Glucocorticoid Target Cells In Human Outflow Pathway: Autopsy and Surgical Specimens

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Reports Glucocorticoid Target Cells In Human Outflow Pathway: Autopsy and Surgical Specimens M. Rosario Hernandez, Eugene J. Wenk, Bernard I. Weinstein, Patricia Abumohor, Steven M. Podos, Michael W.

1 Reports Glucocorticoid Target Cells In Human Outflow Pathway: Autopsy and Surgical Specimens M. Rosario Hernandez, Eugene J. Wenk, Bernard I. Weinstein, Patricia Abumohor, Steven M. Podos, Michael W. Dunn, and A. Louis Southren Target cells for glucocorticoids were found in trabeculectomy specimens obtained surgically from humans with glaucoma (primary and secondary) and from nonglaucomatous autopsy eyes using an autoradiographic technique. Specific nuclear localization of H-dexamethasone was found in cells of the trabecular meshwork, scleral fibroblasts, and in the endothelial lining of both Schlemm's canal and the outflow vessels. Glucocorticoids may alter the outflow facility by a direct effect on the metabolism of these cells. The autoradiographic method is suitable for studying competitors of glucocorticoid binding in small surgical specimens of human tissue involved in the outflow of aqueous humor. Invest Ophthalmol Vis Sci 24: , 198 Primary open angle glaucoma (POAG), the most common form of glaucoma, is unique to humans. Most studies indicate that the elevation of intraocular pressure (IOP) is a consequence of impairment of aqueous humor outflow. Patients with this disorder show a marked increase in intraocular pressure following topical or systemic administration of glucocorticoids. It has been suggested that this disease is caused by an abnormal sensitivity to endogenous glucocorticoids (for review, see references 1 and 2). Thus, the mechanism by which these steroids affect the metabolism of cells of the outflow pathway for aqueous humor may be of significance in the pathogenesis of POAG. In the normal rabbit eye, which does not contain a trabecular meshwork or Schlemm's canal but does have analogous structures, we have detected glucocorticoid receptors in the outflow pathway by both biochemical ' 4 and autoradiographic techniques. 5 Glucocorticoid receptors also have been detected in trabecular cells in culture. 6 In order to study glucocorticoid target cells in human glaucomatous eyes, it is necessary to rely almost entirely on surgical trabeculectomy specimens since it is rare to obtain authentic glaucomatous tissue from autopsy specimens. The minuteness of the surgical specimens requires specialized techniques in order to evaluate hormone responsiveness. The present study was designed to identify specific glucocorticoid target cells in these small surgical specimens and autopsy specimens by steroid autoradiography. Materials and Methods. Discarded specimens from clinically indicated trabeculectomies were obtained from two patients with POAG and two patients with secondary glaucoma associated with exfoliation syndrome. Similar specimens also were obtained from two individuals at autopsy. The specimens were placed in normal saline in the operating room and transported to the laboratory in ice. The tissues were transferred within 60 minutes to an incubation medium (Hepes buffered RPMI 1640) containing 1 /ici/ml of H-dexamethasone (specific activity 21 to 0 Ci/mmol) and incubated at 0 C for 45 minutes. These conditions were judged to be optimal following pilot studies in which the amount of H- dexamethasone was varied from 1-5 ^Ci/ml and the time of incubation was varied from 0-70 minutes. In one case of POAG, one case of secondary glaucoma, and in both autopsy eyes, sufficient tissue was available to permit two parallel incubations. One sample was incubated with H-dexamethasone alone and the other with H-dexamethasone and a 50-fold excess of nonlabeled dexamethasone. A sample of sclera from the posterior segment of an autopsy eye also was incubated with H-dexamethasone with and without an excess of nonlabeled hormone. After incubation the tissues were washed in 90 ml of heat inactivated rabbit serum at 0 C for 90 minutes, either by supervision or with 10-ml aliquots, frozen, and processed for autoradiography as described previously. 7 ' 8 Washing by superfusion or with aliquots both were found to be suitable for removing unbound steroid. After to 10 months of exposure at -20 C, autoradiographs were developed and stained with hematoxylin and eosin (H & E). Three to four months gave a maximum ratio of specific to nonspecific binding. Although exposure for longer times led to increased specific binding, it also resulted in increased nonspecific binding and background. 1612

