Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture

Johnson, Thomas V.; Oglesby, Ericka; Steinhart, Matthew R.; Cone-Kimball, Elizabeth; Jefferys, Joan L.; Quigley, Harry A.

doi:10.1167/iovs.15-17769

Cited by 27 publications

(25 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our new explant model utilizes methods by which fundamental changes in axons can be quantitatively measured, using laser scanning images of the ONH and retrobulbar optic nerve, the site of initial glaucoma damage. Previous models, including dissociated RGC (Welsbie et al, 2013), retinal explant cultures (Bull et al, 2011; Johnson et al, 2016) and eye cup models (Ishikawa et al, 2014), have been reported in which RGC survive for 1-2 weeks without a blood supply, despite transected axons, allowing repeated measures of RGCs over time. These each have advantages, but none preserves the actual tissue relationships of RGC axons in the ONH, and none permits manipulation of IOP in the intact globe to determine its effect in real time on the axons and astrocytes.…”

Section: Discussionmentioning

confidence: 99%

“…The first expresses yellow fluorescent protein (YFP) in a small number of RGCs. Previously, we and others (Danaf and Huberman, 2015; Johnson et al, 2016; Kalesnykas et al, 2012; Leung et al, 2011) have studied changes in RGC bodies and dendrites of the RGC-YFP strain with chronic IOP elevation. Some investigations were performed by cross-sectional study in histological preparations, while others observed sequential events in these cells by ophthalmoscopy.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

Kimball

Pease

Steinhart

et al. 2017

Experimental Eye Research

Self Cite

View full text Add to dashboard Cite

We developed an explant model of the mouse eye and optic nerve that facilitates the study of retinal ganglion cell axons and mitochondria in the living optic nerve head (ONH) in an ex vivo environment. Two transgenic mouse strains were used, one expressing yellow fluorescent protein in selected axons and a second strain expressing cyan fluorescent protein in all mitochondria. We viewed an explanted mouse eye and optic nerve by laser scanning microscopy at and behind the ONH, the site of glaucoma injury. Explants from previously untreated mice were studied with the intraocular pressure (IOP) set artificially at normal or elevated levels for several hours. Explants were also studied from eyes that had undergone chronic IOP elevation from 14 hours to 6 weeks prior to ex vivo study. Image analysis in static images and video of individual mitochondria or axonal structure determined effects of acute and chronic IOP elevation. At normal IOP, fluorescent axonal structure was stable for up to 3 hours under ex vivo conditions. After chronic IOP elevation, axonal integrity index values indicated fragmentation of axon structure in the ONH. In mice with fluorescent mitochondria, the normal density decreased with distance behind the ONH by 45% (p=0.002, t-test). Density increased with prior chronic IOP elevation to 21,300 ± 4,176 mitochondria/mm2 compared to control 16,110 ± 3159 mitochondria/mm2 (p=0.025, t-test), but did not increase significantly after 4 hours, acute IOP elevation (1.5% decrease in density, p=0.83, t-test). Mean normal mitochondrial length of 2.3 ± 1.4 μm became 13% smaller after 4 hours of IOP elevation ex vivo compared to baseline (p=0.015, t-test, N-10). Normal mitochondrial speed of movement was significantly slower in the anterograde direction (towards the brain) than retrograde, but there were more mitochondria in motion and traveling longer lengths in anterograde direction. The percent of mitochondria in motion decreased by >50% with acute IOP increase to 30 mm Hg after 60 minutes. A new ocular explant model implemented with eyes from transgenic mice with fluorescent cellular components provided real time measurement of the early events in experimental glaucoma and quantitative outcomes for neuroprotection therapy experiments.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

Kimball

Pease

Steinhart

et al. 2017

Experimental Eye Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Of note, Thy1-YFP mice have recently been used to develop an organotypic culture model where RGCs are fluorescently labeled. This line provides a useful tool for time-lapse imaging of the outgrowing neurites (Johnson et al 2016). Retinal explants can be harvested from embryonic, postnatal or adult eyes, whether or not primed by optic nerve injury.…”

Section: Ex Vivo Tissue Explant Studies In Rodentsmentioning

confidence: 99%

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

et al. 2017

View full text Add to dashboard Cite

Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient's life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.

show abstract

“…While retinal explants from adult animals of a variety of species including mouse, rat and zebrafish have been utilized for different cell biological approaches 17–20 , live-cell imaging experiments using retinal explants have been restricted to brief periods of time and have not been executed continuously over several hours. 21, 22 Here, we describe a detailed protocol to culture light-damaged adult zebrafish retinas to perform live-cell imaging experiments monitoring INM using multi-photon microscopy 6 . Live-cell imaging approaches are advantageous over immunohistochemical methods when investigating the mechanisms controlling INM, as the dynamics of INM e.g.…”

Section: Introductionmentioning

confidence: 99%

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Lahne

Gorsuch

Nelson

et al. 2017

JoVE

View full text Add to dashboard Cite

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

show abstract

Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture

Cited by 27 publications

References 46 publications

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Contact Info

Product

Resources

About