A functional microbiome catalog crowdsourced from North American rivers.

Predicting elemental cycles and maintaining water quality under increasing anthropogenic influence requires understanding the spatial drivers of river microbiomes. However, the unifying microbial processes governing river biogeochemistry are hindered by a lack of genome-resolved functional insights and sampling across multiple rivers. Here we employed a community science effort to accelerate the sampling, sequencing, and genome-resolved analyses of river microbiomes to create the Genome Resolved Open Watersheds database (GROWdb). This resource profiled the identity, distribution, function, and expression of thousands of microbial genomes across rivers covering 90% of United States watersheds. Specifically, GROWdb encompasses 1,469 microbial species from 27 phyla, including novel lineages from 10 families and 128 genera, and defines the core river microbiome for the first time at genome level. GROWdb analyses coupled to extensive geospatial information revealed local and regional drivers of microbial community structuring, while also presenting a myriad of foundational hypotheses about ecosystem function. Building upon the previously conceived River Continuum Concept 1 , we layer on microbial functional trait expression, which suggests the structure and function of river microbiomes is predictable. We make GROWdb available through various collaborative cyberinfrastructures 2, 3 so that it can be widely accessed across disciplines for watershed predictive modeling and microbiome-based management practices.

Nontarget Chemical Composition of Surface Waters May Reflect Ecosystem Processes More than Discrete Source Contributions.

We investigated environmental, landscape, and microbial factors that could structure the spatiotemporal variability in the nontarget chemical composition of four riverine systems in the Oregon Coast Range, USA. We hypothesized that the nontarget chemical composition in river water would be structured by broad-scale landscape gradients in each watershed. Instead, only a weak relationship existed between the nontarget chemical composition and land cover gradients. Overall, the effects of microbial communities and environmental variables on chemical composition were nearly twice as large as those of the landscape, and much of the influence of environmental variables on the chemical composition was mediated through the microbial community (i.e., environment affects microbes, which affect chemicals). Therefore, we found little evidence to support our hypothesis that chemical spatiotemporal variability was related to broad-scale landscape gradients. Instead, we found qualitative and quantitative evidence to suggest that chemical spatiotemporal variability of these rivers is controlled by changes in microbial and seasonal hydrologic processes. While the contributions of discrete chemical sources are undeniable, water chemistry is undoubtedly impacted by broad-scale continuous sources. Our results suggest that diagnostic chemical signatures can be developed to monitor ecosystem processes, which are otherwise challenging or impossible to study with existing off-the-shelf sensors.

The Roles of Microbes in Stream Restorations.

The goods and services provided by riverine systems are critical to humanity, and our reliance increases with our growing population and demands. As our activities expand, these systems continue to degrade throughout the world even as we try to restore them, and many efforts have not met expectations. One way to increase restoration effectiveness could be to explicitly design restorations to promote microbial communities, which are responsible for much of the organic matter breakdown, nutrient removal or transformation, pollutant removal, and biomass production in river ecosystems. In this paper, we discuss several design concepts that purposefully create conditions for these various microbial goods and services, and allow microbes to act as ecological restoration engineers. Focusing on microbial diversity and function could improve restoration effectiveness and overall ecosystem resilience to the stressors that caused the need for the restoration. Advances in next-generation sequencing now allow the use of microbial 'omics techniques (e.g., metagenomics, metatranscriptomics) to assess stream ecological conditions in similar fashion to fish and benthic macroinvertebrates. Using representative microbial communities from stream sediments, biofilms, and the water column may greatly advance assessment capabilities. Microbes can assess restorations and ecosystem function where animals may not currently be present, and thus may serve as diagnostics for the suitability of animal reintroductions. Emerging applications such as ecological metatranscriptomics may further advance our understanding of the roles of specific restoration designs towards ecological services as well as assess restoration effectiveness.

Early Treatment With a Single Dose of Mesenchymal Stem Cell Derived Extracellular Vesicles Modulates the Brain Transcriptome to Create Neuroprotective Changes in a Porcine Model of Traumatic Brain Injury and Hemorrhagic Shock.

