Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke
Background and Purpose—Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive.
Methods—The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor–deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity.
Results—Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor–deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung.
Conclusions—Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.
- bone marrow chimeric mice
- cholinergic anti-inflammatory pathway
- parasympathetic nervous system
- stroke-induced immunodepression
The management of medical complications is one of the most critical factors in the therapy of stroke because 95% of stroke patients experience at least one relevant medical complication in the first 3 months after stroke.1 In both experimental and clinical studies, infections are the most common complication after stroke.2 Among these, pneumonia is the most relevant poststroke infection,3 accounting for the highest attributable mortality after stroke.4
To maintain body homeostasis in response to challenges from the environment, the nervous and immune systems are closely interconnected in an intense bidirectional communication.5 Recent experimental and clinical evidence indicates that central nervous system injuries like stroke, spinal cord injury, or traumatic brain injury disturb the normally well-balanced interplay between these 2 supersystems, resulting in a profound and long-lasting immunodepression.6,7 Overactivation of the sympathetic nervous system after stroke was shown to mediate suppression of peripheral cellular immune responses and to contribute to development of poststroke bacterial lung infections.8–10 However, changes in the pulmonary immune compartment and the underlying mechanisms of impaired antibacterial lung immune responses after stroke are currently not understood.
Besides resident lung immune cells, such as alveolar macrophages (MΦ), alveolar epithelial cells (AEC) express a variety of receptors recognizing pathogens or their products.11 Rapid activation of MΦ and airway epithelium are of crucial importance for effective host defense during bacterial lung infections.12 Both MΦ and lung epithelial cells have been shown to express the α7 nicotinergic acetylcholine receptor (α7nAChR),13 which has been identified as a crucial downstream element of the so-called cholinergic anti-inflammatory pathway.14 This neuronal pathway, involving the parasympathetic vagus nerve and the neurotransmitter acetylcholine (ACh), was shown to act as a negative feedback loop to prevent a potentially harmful overreaction of the immune system during inflammatory conditions, such as bacterial infections, by suppressing the production of proinflammatory cytokines by activated macrophages on binding of ACh to the α7nAChR.15 Here we demonstrate that stroke directly activates cholinergic anti-inflammatory pathways, which are crucially involved in the development of poststroke lung infections.
Materials and Methods
A detailed description of all materials and methods can be found in the online-only Data Supplement.
Animals and Housing
Male specific pathogen free C57Bl6/J mice (Charles River Laboratories, Sulzfeld, Germany), sex-mixed α7nAChR knockout (KO) mice,16 and wild-type (WT) littermates, respectively (B6.129S7-Chrna7tm1Bay/J; The Jackson Laboratory, Bar Harbor) were housed in cages lined with chip bedding and environmental enrichment (mouse tunnel and igloo) on a 12 h light/dark cycle with ad libitum access to food and water. Experiments were performed using 11- to 14-week-old mice in accordance with the European directive on the protection of animals used for scientific purposes and all other applicable regulations and approved by the relevant authority, Landesamt für Gesundheit und Soziales, Berlin, Germany.
Under general isoflurane anaethesia, a small silicon-coated filament was temporarily introduced via the left common carotid artery into the circle of Willis blocking the origin of the middle cerebral artery for 60 min (middle cerebral artery occlusion [MCAo])17 following the standard operating procedures of our laboratory. Success of MCAo was verified by Bederson score, and animals without neurological deficit after operation were excluded from the study.
Five days before MCAo surgery and under isoflurane anesthesia, the left cervical vagus nerve was carefully and bluntly dissected from the common carotid artery and, in the vagotomy but not sham-operated group, transected.
