Visualization and Analysis of Whole Depot Adipose Tissue Innervation
AbstractAdipose tissue requires neural innervation in order to regulate important metabolic functions. Though seminal work on adipose denervation has underscored the importance of adipose-nerve interactions in both white (energy storing) and brown (energy expending) adipose tissues, much remains a mystery. This is due, in part, to the inability to effectively visualize the various nerve subtypes residing within these tissues and to gain a comprehensive quantitation of neurite density in an entire depot. With the recent surge of advanced imaging techniques such as light sheet microscopy and optical clearing procedures, adipose tissue imaging has been reinvigorated with a focus on three-dimensional analysis of tissue innervation. However, clearing techniques are time consuming, often require solvents caustic to objective lenses, alter tissue morphology, and greatly reduce fluorophore lifespan. Not only are current methods of imaging wholemount adipose tissues inconvenient, but often attempts to quantify neurite density across physiological or pathophysiological conditions have been limited to representative section sampling. We have developed a new method of adipose tissue neurite imaging and quantitation that is faster than current clearing-based methods, does not require caustic chemicals, and leaves the tissue fully intact. Maintenance of a fully intact depot allowed for tiling z-stacks and producing maximum intensity projections of the entire adipose depot, which were then used to quantify neurite density across the tissue. With this processing method we were able to characterize the nerves, nerve-subtypes, and neurovascular interactions within the inguinal subcutaneous white adipose tissue in mice using up to five fluorescent channels at high resolution. We also utilized second harmonic generation, which provides label-free imaging, to investigate collagen fiber abundance in adipose of obese mice.
