Recruitment and derecruitment (R/D) of airways is known to significantly influence mechanical properties of the respiratory system during artificial ventilation, particularly in states of lung injury. The prevailing view of this phenomenon treats airway R/D as a static function of pressure. Recent experimental and clinical data suggests that this is not the case, but rather that R/D is an inherently dynamic process. In order to quantitatively assess the dynamics of lung recruitment during mechanical ventilation we extended a mathematical model by Bates and Irvin (9) for the purpose of fitting experimental data. The model of the lung consists of a parallel network of flow pathways with identical resistive and elastic elements. Each pathway is allowed to be either open, whereby it accumulates flow and decreases overall lung stiffness, or closed, increasing lung elastance and not participating in ventilation. The pathways are characterized by unique critical closing and opening pressures, and opening and closing velocities, each chosen from probability distribution functions. The rate of transition between an open and closed state depends on the magnitude difference between the pressure in the respiratory system and each unit’s critical pressure times the airway’s opening or closing velocity constant. Since the exact form of the pressure dependence governing recruitment and derecruitment remains unknown we explored four model variants to predict how opening or closing behavior is altered in injury. The lung model was coupled with a computational model of a mechanical ventilator in order to simulate elastance changes following deep inflation (DI) at three levels of Positive End Expiratory Pressure (PEEP). Elastance measurements came from healthy or lung injured mice at 4, 14, 24 or 48 hours following intratracheal instillation of saline (control) or hydrochloric acid (injury). The Nelder and Mead simplex optimization method was used to minimize error between model variants and average experimental elastance for each condition. By comparing the residual error of the fits for each model, we have demonstrated that only one variant was able to recreate both the transient response to deep inflations and the response to static PEEP. In fitting the best model to data from individual mice we obtained estimates for parameters governing opening and closing behavior. Statistics and model sensitivity were determined for each parameter in every experimental condition. Comparison of parameter values between groups revealed a significant increase in closing and opening pressures from health to injury, which worsened with increasing injury severity. The progressive increase in critical pressures as injury worsens implicates surfactant deactivation as the likely cause of increased propensity for airway closing during acute lung injury.

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Background: Acute lung injury (ALI) and nosocomial pneumonia are major causes of morbidity and mortality. There are 200,000 cases per year of ALI in the US with a mortality of 40%. On the intensive care unit (ICU), ALI accounts for over 40% of all ventilated patients at any one time. Despite this huge burden on healthcare and the relatively high prevalence, no therapies currently exist in clinical practice that attenuate the condition. The pathophysiology and aetiology of ALI is multifactorial but neutrophilic influx and consequent damage to the endothelial-epithelial interface are regarded as central features. Alongside neutrophils, peripheral blood monocytes (PBMs) are recruited to the acutely inflamed lung. The role played by PBMs in perpetuating the pathogenic neutrophilic influx remains poorly characterised. Nosocomial pneumonia is also a major problem with drug resistant organisms. With the increasing prevalence of antibiotic resistance and the paucity of novel antimicrobials being generated by pharmaceutical companies, there is real concern that the end of the ‘antibiotic era’ may be approaching. AIMS 1) To develop murine models of lung inflammation and infection 2) To establish the role of the PBM in perpetuating the neutrophilic response in ALI 3) To develop non-invasive methodologies to study the trafficking of cells and molecular events within the inflamed lung 4) To apply a novel antimicrobial to prevent and treat nosocomial pneumonia Methods: A murine model of ALI was utilised using direct intratracheal instillation of lipopolysaccharide. To this model 3 different PBM depletion strategies were applied to study the effect on neutrophil recruitment and consequent lung injury. Non invasive optical imaging was utilised to study the effect of PBM depletion on proteolytic events within the murine lung. To understand cellular trafficking, cell labeling strategies were compared for primary murine macrophages with whole body optical imaging in mice. Murine models of Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia were established and a novel antimicrobial agent called the nonalysine like peptoid (NLLP) tested in vitro and in vivo for efficacy. Results: PBM depletion significantly attenuated neutrophil recruitment in an established model of ALI. Near infrared (NIR) optical imaging permitted the non invasive tracking of primary murine cells. A non toxic peptidomimetic agent (NLLP) possessed antimicrobial activity against gram positive and gram negative pathogens with therapeutic and prophylactic efficacy in vivo. Conclusions: PBM depletion is a potential therapeutic strategy for treating ALI. Further studies are required to determine the exact mechanism by which PBMs orchestrate neutrophil recruitment. Optical imaging is a versatile platform for molecular imaging. A novel antimicrobial agent termed NLLP has been discovered with therapeutic and prophylactic efficacy against multi-drug resistant pathogens.

