Excitotoxicity: Still Hammering the Ischemic Brain in 2020

1It has now been 63 years since discovered the ability of parenterally administered glutamate to kill central neurons in the rodent retina, and 51 years since extended this observation to neurons in the hypothalamus and hippocampus. Subsequently finding that a series of structurally related neuroexcitatory amino acids exhibited similar neurotoxicity, with potencies corresponding to known neuroexcitant potencies, proposed that the “necrotizing effect is, in essence, an exaggeration of the excitatory effect” and coined the term “excitotoxic amino acids”. These seminal observations remained relatively fallow until the 1980s, when advances in excitatory amino acid (EAA) pharmacology led to widespread recognition of glutamate’s neurotransmitter function and scientific interest in EAAs burgeoned. Over the next two decades, the subfield of excitotoxicity likewise expanded, driven most prominently by its implication in the pathogenesis of ischemic brain damage, and several pharmaceutical companies developed glutamate receptor antagonists as candidate treatments for stroke. By the early 2000s these drugs had failed in clinical trials and excitotoxicity research lost substantial momentum. A reflection of the excitotoxicity research boom and subsequent slowdown can be found in the number of relevant publications indexed by PubMed (Figure 1).

2However, implication of excitotoxicity as an agent of human diseases has not gone away. The current issue of Frontiers in Neuroscience is timely, marking a half-century of active excitotoxicity research. I thank the editors for my invitation to contribute, and will provide here a brief, semi-chronological, and at times personal, overview of this research, focusing on the still extant path forward for developing an anti-excitotoxic neuroprotective treatment for stroke. Recent reviews on the topic have appeared. Other articles in this issue will likely discuss the possible contribution of excitotoxicity to neurodegeneration in chronic diseases such as amyotrophic lateral sclerosis or Alzheimer’s disease.

3Brain tissue contains high concentrations of free glutamate, in the 5–15 mmol/kg range, but until the 1970s this glutamate was widely assumed to be serving purely metabolic functions. speculated that glutamate might be a neurotransmitter based on convulsant activity on motor cortex, an idea that was strongly supported by direct demonstration of neuroexcitatory properties. Subsequent research on the topic of glutamate signaling proceeded gently for nearly two decades – a lull have dubbed “the dark ages”. But evidence for synaptic synthesis, Ca2+-dependent release and rapid cellular uptake accrued, and pharmacological tools were developed that enabled critical testing of the transmitter hypothesis. Glutamate receptors were classified into N-methyl-D-aspartate (NMDA) and non-NMDA types, the latter subsequently further divided into kainite and quisqualate receptors, and then later, kainate, α-amino-3-methyl-4-isoxazoleproprionic acid (AMPA), and G protein-coupled metabotropic receptors (mGluRs); molecular subunit nomenclature is still evolving. Glutamate antagonists blocked endogenous neural signaling in multiple pathways, identifying glutamate as a neurotransmitter at insect and crustacean neuromuscular junctions, and then throughout the mammalian CNS.

4Olney’s initial considerations of how excitotoxicity might contribute to human disease focused on the possible dangers of exogenous agonists, especially the monosodium glutamate added to food as a flavor enhancer. Recognition of ubiquitous glutamate neurotransmission elevated interest in the possible pathophysiological importance of endogenous stores, and excitotoxicity gained further attention as a laboratory tool, useful for creating “axon-sparing” lesions in brain. These threads came together when injection of kainate into the rat striatum was found to reproduce some of the anatomical and biochemical features of Huntington’s disease, raising speculation that progressive excitotoxic damage might contribute to its pathogenesis. Further, the convulsant properties of kainate and the dependence of kainate neurotoxicity in vivo upon intact glutamatergic afferents supported a role for excitotoxicity in epileptic brain injury. Thus, the stage was well set for three breakthrough studies that leveraged newly available receptor antagonists to implicate endogenous excitotoxicity in ischemic brain damage.

5showed that the glutamate antagonist γ-D-glutamylglycine (DGG) could block glutamate-induced depolarization and cell swelling, as well as anoxic injury in cultured rat hippocampal neurons. This key study followed up on his earlier demonstration that 10 mM MgCl2 could protect the neurons against damage induced by cyanide exposure.

6found that direct hippocampal injection of the selective NMDA antagonist, 2-amino-7-phosphonoheptanoic acid (APH), reduced pathological changes in nearby neurons 2 h after transient global ischemia (TGI) in rats. In retrospect, the study assessed neuronal morphology too soon to determine lasting survival, as it was just becoming recognized that selective neuronal death after TGI can occur days later. Regardless, it demonstrated a neuroprotective effect of NMDA receptor (NMDAR) blockade against a component of ischemic injury in vivo.

