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Characterization of blunt chest trauma in a long-term porcine model of severe multiple trauma
(2016)
Chest trauma has a significant relevance on outcome after severe trauma. Clinically, impaired lung function typically occurs within 72 hours after trauma. However, the underlying pathophysiological mechanisms are still not fully elucidated. Therefore, we aimed to establish an experimental long-term model to investigate physiological, morphologic and inflammatory changes, after severe trauma. Male pigs (sus scrofa) sustained severe trauma (including unilateral chest trauma, femur fracture, liver laceration and hemorrhagic shock). Additionally, non-injured animals served as sham controls. Chest trauma resulted in severe lung damage on both CT and histological analyses. Furthermore, severe inflammation with a systemic increase of IL-6 (p = 0.0305) and a local increase of IL-8 in BAL (p = 0.0009) was observed. The pO2/FiO2 ratio in trauma animals decreased over the observation period (p < 0.0001) but not in the sham group (p = 0.2967). Electrical Impedance Tomography (EIT) revealed differences between the traumatized and healthy lung (p < 0.0001). In conclusion, a clinically relevant, long-term model of blunt chest trauma with concomitant injuries has been developed. This reproducible model allows to examine local and systemic consequences of trauma and is valid for investigation of potential diagnostic or therapeutic options. In this context, EIT might represent a radiation-free method for bedside diagnostics.
Background: Hypothermia has been discussed as playing a role in improving the early phase of systemic inflammation. However, information on the impact of hypothermia on the local inflammatory response is sparse. We therefore investigated the kinetics of local and systemic inflammation in the late posttraumatic phase after induction of hypothermia in an established porcine long-term model of combined trauma.
Materials & Methods: Male pigs (35 ± 5kg) were mechanically ventilated and monitored over the study period of 48 h. Combined trauma included tibia fracture, lung contusion, liver laceration and pressure-controlled hemorrhagic shock (MAP < 30 ± 5 mmHg for 90 min). After resuscitation, hypothermia (33°C) was induced for a period of 12 h (HT-T group) with subsequent re-warming over a period of 10 h. The NT-T group was kept normothermic. Systemic and local (fracture hematoma) cytokine levels (IL-6, -8, -10) and alarmins (HMGB1, HSP70) were measured via ELISA.
Results: Severe signs of shock as well as systemic and local increases of pro-inflammatory mediators were observed in both trauma groups. In general the local increase of pro- and anti-inflammatory mediator levels was significantly higher and prolonged compared to systemic concentrations. Induction of hypothermia resulted in a significantly prolonged elevation of both systemic and local HMGB1 levels at 48 h compared to the NT-T group. Correspondingly, local IL-6 levels demonstrated a significantly prolonged increase in the HT-T group at 48 h.
Conclusion: A prolonged inflammatory response might reduce the well-described protective effects on organ and immune function observed in the early phase after hypothermia induction. Furthermore, local immune response also seems to be affected. Future studies should aim to investigate the use of therapeutic hypothermia at different degrees and duration of application.
