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G.S. Schultz, T.D. Blalock, R. Liu, C. Chen, M.H. Goldstein; Immunohistochemical Localization of TGF, TGFß, and Their Receptors in Rat Corneas During Healing Following PRK Ablation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3815.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We immunolocalized TGFα, EGR-R, TGFß1, TGFß2, TGFß3, TGFß-IR and TGFß-IIR in rat corneas during healing of excimer ablation wounds, and measured the levels of mRNAs for EGF, TGFα and EGF-R in the corneas using Q-RT-PCR. Methods: Bilateral PTK was performed on rat corneas using a 3 mm central ablation zone to a depth of 50 microns. For immunohistochemical analysis, 4 eyes from two rats were fixed in situ with 4% neutral buffered formalin for 10 minutes, then the globe was removed, fixed overnight, and paraffin sections stained with monoclonal antibodies specific for each protein. For Q-RT-PCR, PTK was performed on rat corneas as described above. Rat corneas were then harvested using a central 3 mm corneal zone. At each time point (days 1.5, 7, 21, 42 and 91) eight corneas from four rats were pooled and total RNA was isolated. Normal corneas from rats without PTK served as control. Competition-based quantitative RT-PCR was used to measure the levels of mRNAs for EGF, TGFα and EGF-R genes. Results: Strong immunostaining was present in the basal epithelial cells for TGFß2 on days 0, 7, and 21 while staining for TGFß1, TGFß–IR, and TGFß-IIR proteins increased during wound healing. Faint immunostaining for TGFß3 was present in basal epithelial cells at all three time points. TGFα and EGF-R protein showed strong staining in control corneas with a slight decrease in staining for ablated eyes. Levels of EGF mRNA had a stable expression before and after PTK with a level of 60-80 copies/cell throughout the 91 day time course. Levels of TGFα mRNA showed a slow increase from 50 copies/cell to 300 copies/cell at day 42. Expression of EGF-R decreased drastically from 9 copies/cell in normal corneas, to less than 1 copy/cell at 1.5 days after PRK. Subsequently, levels of mRNA increased rapidly to 571 copies/cell at day 7, and then dropped back to baseline levels at day 42. Conclusions: These results show that there are changes in the TGFß system immunolocalization and protein expression during the corneal wound healing process with a trend toward increasing expression. The TGFα system showed a similar, albeit slower, increase in protein expression which is reflected in mRNA levels.
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