Netrin-1 Ameliorates Postoperative Delirium-like Behavior in Aged Mice by Suppressing Neuroinammation and Restoring Impaired Blood Brain Barrier Permeability

Background Postoperative delirium (POD) is a common and serious postoperative complication in elderly patients, of which the underlying mechanism is elusive and without effective therapy at present. In recent years, the neuroinammatory hypothesis has been developed in the pathogenesis of POD. Netrin-1, an axonal guidance molecule, has been reported to have strong inammatory regulatory and neuroprotective effects. Methods We applied treatment with Netrin-1(45 µg/kg) in aged mice by using the POD model with a simple laparotomy to assess systemic inammatory, neuroinammation by detecting interleukin-6 (IL-6), interleukin-10 (IL-10), high mobility group box chromosomal protein-1(HMGB-1) and assessing the reactive states of microglia, permeability of blood-brain barrier (BBB) by detecting cell junction proteins and leakage of dextran, and behavior of the aged mice. Results We found that a single dose of Netrin-1 prophylaxis decreased the expression of IL-6 and HMGB-1, and upregulated the expression of IL-10 in peripheral blood, hippocampus and prefrontal cortex. Nerin-1 reduced activation of microglia cells in the hippocampus and prefrontal cortex and improved the POD-like behavior. Besides, Netrin-1 also attenuated the anesthesia/surgery-induced increase in BBB permeability by up-regulating the expression of tight junction-associated proteins such as ZO-1, claudin-5, and occludin. Conclusions These ndings conrm the anti-inammatory and BBB protective effects of Netrin-1 in an inammatory environment in vivo and provide better insights into the pathophysiology and potential treatment of POD.

1 reduced activation of microglia cells in the hippocampus and prefrontal cortex and improved the PODlike behavior. Besides, Netrin-1 also attenuated the anesthesia/surgery-induced increase in BBB permeability by up-regulating the expression of tight junction-associated proteins such as ZO-1, claudin-5, and occludin.
Conclusions These ndings con rm the anti-in ammatory and BBB protective effects of Netrin-1 in an in ammatory environment in vivo and provide better insights into the pathophysiology and potential treatment of POD.

Background
Postoperative delirium (POD) is a state of acute cerebral dysfunction characterized by uctuating and concurrent disturbances of attention, cognition, psychomotor behavior, emotion and sleep-wake rhythm [1]. It is a common complication occurring mainly within 1 week after surgery and anesthesia [2].
POD may lead to greater length of hospital stay, increased hospitalization costs, decreased life independence, increased morbidity and mortality, and has potential to induce long-term cognitive dysfunction, and even dementia [3,4]. Advanced age was reported to be an independent risk factor for the development of POD [2,5]. With the increasing aging of the population in global, the number of elderly people who need surgery/anesthesia treatment has been increasing, as well as the incidence of POD.
Unfortunately, there are no effective therapies for this complication for the unde ned underlying pathophysiology.
In recent years, more and more studies have shown that the occurrence of POD is closely related to neuroin ammation [4,6,7]. Aseptic surgical trauma provokes a homeostatic neuroin ammatory response, which when dysregulated, harmful consequences can follow. Surgery can result in an elevated level of proin ammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in systemic circulation [6],which has been strongly linked to the neuroin ammatory cascade that accompanies brain bold barrier failure [8]. In this case,pro-in ammatory cytokines and monocyte derived macrophages enter, leading to the activation of glial cells, including microglia and astrocytes [9,10]. This process is mainly affected by bone marrow-derived macrophages (BMDMs), which enable microglia/macrophages to play a dual role in the microenvironment of brain injury and repair [11,12]. Upon microglial activation, the in ammatory cascade is triggered by the release of pro-in ammatory molecules and concomitant signalling pathways are activated, which causes synaptic damage, neuronal loss and progression.
Reactive astrocytes exhibit neurotoxic effects with loss of neurotrophic functions [13]. The interaction between peripheral immunity and brain caused by systemic in ammation ampli es the in ammatory response in the central nervous system (CNS) [14], while the cascade of neuroin ammation induces synaptic dysfunction and neuronal apoptosis, nally damaging cognitive function [6,15].
Advances on mechanisms in resolution of acute in ammation uncovered a new genus of pro-resolving lipid mediators, called specialized pro-resolving mediators (SPM) [16], which can increased by Netrin-1 (NTN-1) in vivo during acute self-limited in ammation [17]. NTN-1 is an axonal guidance molecule, involved in both physiological and pathological processes such as apoptosis, in ammation and neurogenesis in the nervous system as well as in the lung, heart, and kidneys. NTN-1 has been shown to play a positively regulatory role during in ammatory process recently [18]. It has been demonstrated that NTN-1 can limit in ammatory response through the involvement of in ammatory cascades [19]. In addition, NTN-1 was identi ed as a survival factor for endothelial cells and induced neovascularization and vessel remodeling. Overexpression of NTN-1 promoted angiogenesis and improved long-term neurological functions following ischemic stroke. Recent studies indicated that NTN-1 preserved BBB integrity in model of traumatic brain injury and experimental autoimmune encephalomyelitis [20,21].
However, there are not any reports about the role of NTN-1 in the POD.
Based on these discoveries, we proposed the hypothesis that pretreatment with NTN-1 could improve the POD-like behavior of aged mice through its anti-in ammation effect on the in ammation induced by surgical trauma. To validate this hypothesis, we assessed the effects of NTN-1 on the postoperative behavior of aged mice, and in ammation events both in the periphery and CNS. In addition, we aimed to determine that NTN-1 prevents peripheral in ammatory factors from entering the brain by protecting the tightness of the blood-brain barrier, which plays an important role in preventing peripheral in ammation from metastasizing to CNS.

