Hypothermia prolongs the survival of rats with severe septic shock by inhibiting the splenic release of neutrophils and monocytes Rhett N Willis Jr, MD University of Virginia Health System

Disclosures  I have no disclosures

Introduction  Sepsis is a clinical syndrome characterized by systemic inflammation due to infection.  There is a continuum of severity ranging from sepsis to severe sepsis and septic shock.  Over 1,665,000 cases of sepsis occur in the United States each year 1  Even with optimal treatment, mortality due to severe sepsis or septic shock is approximately 40 percent and can exceed 50 percent in the sickest patients 2-5

Introduction  Surviving Sepsis Campaign advocates for early blood cultures, empiric antibiotics, fluid resuscitation, imaging, etc. all within the first few hour of diagnosis of sepsis 6  Multiple studies have shown that early septic shock is due to a “cytokine storm” inflammatory response due to massive release of activated monocytes and neutrophils into the circulation 7-8  Cytokine dysregulation (elevated IL-6, IL-1, TNF-alpha, and IL- 6:IL-10 ratio) released by activated monocytes leads to end- organ damage and ultimately, death  Recent studies show aseptic inflammatory response is also mitigated by the splenic release of inflammatory cells

Hypothesis  Our Hypothesis is that spleen play a integral role in the inflammatory response in septic shock and that hypothermia prolongs the survival of septic shock by inhibiting this splenic release of activated monocytes and neutrophils into the circulation

Methods  Study include 52 Sprague-Dawley rats  The study involved inducing diffuse peritonitis, and ultimately, severe septic shock, by cecal ligation and incision (CLI).  After CLI, all rats were kept at normothermic temperature for 1 hour with a heat lamp and continuous rectal thermometer  Rats were then assigned to either normothermia or hypothermia  Hypothermia included 4 total hours with 2 additional hours of rewarming.

Methods  Hypothermia was induced using cooling blanket to a goal rectal temperature of 31°C.  Survival was the endpoint.  In additional groups, the spleen was harvested at one hour after hypo- or normothermic treatment.  Spleen imaging was used with the near infared spectroscopy using the IVIS spectrum pre-clinical In Vivo Imaging System  Additional splenic immunohistological staining was performed with anti-rat granulocyte antibodies to activated neutrophils

Results: Survival Curves without splenectomy (p=0.001)  Normothermic rats 2.7±0.79hr (N=8)  Hypothermic rats 8.3±0.42hr (N=8)  No difference in survival with splenectomized rats

Results: Spleen weights / body weight in grams (p= 0.05)

Results: Spleen Fluorescein staining for activated Monocytes and Neutrophils

Results: Immunohistological fluorescence staining Normothermic Sham Normothermic CLI Hypothermic CLI Immunohistological staining of splenic monocytes and neutrophils was performed with mouse anti-rat granulocytes antibody HIS48 (AbD Serotec)

Conclusion  Hypothermia significantly prolongs survival in a septic rat model  Spleen harbors activated monocytes/neutrophils and releases, as shown by both fluorescein staining and histoimmunological staining, these cells into circulation in response to inflammation  Differences in spleen weights between the two groups also signify a substantial release of their activated lymphocytes in the normothermic group  No change in survival times with splenectomy could be attributed to the spleens integral role with regulating IL-10 9

Conclusion: Future work  Further investigate the proper length of hypothermia and rewarming duration to best optimize outcomes  Spleen imaging for cytokine levels before/after hypothermia and normothermia (including IL-10)  Blood cytokine levels  Translational Research – ICU bedside application  Extend the critical window of time to allow targeted therapy in septic shock patients

REFERENCES 1. Elixhauser A, Friedman B, Stranges E. Septicemia in U.S. Hospitals, Agency for Healthcare Research and Quality, Rockville, MD. (Accessed on February 15, 2013). 2. Bernard GR, Wheeler AP, Russell JA, et al. The effects of ibuprofen on the physiology and survival of patients with sepsis. The Ibuprofen in Sepsis Study Group. N Engl J Med 1997; 336: McCloskey RV, Straube RC, Sanders C, et al. Treatment of septic shock with human monoclonal antibody HA-1A. A randomized, double-blind, placebo-controlled trial. CHESS Trial Study Group. Ann Intern Med 1994; 121:1. 4. Zeni F, Freeman B, Natanson C. Anti-inflammatory therapies to treat sepsis and septic shock: a reassessment. Crit Care Med 1997; 25: Sasse KC, Nauenberg E, Long A, et al. Long-term survival after intensive care unit admission with sepsis. Crit Care Med 1995; 23: Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: Crit Care Med 2008; 36: Adrie, C., et al., Successful cardiopulmonary resuscitation after cardiac arrest as a "sepsis-like" syndrome. Circulation, (5): p Adrie, C., et al., Postresuscitation disease after cardiac arrest: a sepsis-like syndrome? Curr Opin Crit Care, (3): p Bachman SL1, et al. The role of the spleen in laparoscopy-associated inflammatory response. Surg Endosc Aug;19(8):

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