engleski

The golden days are back: modern application of gold impregnation technique on rat brain cryosections

Around 150 years ago, the silver impregnation method for histological sections of nervous tissue was published by Camillo Golgi (1). Different impregnation methods were used extensively for some time, and they contributed significantly to the development of neuroscience. With the development of new methods, such as electron and confocal microscopy, silver and gold impregnation methods were used less. However, they can still produce useful data, especially when used in combination with modern image analysis software. In this research, the impregnation technique was used to visualize nerve fibers in the areas of interest in rat brain. The animals were divided into 5 groups (sham-operated control group, ovariectomized untreated group, ovariectomized and treated with alendronate, hop extract and a combination of the two). Rat brains were fixed in 4% paraformaldehyde, cryoprotected and frozen in pre-chilled isopentane. Coronal 35 µm-thick sections were prepared on a cryostat. 0.2% gold chloride solution in 0.02 M neutral phosphate buffer and 0.9% sodium chloride was used for impregnation. After the impregnation, the slides were dehydrated and coverslipped. Samples were photographed and analyzed in a free and open-source program ImageJ (2). For staining intensity quantification, images were separated into 8-bit channels and the blue channel was used for analysis. Parameters chosen for measurement in ImageJ were area, standard deviation, min and max grey value, integrated density and mean grey value. Brightfield microscope examination showed excellent quality of the nerve fiber impregnation. Significant changes in the staining intensity were found in several brain regions of interest between examined groups (primary motor cortex, dentate gyrus and cornu ammonis 3). The impregnation technique was very useful for the quantification of nerve fibers when combined with ImageJ image analysis software. Although impregnation methods in neuroscience are 150 years old, gold impregnation can be effectively used in modern research for the quantification of nerve fibers on brain cryosections using freely available image analysis software.

Hop Extract ; Image Processing, Computer-Assisted ; Impregnation ; Neuroscience ;

nije evidentirano