Cellular Migration and Morphology in Corneal Endothelial Wound

Cellular Migration and Morphology in Corneal Endothelial Wound Repair Mamoru Marsuda,* Mirsuru Sawa,-|- Henry F. Edelhauser,* Stephen P. Barrels,! Arthur H. Neufeld,f and Kenneth R. KenyonjAfter a mechanical denudation of rabbit corneal endothelial cells, the healing process was followed with wide-field specular microscopy. Individual cell migration and morphologic changes were analyzed by computer-assisted morphometry. The cells surrounding the wound migrated to cover the defect without producing intercellular gaps. The greatest cellular migration and morphologic alterations occurred close to the wound edge. As the cells migrated toward the wound, they elongated and increased their surface area in the direction of the migration. As the healing proceeded, the cells lost their original hexagonal pattern, which returned after coverage was complete. The wound was covered completely by large, irregularly shaped cells showing mitotic figures between 24 and 48 hr. During this period, cellular migration decreased and normal cellular morphology began to recover. When mitosis decreased, the normal cellular pattern rearranged towards a more hexagonal shape. During the healing process, the degree and direction of cellular migration varied from cell to cell. Additionally, changes in cell-to-cell contact (positional changes of neighboring cells) occurred in one-third of migrating cells. Such cellular migration can account for monolayered cells sliding without producing gaps between individual cells. Invest Ophthalmol Vis Sci 26:443-449, 1985

The healing process of rabbit corneal endothelium following a variety of mechanical,1"5 chemical,6 and cryothermal injuries7"14 has been studied extensively. It has been demonstrated that a wounded area of rabbit endothelium is covered by migration, elongation, and proliferation of the adjacent cells. The primary method for studying endothelial cell behavior has been to fix the cornea at selected intervals after wounding and to follow the healing process by observing the cellular pattern by light or scanning electron microscopy. Specular microscopy has enabled sequential in vivo observation of the dynamic process of corneal endothelial healing.1516 Sherrard17 and Olson et al18 have shown that a defect of one to six cells in the rabbit endothelium can be healed by pseudopodia-like transformation and migration of surrounding cells, resulting in a rosette, previously defined by Oh and Evans19 as a cluster of radially

arranged cells that cover the wounded area. The healing process of a large defect, however, has not been investigated by specular microscopy because of the limited field of view. Recent development of the wide-field specular microscope, however, permits repeated observation of the same area and identification of the same endothelial cells.20"23 Using this instrument, Ogita et al24 and Honda et al25 have reported the morphologic changes of individual cells in the cat corneal endothelium after wounding. However, their studies were not followed to complete coverage of the wound. In the present study we performed wide-field specular microscopy and computer-assisted morphometry to follow the cellular migration and morphologic changes of the rabbit endothelium that occur after mechanical wounding and during complete coverage of a wounded area.

From the Departments nf Physiology and Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin,* and the Eye Research Institute of Retina Foundation, Boston, Massachusetts.! Dr. Sawa is now at the Department of Ophthalmology, Jichi Medical School, 329-04 Minamikawachi-machi, Kawachi-gun, Tochigi, Japan. Supported in part by National Eye Institute Research Grant EY00933, a research grant from Alcon Laboratories, Inc., and an unrestricted grant from Research to Prevent Blindness. Submitted for publication: June 12, 1984. Reprint requests: Mamoru Matsuda, MD, Departments of Physiology and Ophthalmology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.

Materials and Methods Chinchilla rabbits weighing 2.0 kg were anesthetized with an intramuscular injection of 0.8-1.0 ml of 1:1 ketamine HC1 (30 mg/kg of body weight) and xylazine (6 mg/kg of body weight) before surgery and before each specular microscopic examination. These animal studies conformed to the ARVO Resolution on the Use of Animals in Research. The surgery was performed under an operating microscope in ten

