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Comparison of two different models of sepsis induced by cecal ligation and puncture in rats
j o u r n a l o f s u r g i c a l r e s e a r c h s e p t e m b e r 2 0 1 8 ( 2 2 9 ) 2 7 7 e2 8 2
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Comparison of two different models of sepsis induced by cecal ligation and puncture in rats Linlin Song, MD,a Yanjie Zou, MD,b and Zhongping Cao, MDb,* a
Postgradulate Training Base of Jinzhou Medical University, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force, Tianjin, China b Department of Anesthesia, The Logistics University of Chinese People Armed Police Force Hospital, Tianjin, China
article info
abstract
Article history:
Background: The present study was designed to explore the difference between two rat
Received 11 January 2018
models of sepsis and to establish a more stable rat model.
Received in revised form
Materials and methods: SpragueeDawley rats were randomly divided into three groups:
26 February 2018
sham operation group, simple cecal ligation and perforation group (SCLP), and cecal
Accepted 21 March 2018
ligation perforation plus drainage group (CLP-DS). The general condition of the rats was
Available online xxx
observed, and the time of death and survival rate at 72 h were recorded. The arterial blood and lung tissue were obtained 9 h after the operation.
Keywords:
Results: The mortality of the CLP-DS group was significantly higher than that of the SCLP
Sepsis model
group. The limitation package, intestinal adhesion, and poor drainage were detected in
Simple cecal ligation and puncture
the SCLP rats, whereas intestinal edema and hyperemia, bloody water in the abdominal
Cecal ligation and puncture-
cavity, but no inflammatory package were observed 24 h after the operation in the CLP-
drainage strip
DS rats by autopsy. There were significant differences in interleukin-6 and tumor
Inflammatory cytokine
necrosis factor-alpha levels between the SCLP group and the CLP-DS group. Severe
Smith lung injury score
pulmonary septal thickening, alveolar wall vascular congestion, and protein debris deposition in the alveolar cavity were observed in the SCLP group, whereas pulmonary bullae were observed in the CLP-DS group using light microscopy, and there were significant difference among groups in Smith lung injury score. Conclusions: These results suggested that the cecal ligation combined with puncture drainage model of sepsis is more stable than that of the simple cecal ligation and puncture model of sepsis in the rat, which resolved the problem of puncture wrapped in the traditional CLP model of sepsis in rat. ª 2018 Elsevier Inc. All rights reserved.
Introduction Sepsis is increasingly common in clinical practice and has extremely high mortality; it is a major complication of serious trauma, burn, shock, and major surgery and is also one of the leading causes of death in critically ill patients.1-3 The cecal ligation perforation sepsis model is most similar
to clinical sepsis, and a rat or mouse cecal ligation perforation (CLP) is commonly used to mimic clinical appendicitis perforation or diverticulitis perforation, which has been recognized as the gold standard of sepsis animal modeling.4,5 However, the morbidity and mortality are unstable in the cecal ligation puncture sepsis model due to many factors, which makes these experimental studies
* Corresponding author. Department of Anesthesia, The Logistics University of Chinese People Armed Police Force Hospital, Chenglin Road NO 222, Hedong District, Tianjin 300162, China. Tel./fax: þ86 22 60577658. E-mail address: [email protected] (Z. Cao). 0022-4804/$ e see front matter ª 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2018.03.058
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j o u r n a l o f s u r g i c a l r e s e a r c h s e p t e m b e r 2 0 1 8 ( 2 2 9 ) 2 7 7 e2 8 2
challenging. In this study, we compared the rat cecal ligation perforation sepsis model with ligation perforation and the drainage sepsis model to generate a sepsis research model with more stable morbidity and mortality.
Materials and methods Experimental animal and group The experimental protocol was approved by the Ministry of Science and Technology Committee and Animal Center Committee and Logistics University of Chinese People Armed Police Force Hospital. Animal experiments adhered to the ethical guidelines of the International Association for the Study of Pain, and all efforts were made to minimize animal suffering and to reduce the number of animals used. The experiments in this study were performed on male, SpragueeDawley rats (260 g300 g) housed under approved conditions with a 12-/12-h light/ dark cycle and food and water provided ad libitum. The experiments were performed at the same time period on different days to reduce the effect of circadian rhythms on different individuals. The animals were randomly divided into three groups: sham operation group (sham) (n ¼ 16), simple cecal ligation perforation group (SCLP) (n ¼ 32), and cecal ligation perforation plus drainage group (CLP-DS) (n ¼ 32). Only open abdominal cavity operation was performed on rats in the sham group. After the experiments, the rats were euthanized via decapitation under deep anesthesia by ketamine (intraperitoneal injection).
