The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke

Highlights

• Oxidative stress is an important contributor to brain damage in stroke.

• Despite preclinical data, several tested antioxidant agents failed in human stroke trials.

• We explored a new link between oxidative stress and release of the toxic neurotransmitter glutamate.

• The SOD mimetic tempol reduced glutamate release and brain damage in animal stroke.

• Our findings may help in developing new, more effective antioxidant therapies.

Abstract

The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies have failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit the release of the excitotoxic amino acids glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine, and alanine. Microdialysate delivery of 10 mM tempol reduced the amino acid release by 60–80%, whereas matching levels of edaravone had no effect. In line with these data, an intracerebroventricular injection of tempol but not edaravone (500 nmol each, 15 min before MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior at removing superoxide anion, whereas edaravone was more potent at scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggest that the neuroprotective properties of tempol are probably related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.

Introduction

Stroke is a loss of brain functions due to interruption of cerebral blood flow. In a majority of clinical cases, strokes are initiated by thrombotic or embolic occlusion of one of the major blood vessels in the brain; however, they may also develop from the rupture of a blood vessel (hemorrhagic stroke) or a complete shutdown of cerebral circulation such as in cardiac arrest [1]. Stroke represents the fourth leading cause of death in the United States and the second leading cause of mortality worldwide and is the leading cause of adult long-term disability in the industrialized nations [2], [3]. Despite the large impact on public health, there is only one drug, tissue plasminogen activator (tPA), that is currently approved for acute stroke treatment in the United States. Yet, because of a relatively short therapeutic window and numerous contraindications, tPA has been historically used in no more than 3–5% of stroke patients [4]. Numerous clinical trials that tested other neuroprotective treatments showed no clinical benefits in stroke patients, leaving a large unmet need for the development of new therapeutic interventions [5].

In the ischemic brain, cessation of oxygen and glucose supplies leads to a very rapid depolarization of neural cells followed by a massive release of numerous neurotransmitters via several transport mechanisms [6], [7]. The excitatory neurotransmitters glutamate and aspartate cause activation of highly abundant ionotropic receptor channels. These receptors propagate the rapid onset of tissue depolarization and directly (the NMDA receptor subtype) or indirectly (majority of AMPA and kainate receptors) contribute to a lasting increase in cytosolic Ca²⁺ levels and cell damage and death, termed excitotoxicity [8], [9]. The pathological rise in [Ca²⁺]_i sets in motion multiple damaging cascades culminating in either rapid ischemic cell death via necrosis or delayed cell death via apoptosis and the apoptosis-like process called parthanatos [6], [10], [11].

One of the critical events leading to the excitotoxic demise of neuronal cells is oxidative stress—a redox imbalance stemming from the increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the ischemic tissue (reviewed in [12], [13], [14]). ROS cascades in stroke begin with formation of the superoxide anion (O₂^•−), which is produced in mitochondria as a result of one-electron reduction of oxygen, but also in several additional enzymatic reactions carried by NADPH oxidases, xanthine oxidase, and others. O₂^•− is dismutated to hydrogen peroxide (H₂O₂), either enzymatically or spontaneously. In the presence of transition metals, H₂O₂ decomposition yields the potent oxidant hydroxyl radical (^•OH). In a parallel RNS cascade, ischemia upregulates levels of the free radical signaling molecule nitric oxide (^•NO) and several intermediates of its degradation. Most importantly, ^•NO combines in a rate-limiting reaction with O₂^•− generating the oxidant peroxynitrite (ONOO⁻), which damages cells on its own or via secondary formation of ^•OH and other toxic intermediates [6], [14], [15], [16]. ROS and RNS production is thought to lie downstream of the NMDA receptors because activation of these receptors and ensuing cytosolic Ca²⁺ overload increase mitochondrial formation of O₂^•− and H₂O₂ and stimulate several enzymes producing ROS and RNS [6], [12], [17]. Oxidative, nitrosative, and nitrative damage harms and kills neuronal cells via widespread damage to enzymes, DNA, and lipids. The major role for ROS and RNS in stroke pathology was confirmed in numerous animal studies employing antioxidants and free radical scavengers or manipulating expression levels of enzymes that produce or degrade ROS and RNS (see for example [18], [19], [20], [21], [22], [23] and reviews [12], [13], [17]).

The extensive preclinical data on the role of free radicals and oxidative stress in ischemic brain damage have led to several clinical trials. Two of these trials, which were conducted in Japan, demonstrated modest clinical benefits of the antioxidant edaravone in stroke patients [24], [25]. In contrast, several other trials performed elsewhere showed no neuroprotective properties for other antioxidant molecules [13], [26], [27]. These unsuccessful studies included the most extensive clinical trial to date, SAINT II, which tested the nitrone spin trapping agent NXY-059, or Cerovive [28]. The failure of SAINT II came as a big disappointment and solidified skepticism about the clinical utility of antioxidants as neuroprotective agents [29], [30]. The reasons for the clinical failure of NXY-059 continue to be debated [5], [31].

In the present study we investigated a new hypothetical mechanism that may determine the efficacy of antioxidants in stroke. As mentioned above, the traditional view is that oxidative stress represents one of the terminal steps of ischemic tissue damage, downstream of activation of glutamate receptors and elevation of intracellular Ca²⁺ concentrations. Our recent in vitro and in vivo studies suggest that exogenous and endogenous oxidants, particularly H₂O₂, can also exert “upstream” effects and promote pathological glutamate release from glial and neuronal cells via opening of glutamate-permeable membrane channels [32], [33], [34], [35]. Here, we used the well-established rodent model of stroke induced by microfilament occlusion of the middle cerebral artery (MCAo), to correlate effects of antioxidants on ischemic glutamate release and their ability to reduce ischemic tissue damage. For this purpose, we selected two antioxidants, tempol and edaravone, based on previous animal studies and clinical data (see Discussion for details and references). Our new data may contribute to the understanding of the limited clinical efficacy of some of the previously tested antioxidant agents and provide a blueprint for selecting more effective prototypical drugs for future clinical studies.