This video will illustrate how to capture tissue biopsies from the fovea, macula and peripheral retina for proteomic research. In several steps we'll use a four millimeter skin punch biopsy tool. The human eye is placed in a petri dish after it has been butterflied.
The punch biopsy tool is centered right over the fovea, press down, rolled slightly until an incision is made around the fovea. Center and press an eight millimeter punch biopsy within the arcades of the macula with gentle pressure and rolling. Again, use the four millimeter punch biopsy to make a series or punches in the peripheral retina just outside the arcade.
We get two punches for each flap. After all the punches are made, the eye will look something like this. And we are ready to begin the tissue collection process.
We use a curved 0.12 colibri forcep to grab the edges of the tissue. Here we grasp the edge of the foveal retina. The same forcep is used to grasp the ring of macular tissue.
In this case, it appears to stuck to the disc. Using a pair of westcott scissors, you can trim the edge, so to capture just the retina. Next we turn our attention to the peripheral retinal punches we use a colibri forcep to gently lift and separate them from the underlying RPE and choroid.
In some cases there can be some residual vitreous gel. Try to use the forceps and separate this away before capturing the tissue. This is another specimen with a lot of vitreous gel.
It's just lifted away from separated using the forceps and then the retina is captured. Once the retina has been removed, the RPE-choroid remains. We use the same forceps to grasp the edges of this pigmented tissue and carefully separate it from the sclera.
This tissue represents the RPE-choroid underlying the fovea. Here's the ring of underlying RPE-choroid of the macula. A second instrument in the left hand can be useful for manipulating the tissue.
Similar to the retina, the RPE-choroid complex is fused to the disc and westcott scissors again are useful to separate this tissue from the eye. 12 colibri forceps are used to peel away the RPE-choroid in the peripheral punch area. If the punch biopsy blade was dull, or you didn't push hard enough, there may not be a clean cut surrounding the tissue.
In these cases, pull the tissue away as much as possible and then use westcott scissors to trim and separate. Tissue samples were trypsin digested and analyzed using one dimensional STS page to visualize protein content. The results of this analysis suggested distinct proteins among the different sampled regions of the retina.
The follow up experiment using liquid chromatography tandem mass spectrometry properly identified peptides from rhodopsin, a highly abundant and unique retina protein. In panel B, the representative rhodopsin spectrum obtained from the macular region is shown. Further analysis of the protein content will provide insights into the molecular functions of the anatomically distinct regions of the retina.
The retina and RPE-choroid are complex tissues that demonstrate important regional differences in protein expression and pathological susceptibilities. Here we described the novel method for reliably removing tissue samples from distinct regions of the retina for proteomic analysis. Important considerations for this technique include, proper handling, treatment and storage of samples after collection.
Post-mortem timing of tissue harvesting, considering the use of different skin size punch biopsy tools to adjust the amount and degree of regional specificity of tissue harvested. In considering the use of visual aids, such as a dissecting scope or magnifying glass to aid in the tissue collection process. Particularly in cases of smaller globe sizes.
