Characterization of changes observed in the corneal endothelium with the specular microscope

Transcription

1 Characterization of changes observed in the corneal endothelium with the specular microscope Emil S. Sherrard The specular microscope reveals little of the internal features of the corneal endothelium, but in certain experimental and clinical situations vague markings of four different appearances occur and indicate intracellular disturbances. Comparison of the specular microscopical appearances of the affected cells in vitro with stained flat-mount preparations of the same cells shows that three of the markings are due to intracellular vacuoles and the fourth to cell rupture. The differing specular microscopical, appearances of the vacuoles are discussed. Key words: corneal endothelium, specular microscope I ntracellular changes of the corneal endothelium of rabbits, both in vitro and in vivo, have been observed with the specular microscope to follow various forms of insult to the endothelium, such as might occur in the clinical situation. These include air in the anterior chamber, 1 storage of donor eyes at 4 C, 2 and osmotic dehydration of the cornea. 3 ' 4 Specular microscopy reveals little of the nature of these changes, and they have been termed simply "events" 1 of which four basic types have been recognized. Type i events appear as bright, and type ii as dark, small intracellular "bodies." Type iii are dark and shadowlike, often with a bright central inclusion, and large and tend to obliterate the cell From the Pocklington Eye Transplantation Research Unit (Controller: Barrie R. Jones), Institute of Ophthalmology, London, England. Financial support from the Gift of Thomas Pocklington and the Royal National Institute for the Blind (British Foundation for Research into the Prevention of Blindness. Submitted for publication Sept. 5, Reprint requests: Emil S. Sherrard, Pocklington Unit, Institute of Ophthalmology, Judd Street, London WC1H 9QS, England. margins, whereas type iv are also large but appear coarsely granular and bright. It was suggested previously that these changes were due to displacement of the cell nuclei, 3 but subsequent observations have indicated a more complex nature. Similar changes were later observed with the clinical specular microscope in human corneal endothelium (Sturrock, personal communication, 1977), and it is therefore desirable to characterize them so that they may be recognized and their significance understood when encountered in the clinic. These events have therefore been studied by an alternative technique. Because all four types of event usually occur concurrently within a given insulted endothelium, scattered among normal cells, it was essential that the same event-bearing cells which were observed in the living state by specular microscopy be relocated in the endothelium after fixation and staining in order to avoid confusion of the various types. A method has been devised to mark the position of individual endothelial cells in vitro so that they are readily identifiable in silver-hematoxylin-stained flat mounts /78/ $00.50/0 Assoc. for Res. in Vis. and Ophthal., Inc.

2 Volume 17 Number 4 Comeal endothelium seen with specular microscope 323 Fig. 1. Method of marking position of selected endothelial cell for relocation in stained flat mounts. A, Specular photomicrograph (x320) of rabbit corneal endothelium in vitro, narrow slit. The selected study cell (arrow) is a type iv event. B, Same as A but with mask in slit aperture casting shadow over study cell and neighbours. C, Same as B but with silver deposited on the cells with concentration in the intercellular spaces. D, Same as C but after a longer period of illumination to increase "silvering" on cell surfaces. E, Low-power light photomicrograph of posterior surface of the cornea in A to D. The two heavily silvered rectangles of endothelium are clearly seen as is, in this case, the study cell (arrow) between them. (Shadow to left is bubble trapped between cornea-mounting ring and slide). F, Light photomicrograph (x320) of portion of the endothelium stripped from the cornea and stained (hematoxylin). The study cell (arrow) between the rectangles is unmistakable. In this example the cell itself contains silver particles, indicating cell rupture. Methods To induce intracettular changes in the endothelium 1. Mount freshly excised rabbit cornea under the specular microscope according to the technique of Dikstein and Maurice. 5 Perfuse the endothelial surface with glutathione-bicarbonateringer's solution (GBR) (60 /xl/min), and cover epithelial surface with silicone oil. Maintain a retrocorneal pressure of about 15 cm H2O and a temperature of 35 C. Leave the mounted cornea to equilibrate with its new environment for 1 hr. 2. Examine the corneal endothelium with the specular microscope to ensure its normality. 3. Remove the silicone oil from the epithelial surface with a pipette, and replace with a solution of 20% glycerin. Initially, allow the solution to overflow the corneal mounting chamber to wash away oil residue. Leave the glycerin solution on the epithelial surface for a total time of 5 or 6 min. 4. Suck away glycerin. Wash epithelium briefly with a flow of 0.9% saline, and carefully blot dry. Recover epithelium with silicone oil. 5. After a short recovery (hydration) period, scattered endothelial cells will be seen, with the specular microscope, to possess events of types i to iv. The remainder of the cells will appear to be normal. To mark position of a selected endothelial cell 1. With wide slit illumination, scan the dam-

