Establishment and Evaluation of a "Two-Hit" Mouse Model of Sepsis

Establishment and Evaluation of a "Two-Hit" Mouse Model of Sepsis | 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 Establishment and Evaluation of a "Two-Hit" Mouse Model of Sepsis Fei Li, Chuanxin Wu, Jiahui He, Jie Zhang, Ying Chen, Shuhua Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-474587/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 In recent years, researchers have turned their attention to the study of the mechanism of sepsis-induced immuneparalysis, but there is still a lack of appropriate animal models that reflect the process of sepsis-induced immuneparalysis. The purpose of this study was to explore and evaluate whether a "two-hit" model of sepsis can be used as an appropriate animal model to study the mechanism of sepsis-induced immunoparalysis. Firstly, we established a sepsis model in C57BL/6J mice via cecal ligation and puncture(CLP). Routine blood tests, serum ALT and PCT, etc. were analyzed to evaluate the establishment of a CLP sepsis model on day 1 post-CLP. Secondly, the surviving mice were treated with 40µL suspension of P.aeruginosa ( Pa ) under anesthetic on day 4 post-CLP to establish a "two-hit" model. Gross lung specimens, and pathological examination of the lung tissue, etc. were used to evaluate whether the "two-hit" model was successfully established on days 5 and 11 post-CLP. Finally, the level of serum TNF-α and IL-6, pathological examination of the spleen and the CD4+/CD8 + ratio in blood were detected to evaluate the immune status of the model mice. The above test results proved that the "two-hit" sepsis mouse model was successfully constructed. TNF-α, IL-6, pathological examination of the spleen and the CD4+/CD8 + ratio in blood confirmed that the model was in a state of immunoparalysis. We conclude that this "two-hit" sepsis mouse model is an appropriate animal model, which can successfully replicate the pathophysiological process of in clinical patients with sepsis-induced immunoparalysis. Immunology sepsis two-hit model inflammation immunoparalysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Declarations Ethics A pproval and C onsent to P articipate : The study protocol was approved by the Institutional Review Committee of The Second Affiliated Hospital of Chongqing Medical University. And all applicable institutional and governmental regulations concerning the ethical use of animals were followed. Consent for Publication: All authors contributed to the writings and approved the final version of the manuscript. Availability of Da ta and M aterials : The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conflicts of Interest: The authors declare that they have no competing interests. Funding: This work was supported by a grant from the National Natural Science Foundation of China (No. 81871608). Authors' Contributions: FL, HS and CXW designed performed the study, and draft the manuscript. FL ,JH,JZ established the animal model and collected specimens.YC，SL，LL performed data analysis and statistical analyses. HS made critical revisions and provided guidance for further statistical analysis. All authors read and approved the final manuscript. Introduction Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection[1], and is a complex disease with both high morbidity and mortality. Sepsis is much more dangerous than the infection itself, and there are over 18 million cases of severe sepsis worldwide each year. Although most sepsis patients can survive in the acute phase due to the progress made regarding ICU support treatment, sepsis-induced immunoparalysis leading to secondary infection is becoming a leading cause of death[2] . Recent research has shown that the peak time of sepsis-induced death can be divided into three stages. The early peak is caused by the inflammatory response in the initial days of sepsis and a late peak after several weeks that is due to organ damage or immunoparalysis. The third upswing occurs after 60-90 days due to persistent immune dysfunction and inflammation in patients with other comorbidities or advanced age[3]. The definition of Sepsis 3.0 does not clearly define the nature and mechanism of the disordered host response to infection, which indicates that researchers continue to have an insufficient understanding of the pathophysiological mechanism of the pathogenesis of advanced immunoparalysis in sepsis[4]. The mechanism currently accepted by researchers is that innateand adaptive immune responses of the host are involved in the pathogenesis of sepsis[5]. The early activation of the innate immune response is the first line of defense against infection and plays a central role in the initiation process of adaptive immunity. After the onset of sepsis, a strong pro-inflammatory response accompanied by an anti-inflammatory response will generally last for several days[5], limiting inflammation and protecting the host. However, if such a compensatory response prevails, it will cause a large amount of death of inflammatory mediators and immune cells in the body. This leads to a more lasting disease state of the host, which is likely to cause secondary infection and viral reactivation in the host [6]. Although modern medical strategies have improved the short-term outcomes in patients with sepsis, they have also led to more persistent diseases tates that shift to an immunoparalysis phenotype, increasing the incidence of delayed death. For this reason, immunoparalysis caused by mid-to-late stage sepsis has become an area of keen interest in many laboratories around the world[3]. Therefore, there is an urgent need to study the pathogenesis of mid-to-late stage sepsis and identify more effective treatment methods; however, there remains a lack of appropriate animal models that can reflect the process of mid-to-late stage sepsis-induced immunoparalysis. In this study, we explored a dual model, termed a "two-hit" model, to evaluate whether it can be used to study the pathogenic mechanism of immunoparalysis in patients with mid-to-late stage sepsis. Materials And Methods Animals C57BL/6J mice aged 10-12 weeks old and weighing (24g-26g), were purchased from the Animal Experimental Center of Chongqing Medical University. The mice were housed under specific pathogen-free conditions at least one week before the experiments, with a 12 h dark/light cycle. All animal experimental operations were performed in accordance with Chongqing Management Approach of Laboratory Animal (Chongqing government order NO.195). Selection of an Appropriate Cecal Ligation and Puncture (CLP) Sepsis Model The mice were divided into the following four groups based on the length of cecal ligation: 3/4CLP,1/2CLP, 1/3CLP, and Sham group (n = 10 in each group).The survival rate of the four groups was observed on the fourth day after modeling. The mice were anesthetized with isoflurane (2%3%) and sterilized with iodophor after preparing the skin. Along the middle of the abdominal white line, the skin of the mice was cut approximately 0.8 cm long, the muscle layer was separated, and the abdominal cavity was explored to expose the cecum (the cecum was primarily located in the left lower abdomen of the mice, and damage to the cecum and blood vessels was avoided during exploration). The intestinal content was gently squeezed with forceps to fill the distal cecum, which was then ligated with a 3/0 silk suture. A 21-gauge needle was used to puncture the cecum at the distal end of the ligation (to avoid the blood vessels), which results in a cecal fistula, squeezing out some of the intestinal content, and forming a 1 mm fecal column. Finally, the cecum was carefully placed in the peritoneal cavity, and the abdomen was sutured layer by layer. The 3/4CLP group referred to the distal third-fourth of cecum ligation; the 1/2 CLP group referred to the distal one-half of cecum ligation; the 1/3 CLP group referred to the distal one-third of cecum ligation (Figure 1A); the Sham group only underwent laparotomy, without distal cecum ligation and puncture. After the operation, all mice were subcutaneously injected with 1 mL of saline and were placed back into the cage after waking up, without water and food restriction(Figure 1B). To control pain, buprenorphine s.c.was injected at a level of 0.05 mg/kg 30 min before operation and thereafter was repeated every 8 h. In the hands of skilled researchers, the CLP procedure can be done about 15-20 min for each animal. Additional time is required for postoperative care and specimen and data collection. According to the mortality rate of these groups, 1/3 CLP operation was selected for all subsequent experiments (Figure 2A). Selection of an Appropriate "two-hit" Model of Sepsis The standard strain of Pseudomonas aeruginosa ( P. aeruginosa )(ATCC 27853) was donated by the Department of Microbiology, the Second Affiliated Hospital of Chongqing Medical University. P. aeruginosa were grown overnight in Nalidixic Acid Cetrimide Broth Medium (Qingdao Rishui Bio-technologies Co., Ltd., Qingdao, China)at 37°C with constant shaking to the stationary phase. Different Pa inoculum densities were prepared using sterilized saline. At 4 days post-CLP, the surviving mice were divided into five groups (n = 10) and an esthetized with ether inhalation and raised vertically (Figure 1C). Next, 10 μL of different P. aeruginosa suspensions (3.0×10 9 , 2.5×10 9 , 2.0×10 9 , and 1.0×10 9 colony-forming unit (CFU/mL) was slowly dripped from the left nasal cavity using a pipette, and the operation was repeated four times. It was necessary to observe whether there were bubbles in the nasal cavity of the mice after each nasal drip to ensure that the bacterial liquid was inhaled into the lungs (Figure 1D). The control group was treated identically except that sterile saline was in stilled intranasally instead of the bacterial suspension. Survival was recorded for 11 days after CLP. Based on these mortality rates, 2.0×10 9 CFU/mL was selected for all subsequent experiments (Figure 3A). Study Design C57BL/6J mice were placed into one of four groups: 1) CLP + Pa group received CLP followed by intranasal P. aeruginosa at four days post-surgery; 2) the CLP + NS group received CLP followed by an intranasal administration of saline; 3) the Sham + Pa group received sham surgery followed by an intranasal administration of P. aeruginosa ; and 4) the Sham + NS group received sham surgery followed by intranasal saline. The weight of the mice was recorded, and they were sacrificed at 1, 4, 5, and 11 days post-CLP (n =8 mice/time point). The EDTA anticoagulation tube and disposable coagulation vacuum tube were used to collect blood. The blood in the disposable coagulation vacuum tube was placed at room temperature for 30 min, and centrifuged at 1000´ g at 4°C for 10 min, and the serum was stored at -80°C for later use. After euthanasia, the lungs, spleens, and ceca were removed. The lungs and spleens were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were stained with H&E. Routine Blood Test and ALT Routine blood tests were measured by the Department of Laboratory Medicine, the Second Affiliated Hospital of Chongqing Medical University. The level of serum ALT on days 1 and 4 post-CLP were measured using an Alanine Aminotransferase Assay Kit (Nanjing Jiancheng bioengineering institute, Nanjing, China). Bacterial Culture The blood in an EDTA anticoagulation tube on day1 post-CLP was diluted to 1:100 using sterile saline. The diluted blood was then evenly smeared onto blood agar plates using a sterile cotton swab. On day 1 post-CLP, the abdominal cavity of the mice was washed with 5 mL sterile saline to collect the abdominal cavity lavage fluid. The abdominal cavity lavage fluid was diluted to 1:1000 in the sterile saline and evenly smeared onto a blood agar plate. On day 5 post-CLP, the lungs were flushed with 1 mL sterile saline to collect the bronchoalveolar lavage fluid. Next, 10 μL of the bronchoalveolar lavage fluid was dropped onto the NAC solid medium. All of the above samples were incubated at 37°C for 24 h. Enzyme-linked Immunosorbent Assay (ELISA) Analysis The levels of serum IL-6 and TNF-α at all time points were detected using commercial ELISA kits (4A Biotech Co., Ltd, Beijing, China). The level of serum PCT was detected using a commercial ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) on days 1 and 4 post-CLP. The reagents and methods were operated in accordance with the manufacturer’s instructions. Lung Index and Spleen Index The removed lungs