Hypoxia Limits Inhibitory Effects of Zn2+ on Spreading Depolarizations

Isamu Aiba,C William Shuttleworth,Alexander A Mongin

doi:10.1371/journal.pone.0075739

Abstract

Spreading depolarizations (SDs) are coordinated depolarizations of brain tissue that have been well-characterized in animal models and more recently implicated in the progression of stroke injury. We previously showed that extracellular Zn2+ accumulation can inhibit the propagation of SD events. In that prior work, Zn2+ was tested in normoxic conditions, where SD was generated by localized KCl pulses in oxygenated tissue. The current study examined the extent to which Zn2+ effects are modified by hypoxia, to assess potential implications for stroke studies. The present studies examined SD generated in brain slices acutely prepared from mice, and recordings were made from the hippocampal CA1 region. SDs were generated by either local potassium injection (K-SD), exposure to the Na+/K+-ATPase inhibitor ouabain (ouabain-SD) or superfusion with modified ACSF with reduced oxygen and glucose concentrations (oxygen glucose deprivation: OGD-SD). Extracellular Zn2+ exposures (100 µM ZnCl2) effectively decreased SD propagation rates and significantly increased the initiation threshold for K-SD generated in oxygenated ACSF (95% O2). In contrast, ZnCl2 did not inhibit propagation of OGD-SD or ouabain-SD generated in hypoxic conditions. Zn2+ sensitivity in 0% O2 was restored by exposure to the protein oxidizer DTNB, suggesting that redox modulation may contribute to resistance to Zn2+ in hypoxic conditions. DTNB pretreatment also significantly potentiated the inhibitory effects of competitive (D-AP5) or allosteric (Ro25-6981) NMDA receptor antagonists on OGD-SD. Finally, Zn2+ inhibition of isolated NMDAR currents was potentiated by DTNB. Together, these results suggest that hypoxia-induced redox modulation can influence the sensitivity of SD to Zn2+ as well as to other NMDAR antagonists. Such a mechanism may limit inhibitory effects of endogenous Zn2+ accumulation in hypoxic regions close to ischemic infarcts.

Highlights

Spreading depolarization (SD) is a slowly propagating, feedforward event that initiates from coordinated depolarization of a volume of tissue
We previously showed that SDs generated by localized KCl applications were inhibited by extracellular Zn2+ [7], and the present study extends these findings to other forms of SD that may be more relevant for ischemic injury
Extracellular Zn2+ inhibited normoxic SDs (K-SD and ouabain-SDs generated in 95% and/or 21% O2), but did not inhibit anoxic SDs (OGD-SD, ouabain-SD in 0% O2)

Summary

Introduction

Spreading depolarization (SD) is a slowly propagating, feedforward event that initiates from coordinated depolarization of a volume of tissue. The cumulative metabolic burden of repetitive SDs that occur in the hours and days following injury appears to increase the volume of tissue involved in an infarct, and there is considerable interest in finding effective approaches to limit the incidence of SDs [4,5]. In contrast to the potentially protective effects of extracellular Zn2+, excessive intracellular Zn2+ accumulation contributes to neuronal injury. The results show a dependence of Zn2+ inhibition on oxygen concentration, which could be contributed to by redox modulation. Such a mechanism may provide an additional link between tissue metabolism and the pharmacological sensitivity of SD in ischemic conditions

Experimental Procedures

NMDAR current recording

Results

Resistance of OGD-SD to ZnCl2 reversed by the protein oxidizers

Hypoxic modulation of NMDARs

General