Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits

Abstract

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet’s membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA+ myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60 to 90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10% to 20% of posterior stroma even at four months after keratitis in the central cornea where Descemet’s membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis—similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFβ) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFβ to maintain viability. The endothelium and Descemet’s membrane may serve a similar function modulating TGFβ penetration into the posterior stroma—with the source of TGFβ likely being the aqueous humor.

Injury and defective regeneration of the epithelial basement membrane in corneal fibrosis: A paradigm for fibrosis in other organs?

“…Thus, the TGFβ- and PDGF-dependent immature myofibroblasts that have begun development undergo apoptosis before they produce sufficient disordered extracellular matrix to reduce corneal transparency [13,14]. With more severe injuries, such as bacterial infections or photorefractive keratectomy (PRK) surgery to correct high nearsightedness, normal regeneration of the EBM may be delayed (Fig 2) [13,15]. This defective EBM allows ongoing penetration of high levels of TGFβ1 and PDGF from the epithelium into the stroma to drive development and persistence of large numbers of myofibroblasts, resulting in fibrotic scarring of the cornea (Fig 1C).…”

“…1E). In this situation, the epithelial BM can eventually be repaired, leading to apoptosis of anterior stromal myofibroblasts, while posterior stromal myofibroblasts survive due to persistent damage to Descemet's membrane BM that allows activation and penetration of TGFβ from the aqueous humor within the anterior chamber of the eye into the posterior stroma (Fig 1F) [15]. In some species, in which the corneal endothelium can regenerate, Descemet's BM may also eventually be repaired, leading to apoptosis of the posterior stromal myofibroblasts and restoration of full corneal transparency.…”

“…H. TEM of epithelial-stromal junction at three-months after pseudomonas keratitis. Normal EBM lamina lucida and lamina densa are present (arrows) and likely a keratocyte (k), but possibly a nerve-associated Schwann cell, is present, but myofibroblasts with large amounts of rough endoplasmic reticulum have disappeared [15]. Also note, normal regular collagen fibrils (arrowheads) are now present throughout the anterior stromal shown.…”

“…The other major BM in the cornea, Descemet's membrane, that lies beneath the corneal endothelium on the posterior corneal surface, appears to have a similar role in regulating pro-fibrotic growth factors from the aqueous humor and corneal endothelium that could trigger posterior corneal myofibroblast development and fibrosis (Fig. 1D, 1E, 1G) [15]. …”

Regeneração dos nervos da córnea anterior pós excimer laser e reparação tecidual pós injúria endotelial

“…A ausência dessa regeneração das células do endotélio persistiu até o quarto mês, quando usado um modelo animal para que pudesse ser estudada a fibrose da córnea após infecção por Pseudomonas aeruginosas, cuja profundidade e extensão da lesão chegaram a alcançar a membrana de Descemet. 25 fibrose em 100% dos casos após a cirurgia de PRK sem o uso de MMC para a correção de -9,0D na área de ablação desses animais, enquanto em humanos, essa tendência é de 3-5%. 18,71 A literatura já relata a ocorrência de opacidade após a descemetorréxis sem o posterior transplante endotelial como possíveis complicações da técnica.…”