Marc DeMoya

Fulminant Clostridium difficile colitis: Patterns of care and predictors of mortality

HYPOTHESIS There exist predictors of mortality and the need for colectomy among patients with fulminant Clostridium difficile colitis. DESIGN Retrospective study. SETTING Academic tertiary referral center. PATIENTS We reviewed the records of 4796 inpatients diagnosed as having C difficile colitis from January 1, 1996, to December 31, 2007, and identified 199 (4.1%) with fulminant C difficile colitis, as defined by the need for colectomy or admission to the intensive care unit for C difficile colitis. MAIN OUTCOME MEASURES Risk of inpatient mortality was determined by multivariate analysis according to clinical predictors, colectomy, and medical team. RESULTS The inhospital mortality rate for fulminant C difficile colitis was 34.7%. Independent predictors of mortality included the following: (1) age of 70 years or older, (2) severe leukocytosis or leukopenia (white blood cell count, >or=35 000/microL or <4000/microL) or bandemia (neutrophil bands, >or=10%), and (3) cardiorespiratory failure (intubation or vasopressors). When all 3 factors were present, the mortality rate was 57.1%; when all 3 were absent, the mortality rate was 0%. Patients who underwent colectomy had a trend toward decreased mortality rates (odds ratio, 0.49; 95% confidence interval, 0.21-1.1; P = .08). Among patients admitted primarily for fulminant C difficile colitis, care in the surgical department compared with the nonsurgical department resulted in a higher rate of operation (85.1% vs 11.2%; P < .001) and lower mortality rates (12.8% vs 39.3%; P = .001). Patients admitted directly to the surgical department had a shorter mean (SD) interval from admission to operation (0 vs 1.7 [2.8] days; P = .001). CONCLUSIONS Despite awareness and treatment, fulminant C difficile colitis remains a highly lethal disease. Reliable predictors of mortality exist and should be used to prompt aggressive surgical intervention. Survival rates are higher in patients who were cared for by surgical vs nonsurgical departments, possibly because of more frequent and earlier operations.

Surviving blood loss without blood transfusion in a swine poly-trauma model.

BACKGROUND We have demonstrated previously that valproic acid (VPA), a histone deacetylase inhibitor, can improve survival in lethal models of hemorrhagic shock. This study investigated whether VPA treatment would improve survival in a clinically relevant large animal model of poly-trauma/hemorrhagic shock, and whether the protective effects are executed through the Akt survival pathway. METHODS Yorkshire swine were subjected to a poly-trauma protocol including: (1) Pre-hospital phase- Femur fracture, 60% hemorrhage, 30 min of shock (mean arterial pressure [MAP]: 25-30 mmHg), and infusion of 154mM NaCl (3 x shed blood); (2) Early hospital phase A Grade V liver injury (simulating rupture of a previously contained hematoma) followed by liver packing; (3) Treatment/monitoring phase randomization to 3 treatment groups (n = 6-8/group): no treatment (control), fresh whole blood (FWB), and intravenous VPA (400 mg/kg, given during the pre-hospital phase). Animals were monitored for 4 h, with survival being the primary endpoint. Liver tissue was subjected to Western blot analysis. RESULTS FWB (n = 6) and VPA treatments (n = 7) significantly increased survival (100% and 86%, respectively) compared to control group (n = 8) (25%). The protocol produced significant anemia (Hb<6 g/dL) and lactic acidosis (lactate 3-5 mmol/L). Acidosis improved after blood transfusion and worsened in the other two groups. VPA treatment increased phospho-Akt (activated), phospho-GSK-3beta (Glycogen synthase kinase 3beta), beta-catenin and Bcl-2 (B-cell leukemia/lymphoma 2) protein levels compared to control group (P = .01, .01, .03, and .02, respectively). There was no significant difference in the level of these proteins between the control and FWB groups. CONCLUSION Treatment with VPA without blood transfusion improves early survival in a highly lethal poly-trauma and hemorrhagic shock model. The survival advantage is due not to improvement in resuscitation but to better tolerance of shock by the cells, in part due to the preservation of the Akt survival pathway.

Traumatic brain injury and hemorrhagic shock: evaluation of different resuscitation strategies in a large animal model of combined insults.

