RESUMEN
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.
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Córnea/patología , Lámina Limitante Posterior/lesiones , Endotelio Corneal/fisiología , Regeneración/fisiología , Cicatrización de Heridas/fisiología , Animales , Biomarcadores/metabolismo , Córnea/metabolismo , Queratocitos de la Córnea/metabolismo , Sustancia Propia/metabolismo , Proteínas del Ojo/metabolismo , Femenino , Fibrosis , Inmunohistoquímica , Conejos , Microscopía con Lámpara de Hendidura , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
O carcinoma espinocelular é uma neoplasia maligna dos ceratinócitos que tem como principal causa a exposição a raios ultravioletas. Os felinos constituem a espécie doméstica mais susceptível para o aparecimento desta neoplasia, sendo a face a região mais comum de acometimento, incluindo áreas do plano nasal, pina e pálpebras. No presente trabalho relata-se um felino, sem raça definida, fêmea, 12 anos de idade, 3kg, castrada, de pelagem branca, que foi atendida apresentando lesão extensa de caráter ulcerativo em região periocular de globo ocular esquerdo, com evolução de aproximadamente três meses, envolvendo ambas as pálpebras, superior e inferior. Após realização de avaliação clínica, os achados obtidos por meio da anamnese juntamente com o exame citológico, sugeriram como principal diagnóstico o carcinoma espinocelular. Para tratamento foi preconizado remoção cirúrgica, na qual demonstrou boa efetividade para tratamento dessa afecção. Após, o material foi enviado para análise histopatológica, sendo confirmado o diagnóstico. O paciente apresentou boa recuperação e evolução clínica do quadro, não apresentando nenhuma complicação no decorrer do tratamento. O prognóstico para este caso foi considerado bom, uma vez que não havia indícios de metástase no momento da realização dos exames complementares, e as margens da neoplasia se encontravam livres de células neoplásicas.
Squamous cell carcinoma is a malignant neoplasm of keratinocytes whose main cause is exposure to ultraviolet rays. The original felines are the domestic species most susceptible to the onset of this neoplasm, the face being the most common region of involvement, including areas of the nasal plane, pinna and eyelids. In the present study, we report a feline, mixed breed, female, 12 years old, 3kg, neutered, with white fur, who was treated for an enlarged ulcerative lesion in the periocular region of the left eyeball, with an evolution of approximately three months. involving both the upper and lower eyelids. After conducting a clinical valuation, the findings obtained through the anamnesis together with the cytological examination, suggested squamous cell carcinoma as the main diagnosis. For treatment before surgical removal, in the qualification good effectiveness to treat this condition. Afterwards, the material sent for histopathological analysis, the diagnosis being confirmed. The patient shows good recovery and clinical evolution of the condition, there is no complication or complication during the treatment. The prognosis for this case was considered good, since there was no evidence of metastasis at the time of the complementary tests, and as the neoplasia margins were free of neoplastic cells.
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Animales , Gatos , Neoplasias , Cirugía Veterinaria , Carcinoma de Células Escamosas , Enfermedades de los Gatos , Queratocitos de la CórneaRESUMEN
AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.
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Galectina 1/metabolismo , Galectina 3/metabolismo , Queratocono/metabolismo , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Colágeno/metabolismo , Conjuntiva/metabolismo , Córnea/metabolismo , Queratocitos de la Córnea/efectos de los fármacos , Queratocitos de la Córnea/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Citocinas/metabolismo , Femenino , Humanos , Queratocono/tratamiento farmacológico , Masculino , Fármacos Fotosensibilizantes/farmacología , Estudios Prospectivos , Riboflavina/farmacología , Lágrimas/metabolismo , Rayos UltravioletaRESUMEN
Cell and developmental processes are complex, and profoundly dependent on spatial relationships that change over time. Innovative educational or teaching strategies are always needed to foster deep comprehension of these processes and their dynamic features. However, laboratory exercises in cell and developmental biology at the undergraduate level do not often take into account the time dimension. In this article, we provide a laboratory exercise focused in cell migration, aiming to stimulate thinking in time and space dimensions through a simplification of more complex processes occurring in cell or developmental biology. The use of open-source tools for the analysis, as well as the whole package of raw results (available at http://github.com/danielprieto/keratocyte) make it suitable for its implementation in courses with very diverse budgets. Aiming to facilitate the student's transition from science-students to science-practitioners we propose an exercise of scientific thinking, and an evaluation method. This in turn is communicated here to facilitate the finding of common caveats and weaknesses in the process of producing simple scientific communications describing the results achieved. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):475-482, 2017.
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Escamas de Animales/citología , Movimiento Celular , Queratocitos de la Córnea/citología , Biología Evolutiva/educación , Evaluación Educacional , Peces , Laboratorios/economía , Animales , Técnicas de Cultivo de Célula/economíaRESUMEN
PURPOSE: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. METHODS: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. RESULTS: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. CONCLUSION: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.
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Queratocitos de la Córnea/efectos de los fármacos , Queratocitos de la Córnea/efectos de la radiación , Fármacos Fotosensibilizantes/farmacología , Riboflavina/farmacología , Rayos Ultravioleta , Análisis de Varianza , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Colágeno/farmacología , Sustancia Propia/citología , Reactivos de Enlaces Cruzados/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Formazáns , Humanos , Necrosis , Estadísticas no Paramétricas , Sales de Tetrazolio , Factores de TiempoRESUMEN
ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.
RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.
Asunto(s)
Humanos , Riboflavina/farmacología , Rayos Ultravioleta , Fármacos Fotosensibilizantes/farmacología , Queratocitos de la Córnea/efectos de los fármacos , Queratocitos de la Córnea/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Análisis de Varianza , Técnica del Anticuerpo Fluorescente , Colágeno/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Sustancia Propia/citología , Reactivos de Enlaces Cruzados/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Formazáns , NecrosisRESUMEN
The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or "haze". Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes in corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor ß (TGF-ß) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts produce critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte/corneal fibroblast contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored when the EBM is completely regenerated, myofibroblasts are deprived of TGFß and undergo apoptosis, and the keratocytes re-occupy the anterior stroma and reabsorb disordered extracellular matrix. The aim of this review is to highlight factors involved in the generation of stromal haze and its subsequent removal.
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Lesiones de la Cornea/patología , Opacidad de la Córnea/patología , Sustancia Propia/patología , Epitelio Corneal/patología , Animales , Apoptosis/fisiología , Membrana Basal/patología , Lesiones de la Cornea/metabolismo , Queratocitos de la Córnea/metabolismo , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/fisiopatología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Cicatrización de Heridas/fisiologíaRESUMEN
The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.
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Membrana Basal/metabolismo , Moléculas de Adhesión Celular/metabolismo , Queratocitos de la Córnea/metabolismo , Epitelio Corneal/lesiones , Lesiones Oculares/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio , Sustancia Propia/citología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Persona de Mediana Edad , Donantes de Tejidos , Regulación hacia Arriba/fisiología , Cicatrización de HeridasRESUMEN
La queratitis micótica es una infección de curso subagudo que genera inflamación y ulceración de la córnea. La importancia de las queratitis micóticas en la práctica oftalmológica radica en su dificultad diagnóstica y terapéutica, la falla en el reconocimiento temprano del hongo y la instauración tardía del tratamiento adecuado incrementa la infiltración y ulceración del estroma corneal, lo que puede llegar a tener consecuencias graves en la integridad del globo ocular. Se propuso determinar la frecuencia de ulceras corneales de etiología fúngica en el Hospital Universitario de Caracas durante el periodo julio 2008-julio2012. Se diseñó un estudio retrospectivo, descriptivo, basado en la revisión de las 41 historias médicas de todos los pacientes con cultivo y/o examen directo positivo para hongos en muestras de úlceras corneales. El cultivo fue positivo en 97,56%, representando Fusarium spp el 37,5%, F. solani 20%, F. oxysporum 12,5%, Aspergillus spp 7,5%, y A. terreus, A. fumigatus, A. candidus, Penicillium spp, Candida spp, C. parapsilosis, P. boydii, Curvularia sp y Cladosporium sp representaron 2,5% cada uno. Al 58,54% se les realizo tratamiento quirúrgico. De 41 pacientes 73,17%, eran de sexo masculino, el grupo más afectado estuvo comprendido entre 20 y 59 años representando el 75,61%. El 58,54% procedía de zonas urbanas y el 41,56% de zona rural, y el 78,05% de la población atendida pertenecía al interior del país. Un 48,78% tuvo un evento traumático mientras que el 51,22% no y 87,80% pacientes ya habían recibido tratamiento previo. A pesar de los avances en diagnóstico y tratamiento para las queratomicosis siguen constituyendo un problema en los países subdesarrollados donde el diagnóstico tardío es directamente proporcional al grado de mejoría del paciente.
Fungal keratitis is a subacute infection that produces inflammation and ulceration of the cornea. The importance of fungal keratitis in ophthalmology practice lies in its diagnostic and therapeutic difficulties, failure of the fungus early recognition and appropriate treatment of late-onset increases infiltration and ulceration of the corneal stroma, which can have serious consequences in the integrity of the eyeball. Proposed to determine the frequency of fungal etiology corneal ulcers in Caracas University Hospital during the period July 2008- july 2012. We designed a retrospective descriptive study, based on a review of 41 medical records of all patients with culture and / or direct examination positive for fungal corneal ulcers samples. The culture was positive in 97.56%, representing 37.5% Fusarium spp, 20% F. solani, 12.5% F. oxysporum, Aspergillus spp 7.5%, and A. terreus, A. fumigatus, A. candidus, Penicillium spp, Candida spp, C. parapsilosis, P. boydii, Curvularia sp and Cladosporium sp accounted for 2.5% each. At 58.54% received surgical treatment. Of 41 patients 73.17% were males, the group most affected was between 20 and 59 years old representing 75.61%. , 58.54% were from urban areas and 41.56% in rural area, and 78.05% of the population served came from inside the country. A 48.78% had a traumatic event while 51.22% and 87.80% no patients had received previous treatment. Despite advances in diagnosis and treatment for keratomycosis remains a problem in developing countries where late diagnosis is directly proportional to the degree of patient improvement.
Asunto(s)
Humanos , Masculino , Femenino , Aspergillus , Úlcera de la Córnea , Queratocitos de la Córnea , Fusarium , QueratitisRESUMEN
PURPOSE: To investigate the efficacy and safety of Genipin and UV-riboflavin crosslinking (UV-CLX) in corneal crosslinking. METHODS: Porcine eyes were separated in groups for each crosslinker, genipin 0.25% UV-CLX (clinical crosslinking procedure), glutaraldehyde 0.1% (gold standard crosslinker), and control eyes. Intraocular pressure (IOP) was continuously monitored by a pressure sensor cannulated to the anterior chamber and the volume was changed. The changes in ocular pressure as a function of change of the ocular volume were evaluated. Ocular rigidity was calculated as the exponential of polynomial quadratic fit. Endothelial damage was evaluated in a viability assay with alizarin red staining as the changes in cell counts. RESULTS: Significant changes in IOP were observed in the globes were the cornea was stiffened with genipin and UV-CLX treatment (volume 200 µl: Genipin 19.4 mmHg, UVCRX 18.8 mmHg, glutaraldehide 23.9 mmHg, versus control 14.7 mmHg, and 400 µl genipin 31.5 mmHg, UV-CLX 26.0 mmHg, glutaraldehide 37.3 mmHg versus control 18.7 mmHg). The mean ocular ridigity coefficient was genipin 0.0078 mmHg/µl, UV-CLX 0.0065 mmHg/µl, glutaraldehide 0.0092 mmHg/µl, and 0.0046 mmHg/µl for control eyes. Endothelial cell damage was 5.9±1.8% (control), 10.3±1.7% (UV-CLX), 9.4±1.5% (Genipin 0.25%), and 40.1±6.2% (glutaraldehide). Some granules were observed in the UV-CLX group. Reduction of keratocites was observed in the UV CRX crosslinking. CONCLUSIONS: Corneal crosslinking was similar between UV-CLX and genipin with minimal toxicity to endothelial cells. Stiffened corneas by any method induced substancially higher IOP elevation when ocular volume is increased.