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Fig. J. Sequential photographs of the wound and surrounding endothelial cells. A: 0 hr, B: 3 hr, C: 12 hr, D: 24 hr, E: 48 hr, F: 72 hr, G: 120 hr after wounding. Bar = 100 ^m. The dots show the 34 cells used for orientation of each of the sequential photographs. Outlined area comprised the 123 cells studied, including groups I and II. Lower inset in photograph E showed the cells with mitotic figures in the wound. Bar = 30 ^m. a and b correspond to a and b, respectively. * = the center of the wound.

rabbits. Following an insertion of a 25-gauge needle into the anterior chamber of the eye through the limbus, a 4-0 nylon monofilament was inserted through the needle. An endothelial wound was made with the nylon filament by gently scratching cells, being careful not to collapse the anterior chamber or to damage Descemef s membrane. Of the ten rabbits, seven developed small wounds which healed by sliding of adjacent endothelial cells and had no mitosis; three rabbits with corneal wounds between 0.7 and 2.0 X 105 jim2, which healed by both cell migration and mitosis, were selected in order to study relationships between both of these endothelial healing responses.

The cell analysis of one of these corneas is described in this paper; the other two corneas revealed a similar wound healing response. The defect and surrounding endothelial cells were photographed using a wide-field specular microscope (Keeler-Konan camera) at 0, 3, 12, 24, 48, 72, and 120 hr after wounding. Black and white photographs were printed with a magnification of 240 times. The cell boundaries were traced onto tracing paper. Since spatial distribution of nonhexagonal cells, particularly in regions away from the wound, remained unchanged throughout the period of observation, these cells were used as a reference point for orientation to identify

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CELLULAR MIGRATION AND MORPHOLOGY IN WOUND REPAIR / Morsudo er ol.

24 hrs

I2hrs

3 hrs

0

Group II

4 8 hrs

7 2 hrs

120 hrs

Fig. 2. A drawing of cellular pattern changes of the 123 cells shown in Fig. 1. The dotted line represents the original wound margin. * = the center of wound.

the same individual cells in the sequential photographs. We initially identified 34 cells surrounding the left portion of the wound edge in each of the sequential photographs (Fig. 1, shown as dots). We digitized the apices of these cells with a graphics tablet pen (Hewlett-Packard 9111 A) coupled with a computer (Hewlett-Packard 85-B) and calculated the center of gravity of each cell from the x-y coordinates of the cell apices, with the assumption that each cell is a twodimensional polygon with a linear cell boundary. The center of gravity of the wounded area surrounded by these 34 cells (Fig. 1*) was determined from the x-y coordinates of the center of gravity of the 34 cells in each photograph and served as the reference point for orientation of the sequential photographs (Fig. 1). One hundred twenty-three cells in the region oriented at approximately 120 deg to this arbitrary point were selected for the following study (Fig. 1). Of these, 54 cells which were 50-100 /zm distant from the wound edge (group I) and 26 cells that were 200-250 nm (group II) distant from the wound edge (Fig. 2) were subjected to a computer-assisted digitization to determine the area and shape of each cell.26 The cell shape was determined by digitizing each apex of each cell. Based on x-y coordinates of cell apices, the computer calculated the inertia moment about each pos-

sible axis, which crosses the center of gravity of the cell shape by assuming that a cell is a solid object with a constant thickness and density and that each cell boundary is linear. The minimal inertia moment (Imin) is around the axis where the cell maximally elongates (Fig. 3A). The moment is the greatest (Imax) around the axis, which is perpendicular to the axis of cell elongation (Fig. 3A). Therefore, we can quantitate the degree of cell elongation by using the following equation:

degree of cell elongation = Imax '

The direction of cell elongation was determined by calculating the angle between the axis of elongation (direction of Imin) and the horizontal line formed by the lower edge of the specular photograph (Fig. 3A). When the cell shape is a regular polygon, Imax is equal to Imin; thus, the degree of cell elongation is 0 and the cell has no directional arrangement (Fig. 3B). As the cell elongates toward a linear shape, Imin approaches 0; thus, the degree of cell elongation increases to 1, with its direction varying from 0-180 deg (Fig. 3C). The cellular migration was determined by calculating the distance of the center of gravity of each cell in the sequential photographs, and a shifting

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'-y\Q:

direction of elongation

I max : Imin

26

Group I Group H

Imin,Imin

:

Vol.

