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Nadyr A Damasceno,1 Nadia C Miguel,2 Marcelo Palis Ventura,3 Miguel Burnier Jr,4 Marcos P Avila,5 Eduardo F Damasceno3 1Ophthalmology Department, Hospital Naval Marcílio Dias, 2Laboratory of Neurohistology and Cell Ultrastructure, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 3Ophthalmology Department, Universidade Federal Fluminense, Niterói, Brazil; 4Ophthalmology Department, McGill University, Montreal, QC, Canada; 5Ophthalmology Department, Universidade Federal de Goiás, Goiania, Brazil Purpose: The aim of study was to evaluate the cross-link using riboflavin and ultraviolet A (UVA) for improving scleral wound healing.Materials and methods: This was an experimental study involving four New Zealand rabbits (eight eyes). Therapy procedure was chosen for the right eye and control procedure for the left one. UVA irradiation of 365 nm with a surface irradiance of 3 mW/cm2 and a photosensitizer of riboflavin drops were applied for 30 minutes on the right eye at 2 mm from the limbus. Sclerotomy incision was performed at 2 mm from the limbus in both right (on the cross-link-treated area) and left eye. Then, 30 days after surgery, a morphological analysis and histological staining with hematoxylin–eosin and picrosirius red were performed, and the sclerotomy cicatrization of right and left eyes was compared. The variables investigated were as follows: sclerotomy incision pictures and measurements were made using the ImageJ Software. Scleral thickness was measured (employing the anterior optical coherence tomography and the digital caliper). Collagen fiber density stained with picrosirius red staining was measured using the Image Pro Plus software.Results: The morphological analysis showed that in all samples, the right eye presented sclerotomy closure, and in two eyes, among them, there were no visible edges of the sclerotomies incision. The left eye presented sclerotomy closure and incision edges. The Image Pro Plus demonstrated a higher density of collagen fibers in the right eye when compared to the left one. The statistical analysis was significant when compared to the collagen fiber density in the treated eyes with the control eyes.Conclusion: The cross-link procedure resulted in a better sclerotomy wound healing. Keywords: sclerotomy wound healing, cross-linking, ultraviolet A, riboflavin

