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.
Animals ; Blotting, Western ; Cornea/pathology ; Heat ; Heat-Shock Proteins/biosynthesis ; Heat-Shock Proteins 70/genetics ; Immunohistochemistry ; Keratectomy, Photorefractive, Excimer Laser/*methods ; *Lasers ; Lens, Crystalline/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Support, Non-U.S. Gov't ; Temperature ; Time Factors ; Wound Healing

The purposes of this study are to elucidate the retinal changes of heat shock protein 70.1 (hsp70.1) knockout mice and to compare them between in normal and in retinal degeneration (rd) mice. Eyes of hsp70.1 wild type (+/+) and knockout (-/-) mice in the C57BL/6 or FVB genetic backgrounds respectively, which were reared in the normal environment, were examined by fundus photography, electroretinography, light microscopy, terminal dUTP nick-end labeling (TUNEL) stain, and immunohistochemistry. In C57BL/6 mice, fundus photography showed no changes between hsp70.1+/+ and -/- mice at 1 and 6 months of age. Electroretinographic examination showed a tendency of decreased amplitude of a- and b-wave with aging in both genotype, but there were not different statistically. The ratios of the thickness of inner nuclear and outer nuclear layer to the retinal thickness were respectively decreased with aging in both genotype, but there were not different statstically. TUNEL assay showed a few positively labeled cells in the ganglion cell, inner nuclear and outer nuclear layers and the immunohistochemistry showed no immunopositivity of hsp70 in the inner segments of photoreceptor cell layer in both genotype. In rd mice, fundus photography showed a narrowing of the retinal vessels at the age of 4 weeks, however, there were no differences of retinal changes including pigment epithelial layer in both genotype. Electroretinographic examination at the postnatal 2, 3 and 4 weeks showed no differences between them. Loss of photoreceptor cell and outer nuclear layers showed no differences in both genotype. In conclusion, there were no differences of the retinal changes at least under the normal environmental condition in hsp70.1+/+ and -/- mice. These results show that hsp70.1-/- mice can be used to study the role of hsp70.1 to the external stress to the retina.
Animal ; Electroretinography ; Fundus Oculi ; Heat-Shock Proteins 70/*deficiency/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout/genetics ; Photoreceptors, Vertebrate/*metabolism ; Protozoan Proteins/genetics ; Reference Values ; Retinal Degeneration/metabolism/*pathology/*physiopathology

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.
Cell Survival ; Cells, Cultured ; Cornea/*cytology ; DNA Damage ; Dose-Response Relationship, Drug ; Fibroblasts/cytology/drug effects/*metabolism ; Heat ; Heat-Shock Proteins 70/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Nitric Oxide/metabolism ; Nitric Oxide Donors/pharmacology ; *Oxidative Stress ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't ; S-Nitroso-N-Acetylpenicillamine/pharmacology ; Xanthine/pharmacology ; Xanthine Oxidase/pharmacology