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A diagnosis of pancreatic cancer carries a 5-y survival rate of less than 10%. Furthermore, the detection of pancreatic cancer occurs most often in later stages of the disease due to its location in the retroperitoneum and lack of symptoms (in most cases) until tumors become more advanced. Once diagnosed, cross-sectional imaging techniques are heavily utilized to determine the tumor stage and the potential for surgical resection. However, a major determinant of resectability is the extent of local vascular involvement of the mesenteric vessels and critical tributaries; current imaging techniques have limited capacity to accurately determine vascular involvement. Surrounding inflammation and fibrosis can be difficult to discriminate from viable tumor, making determination of the degree of vascular involvement unreliable. New innovations in fluorescence and optoacoustic imaging techniques may overcome these limitations and make determination of resectability more accurate. These imaging modalities are able to more clearly discern between viable tumor tissue and non-neoplastic inflammation or desmoplasia, allowing clinicians to more reliably characterize vascular involvement and develop individualized treatment plans for patients. This review will discuss the current imaging techniques used to diagnose pancreatic cancer, the barriers that current techniques raise to accurate staging, and novel fluorescence and optoacoustic imaging techniques that may provide more accurate clinical staging of pancreatic cancer.

Trauma remains a leading cause of morbidity and mortality among all age groups in the United States. Hemorrhagic shock and traumatic brain injury (TBI) are major causes of preventable death in trauma. Initial treatment involves fluid resuscitation to improve the intravascular volume. Although crystalloids may provide volume expansion, they do not have any pro-survival properties. Furthermore, aggressive fluid resuscitation can provoke a severe inflammatory response and worsen clinical outcomes. Due to logistical constraints, however, definitive resuscitation with blood products is often not feasible in the pre-hospital setting – highlighting the importance of adjunctive therapies. In recent years, histone deacetylase inhibitors (HDACis) have shown promise as pharmacologic agents for use in both trauma and sepsis. In this review, we discuss the role of histone deacetylases (HDACs) and pharmacologic agents that inhibit them (HDACis). We also highlight the therapeutic effects and mechanisms of action of HDACis in hemorrhagic shock, TBI, polytrauma, and sepsis. With further investigation and translation, HDACis have the potential to be a high-impact adjunctive therapy to traditional resuscitation.

BACKGROUND Trauma is a leading cause of death, and traumatic brain injury is one of the hallmark injuries of current military conflicts. Valproic acid (VPA) administration in high doses (300-400 mg/kg) improves survival in lethal trauma models, but effectiveness of lower doses on survival is unknown. This information is essential for properly designing the upcoming clinical trials. We, therefore, performed the current study to determine the lowest dose at which VPA administration improves survival in a model of lethal injuries. METHODS Swine were subjected to traumatic brain injury (10-mm cortical impact), 40% blood volume hemorrhage, and multiple trauma (femur fracture, rectus crush, and Grade V liver laceration). After 1 hour of shock, animals were randomized (n = 6/group) to four groups: normal saline (NS) resuscitation; or NS with VPA doses of 150 mg/kg (VPA 150) or 100 mg/kg (VPA 100) administered over 3 hours or 100 mg/kg over 2 hours (VPA 100 over 2 hours). Three hours after shock, packed red blood cells were given, and animals were monitored for another 4 hours. Survival was assessed using Kaplan-Meier and log-rank test. RESULTS Without resuscitation, all of the injured animals died within 5 hours. Similar survival rates were observed in the NS (17%) and VPA 100 (0%) resuscitation groups. Survival rates in the 100-mg/kg VPA groups were significantly (p < 0.05) better when it was given over 2 hours (67%) compared to 3 hours (0%). 83% of the animals in the VPA 150 group survived, which was significantly higher than the NS and VPA 100 over 3 hours groups (p < 0.05). CONCLUSION A single dose of VPA (150 mg/kg) significantly improves survival in an otherwise lethal model of multiple injuries. This is a much lower dose than previously shown to have a survival benefit and matches the dose that is tolerated by healthy human subjects with minimal adverse effects. LEVEL OF EVIDENCE Therapeutic, level V.