There is considerable evidence that cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD), and that oxidative stress occurs in COPD with the family of tissue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes playing a significant role in lung pathogenesis. The purpose of this study was to determine the effects of cigarette smoke extract (CSE) on Nox signaling to epithelial sodium channels (ENaCs). Pre-treatment with diphenyleneiodonium (DPI), a pan-Nox inhibitor, prevented stimulatory effects of CSE on ENaC activity; open probability (Po) changed from 0.36 +/- 0.09 to 0.11 +/- 0.02; n=10, p=0.01 following CSE and DPI exposure. Likewise, Fulvene-5 (which inhibits Nox2 and Nox4 isoforms) decreased the number of ENaC per patch (from 2.75 +/- 0.25 to 1 +/- 0.5, n=9, p=0.002) and open probability (0.18 +/- 0.08 to 0.02 +/- 0.08, p=0.04). Cycloheximide chase assays show that CSE exposure prevented alpha-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of alpha-ENaC protein in primary isolated lung cells. In vivo, co-instillation of CSE and Nox inhibitor promoted alveolar flooding in C57Bl6 mice compared to accelerated rates of fluid clearance observed in CSE alone instilled lungs. Real-time PCR indicates that mRNA levels of Nox2 were unaffected by CSE treatment while Nox4 transcript levels significantly increased 3.5 fold in response to CSE. Data indicate that CSE is an agonist of Nox4 enzymatic activity, and that CSE-mediated Nox4 plays an important role in altering lung ENaC activity.

Of the 800,000 suicides recorded globally every year, over a third are due to pesticide ingestion, the majority of which occur in rural Asia with organophosphorus (OP) compounds. These anticholinesterase pesticides cause an acute cholinergic syndrome characterised by decreased consciousness, excessive airway secretions and respiratory failure. A combination of these clinical features is the most common cause of death. Up to 30% of OP pesticide poisoned patients are admitted to the Intensive Care Unit (ICU) for tracheal intubation and lung ventilation, but up to half die. It is not understood why the case fatality for intubated poisoned patients is so high, but one hypothesis is that the patients, when unconscious, aspirate their stomach contents (including the OP and the solvent present in its agricultural formulation) causing a toxic lung injury which contributes to the observed high mortality. In this PhD, I aimed to characterise the lung injury caused by OP pesticide self-poisoning through both indirect (ingestion) and direct (aspiration) means. To achieve this, I analysed data from previous toxicological minipig work and designed and conducted a specific minipig pulmonary aspiration study which was complemented by an experimental OP poisoning ex vivo lung perfusion model and human data from pesticide poisoned patients in Sri Lanka. I first investigated the pulmonary pathophysiology resulting from orogastric administration of OP pesticide without aspiration. Analysis of my group’s Gottingen minipig in vivo work demonstrated that orogastric placement of agricultural OP (dimethoate EC40) produced lung injury via exposure to blood-borne pesticide. Pathological lung changes consisted of alveolar and interstitial oedema, pulmonary haemorrhage and modest neutrophilia with increased concentrations of protein, IL-6 and IL-8 when compared with controls, but with low concentrations of TNF-α and IL-10 in bronchoalveolar lavage fluid (BALF). In a second study, OP poisoned minipigs had increased concentrations of BALF protein, neutrophils, IL-8 and CRP six hours after orogastric poisoning when compared with their baseline values. Electron microscopy images of both studies demonstrated damage to the alveolar capillary membrane secondary to systemic OP poisoning. Prior to conducting the main pulmonary aspiration study in minipigs, there was considerable refinement of the processes involved through use of: (i) pilot aspiration and dose ranging studies; (ii) the development of a specific pulmonary histopathological scoring system; and (iii) employment of modern human anaesthetic equipment and intensive care patient management protocols. After this period of model development, an in vivo 48 hour study using Gottingen minipigs (n=26) was conducted to investigate the pulmonary pathophysiology in animals given either sham bronchoscopy (sham control) or 0.5 mL/kg of: saline (saline control), porcine gastric juice [GJ], OP (dimethoate EC40) + GJ [OP+GJ], or solvent (cyclohexanone) + GJ [Solv+GJ] into the right lung under bronchoscopic guidance. The results showed that in a minipig model OP and GJ placed into one lung created a direct (right) and indirect (left) lung injury significantly different to controls, and in some respects worse than GJ alone 48 hours after poisoning. The direct lung injury caused by OP+GJ was characterised by significantly worse pathology (p=0.0003) in terms of: pulmonary neutrophilia, alveolar haemorrhage, necrosis, oedema and fibrin deposition, when compared with sham controls at 48 hours. Lungs injured directly with OP+GJ also had significantly higher concentrations of BALF neutrophils (p≤0.01), protein (p≤0.05), IL-6 (p≤0.01), IL-8 (p≤0.01) and CRP (p≤0.05) at 24 hours, and BALF protein (p≤ 0.01), and CRP (p≤ 0.05) when compared with sham controls at 48 hours. The BALF from OP+GJ minipigs at 48 hours also had higher numbers of aerobic bacteria than other groups, suggesting