Three-Dimensional Neurite Characterization of Small Incision Lenticule Extraction Derived Lenticules.

Abstract

We study the density and excitatory response of neurites, and Schwann cells (SCs) in fresh and cryopreserved stromal lenticules derived from small incision lenticule extraction (SMILE). Human stromal lenticules (n = 23) were immunostained for β III-tubulin and imaged using spinning disk confocal laser microscopy, followed by three-dimensional reconstruction, to reveal neurite distribution. The lenticule neurite density (LND) was assessed using a validated neurite tracing and length measurement method with NeuronJ. LND was compared among groups of different lenticule thickness (71-165 μm) obtained from -3 to >-6 diopters (D) corrections. SCs were identified by marker expression and the laser effect on SC-neurite interaction was examined under transmission electron microscopy (TEM). Fresh porcine SMILE-lenticules (n = 18) were used for LND comparison among storage conditions and functional excitatory calcium response assay. Using a validated neurite length measurement method, we found an inverse correlation of LND with lenticule thickness. Higher LND was found in thinner lenticules obtained from lower power of correction (r = -0.8925, P < 0.0001), whereas total lenticule neurite lengths did not alter significantly with regards to lenticule thickness. SCs were identified by GAP43 and p75NTR expression and were closely associated with lenticule neurites under TEM. In porcine lenticules, LND and excitatory calcium response were reduced after cold and cryogenic storage, when compared to fresh lenticules. The stromal neurites showed variations in density related to SMILE lenticule thickness and cryopreservation. With the presence of SC support and excitatory response, these neurite residues could retain minimal functionality that might serve as a potential advantage in the event of lenticule implantation.