A. M. Pugh1, L. K. Winer1, V. Nomellini1,2, C. C. Caldwell1 1University Of Cincinnati,Division Of Research, Department Of Surgery,Cincinnati, OH, USA 2University Of Cincinnati,Division Of Trauma, Critical Care, And Acute Care Surgery,Cincinnati, OH, USA
Introduction: Hypothermia is shown to predict persistent lymphopenia in critically ill patients. Although less than 20% of septic patients present with hypothermia, this patient population demonstrates twice the mortality of febrile septic patients. The acute response to sepsis has been extensively investigated with the cecal ligation and puncture (CLP) murine model. However, this model lacks surgical source control and antibiotic administration. Our objective was to develop a sepsis model that included source control and could predict mortality. We hypothesize that a CLP-excision (CLP-E) murine model will mitigate hypothermia and lymphopenia after the acute phase of sepsis.
Methods: Performed CLP on outbred mice with a 50% ligation of the cecum coupled with a through-and-through puncture using a 22-gauge needle. The acute phase of sepsis was determined by the onset of hypothermia, the nadir occurring between 3-4 hours post-CLP. Thus, 4 hours post-CLP, the necrotic cecum was excised, peritoneal wash was performed, and 2.5 mg/kg imipenem was administered. Monitored temperature using Anipills inserted during the CLP. Flow cytometry was used to characterize peritoneal neutrophils and enumerate splenic T cells.
Results: We first hypothesized that source control would improve survival. We demonstrated that CLP-E mortality was 12% compared to 67% in the CLP model. We observed that the persistent hypothermia in CLP mice normalized only after source control (mean = 33.9 vs 36.2?C, p<0.05). Additionally, peritoneal bacterial load was analyzed to verify source control. There was a 2-log decrease in bacterial load after source control in the CLP-E model. We next hypothesized that the activation of neutrophils taken from the infected foci could predict mortality. Peritoneal neutrophil CD11b intensity was significantly different between the mice that survived and those that did not (mean = 1.341×105 vs. 0.7216×105 MFI; p<0.05). A receiver operator characteristic (ROC) curve was then generated using neutrophil CD11b intensity as a predictor of mortality (AUC = 0.85). Lastly, we hypothesized that amelioration of hypothermia in CLP-E mice would prevent lymphopenia observed in the CLP model. Splenic CD4 T cell levels were depleted 32% in CLP mice compared to the untouched, while CLP-E CD4 counts were equivalent to the control group. Splenic CD8 T cell levels were not significantly different between CLP and untouched mice. However, there was a 42% increase in CLP-E mice.
Conclusion: In conclusion, we developed a murine sepsis model that includes surgical and antibiotic source control with the ability to predict mortality. Obtaining source control normalized core body temperature and prevented the lymphopenia seen in septic mice.
