Hippocampal Endoplasmic Reticulum Stress Drives a Pro-inflammatory Microglial Bias and Neuroinflammation in Aged Mice with Perioperative Neurocognitive Disorder

Hippocampal Endoplasmic Reticulum Stress Drives a Pro-inflammatory Microglial Bias and Neuroinflammation in Aged Mice with Perioperative Neurocognitive Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hippocampal Endoplasmic Reticulum Stress Drives a Pro-inflammatory Microglial Bias and Neuroinflammation in Aged Mice with Perioperative Neurocognitive Disorder Hao Wang, Lei Tian, Lu Guo, Yi Zhao, Zhihua Yang, Lin Wu, Xinghua Lv, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8415780/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Perioperative neurocognitive disorder (PND) is a frequent postoperative complication in older individuals and is commonly linked to microglial activation. Endoplasmic reticulum (ER) stress has been implicated in neuroinflammation and postoperative cognitive decline; however, whether hippocampal ER stress acts upstream to bias microglia toward a pro-inflammatory M1 phenotype, rather than serving only as a trigger of cytokine cascades, remains insufficiently defined in PND. Objective To determine whether hippocampal ER stress promotes an M1-like microglial shift and amplifies neuroinflammation after anesthesia and surgery. Methods Eighteen-month-old male C57BL/6 mice underwent aseptic tibial fracture surgery under isoflurane anesthesia. TUDCA, a pharmacological ER stress suppressor, was delivered bilaterally into dorsal hippocampal CA1 as a perioperative mechanistic probe. ER ultrastructure in hippocampal cells was examined using transmission electron microscopy (TEM). ER stress related proteins (GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6) in hippocampus were quantified by Western blotting. Microglial phenotypic signatures were assessed by Western blotting (CD86, iNOS, CD206, ARG-1) and double immunofluorescence (CD86/Iba-1 and CD206/Iba-1). Open-field testing was performed to control for locomotion/anxiety, followed by memory assessments using novel object recognition and Morris water maze starting on postoperative day 3. Results Anesthesia and surgery induced postoperative cognitive deficits, accompanied by heightened hippocampal inflammatory signaling and a shift toward a pro-inflammatory microglial signature characterized by increased CD86 and iNOS with concomitant reductions in CD206 and ARG-1. In parallel, hippocampal ER stress activation was evident, including ER ultrastructural disruption and increased GRP78 with engagement of PERK/eIF2ɑ/ATF4-, IRE1ɑ-, and ATF6-related pathways. hippocampus-targeted delivery of TUDCA attenuated ER stress activation, mitigated the pro-inflammatory microglial shift (decreasing CD86/iNOS and restoring CD206/ARG-1), reduced inflammatory readouts, and improved postoperative memory performance. Conclusions These findings support a mechanistic link between hippocampal ER stress and the pro-inflammatory microglia bias linked to postoperative neuroinflammation and cognitive impairment in aged mice. perioperative neurocognitive disorder endoplasmic reticulum stress microglia M1/M2 polarization neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Perioperative neurocognitive disorder (PND)，characterized by cognitive decline occurring before or after surgery and anesthesia, is a major complication involving central nervous system (CNS) dysfunction. (1) PND disproportionately affects individuals at the extremes of age, with older adults exhibiting particularly high vulnerability. (2; 3) Clinically, it manifests as deficits in memory, attention, and executive functions, often leading to prolonged hospitalization and heightened long-term morbidity. (4) The pathogenesis of PND is complex and multifactorial, involving oxidative stress, impaired synaptic plasticity, blood–brain barrier (BBB) disruption, and neuronal injury. (5) Neuroinflammation is increasingly viewed as a pivotal process that links peripheral surgical insults to postoperative cognitive decline. (6; 7) Microglia, the intrinsic immune cells of the CNS, serve as important mediators linking peripheral injury to central neuroinflammation, ultimately contributing to cognitive dysfunction. (3; 8) Surgical trauma elicits a peripheral inflammatory surge characterized by pro-inflammatory cytokine release and recruitment of bone marrow derived monocytes, which may access the brain in the context of BBB perturbation and activate microglial reactivity. (9) Once activated, microglia undergo polarization toward two functionally distinct phenotypes: the pro-inflammatory M1 type and the anti-inflammatory M2 type. (10) A relative shift toward an M1-like profile can intensify oxidative and inflammatory signaling, disrupt synaptic homeostasis, and compromise neuronal function, whereas M2-like responses are associated with resolution of inflammation and tissue repair. (11) Consequently, targeting microglial phenotypic transition has been explored as a candidate strategy to mitigate neuroinflammation-driven cognitive vulnerability in PND. (12; 13) Endoplasmic reticulum (ER) stress has emerged as a candidate upstream cellular stress signal capable of shaping inflammatory tone in the postoperative brain, particularly within the hippocampus. (14; 15) ER stress is initiated by disturbances in protein-folding equilibrium within the endoplasmic reticulum, resulting in the accumulation of misfolded or unfolded proteins. (16) Through its canonical PERK, IRE1, and ATF6 branches, the unfolded proteins can interface with inflammatory signaling networks and influence neuronal survival under stress. (17) Importantly, the impact of ER stress on neuroinflammation appears to depend on its magnitude and duration: mild or transient ER stress may exert protective preconditioning effects, whereas sustained engagement can promote inflammatory amplification and neuronal injury. (18) Recent studies have implicated hippocampal unfolded protein response (UPR) in the development of PND. Common anesthetics like isoflurane have been shown to induce neuronal apoptosis and cognitive deficits via ER stress-related pathways. (19) Moreover, pharmacological inhibition of ER stress has been shown to suppress NLRP3 inflammasome activity, thereby attenuating neuroinflammation and ameliorating postoperative cognitive dysfunction. (14) Despite these advances, a key mechanistic question remains unresolved: whether hippocampal ER stress acts an upstream signal that biases microglia toward a pro-inflammatory program in aged subjects exposed to anesthesia and surgery, rather than merely accompanying postoperative cellular stress and cytokine cascades. In addition, because most prior studies administered ER stress suppressor systemically, it remains difficult to disentangle indirect neuroprotection secondary to attenuation of surgery-driven peripheral inflammation from a direct central effect after BBB penetration and hippocampal target engagement. We therefore hypothesized that hippocampal ER stress evoked by anesthesia and surgery promotes a shift toward an M1-like microglial profile thereby amplifying neuroinflammation and contributing to postoperative cognitive impairment. To test this hypothesis, we used an aged murine tibial fracture model under isoflurane anesthesia and integrated early ultrastructural and molecular assessment of hippocampal ER stress with quantitative evaluation of microglial phenotypic markers and hippocampus-dependent cognitive performance. Importantly, we delivered TUDCA directly into the hippocampus with perioperative coverage spanning as a mechanistic probe to better interrogate hippocampus local pathways relevant to clinical disease evolution. This work aims to refine the mechanistic framework of PND by linking ER stress to microglial phenotypic regulation as a proximal driver of postoperative neuroinflammation and cognitive dysfunction. Materials and Methods Animals Eighteen-month-old male specific-pathogen-free (SPF) C57BL/6 mice (25-35g) were obtained from the Animal Center of Lanzhou University (Lanzhou, China). All animals were housed under controlled environmental conditions (temperature 22-25℃ humidity 45-65%, 12-h light/dark cycle) with free access to standard food and water. All experimental procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China, (Ethics Approval No. LDYYLL2025-06)). Animal care and handling complied with the National Health Guidelines for the Care and Use of Laboratory Animals and the PREPARE guidelines for ensuring the quality of animal experiments. Establishment of a Tibial Fracture Surgery Model A PND model was established using an aseptic open tibial fracture surgery, as previously described. (20; 21) Briefly, mice were anesthetized with 1.5% isoflurane (in 100% oxygen, 1 L/min) using an animal anesthesia system (RWD Life Science, Shenzhen, China). After sterilization of the surgical site, a 1.5-cm longitudinal incision was made on the skin of the left hind limb to expose the tibia, and surrounding muscles were gently separated. A 0.3-mm stainless steel pin was inserted into the intramedullary canal of the tibia for fixation. The tibia was fractured at the junction between the middle and distal thirds using surgical plie, and the incision was closed with 4-0 absorbable sutures. Throughout the procedure and recovery, body temperature was maintained at 37℃ using a heating pad. The surgical process lasted approximately 20 minutes, after which mice were returned to the induction chamber to maintain anesthesia for a total of 2 hours. To minimize postoperative discomfort, oxybuprocaine hydrochloride gel was applied topically to the surgical wound for local analgesia. Two independent cohorts were used: one for behavioral testing and one sacrificed for biochemical ultrastructural analyses. Drug Preparation To investigate the role of ER stress in tibial fracture–induced PND, tauroursodeoxycholic acid (TUDCA,100㎍/mL in PBS; MCE, catalog number: HY-19696A) was stereotaxically injected bilaterally into the hippocampus (200 nL per side, 50 