A method for constructing a sepsis animal model and application thereof

Abstract

The application discloses a kind of sepsis animal model construction method and its application, belong to animal model construction technical field.The construction method includes the steps of taking donor mouse cecal contents, after dilution and ultrasonic, intraperitoneal injection to recipient mouse.The application successfully overcomes the core defects of CLP sepsis model operation complex, parameter uncontrollable, poor repeatability and insufficient clinical simulation degree by innovative model construction strategy.The sepsis mouse model provided by the application has the following comprehensive advantages: simple operation, high standardization, good consistency, excellent repeatability, significant multi-organ dysfunction characteristics, reasonable natural mortality and closer to clinical characteristics, etc., which provides strong technical support for the basic research, drug development and translational medicine application of sepsis, and has significant scientific and technological progress and practical application value.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing an animal model of sepsis and its application. Background Technology

[0002] Sepsis is a life-threatening syndrome of organ dysfunction caused by dysregulation of the host response to infection, and is one of the leading causes of death in intensive care unit patients. The pathogenesis of sepsis involves complex imbalances in the immune-inflammatory response, coagulation dysfunction, microcirculatory disturbances, and multiple organ dysfunction, and its pathological progression and clinical outcomes are highly heterogeneous. Therefore, establishing standardized, reproducible animal models of sepsis that can simulate clinical characteristics is crucial for elucidating the pathogenesis of sepsis, evaluating the efficacy of therapeutic drugs, and facilitating translational medical research.

[0003] Currently, commonly used animal models of sepsis in laboratories mainly include the following categories: 1. Endotoxin-induced model Systemic inflammatory response is induced by intravenous or intraperitoneal injection of lipopolysaccharide (LPS). This model is simple to operate and has good reproducibility. However, the inflammatory response induced by LPS is a pure endotoxin stimulation, lacking the pathological process of live bacterial infection, and cannot simulate the immunosuppressive state in the later stage of sepsis, showing a significant gap from the true pathological characteristics of clinical sepsis.

[0004] 2. Live bacteria injection model Infection models are established by intravenous or intraperitoneal injection of live bacteria (such as Escherichia coli, Staphylococcus aureus, etc.). Although this model can simulate the bacterial infection process, it is difficult to precisely control the amount of injected bacteria and the virulence of the strains. Furthermore, the distribution of bacteria differs from the natural dissemination pathway of clinical infection foci, which can easily lead to acute septic shock rather than progressive multiple organ dysfunction.

[0005] 3. Cecal Ligation and Puncture (CLP) Model The cecal ligation-percutaneous puncture (CLP) model is currently the most widely used animal model of sepsis. Its principle involves ligating and puncturing the cecum in mice to induce leakage of cecal contents, leading to mixed peritoneal infection and bacteremia, which in turn develops into multiple organ dysfunction syndrome. The CLP model, to a certain extent, simulates the pathway from clinical peritoneal infection to sepsis and is considered one of the "gold standard" models for sepsis research. However, when performing CLP procedures on the cecal ligation-percutaneous puncture sepsis model, significant differences in ligation length, cecal contents, needle size, and the number of punctures can cause substantial variations in the final results within a group. Furthermore, the procedure is difficult to perform and reproducible.

[0006] Therefore, there is an urgent need in this field for a novel animal model that is easy to operate, has controllable parameters, good repeatability, and can more realistically simulate the characteristics of clinical sepsis, so as to promote the development of basic research and translational medicine in sepsis. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing an animal model of sepsis and its application, in order to solve the problems existing in the prior art. The sepsis mouse model provided by this invention has comprehensive advantages such as simple operation, high degree of standardization, good consistency, excellent reproducibility, significant multi-organ dysfunction characteristics, reasonable natural mortality rate and closer to clinical characteristics. It provides strong technical support for basic research, drug development and translational medicine application of sepsis, and has significant scientific and technological progress and practical application value.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for constructing an animal model of sepsis, comprising the steps of taking the cecal contents of a donor mouse, diluting and sonicating them, and then injecting them intraperitoneally into a recipient mouse.

[0009] Optionally, the dilution operation is performed using sterile saline solution, wherein the ratio of sterile saline solution to the cecal contents is 500 μL: 0.3 g.

[0010] Optionally, the ultrasound conditions are 40 Hz ultrasound for 30 minutes.

[0011] Optionally, the injection dose is 80~100μL / animal.

[0012] The present invention also provides the use of the sepsis animal model constructed by the above-described method for screening drugs for the treatment / prevention of sepsis.

[0013] The present invention also provides a method for screening drugs for the treatment / prevention of sepsis, comprising the step of administering the drug to a sepsis animal model constructed as described in the sepsis animal model construction method.

[0014] Optionally, drugs for treating / preventing sepsis can be screened by comparing changes in the animal models of sepsis before and after drug administration.

