Hydrocortisone Stimulates Neurite Outgrowth from Mouse Retinal Explants by Modulating Macroglial Activity

Toops, Kimberly A.; Berlinicke, Cynthia; Zack, Donald J.; Nickells, Robert W.

doi:10.1167/iovs.11-8646

Cited by 13 publications

(16 citation statements)

References 97 publications

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“…Analysis was performed using two commonly used and previously published manual methods 13,22 and a newly developed automated approach, described below and depicted in Figure 1.…”

Section: Microscopy and Morphometric Analysismentioning

confidence: 99%

“…Then, the overlapping signal between the neurite picture (9) and the dilated explant pictures (10-12) is selected, yielding pictures with neurites extending from the explant to the edge of various segments (13)(14)(15). Finally, from these resulting pictures (13)(14)(15) and from the total neurite picture (9), the neurite area is measured. From these measures, the immunodetected areas per segment are calculated by subtraction.…”

Section: Microscopy and Morphometric Analysismentioning

confidence: 99%

“…Although the total neurite area coverage could be detected with this algorithm, the individual neurite length determination failed because the software assigned every neurite to the closest nucleus. 24 As a consequence of the limited availability of automated software for neurite outgrowth analysis of tissue explants, most researchers resort to manual analysis methods, such as the maximum neurite length method 13,14 and the neurite outgrowth area, 22,23 both mentioned above, or to semiautomatic methods, typical for neurite area detection. 17,25,26 Here, we present an automated script for the analysis of the immunodetected neurite area in cultured explants.…”

Section: Morphometric Analysis Of Neurite Outgrowthmentioning

confidence: 99%

“…8 Retinal explants have often been used to study synaptic organization, 9 retinal differentiation, 10,11 cell-cell interaction, and neurite outgrowth. [12][13][14] In contrast to analysis of cell cultures, which is mostly done automatically, 15,16 analysis of neurite outgrowth from explants is typically performed manually. 13,14 However, this is a very labor-intensive and time-consuming process that is prone to subjectivity and fatigue-related errors, which can be circumvented using automated analyses.…”

Section: Introductionmentioning

confidence: 99%

“…[12][13][14] In contrast to analysis of cell cultures, which is mostly done automatically, 15,16 analysis of neurite outgrowth from explants is typically performed manually. 13,14 However, this is a very labor-intensive and time-consuming process that is prone to subjectivity and fatigue-related errors, which can be circumvented using automated analyses. Previous methods often employed semiautomatic approaches, in which a single gray value is used as a threshold to distinguish the outgrowing neurites from the background.…”

Section: Introductionmentioning

confidence: 99%

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Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

2013

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Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti-β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.

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“…Analysis was performed using two commonly used and previously published manual methods 13,22 and a newly developed automated approach, described below and depicted in Figure 1.…”

Section: Microscopy and Morphometric Analysismentioning

confidence: 99%

Section: Microscopy and Morphometric Analysismentioning

confidence: 99%

Section: Morphometric Analysis Of Neurite Outgrowthmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

2013

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Quantitative Assessment of Neurite Outgrowth in Mouse Retinal Explants

Buyens

Gaublomme

Hove

et al. 2014

Methods in Molecular Biology

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Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system (CNS) remains elusive. The discovery of novel neuro-stimulatory agents that promote regeneration is hampered by a gap between high content analysis platforms using neuronal cells and time-consuming preclinical animal models. In this regard, tissue explant cultures, which are easily manageable and more closely resemble the in vivo situation, form an ideal model system to study the effect of compounds on the neuroglial network. Retinal explants have proven to be a useful tool to investigate the effect of molecules on neuronal survival and regeneration. In this chapter, we report a detailed description of how to isolate and culture retinal explants and how to immunolabel the outgrowing neurites. Furthermore, we describe different analysis tools, both manual and automated, to quantify neurite outgrowth from retinal explants.

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In Vitro and In Vivo Methods for Studying Retinal Ganglion Cell Survival and Optic Nerve Regeneration

Yin

Benowitz

2017

Glaucoma

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Glaucoma is marked by a progressive degeneration of the optic nerve and delayed loss of retinal ganglion cells (RGCs), the projection neurons of the eye. Because RGCs are not replaced and because surviving RGCs cannot regenerate their axons, the visual loss in glaucoma is largely irreversible. Here, we describe methods to evaluate treatments that may be beneficial for treating glaucoma using in vitro cell culture models (immunopanning to isolate neonatal RGCs, dissociated mature retinal neurons, retinal explants) and in vivo models that test potential treatments or investigate underlying molecular mechanisms in an intact system. Potentially, use of these models can help investigators continue to improve treatments to preserve RGCs and restore visual function in patients with glaucoma.

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Hydrocortisone Stimulates Neurite Outgrowth from Mouse Retinal Explants by Modulating Macroglial Activity

Abstract: These data suggest that the HC in EN2 indirectly contributed to neurite outgrowth by activating macroglia to produce neurotrophic and neuroprotective molecules.

Cited by 13 publications

References 97 publications

Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Quantitative Assessment of Neurite Outgrowth in Mouse Retinal Explants

In Vitro and In Vivo Methods for Studying Retinal Ganglion Cell Survival and Optic Nerve Regeneration

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