BritishJournal ofOphthalmology 1994; 78: 863-870 863 Improved preservation ofhuman corneal basement membrane following freezing of donor tissue for Br J Ophthalmol: first published as 10.1136/bjo.78.11.863 on 1 November 1994. Downloaded from epikeratophakia Robert D Young, W John Armitage, Paul Bowerman, Stuart D Cook, David L Easty Abstract States, good results continue to be achieved by Current methods for the production of the small number ofBritish surgeons performing lenticules for epikeratophakia involve rapid the technique.4 However, no comprehensive freezing, cryolathing, and slow warming of the account of its long term outcome has yet been donor cornea. We have found that this pro- published. cedure causes structural damage to the Several complications resulting in the failure epithelial basement membrane in the donor of epikeratophakia have been reported, includ- cornea which may subsequently contribute to ing infection, graft dehiscence, persistent inter- poor postoperative re-epithelialisation of the face haze or opacity, ulceration, and imperfect implant, leading to graft failure. Endeavouring re-epithelialisation. Among these, the failure of to overcome these problems, the effects of host epithelial cells to migrate over and re- cryoprotection of donor cornea were investi- surface the anterior face of the grafted tissue gated, using dimethyl sulphoxide, in conjunc- continues to be the major reason for the removal tion with different cooling and warming rates ofepikeratophakia lenticules.'-'0 as part of the protocol for cryolathing. The Epithelial healing is itselfa complex phenome- structural integrity of the epithelial basement non involving mitosis of host cells at the graft membrane zone (BMZ) was then assessed by periphery, centripetal migration, and attach- electron microscopy and by immunofluores- ment. In all of these processes the nature of the cence microscopy using antibodies to types IV underlying substratum must be considered to be and VII coliagen, components of the basal of profound importance, as shown by studies of lamina and anchoring fibrils respectively, and cell growth and attachment in vivo and in an antibody to a component of the anchoring vitro." 12 The integrity ofthe epithelial basement filaments. No differences in the pattern of membrane in the cryolathed donor cornea, immunostaining for these components were which becomes the presumptive substrate for detected, indicating that the composition of host epithelial cells, may therefore be an import- the BMZ was unaltered by the different treat- ant factor influencing the recovery of an intact ment regimens applied. However, electron and durable epithelial sheet over the newly http://bjo.bmj.com/ microscopy showed that preservation of base- grafted lenticule. ment membrane ultrastructure was markedly In preliminary observations both of cryo- improved when cornea was warmed rapidly lathed corneas prepared for epikeratophakia and rather than slowly, both in cryoprotected and of failed grafts, we identified several structural non-cryoprotected tissue. Epithelial cell reten- alterations to the epithelial basement membrane tion and preservation of stromal architecture zone of the (BMZ), including disruption basal on September 26, 2021 by guest. Protected copyright. appeared superior in cryoprotected samples, lamina and disorganisation of anchoring fibril while keratocyte structure was heterogeneous complexes and anterior Bowman's layer. These throughout the experimental groups. Further changes appeared to be attributable to freezing University ofBristol, work is in progress to assess the efficacy of rather than to the lathing procedure itself and Department of these protocols in the preservation of kerato- were sufficiently extensive to warrant considera- Ophthalmology, Muscle cyte viability in association with improved and Collagen Research tion as potential inhibitors ofepithelial healing in Group, Langford, Avon basement membrane structure in donor tissue the immediate postoperative period. The few R D Young for epikeratophakia. studies carried out on the effects of freezing (Br3 Ophthalmol 1994; 78: 863-870) corneal tissue the Bristol Eye Hospital, during preparation ofepikera- Lower Maudlin Street, tophakia lenticules have focused mainly on the Bristol, UK viability of stromal keratocytes.'3 1' Factors such W J Armitage Epikeratophakia was originally introduced in as cooling and warming rates and the presence of S D Cook 1980' as a reversible and simplified approach to D L Easty cryoprotective agents (for example, dimethyl corneal refractive surgery for aphakia, although sulphoxide), not only affect the survival of cells United Kingdom it soon also found application in the surgical during freezing and thawing, but influence