We applied multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) microscopy to monitor corneal wound healing after photorefractive keratectomy (PRK). Our results show that keratocyte activation can be observed by an increase in its MAF, while SHG imaging of corneal stroma can show the depletion of Bowman's layer after PRK and the reticular collagen deposition in the wound healing stage. Furthermore, quantification of the keratocyte activation and collagen deposition in conjunction with immunohistochemistry and histological images demonstrate the effectiveness of mitomycin C (MMC) in suppressing myofibroblast proliferation and collagen regeneration in the post-PRK wound healing process.
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.
The aim of this study is to assess the application of multiphoton autofluorescence and second harmonic generation (SHG) microscopy for investigating corneal wound healing after high myopic (-10.0D) photorefractive keratectomy (PRK) procedures on the rabbit eyes. The effect of PRK on the morphology and distribution of keratocytes were investigated using multiphoton excited autofluorescence imaging, while the effect of PRK on the arrangement of collagen fibers was monitored by second-harmonic generation imaging. Without histological processing, multiphoton microscopy is able to characterize corneal damage and wound healing from PRK. Our results show that this technique has potential application in the clinical evaluation of corneal damage due to refractive surgery, and may be used to study the unwanted side effects of these procedures.
Conductive keratoplasty (CK) is a new refractive surgery for presbyopia and hyperopia patients. By applying radio
frequency current at the peripheral regions of cornea, collagen, the most abundant composition of corneal stroma,
shrinks due to the heat generated. The shrinkage at the periphery alters the corneal architecture and achieves clearer
focus for near vision.
In this work we use multiphoton microscopy to observe the post surgery structure variation at both submicron resolution
and over a large region within the tissue. Since collagen can be induced to generate strong second harmonic generation
(SHG) signal, multiphoton excitation provide direct visualization of collagen orientation within corneal stroma. In
addition, since the SHG intensity of collagen tissue deteriorates with increasing thermal damage [1-3], our methodology
can be used to characterize the extent of corneal stroma damage from the CK procedure. Finally, the influence of CK on
the morphology and distribution of keratocytes can also be investigated by detecting multiphoton excited
autofluorescence from the cells.
In this work we use multiphoton microscopy to observe the post surgery structure variation of rabbit cornea after
photorefractive keratectomy (PRK). In addition, we added mitomycin C (MMC) to the post surgery rabbit cornea in
order to investigate the effect of MMC treatment on the postoperative regeneration.