Epidemiological data show that the degree of active or passive cigarette smoke (CS) exposure is directly related to the development of acute lung injury (ALI) after blunt trauma (1). Moreover, active cigarette smoking increases the susceptibility to develop Acute Respiratory Distress Syndrome (ARDS) despite younger age and better overall general health status (2). In mechanically ventilated rats, CS exposure prior to ventilator-induced lung injury aggravated tissue inflammation and apoptosis (3), and in mice undergoing blunt chest trauma this effect was associated with enhanced nitrosative stress, ultimately leading to more pronounced histological damage (4).
Both chronic (e.g., asthma (5) and CS exposure-induced chronic obstructive pulmonary disease (6)) and acute lung injury (e.g., due to endotoxin (7–9) and blunt chest trauma (10)) are associated with up-regulation of the tissue expression of the purinergic receptors P2XR4 or P2XR7. Pretraumatic CS exposure further enhanced this effect in mice after blunt chest trauma (4). Genetic deletion (7, 11, 12) or pharmacological blockade (9, 11) of P2XR7 reduced pulmonary hyperinflammation and thereby attenuated ALI after administration of endotoxin (7, 9, 11) or cecal-ligation-and-puncture (12). All these data, however, originated from un-resuscitated murine models that did not integrate standard intensive care measures, in particular mechanical ventilation. Due to the significant upregulation of P2XR4 and P2XR7 in lung tissue both after blunt chest trauma (10) and after blunt chest trauma and cigarette smoke exposure (4) in our previous studies, we tested the hypothesis whether genetic P2XR4 deletion would attenuate the acute pulmonary inflammatory response and improve organ function after blunt chest trauma and 4 h of lung-protective mechanical ventilation in mice with and without CS exposure. We studied P2XR4 knock-out (P2XR4−/−) mice rather than P2XR7−/− animals because there is evidence that P2XR4 co-expression enhances the P2XR7-induced inflammatory response (13, 14) and P2XR4 up-regulation compensates for reduced P2XR7 expression (15).
MATERIALS AND METHODS
The study protocol was approved by the University Animal Care Committee and the federal authorities for animal research of the Regierungspräsidium Tübingen, Baden-Württemberg, Germany. The experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. A total of n = 15 C57BL/6J mice (Charles River, Kisslegg, Germany) and n = 17 P2XR4−/− mice (pan-knockout, genetic background C57BL/6, GlaxoSmithKline Research & Development Limited, Stevenage, UK) of either gender at an age of 10 weeks to 16 weeks and weighing 25 g ± 3 g were used, housed in isolated, ventilated cages under a 12-h light–dark cycle, and received food and water ad libitum. The genotype of P2XR4−/− mice was confirmed by polymerase chain reaction of tail snip deoxyribonucleic acid prior to the experiments (see supplementary Figure 1, http://links.lww.com/SHK/A454). Two additional mice of each genetic profile did neither undergo CS exposure, anesthesia, chest trauma, nor surgery, and served as controls for immunoblotting.
Cigarette smoke-induced pulmonary inflammation
In order to address the hypothesis, n = 7 C57BL/6J mice and n = 8 P2XR4−/− mice were exposed to cigarette smoke (“CS”) over 3 to 4 weeks for 5 days per week in an exposure box as described previously (4, 16). Thereafter, 1 week was allowed as a recovery period prior to the blast experiment. This approach was chosen to avoid any acute stress effect induced by the CS-exposure procedure per se(4). Mice received four cigarettes (Roth-Händle without filters, tar 10 mg, nicotine 1.0 mg, carbon monoxide 6 mg, Badische Tabakmanufaktur Roth-Händle, Lahr, Germany) on day 1, six cigarettes on day 2, and eight cigarettes for the following days of the exposure period lasting for 15 min for each cigarette, which was followed by 8 min with fresh air (15 L min−1), and an additional 24-min break after each second cigarette. A semiautomatic cigarette lighter and smoke generator with an electronic timer was used to control the exposure (Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany). Particle concentration was monitored by a real-time ambient particle monitor (MicroDust Pro, Casella, Amherst, NH). In pilot experiments and a previous study (4), this CS-exposure procedure had not caused any effect on the behavior, body weight, or respiratory pattern. Control animals (“Non-CS,” n = 8 C57BL/6J mice, and n = 9 P2XR4−/− mice) were exposed to room air.
