Quantification of Lipid Area within Thermogenic Mouse Perivascular Adipose Tissue Using Standardized Image Analysis in FIJI

Abstract

Quantification of adipocyte size and number is routinely performed for white adipose tissues using existing image analysis software. However, thermogenic adipose tissue has multilocular adipocytes, making it difficult to distinguish adipocyte cell borders and to analyze lipid proportion using existing methods. We developed a simple, standardized method to quantify lipid content of mouse thermogenic adipose tissue. This method, using FIJI analysis of hematoxylin/eosin stained sections, was highly objective and highly reproducible, with ∼99% inter-rater reliability. The method was compared to direct lipid staining of adipose tissue, with comparable results. We used our method to analyze perivascular adipose tissue (PVAT) from C57BL/6 mice on a normal chow diet, compared to calorie restriction or a high fat diet, where lipid storage phenotypes are known. Results indicate that lipid content can be estimated within mouse PVAT in a quantitative and reproducible manner, and shows correlation with previously studied molecular and physiological measures.

Keywords: FIJI; Image analysis; Lipid quantification; Perivascular adipose tissue; Thermogenic adipose tissue.

Figure 1.. Phenotypic diversity of mouse adipose tissue depots.

Adipose tissue was collected from adult C57BL/6 male mice and processing for histology. Shown are hematocxylin/eosin-stained adipose tissue from gonadal white adipose tissue (A), interscapular brown adipose tissue (B), and perivascular adipose tissue surrounding the thoracic aorta (C). Scale bar = 25μm.

Figure 2.. Process for image assessment and quantification of multilocular adipose tissue.

Input images of hematoxylin/eosin stained sections are converted to grayscale (a), and then follow two pathways: identification of the region of interest (b-c) and thresholding of the image (d). The results from both pathways are combined using the «AND» function in the «Image Calculator» tool in ImageJ to produce the final image (e) (this corresponds to step 7 in the protocol). This image is then measured as the final lipid volume, and compared to the entire selected region (c).

Figure 3.. Comparison of lipid quantification methods.

A) Mouse perivascular adipose tissue was obtained from Immunofluorescence images of a frozen PVAT section from a C57BL/6J mouse. Each channel is labeled with its corresponding stain/label. Red arrows on the Brightfield and LipidTox channels indicate lipid that is seen outside of the tissue, that may have been mechanically removed from the tissue during the staining process. Images taken at 100x magnification and digitally zoomed 4x to result in effective 400x magnification, scale bar 50μm. B) Brightfield image of an H&E-stained section of PVAT from the same mouse that was treated with the IF stains. Image taken at 400x magnification, scale bar 25μm. C) Comparison graph of LipidTox and H&E-stained images, each compared based on the percent lipid that was identified based on the images. Comparison occurred between a sample of PVAT and BAT from the same mouse.

Figure 4.. C57BL/6J PVAT phenotype shift under varying diet conditions as quantified using our ImageJ protocol.

A) Images of representative H&E-stained sections for each diet analyzed in this case study. Each image is labeled with the corresponding diet. Scale bar = 25μm for all images. B) Percent lipid measurements of samples images from all studied diets across two raters. Each rater independently quantified each sample image from all diet types, with the results being plotted. Each n=39 per diet. A two-way ANOVA was performed using GraphPad Prism to determine significance between diet types, and multiple t-tests were used for each pair of raters within a diet to determine significance between raters.