Peptide therapies for ocular surface disturbances based on fibronectin–integrin interactions

The condition of the corneal epithelium is a critical determinant of corneal transparency and clear vision. The corneal epithelium serves as a barrier to protect the eye from external insults, with its smooth surface being essential for its optical properties. Disorders of the corneal epithelium include superficial punctate keratopathy, corneal erosion, and persistent epithelial defects (PEDs). The prompt resolution of these disorders is important for minimization of further damage to the cornea. Currently available treatment modalities for corneal epithelial disorders are based on protection of the ocular surface in order to allow natural healing to proceed. PEDs remain among the most difficult corneal conditions to treat, however. On the basis of characterization of the pathobiology of PEDs at the cell and molecular biological levels, we have strived to develop new modes of treatment for these defects. These treatments rely on two key concepts: provision of a substrate, such as the adhesive glycoprotein fibronectin, for the attachment and migration of corneal epithelial cells, and activation of these cells by biological agents such as the combination of substance P and insulin-like growth factor-1 (IGF-1). Central to both approaches is the role of the fibronectin–integrin system in corneal epithelial wound healing. Determination of the minimum amino acid sequences required for the promotion of corneal epithelial wound closure by fibronectin (PHSRN) and by substance P (FGLM-amide) plus IGF-1 (SSSR) has led to the development of peptide eyedrops for the treatment of PEDs that are free of adverse effects of the parent molecules.     

Effects of substance P and IGF-1 in corneal epithelial barrier function and wound healing in a rat model of neurotrophic keratopathy

PURPOSE: To establish a rat model of neurotrophic keratopathy and to examine the effects of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and wound healing in this model. METHODS: Corneal denervation was achieved by thermocoagulation of the ophthalmic branch of the trigeminal nerve. A modified Schirmer test was performed without topical anesthesia. Corneal epithelial barrier function was assessed by measurement of fluorescein permeability with an anterior fluorophotometer. Epithelial wound healing was evaluated by measurement of the area of the defect at various times after removal of the entire epithelium. Eye drops containing both 1 mM SP and IGF-1 (1 micro g/mL) were administered six times daily. RESULTS: The Schirmer test result in eyes subjected to trigeminal denervation was lower than that in control eyes. The fluorescein permeability of the corneal epithelium of denervated eyes was increased relative to that of control eyes. Furthermore, trigeminal denervation induced a delay in corneal epithelial wound healing. Application of eye drops containing SP and IGF-1 to denervated corneas restored the fluorescein permeability of the corneal epithelium to control levels and abolished the delay in epithelial wound healing. CONCLUSIONS: A rat model of neurotrophic keratopathy, characterized by reduced tear secretion, loss of corneal sensation, impaired epithelial barrier function, and delayed epithelial wound healing, was established by trigeminal denervation. Treatment with both SP and IGF-1 improved corneal epithelial barrier function and stimulated corneal epithelial wound healing in this model.     

Promotion of corneal epithelial wound healing by a tetrapeptide (SSSR) derived from IGF-1

PURPOSE: A prior study showed that a tetrapeptide (FGLM-amide) derived from the carboxyl terminus of substance P (SP) and a 12-residue peptide corresponding to the C domain of insulin-like growth factor (IGF)-1 mimic the synergistic effect of the full-length molecules on corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, the current study was conducted to investigate the minimal sequence within the C domain of IGF-1 that is required for such synergism with SP or FGLM-amide. METHODS: The effects of IGF-1-derived peptides on corneal epithelial migration were evaluated with a rabbit corneal organ-culture system. RESULTS: A tetrapeptide (SSSR; Ser(33)-Ser-Ser-Arg) derived from the C domain of IGF-1 was sufficient for the synergistic promotion with FGLM-amide both of corneal epithelial migration in vitro and of wound closure in vivo. The activity of the SSSR peptide was sequence specific and its potency was similar to that of IGF-1. The SSSR peptide by itself also promoted corneal epithelial migration in vitro at higher concentrations. It was devoid, however, of both the mitogenic action of IGF-1 and the ability of the full-length molecule to induce neovascularization. CONCLUSIONS: The SSSR sequence mediates the synergistic effect of IGF-1 with SP on corneal epithelial wound healing. Clinical application of the SSSR peptide would be expected to be free of potentially deleterious side effects associated with treatment with full-length IGF-1. Local administration of the SSSR tetrapeptide, alone or in combination with FGLM-amide, is thus a potential new strategy for the treatment of nonhealing epithelial wounds.     