and spleens were dried with filter paper, weighed, and both the lung index and spleen index were calculated. The following formula was used: lung index = (lung mass/body mass) ×100%, spleen index = (spleen mass/body mass) × 100%. Flow Cytometry The percentage of CD4+ and CD8+ in the whole blood was detected by flow cytometry. The blood in the EDTA anticoagulation tube was dropped into erythrocyte lysis buffer (Tiangen Biotech Co., Ltd, Beijing, China) for 1 min at room temperature, and centrifuged at 2000 g for 5 min. The precipitates were collected and resuspended in 50 μL PBS, the antibodies were conjugated with specific fluorescent dyes, CD3-APC-A750 (Becton, Dickinson and Company), CD4-PerCP5.5 (Becton, Dickinson and Company), and CD8-FITC (Becton, Dickinson and Company)were added and incubated at 4°C for 30 min. Next, they were washed in 1 mL PBS and centrifuged at 2000 g for 5 min. The supernatant was discarded and the cells were resuspended in 200 μL PBS. Finally, the samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Co., Ltd, America). Statistical Analysis GraphPad Prism 8.2.1 software was used for statistical analysis. The survival rate was analyzed using a Log-rank (Mantel-Cox) test. Student’s t-tests and ANOVAs were used as appropriate. The results were displayed as the mean ± SD. A value of P < 0.05 was deemed to be statistically significant. Results Survival Rate s and Identification Results of the CLP Model of Sepsis The survival rate of the mice after CLP surgery was closely related to the length of cecal ligation. The four-day survival rates of the mice in the 3/4 CLP, 1/2CLP, 1/3CLP, and Sham groups were 10%, 40%, 70%, and 100%, respectively. As the mortality rates of the 3/4CLP and 1/2CLP group were too high and non-conducive to subsequent experiments, 1/3CLP was selected as the experimental model for all subsequent experiments (Fig.2a). The routine blood tests on day 1 post-CLP showed that the levels of white blood cells, neutrophils, lymphocytes, and monocytes in the CLP group were 2.25 ± 1.05, 0.47 ± 0.38, 1.56 ± 0.74, and 0.21 ± 0.139 (*10 3 /μL), respectively, which were significantly lower than those in the Sham group (P 0.05, Table1). On day 1 post-CLP, the level of serum ALT in the CLP group was 66.70 ± 36.33 Karu, which was significantly higher than that of Sham group (11.63 ± 2.23 karu, P 0.05, Fig.2b). The level of serum PCT in the CLP group was 958.4 ± 535.2 pg/mL and 607.1 ± 247.5 pg/mL on days 1 and 4 post-surgery, compared to only 124.1 ± 40.9 pg/mL and 156.4 ± 10.0 pg/mL in the Sham group. The level of serum PCT in the CLP group was significantly higher than that of the Sham group (P < 0.05; Fig.2c), indicating a severe infection in the CLP group. A large number of colonies with neat white round edges and smooth surfaces were observed in the blood culture dish and peritoneal lavage fluid culture dish of the CLP group on day 1 post-CLP; however, no such results were observed in the blood culture dish or peritoneal lavage fluid culture dish of the Sham group, and the blood agar medium was bright red (Fig.2d). Therefore, these results demonstrated that the CLP model of sepsis had been successfully established. Survival Rates and Identification Results of the "two-hit" Model of Sepsis According to the survival curve of the "two hit" model, on day 11 following CLP, four mice in the CLP + Pa group died, one mouse in the Sham + Pa group died, and all mice in the CLP + NS and Sham + NS groups survived (Fig.3b). Routines blood tests on day 5 post-CLP showed that the level of white blood cells, neutrophils, lymphocytes, and monocytes were 14.79± 1.28, 8.26± 0.57, 5.46± 0.74, and 1.03± 0.60 (*10 3 /μL) in the CLP + Pa group; 7.02± 1.5, 1.72± 0.55, 4.74± 1.79, and 0.35± 0.05 (*10 3 /μL) in the CLP+NS group; 10.80± 1.67, 2.25± 2.01, 7.14± 1.53, and 1.56± 1.02 (*10 3 /μL) in the Sham + Pa group; and 8.76 ± 0.68, 1.71 ± 0.61,6.20 ± 0.95, and 0.60 ± 0.29 (*10 3 /μL) in the Sham + NS group (Table.2). Compared with the CLP + Pa group, the white blood cells and neutrophils were significantly decreased in the three groups (P < 0.05). On day 5 post-surgery, the bronchoalveolar lavage fluids of the four groups were cultured. Green colonies were observed in the NAC solid medium in the CLP + Pa group and Sham + Pa group, and the number of colonies in the CLP + Pa group was greater than that in the Sham + Pa group. However, no colonies were observed in either the CLP + NS group or Sham + NS group (Fig.3c). We further observed the appearance of the lung tissue. On day 5 post-surgery, there were obvious bleeding points in the lung tissues of the CLP + Pa and Sham + Pa groups, especially in the CLP + Pa group. On day 11 post-surgery, the lung tissues of the surviving mice in the CLP+ Pa group were consolidated dark red in color, and the majority of the lung lobes were affected, whereas there was only partial lung tissue damage in the Sham+ Pa group. The lung tissue of the mice in the CLP+NS and Sham + NS groups was normal at the both time points (Fig.3d). HE slices of the lungs in the CLP + Pa and Sham + Pa groups showed that the lung tissues exhibited widened alveolar septum, damaged alveolar structure, hemorrhage, serous exudation, and a large amount of inflammatory cell infiltration on day 5 after the operation. The degree of lung tissue injury was more severe in the CLP + Pa group. On day 11 post-surgery, there was greater consolidation of the alveolar cavity, focal pulmonary interstitial hemorrhage, and a large number of inflammatory cell infiltration in the connective tissues of the alveoli and alveolar septum in the CLP + Pa group. In the Sham + Pa group, only part of the inflammatory cells had infiltrated into the lung tissue. In the CLP + NS and Sham + NS groups, the lung tissue HE staining showed no significant change (Fig.3e). On day 5 post-CLP, the lung indexes of the CLP + Pa , CLP + NS, Sham + Pa , and Sham + NS groups were calculated respectively: 0.72 ± 0.05%, 0.606 ± 0.02%, 0.70 ± 0.04%, and 0.51 ± 0.02%.The lung index of CLP + Pa group was significantly higher than that of the CLP + NS group and Sham + NS groups (P < 0.01). On day 11 post-CLP, the lung index of the four aforementioned groups were 0.74 ± 0.04%, 0.56 ± 0.02%, 0.66 ± 0.01%, and 0.51 ± 0.02%, respectively. The lung index of the CLP + Pa group was significantly higher than that of the other three groups (P < 0.01), indicating a serious lung infection in the CLP + Pa group (Fig.3f). Together these results prove that the "two-hit" model of sepsis was completely constructed. The Body Weight Results and Abdominal Organ Changes of the "two-hit" Model The body weight changes of the "two-hit" mice were continuously recorded within 11 days post-CLP. The results showed that the weight of the mice in the CLP operation group decreased continuously within 5 days after the operation, and increased slowly. The weight of the mice in the Sham operation group temporarily decreased on the first day after operation and subsequently underwent a gradual increase. The difference between the two groups was statistically significant (P < 0.01). After an intranasal instillation of P. aeruginosa , the weight of the mice in the Sham + Pa group decreased for a short time and gradually increased. On day 11 post-CLP, the weight gain of the mice in the CLP + Pa group was significantly lower than that in the other three groups(P < 0.01), which suggests that the infection in the CLP + Pa group was more serious than that of the other three groups (Fig.4a). In both the CLP + Pa and CLP + NS groups, pale necrosis occurred at the distal end of cecal ligation on day 1 post-CLP; white purulent necrosis, adhesion with surrounding tissues, and edema appeared on day 4; both purulent secretion and the area of abdominal adhesion increased on day 5; and the necrotic area was wrapped and could not be completely separated on day 11. However, in both of the Sham + Pa and Sham + NS groups, the shape and structure of the cecum were intact, and no abnormalities were present throughout the experiment (Fig.4b). The changes in the spleen size of each of the four groups were observed throughout the experimental cycle. From day 4 post-CLP, the spleens in the CLP + Pa and CLP + NS groups were significantly enlarged. On day 11 post-CLP, the size of the spleen in the CLP + Pa group was substantially larger than that of the other three groups, suggesting the inflammatory reaction in the CLP + Pa group was the most severe (Fig.4c). On day 1, 4, 5 and 11 post-CLP, the spleen capsule in the CLP + Pa group gradually thickened, multinucleated macrophages gradually increased, and the number of white pulp decreased significantly, and the white pulp and red pulp boundary in the CLP + Pa group gradually blurred. The number of white pulp in the CLP + NS group was slightly reduced, and the boundary between the white pulp and the red pulp was still distinguishable. The fibrous capsule in the Sham + Pa group was thickened, and the ratio of white pulp to red pulp was normal. The capsule of the spleen tissue in the Sham + NS group was smooth, the Shape and size of the white pulp was regular, the boundary between the white and red pulp was clear and there was no abnormality (Fig.4d). The spleen index was calculated at four different time points. The results showed that the spleen index was 0.26 ± 0.03%, 0.524 ± 0.06%, 0.70 ± 0.04%, and 1.36 ± 0.26%, respectively, in the CLP + Pa group; 0.25 ± 0.04%, 0.50 ± 0.03%, 0.67 ± 0.11%, and 0.89 ± 0.06%, respectively, in the CLP + NS group; 0.23 ± 0.02%, 0.29 ± 0.03%, 0.32 ± 0.03%, and 0.45 ± 0.03%, respectively, in the Sham + Pa group; and 0.24 ± 0.05%, 0.27 ± 0.05%, 0.29 ± 0.05%, and 0.28 ± 0.04%, respectively, in the Sham + NS group. The spleen index of the mice in the CLP operation group was significantly higher than that in Sham operation group(P < 0.01). However, on day 11 after CLP, the spleen index of the mice in the CLP + Pa group also significantly differed from that in the CLP + NS group(P < 0.01), which further highlighted the severity of the infection in the CLP + Pa group (Fig.4e). Our results showed that the spleens were in a state of hypersplenism due to infections and the immune cells were continuously consumed. Immunization Results of the "two-hit" Model On day 11 post-CLP, the white blood cells in the CLP + Pa group were 31.74 ± 7.14*10 3 /μL, which was significantly higher than that in the CLP + NS group , Sham + Pa group and Sham + NS group (P < 0.05 or P < 0.01); the monocytes were 3.11 ± 1.28*10 3 /μL, which was significantly higher than that in the CLP + NS and Sham + NS groups (P < 0.05). However, since the number of neutrophils and lymphocytes in the CLP + Pa group were exceeded the detection range, statistical analysis could not be performed (Table 3). We further observed the changes in the level of TNF-α and IL-6. On day 1 post-CLP, the level of serum TNF-α was 167.00 ± 42.68 pg/mL in the CLP group and 45.90 ± 0.38pg/mL in the Sham group (P < 0.01), indicating that TNF-α was activated following CLP. On day 5 post-CLP, the level of serum TNF-α in the CLP + Pa and Sham + Pa groups were 110.69 ± 9.18pg/mL and 148.43 ± 13.42pg/mL, respectively. These findings were significantly different from those in the CLP + NS group (45.90 ± 0.38pg/mL) and Sham + NS group (45.81 ± 0.77pg/mL) (P < 0.0001), which suggests that serum TNF-α was produced after an intranasal instillation of P. aeruginosa . The level of serum TNF-α in the CLP + Pa group was significantly lower than that in the Sham + Pa group (P < 0.01), suggesting that the CLP operation may inhibit TNF-α reactivation. However, no significant difference was observed regarding the level of serum TNF-α between the four groups on days 4 and 11 post-CLP (Fig.5a). Moreover, the change trend of IL-6 was similar to that of TNF-α (Fig.5b). The ratio of CD4+/CD8+ in whole blood was also detected at four different time points by flow cytometry to observe the cellular immune function. The results showed that the ratio of CD4+/CD8+ in the CLP + Pa group was 0.57 ± 0.10 on day 11 post-CLP, which was significantly lower than that of the other three groups (the ratio of CD4+/CD8+ in the CLP + NS group, Sham + Pa group, and Sham + NS group was 0.91 ± 0.04, 1.17 ± 0.03, and 1.48 ± 0.04, respectively) (P < 0.05 or P < 0.01, Fig.5c,d). These findings indicate that the "two-hit" model of sepsis may lead to the the decreased presence of immune cells . The above results indicate that the "two- hit" model of sepsis could lead to immunoparalysis. Discussion