Establishment of rat model SCLP model The rats were anesthetized by 1% ketamine (100 mg/kg) via an intraperitoneal injection after weighing and then fixed on the small animal operating platform board. After abdominal skin preparation and disinfection, a longitudinal incision (length 2 w 2.5 cm) was performed along the ventral line above the pubic bone to open the celiac. The cecum was identified, and it was then carefully taken out. The contents of the cecum were gently pushed to the cecal distal to reduce the gas of ligation cecum, and the mesentery was carefully separated to avoid injury of the ileocecal artery cecal branch. The free distal cecum was placed in methylene blue solution in a sterile cylinder until a clear blue mark was observed around the cecum, at a volume of 2.5 mL mark. The cecum was ligated with 4-0 silk thread according to the mark. Intestinal obstruction did not occur because the ligation sites were lower than the location of the ileocecal valve. Next, an 18 G (1.2 mm 3.8 mm) needle was applied to take a single pass through the cecum from the mesenteric side to the contralateral side. After the puncture, a small amount of feces was squeezed from the two puncture holes to ensure perforation (supplemental Fig. 1). The cecum was placed back into the abdominal cavity after the feces out of the cecum was wiped off. Next, the muscle and skin were sutured with a silk thread (3-0) to close the abdomen. Preheated saline (50 mL/kg, 37 C) was subcutaneously injected into the back for resuscitation. The rats were placed back into the cage after surgery, and food and water were provided ad libitum.
CLP-DS model The specific steps were performed as follows: the preoperative preparation and abdominal cavity opening were performed similar to the SCLP model. After cecum ligation and puncture, the two puncture holes were kept opened with a drainage bar across (twisted strip of sterile gauze with a width of 2 mm) to make sure that the strip was exposed 5 mm outside the two holes. The remaining length was cut off, and the feces around the area were wiped off (Supplemental Fig. 1).
Indicators and testing methods General postoperative observation, including the appearance, activities, mental state of the rats, among other factors, was performed. The death time point and survival rate of the rats were recorded at 72 h after modeling, and the changes in abdominal cavity were observed 24 h after modeling. Six rats of each group were randomly selected, and 3 mL abdominal aorta blood was obtained 9 h after modeling and placed in an anticoagulant tube for 30 min at 4 C. Next, the samples were centrifuged for 15 min at 3000 r/min, and 200 mL serum was placed in a cryopreserved tube for cryopreservation at 80 C. The serum interleukin (IL)-6 and tumor necrosis factor (TNF)-a levels were subsequently detected using an enzyme-linked immunosorbent assay. Lung tissue observation: The lung tissue was fixed with 4% neutral formaldehyde solution for 48 h, then dehydrated, paraffin-embedded, sliced (5 mm), baked, and HE stained. The histopathological changes were observed under a light microscope, and the lung injury was evaluated by Smith lung injury score. According to the Smith score, the pulmonary edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, pulmonary atelectasis, and hyaline membrane formation were semiquantitative analyzed from 0 to 4 point: noninjury was 0 point, the lesion less than 25% of visual field was 1 point, the lesion from 25% to 50% of visual field was 2 point, the lesion from 50% to 75% of visual field was 3 point, and the lesion in full view was 4 point; the total lung injury score was the sum of the aforementioned. Ten visual fields of each rat were observed, and the average value was adopted.
Statistical analysis The data were expressed as the mean standard deviation ðX sÞ, and statistically analyzed using SPSS 17.0 software. The survival rate of each group was analyzed using KaplaneMeier method (log-rank test). IL-6, TNF-a levels, and Smith lung injury scores among groups were analyzed by single factor analysis of variance. A P-value less than 0.05 was considered statistically different.
Results General condition of the rats after modeling The experimental rats were awakened from anesthesia 1-2 h after operation. The rats in the SCLP and CLP-DS groups
279
song et al comparison of two sepsis models in rat
Table e The rat death number of different time point. Group
Time point (h) 6
12
Sham
0
SCLP
0
CLP-DS
0
72 h survival number
72 h survival rate (%)
18
24
48
0
0
0
0
10
0
12
4
3
6
23.1
10
6
6
4
0
0
exhibited a similar general condition, and the clinical signs of the bacterial infection were less obvious within 6 h after the operation. However, there were symptoms of vertical hairs, bow, curled up, fatigue, mental burnout, and shortness of breath in the rats at 6 h after the operation, and there was even intraocular canthus, nose and mouth bleeding, deep breathing, and stimulation without a response in severe cases. The rats that survived 3 d after the operation in the SCLP group began to recover. Rats in the sham group showed free activity and normal drinking and eating. Twenty-four hours after the operation, an autopsy showed that most of the rat cecum in the SCLP group formed a limited package, closed perforation, and intestinal adhesion, whereas rats in the CLP-DS group, rats showed abdominal organ edema and congestion, intestinal deposition, and cecal ischemia necrosis but no inflammatory package formation.
Survival rate of the rats Most rats in the CLP-DS group died from 8 to 12 h after operation. The rat survival rate of the SCLP group was 23.1% at 72 h after the operation, while no rat survived at 72 h after the operation in the CLP-DS group, and all rats survived after 72 h after the operation in the sham group. There was a statistically significant difference among groups in survival rate (P < 0.01, Table). The survival time curve of the rats is shown in Figure 1, which clearly reflects the difference in survival rates. There were significant differences in survival rates among groups analyzed using log-rank test (P < 0.01).