Materials And Methods 1 Animals
Changsha Tianqin Biotechnology Co. Ltd. Changsha, China. All animals were group-housed ve per cage with free access to food and water. The temperature, humidity, and day-night cycle were maintained according to the standards established by the experimental animal laboratory at Zhongnan Hospital of Wuhan University. The mice were allowed one week to acclimatize the laboratory environment before the experiment.

Experimental protocol
Mice were randomly divided into 4 groups: control group, surgery group, surgery + netrin-1 group, and netrin-1 group. Netrin-1 (R&D Systems, 6419-N1-025) was given at 45 µg/kg in phosphate-buffered saline (PBS), and administered through tail vain with a total volume of 200 µL at 1 hour after surgery, while the equal volume of PBS was given in control group and surgery group. The dose of netrin-1 was based on the researches using other models of acute in ammation with slight modi cation [21]. 3 POD mouse model A simple laparotomy was performed under iso urane anesthesia using the methods described in our previous studies [22]. Speci cally, anesthesia was induced and maintained with 1.4% iso urane in 100% oxygen in a transparent acrylic chamber. Fifteen minutes after the induction, the mouse was moved out of the chamber, and iso urane anesthesia was maintained via a cone device. One 16-gauge needle was inserted into the cone near the nose of the mouse to monitor the concentration of iso urane. A longitudinal midline incision was made from the xiphoid to the 0.5 centimeter proximal pubic symphysis on the skin, abdominal muscles and peritoneum. Then, the incision was sutured layer by layer with 5-0 Vicryl thread. At the end of the procedure, EMLA cream (2.5% lidocaine and 2.5% prilocaine) was applied to the incision wound, and then every eight hours for two days to treat the pain associated with the incision. The procedure for each mouse lasted about ten minutes, and the mouse was put back into the anesthesia chamber for up to 2 hours to receive the rest of the anesthesia consisting of 1.4% iso urane in 100% oxygen. A heat pad was used to keep the mouse body temperature between 36˚C and 37˚C during the surgery. After recovering from the anesthesia, each mouse was returned to a home house with available food and water.

Behavioral tests
The behavioral changes were detected using battery of behavioral tests including buried food test, open eld test, and nally Y maze test-at 24 hours before (baseline) the Surgery/Anesthesia and at 6,9,24 hours postoperatively as described in our previous studies [23]. In all tests, apparatus was cleaned with 75% alcohol after each mouse to remove odors.

