HISTOLOGIC ANALYSIS OF THERMAL EFFECTS OF LASER THERMOKERATOPLASTY AND CORNEAL ABLATION USING SIRIUS-RED POLARIZATION MICROSCOPY

Purpose: To evaluate how well several histologic techniques differenti ate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. Setting: Medical Laser Center Lubeck, Germany. Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 mu m) and a pulsed chromium-thulium-holmium:YAG laser (2.1 mu m) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbiu m:YSGG laser (2.70 mu m), a free-running erbium:YAG laser (2.94 mu m), and an argon-fluoride excimer laser (193 nm). The lesions were evalua ted by tight microscopy (LM) (hematoxylin and eosin, Azan, van Gieson' s, and Masson-Goldner's trichrome stains), transmission electron micro scopy (TEM), and polarization microscopy after Sirius-red staining. Si rius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. Res ults: 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 ce ntral zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new bindi ng sites. The central zone appeared darker under polarization than nor mal collagen because of a loss of birefringence. Intrinsic birefringen ce was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very stro ng lesions, where the collagen was hyalinized, birefringence was compl etely lost because of the complete disintegration of the fibrillar str ucture, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringen ce was largely preserved and enhanced by the increased Sirius-red upta ke. Mechanical stretching between neighboring LTK lesions was manifest ed by increased birefringence. Conclusion: Sirius red offered an impro ved and simple histologic method for analyzing thermal collagen change s. It may contribute to a better understanding of the working mechanis ms of LTK and improve analysis of thermal effects in corneal ablation.