The TREM1-AQP4 Axis Mediates Neuroinflammatory Injury and Brain Edema after Experimental Subarachnoid Hemorrhage

The TREM1-AQP4 Axis Mediates Neuroinflammatory Injury and Brain Edema after Experimental Subarachnoid Hemorrhage | 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 The TREM1-AQP4 Axis Mediates Neuroinflammatory Injury and Brain Edema after Experimental Subarachnoid Hemorrhage Jianxin Chen, Junli Zhang, Wenjing Ning This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7741776/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in European Journal of Medical Research → Version 1 posted 11 You are reading this latest preprint version Abstract Objective: Secondary brain injury following subarachnoid hemorrhage (SAH) is a major factor contributing to poor patient outcomes, with neuroinflammation and cerebral edema representing core pathological mechanisms. Triggering receptor expressed on myeloid cells-1 (TREM1), a major inflammatory amplifier in innate immunity, remains poorly understood in SAH regarding its specific role, cellular targets, and association with aquaporin-4 (AQP4), a key cerebral edema molecule. To elucidate the regulatory role of TREM1 in neuroinflammatory injury following experimental SAH and to investigate whether it functions by driving microglial activation and modulating AQP4 expression. Methods: An SAH model was established in rats via internal carotid artery puncture. TREM1 expression was specifically upregulated or downregulated in vivo through lateral ventricle injection of adeno-associated virus (AAV). Animals were randomly assigned to sham surgery, SAH empty vector control, SAH TREM1 overexpression, and SAH TREM1 knockdown groups. Twenty-four hours post-SAH, neurological function was assessed using the modified Garcia score and balance beam test; brain water content was measured by dry-wet weight method; HE staining was used to observe neuronal morphology; Western Blot and real-time quantitative PCR (qRT-PCR) were employed to detect TREM1, Iba1, GFAP, AQP4, NeuN, Cleaved Caspase-3, IL-6, and IL-13 expression; Immunofluorescence staining was performed for localization and semi-quantitative analysis. Results: Following subarachnoid hemorrhage (SAH), TREM1 expression is significantly upregulated in brain tissue, with its levels negatively correlated with neurological deficits. Functional and molecular studies demonstrate that TREM1 inhibition improves neurological function, reduces cerebral edema, and mitigates neuronal apoptosis, whereas overexpression exacerbates injury. Mechanistic studies reveal that TREM1 exacerbates secondary brain injury by promoting microglial hyperactivation and inflammatory responses, while simultaneously upregulating astrocytic AQP4 expression. Conclusion: This study first reveals the TREM1-AQP4 axis as a critical bridge linking neuroinflammation and cerebral edema after SAH. TREM1 exacerbates brain injury by driving microglia-mediated neuroinflammation and upregulating astrocytic AQP4 expression. Targeting TREM1 inhibition holds promise as a novel therapeutic strategy to improve SAH prognosis by simultaneously alleviating inflammation and reducing edema. Subarachnoid hemorrhage TREM1 AQP4 neuroinflammation cerebral edema. Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease characterized by high mortality and disability rates[ 1 ].Globally, the annual incidence of SAH is approximately 9 cases per 100,000 people. Although it accounts for only 5%-10% of all strokes, it imposes a disproportionately heavy disease burden [ 2 ].Despite significant advances in acute-phase management techniques such as aneurysm clipping and endovascular embolization in recent years, which have effectively reduced the risk of early rebleeding, 30%-50% of SAH survivors still suffer from severe delayed neurological deficits, including cognitive decline, emotional disturbances, and motor impairments. This imposes a heavy economic and caregiving burden on patients' families and society[ 3 ].Therefore, elucidating the pathophysiological mechanisms of brain injury following SAH and identifying effective intervention targets represent critical unresolved challenges in neuroscience and the field of severe cerebrovascular disease. Brain injury following subarachnoid hemorrhage (SAH) is a dynamic process involving multiple stages and mechanisms [ 3 ]. Early brain injury (EBI) occurs within 72 hours post-bleeding and is characterized by a rapid increase in intracranial pressure, a sudden drop in cerebral blood flow, disruption of the blood-brain barrier, and global cerebral ischemia. This phase is considered decisive in determining patient prognosis [ 4 ]. Subsequently, delayed cerebral ischemia (DCI) emerges as the primary complication driving sustained neurological deterioration[ 5 , 6 ].Traditionally attributed to large-vessel spasm, DCI has shown limited improvement in patient outcomes despite spasm-targeted therapies, prompting researchers to explore more intricate cellular and molecular mechanisms [ 7 ].In recent years, mounting evidence indicates that neuroinflammation serves as the core pathological bridge linking EBI and DCI [ 8 , 9 ].Following SAH, blood and its degradation products (e.g., oxyhemoglobin) in the subarachnoid space act as potent stress signals, triggering a robust and persistent innate immune and inflammatory cascade within the central nervous system. This process involves the activation and infiltration of multiple immune cells alongside the release of numerous inflammatory mediators, collectively exacerbating blood-brain barrier permeability, cerebral edema formation, and ultimately neuronal apoptosis and necrosis [ 10 – 12 ]. As resident innate immune cells in the central nervous system (CNS), microglia play a double-edged sword role in neuroinflammation following subarachnoid hemorrhage (SAH) [ 13 , 14 ].Under physiological conditions, microglia exist in a quiescent state, responsible for immune surveillance [ 15 ].Following SAH, they undergo rapid activation. On one hand, appropriate activation aids in clearing cellular debris, red blood cells, and toxic substances, potentially exerting protective effects through the secretion of minor neurotrophic factors [ 16 ];On the other hand, excessive and sustained activation leads to the release of large amounts of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6), chemokines, and reactive oxygen species, further amplifying the inflammatory response and causing secondary brain injury [ 17 ].In recent years, researchers have increasingly recognized that microglia/macrophages are not simply “activated” or “quiescent,” but rather polarize toward distinct functional phenotypes driven by different microenvironmental signals. These primarily include the classically activated pro-inflammatory M1 phenotype and the alternatively activated anti-inflammatory/reparative M2 phenotype, with the dynamic balance between them profoundly influencing disease outcomes[ 17 , 18 ].Therefore, precisely regulating the phenotypic transition of microglia from the harmful M1 state to the beneficial M2 state is considered a highly promising therapeutic strategy for alleviating neuroinflammation following SAH. Triggering receptor expressed on myeloid cells-1 (TREM1) is a key inflammatory amplifier discovered in recent years, primarily expressed on the surface of myeloid cells such as neutrophils and monocytes/macrophages[ 19 ].TREM1 belongs to the immunoglobulin superfamily. By binding to a ligand that remains incompletely characterized and coupling with the adaptor protein DAP12, it activates downstream Syk-PI3K-AKT and MAPK signaling pathways. This process powerfully amplifies the activity of transcription factors such as NF-κB, inducing the explosive release of proinflammatory factors like TNF-α and IL-1β, thereby forming an “inflammatory storm” [ 20 , 21 ].The role of TREM1 in systemic inflammatory response syndromes like septic shock and sepsis has been extensively validated, with TREM1 inhibition significantly improving outcomes. Notably, growing evidence suggests TREM1 also plays a critical role in non-infectious, sterile inflammatory diseases such as atherosclerosis and myocardial ischemia-reperfusion injury [ 22 – 24 ].Recent clinical observations indicate that soluble TREM1 (sTREM1) levels are significantly elevated in the cerebrospinal fluid of SAH patients, positively correlating with clinical severity scores (e.g., WFNS score) and poor prognosis. This strongly suggests TREM1 may be involved in the pathological process of SAH [ 25 ].However, the precise role of TREM1 in neuroinflammation following SAH, its cellular origin within the CNS (whether specifically expressed on microglia), whether it directly drives microglial phenotypic switching, and its key downstream effector molecules and signaling pathways remain incompletely understood. These knowledge gaps significantly impede translational research efforts targeting TREM1 as a potential therapeutic target. Given these research gaps, this study aims to systematically investigate the role of TREM1 in neuroinflammatory damage following experimental SAH and its specific molecular mechanisms. We propose the core hypothesis: TREM1, as a key amplifier of neuroinflammation after SAH, exacerbates neuroinflammation, cerebral edema, and neuronal apoptosis by activating microglia and promoting their polarization toward the pro-inflammatory M1 phenotype, while simultaneously upregulating aquaporin-4 (AQP4) expression, ultimately leading to neurological deficits. To validate this hypothesis, we established a mature rat SAH model and employed adeno-associated virus (AAV)-mediated gene technology to specifically upregulate and knockdown TREM1 expression in the brain. Through a comprehensive approach integrating neurobehavioral assessment, histopathological analysis, protein immunoblotting, real-time quantitative PCR, and immunofluorescence staining, we systematically evaluated TREM1's impact on neurological outcomes, cerebral edema severity, neuronal survival and apoptosis, microglial activation status, inflammatory cytokine profiles, and AQP4 expression. This study aims not only to provide new insights into the pathological mechanisms of SAH but also to establish robust preclinical evidence for TREM1 as a potential therapeutic target through gain-of-function and loss-of-function experiments. This foundation will pave the way for developing novel neuroprotective drugs. 2. Materials and Methods 2.1. Animals Healthy adult male Sprague-Dawley (SD) rats (SPF grade, weighing 250–300 g) were purchased from Jinan Xingkang Biotechnology Co., Ltd. All rats were housed in the barrier environment of the Shandong First Medical University Laboratory Animal Center and maintained under standard conditions (12-hour light/dark cycle, light period 7:00–19:00, ambient temperature 22 ± 2°C, relative humidity 55 ± 10%). They had free access to standard rodent chow and sterile drinking water. All animals underwent at least one week of acclimatization prior to experimentation. 2.2. Experimental Design and Grouping This study employed a completely randomized grouping design. Following successful establishment of the SAH model, surviving rats were randomly assigned to the following four experimental groups (n = 6 per group). Sham group Rats underwent the same surgical procedures as the SAH model group, including anesthesia, skin incision, and vascular dissection, but without internal carotid artery puncture or nylon thread insertion to prevent SAH induction. SAH + AAV2-Vector Control Group (SAH-AAV2-NC Group) Nineteen days prior to SAH modeling, rats received microinjection of empty adeno-associated virus (AAV2) into the lateral ventricle as a viral vector control to exclude non-specific immune reactions or toxic effects potentially caused by the viral vector itself. SAH + AAV2-TREM1 overexpression group (SAH-AAV2-TREM1 group) 19 days prior to SAH modeling, AAV2 virus overexpressing rat TREM1 was microinjected into the lateral ventricle to specifically enhance TREM1 expression in the brain under SAH conditions. SAH + AAV2-shRNA-TREM1 knockdown group (SAH-AAV2-shTREM1 group) 19 days prior to SAH modeling, AAV2 virus carrying short hairpin RNA (shRNA) targeting the rat TREM1 gene was microinjected into the lateral ventricle to specifically suppress TREM1 expression in the brain under SAH conditions. 