Angiofil®-mediated visualization of the vascular system by microcomputed tomography: a feasibility study

Visualization of the vascular systems of organs or of small animals is important for an assessment of basic physiological conditions, especially in studies that involve genetically manipulated mice. For a detailed morphological analysis of the vascular tree, it is necessary to demonstrate the system in its entirety. In this study, we present a new lipophilic contrast agent, Angiofil®, for performing postmortem microangiography by using microcomputed tomography. The new contrast agent was tested in 10 wild-type mice. Imaging of the vascular system revealed vessels down to the caliber of capillaries, and the digital three-dimensional data obtained from the scans allowed for virtual cutting, amplification, and scaling without destroying the sample. By use of computer software, parameters such as vessel length and caliber could be quantified and remapped by color coding onto the surface of the vascular system. The liquid Angiofil® is easy to handle and highly radio-opaque. Because of its lipophilic abilities, it is retained intravascularly, hence it facilitates virtual vessel segmentation, and yields an enduring signal which is advantageous during repetitive investigations, or if samples need to be transported from the site of preparation to the place of actual analysis, respectively. These characteristics make Angiofil® a promising novel contrast agent; when combined with microcomputed tomography, it has the potential to turn into a powerful method for rapid vascular phenotyping

Intravital Multiphoton Microscopy Analysis of Spatial Relationships in Murine Skull Bone Marrow

The BM is a key organ of hematopoiesis and also has an important role in the immune system. The BM microenvironment is a complex, highly vascularized 3D structure composed of different cell types and extracellular matrix. Intense cellular traffic takes place from the peripheral blood to the BM and vice versa. However, the precise arrangement and microscopic dimensions of this environment have only been inferred so far from static imaging of sectioned tissue. We developed a new model to characterize and analyze the 3D microanatomy of murine skull BM in its physiological state using intravital MPM. This technology offers deep tissue penetration, low phototoxicity, superior image contrast and 3D resolution compared to other microscopy techniques. This makes MPM a powerful tool to investigate the BM, overcoming its anatomic inaccessibility. To quantify the dimensions of the BM compartment, we used high molecular weight FITC-dextran and Rhodamine 6G, which delineated the intra- and extravascular space, respectively. Measurements were generated using the 3D visualization and measurement software VoxBlast 3.1 after using a thresholding technique carried out by Adobe Photoshop 6.0. Results were expressed as the ratio of intravascular to extravascular space for different microvascular segments. Moreover, we performed adoptive transfer experiments with isolated naïve B-cells and TCM and studied their location within the BM compartment. The new approach presented here will be a useful tool for further in vivo investigations of cell behavior, trafficking and interactions in the BM

Diagnosis, Rupture Risk Evaluation and Therapeutic Intervention of Abdominal Aortic Aneurysms Using Targeted Nanoparticles

Abdominal aortic aneurysm (AAA) disease causes dilation of the aorta that can lead to aortic rupture and death if not treated early. It is the 14th leading cause of death in the U.S. and is cited as the 10th leading cause of death in men over age 55, affecting thousands of patients and their families. To date, AAA patients have minimal access to safe and efficient imaging modalities for diagnosis as well as pharmacotherapies. AAA is usually detected and monitored with ultrasonography or contrast-enhanced computed tomography (C.T.), which doesn’t provide biomechanical information of the AAAs that are essential for predicting rupture risks. Furthermore, unfortunately, there is no currently known pharmaceutical treatment to cure the AAAs. Key pathological processes occurring within AAAs include inflammation, vascular smooth muscle cell apoptosis, and extracellular matrix (ECM) degradation. The deterioration of the elastic lamina in the aneurysmal wall is a consistent feature of AAAs and the fact that the adult elastic lamina does not remodel in aneurysm progression, making it an ideal target for delivering contrast agents and treatments. In this research, we have delivered gold nanoparticles (AuNPs), a commonly used C.T. contrast agent, and pentagalloyl glucose (PGG) loaded nanoparticles to the AAAs in an angiotensin II (AngII) infusion induced mouse model by conjugating the nanoparticles with antibodies that target degraded elastin. Here, owing to their degraded elastin targeting ability, we have observed a positive correlation between the quantities of the locally accumulated AuNPs in the aneurysmal tissue in C.T. scans and the elastin damage levels of the AAAs. Furthermore, the AuNPs accumulations were found negatively correlated to the mechanical properties of the AAAs, which makes AuNPs a potential non-invasive surrogate marker of AAA rupture risk. Moreover, we have shown that targeted delivery of PGG could reverse the aortic dilation, ameliorate the inflammation, restore the elastin as well as the AAA mechanical properties of the aneurysmal tissue. Therefore, PGG loaded nanoparticles can be an effective treatment option for early to middle stage aneurysms to prevent disease progression