Background Intestinal inflammation is a mediator of multiorgan failure in trauma. We have previously shown that histone deacetylase (HDAC6) inhibitors, including ACY1083, improve survival and preserve intestinal tight junction integrity in a rodent model of hemorrhagic shock (HS). However, mechanisms leading to this alleviation in intestinal injury remain poorly defined. In this study, we sought to determine whether HDAC6 inhibition by ACY1083 can attenuate intestinal inflammation and apoptosis in rats subjected to HS. Methods Sprague Dawley rats were subjected to hemorrhage (40% of total blood volume) followed by intravenous injection of either ACY1083 (30 mg/kg) dissolved in cyclodextrin or cyclodextrin only (vehicle group). Three hours after hemorrhage, blood samples were collected, and small bowel was harvested. Histological effects of ACY1083 on small bowel were examined. Myeloperoxidase (MPO) levels were assessed as a marker for neutrophil infiltration. Whole cell lysates were analyzed for acetylated α-tubulin, metalloproteinase (ADAM) 17, TNF-α, IL-6, and cleaved caspase 3 using Western blot. The levels of ADAM17, TNF-α, and IL-6 in serum were also examined using enzyme-linked immunosorbent assay. Results ACY1083 treatment significantly attenuated HS-induced intestinal injury and MPO production. Both systemic and intestinal TNF-α and IL-6 levels were attenuated following ACY1083 administration. Increased acetylation of α-tubulin was observed in rats treated with ACY1083, along with a significantly decreased expression of cleaved caspase 3 following hemorrhage. Conclusion Inhibition of HDAC6 with ACY1083 provides intestinal protection by attenuating both the inflammatory and apoptotic responses during HS.

BACKGROUND: The use of partial resuscitative endovascular balloon occlusion of the aorta (pREBOA) in combined hemorrhagic shock (HS) and traumatic brain injury (TBI) has not been well studied. We hypothesized that the use of pREBOA in the setting of TBI would be associated with worse clinical outcomes. METHODS: Female Yorkshire swine were randomized to the following groups: HS + TBI; HS + TBI + pREBOA; and HS + pREBOA, (n=5/cohort). Animals in the HS + TBI group were left in shock for a total of 2 hours, whereas animals assigned to pREBOA groups were treated with supraceliac pREBOA deployment (60 minutes) 1 hour into the shock period. All animals were then resuscitated, and physiologic parameters were monitored for six hours. Further fluid resuscitation and vasopressors were administered as needed. At the end of the observation period, brain hemispheric swelling (%) and lesion size (mm(3)) were assessed. RESULTS: Mortality was highest in the HS + TBI + pREBOA group (40% [2/5] vs 0% [0/5] in the other groups, p = 0.1). Severity of shock was greatest in the HS + TBI + pREBOA group, as defined by peak lactate levels and pH nadir (p < 0.05). Fluid resuscitation and norepinephrine requirements were significantly higher in the HS + TBI + pREBOA group (p < 0.05). No significant differences were noted in brain hemispheric swelling and lesion size between the groups. CONCLUSIONS: Prolonged application of pREBOA in the setting of TBI does not contribute to early worsening of brain lesion size and edema. However, the addition of TBI to HS + pREBOA may worsen the severity of shock. Providers should be aware of the potential physiologic sequelae induced by TBI. LEVEL OF EVIDENCE: not applicable (preclinical study).

