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1.
Invest Ophthalmol Vis Sci ; 61(3): 8, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32150250

RESUMO

Purpose: Thymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine that initiates allergic inflammatory reaction between epithelial and dendritic cells (DCs). miR-19b was reported to suppress TSLP expression. The present study aimed to examine miR-19b expression, regulation, and function in allergic conjunctivitis (AC). Methods: A murine model of experimental AC was induced in BALB/c mice by short ragweed pollen. The serum, eye balls, conjunctiva, and cervical lymph nodes (CLN) were used for the study. Gene expression was determined by RT-PCR, whereas protein production and activation were evaluated by immunostaining, ELISA, and Western blotting. Results: In the murine AC model, miR-19b was aberrantly downregulated, whereas the levels of TSLP and p-STAT3, as well as the number of CD11c+ pSTAT3+ DCs were increased. Moreover, Th2 inflammatory cytokine expression was significantly increased. These severe phenotypes could be counteracted by either applying exogenous miR-19b mimic microRNAs or the JAK/STAT inhibitor CYT387. Moreover, overexpression of miR-19b repressed p-STAT3 expression and the number of CD11c+ cells in AC eye and CLN tissues. Conclusions: These findings suggested that miR-19b reduced ocular surface inflammation by inhibiting Stat3 signaling via TSLP downregulation in a murine AC model. Moreover, the present study further demonstrated the clinical potential of applying miR-19b and anti-JAK/STAT therapies in the treatment of AC.


Assuntos
Conjuntivite Alérgica/genética , Janus Quinases/fisiologia , MicroRNAs/genética , Fatores de Transcrição STAT/fisiologia , Animais , Antígenos de Plantas , Antígenos CD11/metabolismo , Vértebras Cervicais , Túnica Conjuntiva/metabolismo , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/metabolismo , Córnea/metabolismo , Citocinas/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Janus Quinases/antagonistas & inibidores , Linfonodos/metabolismo , Camundongos Endogâmicos BALB C , MicroRNAs/biossíntese , Fenótipo , Extratos Vegetais , Fatores de Transcrição STAT/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
2.
Invest Ophthalmol Vis Sci ; 61(2): 7, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031579

RESUMO

Purpose: Confirm that the corneal endothelial pump uses a lactate-coupled water efflux mechanism. Methods: Corneal thickness, lactate efflux, and stromal [lactate] were measured in de-epithelialized swollen and nonswollen ex vivo-mounted rabbit corneas perfused with bicarbonate-rich and bicarbonate-free Ringers, ouabain, or acetazolamide to determine if the relationships among these parameters were similar to previous data using intact corneas. The role of barrier function was tested by perfusion with calcium-free EGTA. Predictions of [lactate] in endothelial dystrophy were examined in the Slc4a11 knock out mouse. Results: De-epithelialized corneal swelling, lactate efflux, and stromal [lactate] in response to bicarbonate-free Ringers, ouabain, and acetazolamide perfusion had the same relationship as in intact corneas. The absolute amount of lactate efflux and stromal [lactate] in the de-epithelialized corneas was about half of intact corneas. De-epithelialized, swollen corneas deswelled fully with bicarbonate-rich, partially in the presence of acetazolamide, but continued to swell with bicarbonate-free or ouabain. The relationship among corneal thickness, lactate efflux, and [lactate] was the same as with nonswollen de-epithelialized corneas. In intact corneas swollen by perfusion with calcium-free EGTA, the relationship between swelling and lactate flux was the inverse of control corneas. The relationship between corneal swelling and [lactate] of intact corneas exposed to ouabain, but perfused with 7 mM lactate to simulate aqueous humor, was the same as without lactate. Corneal [lactate] in Slc4a11 knock out was twice that of wild type. Conclusions: The corneal endothelial pump works via a lactate efflux mechanism that requires an intact osmotic barrier.


Assuntos
Epitélio Posterior/metabolismo , Ácido Láctico/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico Ativo/fisiologia , Córnea/metabolismo , Edema da Córnea/fisiopatologia , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Camundongos Knockout , Ouabaína/farmacologia , Coelhos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Simportadores/metabolismo
3.
Invest Ophthalmol Vis Sci ; 61(2): 25, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084267

RESUMO

Purpose: To determine the role of autophagy in the innate immune response to fungal keratitis (FK) caused by Aspergillus fumigatus infection. Methods: Corneal samples obtained from patients and mice with FK were visualized via transmission electron microscopy (TEM). Autophagy-related proteins LC3B-II, Beclin-1, LAMP-1, and p62 in A. fumigatus-infected corneas of C57BL/6 mice were tested by Western blot. After treatment with autophagy inhibitors 3-methyladenine (3-MA), chloroquine (CQ), or inducer rapamycin, autophagy-related proteins were detected by Western blot. Corneas were photographed with slit lamp microscopy and pathological changes were observed by hematoxylin and eosin staining. Polymorphonuclear neutrophilic leukocytes (PMNs) were assessed by immunofluorescent staining and observed under TEM. The levels of CXCL-1, IL-1ß, HMGB1, IL-18, TNF-α, and IL-10 were tested by reverse transcription polymerase chain reaction and Western blot. The quantification of fungal loads was detected and photographed. Results: The accumulation of autophagosomes in corneas of patients and mice with FK was observed with TEM. The expression of LC3B-II, Beclin-1, and LAMP-1 was elevated in corneas after fungal infection, whereas p62 was reduced. Treatment with 3-MA or CQ upregulated clinical scores, pathological changes, and the expression of CXCL-1, IL-1ß, HMGB1, IL-18, and TNF-α except IL-10. The morphology of PMNs was changed and PMN recruitment was increased in mice corneas treated with 3-MA or CQ, whereas rapamycin reduced the inflammatory response to keratitis. These results were statistically significant. Conclusions: A. fumigatus infection increases the expression of autophagy in corneas. Autophagy plays an anti-inflammatory role in the innate immune response to A. fumigatus keratitis.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Autofagia/imunologia , Infecções Oculares Fúngicas/imunologia , Imunidade Inata/imunologia , Ceratite/imunologia , Animais , Antifúngicos/farmacologia , Aspergilose/metabolismo , Aspergilose/patologia , Autofagia/efeitos dos fármacos , Biomarcadores/análise , Córnea/metabolismo , Córnea/patologia , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/patologia , Ceratite/metabolismo , Ceratite/patologia , Camundongos , Camundongos Endogâmicos C57BL
4.
Invest Ophthalmol Vis Sci ; 61(2): 46, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32106295

RESUMO

Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). Methods: In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro- and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3-/-) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. Results: Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3-/- than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3-/- than B6 mice. Conclusions: Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.


Assuntos
Córnea/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Ceratite Herpética/metabolismo , Animais , Neovascularização da Córnea/patologia , Opacidade da Córnea/patologia , Substância Própria/metabolismo , Herpesvirus Humano 1 , Ceratite Herpética/patologia , Ceratite Herpética/virologia , Camundongos , Camundongos Endogâmicos C57BL
5.
AAPS PharmSciTech ; 21(3): 87, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32016607

RESUMO

This study aims to evaluate the effect of different formulation variables (surfactant type and HLB value) adopting full factorial design (51. 21) using coacervation phase technique on in vitro characterization of dorzolamide hydrochloride (DZ)-loaded proniosomal gels, namely, entrapment efficiency percentage (EE%), vesicle size distribution, polydispersion index (PDI), and in vitro DZ release. The optimum formula F2 with a desirability value of 0.937 composed of 40 mg DZ, 500 mg span 60, 500 mg of L-α-Lethicin, and 55.5 mg cholesterol showing EE% of 84.5 ± 1.5%, PS of 189.5 ± 35.76 nm with PDI 0.8 ± 0.28 and 58.51% ± 1.00 of DZ released after 8 h was further evaluated using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The effect of gamma sterilization on transcorneal permeation and stability of DZ from the selected formulation (F2) revealed that F2 was significantly tolerable, stable, and competent to corneal permeation confirmed by histological examination, confocal laser microscopy, and intraocular pressure (IOP) measurement. Significant corneal bioavailability was attained from formula F2 (370.6 mg. h/m) compared to the market product Trusopt® eye drops (92.59 mg. h/ml) following IOP measurement, thereby proniosomal gels could be considered as tolerable and competent ocular platforms for improving the transcorneal permeation of DZ.


