LYCIA MARIA MARTINS PINHO PEDRAL SAMPAIO
Projetos de Pesquisa
Unidades Organizacionais
LIM/33 - Laboratório de Oftalmologia, Hospital das Clínicas, Faculdade de Medicina
12 resultados
Resultados de Busca
Agora exibindo 1 - 10 de 12
- Standardization of corneal alkali burn methodology in rabbits(2023) VILLABONA-MARTINEZ, Valeria; SAMPAIO, Lycia Pedral; SHIJU, Thomas Michael; WILSON, Steven E.Alkali burns are one of the most common injuries used in corneal wound healing studies. Investigators have used different conditions to produce corneal alkali injuries that have varied in sodium hydroxide concentration, application methods, and duration of exposure. A critical factor in the subsequent corneal healing responses, including myofibroblast generation and fibrosis localization, is whether, or not, Descemet's membrane and the endothelium are injured during the initial exposure. After exposures that produce injuries confined to the epithelium and stroma, anterior stromal myofibroblasts and fibrosis are typical, with sparing of the posterior stroma. However, if there is also injury to Descemet's membrane and the endothelium, then myofibroblast generation and fibrosis is noted full corneal thickness, with predilection to the most anterior and most posterior stroma and a tendency for relative sparring of the central stroma that is likely related to the availability of TGF beta from the tears, epithelium, and the aqueous humor. A method is described where a 5 mm diameter circle of Whatman #1 filter paper wetted with only 30 mu L of alkali solution is applied for 15 s prior to profuse irrigation in rabbit corneas. When 0.6N, or lower, NaOH is used, then the injury, myofibroblasts, and fibrosis generation are limited to the epithelium and stroma. Use of 0.75N NaOH triggers injury to Descemet's membrane and the corneal endothelium with fibrosis throughout the stroma, but rare corneal neovascularization (CNV) and persistent epithelial defects (PED). Use of 1N NaOH with this method produces greater stromal fibrosis and increased likelihood that CNV and PED will occur in individual corneas.
- Transforming growth factor beta-3 localization in the corneal response to epithelial-stromal injury and effects on corneal fibroblast transition to myofibroblasts(2023) SHIJU, Thomas Michael; SAMPAIO, Lycia Pedral; MARTINEZ, Valeria Villabona; HILGERT, Guilherme S. L.; WILSON, Steven E.The purpose of this study was to evaluate the localization of TGF beta-3 in situ in unwounded rabbit corneas and corneas that had epithelial-stromal injuries produced by photorefractive keratectomy (PRK) in rabbits and to evaluate the in vitro effects of TGF beta-3 compared to TGF beta-1 on alpha-smooth muscle actin (alpha-SMA) protein expression and myofibroblast development in corneal fibroblasts. Forty-eight New Zealand white rabbits underwent either -3 diopter (D) or -9D PRK and were studied from one to eight weeks (four corneas in each group at each time point) after surgery with immunohistochemistry for TGF beta-3, laminin alpha-5, and alpha-smooth muscle actin (alpha-SMA). Rabbit corneal fibroblasts were treated with activated TGF beta-1 and/or TGF beta-3 at different concentrations and duration of exposure and studied with immunocytochemistry for myofibroblast development and the expression of alpha-SMA using Jess automated Western blotting. TGF beta-3 was detected at high levels in the stroma of unwounded corneas and corneas at one to eight weeks after -3D or -9D PRK, as well as in the epithelium and epithelial basement membrane (EBM). No difference was noted between corneas that healed with and without myofibroblast-mediated fibrosis, although TGF beta-3 was commonly associated with myofibroblasts. TGF beta-3 effects on corneal fibroblasts in vitro were similar to TGF beta-1 in stimulating transition to alpha-SMA-positive myofibroblasts and promoting alpha-SMA protein expression. The corneal stromal localization pattern of TGF beta-3 protein in unwounded corneas and corneas after epithelial-stromal injury was found to be higher and different from TGF beta-1 and TGF beta-2 reported in previous studies. TGF beta-3 had similar effects to TGF beta-1 in driving myofibroblast development and alpha-SMA expression in corneal fibroblasts cultured in medium with 1% fetal bovine serum.
- Topical Losartan Decreases Myofibroblast Generation But Not Corneal Opacity After Surface Blast-Simulating Irregular PTK in Rabbits(2023) SAMPAIO, Lycia Pedral; VILLABONA-MARTINEZ, Valeria; SHIJU, Thomas Michael; SANTHIAGO, Marcony R.; WILSON, Steven E.Purpose: To evaluate the efficacy of topical losartan after blast injury-simulating irregular phototherapeutic keratectomy (PTK) in rabbits.Methods: Twelve NZW rabbits underwent 100 pulse 6.5 mm diameter PTK over a metal screen to generate severe surface irregularity and inhibit epithelial basement membrane regeneration. Corneas were treated with 0.8 mg/mL losartan in balanced salt solution (BSS) or BSS 50 mu L six times per day for six weeks after PTK. All corneas had slit lamp photography, with and without 1% fluorescein at two, four, and six weeks after PTK, and were analyzed using immunohistochemistry for the myofibroblast marker alpha-smooth muscle actin (alpha-SMA), keratocyte marker keratocan, mesenchymal cell marker vimentin, transforming growth factor (TGF)-beta 1, and collagen type IV.Results: Topical 0.8 mg/mL losartan six times a day significantly decreased anterior stromal alpha-SMA intensity units compared to BSS at six weeks after anterior stromal irregularity-inducing screened PTK (P = 0.009). Central corneal opacity, however, was not significantly different between the two groups. Keratocan, vimentin, TGF-beta 1, or collagen type IV levels in the anterior stroma were not significantly different between the two groups.Conclusions: Topical losartan effectively decreased myofibroblast generation after surface blast simulation irregular PTK. However, these results suggest initial maskingsmoothing PTK, along with adjuvant topical losartan therapy, may be needed to decrease corneal stromal opacity after traumatic injuries that produce severe surface irregularity.Translational Relevance: Topical losartan decreased scar-producing stromal myofibroblasts after irregular PTK over a metal screen but early smoothing of irregularity would also likely be needed to significantly decrease corneal opacity.
conferenceObject Cell biology of spontaneous persistent epithelial defects after photorefractive keratectomy in rabbits(2023) WILSON, Steven; SAMPAIO, Lycia; VILLABONA-MARTINEZ, Valeria; MICHAEL, Shiju; HILGERT, Guilherme; SANTHIAGO, Marcony- Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits(2024) VILLABONA-MARTINEZ, Valeria; DUTRA, Barbara A. L.; SAMPAIO, Lycia P.; SANTHIAGO, Marcony R.; WILSON, Steven E.The purpose of this study was to evaluate transforming growth factor beta (TGFI3) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFI33, myofibroblast marker alpha-smooth muscle actin (alpha-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFI31, TGFI32, or TGFI33 (each with alpha-SMA and vimentin) using isoform-specific antibodies. All three TGFI3 isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFI3 isoform co-localized with alpha-SMA of myofibroblasts, which could be TGFI3 isoform autocrine production by myofibroblasts or TGFI3-1, -2, and -3 binding to these myofibroblasts.
- Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits (vol 216, 108940, 2022)(2022) SAMPAIO, L. P.; HILGERT, G. S. L.; SHIJU, T. M.; SANTHIAGO, M. R.; WILSON, S. E.
- Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits(2021) SAMPAIO, Lycia Pedral; SHIJU, Thomas Michael; HILGERT, Guilherme S. L.; OLIVEIRA, Rodrigo Carlos de; DEDREU, JodiRae; MENKO, A. Sue; SANTHIAGO, Marcony R.; WILSON, Steven E.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 membraneendothelial 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,KATPase beta 1, 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 myofibroblastgenerated fibrosis.
