Corneal Opacity: Cell Biological Determinants of the Transition From Transparency to Transient Haze to Scarring Fibrosis, and Resolution, After Injury.

Purpose: To highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury. Methods: Review of the literature. Results: The regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFß) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet's membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components-resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet's membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFß entry and activity, and triggers TGFß-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency. Conclusions: Injuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet's membrane are key regulators of stromal cellularity through their modulation of TGFß. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.

Epithelial Basement Membrane Regeneration After PRK-Induced Epithelial-Stromal Injury in Rabbits: Fibrotic Versus Non-fibrotic Corneal Healing.

PURPOSE: To study epithelial basement membrane (EBM) regeneration in non-fibrotic and fibrotic corneas after photorefractive keratectomy (PRK). METHODS: Rabbits (120 total) had either epithelial scrape alone, -4.50 diopters (D) PRK, -9.00 D PRK, or no surgery. Immunohistochemistry was performed on cryofixed corneas at time points from unwounded to 8 weeks (four corneas at each time point in each group). Multiplex immunohistochemistry was performed for EBM components, including collagen type IV, laminin beta-3, laminin alpha-5, perlecan, and nidogen-1. Stromal cellular composition was studied by triplex immunohistochemistry for keratocan, vimentin, and alpha-smooth muscle actin (SMA). RESULTS: PRK-injured EBM significantly regenerated by 4 days after surgery. However, early TGF-beta-regulating perlecan incorporation into the nascent EBM declined 4 to 7 days after surgery in fibrotic corneas. Non-fibrotic corneas that had fully regenerated EBM (with all five components incorporated into the EBM) were transparent and had few SMA-positive myofibroblasts in the stroma. Conversely, corneas with defective nascent EBM that lacked perlecan developed many anterior stromal myofibroblasts and fibrosis at 3 to 4 weeks after surgery and had large amounts of collagen type IV in the nascent EBM and anterior stroma. Myofibroblasts synthesized perlecan but were incompetent to incorporate the heparin sulfate proteoglycan into the nascent EBM. Corneal transparency was restored over several months even in fibrotic corneas, and this was associated with a return of EBM perlecan, myofibroblast disappearance, and reabsorption of disordered extracellular matrix. CONCLUSIONS: Defective incorporation of perlecan into the regenerating EBM by subepithelial myofibroblasts, and likely their precursor cells, underlies the development and persistence of stromal fibrosis after PRK corneal injury. [J Refract Surg. 2022;38(1):50-60.].

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits.

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase ß1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane "railroad track" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

TGFß1 and TGFß2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

The Efficacy of Topical HGF on Corneal Fibrosis and Epithelial Healing after Scar-Producing PRK Injury in Rabbits.

Purpose: To determine the effect of topical hepatocyte growth factor (HGF) on myofibroblast development and corneal opacity after fibrosis-producing photorefractive keratectomy (PRK). Methods: Twelve New Zealand rabbits had transepithelial PRK. Six rabbits received topical recombinant human HGF (rhHGF) (50 µL of 0.1 mg/mL) 3 times a day for 1 week beginning 6 hours prior surgery and until full closure of the epithelium, and 6 control rabbits received vehicle by the same schedule. Slit lamp photos were taken immediately and at 43 to 45 hours after surgery to determine the rate of epithelial healing. Slit lamp photographs and immunohistochemistry for α-smooth muscle actin were analyzed at 1 month in masked fashion. Results: The rhHGF group tended to have slower re-epithelization when compared with the controls, but no statistically significant difference was noted (P = 0.62). There was no significant difference in the density of myofibroblasts in the central stroma (P = 0.49) or corneal opacity (P = 0.84) between the HGF and control groups at 1 month after PRK. Conclusions: Topical rhHGF applied three times a day during the early postoperative period prior to epithelial closure did not significantly change the corneal epithelial healing rate, myofibroblast density, or opacity compared with vehicle after transepithelial -9.0 D PRK injury of the central cornea in rabbits. Translational Relevance: HGF has been reported to decrease myofibroblast generation and fibrosis in many organs, but topical HGF applied to the cornea until epithelial healing had no effect on scarring fibrosis in rabbit corneas.

Descemet's membrane development, structure, function and regeneration.

Basement membranes are layers of extracellular matrix which anchor the epithelium or endothelium to connective tissues in most organs. Descemet's membrane- which is the basement membrane for the corneal endothelium- is a dense, thick, relatively transparent and cell-free matrix that separates the posterior corneal stroma from the underlying endothelium. It was historically named Descemet's membrane after Jean Descemet, a French physician, but it is also known as the posterior limiting elastic lamina, lamina elastica posterior, and membrane of Demours. Normal Descemet's membrane ultrastructure in humans has been shown to consist of an interfacial matrix that attaches to the overlying corneal stroma, an anterior banded layer and a posterior non-banded layer-upon which corneal endothelial cells attach. These layers have been shown to have unique composition and morphology, and to contribute to corneal homeostasis and clarity, participate in the control of corneal hydration and to modulate TGF-ß-induced posterior corneal fibrosis. Pathophysiological alterations of Descemet's membrane are noted in ocular diseases such as Fuchs' dystrophy, bullous keratopathy, keratoconus, primary congenital glaucoma (Haab's striae), as well as in systemic conditions. Unrepaired extensive damage to Descemet's membrane results in severe corneal opacity and vision loss due to stromal fibrosis, which may require penetrating keratoplasty to restore corneal transparency. The purpose of this article is to highlight the current understanding of Descemet's membrane structure, function and potential for regeneration.

Pathophysiology and Treatment of Diffuse Lamellar Keratitis.

PURPOSE: To review cytokine- and chemokine-mediated mechanisms of diffuse lamellar keratitis (DLK) after lamellar corneal surgical procedures. METHODS: Review of the basic science and clinical literature. RESULTS: DLK can occur early or late (months to decades) after all lamellar corneal surgeries, including laser in situ keratomileusis, small incision lenticule extraction, anterior lamellar keratoplasty, and Descemet's stripping automated endothelial keratoplasty. It is most commonly triggered by epithelial injury during or after lamellar surgery, which leads to the release of interleukin (IL)-1α, IL-1ß, and tumor necrosis factor (TNF)-α from the epithelium and into the stroma. These chemokines directly attract inflammatory cells into the cornea from the limbal blood vessels and also bind to receptors on keratocytes and corneal fibroblasts where myriad chemokines are upregulated that also chemotactically attract monocytes, macrophages, granulocytes, lymphocytes, and other bone marrow-derived cells into the corneal stroma. Other factors that can trigger DLK include retained blood in the interface, endotoxins and other toxins, and excessive keratocyte necrosis caused by femtosecond lasers. Infiltrating cells show a preference to enter any lamellar interface in the cornea, regardless of the time since surgery, because of the ease of movement toward the chemotactic attractants relative to the surrounding stroma with intact collagen lamellae and stromal cells that serve as relative barriers impeding motility. The mainstay of treatment is topical corticosteroids, but severe cases may also be treated with flap lift irrigation and systemic corticosteroids. CONCLUSIONS: DLK can occur early or late after any lamellar corneal surgical procedure and is most commonly triggered by epithelial-stromal-bone marrow-derived cellular interactions mediated by corneal cytokines and chemokines. [J Refract Surg. 2020;36(2):124-130.].

Fibrocytes, Wound Healing, and Corneal Fibrosis.

Purpose: This review highlights the roles of fibrocytes-their origin, markers, regulation and functions-including contributions to corneal wound healing and fibrosis. Methods: Literature review. Results: Peripheral blood fibroblast-like cells, called fibrocytes, are primarily generated as mature collagen-producing cells in the bone marrow. They are likely derived from the myeloid lineage, although the exact precursor remains unknown. Fibrocytes are identified by a combination of expressed markers, such as simultaneous expression of CD34 or CD45 or CD11b and collagen type I or collagen type III. Fibrocytes migrate into the wound from the blood where they participate in pathogen clearance, tissue regeneration, wound closure and angiogenesis. Transforming growth factor beta 1 (TGF-ß1) and adiponectin induce expression of α-smooth muscle actin and extracellular matrix proteins through activation of Smad3 and adenosine monophosphate-activated protein kinase pathways, respectively. Fibrocytes are important contributors to the cornea wound healing response and there are several mechanisms through which fibrocytes contribute to fibrosis in the cornea and other organs, such as their differentiation into myofibroblasts, production of matrix metalloproteinase, secretion of tissue inhibitor of metalloproteinase, and release of TGF-ß1. In some tissues, fibrocytes may also contribute to the basement membrane regeneration and to the resolution of fibrosis. Conclusions: New methods that block fibrocyte generation, fibrocyte migration, and their differentiation into myofibroblasts, as well as their production of matrix metalloproteinases, tissue inhibitor of metalloproteinase, and TGF-ß1, have therapeutic potential to reduce the accumulation of collagens, maintain tissue integrity and retard or prevent the development of fibrosis.

Practical guidance for the use of cyclosporine ophthalmic solutions in the management of dry eye disease.

Dry eye disease (DED) is a multifactorial disease of ocular surface and tear film, and is a common disorder treated by eye care providers. It is well established that ocular surface inflammation has an important role in the pathophysiology of DED and that anti-inflammatory cyclosporine A (CsA) improves the treatment outcomes of most patients with DED. The purpose of this review is to provide guidance for practitioners in the use of topical CsA for the management of DED to improve patient satisfaction and the quality of life.

Descemet's Membrane Modulation of Posterior Corneal Fibrosis.

Purpose: The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis. Methods: Twelve New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis. Results: Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes. Conclusions: Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.

IL-1 and TGF-ß Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells.

Purpose: To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy. Methods: Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-ß1, IL-1α, IL-1ß, TGF-ß3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK). Results: IL-1α or -1ß significantly upregulated perlecan mRNA expression in keratocytes. TGF-ß1 or -ß3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-ß1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma. Conclusions: IL-1 and TGF-ß1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.