the development of pneumonia could be a source of additional lung injury. Lung damage might also have resulted from a reduction in the surfactant component responsible for the lowering of alveolar surface tension. Direct lung injury with OP+GJ caused a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine (PC) species 16:0/16:0 [29(±4) % vs. 38(±4) %] when compared with sham controls at 48 hours. Unlike the other groups, OP+GJ (direct and indirectly-injured) lungs had type 2 alveolar cell ultrastructural morphological differences in the lamellar bodies that stored the surfactant. The lamellar bodies were more numerous and more dense in the OP+GJ lungs compared with other groups and could signify a failure of surfactant release or some other pathology pertinent to OP aspiration lung injury. Computed tomography analysis showed that direct lung injury with OP+GJ caused significantly more lung tissue to be poorly or non-aerated [77 (±13) % ; p≤0.0001 when compared with sham] as opposed to 62 (±27) % in GJ, 53(±13)% in sham and 47(±0.2)% in saline control animals by 47.5 hours and was mainly due to pulmonary haemorrhage and oedema fluid. The key differences between aspiration of OP+GJ versus GJ alone was that the majority of inflammatory markers (e.g. BALF protein, IL-6 and CRP) appeared to increase from 24-48 hours in OP+GJ treated animals, but decreased in GJ pigs, possibly signifying resolution. Treatment with GJ alone produced less severe histopathological damage, bacterial BALF numbers and percentage of poorly and non-aerated lung tissue. Importantly, there was less evidence of indirect lung injury within the GJ pigs when compared with animals treated with OP+GJ. Solvent placed into the lung seemed to offer some form of protection from the effects of GJ aspiration. This was dramatically demonstrated by the histopathology scores, proportional percentage of beneficial phosphatidylcholine (PC) species 16:0/16:0 and the percentage of poorly and non-aerated lung tissue all approaching control animal levels by 48 hours in minipigs that had Solv+GJ placed in the directly-injured (right) lung. Further evidence of benefit was provided by statistically significant reductions (p≤ 0.05) in BALF concentrations of IL-8, IL-6 and CRP in minipigs which had aspirated Solv+GJ when compared with OP+GJ and/or GJ minipig groups at 24 hours. The pathophysiology of aspirated OP+GJ was also investigated in a pilot ovine ex vivo lung perfusion (EVLP) model (n=4). Lungs directly-injured with OP+GJ had higher concentrations of total protein (4300 mg/L vs. 350 mg/L) with a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine species 16:0/16:0 (27% vs.34%) when compared with control lungs. Analysis of toll-like receptor (TLR) lung tissue expression in the OP+GJ directly and indirectly-injured lungs indicated that inflammatory mechanisms might also involve upregulation of TLR 3 and 5, unlike other lung injuries e.g. those induced with lipopolysaccharide, which typically upregulates TLR 2 and 4. To compare OP-induced lung injury in humans and the minipigs, a small feasibility study was conducted in the ICUs of the University of Peradeniya hospital, Sri Lanka. Unfortunately, ethics review and recruitment proved more difficult than expected and we failed to recruit to target. We did however find raised BALF concentrations of IL-6, IL-8 and CRP and low concentrations of TNF, IL-1β, IL-10 in intubated OP poisoned patients at 24 hours when compared with controls. We also found that two plasma micro-RNA biomarkers thought to be involved in inflammation and lung injury, MiR-21 and MiR-146a, had significantly reduced expression in OP-poisoned patients with aspiration compared to non-intubated control patients from the UK (p=0.008 and p=0.0083 respectively). The work from this thesis has allowed the characterisation of both indirect and direct lung injuries caused by OP pesticide ingestion and aspiration. The minipig model showed that at 48 hours the lung injury created by aspiration of OP+GJ appeared more severe than GJ alone, but the addition of the solvent cyclohexanone seemed protective and even beneficial in the context of GJ aspiration. The cytokine expression profiles from both the human and minipig work, combined with the preliminary TLR lung tissue analysis from the EVLP model, suggest that OP+GJ aspiration is unlike normal GJ aspiration and classic ARDS. / Increased concentrations of aerobic bacteria in the minipig OP+GJ lungs at 48 hours and evidence of suppression of plasma miR-21 and miR-146a in OP poisoned patients could be linked, and may involve cholinergic immune system modulation. These molecular mechanisms need to be investigated further in both in vitro and in vivo models. These discoveries indicate the complex nature of the pulmonary injury that occurs after OP pesticide poisoning, and suggests that damage is not caused by gastric contents alone. Preliminary findings indicate that aspiration of OP+GJ could create favourable conditions for the development of aspiration or ventilator-associated pneumonia but this would need confirmation in larger clinical studies. The potential roles of micro RNA as a biomarker of OP poisoning and lung injury, and solvent as a therapy for aspiration should be explored in further pre-clinical studies.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

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Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.