Background: Cosmetic preservatives are used to protect cosmetic formulations and improve its shelf-life. However, these substances may exert phototoxic effects when used under sunlight., Objective: To assess safety, efficacy and putative phototoxic effects of a sunscreen formulation SPF 30 and its excipients., Materials/methods: Irradiation was performed with solar simulated light (SSL) and the sunscreen from the School of Pharmacy/UFRJ/Brazil. We used albino hairless mice in different groups (control (G1), only irradiated (G2), sunscreen plus irradiation (G3) and vehicle plus irradiation (G4) for morphological assessment and immunefluorescence detection to OKL38. In vitro analyses were with a Saccharomyces cerevisiae (SC) strain plus SSL in the presence of methylparaben, propylparaben, imidazolidinyl urea, aminomethyl propanol and their association., Results: G3 and G4 displayed photosensitization leading to thickening of the epidermis and increased dermal cellularity. G4 displayed strong OKL38 labeling when compared with other groups. Aminomethyl propanol, methylparaben and propylparaben are endowed with phototoxic activity against SC. Propylparaben displayed the highest phototoxic effect, followed by excipients association., Conclusions: The sunscreen's vehicle is endowed with phototoxic activity. Propylparaben was the most phototoxic agent, increasing the overall phototoxicity of excipient association, pointing to a critical concern regarding vehicle associations intended to cosmetic purposes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Extracellular matrix (ECM) and cellular membrane proteoglycans (PGs) play important roles in neural differentiation and cell adhesion. Vascular endothelial growth factor, an important signal protein in vascular and retinal neural cell development, is retained in the ECM due to its high affinity for PG. Bevacizumab, an anti-VEGF agent, has been extensively used for treating retinal diseases in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on neurocan, phosphacan, and syndecan-3 PG levels in newborn rat retina., Methods: Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 48 hours. Immunohistochemical staining was assessed against neurocan, phosphacan, and syndecan-3. Proteoglycan content was quantified based on the intensity of immunohistochemical labeling. Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. The results from the treatment and control groups were compared., Results: No significant difference in the staining intensity and mRNA expression of phosphacan and syndecan-3 was observed between the groups. However, a significant decrease in neurocan content and mRNA expression was observed in bevacizumab-treated retinal explants compared with controls., Conclusions: Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The purpose of the study was to evaluate the effects of a sunscreen formulation on the skin of albino hairless mice subjected to simulated solar light (SSL) in terms of morphological changes. Young adult albino hairless mice HRS/J (n = 36) were used as an experimental model for determining skin photoaging changes. Mice were irradiated with SSL, and the sunscreen (estimated SPF 30, PF-UVA) was obtained from the Pharmacy College/UFRJ, Brazil. The animals were divided into four groups: non-treated (G1), radiation only (G2), sunscreen-treated (G3) and vehicle + radiation (G4). Animals from groups G2, G3 and G4 were irradiated weekly (5 weeks), with no immobilization. One week after the final exposure, the dorsal skin was observed using a dermatoscopic camera. Biopsies were analyzed in order to quantify neovascularization and to evaluate histological aspects of the skin. Neovascularization was also evaluated with immunohistochemical reactions for the Von Willebrand factor. Animals from G2 displayed classical morphological changes denoting skin photoaging: thickening of the epidermis, increased dermal cellularity, follicular keratosis, sebaceous gland hyperplasia, and angiogenesis. Animals from groups G3 and G1 displayed similar morphological profiles, without these changes. Animals from group G4 showed more morphological changes than group G2, emphasizing the relative importance of the putative photosensitizing components present in the vehicle formulation. The extent of the morphological skin changes suggested that the sunscreen formulation was effective against SSL, and showed the importance of assessing the phototoxicity of vehicle formulations.

Purpose: Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina., Methods: Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared., Results: No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls., Conclusions: Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Purpose: To evaluate the efficacy and safety of subconjunctival injection of triamcinolone in the treatment of acute ocular alkali burn in rabbits., Methods: Two groups of 5 rabbits were subjected to alkali burn (1 N NaOH). One group was treated with 1 subconjunctival injection of 0.3 mL of triamcinolone and the other with 1 subconjunctival injection of 0.3 mL of 0.9% saline. The affected corneas were observed for vascularization and opacity approximately 10 minutes after the burn and also after 7, 14, and 21 days. Photographs were taken for observation and statistical analyses. At all time intervals, the corneas were classified according to predetermined scores. Twenty-one days after the treatment, the animals were anesthetized, and their eyes were enucleated and processed for histopathology., Results: Greater vascularization and opacity appeared in the animals that were treated with saline than in those treated with subconjunctival triamcinolone (vascularization: 7 days, P = 0.0107; 14 days, P = 0.0099; and 21 days, P = 0.0088; opacity: 7 days, P = 0.0079; 14 days, P = 0.0112; and 21 days, P = 0.0255). These results were also compatible with the morphological and statistical analyses, which revealed a more intense inflammatory process in the group treated with saline (P = 0.0317). No complications, such as corneal melting, perforation, or infection, were observed., Conclusions: Subconjunctival injection of triamcinolone may be a therapeutic option for the treatment of acute ocular burn because it reduced the corneal inflammatory process, opacity, and vascularization, with no apparent clinical changes in the general state of the animal.