Anesthesia, blast wave, surgery, and experimental protocol
Mice were anesthetized with a mixture of 2.5% sevoflurane (Sevorane, Abbott, Wiesbaden, Germany) in 60% O2 and 40% N2 and buprenorphine i.p. (1 μg·g−1). Blunt chest trauma was induced by a single blast wave centered on the thorax as described previously (4, 17). Briefly, compressed air rapidly ruptures a Mylar polyester film (Du Pont de Nemur, Bad Homburg, Germany), which releases a reproducible single blast wave centered toward the animal's midsternal chest and thus induces a reproducible contusion of the lungs without remote organ damage (see supplementary Figure 2, http://links.lww.com/SHK/A455). Immediately after trauma, animals received i.p. ketamine (85 μg·g−1), midazolam (0.9 μg·g−1), and fentanyl (0.18 μg·g−1) and were placed on a procedure bench equipped with a closed-loop system to control body temperature (4, 17, 18). An incision was made in the anterior neck to expose the trachea. The trachea was intubated, and the lungs were mechanically ventilated with a pressure-controlled, lung-protective ventilation strategy (i.e., application of low tidal volumes of 4–6 μL·g−1 and a positive end-expiratory pressure (PEEP) during ventilation) using a specially designed small animal ventilator (FlexiVentTM, Scireq, Montreal, Canada). After a lung recruitment manoeuver consisting of an inspiratory hold at 18 cm H2O over 5 s, the initial respirator settings were: tidal volume 6 μL·g−1, respiratory rate 150 breaths min−1, inspiratory/expiratory time ratio 1:2, PEEP 3 cm H2O, and FiO2 = 0.21. Recruitment manoeuvers were repeated hourly, because this approach had allowed maintaining thoraco-pulmonary compliance in the physiological range by preventing atelectasis and reduced pulmonary inflammation (19). Catheters were inserted into the right internal jugular vein, the right carotid artery, and the bladder. Anesthesia was maintained with continuous i.v. ketamine (100–150 μg·g−1·h−1), midazolam (0.2–0.3 μg·g−1·h−1), and fentanyl (1–1.5 μg·g−1·h−1). Anesthetic drugs were titrated to reach deep sedation and analgesia as documented by complete tolerance against noxious stimuli. To maintain mean arterial pressure >55 mm Hg, animals received 12 μL·g−1· h−1 of hydroxyethyl starch in a balanced electrolyte solution (Tetraspan 6%, 130, 0.42, Braun Medical, Melsungen, Germany).
All animals were studied over 4 h of mechanical ventilation. Systemic hemodynamics (heart rate, mean blood pressure) and body temperature were recorded hourly. The static thoraco-pulmonary compliance was measured hourly by incrementally increasing the airway pressure up to a maximum inspiratory pressure of 20 cm H2O and using the indwelling software of the respirator that allows automatic recording of the inspiratory and expiratory pressure–volume loop. Arterial blood samples were taken hourly for blood gases and pH. The respiratory rate was titrated to maintain arterial PCO2 at 30 mm Hg to 40 mm Hg, the PEEP level was titrated according to the arterial PO2: if the PaO2/FiO2 ratio was <300 mm Hg PEEP was increased to 5 cmH2O, and if 100 mm Hg <PaO2/FiO2 <200 mm Hg, it was increased to 8 cmH2O. At the end of the observation period, animals were killed through blood withdrawal via the carotid artery. Immediately thereafter the respiratory tubings were clamped at end-expiration, i.e., PEEP level, the thorax was opened, and the lungs were removed.
Plasma and lung tissue preparation
Whole blood was immediately spun, and plasma was stored at −80°C until analysis. The right lung was sampled, immediately frozen in liquid nitrogen and stored at −80°C. At the time of analysis, lung tissue was homogenized and lysed in lysing buffer. For cell extract preparation, cells were resuspended, lysed on ice, and centrifuged. The supernatant fluid (protein extract) was stored at −80°C and used for the measurement of the cytokine profile and for immunoblotting. The left lung was fixed in formalin and paraffinembedded for histology and immunohistochemistry.