As the name suggests, the "two-hit" animal model simulates an attack by two pathogens on the host, induced by CLP surgery and secondary infection. The CLP model of sepsis mainly simulates clinical sepsis induced by acute peritonitis, which currently represents the gold standard in animal models for studying sepsis[7]. The course of sepsis can pass through different stages, which are clearly defined by human physiological parameters. However, no physiological monitoring exists in animal models and the severity of sepsis can only be determined by mortality and time of death[8]. In our experimental model, we reproduced three CLP models by selecting different lengths of cecal ligation. By observing the survival rate and general situation of the mice at four days post-CLP, 1/3 cecal ligation was selected as the experimental model as it caused mild sepsis. Cytokine storm syndrome is induced by innate immune activation during the early stage of sepsis, which may lead to multiple organ failure and early death[9]. Procalcitonin (PCT) is a calcitonin-promoting hormone that is commonly used as a clinical infection index. The level of serum PCT in sepsis patients is significantly increased, and can be detected within 24 h post-infection, peaks at 12-24 h, and is closely related to the severity of infection[10]. PCT was detected in our mouse model of sepsis, which is in accordance with the response of clinical patients. TNF-α and IL-6 are the important mediators of the acute phase of the inflammatory response. TNF-α expression is closely related to the severity of sepsis, which can be used to predict the severity of sepsis[11]. IL-6 is an important pro-inflammatory cytokine in the early stage of inflammation, which increases in blood circulation after infection and trauma[12]. Our findings showed that the levels of PCT, TNF-α, and IL-6 were significantly increased on day 1 post-CLP, which confirmed the presence of a cytokine storm syndrome. Moreover, the level of serum ALT also displayed a significant increase, which coincided with acute organ dysfunction. This phenomenon is consistent with the clinical observation that sepsis can cause multiple organ dysfunction. At present, clinical studies have confirmed that patients with acute sepsis have a high probability of death due to secondary infection after the first three days or longer after the onset of sepsis [13]. One of the main causes of death is secondary infection caused by sepsis-induced immune dysfunction, whereas pneumonia is a common secondary infection[9]. P.aeruginosa is the most common multidrug-resistant gram-negative bacteria that can cause ventilator-associated pneumonia(VAP) and nosocomial pneumonia[9]. In addition, compared with other types of pneumonia, patients with P . aeruginosa pneumonia are more prone to multiple organ failure and death[14-15]. Therefore, P . aeruginosa represents the best choice as a "two-hit" sepsis model strain. It has been reported that the immune dysfunction turning point for sepsis induced by CLP may occur on day 4 post-CLP[16]. Our study also confirmed that CLP mice no longer died from day 4 post-CLP. Thus, we chose to administer the P . aeruginosa suspension via a "nasal drip" infection at day 4 post-CLP as a second hit to observe the general condition and survival rate of the mice. The bronchoalveolar lavage on day 5 post-CLP and the survival rate of the "two-hit" model revealed that this model could successfully simulate sepsis patients with secondary infection. As we all know, neutrophils mainly play the role of the first line of defense in inflammation sites, such as phagocytosis, degranulation and formation of reactive oxygen species[17]. Routines blood tests of the "two-hit" model showed that the number of neutrophils gradually increased from day 5 post-CLP, and it was more than three times of that of the normal mice on day 11 post-CLP. Combined with manifestation of hypersplenism in mice, we thought it was a leukemoid reaction caused by severe infection. Leukemoid reaction refers to the continuous increase of neutrophils, more than 50000 cells per microliter, accompanied by the increase of neutrophil precursors when the cause of leukemia is excluded.The main cause of leukemoid reaction are severe infection, poisoning, malignant tumor, severe hemorrhage or acute hemolysis[18]. However, there is no diagnostic criteria for leukemia reaction of murine. The derangements in the inflammatory response and a state of adaptive immunoparalysis, which are characterized by the decrease of primary humoral immune response[19]. On day 5 post-CLP, the levels of TNF-α and IL-6 in the CLP + Pa group were significantly higher than those in the CLP+NS and Sham + NS groups, but lower than those in the Sham + Pa group. The phenomenon is caused by excessive consumption of inflammatory factors in humoral immune dysfunction after CLP. Splenic pulp includes white pulp(WP,cells with weakly stained nuclei), red pulp(RP) and the marginal zone(MZ).The RP is composed of blood vessels, lining endothelial cells, and macrophages. These macrophages screen and remove pathogens and soluble antigens from the systemic circulation. WP represents lymphoid tissue, which is composed of central arterioles, lymphocytes and antigen-presenting cells (mainly dendritic cells and macrophages).WP produces specific immune responses to antigens[20]. The pathological section of mice found that the white pulp in the CLP+ Pa group was significantly reduced compared with the other three groups, which indicated that the immune function of the "two-hit" model has been compromised. Patients who survive the acute phase of sepsis show long-term damage to immune function due to the decrease in the number and function of many immune cell populations[21]. With the increasing use of antibiotics and various anti-inflammatory factors in recent years, most patients will survive the systemic acute inflammatory reaction period, but subsequently enter a longer-lasting immune dysfunction state [22]. Studies have found that the key cause of immune dysfunction in patients with sepsis is the death of lymphocytes, especially T and B lymphocyte apoptosis, is a major factor in immune dysfunction[23]. CD4+ can help B lymphocytes produce antibodies and induce T lymphocytes to transform into effector cells, and is a surface marker of Ti/Th. CD8 + mediates the destruction and lysis of host cells after pathogen infection, and is a protein in Ts. In mammals, the ratio of CD4 +/CD8 + is normally> 1, depending on the species[24]. And the CD4+/CD8+ ratio is highly correlated with host immune function. An increase in CD4 positive cells and an increase in the CD4/CD8 ratio indicate a strong immune system[25]. Therefore, by continuously monitoring the ratio of CD4+ and CD8+ T cells in the whole blood, we found that the CD4+/CD8+ ratio in the mice in the "two-hit" model was significantly lower than that of the other three groups on day 11 post-surgery, which was consistent with the immune dysfunction status of patients with advanced sepsis. However, there are limitations associated with the "two-hit" model. First, the impact of cecal puncture on humans is a fatal blow but the impact on rodents is not obvious[26]. Secondly, the severity of sepsis caused by CLP is related to multiple factors, including the length of cecal ligation, the puncture needle size, and the number of perforations. Of course, the stability of the model is also closely related to the gender and age of the mice. Thirdly, the "two hits" are administered by “nasal drip”; however, in the process of administering the “nasal drip”, the bacterial solution may be swallowed by the mice rather than inhaled, which may cause different degrees of infection. Fourth, sepsis patients are often accompanied with endocrine, tumor, and other diseases in the clinic. The establishment of a "two-hit" model with only healthy mice could not cover all clinical conditions. Finally, a mouse model cannot completely simulate the changes in the whole blood and dynamic changes in the inflammatory factors in patients with sepsis. Conclusions This "two-hit" model focuses on replicating the process of clinical sepsis patients entering the immune dysfunction stage during the early stages of sepsis. The immune function of the mice was evaluated by the continuous observation of routine blood tests, inflammatory factors, and the CD4+/CD8+ ratio. Together, these findings demonstrate that this "two-hit" model can be used as an appropriate animal model to study the mechanism of immune dysfunction in sepsis and the associated drug development. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81871608). We thank International Science Editing ( http://www.internationalscienceediting.com ) for editing this manuscript. References Singer, M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche J, Coopersmith CM, Hotchkiss RS, Levy MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent J, Angus DC. 2016. The Third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 315: 801-810. Lou, JS, Wang JF, Fei MM, Zhang Y, Wang J, Guo Y, Bian JJ, Deng XM. 2020. Targeting lymphocyte activation gene 3 to reverse T-lymphocyte dysfunction and improve survival in murine polymicrobial sepsis. Journal of Infectious Diseases 222: 1051-1061. Patil, NK, Bohannon JK, Sherwood ER. 2016. Immunotherapy: a promising approach to reverse sepsis-induced immunosuppression. Pharmacological Research 111: 688-702. Rubio, I, Osuchowski MF, Shankar-Hari M, Skirecki T, Winkler MS, Lachmann G, La Rosée P, Monneret G, Venet F, Bauer M, Brunkhorst FM, Kox M, Cavaillon J, Uhle F, Weigand MA, Flohé SB, Wiersinga WJ, Martin-Fernandez M, Almansa R, Martin-Loeches I, Torres A, Giamarellos-Bourboulis EJ, Girardis M, Cossarizza A, Netea MG, van der Poll T, Scherag A, Meisel C, Schefold JC, Bermejo-Martín JF. 2019. Current gaps in sepsis immunology: new opportunities for translational research. The Lancet Infectious Diseases 19: e422-e436. Cao, C, Yu M, Chai Y. 2019. Pathological alteration and therapeutic implications of sepsis-induced immune cell apoptosis. Cell Death & Disease 10:782. Schenz, J, Tamulyte S, Nusshag C, Brenner T, Poschet G, Weigand MA, Uhle F. 2020. Population-specific metabolic alterations in professional antigen-presenting cells contribute to sepsis-associated immunosuppression. Shock 53: 5-15. Wang, Z, Pu Q, Lin P, Li C, Jiang J, Wu M. 2019. Design of cecal ligation and puncture and intranasal infection dual model of sepsis-induced immunosuppression. J Vis Exp (148): 10.3791/59386. Kingsley, SMK, Bhat BV. 2016. Differential paradigms in animal models of sepsis. Current Infectious Disease Reports 18: 26. Carvelli, J, Piperoglou C, Bourenne J, Farnarier C, Banzet N, Demerlé C, Gainnier M, Vély F. 2019. Imbalance of circulating innate lymphoid cell subpopulations in patients with septic shock. Frontiers in Immunology 10: 2179. Wodehouse, T, Demopoulos M, Petty R, Miraki-Moud F, Belhaj A, Husband M, Fulton L, Randive N, Oksche A, Mehta V, Gribben J, Langford R. 2019. A randomized pilot study to investigate the effect of opioids on immunomarkers using gene expression profiling during surgery. Pain 160: 2691-2698. Chen, F, Xu W, Gao H, Wu L, Zhang H, Cheng L, Mei J. 2020. Clinical effect of Changweishu on gastrointestinal dysfunction in patients with sepsis. Journal of International Medical Research 48: 1410562101. Martin, JB, Badeaux JE. 2017. Interpreting laboratory tests in infection: making sense of biomarkers in sepsis and systemic inflammatory response syndrome for intensive care unit patients. Critical Care Nursing Clinics of North America 29: 119-130. Benjamim, CF, Hogaboam CM, Kunkel SL. 2004. The chronic consequences of severe sepsis. Journal of Leukocyte Biology 75: 408-412. Brewer, SC, Wunderink RG, Jones CB, Leeper KV. 1996. Ventilator-associated pneumonia due to pseudomonas aeruginosa. Chest 109: 1019-1029. Taylor, GD, Buchanan-Chell M, Kirkland T, McKenzie M, Wiens R. 1995. Bacteremic nosocomial pneumonia. a 7-year experience in one institution. Chest 108: 786-788. Remick, DG. 2007. Pathophysiology of sepsis. The American Journal of Pathology 170: 1435-1444. Vorobjeva, NV. 2020. Neutrophil extracellular traps: new aspects. Moscow University biological sciences bulletin 75: 173-188. Sakka, V, Tsiodras S, Giamarellos-Bourboulis EJ, Giamarellou H. 2006. An update on the etiology and diagnostic evaluation of a leukemoid reaction. European Journal of Internal Medicine 17: 394-398. Martire-Greco, D, Landoni VI, Chiarella P, Rodriguez-Rodrigues N, Schierloh