Quantitative Analysis of Collagen Produced by Rabbit Keratocytes using Second Harmonic Generation Microscopy

ABSTRACT

Purpose: To quantitatively analyze the collagen produced by rabbit keratocytes treated with mitomycin C (MMC) and/or transforming growth factor-beta (TGF-β₁), and to investigate the collagen arrangement in situ, using second harmonic generation (SHG) microscopy.

Methods: Keratocytes were divided into four groups: The keraotocytes in group rabbit keratocytes were treated with serum free medium containing 1% methylcellulose as controls. The cells in group TGF were received with TGF-β₁ (at 2 ng/ml) and the same medium as the control. After exposure to MMC at 0.02 mg/ml for 5 minutes, the cells in group MMC were received with the same treatment as controls. In group TGF-MMC, the cells were exposed to MMC at 0.02 mg/ml for 5 minutes first, and then treated as group TGF. The signals of collagen were detected by SHG at 2 and 4 weeks post-treatment. The collagen content, as well as the keratocyte numbers, was calculated by MetaMorph Offline.

Results: The collagen arrangement could be detected clearly by SHG. The cell number of group TGF was higher than other groups at 2 and 4 weeks post-treatment. The collagen content in group MMC was the least among all the groups at 2 weeks, while the collagen content in group TGF was higher than in group rabbit keratocytes and MMC at 4 weeks. No significant SHG side effects were found in cell proliferation and collagen deposition.

Conclusions: SHG can provide a highly sensitive method to quantitatively measure collagen content, as well as to analyze its arrangement in situ.

KEYWORDS:

• Collagen
• cornea
• keratocytes
• TGF
• wound healing

Declaration of interest

The authors report no conflicts of interest.

Funding

This research was supported by grants from the National Natural Science Foundation of China (Grant No. 81371039 and 81302683) and grants from the Natural Science Foundation of Shanghai (Grant No. 13ZR1459700). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Additional information

Funding

This research was supported by grants from the National Natural Science Foundation of China (Grant No. 81371039 and 81302683) and grants from the Natural Science Foundation of Shanghai (Grant No. 13ZR1459700). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.