Activated microglia in the human glaucomatous optic nerve head.

To investigate the distribution and potential participation of microglia, the resident defense cells of the central nervous system, in the optic nerve head (ONH) in glaucoma, histological paraffin sections of optic nerves from normal and glaucoma patients with mild to advanced nerve damage were studied using double labeling immunohistofluorescence. A monoclonal antibody for HLA-DR, indicating activated microglia, was colocalized with antibodies for functional proteins. In normal ONHs, microglia do not contain TGF-beta2, COX-2, or TNF-alpha and are not positive for PCNA; however, in glaucomatous ONHs, microglia contain abundant TGF-beta2, TNF-alpha, and PCNA. In glaucomatous eyes, a few microglia are usually positive for COX-2. In normal ONHs, there are rarely microglia containing TGF-beta1, NOS-2, TSP, TIMP-2, and CD68, but, in glaucomatous tissue, a few microglia are positive from the prelaminar to the postlaminar regions. MMP-1, MMP-2, MMP-3, and MMP-14 are constitutively present in the perivascular microglia in normal ONHs and appear to be more abundant in glaucomatous tissue. COX-1, TNF-R1, TIMP-1, and c-fms are constitutively present in normal tissues and appear to be increased in microglia in the glaucomatous ONHs. HSP27 is not present in microglia. In glaucomatous ONHs, microglia become activated and phagocytic and produce cytokines, mediators, and enzymes that can alter the extracellular matrix. Our findings suggest that activated microglia may participate in stabilizing the tissue early in the disease process, but, as the severity of the glaucomatous damage increases, the activities of microglia may have detrimental consequences for the pathological course of glaucomatous optic neuropathy.

Nitric oxide synthase-2 in human optic nerve head astrocytes induced by elevated pressure in vitro.

OBJECTIVE: To determine whether astrocytes of the human optic nerve head can induce nitric oxide synthase-2 (NOS-2) in response to elevated hydrostatic pressure as a mechanism for directly damaging the axons of the retinal ganglion cells in glaucoma. METHODS: Primary cultures of astrocytes from human optic nerve heads were placed in chambers, either pressurized at elevated hydrostatic pressure (60 mm Hg) or maintained at ambient pressure. The induction of NOS-2 was studied by immunocytochemistry, immunoblot, and semiquantitative reverse transcription polymerase chain reaction. RESULTS: In astrocyte cultures under ambient pressure, NOS-2 was almost undetectable. In astrocyte cultures under elevated hydrostatic pressure for 24, 48, and 72 hours, intensive labeling of NOS-2 in the Golgi body and the cytoplasm was observed by immunocytochemistry and intense bands of NOS-2 were detected by immunoblotting. As detected by semiquantitative reverse transcription polymerase chain reaction, the messenger RNA level of NOS-2 increased significantly in the astrocytes under elevated hydrostatic pressure within 12 hours, peaking earlier than the protein level of NOS-2. CONCLUSION: Elevated hydrostatic pressure induces the astrocytes of the human optic nerve head to express NOS-2. CLINICAL RELEVANCE: In glaucoma, the appearance of the neurodestructive NOS-2 in astrocytes of the optic nerve head may be a primary response to elevated intraocular pressure, in vivo, and therefore damaging to the axons of the retinal ganglion cells.

[Expression of nitric oxide synthase-2 in specific cells of human glaucomatous optic nerve head].

OBJECTIVE: To investigate the specific cells expressing nitric oxide synthase-2 (NOS-2) in the optic nerve head of patients with primary open angle glaucoma. METHODS: Double-labeling immunohistochemistry was used for labeling of NOS-2 and one of the specific cell markers. RESULTS: NOS-2 was labeled in the astrocytes. The NOS-2 positive astrocytes were mainly located in the damaged area of nerve fibers. A few arteries had NOS-2 labeling in the endothelial cells. There was no significant labeling of NOS-2 in microglia, vascular smooth muscle cells and pericytes. CONCLUSION: In glaucomatous optic nerve neuropathy, NOS-2 is mainly expressed by astrocytes. Astrocytes may play an important role in the local neurotoxicity of axons of the ganglion cells by producing excessive nitric oxide.

Tumor necrosis factor-alpha: a potentially neurodestructive cytokine produced by glia in the human glaucomatous optic nerve head.