ABSTRACT Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of trauma-related mortality and morbidity. Combination of TBI and HS (TBI + HS) is highly lethal, and the optimal resuscitation strategy for this combined insult remains unclear. A critical limitation is the lack of suitable large animal models to test different treatment strategies. We have developed a clinically relevant large animal model of TBI + HS, which was used to evaluate the impact of different treatments on brain lesion size and associated edema. Yorkshire swine (42–50 kg) were instrumented to measure hemodynamic parameters and intracranial pressure. A computer-controlled cortical impact device was used to create a TBI through a 20-mm craniotomy: 15-mm cylindrical tip impactor at 4 m/s velocity, 100-ms dwell time, and 12-mm penetration depth. Volume-controlled hemorrhage was started (40% blood volume) concurrent with the TBI. After 2 h of shock, animals were randomized to one of three resuscitation groups (n = 5/group): (a) normal saline (NS); (b) 6% hetastarch, Hextend (Hex); and (c) fresh frozen plasma (FFP). Volumes of Hex and FFP matched the shed blood, whereas NS was three times the volume. After 6 h of postresuscitation monitoring, brains were sectioned into 5-mm slices and stained with TTC (2,3,5-triphenyltetrazolium chloride) to quantify the lesion size and brain swelling. Combination of 40% blood loss with cortical impact and a period of shock (2 h) resulted in a highly reproducible brain injury. Total fluid requirements were lower in the Hex and FFP groups. Lesion size and brain swelling in the FFP group (2,160 ± 202.6 mm3 and 22% ± 1.0%, respectively) were significantly smaller than those in the NS group (3,285 ± 130.8 mm3 and 37% ± 1.6%, respectively) (P < 0.05). Hex treatment decreased the swelling (29% ± 1.6%) without reducing the lesion size. Early administration of FFP reduces the size of brain lesion and associated swelling in a large animal model of TBI + HS. In contrast, artificial colloid (Hex) decreases swelling without reducing the actual size of the brain lesion.

Protective effect of suberoylanilide hydroxamic acid against LPS-induced septic shock in rodents.

We have recently found that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improves survival in a lethal model of hemorrhagic shock in rats. The purpose of the present study was to determine whether SAHA treatment would prevent LPS-induced septic shock and improve the survival in a murine model. C57BL/6J mice were randomly divided into two groups. Experimental mice were given intraperitoneal SAHA (50 mg/kg) in vehicle dimethyl sulfoxide fluid (n = 10). The control mice (n = 10) received vehicle dimethyl sulfoxide only. They were injected with LPS (20 mg/kg, i.p.) 2 h later, and the animals from the treatment group were given a second dose of SAHA. Survival was monitored during the next 7 days. In a parallel study, mice treated with or without SAHA were subjected to LPS insult while normal (sham) mice serviced as controls. 1) Lungs were harvested at 3 and 48 h for analysis of gene expression and pathologic changes, respectively; 2) spleens were isolated for analysis of neutrophilic cell population. In addition, RAW264.7 mouse macrophages were cultured to assess the effects of SAHA on LPS-induced inflammation in vitro. All mice in the control group that were subjected to LPS challenge died in less than 48 h. However, SAHA-treated animals displayed a significantly higher 1-week survival rate (87.5%) compared with the control group (0%). Moreover, LPS insult decreased the acetylation of histone proteins (H2A, H2B, and H3), elevated the levels of TNF-&agr; in vivo (circulation) and in vitro (culture medium), increased the neutrophilic cell population in the spleen, enhanced the expression of TNF-&agr; and IL-1&bgr; genes in lung tissue, and augmented the pulmonary neutrophil infiltration. In contrast, SAHA treatment markedly attenuated all of these LPS-induced alterations. We report for the first time that administration of SAHA (50 mg/kg) significantly attenuates a variety of inflammatory markers and improves long-term survival after a lethal LPS insult.