the Transplant Support treatment of keratoconus2 and myopia.3 The Service Authority, location and quantity of ice, and thus the degree Bristol, UK technique involves reshaping a frozen disc of of structural disruption within tissues.'5 We P Bowerman donor cornea on a cryolathe, to form a lenticule, therefore investigated these factors to determine Correspondence to: which is then transplanted onto the de-epithelia- whether the of structural Dr R D Young, Muscle and preservation integrity Collagen Research Group, lised surface ofBowman's layer. ofthe anterior face ofthe prospective graft could Churchill Building, University The results ofepikeratophakia since its incep- be improved. The ultrastructural organisation of of Bristol, Langford, Avon tion have traditionally been considered good,'7 the BMZ was assessed by transmission electron BS18 7DY. and Accepted for publication while, in recent years, the popularity of the microscopy and aspects of its composition were 1 July 1994 procedure may have declined in the United studied by immunofluorescence microscopy 864 Young, Armitage, Bowerman, Cook, Easty with monoclonal antibodies directed against Treatment oftissue samplesfrom paired human corneas specific components of the basal lamina and Br J Ophthalmol: first published as 10.1136/bjo.78.11.863 on 1 November 1994. Downloaded from anchoring fibril structures. Materials and methods COOLING AND WARMING RATES 4 7 8 3 Rapid cooling Samples were cooled at about 20°C/min to - 50°C on the cryolathe. The tissue sample was attached by its epithelial surface to an aluminium arbour, which was clamped in the cryolathe chuck. (3) rapid cool, slow warm (without lathing); (4) rapid cool, rapid warm. Slow cooling Samples 5-8 were from the right cornea, and Samples were cooled at 1°C/min to -50°C in a were incubated in the cryoprotectant medium: programmable controlled rate freezer (Planer (5) addition and removal of cryoprotectant Kryo 10-16). without freezing; (6) slowcool, rapidwarmwithcryoprotectant; (7) rapidcool, slowwarmwithcryoprotectant; Rapid warming (8) rapid cool, rapid warm with cryopro- Frozen samples were immersed directly into tectant. medium at approximately 22°C (ambient The samples were all processed for microscopy temperature), which gave a warming rate of after these treatments. >50°C/min. IMMUNOFLUORESCENCE MICROSCOPY Slow warming Samples from each of the corneal sectors (1-8) Frozen samples were allowed to thaw still were mounted in Tissue-tek medium on attached to the cryolathe chuck, which gave a aluminium stubs and frozen by plunging into warming rate of <20'C/min. liquid nitrogen cooled isopentane. Frozen Corneas routinely processed for epikerato- sections, 7 ,tm thick, were cut on a Bright phakia were cooled and warmed on the cryo- cryostat and mounted on slides coated with lathe - that is, rapid cool/slow warm. Biobond adhesive. Sections were exposed to monoclonal antibodies to type IV collagen (goat anti-human), a component of the basal lamina; CRYOPROTECTANT ADDITION AND REMOVAL type VII collagen (LH7.2, mouse anti-human), a http://bjo.bmj.com/ Samples were incubated in 10% (v/v) dimethyl component of the anchoring fibrils; or to an sulphoxide (DMSO) in Eagle's minimal essential antibody to a component of the anchoring fila- medium (MEM) for 15 minutes at 22°C. The ments (LH39, mouse anti-human). Type IV cryoprotectant was removed by dilution by antibody was an affinity purified reagent sup- transferring corneal tissue into 0-5 mol/litre plied by Southern Biotechnology Associates. sucrose in MEM at 22°C. The sucrose acted as an LH7.2 and LH39 were generously donated by osmotic buffer to limit the cell that Professor I Leigh, Department of Experimental swelling on September 26, 2021 by guest. Protected copyright. would have been induced by the reduction in Dermatology, London Hospital Medical DMSO concentration.'6 After 15 minutes, the College. After 2 h incubations in the primary samples were placed into MEM alone. monoclonal antibody and washing in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), or in PBS/BSA alone in the case EXPERIMENTAL TREATMENTS of control preparations, the sections were The corneas were obtained within 23 h post incubated with secondary antibodies conjugated mortem from a 60-year-old man, with no to fluorescein isothiocyanate (FITC), followed ocular disease, who -had died from Hodgkin's by further washing and mounting in Citifluor. lymphoma, which is a medical contraindication They were then viewed using a Leitz microscope to transplantation of the corneas. Corneoscleral equipped for FITC fluorescence and phase discs were excised and placed in Eagle's MEM. optics. The corneas were then dissected free of sclera, cut into four equal quadrants, and subjected to