Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Summary

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.

Abstract

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.

Introduction

The retina, RPE, and choroid are complex tissues that demonstrate important regional differences in protein expression, physiological function, and pathological susceptibilities^1,2. 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 periphery^1,3,4,6. 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 tissue^{5,7,8,9,10,11,12,13}. 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.

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Protocol

This study was approved by the University of Iowa's Institutional Review Board and adheres to the tenets set forth in the Declaration of Helsinki.

1. Foveal and Macular Biopsy Punch

1. Open and butterfly the human eye, such that it consists of 4 separate flaps of tissue, as described in a previous publication.⁵
2. 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 until an incision is made around the fovea.
3. Next, generate an incision around the macula by centering and pressing down an 8-mm skin punch biopsy tool within the arcades of the macula, applying gentle pressure and rolling. This will produce a second, outer ring of tissue surrounding the first.

2. Peripheral Retinal Biopsy Punch

1. Use the 4-mm skin punch biopsy tool to make a series of punches in the peripheral retina, just outside the arcade. Here, make two punches for each quadrant flap.
   NOTE: After all the punches are made, the eye will have two concentric punches in the center, representing the fovea and macula, and two punches at the base of each flap-totaling 8 punches in the peripheral retina.

3. Fovea and Macula Biopsy Collection

1. As the tissue is now ready for collection, collect all tissue biopsies in separate microfuge tubes and freeze using liquid nitrogen for downstream processing. Store all samples at -80 °C until utilization.
2. Use a curved 0.12 Colibri forceps to grab the edges of the translucent tissue of the retinal fovea. To collect, elevate and separate the foveal tissue from the underlying RPE-choroid.
3. Similarly, use the 0.12 Colibri forceps to grasp the outer ring of the translucent macula tissue. If the tissue is still attached to underlying or adjacent tissue, use a pair of Westcott scissors to carefully trim the edge, capturing just the retinal component and dissecting away any optic nerve tissue included in the punch.

4. Peripheral Retina Collection

1. Use the curved 0.12 Colibri forceps to gently separate the peripheral retinal tissue discs from the underlying RPE-choroid and place within individual microfuge tubes.
2. In the case of residual vitreous gel, use the forceps to lift and separate the gel away as much as possible before collection of the retinal tissue. Once the retina has been removed, the pigmented RPE-choroid remains below.

5. Foveal and Macula RPE-Choroid Collection

1. Use the 0.12 Colibri forceps to grasp the edges of the dark RPE-choroid tissue that lies beneath the area of the removed fovea. Carefully separate this tissue from the sclera and collect.
2. Using the same forceps, grasp the edges of the outer ring of dark RPE-choroid tissue underlying the removed macular area. Carefully separate this tissue from the sclera by grasping the edges at different points around the ring and lightly pulling. Eventually, the ring of RPE-choroid tissue will be dislodged and may be collected.
   NOTE: Secondary forceps in the opposite hand can be helpful in manipulating the RPE-choroid tissue during removal. Like the retinal tissue, Wescott scissors can be used to assist in removing any tissue that was not fully incised using the punch tool.

6. Peripheral Retina RPE-Choroid Collection

1. Use the 0.12 Colibri forceps to peel away the RPE-choroid in the 8 peripheral punch areas.
2. As previously, place the RPE-choroid tissue in microfuge tubes and freeze using liquid nitrogen for downstream processing. Keep all samples at -80 °C until utilization.

7. Scissor Dissection

NOTE: If the punch biopsy blade is dull or the punch biopsy tool is not pushed hard enough, there may not be a clean-cut surrounding the tissue.

1. In these cases, pull the tissue away as much as possible, and then use Westcott scissors to trim and separate.

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Results

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 ...

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Discussion

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 °C or room temperatur...

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Materials

Name	Company	Catalog Number	Comments
4-mm skin punch biopsy tool	Miltex	REF 33-34
8-mm skin punch biopsy tool	Miltex	REF 33-37
0.12 Colibri Forceps	Stephens Instruments	S5-1145
Wescott Scissors	Sklar Surgical Instruments	64-3146
Microfuge tubes	Eppendorf	#022364111	1.5 mL
Liquid Nitrogen	Praxair, Inc.	7727-37-9 [R]