Semi-automated protocol to quantify and characterize fluorescent three-dimensional vascular images

Abstract

The microvasculature facilitates gas exchange, provides nutrients to cells, and regulates blood flow in response to stimuli. Vascular abnormalities are an indicator of pathology for various conditions, such as compromised vessel integrity in small vessel disease and angiogenesis in tumors. Traditional immunohistochemistry enables visualization of tissue cross-sections containing exogenously labeled vasculature. Although this approach can be utilized to quantify vascular changes within small fields-of-view, it is not a practical way to study the vasculature on the scale of whole organs. Three-dimensional (3D) imaging presents a more appropriate method to visualize the vascular architecture in tissue. Here we describe the complete protocol that we use to characterize the vasculature of different organs in mice encompassing the methods to fluorescently label vessels, optically clear tissue, collect 3D vascular images, and quantify these vascular images with a semi-automated approach. To validate the automated segmentation of vascular images, one user manually segmented fifty random regions of interest across different vascular images. The automated segmentation results had an average sensitivity of 80 +/-8% and an average specificity of 90 +/-5% when compared to manual segmentation. Applying this procedure of image analysis presents a method to reliably quantify and characterize vascular networks in a timely fashion. This procedure is also applicable to other methods of tissue clearing and vascular labels that generate 3D images of microvasculature.

Chronic kidney disease promotes cerebral microhemorrhage formation

“…However, these findings should be interpreted with caution, because CMH may develop via a mechanism independent of capillary injury [ 73 – 75 ]. To expand our knowledge on the vascular source of CMH, we have developed a semi-automated approach to characterize microvascular network in three-dimensional (3D) imaging of mouse brains [ 76 ]; this will enable us to visualize the co-localization of fluorescently labeled microvascular network and Prussian blue-positive CMH and therefore, identify the vascular origin of CMH. In addition, the issue of cognitive decline with CKD is important, as we have emphasized [ 77 ].…”