Patrick E. Georgoff

Reduced brain tissue oxygen in traumatic brain injury: Are most commonly used interventions successful?

BACKGROUND Brain tissue oxygenation (PbtO2)-guided management facilitates treatment of reduced PbtO2 episodes potentially conferring survival and outcome advantages in severe traumatic brain injury (TBI). To date, the nature and effectiveness of commonly used interventions in correcting compromised PbtO2 in TBI remains unclear. We sought to identify the most common interventions used in episodes of compromised PbtO2 and to analyze which were effective. METHODS A retrospective 7-year review of consecutive severe TBI patients with a PbtO2 monitor was conducted in a Level I trauma centers intensive care unit or neurosurgical registry. Episodes of compromised PbtO2 (defined as <20 mm Hg for 0.25-4 hours) were identified, and clinical interventions conducted during these episodes were analyzed. Response to treatment was gauged on how rapidly (ΔT) PbtO2 normalized (>20 mm Hg) and how great the PbtO2 increase was (ΔPbtO2). Intracranial pressure (ΔICP) and cerebral perfusion pressure (ΔCPP) also were examined for these episodes. RESULTS Six hundred twenty-five episodes of reduced PbtO2 were identified in 92 patients. Patient characteristics were: age 41.2 years, 77.2% men, and Injury Severity Score and head or neck Abbreviated Injury Scale score of 34.0 ± 9.2 and 4.9 ± 0.4, respectively. Five interventions: narcotics or sedation, pressors, repositioning, FIO2/PEEP increases, and combined sedation or narcotics + pressors were the most commonly used strategies. Increasing the number of interventions resulted in worsening the time to PbtO2 correction. Triple combinations resulted in the lowest ΔICP and dual combinations in the highest ΔCPP (p < 0.05). CONCLUSION Clinicians use a limited number of interventions when correcting compromised PbtO2. Using strategies employing many interventions administered closely together may be less effective in correcting PbO2, ICP, and CPP deficits. Some PbtO2 deficits may be self-limited.

Creating a “Pro-survival Phenotype” Through Histone Deacetylase Inhibition: Past, Present, and Future

ABSTRACT Traumatic injuries and their sequelae represent a major source of mortality in the United States and globally. Initial treatment for shock, traumatic brain injury, and polytrauma is limited to resuscitation fluids to replace lost volume. To date, there are no treatments with inherent prosurvival properties. Our laboratory has investigated the use of histone deacetylase inhibitors (HDACIs) as pharmacological agents to improve survival. This class of drugs acts through posttranslational protein modifications and is a direct regulator of chromatin structure and function, as well as the function of numerous cytoplasmic proteins. In models of hemorrhagic shock and polytrauma, administration of HDACIs offers a significant survival advantage, even in the absence of fluid resuscitation. Positive results have also been shown in two-hit models of hemorrhage and sepsis and in hemorrhagic shock combined with traumatic brain injury. Accumulating data generated by our group and others continue to support the use of HDACIs for the creation of a prosurvival phenotype. With further research and clinical trials, HDACIs have the potential to be an integral tool in the treatment of trauma, especially in the prehospital phase.

Colonic injuries and the damage control abdomen: does management strategy matter?

BACKGROUND The optimal management of colon injury patients requiring damage control laparotomy (DCL) is controversial. The objective of this study was to assess the safety of colonic resection and anastomosis versus fecal diversion in trauma patients requiring DCL. METHODS Patients with traumatic colon injuries undergoing DCL between 2000 and 2010 were identified by the database and chart review. Those who died within 48 h were excluded. Patients were divided into two groups: those undergoing one or more colonic anastomoses with or without distal colostomy (group 1) and those undergoing colostomy only or one or more colonic anastomoses with a protecting proximal ostomy (group 2). Variables were compared using Wilcoxon rank sum, χ2, or Fisher exact tests as appropriate. RESULTS Sixty-one patients were included (group 1, n=28 and group 2, n=33). Fascial closure rates (group 1, 50% versus group 2, 61%; P=0.45), hospital length of stay (29 versus 23 d; P=0.89), and in-patient mortality (11% versus 12%; P=1.0) were similar between groups. There were a total of 11 anastomotic leaks, five of which were related to non-colonic enteric repairs. Colonic anastomosis leak rates were 16% overall (six of the 38 patients), 14% in group 1 (four of the 28 patients), and 20% in group 2 (two of the 10 patients). Compared with patients who did not leak, patients who leaked had a higher median age (37 versus 25 y; P=0.05), greater likelihood of not achieving facial closure before post-injury day 5 (18% versus 2%; P=0.003), and a longer hospital length of stay (46 versus 25 d; P=0.003). CONCLUSIONS Outcomes after colonic injury in the setting of DCL were similar regardless of the surgical management strategy. Based on these findings, a strategy of diversion over anastomosis cannot be strongly recommended.

