动物造模,全身炎症三种动物模型 z-bo 2022-02-06 502

　　Background: Sepsis remains a major clinical challenge in the intensive care unit because it often leads to multiple organ dysfunction and high morbidity, resulting in a mortality rate of about 50%. Although established strategies for the treatment of potential infections have been developed, there is an urgent need to develop new treatment options and identify potential drug candidates to prevent and defeat the disease. For these purposes, animal models are very useful. In order to mimic the course of human sepsis, various rodent models have been developed. These models can be divided into three categories: endotoxin lipopolysaccharide (LPS) exogenous [administration] in vitro administration of exogenous pathogens [inoculation with E. coli], endogenous protective barrier destruction [cecal ligation and perforation model (CLP) ]. These three models have their own advantages and disadvantages, and which animal model is most suitable is still controversial. The LPS animal model has the advantages of simple technology and good reproducibility, and especially plays an important role in inflammation. Soon after administration of LPS, high levels of pro-inflammatory cytokines are released, which can be measured in circulating serum. This led to the rapid development of systemic inflammatory response syndrome (SIRS) and subsequent dose-dependent mortality. However, the LPS model does not fully reproduce the characteristics of human sepsis. LPS can be used to study the pathophysiological process of endotoxemia or SIRS, such as endotoxin shock model. In order to make up for the shortcomings of the LPS model, a variety of microbial sepsis models have been developed. In the abdominal infection and infection model (PCI), various aerobic and anaerobic gram-positive and gram-negative bacteria are isolated from stool samples of healthy, non-vegetarian donors. In addition, the correlation between the PCI model and clinical sepsis is questionable because patients rarely have massive bacteremia. The effect of a single application of high-dose bacteria is close to that observed after intravenous injection of high-dose lipopolysaccharide. The most widely used sepsis is the cecal ligation and perforation (CLP) model, which is recognized as having good compatibility with human sepsis. The purpose of this study is to comprehensively describe the characteristics of LPS, PCI and CLP mouse models within 72 hours, including the acute phase of systemic inflammation. For this reason, a moderate systemic inflammatory response that does not cause acute death is induced. Therefore, there is no need for any antibiotic treatment or fluid resuscitation on animals, which allows us to study the natural course of this disease. The systemic immune response was evaluated by measuring the serum cytokine concentration, measuring the oxidative stress parameters of various organs, and tracking the migration of spleen and liver immune cells. Liver function is critical to the overall prognosis of patients, even more important than kidney and lung function, therefore, liver function parameters are also determined. This is the first time that these three animal models have been directly and comprehensively compared. Within 72 hours, a non-lethal method was used to study the impact of systemic inflammation on organ function. The purpose of this study is to better understand the pathophysiological processes involved in these animal models, to help researchers choose the most suitable model, and to determine which parameters are useful for which model when evaluating candidates for the treatment of systemic inflammation . Methods: Animals and experimental methods: Adult male C57BL/6 mice (12 weeks old, body weight 25-30 g), the animals are placed in plastic cages under standard conditions (light-dark cycle 12/12 hours, temperature 22 ± 2 ° C, humidity 50±10%, pellet feed free drinking water). Mice were treated with lipopolysaccharide LPS. Escherichia coli, 5 mg/kg body weight, dissolved in PBS, intraperitoneal administration volume of 0.1 ml/10 g mouse body weight), cecal ligation and perforation [CLP]. The animals were sacrificed as described after 2, 4, 6, 12, 24, 48 and 72 hours. At the beginning of the experiment (T=0), four control mice in each treatment group were sacrificed. These mice did not receive any treatment. The most appropriate non-lethal LPS dose was determined in the previous (trial) studies. Mice take 1.5μg of stool and dissolve it in PBS intraperitoneally to give 0.1ml/10g of body weight, representing a non-lethal dose. Isoflurane was used to induce anesthesia and the abdominal cavity was cut along the median incision along the white line to expose the cecum, and ligate with non-absorbable suture between the distal end and the cecum floor. Subsequently, a 21 g needle was used to puncture the cecum and gently compress and squeeze out a small amount of cecal contents. Induce the mid-term inflammatory response and suture the abdominal cavity again. Since we are very interested in the natural course of this disease, and because we have induced a moderate systemic inflammation, the possibility of acute death is very small. Among the three animal sepsis models, none of them are treated with antibiotics. In order to track the course of each animal model, investigations were conducted at eight different time points. At 0 h (equivalent to the control group), 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours after the onset of inflammation, the mice were sacrificed (4-6 animals were sacrificed at each time point). At each time point, the body temperature was measured, and the animal’s condition was assessed according to the clinical severity score (CSS), as described above. Evaluate the CSS value every hour and make every effort to reduce the animal's suffering. When the animal's CSS score was 4, the animals were killed by inhaling an overdose of isoflurane. All other mice were sacrificed using the same method at the indicated time points. The brain, kidney, liver, lung, and spleen were taken, weighed, and fixed in 10% paraformaldehyde and quickly frozen in liquid nitrogen for biochemical analysis. After the mice were decapitated, they were bled completely, and the blood was collected in agglutination tubes. Use a commercially available blood glucose meter and corresponding test strips to measure blood glucose levels. Thirty minutes later, the serum obtained by centrifuging 2000 grams of clotted blood for 10 minutes was subjected to ELISA and enzyme activity measurement. No animals with elevated serum cytokine and oxidative stress levels were found in the experiment to ensure that there were no drug-resistant mice in the treatment group. In histological analysis, formalin-fixed organ specimens were implanted in paraffin blocks, and each treatment group was cut into 4μm slices (n=4-6). Interleukin (IL) 6, Interleukin (IL) 10, Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ, CXC Chemokine 12 (CXCL12) and Alanine Aminotransferase (ALAT) ) Method: using IL-6 mouse ELISA kit, TNF-α mouse ELISA kit, IFN-γ mouse ELISA kit, CXCL12 / SDF-1α mouse ELISA kit and alanine aminotransferase assay The kit measures it.

