Why do they die? Comparison of selected aspects of organ injury and dysfunction in mice surviving and dying in acute abdominal sepsis.

BACKGROUND: The mechanisms of sepsis mortality remain undefined. While there is some evidence of organ damage, it is not clear whether this damage alone is sufficient to cause death. Therefore, we aimed to examine contribution of organ injury/dysfunction to early deaths in the mouse abdominal sepsis. METHODS: Female OF-1 mice underwent either medium-severity cecal ligation and puncture (CLP-Only) or non-lethal CLP-ODam (CLP with cisplatin/carbontetrachloride to induce survivable hepatotoxicity and nephrotoxicity). In the first experiment, blood was collected daily from survivors (SUR; CLP-Only and CLP-ODam groups) or until early death (DIED; CLP-Only). In the second experiment (CLP-Only), early outcome was prospectively predicted based on body temperature (BT) and pairs of mice predicted to survive (P-SUR) and die (P-DIE) were sacrificed post-CLP. The overall magnitude of organ injury/dysfunction was compared in retrospectively and prospectively stratified mice. RESULTS: At day 7 post-CLP, survival in CLP-Only was 48%, while CLP-ODam was non-lethal. In CLP-Only mice within 24 h of death, urea increased to 78 (versus 40 mg/dl in SUR), ALT to 166 (vs. 108 U/l), LDH to 739 (vs. 438 U/l) and glucose declined to 43 (vs. 62 mg/dl). In CLP-ODam, hypoglycemia was exacerbated (by 1.5-fold) and ALT and LDH were 20- and 8-fold higher versus DIED (CLP-Only) mice. In CLP-Only, predicted deaths (P-DIE) were preceded by a significant rise only in cystatin C (268 vs. 170 ng/ml in P-SUR) but not in creatinine and troponin I. Respiratory function of mitochondria in the liver and kidney of P-SUR and P-DIE CLP-Only mice was not impaired (vs. controls) and ATP level in organs remained similar among all groups. Histologic injury scores in the liver, kidney, heart and lung showed no major disparities among dying, surviving and control mice. CONCLUSIONS: In CLP-Only mice, although the deregulation of parameters indicative of organ injury/dysfunction was greater in dying versus surviving mice, it never exceeded the changes in surviving CLP-ODam animals, and it was not followed by histopathological damage and/or mitochondrial dysfunction. This shows that interpretation of the contribution of the organ injury/dysfunction to early deaths in the CLP model is not straightforward and depends on the pathophysiological origin of the profiled disturbances.

Mouse model of posttraumatic abdominal sepsis: survival advantage of females over males does not depend on the cecum size.

BACKGROUND: Regardless of whether alone or in combination, cecal ligation and puncture (CLP) is the most commonly used model to emulate human polymicrobial sepsis. Numerous CLP studies have shown that female mice survive better than males. In adult mice, this effect may be partly due to the unequal cecum mass (larger in males), as cecum ligation is frequently not size standardized. Comparing two ligation approaches, we investigated whether cecum size influences gender-specific outcome differences. METHODS: 15-month-old (middle-aged) female and male CD-1 mice underwent sublethal trauma/hemorrhage followed by CLP 48 h later. The evaluation of cecum size (in centimeters) and filling (score) was immediately followed by two ligation protocols: (1) ileocecal ligation (IC-L; n = 28/each gender) below the ileocecal valve, and (2) apex ligation (AP-L; n = 31 males, n = 24 females) 2 cm from the apex ceci - all followed by an identical double puncture and 10-day monitoring. RESULTS: Cecum length (3.4 cm in males vs. 2.65 cm in females) and filling (2.5 score points in males vs. 1.7 in females; both p < 0.05) were always greater in male than female mice (27 vs. 44%, respectively). Compared to AP-L, the 10-day CLP mortality was higher with IC-L (86% in males and 61% in females), and this exacerbation was similar in both genders (by 21% in male and 23% in female mice). Finally, the intergender comparison demonstrated that female mice displayed a significant and similar survival advantage regardless of the ligation approach: the difference reached 25% with IC-L and 24% with AP-L. CONCLUSIONS: Standardization by AP-L did not eliminate the gender-specific difference in mortality due to posttraumatic sepsis. The greater cecum length/filling in middle-aged male mice was not responsible for their inferior survival compared to females.

The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.

Estrus cycle status defined by vaginal cytology does not correspond to fluctuations of circulating estrogens in female mice.

