + NS group, the fluorescence intensity and protein expression of GFAP and S100β were decreased in the S + MaR1 group ( Fig. 4B-C, E-F).
To examine the detailed morphology and microstructure of the astrocytes, TEM was used to observe the structural characteristics of astrocytes in the hippocampal CA1 region of the four groups of rats on the third day after splenectomy (Fig. 4G). The nucleus of astrocytes in group C was oval, with obvious nucleoli and heterochromatin distributed around the nucleus under the nuclear membrane. The cytoplasm was relatively loose with a few normal organelles such as mitochondria and rough endoplasmic reticulum. Astrocytes in group S had irregular nuclei, insignificant nucleoli, and less heterochromatin in the nuclear membrane. The cell body was highly edematous, the mitochondria in the cytoplasm were vacuolated, and the organelles were destroyed and disintegrated. Numerous glial microfilaments were found in the cytoplasm. Astrocytes in the S + NS group had irregular nuclei, the nucleoli were close to the nuclear membrane, and there was less heterochromatin under the nuclear membrane, along with cell body edema, mitochondria swelling in the cytoplasm, and endoplasmic reticulum expansion. Numerous glial microfilaments distributed in bundles were found in the cytoplasm. Astrocytes in the S + MaR1 group had oval nuclei, clear nucleoli, and more heterochromatin in the nuclear membrane, and the distribution tended to be normal. The cell body edema was improved in astrocytes of the S + MaR1 group, the mitochondrial structure tended to be normal, and some endoplasmic reticulum was still expanded.
In summary, these data suggest that anesthesia and surgery can activate astrocytes in the CA1 region of the hippocampus after splenectomy in aged rats, and that pretreatment with MaR1 can inhibit this phenomenon.

Pretreatment with MaR1 may inhibit hippocampal inflammation by regulating the NF-κB pathway
The possible pathways involved in the neuroprotective mechanism of POCD induced by MaR1 pretreatment were further explored. WB ( Fig. 5A-C, F-H), IF (Fig. 5D, E), and RT-qPCR (Fig. 5I-N) were used to detect the key proteins and inflammatory factors of the NF-κB pathway in the hippocampal CA1 region of the four groups on the third day after splenectomy. Compared with group C, the fluorescence intensity and protein and mRNA expression of NF-κB p65 in group S were significantly increased (Fig. 5B, E, G); there was no significant change between S group and S + SN group (Fig. 5B, E, G). Compared with the S + N group, the fluorescence intensity and protein and mRNA expression of NF-κB p65 were decreased in the S + MaR1 group (Fig. 5B, E, G). In addition, compared with group C, splenectomy increased the expression levels of IKKβ protein and mRNA (Fig. 5C, K), and there was no significant change between S group and S + SN group (Fig. 5C, K). Compared with the S + NS group, MaR1 upregulated the expression level of IKKβ protein (Fig. 5C, K). Next, the levels of IL-1β, IL-6, and TNF-α were measured. Compared with group C, the protein and mRNA expression of IL-1β, IL-6, and TNF-α in group S were significantly increased ( Fig. 5F-I, L-N); there was no significant change between S group and S + SN group (Fig. 5F-I, L-N). Compared with S + NS group, the protein and mRNA expressions of the inflammatory factors were decreased in the S + MaR1 group ( Fig. 5F-I, L-N).
These results suggest that anesthesia and surgery can upregulate key proteins of the NF-κB pathway and related inflammatory factors. However, MaR1 pretreatment may inhibit hippocampal inflammation by regulating the NF-κB pathway.

Discussion
The aim of this study was to investigate the mechanisms involved in the neuroprotective effect of MaR1 on POCD in elderly rats. By means of a series of experimental studies, astrocytes in the hippocampal CA1 region were found to be activated after splenectomy, and expression of NF-κB pathway proteins and related inflammatory factors was induced. Pretreatment with MaR1 improved astrocyte activation and hippocampal neuroinflammation induced by anesthesia and surgery. These results suggest that MaR1 pretreatment can improve POCD in rats, and that the neuroprotective mechanism of MaR1 may involve inhibition of hippocampal neuroinflammation and NF-κB-IKKβ signal transduction, which may act by inhibiting the activation degree of astrocytes.
