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of Health, Bethesda, Maryland. Submitted for publication: June glucocorticoid activity by 5/3-dihydrocortisol: its role in glau- 22, 1984. Reprint requests: Bernard I. Weinstein, PhD, Department coma. Science 222:172, 1983. of Medicine, New York Medical College, Cedarwood Hall, Valhalla, 3. Southren AL, Gordon GG, I'Hommedieu D, RaviKumar S, NY 10595-1691. Dunn MW, and Weinstein Bl: 50-Dihydrocortisol: possible mediator of the ocular hypertension in glaucoma. Invest References Ophthalmol Vis Sci 26:393. 1985. 4. Lowry OH, Rosebrough NJ. Farr AL. and Randall RJ: Protein 1. Southren AL, Gordon GG, Munnangi PR, Vittek J, Schwartz measurement with the folin phenol reagent. J Biol Chem 193: J, Monder C, Dunn MW, and Weinstein BI: Altered cortisol 265, 1951. metabolism in cells cultured from trabecular meshwork speci- 5. Tomkins GM: A mammalian 3cv-hydroxysteroid dehydrogenase. mens obtained from patients with primary open angle glaucoma. J Biol Chem 218:437, 1956. Invest Ophthalmol Vis Sci 24:1413, 1983. 6. Marver D and Edelman IS: Dihydrocortisol: a potential miner- 2. Weinstein Bl, Gordon GG, and Southren AL: Potentiation of alocorticoid. J Steroid Biochem 9:1, 1978.

The Production and Mechanism of Ghost Cell Glaucoma in the Cat and Primate

Fred H. Lambrou, Jr, Donald G. Aiken, Wendell D. Woods, and David G. Campbell

Fresh human ghost blood cells (GBCs) have been shown to into the anterior chambers of rabbits and monkeys, cause increased resistance to outflow in enucleated human suggested that endothelial swelling might be important eyes. In addition, glutaraldehyde-fixed GBCs can cause in the pathophysiology of GCG. The purpose of our glaucoma in the rabbit and primate in vivo. The present study was to determine whether GCG is due to study shows for the first time that fresh autologous GBCs primary obstruction of the intertrabecular spaces, as can cause an acute in vivo rise of intraocular pressure when was originally believed, or whether it is due to a injected into the anterior chambers of the cat and primate. secondary effect. A second purpose of this study was This rise was of greater magnitude and longer duration than that caused by the injection of a greater number of pliable, to show for the first time that fresh, autologous GBCs fresh red blood cells. It has been theorized that ghost cell could cause an acute, in vivo rise in IOP, and to glaucoma (GCG) is due to cellular obstruction of the compare the rise with that caused by fresh RBCs. intertrabecular spaces by the nonpliable GBCs. Histologic Materials and Methods. Ghost blood cells were results from the present study confirm this belief. No prepared by incubating washed RBCs in a 37°C water evidence of significant trabecular meshwork degeneration bath for 10 days.5 The tubes were then placed in a or significant GBC phagocytosis was seen. Invest Ophthal- centrifuge at 1000 Xg for 1 hr until the GBCs mol Vis Sci 26:893-897, 1985 separated from the undegenerated RBCs and formed a khaki-colored layer. This layer was then removed, Ghost cell glaucoma is a recently described glau- and GBC formation was verified by phase microscopy. coma that occurs as a consequence of vitreous hem- The concentration of GBCs was determined using a orrhage. Following vitreous hemorrhage, the red blood manual hemocytometer. cells (RBCs) degenerate into GBCs and pass slowly Adult cats and rhesus monkeys were utilized in into the anterior chamber through a disruption in this in vivo study. Eight cat and two monkey anterior the anterior hyaloid face. The cells then flow into the chambers were injected with autologous GBCs. Five outflow pathways, causing an increase in intraocular cat anterior chambers were injected with autologous pressure (IOP).1 When the glaucoma was initially RBCs. In addition, control experiments were con- described, it was felt that the reduced pliability of the ducted on two cats and one primate using injections cells made them effective obstructors of the intertra- of mock aqueous solution. becular spaces. This theory was supported by perfusion Blood cell injection was accomplished by inserting 1 2 studies in enucleated human eyes. ' a 25-gauge needle obliquely through the peripheral Grierson and Lee,3 however, have shown that cornea into the anterior chamber of anesthetized endothelial cells can phagocytose GBCs in traumatized animals (xylazine and ketamine, in the cats; and eyes, leading to speculation that this process may ketamine, in the primates). Approximately 60% of somehow contribute to the rise in IOP. Furthermore, the aqueous humor was withdrawn, and the needle Quigley,4 using glutaraldehyde-fixed GBCs injected was removed. A new needle was inserted through the

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-4 Table I. Summary of IOP response to cellular injection Anterior chamber injection Intraocular pressure Animal Number of cells Duration number Species Material (X106) Baseline Peak (hr)