100
Changes in serum IL-6 and TNF-a levels in rats The levels of IL-6 and TNF-a in CLP-DS and SCLP groups were significantly higher than those in the sham operation group (P < 0.01), and these levels were higher in the CLP-DS group than those in the SCLP group (P < 0.01, Fig. 2).
Pathological changes in lung tissue Rat lung tissue of the sham group showed that the alveolar structure was relatively complete, with no interval thickening and congestion and no inflammatory cell infiltration. The rats’ lung tissue of the SCLP group showed that lung interval was thickening, alveolar cavity was filled with protein fragments, and alveolar septal had a slight rupture. The changes in the CLP-DS group of rats showed that alveolar septal fracture is more serious with pulmonary bullae formation (Fig. 3). The Smith lung injury score in the CLP-DS group of rat was higher than that of the SCLP group (Fig. 4).
Discussion Hospital patients exhibited high mortality due to sepsis, and sepsis caused shock and multiple organ failure; sepsis is one of the main reasons of death in hospital patients.1,6 The Global Sepsis Alliance, which is involved in the control of sepsis pathophysiological processes and nearly all the supportive treatment of all organs or systems, has released guidelines for the treatment of sepsis. However, the incidence and mortality
Fig. 1 e The 72-h survival rate of rat after operation. **P < 0.01 compared with the sham group. ##P < 0.01 compared with the SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLP-DS [ cecal ligation perforation plus drainage group. (Color version of figure is available online.)
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Fig. 2 e Serum IL-6 and TNF-a levels in each group of rat. **P < 0.01 compared with sham group. ##P < 0.01 compared with SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLP-DS [ cecal ligation perforation plus drainage group.
of sepsis remains at a high level, and its pathogenesis is currently still unclear.3,7-9 Therefore, establishment of high stability and good reproducibility of animal models is very important to study the pathogenesis of sepsis and to screen and evaluate the treatment of drugs.10,11 There are three current methods to establish the sepsis model; the first is the endotoxin model induced by intraperitoneal or intravenous injection of exogenous endotoxin or lipopolysaccharide, the infection dose of the endotoxemia model can be artificially controlled, and this model has characteristics of stability, repeatability, and strong controllability.11,12 Animal reactivity is relatively consistent in this model, but transient input of endotoxin can cause animals to quickly tolerate or die; thus, this model can not be a good simulation of sepsis clinical features, and the cost is high.11,12 The second model is a live bacteria model induced by intravenous injection of a single live or mixed bacteria culture. The infectious shock model was obtained when the positive rate of blood culture reach 69%.13,14 The third is a cecal ligation perforation model induced by colonic seepage leakage and bacterial translocation.15,16 Fecal leakage to the abdominal cavity caused a systemic inflammatory response, circulatory and metabolic changes. The disease is not a transient course. Compared with an injection of lipopolysaccharide or live bacteria, the cecal ligation perforation model has characteristics of better clinical relevance and lower cost.15,16 However, the experimental results of this model are diverse, and the repeatability is poor due to the long modeling time and inconsistent experimental factors. Therefore, it is important
to screen a repeatable sepsis animal model according to the experimental factors and steps. The CLP model is considered to be the gold standard for sepsis studies. However, the CLP model still has some difficult problems to be resolved, such as the consistency requirements of animal surgery, cecal ligation length, size and the number of the puncture hole, and the number of feces leaking into the abdominal cavity. In addition, the infection foci are easily wrapped by the omentum and surrounding tissue. All of these factors affect the prognosis of rats and cannot achieve sepsis animal model standards.17,18 Thus, we need to examine an animal model that can quantify feces leaking into the abdominal cavity and to overcome the insufficient intraperitoneal infection of a traditional CLP model. Generally, organ dysfunction or death of rats occurs 6-12 h after sepsis modeling and is mainly caused by systemic inflammatory response.1-3,19 The key to achieve this requirement is to produce an appropriate intensity of infection and to stimulate excessive inflammation. All these conditions were obtained with the rat CLP-DS model of sepsis. The results of this study showed that the survival rates of the two groups were less than 30%, which was consistent with the requirements of the sepsis model.17,18 Importantly, the rat mortality rate in the CLP-DS group was significantly higher than that in the SCLP group, and the rat death time point was focused on 6-12 h after modeling, which indicated that systemic inflammatory response syndrome occurred earlier and more intensely in the CLP-DS group than that in the SCLP
Fig. 3 e Histopathological features of lung tissue in each group (HE X200). Red arrow indicates thickening of the lungs, black arrow indicates protein fragments, and blue arrow indicates lung bullae. (A) Sham group, (B) SCLP group, and (C) CLP-DS group. (Color version of figure is available online.)
song et al comparison of two sepsis models in rat
Fig. 4 e Smith lung injury score in each group of rat. **P < 0.01 compared with sham group. ##P < 0.01 compared with SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLPDS [ cecal ligation perforation plus drainage group.
