A semi-automated image analysis method to quantify neurite preference/axon guidance on a patterned substratum.

Axon outgrowth and guidance are differentially promoted or inhibited by specific extracellular matrix (ECM) molecules. The effects of these molecules can be examined by culturing neuronal explants on patterned substrata consisting of alternating stripes adsorbed with the molecules of interest. While outgrowth on substrata adsorbed with homogenous molecules can be reliably quantified, current methods of quantifying neurite preference on patterned substrata are subjective, labor intensive, and overall less reliable. Here, we present a quick, semi-automated, lowly subjective macro-based method to quantify the effects of a change in substratum on axon extension and guidance. We plated chick dorsal root ganglion explants on a substratum consisting of alternating stripes of laminin-1 (outgrowth supportive) and chondroitin sulfate proteoglycans (CSPGs, outgrowth inhibitory). We evaluated neurite preference for laminin or CSPG-coated regions by measuring total neurite area, and produced an inhibition index. The quantitative data confirmed previous qualitative data showing that increasing concentrations of CSPGs induced increases in inhibition. The methods presented here: (1) require less stringent image capture criteria; (2) are quicker; (3) are less subjective compared to previously described methods; and (4) are versatile in that they can be used to assay neurite preference for any substratum-bound molecules in living or fixed cultures.