Network Reconstruction Reveals that Valproic Acid Activates Neurogenic Transcriptional Programs in Adult Brain Following Traumatic Injury

ObjectivesTo determine the mechanism of action of valproic acid (VPA) in the adult central nervous system (CNS) following traumatic brain injury (TBI) and hemorrhagic shock (HS).MethodsData were analyzed from different sources, including experiments in a porcine model, data from postmortem human brain, published studies, public and commercial databases.ResultsThe transcriptional program in the CNS following TBI, HS, and VPA treatment includes activation of regulatory pathways that enhance neurogenesis and suppress gliogenesis. Genes which encode the transcription factors (TFs) that specify neuronal cell fate, including MEF2D, MYT1L, NEUROD1, PAX6 and TBR1, and their target genes, are induced by VPA. VPA represses genes responsible for oligodendrogenesis, maintenance of white matter, T-cell activation, angiogenesis, and endothelial cell proliferation, adhesion and chemotaxis. NEUROD1 has regulatory interactions with 38% of the genes regulated by VPA in a swine model of TBI and HS in adult brain. Hi-C spatial mapping of a VPA pharmacogenomic SNP in the GRIN2B gene shows it is part of a transcriptional hub that contacts 12 genes that mediate chromatin-mediated neurogenesis and neuroplasticity.ConclusionsFollowing TBI and HS, this study shows that VPA administration acts in the adult brain through differential activation of TFs responsible for neurogenesis, genes responsible for neuroplasticity, and repression of TFs that specify oligodendrocyte cell fate, endothelial cell chemotaxis and angiogenesis.Short title: Mechanism of action of valproic acid in traumatic brain injury

Alterations in the human proteome following administration of valproic acid.

BACKGROUND High doses of the histone deacetylase inhibitor valproic acid (VPA, 150–400 mg/kg) improve outcomes in animal models of lethal insults. We are conducting a US Food and Drug Administration–approved Phase I, double-blind, placebo-controlled trial to evaluate the safety and tolerability of ascending doses of VPA in human volunteers. We hypothesized that VPA would induce significant changes in the proteome of healthy humans when given at doses lower than those used in prior animal studies. METHODS Peripheral blood mononuclear cells were obtained from three healthy subjects randomized to receive VPA (120 mg/kg over 1 hour) at baseline and at 4 and 8 hours following infusion. Detailed proteomic analysis was performed using 1D gel electrophoresis, liquid chromatography, and mass spectrometry. Proteins with differential expression were chosen for functional annotation and pathway analysis using Ingenuity Pathway Analysis (Qiagen GmbH, Hilden, Germany) and Panther Gene Ontology. RESULTS A total of 3,074 unique proteins were identified. The average number of proteins identified per sample was 1,716 ± 459. There were a total of 140 unique differentially expressed proteins (p < 0.05). There was a minor and inconsistent increase in histone and nonhistone protein acetylation. Functional annotation showed significant enrichment of apoptosis (p = 3.5E−43), cell death (p = 9.9E−72), proliferation of cells (p = 1.6E−40), dementia (p = 9.6E−40), amyloidosis (p = 6.3E−38), fatty acid metabolism (p = 4.6E−76), quantity of steroid (p = 4.2E−75), and cell movement (p = 1.9E−64). CONCLUSIONS Valproic acid induces significant changes to the proteome of healthy humans when given at a dose of 120 mg/kg. It alters the expression of key proteins and pathways, including those related to cell survival, without significant modification of protein acetylation. In the next part of the ongoing Phase I trial, we will study the effects of VPA on trauma patients in hemorrhagic shock. LEVEL OF EVIDENCE Therapeutic study, level V.

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 mm3 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.