7injected APH into the rat caudate, and observed markedly improved survival of nearby neurons 1 week after 30 m exposure to hypoglycemia, further implicating NMDARs in acute brain injury.

8Additionally, microdialysis measurements revealed that brain ischemia induced within minutes a large increase in extracellular glutamate emanating from depolarized nerve terminals and astrocytes, indicating that neurons in the ischemic brain would inevitably be exposed to elevated glutamate concentrations.

9It is worth pausing here. The entire cell death field was then a shadow of what it would become. (PubMed papers retrieved by “cell death” and published in 1985 are about 2% of the 14,000 such papers published in 2019). Few people considered either acute or chronic neurodegeneration a worthwhile research topic – cells die, right – and the neurologists of the day managed patients presenting with Alzheimer’s disease or stroke nihilistically, attending mainly to accompanying medical conditions. Early excitotoxicity studies truly changed “normal science”, suggesting that some ischemic neuronal cell death occurred consequential to specific events and pathways accessible to therapeutic interdiction.

10Inspired by Rothman’s in vitro studies, I set out to investigate glutamate neurotoxicity in the mouse cortical cell culture system I had going at the time. Having worked during the 1970s as a graduate student on γ-aminobutyric acid (GABA) signaling and benzodiazepines, I was emerging from clinical training as a neurologist and setting up my own laboratory at Stanford, newly funded to study the electrophysiology of glutamate receptors. Shifting research focus on the fly was entirely feasible – thank you, NIH.

11I found that bath exposure to 500 μM glutamate caused cultured cortical neurons to swell immediately and then proceed to disintegrate, similar to the “intracellular edema and neuronal necrosis” observed by in mouse brains after glutamate injection. However, I wondered if leaving glutamate in the bath, as Rothman had done, might not exaggerate cell swelling and attendant damage over what would occur in vivo within a three-dimensional brain and closed skull. I decided therefore to terminate the bath exposure after 5 m, an exposure still widely lethal by the next day. Replacing extracellular Na+ with an impermeant cation eliminated acute neuronal swelling, but most neurons still went on to degenerate over the next hours. In contrast, removing extracellular Ca2+ increased early cell swelling, and yet most cells recovered and survived. This and other experiments suggested that glutamate neurotoxicity at lower exposure levels was predominantly driven by delayed, Ca2+ -dependent processes rather than by the immediate entry of Na+ responsible for neuroexcitation and, together with Cl– and water, immediate excitotoxic swelling.

12A Ca2+ -dependent death fit with observations of EAA-induced Ca2+ movement into brain tissue, and aligned excitotoxic death with a larger theme of Ca2+ overload in other types of cell death, including the toxin-induced death of hepatocytes and agonist- or mechanical injury-induced muscle cell death. It also meshed with prior implications of Ca2+ overload in the neuronal death induced by ischemia or prolonged seizures. Measurements with newly available Ca2+-sensitive microelectrodes revealed a rapid and large drop in brain extracellular Ca2+ after the onset of ischemia. The toxic EC50 for glutamate with 5 m exposure was 50–100 μM in mixed astrocyte + neuron cultures, dropping to 5 μM with 30 m exposure in astrocyte-poor cultures lacking protective cellular uptake.

13The exciting discovery that NMDA but not kainate gated Ca2+-permeable channels suggested that NMDARs would play a foreground role in glutamate neurotoxicity. This was borne out. The selective NMDA antagonist 2-amino-5-phophonovalerate (APV) only modestly reduced the neuroexcitation or acute neuronal cell swelling induced by brief glutamate exposure, but markedly reduced later cell death. Selective NMDAR block was also effective in reducing the neuronal death induced by hypoxia, glucose deprivation or mechanical trauma.

