- Meeting Abstracts
- Open Access
37th International Symposium on Intensive Care and Emergency Medicine (part 3 of 3)
Brussels, Belgium. 21-24 March 2017
Critical Care volume 21, Article number: 58 (2017)
P349 Muscle mitochondrial function and N+/K+ -ATPase activity are unaffected by sepsis in pigs
M Von Seth, L Hillered, A Otterbeck, K Hanslin, A Larsson, J Sjölin, M Lipcsey
Uppsala University, Uppsala, Sweden
Imbalance in cellular energetics has been suggested to be an important mechanism for organ failure in sepsis and septic shock. We hypothesized that such energy imbalance would either be caused by metabolic changes leading to decreased energy production or by increased energy consumption. Thus, we set out to investigate if mitochondrial dysfunction or decreased energy consumption alters cellular metabolism in muscle tissue in experimental sepsis.
We submitted anesthetized piglets to sepsis (n = 12) or placebo (n = 4) and monitored them for 3 hours. Plasma lactate and markers of organ failure were measured hourly, as was muscle metabolism by microdialysis. Energy consumption was intervened locally by infusing ouabain through one microdialysis catheter to block major energy expenditure of the cells, by inhibiting the major energy consuming enzyme, N+/K + -ATPase. Similarly, energy production was blocked infusing sodium cyanide (NaCN), in a different region, to block the cytochrome oxidase in muscle tissue mitochondria.
All animals submitted to sepsis fulfilled sepsis criteria as defined in Sepsis-3, whereas no animals in the placebo group did. Muscle glucose decreased during sepsis independently of N+/K + -ATPase or cytochrome oxidase blockade. Muscle lactate did not increase during sepsis in naïve metabolism. However, during cytochrome oxidase blockade, there was an increase in muscle lactate that was further accentuated during sepsis. Muscle pyruvate did not decrease during sepsis in naïve metabolism. During cytochrome oxidase blockade, there was a decrease in muscle pyruvate, independently of sepsis. Lactate to pyruvate ratio increased during sepsis and was further accentuated during cytochrome oxidase blockade. Muscle glycerol increased during sepsis and decreased slightly without sepsis regardless of N+/K + -ATPase or cytochrome oxidase blocking. There were no significant changes in muscle glutamate or urea during sepsis in absence/presence of N+/K + -ATPase or cytochrome oxidase blockade.
These results indicate increased metabolism of energy substrates in muscle tissue in experimental sepsis. Our results do not indicate presence of energy depletion or mitochondrial dysfunction in muscle and should similar physiologic situation be present in other tissues, other mechanisms of organ failure must be considered.
P350 Pilot study showing reduced bone strength at 96 hours in rodent sepsis
ME Cove1, NS Chew2, LH Vu2, RZ Lim2, Z Puthucheary3
1National University Hospital, Singapore, Singapore; 2Yong Loo Lin School of Medicine, National University Singapore, Singapore, Singapore; 3University College London, London, United Kingdom
Bone mineral density (BMD) is reduced in critical care survivors , and long-term follow up has shown increased fracture risk . It is unclear if these changes are a consequence of acute critical illness, or reduced activity afterwards. Bone health assessment during critical illness is challenging, and direct bone strength measurement is not possible. We used a rodent sepsis model to test the hypothesis that critical illness causes early reduction in bone strength and changes in bone architecture.
20 Sprague-Dawley rats (350 ± 15.8g) were anesthetised and randomised to receive cecal ligation and puncture (CLP) (50% cecum length, 18G needle single pass through anterior and posterior walls) or sham surgery (cecum mobilised, no CLP), and then returned to their cages. 10 rodents (5 CLP, 5 sham) were sacrificed at 24 hours, and the remaining 10 at 96 hours. Femur bones were harvested and bone strength testing was conducted using the Instron 5543 (Instron Corp, USA). Trabecular bone strength was measured using a femoral neck break and cortical bone strength tested using a femoral shaft 3-point bending test. Bone architecture was assessed using micro-computerised tomography (microCT) imaging (PerkinElmer, USA), and images analysed with BoneJ .
