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 independ....

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 ....

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 .......

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.......

Materials

List of materials used in this article

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]