参见作者原研究论文

本实验方案简略版
Jan 2020

本文章节


 

Using the Cecal Ligation and Puncture Model of Sepsis to Induce Rats to Multiple Organ Dysfunction
采用败血症盲肠结扎穿刺模型诱导大鼠多脏器功能障碍   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Sepsis is a dysregulated hyperinflammatory disease caused by infection. Sepsis leads to multiple organ dysfunction syndrome (MODS), which is associated with high rates of mortality. The cecal ligation and puncture (CLP) model has been widely used in animals and has become the gold-standard method of replicating features of sepsis in humans. Despite several studies and modified CLP protocols, there are still open questions regarding the multifactorial determinants of its reproducibility and medical significance. In our protocol, which is also aimed at mimicking the sepsis observed in clinical practice, male Wistar rats are submitted to CLP with adequate fluid resuscitation (0.15 M NaCl, 25 ml/kg BW i.p.) immediately after surgery. At 6 h after CLP, additional fluid therapy (0.15 M NaCl, 25 ml/kg BW s.c.) and antibiotic therapy with imipenem-cilastatin (single dose of 14 mg/kg BW s.c.) are administered. The timing of the fluid and antibiotic therapy correspond to the initial care given when patients are admitted to the intensive care unit. This model of sepsis provides a useful platform for simulating human sepsis and could lay the groundwork for the development of new treatments.

Keywords: Sepsis (败血症), Rats (大鼠), Cecal ligation and puncture (CLP) (盲肠结扎穿孔术), Organ dysfunction (器官功能障碍), Animal Model (动物模型), Acute Kidney Injury (急性肾损伤)

Background

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (Singer et al., 2016). It is a major public health problem worldwide and is the leading cause of multiple organ dysfunction syndrome (MODS), which is associated with high in-hospital mortality (Torio and Moore, 2016; Kellum et al., 2019). When sepsis was first defined in 1991, the focus was on the systemic inflammatory response (ACCP/SCCMCC et al., 1992). Since then, studies of pro- and anti-inflammatory molecules have advanced and have begun to reveal that the host adaptive responses go beyond immunological phenomena (Singer et al., 2016). In the early or acute phase, sepsis is characterized by a hyperinflammatory host response accompanied by pro- and anti-inflammatory processes that lead to cell death by apoptosis. In the late or immunosuppressive phase, it is characterized by depletion of immune cells (Hotchkiss et al., 2013). In the early phase, many physiological systems are compromised, hemodynamic and cardiopulmonary alterations being the most clinical significant symptoms (Rello et al., 2017). Despite the use of several modern treatment strategies, including fluid and antimicrobial therapy, the mortality rates associated with sepsis remain high. Therefore, experimental animal models have been developed apace with the evolution of the knowledge of sepsis and advances in its treatment. The establishment of an animal model that replicates the features of sepsis in humans is essential for understanding the pathogenic mechanisms and for developing new treatment strategies. Various experimental rodent models have been described, including intraperitoneal or intravenous infusion of purified endotoxin (lipopolysaccharide), intravascular infusion of certain bacterial species, the abscess model, and the cecal ligation and puncture (CLP) model (Baker et al., 1983; Parker et al., 2001; Fink, 2014).


The CLP model is a polymicrobial model of bacterial peritonitis that is considered the gold-standard method of replicating features of sepsis in humans, which is easily reproducible, and can be adapted to mimic specific clinical events. It has been widely used as a research tool for more than 30 years (Wichterman et al., 1980). The use of this model produces a hyperdynamic circulatory state, mitochondrial dysfunction, and endothelial alterations, as well as having acute renal, pulmonary, and cardiac effects. It also stimulates receptor molecules present in immune cells, such as Toll-like receptors (TLRs), and cytokine production, as observed in pre-clinical studies (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016; Capcha et al., 2020). The CLP model can be modified to produce varying degrees of severity, depending on the cecum ligation site and the number of punctures made (Singleton et al., 2003).


In our version of the CLP model, antibiotics and crystalloid solution are administered at 6 h after the procedure, in accordance with intensive care unit (ICU) guidelines for the restoration of blood volume and pressure (Singer et al., 2016). Although early recognition of sepsis is particularly important for appropriate patient management, as well as to achieve favorable outcomes, not all patients have timely access to treatment. Therefore, the time to the start of treatment in our model is intended to simulate the delay that occurs when patients have limited access to health care. In this rat model, 5-day mortality is typically 30-60%.


Here, we describe the CLP model in detail in order to disseminate the methodology, as well as to contribute to a better understanding of the mechanisms involved in the development of sepsis.


