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Investigative Ophthalmology & Visual Science, Vol. 33, No. 11, October 1992 Copyright © Association for Research in Vision and Ophthalmology A New In Vitro Corneal Preparation to Study Epithelial Wound Healing Darrell L. Tanelian and Kammi Bisla 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 jum/hr and 38 ± 7 Atm/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. Invest Ophthalmol Vis Sci 33:3024-3028,1992 Corneal epithelium forms a protective barrier1 and modulates fluid transport to maintain normal stromal hydration.2 Because of the epithelium's functional importance, reepithelialization after injury from trauma or surgery has been studied extensively.3"8 Reepithelialization occurs by epithelial cell migration and proliferation to cover the denuded area,9"13 and healing rates for this process have been determined.14"17 Effects of therapeutic agents or growth factors on corneal wound healing also have been examined. However, the effects of these agents on wound healing has produced inconsistent results that may result from differences in the wound healing models used.18"24 In vivo studies have difficulty establishing steady-state delivery of agents, such that applied concentrations are equivalent to effector site concentrations. These studies also have difficulty manipulating environmental conditions. In vitro experiments, while controlling experimental variables, have not maintained normal corneal shape or tissue pressure, and in the case of epithelial tissue culture, other corneal layers are not present. Also, fetal calf serum (FCS), containing an unknown composition of growth factors and other products, has been added to the tissue culture medium of some in vitro studies, '7>25"30 potentially altering the effect of applying further therapeutic agents. The present study introduces a new in vitro whole mount corneal preparation to study the effects of therapeutic agents on epithelial wound healing. This organ culture preparation combines attributes of current in vivo and in vitro systems, allowing reepithelialization to occur without the addition of supplemental growth factors or FCS to the culture medium. Furthermore, normal corneal shape, tissue pressure, and tissue clarity are maintained. Wound healing rates comparable to in vivo preparations are achieved. Materials and Methods From the Pain Research Center, Department of Anesthesiology, University of Texas, Southwestern Medical Center at Dallas. Supported by NIH grant 1RO1 NS28646-01A1. Submitted for publication: January 10, 1992; accepted April 15, 1992. Reprint requests: Darrell L. Tanelian, University of Texas, Southwestern Medical Center, Department of Anesthesiology, 5323 Harry Hines Blvd., Dallas, TX 75235-9068. Animal care and treatment in this study complied with the Institutional Animal Care Review Committee at Stanford University School of Medicine and the ARVO resolution on the use of laboratory animals. New Zealand white rabbits, weighing 2-3 kg, were killed with an intravenous injection of Beuthanasia (pentobarbital/phenytoin; Schering Corp., Kenilworth, NJ) after anesthesia with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/ kg). The eye was proptosed and rinsed with corneal Ringer's solution equilibrated with 95% O2/5% CO2 by bubbling with this gas mixture at 21 °C. The cornea and a 2 mm rim of sclera were excised, and the iris and lens were removed. The cornea then was mounted onto an in vitro perfusion chamber made 3024 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933389/ on 06/11/2017 No. 11 VITRO CORNEAL EPITHELIAL WOUND HEALING / Tonelion ond Dislo WOUNDED AREA TEMPERATURE PROBE 3025 wound, the epithelium within the marked area was scraped away and the basement membrane was left intact (Fig. 2A). In the subepithelial wound, the epithelium, basement membrane, and part of the anterior stroma were scraped away (Fig. 2B). Wounding was confirmed by staining with 0.5% fluorescein and by histologic studies. Analysis of Wound Area Fig. 1. Schematic of in vitro whole mount corneal chamber with mounted, wounded cornea. The corneal epithelium is kept in a humidified air environment, while the endothelium is perfused with tissue culture medium entering through the inlet valve. Temperature and intraocular pressure may be constantly monitored as indicated. There is constant flow through the system at 5 ml/hr. out of Plexiglas, which was designed and built by the authors (Fig. 1). The epithelial side of the cornea was kept in a warmed (35°C), humidified air environment to prevent drying. This was accomplished by