,(Q

(Horizontal line of Photograph)

Fig. 3. Diagrams of cell elongation and the inertia moment of cell shape. 6 = direction of cell elongation (direction of Imin).

velocity (jum/day) was calculated for the individual cells.

Results Originally the endothelial cells were uniform in area and shape. The mean cell area and relative frequency of hexagonal cells were 237 ± 42.4 /im2 (mean ± SD) and 73%, respectively. The wounded area in this rabbit was approximately 1.24 X 105 /urn2, roughly oval in shape, corresponding to a loss of about 500 cells (Fig. 1A). The wound decreased to approximately 70% of its initial size at 24 hr (Fig. ID) and was completely healed within 48

wound edge Fig. 4. Line drawing of movements of individual cells. Solid circles and triangles show the center of gravity of cell shape, representing position of cells. Each capital represents each cell shown in Figure 2. Each number shows the time after wounding. * = the center of wound.

0 3

12

24

48 72 Time After Scratching (hr)

120

Fig. 5. Shifting velocity (^m/day) of individual cells in groups I and II. Bar: SEM.

hr after wounding (Fig. IE). The cells covering the wound were large, irregular, and pleomorphic with most of these cells showing mitotic figures (Fig. 1E inset). Between 48 and 72 hr, the number of cells with mitotic figures decreased markedly (Fig. IF). The cells became smaller and uniform as the cellular pattern approached a more hexagonal pattern by 120 hr (Fig. 1G). However, a considerable deviation from the original hexagonal pattern still remained. Figure 2 illustrates the cellular pattern changes of a cluster of the 123 cells, including groups I and II. As time elapsed, these cells migrated and changed their shape but still maintained contact between each other. No mitotic figures were found among these cells. The migration of individual cells is shown in Figure 4. Not all of the cells migrated toward the wound to the same distance and in the same direction. Some cells migrated a long distance, while others migrated very little; with some cells the migration occurred in a different direction. As early as 3 hr, the cells in group I migrated (84 ^in/day) toward the wound (Fig. 5). The cellular migration was more pronounced toward the wound within 12 hr (94 ftm/ day) and then rapidly decreased to 15 ^m/day between 24 and 48 hr. After 48 hr these cells showed only slight migration. The cells in group II also migrated toward the wound in a similar manner as those in group I. Cellular migration in group II occurred more slowly and to a lesser extent, reaching its peak (69 /um/day) during the first 24 hr after wounding. The cells in group I did not show elongation during the first 3 hr although migration was already apparent (Fig. 6). Between 3 and 12 hr these cells showed remarkable elongation toward the wound, which decreased considerably by 24 hr and continued to decrease with time. In contrast, the cells in group II did not show any degree of elongation throughout the observation period.

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Group I Group H.

Group I Group II

0 3

12

24

48

72

120

Time After Scratching (hr)

0 3

12

24

48

72


Fig. 6. Degree of cell elongation of individual cells in groups I and II. Bar: SEM.

Fig. 7. Changes of individual cell areas in groups I and II.

The group I cells showed minimal changes in their area within 3 hr but their area increased by 24 hr, the time period of maximal cell migration and elongation (Fig. 7). After this period the individual cell areas decreased with time. The cells in group II showed a similar area change but to a lesser extent. Alterations in the cell-to-cell contact (positional changes of neighboring cells) occurred in 15 of 54 cells (28%) in group I and 8 of 26 cells (31%) in group II. This was associated with celi migration and with alterations in the number of individual cell apices. As a result, the number of hexagonal cells in group I decreased within the first 48 hr after wounding (Fig. 8) with an increase of cells with other distinct shapes. During the following period, the hexagonal cells increased. By comparison, the total number of hexagonal cells in group II did not change markedly.

