Abstract
Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.
Highlights
The vitreous humor (VH) is a transparent gel-like extracellular matrix that occupies the cavity between the lens and the retina
Two sets of freshly enucleated eyes of New Zealand white (NZW) (n = 8/set), young (8 weeks old, n = 4), and mature (6 months and older, n = 4; rabbits reach sexual maturity at 6 month [13]) rabbits were obtained from Pel-freeze Biologicals (n = 8, Rogers, AR) and Johns Hopkins University (n = 8, JHU) within 10 min of sacrifice with euthanasia using pentobarbital containing solution (80–100 mg/kg, Fatal plus, Butler Schein Animal Health, Dublin, OH) intravenously under a different non ocular protocol approved by the Institutional Animal Care and Use Committee at JHU
A total of 16 independent vitreous samples were divided into 4 groups according to age and location of vitreous: anterior and posterior vitreous compartments from young rabbits; anterior and posterior vitreous compartments from mature rabbits
Summary
The vitreous humor (VH) is a transparent gel-like extracellular matrix that occupies the cavity between the lens and the retina. In addition to its physical functions, the VH contains many proteins accumulated by local secretion, filtration from the blood, or diffusion from the surrounding tissues and vasculature[2,3,4] These proteins may alter the physiochemical properties of this matrix and affect processes occurring in the structures in contact with or adjacent to the VH[5]. Different compartments of VH exist that include the vitreous base, core, cortex, and anterior hyaloid[6]. These various compartments are either in contact with the lens/ciliary body anteriorly, or with the retinal surface posteriorly. The authors suggested that there are differentially expressed proteins in the various vitreous body substructures Identification of specific proteins may provide greater insight into the clinically identified vitreous compartments and reveal candidate molecules underlying various vitreoretinal or intraocular inflammatory diseases
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