Dynamics of corneal epithelial healing after an alkali burn. A statistical analysis

A precise definition of epithelial healing kinetics following chemical injury is necessary to aid the investigation of control mechanisms, potential therapeutic intervention and ophthalmic drug toxicity. Wound healing was studied photographically at frequent intervals in rabbits following 1N or 4N alkali burns. Planar wound areas were determined by computerized planimetry and transformed mathematically to curved surface data. The decrease in equivalent wound radius with time was computer-modelled using two linear and three nonlinear regressions. A periodic function was also investigated. Serial photographs showed that intermittent attrition of small areas of the migrating wound edge was a common confounding variable. Although excellent coefficients of determination were found for all models, the addition of nonlinear factors gave a small advantage. A mean lag phase of 3.96 and 6.52 hr occurred after 1N and 4N alkali burns, respectively; wound edge attrition was notably prevalent in early healing. Epithelial healing in the rabbit had a fundamental linear component, with mean epithelial migration rates of 76 and 80 microns/hr after 1N and 4N alkali burns, respectively, and which continued to closure. A quadratic nonlinear component was also suggested. No significant circadian component was detected.     

Role of fibronectin and fibrinogen in healing of corneal epithelial scrape wounds

A provisional fibronectin (Fn)-fibrinogen (Fg) matrix forms de novo on the bare basement membrane (BM) surface of superficial epithelial scrape wounds in rabbit and guinea pig (GP) corneas. We determined whether such a substrate is essential for epithelial cell adhesion and migration, using three different approaches. (1) Polyclonal and monoclonal IgG reactive against GP Fn were topically administered to inhibit Fn formation in a GP epithelial scrape wound model. Immunofluorescence studies showed that deposition of both Fn and Fg was inhibited in antibody-treated corneas. During the first 38 hr after wounding, the healing rates were 0.46 +/- 0.06 mm2/hr in control eyes, 0.43 +/- 0.05 mm2/hr in those treated with rabbit polyclonal IgG anti-GP Fn and 0.45 +/- 0.09 mm2/hr in those treated with murine monoclonal IgG anti-GP Fn (P greater than 0.4). (2) In a rabbit epithelial scrape wound model, ancrod was administered intravenously to induce systemic Fg depletion. Fg deposition was completely inhibited on the wound surface, but Fn deposition was not suppressed. The healing rate was 1.24 +/- 0.41 mm2/hr in ancrod-treated corneal wounds and 1.19 +/- 0.29 mm2/hr in control eyes during the first 48 hr after wounding (P greater than 0.5). These data from the antibody and ancrod inhibition indicate that Fg binds to Fn and that Fn binds to components other than Fg.(ABSTRACT TRUNCATED AT 250 WORDS)     

A larger corneal epithelial wound closes at a faster rate

In order to evaluate relationships between healing rates and initial wound area, epithelial wounds were made on rabbit corneas by scraping the epithelium within a 4-, 6.5-, or 8-mm trephine mark. The wounds were stained with fluorescein and photographed during healing. The wounded areas were measured by planimetry. Although larger wounds closed later than smaller wounds, all of the healing curves appeared to be linear. The mean healing rate of the 8-mm diameter wounds (0.91 mm2/hr) was significantly greater than that of the 6.5-mm diameter wounds (0.80 mm2/hr). The 4-mm diameter wounds healed at a significantly slower rate (0.37 mm2/hr) when compared to the 6.5-mm diameter wounds. The authors found a strong positive correlation between the healing rates and the initial wound areas. By comparison, regardless of the initial wound area, the wound diameter decreased at a rate of approximately 0.1 mm/hr, which may explain the dependency of the healing rate on the initial wound area. The healing rate varied considerably between animals with the same diameter wounds, but both eyes of each animal showed a similar healing rate.     