poration occurs during the DNA synthetic phase (Sphase) of the cell cycle, 45 followed by several hours of G-2 and M (dividing) phase,20 there are few daughter cells formed by cell division within 24 hr after wounding. Thus, cell behavior observed in this study within the first 24 hr is not affected by cell division and can be comparable to the endothelial response of cat, monkey, and humans. The first response of endothelial cells after wounding is cellular migration, 3 ' 8111218 which was similarly confirmed in vivo in this study. As early as 3 hr after wounding, the cells migrated toward the wound with few changes in their morphologic appearance. The cells close to the wound showed a remarkable elongation between 3 and 12 hr, which was oriented in the direction of migration. There was a significant correlation between the degree of cell elongation and the shifting distance of the cells during this period (r

Discussion The present study documents the in vivo endothelial cell migration following wounding. Wide-field specular microscopy has provided a method for accurate identification and localization of the same endothelial cells, enabling one to quantitate migration and morphologic alteration of individual cells during healing. As shown in previous- time-series studies, 37 ' 11131418 the closer the cells to the wound, the greater the changes in cellular migration and morphology. The rabbit corneal endothelium has a marked proliferative capacity when compared with cat,27 monkey,28 and humans. 29 By autoradiography with tritiated thymidine, Yano and Tanishima 14 have shown that labeling of rabbit endothelial cells was low within the first 16 hr after wounding, reaching a peak between 24 and 48 hr. Since thymidine incor-

Group 1 Group I I

0 3

12

24

48

72

120


Fig. 8. Changes of the number of hexagonal cells in groups I and II. ( ) shows the number of cells that changed their number of apices by sliding past one another (positional changes of neighboring cells).

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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1985

= .854; P < .01). These results suggest that the degree of elongation of the cell can reflect its migration. A similar finding has been reported in the healing cat corneal endothelium. 25 Endothelial cell areas were enlarged in the area close to the wound as well as in the region distant from it. Such an increase in cell area can coincide with flattening of cells reported in previous histologic studies. 1 ' 3 ' 8 ' 1011 ' 1318 The cells adjacent to the wound also increased in area as they elongated toward the wound. Between 12 and 24 hr, the cells increased in area, while cell elongation decreased. These changes may be explained in that the cells adjacent to the wound expand initially toward the wound, resulting in a marked elongation, and then spread perpendicularly to the wound. After 48 hr the cell area decreased with time. Such a decrease in area may be due to the migration of the peripheral cells toward the wound or the appearance of newly formed cells by cell division in the wound, or both. Of particular interest is the observation that the cells retained surface contact between each other during migration, elongation, and surface area expansion. The mechanism for this is unclear but may be closely related to the positional changes of neighboring cells sliding past one another. During this sliding, breakage and reformation of cell junctions may possibly occur. Similar cell behavior has been reported in the monolayered cell sheet of other tissue31 and the corneal endothelium of cat25 and humans. 23 ' 32 It should be noted that the cellular migration did not occur in the same manner in all of the cells; the degree and direction of cell migration varied from cell to cell. This type of cellular migration also can be the reason why monolayered cells slide without producing any gaps between them. After the initial coverage of the wound, the number of cells with mitotic figures and cellular migration decreased abruptly. The former may correspond to "contact inhibition of cell growth," 3033 and the latter may correspond to "contact inhibition of cell movement" 33 as observed in cell cultures. In the region close to the wound, the original hexagonal pattern was considerably compromised during the first 48 hr after wounding. However, after the initial coverage of the wound, the rearrangement of cellular pattern occurred toward a more regular hexagonal pattern. Such a rearrangement also has been observed in human corneal endothelium.34'35 Our results clearly demonstrate the in vivo wound healing process of the rabbit corneal endothelium by sequentially observing the same individual cells. Several investigators have shown that repeated photography of the same endothelial cells is also possible in

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humans with use of the wide-field specular microscope and by making an endothelial montage.20"23 Thus, the method outlined in this study may be applied to evaluate the actual cellular migration of human corneal endothelium following ocular trauma and intraocular surgery when corneal edema is not extensive and sufficient cooperation of patients is obtained. Key words: cellular migration, rabbit, corneal endothelium, wide-field specular microscopy, computer-assisted morphometry

Acknowledgment The authors are very grateful to Richard W. Yee, MD for his valuable discussions and suggestions regarding this work.