2 Reporrs No. 12,..; 161 Table 1. The distribution of H-dexamethasone in trabecular meshwork cells obtained at surgery and autopsy.*.. Diagnosis v.. loum '. Primary open angle glaucoma (n = ) Primary open angle glaucoma (n = 45) (exfoliation syndrome) (n = 1) (exfoliation syndrome) (n = 0) Nonglaucomatous autopsy eye (n = 40) Nonglaucomatous autopsy eye (n = 45) Nuclear S.G./25 fim1 Extranuclear S.G./25 pm ± ± 0.4* 14.5 ± ± 0,2* 14.0 ± ± 0.2* 9.8 ± ±0.2* 10.9 ± ± 0.4* 7.8 ± ± 0.2* Values are expressed as means ± SE. Exposure times varied from to 10 months and, therefore, quantitative comparisons cannot be made from case to case. S.G. = silver grains; n = number of nuclear or extranuclear areas included in each determination. * Indicates that the difference between nuclear and extranuclear S.G. are statistically significant (P < 0.01). V Fig. 1. Autoradiographs of human aqueous humor outflow region in a surgical specimen from a patient with glaucoma associated with exfoliation syndrome. The specimen was incubated with 1 jici/ml H-dexamethasone (H & E), Exposure time, 10 months. Scale bar, 10 nm. Focus is at the level of the silver grains. A. Trabecular meshwork cells showing nuclear localization of H-dexamethasone (arrows). B. Schlemm's canal (SC) showing localization of the label to endothelial cells. Trabecular meshwork (TM) cells also show labeling (arrows). C. Outflow vessel (OV) shows localization of the label to endothelial cells. Arrows point to labeled trabecular meshwork (TM) cells. Quantitation of the silver grain densities was performed using photomicrographs of the trabecular meshwork at a magnification of 2,000. Grains were counted in a 25-^irn2 area superimposed to the cell nucleus and to equivalent immediately adjacent areas of extranuclear space. Because of the shape of the trabecular meshwork cells, the extranuclear space includes cytoplasm and intervening extracellular space. All trabecular meshwork cell nuclei contained in all sections prepared for autoradiography were counted. At least 0 nuclear areas and 0 extranuclear areas were counted in each case. The final values were expressed as the mean of silver grains per nuclear or extranuclear area ± standard error and submitted to a Student's ttest for significance. Results, Nuclear localization of H-dexamethasone was readily apparent in the cells of the trabecular meshwork in samples incubated with the labeled hormone (Figs. 1A and 2A), Nuclear localization also was found in the endothelial lining of Schlemm's canal and the outflow vessels (Figs. IB and 1C). Table 1 shows the distribution of silver grains in the trabecular meshwork of six different specimens after incubation with H-dexamethasone. The concentration of silver grains over the nuclei in each of the specimens is significantly higher than over the extranuclear space {P < 0.01). When samples were incubated with H-dexamethasone and an excess of nonlabeled hormone, nuclear localization of the silver grains in the trabecular meshwork cells was suppressed (Fig. 2B). Table 2 shows the quantitation and statistical analysis of these data. Nuclear localization of the silver grains was decreased significantly in the presence of an excess of the nonlabeled steroid (P < 0.01), indicating that this binding

$1614 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 198 Vol. 24 7 a b d \ rig. L.$

3 1614 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 198 Vol a b d \ rig. L. Auioraaiograpns or numan trabecuiar meshwork, antenor, and pumcnur sciera irum a nongiaucomaious eye ootained at autopsy (H & E). Exposure time, months. Scale bar 10 jtm. Focus at the level of the silver grains. A. Trabecular meshwork cells from a sample incubated with H-dexamethasone showing localization of the silver grains to the cell nuclei (arrows). B. Trabecular meshwork cells from a sample incubated with H-dexamethasone and an excess of nonlabeled hormone. Note the lack of nuclear localization of the silver grains to the nuclei of the trabecular meshwork cells (arrows). C. Sclera adjacent to the outflow region from a sample incubated with H-dexamethasone. Note the localization of the silver grains to the nuclei of the stromal fibroblasts. D. Fibroblasts from the same region as in C but from a sample incubated with H-dexamethasone and an excess of nonlabeled hormone. Note the lack of nuclear localization of the silver grains. E. Small blood vessel from the anterior sclera from a sample incubated with 'H-dexamethasone showing localization of the label to the endothelial cells (arrows). F. Sclera of the posterior segment of the same eye showing lack of nuclear concentration of the silver grains in a sample incubated with H-dexamethasone alone.