Gender-oriented studies in shock, trauma, and/or sepsis require accurate monitoring of hormonal fluctuations as estrogens may influence various end points. Yet, monitoring is challenging in small laboratory animals: e.g., despite its subjectivity, vaginal smears are the major method for determination of estrus cycle phases in mice. Using female mice of different age, we aimed to (a) characterize general age-related changes in systemic estrogens and (b) examine the utility of determination of the estrus cycle by vaginal smears and/or impedance simultaneously comparing them with oscillation of systemic estrogens. In this study, 3-, 15-, and 20-month-old mice underwent vaginal smear and impedance examination each morning for 22 days. Ten hours after each morning checkup, feces were collected, and a second vaginal smear performed. Blood was collected on days 15 and 22. In 3-month-old females, estrus (by smears) was three times more frequent than in older mice, but mean concentrations of plasma and fecal estrogens never decreased with age. Collectively (not individually) plotted fecal estrogens values increased in the proestrus/estrus interphase (by smears) in 3-month-old mice only. Impedance typically peaked (4.5 Ω in 3-month-old mice) in the estrus phase, and only the prediction of estrus (highest area under the curve = 0.87 in 3-month-old) but not of other phases was possible. Regardless of age, individual cycle phase (by smears) never correlated with corresponding fecal estrogens, and estrus could not be predicted. In conclusion, while the fecal estrogens oscillation and frequency of estrus phase were affected by age, the systemic hormone release persisted. In mice, vaginal cytology did not reflect changes of systemic (fecal) estrogens, whereas impedance accurately identified estrus. The flaws and advantages of the examined monitoring methods should be considered in the design of future shock studies.

A non-lethal traumatic/hemorrhagic insult strongly modulates the compartment-specific PAI-1 response in the subsequent polymicrobial sepsis.

INTRODUCTION: Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes. METHODS: Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE). RESULTS: In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2-4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction. CONCLUSIONS: Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.

Relationship between age/gender-induced survival changes and the magnitude of inflammatory activation and organ dysfunction in post-traumatic sepsis.

Age/gender may likely influence the course of septic complications after trauma. We aimed to characterize the influence of age/gender on the response of circulating cytokines, cells and organ function in post-traumatic sepsis. We additionally tested whether post-traumatic responses alone can accurately predict outcomes in subsequent post-traumatic sepsis. A mouse 2-hit model of trauma/hemorrhage (TH, 1(st) hit) and cecal ligation and puncture (CLP, 2(nd) hit) was employed. 3, 15 and 20 month (m) old female (♀) and male (♂) CD-1 mice underwent sublethal TH followed by CLP 2 days later. Blood was sampled daily until day 6 post-TH and survival was followed for 16 days. To compare general response patterns among groups, we calculated two scores: the inflammatory response (including KC, MIP-1α, TNFα, MCP-1, IFNγ, IL-1ß,-5,-6,-10) and the organ dysfunction score (Urea, ALT, AST and LDH). Moreover, mice were retrospectively divided into survivors (SUR) and dying (DIE) based on post-CLP outcome. In general, females survived better than males and their survival did not correspond to any specific estrus cycle phase. Pre-CLP phase: the post-TH inflammatory score was weakest in 3 m♂ but there were no changes among remaining groups (similar lack of differences in the organ dysfunction score). TH induced a 40% increase of IFNγ, MIP-1α and IL-5 in 15 m♂ SUR (vs. DIE) but predictive accuracy for post-CLP outcomes was moderate. Post-CLP phase: while stable in males, inflammatory response score in 15 m and 20 m females decreased with age at day 1 and 2 post-CLP. SUR vs. DIE differences in inflammatory and organ dysfunction score were evident but their magnitude was comparable across age/gender. Nearly identical activation of the humoral inflammatory and organ function compartments, both across groups and according to sepsis severity, suggests that they are not directly responsible for the age/gender-dependent disparity in TH-CLP survival in the studied young-to-mature population.

Sepsis chronically in MARS: systemic cytokine responses are always mixed regardless of the outcome, magnitude, or phase of sepsis.

The paradigm of systemic inflammatory response syndrome-to-compensatory anti-inflammatory response syndrome transition implies that hyperinflammation triggers acute sepsis mortality, whereas hypoinflammation (release of anti-inflammatory cytokines) in late sepsis induces chronic deaths. However, the exact humoral inflammatory mechanisms attributable to sepsis outcomes remain elusive. In the first part of this study, we characterized the systemic dynamics of the chronic inflammation in dying (DIE) and surviving (SUR) mice suffering from cecal ligation and puncture sepsis (days 6-28). In the second part, we combined the current chronic and previous acute/chronic sepsis data to compare the outcome-dependent inflammatory signatures between these two phases. A composite cytokine score (CCS) was calculated to compare global inflammatory responses. Mice were never sacrificed but were sampled daily (20 µl) for blood. In the first part of the study, parameters from chronic DIE mice were clustered into the 72, 48, and 24 h before death time points and compared with SUR of the same post-cecal ligation and puncture day. Cytokine increases were mixed and never preceded chronic deaths earlier than 48 h (3- to 180-fold increase). CCS demonstrated simultaneous and similar upregulation of proinflammatory and anti-inflammatory compartments at 24 h before chronic death (DIE 80- and 50-fold higher versus SUR). In the second part of the study, cytokine ratios across sepsis phases/outcomes indicated steady proinflammatory versus anti-inflammatory balance. CCS showed the inflammatory response in chronic DIE was 5-fold lower than acute DIE mice, but identical to acute SUR. The systemic mixed anti-inflammatory response syndrome-like pattern (concurrent release of proinflammatory and anti-inflammatory cytokines) occurs irrespective of the sepsis phase, response magnitude, and/or outcome. Although different in magnitude, neither acute nor chronic septic mortality is associated with a predominating proinflammatory and/or anti-inflammatory signature in the blood.