Buried food test
The buried food test was performed as described in previous studies [22,24] with modi cations. Speci cally, two days before buried food test, each mouse was received 2 pieces of sweetened cereal. On the test days, we had the each mouse acclimatize for one hour by placing the home cage with mice in the testing room. The test cage was prepared with clean paddling of 3 centimeters high in which we buried 1 piece of sweetened cereal below the padding. Its location was freely chosen and it was not visible. We placed the mouse in the center of the cage and measured the latency of eating the food. The latency was de ned as the time from when the mouse was placed in the case to the mouse uncovered the food and grasped it with forepaws and/or teeth. When mice found the food pellet within 5 minutes, they were allowed to eat up the food and then returned their home cage. If they couldn't nd the pellet within 5 minutes, they would be taken back when up to 5 minutes and the latency was recorded as 300 seconds.

Open eld test
The open eld test was performed as described in previous studies [23,25]

Y maze test
The Y maze test was performed as described in previous studies with modi cations [26,27]. Speci cally, the Y maze was placed in a quiet and illuminated room, and consisted of three arms (8 × 30 × 15 centimeters) with an angle of 120 degrees between each arm. The three arms included the start arm, in which the mouse started to explore (always open), novel arm, which was blocked at the rst trial, but opened at the second trial, and the other arm (always open). The start arm and other arm were designed randomly to avoid the spatial memory error. The Y maze test consisted of two trials separated by an intertrial interval (ITI). The rst trial (training) was 10 minutes in duration and allowed the mouse to explore start arm and other arm. After 2 hours (for the studies of 6 and 24 hours after the surgery) or 4 hours (for the study of 9 hours after surgery) ITI, the second trial was conducted. For the second trial, the mouse was placed back in the maze in the same start arm with free access to all arms for 5 minutes. A video camera, which was linked to the Any-Maze animal tracking system software, was installed 60 centimeters above the chamber to monitor and analyze the number of entries and the time spent in each arm. The time spent in and entries into the novel arms indicated the spatial recognition memory (learned behavior).

BBB Permeability Assay
Dextran was used to Dextran was used to measure BBB permeability as described in previous studies with modi cations [28,29]. Speci cally, 6 hours after surgery, each mouse was injected intravenously with 100 µl 10-kDa dextran Texas Red lysine xable (4 mg/ml, Invitrogen, D1863). Fifteen minutes after injection, each mouse was anesthetized with 1.4% iso urane and decapitated. The brain tissue was harvested and xed by 4% paraformaldehyde overnight at 4 ℃, then cryopreserved in 30% sucrose and Spectrophotometric quanti cation of 10-kDa dextran Texas Red from extracts of hippocampus and prefrontal cortex was carried out at 24 hours after surgery. Speci cally, each mouse was injected intravenously with 100 µl 10-kDa dextran Texas Red lysine xable (4 mg/ml, Invitrogen, D1863) 24 hours postoperatively. Fifteen minutes after injection, each mouse was deeply anesthetized and perfused with PBS transcardially (150 mL for 5 min). Then the mice were decapitated, and the hippocampus and prefrontal cortex were harvested. Then we used 1% Triton X-100 in PBS to homogenize the brain tissue (100 µL/100 mg brain tissue). Tissue lysates were centrifuged at 16000 r.p.m. for 20 minutes and the uorescence of the supernatant was measured on a uorometer POLAR star Omega (BMG Labtech) (ex/em 595/615 nm).
8 Immuno uorescence 24 hours after surgery, each mouse was anesthetized with 1.4% iso urane and perfused transcardially with ice-cold 0.1 M PBS followed by 4% PFA in 0.1 M PBS at pH 7.4. Brains were harvested and xed in 4% PFA in 0.1 M PBS at 4 ℃, then cryoprotected in 30% sucrose for 72 hours, and frozen in TissueTek OCT (Sakura), were cut sequentially to 20 µm. Washed in PBS and permeabilized in 0.5% Triton X-100, the section was blocked with 10% goat serum for 2 hours at room temperature in order to block non-speci c bindings, washed in PBS, then the following primary antibody rabbit anti-Iba-1 (1:200, Abcam, ab178847) at 4 ℃ overnight. After washing, the sections were incubated with secondary antibody (goat anti-rabbit) conjugating with Alexa Fluor dyes 488 from Invitrogen (1:500) at room temperature for 2 hours at dark. Immunolabelled sections were coverslipped with 40,6-diamidino-2-phenylindole (DAPI; Invitrogen) and analyzed by microscope (Olympus, Tokyo, Japan) equipped with an imaging system. Five high magni cations were chosen in three non-overlapping elds randomly acquired in hippocampus and prefrontal cortex subregions using a counting frame size of 0.4mm 2 . Images were processed and the area of the microglia quanti ed using ImageJ software (NIH). The area of the selected cells was converted into a immunoreactivity was calculated as percentage area density de ned as the number of pixels (positively stained area) divided by the total number of pixels (sum of positively and negatively stained area) in the imaged eld.

