archives@tulane.edu / Acute respiratory distress syndrome (ARDS) is a life-threatening, non-carcinogenic inflammatory pulmonary conditions characterized by the collection of fluids in the air sacs of the lungs. When fluid-filled airways are ventilated, the stresses of repetitive recruitment-decruitment (Rec-Der) causes cellular damage to the epithelial surface, leading to ventilator induced lung injury (VILI). The objective of this study was to establish a foundation for use of electric cell-substance impedance sensing (ECIS) in real-time analysis of cell membrane morphology and RecDer-induced damage. NCI H441 papillary adenocarcinoma human pulmonary epithelial cells are cultured onto custom 1F8x10E PC Flow Array. 10mM cysteine and 1% gelatin surface treatments demonstrated strong results for improved cell-substrate adhesion strength. RecDer insults were introduced at a velocity of 0.5mm/s through FBS-enhanced RPMI164 growth media. Experimental trials for 0 (n=1), 1 (n=1), 5 (n=1), 10 (n=1), 20 (n=7), and 50 (n=1) RecDer insults were analyzed using Annexin-V/PI flow cytometry; results showed monolayer health of 97.76%, 93.152%, 91.801%, 72.495%, 66.88% and 60.812% respectively. Trials for 20 (n=1), 30 (n=1), and 40 (n=1) RecDer insults were analyzed using ECIS; Frequency-dependent impedance modeling of the acquired data suggested increased damage to both cell-cell junction health and cell membrane integrity with increased RecDer insults. Results established a strong foundation for ECIS analysis of RecDer-induced monolayer damage. / 1 / Joshua Erwa Yao

Mechanical ventilation (MV) is a life-saving therapy in acute respiratory distress syndrome (ARDS), a condition that affects 3000 patients/year in Sweden with a mortality rate of about 40%. However, MV may induce or worsen lung injury causing “ventilator-induced lung injury (VILI)”. From a mechanical perspective strain (deformation, or relative change in lung volume) and stress (tension) have been postulated as main determinants of VILI. High respiratory rate is potentially another factor that may exacerbate VILI by amplifying the total energy transmitted to the lungs during MV. In this thesis in animal ARDS models the hypotheses were that 1) lung parenchyma inhomogeneities concentrate stress and amplify lung damage and inflammation, 2) higher respiratory rates increase lung inflammation and lung edema in heterogeneous ARDS, and 3) local lung deformation is related to local inflammation. First, in a rat model the effect on inflammation and structural damage of regional lung collapse on the healthy surrounding lung tissue was assessed. Second, in porcine models the effect of respiratory rate on lung edema and inflammation was studied during two ventilatory modes; a) a permissive collapse mode and b) a homogenized lung parenchyma mode. Finally, lung deformation was correlated with lung inflammation assessed by positron emission tomography using 18F-FDG uptake. It was found that; 1) local inhomogeneities can act as stress amplifiers, increasing lung tissue inflammation and damage in the healthy surrounded lung. 2) high respiratory rate increases lung edema but decreases lung inflammation when permissive lung collapse is used and that these effects are prevented with lung parenchyma homogenization; 3) local lung deformation and inflammation are well correlated. In conclusion, lung inhomogeneities may aggravate VILI, respiratory rate may affect in different ways VILI progression depending on the ventilatory strategy, and finally, lung deformation is closely related to lung inflammation. With the caveat that the studies are performed in animal models, the results suggest that using ventilator strategies that homogenize the lungs, i.e., open collapsed lung regions and prevent re-collapse in ARDS will reduce VILI and in the end may decrease morbidity and the high mortality in this condition.

No description available.

