A new R,R-RvD6 isomer with protective actions following corneal nerve injury.

The transparent cornea is the most densely innervated tissue in the body, primarily by sensory nerves originating from the trigeminal ganglia (TG). Damage to corneal nerves reduces sensitivity and tear secretion and results in dry eye. Consequently, ocular pain, for which no satisfactory therapies exist, arises in many cases. Treatment of injured corneas with pigment epithelium-derived factor (PEDF) combined with docosahexaenoic acid (DHA) stimulates nerve regeneration in models of refractive surgery, which damages nerves. The mechanism involves the synthesis of a stereoisomer of resolvin D6 (R,R-RvD6) formed after incorporating DHA into membrane lipids. Activation of a PEDF receptor (PEDF-R) with phospholipase activity releases DHA to synthesize the new resolvin isomer, which is secreted via tears. Topical treatment of mice corneas with R,R-RvD6 shows higher bioactivity in regenerating nerves and increasing sensitivity compared to PEDF+DHA. It also stimulates a transcriptome in the TG that modulates genes involved in ocular pain. Our studies suggest an important therapeutic role for R,R-RvD6 in regenerating corneal nerves and decreasing pain resulting from dry eye.

Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome.

PURPOSE: To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. METHODS: To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 µL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. RESULTS: Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. CONCLUSIONS: LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Neuroanatomy of Adult and Aging Chicken Cornea.

PURPOSE: To provide a complete nerve architecture and main sensory neuropeptide distribution in the chicken cornea. METHODS: Adult chickens aged 6 months and 4 years were used. The whole cornea was stained with protein gene product (PGP) 9.5 antibody-a pan marker for nerve fibers, calcitonin gene-related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. Relative corneal epithelial nerve fiber densities, including subbasal bundles and superficial terminals, were assessed by computer-assisted analysis. RESULTS: An average of about 76.3 ± 5.7 (n = 8 corneas, 4 M/4F) stromal nerve trunks enter the cornea radially and are evenly distributed around the limbus with no significant difference between male and female chickens. The subbasal nerve bundles do not extend in a given direction and, as a result, do not form a vortex in the center of the cornea. Furthermore, the chicken cornea contains more SP-positive nerves than CGRP-positive nerves. It is also shown that aging significantly reduces corneal epithelial nerve density in chickens. CONCLUSIONS: This is the first study to provide a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the adult chicken cornea. The findings show chicken corneal innervation has many differences to human and mammal cornea.

Elucidating the structure and functions of Resolvin D6 isomers on nerve regeneration with a distinctive trigeminal transcriptome.

Innervation sustains cornea integrity. Pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15- or 5-LOX inhibitors, suggesting that the 4- and 17-hydroxy of DHA have an RR- or SR-configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1-4 from tears were resolved. The RR-RvD6 standard eluted as a single peak with RvD61 while pure SR-RvD6 eluted with RvD63 . The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that RR-RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. RR-RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

ELV-N32 and RvD6 isomer decrease pro-inflammatory cytokines, senescence programming, ACE2 and SARS-CoV-2-spike protein RBD binding in injured cornea.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic collapse around the world. Effective treatments to mitigate this viral infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells (HCEC) in culture challenged with IFNγ as models of the eye surface to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein to angiotensin-converting enzyme 2 (ACE2). We found that the lipid mediators, elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i) decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ-stimulated HCEC. There was also a concomitant decrease in the binding of Spike RBD with the lipid treatments. Using RNA-seq analysis, we uncovered that the lipid mediators also attenuated the expression of pro-inflammatoy cytokines participating in hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to open therapeutic avenues to counteract virus attachment and entrance to the body.

Neuroanatomy and neurochemistry of rat cornea: Changes with age.

PURPOSE: To characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution. METHODS: Sprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis. RESULTS: Thick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% ± 3.5% and 34.9% ± 5.1%, respectively, of the total nerves. VIP was 18.4% ± 5.7%, and NPY and TH positive fibers took up 6.92% ± 2.66% and 2.92% ± 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks. CONCLUSION: This study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.

Docosanoid signaling modulates corneal nerve regeneration: effect on tear secretion, wound healing, and neuropathic pain.

The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using ω-3 supplementation show unclear and sometimes negative results. Using animal models of corneal nerve damage, we show that treating corneas with pigment epithelium-derived factor plus DHA increases nerve regeneration, wound healing, and tear secretion. The mechanism involves the activation of a calcium-independent phospholipase A2 that releases the incorporated DHA from phospholipids and enhances the synthesis of the docosanoids, neuroprotectin D1 (NPD1) and a new resolvin stereoisomer, resolvin D6i (RvD6i). NPD1 stimulates the synthesis of brain-derived neurotrophic factor, nerve growth factor, and semaphorin 7A. RvD6i treatment of injured corneas modulates gene expression in the TG resulting in enhanced neurogenesis, decreased neuropathic pain, and increased sensitivity. Taken together, these results represent a promising therapeutic option to reestablish the homeostasis of the cornea.

Elovanoid-N32 or RvD6-isomer decrease ACE2 and binding of S protein RBD after injury or INFγ in the eye.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic tall around the world. Effective treatments to mitigate this virus infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells in culture challenged with IFNγ to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). Elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i), among the lipid mediators studied, consistently decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ stimulated human corneal epithelial cells (HCEC). There was also a concomitant decrease in the binding of spike RBD with the lipid treatments. Concurrently, we uncovered that the lipid mediators also attenuated the expression of cytokines that participate in the cytokine storm, hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to opening therapeutic avenues for COVID-19 by counteracting virus attachment and entrance to the eye and other cells and the ensuing disruptions of homeostasis.

Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.

The high-density corneal innervation plays a pivotal role in sustaining the integrity of the ocular surface. We have previously demonstrated that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) promotes corneal nerve regeneration; here, we report the mechanism involved and the discovery of a stereospecific Resolvin D6-isomer (RvD6si) that drives the process. RvD6si promotes corneal wound healing and functional recovery by restoring corneal innervation after injury. RvD6si applied to the eye surface elicits a specific transcriptome signature in the trigeminal ganglion (TG) that includes Rictor, the rapamycin-insensitive complex-2 of mTOR (mTORC2), and genes involved in axon growth, whereas genes related to neuropathic pain are decreased. As a result, attenuation of ocular neuropathic pain and dry eye will take place. Thus, RvD6si opens up new therapeutic avenues for pathologies that affect corneal innervation.

Remodeling of Substance P Sensory Nerves and Transient Receptor Potential Melastatin 8 (TRPM8) Cold Receptors After Corneal Experimental Surgery.

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.

Mapping the entire nerve architecture of the cat cornea.

OBJECTIVE: To provide a complete nerve architecture and neuropeptide distribution in the cat cornea. ANIMALS STUDIED: Two adult domestic cats. PROCEDURE: The cat corneas were stained with protein gene product (PGP) 9.5 antibody-a pan marker for nerve fibers-and then divided into four quarters and double labeled with calcitonin gene-related peptide (CGRP) or substance P (SP) antibodies. Relative corneal nerve fiber densities and nerve terminals were evaluated in whole mount images by computer-assisted analysis. RESULTS: An average of 21.5 ± 2.1 thick stromal nerves enters the cornea around the limbus where they split into many branches going up to the anterior stroma. Some branches link to each other, but most of them penetrate the basement membrane in the periphery to give origin to subbasal bundles, which run centripetally and merge to form a whirl-like structure (vortex) at the center. These nerve bundles send out many fine terminals that innervate the epithelial cells. Subbasal nerve density and nerve terminals were greater in the center than in the periphery of the cornea. Additionally, CGRP-positive central epithelial nerve fibers and terminals were more abundant than SP-positive nerves and terminals. CONCLUSION: The architecture of cat corneal nerves shows similarities to human and mouse cornea innervation. This study provides useful data for researchers who use the cat model to assess corneal nerve pathological alterations, as well as in the veterinary field where corneal opacities, ulcerations, and infections damage the nerves and decrease sensitivity.

Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

A variety of mouse strains and sexes are used in studies of corneal wound healing and nerve regeneration. However, there is a gap of knowledge about corneal nerve density and its function in different mouse strains and sexes. In this study, we report a strain divergence of total and substance P (SP) sensory corneal nerves in uninjured mice. The BALB/c mouse showed the highest nerve density, corneal sensitivity, and tear volume followed by CFW and then C57BL/6. No differences were found in total nerves and SP-positive nerves between sexes. After injury damaged the corneal nerves, an important role for mouse strains, biologic sex, and their association to corneal nerve regeneration was identified. All female mice have a faster nerve regeneration rate than males. The molecular mechanism of this sexual divergence involves higher secretion neurotrophic factors in tears, which in turn modulate gene expression in trigeminal ganglion neurons. An important upstream signaling regulator was ß-estradiol, and topical treatment with ß-estradiol confirmed its function in corneal nerve regeneration. In conclusion, our study shows that the strain and sex of laboratory mice significantly affect the different indicators of corneal innervation and nerve regeneration. Researchers investigating corneal diseases should carefully consider these factors.-Pham, T. L., Kakazu, A., He, J., Bazan, H. E. P. Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea-TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Recovery of Corneal Sensitivity and Increase in Nerve Density and Wound Healing in Diabetic Mice After PEDF Plus DHA Treatment.

Diabetic keratopathy decreases corneal sensation and tear secretion and delays wound healing after injury. In the current study, we tested the effect of treatment with pigment epithelium-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) on corneal nerve regeneration in a mouse model of diabetes with or without corneal injury. The study was performed in streptozotocin-induced diabetic mice (C57BL/6). Ten weeks after streptozotocin injection, diabetic mice showed significant decreases of corneal sensitivity, tear production, and epithelial subbasal nerve density when compared with age-matched normal mice. After diabetic mice were wounded in the right eye and treated in both eyes with PEDF+DHA for 2 weeks, there was a significant increase in corneal epithelial nerve regeneration and substance P-positive nerve density in both wounded and unwounded eyes compared with vehicle-treated corneas. There also was elevated corneal sensitivity and tear production in the treated corneas compared with vehicle. In addition, PEDF+DHA accelerated corneal wound healing, selectively recruited type 2 macrophages, and prevented neutrophil infiltration in diabetic wounded corneas. These results suggest that topical treatment with PEDF+DHA promotes corneal nerve regeneration and wound healing in diabetic mice and could potentially be exploited as a therapeutic option for the treatment of diabetic keratopathy.

PEDF plus DHA modulate inflammation and stimulate nerve regeneration after HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) infection leads to impaired corneal sensation and, in severe cases, to corneal ulceration, melting and perforation. Here, we explore the potential therapeutic action of pigment epithelial-derived factor (PEDF) plus docosahexaenoic acid (DHA) on corneal inflammation and nerve regeneration following HSV-1 infection. Rabbits inoculated with 100,000 PFU/eye of HSV-1 strain 17Syn+ were treated with PEDF + DHA or vehicle. PEDF + DHA treatment resulted in a biphasic immune response with stronger infiltration of CD4+T cells, neutrophils and macrophages at 7-days post-treatment (p.t.) that was significantly decreased by 14 days, compared to the vehicle-treated group. Screening of 14 immune-related genes by q-PCR showed that treatment induced higher expression of IFN-γ and CCL20 and inhibition of IL-18 by 7 days in the cornea. PEDF + DHA-treated animals developed less dendritic corneal lesions, opacity and neovascularization. Corneal nerve density increased at 12-weeks p.t. with functional recovery of corneal sensation. Treatment with PEDF + DHA that was postponed by 3 weeks also showed increased nerve density when compared to vehicle. Our data demonstrate that PEDF + DHA promotes resolution of the inflammatory response to the virus and, most importantly, induces regeneration of damaged corneal nerves vital for maintaining ocular surface homeostasis.

Changes in Corneal Innervation after HSV-1 Latency Established with Different Reactivation Phenotypes.

