Tumour irradiation combined with vascular-targeted photodynamic therapy enhances antitumour effects in pre-clinical prostate cancer.

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data.

Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox - 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.

Whole brain vascular imaging in a mouse model of Alzheimer's disease with two-photon microscopy.

Given known correlations between vascular health and cognitive impairment, the development of tools to image microvasculature in the whole brain could help investigate these correlations. We explore the feasibility of using an automated serial two-photon microscope to image fluorescent gelatin-filled whole rodent brains in three-dimensions (3-D) with the goal of carrying group studies. Vascular density (VD) was computed using automatic segmentation combined with coregistration techniques to build a group-level vascular metric in the whole brain. Focusing on the medial prefrontal cortex, cerebral cortex, the olfactory bulb, and the hippocampal formation, we compared the VD of three age groups (2-, 4.5-, and 8-months-old), for both wild type mice and a transgenic model (APP/PS1) with pathology resembling Alzheimer's disease (AD). We report a general loss of VD caused by the aging process with a small VD increase in the diseased animals in the somatomotor and somatosensory cortical regions and the olfactory bulb, partly supported by MRI perfusion data. This study supports previous observations that AD transgenic mice show a higher VD in specific regions compared with WT mice during the early and late stages of the disease (4.5 to 8 months), extending results to whole brain mapping.

Comparing three-dimensional serial optical coherence tomography histology to MRI imaging in the entire mouse brain.

An automated serial histology setup combining optical coherence tomography (OCT) imaging with vibratome sectioning was used to image eight wild type mouse brains. The datasets resulted in thousands of volumetric tiles resolved at a voxel size of (4.9×4.9×6.5) µm3 stitched back together to give a three-dimensional map of the brain from which a template OCT brain was obtained. To assess deformation caused by tissue sectioning, reconstruction algorithms, and fixation, OCT datasets were compared to both in vivo and ex vivo magnetic resonance imaging (MRI) imaging. The OCT brain template yielded a highly detailed map of the brain structure, with a high contrast in white matter fiber bundles and was highly resemblant to the in vivo MRI template. Brain labeling using the Allen brain framework showed little variation in regional brain volume among imaging modalities with no statistical differences. The high correspondence between the OCT template brain and its in vivo counterpart demonstrates the potential of whole brain histology to validate in vivo imaging.

Fully automated dual-resolution serial optical coherence tomography aimed at diffusion MRI validation in whole mouse brains.

An automated dual-resolution serial optical coherence tomography (2R-SOCT) scanner is developed. The serial histology system combines a low-resolution ( 25 µ m / voxel ) 3 × OCT with a high-resolution ( 1.5 µ m / voxel ) 40 × OCT to acquire whole mouse brains at low resolution and to target specific regions of interest (ROIs) at high resolution. The 40 × ROIs positions are selected either manually by the microscope operator or using an automated ROI positioning selection algorithm. Additionally, a multimodal and multiresolution registration pipeline is developed in order to align the 2R-SOCT data onto diffusion MRI (dMRI) data acquired in the same ex vivo mouse brains prior to automated histology. Using this imaging system, 3 whole mouse brains are imaged, and 250 high-resolution 40 × three-dimensional ROIs are acquired. The capability of this system to perform multimodal imaging studies is demonstrated by labeling the ROIs using a mouse brain atlas and by categorizing the ROIs based on their associated dMRI measures. This reveals a good correspondence of the tissue microstructure imaged by the high-resolution OCT with various dMRI measures such as fractional anisotropy, number of fiber orientations, apparent fiber density, orientation dispersion, and intracellular volume fraction.

Serial optical coherence scanning reveals an association between cardiac function and the heart architecture in the aging rodent heart.

Normal aging is accompanied by structural changes in the heart architecture. To explore this remodeling, we used a serial optical coherence tomography scanner to image entire mouse hearts at micron scale resolution. Ex vivo hearts of 7 young (4 months) and 5 old (24 months) C57BL/6 mice were acquired with the imaging platform. OCT of the myocardium revealed myofiber orientation changing linearly from the endocardium to the epicardium. In old mice, this rate of change was lower when compared to young mice while the average volume of old mice hearts was significantly larger (p<0.05). Myocardial wall thickening was also accompanied by extracellular spacing in the endocardium, resulting in a lower OCT attenuation coefficient in old mice endocardium (p<0.05). Prior to serial sectioning, cardiac function of the same hearts was imaged in vivo using MRI and revealed a reduced ejection fraction with aging. The use of a serial optical coherence tomography scanner allows new insight into fine age-related changes of the heart associated with changes in heart function.

