Measurement of hepatocellular glycogen is an appropriate exercise in determining level of gluconeogenesis in rodent efficacy studies for evaluating potential antidiabetic compounds. However, accuracy in glycogen quantitation requires strict control of the fasting period in all study animals because intracellular levels can vary greatly depending on postprandial time. Technical challenges in visualizing glycogen also exist because it is highly soluble, and can quickly dissipate with use of common soluble fixatives such as 10% formalin.
The periodic acid Schiff (PAS) stain is commonly utilized for visualizing glycogen, but its use suffers from the fact that the blue hematoxylin component of the stain tends to obscure the red Schiff stain in the hepatocyte cytoplasm. More accurate quantitation is enabled by using a color deconvolution algorithm which “deconvolves” or separates the various stain components (ie. blue hematoxylin and red Schiff). Effectively, then the pathologist can actually look “under” the obscuring hematoxylin to quantitate intensity and area of the red-stained glycogen.
The micrographs below depict a PAS stain (left pane) and its corresponding mark-up image (right pane) in which measurable glycogen is revealed by a red mark-up color. The bar graph insert illustrates glycogen increases as a consequence of treatment in a diabetic DIO mouse study with a prospective antidiabetic compound.