Effect of a collagen shield on cat corneal epithelial wound healing

We have previously demonstrated that a corneal bandage lens made from porcine scleral collagen may be useful in treating various ocular surface problems. In order to determine whether the collagen shield would accelerate epithelial healing, a 7 mm diameter circular area in the center of the left cornea of ten domestic cats was mechanically deepithelialized. In five of the cats, a 14.5 mm non-cross-linked collagen shield was then placed on the cornea covering the wound. Another shield was applied 24 hr after surgery. The wound size was determined immediately after surgery and at 8-hr intervals until wound closure. Using analysis of variance for experiments with repeated observations, there was a significantly greater healing response in the treated group than in the control group. There was, however, no significant difference in slope between the two groups, suggesting that the shield did not increase the speed of epithelial cell migration. Rather, the effect of the shield was most pronounced during the first 8 hr after wounding. In contrast to that of the treated group, the mean defect radius of the control group was larger at t = 8 hr than at t = 0 hr. The earlier wound closure exhibited by the treated group, which may be due to protection and lubrication of the epithelial cells at the margins of the fresh wound, suggests that the collagen shield may be useful in treating corneal surface conditions of which deepithelialization is a component.     

Role of fibronectin in the healing of superficial keratectomies in vitro

A fibronectin (Fn)-fibrinogen (Fg) surface matrix is not essential for epithelial cell migration in a corneal epithelial scrape wound model, in which the basement membrane is preserved. We have therefore tested whether such a provisional scaffolding becomes more critical in a superficial keratectomy model, when the basement membrane is surgically removed. Exogeneous Fn at 0.3 mg/ml was added to the medium of organ cultures of rabbit superficial keratectomies. At 48 hr after wounding, the healing rate was 1.12 +/- 0.03 mm2/hr in control corneas and 1.11 +/- 0.03 mm2/hr in those cultured with Fn. At 64 hr after wounding, the healing rates were also not significantly different (P greater than 0.5). Immunofluorescence studies showed that Fn was not detectable on the surface of control corneas but could be depicted under the migrating epithelium. In corneas cultured with Fn, it diffused throughout the entire stroma but did not deposit as a surface matrix. We therefore attempted to obtain formation of a provisional Fn-Fg surface matrix before establishment of the in vitro organ culture by leaving the superficial keratectomies in vivo for 8 hr, 24 hr or 64 hr. At 64 hr after wounding, their healing rate was 0.80 +/- 0.04 mm2/hr, 0.86 +/- 0.04 mm2/hr and 0.85 +/- 0.06 mm2/hr, respectively, which were not significantly different from that of contralateral ex vivo-wounded cultured corneas (0.83 +/- 0.04 mm2/hr, P greater than 0.5). Immunofluorescence studies revealed a Fn matrix on the bare surface of in vivo-wounded specimen, which was not detectable on ex vivo-wounded cultured corneas; there was also no diffuse stromal Fn.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

New fixation device for photorefractive surgery and its effect on corneal epithelial wound healing.

PURPOSE: A new eyeball fixation device during photorefractive surgery was designed and tested. The device fixates the eyeball by means of a suction ring, and is then fixated to the headrest of the patient's chair via clipper and metal frames. METHODS: Photorefractive keratectomy (PRK) was performed on PMMA contact lenses placed over the patient's cornea (n=6) to evaluate smoothness of the ablated surface and on rabbit (n=24) and patient (n=30) corneas for evaluation of wound healing time. Decentration with fixation was examined using videokeratography after PRK. RESULTS: After fixation, only small amounts of corneal movement from the patient's pulsating heart were noted. The mean smoothness (root mean square) of the PMMA contact lens ablated surface was 0.43 +/- 0.16 microm in non-fixated eyes and 0.26 +/-0.05 microm in fixated eyes. Mean epithelial healing rate was 47.93 +/- 21.80 microm/hr in non-fixated rabbit eyes and 66.49 +/- 20 microm/hr in fixated rabbit eyes. Mean epithelial healing time was 3.47 +/- 1.11 days in non-fixated human eyes and 2.53 +/- 0.51 days in fixated human eyes. Mean decentration after PRK was 0.30 +/- 0.28 mm in fixated human eyes. CONCLUSION: Fixating the eyeball allows less movement of the eye and achieves a smoother ablation surface for more rapid epithelial healing after PRK.     

