J. Jason Hoth

Blunt chest trauma resulting in pulmonary contusion is a common but poorly understood injury. We previously demonstrated that lung contusion activates localized and systemic innate immune mechanisms and recruits neutrophils to the injured lung. We hypothesized that the innate immune and inflammatory activation of neutrophils may figure prominently in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function and pulmonary neutrophil recruitment. Comparisons were made between injured mice with and without neutrophil depletion. We further examined the role of chemokines and adhesion receptors in neutrophil recruitment to the injured lung. We found that lung injury and resultant physiological dysfunction after contusion were dependent on the presence of neutrophils in the alveolar space. We show that CXCL1, CXCL2/3, and CXCR2 are involved in neutrophil recruitment to the lung after injury and that intercellular adhesion molecule 1 is locally expressed and actively participates in this process. Injured gp91^phox-deficient mice showed improved lung function, indicating that oxidant production by neutrophil NADPH oxidase mediates lung dysfunction after contusion. These data suggest that both neutrophil presence and function are required for lung injury after lung contusion.

Introduction: Traumatic injury may result in an exaggerated response to subsequent immune stimuli such as nosocomial infection. This “second hit” phenomenon and molecular mechanism(s) of immune priming by traumatic lung injury, specifically, pulmonary contusion, remain unknown. We used an animal model of pulmonary contusion to determine whether the injury resulted in priming of the innate immune response and to test the hypothesis that resuscitation fluids could attenuate the primed response to a second hit. Methods: Male, 8 to 9 weeks, C57/BL6 mice with a pulmonary contusion were challenged by a second hit of intratracheal administration of the Toll-like receptor 4 agonist, lipopolysaccharide (LPS, 50 μg) 24 hours after injury (injury + LPS). Other experimental groups were injury + vehicle or LPS alone. A separate group was injured and resuscitated by 4 cc/kg of hypertonic saline (HTS) or Lactated Ringer’s (LR) resuscitation before LPS challenge. Mice were killed 4 hours after LPS challenge and blood, bronchoalveolar lavage, and tissue were isolated and analyzed. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest for significant differences (*p ≤ 0.05). Results: Injury + LPS showed immune priming observed by lung injury histology and increased bronchoalveolar lavage neutrophilia, lung myeloperoxidase and serum IL-6, CXCL1, and MIP-2 levels when compared with injury + vehicle or LPS alone. After injury, resuscitation with HTS, but not Lactated Ringer’s was more effective in attenuating the primed response to a second hit. Conclusion: Pulmonary contusion primes innate immunity for an exaggerated response to a second hit with the Toll-like receptor 4 agonist, LPS. We observed synergistic increases in inflammatory mediator expression in the blood and a more severe lung injury in injured animals challenged with LPS. This priming effect was reduced when HTS was used to resuscitate the animal after lung contusion.

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We previously demonstrated that toll-like receptor 2 (TLR-2) participates in the inflammatory response to lung injury. We hypothesized that the TLR-4, in an MyD88-dependent manner, may also participate in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function, pulmonary neutrophil recruitment, and the systemic innate immune response. Comparisons were made between wild-type mice and mice deficient in TLR-4 or MyD88. We found TLR-4-dependent responses to pulmonary contusion that include hypoxemia, edema, and neutrophil infiltration. Increased expression of IL-6 and chemokine (C-X-C motif) ligand 1 in the bronchoalveolar lavage and serum was also dependent on TLR-4 activation. We further demonstrated that these responses to pulmonary contusion were dependent on MyD88, an adapter protein in the signal transduction pathway mediated by TLRs. These results show that TLRs have a primary role in the response to acute lung injury. Lung inflammation and systemic innate immune responses are dependent on TLR activation by pulmonary contusion.

This study presents an image analysis algorithm for quantifying the volume fraction of pathology in serial histology of the rat lung. The right lung of male Sprague-Dawley rats (n=4) was contused using a controlled insult applied via an instrumented, electronic piston. The right (struck) and left lungs were excised at 48 hours post-insult, fixed in 10% formalin. Serial histology was taken at 1.5 mm intervals on the struck lung and 3 mm intervals on the left lung. Sections were stained with H&E and digitally imaged using a fixed, high-resolution digital camera. Immediately prior to sacrifice, computed tomography images of all subjects were also acquired. An automated image analysis program was applied to each image (8-bit, RGB) using morphological (dilatation and erosion), filtering (circular averaging), and thresholding techniques to sort pixels representing healthy lung tissue, pathologic lung tissue, and blood into separate masks. Blood was determined by multi-channel thresholding in the following inclusive ranges: Red (197-240), Green (0-90) and Blue (115-154). After applying the morphological and filtering operations, lung pathology was identified as all pixels exceeding a grayscale value of 85, with 0 being black and 255 being white. The average normalized volume of pathology across the data set was 12.63 +/- 11.40 %. The results showed greater volumes of tissue pathology than blood in all subjects indicating a good model for blunt lung trauma. CT segmentation data from the same four animals was used for a related study but was included for the purposes of comparison. These values were determined through a semi-automated approach and resulted in an average pathology volume of 16.92 +/- 9.66 %. Normalized pathology did not differ significantly between histology and CT (p = 0.18, paired t-test for sample means, a = 0.05). In general, the histology analysis algorithm and CT data resulted in similar trends across the data set. This approach can be used to improve the accuracy of both histology-based and CT-based segmentation processes.