Surviving blood loss without fluid resuscitation

BACKGROUND Patients with massive blood loss often die before delivery of definitive care, especially in austere environments. Strategies that can maintain life during evacuation and transport to higher levels of care may be lifesaving. We have previously shown that administration of histone deacetylase inhibitors (HDACI) enhance gene transcription through specific modifications of DNA-associated histone proteins. Furthermore, it protects against organ damage when given before hemorrhage. The current experiment was done to test whether administration of HDACI after lethal hemorrhage, without fluid resuscitation, would improve outcome by creating a pro-survival phenotype. METHODS Seventy-two male Wistar-Kyoto rats (n = 12 per group) were subjected to 60% blood volume loss for 1 hour (40% arterial bleed for 10 minutes and 20% venous bleed for 50 minutes). After hemorrhage, animals were randomized to receive one of two HDACI: (1) valproic acid (VPA, 300 mg/kg in 0.25 mL saline), or (2) suberoyanilide hydroxamic acid (SAHA, 7.5 mg/kg in 0.25 mL saline). Control groups included (3) no hemorrhage (Sham), (4) no resuscitation (NR), (5) 0.9% saline resuscitation, 3 times the volume of shed blood (NS), and (6) vehicle control, 0.25 mL 0.9% saline (VEH). Hemodynamic data were recorded continuously, and physiologic parameters were measured serially. Survival for 3 hours was the primary endpoint for this experiment. RESULTS Nonresuscitated shock (NR group) was highly lethal and only 25% of the animals survived for 3 hours. Administration of HDACI after hemorrhage (without fluid resuscitation) significantly improved survival (75% and 83% in VPA and SAHA groups, respectively, p < 0.05 vs. NR). Survival was 40%, 100%, and 100% in the VEH, Sham, and NS resuscitation groups, respectively. CONCLUSIONS This study demonstrates that post-shock administration of HDACI can significantly improve early survival in a highly lethal model of hemorrhagic shock, even in the absence of conventional fluid resuscitation. This approach may be especially relevant for austere environments where fluids are in limited supply, such as a battlefield.

Cell protective mechanism of valproic acid in lethal hemorrhagic shock

BACKGROUND We have demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve animal survival after hemorrhagic shock and protect neurons from hypoxia-induced apoptosis. This study investigated whether VPA treatment works through the beta-catenin survival pathways. METHODS Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss) followed by treatment with or without VPA (300 mg/kg). Brains were harvested after 1, 6, and 24 hours and analyzed for acetylated histone-H3 at lysine-9 (Ac-H3K9), acetylated and total beta-catenin, and Bcl-2 by Western blot. In addition, primary neurons dissociated from E18 rat embryos were exposed to hypoxia (0.5% O(2)) for 16 hours with or without VPA (1 mmol/L) and analyzed using confocal microscopy. RESULTS After treatment of hemorrhaged animals with VPA, acetylated beta-catenin was found in both the cytosol and nucleus, along with Ac-H3K9. Bcl-2 transcript increased after 1 hour followed by an increase in Bcl-2 protein at 6 hours. Confocal imaging demonstrated that after VPA treatment, beta-catenin translocated into the nucleus and colocalized with Ac-H3K9. CONCLUSION VPA treatment acetylates H3K9 and beta-catenin and enhances translocation of beta-catenin into the nucleus, where it colocalizes with Ac-H3K9 and stimulates the transcription of survival gene bcl-2. This finding suggests that VPA protects cells after severe insult through the beta-catenin survival pathway.

Effect of valproic acid on acute lung injury in a rodent model of intestinal ischemia reperfusion

