Journal of Advanced Research

Journal of Advanced Research

Volume 38, May 2022, Pages 275-284
Journal of Advanced Research

Effect of corneal stromal lenticule customization on neurite distribution and excitatory property

https://doi.org/10.1016/j.jare.2021.09.004Get rights and content
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Highlights

  • Refractive SMILE-derived stromal lenticules are useful in various tissue-engineering approach for therapeutics, of which they are required to be customized before implantation.

  • Excimer laser-mediated reshaping, riboflavin-UVA-induced collagen crosslinking and chemical decellularization significantly removed lenticule neurites, but the residual neurites retained excitatory response.

  • Reinnervation occurred in the decellularized lenticules, indicating a potential of nerve regeneration.

  • Stromal lenticules, as a unique collagen-rich biomaterial with high transparency, refractivity and mechanically robust, together with the ability of neurite regeneration, could hold a potential for various ophthalmic applications.

Abstract

Introduction

Refractive stromal lenticules from Small Incision Lenticule Extraction (SMILE), though usually discarded, hold a potential for various ophthalmic applications, including refractive correction, stromal volume expansion, and biomechanical strengthening of the cornea.

Objectives

To investigate the effect of lenticule customization on lenticule neurite length profile and the excitatory response (calcium signaling) and the potential of reinnervation.

Methods

Human and porcine stromal lenticules were treated by (1) excimer laser reshaping, (2) ultraviolet A-riboflavin crosslinking (CXL), and (3) decellularization by sodium dodecyl sulfate (SDS), respectively. The overall neurite scaffold immuno-positive to TuJ1 (neuron-specific class III β-tubulin) expression and population of active neurite fragments with calcium response revealed by L-glutamate-induced Fluo-4-acetoxymethyl ester reaction were captured by wide-field laser-scanning confocal microscopy, followed by z-stack image construction. The NeuronJ plugin was used to measure neurite lengths for TuJ1 (NL-TuJ1) and calcium signal (NL-Ca). Reinnervation of lenticules was examined by the ex vivo grafting of chick dorsal root ganglia (DRG) to the decellularized human lenticules. Differences between groups and controls were analyzed with ANOVA and Mann-Whitney U test.

Results

The customization methods significantly eliminated neurites inside the lenticules. NL-TuJ1 was significantly reduced by 84% after excimer laser reshaping, 54% after CXL, and 96% after decellularization. The neurite remnants from reshaping and CXL exhibited calcium signaling, indicative of residual excitatory response. Re-innervation occurred in the decellularized lenticules upon stimulation of the grafted chick embryo DRG with nerve growth factor (NGF 2.5S).

Conclusion

All of the lenticule customization procedures reduced lenticule neurites, but the residual neurites still showed excitatory potential. Even though these neurite remnants seemed minimal, they could be advantageous to reinnervation with axon growth and guidance after lenticule reimplantation for refractive and volume restoration of the cornea.

Graphical abstract

A Schematic overview of this study from the SMILE lenticule creation and extraction to lenticule customization: excimer laser reshaping/thinning, ultraviolet A (UVA)-riboflavin crosslinking and sodium dodecyl sulfate (SDS) decellularization, as well as the reinnervation of customized lenticules. Stromal neurites in lenticules (black color) sand regenerating neuritis (red color).

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Keywords

Corneal refractive surgery
SMILE
Stromal lenticules
Customization
Neurite profile
Excitatory response

Cited by (0)

Peer review under responsibility of Cairo University.

1

Yam GH and Bandeira F had an equal contribution to this work.