Sensitizing effect of substance P on corneal epithelial migration induced by IGF-1, fibronectin, or interleukin-6

PURPOSE: Substance P (SP) is present in the sensory nerve fibers of the corneal epithelium. Various biological agents, including epidermal growth factor, fibronectin, interleukin-6, and the combination of SP and insulin-like growth factor (IGF)-1, promote the healing of corneal epithelial wounds. The role of SP in corneal epithelial migration was examined. METHODS: The effects of various agents on corneal epithelial migration were investigated with the rabbit cornea in an organ culture system. RESULTS: An SP-derived tetrapeptide, FGLM-amide, shifted the dose-response relations for the induction of corneal epithelial migration not only by an IGF-1-derived peptide (C-domain peptide) but also by fibronectin or interleukin-6 to lower concentrations. This action of SP was prevented by inhibitors of phospholipase C, of the inositol 1,4,5-trisphosphate receptor-mediated release of Ca(2+) from intracellular stores, and of Ca(2+)- and calmodulin-dependent protein kinase II (CaM-PK II). CONCLUSIONS: These results indicate that SP, acting at the neurokinin type 1 receptor, functions as an important modulator of corneal epithelial wound healing by activating CaM-PK II in epithelial cells and thereby sensitizing them to the induction of migration by various biological agents. They also provide important insight into a new strategy for the treatment of corneal wounds.     

Role of the C domain of IGFs in synergistic promotion, with a substance P-derived peptide, of rabbit corneal epithelial wound healing

PURPOSE: Insulin-like growth factors (IGFs) and either substance P (SP) or an SP-derived peptide (FGLM-amide) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. The mechanism of this synergism and the clinical potential of these agents were further investigated by determination of the relevant functional domain of IGFs. METHODS: The effects of IGF-derived peptides on corneal epithelial cell migration were evaluated with the rabbit cornea in an organ culture system. Corneal epithelial wound closure in vivo was also evaluated in rabbits after epithelial debridement with n-heptanol. RESULTS: In the presence of FGLM-amide, peptides corresponding to the C domain of IGF-1 or -2 significantly promoted corneal epithelial migration in vitro to an extent similar to that apparent with the full-length molecules. In contrast, peptides corresponding to the D domain of these growth factors had no such effect. Mutation of serine-34 in the C domain of IGF-1 to alanine abolished the synergistic effect with FGLM-amide on corneal epithelial migration. The C peptide of proinsulin did not affect corneal epithelial migration in the absence or presence of FGLM-amide. The administration of eye drops containing both the C-domain peptide of IGF-1 and FGLM-amide significantly promoted corneal epithelial wound closure in vivo. CONCLUSIONS: The C domain of IGF-1 or -2, for which no biological function has previously been identified, is essential for the synergistic effect of these growth factors with SP on corneal epithelial migration.     

Characterization of insulin-like growth factor-1 receptors in rabbit corneal epithelial cells

Substance P (SP) and insulin-like growth factor-1 (IGF-1) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. This synergism is mediated through the NK-1 receptors for SP, and IGF-1 does not modulate the binding affinity of NK-1 receptors. To clarify the effect of SP on the binding characteristics of IGF-1 receptors, the binding affinity and number of binding sites for IGF-1 in rabbit corneal epithelial cells were studied using a binding assay for(125)I-IGF-1. The binding affinity and number of binding sites for IGF-1 were determined by Scatchard plot analysis. Cultured rabbit corneal epithelial cells bound specifically to IGF-1. For IGF-1 in corneal epithelial cells, the binding affinity was 4 n m and the number of binding sites was 1x10(5)binding sites cell(-1). Although IGF-2 and insulin also bind to IGF-1 receptors, their affinities were, respectively, eight- and 300-fold lower than that of IGF-1. IGF-1 and IGF-2 stimulated corneal epithelial migration in the presence of SP, but insulin did not. Pretreatment of the corneal epithelial cells with SP (2x10(-5)m) failed to change the binding affinity or number of binding sites for IGF-1. These results demonstrated that corneal epithelial cells possess specific receptors for IGF-1. The synergistic effect of SP and IGF-1 on corneal epithelial wound healing does not result from regulation at the receptor level.     

