Diurnal Control of Sensory Axon Growth and Shedding in the Mouse Cornea.

Purpose: The circadian clock plays an important role in the expression and regulation of various genes and cellular processes in the body. Here, we study diurnal regulation of the growth and shedding of the sensory axons in the mouse cornea. Methods: Male and female BALB/cN mice were euthanized 90 minutes before and after the lights are turned on and off; at 5:30 AM, 8:30 AM, 5:30 PM, and 8:30 PM. Nerve terminal growth, shedding and overall axon density were assessed at these four time points using confocal imaging after staining axons in en face whole mount corneas with antibodies against ßIII tubulin, GAP43, and L1CAM. In addition, corneal epithelial cell proliferation, thickness, and desquamation were assessed using ki67, LAMP1, Involucrin, and ZO1. Results: Nerve terminal shedding took place between 5:30 AM and 8:30 AM and correlated positively with the timing of apical cell desquamation. After shedding the tips of the nerve terminals, axonal growth increased as indicated by increased axonal GAP43 expression. At 5:30 PM and 8:30 PM before and after the lights are turned off, cell proliferation was reduced, and epithelial thickness was maximal. Conclusions: Intraepithelial corneal nerve growth and shedding are under diurnal control regulated by the time of day and whether lights are on or off. Axons extend during the day and are shed within 90 minutes after lights are turned on. The data presented in this article shed light on the potential role that circadian clock plays in corneal pain and discomfort.

The impact of euthanasia and enucleation on mouse corneal epithelial axon density and nerve terminal morphology.

INTRODUCTION: Here we study the impact of using either CO2 gas or cervical dislocation (CD) for euthanasia and using different techniques to enucleate the eye on preserving axonal density and morphology of the intraepithelial corneal nerves (ICNs). OBJECTIVES: To determine whether using CO2 gas or CD for euthanasia and enucleating by cutting or pulling eyes out impacts axon density and nerve terminal morphology in the mouse cornea. METHODS: Mice were euthanized by CO2 gas or CD; the impact of delaying fixation for 5 min post-euthanasia was also assessed. We tested two different techniques to enucleate the eyes: cutting the optic nerve by curved scissors or pulling the eye out. A minimum of 10 corneas from 5 male and female BALB/c mice were used for each variable. Axons and intraepithelial corneal nerve terminals (ICNTs) were visualized utilizing ßIII tubulin and L1CAM and quantified using confocal microscopy. RESULTS: The variations seen in axon density between individual mice are not gender- or euthanasia-dependent. A significant reduction in axon density and loss of ICNT morphology are observed in eyes enucleated by pulling the optic nerve out. Similar results are obtained in male and female mice. CONCLUSION: While the variations tested in euthanasia do not affect axon density in male and female mouse corneas, enucleation by proptosing and gently cutting out the eyes yields increased axon density and improved ICNT morphology compared to pulling eyes out and leaving the optic nerve attached.

Parity Attenuates Intraepithelial Corneal Sensory Nerve Loss in Female Mice.

Aging impacts the ocular surface and reduces intraepithelial corneal nerve (ICN) density in male and female mice. Many researchers use retired breeders to study naturally aged female mice. Yet, the impact of parity and the length of time since breeders were retired on age-related changes in the intraepithelial corneal nerves is not known. Here we study 2 month (M) nulliparous (NP) females as well as 9M, 10M, and 11M NP and multiparous (MP) female mice to determine whether parity impacts the age-related decline seen in corneal axon density; 9M male mice are also included in these assessments. After showing that parity attenuates age-related loss in axon density, we also assess the impact of parity on corneal epithelial cell proliferation and find that it impacts cell proliferation and axon density normalized by cell proliferation. Stromal nerve arborization is also impacted by aging with parity enhancing stromal nerves in older mice. qPCR was performed on 20 genes implicated in ICN density using corneal epithelial RNA isolated from 10M NP and MP mice and showed that NGF expression was significantly elevated in MP corneal epithelium. Corneal sensitivity was significantly higher in 9M MP mice compared to NP mice and increased sensitivity in MP mice was accompanied by increased nerve terminals in the apical and middle cell layers. Together, these data show that parity in mice attenuates several aspects of the age-related decline seen on the ocular surface by retaining sensory axons and corneal sensitivity as mice age.

Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study.