[0015] The present invention also provides a method for studying the pathogenesis of sepsis, comprising the step of administering a candidate drug to a sepsis animal model constructed by the method described above.

[0016] The present invention discloses the following technical effects: This invention is simple to operate and lowers the technical threshold: it eliminates the complex open abdominal surgery procedure of CLP models, avoiding highly complex microsurgical operations such as cecal identification, ligation, puncture, and layer-by-layer suturing. Experimenters only need simple training to master the standardized operating procedure, significantly shortening the learning curve and reducing reliance on individual operator experience. This improvement allows experimenters of varying experience levels to achieve consistent modeling results, significantly improving the accessibility and widespread applicability of the experiment.

[0017] The parameters of this invention are highly standardized to ensure intragroup consistency: by replacing the difficult-to-quantify operational variables in the CLP model with preset, precise and controllable experimental parameters, intragroup heterogeneity caused by individual differences of operators, individual anatomical variations of animals and differences in gut microbiota is effectively eliminated, ensuring the statistical reliability and biological repeatability of experimental data.

[0018] The model of this invention exhibits excellent reproducibility, enhancing the comparability of cross-laboratory data: the standardized construction method ensures high consistency of model parameters across different laboratories, batches, and operators. Key indicators such as mortality curves, dynamic changes in inflammatory factors, and organ damage scores demonstrate good batch-to-batch reproducibility, providing a solid technical foundation for large-scale multi-center studies, horizontal comparisons in drug screening projects, and longitudinal tracking of historical data. This characteristic effectively overcomes the industry pain point of poor cross-laboratory data comparability caused by operator dependence in CLP models.

[0019] In summary, this invention, through an innovative model construction strategy, successfully overcomes the core shortcomings of CLP sepsis models, such as complex operation, uncontrollable parameters, poor reproducibility, and insufficient clinical simulation. The sepsis mouse model provided by this invention has comprehensive advantages, including ease of operation, high standardization, good consistency, excellent reproducibility, significant multi-organ dysfunction characteristics, reasonable natural mortality rate, and closer resemblance to clinical characteristics. It provides strong technical support for basic research, drug development, and translational medicine applications of sepsis, demonstrating significant scientific and technological advancement and practical application value. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Schematic diagram of cecal ligation and puncture in mice; A: Anesthesia and fixation; B: Hair removal; C: Locating the cecum; D: Cecal ligation; E: Puncture; F: Postoperative suturing; Figure 2 The following data pertain to postoperative peritoneal inflammation in mice of each group: A: Blank group; B: Sham-operated group; C: CLP group; D: Modified group; Figure 3 Postoperative blood routine and blood biochemical index results for each group of mice; A: White blood cells; B: Neutrophils; C: Lymphocytes; D: Platelets; E: AST; F: ALT; G: BUN; H: Cr; Figure 4 The results of postoperative TNF-α (A) and IL-6 (B) detection in mice of each group; Figure 5 HE staining results of intestines, liver, kidneys, and lungs of mice in each group; Figure 6 Postoperative survival curves for mice in each group. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1 1. Laboratory animals Eighty-four healthy SPF-grade male KM mice, aged 8-9 weeks and weighing 23-25g, were provided by the Experimental Animal Center of Three Gorges University. All mice were housed for one week in the SPF-grade housing at the Experimental Animal Platform of the Institute of Pharmaceutical Research of Three Gorges University before the experiment (room temperature controlled at around 25℃, air humidity controlled at around 50%, 12 hours of light per day, and free access to food and water). Animal handling methods complied with animal ethics standards.

[0028] 2. Grouping of experimental animals and establishment of experimental models The mice were randomly divided into four groups using a random number method: a control group (NC), a sham-operated group (Sham), a CLP group (cecal ligation and puncture), and a modified group (Pro, the method described in this invention), with twenty mice in each group. Ten mice in each group were used for survival analysis. Four mice were euthanized by cervical dislocation, and their cecal contents were collected. The cecal contents (0.3g) of KM mice were diluted with 500μL of physiological saline and sonicated at 40Hz for 30 minutes for subsequent use.

[0029] No processing is performed on the blank group; The CLP group underwent cecal ligation and puncture experiments; The sham surgery group underwent the same procedures as the CLP group, except that cecal ligation and perforation were not performed. Modified group: Each animal was injected intraperitoneally with 80-100 μL of cecal contents diluted with physiological saline and sonicated. In this example, 100 μL was selected.