Tumor necrosis factor-alpha (TNF-alpha) mediates a range of cellular responses, which have potentially detrimental consequences that affect multiple cell types. To determine whether TNF-alpha contributes to glaucomatous optic neuropathy, we have studied the expression of this cytokine and its receptor, tumor necrosis factor receptor-1 (TNF- R1), in human glaucomatous optic nerve heads from patients with different stages of disease using double labeling fluorescence immunohistochemistry. We have also investigated the ability of this cytokine to induce nitric oxide synthase (NOS-2) in cultured human optic nerve astrocytes by immunocytochemistry and immunoblot. Normal tissue showed constitutive expression of TNF-R1 in the vasculature of the optic nerve heads but no positive labeling for TNF-alpha. In the glaucomatous optic nerve heads, the expression of both TNF-alpha and TNF-R1 were apparently upregulated, primarily in glial fibrillary acidic protein (GFAP)-positive astrocytes, and appeared to parallel the progression of optic nerve degeneration. In eyes with severe glaucomatous damage, some HLA-DR positive microglia also contained TNF-alpha and TNF-R1. In the most severely damaged optic nerve heads, the axons of the retinal ganglion cells contained TNF-R1 and, therefore, are direct targets for neurodegeneration caused by TNF-alpha. In vitro astrocytes constitutively express TNF-R1 and TNF-alpha stimulation induces expression of NOS-2. We hypothesize that TNF-alpha contributes to the progression of optic nerve degeneration in glaucoma by both a direct effect on the axons of the retinal ganglion cells and by inducing NOS-2 in astrocytes.

Expression of nitric oxide synthase-2 (NOS-2) in reactive astrocytes of the human glaucomatous optic nerve head.

Inducible nitric oxide synthase (NOS-2) is abundantly present in the optic nerve heads of glaucoma patients. To determine the regulation of NOS-2 expression in the glaucomatous optic nerve head, the specific cells that express NOS-2 in the optic nerve heads of patients with primary open-angle glaucoma were studied by immunohistochemical double-labeling of NOS-2 and one of the characteristic cell markers for different cell types. Most of the labeling for NOS-2 was identified in reactive astrocytes that were clustered in the areas of nerve damage in the prelaminar and lamina cribrosa regions of the glaucomatous optic nerve heads. In vitro, the expression of GFAP and NOS-2 by reactive astrocytes of human optic nerve heads was demonstrated by immunocytochemistry and Western blot. In primary cultures of human lamina cribrosa astrocytes, stimulation by interferon-gamma and interleukin-1beta upregulated GFAP and induced expression of NOS-2 protein. At 24, 48 and 72 h of stimulation, NOS-2 appeared first in the Golgi body and then was sent out into the cytoplasm in granules. These results demonstrated that the astrocytes of human optic nerve head are capable of inducing the expression of NOS-2. Reactive astrocytes in the glaucomatous optic nerve heads apparently play an important role in local neurotoxicity to the axons of the retinal ganglion cells by producing excessive nitric oxide in glaucomatous optic neuropathy.

Cellular localization of neuronal nitric oxide synthase (NOS-1) in the human and rat retina.

The neuronal form of nitric oxide synthase (NOS-1) has been localized to several cell types in the retinas of experimental animals; however, localization in the human retina has not been definitive. By using in situ hybridization and immunohistochemistry, we have compared the cellular expression and localization of NOS-1 in the rat and human retinas. In both rat and human retinas, NOS-1 is expressed in the inner segments of photoreceptors, cells in the inner nuclear layer, particularly amacrine cells, and retinal ganglion cells. In human cones, NOS-1 is abundantly present in the outer segments. In the rat, optic nerve transection caused a loss of cells that were positive for NOS-1 in the ganglion cell layer. Although a retinal ganglion cell localization has not been reported consistently in the literature, our data clearly localize NOS-1 to the retinal ganglion cells of the rat and human retinas.

Confirmation of the rat model of chronic, moderately elevated intraocular pressure.

We have confirmed the usefulness of the rat model of chronic, moderately elevated intraocular pressure (IOP) for studying loss of retinal ganglion cells, and as a model for pharmacological neuroprotection studies that may be relevant to treating human glaucoma. By unilaterally cauterizing three episcleral vessels, as described previously in the literature by another laboratory, we observed an approximately 1.6-fold increase in IOP compared to the contralateral eye (18.6 vs 11.5 mm Hg, respectively). Elevated IOP persisted for 6 months without re-treatment. Cupping of the optic disk was observable by examination, in vivo. In 6 months, there was an approximately 40% loss of retinal ganglion cells in the peripheral retina. This model provides a reproducible and quantitative model for pharmacological experiments using neuroprotective agents.

