Epithelial phenotype in total sclerocornea

Patients

From 2008 to 2011, four patients with total sclerocornea (male:female = 1:3, mean age = 5.4±4.3; 1–11 years old) received PKP in our hospital. The demographic data of the patients are listed in Table 1. There was no family history of hereditary eye diseases in Patients 1, 3, and 4, but CCO and glaucoma were found in the father and the grandmother of Patient 2. Measured preoperative visual acuity ranged from hand motion (Patient 3) to finger counting at 50 cm (Patient 1). All transplantations were performed under general anesthesia. Donor corneas (6.5 to 7.5 mm in diameter; all 0.5 mm larger than the recipient bed) were punched with a superblade trephine (Pharmacia Production BV, the Netherlands), and the diseased corneas were trephined with the Hessburg-Barron vacuum trephine (Jedmed Instrument, St. Louis, MO). During the operation, the corneas were carefully trephined to avoid cutting through the iris. Extensive iris adhesion to the cornea were noted in Patients 2 and 3, which had to be separated meticulously; while an atrophic iris and lens were seen in Patient 1. The graft was sutured with 16 interrupted sutures of 10–0 nylon suture. Postoperative medications included a topical steroid and antibiotic solution (Tobradex solution, Alcon, Fort Worth, TX), but no systemic prednisolone (5 mg per day) was given except during PKP for the second eye of Patient 4 when the first eye failed due to rejection.

Transmission electron microscopy

Corneal samples from Patients 2 and 3 were fixed in 50 mM phosphate buffer (pH 7.2) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 24 h at room temperature. After refixation in 1% osmium tetraoxide for 4 h at room temperature, the corneal samples were washed in PBS (1X; 120 mM NaCl, 20 mM KCl, 10 mM Na₂PO₄, 5 mM K₂PO₄, pH 7.4), dehydrated, and embedded in Epon 812 epoxy resin. The 50 nm ultrathin sections were stained with uranyl acetate and lead citrate, and images were photographed with a Hitachi 7100 transmission electron microscope (Hitachi, Tokyo, Japan) equipped with an AMT Digital camera.

Immunohistochemistry and confocal laser scanning microscopy

Surgically excised scleralized corneas were embedded in optimum cutting temperature (OCT) compound and then were frozen in a −70 °C refrigerator. Immunofluorescent staining was modified from our previous report [14]. Briefly, 5–7 μm frozen sections were rinsed with PBS and then fixed with 100% methanol at 4 °C for 10 min. Nonspecific reactions were blocked by incubating the sections with PBS containing 2.5% bovine serum albumin (BSA) for 30 min. The slides were then incubated overnight at 4 °C with the appropriate primary antibodies (Table 2), which included antibodies against epithelial differentiation markers keratin 3 and 12 (for the corneal epithelium), keratin 13 (for the conjunctival epithelium), gap junction protein connexin 43 (Cx43), MUC5AC (goblet cell-specific mucin), stem cell markers transcription factor p63 and ATP-binding cassette sub-family G member 2 (ABCG2), laminin-5, and fibronectin. Sections incubated with irrelevant mouse or goat immunoglobulin G (IgG) were used as negative controls. After the sections were washed three times in Tris-buffered saline containing 0.5% Tween-20 for 5 min, they were incubated at room temperature for 1 h with the appropriate secondary antibodies, including cyanine-3 (Cy3)-conjugated donkey anti-mouse IgG (for p63 and ABCG2), fluorescein isothiocyanate (FITC)–conjugated donkey anti-goat IgG (for K12), or FITC-conjugated donkey anti-mouse IgG (for Cx43; keratin 3 and 13; MUC5AC, laminin-5, and fibronectin). Finally, sections were washed three times in Tris-buffered saline with Tween-20 for 5 min and covered with antifade mounting medium containing propidium iodide (PI) or 4',6-diamidino-2-phenylindole (DAPI). Laser confocal microscopy (TCS SP2-MP system; Leica, Heidelberg, Germany) was performed with filters for FITC (excitation 488 nm, emission 500–535 nm), DAPI (excitation 359 nm, emission 450–460 nm), and PI (excitation 514 nm, emission 595–633 nm). The image was averaged from 14 scans within a thickness of 5 to 7 μm. Hematoxylin-eosin (HE) staining was also performed for morphological comparison. Unless particularly mentioned, photos taken in the central region of the specimens are shown.

