Pre-Clinical Assessment of Cardiac and Lung Fibrosis Using Next Generation Imaging Modalities
by Amara Khan
Date of Examination:2021-09-01
Date of issue:2022-03-08
Advisor:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Elisabeth Zeisberg
Referee:Prof. Dr. Ralf Dressel
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EnglishFibrosis, defined as an excessive deposition of connective tissue components causing disruption of the physiological architecture and organ malfunction, is commonly associated with high morbidity and mortality. The global incidence of fibrosis is increasing rapidly making fibrosis one of today’s major health-care challenges. Despite this recognition, fibrotic diseases are underdiagnosed in clinical practice mainly due to a lack of diagnostic tools for organ-specific detailed characterization. To establish innovative and unique imaging techniques, I chose two organ systems, i.e. heart and lung for structural and functional assessment of fibrosis in pre-clinical animal models. By performing high-resolution and label-free imaging of cardiac tissue of mouse and marmosets, we aimed to identify sub-cellular fibrosis induced structural remodeling which is not feasible by currently used histology and non-invasive methods. To this end, we implemented comparative second harmonic generation (SHG) and x-ray diffraction imaging to analyse myocardial tissue in a mouse model of cardiac pressure overload. SHG emission from myosin and collagen was differentiated in tissue sections based on their distinct morphology. Heterogeneously distributed fibrotic lesions and micro-level distortion of myofibrils were detected and quantified by SHG imaging. These findings were corroborated with x-ray diffraction data which presented spatial maps of cardiac tissue and revealed increased lattice spacing, low anisotropy and peak intensity at the site of tissue remodeling. Regions that lacked both SHG and x-ray diffraction signals were identified as sites of active fibrosis due to high-immune cell infiltration. These findings show that the combined use of these imaging modalities allows detecting the different stages during cardiac fibrosis progression. Our label-free imaging approach was further augmented by acquiring SHG signals in combination with two-photon-excitation fluorescence (TPEF) in three-dimensions (3D) to investigate age-related structural modulation in non-human primate model which reflects a comparable life span as humans. The hearts were obtained from common marmoset monkeys (Callithrix jacchus) of three different age groups including neonatal, young adult and old/geriatric. By devising a unique strategy for segregating collagen and myosin emitted SHG signals, I performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodeling indicating the presence of age-related cardiac fibrosis. Our finding of marked low tissue volume in neonatal marmoset hearts was attributed to a lack of banding between the myofibrils in maturing cardiac tissue. By applying semi-automated analysis, significant differences were revealed in parameters such as total tissue volume, myofibril length, alignment, curvature and angulation between all three age groups. Overall, our imaging approach highlights the unrivalled potential of TPM for detailed evaluation and characterization of age-related cardiac structural remodeling in the marmoset heart that may provide insight into pathological processes. Pre-clinical monitoring of the degree of lung fibrosis will mostly profit from a reliable and standardized method for functional imaging. Therefore, I improved the x-ray based lung function (XLF) which uses dramatically lower x-ray doses and acquisition times in comparison to micro-CT and has been previously reported to be more sensitive than whole-body plethysmography (WBP) in allergic airway inflammation mouse models. Because XLF so far was unable to relate its parameters to pulmonary air volume, we designed an experimental set-up to perform correlative lung function measurements using either XLF or micro-CT with WBP on healthy mice. Using micro-CT as a gold-standard, our results reveal a strong correlation of lung volumes obtained from radiographic XLF and micro-CT and demonstrate that XLF is superior to WBP in precision and sensitivity to assess lung volumes. We thus present XLF as a promising tool for future pre-clinical studies on longitudinal assessment of lung fibrosis during the course of the disease and in response to therapy. Furthermore, XLF as a biomedically relevant non-invasive technique has a high potential for clinical translation.
Keywords: Cardiac fibrosis, Lung fibrosis, Diagnostics, High-resolution imaging, Non-linear Optical Microscopy, X-ray diffraction imaging, X-ray based lung function