placing the corneal mounting chamber (Fig. 1) inside an electronically heated and temperature-controlled covered Plexiglas box partially filled with normal saline. The endothelial side was perfused at 5 ml/hr with Gibco's (Grand Island, NY) Medium 199 and 50 tig/ml gentamicin equilibrated with 95% O2/5% CO2 and brought to a pH of 7.4. Corneas were maintained at 35°C, and the intraocular pressure was 15 mmHg. Rates of wound closure were monitored by staining with fluorescein every 5 hr and recording the average radius of the remaining wound. Corneas were allowed to heal until fluorescein failed to stain the cornea, which was reported as the time of wound closure. The quality and extent of reepithelialization was evaluated by stereo microscopic (M3Z; Wild Leitz, Heerbrugg, Switzerland) visualization after fluorescein staining. The wound radius was measured with a calibrated grid. The wound area and radius were calculated using the methods of Crosson, Klyce, and Beuerman.14 Wound area was determined from wound radius by considering corneal curvature and comparing wound radius to the entire corneal radius. The equation is: As = 2 R[R - (R2 - r2)*], where As is the remaining wound area, R is corneal radius, and r is the radius of the remaining wound area. Data were statistically analyzed using repeated measures analysis of variance, and the mean ± standard error of the mean was plotted on the graphs. Histology Corneal Wounding A Bard-Parker No. 15 blade was used to create one of two types of corneal wounds: a superficial epithelial or an anterior keratectomy subepithelial wound. A 5 mm circular trephine mark was made in the center of the cornea to mark the wound area. In the epithelial Fig. 2. (A) Cross-section of corneal tissue immediately after epithelial wounding confirms that the epithelium has been removed from the wounded area. Arrow indicates wound margin. (B) Subepithelia! wounding of cornea extends through the epithelium and basal lamina and into the anterior stroma, as this photograph confirms. The arrow specifies the wound margin. (All bars= 100 Mm.) After wound closure, corneas were fixed in 10% buffered formalin fixative for histologic studies. The corneas then were stained with hematoxylin-eosin/ phloxine counterstain as well as periodic acid-Schiff stain to highlight the basal lamina. Corneas then were sectioned (6 /xm) and mounted with Permount B Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933389/ on 06/11/2017 3026 Vol. 33 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1992 Fig. 3. Photographs of corneal epithelial wound healing from initial subepithelial wounding through 70 hours. Time 0 is the time of initial wounding. Wounds have been stained with 2% fluorescein for visualization, and wound edges are marked with arrows. Complete wound closure for this type of wound occurred on average in 69 ± 4.5 hr (mean ± standard deviation). O hr 10 hr 20 hr 30 hr 40 hr 50 hr 6 0 hr 70 hr (Fisher Chemical, Fair Lawn, NJ). Sections were viewed with a Nikon Diaphot inverted microscope. Results Wound healing occurred in a circularly symmetrical manner in both types of corneal wounds, and corneas remained transparent throughout the healing process (Fig. 3). Graphs summarizing the mean decrease in epithelial wound area with time (n = 5) for a 5 mm diameter epithelial wound are shown in Figure 4. The subepithelial wound healing experiments (n = 10) have been plotted on the same graph (Fig. 4). Examination of this graph reveals the epithelial wound healed more rapidly than the subepithelial wound. Complete wound closure occurred in 51 ± 5.5 hr for the epithelial wound and in 69 ± 4.5 hr for the subepithelial wound. These curves reveal a nonlinear decline in wound area with time. In the early phase of healing (0-25 hr), the epithelial wound healing rate was 0.57 ± 0.07 mm2/hr, and later (30-55 hr) the rate decreases to 0.21 ± 0.06 mm2/hr. The subepithelial curve shows a similar pattern with a fast rate in the first part of the healing curve (0-25 hr) of 0.42 ± 0.07 mm2/hr, followed by a decrease to 0.25 ± 0.05 mm2/ hr (30-55 hr). Another way to quantitate wound healing is to measure the velocity of epithelial cell migration. This method, initially described by Crossen, Klyce, and Beuerman,14 measures the actual epithelial migration rate independent of wound size and allows for comparison between different wound healing models. The results of the epithelial and subepithelial wound healing experiments are plotted as a function of wound radius in Fig. 5. This plot approximates a linear function from which linear regression analysis can be used to