An organ culture system for study of adherence of Pseudomonas aeruginosa to normal and wounded corneas

An organ culture system has been developed to study the adherence of Pseudomonas aeruginosa to unwounded corneas and to corneas healing after a 3 mm central epithelial debridement. The Pseudomonas strain was isolated from a human corneal ulcer; suspensions containing 1 X 10(8) colony-forming units/ml (CFU/ml) of bacteria were incubated with the corneas for the last 30 min of the 18 hr culture period. The distribution pattern and number of adherent bacteria on the ocular surface were determined by morphometric analysis of scanning electron micrographs. Few bacteria (25 +/- 15/mm2) adhered to the apical cells of unwounded corneas. There was a definite region-specific distribution of adherent bacteria on healing corneas. There was a definite region-specific distribution of adherent bacteria on healing corneas. Most bacteria were found on the denuded basal lamina in front of the leading edge of the migrating epithelium (360,700 +/- 49,000/mm2). Appreciable but lower numbers adhered to the apical membrane of leading-edge cells (37,700 +/- 6,100/mm2) and to the central portion of the denuded basal lamina (28,800 +/- 10,700/mm2). No bacteria were found adherent to the apical cells of the stratified epithelium behind the leading edge of the epithelium migrating to cover the wound. A similar region-specific distribution of adherent bacteria was found when corneas were inverted in the bacterial suspension and when corneas were incubated in the bacterial suspension for 15 rather than 30 min. Corneas preincubated with the lectin, succinyl-concanavalin A, showed significantly decreased bacterial adherence, indicating a possible role for mannose moieties of wound surface glycoconjugates in bacterial adherence.     

Corneal endothelial healing rate and the effect of topical retinoic acid

These studies were undertaken to evaluate wound healing rates of the corneal endothelium in vivo. After insertion of a 26-gauge needle into the anterior chamber of the rabbit eye through the limbus, a 5-0 nylon monofilament was introduced through the needle, and endothelial wounds were made by scratching the cells with the filament. The wounds were photographed with a wide-field specular microscope at various intervals. Montages of the wounds were made, and the areas of the wounds were determined by planimetry. Wound closure occurred rapidly in a linear manner during the first 6 hr after wounding, after which the rate of cell migration decreased. Healing rates (micron2/hr) during the first 6 hr were calculated by linear regression analysis. There was a direct linear correlation between the healing rate and initial wound area. The slope of this line for nine normal (untreated) corneas was 0.093 hr-1. Nine corneas were treated with 0.1% retinoic acid in petrolatum ointment, while eight control corneas received vehicle alone. The slope of healing rate versus initial wound area for treated corneas (0.11 hr-1) was significantly greater than control (0.097 hr-1). This was interpreted as a stimulation of corneal endothelial migration during healing by retinoic acid. As a result of this study, a method for analysis of corneal endothelial healing rate has been developed which can be used for comparison of healing rates among treatments when initial wound area cannot be standardized.     

Delay in migration of the corneal epithelium after incorporation of [3H]-mannose in organ cultures

PURPOSE: Corneal epithelial cell surface carbohydrate moieties influence migration during wound healing. The purpose of the present study was to investigate the incorporation of galactose and mannose sugar during corneal epithelial migration and their effect on the rate of wound healing. METHODS: Organ cultures of non-migrating (n = 6) and migrating (n = 30) corneal epithelia were prepared. The incorporation of [3H]-galactose and [3H]-mannose was followed in the migrating samples after 18, 24, 48 and 72h incubation and at 18h in the non-migrating samples. Wound size was documented at the same intervals and the rate of healing was established. RESULTS: The migrating corneal epithelium of the galactose and mannose treated samples incorporated more radioactive sugar than the non-migrating samples. Mannose incorporation decreased with time whereas the rate of healing was significantly delayed (41 +/- 0.01 microm/h) compared to galactose treated samples (63 +/- 0.01 microm/h) and controls (61 +/- 0.01 microm/h) during the active healing phase (18-48h). Galactose incorporation increased with time. CONCLUSIONS: Incorporation of mannose sugar has an inhibitory effect on the rate of migration during the linear healing phase of the corneal epithelium.     

