Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Abstract

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.

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

“…This categorization can be used to study the effects of compounds on axonal length, by comparing the immunodetected neurite area of the outer segments, which are comprised solely of long axons, to that of the inner segments, which include all axons (Gaublomme et al . ). Both INA and NOA measures were first normalized for explant size by dividing by the perimeter of the 4′,6‐diamidino‐2‐phenylindole‐stained explants, and normalized further toward an average value of 100% for all control explants per experiment.…”

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

“…Our research group recently established a postnatal murine retinal explant model to study axonal outgrowth (Gaublomme et al 2013;Buyens et al 2014; Van de Velde et al 2015;Van Hove et al 2015). This model is based on a previously described rat explant model, in which retinas from rat pups of 10 days old (postnatal day 10, P10) were used and neurite outgrowth was quantified after 3 days in culture (Lagreze et al 2005).…”

“…1), conform previous reports (Goldberg et al 2002), we have used retinal explants from mice at day of birth (P0) or P3, to obtain a better and more consistent neurite outgrowth profile. Axonal outgrowth from explants can easily be visualized via immunostaining for the axonal marker β-tubulin, and automatically quantified as previously described (Gaublomme et al 2013; Van de Velde et al Mouse retinal explants were dissected at postnatal day 0 (P0, day of birth) (a), P3 (b) and P7 (c) and their neurite outgrowth was quantified after 72 h using a β-tubulin immunostaining. P0 retinal explants showed significantly more neurite outgrowth as compared to explants harvested at P3 and P7.…”

“…These scripts allow for fast detection of neurite outgrowth, with the possibility to distinguish between neurite outgrowth initiation and neurite elongation by dividing the neurite outgrowth area into four segments. Of note, it is also worthwhile to visualize glial processes extending from the retinal explants via immunolabeling for glial fibrillary acidic protein (GFAP) (Gaublomme et al 2013). As mentioned above, the expression of growth factors and guidance molecules by activated retinal glia highly contributes to RGC axonal lengthening (Muller et al 2007;Lorber et al 2009Lorber et al , 2012.…”

Short-term Alteration of Developmental Neural Activity Enhances Neurite Outgrowth of Retinal Explants

“…Note that the neurite area < 100 lm can be considered as a measure of neurite density, whereas the neurite area > 200 lm represents the amount of elongated axons. A similar approach was used by Gaublomme et al 28 All image analyses were performed using ImageJ software (http:// imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).…”