P, Rearte B, Isturiz MA, Fernandez GC. 2014. All-trans-retinoic acid improves immunocompetence in a murine model of lipopolysaccharide-induced immunosuppression. Clinical Science 126: 355-365. Duarte-Neto, AN, Croda J, Pagliari C, Soriano FG, Nicodemo AC, Duarte MIS. 2019. Severe leptospirosis features in the spleen indicate cellular immunosuppression similar to that found in septic shock. Frontiers in Immunology 10: 920. Martin, MD, Badovinac VP, Griffith TS. 2020. CD4 T cell responses and the sepsis-induced immunoparalysis state. Frontiers in Immunology 11: 1364. Suzuki, T, Inoue K, Igarashi T, Kato J, Nagata H, Yamada T, Minamishima S, Morisaki H. 2019. Beta-blocker therapy preserves normal splenic T-lymphocyte numbers reduced in proportion to sepsis severity in a sepsis model. Critical Care Research and Practice 2019:8157482. Cheng, Z, Abrams ST, Toh J, Wang SS, Wang Z, Yu Q, Yu W, Toh C, Wang G. 2020. The critical roles and mechanisms of immune cell death in sepsis. Frontiers in Immunology 11:1918. Tian, H, Zhang M, Du C, Li D, Zhou Q, Wu L, Meng F, Song S, Wang L, Lu P, Zhao Z, Yang X. 2015. Effects of rhubarb combined with ulinastatin on T-cell subsets in sepsis rats. International Journal of Clinical and Experimental Medicine 8: 1234-1240. Wang, J, Zhou J, Bai S. 2020. Combination of glutamine and ulinastatin treatments greatly improves sepsis outcome. Journal of Inflflammation Research 13: 109-115. Li, W, Zhu S, Zhang Y, Li J, Sama AE, Wang P, Wang H. 2012. Use of animal model of sepsis to evaluate novel herbal therapies. Journal of Visualized Experiments (62):3926. Tables Table 1. Changes in routine blood test s on days 1 and 4 post-CLP Group Day 1 Day 4 CLP Sham CLP Sham WBC （ *10 3 /μl) 2.25±1.05 10.3±1.43 *** 6.55±1.6 7.11±1.56 NEUT#(*10 3 /μl) 0.47±0.38 1.30±0.37 * 0.98±0.69 1.09±0.14 LYMPH# (*10 3 /μl) 1.56±0.74 8.16±1.33 *** 3.99±1.15 5.37±1.42 MONO#(*10 3 /μl) 0.21±0.139 0.65±0.312 * 1.55±1.18 0.38±0.1 Compared with the CLP group , * P < 0.05; *** P < 0.001. WBC, white blood cell; NEUT#, neutrophil count; LYMPH#, lymphocyte count; MONO#, monocyte count . Table 2 . Changes in routine blood test s on day 5 post-CLP Group CLP+ Pa CLP+NS Sham+ Pa Sham+NS WBC （ *10 3 /μl) 14.79±1.28 7.02±1.5 *** 10.80±1.67 * 8.76±0.68 **** NEUT#(*10 3 /μl) 8.26±0.57 1.72±0.55 **** 2.25±2.01 ** 1.71±0.61 **** LYMPH# (*10 3 /μl) 5.46±0.74 4.74±1.79 7.14±1.53 6.20±0.95 MONO#(*10 3 /μl) 1.03±0.60 0.35±0.05 1.56±1.02 0.60±0.29 Compared with the CLP + Pa group, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. WBC, white blood cell ; NEUT#, neutrophil count; LYMPH#, lymphocyte count; MONO#, monocyte count. Table 3. Changes in routine blood test s on day 11 post-CLP Group CLP+ Pa CLP+NS Sham+ Pa Sham+NS WBC （ *10 3 /μl) 31.74±7.14 11.09±0.57 ** 14.4±3.98 * 9.23±0.44 ** NEUT#(*10 3 /μl) ━ 1.88±0.18 5.23 ±0.07 3.92±1.23 LYMPH# (*10 3 /μl) ━ 7.97±0.61 4.30 ± 2.11 4.68±1.00 MONO#(*10 3 /μl) 3.11±1.28 0.88±0.25 * 2.04±0.94 0.21±0.02 * Compared with the CLP + Pa group, * P < 0.05; ** P < 0.01; ━ : Above the measuring range. WBC, white blood cell ; NEUT#, neutrophil count; LYMPH#, lymphocyte count; MONO#, monocyte count. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-474587","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":29584487,"identity":"ebbfb6ab-a228-4cb0-8d91-79a4aa07b29d","order_by":0,"name":"Fei Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Li","suffix":""},{"id":29584488,"identity":"4e509cbd-77a0-437d-9131-a4d6e178bf19","order_by":1,"name":"Chuanxin Wu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chuanxin","middleName":"","lastName":"Wu","suffix":""},{"id":29584489,"identity":"871abe48-f60d-4951-a3bb-2e7d4c09986d","order_by":2,"name":"Jiahui He","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"He","suffix":""},{"id":29584490,"identity":"4cd001db-3ead-424a-b1c7-8a756f40e017","order_by":3,"name":"Jie Zhang","email":"","orcid":"","institution":"Chengdu Shuangliu District Maternal and Child Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhang","suffix":""},{"id":29584491,"identity":"ad874390-cedc-48a8-8e48-20315c5b3583","order_by":4,"name":"Ying Chen","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":29584492,"identity":"6e8a097c-1840-4e86-9c42-5b11d2e9e046","order_by":5,"name":"Shuhua Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuhua","middleName":"","lastName":"Li","suffix":""},{"id":29584493,"identity":"3b9a021b-3b5c-4fcf-994d-7d1064cba6ef","order_by":6,"name":"Li Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Li","suffix":""},{"id":29584494,"identity":"6b4d2c0d-022a-4883-9cee-86a6973351bf","order_by":7,"name":"Hang Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACeWbGxgcJP2ygXDYitBi2Nx82eNiTRoIWhjPH0gQfsB0mQQvjjBwzhgSe8/J8184YMHwoO8zAP7sBvxZ2iRyzBwkWtw1n3s4xYJxx7jCDxJ0DBG0xN0jguZ1gANTCzNt2mMFAIoGAy27kmEkksJ2DaPlLlBag94FaDkC0MBKjBRzIiT3JQL+kFRzsOZfOI3GDgBZQVD788cNOnu928sYHP8qs5fhnEHIYHBwAIwYeYtVDtIyCUTAKRsEowAoAruJHJG98c9oAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2302-1993","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2021-04-29 00:43:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-474587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-474587/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9719027,"identity":"0d6fef9b-92d1-4e2c-940e-82df597c09e4","added_by":"auto","created_at":"2021-05-28 17:52:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":491043,"visible":true,"origin":"","legend":"The detailed process of establishing the \"two-hit\"sepsis mouse model. a.Schematic illustration of characterized positions of cecal ligation to induce 3/4 CLP,1/2 CLP and 1/3 CLP in mice. b. CLP and Sham operations were performed. A median laparotomy of 0.8 cm was performed and the cecum was exposed. The distal one-third of the cecum was ligated with 3/0 silk suture and punctured once with a 21-gaugeneedle. The cecum was gently squeezed to extrude some feces before returning it to the abdomen. The Sham group only underwent a laparotomy, without distal cecum ligation and puncture.The abdominal wall and skin were closed in two layers and 1mL saline was administered intraperitoneally after surgery. c.The surviving mice on day 4 post-CLP were anesthetized with ether inhalation. d. The mice were infected with Pseudomonas aeruginosa via \"nasal drops\" under anesthesia.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/bd0cf315b8c19b1c1553df3f.png"},{"id":9719028,"identity":"559f612d-eee6-49cf-b570-73ae3923f080","added_by":"auto","created_at":"2021-05-28 17:52:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265246,"visible":true,"origin":"","legend":"The survival rates and identification results associated with the CLP model of sepsis. a. The survival rate of the mice was observed and recorded within 4 days(n = 10). Based on the survival curves, a 1/3 CLP operation was selected for subsequent experiments. b.The changes in the level of serum ALT in the CLP and Sham group on days 1 and 4 post-CLP. c.Changes in the level of serum PCT in the CLP and Sham groups on days 1 and 4 post-CLP. d. The results of blood and peritoneal lavage fluid cultures in the CLP and Sham groups on day 1 post-CLP. Compared with the CLP group,*P \u003c 0.05.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/112ccf6c5510baef0d06c569.png"},{"id":9718854,"identity":"a25de5bd-8911-4edd-96ad-d514f9d2839d","added_by":"auto","created_at":"2021-05-28 17:49:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326789,"visible":true,"origin":"","legend":"Survival curves and identification results of the\"two-hit\" model of sepsis. a. Selection of the optimal suspension concentration of P.aeruginosa(Pa). At 4 days post-CLP, the surviving mice were divided into five groups (n=10) and administered different concentrations of Pa suspension (3.0×109, 2.5×109, 2.0×109, or 1.0×109 CFU/mL) via a 40 μL intranasal drip.The survival rates were observed and recorded for 11 days post-CLP. According to the survival curve, 2×109 CFU/mL was selected for all subsequent experiments. b.The survival rates between the four groups (n = 8). c. The results of the bronchoalveolar lavage fluid culture of the different groups of mice on day 5 post-CLP. d. Morphological characteristics of the lungs in the mice from each group on days 5 and 11 post-CLP. e. H\u0026E staining of the lungs of mice from each group on days 5 and 11 post-CLP. Magnification: 200× and 400×. F.Comparison of the lung index of mice in each group on days 5 and 11 post-CLP. Compared with the CLP + Pa group. ** P \u003c 0.01;**** P \u003c 0.0001","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/ccc3464a2185f5516e87800b.png"},{"id":9718856,"identity":"a3eb3fdd-e174-43ed-bd29-a3ce9a8d927e","added_by":"auto","created_at":"2021-05-28 17:49:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":439325,"visible":true,"origin":"","legend":"Clinical manifestations and tissue damage in the \"two-hit\" model mice. a. Changes in the body weight of mice inthe CLP + Pa, CLP + NS, Sham + Pa, and Sham + NS groups. Compared with the CLP+Pa group, *P \u003c 0.0001. b. Morphological characteristics of the cecum in the \"two-hit\" model at different time points post-CLP. c. Morphological characteristics of the spleens in the \"two-hit\" model at different time points post-CLP. d. H\u0026E staining of the spleens of mice from each group at different time. Magnification: 200× and 400×. e.The spleen index of the \"two-hit\" model at different time points post-CLP. Compared with the CLP+Pa group, ** P \u003c 0.01; *** P \u003c 0.001; **** P \u003c 0.0001.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/f7de1c1b7a89a37721ea99c1.png"},{"id":9718857,"identity":"386f38d6-2fed-46c2-aed5-c03b1bae4822","added_by":"auto","created_at":"2021-05-28 17:49:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":189622,"visible":true,"origin":"","legend":"Immunization damage in the \"two-hit\" model mice. Changes in the level of serum cytokines TNF-α (a),IL-6 (b), and whole blood CD4+/CD8+ ratio (c) in mice that received the \"two-hit\" model at different time points post-CLP. The levels of TNF-α and IL-6 were determined by an ELISA. (d) The percentage of CD4+ and CD8+ in the whole blood were detected by flow cytometry. Data are presented as the mean±SD (n = 8 mice per group). Compared with the CLP + Pa group, * P \u003c 0.05; ** P \u003c 0.01; **** P \u003c 0.0001.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/c0625e3f61b30b9e1158b658.png"},{"id":13695780,"identity":"c4467d3c-7577-4c06-b460-c18a1cd37abe","added_by":"auto","created_at":"2021-09-17 12:59:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1974223,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-474587/v1/092917cb-0617-4572-8721-f675a0a9f549.pdf"}],"financialInterests":"","formattedTitle":"Establishment and Evaluation of a \"Two-Hit\" Mouse Model of Sepsis","fulltext":[{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics \u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003epproval and \u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eonsent to \u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003earticipate\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The study protocol was approved by the Institutional Review Committee of The Second Affiliated Hospital of Chongqing Medical University. And all applicable institutional and governmental regulations concerning the ethical use of animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e All authors contributed to the writings and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Da\u003c/strong\u003e\u003cstrong\u003eta \u003c/strong\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eaterials\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest: \u003c/strong\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u003cbr /\u003e Funding: \u003c/strong\u003eThis work was supported by a grant from the National Natural Science Foundation of China (No. 81871608).\u003cbr /\u003e \u003cstrong\u003eAuthors' Contributions: \u003c/strong\u003eFL, HS and CXW designed performed the study, and draft the manuscript. FL ,JH,JZ established the animal model and collected specimens.YC，SL，LL performed data analysis and statistical analyses. HS made critical revisions and provided guidance for further statistical analysis. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection[1], and is a complex disease with both high morbidity and mortality. Sepsis is much more dangerous than the infection itself, and there are over 18 million cases of severe sepsis worldwide each year. Although most sepsis patients can survive in the acute phase due to the progress made regarding ICU support treatment, sepsis-induced immunoparalysis leading to secondary infection is becoming a leading cause of death[2] .\u003c/p\u003e\n\u003cp\u003eRecent research has shown that the peak time of sepsis-induced death can be divided into three stages. The early peak is caused by the inflammatory response in the initial days of sepsis and a late peak after several weeks that is due to organ damage or immunoparalysis. The third upswing occurs after 60-90 days due to persistent immune dysfunction and inflammation in patients with other comorbidities or advanced age[3].\u003c/p\u003e\n\u003cp\u003eThe definition of Sepsis 3.0 does not clearly define the nature and mechanism of the disordered host response to infection, which indicates that researchers continue to have an insufficient understanding of the pathophysiological mechanism of the pathogenesis of advanced immunoparalysis in sepsis[4]. The mechanism currently accepted by researchers is that innateand adaptive immune responses of the host are involved in the pathogenesis of sepsis[5]. The early activation of the innate immune response is the first line of defense against infection and plays a central role in the initiation process of adaptive immunity. After the onset of sepsis, a strong pro-inflammatory response accompanied by an anti-inflammatory response will generally last for several days[5], limiting inflammation and protecting the host. However, if such a compensatory response prevails, it will cause a large amount of death of inflammatory mediators and immune cells in the body. This leads to a more lasting disease state of the host, which is likely to cause secondary infection and viral reactivation in the host [6]. Although modern medical strategies have improved the short-term outcomes in patients with sepsis, they have also led to more persistent diseases tates that shift to an immunoparalysis phenotype, increasing the incidence of delayed death. For this reason, immunoparalysis caused by mid-to-late stage sepsis has become an area of keen interest in many laboratories around the world[3].\u003c/p\u003e\n\u003cp\u003eTherefore, there is an urgent need to study the pathogenesis of mid-to-late stage sepsis and identify more effective treatment methods; however, there remains a lack of appropriate animal models that can reflect the process of mid-to-late stage sepsis-induced \u0026nbsp;immunoparalysis. In this study, we explored a dual model, termed a \"two-hit\" model, to evaluate whether it can be used to study the pathogenic mechanism of immunoparalysis in patients with mid-to-late stage sepsis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J mice aged 10-12 weeks old and weighing (24g-26g), were purchased from the Animal Experimental Center of Chongqing Medical University. The mice were housed under specific pathogen-free conditions at least one week before the experiments, with a 12 h dark/light cycle. All animal experimental operations were performed in accordance with Chongqing Management Approach of Laboratory Animal (Chongqing government order NO.195).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of an Appropriate Cecal Ligation and Puncture (CLP) Sepsis Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were divided into the following four groups based on the length of cecal ligation: 3/4CLP,1/2CLP, 1/3CLP, and Sham group (n = 10 in each group).The survival rate of the four groups was observed on the fourth day after modeling.\u003c/p\u003e\n\u003cp\u003eThe mice were anesthetized with isoflurane (2%3%) and sterilized with iodophor after preparing the skin. Along the middle of the abdominal white line, the skin of the mice was cut approximately 0.8 cm long, the muscle layer was separated, and the abdominal cavity was explored to expose the cecum (the cecum was primarily located in the left lower abdomen of the mice, and damage to the cecum and blood vessels was avoided during exploration). The intestinal content was gently squeezed with forceps to fill the distal cecum, which was then ligated with a 3/0 silk suture. A 21-gauge needle was used to puncture the cecum at the distal end of the ligation (to avoid the blood vessels), which results in a cecal fistula, squeezing out some of the intestinal content, and forming a 1 mm fecal column. Finally, the cecum was carefully placed in the peritoneal cavity, and the abdomen was sutured layer by layer. The 3/4CLP group referred to the distal third-fourth of cecum ligation; the 1/2 CLP group referred to the distal one-half of cecum ligation; the 1/3 CLP group referred to the distal one-third of cecum ligation (Figure 1A); the Sham group only underwent laparotomy, without distal cecum ligation and puncture. After the operation, all mice were subcutaneously injected with 1 mL of saline and were placed back into the cage after waking up, without water and food restriction(Figure 1B). To control pain, buprenorphine s.c.was injected at a level of 0.05 mg/kg 30 min before operation and thereafter was repeated every 8 h. In the hands of skilled researchers, the CLP procedure can be done about 15-20 min for each animal. Additional time is required for postoperative care and specimen and data collection. According to the mortality rate of these groups, 1/3 CLP operation was selected for all subsequent experiments (Figure 2A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of an Appropriate \"two-hit\" Model of Sepsis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe standard strain of \u003cem\u003ePseudomonas aeruginosa \u003c/em\u003e(\u003cem\u003eP. aeruginosa\u003c/em\u003e)(ATCC 27853) was donated by the Department of Microbiology, the Second Affiliated Hospital of Chongqing Medical University. \u003cem\u003eP. aeruginosa \u003c/em\u003ewere grown overnight in Nalidixic Acid Cetrimide Broth Medium (Qingdao Rishui Bio-technologies Co., Ltd., Qingdao, China)at 37\u0026deg;C with constant shaking to the stationary phase. Different\u003cem\u003e Pa \u003c/em\u003einoculum densities were prepared using sterilized saline.\u003c/p\u003e\n\u003cp\u003eAt 4 days post-CLP, the surviving mice were divided into five groups (n = 10) and an esthetized with ether inhalation and raised vertically (Figure 1C). Next, 10 \u0026mu;L of different\u003cem\u003e P. aeruginosa\u003c/em\u003e suspensions (3.0\u0026times;10\u003csup\u003e9\u003c/sup\u003e, 2.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e, 2.0\u0026times;10\u003csup\u003e9\u003c/sup\u003e, and 1.0\u0026times;10\u003csup\u003e9\u003c/sup\u003ecolony-forming unit (CFU/mL) was slowly dripped from the left nasal cavity using a pipette, and the operation was repeated four times. It was necessary to observe whether there were bubbles in the nasal cavity of the mice after each nasal drip to ensure that the bacterial liquid was inhaled into the lungs (Figure 1D). The control group was treated identically except that sterile saline was in stilled intranasally instead of the bacterial suspension. Survival was recorded for 11 days after CLP. Based on these mortality rates, 2.0\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL was selected for all subsequent experiments (Figure 3A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J mice were placed into one of four groups: 1) CLP + \u003cem\u003ePa \u003c/em\u003egroup received CLP followed by intranasal \u003cem\u003eP. aeruginosa\u003c/em\u003e at four days post-surgery; 2) the CLP + NS group received CLP followed by an intranasal administration of saline; 3) the Sham + \u003cem\u003ePa\u003c/em\u003e group received sham surgery followed by an intranasal administration of \u003cem\u003eP. aeruginosa\u003c/em\u003e; and 4) the Sham + NS group received sham surgery followed by intranasal saline.\u003c/p\u003e\n\u003cp\u003eThe weight of the mice was recorded, and they were sacrificed at 1, 4, 5, and 11 days post-CLP (n =8 mice/time point). The EDTA anticoagulation tube and disposable coagulation vacuum tube were used to collect blood. The blood in the disposable coagulation vacuum tube was placed at room temperature for 30 min, and centrifuged at 1000\u0026acute; g at 4\u0026deg;C for 10 min, and the serum was stored at -80\u0026deg;C for later use. After euthanasia, the lungs, spleens, and ceca were removed. The lungs and spleens were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were stained with H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoutine Blood Test and ALT \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRoutine blood tests were measured by the Department of Laboratory Medicine, the Second Affiliated Hospital of Chongqing Medical University. The level of serum ALT on days 1 and 4 post-CLP were measured using an Alanine Aminotransferase Assay Kit (Nanjing Jiancheng bioengineering institute, Nanjing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial Culture\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood in an EDTA anticoagulation tube on day1 post-CLP was diluted to 1:100 using sterile saline. The diluted blood was then evenly smeared onto blood agar plates using a sterile cotton swab. On day 1 post-CLP, the abdominal cavity of the mice was washed with 5 mL sterile saline to collect the abdominal cavity lavage fluid. The abdominal cavity lavage fluid was diluted to 1:1000 in the sterile saline and evenly smeared onto a blood agar plate. On day 5 post-CLP, the lungs were flushed with 1 mL sterile saline to collect the bronchoalveolar lavage fluid. Next, 10 \u0026mu;L of the bronchoalveolar lavage fluid was dropped onto the NAC solid medium. All of the above samples were incubated at 37\u0026deg;C for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked Immunosorbent Assay (ELISA) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of serum IL-6 and TNF-\u0026alpha; at all time points were detected using commercial ELISA kits (4A Biotech Co., Ltd, Beijing, China). The level of serum PCT was detected using a commercial ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) on days 1 and 4 post-CLP. The reagents and methods were operated in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLung Index and Spleen Index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe removed lungs and spleens were dried with filter paper, weighed, and both the lung index and spleen index were calculated. The following formula was used: lung index = (lung mass/body mass) \u0026times;100%, spleen index = (spleen mass/body mass) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage of CD4+ and CD8+ in the whole blood was detected by flow cytometry. The blood in the EDTA anticoagulation tube was dropped into erythrocyte lysis buffer (Tiangen Biotech Co., Ltd, Beijing, China) for 1 min at room temperature, and centrifuged at 2000 g for 5 min. The precipitates were collected and resuspended in 50 \u0026mu;L PBS, the antibodies were conjugated with specific fluorescent dyes, CD3-APC-A750 (Becton, Dickinson and Company), CD4-PerCP5.5 (Becton, Dickinson and Company), and CD8-FITC (Becton, Dickinson and Company)were added and incubated at 4\u0026deg;C for 30 min. Next, they were washed in 1 mL PBS and centrifuged at 2000 g for 5 min. The supernatant was discarded and the cells were resuspended in 200 \u0026mu;L PBS. Finally, the samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Co., Ltd, America).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8.2.1 software was used for statistical analysis. The survival rate was analyzed using a Log-rank (Mantel-Cox) test. Student\u0026rsquo;s t-tests and ANOVAs were used as appropriate. The results were displayed as the mean \u0026plusmn; SD. A value of P \u0026lt; 0.05 was deemed to be statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSurvival \u003c/strong\u003e\u003cstrong\u003eRate\u003c/strong\u003e\u003cstrong\u003es and \u003c/strong\u003e\u003cstrong\u003eIdentification \u003c/strong\u003e\u003cstrong\u003eResults of the CLP\u003c/strong\u003e\u003cstrong\u003e Model of \u003c/strong\u003e\u003cstrong\u003eSepsis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival rate of the mice after CLP surgery was closely related to the length of cecal ligation. The four-day survival rates of the mice in the 3/4 CLP, 1/2CLP, 1/3CLP, and Sham groups were 10%, 40%, 70%, and 100%, respectively. As the mortality rates of the 3/4CLP and 1/2CLP group were too high and non-conducive to subsequent experiments, 1/3CLP was selected as the experimental model for all subsequent experiments (Fig.2a).