Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome

Due to spectral overlap, the number of fluorescent labels for imaging cryomicrotome detection was limited to 4. The aim of this study was to increase the separation of fluorescent labels. In the new imaging cryomicrotome, the sample is cut in slices of 40 μm. Six images are taken for each cutting plane. Correction for spectral overlap is based on linear combinations of fluorescent images. Locations of microspheres are determined by using the system point spread function. Five differently colored microspheres were injected in vivo distributed over two major coronaries, the left anterior descending and left circumflex artery. Under absence of collateral flow, microspheres outside of target perfusion territories were not found and the procedure did not generate false positive detection when spectral overlap was relevant. In silico-generated microspheres were used to test the effect of background image, transparency correction, and color separation. The percentage of microspheres undetected was 2.3 ± 0.8% in the presence and 1.5 ± 0.4% in the absence of background structures with a density of 900 microspheres per color per cm3. The image analysis method presented here, allows for an increased number of experimental conditions that can be investigated in studies of regional myocardial perfusion

Bronchial Artery Angiogenesis Drives Lung Tumor Growth

Lung cancer is the leading cause of cancer related deaths and is responsible for over one million deaths worldwide each year. While it is widely acknowledged that angiogenesis plays an integral role in tumor growth, and therapeutic approaches have been taken to inhibit angiogenesis, clinical results have been unexceptional at best. Current research models discount the dual lung circulations that create a unique growth environment for tumors, by utilizing subcutaneous xenograft models that have little relevance to the lung, or orthotopic models in mice, which lack a bronchial circulation. In an effort to bridge the gap between animal models of questionable relevance, and clinical trials, we developed an orthotopic model of lung cancer in nude rats to examine the role of the bronchial artery in tumor growth. Using two methods of quantifying tumor perfusion in vivo we measured an increase in bronchial artery perfusion quantified by fluorescent microsphere injection (206%) and HRCT scan (276%), that paralleled the growth in tumor volume, while pulmonary perfusion remained unchanged. When ablating the bronchial artery after the initiation of tumor growth, we observed a 76% decrease in final tumor volumes at 4 weeks post ablation. In an effort to examine the innate differences in the pulmonary and bronchial circulations’ response to tumor growth, primary endothelial cell lines were isolated from the bronchial artery, pulmonary artery, and pulmonary microvasculature of nude rats for the determination of their angiogenic potential. Bronchial artery endothelial cells uniquely showed increased proliferation, tube formation, and chemotaxis when exposed to angiogenic stimuli (VEGF, CINC-3, Adenocarcinoma Supernatant). We conclude that the pulmonary circulation initially sustains lung tumor establishment. As a tumor increases in size it is the bronchial circulation that proliferates to sustain tumor growth beyond the point at which a tumor can be supported by the pulmonary circulation alone. The increased angiogenic potential of bronchial artery endothelial cells, suggests innate differences between lung circulations is due to its unique vascular niche

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

INTRODUCTION: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. METHODS: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. RESULTS: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells. CONCLUSIONS: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration