Cellular Incursion into Bowman's Membrane in the Peripheral Cone of the Keratoconic Cornea T. SHERWIN a *, N. H. BROOKES a ,I.-P. LOH a , C. A. POOLE b AND G. M. CLOVER a a Discipline of Ophthalmology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand and b Division of Anatomy with Radiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand Received Seattle 26 July 2001 and accepted in revised form 21 November 2001) Analysis of corneal tissue from normal and keratoconic donors has revealed differences which may represent early signs in the pathogenesis of keratoconus. Peripheral areas of keratoconic tissue obtained from transplant surgery were targeted to ascertain cellular disposition and morphological changes which may be masked within the extensive damage of the central keratoconic cone. Peripheral keratoconic corneae exhibited discrete incursion of ®ne cellular processes into Bowman's membrane. These processes originated from keratocytes and were often observed in conjunction with a de®ned indentation from the basal epithelium. Comparison of the lysosomal enzymes cathepsin B and G with constitutively expressed cytoplasmic esterase determined that both cathepsins were elevated within keratocytes of keratoconic tissue compared with normal tissue. Some clusters of keratoconic keratocytes had elevated levels of cathepsin exceeding all others. Cathepsin-rich keratocytes localized with morphologically compromised regions of Bowman's membrane. The presence of cell nests deeper within the stroma indicated that the catabolic changes, which are visible within the acellular Bowman's membrane, are probably also occurring deeper within the stroma, but are masked and not readily detectable. # 2002 Elsevier Science Ltd. Key words: cornea; keratoconus; keratocyte; epithelium; Bowman's membrane; cathepsin B; cathepsin G. 1. Introduction Corneal Structure The stroma of normal corneae is a highly ordered complex structure comprising interspersed lamellae of cells and collagen. Each lamella is 2±3 mm thick and the number of lamellae is uniform. The nature of the keratocyte layers has been re-evaluated in the labo- ratory using ®xable cytoplasmic dyes which enabled identi®cation of three morphologically distinct cell populations in the anterior, central and posterior stroma Poole et al., 1996). Ramping cell body and cell process connections between adjacent cell layers were also visualized. Evidence for the functionality of anatomically identi®ed cell contacts was provided by the imaging of immunohistochemically labelled gap junction proteins connexins) in stromal keratocytes Clover et al., 1996a, Clover et al., 1996b). The distribution of gap junction proteins within the normal corneal epithelium has also been shown previously Dong et al., 1994). The disease state of keratoconus is characterized by progressive thinning of the cornea for review see Rabinowitz, 1998). Transmission electron microscopy TEM) studies of diseased tissue have revealed that the thickness of collagen lamellae in keratoconus is unaltered, but the number of lamellae appears to be signi®cantly less in normal tissue Takahashi et al., 1990). Synchrotron X-ray diffraction studies indicate that there is no difference in inter®brillar collagen spacing, hence thinning of the cornea is not due to closer packing of the collagen ®brils within the stroma Fullwood et al., 1992). This suggests progressive loss of lamellae from the stroma, but how this loss is initiated and the fate of the collagen and keratocytes are unknown. However, low angle X-ray scattering has shown that the orientation of collagen ®brils within the lamellae is altered in keratoconus Daxer and Fratzl, 1997), suggesting that loss of structural integrity, degradation and/or insuf®cient repair mech- anisms may all be important in the disease process. Cellular involvement in these degradative processes remains unclear, although differences in keratocyte morphology and cell±matrix interactions have been shown by high voltage electron microscopy Rock et al., 1995). Extracellular Matrix Proteins The literature describing collagen levels in kerato- conic corneae is inconclusive: Critch®eld et al. 1988) described decreased collagen and total protein levels Exp. Eye Res. 2002) 74, 473±482 doi:10.1006/exer.2001.1157, available online at http://www.idealibrary.com on 0014-4835/02/ $35.00/0 # 2002 Elsevier Science Ltd. * Address correspondence to: Trevor Sherwin, Discipline of Ophthalmology, Faculty of Medicine and Health Sciences, Private Bag 92019, University of Auckland, Auckland, New Zealand. E-mail: t.sherwin@auckland.ac.nz