Broncho-Alveolar Lavage and Measurement of Bacterial Burden
Under anaethesia with midazolam (5 mg/kg body weight) and medetomidin (0.5 mg/kg body weight) IP (antagonization with flumazenil 0.5 mg/kg body weight and atipamezol 5 mg/kg body weight, if needed), mice were intubated with a 22G peripheral venous catheter. Afterward, 0.4 mL of saline plus 0.2 mL air were applied over the tubus and immediately withdrawn.18 For the determination of colony-forming units, broncho-alveolar lavage (BAL) fluid was serially diluted, plated on LB agar plates, and incubated at 37°C for 18 h, and bacterial colonies were counted.8 The cut-off value (<104 colony-forming units/mL) has been determined by investigating BAL fluid of naïve mice.
Drug Preparation and Administration
Nicotine (Sigma Aldrich) was given via drinking water in a concentration of 100 μg/mL. The effects on heart rate (HR) were monitored by pulse oximetry (MouseOx; STARR life sciences Corp.).
Creation of Bone Marrow Chimeric Mice
For generation of bone marrow (BM) chimeric mice, BM cells were isolated under sterile conditions from the tibias and femurs of donor mice. 5×106 BM cells were IV injected into 6- to 8-week-old lethally irradiated recipient mice 24 h after total body irradiation (1100 cGy) using a 137Caesium Gammacell 40 Exactor (Theratronics).
Blood pressure (BP) and HR were measured by telemetry combined with fast Fourier transform analysis of BP and HR, as described elsewhere.19 In mice aged 8 to 10 weeks, transmitters (TA11PA-C10 BP; Data Sciences International) were implanted in a subcutaneous pocket along the right flank with the pressure-sensing catheter in the abdominal aorta. After 7 days recovery, baseline was recorded using DATAQUEST software (A.R.T. 2.1, Data Sciences International), and sympathetic and parasympathetic activities were verified pharmacologically by applying metoprolol (4 mg/kg) and atropine (2 mg/kg), respectively. Parasympathetic activity was assessed via spectral analysis using PV-wave software (Visual Numerics). The power spectra of systolic BP, pulse interval time series, and the cross spectra were calculated using fast Fourier transformation. Low-frequency components of pulse intervals spectrum (LF), root mean square of successive differences between adjacent normal pulse intervals, total power, and the baroreflex sensitivity (BRS-LF) were calculated. The baroreceptor HR reflex was investigated using sequence technique and spectral function analysis of spontaneous changes in systolic BP and HR, and BRS was defined as the mean magnitude value of transfer function between systolic BP and R-R interval in the low-frequency band (BRS-LF).
Cell phenotyping was performed on an LSRII flow cytometer using FACSDiva software (BD Biosciences) and FlowJo software (Tree Star Inc.) with the following anti-mouse monoclonal antibodies: CD45-PE-Cy7, CD3-APC, CD4-A700, CD8-PB, NK1.1-PE, CD11b-APC-Cy7, B220-FITC, SiglecF-PE, F4/80-A700, CD11c-FITC, CD11b-PE-TR, Gr1-PB, and MHCII-APC-Cy7 (Biolegend). Fluorescence-activated cell sorter–based cell sorting was performed on a FACSAria III cell sorter (BD Biosciences).
Isolation of Primary Alveolar Macrophages and Alveolar Epithelial Cells
MΦ were obtained by repeated gentle flushing of tracheotomized lungs from naïve C57Bl6/J mice with NaCl, centrifugation (300g, 10 min), and resuspension in Roswell Park Memorial Institute medium. For purification of AEC,20 lungs were flushed after perfusion and BAL with 1500 μL dispase followed by intratracheal injection of 500 μL of 1% low melting temperature agarose. Lung single-cell suspension (homogenization in DMEM) underwent magnetic cell separation for depletion of nonepithelial cells using anti-CD45, anti-CD31, and anti-CD16/CD32 mAb (BioLegend). Purity of macrophages and epithelial cells was confirmed by flow cytometry (>95% and >90%, respectively).
