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

Area of Science:

• Ophthalmology
• Cell Biology
• Biomaterials Science

Background:

• Keratocytes are crucial for corneal structure and collagen production.
• Understanding factors influencing keratocyte collagen synthesis is vital for corneal wound healing research.
• Mitomycin C (MMC) and transforming growth factor-beta (TGF-β1) are known modulators of cellular processes.

Purpose of the Study:

• To quantitatively analyze collagen production by rabbit keratocytes treated with MMC and/or TGF-β1.
• To investigate the in situ collagen arrangement using second harmonic generation (SHG) microscopy.

Main Methods:

• Rabbit keratocytes were divided into four groups: control, TGF-β1, MMC, and TGF-β1-MMC.
• Cells were treated with specific agents (MMC 0.02 mg/ml for 5 min, TGF-β1 2 ng/ml).
• Collagen content and cell numbers were measured using SHG microscopy and MetaMorph Offline at 2 and 4 weeks.

Main Results:

• SHG microscopy clearly visualized collagen arrangement.
• TGF-β1 treatment significantly increased keratocyte numbers at 2 and 4 weeks.
• MMC treatment resulted in the least collagen content at 2 weeks, while TGF-β1 showed higher collagen content at 4 weeks compared to control and MMC groups.

Conclusions:

• SHG microscopy is a sensitive tool for quantitative collagen analysis and in situ arrangement studies.
• TGF-β1 promotes keratocyte proliferation and collagen deposition.
• MMC inhibits collagen production in rabbit keratocytes.