Asthma ist eine Erkrankung die das komplette Immunsystems involviert, ein System so komplex, dass es sich nur unzureichend in-vitro studieren lässt. Daher haben sich Mausmodelle als ein unverzichtbares Werkzeug in der präklinischen Asthmaforschung etabliert. Da es sich weiterhin bei Asthma um eine Erkrankung handelt, die durch eine schnelle Änderung der Symptome gekennzeichnet ist, wäre longitudinale vorzugsweise nicht-invasive Bildgebung, insbesondere bei der Entwicklung und Bewertung neuer Therapiekonzepte von großem Interesse. Nachteilig hingegen ist, dass die Darstellung der Mauslunge in der Praxis auf Grund der Größe des Organs und, im Falle einer in vivo Bildgebung, durch die Bewegung des Brustkorbes sich als äußerst schwierig herausstellt. Die Vielzahl der Luft-Gewebe-Grenzflächen erzeugt starke Streuung in der optischen Bildgebung, der große Hohlraum der Lunge verursacht Suszeptibilitätsartefakte bei der MRT und die Rippen erschweren eine Ultraschallbildgebung. Aus diesen Gründen besteht ein großer Bedarf an neuen Bildgebungsverfahren, um die durch Asthma verursachten anatomischen, funktionalen und molekularen Veränderungen darstellen zu können. Um die Schwierigkeiten in der Lungenbildgebung bei Mäusen zu umgehen, habe ich mich auf drei wesentliche Bildgebungsstrategien fokussiert: A) anatomische Bildgebung durch “inline free propagation phase contrast computed tomography”, B) direkte Messung der Lungenfunktion durch “low dose planar cinematic x-ray imaging” und C) funktionale Bildgebung mit Hilfe der „near infrared fluorescence imaging“ in Kombination mit Antikörpern, die mit einem Fluoreszenzfarbstoff markiert wurden, oder “smart probes”, die in Gegenwart von Entzündungen aktiviert werden. Durch die Anwendung von “phase contrast computed tomography” für die anatomische Bildgebung war ich in der Lage morphologische Veränderung des Lungengewebes zu quantifizieren, indem ich lokal das Verhältnis zwischen Weichgewebe und Luft, das Zusammenziehen der Luftwege sowie das Anschwellen der Bronchialwände im asthmatischen Lungengewebe ausgewertet habe. Diese Parameter erlaubten es zwischen Mäusen von Asthmamodellen unterschiedlicher Schweregrade, therapierten und gesunden Mäusen zu unterscheiden. Zusätzlich ermöglichte diese Technik die Darstellung intra-tracheal applizierter Bariumsulfat markierter Makrophagen im Lungengewebe. Dies stellt meines Wissens die erste Kombination einer funktionalisierten Kontrastierung und hochauflösender Lungenbildgebung mittels CT unter in vivo ähnlichen Bedingungen dar. Um diese Ergebnisse mit dem Grad der asthmabedingten Kurzatmigkeit zu korrelieren, habe ich eine einfache und verlässige Methode entwickelt die es, basierend auf 2D Röntgen-videos niedriger Röntgendosis (~6,5mGy) erlaubt, in narkotisierten Mäusen die Lungenfunktion zu bewerten. Mit Hilfe dieser neuen Methode gelang es mir charakteristische Unterschiede in der Lungenfunktion von asthmatischen, therapierten und gesunden Mäusen in vivo über die Zeit nachzuweisen, und diese Resultate mit den Ergebnissen von CT und Histologie zu korrelieren. Das Verfahren wird derzeit von mir für die Anwendung an frei beweglichen und nicht narkotisierten Mäusen weiterentwickelt. Dies sollte zu einer deutlichen Stressreduktion für die Maus bei der Untersuchung führen und somit, vor allem in Asthma, im Gegensatz zu etablierten Verfahren wie Plethysmographie, die Erhebung validerer Messdaten erlauben. Mit Hilfe von „near infrared fluorescence imaging“ konnten wir in vivo und longitudinal erfolgreich verschiedene durch Asthma ausgelöste molekulare Veränderungen in der Mauslunge verfolgen. Erstens erlaubte die Verwendung einer neuen Polyglyzerol Probe mit dendritischer Struktur (MN2012) die spezifisch an Selektine bindet, die Darstellung der durch Asthma verursachten Entzündung der Lunge. Im Zuge dessen konnten wir nachweisen, dass sich MN2012  zur Darstellung von Enzymkinetiken bei Entzündungsreaktionen durch eine schnellere Kinetik und höher Spezifität als kommerziell erhältliche Proben auszeichnet. Zweitens haben wir gezeigt, dass in Kombination mit einem Fluoreszenz markiertem Antikörper gegen SiglecF, einem Antigen das hauptsächlich auf Eosinophilen exprimiert ist, Eosinophilie in asthmatischen Mäusen verfolgt und der Effekt einer Dexamethason Behandlung  ebenso dargestellt werden kann. Drittens konnten wir den Verbleib inhalierter fluoreszierender Nanopartikel in der Lunge der Maus in vivo untersuchen und dabei nachweisen, dass diese hauptsächlich von endogenen Makrophagen im Lungengewebe aufgenommen werden. Alle diese Techniken wurden gegeneinander und mittels histologischer Analyse und Fluoreszenzmikroskopie korreliert und validiert.  Zusammenfassend bilden die in meiner Dissertation entwickelten Lungenbildgebungsstrategien für Asthmamausmodelle eine Bildgebungsplattform, um sowohl spezifische Effekte in asthmatischen Mäusen unterschiedlichen Schweregrades als auch die Auswirkungen neuer Therapien abzubilden und im Detail zu untersuchen.

