Jason Luciano

Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation

BACKGROUND Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. METHODS Wild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. RESULTS AICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. CONCLUSIONS In this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

Benzyl alcohol attenuates acetaminophen‐induced acute liver injury in a Toll‐like receptor‐4‐dependent pattern in mice

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP‐induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP‐induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP‐induced liver injury by modulation of danger signaling. APAP‐induced liver injury was dependent, in part, on Toll‐like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135‐540 μg/g body weight or when delivered as a pre‐, concurrent, or post‐APAP therapeutic. Furthermore, BA abrogated APAP‐induced cytokines and chemokines as well as high‐mobility group box 1 release. Moreover, BA prevented APAP‐induced inflammasome signaling as determined by interleukin (IL)‐1β, IL‐18, and caspase‐1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell‐type–specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre‐tlr4−/−) were necessary for the protective effects of BA. Conclusion: BA protects against APAP‐induced acute liver injury and reduced inflammasome activation in a TLR4‐dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury. (Hepatology 2014;60:990–1002)

Carbon Monoxide Protects Against Hemorrhagic Shock and Resuscitation-Induced Microcirculatory Injury and Tissue Injury

ABSTRACT Traumatic injury is a significant cause of morbidity and mortality worldwide. Microcirculatory activation and injury from hemorrhage contribute to organ injury. Many adaptive responses occur within the microcirculatory beds to limit injury including upregulation of heme oxygenase (HO) enzymes, the rate-limiting enzymes in the breakdown of heme to carbon monoxide (CO), iron, and biliverdin. Here we tested the hypothesis that CO abrogates trauma-induced injury and inflammation protecting the microcirculatory beds. Methods: C57Bl/6 mice underwent sham operation or hemorrhagic shock to a mean arterial pressure of 25 mmHg for 120 minutes. Mice were resuscitated with lactated Ringer’s at 2× the volume of maximal shed blood. Mice were randomized to receive CO-releasing molecule or inactive CO-releasing molecule at resuscitation. A cohort of mice was pretreated with tin protoporphyrin-IX to inhibit endogenous CO generation by HOs. Primary mouse liver sinusoidal endothelial cells were cultured for in vitro experiments. Results: Carbon monoxide–releasing molecule protected against hemorrhagic shock/resuscitation organ injury and systemic inflammation and reduced hepatic sinusoidal endothelial injury. Inhibition of HO activity with tin protoporphyrin-IX exacerbated liver hepatic sinusoidal injury. Hemorrhagic shock/resuscitation in vivo or cytokine stimulation in vitro resulted in increased endothelial expression of adhesion molecules that was associated with decreased leukocyte adhesion in vivo and in vitro. Conclusions: Hemorrhagic shock/resuscitation is associated with endothelial injury. Heme oxygenase enzymes and CO are involved in part in diminishing this injury and may prove useful as a therapeutic adjunct that can be harnessed to protect against endothelial activation and damage.

Clostridium Difficile Infection: Prevention, Treatment, and Surgical Management

Clostridium difficile is increasing in both incidence and severity. Although metronidazole and vancomycin remain the gold standard for medical management, and surgical colectomy the gold standard for surgical management, new treatment alternatives, including the creation of a diverting loop ileostomy along with colonic lavage and vancomycin enemas, are being investigated that may lead to changes in the current treatment algorithms. The most exciting development in the treatment options for C difficile infection, however, is likely to be novel immunologic agents, which hold the potential to reduce the incidence, mortality, and costs associated with C difficile.

Inhaled Carbon Monoxide Protects against the Development of Shock and Mitochondrial Injury following Hemorrhage and Resuscitation

Aims Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation. Results Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01). Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05). A dose response of CO (25–500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively. CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100–500ppm). Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation. In porcine HS/R, CO did not influence hemodynamics. However, CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets). Conclusion These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R; however, further clinical studies are warranted.

Sirtuin 1 Agonist Minimizes Injury and Improves the Immune Response Following Traumatic Shock.