Statistical Analysis
The statistical analysis were performed with SPSS 23.0 (IBM, New York, USA) or GraphPad Prism 6 (GraphPad, New York, USA). The normality of the data was analyzed using the Shapiro-Wilk test, and the data was found to be normally distributed. The quantitative data are expressed as the mean ± standard error of the mean (SEM), with the error bars indicating the SEM. Different groups were compared using the one-way analysis of variance, followed by Bonferroni post hoc test. A value of p < 0.05 was considered statistically signi cant.

Administration of exogenous NTN-1 improves POD-like behavior induced by surgery/anesthesia in aged mice
To assess whether surgery/anesthesia affects general and cognitive behavior of aged mice, we performed a battery of behavioral tests with food buried test, open eld test and Y maze test, at 24 hours before surgery and 6, 9, 24 hours after surgery in the present study as we previously reported [22,23]. Composite Z scores for each of the 40 mice in the four groups were calculated at 6, 9 and 24 hours after surgery (P < 0.05, Fig. 1A-C).
At rst we executed the buried food test to explore whether surgery/anesthesia affected the mice's ability to associate odorant with food reward [30]. The latency to eat food was markedly increased in the Surgery group compared to the Control group at 9 hours after surgery (P < 0.01, Fig. 1D), while administration with NTN-1 improved the impaired ability of nding and eating food induced by surgery/anesthesia (P < 0.05, Fig. 1D). No signi cant changes were observed between the NTN-1group and Control group. Surgery/anesthesia-induced impairment in mice's ability to search for and eat food suggests that the surgery/anesthesia might cause the mice to develop the changes in behaviors (inattention, disorganized thinking and altered level of consciousness) associated with delirium.
Then we executed the open eld test to examine the locomotor ability and exploratory behavior of mice. There were no signi cant differences in total distance traveled by mice between four groups at 9 hours after surgery, indicating that surgery/anesthesia did not affect the motor function of aged mice (Fig. 1E). Surgery/anesthesia signi cantly decreased the time spent in the center at 9 hours after surgery (P < 0.05, Fig. 1F), and preemptive administration of NTN-1 ameliorated this phenomenon at 9 hours after surgery (P < 0.05, Fig. 1F). Besides, surgery/anesthesia signi cantly decreased the freezing time at 9 hours after surgery (P < 0.05, Fig. 1G), while preoperative treatment with NTN-1 increased the freezing time at 9 a hours after surgery (P < 0.05, Fig. 1G). It's worth noting that NTN-1 administration did not change these parameters compared to the control condition ( Fig. 1E-G). These ndings suggest that the surgery/anesthesia altered the natural behavior of the mice such as anxiety (time spent in the center) and natural reaction (freezing time).
At last we conducted Y maze for assessing the spatial memory in aged mice as previously validated [31]. Surgery/anesthesia signi cantly reduced the number of entries in the novel arm at 9 hours after surgery (P < 0.05, Fig. 1H) and the duration in the novel arm at 9 hours after surgery (P < 0.05, Fig. 1I), as compared to the control condition. Pretreatment with NTN-1 increased the number of entries in the novel arm and duration in the novel arm at 9 hours after surgery (P < 0.05, Fig. 1H-I). However, NTN-1 administration alone did not affect the performance of aged mice in the Y maze test at 9 hours after surgery ( Fig. 1H-I).
Taken together, no signi cant changes were observed between the NTN-1 group and Control group but prophylaxis with NTN-1 attenuated the impairment of POD-behavior by surgery/anesthesia of aged mice in a uctuating way.