Materials and Reagents

  1. Gauze pads (Dentalcremer)

  2. 16 G needles (Becton Dickinson, catalog number: 305198)

  3. 21 G needles (Becton Dickinson, catalog number: 305165)

  4. Razor blades (no specific brand)

  5. Liquid soap (no specific brand)

  6. Syringes (5 ml and 10 ml)

  7. Operator protective equipment: surgical gloves and face mask

  8. Mononylon 3.0 suture (Ethicon, Johnson & Johnson Medical Device Companies)

  9. Mononylon 4.0 suture (Ethicon, Johnson & Johnson Medical Device Companies)

  10. Male Wistar rats (200-300 g), obtained from institutional animal facility

  11. Isoflurane 100% (Cristália, Isoforine®), stored at room temperature

  12. Imipenem-cilastatin (Merck Sharp & Dohme Corp, Tienam®), stored at 4 °C

  13. Tramadol hydrochloride 50 mg/ml

  14. Ketamine (BioChimico, cloridrato de cetamina), stored at room temperature

  15. Xylazine 2% (Syntec, xilazin), stored at room temperature

  16. Alcohol 70% (Rioquimica, rialcool 70), stored at room temperature

  17. Sterile saline solution 0.9% wt/vol (Fresenius Medical Care Ltda)

  18. Povidone-iodine (Rioquimica, riodeinetopico), stored at room temperature

Equipment

  1. Surgical instruments: dissection scissors, microdissection scissors, straight surgical forceps, straight anatomical forceps, and needle holder (IncolInstrumentosCirúrgicos)

    Note: Sterilize these instruments by autoclaving before use.

  2. Baby bed warmer (Sunbeam Heating Pad, catalog number: 000756-500-000U)

  3. Isoflurane vaporizer (Harvard Apparatus, 75-0239 INT, catalog number: 1073/216)

Procedure

  1. Preoperative settings

    1. Design the study and experiments in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

    2. Weigh animals to verify that all weights are within the 200-300 g range and use the individual weights to determine the dose of antibiotic and the amount of fluid resuscitation to be used, as well as the doses of ketamine and xylazine in case anesthesia is necessary.

    3. Clean and disinfect the operating table with disinfectant wipes or alcohol.


  2. Anesthesia

    1. Anesthetize the animals with isoflurane (0.25-2.5%/kg BW gas inhalation) or with ketamine (80-100 mg/kg BW i.p.) with xylazine (5-15 mg per kg body weight i.p.) for general tests, such as cardiovascular, pulmonary or renal studies.

      Note: Commonly, rats weighing around 300 g present more tolerance to anesthetics, therefore we suggest increasing the dose according to the ranges mentioned above.

    2. Verify the effect of the anesthesia by pinching the tail and a toe. Typically, the extremities will be unresponsive within 1-2 min after administration of the anesthetic agent(s). In our experience, isoflurane can be used for all procedures.

    3. Shave the lower abdominal region using liquid soap and razor blades. Disinfect the region with gauze, alcohol, and povidone-iodine (Figure 1A).

    4. Place rats in the supine position on a flat surface (Figures 1A-1I).

      Note: In case of training or some reasons where the surgery take much time, we recommend using surgical eye drops to protect rat eyes.


  3. Cecal ligation and puncture

    1. Make a longitudinal incision (3-4 cm long) in the disinfected region with scissors and tweezers (Figure 1B).

    2. Dissect the muscle fascia from the abdominal musculature and carefully make a 3- to 4-cm incision (Figure 1B).

    3. Find the cecum, using anatomical tweezers to expose it (i.e., bring it out of the abdominal cavity). In many cases, the cecum is located on the left side of the abdomen (Figure 1C).

      Note: Expose only the cecum and leave the bowels inside peritoneal cavity, taking care to do not damage the mesenteric blood vessels (Figure 1C).

      Sham group: After completing the Step C3, return the cecum to the abdominal cavity, then close the abdominal musculature and skin as indicated in the Steps C8 and C9.

    4. Use thin tweezers to cross the cecal mesentery with nonabsorbable silk suture 3.0 to ligate the cecum (1.5-cm from the cecal tip or 50% of the cecum; Figure 1D).

      Note: Keep the cecal contents near the cecal tip and keep the intestinal passage (through the ileocecal valve) free (Figure 1E).

    5. Puncture the distal cecum twice with a 16 G needle. The perforations should traverse the cecum (Figure 1F).

    6. Squeeze the punctured distal cecum in order to extract a small amount of feces (approximately 2 drops) and distribute it around the cecum and peritoneal cavity (Figure 1G).

      Note: Extract the same amount of feces and distribute it in the same regions in all animals. Use tweezers to distribute it.

    7. Return the cecum to the abdominal cavity carefully (i.e., without extracting more feces on the abdomen).

    8. Close the abdominal musculature by simple suturing. Before completing the closure, administer fluid resuscitation with pre-warmed saline solution 0.9% or 0.15 M NaCl at 37 °C (25 ml/kg BW i.p.), as illustrated in Figure 1H.

    9. Close the abdominal skin with simple sutures (Figure 1I).

      Note: Ideally all these steps should be performed within 10 min, controlled from the first surgical step (Video 1).



      Figure 1. Cecal Ligation and Puncture Model in Rats. A. Disinfection and shaving of the lower abdominal region. B. Longitudinal incision in the skin and muscle fascia. C. Cecum exposure. D-E. Cecal ligation (in the mid-cecum). F. Puncture of the distal cecum with a 16 G needle. G. Squeezing and distribution of feces around the cecum and peritoneal cavity. H-I. Closure of the abdominal muscle fascia and skin.


