| Autor: |
Chintalapudi SR; Department of Ophthalmology, The University of Tennessee Health Science Center Memphis, TN, USA., Djenderedjian L; Department of Ophthalmology, The University of Tennessee Health Science Center Memphis, TN, USA., Stiemke AB; Department of Ophthalmology, The University of Tennessee Health Science Center Memphis, TN, USA., Steinle JJ; Department of Anatomy and Cell Biology, Wayne State UniversityDetroit, MI, USA; Department of Ophthalmology, Wayne State UniversityDetroit, MI, USA., Jablonski MM; Department of Ophthalmology, The University of Tennessee Health Science CenterMemphis, TN, USA; Department of Anatomy and Neurobiology, The University of Tennessee Health Science CenterMemphis, TN, USA; Department of Pharmaceutical Sciences, The University of Tennessee Health Science CenterMemphis, TN, USA., Morales-Tirado VM; Department of Ophthalmology, The University of Tennessee Health Science CenterMemphis, TN, USA; Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science CenterMemphis, TN, USA. |
| Abstrakt: |
Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. |
