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Purpose To investigate changes in corneal nerves positive to substance P (SP) and transient receptor potential melastatin 8 (TRPM8) and gene expression in the trigeminal ganglia (TG) following corneal surgery to unveil peripheral nerve mechanism of induced dry eye-like pain (DELP). Methods Surgery was performed on mice by removing the central epithelial and anterior stromal nerves. Mice were euthanized at different times up to 15 weeks. Immunostaining was performed with TRPM8, SP, or protein gene product 9.5 (PGP9.5) antibodies, and epithelial nerve densities were calculated. The origin of TRPM8- and SP-TG neurons were analyzed by retrograde tracing. Gene expression in TG was studied by real-time PCR analysis. Results SP-positive epithelial corneal nerves were more abundant than TRPM8 and were expressed in different TG neurons. After injury, epithelial nerve regeneration occurs in two distinct stages. An early regeneration of the remaining epithelial bundles reached the highest density on day 3 and then rapidly degraded. From day 5, the epithelial nerves originated from the underlying stromal nerves were still lower than normal levels by week 15. The SP- and TRPM8-positive nerve fibers followed the same pattern as the total nerves. TRPM8-positive terminals increased slowly and reached only half of normal values by 3 months. Corneal sensitivity gradually increased and reached normal values on day 12. Corneal injury also induced significant changes in TG gene expression, decreasing trpm8 and tac1 genes. Conclusions Abnormal SP expression, low amounts of TRPM8 terminals, and hypersensitive nerve response occur long after the injury and changes in gene expression in the TG suggest a contribution to the pathogenesis of corneal surgery-induced DELP.

The cornea is densely innervated, and several studies have shown that the health of corneal nerves is vital to maintain homeostasis of the ocular surface and tissue clarity.1–4 Many diseases that affect the cornea can compromise corneal innervation, leading to a decrease in tear production and blink reflex as well as impaired epithelial wound healing.4–8 As a result, neurotrophic keratitis and corneal opacification may ensue. It is estimated that 1.57 million people suffer from corneal blindness worldwide.9 In turn, some of surgical interventions such as refractive surgery and corneal transplantation sever corneal nerves, and studies have shown that corneal sensation can take years to recover, and in some cases it is permanently reduced.10,11 Unfortunately, there are few therapeutic interventions available today that can successfully promote the recovery of corneal sensation. Acknowledging our current limitations, the National Eye Institute (NEI) has identified the need for development of therapeutic agents that can stimulate corneal nerve regeneration as one of the highest priorities in vision research.12 Several molecules have been studied that may have a positive effect on corneal nerve regeneration, including nerve growth factor (NGF), VEGF, semaphorins, neurotrophins 3 and 4 (NT-3; NT-4), growth associated protein-43 (GAP-43), and others, all with variable success.13–17 Neuropeptides, such as substance P (SP) and insulin-like growth factor-1 (IGF-1), have been used to treat delayed wound healing due to corneal nerve damage when traditional treatments have failed.18 Pigment epithelial-derived factor (PEDF) is a glucoprotein discovered in the culture media of retinal pigment epithelial (RPE) cells and is widely expressed in different tissues, including the cornea.19,20 Pigment epithelial-derived factor has broad neurotrophic, neuroprotective, and antiangiogenic activity.21 Work in our laboratory has previously shown that treatment with PEDF in association with the ω-3 fatty acid, docosahexaenoic acid (DHA), after lamellar keratectomy increases regeneration of rabbit corneal nerves.22 We have also shown that corneal sensation returns to normal levels in treated animals only 8 weeks after surgical injury.23 The mechanisms of the neuroregenerative action of PEDF and DHA is not completely understood, but our studies suggest that it involves the synthesis of neuroprotectin D1 (NPD1), a docosanoid synthetized from DHA with strong anti-inflammatory and neuroprotective activity.24–26 Pigment epithelial-derived factor is a 50-kDa protein, and synthetic peptides derived from the sequence of human PEDF, which maintain part of its bioactivity, have been tried in models of choroid and corneal neovascularization, and in diabetic retinopathy.27–29 A 44-amino-acid fragment of PEDF, corresponding to positions Val78-Thr121 of the 418 amino acids of PEDF, has neuroprotective activity and has been shown to stimulate survival and differentiation of spinal motor neurons.30 However, the adjacent 34-amino-acid peptide, corresponding to positions Asp44–Asn77, has been shown to be antiangiogenic.31 Some therapeutic advantages of using small peptides include better tissue penetration, a narrower spectrum of action with reduced side effects, and ease of synthesis in reproducibly large-scale quantities, while a possible disadvantage could be faster biodegradation and lower efficacy of the drug. These are all important considerations in the development of therapeutic agents. In this study we compare the effect of the synthetic 44-mer PEDF and 34-mer PEDF peptides and the whole PEDF molecule in association with DHA on the regeneration of corneal nerves.

