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P.G. Layer, J.B. Mocko, L.E. Paraoanu; Formation of the IPL in Retinal Reaggregates of the Chick Embryo Is Inhibited by the Organophosphate Diazinon . Invest. Ophthalmol. Vis. Sci. 2005;46(13):558.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Developing embryos are more vulnerable than adults to acute cholinergic intoxication by anticholinesterases, including organophosphorus pesticides. These agents affect the process of neural development itself, leading to permanent deficits in the architecture of the nervous system. Recent evidence on direct roles of acetylcholinesterase (AChE) on neuronal differentiation provides additional grounds for investigating the developmental toxicity of anticholinesterases. Therefore, the effect of the organophosphate diazinon on the development of chick retinal differentiation was studied by an in vitro reaggregate approach. Methods: Reaggregated spheres from dissociated retinal cells of the E6 chick embryo were produced in rotation culture. During the whole culture period of ten days, experimental cultures were supplemented with different concentrations of pesticides, from 6.25 to 31.25 µg/ml diazinon. After this time, the spheres were fixed and cryo–sectioned at 12–20 µm thickness. AChE and BChE activity were revealed by the Karnovsky–Roots technique. TUNEL staining was used to show apoptotic nuclei. Furthermore, immunostainings were performed using antibodies to calretinin, CERN901 and glutamine synthetase. Calretinin stains a subpopulation of amacrine cells, CERN 901 labels all rod photoreceptors, and glutamine synthetase is a marker for Müller glial cells. Results: The pesticide–treated spheres were reduced in size, and their outer surface was irregular. More importantly, inner structural distortions could be easily traced, since the structure of control spheroids can be well characterised by a histotypical arrangement of laminar parts homologous to the normal retina. AChE activity in diazinon–treated spheres was reduced when compared with controls. As a dramatic effect of exposure to the pesticide, IPL–like areas in spheroids were not distinguishable anymore. Similarly, photoreceptor rosettes and Müller radial glia were strongly affected, and apoptosis was stimulated. Conclusions: Diazinon was deleterious to the growth and histogenesis of reaggregated spheres from chicken embryonic retinae. In particular, neurite growth into neuropil areas (IPL) was completely inhibited. This further proves the significance of cholinesterases and the cholinergic system for proper nervous system development. This project is supported within the EU consortium Sens–Pesti (Contract No. QLK4–CT–2002–02264).
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