Nádia Campos de Oliveira Miguel

Ultrastructural study of first and second order neurons in the visual system of the crab Ucides cordatus following exposure to ultraviolet radiation.

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.

Clinical and histopathological outcomes of subconjunctival triamcinolone injection for the treatment of acute ocular alkali burn in rabbits.

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.

A structural study of the retinal photoreceptor, plexiform and ganglion cell layers following exposure to UV-B and UV-C radiation in the albino rat

Over the last two decades, ultraviolet radiation levels (UV), reaching the Earths 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 retinas proper functioning.

Trypan blue staining for capsulorhexis: Ultrastructural effect on lens epithelial cells and capsules

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.

Reduced occurrence of programmed cell death and gliosis in the retinas of juvenile rabbits after shortterm treatment with intravitreous bevacizumab

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.

Catalase, Bax and p53 expression in the visual system of the crab Ucides cordatus following exposure to ultraviolet radiation.

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.

Bevacizumab reduces neurocan content and gene expression in newborn rat retina in vitro.

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.

α-Bisabolol improves 5-aminolevulinic acid retention in buccal tissues: Potential application in the photodynamic therapy of oral cancer

CONTEXT 5-Aminolevulinic acid (5-ALA) is a prodrug used in photodynamic therapy (PDT) of tumors, including cancer of the oral mucosa. 5-ALA poorly penetrates oral tissues due to its high hydrophilicity, which impairs its local effects in PDT. OBJECTIVES To examine whether α-bisabolol (α-Bis) influences the 5-ALA permeability in the porcine buccal mucosa, to an extent that improves its application in PDT (which requires low permeation and high retention in the buccal mucosa). METHODS In vitro permeability studies with 5-ALA (1% and 10% w/w) associated with α-Bis (1% to 20% w/w) in propylene glycol were carried out at 4h and 24h using porcine buccal mucosa in a modified Franz cell system. The in vitro release profiles (0.5 to 48h) of the selected formulation and its respective control were determined using artificial membranes. Samples of buccal mucosa treated with the formulation were submitted to histopathological analysis, using a routine optical microscopy technique. RESULTS The association of 1% 5-ALA and 5% α-Bis provided the best results; after 4h of treatment with this formulation, the 5-ALA permeation was low and its retention in the mucosa was six-fold higher than that promoted by the control formulation (5-ALA alone). Histological analysis of the porcine buccal mucosa evidenced that 5% α-Bis altered the tissue morphology, which probably promoted 5-ALA retention. We concluded that 5% α-Bis is a potential adjuvant in formulations containing 5-ALA that could improve its retention after topical oral administration for the PDT treatment of cancer.

Phototoxic assessment of a sunscreen formulation and its excipients: An in vivo and in vitro study

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 sunscreens 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.

Cellular stress response in human Müller cells (MIO-M1) after bevacizumab treatment

&NA; Bevacizumab, an anti‐vascular endothelial growth factor (VEGF) agent, is widely used in the treatment of retinal vascular diseases. However, due to the essential role Müller cell derived‐VEGF plays in the maintenance of retinal neurons and glial cells, cell viability is likely to be affected by VEGF inhibition. We therefore evaluated the effect of bevacizumab‐induced VEGF inhibition on Müller cells (MIO‐M1) in vitro. MIO‐M1 cells were cultured for 12 or 24 h in media containing bevacizumab at 0.25 or 0.5 mg/mL. Controls were cultured in medium only. Cell viability was determined with the trypan blue exclusion test and MTT assay. Caspase‐3, beclin‐1, glial fibrillary acidic protein (GFAP) and vimentin content were quantified by immunohistochemistry. Gene expression was evaluated by real‐time quantitative PCR. Treatment with bevacizumab did not reduce MIO‐M1 cell viability, but increased metabolic activity at 24 h (0.5 mg/mL) and induced apoptosis and autophagy, as shown by the increased caspase‐3 levels at 12 h (0.25 and 0.5 mg/mL) and the increased beclin levels at 24 h (0.5 mg/mL). Caspase‐3 mRNA was upregulated at 12 h and downregulated at 24 h in cells treated with bevacizumab at 0.25 mg/mL. Bevacizumab treatment was also associated with structural protein abnormalities, with decreased GFAP and vimentin content and upregulated GFAP and vimentin mRNA expression. Although bevacizumab did not significantly affect MIO‐M1 cell viability, it led to metabolic and molecular changes (apoptosis, autophagy and structural abnormalities) suggestive of significant cellular toxicity. HighlightsAnti‐VEGF drugs are used as treatment of vascular retinopathies but its affect on retinal glial cells survival is unknown.Anti‐VEGF bevacizumab did not reduce Müller cell viability but caused increased metabolic activity, apoptosis and autophagy.Bevacizumab treatment was also associated with structural protein abnormalities, including GFAP and vimentin.We conclude that bevacizumab led to metabolic and molecular changes suggestive of cellular toxicity to Müller cells.Exposure to bevacizumab seems to triggers an adaptive mechanism in Müller cells capable of reducing cellular damage.