Isoforms of nitric oxide synthase in the optic nerves of rat eyes with chronic moderately elevated intraocular pressure.

PURPOSE: To investigate the hypothesis that nitric oxide (NO) in the optic nerve heads of rats with chronic moderately elevated intraocular pressure (IOP) contributes to neurotoxicity of the retinal ganglion cells, the presence of the three isoforms of nitric oxide synthase (NOS) was determined in the tissue. METHODS: Unilateral chronic moderately elevated IOP was produced in rats by cautery of three episcleral vessels. Histologic sections of optic nerves from eyes with normal IOP and with chronic moderately elevated IOP were studied by immunohistochemistry and by immunoblot analysis. Polyclonal antibodies to NOS-1, NOS-2, NOS-3, and glial fibrillary acidic protein (GFAP) were localized with immunoperoxidase. RESULTS: In the optic nerve of rat eyes with normal IOP, NOS-1 was constitutively present in astrocytes, pericytes and nerve terminals in the walls of the central artery. NOS-2 was not present in eyes with normal IOP. In these eyes, NOS-3 was constitutively present in the vascular endothelia of large and small vessels. Rat eyes treated with three-vessel cautery had sustained elevated IOP (1.6 fold) for at least 3 months. In these eyes, no obvious changes in NOS-1 or NOS-3 were noted. However, at time points as early as 4 days of chronic moderately elevated IOP, NOS-2 appeared in astrocytes in the optic nerve heads of these eyes and persisted for up to 3 months. Immunoblot analysis did not detect differences in NOS isoforms. CONCLUSION: The cellular distributions of constitutive NOS isoforms in the rat optic nerve suggest physiological roles for NO in this tissue. NOS-1 in astrocytes may produce NO as a mediator between neighboring cells. NO, produced by NOS-1 in pericytes and nitrergic nerve terminals and by NOS-3 in vascular endothelia, is probably a physiological vasodilator in this tissue. In eyes with chronic moderately elevated IOP, NOS-2 is apparently induced in astrocytes. The excessive NO production that is associated with this isoform may contribute to the neurotoxicity of the retinal ganglion cells in eyes with chronic moderately elevated IOP.

Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma.

Glaucoma is an optic neuropathy with cupping of the optic disk, degeneration of retinal ganglion cells, and characteristic visual field loss. Because elevated intraocular pressure (IOP) is a major risk factor for progression of glaucoma, treatment has been based on lowering IOP. We previously demonstrated inducible nitric-oxide synthase (NOS-2) in the optic nerve heads from human glaucomatous eyes and from rat eyes with chronic, moderately elevated IOP. Using this rat model of unilateral glaucoma, we treated a group of animals for 6 months with aminoguanidine, a relatively specific inhibitor of NOS-2, and compared them with an untreated group. At 6 months, untreated animals had pallor and cupping of the optic disks in the eyes with elevated IOP. Eyes of aminoguanidine-treated animals with similar elevations of IOP appeared normal. We quantitated retinal ganglion cell loss by retrograde labeling with Fluoro-Gold. When compared with their contralateral control eyes with normal IOP, eyes with elevated IOP in the untreated group lost 36% of their retinal ganglion cells; the eyes with similarly elevated IOP in the aminoguanidine-treated group lost less than 10% of their retinal ganglion cells. Pharmacological neuroprotection by inhibition of NOS-2 may prove useful for the treatment of patients with glaucoma.

Microglia in the optic nerve head and the region of parapapillary chorioretinal atrophy in glaucoma.

BACKGROUND: Microglia, the macrophages and immune surveillance cells of the central nervous system, are quiescent normally but become activated in injured neural tissue. We have determined the distribution and potential participation of microglia in glaucomatous optic nerve degeneration. METHODS: Microglia were localized by immunohistochemistry on paraffin sections of age-matched normal and glaucomatous human eyes obtained within 24 hours after death. Monoclonal and polyclonal antibodies that recognize specific epitopes on microglia and other cell types were localized by immunoperoxidase and immunofluorescence. RESULTS: Stellate cells with thin, ramified processes, positive for HLA-DR and CD45 but negative for glial fibrillary acid protein, were identified as quiescent microglia. These cells were found throughout the normal optic nerve head in the walls of large blood vessels and surrounding capillaries in glial columns and cribriform plates. In glaucomatous eyes with moderate and severe optic nerve head damage, microglia were present as clusters of large ameboid, activated cells in the compressed lamina cribrosa and as formations of concentric circles surrounding blood vessels. In the parapapillary chorioretinal region of glaucomatous optic nerve heads, large, activated microglia were present as single cells or clusters on the termination of the Bruch's membrane. In addition, along the optic nerve/choriocapillaris-scleral interface, activated microglia appeared to form linear arrays near the choriocapillaris vessels. These cells were parenchymal and not in close association with the vasculature. CONCLUSIONS: In glaucoma, microglia in the optic nerve head become activated and redistributed. Enlarged, activated microglia appear in the parapapillary chorioretinal region, perhaps due to migration from the disorganized prelaminar and laminar tissue. Strategically positioned microglia may also serve a neuroprotective function in relation to a damaged blood-retinal barrier. The activity of microglia in the parapapillary chorioretinal region in glaucoma may be responsible for some of the biomicroscopic and histological changes that are associated with parapapillary chorioretinal atrophy.

Nitric oxide: a potential mediator of retinal ganglion cell damage in glaucoma.

In the glaucomatous optic nerve head, excessive nitric oxide (NO) may be responsible, at least in part, for degeneration of axons of retinal ganglion cells. We have demonstrated the apparent up-regulation and induction of certain isoforms of nitric oxide synthase (NOS), the enzyme that synthesizes NO, in astrocytes in the prelaminar and lamina cribrosa regions of optic nerve heads of patients with glaucoma. Evidence of NO toxicity, histochemical staining for nitrotyrosine, is present in these damaged optic nerve heads. In rats with experimentally induced, moderately elevated intraocular pressure, the isoforms of NOS were also identified.

Effect of ticrynafen on aqueous humor dynamics in monkeys.

OBJECTIVE: To determine the effect of ticrynafen, a nonsulfhydryl-reactive compound similar to ethacrynic acid, on outflow facility in normotensive monkey eyes and on intraocular pressure (IOP) in monkey eyes with laser-induced glaucoma. METHODS: In normotensive eyes, facility (perfusion) was measured shortly before and after bolus or exchange intracameral infusion of ticrynafen or vehicle in opposite eyes, and 3.5 to 4.5 hours after 5 days of twice-daily 2% ticrynafen or vehicle ointment. In glaucomatous eyes, baseline and vehicle diurnal IOP curves were established, 2% ticrynafen ointment was given twice daily for 5 days, and IOP was measured immediately before and 0.5 to 6 hours after each morning treatment. RESULTS: In normotensive eyes, exchange 2-mL influsion of 0.2-, 1-, or 4-mmol/L ticrynafen increased facility by 33% +/- 6% (mean +/- SEM), 73% +/- 18%, and 60% +/- 11%, respectively. Day 5 posttreatment facility was higher in the ticrynafen group than in controls by 28% +/- 9%. In glaucomatous eyes, maximum IOP decline, from approximately 35 mm Hg, was 7.5 +/- 2.0 mm Hg on day 4 and 9.8 +/- 2.4 mm Hg on day 5 of twice-daily ticrynafen treatment. CONCLUSION: The facility-increasing, IOP-lowering action of ticrynafen, ethacrynic acid, and derivatives may not depend entirely on sulfhydryl reactivity. CLINICAL RELEVANCE: Whether such drugs as ethacrynic acid and ticrynafen prove valuable for glaucoma therapy, at the least they are useful probes to study aqueous outflow mechanisms.

Nitric oxide synthase in the human glaucomatous optic nerve head.

