Andres Torres

A dynamic view of trauma/hemorrhage-induced inflammation in mice: principal drivers and networks.

Background Complex biological processes such as acute inflammation induced by trauma/hemorrhagic shock/ (T/HS) are dynamic and multi-dimensional. We utilized multiplexing cytokine analysis coupled with data-driven modeling to gain a systems perspective into T/HS. Methodology/Principal Findings Mice were subjected to surgical cannulation trauma (ST) ± hemorrhagic shock (HS; 25 mmHg), and followed for 1, 2, 3, or 4 h in each case. Serum was assayed for 20 cytokines and NO2 −/NO3 −. These data were analyzed using four data-driven methods (Hierarchical Clustering Analysis [HCA], multivariate analysis [MA], Principal Component Analysis [PCA], and Dynamic Network Analysis [DyNA]). Using HCA, animals subjected to ST vs. ST + HS could be partially segregated based on inflammatory mediator profiles, despite a large overlap. Based on MA, interleukin [IL]-12p40/p70 (IL-12.total), monokine induced by interferon-γ (CXCL-9) [MIG], and IP-10 were the best discriminators between ST and ST/HS. PCA suggested that the inflammatory mediators found in the three main principal components in animals subjected to ST were IL-6, IL-10, and IL-13, while the three principal components in ST + HS included a large number of cytokines including IL-6, IL-10, keratinocyte-derived cytokine (CXCL-1) [KC], and tumor necrosis factor-α [TNF-α]. DyNA suggested that the circulating mediators produced in response to ST were characterized by a high degree of interconnection/complexity at all time points; the response to ST + HS consisted of different central nodes, and exhibited zero network density over the first 2 h with lesser connectivity vs. ST at all time points. DyNA also helped link the conclusions from MA and PCA, in that central nodes consisting of IP-10 and IL-12 were seen in ST, while MIG and IL-6 were central nodes in ST + HS. Conclusions/Significance These studies help elucidate the dynamics of T/HS-induced inflammation, complementing other forms of dynamic mechanistic modeling. These methods should be applicable to the analysis of other complex biological processes.

An adequately robust early TNF-α response is a hallmark of survival following trauma/hemorrhage

Background Trauma/hemorrhagic shock (T/HS) results in cytokine-mediated acute inflammation that is generally considered detrimental. Methodology/Principal Findings Paradoxically, plasma levels of the early inflammatory cytokine TNF-α (but not IL-6, IL-10, or NO2 -/NO3 -) were significantly elevated within 6 h post-admission in 19 human trauma survivors vs. 4 non-survivors. Moreover, plasma TNF-α was inversely correlated with Marshall Score, an index of organ dysfunction, both in the 23 patients taken together and in the survivor cohort. Accordingly, we hypothesized that if an early, robust pro-inflammatory response were to be a marker of an appropriate response to injury, then individuals exhibiting such a response would be predisposed to survive. We tested this hypothesis in swine subjected to various experimental paradigms of T/HS. Twenty-three anesthetized pigs were subjected to T/HS (12 HS-only and 11 HS + Thoracotomy; mean arterial pressure of 30 mmHg for 45–90 min) along with surgery-only controls. Plasma obtained at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min (in the HS only group) or 90 min (in the HS + Thoracotomy group) was assayed for TNF-α, IL-6, IL-10, and NO2 -/NO3 -. Mean post-surgery±HS TNF-α levels were significantly higher in the survivors vs. non-survivors, while non-survivors exhibited no measurable change in TNF-α levels over the same interval. Conclusions/Significance Contrary to the current dogma, survival in the setting of severe, acute T/HS appears to be associated with an immediate increase in serum TNF-α. It is currently unclear if this response was the cause of this protection, a marker of survival, or both. This abstract won a Young Investigator Travel Award at the SHOCK 2008 meeting in Cologne, Germany.

