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

Abstract

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.

Figure 1

Survival curve and outcome prediction based on body temperature. (A) Seven-day mortality in 4-week-old OF-1 mice subjected to CLP-Only and daily low-volume blood sampling for days 1 to 5 post-CLP. (B) Experimental setup for prospective BT-based outcome prediction. A septic mouse was predicted to die (P-DIE) whenever its BT dropped below 28°C within days 1 to 5 post-CLP. Upon detection, each P-DIE mouse was always sacrificed with a matching predicted to survive (P-SUR) mouse (i.e. with BT ≥35°C). (C) ROC curve and corresponding area under the curve (AUC) for the prediction of outcome based on BT in acute CLP sepsis (days 1 to 5 post-CLP). BT ≤28°C identified dying mice with 94% specificity, while a BT ≥35°C identified surviving mice with 100% specificity. The BT cutoffs for outcome prediction utilized in this study were predetermined based on the internal BT measurement database compiled in previous studies.

Figure 2

Retrospective comparison of organ function between dying and surviving septic mice using CLP as reference point. Plasma levels of (A) ALT (B) LDH (C) urea and (D) glucose in mice that died (CLP-Only DIED) or survived (CLP-Only SUR) post-CLP were compared to surviving CLP-ODam mice (CLP-ODam SUR). For (A) to (D) in CLP-Only: at 6 h, DIED n = 45 and SUR n ≥ 36; at 24 h, DIED n = 37 and SUR n = 38; at 48 h, DIED n = 13 and SUR n ≥ 37; at 72 h, DIED n = 7 and SUR n ≥ 34; at 96 h, DIED n = 3, SUR n = 36. In CLP-ODam SUR n = 3 at all measured time points (24, 48, 72 h); *p < 0.05 between CLP-Only DIED and CLP-ODam SUR. Dotted lines indicate normal range. Data points shown as mean ± SD.

Figure 3

Retrospective comparison of organ function between dying and surviving septic mice using death as reference point. Plasma levels of (A) ALT, (B) LDH, (C) urea and (D) glucose in mice subjected to CLP were compared between those that either died (CLP-Only DIED) or survived (CLP-Only SUR) and additionally to surviving CLP-ODam mice (CLP-ODam SUR). For (A) to (D) in the CLP-Only group: at 72 h, DIED n = 9 and SUR n = 35; at 48 h, DIED n = 33 and SUR n ≥ 30; at 24 h, DIED n = 42 and SUR n = 40. In the CLP-ODam SUR group, an average value of all combines measurements (i.e. taken at 24, 48 and 72 h post-CLP; n = 9) is shown at the 24 h prior death time point; *p < 0.05 between CLP-Only DIED and SUR #p < 0.05 between CLP-Only DIED and CLP-ODam SUR. Dotted lines indicate normal range. Data points shown as mean ± SD.

Figure 4

Comparison of organ function between dying and surviving septic mice using prospective stratification of outcome. Mice were subjected to CLP, monitored for BT and stratified into either predicted to die (P-DIE) or predicted to survive (P-SUR). Upon identification, P-DIE and P-SUR mice were always sacrificed in pairs additionally compared to the healthy animals (CTRL). (A) Cystatin C: P-DIE n = 11, P-SUR n = 14 and CTRL n = 14. (B) Creatinine: P-DIE n = 7, P-SUR n = 10 and CTRL n = 13. C. Troponin I: P-DIE n = 7, P-SUR n = 7 and CTRL n = 10. *p < 0.05 versus all other groups. Solid horizontal bars indicate mean ± standard error of means (SEM).

Figure 5

Comparison of mitochondrial function between dying and surviving septic mice using prospective stratification of outcome. Mice were subjected to CLP, monitored for BT and stratified into either predicted to die (P-DIE) or predicted to survive (P-SUR). Upon identification, P-DIE and P-SUR mice were always sacrificed in pairs and additionally compared to the healthy animals (CTRL). (A,B) Succinate state II. (C,D) Succinate state III. (E,F) Succinate RC. For all parameters: P-DIE n = 5, P-SUR n = 6 and CTRL n = 5. Solid horizontal bars indicate mean ± SEM.

Figure 6

Comparison of mitochondrial ATP level between dying and surviving septic mice using prospective stratification of outcome. Mice were subjected to CLP, monitored for BT and stratified into either predicted to die (P-DIE) or predicted to survive (P-SUR). Upon identification, P-DIE and P-SUR mice were always sacrificed in pairs (days 1 to 3 post-CLP) and additionally compared to the healthy animals (CTRL). (A) Liver. (B) Heart. (C) Kidney. For all parameters: P-DIE n = 4, P-SUR n = 6 and CTRL n = 2. Solid horizontal bars indicate mean ± SEM.

Figure 7

Comparison of pathomorphologic changes mitochondrial function between dying and surviving septic mice using prospective stratification of outcome. Mice were subjected to CLP, monitored for BT and stratified into either predicted to die (P-DIE) or predicted to survive (P-SUR). Upon identification, P-DIE and P-SUR mice were always sacrificed in pairs (days 1 to 3 post-CLP) and additionally compared to the healthy animals (CTRL). HE-staining of (A) lung, (B) liver, (C) kidney and (D) heart was performed. P-DIE and P-SUR n = 11 and CTRL n = 7. Representative photographs are shown (original magnification ×10).