Eye (1989) 3, 678-684

How Clean is Your Capsule?

W. T. GREEN and D. L. BOASE Portsmouth

Summary Proliferation of residual lens epithelial cells is believed to be the major cause of pos­ terior capsule opacification following extracapsular cataract extraction. During sur­ gery these cells can be visualised with appropriate illumination facilitating their mechanical removal with the McIntyre cannula. When flat preparations of the anterior capsule are examined by light microscopy, the areas 'cleaned' of cells in this way appear transparent but scanning electron microscopy reveals tufts of remaining debris which may represent points of cellular attachment to the capsule. Control of lens epithelial cell proliferation is important for the future development of cataract surgery.

The undoubted advantages of extracapsular and also on human cadaver eyes. A horizontal cataract extraction are offset in many patients capsulotomy in the upper part of the lens by posterior capsule opacification requiring allowed nucleus removal. Irrigation and caps ulotomy. Not only is this disappointing aspiration of the cortical lens material was for the patient, but the procedure carries a then carried out using a McIntyre cannula risk of serious complications. with Hartman's irrigation solution. During in The major cause of posterior capsule opac­ vitro surgery this was aided by first removing ification is proliferation of residual lens epi­ the entire cornea and iris to improve visual­ thelial cells. I If these cells could be removed at isation and explore different methods of the time of surgery we believe that the inci­ illumination. dence of posterior capsule opacification and The importance of illumination was first the need for subsequent capsulotomy would suspected when it was observed, during rou­ be reduced. A study was therefore under­ tine intercapsular cataract extraction, that the taken to determine whether these cells could lens epithelial cells lining the inner aspect of be visualised and physically removed during the anterior capsule could be visualised much surgery. more clearly with the assistant microscope In one case an attempt was made to esti­ attachment (at 30° of incidence) on a Zeiss op­ mate the number of cells remaining after an erating microscope (OMPI 6) than by the op­ extracapsular cataract extraction in a cadaver erating surgeon with his coaxial view. We eye with a canopener caps ulotomy. therefore investigated the use of oblique illu­ mination using fibreoptic light sources with focusing lenses held by adjustable clamps to Methods and Results provide light from any angle. Illumination and visualisation during surgery It was found that oblique illumination Intercapsular (endocapsular) cataract extrac­ during in vitro surgery allowed visualisation of tion was performed during cataract surgery details on the anterior capsule with the lens

Correspondence to: Eye Department. Queen Alexandra Hospital, Cosham, Portsmouth P06 3LY. HOW CLEAN IS YOUR CAPSULE 679

Figs. 1,2,3. In vitro surgery; cornea and iris removed demonstrating streaks of removed lens epithelial cells in each case with oblique (sixty degrees from the vertical) illumination.

epithelial cells standing out more clearly well dilated the pupil) obscures details of the against the dark background (Figs. 1,2,3) fornix of the capsule bag. than with the red reflexprovided by coaxial il­ lumination (Figs.1a,2a,3a). Histology The best arrangement was found �o be one Light Microscopy fibreopticlight source on either side at an an­ For the purposes of demonstrating histologi­ gle of 30° to the horizontal with the light cally that it is indeed cdl removal which is focused as much as possible onto the lens cap­ being witnessed, an area of anterior capsule sule (Fig. 4). was cleaned in cadaver eyes. The capsule bag Having illuminated the lens epithelial cells was then stained with toludine blue (which in this way it was found that they could be stains cells but not 'cleaned' capsule) and the removed mechanically by applying the port of anterior capsule excised. The junction the McIntyre cannula to the inner aspect of between cell laden, and 'cleaned' capsule was the anterior capsule and moving it gently from then examined by light microscopy at various side to side while applying a gentle vacuum. magnifications. However, cleaning cells from a free edge of These preparations showed clearly that the capsule is extremely difficultdue to loss of the presumed areas of 'clean' capsule were indeed vacuum (which keeps the port applied to the free of cells, and the capsule appeared trans­ capsule) as the capsule edge is approached. parent in these areas using this stain (Fig. 5). Once learned during in vitro surgery, this technique can be applied to live intercapsular Scanning Electron Microscopy surgery, even though the iris (no matter how No suitable means was found to prevent the 680 W. T. GREEN AND D. L. BOASE

Figs. la,2a,3a. The same three cadaver eyes viewed with coaxial illumination.

anterior capsule specimens from rolling up placed in 2% aqueous osmium tetroxide at 4 into a 'scroll' prior to EM since any substance degree C in the dark for 16 hours; allowed to introduced to hold the specimen flat could attain room temperature and washed in dis­ produce artefacts. A number of rolled up tilled water; dehydrated through a 10% specimens were therefore studied and it was graded ethanol series and transferred to abso­ soon apparent that some had lens epithelial lute ethanol for 30 minutes. cells on the outside whereas others had rolled The specimens were then infiltrated with a up with the cells inside and these latter speci­ series of increasing concentrations of acetone; mens were difficult to study. Specimens were dried at critical point (Polaron E3000 appara-