Assuntos
Córnea/metabolismo , Glaucoma/tratamento farmacológico , Sulfonamidas/administração & dosagem , Tiofenos/administração & dosagem , Animais , Composição de Medicamentos , Estabilidade de Medicamentos , Raios gama , Géis/química , Lipossomos/química , Masculino , Permeabilidade , Coelhos , Esterilização , Sulfonamidas/farmacocinética , Tiofenos/farmacocinética
6.
Ocul Immunol Inflamm ; 28(3): 384-390, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-31120777

RESUMO

We studied the production of PGE2 by human conjunctival and corneal cells in response to inflammation, and reduction of inflammation with non-steroidal anti-inflammatory drugs. Primary cultures of human conjunctival epithelial cells, fibroblasts, corneal epithelial cells, and keratocytes were incubated with IL-4 and TNF-α. PGE2 and COX-2 levels were analyzed. Effects of anti-inflammatory and anti-immune drugs on PGE2 production were also investigated. IL-4 and TNF-α induced the generation of PGE2 and COX-2 in conjunctival and corneal cells. Epithelial PGE2 production was significantly lower than in keratocytes and fibroblasts, which was down-regulated by aspirin. IL-4 and TNF-α enhanced the inflammatory response via prostaglandin production which contributed to ocular surface inflammation. Prostaglandin production was higher in stromal cells than epithelial cells. These results suggest that the epithelial barrier disruption may contribute to ocular allergic inflammation by the PGE2 production from stromal cells. Moreover, NSAIDs were effective in suppressing PGE2 production in our experiment.


Assuntos
Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Ceratócitos da Córnea/metabolismo , Citocinas/farmacologia , Dinoprostona/biossíntese , Células Epiteliais/metabolismo , Tacrolimo/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Western Blotting , Células Cultivadas , Túnica Conjuntiva/citologia , Córnea/citologia , Ceratócitos da Córnea/citologia , Ceratócitos da Córnea/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Imunossupressores/farmacologia , Interleucina-4/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
7.
Cornea ; 39(1): 132-135, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31403529

RESUMO

PURPOSE: To describe a new method to distinguish between normal versus lipid-deficient dry eye using a Tear Film Imager (TFI). METHODS: Two groups of study subjects, controls versus lipid-deficient dry eye, were tested using the TFI. This instrument provides an accurate measurement of the thickness and spatial distribution of the muco-aqueous and lipid layers of the tear film. The nanometer thickness resolution of the TFI enables the creation of detailed maps of the lipid layer thickness (LLT) across the corneal surface. These maps are captured with a large field of view of 6.5 mm diameter. RESULTS: A LLT map taken at 1 second from a blink end in the controls appears uniform, whereas a nonuniform layer was measured in the lipid-deficient dry eye. Lipid map uniformity can quantify the spatial variation of lipid across the cornea. A case study showed the ability to distinguish between controls [lipid map uniformity (LMU) = 14 nm] and lipid-deficient dry eye (LMU = 125 nm) through characterization of the LLT distribution. CONCLUSIONS: High-resolution lateral LLT maps demonstrate the significance of the lipid layer uniformity, which may play an important role in maintaining tear film health. LLT maps and the quantitative LMU could be used to diagnose and treat patients with dry eye.


Assuntos
Córnea/metabolismo , Técnicas de Diagnóstico Oftalmológico/instrumentação , Síndromes do Olho Seco/diagnóstico , Metabolismo dos Lipídeos , Lipídeos/análise , Lágrimas/química , Piscadela/fisiologia , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/fisiopatologia , Desenho de Equipamento , Humanos , Reprodutibilidade dos Testes
8.
Invest Ophthalmol Vis Sci ; 60(15): 5035-5044, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31800960