Nonselective histone deacetylase (pan-HDAC) inhibitors, such as valproic acid (VPA), have demonstrated neuroprotective properties in trauma models. However, isoform-specific HDAC inhibitors may provide opportunity for more effective drug administration with fewer adverse effects. We investigated HDAC6 inhibition with ACY-1083 in an in vitro and an in vivo large animal model of injury.Mouse hippocampal cells were subjected to oxygen-glucose deprivation (0% O2, glucose-free and serum-free medium, 18 hours) and reoxygenation (21% O2, normal culture media, 4 hours) with/without VPA (4 mmol/L) or ACY-1083 (30 nmol/L, 300 nmol/L). Cell viability was measured by methylthiazolyl tetrazolium assay. Expression of hypoxia-inducible factor-1α, heat shock protein 70, and effectors in the phosphoinositide-3 kinase/mammalian target of rapamycin pathway were measured by Western blot analysis. Additionally, swine were subjected to combined traumatic brain injury and hemorrhagic shock and randomized to three treatment groups (n = 5/group): (i) normal saline (NS; 3× hemorrhage volume); (ii) NS + VPA (NS; 3× hemorrhage volume, VPA; 150 mg/kg), and (iii) NS + ACY-1083 (NS; 3× hemorrhage volume, ACY-1083; 30 mg/kg). After 6 hours, brain tissue was harvested to assess lesion size and brain swelling.Significant improvement in cell viability was seen with both HDAC inhibitors in the in vitro study. ACY-1083 suppressed hypoxia-inducible factor-1α expression and up-regulated phosphorylated mammalian target of rapamycin and heat shock protein 70 in a dose-dependent manner. Lesion size and brain swelling in animals treated with pharmacologic agents (VPA and ACY-1083) were both smaller than in the NS group. No differences were observed between the VPA and ACY-1083 treatment groups.In conclusion, selective inhibition of HDAC6 is as neuroprotective as nonselective HDAC inhibition in large animal models of traumatic brain injury and hemorrhagic shock.

OBJECTIVE Traumatic brain injury (TBI) is a leading cause of trauma-related morbidity and mortality. Valproic acid (VPA) has been shown to attenuate brain lesion size and swelling within the first few hours following TBI. Because injured neurons are sensitive to metabolic changes, we hypothesized that VPA treatment would alter the metabolic profile in the perilesional brain tissues to create a neuroprotective environment. METHODS We subjected swine to combined TBI (12-mm cortical impact) and hemorrhagic shock (40% blood volume loss and 2 hours of hypotension) and randomized them to two groups (n = 5/group): (1) normal saline (NS; 3× hemorrhage volume) and (2) NS-VPA (NS, 3× hemorrhage volume; VPA, 150 mg/kg). After 6 hours, brains were harvested, and 100 mg of the perilesional tissue was used for metabolite extraction. Samples were analyzed using reversed-phase liquid chromatography-mass spectrometry in positive and negative ion modes, and data were analyzed using MetaboAnalyst software (McGill University, Quebec, Canada). RESULTS In untargeted reversed-phase liquid chromatography-mass spectrometry analysis, we detected 3,750 and 1,955 metabolites in positive and negative ion modes, respectively. There were no significantly different metabolites in positive ion mode; however, 167 metabolite features were significantly different (p < 0.05) in the negative ion mode, which included VPA derivates. Pathway analysis showed that several pathways were affected in the treatment group, including the biosynthesis of unsaturated fatty acids (p = 0.001). Targeted amino acid analysis on glycolysis/tricarboxylic acid (TCA) cycle revealed that VPA treatment significantly decreased the levels of the excitotoxic amino acid serine (p = 0.001). CONCLUSION Valproic acid can be detected in perilesional tissues in its metabolized form. It also induces metabolic changes in the brains within the first few hours following TBI to create a neuroprotective environment.

Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) remains a leading cause of preventable death worldwide. Mesenchymal stem cell-derived exosomes have demonstrated promise in small animal models of neurologic injury. To investigate the effects of exosome treatment in a clinically realistic large animal model, Yorkshire swine underwent TBI and HS. Animals were maintained in shock for 2 h before resuscitation with normal saline (NS). Animals were then resuscitated either with NS (3 × volume of shed blood) or with the same volume of NS with delayed exosome administration (1 × 10

