Current Research in Ophthalmic Electron Microscopy 4 Edited by William R. Lee With 88 Figures Springer-Verlag Berlin Heidelberg New York 1981 Transactions of the 8th Annual Meeting of the European Club for Ophthalmic Fine Structure in West Berlin, March 28 and 29, 1980 Editor Professor WILLIAM R. LEE University Departments of Ophthalmology and Pathology, The University of Glasgow, Scotland ISBN-13: 978-3-540-10651-7 e-ISBN-13: 978-3-642-81614-7 DOl: 10.1007/978-3-642-81614-7 Library of Congress Cataloging in Publication Data' European Club for Ophthalmic Fine Structure. Current research in ophthalmic electron microscopy. 4. "Transactions of the 8th annual meeting of the European Club for Ophthalmic Fine Structure in West Berlin. March 28-29. 1980"-Verso t.p. Bibliography: p. Includes index. 1. Eye-Anatomy-Con gresses. 2. Ultrastructure (Biology)-Congresses. 3. Electron microscopy-Congresses. 4. Ophthalmology-Congresses. 1. Lee, William R., 1932- . II. Title. QM511.E971981 599.01'823 81-1894 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation. reprinting, re-use of illustrations, broadcasting. reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of German Copyright Law where copies are made for other than private use, a fee is payable to 'Verwertungsgesellschaft Wort', Munich. © by Springer-Verlag Berlin· Heidelberg 1981 The use of registered names. trademarks. etc. in this publication does not imply. even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Universitiitsdruckerei H. Sturtz AG. Wurzburg 2121/3130-543210 Contents Human Conjunctival Surface Mucins: A Quantitative Study of Normal and Diseased (KCS) Tissue W.R. LEE, S.B. MURRAY, J. WILLIAMSON, and D.L. McKEAN. With 8 Figures . . . . . . . . . . . . . The Basement Membrane Complex of the Human Corne al EpithelIum H. BREWITT and E. REALE. With 4 Figures . . . . .. 15 Shrmkage m Preparatory Steps for SEM. A Study on Rabbit Corneal Endothelium O.A. JENSEN, J.U. PRAUSE, and H. LAURSEN. With 6 Figures 25 Effects of Freezing on the Corneal Stroma of the Rabbit After Keratophakia F. HOFFMANN and J.-P. HARNISCH. With 6 Figures 35 The Post-Mortem Vacuoles of Schlemm's Canal 1. GRIERSON and N.F. JOHNSON. With 11 Figures 41 Immotile-ciha Syndrome and the Cilia of the Eye B. SVEDBERGH, V. JONSSON, and B. AFZELIUS. With 3 Figures 57 Identification of a Basement Membrane Proteoglycan m Exfoliation Material J.-P. HARNISCH, H.J. BARRACH. J.R. HASSELL, and P.K. SI~HA. With 6 Figures . . . . . . . . . . . . . . . 65 VI Contents Cellular Composition of Post-haemorrhagic Opacities in the Human Vitreous J.V. FORRESTER and W.R. LEE. With 9 Figures .... 71 Experimentally Induced Lipidosis in Rat Retinal Pigment Epithelium. A Brief Review R. LULLMANN-RAUCH. With 4 Figures ...... . 89 Qualitative Observations on the Variation of Light In duced Damage to the Rabbit Retina N.M. McKECHNIE and W.S. FOULDS. With 14 Figures . 97 An Electron MicroscopIc Study of the Epiretinal Mem brane of Human Eye~ T. HARADA, D. CHAUVAUD, and Y. POULIQUEN. With 11 Figures . 119 Regeneration of the Human Corneal Endothelium. A S.E.M. Study G. RENARD, Y. POULIQUEN, and M. HIRSCH. With 3 Fig- ures .......... . 133 Indexed in Current Contents Human Conjunctival Surface Mucins: A Quantitative Study of Normal and Diseased (KCS) Tissue W.R. Lee1**, S.B. Murray 2, 1. Williamson2 and D.L. McKean 3 I Tennent Instltute of Ophthalmology (Director Professor W S Foulds). Glasgow Umversity. Scotland 2 Southern General HospItal. Glasgow. Scotland 3 PharmacologIcal Research Dept. Burton Parsons & Co . Washmgton DC, USA Abstract. The archItecture and distribution of surface mucins were studied qualitatively and quantitatively by transmission electron microscopy in ruthe nium red stained biopSIes of the human conjunctiva. Six control specImens were compared with three from early keratoconjunctivitis sicca (KCS) and with three specimens from severe KCS cases. The area of the ruthenium red/Os04/mucin reaction product on the conjunctival epithelial surface was measured by image analysis and the values were expressed in 11m2 per 11m length of epithelium. The total area of surface mucins was differentiated from the narrow zone of mucins in close contact with