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Background: As the meningeally derived, fibroblast-rich, mass-produced by intrathecal morphine infusion is not produced by all opiates, but reduced by mast cell stabilizers, the authors hypothesized a role for meningeal mast cell/fibroblast activation. Using the guinea pig, the authors asked: (1) Are intrathecal morphine masses blocked by opiate antagonism?; (2) Do opioid agonists not producing mast cell degranulation or fibroblast activation produce masses?; and (3) Do masses covary with Mas-related G protein-coupled receptor signaling thought to mediate mast cell degranulation?, Methods: In adult male guinea pigs (N = 66), lumbar intrathecal catheters connected to osmotic minipumps (14 days; 0.5 µl/h) were placed to deliver saline or equianalgesic concentrations of morphine sulfate (33 nmol/h), 2',6'-dimethyl tyrosine-(Tyr-D-Arg-Phe-Lys-NH2) (abbreviated as DMT-DALDA; 10 pmol/h; μ agonist) or PZM21 (27 nmol/h; biased μ agonist). A second pump delivered subcutaneous naltrexone (25 µg/h) in some animals. After 14 to 16 days, animals were anesthetized and perfusion-fixed. Drug effects on degranulation of human cultured mast cells, mouse embryonic fibroblast activation/migration/collagen formation, and Mas-related G protein-coupled receptor activation (PRESTO-Tango assays) were determined., Results: Intrathecal infusion of morphine, DMT-DALDA or PZM21, but not saline, comparably increased thermal thresholds for 7 days. Spinal masses proximal to catheter tip, composed of fibroblast/collagen type I (median: interquartile range, 0 to 4 scale), were produced by morphine (2.3: 2.0 to 3.5) and morphine plus naltrexone (2.5: 1.4 to 3.1), but not vehicle (1.2: 1.1 to 1.5), DMT-DALDA (1.0: 0.6 to 1.3), or PZM21 (0.5: 0.4 to 0.8). Morphine in a naloxone-insensitive fashion, but not PZM21 or DMT-DALDA, resulted in mast cell degranulation and fibroblast proliferation/collagen formation. Morphine-induced fibroblast proliferation, as mast cell degranulation, is blocked by cromolyn. Mas-related G protein-coupled receptor activation was produced by morphine and TAN67 (∂-opioid agonist), but not by PZM21, TRV130 (mu biased ligand), or DMT-DALDA., Conclusions: Opiates that activate Mas-related G protein-coupled receptor will degranulate mast cells, activate fibroblasts, and result in intrathecal mass formation. Results suggest a mechanistically rational path forward to safer intrathecal opioid therapeutics.

The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process.

Background: Vagal nerve stimulation (VNS) can have a marked anti-inflammatory effect. We have previously shown that preinjury VNS prevented intestinal barrier breakdown and preserved epithelial tight junction protein expression. However, a pretreatment model has little clinical relevance for the care of the trauma patient. Therefore, we postulated that VNS conducted postinjury would also have a similar protective effect on maintaining gut epithelial barrier integrity., Methods: Male balb/c mice were subjected to a 30% total body surface area, full-thickness steam burn followed by right cervical VNS at 15, 30, 60, 90, 120, and 150 minutes postinjury. Intestinal barrier dysfunction was quantified by permeability to 4 kDa fluorescein isothiocyanate-Dextran, histologic evaluation, gut tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay, and expression of tight junction proteins (myosin light chain kinase, occludin, and ZO-1) using immunoblot and immunoflourescence., Results: Histologic examination documented intestinal villi appearance similar to sham if cervical VNS was performed within 90 minutes of burn insult. VNS done after injury decreased intestinal permeability to fluorescein isothiocyanate-Dextran when VNS was ≤90 minutes after injury. Burn injury caused a marked increase in intestinal TNF-α levels. VNS-treated animals had TNF-α levels similar to sham when VNS was performed within 90 minutes of injury. Tight junction protein expression was maintained at near sham values if VNS was performed within 90 minutes of burn, whereas expression was significantly altered in burn., Conclusion: Postinjury VNS prevents gut epithelial breakdown when performed within 90 minutes of thermal injury. This could represent a therapeutic window and clinically relevant strategy to prevent systemic inflammatory response distant organ injury after trauma.

Loss of intestinal barrier function after burn injury allows movement of intraluminal contents across the mucosa, which can lead to the development of distant organ injury and multiple organ failure. Tight junction function is highly regulated by membrane-associated proteins including occludin and zonula occludens protein 1 (ZO-1), which can be modulated by systemic inflammation. We hypothesized that (1) burn injury leads to gut barrier injury, and (2) phosphodiesterase inhibition will attenuate these burn-induced changes. Male balb/c mice undergoing a 30% steam burn were randomized to resuscitation with normal saline or normal saline + pentoxifylline (PTX; 12.5 mg/kg). Intestinal injury was assessed by histological diagnosis and TNF-alpha levels using enzyme-linked immunosorbent assay. Intestinal permeability was assessed by measuring the plasma concentration of fluorescein isothiocyanate-dextran after intraluminal injection in the distal ileum. Occludin and ZO-1 levels were analyzed by immunoblotting and immunohistochemistry. Thirty percent total body surface area (TBSA) burn results in a significant increase in intestinal permeability. Treatment with PTX after burn attenuates intestinal permeability to sham levels. Burn injury resulted in a marked decrease in the levels of tight junction proteins occludin and ZO-1 at 6 and 24 h. The use of PTX after burn significantly decreases the breakdown of occludin and ZO-1. Pentoxifylline also attenuates the burn-induced increase in plasma and intestinal TNF-alpha. Confocal microscopy demonstrates that PTX attenuates the burn-induced reorganization of occludin and ZO-1 away from the tight junction. Pentoxifylline attenuates burn-induced intestinal permeability and decreases the breakdown and reorganization of intestinal occludin and ZO-1. Therefore, phosphodiesterase inhibition may be a useful adjunct strategy in the attenuation of burn-induced gut barrier injury.