2.3. Adeno-associated virus (AAV) vector construction and lateral ventricle injection The AAV2 vector overexpressing TREM1 (AAV2-CMV-TREM1-3xFLAG), the AAV2 vector expressing TREM1-specific shRNA (AAV2-U6-shTREM1), and the corresponding empty vector control (AAV2-CMV-NC) were designed and packaged by Shanghai GK Gene Chemistry Technology Co., Ltd. The shRNA target sequence used was: 5'-GCACAAGATCAACCAGTATTT-3'. All viral titers were adjusted to 2×10¹² viral genomes/mL (vg/mL). Viral injections were performed 19 days prior to modeling to ensure sufficient time for viral expression and effect in target cells. Rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, intraperitoneal injection) and fixed in a stereotaxic brain stereotaxic apparatus (Ruiwo, China). The scalp was shaved and disinfected, and a skin incision was made along the coronal suture to expose the anterior fontanelle. Referencing a rat stereotaxic atlas, the coordinates for the left lateral ventricle were determined: 0.8 mm posterior to the anterior fontanelle, 1.5 mm lateral to the midline, and 3.5 mm subdural. A microinjector (Hamilton, USA) was used to inject 2 µL of viral solution at a slow rate of 0.2 µL/min. After injection, the needle was left in place for 10 minutes to allow complete diffusion of the virus before being slowly withdrawn. The skin incision was sutured, and the rat was placed on a warming pad for recovery. Once fully awake, the animal was returned to its housing cage. 2.4. Establishment of Subarachnoid Hemorrhage (SAH) Model The SAH model was established using the classic internal carotid artery puncture method, which effectively mimics clinically induced SAH from ruptured aneurysms. The procedure is briefly described as follows: Rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, intraperitoneal injection) and fixed in a supine position. The neck region was shaved and rigorously disinfected. A 2–3 cm skin incision was made along the midline of the neck. Subcutaneous tissue and glands were bluntly dissected to expose the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The distal end of the ECA was ligated and transected, temporarily occluding blood flow in the CCA and ICA. A 4 − 0 monofilament nylon suture (approximately 0.28 mm diameter, with the tip heated and blunted to minimize vascular injury) was inserted through the ECA stump. It was gently advanced cranially through the ICA bifurcation until slight resistance was felt (indicating the suture tip had reached the anterior portion of the circle of Willis, typically near the origin of the middle cerebral artery). then advance approximately 3 mm further to puncture the anterior cerebral artery-anterior communicating artery complex, inducing bleeding. Leave the thread in place for 10 seconds before slowly withdrawing it. Immediately ligate the ECA stump and release the arterial clamps on the CCA and ICA to restore blood flow. In the sham-operated group, rats underwent identical vascular dissection procedures except for the absence of nylon thread puncture. Postoperatively, all rats were housed individually, with vital signs closely monitored while receiving thermal regulation and analgesia. 2.5. Neurobehavioral Assessment Twenty-four hours after SAH, two experienced investigators blinded to experimental group assignments independently conducted neurobehavioral assessments. Final scores were averaged to ensure objectivity and reliability. This time point was selected as it represents the peak of early brain injury (EBI), enabling optimal evaluation of TREM1 regulation's impact on acute-phase neurological outcomes. Modified Garcia Neurological Score: This scoring system comprehensively evaluates neurological function across six domains: spontaneous activity, symmetry of limb movements, forelimb extension ability, climbing ability, tactile response, and whisker response. Each domain is scored from 1 to 3 points, yielding a total score ranging from 3 to 18 points. A lower score indicates more severe neurological deficits. Balance Beam Test: Used to assess rats' motor coordination, balance ability, and fine motor control. The apparatus consists of a circular wooden beam measuring 120 cm in length and 2.5 cm in width, suspended 50 cm above the ground. All rats underwent three days of adaptation training prior to testing to ensure proficiency in traversing the beam. During formal testing, the time required for the rat to traverse the beam from one end to the other (traverse time, in seconds) and the number of times the rat slipped off the beam during the process (slip count) were recorded. Each rat underwent three trials, with the average value recorded. 2.6. Brain Water Content Measurement Twenty-four hours post-modeling and following behavioral testing, rats were rapidly decapitated under deep anesthesia to harvest brains. The entire brain was carefully dissected and removed. On an ice plate, the left cerebral hemisphere (i.e., the ipsilateral hemisphere), right cerebral hemisphere (contralateral hemisphere), cerebellum, and brainstem were promptly separated. Immediately weigh the wet weight of each section using a precision electronic analytical balance (accuracy 0.1 mg). Subsequently, place the brain tissue samples in a preheated 105°C oven and dry continuously for 72 hours until constant weight (difference between two consecutive weighings < 0.2 mg). Then, weigh the dry weight again. The formula for calculating brain water content (%) is: [(wet weight - dry weight) / wet weight] × 100%. 2.7. Tissue Processing and Hematoxylin-Eosin (HE) Staining Twenty-four hours after modeling, rats were deeply anesthetized and rapidly perfused via the left ventricle with approximately 200 mL of pre-chilled 0.9% saline until the effluent became clear. This was followed by perfusion with approximately 300 mL of 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for fixation. The brain was removed and post-fixed in 4% paraformaldehyde for 24 hours. It was then sequentially dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Continuous coronal sections of 5µm thickness were prepared using a paraffin microtome (Leica, Germany), targeting brain regions including the hippocampus and cortex. Sections were dewaxed with xylene, rehydrated with graded ethanol, and routinely stained with hematoxylin and eosin. Following staining, sections were mounted with neutral binder and examined under an optical microscope (Nikon Eclipse Ci, Japan) to observe the morphological structure, arrangement density, and nuclear shrinkage of neurons in the cortex and hippocampal CA1 region, with images captured. 2.8. Immunofluorescence Staining Paraffin sections were deparaffinized to water and placed in citrate buffer (pH 6.0) for microwave-assisted antigen retrieval. After cooling, sections were permeabilized with PBS containing 0.3% Triton X-100 for 10 minutes, followed by blocking with 5% donkey serum at room temperature for 1 hour. Discard the blocking solution without washing. Directly add appropriately diluted primary antibody working solutions, including: rabbit anti-rat TREM1 polyclonal antibody (1:200), mouse anti-rat AQP4 monoclonal antibody (1:200), and rabbit anti-rat Iba1 polyclonal antibody (1:500). Incubate overnight at 4°C in a humidified chamber. The following day, wash three times with PBS for 5 minutes each. Add the corresponding fluorescein-labeled secondary antibody (Alexa Fluor 488-labeled donkey anti-rabbit IgG or Alexa Fluor 594-labeled donkey anti-mouse IgG, 1:500) and incubate at room temperature in the dark for 1 hour. After washing with PBS, mount with DAPI-containing mounting medium. Images were acquired using a fully automated fluorescence upright microscope (Zeiss Axio Imager Z2, Germany) or a laser confocal microscope (Leica TCS SP8, Germany). Semi-quantitative analysis of fluorescence intensity in regions of interest (ROIs) was performed using ImageJ software (NIH, USA). Morphological skeleton analysis of microglia was conducted using ImageJ plugins to quantify branch length and node count. 2.9. Real-Time Quantitative PCR (qRT-PCR) Twenty-four hours after modeling, rat hippocampal tissue was rapidly isolated, flash-frozen in liquid nitrogen, and then transferred to a -80°C ultra-low temperature freezer for storage. Total RNA was extracted from the tissue using the TRIzol reagent method. RNA concentration and purity were assessed using a micro-volume spectrophotometer (A260/A280 ratio between 1.8 and 2.0). Following the Prime Script RT Master Mix protocol, 1 µg of total RNA was reverse transcribed into cDNA. Amplification was performed using the SYBR Premix Ex Taq II kit on a real-time quantitative PCR instrument (Applied Biosystems Quant Studio 5, USA). Reaction program: 95°C pre-denaturation for 30 seconds; 40 cycles of 95°C for 5 seconds, 60°C for 30–34 seconds. β-Actin was used as the internal reference gene, and the relative expression levels of target mRNA genes were calculated using the 2^(-ΔΔCt) method. Genes detected included TREM1 and AQP4 . Primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Sequences are as follows: TREM1 upstream:5‘-CTGGGCTCTGTCATCGTCTTG-3’, downstream:5‘-CAGGTAGGTGTCAAAGGCAGC-3’; AQP4 upstream:5‘-TCAACCTGGGCATCGTCTAC-3’, downstream:5‘-AGCCAGCACATCAAAGACGA-3’; β-Actin upstream:5‘-CCCATCTATGAGGGTTACGC-3’, downstream:5‘-TTTAATGTCACGCACGATTTC-3’. 2.10. Western Blot Take hippocampal or cortical tissue, add pre-chilled RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor cocktail), and homogenize thoroughly on ice using an electric homogenizer. Centrifuge at 4°C, 12,000 rpm for 15 minutes, then collect the supernatant as the total protein extract. Determine protein concentration using the BCA Protein Concentration Assay Kit. Mix equal volumes of protein sample (typically 30 µg) with 5× loading buffer and denature proteins by boiling at 100°C for 10 minutes. Separate proteins via 10% or 12% SDS-polyacrylamide gel electrophoresis, then transfer proteins to a PVDF membrane using wet transfer. After transfer, membranes were blocked with 5% nonfat milk at room temperature for 1 hour. Overnight incubation at 4°C was performed with the following specific primary antibodies: rabbit anti-TREM1 (1:1000), rabbit anti-Iba1 (1:1000), mouse anti-GFAP (1:2000), rabbit anti-AQP4 (1:1000), rabbit anti-Cleaved Caspase-3 (1:1000), mouse anti-NeuN (1:1000), rabbit anti-IL-6 (1:1000), rabbit anti-IL-13 (1:1000), mouse anti-β-Actin (1:5000, as internal control). The following day, the membranes were washed three times with TBST for 10 minutes each. They were then incubated at room temperature for 1 hour with the corresponding HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000). After thorough washing with TBST, the membrane was developed using a high-sensitivity chemiluminescent substrate and imaged on an Amersham Imager 600 chemiluminescence imaging system. ImageJ software was used to quantitatively analyze the gray values of target bands, with protein expression levels expressed as the ratio of gray values relative to the β-Actin internal control. 2.11. Statistical Analysis All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 9.0 software. All data underwent normality testing (Shapiro-Wilk test) and homogeneity of variance testing (Brown-Forsythe test). For data meeting normality and homogeneity of variance criteria, comparisons among multiple groups were conducted using one-way ANOVA. If ANOVA results indicated significant differences, pairwise comparisons were further performed using Tukey's post hoc test. Comparisons between two groups were performed using the unpaired Student's t-test. Differences were considered statistically significant at P < 0.05. Significance in all figures is denoted as: *P < 0.05 vs. Sham group; #P < 0.05 vs. SAH-AAV2-NC group. . 