\u003c/p\u003e\n\u003cp\u003eThe routine blood tests on day 1 post-CLP showed that the levels of white blood cells, neutrophils, lymphocytes, and monocytes in the CLP group were 2.25 \u0026plusmn; 1.05, 0.47 \u0026plusmn; 0.38, 1.56 \u0026plusmn; 0.74, and 0.21 \u0026plusmn; 0.139 (*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L), respectively, which were significantly lower than those in the Sham group (P \u0026lt; 0.05). However, there was no significant difference in routine blood tests between the two groups on day 4 post-CLP (P \u0026gt; 0.05, Table1).\u003c/p\u003e\n\u003cp\u003eOn day 1 post-CLP, the level of serum ALT in the CLP group was 66.70 \u0026plusmn; 36.33 Karu, which was significantly higher than that of Sham group (11.63 \u0026plusmn; 2.23 karu, P \u0026lt; 0.05); however, there was no significant difference between the two groups on day 4 post-CLP (P \u0026gt; 0.05, Fig.2b).\u003c/p\u003e\n\u003cp\u003eThe level of serum PCT in the CLP group was 958.4 \u0026plusmn; 535.2 pg/mL and 607.1 \u0026plusmn; 247.5 pg/mL on days 1 and 4 post-surgery, compared to only 124.1 \u0026plusmn; 40.9 pg/mL and 156.4 \u0026plusmn; 10.0 pg/mL in the Sham group. The level of serum PCT in the CLP group was significantly higher than that of the Sham group (P \u0026lt; 0.05; Fig.2c), indicating a severe infection in the CLP group.\u003c/p\u003e\n\u003cp\u003eA large number of colonies with neat white round edges and smooth surfaces were observed in the blood culture dish and peritoneal lavage fluid culture dish of the CLP group on day 1 post-CLP; however, no such results were observed in the blood culture dish or peritoneal lavage fluid culture dish of the Sham group, and the blood agar medium was bright red (Fig.2d). Therefore, these results demonstrated that the CLP model of sepsis had been successfully established.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival Rates and Identification Results of the \"two-hit\" Model of Sepsis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the survival curve of the \"two hit\" model, on day 11 following CLP, four mice in the CLP + \u003cem\u003ePa\u003c/em\u003e group died, one mouse in the Sham + \u003cem\u003ePa\u003c/em\u003e group died, and all mice in the CLP + NS and Sham + NS groups survived (Fig.3b).\u003c/p\u003e\n\u003cp\u003eRoutines blood tests on day 5 post-CLP showed that the level of white blood cells, neutrophils, lymphocytes, and monocytes were 14.79\u0026plusmn; 1.28, 8.26\u0026plusmn; 0.57, 5.46\u0026plusmn; 0.74, and 1.03\u0026plusmn; 0.60 (*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L) in the CLP + \u003cem\u003ePa\u003c/em\u003e group; 7.02\u0026plusmn; 1.5, 1.72\u0026plusmn; 0.55, 4.74\u0026plusmn; 1.79, and 0.35\u0026plusmn; 0.05 (*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L) in the CLP+NS group; 10.80\u0026plusmn; 1.67, 2.25\u0026plusmn; 2.01, 7.14\u0026plusmn; 1.53, and 1.56\u0026plusmn; 1.02 (*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L) in the Sham + \u003cem\u003ePa\u003c/em\u003e group; and 8.76 \u0026plusmn; 0.68, 1.71 \u0026plusmn; 0.61,6.20 \u0026plusmn; 0.95, and 0.60 \u0026plusmn; 0.29 (*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L) in the Sham + NS group (Table.2). Compared with the CLP + \u003cem\u003ePa\u003c/em\u003e group, the white blood cells and neutrophils were significantly decreased in the three groups (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eOn day 5 post-surgery, the bronchoalveolar lavage fluids of the four groups were cultured. Green colonies were observed in the NAC solid medium in the CLP + \u003cem\u003ePa\u003c/em\u003e group and Sham + \u003cem\u003ePa\u003c/em\u003e group, and the number of colonies in the CLP + \u003cem\u003ePa\u003c/em\u003e group was greater than that in the Sham + \u003cem\u003ePa\u003c/em\u003e group. However, no colonies were observed in either the CLP + NS group or Sham + NS group (Fig.3c).\u003c/p\u003e\n\u003cp\u003eWe further observed the appearance of the lung tissue. On day 5 post-surgery, there were obvious bleeding points in the lung tissues of the CLP + \u003cem\u003ePa\u003c/em\u003e and Sham + Pa groups, especially in the CLP + \u003cem\u003ePa\u003c/em\u003e group. On day 11 post-surgery, the lung tissues of the surviving mice in the CLP+\u003cem\u003ePa\u003c/em\u003e group were consolidated dark red in color, and the majority of the lung lobes were affected, whereas there was only partial lung tissue damage in the Sham+\u003cem\u003ePa\u003c/em\u003e group. The lung tissue of the mice in the CLP+NS and Sham + NS groups was normal at the both time points (Fig.3d).\u003c/p\u003e\n\u003cp\u003eHE slices of the lungs in the CLP + \u003cem\u003ePa\u003c/em\u003e and Sham + \u003cem\u003ePa\u003c/em\u003e groups showed that the lung tissues exhibited widened alveolar septum, damaged alveolar structure, hemorrhage, serous exudation, and a large amount of inflammatory cell infiltration on day 5 after the operation. The degree of lung tissue injury was more severe in the CLP + \u003cem\u003ePa\u003c/em\u003e group. On day 11 post-surgery, there was greater consolidation of the alveolar cavity, focal pulmonary interstitial hemorrhage, and a large number of inflammatory cell infiltration in the connective tissues of the alveoli and alveolar septum in the CLP + \u003cem\u003ePa\u003c/em\u003e group. In the Sham + \u003cem\u003ePa\u003c/em\u003e group, only part of the inflammatory cells had infiltrated into the lung tissue. In the CLP + NS and Sham + NS groups, the lung tissue HE staining showed no significant change (Fig.3e).\u003c/p\u003e\n\u003cp\u003eOn day 5 post-CLP, the lung indexes of the CLP + \u003cem\u003ePa\u003c/em\u003e, CLP + NS, Sham + \u003cem\u003ePa\u003c/em\u003e, and Sham + NS groups were calculated respectively: 0.72 \u0026plusmn; 0.05%, 0.606 \u0026plusmn; 0.02%, 0.70 \u0026plusmn; 0.04%, and 0.51 \u0026plusmn; 0.02%.The lung index of CLP + \u003cem\u003ePa\u003c/em\u003e group was significantly higher than that of the CLP + NS group and Sham + NS groups (P \u0026lt; 0.01). On day 11 post-CLP, the lung index of the four aforementioned groups were 0.74 \u0026plusmn; 0.04%, 0.56 \u0026plusmn; 0.02%, 0.66 \u0026plusmn; 0.01%, and 0.51 \u0026plusmn; 0.02%, respectively. The lung index of the CLP + \u003cem\u003ePa\u003c/em\u003e group was significantly higher than that of the other three groups (P \u0026lt; 0.01), indicating a serious lung infection in the CLP + \u003cem\u003ePa\u003c/em\u003e group (Fig.3f). Together these results prove that the \"two-hit\" model of sepsis was completely constructed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Body Weight Results and Abdominal Organ Changes of the \"two-hit\" Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe body weight changes of the \"two-hit\" mice were continuously recorded within 11 days post-CLP. The results showed that the weight of the mice in the CLP operation group decreased continuously within 5 days after the operation, and increased slowly. The weight of the mice in the Sham operation group temporarily decreased on the first day after operation and subsequently underwent a gradual increase. The difference between the two groups was statistically significant (P \u0026lt; 0.01). After an intranasal instillation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, the weight of the mice in the Sham + \u003cem\u003ePa\u003c/em\u003e group decreased for a short time and gradually increased. On day 11 post-CLP, the weight gain of the mice in the CLP + \u003cem\u003ePa\u003c/em\u003e group was significantly lower than that in the other three groups(P \u0026lt; 0.01), which suggests that the infection in the CLP + \u003cem\u003ePa\u003c/em\u003e group was more serious than that of the other three groups (Fig.4a).\u003c/p\u003e\n\u003cp\u003eIn both the CLP + \u003cem\u003ePa \u003c/em\u003eand CLP + NS groups, pale necrosis occurred at the distal end of cecal ligation on day 1 post-CLP; white purulent necrosis, adhesion with surrounding tissues, and edema appeared on day 4; both purulent secretion and the area of abdominal adhesion increased on day 5; and the necrotic area was wrapped and could not be completely separated on day 11. However, in both of the Sham + \u003cem\u003ePa\u003c/em\u003e and Sham + NS groups, the shape and structure of the cecum were intact, and no abnormalities were present throughout the experiment (Fig.4b).\u003c/p\u003e\n\u003cp\u003eThe changes in the spleen size of each of the four groups were observed throughout the experimental cycle. From day 4 post-CLP, the spleens in the CLP + \u003cem\u003ePa\u003c/em\u003e and CLP + NS groups were significantly enlarged. On day 11 post-CLP, the size of the spleen in the CLP + \u003cem\u003ePa\u003c/em\u003e group was substantially larger than that of the other three groups, suggesting the inflammatory reaction in the CLP + \u003cem\u003ePa\u003c/em\u003e group was the most severe (Fig.4c).\u003c/p\u003e\n\u003cp\u003eOn day 1, 4, 5 and 11 post-CLP, the spleen capsule in the CLP + \u003cem\u003ePa\u003c/em\u003e group gradually thickened, multinucleated macrophages gradually increased, and the number of white pulp decreased significantly, and the white pulp and red pulp boundary in the CLP + \u003cem\u003ePa\u003c/em\u003e group gradually blurred. The number of white pulp in the CLP + NS group was slightly reduced, and the boundary between the white pulp and the red pulp was still distinguishable. The fibrous capsule in the Sham + \u003cem\u003ePa\u003c/em\u003e group was thickened, and the ratio of white pulp to red pulp was normal. The capsule of the spleen tissue in the Sham + NS group was smooth, the Shape and size of the white pulp was regular, the boundary between the white and red pulp was clear and there was no abnormality (Fig.4d).\u003c/p\u003e\n\u003cp\u003eThe spleen index was calculated at four different time points. The results showed that the spleen index was 0.26 \u0026plusmn; 0.03%, 0.524 \u0026plusmn; 0.06%, 0.70 \u0026plusmn; 0.04%, and 1.36 \u0026plusmn; 0.26%, respectively, in the CLP + \u003cem\u003ePa\u003c/em\u003e group; 0.25 \u0026plusmn; 0.04%, 0.50 \u0026plusmn; 0.03%, 0.67 \u0026plusmn; 0.11%, and 0.89 \u0026plusmn; 0.06%, respectively, in the CLP + NS group; 0.23 \u0026plusmn; 0.02%, 0.29 \u0026plusmn; 0.03%, 0.32 \u0026plusmn; 0.03%, and 0.45 \u0026plusmn; 0.03%, respectively, in the Sham + \u003cem\u003ePa\u003c/em\u003e group; and 0.24 \u0026plusmn; 0.05%, 0.27 \u0026plusmn; 0.05%, 0.29 \u0026plusmn; 0.05%, and 0.28 \u0026plusmn; 0.04%, respectively, in the Sham + NS group. The spleen index of the mice in the CLP operation group was significantly higher than that in Sham operation group(P \u0026lt; 0.01). However, on day 11 after CLP, the spleen index of the mice in the CLP + \u003cem\u003ePa\u003c/em\u003e group also significantly differed from that in the CLP + NS group(P \u0026lt; 0.01), which further highlighted the severity of the infection in the CLP + \u003cem\u003ePa\u003c/em\u003e group (Fig.4e). Our results showed that the spleens were in a state of hypersplenism due to infections and the immune cells were continuously consumed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunization Results of the \"two-hit\" Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn day 11 post-CLP, the white blood cells in the CLP + \u003cem\u003ePa\u003c/em\u003e group were 31.74 \u0026plusmn; 7.14*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L, which was significantly higher than that in the CLP + NS group , Sham + \u003cem\u003ePa\u003c/em\u003e group and Sham + NS group (P \u0026lt; 0.05 or P \u0026lt; 0.01); the monocytes were 3.11 \u0026plusmn; 1.28*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;L, which was significantly higher than that in the CLP + NS and Sham + NS groups (P \u0026lt; 0.05). However, since the number of neutrophils and lymphocytes in the CLP + \u003cem\u003ePa\u003c/em\u003e group were exceeded the detection range, statistical analysis could not be performed (Table 3).