BACKGROUND Hemorrhage is a leading preventable cause of death. Nonselective histone deacetylase inhibitors (HDACIs), such as valproic acid (VPA), have been shown to improve outcomes in hemorrhagic shock (HS). The HDACs can be divided into four functional classes (I, IIa/IIb, III, and IV). Classes I, IIa/IIb, and III have previously been implicated in the pathophysiology of HS. This study aimed to determine which HDAC class, or classes, are responsible for the survival benefit observed with nonselective HDACIs. METHODS Survival study: Sprague-Dawley rats were subjected to lethal HS (50% hemorrhage) and randomized to the following groups (n = 8): (1) no treatment, (2) normal saline vehicle, (3) cyclodextrin vehicle, (4) MS275 (class I HDACI), (5) VPA (class I/IIa HDACI), (6) MC1568 (class IIa HDACI), (7) ACY1083 (class IIb HDACI), and (8) EX527 (class III HDACI). Survival was monitored for 24 hours. Mechanistic study: Sprague-Dawley rats were subjected to sublethal HS (40% hemorrhage) and randomized to the same groups (n = 3), excluding EX527, based on results of the survival study. Tissues were harvested at 3 hours posttreatment, and expression of phosphorylated-AKT, β-catenin, acetylated histones H3 and H4, and acetylated α-tubulin were analyzed in myocardial tissue. RESULTS Survival rate was 12.5% in the untreated group, and did not improve with vehicle or MS275 treatment. EX527 improved survival to 50%, although this did not achieve statistical significance (p = 0.082). However, treatment with VPA, MC1568, and ACY1083 improved survival rates to 87.5%, 75%, and 75%, respectively (p < 0.05). The VPA-induced acetylation of both histones H3 and H4, while MC1568 and ACY1083 increased acetylation of histone H4. ACY1083 also induced acetylation of α-tubulin. All treatment groups, except MS275, increased phosphorylated-AKT, and β-catenin. CONCLUSION Inhibition of HDAC classes IIa or IIb, but not class I, activates prosurvival pathways, which may be responsible for the improved outcomes in rodent models of HS.

Background Early treatment with valproic acid (VPA) has demonstrated benefit in preclinical models of traumatic brain injury, including smaller brain lesion size, decreased edema, reduced neurologic disability, and faster recovery. Mechanisms underlying these favorable outcomes are not fully understood. We hypothesized that VPA treatment would upregulate genes involved in cell survival and proliferation and downregulate those associated with cell death and the inflammatory response. Methods Ten female swine were subjected to a protocol of traumatic brain injury and hemorrhagic shock. They were assigned to two groups (n = 5): normal saline (NS; 3× volume of shed blood), or NS + VPA (150 mg/kg). Following 6 hours of observation, brain tissue was harvested to evaluate lesion size and edema. Brain tissue was processed for RNA sequencing. Gene set enrichment and pathway analysis was performed to determine the differential gene expression patterns following injury. Results Animals treated with VPA were noted to have a 46% reduction in brain lesion size and a 57% reduction in ipsilateral brain edema. Valproic acid significantly upregulated genes involved in morphology of the nervous system, neuronal development and neuron quantity. The VPA treatment downregulated pathways related to apoptosis, glial cell proliferation, and neuroepithelial cell differentiation. Ingenuity Pathway Analysis identified VPA as the top upstream regulator of activated transcription, supporting it as a direct cause of these transcriptional changes. Master transcriptional regulator NEUROD1 was also significantly upregulated, suggesting that VPA may induce additional transcription factors. Conclusion Administration of VPA attenuated brain lesion size, reduced brain edema, and induced significant changes in the transcriptome of injured brain within 6 hours. Patterns of differential expression were consistent with the proposed neurogenic and prosurvival effects of VPA treatment.