      Video 1. Cecal Ligation and Puncture Model in Rats. This video was made at University of São Paulo (USP) according to guidelines from the “USP” on Animal Care and approved by the Research Ethics Committee at University of São Paulo School of Medicine under protocol # 378 603.

  4. Postoperative care

    1. After abdominal closure, inject tramadol hydrochloride 50 mg/ml (dose: 5 mg/kg i.m., every 8 h) for postoperative analgesia and continue as necessary.

    2. Return rats immediately to pre-warmed cages with a baby bed warmer (25 °C) below each cage.
      Note: Food and water should be made available.

    3. At 6 h after the procedure (sham or CLP), under anesthesia with isoflurane, administer imipenem-cilastatin (14 mg/kg BW in saline solution s.c.). Then inject saline solution 0.9% at 25 °C (25 ml/kg BW in saline solution s.c.).

      Note: Subcutaneous administration (s.c.) for saline solution should be distributed in different abdominal regions, we suggest at the least 5 places. Be careful and avoid administering high volume of solution in a specific zone

    4. Return rats immediately to cages in a temperature-controlled environment (20 °C), on a 12/12-h light/dark cycle, with ad libitum access to food and water. The animals should be monitored every 6 h, and outcomes should be evaluated at 24 h or 48 h.

Notes

This rat CLP protocol mirrors the clinical conditions seen in patients with sepsis. Despite the fact that fluid and antimicrobial therapy are administered at 6 h after sepsis induction, the rats develop MODS and the overall mortality rate is 30-60% (Figure 2). The results are reproducible, as has been demonstrated in pre-clinical studies (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016; Capcha et al., 2020). However, deviation from the protocol, especially in the cecal ligation or cecal puncture steps, can result in some variability. Volume resuscitation is extremely important to maintain the central blood volume of the rats. The fluid resuscitation is performed in order to mimic the treatment given to patients in the early (hyperdynamic) phase of sepsis. It is also important that the saline solution be pre-warmed (to 37 °C) to avoid iatrogenic hypothermia, which is likely to affect the outcome of the CLP procedure. Precautions should be taken to avoid cecal bleeding and ileocecal obstruction, which could cause the rats to stop drinking water and go into hypovolemic shock.



Figure 2. Survival curve in rats. Survival rates in rats induced to sepsis by cecal ligation and puncture (CLP, n = 12) or submitted to a sham procedure (n = 16), with administration of fluid and antimicrobial therapy at 6 h after the procedure.


    In patients with sepsis, fluids such as saline or crystalloid solution (i.e., Ringer’s lactate solution) are administered in accordance with established guidelines (Singer et al., 2016). Although the total volume administered varies depending on the condition of each patient, it should be sufficient to restore normal blood volume and pressure, thus ensuring organic perfusion. In clinical practice, the initial antimicrobial should be a broad-spectrum antibiotic and should be re-evaluated after the microbiology results are known. Early antimicrobial therapy and focused control are associated with favorable outcomes (Rello et al., 2017). At our institution, we use imipenem-cilastatin, a broad-spectrum antibiotic, as the first-line treatment for patients with sepsis. As in patients, all of these points should be borne in mind in experimental models of sepsis, in order to induce MODS while controlling the disease slightly.

    In our pre-clinical studies (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016; Capcha et al., 2020), MODS was observed at 24 h after CLP. Studies employing our version of the CLP protocol have reported that the main effects are renal, cardiovascular, and pulmonary dysfunction (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016; Capcha et al., 2020). Such studies have also reported sepsis-induced acute kidney injury, as identified by evaluating inulin clearance, tubular transport, urinary osmolality, and mitochondrial morphology, as well as glomerular dysfunction, tubular lesion (urine concentrating defect and reduced fractional excretion of electrolytes), and inflammation (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016). Our version of the CLP protocol also promotes pulmonary inflammation, as demonstrated by greater macrophage infiltration and expression of TLR4 on the surface of macrophages (Cóndor et al., 2016; Capcha et al., 2020).

    After CLP, there is a significant increase in the serum levels of cytokines, leading to damage of the microvascular endothelium. It has also been demonstrated that CLP leads to downregulation of the expression of endothelial nitric oxide synthase, Slit2, and Robo4, proteins that are responsible for maintaining the integrity of the endothelial barrier (Moreira et al., 2014). In addition, there is considerable evidence that the procedure results in cardiac injury, manifesting as diastolic dysfunction, a reduction in the left ventricular end-diastolic diameter and pressure, and sustained increase in heart rate (Moreira et al., 2014). Sepsis can reduce renal expression of Klotho, which has been be associated with worse outcomes (Cóndor et al., 2016). Sepsis is characterized by a hyperinflammatory mechanism, TLR4/nuclear factor-kappa B signaling being one of the mechanisms most often defined as a parameter to evaluate treatment outcomes. Furthermore, overexpression of pro-inflammatory cytokines in the kidney, spleen, heart, and serum has been reported in animals induced to sepsis. As a consequence of this inflammatory disorder, an apoptotic mechanism involving BAX and Bcl-2 expression, together with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells, has also been reported (Rodrigues et al., 2012; Moreira et al., 2014; Cóndor et al., 2016). Our version of the CLP protocol can elicit these major mechanisms of sepsis.

