Effect of the PARP-1 inhibitor PJ 34 on excitotoxic damage evoked by kainate on rat spinal cord organotypic slices

Abstract

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.

Fig. 1

Time-dependence of PAR immunofluorescence in organotypic slices after treatment with kainate. a Examples of PAR staining in control condition (left) and after 1 or 6 h after washing out kainate (0.1 mM for 1 h). Immunopositivity signals are almost absent in control condition. The inset to the middle panel shows an example of the PAR immunoreactivity detected as a cytoplasmic rim around the nucleus. All calibration bars = 75 μm. b Histograms showing the increase (with respect to control) in the number of PAR positive nuclei at various times after washing out kainate. The data are from three experiments with six slices; *P < 0.05, **P < 0.01 vs. control. c Plots of percentage of cells (in the central region of the slice) with condensed chromatin nucleus (pyknosis; left; filled squares) or percent of NeuN positive cells (neurons; right; filled diamonds) for various concentrations of kainate (log scale) in the range from 1 μM to 1 mM (applied for 1 h). Data were collected after 24 h washout from at least three different experiments, n = 4–12; **P < 0.01, ***P < 0.001 vs. control

Fig. 2

The PARP-1 inhibitor PJ 34 could counteract the toxic effect of kainate. a Example of the ventral region with cells manifesting condensed chromatin 24 h after 1 h application of kainate (10 μM). Fewer pyknotic nuclei were apparent when PJ 34 was co-applied with kainate or applied at the washout time. b Histograms showing average percent of pyknotic cells in the three regions of interests after 10 μM kainate alone, or kainate together with PJ 34, or kainate plus PJ 34 applied 1 h later at washout. c Histograms showing average percent of pyknotic cells in the three regions of interests after 0.1 mM kainate alone, or kainate together with PJ 34, or kainate plus PJ 34 applied 1 h later at washout. Average data are from three experiments in which 7–12 slices were used. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. kainate treatment

Fig. 3

Neuronal loss evoked by kainate was inhibited by PJ 34 co-treatment. a Example of how neuronal loss (in the central region) evoked by kainate (10 μM; 1 h) was counteracted by co-application of PJ 34 (30 μM). Images were collected after 24 h washout. b Histograms showing the average number of NeuN positive cells in the three regions analyzed. Each bar represents mean data from three experiments in which 6–11 slices were used. ^# P < 0.05 vs. kainate treatment

Fig. 4

Characterization of kainate effects on S100 positive astrocytes with or without application of PJ 34. a Examples of S100 immunopositive astrocytes in the ventral region after kainate (0.1 mM; 1 h; middle) and 24 h washout with PJ 34 (right). Note good expression of S100 immunoreactivity in the three panels. b On the same preparations shown in Fig. 3, S100 immunostaining was quantified (with densitometry analysis of a 500 × 500 μm area) to provide mean data for at least three experiments in which 4–12 slices were used. Note no significant change versus control