Objective: Bevacizumab has been widely used as a vascular endothelial growth factor antagonist in the treatment of retinal vasoproliferative disorders in adults and, more recently, in infants with retinopathy of prematurity. Recently, it has been proposed that vascular endothelial growth factor acts as a protective factor for neurons and glial cells, particularly in developing nervous tissue. The purpose of this study was to investigate the effects of bevacizumab on the developing retinas of juvenile rabbits., Methods: Juvenile rabbits received bevacizumab intravitreously in one eye; the other eye acted as an untreated control. Slit-lamp and fundoscopic examinations were performed both prior to and seven days after treatment. At the same time, retina samples were analyzed using immunohistochemistry to detect autophagy and apoptosis as well as proliferation and glial reactivity. Morphometric analyses were performed, and the data were analyzed using the Mann-Whitney U test., Results: No clinical abnormalities were observed in either treated or untreated eyes. However, immunohistochemical analyses revealed a reduction in the occurrence of programmed cell death and increases in both proliferation and reactivity in the bevacizumab-treated group compared with the untreated group., Conclusions: Bevacizumab appears to alter programmed cell death patterns and promote gliosis in the developing retinas of rabbits; therefore, it should be used with caution in developing eyes.

Purpose: To evaluate the ultrastructural effect of trypan blue 0.1% staining for capsulorhexis on lens epithelial cells (LECs) and capsules., Setting: Division of Ophthalmology, University of São Paulo, São Paulo, Brazil., Methods: Before capsulorhexis, patients were randomly assigned to 1 of 2 groups. Trypan blue 0.1% staining was performed in the treatment group. No trypan blue was used in the control group. Samples of capsules with LECs were fixed and analyzed with routine optical microscopy techniques, immunohistochemistry for beclin-1 expression (a marker of autophagy), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling to detect apoptosis, and transmission electron microscopy (TEM). Morphometric analyses were performed, and the 2 sets of data were compared., Results: Each group comprised 15 patients. Cell death by autophagy and apoptosis was observed in the treatment group but not in the control group. The TEM images of subcapsular epithelium cells showed mitochondrial rupture, dilation of the cisterns of the endoplasmic reticulum, increased cytoplasmic and nuclear electron density, and abnormalities in the nuclear profile of trypan blue-stained cells. Morphometric analysis showed statistically significant differences between the 2 groups in the longest nuclear axes and the ratio between the total nuclear perimeter and the cell area (P = .03). The difference in capsule thickness between groups was not significant., Conclusion: Trypan blue caused LEC death, which supports the hypothesis that staining with trypan blue 0.1% can help reduce the incidence of posterior capsule opacification after cataract surgery., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright (c) 2010 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

In invertebrates, a few studies have suggested apoptosis as the mechanism of choice to protect the retina after exposure to ultraviolet (UV) radiation. We demonstrated previously, by electron microscopy, that the retina and lamina ganglionaris (or lamina) cells of the crab Ucides cordatus displayed subcellular signs of apoptosis after exposure to UVB and UVC. Here, we first ascertained, by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, that UV irradiation indeed produced the previously reported results. We next tested, in the visual system of U. cordatus, whether the expression (as analyzed by immunohistochemistry and observed with laser scanning microscopy) and levels (as examined by Western blotting) of catalase, Bax, and p53 were affected by the same dose of UV radiation as that used previously. Our data revealed that the intensity of catalase, Bax, and p53 labeling was stronger in irradiated retina and lamina cells than in non-irradiated retina and lamina. However, no significant difference was observed in the concentrations of these proteins isolated from the whole optic lobe. The results thus suggest that UVB and UVC induce apoptosis in the crustacean retina and lamina by increasing catalase expression and activating the Bax- and p53-mediated apoptosis pathways.

