Three-Dimensional Scanning Electron Microscopic Study of Keratoconus Corneas FREE

KERATOCONUS is a noninflammatory disease characterized by thinning and scarring of the central portion of the cornea.¹ Histopathologic and ultrastructural studies have demonstrated that in early stages of the disease, fragmentation of the epithelial basement membrane occurs with disintegration of the Bowman layer and fibrillation of the anterior stroma.^{2 - 5} The central cornea then becomes thinned, with destruction of the Bowman layer and stromal scarring. A loss of collagen lamellae occurs, but the collagen fibrils are of normal diameter.^{6 - 7} The lamellae are surrounded by granular materials, which are shown to be rich in neutral polysaccharides and glycoproteins.^{1 ,4} In advanced stages of the disease, the central portion of the Descemet membrane may rupture, resulting in acute hydrops.

Electron microscopic studies aimed at 2-dimensional examination of keratoconus corneas were performed more than 20 years ago. Since then, the technology, resolution, and machinery have vastly improved.

Our investigation sought to revisit the structure of keratoconus corneas, focusing particularly on the 3-dimensional collagen architecture in the Bowman layer. Direct observation of this structure by conventional techniques has been infeasible because of the overlying corneal epithelium. With a recently developed cell maceration and tissue conductive method,^{8 - 11} we removed the corneal epithelium and visualized the 3-dimensional collagenous architecture of the Bowman layer under scanning electron microscopy. Multiple ruptures in the Bowman layer of keratoconus corneas and the possible sequence of events were demonstrated.

Eight corneal buttons from patients with typical clinical features of keratoconus (Table 1) were obtained at the time of penetrating keratoplasty from the Cornea Service of University of Illinois at Chicago College of Medicine or Seirei Hamamatsu Hospital, Shizuoka, Japan. Patients ranged in age from 18 to 46 years at the time of surgery. Five normal human corneas from donors (aged 2, 19, 43, 53, and 66 years) were obtained from the Illinois Eye Bank, Chicago, within 24 hours of death. The corneas were clear and the donors did not have known ocular disease.

To serve as another set of controls, corneas were obtained from 8 patients with other corneal diseases. They were from 2 patients (aged 69 and 70 years) with pseudophakic bullous keratopathy, 1 patient (aged 18 years) with bullous keratopathy associated with absolute glaucoma, 1 patient (aged 55 years) with corneal scar after ulceration, 2 patients (aged 55 and 58 years) with lattice corneal dystrophy, 1 patient (aged 48 years) with granular corneal dystrophy, and 1 patient (aged 51 years) with herpetic interstitial keratitis.

Corneas obtained were fixed immediately in 2.5% glutaraldehyde and 2% formalin in Sorensen phosphate buffer solution for 2 to 5 days and transferred to buffered formalin until processing.

The cell maceration method was carried out as previously described.^{4 - 8} Briefly, the fixed specimens were rinsed in distilled water overnight, immersed at room temperature for 5 days in a 10% sodium hydroxide solution, and rinsed thoroughly with several changes of distilled water for 24 hours. They were then subjected to a conductive staining method by soaking in 2% tannic acid for 3 hours, washed in distilled water for 1 hour, and postfixed in aqueous 1% osmium tetroxide for 3 hours. After dehydration through a graded ethyl alcohol series, the specimens were transferred to isoamyl alcohol, critical-point dried, mounted on aluminum stabs, and coated with gold in an ion coater (Hitachi, Tokyo, Japan). Scanning electron microscopy was performed with a scanning electron microscope (Hitachi S-2300) at an accelerated voltage of 25 kV.

To ensure that the corneal specimens were processed properly, pieces of the tissue were embedded in epoxy resin after cell maceration and conductive staining. Ultrathin sections were double stained with uranyl acetate–lead citrate and were observed under a transmission electron microscope (Hitachi H-7000).

Table 1 summarizes the clinical features of 8 patients with keratoconus, including age at onset, age at surgery, and ocular manifestations. Patients 5 and 6 had a history of acute hydrops and patients 4 and 8 had relatively mild cases. None of the patients had a family history of the disease except for patient 7, whose brother had keratoconus. Routine pathologic examinations showed the typical features of keratoconus, including breaks in the Bowman layer, scarring, and iron ring.

