Abstract
Purpose :
A thorough understanding of the process of retinal regeneration in zebrafish is critical to our goal to regenerate damaged retinas in humans, as mammals are not able to achieve successful regeneration of the retina. During peripheral nerve regeneration and wound healing, immune cells such as macrophages, along with key immune factors, coordinate a successful regenerative response. In the case of retinal regeneration in zebrafish, the roles of immune cells and factors during the course of a successful regenerative response remain poorly characterized. Here we examine microglia and expression of selected immune genes during retinal regeneration.
Methods :
Intravitreal injection of the neurotoxin ouabain was used to induce death of inner retinal neurons, while control retinas were injected with saline. At selected times post injury, microglial numbers, expression of specific markers, morphology, and location were analyzed in whole retinas by confocal microscopy. Characteristics of microglia were correlated with the reemergence of histological features and reexpression of bipolar cell markers. Expression of selected immune genes was measured by qPCR at selected times relative to saline injected controls.
Results :
Ouabain-induced neuronal cell death results in a robust increase of macrophages/microglia in the retina. Elevated microglial numbers and morphologies associated with activation remain for several weeks after initial injury, even when microglia begin to return to their initial retinal locations. Extensions of microglial processes suggest increased motility and contact with regenerating neurons compared to those in controls. Transcripts corresponding to putative zebrafish C1q proteins and complement receptor subunits were amplified in both undamaged and regenerating whole eyes, with a significant increase in expression at 3 days post lesion (p< 0.001). Further, complement C9 expression was detected in whole eyes at day 21 of regeneration (Ct=31.0+/-1.94, n=3), whereas C9 transcript was undetectable in undamaged and saline controls.
Conclusions :
Characteristics of microglia during retinal regeneration suggest that they play active roles in the regenerative process well beyond time of neuronal cell death and progenitor proliferation. Further, expression of C9 during regeneration suggests that complement components could be involved in the regenerative process.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.