nL/min) 30 minutes before surgery and repeated once daily for two consecutive days after surgery. Mice were anesthetized and positioned in a stereotaxic apparatus (RWD Life Science, China). A burr hole was drilled in the skull, and a glass microelectrode injector pump (RWD Life Science, China) was used to deliver 200 nL of TUDCA solution into each hippocampal site. After the first injection, an indwelling guide cannula was left in place at the injection site and sealed with sterile bone wax. The subsequent two injections were administered through the implanted cannula. The stereotaxic coordinates were as follows: anteroposterior (AP)-2.0 mm, mediolateral (ML)±1.6 mm, and dorsoventral (DV) -1.8 mm, at an injection rate of 50 nL/min. After injection, the needle was left in place for 10 minutes to prevent reflux before being slowly withdrawn. Following injection, mice were then kept on a heating pad until full recovery from anesthesia. Western Blotting Mice were euthanized with 8% isoflurane, and bilateral hippocampal tissues were rapidly dissected. Tissues were homogenized and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Lysates were clarified by centrifugation (12,000 rpm, 30 min, 4℃), and the supernatant was collected for protein quantification using a BCA protein assay kit (Solarbio, Beijing, China). Samples were denatured by boiling in 1× loading buffer at 60℃ for 15 minutes. Approximately 30㎍ of protein per lane was separated by 7.5-10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in TBST for 2 hours at room temperature and then incubated overnight at 4℃ with primary antibodies. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and quantified with ImageJ 1.53e software (NIH, Wayne Rasband, MD, USA). Band analysis of GRP78, ATF4, ATF6, CD86, CD206, iNOS, and Arg-1 were normalized to β-actin, whereas phosphorylated PERK, eIF2ɑ, and IRE1ɑ were normalized to their corresponding total protein levels. Transmission Electron Microscopy (TEM) Fresh hippocampal tissue blocks (approximately 1 mm 3 ) were immediately placed into EP tubes containing TEM fixative solution (Servicebio, G1102) following euthanasia. After post-fixation in 1% osmium tetroxide, tissues were dehydrated through graded ethanol and embedded in EMBed-812 resin. Ultrathin sections (60 nm) were cut using an ultramicrotome and mounted on 150-mesh copper grids coated with Formvar film. Sections were stained with 2% uranyl acetate and 2.6% lead citrate and examined under a HITACHI transmission electron microscope system(Tokyo, Japan). Enzyme-Linked Immunosorbent Assay (ELISA) Concentrations of inflammatory cytokines in hippocampal tissues, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-ɑ (TNF-ɑ), were determined with commercial ELISA kits. Total protein concentrations were quantified, and cytokine levels were normalized to standard calibration curves. ELISA kits for mouse IL-1β (CME0015-048), IL-6 (CME0006-048), and TNF-ɑ (CME0004-048) ELISA kits were purchased from 4A BIOTECH (Suzhou, China). RNA extraction and qRT-PCR analysis Total RNA was extracted from mouse hippocampal tissue using RNAiso Plus (9109, Takara, Japan). RNA quantity and purity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). For qRT-PCR, cDNA was synthesized with the PrimeScript RT Reagent Kit (RR036A, Takara, Japan), and quantitative PCR was conducted using TB Green® Premix Ex Taq TM II (FAST qPCR; CN830S, Takara, Japan). Relative gene expression was normalized to an internal reference (β-ACTIN). Primer sequences are provided in Table 1. Immunofluorescence (IF) On postoperative day 3, mice were deeply anesthetized and transcardially perfused with heparinized saline followed by 4% ice-cold paraformaldehyde (PFA). Brains were carefully removed and post-fixed in 4% PFA overnight at 4℃ Fixed tissues were cryoprotected in 30% sucrose at 4℃ until they sank. Coronal sections (20㎛ thick) were prepared using a cryostat and mounted onto glass slides. Sections were washed three times in PBS, permeabilized with 0.2% Triton X-100 for 30 minutes, and blocked in 5% bovine serum albumin (BSA) blocking buffer for 1 hour at room temperature. They were then incubated overnight at 4℃ with primary antibodies against Iba-1 (1:200), GFAP (1:200), GRP78 (1:200), ATF6((1:100), and p-IRE1ɑ (1:100)). After washing, sections were incubated with appropriate fluorescent secondary antibodies for 2 hours at room temperature. Following countertaining with DAPI, hippocampal images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan). Open Field Test (OFT) Behavioral activities were recorded and analyzed using a video tracking system (SMART PREMIUM, Panlab, Barcelona, Spain). All behavioral tests were conducted daily between 12:00 and 21:00. Mice were acclimated to the testing room for approximately 1 hour before testing, and adequate intervals were provided between tasks to avoid carryover effects. Each mouse was gently placed in the center of a gray open-field chamber (40 × 40 × 40 cm) and allowed to explore freely for 5 minutes. Movements were recorded by a top-mounted camera, and the apparatus was cleaned with 75% ethanol between trials to remove olfactory cues. Total distance traveled and time spent in the central zone were quantified to assess locomotor activity and anxiety-like behavior. Novel Object Recognition Test (NORT) The novel object recognition test (NORT) was performed 24 hours after the OFT according to established protocols. Mice were habituated to the test chamber (40 × 40 × 40 cm) for 5 minutes before the familiarization phase. During familiarization, two identical objects (A + A) were placed in adjacent corner, 5 cm from the walls, and mice were allowed to explore freely for 10 minutes. After 24 hours, one familiar object (A) was replaced with a novel object (B) of similar size but different shape and color. Exploration was defined as the mouse’s nose oriented toward the object within 2 cm. Exploration time for each object was measured, and the recognition index was calculated as the ratio of the time spent exploring the novel object to the total exploration time for both objects. Between trials, the apparatus was cleaned with 75% ethanol to remove residual olfactory cues. Morris Water Maze (MWM) The Morris water maze (MWM) test was conducted to assess spatial learning and memory performance in mice. A circular pool (diameter 120 cm, height 50 cm) was filled with water (22±1℃) to a depth of 40 cm, and white tempera paint was added to render the water opaque. A hidden circular platform (diameter 10 cm) was submerged 1 cm below the water surface, and the pool was virtually divided into four equal quadrants. Mice underwent four acquisition trials per day for five consecutive days, followed by a probe test on day six. In each trial, mice were gently placed into the pool facing the wall and allowed 60 seconds to locate the hidden platform. Swimming trajectories were recorded using an automated video tracking system (SMART PREMIUM, Panlab, Spain). Mice that successfully located the platform were allowed to remain on it for 15 seconds, whereas those that failed were guided to the platform and kept there for the same duration. Escape latency (the time required to locate the hidden platform) was recorded for each trial. On day six, a 60-second probe trial was performed to evaluate spatial reference memory, during which the platform was removed. Platform crossings, and time spent in the target quadrant were analyzed. Statistical Analysis All statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean±standard error of the mean (SEM). Data normality was assessed using the Shapiro-Wilk test. Sample sizes for each experiment are indicated in the corresponding figure legends. For comparisons between two groups with normally distributed data, an unpaired Student’s t-test was applied. For multiple-group comparisons, escape latency data were analyzed using two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post hoc test. Western blot and behavioral data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. For non-normally distributed data, the Kruskal-Wallis test was used. A value of P < 0.05 was considered statistically significant. Results Cognitive impairment and hippocampal glial activation after anesthesia and surgery A PND model was established using tibial fracture surgery under isoflurane anesthesia, and postoperative behavior was assessed starting on postoperative day 3(POD3) (Fig. 1A). In the open field test (OFT), total distance traveled and time spent in the central zone did not differ significantly between sham and surgery groups, suggesting preserved locomotion and anxiety-like behavior. In contrast, the surgery group exhibited a reduced recognition index in the novel object recognition test (NORT), consistent with impaired recognition memory. Similarly, in the Morris water maze (MWM), surgery significantly decreased platform crossings and reduced time spent in the target quadrant. Immunofluorescence staining further demonstrated increased Iba1 and GFAP signals in the hippocampus after surgery, indicating activation of microglia and astrocytes. Collectively, these data show that anesthesia plus tibial fracture surgery induces PND accompanied by hippocampal glial activation. Anesthesia and surgery activate hippocampal ER stress Transmission electron microscopy performed on postoperative day 3 revealed ER swelling and luminal dilation in hippocampal cells, consistent with early ER stress activation (Fig. 2A). In parallel, Western blot showed increased GRP78 expression and activation of canonical ER stress related signaling, including elevated p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6 in the surgery group compared with sham controls (Fig. 2B-G). Immunofluorescence further confirmed enhanced hippocampal immunoreactivity for GRP78, p-IRE1ɑ, and ATF6 after surgery (Fig. 2H-J). Together, these POD3 ultrastructural, molecular, and immunostaining data indicate an early engagement of hippocampal ER stress signaling following anesthesia and tibial fracture surgery. Anesthesia and surgery