In their post-traumatic course, trauma patients suffering from multiple injuries have a high risk for immune dysregulation, which may contribute to post-injury complications and late mortality. Monocytes as specific effector cells of the innate immunity play a crucial role in inflammation. Using their Pattern Recognition Receptors (PRRs), notably Toll-Like Receptors (TLR), the monocytes recognize pathogens and/or pathogen-associated molecular patterns (PAMPs) and organize their clearance. TLR2 is the major receptor for particles of gram-positive bacteria, and initiates their phagocytosis. Here, we investigated the phagocytizing capability of monocytes in a long-term porcine severe trauma model (polytrauma, PT) with regard to their TLR2 expression. Polytrauma consisted of femur fracture, unilateral lung contusion, liver laceration, hemorrhagic shock with subsequent resuscitation and surgical fracture fixation. After induction of PT, peripheral blood was withdrawn before (-1 h) and directly after trauma (0 h), as well as 3.5 h, 5.5 h, 24 h and 72 h later. CD14+ monocytes were identified and the expression levels of H(S)LA-DR and TLR2 were investigated by flow cytometry. Additionally, the phagocytizing activity of monocytes by applying S. aureus particles labelled with pHrodo fluorescent reagent was also assessed by flow cytometry. Furthermore, blood samples from 10 healthy pigs were exposed to a TLR2-neutralizing antibody and subsequently to S. aureus particles. Using flow cytometry, phagocytizing activity was determined. P below 0.05 was considered significant. The number of CD14+ monocytes of all circulating leukocytes remained constant during the observational time period, while the percentage of CD14+H(S)LA-DR+ monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of TLR2+ expressing cells out of all monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of phagocytizing monocytes decreased immediately and remained lower during the first 3.5 h after trauma, but increased after 24 h. Antagonizing TLR2 significantly decreased the phagocytizing activity of monocytes. Both, decreased percentage of activated as well as TLR2 expressing monocytes persisted as long as the reduced phagocytosis was observed. Moreover, neutralizing TLR2 led to a reduced capability of phagocytosis as well. Therefore, we assume that reduced TLR2 expression may be responsible for the decreased phagocytizing capacity of circulating monocytes in the early post-traumatic phase.
Background: Recognizing patients at risk for pulmonary complications (PC) is of high clinical relevance. Migration of polymorphonuclear leukocytes (PMN) to inflammatory sites plays an important role in PC, and is tightly regulated by specific chemokines including interleukin (IL)−8 and other mediators such as leukotriene (LT)B4. Previously, we have reported that LTB4 indicated early patients at risk for PC after trauma. Here, the relevance of LTB4 to indicating lung integrity in a newly established long-term porcine severe trauma model (polytrauma, PT) was explored.
Methods: mTwelve pigs (3 months old, 30 ± 5 kg) underwent PT including standardized femur fracture, lung contusion, liver laceration, hemorrhagic shock, subsequent resuscitation and surgical fracture fixation. Six animals served as controls (sham). After 72 h lung damage and inflammatory changes were assessed. LTB4 was determined in plasma before the experiment, immediately after trauma, and after 2, 4, 24 or 72 h. Bronchoalveolar lavage (BAL)-fluid was collected prior and after the experiment.
Results: Lung injury, local gene expression of IL-8, IL-1β, IL-10, IL-18 and PMN-infiltration into lungs increased significantly in PT compared with sham. Systemic LTB4 increased markedly in both groups 4 h after trauma. Compared with declined plasma LTB4 levels in sham, LTB4 increased further in PT after 72 h. Similar increase was observed in BAL-fluid after PT.
Conclusions: In a severe trauma model, sustained changes in terms of lung injury and inflammation are determined at day 3 post-trauma. Specifically, increased LTB4 in this porcine long-term model indicated a rapid inflammatory alteration both locally and systemically. The results support the concept of LTB4 as a biomarker for PC after severe trauma and lung contusion.
Background: Severely injured patients experience substantial immunological stress in the aftermath of traumatic insult, which often results in systemic immune dysregulation. Regulatory T cells (Treg) play a key role in the suppression of the immune response and in the maintenance of immunological homeostasis. Little is known about their presence and dynamics in blood after trauma, and nothing is known about Treg in the porcine polytrauma model. Here, we assessed different subsets of Treg in trauma patients (TP) and compared those to either healthy volunteers (HV) or data from porcine polytrauma.
Methods: Peripheral blood was withdrawn from 20 TP with injury severity score (ISS) ≥16 at the admittance to the emergency department (ED), and subsequently on day 1 and at day 3. Ten HV were included as controls (ctrl). The porcine polytrauma model consisted of a femur fracture, liver laceration, lung contusion, and hemorrhagic shock resulting in an ISS of 27. After polytrauma, the animals underwent resuscitation and surgical fracture fixation. Blood samples were withdrawn before and immediately after trauma, 24 and 72 h later. Different subsets of Treg, CD4+CD25+, CD4+CD25+FoxP3+, CD4+CD25+CD127−, and CD4+CD25+CD127−FoxP3+ were characterized by flow cytometry.