References 1. Binder RF and Binder HF: Regenerative process in the endothelium of the cornea. Arch Ophthalmol 57:11, 1957. 2. Morton PL and Ormsby HL: Healing of endothelium and Descemet's membrane of rabbit cornea. Am J Ophthalmol 46: 62, 1958. 3. Chi HH, Teng CC, and Katzin HM: Healing process in the mechanical denudation of the corneal endothelium. Am J Ophthalmol 49:693, 1960. 4. Mills NL and Donn A: Incorporation of tritium-labeled thymidine by rabbit corneal endothelium. Arch Ophthalmol 64: 443, 1960. 5. Bito LZ and Harding CV: Tritium retention by corneal endothelium after incorporation of H3-thymidine. Arch Ophthalmol 66:553, 1961. 6. Matsuda H and Smelser GK: Endothelial cells in alkali-burned corneas. Arch Ophthalmol 89:402, 1973. 7. Chi HH and Kelman CD: Effect of freezing on ocular tissues: I. Clinical and histologic study of corneal endothelium. Am J Ophthalmol 61:630, 1966. 8. Faure JP, Kim YZ, and Graf B: Formation of giant cells in the corneal endothelium during its regeneration after destruction by freezing. Exp Eye Res 12:6, 1971. 9. Capella JA: Regeneration of endothelium in diseased and injured corneas. Am J Ophthalmol 74:810, 1972. 10. Kanai A: The regeneration of corneal endothelial cells. Acta Soc Ophthalmol Jpn 76:1163, 1972. 11. Hirsh M, Renard G, Faure JP, and Pouliquen Y: Formation of intracellular spaces and junctions in regenerating rabbit corneal endothelium. Exp Eye Res 23:385, 1976. 12. Khodadoust AA and Green K: Physiological function of regenerating endothelium. Invest Ophthalmol 15:96, 1976. 13. Renard G, Hirsh M, Faure JP, Usui M, and Pouliquen Y: Ultrastructural study on the regeneration of the corneal endothelial cells. Jpn J Ophthalmol 20:243, 1976. 14. Yano M and Tanishima T: Wound healing in rabbit corneal endothelium. Jpn J Ophthalmol 24:297, 1980. 15. Bourne WM and Kaufman HE: Specular microscopy of human corneal endothelium in vivo. Am J Ophthalmol 81:319, 1976. 16. Laing RA, Sandstrom MM, Berrospi AR, and Leibowitz HM: Changes in the corneal endothelium as function of age. Exp Eye Res 22:587, 1976. 17. Sherrard ES: The corneal endothelium in vivo: its response to mild trauma. Exp Eye Res 22:347, 1976. 18. Olson LE, Marshall J, Rice NSC, and Andrews R: Effects of

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KJ: Corneal endothelial changes in type I and type II diabetes mellitus. Am J Ophthalmol 98:401, 1984. Van Horn DL, Sendele DD, Seideman S, and Buco PJ: Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci 16:597, 1977. Van Horn DL and Hyndiuk RA: Endothelial wound repair in primate cornea. Exp Eye Res 21:113, 1975. Treffers WF: Human corneal endothelial wound repair. In vitro and in vivo. Ophthalmology 89:605, 1982. Todaro GJ, Lazar GK, and Green H: The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Comp Physiol 66:325, 1965. Armstrong PB: Time-lapse cinemicrographic studies of cell motility during morphogenesis of the embryonic yolk sac of Fundulus heteroclitus (Pisces. Teleosti). J Morphol 105:13, 1980. Sperling S: Early morphological changes in organ cultured human corneal endothelium. Acta Ophthalmol 56:785, 1978. Martz E and Steinberg MS: The role of cell-cell contact in "contact" inhibition of cell division. A review and new evidence. J Cell Physiol 79:189, 1972. Olsen T: The specular microscopic appearance of corneal graft endothelium during an acute rejection episode. A case report. Acta Ophthalmol 59:882, 1979. Matsuda M, Suda T, and Manabe R: Serial alterations in corneal endothelial cellular pattern after intraocular surgery. Am J Ophthalmol 98:313, 1984.