Abstract
Understanding vascular structures and dysfunction is a fundamental challenge. This task has been approached by using traditional methodologies such as microscopic computed tomography and magnetic resonance imaging. Both techniques are not only expensive but also time-consuming. Here, we present a new method for visualizing vascular structures in different organs in an efficient manner. A cationic near infrared (NIR) fluorescent dye was developed with attractive features to specifically stain blood vessels. Furthermore, we refined the process of organ staining and harvesting by retrograde perfusion and optimized the subsequent dehydration and clearing process by the use of an automatic tissue processor and a non-toxic substance, ethyl-cinnamate. Using this approach, the time interval between organ harvesting and microscopic analysis can be reduced from day(s) or weeks to 4 hours. Finally, we have demonstrated that the new NIR fluorescent agent in combination with confocal or light-sheet microscopy can be efficiently used for visualization of vascular structures, such as the blood vessels in different organs e.g. glomeruli in kidneys, with an extremely high resolution. Our approach facilitates the development of automatic image processing and the quantitative analysis to study vascular and kidney diseases.
Highlights
Changes in vascularization and vascular dysfunction have been associated with many diseases, including cardiovascular and kidney disease, making this an area of intense interest[1]
We have proven that a new cationic near infrared (NIR) fluorescent dye stains blood vessels
MHI148-PEI exhibits several attractive features: (i) the NIR wavelength provides a deep penetration depth and minimizes the interference of auto-fluorescence with biological tissues; (ii) the large molecular weight (50–100 kDa) prevents it from passing through the glomeruli into tubules; (iii) the positive charges derived from numerous cationic amino groups leads to its electrostatic attachment to GAGs and proteoglycans in vessels as well as the glomerular basement membrane
Summary
Changes in vascularization and vascular dysfunction have been associated with many diseases, including cardiovascular and kidney disease, making this an area of intense interest[1]. The majority of GAGs are found in the body as proteoglycan components and are abundant in vessels, basement membranes of glomeruli in kidneys, cartilage and bone[8] This MRI technique requires instrumentation (i.e. not available), expensive contrast agents, and suffers from relatively poor resolution that limits distinction between capillary tuft and Bowman’s capsule[9]. We accelerated the ethyl-cinnamate clearing process significantly This approach combined with confocal or light-sheet microscopy allows a rapid visualization of vascular structures in different organs e.g. in kidney or heart
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