Ventilation with "clinically relevant" high tidal volumes does not promote stretch-induced injury in the lungs of healthy mice

OBJECTIVE: Ventilator-induced lung injury is a crucial determinant of the outcome of mechanically ventilated patients. Increasing numbers of mouse studies have identified numerous pathways and mediators that are modulated by ventilation, but it is conceptually difficult to reconcile these into a single paradigm. There is substantial variability in tidal volumes used in these studies, and no certainty about the pathophysiology that such varied models actually represent. This study was designed to investigate whether ventilation strategies ranging from ‘very high’ to more ‘clinically-relevant’ tidal volumes induce similar pathophysiologies in healthy mice, or represent distinct entities. DESIGN: In vivo study. SETTING: University research laboratory. SUBJECTS: C57/Bl6 mice. INTERVENTIONS: Anesthetised mice were ventilated with various tidal volumes up to 40ml/kg. MEASUREMENTS AND MAIN RESULTS: Respiratory system compliance and arterial blood gases were used to evaluate physiological parameters of injury. Lung wet:dry weight ratio, lavage fluid protein and cytokines were used to assess pulmonary edema and inflammation. All ventilation strategies induced changes in respiratory system compliance, although the pattern of change was unique for each strategy. 10ml/kg and 40ml/kg ventilation also induced decreases in arterial pO(2) and blood pressure. Any physiological changes induced during the 10, 20 and 30ml/kg strategies were largely reversed by recruitment maneuvers at the end of the protocol. Markers of pulmonary edema and inflammation indicated that only 40ml/kg induced substantial increases in both, consistent with development of lung injury. CONCLUSIONS: Tidal volumes up to 20ml/kg are unlikely to induce substantial lung over-stretch in models using healthy, young mice. Signs of injury/inflammation using such models are likely to result from other factors, particularly alveolar derecruitment and atelectasis. The results of such studies may need to be re-evaluated before clinical relevance can be accurately determined

Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.

Extracellular microvesicles (MVs) are released into the circulation in large numbers during acute systemic inflammation, yet little is known of their intravascular cell/tissue-specific interactions under these conditions. We recently described a dramatic increase in the uptake of intravenously injected MVs by monocytes marginated within the pulmonary vasculature, in a mouse model of low-dose lipopolysaccharide-induced systemic inflammation. To investigate the mechanisms of enhanced MV uptake by monocytes, we developed an in vitro model using in vivo derived monocytes. Although mouse blood is a convenient source, monocyte numbers are too low for in vitro experimentation. In contrast, differentiated bone marrow monocytes are abundant, but they are rapidly mobilized during systemic inflammation, and thus no longer available. Instead, we developed a protocol using marginated monocytes from the pulmonary vasculature as an anatomically relevant and abundant source. Mice are sacrificed by terminal anesthesia, the lungs inflated and perfused via the pulmonary artery. Perfusate cell populations are evaluated by flow cytometry, combined with in vitro generated fluorescently labelled MVs, and incubated in suspension for up to one hour. Washed cells are analyzed by flow cytometry to quantify MV uptake and confocal microscopy to localize MVs within cells (O'Dea et al., 2020). Using this perfusion-based method, substantial numbers of marginated pulmonary vascular monocytes are recovered, allowing multiple in vitro tests to be performed from a single mouse donor. As MV uptake profiles were comparable to those observed in vivo, this method is suitable for physiologically relevant high throughput mechanistic studies on mouse monocytes under in vitro conditions. Graphic abstract: Figure 1. Schematic of lung perfusate cell harvest and co-incubation with in vitro generated MVs. Created with BioRender.com

In vivo compartmental analysis of leukocytes in mouse lungs

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined “interstitial” leukocyte populations during models of inflammatory lung diseases

Acridine Orange Fluroscence Study of Lung - Histopathology in Autopsy Cases of Burns

Background: The major cause of death in the burn patients includes multiple organ failure and septicemia but, sometimes the exact cause of death in many fatally burn patients is difficult to detect. Aim: The aim was to study various histopathological changes in lung in the post-mortem cases of burns, by using routine Haematoxylin and Eosin stain (H&E stain). Periodic and Schiff ’s Stain (PAS) stain to study the role of acridine orange fluorescence study, to explore the forensic utility of this study and to find out the relationship between duration of survival and histopathological changes observed. Material & Methods: Total 32 cases of death due to burns were autopsied at mortuary from october 2010 to september 2012, department of Forensic Medicine and Toxicology in our hospital. These were forwarded to Department of Pathology for histopathological examination. Result: In the present study, maximum number of burns cases in 21-30 years of age group & female predominance. Grossly, 19 cases (59.38%) showed congestion while microscopy showed diffuse alveolar damage (34.38%). The sections stained by acridine orange and observed under fluorescent microscope were negative in 28 cases (87.50%) and lightly positive in 04 cases (12.50%). Conclusion: Routine microscopy does help us in getting specific lesions in lung due to burns. But PAS and Acridine orange fluorescence do not add anything further in our knowledge of pathology due to burns. However, none of these add any new tool to resolve any forensic issues of burns. Therefore, microscopy (including PAS and fluorescent), if done would be redundant

Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health

An approach to provide dynamic, illustrative, video-based guidance within a goal-driven smart home

The global population is aging in a never-before seen way, introducing an increasing ageing-related cognitive ailments, such as dementia. This aging is coupled with a reduction in the global support ratio, reducing the availability of formal and informal support and therefore capacity to care for those suffering these aging related ailments. Assistive Smart Homes (SH) are a promising form of technology enabling assistance with activities of daily living, providing support of suffers of cognitive ailments and increasing their independence and quality of life. Traditional SH systems have deficiencies that have been partially addressed by through goal-driven SH systems. Goal-driven SHs incorporate flexible activity models, goals, which partially address some of these issues. Goals may be combined to provide assistance with dynamic and variable activities. This paradigm-shift, however, introduces the need to provide dynamic assistance within such SHs. This study presents a novel approach to achieve this through video based content analysis and a mechanism to facilitate matching analysed videos to dynamic activities/goals. The mechanism behind this approach is detailed and followed by the presentation of an evaluation where showing promising results were shown

Inhibition of TNF receptor p55 by a domain antibody attenuates the initial phase of acid-induced lung injury in mice

Background: Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator36 induced lung injury. In the current study we explored the efficacy of this antibody in mouse models of acid-induced lung injury, to investigate the longer consequences of treatment. Methods: We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting ‘dummy’ dAb, 1 or 4 hours before acid instillation. Results: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. Conclusions: These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 hours. Together with our previous data, the current study lends support towards the clinical targeting of p55 for patients with, or at risk of ARDS