    The CLP model can be adapted to the needs/goals of each laboratory. In our version of the CLP protocol, the end point of MODS is achieved, as evidenced by the mortality rate. This novel CLP protocol was established in order to mimic the conditions seen in ICU patients with sepsis. This protocol, involving controlled MODS, has proven to be reproducible across studies if all of the technical parameters are carefully managed.

Acknowledgments

This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo Research Foundation; Grant no. 2010/19012-0). JMCC is the recipient of a FAPESP grant (Grant no. 2015/21308-9). LA is the recipient of a grant from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (National Council for Scientific and Technological Development; Grant no. 301193/2016-9).

    The present protocol was adapted from Wichterman et al. (1980).

Competing interests

The authors declare that they have no conflicts of interest or competing interests.

Ethics

The Research Ethics Committee of the University of São Paulo School of Medicine Hospital das Clínicas, in the city of São Paulo, Brazil, has approved the use of this protocol (Reference no. 378 603). All procedures are in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

References

  1. ACCP/SCCMCC. (1992). American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 20(6): 864-874.
  2. Baker, C. C., Chaudry, I. H., Gaines, H. O. and Baue, A. E. (1983). Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery 94(2): 331-335.
  3. Capcha, J. M. C., Rodrigues, C. E., Moreira, R. d. S., Silveira, M. D., Dourado, P., Dos Santos, F., Irigoyen, M. C., Jensen, L., Garnica, M. R., Noronha, I. L., Andrade, L. and Gomes, S. A. (2020). Wharton's jelly-derived mesenchymal stem cells attenuate sepsis-induced organ injury partially via cholinergic anti-inflammatory pathway activation. Am J Physiol Regul Integr Comp Physiol 318(1): R135-R147.
  4. Cóndor, J. M., Rodrigues, C. E., Sousa Moreira, R., Canale, D., Volpini, R. A., Shimizu, M. H., Camara, N. O., Noronha Ide, L. and Andrade, L. (2016). Treatment With Human Wharton's Jelly-Derived Mesenchymal Stem Cells Attenuates Sepsis-Induced Kidney Injury, Liver Injury, and Endothelial Dysfunction. Stem Cells Transl Med 5(8): 1048-1057.
  5. Fink, M. P. (2014). Animal models of sepsis. Virulence 5(1): 143-153.
  6. Hotchkiss, R. S., Monneret, G. and Payen, D. (2013). Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol 13(12): 862-874.
  7. Kellum, J. A., Wen, X., de Caestecker, M. P. and Hukriede, N. A. (2019). Sepsis-Associated Acute Kidney Injury: A Problem Deserving of New Solutions. Nephron 143(3): 174-178.
  8. Moreira, R. S., Irigoyen, M., Sanches, T. R., Volpini, R. A., Camara, N. O., Malheiros, D. M., Shimizu, M. H., Seguro, A. C. and Andrade, L. (2014). Apolipoprotein A-I mimetic peptide 4F attenuates kidney injury, heart injury, and endothelial dysfunction in sepsis. Am J Physiol RegulIntegr Comp Physiol 307(5): R514-524.
  9. National Research Council (US). (2011). Committee for the Update of the Guide for the Care and Use of L. Guide for the Care and Use of Laboratory Animals [Online]. 8th edition.
  10. Parker, S. J. and Watkins, P. E. (2001). Experimental models of gram-negative sepsis. Br J Surg 88(1): 22-30.
  11. Rello, J., Valenzuela-Sanchez, F., Ruiz-Rodriguez, M. and Moyano, S. (2017). Sepsis: A Review of Advances in Management. Adv Ther 34(11): 2393-2411.
  12. Rodrigues, C. E., Sanches, T. R., Volpini, R. A., Shimizu, M. H. M., Kuriki, P. S., Camara, N. O. S., Seguro, A. C. and Andrade, L. (2012). Effects of continuous erythropoietin receptor activator in sepsis-induced acute kidney injury and multi-organ dysfunction. PloS one 7(1): e29893-e29893.
  13. Singer, M., Deutschman, C. S., Seymour, C. W., Shankar-Hari, M., Annane, D., Bauer, M., Bellomo, R., Bernard, G. R., Chiche, J. D., Coopersmith, C. M., Hotchkiss, R. S., Levy, M. M., Marshall, J. C., Martin, G. S., Opal, S. M., Rubenfeld, G. D., van der Poll, T., Vincent, J. L. and Angus, D. C. (2016). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315(8): 801-810.
  14. Singleton, K. D. and Wischmeyer, P. E. (2003). Distance of cecum ligated influences mortality, tumor necrosis factor-alpha and interleukin-6 expression following cecal ligation and puncture in the rat. Eur Surg Res 35(6): 486-491.
  15. Torio, C. M. and Moore, B. J. (2016). National Inpatient Hospital Costs: The Most Expensive Conditions by Payer, 2013. Statistical Brief #204. In: Healthcare Cost and Utilization Project (HCUP) Statistical Briefs [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US).
  16. Wichterman, K. A., Baue, A. E. and Chaudry, I. H. (1980). Sepsis and septic shock--a review of laboratory models and a proposal.J Surg Res 29(2): 189-201.