group. When the body has developed systemic inflammatory response syndrome, a large number of proinflammatory factors are released into circulation. IL-6 and TNF-a are important indicators of the degree of systemic inflammatory syndrome and the severity and prognosis of sepsis.20,21 The levels of IL-6 and TNF-a in the CLP-DS group were significantly higher than those in the SCLP group. The symptoms of the CLP-DS group were similar to acute peritonitis due to the intestinal feces that continued to leak into the abdominal cavity, whereas symptoms of the SCLP rats were similar to abdominal abscess formation due to the severe infection that was more likely to occur after necrosis of the cecal bowel ligation. This difference could also be proven by the rat abdominal anatomy, which demonstrated that the edema and congestion of abdominal organs of the CLP-DS group was more severe than that of the SCLP, whereas the majority of SCLP survivors demonstrated localized abscess formation. The length of ligation cecum did not accurately reflect the total amount of fecal leakage into the abdominal cavity to cause infection, due to the variety of shapes of the experimental rats’ cecum. At the same time, as the cecum ligation was performed only according to the length of the surface of one side of the cecum, the mark was not complete. There was a deviation among the cecum length of experimental rats.17,18 Although the species, strain, sex, and weight of the experimental rats were similar, the size of the cecum between different individuals and the amount of their contents were significantly different. The volume-controlled ligation method used in the CLP model in this experiment was not limited by the cecal geometry, which eliminated the difference in cecal size between different individuals and reflected the equal amount of intestinal contents. It has been reported that endotoxin levels in rats at 2 h after CLP began to increase, reached a peak at 12 h and a sharp increase at 6-12 h, and the systemic inflammatory response had the same time course.10,17,18 The blood and lung samples were then taken at 9 h after the modeling. Pulmonary pathology observed by light microscopy and evaluated by Smith score also showed that pathological changes of the CLP-DS group were more severe than those of the SCLP group. In this study, the procedure was performed by the same operator on the same time point on a different day to reduce the effect of operator and circadian
281
rhythm on the inflammatory response between different individuals. The same puncture needles were applied. These results showed that the model was stable and reliable and had good controllability and repeatability. The deficiency of this experiment is that the experimental animals did not have antibiotic and lesion excision treatment, which could not completely simulate the pathophysiological process of the patients with septic shock, but it may serve as a stable septic shock model for a variety of acute animal experiments to study the mechanism of sepsis and to evaluate the efficacy of drugs.
Conclusions Our study suggests that cecal ligation combined with puncture drainage model of sepsis can quantify feces leakage into the abdominal cavity and generate an insufficient intraperitoneal infection of traditional CLP model, which demonstrates greater morbidity and mortality, inflammatory mediators, and severe lung injury compared with the simple cecal ligation and puncture model of sepsis in rats. Thus it is more suitable than other models for sepsis studies.
Acknowledgment Funding: This work was supported by the National Natural Science Foundation of China (81270560). Authors’ contribution: Z.C. contributed to the study design and acquisition of the data. L.S. contributed to the analysis and interpretation of the data and drafting of the manuscript. Y.Z. contributed to revision of the manuscript and statistical analysis.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jss.2018.03.058.
references
1. Derek CA, Tom VDP. Severe sepsis and septic shock. N Engl J Med. 2013;369:840e851. 2. The PRISM Investigators. Early, goal-directed therapy for septic shock d a patient-level meta-analysis. N Engl J Med. 2017;376:2223e2234. 3. Rhodes A, Evans LE, Alhazzani W, et al. Surviving sepsis campaign: international guidelines for management of sepsis and septic shock 2016. Crit Care Med. 2017;45:486e552. 4. Bastarache JA, Matthay MA. Cecal ligation model of sepsis in mice: new insights. Crit Care Med. 2017;41:356e357.