[0030] CLP modeling ( Figure 1): Administer an intraperitoneal injection of 0.1 mL / 10 g of 0.3% sodium pentobarbital solution according to the mouse's body weight. After the mouse becomes paralyzed, check the anesthesia effect by clamping the skin of the lower limbs with forceps. When the limbs are unresponsive, breathing is shallow, slow, and rhythmic, and there is no righting reflex, the anesthesia is considered successful. Use an electric epidermal hair removal machine to shave the hair in the lower abdominal quadrant and disinfect the area with alcohol. Make a 1.5 cm longitudinal incision 1 cm to the right of the abdominal midline, separate the epidermis and muscle layer, and enter the abdominal cavity layer by layer to locate the cecum. Carefully separate the cecum, taking care to avoid damaging the ileocecal vessels. Gently squeeze the contents of the cecum towards the free end of the cecum. Ligate the middle section of the cecum with 4.0 silk suture. Puncture the cecum with a 20 mL syringe needle between the ligated cecum and the end of the cecum, squeezing gently to observe whether intestinal contents leak out from the needle holes on both sides. Carefully return the cecum to the abdominal cavity, taking care not to let the intestinal contents adhere to the surgical incision, and suture the muscle and skin layers layer by layer. After closing the abdomen, apply iodine solution to the surgical incision for disinfection and gently massage to ensure the intestines are back in place and the sutures are smooth, which is beneficial for the mouse's recovery. All postoperative mice were kept warm with an electric heater until they regained consciousness. After recovery, the mice were transferred to marked cages for routine care.

[0031] 3. Postoperative clinical symptom observation The appearance, activity level, food and water intake, and defecation status of rats in each group were observed, and the survival status of mice in each group was recorded in a timely manner.

[0032] 4. Complete blood count (CBC) Twelve hours post-surgery, mice were anesthetized using the same anesthesia method as during model establishment. The abdominal cavity was opened along the original surgical incision, and the intestines and peritoneum were carefully separated with sterile forceps to expose the abdominal aorta. One mL of blood was collected by puncturing the abdominal aorta using a disposable blood collection needle and collected in a sterile tube. 20 μL of whole blood was added to a sterile EP tube containing diluent, and a complete blood count was performed using a hematology analyzer to analyze changes in white blood cell, neutrophil, and lymphocyte counts.

[0033] 5. Blood biochemical index testing The remaining whole blood was allowed to coagulate naturally at room temperature for 10-20 minutes. Then, it was centrifuged at 3000 rpm for 20 minutes at 4°C. The supernatant serum was carefully collected, and the changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a blood biochemistry analyzer.

[0034] 6. Cytokine detection The collected serum was tested for the expression levels of IL-6 and TNF-α using a standard diagnostic kit, following the instructions of the kit.

[0035] 7. Histopathological examination After blood collection from each mouse, intestinal, liver, lung, and kidney tissues were taken, fixed with 4% neutral formaldehyde solution, dehydrated, embedded in paraffin, sectioned in paraffin, stained with hematoxylin and eosin (HE), and observed under an optical microscope to assess the degree of inflammation and tissue damage.

[0036] 8. Survival Analysis SPSS 20.0 software was used for statistical analysis. The Log-rank (Mantel-Cox) test was used for survival statistics. The t-test was used to compare numerical differences between groups. GraphPad Prism 6.0 software was used for plotting. P < 0.05 was used to indicate statistical difference.

[0037] 9. Results 9.1 Postoperative condition and peritoneal inflammation in mice No mice in any group died before the samples were collected.

[0038] Control group: Everything was normal as usual, and no abnormalities were found in any organs after dissection. Figure 2 (A) Sham-operated group: Mice were agile, had uniform fur, and could eat and drink normally. They occasionally fought and climbed, but showed no lethargy, curling up, or piloerection. Upon dissection, the small intestine in the abdominal cavity showed no obvious signs of congestion, edema, or other inflammation; the intestines were not dilated; and there were no significant changes in any organs. Figure 2 (Middle B) CLP group: Mice showed sluggish response to external stimuli, erect back fur, lethargy, reduced activity, slow gait, thick discharge from the eyelids, decreased appetite, and a tendency to huddle together for warmth in the corners of their cages. Upon dissection, the abdominal cavity revealed: edematous and distended intestines filled with gas, a small amount of peritoneal effusion, and moderate congestion of the cecal tissue at the ligation site. Figure 2 (C) Improved group: Mice were also less responsive to external stimuli, often resting with their eyes half-closed, huddling together for warmth, had loose stools, upright and messy fur, and increased water intake. Dissection revealed increased peritoneal effusion, severe visceral adhesions, blackened and swollen distal cecum with a slight foul odor. Figure 2 (D).

[0039] 9.2 Postoperative blood routine test and blood biochemical index test Blood routine tests were performed on mice in each group, and the results are as follows: Figure 3 As shown, compared with the NC and Sham groups, the CLP and Pro groups had lower white blood cell counts ( Figure 3 (A) Neutrophils ( Figure 3 B), platelets ( Figure 3 Both D and lymphocytes were significantly reduced (P<0.001), and lymphocytes ( Figure 3The levels of white blood cells, neutrophils, lymphocytes, and platelets in the modified group also decreased to varying degrees (P<0.001). The standard deviations of white blood cells, neutrophils, lymphocytes, and platelets in the modified group were lower than those in the CLP group, indicating that the differences among samples within the group were smaller, the amplitudes were more stable, and the homogeneity was better.

