Reducing conditions produce a loss of neuroprotective efficacy of competitive but not non-competitive antagonists in a model of NMDA-mediated excitotoxicity in organotypic hippocampal slice cultures

Experimental data indicate that NMDA receptor activation is strongly implicated in the pathogenesis of cerebral ischaemia. However, the results from in vivo studies are equivocal, with NMDA antagonists being active in only some models. It has recently been demonstrated that competitive and non-competitive NMDA antagonists behave differently under normal and ischaemic conditions. These studies have used organotypic hippocampal slice cultures to investigate whether this disparity is due to redox-modulation of the NMDA receptor which occurs in ischaemia. NMDA-mediated toxicity was concentration dependent with little damage occurring with less than 10 µM NMDA and maximal damage produced by 30 µM. NMDA toxicity was significantly enhanced by pre-treatment with 1 mM dithiothreitol, a reducing agent, such that damage occurred at 1 nM NMDA, and maximal damage was produced by 10 µM. The efficacy of MK-801 was not altered by reducing conditions, but the EC50 of the competitive antagonist APV was increased by 20-fold. These data strongly suggest that the neuroprotective efficacy of NMDA antagonists is significantly altered under ischaemic conditions, and that more beneficial effects will be obtained with antagonists having a higher affinity for the receptor in the reduced configuration

Reducing conditions significantly attenuate the neuroprotective efficacy of competitive, but not other NMDA receptor antagonists in vitro

Inappropriate activation of NMDA receptors during a period of cerebral ischaemia is a crucial event in the pathway leading to neuronal degeneration. However, significant research has failed to deliver a clinically active NMDA receptor antagonist, and competitive NMDA antagonists are ineffective in many experimental models of ischaemia. The NMDA receptor itself has a number of modulatory sites which may affect receptor function under ischaemic conditions. Using rat organotypic hippocampal slice cultures we have investigated whether the redox modulatory site affects the neuroprotective efficacy of NMDA receptor antagonists against excitotoxicity and experimental ischaemia (OGD). NMDA toxicity was significantly enhanced in cultures pretreated with a reducing agent. The noncompetitive antagonist MK-801 and a glycine-site blocker were equally neuroprotective in both normal and reduced conditions, but there was a significant rightward shift in the dose–response curves of the competitive antagonists APV and CPP and the uncompetitive antagonist memantine. OGD produced neuronal damage predominantly in the CA1 region, which was prevented by MK-801 and memantine, but not by APV or CPP. Inclusion of an oxidizing agent during the period of OGD had no effect alone, but significantly enhanced the neuroprotective potency of the competitive antagonists. These data clearly demonstrate that chemical reduction of the redox modulatory site of the NMDA receptor decreases the ability of competitive antagonists to block NMDA receptor-mediated neuronal damage, and that the reducing conditions which occur during simulated ischaemia are sufficient to produce a similar effect. This may have important implications for the design of future neuroprotective agents

Interleukin-1? exacerbates hypoxia-induced neuronal damage, but attenuates toxicity produced by simulated ischaemia and excitotoxicity in rat organotypic hippocampal slice cultures

Using organotypic hippocampal slice cultures we have investigated the actions of Interleukin-1 (IL-1) in a number of injury paradigms. Low concentrations of IL-1 potentiated hypoxia-induced neurodegeneration whilst high concentrations had no effect. In contrast, higher concentrations of IL-1 were strongly neuroprotective in models of combined oxygen/glucose deprivation and N-methyl-D-aspartate toxicity, but no potentiation was observed at low IL-1 concentrations. Both protective and toxic effects of IL-1 were fully antagonized by IL-1 receptor antagonist. These data demonstrate that the effects of IL-1 on neuronal injury are complex, and may be directly related to the injury paradigm studied

Characterisation of a novel class of polyamine-based neuroprotective compounds

Prolonged cerebral ischaemia initiates complex intra- and inter-cellular signalling cascades ultimately resulting in neuronal death. Well-characterised mediators of ischaemic cell death are glutamate, free radicals and nitric oxide. Many drugs that block these mechanisms are neuroprotective in vitro, but have unfavourable side-effect profiles in man. We have recently demonstrated that the compound L-arginyl-3,4-spermidine (L-Arg3,4) is neuroprotective in vitro through an interaction with several of these mechanisms, and prevents ischaemic neurodegeneration in vivo with no gross side effects. In this study, we have used solid-phase combinatorial chemistry, to synthesise a number of analogues of L-Arg3,4, and investigate the structure-activity relationship using an in vitro, organotypic hippocampal slice culture model of cerebral ischaemia. A number of molecular features were identified which were essential for the neuroprotective activity including the requirement for a positive charge and an amino acid in the L-configuration. Relatively minor alterations to both the terminal arginine and polyamine moieties significantly attenuated neuroprotective efficacy. Our data implies that these compounds are neuroprotective through a currently undefined mechanism rather than non-specific ionic interactions described previously for other polyamine-containing compounds

Polymorphisms in matrix metalloproteinase-1, -3, -9, and -12 genes in relation to subarachnoid hemorrhage

Background and Purpose: Intracranial aneurysm, which underlies the vast majority of subarachnoid hemorrhage incidences, has a multifactorial etiology, and the importance of genetic factors is increasingly recognized. Development and rupture of intracranial aneurysms involve degradation and remodeling of the vascular wall matrix in which the matrix metalloproteinases (MMPs) play an important role. The possible impact of MMP gene polymorphisms on susceptibility to intracranial aneurysms is still controversial, with conflicting data from different reported studies. Methods: In this study we analyzed 5 different functional promoter polymorphisms in the MMP-1, MMP-3, MMP-9, and MMP-12 genes in a sample of 92 patients with aneurysmal subarachnoid hemorrhage and 158 healthy control subjects, all from southern England. Results: No significant difference was detected between the patient and control groups in genotype distribution of any of the polymorphisms studied. Conclusions: The data do not support the hypothesis that MMP gene variations influence the development of intracranial aneurysms in the population studied

A microdialysis method for the recovery of IL-1beta, IL-6 and nerve growth factor from human brain in vivo

Intracerebral microdialysis is used extensively as a research tool in the investigation of the neurochemical and metabolic changes that occur following acute brain injury. Microdialysis has enabled elucidation of intra-cerebral levels of substances such as lactate, pyruvate and glycerol but, as yet, has not been used effectively to recover macromolecules from the human brain. Traumatic brain injury is known to result in the generation of cytokines and neurotrophins into extracellular fluid compartment of the brain, with effects on neuronal damage and repair. We have developed a technique of in vivo sampling of the interstitial fluid of the brain of patients with severe head injuries which has allowed the measurement of IL-1?, IL-6 and nerve growth factor. This report confirms the safety and effectiveness of this modified microdialysis method in the clinical setting of a neurological intensive care unit. The technique provides a timely addition to the armamentarium of the clinical scientist and will potentially lead to a greater understanding of neuroinflammation following acute traumatic brain injury