Plasma and lung tissue levels of the cytokines and chemokines tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, IL-10, IL-18, keratinocyte chemoattractant (KC), and monocyte chemotactic protein-1 (MCP-1) were measured by a mouse multiplex cytokine kit (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, Calif) in accordance with the manufacturer's instructions (4, 17, 18). In brief, the appropriate standards and samples (50 μL protein extract per sample) were added to a filter plate. The samples were incubated with antibodies chemically attached to fluorescent-labeled micro beads. Thereafter, premixed detection antibodies were added to each well, and streptavidinphycoerythrin was added. Beads were then resuspended, and the cytokines reaction mixture was quantified using the Bio-Plex protein array reader. Data were automatically processed and analyzed by Bio-Plex Manager Software 4.1 using the standard curve produced from recombinant cytokine standards. Levels below the detection limit of the assays were set to zero for statistical purposes.
For the assessment of the expression of heme oxygenase-1 (HO-1), B-cell lymphoma extra large (Bcl-xL) and natural inhibitor IκBα of the nuclear transcription factor kappa-B, protein concentrations were determined, and equal total protein aliquots (20–60 μg) were separated by SDS-PAGE and transferred by Western blotting (4, 17, 18).
After blocking, the membranes were incubated with commercially available primary antibodies (anti-HO-1, Abcam, Cambridge, NY; anti-Bcl-xL, Cell Signaling, Danvers, Mass; anti-IκBα, Thermo Fisher Scientific, Waltham, Mass). The primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, Mass or Santa Cruz, Dallas, Tex). The membranes were subjected to chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Exposed films were scanned, and intensity of immunoreactivity was measured using NIH ImageJ software (http://rsb.info.nih.gov/nih-image). For comparison between individual blots, the intensity of each band was related to that of simultaneously loaded protein extracts from control animals that had not undergone surgical instrumentation and trauma. Therefore, the immunoblot data are expressed as fold increase over control values.
Histology and immunohistochemistry
The left lung was formalin fixed for 6 days and embedded in paraffin for histological and immunohistochemical analysis. Adapting recently published scoring systems (19), the hematoxylin eosin-stained lung sections were analyzed by two experienced pathologist (AS, PM) blinded for the group assignment. The readouts consisted of alveolar collapse (i.e., dystelectasis), emphysematous over-distension, inflammatory cell infiltration (i.e., lymphocytes, neutrophils, and macrophages, respectively), thickening of the alveolar wall, alveolar edema, and the total intensity of the inflammatory alterations (4). In the hematoxylin eosin-stained lung sections, alveolar edema could be determined as slightly foamy, eosinophilic material due to the fact that edema always contains some amount of protein and not exclusively H2O. All findings were summarized in a total histopathology score. Immunohistochemical detection of nitrotyrosine formation and expression of the purinergic receptors P2XR4 and P2XR7 was performed as follows: the paraffin sections were deparaffinized in xylene and graded ethanols, boiled twice in sodium citrate buffer for heat-induced epitope retrieval before being exposed to the primary antibody (anti-nitrotyrosine, Millipore, Schwalbach, Germany; anti-P2X4 and anti-P2X7-Receptor, Alomone Labs, Jerusalem, Israel). Primary antibody detection was performed by a secondary antibody and visualized with a red chromogen (alkaline phosphatase-conjugated goat-anti-rabbit IgG, Jackson Immuno Research, West Grove, Pa; chromogen [red], Dako REAL detection system, Dako, Denmark). Slides were visualized using a Zeiss Axio Imager A1 microscope using a ×10 objective (EC Plan-NEOFLUAR). Four distinct random 800 μm2 square regions were quantified for intensity of signal using the image analysis AxioVision 4.8 software (Zeiss, Jena, Germany). Therefore, results are presented as densitometric sum red (4, 17, 18). We elected to detect P2XR4 and P2XR7 expression with immunohistochemistry, as purinergic receptor expression is linked to different anatomic regions in lung tissue (4, 10) and this methodology offers the opportunity to allow for discrimination between these regions (e.g., alveolar cells and bronchi).
All data are presented as median (quartiles) unless otherwise stated. The sample sizes were based on our previous experiments (4), for which a statistical power analysis using thoraco-pulmonary compliance and lung tissue NF-κB activation as main criteria had yielded a minimum of n = 8 to 10 for eight experimental groups. The scope of the present study was to test whether a genetic P2XR4 deletion would attenuate the acute pulmonary inflammatory response in mice with and without CS exposure after blunt chest trauma. Therefore, due to a preliminary approach and an attempt to reduce the number of animals according to the principles of replacement, reduction, and refinement in animal research, we did not study sham-operated mice and used n = 7 to 9 in each of the four experimental groups. After exclusion of normal distribution using the Kolmogorov–Smirnov test, intergroup differences were analyzed with a Kruskal–Wallis one way ANOVA on ranks and a subsequent Dunn's test for multiple comparisons using a two-tailed hypothesis testing or a Mann–Whitney test where appropriate. Differences were considered statistically significant when P < 0.05. All quantitative graphical presentations and statistical analyses were performed using the GraphPad Prism 5, version 5.04, software (GraphPad Software Inc, La Jolla, Calif).
