Ovalbumin-Induced Allergic Inflammation Diminishes Cross-Linked Collagen Structures in an Experimental Rabbit Model of Corneal Cross-Linking

Abstract

Background: Allergic conjunctivitis (AC) is one of the reported potential risk factors of progression in keratoconus patients after corneal cross-linking surgery; however, the causal relationship is still inconclusive. Recent studies have indicated that various inflammatory cytokines play a vital role in the development of primary keratoconus. It is still unclear whether these inflammatory mediators also trigger CXL failures. This study aimed to investigate the impact of AC on the rabbit corneas after trans-epithelial corneal cross-linking (TCXL).

Methods: A total of six rabbits were kept untreated as the normal control (NC) group. A total of 18 rabbits were treated by TCXL and divided into three groups (six in each group), namely, no treatment (TCXL group); induction of AC (TCXL + AC group); and induction of AC plus topical prednisolone acetate (TCXL + AC + PA group), according to additional treatment. AC was induced by topical application of ovalbumin after intraperitoneal pre-sensitization with ovalbumin. Rabbits were evaluated by slit lamp, in vivo laser scanning confocal microscopy, anterior segment optical coherence tomography, and measurement of corneal biomechanics. The cornea specimens were collected for the transmission electron microscope, the collagenase I digestion test, and PCR assay for TNF-α, IL-6, IL-1β, matrix metalloproteinase 9 (MMP-9), lysyl oxidase (LOX), and tissue inhibitor of metalloproteinases 1 (TIMP-1) on the day (D) 28.

Results: On D28, the TNF-α, IL-6, IL-1β, MMP-9, and LOX levels were significantly increased while the TIMP-1 was decreased in the TCXL + AC group when compared with the TCXL and TCXL + AC + PA groups. In vivo confocal microscopy revealed that at a depth of 150-210 μm, a trabecular patterned hyperdense structure surrounded by elongated needle-like processes could be observed in the TCXL and TCXL + AC + PA groups, but hardly seen in the TCXL + AC group. The demarcation lines were indistinct and blurred in the TCXL + AC group. An electron microscope demonstrated less interlacing fibril lamellae and higher interfibrillar spacing in the TCXL + AC group. The stability of corneal biomechanics and resistance to collagenase were decreased in the TCXL + AC group.

Conclusion: The corneal microstructures induced by TCXL and biomechanical stability were diminished in rabbits with AC but could be maintained by topical anti-inflammatory treatment. Our results supported the causal relationship between altered cytokine profiles and corneal microstructure after primary corneal cross-linking.

Keywords: allergic conjunctivitis; corneal collagen cross-linking; in vivo confocal microscopy (IVCM); inflammatory cytokine; matrix metalloproteinase.

FIGURE 1

Representative slit-lamp photographs on D28 show the palpebral conjunctival hyperemia (row 1), corneal fluorescein sodium staining (row 2), central corneal edema (row 3) and demarcation line in the anterior stroma (row 4) in different groups. The degree of conjunctival hyperemia in the TCXL + AC group was apparently higher than that in the TCXL group or TCXL + AC + PA group, while no obvious hyperemia was observed in the NC group. No obvious corneal epithelium staining or corneal edema was observed in any group on D28. The white arrows indicated the conjunctival papillae, which were the signs of allergic conjunctivitis. The black arrows indicated the clear demarcation lines in the middle stroma of TCXL and TCXL + AC + PA group, as well as an indistinct hyper-reflective structure in the superficial stroma of TCXL + AC group.

FIGURE 2

Quantitative analysis of TNF-α (A), IL-6 (B), IL-1β (C), MMP9 (D), LOX (E), and TIMP-1 (F) mRNA transcripts in the corneal stroma on D28. The mRNA levels of TNF-α, IL-6, IL-1β, and MMP-9 were significantly increased in the TCXL + AC group when compared with the TCXL and TCXL + AC + PA groups. In contrast, the TIMP-1 level was significantly lower in the TCXL + AC group when compared with the TCXL and TCXL + AC + PA groups. Data are presented as mean ± SD, n = 6/group (**P < 0.01; ***P < 0.001).

FIGURE 3

Representative IVCM images showing collagen structures in the anterior corneal stroma on D28 at different depths (row A, 50–60 μm; row B, 100–110 μm; row C, 15–160 μm; and row D, 200–210 μm). At the depths of 150–160 μm and 200–210 μm, trabecular patterned hyperdense structure surrounded by elongated needle-like processes could be observed in the TCXL and TCXL + AC + PA groups (white arrows), but hardly seen in the TCXL + AC group.

FIGURE 4

Representative images of anterior segment optical coherence tomography showing the demarcation line (white arrows) in the corneal stroma on D28. No demarcation line could be observed in the normal control. The demarcation lines in the TCXL and TCXL + AC + PA groups were distinct and homogeneous, but indistinct and blurred in the TCXL + AC group.

FIGURE 5

Representative images of transmission electron microscopy showing the ultrastructural changes in corneal stroma on D28. Parallel running collagen fibril lamellae were present in the anterior stroma of normal control (A), while obviously interlacing lamellae were present in the TCXL group (B). In TCXL + AC group, parallel running and interlacing fibril lamellae were both present (C), but the fibril lamellae were still obviously interlacing in TCXL + AC + PA group (D). Cross section of the collagen fibrils showing the interfibrillar spacing in the TCXL cornea (F,I) was lower than that of normal control (E,I) and TCXL+AC group (G,I), but was comparable with that of TCXL+AC+PA group (H,I). (A–D) Magnification 5000×; (E–H) magnification 45000×.

FIGURE 6

Representative images showing the DA Ratio Max (2 mm) (A) and corneal resistance to collagenase digestion (B) on D28. The DA Ratio Max of the TCXL group was significantly increased when compared with the NC group, while the DA Ratio Max of the TCXL + AC group significantly decreased when compared with the TCXL group. In the collagenase digestion assay, the final relative weight of the cornea in the TCXL group was significantly higher than those in the NC and TCXL + AC groups. Data were presented as mean ± standard deviation (n = 6; *P < 0.05; **P < 0.01; ***P < 0.001).