During the course of studies on local graft-vs.-host reactions in the corneal endothelium 2 we had occasion to

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1 Desquamation of corneal endothelial cells Arthur M. Silverstein, Ali A. Khodadoust, and Robert A. Prendergast The process of wound healing of rabbit corneal endothelium involves the desquamation of significant numbers of endothelial cells, which may be found floating freely in the aqueous. It is suggested that this may be a general phenomenon of corneal endothelial wound healing, which accompanies the release of endothelial-cell attachments to Descemet's membrane and to neighboring cells during mitosis, allowing some of the cells to "fall off into the anterior chamber. The possibility is discussed that such endothelial desquamation may contribute to host sensitization to the histocompatibility antigens of penetrating corneal grafts. (INVEST OPHTHALMOL VIS SCI 22: , 1982.) Key words: corneal endothelium, desquamation, corneal wound healing, tissue culture, corneal graft, allograft reaction During the course of studies on local graft-vs.-host reactions in the corneal endothelium of rabbits, 1 ' 2 we had occasion to do aqueous taps for study of the cytology of inflammatory cells. Much to our surprise, these preparations often revealed the presence of corneal endothelial cells floating freely in the anterior chamber. We were at first excited by the possibility that in addition to in situ specific immunologic destruction of endothelium by sensitized lymphoid cells, an immunologically mediated desquamation of endothelium might also contribute to its damage during the rejection process. It was found, however, that a similar desquamation of corneal endothelial cells occurs during the healing phase after experimental destruction of the rabbit's endothelium by freezing. Thus From The Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Md. This work was supported in part by National Institutes of Health research grant EY and by an Independent Order of Odd Fellows Research Professorship. Submitted for publication Oct. 23, Reprint requests: Dr. Arthur M. Silverstein, The Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Md the phenomenon may be a general component of the endothelial repair process. We are unaware of any previous reports in the literature on corneal endothelial cell desquamation. This article will describe the phenomenon and will introduce some conjectures about its cause and its implications. Materials and methods These studies were carried out on 30 young adult (3 to 4 months) New Zealand white rabbits, often using both eyes of an animal. Central endothelial lesions were produced by twice freezing the anesthetized cornea. The end point used was the formation of a 2 to 3 mm hemisphere of ice on the posterior surface of the cornea, and thawing was permitted between freezings. A large brass probe with a 3 mm tip and prechilled in a Dry Ice-alcohol bath was employed. At daily intervals thereafter, 0.05 ml aqueous taps were withdrawn through the peripheral cornea into a tuberculin syringe wetted with heparin and containing 0.05 ml of 10% fetal calf serum in Hanks' balanced salt solution. The taps were immediately mixed and placed into the chamber of a cytocentrifuge, from which they were deposited on microscope slides. These slides were stained with hematoxylin and eosin and examined for the presence of cells with typical corneal endothelial morphologic characteristics. Primary cultures of endothelium from normal /82/ $00.80/ Assoc. for Res. in Vis. and Ophthal., Inc. 351

2 352 Silverstein et al. Invest. Ophthaltnol. Vis. Sci. March 1982 Fig. 1. Flat preparation of corneal endothelium 6 days alter freezing. Descemet's membrane is completely covered, although many of the cells are abnormal some multinucleated, some polyploid, and some with bizarre nuclear form. (Silver-hematoxylin; x95.) rabbit corneas were prepared by the method of Perlman and Baum 3 on Labtech four-chamber microscope slides, with trypsin used to establish a free-cell primary culture. Liebovitz's L-15 medium (Grand Island Biological Co.) was used to avoid CO 2 -dependent ph changes during cinematography. The cultures were maintained on the heated stage of a phase microscope for time-lapse cinematography, with exposures made at intervals of 6 to 10 sec. At daily intervals after corneal freezing, groups of usually three animals had aqueous taps and were sacrificed. The corneas were removed, and total flat-mount preparations of the cornea! endothelium were made by the method of Smolin, 4 employing a silver-hematoxylin stain. Examination of these preparations permitted us to follow the course of healing of the endothelial defect and to