Table 1 demonstrates that heart rate, mean arterial pressure, lactate concentrations, thoraco-pulmonary compliance, PEEP, and arterial carbon dioxide partial pressure (PaCO2) did not differ between groups. However, animals in the CS-exposed wildtype group had significantly lower glucose levels compared with the non-CS wildtype group, coinciding with a significantly lower pH and base excess (P < 0.05 vs. non-CS wildtype). Furthermore, animals in the CS P2XR4−/− group had a significantly lower arterial oxygen partial pressure (PaO2) and a significantly lower PaO2/fraction of inspired oxygen (FiO2) ratio compared with the non-CS P2XR4−/− group. Animals in the CS-exposed P2XR4−/− group needed significantly lower minute ventilation to maintain normocapnia (P < 0.05 vs. non-CS wildtype).
Table 2 shows plasma and lung tissue cytokine and chemokine concentrations at the end of the 4-h observation period of all groups. There was no significant intergroup difference, except for a significantly higher level of plasma MCP-1 in the CS-exposed wildtype group (P < 0.05 vs. non-CS wildtype) and significantly lower levels of lung TNF in the CS-exposed wildtype and the non-CS-exposed P2XR4−/− group (P < 0.05 vs. non-CS wildtype).
The histopathology findings are summarized in Table 3. The level of lung tissue damage in both CS-exposed groups was significantly increased compared with the non-CS wildtype group, indicated by an elevated level of lymphocyte infiltration and a significant increase of the total histopathology score (P < 0.05 vs. non-CS wildtype). Moreover, infiltration of macrophages in lung tissue and the thickness of the alveolar wall were significantly higher in the CS wildtype group compared with the non-CS wildtype group.
Results of the immuno-blotting are given in Figure 1. Blunt chest trauma led to a marked increase of HO-1-expression in the CS-exposed P2XR4−/− group (P < 0.05 vs. non-CS wildtype and vs. non-CS P2XR4−/−, Fig. 1A). In addition, mice in the non-CS-exposed P2XR4−/− group presented with the lowest levels of Bcl-xL-expression after chest trauma (P < 0.05 vs. non-CS wildtype, Fig. 1B). Finally, no significant intergroup difference could be detected concerning the expression of IκBα (Fig. 1C).
Lung tissue nitrotyrosine formation (Fig. 2B) did not significantly differ between groups, whereas lung tissue P2XR7 expression was significantly decreased in the CS-exposed WT group (P < 0.05 vs. non-CS wildtype, Fig. 2D). In contrast, P2XR4-expression was significantly increased in the CS-exposed WT group after trauma (P < 0.05 vs. non-CS wildtype, Fig. 2F).
In this study, we tested the hypothesis whether genetic P2XR4 deletion would attenuate the acute pulmonary inflammatory response and organ dysfunction after blunt chest trauma in mice with and without CS exposure. Our main findings were that neither short-term CS-exposure nor P2XR4 deletion had any significant effect on pulmonary function and acute inflammatory response, despite a significant increase of the histopathology score in both CS-exposed groups after blunt chest trauma. However, P2XR4 deletion was associated with attenuated impairment of glucose homeostasis and acid-base status after CS exposure and chest trauma, possibly due to less alveolar hypoxia-induced right ventricular remodeling resulting in preserved liver metabolic capacity.
Minor effects of cigarette smoke exposure on the acute trauma response
In our previous study, we reported a significant reduction of the PaO2/FiO2 ratios of CS-exposed mice after blunt chest trauma, coinciding with increased lung nitrotyrosine formation, NF-κB-activation, purinergic receptor expression, and higher inflammatory cell infiltration (4).
Our present study could partially confirm previous findings: while CS exposure did not significantly influence pulmonary function or acute inflammatory response after blunt chest trauma, CS exposure was associated with a significant increase of the parameters of lung histopathology, i.e., inflammatory cell infiltration and the total histopathology score compared with the non-CS wildtype group. We can only speculate on the lack of effect of CS on gas exchange and lung mechanics in the present study, but this may be due to less severe effects of CS exposure and a better tolerance in individual animals in the present trial.