promote microglial polarization toward an M1 phenotype and neuroinflammation To determine whether anesthesia and surgery bias microglial phenotype, we quantified proteins associated with M1-like and M2-like programs in the hippocampus. Western blotting showed increased expression of the M1-associated markers CD86 and iNOS, accompanied by reduced expression of the M2-associated markers CD206 and Arg-1 in the surgery group versus sham controls (Fig. 3A-D). Consistently, double immunofluorescence staining revealed increased CD86/Iba1 and reduced CD206/Iba1 immunoreactivity in the hippocampus (Fig. 3E-F). Meanwhile, levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-ɑ) were elevated after surgery (Fig. 3G-L). These findings support a postoperative shift toward a pro-inflammatory (M1-like) microglial signature associated with increased hippocampal inflammatory signaling. TUDCA rescues cognitive deficits and attenuates hippocampal ER stress To probe the functional relevance of ER stress to postoperative cognitive impairment, TUDCA was bilaterally microinjected into the hippocampus 30 min before tibial fracture surgery. TUDCA did not alter OFT measures of locomotion or center time compared with vehicle controls (Fig. 4E,F). In contrast, TUDCA improved memory-related performance: the reduced recognition index in the Surgery+Vehicle group was restored by TUDCA in the NORT (Fig. 4G), and MWM deficits (fewer platform crossings and reduced target quadrant time) were attenuated by TUDCA (Fig. 4H-J). At the molecular level, TUDCA reduced surgery associated activation of UPR signaling, decreasing GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6 (Fig. 5A-F), and lowering GRP78, p-IRE1ɑ, and ATF6 immunoreactivity (Fig. 5G-I). TEM analysis further showed that TUDCA alleviated ER dilation of hippocampal microglia relative to vehicle treated surgical mice (Fig. 5J). Collectively, these data indicate that hippocampus targeted ER stress suppression is accompanied by improved postoperative cognition and reduced hippocampal ER stress. ER stress inhibition reverses microglial polarization and dampens neuroinflammation Iba1 immunofluorescence revealed marked microglial activation in hippocampal CA1, CA3, and DG regions after surgery, whereas TUDCA reduced Iba1 signal intensity relative to Surgery+Vehicle controls (Fig. 6). Consistent with a shift in microglial phenotypic signature, TUDCA attenuated the surgery induced increase in CD86 and iNOS and restored CD206 and Arg-1 expression on Western blotting (Fig. 7A-D). Immunofluorescence similarly showed reduced CD86/Iba1 and increased CD206/Iba1 immunoreactivity in TUDCA treated mice compared with vehicle-treated surgical mice (Fig. 7E,F). In parallel, surgery increased IL-1β, IL-6, and TNF-α at both protein and transcript levels, whereas TUDCA prevented these increases (Fig. 7G-L). Collectively these findings suggest hippocampal ER stress suppression is associated with reduced pro-inflammatory microglial bias and dampened hippocampal inflammatory signaling after anesthesia and surgery. ER stress inhibition mitigated neuron loss in mice hippocampus following anesthesia and surgery Nissl staining revealed reduced neuronal density and disrupted cytoarchitecture in hippocampal CA1, CA3, and DG regions after surgery (Fig. 8A). Compared with sham controls, neurons in the surgery group appeared shrunken with pyknotic features and disorganized lamination.. In contrast, TUDCA treated mice exhibited improved neuronal preservation, with higher apparent neuronal density and more intact hippocampal architecture, approaching sham levels. Together with the observed attenuation of ER stress, microglial pro-inflammatory bias, and inflammatory signaling, these results are consistent with a neuroprotective effect of hippocampus targeted ER stress suppression in this postoperative model. Discussion In this study, we provide evidence that anesthesia and surgery engage hippocampal ER stress and are accompanied by a shift toward a pro-inflammatory microglial phenotype, heightened inflammatory signaling, and Perioperative neurocognitive disorder. Using hippocampus targeted delivery of TUDCA as a mechanistic probe, we found that dampening ER stress coincided with improved performance in hippocampus-dependent memory tasks, reduced CD86/iNOS with restoration of CD206/Arg-1, lower pro-inflammatory cytokine levels, and better neuronal preservation. Collectively, these findings support a model in which hippocampal ER stress is linked to microglial phenotypic bias and neuroinflammation associated with postoperative cognitive deficits. Neuroinflammation is widely implicated in postoperative cognitive vulnerability, and microglia are key cellular effectors that translate peripheral inflammatory cues into brain-resident immune responses. (22) Microglia are central to CNS inflammatory and help maintain homeostasis through crosstalk with neurons and astrocyte. (23; 24) In this context, a relative bias toward an M1-like microglial program can amplify cytokine and oxidative signaling and disturb synaptic homeostasis, whereas M2-like programs are more consistent with inflammatory resolution and repair. (25; 26) Specifically, cytokines produced by M1 polarization microglia can impair neuronal function. Additionally, microglia TGF-β can up-regulate neuronal excitability by promote the expression of Kir4.1 in astrocytes, highlinghting the importance of microglia-astrocyte cross-dialogue in the development of neural excitability. (27) Prior studies have linked microglial activation or phenotypic skewing to postoperative cognitive deficits in aged animals. (28; 29) However, the upstream signals that bias microglial phenotype in the postoperative hippocampus remain incompletely defined. Our data nominate hippocampal ER stress engagement as a candidate upstream driver that is closely associated with this pro-inflammatory microglial shift. ER stress represents a conserved adaptive response aimed at restoring proteostasis, yet sustained UPR activation can become maladaptive and intersect with inflammatory pathways. (30) In perioperative settings, anesthetic and surgical stressors have been associated with hippocampal ER stress and cognitive impairment. (31; 32) However, a frequent limitation of prior work is the focus on a single UPR arm or reliance on molecular markers without ultrastructural validation. Here, we profiled PERK, IRE1ɑ, and ATF6-associated signaling in parallel and complemented these data with TEM based assessment of ER morphology, providing convergent molecular and ultrastructural evidence for anesthesia/surgery-evoked hippocampal ER stress. Another important limitation of previous studies is that TUDCA has predominantly been administered systemically (intravenous or intraperitoneal injection) to evaluate its effects on postoperative cognition and neuroinflammation. This design may complicate its interpretation. Specifically, it is difficult to distinguish whether the benefit arises from dampening surgery induced peripheral inflammation (an indirect brain-protective effect) or from TUDCA crossing the BBB and directly suppressing hippocampal ER stress and local inflammatory injury. In the present study, we delivered TUDCA directly into the hippocampus and maintained perioperative coverage before and after surgery. This design better matches the temporal evolution of postoperative pathology and enables a more direct assessment of hippocampus local mechanisms relevant to clinical disease progression. How ER stress translates into cognitive vulnerability after anesthesia and surgery likely involves bidirectional coupling between UPR signaling and innate immune programs. In multiple neurological disorders, ER stress can modulate inflammatory signaling and influence microglial activation states. (33; 34) A recent study suggested that pharmacological modulation of hippocampal ER stress can alleviate LPS-induced cognitive dysfunction (35) . ER stress may converge on multiple inflammatory programs, including inflammasome pathways. (14) However, our results highlight microglial phenotypic bias as an additional, proximal cellular layer. In the present study, hippocampus-targeted TUDCA administration attenuated UPR activation and was accompanied by a coordinated reversal of microglial phenotypic markers (reduced CD86/iNOS with restoration of CD206/Arg-1), suppression of pro-inflammatory cytokine production, and improved hippocampus-dependent memory. These findings are consistent with a pathway in which postoperative ER stress biases microglia toward a pro-inflammatory program that amplifies inflammatory signaling and contributes to neuronal vulnerability and cognitive deficits. Our study has limitations. First, the molecular determinants by which ER stress drives microglial M1 polarization remain unresolved and future studies using pathway-selective perturbations would help define specificity. Second, although hippocampal TUDCA delivery supports a mechanistic association, pharmacologic interventions may have off-target effects and do not establish microglia specific causality. Future work should centered on genetic or microglia targeted strategies to rigorously test the ER stress–microglia axis. Third, hippocampal microinfusion was used to probe mechanism and does not model clinically feasible delivery, future work should test systemic dosing, timing windows, and safety. In conclusion, our data support a model in which anesthesia and surgery engage hippocampal UPR signaling that is associated with a pro-inflammatory microglial phenotypic bias, heightened neuroinflammatory signaling, and postoperative cognitive impairment in aged mice. Targeting ER stress, potentially to modulate microglial phenotype and inflammatory tone, may represent a promising direction for mitigating PND, although further work is required to establish pathway and cell-type specificity and to evaluate clinically relevant routes and timing of intervention. Declarations Supplementary Information All data generated or analysed during this study are included in this published article. Ethics approval and consent to participate All experimental procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China, (Ethics Approval No. LDYYLL2025-06)). Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing Interests The authors declare that they have no competing interests Funding This project was supported by Natural Science Joint Fund of Gansu Province (No:24JRRA914). Authors ’ contributions HW, YLL and XHL contributed to study conception and design. HW, LG and XHY performed the experiments. HW , YZ and LW performed the statistical analysis and interpreted the data. HW wrote the first draft of the manuscript. YLL and XHL revised the manuscript for important intellectual content. All authors read and approved the final manuscript. Acknowledgements The authors thank all participants involved in this study. We also thank Figdraw (www.figdraw.com) for the assistance in creating Scheme and Abstract Scheme. Author details 1 The First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, China. 2 Ambulatory Surgery Center, the First Hospital of Lanzhou University, Lanzhou, 730000, China 3 Department of Anesthesiology, The First Hospital of Lanzhou University, Lanzhou, 730000, China. References Evered L, Silbert B, Knopman DS et al. (2018) Recommendations for the Nomenclature of Cognitive Change Associated with Anaesthesia and Surgery-2018. Anesthesiology 129 , 872-879. Vacas S, Cole DJ, Cannesson M (2021) Cognitive Decline Associated With Anesthesia and Surgery in Older Patients. LID - 10.1001/jama.2021.4773 [doi] FAU - Vacas, Susana. Evered L (2022) Peri-operative neurocognitive disorders: a narrative review. Anaesthesia . Dilmen OK, Meco BC, Evered LA et al. 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Zhang M, Yin Y Dual roles of anesthetics in postoperative cognitive dysfunction: Regulation of microglial activation through inflammatory signaling pathways. Liu Y, Fu H, Wang TA-O Neuroinflammation in perioperative neurocognitive disorders: From bench to the bedside. NT S, SG S, CL S et al. (2017) Endoplasmic reticulum stress and inflammation in the central nervous system. Molecular neurodegeneration 12 , 42. Neubrand VE, Sepúlveda MR (2024) New insights into the role of the endoplasmic reticulum in microglia. Neural Regeneration Research 19 , 1397-1398. Wang B, Ge S, Xiong W et al. (2018) Effects of resveratrol pretreatment on endoplasmic reticulum stress and cognitive function after surgery in aged mice. BMC anesthesiology 18 . Yi H, Duan Y, Song R et al. (2023) Activation of glucagon-like peptide-1 receptor in microglia exerts protective effects against sepsis-induced encephalopathy via attenuating endoplasmic reticulum stress-associated inflammation and apoptosis in a mouse model of sepsis. Experimental Neurology 363 . Jiao B, Zhang W, Zhang C et al. (2024) Protein tyrosine phosphatase 1B contributes to neuropathic pain by aggravating NF‐κB and glial cells activation‐mediated neuroinflammation via promoting endoplasmic reticulum stress. CNS Neuroscience & Therapeutics 30 . Zhang D-Q, Dong X, Su S et al. (2024) Temporin-GHaR Peptide Alleviates LPS-Induced Cognitive Impairment and Microglial Activation by Modulating Endoplasmic Reticulum Stress. Probiotics and Antimicrobial Proteins . Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.doc SupplementaryFig.S2.doc AbstractFigure.doc SupplementaryFig.S3.doc SupplementaryFig.S5.doc SupplementaryFig.S7.doc Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Jan, 2026 Reviewers agreed at journal 23 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Editor invited by journal 29 Dec, 2025 Submission checks completed at journal 28 Dec, 2025 First submitted to journal 28 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":121810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAged mice exhibited cognitive impairment and glial activation.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Experimental scheme of the research. \u003cstrong\u003eB\u003c/strong\u003e The representative OFT trajectories of mice of day 3. \u003cstrong\u003eC\u003c/strong\u003e The representative NOR exploration heat maps of mice of day 4. \u003cstrong\u003eD\u003c/strong\u003e The representative MWM tracing in the swimming pool on the test day. \u003cstrong\u003eE\u003c/strong\u003e The total distance of OFT. \u003cstrong\u003eF\u003c/strong\u003e Time in centers of OFT. \u003cstrong\u003eG\u003c/strong\u003e The recognition index of NOR test. \u003cstrong\u003eH\u003c/strong\u003e Escape latency to reach the target platform in MWM test. \u003cstrong\u003eI\u003c/strong\u003e Number of crossing the target platform in the probe test of MWM test. \u003cstrong\u003eJ\u003c/strong\u003e The time in target area of MWM test. \u003cstrong\u003eK \u003c/strong\u003eImmunoflurescence staining of IBA-1 on mice hippocampus. \u003cstrong\u003eL\u003c/strong\u003e Immunoflurescence staining of GFAP on mice hippocampus. Data are presented as mean±SEM, n=8-10, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham group.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/b324c212521a940c1861512b.jpg"},{"id":100952142,"identity":"5133d358-d006-42ff-9f0d-143da6443ed0","added_by":"auto","created_at":"2026-01-23 07:12:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnesthesia and surgery activated ER stress in the hippocampus of PND mice. A \u003c/strong\u003eElectron microscopic observation of the subcellular morphological change of the hippocampal cells(Neuron, Microglia and Astrocyte) on day 11 after anesthesia and surgery. The orange rows indicate swollen endoplasmic reticulum. Scale bars = 0.5㎛. \u003cstrong\u003eB-G\u003c/strong\u003e Protein expression of ER stress markers, including GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4 and ATF6 in mice hippocampus were analyzed by Western blot. \u003cstrong\u003eH-J\u003c/strong\u003e Immunofluorescence staining of GRP78, p-IRE1ɑ and ATF6 on mice hippocampal CA1 regions. Data are presented as mean±SEM, n=6, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/52ce5ac51018024a13c192b7.jpg"},{"id":100930061,"identity":"64a902db-1ea7-4cfb-b663-e8408edd0de4","added_by":"auto","created_at":"2026-01-23 00:39:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnesthesia and surgery promoted microglial M1 polarization and increased pro-inflammatory cytokines in mice hippocampus. A-D \u003c/strong\u003eProtein levels of CD86, iNOS, CD206 and ARG-1 in mice hippocampus were detected by Western blot. \u003cstrong\u003eE-F\u003c/strong\u003e Double immunofluorescence of CD86, CD206 and IBA-1 on mice hippocampal CA1 regions. \u003cstrong\u003eG-I\u003c/strong\u003e The levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-ɑ in mice hippocampal tissues were detected by ELISA. \u003cstrong\u003eJ-L\u003c/strong\u003e mRNA expression of IL-1β, IL-6, and TNF-ɑ genes in the hippocampus were measured by qRT-PCR. Data are presented as mean±SEM, n=6, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/80081e3d2f64dcdba589e736.jpg"},{"id":100952130,"identity":"f6107054-80a1-4f46-be30-e383d16288c3","added_by":"auto","created_at":"2026-01-23 07:11:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTUDCA improves cognitive deficits after anesthesia and surgery. A \u003c/strong\u003eFlow chart of the experimental protocol in mice. B The representative OFT trajectories of mice. \u003cstrong\u003eC\u003c/strong\u003e The representative NOR exploration heat maps of mice. \u003cstrong\u003eD\u003c/strong\u003e The representative MWM tracing in the swimming pool on the probe test. \u003cstrong\u003eE\u003c/strong\u003e The total distance of OFT. \u003cstrong\u003eF\u003c/strong\u003e Time in centers of OFT. \u003cstrong\u003eG\u003c/strong\u003e The recognition index of NOR test. \u003cstrong\u003eH\u003c/strong\u003e Escape latency to reach the target platform in MWM test. \u003cstrong\u003eI\u003c/strong\u003e Number of crossing the target platform in the probe test of MWM test. \u003cstrong\u003eJ\u003c/strong\u003e The time in target area of MWM test. Data are presented as mean±SEM, n=6, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001 compared with Sham+Vehicle group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared with Sham+Vehicle group\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/e529a63eb952fbc993d385ca.jpg"},{"id":100930066,"identity":"59c5687a-a012-4b25-9233-fcf5da4e4d2f","added_by":"auto","created_at":"2026-01-23 00:39:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTUDCA attenuates hippocampal ER stress. A-F \u003c/strong\u003eProtein levels of GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4 and ATF6 in mice hippocampus were analyzed by Western blot. \u003cstrong\u003eG-I\u003c/strong\u003e Protein expression of GRP78, p-IRE1ɑ and ATF6 in hippocampal CA1 regions were detected by immunofluorescence staining. \u003cstrong\u003eJ\u003c/strong\u003e Endoplasmic reticulum morphology in mice hippocampal microglia was visualized by TEM. Data are presented as mean±SEM, n=6, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham+Vehicle group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham+Vehicle group\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/6f6e818c2a34afe4f1e0c233.jpg"},{"id":100952144,"identity":"6fdc462f-082e-4993-a673-2b1a929d189c","added_by":"auto","created_at":"2026-01-23 07:12:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress inhibition inhibited microglial activation in mice hippocampal CA1, CA3 and DG regions. A \u003c/strong\u003eImmunofluorescence of IBA-1 on mice hippocampal CA1 region. \u003cstrong\u003eB\u003c/strong\u003e Immunofluorescence of IBA-1 on mice hippocampal CA3 region. \u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence of IBA-1 on mice hippocampal DG region.