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5. Song JC, Hu DZ, He C, et al. Novel biomarkers for early prediction of sepsis-induced disseminated intravascular coagulation in a mouse cecal ligation and puncture model. J Inflam. 2013;10:202e215. 6. Uhle F, Lichtenstern C, Brenner T, Weigand MA. Pathophysiology of sepsis. Anasthesiol Intensivmed Notfallmed Schmerzther. 2015;50:114e122. 7. Dellinger RP, Carlet JM, Masur H, et al. Surviving sepsis campaign guidelines for management of severe sepsis and septic shock. Crit Care Med. 2004;32:858e873. 8. Dellinger RP, Levy MM, Carlet JM, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med. 2008;36:296e327. 9. Dellinger RP, Levy MM, Rhodes A, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med. 2013;41:580e637. 10. DeLuca L, Hendryx J, Ririe T, et al. Rapid development of septic shock in a swine model using cecal ligation and perforation plus fecal inoculation. Crit Care Med. 2012;40:13e28. 11. Yu CY, Chun YW. Effects of dexmedetomidine on intestinal microcirculation and intestinal epithelial barrier in endotoxemic rats. Anesthesiol. 2016;125:355e367. 12. Taylor FB. Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants. Crit Care Med. 2001;29:78e89. 13. Garrido AP, Cruz RJ, Figueiredo LFPD, Silva MRE. Small volume of hypertonic saline as the initial fluid
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replacement in experimental hypodynamic sepsis. Crit Care. 2006;10:R62. Rahall L, Garrido AG, Cruz RJ, Rocha SM, Figueiredo LFPD. Systemic and regional hemodynamic effects of enalaprilat infusion in experimental normotensive sepsis. Braz J Med Bio Res. 2006;39:1205e1215. Miranda ML, Balarini MM, Balthazar DS, Paes LS, Santos MS, Bouskela E. Ivabradine attenuates the microcirculatory derangements evoked by experimental sepsis. Anesthesiol. 2017;126:140e149. Chang R, Holcomb JB, Johansson PI, Pati S, Schreiber MA, Wade CE. Plasma resuscitation improved survival in a cecal ligation and puncture rat model of sepsis. Shock. 2018;49:53e61. Figueiredo LFPD, Garrido AG, Nakagawa N, Sannomiya P. Experimental models of sepsis and their clinical relevance. Shock. 2008;30:53e59. Maier S, Traeger T, Entleutner M, et al. Cecal ligation and puncture versus colon ascendens stent peritonitis: two distinct animal models for polymicrobial sepsis. Shock. 2004;21:505e511. Quenot JP, Binquet C, Vinault S, Pavon A. Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock. Crit Care. 2013;17:71. Oliver HV, Yousuke M, Mirna YP, et al. Lipopolysaccharideinduced binding of receptor CD300b to Toll-like receptor 4 alters signaling to drive lethal cytokine responses that enhance septic shock. Immunity. 2016;44:1365e1378. Campos LM, Tomas MA, Barrachina LG, et al. Cytokine levels is septic shock. Intens Care Med Exp. 2015;3:A296.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Comparison of two different models of sepsis induced by cecal ligation and puncture in rats Linlin Song, MD,a Yanjie Zou, MD,b and Zhongping Cao, MDb,* a
Postgradulate Training Base of Jinzhou Medical University, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force, Tianjin, China b Department of Anesthesia, The Logistics University of Chinese People Armed Police Force Hospital, Tianjin, China
article info
abstract
Article history:
Background: The present study was designed to explore the difference between two rat
Received 11 January 2018
models of sepsis and to establish a more stable rat model.
Received in revised form
Materials and methods: SpragueeDawley rats were randomly divided into three groups:
26 February 2018
sham operation group, simple cecal ligation and perforation group (SCLP), and cecal
Accepted 21 March 2018
ligation perforation plus drainage group (CLP-DS). The general condition of the rats was
Available online xxx
observed, and the time of death and survival rate at 72 h were recorded. The arterial blood and lung tissue were obtained 9 h after the operation.
Keywords:
Results: The mortality of the CLP-DS group was significantly higher than that of the SCLP
Sepsis model
group. The limitation package, intestinal adhesion, and poor drainage were detected in
Simple cecal ligation and puncture
the SCLP rats, whereas intestinal edema and hyperemia, bloody water in the abdominal
Cecal ligation and puncture-
cavity, but no inflammatory package were observed 24 h after the operation in the CLP-
drainage strip
DS rats by autopsy. There were significant differences in interleukin-6 and tumor
Inflammatory cytokine
necrosis factor-alpha levels between the SCLP group and the CLP-DS group. Severe
Smith lung injury score
pulmonary septal thickening, alveolar wall vascular congestion, and protein debris deposition in the alveolar cavity were observed in the SCLP group, whereas pulmonary bullae were observed in the CLP-DS group using light microscopy, and there were significant difference among groups in Smith lung injury score. Conclusions: These results suggested that the cecal ligation combined with puncture drainage model of sepsis is more stable than that of the simple cecal ligation and puncture model of sepsis in the rat, which resolved the problem of puncture wrapped in the traditional CLP model of sepsis in rat. ª 2018 Elsevier Inc. All rights reserved.
Introduction Sepsis is increasingly common in clinical practice and has extremely high mortality; it is a major complication of serious trauma, burn, shock, and major surgery and is also one of the leading causes of death in critically ill patients.1-3 The cecal ligation perforation sepsis model is most similar
to clinical sepsis, and a rat or mouse cecal ligation perforation (CLP) is commonly used to mimic clinical appendicitis perforation or diverticulitis perforation, which has been recognized as the gold standard of sepsis animal modeling.4,5 However, the morbidity and mortality are unstable in the cecal ligation puncture sepsis model due to many factors, which makes these experimental studies
* Corresponding author. Department of Anesthesia, The Logistics University of Chinese People Armed Police Force Hospital, Chenglin Road NO 222, Hedong District, Tianjin 300162, China. Tel./fax: þ86 22 60577658. E-mail address: [email protected] (Z. Cao). 0022-4804/$ e see front matter ª 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2018.03.058
278
j o u r n a l o f s u r g i c a l r e s e a r c h s e p t e m b e r 2 0 1 8 ( 2 2 9 ) 2 7 7 e2 8 2
challenging. In this study, we compared the rat cecal ligation perforation sepsis model with ligation perforation and the drainage sepsis model to generate a sepsis research model with more stable morbidity and mortality.