OBJECTIVES: To investigate the hypothesis that nitric oxide contributes to neurotoxicity in the optic nerve heads of patients with primary open-angle glaucoma, we have determined the presence of the 3 isoforms of nitric oxide synthase (NOS) in the tissue. METHODS: Histological sections of optic nerve heads from normal subjects and patients with glaucoma who have moderate to advanced nerve damage were studied by immunohistochemistry. Polyclonal antibodies to NOS-1, NOS-2, and NOS-3 were localized with immunoperoxidase staining. RESULTS: In normal eyes, NOS-1 is sparsely present in astrocytes throughout the optic nerve head. In glaucomatous optic nerve heads, almost every astrocyte is positive for NOS-1. Nitric oxide synthase-1 immunoreactivity is abundantly present throughout the prelaminar region and the lamina cribrosa and is localized inside the diminished nerve fiber bundles. Nitric oxide synthase-2 is present in a few cells in the disorganized lamina cribrosa of the glaucomatous eye and is not present at all in normal tissue. Nitric oxide synthase-3 is present in normal eyes in the vascular endothelia of small blood vessels of the prelaminar region. In glaucomatous tissue, NOS-3 is present in astrocytes and in the vascular endothelia of large and small vessels. CONCLUSIONS: The 3 isoforms of NOS are present in apparently increased amounts in the optic nerve head of patients with primary open-angle glaucoma. The increased presence of NOS-1 and the induction of NOS-2 in astrocytes of the lamina cribrosa suggest that the glaucomatous optic nerve head is exposed to excessive levels of nitric oxide, which may be neurodestructive, locally, to the axons of the retinal ganglion cells. Conversely, the increased presence of NOS-3 in vascular endothelia may be neuroprotective by causing vasodilation and increased blood flow in the tissue.

Cyclooxygenase-1 and cyclooxygenase-2 in the human optic nerve head.

To investigate the hypothesis that eicosanoids act as cellular mediators in the optic nerve head of normals and of patients with glaucoma, we have determined the presence of the two cyclooxygenase (COX) isoforms in human tissue. Histological sections of optic nerve heads were studied by immunohistochemistry. Age matched normal donors were compared with eyes from glaucoma patients with moderate to severe nerve damage. Polyclonal antibodies to human COX-1 and COX-2 were localized with immunoperoxidase staining. Specific antibodies for vascular endothelia and microglia were also colocalized. In normal and glaucomatous eyes. COX-1 was localized exclusively to the prelaminar and lamina cribrosa regions of the optic nerve head. No staining for COX-1 was observed in the nerve fiber layer or the myelinated optic nerve. COX-1 was associated with the astrocytes of the glial columns and the cribriform plates, but not with the endothelia lining the capillaries. In glaucoma, more astrocytes appeared to be stained with antibody to COX-1 than in normals and staining was intensely perinuclear. There was no staining for COX-2 in normal tissue. A few COX-2 positive cells were found in the prelaminar, lamina cribrosa and postlaminar regions of the glaucomatous optic nerves. Positive staining for COX-2 was not associated with microglia. COX-1 is constitutively present in astrocytes that are localized exclusively to the prelaminar and lamina cribrosa regions of the human optic nerve head. Eicosanoids, synthesized by COX-1 in this tissue, may have a homeostatic and a neuroprotective role related to the axons of the retinal ganglion cells. The sparse presence of COX-2 in glaucomatous tissue probably reflects the lack of inflammation associated with glaucomatous optic neuropathy.

Effects of topical ethacrynic acid ointment vs timolol on intraocular pressure in glaucomatous monkey eyes.

OBJECTIVE: To evaluate the long-term effects of ethacrynic acid (ECA) ointment, compared with timolol maleate on intraocular pressure (IOP) in cynomolgus monkey eyes with argon laser-induced glaucoma. METHODS: In a 5-day study, IOP was measured for 7 hours after once-daily topical applications of ECA ointment to four glaucomatous monkey eyes. For this study, ECA ointment was given in 0.5%, 1.0%, 1.5%, or 2.5% concentrations. In a separate 30-day study, IOP was measured after once-daily topical applications of ECA ointment in concentrations of 0.75% or 1.5%. The results were compared with IOP after the application of 0.5% timolol maleate administered twice daily on weekdays and once daily on weekends. RESULTS: In the 5-day study, 2.5% ECA ointment had the greatest effect on lowering IOP, with a maximum reduction of 8.5 +/- 2.9 mm Hg (mean +/- SEM). A more pronounced reduction in IOP was observed on the fifth day of treatment for each of the four concentrations. In the 30-day study, 1.5% ECA ointment or 0.5% timolol maleate reduced IOP as much as 11.5 +/- 3.7 mmHg and 14.0 +/- 4.5 mmHg, respectively. With repetitive dosing, the effect on IOP after using 1.5% ECA ointment increased with time. Mild eyelid edema, conjunctival hyperemia, and discharge were observed in some eyes treated with the highest concentrations. One eye of four treated with 1.5% ECA ointment for 30 days developed a superficial corneal erosion in the 30-day study. CONCLUSIONS: The ECA ointment reduced IOP in glaucomatous monkey eyes. This reduction was evident by the fifth day of treatment with all the concentrations tested. The reduction in IOP produced by once-daily treatment with 1.5% ECA ointment was comparable with that of 0.5% timolol maleate administered twice daily. Therefore, drugs in this class of compound may prove to be useful in glaucoma therapy.