Effects of capsaicin on corneal wound healing.

This study examined whether the depletion of neuropeptides from sensory nerve terminals induced by capsaicin modifies the healing rate of experimental corneal wounds in adult rabbits. Capsaicin (33 or 3.3 mM solutions) was administered topically and/or by a single retrobulbar injection to one eye while the fellow eye, treated with the vehicle, served as a control. After 1-3 weeks of treatment, an epithelial wound was made in the center of the cornea of both eyes with n-heptanol. Migration rates of epithelial cells surrounding the wound and estimated wound closure times were calculated by measuring the reduction in wound size. Combined treatment with 33 mM retrobulbar and 3.3 mM topical capsaicin for 3 weeks induced a significant delay in epithelial migration rates and in wound closure times (P less than 0.05). Topical or retrobulbar capsaicin alone for 3 weeks and combined treatment lasting only 1 week were not sufficient to modify wound healing times. The substance P antagonist, spantide (3 mM), applied topically for 1-3 weeks before or immediately after corneal wounding was also ineffective in changing wound closure rates. These findings suggest that the delayed wound healing observed after prolonged treatment with capsaicin could be due to a sustained depletion of neuropeptides from corneal sensory endings, supporting the hypothesis that trophic effects of sensory nerves on corneal epithelium are, at least in part, mediated by neuropeptides contained in peripheral nerve terminals.     

Participation of p38 MAP kinase, but not p44/42 MAP kinase, in stimulation of corneal epithelial migration by substance P and IGF-1

PURPOSE: Substance P and insulin-like growth factor-1 (IGF-1) synergistically promote corneal epithelial migration both in vitro and in vivo. The mechanism of this action was investigated. METHODS: The effects of various inhibitors and activators of intracellular signaling pathways on corneal epithelial migration were examined by measuring the length of the migration path in rabbit corneal blocks in culture. RESULTS: Inhibitors of signaling by p38 or p44/42 isoforms of mitogen-activated protein (MAP) kinase or of phosphatidylinositol (PI) 3-kinase reduced the extent of spontaneous migration of the corneal epithelium, whereas modulators of signaling by cyclic AMP- or cyclic GMP-dependent protein kinases had no effect. The inhibitors of p38 MAP kinase and of PI 3-kinase also abolished the stimulatory effect of substance P and IGF-1 on epithelial migration, whereas inhibitors of signaling by p44/42 MAP kinase or modulators of cyclic nucleotide-dependent signaling did not. CONCLUSIONS: These results suggest that various signal transduction systems participate in spontaneous corneal epithelial migration as well as in the combined effect of substance P and IGF-1 on this process. In particular, although both p38 and p44/42 isoforms of MAP kinase appear to regulate spontaneous corneal epithelial migration, the stimulatory effect of substance P and IGF-1 appears to be mediated by p38 MAP kinase but not by p44/42 MAP kinase.     

Congenital Hypoplastic Trigeminal Nerve Revealed by Manifestation of Corneal Disorders Likely Caused by Neural Factor Deficiency

Purpose

To report a case of hypoplastic trigeminal nerve associated with corneal epithelial disorders that were successfully treated with peptides derived from substance P and insulin-like growth factor-1 (IGF-1).

Case Report

A 16-month-old boy was referred for treatment of a persistent corneal epithelial defect on his left eye. Magnetic resonance imaging revealed the apparent absence of the trigeminal nerve on the left side, and the patient was therefore diagnosed with neurotrophic keratopathy. Treatment with eye drops containing the tetrapeptides FGLM-NH₂ and SSSR derived from the neuropeptide substance P and the growth factor IGF-1, respectively, resulted in resurfacing of the corneal epithelial defect.