ABSTRACT Survival from traumatic injury requires a coordinated and controlled inflammatory and immune response. Mitochondrial and metabolic responses to stress have been shown to play a role in these inflammatory and immune responses. We hypothesized that increases in mitochondrial biogenesis via a sirtuin 1 agonist would decrease tissue injury and partially ameliorate the immunosuppression seen following trauma. C57Bl/6 mice were subjected to a multiple trauma model. Mice were pretreated with either 100 mg/kg per day of the sirtuin 1 agonist, Srt1720, via oral gavage for 2 days prior to trauma and extended until the day the animals were killed, or they were pretreated with peroxisome proliferator-activated receptor &ggr; coactivator 1&agr; (PGC1&agr;) siRNA via hydrodynamic tail vein injection 48 h prior to trauma. Markers for mitochondrial function and biogenesis were measured in addition to splenocyte proliferative capacity and bacterial clearance. Srt1720 was noted to improve mitochondrial biogenesis, mitochondrial function, and complex IV activity following traumatic injury (P < 0.05), whereas knockdown of PGC1&agr; resulted in exacerbation of mitochondrial dysfunction (P < 0.05). These changes in mitochondrial function were associated with altered severity of hepatic injury with significant reductions in serum alanine aminotransferase levels seen in mice treated with srt1720. Splenocyte proliferative capacity and intraperitoneal bacterial clearance were evaluated as markers for overall immune function following trauma-hemorrhage. Treatment with Srt1720 minimized the trauma-induced decreases in splenocyte proliferation (P < 0.05), whereas treatment with PGC1&agr; siRNA led to diminished bacterial clearance. The PGC1&agr; signaling pathway is an important regulator of mitochondrial function and biogenesis, which can potentially be harnessed to protect against hepatic injury and minimize the immunosuppression that is seen following trauma-hemorrhage.

Acute kidney injury in the setting of knee arthroplasty: a case report and discussion investigating Angiotensin-converting enzyme inhibitors as the culprit.

Total knee arthroplasty (TKA) has become the predominant treatment modality for severe degenerative joint disease. With recent advancements in surgical and anesthetic technique, patients with severe comorbidities are able to have this procedure; they would have been precluded from TKA only a matter of years ago. Although many studies have investigated risk factors and the causes of perioperative morbidity and mortality in the arthroplasty patient, few have linked risk factors with specific outcomes. We present a case report that illustrates the link between the use of angiotensin-converting enzyme inhibitors and the development of postoperative acute kidney injury. While this relationship has been extensively studied in cardiac and gastric bypass patient populations, it has never been examined in the setting of joint replacement.

Heme Oxygenase-2 Localizes to Mitochondria and Regulates Hypoxic Responses in Hepatocytes

Hypoxia occurs as a part of multiple disease states, including hemorrhagic shock. Adaptive responses occur within the cell to limit the consequences of hypoxia. This includes changes in mitochondrial respiration, stress-induced cell signaling, and gene expression that is regulated by hypoxia inducible factor-1α (HIF-1α). Heme oxygenase-2 (HO-2) has been shown to be involved in oxygen sensing in several cell types. The purpose of these experiments was to test the hypothesis that HO-2 is a critical regulator of mitochondrial oxygen consumption and reactive oxygen species (ROS) production to influence hypoxia-adaptive responses such as HIF-1α protein levels and JNK signaling. Methods and Results. In vitro studies were performed in primary mouse hepatocytes. HO-2, but not HO-1, was expressed in mitochondria at baseline. Decreased oxygen consumption and increased mitochondrial ROS production in response to hypoxia were dependent upon HO-2 expression. HO-2 expression regulated HIF-1α and JNK signaling in a mitochondrial ROS-dependent manner. Furthermore, knockdown of HO-2 led to increased organ damage, systemic inflammation, tissue hypoxia, and shock in a murine model of hemorrhage and resuscitation. Conclusion. HO-2 signaling plays a role in hypoxic signaling and hemorrhagic shock. This pathway may be able to be harnessed for therapeutic effects.

Operative Risk Stratification of the Geriatric Patient

The preoperative evaluation of the geriatric patient requires a thorough assessment of the patient’s overall health in addition to their cognitive and functional assessment. While risk stratification has historically focused on assessment of individual organ systems, the assessment of the geriatric patient requires a more comprehensive evaluation. In addition to evaluation of patient’s comorbid conditions, the presence of malnutrition, frailty, and cognitive dysfunction are all predictors of poor outcome following surgery. A thorough geriatric evaluation allows a more complete discussion with the patient and allows the prediction of mortality, functional recovery, and potential for return to home following discharge from the hospital.