Keratocyte-Derived Myofibroblasts: Functional Differences With Their Fibroblast Precursors.

Purpose: In this study, we aim to elucidate functional differences between fibroblasts and myofibroblasts derived from a keratocyte lineage to better understand corneal scarring. Methods: Corneal fibroblasts, derived from a novel triple transgenic conditional KeraRT/tetO-Cre/mTmG mouse strain that allows isolation and tracking of keratocyte lineage, were expanded, and transformed by exposure to transforming growth factor (TGF)-ß1 to myofibroblasts. The composition and organization of a fibroblast-built matrix, deposited by fibroblasts in vitro, was analyzed and compared to the composition of an in vitro matrix built by myofibroblasts. Second harmonic generation microscopy (SHG) was used to study collagen organization in deposited matrix. Different extracellular matrix proteins, expressed by fibroblasts or myofibroblasts, were analyzed and quantified. Functional assays compared latent (TGF-ß) activation, in vitro wound healing, chemotaxis, and proliferation between fibroblasts and myofibroblasts. Results: We found significant differences in cell morphology between fibroblasts and myofibroblasts. Fibroblasts expressed and deposited significantly higher quantities of fibril forming corneal collagens I and V. In contrast, myofibroblasts expressed and deposited higher quantities of fibronectin and other non-collagenous matrix components. A significant difference in the activation of latent TGF-ß activation exists between fibroblasts and myofibroblasts when measured with a functional luciferase assay. Fibroblasts and myofibroblasts differ in their morphology, extracellular matrix synthesis, and deposition, activation of latent TGF-ß, and chemotaxis. Conclusions: The differences in the expression and deposition of extracellular matrix components by fibroblasts and myofibroblasts are likely related to critical roles they play during different stages of corneal wound healing.

Stromal matrix directs corneal fibroblasts to re-express keratocan after injury and transplantation.

Every tissue has an extracellular matrix (ECM) with certain properties unique to it - the tissue 'niche' - that are necessary for normal function. A distinct specific population of quiescent keratocan-expressing keratocytes populate the corneal stroma during homeostasis to maintain corneal function. However, during wound healing, when there is alteration of the niche conditions, keratocytes undergo apoptosis, and activated corneal fibroblasts and myofibroblasts attempt to restore tissue integrity and function. It is unknown what the fate of activated and temporary fibroblasts and myofibroblasts is after the wound healing process has resolved. In this study, we used several strategies to elucidate the cellular dynamics of corneal wound healing and the fate of corneal fibroblasts. We injured the cornea of a novel mouse model that allows cell-lineage tracing, and we transplanted a cell suspension of in vitro-expanded corneal fibroblasts that could be tracked after being relocated into normal stroma. These transplanted fibroblasts regained expression of keratocan in vivo when relocated to a normal stromal niche. These findings suggest that transformed fibroblasts maintain plasticity and can be induced to a keratocyte phenotype once relocated to an ECM with normal signaling ECM.

Primary cutaneous lymphoma in Argentina: a report of a nationwide study of 416 patients.

BACKGROUND: The aim of this study was to determine the relative frequency of primary cutaneous lymphoma (PCL) in Argentina according to the new World Health Organization (WHO)-European Organization for the Research and Treatment of Cancer (EORTC) classification system. METHODS: A total of 416 patients from 21 dermatology services were included during a 5-year period (2010-2015); these patients were classified using WHO-EORTC criteria. RESULTS: There were 231 (55.2%) males and 185 (44.8%) females; the male-to-female ratio was 1.35. The median age of the patients was 57 years (range, 0-90 years). Most patients were Caucasian (79%), and only 16% of patients were registered as Amerindian. Most patients (387/416, 93%) had cutaneous T-cell lymphoma (CTCL); 28 patients (6.7%) were diagnosed with cutaneous B-cell lymphoma (CBCL). The most frequent CTCL subtypes, in decreasing order of prevalence, were mycosis fungoides (MF), including its variants (75.7%); CD30+ primary cutaneous lymphoproliferative disorders (7.2%); and Sézary syndrome (SS) (3.1%). Cutaneous follicle center lymphoma was the most common CBCL subtype (2.9%). In the subset of patients ≤20 years of age, the most common condition was MF (57%), followed by extranodal NK-T nasal-type lymphoma (14%). CONCLUSIONS: This study revealed relatively higher rates of MF and lower rates of CBCL in Argentinean patients that have been reported in American and European countries.