Fig. 4. Oblique illumination provided at sixtydegrees Fig. 5. Light micrograph showing junction between from the vertical by an adjustable fibre-optic cable lens epithelial cells (left), and 'cleaned' anterior capusle (covered with sterile plastic tubing during surgery). (right), the latter being transparent with this stain Second cable held with clamp (lower right). (toludine blue). (Bar=511). HOW CLEAN IS YOUR CAPSULE 681 tus) using liquid carbon dioxide; mounted on to further define these apparent areas of stubs with double-sided adhesive and gold adhesion. coated (25 nm thickness) in a Polaron 5000 Diode Sputtering system. The specimens Discussion were then examined in a lEOL 35C scanning Coaxial illumination is considered desirable electron microscope at a range of accelerating for cataract surgery as it provides a bright red voltages. reflexwhich is especially helpful during aspir­ Using this method, the surface of appar­ ation of cortical lens material. However, ently 'cleaned' capsule was found to have methods of oblique illumination highlight small tufts of retained debris (Fig. 6). These details of the surgical anatomy and allow bet­ tufts may represent firm attachments of the ter visualisation of the lens epithelial cells lens epithelial cells to the capsule, although it which can then be removed by simple me­ is interesting to note that these were not pres­ chanical means. ent in a ratio of one per cell (Fig. 7). An 'edge' The layer of lens epithelial cells probably view of these tufts was obtained by studying a reflects light by specular reflection with the region where a split in one specimen fortui­ arrangement of light described here, allowing tously passed through some of these tufts the cells to be clearly seen against what is now (Fig. 8). The layers of capsule adjacent to a black background. With recent concern each tuft are seen to be elevated providing about incident light upon the macula reaching further evidence of cellular adhesion. photo toxic levels during surgery, this tech­ Further studies using scanning and trans­ nique offers the advantage of reduced direct mission EM with serial sections are underway illumination of the macula.

Fig. 6. Scanning electron micrograph showing the 'inner' surface of one anterior capsule specimen previously 'cleaned' of cells using the McIntyre cannula. Patches of debris remain attached to the capsule. (Bar=lOOu). 682 W. T. GREEN AND D. L. BOASE

Fig. 7. Scanning electron micrograph of junction between cells (above) and 'cleaned' capsule (below) with retained debris. (8ar=1Ou.). Attention is frequently directed to cleaning tomy (for the reasons given above). It is the posterior capsule during cataract surgery, sometimes erroneously assumed that a wide and the lens epithelial cells ignored. This caps ulotomy of this type will remove all the paper describes a method of visualising the cells. Unfortunately the cells extend into the lens epithelial cells during in vitro surgery and far recesses ('equator') of the capsule fornix their mechanical removal. These methods and in cataractous lenses frequently onto the have been applied to live intercapsular peripheral posterior capsule. 2 Therefore even surgery and the equipment used to produce the widest surgical capsulotomy would leave oblique illumination for this study was found an extensive area of lens epithelium. to provide excellent visualisation, but would To demonstrate the extent of residual lens require further refinements to be practical in epithelium with this type of caps ulotomy, we the context of a sterile operating field. Ideally, examined a capsule bag preparation following one should have the ability to vary the the widest possible canopener capsulotomy in direction of one or more sources of illumi­ one cadaver eye. The specimen was stained nation during surgery to suit the needs of the with toludine blue, carefully dissected free of moment, and newly available microscopes all attachments and radial cuts made in the with a range of fibreoptic attachments to anterior capsule (Figs. 9,10) so that the speci­ various light sources will hopefully provide men could be laid flaton a Neubauer counting this. chamber (consisting of a slide with a grid of This method of lens epithelial cell removal known dimensions marked on its surface), is not practical with a 'canopener' capsulo- and the total number of remaining lens epi- HOW CLEAN IS YOUR CAPSULE 683

Fig. 8. Scanning electron micrograph view of anterior capsule edge formed by an artefactual split (seen in Fig. 6) which has fortuitously passed through retained 'tufts' on the capsule surface from which cells have been mechanically removed. (Bar=l!J.). theliaI cells estimated,3 In this case the tomy for cataract extraction would normally number of remaining cells was found to be be smaller than in this case and with recent approxiamtely 100,000 (4000 per square mm trends towards leaving larger anterior capsule with a 25 square mm total stained area of capsule). This represents a large pool of potentially proliferating lens epithelial cells living in a natural culture medium. Surgical capsulo-

Fig. 9. Diagram to show the excised capsule bag in Fig. 10. Photograph of specimen prepared as in Fig. 9 which radial incisions are made in the anterior capsule and stained with toludine blue (which stains cells but not 'frill' which remains following a canopener the central cell-free posterior capsule). The small capsulotomy in a cadaver eye. squares of the grid are 1 mm2• 684 W. T. GREEN AND D. L. BOASE flaps in intercapsular surgery, still greater incidence of posterior capsule opacificationis numbers of lens epithelial cells must the subject of further investigation. remain. It has been shown that lens epithelial cells References 1 Green WR and McDonnell PJ: Opacification of the grow predominantly from a cut edge of posterior capsule. Trans Ophthalmol Soc UK capsule in tissue culture, presumably due to 1985, 104: 727-39. lack of contact inhibition. 4 It may therefore be 2 Marshall J, BeaconsfieldM, Rothery S: The anatomy important to remove lens epithelial cells from and development of the human lens and zonules. Trans Ophthalmol Soc UK 1982, 102: 423-40. the area of capsule through which one will 3 Dacie JV and Lewis SM, eds: Counting red blood subsequently perform the capsulotomy and cells with a Neubauer counting chamber In: Prac­ capsulorrhexis during intercapsular cataract tical Haematology, 3rd ed., Churchill, London surgery (to leave a 'cell free' capsule edge), 1963, 18-27. 4 particularly since relatively large capsule flaps Jacob TJC, Humphrey RC, Davies EG, Thompson GM: Cytological factors relating to posterior cap­ often remian following this surgery. Whether sule opacification following cataract surgery. Br J this is of clinical benefit in reducing the Ophthalmol1987, 71: 659-63.