Materials and methods Experimental animal and group The experimental protocol was approved by the Ministry of Science and Technology Committee and Animal Center Committee and Logistics University of Chinese People Armed Police Force Hospital. Animal experiments adhered to the ethical guidelines of the International Association for the Study of Pain, and all efforts were made to minimize animal suffering and to reduce the number of animals used. The experiments in this study were performed on male, SpragueeDawley rats (260 g300 g) housed under approved conditions with a 12-/12-h light/ dark cycle and food and water provided ad libitum. The experiments were performed at the same time period on different days to reduce the effect of circadian rhythms on different individuals. The animals were randomly divided into three groups: sham operation group (sham) (n ¼ 16), simple cecal ligation perforation group (SCLP) (n ¼ 32), and cecal ligation perforation plus drainage group (CLP-DS) (n ¼ 32). Only open abdominal cavity operation was performed on rats in the sham group. After the experiments, the rats were euthanized via decapitation under deep anesthesia by ketamine (intraperitoneal injection).
Establishment of rat model SCLP model The rats were anesthetized by 1% ketamine (100 mg/kg) via an intraperitoneal injection after weighing and then fixed on the small animal operating platform board. After abdominal skin preparation and disinfection, a longitudinal incision (length 2 w 2.5 cm) was performed along the ventral line above the pubic bone to open the celiac. The cecum was identified, and it was then carefully taken out. The contents of the cecum were gently pushed to the cecal distal to reduce the gas of ligation cecum, and the mesentery was carefully separated to avoid injury of the ileocecal artery cecal branch. The free distal cecum was placed in methylene blue solution in a sterile cylinder until a clear blue mark was observed around the cecum, at a volume of 2.5 mL mark. The cecum was ligated with 4-0 silk thread according to the mark. Intestinal obstruction did not occur because the ligation sites were lower than the location of the ileocecal valve. Next, an 18 G (1.2 mm 3.8 mm) needle was applied to take a single pass through the cecum from the mesenteric side to the contralateral side. After the puncture, a small amount of feces was squeezed from the two puncture holes to ensure perforation (supplemental Fig. 1). The cecum was placed back into the abdominal cavity after the feces out of the cecum was wiped off. Next, the muscle and skin were sutured with a silk thread (3-0) to close the abdomen. Preheated saline (50 mL/kg, 37 C) was subcutaneously injected into the back for resuscitation. The rats were placed back into the cage after surgery, and food and water were provided ad libitum.
CLP-DS model The specific steps were performed as follows: the preoperative preparation and abdominal cavity opening were performed similar to the SCLP model. After cecum ligation and puncture, the two puncture holes were kept opened with a drainage bar across (twisted strip of sterile gauze with a width of 2 mm) to make sure that the strip was exposed 5 mm outside the two holes. The remaining length was cut off, and the feces around the area were wiped off (Supplemental Fig. 1).
Indicators and testing methods General postoperative observation, including the appearance, activities, mental state of the rats, among other factors, was performed. The death time point and survival rate of the rats were recorded at 72 h after modeling, and the changes in abdominal cavity were observed 24 h after modeling. Six rats of each group were randomly selected, and 3 mL abdominal aorta blood was obtained 9 h after modeling and placed in an anticoagulant tube for 30 min at 4 C. Next, the samples were centrifuged for 15 min at 3000 r/min, and 200 mL serum was placed in a cryopreserved tube for cryopreservation at 80 C. The serum interleukin (IL)-6 and tumor necrosis factor (TNF)-a levels were subsequently detected using an enzyme-linked immunosorbent assay. Lung tissue observation: The lung tissue was fixed with 4% neutral formaldehyde solution for 48 h, then dehydrated, paraffin-embedded, sliced (5 mm), baked, and HE stained. The histopathological changes were observed under a light microscope, and the lung injury was evaluated by Smith lung injury score. According to the Smith score, the pulmonary edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, pulmonary atelectasis, and hyaline membrane formation were semiquantitative analyzed from 0 to 4 point: noninjury was 0 point, the lesion less than 25% of visual field was 1 point, the lesion from 25% to 50% of visual field was 2 point, the lesion from 50% to 75% of visual field was 3 point, and the lesion in full view was 4 point; the total lung injury score was the sum of the aforementioned. Ten visual fields of each rat were observed, and the average value was adopted.
Statistical analysis The data were expressed as the mean standard deviation ðX sÞ, and statistically analyzed using SPSS 17.0 software. The survival rate of each group was analyzed using KaplaneMeier method (log-rank test). IL-6, TNF-a levels, and Smith lung injury scores among groups were analyzed by single factor analysis of variance. A P-value less than 0.05 was considered statistically different.