NTN-1 regulates the expression of in ammatory cytokines after surgery
To evaluate the effects of NTN-1 on the systemic in ammation, we rstly measured the changes of IL-6, IL-10 and HMGB-1 in blood plasma at 6 hours after surgery [32]. Surgery/anesthesia signi cantly increased the level of IL-6 and HMGB-1(P 0.05, Fig. 2A,2C)but did not change the expression of IL-10 after surgery (P 0.05, Fig. 2B). Though a single dose of NTN-1 did not completely reverse the increase of proin ammatory cytokines to the control condition, it markedly reduced the levels of IL-6 and HMGB-1 after surgery(P 0.05, Fig. 2A,2C). Besides, pretreatment of NTN-1 increased the expression of IL-10, a crucial cytokine during the resolution phase of in ammation after surgery(P 0.05, Fig. 2B). Secondly, we measured these cytokines above in the hippocampus and prefrontal cortex which are two key brain regions related to memory network [33,34] to evaluate the effects of NTN-1 on neuroin ammation at 6 hours after surgery. Surgery/anesthesia induced a marked increase in the expression of IL-6 after surgery both in the hippocampus and prefrontal cortex compared to the control condition (P 0.05, Fig. 3A, 3D). Pretreatment with NTN-1 signi cantly decreased the expression of IL-6 compared to the Surgery group in these brain regions(P 0.05, Fig. 3A,3D). Besides, pretreatment with NTN-1 increased the expression of IL-10 not only in the hippocampus after surgery(P 0.05, Fig. 3B), but also in the prefrontal cortex after surgery(P 0.05, Fig. 3E).
Surgery/anesthesia decreases the endogenous NTN-1 in the hippocampus and the prefrontal cortex in aged mice To investigate whether the endogenous NTN-1 was involved in anti-in ammatory and neuroprotective effects, we measured the changes of the endogenous NTN-1 in the hippocampus and the prefrontal cortex at 6 hours after surgery. Our result suggested that surgery/anesthesia signi cantly decreased the level of NTN-1 in the hippocampus and the prefrontal cortex after surgery (P 0.05, Fig. 3C,3F). NTN-1 prevents neuroin ammation in the hippocampus and prefrontal cortex We measured the changes of immunoreactivity of Iba-1 in the hippocampus and prefrontal cortex to assess the reactive states of microglia, which represent the major pathological manifestation of neuroin ammation [35,36]. NTN-1 attenuated microglial activation as measured by changes in the expression of Iba-1.Surgery induced the amoeba-like morphology of microglia and increased Iba-1 immunoreactive area in the hippocampus and prefrontal cortex compared with the control condition (P 0.05, Fig. 4A-D), while preemptive administration of NTN-1 signi cantly restored the rami ed shape of microglia and reduced cellular area (P 0.05, Fig. 4A-D). No signi cant changes in Iba-1 were observed in the NTN-1 group.

NTN-1 prophylaxis alleviates the leakage of BBB induced by surgery/anesthesia
The breakdown of blood-brain barrier (BBB) has been reported to be associated with delirium and perioperative neurocognitive disorders [37,38], so we employed a well-established dye injection assay to investigate the integrity of BBB [32,39] under the treatment of surgery/anesthesia with or without administration of NTN-1.
The immuno uorescence images revealed that 10-kDa dextran was primarily con ned to vessels in the four groups. By contrast, the signal of dextran was detected in the brain parenchyma around vessels of mice in the Surgery group (Fig. 5A). To quantitate the extravascular dextran, spectrophotometric quanti cation of 10-kDa dextran-Texas Red from brain tissue extracts was performed. In the hippocampus, we found that surgery/anesthesia increased the level of extravascular 10-kDa dextran as compared to the control condition, while NTN-1 prophylaxis decreased the leakage of dextran induced by surgery/anesthesia (P < 0.05, Fig. 5B).
We next examined the effects of NTN-1 on the expression of occludin, ZO-1 and claudin-5 after surgery ( Fig. 6D-F, Fig. 7D-F), which are the tight junction (TJ) associated proteins to maintain the integrity of BBB [40,41]. By quantitative western blot we found that there was a marked decrease in the expression of occludin, ZO-1 and claudin-5 both in the hippocampus and prefrontal cortex at 9 hours after surgery, while pretreatment with NTN-1 signi cantly attenuated the reduction of these proteins (P < 0.05, Fig. 6A-C, Fig. 7A-C,). Preemptive administration of NTN-1 alone did not have any effects on BBB.