简介

[摘要]败血症是由感染引起的失调的高炎症性疾病。败血症导致多器官功能障碍综合征(MODS),其与高死亡率相关。盲肠结扎穿刺(CLP)模型已在动物中广泛使用,并已成为在人类中复制败血症特征的金标准方法。尽管进行了多项研究并修改了CLP协议,但关于其可重复性和医学意义的多因素决定因素仍存在未解决的问题。在我们的协议中,其目的还在于模仿在临床实践中观察到的败血症,雄性Wistar大鼠在手术后立即接受CLP并进行足够的液体复苏(0.15 M NaCl,25 ml / kg BW ip)。CLP后6小时,进行额外的液体疗法(0.15 M NaCl,25 ml / kg BW sc)和用亚胺培南-西司他丁的抗生素疗法(单剂量14 mg / kg BW sc)。液体疗法和抗生素疗法的时机与患者进入重症监护室时的初始护理相对应。这种脓毒症模型为模拟人类脓毒症提供了有用的平台,并可能为开发新疗法奠定基础。

[背景]败血症的定义是宿主对感染的反应失调导致的危及生命的器官功能障碍(Singer等,2016)。它是全球主要的公共卫生问题,是多器官功能不全综合征(MODS)的主要原因,多器官功能不全综合征与医院内高死亡率相关(Torio and Moore ,201 6 ; Kellum et al。,2019)。1991年首次定义败血症时,重点是全身性炎症反应(ACCP / SCCMCC等,1992)。从那时起,对促炎和抗炎分子的研究已经取得进展,并开始揭示宿主的适应性反应已经超越了免疫学现象(Singer等人,2016)。在早期或急性期,败血症的特征在于高炎症宿主反应,并伴有促炎症过程和消炎过程,这些过程通过凋亡导致细胞死亡。在晚期或免疫抑制阶段,其特征在于免疫细胞的耗竭(Hotchkiss等,2013)。在早期阶段,许多生理系统受到损害,血液动力学和心肺改变是临床上最明显的症状(Rello等人,2017)。尽管使用了包括液体疗法和抗菌疗法在内的几种现代治疗策略,败血症相关的死亡率仍然很高。因此,随着脓毒症知识的发展和治疗的进展,已经迅速开发了实验动物模型。建立在人类中复制败血症特征的动物模型对于理解致病机理和开发新的治疗策略至关重要。已经描述了各种实验性啮齿动物模型,包括腹膜内或静脉内输注纯化的内毒素(脂多糖),某些细菌种类的血管内输注,脓肿模型以及盲肠结扎和穿刺(CLP)模型(Baker等,1983; Parker。等人,2001; Fink,2014)。

CLP模型是细菌性腹膜炎的一种多菌种模型,被认为是在人类中复制败血症特征的金标准方法,该方法易于复制,并且可以适应特定的临床事件。30多年来,它已被广泛用作研究工具(Wichterman等,1980)。使用此模型会产生高动力循环状态,线粒体功能障碍和内皮改变,并具有急性肾,肺和心脏功能。这也刺激受体分子存在于免疫细胞,如Toll样受体(TLR)和细胞因子产生,如在临床前研究中观察到(Rodrigues的等人,2012;莫雷拉等人,2014;Ç ó ndor等等人,2016; Capcha等人,20 20 )。可以修改CLP模型,以产生不同程度的严重性,具体取决于盲肠结扎部位和穿刺次数(Singleton等,2003)。

在我们的CLP模型中,根据重症监护病房(ICU)指导的恢复血容量和血压的操作,在术后6小时使用抗生素和晶体溶液(Singer等人,2016)。尽管败血症的早期识别对于适当的患者管理以及取得良好的结局特别重要,但并非所有患者都能及时获得治疗。因此,在我们的模型中开始治疗的时间旨在模拟当患者无法获得医疗服务时发生的延迟。在该大鼠模型中,5天死亡率通常为30-60%。

在这里,我们将详细描述CLP模型,以传播方法论,并有助于更好地了解败血症发展中涉及的机制。

关键字:败血症, 大鼠, 盲肠结扎穿孔术, 器官功能障碍, 动物模型, 急性肾损伤



材料和试剂


1.纱布(Dentalcremer)     

2. 16 G针(Becton Dickinson,目录号:305198 )     

3. 21克针(Becton Dickinson,目录号:305165 )     

4.剃须刀片(无特定品牌)     

5.液体肥皂(无特定品牌)     

6.注射器(5毫升和10毫升)     

7.操作员防护装备:手术手套和口罩     

8. Mononylon 3.0缝合线(Ethicon,强生医疗设备公司)     

9. Mononylon 4.0缝合线(Ethicon,强生医疗设备公司)     

10.从机构动物设施获得的雄性Wistar大鼠(200-300 g) 

11.异氟烷100%(Cristália,Isoforine ® ),在室温下保存 

12.亚胺培南西司他丁(默沙东公司,泰能® ),保存于4 ℃下 

13.盐酸曲马多50 mg / ml 

14.氯胺酮(BioChimico,cloridrato de cetamina),在室温下保存 

15. 2%的甲苯噻嗪(Syntec,xilazin),在室温下保存 

16.酒精70%(Rioquimica,rialcool 70),在室温下保存 

17.无菌盐溶液0.9%wt / vol(Fresenius Medical Care Ltda) 