OBJECTIVES Acute lung injury (ALI) can develop during the course of many clinical conditions, and is associated with significant morbidity and mortality. Valproic acid (VPA), a well-known anti-epileptic drug, has been shown to have anti-oxidant and anti-inflammatory effects in various ischemia/reperfusion (I/R) models. The purpose of this study was to investigate whether VPA could affect survival and development of ALI in a rat model of intestinal I/R. METHODS Two experiments were performed. Experiment I: Male Sprague-Dawley rats (250-300 g) were subjected to intestinal ischemia (1h) and reperfusion (3h). They were randomized into 2 groups (n=7 per group) 3 min after ischemia: Vehicle (Veh) and VPA (300 mg/kg, IV). Primary end-point for this study was survival over 4h from the start of ischemia. Experiment II: The histological and biochemical effects of VPA treatment on lungs were examined 3h (1h ischemia+2h reperfusion) after intestinal I/R injury (Veh vs. VPA, n=9 per group). An objective histological score was used to grade the degree of ALI. Enzyme linked immunosorbent assay (ELISA) was performed to measure serum levels of interleukins (IL-6 and 10), and lung tissue of cytokine-induced neutrophil chemoattractant (CINC) and myeloperoxidase (MPO). In addition, the activity of 8-isoprostane was analyzed for pulmonary oxidative damage. RESULTS In Experiment I, 4-h survival rate was significantly higher in VPA treated animals compared to Veh animals (71.4% vs. 14.3%, p=0.006). In Experiment II, ALI was apparent in all of the Veh group animals. Treatment with VPA prevented the development of ALI, with a reduction in the histological score (3.4 ± 0.3 vs. 5.3 ± 0.6, p=0.025). Moreover, compared to the Veh control group the animals from the VPA group displayed decreased serum levels of IL-6 (952 ± 213 pg/ml vs. 7709 ± 1990 pg/ml, p=0.011), and lung tissue concentrations of CINC (1188 ± 28 pg/ml vs. 1298 ± 27 pg/ml, p<0.05), MPO activity (368 ± 23 ng/ml vs. 490 ± 29 ng/ml, p<0.05) and 8-isoprostane levels (1495 ± 221 pg/ml vs. 2191 ± 177 pg/ml, p<0.05). CONCLUSION VPA treatment improves survival and attenuates ALI in a rat model of intestinal I/R injury, at least in part, through its anti-oxidant and anti-inflammatory effects.

Assessment of coagulopathy, endothelial injury, and inflammation after traumatic brain injury and hemorrhage in a porcine model

BACKGROUND Traumatic brain injury (TBI) and hemorrhagic shock (HS) can be associated with coagulopathy and inflammation, but the mechanisms are poorly understood. We hypothesized that a combination of TBI and HS would disturb coagulation, damage the endothelium, and activate inflammatory and complement systems. METHODS A total of 33 swine were allocated to either TBI + HS (n = 27, TBI and volume-controlled 40% blood loss) or controls (n = 6, anesthesia and instrumentation). TBI + HS animals were left hypotensive (mean arterial pressure, 30–35 mm Hg) for 2 hours. Blood samples were drawn at baseline, 3 minutes and 15 minutes after injury, as well as following 2 hours of hypotension. Markers of coagulation, anticoagulation, endothelial activation/glycocalyx shedding, inflammation, complement, and sympathoadrenal function were measured. RESULTS The TBI + HS group demonstrated an immediate (3 minutes after injury) activation of coagulation (prothrombin fragment 1 + 2, 289 ng/mL vs. 232 ng/mL, p = 0.03) and complement (C5a, 2.83 ng/mL vs. 2.05 ng/mL, p = 0.05). Shedding of the endothelial glycocalyx (syndecan 1) was evident 15 minutes after injury (851.0 ng/ml vs. 715.5 ng/ml, p = 0.03) while inflammation (tumor necrosis factor &agr; [TNF-&agr;], 81.1 pg/mL vs. 50.8 pg/mL, p = 0.03) and activation of the protein C system (activated protein C, 56.7 ng/mL vs. 26.1 ng/mL, p = 0.01) were evident following the 2-hour hypotension phase. CONCLUSION The combination of TBI and shock results in an immediate activation of coagulation and complement systems with subsequent endothelial shedding, protein C activation, and inflammation.

Pharmacologic resuscitation for hemorrhagic shock combined with traumatic brain injury.

