S,Ipecular microscopy has added a new

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1 Functional and structural changes in the corneal endothelium during in vitro perfusion Bernard E. McCarey,* Henry F. Edelhauser, and Diane L. Van Horn The endothelium of isolated rabbit corneas was perfused with either a bicarbonate Ringer's solution, Kinsey Medium (KEI), or a glutathionebicarbonate Ringer's solution (GBR) in the perfusion chamber of the specular microscope. Changes in corneal thickness and endothelial pattern were monitored throughout the perfusion, and the corneas were fixed at the end of the perfusion for electron microscopic observation of the endothelial cells. Perfusion of bicarbonate Ringer's resulted in a mean increase in corneal thickness of 54 n per hour, alteration of the endothelial cell pattern, and disruption of the endothelial cells by the end of the three-hour perfusion. In contrast, corneal thickness and normal endothelial pattern could be maintained during perfusion of KEI or GBR for three hours. Electron microscopy of the same corneas revealed no ultrastructural damage in the endothelium. The loss of endothelial pattern could be correlated not only with differences in the rate of corneal swelling but also with the extent of ultrastructural damage in the endothelial cells. Key words: cornea, corneal thickness, corneal hydration, corneal endothelium, glutathione bicarbonate Ringer's, specular microscope. From the Departments of Physiology and Ophthalmology, The Medical College of Wisconsin, Milwaukee, and Veterans Administration Center, Wood, Wise. This investigation was supported in part by a National Defense Education Act Fellowship, a Medical College of Wisconsin Traineeship to Bernard E. McCarey, United States Public Health Service Research Grants Nos. EY and EY-00625, and an unrestricted grant from Research for Prevention of Blindness. Manuscript submitted for publication Nov. 1, 1972; manuscript accepted for publication March 26, Reprint requests: Dr. Henry F. Edelhauser, Department of Physiology, The Medical College of Wisconsin, 561 N. 15 St., Milwaukee, Wise ''In partial fulfillment of the requirements for the degree of Doctor of Philosophy at Marquette University, Milwaukee, Wise. Present address: Department of Ophthalmology, University of Florida, Gainesville, Fla S,Ipecular microscopy has added a new dimension to in vitro perfusion studies of the cornea because the endothelial cells can be directly and continuously observed during perfusion. 1 ' 2 The important role of the endothelium in maintaining normal corneal thickness has been documented by Mishima and Kudo, 3 Dickstein and Maurice, 4 and Maurice, 2 who demonstrated that the corneal endothelium is responsible for pumping fluid out of the stroma by an active transport process. Hodson, 5 using the specular microscope, has recently shown that corneal thickness is maintained in rabbit cornea by a sodium bicarbonate pumping mechanism within the endothelium. It has been suggested that changes in the appearance of the endothelial cell pattern may be correlated with functional changes of the cells. For example, Hoefle,

2 Volume 12 Number 6 Changes in corneal endothelium 411 Microscope Objective Cornea Endothelial Rerfusion Chambf (.25 ml Vol) Water Jacket I = inflow from perfusion pump P= connection to pressure transducer t = temperture monitored O = outflow from chamber Fig. 1. Diagram of the specular microscope perfusion chamber showing the isolated cornea clamped between the perfusion disk and the corneal support ring to which it is tied. Note the outlets on the perfusion disc for measurement of temperature and pressure. Maurice, and Sibley 0 have observed folds and nuclear changes in the endothelium which become more severe with increased time of storage of human donor eyes. Whether these folds or nuclear changes are of functional significance has yet to be determined; however, the specular microscope may some day be utilized routinely in eyebank management to determine the state of the endothelium prior to keratoplasty. The purpose of this investigation was to assess corneal endothelial function by measuring corneal thickness in the specular microscope during perfusion with different media and to correlate the changes in thickness and swelling rate to the appearance of the endothelial cells in the specular microscope and to their ultrastructural appearance at the end of the perfusion. Methods Albino rabbits weighing approximately 2 kilograms were killed by a blow to the back of the neck. The eyes were enucleated and the isolated corneas were mounted in the specular microscope without distorting the corneal curvature. The temperature of the perfusion medium was maintained in the perfusion chamber at 34 C. by monitoring with a Tele-Thermometer attached to a 24-gauge needle tennistor (Yellow Springs Instrument Co., Yellow Springs, Ohio). Perfusion pressure was maintained throughout the experiment at 15 mm. Hg by monitoring with a 70 mm. Hg pressure transducer (Statham Instrument Inc., Oxnard, Calif.) and recording on a Grass Model 5 Polygraph (Fig. 1). Corneas were perfused at a constant rate of 30 fj-l per minute with bicarbonate Ringer's (Table I), Kinsey Medium (KEI), 7 or a glutathionebicarbonate Ringer's (GBR) which contained 0.3 mm. reduced glutathione and 5.0 mm. adenosine (Table I). The corneal epithelium in all experiments was intact and covered with medical grade silicone oil (No. 20 CSKS Dow Coming, Midland, Mich.). In order to determine the rate of corneal swelling, the corneal thickness measurements (mean of five measurements per point) were statistically fit to a line by regression analysis. The corneas were fixed for electron microscopy by perfusion of 2 per cent glutaraldehyde buffered to ph 7.4 in 0.15 M. sodium cacodylate at 15 mm. Hg perfusion pressure. They were then

