Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy

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Abstract
Purpose: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. Setting: Medical Laser Center Lübeck, Germany. Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 μm) and a pulsed chromium-thulium-holmium:YAG laser (2.1 μm) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbium:YSGG laser (2.70 μm), a free-running erbium:YAG laser (2.94 μm), and an argon-fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson's, and Masson-Goldner's trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius-red staining. Sirius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. Results: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius-red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius-red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence. Conclusion: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.