Collagen type I mRNA levels in cultured human lamina cribrosa cells: effects of elevated hydrostatic pressure.

In primary open-angle glaucoma, elevated intraocular pressure is associated with distortion and reorganization of the connective tissue plates of the lamina cribrosa. We have previously postulated that the resident cells of the lamina cribrosa may respond to elevated intraocular pressure by altering the biosynthesis or degradation of extracellular matrix. To determine the response of lamina cribrosa cells to increased pressure, we have compared cultures of human lamina cribrosa cells, from five individuals, maintained under control and pressurized conditions in vitro. Cells from third to fifth passage cultures of human lamina cribrosa subjected to elevated hydrostatic pressure (50 mm Hg) for 7 days changed shape from flat and polygonal to elongated, synthesized and secreted increased amounts of collagen type I as shown by immunofluorescent localization, and exhibited increased mRNA levels of collagen type I (199 +/- 36% of control) (mean +/- S.D.), as determined by slot-blot hybridization. In contrast, beta-actin mRNA levels were unchanged, indicating that the effects of elevated pressure are probably relatively selective. Our data indicate that elevated pressure increases the synthesis of collagen Type I by human lamina cribrosa cells in vitro. In vivo, lamina cribrosa cells may react to changes in their environment by modulating, specifically, changes in the mRNA levels, production, and secretion of extracellular matrix macromolecules. The relationship of these changes in extracellular matrix to those observed in glaucoma remains to be determined.

Localization of collagen types I and IV mRNAs in human optic nerve head by in situ hybridization.

Using in situ hybridization, individual cells expressing mRNAs for collagen types I and IV were localized in fixed-tissue sections of adult and fetal human optic nerve heads. Astroglial cells lining the cribriform plates and cells inside the cribriform plates of the lamina cribrosa had mRNA for collagen type IV. Cells in the glial columns, pial septa, and vascular wall also contained mRNA collagen type IV. Collagen type I mRNA was expressed by cells of the cribriform plates of the lamina cribrosa of adults. Few cells in the glial columns, pial septa, and blood vessels had mRNA for collagen type I. Scleral fibroblasts contained mRNA for collagen type I. These results indicated that the expression of mRNA for both collagen types I and IV paralleled the localization of these extracellular matrix proteins in the optic nerve head and suggested that both collagen types were synthesized in this tissue throughout life.

In vitro pharmacologic separation of corneal endothelial migration and spreading responses.

Repair of corneal endothelial wounds involves two forms of cell translocation: (1) "migration," in which individual cells at the wound edge break contacts with neighboring cells and move as individuals into the wound defect, and (2) "spreading," in which cells within the confluent monolayer adjacent to the wound move as a group into the wound area. The authors combined morphometric analysis of Giemsa-stained cultures, phase-contrast video microscopy, and Rh-phalloidin staining of actin filaments to study the effects of epidermal growth factor (EGF) and indomethacin on the migratory and spreading responses to wounding using an in vitro wound-closure model which mimics the amitotic state and general behavior of human corneal endothelium. They found that EGF stimulated the migration of individual cells from the wound edge, induced cellular elongation, and promoted a diffuse distribution of actin filaments. Indomethacin promoted spreading of the confluent monolayer into the wound defect, induced enlargement and flattening of cells, and promoted the formation of long, thick actin stress fibers. These results provide evidence that the migration and spreading responses of corneal endothelial cells to wounding can be pharmacologically separated. The findings suggest that migration of individual cells during wound repair may result from an endogenous form of EGF-like stimulation and that the elongated shape associated with this form of translocation results, at least in part, from an EGF-like alteration in actin-filament organization. Spreading of the confluent monolayer to cover the wound defect may result from a decrease in cyclic adenosine monophosphate induced by a transient reduction in prostaglandin E2 synthesis. This form of translocation may result, in part, from enlargement and flattening of corneal endothelial cells secondary to an enhancement of actin stress-fiber formation.