Corneal stromal changes following reconstruction by ex vivo expanded limbal epithelial cells in rabbits with total limbal stem cell deficiency

Abstract

Aim: To study corneal stromal changes and the presence of myofibroblasts after transplantation of ex vivo expanded limbal epithelium.

Methods: A state of limbal deficiency was induced in 16 rabbits. After transplantation with autologous ex vivo expanded limbal epithelium on amniotic membrane (AM), their clinical outcomes were classified as success, partial success or failure according to surface smoothness, stromal clarity, and vascularisation. Clinical outcomes were correlated with phenotypic outcomes of corneal, conjunctival, or mixed epithelium, defined by expression of K3 keratin or MUC5AC. Immunostaining was performed with antibodies against collagen IV, fibronectin, and alpha-smooth muscle actin (alpha-SMA) to assess stromal wound remodelling.

Results: Rabbits were sacrificed after a mean follow up of 10 (SD 3.3) months. Collagen IV, expressed in the basement membrane of all three groups, was found in the stroma of the partial success, but not in that of the success or the failure. Fibronectin was absent in the success and the failure, but expressed in the stroma of the partial success. Alpha-SMA was expressed in superficial stroma of the partial success, but suppressed in areas with AM remnants.

Conclusion: Restoration of a clear and transparent cornea is associated with a normal corneal epithelium and complete wound remodelling. In contrast, wound healing remains active and incomplete in conjunctivalised corneas, which remain opaque with myofibroblasts.

Figure 1

Normal control cornea. Haematoxylin staining (A). Keratin 3 (K3) is expressed in the full thickness of the corneal epithelium (B). Collagen IV staining in the basement membrane of corneal epithelium was weak and thin (C) while thicker and wavy in the conjunctival region and the underlying stromal blood vessels (D). Fibronectin (Fn) staining was negative in the entire cornea (E) and conjunctiva (F) except for the lumen of blood vessels (arrow). Bar 50 µm.

Figure 2

Cornea with success. External appearance shows that the reconstructed surface is smooth and clear without vascularisation (A). Keratin 3 (K3) is expressed in the reconstructed corneal epithelium (B, arrow marks the limbus). MUC5AC positive goblet cells are limited to the conjunctival region (C, arrow marks the limbus). Coll-IV is expressed in the corneal basement membrane and Descemet’s membrane but not in the stroma (D). Coll-IV is also expressed in the stroma of peripheral cornea and limbal region (E). Immunoreactivity against fibronectin (Fn) was negative (F). α-SMA was expressed in the smooth muscle of blood vessels of the limbal and conjunctival stroma (G). Bar 50 µm.

Figure 3

Corneas with partial success. Slit lamp appearance shows that part of these corneas is smooth and clear and the other is vascularised and opaque (A, F). Keratin 3 (K3) is expressed only in the smooth and clear area (arrow marks the border between corneal and conjunctival phenotypes) (B). Haematoxylin and eosin staining shows AM remnant (marked by asterisks) (C). Coll-IV is expressed in the corneal basement membrane, AM remnants and the superficial corneal stroma (D). α-SMA was only expressed in the smooth muscles of blood vessels in the limbal stroma (arrowhead marks a blood vessel) (E). Coll-IV is expressed in superficial stroma and intrastromal blood vessels (G). Fibronectin (Fn) was highly expressed in the stroma and blood vessels of granuloma (H). α-SMA expression suggests the presence of myofibroblasts in the granuloma (I). Bar 50 µm.

Figure 4

Cornea with failure following ex vivo expansion. Slit lamp appearance shows corneal haze, vascularisation, and inflammation (A). MUC5AC staining of the goblet cells confirms conjunctival invasion of the corneal surface (B). Coll-IV is expressed in the corneal basement membrane and intrastromal blood vessels. Inset shows the higher magnification of blood vessel wall (C). Fibronectin (Fn) and α-SMA were expressed only in stromal blood vessels (D and E, respectively). Bar 50 µm.

Figure 5

Cornea with failure following AM transplantation alone. Slit lamp appearance shows corneal haze and vascularisation. AM remnants are seen in the stroma (enclosed by brackets) (A). Haematoxylin stain shows the presence of AM remnants in the cornea (enclosed by brackets) (B). Coll-IV is highly expressed in the corneal basement membrane and stroma (marked by arrows) (C). Coll-IV is also found in AM remnants (enclosed by brackets) (D). (Fibronectin (Fn) is also found in the corneal stroma with AM remnants (E). α-SMA is not expressed in this area (F). Bar 50 µm.