A cationic near infrared fluorescent agent and ethyl-cinnamate tissue clearing protocol for vascular staining and imaging

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.

Mapping vascular and glomerular pathology in a rabbit model of neonatal acute kidney injury using MRI

“…CF-enhanced MRI (CFE-MRI) has been used to map glomerular microstructure in various animal models of kidney disease, both ex vivo and in vivo, providing a unique view of renal microstructure and its relationship to the onset and development of tissue pathology. The development of CFE-MRI is preceded by decades of stereological work and coincides with recent investigations of nephron number in human kidneys (Bertram, 1995;Bertram et al, 2011Bertram et al, , 2014Kanzaki et al, 2017), but with the potential for in vivo preclinical and clinical translation. In addition to its potential for in vivo and clinical use, CFE-MRI provides a wide range of soft tissue contrast that could enable a fully integrated view of the kidney during disease progression, reporting not only nephron loss, but also how glomerular loss is related to other vascular and tubular structural changes.…”

“…Light sheet microscopy performed in optically cleared organs is beginning to J.R. Charlton and N. Parvin contributed equally to this work. provide three dimensional (3D) maps of molecular signaling in targeted cells in conjunction with the surrounding tissue morphology in small samples (Chung et al, 2013;Huang et al, 2019). Several new radiological imaging techniques are being developed, including x-ray computed tomography (CT) (Xie et al, 2019) and magnetic resonance imaging (MRI) (Xie et al, 2016), to connect the molecular and cellular levels in the whole, intact kidney.…”

Advances in Ex Situ Tissue Optical Clearing

“…Application of ECi has already allowed the researchers to visualize morphological and molecular remodeling of distal convoluted tubules upon low‐potassium diet and to reveal new 3D quantitative endpoints in the model of crescentic nephritis . ECi‐based clearing was also applied to present utility of new cationic near infrared fluorescent agent, MHI148‐PEI, for specific staining of vascular structures in variety of organs, for example, skin, liver and lung . Altogether, ECi methods are rapid, inexpensive, not toxic and not hazardous for imaging equipment, it yields full transparency of many organs, preserves XFP signal for reasonable time and as such should be perceived as a very promising method to be applied in further studies.…”

New technical approaches for 3D morphological imaging and quantification of measurements

“…It is an FDA approved food flavor and additive for cosmetic products (Wang, Zhang, Zhang, Chen, & Zhi, 2016). We described a new ECi-based OTC (ECi-OTC) protocol by using an automatic tissue processor and performing the whole sample processing at room temperature which optimizes the diffusion coefficient (Huang et al, 2019). Thereby, the time interval between organ harvesting and microscopic analysis (confocal or lightsheet microscopy) was reduced from 18 to 5 hr.…”

“…Thereby, the time interval between organ harvesting and microscopic analysis (confocal or lightsheet microscopy) was reduced from 18 to 5 hr. Moreover, a cationic near-infrared fluorescent dye was developed for the staining of blood vessels, for example, in kidney, heart, liver, and lung (Huang et al, 2019).…”