14Non-NMDA agonists were also potently neurotoxic on cortical neurons, but prolonged exposures of several hours was required to produce widespread cell death. Consistent with the effects of glutamate + APV, kainate induced immediate excitotoxic neuronal swelling, but if exposure was terminated at 5 m, most cells recovered and survived. We considered it likely that this more slowly triggered neurotoxicity mediated by kainate or AMPA receptors (KARs, AMPARs) was mediated by slower Ca2+ overload secondary to excessive Na+ entry, involving voltage-gated Ca2+ channels and reverse operation of the electrogenic Na+/Ca2+ exchanger, NCX. In that mode, favored under conditions of membrane depolarization, high internal [Na+] and low external [Na+], NCXs provide high capacity transport for 3 Na+ ions out coupled to 1 Ca2+ ion in. The importance of NCXs to excitotoxicity was supported by implication in anoxic optic nerve injury. Later we showed that net cellular 45Ca2+ accumulation induced by NMDA or glutamate was much larger/faster than that induced by high K+, kainate, or AMPA, and found a similar relationship for [Ca2+]i.

15Identification of excessive Ca2+ entry as the primary mediator of excitotoxicity, likely augmented by Ca2+ release from endoplasmic reticulum (ER, triggered by mGluR activation; see below), also bridged to prior studies implicating free radical generation in the pathogenesis of ischemic brain injury. Multiple Ca2+-dependent enzymes including calpains, endonucleases and lipases, were known to be capable of damaging cells, and Ca2+ overload would impair mitochondrial energy production and perturb Ca2+-dependent signaling pathways. But the generation of free radicals seemed especially well positioned to drive lethal cytodegeneration. Consequent to cellular Ca2+ overload, breakdown of lipid membranes into fatty acids mediated by phospholipase A2 and further metabolism via prostaglandin and leukotriene pathways, damage to mitochondrial electron transport and conversion of xanthine dehydrogenase to xanthine oxidase all generate free radicals, promoting lipid peroxidation and membrane failure. Testing the hypothesis, 21-aminosteroid lipid peroxidation inhibitors attenuated both glutamate neurotoxicity and oxygen-glucose deprivation (OGD)-induced neuronal death. Toxic NMDA exposure increased superoxide formation in cultured cerebellar neurons, and trapping this superoxide was neuroprotective. Subsequent studies used electron paramagnetic resonance spectroscopy and oxidation state-sensitive fluorescent dyes to detect mitochondrial production of reactive oxygen species (ROS) after NMDAR-mediated Ca2+ overload, and demonstrated that this production could be substantially reduced by inhibition of mitochondrial electron transport or dissipation of the mitochondrial membrane potential. Figure 2 shows a diagram of excitotoxicity mechanisms as we saw it in 1988.

16The discovery that NMDAR-mediated Ca2+ influx activated neuronal nitric oxide synthase (NOS) suggested that nitric oxide (NO) might be both a normal neuronal signaling molecule and a free radical mediator of glutamate neurotoxicity. Freely diffusible through membranes, NO released from neurons activates soluble guanylate cyclase in neighboring cells including smooth muscle. Cytotoxicity at higher concentrations was harnessed by macrophages to kill target cells, with NO reacting with superoxide to form the highly reactive nitrogen species (RNS), peroxynitrite, and promoting destructive hydroxyl radical formation. Pivotal experiments were carried out by, demonstrating that inhibition of neuronal NOS (nNOS) selectively attenuated NMDAR-mediated neurotoxicity but not kainate neurotoxicity. Later, they would show that a major toxic consequence of NMDA-induced NO formation was the hyperactivation of poly(ADP-ribose) polymerase (PARP), likely consequential to peroxynitrite-induced DNA damage and resulting in cellular energy depletion and nuclear translocation of apoptosis-inducing factor (AIF), triggering a caspase-independent death (“parthanatos”). Subsequent studies from other labs would raise a possible role for oxidative intracellular Zn2+ release in connecting NO to PARP overactivation (see below).

17As a sidebar, inhibiting nNOS did not reduce glutamate neurotoxicity in the cortical cultures my lab was using in the 1990s. We came to regard this as an idiosyncrasy, likely reflecting low baseline nNOS expression and/or a large astrocyte presence sopping up NO. Raising inducible NOS (iNOS) expression in the astrocyte layer did add a NOS-dependent component to NMDAR-mediated toxicity in the cultures. In any case, this culture system difference illustrates that more than one potentially lethal downstream cascade can be triggered in parallel by glutamate receptor overactivation and robustly kill neurons. A messy “and”, not an “or”: a race to death. Whether a specific cascade ends up directly responsible for cell death such that its inhibition improves survival, and the exact features of that death, may depend on where that cascade lies in an injury hierarchy, as well as on the identity and closeness of competitive injury pathways that may leave behind their own incomplete molecular and morphological signatures.