All 20 rats survived to the end of the protocol. The load required to fracture the femoral neck and shaft was not significantly different for CLP and sham groups at 24 hours (97 ± 19N vs 81 ± 10N p = 0.12 and 127 ± 8N vs 119 ± 18N p = 0.35, respectively). However, at 96 hours there was a significant reduction in the fracture force at both the femoral neck and shaft in the CLP group, compared to sham (75 ± 11N vs 97 ± 13N p = 0.02 and 102 ± 20N vs 139.9 ± 28N p = 0.04). In contrast, there were no bone architecture differences, as measured by bone volume/total volume, trabecular thickness/separation, connectivity density, anisotropy and BMD (all p > 0.20) using microCT at 24 or 96 hours.
In this rodent model of sepsis, there is a significant reduction in trabecular and cortical bone strength at 96 hours. In the absence of changes in bone architecture, these findings suggest sepsis may induce early biochemical changes affecting bone strength. We plan further rodent experiments to confirm these results, increase our power, assess nano-mechanics and complete a histological analysis.
1 Orford NR et al: Am J Resp Crit Care 2016,193:736–744
2 Rawal J et al: Crit Care 2015,19:165
3 Doube M et al: Bone 2010, 47:1076–1079
P351 Endotoxin clearance by the spleen is unaffected by pre-existing systemic inflammation in porcine septic shock
K Hanslin1, F Wilske2, P Skorup2, E Tano2, J Sjölin2, M Lipcsey1
1Uppsala University, Uppsala, Sweden; 2Uppsala University, Department of Medical Sciences, Uppsala, Sweden
As part of the mononuclear phagocytic system, the spleen participates in bacterial and endotoxin clearance. In our previous study we saw decreased endotoxin clearance by the liver in pigs with pre-existing systemic inflammatory response (SIR). We therefore hypothesized that immunosuppression induced by SIR may also lead to decreased trans-splenic endotoxin clearance, and set out to investigate this in a porcine model of sepsis.
15 anesthetized pigs received an [i]E. coli[/i] infusion intravenously (i.v.) for 3 hours (h). In group Pre-existing SIR (n = 6), SIR was induced by 24 h of i.v. endotoxin infusion prior to the [i]E. coli[/i] infusion. Group Non-Pre-existing SIR (n = 6) received the bacterial infusion without prior endotoxin exposure. To study the effects of 24 h of anesthesia alone, “Controls” (n = 3) received saline instead of endotoxin for 24 h prior to the bacterial infusion (not included in the primary analysis). The kinetic chromogenic LAL-test was used to analyze endotoxin in arterial and splenic venous blood samples.
All animals receiving endotoxin developed SIR prior to the [i]E. coli[/i] infusion. The amounts of [i]E. coli[/i] given to the groups were comparable. Endotoxin levels were similar at 3 h, just before the end of the [i]E. coli[/i] infusion, in the Pre-existing SIR and Non-Pre-existing SIR groups in arterial (Fig. 1) and splenic venous blood (2.40 (1.94-2.60) vs. 2.81 EU/mL (2.73-2.91) median (IQR)). Furthermore, endotoxin levels at 4 h, one hour after completed [i]E. coli[/i] infusion, were lower in Pre-existing SIR vs. Non-Pre-existing SIR group both in arterial (Fig. 1) and splenic venous blood (0.38 (0.31-0.42) vs. 0.51 EU/mL (0.47-0.71); p < 0.05). There was no difference in the ratio of splenic venous to arterial endotoxin levels between Pre-existing SIR and Non-Pre-existing SIR groups.
In our model, the endotoxin clearance by the spleen is not affected by pre-existing inflammatory response in porcine [i]E. coli[/i] septic shock.
P352 The role of autophagy in critical illness-induced organ failure
I Derese, S Thiessen, S Derde, T Dufour, L Pauwels, Y Bekhuis, G Van den Berghe, I Vanhorebeek
University Hospital, Leuven, Belgium
Increasing evidence implicates mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR), as contributors to critical illness-induced organ failure. Both can be alleviated by autophagy, a cellular defense mechanism. However, a phenotype of insufficiently activated autophagy has been observed during critical illness. We hypothesized that insufficient hepatic autophagy during critical illness aggravates liver damage/failure, hallmarked by mitochondrial dysfunction and ER stress.