Investigation of factors controlling cutaneous circulation in flaps

The aim of this research was to investigate the blood supply to the lower abdomen. This is a commonly used donor site for autologous reconstruction following breast cancer and the flap of tissue used is based on the deep inferior epigastric circulation (DIEP flap) or the superficial inferior epigastric circulation (SIEA flap). A pilot study investigated the feasibility of assessing the vascular territory of multiple blood vessels in the lower abdomen, and also observed the timing of changes in skin blood supply after free flap transfer. Further studies included sampling using microdialysis catheters, from different areas of the flap, around the theoretical time of opening of choke vessels between angiosomes. Manipulation of skin blood flow was initially investigated using capillary malformations as a model, observing current clinical use of EMLA and AMETOP topical anaesthetic pre-laser treatment

Investigation of high-frequency ultrasound biomicroscopy for examining arteriopathy in hand transplant patients.

The fourth hand transplant recipient in Louisville lost his graft at nine months post transplant from ischemia due to severe graft arteriopathy. Conventional imaging techniques and clinical measurements, including CT angiography and measurements of brachial indices via ultrasound, did not indicate that the patient was in such a severe condition in the days prior to amputation. Histological analysis of tissue from the amputated graft revealed massive intimal hyperplasia, a hallmark of chronic rejection. The purpose of this study was to describe a method to allow non-invasive monitoring of wall thicknesses in the radial, ulnar, palmar arch, and digital arteries of the transplanted hand. A novel ultrasound biomicroscopy unit (Vevo 2100) was used to image the blood vessels of the transplanted and native hands in four hand transplant recipients, ranging from 16 months to 11 years post transplant. The unit has a potential resolution of 30 µm, which allowed measurement of the intima and media layers of the arteries. The intima, media, and lumen measurements were compared in the native and transplanted hand in all four patients. Significant increases in intima thickness were found in the transplanted hands. In addition, the transplanted hand had significantly higher intima to lumen diameter ratios. From Doppler waveforms, it was found that that peak velocities were lower in the transplanted hand. Finally, hoop stress calculations revealed that the hoop stress seen in the arterial walls was lower in the transplanted hand. The ultrasound biomicroscopy unit provided an accurate, repeatable, and real-time method to investigate the arteriopathy of hand transplant patients. In the future, it will provide a way for clinicians to monitor the progression of arteriopathy in the hand, including intimal hyperplasia

The role of tumour vasculature in fluid flow and drug transport in solid tumours

The aberrance of the vasculature in tumours has been linked to increased aggressiveness and poor drug delivery in tumours. Complexities in the microarchitecture of tumour vasculature occurring on microscopic scales can affect fluid flow and drug transport making it difficult to predict tumour response to treatment. Given this, mathematical models can play an important role in understanding the various aspects of the tumour vasculature that can promote invasiveness and limit drug delivery. In this work, computational models are developed to investigate the effect of tumour vasculature on fluid flow and drug distribution and novel imaging methods are assessed for their ability to characterise the tumour vasculature in whole human tumours. A mathematical angiogenesis model is used to generate microscopic details including individual vessel properties on a whole vascular network scale which are coupled with a fluid flow and drug transport model. The interstitial fluid pressure (IFP) in the tumour model was found to be elevated with increased heterogeneity caused by the presence of a necrotic core and heterogenous vessel permeability. Subtle changes to the network on a microscopic scale significantly influenced fluid flow in the tumour vessels and tissue. Delivery of doxorubicin to tumours was found to be highly dependent on the properties of tumour vasculature and blood flow, where regions with excessive branching and vessel tortuosity had reduced drug concentrations due to poor blood flow. Hence, the vascular density was not found to be the main factor in the accumulation of the drug within the tissue space and it’s uptake by cancer cells. An interplay between treatment strategy including dose and administration mode and properties of the vasculature was found by evaluating the spatial intracellular concentration. The fluid flow and drug transport models showed the significant effect of incorporating the microscopic properties of the tumour vasculature which can influence fluid flow and drug distribution on a macroscopic scale. The imaging methods assessed in this work shows that Optical projection tomography combined with fluorescent Immunohistochemistry labelling methods can be used to extract angiogenesis related parameters in whole human tumours. Additionally, the method was able to extract clean network topologies that show promise in application to understanding fluid flow and drug transport in real tumours.Open Acces