the microplicae. The value for total mucins in two cases of early KCS was higher (0.4 11m2 111m) than the control range (0.1-0.3 11m2 111m) due to the presence of large clumps of mucins on the surface. The values for microplical mucins in early and severe KCS were within the control range, but were an overestimate owing to the presence of abnormal structures, e.g. veSIcles, and abnormal clumps on the surface. This morphometric technique has limitations, but with appro priate material it might be a useful tool for the identification of conjunctival surface mucins. Introduction The ultrastructural features of the human and animal conjunctival epithelium are well documented (see Abdel-Khalek Williamson and Lee 1978a and Latkovic and Nilsson 1979 for recent review articles). There is general agreement that the numerous small processes which project from the epithelial surface (micro pi i cae being a more accurate term than microvilli) serve as a support for the hydrophilic mucosubstances which are essential for the stability of the tear ** Correspondmg author W.R. Lee et al. film. The origin and chemical nature of the mucosubstances, which hereafter for the sake of brevity will be referred to as mucins, is not yet established (see Holly and Lemp 1977, for review), although it has been suggested that glycoproteins (Moore and Tiffany 1979), and in particular sialomucins (Srini vasan, Borgul, Iwamoto and Merriam 1977) form an important component. If the ultrastructure of surface mucins could be reliably demonstrated, their chemical nature could be investigated by pretreatment of the tissue with e.g. sialidase. This would however, require a quantitative form of analysis which could differentiate between biological variation and enzyme effect. As part of a preliminary study of the potential of this approach to the conjunctival surface/ tear film relationships, we attempted to measure surface mucins by image analy sis (Fisher 1971) after staining with ruthenium red. Human tissue (control and keratoconjunctivitis sicca) was used for the investigation and it serves to illustrate the disadvantages and the potential of the method. Material and Methods Conjunctival tissue was obtained from 12 middle aged and elderly mdlviduals who volunteered for the mvestigatlOn Elliptical bIOpSies (4 x 3 mm) were taken from the temporal bulbar conjunctiva SIX mdlVlduals, four female and two male aged 58, 64, 69, 71, 76 and 78 years, did not show any clinical evidence of tear defiCiency and the bIOpsy was taken pnor to cataract surgery. BIOpSies were taken after applicatIOn of local anaesthetic (benoxenate hydrochloride 04%) from SIX patients m the KCS climc. Three females, aged 48, 63 and 67 years, were conSidered to be early cases of KCS. In three severe KCS cases, the patients, one male and two female, were 66, 67 and 68 years of age. The conjunctival biopSies were fixed immediately after eXCISIOn m a mixture of one part glutaral dehyde (3%), one part cacodylate buffer and one part ruthemum red solution (1,500 ppm) The solutIOns were refngerated at 4° C Half of each specimen was processed for paraffin embeddmg and light microscopy The remamder was processed according to the method deSCribed by Luft (J97Ja) for Araldlte embeddmg SectIOns of 60-70 nm A (silver-gold) thickness were cut on an LKB Ultratome III and, without counterstam, were exammed m a Philips 301 transmiSSIOn electron microscope. An acceleratmg voltage of 60 KV was used with standard apertures throughout the mvestigatlOn and the ruthemum red/Os0 /reactlOn product on the epithelial surface was photographed onto 4 Kodak cut film 4463 at a magmficatlOn of X 9,100 under standard exposure conditions. Where there was any suggestIOn that the surface epithelium had been sectIOned obliquely, the regIOn was Ignored Thirty two evenly spaced negatives were taken from each specimen ImtJaI studies showed that negative denSity could be vaned accordmg to the pattern of tissue and embeddmg medIUm m relation to the light sensors on the vlewmg plate. Therefore, for the major part of the study, the same orientatIOn was chosen whenever pOSSible. The procedure for development (Kodak D19) of the negatives was made as umform as IS possible wlthm