PURPOSE OF THE STUDY: We used a rabbit model infected with high phenotypic reactivators (HPRs) as well as recombinant HSV-1 (herpes simplex virus 1) with deletions to study their effect on corneal innervations after latency was established. MATERIALS AND METHODS: Corneas from noninfected New Zealand white rabbits were used to obtain the entire map of corneal innervation. Others were inoculated with the HSV-1 strains McKrae, 17Syn+, or recombinant mutants with glycoprotein K (gK) deletion, or with infected early protein 0 (ICP0) deletion. The animals were euthanized at 124 to 125 days postinfection and the corneas were immunostained with a mouse monoclonal anti-ßIII tubulin antibody. Images were acquired with a fluorescence microscope and corneal sub-basal nerve density was calculated on the basis of the whole mount images. Differences between the HSV-infected eyes, and comparison with normal control, were analyzed. RESULTS: In the noninfected rabbit, the stroma was densely innervated in the central area and as a consequence the sub-basal epithelial nerve bundles were shorter, and no vortex was found. The HSV-infected corneas showed nerve damage in both epithelial and stromal nerves. Corneas infected with ICP0 and gK deletion mutants showed mild to moderate damage, while those infected with 17Syn+ and McKrae strains were seriously damaged. In the eyes infected with ICP0 and gK deletion, there were reduced numbers of sub-basal nerve bundles, but most of the corneas retained a normal stromal network. Corneas infected with 17 Syn+ and McKrae displayed destroyed nerve structures and formation of a scar tissue in the central cornea, in which only a few nerve fibers could be detected. CONCLUSION: HSV-1 primary corneal infection seriously damages the corneal nerves, persisting for more than 4 months. Reduction of axonal transport (by gK deletion) or virus replication (by ICP0 deletion) significantly attenuated the nerve damage induced by the virus.

Neuroanatomy and Neurochemistry of Mouse Cornea.

PURPOSE: To investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation. METHODS: Mice aged 1 to 24 weeks were used. The corneas were stained with neuronal-class ßIII-tubulin, calcitonin gene-related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. To test the origin of CGRP and SP, trigeminal ganglia (TG) were processed for immunofluorescence. Relative corneal nerve fiber densities or neuron numbers were assessed by computer-assisted analysis. RESULTS: Between 1 and 3 weeks after birth, mouse cornea was mainly composed of a stromal nerve network. At 4 weeks, a whorl-like structure (or vortex) appeared that gradually became more defined. By 8 weeks, anatomy of corneal nerves had reached maturity. Epithelial bundles converged into the central area to form the vortex. The number and pattern of whorl-like structures were different. Subbasal nerve density and nerve terminals were greater in the center than the periphery. Nerve fibers and terminals that were CGRP-positive were more abundant than SP-positive nerves and terminals. In trigeminal ganglia, the number of CGRP-positive neurons significantly outnumbered those positive for SP. CONCLUSIONS: This is the first study to show a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the mouse cornea. This finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves.

The PEDF Neuroprotective Domain Plus DHA Induces Corneal Nerve Regeneration After Experimental Surgery.

PURPOSE: To compare a 44-mer pigment epithelial-derived factor (PEDF) peptide with neurotrophic activity, and a 34-mer PEDF with antiangiogenic properties in association with docosahexaenoic acid (DHA) in corneal nerve regeneration after experimental surgery. METHODS: A corneal stromal dissection was performed in rabbits. Treatment groups received topical 44-mer, 34-mer, or full PEDF plus DHA. Corneal sensitivity and Schirmer's test were performed weekly. Rabbits were euthanized at 2 and 4 days and 8 weeks. Two- and 4-day samples were stained for neutrophils and CD11b+ cells. Corneal nerves were stained with ßIII tubulin and calcitonin gene-related peptide (CGRP) antibodies in specimens collected at 8 weeks. Subepithelial nerve plexus density was calculated. A PEDF-receptor (PEDF-R) was analyzed in rabbit corneal epithelial cells (RCEC) by Western blot and immunofluorescence. RESULTS: Infiltration of CD11b+cells and neutrophils was reduced by treatment with 44-mer PEDF+DHA. A 3-fold increase in subepithelial corneal nerves and CGRP-positive nerves was found in the 44-mer PEDF+DHA-treated group compared with the 34-mer PEDF+DHA- and vehicle-treated groups. There was a 75% recovery of corneal sensitivity by week 7, and Schirmer's test reached control values in the 44-mer PEDF+DHA-treated corneas at 7 weeks. A PEDF-R protein with homology to calcium-independent phospholipase A2ς was expressed in RCEC. CONCLUSIONS: The 44-mer PEDF+DHA, but not the 34-mer PEDF+DHA, promotes functional regeneration of damaged corneal nerves. Forty four-mer PEDF, by activating a corneal epithelial receptor, in conjunction with DHA could be a novel therapeutic agent for the treatment of neurotrophic keratitis and dry eye that develops as a result of corneal nerve damage.

