- Accueil
- Volume 18 (1999)
- Number 3 - Nov. 1999
- Densitometric and stereological determination of zonal gene expression in liver
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Densitometric and stereological determination of zonal gene expression in liver
Abstract
The expression of many hepatic genes depends on the position of the hepatocyte along the sinusoids connecting the terminal branches of the portal vein and the central vein. The expression pattern along this porto-central axis may differ strongly for different genes or under varying metabolic conditions. Hepatocyte gene expression can be visualized by in situ hybridization on tissue sections. Previous tests have shown that the optical density (OD) of the resulting sections is proportional to the amount of gene product present in the tissue. To obtain a graphical representation of this expression pattern a measurement procedure based on a combination of densitometry and a stereological model was developed. In this model the liver lobulus is considered to be a convoluted cylindrical tube which is randomly oriented with respect to the plane of sectioning; different gene expression levels can then be pictured as concentric circles or ellipses of hepatocytes with increasing or decreasing OD. The cumulative area occupied by a class of hepatocytes with similar OD and all classes between this class and the centre of the lobulus, expressed as fraction of the total area, is equal to the square of the outer limit of this class, expressed as a fraction of the radius of the lobulus.
Applying this model to actual OD images of liver sections involves segmentation of the OD values in the image into a number of zones. This segmentation is carried out with an automatic procedure that removes spatial noise. The area of the resulting concentric zones and the mean OD of the same area, masked in the original image, are measured. The area occupied by the portal- and central veins is measured separately. The relative position of the outer border of each zone on the radius of the liver lobulus can then be calculated by taking the square root of the values in the relative cumulative area distribution. The resulting graphs show on the Y-axis the mean optical density per class and on the X-axis the position of this zone on the porto-central radius of the lobulus. The results obtained by this approach are reproducible and fitting the data to a model for the regulation of gene expression in the liver can be used to extract kinetic parameters.