ABSTRACT
Purpose of this study was to investigate firstly specific proteomic changes within the retina in the course of an animal glaucoma model and to identify secondly new approaches for neuroprotective, therapeutic options in glaucoma by addressing those specific changes. Intraocular pressure was elevated through cauterization of episcleral veins in adult Sprague Dawley rats. Molecular and morphological changes were surveyed using mass spectrometry, optical coherence tomography as well as immunohistochemical cross section- and flat mount stainings. By quantifying more than 1500 retinal proteins, it was found that the HspB5 protein and numerous beta-crystallins showed a uniform and unique shifting expression pattern as a result of different periods of elevated IOP exposure. Crystallins showed a significant downregulation (p<0.05) after 3 weeks of elevated IOP and an upregulation after 7 weeks. Counteracting those typical changes, an intravitreal injection of ß-crystallin B2 at the time of IOP elevation was found to reduce retinal ganglion cell loss (p<0.05), decrease of the retinal nerve fiber layer (p<0.05) and impairment of the optic nerve. Ultimately, proteomic data revealed that ß-crystallin B2 might influence calcium-depended cell signaling pathways with severe effect on apoptosis and gene regulation. In this context especially annexin A5, calcium-transporting ATPase 1 and various histone proteins seem to play a major role.
Subject(s)
Disease Models, Animal , Glaucoma/pathology , Retinal Ganglion Cells/drug effects , beta-Crystallin B Chain/administration & dosage , Animals , Cell Survival/drug effects , Glaucoma/physiopathology , Intraocular Pressure , Intravitreal Injections , Male , Rats , Retinal Ganglion Cells/pathology , beta-Crystallin B Chain/pharmacologyABSTRACT
The purpose of the study was to further scrutinize the potential of ßB2-crystallin in supporting regeneration of injured retinal ganglion cell axons both in vitro and in vivo. Retinal explants obtained from animals after treatment either with lens injury (LI) alone or with combined LI 5 days or 3 days before or simultaneously with an optic nerve crush (ONC) were cultured for 96 h under regenerative conditions, and the regenerating axons were quantified and compared with untreated controls. These measurements were then repeated with LI replaced by intravitreal injections of γ-crystallin and ß-crystallin at 5 days before ONC. Finally, ßB2-crystallin-overexpressing transfected neural progenitor cells (ßB2-crystallin-NPCs) in the eye were studied after crushing the optic nerve in vivo. Regeneration was monitored with the aid of immunoblotting of the retina and optic nerve both distal and proximal to the lesion site, and this was compared with controls that received injections of phosphate buffer only. LI performed 5 days or 3 days before ONC significantly promoted axonal outgrowth in vitro (p < 0.001), while LI performed alone before explantation did not. Intravitreal injections of ß-crystallin and γ-crystallin mimicked the effects of LI and significantly increased axonal regeneration in culture at the same time intervals (p < 0.001). Western blot analysis revealed that crystallins were present in the proximal optic nerve stump at the lesion site in ONC, but were neither expressed in the undamaged distal optic nerve nor in uninjured tissue. ßB2-crystallin-NPCs supported the regeneration of cut optic nerve axons within the distal optic nerve stump in vivo. The reported data suggest that ßB2-crystallin-producing "cell factories" could be used to provide novel therapeutic drugs for central nervous system injuries.