\u003c/p\u003e\n\u003cp\u003eWe further observed the changes in the level of TNF-\u0026alpha; and IL-6. On day 1 post-CLP, the level of serum TNF-\u0026alpha; was 167.00 \u0026plusmn; 42.68 pg/mL in the CLP group and 45.90 \u0026plusmn; 0.38pg/mL in the Sham group (P \u0026lt; 0.01), indicating that TNF-\u0026alpha; was activated following CLP. On day 5 post-CLP, the level of serum TNF-\u0026alpha; in the CLP + \u003cem\u003ePa\u003c/em\u003e and Sham + \u003cem\u003ePa\u003c/em\u003e groups were 110.69 \u0026plusmn; 9.18pg/mL and 148.43 \u0026plusmn; 13.42pg/mL, respectively. These findings were significantly different from those in the CLP + NS group (45.90 \u0026plusmn; 0.38pg/mL) and Sham + NS group (45.81 \u0026plusmn; 0.77pg/mL) (P \u0026lt; 0.0001), which suggests that serum TNF-\u0026alpha; was produced after an intranasal instillation of \u003cem\u003eP. aeruginosa\u003c/em\u003e. The level of serum TNF-\u0026alpha; in the CLP + \u003cem\u003ePa\u003c/em\u003e group was significantly lower than that in the Sham + \u003cem\u003ePa\u003c/em\u003e group (P \u0026lt; 0.01), suggesting that the CLP operation may inhibit TNF-\u0026alpha; reactivation. However, no significant difference was observed regarding the level of serum TNF-\u0026alpha; between the four groups on days 4 and 11 post-CLP (Fig.5a). Moreover, the change trend of IL-6 was similar to that of TNF-\u0026alpha; (Fig.5b).\u003c/p\u003e\n\u003cp\u003eThe ratio of CD4+/CD8+ in whole blood was also detected at four different time points by flow cytometry to observe the cellular immune function. The results showed that the ratio of CD4+/CD8+ in the CLP + \u003cem\u003ePa\u003c/em\u003e group was 0.57 \u0026plusmn; 0.10 on day 11 post-CLP, which was significantly lower than that of the other three groups (the ratio of CD4+/CD8+ in the CLP + NS group, Sham + \u003cem\u003ePa\u003c/em\u003e group, and Sham + NS group was 0.91 \u0026plusmn; 0.04, 1.17 \u0026plusmn; 0.03, and 1.48 \u0026plusmn; 0.04, respectively) (P \u0026lt; 0.05 or P \u0026lt; 0.01, Fig.5c,d). These findings indicate that the \"two-hit\" model of sepsis may lead to the the decreased presence of immune cells . The above results indicate that the \"two- hit\" model of sepsis could lead to immunoparalysis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the name suggests, the \"two-hit\" animal model simulates an attack by two pathogens on the host, induced by CLP surgery and secondary infection. The CLP model of sepsis mainly simulates clinical sepsis induced by acute peritonitis, which currently represents the gold standard in animal models for studying sepsis[7]. The course of sepsis can pass through different stages, which are clearly defined by human physiological parameters. However, no physiological monitoring exists in animal models and the severity of sepsis can only be determined by mortality and time of death[8]. In our experimental model, we reproduced three CLP models by selecting different lengths of cecal ligation. By observing the survival rate and general situation of the mice at four days post-CLP, 1/3 cecal ligation was selected as the experimental model as it caused mild sepsis.\u003c/p\u003e\n\u003cp\u003eCytokine storm syndrome is induced by innate immune activation during the early stage of sepsis, which may lead to multiple organ failure and early death[9]. Procalcitonin (PCT) is a calcitonin-promoting hormone that is commonly used as a clinical infection index. The level of serum PCT in sepsis patients is significantly increased, and can be detected within 24 h post-infection, peaks at 12-24 h, and is closely related to the severity of infection[10]. PCT was detected in our mouse model of sepsis, which is in accordance with the response of clinical patients. TNF-\u0026alpha; and \u0026nbsp;IL-6 are the important mediators of the acute phase of the inflammatory response. TNF-\u0026alpha; expression is closely related to the severity of sepsis, which can be used to predict the severity of sepsis[11]. IL-6 is an important pro-inflammatory cytokine in the early stage of inflammation, which increases in blood circulation after infection and trauma[12]. Our findings showed that the levels of PCT, TNF-\u0026alpha;, and IL-6 were significantly increased on day 1 post-CLP, which confirmed the presence of a cytokine storm syndrome. Moreover, the level of serum ALT also displayed a significant increase, which coincided with acute organ dysfunction. This phenomenon is consistent with the clinical observation that sepsis can cause multiple organ dysfunction.\u003c/p\u003e\n\u003cp\u003eAt present, clinical studies have confirmed that patients with acute sepsis have a high probability of death due to secondary infection after the first three days or longer after the onset of sepsis [13]. One of the main causes of death is secondary infection caused by sepsis-induced immune dysfunction, whereas pneumonia is a common secondary infection[9]. \u003cem\u003eP.aeruginosa\u003c/em\u003e is the most common multidrug-resistant gram-negative bacteria that can cause ventilator-associated pneumonia(VAP) and nosocomial pneumonia[9]. In addition, compared with other types of pneumonia, patients with \u003cem\u003eP\u003c/em\u003e\u003cem\u003e. aeruginosa\u003c/em\u003e pneumonia are more prone to multiple organ failure and death[14-15]. Therefore, \u003cem\u003eP\u003c/em\u003e\u003cem\u003e. aeruginosa\u003c/em\u003e represents the best choice as a \"two-hit\" sepsis model strain.\u003c/p\u003e\n\u003cp\u003eIt has been reported that the immune dysfunction turning point for sepsis induced by CLP may occur on day 4 post-CLP[16]. Our study also confirmed that CLP mice no longer died from day 4 post-CLP. Thus, we chose to administer the \u003cem\u003eP\u003c/em\u003e\u003cem\u003e. aeruginosa\u003c/em\u003e suspension via a \"nasal drip\" infection at day 4 post-CLP as a second hit to observe the general condition and survival rate of the mice. The bronchoalveolar lavage on day 5 post-CLP and the survival rate of the \"two-hit\" model revealed that this model could successfully simulate sepsis patients with secondary infection.\u003c/p\u003e\n\u003cp\u003eAs we all know, neutrophils mainly play the role of the first line of defense in inflammation sites, such as phagocytosis, degranulation and formation of reactive oxygen species[17]. Routines blood tests of the \"two-hit\" model showed that the number of neutrophils gradually increased from day 5 post-CLP, and it was more than three times of that of the normal mice on day 11 post-CLP. Combined with manifestation of hypersplenism in mice, we thought it was a leukemoid reaction caused by severe infection. Leukemoid reaction refers to the continuous increase of neutrophils, more than 50000 cells per microliter, accompanied by the increase of neutrophil precursors when the cause of leukemia is excluded.The main cause of\u0026nbsp; leukemoid reaction are severe infection, poisoning, malignant tumor, severe hemorrhage or acute hemolysis[18]. However, there is no diagnostic criteria for leukemia reaction of murine.\u003c/p\u003e\n\u003cp\u003eThe derangements in the inflammatory response and a state of adaptive immunoparalysis, which are characterized by the decrease of primary humoral immune response[19]. On day 5 post-CLP, the levels of TNF-\u0026alpha; and IL-6 in the CLP + \u003cem\u003ePa\u003c/em\u003e group were significantly higher than those in the CLP+NS and Sham + NS groups, but lower than those in the Sham + \u003cem\u003ePa\u003c/em\u003e group. The phenomenon is caused by excessive consumption of inflammatory factors in humoral immune dysfunction after CLP.\u003c/p\u003e\n\u003cp\u003eSplenic pulp includes white pulp(WP,cells with weakly stained nuclei), red pulp(RP) and the marginal zone(MZ).The RP is composed of blood vessels, lining endothelial cells, and macrophages. These macrophages screen and remove pathogens and soluble antigens from the systemic circulation. WP represents lymphoid tissue, which is composed of central arterioles, lymphocytes and antigen-presenting cells (mainly dendritic cells and macrophages).WP produces specific immune responses to antigens[20]. The pathological section of mice found that the white pulp in the CLP+\u003cem\u003ePa\u003c/em\u003e group was significantly reduced compared with the other three groups, which indicated that the immune function of the \"two-hit\" model has been compromised.\u003c/p\u003e\n\u003cp\u003ePatients who survive the acute phase of sepsis show long-term damage to immune function due to the decrease in the number and function of many immune cell populations[21]. With the increasing use of antibiotics and various anti-inflammatory factors in recent years, most patients will survive the systemic acute inflammatory reaction period, but subsequently enter a longer-lasting immune dysfunction state [22]. Studies have found that the key cause of immune dysfunction in patients with sepsis is the death of lymphocytes, especially T and B lymphocyte apoptosis, is a major factor in immune dysfunction[23]. CD4+ can help B lymphocytes produce antibodies and induce T lymphocytes to transform into effector cells, and is a surface marker of Ti/Th. CD8 + mediates the destruction and lysis of host cells after pathogen infection, and is a protein in Ts. In mammals, the ratio of CD4 +/CD8 + is normally\u0026gt; 1, depending on the species[24]. And the CD4+/CD8+ ratio is highly correlated with host immune function. An increase in CD4 positive cells and an increase in the CD4/CD8 ratio indicate a strong immune system[25]. Therefore, by continuously monitoring the ratio of CD4+ and CD8+ T cells in the whole blood, we found that the CD4+/CD8+ ratio in the mice in the \"two-hit\" model was significantly lower than that of the other three groups on day 11 post-surgery, which was consistent with the immune dysfunction status of patients with advanced sepsis.\u003c/p\u003e\n\u003cp\u003eHowever, there are limitations associated with the \"two-hit\" model. First, the impact of cecal puncture on humans is a fatal blow but the impact on rodents is not obvious[26]. Secondly, the severity of sepsis caused by CLP is related to multiple factors, including the length of cecal ligation, the puncture needle size, and the number of perforations. Of course, the stability of the model is also closely related to the gender and age of the mice. Thirdly, the \"two hits\" are administered by \u0026ldquo;nasal drip\u0026rdquo;; however, in the process of administering the \u0026ldquo;nasal drip\u0026rdquo;, the bacterial solution may be swallowed by the mice rather than inhaled, which may cause different degrees of infection. Fourth, sepsis patients are often accompanied with endocrine, tumor, and other diseases in the clinic. The establishment of a \"two-hit\" model with only healthy mice could not cover all clinical conditions. Finally, a mouse model cannot completely simulate the changes in the whole blood and dynamic changes in the inflammatory factors in patients with sepsis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis \"two-hit\" model focuses on replicating the process of clinical sepsis patients entering the immune dysfunction stage during the early stages of sepsis. The immune function of the mice was evaluated by the continuous observation of routine blood tests, inflammatory factors, and the CD4+/CD8+ ratio. Together, these findings demonstrate that this \"two-hit\" model can be used as an appropriate animal model to study the mechanism of immune dysfunction in sepsis and the associated drug development.\u003c/p\u003e"},{"header":"Acknowledgements","content":"\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 81871608). We thank International Science Editing ( http://www.internationalscienceediting.com ) for editing this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSinger, M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche J, Coopersmith CM, Hotchkiss RS, Levy MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent J, Angus DC. 2016. The Third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 315: 801-810.\u003c/li\u003e\n\u003cli\u003eLou, JS, Wang JF, Fei MM, Zhang Y, Wang J, Guo Y, Bian JJ, Deng XM. 2020. Targeting lymphocyte activation gene 3 to reverse T-lymphocyte dysfunction and improve survival in murine polymicrobial sepsis. Journal of Infectious Diseases 222: 1051-1061.\u003c/li\u003e\n\u003cli\u003ePatil, NK, Bohannon JK, Sherwood ER. 2016. Immunotherapy: a promising approach to reverse sepsis-induced immunosuppression. 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Journal of Visualized Experiments (62):3926.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:15.6pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTable 1.