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/82f664b3d8e7e70d6fda2cf2.jpg"},{"id":100952150,"identity":"73783b65-ab58-4e4c-bb82-39829a1c7f69","added_by":"auto","created_at":"2026-01-23 07:12:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress inhibition reverses microglial polarization and dampens neuroinflammation. A-D \u003c/strong\u003eProtein levels of hippocampal CD86, iNOS, CD206 and ARG-1 in mice were detected by Western blot. \u003cstrong\u003eE-F\u003c/strong\u003e Double immunofluorescence of CD86, CD206 and IBA-1 on mice hippocampal CA1 regions. \u003cstrong\u003eG-I\u003c/strong\u003e The levels of pro-inflammatory cytokines of IL-1β, IL-6, and TNF-ɑ in mice hippocampal tissues were detected by ELISA. \u003cstrong\u003eJ-L\u003c/strong\u003e mRNA expression of IL-1β, IL-6, and TNF-ɑ genes in the hippocampus were measured by qRT-PCR. Data are presented as mean±SEM, n=6, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham+Vehicle group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham+Vehicle group\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/7ed3421f953a616de66605cd.jpg"},{"id":100930049,"identity":"f29e9681-a069-4793-8f22-5ed5406b7399","added_by":"auto","created_at":"2026-01-23 00:39:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":185303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress inhibition mitigated neuron loss in mice hippocampus following anesthesia and surgery. A \u003c/strong\u003eRepresentative images of Nissl staining in mice hippocampal CA1, CA3, and DG regions. \u003cstrong\u003eB \u003c/strong\u003eQuantification of Nissl staining neurons numbers. Data are presented as mean±SEM, n=6, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 compared with Sham+Vehicle group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared with Surgery+Vehicle\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/426eab4268150e1a846fd35d.jpg"},{"id":101296895,"identity":"2a3b3d17-4ae2-4ed2-bedb-1830576a22b5","added_by":"auto","created_at":"2026-01-28 09:22:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2299758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/50a10127-6f6e-4002-b119-7a7a45655408.pdf"},{"id":100930091,"identity":"31958afc-426f-45ee-98d0-15da7bf092eb","added_by":"auto","created_at":"2026-01-23 00:39:30","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17408,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/12a001a2eb94e9902186204f.doc"},{"id":100930053,"identity":"65ec22a0-6d1e-4f1f-bead-3482c8a52d8f","added_by":"auto","created_at":"2026-01-23 00:39:27","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5026304,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S2.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/03e6aac306c0c1d0abbe8d19.doc"},{"id":100951593,"identity":"939107b2-9d2d-411c-8336-303d89288666","added_by":"auto","created_at":"2026-01-23 07:10:55","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":270336,"visible":true,"origin":"","legend":"","description":"","filename":"AbstractFigure.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/c2d3ddc521156e47deb6ff92.doc"},{"id":100930088,"identity":"fab58a13-2d63-49b6-89fb-e4a11c710fec","added_by":"auto","created_at":"2026-01-23 00:39:29","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3047424,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S3.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/7973f41b3e85b4f23b3ac462.doc"},{"id":100930077,"identity":"041e9753-70c6-4d82-b404-14ac2ee9d4d6","added_by":"auto","created_at":"2026-01-23 00:39:28","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5257216,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S5.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/d6a33ddd4a149e9ee45d7f84.doc"},{"id":100951272,"identity":"9d0a3694-9950-4af3-a6f8-3d53640cb57e","added_by":"auto","created_at":"2026-01-23 07:10:22","extension":"doc","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3151872,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S7.doc","url":"https://assets-eu.researchsquare.com/files/rs-8415780/v1/85c4a6d37a743e6025c623c5.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hippocampal Endoplasmic Reticulum Stress Drives a Pro-inflammatory Microglial Bias and Neuroinflammation in Aged Mice with Perioperative Neurocognitive Disorder","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePerioperative neurocognitive disorder (PND)，characterized by cognitive decline occurring before or after surgery and anesthesia, is a major complication involving central nervous system (CNS) dysfunction.\u003csup\u003e(1)\u003c/sup\u003e PND disproportionately affects individuals at the extremes of age, with older adults exhibiting particularly high vulnerability.\u003csup\u003e(2; 3)\u003c/sup\u003e Clinically, it manifests as deficits in memory, attention, and executive functions, often leading to prolonged hospitalization and heightened long-term morbidity.\u003csup\u003e(4)\u003c/sup\u003e The pathogenesis of PND is complex and multifactorial, involving oxidative stress, impaired synaptic plasticity, blood–brain barrier (BBB) disruption, and neuronal injury.\u003csup\u003e(5)\u003c/sup\u003e Neuroinflammation is increasingly viewed as a pivotal process that links peripheral surgical insults to postoperative cognitive decline.\u003csup\u003e(6; 7)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMicroglia, the intrinsic immune cells of the CNS, serve as important mediators linking peripheral injury to central neuroinflammation, ultimately contributing to cognitive dysfunction.\u003csup\u003e(3; 8)\u003c/sup\u003e Surgical trauma elicits a peripheral inflammatory surge characterized by pro-inflammatory cytokine release and recruitment of bone marrow derived monocytes, which may access the brain in the context of BBB perturbation and activate microglial reactivity.\u003csup\u003e(9)\u003c/sup\u003e Once activated, microglia undergo polarization toward two functionally distinct phenotypes: the pro-inflammatory M1 type and the anti-inflammatory M2 type.\u003csup\u003e(10)\u003c/sup\u003e A relative shift toward an M1-like profile can intensify oxidative and inflammatory signaling, disrupt synaptic homeostasis, and compromise neuronal function, whereas M2-like responses are associated with resolution of inflammation and tissue repair.\u003csup\u003e(11)\u003c/sup\u003e Consequently, targeting microglial phenotypic transition has been explored as a candidate strategy to mitigate neuroinflammation-driven cognitive vulnerability in PND.\u003csup\u003e(12; 13)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eEndoplasmic reticulum (ER) stress has emerged as a candidate upstream cellular stress signal capable of shaping inflammatory tone in the postoperative brain, particularly within the hippocampus.\u003csup\u003e(14; 15)\u003c/sup\u003e ER stress is initiated by disturbances in protein-folding equilibrium within the endoplasmic reticulum, resulting in the accumulation of misfolded or unfolded proteins.\u003csup\u003e(16)\u003c/sup\u003e Through its canonical PERK, IRE1, and ATF6 branches, the unfolded proteins can interface with inflammatory signaling networks and influence neuronal survival under stress.\u003csup\u003e(17)\u003c/sup\u003e Importantly, the impact of ER stress on neuroinflammation appears to depend on its magnitude and duration: mild or transient ER stress may exert protective preconditioning effects, whereas sustained engagement can promote inflammatory amplification and neuronal injury.\u003csup\u003e(18)\u003c/sup\u003e Recent studies have implicated hippocampal unfolded protein response (UPR) in the development of PND. Common anesthetics like isoflurane have been shown to induce neuronal apoptosis and cognitive deficits via ER stress-related pathways.\u003csup\u003e(19)\u003c/sup\u003e Moreover, pharmacological inhibition of ER stress has been shown to suppress NLRP3 inflammasome activity, thereby attenuating neuroinflammation and ameliorating postoperative cognitive dysfunction.\u003csup\u003e(14)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eDespite these advances, a key mechanistic question remains unresolved: whether hippocampal ER stress acts an upstream signal that biases microglia toward a pro-inflammatory program in aged subjects exposed to anesthesia and surgery, rather than merely accompanying postoperative cellular stress and cytokine cascades. In addition, because most prior studies administered ER stress suppressor systemically, it remains difficult to disentangle indirect neuroprotection secondary to attenuation of surgery-driven peripheral inflammation from a direct central effect after BBB penetration and hippocampal target engagement. We therefore hypothesized that hippocampal ER stress evoked by anesthesia and surgery promotes a shift toward an M1-like microglial profile thereby amplifying neuroinflammation and contributing to postoperative cognitive impairment. To test this hypothesis, we used an aged murine tibial fracture model under isoflurane anesthesia and integrated early ultrastructural and molecular assessment of hippocampal ER stress with quantitative evaluation of microglial phenotypic markers and hippocampus-dependent cognitive performance. Importantly, we delivered TUDCA directly into the hippocampus with perioperative coverage spanning as a mechanistic probe to better interrogate hippocampus local pathways relevant to clinical disease evolution. This work aims to refine the mechanistic framework of PND by linking ER stress to microglial phenotypic regulation as a proximal driver of postoperative neuroinflammation and cognitive dysfunction.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEighteen-month-old male specific-pathogen-free (SPF) C57BL/6 mice (25-35g) were obtained from the Animal Center of Lanzhou University (Lanzhou, China). All animals were housed under controlled environmental conditions (temperature 22-25℃\u0026nbsp;humidity 45-65%, 12-h light/dark cycle) with free access to standard food and water. All experimental procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China, (Ethics Approval No. LDYYLL2025-06)). Animal care and handling complied with the National Health Guidelines for the Care and Use of Laboratory Animals and the PREPARE guidelines for ensuring the quality of animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of a Tibial Fracture Surgery Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA PND model was established using an aseptic open tibial fracture surgery, as previously described.