RESUMO
The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs.
Assuntos
Membrana Basal/química , Retinopatia Diabética/metabolismo , Proteínas da Matriz Extracelular/análise , Retina/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/ultraestrutura , Western Blotting , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Elasticidade , Feminino , Humanos , Masculino , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Retina/ultraestruturaRESUMO
PURPOSE: To determine corneal cross-linking (CXL) efficacy and chromophore penetration after excimer laser-assisted patterned de-epithelialization. METHODS: Two-hundred-twenty porcine eyes were de-epithelialized ex vivo, either fully (mechanical; n = 88) or patterned (excimer laser; n = 132). Consecutively, corneas were impregnated with hypo- or hyperosmolar riboflavin (RF; n = 20, RF-D; n = 40, respectively) or water-soluble taurine (WST11; n = 40, and WST-D; n = 40, respectively), or kept unimpregnated (n = 80). Sixty corneas were subsequently irradiated, inducing CXL, with paired contralateral eyes serving as controls. Outcome measurements included strip extensiometry to assess CXL efficacy, and spectrophotometry and fluorescence microscopy to determine stromal chromophore penetration. RESULTS: All tested chromophores induced significant CXL (p < 0.001), ranging from 7.6% to 14.6%, with similar stiffening for all formulations (p = 0.60) and both de-epithelialization methods (p = 0.56). Light transmittance was significantly lower (p < 0.001) after full compared with patterned de-epithelialization. Stromal chromophore penetration was comparable between fully and patterned de-epithelialized samples, with full penetration in RD and RF-D samples and penetration depths measuring 591.7 ± 42.8 µm and 592.9 ± 63.5 µm for WST11 (p = 0.963) and 504.2 ± 43.2 µm and 488.8 ± 93.1 µm for WST-D (p = 0.669), respectively. CONCLUSIONS: Excimer laser-assisted patterned de-epithelialization allows for effective CXL. Stromal chromophore concentration is, however, reduced, which may have safety implications given the need for sufficient UVA attenuation in RF/UVA CXL. The different safety profile of near-infrared (NIR) may allow safe WST11/NIR CXL even with reduced stromal chromophore concentration values. In vivo studies are needed to evaluate the benefits and further assess safety of excimer laser-assisted patterned de-epithelialization for corneal CXL.
Assuntos
Substância Própria , Lasers de Excimer , Animais , Colágeno/farmacologia , Córnea/cirurgia , Substância Própria/cirurgia , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Lasers de Excimer/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Riboflavina/farmacologia , Riboflavina/uso terapêutico , Suínos , Raios UltravioletaRESUMO
Purpose: To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10- and 30-minute impregnation. Methods: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. Results: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)â¢10-4% and (5.4 ± 0.21)â¢10-4%, and trans-corneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10- and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. Conclusions: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. Translational Relevance: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients' burden and costly operation room time.
Assuntos
Células Endoteliais , Fármacos Fotossensibilizantes , Animais , Colágeno , Córnea , Reagentes de Ligações Cruzadas/efeitos adversos , Endotélio , Humanos , Fármacos Fotossensibilizantes/efeitos adversos , Coelhos , RiboflavinaRESUMO
Purpose: To determine the long-term safety and efficacy of WST-D/near-infrared (NIR) corneal stiffening. Methods: One eye of 23 New Zealand White rabbits was de-epithelialized mechanically followed by topical application of 2.5 mg/mL WST11, combined with dextran-500 (WST-D) for 20 minutes. Subsequently, samples were irradiated with a NIR (755 nm) laser at 10 mW/cm2 for 30 minutes. Untreated fellow eyes served as controls. One week (n = 4), 1 month (n = 6), 4 months (n = 9), or 8 months (n = 4) after treatment rabbits were euthanized. Corneal strips were cut in superior-inferior direction for extensiometry testing (1, 4, and 8 months), and histologic sections were prepared for evaluation of keratocyte distribution (1 week and 8 months). Results: Elastic modulus after treatment was significantly higher than in paired controls (16.0 ± 2.3 MPa versus 9.6 ± 3.6 MPa [P = 0.008], 18.1 ± 4.5 MPa versus 12.6 ± 2.3 MPa [P = 0.003], and 18.6 ± 3.6 MPa versus 14.2 ± 3.6 MPa [P = 0.010], at 1, 4, and 8 months, respectively). A significant decrease in keratocyte count at the anterior stroma was observed directly after treatment (1.5 ± 1.7 vs. 19.0 ± 4.1 [P = 0.002]). At 8 months keratocyte repopulation appeared completed, with similar distribution in treated and untreated corneas (15.9 ± 1.1 vs. 14.5 ± 2.5 [P = 0.562]). Corneal thickness was comparable between treated and untreated corneas at all time points. Conclusions: WST-D/NIR treatment resulted in significant and persistent long-term increase in corneal stiffness. Initial keratocyte apoptosis in the anterior stroma is followed by repopulation to normal level at 8 months after treatment. The safe nature of NIR light allows treatment of corneas of any thickness without endangering corneal endothelium or deeper ocular structures, potentially benefiting patients deemed unsuitable for riboflavin/UV-A cross-linking.