standard laboratory practice. Image Analysis Careful attentIOn to the preparatIOn of negatives was essential for image analYSIS which reqUires uniform stam, umform grain density and umform contrast, If valid comparisons are to be made (Bradbury 1979). The study of human material, which was obtamed and mvestlgated during a period of 12 months, presented many problems With regard to the fulfilment of the aforementIOned rigid Criteria and prehmmary studies were necessary to assess the scale of potential maccuracies The negatives were analysed With an Optomax Image Analyser MICfomeasurements Ltd., Saffron Walden, Essex, CBII 3AQ, UK) which rapidly measures the area of a component of a given denSity on a teleVISIOn Image The teleVISIOn camera was posItioned above the transillummated negative to give a x 3 magmflcatlOn to the screen and a total tissue magmflcatlOn of x 27,300. 2 ConJunctival Surface M UCIns ·b +.t ~ " - ~ Fig. 1 a, b. ConJunctIval surface mucms stamed en bloc with collOIdal Iron and counterstamed with uranyl acetate Note the relatIvely low density of the reaction product which has a delicate framework (arro\\') Baboon a x 26,000 b x 90,000 The negatIve Image wa, reversed electronIcally and a frame selected and calibrated so that the total area of reaction product m ~m2 was measured per ~m length of epithelium In the KCS materIal, high values were obtamed as a result of the presence of large clumps of reaction product on the surface As these serve no functIOn m tear film stability, values were obtamed (m additIOn to total mUClns) for nlIcropiLcai mUClns. These were obtamed by restrIctmg the frame to a distance of 0 25 ~m from the mlcrophcal surface The output from the Image analyser was mterfaced with a Hewlett-Packard desk-top computer (98 I 5A) which proVided a statistIcal analysIs of the data 3 W R Lee et al. Fig. 2. Conjunctival surface muclns demonstrated by the reactIOn product of ruthellium red; no counterstain. Note the fine strands (arrows) connecting the electron dense clumps on the surface of the mlcroplicae. Normal female, 76 years. x 65,000 Results Preliminary Studies Ruthenium red is a complex and unstable substance which has, as yet, undefined specificities (Luft 1971 a, b), so that it was not the stain of initial choice. Colloidal thorium and colloidal iron are to be preferred, but the former is no longer available in the U.K. owing to the health hazard. Colloidal iron-stained baboon conjunctival tissue was available in the laboratory files and had been prepared according to the technique recommended by Curran, Clark and Lovell (1965). The stain demonstrated mucins on and between the microplicae (Fig. 1) as fine strands and clumps, but the density of the crystals was such that discrimina tion of the crystal grey level against the cytoplasmic grey level was inaccurate when the image analyser was used to quantify the negatives. The reaction product of ruthenium red was much denser by comparison. In the normal human con junctiva (Figs. 2 and 3), it appeared as a dense line along the glycocalyx of the cell membrane and as a clump on the surface of the microplicae which in some cases were linked by fine strands. The density of the components of the reaction product was unequal and the differences were detectable by variation in the selector levels for grey level detection in the image analyser. To test the importance of subjective variation in selector level adjustment, the levels between the two extreme ranges of a) under detection (81) and b) over 4 ConjunctIval Surface M UClns b c d e Fig.3a-e. IllustratIons of the vanatIOn In appearance and dlstnbutIon of surface mUCInS In the normal conjunctIva For explanatIon see text RuthenIUm red, wIthout counterstaIn. a female, 69 years. b female, 58 years, c, d, e female 76 years. a x 25,000 b, c, d, e x 23,500 detection (85) were used to measure the area of the reaction product at one site. The graph obtained (Fig. 4) showed a linear relationship between the area measured and the selector setting, the highest value being some 30% greater than the lower. The standard deviations of the means were less than I %, which is an indicatIOn of the repeatability of the image analyser. Thus errors are 5