Adenosine 5’-Monophosphate Protects From Hypoxia By Lowering Mitochondrial Metabolism and Oxygen Demand ABSTRCT Ischemia and reperfusion injury following severe trauma or cardiac arrest are major causes of organ damage in intensive care patients. The brain is particularly vulnerable because hypoxia rapidly damages neurons due to their heavy reliance on oxidative phosphorylation. Therapeutic hypothermia can reduce ischemia-induced brain damage, but cooling procedures are slow and technically difficult to perform in critical care settings. It has been previously reported that injection of naturally occurring adenosine 5’-monophosphate (AMP) can rapidly induce hypothermia in mice. We studied the underlying mechanisms and found that AMP transiently reduces the heart rate, respiratory rate, body temperature, and the consciousness of adult male and female C57BL/6J mice. Adding AMP to mouse or human neuronal cell cultures dose-dependently reduced the membrane potential (ΔΨm) and Ca2+ signaling of mitochondria in these cells. AMP treatment increased intracellular AMP levels and activated AMP-activated protein kinase (AMPK), which resulted in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and of mitochondrial and cytosolic Ca2+ signaling in resting and stimulated neurons. Pretreatment with an intraperitoneal injection of AMP almost doubled the survival time of mice under hypoxic (6% O2) or anoxic (<1% O2) conditions when compared to untreated mice. These findings suggest that AMP induces a hypometabolic state that slows mitochondrial respiration, reduces oxygen demand, and delays the processes that damage mitochondria in the brain and other organs following hypoxia and reperfusion. Further examination of these mechanisms may lead to new treatments that preserve organ function in critical care patients. Address reprint requests to Wolfgang G. Junger, PhD, Harvard Medical School, Beth Israel Deaconess Medical Center, Department of Surgery, 330 Brookline Avenue, Boston, MA 02215, USA. E-mail: wjunger@bidmc.harvard.edu Received 10 July, 2019 Revised 24 July, 2019 Accepted 19 August, 2019 Source of Funding: The study was supported in part by grants from the National Institutes of Health, GM-51477, GM-60475, GM-116162, AI-080582, and T32 GM-103702 (to W.G.J.), and the National Natural Science Foundation of China (81701564) and the Applied Basic Research Program of the Science and Technology Department of Sichuan Province (2018JY0676; to X.L.). Conflicts of interest: The authors have no conflicts of interest to declare. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

Conditioned Contextual Freezing is A Neurobehavioral Biomarker of Axonal Injury Indicated by Reduced Fractional Anisotropy In A Mouse Model of Blast-Induced Mild Traumatic Brain Injury Mild Traumatic Brain Injury (TBI) is an important public health problem generated by closed head injury. This study is focused on the impact of blast-induced mild TBI on auditory trace and delay fear conditioning, models of declarative and non-declarative memory, respectively, and the correlation of conditioned freezing and fractional anisotropy, a measure of axonal state. A supersonic helium pressure wave was generated by a shock tube to blast 8 week old male mice on Day 1 for 1.4 msec with an incident pressure of 16 psi, corresponding to a reflected pressure of 56.5 psi at the mouse head. On Day 3, the mice were subjected to auditory trace- or delay- fear conditioning. On Day 4, contextual freezing in the trained context, and pre-cue and cued freezing in a novel context were determined. After cardiac perfusion on Day 5, ex vivo images were obtained with diffusion tensor imaging at 14.1 Tesla. We observed that delay fear conditioning prevented or reversed the decrease in fractional anisotropy in both the medial and lateral corpus callosum suggesting axonal stabilization of potentially behavioral therapeutic significance. Moderately strong and statistically significant Pearson correlations were found between fractional anisotropy and contextual freezing in the medial and lateral corpus callosum of blasted and sham-blasted delay- or trace- fear conditioned mice. Thus, contextual freezing is a neurobehavioral biomarker for axonal injury in mild TBI and is a reliable and high-throughput behavioral assay for the evaluation of potential therapeutics to treat mild TBI. Address reprint requests to Joachim Spiess, PhD, MD, Cortrop Incorporated, Encinitas, CA 92024. E-mail: joachim.spiess@gmail.com Received 29 May, 2019 Revised 14 June, 2019 Accepted 23 July, 2019 Support: NIH grants RO1NS059879 (CW), NS10RR13880 (AMW), MH047340 (JFD), and Max Planck Society (JS). Author contributions: J.S. initiated the project, designed the program, participated in the design of the behavior and DTI experiments including the effect of behavior therapy, worked out the details of the blast procedure, interpreted all data, carried out the blast experiments, wrote most of the manuscript. C.W. participated in the design of the program, designed the behavior experiments, participated in the design of the behavior experiments modifying FA, performed data analysis of the behavior and DTI experiments, interpreted the behavior and DTI data and integrated them by determining their correlation, interpreted the relationship of the behavior experiments and the protective effect of the behavior paradigms, participated in writing the manuscript, carried out the behavior experiments and the cardiac perfusion. P.H.R. participated in the overall design of the program, designed the shock tube, worked out all physical details of the shock tube function, backed the day to day operation of the blast procedure, designed and performed all pressure measurements, analyzed the pressure curve of each mouse. F.R.L. participated in the design of the DTI experiments, carried out these experiments, analyzed them and compared them with the behavior experiments. A.M.W. participated in the design of the program, designed the DTI experiments, analyzed the DTI data, interpreted the DTI experiments and interpreted their relationship to the behavior experiments and the protective effect of the behavior paradigms. J.F.D. participated in the design of the program, participated in the design of the behavior and DTI experiments, interpreted all data and contributed to potential effects of behavior therapy. © 2019 by the Shock Society

