DCPIB, a specific inhibitor of volume regulated anion channels (VRACs), reduces infarct size in MCAo and the release of glutamate in the ischemic cortical penumbra

Abstract

Previous studies have indicated that volume regulated anion channels (VRACs) may be involved in the pathology of the ischemic brain cortical penumbra due to activation of VRAC-mediated excitatory amino-acid (EAA) release. To assess this we had studied neuroprotection and EAA release inhibition by a potent VRAC inhibitor, tamoxifen. However, tamoxifen inhibits several other neurodamaging processes. In the present study we use an ethacrynic acid derivative, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), that has recently been shown to be a specific antagonist of volume regulated anion channels (VRAC), to measure the extent of neuroprotection provided and thus to better assess the role of VRAC-mediated release of excitatory amino acids in an intraluminal suture, reversible middle cerebral artery occlusion (rMCAO) model in adult rats. Rats given DCPIB intracisternally had significantly better neurobehavioral scores after 24 h and showed significantly reduced infarct volumes. Mean infarct volumes were 208.0 (SD = 38.3) mm³ for the vehicle groups, compared with 68.5 (SD = 22.7) mm³ for intracisternally DCPIB-treated groups (p = 0.02, Mann–Whitney test), a reduction of around 75%. However, a 500-fold higher dose of DCPIB given intravenously did not reduce infarct volume or improve behavior. The microdialysis study demonstrated statistically significant reduced brain extracellular fluid glutamate when DCPIB was present in the probe. Thus DCPIB, a specific inhibitor of VRACs, given i.c., provides strong neuroprotection in brain ischemia, but it appears to not cross the blood brain barrier as it is not effective when given i.v. These experiments support the hypothesis that EAA released via VRACs contributes to later ischemic-induced damage.

Introduction

Astrocytes swell during ischemia and as a result can release excitatory amino acids (EAAs) via activation of volume regulated anion channels (VRACs). VRACs may be a major contributor to neuronal damage in ischemia (Kimelberg, 2000, Kimelberg, 2005, Phillis et al., 2000, Ochoa De La Paz et al., 2002, Law, 1991). Excess EAAs, primarily extracellular glutamate, initiate a cascade of events progressing over hours to many days (Lipton, 1999). The increase in extracellular EAAs may occur through a number of routes, including exacerbation by decreased reuptake into neurons and astrocytes, exocytotic release from neurons, reversal of the high affinity uptake system and swelling-induced opening of anion channels, especially in astrocytes (Nicholls and Attwell, 1990, Kimelberg and Mongin, 1998, Mongin and Kimelberg, 2004, Law, 1994, Quesada et al., 1998, Pasantes-Morales et al., 1990, Phillis et al., 2000, Phillis et al., 1997, Strange et al., 1994, Liu et al., 2006). Prevention or inhibition of ischemia-induced EAA release, either acute or prolonged, may protect neurons in ‘at risk’ locations. Swelling-activated VRACs play a major role in excitatory amino acids (EAAs) release in ischemic injury (reviewed in Kimelberg, 2005), especially in the ischemic cortical penumbra (Feustel et al., 2004). The inhibition of the VRACs seems a suitable target because VRACs are normally closed and their blockade presumably would not affect normal brain function.

Some years ago our laboratory in collaboration with E. Cragoe of Merck & Co., developed a series of compounds based on ethacrynic acid which were able to confer protection in a closed head injury model. Synthesis of these compounds was guided by the principle of increasing their efficacy in inhibiting brain edema and reducing their renal saludiuretic effects (Nelson et al., 1982, Kimelberg et al., 1987, Kimelberg et al., 1990, Bourke et al., 1979, Cragoe et al., 1982, Cragoe et al., 1986, Cragoe, 1987). Since that time one of these compounds, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid and therefore given the acronym DCPIB (Cragoe et al., 1982, Bourke et al., 1981, Nelson et al., 1979; and see Fig. 1a), has been shown to specifically inhibit VRAC activity, but not a number of expressed chloride channels in Xenopus oocytes (Decher et al., 2001). VRACs are an important class of anion channels that are widely distributed, but whose molecular identity has so far eluded investigators' best efforts (Nilius and Droogmans, 2003, Strange, 1998 Okada, 1997). DCPIB has recently been shown by us to completely inhibit cell-swelling-induced, EAA release and Cl⁻ currents in primary astrocyte cultures (Abdullaev et al., 2006). In the current study we have examined the effects of DCPIB in a rat model of reversible middle cerebral artery occlusion (rMCAo) and show that it results in robust reduction of infarct volume when given intracisternally (i.c.), but no effect when given intravenously (i.v.). Modest correlative improvements in behavior scores were also found. We also show that local administration of DCPIB via a microdialysis probe results in lower glutamate levels during ischemia.