VBM is supported by NIH grants [K08EY020530, R01EY024665, R01EY025225, R01EY024698 and R21AG050437], Doris Duke Charitable Foundation Grant #: 2013103, and Research to Prevent Blindness (RPB), New York, NY. MT and GV are supported by NIH grant T32GM007337.

This study was approved by the University of Iowa&#39;s Institutional Review Board and adheres to the tenets set forth in the Declaration of Helsinki.
1. Foveal and Macular Biopsy Punch
<ol>
	<li>Open and butterfly the human eye, such that it consists of 4 separate flaps of tissue, as described in a previous publication.5</li>
	<li>Beginning with a butterflied human eye placed in a Petri dish, center a 4-mm punch biopsy tool over the fovea, press down, and roll gently...

<ol>
	<li>Chirco, K. R., Sohn, E. H., Stone, E. M., Tucker, B. A., Mullins, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+molecular+changes+in+the+aging+choroid:+implications+for+age-related+macular+degeneration.">Structural and molecular changes in the aging choroid: implications for age-related macular degeneration.</a> Eye (Lond). , (2016).</li><li>Zhang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+proteome+of+human+iris,+ciliary+body,+retinal+pigment+epithelium,+and+choroid.">Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.</a> Proteomics. 16 (7), 1146-1153 (2016).</li><li>Funke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glaucoma+related+Proteomic+Alterations+in+Human+Retina+Samples.">Glaucoma related Proteomic Alterations in Human Retina Samples.</a> Sci Rep. 6, 29759 (2016).</li><li>Decanini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+retinal+pigment+epithelium+proteome+changes+in+early+diabetes.">Human retinal pigment epithelium proteome changes in early diabetes.</a> Diabetologia. 51 (6), 1051-1061 (2008).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+human+vitreous+body+elements+for+proteomic+analysis.">Dissection of human vitreous body elements for proteomic analysis.</a> J Vis Exp. (47), (2011).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+landscape+of+the+human+choroid-retinal+pigment+epithelial+complex.">Proteomic landscape of the human choroid-retinal pigment epithelial complex.</a> JAMA Ophthalmol. 132 (11), 1271-1281 (2014).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+mouse+vitreous+and+retina+for+proteomic+analyses.">Evisceration of mouse vitreous and retina for proteomic analyses.</a> J Vis Exp. (50), (2011).</li><li>Skeie, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+vitreous+biopsy+techniques.">Proteomic analysis of vitreous biopsy techniques.</a> Retina. 32 (10), 2141-2149 (2012).</li><li>Skeie, J. M., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+interactions+in+the+mouse+vitreous-retina+complex.">Proteomic interactions in the mouse vitreous-retina complex.</a> PLoS One. 8 (11), e82140 (2013).</li><li>Mahajan, V. B., Skeie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+vitreous+proteomics.">Translational vitreous proteomics.</a> Proteomics Clin Appl. 8 (3-4), 204-208 (2014).</li><li>Skeie, J. M., Roybal, C. N., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+insight+into+the+molecular+function+of+the+vitreous.">Proteomic insight into the molecular function of the vitreous.</a> PLoS One. 10 (5), e0127567 (2015).</li><li>Velez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Medicine:+Personalized+Proteomics+for+the+Diagnosis+and+Treatment+of+Idiopathic+Inflammatory+Disease.">Precision Medicine: Personalized Proteomics for the Diagnosis and Treatment of Idiopathic Inflammatory Disease.</a> JAMA Ophthalmol. 134 (4), 444-448 (2016).</li><li>Velez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+elevated+intraocular+pressure+with+retinal+detachment.">Proteomic analysis of elevated intraocular pressure with retinal detachment.</a> Am J Ophthalmol Case Rep. 5, 107-110 (2017).</li><li>Skeie, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biorepository+for+ophthalmic+surgical+specimens.">A biorepository for ophthalmic surgical specimens.</a> Proteomics Clin Appl. 8 (3-4), 209-217 (2014).</li><li>Ferrer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+protein+preservation+largely+depends+on+the+postmortem+storage+temperature:+implications+for+study+of+proteins+in+human+neurologic+diseases+and+management+of+brain+banks:+a+BrainNet+Europe+Study.">Brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a BrainNet Europe Study.</a> J Neuropathol Exp Neurol. 66 (1), 35-46 (2007).</li><li>Ahmed, M. M., Gardiner, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+protein+profiles+in+tissue+samples:+differing+outcomes+with+and+without+heat+stabilization.">Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization.</a> J Neurosci Methods. 196 (1), 99-106 (2011).</li><li>Kanshin, E., Tyers, M., Thibault, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+Collection+Method+Bias+Effects+in+Quantitative+Phosphoproteomics.">Sample Collection Method Bias Effects in Quantitative Phosphoproteomics.</a> J Proteome Res. 14 (7), 2998-3004 (2015).</li><li>Crecelius, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+quantitative+post-mortem+changes+in+the+gray+matter+of+the+human+frontal+cortex+proteome+by+2-D+DIGE.">Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE.</a> Proteomics. 8 (6), 1276-1291 (2008).</li><li>Oka, T., Tagawa, K., Ito, H., Okazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+of+the+phosphoproteome+in+postmortem+mouse+brains.">Dynamic changes of the phosphoproteome in postmortem mouse brains.</a> PLoS One. 6 (6), e21405 (2011).</li><li>Nagy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+postmortem+interval+on+biomolecule+integrity+in+the+brain.">Effects of postmortem interval on biomolecule integrity in the brain.</a> J Neuropathol Exp Neurol. 74 (5), 459-469 (2015).</li><li>Mukherjee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+frozen+tissue+samples+using+laser+capture+microdissection.">Proteomic analysis of frozen tissue samples using laser capture microdissection.</a> Methods Mol Biol. 1002, 71-83 (2013).</li></ol>