$1615 [\eport5 No. 12 0: Fig. 2. {continued) The fibroblasts of the sclera from the posterior segment of the eye do not show any concentration of silver grains in the nuclei (Fig. 2F). Discussion.$

4 1615 [\eport5 No. 12 0: Fig. 2. {continued) The fibroblasts of the sclera from the posterior segment of the eye do not show any concentration of silver grains in the nuclei (Fig. 2F). Discussion. This study demonstrates localization of H-dexamethasone to the nucleus of human trabecular meshwork cells, scleral fibroblasts adjacent to the outflow pathway, and the endothelial lining of both Schlemm's canal and outflow vessels. The localization was suppressed by the addition of an excess of nonlabeled steroid to the incubation medium indicating was saturable. By contrast, the number of silver grains in the extranuclear space was not altered significantly. In all samples, the fibroblasts of the sclera adjacent to both Schlemm's canal and the trabecular meshwork showed clear nuclear localization of H-dexamethasone (Fig. 2C). Addition of an excess of nonlabeled steroid suppressed nuclear localization of H-dexamethasone in these adjacent scleral fibroblasts (Fig. 2D). Small blood vessels in the sclera also showed localization of the steroid in the nuclei of endothelial cells (Fig. 2E). Table 2. The distribution of H-dexamethasone in trabecular meshwork cells from four individuals. Each specimen was incubated with H-dexamethasone with or without a 50-fold excess of nonlabeled steroid. Extranuclear S.G./25 nm2 Nuclear S.G./25 urn2 Diagnosis Primary open angle glaucoma (n = ) (exfoliation syndrome) (n = 0) Nonglaucomatous autopsy eye (n = 40) Nonglaucomatous autopsy eye (n = 45) H-dexamethasone *H-dexamethasone with excess nonlabeled steroid s H-dexamethasone H-dexamethasone with excess nonlabeled steroid 12.5 ± ± 0.2".8 ± ± ± ± 0.2* 1.8 ± ± ± ± 0.2*.4 ± ± ± ±0.1* 2.6 ± ± 0.2 Values are expressed as means ± SE. Exposure times varied from to 6 months and, therefore, quantitative comparisons cannot be made from case to case. S,G. = silver grains; n = nuclear or extranuclear areas included in each determination. * Indicates that the differences between the nuclear localization of S.G. in the tissues incubated with and without an excess of nonlabeled steroid was significant (P < 0.01).

1616 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 198 Vol. 24 that the binding sites are saturable, characteristic of steroid receptor binding.

