A Propagated Skeleton Approach to High Throughput Screening of Neurite Outgrowth for In Vitro Parkinson’s Disease Modelling

Abstract

Neuronal models of neurodegenerative diseases such as Parkinson’s Disease (PD) are extensively studied in pathological and therapeutical research with neurite outgrowth being a core feature. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. In this study, we describe an autonomous computational assay for a high throughput skeletonization approach allowing for quantification of neurite outgrowth in large data sets from fluorescence microscopic imaging. Development and validation of the assay was conducted with differentiated SH-SY5Y cells and primary mesencephalic dopaminergic neurons (MDN) treated with the neurotoxic lesioning compound Rotenone. Results of manual annotation using NeuronJ and automated data were shown to correlate strongly (R2-value 0.9077 for SH-SY5Y cells and R2-value 0.9297 for MDN). Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula (y=0.5410·x+1792; y=0.8789·x+0.09191 for normalized results) with y depicting automated and x depicting manual data. This automated neurite length algorithm constitutes a valuable tool for modelling of neurite outgrowth that can be easily applied to evaluate therapeutic compounds with high throughput approaches.

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

“…Addition of a chemical stimulus after differentiation of the SH-SY5Y cell line has been previously reported in the literature 13 . In our study, neurotrophic stimulation of terminally differentiated cells (Fig.…”

“…A number of studies have differentiated the SH-SY5Y cell line with RA for a short duration of 2–4 days, at which point the cells are then treated with a chemical factor, to evaluate the inhibitory or stimulatory effect on neurite outgrowth over a specified period 13 , 29 , 30 . However, such short differentiation protocols would be insufficient to produce a population of terminally differentiated cells 31 .…”

“…This paper focuses on the human SH-SY5Y cell line, which is a subclone of the parental neuroblastoma SK-N-SH cell line. SH-SY5Y cells have been widely employed as an in vitro neuronal model for neurodegeneration 10 , neurotrauma 11 , developmental neurotoxicity 12 and neurite outgrowth 13 . However, they have most commonly been used in an undifferentiated state 14 .…”

“…Such properties include the ability to fire action potentials, express neuronal receptors, and exhibit a dependency on neurotrophic factors 15 , 16 . There are a number of different protocols reported in the literature to differentiate SH-SY5Y cells into a neuronal phenotype 13 , 15 – 18 . Depending on the reagents used, these cells can be differentiated to possess dopaminergic, cholinergic or adrenergic properties 3 .…”

Differentiated SH-SY5Y Cells for High-Throughput Screening in a 96-Well Plate Format: Protocol Optimisation and Application for Neurite Outgrowth Assays

“…Such properties include the ability to fire action potentials, express neuronal receptors, and exhibit a dependency on neurotrophic factors 15,16 . There are a number of different protocols reported in the literature to differentiate SH-SY5Y cells into a neuronal phenotype 13,[15][16][17][18] . Depending on the reagents used, these cells can be differentiated to possess dopaminergic, cholinergic or adrenergic properties 3 .…”