3 412 McCarey, Edelhauser, and Van Horn Investigative Ophthalmology June 1973 Table I. Chemical composition of Ringer's solutions 400',300 3 Time (hr n. 7 J (.05* -.008) X Fig. 2. Increase in corneal thickness during perfusion with bicarbonate Ringer's solution. Plotted line was calculated by regression analysis of seven experiments. The mean swelling rate was 54 fi per hour. B Fig. 3. Progressive loss of comeal endothelial cell pattern during perfusion in the specular microscope with bicarbonate Ringer's: A, initial or control endothelial cell pattern; B, poor endothelial cell pattern; and C, loss of the endothelial cell pattern. postfixed in 2 per cent osmium tetroxide for one hour, and embedded in Epon 812. Thin sections were cut with a diamond knife on an LKB ultramicrotome. Results Perfusion of bicarbonate Ringer's to the corneal endothelium resulted in a mean Glucose NaCl KCI CaCh MgCU 6 H,0 NaHPO* NaHCO 3 Adenosine Reduced glutathione Total osmolarityf Bicarbonate Ringer's 0 (Gm./L.) Glutathione bicarbonate 0 Ringer's (Gm./L.) "Adjusted to ph 7.4 by bubbling with 95 per cent air/5 per cent CO2. f Measured with a Fiske Osmometer. increase in corneal thickness of 54 ju, per hour (Fig. 2) with changes in the endothelial pattern (Table II) and ultrastructural damage that could be directly related to the swelling rate. In corneas with a swelling rate of < 25 ju. per hour there were no observable changes in the endothelial pattern (Fig. 3, A, Table II), and the endothelial cells were not ultrastructurally damaged (Fig. 4, A). Those corneas with a swelling rate of > 33 ju per hour had a poor endothelial pattern (Fig. 3,";B, Table II) and loss of ultrastructural organization (Fig, 4, B). At greater rates of/welling, the endothelial pattern was lost,(fig. 3, C, Table II) and the cells wer^ disrupted (Fig. 4, C). In experimental corneas perfused with KEI, the mean swelling rate was 4 /*. per hour during the first 3.5 hours (Fig. 5). From 3.5 hours to 6.5 hours, a second slope was calculated to be 11 ju, per hour. The endothelial pattern was' 1 maintained throughout the perfusion period of 6.5 hours and cellular ultrastructure remained normal (Fig. 6). Corneas from eyes stored in moist chambers at 4 C. for 24 hours were also mounted in the specular microscope and perfused with KEI for temperature reversal studies (Fig. 7). At the end of 3.5 hour perfusion period, the corneal thick-

