Immunohistochemical localization of EGF, TGF-α, TGF-β, and their receptors in rat corneas during healing of excimer laser ablation

Purpose: Members of the epidermal growth factor (EGF) and transforming growth factor beta (TGF-β) families of growth factors and receptors are known to regulate key aspects of corneal wound healing, including epithelial migration and scar formation. To further understand their roles, mRNA levels were measured and proteins were immunolocalized in rat corneas at multiple time points during healing of excimer laser ablation injury. Methods: Excimer laser photoablation was performed to a depth of 50 μm on rat corneas. Levels of mRNAs for EGF, TGF-α, TGF-β isoforms 1, 2, and 3, and their receptors (EGF-R and TGFβ-IIR) were measured by quantitative RT-PCR on days 0, 1.5, 7, 21, 42, and 91 after ablation. Immunohistochemical localization of the growth factors and their receptors was performed on days 0, 7, and 21 in corneal sections. Results: Levels of EGF mRNA remained stable in rat corneas after ablation (68 ± 12 copies/cell, mean ± SD), whereas levels of TGF-α mRNA progressively increased sixfold to a maximum at day 42 (300 copies/cell) then slightly decreased on day 91. Levels of EGF-R mRNA rapidly increased 60-fold on day 7 compared with day 0 (571 vs. 9 copies/cell) then decreased sixfold above baseline at day 91. Levels of TGF-β1 mRNA remained stable (36 ± 10 copies/cell), whereas levels of TGF-β2 and TGF-β3 mRNAs peaked on day 21 (300-fold and 25-fold increase) and remained elevated through day 91. Levels of TGFβ-IIR mRNA showed a similar pattern. Immunostaining of all the growth factors and receptors was primarily in basal layers of epithelial cells in uninjured cornea and during healing. Intensity of immunostaining for TGF-β1, TGFβ-IR, and TGFβ-IIR increased appreciably in the basal epithelial layers after ablation. Conclusions: Levels of mRNAs for several key members of the EGF and TGF-β systems increase during corneal wound healing. In addition, the proteins are primarily localized in basal layers of epithelial cells, which suggest these cells are active in synthesizing autocrine and paracrine growth factors that modulate corneal wound healing.