Analysis of Ex Vivo Cytokine Production
Lung single-cell suspensions (2×106/mL), isolated primary MΦ (1×105/mL), or primary AEC (1×10E5/mL) were stimulated with lipopolysaccharide (LPS) (1 μg/mL; Sigma) or Pam3CSK4 (200 ng/mL; Invivogen) in the absence or presence of different concentrations of nicotine or PNU282987 (both from Sigma) for 12 h at 37°C.
Analysis of Cytokines in BAL and Cell Culture Supernatants
Cytokines in BAL and cell culture supernatant were measured by magnetic bead–based assay on a Bio-Plex 200 System (Luminex xMAP Technology) with Bio-Plex Manager 6.0 software (Bio-Rad) according to the manufacturer’s recommendations.
Quantitative Reverse Transcriptase Polymerase Chain Reaction
RNA was extracted from fluorescence-activated cell–sorted or Dynabead-purified lung cells using the NucleoSpin RNA II Kit (Macherey-Nagel). cDNA synthesis was done using the QuantiTect kit (Qiagen) and measured by real-time quantitative reverse transcriptase polymerase chain reaction (7500 Sequence Detection System, Applied Biosystems) with β-Actin as housekeeping gene.
Treatment allocation was performed randomly and remained completely blinded for the examiners. Appropriate 2-sided statistical tests were used to compare groups for significant differences (P<0.05; SPSS Statistics 18.0, IBM). Closed testing procedure was applied in telemetry experiments, putting the comparisons at different time points in a hierarchical order. A priori sample size calculation (G*Power3.021) assumed a change in bacterial burden by 100-fold as relevant or a doubling of HR variability (HRV) parameters, respectively. Prespecified inclusion and exclusion criteria and detailed report on flow of animals through the experiments are reported in the online-only Data Supplement (Tables I and II in the online-only Data Supplement).
Middle Cerebral Artery Occlusion Reduces HR and Increases Blood Pressure
To obtain unbiased data on physiological responses after experimental stroke in mice, we established a telemetric approach to measure HR and BP in a quiet, undisturbed environment. In MCAo mice, HR was decreased during the first 3 days after surgery compared with baseline and sham-operated animals. BP rose in both groups, but remained significantly elevated for 5 days in MCAo animals compared with sham controls (Figure IA and IB in the online-only Data Supplement). Physical activity was slightly reduced in both groups compared with baseline with no group differences in the first 2 weeks (Figure IC in the online-only Data Supplement).
Increased HRV and Baroreflex Sensitivity Indicates Parasympathetic Activation After Experimental Stroke
In contrast to sham-operated animals, we observed a strong increase in the LF power component of HRV reflecting increased parasympathetic activity in MCAo-treated mice on day 1, 3, and 5 after surgery (Figure 1A). These findings were corroborated by changes in 2 time domain parameters describing parasympathetic activity in HRV, the standard deviation of RR intervals (Figure 1B), and the root mean square of successive RR differences (Figure 1C).
BRS is the ability to adapt the sinus frequency in response to BP changes. Cross spectral analysis of the BRS-LF, calculated as mean value of the transfer function between systemic BP and pulse intervals in the LF band, revealed a significant increase within the first 5 days after experimental stroke (Figure 1D). One, 3, and 5 days after experimental stroke, the adaptation time for up slopes and down slopes of systemic BP were increased compared with baseline and sham controls (Figure II in the online-only Data Supplement).
Unilateral Vagotomy Reduces Bacterial Burden by Restoring Pulmonary Immune Function
MCAo animals develop spontaneous bacterial pneumonia between day 1 and day 3 after experimental cerebral ischemia.8 To investigate the influence of parasympathetic activation on poststroke infections, we first performed unilateral cervical vagotomy. In mice that underwent vagotomy 5 days before MCAo, we observed a significant reduction in bacterial load on day 3 after MCAo compared with sham vagotomized stroke animals (Figure 2A). Importantly, administration of nicotine in drinking water reversed the beneficial effect of vagotomy on bacterial pneumonia after stroke (Figure 2A). Infarct volumes and HR did not significantly differ between the groups (Figure III in the online-only Data Supplement).