Alternative Complement Pathway Activation Provokes A Hypercoagulable State with Diminished Fibrinolysis Introduction: Several disease processes trigger prolonged activation of the alternative complement pathway. Crosslinks between complement activation and physiologic changes in platelets and neutrophils have been identified, but how this interplay alters the hemostatic potential in humans remains undefined. We hypothesize that activation of the alternative pathway triggers a hypercoagulable state. Methods: C3/C5 convertase Cobra Venom Factor (CVF, 10 Units/mL) was employed to activate the alternative complement pathway in whole blood. Complement inhibition was completed with inhibitors for C3/C3b (Compstatin, 25 and 50 μM), C3a receptor (SB290157, 300 nM, C3aR), and C5a receptor (W54011, 6 nM, C5aR). Coagulation was assessed using native thrombelastography which produces the following: reaction time (R time); angle; maximum amplitude (MA); percent fibrinolysis at 30-minutes post-MA (LY30). Results: Inhibition with C3aR and C5aR inhibitors did not alter clot formation (R time, 11.2 vs 11.6 min, p = 0.36), clot strength (MA, 52.0 vs 52.3 mm, p = 0.43) or fibrinolysis (LY30, 1.6 vs 4.0%, p = 0.19). Compstatin did not influence clot formation or clot strength but did induce a dose-dependent increase in fibrinolysis (control LY30 3.0 vs 7.8% and 12.4% for 25 and 50 μM respectively, p = 0.0002). CVF increased MA (58.0 vs 62.8 mm, p < 0.0001), decreased LY30 (2.3 vs 1.4%, p = 0.004), and increased R time (8.4 vs 9.9 min, p = 0.008). Compstatin reversed the effects of CVF, while C5a reversed only the change in LY30. Conclusions: C3 contributes to fibrinolysis, as inhibition with Compstatin enhanced fibrinolysis, and CVF cleavage of C3 decreased fibrinolysis. CVF also induced a hypercoagulable state with increased clot strength. Address reprint requests to Christopher C. Silliman, Research Laboratory, Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230, USA. E-mail: Christopher.Silliman@ucdenver.edu; Jason M. Samuels, MD, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO United States. E-mail: Jason.Samuels@ucdenver.edu Received 28 June, 2019 Revised 18 July, 2019 Accepted 7 August, 2019 Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). Financial Disclosures: The authors appreciate research support from Haemonetics with shared intellectual property. Funding: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (T32 GM008315 and P50 GM049222) and Department of Defense (USAMRAA, W81XWH-12-2-0028). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other sponsors of the project. Presentation history: Portions of this data were presented at the 64th Annual International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee meeting Dublin, Ireland, July 18-21, 2018. © 2019 by the Shock Society

