Valproic Acid Protects Against Acute Kidney Injury in Hemorrhage and Trauma.

INTRODUCTION: Trauma is the leading cause of death among young people. These patients have a high incidence of kidney injury, which independently increases the risk of mortality. As valproic acid (VPA) treatment has been shown to improve survival in animal models of lethal trauma, we hypothesized that it would also attenuate the degree of acute kidney injury. METHODS: We analyzed data from two separate experiments where swine were subjected to lethal insults.  Model 1: hemorrhage (50% blood volume hemorrhage followed by 72-h damage control resuscitation). Model 2: polytrauma (traumatic brain injury, 40% blood volume hemorrhage, femur fracture, rectus crush and grade V liver laceration). Animals were resuscitated with normal saline (NS) +/- VPA 150 mg/kg after a 1-h shock phase in both models (n = 5-6/group). Serum samples were analyzed for creatinine (Cr) using colorimetry on a Liasys 330 chemistry analyzer. Proteomic analysis was performed on kidney tissue sampled at the time of necropsy. RESULTS: VPA treatment significantly (P < 0.05) improved survival in both models. (Model 1: 80% vs 20%; Model 2: 83% vs. 17%). Model 1 (Hemorrhage alone): Cr increased from a baseline of 1.2 to 3.0 in NS control animals (P < 0.0001) 8 h after hemorrhage, whereas it rose only to 2.1 in VPA treated animals (P = 0.004). Model 2 (Polytrauma): Cr levels increased from baseline of 1.3 to 2.5 mg/dL (P = 0.01) in NS control animals 4 h after injury but rose to only 1.8 in VPA treated animals (P = 0.02). Proteomic analysis of kidney tissue identified metabolic pathways were most affected by VPA treatment. CONCLUSIONS: A single dose of VPA (150 mg/kg) offers significant protection against acute kidney injury in swine models of polytrauma and hemorrhagic shock.

Assessment of the Cytoprotective Effects of High-Dose Valproic Acid Compared to a Clinically Used Lower Dose.

OBJECTIVE: Valproic acid (VPA) treatment improves survival in animal models of injuries on doses higher than those allowed by Food and Drug Administration (FDA). We investigated the proteomic alterations induced by a single high-dose (140mg/kg) of VPA (VPA140) compared to the FDA-approved dose of 30mg/kg (VPA30) in healthy humans. We also describe the proteomic and transcriptomic changes induced by VPA140 in an injured patient. We hypothesized that VPA140 would induce cytoprotective changes in the study participants. METHODS: Serum samples were obtained from healthy subjects randomized to two groups; VPA140 and VPA30 at 3 timepoints: 0h(baseline), 2h, and 24h following infusion(n = 3/group). Samples were also obtained from an injured patient that received VPA140 at 0h, 6h and 24h following infusion. Proteomic analyses were performed using liquid chromatography-mass spectrometry (LC-MS/MS), and transcriptomic analysis was performed using RNA-sequencing. Differentially expressed (DE) proteins and genes were identified for functional annotation and pathway analysis using iPathwayGuide and gene set enrichment analysis (GSEA), respectively. RESULTS: For healthy individuals, a dose comparison was performed between VPA140 and VPA30 groups at 2 and 24 h. Functional annotation showed that top biological processes in VPA140 versus VPA30 analysis at 2 h included regulation of fatty acid (P = 0.002) and ATP biosynthesis (P = 0.007), response to hypoxia (P = 0.017), cell polarity regulation (P = 0.031), and sequestration of calcium ions (P = 0.031). Top processes at 24 h in VPA140 versus VPA30 analysis included amino acid metabolism (P = 0.023), collagen catabolism (P = 0.023), and regulation of protein breakdown (P = 0.023). In the injured patient, annotation of the DE proteins in the serum showed that top biological processes at 2 h included neutrophil chemotaxis (P = 0.002), regulation of cellular response to heat (P = 0.008), regulation of oxidative stress (P = 0.008) and regulation of apoptotic signaling pathway (P = 0.008). Top biological processes in the injured patient at 24 h included autophagy (P = 0.01), glycolysis (P = 0.01), regulation of apoptosis (P = 0.01) and neuron apoptotic processes (P = 0.02). CONCLUSIONS: VPA140 induces cytoprotective changes in human proteome not observed in VPA30. These changes may be responsible for its protective effects in response to injuries.

