Vahagn C. Nikolian

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.

Anastomotic leak after colorectal resection: A population-based study of risk factors and hospital variation

Background: Anastomotic leak is a major source of morbidity in colorectal operations and has become an area of interest in performance metrics. It is unclear whether anastomotic leak is associated primarily with surgeons’ technical performance or explained better by patient characteristics and institutional factors. We sought to establish if anastomotic leak could serve as a valid quality metric in colorectal operations by evaluating provider variation after adjusting for patient factors. Methods: We performed a retrospective cohort study of colorectal resection patients in the Michigan Surgical Quality Collaborative. Clinically relevant patient and operative factors were tested for association with anastomotic leak. Hierarchical logistic regression was used to derive risk‐adjusted rates of anastomotic leak. Results: Of 9,192 colorectal resections, 244 (2.7%) had a documented anastomotic leak. The incidence of anastomotic leak was 3.0% for patients with pelvic anastomoses and 2.5% for those with intra‐abdominal anastomoses. Multivariable analysis showed that a greater operative duration, male sex, body mass index >30 kg/m2, tobacco use, chronic immunosuppressive medications, thrombocytosis (platelet count >400 × 109/L), and urgent/emergency operations were independently associated with anastomotic leak (C‐statistic = 0.75). After accounting for patient and procedural risk factors, 5 hospitals had a significantly greater incidence of postoperative anastomotic leak. Conclusion: This population‐based study shows that risk factors for anastomotic leak include male sex, obesity, tobacco use, immunosuppression, thrombocytosis, greater operative duration, and urgent/emergency operation; models including these factors predict most of the variation in anastomotic leak rates. This study suggests that anastomotic leak can serve as a valid metric that can identify opportunities for quality improvement.

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.

OpTrust: Validity of a Tool Assessing Intraoperative Entrustment Behaviors

Objective: The aim of this study is to establish evidence to support the validity of a novel faculty-resident intraoperative assessment tool for entrustment known as OpTrust. Background: Recently, the landscape of surgical training has been altered, in part, because of resident work-hour changes and increased supervision requirements. To address these concerns, a new model for assessment of teaching and learning in surgical residencies must be anchored on progression through milestones and entrustment. Methods: OpTrust was designed to assess the faculty-resident dyad in the operating room and measure the entrustment exhibited during intraoperative interactions across 5 domains: (i) types of questions asked, (ii) operative plan, (iii) instruction, (iv) problem solving, and (v) leadership by the surgical resident. After initial pilot testing and refinement of OpTrust, 5 individual raters underwent rater training sessions; 49 individual operating room observations were completed based on 28 cases. Results: OpTrust, as a tool for assessing intraoperative entrustment, is supported by strong validity evidence. In part, it demonstrates strong interrater reliability across all faculty domains as measured by intraclass correlation 1 (ICC1) (0.81–0.93). For resident domains the results were similar with ICC1 (0.84–0.94). Cronbach alpha was 0.89 and 0.87 for faculty and resident entrustment respectively, signifying the 5 domains could be combined into a single construct of entrustment. A high correlation existed between faculty and resident scores (Pearson r = 0.94, P < 0.001) indicating a strong positive linear relationship between faculty and resident mean entrustment scores across all scale domains. Conclusions: OpTrust successfully assesses behaviors associated with entrustment during intraoperative faculty-resident interactions, and has the potential to be adopted across other procedural-based specialties to promote autonomous training progression.

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.

Association of Faculty Entrustment With Resident Autonomy in the Operating Room

Importance A critical balance is sought between faculty supervision, appropriate resident autonomy, and patient safety in the operating room. Variability in the release of supervision during surgery represents a potential safety hazard to patients. A better understanding of intraoperative faculty-resident interactions is needed to determine what factors influence entrustment. Objective To assess faculty and resident intraoperative entrustment behaviors and to determine whether faculty behaviors drive resident entrustability in the operating room. Design, Setting, and Participants This observational study was conducted from September 1, 2015, to August 31, 2016, at Michigan Medicine, the University of Michigan’s health care system. Two surgical residents, 1 medical student, 2 behavioral research scientists, and 1 surgical faculty member observed surgical intraoperative interactions between faculty and residents in 117 cases involving 28 faculty and 35 residents and rated entrustment behaviors. Without intervening in the interaction, 1 or 2 researchers observed each case and noted behaviors, verbal and nonverbal communication, and interaction processes. Immediately after the case, observers completed an assessment using OpTrust, a validated tool designed to assess progressive entrustment in the operating room. Purposeful sampling was used to generate variation in type of operation, case difficulty, faculty-resident pairings, faculty experience, and resident training level. Main Outcomes and Measures Observer results in the form of entrustability scores (range, 1-4, with 4 indicating full entrustability) were compared with resident- and faculty-reported measures. Difficulty of operation was rated on a scale of 1 to 3 (higher scores indicate greater difficulty). Path analysis was used to explore direct and indirect effects of the predictors. Associations between resident entrustability and observation duration, observation month, and faculty entrustment scores were assessed by pairwise Pearson correlation coefficients. Results Twenty-eight faculty and 35 residents were observed across 117 surgical cases from 4 surgical specialties. Cases observed by postgraduate year (PGY) of residents were distributed as follows: PGY-1, 21 (18%); 2, 15 (13%); 3, 17 (15%); 4, 27 (23%); 5, 28 (24%); and 6, 9 (8%). Case difficulty was evenly distributed: 36 (33%) were rated easy/straightforward; 43 (40%), moderately difficult; and 29 (27%), very difficult by attending physicians. Path analysis showed that the association of PGY with resident entrustability was mediated by faculty entrustment (0.23 [.03]; P < .001). At the univariate level, case difficulty (mean [SD] resident entrustability score range, 1.97 [0.75] for easy/straightforward cases to 2.59 [0.82] for very difficult cases; F = 6.69; P = .01), PGY (range, 1.31 [0.28] for PGY-1 to 3.16 [0.54] for PGY-6; F = 22.85; P < .001), and faculty entrustment (2.27 [0.79]; R2 = 0.91; P < .001) were significantly associated with resident entrustability. Mean (SD) resident entrustability scores were highest for very difficult cases (2.59 [0.82]) and PGY-6 (3.16 [0.54]). Conclusions and Relevance Faculty entrustment behaviors may be the primary drivers of resident entrustability. Faculty entrustment is a feature of faculty surgeons’ teaching style and could be amenable to faculty development efforts.

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).