Noninvasive Imaging of Cone Ablation and Regeneration in Zebrafish

Abstract

To observe and characterize cone degeneration and regeneration in a selective metronidazole-mediated ablation model of ultraviolet-sensitive (UV) cones in zebrafish using in vivo optical coherence tomography (OCT) imaging. Methods: Twenty-six sws1:nfsB-mCherry;sws2:eGFP zebrafish were imaged with OCT, treated with metronidazole to selectively kill UV cones, and imaged at 1, 3, 7, 14, 28, or 56 days after ablation. Regions 200 × 200 μm were cropped from volume OCT scans to count individual UV cones before and after ablation. Fish eyes were fixed, and immunofluorescence staining was used to corroborate cone density measured from OCT and to track monocyte response. Results: Histology shows significant loss of UV cones after metronidazole treatment with a slight increase in observable blue cone density one day after treatment (Kruskal, Wallis, P = 0.0061) and no significant change in blue cones at all other timepoints. Regenerated UV cones measured from OCT show significantly lower density than pre-coneablation at 14, 28, and 56 days after ablation (analysis of variance, P < 0.01, P < 0.0001, P < 0.0001, respectively, 15.9% of expected nonablated levels). Histology shows significant changes to monocyte morphology (mixed-effects analysis, P < 0.0001) and retinal position (mixed-effects analysis, P < 0.0001). Conclusions: OCT can be used to observe loss of individual cones selectively ablated by metronidazole prodrug activation and to quantify UV cone loss and regeneration in zebrafish. OCT images also show transient changes to the blue cone mosaic and inner retinal layers that occur concomitantly with selective UV cone ablation. Translational Relevance: Profiling cone degeneration and regeneration using in vivo imaging enables experiments that may lead to a better understanding of cone regeneration in vertebrates.

Brd activity regulates Müller glia‐dependent retinal regeneration in zebrafish

“…(k, m, o) zrf1 staining (yellow) and DAPI staining (blue) are merged. Scale bar, 100 μm and applies to all images.3.5 | Brd-dependent recognition of acetylated histones is required for regeneration of UV conesTransgene-mediated ablation of UV cones and other photoreceptors in larvae leads to regeneration of these cells in larvae(D'Orazi et al, 2020;White et al, 2017;Yoshimatsu et al, 2016), and adults(Huckenpahler et al, 2020;Montgomery et al, 2010). We first confirmed this using our ablation paradigm.…”

“…Transgene‐mediated ablation of UV cones and other photoreceptors in larvae leads to regeneration of these cells in larvae (D'Orazi et al, 2020; White et al, 2017; Yoshimatsu et al, 2016), and adults (Huckenpahler et al, 2020; Montgomery et al, 2010). We first confirmed this using our ablation paradigm.…”

Visual Function is Gradually Restored During Retina Regeneration in Adult Zebrafish

“…Hence, we investigated the underlying morphological processes at the critical time points of vision recovery. Optical coherence tomography (OCT) imaging has proven useful to monitor retinal regeneration before (Bailey et al, 2012;Bell et al, 2016;Huckenpahler et al, 2020) as it allows a detailed analysis of retinal structures in vivo (Figure 7). We found a close correlation between the kinetics of morphological regeneration and functional restoration of vision.…”

In vivo imaging reveals mature Oligodendrocyte division in adult Zebrafish

“…S 2 ; Movies S 1 , S 2 and S 3 ). In the future, novel and recently developed methods (such as optical coherence tomography imaging) will help confirm whether newly generated cells could mature into myelinated oligodendrocytes over a long period of time (Huckenpahler et al 2020 ).…”