A Randomized Trial Comparing Antibiotics with Appendectomy for Appendicitis.

BACKGROUND: Antibiotic therapy has been proposed as an alternative to surgery for the treatment of appendicitis. METHODS: We conducted a pragmatic, nonblinded, noninferiority, randomized trial comparing antibiotic therapy (10-day course) with appendectomy in patients with appendicitis at 25 U.S. centers. The primary outcome was 30-day health status, as assessed with the European Quality of Life-5 Dimensions (EQ-5D) questionnaire (scores range from 0 to 1, with higher scores indicating better health status; noninferiority margin, 0.05 points). Secondary outcomes included appendectomy in the antibiotics group and complications through 90 days; analyses were prespecified in subgroups defined according to the presence or absence of an appendicolith. RESULTS: In total, 1552 adults (414 with an appendicolith) underwent randomization; 776 were assigned to receive antibiotics (47% of whom were not hospitalized for the index treatment) and 776 to undergo appendectomy (96% of whom underwent a laparoscopic procedure). Antibiotics were noninferior to appendectomy on the basis of 30-day EQ-5D scores (mean difference, 0.01 points; 95% confidence interval [CI], -0.001 to 0.03). In the antibiotics group, 29% had undergone appendectomy by 90 days, including 41% of those with an appendicolith and 25% of those without an appendicolith. Complications were more common in the antibiotics group than in the appendectomy group (8.1 vs. 3.5 per 100 participants; rate ratio, 2.28; 95% CI, 1.30 to 3.98); the higher rate in the antibiotics group could be attributed to those with an appendicolith (20.2 vs. 3.6 per 100 participants; rate ratio, 5.69; 95% CI, 2.11 to 15.38) and not to those without an appendicolith (3.7 vs. 3.5 per 100 participants; rate ratio, 1.05; 95% CI, 0.45 to 2.43). The rate of serious adverse events was 4.0 per 100 participants in the antibiotics group and 3.0 per 100 participants in the appendectomy group (rate ratio, 1.29; 95% CI, 0.67 to 2.50). CONCLUSIONS: For the treatment of appendicitis, antibiotics were noninferior to appendectomy on the basis of results of a standard health-status measure. In the antibiotics group, nearly 3 in 10 participants had undergone appendectomy by 90 days. Participants with an appendicolith were at a higher risk for appendectomy and for complications than those without an appendicolith. (Funded by the Patient-Centered Outcomes Research Institute; CODA ClinicalTrials.gov number, NCT02800785.).

Valproic acid treatment rescues injured tissues after traumatic brain injury.

BACKGROUND: No agents that are specifically neuroprotective are currently approved to emergently treat patients with traumatic brain injury (TBI). The histone deacetylase inhibitor, high-dose valproic acid (VPA) has been shown to have cytoprotective potential in models of combined TBI and hemorrhagic shock, but it has not been tested in an isolated TBI model. We hypothesized that VPA, administered after isolated TBI, will penetrate the injured brain, attenuate the lesion size, and activate prosurvival pathways. METHODS: Yorkshire swine were subjected to severe TBI by cortical impact. One hour later, animals were randomized to VPA treatment (150 mg/kg delivered intravenously for 1 hour; n = 4) or control (saline vehicle; n = 4) groups. Seven hours after injury, animals were sacrificed, and brain lesion size was measured. Mass spectrometry imaging was used to visualize and quantitate brain tissue distribution of VPA. Sequential serum samples were assayed for key biomarkers and subjected to proteomic and pathway analysis. RESULTS: Brain lesion size was 50% smaller (p = 0.01) in the VPA-treated animals (3,837 ± 948 mm) compared with the controls (1,900 ± 614 mm). Endothelial regions had eightfold higher VPA concentrations than perivascular regions by mass spectrometry imaging, and it readily penetrated the injured brain tissues. Serum glial fibrillary acid protein was significantly lower in the VPA-treated compared with the control animals (p < 0.05). More than 500 proteins were differentially expressed in the brain, and pathway analysis revealed that VPA affected critical modulators of TBI response including calcium signaling pathways, mitochondria metabolism, and biosynthetic machinery. CONCLUSION: Valproic acid penetrates injured brain tissues and exerts neuroprotective and prosurvival effects that resulted in a significant reduction in brain lesion size after isolated TBI. Levels of serum biomarkers reflect these changes, which could be useful for monitoring the response of TBI patients during clinical studies.