Morphology and neurochemistry of rabbit iris innervation.

The aim of this study was to map the entire nerve architecture and sensory neuropeptide content of the rabbit iris. Irises from New Zealand rabbits were stained with antibodies against neuronal-class ßIII-tubulin, calcitonin gene-related peptide (CGRP) and substance P (SP), and whole-mount images were acquired to build a two-dimensional view of the iridal nerve architecture. After taking images in time-lapse mode, we observed thick nerves running in the iris stroma close to the anterior epithelia, forming four to five stromal nerve rings from the iris periphery to the pupillary margin and sub-branches that connected with each other, constituting the stromal nerve plexus. In the anterior side, fine divisions derivated from the stromal nerves, forming a nerve network-like structure to innervate the superficial anterior border layer, with the pupillary margin having the densest innervation. In the posterior side, the nerve bundles ran along with the pupil dilator muscle in a radial pattern. The morphology of the iris nerves on both sides changed with pupil size. To obtain the relative content of the neuropeptides in the iris, the specimens were double stained with ßIII-tubulin and CGRP or SP antibodies. Relative nerve fiber densities for each fiber population were assessed quantitatively by computer-assisted analysis. On the anterior side, CGRP-positive nerve fibers constituted about 61%, while SP-positive nerves constitute about 30.5%, of the total nerve content, which was expressed as ßIII tubulin-positive fibers. In addition, in the anterior stroma of the collarette region, there were non-neuronal cells that were positive for SP. On the posterior side, CGRP-positive nerve fibers were about 69% of total nerve content, while SP constituted only up to 20%. Similarly, in the trigeminal ganglia (TG), the number of CGRP-positive neurons significantly outnumbered those that were positive for SP. Also, all the SP-positive neurons were labeled with CGRP. This is the first study to provide a two-dimensional whole mount and a cross-sectional view of the entire iris nerve architecture. Considering the anatomical location, the high expression of CGRP and SP suggests that these neuropeptides may play a role in the pathogenesis of anterior uveitis, glaucoma, cataracts and chronic ocular pain.

Differential effects of hepatocyte growth factor and keratinocyte growth factor on corneal epithelial cell cycle protein expression, cell survival, and growth.

PURPOSE: Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are secreted in the cornea in response to injury. In this study, we investigated the HGF- and KGF-mediated effect on the expression of cell cycle and apoptosis controlling proteins, cell survival, and growth in the corneal epithelium to better understand the possible role of their signaling mechanisms in repairing epithelial injuries. METHODS: The cell survival capability of HGF and KGF in epithelial primary cultures was evaluated by using a staurosporine-induced apoptosis model. Apoptosis was quantified with image analysis following nuclear staining with Hoechst fluorescent dye and DNA laddering. Western immunoblotting was used to study the effect of growth factors on the expression of cell cycle- and apoptosis-regulating proteins. RESULTS: HGF and KGF protected cells from apoptosis for a short duration (10 h), but only KGF exhibited cell survival capability and maintained cell growth for a longer period (24 h). The onset of apoptosis was accompanied by a significant increase in cell cycle inhibitor p27(kip). HGF and KGF suppressed p27(kip) levels in the apoptosis environment; however, KGF- but not HGF-dependent downregulation in p27(kip) expression was sustained for a longer duration. Inhibition of phosphatidylinositol 3-kinase/Akt activation blocked HGF- and KGF-mediated control of p27(kip) expression. Further, when compared to HGF, the presence of KGF produced significant downregulation of p53 and poly(adenosine diphosphate-ribose) polymerase, the key proteins involved in apoptosis and blocked the degradation of G1/S cell cycle progression checkpoint protein retinoblastoma. HGF and KGF upregulated the levels of p21(cip), cyclins A, D, and E and cyclin-dependent kinases (CDK2 and CDK4) as well, but the KGF-mediated effect on the expression of these molecules lasted longer. CONCLUSIONS: Sustained effect of KGF on cell survival and proliferation could be attributed to its ability to inhibit p53, retinoblastoma, caspases, and p27(kip) functions in apoptosis and cell cycle arrest and promote the expression of cell cycle progressing molecules for longer duration. Designing therapeutic strategies targeting cell cycle control through KGF may be beneficial for repairing difficult-to-heal corneal epithelial injuries that require sustained growth and cell survival promoting signals.