Inhibition of Lipolysis with Acipimox Attenuates Post-Burn White Adipose Tissue Browning and Hepatic Fat Infiltration Extensive burn injuries promote an increase in the lipolysis of white adipose tissue (WAT), a complication that enhances post-burn hypermetabolism, contributing to hyperlipidemia and hepatic steatosis. The systemic increase of free fatty acids (FFA) due to burn-induced lipolysis and subsequent organ fatty infiltration may culminate in multiple organ dysfunction and ultimately, death. Thus, reducing WAT lipolysis to diminish the mobilization of FFAs may render an effective means to improve outcomes post-burn. Here, we investigated the metabolic effects of Acipimox, a clinically approved drug that suppresses lipolysis via inhibition of hormone-sensitive lipase (HSL). Using a murine model of thermal injury, we show that specific inhibition of HSL with Acipimox effectively suppresses burn-induced lipolysis in the inguinal WAT leading to lower levels of circulating FFAs at 7 days post-burn (p < 0.05). The FFA substrate shortage indirectly repressed the thermogenic activation of adipose tissue following injury, reflected by the decrease in protein expression of key browning markers, UCP-1 (p < 0.001) and PGC-1α (p < 0.01). Importantly, reduction of FFA mobilization by Acipimox significantly decreased liver weight and intracellular fat accumulation (p < 0.05), suggesting that it may also improve organ function post-burn. Our data validate the pharmacological inhibition of lipolysis as a potentially powerful therapeutic strategy to counteract the detrimental metabolic effects induced by burn. Address reprint requests to Marc G. Jeschke, MD, PhD, Director Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Division of Plastic Surgery, Department of Surgery, Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Ave., Rm. D704, Toronto, ON, CANADA, M4N 3M5. E-mail: marc.jeschke@sunnybrook.ca. Received 24 May, 2019 Revised 12 June, 2019 Accepted 9 August, 2019 Address for Reprints: Same as corresponding author, please see above. Conflicts of Interest and Source of Funding: This study was supported by – Canadian Institutes of Health Research # 123336. CFI Leaders Opportunity Fund: Project # 25407 NIH RO1 GM087285-01 Author Contributions: DB, RV, and CK performed experiments and wrote portions of the manuscript; CA and AA guided experiments and wrote portions of the manuscript; MJ guided the experiments and wrote portions of the manuscript. Competing Financial Interests statement: The authors have no competing financial interests to declare. © 2019 by the Shock Society

Pulmonary Vasodilation by Intravenous Infusion of Organic Mononitrites of 1,2-Propanediol in Acute Pulmonary Hypertension Induced by Aortic Cross Clamping and Reperfusion: A Comparison with Nitroglycerin in Anesthetised Pigs Introduction: Suprarenal aortic cross clamping (SRACC) and reperfusion may cause acute pulmonary hypertension and multiple organ failure. Hypothesis: The organic mononitrites of 1,2-propanediol (PDNO), an NO donor with a very short half-life, is a more efficient pulmonary vasodilator and attenuator of end-organ damage and inflammation without significant side effects compared to nitroglycerin and inorganic nitrite in a porcine SRACC model. Methods: Anesthetised and instrumented domestic pigs were randomised to either of four IV infusions until the end of the experiment (n = 10 per group): saline (control), PDNO (45 nmol kg−1 min−1), nitroglycerin (44 nmol kg−1 min−1), or inorganic nitrite (a dose corresponding to PDNO). Thereafter, all animals were subjected to 90 minutes of SRACC and 10 hours of reperfusion and protocolised resuscitation. Hemodynamic and respiratory variables as well as blood samples were collected and analysed. Results: During reperfusion, mean pulmonary arterial pressure and pulmonary vascular resistance were significantly lower, and stroke volume was significantly higher in the PDNO group compared to the control, nitroglycerin, and inorganic nitrite groups. In parallel, mean arterial pressure, arterial oxygenation, and fraction of methaemoglobin were similar in all groups. The serum concentration of creatinine and tumour necrosis factor alpha were lower in the PDNO group compared to the control group during reperfusion. Conclusions: PDNO was an effective pulmonary vasodilator and appeared superior to nitroglycerin and inorganic nitrite, without causing significant systemic hypotension, impaired arterial oxygenation, or methaemoglobin formation in an animal model of SRACC and reperfusion. Also, PDNO may have kidney-protective effects and anti-inflammatory properties. Address reprint requests to Kristofer F. Nilsson, Department of Cardiothoracic and Vascular Surgery, Örebro University Hospital, SE-70185 Örebro, Sweden. E-mail: kristofer-bo-ingemar.nilsson@regionorebrolan.se Received 13 June, 2019 Revised 6 August, 2019 Accepted 6 August, 2019 Conflicts of Interest and Source of Funding: Author KFN wishes to declare competing financial interest in the subject matter. Author CF wishes to declare financial interest in the clinical use of inhaled nitric oxide. The other authors declare no competing interests. This study was supported by grants from the Swedish Heart-Lung Foundation, the European Space Agency, the Fraenckel Foundation, the Lars Hierta Foundation, Karolinska Institutet, Region Örebro County ALF grants, The Swedish Society for Medical Research, and an unrestricted educational grant from CF Research and Consulting AB, Stockholm, Sweden. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0 © 2019 by the Shock Society