As evidenced by transmission electron microscopy, treatment of corneas with 10% sodium hydroxide solution removed most of the cellular elements, basal laminae, and other extracellular materials, leaving collagen fibrils relatively intact (Figure 1). The scanning electron micrographs shown in Figure 2 depict the surface of the Bowman layer from normal human corneas at varying magnifications. At lower magnifications (Figure 2, A and B), smooth surface of the Bowman layer was observed. At higher magnifications (Figure 2, C and D), the Bowman layer displayed a honeycomblike porous structure, made up of a dense meshwork of collagen fibrils. Similar features were found in all 5 normal human corneas studied.

In contrast, multiple sharply edged defects in the Bowman layer (Figure 3) were noted in all the keratoconus corneas examined. A lattice-like configuration of ruptured Bowman layer was found. Hypertrophic collagenous proliferation, which partially or totally replenished the ruptures, was observed. The fact that patients 5 and 6 (Figure 3, D and E) had a history of acute hydrops may explain why much more collagenous proliferation was observed.

Under a higher magnification, the proliferation of collagenous tissue and ruptures in the Bowman layer could be viewed more clearly (Figure 4, A). The ruptured areas of the Bowman layer were filled by proliferated collagenous tissue that appeared to derive from the anterior stroma just beneath the Bowman defects (Figure 4, B).

When specimens were observed from a lateral view, normal human corneas (Figure 5, A) showed well-packed collagenous lamellae with spindle-shaped remains of sodium hydroxide–digested keratocytes and the Bowman layer (Figure 5, A, between white arrows) uniformly covering the stroma. In keratoconus corneas, misaligned Bowman layer (Figure 5, B, arrow), irregularly thinned Bowman layer (Figure 5, C) and defects (Figure 5, D) were demonstrated. With increasing severity of damage in the Bowman layer, the arrangement of collagenous lamellae also seemed to be more distorted into the deeper stroma (Figure 5, B through D) of keratoconus corneas. Under a higher magnification, the well-packed and well-aligned collagenous lamellae observed in normal human corneas (Figure 5, E) were replaced by the loose and randomly oriented collagen fibrils in keratoconus corneas (Figure 5, F). Multiple pores (Figure 5, F, asterisks) were seen.

None of the other diseased corneas (Figure 6) exhibited these alterations in the Bowman layer under scanning electron microscopy. No multiple breaks were observed. The surface appearance was unlike that of the keratoconus cases even when prominent collagenous scar tissue could be seen, such as those in the cases of bullous keratopathy (Figure 6, A through C) and corneal scarring (Figure 6, D). Corneas from the patient with lattice corneal dystrophy (Figure 6, E) and the patient with granular corneal dystrophy (Figure 6, F) had a characteristic lattice or granular appearance, respectively.

The current scanning electron microscopic study after a cell maceration and tissue conductive procedure distinctly demonstrated the 3-dimensional organization of collagen fibrils in the Bowman layer of normal human, keratoconus, and other diseased corneas. This method, previously used successfully for examination of peripheral nerve,^{8 - 9} pial septa,¹⁰ cornea,^{11 - 13} sclera,¹¹ and the Bruch membrane,¹⁴ allows a direct visualization of the architecture of collagen fibrils, which, to our knowledge, had not previously been attainable for the Bowman layer.

Keratoconus specimens in this study, depending on disease severity, showed varying degrees of lattice-like, sharply edged ruptures and fragmentation of the Bowman layer. In more advanced areas, hypertrophic collagenous proliferation partially or totally filled the ruptures. The observed characteristic breaks and changes in the Bowman layer confirm and expand findings reported previously by light and electron microscopy.^{2 - 5 ,12 - 13} When the changes in the Bowman layer were minimal to mild, the corneal stroma underlying the Bowman layer correspondingly showed only minimal changes. However, the corneal stromal alteration advanced when damage in the Bowman layer became more evident in keratoconus.

In 2 earlier light and electron microscopic studies, Chi et al² and Teng³ postulated that the ruptures of the Bowman layer were most likely to be replaced by tissue from stroma underneath the Bowman layer. Such a possibility was substantiated by this study. The ruptured areas appeared to be filled with proliferated collagenous tissue from the anterior stroma just beneath the ruptured Bowman layer.

By slitlamp microscopy, anterior clear spaces have been observed within the thin portion of the conical protrusion in both early and advanced cases of keratoconus.¹⁵ Histologic examinations further revealed that the clinical finding correlated with breaks in the Bowman layer. It was suggested that the clear zones observed may represent breaks in the Bowman layer before the scar formation. In the current study, we are unable to determine whether our scanning electron microscopic findings can be correlated with the clinical finding, as slitlamp photographs were unavailable.