Assessment of ex vivo cytokine secretion by lung cells isolated 1 day after MCAo revealed reduced release of proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α after stimulation with the Toll-like receptor 4 (TLR4) agonist LPS (Figure 2B and 2C) and the TLR1/2 agonist Pam3CSK4 (Figure IV in the online-only Data Supplement) in MCAo mice compared with sham controls. In contrast, lung cells isolated from mice vagotomized 5 days before MCAo released similar amounts of IL-6 and TNF-α after Pam3CSK4 and LPS stimulation as lung cells from sham-operated controls. Treatment of MCAo animals with nicotine in drinking water decreased ex vivo proinflammatory cytokine production after TLR stimulation of lung cells isolated from vagotomized mice to levels observed in non- or sham-vagotomized animals (Figure 2B and 2C; Figure IV in the online-only Data Supplement).
Role of α7nAChR in Diminished Antibacterial Defense in Lung After Experimental Stroke
The α7nAChR receptor has been described as mediator of cholinergic anti-inflammatory effects on macrophages.14 Similar to the effects of vagotomy in WT animals, mice lacking the α7nAChR showed significantly less bacterial load in lungs after MCAo compared with WT littermates (Figure 3A). Nicotine did not further increase lung bacterial burden in WT mice. In α7nAChR KO mice, bacterial loads in lung increased only slightly by nicotine treatment but remained at lower levels compared with WT animals. Infarct volumes and HR did not significantly differ between the groups (Figure 3B and 3C). The rather small number of male compared with female animals per group does not allow a definitive conclusion on sex-related differences of α7nAChR KO mice in response to stroke. However, subgroup analysis of infarct volumes and bacterial burden in lung does not suggest sex-specific differences (data not shown).
Cholinergic Signaling Attenuates Antibacterial Defense of Lung Myeloid and Epithelial Cells After Experimental Stroke
To differentiate between cholinergic effects on BM-derived immune cells and resident lung cells contributing to poststroke immunodepression, we investigated the development of poststroke pneumonia in mice responding to α7nAChR-mediated cholinergic signaling either in BM-derived cells (WT→KO) or resident lung cells (KO→WT). For control, we transplanted BM cells from WT into WT animals (WT→WT) and from KO into KO animals (KO→KO). After experimental stroke, bacterial burden in lung was significantly reduced in KO→KO mice compared with WT→WT mice (Figure 4A), corroborating findings observed in α7nAChR KO and WT mice (compare Figure 3A). Both KO→WT and WT→KO chimeric mice showed an intermediate phenotype regarding susceptibility to pneumonia compared with syngeneic controls (Figure 4A).
Lung cells isolated 3 days after MCAo from ischemic WT→WT mice produced significantly lower TNF-α levels after LPS stimulation than lung cells from KO→KO mice, whereas lung cells from WT→KO and KO→WT chimeric mice again exhibited an intermediate phenotype compared with KO→KO and WT→WT mice (Figure 4B).
mRNA Expression Pattern of α7nAChR in Alveolar Macrophages and Alveolar Epithelial Cells
To determine the cellular expression pattern of α7nAChR in lung tissue, we sorted single cell suspensions from WT mice by magnetic cell sorting. α7nAChR was ≈50-fold higher expressed in AEC compared with MΦ. In contrast, α7nAChR expression was not observed in endothelial cells, NK-, B-, and T-lymphocytes, dendritic cells, or granulocytes (Figure 5).