Persistent Neuroinflammation and Brain Specific Immune Priming in A Novel Survival Model of Murine Pneumosepsis Pneumonia is the leading cause of sepsis and septic shock. Patients who survive pneumonia are vulnerable to long-term complications including increased risk of neurocognitive dysfunction. This study investigated the immune response and long-term complications of a non-surgical mouse model of Klebsiella pneumoniae pneumosepsis with antibiotic treatment. Pneumosepsis resulted in acutely enhanced expression of inflammatory cytokines, chemokines, and damage associated molecular patterns in the brain and spleen. Despite resolution of infection, murine pneumosepsis survivors demonstrated a deficit in exploratory locomotor behavior at two weeks. This was associated with brain specific persistent inflammatory gene expression and infiltrating myeloid cells in the brain. The brain inflammatory response was also primed in response to secondary challenge with lipopolysaccharide. The findings of this study demonstrate behavioral and inflammatory outcomes that parallel observations in other models of sepsis, but that have not previously been described in antibiotic treated pneumonia models, highlighting a common pathway to the development of chronic brain dysfunction in sepsis survival. Address reprint requests to Scott J. Denstaedt, MD, 4868 Biomedical Science Research Building 109 Zina Pitcher Pl. Ann Arbor, MI 48103. E-mail: sdenstae@med.umich.edu Received 19 June, 2019 Revised 10 July, 2019 Accepted 5 August, 2019 Financial support: National Institutes of Health grants T32HL00774921 (to SJD), MH116267 (to JLSS), K08NS101054 (to BHS), R01HL123515 (to TJS). Conflict of interest: The authors have no conflicts relevant to the present study. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