Safety and Tolerability of Intravenous Valproic Acid in Healthy Subjects: A Phase I Dose-Escalation Trial.

BACKGROUND: Valproic acid, a histone deacetylase inhibitor, has beneficial effects in the setting of cancer, neurologic diseases, and traumatic injuries. In animal models of traumatic injury, a single dose of valproic acid has been shown to reduce mortality. The purpose of this trial was to determine the maximum tolerated single dose of intravenous valproic acid in healthy humans. METHODS: A double-blinded, placebo-controlled, dose-escalation trial design was used to identify dose-limiting toxicities in healthy subjects who received a single dose of intravenous valproic acid. Patients were monitored for adverse events and data were collected for pharmacokinetic, pharmacodynamic, and safety profiling of valproic acid. RESULTS: Fifty-nine healthy subjects (mean 30 ± 12 years) were enrolled. Forty-four subjects received valproic acid in doses from 15 to 150 mg/kg. The most common adverse events were hypoacusis (n = 19), chills (n = 18), and headache (n = 16). The maximum tolerated dose was 140 mg/kg. Dose-limiting toxicities included headache and nausea lasting longer than 12 h. No drug-related abnormalities were seen in other safety measures including laboratory tests, hemodynamic parameters, cardiac rhythm monitoring, and cognitive testing. A two-compartment model was predictive of valproic acid concentration-time profiles, with a strong correlation (R 2 = 0.56) observed between the number of reported adverse events and the dose level. CONCLUSIONS: The maximum tolerated dose of intravenous valproic acid in healthy subjects is 140 mg/kg. This is significantly higher than the previously established maximum tolerated dose of 60-75 mg/kg. Next, the safety and tolerability of high-dose valproic acid will be tested in trauma patients in hemorrhagic shock. ClinicalTrials.gov Identifier: NCT01951560.

Addition of low-dose valproic acid to saline resuscitation provides neuroprotection and improves long-term outcomes in a large animal model of combined traumatic brain injury and hemorrhagic shock.

BACKGROUND: Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In a nonsurvival model of TBI + HS, addition of high-dose valproic acid (VPA) (300 mg/kg) to hetastarch reduced brain lesion size and associated swelling 6 hours after injury; whether this would have translated into better neurologic outcomes remains unknown. It is also unclear whether lower doses of VPA would be neuroprotective. We hypothesized that addition of low-dose VPA to normal saline (NS) resuscitation would result in improved long-term neurologic recovery and decreased brain lesion size. METHODS: TBI was created in anesthetized swine (40-43 kg) by controlled cortical impact, and volume-controlled hemorrhage (40% volume) was induced concurrently. After 2 hours of shock, animals were randomized (n = 5 per group) to NS (3× shed blood) or NS + VPA (150 mg/kg). Six hours after resuscitation, packed red blood cells were transfused, and animals were recovered. Peripheral blood mononuclear cells were analyzed for acetylated histone-H3 at lysine-9. A Neurological Severity Score (NSS) was assessed daily for 30 days. Brain magnetic resonance imaging was performed on Days 3 and 10. Cognitive performance was assessed by training animals to retrieve food from color-coded boxes. RESULTS: There was a significant increase in histone acetylation in the NS + VPA-treated animals compared with NS treatment. The NS + VPA group demonstrated significantly decreased neurologic impairment and faster speed of recovery as well as smaller brain lesion size compared with the NS group. Although the final cognitive function scores were similar between the groups, the VPA-treated animals reached the goal significantly faster than the NS controls. CONCLUSION: In this long-term survival model of TBI + HS, addition of low-dose VPA to saline resuscitation resulted in attenuated neurologic impairment, faster neurologic recovery, smaller brain lesion size, and a quicker normalization of cognitive functions.

Protective effect of suberoylanilide hydroxamic acid against lipopolysaccharide-induced liver damage in rodents.

