Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa
Skeie, J. M., Mahajan, V. B. Dissection of Human Vitreous Body Elements for Proteomic Analysis. J. Vis. Exp. (47), e2455, doi:10.3791/2455 (2011).
The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.
1. Anterior Segment Dissection.
2. Vitreous Core Aspiration.
3. Anterior Hyaloid Dissection.
4. Vitreous Base Dissection.
5. Vitreous Cortex Removal.
6. Representative Results
Tissue samples can be processed by a variety of methods for specific experiments. In our case, samples were submitted for protein analysis by SDS-PAGE (Figure 3).
Figure 1. Cross-sectional view of the human eye depicting different substructures of the vitreous body. The most anterior vitreous is a thin collagenous layer called the anterior hyaloid. The vitreous core comprises the entire central region of the vitreous body. This portion of the vitreous is more aqueous in contrast to the vitreous base, which is viscous enough to be grasped by forceps and is firmly attached to the underlying ciliary body and retina. Encompassing the vitreous core is a very thin collagenous shell called the vitreous cortex.
Figure 2. Vitreous base anatomy. The vitreous base is a semi-transparent substructure of the vitreous body located along the ora serrata (white arrows), which is the dividing line separating the ciliary body and retina. The anterior border of the vitreous base extends over the pars plana (white line) of the ciliary body. The posterior border of the vitreous base extends 2-3-mm posterior to the ora serrata (white dash). To excise the vitreous base, forceps are used to grasp the tissue and pull it away from the underlying ciliary body and retina. Once elevated, Westcott scissors are used to cut along the base.
Figure 3. One-dimensional SDS-PAGE of Vitreous Body Elements. Total protein concentrations for the anterior hyaloid, vitreous base, vitreous core, and vitreous cortex were 11.24, 20.1, 16.61, 14.24 mg/mL, respectively. Gel electrophoresis was performed at 200 kV for 45 minutes, stained with Flamingo (Bio-Rad), and visualized using a VersaDoc Imaging system (Bio-Rad). The profiles for the different tissues show several similar bands, indicating either conserved or cross-contaminating proteins, as well as unique bands (asterisk), indicating differentially localized proteins.
The vitreous body is a semi-transparent gel whose molecular composition is poorly understood, especially at the level of its substructures: the vitreous base, core, cortex, and anterior hyaloid. The vitreous core contains collagens II, V, IX, and XI, along with chondroitin sulphate proteoglycans, heparan sulphate proteoglycans, and hyaluronan.1,2 Protein biomarkers in the vitreous core have been associated with diseases such as diabetic retinopathy.3-5 How these proteins are differentially expressed in each of the substructures, and in many cases the specific protein identities, are not known. These details may give insight to the origin of proteins associated with specific vitreoretinal diseases and help target future therapies. Although the optimum post mortem interval for tissue dissection has not been determined, protein degradation may affect downstream experiments. For example, immunohistochemistry is affected in 12-hour postmortem eyes and some specific enzyme activities may be reduced within a few hours (unpublished observation). All tissues in this study were collected between 2 and 8 hours of death without significant changes in protein expression or suitability for proteomics analsyis. The liquid nitrogen freezing method of preservation is chosen over fixation in order to prevent small changes in protein structure caused by fixative cross linking, which has been demonstrated in other tissues by LC-MS/MS.6 Proteomic studies will depend on the ability to accurately dissect the different compartments of the vitreous, as demonstrated in this video experiment. We have validated the dissection technique using 1-dimensional SDS-PAGE. As our results suggest, there are differentially expressed proteins in the various vitreous body substructures. Identifying these proteins will provide a more detailed understanding of vitreous compartmentalization.
No conflicts of interest declared.
Funding was provided by Fight for Sight. Tissues were obtained from the Iowa Lions Eye Bank.
|0.12 forceps||Storz Ophthalmics||E1502|
|5-cc syringe||BD Biosciences||309603|
|Straight Dressing Forceps With Serrations||Storz Ophthalmics||E1400|
|23 gauge needle||BD Biosciences||305145|
|Colibri forceps||Storz Ophthalmics||2/132|
|Castroviejo angled corneal scissors||Storz Ophthalmics||E3223|
|Vannas Curved Capsulotomy Scissors||Storz Ophthalmics||E3387|
|Weck-Cel surgical spears||Medtronic Inc.||0008680|
|Westcott Curved Tenotomy Scissors||Storz Ophthalmics||E3320|