title: Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish.
abstract: We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL) with minimal photoreceptor cell damage. By 24 h after ouabain injection, maximal numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer. Immunolabeling revealed that approximately 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 d post-ouabain injection (dpi). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased proliferating cell nuclear antigen (PCNA) expression in both the INL and GCL. By 3 dpi, PCNA expression is primarily restricted to the Müller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the Müller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the Müller glial cells reentering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina.
authors.affiliation: Department of Biological Sciences and Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Shane M
authors.initials: SM
authors.lastname: Fimbel
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Montgomery
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Burket
authors.forename: David R
authors.initials: DR
authors.lastname: Hyde
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D015153:Blotting, Western / Q000379:methods; D004305:Dose-Response Relationship, Drug; D051959:ELAV Proteins / Q000378:metabolism; D000067778:ELAV-Like Protein 3; D004791:Enzyme Inhibitors / Q000633:toxicity*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology; D000076104:Oligodendrocyte Transcription Factor 2; D010042:Ouabain / Q000633:toxicity*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012038:Regeneration / Q000502:physiology*; D012160:Retina / Q000473:pathology*; D012165:Retinal Ganglion Cells / Q000187:drug effects*; D013997:Time Factors; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; D000076104:Oligodendrocyte Transcription Factor 2; D010042:Ouabain; D018809:Proliferating Cell Nuclear Antigen; D029961:Zebrafish Proteins; C088635:elavl3 protein, zebrafish; C465361:olig2 protein, zebrafish