Surgical outcome

The measured preoperative visual acuity ranged from hand motion (Patient 3) to counting fingers at 50 cm (Patient 1). Postoperatively, elevated intraocular pressure (IOP) was controlled by antiglaucoma agents in all patients. In addition, trabeculectomy was performed in both eyes of Patient 2, but both grafts failed subsequently, and the vision in the oculus dexter (OD) was permanently damaged. After PKP, Patient 3 received lens aspiration and intraocular lens implantation in both eyes. After a mean follow-up period of 38.8±14.0 (12–54) months, grafts remained clear in half of the eyes: Two grafts failed due to glaucoma, one due to allograft rejection, and one to progressive graft edema. For the three patients who were verbally communicative, vision improved in two (Patients 1 and 3; 20/1,000 and 20/400, respectively) but remained the same (ocular sinister, OS) or worse (OD) in one (Patient 2).

Histology

All four patients presented with diffuse corneal opacity that obscured the iris, and no clear demarcation separated the limbus from the cornea. Palisades of Vogt were identified only in Patient 1 (Figure 1A,E), and various degrees of corneal NV reaching the pupil zone were seen in Patients 2 to 4, most prominently in Patient 4 (Figure 1D,H). Under light microscopy, the stratification of the corneal epithelium and the stromal thickness varied among cases. In general, the epithelia in the central cornea in Patients 1 to 3 resembled the normal corneal epithelium, which contained four to seven layers of superficial squamous and basal cuboidal epithelial cells, but the Bowman’s layer was invisible (Figure 1U–X). Increased epithelial stratification from five to 12 layers was noted in the central cornea of Patient 4, and most basal cells were cuboidal or columnar (Figure 1T,X). No goblet cells were seen in any specimen, but with immunohistochemistry, the hyperstratified epithelial cells in Patient 4 were identified as conjunctival (Figure 1X, to the right of the arrow). In Patients 2 to 4, the collagen lamellae in the superficial stroma were more irregularly arranged, while the alignment became more uniform posteriorly (Figure 1Q-T). In Patients 2 and 3, NV was prominent in the anterior corneal stroma (Figure 1R,S; arrowhead), while NV was seen in the entire thickness of the stroma in Patient 4 (Figure 1T; arrowhead). In Patients 2 to 4, stromal hypercellularity was also evident in areas containing corneal NV (Figure 1R-T). Descement’s membrane (DM)-like structure was seen only in Patient 1 (Figure 1Q); in the other three cases, there was no DM, and adhesion of the iris pigment to the posterior stroma was seen in Patients 3 and 4 (Figure 1S,T; arrows). In all four cases, no corneal endothelial cells were seen in the specimens.

Transmission electron microscopy

The ultrastructures of a normal corneal stromal layer consist of regularly arranged collagen fibers along with sparsely distributed interconnected keratocytes (Figure 2A). The collagen fibrils in the stroma are uniformly organized in a lamellar arrangement. Figure 2B,C exhibited the regular arrangement of collagen fibrils in the longitudinal and cross-sectional view, with an average fiber diameter of 35 nm. Figure 2E,I shows the irregular and variably thinned collagen lamellar layers in Patients 2 and 3, respectively. Furthermore, disorganized, nonlinear, and random distribution of collagen fibrils was found in the scleralized stromal tissue (Figure 2F,G,J,K). In contrast to a normal cornea (Figure 2C), the collagen fibers in Patient 3 varied considerably in diameter, ranging from 46 to 78 nm (Figure 2K).

Normal corneal basal epithelial cells with Bowman’s membrane are shown in Figure 2D (arrowhead). Note that the anterior stromal collagen is loosely organized. Figure 2H,L shows the irregular and nearly invisible Bowman’s membrane in Patients 2 and 3, respectively. In contrast to a normal cornea, collagen fibers in the anterior stroma of Patient 2 are more densely arranged (Figure 2H).