Research on POCD has made great progress in recent years, but there are differing opinions about the construction of animal models for POCD and how to verify the success of the models (Safavynia and Goldstein, 2018). The behavioral correlation of hippocampal dysfunction caused by experimental damage has become the gold standard for recording cognitive changes in laboratory rodents (Voikar et al., 2018). In the present study, rats aged 18-20 months were selected and the POCD model was prepared by sevoflurane anesthesia and splenectomy. Two behavioral methods, MWM and IntelliCage, were used to evaluate the cognition of the rats. MWM is a well-known and reliable traditional behavioral task used to monitor spatial memory performance in rodents (Haider and Tabassum, 2018). The MWM results in the current study revealed that the elderly rats exhibited cognitive decline after splenectomy, with the cognitive impairment appearing most severe on the third day after splenectomy ( Fig. 2A-M). MWM has been widely used in neurobiological and neuropharmacological studies of spatial learning and memory (Bromley-Brits et al., 2011); however, there are some disadvantages to MWM, such as concerns about stress during swimming, which may not be sensitive to working memory, etc. (Vorhees and Williams, 2014). Therefore, to validate the MWM findings in the current study, an IntelliCage experiment was also employed. In contrast to other behavioral sciences, IntelliCage is a fully automated system for behavioral assessment of rats living in social groups (Kiryk et al., 2020). Importantly, automation can improve repeatability, thereby reducing the number of animals and experimental replicates needed to obtain reliable results, and reducing human error. At present, there are no reports of IntelliCage as a POCD model mouse for behavioral verification. In the current study, IntelliCage was used to continuously monitor the spontaneous behavior of rats and observe the continuous cognitive status of rats after splenectomy. Cognitive function of the rats decreased in the first three days after splenectomy, and then gradually recovered ( Fig. 2D-E, G, L), which is consistent with previous studies (Liu et al., 2021;Feng et al., 2021). This laid the foundation for the next experiment. In addition, Ge and colleagues found that mice that inhaled 2 % sevoflurane for 6 h would suffer from cognitive impairment (Ge et al., 2021;Guo et al., 2021). However, the current study showed that the elderly rats with 5 % sevoflurane induction for 2 min and 2 % sevoflurane maintenance for 30 min did not suffer from postoperative cognitive impairment ( Fig. 2A-M). These discrepancies between studies may be due to differences in the duration of anesthesia. Combined with the above results, we believe that simple temporary anesthesia does not (D) IF of GFAP and S100β in the hippocampal CA1 region of each group (blue, DAPI; red, GFAP; green, S100β). Scale bar, 100 μm. Three rats in each of the four groups were used for IF staining, and six images were selected for the fluorescence intensity analysis. (E) Fluorescence quantitative analysis of GFAP. (F) Quantitative fluorescence analysis of S100β. (G) Morphology of astrocytes in CA1 region of the hippocampus of each group under the electron microscope (blue arrow, nuclear envelope; red arrow, nucleolus; orange arrow, mitochondria; green arrow, endoplasmic reticulum; purple arrow, glial microfilaments). Higher magnification images of the depicted areas (yellow boxes) are shown to the right; left column scale bar, 5 μm; right column scale bar, 1 μm. Data are expressed as mean ± SD. n = 3 rats per experimental group. Significance was assessed by one-way ANOVA and Tukey's post-hoc test. *P < 0.05, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) lead to cognitive function changes. POCD may be the result of comprehensive factors, which are jointly affected by surgical trauma and anesthesia.
POCD is generally considered to be a neurodegenerative disease occurring during the perioperative period. The reason for its occurrence may be due to the activation of astrocytes by perioperative trauma, ischemia, hypoxia, and other factors (Jha et al., 2018). Subsequently, various soluble small molecules and proteins are secreted, resulting in a unique regulation of neuroinflammatory response that ultimately leads to brain tissue damage (Jha et al., 2018). In the present study, IF ( Fig. 3D-F), WB ( Fig. 3A-C), and TEM (Fig. 3G) analyses demonstrated that astrocytes were activated in the hippocampal CA1 region of aged rats after splenectomy, which again confirmed the activation of astrocytes during the development of POCD (Liu et al., 2019;Huang and Lu, 2021). Activated astrocytes can synthesize and release various inflammatory factors, such as IL-6, IL-1β, and TNF-α (Kery et al., 2020). An excessive inflammatory response can trigger the inflammatory process of the brain, produce a neurotoxic reaction, affect neurological function, and lead to cognitive impairment. This inflammatory state will destroy the integrity of the blood-brain barrier, resulting in an increase in the level of inflammatory factors and aggravating the injury (Kery et al., 2020). Consequently, exploring the expression level of proinflammatory cytokines can facilitate evaluation of the neuroinflammatory response induced by splenectomy in aged rats. In the current study, the expression levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were increased in the hippocampus of aged rats after splenectomy ( Fig. 4F-I, L-N). This is consistent with the studies of Ruth