Results: Absolute cell counts of leukocytes were significantly increasing after trauma, and again decreasing in the follow-up in human and porcine samples. The proportion of human Treg in the peripheral blood of TP admitted to the ED was lower when compared to HV. Their numbers did not recover until 72 h after trauma. Comparable data were found for all subsets. The situation in the porcine trauma model was comparable with the clinical data. In porcine peripheral blood before trauma, we could identify Treg with the typical immunophenotype (CD4+CD25+CD127−), which were virtually absent immediately after trauma. Similar to the human situation, most of these cells expressed FoxP3, as assessed by intracellular FACS stain.
Conclusion: Despite minor percental differences in the recovery of Treg populations after trauma, our findings show a comparable decrease of Treg early after polytrauma, and strengthen the immunological significance of the porcine polytrauma model. Furthermore, the Treg subpopulation CD4+CD25+CD127− was characterized in porcine samples.
Blunt thoracic trauma (TxT) deteriorates clinical post-injury outcomes. Ongoing inflammatory changes promote the development of post-traumatic complications, frequently causing Acute Lung Injury (ALI). Club Cell Protein (CC)16, a pulmonary anti-inflammatory protein, correlates with lung damage following TxT. Whether CC16-neutralization influences the inflammatory course during ALI is elusive. Ninety-six male CL57BL/6N mice underwent a double hit model of TxT and cecal ligation puncture (CLP, 24 h post-TxT). Shams underwent surgical procedures. CC16 was neutralized by the intratracheal application of an anti-CC16-antibody, either after TxT (early) or following CLP (late). Euthanasia was performed at 6 or 24 h post-CLP. Systemic and pulmonary levels of IL-6, IL-1β, and CXCL5 were determined, the neutrophils were quantified in the bronchoalveolar lavage fluid, and histomorphological lung damage was assessed. ALI induced a significant systemic IL-6 increase among all groups, while the local inflammatory response was most prominent after 24 h in the double-hit groups as compared to the shams. Significantly increased neutrophilic infiltration upon double hit was paralleled with the enhanced lung damage in all groups as compared to the sham, after 6 and 24 h. Neutralization of CC16 did not change the systemic inflammation. However, early CC16-neutralization increased the neutrophilic infiltration and lung injury at 6 h post-CLP, while 24 h later, the lung injury was reduced. Late CC16-neutralization increased neutrophilic infiltration, 24 h post-CLP, and was concurrent with an enhanced lung injury. The data confirmed the anti-inflammatory potential of endogenous CC16 in the murine double-hit model of ALI.
Short Summary: Extracellular vesicles (EVs), released during tissue/cell injury, contain a "barcode" indicating specific microRNAs (miRs) that can uncover their origin. We examined whether systemic EVs possessing hepatic miR-signatures would indicate ongoing liver injury and clinical complications in trauma patients (TP). We grouped the patients of alcoholic drinkers into "alcohol-drinkers with liver injury (LI)" (EtOH with LI) or "alcohol-drinkers without LI" (EtOH w/o LI) and we compared these groups to "non-drinkers" (no EtOH). When we examined patient blood from the EtOH with LI group we found the total number of EVs to be increased, along with an increase in miR-122 and let7f—two EV-associated miRNAs—and several inflammation-associating cytokines, such as interleukin (IL)-6 and IL-33. In contrast, all of the aforementioned readouts were found to be decreased in the EtOH w/o LI group. These novel data demonstrate that hepatocyte damage in alcohol-intoxicated trauma patients presenting with liver injury can be reflected by an increase in circulating serum EVs, their specific miR-"barcode" and the concomitant increase of systemic inflammatory markers IL-6 and IL-33. Anti-inflammatory effect of alcohol-drinking in EtOH w/o LI can be presented by a reduced number of hepato-derived EVs, no upregulation of IL-6 and IL-33, and a miR "barcode" different from patients presenting with liver injury.