3. Results 3.1. TREM1 Expression Is Upregulated After SAH and Leads to Neurological Deficits To validate the SAH model and investigate the role of TREM1, we first assessed its expression levels in brain tissue. As shown in Fig. 1 A-C, compared with the Sham group, both TREM1 protein and mRNA expression levels were significantly increased in hippocampal tissue of SAH-AAV2-NC rats (P < 0.05), confirming that SAH successfully induced endogenous TREM1 expression. Correspondingly, neurobehavioral results (Table 1 ) revealed that SAH-AAV2-NC rats exhibited the most severe neurological deficits, with significantly reduced modified Garcia scores (9.2 ± 1.3 vs. Sham group 17.8 ± 0.8), significantly prolonged balance beam crossing times (18.3 ± 2.5 s vs. Sham group 4.5 ± 0.8 s), and significantly increased slip frequency (4.8 ± 1.1 vs. 0.4 ± 0.5 in the Sham group) (all P < 0.05). Importantly, specific modulation of TREM1 expression via viral intervention effectively regulated neurological outcomes: Compared with the SAH-AAV2-NC group, TREM1 knockdown (SAH-AAV2-shTREM1 group) significantly improved neurological function in rats, manifested as elevated Garcia scores, reduced balance beam crossing time, and decreased slip frequency (all P < 0.05). Conversely, TREM1 overexpression (SAH-AAV2-TREM1 group) further exacerbated neurological deficits, with all behavioral metrics worse than the SAH-AAV2-NC group (all P < 0.05). These findings clearly demonstrate that TREM1 is not only an upregulated biomarker after SAH but also a key functional factor regulating neurological prognosis following SAH. Table 1 Garcia scores and balance beam test results for each group of rats Group Modified Garcia Score(min) Beam Traversal Time(s) Beam Slip Counts Sham 17.8 ± 0.8 4.5 ± 0.8 0.4 ± 0.5 SAH-AAV2-NC 9.2 ± 1.3 18.3 ± 2.5 * 4.8 ± 1.1 * SAH-AAV2-TREM1 6.5 ± 1.0 *# 25.6 ± 3.1 *# 6.5 ± 1.3 *# SAH-AAV2-shTREM1 13.5 ± 1.2 *# 10.2 ± 1.9 *# 2.2 ± 0.7 *# 3.2. TREM1 Inhibition Alleviates Cerebral Edema and Neuronal Injury Following SAH We further evaluated the impact of TREM1 on pathological brain tissue damage. Brain water content measurements (Table 2) revealed significantly increased water content in the left hemisphere (injured side) of the SAH-AAV2-NC group compared to the Sham group (82.65 ± 0.72% vs. 78.20 ± 0.41%, P < 0.05), indicating marked cerebral edema. HE staining (Fig. 2 F) revealed that neurons in the cortex and hippocampal CA1 region of the Sham group exhibited regular morphology, tightly packed arrangement, and clearly visible nucleoli. In contrast, the SAH-AAV2-NC group showed disorganized and loosely arranged neurons with widened intercellular spaces. Numerous neuronal cell bodies appeared shrunken and deeply stained (nuclear condensation), with a marked reduction in cell numbers. At the molecular level, Western Blot results (Fig. 2 B and D) revealed significantly downregulated expression of the neuronal marker NeuN protein in the SAH-AAV2-NC group, while expression of Cleaved Caspase-3 (Fig. 2 C and E), a key executor of apoptosis, was significantly upregulated. Notably, intervention targeting TREM1 expression significantly reversed these pathological alterations. Knockdown of TREM1 (SAH-AAV2-shTREM1 group) markedly reduced brain water content, improved neuronal morphology and alignment, increased the number of normal neurons, and simultaneously significantly upregulated NeuN protein expression while downregulating Cleaved Caspase-3 expression (both P < 0.05 compared to the SAH-AAV2-NC group). Conversely, TREM1 overexpression (SAH-AAV2-TREM1 group) further exacerbated cerebral edema, neuronal structural disruption, and abnormal expression of NeuN and Cleaved Caspase-3 (all P < 0.05 compared to the SAH-AAV2-NC group). These findings indicate that TREM1 actively participates in the formation of cerebral edema and neuronal apoptosis following SAH. 3.3. TREM1 Exerts Effects by Driving Microglial Activation and Neuroinflammation To elucidate the molecular mechanisms of TREM1, we focused on neuroinflammatory responses. As shown in Fig. 3 A and B, the protein levels of the microglial marker Iba1 were significantly higher in the SAH-AAV2-NC group compared to the Sham group (P < 0.05), indicating widespread microglial activation following SAH. Immunofluorescence staining further revealed morphological changes in microglia: Iba1-positive cells in the Sham group exhibited a quiescent state with small cell bodies and thin, elongated processes. In contrast, microglia in the SAH-AAV2-NC group displayed an activated state characterized by enlarged, rounded cell bodies and shortened, thickened processes with an amoeboid morphology. Concurrently, inflammatory cytokine analysis showed significantly increased protein expression of the proinflammatory cytokine IL-6 in the SAH-AAV2-NC group (Fig. 3 C and D), while expression of the anti-inflammatory factor IL-13 was markedly reduced (Fig. 3 E and F), indicating a severe proinflammatory shift in the inflammatory balance. Modulating TREM1 effectively intervened in this process. Knockdown of TREM1 not only suppressed the upregulation of Iba1 protein expression in microglia (Fig. 3 A and B) but also partially restored their resting-state morphology. It further shifted the inflammatory balance toward an anti-inflammatory direction, manifested by significantly reduced IL-6 levels and markedly elevated IL-13 levels (both P < 0.05 compared to the SAH-AAV2-NC group). Overexpression of TREM1 produced the opposite effect, further enhancing microglial activation and exacerbating the imbalance between pro-inflammatory and anti-inflammatory factors (both P < 0.05). Furthermore, Fig. 3 G and F show that astrocyte marker GFAP expression is also upregulated after SAH and similarly regulated by TREM1 expression, indicating that astrocytes participate in the TREM1-mediated inflammatory response network. 3.4. TREM1 Regulates Expression of AQP4, a Key Protein in Cerebral Edema Given TREM1's potent impact on cerebral edema, we investigated its association with aquaporin 4 (AQP4), a key molecule regulating water transport in the brain and closely linked to vasogenic cerebral edema. As shown in Fig. 4 A-C, changes in AQP4 expression at both mRNA and protein levels highly correlated with TREM1: SAH induced significant upregulation of AQP4, which was markedly suppressed by TREM1 knockdown and further enhanced by TREM1 overexpression (both P < 0.05 compared to the SAH-AAV2-NC group). Immunofluorescence staining in Fig. 4 D and E morphologically confirmed this finding, revealing markedly enhanced TREM1 and AQP4 fluorescence signals in SAH-AAV2-NC group brain tissue, particularly in the terminal processes of perivascular astrocytes. More importantly, the changes in TREM1 and AQP4 fluorescence intensity showed remarkable synchrony across different intervention groups: the strongest signals were observed in the SAH-AAV2-TREM1 group, while signals were markedly attenuated in the SAH-AAV2-shTREM1 group. These findings strongly suggest that TREM1 may regulate AQP4 expression on astrocytes through direct or indirect pathways, thereby contributing to the development of cerebral edema following SAH. 4. Discussion This study systematically reveals for the first time the core driving role of the TREM1-AQP4 axis in neuroinflammatory injury and cerebral edema following experimental subarachnoid hemorrhage (SAH), integrating multidimensional evidence from neurobehavioral, histopathological, and molecular biological analyses. Previous studies have established TREM1 as a key inflammatory amplifier in various systemic inflammatory diseases, such as sepsis and atherosclerosis [ 22 , 26 , 27 ].Within the neurological domain, particularly in SAH, prior observational clinical studies have identified elevated sTREM1 levels in the cerebrospinal fluid of SAH patients, correlating with clinical severity. However, this merely suggested TREM1's potential as a biomarker of disease severity [ 25 ].Our study significantly expands this understanding. We not only confirmed in animal models that TREM1 mRNA and protein expression are significantly upregulated in brain parenchyma (especially hippocampal tissue) after SAH, but more importantly, we conducted rigorous functional validation through AAV-mediated in vivo genetic manipulation. We found that knocking down TREM1 significantly improved neurological function scores and motor coordination after SAH, while reducing cerebral edema and neuronal apoptosis. Conversely, TREM1 overexpression produced the opposite, exacerbating effects. This perfect correspondence between “gain-of-function” and “loss-of-function” experiments compellingly demonstrates that TREM1 is causally implicated in post-SAH brain injury, rather than merely a concomitant phenomenon. This elevates its role in SAH from a passive ‘indicator’ to an active “functional therapeutic target.” This finding resonates with TREM1's role in ischemic stroke [ 28 ], but our study provides unique evidence for its therapeutic potential in the distinct pathophysiology of SAH—characterized by rapid intracranial pressure elevation and direct stimulation from subarachnoid hemorrhage. Microglia-mediated neuroinflammation is a core component of EBI and DCI following SAH [ 9 , 14 , 29 ].Our study demonstrates that TREM1 is a key upstream regulator of microglial state. Following SAH, TREM1 upregulation is accompanied by a significant increase in expression of the microglial marker Iba1, alongside a morphological shift from the resting dendritic state to the activated amoeboid state. More profoundly, we discovered that TREM1 not only regulates the “quantity” (number of activated cells) of microglia but also determines their “quality” (functional phenotype). Imbalance between M1 and M2 phenotypes constitutes the molecular basis of neuroinflammatory injury [ 30 ].Our data demonstrate that TREM1 overexpression exacerbates proinflammatory cytokine IL-6 release while suppressing anti-inflammatory cytokine IL-13 production, tilting the inflammatory balance toward the destructive M1 phenotype. Conversely, TREM1 knockdown effectively reverses this imbalance, promoting a shift toward the reparative M2 phenotype in the inflammatory microenvironment. This suggests TREM1 likely serves as an upstream “switch” for microglial/macrophage phenotype conversion. The underlying mechanism may involve TREM1 activation downstream of the Syk-PI3K-AKT and MAPK signaling pathways, thereby potently amplifying NF-κB transcriptional activity [ 20 , 31 ].NF-κB serves as a key transcription factor for numerous proinflammatory genes associated with the M1 phenotype, while its regulatory influence on M2-related genes is relatively weaker [ 32 , 33 ].Thus, TREM1 inhibition may suppress M1 polarization by attenuating NF-κB signaling while simultaneously creating a favorable microenvironment for M2 polarization. Furthermore, recent studies suggest that TREM1 drives proinflammatory phenotypes by influencing metabolic reprogramming (e.g., promoting aerobic glycolysis) [ 27 ],offering new avenues for future investigations into the precise mechanisms by which TREM1 regulates phenotypic switching The most illuminating finding of this study lies in revealing the intrinsic connection between TREM1 and AQP4, a key molecule in cerebral edema. Cerebral edema, particularly vasogenic edema, is one of the primary features of EBI following SAH and is directly correlated with patient prognosis[ 34 ].AQP4, the primary aquaporin expressed in astrocytic end-feet, is widely recognized for its dual role in both the formation and resolution of cerebral edema [ 35 – 37 ],However, the mechanisms regulating its expression following SAH remain incompletely understood. We confirmed at both mRNA and protein levels that TREM1 expression changes are highly synchronized with AQP4. This strong positive correlation suggests a potential direct regulatory pathway between the two. We propose the following potential mechanisms: First, indirect cell-cell