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Experimental Protocol for Cecal Ligation and Puncture Model of Polymicrobial Sepsis and Assessment of Vascular Functions in Mice

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1717)

Abstract

Sepsis is the systemic inflammatory response syndrome that occurs during infection and is exacerbated by the inappropriate immune response encountered by the affected individual. Despite extensive research, sepsis in humans is one of the biggest challenges for clinicians. The high mortality rate in sepsis is primarily due to hypoperfusion-induced multiorgan dysfunctions , resulting from a marked decrease in peripheral resistance. Vascular dysfunctions are further aggravated by sepsis-induced impairment in myocardial contractility. Circulatory failure in sepsis is characterized by refractory hypotension and vascular hyporeactivity (vasoplegia) to clinically used vasoconstrictors. To investigate the complex pathophysiology of sepsis and its associated multiple organ dysfunction, several animal models have been developed. However, cecal ligation and puncture (CLP) model of murine sepsis is still considered as ‘gold standard’ in sepsis research. In this protocol we have described the standard surgical procedure to induce polymicrobial sepsis by cecal ligation and puncture. Further, we have described the protocol to study the molecular mechanisms underlying vascular dysfunctions in sepsis.

Key words

Cecal ligation and puncture Polymicrobial sepsis Vascular reactivity 
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References