BACKGROUND: Cell-based therapies using mesenchymal stem cell derived extracellular vesicles (EVs) improve neurologic outcomes in animal models of traumatic brain injury (TBI), stroke, and hemorrhage. Using a porcine 7-day survival model of TBI and hemorrhagic shock (HS), we previously demonstrated that EV-treatment was associated with reduced brain lesion size, neurologic severity score, and cerebral inflammation. However, the underlying cellular and genomic mechanisms remain poorly defined. We hypothesize that EV treatment modulates the brain transcriptome to enhance neuroprotection and neurorestoration following TBI + HS. METHODS: Swine were subjected to severe TBI (8-mm cortical impact) and HS (40% blood volume). After 1 h of shock, animals were randomized (n = 4/group) to treatment with either lactated Ringer's (LR) or LR + EV. Both groups received fluid resuscitation after 2 h of shock, and autologous packed red blood cells 5 h later.After 7-days, brains were harvested and RNA-sequencing was performed. The transcriptomic data were imported into the iPathway pipeline for bioinformatics analyses. RESULTS: 5,273 genes were differentially expressed in the LR + EV group versus LR alone (total 9,588 measured genes). Genes with the greatest upregulation were involved in synaptic transmission and neuronal development and differentiation, while downregulated genes were involved in inflammation. GO-terms experiencing the greatest modulation were involved in inflammation, brain development, and cell adhesion. Pathway analysis revealed significant modulation in the glutamatergic and GABAergic systems. Network analysis revealed downregulation of inflammation, and upregulation of neurogenesis, and neuron survival and differentiation. CONCLUSIONS: In a porcine model of TBI + HS, EV treatment was associated with an attenuation of cerebral inflammatory networks and a promotion of neurogenesis and neuroplasticity. These transcriptomic changes could explain the observed neuroprotective and neurorestorative properties associated with EV treatment.

Modulation of Brain Transcriptome by Combined Histone Deacetylase Inhibition and Plasma Treatment Following Traumatic Brain Injury and Hemorrhagic Shock.

INTRODUCTION: We previously showed that the addition of valproic acid (VPA), a histone deacetylase inhibitor, to fresh frozen plasma (FFP) resuscitation attenuates brain lesion size and swelling following traumatic brain injury (TBI) and hemorrhagic shock (HS). The goal of this study was to use computational biology tools to investigate the effects of FFP+VPA on the brain transcriptome following TBI+HS. METHODS: Swine underwent TBI+HS, kept in shock for 2 h, and resuscitated with FFP or FFP + VPA (n = 5/group). After 6 h of observation, brain RNA was isolated and gene expression was analyzed using a microarray. iPathwayGuide, Gene Ontology (GO), Gene-Set Enrichment Analysis, and Enrichment Mapping were used to identify significantly impacted genes and transcriptomic networks. RESULTS: Eight hundred differentially expressed (DE) genes were identified out of a total of 9,118 genes. Upregulated genes were involved in promotion of cell division, proliferation, and survival, while downregulated genes were involved in autophagy, cell motility, neurodegenerative diseases, tumor suppression, and cell cycle arrest. Seven hundred ninety-one GO terms were significantly enriched. A few major transcription factors, such as TP53, NFKB3, and NEUROD1, were responsible for modulating hundreds of other DE genes. Network analysis revealed attenuation of interconnected genes involved in inflammation and tumor suppression, and an upregulation of those involved in cell proliferation and differentiation. CONCLUSION: Overall, these results suggest that VPA treatment creates an environment that favors production of new neurons, removal of damaged cells, and attenuation of inflammation, which could explain its previously observed neuroprotective effects.

Different resuscitation strategies and novel pharmacologic treatment with valproic acid in traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of death in young adults, and effective treatment strategies have the potential to save many lives. TBI results in coagulopathy, endothelial dysfunction, inflammation, cell death, and impaired epigenetic homeostasis, ultimately leading to morbidity and/or mortality. Commonly used resuscitation fluids such as crystalloids or colloids have several disadvantages and might even be harmful when administered in large quantities. There is a need for next-generation treatment strategies (especially in the prehospital setting) that minimize cellular damage, improve survival, and enhance neurological recovery. Pharmacologic treatment with histone deacetylase inhibitors, such as valproic acid, has shown promising results in animal studies of TBI and may therefore be an excellent example of next-generation therapy. This review briefly describes traditional resuscitation strategies for TBI combined with hemorrhagic shock and describes preclinical studies on valproic acid as a new pharmacologic agent in the treatment of TBI. It finally discusses limitations and future directions on the use of histone deacetylase inhibitors for the treatment of TBI.

