Optimization of a Fresh Fecal Intraperitoneal Injection Sepsis Model and Its Divergent Dynamics from Cecal Ligation and Puncture in Mice

Optimization of a Fresh Fecal Intraperitoneal Injection Sepsis Model and Its Divergent Dynamics from Cecal Ligation and Puncture in Mice | 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 Optimization of a Fresh Fecal Intraperitoneal Injection Sepsis Model and Its Divergent Dynamics from Cecal Ligation and Puncture in Mice Lixiang Zhao, Zhiwen Wu, Zetian Zhong, Xiaoling Lu, Ruonan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9124307/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Sepsis remains a critical challenge in intensive care, necessitating reliable animal models that accurately mimic human pathophysiological responses. While cecal ligation and puncture (CLP) is widely considered the gold standard, its inherent variability often limits reproducibility. This study aimed to optimize a fecal intraperitoneal injection (FIP) murine model by evaluating the impact of fecal preparation (fresh vs. lyophilized) and dosage (0.5–1.0 g/kg) on model stability. We systematically compared the optimized FIP model with the conventional CLP method in male BALB/c mice to define their respective pathophysiological characteristics and suitability for therapeutic screening. Results Our findings demonstrate that fresh fecal suspensions significantly enhance model reproducibility compared to dried preparations, which showed inconsistent virulence. An optimized FIP dose of 0.7 g/kg induced a hyperacute sepsis phenotype, characterized by rapid systemic bacterial dissemination and significant acute lung and kidney injury within 24 hours. In contrast, the CLP model exhibited a more protracted progression of organ dysfunction, with more pronounced and sustained intestinal mucosal damage and evolving infectious dynamics. Hematological analysis confirmed that while both models induced systemic inflammation, the FIP model provided a more synchronized and predictable onset of multi-organ failure. Conclusions The optimized FIP model, characterized by its procedural simplicity, high controllability, and superior reproducibility, serves as a robust platform for investigating the early, fulminant pathophysiological mechanisms of unmitigated sepsis. Conversely, the CLP model remains the preferred choice for studies focusing on protracted infection and chronic organ dysfunction. These findings provide a methodological framework for selecting appropriate sepsis models based on specific research objectives in experimental medicine. Sepsis Fresh fecal suspension Fecal intraperitoneal injection Cecal ligation and puncture Animal model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Sepsis is characterized by life-threatening organ dysfunction resulting from a dysregulated host response to infection [ 1 ]. This complex clinical syndrome involves intertwined physiological, pathological, and biochemical disturbances. Despite advancements in supportive care, sepsis imposes a substantial global burden, with approximately 48.9 million cases and 11.0 million deaths annually [ 2 ]. Effective therapies remain limited, necessitating reliable animal models to elucidate disease mechanisms and translate interventions into clinical practice [ 3 ]. Experimental sepsis models primarily include exogenous endotoxin administration (e.g., lipopolysaccharide, LPS), exogenous live pathogen inoculation (e.g., fecal intraperitoneal injection, FIP), and endogenous host barrier disruption (e.g., cecal ligation and puncture, CLP) [ 4 ]. While LPS models are highly reproducible, they fail to mimic the polymicrobial nature or the full clinical course of human sepsis [ 5 ]. Similarly, CLP is considered the gold standard but suffers from high inter-operator variability and challenges in standardization [ 6 , 7 ]. In contrast, FIP offers operational simplicity, high standardization potential, and the ability to induce polymicrobial sepsis. However, its implementation is often hindered by a lack of unified protocols for fecal preparation and dosing. Crucially, while the Minimum Quality Threshold in Pre-clinical Sepsis Studies (MQTiPSS) emphasizes the importance of clinical mimicry [ 8 ], the foundational reliability of the induction method itself remains the primary bottleneck to achieving these standards. To address these limitations, the present study systematically optimized the FIP procedure—including fecal processing, filtration, and standardized dosing—to establish a stable protocol. We further conducted a multi-dimensional comparison between this optimized FIP model and the traditional CLP model to provide a rational basis for model selection in sepsis research. Methods Experimental Animals Specific pathogen-free (SPF) male BALB/c mice (8–10 weeks old; 25–30 g) were purchased from the Guangdong Medical Laboratory Animal Center (license SCXK (Yue) 2020-0051) and housed under previously described conditions [ 9 ]. Briefly, animals were acclimated for one week under SPF conditions, maintained at a constant temperature of 22 ± 2°C and 50–60% humidity, with a 12-hour light/dark cycle. They were provided with standard laboratory chow and water ad libitum. All experimental procedures and this study were reported in accordance with ARRIVE guidelines. The study was approved by the Ethics Committee for Animal Experiments of Zunyi Medical University (approval no. ZHSC-2-[2024]078). All methods were performed in accordance with the relevant guidelines and regulations. At the designated time points (24, 48, or 72 h) or upon reaching humane endpoints, mice were euthanized by cervical dislocation under deep anesthesia with Zoletil 50 (Virbac, Carros, France) to minimize suffering. Establishment of the CLP model The CLP procedure was performed according to previously described methods [ 10 ]. Briefly, mice were fasted for 12 h and anesthetized with Zoletil 50 (Virbac, Carros, France). Following a ~ 1 cm midline laparotomy, the distal half of the cecum was ligated with a 4 − 0 suture and punctured twice with a 21-gauge needle. A minimal amount of fecal content was extruded to ensure patency. Following closure, mice received subcutaneous fluid resuscitation (37°C sterile saline, 5 mL/100 g). Sham-operated mice underwent the same surgical steps excluding ligation and puncture. To minimize inter-operator variability, all procedures were performed by a single investigator. Establishment of the Optimized FIP model To minimize variability driven by moisture content and circadian rhythms, feces were collected between 9:00 and 11:00 AM. To further minimize inter-individual variability in gut microbiota, healthy donor mice of the same batch, sex, and age were selected. Donors were placed in empty cages lined with sterile filter paper to collect fresh feces excreted within a strict 30-min window. Fecal pellets that were urine-contaminated, discolored, or desiccated were excluded; only moist, well-formed pellets were retained. Subsequently, all eligible samples were pooled in a sterile dish and gently mixed to achieve a homogenized microbial composition, thereby eliminating specific deviations from individual mice. We compared suspensions derived from oven-dried (37°C) feces versus fresh feces. Crude suspensions were prepared in sterile saline to target doses of 0.5–0.8 g/kg. To ensure homogeneity, a two-step filtration was employed: a 0.5 mm mesh followed by a 70-µm mesh. Filtrates were maintained on ice and used within 2 h. Mice received intraperitoneal injections at a fixed volume of 10 mL/kg. Control animals (normal saline [NS] group) received an equivalent volume of sterile saline. In this baseline optimization study, additional fluid resuscitation and analgesics were not administered to FIP mice to isolate the pathophysiological effects of the bacterial challenge without pharmacological confounders. Monitoring and Assessment Sepsis induction was confirmed by clinical manifestations, including piloerection, reduced activity, and labored breathing. Survival was monitored at 12-hour intervals, while disease severity was evaluated using the Murine Sepsis Score (MSS). As detailed in Supplementary Table S1 , this scoring system assesses several clinical parameters, such as appearance, level of consciousness, activity, response to stimuli, ocular discharge, and respiratory quality [ 11 , 12 ]. Bacterial