M Barrientos, who showed that there is inflammatory activation in the hippocampus after peripheral surgery and that the expression level of proinflammatory cytokines in the hippocampus is upregulated (Barrientos et al., 2012). The NF-κB signaling pathway has been confirmed to be involved in the activation of astrocytes in numerous neuroinflammatory studies (Cannella and Raine, 1989;Yuan et al., 2017). In POCD model rats undergoing partial liver resection, NF-κB p65 and inflammatory factors in hippocampal increased on day 1 and day 3 after surgery (Wei et al., 2018). In the present study, the expression of NF-κB p65 and IKKβ increased in the hippocampus of aged rats after splenectomy ( Fig. 5A-E, J-K). This indicated that astrocyte activation during POCD development may be accomplished through regulation of the NF-κB pathway. MaR1 is a specialized pro-resolving mediator synthesized by macrophages during the resolution phase of inflammation, providing unique anti-inflammatory and pro-resolution effects by limiting excessive neuroinflammation and promoting postoperative recovery (Medzhitov, 2008). Our behavioral tests showed that the postoperative cognitive function of rats was improved after MaR1 pretreatment (Fig. 3A-M), suggesting a unique neuroprotective effect of MaR1 on POCD. Both fluorescence intensity and protein expression of GFAP and S100β were decreased in the hippocampal CA1 region of aged rats after splenectomy ( Fig. 3A-F), which suggested that the activation of astrocytes was inhibited after MaR1 pretreatment (Wei et al., 2022). Simultaneously, the release of related proinflammatory cytokines from astrocytes, including IL-6, IL-1β and TNF-α, was attenuated ( Fig. 4F-I, L-N), further strengthening the evidence that MaR1 pretreatment inhibited the inflammatory response (Wang et al., 2021). POCD is a manifestation of CNS inflammation in the systemic inflammatory response induced by surgical trauma and anesthesia (Liu and Yin, 2018). So does MaR1 treatment of POCD improve CNS symptoms indirectly by inhibiting systemic inflammation or directly by inhibiting CNS inflammation? In our study, aged rats showed only cognitive decline on behavioral tests after A&S. Because the speed in MWM probe trial, which reflects motor ability, did not show any difference between the groups (Figs. 2G, 3F). It was suggested that S group had no effect on the function of the motor system. When the cognitive impairment rats were pretreated with MaR1, the cognitive performance was improved (Fig. 3D, E, G and L). This reflects that MaR1 pretreatment can act directly on the CNS to ameliorate neuroinflammation in the absence of overt peripheral inflammatory effects. Moreover, Yang's report further supports our findings (Yang et al., 2019). Yang examined microglial in the CNS within 24 h after surgery and found that pretreatment with MaR1 inhibited microglial infiltration in the brain parenchyma (Yang et al., 2019). This finding is very significant and reflects that MaR1 has a direct effect on the CNS. In a rat model of liver ischemia-reperfusion injury, MaR1 plays a protective role by reducing NF-κB activity through nuclear translocation of Nrf-2 (Soto et al., 2020). In addition, MaR1 (1 ng per mouse) reduced neuronal degeneration and cerebral infarction size by upregulating SIRT1 and Bcl-2, downregulating NF-κB and Bax, and reducing inflammatory factors (Xian et al., 2019). To investigate changes in the NF-κB signaling pathway after MaR1 pretreatment, WB and IF experiments were performed and indicated that expression of NF-κB p65 and IKKβ was decreased in the rats pretreated with MaR1 prior to splenectomy ( Fig. 5A-D, J-K). All these factors are key components in the regulation of inflammation, suggesting that MaR1 pretreatment can inhibit inflammation caused by astrocyte activation by inhibiting the NF-κB pathway (Qiu et al., 2020;Rodriguez et al., 2021).
There are several limitations to the current study. Firstly, cognitive function was only examined in the early postoperative period. Given the occasional long duration of POCD reported in the clinic, the impact on long-term outcomes of brain cognitive function should be investigated in future studies. Secondly, the specific mechanism by which MaR1 affects astrocyte activation needs to be further explored. Thirdly, as an antiinflammatory mediator originally existing in the human body, the content of MaR1 at different stages after anesthesia and surgery needs to be observed to better confirm the protective effect of MaR1 on POCD. Finally, other potential molecular mechanisms that may be involved in MaR1-mediated POCD in aged rats cannot be excluded.
In conclusion, MaR1 may inhibit the activation of astrocytes by participating in the regulation of the NF-κB pathway, so as to exert its unique anti-inflammatory and pro-regression effects, thereby improving the effect of POCD in aged rats. The findings from this study highlight a new idea of using pro-decomposing lipid mediators in the treatment of neuroinflammation and POCD.
CRediT authorship contribution statement X Li, Y Gao, and X Ni contributed to the conception and design of the review. X Li and Y Gao contributed equally to the overall text and figs. X Li, Y Gao, X Han and S Tang performed experiments. X Liu and N Li helped the statistical analysis of data. X Ni contributed the revising of the manuscript and provided critical advice on the content. All authors contributed to the article and approved the final version.

Conflict of interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in, or the review of, the manuscript.