Long-term follow-up of a supradescemetic keratoprosthesis in rabbits: an immunofluorescence study.

OBJECTIVE: To evaluate the long-term clinical and immunohistological outcome of two different non-penetrating keratoprosthesis (KPro) implanted in non-injured rabbit corneas. MATERIALS AND METHODS: Three rabbits underwent implantation of a pHEMA-MMA(34) synthetic cornea in the supradescemetic space, and PMMA synthetic corneas in the supradescemetic space and within the central stroma. Animals were followed for at least 24 months before euthanasia. Periodic evaluation was performed with slit-lamp examination and photography. At the end of the follow-up, histological examination including hematoxylin eosin staining and immunocharacterization against collagen IV, alpha smooth muscle actin (α-SMA) and macrophages was performed. RESULTS: The pHEMA-MMA(34) implant was not extruded, and remained transparent until the end of follow-up. This material did not induce any cell infiltration, corneal scarring or tissue remodeling in the surrounding stroma as shown by immunofluorescence. In contrast, synthetic corneas made of PMMA-induced myofibroblast differentiation, stromal remodeling and macrophage infiltration. This reaction was even more significant in the rabbit with the PMMA implant within the corneal stroma. CONCLUSION: pHEMA-MMA(34) was clinically biocompatible, and did not induce any inflammatory reaction or scarring when implanted in the supradescemetic space. This material showed more promising biocompatibility results than for PMMA, whether implanted within the central cornea stroma or in the supradescemetic space.

Estimation of intraocular pressure in rabbits with commonly used tonometers.

BACKGROUND AND OBJECTIVES: To validate accuracy and reproducibility of the Perkins tonometer, pneumatonometer, and Tono-Pen XL (Medtronic Solan, Jacksonville, FL) in estimating intraocular pressure (IOP) in rabbits. MATERIALS AND METHODS: IOP was increased from 5 to 50 mm Hg in 5-mm increments. Measurements were compared to readings of two digital manometers simultaneously measuring real IOP in the anterior chamber and vitreous cavity. Interobserver accuracy was evaluated using 4 eyes with the Perkins tonometer. RESULTS: The Perkins tonometer and Tono-Pen XL underestimated IOP and were more accurate at pressures less than 30 mm Hg. No statistically significant difference was found between real IOP and Tono-Pen XL readings. The pneumatonometer overestimated pressures in the low ranges but was accurate at pressures greater than 40 mm Hg. The Tono-Pen XL had more variability than the Perkins tonometer and pneumatonometer at high IOP. CONCLUSIONS: None of the tonometers are accurate or reproducible in estimating IOP in rabbits over the tested range. Pneumatonometry, although not very accurate, has the advantage of having acceptable variability.

A newly designed glaucoma drainage implant made of poly(styrene-b-isobutylene-b-styrene): biocompatibility and function in normal rabbit eyes.