communication (paracrine pathway): This is the most likely mechanism. TREM1 is primarily expressed on myeloid cells (e.g., microglia) [ 38 , 39 ].TREM1 activation leads to the release of proinflammatory cytokines such as IL-6 [ 38 ].Conclusive evidence demonstrates that IL-6 and other inflammatory mediators significantly upregulate AQP4 expression in astrocytes [ 40 ].Thus, we hypothesize a paracrine signaling axis: “glial TREM1 activation → IL-6 release → action on astrocytes → AQP4 upregulation → exacerbated cerebral edema.” Second, potential direct regulation (autocrine/other pathways): Although less frequently reported, it cannot be ruled out that some astrocytes may also express TREM1 under pathological conditions [ 41 , 42 ].If so, TREM1 could directly regulate its own AQP4 expression via autocrine mechanisms. Furthermore, TREM1 activates downstream signaling pathways such as NF-κB [ 43 ],which itself is a key transcriptional regulator of AQP4, suggesting potential shared transcriptional regulatory networks. This study also observed TREM1 regulation of the astrocyte marker GFAP, further supporting TREM1's profound influence on astrocytes within the neurovascular unit. Therefore, we propose for the first time that the “TREM1-neuroinflammation-AQP4” axis represents a core link connecting excessive immune responses to vasogenic cerebral edema following SAH. This proposed axis offers an integrated perspective for understanding the complexity of brain injury after SAH, revealing that inflammation and edema are not independent parallel pathways but rather a unified pathological process tightly intertwined and mutually amplified through the critical node of TREM1. Admittedly, this study has certain limitations, which also represent directions for future research. First, while the AAV2 vector we employed efficiently infected multiple CNS cell types, it lacked cell specificity. Consequently, we could not precisely determine whether TREM1 in the SAH pathological context primarily originates from microglia, infiltrating macrophages, or partially activated astrocytes. Future studies utilizing Cx3cr1-CreERT2 (glia/macrophage-specific) or GFAP-Cre (astrocyte-specific) mice crossed with TREM1 floxed mice to generate cell-specific knockout models will enable precise analysis of the relative contributions of TREM1 from different cellular origins. Second, the absence of TREM1/Iba1/GFAP/AQP4 immunofluorescence multiplex labeling precludes direct morphological evidence of spatial proximity between TREM1-positive and AQP4-positive cells. Future confocal microscopy analysis will help validate our proposed paracrine hypothesis. Third, the direct downstream signaling pathway by which TREM1 regulates AQP4 expression remains unelucidated. Does it involve the classical Syk-PI3K-AKT-NF-κB pathway, or does it incorporate other unknown signaling molecules? Utilizing an in vitro co-culture system of astrocytes and microglia, combined with specific pathway inhibitors, will enable detailed dissection of this regulatory network. Finally, from a translational medicine perspective, exploring the therapeutic efficacy of specific TREM1-targeting inhibitors (such as the known soluble TREM1 antagonist peptide LR12 or developing neutralizing antibodies) during different time windows after SAH (e.g., the EBI phase or DCI phase) is an essential step toward clinical application. Assessing synergistic effects with existing therapies (e.g., nimodipine) also holds significant clinical value. In summary, this study establishes TREM1 as a core driver in the pathological progression of SAH. It coordinates microglia-mediated neuroinflammation, disrupts cytokine homeostasis, and innovatively upregulates astrocytic AQP4 expression—collectively forming a vicious cycle that exacerbates brain injury. The proposed “TREM1-Neuroinflammation-AQP4” axis offers a novel integrated explanation for the pathophysiology of SAH. From a therapeutic perspective, targeting TREM1 presents unique advantages: it holds promise for dual intervention through a single target—simultaneously alleviating neuroinflammation and reducing vasogenic cerebral edema. This “two birds with one stone” effect may yield greater neuroprotective benefits and a broader therapeutic time window compared to traditional therapies targeting single pathological pathways. Consequently, TREM1 represents a highly promising therapeutic target with significant translational potential. Developing specific inhibitors against it holds the promise of opening a hopeful new avenue for improving the prognosis of patients with SAH, a critical cerebrovascular disease. 5. Conclusion In summary, this study establishes TREM1 as a central player in the pathological progression of subarachnoid hemorrhage (SAH). It mediates neuroinflammation primarily driven by microglia and potentially exacerbates cerebral edema by upregulating AQP4, ultimately leading to neuronal death and neurological deficits. Targeting TREM1 inhibition holds promise as a novel combined therapeutic strategy to improve SAH prognosis by simultaneously alleviating inflammation and reducing edema. Declarations Author contributions Jianxin Chen, Junli Zhang, Wenjing Ning All authors made a considerable contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be submitted for publication; have agreed on the journal to which the article has been submit ted; and agree to be accountable for all aspects of the work. Funding Statement Funding: This work was supported by grants from the Jinan Health Commission Science and Technology Development Plan (No. 2024301006); the Jinan Health Commission Science and Technology Development Plan (No. 2025301007); and the Taian Science and Technology Development Plan (No. 2023NS164) Data availability Data from this study are available from the corresponding author upon request. Ethics approval All animal procedures in this study strictly adhered to national regulations governing the management and use of laboratory animals. They were reviewed and approved by the Animal Ethics Committee of Jinan First People's Hospital (Approval No. 2025-03-01-17), maximizing compliance with the 3Rs principle (reduction, replacement, refinement) to minimize both the number of animals used and their suffering. Competing interest The authors declare no competing interests. References Claassen, J. and S. Park, Spontaneous subarachnoid haemorrhage . 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Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in European Journal of Medical Research → Version 1 posted Editorial decision: Revision requested 13 Oct, 2025 Reviews received at journal 13 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviews received at journal 08 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 07 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 First submitted to journal 29 Sep, 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7741776","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528690213,"identity":"176ca3a3-a3bc-46ac-91ad-15a57fd4f06d","order_by":0,"name":"Jianxin Chen","email":"","orcid":"","institution":"The First People's Hospital of Jinan","correspondingAuthor":false,"prefix":"","firstName":"Jianxin","middleName":"","lastName":"Chen","suffix":""},{"id":528690214,"identity":"f78dc235-9715-4383-83b3-524da82a1428","order_by":1,"name":"Junli Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Shandong First Medical 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1","display":"","copyAsset":false,"role":"figure","size":92080,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7741776/v1/896eaebd05abb7f99efd91bf.png"},{"id":94118672,"identity":"ce506f31-9770-4846-97c6-6f2830106739","added_by":"auto","created_at":"2025-10-22 14:46:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":595948,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7741776/v1/e48d491e251fdca8d1196343.png"},{"id":94118671,"identity":"324d0ab7-a303-41ea-bbf1-ab0c4c9bc5d7","added_by":"auto","created_at":"2025-10-22 14:46:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148204,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7741776/v1/5801115c5488590fba5bc3ee.png"},{"id":94118677,"identity":"3336d507-0940-4145-ba33-1053215efde5","added_by":"auto","created_at":"2025-10-22 14:46:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":434068,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7741776/v1/c11e93dd646ec483a4a7f2b8.png"},{"id":97179392,"identity":"a81cf8f6-22a8-4330-baa4-b01b08e080f3","added_by":"auto","created_at":"2025-12-01 16:15:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2029911,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7741776/v1/41b1ed22-b806-45e1-af0e-6bee6d7195aa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The TREM1-AQP4 Axis Mediates Neuroinflammatory Injury and Brain Edema after Experimental Subarachnoid Hemorrhage","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSubarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease characterized by high mortality and disability rates[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].Globally, the annual incidence of SAH is approximately 9 cases per 100,000 people. Although it accounts for only 5%-10% of all strokes, it imposes a disproportionately heavy disease burden [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].Despite significant advances in acute-phase management techniques such as aneurysm clipping and endovascular embolization in recent years, which have effectively reduced the risk of early rebleeding, 30%-50% of SAH survivors still suffer from severe delayed neurological deficits, including cognitive decline, emotional disturbances, and motor impairments. This imposes a heavy economic and caregiving burden on patients' families and society[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Therefore, elucidating the pathophysiological mechanisms of brain injury following SAH and identifying effective intervention targets represent critical unresolved challenges in neuroscience and the field of severe cerebrovascular disease.\u003c/p\u003e\u003cp\u003eBrain injury following subarachnoid hemorrhage (SAH) is a dynamic process involving multiple stages and mechanisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Early brain injury (EBI) occurs within 72 hours post-bleeding and is characterized by a rapid increase in intracranial pressure, a sudden drop in cerebral blood flow, disruption of the blood-brain barrier, and global cerebral ischemia. This phase is considered decisive in determining patient prognosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Subsequently, delayed cerebral ischemia (DCI) emerges as the primary complication driving sustained neurological deterioration[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].Traditionally attributed to large-vessel spasm, DCI has shown limited improvement in patient outcomes despite spasm-targeted therapies, prompting researchers to explore more intricate cellular and molecular mechanisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].In recent years, mounting evidence indicates that neuroinflammation serves as the core pathological bridge linking EBI and DCI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].Following SAH, blood and its degradation products (e.g., oxyhemoglobin) in the subarachnoid space act as potent stress signals, triggering a robust and persistent innate immune and inflammatory cascade within the central nervous system. This process involves the activation and infiltration of multiple immune cells alongside the release of numerous inflammatory mediators, collectively exacerbating blood-brain barrier permeability, cerebral edema formation, and ultimately neuronal apoptosis and necrosis [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs resident innate immune cells in the central nervous system (CNS), microglia play a double-edged sword role in neuroinflammation following subarachnoid hemorrhage (SAH) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].Under physiological conditions, microglia exist in a quiescent state, responsible for immune surveillance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].Following SAH, they undergo rapid activation. On one hand, appropriate activation aids in clearing cellular debris, red blood cells, and toxic substances, potentially exerting protective effects through the secretion of minor neurotrophic factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e];On the other hand, excessive and sustained activation leads to the release of large amounts of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6), chemokines, and reactive oxygen species, further amplifying the inflammatory response and causing secondary brain injury [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].In recent years, researchers have increasingly recognized that microglia/macrophages are not simply \u0026ldquo;activated\u0026rdquo; or \u0026ldquo;quiescent,\u0026rdquo; but rather polarize toward distinct functional phenotypes driven by different microenvironmental signals. These primarily include the classically activated pro-inflammatory M1 phenotype and the alternatively activated anti-inflammatory/reparative M2 phenotype, with the dynamic balance between them profoundly influencing disease outcomes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].Therefore, precisely regulating the phenotypic transition of microglia from the harmful M1 state to the beneficial M2 state is considered a highly promising therapeutic strategy for alleviating neuroinflammation following SAH.