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eChanges in\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eroutine\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eblood\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003etest\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003es\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;on\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003edays 1 and 4 post-CLP\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"width: 4.1e+2pt;border: none;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:102.8pt;border-top:solid black 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 0in 0in 0in;height:18.7pt;\"\u003e\n \u003cp style='margin-top:.25pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGroup\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:169.8pt;border:none;border-top:solid black 1.0pt;padding:0in 0in 0in 0in;height:18.7pt;\"\u003e\n \u003cp style='margin-top:.9pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eDay\u0026nbsp;1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:135.0pt;border:none;border-top:solid black 1.0pt;padding:0in 0in 0in 0in;height:18.7pt;\"\u003e\n \u003cp style='margin-top:.9pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eDay\u0026nbsp;4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:84.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:21.4pt;\"\u003e\n \u003cp style='margin-top:4.1pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;text-indent:16.5pt;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:21.4pt;\"\u003e\n \u003cp style='margin-top:4.1pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;text-indent:12.0pt;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eSham\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:21.4pt;\"\u003e\n \u003cp style='margin-top:4.1pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;text-indent:11.0pt;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e5.37\u0026plusmn;1.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:102.8pt;border:none;border-bottom:solid black 1.0pt;padding:0in 0in 0in 0in;height:24.8pt;\"\u003e\n \u003cp style='margin-top:.8pt;margin-right:0in;margin-bottom: 8.0pt;margin-left:1.25pt;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eMONO#(*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:84.75pt;border:none;border-bottom:solid black 1.0pt;padding:0in 0in 0in 0in;height:24.8pt;\"\u003e\n \u003cp style='margin-top:7.6pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.21\u0026plusmn;0.139\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid black 1.0pt;padding:0in 0in 0in 0in;height:24.8pt;\"\u003e\n \u003cp style='margin-top:7.6pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.65\u0026plusmn;0.312\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003e*\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid black 1.0pt;padding:0in 0in 0in 0in;height:24.8pt;\"\u003e\n \u003cp style='margin-top:7.6pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e1.55\u0026plusmn;1.18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:64.15pt;border:none;border-bottom:solid black 1.0pt;padding:0in 0in 0in 0in;height:24.8pt;\"\u003e\n \u003cp style='margin-top:7.6pt;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.38\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n 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WBC, white blood cell; NEUT#, neutrophil\u0026nbsp;count; LYMPH#,\u0026nbsp;lymphocyte count; MONO#, monocyte\u0026nbsp;count\u003c/span\u003e\u003c/span\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:15.6pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New 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Roman\",serif;'\u003etest\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003es\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;on\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eday 5 post-CLP\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:21.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGroup\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:21.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP+\u003cem\u003ePa\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:21.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP+NS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:21.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eSham+\u003cem\u003ePa\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:21.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eSham+NS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eWBC\u003c/span\u003e\u003cspan style=\"font-size:16px;line-height:107%;font-family:SimSun;\"\u003e（\u003c/span\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e14.79\u0026plusmn;1.28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e7.02\u0026plusmn;1.5\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e***\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e10.80\u0026plusmn;1.67\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003e*\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.55pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e8.76\u0026plusmn;0.68\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e****\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:28.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eNEUT#(*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:28.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e8.26\u0026plusmn;0.57\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:28.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.72\u0026plusmn;0.55\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003e****\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:28.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e2.25\u0026plusmn;2.01\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e**\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:28.45pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.71\u0026plusmn;0.61\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e****\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eLYMPH#\u003c/span\u003e\u003cspan style='font-size:16px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003e(*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e5.46\u0026plusmn;0.74\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e4.74\u0026plusmn;1.79\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e7.14\u0026plusmn;1.53\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:27.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e6.20\u0026plusmn;0.95\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eMONO#(*10\u003csup\u003e3\u003c/sup\u003e/\u0026mu;l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.03\u0026plusmn;0.60\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:79.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.35\u0026plusmn;0.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.56\u0026plusmn;1.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:83.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.60\u0026plusmn;0.29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003eCompared with the CLP\u003c/span\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;+ \u003cem\u003ePa\u003c/em\u003e group,\u0026nbsp;*\u0026nbsp;P \u0026lt; 0.05;\u0026nbsp;**\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eP\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026lt; 0.01;\u0026nbsp;***\u0026nbsp;\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001;\u0026nbsp;****\u0026nbsp;\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001. WBC,\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif;'\u003ewhite blood cell\u003c/span\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e; NEUT#, neutrophil count; LYMPH#, lymphocyte count; MONO#, monocyte count.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:15.6pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTable 3. Changes in\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eroutine\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eblood\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003etest\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003es on day 11 post-CLP\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:100.4pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGroup\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:81.6pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP+\u003cem\u003ePa\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:80.05pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCLP+NS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:1.15in;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.35pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eSham+\u003cem\u003ePa\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e2.04\u0026plusmn;0.94\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:81.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.2pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.21\u0026plusmn;0.02\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-size:14px;line-height: 107%;font-family:\"Times New Roman\",serif;'\u003e*\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:107%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003eCompared with the CLP\u003c/span\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;+ \u003cem\u003ePa\u003c/em\u003e group, * \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01;\u0026nbsp;\u003c/span\u003e\u003cspan style=\"font-size:14px;line-height:107%;font-family:SimSun;\"\u003e━\u003c/span\u003e\u003cspan style='font-size:14px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e: Above the measuring range. WBC,\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif;'\u003ewhite blood cell\u003c/span\u003e\u003c/span\u003e\u003cspan style='font-size: 14px; line-height: 107%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003e; NEUT#, neutrophil count; LYMPH#, lymphocyte count; MONO#, monocyte count.\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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, two-hit model, inflammation, immunoparalysis","lastPublishedDoi":"10.21203/rs.3.rs-474587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-474587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, researchers have turned their attention to the study of the mechanism of sepsis-induced immuneparalysis, but there is still a lack of appropriate animal models that reflect the process of sepsis-induced immuneparalysis. The purpose of this study was to explore and evaluate whether a \"two-hit\" model of sepsis can be used as an appropriate animal model to study the mechanism of sepsis-induced immunoparalysis. Firstly, we established a sepsis model in C57BL/6J mice via cecal ligation and puncture(CLP). Routine blood tests, serum ALT and PCT, etc. were analyzed to evaluate the establishment of a CLP sepsis model on day 1 post-CLP. Secondly, the surviving mice were treated with 40\u0026micro;L suspension of \u003cem\u003eP.aeruginosa\u003c/em\u003e (\u003cem\u003ePa\u003c/em\u003e) under anesthetic on day 4 post-CLP to establish a \"two-hit\" model. Gross lung specimens, and pathological examination of the lung tissue, etc. were used to evaluate whether the \"two-hit\" model was successfully established on days 5 and 11 post-CLP. Finally, the level of serum TNF-α and IL-6, pathological examination of the spleen and the CD4+/CD8\u0026thinsp;+\u0026thinsp;ratio in blood were detected to evaluate the immune status of the model mice. The above test results proved that the \"two-hit\" sepsis mouse model was successfully constructed. TNF-α, IL-6, pathological examination of the spleen and the CD4+/CD8\u0026thinsp;+\u0026thinsp;ratio in blood confirmed that the model was in a state of immunoparalysis. We conclude that this \"two-hit\" sepsis mouse model is an appropriate animal model, which can successfully replicate the pathophysiological process of in clinical patients with sepsis-induced immunoparalysis.\u003c/p\u003e","manuscriptTitle":"Establishment and Evaluation of a \"Two-Hit\" Mouse Model of Sepsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-28 17:49:50","doi":"10.21203/rs.3.rs-474587/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":"14ddeb7a-a8e5-4eed-9ce2-a4493748f0c9","owner":[],"postedDate":"May 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":4635259,"name":"Immunology"}],"tags":[],"updatedAt":"2021-06-03T12:28:36+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-28 17:49:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-474587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-474587","identity":"rs-474587","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}