Page 103 - Live-cellanalysis handbook

Kinetic Neurite Analysis Assays

Quantification and visualization of neurite
outgrowth, disruption and cell health

Introduction

Neurite dynamics are central to the study of neuropathological temporal insight, but require large cell numbers and are subject
disorders, neuronal injury and regeneration, embryonic to further artifacts caused by differences in cell plating or other
development and neuronal differentiation. During these processes, variable culture conditions.
the integrity of neuronal networks is slowly altered, accompanied
by changes in neurite outgrowth and disruption that may lead Continuous real-time imaging and analysis offers a significant
to impaired neuronal connectivity. Characterizing the dynamic advantage over end-point assays in that it provides a more
changes of neurites in vitro, and how they interact with other physiologically relevant and dynamic approach to visualizing and
cells or environmental stimuli, can be invaluable for optimizing analyzing neurite outgrowth and disruption. It is achieved using
in vitro models, identifying diseased cells from a heterogeneous non-destructive, cell-sparing, repeated measurements of the same
population, studying phenotypic effects from genetic neuronal networks over extended periods of time (days or weeks),
manipulation, or performing drug pharmacology studies. without perturbing cell health or delicate neurite structures.
Conducting experiments at these time scales offers a significant
Traditional in vitro approaches for analyzing neuronal structures advantage when investigating chronic toxicity, as toxic effects
rely on endpoint assays and imaging techniques that require develop unpredictably over time.
immunochemical staining. Neuronal cultures are exposed to
a treatment condition, fixed and stained at a pre-determined In this chapter we will examine kinetic approaches for measuring
time post-treatment, and then imaged using either high content changes in neurite outgrowth or disruption in both mono- and
imaging or traditional fluorescent microscopes. Images are then co-culture cell models. Cultures can be measured and visually
analyzed to generate measurements of neurite length and branch validated for days, weeks, or even months, and at microplate
points. Although this method has been an important tool in throughput, making these approaches particularly well-suited for
neurobiology, the process is labor-intensive, perturbing to fragile the optimization and analysis of neuronal cell models as well as
neurites, and produces only a single time point of data. In sum, for conducting pharmacological studies and investigations of drug
the approach lacks temporal insight and is subject to artifact. mechanisms of action.
Concatenated endpoints may be utilized to address the lack of

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