Maschinelle Beatmung kann neben den gewünschten Effekten eine vorbestehende Lungenerkrankung weiter aggravieren und sogar das Lungenparenchym zuvor lungengesunder Patienten schädigen. Mit Hilfe der quantitativen Computertomographie (qCT) können pathologische Belüftungszustände und gegebenenfalls durch maschinelle Beatmung verursachte Schäden analysiert werden. Solche auf der qCT basierende Analysen der Lungenbelüftung werden jedoch potentiell durch CT-Akquisitions- und Bildrekonstruktionsparameter beeinflusst. Um die Ergebnisse vor allem von Analysen des überbelüfteten Lungenvolumens richtig bewerten zu können, müssen solche Einflüsse untersucht werden. Bei 10 Versuchstieren (Schweine) wurden bei einem konstanten Atemwegsdruck von 25 cm H2O zuerst bei gesunder Lunge und dann erneut nach experimenteller Lungenschädigung CT-Bildserien mit zwei unterschiedlichen Strahlendosen angefertigt. Von diesen Rohdaten wurden Bildserien mit unterschiedlichen Rekonstruktionsparametern angefertigt und in jeder dieser Bildserien das überbelüftete Lungenvolumen bestimmt. Sowohl die Schichtdicke, der Filter als auch die Stromstärke hatten einen signifikanten Einfluss auf das eigentlich konstante überbelüftete Lungenvolumen, der jedoch nur teilweise klinisch relevant war. Bei der Interpretation von Messungen des überbelüfteten Lungenvolumens sollten dennoch die Einflüsse der genannten Parameter beachtet und für Vergleichsuntersuchungen gleiche Parametereinstellungen verwendet werden. Eine Dosisreduktion scheint dabei für Messungen des überbelüfteten Lungenvolumens praktikabel.

Abstract Background The majority of patients requiring mechanical ventilation are over the age of 65 and advanced age is known to increase the severity of ventilator-induced lung injury (VILI) and mortality. However, the mechanisms which predispose aging ventilator patients to increased mortality rates are not fully understood. Pulmonary edema is a hallmark of VILI and the severity of edema increases with age. Ventilation with conservative fluid management decreases mortality rates in acute respiratory distress (ARDS) patients, but has not been investigated in VILI. We hypothesized that age-associated increases in pulmonary edema promote age-related increases in ventilator-associated mortality. Endoplasmic reticulum (ER) stress can disrupt cellular functions and plays a key role in many disease states. The severity of ER stress also increases with age. We hypothesized that age-associated increases in ER stress also increase in the severity of VILI. Finally, serum Vitamin C (VitC) levels also decrease with age. VitC treatments have been shown to decrease mortality rates in murine models of ARDS by and attenuate pulmonary edema. We hypothesize that VitC treatments will attenuate ventilator induced pulmonary edema in our aged murine subjects. Methods Mechanical Ventilation: Young and old mice were mechanically ventilated with either high tidal volume (HVT) or low tidal volume (LVT) for with either liberal or conservative fluid support. One group received VitC treatment prior to ventilation. Cell Stretch: Alveolar epithelial cells (ATIIs) from young and old mice were harvested, cultured, and mechanically stretched. Treatment groups received ER stress inhibitor 4-PBA. Results Both advanced age and HVT ventilation significantly increased inflammation, injury, and decreased survival rates. Conservative fluid support significantly diminished pulmonary edema decreased mortality rates. VitC treatments significantly decreased pulmonary edema and improved pulmonary mechanics. Mechanical stretch promoted ER Stress and upregulated proinflammatory gene expression and secretion in aged ATIIs. ER stress inhibition attenuated all of these effects. Conclusion Conservative fluid management alone attenuated age-associated increases in ventilator-associated mortality. VitC treatments decreased pulmonary edema and partially restore pulmonary mechanics in old mice ventilated with HVT. ER stress inhibition decreased stretch induced proinflammatory gene expression and protein secretion in aged mechanically stretched ATII cells.