　　Result: Health conditions, including blood pressure, body temperature and weight measurements are affected by LPS, PCI and CLP treatments: In each animal model, we induced a moderate, non-fatal systemic inflammatory state to study the course of the disease. However, in the CLP model, one after 24 hours, one after 40 hours and one after 70 hours had to be euthanized due to a CSS score of 4. Because these animals did not meet the specified time point, they were not included in further analysis. Mice receiving LPS or PCI showed impaired health, and the clinical symptom severity score (CSS) was more obvious than that of the control group. The infection reached a maximum 6 to 12 hours after induction, and then dropped to the baseline level. After 48-72 hours, there was no difference in CSS values between the treated mice and the control group. CLP-treated mice also showed CSS peaks after 6-12 hours, but compared with the other two groups of animals, these values still increased until 72 hours, indicating that the disease course was longer without recovery. Changes in blood pressure and heart rate are crucial during systemic inflammation. In all three animal models, blood pressure values decreased compared to the control group (control: 115•6 mmHg, LPS: 80•8 mmHg, PCI: 90•16 mmHg, CLP: 79•16 mm Hg), while the average heart rate increased (control: 446•51 BPM, LPS: 591•33 BPM, PCI: 514•60 BPM, CLP: 554•43 BPM). Compared with the control group, systemic inflammation also affected body temperature and weight. LPS, PCI and CLP treatments can reduce body temperature and weight. The body temperature dropped significantly between 2 and 12 hours, and the body temperature of CLP-treated mice dropped the most. After 6 hours, the animal's body temperature dropped. The effects of different treatments on body weight are very similar, because the mice in all three animal models lost weight within 24 hours. After 24 hours, the PCI and CLP-treated mice recovered their body weight, while the LPS-treated group lost weight compared with the control group after 48 and 72 hours.