Results General condition of the rats after modeling The experimental rats were awakened from anesthesia 1-2 h after operation. The rats in the SCLP and CLP-DS groups
279
song et al comparison of two sepsis models in rat
Table e The rat death number of different time point. Group
Time point (h) 6
12
Sham
0
SCLP
0
CLP-DS
0
72 h survival number
72 h survival rate (%)
18
24
48
0
0
0
0
10
0
12
4
3
6
23.1
10
6
6
4
0
0
exhibited a similar general condition, and the clinical signs of the bacterial infection were less obvious within 6 h after the operation. However, there were symptoms of vertical hairs, bow, curled up, fatigue, mental burnout, and shortness of breath in the rats at 6 h after the operation, and there was even intraocular canthus, nose and mouth bleeding, deep breathing, and stimulation without a response in severe cases. The rats that survived 3 d after the operation in the SCLP group began to recover. Rats in the sham group showed free activity and normal drinking and eating. Twenty-four hours after the operation, an autopsy showed that most of the rat cecum in the SCLP group formed a limited package, closed perforation, and intestinal adhesion, whereas rats in the CLP-DS group, rats showed abdominal organ edema and congestion, intestinal deposition, and cecal ischemia necrosis but no inflammatory package formation.
Survival rate of the rats Most rats in the CLP-DS group died from 8 to 12 h after operation. The rat survival rate of the SCLP group was 23.1% at 72 h after the operation, while no rat survived at 72 h after the operation in the CLP-DS group, and all rats survived after 72 h after the operation in the sham group. There was a statistically significant difference among groups in survival rate (P < 0.01, Table). The survival time curve of the rats is shown in Figure 1, which clearly reflects the difference in survival rates. There were significant differences in survival rates among groups analyzed using log-rank test (P < 0.01).
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Changes in serum IL-6 and TNF-a levels in rats The levels of IL-6 and TNF-a in CLP-DS and SCLP groups were significantly higher than those in the sham operation group (P < 0.01), and these levels were higher in the CLP-DS group than those in the SCLP group (P < 0.01, Fig. 2).
Pathological changes in lung tissue Rat lung tissue of the sham group showed that the alveolar structure was relatively complete, with no interval thickening and congestion and no inflammatory cell infiltration. The rats’ lung tissue of the SCLP group showed that lung interval was thickening, alveolar cavity was filled with protein fragments, and alveolar septal had a slight rupture. The changes in the CLP-DS group of rats showed that alveolar septal fracture is more serious with pulmonary bullae formation (Fig. 3). The Smith lung injury score in the CLP-DS group of rat was higher than that of the SCLP group (Fig. 4).
Discussion Hospital patients exhibited high mortality due to sepsis, and sepsis caused shock and multiple organ failure; sepsis is one of the main reasons of death in hospital patients.1,6 The Global Sepsis Alliance, which is involved in the control of sepsis pathophysiological processes and nearly all the supportive treatment of all organs or systems, has released guidelines for the treatment of sepsis. However, the incidence and mortality
Fig. 1 e The 72-h survival rate of rat after operation. **P < 0.01 compared with the sham group. ##P < 0.01 compared with the SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLP-DS [ cecal ligation perforation plus drainage group. (Color version of figure is available online.)
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Fig. 2 e Serum IL-6 and TNF-a levels in each group of rat. **P < 0.01 compared with sham group. ##P < 0.01 compared with SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLP-DS [ cecal ligation perforation plus drainage group.
of sepsis remains at a high level, and its pathogenesis is currently still unclear.3,7-9 Therefore, establishment of high stability and good reproducibility of animal models is very important to study the pathogenesis of sepsis and to screen and evaluate the treatment of drugs.10,11 There are three current methods to establish the sepsis model; the first is the endotoxin model induced by intraperitoneal or intravenous injection of exogenous endotoxin or lipopolysaccharide, the infection dose of the endotoxemia model can be artificially controlled, and this model has characteristics of stability, repeatability, and strong controllability.11,12 Animal reactivity is relatively consistent in this model, but transient input of endotoxin can cause animals to quickly tolerate or die; thus, this model can not be a good simulation of sepsis clinical features, and the cost is high.11,12 The second model is a live bacteria model induced by intravenous injection of a single live or mixed bacteria culture. The infectious shock model was obtained when the positive rate of blood culture reach 69%.13,14 The third is a cecal ligation perforation model induced by colonic seepage leakage and bacterial translocation.15,16 Fecal leakage to the abdominal cavity caused a systemic inflammatory response, circulatory and metabolic changes. The disease is not a transient course. Compared with an injection of lipopolysaccharide or live bacteria, the cecal ligation perforation model has characteristics of better clinical relevance and lower cost.15,16 However, the experimental results of this model are diverse, and the repeatability is poor due to the long modeling time and inconsistent experimental factors. Therefore, it is important