Copper-Alkyne Complexation Responsible for the Nucleolar Localization of Quadruplex Nucleic Acid Drugs Labeled by Click Reactions.

G-Quadruplex(es) (G4) are noncanonical nucleic-acid structures found in guanine-rich sequences. They can be targeted with small molecules (G4 ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localization of PhenDC3 , one of the most powerful G4 ligands, by synthesizing two clickable azide and alkyne derivatives (PhenDC3 -alk, PhenDC3 -az) and labeling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper-based CuAAC and copper-free SPAAC methodologies in fixed cells implicated CuI /alkyne intermediates in the nonspecific localization of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labeling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localize drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.

Whole mouse brain imaging using optical coherence tomography: reconstruction, normalization, segmentation, and comparison with diffusion MRI.

An automated massive histology setup combined with an optical coherence tomography (OCT) microscope was used to image a total of [Formula: see text] whole mouse brains. Each acquisition generated a dataset of thousands of OCT volumetric tiles at a sampling resolution of [Formula: see text]. This paper describes techniques for reconstruction and segmentation of the sliced brains. In addition to the measured OCT optical reflectivity, a single scattering photon model was used to compute the attenuation coefficients within each tissue slice. Average mouse brain templates were generated for both the OCT reflectivity and attenuation contrasts and were used with an [Formula: see text]-tissue segmentation algorithm. To better understand the brain tissue OCT contrast origin, one of the mouse brains was acquired using dMRI and coregistered to its corresponding assembled brain. Our results indicate that the optical reflectivity in a fiber bundle varies with its orientation, its fiber density, and the number of fiber orientations it contains. The OCT mouse brain template generation and coregistration to dMRI data demonstrate the potential of this massive histology technique to pursue cross-sectional, multimodal, and multisubject investigations of small animal brains.

Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

Quantifying the microvascular origin of BOLD-fMRI from first principles with two-photon microscopy and an oxygen-sensitive nanoprobe.

The blood oxygenation level-dependent (BOLD) contrast is widely used in functional magnetic resonance imaging (fMRI) studies aimed at investigating neuronal activity. However, the BOLD signal reflects changes in blood volume and oxygenation rather than neuronal activity per se. Therefore, understanding the transformation of microscopic vascular behavior into macroscopic BOLD signals is at the foundation of physiologically informed noninvasive neuroimaging. Here, we use oxygen-sensitive two-photon microscopy to measure the BOLD-relevant microvascular physiology occurring within a typical rodent fMRI voxel and predict the BOLD signal from first principles using those measurements. The predictive power of the approach is illustrated by quantifying variations in the BOLD signal induced by the morphological folding of the human cortex. This framework is then used to quantify the contribution of individual vascular compartments and other factors to the BOLD signal for different magnet strengths and pulse sequences.

Large arteriolar component of oxygen delivery implies a safe margin of oxygen supply to cerebral tissue.

What is the organization of cerebral microvascular oxygenation and morphology that allows adequate tissue oxygenation at different activity levels? We address this question in the mouse cerebral cortex using microscopic imaging of intravascular O2 partial pressure and blood flow combined with numerical modelling. Here we show that parenchymal arterioles are responsible for 50% of the extracted O2 at baseline activity, and the majority of the remaining O2 exchange takes place within the first few capillary branches. Most capillaries release little O2 at baseline acting as an O2 reserve that is recruited during increased neuronal activity or decreased blood flow. Our results challenge the common perception that capillaries are the major site of O2 delivery to cerebral tissue. The understanding of oxygenation distribution along arterio-capillary paths may have profound implications for the interpretation of blood-oxygen-level dependent (BOLD) contrast in functional magnetic resonance imaging and for evaluating microvascular O2 delivery capacity to support cerebral tissue in disease.

Aging-related differences in cerebral capillary blood flow in anesthetized rats.

Age-related decreases in baseline cerebral blood flow have been measured with various imaging modalities, however, the contribution of capillary flow to this phenomenon remain to elucidate. This study used 2-photon laser scanning fluorescence microscopy to measure capillary diameter, red blood cell speed, and flux in individual capillaries in the sensory-motor cortex of 12 adult (3-month-old) and 12 old (24-month-old) male Long-Evans rats under isoflurane anesthesia. The average (± standard deviation) diameter and speed over 921 capillaries were 6.4 ± 1.4 µm and 1.3 ± 1.1 mm/s, respectively. Red blood cell speed and flux were significantly higher, by 48% and 15%, respectively, in old compared with young animals (p < 5%). The diameter also showed a similar tendency (7% higher, p = 5.7%). Furthermore, capillary hematocrit and density were significantly lower in the older group (p < 5%), by 32% and 20%, respectively.