Discussion

The trigeminal nerve anomaly of the patient likely gave rise to neurotrophic keratopathy as a result of a deficiency of neural factors, emphasizing the importance of neural regulation in corneal epithelial homeostasis.Key words: Neurotrophic keratopathy, Trigeminal nerve, Corneal epithelial disorder     

Neurotrophe Keratopathie

Neurotrophic keratitis is a degenerative disease of the cornea caused by reduced corneal innervation. Trauma, tumors, inflammatory lesions and surgical procedures can damage the first branch of the trigeminal nerve on its entire course from brainstem to and within the cornea. Loss or reduction of corneal innervation leads to a reduced aqueous phase of the tear film and due to reduced supply with neurotransmitters/trophic factors also to reduced epithelial healing capacity (impaired mitosis and migration). Combined existence of tear film deficiency and impaired epithelial healing capacity predispose to persistent epithelial defects, corneal ulcers and perforation. Early diagnosis and adequate treatment may prevent this catastrophic chain of events.     

Restoration of corneal epithelial barrier function and wound healing by substance P and IGF-1 in rats with capsaicin-induced neurotrophic keratopathy

PURPOSE: To investigate the effects of topical application of the combination of substance P (SP) and insulin-like growth factor (IGF)-1 on corneal epithelial barrier function and epithelial wound closure in rats with capsaicin-induced neurotrophic keratopathy. METHODS: Neonatal rats were injected subcutaneously with a single dose of capsaicin to induce neurotrophic keratopathy. Corneal epithelial barrier function was evaluated with an anterior fluorophotometer. Tear fluid secretion was measured by the Schirmer test. Corneal epithelial wound healing was determined by measurement of the size of the epithelial defect after debridement of the entire epithelium. The combination of SP (1 mM) and IGF-1 (1 micro g/mL) in phosphate-buffered saline was administered in eye drops six times daily. RESULTS: Corneal epithelial barrier function was impaired and corneal epithelial wound healing was delayed in rats injected with capsaicin. The application of eye drops containing the combination of SP and IGF-1 to capsaicin-injected rats resulted in a significant improvement in corneal epithelial barrier function compared with that apparent in capsaicin-injected animals that received eye drops containing vehicle alone. Such treatment with SP and IGF-1 also significantly increased the rate of corneal epithelial wound closure in capsaicin-injected animals. CONCLUSIONS: Topical application of the combination of SP and IGF-1 improved both corneal epithelial barrier function and epithelial wound healing in an animal model of neurotrophic keratopathy.     

The two-faced effects of nerves and neuropeptides in corneal diseases

Corneal nerves are instrumental to maintain cornea integrity through regulation of key physiological functions such as tear secretion, blink reflex, and neuropeptide turnover. Corneal nerve injury/stimulation can follow many insults including mechanical/chemical trauma, infections and surgeries. Nerve disruption initiates a process named neurogenic inflammation which leads to edema, pain, and recruitment and activation of leukocytes. Interestingly, leukocyte influx in the cornea can further damage nerves by releasing inflammatory mediators-including neuropeptides. The clinical outcome of neuroinflammation can be beneficial or detrimental to corneal integrity. On one side, it ensures prompt wound healing and prevents infections. On the other, prolonged and/or deranged neuroinflammation can permanently disrupt corneal integrity and impair vision.The cornea is an ideal site to study peripheral neuroinflammation and neurogenic inflammation since it receives the highest density of sensory nerves of the entire body.We will review the corneal nerve anatomy and neurochemistry, discuss the beneficial and detrimental effects of neurogenic inflammation in corneal wound healing, inflammatory processes, and pain. We will also examine the emerging remote impact of corneal nerve disruption on the trigeminal ganglion and the brain, highlighting the key role of neuropeptide Substance P. Finally, we will discuss the clinical relevance of such neuroinflammatory network in the context of severe and highly prevalent ocular diseases, including potential treatments.     

Use of nerve growth factor to treat congenital neurotrophic corneal ulceration

PURPOSE: This study was designed to assess the efficacy of nerve growth factor in the treatment of neurotrophic corneal ulceration in a child with bilateral congenital corneal anesthesia secondary to trigeminal insufficiency. METHODS: A 5-month-old child presented to the casualty department with a 2-week history of red eyes and right corneal ulceration. Slit-lamp examination showed a central defect in the right corneal epithelium with underlying stromal opacification, only mild conjunctival inflammation with slight decreased tear production, and otherwise apparently normal eyes. Initially this was investigated as an infected ulcer and treated for several weeks as herpetic ulceration with no beneficial effect. Further clinical examination demonstrated bilateral decreased corneal sensation along with decreased facial sensation in keeping with congenital trigeminal nerve insufficiency. Investigation with magnetic resonance imaging showed no obvious abnormality. Conservative treatment with lubricants resulted in progressive right corneal stromal loss, and no healing occurred in the left corneal ulcer. Bilateral large lateral tarsorrhaphies were performed. Despite this, the left corneal ulcer demonstrated no improvement and increasing stromal opacification was noted. Topical nerve growth factor (NGF) was then used to treat the left cornea and resulted in epithelial healing within 1 week. Treatment was continued for a further 10 days after epithelial healing. Despite conventional treatment on 3 separate occasions, further epithelial breakdown occurred. Topical NGF treatment resulted in a rapid improvement and healing of the epithelial defect.'At present, the patient is receiving a 6-month continuous treatment plan of NGF. RESULTS: Persistent epithelial defects (PED) secondary to neurotrophic ulceration have responded to topical NGF on 3 separate occasions during a 2-year period. The corneal epithelium now remains intact, and the cornea has no vascularization; however, mild anterior stromal opacification has gradually increased despite prolonged NGF treatment. CONCLUSION: NGF seems to represent a safe and efficacious treatment option to restore the integrity of corneal epithelium in which there is congenital corneal anesthesia because of trigeminal insufficiency. However, this treatment alone is insufficient to prevent progressive anterior stromal opacification.     

Signaling regulation for synergistic effects of substance P and insulin-like growth factor-1 or epidermal growth factor on corneal epithelial migration

PURPOSE: In a previous report we showed that substance P (SP) and insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) synergistically stimulate corneal epithelial migration. In this study, we used an organ culture system of rabbit cornea to identify which signal transduction system affects corneal epithelial migration. METHODS: Rabbit corneal blocks were cultured in TC-199 culture medium containing various reagents for 24 hours. After the end of cultivation, the length of the path of epithelial migration was measured. RESULTS: Acting alone, protein kinase C (PKC) inhibitors, calphostin C and H-7, each reduced the length of epithelial migration. Tyrosine kinase (TK) inhibitors, genistein and herbimycin A, also acted individually to inhibit epithelial migration. The synergistic stimulatory effects of SP and IGF-1 on corneal epithelial migration were eliminated when PKC inhibitors or TK inhibitors were added. The synergistic effect of SP and EGF was eliminated by TK inhibitors, but only partly suppressed by PKC inhibitors. CONCLUSIONS: These results suggest that the synergistic effect of SP and EGF might require a TK pathway, and that the synergistic effect of SP and IGF-1 might require both PKC and TK pathways.     

A new in vitro corneal preparation to study epithelial wound healing.

Corneal epithelial wound healing is an important process necessary for maintenance of visual integrity. Corneal epithelial wound healing occurs by cellular migration and proliferation. However, the molecular basis of reepithelialization is not known. To investigate individual molecular contributions to the wound healing process, an in vitro corneal preparation comparable to the in vivo condition is needed. This investigation developed a new whole mount in vitro rabbit cornea preparation and studied epithelial wound healing rates for epithelial and subepithelial wounds. The wound closure rates obtained in this study for epithelial and subepithelial wound healing (52 +/- 14 microns/hr and 38 +/- 7 microns/hr, respectively) are comparable to in vivo rates of wound healing determined by other laboratories for rabbits. This preparation, achieved by functionally separating the epithelial and endothelial sides of the cornea, allows application of agents to the cornea in a manner that approximates the in vivo condition. This in vitro system is promising for future studies designed to investigate corneal wound healing while reducing potential ocular discomfort associated with in vivo corneal wounding.     

The effect of substance P with insulin-like growth factor-1 on corneal epithelial cell proliferation]

PURPOSE: To elucidate the pathogenesis of neuroparalytic keratopathy, we examined the effect of substance P (SP) on corneal epithelial cell proliferation in comparison with that of insulin-like growth factor-1 (IGF-1) in vivo and in vitro. METHODS: In the in vivo study, the epithelium of rabbit cornea was removed mechanically and treated with eye drops containing SP, IGF-1, SP + IGF-1, or physiological saline as a control. Corneas were excised 48 hours after the removal of the epithelium, labeled with 3H-thymidine, and subjected to autoradiography. An in vitro study was also performed by culturing rabbit corneal epithelial cells in culture medium containing SP with or without IGF-1 and labeling cells with 3H-thymidine for a subsequent autoradiographical study. FINDINGS: In the in vivo study, SP in combination with IGF-1 enhanced the healing of rabbit corneal epithelial defect. 3H-thymidine autoradiography revealed that in both in vivo and in vitro studies, SP + IGF-1 stimulated corneal epithelial cell proliferation, but SP or IGF-1 alone did not. CONCLUSION: These results indicate that SP enhances the stimulatory effect of IGF-1 on corneal epithelial cell proliferation and accelerates corneal epithelial healing.     

New therapeutic approaches in the treatment of diabetic keratopathy: a review

The cornea is densely innervated, and the integrity of these nerve fibres is critical in maintaining the refractive and protective functions of the cornea. Many ocular and systemic diseases can adversely affect corneal sensory nerves and consequently impair their function, with vision loss being the inevitable consequence of severe corneal neurotrophic ulceration. However, current standard treatments regimens are often ineffective. Over the past three decades, the role of growth factors in maintaining the normal structure and function of the cornea, and in corneal epithelial healing, has become increasingly evident. Many preclinical and clinical trials have shown that growth factors and cytokines can significantly enhance epithelialization (epithelial proliferation and migration) and consequently accelerate wound healing. More recently, local/topical administration of insulin, naltrexone (opioid antagonist) and nicergoline (ergoline derivatives) were found to improve, and significantly increase, the corneal wound healing rate. This report reviews the major attributes of these growth factors and therapeutic agents that may be used in ameliorating impaired corneal wound healing, and presents a perspective on the potential clinical use of these agents as a new generation of ophthalmic pharmaceuticals for the treatment of diabetic keratopathy.     

Nerve growth factor and corneal wound healing in dogs

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.     

角膜神经调控眼表微环境的研究进展

角膜是眼前段透明的外层结构，由高密度的神经组织支配。在角膜神经支配过程中，三叉神经节起源的角膜神经穿过上皮层和基质层中不同类型的角膜细胞。角膜基质细胞、上皮细胞、免疫细胞等多种细胞和角膜神经之间发生密切的相互作用，共同维持角膜微环境稳态。此外，角膜神经参与许多眼表疾病的发生发展过程。角膜神经释放多种活性肽物质，参与调控角膜感觉、维持上皮完整性和增殖、促进伤口愈合及调控角膜局部炎症和免疫反应等。本文对角膜神经在眼表微环境调控作用的研究进展进行综述，为角膜神经相关疾病的研究及治疗提供新的思路。     