RESUMO

Purpose: To compare the changes in human tear proteome and clinical effects following topical cyclosporine A (CsA) 0.05% or diquafosol tetrasodium (DQS) 3% treatment of dry eye disease (DED), and to identify biomarkers for determining disease severity and treatment effectiveness in DED. Methods: A total of 18 patients were diagnosed with non-Sjögren DED. Nine patients in each group were treated with topical CsA 0.05% or DQS 3% for 4 weeks. Tear samples were collected after evaluation of tear breakup time, corneal and conjunctival erosion staining, and results of Schirmer's test 1 before and after treatment. Proteomes were characterized using liquid chromatography mass spectrometry, and proteins exhibiting a fold change >1.5 or <0.67 (P < 0.05) were considered differentially expressed (DEP). Results: A total of 794 proteins were identified, with no significant difference observed between pretreatment and posttreatment conditions. Proteomic analysis identified 54 and 106 DEPs between treatment groups (CsA and DQS, respectively), with gene ontology analysis indicating that both treatments enhanced innate and adaptive immune responses and cellular detoxification. Protein-network analysis showed that inflammation associated with the immune response was primarily responsible for the therapeutic process in both groups. Conclusions: These results provide insight into the broad scope of changes at the ocular surface in DED and indicated that although both drugs improved the clinical parameters, the activated tear-specific biomarkers differed significantly between treatments. Our findings suggest that the DEPs identified here and those correlated with the clinical parameters might represent candidate biomarkers for DED.


Assuntos
Ciclosporina/administração & dosagem , Síndromes do Olho Seco/tratamento farmacológico , Polifosfatos/administração & dosagem , Proteoma/metabolismo , Lágrimas/metabolismo , Nucleotídeos de Uracila/administração & dosagem , Administração Tópica , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Córnea/metabolismo , Córnea/patologia , Relação Dose-Resposta a Droga , Síndromes do Olho Seco/metabolismo , Feminino , Seguimentos , Humanos , Imunossupressores/administração & dosagem , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas/administração & dosagem , Estudos Prospectivos , Método Simples-Cego , Lágrimas/efeitos dos fármacos , Resultado do Tratamento
9.
Nat Commun ; 10(1): 5678, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831729

RESUMO

As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8+ cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8+ neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8+ cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8+ sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8+ cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.


Assuntos
Córnea/metabolismo , Nociceptividade/fisiologia , Células Receptoras Sensoriais/metabolismo , Substância P/metabolismo , Canais de Cátion TRPV/metabolismo , Sensação Térmica/fisiologia , Animais , Temperatura Baixa , Síndromes do Olho Seco , Regulação da Expressão Gênica , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/genética , Tamoxifeno/farmacologia
10.
Medicine (Baltimore) ; 98(51): e18256, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31860972

RESUMO

BACKGROUND: We compared the clinical outcomes of accelerated corneal collagen crosslinking (CXL) and 5% NaCl hypertonic saline (HS) for the treatment of symptomatic bullous keratopathy (BK). METHODS: A randomized controlled trial was held at Department of Ophthalmology, Tokyo Dental College Ichikawa General Hospital, Chiba, Japan. Twenty-three eyes of 23 consecutive patients with symptomatic BK were enrolled. The etiology of BK included pseudophakic BK, previous keratoplasty, previous endotheliitis, previous glaucoma surgery, trauma, herpes infection, as well as unknown causes. Eleven eyes received epi-off accelerated CXL (with epithelial abrasion and 18 mW/cm ultraviolet A irradiation for 5 minutes) and 12 eyes received HS instillation. In addition to the usual ophthalmic examination, the best-corrected visual acuity (BCVA) and central corneal thickness (CCT) were determined. The CCT was measured using anterior segment optical coherence tomography before and up to 6 months after treatments. Subjective symptoms of pain, blurred vision, photophobia, and irritation were also recorded. RESULTS: The follow-up was completed for all patients in the CXL group. However, 6 patients in the HS group requested CXL treatments after 3 months. The BCVA was not significantly changed during the study periods in both groups. The CCT was significantly thinner in the CXL group compared to the HS group at 1 and 6 months (P = .015 and 0.144, respectively). Among the subjective symptoms recorded, irritation was significantly lower in the CXL group at 1 month (P = .013). CONCLUSIONS: Accelerated CXL may produce transient improvement in pain and corneal edema in patients with BK.