Kinetic study of cell proliferation in a new wound healing model using tissue cultured corneal endothelial cells

We studied cell kinetics in the wound healing process using cultured bovine corneal endothelium. We cultured bovine endothelial cells on coverslips coated by hydroxyethyl methacrylate (HEMA) which had on 8 mm HEMA-free zone in diameter, and produced wounds at the center of the monolayer cell sheets using a rotating silicone tip. At various time periods after injury, we added bromodeoxyuridine (BrdU) to the medium and incubated for 12 hours. Then, the specimens with fixed 10% phosphate-buffered formalin and were incubated with a monoclonal antibody against BrdU. The cells incorporating BrdU into DNA were stained by the Avidin Biotin Peroxidase Complex (ABC) method. At 12 hours, no labeled cells were observed. At 24 hours, 14.6 +/- 4.0 cells were stained. The maximum labeling occurred during 48-60 hours after the wound. Seventy two hours after the wound, labeled cells decreased rapidly. Labeled cells were localized within 0.6mm from the wound edge throughout the wound healing process. It can be thought that the wound healing process comprised four phases, i.e., latent, migration, migration plus mitosis, and contact inhibition phases. The first is the latent phase observed during the first six hours after wound infliction. The cells respond to external expansion. The second phase in the migration phase which is last until 24 hours after wound infliction, and occurs mainly through cellular migration, while proliferation has only a minor contribution in this phase. The third is the migration plus mitosis phase. The cell proliferation shows a rapid increase after 72 hours after wound infliction.(ABSTRACT TRUNCATED AT 250 WORDS)     

EGF does not enhance corneal epithelial cell motility

Although it is well known that epidermal growth factor (EGF) accelerates corneal epithelial wound healing by stimulating mitosis, it is also believed that EGF may directly stimulate the motility of individual corneal epithelial cells. We employed three different experimental methods to determine if EGF does indeed enhance the motility of corneal epithelial cells (independent of mitotic effects). First, the effects of EGF on the motility of tissue-cultured rat and rabbit corneal epithelial cells were investigated by a Boyden chamber assay. In rat corneal epithelium, these effects were further investigated by a second method, the agarose drop assay. Both assay techniques demonstrated no increase in corneal epithelial cell motility in the presence of EGF. These findings were corroborated by a third method which consisted of measuring the closure rate of epithelial wounds in organ-cultured rat corneas in the presence and absence of EGF, while concurrently arresting mitosis with colchicine. The wound closure rate before addition of any drug was 0.46 +/- 0.03 mm2/hr. The wound closure rate with EGF (50 ng/ml) was 0.55 +/- 0.03 mm2/hr, significantly (P less than 0.005) more rapid than the drug-free controls. However, when EGF (50 ng/ml) and colchicine (40 micrograms/ml) were used simultaneously, the acceleration of wound closure by EGF was completely negated by the presence of colchicine, resulting in a wound closure rate (0.46 +/- 0.06 mm2/hr) that did not differ significantly (P greater than 0.50) from that of the drug-free control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human recombinant epidermal growth factor in experimental corneal wound healing

Human recombinant epidermal growth factor (hEGF) was evaluated in various corneal wound healing models in the rabbit. Human EGF accelerated epithelial wound healing in corneal reepithelialization, anterior-keratectomy, and alkali-burn models at concentrations of 10-500 micrograms/ml given four times daily (qid). In the corneal reepithelialization model, 100 micrograms/ml of hEGF qid produced a 45% increase in the wound-healing rate compared with control (0.13 versus 0.09 mm/hr) with a similar response at 500 micrograms/ml qid. In the anterior-keratectomy model, 500 micrograms/ml of hEGF qid accelerated healing by 40% (0.07 versus 0.05 mm/hr), although the 100 micrograms/ml dose was not active in this model, and 1 microgram/ml of hEGF actually slowed the healing rate. In the alkali-burn model, 10 and 100 micrograms/ml of hEGF qid for 32 days appeared to produce faster initial healing of the wound compared with control, although the wound recurred in both hEGF and control groups. These results suggest that hEGF may be helpful in some epithelial disorders in humans, although considerations of dose response and optimal dosing regimens must be addressed.     

