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Geraint P Williams, Gary HF Yam, Xiao-Wen Lee, Jodhbir S Mehta; Validation and quantification of neurotaxis effect in a chick dorsal root ganglion/corneal stromal cell model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1920.
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© ARVO (1962-2015); The Authors (2016-present)
Nerve regeneration following disease or surgery could be optimised in a microenvironment that mimics the healthy corneal stromal. We developed an in-vitro system to study the effect of corneal stromal cells on nerve outgrowth from chick dorsal root ganglion (DRG). In order to reliably quantify neurite growth, we validated a concentric circle intersection (CCI) quantification assay in a primary DRG culture system, employing stromal cell conditioned medium (CM) as a stimulant to nerve growth.
Primary human corneal stromal cells were obtained from research grade cadaveric corneal stroma under full consent and propagated to P5 in serum culture. After washing of serum factors from culture for 24 hours using serum-free media (SFM), cells were incubated in fresh SFM for 48 hours for medium conditioning. The collected CM was applied to single chick DRG culture, harvested from E10 fetuses. After 72 hours, DRG neurite explants were fixed in situ and TuJ1-positive neurites were imaged. Using CCI assay performed by two masked observers, the intersections of neurite processes with concentric circles at every 100μm interval were quantified as total count per DRG. Agreement was calculated using Bland-Altman plots together with mean difference and level of agreement and by correlation.
Using 12 DRGs incubated with CM, quantification of CCI showed a reduction in neurites with increasing distance from DRG centre, with an average of 44.5 (SD 21.8) intersections at 500um through to 2.3 (SD 4.7) at 1500um. Individual intra-observer agreement was high for both observer 1 and 2 with a mean difference of <1.9 and <1.5 intersections, respectively, and a correlation of 0.96 to 1.0 (p<0.0001), respectively. Inter-observer agreement was also high with a mean difference of <2.8 intersections and a correlation of 0.98 to 1.0 (p<0.0001).
CCI quantification assay measuring DRG neurite extension demonstrated a predictable decline in neurite outgrowth from DRG centre. A masked validation exercise revealed that this means of assessing neurites intersections at defined distance from DRG centre could offer an objective, simple and reliable method for measuring nerve outgrowth ex vivo. This method can be applied to study neurotaxis effect exerted by molecules, such as growth factors, inhibitors, and in co-culture models.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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