Fine Structure of the Interface between the Anterior Limiting Lamina and the Anterior Stromal Fibrils of the Human Cornea

Abstract

purpose. To use transmission electron microscopy (TEM) to investigate further the ultrastructural details of the collagen fibrils linking the anterior limiting lamina (ALL; Bowman’s membrane) of the human cornea to the anterior stromal lamellae.

methods. Six disease-free corneas from donors aged 42 to 82 years were fixed (2% glutaraldehyde in 80 mM sodium cacodylate) and processed for TEM within 72 hours postmortem. A series of overlapping images, at 10,204× magnification, of the central corneal ALL–stroma interface were assembled. The features of the terminal ends of fibril bundles at the interface with the anterior stroma were quantitatively assessed.

results. TEM revealed apparently terminating anterior stromal fibril bundles adjacent to the ALL. These terminating lamellae (7.8 per 100 μm) were embedded in an electron-dense material within the surrounding stromal matrix and were termed electron-dense formations (EDFs). The mean width of these stromal features was 1.6 μm. At intervals, anterior stromal lamellae approached the ALL and, in a shallow manner, inserted into the ALL. Such projections (5.4 per 100 μm) into the ALL were, on average, less than 1 μm. Numerous fibrils (29.8 per 100 μm) extended from the ALL into the stroma with a mean length of 0.8 μm.

conclusions. The interface the ALL forms with the anterior stroma is complex, and TEM revealed at least three different types of fibrillar arrangements, which may serve optical requirements rather than provide a structural function.

The human cornea is organized into five main layers—namely, the outer epithelium, the anterior limiting lamina (ALL), the collagen lamellae and keratocyte matrix of the stroma, the posterior limiting lamina, and the inner corneal endothelial cell layer. ¹ Although this essential organization has been well known for many years, ^{2 3 4 5 6} increasing attention has been given more recently to the lamellar organization of the anterior corneal stroma and its role in the mechanical properties related to the maintenance of the shape of the anterior surface of the cornea. ^{7 8 9}  

A structural aspect of the anterior cornea that has received recent attention is the ALL (often referred to by its eponymous name, Bowman’s membrane) and, in particular, how this layer is linked to the anterior stromal lamellae. Special features of this interface were first noted by light microscopy methods by Bowman, ¹⁰ who observed that there are fibers (from the ALL) that “penetrate the (anterior) lamellae, divide and expand in such a manner as to take a firm hold on them… .” Clareus ² noted that, unlike at the interface with the posterior (P)LL, there is no clear borderline between the ALL and anterior stroma, but rather they are continuous with each other. Duke-Elder and Wybar ³ summarize many older ideas by stating that the posterior boundary of the ALL “appears to send prolongations into the substantia propria so that the fibrillar systems of the two are continuous.” In contrast, early transmission electron microscopy (TEM) studies of the human cornea noted that the posterior border of the ALL is “marked by well defined bands of oriented fibers entering an area of unoriented fibers.” It was also noted that these fibers, from the anterior stroma, are sometimes parallel to the corneal surface and sometimes almost at right angles to the surface. ¹¹ In chicken corneas, which also have a fairly well-defined ALL, the conclusion drawn from TEM studies was that some fibril bundles are derived from the ALL and projected into the anterior lamellae, whereas a small contribution come from some terminal ends of anterior lamellae inserting into the ALL particularly in proximity to anteriorly located keratocytes. ¹² Studies in which scanning electron microscopy (SEM) of freeze-fractured corneal specimens was used revealed the presence of perpendicular coarse fibril bundles at the ALL–stroma interface, and it was concluded that these fibril bundles merge into the ALL and that the ALL is actually a “condensation of the superficial layers of the stroma.” ¹³ Recently, second-harmonic imaging microscopy has been used to demonstrate the presence of anterior lamellar and fibril bundles that appeared to insert into the ALL. ¹⁴  

Whether from the earliest light microscopy observations ¹⁰ or much later assessments with TEM, ^{11 12} there is still considerable uncertainty about the fine structure of the interface between the anterior stroma and the ALL. The intent of the present study was to define better the ultrastructure of the ALL–stromal interface at the resolution of the electron microscope, to assess details not resolved in recent second-harmonic laser imaging studies of this interface. ¹⁴ This higher resolution imaging was needed to identify the nature of the terminal ends of these collagen fibril bundles and indicated that there are distinct differences that could be related to the structural roles that this interface may play. 

Materials and Methods

Corneas, with a scleral rim, were obtained from the National Disease Research Interchange (NDRI; Philadelphia, PA) and Lions Eye Bank (Houston and Dallas, TX) and were handled in accordance with the guidelines of the Declaration of Helsinki regarding research on human tissue. The eyes were from six individuals, aged 42 to 82 years, but had been rejected for use in corneal transplantation for reasons other than corneal disease or previous ocular surgery. 