18.聚维酮碘(Rioquimica,riodeinetopico),在室温下保存 



设备


手术器械:解剖剪刀,显微解剖剪刀,笔直的手术钳,笔直的解剖钳和持针器(IncolInstrumentosCirúrgicos)。
注意:使用前,请通过高压灭菌器对这些器械进行灭菌。


婴儿床暖床(阳光加热垫,目录号:000756-500-000U)
异氟烷气化器(Harvard Apparatus,75-0239 INT,目录号:1073/216)


程序


术前设定
根据《国家研究委员会实验动物的护理和使用指南》(国家研究委员会,2011年)设计研究和实验。
称重动物以确认所有重量都在200-300 g范围内,并使用各个重量来确定抗生素的剂量和要使用的液体复苏的量,以及在需要麻醉的情况下氯胺酮和甲苯噻嗪的剂量。 。
用消毒湿巾或酒精清洁和消毒手术台。


麻醉
用异氟烷(0.25-2.5%/ kg BW气体吸入)或氯胺酮(80-100 mg / kg BW ip ip)和甲苯噻嗪(5-15 mg / kg体重ip)麻醉动物,以进行常规测试,例如心血管疾病,肺部或肾脏研究。
注意:通常,体重约300 g的大鼠对麻醉药的耐受性更高,因此我们建议根据上述范围增加剂量。


捏住尾巴和脚趾,验证麻醉效果。通常,在施用麻醉剂后1-2分钟内,四肢无反应。根据我们的经验,异氟烷可用于所有程序。
使用液体肥皂和剃须刀片刮擦下腹部区域。用纱布,酒精和聚维酮碘消毒该区域(图1A)。
将大鼠平卧在仰卧位置(图s 1A-1I)。
注意:如果需要培训或出于某些原因而需要大量的手术时间,我们建议使用手术滴眼液保护大鼠的眼睛。


盲肠结扎和穿刺
用剪刀和镊子在消毒区域做一个纵向切口(3-4厘米长)(图1B)。
从腹部肌肉解剖肌肉筋膜,并小心地切一个3至4厘米的切口(图1B)。
找到盲肠,使用解剖镊揭露它(即,把它拿出来腹腔)。在许多情况下,盲肠位于腹部的左侧(图1C)。
注意:仅暴露盲肠并将肠留在腹膜腔内,注意不要损坏肠系膜血管(图1C)。


假手术组:完成步骤C3后,将盲肠放回腹腔,然后按照步骤C8和C9中的d所示,关闭腹部肌肉和皮肤。


用细镊子用不可吸收的丝线3.0穿过盲肠肠系膜以结扎盲肠(距盲肠尖端1.5厘米或盲肠的50%;图1D)。
注意:将盲肠内容物留在盲肠尖端附近,并保持肠道通道(通过回盲瓣)畅通(图1E)。


用16 G针刺穿盲肠远端盲肠两次。穿孔应穿过盲肠(图1F)。
挤压穿刺的盲肠远端,以提取少量粪便(约2滴)并将其分布在盲肠和腹膜腔周围(图1G)。
注意:提取相同数量的粪便,并将其分布在所有动物的相同区域。使用镊子分发


小心地将盲肠放回腹腔中(即不要在腹部抽出更多的粪便)。
通过简单的缝合即可闭合腹部肌肉。在完成封闭之前,在37°C(25 ml / kg BW ip ip)下用预热的0.9%盐水溶液或0.15 M NaCl进行液体复苏,如图1H所示。
用简单的缝合线闭合腹部皮肤(图1I)。
注意:理想情况下,所有这些步骤均应在10分钟内执行,并应从第一个手术步骤开始控制(视频1 )。




图1.大鼠盲肠结扎和穿刺模型。A.下腹部消毒和剃须。B.在皮肤和肌肉筋膜中的纵向切口。C.盲肠暴露。DE。盲肠结扎(盲肠中)。F.用16 G针刺穿盲肠远端。G.在盲肠和腹膜腔周围挤压和分布粪便。你好。腹部肌肉筋膜和皮肤关闭。




视频1.大鼠的盲肠结扎和穿刺模型。该视频是根据圣保罗大学动物护理指南从圣保罗大学(USP)制作的,并由圣保罗大学医学院研究伦理委员会批准,协议编号为378603 。


术后护理
腹部闭合后,注射盐酸曲马多50 mg / ml(剂量:5 mg / kg im,每8小时一次)用于术后镇痛,并在必要时继续进行。
立即将大鼠放回预热的笼子中,并在每个笼子下面放一个婴儿床(25°C)。注意:应提供食物和水。
手术后6小时(假手术或CLP),在异氟烷麻醉下,施用亚胺培南-西司他丁(13 mg / kg体重的生理盐水,皮下注射)。然后在25°C下注入0.9%的盐溶液(在盐溶液sc中为25 ml / kg BW)。
注:生理盐水皮下注射(SC),应分配ð在不同的腹部地区,我们建议在至少5个名额。注意并避免在特定区域内使用大量溶液