1 eat GBC 1.90 13.0 36 18 2 cat GBC 1.39 19.5 60 18 3 cat GBC 2.02 17.0 36 18 4 cat GBC 0.21 13.0 60 18 5 cat GBC 6.75 18.7 >60 24 cat GBC 4.9 13.0 >60 5 7 cat GBC 9.48 15.0 >60 * 8 cat QIC 9.6 15.0 >60 * Mean 4.5 15.5 54 17 5 cat RBC 13,9 14.0 23 5 6 cat RBC 25.5 17.7 54 5 7 cat RBC 17.4 14.0 34 * 8 cat RBC 18.8 16.0 54 5 9 cat RBC — 6.7 I 0 Mean 18.9 13.7 34.6 4

1 monkey GBC 0.408 16.0 >60 24 J5 2 monkey GBC — 16.0 >60 • * Elevated at lime of killing.

same tract, and the appropriate GBC or RBC autol- was removed after injection, and gentle pressure was ogous solution was injected; cats received 600 JAI, and applied to the exit wound. the primates were injected with 200 jA. The needle Intraocular pressure was determined using a Kowa

Fig. 1. Phase micrograph of the cat outflow pathway 15 hr after injection of GBCs into the anterior chamber. The intertrabecular spaces can be seen to be occluded with GBCs. AP: aqueous plexus; TM: trabecular meshwork.

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Fig. 2. Electron micrograph of the cat outflow pathways 15 hr after injection of GBCs into the anterior chamber. The trabecular beams are intact, and the irabecular endothelial cells appear normal. AP: aqueous plexus; E: endothelial cell; arrow: GBC. Inset, Trabecular endothelial cell phagocytosis of a GBC.

hand-held applanation tonometer (Kowa; Japan) on Stockholm, Sweden), stained with uranyi acetate and anesthetized animals. The tonometer was calibrated lead citrate, and examined with a Zeiss S-2 electron in our laboratory using intraocular manometry. A microscope. This investigation conformed to the baseline pressure was obtained for each animal over ARVO Resolution on the Use of Animals in Research. a period of several days. Pressure was then measured Results. Injections of the GBCs in the cat anterior at the time of paracentesis and at 5, 15, 24, and 72 chamber initially produced a 75% GBC hyphema hr, until enucleation. Statistical analysis utilized the which cleared rapidly over 24-36 hr and was com- t-test for independent samples. pletely cleared on gross visual examination at 48 hr. Histologic studies were performed on normal, con- This GBC injection caused an acute rise in IOP in trol, 15-hr/GBC, 3-day/GBC and 15-hr/RBC cats, all eight cats, with pressure elevating from a mean and on a 17-hr/GBC monkey. The eyes were imme- baseline of 15.5 ± 3.1 mmHg to a mean peak IOP diately enucleated at the time of killing, rapidly of greater than 54 mmHg (P < 0.01; Table 1). In six trephined, and fixed in a solution of 2.5% glutaral- cats, the IOP exceeded 60 mmHg, the highest reading dehyde in O.I M cacodylate bufFer. A sample of the on the tonometer, and 60 mmHg was therefore used outflow pathway was dissected from the inferior for calculation of the mean. The pressure rise was of portion of the anterior chamber and refixed in glu- shorter duration than the observed hyphema, with a taraldehyde for 12 hr at 4°C. After rinsing in caco- mean length of 17 hr. The GBCs also caused an dylate buffer, the sample was postfixed for 1 hr at acute rise in IOP when injected into two rhesus 4°C in 1% osmium tetroxide, then dehydrated through monkeys, with a mean rise from 16 mmHg to a peak graded acetone and embedded in epon-araldite. Sec- exceeding 60 mmHg and a duration of 24 hr. In one tions measuring 1.5 jt/m were stained with 1% toluidine monkey, this pressure rise was followed by a period blue for light microscopy. Additional ultrathin sections of hypotony lasting 10 days. (60-70 nm) were cut on an LKB Ultratome IV (LKB; Red blood cell injection into five cats also caused

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Fig. 3. Electron micrograph of the primate outflow pathways 17 hr after injection of GBCs into the anterior chamber. The trabecular meshwork architecture appears intact. No migration or degeneration of endothelial cells can be seen. Because ghost cells consist of only a thin cell membrane lined with Heinz bodies, they are difficult to distinguish in this micrograph but are occluding the intertrabecular spaces. SC: Schlemm's canal; TB: trabecular beam; arrows: endothelial cells.

