Bashkim Misini1, Wolfhardt Freinbichler1, Maria Alessandra Colivicchi2, Kemajl Bisilimi3, Wolfgang Linert1, Keith F Tipton4, Laura Della Corte5. 1. Institute for Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria. 2. Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy. 3. Department of Biology, University of Prishtina, Kosovo. 4. School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. 5. Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy. Electronic address: laura.dellacorte@unifi.it.
Abstract
BACKGROUND: Terephthalate (TA(2-)), which reacts with highly reactive oxygen species (hROS) to form the fluorophor 2-hydroxy terephthalic acid (OH-TA) with a high selectivity, has been used for determining hROS formation during in vivo microdialysis. Previously this involved collecting fractions of the microdialysate and determining the OH-TA formed after HPLC (the batch method). NEW METHOD: This work reports the development and validation of a procedure for continuously determining hROS formation during microdialysis. TA(2-) was added to the artificial cerebrospinal fluid (aCSF) perfusing medium to trap hROS. OH-TA formation was detected in real time with a sensitive fluorescence detector equipped with a capillary flow cell that was coupled directly to the effluent stream of the microdialysis system. RESULTS: The behaviour of the system was assessed by comparison with the batch method and using a well-characterized animal model of excitotoxic damage, based on the application of high concentrations (1mM and 500μM) of the non-NMDA glutamate receptor agonist kainate (KA) to the neostriatum. Data for the evoked release of taurine were also determined in these samples. No temporal difference between hROS and taurine release could be detected. COMPARISON WITH EXISTING METHOD(S): The flow method had a comparable sensitivity of hROS detection to the batch method. It was simpler, cheaper and less time-consuming than the batch method. CONCLUSIONS: This direct system is convenient and technically undemanding. It should be useful for the rapid assessment of the hROS responses to neurotoxins and other compounds in microdialysis experiments in vivo.
BACKGROUND:Terephthalate (TA(2-)), which reacts with highly reactive oxygen species (hROS) to form the fluorophor 2-hydroxy terephthalic acid (OH-TA) with a high selectivity, has been used for determining hROS formation during in vivo microdialysis. Previously this involved collecting fractions of the microdialysate and determining the OH-TA formed after HPLC (the batch method). NEW METHOD: This work reports the development and validation of a procedure for continuously determining hROS formation during microdialysis. TA(2-) was added to the artificial cerebrospinal fluid (aCSF) perfusing medium to trap hROS. OH-TA formation was detected in real time with a sensitive fluorescence detector equipped with a capillary flow cell that was coupled directly to the effluent stream of the microdialysis system. RESULTS: The behaviour of the system was assessed by comparison with the batch method and using a well-characterized animal model of excitotoxic damage, based on the application of high concentrations (1mM and 500μM) of the non-NMDA glutamate receptor agonist kainate (KA) to the neostriatum. Data for the evoked release of taurine were also determined in these samples. No temporal difference between hROS and taurine release could be detected. COMPARISON WITH EXISTING METHOD(S): The flow method had a comparable sensitivity of hROS detection to the batch method. It was simpler, cheaper and less time-consuming than the batch method. CONCLUSIONS: This direct system is convenient and technically undemanding. It should be useful for the rapid assessment of the hROS responses to neurotoxins and other compounds in microdialysis experiments in vivo.