We have previously shown that treatment with valproic acid (VPA) decreases brain lesion size in swine models of traumatic brain injury (TBI) and controlled hemorrhage. To translate this treatment into clinical practice, validation of drug efficacy and evaluation of pharmacologic properties in clinically realistic models of injury are necessary. In this study, we evaluate neurologic outcomes and perform pharmacokinetic analysis of a single dose of VPA in swine subjected to TBI, hemorrhagic shock, and visceral hemorrhage.Yorkshire swine (n = 5/cohort) were subjected to TBI, hemorrhagic shock, and polytrauma (liver and spleen injury, rib fracture, and rectus abdominis crush). Animals remained in hypovolemic shock for 2 hours before resuscitation with isotonic sodium chloride solution (ISCS; volume = 3× hemorrhage) or ISCS + VPA (150 mg/kg). Neurologic severity scores were assessed daily for 30 days, and brain lesion size was measured via magnetic resonance imaging on postinjury days (PID) 3 and 10. Serum samples were collected for pharmacokinetic analysis.Shock severity and response to resuscitation were similar in both groups. Valproic acid-treated animals demonstrated significantly less neurologic impairment between PID 1 to 5 and smaller brain lesions on PID 3 (mean lesion size ± SEM, mm: ISCS = 4,956 ± 1,511 versus ISCS + VPA = 828 ± 279; p = 0.047). No significant difference in lesion size was identified between groups at PID 10 and all animals recovered to baseline neurologic function during the 30-day observation period. Animals treated with VPA had faster neurocognitive recovery (days to initiation of testing, mean ± SD: ISCS = 6.2 ± 1.6 vs ISCS + VPA = 3.6 ± 1.5; p = 0.002; days to task mastery: ISCS = 7.0 ± 1.0 vs ISCS + VPA = 4.8 ± 0.5; p = 0.03). The mean ± SD maximum VPA concentrations, area under the curve, and half-life were 145 ± 38.2 mg/L, 616 ± 150 hour·mg/L, and 1.70 ± 0.12 hours.In swine subjected to TBI, hemorrhagic shock, and polytrauma, VPA treatment is safe, decreases brain lesion size, and reduces neurologic injury compared to resuscitation with ISCS alone. These benefits are achieved at clinically translatable serum concentrations of VPA.Therapeutic (preclinical study).

BACKGROUND Although damage control resuscitation (DCR) is routinely performed for short durations, prolonged DCR may be required in military conflicts as a component of prolonged field care. Valproic acid (VPA) has been shown to have beneficial properties in lethal hemorrhage/trauma models. We sought to investigate whether the addition of a single dose of VPA to a 72-hour prolonged DCR protocol would improve clinical outcomes. METHODS Fifteen Yorkshire swine (40-45 kg) were subjected to lethal (50% estimated total blood volume) hemorrhagic shock (HS) and randomized to three groups: (1) HS, (2) HS-DCR, (3) HS-DCR-VPA (150 mg/kg over 3 hours) (n = 5/cohort). In groups assigned to receive DCR, Tactical Combat Casualty Care guidelines were applied (1 hour into the shock period), targeting a systolic blood pressure of 80 mm Hg. At 72 hours, surviving animals were given transfusion of packed red blood cells, simulating evacuation to higher echelons of care. Survival rates, physiologic parameters, resuscitative fluid requirements, and laboratory profiles were used to compare the clinical outcomes. RESULTS This model was 100% lethal in the untreated animals. DCR improved survival to 20%, although this was not statistically significant. The addition of VPA to DCR significantly improved survival to 80% (p < 0.01). The VPA-treated animals also had significantly (p < 0.05) higher systolic blood pressures, lower fluid resuscitation requirements, higher hemoglobin levels, and lower creatinine and potassium levels. CONCLUSION VPA administration improves survival, decreases resuscitation requirements, and improves hemodynamic and laboratory parameters when added to prolonged DCR in a lethal hemorrhage model.

A single-dose (150 mg/kg) of valproic acid (VPA) has been shown to decrease brain lesion size and improve neurologic recovery in preclinical models of traumatic brain injury (TBI). However, the longer-term (30 days) impact of single-dose VPA treatment after TBI has not been well evaluated.Yorkshire swine were subjected to TBI (cortical impact), hemorrhagic shock, and polytrauma. Animals remained in hypovolemic shock for 2 hours before resuscitation with normal saline (NS; volume = 3× hemorrhaged volume) or NS + VPA (150 mg/kg) (n = 5/cohort). Brain samples were harvested 30 days after injuries. The cerebral cortex adjacent to the site of cortical impact was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunohistochemistry, and Western blot analysis. Neural apoptosis, inflammation, degeneration, plasticity, and signaling pathways were evaluated.For apoptosis, VPA treatment significantly decreased (p0.05) the number of TUNEL (+) cells and expression of cleaved-caspase 3. For inflammation and degeneration, expression of ionized calcium binding adaptor molecule-1, glial fibrillary acid protein, amyloid-β, and phosphorylated-Tau protein were significantly attenuated (p0.05) in the VPA-treated animals compared with the NS group. For, plasticity, VPA treatment also increased expression of brain-derived neurotrophic factor significantly (p0.05) compared with the NS group. For signaling pathways, nuclear factor-κB was decreased significantly (p0.05) and cytosolic IκBα expression was increased significantly (p0.05) in the VPA-treated animals compared with the NS group.Administration of a single dose of VPA (150 mg/kg) can decrease neural apoptosis, inflammation, and degenerative changes, and promote neural plasticity at 30 days after TBI. In addition, VPA acts, in part, via regulation of nuclear factor-κB and IκBα pathways.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