BACKGROUND: Lipopolysaccharide (LPS) has a deleterious effect on several organs, including the liver, and eventually leads to endotoxic shock and death. LPS-induced hepatotoxicity is characterized by disturbed intracellular redox balance and excessive reactive oxygen species (ROS) accumulation, leading to liver injury. We have shown that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improves survival in a murine model of LPS-induced shock, but the protective effect of SAHA against liver damage remains unknown. The goal of this study was to investigate the mechanism underlying SAHA action in murine livers. METHOD: Male C57BL/6J mice (6-8 wk), weighing 20-25 g, were randomly divided into three groups: (A) a sham group was given isotonic sodium chloride solution (10 µL/g body weight, intraperitoneal, i.p.) with dimethyl sulfoxide (DMSO; 1 µL/g body weight, i.p.); (B) an LPS group was challenged with LPS (20 mg/kg, i.p.) dissolved in isotonic sodium chloride solution with DMSO; (C) and an LPS plus SAHA group was treated with SAHA (50 mg/kg, i.p.) dissolved in DMSO immediately after injection of LPS (20 mg/kg, i.p.). Mice were anesthetized, and their livers were harvested 6 or 24 h after injection to analyze whether SAHA affected production of ROS and activation of apoptotic proteins in the liver cells of challenged mice. RESULTS: SAHA counteracted LPS-induced production of ROS (thiobarbituric acid reactive substances and nitrite) and reversed an LPS-induced decrease in antioxidant enzyme, glutathione. SAHA also attenuated LPS-induced hepatic apoptosis. Moreover, SAHA inhibited activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1, and the mitogen-activated protein kinases, p38 and Jun N-terminal kinase. CONCLUSIONS: Our data indicate, for the first time, that SAHA is capable of alleviating LPS-induced hepatotoxicity and suggest that a blockade of the upstream events required for apoptosis signal-regulating kinase-1 action may serve as a new therapeutic option in the treatment of LPS-induced inflammatory conditions.

Pharmacologic modulation of cerebral metabolic derangement and excitotoxicity in a porcine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Cerebral metabolic derangement and excitotoxicity play critical roles in the evolution of traumatic brain injury (TBI). We have shown previously that treatment with large doses of valproic acid (VPA) decreases the size of brain lesion. The goal of this experiment was to determine whether this effect was owing to metabolic modulation. METHODS: Yorkshire swine (n = 9) underwent a protocol of computer-controlled TBI and 40% hemorrhage and were resuscitated randomly with either fresh frozen plasma equal to the volume of shed blood (FFP; n = 4) or VPA (300 mg/kg) and FFP (FFP+VPA; n = 5). Hemodynamics, brain oxygenation, and blood glucose were monitored continuously for 6 hours after resuscitation. Cerebral microdialysis was used to measure glucose, lactate, pyruvate, glutamate, and glycerol levels at baseline, 1 and 2 hours post-shock, post-resuscitation (PR), and at 2, 4, and 6 hours PR. Brain samples from the injured side were then separated into mitochondrial and cytosolic fractions, and activity of pyruvate dehydrogenase complex (PDH) was measured using a dipstick assay kit. RESULTS: At baseline, there was no difference in brain lactate, pyruvate, glycerol, and glutamate concentrations between the groups. At all time points, there were no differences between the groups in brain oxygenation, cerebral perfusion pressure, or blood and brain glucose concentrations. After VPA infusion (PR time point), however, there was sustained decrease in lactate (0.91 ± 0.47 vs 2.54 ± 0.59 mmol/L; P < .01) and pyruvate (12.80 ± 4.89 vs 46.25 ± 9.22; P < .001) concentrations compared with the FFP alone group, implying superior glucose utilization for ATP production. There was also a decrease in concentrations of glutamate (6.64 ± 3.68 vs 42.25 ± 27.07 mmol/L; P = .02) and glycerol (19.20 ± 6.76 vs 69.75 ± 30.07 mmol/L; P = .01), in the FFP+VPA group, signifying lesser degree of excitotoxicity and brain damage, respectively. Brain PDH activity was greater in the mitochondrial fractions (5,984 ± 504 adjusted volume intensity [INT] × mm(2) vs 4,332 ± 1,055 INT × mm(2); P = .04) and lower in cytosolic fractions in the FFP+VPA group (1,597 ± 1,395 vs 4,026 ± 1,067 INT × mm(2); P = .03), indicating better mitochondrial membrane function and enhanced mitochondrial PDH retention. CONCLUSION: VPA treatment attenuates perturbation of post-traumatic cerebral metabolism by mitigating mitochondrial dysfunction, and decreases glutamate-mediated excitotoxic damage. These properties could explain its effectiveness in decreasing lesion size and post-traumatic cerebral edema.

Synergistic effects of hypertonic saline and valproic acid in a lethal rat two-hit model.

BACKGROUND: Hemorrhagic shock (HS) followed by an infection ("second hit") can lead to severe systemic inflammatory response and multiple-organ failure. Studies have shown that resuscitation with hypertonic saline (HTS) can blunt the inflammatory response. We demonstrated that large doses of valproic acid (VPA, 300 mg/kg), a histone deacetylase inhibitor, improves survival in a rodent two-hit model (HS followed by cecal ligation and puncture [CLP]). In the present study, we examined whether combination of HTS with VPA would allow us to achieve survival advantage at a lower dose of VPA (200 mg/kg). METHODS: Male Sprague-Dawley rats were subjected to HS (50% blood loss) and randomized into five groups (n = 7-8 per group) as follows: (1) isotonic sodium chloride solution (ISCS), (2) 7.5% saline, (3) VPA, (4) ISCS + VPA, and (5) HTS + VPA. After 24 hours, they underwent CLP, followed by the same doses of ISCS, HTS, and/or VPA and were monitored for 10 days. In a parallel experiment, blood, peritoneal irrigation fluid and lung homogenate were subjected to enzyme-linked immunosorbent assay 3 hours and 24 hours after CLP to measure myeloperoxidase activity and proinflammatory cytokines tumor necrosis factor α and interleukin 1ß levels. Western blotting was performed to investigate the expression of pentraxin 3 protein in the lung homogenate at 24 hours after CLP. Hematoxylin and eosin staining of lungs at the 24 hours were performed to quantify the degree of acute lung injury. RESULTS: HTS + VPA treatment significantly improved survival (87.5%), compared with the other groups (14.3%; p < 0.05), while attenuating peritoneal myeloperoxidase levels and proinflammatory cytokine tumor necrosis factor α and interleukin 1ß levels in the serum, peritoneal cavity, and lung. The degree of acute lung injury and expression of pentraxin 3 in the lung were significantly reduced in the HTS + VPA group. CONCLUSION: This is the first study to show that VPA and HTS can work synergistically to attenuate inflammation and improve survival in a lethal two-hit model.

Testing of blood products in a polytrauma model: results of a multi-institutional randomized preclinical trial.

INTRODUCTION: Trauma-induced coagulopathy, acidosis, and hypothermia form a "lethal triad" that is difficult to treat and is associated with extremely high mortality. This study was performed at three academic centers to evaluate whether resuscitation with blood components could reverse the coagulopathy in a complex polytrauma model. METHODS: Yorkshire swine (40 +/- 5 kg) were subjected to a three-phase protocol: (a) "Prehospital" phase = femur fracture, hemorrhage (60% blood volume), and 30 minutes shock + infusion of saline (3x shed blood) + induction of hypothermia (33 degrees C); (b) "Early hospital" phase = grade V liver injury; and (c) "Operative" phase= liver packing. After liver packing, the animals (n = 60) were randomized to the following groups: (1) Sham-instrumentation and anesthesia without hemorrhage/injuries, (2) fresh whole blood (FWB), (3) 6% hetastarch (Hextend), (4) fresh frozen plasma/packed RBCs in 1:1 ratio (1:1 FFP/PRBC), and (5) FFP alone. Treatment volumes were equal to the volume of shed blood. Hemodynamic and physiologic parameters and coagulation profile (thrombelastography, prothrombin time, activated partial thromboplastin time, international normalized ratio, and platelets) were monitored during the experiment and for 4 hours posttreatment. RESULTS: At the end of prehospital phase, animals had developed significant acidosis (lactate >5 mmol/L and base deficit >9 mmol/L) and coagulopathy. Posttreatment mortality rates were 85% and 0% for the Hextend and blood component treated groups, respectively (p < 0.05). Hemodynamic parameters and survival rates were similar in groups that were treated with blood products (FWB, FFP, and FFP:PRBC). Animals treated with FFP and Hextend had significant anemia compared with the groups that received red blood cells (FWB and FFP:PRBC). Treatment with FFP and FFP:PRBC corrected the coagulopathy as effectively as FWB, whereas Hextend treatment worsened coagulopathy. CONCLUSIONS: In this reproducible model, we have shown that trauma-associated coagulopathy is made worse by hetastarch, but it can be rapidly reversed with the administration of blood components. Impressively, infusion of FFP, even without any red blood cells, can correct the coagulopathy and result in excellent early survival.