18Demonstration of the neuroprotective effects of glutamate antagonists against hypoxia/ischemia/hypoglycemia in vivo and in vitro, together with identification of NMDAR overactivation and Ca2+ overload as primary mediators of acute glutamate toxicity, encouraged global efforts to test NMDA antagonist drugs in various animal models of brain ischemia and develop drugs suitable for human use. The first drug candidate out of the gate was Merck’s MK-801, a potent and selective NMDA antagonist with excellent brain penetrance. reported that pretreatment with MK-801 reduced neuronal death in gerbil brains subjected to either bilateral or unilateral forebrain ischemia. Benefits of NMDA antagonists later proved variable in models of TGI, in some cases perhaps confounded by hypothermia, but reduction of infarction or disability was robustly observed with NMDA antagonists given up to 2 h after focal brain ischemia in rodents or larger animals. By 1992 close to 20 published studies reported this finding. AMPA/kainate antagonists also were neuroprotective in brain ischemia studies, especially after TGI. In 1994, Huang et al. reported that mice lacking nNOS exhibited reduced infarcts after permanent occlusion of the middle cerebral artery (pMCAO).

19However, the NMDA antagonist drug candidates brought into development by several companies for use in stroke all failed in clinical testing. Some were abandoned because of side effects; others lacked efficacy. In two cases (CNS-1102, CGS 19755), there were worrisome trends toward worse outcome in the treated group. As those reviewers noted, clinical studies did not match up well with the earlier animal studies. Some drug dosing was too low (limited by mechanism-associated behavioral side effects such as hallucinations) or too late (most dosing was > 3 h post stroke onset, and in some trials up to 48 h post onset, as enrolling patients at earlier time points was not practical at that time). A smaller number of pilot studies with AMPA antagonists were also disappointing, in one case (ZK20075) unsurprisingly depressing consciousness.

20The first wave of NMDA antagonist stroke trials was followed by two significant improvements, but these second-generation efforts also failed. The first improvement, informed by insights into the molecular biology and subunit composition of glutamate receptors, was the use of antagonists selective for NR2B/GluN2B. The predominant expression of NR2B in forebrain vs. cerebellum and limbic areas raised hopes of achieving neuroprotection with less dose-limiting side effects. Furthermore, as discussed below, consideration of Ca2+ source specificity and NMDAR signaling relationships suggested that NR2B contributes more to excitotoxic death than the other major forebrain subtype, NR2A. A older antagonist, ifenprodil, was discovered to be conveniently NR2B selective, attractively use-dependent and neuroprotective in multiple animal models of global and focal brain ischemia. New molecular entity congeners of ifenprodil, SL-82.0715 and CP-101,606 were developed by Synthélabo and Pfizer, respectively, and exhibited reduced side effects compared to pan NMDAR antagonists. But the former failed to show efficacy in a stroke trial and the latter was felled by electrocardiographic toxicity (QT prolongation) after showing some promise against severe traumatic brain injury.

21The second improvement was in stroke trial methodology. Recognizing that treatment delay was problematic in prior stroke neuroprotection trials, completed a remarkable multicenter study (FAST-MAG) in which intravenous magnesium sulfate was given to 1700 acute stroke patients by paramedics within 2 h (and often within 1 h) of stroke onset. Still no improvement in outcome measures was observed. An earlier study of Mg2+ treatment for stroke (IMAGES) with a 12 h treatment window was also negative. While the methodological advance represented by FAST-MAG is clear, direct relevance of FAST-MAG and IMAGES to the hypothesis of NMDAR-mediated excitotoxicity in human stroke is less so. Mg2+ has multiple actions that might contribute to its modest neuroprotective effects in animal stroke models, and it is likely that its block of NMDA channels in the ischemic brain would be substantially relieved by cellular depolarization.

22Enthusiasm for developing anti-excitotoxic therapies for stroke was also progressively dampened by the lack of efficacy in stroke trials demonstrated by several other drugs targeting related mechanisms, including voltage-gated Ca2+ channels (nimodipine), free radicals (tirilazad mesylate, ebselen, NXY-059), phospholipid hydrolysis (citicoline), and nitric oxide synthase (lubeluzole). These trials also had shortcomings in concept or design, commonly involving the use of doses and delay time windows that were not supported by the enabling preclinical studies. Administration of a lipophilic Ca2+/Zn2+ chelator, DP-b99, within 9 h of stroke onset also failed in Phase III testing.

