TNF-α and glutamate preconditioning effects on calcium dynamics in rat hippocampal cultures

Orla Watters, John O’Connor. UCD School Biomol and Biomed Sci, Conway Institute, Dublin 4, Ireland.

Ischemic tolerance is a phenomenon which results in preconditioning of the affected area to become more resilient against a subsequent more severe ischemic insult. As TNF-α/glutamate are elevated during a mild transient ischemic attack (TIA), their potential involvement in the development of ischemic tolerance is undergoing extensive research.

As calcium is a well-established mediator of cell death, we investigated the individual calcium-mediated effects of acute mild preconditioning with TNF-α or glutamate within the CA region of organotypic hippocampal cultures2. Hippocampal cultures (400 µm) were prepared from P6-9 Wistar rats (humanely killed – decapitated after isoflurane anaesthesia). All procedures were carried out in accordance with the animals ethics committee of University College Dublin. After 6 days in vitro the cultures were exposed to 30 µM glutamate/5 ng/ml TNF-α, for 30 min followed by 24 h recovery before experimentation.

We then assessed the effect of the preconditioning treatments on resting cellular calcium levels, using Indo-1. The proportion of spontaneously active cells at rest and the frequency of the Ca2+ events of these cells were analysed using Fluo-4. We also assessed changes in glutamate-evoked Ca2+ influx in the preconditioned cultures.

Pretreatment with TNF-α/glutamate caused a similar level of reduction in subsequent glutamate-induced Ca2+ influx 24 h post treatment (control: 100.0±0.8%, n = 7362 cells from > 3 animals and in all subsequent values; TNF-α: 76.8±1.0%, n = 5543; glutamate: 75.3±1.4%, n = 3859; p<0.001). Both preconditioning agents also resulted in a reduction in the proportion of responsive cells within the CA region (control; p = 0.459, n = 14968, TNF-α; p = 0.400, n = 15218; glutamate; p = 0.388, n = 13919) along with a depression of the frequency of spontaneous Ca2+ events within these cells (Vs. control: TNF-α: p>0.00001, D = 0.0454; glutamate: p>0.0001, D = 0.0534) after the 24 h recovery period. However, TNF-α and glutamate induced opposite effects on calcium levels at rest. Inhibition of the p38 MAP kinase pathway (10 µM SB 203580) reversed the effects seen by TNF-α (TNF-α+SB: 104.7±1.3%, n = 1579, p>0.05) but not that by glutamate (glut + SB: 76.4±1.6%, n = 811; p>0.05). The mode of action of glutamate-mediated preconditioning remains inconclusive.

Overall these results suggest that TNF-α/glutamate preconditioning reduce overall calcium-associated responsiveness of the cells of the CA region of the hippocampal cultures, which may in turn reduce Ca2+-mediated cell injury to a subsequent ischemic or excitotoxic insult.