Joshua W. Kuethe

Microparticles impact coagulation after traumatic brain injury

BACKGROUND The pathophysiology that drives the subacute hypercoagulable state commonly seen after traumatic brain injury (TBI) is not well understood. Alterations caused by TBI in platelet and microparticle (MP) numbers and function have been suggested as possible causes; however, the contributions of platelets and MPs are currently unknown. MATERIALS AND METHODS A weight-drop technique of TBI using a murine model of moderate head injury was used. Blood was collected at intervals after injury. MP enumeration and characterization were performed using Nanoparticle Tracking Analysis, and platelet counts and coagulation parameters were determined using thromboelastometry. A MP procoagulant assay was used to compare activity between injured and sham mice. RESULTS At 24 h after injury, there were no changes in circulating platelet numbers. However, there was a decrease in platelet contribution to clot formation. In contrast, there was a decline in circulating total MP numbers. When MPs from sham mice were added to the blood from head-injured animals, there was a normalization of platelet contribution to clot formation. Conversely, when MPs from TBI mice were added to sham blood, there was a significant decrease in platelet contribution to clot formation. Notably, there was an increase in MP procoagulant activity in head-injured mice. CONCLUSIONS MPs generated after TBI likely contribute to altered coagulation after head injury and may play a key role in the development of a posttraumatic hypercoagulable state in TBI patients.

Fecal Microbiota Transplant Restores Mucosal Integrity in a Murine Model of Burn Injury.

ABSTRACT The gut microbiome is a community of commensal organisms that are known to play a role in nutrient production as well as gut homeostasis. The composition of the gut flora can be affected by many factors; however, the impact of burn injury on the microbiome is not fully known. Here, we hypothesized that burn-induced changes to the microbiome would impact overall colon health. After scald-burn injury, cecal samples were analyzed for aerobic and anaerobic colony forming units, bacterial community, and butyrate levels. In addition, colon and total intestinal permeabilities were determined. These parameters were further determined in a germ-reduced murine model. Following both burn injury and germ reduction, we observed decreases in aerobic and anaerobic bacteria, increased colon permeability and no change to small intestinal permeability. After burn injury, we further observed a significant decrease in the butyrate producing bacteria R. Gnavus, C. Eutactus, and Roseburia species as well as decreases in colonic butyrate. Finally, in mice that underwent burn followed by fecal microbiota transplant, bacteria levels and mucosal integrity were restored. Altogether our data demonstrate that burn injury can alter the microbiome leading to decreased butyrate levels and increased colon permeability. Of interest, fecal microbiota transplant treatment was able to ameliorate the burn-induced changes in colon permeability. Thus, fecal transplantation may represent a novel therapy in restoring colon health after burn injury.

CXC chemokine receptor-4 signaling limits hepatocyte proliferation after hepatic ischemia-reperfusion in mice

The role of stromal cell-derived factor-1 (SDF-1 or CXCL12) and its receptor CXC chemokine receptor-4 (CXCR4) in ischemic liver injury and recovery has not been studied. Some reports suggest that this chemokine may aid in liver regeneration, but others suggest that it may be profibrotic through its activation of hepatic stellate cells. In this study we sought to elucidate the role of SDF-1 and its receptor CXCR4 during liver injury, recovery, and regeneration after ischemia-reperfusion (I/R). A murine model of partial (70%) I/R was used to induce liver injury and study the reparative and regenerative response. CXCR4 was expressed constitutively in the liver, and hepatic levels of SDF-1 peaked 8 h after reperfusion but remained significantly increased for 96 h. Treatment of mice with the CXCR4 antagonist AMD3100 or agonist SDF-1 had no effect on acute liver injury assessed 8 h after I/R. However, treatment with AMD3100 increased hepatocyte proliferation after 72 and 96 h of reperfusion and reduced the amount of liver necrosis. In contrast, treatment with SDF-1 significantly decreased hepatocyte proliferation. These effects appeared to be dependent on the presence of liver injury, as AMD3100 and SDF-1 had no effect on hepatocyte proliferation or liver mass in mice undergoing 70% partial hepatectomy. The data suggest that signaling through CXCR4 is detrimental to liver recovery and regeneration after I/R and that clinical therapy with a CXCR4 antagonist may improve hepatic recovery following acute liver injury.

Assessing the immune status of critically ill trauma patients by flow cytometry.