Mathematical modeling of posthemorrhage inflammation in mice: studies using a novel, computer-controlled, closed-loop hemorrhage apparatus.

Hemorrhagic shock (HS) elicits a global acute inflammatory response, organ dysfunction, and death. We have used mathematical modeling of inflammation and tissue damage/dysfunction to gain insight into this complex response in mice. We sought to increase the fidelity of our mathematical model and to establish a platform for testing predictions of this model. Accordingly, we constructed a computerized, closed-loop system for mouse HS. The intensity, duration, and time to achieve target MAP could all be controlled using a software. Fifty-four male C57/black mice either were untreated or underwent surgical cannulation. The cannulated mice were divided into 8 groups: (a) 1, 2, 3, or 4 h of surgical cannulation alone and b) 1, 2, 3, or 4 h of cannulation + HS (25 mmHg). MAP was sustained by the computer-controlled reinfusion and withdrawal of shed blood within ±2 mmHg. Plasma was assayed for the cytokines TNF, IL-6, and IL-10 as well as the NO reaction products NO2−/NO3−. The cytokine and NO2−/NO3− data were compared with predictions from a mathematical model of post-hemorrhage inflammation, which was calibrated on different data. To varying degrees, the levels of TNF, IL-6, IL-10, and NO2−/NO3− predicted by the mathematical model matched these data closely. In conclusion, we have established a hardware/software platform that allows for highly accurate, reproducible, and mathematically predictable HS in mice.ABBREVIATIONS-HS-hemorrhagic shock; HR-heart rate

Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Physiologic responses to severe hemorrhagic shock and the genesis of cardiovascular collapse: can irreversibility be anticipated?

BACKGROUND The causes of cardiovascular collapse (CC) during hemorrhagic shock (HS) are unknown. We hypothesized that vascular tone loss characterizes CC, and that arterial pulse pressure/stroke volume index ratio or vascular tone index (VTI) would identify CC. METHODS Fourteen Yorkshire-Durock pigs were bled to 30 mmHg mean arterial pressure and held there by repetitive bleeding until rendered unable to compensate (CC) or for 90 min (NoCC). They were then resuscitated in equal parts to shed volume and observed for 2 h. CC was defined as a MAP < 30 mmHg for 10 min or <20 mmHg for 10 s. Study variables were recorded at baseline (B0), 30, 60, 90 min after bleeding and at resuscitation (R0), 30, and 60 min afterward. RESULTS Swine were bled to 32% ± 9% of total blood volume. Epinephrine (Epi) and VTI were low and did not change in NoCC after bleeding compared with CC swine, in which both increased (0.97 ± 0.22 to 2.57 ± 1.42 mcg/dL, and 173 ± 181 to 939 ± 474 mmHg/mL, respectively), despite no differences in bled volume. Lactate increase rate (LIR) increased with hemorrhage and was higher at R0 for CC, but did not vary in NoCC. VTI identified CC from NoCC and survivors from non-survivors before CC. A large increase in LIR was coincident with VTI decrement before CC occurred. CONCLUSIONS Vasodilatation immediately prior to CC in severe HS occurs at the same time as an increase in LIR, suggesting loss of tone as the mechanism causing CC, and energy failure as its probable cause.

A simple, rapid, and convenient Luminex-compatible method of tissue isolation.

Various pathological conditions are associated with changes in multiple protein biomarkers, and these changes can be assessed using xMAP™ beads and the Luminex™ platform. Although this platform is most commonly utilized in the analysis of biomarkers in serum, plasma, or urine, it is often desirable to measure these analytes in tissues (e.g., biopsy specimens). We have developed a simple, rapid method of tissue isolation in which excised tissues are permeabilized in RNAlater™ at 4°C overnight, followed by Luminex™ analysis for inflammatory biomarkers (cytokines). This method was compared with flash‐freezing in both mouse liver and human debrided wound specimens, and may be of utility in other clinical settings. J. Clin. Lab. Anal. 22:278–281, 2008.