Fletcher Lab ~ Techniques

We use rats and mice to examine the structure and function of the normal retina, and transgenic or chemically induced rodent models of diabetes and retinal degeneration.

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High resolution immunocytochemistry and electron microscopy.

Transmission electron micrograph of a cone photoreceptor synapse from the rabbit retina.

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Identification of specific neurons using multispectral analysis.

This is a high powered form of computer analysis that allows us to identify different retinal cell types and changes in these cells during disease. It is based on software used to examine satellite images. A satellite takes sequential photographs of the ground through different filters. Houses, crops or other features of interest will appear differently through the different filters (eg have a different colour). Computer software can then be used to examine the unique "signature" of each feature of interest. In the same way, sections of retina that have been labeled with different markers can be analysed and unique signatures generated using this software. This allows us to evaluate objectively major alterations in retinal neurons and glia during disease.

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Functional marking of neuronal activity.

A vertical section of a normal control and diabetic rat retina that has been labeled with agmatine. Retinae were incubated in agmatine (decarboxylated arginine), and subsequently fixed, sectioned and labeled for agmatine. Because agmatine passes through non-specific cation channels it localizes within cells that are depolarized. There is a dramatic difference in agmatine labeling in control and diabetic retinae indicating that neuronal function is altered during diabetes.

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Electrophysiology: Electroretinogram recording.

The electroretinogram (ERG) is a measure of the electrical signals from the retina. Like the ECG or EEG, activity from neurons within the retina generate a signal that can be detected from electrodes on the surface of the eye. We use this clinical tool to assess the gross retinal function of control and disease animals to determine whether neurons are abnormally functioning.

ERG waveforms recorded from a normal rat, a non diabetic (mRen2)27 rat and a diabetic (mRen2)27 rat. The ERG recorded from the diabetic rat shows a dramatic decrease in amplitude compared to the other two strains of rat. This demonstrates that there is neuronal dysfunction early in diabetes.

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Analysis of gene expression using real time PCR.

A PCR gel showing the expression of the five glutamate transporters (EAAT1-EAAT5) in rat retina and brain.