Mining the topography and dynamics of the 4D Nucleome to identify novel CNS drug pathways

The pharmacoepigenome can be defined as the active, noncoding province of the genome including canonical spatial and temporal regulatory mechanisms of gene regulation that respond to xenobiotic stimuli. Many psychotropic drugs that have been in clinical use for decades have ill-defined mechanisms of action that are beginning to be resolved as we understand the transcriptional hierarchy and dynamics of the nucleus. In this review, we describe spatial, temporal and biomechanical mechanisms mediated by psychotropic medications. Focus is placed on a bioinformatics pipeline that can be used both for detection of pharmacoepigenomic variants that discretize drug response and adverse events to improve pharmacogenomic testing, and for the discovery of novel CNS therapeutics. This approach integrates the functional topology and dynamics of the transcriptional hierarchy of the pharmacoepigenome, gene variant-driven identification of pharmacogenomic regulatory domains, and mesoscale mapping for the discovery of novel CNS pharmacodynamic pathways in human brain. Examples of the application of this pipeline are provided, including the discovery of valproic acid (VPA) mediated transcriptional reprogramming of neuronal cell fate following injury, and mapping of a CNS pathway glutamatergic pathway for the mood stabilizer lithium. These examples in regulatory pharmacoepigenomics illustrate how ongoing research using the 4D nucleome provides a foundation to further insight into previously unrecognized psychotropic drug pharmacodynamic pathways in the human CNS.

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 &agr;-tubulin (Ac-tubulin), total tubulin, acetylated histone 3 at lysine 9 (Ac-H3K9), &bgr;-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.

Inhibition of histone deacetylase 6 restores innate immune cells in the bone marrow in a lethal septic model.

BACKGROUND We have previously demonstrated that Tubastatin A, a selective inhibitor of histone deacetylase 6 (HDAC6), improves survival and increases circulating monocyte count and bacterial clearance in a lethal model of cecal ligation and puncture (CLP) in mice. The aim of the present study was to characterize the effects of inhibition of HDAC6 on the bone marrow cell population. METHODS C57BL/6J mice were subjected to CLP and, 1 hour later, given an intraperitoneal injection of either Tubastatin A (70 mg/kg) dissolved in DMSO or DMSO alone (n = 9 per group). Sham-operated animals were treated in an identical fashion, without CLP. Forty-eight hours later, bone marrow cells were flushed out from the femurs and tibias. Erythrocytes were lysed, and a single-cell suspension was made for analysis. Cells were washed; blocked with antimouse CD16/32; stained with antimouse B220 PE-Cy7, CD3 APC-eFluor 780, CD11b FITC, Gr-1 PerCP-Cy5.5, and F4/80 Antigen APC; and subjected to flow cytometry. Data were acquired on an LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Flowjo, LLC, Ashland, OR). RESULTS In comparison with the sham group, CLP animals showed decreased percentage of innate immune cells (CD11b+, 62.1% ± 3.1% vs. 32.9% ± 4.9%, p = 0.0025) and macrophages (CD11b+F4/80+, 44.6% ± 3.4% vs. 19.8% ± 2.6%, p = 0.0002) as well as increased percentage of T lymphocytes (CD3+, 1.1% ± 0.2% vs. 3.3% ± 0.4%, p = 0.0082) in the bone marrow 48 hours after CLP. Treatment with Tubastatin A restored the innate immune cells (32.9% ± 4.9% vs. 54.0% ± 4.1%, p = 0.0112) and macrophages (19.8% ± 2.6% vs. 47.1% ± 4.6%, p = 0.0001) and increased the percentage of neutrophils (CD11b+Gr-1+, 28.4% ± 3.9% vs. 48.0% ± 4.0%, p = 0.0075). The percentages of B (B220+) and T lymphocytes were not significantly altered by Tubastatin A, compared with the vehicle-treated CLP animals. CONCLUSION Selective inhibition of HDAC6 in this lethal septic model restored the innate immune cell and macrophage populations and increased the neutrophil composition in the bone marrow. These results may explain the previously reported beneficial effects of Tubastatin A treatment in a septic model.

Valproic acid decreases brain lesion size and improves neurologic recovery in swine subjected to traumatic brain injury, hemorrhagic shock, and polytrauma

BACKGROUND 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. METHODS 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. RESULTS 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, mm3: 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. CONCLUSIONS 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. LEVEL OF EVIDENCE Therapeutic (preclinical study).