Histological changes of fetal lower limb musculoskeletal layout after clearing with Dawson's technique

Abstract

Dawson's clearing technique is widely used for the study of skeletal embryology, particularly at examining endochondral ossification in human developmental stages (Fig 1). While it has proven to be a useful tool for visualizing bony structures through cleared flesh, little is known about the histological changes suffered by both bony and muscular tissue.Our aim is to contrast cleared and formaldehyde fixated musculoskeletal tissue in fetal lower limbs at cellular and tissular levels. Two previously hard‐fixated fetuses' lower limbs were treated with 1) Dawson's clearing method based on Alizarin Red staining and immersion in aqueous KOH at 1% weight/volume, until complete translucency was obtained and 2) immersion in 10% volume/volume formaldehyde. For both, transversal cuts of tarsus, metatarsus and phalanges were obtained and stained in Hematoxylin and Eosin (H&E), Periodic acid‐Schiff (PAS) stain and Masson's Trichrome (MT) stain.Samples were observed under a light microscope at 4×, 10× and 20×. Cleared tissue slides in MT stain showed preserved collagen structures in connective tissue while H&E stain showed absence of cell membranes, nuclear structures, and epithelia. Bony structures were partially degraded while cartilaginous tissue maintained a similar but dilated resemblance to formaldehyde preserved tissue. PAS stain did not show any relevant change between specimens.Many of these observable changes are aligned to the alkaline hydrolysis process occurring between KOH and macromolecules. This interaction is widely reported in the literature. Said variations, in addition to studies proving a decrease of weight and volume in cleared specimens, show that microscopic changes in cleared tissue may be affecting its macroscopic observable layout. While clearing process is useful for examining endochondral ossification, it is important to take caution in quantitative measurements as the process may change the original characteristics of the tissue.Support or Funding InformationUniversidad de los Andes School of Medicine, Anatomy Laboratory.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.