5 1616 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 198 Vol. 24 that the binding sites are saturable, characteristic of steroid receptor binding. The nuclear localization of H-dexamethasone is probably the result of binding to the cytoplasmic glucocorticoid receptor with subsequent translocation to the cell nucleus during incubation (0 C for 45 minutes), as is seen in virtually all steroid hormone target cells. 910 The lack of nuclear localization of H-dexamethasone in the scleral fibroblasts of the posterior segment of the eye, as opposed to those adjacent to the outflow pathway, suggests a functional difference between these two groups of scleral cells. Since this is a single observation, however, it requires confirmation with additional specimens. The present study demonstrates the presence of glucocorticoid target cells in the outflow region of both glaucomatous and nonglaucomatous eyes. Additional studies will be required in order to determine whether or not there are disease-related differences in the distribution of glucocorticoid binding sites. It is clear, however, that this autoradiographic technique is applicable to small surgical specimens and will permit the testing of various competitors of glucocorticoid binding directly in the human tissue involved in the outflow of aqueous humor. Some of these competitors may be glucocorticoid antagonists of potential use in the clinical management of POAG. Key words: glucocorticoids, target cells, glaucoma, autoradiography Acknowledgments. The authors gratefully acknowledge the cooperation of Claudio Yaluff, M.D. in supplying some of the surgical specimens. The authors also wish to thank Auvra Kartub and Teresa Navarro for their secretarial work and Susana Dominguez for her technical assistance. From the Departments of Ophthalmology, Medicine and Anatomy of New York Medical College, Valhalla, New York, the Department of Experimental Morphology and the Unit of Biostructure, School of Medicine, University of Chile, and the Department of Ophthalmology, Mount Sinai School of Medicine, The City University of New York, New York, New York. Supported by USPHS Grant EY- 011 and in part, by Grant B of D.D.I., University of Chile. Submitted for publication March 7, 198. Reprint requests: A. Louis Southren, MD, New York Medical College, Valhalla, NY References 1. Wilensky JT and Podos SM: Prognostic parameters in primary open angle glaucoma. In Symposium on Glaucoma, Transactions of the New Orleans Academy of Ophthalmology, St. Louis, The C. V. Mosby Company, 1975, pp Kolker AE and Hetherington J Jr: Becker-Shaffer's Diagnosis and Therapy of the Glaucomas. St. Louis, The C. V. Mosby Company, 1976, pp , Weinstein BI, Altman K, Gordon GG, Dunn M, and Southren AL: Specific glucocorticoid receptor in the iris-ciliary body of the rabbit. Invest Ophthalmol Vis Sci 16:97, Southren AL, Gordon GG, Yeh HS, Dunn MW, and Weinstein BI: Nuclear translocation of the cytoplasmic glucocorticoid receptor in the iris-ciliary body of the rabbit. Invest Ophthalmol Vis Sci 18:517, Tchernitchin A, Wenk EJ, Hernandez MR, Weinstein BI, Dunn MW, Gordon GG, and Southren AL: Glucocorticoid localization by radioautography in the rabbit eye following systemic administration of H-dexamethasone. Invest Ophthalmol Vis Sci 19:121, Weinreb RN, Bloom E, Baxter JD, Alvarado J, Lan N, O'Donnell J, and Polansky JR: Detection of glucocorticoid receptors in cultured human trabecular cells. Invest Ophthalmol Vis Sci 21:40, Wenk EJ, Hernandez MR, Weinstein BI, Gordon GG, Dunn MW, and Southren AL: Glucocorticoid receptor binding in bovine lens. Invest Ophthalmol Vis Sci 22:599, Stumpf WE and Roth LJ: High resolution autoradiography with dry mounted, freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds H-estradiol and H-mesobilirubinogen. J Histochem Cytochem 14:274, Jensen EV, Suzuki T, Kawashima T, Stumpf WE, Jungblut PW, and DeSombre ER: A two-step mechanism for the interaction of estradiol with rat uterus. Proc Natl Acad Sci USA 59:62, Southren AL, Dominguez MO, Gordon GG, Wenk EJ, Hernandez MR, Dunn MW, and Weinstein BI: Nuclear translocation of the cytoplasmic glucocorticoid rceptor in the iris-ciliary body and adjacent corneoscleral tissue of the rabbit following topical administration of various glucocorticoids. Invest Ophthalmol Vis Sci 24:147, 198. Coogulotion Properties of Introoculor Humors ond Cerebrospinol Fluid Ali A. Khododousr, Wolrer J. Stork, and William R. Dell Clinical observations indicated a prompt cessation of episcleral capillary bleeding after exposure of those tissues to aqueous humor during intraocular surgery. Based on this observation, both aqueous and vitreous humors were obtained from eyes provided by an eye bank and from human eyes prior to intraocular surgery. Samples of cerebrospinal fluid also were obtained from two patients, one with optic atrophy and another with nutritional amblyopia. A total of 49 specimens were studied. Aqueous humor shortened the average ear lobe puncture bleeding time from 4.74 ± 0.2 to 1.1 ± 0.1 minutes. Varying dilutions of aqueous humor, up to a dilution of 1:16, shortened prothrombin and partial thromboplastin

Reports ROSARIO HERNANDEZ, BERNARD I. WEIN- DON, AND A. Louis SOUTHREN.

Reports ROSARIO HERNANDEZ, BERNARD I. WEIN- DON, AND A. Louis SOUTHREN. Reports Glucocorticoid localization by radioautography in the rabbit eye following systemic administration of 3 H-dexamethasone. AN- DRES TCHERNITCHIN, EUGENE J. WENK, M. ROSARIO HERNANDEZ, BERNARD I.