A fairly large portion of patients receiving surgical or intensive care will need mechanical ventilation at some point. The potential ventilator-induced lung injury (VILI) is thus of interest. One of the main causal factors in VILI is the cyclic energy shifts, i.e. tidal volumes, in the lung during mechanical ventilation. The problem can be approached in two ways. Firstly, one can utilize apneic oxygenation and thus not cause any tidal injuries at all. Secondly, and more traditionally, one can simply lower the tidal volumes and respiratory rates used. The following describes a series of animal experiments exploring these options. In the first two papers, I explored and improved upon the methodology of apneic oxygenation. There is a generally held belief that it is only possible to perform apneic oxygenation by prior denitrogenation and by using 100% oxygen during the apnea. As 100% oxygen is toxic, this has prevented apneic oxygenation from more widespread use. The first paper proves that it is indeed possible to perform apneic oxygenation with less than 100% oxygen. I also calculated the alveolar nitrogen concentration which would conversely give the alveolar oxygen concentration. The second paper addresses the second large limitation of apneic oxygenation, i.e. hypercapnia. Using a high dose infusion of tris(hydroxymethyl)aminomethane (THAM) buffer, a pH > 7.2 could be maintained during apneic oxygenation for more than 4.5 hours. In the last two papers, THAM’s properties as a proton acceptor are explored during respiratory acidosis caused by very low volume ventilation. In paper III, I found that THAM does not, in the long term, affect pH in respiratory acidosis after stopping the THAM infusion. It does, however, lower PVR, even though the PaCO2 of THAM-treated animals had rebounded to levels higher than that of the controls. In the last experiment, I used volumetric capnography to confirm our hypothesis that carbon dioxide elimination through the lungs was lower during the THAM infusion. Again, the PaCO2 rebounded after the THAM infusion had stopped and I concluded that renal elimination of protonated THAM was not sufficient.

Maschinelle Beatmung kann neben den gewünschten Effekten eine vorbestehende Lungenerkrankung weiter aggravieren und sogar das Lungenparenchym zuvor lungengesunder Patienten schädigen. Mit Hilfe der quantitativen Computertomographie (qCT) können pathologische Belüftungszustände und gegebenenfalls durch maschinelle Beatmung verursachte Schäden analysiert werden. Solche auf der qCT basierende Analysen der Lungenbelüftung werden jedoch potentiell durch CT-Akquisitions- und Bildrekonstruktionsparameter beeinflusst. Um die Ergebnisse vor allem von Analysen des überbelüfteten Lungenvolumens richtig bewerten zu können, müssen solche Einflüsse untersucht werden. Bei 10 Versuchstieren (Schweine) wurden bei einem konstanten Atemwegsdruck von 25 cm H2O zuerst bei gesunder Lunge und dann erneut nach experimenteller Lungenschädigung CT-Bildserien mit zwei unterschiedlichen Strahlendosen angefertigt. Von diesen Rohdaten wurden Bildserien mit unterschiedlichen Rekonstruktionsparametern angefertigt und in jeder dieser Bildserien das überbelüftete Lungenvolumen bestimmt. Sowohl die Schichtdicke, der Filter als auch die Stromstärke hatten einen signifikanten Einfluss auf das eigentlich konstante überbelüftete Lungenvolumen, der jedoch nur teilweise klinisch relevant war. Bei der Interpretation von Messungen des überbelüfteten Lungenvolumens sollten dennoch die Einflüsse der genannten Parameter beachtet und für Vergleichsuntersuchungen gleiche Parametereinstellungen verwendet werden. Eine Dosisreduktion scheint dabei für Messungen des überbelüfteten Lungenvolumens praktikabel.:Inhaltsverzeichnis 0 Abkürzungsverzeichnis 1 1 Einleitung 3 1.1 Ventilator-associated Lung Injury (VALI) 3 1.2 Computertomographie und Diagnostik von Lungenerkrankungen 5 1.3 Spiral-CT 9 1.4 Datenerfassung und Bildrekonstruktion 10 1.5 Grundlagen zur Dichtemessung 12 1.6 Einfluss von Filter und Schichtdicke auf das Bild 13 1.7 Einfluss von Filter und Schichtdicke auf die Analyse der pulmonalen Überbelüftung 15 1.8 Zielstellung 17 2 Materialien und Methodik 18 2.1 Versuchstiere 18 2.2 Überblick über den Versuchsablauf 18 2.2.1 Prämedikation und Narkoseführung 18 2.2.2 Induktion des Lungenschadens 20 2.2.3 CT-Scans und Bildrekonstruktionen 21 2.3 Segmentierung und volumetrische Analyse 22 2.4 Statistische Analyse 24 3 Ergebnisse 26 3.1 Einfluss von Schichtdicke, Filter und Stromstärke auf normale Lungen 26 3.2 Einfluss von Schichtdicke, Filter und Stromstärke auf geschädigte Lungen 34 3.3 Vergleich der automatischen und manuellen Segmentierung 38 4 Diskussion 40 4.1 Einfluss von Schichtdicke und Filter 42 4.2 Einfluss der Stromstärke 49 4.3 Einfluss der experimentell induzierten Lungenschädigung 53 4.4 Vergleich der Segmentierungssoftware 55 4.5 Diskussion der Methodik 55 4.6 Schlussfolgerung 58 5 Zusammenfassung der Arbeit 60 6 Literaturverzeichnis 63 7 Danksagung 77 8 Erklärung über die eigenständige Abfassung der Arbeit 78