Diminished Cytokine Production in AEC and MΦ upon α7nAChR Stimulation
To investigate cell type–dependent α7nAChR-mediated effects, we assessed TLR ligand–induced IL-6 release in primary AEC and MΦ. Nicotine diminished LPS-induced IL-6 production in AEC isolated from WT but not α7nAChR KO mice in a dose–dependent manner (Figure 6A). Likewise, α7nAChR-specific agonist PNU282987 suppressed IL-6 release in LPS-stimulated AEC from WT but not α7nAChR KO mice (Figure 6C). In contrast, nicotine and PNU282987 dose-dependently reduced LPS-induced IL-6 secretion in MΦ from WT and to lesser extent also in α7nAChR KO mice (Figure 6B and 6D). These data suggest that cholinergic control of TLR-induced proinflammatory cytokine release by MΦ is partly independent from α7nAChR.
Besides neurological deficits leading to dysphagia and aspiration, suppression of peripheral immune responses because of an overactivation of stress pathways has been recognized as a critical risk factor for the development of infectious complications after stroke. Previous studies in experimental stroke models have provided evidence that an increased activity of the sympathetic nervous system after cerebral ischemia leads to rapid impairment of peripheral cellular immune responses, in particular natural killer cell and T cell responses. These experimental findings were corroborated by clinical studies showing increased levels of catecholamines and reduced T cell counts in peripheral blood, as well as impaired ex vivo T cell function in stroke patients before the onset of infectious complications.6,10 Thus, although pathophysiological responses in human diseases and respective mouse models may markedly differ as recently shown for the genomic response in acute inflammatory conditions,22 the MCAo model in mice has proven to be clinically relevant to elucidate mechanisms of increased susceptibility to infection and to develop preventive treatment strategies.23 However, it remained unclear whether cerebral ischemia affects resident lung cells, which mediate first-line pulmonary immune responses during respiratory infections. Our data suggest that lung innate immune responses are suppressed after stroke by increased cholinergic signaling, which is relayed by the parasympathetic vagus nerve and the α7nAChR expressed on AEC and MΦ. Hence, both an impaired early T/NK-cell dysfunction mediated by sympathetic nervous system and an impaired innate immune response in the lung mediated by the parasympathetic nervous system seem to be important additive mechanisms leading to decreased antibacterial defense and poststroke bacterial infections.
The cholinergic anti-inflammatory pathway has been described as a protective mechanism that prevents potentially harmful overactivation of the immune system in response to infections or inflammation-induced tissue damage. Sensing of peripheral inflammatory processes via vagal afferent fibers was initially proposed to trigger an anti-inflammatory response via release of acetylcholine from vagal efferent fibers, which acts on α7nAChR on macrophages to suppress the release of proinflammatory cytokines.14 Recent evidence suggests, however, that the neural reflex that is triggered by and dampens systemic inflammatory responses may be more complex and involve non-neural cholinergic and noradrenergic sympathetic mechanisms.24 Nevertheless, vagus nerve stimulation by centrally acting muscarinic agonists, such as CNI-1493, has been shown to suppress peripheral inflammatory responses that can be prevented by vagotomy, demonstrating that the vagus is an essential efferent pathway of the central cholinergic anti-inflammatory pathway.25
Assessment of HRV has been recognized to provide reliable, noninvasive information on the activity of the autonomic nervous system, including its vagal and sympathetic components. Using a telemetry system, we observed an immediate and long-lasting activation of the parasympathetic nervous system after cerebral ischemia, which is in line with previous observations showing a similar increase in root mean square of successive RR differences and LF-HRV after experimental stroke in mice.26 Because HRV parameters have been shown to differ between species with respect to the influence of parasympathetic and sympathetic outflow,27 we have verified the specificity of selected HRV measures by pharmacological blockade of sympathetic and parasympathetic input using the selective antagonists metoprolol and atropine, respectively.19 According to the few clinical data, autonomic dysfunctions in stroke patients occur in the acute phase with a predominance of parasympathetic rather than sympathetic dysfunction.28–30