Effects of amniotic membrane on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea

AIMS: To investigate if the amniotic membrane (AM) promotes epithelial migration while inhibiting stromal remodelling associated with corneal haze after excimer laser keratectomy. METHODS: A wound 150 microm in depth and 6.0 mm in diameter was produced in 40 rabbits using an excimer laser. One eye was randomly chosen to be covered by the AM while the other eye served as a control. Epithelial wound healing was evaluated, together with any morphological changes of the anterior stroma connected with corneal haze. These morphological changes were histopathologically analysed using dichlortriazinyl aminofluorescein (DTAF), Masson trichrome staining, and an image analyser. RESULTS: The AM group had a short latent phase followed by fast epithelial healing (p<0.001) during the early wound healing period and a significant decrease in the inflammatory response, together with a smaller change in the number of keratocytes than the control group. The mean thickness of the regenerated stroma was significantly thinner in the AM group than in the control group at 8 weeks (p<0.0001). The AM group had a more regular architecture of regenerated stromal lamella at 8 weeks and significantly less haze after 4 weeks than the control group (p<0.05). CONCLUSION: Use of the AM as a dressing on a corneal wound created by excimer laser surgery, in which severe haze is expected, may induce rapid epithelial healing with less inflammatory response. The AM may inhibit the irregular synthesis of stromal collagen that is associated with corneal haze.     

The epidermal growth factor receptor (EGFR): role in corneal wound healing and homeostasis

To evaluate the role of the epidermal growth factor receptor (EGFR) in corneal epithelial wound healing, the effect of an EGFR inhibitor on epithelial cell proliferation and cell stratification during wound healing was investigated. From 3 days prior to wounding until wound healing was complete, rats were systemically treated with either an EGFR tyrosine kinase inhibitor (ZD1839) at 40 mg kg(-1) day(-1)or 80 mg kg(-1) day(-1), or with vehicle only (control). A single corneal wound was made in the center of 66 rat corneas, using a 6.0 mm glass tube wrapped in tissue paper soaked in n-heptanol. Subsequently, each wound was photographed and measured by a computer-assisted digitizer every 12 hr. To determine the number of cells in S phase, entire corneas were labelled with (3)H-thymidine and subjected to autoradiography at 0, 12, 24 and 48 hr after wounding. Epithelial thickness was also measured at these time points by microscopy. Epithelial wound healing was significantly and dose-dependently delayed following administration of ZD1839. At 24 hr after wounding, the number of S-phase cells in the limbal corneal epithelium was significantly lower in both the treated groups compared with the control group (P < 0.05). In the cornea before wounding (0 hr) and at 48 hr post-wounding, epithelial thickness was also significantly less in treated rats compared with controls (P < 0.05). These results indicate that EGFR inhibition affects epithelial cell proliferation and stratification during corneal epithelial wound healing and may play a role in maintaining normal corneal epithelial thickness.     

Localization of integrin and syndecan in vivo in a corneal epithelial abrasion and keratectomy