\u003csup\u003e(20; 21)\u003c/sup\u003e Briefly, mice were anesthetized with 1.5% isoflurane (in 100% oxygen, 1 L/min) using an animal anesthesia system (RWD Life Science, Shenzhen, China). After sterilization of the surgical site, a 1.5-cm longitudinal incision was made on the skin of the left hind limb to expose the tibia, and surrounding muscles were gently separated. A 0.3-mm stainless steel pin was inserted into the intramedullary canal of the tibia for fixation. The tibia was fractured at the junction between the middle and distal thirds using surgical plie, and the incision was closed with 4-0 absorbable sutures. Throughout the procedure and recovery, body temperature was maintained at 37℃\u0026nbsp;using a heating pad. The surgical process lasted approximately 20 minutes, after which mice were returned to the induction chamber to maintain anesthesia for a total of 2 hours. To minimize postoperative discomfort, oxybuprocaine hydrochloride gel was applied topically to the surgical wound for local analgesia. Two independent cohorts were used: one for behavioral testing and one sacrificed for biochemical ultrastructural analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of ER stress in tibial fracture–induced PND, tauroursodeoxycholic acid (TUDCA,100㎍/mL in PBS; MCE, catalog number: HY-19696A) was stereotaxically injected bilaterally into the hippocampus (200 nL per side, 50 nL/min) 30 minutes before surgery and repeated once daily for two consecutive days after surgery. Mice were anesthetized and positioned in a stereotaxic apparatus (RWD Life Science, China). A burr hole was drilled in the skull, and a glass microelectrode injector pump (RWD Life Science, China) was used to deliver 200 nL of TUDCA solution into each hippocampal site. After the first injection, an indwelling guide cannula was left in place at the injection site and sealed with sterile bone wax. The subsequent two injections were administered through the implanted cannula. The stereotaxic coordinates were as follows: anteroposterior (AP)-2.0 mm, mediolateral (ML)±1.6 mm, and dorsoventral (DV) -1.8 mm, at an injection rate of 50 nL/min. After injection, the needle was left in place for 10 minutes to prevent reflux before being slowly withdrawn. Following injection, mice were then kept on a heating pad until full recovery from anesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were euthanized with 8% isoflurane, and bilateral hippocampal tissues were rapidly dissected. Tissues were homogenized and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Lysates were clarified by centrifugation (12,000 rpm, 30 min, 4℃), and the supernatant was collected for protein quantification using a BCA protein assay kit (Solarbio, Beijing, China). Samples were denatured by boiling in 1×\u0026nbsp;loading buffer at 60℃\u0026nbsp;for 15 minutes. Approximately 30㎍\u0026nbsp;of protein per lane was separated by 7.5-10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in TBST for 2 hours at room temperature and then incubated overnight at 4℃\u0026nbsp;with primary antibodies. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and quantified with ImageJ 1.53e software (NIH, Wayne Rasband, MD, USA). Band analysis of GRP78, ATF4, ATF6, CD86, CD206, iNOS, and Arg-1 were normalized to\u0026nbsp;β-actin, whereas phosphorylated PERK, eIF2ɑ, and IRE1ɑ\u0026nbsp;were normalized to their corresponding total protein levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh hippocampal tissue blocks (approximately 1 mm\u003csup\u003e3\u003c/sup\u003e) were immediately placed into EP tubes containing TEM fixative solution (Servicebio, G1102) following euthanasia. After post-fixation in 1% osmium tetroxide, tissues were dehydrated through graded ethanol and embedded in EMBed-812 resin. Ultrathin sections (60 nm) were cut using an ultramicrotome and mounted on 150-mesh copper grids coated with Formvar film. Sections were stained with 2% uranyl acetate and 2.6% lead citrate and examined under a HITACHI transmission electron microscope system(Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConcentrations of inflammatory cytokines in hippocampal tissues, including interleukin-1β\u0026nbsp;(IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-ɑ\u0026nbsp;(TNF-ɑ), were determined with commercial ELISA kits. Total protein concentrations were quantified, and cytokine levels were normalized to standard calibration curves. ELISA kits for mouse IL-1β (CME0015-048), IL-6 (CME0006-048), and TNF-ɑ (CME0004-048) ELISA kits were purchased from 4A BIOTECH (Suzhou, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and qRT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from mouse hippocampal tissue using RNAiso Plus (9109, Takara, Japan). RNA quantity and purity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). For qRT-PCR, cDNA was synthesized with the PrimeScript RT Reagent Kit (RR036A, Takara, Japan), and quantitative PCR was conducted using TB Green® Premix Ex Taq\u003csup\u003eTM\u003c/sup\u003e II (FAST qPCR; CN830S, Takara, Japan). Relative gene expression was normalized to an internal reference (β-ACTIN). Primer sequences are provided in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence (IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn postoperative day 3, mice were deeply anesthetized and transcardially perfused with heparinized saline followed by 4% ice-cold paraformaldehyde (PFA). Brains were carefully removed and post-fixed in 4% PFA overnight at 4℃\u0026nbsp;Fixed tissues were cryoprotected in 30% sucrose at 4℃\u0026nbsp;until they sank. Coronal sections (20㎛\u0026nbsp;thick) were prepared using a cryostat and mounted onto glass slides. Sections were washed three times in PBS, permeabilized with 0.2% Triton X-100 for 30 minutes, and blocked in 5% bovine serum albumin (BSA) blocking buffer for 1 hour at room temperature. They were then incubated overnight at 4℃\u0026nbsp;with primary antibodies against Iba-1 (1:200), GFAP (1:200), GRP78 (1:200), ATF6((1:100), and p-IRE1ɑ\u0026nbsp;(1:100)). After washing, sections were incubated with appropriate fluorescent secondary antibodies for 2 hours at room temperature. Following countertaining with DAPI, hippocampal images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Field Test (OFT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioral activities were recorded and analyzed using a video tracking system (SMART PREMIUM, Panlab, Barcelona, Spain). All behavioral tests were conducted daily between 12:00 and 21:00. Mice were acclimated to the testing room for approximately 1 hour before testing, and adequate intervals were provided between tasks to avoid carryover effects. Each mouse was gently placed in the center of a gray open-field chamber (40\u0026nbsp;×\u0026nbsp;40\u0026nbsp;×\u0026nbsp;40 cm) and allowed to explore freely for 5 minutes. Movements were recorded by a top-mounted camera, and the apparatus was cleaned with 75% ethanol between trials to remove olfactory cues. Total distance traveled and time spent in the central zone were quantified to assess locomotor activity and anxiety-like behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovel Object Recognition Test (NORT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe novel object recognition test (NORT) was performed 24 hours after the OFT according to established protocols. Mice were habituated to the test chamber (40\u0026nbsp;×\u0026nbsp;40\u0026nbsp;×\u0026nbsp;40 cm) for 5 minutes before the familiarization phase. During familiarization, two identical objects (A + A) were placed in adjacent corner, 5 cm from the walls, and mice were allowed to explore freely for 10 minutes. After 24 hours, one familiar object (A) was replaced with a novel object (B) of similar size but different shape and color. Exploration was defined as the mouse’s nose oriented toward the object within 2 cm. Exploration time for each object was measured, and the recognition index was calculated as the ratio of the time spent exploring the novel object to the total exploration time for both objects. Between trials, the apparatus was cleaned with 75% ethanol to remove residual olfactory cues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorris Water Maze (MWM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Morris water maze (MWM) test was conducted to assess spatial learning and memory performance in mice. A circular pool (diameter 120 cm, height 50 cm) was filled with water (22±1℃) to a depth of 40 cm, and white tempera paint was added to render the water opaque. A hidden circular platform (diameter 10 cm) was submerged 1 cm below the water surface, and the pool was virtually divided into four equal quadrants. Mice underwent four acquisition trials per day for five consecutive days, followed by a probe test on day six.