<p>Radiation-induced lung injury (RILI) is a common condition in the setting of lung and breast cancer. Often, patients who suffer from RILI experience pneumonitis and pulmonary fibrosis months after treatment. These pathologies have commonly been modeled using mice and observing their deterioration until mortality and quantifying pathology on histological sections.<b> </b></p> <p>With this study, we used a longitudinal microCT and a 7T MRI to characterize male C57Bl/6 mice irradiated with a single dose of 20 Gy to the whole thoracic area delivered by an X-Rad cabinent irradiator. CT was performed with a respiratory gating sequence at 2 week timepoints to construct an RIPF model. The fraction of RIPF to total lung volume was calculated at each time point from images, and the data was anaylzed using one-way ANOVA Welch and Dunnett’s T3 multiple comparisons tests. Tidal lung volumes were also calculated and anlyazed in a simlar manner. Mice were then imaged using MRI and CT at 0, 5, and 8 week timepoints to compare results. These results were analyzed for comparison (ANOVA and Dunnett’s T3) and correlation (Pearson’s r) with each other. Histology was later performed using H&E and Trichrome stains to provide ex-vivo verification of pathology.<b> </b>At the 10-12 week time point ( ) significant RIPF formed. Weeks proceeding showed increased significance until the 22+ week timepoint, which showed less statistical significance ( ) due to increased variance at this timepoint. Dunnett’s T3 test showed no significant differences between tidal lung volumes over time. Tests also showed no significant differences between CT and MRI results with a correlation coefficient of .<b> </b>Early in the study, problems arose when pre-marture mortality was occurring to a significant portion of our subjects. Analysis later showed issues during irradiation that resulted in significant dose being absorbed by the stomach. Adjusting our shiedling lead to increased early survival of our subjects enabling us to contine our study. Significant RIPF development was not significant until 10-12 weeks post-irradiation, then RIPF became more severe at proceeding timepoints. Tidal lung volume showed no significant deviation over the development of RIPF. This result is most likely affected by the variation of results at later timepoints, since several mice with severe RIPF were significantly hindered in their ability to breathe during the study. MRI results showed close correlation with CT results and prodcued similar values at early timepoints. However, noticeable differences were seen at later timepoints when significant RIPF developed ( ). <b></b></p>

<p>Since the inception of hyperpolarized 129Xe MRI, the field has yearned for more efficient production of more highly polarized 129Xe. For nearly all polarizers built to date, both peak 129Xe polarization and production rate fall far below theoretical predictions. This thesis sought to develop a fundamental understanding of why the observed performance of large-scale 129Xe hyperpolarization lagged so badly behind theoretical predictions.</p><p> This is done by thoroughly characterizing a high-volume, continuous-flow polarizer using optical cells having three different internal volumes, and employing two different laser sources. For each of these 6 combinations, 129Xe polarization was carefully measured as a function of production rate across a range of laser absorption levels. The resultant peak polarizations were consistently a factor of 2-3 lower than predicted across a range of absorption levels, and scaling of production rates deviated badly from predictions based on spin exchange efficiency.</p><p> To bridge this gap, we propose that paramagnetic, activated Rb clusters form during spin exchange optical pumping (SEOP), and depolarize Rb and 129Xe, while unproductively scattering optical pumping light. When a model was built that incorporated the effects of clusters, its predictions matched observations for both polarization and production rate for all 6 systems studied. This permits us to place a limit on cluster number density of <2 × 109 cm-3.</p><p> The work culminates with deploying this framework to identify methods to improve polarization to above 50%, leaving the SEOP cell. Combined with additional methods of preserving polarization, the polarization of a 300-mL batch of 129Xe increased from an average of 9%, before this work began, to a recent value of 34%.</p><p> We anticipate that these developments will lay the groundwork for continued advancement and scaling up of SEOP-based hyperpolarization methods that may one day permit real-time, on-demand 129Xe MRI to become a reality.</p> / Dissertation