Background: Mechanical ventilation (MV) inflicts stress on the lungs, initiating or increasing lung inflammation, so-called ventilator-induced lung injury (VILI). Besides overdistention, cyclic opening-and-closing of alveoli (atelectrauma) is recognized as a potential mechanism of VILI. The dynamic stretch may be reduced by positive endexpiratory pressure (PEEP), which in turn increases the static stretch. We investigated whether static stretch modulates the inflammatory response of rat type 2 alveolar epithelial cells (AECs) at different levels of dynamic stretch and hypothesized that static stretch increases pro-inflammatory response of AECs at given dynamic stretch. - Methods: AECs, stimulated and not stimulated with lipopolysaccharide (LPS), were subjected to combinations of static (10, 20, and 30%) and dynamic stretch (15, 20, and 30%), for 1 and 4 h. Non-stretched AECs served as control. The gene expression and secreted protein levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) were studied by real-time polymerase chain reaction (RTqPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The effects of static and dynamic stretch were assessed by two-factorial ANOVA with planned effects post-hoc comparison according to Šidák. Statistical significance was considered for p < 0.05. - Results: In LPS-stimulated, but not in non-stimulated rat type 2 AECs, compared to nonstretched cells: 1) dynamic stretch increased the expression of amphiregulin (AREG) (p < 0.05), MCP-1 (p < 0.001), and MIP-2 (<0.05), respectively, as well as the protein secretion of IL-6 (p < 0.001) and MCP-1 (p < 0.05); 2) static stretch increased the gene expression of MCP-1 (p < 0.001) and MIP-2, but not AREG, and resulted in higher secretion of IL-6 (p < 0.001), but not MCP-1, while MIP-2 was not detectable in the medium. - Conclusion: In rat type 2 AECs stimulated with LPS, static stretch increased the proinflammatory response to dynamic stretch, suggesting a potential pro-inflammatory effect of PEEP during mechanical ventilation at the cellular level.