Background: Alcohol abuse is associated with (neuro)protective effects related to (head) injuries, and with negative effects regarding infection rates and survival in severely injured trauma patients (TP). Extracellular vesicles (EVs), which are released during tissue and/or cell injury, can contain a "barcode" including specific microRNAs (miRs) that uncover their origin. We examined whether EVs with a hepatic miR signature can be systemically measured, and whether they can indicate ongoing liver injury in alcohol-intoxicated TP and foretell clinical complications.
Patients/Methods: We enrolled 35 TP and measured blood EVs, IL-6, TNF-alpha, IL-1beta, IL-10 and IL-33, alcohol (ethanol, EtOH) concentration (BAC), GLDH, GGT, AST, ALT, leukocytes, platelets, and bilirubin. Within circulating EVs we measured the expression levels of miR-122, let7f, miR21, miR29a, miR-155, and miR-146a. Patients of alcohol-drinkers were grouped into "alcohol drinkers with liver injury (LI)" (EtOH with LI) or "alcohol drinkers without LI" (EtOH w/o LI) and compared to "non-drinkers" (no EtOH). We assessed systemic injury characteristics and the outcome of hospitalization with regard to sepsis, septic shock, pneumonia, or mortality.
Results: EtOH with LI patients had significantly increased rates of pneumonia vs. the EtOH w/o LI group. EVs, IL-6, and IL-33 levels were significantly increased in EtOH with LI vs. EtOH w/o LI group (p < 0.05). EV number correlated positively with ALT and IL-6 (p < 0.0001). Two miRs, miR-122 and let7f, were increased only in the blood EVs from the EtOH with LI group (p < 0.05). Five miRs, miR-122, let7f, miR-21, miR-29a, and miR-146a, were reduced in the blood EVs from the EtOH w/o LI group, vs. no EtOH (p < 0.05). Notably miR-122 correlated significantly with increased bilirubin levels in the EtOH with LI group (p < 0.05).
Conclusions: Liver injury in alcohol-intoxicated TP is reflected by increased EV numbers, their specific miR barcode, and the correlated increase of systemic inflammatory markers IL-6 and IL-33. Interestingly, severely injured TP without liver injury were found to have a reduced number of liver-derived EVs, no observed inflammatory infiltration and reduced specific miR "barcode."
Sepsis is a serious clinical condition which can cause life-threatening organ dysfunction, and has limited therapeutic options. The paradigm of limiting excessive inflammation and promoting anti-inflammatory responses is a simplified concept. Yet, the absence of intrinsic anti-inflammatory signaling at the early stage of an infection can lead to an exaggerated activation of immune cells, including monocytes and macrophages. There is emerging evidence that endogenous molecules control those mechanisms. Here we aimed to identify and describe the dynamic changes in monocyte and macrophage subsets and lung damage in CL57BL/6N mice undergoing blunt chest trauma with subsequent cecal ligation and puncture. We showed that early an increase in systemic and activated Ly6C+CD11b+CD45+Ly6G− monocytes was paralleled by their increased emigration into lungs. The ratio of pro-inflammatory Ly6ChighCD11b+CD45+Ly6G− to patrolling Ly6ClowCD11b+CD45+Ly6G− monocytes significantly increased in blood, lungs and bronchoalveolar lavage fluid (BALF) suggesting an early transition to inflammatory phenotypes during early sepsis development. Similar to monocytes, the level of pro-inflammatory Ly6ChighCD45+F4/80+ macrophages increased in lungs and BALF, while tissue repairing Ly6ClowCD45+F4/80+ macrophages declined in BALF. Levels of inflammatory mediators TNF-α and MCP-1 in blood and RAGE in lungs and BALF were elevated, and besides their boosting of inflammation via the recruitment of cells, they may promote monocyte and macrophage polarization, respectively, toward the pro-inflammatory phenotype. Neutralization of uteroglobin increased pro-inflammatory cytokine levels, activation of inflammatory phenotypes and their recruitment to lungs; concurrent with increased pulmonary damage in septic mice. In in vitro experiments, the influence of uteroglobin on monocyte functions including migratory behavior, TGF-β1 expression, cytotoxicity and viability were proven. These results highlight an important role of endogenous uteroglobin as intrinsic anti-inflammatory signal upon sepsis-induced early lung injury, which modules the early monocyte/macrophages driven inflammation.