Burden and Biochemical Analysis Bacterial loads in whole blood and peritoneal lavage fluid were quantified via colony-forming unit (CFU) counts on blood agar plates (Huankai Microbial, Guangzhou, China) after 24 h of incubation at 37℃. Serum biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr), were measured using a cobas 8000-c701 automated biochemical analyzer (Roche Diagnostics, Basel, Switzerland). Complete blood counts, encompassing white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MON), and platelets (PLT), were determined using a Mindray BC-7500 series automated hematology analyzer (Mindray, Shenzhen, China). Histopathology Tissues (heart, liver, spleen, lung, kidney, and small intestine) were collected from experimental mice, fixed in 4% paraformaldehyde, paraffin-embedded, and stained with hematoxylin and eosin (H&E; Servicebio, Wuhan, China). Sections were evaluated by light microscopy for pathological changes. Statistical analysis Statistical analyses were performed using SPSS version 29.0 (IBM Corp., Armonk, NY, USA). Normality was assessed prior to hypothesis testing. Survival rates were evaluated using the Log-rank (Mantel-Cox) test. Continuous variables with normal distribution are expressed as mean ± standard deviation (SD) and were compared using one-way ANOVA followed by Dunnett’s post hoc test. Discrete or non-normally distributed data, such as the Murine Sepsis Score (MSS), are presented as median with interquartile range (IQR); these were analyzed using the Kruskal-Wallis test (or repeated measures ANOVA on ranks) with Dunn’s test for post hoc comparisons. A two-sided P < 0.05 was considered statistically significant. Results Impact of Fecal State on Model Reproducibility The standardization of the inoculum substrate is a fundamental prerequisite for ensuring the consistency and physiological relevance of the fecal intraperitoneal injection (FIP) model. To determine the influence of fecal state on reproducibility, survival outcomes were evaluated following the administration of either oven-dried or fresh fecal suspensions. In experiments utilizing dried fecal preparations, survival curves demonstrated substantial variability even at identical doses (0.5–0.8 g/kg) (Fig. 1 a, b). This poor reproducibility was likely associated with altered microbial viability and reduced suspension uniformity, which manifested operationally as particle aggregation and frequent syringe clogging. Conversely, suspensions prepared from fresh feces yielded highly consistent survival profiles across independent experimental trials (Fig. 1 c, d). Consequently, fresh fecal suspensions were adopted for all subsequent standardized FIP modeling procedures. Identification of an optimal FIP dose and comparison with CLP Establishing a precise dose-response relationship is essential for facilitating meaningful head-to-head comparisons between the optimized FIP model and the established cecal ligation and puncture (CLP) protocol. To identify an optimal induction dose, survival was monitored across a dose range of fresh fecal suspensions (0.5–1.0 g/kg) (Fig. 2 a). A clear dose-response relationship was observed; all mice survived at a dose of 0.5 g/kg, whereas complete mortality occurred within 24 h at doses ≥ 0.8 g/kg (Fig. 2 a). The survival trajectory of the 0.7 g/kg FIP group most closely resembled that of the CLP group, leading to its selection as the standard dose for comparative evaluation. Monitoring of the murine sepsis score (MSS) revealed that the FIP group consistently exhibited higher disease severity than the CLP group at 12, 24, and 36 h (P < 0.05) (Fig. 2 b). Furthermore, significant body weight reductions were recorded in the CLP group at 24 and 48 h (P < 0.05) and in the 0.7 g/kg FIP group (P < 0.05) compared to their respective controls (Fig. 2 c). Comparison of Bacterial Loads in Blood and Peritoneal Lavage Fluid Quantification of bacterial dissemination serves as a direct indicator of infectious severity and the efficacy of host clearance mechanisms across different modeling strategies. Substantial bacterial dissemination was observed in both models, with peritoneal bacterial loads (10 7 –10 8 CFU/mL) consistently exceeding circulating concentrations. In the blood, bacterial burdens were significantly increased in the CLP group compared with sham controls ( P < 0.05) and in the FIP group compared with NS controls ( P < 0.05) at both 24 and 48 h. However, blood bacterial loads were significantly higher in the FIP group than in the CLP group at these same time points (P < 0.05) (Fig. 3 a). This disparity likely stems from the different infection dynamics of the two models: FIP involves an immediate, high-density bacterial bolus injection, leading to rapid systemic translocation, whereas CLP represents a progressively evolving infection through gradual leakage from the punctured cecum. This characteristic suggests that the optimized FIP model is particularly effective for simulating acute, fulminant sepsis. Furthermore, peritoneal lavage bacterial loads were significantly elevated in the CLP group relative to sham controls ( P < 0.05) and in the FIP group relative to NS controls (P < 0.05) (Fig. 3 b). Comparison between the models indicated that peritoneal bacterial loads were significantly higher in the FIP group than in the CLP group at 24 h (P < 0.05) but became significantly lower by 48 h (P < 0.05) (Fig. 3 b). This crossover likely reflects the host’s immune clearance of the initial FIP bolus, contrasted with the persistent microbial influx from the unsealed cecal leak in the CLP model, which serves as a continuous source of infection. Systemic Inflammatory Response and Organ Dysfunction The characterization of hematological and biochemical markers is necessary to validate systemic injury and to delineate the extent of multi-organ failure induced by sepsis. To this end, hematological parameters, including WBC, NEU, LYM, and MON counts, were analyzed to evaluate systemic inflammation and immune status, while serum biochemical markers, specifically ALT and AST for hepatic function and BUN and Scr for renal function, were measured to assess organ-specific injury. Both models significantly altered peripheral blood cell counts, including a marked reduction in WBC counts in CLP mice at 48 h (P < 0.05) and in FIP mice at all assessed time points (P < 0.05) (Fig. 4 a). Significant increases in NEU counts at 24 h (P < 0.05), suppression of LYM counts (P < 0.05), and elevations in MON counts at 24 h (P < 0.05) were observed in both groups, with FIP mice exhibiting significantly higher MON counts than CLP mice at both 24 and 48 h (P < 0.05) (Fig. 4 b, c, d). PLT counts were also significantly decreased in both models (P < 0.05) (Fig. 4 e). Serum biochemical analysis revealed significant elevations in ALT, AST, BUN, and Scr in both models compared to controls (P < 0.05) (Fig. 4 f-i). Notably, Scr was significantly lower in FIP mice than in CLP mice at 24 h (P < 0.05), whereas ALT and AST levels were significantly higher in the CLP group than in the FIP group by 48 h (P < 0.05) (Fig. 4 f-i). Taken together, these findings indicate that the optimized FIP model induces a more rapid and acute systemic inflammatory response and hematological disruption. Conversely, the CLP model leads to more progressive and sustained hepatic and renal impairment, reflecting the different pathological dynamics of a single-bolus infection versus a continuous infectious leak. Multi-organ Histopathological Injury Histopathological evaluation of major organs, including the heart, liver, lung, kidney, and intestine, provides direct morphological evidence of systemic tissue damage. H&E staining revealed characteristic multi-organ injuries in both models (Fig. 5 and Fig. 6 ). In the heart, we observed myocardial fiber fragmentation and interstitial edema. In the liver, significant hepatocellular swelling and vacuolar degeneration were evident. Myocardial and hepatic injuries were more prominent during the acute phase (24 h) in the FIP group, whereas they appeared more severe and persistent at 48 h in the CLP group. Pulmonary pathology in the FIP group was characterized by early-onset alveolar wall thickening and inflammatory infiltration at 24 h; in contrast, lung injury in the CLP group was more protracted. Renal damage, manifested by glomerular shrinkage and acute tubular