Beatmungsbedingte Lungenschäden in der Therapie des akuten Atemnotsyndroms (ARDS) sind aufgrund der inhomogenen Vorschädigung der Lunge praktisch unvermeidbar. Die unphysiologische mechanische Belastung der Lunge führt über Volutrauma, Atelektotrauma und Biotrauma nicht selten zur Exazerbation des Syndroms und trägt zur hohen Mortalität des ARDS bei. Pharmakologische Interventionsmöglichkeiten sind Gegenstand der aktuellen Forschung. Diesbezüglich vielversprechend ist die zentrale Komponente des Kinin-Kallikrein-Systems, namentlich Bradykinin, das über seinen B2-Rezeptor anti-apoptotische Signalwege aktivieren kann und somit zellprotektive Wirkung besitzt. In der vorliegenden Arbeit wurde untersucht, in welcher Weise zyklische mechanische Dehnung die Konzentration einzelner Komponenten des Kinin-Kallikrein-Systems in isolierten alveolären Epithelzellen (Typ II) der Ratte beeinflusst. Dafür wurden die alveolären Typ-II-Zellen auf speziellen BioFlex®-Membranen kultiviert und für 24 Stunden zyklisch mit hoher Dehnungsamplitude gedehnt. Anschließend wurden mit etablierten Analysemethoden in Zellüberständen bzw. Zelllysaten die Konzentrationen von Kininogen 1, Bradykinin und vom B2-Rezeptor gemessen, sowie die Aktivität des Enzyms Kallikrein und des Bradykinin-Abbaus bestimmt - jeweils im Vergleich mit Überständen bzw. Lysaten ungedehnter AT-II-Zellkulturen. Es zeigte sich dehnungs-bedingt eine Zunahme der Bradykinin-Produktion durch Kininogen und Kallikrein und eine stark gesteigerte Bradykinin-abbauende Aktivität, sodass sich der Bradykininspiegel insgesamt verringerte. Die Konzentration des B2-Rezeptors blieb unverändert. Detailliertes Wissen über den Einfluss zyklischer mechanischer Dehnung auf die Einzelkomponenten des Kinin-Kallikrein-Systems ist eine Grundvoraussetzung, um die zellprotektive Wirkung von Bradykinin im Sinne einer pharmakologischen Interventionsmöglichkeit bei ARDS nutzbar machen zu können.

Background: O câncer de colo uterino é um grande problema de saúde pública no Brasil e no mundo. Atinge cerca de meio milhão de mulheres, sendo que 50 % destas morrem. Os métodos visuais, alternativos á citologia e viáveis para países em desenvolvimento, têm sido avaliados mais intensamente na última década. Objetivos: Avaliar o desempenho da Fotografia Cervical Digital (FCD) na detecção do câncer do colo de útero e de suas lesões precursoras. Métodos: 176 mulheres foram avaliadas pelos métodos de Inspeção Visual com ácido acético (VIA), Inspeção Visual com lugol (VILI), Fotografia Cervical Digital com ácido acético (FCDA) e Fotografia Cervical Digital com lugol (FCDL). Destas, 36 foram classificadas pela histologia. Resultados: A concordância entre avaliadores observou valores de Kappa: Fotografia Cervical Digital com ácido acético, K=0,441, e Fotografia Cervical Digital com lugol, K=0,533. A concordância entre as inspeções a olho nu e as fotografias cervicais digitais após a utilização de ácido acético no colo uterino, K=0,559, e a concordância após a utilização da solução de lugol, K=0,507, considerada como concordância moderada em ambos os casos. Das 36 pacientes com avaliações histológicas, 20 dos 25 casos positivos foram corretamente avaliados tanto pela FCDA, como pela FCDL, demonstrando resultados similares aos descritos anteriormente. Conclusão: A FCD é um método promissor para o rastreamento do câncer de colo de útero e suas lesões precursoras, para países em desenvolvimento. / Background: Uterine cervix cancer is a major public health problem in Brazil and in the world. It affects half a million women with a death rate of 50%. The visual, alternative methods to cytology, considered viable for developing countries, are being assessed more intensively in the last decade. Objective: To evaluate the performance of Cervical Digital Photography (CDP) in detecting cervical cancer and its precursory lesions. Methods: A total of 176 women were evaluated by the following methods: Visual Inspection with Acetic Acid (VIA), Visual Inspection with Lugol’s Iodine (VILI), Cervical Digital Photography with Acetic Acid (CDPA) and Cervical Digital Photography with Lugol’s Iodine (CDPL). Among these, 36 were classified by histology. Results: The correlation between evaluators observed Kappa values: Cervical Digital Photography with acetic acid, K = 0.441, and Cervical Digital Photography with Lugol's Iodine, K = 0.533. The correlation between the naked eye and cervical digital photography inspection after using acetic acid in the uterine cervix, K = 0.559, and the correlation after using Lugol's Iodine solution, K = 0.507, were regarded as moderate in both cases. Of the 36 patients with histological evaluation, 20 of the 25 positive cases were correctly assessed both by the CDPA as well as by CDPL, showing similar results than those described previously. Conclusion: CDP is considered a more promising method for screening the uterine cervix cancer and its precursory lesions in developing countries.