  1. 1.
    Angus DC, Wax RS (2001) Epidemiology of sepsis: an update. Crit Care Med 29:S109–S116CrossRefPubMedGoogle Scholar
  2. 2.
    Kochanek KD, Smith BL (2004) Deaths: preliminary data for 2002. Natl Vital Stat Rep 52:1–47PubMedGoogle Scholar
  3. 3.
    Finfer S, Bellomo R, Lipman J, French C, Dobb G, Myburgh J (2004) Adult population incidence of severe sepsis in Australian and New Zealand intensive care units. Intensive Care Med 30:589–596CrossRefPubMedGoogle Scholar
  4. 4.
    Todi S, Chatterjee S, Sahu S, Bhattacharyya M (2010) Epidemiology of severe sepsis in India: an update. Crit Care 14:382CrossRefGoogle Scholar
  5. 5.
    Taylor FB (2001) 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 29:S78–S89CrossRefPubMedGoogle Scholar
  6. 6.
    Silverstein R, Wood JG, Xue Q, Norimatsu M, Horn D, Morrison DC (2000) Differential host inflammatory responses to viable versus antibiotic-killed bacteria in experimental microbial sepsis. Infect Immun 68:2301–2308CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lang CH, Bagby GJ, Bornside GH, Vial LJ, Spitzer JJ (1983) Sustained hypermetabolic sepsis in rats: characterization of the model. J Surg Res 35:201–210CrossRefPubMedGoogle Scholar
  8. 8.
    Wicherman KA, Baue AE, Chaudry IH (1980) Sepsis and septic shock-A review of laboratory models and a proposal. J Surg Res 29:189–201CrossRefGoogle Scholar
  9. 9.
    Durkot MJ, Wolfe RR (1989) Hyper and hypodynamic models of sepsis in guinea pigs. J Surg Res 46:118–122CrossRefPubMedGoogle Scholar
  10. 10.
    Karzai W, Cui X, Mehlhom B, Straube E, Hartung T, Gerstenberger E, Banks SM, Natanson C, Reinhart K, Eichacker PQ (2003) Protection with antibody to tumor necrosis factor differs with similarly lethal Escherichia coli versus Staphylococcus aureus pneumonia in rats. Anesthesiology 9:81–89CrossRefGoogle Scholar
  11. 11.
    Ribes S, Domenech A, Cabellos C, Tubau F, Linares J, Viladrich PF, Gudiol F (2003) Experimental meningitis due to a high-level cephalosporin-resistant strain of Streptococcus pneumoniae serotype 23F. Enferm Infecc Microbiol Clin 21:329–333CrossRefPubMedGoogle Scholar
  12. 12.
    Fink MP, Heard SO (1990) Laboratory models of sepsis and septic shock. J Surg Res 49:186–196CrossRefPubMedGoogle Scholar
  13. 13.
    Wang P, Ba ZF, Chaudry IH (1995) Endothelium-dependent relaxation is depressed at the macro- and microcirculatory levels during sepsis. Am J Physiol 269:R988–R994PubMedGoogle Scholar
  14. 14.
    Hubbard WJ, Choudhry M, Schwacha MG, Kerby JD, Rue LW, Bland KI, Chaudry IH (2005) Cecal ligation and puncture. Shock 24:52–57CrossRefPubMedGoogle Scholar
  15. 15.
    Ayala A, Chaudry IH (1996) Immune dysfunction in murine polymicrobial sepsis: mediators, macrophages, lymphocytes and apoptosis. Shock 6:S27–S38CrossRefPubMedGoogle Scholar
  16. 16.
    Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis. N Engl J Med 348:138–150CrossRefPubMedGoogle Scholar
  17. 17.
    Buras JA, Holzmann B, Sitkovsky M (2005) Animal models of sepsis: setting the stage. Nat Rev Drug Discov 4:854–865CrossRefPubMedGoogle Scholar
  18. 18.
    Xiao H, Siddiqui J, Remick DG (2006) Mechanisms of mortality in early and late sepsis. Infect Immun 74:5227–5235CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Latifi SQ, O’Riordan MA, Levine AD (2002) Interleukin-10 controls the onset of irreversible septic shock. Infect Immun 70:4441–4446CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Zantl N, Uebe A, Neumann B, Wagner H, Siewert J-R, Holzmann B, Heidecke C-D, Pfeffer K (1998) Essential role of gamma interferon in survival of colon ascendens stent peritonitis, a novel murine model of abdominal sepsis. Infect Immun 66:2300–2309PubMedPubMedCentralGoogle Scholar
  21. 21.
    Shelley O, Murphy T, Paterson H, Mannick JA, Lederer JA (2003) Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury. Shock 20:123–129CrossRefPubMedGoogle Scholar
  22. 22.
    Cobb JP, Danner RL (1996) Nitric oxide and septic shock. JAMA 275:1992–1996CrossRefGoogle Scholar
  23. 23.
    da Silva-Santos JE, Terluk MR, Assreuy J (2002) Differential involvement of guanylate cyclase and potassium channels in nitric oxide-induced hyporesponsiveness to phenylephrine in endotoxemic rats. Shock 17:70–76CrossRefPubMedGoogle Scholar
  24. 24.
    Chen SJ, Chen KH, CC W (2005) Nitric oxide-cyclic GMP contributes to abnormal activation of Na+-K+-ATPase in the aorta from rats with endotoxic shock. Shock 23:179–185CrossRefPubMedGoogle Scholar
  25. 25.
    Secco DD, Olivon V, Correa T, Celes MR, Abreu M, Rossi M, Oliveira AM, Cunha F, Assreuy J (2010) Cardiovascular hyporesponsiveness in sepsis is associated with G-protein receptor kinase expression via a nitric oxide-dependent mechanism. Crit Care 14:P34CrossRefGoogle Scholar
  26. 26.
    Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 4:31–36CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hollenberg S, Dumasius A, Easington C, Colilla S, Neumann A, Parrillo J (2001) Characterization of a hyperdynamic murine model of resuscitated sepsis using echocardiography. Am J Respir Crit Care Med 32:2589–2597Google Scholar
  28. 28.
    Hugunin KM, Fry C, Shuster K, Nemzek JA (2010) Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model. Shock 34:250–260CrossRefPubMedGoogle Scholar
  29. 29.
    Toscano MG, Ganea D, Gamero AM (2011) Cecal ligation puncture procedure. J Vis Exp 51:e2860.  https://doi.org/10.3791/2860. Google Scholar
  30. 30.
    Baker CC, Chaudry IH, Gaines HO, Baue AE (1983) Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery 94:331–335PubMedGoogle Scholar