necrosis, progressed over time in the CLP group and peaked at 48 h, while FIP induced substantial injury as early as 24 h. Furthermore, intestinal injury—consisting of mucosal edema, villi blunting, and epithelial necrosis—tended to be more severe and sustained in the CLP group, likely driven by the continuous leakage of infectious material from the cecum. Discussion In the present study, an optimized murine model of polymicrobial sepsis was established and systematically evaluated using fecal intraperitoneal injection. Our central findings indicate that a standardized fresh fecal suspension administered at a dose of 0.7 g/kg generates key outcomes—including mortality, multi-organ dysfunction, and systemic bacterial burdens—that are broadly comparable to those produced by the conventional CLP model. Crucially, the optimized protocol demonstrates substantial advantages in terms of reproducibility, procedural simplicity, and controllability of disease severity, thereby supporting its utility as a reliable and efficient preclinical platform for sepsis research and therapeutic evaluation. Although CLP is widely regarded as the gold standard for modeling polymicrobial sepsis, its primary limitation remains high inter-individual variability and a strong dependency on surgical technique. In the present study, mice in the CLP group exhibited pronounced disease severity. This observation aligns with the findings of Jain et al., who reported that mortality in the CLP control group reached 100% within 24 h in the absence of effective therapeutic intervention [ 10 ]. Such high lethality further underscores the stringency of the CLP model in simulating severe sepsis and assessing pharmacological efficacy. However, consistency in outcomes is often complicated by variations in the extent of ischemic necrosis in the ligated cecal segment [ 13 ]. Furthermore, variability arises from multiple factors that are difficult to standardize, such as the exact ligation position, needle gauge, number of punctures, and operator proficiency, all of which directly determine the initial infectious burden. Additionally, anatomical and local pathological differences among mice can lead to the partial occlusion of puncture sites by adjacent tissues, thereby altering the leakage dynamics of infectious material [ 14 ]. These complex local interactions collectively make the strict standardization of the CLP model challenging. By contrast, the optimized FIP approach mitigates these sources of variability through the systematic control of the infectious source, the preparation process, and the dosing strategy. First, the immediate collection of fresh feces from donor mice maximizes the preservation of microbial viability and the community complexity, which more faithfully models polymicrobial infection while remaining ethically compatible [ 15 ]. Second, the implementation of a two-step filtration workflow—utilizing a 0.5 mm mesh followed by 70 µm filtration—improves suspension homogeneity and injectability. This refinement significantly reduces the risk of syringe clogging and potential particulate-related complications, thereby increasing overall procedural reliability. Third, graded dosing experiments identified 0.7 g/kg as an optimal dose that yields moderate mortality (approximately 50%) and a severity comparable to CLP, thus providing a practical therapeutic intervention window. Regarding disease assessment, the Murine Sepsis Score (MSS) effectively differentiates healthy from septic states and captures temporal changes in severity; however, its predictive value is intrinsically limited. Notably, a subset of mice died before reaching peak MSS, a finding consistent with multicenter reports of fecal-induced peritonitis models [ 16 ]. This likely reflects fatal physiological derangements, such as malignant arrhythmias, abrupt hypotension, or catastrophic homeostatic collapse, which can precede the overt behavioral deterioration captured by the MSS [ 17 ]. Therefore, while the MSS remains a valuable tool, it should be integrated with objective laboratory indices and, where feasible, continuous physiological monitoring to improve phenotyping accuracy in sepsis studies. Beyond procedural differences, our data suggest that FIP and CLP model distinct sepsis trajectories. The CLP model establishes a persistent infectious focus with ongoing leakage, thereby mimicking sepsis driven by unresolved source control. Consistently, CLP produces a more protracted clinical course characterized by sustained or worsening organ injury at later time points [ 18 ]. In contrast, the FIP model delivers a single, high-load polymicrobial challenge, leading to earlier bacteremia peaks and rapid multi-organ injury within 24 h. Notably, the FIP group exhibited lung edema and inflammatory infiltration as early as 24 h, whereas CLP-induced damage was more protracted. This rapid onset aligns with the biological vulnerability of the lung as the most critical organ during sepsis [ 19 ]. Our observation of sustained high bacterial loads contrasts with reports of declining counts in similar fresh-fecal models [ 20 ]. This discrepancy is likely attributable to differences in therapeutic intervention and animal species. Tallósy et al. utilized a rat model and administered fluid resuscitation and analgesics following induction, interventions known to bolster hemodynamic stability and facilitate host immune clearance of pathogens. In contrast, our study employed a mouse model without fluid resuscitation to establish a baseline of unmitigated sepsis. Consequently, the compromised host defense in our model likely failed to limit bacterial proliferation, resulting in persistently elevated peritoneal bacterial burdens typical of fulminant, untreated sepsis. Accordingly, CLP and FIP should be viewed as complementary rather than interchangeable models: FIP may be preferable for studying early inflammatory storms and rapid innate immune activation (supported by the acute hematological shifts in Fig. 4 ), whereas CLP is more suitable for investigating sustained infection, sepsis-associated immunosuppression, prolonged organ dysfunction, or secondary infections (consistent with the progressive hepatic and renal damage observed at 48 h in Fig. 4 and Fig. 6 ). Multi-dimensional evaluation further confirmed that the optimized FIP model successfully reproduces the core features of sepsis, including systemic bacterial dissemination, organ dysfunction (indicated by elevated ALT, AST [ 21 ], BUN, and Scr [ 22 ]), and multi-organ histopathological injury. These concordant functional and morphological abnormalities support the construct validity of the optimized FIP model as a clinically relevant tool for polymicrobial sepsis research. However, several limitations should be acknowledged. First, to isolate the variability of the induction method itself, this study purposefully excluded therapeutic interventions (e.g., fluid resuscitation, antibiotics). While necessary for standardization, this absence of circulatory support means the model represents an unmitigated, fulminant sepsis trajectory, differing from the managed clinical course seen in human patients. Second, regarding histopathology, our evaluation was restricted to qualitative morphological description to confirm the presence of organ injury, rather than applying semi-quantitative scoring systems for each organ. Third, the study was limited to young male mice and quantitative CFU analysis. Future research should validate this protocol in aged and female cohorts and incorporate metagenomic sequencing to better characterize the specific 'sepsis-inducing' microbiome components. Conclusions Collectively, this study successfully establishes and validates a highly standardized and reproducible fresh FIP murine model of polymicrobial sepsis. Characterized by operational simplicity and tunable severity, this model offers a robust and cost-effective platform for investigating the complex pathophysiology of moderately severe sepsis. Furthermore, by delineating the fundamental temporal and pathological divergence between the FIP and CLP models, these findings provide critical practical guidance for selecting the most appropriate experimental framework based on specific research objectives. Abbreviations ALT Alanine aminotransferase AST Aspartate aminotransferase BUN Blood urea nitrogen CFU Colony forming unit CLP Cecal ligation and puncture FIP Fecal intraperitoneal injection LPS Lipopolysaccharide LYM Lymphocyte MON Monocyte MSS Murine Sepsis Score NEU Neutrophil PCF Peritoneal cavity fluid PLT Platelet Scr Serum creatinine WBC White blood cell Declarations Ethics approval and consent to participate All experimental procedures and this study were reported in accordance with ARRIVE guidelines. The animal study protocol was approved by the Academic Ethics Committee of Zhuhai Campus of Zunyi Medical University (Protocol No: ZHSC-2-[2024]078, approved on 26 September 2024). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was funded by the Zhuhai City Medical Research Project of Guangdong Province (Grant No. 2520009000090). Authors’ contributions Y.L. and X.Y. conceptualized and supervised the study. L.Z. contributed to the methodology, software, formal analysis, and visualization. L.Z., Z.W., and Z.Z. performed the validation. L.L. conducted the investigation. Y.L. provided resources. L.Z., Z.W., X.L., and L.L. curated the data. L.Z. and R.Z. drafted the original manuscript. Y.L. reviewed and edited the manuscript. X.Y. administered the project. All authors read and approved the final manuscript. Acknowledgements Not applicable. Author’s information Not applicable. References Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 2016;315(8):801–10. Meyer NJ, Prescott HC. Sepsis and septic shock. N Engl J Med. 2024;391(22):2133–46. Huang M, Cai S, Su J. 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The microbial composition of the initial insult can predict the prognosis of experimental sepsis. Sci Rep. 2021;11(1):22772. Jin GL, Liu HP, Huang YX, et al. Koumine regulates macrophage M1/M2 polarization via TSPO, alleviating sepsis-associated liver injury in mice. Phytomedicine. 2022;107:154484. Luo M, Zhu Q, Xu G, et al. Intervention effect of curcumin on sepsis-associated acute kidney injury via regulation of p300 expression and protein lactylation. BMC Immunol. 2025;26(1):67. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx File name: Supplementary_Material File format: .docx Title of data: Table S1. Murine Sepsis Score (MSS) assessment criteria Description of data: This table details the clinical parameters and the scoring system utilized to evaluate disease severity in the murine sepsis models. 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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-9124307","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614969180,"identity":"cc2f98f4-49c9-4d82-bf82-670085a285a9","order_by":0,"name":"Lixiang Zhao","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lixiang","middleName":"","lastName":"Zhao","suffix":""},{"id":614969181,"identity":"52eb1884-67f5-4f0a-819d-fe8fa5bf8f9c","order_by":1,"name":"Zhiwen Wu","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiwen","middleName":"","lastName":"Wu","suffix":""},{"id":614969182,"identity":"66107535-5247-4b14-8801-f2cffe6b7d34","order_by":2,"name":"Zetian Zhong","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zetian","middleName":"","lastName":"Zhong","suffix":""},{"id":614969183,"identity":"2ecba22e-380d-4c38-8cb3-555ca923e208","order_by":3,"name":"Xiaoling Lu","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Lu","suffix":""},{"id":614969184,"identity":"0d4a5ad1-d2ce-4995-8fb6-064eeb73bf56","order_by":4,"name":"Ruonan Zhang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruonan","middleName":"","lastName":"Zhang","suffix":""},{"id":614969185,"identity":"2c89f564-4907-4c97-9408-53db459da9d7","order_by":5,"name":"Li Luo","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Luo","suffix":""},{"id":614969186,"identity":"de61b05a-dde5-459e-ba34-84eadfadc23f","order_by":6,"name":"Yanxin Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYLCCBAYLBn4Ik5loLRIMkg0kaWEAajE4QKwWg+Nnj0k83CGRuPn86TQJhgrrxAb2swfwazmTl2yQeEYicduBs9skGM6kJzbw5CXg13Igx/BBYhtQy8HebRKMbYcTGyR4DPBrOf/G4ABIy+ZmXqCWf8RouQG1ZQMbSEsDEVokb7wxNgBqMZ5xhnezRcKxdOM2nhz8WvjO55hJ/myzke3vP7vxxocaa9l+9jP4tSgcQOYlADEbXvVAIN9ASMUoGAWjYBSMAgCnt0Y+q5r6+AAAAABJRU5ErkJggg==","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yanxin","middleName":"","lastName":"Lu","suffix":""},{"id":614969187,"identity":"f4014bbc-7420-4ccf-b394-81dcfbde4793","order_by":7,"name":"Xupeng Yue","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xupeng","middleName":"","lastName":"Yue","suffix":""}],"badges":[],"createdAt":"2026-03-14 17:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9124307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9124307/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106094100,"identity":"a8bb495f-d0d1-4acd-803d-db6d941f20b4","added_by":"auto","created_at":"2026-04-03 11:40:59","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214369,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of model reproducibility between dry and fresh fecal suspensions in the fecal intraperitoneal injection (FIP) model. a, b Survival curves from two independent experiments using dry fecal suspension. c, d Survival curves from two independent experiments using fresh fecal suspension. Mice were injected at doses of 0.5, 0.6, 0.7, or 0.8 g/kg (n = 6 per group). Survival was monitored for 72 h post-injection. NS: normal saline\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/c3fae0833d7cef54f03dc006.jpeg"},{"id":106094047,"identity":"e5a87a06-415e-4c18-9aef-af6073230e76","added_by":"auto","created_at":"2026-04-03 11:40:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146188,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of survival trajectories, disease severity, and body weight changes between the CLP and optimized fresh FIP models. a Survival curves of mice in Sham, CLP, NS, and FIP groups across a dosage range of fresh fecal suspensions (0.5–1.0 g/kg). b Dynamic assessment of the Murine Sepsis Score (MSS) at 12-h intervals over 72 h. Data for MSS are presented as median with interquartile range (IQR) and analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test. c Changes in body weight at 24 and 48 h post-modeling. \"Endpoint\" refers to the time of euthanasia (24 or 48 h). Data for body weight are presented as mean ± SD and analyzed by one-way ANOVA. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. CLP group at the same time point. \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. Sham group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. NS group; CLP: cecal ligation and puncture; FIP: fecal intraperitoneal injection; NS: normal saline\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/040a767a21aaa74cc8c86179.jpeg"},{"id":106035531,"identity":"b69939ed-fc74-4b7d-9752-2473379c706b","added_by":"auto","created_at":"2026-04-02 16:19:11","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114458,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative bacterial burdens in the systemic circulation and peritoneal cavity at 24 and 48 h post-modeling . a Bacterial loads in whole blood. b Bacterial loads in the peritoneal lavage fluid (PLF). Data are presented as mean ± SD (n = 3). \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. Sham group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. NS group; \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. CLP 24-h group. \u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. CLP 48-h group. CLP: cecal ligation and puncture; FIP: fecal intraperitoneal injection; PLF: peritoneal lavage fluid\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/92a243409e85ac0ee27bad35.jpeg"},{"id":106094086,"identity":"4a6e1090-7c50-47f0-8307-f9fcf581e448","added_by":"auto","created_at":"2026-04-03 11:40:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293661,"visible":true,"origin":"","legend":"\u003cp\u003eHematological parameters and systemic biomarkers of hepatic and renal dysfunction. a-e Complete blood count analysis, including white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MON), and platelets (PLT). f-i Serum biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), and blood urea nitrogen (BUN). Data are presented as mean ± SD (n = 6). \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. Sham group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. NS group; \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. CLP 24-h group; \u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. CLP 