estimate the time required to re-cover Descemet's membrane with sliding endothelium and for the endothelial cells to regain their normal size. In most instances each test eye provided only a single aqueous tap. To confirm that the cells in question were indeed corneal endothelial and not inflammatory cells of similar structure, taps from three animals on days 3 to 4 were used to seed primary tissue cultures, as described above. These were cultured for 4 to 6 days, fixed and stained on the slide with hematoxylin and eosin, and examined to determine their growth characteristics. These aqueous samples were estimated to contain from 20 to 100 endothelial-like cells. To confirm that small numbers of rabbit cornea! endothelial cells are capable of establishing primary cultures, culture chambers were seeded with 20, 40, and 80 cells from trypsinized normal corneal endothelium and were similarly assessed. To demonstrate further that the putative endothelial cells found in the aqueous were not macrophages (blood-derived monocytes), a nonspecific esterase stain 5 was used for comparison of peritoneal macrophages (which stain brick red) with suspensions of cells from trypsinized corneal endothelium and with cells from the aqueous of eyes subjected to corneal freezing. In addition, preparations of peritoneal macrophages and aqueous

3 Volume 22 Number 3 Desquamation of corneal endothelial cells 353 m 4 v Fig. 2. CytocentrifLige preparation of cells (composite picture of six fields) from aqueous taps 5 days after freezing. Although there is some vacuolization, the cells appear reasonably normal, with morphologic traits consistent with those of corneal endothelial cells. (H & E: X320.) taps, prepared as for primary culture, were assessed for their ability to give positive nonspecific esterase reactions. Results Repeated freezing of the central cornea results in almost complete destruction of endothelial cells within the very discrete circle described by the freezing process. 6 Within 48 to 72 hr after freezing of the central cornea, during which a 2 to 3 mm defect had been produced in the endothelium, Descemet's membrane was once again re-covered, primarily by very large attenuated endothelial cells that had slid in from the periphery. This correlates well with the return of normal physiologic parameters of the cornea. 7 The size of the cells rapidly decreased, presumably by addition to their numbers through mi to tic activity, 6 so that within an additional 4 to 7 days the average cell size was near normal, despite the usual presence of numerous abnormal cells, some multinucleated and some with atypical nuclei (Fig. I). 8 Cytologic examination of the aqueous humor revealed much debris but few healthyappearing endothelial cells during the first 24 hr after freezing. A rapid increase in the number of endothelial cells found free in the anterior chamber started on the second day after freezing, peaked on about the fourth or fifth day, and then fairly rapidly waned, although an occasional endothelial cell could still be found floating in the anterior chamber as late as 2 weeks after freezing. At its peak the occasional animal (two of 10) might show as many as 100 or more endothelial cells per 0.05 ml of aqueous* although most showed only 10 to 50 such cells deposited over the surface of the cytocentrifuge slide. Aqueous taps of normal eyes showed no free cells, ruling out the trauma of the tap itself as the cause of desquamation. Most of the endothelial cells from the anterior chamber appeared morphologically normal on light microscopic examination (Fig. 2), given the changes that might occur consequent to the cells having floated for a

4 354 Silverstein et al. invest. Ophthalmol. Vis. Sci. March 1982 Fig. 3. Photomicrograph of 6-day tissue culture of cells from an aqueous tap 4 days after freezing. The cells grow well and show typical nuclear and nucleolar characteristics of rabbit cornea] endothelium. (H & E; X180.) time freely in the aqueous away from their normal attachment to Descemet's membrane and any trauma that they might have received in the cytocentrifuge. It should be noted that these free cells are almost invariably single, although the rare double cell can be found. Most of the aqueous taps revealed modest numbers of inflammatory cells, although two animals showed significant numbers (hundreds) of inflammatory cells and one animal also had erythrocytes. There was, however, no relationship between inflammatory cell numbers and the number of cells we identified as corneal endothelium. In addition, there was no difficulty in distinguishing these cells from either lymphocytes or polymorphonuclear leukocytes, and we were also able to differentiate most of them from monocytes or histiocytes on the basis of the morphologic appearance of their nuclei and nucleoli (Fig. 2). To further confirm that these were indeed not inflammatory cells, they were seeded into primary tissue cultures. In all three tests islands of cells grew out by mitotic division, with morphologic characteristics, especially nuclear, typical of rabbit corneal endothelium during the active growth phase (Fig. 3). Since it has been suggested that primary cultures of corneal endothelium are difficult to obtain with small numbers of seed cells, dilutions of a suspension of trypsinized normal rabbit endothelium were cultured, using 40 to 160 cells per culture. In every case, at least one island and often up to 8 to 10 islands of replicating endothelium were found after 6 days in culture, with a morphologic appearance identical to that shown in Fig. 3 for cells derived from aqueous taps. Use of the nonspecific esterase stain revealed that cytospin preparations of peritoneal macrophages stained a deep red, whereas preparations made from suspensions of

5 Volume 22 Number 3 Desquamation of corneal endothelial cells 355 Fig. 4. Endothelial cell undergoing mitosis in tissue culture. A time-lapse cinematographic sequence taken with a phase microscope shows: A, four cells are attached to the bottom of the dish, the cell with the arrow preparing for mitosis; B, this cell then rounds up and lifts off the bottom, as shown by the refractive halo; C, the mitotic spindle has formed and the cell is undergoing karyokinesis; D, mitosis is complete, and both daughter cells are still off the bottom; E, the upper cell (arrow) is attaching to the bottom and spreading; and F, the second daughter cell has regained its attachment. rabbit corneal endothelial cells did not stain at all. Although both staining and nonstaining large mononuclear cells may be found in aqueous taps from eyes with endothelial damage (suggesting a macrophage contamination), the cells that grow out in islands in culture did not stain for nonspecific esterase, but adherent preparations of peritoneal macrophages took up the stain well. In line with the observation of numerous other investigators, we have found it fairly simple to establish primary cultures of rabbit corneal endothelium. During the first few days of culture, prior to the attainment of a

6 356 Silverstein et al. Invest. Ophthalmol. Vis. Set. March 1982 confluent monolayer at the bottom of the culture chamber, time-lapse cinematography has permitted a study of endothelial cell attachment and spreading on the floor of the chamber, its mobility, and especially its mitotic activity. We have observed repeatedly (as have many other investigators) that endothelialcell mitosis involves the following sequence of events (Fig. 4): (1) the endothelial cell rounds up while still attached to its subjacent support; (2) the cell lifts off its support into the fluid above, presumably releasing or attenuating any attachments to adjacent cells and to its underlying support structure; (3) the cell engages in a frenzy of cytoplasmic activity and then undergoes mitotic division in its elevated position; (4) the two daughter cells settle down on the bottom of the chamber, spread out to regain normal cytologic features, and presumably reattach all around. The lifting process is readily visualized by changes in the refraction of light in the phase microscope, with all cells not attached to the chamber floor showing a pronounced halo of light. Discussion It appears to be generally accepted that the corneal endothelial cell never undergoes desquamation, and this is probably true of the normal cornea, given the modest adhesion of the endothelial cell to Descemet's membrane and the strong junctions that exist among neighboring endothelial cells. We have found, however, that desquamation of corneal endothelial cells into the aqueous is apparently a normal accompaniment of the endothelial healing process in the rabbit after damage. After production of an endothelial lesion by freezing the rabbit eye, cytologic analysis of the aqueous r.eveals the presence of small numbers of apparently normal endothelial cells within a day or two after damage. The number of desquamated endothelial cells floating freely in the anterior chamber increases rapidly, reaching a peak at about 4 to 5 days after damage, and then declines, but small numbers of free endothelial cells are still present in the aqueous of most eyes 2 weeks after the insult. The critical question is whether these cells are in fact corneal endothelium or perhaps only inflammatory cells that migrate into the eye as a consequence of the trauma of freezing. Morphologically, they appear to be endothelial, especially in their nuclear and