Morphometric Analyses of Retinal Sections

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 excitotoxicity 1 , retinal ischemia-reperfusion injury 2 and glaucoma 3 . Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity 4 . 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.

A Novel ImageJ Macro for Automated Cell Death Quantitation in the Retina

“…These premises argue in favor of the high level of accuracy of this measurement tool. 16 On a side note, TUNEL þ cell count variability was increased in INL images with more TUNEL þ cells, probably owing to the presence of cells of different sizes 19 and staining patterns. Nonetheless, because there is no absolute reference value in TUNEL þ cell counting, we believe it is crucial to follow consistent criteria when counting cells, in order to detect consistent differences between groups.…”

Alternative transcribed 3' isoform of long non-coding RNA Malat1 inhibits mouse retinal oxidative stress

“…Retinal sections were hydrated in graded ethanol solutions (95%, 75%, 50%) for 30 s each. The retinal sections were then stained with Hematoxylin and Eosin stain as previously described 93 and images were taken using confocal microscopy (Olympus IX73, Japan).…”

Preservation of Retinal Function Through Synaptic Stabilization in Alzheimer's Disease Model Mouse Retina by Lycium Barbarum Extracts

“…Images were captured with a light microscope (Eclipse80i, Nikon) under 40x magnification. Morphometric analysis was carried out as in our previous report (Chan et al, 2012 ). The thickness of the inner retinal layer (IRL) was measured from inner limiting membrane to the outermost point of the inner nuclear layer (INL), and the outer nuclear layer (ONL), as well as the number of RGC both in the central or peripheral retina, were calculated using Fiji software (NIH, MD, USA).…”