Use of Osmium Tetroxide Staining with Microcomputerized Tomography to Visualize and Quantify Bone Marrow Adipose Tissue In Vivo

Abstract

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

Marrow Adipose Tissue: Trimming the Fat

“…humans > rabbits > rats > mice) [2]. Mice have a significantly reduced proportion of MAT in areas of red marrow, as quantified in three-dimensions using osmium tetroxide staining combined with micro-computed tomography [5,8,12,13]. For example, 12-week-old C57Bl/6J (B6) male mice have on average 0.7% MAT by volume in the bone marrow of the tibial diaphysis [5].…”

Exercise Regulation of Marrow Fat in the Setting of PPARγ Agonist Treatment in Female C57BL/6 Mice

“…Our method for volumetric assessment of osmium staining of lipid in mouse bones has been previously described (7,36). Briefly, femurs were fixed, then decalcified, and subsequently incubated with 1% osmium tetroxide/2.5% potassium dichromate for 48 hours.…”

Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair

“…2012). But an advantage of osmium it has been used to combine micro-CT imaging with post-imaging histology at both the light and electron microscopic levels (Handschuh et al 2013; Sengle et al 2013; Scheller et al 2014). With iodine, post-imaging histological studies have been limited, except for very superficial analyses of tissue structure, because iodine treatment renders soft tissues dense and brittle and imparts a yellow color (Jeffery et al 2011).…”