Assuntos
Colágeno/metabolismo , Doenças da Córnea/terapia , Reagentes para Ligações Cruzadas/uso terapêutico , Solução Salina Hipertônica/uso terapêutico , Idoso , Córnea/metabolismo , Córnea/patologia , Doenças da Córnea/patologia , Feminino , Humanos , Masculino , Fotoquimioterapia/métodos , Riboflavina/uso terapêutico , Raios Ultravioleta
11.
Appl Opt ; 58(29): 7987-7995, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31674351

RESUMO

Dry eye (DE) disease is a multifactorial disease of the outer ocular surface characterized by several ocular symptoms and mainly by tear film instability. We have developed an optical imaging system, the tear film imager (TFI), which is the first instrument that can directly image the muco-aqueous tear layer physical dimension in vivo and evaluate its parameters in a noninvasive mode with nanometer axial resolution. This instrument provides quantified information about many attributes of the tear film, including muco-aqueous layer thickness, lipid layer thickness, thickness change rate, and the break-up time. The TFI performances are based on simultaneous acquisition of large field of view (FOV) imagery and fast spectrometric measurement of the interference from the thin tear film sublayers. Herein, after describing the instrument and the methodology of the measurements, we use a tear film mock-up to quantify device accuracy (2.2 nm) and repeatability (0.25 nm standard deviation). In conclusion, we present a new technology for the assessment of the tear film with an unprecedented axial resolution and excellent accuracy and reproducibility.


Assuntos
Síndromes do Olho Seco/diagnóstico por imagem , Síndromes do Olho Seco/metabolismo , Lipídeos/química , Lágrimas/química , Córnea/metabolismo , Desenho de Equipamento , Humanos , Pessoa de Meia-Idade
12.
Biomater Sci ; 7(12): 5506-5515, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31667480

RESUMO

The regeneration of the epithelium, covering the avascular cornea, involves the processes of differentiation, proliferation and migration of cells originating from the corneal epithelial stem cells. We ask the question if these energy-consuming processes can be fueled by the physiological, inorganic polyphosphate (polyP), the main energy storage/donor molecule in the extracellular space. The ex vivo results reveal that addition of polyP, in the form of soluble Na-polyP, to the culture medium elicits a strong stimulatory effect on cell viability/growth and migration of corneal epithelial cells. Microscopic analyses of partially denuded cornea specimens show that in the presence of polyP, but not in polyP-free controls, cells are migrating centripetally from the corneal limbus towards the corneal center. In vitro experiments using human corneal epithelial cells reveal an even stronger stimulatory effect on growth rates by calcium-polyP microparticles (Ca-polyP-MP) that mimic the physiological form of polyP in the platelets compared to particle-free Na-polyP, most likely by the transformation of Ca-polyP-MP into a biologically active coacervate. The increase in cell proliferation is paralleled with an increased expression of mucin 1. Our results suggest that polyP, in the form of the water-soluble Na-polyP, has the potential to be applicable as a biomimetic tear fluid to maintain and restore the integrity of the corneal epithelium.


Assuntos
Cálcio/química , Córnea/citologia , Meios de Cultura/química , Mucina-1/metabolismo , Polifosfatos/farmacologia , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Córnea/efeitos dos fármacos , Córnea/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Polifosfatos/química , Regeneração , Solubilidade , Tecidos Suporte
13.
Drug Deliv ; 26(1): 1235-1242, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31752553

RESUMO

Objective: To prepare everolimus nanoformulations and increase their solubility to suit their application in the eye. Methods: The everolimus micelles was prepared by thin film dispersion method using Tween-80 (P80) and polyoxyethylene stearate (P40S) as carriers. In addition, the everolimus nanosuspension was prepared by injection method using poloxamer 407 (P407), hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA) as stabilizers. It was characterized in terms of particle size, PDI and encapsulation efficiency or drug loading. The in vitro release and in vitro rabbit scleral permeability characteristics were investigated, and the pharmacokinetics of anterior chamber drug in rabbit eyes were studied. Results: The average particle size of the micelles was (8.74 ± 0.21) nm, the encapsulation efficiency and drug loading were (90.12 ± 1.18)% and (2.14 ± 0.028)%, while the average particle size of the nanosuspension was (156.47 ± 1.10) nm, and the drug loading was (16.51 ± 0.21)%, respectively. Both in vitro release and rabbit scleral permeation models were consistent with the Higuchi equation. The pharmacokinetic experiments of aqueous humor showed that area under the curve of everolimus nanosuspension was about 3 times higher than that of micelles. Micelles could be achieved in the eye and maintained for a long time. Conclusion: The preparation of everolimus micelles or nanosuspension for eye are suitable for ocular administration and expected to be new dosage form for corneal transplantation immunological rejection or other ocular disease.


Assuntos
Córnea/efeitos dos fármacos , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Everolimo/administração & dosagem , Imunossupressores/administração & dosagem , Nanopartículas/química , Esclera/efeitos dos fármacos , Administração Oftálmica , Animais , Córnea/metabolismo , Liberação Controlada de Fármacos , Everolimo/farmacocinética , Imunossupressores/farmacocinética , Micelas , Tamanho da Partícula , Permeabilidade , Coelhos , Esclera/metabolismo
14.
Int J Exp Pathol ; 100(5-6): 330-336, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777145

RESUMO

One of the most important causes of visual loss (blindness) is glaucoma, which occurs due to the degeneration of the ganglion cells in retina. It has been shown that hydrogen sulphide (H2 S) acts an antioxidant, neuroprotective and neuromodulator and provides protection against oxidative stress and apoptosis. This study aims to examine through which apoptotic pathway H2 S acts in experimental glaucoma model. Twenty-two male wistar albino rats were used in this study. Group 1 (n = 6, control group): Intravitreal saline was given in the third week without inducing ocular hypertension (OHT) with laser photocoagulation. Group 2 (n = 8): After the induction of OHT with laser photocoagulation, intravitreal saline was given in the third week. Group 3 (n = 8): After the induction of OHT with laser photocoagulation, intravitreal H2 S's donor sodium hydrosulphide (NaSH) 100 nmol/L was given in the third week. At the end of the 6th week, the eyes of the rats were sacrified under anaesthesia and extracted and then routine tissue follow-up was undertaken. Besides haematoxylin & eosin (H&E) staining, Bax, Bcl-2, p53 and caspase-3 activation were examined immunohistochemically in the retina and the cornea. This showed that ocular hypertension caused apoptosis through the intrinsic pathway, due to Bax and caspase-3 activation, in both retina and cornea, and that this led to DNA damage due to p53 activation. Also, we found that H2 S exposure in glaucoma distinctly suppressed Bax, caspase-3 and p53 activations in retina but that it has a limited effect on the cornea. According to these results, glaucoma caused apoptosis in the retina through intrinsic pathway, and the damage to the retina could be compensated partially by H2 S but would have limited on the cornea.


Assuntos
Apoptose/efeitos dos fármacos , Córnea/diagnóstico por imagem , Glaucoma/tratamento farmacológico , Sulfeto de Hidrogênio/farmacologia , Substâncias Protetoras/farmacologia , Retina/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Córnea/metabolismo , Córnea/fisiopatologia , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Sulfeto de Hidrogênio/administração & dosagem , Sulfeto de Hidrogênio/uso terapêutico , Injeções Intravítreas , Masculino , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/uso terapêutico , Ratos , Ratos Wistar , Retina/metabolismo , Retina/fisiopatologia
15.
Dis Markers ; 2019: 8781524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781308

RESUMO

Background: Sclerocornea is a rare congenital disorder characterized with the opacification of the cornea. Here, we report a nonconsanguineous Chinese family with multiple peripheral sclerocornea patients spanning across three generations inherited in an autosomal dominant manner. Methods: This is a retrospective case series of a peripheral sclerocornea pedigree. Comprehensive ophthalmic examinations were conducted and assessed on 14 pedigree members. Whole-exome sequencing was used to identify the genetic alterations in the affected pedigree members. Lymphoblastoid cell lines (LCLs) were established using blood samples from the family members. Functional tests were performed with these cell lines. Results: Six affected and eight unaffected members of a family with peripheral sclerocornea were examined. All affected individuals showed features of scleralization over the peripheral cornea of both eyes. Mean horizontal and vertical corneal diameter were found significantly decreased in the affected members. Significant differences were also observed on the mean apex pachymetry between affected and unaffected subjects. These ophthalmic parameters did not resemble that of cornea plana. A RAD21C1348T variant was identified by whole-exome sequencing. Although this variant causes RAD21 R450C substitution at the separase cleavage site, cells from peripheral sclerocornea family members had no mitosis and ploidy defects. Conclusion: We report a family of peripheral sclerocornea with no association with cornea plana. A RAD21 variant was found cosegregating with peripheral sclerocornea. Our results suggest that RAD21 functions, other than its cell cycle and chromosome segregation regulations, could underline the pathogenesis of peripheral sclerocornea.


Assuntos
Biomarcadores/análise , Proteínas de Ciclo Celular/genética , Córnea/anormalidades , Córnea/patologia , Doenças da Córnea/genética , Doenças da Córnea/patologia , Proteínas de Ligação a DNA/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Criança , Córnea/metabolismo , Feminino , Seguimentos , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Prognóstico , Subunidades Proteicas , Estudos Retrospectivos , Adulto Jovem
16.
Artif Cells Nanomed Biotechnol ; 47(1): 4097-4108, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31663388

RESUMO

Aim: 2-HP-ß-cyclodextrin-PLGA nanoparticle complexes were prepared to enhance the aqueous humour delivery of Triamcinolone acetonide.Materials & methods: Drug-loaded 2-HP-ß-CD/PLGA nanoparticle complexes prepared by adapting a quasi-emulsion solvent evaporation technique. In vitro drug release, in vitro transcorneal permeation study, histopathological study and in vivo transcorneal penetration of PLGA nanoparticles and 2-HP-ß-CD/PLGA nanoparticle complexes were evaluated. Results: Particle size distributions of 2-HP-ß-CD/PLGA nanoparticle complexes were 149.4 ± 3.7 nm and presented stable system. Corneal penetration studies revealed steady sustained drug release (First-order); 2-HP-ß-CD/PLGA nanoparticle complexes increased ocular bioavailability by increasing dispersion in the tear film and improving drug release. Conclusion: 2-HP-ß-CD/PLGA nanoparticle complex formulation is a promising alternative to conventional eye drops.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Portadores de Fármacos/química , Olho/metabolismo , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Córnea/metabolismo , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Coelhos
17.
Middle East Afr J Ophthalmol ; 26(3): 148-152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31619902

RESUMO

PURPOSE: Glaucoma drainage device surgery (GDDS) has gained popularity, with outcomes equivalent to trabeculectomy. Erosion of the tube through the overlying conjunctiva may occur in 5%-10% of eyes. Donor corneal tissue has been used as a patch graft for GDDS. MATERIALS AND METHODS: This was a prospective proof of concept study in 10 patients undergoing GDDS. From patients undergoing endothelial keratoplasty, the donor tissue (approximately 300 µ in thickness) was placed epithelial side down in a well and was allowed to soak in riboflavin solution (VibeX, Avedro, Waltham, MA, USA) for 15 min. This anterior corneal lenticule received 8 mW/cm2 ultraviolet (UV) irradiation applied for 15 min (total energy of 7.2 J/cm2). Each lenticule was then bisected and utilized for the two study participants. The tissue was sutured over the tube during the GDDS and then was covered with recipient conjunctiva as per the usual technique. Representative graft tissues were fixed and examined to determine the depth of cross-linking effect. The patients were followed for 1 year. RESULTS: Histology revealed no apparent demarcation line in the cross-linked grafts; this supported a full-thickness cross-linking treatment effect. There were no intra- or postoperative complications attributed to the graft tissue. No patient developed erosion or exposure of the tube during the 1-year follow-up. CONCLUSIONS: UV-riboflavin cross-linking of the corneal tissue patch graft material appears to be a safe modification when used in GDDS and warrants ongoing study. This method of patch graft can replace other costy methods used with GDD.


Assuntos
Colágeno/metabolismo , Córnea/efeitos dos fármacos , Transplante de Córnea/métodos , Reagentes para Ligações Cruzadas , Implantes para Drenagem de Glaucoma , Glaucoma de Ângulo Aberto/cirurgia , Adulto , Idoso , Córnea/metabolismo , Feminino , Humanos , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Fármacos Fotossensibilizantes/uso terapêutico , Projetos Piloto , Complicações Pós-Operatórias , Estudo de Prova de Conceito , Estudos Prospectivos , Riboflavina/uso terapêutico , Doadores de Tecidos , Raios Ultravioleta , Adulto Jovem
18.
ACS Appl Mater Interfaces ; 11(43): 39603-39612, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31580053

RESUMO

The sealed anatomical features of the eye and its physiological activity that rapidly removes drugs are called anatomical and physiological barriers, which are the cause of more than 90% of drug loss. This aspect remains a critical issue in eye surface medication. Thus, promoting tissue permeability of drugs as well as prolonging their retention on the eye surface can improve their bioavailability and enhance their therapeutic effects. Thanks to the existence of a negatively charged mucin layer on the eye surface, several peptide-decorated polymeric micelles were prepared to enhance the interaction between the micelle and eye surface, thus prolonging the drug retention on the eye surface and promoting its tissue permeability. Tacrolimus (also known as FK506) is a hydrophobic macrolide immunosuppressant used to treat dry eye syndrome and other eye diseases. However, its hydrophobic nature makes its delivery as a topical eye surface medication difficult, with the risk of side effects due to overdoses. Therefore, the aim of this work is to evaluate the ability of FK506 micelles in promoting their permeability on the eye surface. Our results showed that the positively charged nanomicelles could significantly prolong FK506 retention on the eye surface and enhance its corneal permeability in ex vivo and in vivo conditions. FK506 nanomicelles exhibited superior curing effects against dry eye diseases than the FK506 suspension and a commercial FK506 formula. It exerted better inhibitory effects on eye surface inflammation and corneal epithelium apoptosis when examined by a slip lamp and a transferase-mediated dUTP nick end labeling assay, respectively. Further assays revealed the higher suppressive effects on the expression of several inflammation-related factors at an mRNA and protein level. Hence, our results suggested that these positively charged nanomicelles might be a good drug delivery system for ocular surface medication.


Assuntos
Córnea/metabolismo , Síndromes do Olho Seco , Micelas , Soluções Oftálmicas , Peptídeos , Tacrolimo , Administração Tópica , Animais , Córnea/patologia , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Soluções Oftálmicas/química , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/farmacologia , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologia , Permeabilidade , Coelhos , Tacrolimo/química , Tacrolimo/farmacocinética , Tacrolimo/farmacologia
19.
In Vivo ; 33(6): 1851-1855, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662512

RESUMO

AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.


Assuntos
Movimento Celular/fisiologia , Córnea/fisiologia , Epitélio Anterior/fisiologia , Fluoresceínas/metabolismo , Células-Tronco/fisiologia , Succinimidas/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Córnea/metabolismo , Epitélio Anterior/metabolismo , Humanos , Células-Tronco/metabolismo
20.
OMICS ; 23(11): 583-597, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31651220

RESUMO

Keratoconus (KCN) is a leading cause for cornea grafting worldwide. Keratoconus is a multifactorial disease that causes progressive thinning of the cornea and whose etiology is poorly understood. Several studies have used proteomics on patient tear fluids to identify potential biomarkers. However, proteome of the cornea itself has not been investigated fully. We report here new findings from a case-control study using multiplexed mass spectrometry (MS) on individual (unpooled) corneas to gain deeper insights into proteins and biomarkers relevant to keratoconus. We employed a high-pressure approach to extract total protein from individual corneas from five cases and five controls, followed by trypsin digestion and tandem mass tag (TMT) labeling. The MS-derived data were searched using the Human NCBI RefSeq protein database v92, with peptides and proteins filtered at 1% false discovery rate. A total of 3132 proteins were detected, of which 627 were altered significantly (p ≤ 0.05) in keratoconus corneas. The increases were overwhelmingly in the mTOR/PI3/AKT signal-mediated regulations of cell survival and proliferation, nonsense-mediated decay of transcripts, and proteasomal pathways. The decreases were in several extracellular matrix proteins and in many members of the complement system. Importantly, this multiplexed proteomic study of keratoconus corneas identified, to our knowledge, the largest number of corneal proteins. The novel findings include changes in pathways that regulate transcript stability, proteasomal degradation, and the complement system in corneas with keratoconus. These observations offer new prospects toward future discovery of novel molecular targets for diagnostic and therapeutic innovations for patients with keratoconus.


Assuntos
Córnea/metabolismo , Ceratocone/etiologia , Ceratocone/metabolismo , Proteoma , Proteômica , Transporte Biológico , Biomarcadores , Estudos de Casos e Controles , Cromatografia Líquida , Córnea/patologia , Suscetibilidade a Doenças , Matriz Extracelular , Espectrometria de Massas , Degradação do RNAm Mediada por Códon sem Sentido , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Ubiquitina/metabolismo , Fluxo de Trabalho
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