3 324 Invest. Ophthalmol. Visual Sci. April 1978 Sherrard Fig. 2. a, Specular photomicrograph of rabbit corneal endothelium in vitro after dehydration of the cornea with 20% glycerin on epithelial surface. Cells show events of types i and ii. b, Flat mount of same field, fixed and stained (silver and hematoxylin). Intracellular vacuoles coincide with events in a. Fig. 3. a, Specular photomicrograph of rabbit corneal endothelium in vitro after dehydration of the cornea with 20% glycerin on epithelial surface. Cells show events of types i and iii. b, Same field, fixed and stained (silver and hematoxylin). Type iii event in a also caused by vacuole in b. aged endothelium with the specular microscope, and select a suitable cell for study. 2. Center the study cell in the visible field, reduce width of the slit, and photograph (Fig. 1 A). 3. Remove reducing agents from perfusion system to prevent later generalized precipitation of silver salts: clamp off inlet tube, cut it, and connect hand-operated 20 ml hypodermic syringe containing 10% sucrose solution. Unclamp and gently flush out all GBR from system with the sucrose 1 ml/min. Maintain visual check on cells to ensure that there is no change due to increase of intraocular pressure during flushing or due to change of medium. 4. Place mask against slit apperture of microscope to cast a shadow over the study cell and its neighbors, but leave several cells at the edges of the visible field illuminated (Fig. 1 B); these marginal cells are to be "marked." 5. Exchange sucrose-filled syringe with another containing 0.25% silver nitrate solution in 10% sucrose, and gently introduce into system as before. When the AgN03 contacts the endothelium, the borders of the illuminated marginal cells suddenly become very bright (Fig. 1 C), reversing their usual appearance, as a result of the deposition of metallic silver particles under the influence of the microscope light. 6. Leaving.the light on, flush the silver nitrate solution from the system with more sucrose, meanwhile observing the marginal cells which gradually acquire a glistening, golden brown coloration (Fig. 1 D): When this occurs, switch off the light. Prolonged illumination results in overdarkening of the entire endothelium due to light reflected and scattered within the perfusion chamber. Continue flushing for some minutes to ensure removal of silver nitrate and its reduction products. 7. Clamp off inlet and outlet tubes. Remove brass mount with cornea from microscope, and wash oil from epithelial surface under running tap. Free oil globules may later become adherent to the endothelium and retard fixation. 8. Remove cornea with its plastic mounting ring from brass mount, and wash well but swiftly in large volume of sucrose solution. 9. Place cornea and mounting ring into absolute methanol, and leave to "fix" 1 hr in dark. 10. Examine retrocorneal surface, under methanol, with staining microscope; observe two brown-black rectangular areas of heavily silvered endothelium (Fig. 1, E) the marginal cells; the study cell lies between them. 11. With small-diameter trephine (5 mm) cut posterior layers of cornea, to include marked rectangles, against wax block. The remaining cornea and mounting ring provide an anchor which facilitates removal of the endothelial disc. 12. Submerge cornea and ring in tap water. 13. Assist flotation of trephined endothelial disc from cornea with fine brush; catch floating disc of endothelium on glass slide; dry in air in dark; stain with hematoxylin, dehydrate, clear, and mount Examine with light microscope. The study