Prevention of hypoxia-induced neuronal apoptosis through histone deacetylase inhibition.

BACKGROUND: We have recently discovered that administration of valproic acid (VPA), a histone deacetylase inhibitor, enhances nuclear histone acetylation and improves survival after lethal hemorrhage in rats. In the present study, neurons were subjected to severe hypoxic condition in vitro to test whether VPA would prevent hypoxia-induced apoptosis, and to explore the possible mechanisms. METHODS: Primary hippocampal and cortical cultures dissociated from E18 rat embryos were plated in quadruplicate at a density of 2 x 10/well in neurobasal medium supplemented with B-27 on glass cover-slips coated with poly-l-lysine. On the 10th day after plating, cells were incubated in a hypoxia chamber (0.5% O2, 10% CO2, 89.5% N2) at 37 degrees C for 6 hour and 16 hour in the presence or absence of VPA (1 mmol/L). The cells were then fixed, stained with antiactivated caspase-3 and antiacetyl histone H3 lysine 9 (Ac H3 K9) antibodies and visualized under confocal microscope. The caspase-3 positive cells were counted as apoptotic. Ratio of the apoptotic to total cells stained with 4',6-diamidino-2-phenylindole was determined. Numerical data were subjected to t test analysis. p < 0.05 was considered statistically significant. Western blot was performed to determine the level of acetylation of nuclear factor-kappa B (NF-kappaB) and phospho-JNK (c-Jun N-terminal kinase) in cells treated with or without VPA. Luciferase report assay was employed to analyze the activation of NF-kappaB after the cells were transfected with NF-kBLuc with or without VPA treatment. RESULTS: Exposure of neurons to VPA prevented apoptotic cell death under hypoxic conditions (20% apoptosis). In contrast, about 95% cells underwent apoptosis at the same level of hypoxia. VPA treatment induced acetylation of histone H3 K9 and NF-kappaB lysine 310. NF-kappaB was activated at the same time as the protein acetylation. Moreover, JNK phosphorylation was inhibited after the cells were treated with VPA under hypoxia condition. CONCLUSION: VPA enhances acetylation of histone 3 at lysine 9 and NF-kappaB at 310, induces NF-kappaB activation, reduces JNK activation, and protects the neurons from hypoxia-induced apoptosis in vitro.

Making sense of the preclinical literature on advanced hemostatic products.

A number of new hemostatic products have been developed recently for use in trauma settings of severe uncontrolled bleeding. Currently, the literature on these products is controversial, with efficacy demonstrated under some circumstances but not others. In this review, we analyze the current literature pertaining to four of the most promising products (dry fibrin sealant dressing, Rapid Deployment Hemostat, HemCon chitosan dressing, and QuikClot) that have been suggested for use in combat casualty care applications. In particular, this analysis takes into account the characteristics of the animal models used for efficacy testing of these products, the desired characteristics of hemostatic dressings, and specific safety considerations. Animal models ranged from those featuring low-pressure/low-flow bleeding to those featuring high-pressure/high-flow bleeding. When data are viewed in the context of the specific characteristics of the differing animal models used, seemingly disparate experimental results related to efficacy and safety become quite complementary and lead to recommendations for the use of different products in different injury scenarios. Mission and training requirements will dictate the use of these products by military and civilian prehospital care providers.

D-lactate increases pulmonary apoptosis by restricting phosphorylation of bad and eNOS in a rat model of hemorrhagic shock.

