Producción CyT
Congreso
Autoría
Fecha
2012
Editorial y Lugar de Edición
Sarissa Biomedical Ltd
Resumen
Información suministrada por el agente en
SIGEVA
Full understanding of the complex mechanisms that lead to neuronal death after lesion is important to devise neuroprotective treatments. Amongst the factors proposed to strongly contribute to neuronal loss is the substantially raised concentration of extracellular glutamate evoked by excitotoxic damage (Lau and Tymianski, 2010). This process is though to be linked to the onset of the secondary injury that can amplify the initial damage to distant areas. An ample literature exists on the robust ...
Full understanding of the complex mechanisms that lead to neuronal death after lesion is important to devise neuroprotective treatments. Amongst the factors proposed to strongly contribute to neuronal loss is the substantially raised concentration of extracellular glutamate evoked by excitotoxic damage (Lau and Tymianski, 2010). This process is though to be linked to the onset of the secondary injury that can amplify the initial damage to distant areas. An ample literature exists on the robust release of glutamate during excitotoxicity in brain or spinal cord preparations (Park et al., 2004). These data were, however, obtained with rather slow sampling procedures that comprise microdialysis coupled to HPLC (Park et al., 2004) and/or measurements based on radiolabelled glutamate (Milanese et al., 2010). A recently-developed glutamate biosensor (Tian et al., 2009) can be employed for real-time monitoring of endogenous glutamate release from an in vitro model of acute spinal cord injury applied to organotypic spinal slices that retain the basic cytoarchitecture of the spinal cord. This method can, therefore, be useful to study the cellular action of neuroprotective drugs targeting glutamate release mechanisms. For this purpose, we used Sarissa Biomedical glutamate biosensors. A calibration curve for glutamate biosensors was first constructed to demonstrate a highly linear correlation (r=0.9902) between recorded current generated by glutamate oxidation and glutamate concentrations in the 0.1-100 mM range. These calibrations were stable and reproducible. We then analysed the real-time glutamate release evoked by kainic acid application, a stable glutamate analogue, as a reliable method to produce a moderate excitotoxic insult (Mazzone and Nistri, 2011c). No interference was found between the glutamate and the null sensors with or without excitotoxic treatment. Furthermore, we next tested if co-application of kainic acid with riluzole, a drug prescribed for the symptomatic treatment of amyotrophic lateral sclerosis (ALS; National Institute of Clinical Excellence, www.nice.org.uk), and that appears to slow down neurodegenerative progression (Bensimon et al., 1994;Orrell, 2010), could afford early neuroprotection of in vitro motoneurons. Our protocol involved triggering excitotoxicity with 1 h application of kainic acid and studying endogenous glutamate release with a biosensor placed on the ventral horn area of such slices bathed with a basic solution containing (mM) NaCl, 152; KCl, 5; CaCl2, 2; MgCl2, 1; HEPES, 10; glucose, 10 (pH 7.4; 300?320 mOsm) (Figure 1 A, Mazzone and Nistri, 2011c). As indicated in Fig. 1 B, kainic acid (0.5 mM) elicited Ca2+-dependent glutamate release (presumably from neurons) as well as Ca2+-independent glutamate release (presumably from glia) when extracellular Ca2+ was omitted. It is noteworthy that the peak release of glutamate was similar in both conditions, although characterized by much slower onset in the Ca2+ free medium. This observation suggests distinct kinetics dependent on the source of glutamate. Unlike neurons, glia is not rapidly destroyed by kainic acid (Mazzone and Nistri, 2011b). We also enquired if riluzole, which is reported to be a strong inhibitor of glutamate release (Doble, 1996;Dzahini et al., 2010), could modulate the action of kainic acid. The glutamate release peak was largely inhibited by 5 μM riluzole co-applied with kainic acid (Figure 1B; Mazzone and Nistri, 2011a), validating the usefulness of electrochemical detection of endogenous glutamate as an approach to study excitotoxicity. Immunohistochemical analysis of kainic acid-treated cultures demonstrated substantial preservation of cell numbers after 1 h treatment as cell death gradually progressed over a time span of 24 h (Mazzone and Nistri, 2011a). Thus, released glutamate caused only a small loss of cells at an early stage of the excitotoxic process. In conclusion, glutamate release monitoring can offer an advantageous approach to test the mechanism of action and time-dependence of neuroprotective drugs aimed at blocking the in vitro onset of excitotoxicity as early as possible. This method can also be combined with immunohistochemical identification of specific cell target populations to study selective vulnerability and neuroprotection.
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Palabras Clave
spinal cord injuryendogenous glutamate release