4 Volume 12 Number 6 Changes in corneal endothelium 413 N M U V ER ER M Fig. 4. Electron micrographs of the endothelium of corneas perfused with bicarbonate Ringer's solution for 3.5 hours. A, swelling rate of 22.8 n per hour. The endothelial cell has minimal ultrastructural damage. The chromatin in the nucleus (N) is evenly dispersed. The intercellular junction (IJ) is of normal width and the posterior cell membrane is flat. The mitochondria (M) contain concentric cristae and the profiles of endoplasmic reticulum {ER) are linear. A few small vacuoles (V) are present (xl3,132). B, swelling rate of 33.5 fi per hour. Some loss of cellular organization has occurred. The nucleus (N) is swollen. Mitochondria (M) are distorted and the endoplasmic reticulum (ER) is swollen. A large vacuole (V) is present in the cytoplasm and the posterior membrane of the cell is not flat (x7,616). C, swelling rate of 88.7 ix per hour. Complete loss of cellular organization. The nucleus (N) is pyknotic and the perinuclear space is enlarged. The posterior plasma membrane is discontinuous and the cytoplasm is disrupted (xl 1,293). ness measurement was 74 /A lower than the initial measurement, a thinning rate of 21 ju per hour. The endothelium pattern of corneas which exhibited temperature reversal was virtually unchanged throughout the perfusion. Fig. 8 is a series of specular micrographs taken from one cornea during its temperature reversal covering 187 minutes of perfusion. The final photograph at 205 minutes is from the cornea indicated by closed circles in Fig. 7 which did not temperature reverse and illustrates loss of the endothelial pattern. Freshly excised corneas perfused with GBR (Table I) maintained a constant thickness during the 3.5 hour experimental period (Fig. 9) and compare to the KEIperfused corneas. The corneal endothelial cell pattern remained undamaged throughout these perfusions (Fig. 10), and normal endothelial ultrastructure was maintained (Fig. 11). Discussion Mishima and Kudo, 3 and Hodson 5 have reported that bicarbonate Ringer's solution is inadequate to maintain constant corneal thickness during in vitro perfusion. In the

5 <m 414 McCarey, Edelhauser, and Van Horn Investigative Ophthalmology June 1973 KEI Perfusion n =5 y = y = ± X X Time Fig. 5. Changes in corneal thickness during perfusion with KEI. The measurements were taken at 30-minute intervals. The regression line calculated for five corneas, shows a swelling rate of 4 n per hour for the first 3.5 hours and 11 /* per hour for the next 3.5 hours. Fig. 6. Electron micrograph of endothelial cells from a rabbit cornea perfused with KEI for 3,5 hours. The cells were intact (xl5,600) and little evidence of ultrastructural damage was seen. J.50CH, ) Time (hr) ao ( )x Fig. 7. Temperature reversal of corneal thickness. The corneas were perfused with KEI at 34 C. following 24-hour storage of the enucleated eye in a moist chamber at 4 C. The open circles represent swollen corneas that temperature reversed at a mean rate of 21 n per hour; whereas, the closed circles represent a cornea that did not temperature reverse. present study, individual differences in the rate of swelling could be correlated to changes in endothelial pattern and ultrastructure. For example, at swelling rates of > 57 ju. per hour the endothelial pattern was completely lost and severe ultrastructural damage was present in the endothelium. In contrast, corneas with lesser swelling rates had minimal ultrastructure damage in the endothelial cells. The successful use of KEI for maintaining the in vitro cornea at constant thickness for more than 10 hours has been previously reported by Mishima and Kudo. 3 Of the corneas perfused with KEI in our study, only one cornea had a calculated swelling rate of < 3 p per hour for 6.5

6 Volume 12 Number 6 Changes in corneal endothelium m.n 52mm 87m,, Fig. 8. Sequential specular micrographs of endothelial cell pattern in a temperature-reversing cornea (18 to 187 minutes). The cellular pattern was unchanged throughout the perfusion period. Endothelial pattern after 205 minutes from a cornea which did not temperature reverse. The cell pattern is lost. hours. Despite the slow swelling in isolated corneas perfused with KEI, cold-stored corneas perfused with KEI were able to temperature reverse at rates similar to those prevously reported *! 11 and with no loss of the endothelial cell pattern or ultrastructural damage. The one cornea that did not temperature reverse had a loss of endothelial pattern and ultrastructural damage within the cells. The maintenance of isolated perfused corneas at constant thickness for up to seven hours by the addition of reduced glutathione to bicarbonate Ringer's has also been previously reported 4 ' s ; in the present study we showed that there was no significant difference between the swelling rates of corneas perfused with GBR or KEI for the experimental period of 3.5 hours. The specific function of glutathione in this chemically defined media has not been studied, and it has not been isolated from the aqueous humor nor the corneal endothelium. Glutathione has been found, however, in lens, corneal stroma, and epithelium. 0 An adequate supply of reduced glutathione is apparently necessary for many of the normal activities of cells. Kosower and Kosower 10 have suggested that the concentration of reduced glutathione within cells plays some role in the regulation of protein synthesis, and in maintaining cell membrane integrity by protecting against chemical challenges or oxidizing agents. Whether one or both of these roles is involved in the normal corneal endothelial function is unknown. However, the addition of glutathione to the perfusion media in our studies not only enabled the cornea to maintain constant thickness, but also maintained the endothelial cell morphology equally as well as KEI for the extended perfusion period. Although the loss of endothelial cell pattern correlates with the inability of a cornea to maintain normal thickness during perfusion (e.g., with bicarbonate Ringer's solution), it was not known if the pattern

7 416 McCarey, Edelhauser, and Van Horn Investigative Ophthalmology June f =.398+ (.002 ±.002 )X Time hr Fig, 9. Cornea] thickness changes during perfusion with a GBR, The upper dashed line represents a single cornea during a ten-hour perfusion. The other line is the regression line calculated from six experiments. The mean swelling rate was 2 /x per hour. Fig. 10. A montage of overlapping specular photomicrographs from a cornea perfused for 3.5 hours with GBR. The normal endothelial pattern was maintained. Fig. 11. Electron micrograph of the endothelium of a cornea perfused for 3.5 hours with GBR. Little evidence of ultrastructural damage is present (xl7,897).

8 Volume 12 Number 6 Changes in corneal endothelium 417 Table II. Correlation of corneal swelling rate to specular microscope endothelial pattern of corneas perfused with a bicarbonate Ringer's Experiment No Swelling rate (p/hr.) Specular microscopic appearance of the endothelium Normal pattern Normal pattern Poor pattern Loss of pattern Loss of pattern Loss of pattern Loss of pattern loss was due to stromal swelling or disruption of the cells. We have used electron microscopy to show that minimal ultrastructural changes are present in the endothelial cells of corneas that maintain constant thickness or have a slow rate of swelling (e.g., during KEI or GBR perfusion); whereas, in the corneas that have a high rate of swelling or are unable to temperature reverse, the endothelial cells have loss of cellular organization or are completely disrupted. The results of this investigation show that a correlation exists between corneal swelling rate, endothelial pattern, and ultrastructure and suggest that changes in the corneal endothelial pattern can be used to provide a direct assessment of cellular integrity. REFERENCES 1. Maurice, D. M.: Cellular membrane activity in the corneal endothelium of the intact eye, Experientia 24: 1094, Maurice, D. M.: The location of the fluid pump in the cornea, J. Physiol. 221: 43, Mishima, S., and Kudo, T.: In vitro incubation of rabbit cornea, INVEST. OPHTHALMOL. 6: 329, Dickstein, S., and Maurice, D. M.: The metabolic basis to the fluid pump in the cornea, J. Physiol. 221: 29, Hodson, S.: Evidence for a bicarbonate-dependent sodium pump in corneal endothelium, Exp. Eye Res. 11: 20, Hoefle, F. B., Maurice, D. M., and Sibley, R. C: Human corneal donor material, Arch. Ophthalmol. 84: 741, Kinsey, V. E., and Reddy, D. V. N.: Studies on crystalline lens. XI. The relative role of the epithelium and capsule in transport, IN- VEST. OPHTHALMOL. 4: 104, Harris, J. E., and Nordquist, L. T.: The hydration of the cornea. I. Transport of water from the cornea, Am. J. Ophthalmol. 40: 100, Anderson, E. I., and Spector, A.: Oxidationreduction reactions involving ascorbic acid and the hexosemonophosphate shunt in corneal epithelium. INVEST. OPHTHALMOL. 10: 41, Kosower, E. M., and Kosower, N. W.: Lest I forget thee, glutathione, Nature 224: 117, 1969.