We have found earlier that Tubastatin A (TubA), a selective inhibitor of histone deacetylase 6 (HDAC6), improves survival in a mouse model of lethal cecal ligation and puncture (CLP)-induced sepsis. However, the underlying mechanisms have not been fully established. This study sought to test the hypothesis that TubA could affect both lung and splenic function. C57BL/6J mice were subjected to CLP, and randomized to receive either TubA (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO), or DMSO alone, 1 hour following CLP. Sham animals acted as a control. Twenty-four hours later, lung tissue was harvested for pathological examination, and splenic tissue was harvested for bacterial colonization. In a parallel study, the spleen was collected 48 hours following CLP, and single cell suspension was prepared. Splenocytes then underwent flow cytometry to analyze the immune cell population. RAW264.7 macrophages were treated with lipopolysaccharide (LPS) with or without the presence of TubA (10 μΜ) at 37°C for 3h to assess the effect on macrophage phagocytosis. We found that acute lung injury secondary to lethal sepsis was attenuated by TubA. Treatment with TubA restored the percentage of B lymphocytes, and significantly increased percentages of innate immune cells and macrophages compared to the vehicle-treated CLP group. Moreover, TubA significantly decreased the bacterial load in spleen, and improved the phagocytic ability of RAW264.7 murine macrophages in vitro. Such findings may help to explain the beneficial effects of TubA treatment in a model of lethal sepsis, as previously reported.

The primary aim of this study was to examine the effects of valproic acid (VPA) treatment on serum glial fibrillary acidic protein (GFAP) and neurofilament light chain (NF-L) levels. To achieve this aim, we obtained serum samples from: 1) 10 Yorkshire swine subjected to controlled cortical impact traumatic brain injury (CCI TBI) + polytrauma and randomized to receive either normal saline (NS) + VPA (n = 5) or NS alone (n = 5) and 2) five additional swine subjected to CCI TBI without polytrauma and treated with VPA. GFAP and NF-L levels were measured in samples obtained from baseline until 10 days post-injury using a digital immunoassay from Quanterix Corporation. We found that elevated GFAP and NF-L levels were first detected at 2 h post-injury; and peaked at 24 h and 72 h respectively. GFAP levels returned to baseline levels by Day 10, while NF-L remained elevated at Day 10. In TBI + polytrauma swine, the magnitude and duration of biomarker elevation, quantified by the area under the biomarker-concentration-versus-time curve during the first 10 days (AUC

Combined traumatic brain injury and hemorrhagic shock are highly lethal. Following injuries, the integrity of the blood-brain barrier can be impaired, contributing to secondary brain insults. The status of the blood-brain barrier represents a potential factor impacting long-term neurologic outcomes in combined injuries. Treatment strategies involving plasma-based resuscitation and valproic acid therapy have shown efficacy in this setting. We hypothesize that a component of this beneficial effect is related to blood-brain barrier preservation.Following controlled traumatic brain injury, hemorrhagic shock, various resuscitation and treatment strategies were evaluated for their association with blood-brain barrier integrity. Analysis of gene expression profiles was performed using Porcine Gene ST 1.1 microarray. Pathway analysis was completed using network analysis tools (Gene Ontology, Ingenuity Pathway Analysis, and Parametric Gene Set Enrichment Analysis).Female Yorkshire swine were subjected to controlled traumatic brain injury and 2 hours of hemorrhagic shock (40% blood volume, mean arterial pressure 30-35 mmHg).Subjects were resuscitated with 1) normal saline, 2) fresh frozen plasma, 3) hetastarch, 4) fresh frozen plasma + valproic acid, or 5) hetastarch + valproic acid (n = 5 per group). After 6 hours of observation, brains were harvested for evaluation.Immunofluoroscopic evaluation of the traumatic brain injury site revealed significantly increased expression of tight-junction associated proteins (zona occludin-1, claudin-5) following combination therapy (fresh frozen plasma + valproic acid and hetastarch + valproic acid). The extracellular matrix protein laminin was found to have significantly improved expression with combination therapies. Pathway analysis indicated that valproic acid significantly modulated pathways involved in endothelial barrier function and cell signaling.Resuscitation with fresh frozen plasma results in improved expression of proteins essential for blood-brain barrier integrity. The addition of valproic acid provides significant improvement to these protein expression profiles. This is likely secondary to activation of key pathways related to endothelial functions.