Purpose The purpose of this study was to investigate the expression and activity of protein tyrosine phosphatases (PTPs) in epithelium during corneal wound healing and to investigate how PTPs regulate activation of the c-Met receptor and the receptor's proximal signaling. Methods Rabbit corneas were injured by gentle scraping of the surface, leaving the limbal epithelium intact, and epithelium was collected at 1, 2, 3, and 7 days after injury. In organ culture models, epithelium was removed and corneas were incubated with hepatocyte growth factor (HGF), with or without the PTP inhibitor bpV(phen), and the PI-3K inhibitors wortmannin and LY294002. Human corneal epithelial (HCE) cells were stimulated with HGF with or without bpV(phen). Total cell lysates and cytosolic and membrane fractions were analyzed by Western blot. PTP activities were measured with specific substrates. PTP1B and SHP-2 genes were knocked down by interference RNA (siRNA). Results PTP activity and expression increased during wound healing. The most abundant were SHP-2, PTP1B, and PTEN. HGF activated the c-Met receptor in HCE cells up to 30 minutes and was downregulated by 2 hours. Inhibition of PTPs increased HGF-promoted wound healing, HGF-activated phosphorylation of c-Met, and its downstream signal PI-3K/Akt, but not ERK1/2 or p70S6K. PTP1B and SHP-2 were bound to the c-Met. Part of the c-Met was colocalized in the endoplasmic reticulum with PTP1B. PTP1B phosphorylation increased when the c-Met receptor was deactivated, and gene knockdown of PTP1B increased c-Met activation. SHP-2 phosphorylation and binding to c-Met was higher during receptor activation, and SHP-2 gene silencing decreased receptor phosphorylation. Conclusions Inhibition of PTP activity mimics the effect of HGF by activating the PI-3K/Akt signal involved in wound healing. PTP1B and SHP-2 are bound to the c-Met receptor to control its activity. Although the binding of PTP1B increases when there is a decrease in c-Met activation and acts as a negative regulator of the receptor, the increased binding and phosphorylation of SHP-2 coincide with maximal stimulation of c-Met, acting as a positive regulator.

PURPOSE Cell survival is critical during corneal epithelial regeneration after injury, and growth factors could be fundamental in cytoprotection. The goal of this study was to investigate the involvement of the paracrine hepatocyte growth factor (HGF) in the prevention of corneal epithelial cell apoptosis and to identify signal transducers in this process. METHODS Apoptosis in human and rabbit corneal epithelial (HCE and RCE) cells was induced with a nutrient-deprived exhausted medium (ExM) or by treatment with staurosporine (20-100 ng/mL) or the calcium ionophore A23187 (0.5 microM). Apoptotic cells were identified by DNA fragmentation in agarose gels and by Hoechst staining. Active Akt-1 overexpression (Akt-1 pUSEamp cDNA) and small interfering RNA (siRNA) specific for Akt mRNA were used. Immunofluorescence, Western immunoblot analysis, and Akt kinase assays were also used. RESULTS Staurosporine, ExM, and A23187 induced DNA fragmentation in HCE and RCE cells. HGF (20 ng/mL) in combination with the apoptotic agents greatly reduced DNA breakdown and the number of Hoechst-positive cells. In the presence of phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin and LY294002), HGF did not overcome apoptosis. However, PD98059, the ERK1/2 cascade pathway inhibitor, was ineffective in preventing HGF protection. HGF induced a sustained activation of Akt-1, and overexpression of active Akt-1 reduced apoptosis. HGF stimulated the downstream targets of Akt, glycogen synthase kinase (GSK-3), and Bad, a proapoptotic member of the Bcl-2 family, an effect that was blocked by PI-3K inhibitors but not by ERK1/2 inhibition. Suppressing the expression of Akt by Akt siRNA led to a decrease in the phosphorylation of Bad and GSK-3. Translocation of Bad to the mitochondria, a critical stage in apoptosis, was prevented by HGF when apoptosis was induced. Moreover, in epithelial cells overexpressing active Akt-1, Bad translocation was also prevented. CONCLUSIONS HGF modulates multiple signaling cascades in corneal epithelial cells. The results demonstrated that HGF, in a paracrine fashion, protects cells from apoptosis through a PI-3K/Akt/Bad pathway but not through an ERK1/2 pathway. It was also demonstrated that GSK-3 is a target of PI-3K/Akt-1.