In a centrally catheterized mouse model of critical illness, induced by cecal ligation and puncture, the effect of genetic inactivation of hepatic autophagy (via inducible deletion of autophagy gene 7 in liver) on survival, markers of organ damage, apoptosis, UPR and mitochondrial content and function was evaluated in the acute (30 hrs) and prolonged (3 days) phase. For each time point, 2 groups of critically ill mice and 2 groups of healthy pair-fed mice were included (at least 10 surviving mice per group), where each time autophagy was inactivated in one group but not in the other.
Hepatic autophagy deficiency during critical illness did not affect survival, but increased hepatic damage/dysfunction. In the acute phase, this was illustrated by higher plasma ALT (P = 0.0001), and by elevated markers of apoptosis (P = 0.001) and more mitochondrial dysfunction (Complex V activity, P = 0.02) in liver. In the prolonged phase, hepatic autophagy inactivation increased apoptosis (P = 0.01) and aggravated mitochondrial dysfunction (Complex V activity, P = 0.005) in liver. Autophagy deficiency did not affect mitochondrial DNA content (day 1 P = 0.98, day 3 P = 0.57). Autophagy deficiency time-dependently modulated several branches of the UPR in liver. On day 1, it decreased activation of the IRE1alpha-XBP1s (P = 0.003) and ATF6-CREB3L3 pathway (P = 0.003), coinciding with a diminished inflammatory response as shown by lower C-reactive protein gene expression (P = 0.006), but did not affect the p-eIF2alpha pathway (P = 0.26). At day 3, autophagy deficiency increased the activation of the p-eIF2alpha pathway (P = 0.03), but not the IRE1alpha-XBP1s (P = 0.37) or ATF6-CREB3L3 (P = 0.14) pathway.
Insufficient hepatic autophagy during critical illness aggravates liver damage, coinciding with more mitochondrial dysfunction and a time-dependent modulation of the UPR, hereby likely aggravating liver failure.
P353 Role of leptin and proprotein convertase subtilisin/kexin type 9 in modulating pulmonary inflammation in a murine model of early sepsis
M Khan1, D Dwivedi1, J Zhou1, A Prat2, NG Seidah2, PC Liaw1, AE Fox-Robichaud1
1McMaster University, Hamilton, Canada; 2Montreal Clinical Research Institute, Montreal, Canada
Obesity increases the risk of sepsis but how obesity shapes the immune responses to infection is unknown. Similar to patients, we previously demonstrated that Western diet fed obese mice have reduced lung inflammation during early sepsis. In this study we explore the potential mechanisms to explain this finding. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a protein involved in cholesterol homeostasis that is implicated in sepsis survival. Leptin is a hormone produced by adipocytes that regulates energy homeostasis and is increased in obesity and sepsis. We hypothesized that either PCSK9 and/or leptin contributes to the obesity-associated lung protection in sepsis.
PCSK9 deficient, PCSK9 overexpressing and wild type mice on a C57/Bl6 background were fed either a high fat Western diet (WD) or a normal chow diet (NCD) for 15 weeks (n = 5/group). Sepsis was induced by cecal ligation and puncture (CLP). Tissues were harvested six hours post surgery. For the leptin studies mice were housed in static cages for 10-12 weeks. Mice were injected with recombinant leptin protein (1mg/kg)) one hour prior to CLP, then re-anesthetized and tissues collected at 6 hours. All mice were resuscitated with 2ml of lactated Ringers SQ pre surgery, and 1ml IV post surgery. Lung injury was assessed by myeloperoxidase (MPO in U/mg of tissue) assay of lung tissues and histopathology scores. Data are expressed as mean ± SEM and analyzed using ANOVA or t-test.