The eyes were delivered to the laboratory within 72 hours after death, in an iced container and individually immersed in an eye bank storage medium (Optisol GS; Bausch and Lomb, Irvine, CA). In accordance with an established protocol developed to minimize tissue distortion and dimensional change, ^{15 16 17} the preparations were first thoroughly rinsed with 0.1 M sodium cacodylate buffer (pH 7.4) and then immersed overnight at 4°C in a fixative solution of 2% glutaraldehyde in 80 mM sodium cacodylate (pH 7.4, 320–340 mOsm/kg). After primary fixation, small pieces (1 × 3 mm) were cut from the central region of the cornea and then rinsed in cacodylate buffer at room temperature (RT) and subsequently postfixed by immersion for 1 hour, under subdued lighting, in a freshly prepared 1% solution of osmium tetroxide (OsO₄) in 0.1 M cacodylate buffer. The samples were again washed in buffer and then dehydrated through a graded alcohol series (30%–100% in six steps) at RT. Then the samples were infiltrated with propylene oxide (three changes at 10-minute intervals) and then with a 3:1 vol/vol mixture of propylene oxide and Spurr’s resin (product number 4300; Electron Microscopy Sciences, Fort Washington, PA) for 3 hours. This was followed by overnight immersion in a 1:1 vol/vol mixture of propylene oxide and Spurr’s resin, followed by transfer to 100% Spurr’s resin overnight. The tissue samples were then oriented in embedding molds and left for overnight polymerization at 60°C. 

The corneal samples were prepared for light microscopy by first cutting thick transverse sections (0.5–1 μm) and then staining with toluidine blue. For electron microscopy, parallel bar copper grids (product no. 2415C-XA SPI Supplies, West Chester, PA) with a 125 μm spacing were used, and the ultrathin sections (∼60–90 nm) were stained first in 3.5% uranyl acetate (dissolved in water) for 20 minutes at 60°C, followed by Reynold’s lead citrate for 10 minutes at RT. The grids were examined in a transmission electron microscope (model 100C; JEOL USA, Peabody, MA) operating at 60 kV. A sequence of slightly overlapping micrographs was taken at 6600× magnification along the ALL–stroma interface. Sections free of gross sectioning artifacts were selected as suitable for such a montage where the epithelial basement membrane formed a flat and uninterrupted interface with the basal cells. 

The negatives were printed to a final magnification of 10,204×, and then assembled to form a continuous montage from each cornea, which was then placed on a flat surface and viewed under cool white fluorescent lighting. The corneal montages measured between 115 and 259 cm in width and represented a tissue range of 125 to 280 μm. Detailed measurements were taken, with a ruler placed directly on the prints, of the cross-section length and width of fibril features and relevant lamellar characteristics in five representative locations of each montage. The total number of fibrillar bundles (lamellae terminating intrastromally, stromal lamellar projections into ALL, and extensions from ALL into stroma) per montage was also counted. All the data were entered into a statistics software package (Systat, ver. 8.0; Systat Inc., Evanston, IL) for generation of descriptive statistics and graphical output. Data sets were checked for normality with the Lilliefors variant of the one-sample Kolmogorov-Smirnov test as included in the software, and then appropriate parametric or nonparametric tests were used to compare measures. A level of statistical significance was set at P = 0.05. 

Results

General Features of the Fine Structure of the ALL–Stromal Interface as Viewed by TEM

For all six specimens, TEM showed the ALL to be a well-defined and uniformly staining amorphous zone immediately under the basement membrane of the corneal epithelium (Fig. 1) . The anterior margin of the ALL appeared smooth, whereas the posterior edge was irregular. This appearance is primarily explained by the fact that the anterior ALL lines the flat, epithelial basement membrane, whereas its posterior extreme borders the anterior stroma with its interwoven, banding, and terminating lamellae that harbor anterior keratocytes, predominantly between the lamellae (Fig. 1) . 

Large stretches of the ALL–stromal interface followed an irregular outline because of the fibrillar interaction and overlapping between these two layers at this central location. A narrow transect along this interface, between the posterior aspect of the ALL and the anterior stroma, indicated a substantial number of crossings of fibril bundles in both directions, but fibrils predominantly originated from the ALL, with some emanating from terminating anterior stromal lamellae. However, despite the numerous crossings, the true overlap between these two corneal layers appeared to be slight and less than 1 μm in either direction (Fig. 2) . 

Detailed Analysis of Projections from the ALL and Lamellar Insertions into the ALL

Many of these fibril bundles in the interface region stained relatively intensely and were usually oriented obliquely or tangentially, but sometimes almost perpendicularly to the main bulk of the ALL. These rather numerous but finer bundles of fibrils were clearly distinct from the thicker, but still densely staining, lamellae of the most anterior stroma. 