\u003c/p\u003e\u003cp\u003eTriggering receptor expressed on myeloid cells-1 (TREM1) is a key inflammatory amplifier discovered in recent years, primarily expressed on the surface of myeloid cells such as neutrophils and monocytes/macrophages[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].TREM1 belongs to the immunoglobulin superfamily. By binding to a ligand that remains incompletely characterized and coupling with the adaptor protein DAP12, it activates downstream Syk-PI3K-AKT and MAPK signaling pathways. This process powerfully amplifies the activity of transcription factors such as NF-κB, inducing the explosive release of proinflammatory factors like TNF-α and IL-1β, thereby forming an \u0026ldquo;inflammatory storm\u0026rdquo; [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].The role of TREM1 in systemic inflammatory response syndromes like septic shock and sepsis has been extensively validated, with TREM1 inhibition significantly improving outcomes. Notably, growing evidence suggests TREM1 also plays a critical role in non-infectious, sterile inflammatory diseases such as atherosclerosis and myocardial ischemia-reperfusion injury [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Recent clinical observations indicate that soluble TREM1 (sTREM1) levels are significantly elevated in the cerebrospinal fluid of SAH patients, positively correlating with clinical severity scores (e.g., WFNS score) and poor prognosis. This strongly suggests TREM1 may be involved in the pathological process of SAH [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].However, the precise role of TREM1 in neuroinflammation following SAH, its cellular origin within the CNS (whether specifically expressed on microglia), whether it directly drives microglial phenotypic switching, and its key downstream effector molecules and signaling pathways remain incompletely understood. These knowledge gaps significantly impede translational research efforts targeting TREM1 as a potential therapeutic target.\u003c/p\u003e\u003cp\u003eGiven these research gaps, this study aims to systematically investigate the role of TREM1 in neuroinflammatory damage following experimental SAH and its specific molecular mechanisms. We propose the core hypothesis: TREM1, as a key amplifier of neuroinflammation after SAH, exacerbates neuroinflammation, cerebral edema, and neuronal apoptosis by activating microglia and promoting their polarization toward the pro-inflammatory M1 phenotype, while simultaneously upregulating aquaporin-4 (AQP4) expression, ultimately leading to neurological deficits. To validate this hypothesis, we established a mature rat SAH model and employed adeno-associated virus (AAV)-mediated gene technology to specifically upregulate and knockdown TREM1 expression in the brain. Through a comprehensive approach integrating neurobehavioral assessment, histopathological analysis, protein immunoblotting, real-time quantitative PCR, and immunofluorescence staining, we systematically evaluated TREM1's impact on neurological outcomes, cerebral edema severity, neuronal survival and apoptosis, microglial activation status, inflammatory cytokine profiles, and AQP4 expression. This study aims not only to provide new insights into the pathological mechanisms of SAH but also to establish robust preclinical evidence for TREM1 as a potential therapeutic target through gain-of-function and loss-of-function experiments. This foundation will pave the way for developing novel neuroprotective drugs.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals\u003c/h2\u003e\u003cp\u003eHealthy adult male Sprague-Dawley (SD) rats (SPF grade, weighing 250\u0026ndash;300 g) were purchased from Jinan Xingkang Biotechnology Co., Ltd. All rats were housed in the barrier environment of the Shandong First Medical University Laboratory Animal Center and maintained under standard conditions (12-hour light/dark cycle, light period 7:00\u0026ndash;19:00, ambient temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%). They had free access to standard rodent chow and sterile drinking water. All animals underwent at least one week of acclimatization prior to experimentation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental Design and Grouping\u003c/h2\u003e\u003cp\u003eThis study employed a completely randomized grouping design. Following successful establishment of the SAH model, surviving rats were randomly assigned to the following four experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSham group\u003c/strong\u003e\u003cp\u003eRats underwent the same surgical procedures as the SAH model group, including anesthesia, skin incision, and vascular dissection, but without internal carotid artery puncture or nylon thread insertion to prevent SAH induction.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSAH\u0026thinsp;+\u0026thinsp;AAV2-Vector Control Group (SAH-AAV2-NC Group)\u003c/strong\u003e\u003cp\u003eNineteen days prior to SAH modeling, rats received microinjection of empty adeno-associated virus (AAV2) into the lateral ventricle as a viral vector control to exclude non-specific immune reactions or toxic effects potentially caused by the viral vector itself.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSAH\u0026thinsp;+\u0026thinsp;AAV2-TREM1 overexpression group (SAH-AAV2-TREM1 group)\u003c/strong\u003e\u003cp\u003e19 days prior to SAH modeling, AAV2 virus overexpressing rat TREM1 was microinjected into the lateral ventricle to specifically enhance TREM1 expression in the brain under SAH conditions.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSAH\u0026thinsp;+\u0026thinsp;AAV2-shRNA-TREM1 knockdown group (SAH-AAV2-shTREM1 group)\u003c/strong\u003e\u003cp\u003e19 days prior to SAH modeling, AAV2 virus carrying short hairpin RNA (shRNA) targeting the rat TREM1 gene was microinjected into the lateral ventricle to specifically suppress TREM1 expression in the brain under SAH conditions.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Adeno-associated virus (AAV) vector construction and lateral ventricle injection\u003c/h2\u003e\u003cp\u003eThe AAV2 vector overexpressing TREM1 (AAV2-CMV-TREM1-3xFLAG), the AAV2 vector expressing TREM1-specific shRNA (AAV2-U6-shTREM1), and the corresponding empty vector control (AAV2-CMV-NC) were designed and packaged by Shanghai GK Gene Chemistry Technology Co., Ltd. The shRNA target sequence used was: 5'-GCACAAGATCAACCAGTATTT-3'. All viral titers were adjusted to 2\u0026times;10\u0026sup1;\u0026sup2; viral genomes/mL (vg/mL). Viral injections were performed 19 days prior to modeling to ensure sufficient time for viral expression and effect in target cells. Rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, intraperitoneal injection) and fixed in a stereotaxic brain stereotaxic apparatus (Ruiwo, China). The scalp was shaved and disinfected, and a skin incision was made along the coronal suture to expose the anterior fontanelle. Referencing a rat stereotaxic atlas, the coordinates for the left lateral ventricle were determined: 0.8 mm posterior to the anterior fontanelle, 1.5 mm lateral to the midline, and 3.5 mm subdural. A microinjector (Hamilton, USA) was used to inject 2 \u0026micro;L of viral solution at a slow rate of 0.2 \u0026micro;L/min. After injection, the needle was left in place for 10 minutes to allow complete diffusion of the virus before being slowly withdrawn. The skin incision was sutured, and the rat was placed on a warming pad for recovery. Once fully awake, the animal was returned to its housing cage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Establishment of Subarachnoid Hemorrhage (SAH) Model\u003c/h2\u003e\u003cp\u003eThe SAH model was established using the classic internal carotid artery puncture method, which effectively mimics clinically induced SAH from ruptured aneurysms. The procedure is briefly described as follows: Rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, intraperitoneal injection) and fixed in a supine position. The neck region was shaved and rigorously disinfected. A 2\u0026ndash;3 cm skin incision was made along the midline of the neck. Subcutaneous tissue and glands were bluntly dissected to expose the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The distal end of the ECA was ligated and transected, temporarily occluding blood flow in the CCA and ICA. A 4\u0026thinsp;\u0026minus;\u0026thinsp;0 monofilament nylon suture (approximately 0.28 mm diameter, with the tip heated and blunted to minimize vascular injury) was inserted through the ECA stump. It was gently advanced cranially through the ICA bifurcation until slight resistance was felt (indicating the suture tip had reached the anterior portion of the circle of Willis, typically near the origin of the middle cerebral artery). then advance approximately 3 mm further to puncture the anterior cerebral artery-anterior communicating artery complex, inducing bleeding. Leave the thread in place for 10 seconds before slowly withdrawing it. Immediately ligate the ECA stump and release the arterial clamps on the CCA and ICA to restore blood flow. In the sham-operated group, rats underwent identical vascular dissection procedures except for the absence of nylon thread puncture. Postoperatively, all rats were housed individually, with vital signs closely monitored while receiving thermal regulation and analgesia.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Neurobehavioral Assessment\u003c/h2\u003e\u003cp\u003eTwenty-four hours after SAH, two experienced investigators blinded to experimental group assignments independently conducted neurobehavioral assessments. Final scores were averaged to ensure objectivity and reliability. This time point was selected as it represents the peak of early brain injury (EBI), enabling optimal evaluation of TREM1 regulation's impact on acute-phase neurological outcomes.\u003c/p\u003e\u003cp\u003eModified Garcia Neurological Score: This scoring system comprehensively evaluates neurological function across six domains: spontaneous activity, symmetry of limb movements, forelimb extension ability, climbing ability, tactile response, and whisker response. Each domain is scored from 1 to 3 points, yielding a total score ranging from 3 to 18 points. A lower score indicates more severe neurological deficits.