a rise in IOP (P < 0.05), but this was of lesser intact and that the collagenous beams were not magnitude (P < 0.025) and duration (P < 0.01) than denuded (Fig. 2). Although it was a rare finding, an that caused by GBC injection. The baseline was 13.7 occasional endothelial cell could be seen phagocytizing ± 4.7 mmHg and rose to an average peak of 34.6 a GBC, but the former did not appear swollen or ± 19.9 mmHg. In fact, it should be noted that one degenerated (Fig. 2, inset). After 3 days, with the IOP cat did not show a rise in IOP, and no IOP rose returned to normal, no GBCs were seen in the above 54 mmHg. Moreover, the mean duration was trabecular meshwork. The meshwork appeared nor- only 4 hr. The number of RBCs injected was approx- mal, with some neutrophils present. imately four times greater than the number of GBCs Similar changes occurred in the 17-hr/GBC mon- (18.9 ±4.2 X 106 as compared to 4.5 ± 3.7 X I06 key. The IOP was elevated, and the trabecular mesh- GBCs). This number of RBCs produced an initial work appeared completely occluded with intertrabec- 75% hyphema which cleared rapidly by 5 hr. There ular GBCs. Electron microscopy confirmed that the was no pressure rise seen in the control animals. intertrabecular spaces were filled with GBCs (Fig. 3). Light microscopy showed no difference between The endothelial cells remained intact, and there was the control and normal meshworks. In the 15-hr/ no evidence of migration, degeneration or phagocy- GBC cat with elevated IOP, the meshwork appeared tosis. totally occluded with GBCs (Fig. 1). Some GBCs Discussion. We have shown for the first time that were seen in the aqueous plexus, and although a few fresh autologous GBCs can cause IOP elevation when neutrophils were seen, there was little inflammatory injected into in vivo cat and primate eyes. This reaction. Electron microscopy supported these obser- pressure rise is due to primary obstruction of the vations and showed that the endothelial cells were intertrabecular spaces by the nonpliable GBCs. In the

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present study, little phagocytosis of the fresh autolo- secondary effects may mean that future treatment gous GBCs was seen, and in the rare finding of GBC could be aimed at dislocation of the cells, perhaps by phagocytosis, the endothelial cells appeared normal, ultrasound or biochemical means, to render the cells indicating little role of a secondary effect. This con- more pliable. trasts with earlier studies in which glutaraldehyde- fixed GBCs caused some phagocytosis and endothelial 4 Key words: ghost cell glaucoma, trabecular meshwork, swelling. Perhaps the fixation of the cells accounts glaucoma mechanisms, blood cell deformability for this difference. The acute, one-time injection of GBCs into the anterior chamber to create GCG differs from the actual clinical setting, where ghost From the Glaucoma Research Laboratory and the Yerkes Regional Primate Research Center, Emory University School of Medicine, cells present more slowly. This difference could mean Atlanta, Georgia. Presented at the ARVO Meeting, Sarasota, Florida, that the experimental findings may differ from the May 5, 1983. Supported in part by NIH grant #5-R01-EY03230- human and pathologic findings. From the present 05 and by an unrestricted grant to the Department of Ophthalmology study, however, it is clear that the intertrabecular from Research to Prevent Blindness, Inc., New York, New York. spaces become acutely blocked by GBCs with little Additional support provided by NIH grant #RR-00165 of the phagocytic response. The effect of chronic exposure Animal Resources Program to the Yerkes Regional Primate Research Center of Emory University, Atlanta, Georgia. Submitted for has yet to be studied. publication: June 5, 1984. Reprint requests: David G. Campbell, GBCs were found to be far more effective in MD, Department of Ophthalmology, Emory University School of causing trabecular meshwork obstruction in vivo than Medicine, 1365 Clifton Road NE, Atlanta, GA 30322. RBCs. Although four times more RBCs than GBCs were injected, GBCs caused a higher and longer References elevation of IOP. As has been shown, this is most likely due to decreased pliability.1 Previous studies 1. Campbell DG, Simmons RJ, and Grant WM: Ghost cells as a using glutaraldehyde-fixed GBCs showed an in vivo cause of glaucoma. Am J Ophthalmol 81:441, 1976. pressure elevation lasting many days in the rabbit 2. Campbell DG and Essigman EM: Hemolytic ghost cell glau- coma: further studies. Arch Ophthalmol 97:2141, 1979. and cynomologous monkey.4 This difference in du- 3. Grierson I and Lee WR: Further observations on the process ration appears to be due to the fixation of the GBCs; of haemopgocytosis in the human outflow system. Albrecht it has been shown that glutaraldehyde can cause RBC Von Graefes Arch Klin Exp Ophthalmol 208:49, 1978. 6 7 membranes to become rigid. ' Specifically, in the 4. Quigley HA and Addicks EM: Chronic experimental glaucoma progression from fresh RBCs to GBCs and fixed in primates. Invest Ophthalmol Vis Sci 19:126, 1980. GBCs, decreased pliability parallels a decrease in 5. Jandl JH, Engle LK, and Allen DW: Oxidative hemolysis and ability to pass through the small intertrabecular spaces precipitation of hemoglobin. J Clin Invest 39:1818, I960. of the meshwork, resulting in a longer duration of 6. Stolz JF: Drugs affecting blood rheology. Scand J Clin Lab Invest 41 (Suppl):287, 1981. elevated IOP. 7. Corry WD and Seaman GVF: Alteration of RBC physiological The finding that GCG may be due to primary properties by t-butyldroperoxide and glutaraldehyde. Scand J obstruction of the intertrabecular spaces rather than Clin Lab Invest 41(Suppl):275, 1981.

A Larger Corneal Epithelial Wound Closes at a Faster Rate

Momoru Marsuda,* John L. libels,f and Henry F. Edelhauser|

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

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