In the present paper, the modulation of the basolateral membrane (BLM) Na+ -ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) was evaluated. Ang II and Ang-(1-7) inhibit the Na+ -ATPase activity in a dose-dependent manner (from 10(-11) to 10(-5) M), with maximal effect obtained at 10(-7) M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1-7) are mediated by AT2 receptor: The effect of both polypeptides is completely reversed by 10(-8) M PD 123319, a selective AT2 receptor antagonist, but is not affected by either (10(-12) - 10(-5) M) losartan or (10(-10)-10(-7) M) A779, selective antagonists for AT1 and AT(1-7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3',5'-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10(-9) M guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; an inhibitor of the G protein activity), and mimicked by 10(-10) M guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the Gs protein in the isolated basolateral membrane fraction; (4) (10(-10)-10(-6) M) cAMP has a similar and non-additive effect to Ang II and Ang-(1-7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1-7); and (6) both angiotensins stimulate PKA activity.

Over the last two decades, ultraviolet radiation levels (UV), reaching the Earth's surface, have been increasing at a rate of 1.5% per each 1% loss of the ozone layer. Moreover, artificial UV-sources have also proliferated and contributed to the rising UV-stress that many organisms have to face. To assess how the vertebrate retina responds to an exposure of short wavelength UV, we focused our attention on the rat retina, observing photoreceptor (containing outer and inner segments of rods and cones), inner plexiform, and ganglion cell layers by light and transmission electron microscopy using conventional and cytochemical techniques. We analyzed how cells of the layers in question responded to a 30 min exposure to UV-C and UV-B radiation with doses of 7200 and 590 J/cm(2), respectively. The results show that there are significant changes in the nuclei and cytoplasmic organelles of the exposed retinae when compared with those of the unexposed controls. The changes include an increase in heterochromatin, distension of rough endoplasmic reticulum, mitochondrial disruptions, and increases in the number of myelin bodies. The recorded morphological changes, especially those of the ganglion cells, are suggestive of apoptotic processes and show that the exposure of vertebrate retina to wavelengths ranging from 254 to 312 nm can produce alterations that are likely to impact negatively on the retina's proper functioning.

The visual system as an interface between the environment and the living organism can serve as a sensitive indicator especially in studies that deal with effects of radiation. The crab retina as the seat of the photoreceptors and the lamina ganglionaris as the place of second order neurons were the targets of our study. Conventional specimen preparation techniques for transmission electron microscopy (TEM) are adequate to preserve any modifications that may occur as a consequence of the experimental treatment. In this study we analyzed by TEM how retinal and lamina ganglionaris cells of the crab Ucides cordatus responded to a 30 min exposure to ultraviolet C (UV-C) and ultraviolet B (UV-B) radiation with doses of 7200 and 590J/cm(2), respectively. The results show that damaged cells occurred in both retina and lamina ganglionaris, but that the retinal cells were affected to a greater extent. Morphological alterations of the pigment granules and an increase in the quantity of lipid droplets of the retinal cells were also observed. Additional changes include an increase in heterochromatin, nuclear karyolyses and karyorrhexes, distention of rough endoplasmic reticulum and mitochondrial disruptions. The observed morphological changes are indicative of apoptotic processes and show that an exposure to light of wavelengths of 254 and 312 nm may be injurious to the visual system of invertebrates.

We examined the effect of aldrin on hepatocyte ultrastructure in liver of Rana catesbeiana. The frogs were experimentally exposed to chemical substance and liver fragments processed for routine transmission electron microscopy. Hepatic peroxisomes were visualized after incubation with alkaline 3,3'-diaminobezidine (DAB) method. Ultrastructural analysis revealed progressive hepatocyte changes induced by this drug. After 2-weeks, in the hepatocytes the nuclear envelop and the cisternae of both smooth and rough endoplasmic reticulum (SER und RER, respectively) were unusually enlarged. Reduction of glycogen granules associated with an increased frequency of lysosomes was observed. Normal appearing peroxisomes were present in clusters. Lipid droplets were also visualuzed. After 4-weeks, there was a new increase of glcogen associated with a great number of mitochondria and peroxisomes. Moreover, SER und RER were still dilated. Intracellular lipid inclusions became more abundant. These results suggest that the aldrin 250 induces ultrastructural changes in the hepatocyte of Rana catesbeiana.