立即将大鼠放回到温度受控的环境(20°C)中的笼子中,进行12/12小时的明暗循环,可以随意获取食物和水。应每6小时对动物进行一次监测,并应在24 h或48 h评估结果。


笔记


该大鼠CLP协议反映了败血症患者的临床情况。尽管在脓毒症诱发后6小时进行了液体和抗菌疗法,但大鼠仍会发展为MODS,总死亡率为30-60%(图2)。结果是可再现的,如已经在临床前研究中得到证实(Rodrigues的等人,2012;莫雷拉等人,2014; COND或等人,2016; CAPCHA等人,20 20 )。但是,与规程的偏离,特别是在盲肠结扎或盲肠穿刺步骤中,可能会导致某些可变性。容积复苏对于维持大鼠的中心血容量极为重要。进行液体复苏是为了模拟败血症早期(超动力)阶段给予患者的治疗。将盐溶液预热(至37°C)以避免医源性体温过低,这很可能会影响CLP程序的结果,这一点也很重要。应采取预防措施,避免盲肠出血和回盲肠阻塞,这可能导致大鼠停止饮水并进入低血容量性休克。




图2.大鼠的生存曲线。通过盲肠结扎和穿刺(CLP,n = 12)或进行假手术(n = 16)诱发败血症的大鼠的存活率,在手术后6 h进行液体和抗菌治疗。


在脓毒症患者,流体如盐水或晶体液(即,林格氏乳酸盐溶液)按照制定的准则被施用(歌手等人,2016)。尽管总给药量根据每个患者的病情而有所不同,但应足以恢复正常的血液量和压力,从而确保器质性灌注。在临床实践中,初始抗微生物应该是一种广谱抗生素,应该重新-微生物学结果是已知的后评价。早期的抗微生物治疗和重点控制与良好的预后相关(Rello et al。,2017)。在我们的机构中,我们使用广谱抗生素亚胺培南-西司他丁作为败血症患者的一线治疗药物。与患者一样,在脓毒症的实验模型中应牢记所有这些要点,以便在轻微控制疾病的同时诱导MODS。


在我们的临床前研究(Rodrigues的等人,2012;莫雷拉。等人,2014;Ç ó ndor 。等人,2016; CAPCHA 。等人,20 20 ),MODS在24小时观察到的CLP后。已经采用我们的CLP协议的版本研究报道,主要的作用是肾,心血管和肺功能障碍(罗德里格斯等人。,2012;莫雷拉等人2014;Ç ó ndor等人,2016年CAPCHA等。,20 20 )。此类研究还报告了败血症诱发的急性肾损伤,可通过评估菊粉清除率,肾小管转运,尿渗透压和线粒体形态以及肾小球功能障碍,肾小管病变(尿液浓缩缺陷和电解质分数排泄减少)来确定,以及炎症(Rodrigues的等人,2012;莫雷拉等人,2014;ç ó ndor 。等人,2016)。我们的CLP协议的版本还促进肺部炎症,由较大的巨噬细胞浸润和TLR4的表达所证明巨噬细胞的表面上的(C ó ndor等人,2016; CAPCHA 。等人,20 20 )。


CLP后,血清中细胞因子水平显着增加,导致微血管内皮细胞受损。还已经证明,CLP导致内皮一氧化氮合酶Slit2和Robo4的表达下调,这些蛋白负责维持内皮屏障的完整性(Moreira等,2014)。此外,有相当多的证据表明该手术会导致心脏损伤,表现为舒张功能障碍,左心室舒张末期直径和压力降低以及心率持续升高(Moreira等人,2014)。脓毒症可以减少的Klotho,其已经用更糟糕的结果相关联的肾表达(C ó ndor等人,2016)。脓毒症的特征在于高炎症机制,TLR4 /核因子-κB信号传导是最常被定义为评估治疗结果的参数的机制之一。此外,据报道在诱发败血症的动物中肾脏,脾脏,心脏和血清中促炎性细胞因子的过表达。由于这种炎症性疾病,还报道了涉及BAX和Bcl-2表达以及末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记阳性细胞的凋亡机制(Rodrigues等,2012; Moreira等)人,2014;ç ó ndor 。等人,2016)。我们的CLP协议版本可以引起这些败血症的主要机制。


CLP模型可以适应每个实验室的需求/目标。在我们的CLP协议版本中,死亡率达到了MODS的终点。建立这种新颖的CLP方案是为了模拟ICU败血症患者的病情。如果仔细管理所有技术参数,则涉及受控MODS的该方案已被证明可重复进行。


致谢


这项研究得到了圣保罗埃斯帕多萨·安帕罗基金会的资助(圣保罗研究基金会,FAPESP;批准号:2010 / 19012-0)。JMCC是FAPESP赠款的获得者(赠款编号2015 / 21308-9)。LA是来自巴西的赠款的接受者Conselho国立DesenvolvimentoCientíficoËTecnológico (国家委员会科学和技术发展;授予第301193 / 2016-9)。


