Perilipin-1 immunostaining improves semi-automated digital quantitation of bone marrow adipocytes in histological bone sections

ABSTRACT

Bone marrow adipocytes (BMAds) are not just passive fillers inside the bone marrow compartment but respond to various metabolic changes. Assessment of those responses requires evaluation of the number of BMAds and their morphology for which laborious and error-prone manual histological analysis remains the most widely used method. Here, we report an alternative image analysis strategy to semi-automatically quantitate and analyse the morphology of BMAds in histological bone sections. Decalcified, formalin-fixed paraffin-embedded histological sections of long bones of Sprague-Dawley rats were stained with either haematoxylin and eosin (HE) or by immunofluorescent staining for adipocyte-specific protein perilipin-1 (PLIN1). ImageJ-based commands were constructed to detect BMAds sized 200 µm² or larger from standardized 1 mm² analysis regions by either classifying the background colour (HE) or the positive and circular PLIN1 fluorescent signal. Semi-automated quantitation strongly correlated with independent, single-blinded manual counts regardless of the staining method (HE-based: r=0.85, p<0.001; PLIN1 based: r=0.95, p<0.001). The detection error was higher in HE-stained sections than in PLIN1-stained sections (14% versus 5%, respectively; p<0.001), which was due to false-positive detections of unstained adipocyte-like circular structures. In our dataset, the total adiposity area from standardised ROIs in PLIN-1-stained sections correlated with that in whole-bone sections (r=0.60, p=0.02).

KEYWORDS:

• Bone marrow adipocytes
• image analysis
• adiposity
• adipocyte quantitation
• perilipin-1

Acknowledgments

The authors thank Histology Core unit at the University of Turku, Finland, for excellent technical assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

KKI conceptualized the study, acquired funding, designed the methods and supervised the study. NW designed the image analysis workflows. NW, YM and NJ conducted the laboratory work and analysed images. NW and KKI provided the first version of the manuscript. All the authors approved the final version of the manuscript.

Data availability statement

The workflow presented in the supplementary material can process and analyse any images containing stained adipocytes. Original data is available upon request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21623945.2023.2252711

Additional information

Funding

This work was supported by a grant from Academy of Finland (grant 325498).