Intracellular Calcium and Cell Death during Ischemia in Neonatal Rat White Matter AstrocytesIn Situ

Abstract

The major pathological correlate of cerebral palsy is ischemic injury of CNS white matter. Histological studies show early injury of glial cells and axons. To investigate glial cell injury, I monitored intracellular Ca2+and cell viability in fura-2-loaded neonatal rat white matter glial cells during ischemia. Fura-2 fixation combined with immunohistochemistry revealed that fura-2-loaded cells were GFAP+/O4−and were therefore a population of neonatal white matter astrocytes.Significant ischemic Ca2+influx was found, mediated by both L- and T-type voltage-gated Ca2+channels. Ca2+influx via T-type channels was the most important factor during the initial stage of ischemia and was associated with significant cell death within 10–20 min of the onset of ischemia. The Na+–Ca2+exchanger acted to remove cytoplasmic Ca2+throughout the ischemic and recovery periods. Neither the release of Ca2+from intracellular stores nor influx via glutamate-gated channels contributed to the rise in intracellular Ca2+during ischemia. Ischemic cell death was reduced significantly by removing extracellular Ca2+or by blocking voltage-gated Ca2+channels. The exclusively voltage-gated Ca2+channel nature of the Ca2+influx, the role played by T-type Ca2+channels, the protective effect of the Na+–Ca2+exchanger, and the lack of significant Ca2+release from intracellular stores are features of ischemia that have not been reported in other CNS cell types.

The Mechanisms of Acute Ischemic Injury in the Cell Processes of Developing White Matter Astrocytes

“…While ischemic cell death of pre-myelinating white matter astrocytes is Ca 2 + -dependent (Fern, 1998), we have recently shown that Ca 2 + does not play a role in the death of astrocytes in white matter at a similar developmental stage to those studied here, at least during relatively short periods of ischemia ). In the current study, initial experiments showed that removing Ca 2 + from the extracellular space by perfusing with aCSF to which no Ca 2 + had been added and which contained 70 mmol/L EGTA ('zero-Ca 2 + '), resulted in significant damage to processes at 40 mins (48.374.1% decrease in pixel intensity; n = 5 nerves; P < 0.001) and 60 mins (76.973.1%; P < 0.001; Figures 7A and 7D).…”

Augmented Vasoconstriction and Thromboxane Formation by 15-F 2t -Isoprostane (8-Iso-Prostaglandin F 2α ) in Immature Pig Periventricular Brain Microvessels

“…Astrocytes contain various calcium channels. 37,38,43 The increase in thromboxane formation and calcium signals in astrocytes was inhibited by the selective N-type voltage-gated calcium channel blocker -conotoxin 35 but not by L-type voltage-gated channel blocker nifedipine and putative inhibitor of non-voltage-gated calcium channels SK&F96365. 34 This finding would suggest that in astrocytes, 15-F 2t -IsoP stimulates thromboxane formation by enhancing entry of calcium mainly via N-type voltage-gated calcium channels.…”

“…We focused on receptor-operated and N-as well as L-type voltage-gated Ca 2ϩ channels since endothelial cells are not excitable and are mostly devoid of voltage-gated Ca 2ϩ channels, 36 whereas astrocytes contain voltage-gated channels, primarily of the N-and L-types. 37,38,43 The putative receptoroperated Ca 2ϩ channel blocker SK&F96365 34 selectively reduced TXB 2 formation in endothelial cells, and the selective N-voltage-gated Ca 2ϩ channel blocker -conotoxin 35 caused a similar effect only in astrocytes ( Figure 4); Ca 2ϩ chelator EGTA was effective in both cells. Therefore, TXB 2 formation induced by 15-F 2t -IsoP is dependent on extracellular Ca 2ϩ , which seems to influx through activation of distinct Ca 2ϩ channels in endothelial and astroglial cells.…”

Acute Lipopolysaccharide-Mediated Injury in Neonatal White Matter Glia: Role of TNF-α, IL-1β, and Calcium

“…The atmosphere in the recording chamber was switched simultaneously to 95% N 2 /5% CO 2 (see Ref. 26 for further details). Ischemia was maintained for 60 min, at which point normal conditions were re-established for a further 15 min.…”

“…RONs were maintained in hydrated 95% O 2 /5% CO 2 atmosphere during the incubation period and were washed in aCSF before being mounted in the perfusion chamber. The ends of the optic nerves were fixed to a 22 ϫ 40 mm glass coverslip with small amounts of cyanoacrylate glue, leaving the majority of the nerve completely free of glue (26). The coverslip was sealed onto a Plexiglas perfusion chamber (atmosphere chamber; Warner Instruments) with silicone grease.…”