Assuntos
Colágeno/farmacologia , Córnea/patologia , Ceratocone/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Raios Ultravioleta , Animais , Apoptose , Fenômenos Biomecânicos , Córnea/fisiopatologia , Reagentes de Ligações Cruzadas , Modelos Animais de Doenças , Seguimentos , Ceratocone/patologia , Ceratocone/fisiopatologia , Coelhos , Fatores de TempoRESUMO
PURPOSE: The aim of this study is to determine the effect of variation of the exposure time of near-infrared irradiation on corneal stiffening after a bacteriochlorophyll derivative (WST11) with dextran (WST-D) application. METHODS: One hundred four paired eyes of 3-month-old New Zealand White rabbits were included in this study. Fifty-two eyes (ex vivo n = 34, in vivo n = 18) were mechanically deepithelialized, treated topically with WST-D, and irradiated at 10 mW/cm using a diode laser at 755 nm for 1, 5, or 30 minutes. Untreated fellow eyes served as controls. Corneoscleral rings were removed immediately after treatment (ex vivo), or 1 month after treatment (in vivo). Corneal strips were cut and underwent biomechanical stress-strain measurements. RESULTS: Ex vivo, the mean tangent elastic modulus was significantly higher in the treatment groups than in the control groups for 1, 5, and 30 minutes of irradiation, respectively, 6.06 MPa, 95% confidence interval (CI, 4.5-7.6) versus 14.02 MPa, 95% CI (10.2-17.8), n = 11, 4.8 MPa, 95% CI (3.9-5.7) versus 15.03 MPa, 95% CI (12-18.1), n = 11, and 7.8 MPa, 95% CI (5.6-10.02) versus 16.2 MPa, 95% CI (13.6-18.9), n = 11; P < 0.001 for all comparisons. In vivo, the mean elastic moduli in the treatment groups were significantly higher for 5 and 30 minutes of irradiation but not for 1 minute of irradiation, respectively, 11.4 MPa, 95% CI (8.5-14.2), versus 17.1 MPa, 95% CI (14.5-19.7), n = 5; P < 0.001, and 9.4 MPa, 95% CI (5.1-13.8) versus 16 MPa, 95% CI (13.1-19), n = 5; P < 0.01, and 11.3 MPa, 95% CI (6-16.6) versus 12.2 MPa, 95% CI (7.5-16.8), n = 5; P = 0.7. CONCLUSIONS: WST-D/near-infrared treatment using shortened irradiation time (1 minute ex vivo and 5 minutes in vivo) results in significant corneal stiffening, and this might provide an alternative to the currently applied riboflavin/ultraviolet A cross-linking.
Assuntos
Bacterioclorofilas/farmacologia , Córnea/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Dextranos/farmacologia , Raios Infravermelhos , Fármacos Fotossensibilizantes/farmacologia , Animais , Fenômenos Biomecânicos , Colágeno/metabolismo , Córnea/metabolismo , Córnea/fisiopatologia , Paquimetria Corneana , Substância Própria/metabolismo , Combinação de Medicamentos , Coelhos , Fatores de TempoRESUMO
PURPOSE: Cataract surgery requires the removal of a circular segment of the anterior lens capsule (LC) by manual or femtosecond laser (FL) capsulotomy. Tears in the remaining anterior LC may compromise surgical outcome. We investigated whether biophysical differences in the rim properties of the LC remaining in the patient after manual or FL capsulotomy (FLC) lead to different risks with regard to anterior tear formation. METHODS: Lens capsule samples obtained by either continuous curvilinear capsulorhexis (CCC) or FLC were investigated by light microscopy, laser scanning confocal microscopy, and scanning electron microscopy; atomic force microscopy (AFM) was used to test the biomechanical properties of the LC. The mechanical stability of the LC following either of the two capsulotomy techniques was simulated by using finite-element modeling. RESULTS: Continuous curvilinear capsulorhexis produced wedge-shaped, uniform rims, while FLC resulted in nearly perpendicular, frayed rims with numerous notches. The LC is composed of two sublayers: a stiff epithelial layer that is abundant with laminin and a softer anterior chamber layer that is predominantly made from collagen IV. Computer models show that stress is uniformly distributed over the entire rim after CCC, while focal high stress concentrations are observed in the frayed profiles of LC after FLC, making the latter procedure more prone to anterior tear formation. CONCLUSIONS: Finite-element modeling based on three-dimensional AFM maps indicated that CCC leads to a capsulotomy rim with higher stress resistance, leading to a lower propensity for anterior radial tears than FLC.