Discussion
In the present study, we demonstrate that the exogenous NTN-1, an axonal guidance molecule, improve the postoperative of POD-like behavior in aged mice by its anti-in ammatory and BBB-protecting effect.
Our results indicate that pretreatment with NTN-1 given through the caudal vein alleviates systemic in ammatory response and protects BBB integrity after surgery/anesthesia. In addition, the exogenous NTN-1limits neuroin ammation both in the hippocampus and prefrontal cortex, according to the expression of in ammatory cytokines and reactive states of microglia in these brain regions. As far as we know,this is the rst report about the neuroprotective effect of NTN-1 in mice model of POD.
A large amount of evidence indicated that neuroin ammation plays an important role in POD. Peripheral aseptic in ammation activates innate immune system, which starts the in ammatory process and eventually lead to POD. [7,15,42]. In the aseptic surgery setting, cell trauma releases damage associated molecular patterns (DAMPs) that bind to Toll-like receptors (TLRs) via high mobility group box-1 (HMGB1) to activate BMDMs, which then upregulating the expression of pro-in ammatory cytokines such as TNF-α, IL-1 and IL-6 [43,44]. These cytokines can cause further activation of DAMPs in positive feedback [45,46]and be released into the circulation and disrupt the integrity of the blood brain barrier (BBB) [42,47]. Our results show that NTN-1 attenuates the systemic release of proin ammatory factor IL-6 and increases of anti-in ammation cytokine IL-10 after surgery, which are the vital cytokines after trauma. At the same time, NTN-1 reduced the release of HMGB-1, which is passively released from cells damaged by aseptic trauma and targets circulating BM-DMs. These ndings are consistent with the potent anti-in ammatory activity of NTN-1 in many other disease models that associated with in ammation such as renal ischemia reperfusion injury [48], acute peritonitis [49], acute pancreatitis [50]. The migration and aggregation of white blood cells to the in ammatory site is the central link of the whole in ammatory response. Early studies found that NTN-1 interacts with the UNC-5B receptor expressed on the surface of white blood cells and inhibits the migration of white blood cells [19]. In Alzheimer's disease (AD) rats [51], it has been demonstrated that NTN-1 concentrations in the serum were positively correlated with the systemic expression of IL-10, one of the most important mediators in the anti-in ammatory activity [52]. What is more, in acute peritonitis and acute colitis models, NTN-1 inhibits the migration of in ammatory cells and induces the M2 polarization phenotype of macrophages [53,54].
This further indicates that the changes of peripheral in ammatory factors may be related to powerful anti-in ammatory effect of NTN-1.
An intact functioning blood-brain barrier (BBB) is fundamental to proper homoeostatic maintenance and perfusion of the CNS. In ammatory damage to the unique microvascular endothelial cell monolayer that constitutes the luminal BBB surface, leading to elevated capillary permeability, has been linked to various neurological disorders ranging from ischaemic stroke and traumatic brain injury, to neurodegenerative disease and CNS infections [55]. Moreover, the neuroin ammatory cascade that typically accompanies BBB failure in these circumstances has been strongly linked to elevated levels of proin ammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) [8]. In models of subarachnoid hemorrhage [21], multiple sclerosis [56] and stroke [57], NTN-1 has been shown to have a protective effect on BBB and improve neurocognitive function, which was also noted in our model. There is compelling evidence that exogenous NTN-1 signi cantly diminished the diffusion of dextran across mouse brainderived endothelial cells in vitro. The barrier tightening induced by NTN-1, at least in part, is the consequence of netrin-induced tight junction molecule upregulation. It has been reported that levels of both transmembrane and intracellular components of the junctional complex increased in response to NTN-1. In addition, treatment of human brain-derived endothelial cells with NTN-1 enriched junctional proteins in lipid raft membrane microdomains, where proteins effectively interact to form functional clusters that support barrier integrity [20]. Thus, NTN-1 reduces the incidence of POD by reducing the entry of peripheral in ammatory cytokines through impaired ow barriers.
