Protection by taurine of rat brain cortical slices against oxygen glucose deprivation- and reoxygenation-induced damage

Abstract

Taurine neuroinhibitory features have suggested its potential for neuroprotection. The aim of the present study was to assess whether it prevents or counteracts brain ischemia and reperfusion-induced cell injury. Rat brain cortical slices were subjected to oxygen/glucose deprivation and reperfusion. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, taken as an index of cell swelling. When added during the reperfusion period taurine did not significantly affect oxygen/glucose deprivation-induced LDH and glutamate release, while it antagonised tissue water gain in a concentration-dependent manner (IC₅₀ = 46.5 µM). The latter effect was antagonised by 50% when a taurine transport inhibitor, 2-(guanidino)ethanesulphonic acid (GES), or a GABA_A receptor antagonist, bicuculline, was added together with taurine, while it was completely abolished when both GES and bicuculline or the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), was used. On the contrary, when present throughout the entire experiment, taurine significantly reduced oxygen/glucose deprivation-induced LDH and glutamate release with a maximal effect (45% reduction) between 5 and 20 mM. Taurine antagonised also tissue water gain according to a “U-shaped” concentration–response curve, which was significant within the range of 0.01–1.0 mM concentration. This effect was partially counteracted by GES as well as by bicuculline and fully reverted by NPPB. In conclusion, since brain edema is a major contributing factor to morbidity and mortality in stroke, the present findings give the rational basis for assessing taurine efficacy in reducing brain edema in vivo.

Introduction

Excitatory amino acids are massively released from neurons during hypoxia and ischemia both in vitro (Pellegrini-Giampietro et al., 1990, Collard and Menon-Johansson, 1993, Nelson et al., 2003) and in vivo (Hagberg et al., 1985, Globus et al., 1988, Takagi et al., 1993) and overstimulation of their receptors leads to neuronal death (for a review see Bano and Nicotera (2007)). Other critical pathophysiological processes that contribute to cell injury in ischemic stroke include formation of ionic- as well as vasogenic-edema (Simard et al., 2007a). Molecular mechanisms involved in these processes are only beginning to be elucidated although those largely independent of continuous expenditure of energy such as the activity of nonselective cation channels are likely to be markedly involved (Simard et al., 2007a, Simard et al., 2007b, Inoue and Okada, 2007).

Taurine (2-aminoethanesulphonic acid), one of the most abundant free amino acids in the central nervous system, is released in high concentrations from brain cells under a variety of damaging conditions, including osmotic imbalance, ischemia or hypoxia (Saransaari and Oja, 1999, Saransaari and Oja, 2007, Ritz et al., 2006, Shennan, 2008) exposure to hot environment and fever (Frosini, 2007). In the case of ischemia, the release occurs simultaneously with the ischemic-induced raise of excitatory amino acid levels, and this might represent an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death (Saransaari and Oja, 2007, Saransaari and Oja, 2000, Oja and Saransaari, 2000). Furthermore, it has been demonstrated that taurine-containing neurons are fairly resistant to cerebral ischemia induced by four-vessel occlusion in rats (Matsumoto et al., 1991, Wu et al., 1994) and that this amino acid protects rat cerebellar granule cells exposed to kainate without affecting the production of reactive oxygen species (Boldyrev et al., 1999). However, the mechanism of neuroprotection has not been so far clarified, and the exact molecular target or signal chain activated by taurine remains unknown. It has been demonstrated that taurine interferes with the function of GABA and glycine receptors (Kontro and Oja, 1987a, Frosini et al., 2003a) and a putative specific taurine recognition site has been described (Frosini et al., 2003a, Kontro and Oja, 1987b, Frosini et al., 2003b). However, whether the protective effect of taurine is mediated by GABA receptors or by intracellular mechanisms during excitotoxic-induced brain injury remains unresolved.

The aim of the present investigation was to assess whether taurine could prevent or counteract neuronal injury induced by oxygen/glucose deprivation and reperfusion, in rat brain cortical slices. Tissue damage and protection were assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) into bathing artificial cerebrospinal fluid (aCSF) during reperfusion and by determining at the end of the experiment tissue water gain taken as an index of tissue edema (Hrabetová et al., 2002, MacGregor et al., 2003). Results demonstrated that taurine could fully antagonise oxygen/glucose deprivation and reperfusion-induced edema. This effect was dependent on taurine transport into the cells as well as on GABA_A receptor activation and involved the activity of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels.