Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord

Mazzone, Graciela L.; Margaryan, G.; Kuzhandaivel, Anujaianthi; Nasrabady, Sara Ebrahimi; Mladinić, Miranda; Nistri, Andrea

doi:10.1016/j.neuroscience.2010.03.055

Cited by 61 publications

(124 citation statements)

References 51 publications

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“…Organotypic cultures have recently been used, as a model, to mimic early pathological processes undergoing traumatic injury (Cater et al 2007). In keeping with this notion, a recent study from our laboratory has shown that such cultures undergo very similar processes of excitotoxicity like those observed in the spinal cord (Mazzone et al 2010), thus offering an advantageous system to test the efficiency of neuroprotective agents on spinal networks. The present study, therefore, aimed at exploring whether PJ 34, employed at concentrations used for cell protection studies in vitro ), could spare neurons from either strong or threshold excitotoxic stimuli evoked by kainate.…”

Section: Introductionmentioning

confidence: 72%

“…Embryonic organotypic slice cultures of spinal cord were prepared from pregnant Wistar rat, at 13 days of gestation, in accordance with our standard procedure as recently published (Mazzone et al 2010). The foetuses were delivered by caesarean section from timed-pregnant rats, in accordance with the National Institutes of Health guidelines and the Italian act Decreto Legislativo 27/1/92 n. 116 (implementing the European Community directives n. 86/ 609 and 93/88).…”

Section: Preparation and Maintenance Of Organotypic Culturesmentioning

confidence: 99%

“…Cultures were processed for immunofluorescence analysis in accordance with our previous report (Mazzone et al 2010). Briefly, slices were blocked with 3% foetal calf serum (FCS), 3% bovine serum albumin (BSA), 0.3% Triton in PBS (blocking solution) for 1 h at room temperature, followed by overnight incubation at 4°C in a blocking solution containing the mouse NeuN antibody (1:250 dilution; clone A60; Millipore, Milan, Italy), and the rabbit polyclonal anti-S100 (1:1000; Dako, Glostrup, Denmark) or the mouse monoclonal against poly ADP ribose (PAR, 1:50; Serotec, Kidlington, UK).…”

Section: Immunofluorescence Organotypic Culturesmentioning

confidence: 99%

“…A number of investigations have, however, indicated that massive release of glutamate is a major contributor to trigger such a damage via a process termed excitotoxicity (Lau and Tymianski 2010). To elucidate the downstream effector systems activated by excitotoxicity, we have recently employed a large dose of the metabolically stable glutamate agonist kainate to produce extensive neuronal loss in the isolated rat spinal cord in vitro, which is a useful model to investigate the basic mechanisms of spinal injury (Taccola et al 2008;Mazzone et al 2010). Such a treatment evokes hyperactivation of poly(ADP-ribose) polymerase (PARP) enzyme 1 (PARP-1) that is responsible for about 90% of the cell poly(ADP-ribosylation) activity (Giansanti et al 2010) and whose pharmacological inhibition has been successfully employed as a novel therapeutic strategy to counteract the cytotoxic effects of DNA-damaging agents (Sandhu et al 2010).…”

Section: Introductionmentioning

confidence: 99%

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Effect of the PARP-1 Inhibitor PJ 34 on Excitotoxic Damage Evoked by Kainate on Rat Spinal Cord Organotypic Slices

Mazzone

Nistri

2010

Cell Mol Neurobiol

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Excitotoxicity triggered by over-activation of glutamate receptors is thought to be an early mechanism of extensive neuronal death with consequent loss of function following lesion of spinal networks. One important process responsible for excitotoxic death is 'parthanatos' caused by hyperactivation of poly(ADP-ribose) polymerase (PARP) enzyme 1. Using rat organotypic spinal slices as in vitro models, the present study enquired if 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2yl)acetamide (PJ 34), a pharmacological inhibitor of PARP-1, could counteract the excitotoxic damage evoked by transient application (1 h) of kainate, a potent analogue of glutamate. Kainate induced dose-dependent (1 μM threshold) neuronal loss (without damage to astrocytes) detected 24 h later via a PARP-1 dependent process that had peaked at 4 h after washout kainate. All spinal regions (ventral, central and dorsal) were affected, even though the largest damage was found in the dorsal area. Whereas PJ 34 did not protect against a large concentration (100 μM) of kainate, it significantly inhibited neuronal losses evoked by 10 μM kainate as long as it was co-applied with this glutamate agonist. When the application of PJ 34 was delayed to the washout time, neuroprotection was weak and regionally restricted. These data suggest that kainate-induced parthanatos developed early and was prevented by PJ 34 only when it was co-applied together with excitotoxic stimulus. Our results highlight the difficulty to arrest parthanatos as a mechanism of spinal neuron death in view of its low threshold of activation by kainate, its widespread distribution, and relatively fast development.

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Section: Introductionmentioning

confidence: 72%

Section: Preparation and Maintenance Of Organotypic Culturesmentioning

confidence: 99%

Section: Immunofluorescence Organotypic Culturesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Effect of the PARP-1 Inhibitor PJ 34 on Excitotoxic Damage Evoked by Kainate on Rat Spinal Cord Organotypic Slices

Mazzone

Nistri

2010

Cell Mol Neurobiol

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“…Notwithstanding the fact that the detailed topography of neurons recruited during stimulation with FListim remains to be explored, the increase in cycle amplitude due to stimulation with higher frequencies may correspond to the involvement of a larger number of motoneurons, as a previous study has reported a relation between number of motoneurons and cycle amplitude (Mazzone et al 2010). On the other hand, the larger cycle amplitude due to stimulation with high frequency waveforms might also be determined by the recruitment of an additional pool of motoneurons within the same segment.…”

Section: Discussionmentioning

confidence: 99%

Coapplication of noisy patterned electrical stimuli and NMDA plus serotonin facilitates fictive locomotion in the rat spinal cord

Dose

Taccola

2012

Journal of Neurophysiology

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, consisting of intrinsically variable weak waveforms applied to a single dorsal root is very effective (though not optimal as it eventually wanes away) in activating the locomotor program of the isolated rat spinal cord. The present study explored whether combination of FListim with low doses of pharmacological agents that raise network excitability might further improve the functional outcome, using this in vitro model. FListim was applied together with N-methyl-D-aspartate (NMDA) ϩ serotonin, while fictive locomotion (FL) was electrophysiologically recorded from lumbar ventral roots. Superimposing FListim on FL evoked by these neurochemicals persistently accelerated locomotorlike cycles to a set periodicity and modulated cycle amplitude depending on FListim rate. Trains of stereotyped rectangular pulses failed to replicate this phenomenon. The GABA B agonist baclofen dose dependently inhibited, in a reversible fashion, FL evoked by either FListim or square pulses. Sustained episodes of FL emerged when FListim was delivered, at an intensity subthreshold for FL, in conjunction with subthreshold pharmacological stimulation. Such an effect was, however, not found when high potassium solution instead of NMDA ϩ serotonin was used. These results suggest that the combined action of subthreshold FListim (e.g., via epidural stimulation) and neurochemicals should be tested in vivo to improve locomotor rehabilitation after injury. In fact, reactivation of spinal locomotor circuits by conventional electrical stimulation of afferent fibers is difficult, while pharmacological activation of spinal networks is clinically impracticable due to concurrent unwanted effects. We speculate that associating subthreshold chemical and electrical inputs might decrease side effects when attempting to evoke human locomotor patterns. fictive locomotion; central pattern generator; dorsal root train; FListim IN THE ISOLATED SPINAL CORD of the neonatal rat, episodes of locomotor-like oscillations can be recorded from lumbar (L) 2 and L5 ventral roots (VRs), alternating between the left and right sides of the cord, due to rhythmic activation of flexor and extensor motor pools, respectively

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Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures

Cifra

Mazzone

Nani

et al. 2012

Developmental Neurobiology

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In vitro preparations of the neonatal rat spinal cord or brainstem are useful to investigate the organization of motor networks and their dysfunction in neurological disease models. Long-term spinal cord organotypic cultures can extend our understanding of such pathophysiological processes over longer times. It is, however, surprising that detailed descriptions of the type (and number) of neurons and glia in such preparations are currently unavailable to evaluate cell-selectivity of experimental damage. The focus of the present immunohistochemical study is the novel characterization of the cell population in the lumbar locomotor region of the rat spinal cord and in the brainstem motor nucleus hypoglossus at 0-4 postnatal days, and its comparison with spinal organotypic cultures at 2-22 days in vitro. In the nucleus hypoglossus, neurons were 40% of all cells and 80% of these were motoneurons. Astrocytes (35% of total cells) were the main glial cells, while microglia was <10%. In the spinal gray matter, the highest neuronal density was in the dorsal horn (>80%) and the lowest in the ventral horn (≤57%) with inverse astroglia numbers and few microglia. The number of neurons (including motoneurons) and astrocytes was stable after birth. Like in the spinal cord, motoneurons in organotypic spinal culture were <10% of ventral horn cells, with neurons <40%, and the rest made up by glia. The present report indicates a comparable degree of neuronal and glial maturation in brainstem and spinal motor nuclei, and that this condition is also observed in 3-week-old organotypic cultures.

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Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord

Cited by 61 publications

References 51 publications

Effect of the PARP-1 Inhibitor PJ 34 on Excitotoxic Damage Evoked by Kainate on Rat Spinal Cord Organotypic Slices

Effect of the PARP-1 Inhibitor PJ 34 on Excitotoxic Damage Evoked by Kainate on Rat Spinal Cord Organotypic Slices

Coapplication of noisy patterned electrical stimuli and NMDA plus serotonin facilitates fictive locomotion in the rat spinal cord

Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures

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