Abstract
Purpose :
Intraepithelial corneal basal nerves (ICBNs) are captured by in vivo confocal microscopy; however, this mode of visualization lacks resolution. Corneas, immunostained and imaged ex vivo solve this problem, however comprehensive analyses of nerve parameters is sub-optimal. This study compared current methods of analyzing intraepithelial ICBNs to a new noise-based segmentation (NoBS) approach in mouse models of aging (n=49), wound-healing (n=32), and limbal stem cell deficiency (n=32).
Methods :
For aging, eyes were collected from 2-52 week-old normal C57BL/6 mice (n≥4/time-point). For wound-healing and limbal stem cell deficiency, 7 week-old mice received a limbal-sparing or limbal-to-limbal epithelial debridement to their right cornea, respectively. Eyes were collected 2-16 weeks post-injury (n=4/group/time-point), corneas procured, immunolabelled with βIII-tubulin, flat-mounted and imaged by scanning confocal microscopy.
Results :
A subset of images (n=15) from each model was analyzed for ICBN density using various analytical platforms, and an agreement to manual-tracing (gold-standard) was established for all methods. NoBS had the strongest Pearson’s correlation (r=0.97), followed by manual-counting (r=0.93), manual-thresholding (r=0.89), Imaris-bitplane (r=0.57) and automated-thresholding with ImageJ (r=0.18). NoBS was used to compute all measurements as percentage (%) area occupied by nerves. Between 3-12 weeks of age, ICBN density increased from 5.4% to 17.8% centrally (p<0.01) and 13.0% to 24.9% peripherally (p<0.01), then by 20-weeks (and beyond) decreased to 10.5% centrally (p<0.01) and 18.3% peripherally (p<0.01). During wound-healing, ICBN density was gradually restored, and at 8-weeks reached control levels in the centre (p>0.1) and periphery (p>0.1) but failed to reform the central whorl even after 16-weeks. In limbal stem cell deficient mice, ICBNs regeneration was observed over time and was not statistically different to controls in the centre or periphery at 16-weeks (p>0.1), however the curvilinear pattern that characterizes ICBNs in normal corneas, was not restored.
Conclusions :
Our new analytical modality affords the potential to rapidly and reliably detect microstructural changes to ICBNs. This will enable the refinement of non-invasive diagnostic and surveillance tests for patients with ocular and/or systemic diseases.
This is a 2020 ARVO Annual Meeting abstract.