Although measurement of HRV reflects primarily autonomic influences on the cardiovascular system, several human studies have linked changes in HRV with markers of inflammation and immunity.31,32 An increase in sympathetic and parasympathetic HRV parameters is associated with worse outcome after stroke.9,33,34
In the present study, we demonstrated that increased parasympathetic activity after cerebral ischemia plays a crucial role in mediating increased susceptibility to bacterial pneumonia after stroke. Although MCAo animals developed spontaneous bacterial infections, either disruption of parasympathetic signaling in WT mice by vagotomy or inhibition of cholinergic effects by using α7nAChR-deficient mice reduced bacterial burden in BAL fluid almost completely to levels observed in naïve mice (<104 colony-forming units/mL).35 Furthermore, vagotomy resulted in the restoration of inflammatory responses triggered by TLR agonist in lung cells in stroke animals. Moreover, treatment of MCAo animals with the nonselective nAChR agonist nicotine prevented the beneficial effect of vagotomy on lung bacterial burden and on ex vivo cytokine secretion by lung cells in WT mice but only slightly increased bacterial load in α7nAChR mice, indicating that suppression of pulmonary antibacterial responses after stroke by cholinergic signals involves α7nAChR. Because we have not assessed all physiological parameters under parasympathetic control in these experiments, other than immune mechanisms might be involved in poststroke pneumonia. However, vagotomy resulted in the restoration of inflammatory responses triggered by TLR agonist in lung cells in stroke animals. Recently, Lafargue et al made the important observation that mice deficient in α7nAChR showed reduced lung injury and mortality compared with WT mice in an aspiration-induced Pseudomonas aeruginosa pneumonia model after experimental stroke.36 However, the Pseudomonas aspiration pneumonia model does not reflect the pathophysiological course of lung infections in stroke patients because it requires a high bacterial inoculum, which by itself may trigger (neural) mechanisms to perturb pulmonary immune defense other than those induced by central activation of cholinergic anti-inflammatory mechanisms after acute central nervous system -injury.37 Our study provides direct evidence for the crucial role of the cholinergic anti-inflammatory pathway in the development of spontaneous pneumonia after MCAo in mice.
Both, MΦ and AEC play a crucial role in the first-line defense against pulmonary infections.11,12 Cholinergic signals transmitted via the α7nAChR have been previously shown to limit inflammatory responses in these cells.38 Accordingly, we examined whether resident lung immune competent cells or BM-derived immune cell are predominantly targeted by cholinergic anti-inflammatory signaling after stroke. Our results using a model of BM chimerism between WT and α7nAChR-deficient mice suggest that the inhibitory effects of α7nAChR-mediated cholinergic signaling are exerted on both lung-resident cells and BM-derived immune cells because chimeric mice showed an intermediate phenotype in terms of bacterial burden and ex vivo cytokine secretion capacity of whole lung cells compared with syngeneic WT→WT and KO→KO controls.
To further elucidate pulmonary cellular targets of cholinergic anti-inflammatory signaling, we determined α7nAchR expression in sorted cells isolated from lung. We observed α7nAChR expression only in AEC and MΦ but not in endothelial cells or other leukocyte subsets. Ex vivo functional analysis revealed that the anti-inflammatory effects of cholinergic signaling on AEC were largely dependent on the presence of the α7-subunit nAChR because suppression of TLR-induced proinflammatory cytokine production by the nonselective nAchR agonist nicotine and the selective α7 agonist PNU282987 was observed in primary AEC from WT but not α7nAChR-deficient mice. In contrast, although PNU282987 showed α7nAChR-dependent inhibitory effects on MΦ, nicotine dose-dependently suppressed IL-6 production in these cells also in the absence of α7nAChR. Although α7nAChR was consistently detectable in MΦ, expression levels were ≈100-fold lower compared with primary AEC. These data suggest that nicotinic receptors other than α7nAchR may be involved in cholinergic anti-inflammatory signaling in MΦ.