Improvement of Blood-Brain Barrier Integrity in Traumatic Brain Injury and Hemorrhagic Shock Following Treatment With Valproic Acid and Fresh Frozen Plasma.

OBJECTIVE: 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. DESIGN: 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). SUBJECTS: 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). INTERVENTIONS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

Valproic acid modulates platelet and coagulation function ex vivo.

: Trauma-induced coagulopathy is associated with adverse patient outcome. Animal models demonstrate that histone deacetylase inhibitors, such as valproic acid (VPA), improve survival following injury. While in-vivo data suggest that improved survival may in part be because of an attenuation of coagulopathy, it remains unknown whether this is a direct effect of the drug, or the establishment of an overall prosurvival phenotype. We thus conducted an ex-vivo experiment to determine if VPA has an effect on coagulation and platelet function. Ten swine were subjected to traumatic brain injury (TBI) and hemorrhagic shock (HS). Blood samples were drawn prior to TBI+HS insult (Healthy group) and 2 h following TBI+HS (Shock group). Samples were incubated with VPA or vehicle controls for 1 h. Platelet aggregation was analyzed via impedance aggregometry and coagulation was measured using thromboelastography. Addition of VPA to the healthy blood did not affect platelet aggregation or coagulation parameters. In shock blood, incubation with VPA significantly reduced collagen-(P = 0.050), arachidonic acid-(P = 0.005), and adenosine diphosphate-(P = 0.023) induced platelet aggregation. VPA also significantly increased the clot strength (P = 0.002) and clot formation rate (P = 0.011). This is the first study to investigate the effect of VPA on platelet function ex vivo. Our results suggest that VPA has no effect on normal blood, but it decreases platelet activation and improves clot dynamics (strength and rate of formation) in blood from shocked animals. This suggests that VPA is capable of exerting a selective platelet sparing effect while enhancing the clot integrity.

Fresh Frozen Plasma Modulates Brain Gene Expression in a Swine Model of Traumatic Brain Injury and Shock: A Network Analysis.

BACKGROUND: Resuscitation with fresh frozen plasma (FFP) decreases brain lesion size and swelling in a swine model of traumatic brain injury and hemorrhagic shock. We hypothesized that brain gene expression profiles after traumatic brain injury and hemorrhagic shock would be modulated by FFP resuscitation. STUDY DESIGN: Fifteen swine underwent a protocol of traumatic brain injury and hemorrhagic shock and 2 hours of shock followed by resuscitation with FFP, normal saline, or hetastarch (5/group). After 6 hours, brain RNA was isolated and hybridized onto a porcine gene ST 1.1 microarray. Weighted gene correlation network analysis was used to identify clusters of highly coexpressed genes. Principal component analysis identified cluster eigenvectors, indicating overall direction and magnitude of cluster gene expression. Using linear regression, cluster eigenvectors were associated with treatment as well as brain lesion size and swelling. Results were post-hoc corrected using false discovery rate. Relevant gene clusters were subjected to pathway analysis using the Reactome tool. RESULTS: Network analysis identified 322 gene expression clusters (total of 12,462 coexpressed genes). Fresh frozen plasma resuscitation (but not normal saline or hetastarch) was positively associated with 2 distinct gene clusters (termed A and B) comprising 493 genes. Gene expression in both clusters was negatively associated with brain swelling, and cluster B was also negatively associated with lesion size. Pathway analysis revealed an upregulation of genes involved in metabolic and platelet signaling, as well as collagen formation and downregulation of inflammation. CONCLUSIONS: Fresh frozen plasma resuscitation in this model was associated with downregulation of inflammatory pathway genes and expression of gene clusters mapping to increased metabolic and platelet signaling, which, in turn, was reversely associated with brain swelling.

