ABSTRACT
Title
Characterization of the effects induced by hypoxic ischemia and reperfusion in rodent organotypic brain slices and involvement of purinergic signalling
Authors
L. Colombo
Dept. of Pharmacological Sciences, Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, University of Milan, Italy
Dept. of Biotechnologies and Biosciences, Laboratory of Neurophysiology, University of Milan-Bicocca, Italy
Doctorate School in Pharmacological Sciences, Ph.D. Program in Pharmacotoxicology, Pharmacognosy and Pharmacological Biotechnologies, XXIV cycle
Dept. of Pharmacological Sciences, Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, University of Milan, Italy
Dept. of Biotechnologies and Biosciences, Laboratory of Neurophysiology, University of Milan-Bicocca, Italy
Doctorate School in Pharmacological Sciences, Ph.D. Program in Pharmacotoxicology, Pharmacognosy and Pharmacological Biotechnologies, XXIV cycle
Abstract
Besides acting as metabolic intermediates, ATP and extracellular nucleotides are key extracellular signalling molecules in many organs and systems, including the central nervous system (CNS), profoundly influencing cell survival under acute or chronic degenerative conditions. Their actions are mediated by 7 ionotropic P2X [1] and 8 metabotropic P2Y purinergic receptors [2] and terminated by various ATP metabolizing enzymes [3].We are interested in verifying if and how purinergic signalling can contribute to the survival, repair and regeneration of neurons and neuronal circuitries in organotypic brain slices after oxygen-glucose deprivation (OGD), which mimics in vitro the cytotoxicity induced by ischemia in vivo.
Our experimental model consists of organotypic co-cultures of rodent brain slices from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC), maintained in culture up to 3 weeks. In these cultures it has been demonstrated that dopaminergic mesencephalic neurons at the VTA-SN are able to properly and spontaneously innervate the target areas in the PFC, as it occurs in vivo [4]. These new-born projections are functional: electrophysiological recordings, performed in collaboration with prof. E. Wanke’s laboratory at University of Milan-Bicocca using the Multi Electrode Array (MEA) technique, showed that co-cultures display a spontaneous activity in the form of bursts and local field potentials, which are able to propagate from VTA to PFC and back [5]. Furthermore, different purinergic receptors subtypes are regionally and cell type-dependent expressed, suggesting an important role of purinergic signalling in the development and growth of the CNS [6].
The main topic of this work is the characterization of the OGD and reperfusion induced damages in order to further study the effect of purinergic agents on nerve fibres regeneration and on the reconstruction of damaged neuronal circuitries. Cytotoxicity assay showed significant tissue damage after a 1h OGD followed by 1-6-18-24h reperfusion times and this data correlated with the increased number of Propidium Iodide-positive cells in OGD-reperfused cultures. We have then focussed on the effects on neurons and on the glial population (namely, astroglia, microglia and oligodendroglia), in order to understand the modifications of the glial response after injury and their possible role in damage propagation or recovery. As expected, the number of neuron is strongly reduced after OGD, microglial cells were activated at longer times of reperfusion, while a trend to decreased oligodendroglia cell number was highlighted. The number of astroglial cells is not affected by OGD, even if the staining pattern for GFAP, a typical astrocyte marker, appears to be more diffuse after 1h OGD.
As a first step to verify how purinergic signals affect survival and regeneration of neural cells consequent to OGD [7], we applied Pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS), a non-selective P2Y receptor antagonist, during the 24h reperfusion time: preliminary results showed an interesting trend to a reduction in the amount of damaged neurons. We are evaluating the effects of this agent on microglial cells activation.
Sponsored by Fondazione Cariplo 2008 “Functional regeneration of the mesocorticolimbic dopaminergic system as a model to study novel neuroreparative strategies”.
1) Jarvis MF and Khakh BS (2009) Neuropharmacology, 56, 208-15
2) Abbracchio M et al. (2006) Pharmacol Rev, 58, 281‐341
3) Yegutkin G (2003) Biochim Biophys Acta, 1783, 673-694
4) Franke H et al. (2003) Neurochem Int, 42, 431-439
5) Dossi E et al. (2010) Proceedings of the 40th Annual Neuroscience Meeting, 318.5
6) Heine C et al. (2007) Neuroscience, 149, 165-181
7) Franke H et al. (2006) Pflugers Archiv, 452, 622-644
Our experimental model consists of organotypic co-cultures of rodent brain slices from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC), maintained in culture up to 3 weeks. In these cultures it has been demonstrated that dopaminergic mesencephalic neurons at the VTA-SN are able to properly and spontaneously innervate the target areas in the PFC, as it occurs in vivo [4]. These new-born projections are functional: electrophysiological recordings, performed in collaboration with prof. E. Wanke’s laboratory at University of Milan-Bicocca using the Multi Electrode Array (MEA) technique, showed that co-cultures display a spontaneous activity in the form of bursts and local field potentials, which are able to propagate from VTA to PFC and back [5]. Furthermore, different purinergic receptors subtypes are regionally and cell type-dependent expressed, suggesting an important role of purinergic signalling in the development and growth of the CNS [6].
The main topic of this work is the characterization of the OGD and reperfusion induced damages in order to further study the effect of purinergic agents on nerve fibres regeneration and on the reconstruction of damaged neuronal circuitries. Cytotoxicity assay showed significant tissue damage after a 1h OGD followed by 1-6-18-24h reperfusion times and this data correlated with the increased number of Propidium Iodide-positive cells in OGD-reperfused cultures. We have then focussed on the effects on neurons and on the glial population (namely, astroglia, microglia and oligodendroglia), in order to understand the modifications of the glial response after injury and their possible role in damage propagation or recovery. As expected, the number of neuron is strongly reduced after OGD, microglial cells were activated at longer times of reperfusion, while a trend to decreased oligodendroglia cell number was highlighted. The number of astroglial cells is not affected by OGD, even if the staining pattern for GFAP, a typical astrocyte marker, appears to be more diffuse after 1h OGD.
As a first step to verify how purinergic signals affect survival and regeneration of neural cells consequent to OGD [7], we applied Pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS), a non-selective P2Y receptor antagonist, during the 24h reperfusion time: preliminary results showed an interesting trend to a reduction in the amount of damaged neurons. We are evaluating the effects of this agent on microglial cells activation.
Sponsored by Fondazione Cariplo 2008 “Functional regeneration of the mesocorticolimbic dopaminergic system as a model to study novel neuroreparative strategies”.
1) Jarvis MF and Khakh BS (2009) Neuropharmacology, 56, 208-15
2) Abbracchio M et al. (2006) Pharmacol Rev, 58, 281‐341
3) Yegutkin G (2003) Biochim Biophys Acta, 1783, 673-694
4) Franke H et al. (2003) Neurochem Int, 42, 431-439
5) Dossi E et al. (2010) Proceedings of the 40th Annual Neuroscience Meeting, 318.5
6) Heine C et al. (2007) Neuroscience, 149, 165-181
7) Franke H et al. (2006) Pflugers Archiv, 452, 622-644