Abstract
Purpose:
The zebrafish retina regenerates following injury. Previous studies showed Müller cells become retinal progenitors to repopulate the damaged areas. However, little is known about the role of microglia during regeneration. We hypothesize microglia are the earliest responders to injury. To test this hypothesis we characterized the early cellular response following targeted injury to photoreceptors in the adult zebrafish retina.
Methods:
Focal lesions, localized to the photoreceptor layer, were created in fish retina using our optical coherence tomography (OCT)-guided laser injury model (DiCicco et al., IOVS 2014). Each laser-injured retina was imaged with both OCT and confocal scanning laser ophthalmoscopy (cSLO) at intervals starting at 0 days post lesion (dpl) and continuing through 6 weeks post lesion (wpl) to monitor the progression of the lesions in vivo. At each interval, fish were euthanized and the eyes cryosectioned. Immunohistochemistry was used to identify microglia (4c4), apoptotic cells (TUNEL), and proliferating cells (PCNA) within the injury site and surrounding tissue.
Results:
Following laser injury, there was a rapid (1 dpl) migration of microglia to the outer nuclear layer (ONL) and outer segment zone (OSZ) from their baseline locations. There was also focal microglia migration to the inner plexiform layer (IPL) adjacent to the injury site. Before injury the microglia were found mainly in the outer plexiform layer (OPL) and inner retina. Apoptosis followed the migration of microglia and was predominantly found in the ONL and OSZ with some cells in the INL. Apoptosis increased through 3 dpl then declined. Proliferating cells were located mainly in the INL and ONL, and increased through 3 dpl then declined. A few proliferation cells were also found in the IPL and GCL.
Conclusions:
Laser injury to the zebrafish retina produces a rapid migration of microglia to the lesion site. This response from the microglia occurs prior to the increase in cellular proliferation and apoptosis, and thus supports our hypothesis that microglia are the earliest responders to injury in zebrafish. Cellular proliferation and apoptosis begin to increase after the appearance of the microglia within the injury zone.