nucleolar pattern, and could not be confused with lymphocytes, polymorphonuclear leukocytes, eosinophils, or macrophage-histiocytic cells. This is further confirmed by their ability to grow by mitosis in tissue culture to yield daughters with typical corneal endothelial structure, a capability not possessed by macrophages. In addition, rabbit peritoneal macrophages stain positively for nonspecific esterase, both in cytospin preparations and after adherence and spreading on glass. Neither rabbit corneal endothelium nor the putative endothelial cells derived from the aqueous of wounded eyes, take up this stain. Thus, by tissue culture growth and enzyme markers, these cells appear not to be derived from blood monocytes, the only plausible blood leukocyte candidate. At least tentatively, then, we may conclude that corneal endothelial cells may be desquamated into the aqueous. There are a number of possible explanations for this phenomenon. First, it may be argued that the desquamated cells represent those that were only partly damaged during the freezing process, expressing this damage later by giving up their attachments to Descemet's membrane and to surrounding cells. Although the cellsfloatingin the aqueous appear reasonably normal cytologically, this may not constitute an adequate criterion of minor damage. A more convincing argument against this possibility is based on our observation that after adequate freezing, few if any surviving endothelial cells can be found within the area of destruction during the next day or two, as determined by examination of flat preparations of endothelium (see also ref. 9). Within 3 to 4 days after production of the lesion, a 2 to 3 mm defect will be completely re-covered by sliding endothelium from the

7 Volume 22 Number 3 Desquamation of corneal endothelial cells 357 periphery, 8 ' 9 and the persistence of desquamation for as long as 2 weeks would seem to argue strongly against a "modest-damage" hypothesis. A second explanation for this phenomenon lies in the possibility of endothelial cell loss during the reparative sliding process. The cells at the periphery of an endothelial defect can probably move at the rate of 0.5 to 1 mm per day. 7 During this movement they must continuously break and remake their attachments to Descemet's membrane and to adjacent endothelial cells, at which time some of them, transiently unattached, could possibly "fall off" Descemet's membrane. Again, this would appear to be an unlikely explanation, since the peak of desquamation comes after the sliding process has completely re-covered the defect, and desquamation persists long after endothelial cell sliding has presumably ended. 6 We currently favor a third hypothesis to explain endothelial desquamation, suggested by time-lapse cinematographic studies on corneal endothelium in tissue culture. This approach shows (Fig. 4) that in preparation for the mitotic event, the corneal endothelial cell rounds up and at least partially lifts off its underlying support. After mitosis, both daughter cells reattach to the chamber floor and spread. If endothelial cell mitosis in vivo follows a course similar to that seen in vitro, then the endothelial cell during mitosis may be at risk of either floating away in the aqueous currents or of falling off the posterior surface of the cornea by gravitational forces. Until more information on this phenomenon is collected, we would favor the latter explanation and suggest that it may be a more general phenomenon associated with any wound healing of the corneal endothelium. We have thus far observed endothelial desquamation into the aqueous humor during repair of frozen corneas and after specific immunologic damage involving the rejection of corneal endothelium. The time course of desquamation appears to follow fairly closely the kinetics of endothelial mitotic activity during wound healing. It is not suggested here that every endothelial cell that undergoes mitosis is lost in the process; the fact that the rabbit cornea does heal and that tritiated thymidinelabeled cells can be found within the healed endothelial lattice 6 ' 10 ~ 12 disproves this. But during mitosis the attachments of the cells may be so reduced that some of them are lost to the endothelial monolayer, and it is these that we would find floating in the anterior chamber. It is of great interest that certain species such as man, 13 " 16 the subhuman primate, 9 and the cat 6 are far less able to repair endothelial defects than is the rabbit. It is thought that this is because of an inability of their endothelial cells to divide, so that although small defects can be covered by cell sliding and attenuation, normal cell size is not regained by mitotic replacement. While