calculate the epithelial migration rates. The rate of epithelial migration is 52 ± 14 /im/hr for epithelial wounds and 38 ± 7 j^m/hr for subepithelial wounds. A brief latency period was noted for the epithelial and subepithelial wounds. However, the initial measurement intervals used in these experiments were not short enough to accurately evaluate this early phase of healing. IN VITRO WOUND HEALING SUBEPITHELIAL WOUND EPITHELIAL WOUND 30 TIME 45 (hr) Fig. 4. The average epithelial (n = 5; circles) and subepithelial (n = 10; squares) wound area at specific times after wounding. This curve appears to show two separate healing phases (0-25 hr and 30-55 hr). The average rate of decrease in wound area for the first phase is 0.57 ± 0.07 mm 2 /hr and 0.42 ± 0.07 mm 2 /hr (mean ± standard error of the mean) for the epithelial and subepithelial wounds, respectively. Both rates decrease in the second half of the healing process. The epithelial wound heals at a rate of 0.21 ± 0.06 mm 2 /hr (30-55 hr), and the subepithelial wound healing rate decreases to 0.25 ± 0.05 mm 2 /hr (mean ± SEM). Time to complete wound closure is 51 ± 5.5 and 69 ± 4.5 hr(mean ± standard deviation) for the epithelial and subepithelial wounds, respectively. Error bars indicate SEM. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933389/ on 06/11/2017 No. 11 VITRO CORNEAL EPITHELIAL WOUND HEALING / Tonelion and Dislo IN VITRO WOUND HEALING • • SUBEPITHELIAL WOUND EPITHELIAL WOUND Fig. 5. Average decrease in wound radius for the epithelial wound (n = 5; circles) and for the subepithelial wound (n = 10; squares). The average rate of epithelial wound closure was 52 ± 14 jum/hr (mean ± standard error of the mean), and the average rate of subepithelial wound closure was 38 ± 7 (mean ± SEM). Error bars indicate SEM. Histology of healed epithelial and subepithelial corneas revealed a continuous layer of epithelial cells covering the wounded area. The epithelial lining the original wound and the area immediately surrounding was thinned after wound closure. Normal corneal thickness was maintained throughout the entire wound healing process. Discussion Epithelial cells at the wound margin were shown to migrate toward the center of the corneal wound to cover the denuded region, consistent with other investigations of corneal epithelial healing.910'31 During this migration phase, DNA synthesis ceases in the migrating basal epithelial cells, and cell mitosis does not occur until after wound closure.32 A temporary increase in mitotic rate is seen several days after initial wounding.9 The molecular mechanisms underlying corneal wound healing are not known, except that functional actin filaments are required for wound closure. The in vitro whole mount preparation was used in this study to monitor wound healing for epithelial and subepithelial wounds. Corneal transparency, normal shape, and thickness were maintained for up to 94 hr, although a maximum limit of viability was not established. The present study indicates that cellular components vital to wound healing are present in the intact in vitro cornea. Also, with the addition of a minimal medium, wound healing was able to proceed in a normal fashion. The wound closure rates obtained in this study for epithelial and subepithelial wound heal- 3027 ing (52 ± 14 /um/hr and 38 ± 7 /xm/hr, respectively) are comparable to in vivo rates of wound healing for rabbits, which are 64 ± 2 ^tm/hr for epithelial wounds14 and approximately 50 jim/hr for subepithelial wounds.15 The in vitro condition does not exactly replicate the in vivo state because factors such as plasminogen and plasmin in the tear film, which aid corneal epithelial migration,33 are not present. This may explain the slightly decreased rates with this in vitro model compared to in vivo studies. Other in vitro organ culture assays have measured epithelial wound healing rates ranging from 7.4 mm2/day (0.38 mm2/ hr) 34 to0.83mm 2 /hr. 16 This new in vitro corneal preparation is promising as a model for investigating the epithelial wound healing process. The ability to maintain steady-state drug concentrations is a great advantage for evaluation of therapeutic agents and growth factors. Manipulation of environmental conditions also may reveal valuable insights into the mechanisms of corneal epithelial wound healing. Key words: cornea, epithelial, in vitro, organ culture, wound healing References 1. KJyce SD and McCarey BE: Physiology of the cornea. Contact Lenses, Chapter 5, 5.1, 1986. 2. Klyce SD: Transport of Na, Cl and water by the rabbit corneal epithelium at resting potential. Am J Physiol 228:1446, 1975. 