Septic PCSK9 over expressing mice fed NCD had greater lung MPO levels (46.5 ± 4.5) compared to PCSK9 deficient mice (31.1 ± 1.7) on NCD (p < 0.01). In mice fed the WD for 15 wks the protection from the loss of PCSK9 was no longer present, however the injury was reduced. Septic PCSK9 deficient (14.4 ± 1.4), wildtype (17.6 ± 0.9) and overexpressing (17.9 ± 1.0) mice on WD had no significant differences in lung MPO levels. This correlated with histopathology scores for PCSK9 deficient (0.7 ± 0.2), wildtype (1.1 ± 0.2) and PCSK9 overexpressing (1.3 ± 0.7) septic mice. We found that leptin treated septic mice had lower lung MPO (32.6 ± 1.6) levels compared to saline treated septic mice (46.6 ± 3.5) (p < 0.001). Sham operated mice had significantly lower MPO levels (12.7 ± 1.7 for leptin and 11.8 ± 1.2 for saline) compared to septic counterparts.
Our data suggests that both lack of PCSK9 and increases in leptin contribute to the lung protection in early sepsis. However, when exposed to a WD the potential benefits of PCSK9 deficiency to further reduce lung injury are no longer evident. These findings have implications for potential therapeutic strategies to reduce sepsis-induced lung injury.
P354 The role of oxygen delivery on plasma lactate and organ failure in experimental septic shock
M Von Seth, P Skorup, L Hillered, A Larsson, J Sjölin, M Lipcsey
Uppsala University, Uppsala, Sweden
The concept resuscitation of patients with septic shock, aiming at normalization of oxygen delivery (DO2), to limit tissue dysoxia and organ failure has not been confirmed in recent trials. Elevated plasma lactate in septic shock is considered as a key marker of inadequate DO2. We hypothesized that, apart from severely decreased levels, DO2 is not associated to plasma lactate in a model of septic shock.
We investigated the effects of circulatory shock and inflammation on plasma lactate in a retrospective analysis of 105 anesthetized endotoxemic (N = 61) or bacteremic (N = 44) piglets in shock. Tumor Necrosis Factor alpha (TNF-α) and Interleukin-6 (IL-6) were measured hourly during 6 hours (h) of shock. Muscle metabolism was monitored by microdialysis. The animals were stratified per degree of shock by DO2. The primary analysis was the breakpoint of insufficient DO2 to yield an elevated plasma lactate. ANOVA and regression models were used.
All animals developed macrocirculatory shock, elevated plasma and muscle lactate levels, elevated levels of cytokines in plasma, as well as renal and pulmonary failure. At 3 h, DO2 was 289 ± 68 mL x min-1 x m-2 (mean ± SD) and plasma lactate levels were 2.7 (2.0-3.6) mmol x L-1 (median(IQR)). Mixed venous saturation (SvO2) decreased and oxygen extraction increased linearly with DO2 (p > 0.001). Oxygen consumption (VO2) was not DO2 dependent.
Plasma lactate increased at DO2 < 250 mL x min-1 x m-2 (p < 0.001). Urinary output decreased at DO2 < 250 mL x min-1 x m-2 (p < 0.01), but static lung compliance was not DO2-dependent. Muscle glucose, lactate and pyruvate, urea and glutamate were not DO2-dependent. Muscle glycerol was DO2-dependent without breakpoint.
Plasma lactate correlated to Mean Arterial Blood Pressure (MAP), DO2 and peak IL-6, but not Systemic Vascular Resistance Index (SVRI) and peak TNF-α.Urinary output correlated to DO2 and MAP. Static lung compliance did not correlate to any parameters above.
Over time, muscle pyruvate increased and muscle glycerol and glucose decreased but no changes in muscle lactate and glutamate were seen. Muscle pyruvate correlated to MAP. Muscle glycerol correlated to MAP and to TNF-α.
In porcine experimental sepsis, elevated plasma lactate was only associated with very low DO2 while oxygen consumption was unaffected by low DO2 despite development of organ failure. Tissue metabolism was associated with both inflammatory and circulatory changes. Our findings suggest that the current concepts of resuscitation focusing on restoration of oxygen delivery must be combined with measures to limit the inflammatory response.