Background: Extremity fracture is frequently seen in multiple traumatized patients. Local post-traumatic inflammatory reactions as well as local and systemic interactions have been described in previous studies. However, trauma severity and its impact on the local immunologic reaction remains unclear. Therefore, fracture-associated local inflammation was investigated in a porcine model of isolated and combined trauma to gain information about the early inflammatory stages.
Material and Methods: Polytrauma (PT) consisted of lung contusion, liver laceration, femur fracture, and controlled hemorrhage. Monotrauma (MT) consisted of femur fracture only. The fracture was operatively stabilized and animals were monitored under ICU-standard for 72 h. Blood, fracture hematoma (FH) as well as muscle samples were collected throughout the experimental period. Levels of local and systemic pro- and anti-inflammatory as well as angiogenetic cytokines were measured by ELISA.
Results: Both groups showed a significant decrease in pro-inflammatory IL-6 in FH over time. However, concentrations in MT were significantly higher than in PT. The IL-8 concentrations initially decreased in FH, but recovered by the end of the observation period. These dynamics were only statistically significant in MT. Furthermore, concentrations measured in muscle tissue showed inverse kinetics compared to those in FH. The IL-10 did not present statistical resilient dynamics over time, although a slight increase in FH was seen by the end of the observation time in the MT group.
Conclusions: Time-dependent dynamics of the local inflammatory response were observed. Trauma severity showed a significant impact, with lower values in pro- as well as angiogenetic mediators. Fracture repair could be altered by these trauma-related changes of the local immunologic milieu, which might serve as a possible explanation for the higher rates of delayed or non-union bone repair in polytraumatised patients.
Background: Polytraumatized patients undergo a strong immunological stress upon insult. Phagocytes (granulocytes and monocytes) play a substantial role in immunological defense against bacteria, fungi and yeast, and in the clearance of cellular debris after tissue injury. We have reported a reduced monocytes phagocytic activity early after porcine polytrauma before. However, it is unknown if both phagocyte types undergo those functional alterations, and if there is a pathogen-specific phagocytic behavior. We characterized the phagocytic activity and capacity of granulocytes and monocytes after polytrauma.
Methods: Eight pigs (Sus scrofa) underwent polytrauma consisting of lung contusion, liver laceration, tibial fracture and hemorrhagic shock with fluid resuscitation and fracture fixation with external fixator. Intensive care treatment including mechanical ventilation for 72 h followed. Phagocytic activity and capacity were investigated using an in vitro ex vivo whole blood stimulation phagocytosis assays before trauma, after surgery, 24, 48, and 72 h after trauma. Blood samples were stimulated with Phorbol-12-myristate-13-acetate and incubated with FITC-labeled E. coli, S. aureus or S. cerevisiae for phagocytosis assessment by flow cytometry.
Results: Early polytrauma-induced significant increase of granulocytes and monocytes declined to baseline values within 24 h. Percentage of E. coli-phagocytizing granulocytes significantly decreased after polytrauma and during further intensive care treatment, while their capacity significantly increased. Interestingly, both granulocytic phagocytic activity and capacity of S. aureus significantly decreased after trauma, although a recovery was observed after 24 h and yet was followed by another decrease. The percentage of S. cerevisiae-phagocytizing granulocytes significantly increased after 24 h, while their impaired capacity after surgery and 72 h later was detected. Monocytic E. coli-phagocytizing percentage did not change, while their capacity increased after 24–72 h. After a significant decrease in S. aureus-phagocytizing monocytes after surgery, a significant increase after 24 and 48 h was observed without capacity alterations. No significant changes in S. cerevisiae-phagocytizing monocytes occurred, but their capacity dropped 48 and 72 h.
Conclusion: Phagocytic activity and capacity of granulocytes and monocytes follow a different pattern and significantly change within 72 h after polytrauma. Both phagocytic activity and capacity show significantly different alterations depending on the pathogen strain, thus potentially indicating at certain and possibly more relevant infection causes after polytrauma.