Morphometric Analyses of Retinal Sections

Summary

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Abstract

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)–induced excitotoxicity¹, retinal ischemia-reperfusion injury² and glaucoma³. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity⁴. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure^3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.

The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.

This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience — MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

Protocol

1. Tools

Microscope, Nikon

Stereo Investigator, MBF Bioscience – MicroBrightField, Inc.

2. Preparation

1. Before working on any morphometric analysis, each retina sample is sectioned at 4 micron thickness and undergoes the H&E staining.
2. The retina section is divided into 4 regions – upper peripheral region, the upper central region, the lower peripheral region, and the lower central region.
3. After defining the regions, an image of each region is acquired at 10x40 magnifications under the bright beam microscope of Nikon.
4. Now, we are ready to ana....

Discussion

1. How to obtain a more accurate thickness measurement?

You can enlarge the image by clicking "Zoom in" button and left click in the tracing window. The INL border can be seen clearly. If the tip of the line is not at the outer border of the INL, we can adjust the line under the Editing mode. After picking the concerned line, right click the tracing window and choose "Insert Point in Selected Contour". A point can be added at the outer border while the point added should be along the same line. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo Investigator		MicroBright Field
Microscope		Olympus Corporation	BX51