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We report that Toll-like receptor (TLR) 2 participates in the inflammatory response to lung injury. To show this, we use a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans based on histologic, morphologic, and biochemical criteria of acute lung injury. The inflammatory response to pulmonary contusion in our mouse model is characterized by pulmonary edema, neutrophil transepithelial migration, and increased expression of the innate immunity proinflammatory cytokines IL 1β and IL 6, the adhesion intracellular adhesion molecule 1, and chemokine (CXC motif) ligand 1. Compared with wild-type animals, contused Tlr2(−/−) mice have significantly reduced pulmonary edema and neutrophilia. These findings are associated with decreased levels of circulating chemokine (CXC motif) ligand 1. In contrast, systemic IL 6 levels remain elevated in the TLR2-deficient phenotype. These results show that TLR2 has a primary role in the neutrophil response to acute lung injury. We suggest that an unidentified noninfectious ligand generated by pulmonary contusion acts via TLR2 to generate inflammatory responses.

The purpose of this study was to establish a semi-automated method for determining the extent of damage to the spleen from computed tomography (CT) scans. This method uses a software program to evaluate the differences in splenic tissue based on Hounsfield units. In this study, five patients that suffered blunt abdominal injury with splenic injury from a motor vehicle crash were evaluated. First, a trained radiologist identified the damaged areas of the spleens of each patient. From this assessment a semi-automated method of assessment was created using a wide range of Hounsfield units to create a mask of the entire spleen. The contrast range of the injured tissue, consisting of laceration, hematoma, contusion, or other injury was also identified. A percent injured tissue was created by dividing the injured tissue mask volume by the total spleen mask volume. The results demonstrated as the AIS grade increased, the percent injury volume increased. In conclusion, it is possible to calculate injury volumes from abdominal CT scans with minimal user inputs.

A comparison between two stochastic optimization techniques, adaptive simulated annealing (ASA) and the multi-island genetic algorithm (MIGA) is conducted to investigate which is the more attractive option for tuning a finite element model to match experimental data. The study probes the repeatability, robustness and sensitivity of each algorithm. While both algorithms produced FE results within one standard deviation of the experimental mean, the ASA approach proved more effective than the MIGA. The ASA algorithm demonstrated better repeatability after 1000 trials, with an average parameter value change of 9%. The ASA algorithm also probed a wider range of the solution space and produced results with significantly lower sum-of-squares error than the MIGA (t-test, one tailed, p < 0.001).

Chemokines direct leukocytes to areas of inflammation or injury. In general, CC chemokines (MCP-1, MIP-1α, RANTES) are chemoattractants for mononuclear cells and CXC (CINC-1, MIP-2α) for polymorphonuclear cells (PMNs). Herein we describe an open chest model of pulmonary contusion (PC) in a rodent (rat) and have identified a possible role for CC and CXC chemokines in the pathogenesis of PC. Sprague-Dawley rats (350 g) underwent thoracotomy. The exposed lung was struck with a piston at 5.2 m/s (150 J/M2). Blood, bronchoalveolar lavage (BAL), and lung tissue were collected at 3 hours and 24 hours after injury. PaO2/FiO2 (P/F) ratio was calculated at 15-minute intervals for 3 hours after contusion. Serum was evaluated for cytokine and chemokine expression using ELISA. Cell count/differential was performed on BAL, and lung tissue was obtained for histologic analysis, protein expression and wet to dry weights. Data are reported as pg/mL ± SE. Data were analyzed using Student's t test to identify significant differences (p ≤ 0.05 significant) between sham and injured animals. Piston impact caused PC based upon morphologic and histologic criteria. BAL cell count and lung wet to dry weights were increased and P/F ratio was decreased after PC. Systemic levels of IL-ra, MCP-1, and the CXC chemokines MIP-2α and CINC-1 were significantly elevated at 3 hours when sham and injured animals were compared. All chemokines were found to be significantly elevated at 24 hours, consistent with the early PMN and subsequent mononuclear infiltration observed in the contused lung. Pulmonary expression of TNF-α, IL-1β, CINC-1, MIP-2α, ICAM-1, and elastase were increased and activated systemic neutrophils showed increased CD-11b. A model of PC is described in which innate inflammation is activated locally and systemically. Systemic levels of CC and CXC chemokines are increased after PC. This correlates with elevated PMN CD-11b expression, enhanced pulmonary ICAM-1 expression, and mononuclear and PMN infiltration into the lung and alveolar space. Elevated levels of CC and CXC chemokines are seen after PC and may be involved in the lung's inflammatory response to injury.