Compartment-specific expression of plasminogen activator inhibitor-1 correlates with severity/outcome of murine polymicrobial sepsis.

INTRODUCTION: Plasminogen activator inhibitor type 1 (PAI-1) co-induces septic coagulopathy. We aimed to characterize spatiotemporal PAI-1 gene/protein changes occurring in acute sepsis and tested whether PAI-1 fluctuations correlate with sepsis severity and early outcome. MATERIALS AND METHODS: Female mice underwent cecal ligation and puncture (CLP) in three experiments. I: mild (23 G needle) CLP to compare circulating PAI-1 to its organ gene expression within 0-24h. II: mild or severe (17 G) CLP to asses differences in PAI-1 organ-specific expression and in coagulation/fibrinolysis. III: moderate (18 G) CLP to characterize circulating PAI-1 in survivors (SUR), and to retrospectively compare it to dying (DIE) mice. RESULTS: In mild sepsis, the trajectory of circulating PAI-1 (1089 ng/ml peak at 24h) was identical to PAI-1 gene expression in the left cranial vena cava (LCVC; 39-fold peak at 24h). PAI-1 expression rise was immediate (60-fold at 6h) and sustained in the liver, but marginal in the kidney, lungs and heart. Body temperature decrease correlated with the PAI-1 expression increase in the liver (rho = -0.79), and blood (protein, rho = -0.53). Regardless of severity, PAI-1 gene expression remained unaltered except the LCVC where it was >3-fold higher in 17G (vs. 23 G). Severe sepsis extended activated partial thromboplastin/pro-thrombin time and increased circulating PAI-1, while antithrombin and fibrinogen decreased at 6 and/or 24h (vs. 23 G). Within 24h of death, circulating PAI-1 in DIE was >3-fold higher versus SUR. CONCLUSIONS: Polymicrobial sepsis caused a gradual circulating PAI-1 release and highly variable gene expression response pattern in organs. Only circulating PAI-1 and PAI-1 expression in the LCVC correlated with response severity and/or outcome.

Experimentally approaching the ICU: monitoring outcome-based responses in the two-hit mouse model of posttraumatic sepsis.

To simulate and monitor the evolution of posttraumatic sepsis in mice, we combined a two-hit model of trauma/hemorrhage (TH) followed by polymicrobial sepsis with repetitive blood sampling. Anesthetized mice underwent femur fracture/sublethal hemorrhage and cecal ligation and puncture (CLP) 48 h later. To monitor outcome-dependent changes in circulating cells/biomarkers, mice were sampled daily (facial vein) for 7 days and retrospectively divided into either dead (DIE) or surviving (SUR) by post-CLP day 7. Prior to CLP, AST was 3-fold higher in DIE, while all other post-TH changes were similar between groups. There was a significant post-CLP intergroup separation. In SUR, RBC and Hb were lower, platelets and neutrophils higher, and lymphocytes mixed compared to DIE. In DIE, all organ function markers except glucose (decrease) were few folds higher compared to SUR. In summary, the combination of daily monitoring with an adequate two-hit model simulates the ICU setting, allows insight into outcome-based responses, and can identify biomarkers indicative of death in the acute posttraumatic sepsis in mice.

Repetitive low-volume blood sampling method as a feasible monitoring tool in a mouse model of sepsis.

Blood-based monitoring of immunoinflammatory and organ function fluctuations is essential in models of critical illness. This is challenging in diseased mice as repetitive blood collection may be harmful and/or affect end points. We studied the influence of daily sampling in acutely septic (days 1-5) mice upon survival and selected hematologic and organ function parameters. In addition, we tested the reliabilty of complete blood cell (CBC) count using resuspended blood cells. Female OF-1 and CD-1 mice underwent cecal ligation and puncture (CLP) and were subdivided into Daily and Day 5 groups. Blood was collected daily for 5 days in the Daily group and only on day 5 post-CLP in the Day 5 group. We tested 20 µL (both strains) and 35 µL (OF-1 mice) sampling volumes. The 35-µL volume simultaneously served to test the CBC reliabilty in resuspended versus unprocessed blood. Daily sampling did not affect the 14-day CLP mortality. Compared with the Day 5 group, daily 35-µL sampling in OF-1 mice decreased the red blood cell count and hemoglobin concentration by 22% and 23% (P < 0.05). In neither strain did daily 20-µL sampling affect the red blood cell count, whereas there was a 9% hemoglobin decrease (P < 0.05) in OF-1 mice. Although alanine aminotransferase, lactate dehydrogenase, and glucose levels were comparable, urea significantly increased by 24% in the Daily group (20-µL volume, OF-1 mice). Interleukin 6, platelets, and white blood cell counts remained unaffected. There was an excellent correlation between regular and resuspended CBC for all cell types (r ≥ 0.9; slope, ≥0.9), except lymphocytes (r > 0.5; slope, >0.5). This method provides a feasible and safe translation of clinically relevant daily immunomonitoring to the mouse sepsis model.