Acute Ischemic Injury of Astrocytes Is Mediated by Na-K-Cl Cotransport and not Ca²⁺Influx at a Key Point in White Matter Development

Abstract

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca2+]i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca2+ prevented increases in [Ca2+]i but increased the amount of cell death. Astrocytes showed rapid [Na+]i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na+ or Cl- or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca2+, which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore, NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca2+-dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.

Functional glutamate transport in rodent optic nerve axons and glia

“…In addition, the presence of functional GLT1 demonstrates the potential for transport-mediated glutamate release from these cells. Thomas et al (2004) also demonstrated a high resting intracellular glutamate levels in oligodendrocytes that was elevated following ischemia, suggesting both effective glutamate uptake under normal conditions and the absence of uptake reversal during ischaemia. It is not known how [Na + ] i , pH i and membrane potential are affected by ischemia in these cells but it would appear that the conditions are not met for significant reversal of glutamate uptake in this preparation (but see Back et al 2006).…”

“…Average Daspartate uptake into astrocytes was higher than into any other cell compartment, although the degree of uptake varied considerably from cell to cell. Recent studies have shown rapid astrocyte swelling leading to necrosis in ischemic neonatal white matter, F o r P e e r R e v i e w 13 which will liberate astrocyte glutamate into the extracellular space in a glutamate transporter independent fashion (Thomas et al, 2004). In addition, the presence of functional GLT1 demonstrates the potential for transport-mediated glutamate release from these cells.…”

“…Nerves were post-fixed with 2% osmium tetroxide and dehydrated prior to infiltration in epoxy. Sections were counterstained with uranyl acetate and lead citrate and examined with a Jeol 100CX electron microscope, see (Thomas et al, 2004) for further details. Antibodies were the same as listed above for light immunohistochemistry.…”

“…Ischemia evokes significant rises in extracellular glutamate concentration in white matter (Shimada et al, 1993;Chiu and Kriegler, 1994), and recent immuno-electron microscopy studies suggest that glutamate is released from both astrocytes and axons in ischemic neonatal white matter (Li et al, 1999;Wilke et al, 2004). There is little information about the type of transporters mediating glutamate uptake in white matter tracts of the central nervous system.…”

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

“…At this point in development the mouse optic nerve was at a similar developmental point to the P10 rat optic nerves we have previously examined , with the majority of axons pre-myelinated ( Figures 3A and 3B). In most cases, glial cells could be readily identified using established criteria (see Materials and methods and Thomas et al, 2004). In control nerves perfused for 60 mins in aCSF before fixation, astrocyte processes radiated from the somata in the form of either thick primary processes or finer processes that originated directly from the somata or derived from branching of the primary processes.…”