本协议改编自Wichterman等人。(1980)。


利益争夺


作者宣称他们没有利益冲突或利益冲突。


伦理


位于巴西圣保罗市的圣保罗大学医学院附属医院dasClínicas的研究伦理委员会已批准使用此协议(参考号378603)。所有程序均符合《美国国家研究委员会实验动物的护理和使用指南》(国家研究委员会,2011年)。


参考


ACCP / SCCMCC。(1992)。美国胸科医师学会/重症医学会共识会议:败血症和器官衰竭的定义以及败血症中使用创新疗法的指南。Crit Care Med 20(6):864-874。              
贝克,抄送,乔杜里,IH,盖恩斯,何和鲍伊,AE(1983年)。在鼠盲肠结扎和穿刺模型中评估败血症后影响死亡率的因素。手术94(2):331-335。              
Capcha,JMC,Rodrigues,CE,Moreira,R。d。S.,Silveira,MD,Dourado,P.,Dos Santos,F.,Irigoyen,MC,Jensen,L.,Garnica,MR,Noronha,IL,Andrade,L.和Gomes,SA(2020)。沃顿商学院的果冻来源的间充质干细胞可通过胆碱能抗炎途径激活部分地减轻败血症诱导的器官损伤。Am J生理学法规综合比较318(1):R135-R147。
康多(JM),罗德里格斯(CE),索萨·莫雷拉(R.Sousa Moreira),卡纳莱(Canale),D。沃尔皮尼(RA),清水(H.S。),MH,卡马拉(Camara),诺罗尼亚·艾德(Noronha Ide)和安德拉德(L.)(2016)。人沃顿氏胶质间充质干细胞的治疗可减轻败血症诱发的肾脏损伤,肝损伤和内皮功能障碍。干细胞Transl Med 5(8):1048-1057。
芬克议员(2014)。败血症的动物模型。毒力5(1):143-153。
Hotchkiss,RS,Monneret,G.和Payen,D.(2013)。败血症诱导的免疫抑制:从细胞功能障碍到免疫治疗。Nat Rev Immunol 13(12):862-874。
              Kellum,JA,Wen,X.,de Caestecker,MP和Hukriede,NA(2019)。败血症相关的急性肾损伤:值得新解决方案解决的问题。Nephron 143(3):174-178。
莫雷拉(RS),伊里戈延(Irigoyen),密歇根州(Sanches),TR,沃尔皮尼(RA),卡马拉(Camara),否,马尔希罗斯(Malheiros),马克(DM),清水(MH),塞古罗(Seguro),AC和安德拉德(L.)(2014)。载脂蛋白AI模拟肽4F可减轻败血症中的肾脏损伤,心脏损伤和内皮功能障碍。Am J生理学法规综合生理学307(5):R514-524。              
国家研究委员会(美国)。(2011)。L.护理和使用指南更新委员会。实验动物的护理和使用指南[在线]。第八版。
Parker,SJ和Watkins,PE(2001)。革兰氏阴性脓毒症的实验模型。Br J Surg 88(1):22-30。              
              Rello,J.,Valenzuela-Sanchez,F.,Ruiz-Rodriguez,M.和Moyano,S.(2017年)。败血症:管理进展综述。Adv Ther 34(11):2393-2411。
Rodrigues,CE,Sanches,TR,Volpini,RA,清水,MHM,Kuriki,PS,Camara,NOS,Seguro,AC和Andrade,L。(2012年)。持续促红细胞生成素受体激活剂在败血症诱发的急性肾损伤和多器官功能障碍中的作用。一项7(1):e29893-e29893。              
Singer,M.,Deutschman,CS,Seymour,CW,Shankar-Hari,M.,Annane,D.,Bauer,M.,Bellomo,R.,Bernard,GR,Chiche,JD,Coopersmith,CM,Hotchkiss,RS ,列维(Levy),MM,马歇尔(Marshall),JC,马丁(Martin),GS,Opal,SM,鲁宾菲尔德(Rubenfeld),GD,范德波尔(T.Van der Poll),文森特(Vincent),JL和安格斯(DC)(2016)。败血症和败血性休克的第三国际共识定义(Sepsis-3)。JAMA 315(8):801-810。              
Singleton,KD和Wischmeyer,PE(2003)。盲肠结扎和穿刺后盲肠结扎的距离影响死亡率,肿瘤坏死因子-α和白介素6的表达。Eur Surg Res 35(6):486-491。
加利福尼亚州的托里奥(Torio)和北京的摩尔(Moore,BJ)(20 16 )。国家住院医院费用:付款人最昂贵的条件,2013年。统计摘要#204。见:医疗保健成本和利用项目(HCUP)统计摘要[Internet]。罗克维尔(MD):医疗研究与质量局(美国)。
Wichterman,KA,Baue,AE和Chaudry,IH(1980年)。败血症和败血性休克-实验室模型和建议的审查。J Surg Res 29(2):189-201。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Capcha, J. M., Moreira, R. S., Rodrigues, C. E., Silveira, M. A., Andrade, L. and Gomes, S. A. (2021). Using the Cecal Ligation and Puncture Model of Sepsis to Induce Rats to Multiple Organ Dysfunction. Bio-protocol 11(7): e3979. DOI: 10.21769/BioProtoc.3979.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。