Introdução: A atelectasia intraoperatória ocorre imediatamente após a indução anestésica e pode ser detectada por ultrassom pulmonar (LUS). No entanto, até o momento o LUS não é utilizado para avaliar a hiperdistensão pulmonar. Neste estudo, descreveu-se um método para detectar hiperdistensão pulmonar usando LUS. A tomografia de impedância elétrica (TIE) foi a referência para comparação dos métodos. Métodos: Dezoito (18) pacientes, com 63 ± 6 anos de idade, com pulmões normais, submetidos à cirurgia abdominal inferior. O TIE foi calibrado, realizada a indução anestésica, intubação e ventilação mecânica. Para reverter a atelectasia posterior, realizou-se uma manobra de recrutamento alveolar com o uso de pressão expiratória final positiva (PEEP) 20 cmH20 e pressão aérea do platô 40 cmH2O durante 120 segundos. A titulação PEEP foi então obtida com valores descendentes: 20, 18, 16, 14,12,10, 8, 6 e 4 cmH2O. Os dados de ultrassom e TIE foram coletados em cada nível PEEP e interpretados por dois observadores independentes. O número de linhas H foi contado usando um filtro especial. O teste de correlação de Spearman e a curva ROC foram utilizados para comparar os dados do LUS e TIE. Resultados: O número de linhas H aumentou linearmente com PEEP: de 3 em PEEP 4 cmH2O a 10 em PEEP 20 cmH2O. Cinco linhas H foram o limiar para a detecção de hiperdistensão pulmonar, definida como hiperdistensão na TIE >= 24,5%. A área sob a curva ROC foi 0,947 (IC 95% 0.901-0.976). Conclusão: O LUS intraoperatório detectou hiperdistensão pulmonar em valores descendentes de PEEP. A presença de cinco ou mais linhas H podem ser consideradas como indicando hiperdistensão pulmonar / Purpose: Intraoperative atelectasis occurs immediately after anaesthetic induction and can be detected by lung ultrasound (LUS). However, LUS is considered as unable to assess pulmonary hyperinflation. In this study, we propose a method to detect pulmonary hyperinflation using LUS. Electrical impedance tomography (EIT) was the reference method. Methods: We included 18 patients, 63 ± 6-year old, with normal lungs, undergoing lower abdominal surgery. The following protocol was used: EIT was calibrated, followed by anaesthetic induction, intubation and mechanical ventilation. To reverse posterior atelectasis, a recruitment maneuver - positive end-expiratory pressure (PEEP) 20 cmH20 and plateau airway pressure 40 cmH2O during 120 sec was performed. PEEP titration was then obtained during a descending trial: 20, 18, 16, 14,12,10, 8, 6 and 4 cmH2O. Ultrasound and EIT data were collected at each PEEP level and analyzed by two independent observers. The number of H lines was counted using a special filter. Spearman correlation test and ROC curve were used to compare LUS and EIT data. Results: The number of H lines increased linearly with PEEP: from 3 at PEEP 4 cmH2O to 10 at PEEP 20 cmH2O. Five H lines was the threshold for detecting pulmonary hyperinflation, defined as a mean decrease in maximum EIT compliance >= 24,5 %. The area under the ROC curve was 0.947 (CI 95% 0.901-0.976). Conclusion: Intraoperative transthoracic LUS can detect pulmonary hyperinflation during a PEEP descending trial. Five or more H lines can be considered as indicating pulmonary hyperinflation in normally aerated lung regions