Background Histone deacetylase inhibitors such as valproic acid (VPA) improve survival in lethal models of hemorrhagic shock and polytrauma. Although VPA is known to modulate transcription, its ability to reduce mortality within minutes of administration suggests involvement of a rapid, posttranslational mechanism. We hypothesized that VPA treatment would cause proteomic changes within minutes of treatment including quantitative and/or posttranslational differences in structural and/or effector proteins. Materials and methods We used a porcine model of traumatic brain injury (computer-controlled cortical impact, 12 mm depth) and hemorrhagic shock (40% hemorrhage). Animals were kept in shock for 2 h and randomized to two groups (n = 3): normal saline (volume = 3:1 hemorrhage volume) or normal saline + VPA (150 mg/kg, single dose). Peripheral blood mononuclear cells were collected at baseline, postshock, and postresuscitation. Intracellular protein profiles were assessed using 1 dimensional gel electrophoresis, liquid chromatography, mass spectrometry, and analyzed with Ingenuity Pathway Analysis software. Results Animals treated with VPA demonstrated significant proteomic changes. Quantitative differences were found in over 200 proteins including effector, regulatory, and structural proteins in critical cell signaling pathways. Posttranslational modification analysis demonstrated differential VPA-induced acetylation of lysine residues in histone and nonhistone proteins. Pathway analysis correlated these changes with significant increases in numerous prosurvival and cytoskeletal intracellular pathways, including Rho GTPase signaling (P = 1.66E-11), integrin signaling (P = 4.19E-21), and a decrease in Rho guanosine nucleotide dissociation inhibitor signaling (P = 4.83E-12). Conclusions In a porcine model of severe injuries, a single dose of VPA is associated with protective changes in the proteome that are measurable within minutes of treatment.

Hemorrhage is a common cause of death in the battlefield. Valproic acid (VPA) has been associated with improved outcomes in multiple models of trauma, when combined with isotonic fluid resuscitation. However, isotonic fluid administered in this setting is logistically impractical and may be associated with complications. In this study, we sought to evaluate the feasibility and immunologic impact of combining VPA treatment with low-volume hypertonic saline (HTS). In vivo: female Yorkshire swine were subjected to hemorrhage (40% total blood volume) and polytrauma (rib fracture and delayed liver injury). Animals were kept in shock for 30 minutes and resuscitated with (1) normal saline (NS, 3× hemorrhaged volume), (2) HTS (7.5% saline, 4 mL/kg), or (3) HTS + VPA (4 mg/kg; 150 mg/kg; n = 3/cohort). After 18 hours of observation, animals were euthanized and the lungs evaluated for acute injury and expression of myeloperoxidase (MPO) and caveolin-1 (Cav-1). In vitro: human umbilical vein endothelial cells (HUVECs) were exposed to anoxic conditions (5% CO2, 95% N2) for 16 hours in (1) normosmotic, (2) hyperosmotic (400 mOsm), or (3) hyperosmotic + VPA (4 mM) media. Immunohistochemistry and Western blots were performed to determine Cav-1 expression. Lungs from VPA-treated animals demonstrated decreased acute injury, MPO expression, and endothelial expression of Cav-1 when compared to lungs from animals resuscitated with NS or HTS alone. Similarly, HUVECs cultured in hyperosmotic media containing VPA demonstrated decreased expression of Cav-1. This study demonstrates that combined treatment with VPA and HTS is a viable strategy in hemorrhagic shock and polytrauma. Attenuation of lung injury following VPA treatment may be related to modulation of the inflammatory response.