Several studies suggested that stimulation of the cholinergic anti-inflammatory pathway via vagus nerve and α7nAChR may also be a neuroprotective strategy.39–41 However, our data strongly suggest that inhibition of peripheral parasympathetic actions is beneficial in terms of medical complications after stroke, whereas an additional stimulation of the cholinergic pathway might exacerbate the risk for life-threatening bacterial infections. Thus, immunomodulatory therapeutic strategies will have to consider and keep the balance between reversal of the peripheral stroke-induced immunodepression and concurrent preservation of its putative neuroprotective effects.
In summary, although activation of cholinergic anti-inflammatory mechanisms during local and systemic inflammatory responses may be a protective feedback mechanism to limit potentially harmful effects of overt inflammation, central nervous system injury–induced activation of such pathway in the absence of an inflammatory stimulus may inappropriately inhibit first-line antimicrobial responses and increase susceptibility to infectious complications after acute central nervous system injury.
We thank Sabine Kolodziej, Carena Teufelhart, Verena Wörtmann, Yvonne Amoneit, Claudia Muselmann-Genschow (Charité), and Ilona Kamer (MDC) for excellent technical assistance.
Sources of Funding
This study was supported by German Research Foundation (Exc257, SFB-TRR84, SFB-TRR43, FOR1336, SFB1021 C05, Excellence Cluster Cardio-Pulmonary System), German Federal Ministry of Education and Research (01EO0801, German Center for Lung Research), European Community FP7 (201024), and Hessen State Ministry of Higher Education, Research and the Arts (Universities Giessen & Marburg Lung Center).
A patent on preventive antibacterial therapy after stroke has been filed to the European Patent Office (PCT/EP03/02246) by Drs Volk, Dirnagl, C. Meisel, and A. Meisel. The other authors report no conflicts.
Guest Editor for this article was Malcolm R. Macleod, PhD.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.115.008989/-/DC1.
- Received March 11, 2015.
- Revision received August 19, 2015.
- Accepted August 24, 2015.
- © 2015 American Heart Association, Inc.
- Davenport RJ,
- Dennis MS,
- Wellwood I,
- Warlow CP.
- Kalra L,
- Yu G,
- Wilson K,
- Roots P.
- Guilliams M,
- De Kleer I,
- Henri S,
- Post S,
- Vanhoutte L,
- De Prijck S,
- et al
- Prass K,
- Meisel C,
- Höflich C,
- Braun J,
- Halle E,
- Wolf T,
- et al
- Orr-Urtreger A,
- Göldner FM,
- Saeki M,
- Lorenzo I,
- Goldberg L,
- De Biasi M,
- et al
- Engel O,
- Kolodziej S,
- Dirnagl U,
- Prinz V.
- Walters DM,
- Wills-Karp M,
- Mitzner W.
- Seok J,
- Warren HS,
- Cuenca AG,
- Mindrinos MN,
- Baker HV,
- Xu W,
- et al
- Dirnagl U,
- Endres M.
- Gehrmann J,
- Hammer PE,
- Maguire CT,
- Wakimoto H,
- Triedman JK,
- Berul CI.
- Tokgözoglu SL,
- Batur MK,
- Top uoglu MA,
- Saribas O,
- Kes S,
- Oto A.
- Korpelainen JT,
- Sotaniemi KA,
- Huikuri HV,
- Myllyä VV.
- Xiong L,
- Leung HH,
- Chen XY,
- Han JH,
- Leung TW,
- Soo YO,
- et al
- Thayer JF.
- Marsland AL,
- Gianaros PJ,
- Prather AA,
- Jennings JR,
- Neumann SA,
- Manuck SB.
- Sykora M,
- Diedler J,
- Poli S,
- Rizos T,
- Turcani P,
- Veltkamp R,
- et al
- Lafargue M,
- Xu L,
- Carlès M,
- Serve E,
- Anjum N,
- Iles KE,
- et al