- Topical losartan inhibits corneal scarring fibrosis and collagen type IV deposition after Descemet's membrane-endothelial excision in rabbits(2022) SAMPAIO, Lycia Pedral; HILGERT, Guilherme S. L.; SHIJU, Thomas Michael; MUEILLO, Sofia E.; SANTHIAGO, Marcony R.; WILSON, Steven E.The purpose of this study was to examine the effect of topical and/or oral angiotensin converting enzyme II inhibitor and TGF-beta signaling blocker losartan on corneal stromal fibrosis that developed in rabbit corneas after Descemetorhexis removal of central Descemet's membrane and corneal endothelium. Twenty-eight New Zealand white rabbits were included and either had 8 mm central Descemetorhexis or sham control surgery without Descemetorhexis in one eye. Groups of 4 eyes without Descemetorhexis were treated for one month with no medications, topical losartan or oral losartan. Groups of 4 eyes with Descemetorhexis were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical losartan and oral losartan for one month. Standardized slit lamp photos were obtained with central opacity intensity measured with ImageJ. The posterior fibrotic zone of corneas was measured on immunohistochemistry for alpha-smooth muscle actin (SMA) and keratocan using QuPath analysis. Collagen type IV expression in the posterior cornea was quantitated with ImageJ and duplex immunohistochemistry for collagen type IV and TGF beta-1. After Descemetorhexis, topical, but not oral, losartan decreased the intensity of central stromal opacity, reduced peripheral corneal scarring, and decreased alpha-smooth muscle actin myofibroblast fibrosis area compared to corneas that had Descemetorhexis and treatment with vehicles alone. Topical losartan decreased posterior stromal cellular, non-Descemet's membrane, collagen type IV production, that is likely stimulated by TGF beta as part of a negative regulatory feedback mechanism, compared to vehicle treatment at one month after Descemetorhexis. Topical losartan is likely to be effective in reducing corneal scarring fibrosis produced by traumatic injury, microbial infection, and some corneal diseases and surgeries.
- Corneal Opacity: Cell Biological Determinants of the Transition From Transparency to Transient Haze to Scarring Fibrosis, and Resolution, After Injury(2022) WILSON, Steven E.; SAMPAIO, Lycia Pedral; SHIJU, Thomas Michael; HILGERT, Guilherme S. L.; OLIVEIRA, Rodrigo Carlos dePURPOSE. 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 (TGF beta) 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 TGF beta entry and activity, and triggers TGF beta-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 TGF beta. 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.
- Topical Losartan and Corticosteroid Additively Inhibit Corneal Stromal Myofibroblast Generation and Scarring Fibrosis After Alkali Burn Injury(2022) SAMPAIO, Lycia Pedral; HILGERT, Guilherme S. L.; SHIJU, Thomas Michael; SANTHIAGO, Marcony R.; WILSON, Steven E.Purpose: To evaluate the efficacy of losartan and prednisolone acetate in inhibiting corneal scarring fibrosis after alkali burn injury in rabbits. Methods: Sixteen New Zealand White rabbits were included. Alkali injuries were produced using 1N sodium hydroxide on a 5-mm diameterWhatman #1 filter paper for 1 minute. Four corneas in each group were treated six times per day for 1 month with 50 mu L of (1) 0.2 mg/mL losartan in balanced salt solution (BSS), (2) 1% prednisolone acetate, (3) combined 0.2 mg/mL losartan and 1% prednisolone acetate, or (4) BSS. Area of opacity and total opacitywere analyzed in standardized slit-lamp photoswith ImageJ. Corneas in both groupswere cryofixed in Optimal cutting temperature (OCT) compound at 1 month after surgery, and immunohistochemistry was performed for alpha-smooth muscle actin (alpha-SMA) and keratocan or transforming growth factor beta 1 and collagen type IV with ImageJ quantitation. Results: Combined topical losartan and prednisolone acetate significantly decreased slit-lamp opacity area and intensity, as well as decreased stromal myofibroblast alpha-SMA area and intensity of staining per section and confinedmyofibroblasts to only the posterior stroma with repopulation of the anterior and mid-stroma with keratocan-positive keratocytes after 1 month of treatment. Corneal fibroblasts produced collagen type IV not associated with basement membranes, and this production was decreased by topical losartan. Conclusions: Combined topical losartan and prednisolone acetate decreased myofibroblast-associated fibrosis after corneal alkali burns that produced full-thickness injury, including corneal endothelial damage. Increased dosages and duration of treatment may further decrease scarring fibrosis. Translational Relevance: Topical losartan and prednisolone acetate decrease myofibroblast-mediated scarring fibrosis after corneal injury.