Early resuscitation with lyophilized plasma provides equal neuroprotection compared with fresh frozen plasma in a large animal survival model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In previous models of combined TBI + HS, we showed that early resuscitation with fresh frozen plasma (FFP) improves neurologic outcomes. Delivering FFP, however, in austere environments is difficult. Lyophilized plasma (LP) is a logistically superior alternative to FFP, but data are limited regarding its efficacy for treatment of TBI. We conducted this study to determine the safety and long-term outcomes of early treatment with LP in a large animal model of TBI + HS. METHODS: Adult anesthetized swine underwent TBI and volume-controlled hemorrhage (40% blood volume) concurrently. After 2 hours of shock, animals were randomized (n = 5 per /group) to FFP or LP (1× shed blood) treatment. Serial blood gases were drawn, and thromboelastography was performed on citrated, kaolin-activated whole-blood samples. Five hours after treatment, packed red blood cells were administered, and animals recovered. A 32-point Neurologic Severity Score was assessed daily for 30 days (0 = normal, 32 = most severe injury). Cognitive functions were tested by training animals to retrieve food from color-coded boxes. Brain lesion size was measured on serial magnetic resonance imaging, and an autopsy was performed at 30 days. RESULTS: The severity of shock and the degree of resuscitation were similar in both groups. Administration of FFP and LP was well tolerated with no differences in reversal of shock or thromboelastography parameters. Animals in both groups displayed the worst Neurologic Severity Score on postoperative Day 1 with rapid recovery and return to baseline within 7 days of injury. Lesion size on Day 3 in FFP-treated animals was 645 ± 85 versus 219 ± 20 mm in LP-treated animals (p < 0.05). There were no differences in cognitive functions or delayed treatment-related complications. CONCLUSIONS: Early treatment with LP in TBI + HS is safe and provides neuroprotection that is comparable to FFP.

Inhibition of histone deacetylase 6 restores intestinal tight junction in hemorrhagic shock.

BACKGROUND: We recently discovered that Tubastatin-A, a histone deacetylase (HDAC6) inhibitor, can improve survival in a rodent model of hemorrhagic shock (HS), but mechanisms remain poorly defined. In this study, we investigated whether Tubastatin-A could protect intestinal tight junction (TJ) in HS. METHODS: In an in-vivo study with Wistar-Kyoto rats, the rats underwent HS (40% blood loss) followed by Tubastatin-A (70 mg/kg) treatment, without fluid resuscitation. The experimental groups were (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, without treatment), and (3) treatment (hemorrhage with Tubastatin-A administration). Six hours after hemorrhage, ileum was harvested. Whole cell lysate were analyzed for acetylated α-tubulin (Ac-tubulin), total tubulin, acetylated histone 3 at lysine 9 (Ac-H3K9), ß-actin, claudin-3 and zonula occludens 1 (ZO-1) proteins by Western blot. Histological effects of Tubastatin-A on small bowel were examined. In an in-vitro study, human intestinal epithelial cells (Caco-2) were divided into three groups: (1) sham (normoxia), (2) control (anoxia, no treatment), and (3) treatment (anoxia, treatment with Tubastatin-A). After 12 hours in an anoxia chamber, the cells were examined for Ac-tubulin and Ac-H3K9, cellular viability, cytotoxicity, claudin-3 and ZO-1 protein expression, and transwell permeability study. RESULTS: Tubastatin-A treatment significantly attenuated HS-induced decreases of Ac-tubulin, Ac-H3K9, ZO-1 and claudin-3 proteins in small bowel in-vivo (p < 0.05). In cultured Caco-2 cells, anoxia significantly decreased cellular viability (p < 0.001) and increased cytotoxicity (p < 0.001) compared to the sham group, while Tubastatin-A treatment offered significant protection (p < 0.0001). Moreover, expression of claudin-3 was markedly decreased in vitro compared to the sham group, whereas this was significantly attenuated by Tubastatin-A (p < 0.05). Finally, anoxia markedly increased the permeability of Caco-2 monolayer cells (p < 0.05), while Tubastatin-A significantly attenuated the alteration (p < 0.05). CONCLUSION: Inhibition of HDAC6 can induce Ac-tubulin and Ac-H3K9, promote cellular viability, and prevent the loss of intestinal tight junction proteins during HS and anoxia.

Resuscitation with Valproic Acid Alters Inflammatory Genes in a Porcine Model of Combined Traumatic Brain Injury and Hemorrhagic Shock.

