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

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

J Neurosci Methods. 2021 Nov 1:363:109341. doi: 10.1016/j.jneumeth.2021.109341. Epub 2021 Aug 31.

Authors

Dominik Schmidbauer¹, Stefan Fink², Francis Rousset³, Pascal Senn⁴, Marcus Müller², Youssef Adel², Rudolf Glueckert¹

Affiliations

¹ Inner Ear Laboratory, Department of Otolaryngology, Medical University of Innsbruck, Innsbruck, Austria.
² Translational Hearing Research, Tübingen Hearing Research Center, Department of Otolaryngology, Head and Neck Surgery, University of Tübingen, Tübingen, Germany.
³ The Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
⁴ The Inner Ear and Olfaction Lab, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Department of Clinical Neurosciences, Service of ORL & Head and Neck Surgery, University Hospital of Geneva, Geneva, Switzerland.

PMID: 34474047
DOI: 10.1016/j.jneumeth.2021.109341

Abstract

Background: 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. Thus, we developed ExplantAnlayzer, a time-saving high-throughput evaluation method, yielding numerous metrics to objectively describe neuronal outgrowth.

New method: Spiral 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.

Results: After 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.

Conclusions: ExplantAnalyzer is a time-saving and objective method for an in-depth evaluation of organotypic explant outgrowth.

Keywords: Automated image analysis; High throughput; MATLAB script; Neurite outgrowth; Organotypic explant culture; Spiral ganglion neuron culture.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cells, Cultured
Nerve Growth Factors
Neurites
Neuronal Outgrowth*
Neurons*

Substances

Nerve Growth Factors

Grants and funding

I 3154/FWF_/Austrian Science Fund FWF/Austria