Some of the most anterior lamellae had an orientation that was essentially parallel to the ALL. However, other anterior lamellae appeared to have both branched into thinner lamellae and then approached the ALL at a slight angle before apparently coming to an abrupt end near the posterior boundary of the ALL in the central corneal region. This phenomenon was consistently observed in all samples. These apparent terminal lamellae were embedded in an electron-dense, granular material resident in the surrounding stromal matrix, and, will herein be described as electron-dense formations (EDFs; Fig. 3 ). The width of these terminal ends appeared to be substantial, indicating that they were not just occasional and/or spurious splayed ends of lamellae. In each montage, there was a reasonably consistent incidence of EDFs (average, 7.8 ± 1.3 per 100 μm; Table 1 ). Measures of the width of these EDFs showed a range of values from 0.25 to 2.75 μm (Fig. 4) , and across the six corneal samples the group mean width was 1.56 ± 0.55 μm. 

Anterior lamellae approaching the ALL, at intervals, leveled off and minimally engaged with the ALL at a shallow angle. These lamellae were not surrounded by electron-dense material as were the EDFs and did not project beyond 1 μm into the ALL. These were considered to be lamellar projections or insertions into the ALL (Fig. 5) . The lamellar projections were present at a slightly lower frequency than the EDFs, with an average incidence of 5.4 ± 0.8 insertions per 100 μm (Table 1) . The broad endings on the fibril insertions appeared somewhat stubby, and measures of the length for these stroma-originating insertions across the six corneas yielded an average value of 0.52 ± 0.38 μm (median, 0.29 μm). Many of these stromal insertions were only 0.25 μm in length, and the median value was just 0.29 μm (Fig. 6) . 

Many of the other fibril bundles that crossed this zone had a distinctly different configuration, suggesting that these emanated from the ALL (Fig. 7) . These latter bundles had a wide profile to their posterior aspect and were generally splayed out. The splayed ends were located close to the amorphous aspect of the ALL and were characteristically very fine indeed, as they appeared to approach the anterior lamellae of the stroma. The splayed portions of these fine fibril bundles, which were considered to be ALL-originating extensions (and are referred to as ALL extensions), were generally oriented at a steep angle to the plane of the anterior lamellae, sometimes all but perpendicularly. These ALL extensions occurred at a frequency of 29.8 ± 15.6 per 100 μm (Table 1) . Measures of the overall length of these finer fibril extensions gave a mean value of 0.81 ± 0.58 μm (median, 0.64 μm), but with some as long as 2.65 μm (Fig. 6) . Overall, these ALL projections were slightly longer than the stubby lamellar extensions from the stroma (Fig. 6) , and this difference was statistically significant (P = 0.031). 

Discussion

The intent of the present study was to provide a better definition of the structure of the ALL–stromal interface at the electron microscope level, while investigating lamellar organization, continuity, and discontinuity in the central cornea just under the ALL. As viewed by TEM, the ALL has long been noted to be formed by a dense network of fine, randomly oriented collagen fibers, which are slightly smaller in cross section than the underlying stromal collagen. ¹¹ Such general features have also been noted in more contemporary TEM studies of the ALL, ¹⁸ yet provide few clues as to the orientation and/or origin of various fibril bundles. Orientation and origin were more the goal in the present study. In initial ultrastructural observations using TEM (Horne J, et al. IOVS 2003;44:ARVO E-Abstract 885), different types of terminal endings of these anterior lamellae were noted. The present report is a completion of the first stage of these analyses which are presented as further evidence for there being both insertions into the ALL and extensions from the ALL to the anterior stroma. The TEM methodology, with its associated ultrathin sectioning, has the resolution to demonstrate the existence of the fibrillar overlapping occurring at this interface but cannot reliably reveal the total length of the fibrillar projections from either the ALL or the stroma into its neighboring tissue. High-voltage TEM (HVTEM), or TEM with a goniometer, may provide a better view of the extent of projection. The former (HVTEM) has demonstrated thicker fibrils crossing this interface that “could be followed for up to 1.5 μm,” ¹⁹ but it is as yet unclear whether these images represent the total length of such fibrils. In recent second-harmonic laser imaging of the same region of cornea, it was concluded that prominent lamellae inserts into the ALL, ¹⁴ but such imaging does not have the resolution to see the detail observable with TEM. However, the laser imaging of what appears to be lamellar insertions show features similar to those seen in light microscopy (not shown), very low magnification TEM images (e.g., Fig. 1of this article), or even SEM, ¹³ as well as showing remarkable similarity to the diagrams based on light microscopy provided by Sir William Bowman. ¹⁰  