\u003c/p\u003e\u003cp\u003eBalance Beam Test: Used to assess rats' motor coordination, balance ability, and fine motor control. The apparatus consists of a circular wooden beam measuring 120 cm in length and 2.5 cm in width, suspended 50 cm above the ground. All rats underwent three days of adaptation training prior to testing to ensure proficiency in traversing the beam. During formal testing, the time required for the rat to traverse the beam from one end to the other (traverse time, in seconds) and the number of times the rat slipped off the beam during the process (slip count) were recorded. Each rat underwent three trials, with the average value recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Brain Water Content Measurement\u003c/h2\u003e\u003cp\u003eTwenty-four hours post-modeling and following behavioral testing, rats were rapidly decapitated under deep anesthesia to harvest brains. The entire brain was carefully dissected and removed. On an ice plate, the left cerebral hemisphere (i.e., the ipsilateral hemisphere), right cerebral hemisphere (contralateral hemisphere), cerebellum, and brainstem were promptly separated. Immediately weigh the wet weight of each section using a precision electronic analytical balance (accuracy 0.1 mg). Subsequently, place the brain tissue samples in a preheated 105\u0026deg;C oven and dry continuously for 72 hours until constant weight (difference between two consecutive weighings\u0026thinsp;\u0026lt;\u0026thinsp;0.2 mg). Then, weigh the dry weight again.\u003c/p\u003e\u003cp\u003eThe formula for calculating brain water content (%) is: [(wet weight - dry weight) / wet weight] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Tissue Processing and Hematoxylin-Eosin (HE) Staining\u003c/h2\u003e\u003cp\u003eTwenty-four hours after modeling, rats were deeply anesthetized and rapidly perfused via the left ventricle with approximately 200 mL of pre-chilled 0.9% saline until the effluent became clear. This was followed by perfusion with approximately 300 mL of 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for fixation. The brain was removed and post-fixed in 4% paraformaldehyde for 24 hours. It was then sequentially dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Continuous coronal sections of 5\u0026micro;m thickness were prepared using a paraffin microtome (Leica, Germany), targeting brain regions including the hippocampus and cortex. Sections were dewaxed with xylene, rehydrated with graded ethanol, and routinely stained with hematoxylin and eosin. Following staining, sections were mounted with neutral binder and examined under an optical microscope (Nikon Eclipse Ci, Japan) to observe the morphological structure, arrangement density, and nuclear shrinkage of neurons in the cortex and hippocampal CA1 region, with images captured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eParaffin sections were deparaffinized to water and placed in citrate buffer (pH 6.0) for microwave-assisted antigen retrieval. After cooling, sections were permeabilized with PBS containing 0.3% Triton X-100 for 10 minutes, followed by blocking with 5% donkey serum at room temperature for 1 hour. Discard the blocking solution without washing. Directly add appropriately diluted primary antibody working solutions, including: rabbit anti-rat TREM1 polyclonal antibody (1:200), mouse anti-rat AQP4 monoclonal antibody (1:200), and rabbit anti-rat Iba1 polyclonal antibody (1:500). Incubate overnight at 4\u0026deg;C in a humidified chamber. The following day, wash three times with PBS for 5 minutes each. Add the corresponding fluorescein-labeled secondary antibody (Alexa Fluor 488-labeled donkey anti-rabbit IgG or Alexa Fluor 594-labeled donkey anti-mouse IgG, 1:500) and incubate at room temperature in the dark for 1 hour. After washing with PBS, mount with DAPI-containing mounting medium. Images were acquired using a fully automated fluorescence upright microscope (Zeiss Axio Imager Z2, Germany) or a laser confocal microscope (Leica TCS SP8, Germany). Semi-quantitative analysis of fluorescence intensity in regions of interest (ROIs) was performed using ImageJ software (NIH, USA). Morphological skeleton analysis of microglia was conducted using ImageJ plugins to quantify branch length and node count.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Real-Time Quantitative PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTwenty-four hours after modeling, rat hippocampal tissue was rapidly isolated, flash-frozen in liquid nitrogen, and then transferred to a -80\u0026deg;C ultra-low temperature freezer for storage. Total RNA was extracted from the tissue using the TRIzol reagent method. RNA concentration and purity were assessed using a micro-volume spectrophotometer (A260/A280 ratio between 1.8 and 2.0). Following the Prime Script RT Master Mix protocol, 1 \u0026micro;g of total RNA was reverse transcribed into cDNA. Amplification was performed using the SYBR Premix Ex Taq II kit on a real-time quantitative PCR instrument (Applied Biosystems Quant Studio 5, USA). Reaction program: 95\u0026deg;C pre-denaturation for 30 seconds; 40 cycles of 95\u0026deg;C for 5 seconds, 60\u0026deg;C for 30\u0026ndash;34 seconds. β-Actin was used as the internal reference gene, and the relative expression levels of target mRNA genes were calculated using the 2^(-ΔΔCt) method.\u003c/p\u003e\u003cp\u003eGenes detected included \u003cem\u003eTREM1\u003c/em\u003e and \u003cem\u003eAQP4\u003c/em\u003e. Primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Sequences are as follows:\u003c/p\u003e\u003cp\u003e\u003cem\u003eTREM1\u003c/em\u003e upstream:5\u0026lsquo;-CTGGGCTCTGTCATCGTCTTG-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003edownstream:5\u0026lsquo;-CAGGTAGGTGTCAAAGGCAGC-3\u0026rsquo;;\u003c/p\u003e\u003cp\u003e\u003cem\u003eAQP4\u003c/em\u003e upstream:5\u0026lsquo;-TCAACCTGGGCATCGTCTAC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003edownstream:5\u0026lsquo;-AGCCAGCACATCAAAGACGA-3\u0026rsquo;;\u003c/p\u003e\u003cp\u003e\u003cem\u003eβ-Actin\u003c/em\u003e upstream:5\u0026lsquo;-CCCATCTATGAGGGTTACGC-3\u0026rsquo;,\u003c/p\u003e\u003cp\u003edownstream:5\u0026lsquo;-TTTAATGTCACGCACGATTTC-3\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Western Blot\u003c/h2\u003e\u003cp\u003eTake hippocampal or cortical tissue, add pre-chilled RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor cocktail), and homogenize thoroughly on ice using an electric homogenizer. Centrifuge at 4\u0026deg;C, 12,000 rpm for 15 minutes, then collect the supernatant as the total protein extract. Determine protein concentration using the BCA Protein Concentration Assay Kit. Mix equal volumes of protein sample (typically 30 \u0026micro;g) with 5\u0026times; loading buffer and denature proteins by boiling at 100\u0026deg;C for 10 minutes. Separate proteins via 10% or 12% SDS-polyacrylamide gel electrophoresis, then transfer proteins to a PVDF membrane using wet transfer. After transfer, membranes were blocked with 5% nonfat milk at room temperature for 1 hour. Overnight incubation at 4\u0026deg;C was performed with the following specific primary antibodies: rabbit anti-TREM1 (1:1000), rabbit anti-Iba1 (1:1000), mouse anti-GFAP (1:2000), rabbit anti-AQP4 (1:1000), rabbit anti-Cleaved Caspase-3 (1:1000), mouse anti-NeuN (1:1000), rabbit anti-IL-6 (1:1000), rabbit anti-IL-13 (1:1000), mouse anti-β-Actin (1:5000, as internal control). The following day, the membranes were washed three times with TBST for 10 minutes each. They were then incubated at room temperature for 1 hour with the corresponding HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000). After thorough washing with TBST, the membrane was developed using a high-sensitivity chemiluminescent substrate and imaged on an Amersham Imager 600 chemiluminescence imaging system. ImageJ software was used to quantitatively analyze the gray values of target bands, with protein expression levels expressed as the ratio of gray values relative to the β-Actin internal control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Statistical analysis was performed using GraphPad Prism 9.0 software. All data underwent normality testing (Shapiro-Wilk test) and homogeneity of variance testing (Brown-Forsythe test). For data meeting normality and homogeneity of variance criteria, comparisons among multiple groups were conducted using one-way ANOVA. If ANOVA results indicated significant differences, pairwise comparisons were further performed using Tukey's post hoc test. Comparisons between two groups were performed using the unpaired Student's t-test. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significance in all figures is denoted as: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. Sham group; #P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. SAH-AAV2-NC group.\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. TREM1 Expression Is Upregulated After SAH and Leads to Neurological Deficits\u003c/h2\u003e\u003cp\u003eTo validate the SAH model and investigate the role of TREM1, we first assessed its expression levels in brain tissue. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, compared with the Sham group, both TREM1 protein and mRNA expression levels were significantly increased in hippocampal tissue of SAH-AAV2-NC rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming that SAH successfully induced endogenous TREM1 expression. Correspondingly, neurobehavioral results (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed that SAH-AAV2-NC rats exhibited the most severe neurological deficits, with significantly reduced modified Garcia scores (9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 vs. Sham group 17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8), significantly prolonged balance beam crossing times (18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 s vs. Sham group 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 s), and significantly increased slip frequency (4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 vs. 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 in the Sham group) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eImportantly, specific modulation of TREM1 expression via viral intervention effectively regulated neurological outcomes: Compared with the SAH-AAV2-NC group, TREM1 knockdown (SAH-AAV2-shTREM1 group) significantly improved neurological function in rats, manifested as elevated Garcia scores, reduced balance beam crossing time, and decreased slip frequency (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, TREM1 overexpression (SAH-AAV2-TREM1 group) further exacerbated neurological deficits, with all behavioral metrics worse than the SAH-AAV2-NC group (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings clearly demonstrate that TREM1 is not only an upregulated biomarker after SAH but also a key functional factor regulating neurological prognosis following SAH.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGarcia scores and balance beam test results for each group of rats\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModified Garcia Score(min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBeam Traversal Time(s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBeam Slip Counts\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSham\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAH-AAV2-NC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 *\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 *\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAH-AAV2-TREM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 *#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 *#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 *#\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAH-AAV2-shTREM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 *#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 *#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 *#\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. TREM1 Inhibition Alleviates Cerebral Edema and Neuronal Injury Following SAH\u003c/h2\u003e\u003cp\u003eWe further evaluated the impact of TREM1 on pathological brain tissue damage. Brain water content measurements (Table\u0026nbsp;2) revealed significantly increased water content in the left hemisphere (injured side) of the SAH-AAV2-NC group compared to the Sham group (82.