After tissue collection, sample handling and treatment are crucial considerations14. Preservation in liquid nitrogen is preferred over chemical fixation, as the latter may result in damage to protein structure, which could skew downstream analysis. Additionally, liquid nitrogen preservation is preferred to methods which do not involve freezing of samples. Notably, Ferrer et al. showed significant differences in protein levels between brain samples preserved at 4 &#176;C or room temperatur...

The retina, RPE, and choroid are complex tissues that demonstrate important regional differences in protein expression, physiological function, and pathological susceptibilities1,2. For example, diseases such as Age-Related Macular Degeneration (AMD), retinitis pigmentosa, and central serous retinopathy each demonstrate characteristic localization within the fovea, macula, or retina periphery1,3,4,6. Here, we present a method demonstrating how distinct retinal regions can be independently sampled. The overall goal of this method is to provide a reliable guide for collection of tissue samples from the foveal, macular, and peripheral regions of the human retina and RPE-choroid for proteomic analysis. The rationale for the development and use of this technique is that through proteomic analysis of these specific retinal regions, important molecular insights may be gained into the physiological and pathophysiological functions of these regions.
This approach promises to reveal the proteomic basis for relative regional disease susceptibilities, and to facilitate the identification of new specific therapeutic targets. Indeed, proteomic investigations of the vitreous and its interactions with the retina have provided key insights into the molecular composition and function of healthy and diseased tissue5,7,8,9,10,11,12,13. However, clear comparative proteomic analyses of distinct retinal regions are lacking. The technique will help to support these much-needed studies, providing advantages over other methods by demonstrating a reliable and reproducible tissue collection approach. More so, the approach is very accessible, taking advantage of standard-sized and readily available tissue punch biopsy tools. Our technique emphasizes the appropriate collection and storage of tissues for proteomic processing, making important considerations for protein stability and degradation. Thus, this method is most appropriate for investigators considering downstream molecular analysis of proteomic factors.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-mm skin punch biopsy tool</td>
			<td style="width:189px;">Miltex</td>
			<td style="width:119px;">REF 33-34</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-mm skin punch biopsy tool</td>
			<td style="width:189px;">Miltex</td>
			<td style="width:119px;">REF 33-37</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.12 Colibri Forceps</td>
			<td style="width:189px;">Stephens Instruments</td>
			<td style="width:119px;">S5-1145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wescott Scissors</td>
			<td style="width:189px;">Sklar Surgical Instruments</td>
			<td style="width:119px;">64-3146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfuge tubes</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:119px;">#022364111</td>
			<td>1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid Nitrogen</td>
			<td style="width:189px;">Praxair, Inc.</td>
			<td style="width:119px;">7727-37-9 [R]</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of human retina and rpe-choroid for proteomic analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

Retinal and RPE-choroid tissue can be processed in various ways to suit an individual investigation. After collection, the researcher will possess samples of retinal and RPE-choroid tissue from the foveal region, outer macula, and peripheral retina (Figure 1). Specifically, the foveal region punch will include the fovea, the parafovea, and a small amount of the adjacent perifovea. The macular punch includes the remainder of the perifoveal region as well as a ...

Watch this Scientific Journal Video about Dissection of Human Retina and RPE-Choroid for Proteomic Analysis at JoVE.com

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

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.

dissection-of-human-retina-and-rpe-choroid-for-proteomic-analysis

Research

JoVE Journal

Biochemistry

10.7K visualizações.  Columbia University. 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 p...