Traumatic brain injury and hemorrhagic shock (TBI+HS) elicit a complex inflammatory response that contributes to secondary brain injury. There is currently no proven pharmacologic treatment for TBI+HS, but modulation of the epigenome has been shown to be a promising strategy. The aim of this study was to investigate whether valproic acid (VPA), a histone deacetylase inhibitor, modulates the expression of cerebral inflammatory gene profiles in a large animal model of TBI+HS. Ten Yorkshire swine were subjected to computer-controlled TBI+HS (40% blood volume). After 2 h of shock, animals were resuscitated with Hextend (HEX) or HEX+VPA (300 mg/kg, n = 5/group). Six hours after resuscitation, brains were harvested, RNA was isolated, and gene expression profiles were measured using a porcine microarray. Ingenuity Pathway Analysis® (IPA), gene ontology (GO), Parametric Gene Set Enrichment Analysis (PGSEA), and DAVID (Database for Annotation, Visualization, and Integrated Discovery) were used for pathway analysis. Key microarray findings were verified using real-time polymerase chain reaction (PCR). IPA analysis revealed that VPA significantly down-regulated the complement system (p < 0.001), natural killer cell communication (p < 0.001), and dendritic cell maturation (p < 0.001). DAVID analysis indicated that a cluster of inflammatory pathways held the highest rank and gene enrichment score. Real-time PCR data confirmed that VPA significantly down-expressed genes that ultimately regulate nuclear factor-kB (NF-kB)-mediated production of cytokines, such as TYROBP, TREM2, CCR1, and IL-1ß. This high-throughput analysis of cerebral gene expression shows that addition of VPA to the resuscitation protocol significantly modulates the expression of inflammatory pathways in a clinically realistic model of TBI+HS.

Histone deactylase gene expression profiles are associated with outcomes in blunt trauma patients.

BACKGROUND: Treatment with histone deacetylase (HDAC) inhibitors, such as valproic acid, increases survival in animal models of trauma and sepsis. Valproic acid is a pan-inhibitor that blocks most of the known HDAC isoforms. Targeting individual HDAC isoforms may increase survival and reduce complications, but little is known of the natural history of HDAC gene expression following trauma. We hypothesized that distinct HDAC isoform gene expression patterns would be associated with differences in outcomes following trauma. METHODS: Twenty-eight-day longitudinal HDAC leukocyte gene expression profiles in 172 blunt trauma patients were extracted from the Inflammation and the Host Response to Injury (Glue Grant) data set. Outcome was classified as complicated (death or no recovery by Day 28, n = 51) or uncomplicated (n = 121). Mixed modeling was used to compare the HDAC expression trajectories between the groups, corrected for Injury Severity Score (ISS), base deficit, and volume of blood products transfused during the initial 12 hours following admission. Weighted gene correlation network analysis identified modules of genes with significant coexpression, and HDAC genes were mapped to these modules. Biologic function of these modules was investigated using the Gene Ontology database. RESULTS: Elevated longitudinal HDAC expression trajectories for HDAC1, HDAC3, HDAC6, and HDAC11 were associated with complicated outcomes. In contrast, suppressed expression of Sirtuin 3 (SIRT3) was associated with adverse outcome (p < 0.01). Weighted gene correlation network analysis identified significant coexpression of HDAC and SIRT genes with genes involved in ribosomal function and down-regulation of protein translation in response to stress (HDAC1), T-cell signaling, and T-cell selection (HDAC3) as well as coagulation and hemostasis (SIRT3). No coexpression of HDAC11 was identified. CONCLUSION: Expression trajectories of HDAC1, HDAC3, HDAC6, HDAC11, and SIRT3 correlate with outcomes following trauma and may potentially serve as biomarkers. They may also be promising targets for pharmacologic intervention. The effects of HDAC and SIRT gene expression in trauma may be mediated through pathways involved in ribosomal and T-cell function as well as coagulation and hemostasis. LEVEL OF EVIDENCE: Prognostic study, level III.

Selective inhibition of histone deacetylase 6 promotes survival in a rat model of hemorrhagic shock.

