The Mechanisms of Energy Crisis in Human Astrocytes After Subarachnoid Hemorrhage

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Abstract

BACKGROUND:

Calcium (Ca2+) is a cofactor of multiple cellular processes. The mechanisms that lead to elevated cytosolic Ca2+ concentration are unclear.

OBJECTIVE:

To illuminate how bloody cerebrospinal fluid (bCSF) from patients with intraventricular hemorrhage causes cell death of cultured human astrocytes.

METHODS:

Cultured astrocytes were incubated with bCSF. In control experiments, native CSF was used. Cytosolic Ca2+ concentration was measured by fura-2 fluorescence. Apoptosis and necrosis were evaluated by staining with Hoechst-3342 and propidium iodide.

RESULTS:

Incubation of astrocytes with bCSF provoked a steep Ca2+ concentration peak that was followed by a slow Ca2+ rise during the observation period of 50 minutes. Necrosis, but not apoptosis, was induced. Blockade of ATP-sensitive P2 receptors with suramin inhibited the bCSF-induced initial Ca2+ peak and necrosis. Blockade of P1 receptors with 8-phenyltheophylline or of N-methyl-D-aspartate receptors with D(−)-2-amino-5-phosphopentanoic acid had no significant effect. Preincubation with xestospongin D, a blocker of inositol 1,4,5-trisphosphate receptors, prevented the initial Ca2+ rise and reduced the rate of necrosis. Preemptying of the endoplasmic reticulum with thapsigargin protected astrocytes from the bCSF-induced Ca2+ peak. Inhibition of mitochondrial permeability transition pores opening with cyclosporin A reduced the rate of astrocytic necrosis significantly, although it did not influence the initial Ca2+ peak.

CONCLUSION:

bCSF elicits a steep, transient Ca2+ rise when administered to human astrocytes by activation of ATP-sensitive P2 receptors and subsequent inositol 1,4,5-trisphosphate-dependent Ca2+ release from endoplasmic reticulum. This massive Ca2+ overload leads to subsequent mitochondrial permeability transition pores opening and necrosis of the cells.

ABBREVIATIONS:

bCSF, bloody cerebrospinal fluid

ABBREVIATIONS:

ER, endoplasmic reticulum

ABBREVIATIONS:

HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

ABBREVIATIONS:

HOE, Hoechst 33342

ABBREVIATIONS:

nCSF, native cerebrospinal fluid

ABBREVIATIONS:

NMDA,N-methyl-D-aspartate

ABBREVIATIONS:

PI, propidium iodide

ABBREVIATIONS:

SAH, subarachnoid hemorrhage

ABBREVIATIONS:

SERCA, endoplasmic reticulum calcium ATPase

ABBREVIATIONS:

TBI, traumatic brain injury

ABBREVIATIONS:

XeD, xestospongin D

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