Francisco Bandeira1,2,3, Gary Hin-Fai Yam1,4,5, Yu-Chi Liu1,4,6, Kavya Devarajan1, Jodhbir S Mehta1,4,6,7,8. 1. Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore. 2. Federal University of São Paulo, Sao Paulo, Brazil. 3. São Gonçalo Eye Hospital, Rio de Janeiro, Brazil. 4. Eye-Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore. 5. Experimental Microscopy Platform, Singapore Eye Research Institute, Singapore. 6. Singapore National Eye Centre, Singapore. 7. Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 8. School of Material Science and Engineering, Nanyang Technological University, Singapore.
Abstract
Purpose: 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). Methods: 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. Results: 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. Conclusions: 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.
Purpose: 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). Methods: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. Results: 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. Conclusions: 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.
Authors: Martina Nemcokova; Jakub Dite; Yun Min Klimesova; Magdalena Netukova; Pavel Studeny Journal: Cell Tissue Bank Date: 2022-02-06 Impact factor: 1.522
Authors: Gary Hin-Fai Yam; Francisco Bandeira; Yu-Chi Liu; Kavya Devarajan; Nur Zahirah Binte M Yusoff; Hla-Myint Htoon; Jodhbir S Mehta Journal: J Adv Res Date: 2021-09-16 Impact factor: 12.822