\u003c/p\u003e\n\u003cp\u003eIn each trial, mice were gently placed into the pool facing the wall and allowed 60 seconds to locate the hidden platform. Swimming trajectories were recorded using an automated video tracking system (SMART PREMIUM, Panlab, Spain). Mice that successfully located the platform were allowed to remain on it for 15 seconds, whereas those that failed were guided to the platform and kept there for the same duration. Escape latency (the time required to locate the hidden platform) was recorded for each trial. On day six, a 60-second probe trial was performed to evaluate spatial reference memory, during which the platform was removed. Platform crossings, and time spent in the target quadrant were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean±standard error of the mean (SEM). Data normality was assessed using the Shapiro-Wilk test. Sample sizes for each experiment are indicated in the corresponding figure legends. For comparisons between two groups with normally distributed data, an unpaired Student’s t-test was applied. For multiple-group comparisons, escape latency data were analyzed using two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post hoc test. Western blot and behavioral data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. For non-normally distributed data, the Kruskal-Wallis test was used. A value of P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCognitive impairment and hippocampal glial activation after anesthesia and surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA PND model was established using tibial fracture surgery under isoflurane anesthesia, and postoperative behavior was assessed starting on postoperative day 3(POD3) (Fig. 1A). In the open field test (OFT), total distance traveled and time spent in the central zone did not differ significantly between sham and surgery groups, suggesting preserved locomotion and anxiety-like behavior. In contrast, the surgery group exhibited a reduced recognition index in the novel object recognition test (NORT), consistent with impaired recognition memory. Similarly, in the Morris water maze (MWM), surgery significantly decreased platform crossings and reduced time spent in the target quadrant. Immunofluorescence staining further demonstrated increased Iba1 and GFAP signals in the hippocampus after surgery, indicating activation of microglia and astrocytes. Collectively, these data show that anesthesia plus tibial fracture surgery induces PND accompanied by hippocampal glial activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnesthesia and surgery activate hippocampal ER stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy performed on postoperative day 3 revealed ER swelling and luminal dilation in hippocampal cells, consistent with early ER stress activation (Fig. 2A). In parallel, Western blot showed increased GRP78 expression and activation of canonical ER stress related signaling, including elevated p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6 in the surgery group compared with sham controls (Fig. 2B-G). Immunofluorescence further confirmed enhanced hippocampal immunoreactivity for GRP78, p-IRE1ɑ, and ATF6 after surgery (Fig. 2H-J). Together, these POD3 ultrastructural, molecular, and immunostaining data indicate an early engagement of hippocampal ER stress signaling following anesthesia and tibial fracture surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnesthesia and surgery promote microglial polarization toward an M1 phenotype and neuroinflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether anesthesia and surgery bias microglial phenotype, we quantified proteins associated with M1-like and M2-like programs in the hippocampus. Western blotting showed increased expression of the M1-associated markers CD86 and iNOS, accompanied by reduced expression of the M2-associated markers CD206 and Arg-1 in the surgery group versus sham controls (Fig. 3A-D). Consistently, \u0026nbsp;double immunofluorescence staining revealed increased CD86/Iba1 and reduced CD206/Iba1 immunoreactivity in the hippocampus (Fig. 3E-F). Meanwhile, levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-ɑ) were elevated after surgery (Fig. 3G-L). These findings support a postoperative shift toward a pro-inflammatory (M1-like) microglial signature associated with increased hippocampal inflammatory signaling. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUDCA rescues cognitive deficits and attenuates hippocampal ER stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo probe the functional relevance of ER stress to postoperative cognitive impairment, TUDCA was bilaterally microinjected into the hippocampus 30 min before tibial fracture surgery. TUDCA did not alter OFT measures of locomotion or center time compared with vehicle controls\u0026nbsp;(Fig. 4E,F). In contrast, TUDCA improved memory-related performance: the reduced recognition index in the Surgery+Vehicle group was restored by TUDCA in the NORT (Fig. 4G), and MWM deficits (fewer platform crossings and reduced target quadrant time) were attenuated by TUDCA (Fig. 4H-J).\u003c/p\u003e\n\u003cp\u003eAt the molecular level, TUDCA reduced surgery associated activation of UPR signaling, decreasing GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6\u0026nbsp;(Fig. 5A-F), and lowering GRP78, p-IRE1ɑ, and ATF6 immunoreactivity\u0026nbsp;(Fig. 5G-I). TEM analysis further showed that TUDCA alleviated ER dilation of hippocampal microglia relative to vehicle treated surgical mice (Fig. 5J). Collectively, these data indicate that hippocampus targeted ER stress suppression is accompanied by improved postoperative cognition and reduced hippocampal ER stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER stress inhibition reverses microglial polarization and dampens neuroinflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIba1 immunofluorescence revealed marked microglial activation in hippocampal CA1, CA3, and DG regions after surgery, whereas TUDCA reduced Iba1 signal intensity relative to Surgery+Vehicle controls\u0026nbsp;(Fig. 6). Consistent with a shift in microglial phenotypic signature, TUDCA attenuated the surgery\u0026nbsp;induced increase in CD86 and iNOS and restored CD206 and Arg-1 expression on Western blotting\u0026nbsp;(Fig. 7A-D). Immunofluorescence similarly showed reduced CD86/Iba1 and increased CD206/Iba1 immunoreactivity in TUDCA treated mice compared with vehicle-treated surgical mice (Fig. 7E,F). In parallel, surgery increased IL-1β, IL-6, and TNF-α at both protein and transcript levels, whereas TUDCA prevented these increases (Fig. 7G-L). Collectively these findings suggest hippocampal ER stress suppression is associated with reduced pro-inflammatory microglial bias and dampened hippocampal inflammatory signaling after anesthesia and surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER stress inhibition mitigated neuron loss in mice hippocampus following anesthesia and surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNissl staining revealed reduced neuronal density and disrupted cytoarchitecture in hippocampal CA1, CA3, and DG regions after surgery (Fig. 8A). Compared with sham controls, neurons in the surgery group appeared shrunken with pyknotic features and disorganized lamination.. In contrast, TUDCA treated mice exhibited improved neuronal preservation, with higher apparent neuronal density and more intact hippocampal architecture, approaching sham levels. Together with the observed attenuation of ER stress, microglial pro-inflammatory bias, and inflammatory signaling, these results are consistent with a neuroprotective effect of hippocampus targeted ER stress suppression in this postoperative model.\u003c/p\u003e\n\n\n\n\n\n\n"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we provide evidence that anesthesia and surgery engage hippocampal ER stress and are accompanied by a shift toward a pro-inflammatory microglial phenotype, heightened inflammatory signaling, and Perioperative neurocognitive disorder. Using hippocampus targeted delivery of TUDCA as a mechanistic probe, we found that dampening ER stress coincided with improved performance in hippocampus-dependent memory tasks, reduced CD86/iNOS with restoration of CD206/Arg-1, lower pro-inflammatory cytokine levels, and better neuronal preservation. Collectively, these findings support a model in which hippocampal ER stress is linked to microglial phenotypic bias and neuroinflammation associated with postoperative cognitive deficits.\u003c/p\u003e\u003cp\u003eNeuroinflammation is widely implicated in postoperative cognitive vulnerability, and microglia are key cellular effectors that translate peripheral inflammatory cues into brain-resident immune responses.\u003csup\u003e(22)\u003c/sup\u003e Microglia are central to CNS inflammatory and help maintain homeostasis through crosstalk with neurons and astrocyte.\u003csup\u003e(23; 24)\u003c/sup\u003e In this context, a relative bias toward an M1-like microglial program can amplify cytokine and oxidative signaling and disturb synaptic homeostasis, whereas M2-like programs are more consistent with inflammatory resolution and repair.\u003csup\u003e(25; 26)\u003c/sup\u003e Specifically, cytokines produced by M1 polarization microglia can impair neuronal function. Additionally, microglia TGF-β\u0026nbsp;can up-regulate neuronal excitability by promote the expression of Kir4.1 in astrocytes, highlinghting the importance of microglia-astrocyte cross-dialogue in the development of neural excitability.\u003csup\u003e(27)\u003c/sup\u003e Prior studies have linked microglial activation or phenotypic skewing to postoperative cognitive deficits in aged animals.\u003csup\u003e(28; 29)\u003c/sup\u003e However, the upstream signals that bias microglial phenotype in the postoperative hippocampus remain incompletely defined. Our data nominate hippocampal ER stress engagement as a candidate upstream driver that is closely associated with this pro-inflammatory microglial shift.\u003c/p\u003e\u003cp\u003eER stress represents a conserved adaptive response aimed at restoring proteostasis, yet sustained UPR activation can become maladaptive and intersect with inflammatory pathways.\u003csup\u003e(30)\u003c/sup\u003e In perioperative settings, anesthetic and surgical stressors have been associated with hippocampal ER stress and cognitive impairment.\u003csup\u003e(31; 32)\u003c/sup\u003eHowever, a frequent limitation of prior work is the focus on a single UPR arm or reliance on molecular markers without ultrastructural validation. Here, we profiled PERK, IRE1ɑ, and ATF6-associated signaling in parallel and complemented these data with TEM based assessment of ER morphology, providing convergent molecular and ultrastructural evidence for anesthesia/surgery-evoked hippocampal ER stress.