We compared the localization of integrin, a glycoprotein adhesion receptor, and syndecan, a transmembrane proteoglycan receptor, in vivo during the process of corneal epithelial wound healing to determine if the contact between migrating epithelium and substrata influence the expression of these two adhesion proteins. The expression of three integrin subunits of the alpha/beta heterodimer complex, beta 1, alpha 5, and alpha 6, were examined over a 48 hr in response to an abrasion. We also examined their expression in a keratectomy and compared their two conditions. The beta 1 subunit, which is expressed basolaterally in a normal cornea, was localized during epithelial migration over the basal lamina and keratectomy. Negligible levels of the fibronectin binding receptor subunit, alpha 5, were detected in a normal cornea. The levels of staining between a normal cornea, an abrasion and a keratectomy did not differ. Detection of alpha 6, the laminin binding receptor subunit, was expressed most intensely along the basal side of basal cells in a normal cornea. During the course of wound healing, alpha 6 was only detected in the basal cells and by two days the localization resembled that seen in the normal cornea. Epithelial staining for the alpha 6 subunit was also detected during the migration of cells over the keratectomy, more intensely than the other two subunits. Syndecan, which acts as a more permanent adhesion receptor in stratified epithelium, was only detected 48 hr post-abrasion, throughout all layers of epithelium, as was expected. These results indicate a predominance of alpha 6 expression by corneal epithelial cells at all times of in vivo wound healing and the presence of syndecan only upon restratification of the epithelium.     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

The behaviour of corneal epithelium following a standardized alkali wound

The short and long-term healing of the rabbit corneal epithelium was studied after a standardized alkali wound. The wound was inflicted by applying a round filter paper, 5.5 mm in diameter, soaked in 1 N NaOH, for 60 seconds on the central cornea. The wound size and intensity was chosen not to cause melting and perforation, and not to cause vascular ingrowth. n-Heptanol corneal wounds of the same size were used as control. The eyes were followed for 8 weeks. Two phases of epithelial healing were discerned. The initial healing phase lasted 48 h during which the would was completely resurfaced. In spite of the more extensive tissue damage caused by alkali, the initial epithelial healing rate was faster than in n-heptanol wounds. The late healing phase consisted of recurrent epithelial break down, sometimes seen preceded by epithelial blister formation. Four weeks after trauma the state of epithelial healing was at its worst.     

Radial keratotomy. 1. The wound healing process and measurement of incisional gape in two animal models using in vivo confocal microscopy.

Using in vivo confocal microscopy, corneal wound healing was evaluated in both rabbit and cat eyes after radial keratotomy. A total of six rabbit and six cat eyes were evaluated sequentially over time for 1 mo after surgery by in vivo confocal microscopy, and quantitative measurements of changes in incisional wound gape were determined. In vivo histopathologic changes were correlated with conventional histopathologic findings in 18 rabbit and 4 cat eyes; the animals were killed at various intervals from 0-30 days after surgery. In the rabbit, in vivo corneal wound healing was characterized by the initial ingrowth of corneal epithelium followed by persistence within the wound without a marked fibrotic response. Measurement of incisional wound gape showed increasing gape from 144 +/- 32 microns on day 0 to 976 +/- 155 microns on day 26 at a distance of 2.4 mm from the optical zone. These in vivo measurements were not significantly different (P = 0.996) from those obtained using conventional histopathologic techniques which showed an incisional wound gape of 252 +/- 112 microns on day 0 and 917 +/- 216 microns on day 26 at 2.5 mm from the optical zone. In the cat eyes, healing of radial keratotomy wounds showed an initial increase in incisional wound gape from 135 +/- 56 microns on day 0 to 245 +/- 88 microns on day 7 at a distance of 2.4 mm from the optical zone. Starting at day 14 and continuing to day 30, there was a progressive decrease in incisional wound gape from 198 +/- 41 microns to 92 +/- 35 microns. Sequential, in vivo histopathologic analyses indicated that increasing incisional wound gape correlated with the retention of corneal epithelium in the wound. Initiation of decreasing incisional wound gape was associated with replacement of the incisional epithelial plug with fibroblastic tissue. These changes in the incisional wound gape observed in the cat suggest that healing of radial keratotomy wounds involves contraction of the wound in response to the ingrowth of fibroblastic cells. Furthermore, the contractile response appears to be biphasic involving a precontractile and contractile phase. Overall these data indicated that in vivo confocal microscopy provides quantitative histopathologic data on living tissue comparable with that obtained with conventional techniques on dead, fixed, and sectioned tissue. Additionally, the absence of wound fibrosis in the rabbit radial keratotomy model raises important questions as to the appropriateness of this experimental model for human radial keratotomy.