48-h group. NS: normal saline; CLP: cecal ligation and puncture; FIP: fecal intraperitoneal injection; NS: normal saline\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/bd4de896e6ae65fb1f5c9f38.jpeg"},{"id":106035532,"identity":"35152c5f-fce8-4864-af07-2d3719907106","added_by":"auto","created_at":"2026-04-02 16:19:11","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1591705,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological evaluation of the heart, liver, and spleen in CLP and FIP sepsis models. Representative hematoxylin and eosin (H\u0026amp;E)-stained sections from Sham, CLP (24 and 48 h), NS, and FIP (24 and 48 h) groups are shown. Arrows highlight characteristic septic injuries, including myocardial fiber disorganization, hepatocellular vacuolar degeneration, and splenic red–white pulp blurring. Scale bar = 50 µm. CLP: cecal ligation and puncture; FIP: fecal intraperitoneal injection; NS: normal saline\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/e61fa4d67e2a7c9d1b1a2ef3.jpeg"},{"id":106035533,"identity":"6d446cb9-da25-4b9f-a90f-470ee8e8c37f","added_by":"auto","created_at":"2026-04-02 16:19:11","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1529779,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological evaluation of the lung, kidney, and intestine in CLP and FIP sepsis models . Representative H\u0026amp;E-stained sections from Sham, CLP (24 and 48 h), NS, and FIP (24 and 48 h) groups are shown. Arrows highlight pathological features such as alveolar septal thickening, tubular epithelial necrosis, and intestinal mucosal sloughing. Scale bar = 50 µm. CLP: cecal ligation and puncture; FIP: fecal intraperitoneal injection; NS: normal saline\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/0845061fe24fd2c835c99454.jpeg"},{"id":106723653,"identity":"c853c08b-e186-4fd1-9282-dfc2b440f7b8","added_by":"auto","created_at":"2026-04-12 18:10:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4613206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/be4f4c9b-3bf4-4e06-9c31-3add4b8ad6eb.pdf"},{"id":106035528,"identity":"27e2abb8-0210-461d-a3e7-48f01c061db5","added_by":"auto","created_at":"2026-04-02 16:19:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFile name:\u003c/strong\u003e Supplementary_Material\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFile format:\u003c/strong\u003e .docx\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTitle of data:\u003c/strong\u003e Table S1. Murine Sepsis Score (MSS) assessment criteria\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of data:\u003c/strong\u003e This table details the clinical parameters and the scoring system utilized to evaluate disease severity in the murine sepsis models.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9124307/v1/a5b4371dbbf600e21511c6ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of a Fresh Fecal Intraperitoneal Injection Sepsis Model and Its Divergent Dynamics from Cecal Ligation and Puncture in Mice","fulltext":[{"header":"Background","content":"\u003cp\u003eSepsis is characterized by life-threatening organ dysfunction resulting from a dysregulated host response to infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This complex clinical syndrome involves intertwined physiological, pathological, and biochemical disturbances. Despite advancements in supportive care, sepsis imposes a substantial global burden, with approximately 48.9\u0026nbsp;million cases and 11.0\u0026nbsp;million deaths annually [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Effective therapies remain limited, necessitating reliable animal models to elucidate disease mechanisms and translate interventions into clinical practice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperimental sepsis models primarily include exogenous endotoxin administration (e.g., lipopolysaccharide, LPS), exogenous live pathogen inoculation (e.g., fecal intraperitoneal injection, FIP), and endogenous host barrier disruption (e.g., cecal ligation and puncture, CLP) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While LPS models are highly reproducible, they fail to mimic the polymicrobial nature or the full clinical course of human sepsis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, CLP is considered the gold standard but suffers from high inter-operator variability and challenges in standardization [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, FIP offers operational simplicity, high standardization potential, and the ability to induce polymicrobial sepsis. However, its implementation is often hindered by a lack of unified protocols for fecal preparation and dosing. Crucially, while the Minimum Quality Threshold in Pre-clinical Sepsis Studies (MQTiPSS) emphasizes the importance of clinical mimicry [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the foundational reliability of the induction method itself remains the primary bottleneck to achieving these standards.\u003c/p\u003e \u003cp\u003eTo address these limitations, the present study systematically optimized the FIP procedure\u0026mdash;including fecal processing, filtration, and standardized dosing\u0026mdash;to establish a stable protocol. We further conducted a multi-dimensional comparison between this optimized FIP model and the traditional CLP model to provide a rational basis for model selection in sepsis research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animals\u003c/h2\u003e \u003cp\u003eSpecific pathogen-free (SPF) male BALB/c mice (8\u0026ndash;10 weeks old; 25\u0026ndash;30 g) were purchased from the Guangdong Medical Laboratory Animal Center (license SCXK (Yue) 2020-0051) and housed under previously described conditions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Briefly, animals were acclimated for one week under SPF conditions, maintained at a constant temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 50\u0026ndash;60% humidity, with a 12-hour light/dark cycle. They were provided with standard laboratory chow and water ad libitum. All experimental procedures and this study were reported in accordance with ARRIVE guidelines. The study was approved by the Ethics Committee for Animal Experiments of Zunyi Medical University (approval no. ZHSC-2-[2024]078). All methods were performed in accordance with the relevant guidelines and regulations. At the designated time points (24, 48, or 72 h) or upon reaching humane endpoints, mice were euthanized by cervical dislocation under deep anesthesia with Zoletil 50 (Virbac, Carros, France) to minimize suffering.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEstablishment of the CLP model\u003c/h3\u003e\n\u003cp\u003eThe CLP procedure was performed according to previously described methods [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, mice were fasted for 12 h and anesthetized with Zoletil 50 (Virbac, Carros, France). Following a\u0026thinsp;~\u0026thinsp;1 cm midline laparotomy, the distal half of the cecum was ligated with a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 suture and punctured twice with a 21-gauge needle. A minimal amount of fecal content was extruded to ensure patency. Following closure, mice received subcutaneous fluid resuscitation (37\u0026deg;C sterile saline, 5 mL/100 g). Sham-operated mice underwent the same surgical steps excluding ligation and puncture. To minimize inter-operator variability, all procedures were performed by a single investigator.\u003c/p\u003e\n\u003ch3\u003eEstablishment of the Optimized FIP model\u003c/h3\u003e\n\u003cp\u003eTo minimize variability driven by moisture content and circadian rhythms, feces were collected between 9:00 and 11:00 AM. To further minimize inter-individual variability in gut microbiota, healthy donor mice of the same batch, sex, and age were selected. Donors were placed in empty cages lined with sterile filter paper to collect fresh feces excreted within a strict 30-min window. Fecal pellets that were urine-contaminated, discolored, or desiccated were excluded; only moist, well-formed pellets were retained. Subsequently, all eligible samples were pooled in a sterile dish and gently mixed to achieve a homogenized microbial composition, thereby eliminating specific deviations from individual mice. We compared suspensions derived from oven-dried (37\u0026deg;C) feces versus fresh feces. Crude suspensions were prepared in sterile saline to target doses of 0.5\u0026ndash;0.8 g/kg.