We¹⁶ and other investigators^{17 - 18} have reported that the collagen content in some cases of keratoconus is reduced compared with normal human corneas. In this study, loosely packed and randomly oriented collagen fibrils were demonstrated in some of the keratoconus corneal stroma, which may reflect the reduced collagen density. Consistent with the earlier histologic findings,⁴ abnormal chondroitin and dermatan sulfate–type proteoglycans were more recently found to accumulate in keratoconus corneas around collagen fibrils and collagen lamellae.¹⁹ The numerous pores noted in the stroma of keratoconus corneas in this study may thus represent areas occupied by keratocytes and by the abnormal proteoglycan molecules that are dislodged during the sodium hydroxide treatment. In nonkeratoconus diseased corneas, we noted no alterations in the Bowman layer characteristic of keratoconus changes.

The Bowman layer is an acellular matrix at the interface between the corneal epithelium and the stroma. It links the epithelial basement membrane and the stroma proper and may be crucial for the epithelial attachment and function. During human corneal epithelial development, a distinct Bowman layer is formed at 19 weeks.²⁰ After birth, the thickness of the Bowman layer remains unchanged. Components of Bowman layer are believed to be synthesized by both corneal epithelial and stromal cells and the epithelial-stromal interaction may be a major factor in the formation of the Bowman layer.²¹ Several years after radial keratotomy in human corneas, a Bowman layer–like structure was formed underneath epithelial plugs that extended into the stroma.²² The collagen fibrils in the Bowman layer are of relatively small diameter and are randomly arranged. It is unclear however how these collagens are organized or how they are maintained. In the underlying corneal stroma, the resident stromal cells are responsible for the maintenance and organization of the collagens. However, the Bowman layer is acellular. One possibility is that the maintenance is performed by the sparse stromal cells that transverse into the Bowman layer. Alternatively, cytokines may also be involved. In keratoconus, the maintenance function of the stromal cells or the cytokines may be disturbed both for the Bowman layer and for the stroma.

The etiologic mechanism for the development of keratoconus is not fully clear. One hypothesis, based on data from our laboratory^{23 - 26} and others,²⁷ is that the degradation processes in keratoconus may be aberrant. Supporting evidence includes elevated lysosomal hydrolase activities²³ and decreased levels of proteinase inhibitors^{24 - 25} in keratoconus corneas. Through the course of enzyme and inhibitor studies, the corneal epithelium consistently shows the most dramatic biochemical abnormalities.^{23 - 25} It thus has been suggested that even though the thinning in keratoconus occurs primarily in the stroma, the corneal epithelium may also be involved in the disease development.

The corneal epithelial theory was proposed based on an electron microscopic examination by Teng³ in the early 1960s. Lacking convincing evidence, however, this suggestion has since been considered as only conjecture.⁶ Our studies^{23 - 25} showing biochemical alterations mostly in the corneal epithelium provide more direct support for the theory. It is also corroborated by our finding that conjunctival epithelial cells from patients with keratoconus contain higher than normal lysosomal hydrolase activities.²⁸

The sequence of events noted in this study further indicates that the changes in the Bowman layer precede those in the corneal stroma, suggesting that the corneal epithelium may be an important factor at the initial or early stage of keratoconus development. One scenario may be that the increased degradative enzymes and reduced inhibitors in the corneal epithelium trigger rupture and fragmentation of the Bowman layer. The subsequent environmental changes or the interactions between the epithelial cells and the genetically predisposed stromal cells may ultimately induce the thinning and scarring manifested in keratoconus.

The epithelial-stromal interaction has been considered to be a factor involved in the development of keratoconus. Wilson et al²⁹ have postulated that interleukin 1 (IL-1) may be a modulator of epithelial and stromal interactions, regulating the corneal cell proliferation, differentiation, and death. They have also proposed a role of the IL-1 system in the causes of keratoconus. Interestingly, cultured keratoconus stromal cells have been shown to contain 4-fold higher binding sites for IL-1.³⁰ An enhanced expression of IL-1 receptor has also been noted in keratoconus corneas.³¹ The IL-1 hypothesis is consistent with the degradation hypothesis because IL-1 is known to regulate the expression of matrix metalloproteinases in the cornea.³²

Accepted for publication July 25, 1997.

This investigation was supported in part by research grants EY 03890 and EY 05628 and core grant EY 01792 from the National Eye Institute, National Institutes of Health, Bethesda, Md, and by the Research to Prevent Blindness (New York, NY) Senior Scientific Investigator Award (Dr Yue).

Reprints: Beatrice Y. J. T. Yue, PhD, University of Illinois at Chicago College of Medicine, Department of Ophthalmology and Visual Sciences, 1855 W Taylor St, Chicago, IL 60612.