In this study, noninvasive imaging of second-harmonic– generated signals was used to visualize the structural lamellar organization of the anterior cornea. This imaging approach identified a high degree of lamellar interweaving in the anterior cornea with a distinct population of lamellae appearing to insert into Bowman’s layer and run transverse to the corneal surface to a depth of approximately 120 μ
m. In a recent report, these lamellae that are identified by second-harmonic imaging have been shown to be distinct to the human and not observed in mouse or rabbit corneas.12
Of note, the transverse orientation with insertion into Bowman’s layer suggests that the lamellae have an important mechanical function. In dogfish, “sutural” lamellae that insert into Bowman’s layer and Descemet’s membrane are thought to be responsible for the maintenance of corneal deturgescence in the absence of a functioning corneal endothelium.16,17
In the human cornea, such sutural lamellae may play a similar role and provide rigidity to the anterior corneal stroma as well as define the corneal shape and curvature.
More important, second harmonic imaging was able to detect distinct differences in the organizational pattern of lamellae in 12 of 13 keratoconus samples including a marked loss or decrease in anterior lamellar interweaving and lamellae that inserted into Bowman’s layers. The remaining case, which did not show loss of sutural lamellae or anterior lamellar interweaving, was from a keratoconus patient from which only half of the corneal button was available for analysis, the remaining half having been submitted to pathology. These findings provided by a noninvasive imaging technique are consistent with previous light and electron microscopic studies of keratoconus corneas showing uniform, orthogonally arranged lamellar layers in the anterior stroma, with little or no lamellar interweaving.10
In addition, studies using x-ray scattering have demonstrated that keratoconus corneas show displacement of collagen orientation suggestive of lamellar slippage, which for anterior lamellae may involve disinsertion of lamellae from Bowman’s layer.11
Overall, these findings suggest that the mechanical weakness of keratoconus corneas that lead to corneal thinning and cone formation may be associated with the loss of a distinct population of anterior stromal sutural lamellae that insert into Bowman’s layer and anchor deeper within the stroma. Clearly, future development of second-harmonic imaging may provide a clinical tool to assess further this mechanism as well as help in diagnosing and observing the progression of this disorder.
Since this study was performed on pathologic specimens obtained after penetrating keratoplasty, the importance of these findings remains unclear. Although it is possible that the changes identified are associated with the end-stage disease, the finding that there was marked loss of lamellae inserting into Bowman’s layer in at least one whole corneal button showing no breaks in Bowman’ layer or corneal scarring suggests that the loss of these lamellae may precede end-stage disease. In support of this conclusion, a recent 3-D SEM study of keratoconus showed sharply edged defects in Bowman’s layer.18
These defects may be associated with the loss of lamellae that directly insert into the edge of Bowman’s layer. Although these investigators have suggested that the changes in Bowman’s layer may be the first signs of keratoconus,18
in our study, four patients without any evidence of breaks in Bowman’s layer exhibited marked changes in the number of lamellae that inserted into Bowman’s layer, presumably occurring in conjunction with paracentral corneal thinning and steepening. We therefore suggest that the changes observed in these structures may be responsible in part for the changes in the mechanical properties of keratoconus corneas that lead directly to altered corneal shape. Such a hypothesis is consistent and supportive of the hypothesis proposed by Bron19
and Muller et al.20
that anterior lamellar interweaving controls corneal shape.
The loss of lamellae inserting into Bowman’s layer may also explain in part the findings by x-ray scattering that the preferred horizontal and vertical orthogonal orientation of stromal collagen is altered in keratoconus.11,21
In the more recent report, scattering data indicate that collagen orientation in keratoconus assumes a more curved or tangential alignment to the cone, suggesting slippage of lamellae during progression of disease.11
This finding supports a long-postulated alternative view of the pathogenesis of keratoconus involving slippage with no degradation of tissue,22
as well as the more recent observations that surface topographic changes in keratoconus suggest that the disease is an extreme form of corneal warpage rather than ectasia or corneal stretching perhaps caused by lack of structural integrity brought on by stromal degeneration and external forces.23
Since insertion of lamellae into Bowman’s layer and extensive interweaving of these lamellae deeper within the anterior cornea may greatly increase the mechanical strength of the anterior cornea, the selective loss of these structures may markedly reduce the interlamellar cohesion and facilitate slippage of lamellae. Indeed, Meek et al.11
have suggested such a mechanism of slippage in the anterior cornea with disinsertion of lamellae from Bowman’s layer in a recent report.
Although the data may suggest degradation of lamellae that insert into Bowman’s layer, it should be noted that in keratoconus corneas, total collagen content is generally not substantially altered. Because these lamellae most likely represent a minor population of the overall collagen lamellae, it is not likely that their selective removal or loss would be detected in bulk collagen assays. Furthermore, it is possible that these lamellae may be replaced by orthogonally arranged collagen lamellae running parallel to the corneal surface with no overall decrease in collagen content.
Several questions remain concerning the disappearance of lamellae that insert into Bowman’s layer. Since in this study we did not have age-matched control eyes, we do not know if these structures develop in older eyes or are developmentally regulated. In support of a developmental regulatory program is the finding that younger normal corneas aged 36 and 38 years showed a similar distribution of lamellae inserting into Bowman’s layer to those of older individuals, 60 to 84 years. Therefore, at least after adolescence, there does not appear to be any changes in the organization of these structures. However, further study is necessary to address this question. Second, the inability to detect these structures in keratoconus does not necessarily mean that these structures have been lost. It is possible that changes in the collagen organization or binding of other extracellular matrix protein and glycoprotein may block the ability of the collagen to generate second-harmonic signals. However, it should be noted that in the x, z projections from the backscattered signal, the organization of the collagen appeared predominantly orthogonal () in contrast to the interwoven pattern seen in normal (compare with ). Further, there were no void regions in the reconstructions from the keratoconus corneas that suggested the presence of lamellae inserting into Bowman’s layer and extending deeper within the cornea. Taken together, these findings suggest that the loss of second-harmonic signals is related to the disappearance or absence of these structures.
Certainly, additional study is warranted to evaluate the lack of these structures in keratoconus corneas. Imaging of second-harmonic signals is a noninvasive procedure that theoretically can be used in patients, at least to detect backscattered SHG signals. Further development of this technology may provide important clues to the pathogenesis of keratoconus, as well as the detection of early or suspect keratoconus and the structural organization of the cornea in normal individuals. In addition, identifying the contribution of sutural lamellae to the corneal biomechanical strength may be important to understanding how collagen organization controls corneal shape, which may lead to better insights into the basic mechanisms underlying refractive errors such as myopia, hyperopia, and astigmatism, and may help to understand the effects of refractive surgery and the potential risk of corneal ectasia after LASIK.