4 Volume 17 Number 4 Corneal endothelium seen with specular microscope 325 Fig. 4. a, Specular photomicrograph of rabbit corneal endothelium in vitro after dehydration of the cornea with 20% glycerin on epithelial surface. One cell shows a type iv event, b. Same field after fixation and staining (silver and hematoxylin). The type iv event in a appears as a ruptured cell in b, cell is readily located between the two rectangles of heavily silvered cells (Fig. 1, F) by comparison with the original specular photomicrograph (Fig. 1 A). Results The paired photomicrographs in Figs. 2 to 4 represent (a) the specular microscopical appearances of portions of insulted rabbit corneal endothelia in vitro and (b) the appearance of the same fields of endothelium as seen with the ordinary light microscope after fixation and staining. The linking lines, between (a) and (b) identify the same eventbearing cells. Fig. 2, a, shows events of types i and ii, and in 2 b, these are both seen to be due to intracellular vacuoles. Fig. 3, a, shows events of types i and iii, and in Fig. 3, b, the latter also appears as an intracellular vacuole. Fig. 4, a, includes a type iv event which in the stained preparation (Fig. 4, b) is seen to be a collapsed cell with folded membranes and vacuolated cytoplasm. It is a ruptured cell. Similarly, Fig. 1, A, shows a type iv change which in Fig. 1, F, after staining, contains a dense deposit of silver particles indicating rupture of the membrane. Discussion The specular microscopical appearances of the events induced in the endothelium by some forms of environmental change suggest that the cells respond in four different ways resulting in four types of intracellular alteration. However, stained preparations of event-bearing cells show that types i to iii are all due to the presence of cytoplasmic vacuoles whereas type iv represents a ruptured cell. This is consistent with the fate of the events as seen in in vivo studies; types i and ii, and occasionally type iii interchange one with another, usually diminish in apparent size, and finally disappear, restoring to the affected cell a normal appearance, but cells with type iv changes are sloughed from Descemet's membrane and replaced by protrusions from the neighboring cells.3 The stained preparations indicate that the different specular microscopical appearances of the events are unrelated to the size of the causative vacuoles (e.g., types i and iii, Fig. 3, are both due to large vacuoles), but it seems possible that event type is governed by the shape of a vacuole and its relationship to the cell margins. Spherical vacuoles will, scatter light in all directions away from the microscope and hence appear dark, whereas vacuoles which are flattened against the anterior and/or posterior cell membranes will enhance the reflectability of the endothelium-aqueous humour-interface and increase specular reflection and appear bright. Events of types i and ii move in the living cell

5 326 Sherrard Invest. Ophthalmol. Visual Sci. April 1978 and alternate between bright (type i) and dark (type ii) as they do so. 3 This is interpreted as their moving into and out of contact with the cell membrane. It is difficult to explain the brightness of ruptured (type iv) cells in this way, but it is possible that their collapsed membranes are denser than usual and so reflect more light. The intracellular changes ("events") which often occur in the living corneal endothelium in response to environmental alterations and are seen with the specular microscope are vacuoles (types i to iii) and ruptured cells (type iv). Comment The characterization of events i and ii as intracellular vacuoles and of type iv as ruptured cells is totally repeatable by the technique described, but some difficulty has been experienced with the type iii events, which, in most instances, show as intracellular vacuoles after staining (Fig. 3) but occasionally appear to be ruptured. Since, however, in vivo observations have shown that type iii's may metamorphose to types ii or iv, and if to the latter will then be evicted from the endothelium, 3 it seems probable that cells containing type iii events are extrafragile and are thus sometimes ruptured during the silvering process. I thank William Ng for technical assistance. REFERENCES 1. Leibowitz, H. M., Laing, R. A., and Sandstrom, M.: Corneal endothelium: the effect of air in the anterior chamber, Arch. Ophthalmol. 92:227, Sherrard, E. S.: The corneal endothelium in vitro: its survival during banking at 4 C, Trans. Ophthalmol. Soc. U. K. 94:80, Sherrard, E. S.: The corneal endothelium in vivo: its response to mild trauma, Exp. Eye Res. 22:347, Sherrard, E. S.: Specular microscopy of the corneal endothelium, Ophthalmic Optician 17(19): Dikstein, S., and Maurice, D. M.: The metabolic basis to the fluid pump in the cornea, J. Physiol. 221:29, Oh, J. O.: Changes with age in the corneal endothelium of normal rabbits, Acta Ophthalmol. 41:568, Copyright information The appearance of a code at the bottom of the first page of an original article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., P.O. Box 765, Schenectady, N.Y , /518/ , for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale.