Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia.

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Protection with metabotropic glutamate 1 receptor antagonists in models of ischemic neuronal death: time-course and mechanisms.

In order to study the role of metabotropic glutamate 1 (mGlu1) receptors in ischemic neuronal death, we examined the effects of the recently characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) in murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen glucose deprivation (OGD) and in vivo, following transient global ischemia in gerbils. AIDA and CBPG significantly reduced neuronal death when added to the incubation medium during the OGD insult and the subsequent recovery period. Neuroprotection was observed even when these compounds were added up to 60 min (in cortical neurons) or 30 min (in hippocampal slices) after OGD. In vivo, i.c.v. administration of AIDA and CBPG reduced hippocampal CA1 pyramidal cell injury following transient global ischemia. Neuroprotection was also observed when AIDA was added to the hippocampal perfusion fluid in microdialysis experiments, and this effect was associated with an increase in the basal output of GABA. These findings demonstrate that AIDA and CBPG are neuroprotective when administered during the maturation of ischemic damage and that different mechanisms are likely to be involved in mediating their effects following blockade of mGlu1 receptors in cortical and hippocampal neurons.

Neuroprotective effects of kynurenine-3-hydroxylase inhibitors in models of brain ischemia.

The neuroprotective effects of two kynurenine hydroxylase inhibitors, (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfona mide (Ro 61-8048), were studied in vitro and in vivo. In organotypic hippocampal slice cultures deprived of oxygen and glucose, these inhibitors significantly reduced neuronal damage. In gerbils subjected to bilateral carotid occlusion for 5 min, the administration of mNBA (400 mg/kg i.p., 3 times) or Ro 61-8048 (40 mg/kg i.p., 3 times) dramatically decreased the percentage of damaged pyramidal neurones in the hippocampal CA1 region. Finally, in rats with permanent occlusion of the middle cerebral artery, mNBA (200-400 mg/kg i.p.) and Ro 61-8048 (40 mg/kg i.p.) administration reduced the infarct volume. Our results demonstrate that ischemic neuronal damage may be significantly decreased by inhibiting kynurenine hydroxylase.

Type 2 metabotropic glutamate (mGlu) receptors tonically inhibit transmitter release in rat caudate nucleus: in vivo studies with (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, a new potent and selective antagonist.

Anatomical, biochemical and electrophysiological studies have previously shown that cortico-striatal terminals contain abundant presynaptic group 2 metabotropic glutamate (mGlu) receptors. Using brain slices we have previously shown that these receptors inhibit depolarization-induced transmitter release. Using microdialysis in freely moving rats, we now report the effects of group 2 mGlu receptor agonists and antagonists on glutamate concentration in the caudate extracellular fluid. A mild decrease (20-30%) in glutamate concentration in caudate dialysates was observed when 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid or (2S,3S,4S)-alpha-carboxycyclopropyl-glycine (L-CCG-1), mGlu receptor agonists, was locally administered. On the contrary, alpha-methyl-4-carboxyphenylglycine, an antagonist of type 1 and type 2 mGlu receptors, increased the glutamate concentration in dialysates by up to 3.5-fold, and its effects were prevented by the simultaneous administration of L-CCG-1, a preferential type 2 mGlu receptor agonist. A significant increase of glutamate output in striatal dialysate was also found after local administration of (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, another structurally unrelated, relatively selective and potent type 2 mGlu receptor antagonist. The results suggest that type 2 mGlu receptors tonically inhibit transmitter release from cortico-striatal terminals. Since the cortico-striatal pathway profoundly affects the function of a large percentage of caudate neurons, it is reasonable to predict that the use of selective type 2 mGlu receptor agents will be helpful for scientific and therapeutic studies on the physiopathology of basal ganglion disorders.

Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent mGluR1 antagonist.

We examined the pharmacological profile of 1-aminoindan-1,5-dicarboxylic acid (AIDA), a rigid (carboxyphenyl)glycine derivative acting on metabotropic glutamate receptors (mGluRs). In cells transfected with mGluR1a, AIDA competitively antagonized the stimulatory responses of glutamate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] on phosphoinositide hydrolysis (pA2 = 4.21). In cells transfected with mGluR5a, AIDA displayed a much weaker antagonist effect. In transfected cells expressing mGluR2, AIDA (< or = 1 mM) did not affect the inhibition of forskolin-stimulated adenylate cyclase activity induced by (1S,3R)-ACPD, but at large concentrations, it displayed a modest agonist activity. In rat hippocampal or striatal slices, AIDA (0.1-1 mM) reduced the effects of (1S,3R)-ACPD on phospholipase C but not on adenylate cyclase responses, whereas (+)-alpha-methyl-4-carboxyphenylglycine (0.3-1 mM) was an antagonist on both transduction systems. In addition, AIDA (0.3-1 mM) had no effect on mGluRs coupled to phospholipase D, whereas (+)-alpha-methyl-4-carboxy-phenylglycine (0.5-1 mM) acted as an agonist with low intrinsic activity. In rat cortical slices, AIDA antagonized the stimulatory (mGluR1-mediated) effect of (1S,3R)-ACPD on the depolarization-induced outflow of D-[3H]aspartate, disclosing an inhibitory effect ascribable to (1S,3R)-ACPD activating mGluR2 and/or mGluR4. Finally, mice treated with AIDA (0.1-10 nmol i.c.v.) had an increased pain threshold and difficulties in initiating a normal ambulatory behavior. Taken together, these data suggest that AIDA is a potent, selective and competitive mGluR1 a antagonist.