Daily Changes in The Expression of Clock Genes in Sepsis and Their Relation with Sepsis Outcome and Urinary Excretion of 6-Sulfatoximelatonin Background: Whereas the circadian system controls the daily production of melatonin and the daily activity of the immune system, increasing evidences support the association between circadian misalignment with the alterations in the immune response and melatonin rhythm during sepsis. The aim of this study was to analyze the daily changes in clock genes expression and the urinary excretion of 6-SM (6-sulfatoxymelatonin, the major melatonin metabolite), and their connection with the innate immune activity, oxidative status in blood, and clinical outcome during sepsis. Methods: Healthy volunteers, non-septic ICU patients, and septic ICU patients, were evaluated. The expression of bmal1, per2, clock, and cry1 genes was determined by PCR in blood; 6-SM was assessed in urine by ELISA; plasma cytokines IL-1β, IL-6, IL-8, TNFα, and IL-10 were determined by a multiplex array method, and lipid peroxidation (LPO) and protein oxidation (AOPP) by spectrophotometry. Hematological and biochemical data, and clinical scores of the patients, were also recorded. Results: Clock gene rhythm was maintained in non-septic patients but blunted in septic ones, whereas the innate immune and the oxidative stress responses were significantly higher in the latter. 6-SM excretion was also more elevated in septic than in non-septic patients, and it correlated with the degree of the immune response and oxidative status. 6-SM also correlated with SOFA and procalcitonin in the patients. Proinflammatory cytokines, LPO, and AOPP were normalized in the patients once recovered from sepsis. Conclusion: Our data suggest a relationship between clock genes rhythm disruption, the immune response and the oxidative status, with 6-SM acting as a compensatory response. ICU conditions are not a main clock disrupter because the significant differences found in the responses of septic versus non-septic patients under the same ICU environment. Address reprint requests to Carlos Acuña-Fernández, MD, Unidad of Anestesiology y Reanimación, Hospital Universitario de Canarias, Carreterra de Ofra, s/n, 38320 San Cristóbal de La Laguna, Santa Cruz de Tenerife, Spain. E-mail: cacufer89@gmail.com; Darío Acuña-Castroviejo, MD, PhD, Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avenida del Conocimiento s/n; 18016 Granada, Spain. E-mail: dacuna@ugr.es Received 9 July, 2019 Revised 18 July, 2019 Accepted 26 July, 2019 Funding statement: This study has been funded by Instituto de Salud Carlos III through the projects PI13-00981, PI16–00519, and CB/10/00238 (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”); the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, Spain (CTS-101), and from Pfizer S.L.U, Madrid, Spain. Conflicts of Interest: The authors declare no competing interests. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com). © 2019 by the Shock Society

SIRT1 Mediates Septic Cardiomyopathy in a Murine Model of Polymicrobial Sepsis Background: Cardiac dysfunction, a common complication from severe sepsis, is associated with increased morbidity and mortality. However, the molecular mechanisms of septic cardiac dysfunction are poorly understood. SIRT1, a member of the sirtuin family of NAD+-dependent protein deacetylases, is an important immunometabolic regulator of sepsis, and sustained SIRT1 elevation is associated with worse outcomes and organ dysfunction in severe sepsis. Herein, we explore the role of SIRT1 in septic cardiac dysfunction using a murine model of sepsis. Methods: An in vitro model of inflammation in isolated H9c2 cardiomyocytes was used to confirm SIRT1 response to stimulation with lipopolysaccharide (LPS), followed by a murine model of cecal ligation and puncture (CLP) to investigate the molecular and echocardiographic response to sepsis. A selective SIRT1 inhibitor, EX-527, was employed to test for SIRT1 participation in septic cardiac dysfunction. Results: SIRT1 mRNA and protein levels in cultured H9c2 cardiomyocytes were significantly elevated at later time points after stimulation with LPS. Similarly, cardiac tissue harvested from C57BL/6 mice 36 hours after CLP demonstrated increased expression of SIRT1 mRNA and protein compared to sham controls. Administration of EX-527 18 hours after CLP reduced SIRT1 protein expression in cardiac tissue at 36 hours. Moreover, treatment with EX-527 improved cardiac performance with increased global longitudinal strain and longitudinal strain rate. Conculsions: Our findings reveal that SIRT1 expression increases in isolated cardiomyocytes and cardiac tissue after sepsis-inflammation. Moreover, rebalancing SIRT1 excess in late sepsis improves cardiac performance suggesting that SIRT1 may serve as a therapeutic target for septic cardiomyopathy. Address reprint requests to Lane M. Smith, MD, PhD, Department of Emergency Medicine, Meads Hall 2nd Floor, Medical Center Blvd., Winston Salem, NC 27157. E-mail: lmsmith@wakehealth.edu. Received 22 May, 2019 Revised 11 June, 2019 Accepted 26 July, 2019 © 2019 by the Shock Society

Letter to the Editor Regarding the Manuscript of Kasotakis et al “Histone Deacetylase 7 Inhibition in a Murine Model of Gram-negative Pneumonia-induced Acute Lung Injury” Shock. 2019 May 2. doi: 10.1097/SHK.0000000000001372. [Epub ahead of print] No abstract available

Histone Deacetylase 7 In Murine Gram-Negative Acute Lung Injury No abstract available