At the present time, it is only possible to speculate on the possible origins and/or functions of insertions into the ALL. The present TEM imaging suggests that some of these insertions arise from branches of anterior lamellae and this then raises the important issue of the course and orientation of any of the lamellae in the anterior stroma. One perspective on the physiology of the corneal lamellae is that they are essentially flat and very elongated bundles that follow a course from one side of the cornea to the other. However, it was long ago considered and in quite some detail, that some lamellae may well bridge the maximum diameter of the cornea, whereas others are arranged across much shorter distances. ²⁰ More recent observations have indicated that the anterior lamellae actually overlap each other quite extensively, and the extent of this so-called interlacing between the most anterior lamellae is more substantial than in the mid- or posterior stroma. ^{8 18 21 22} In addition, evidence has been presented that the lamellae also branch and even insert through one another. ²³  

At least in the normal human cornea, there appear to be stromal fibril projections into the ALL that are associated with slender ends or branches of anterior lamellae. Such branches, rather than following a course parallel to the ALL interface, are clearly oriented anteriorly. Some appear to broaden out as they become immersed in the network of very fine fibrils that make up the ALL, although others terminate in characteristic electron dense structures (EDFs) that are located posteriorly to the main elements of the ALL (i.e., they do not enter the ALL in a substantial fashion). Because these insertions appear to arise from a subgroup of anterior lamellae, it is possible that these lamellae are those that do not cross the entire cornea but essentially terminate in the central region. The electron-dense material, in which the collagen fibers were embedded may provide the means for securely anchoring the anteriorly terminating lamellae. This lamellar arrangement and configuration is consistent with a report that there are fewer lamellae in the central region of the cornea compared with the most peripheral regions. ^{15 24} In addition to these special configurations of anterior lamellae, a unique group of splayed fibrils appear to originate within the ALL, are directed toward the branched anterior lamellae, but are clearly separated and distinct from these. 

The ALL–stromal interface is complex, involving multiple fibrillar arrangements, each showing a different incidence but together occurring at a relatively high density of 43 per 100 μm. The combined structural and mechanical effects of these three types of fibrillar organizations at the posterior aspect of the ALL (that can only be seen at the resolution of the TEM) presumably account for the anecdotal observations that the ALL cannot be detached from the anterior stroma in the normal healthy cornea. ^{2 3 5 6 10} Logically, such a continuity or shallow blending between the two corneal layers does not so much provide structural strength to the most anterior part of the cornea as it serves an optical function. For instance, the gradual transition of one tissue into the other would serve optical transparency better than a distinct interface. In addition, we propose that the aspheric curvature of the corneal surface may be explained by the lamellar organization of the central anterior stroma where lamellae appear to terminate rather than stretching from limbus to limbus. The present observations and data will help define the normal morphology of this region and will be most helpful in assessing the pathologic consequences of corneal thinning diseases such as keratoconus and pellucid marginal degeneration. 

 Supported by The Eye Birth Defects Research Foundation (JPGB), National Eye Institute CORE Grant P30 EY07551 to the University of Houston College of Optometry, short-term NEI training grant T35 EY07088 for professional students, and National Institutes of Health (NIH) Loan Repayment Grant (JHM).

 Submitted for publication June 12, 2007; revised November 2, 2007, and April 1, 2008; accepted July 22, 2008.

 Disclosure: J. H. Mathew, None; J.P.G. Bergmanson, None; M.J. Doughty, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Low magnification of the anterior cornea, showing basal epithelial cells (E), the ALL, and the anterior stroma, with regions of flattened, stellate keratocytes (arrow).

Figure 2.

 

Example of a montage from the central region, approximately 125 μm in length, of a cornea showing the ALL–stromal interface (I).

Figure 3.

 

Example of the terminal end of a branch from an anterior lamella in an electron-dense formation (arrow).

Table 1.

 

Incidence of Fibrillar Features at the ALL–Stromal Interface

Montage	EDF	Stromal Lamellar Projection/Insertion into ALL	ALL Extension into Stroma
1	8	4.8	61
2	6.1	5	21.4
3	6.6	6.1	21
4	7.6	5.6	22.2
5	9.6	6.4	23.2
6	8.9	4.5	30
Average ± SD	7.8 ± 1.3	5.4 ± 0.8	29.8 ± 15.6

 Data are the number of fibrils per 100 μm of tissue and were collected from montages of six separate corneas.

Figure 4.

 

Distribution of sizes (in micrometers), of the EDFs noted at the posterior boundary of the ALL.

Figure 5.

 

Example of a stromal lamellar projection/insertion (arrow) into the ALL without a terminal electron-dense formation.

Figure 6.

 

Box plot showing the difference in length (in micrometers) of the fibril bundles that make up the lamellar stromal insertions (INSERT) versus those that constitute fibril extensions (EXTEN) from the anterior limiting lamina.

Figure 7.

 

Example of a fine fibrillar extension (arrow) from the ALL into the anterior stroma.