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    • br Materials and methods All animal experiments were perform

    2023-12-21


    Materials and methods All animal experiments were performed using the recommendations of the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and approved by the University of Colorado—Denver Institutional Animal Care and Use Committee. Adult male Sprague—Dawley rats (Harlan Laboratories, Indianapolis IN) of uniform weight (range 350-425 g) were supplied with food and water ad libitum, maintained in climate-controlled facilities with 12-h light/dark cycles, and allowed to acclimate for at least 1 wk before experimentation. No female rats were included in this study due to their resistance to end organ dysfunction following T/HS. Sample size calculation was performed with two-tailed tests with α set to 0.05, power at 80%, and an estimated standard deviation of 20%. We wanted to be able to detect an effect size of 35%-40%, giving us a sample size of 6-8 animals per group.
    Results
    Discussion Post-traumatic lung injury is the most common type of organ dysfunction following trauma. Despite a decreasing incidence over time, lung injury still carries a high cost to the trauma system and substantial mortality. Recent work has implicated crystalloid as a risk factor for development of ARDS following trauma. As trauma centers continue to work on limiting crystalloid infusion, this may be responsible for the decreased incidence of ARDS in this population. The role of blood products in the development of post-traumatic lung injury is less clear, although some studies have implicated increased blood product transfusion with the development of lung injury; others did not find this association.13, 14, 15 This may be confounded by the role of shock, as patients with higher degrees of shock will need more blood products. Shock appears to be central to the development of an inflammatory state through production of the bioactive eicosanoids. This contributes to post-traumatic lung injury secondary to the disruption of blood flow to the splanchnic vascular beds.5, 16, 17, 18 Specifically, postshock mesenteric peficitinib has been found to play an integral role in the pathogenesis of lung injury following shock, likely due to its high concentration of AA.2, 4, 19, 20 AA has been found to prime the PMN for increased superoxide anion and elastase release to facilitate the respiratory burst. In addition, primed PMNs can convert free AA to acute-phase proinflammatory eicosanoids that include the synthesis of the leukotrienes via the 5-lipoxygenase pathway. These include LTB4, an effective PMN chemoattractant and potentiator of PMN-mediated cytotoxicity, and LTC4, the precursor to the downstream vasoactive cysteinyl leukotrienes, which have peficitinib been shown to increase vascular permeability allowing PMN extravasation.21, 22, 23, 24 ALOX5 catalyzes the first step in leukotriene biosynthesis. In its inactive state, ALOX5 is a soluble protein found in both the cytoplasm and nuclear matrix.25, 26 With PMN activation, ALOX5 is regulated through several distinct mechanisms that serve to augment leukotriene A4 (LTA4) synthesis and localize the enzyme to the nuclear membrane.27, 28, 29, 30 Furthermore, ALOX5 interacts with its cofactor ALOX5AP and further potentiates its catalytic activity. The ALOX5/ALOX5AP complex has a concomitant interaction with membrane-bound LTC4 synthase resulting in localized cysteinyl leukotriene synthesis.6, 25 These data show that ALOX5 increases following T/HS and closely associates with ALOX5AP. Because of the use of the fluorescent secondary antibodies in these experiments, a positive FRET can only be achieved when the maximal distance between ALOX5 and ALOX5AP is < 30 nm. This close association is essential for post-traumatic lung damage as elimination of ALOX5/ALOX5AP complex formation by MK-886 reduces that damage. Prior studies have suggested that ALOX5AP has two primary functions: (1) a protein anchor localizing ALOX5 to the nuclear membrane6, 26, 32 and (2) a nonenzymatic carrier for AA.6, 32, 33 This study showed no change in ALOX5AP activity after treatment with MK-886. However, we did show a decrease in ALOX5. This is likely a consequence of the decreased association of ALOX5/ALOX5AP (as seen in the FRET signal) and the now free ALOX5 being more susceptible to degradation than when complexed and localized to the nuclear membrane. Prior studies have shown that ALOX5 and ALOX5AP colocalize to the nuclear and perinuclear domains; this study demonstrated association of ALOX5/ALOX5AP in all compartments. While perinuclear FRET signal was highest, the increased levels seen in the cytoplasmic and nuclear compartments may be due to local membrane and organelle destruction with diffusion of ALOX5/ALOX5AP into adjacent compartments.