Abstract
Purpose :
During glaucoma, retinal ganglion cell (RGC) axons are damaged, causing the RGCs to die, resulting in the irreversible loss of visual function. There are no FDA-approved drugs or therapies to protect RGCs from death in glaucoma. Unlike in mammals, zebrafish maintain most RGCs after optic nerve injury; however, the molecular underpinnings of RGC neuroprotection are unknown. Here, we sought to identify neuroprotective factors in zebrafish RGCs after injury and determine if macrophages/microglia are required to survive RGCs after optic nerve transection.
Methods :
isl2b:GFP zebrafish, in which ~60% of RGCs are labeled, was utilized at ~3 months. Animals underwent an optic nerve transection surgery in the left eye, while the right eye of the same fish went through a sham surgery. To quantify RGC survival after transection, retinal flat mounts were made and imaged using confocal microscopy at 1-14 days post-injury (dpi) and RGC survival over time was quantified using ImageJ software. Retinae were dissociated at 6,12 and 24 hours post transection and RGCs were sorted and collected by fluorescence-activated cell sorting. RNA-Seq was performed in biological triplicate, and gene expression changes after injury were quantified. After analyzing the RNA-seq data, several pathways were identified to be possibly involved in RGC survival. Pharmacological experiments were performed and the results on RGC survival quantified. Immunofluorescence imaging of transgenic lines labeling specific leukocyte populations was performed to assess the interaction of immune responses and RGC survival after optic nerve transection.
Results :
After injury, RGC survival was: 94.44 ± 5.45% (1dpi); 92.52 ± 3.54% (3dpi) and 76.35 ± 2.58% (7dpi), with levels returning to 98.71 ± 4.82% at 14dpi. Analysis of RNA-seq data indicate JAK/STAT signaling pathway and immune responses were activated after optic nerve transection, amongst other pathways. Local inhibition of the JAK/STAT pathway leads to the reduction of RGC survival, while local suppression of immune responses by dexamethasone rescued RGCs after injury. Furthermore, depletion of macrophages/microglia increased RGC survival after injury.
Conclusions :
Zebrafish maintain most RGCs after optic nerve injury. JAK/STAT signaling pathway regulates RGC survival after optic nerve injury. Immune responses play a critical role in RGC survival after injury.
This is a 2021 ARVO Annual Meeting abstract.