BACKGROUND: Hemorrhage is the leading cause of preventable trauma-related deaths. We have previously shown that treatment with Tubastatin A (Tub A), a histone deacetylase 6 (HDAC6) inhibitor, can improve survival in a rodent model of septic shock. The aims of the present study were to determine whether selective inhibition of HDAC6 can promote survival in a model of hemorrhagic shock (HS). METHODS: In Experiment I (survival study), Wistar-Kyoto rats were subjected to HS (55% volume blood loss), followed by intraperitoneal injection of either Tub A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO) or DMSO only (vehicle group) (n = 8 per group). Survival was monitored for 24 hours. In Experiment II (physiologic study), rats were subjected to a sublethal HS (40% blood loss), followed by the same treatment with Tub A (treatment group) or DMSO only (vehicle group, n = 5 per group). All animals were sacrificed 6 hours after hemorrhage, and the heart and liver tissues were harvested. Sham animals were not subjected to hemorrhage and treatment (sham group, n = 5 per group). Cardiac mitochondria were isolated to study the pyruvate dehydrogenase (PDH, an essential enzyme for adenosine triphosphate production) activity. Liver tissue lysates were analyzed for markers of apoptosis (cytochrome c, cleaved caspase 3) and inflammation (high-mobility group box 1) by Western blotting. RESULTS: Severe HS (55% blood loss) was associated with 75% mortality, which was significantly improved by Tub A treatment (37.5% mortality in 24 hours, p = 0.048). Tub A also significantly enhanced the cardiac PDH activity compared with the vehicle group, while suppressing the hepatic high-mobility group box 1 expression, cytochrome c release, and caspase 3 activation. CONCLUSION: Our study has demonstrated for the first time that selective inhibition of HDAC6 can improve survival in a rodent model of HS. The potential mechanisms include enhanced PDH activity, decreased inflammatory drive, and attenuated cellular apoptosis.

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.

Early resuscitation with fresh frozen plasma for traumatic brain injury combined with hemorrhagic shock improves neurologic recovery.

BACKGROUND: We have shown that early administration of fresh frozen plasma (FFP) reduces the size of brain lesions 6 hours after injury in a large animal model of traumatic brain injury (TBI) and hemorrhagic shock (HS). To examine long-term outcomes, we hypothesized that early treatment with FFP would result in faster neurologic recovery and better long-term outcomes in a combined TBI and HS model. STUDY DESIGN: Anesthetized Yorkshire swine underwent combined TBI and volume-controlled hemorrhage (40% blood volume). After 2 hours of shock, animals were randomized (n = 5/group) to normal saline (3× shed blood) or FFP (1× shed blood) treatment. A neurologic severity score was assessed for 30 days. Magnetic resonance imaging of the brain was performed at days 3, 10, and 24. Cognitive function was tested by training animals to retrieve food from color-coded boxes. RESULTS: Neurologic impairment was lower and speed of recovery was considerably faster in the FFP-treated animals. There was a trend toward a smaller lesion size in FFP-treated animal at days 3 and 10, but this did not reach statistical significance. Both groups reached baseline performance on the cognitive testing; however, FFP-treated animals were able to participate, on average, 8 days earlier due to quicker recovery. CONCLUSIONS: This is the first study to demonstrate the beneficial effects of FFP treatment in a long-term survival model of combined TBI and HS. Our data show that early treatment with FFP substantially attenuates the degree of neurologic impairment, improves the rate of recovery, and preserves the cognitive functions.

Hypothermia and valproic acid activate prosurvival pathways after hemorrhage.

BACKGROUND: Therapeutic hypothermia (hypo) and valproic acid (VPA, a histone deacetylase inhibitor) have independently been shown to be protective in models of trauma and hemorrhagic shock but require logistically challenging doses to be effective. Theoretically, combined treatment may further enhance effectiveness, allowing us to use lower doses of each modality. The aim of this study was to determine whether a combination of mild hypo and VPA treatments would offer better cytoprotection compared with that of individual treatments in a hemorrhage model. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to 40% volume-controlled hemorrhage, kept in shock for 30 min, and assigned to one of the following treatment groups: normothermia (36°C-37°C), hypo (30 ± 2°C), normothermia + VPA (300 mg/kg), and hypo + VPA (n = 5 per group). After 3 h of observation, the animals were sacrificed, liver tissue was harvested and subjected to whole cell lysis, and levels of key proteins in the prosurvival Akt pathway were measured using Western blot. RESULTS: Activation of the proapoptotic protein cleaved caspase-3 was significantly lower in the combined treatment group relative to normothermia (P < 0.05). Levels of the prosurvival Bcl-2 was significantly higher in the combined treatment group relative to sham, normothermia, and normothermia + VPA groups (P < 0.005). The downstream prosurvival protein phospho-GSK-3ß was significantly higher in the sham, hypo, and combined treatment groups compared with that in normothermia groups with or without VPA (P < 0.05). Levels of the prosurvival ß-catenin were significantly higher in the combined treatment group relative to normothermia (P < 0.01). CONCLUSIONS: This is the first in vivo study to demonstrate that combined treatment with VPA and hypo offers better cytoprotection than these treatments given independently.