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72% vs. 78.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating marked cerebral edema. HE staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) revealed that neurons in the cortex and hippocampal CA1 region of the Sham group exhibited regular morphology, tightly packed arrangement, and clearly visible nucleoli. In contrast, the SAH-AAV2-NC group showed disorganized and loosely arranged neurons with widened intercellular spaces. Numerous neuronal cell bodies appeared shrunken and deeply stained (nuclear condensation), with a marked reduction in cell numbers. At the molecular level, Western Blot results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and D) revealed significantly downregulated expression of the neuronal marker NeuN protein in the SAH-AAV2-NC group, while expression of Cleaved Caspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and E), a key executor of apoptosis, was significantly upregulated.\u003c/p\u003e\u003cp\u003eNotably, intervention targeting TREM1 expression significantly reversed these pathological alterations. Knockdown of TREM1 (SAH-AAV2-shTREM1 group) markedly reduced brain water content, improved neuronal morphology and alignment, increased the number of normal neurons, and simultaneously significantly upregulated NeuN protein expression while downregulating Cleaved Caspase-3 expression (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the SAH-AAV2-NC group). Conversely, TREM1 overexpression (SAH-AAV2-TREM1 group) further exacerbated cerebral edema, neuronal structural disruption, and abnormal expression of NeuN and Cleaved Caspase-3 (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the SAH-AAV2-NC group). These findings indicate that TREM1 actively participates in the formation of cerebral edema and neuronal apoptosis following SAH.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. TREM1 Exerts Effects by Driving Microglial Activation and Neuroinflammation\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanisms of TREM1, we focused on neuroinflammatory responses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B, the protein levels of the microglial marker Iba1 were significantly higher in the SAH-AAV2-NC group compared to the Sham group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating widespread microglial activation following SAH. Immunofluorescence staining further revealed morphological changes in microglia: Iba1-positive cells in the Sham group exhibited a quiescent state with small cell bodies and thin, elongated processes. In contrast, microglia in the SAH-AAV2-NC group displayed an activated state characterized by enlarged, rounded cell bodies and shortened, thickened processes with an amoeboid morphology. Concurrently, inflammatory cytokine analysis showed significantly increased protein expression of the proinflammatory cytokine IL-6 in the SAH-AAV2-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D), while expression of the anti-inflammatory factor IL-13 was markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F), indicating a severe proinflammatory shift in the inflammatory balance.\u003c/p\u003e\u003cp\u003eModulating TREM1 effectively intervened in this process. Knockdown of TREM1 not only suppressed the upregulation of Iba1 protein expression in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B) but also partially restored their resting-state morphology. It further shifted the inflammatory balance toward an anti-inflammatory direction, manifested by significantly reduced IL-6 levels and markedly elevated IL-13 levels (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the SAH-AAV2-NC group). Overexpression of TREM1 produced the opposite effect, further enhancing microglial activation and exacerbating the imbalance between pro-inflammatory and anti-inflammatory factors (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and F show that astrocyte marker GFAP expression is also upregulated after SAH and similarly regulated by TREM1 expression, indicating that astrocytes participate in the TREM1-mediated inflammatory response network.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4. TREM1 Regulates Expression of AQP4, a Key Protein in Cerebral Edema\u003c/h2\u003e\u003cp\u003eGiven TREM1's potent impact on cerebral edema, we investigated its association with aquaporin 4 (AQP4), a key molecule regulating water transport in the brain and closely linked to vasogenic cerebral edema. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, changes in AQP4 expression at both mRNA and protein levels highly correlated with TREM1: SAH induced significant upregulation of AQP4, which was markedly suppressed by TREM1 knockdown and further enhanced by TREM1 overexpression (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the SAH-AAV2-NC group). Immunofluorescence staining in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E morphologically confirmed this finding, revealing markedly enhanced TREM1 and AQP4 fluorescence signals in SAH-AAV2-NC group brain tissue, particularly in the terminal processes of perivascular astrocytes. More importantly, the changes in TREM1 and AQP4 fluorescence intensity showed remarkable synchrony across different intervention groups: the strongest signals were observed in the SAH-AAV2-TREM1 group, while signals were markedly attenuated in the SAH-AAV2-shTREM1 group. These findings strongly suggest that TREM1 may regulate AQP4 expression on astrocytes through direct or indirect pathways, thereby contributing to the development of cerebral edema following SAH.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study systematically reveals for the first time the core driving role of the TREM1-AQP4 axis in neuroinflammatory injury and cerebral edema following experimental subarachnoid hemorrhage (SAH), integrating multidimensional evidence from neurobehavioral, histopathological, and molecular biological analyses.\u003c/p\u003e\u003cp\u003ePrevious studies have established TREM1 as a key inflammatory amplifier in various systemic inflammatory diseases, such as sepsis and atherosclerosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].Within the neurological domain, particularly in SAH, prior observational clinical studies have identified elevated sTREM1 levels in the cerebrospinal fluid of SAH patients, correlating with clinical severity. However, this merely suggested TREM1's potential as a biomarker of disease severity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].Our study significantly expands this understanding. We not only confirmed in animal models that TREM1 mRNA and protein expression are significantly upregulated in brain parenchyma (especially hippocampal tissue) after SAH, but more importantly, we conducted rigorous functional validation through AAV-mediated in vivo genetic manipulation.\u003c/p\u003e\u003cp\u003eWe found that knocking down TREM1 significantly improved neurological function scores and motor coordination after SAH, while reducing cerebral edema and neuronal apoptosis. Conversely, TREM1 overexpression produced the opposite, exacerbating effects. This perfect correspondence between \u0026ldquo;gain-of-function\u0026rdquo; and \u0026ldquo;loss-of-function\u0026rdquo; experiments compellingly demonstrates that TREM1 is causally implicated in post-SAH brain injury, rather than merely a concomitant phenomenon. This elevates its role in SAH from a passive \u0026lsquo;indicator\u0026rsquo; to an active \u0026ldquo;functional therapeutic target.\u0026rdquo; This finding resonates with TREM1's role in ischemic stroke [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but our study provides unique evidence for its therapeutic potential in the distinct pathophysiology of SAH\u0026mdash;characterized by rapid intracranial pressure elevation and direct stimulation from subarachnoid hemorrhage.\u003c/p\u003e\u003cp\u003eMicroglia-mediated neuroinflammation is a core component of EBI and DCI following SAH [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].Our study demonstrates that TREM1 is a key upstream regulator of microglial state. Following SAH, TREM1 upregulation is accompanied by a significant increase in expression of the microglial marker Iba1, alongside a morphological shift from the resting dendritic state to the activated amoeboid state. More profoundly, we discovered that TREM1 not only regulates the \u0026ldquo;quantity\u0026rdquo; (number of activated cells) of microglia but also determines their \u0026ldquo;quality\u0026rdquo; (functional phenotype).\u003c/p\u003e\u003cp\u003eImbalance between M1 and M2 phenotypes constitutes the molecular basis of neuroinflammatory injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].Our data demonstrate that TREM1 overexpression exacerbates proinflammatory cytokine IL-6 release while suppressing anti-inflammatory cytokine IL-13 production, tilting the inflammatory balance toward the destructive M1 phenotype. Conversely, TREM1 knockdown effectively reverses this imbalance, promoting a shift toward the reparative M2 phenotype in the inflammatory microenvironment. This suggests TREM1 likely serves as an upstream \u0026ldquo;switch\u0026rdquo; for microglial/macrophage phenotype conversion. The underlying mechanism may involve TREM1 activation downstream of the Syk-PI3K-AKT and MAPK signaling pathways, thereby potently amplifying NF-κB transcriptional activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].NF-κB serves as a key transcription factor for numerous proinflammatory genes associated with the M1 phenotype, while its regulatory influence on M2-related genes is relatively weaker [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].Thus, TREM1 inhibition may suppress M1 polarization by attenuating NF-κB signaling while simultaneously creating a favorable microenvironment for M2 polarization. Furthermore, recent studies suggest that TREM1 drives proinflammatory phenotypes by influencing metabolic reprogramming (e.g., promoting aerobic glycolysis) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e],offering new avenues for future investigations into the precise mechanisms by which TREM1 regulates phenotypic switching\u003c/p\u003e\u003cp\u003eThe most illuminating finding of this study lies in revealing the intrinsic connection between TREM1 and AQP4, a key molecule in cerebral edema. Cerebral edema, particularly vasogenic edema, is one of the primary features of EBI following SAH and is directly correlated with patient prognosis[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].AQP4, the primary aquaporin expressed in astrocytic end-feet, is widely recognized for its dual role in both the formation and resolution of cerebral edema [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e],However, the mechanisms regulating its expression following SAH remain incompletely understood.\u003c/p\u003e\u003cp\u003eWe confirmed at both mRNA and protein levels that TREM1 expression changes are highly synchronized with AQP4. This strong positive correlation suggests a potential direct regulatory pathway between the two. We propose the following potential mechanisms:\u003c/p\u003e\u003cp\u003eFirst, indirect cell-cell communication (paracrine pathway): This is the most likely mechanism. TREM1 is primarily expressed on myeloid cells (e.g., microglia) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].TREM1 activation leads to the release of proinflammatory cytokines such as IL-6 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].Conclusive evidence demonstrates that IL-6 and other inflammatory mediators significantly upregulate AQP4 expression in astrocytes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].Thus, we hypothesize a paracrine signaling axis: \u0026ldquo;glial TREM1 activation \u0026rarr; IL-6 release \u0026rarr; action on astrocytes \u0026rarr; AQP4 upregulation \u0026rarr; exacerbated cerebral edema.