In vivo lung micro-computed tomography (micro-CT) is being increasingly embraced in pulmonary research because it provides longitudinal information on dynamic disease processes in a field in which ex vivo assessment of experimental disease models is still the gold standard. To optimize the quantitative monitoring of progression and therapy of lung diseases, we evaluated longitudinal changes in four different micro-CT-derived biomarkers [aerated lung volume, lung tissue (including lesions) volume, total lung volume and mean lung density], describing normal development, lung infections, inflammation, fibrosis and therapy. Free-breathing mice underwent micro-CT before and repeatedly after induction of lung disease (bleomycin-induced fibrosis, invasive pulmonary aspergillosis, pulmonary cryptococcosis) and therapy (imatinib). The four lung biomarkers were quantified. After the last time point, we performed pulmonary function tests and isolated the lungs for histology. None of the biomarkers remained stable during longitudinal follow-up of adult healthy mouse lungs, implying that biomarkers should be compared with age-matched controls upon intervention. Early inflammation and progressive fibrosis led to a substantial increase in total lung volume, which affects the interpretation of aerated lung volume, tissue volume and mean lung density measures. Upon treatment of fibrotic lung disease, the improvement in aerated lung volume and function was not accompanied by a normalization of the increased total lung volume. Significantly enlarged lungs were also present in models of rapidly and slowly progressing lung infections. The data suggest that total lung volume changes could partly reflect a compensatory mechanism that occurs during disease progression in mice. Our findings underscore the importance of quantifying total lung volume in addition to aerated lung or lesion volumes to accurately document growth and potential compensatory mechanisms in mouse models of lung disease, in order to fully describe and understand dynamic processes during lung disease onset, progression and therapy. This is highly relevant for the translation of therapy evaluation results from preclinical studies to human patients.

Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard.

Cryptococcus neoformans is a leading cause of fungal brain infection, but the mechanism of dissemination and dynamics of cerebral infection following pulmonary disease are poorly understood. To address these questions, non-invasive techniques that can study the dynamic processes of disease development and progression in living animal models or patients are required. As such, bioluminescence imaging (BLI) has emerged as a powerful tool to evaluate the spatial and temporal distribution of infection in living animals. We aimed to study the time profile of the dissemination of cryptococcosis from the lung to the brain in murine models by engineering the first bioluminescent C. neoformans KN99α strain, expressing a sequence-optimized red-shifted luciferase. The high pathogen specificity and sensitivity of BLI was complemented by the three-dimensional anatomical information from micro-computed tomography (μCT) of the lung and magnetic resonance imaging (MRI) of the brain. These non-invasive imaging techniques provided longitudinal readouts on the spatial and temporal distribution of infection following intravenous, intranasal or endotracheal routes of inoculation. Furthermore, the imaging results correlated strongly with the fungal load in the respective organs. By obtaining dynamic and quantitative information about the extent and timing of brain infections for individual animals, we found that dissemination to the brain after primary infection of the lung is likely a late-stage event with a timeframe that is variable between animals. This novel tool in Cryptococcus research can aid the identification of host and pathogen factors involved in this process, and supports development of novel preventive or therapeutic approaches.

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these noninvasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.

Magnetic resonance imaging sensitively visualizes and quantifies lung fibrosis in vivo, which makes it a noninvasive, translatable, safe, and potentially more versatile alternative to invasive methods or μCT, thereby stimulating pathogenesis and preclinical research.

causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the efficacies of antifungals. In recent years, there has been an increasing interest in the use of noninvasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leukopenic mouse model of invasive pulmonary aspergillosis (IPA). This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. The results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that a reduction in fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows an investigation of different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner.

Respiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.

Many different types of nanoparticles have been shown to possess immunomodulatory properties, depending on their physicochemical parameters. This can potentially be harnessed as a possible antitumor therapy. However, in the current work we show that inflammation elicited by nanomaterials can have grave effects in pathophysiological conditions, where non-specific inflammation was found to increase cancer cell mobility and tumor malignancy. These data show that immunomodulatory properties of nanomaterials must be carefully controlled to avoid any undesired side-effects.