BackgroundUnintentional injury or trauma remains the leading cause of death among young adults. About one fifth of these trauma patients require care in an intensive care unit (ICU) because of severity of injuries and comorbidities. Patients hospitalized in an ICU are at increased risk for nosocomial infections, such as urinary tract infections, pneumonia, bacteremia, and wound infections. Many of these patients will develop sepsis or septic shock, and some will progress to multiple organ failure and death. The balance between the proinflammatory and counterinflammatory immune response appears to be a driving factor in this progression. At present, there is no proposed method for the timely detection of the immune status in trauma patients, making rational decisions to use immune-altering therapies difficult. ObjectiveWe demonstrate that flow cytometry, with its capabilities to characterize and/or enumerate (a) leukocyte subtypes, (b) leukocyte activation markers, (c) leukocyte-derived cytokines and microvesicles, and (d) leukocyte function is well suited to monitor the immune status of critically ill trauma patients. MethodsInformation for the review was obtained from the extant literature. DiscussionWe suggest that flow cytometry is a research method that might aid nurse scientists in investigating the immune status of critically ill patients, the recovery status of conditions such as hemorrhagic shock and tissue injury and the relationship between cancer disease progression and symptoms. Therefore, flow cytometry has the potential to broaden nursing research priority areas so that a comprehensive approach to understanding the cellular response is attained.

Burn injury influences the T cell homeostasis in a butyrate-acid sphingomyelinase dependent manner

Following burn injury, a key factor for patients susceptible to opportunistic infections is immune suppression. Butyrate levels are important in maintaining a functional immune system and these levels can be altered after injury. The acid sphingomyelinase (Asm) lipid signaling system has been implicated in a T cell actions with some evidence of being influenced by butyrate. Here, we hypothesized that burn-injury changes in butyrate levels would mediate Asm activity and, consequently, T cell homeostasis. We demonstrate that burn injury temporally decreases butyrate levels. We further determined that T cell Asm activity is increased by butyrate and decreased after burn injury. We additionally observed decreased T cell numbers in Asm-deficient, burn-injured, and microbiota-depleted mice. Finally, we demonstrate that butyrate reduced T cell death in an Asm-dependent manner. These data suggest that restoration of butyrate after burn injury may ameliorate the T cell lost observed in burn-injured patients by Asm regulation.

Impact of Platelets and Platelet-Derived Microparticles on Hypercoagulability Following Burn Injury.

ABSTRACT An acute burn induced coagulopathy develops after scald injury, which evolves into a subacute, hypercoagulable state. Microparticles, specifically platelet-derived MPs (PMPs), have been suggested as possible contributors. We first developed a model of burn-induced coagulopathy and then sought to investigate the role of platelets and PMPs in coagulation after burn. We hypothesized that changes in circulating platelet and PMP populations after injury would contribute to the post-burn, hypercoagulable state. A murine scald model with 28% TBSA full thickness burn injury was utilized and blood samples were collected at intervals after injury. Circulating MP populations, platelet counts, overall coagulation, and platelet function were determined. Burn injury led to hypercoagulability on post-burn day one (PBD1), which persisted 6 days after injury (PBD6). On PBD1, there was a significant decrease in platelet numbers and a decline in platelet contribution to clot formation with a concomitant increase in circulating procoagulant PMPs. On PBD6, there was a significant increase in platelet numbers and in platelet activation with no change in PMPs compared with sham. Further, on PBD1 decreased ADP-induced platelet activation was observed with a contrasting increase in ADP-induced platelet activation on PBD6. We therefore concluded that there was a temporal change in the mechanisms leading to a hypercoagulable state after scald injury, that PMPs are responsible for changes seen on PBD1, and finally that ADP-induced platelet activation was key to the augmented clotting mechanisms 6 days after burn.

Matrix Metalloproteinase-8 Augments Bacterial Clearance in a Juvenile Sepsis Model

Genetic ablation or pharmacologic inhibition of matrix metalloproteinase-8 (MMP8) improves survival in an adult murine sepsis model. Because developmental age influences the host inflammatory response, we hypothesized that developmental age influences the role of MMP8 in sepsis. First, we compared sepsis survival between wild-type (WT, C57BL/6) and MMP8 null juvenile-aged mice (12–14 d) after intraperitoneal injection of a standardized cecal slurry. Second, peritoneal lavages collected 6 h and 18 h after cecal slurry injection were analyzed for bacterial burden, leukocyte subsets and inflammatory cytokines. Third, juvenile WT mice were pretreated with an MMP8 inhibitor prior to cecal slurry injection; analysis of their bacterial burden was compared with vehicle-injected animals. Fourth, the phagocytic capacity of WT and MMP8 null peritoneal macrophages was compared. Finally, peritoneal neutrophil extracellular traps (NETs) were compared using immunofluorescent imaging and quantitative image analysis. We found that juvenile MMP8 null mice had greater mortality and higher bacterial burden than WT mice. Leukocyte counts and cytokine concentrations in the peritoneal fluid were increased in the MMP8 null mice relative to the wild-type mice. Peritoneal macrophages from MMP8 null mice had reduced phagocytic capacity compared to WT macrophages. There was no quantitative difference in NET formation, but fewer bacteria were adherent to NETs from MMP8 null animals. In conclusion, in contrast to septic adult mice, genetic ablation of MMP8 increased mortality following bacterial peritonitis in juvenile mice. This increase in mortality was associated with reduced bacterial clearance and reduced NET efficiency. We conclude that developmental age influences the role of MMP8 in sepsis.