Visualization and Analysis of Whole Depot Adipose Tissue Neural Innervation

Abstract

53Adipose tissue requires neural innervation in order to regulate important metabolic functions. 54Though seminal work on adipose denervation has underscored the importance of adipose-nerve 55 interactions in both white (energy storing) and brown (energy expending) adipose tissues, much 56 remains a mystery. This is due, in part, to the inability to effectively visualize the various nerve 57 subtypes residing within these tissues and to gain a comprehensive quantitation of neurite 58 density in an entire depot. With the recent surge of advanced imaging techniques such as light 59 sheet microscopy and optical clearing procedures, adipose tissue imaging has been 60 reinvigorated with a focus on three-dimensional analysis of tissue innervation. However, 61 clearing techniques are time consuming, often require solvents caustic to objective lenses, alter 62 tissue morphology, and greatly reduce fluorophore lifespan. Not only are current methods of 63 imaging wholemount adipose tissues inconvenient, but often attempts to quantify neurite density 64 across physiological or pathophysiological conditions have been limited to representative 65 section sampling. We have developed a new method of adipose tissue neurite imaging and 66 quantitation that is faster than current clearing-based methods, does not require caustic 67 chemicals, and leaves the tissue fully intact. Maintenance of a fully intact depot allowed for tiling 68 z-stacks and producing maximum intensity projections of the entire adipose depot, which were 69 then used to quantify neurite density across the tissue. With this processing method we were 70 able to characterize the nerves, nerve-subtypes, and neurovascular interactions within the 71 inguinal subcutaneous white adipose tissue in mice using up to five fluorescent channels at high 72 resolution. We also utilized second harmonic generation, which provides label-free imaging, to 73 investigate collagen fiber abundance in adipose of obese mice. 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Introduction: 88Historically overlooked as a location of diverse peripheral innervation [1], the adipose organ 89 was most prominently inspected for innervation in the mid 1960's when sympathetic nerve fibers 90 were visualized within brown adipose tissue (BAT) [2]. BAT nerves were later comprehensively 91 investigated by T.J. Bartness [3]. Energy expending BAT was the first adipose tissue to be 92 identified as being highly innervated due to its important role in thermogenesis [4], which 93 requires significant sympathetic input [2, 5, 6]. Sensory innervation has also been documented 94 in BAT, particularly around vasculature, and has been proposed to play a role in lipolysis [7]. 95More recently white adipose tissue (WAT), associated more with energy storage, was 96 demonstrated to be highly innervated by sympathetic [8, 9] and sensory nerves [5, 10], but not 97 parasympathetic [11] nerves. 98In order to visualize adipose innervation, it had been common practice to slice adipose 99 tissue into 7um-10um thick sections and...

A single-cell atlas of human and mouse white adipose tissue

“…Nonetheless, we estimated the degree of innervation using the presence of neuron-specific gene expression in the ambient RNA of our visceral sNuc-seq samples. Indeed, the amount of pan-neuronal markers like TUBB3 and UCHL1 32 strongly correlate with hAd6 proportion (Extended Data Figure 11h), further supporting a role for hAd6 as a novel visceral adipocyte subtype with thermogenic potential.…”

The involvement of neuroimmune cells in adipose innervation

“…# 5013) and 1 1/2 coverslips, and imaged on a Nikon Eclipse E400 epifluorescent microscope equipped with Nikon DS-fi2 camera. Wholemount adipose staining was performed as previously described (Blaszkiewicz et al 2019;Willows et al 2019) and imaged by widefield and confocal microscopy. Widefield imaging was performed on a Nikon E400 epifluorescence microscope with Hamamatsu ORCA-Flash4.0 V2 Digital CMOS monochrome camera (Hamamatsu Photonics K.K., C11440-22CU).…”

The involvement of neuroimmune cells in adipose innervation 

“…# 5013) and 1 1/2 coverslips, and imaged on a Nikon Eclipse E400 epi uorescent microscope equipped with Nikon DS-2 camera. Wholemount adipose staining was performed as previously described (8,48) and imaged by wide eld and confocal microscopy. Wide eld imaging was performed on a Nikon E400 epi uorescence microscope with Hamamatsu ORCA-Flash4.0 V2 Digital CMOS monochrome camera (Hamamatsu Photonics K.K., C11440-22CU).…”