Effects of genetic P2XR deletion on the acute trauma response
Genetic P2XR7 deletion attenuated the endotoxin-induced release of IL-1β and thereby attenuated the impairment of lung mechanics and the histological organ damage in a model of LPS-induced acute lung injury in mice (7), and P2XR7 activation led to a more pronounced endotoxin-related vascular hyporeactivity in vitro(20). Co-expression of the P2XR4 receptor enhanced the P2XR7-related inflammatory response (21), and up-regulation of P2XR4 was reported to compensate for P2XR7 depletion (15). Finally, in addition to their role for the development of acute respiratory distress syndrome, P2XR7 and P2XR4 activation were shown to play an important role in the inflammatory pathways of CS-related lung injury: CS exposure caused an up-regulation of both the P2XR4 and P2XR7 receptors (22), and either pharmacological blockade or genetic deletion of the P2XR7 receptor decreased pulmonary IL-1β and IL-18 concentrations after CS exposure (11).
In contrast to the existing literature (7), purinergic receptor deletion was not able to modify pulmonary (dys)function or the inflammatory response after blunt chest trauma and 4 h of lung-protective mechanical ventilation in our study. We can only speculate about the reasons: as our immunohistochemistry results demonstrated that P2XR7 was expressed in the lung tissue of both wildtype and P2XR4−/− animals, the inflammatory response could have been triggered solely by the presence of P2XR7 in lung tissue of the corresponding animals, despite genetic deletion of P2XR4. Concerning the significantly higher P2XR4-expression in wildtype animals with CS exposure after trauma, we could confirm the findings of our previous study (4).
Interestingly, CS-exposed P2XR4−/− mice had significantly higher glycemia levels than the corresponding wildtype animals, indicating a higher rate of gluconeogenesis. We have previously demonstrated in resuscitated septic shock that well-preserved gluconeogenesis mirrors maintenance of liver metabolic capacity (23–25). In this context, it is of particular interest that Ohata et al. (26) have already demonstrated that the P2X receptors, e.g., P2XR4 and P2XR1, may play an important role in the pathways inducing right ventricular hypertrophy with consecutive remodeling due to pulmonary hypertension and possibly subsequent liver congestion.
Our findings also coincide with a significantly increased expression of HO-1 in lung tissue of CS-exposed P2XR4−/− mice. HO-1 catalyzes the degradation of heme to biliverdin and subsequently to bilirubin, and is the main endogenous source of carbon monoxide (CO) in the body. CO has been recognized as a physiologically important signaling molecule, acting as a direct and indirect vasodilator (27). Christou et al. (28) already showed that pharmacologically induced enhancement of HO-1 expression in a rat model of chronic hypoxia attenuated the development of structural remodeling and pulmonary hypertension. Furthermore, treatment with a CO-releasing molecule for 3 weeks prevented pulmonary hypertension, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia (29). Finally, we have already shown in a model of long-term hyperdynamic porcine endotoxemia that pharmacological inhibition of HO-1 was associated with increased mean pulmonary artery pressure and consecutive evidence of right-ventricular failure (30).
Limitations of the study
It can be argued that lung damage was only mild to moderate in our study, highlighted by the fact that PaO2/FiO2 ratios remained above the threshold of 300 mm Hg, i.e., the cutoff for the definition of ARDS. This could have been due to the well-titrated intensity of blunt chest trauma or the pressure-controlled, lung-protective ventilation applied in our study, avoiding any additional damage induced by mechanical ventilation beyond the effect of lung contusion per se. Only few studies reported Horovitz indices compatible with the definition of ARDS (<300 mm Hg) in mechanically ventilated mice: animals were either ventilated with injurious tidal volumes (12–40 mL·kg−1) (31–34), or 24 h after injection of endotoxin, i.e., in the presence of prolonged, severe ALI (35).
Moreover, the short duration of the mechanical ventilation precludes any conclusion on the long-term effects. Concerning this issue, a recent review article highlighted that only two studies described mechanical ventilation in mice up to 8 h, while in the other reports only 4 to 6 h of mechanical ventilation was used (19).
The authors are indebted to Rosemarie Mayer and Rosa Engelhardt for skillful technical assistance. The authors also thank Professor Paul Dietl, PhD, for valuable discussions about the manuscript. Furthermore, they thank Glaxo SmithKline Research & Development Limited, Stevenage, UK, for providing the P2XR4−/− mice.
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