In addition to mitigate peripheral in ammatory response, NTN-1 reduces the activation of glia cells and the expression of in ammatory cytokines in the hippocampus and prefrontal cortex. Microglia are crucially important during development involved in the phagocytosis of neural precursor cells [58]. Under non-injurious conditions, microglia subserve important functions involved in surveillance of brain parenchyma in order to maintain homeostasis [59]. Following release of pro-in ammatory cytokines by the innate immune response, microglia are activated by one or more pathways. Activated microglia rapidly switch to a proin ammatory phenotype with stout morphology, and enhance the production of proin ammatory molecules [42]. These pro-in ammatory cytokines and the debris released by activated microglia can convert astrocytes into a neurotoxic A1 reactive subtype [60,61], which cause astrocytes to lose their normal synaptic maintenance and phagocytosis along with induce rapid death of neurons and oligodendrocytes [60,61]. In our model of POD, NTN-1 reverts the morphological changes of microglia both in the hippocampus and prefrontal cortex to their original forms, representing the transformation of the in ammatory phenotype to the resting state, thereby changing the pro-in ammatory environment by regulating the secretion of in ammatory cytokines. Herein, it is reasonable that pre-treatment with NTN-1 facilitates the improve of POD-like behavior in aged mice because hippocampus and prefrontal cortex which are in charge of shaping emotion, learning and organizing memory [62,63].Ì n addition, the regulation of lipid mediators by neuronal circuits might be an important part in the control of in ammation to sterile injury. The vagus nerve regulates the expression of the axonal guidance molecule NTN-1 can increases SPM production in vivo during acute-self limited in ammation, were this protein upregulates exudate RvD5 and PD1 concentrations [17,64]. Our previous research has veri ed the anti -in ammatory and proresolving activities of PD1 in the in ammatory milieu both in vivo and in vitro and identi ed the role of PD1 in regulating postoperative in ammation and ensuing POD-like behavior of mice. In our study, compared with the control group, the concentration of the endogenous NTN-1 in hippocampus and prefrontal cortex at 6 hours postoperatively signi cantly reduced. This is most likely the result of the endogenous NTN-1 being consumed after participating in pro-resolution of in ammation by regulating SPM. So, the neuroprotective effect of Netrin-1 may be related to this mechanism. What is more, NTN-1 is involved in regulating in ammatory signaling pathways and inhibiting the production of pro-in ammatory cytokines. In previous studies, it has been observed that NTN-1 can promote the production of cAMP in immune cells and activate the cAMP/ protein kinase A (PKA) signaling pathway to inhibit the production of pro-in ammatory cytokines [65,66]. Ranganathan found that inhibits the ischemia-reperfusion(I/R) induced acute kidney injury (AKI) NTN-1 model of renal tubular epithelial cells, polymorphonuclear neutrophils(PMN) and mononuclear cells in an enzyme called cyclooxygenase 2 (cox-2) expression. NTN-1 May inhibit the NF-κB activation lowered cox-2 expression, thus reduce the in ammatory response [67]. Does NTN-1 also inhibit the production of pro-in ammatory mediators by other means? It is thus essential to explore the underlying mechanism of NTN-1 on in ammation in further investigation.
There are several limitations to our research. First of all, there are a number of signaling pathways that have been shown to be involved in anti-in ammatory and vascular endothelial cell protection. An in-depth study of the mechanism of NTN-1that we need to search will open up a new way for the prevention and treatment of in ammation-related lesions. Secondly, we have only demonstrated that exogenous prophylactic NTN-1 can improve POD by providing positive anti-in ammatory responses and protective BBB functions after surgery in elderly mice. However, how endogenous NTN-1 changes during this process has not been studied, NTN-1 small interfering RNA (siRNA) can be used in later study.

Conclusions
In conclusion, the present study identi es the administration of exogenous NTN-1 could regulate postoperative in ammation and protect the integrity of BBB to improve POD of aged mice. These ndings indicate the potential of NTN-1 to be a novel therapy for POD.

Consent for publication
Not applicable.

Availability of data and materials
All data generated during this study are included in this published article. Further details regarding the presented datasets are available from the corresponding author upon request.

Competing interests
The authors declare no competing interests.

Funding
This research was supported by the grants from National Natural Science Foundation of China (81371195, 81870851 and 82071208), and the Outstanding Talented Young Doctor Program of Hubei Province (HB20200407).
Authors' contributions KL and JW designed and performed the experiment, collected and analyzed the data, and prepared the manuscript. MG and XL were involved in preparing the animal models and participated in interpreting the results. LC contributed to behavioral testing. YZ was involved in biochemical analysis. KL and JW participated in the statistical analysis. MP contributed to the study concept and design, secured funding for the project, and prepared and critically revised the manuscript. All authors reviewed the manuscript.