Effect of thermal preconditioning before excimer laser photoablation

Abstract

The purposes of this study were to assess the expression patterns of heat shock proteins (Hsps), after eyeball heating or cooling, and to elucidate their relationships with corneal wound healing and intraocular complications after excimer laser treatment. Experimental mice were grouped into three according to local pretreatment type: heating, cooling, and control groups. The preconditioning was to apply saline eyedrops onto the cornea prior to photoablation. Following photoablation, we evaluated corneal wound healing, corneal opacity and lens opacity. Hsp expression patterns were elucidated with Western blot and immunohistochemical staining. The heating and cooling groups recovered more rapidly, and showed less corneal and lens opacity than the control group. In the heating and cooling groups, there were more expressions of Hsps in the cornea and lens than in the control group. These results were confirmed in the Hsp 70.1 knockout mouse model. Our study showed that Hsps were induced by the heating or cooling preconditioning, and appeared to be a major factor in protecting the cornea against serious thermal damage. Induced Hsps also seemed to play an important role in rapid wound healing, and decreased corneal and lens opacity after excimer laser ablation.

Fig. 1

Corneal wound after laser treatment by time interval. At 72 hr after laser treatment, in Group H (A-C), the wounds are completely healed with no visible fluorescein-stained area. In Group C (D-F), the wounds are largely healed, but small wounds remain. In Group N (G-I), and the knockout mice (J-L), large, incompletely healed wounds remain.

Fig. 2

Corneal opacity (two weeks after laser treatment). The pictures above are representative cases of Grade 0.5 (A), Grade 1 (B), and Grade.

Fig. 3

Lens opacity. Biomicroscopic pictures of mouse lenses, two weeks after excimer laser application. The pictures above are representative cases of Grade 0 (A), Grade 1 (B), Grade 2 (C), Grade 3 (D), Grade 4 (E), and the knockout mice (Grade 4 (F)).

Fig. 4

Western blot. In heating group (A), Hsp 70 is seen normally but increased progressively by 3 hr after heating and peak at 12 hr and then decreased until 24 hr. And Hsp 47 is not visible in normal cornea. Three hours after heat treatment, however, Hsp 47 is at the peak level and then decreased progressively. In cooling group (B), Hsp 70 is seen normally but increased progressively at 3 hr after cooling and maintained this level at 24 hr. And Hsp 47 is not visible at normal cornea, however, 12 hr after cooling, small amount of Hsp 47 is seen and reached the peak level at 24 hr. The resulting values (C, D) are obtained by densitometry.

Fig. 5

Immunohistochemical localization of Hsps in the cornea and lens after excimer laser photoablation. Hsp 25 in the control group shows weak expression in keratocytes (A) and no expression in lens epithelium (data not shown), but shows increased expression in keratocytes (arrow) of the limbus (B) and the lens epithelium (arrow) at 12 hr after heating (C). Although Hsp 45 is normally expressed faintly in fibroblasts of the limbus, it was not expressed in the central cornea and lens epithelium of the control group (data not shown). Hsp 45 is expressed strongly in both the central cornea and limbus 24 hr after cooling (D) and 6 hr after heating (E). Hsp 45 is expressed strongly in both the central cornea and limbus 24 hr after cooling (D) and 6 hr after heating (E), however, is also expressed in the lens epithelium (arrow) (F). Hsp70 is normally expressed in keratocytes of the limbus and endothelium (data not shown), and increased in overall level of expression 12 hr after heating (G) and cooling (H).