\u003c/p\u003e \u003cp\u003eTo ensure homogeneity, a two-step filtration was employed: a 0.5 mm mesh followed by a 70-\u0026micro;m mesh. Filtrates were maintained on ice and used within 2 h. Mice received intraperitoneal injections at a fixed volume of 10 mL/kg. Control animals (normal saline [NS] group) received an equivalent volume of sterile saline. In this baseline optimization study, additional fluid resuscitation and analgesics were not administered to FIP mice to isolate the pathophysiological effects of the bacterial challenge without pharmacological confounders.\u003c/p\u003e\n\u003ch3\u003eMonitoring and Assessment\u003c/h3\u003e\n\u003cp\u003eSepsis induction was confirmed by clinical manifestations, including piloerection, reduced activity, and labored breathing. Survival was monitored at 12-hour intervals, while disease severity was evaluated using the Murine Sepsis Score (MSS). As detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, this scoring system assesses several clinical parameters, such as appearance, level of consciousness, activity, response to stimuli, ocular discharge, and respiratory quality [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBacterial Burden and Biochemical Analysis\u003c/h3\u003e\n\u003cp\u003eBacterial loads in whole blood and peritoneal lavage fluid were quantified via colony-forming unit (CFU) counts on blood agar plates (Huankai Microbial, Guangzhou, China) after 24 h of incubation at 37℃. Serum biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr), were measured using a cobas 8000-c701 automated biochemical analyzer (Roche Diagnostics, Basel, Switzerland). Complete blood counts, encompassing white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MON), and platelets (PLT), were determined using a Mindray BC-7500 series automated hematology analyzer (Mindray, Shenzhen, China).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eTissues (heart, liver, spleen, lung, kidney, and small intestine) were collected from experimental mice, fixed in 4% paraformaldehyde, paraffin-embedded, and stained with hematoxylin and eosin (H\u0026amp;E; Servicebio, Wuhan, China). Sections were evaluated by light microscopy for pathological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS version 29.0 (IBM Corp., Armonk, NY, USA). Normality was assessed prior to hypothesis testing. Survival rates were evaluated using the Log-rank (Mantel-Cox) test. Continuous variables with normal distribution are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were compared using one-way ANOVA followed by Dunnett\u0026rsquo;s post hoc test. Discrete or non-normally distributed data, such as the Murine Sepsis Score (MSS), are presented as median with interquartile range (IQR); these were analyzed using the Kruskal-Wallis test (or repeated measures ANOVA on ranks) with Dunn\u0026rsquo;s test for post hoc comparisons. A two-sided \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImpact of Fecal State on Model Reproducibility\u003c/h2\u003e \u003cp\u003eThe standardization of the inoculum substrate is a fundamental prerequisite for ensuring the consistency and physiological relevance of the fecal intraperitoneal injection (FIP) model. To determine the influence of fecal state on reproducibility, survival outcomes were evaluated following the administration of either oven-dried or fresh fecal suspensions. In experiments utilizing dried fecal preparations, survival curves demonstrated substantial variability even at identical doses (0.5\u0026ndash;0.8 g/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). This poor reproducibility was likely associated with altered microbial viability and reduced suspension uniformity, which manifested operationally as particle aggregation and frequent syringe clogging. Conversely, suspensions prepared from fresh feces yielded highly consistent survival profiles across independent experimental trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Consequently, fresh fecal suspensions were adopted for all subsequent standardized FIP modeling procedures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of an optimal FIP dose and comparison with CLP\u003c/h2\u003e \u003cp\u003eEstablishing a precise dose-response relationship is essential for facilitating meaningful head-to-head comparisons between the optimized FIP model and the established cecal ligation and puncture (CLP) protocol. To identify an optimal induction dose, survival was monitored across a dose range of fresh fecal suspensions (0.5\u0026ndash;1.0 g/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A clear dose-response relationship was observed; all mice survived at a dose of 0.5 g/kg, whereas complete mortality occurred within 24 h at doses\u0026thinsp;\u0026ge;\u0026thinsp;0.8 g/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The survival trajectory of the 0.7 g/kg FIP group most closely resembled that of the CLP group, leading to its selection as the standard dose for comparative evaluation. Monitoring of the murine sepsis score (MSS) revealed that the FIP group consistently exhibited higher disease severity than the CLP group at 12, 24, and 36 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Furthermore, significant body weight reductions were recorded in the CLP group at 24 and 48 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and in the 0.7 g/kg FIP group \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparison of Bacterial Loads in Blood and Peritoneal Lavage Fluid\u003c/h2\u003e \u003cp\u003eQuantification of bacterial dissemination serves as a direct indicator of infectious severity and the efficacy of host clearance mechanisms across different modeling strategies. Substantial bacterial dissemination was observed in both models, with peritoneal bacterial loads (10\u003csup\u003e7\u003c/sup\u003e\u0026ndash;10\u003csup\u003e8\u003c/sup\u003e CFU/mL) consistently exceeding circulating concentrations. In the blood, bacterial burdens were significantly increased in the CLP group compared with sham controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and in the FIP group compared with NS controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at both 24 and 48 h. However, blood bacterial loads were significantly higher in the FIP group than in the CLP group at these same time points \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This disparity likely stems from the different infection dynamics of the two models: FIP involves an immediate, high-density bacterial bolus injection, leading to rapid systemic translocation, whereas CLP represents a progressively evolving infection through gradual leakage from the punctured cecum. This characteristic suggests that the optimized FIP model is particularly effective for simulating acute, fulminant sepsis. Furthermore, peritoneal lavage bacterial loads were significantly elevated in the CLP group relative to sham controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and in the FIP group relative to NS controls \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Comparison between the models indicated that peritoneal bacterial loads were significantly higher in the FIP group than in the CLP group at 24 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but became significantly lower by 48 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This crossover likely reflects the host\u0026rsquo;s immune clearance of the initial FIP bolus, contrasted with the persistent microbial influx from the unsealed cecal leak in the CLP model, which serves as a continuous source of infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSystemic Inflammatory Response and Organ Dysfunction\u003c/h2\u003e \u003cp\u003eThe characterization of hematological and biochemical markers is necessary to validate systemic injury and to delineate the extent of multi-organ failure induced by sepsis. To this end, hematological parameters, including WBC, NEU, LYM, and MON counts, were analyzed to evaluate systemic inflammation and immune status, while serum biochemical markers, specifically ALT and AST for hepatic function and BUN and Scr for renal function, were measured to assess organ-specific injury.