Ex vivo multiphoton analysis of rabbit corneal wound healing following conductive keratoplasty

Abstract

Ex vivo multiphoton imaging is used to characterize rabbit corneal wound healing after conductive keratoplasty (CK) procedures. CK is performed on the right eyes from eight New Zealand albino rabbits while the left eyes are punctured by a keratoplast tip without energy application. Rabbits are humanely sacrificed 1 day, 1, 2, and 4 weeks after the CK procedure. Eye balls are enucleated and placed on the microscope for multiphoton imaging. Multiphoton imaging reveals damage of corneal epithelium and stroma caused by the CK procedure and the subsequent wound healing process can be followed without histological procedures. Multiphoton excited autofluorescence images demonstrate that re-epithelilialization is accomplished within 1 week in both CK and control groups. However, epithelial hyperplasia is observed in CK corneas. In addition, stromal wounds in the control group become inconspicuous within 1 week while obvious wounds still exist in CK corneas for at least 4 weeks. Postconductive keratoplasty corneal damage and wound healing can be characterized by multiphoton microscopy without histological procedures. Our results suggest that multiphoton microscopy has potential in the clinical evaluation of corneal damage due to refractive surgery, and can be used to study and reduce the unwanted side effects of these procedures.

Second harmonic generation microscopy: principles and applications to disease diagnosis

“…Recently Pavone and coworkers showed a new method to quantify the similar resulting disorder in the collagen structure in the cornea following laser welding [63]. Collectively these results demonstrate the feasibility of using SHG/MAF imaging in probing the effects of refractive surgical procedures and provide insights into the subsequent wound healing process [64].…”

Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

“…The flap creation is performed by fs lasers with low repetition rate (in the range from Hz to kHz) by inducing photodisruption and destructive optical breakdown in the more external corneal tissue. The use of fs lasers in conductive keratoplasty has also a great potential in clinical environments [21,22,42]. The main advantage originates from the feasibility for precise cutting of any arbitrary geometry (zig-zag, top hat patterns,…) on both the donor and the recipient eye, increasing resistance and accelerating the healing process.…”

Second Harmonic Generation Microscopy: A Tool for Quantitative Analysis of Tissues

“…Although images showed different information, collagen fibers always showed a regular packaging [36,37]. However, this regular pattern has been shown to change with pathologies [40,41] or after surgical procedures [42][43][44]. The corneal stroma also suffers alterations due to scars [45] or changes in the intraocular pressure [46] that have been explored through SHG imaging.…”