BACKGROUND We have previously demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve survival after hemorrhagic shock (HS), protect neurons from hypoxia-induced apoptosis, and attenuate the inflammatory response. We have also shown that administration of 6% hetastarch (Hextend [Hex]) after traumatic brain injury (TBI) decreases brain swelling, without affecting size of the lesion. This study was performed to determine whether addition of VPA to Hex would decrease the lesion size in a clinically relevant large animal model of TBI + HS. METHODS Yorkshire swine (42–50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. A custom-designed, computer-controlled cortical impact device was used to create a TBI through a 20-mm craniotomy: 15-mm cylindrical tip impactor at 4-m/s velocity, 100-millisecond dwell time, and 12-mm penetration depth. Volume-controlled hemorrhage was started (40% blood volume) concurrent with the TBI. After 2 hours of shock, animals were randomized to one of three resuscitation groups (n = 7 per group) as follows: (1) isotonic sodium chloride solution; (2) 6% hetastarch, Hex; and (3) Hex and VPA 300 mg/kg (Hex + VPA). Volumes of Hex matched the shed blood, whereas that of the isotonic sodium chloride solution was three times the volume. VPA treatment was started after an hour of shock. After 6 hours of postresuscitation monitoring, brains were sectioned into 5-mm slices and stained with 2, 3, 5-Triphenyltetrazolium chloride to quantify the lesion size (mm3) and brain swelling (percent change compared with uninjured side). Levels of acetylated histone H3 were determined to quantify acetylation, and myeloperoxidase and interleukine-1&bgr; (IL-1&bgr;) levels were measured as markers of brain inflammation. RESULTS Combination of 40% blood loss with cortical impact and a period of shock (2 hours) and resuscitation resulted in a highly reproducible brain injury. Lesion size and brain swelling in the Hex + VPA group (1,989 [156.8] mm3, and 19% [1.6%], respectively) were significantly smaller than the isotonic sodium chloride solution group (3,335 [287.9] mm3 and 36% [2.2%], respectively). Hex alone treatment significantly decreased the swelling (27% [1.6%]) without reducing the lesion size. The number of CD11b-positive cells as well as myeloperoxidase and IL-1 levels in the brains were significantly reduced by the VPA treatment. CONCLUSION In a combined HS and TBI model, treatment with artificial colloid (Hex) improves hemodynamic parameters and reduces swelling, without affecting the actual size of the brain lesion. Addition of VPA effectively reduces both the size of brain lesion and associated swelling by attenuating the inflammatory response.

Identification of citrullinated histone H3 as a potential serum protein biomarker in a lethal model of lipopolysaccharide-induced shock

BACKGROUND Circulating proteins may serve as biomarkers for the early diagnosis and treatment of shock. We have recently demonstrated that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, significantly improves survival in a rodent model of lipopolysaccharide (LPS)-induced septic shock. Preliminary proteomic data showed that LPS-induced shock altered a number of proteins in circulation, including histone H3 (H3) and citrullinated histone H3 (Cit H3). The present study was designed to confirm these findings and to test whether the pro-survival phenotype could be detected by an early alteration in serum biomarkers. METHODS Three experiments were performed. In experiment I, Western blotting was performed on serum samples from male C57B1/6J mice (n = 9, 3/group) that belonged to the following groups: (a) LPS (20 mg/kg)-induced septic shock, (b) SAHA-treated septic shock, and (c) sham (no LPS, no SAHA). In experiment II, HL-60 granulocytes were cultured and treated with LPS (100 ng/m1) in the absence or presence of SAHA (10 μmol/L). Sham (no LPS, no SAHA) granulocytes served as controls. The medium and cells were harvested at 3 hours, and proteins were measured with Western blots. In experiment III, a large dose (LD, 35 mg/kg) or small dose (SD, 10 mg/kg) of LPS was injected intraperitoneally into the C57B1/6J mice (n = 10 per group). Blood was collected at 3 hours, and serum proteins were determined by Western blots or enzyme-linked immunosorbent assay (ELISA). All of the Western blots were performed with antibodies against H3, Cit H3, and acetylated H3 (Ac H3). ELISA was performed with antibody against tumor necrosis factor (TNF)-α. Survival rates were recorded over 7 days. RESULTS In experiment I, intraperitoneal (IP) injection of LPS (20 mg/kg) significantly increased serum levels of H3, which was prevented by SAHA treatment. In experiment II, LPS (100 ng/mL) induced expression and secretion of Cit H3 and H3 proteins in neutrophilic HL-60 cells, which was decreased by SAHA treatment. In experiment III, administration of LPS (LD) caused a rise in serum H3 and Cit H3 but not Ac H3 at 3 hours, and all of these animals died within 23 hours (100% mortality). Decreasing the dose of LPS (SD) significantly reduced the mortality rate (10% mortality) as well as the circulating levels of Cit H3 (non detectable) and H3. An increase in serum TNF-α was found in both LPS (LD) and (SD) groups, but in a non-dose-dependent fashion. CONCLUSION Our results reveal for the first time that Cit H3 is released into circulation during the early stages of LPS-induced shock. Moreover, serum levels of Cit H3 are significantly associated with severity of LPS-induced shock. Therefore, Cit H3 could serve as a potential protein biomarker for early diagnosis of septic shock, and for predicting its lethality.