The intraepithelial corneal nerves (ICNs) that innervate the corneal epithelium are maintained through interactions with corneal epithelial cells and the extracellular matrix they produce. One to several axons bundle together within the basal cell layer and extend parallel to the ocular surface or branch and extend apically. Here we use 3-dimentional (3D) ultrastructural reconstructions of control and trephine injured mouse corneal epithelium and stroma produced using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to determine whether corneal epithelial or immune cells resident in the epithelium remove axonal debris and degrade it in their lysosomes after trephine injury to the cornea. We demonstrate that axonal fragments are internalized in the corneal epithelium and accumulate within electron dense structures consistent with lysosomes 3 h after trephine injury in both epithelial and immune cells located among the basal cells of the trephine injured cornea. Confocal imaging showed fewer CD45+ immune cells within the corneal epithelium after trephine injury compared to controls. The resolution obtained using FIB-SEM also allowed us to show that the presence of sensory axons at the basal aspect of the epithelial basal cells close to the anterior aspect of the epithelial basement membrane (EBM) is associated with a focal reduction in EBM thickness. In addition, we show using FIB-SEM and confocal imaging that superficial trephine injuries that do not penetrate the stroma, damage the integrity of anterior stromal nerves. These studies are the first to look at the mouse cornea following nerve injury using FIB-SEM.

Transient Mitomycin C-treatment of human corneal epithelial cells and fibroblasts alters cell migration, cytokine secretion, and matrix accumulation.

A single application of Mitomycin C (MMC) is used clinically in ophthalmology to reduce scarring and enhance wound resolution after surgery. Here we show in vitro that a 3-hour MMC treatment of primary and telomerase immortalized human corneal limbal epithelial (HCLE) cells impacts their migration and adhesion. Transient MMC treatment induces HCLE expression of senescence associated secretory factors, cytokine secretion, and deposition of laminin 332 for several days. Transient MMC treatment also reduces migration and deposition of transforming growth factor-ß1 (TGFß1)-stimulated collagen by corneal fibroblasts. Using conditioned media from control and MMC treated cells, we demonstrate that factors secreted by MMC-treated corneal epithelial cells attenuate collagen deposition by HCFs whereas those secreted by MMC-treated HCFs do not. These studies are the first to probe the roles played by corneal epithelial cells in reducing collagen deposition by corneal fibroblasts in response to MMC.

Desmin deficiency is not sufficient to prevent corneal fibrosis.

The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model.

Reduced Corneal Innervation in the CD25 Null Model of Sjögren Syndrome.

Decreased corneal innervation is frequent in patients with Sjögren Syndrome (SS). To investigate the density and morphology of the intraepithelial corneal nerves (ICNs), corneal sensitivity, epithelial cell proliferation, and changes in mRNA expression of genes that are involved in autophagy and axon targeting and extension were assessed using the IL-2 receptor alpha chain (CD25 null) model of SS. ICN density and thickness in male and female wt and CD25 null corneas were assessed at 4, 6, 8, and 10/11 wk of age. Cell proliferation was assessed using ki67. Mechanical corneal sensitivity was measured. Quantitative PCR was performed to quantify expression of beclin 1, LC3, Lamp-1, Lamp-2, CXCL-1, BDNF, NTN1, DCC, Unc5b1, Efna4, Efna5, Rgma, and p21 in corneal epithelial mRNA. A significant reduction in corneal axon density and mechanical sensitivity were observed, which negatively correlate with epithelial cell proliferation. CD25 null mice have increased expression of genes regulating autophagy (beclin-1, LC3, LAMP-1, LAMP-2, CXCL1, and BDNF) and no change was observed in genes that were related to axonal targeting and extension. Decreased anatomic corneal innervation in the CD25 null SS model is accompanied by reduced corneal sensitivity, increased corneal epithelial cell proliferation, and increased expression of genes regulating phagocytosis and autophagy.

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding.

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.

Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells.

Cold atmospheric plasma (CAP), an ionized gas operated at near-ambient temperatures, has been introduced as a new therapeutic opportunity for treating cancers. The effectiveness of the therapy has been linked to CAP-generated reactive oxygen and nitrogen species such as hydrogen peroxide and nitrite. In this study, we monitor in real-time cancer cell response to CAP over the course of 48 h. The results demonstrate a correlation between cell viability, exposure time (30, 60, 90, and 180 s), and discharge voltage (3.16 and 3.71 kV), while stressing the likely therapeutic role of plasma-generated reactive species. A 30-60 s increase in CAP exposure time and/or a discharge voltage adjustment from 3.16 to 3.71 kV is consistently accompanied by a significant reduction in cell viability. Comparably, levels of hydrogen peroxide and nitrite vary as a function of voltage with elevated levels detected at the highest tested voltage condition of 3.71 kV. CAP ultimately initiates a reduction in cell viability and triggers apoptosis via damage to the mitochondrial membrane, while also deregulating protein synthesis. The findings presented in this study are discussed in the context of facilitating the development of an adaptive CAP platform which could improve treatment outcomes.

Reduced intraepithelial corneal nerve density and sensitivity accompany desiccating stress and aging in C57BL/6 mice.

Dry Eye disease causes discomfort and pain in millions of patients. Using a mouse acute desiccating stress (DS) model we show that DS induces a reduction in intraepithelial corneal nerve (ICN) density, corneal sensitivity, and apical extension of the intraepithelial nerve terminals (INTs) that branch from the subbasal nerves (SBNs). Topical application of 0.02% Mitomycin C (MMC) or vehicle alone has no impact on the overall loss of axon density due to acute DS. Chronic dry eye, which develops progressively as C57BL/6 mice age, is accompanied by significant loss of the ICNs and corneal sensitivity between 2 and 24 months of age. QPCR studies show that mRNAs for several proteins that regulate axon growth and extension are reduced in corneal epithelial cells by 24 months of age but those that regulate phagocytosis and autophagy are not altered. Taken together, these data demonstrate that dry eye disease is accompanied by alterations in intraepithelial sensory nerve morphology and function and by reduced expression in corneal epithelial cells of mRNAs encoding genes mediating axon extension. Précis: Acute and chronic mouse models of dry eye disease are used to evaluate the pathologic effects of dry eye on the intraepithelial corneal nerves (ICNs) and corneal epithelial cells. Data show reduced numbers of sensory nerves and alterations in nerve morphology, sensitivity, corneal epithelial cell proliferation, and expression of mRNAs for proteins mediating axon extension accompany the pathology induced by dry eye.

Alterations in Corneal Sensory Nerves During Homeostasis, Aging, and After Injury in Mice Lacking the Heparan Sulfate Proteoglycan Syndecan-1.

Purpose: To determine the impact of the loss of syndecan 1 (SDC1) on intraepithelial corneal nerves (ICNs) during homeostasis, aging, and in response to 1.5-mm trephine and debridement injury. Methods: Whole-mount corneas are used to quantify ICN density and thickness over time after birth and in response to injury in SDC1-null and wild-type (WT) mice. High-resolution three-dimensional imaging is used to visualize intraepithelial nerve terminals (INTs), axon fragments, and lysosomes in corneal epithelial cells using antibodies against growth associated protein 43 (GAP43), ßIII tubulin, and LAMP1. Quantitative PCR was performed to quantify expression of SDC1, SDC2, SDC3, and SDC4 in corneal epithelial mRNA. Phagocytosis was assessed by quantifying internalization of fluorescently labeled 1-µm latex beads. Results: Intraepithelial corneal nerves innervate the corneas of SDC1-null mice more slowly. At 8 weeks, ICN density is less but thickness is greater. Apically projecting intraepithelial nerve terminals and lysosome-associated membrane glycoprotein 1 (LAMP1) are also reduced in unwounded SDC1-null corneas. Quantitative PCR and immunofluorescence studies show that SDC3 expression and localization are increased in SDC1-null ICNs. Wild-type and SDC1-null corneas lose ICN density and thickness as they age. Recovery of axon density and thickness after trephine but not debridement wounds is slower in SDC1-null corneas compared with WT. Experiments assessing phagocytosis show reduced bead internalization by SDC1-null epithelial cells. Conclusions: Syndecan-1 deficiency alters ICN morphology and homeostasis during aging, reduces epithelial phagocytosis, and impairs reinnervation after trephine but not debridement injury. These data provide insight into the mechanisms used by sensory nerves to reinnervate after injury.

Corneal epithelial cells function as surrogate Schwann cells for their sensory nerves.

The eye is innervated by neurons derived from both the central nervous system and peripheral nervous system (PNS). While much is known about retinal neurobiology and phototransduction, less attention has been paid to the innervation of the eye by the PNS and the roles it plays in maintaining a functioning visual system. The ophthalmic branch of the trigeminal ganglion contains somas of neurons that innervate the cornea. These nerves provide sensory functions for the cornea and are referred to as intraepithelial corneal nerves (ICNs) consisting of subbasal nerves and their associated intraepithelial nerve terminals. ICNs project for several millimeters within the corneal epithelium without Schwann cell support. Here, we present evidence for the hypothesis that corneal epithelial cells function as glial cells to support the ICNs. Much of the data supporting this hypothesis is derived from studies of corneal development and the reinnervation of the ICNs in the rodent and rabbit cornea after superficial wounds. Corneal epithelial cells activate in response to injury via mechanisms similar to those induced in Schwann cells during Wallerian Degeneration. Corneal epithelial cells phagocytize distal axon fragments within hours of ICN crush wounds. During aging, the proteins, lipids, and mitochondria within the ICNs become damaged in a process exacerbated by UV light. We propose that ICNs shed their aged and damaged termini and continuously elongate to maintain their density. Available evidence points to new unexpected roles for corneal epithelial cells functioning as surrogate Schwann cells for the ICNs during homeostasis and in response to injury. GLIA 2017;65:851-863.

K14 + compound niches are present on the mouse cornea early after birth and expand after debridement wounds.

BACKGROUND: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re-epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. RESULTS: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q-PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. CONCLUSIONS: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds.

Topical Mitomycin-C enhances subbasal nerve regeneration and reduces erosion frequency in the debridement wounded mouse cornea.

Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.

Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea.

Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14 days after corneal debridement may destabilize newly reinnervated sub-basal axons and lead to their retraction toward the periphery.

Syndecan-1 and Its Expanding List of Contacts.

Significance: The binding of cytokines and growth factors to heparan sulfate (HS) chains on proteoglycans generates gradients that control development and regulate wound healing. Syndecan-1 (sdc1) is an integral membrane HS proteoglycan. Its structure allows it to bind with cytosolic, transmembrane, and extracellular matrix (ECM) proteins. It plays important roles in mediating key events during wound healing because it regulates a number of important processes, including cell adhesion, cell migration, endocytosis, exosome formation, and fibrosis. Recent Advances: Recent studies reveal that sdc1 regulates wound healing by altering integrin activation. Differences in integrin activation lead to cell-type-specific changes in the rate of cell migration and ECM assembly. Sdc1 also regulates endocytosis and the formation and release of exosomes. Critical Issues: Understanding how sdc1 facilitates wound healing and resolution will improve treatment options for elderly and diabetic patients with delayed wound healing. Studies showing that sdc1 function is altered in cancer are relevant to those interested in controlling fibrosis and scarring. Future Directions: The key to understanding the various functions ascribed to sdc1 is resolving how it interacts with its numerous binding partners. The role played by chondroitin sulfate glycosaminoglycan (GAG) chains on the ability of sdc1 to associate with its ligands needs further investigation. At wound sites heparanase can cleave the HS GAG chains of sdc1, alter its ability to bind cytokines, and induce shedding of the ectodomain. This review will discuss how the unique structure of sdc1 allows it to play key roles in cell signaling, ECM assembly, and wound healing.

Inhibition of amyloid precursor protein secretases reduces recovery after spinal cord injury.

Amyloid-ß (Aß) is produced through the enzymatic cleavage of amyloid precursor protein (APP) by ß (Bace1) and γ-secretases. The accumulation and aggregation of Aß as amyloid plaques is the hallmark pathology of Alzheimer׳s disease and has been found in other neurological disorders, such as traumatic brain injury and multiple sclerosis. Although the role of Aß after injury is not well understood, several studies have reported a negative correlation between Aß formation and functional outcome. In this study we show that levels of APP, the enzymes cleaving APP (Bace1 and γ-secretase), and Aß are significantly increased from 1 to 3 days after impact spinal cord injury (SCI) in mice. To determine the role of Aß after SCI, we reduced or inhibited Aß in vivo through pharmacological (using DAPT) or genetic (Bace1 knockout mice) approaches. We found that these interventions significantly impaired functional recovery as evaluated by white matter sparing and behavioral testing. These data are consistent with a beneficial role for Aß after SCI.

Wounding the cornea to learn how it heals.

Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors' expertise.

Cytokine deposition alters leukocyte morphology and initial recruitment of monocytes and γδT cells after corneal injury.

PURPOSE: An in vivo mouse model reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously 2 weeks after a single 1.5-mm corneal debridement wound made with a dulled blade. When 1.5-mm wounds are made by a rotating burr so that the corneal epithelial basement membrane is removed, corneas heal without developing erosions. Here, we characterize differences in cytokine deposition and changes in leukocytes between 0 and 6 hours after dulled-blade and rotating-burr wounding. METHODS: BALB/c mice were used to study 1.5-mm corneal wounds made using a dulled blade or a rotating burr. Mice were studied immediately after wounding (0 hour) and at 6 hours in vivo and in vitro in organ culture. Corneas, corneal extracts, and collagenase digests from naïve and wounded mice were used for three-dimensional (3D) confocal imaging, cytokine arrays, and flow cytometry. RESULTS: Confocal imaging showed CD45, a protein derived from leukocytes, accumulates at the wound edge by 3 and 6 hours after wounding in vivo but not in vitro with more CD45 accumulating after dulled-blade compared with rotating-burr wounds. Morphologic changes occurred in CD45+ leukocytes and higher levels for several cytokines were detected in the stromal wound bed within minutes following dulled-blade wounds. Flow cytometry showed significantly more monocytes (CD45+/CD11b+/Ly6C+) and γδT cells (CD45+/GL3+) recruited into the corneas of mice with dulled-blade wounds by 6 hours. CONCLUSIONS: Differences in cytokine-driven leukocyte responses are seen after dulled-blade debridement compared with rotating-burr injury.

Corneal goblet cells and their niche: implications for corneal stem cell deficiency.

Goblet cells are terminally differentiated cells secreting mucins and antibacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, nonproliferating cells, and postmitotic differentiated K12+Muc5ac+ goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.