this may in fact be the actual mechanism involved, the possibility exists (and deserves testing) that endothelial mitosis does occur in these species but that most of these cells are lost to the monolayer by the process described above. Indeed, Van Horn et al. 6 and Schutten et al. 17 have shown that some tritiated thymidine incorporation can be found in healing feline corneal endothelium. The normal desquamation of corneal endothelial cells during wound healing carries with it an interesting implication for corneal transplantation. It has always been difficult to understand the mechanisms of host sensitization after penetrating allokeratoplasty, wherein a technically successful graft in an avascular corneal bed occasionally will undergo spontaneous rejection. If, indeed, endothelial cells from the graft find their way into the anterior chamber and thence into the blood while the donor tissue collaborates with the recipient in healing the endothelial defect at the graft margin, this may suffice to sensitize the occasional individual to the histocompatibility antigens of the donor. I8 It is interesting that Aviner et al. 19 observed a rather consistent but transient sensitization of human corneal graft recipients during the third postoperative week, whereas MacDonald and Basu 20 showed sensitization in grafted

8 358 Silverstein et al. Invest. Ophthalmol. Vis. Sci. March 1982 rabbits within 5 to 7 days. However, these investigators employed a leukocyte migration inhibition test with corneal extracts, and it is not clear that they were in fact measuring sensitization to histocompatibility antigens. The situation is further complicated by the suggestion of Streilein and Kaplan 21 that histocompatibility antigens introduced via the anterior chamber may produce an aberrant and protective immune response to tissue transplants. It would be interesting to apply some sensitive test for histocompatibility sensitization to a series of graft recipients, for further evaluation of the significance of the release of small numbers of donor corneal endothelial cells, such as appears probable from these studies. REFERENCES 1. Khodadoust AA and Silverstein AM: Local graft versus host reactions within the anterior chamber of the eye: formation of corneal endothelial pocks. INVEST OPHTHALMOL 14:640, Khodadoust AA and Silverstein AM: Induction of corneal graft rejection by passive cell transfer. IN- VEST OPHTHALMOL 15:89, Perl man M and Baum JL: The mass culture of rabbit conieal endothelial cells. Arch Ophthalmol 92:235, Smolin G: A technique for staining and separating corneal endothelium. Am J Ophthalmol 65:232, Li CY, Lam KVV, and Yam LT: Esterases in human leukocytes. J Histochem Cytochem 21:1, 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, Khodadoust AA and Green K: Physiologic function of regenerating endothelium. INVEST OPHTHALMOL 15:96, Faure JP, Kim YZ, and Graf B: Formation of giant cells in the conieal endothelium during its regeneration after destruction by freezing. Exp Eye Res 12:6, Van Horn DL and Hyndiuk RA: Endothelial wound repair in primate cornea. Exp Eye Res 21:113, Hanna C and Irwin ES: Fate of cells in the corneal graft. Arch Ophthalmol 68:810, Polack FM, Smelser CK, and Rose J: Long-term survival of isotopically labeled stromal and endothelial cells in corneal homografts. Am J Ophthalmol 57:67, Staatz WD and Van Horn DL: The effect of aging and inflammation on corneal endothelial wound healing in rabbits. INVEST OPHTHALMOL VIS SCI 19:983, Irvine AR and Irvine AR Jr: Variations in normal human corneal endothelium; preliminary report of pathologic human corneal endothelium. Am J Ophthalmol 36:1279, Kaufman HE, Capella JA, and Robbins JE: The human corneal endothelium. Am J Ophthalmol 61:835, Capella JA: The pathology of the corneal endothelium. Ann Ophthalmol 3:397, Stacker FW: The Endothelium of the Cornea and Its Clinical Implications, ed. 2. Springfield, 111., 1971, Charles C Thomas, Publisher. 17. Schutten WH, Van Horn DL, Bade BJ, and Faculjak ML: Corneal endothelial autoradiography with the scanning electron microscope. INVEST OPH- THALMOL VIS SCI 19:417, Raju S and Grogan JB: Immunology of anterior chamber of the eye. Transplant Proc 3:605, Aviner Z, Henley WL, Okas S, Castroviejo R, and Meltzer M: Leucocyte migration test in patients after corneal transplantation. Can J Ophthalmol 11:165, MacDonald AL and Basu PK: Systemic sensitization of corneal allograft recipients before the clinical onset of graft reaction. Can J Ophthalmol 12:60, Streilein JW and Kaplan HJ: Immunologic privilege in the anterior chamber. In The Immunology and Immunopathology of the Eye, Silverstein AM and O'Connor GR, editors. New York, 1979, Masson Publishing USA, Inc., pp