\u0026rdquo;\u003c/p\u003e\u003cp\u003eSecond, potential direct regulation (autocrine/other pathways): Although less frequently reported, it cannot be ruled out that some astrocytes may also express TREM1 under pathological conditions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].If so, TREM1 could directly regulate its own AQP4 expression via autocrine mechanisms. Furthermore, TREM1 activates downstream signaling pathways such as NF-κB [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e],which itself is a key transcriptional regulator of AQP4, suggesting potential shared transcriptional regulatory networks.\u003c/p\u003e\u003cp\u003eThis study also observed TREM1 regulation of the astrocyte marker GFAP, further supporting TREM1's profound influence on astrocytes within the neurovascular unit. Therefore, we propose for the first time that the \u0026ldquo;TREM1-neuroinflammation-AQP4\u0026rdquo; axis represents a core link connecting excessive immune responses to vasogenic cerebral edema following SAH. This proposed axis offers an integrated perspective for understanding the complexity of brain injury after SAH, revealing that inflammation and edema are not independent parallel pathways but rather a unified pathological process tightly intertwined and mutually amplified through the critical node of TREM1.\u003c/p\u003e\u003cp\u003eAdmittedly, this study has certain limitations, which also represent directions for future research.\u003c/p\u003e\u003cp\u003eFirst, while the AAV2 vector we employed efficiently infected multiple CNS cell types, it lacked cell specificity. Consequently, we could not precisely determine whether TREM1 in the SAH pathological context primarily originates from microglia, infiltrating macrophages, or partially activated astrocytes. Future studies utilizing Cx3cr1-CreERT2 (glia/macrophage-specific) or GFAP-Cre (astrocyte-specific) mice crossed with TREM1 floxed mice to generate cell-specific knockout models will enable precise analysis of the relative contributions of TREM1 from different cellular origins.\u003c/p\u003e\u003cp\u003eSecond, the absence of TREM1/Iba1/GFAP/AQP4 immunofluorescence multiplex labeling precludes direct morphological evidence of spatial proximity between TREM1-positive and AQP4-positive cells. Future confocal microscopy analysis will help validate our proposed paracrine hypothesis.\u003c/p\u003e\u003cp\u003eThird, the direct downstream signaling pathway by which TREM1 regulates AQP4 expression remains unelucidated. Does it involve the classical Syk-PI3K-AKT-NF-κB pathway, or does it incorporate other unknown signaling molecules? Utilizing an in vitro co-culture system of astrocytes and microglia, combined with specific pathway inhibitors, will enable detailed dissection of this regulatory network.\u003c/p\u003e\u003cp\u003eFinally, from a translational medicine perspective, exploring the therapeutic efficacy of specific TREM1-targeting inhibitors (such as the known soluble TREM1 antagonist peptide LR12 or developing neutralizing antibodies) during different time windows after SAH (e.g., the EBI phase or DCI phase) is an essential step toward clinical application. Assessing synergistic effects with existing therapies (e.g., nimodipine) also holds significant clinical value.\u003c/p\u003e\u003cp\u003eIn summary, this study establishes TREM1 as a core driver in the pathological progression of SAH. It coordinates microglia-mediated neuroinflammation, disrupts cytokine homeostasis, and innovatively upregulates astrocytic AQP4 expression\u0026mdash;collectively forming a vicious cycle that exacerbates brain injury. The proposed \u0026ldquo;TREM1-Neuroinflammation-AQP4\u0026rdquo; axis offers a novel integrated explanation for the pathophysiology of SAH.\u003c/p\u003e\u003cp\u003eFrom a therapeutic perspective, targeting TREM1 presents unique advantages: it holds promise for dual intervention through a single target\u0026mdash;simultaneously alleviating neuroinflammation and reducing vasogenic cerebral edema. This \u0026ldquo;two birds with one stone\u0026rdquo; effect may yield greater neuroprotective benefits and a broader therapeutic time window compared to traditional therapies targeting single pathological pathways. Consequently, TREM1 represents a highly promising therapeutic target with significant translational potential. Developing specific inhibitors against it holds the promise of opening a hopeful new avenue for improving the prognosis of patients with SAH, a critical cerebrovascular disease.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, this study establishes TREM1 as a central player in the pathological progression of subarachnoid hemorrhage (SAH). It mediates neuroinflammation primarily driven by microglia and potentially exacerbates cerebral edema by upregulating AQP4, ultimately leading to neuronal death and neurological deficits. Targeting TREM1 inhibition holds promise as a novel combined therapeutic strategy to improve SAH prognosis by simultaneously alleviating inflammation and reducing edema.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJianxin Chen, Junli Zhang, Wenjing Ning\u003c/p\u003e\n\u003cp\u003eAll authors made a considerable contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be submitted for publication; have agreed on the journal to which the article has been submit ted; and agree to be accountable for all aspects of the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Jinan Health Commission Science and Technology Development Plan (No. 2024301006); the Jinan Health Commission Science and Technology Development Plan\u0026nbsp;(No. 2025301007); and the Taian Science and Technology Development Plan (No. 2023NS164)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures in this study strictly adhered to national regulations governing the management and use of laboratory animals. They were reviewed and approved by the Animal Ethics Committee of Jinan First People\u0026apos;s Hospital (Approval No. 2025-03-01-17), maximizing compliance with the 3Rs principle (reduction, replacement, refinement) to minimize both the number of animals used and their suffering.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClaassen, J. and S. Park, \u003cem\u003eSpontaneous subarachnoid haemorrhage\u003c/em\u003e. Lancet, 2022. 400(10355): p. 846\u0026ndash;862.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacdonald, R.L. and T.A. Schweizer, \u003cem\u003eSpontaneous subarachnoid haemorrhage.\u003c/em\u003e Lancet, 2017. 389(10069): p. 655\u0026ndash;666.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Gijn, J., R.S. 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Huang, and S. Ye, \u003cem\u003eTranscription factor Yy1 modulates Trem1 to control LPS-triggered neuroinflammation and oxidative stress in mouse astrocytes via the NF-κB pathway\u003c/em\u003e. Gen Physiol Biophys, 2025. 44(1): p. 81\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, S., Z. Fan, X. Liu, L. Wang, and Z. Ge, \u003cem\u003eMicroglia TREM1-mediated neuroinflammation contributes to central sensitization via the NF-κB pathway in a chronic migraine model\u003c/em\u003e. J Headache Pain, 2024. 25(1): p. 3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Subarachnoid hemorrhage, TREM1, AQP4, neuroinflammation, cerebral edema.","lastPublishedDoi":"10.21203/rs.3.rs-7741776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7741776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSecondary brain injury following subarachnoid hemorrhage (SAH) is a major factor contributing to poor patient outcomes, with neuroinflammation and cerebral edema representing core pathological mechanisms. Triggering receptor expressed on myeloid cells-1 (TREM1), a major inflammatory amplifier in innate immunity, remains poorly understood in SAH regarding its specific role, cellular targets, and association with aquaporin-4 (AQP4), a key cerebral edema molecule.\u003c/p\u003e\n\u003cp\u003eTo elucidate the regulatory role of TREM1 in neuroinflammatory injury following experimental SAH and to investigate whether it functions by driving microglial activation and modulating AQP4 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn SAH model was established in rats via internal carotid artery puncture. TREM1 expression was specifically upregulated or downregulated in vivo through lateral ventricle injection of adeno-associated virus (AAV). Animals were randomly assigned to sham surgery, SAH empty vector control, SAH TREM1 overexpression, and SAH TREM1 knockdown groups. Twenty-four hours post-SAH, neurological function was assessed using the modified Garcia score and balance beam test; brain water content was measured by dry-wet weight method; HE staining was used to observe neuronal morphology; Western Blot and real-time quantitative PCR (qRT-PCR) were employed to detect TREM1, Iba1, GFAP, AQP4, NeuN, Cleaved Caspase-3, IL-6, and IL-13 expression; Immunofluorescence staining was performed for localization and semi-quantitative analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing subarachnoid hemorrhage (SAH), TREM1 expression is significantly upregulated in brain tissue, with its levels negatively correlated with neurological deficits. Functional and molecular studies demonstrate that TREM1 inhibition improves neurological function, reduces cerebral edema, and mitigates neuronal apoptosis, whereas overexpression exacerbates injury. Mechanistic studies reveal that TREM1 exacerbates secondary brain injury by promoting microglial hyperactivation and inflammatory responses, while simultaneously upregulating astrocytic AQP4 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study first reveals the TREM1-AQP4 axis as a critical bridge linking neuroinflammation and cerebral edema after SAH. TREM1 exacerbates brain injury by driving microglia-mediated neuroinflammation and upregulating astrocytic AQP4 expression. Targeting TREM1 inhibition holds promise as a novel therapeutic strategy to improve SAH prognosis by simultaneously alleviating inflammation and reducing edema.\u003c/p\u003e","manuscriptTitle":"The TREM1-AQP4 Axis Mediates Neuroinflammatory Injury and Brain Edema after Experimental Subarachnoid Hemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 14:46:41","doi":"10.21203/rs.3.rs-7741776/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T08:51:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T04:33:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T14:59:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169370821329668006806549010942048707791","date":"2025-10-09T12:50:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T05:52:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180266761457748152266183685887621034149","date":"2025-10-07T08:29:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39144496673733968663950162096745608148","date":"2025-10-07T06:10:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T05:51:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T13:45:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T13:32:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2025-09-29T11:51:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c882334-37cd-4218-a32d-23bfc47bb38f","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:11:26+00:00","versionOfRecord":{"articleIdentity":"rs-7741776","link":"https://doi.org/10.1186/s40001-025-03462-x","journal":{"identity":"european-journal-of-medical-research","isVorOnly":false,"title":"European Journal of Medical Research"},"publishedOn":"2025-11-26 15:57:24","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-10-22 14:46:41","video":"","vorDoi":"10.1186/s40001-025-03462-x","vorDoiUrl":"https://doi.org/10.1186/s40001-025-03462-x","workflowStages":[]},"version":"v1","identity":"rs-7741776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7741776","identity":"rs-7741776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}