ExplantAnalyzer: An advanced automated neurite outgrowth analysis evaluated by means of organotypic auditory neuron explant cultures

Dominik Schmidbauer,Stefan Fink,Francis Rousset,Pascal Senn,Marcus Müller,Youssef Adel,Rudolf Glueckert

doi:10.1016/j.jneumeth.2021.109341

Abstract

BackgroundNeuronal outgrowth assays using organotypic explant cultures are commonly utilized to study neuroregenerative and -protective effects of drugs such as neurotrophins. While this approach offers higher organized tissue compared to single cell cultures and less experimental effort than in-vivo studies, quantitative evaluation of the neuronal network is often time consuming. Thus, we developed ExplantAnlayzer, a time-saving high-throughput evaluation method, yielding numerous metrics to objectively describe neuronal outgrowth. New methodSpiral ganglion explants were cultured in 24-well plates, mechanically fixed in a collagen matrix and immunolabeled against beta-III-tubulin. The explants were imaged using a fluorescent tile-scan microscope and resulting images were stitched. The evaluation was developed as an open-source MATLAB routine and involves several image processing steps, including adaptive thresholding. The neurite network was eventually converted to a graph to track neurites from their terminals back to the explant body. Comparison with existing method(s)We compared ExplantAnlayzer quantitatively and qualitatively to common existing methods, such as Sholl analyses and manual fiber tracing, using representative explant images. ExplantAnlayzer is able to achieve similar and as detailed results as manual tracing while decreasing manual interaction and required time dramatically. ResultsAfter an initial setup phase, the explant images could be batch-processed altogether. Bright bundles as well as faint fibers were reliably detected. Several metrics describing the outgrowth morphology, including total outgrowth, neurite numbers and length estimations, as well as their growth directions, were computed. ConclusionsExplantAnalyzer is a time-saving and objective method for an in-depth evaluation of organotypic explant outgrowth.

Highlights

IntroductionOne of the applications of organotypic cultures is the study of nerve regeneration and -growth
In organotypic cultures, primary ex-vivo tissue is used to address numerous scientific questions
Organotypic explant cultures present a valuable balance of these factors, as differentiated tissue can be cultured in its local cellular environment while external conditions can be pre cisely controlled (Gahwiler, 1988)

Summary

Introduction

One of the applications of organotypic cultures is the study of nerve regeneration and -growth Tissues used for these studies include dorsal root ganglia (Gomes et al, 2016; Kim et al, 2021; Livni et al, 2019), spinal cord slices (Romeo-Guitart et al, 2020; Santos et al, 2016), the spiral ganglion (Sun et al, 2020; Szobota et al, 2019) and retinal gan glion cells (Binley et al, 2016; Schaeffer et al, 2020). Neuronal outgrowth assays using organotypic explant cultures are commonly utilized to study neuroregenerative and -protective effects of drugs such as neurotrophins While this approach offers higher organized tissue compared to single cell cultures and less experimental effort than in-vivo studies, quantitative evaluation of the neuronal network is often time consuming. Conclusions: ExplantAnalyzer is a time-saving and objective method for an in-depth evaluation of organotypic explant outgrowth

Methods

Results

Discussion

Conclusion