\u003c/p\u003e \u003cp\u003eBoth models significantly altered peripheral blood cell counts, including a marked reduction in WBC counts in CLP mice at 48 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and in FIP mice at all assessed time points \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Significant increases in NEU counts at 24 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suppression of LYM counts \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and elevations in MON counts at 24 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed in both groups, with FIP mice exhibiting significantly higher MON counts than CLP mice at both 24 and 48 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c, d). PLT counts were also significantly decreased in both models \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Serum biochemical analysis revealed significant elevations in ALT, AST, BUN, and Scr in both models compared to controls \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-i). Notably, Scr was significantly lower in FIP mice than in CLP mice at 24 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas ALT and AST levels were significantly higher in the CLP group than in the FIP group by 48 h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-i).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these findings indicate that the optimized FIP model induces a more rapid and acute systemic inflammatory response and hematological disruption. Conversely, the CLP model leads to more progressive and sustained hepatic and renal impairment, reflecting the different pathological dynamics of a single-bolus infection versus a continuous infectious leak.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMulti-organ Histopathological Injury\u003c/h2\u003e \u003cp\u003eHistopathological evaluation of major organs, including the heart, liver, lung, kidney, and intestine, provides direct morphological evidence of systemic tissue damage. H\u0026amp;E staining revealed characteristic multi-organ injuries in both models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the heart, we observed myocardial fiber fragmentation and interstitial edema. In the liver, significant hepatocellular swelling and vacuolar degeneration were evident. Myocardial and hepatic injuries were more prominent during the acute phase (24 h) in the FIP group, whereas they appeared more severe and persistent at 48 h in the CLP group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePulmonary pathology in the FIP group was characterized by early-onset alveolar wall thickening and inflammatory infiltration at 24 h; in contrast, lung injury in the CLP group was more protracted. Renal damage, manifested by glomerular shrinkage and acute tubular necrosis, progressed over time in the CLP group and peaked at 48 h, while FIP induced substantial injury as early as 24 h. Furthermore, intestinal injury\u0026mdash;consisting of mucosal edema, villi blunting, and epithelial necrosis\u0026mdash;tended to be more severe and sustained in the CLP group, likely driven by the continuous leakage of infectious material from the cecum.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, an optimized murine model of polymicrobial sepsis was established and systematically evaluated using fecal intraperitoneal injection. Our central findings indicate that a standardized fresh fecal suspension administered at a dose of 0.7 g/kg generates key outcomes\u0026mdash;including mortality, multi-organ dysfunction, and systemic bacterial burdens\u0026mdash;that are broadly comparable to those produced by the conventional CLP model. Crucially, the optimized protocol demonstrates substantial advantages in terms of reproducibility, procedural simplicity, and controllability of disease severity, thereby supporting its utility as a reliable and efficient preclinical platform for sepsis research and therapeutic evaluation.\u003c/p\u003e \u003cp\u003eAlthough CLP is widely regarded as the gold standard for modeling polymicrobial sepsis, its primary limitation remains high inter-individual variability and a strong dependency on surgical technique. In the present study, mice in the CLP group exhibited pronounced disease severity. This observation aligns with the findings of Jain et al., who reported that mortality in the CLP control group reached 100% within 24 h in the absence of effective therapeutic intervention [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Such high lethality further underscores the stringency of the CLP model in simulating severe sepsis and assessing pharmacological efficacy. However, consistency in outcomes is often complicated by variations in the extent of ischemic necrosis in the ligated cecal segment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, variability arises from multiple factors that are difficult to standardize, such as the exact ligation position, needle gauge, number of punctures, and operator proficiency, all of which directly determine the initial infectious burden. Additionally, anatomical and local pathological differences among mice can lead to the partial occlusion of puncture sites by adjacent tissues, thereby altering the leakage dynamics of infectious material [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These complex local interactions collectively make the strict standardization of the CLP model challenging.\u003c/p\u003e \u003cp\u003eBy contrast, the optimized FIP approach mitigates these sources of variability through the systematic control of the infectious source, the preparation process, and the dosing strategy. First, the immediate collection of fresh feces from donor mice maximizes the preservation of microbial viability and the community complexity, which more faithfully models polymicrobial infection while remaining ethically compatible [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Second, the implementation of a two-step filtration workflow\u0026mdash;utilizing a 0.5 mm mesh followed by 70 \u0026micro;m filtration\u0026mdash;improves suspension homogeneity and injectability. This refinement significantly reduces the risk of syringe clogging and potential particulate-related complications, thereby increasing overall procedural reliability. Third, graded dosing experiments identified 0.7 g/kg as an optimal dose that yields moderate mortality (approximately 50%) and a severity comparable to CLP, thus providing a practical therapeutic intervention window.\u003c/p\u003e \u003cp\u003eRegarding disease assessment, the Murine Sepsis Score (MSS) effectively differentiates healthy from septic states and captures temporal changes in severity; however, its predictive value is intrinsically limited. Notably, a subset of mice died before reaching peak MSS, a finding consistent with multicenter reports of fecal-induced peritonitis models [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This likely reflects fatal physiological derangements, such as malignant arrhythmias, abrupt hypotension, or catastrophic homeostatic collapse, which can precede the overt behavioral deterioration captured by the MSS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, while the MSS remains a valuable tool, it should be integrated with objective laboratory indices and, where feasible, continuous physiological monitoring to improve phenotyping accuracy in sepsis studies.