\u0026nbsp;Another important limitation of previous studies is that TUDCA has predominantly been administered systemically (intravenous or intraperitoneal injection) to evaluate its effects on postoperative cognition and neuroinflammation. This\u0026nbsp;design may\u0026nbsp;complicate\u0026nbsp;its\u0026nbsp;interpretation. Specifically, it is difficult to distinguish whether the benefit arises from dampening surgery\u0026nbsp;induced\u0026nbsp;peripheral inflammation (an indirect brain-protective effect) or from TUDCA crossing the\u0026nbsp;BBB\u0026nbsp;and directly suppressing hippocampal ER stress and local inflammatory injury. In the present study, we delivered TUDCA directly into the hippocampus and maintained perioperative coverage before and after surgery. This design better matches the temporal evolution of postoperative pathology and enables a more direct assessment of hippocampus\u0026nbsp;local mechanisms relevant to clinical disease progression.\u003c/p\u003e\u003cp\u003eHow ER stress translates into cognitive vulnerability after anesthesia and surgery likely involves bidirectional coupling between UPR signaling and innate immune programs. In multiple neurological disorders, ER stress can modulate inflammatory signaling and influence microglial activation states.\u003csup\u003e(33; 34)\u003c/sup\u003e A recent study suggested that pharmacological modulation of hippocampal ER stress can alleviate LPS-induced cognitive dysfunction\u003csup\u003e(35)\u003c/sup\u003e.\u0026nbsp;ER stress may converge on multiple inflammatory programs,\u0026nbsp;including inflammasome pathways.\u003csup\u003e(14)\u003c/sup\u003e However,\u0026nbsp;our results highlight microglial phenotypic bias as an additional, proximal cellular layer.\u0026nbsp;In the present study, hippocampus-targeted TUDCA administration attenuated UPR activation and was accompanied by a coordinated reversal of microglial phenotypic markers (reduced CD86/iNOS with restoration of CD206/Arg-1), suppression of pro-inflammatory cytokine production, and improved hippocampus-dependent memory. These findings are consistent with a pathway in which postoperative ER stress biases microglia toward a pro-inflammatory program that amplifies inflammatory signaling and contributes to neuronal vulnerability and cognitive deficits.\u003c/p\u003e\u003cp\u003eOur study has limitations. First, the molecular determinants by which ER stress drives microglial M1 polarization remain unresolved and future studies using pathway-selective perturbations would help define specificity. Second, although hippocampal TUDCA delivery supports a mechanistic association, pharmacologic interventions may have off-target effects and do not establish microglia specific causality. Future work should centered on genetic or microglia targeted strategies to rigorously test the ER stress–microglia axis. Third, hippocampal microinfusion was used to probe mechanism and does not model clinically feasible delivery, \u0026nbsp;future work should test systemic dosing, timing windows, and safety.\u003c/p\u003e\u003cp\u003eIn conclusion, our data support a model in which anesthesia and surgery engage hippocampal UPR signaling that is associated with a pro-inflammatory microglial phenotypic bias, heightened neuroinflammatory signaling, and postoperative cognitive impairment in aged mice. Targeting ER stress, potentially to modulate microglial phenotype and inflammatory tone, may represent a promising direction for mitigating PND, although further work is required to establish pathway and cell-type specificity and to evaluate clinically relevant routes and timing of intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China, (Ethics Approval No. LDYYLL2025-06)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by Natural Science Joint Fund of Gansu Province (No:24JRRA914).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e’\u003c/strong\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHW, YLL and XHL contributed to study conception and design. HW, LG and XHY\u003c/p\u003e\n\u003cp\u003eperformed the experiments. HW , YZ and LW performed the statistical analysis and interpreted the data. HW wrote the first draft of the manuscript. YLL and XHL revised the manuscript for important intellectual content. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all participants involved in this study. We also thank Figdraw (www.figdraw.com) for the assistance in creating Scheme and Abstract Scheme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e The First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Ambulatory Surgery Center, the First Hospital of Lanzhou University, Lanzhou, 730000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Department of Anesthesiology, The First Hospital of Lanzhou University, Lanzhou, 730000, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEvered L, Silbert B, Knopman DS\u003cem\u003e et al.\u003c/em\u003e (2018) Recommendations for the Nomenclature of Cognitive Change Associated with Anaesthesia and Surgery-2018. \u003cem\u003eAnesthesiology\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 872-879.\u003c/li\u003e\n\u003cli\u003eVacas S, Cole DJ, Cannesson M (2021) Cognitive Decline Associated With Anesthesia and Surgery in Older Patients. 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Therapeutics\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eZhang D-Q, Dong X, Su S\u003cem\u003e et al.\u003c/em\u003e (2024) Temporin-GHaR Peptide Alleviates LPS-Induced Cognitive Impairment and Microglial Activation by Modulating Endoplasmic Reticulum Stress. \u003cem\u003eProbiotics and Antimicrobial Proteins\u003c/em\u003e.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-anesthesiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bane","sideBox":"Learn more about [BMC Anesthesiology](http://bmcanesthesiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bane","title":"BMC Anesthesiology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"perioperative neurocognitive disorder, endoplasmic reticulum stress, microglia, M1/M2 polarization, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-8415780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8415780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePerioperative neurocognitive disorder (PND) is a frequent postoperative complication in older individuals and is commonly linked to microglial activation. Endoplasmic reticulum (ER) stress has been implicated in neuroinflammation and postoperative cognitive decline; however, whether hippocampal ER stress acts upstream to bias microglia toward a pro-inflammatory M1 phenotype, rather than serving only as a trigger of cytokine cascades, remains insufficiently defined in PND.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo determine whether hippocampal ER stress promotes an M1-like microglial shift and amplifies neuroinflammation after anesthesia and surgery.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEighteen-month-old male C57BL/6 mice underwent aseptic tibial fracture surgery under isoflurane anesthesia. TUDCA, a pharmacological ER stress suppressor, was delivered bilaterally into dorsal hippocampal CA1 as a perioperative mechanistic probe. ER ultrastructure in hippocampal cells was examined using transmission electron microscopy (TEM). ER stress related proteins (GRP78, p-PERK, p-eIF2ɑ, p-IRE1ɑ, ATF4, and ATF6) in hippocampus were quantified by Western blotting. Microglial phenotypic signatures were assessed by Western blotting (CD86, iNOS, CD206, ARG-1) and double immunofluorescence (CD86/Iba-1 and CD206/Iba-1). Open-field testing was performed to control for locomotion/anxiety, followed by memory assessments using novel object recognition and Morris water maze starting on postoperative day 3.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAnesthesia and surgery induced postoperative cognitive deficits, accompanied by heightened hippocampal inflammatory signaling and a shift toward a pro-inflammatory microglial signature characterized by increased CD86 and iNOS with concomitant reductions in CD206 and ARG-1. In parallel, hippocampal ER stress activation was evident, including ER ultrastructural disruption and increased GRP78 with engagement of PERK/eIF2ɑ/ATF4-, IRE1ɑ-, and ATF6-related pathways. hippocampus-targeted delivery of TUDCA attenuated ER stress activation, mitigated the pro-inflammatory microglial shift (decreasing CD86/iNOS and restoring CD206/ARG-1), reduced inflammatory readouts, and improved postoperative memory performance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings support a mechanistic link between hippocampal ER stress and the pro-inflammatory microglia bias linked to postoperative neuroinflammation and cognitive impairment in aged mice.\u003c/p\u003e","manuscriptTitle":"Hippocampal Endoplasmic Reticulum Stress Drives a Pro-inflammatory Microglial Bias and Neuroinflammation in Aged Mice with Perioperative Neurocognitive Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 00:39:13","doi":"10.21203/rs.3.rs-8415780/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-27T04:32:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186084577874363971267616319270138686789","date":"2026-01-23T14:32:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101587000997550415247304905906784571492","date":"2026-01-21T04:04:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T02:57:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T07:58:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-29T06:59:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-28T14:03:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Anesthesiology","date":"2025-12-28T13:56:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-anesthesiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bane","sideBox":"Learn more about [BMC Anesthesiology](http://bmcanesthesiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bane","title":"BMC Anesthesiology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89a7eb9a-14a7-4195-bd3a-41b97612abdb","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T00:39:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 00:39:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8415780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8415780","identity":"rs-8415780","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}