Three-dimensional volume fluorescence-imaging of vascular plasticity in adipose tissues

Abstract

ObjectiveThe vascular system is central to sustaining tissue survival and homeostasis. Blood vessels are densely present in adipose tissues and exert essential roles in their metabolism. However, conventional immunohistochemistry methods have intrinsic limitations in examining the 3D vascular network in adipose tissues as well as other organs in general.MethodsWe established a 3D volume fluorescence-imaging technique to visualize the vasculatures in mouse adipose tissues by combining the optimized steps of whole-mount immunolabeling, tissue optical clearing, and lightsheet volume fluorescence-imaging. To demonstrate the strength of this novel imaging procedure, we comprehensively assessed the intra-adipose vasculatures under obese conditions or in response to a cold challenge.ResultsWe show the entirety of the vascular network in mouse adipose tissues on the whole-tissue level at a single-capillary resolution for the first time in the field. We accurately quantify the pathological changes of vasculatures in adipose tissues in wild-type or obese mice (ob/ob, db/db, or diet-induced obesity). In addition, we identify significant and reversible changes of the intra-adipose vasculatures in the mice subjected to cold challenge (i.e., 4°). Furthermore, we demonstrate that the cold-induced vascular plasticity depends on the sympathetic-derived catecholamine signal and is involved in the beiging process of white adipose tissues.ConclusionsWe report a 3D volume fluorescence-imaging procedure that is compatible with many areas of vascular research and is poised to serve the field in future investigations of the vascular system in adipose tissues or other research scenarios.

Morphology of the Vasculature and Blood Supply of the Brown Adipose Tissue Examined in an Animal Model by Micro‐CT

“…The advantages of micro-CT scanning with Aurovist™ are more precise and detailed tissue samplings which in turn could serve for further histological and immunohistochemical studies of I-BAT blood supply [24]. When enlarging the ROIs of the scans, specifically, I-BAT subregions with the most dense microcirculation (the most metabolically active part of the I-BAT) can be targeted which enables (i) local perfusion of the tissue, (ii) changes in its thermodynamics, and (iii) levels of various chemical substances in the intra-or extravascular space.…”

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures

“…We also tested another popular probe for endothelial cells, anti-CD31 antibody. This antibody is frequently used to carry out 3D imaging of blood vessels in various organs [31][32][33] , but the expression level of CD31 can vary among different endothelial populations 34 . Although the renal vasculature labelling patterns for anti-CD31 antibody and vessel painting were comparable, the signal intensity was much stronger for vessel painting.…”

Advances in Ex Situ Tissue Optical Clearing

“…It should be also noted that tissue treatment with methanol, a key Adipo‐Clear reagent, might impair the structure of particular epitopes, as it was already shown for CD31 and CD105, common vascular epitopes. This encouraged Cao et al . to search for alternatives and resulted in development of modified iDISCO technique for adipose tissue clearing, in which methanol‐based permeabilization is replaced by detergent‐based steps.…”

“…c) Isolectin staining is compatible with such clearing, thereby it serves as a potent alternative when, for example, vascular remodeling in adipose tissue is being studied; d) cold‐induced vascular plasticity. Reproduced with permission . Copyright 2018, Elsevier.…”