3. Friedenwald JS and Buschke W: The influence of some experimental variables on epithelial movements in the healing of corneal wounds. J Cell Comp Physiol 23:95, 1944. 4. Soong HK and Cintron C: Different corneal epithelial healing mechanisms in the rat and rabbit: Role of actin and calmodulin. Invest Ophthalmol Vis Sci 26:838, 1985. 5. Zieske JD and Gipson IK: Protein synthesis during corneal wound healing. Invest Ophthalmol Vis Sci 27:1, 1986. 6. Medin W and Davanger M: Wound healing of rabbit cornea in organ culture. Acta Ophthalmol 65:257, 1987. 7. Huang AJ and Tseng SC: Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 32:96, 1991. 8. Matsuda M, Ubels JL, and Edelhauser HF: A larger corneal epithelial wound closes at a faster rate. Invest Ophthalmol Vis Sci 26:897, 1985. 9. Hanna C: Proliferation and migration of epithelial cells during corneal wound repair in the rabbit and the rat. Am J Ophthalmol 61:55, 1966. 10. KuwabaraT, Perkin DG, andCogan DG: Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol Vis Sci 15:4, 1976. 11. 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Brazzell RK, Stern ME, Aquavella JV, Beuerman RW, and Baird L: Human recombinant epidermal growth factor in experimental corneal wound healing. Invest Ophthalmol Vis Sci 32:336, 1991. ZieskeJD, HigashijimaSC, Spurr-MichaudSJ,andGipsonIK: Biosynthetic responses of the rabbit cornea to a keratectomy wound. Invest Ophthalmol Vis Sci 28:1668, 1987. Jumblatt MM and Neufeld AH: A tissue culture assay of corneal epithelial wound closure. Invest Ophthalmol Vis Sci 27:8, 1986. Petroutsos G, Guimaraes R, and Pouliquen Y: The effect of concentrated antibiotics on the rabbit's corneal epithelium. Int Ophthalmol 7:65, 1984. Petroutsos G, Courty J, Guimaraes R, Pouliquen Y, Barritault D, Plonet J, and Courtois Y: Comparison of the effect of EGF, pFGF and EDGF on corneal epithelium wound healing. Curr Eye Res 3:593, 1984. Alfonso E, Denyon K.R, D'Amico DJ, Saulenas AM, and Albert DM: Effects of gentamicin on healing of transdifferentiating conjunctival epithelium in rabbit eyes. Am J Ophthalmol 105:198, 1988. Fredj-Reygrobellet D, Plouet J, Delayre T, Baudouin C, Bourret F, and Lapalus P: Effects of aFGF and bFGF on wound healing in rabbit corneas. Curr Eye Res 6:1205, 1987. Chandler LP, Chandler CE, Hosang M, and Shooter EM: A monoclonal antibody which inhibits epidermal growth factor binding has opposite effects on the biological action of epidermal growth factor in different cells. J Biol Chem 260:3360, 1985. Soong HK, McClenic B, Varani J, Hassan T, Huang S, and 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. Vol. 33 Brenz R: EGF does not enhance corneal epithelial cell motility. Invest Ophthalmol Vis Sci 30:1808, 1989. Burstein NL: Review: Growth factor effects on corneal wound healing. J Ocul Pharmacol 3:263, 1987. Nelson JD, Silverman V, Lima PH, and Beckman G: Corneal epithelial wound healing: A tissue culture assay on the effects of antibiotics. Curr Eye Res 9:277, 1990. Medin H and Davanger M: Endothelial repair of posterior corneal wounds in rabbits, influence of foetal calf serum in the culture medium. Acta Ophthalmol 68:195, 1990. Raymond GM, Jumblatt MM, Bartels SP, and Neufeld AH: Rabbit corneal endothelial cells in vitro: Effects of EGF. Invest Ophthalmol Vis Sci 27:474, 1986. Hyldahl L, Engstrom W, and Scholfield PN: Stimulatory effects of insulin-like growth factors on DNA synthesis in the human embryonic cornea. Journal of Embryology and Experimental Morphology 98:71, 1986. Gospodarowicz D, Mescher AL, Brown KD, and Birdwell CR: The role offibroblastgrowth factor and epidermal growth fac: tor in the proliferative response of the corneal and lens epithelium. Exp Eye Res 25:631, 1977. Woost PG, Brightwell J, Eiferman RA, and Schultz GS: Effect of growth factors with dexamethasone on healing of rabbit corneal stromal incisions. Exp Eye Res 40:47, 1985. Chan KY, Patton DL, and Cosgrove YT: Time-lapse videomicroscopic study of in vitro wound closure in rabbit corneal cells. Invest Ophthalmol Vis Sci 30:2488, 1989. Essepian JP, Feng W, Hildesheim J, and Jester JV: Comparisons of corneal epithelial wound healing rates in scrape vs. lamellar keratectomy injury. Cornea 9(4):294, 1990. Van Setten G, Salonen E, Vaheri A, Beuerman RW, Hietanen J, Tarkkanen A, and Tervo T: Plasmin and plasminogen activator activities in tear fluid during corneal wound healing after anterior keratectomy. Curr Eye Res 8:1293, 1989. Chung J: Healing of rabbit corneal alkali wounds in vitro. Cornea 9:36, 1990. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933389/ on 06/11/2017