OBJECTIVE: To report clinical evaluation, flow patency, and histopathological findings of a novel glaucoma drainage implant (GDI) made of poly(styrene-b-isobutylene-b-styrene) (SIBS) in rabbits. METHODS: In 16 normal eyes, the proximal end of the SIBS GDI was inserted into the anterior chamber while the distal end was placed in the subconjunctival space. A control group underwent implantation of a similarly designed silicone GDI. Slitlamp follow-up and intraocular pressure measurements were recorded. Flow patency was evaluated by injecting 0.01% fluorescein into the anterior chamber. Immunostaining against collagen IV, macrophages, and alpha smooth muscle actin was performed. RESULTS: Slitlamp examination suggested adequate biocompatibility. A low and diffuse bleb was observed in the SIBS group. All SIBS tubes were patent 6 months after insertion. Immunostaining demonstrated noncontinuous collagen deposition. No macrophages or myofibroblasts were visible around the SIBS tubes. In contrast, silicone induced collagen deposition and myofibroblast differentiation. CONCLUSION: This new GDI is clinically biocompatible in the rabbit and maintained 100% patency at 6 months. A remarkable difference was the absence of myofibroblasts in the surrounding tissue in the SIBS group. CLINICAL RELEVANCE: This novel GDI made of SIBS would prevent the feared complication of hypotony and will decrease the amount of subconjunctival fibrosis.

Corneal stroma regeneration in felines after supradescemetic keratoprosthesis implantation.

PURPOSE: To show corneal regeneration in 3 cats that underwent lamellar keratectomy (90%) depth during supradescemetic keratoprosthetic implantation. METHODS: Three 2-year-old cats that underwent spontaneous keratoprosthesis extrusion between 15 and 150 days after implanting a supradescemetic prosthesis into their right eyes were studied. Corneal structures and stroma thickness were evaluated by slit-lamp photographs, pachymetry, and confocal microscopy. Regenerated corneal epithelial cells, stroma matrix, and keratocyte morphology were studied with histology and transmission electron microscopy. Epithelial and stromal cell immunocharacterization was performed. RESULTS: Corneas progressively regained normal thickness and improved clarity within 40 to 60 days. Slit-lamp photographs and pachymetry showed gains in stromal thickness until 600 microm or more. In vivo confocal microscopy showed the restoration of normal epithelium and stroma in all cats. Corneal nerves were seen in the regenerated stroma of 2 cats. Immunostaining showed absent alpha-smooth muscle actin (SMA) expression and a keratin K3-expressing epithelium. Electron microscopy showed regeneration of normal epithelium with a well-formed basement membrane, organized corneal lamellae, and the presence of normal keratocytes. CONCLUSION: Felines are capable of regenerating corneal structures including epithelium and reinnervated stroma matrix after deep lamellar keratectomy. The use of feline models in corneal keratoprosthesis is therefore questionable.

Validation of the Barraquer tonometer for high intraocular pressure estimation.

PURPOSE: To compare the pneumatonometer and the Tono-Pen XL in a closed ex-vivo system in human eye bank eyes at high intraocular pressures (IOP) and evaluate the validity of high IOP measurements with the Barraquer tonometer. METHODS: Intraocular pressure was monitored by cannulation of the anterior chamber and vitreous cavity in eight human cadaver eyes (mean donor age: 77.3 +/- 4.9 years, range: 72 to 84 years). Intraocular pressure measurements were taken at 50, 65, and 90 mmHg with the Tono-Pen XL and pneumatonometer. Intraocular pressure was raised to 110 mmHg and then the eyes were deflated slowly until they reached 50 mmHg. Pressure readings with the Barraquer tonometer were recorded when the corneal tonometer interface reached the inner and outer rings. RESULTS: The Tono-Pen XL underestimated IOP, a tendency that was more evident at higher IOP In contrast, the pneumatonometer was more accurate and reliable at IOP of 50 and 65 mmHg but its readings underestimated IOP at 90 mmHg. The Barraquer tonometer used in this experiment accurately estimated high IOP A variability of 5.9 mmHg and 5.8 mmHg were recorded for the inner and outer ring, respectively. CONCLUSIONS: The Tono-pen XL is an inadequate instrument to assess pressures normally encountered during LASIK flap creation in an ex vivo model using human cadaver eyes. The pneumatonometer and the Barraquer tonometer are accurate instruments at high IOP; however, the pneumatonometer underestimated pressures around 90 mmHg.