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ExplantAnalyzer: An advanced automated neurite outgrowth analysis evaluated by means of organotypic auditory neuron explant cultures
Journal of Neuroscience Methods ( IF 3 ) Pub Date : 2021-08-31 , DOI: 10.1016/j.jneumeth.2021.109341
Dominik Schmidbauer 1 , Stefan Fink 2 , Francis Rousset 3 , Pascal Senn 4 , Marcus Müller 2 , Youssef Adel 2 , Rudolf Glueckert 1
Affiliation  

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.



中文翻译:

ExplantAnalyzer:一种先进的自动化神经突生长分析,通过器官型听觉神经元外植体培养物进行评估

背景

使用器官型外植体培养物的神经元生长测定通常用于研究神经营养因子等药物的神经再生和保护作用。虽然与单细胞培养相比,这种方法提供了更高的组织组织,并且比体内研究更少的实验工作,但神经元网络的定量评估通常很耗时。因此,我们开发了ExplantAnlayzer,这是一种省时的高通量评估方法,产生了许多指标来客观地描述神经元的生长。

新方法

螺旋神经节外植体在 24 孔板中培养,机械固定在胶原基质中并针对 β-III-微管蛋白进行免疫标记。使用荧光平铺扫描显微镜对外植体进行成像,并将所得图像缝合。该评估是作为开源MATLAB例程开发的,涉及多个图像处理步骤,包括自适应阈值。神经突网络最终被转换为图表,以跟踪神经突从它们的末端返回到外植体。

与现有方法的比较

我们使用具有代表性的外植体图像将ExplantAnlayzer与常见的现有方法(例如 Sholl 分析和手动纤维追踪)进行了定量和定性比较。ExplantAnlayzer能够实现与手动跟踪类似且详细的结果,同时显着减少手动交互和所需时间。

结果

在初始设置阶段之后,外植体图像可以完全进行批处理。可靠地检测到明亮的束以及微弱的纤维。计算了描述生长形态的几个指标,包括总生长、神经突数量和长度估计,以及它们的生长方向。

结论

ExplantAnalyzer是一种用于深入评估器官型外植体生长的既省时又客观的方法。

更新日期:2021-09-16
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