UNLABELLED: Resuscitation with racemic lactated Ringer's solution (containing equal amounts of D and L isomers of lactate) has been shown to induce pulmonary apoptosis. Substitution of DL-isomer lactate with ketone bodies (beta-hydroxybutyrate, BHB), sodium pyruvate, or L-isomer of lactate decrease this injury without changing the energy status of the tissues or the expression of apoptotic genes. These modified solutions however alter the function of apoptotic proteins through an unknown mechanism. We postulated that DL-LR induces apoptosis by restricting the phosphorylation of key apoptotic proteins. METHODS: Male Sprague Dawley rats (n = 30, 5/group) were subjected to a three stage, volume-controlled hemorrhage and randomized to the following groups. 1) No hemorrhage (Sham); 2) Hemorrhage and no resuscitation (NR); 3) Resuscitation with 3x shed blood volume of racemic LR (DL-LR); 4) Resuscitation with 3x shed blood volume of LR containing only the L-isomer of lactate (L-LR); 5) Resuscitation with 3s shed blood volume of pyruvate Ringer's (PR); 6) Resuscitation with 3s shed blood volume of ketone Ringer's (KR). The modified Ringer's solutions were identical to racemic LR except for equimolar substitution of DL-lactate for L-lactate, pyruvate and BHB respectively. Lung tissue was obtained 2 hours later and subjected to Western Blotting. The levels of Akt, Bad, and eNOS (total and phosphorylated) proteins were measured. Finally, the expression of gene coding for protein 14-3-3 was measured using RT-PCR. RESULTS: Resuscitation with DL-LR caused a significant (p < 0.05) increase in the total Bad and a decrease in phosphorylated Bad protein expression in the lung. It also caused an increase in the phosphorylated Akt levels and a decrease in gene coding for protein 14-3-3. These changes were consistent with signaling imbalances that favor apoptosis. Modified LR solutions, on the other hand, did not cause these alterations. Phosphorylation pattern of eNOS supported the involvement of PI3K/Akt pathway in this process. CONCLUSION: Racemic lactate plays a role in the induction of pulmonary apoptosis by restricting phosphorylation of Bad and eNOS proteins.

Application of a zeolite hemostatic agent achieves 100% survival in a lethal model of complex groin injury in Swine.

BACKGROUND: Techniques for better hemorrhage control after injury could change outcome. We have previously shown that a zeolite mineral hemostatic agent (ZH) can control aggressive bleeding through adsorption of water, which is an exothermic process. Increasing the residual moisture content (RM) of ZH can theoretically decrease heat generation, but its effect on the hemostatic properties is unknown. We tested ZH with increasing RM against controls and other hemostatic agents in a swine model of battlefield injury. METHODS: A complex groin injury was created in 72 swine (37 +/- 0.8 kg). This included semitransection of the proximal thigh and complete division of the femoral artery and vein. After 3 minutes, the animals were randomized to 1 of 10 groups: group 1, no dressing (ND); group 2, standard dressing (SD); group 3, SD + 3.5 oz ZH with 1% RM (1% ZH); group 4, SD + 3.5 oz ZH with 4% RM (4% ZH); group 5, SD + 2 oz ZH with 1% RM (1% ZH 2oz); group 6, SD + 3.5 oz ZH with 8% RM (8% ZH); group 7, SD + chitosan-based hemostat, HemCon (HC); group 8, SD + 3.5 oz nonzeolite mineral hemostat, Quick Relief (NZH); group 9, SD + bovine clotting factors-based hemostat, Fast Act (FA); and group 10, SD + 30 g of starch-based hemostat, TraumaDex (TDex). Resuscitation (500 mL of Hespan over 30 minutes) was started 15 minutes after injury and hemodynamic monitoring was performed for 180 minutes. Primary endpoints were survival for 180 minutes and blood loss. In addition, maximum wound temperatures were recorded, and histologic damage to artery, vein, nerve, and muscle was documented. RESULTS: Use of 1% ZH decreased blood loss and reduced mortality to 0% (p < 0.05). Increasing the RM adversely affected efficacy without any significant decrease in wound temperatures. Minimal histologic tissue damage was seen with ZH independent of the percentage of RM. CONCLUSION: The use of zeolite hemostatic agent (1% residual moisture, 3.5 oz) can control hemorrhage and dramatically reduce mortality from a lethal groin wound.