Introdução: A atelectasia intraoperatória ocorre imediatamente após a indução anestésica e pode ser detectada por ultrassom pulmonar (LUS). No entanto, até o momento o LUS não é utilizado para avaliar a hiperdistensão pulmonar. Neste estudo, descreveu-se um método para detectar hiperdistensão pulmonar usando LUS. A tomografia de impedância elétrica (TIE) foi a referência para comparação dos métodos. Métodos: Dezoito (18) pacientes, com 63 ± 6 anos de idade, com pulmões normais, submetidos à cirurgia abdominal inferior. O TIE foi calibrado, realizada a indução anestésica, intubação e ventilação mecânica. Para reverter a atelectasia posterior, realizou-se uma manobra de recrutamento alveolar com o uso de pressão expiratória final positiva (PEEP) 20 cmH20 e pressão aérea do platô 40 cmH2O durante 120 segundos. A titulação PEEP foi então obtida com valores descendentes: 20, 18, 16, 14,12,10, 8, 6 e 4 cmH2O. Os dados de ultrassom e TIE foram coletados em cada nível PEEP e interpretados por dois observadores independentes. O número de linhas H foi contado usando um filtro especial. O teste de correlação de Spearman e a curva ROC foram utilizados para comparar os dados do LUS e TIE. Resultados: O número de linhas H aumentou linearmente com PEEP: de 3 em PEEP 4 cmH2O a 10 em PEEP 20 cmH2O. Cinco linhas H foram o limiar para a detecção de hiperdistensão pulmonar, definida como hiperdistensão na TIE >= 24,5%. A área sob a curva ROC foi 0,947 (IC 95% 0.901-0.976). Conclusão: O LUS intraoperatório detectou hiperdistensão pulmonar em valores descendentes de PEEP. A presença de cinco ou mais linhas H podem ser consideradas como indicando hiperdistensão pulmonar / Purpose: Intraoperative atelectasis occurs immediately after anaesthetic induction and can be detected by lung ultrasound (LUS). However, LUS is considered as unable to assess pulmonary hyperinflation. In this study, we propose a method to detect pulmonary hyperinflation using LUS. Electrical impedance tomography (EIT) was the reference method. Methods: We included 18 patients, 63 ± 6-year old, with normal lungs, undergoing lower abdominal surgery. The following protocol was used: EIT was calibrated, followed by anaesthetic induction, intubation and mechanical ventilation. To reverse posterior atelectasis, a recruitment maneuver - positive end-expiratory pressure (PEEP) 20 cmH20 and plateau airway pressure 40 cmH2O during 120 sec was performed. PEEP titration was then obtained during a descending trial: 20, 18, 16, 14,12,10, 8, 6 and 4 cmH2O. Ultrasound and EIT data were collected at each PEEP level and analyzed by two independent observers. The number of H lines was counted using a special filter. Spearman correlation test and ROC curve were used to compare LUS and EIT data. Results: The number of H lines increased linearly with PEEP: from 3 at PEEP 4 cmH2O to 10 at PEEP 20 cmH2O. Five H lines was the threshold for detecting pulmonary hyperinflation, defined as a mean decrease in maximum EIT compliance >= 24,5 %. The area under the ROC curve was 0.947 (CI 95% 0.901-0.976). Conclusion: Intraoperative transthoracic LUS can detect pulmonary hyperinflation during a PEEP descending trial. Five or more H lines can be considered as indicating pulmonary hyperinflation in normally aerated lung regions

As novel engineered nanoparticles such as single-walled carbon nanotubes (SWCNT) are extensively used in nanotechnology due to their superior properties, it becomes critical to fully understand their biodistribution and effect when accidently inhaled. There fore, development of animaging technique which allow longitudinal in vivo follow-up of SWCNT effect based on their intrinsic properties is highly desirable. Non invasive free-breathing hyperpolarized 3He lung MRI protocol was developed complementary to proton systemic MR protocol to allow monitoring SWCNT based on their intrinsic iron impurities after intrapulmonary exposition. Combined toproton lung MRI and ex vivo optical and electron microscopy at different time points, this protocol represents a powerful multimodality imaging techniques which allows a full characterization of the biodistribution and biological impacts of iron containing SWCNT. SWCNT was found to produce granulomatous and inflammatory reactions in a time and dose dependent manner with their bio persistenc eafter intrapulmonary exposition.From biological impact evaluations after intrapulmonary exposition towards biomedical applications, SWCNT hold promise for applications in nanomedicine field with their distinct architecture and their novel physicochemical properties. The biodistribution and pharmacological profile of various well-dispersed pristine and functionalized SWCNT were assessed in blood and target tissues after their intra venous administration by longitudinal in vivo susceptibility weighted MRI and their potential effect on liver metabolism by ex vivo HRMAS 1H NMR. No presence ofacute toxicological effect (variation in liver metabolism) was observed confirmed by the absence of clustering in NMR spectra using Principal Component Analysis (specific biomarkers of toxicity).

