title: Preconditioning effects of tumor necrosis factor-α and glutamate on calcium dynamics in rat organotypic hippocampal cultures.
abstract: During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels in the hippocampus may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. In this study we have investigated the individual contribution of pre-exposure to TNF-α or glutamate in the development of 'ischemic tolerance' to a subsequent insult, using organotypic hippocampal cultures. At 6 days in vitro (DIV), cultures were exposed to an acute concentration of glutamate (30 μM) or TNF-α (5 ng/ml) for 30 min, followed by 24h recovery period. We then examined the effect of the pretreatments on calcium dynamics of the cells within the CA region. We found that pretreatment with TNF-α or glutamate caused in a significant reduction in subsequent glutamate-induced Ca(2+) influx 24h later (control: 100.0 ± 0.8%, n=7769 cells; TNF-α: 76.8 ± 1.0%, n=5543 cells; glutamate: 75.3 ± 1.4%, n=3859 cells; p<0.001). Antagonism of circulating TNF-α (using infliximab, 25 μg/ml), and inhibition of the p38 MAP kinase pathway (using SB 203580, 10 μM) completely reversed this effect. However glutamate preconditioning did not appear to be mediated by p38 MAP kinase signalling, or NMDAR activation as neither SB 203580 nor D-AP5 (100 μM) altered this effect. Glutamate and TNF-α preconditioning resulted in small yet significant alterations in resting Ca(2+) levels (control: 100.0 ± 0.9%, n=2994 cells; TNF-α: 109.7 ± 1.0%, n=2884 cells; glutamate; 93.3 ± 0.8%, n=2899 cells; p<0.001), TNF-α's effect reversed by infliximab and SB 203580. Both TNF-α and glutamate also resulted in the reduction of the proportion (P) of responsive cells within the CA region of the hippocampus (control; P=0.459, 0.451 ≤ x ≥ 0.467, n=14,968 cells, TNF-α; P=0.40, 0.392 ≤ x ≥ 0.407, n=15,218; glutamate; P=0.388, 0.303 ≤ x ≥ 0.396, n=13,919 cells), and in the depression of the frequency of spontaneous Ca(2+) events (vs. control: TNF-α: p>0.00001, D=0.0454; glutamate: p>0.0001, D=0.0534). Our results suggest that attenuation in resting Ca(2+) activity and Ca(2+) related responsiveness of cells within the CA region as a result of glutamate or TNF-α pre-exposure, may contribute to the development of ischemic tolerance.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Orla
authors.initials: O
authors.lastname: Watters
authors.forename: Mark
authors.initials: M
authors.lastname: Pickering
authors.forename: John J
authors.initials: JJ
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004334:Drug Administration Schedule; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000494:pharmacology* / Q000633:toxicity; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D008297:Male; D017711:Nonlinear Dynamics; D009924:Organ Culture Techniques; D011419:Propidium; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D007093:Imidazoles; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D018698:Glutamic Acid; C056639:2-amino-5-phosphopentanoic acid; D014633:Valine; C093642:SB 203580; D002118:Calcium