Polymicrobial sepsis induces organ changes due to granulocyte adhesion in a murine two hit model of trauma.

INTRODUCTION: Polytrauma patients, who develop organ dysfunction, have often undergone multiple subsequent insults ("hits"). The sequence of organs that show a dysfunction mostly is lung, liver, kidney and heart. The aim of the present study was to investigate whether a second hit after trauma induces organ changes. Furthermore, it was of interest to identify possible pathogenic mediators such as polymorphonuclear granulocytes (PMN) and cytokines. For this purpose, a two hit model of systemic damage in mice was developed. Sepsis was induced by caecal ligation and puncture (CLP), which was preceded 48 hours by a femur fracture, the most common fracture of long bones in trauma patients. This fracture was combined with a haemorrhagic shock. METHODS: In both mouse groups studied, a standardized femur fracture was produced using a blunt guillotine device with a weight of 500 g. This was followed by a haemorrhagic shock with substitution of ringer's lactate after 1 hour. In the study group, CLP was induced by puncturing the caecum using a 21G needle. As a control, sham animals underwent a laparotomy without CLP. Both groups were sacrificed after 48 or 96 hours. Clinical parameters were investigated on a daily basis to evaluate the animals' status. Lung, liver and kidney morphology was studied by light microscopy. PMN adhesion was determined by counting the number of adherent PMN per 100 microm of endothelium. Serum levels of TNF-alpha were measured after 48 and 96 hours. RESULTS: In the group submitted to laparotomy, all animals survived. The induction of polymicrobial sepsis by CLP resulted in an 85% (34/40) mortality within 96 hours after surgery (p < 0.05). The induction of a polymicrobial sepsis resulted in a significantly steady worsening of the clinical situation compared to the sham animals (p < 0.05). Lung morphology demonstrated significant changes at the end of the experimental period after 96 h in the two hit group. The alveolar septa were thickened and in all lungs haemorrhagic foci were observed. The number of PMN adhering to the pulmonary endothelium significantly increased at 96 hours. Some of the liver specimens in the two hit group showed focal hydropic degeneration and PMN infiltration. No kidney pathology was observed. This result coincided with an increase in TNF-alpha serum levels. DISCUSSION: A new rodent model mimicking the situation in the polytraumatized patient was developed. Although the animals showed minimal organ manifestation, a high percentage died probably due to cytokinemia. Furthermore, the increased TNF-alpha levels may lead to increased adhesion of PMN in the lung venules. This adhesion developed four days after the second hit. This might be the initial step for the development of extensive lung lesions in later phases. This model represents the SIRS more than MODS. This is a model for devolopment of posttraumatic disease due to cytokinemia and less for chronic multiple organ dysfunction and failure.

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture.

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.