En raison de leurs propriétés uniques, des nanoparticules industriellement fabriquées comme les nanotubes de carbone (NTC) ont révolutionné le domaine de la nanotechnologie. Il apparait nécessaire de développer des techniques d’investigation in vivo basées sur les propriétés intrinsèques de ces particules et permettant un suivi longitudinal pour évaluer leur risque après inhalation accidentelle par voie respiratoire. Un protocole d’IRM pulmonaire non-invasive utilisant l’hélium-3 hyper polarisé sous respiration spontanée a été développé en complément d’un protocole d’IRM systémique proton pour permettre la détection des NTC grâce à l’effet de susceptibilité magnétique induit par les impuretés de fer, associées aux nanotubes après leur exposition intra pulmonaire. Combiné avec l’IRM pulmonaire proton et des analyses en microscopie optique et électronique à différents temps d’investigation, ce protocole d’imagerie multimodale permet d’évaluer la biodistribution et l’impact biologique des NTC bruts après exposition intra pulmonaire.Une accentuation des réactions inflammatoires (granulomes multifocaux, dépôt de fibres de collagène…) avec le temps et la dose administrée a été observée.De l’évaluation de l’impact biologique des NTC après une exposition intra pulmonaire vers leurs applications biomédicales, les nanotubes de carbone avec leurs propriétés physicochimiques fascinantes et leur forme spécifique laissent entrevoir des applications potentielles en nanomédecine. La bio distribution et le profil pharmacologique des différents types de NTC ont été évalués longitudinalement par IRM et dosage dans le sang et les organes cibles après une injection intraveineuse, et leur impact biologique sur le métabolisme du foie a été examiné ex vivo par RMN haute résolution à l’angle magique (HR-MAS). Aucun signe de toxicité aiguë (variation du métabolisme du foie) n’a été observé et les analyses statistiques conduits sur les spectres RMN (tests PCA) ne montrent aucune différence entre les échantillons analysés et donc l’absence de discrimination entre les différents groupes par rapport aux animaux contrôles. / As novel engineered nanoparticles such as single-walled carbon nanotubes (SWCNT) are extensively used in nanotechnology due to their superior properties, it becomes critical to fully understand their biodistribution and effect when accidently inhaled. There fore, development of animaging technique which allow longitudinal in vivo follow-up of SWCNT effect based on their intrinsic properties is highly desirable. Non invasive free-breathing hyperpolarized 3He lung MRI protocol was developed complementary to proton systemic MR protocol to allow monitoring SWCNT based on their intrinsic iron impurities after intrapulmonary exposition. Combined toproton lung MRI and ex vivo optical and electron microscopy at different time points, this protocol represents a powerful multimodality imaging techniques which allows a full characterization of the biodistribution and biological impacts of iron containing SWCNT. SWCNT was found to produce granulomatous and inflammatory reactions in a time and dose dependent manner with their bio persistenc eafter intrapulmonary exposition.From biological impact evaluations after intrapulmonary exposition towards biomedical applications, SWCNT hold promise for applications in nanomedicine field with their distinct architecture and their novel physicochemical properties. The biodistribution and pharmacological profile of various well-dispersed pristine and functionalized SWCNT were assessed in blood and target tissues after their intra venous administration by longitudinal in vivo susceptibility weighted MRI and their potential effect on liver metabolism by ex vivo HRMAS 1H NMR. No presence ofacute toxicological effect (variation in liver metabolism) was observed confirmed by the absence of clustering in NMR spectra using Principal Component Analysis (specific biomarkers of toxicity).