Fresh frozen plasma resuscitation provides neuroprotection compared to normal saline in a large animal model of traumatic brain injury and polytrauma.

We have previously shown that early treatment with fresh frozen plasma (FFP) is neuroprotective in a swine model of hemorrhagic shock (HS) and traumatic brain injury (TBI). However, it remains unknown whether this strategy would be beneficial in a more clinical polytrauma model. Yorkshire swine (42-50 kg) were instrumented to measure hemodynamic parameters, brain oxygenation, and intracranial pressure (ICP) and subjected to computer-controlled TBI and multi-system trauma (rib fracture, soft-tissue damage, and liver injury) as well as combined free and controlled hemorrhage (40% blood volume). After 2 h of shock (mean arterial pressure, 30-35 mm Hg), animals were resuscitated with normal saline (NS; 3×volume) or FFP (1×volume; n=6/group). Six hours postresuscitation, brains were harvested and lesion size and swelling were evaluated. Levels of endothelial-derived vasodilator endothelial nitric oxide synthase (eNOS) and vasoconstrictor endothelin-1 (ET-1) were also measured. FFP resuscitation was associated with reduced brain lesion size (1005.8 vs. 2081.9 mm(3); p=0.01) as well as swelling (11.5% vs. 19.4%; p=0.02). Further, FFP-resuscitated animals had higher brain oxygenation as well as cerebral perfusion pressures. Levels of cerebral eNOS were higher in the FFP-treated group (852.9 vs. 816.4 ng/mL; p=0.03), but no differences in brain levels of ET-1 were observed. Early administration of FFP is neuroprotective in a complex, large animal model of polytrauma, hemorrhage, and TBI. This is associated with a favorable brain oxygenation and cerebral perfusion pressure profile as well as higher levels of endothelial-derived vasodilator eNOS, compared to normal saline resuscitation.

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.

Effect of pharmacologic resuscitation on the brain gene expression profiles in a swine model of traumatic brain injury and hemorrhage.

BACKGROUND: We have previously shown that addition of valproic acid (VPA; a histone deacetylase inhibitor) to hetastarch (Hextend [HEX]) resuscitation significantly decreases lesion size in a swine model of traumatic brain injury (TBI) and hemorrhagic shock (HS). However, the precise mechanisms have not been well defined. As VPA is a transcriptional modulator, the aim of this study was to investigate its effect on brain gene expression profiles. METHODS: Swine were subjected to controlled TBI and HS (40% blood volume), kept in shock for 2 hours, and resuscitated with HEX or HEX + VPA (n = 5 per group). Following 6 hours of observation, brain RNA was isolated, and gene expression profiles were measured using a Porcine Gene ST 1.1 microarray (Affymetrix, Santa Clara, CA). Pathway analysis was done using network analysis tools Gene Ontology, Ingenuity Pathway Analysis, and Parametric Gene Set Enrichment Analysis. Real-time polymerase chain reaction was used to verify the key microarray findings. RESULTS: A total of 1,668 probe sets mapping to 370 known genes were differentially expressed between the HEX and HEX + VPA groups. Expression of apoptotic genes differed between groups, and biologic function analysis predicted a significant downregulation of apoptosis (p = 1.29 × 10), cell death (p = 8.46 × 10), and necrosis (p = 9.07 × 10). Pathway analysis indicated a significant modulation of pathways involved in cell signaling, dendritic cell response, and the complement system. CONCLUSION: This is the first high-throughput analysis of cerebral gene profiling following TBI + HS. It shows that treatment with VPA significantly alters early transcription of pathways related to cell survival, which may explain its neuroprotective effects.