\u003c/p\u003e \u003cp\u003eBeyond procedural differences, our data suggest that FIP and CLP model distinct sepsis trajectories. The CLP model establishes a persistent infectious focus with ongoing leakage, thereby mimicking sepsis driven by unresolved source control. Consistently, CLP produces a more protracted clinical course characterized by sustained or worsening organ injury at later time points [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, the FIP model delivers a single, high-load polymicrobial challenge, leading to earlier bacteremia peaks and rapid multi-organ injury within 24 h. Notably, the FIP group exhibited lung edema and inflammatory infiltration as early as 24 h, whereas CLP-induced damage was more protracted. This rapid onset aligns with the biological vulnerability of the lung as the most critical organ during sepsis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our observation of sustained high bacterial loads contrasts with reports of declining counts in similar fresh-fecal models [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This discrepancy is likely attributable to differences in therapeutic intervention and animal species. Tall\u0026oacute;sy et al. utilized a rat model and administered fluid resuscitation and analgesics following induction, interventions known to bolster hemodynamic stability and facilitate host immune clearance of pathogens. In contrast, our study employed a mouse model without fluid resuscitation to establish a baseline of unmitigated sepsis. Consequently, the compromised host defense in our model likely failed to limit bacterial proliferation, resulting in persistently elevated peritoneal bacterial burdens typical of fulminant, untreated sepsis. Accordingly, CLP and FIP should be viewed as complementary rather than interchangeable models: FIP may be preferable for studying early inflammatory storms and rapid innate immune activation (supported by the acute hematological shifts in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), whereas CLP is more suitable for investigating sustained infection, sepsis-associated immunosuppression, prolonged organ dysfunction, or secondary infections (consistent with the progressive hepatic and renal damage observed at 48 h in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMulti-dimensional evaluation further confirmed that the optimized FIP model successfully reproduces the core features of sepsis, including systemic bacterial dissemination, organ dysfunction (indicated by elevated ALT, AST [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], BUN, and Scr [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]), and multi-organ histopathological injury. These concordant functional and morphological abnormalities support the construct validity of the optimized FIP model as a clinically relevant tool for polymicrobial sepsis research.\u003c/p\u003e \u003cp\u003eHowever, several limitations should be acknowledged. First, to isolate the variability of the induction method itself, this study purposefully excluded therapeutic interventions (e.g., fluid resuscitation, antibiotics). While necessary for standardization, this absence of circulatory support means the model represents an unmitigated, fulminant sepsis trajectory, differing from the managed clinical course seen in human patients. Second, regarding histopathology, our evaluation was restricted to qualitative morphological description to confirm the presence of organ injury, rather than applying semi-quantitative scoring systems for each organ. Third, the study was limited to young male mice and quantitative CFU analysis. Future research should validate this protocol in aged and female cohorts and incorporate metagenomic sequencing to better characterize the specific 'sepsis-inducing' microbiome components.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCollectively, this study successfully establishes and validates a highly standardized and reproducible fresh FIP murine model of polymicrobial sepsis. Characterized by operational simplicity and tunable severity, this model offers a robust and cost-effective platform for investigating the complex pathophysiology of moderately severe sepsis. Furthermore, by delineating the fundamental temporal and pathological divergence between the FIP and CLP models, these findings provide critical practical guidance for selecting the most appropriate experimental framework based on specific research objectives.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColony forming unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCecal ligation and puncture\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFecal intraperitoneal injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLYM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLymphocyte\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMON\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonocyte\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMurine Sepsis Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNEU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutrophil\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeritoneal cavity fluid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eScr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSerum creatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhite blood cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures and this study were reported in accordance with ARRIVE guidelines. The animal study protocol was approved by the Academic Ethics Committee of Zhuhai Campus of Zunyi Medical University (Protocol No: ZHSC-2-[2024]078, approved on 26 September 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Zhuhai City Medical Research Project of Guangdong Province (Grant No. 2520009000090).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L. and X.Y. conceptualized and supervised the study. L.Z. contributed to the methodology, software, formal analysis, and visualization. L.Z., Z.W., and Z.Z. performed the validation. L.L. conducted the investigation. Y.L. provided resources. L.Z., Z.W., X.L., and L.L. curated the data. L.Z. and R.Z. drafted the original manuscript. Y.L. reviewed and edited the manuscript. X.Y. administered the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSinger M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). 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BMC Immunol. 2025;26(1):67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sepsis, Fresh fecal suspension, Fecal intraperitoneal injection, Cecal ligation and puncture, Animal model","lastPublishedDoi":"10.21203/rs.3.rs-9124307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9124307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSepsis remains a critical challenge in intensive care, necessitating reliable animal models that accurately mimic human pathophysiological responses. While cecal ligation and puncture (CLP) is widely considered the gold standard, its inherent variability often limits reproducibility. This study aimed to optimize a fecal intraperitoneal injection (FIP) murine model by evaluating the impact of fecal preparation (fresh vs. lyophilized) and dosage (0.5\u0026ndash;1.0 g/kg) on model stability. We systematically compared the optimized FIP model with the conventional CLP method in male BALB/c mice to define their respective pathophysiological characteristics and suitability for therapeutic screening.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings demonstrate that fresh fecal suspensions significantly enhance model reproducibility compared to dried preparations, which showed inconsistent virulence. An optimized FIP dose of 0.7 g/kg induced a hyperacute sepsis phenotype, characterized by rapid systemic bacterial dissemination and significant acute lung and kidney injury within 24 hours. In contrast, the CLP model exhibited a more protracted progression of organ dysfunction, with more pronounced and sustained intestinal mucosal damage and evolving infectious dynamics. Hematological analysis confirmed that while both models induced systemic inflammation, the FIP model provided a more synchronized and predictable onset of multi-organ failure.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe optimized FIP model, characterized by its procedural simplicity, high controllability, and superior reproducibility, serves as a robust platform for investigating the early, fulminant pathophysiological mechanisms of unmitigated sepsis. Conversely, the CLP model remains the preferred choice for studies focusing on protracted infection and chronic organ dysfunction. These findings provide a methodological framework for selecting appropriate sepsis models based on specific research objectives in experimental medicine.\u003c/p\u003e","manuscriptTitle":"Optimization of a Fresh Fecal Intraperitoneal Injection Sepsis Model and Its Divergent Dynamics from Cecal Ligation and Puncture in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 16:19:06","doi":"10.21203/rs.3.rs-9124307/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b6718aea-dec1-43dd-b3bf-3effb1263e21","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T11:40:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 16:19:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9124307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9124307","identity":"rs-9124307","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}