　　 Different levels of blood glucose, serum cytokines, and liver enzymes in sepsis models: To further evaluate the systemic results after LPS, PCI, and CLP treatment, we evaluated the levels of glucose in whole blood and the levels of inflammatory cytokines in serum. LPS and PCI-treated mice showed hypoglycemia, reaching a peak 12 hours after infection. After 12 hours, the blood sugar level continued to rise and eventually returned to a controlled level. In the CLP model, hypoglycemia also appeared, but compared with the LPS and PCI models, the blood glucose level remained low 72 hours after infection. After LPS administration, the tumor necrosis factor (TNF) alpha level increased by about 4500% after only 2 hours compared with the control group. Compared with the control group, the PCI and CLP groups increased the tumor necrosis factor alpha concentration by approximately 1500% and 1000%, respectively. However, the CLP group increased serum TNF-α levels at subsequent time points. Similar to TNFα, LPS treatment has the strongest effect on serum interferon (IFN) gamma levels. Compared with the control value, the levels of each group increased significantly at 4, 6, and 12 hours (25000%, 45000%, and 22000%, respectively). CLP group can moderately increase IFN-γ, but PCI group has almost no effect on serum IFN-γ concentration. Check the concentration of interleukin (IL)-6. LPS administration leads to an early increase in serum IL-6 levels, especially at 2, 4 and 6 hours after infection. The serum levels of PCI-treated mice also increased similarly. The CLP group also led to an early increase of cytokines, which was less than that in the LPS or PCI model. The IL-6 concentration of the CLP group was still about 5000% higher than that of the control group. In order to explore the compensation mechanism, the serum IL-10 concentration was also measured. Here, the three animal models caused different results. LPS treatment induced a biphasic IL-10 response, which increased significantly after 2 hours, followed by a decrease to 12 hours, and a second increase after 24 hours. PCI treatment resulted in a continuous increase in IL-10 levels for 6 hours, followed by a gradual decrease, while mice in the CLP group showed increased IL-10 levels at a later time point. In LPS-administered mice, the level of CXCL12 continued to decrease, and after 24 hours, inflammation had onset, followed by an increase at a later time point. Compared with the control group, the PCI group induced a biphasic response, the CXCL12 level was lower after 4 h, and increased after 24 h. CLP treatment resulted in increased serum CXCL12 levels at 24 and 48 h.

　　 The three models of systemic inflammation all affect liver function: because ALAT levels in all groups are elevated, liver function needs to be further checked. He and periodic acid Schiff staining (PAS) staining were used to analyze liver tissue sections. HE staining showed a large amount of fat accumulation and inflammatory cell infiltration in the liver of LPS-treated mice after 24 hours. Although PCI and CLP treated mice did not show liver fat accumulation, infiltrating cells were also found. However, compared with the LPS group, the infiltration was less obvious. When determining the liver protein content as a reference value for the cytochrome P450 (CYP) model response, the turbidity value of each sample was evaluated as an approximate value for the liver fat content. Consistent with histological examination, the turbidity value after LPS treatment increased from 4 to 24 hours. As a measure of liver glycogen content, PAS staining showed similar responses in all three animal models. LPS, PCI, and CLP caused significant loss of liver glycogen after 6 hours, but recovered after 24 hours and reached the baseline value in 72 hours. As an additional indicator of liver oxidative stress, immunohistochemistry detects the expression of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS). With the prolongation of treatment time, the expression of HO-1 increased, and obvious expression appeared after 72 hours. In contrast, after CLP treatment, the expression of HO-1 was not affected. When detecting the expression of iNOS, there was no significant difference between the LPS group and the PCI group, but the effect of PCI was not as obvious as that of the LPS group. Two hours after infection, LPS caused iNOS-positive neutrophils to infiltrate the surrounding area of liver lobules. After 12 hours, the largest number of infiltrating granulocytes was observed and distributed throughout the liver lobules. At a later time point, granulocytes were rarely detected. The LPS and PCI groups had more influence on neutrophil migration than the CLP group. Only a few neutrophils were observed at 6 hours, and there was no further increase in the subsequent. A core parameter of liver function is its biotransformation ability. The treatment of LPS, PCI and CLP resulted in a significant and persistent decrease in liver CYP enzyme activity.

　　 Conclusion: This study showed that compared with the CLP group, LPS and PCI treatments caused non-fatal systemic inflammatory responses to have different effects on organ function, cytokine response, oxidative stress levels and overall health. LPS and PCI models can cause a rapid inflammatory response, including early serum pro-inflammatory cytokines and oxidative stress in organs and tissues, rapid reduction in blood glucose and biotransformation capacity, and immune cells circulating from the blood to the liver and spleen, and apoptosis in the spleen. After LPS administration, the inflammatory response was the strongest in all models. Interestingly, mice treated with LPS and PCI recovered after 72 hours, possibly by inducing protective mechanisms such as HO-1 upregulation. This study shows that attention must be paid to differences in systemic inflammation models to ensure that the experimental goals are properly addressed. This includes assessing the time required for research when testing new anti-inflammatory drugs. In assessing the impact on acute inflammation, the LPS model is the most appropriate because the systemic impact is easy to identify and measure. In addition, LPS is easy to control and the model is reproducible. Of course, LPS injection does not fully replicate the process of human sepsis, and CLP is the "gold standard" in this regard. However, the time period of this mode must be chosen to be long enough because of the delayed process of inducing inflammation. In short, each model has benefits, and the experimental parameters to be studied must be weighed against the overall goal of each study.