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Monique Matsuda, Paloma G. Krempel, Thiago Puntar, Alfred Scholl-Franco, André Luis F. Portes, Silvana Allodi, Nádia Campos O. Miguel, Mario Luiz R. Monteiro; In Vitro Effect Of Bevacizumab Treatment On Cell Proliferation, Death And Differentiation In The Developing Retina Of Rats. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2969.
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The vascular endothelial growth factor (VEGF) is essential of the development central nervous system of vertebrates by promoting neurogenesis, neuronal patterning, neuroprotection and gliogenesis. Bevacizumab (BVZ) has been extensively used as an anti-VEGF agent for controlling pathological neovascularization in the retina of both adult patients with a wide array of retinal diseases and more recently, in newborn infants with retinopathy of prematurity. However, our hypothesis is that BVZ may also interfere with essential cellular processes in the development of retina since VEGF is important in protecting and maintenance of neurons. The aim of our study was to investigate the effects of BVZ in the differentiation, cell death and proliferation in the developing retina of rats.
Sixty retinas of 2-day-old Lister Hooded rats were dissected after enucleation of the eyes and the retinal explants were raised in culture medium with (experimental group) or without (controls) use of 0.5 mg/mL of BVZ during 48 hours. After the treatment, retinal explants were separated for analyses by immunohistochemistry (IHC) and gene expression by real-time polymerase chain reaction (RT-PCR) for the markers of proliferating cell nuclear antigen (PCNA) (to detect retinal cell proliferation), caspase-3 (a marker of cell death by apoptosis), beclin-1 (a marker of cell death by autophagy), vimentin (for labeling undifferentiated glia) and glial fibrillary acidic protein (GFAP) (for labeling astrocytes and Müller cells). Photographs of the retina for observation and quantification were taken using a confocal microscope (Leica) TCS-SP5 and the mRNA content was measured by SYBR Green fluorescence. Comparisons between the groups were performed using the Student’s unpaired or Mann-Whitney.
No abnormalities were observed both in the IHC material and in the real time RT-PCR analysis of retina explants regarding programmed cell death, as well as proliferating cells. However, a significant increase in the vimentin protein content and a decrease in the GFAP mRNA were observed in the BVZ-treated group compared to controls.
Our study indicates that BVZ leads to abnormalities in the content of vimentin filament protein and mRNA expression of GFAP. BVZ can therefore interfere with the processes of differentiation of retinal cells. These data show the importance of further studies to evaluate the effects of BVZ and suggest caution in its use, particularly in the developing retina, as currently used in children with retinopathy of prematurity.
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