Immunoconfocal microscopy

Using a panel of antibodies against differentiation-related cytokeratins, we showed that immunoreactivity to keratin 3 was homogenously expressed by the entire layer of the corneal epithelium in Patients 1 and 2, and in the peripheral corneal button in Patient 4 (Figure 3A,B,D). However, signals for keratin 3 were negative in the basal epithelial layer in Patient 3 and the central corneal button in Patient 4 (Figure 3C,D insert). Keratin 12, the acidic cytokeratin pair of keratin 3, and a more specific corneal epithelial marker, was expressed homogenously in Patients 1 and 2 (Figure 3E,F), but was negative in some basal epithelial cells in Patient 3 (Figure 3G). The immunostaining photos shown in Patients 1 to 3 were from the central cornea, and the expression of keratin or other proteins was similar in either the central or peripheral cornea. In Patient 4, however, because of the conjunctival epithelium invasion, different areas were compared, as shown in Figure 3I. In Patient 4, although the peripheral epithelium was keratin 12 positive (Figure 3H, the right peripheral corneal button), keratin 12 was entirely negative in the central cornea except in the focal areas (Figure 3I, insert). In Patient 4, when the specimen was dual-stained with keratin 12 (green) and keratin 13 (red), the peripheral corneal button was keratin 12 positive, while the central cornea was keratin 13 positive, and these two keratins were expressed in a mutually exclusive pattern (Figure 3I). Judging from the keratin expression pattern, this specimen must have contained invading conjunctival epithelium in the center. In Patient 4, the central cornea was covered by the conjunctival epithelium while the peripheral cornea was covered by the corneal epithelium is because the invasion of the conjunctival epithelium was predominantly from the superior and inferior side, while the corneal button was bisected horizontally (Figure 1T, Figure 3I).

Keratin 13 is expressed by differentiated mucosal epithelia, including conjunctiva and oral mucosa [14,15]. In Patients 1 to 3, staining for keratin 13 was negative (Figure 4A–C); however, in Patient 4, strong suprabasal staining for keratin 13 was evident in the central cornea, while staining at the periphery was negative (Figure 3I, Figure 4D; both photos taken at the right peripheral corneal button). MUC5AC is the mucin secreted by goblet cells. The insert in Figure 4L shows normal conjunctiva containing goblet cells, which were MUC5AC positive. In Patients 1 to 4, the signal for MUC5AC was universally negative, suggesting that none contained goblet cells (Figure 4I-L). Connexin 43 (Cx43) is a gap junction protein often considered a keratinocyte differentiation marker. In Patients 1 to 3, Cx43 staining was present in the entire epithelial layer (Figure 4M-P), similar to the pattern in a normal central cornea. However, Cx43 staining was predominantly in the superficial and suprabasal epithelial layer but less in the basal epithelial layer in Patient 4 (Figure 4P).

Transcription factor p63, notably the deltaNp63α isoform, and ATP binding cassette (ABC) transporter ABCG2 have been proposed as markers for corneal epithelial stem cells [16,17]. In all patients, staining for p63 and ABCG2 was invariably negative (Figure 5A-D, E-H), suggesting that the epithelia were more differentiated, similar to the corneal epithelium but not the limbal epithelium. Laminin-5 (LN5; laminin α3β3γ2) is a component of the epithelial basement membrane proteins that facilitate the attachment and migration of basal keratinocytes. In all four specimens, homogenous linear staining of LN5 in the epithelial basement membrane (EBM) zone was evident (Figure 5I-L), suggesting that in these patients the EBM zone was intact. Finally, fibronectin is an extracellular glycoprotein involved in corneal wound healing and epithelial migration. Normally, fibronectin is present in the EBM zone of the corneal, limbal, and conjunctival epithelium [18,19], but during corneal wounding stromal staining of fibronectin can be seen [20]. Linear fibronectin staining was evident along the EBM in Patients 1 to 3, with only sparse staining in the stroma (Figure 5M-O). Staining of fibronectin in the EBM zone was absent in Patient 4; however, scattered staining was seen in the stroma (Figure 5P).