to screen a repeatable sepsis animal model according to the experimental factors and steps. The CLP model is considered to be the gold standard for sepsis studies. However, the CLP model still has some difficult problems to be resolved, such as the consistency requirements of animal surgery, cecal ligation length, size and the number of the puncture hole, and the number of feces leaking into the abdominal cavity. In addition, the infection foci are easily wrapped by the omentum and surrounding tissue. All of these factors affect the prognosis of rats and cannot achieve sepsis animal model standards.17,18 Thus, we need to examine an animal model that can quantify feces leaking into the abdominal cavity and to overcome the insufficient intraperitoneal infection of a traditional CLP model. Generally, organ dysfunction or death of rats occurs 6-12 h after sepsis modeling and is mainly caused by systemic inflammatory response.1-3,19 The key to achieve this requirement is to produce an appropriate intensity of infection and to stimulate excessive inflammation. All these conditions were obtained with the rat CLP-DS model of sepsis. The results of this study showed that the survival rates of the two groups were less than 30%, which was consistent with the requirements of the sepsis model.17,18 Importantly, the rat mortality rate in the CLP-DS group was significantly higher than that in the SCLP group, and the rat death time point was focused on 6-12 h after modeling, which indicated that systemic inflammatory response syndrome occurred earlier and more intensely in the CLP-DS group than that in the SCLP
Fig. 3 e Histopathological features of lung tissue in each group (HE X200). Red arrow indicates thickening of the lungs, black arrow indicates protein fragments, and blue arrow indicates lung bullae. (A) Sham group, (B) SCLP group, and (C) CLP-DS group. (Color version of figure is available online.)
song et al comparison of two sepsis models in rat
Fig. 4 e Smith lung injury score in each group of rat. **P < 0.01 compared with sham group. ##P < 0.01 compared with SCLP group. Sham [ sham operation group; SCLP [ simple cecal ligation perforation group; CLPDS [ cecal ligation perforation plus drainage group.
group. When the body has developed systemic inflammatory response syndrome, a large number of proinflammatory factors are released into circulation. IL-6 and TNF-a are important indicators of the degree of systemic inflammatory syndrome and the severity and prognosis of sepsis.20,21 The levels of IL-6 and TNF-a in the CLP-DS group were significantly higher than those in the SCLP group. The symptoms of the CLP-DS group were similar to acute peritonitis due to the intestinal feces that continued to leak into the abdominal cavity, whereas symptoms of the SCLP rats were similar to abdominal abscess formation due to the severe infection that was more likely to occur after necrosis of the cecal bowel ligation. This difference could also be proven by the rat abdominal anatomy, which demonstrated that the edema and congestion of abdominal organs of the CLP-DS group was more severe than that of the SCLP, whereas the majority of SCLP survivors demonstrated localized abscess formation. The length of ligation cecum did not accurately reflect the total amount of fecal leakage into the abdominal cavity to cause infection, due to the variety of shapes of the experimental rats’ cecum. At the same time, as the cecum ligation was performed only according to the length of the surface of one side of the cecum, the mark was not complete. There was a deviation among the cecum length of experimental rats.17,18 Although the species, strain, sex, and weight of the experimental rats were similar, the size of the cecum between different individuals and the amount of their contents were significantly different. The volume-controlled ligation method used in the CLP model in this experiment was not limited by the cecal geometry, which eliminated the difference in cecal size between different individuals and reflected the equal amount of intestinal contents. It has been reported that endotoxin levels in rats at 2 h after CLP began to increase, reached a peak at 12 h and a sharp increase at 6-12 h, and the systemic inflammatory response had the same time course.10,17,18 The blood and lung samples were then taken at 9 h after the modeling. Pulmonary pathology observed by light microscopy and evaluated by Smith score also showed that pathological changes of the CLP-DS group were more severe than those of the SCLP group. In this study, the procedure was performed by the same operator on the same time point on a different day to reduce the effect of operator and circadian
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rhythm on the inflammatory response between different individuals. The same puncture needles were applied. These results showed that the model was stable and reliable and had good controllability and repeatability. The deficiency of this experiment is that the experimental animals did not have antibiotic and lesion excision treatment, which could not completely simulate the pathophysiological process of the patients with septic shock, but it may serve as a stable septic shock model for a variety of acute animal experiments to study the mechanism of sepsis and to evaluate the efficacy of drugs.
Conclusions Our study suggests that cecal ligation combined with puncture drainage model of sepsis can quantify feces leakage into the abdominal cavity and generate an insufficient intraperitoneal infection of traditional CLP model, which demonstrates greater morbidity and mortality, inflammatory mediators, and severe lung injury compared with the simple cecal ligation and puncture model of sepsis in rats. Thus it is more suitable than other models for sepsis studies.
Acknowledgment Funding: This work was supported by the National Natural Science Foundation of China (81270560). Authors’ contribution: Z.C. contributed to the study design and acquisition of the data. L.S. contributed to the analysis and interpretation of the data and drafting of the manuscript. Y.Z. contributed to revision of the manuscript and statistical analysis.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jss.2018.03.058.
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