  31. 31.
    Cuenca AG, Delano MJ, Kelly-Scumpia KM, Moldawer LL, Efron PA (2010) Current protocols in immunology: cecal ligation and puncture. Curr Protoc Immunol.  https://doi.org/10.1002/0471142735.im1913s91
  32. 32.
    Diodato MD, Knoferl MW, Schwacha MG, Bland KI, Chaudry IH (2001) Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis. Cytokine 14:162–169CrossRefPubMedGoogle Scholar
  33. 33.
    Turnbull IR, Wlzorek JJ, Osborne D, Hotchkiss RS, Coopersmith CM, Buchman TG (2003) Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture. Shock 19:310–313CrossRefPubMedGoogle Scholar
  34. 34.
    Watanabe H, Numata K, Ito T, Takagi K, Matsukawa A (2004) Innate immune response in Th1- and Th2-dominant mouse strains. Shock 22:460–466CrossRefPubMedGoogle Scholar
  35. 35.
    De Maio A, Torres MB, Reeves RH (2005) Genetic determinants influencing the response to injury, inflammation, and sepsis. Shock 23:11–17CrossRefPubMedGoogle Scholar
  36. 36.
    Hosoda C, Tanoue A, Shibano M, Tanaka Y, Hiroyama M, Koshimizu TA, Cotecchia S, Kitamura T, Tsujimoto G, Koike K (2005) Correlation between vasoconstrictor roles and mRNA expression of α1-adrenoceptor subtypes in blood vessels of genetically engineered mice. Br J Pharmacol 146:456–466CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Yamamoto Y, Koike K (2001) Characterization of α1-adrenoceptor-mediated contraction in the mouse thoracic aorta. Eur J Pharmacol 424:131–140CrossRefPubMedGoogle Scholar
  38. 38.
    Tanoue A, Nasa Y, Koshimizu T, Shinoura H, Oshikawa S, Kawai T, Sunada S, Takeo S, Tsujimoto G (2002) The α1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction. J Clin Invest 109:765–775CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Cohn HI, Harris DM, Pesant S, Pfeiffer M, Rui-Hai Z, Koch WJ, Dorn GW, Eckhart AD (2008) Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances α1D-adrenergic receptor constriction. Am J Physiol Heart Circ Physiol 295:H1695–H1704CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kandasamy K, Prawez S, Choudhury S, More AS, Ahanger AA, Singh TU, Parida S, Mishra SK (2011) Atorvastatin prevents vascular hyporeactivity to norepinephrine in sepsis: role of nitric oxide and α1D-adrenoceptor mRNA expression. Shock 36:76–82CrossRefPubMedGoogle Scholar
  41. 41.
    Reddy AK, Taffet GE, Madala S, Michael LH, Entman ML, Hartley CJ (2003) Noninvasive blood pressure measurement in mice using pulsed Doppler ultrasound. Ultrasound Med Biol 29:379–385CrossRefPubMedGoogle Scholar
  42. 42.
    Kurtz TW, Griffin KA, Bidani AK, Davisson RL, Hall JE (2005) Recommendations for blood pressure measurement in humans and experimental animals. Part 2: Blood pressure measurement in experimental animals. Hypertension 45:299–310CrossRefPubMedGoogle Scholar
  43. 43.
    Tsukamoto A, Serizawa K, Sato R, Yamazaki J, Inomata T (2015) Vital signs monitoring during injectable and inhalant anesthesia in mice. Exp Anim 64(1):57–64CrossRefPubMedGoogle Scholar
  44. 44.
    Parasuraman S, Raveendran R (2012) Measurement of invasive blood pressure in rats. J Pharmacol Pharmacother 3:172–177CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Kurowski SZ, Slavik KJ, Szilagyi JE (1991) A method for maintaining and protecting chronic arterial and venous catheters in conscious rats. J Pharmacol Methods 26:249–256CrossRefPubMedGoogle Scholar
  46. 46.
    Ordodi VL, Mic FA, Mic AA, Toma O, Sandesc D, Paunescu V (2005) A simple device for invasive measurement of arterial blood pressure and ECG in the anesthesized rat. Timisoara Med J 55:35–37Google Scholar
  47. 47.
    Bardelmeijer HA, Buckle T, Ouwehand M, Beijnen JH, Schellens JH, van Tellingen O (2003) Cannulation of the jugular vein in mice: a method for serial withdrawal of blood samples. Lab Anim 37:181–187CrossRefPubMedGoogle Scholar
  48. 48.
    McLachlan RS (1993) Suppression of interictal spikes and seizures by stimulation of the vagus nerve. Epilepsia 34:918–923CrossRefPubMedGoogle Scholar
  49. 49.
    Hatton KW, McLarney JT, Pittman T, Fahy BG (2006) Vagal nerve stimulation: overview and implications for anesthesiologists. Anesth Analg 103:1241–1249CrossRefPubMedGoogle Scholar
  50. 50.
    Choudhury S, Kannan K, Pule AM, Darzi SA, Singh V, Singh TU, Thangamalai R, Dash JR, Parida S, Debroy B, Paul A, Mishra SK (2015) Combined treatment with atorvastatin and imipenem improves survival and vascular functions in mouse model of sepsis. Vascul Pharmacol 71:139–150.  https://doi.org/10.1016/j.vph.2015.03.012
  51. 51.
    Kandasamy K, Choudhury S, Singh V, Addison MP, Darzi SA, Kasa JK, Thangamalai R, Dash JR, Kumar T, Sultan F, Singh TU, Parida S, Mishra SK (2016) Erythropoietin reverses sepsis-induced vasoplegia to norepinephrine through preservation of α1D-adrenoceptor mRNA expression and inhibition of GRK2-mediated desensitization in mouse aorta. J Cardiovasc Pharmacol Ther 21(1):100–113.  https://doi.org/10.1177/1074248415587968
  52. 52.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007CrossRefGoogle Scholar
  53. 53.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔct Method. Methods 25:402–408CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Division of Pharmacology & ToxicologyIndian Veterinary Research InstituteBareillyIndia
  2. 2.BhubaneswarIndia
  3. 3.Department of Pharmacology and Toxicology, College of Veterinary Science & Animal HusbandryU.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan SansthanMathuraIndia

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