The cornea: stasis and dynamics

The physiological roles of the cornea are to conduct external light into the eye, focus it, together with the lens, onto the retina, and to provide rigidity to the entire eyeball. Good vision thus requires maintenance of the transparency and proper refractive shape of the cornea. Although the cornea appears to be a relatively static structure, dynamic processes operate within and around the cornea at the tissue, cell, and molecular level. In this article, I review the mechanisms responsible for maintenance of corneal homeostasis as well as the development of new modes of treatment for various corneal diseases. I. The static cornea: structure and physiological functions. The cornea is derived from ectoderm, so that it can be considered as transparent skin. It is devoid of blood vessels and manifests the highest sensitivity in the entire body. The surface of the cornea is covered by tear fluid, which serves both as a lubricant and as a conduit for regulatory molecules. The cornea is also supplied with oxygen and various nutrients by the aqueous humor and a loop vascular system in addition to tear fluid. The cornea interacts with its surrounding tissues directly as well as indirectly through tear fluid or aqueous humor, with such interactions playing an important role in the regulation of corneal structure and functions. The resident cells of the cornea-epithelial cells, fibroblasts (keratocytes), and endothelial cells--also engage in mutual interactions through network systems. These interactions as well as those with infiltrated cells and regulation by nerves contribute to the maintenance of the normal structure and functions of the cornea as well as to the repair of corneal injuries. II. The dynamic cornea: maintenance of structure and functions by network systems. Developments in laser and computer technology have allowed observation of the cells and collagen fibers within the cornea. Furthermore, progress in cell and molecular biology has allowed characterization of dynamic network systems-including cell-cell and cell-extracellular matrix interactions as well as cytokines and neural factors-that contribute to the maintenance of corneal transparency and shape. III. Disruption of network systems: persistent corneal epithelial defects and corneal ulcer. Selection of the appropriate treatment for pathologic lesions of the cornea and the accompanying decrease in visual acuity requires localization of the lesion with regard to the epithelium, stroma, or endothelium of the cornea. In certain instances, however, it is not possible to determine the cause of the problem within the cornea. In such cases, the cause of the pathologic lesion and the target for treatment may lie in the surrounding tissues or environment. For example, corneal epithelial wound healing may be delayed, leading to the development of persistent epithelial defects, as a result of disruption of intercellular junctions between epithelial cells, an abnormality of the corneal basement membrane, altered concentrations of various cytokines in tear fluid, a lowered corneal sensation, or allergic reactions in the lid conjunctiva. Loss of corneal epithelial barrier function can further allow inflammatory cytokines present in tear fluid, together with infiltrated cells, to activate keratocytes and elicit excessive degradation of collagen in the stroma, thereby giving rise to corneal ulcer. IV. Development of new drugs for corneal diseases. We have attempted to apply the results of basic scientific research to the development of new drugs for corneal diseases that remain difficult to treat. The process of authorization for new drugs from the Ministry of Health, Labor, and Welfare takes more than two decades, however. The path from the bench to clinical practice is thus a long one. 1. Development of eyedrops for treatment of persistent corneal epithelial defects. We demonstrated the clinical efficacy of fibronectin eyedrops for the treatment of persistent epithelial defects of the cornea. However, the possibility of blood-borne infections has interfered with the development of serum-derived fibronectin as a drug. An automated machine for the preparation of autologous fibronectin eyedrops has therefore recently been developed. Furthermore, in seeking an alternative to fibronectin eyedrops, we are investigating the effects of a peptide corresponding to the second cell-binding domain of fibronectin on corneal epithelial wound healing. Considering that urokinase-type plasminogen activator may be expressed at the site of corneal epithelial defects and facilitates epithelial migration, the potential clinical application of annexin V, which stimulates the secretion of urokinase-type plasminogen activator for the treatment of persistent corneal epithelial defects is also now under investigation in Japan. 2. Development of eyedrops for treatment of neurotrophic keratopathy. Substance P, a neurotransmitter, stimulates corneal epithelial migration in a synergistic manner with insulin-like growth factor (IGF)--1. We have shown that eyedrops containing both the substance P-derived peptide FGLM-amide and the IGF-1--derived peptide SSSR are effective for the treatment of persistent corneal epithelial defects in individuals with diabetic keratopathy or neurotrophic keratopathy, both of which are associated with a reduction in corneal sensation. 3. Development of drugs for corneal ulcer. Treatment of corneal infection with antibiotics does not necessarily halt the process of corneal ulceration, which is characterized by excessive degradation of stromal collagen, or resolve persistent corneal epithelial defects. In addition to eyedrops for the treatment of persistent corneal epithelial defects, we have therefore also been working on the development of new drugs for the treatment of corneal ulcer. To this end, we have established an experimental system in which corneal fibroblasts are cultured in a three-dimensional collagen gel. With this system, we have shown that triptolide and steroids inhibit collagen degradation by corneal fibroblasts. Triptolide or its derivatives are thus potential drugs for the treatment of corneal ulcer and would work by acting directly on corneal fibroblasts rather than by inhibiting the secreted enzymes(matrix metalloproteinases) responsible for collagen degradation.