[0040] Blood biochemical parameters in mice of the CLP and Pro groups showed that, compared with the NC and Sham groups, the CLP and Pro groups had lower levels of AST ( Figure 3 E), ALT ( Figure 3 Medium F), BUN ( Figure 3 G), Cr ( Figure 3 The levels of H in the modified group were significantly increased (P<0.0001), and the standard deviations of AST, Cr, and BUN in the modified group were lower than those in the CLP group, indicating that the differences among samples within the group were small and the amplitudes were relatively stable, reflecting the good homogeneity of the samples within the group.

[0041] 9.3 Postoperative cytokine detection Cytokine levels were measured in mice of each group. Compared with the NC and Sham groups, the CLP group and the modified group had lower levels of TNF-α ( Figure 4 (A) and IL-6 ( Figure 4 The levels of B in the CLP group were significantly increased (P<0.001), with the standard deviation of the improved group being lower than that of the CLP group.

[0042] 9.4 Histopathological examination like Figure 5 As shown, no obvious pathological changes were observed in the intestines, liver, kidneys, and lungs of the NC and Sham groups. In the CPL group, the intestinal mucosa showed an uneven surface and unclear structure, with disordered glandular arrangement, hyperplasia, and atrophy, and extensive inflammatory cell infiltration. The liver tissue showed morphological changes, with loss of lobular structure, accompanied by extensive vacuolar fatty degeneration, edema and necrosis in some cells, and significant congestion of the hepatic sinusoids. The main manifestations in the lungs were interstitial edema and thickening of the alveolar-capillary membrane, with extensive inflammatory cell infiltration and erythrocyte leakage in the alveoli and interstitium. In the kidney tissue, the main morphological changes were extensive inflammatory cell infiltration in the renal interstitium and glomeruli, thickening of glomerular epithelial cells and widening of the capsule, and disordered renal tubular structure. In addition to exhibiting the same symptoms in all organs as in the CLP group mice, the Pro group mice also showed extensive hepatocellular necrosis in the liver, more severe inflammatory cell infiltration and erythrocyte leakage in the alveoli and interstitial lungs, and more pronounced interstitial edema and thickening of alveolar capillaries.

[0043] 9.5 Postoperative survival curve Survival curves of mice in each group ( Figure 6The results showed that the CLP and Pro groups were significantly different from the NC and sham surgery groups (P<0.0001), with both models exhibiting higher mortality rates, and the survival rates of the CLP and Pro groups differed (P<0.05). The median survival time in the CLP group was 3.5 days, while the median survival time in the Pro group was 2 days.

[0044] In summary, this invention, through an innovative model construction strategy, successfully overcomes the core shortcomings of CLP sepsis models, such as complex operation, uncontrollable parameters, poor reproducibility, and insufficient clinical simulation. The sepsis mouse model provided by this invention has comprehensive advantages, including ease of operation, high standardization, good consistency, excellent reproducibility, significant multi-organ dysfunction characteristics, reasonable natural mortality rate, and closer resemblance to clinical characteristics. It provides strong technical support for basic research, drug development, and translational medicine applications of sepsis, demonstrating significant scientific and technological advancement and practical application value.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing an animal model of sepsis, characterized in that, The procedure includes taking the cecal contents of a donor mouse, diluting and sonicating them, and then injecting them intraperitoneally into a recipient mouse.

2. The method for constructing an animal model of sepsis as described in claim 1, characterized in that, The dilution was performed using sterile saline solution at a ratio of 500 μL to 0.3 g of the cecal contents.

3. The method for constructing an animal model of sepsis as described in claim 1, characterized in that, The ultrasound conditions were 40 Hz ultrasound for 30 minutes.

4. The method for constructing an animal model of sepsis as described in claim 1, characterized in that, The injection dose is 80~100μL / animal.

5. The use of the sepsis animal model constructed by the method for constructing a sepsis animal model as described in any one of claims 1-4 in screening drugs for the treatment / prevention of sepsis.

6. A method for screening drugs for the treatment / prevention of sepsis, characterized in that, The method includes the step of administering the drug to an animal model of sepsis constructed by the method for constructing an animal model of sepsis as described in any one of claims 1-4.

7. The method as described in claim 6, characterized in that, By comparing the changes in the animal models of sepsis before and after drug administration, drugs for the treatment / prevention of sepsis were screened.

8. A method for studying the pathogenesis of sepsis, characterized in that, The method includes the step of administering a candidate drug to an animal model of sepsis constructed by the method for constructing an animal model of sepsis as described in any one of claims 1-4.

Images