23The free radical drugs tested would be considered less than compelling candidates today in updated comparisons. Neither tirilazad nor NXY-059 penetrate well into brain parenchyma, and NYX-059 exhibited little antioxidant activity on cultured neurons. Ebselen is a mimic of glutathione peroxidase that would be expected to decompose only a subset of harmful radicals (hydroperoxides); it has little aqueous solubility and requires a thiol co-substrate like glutathione to maintain activity. A fourth antioxidant drug, edaravone, has a favorable chemical profile, being amphiphilic and able to directly scavenge a range of harmful lipid- and water-soluble radicals. It has protective effects in multiple animal models of brain ischemia, and showed sufficient evidence of neuroprotective efficacy in clinical testing to gain approval for use in stroke in Japan. Available published data documented a just significant (p = 0.048) beneficial effect on 90 d clinical outcome in patients treated within 72 h of stroke onset; clear benefit was suggested in an exploratory analysis including only patients treated within 24 h. Subsequent clinical trials demonstrating ability to slow the progression of amyotrophic lateral sclerosis supported the premise of human neuroprotective activity, and led to US FDA approval in 2017 for that indication (Radicava).

24By 2001, I had moved to work in the pharmaceutical industry and was in a position to champion an improved NMDA antagonist/stroke trial, but I had become worried that there was potentially a conceptual problem with hard blocking NMDARs in the ischemic brain: apoptosis. Programmed cell death had come to center stage in biological research, spearheaded by elegant genetic studies in C. elegans and growing appreciation that apoptosis occurs in a wide range of disease states outside of normal development. Hypoxic-ischemic cell death was long considered to be a defining example of a non-apoptotic, “accidental” death – necrosis – triggered by “violent and non-physiological” environmental changes. It, like classic excitotoxicity, was associated with prominent cellular and organellar swelling and membrane rupture, in contrast to the controlled cellular condensation characteristic of apoptosis. Yet evidence was steadily emerging suggesting that some neurons underwent apoptosis after ischemia, in particular selectively vulnerable neurons dying in delayed fashion after TGI; but also after focal ischemia, especially threshold insults triggering “very delayed infarction” days later.

25As expected, intense excitotoxic death appeared typically not to be apoptotic. However, slowly triggered AMPAR/KAR-mediated neuronal death was associated with internucleosomal DNA cleavage, consistent with incomplete activation of apoptosis pathways, and neuronal apoptosis occurred after mild excitotoxic insults, either in young cultures with limited EAA receptor expression or with lower concentrations of NMDA. These studies fit with findings that apoptosis could be induced by Ca2+ overload or oxidative stress, including NO. More recent studies have indicated that Ca2+ and oxidative surges can interact at the ER-mitochondrial signaling interface to trigger mitochondrial membrane permeabilization, permitting cytochrome c and AIF release, and the activation of caspase-dependent or caspase-independent apoptosis pathways. Or resulting in necrosis, if mitochondrial and cellular failure is fulminant.

26Together, these observations suggested that excitotoxicity, like many other insults, has the potential to trigger regulated cell death programs; but when intense, membrane, energy and protein synthesis failure destroy neurons before these programs can complete. Ca2+ ionophores likewise can induce neuronal apoptosis at low concentrations, and necrosis at high concentrations. A death race hierarchy was already apparent in cultured cortical neurons deprived of oxygen and glucose: blockade of rapidly triggered NMDAR-mediated death was necessary to reveal AMPAR/KAR-dependent death. Blocking both NMDAR and AMPAR/KAR-mediated neurotoxicity rendered neurons resistant to prolonged OGD, but then a further increase in OGD duration (“blocked OGD”) drove neurons into apoptosis.

27Consideration of ischemic apoptosis suggested that a sustained high degree of NMDAR blockade could be harmful. While presumably beneficial initially in reducing acute excitotoxic necrosis and the triggering of ischemic apoptosis, sustained blockade risks keeping [Ca2+]i below a necessary survival “set-point”. U-shaped curves are ubiquitous in cell and organismal biology, with deleterious effects induced by both too little and too much of many metabolites, messengers, drugs, etc. Apoptosis of young sympathetic neurons induced by nerve growth factor (NGF) withdrawal can be attenuated by raising [Ca2+]i from a basal level of 90 nM to about 240 nM, the same higher basal level found in older neurons capable of surviving without NGF. This survival-promoting level of [Ca2+]i is considerably lower than the levels associated with excitotoxic exposure to glutamate, which can exceed 10 μM and are associated with much larger amounts of net cellular calcium loading than that induced by survival-promoting activation of voltage-gated Ca2+ channels. Lowering extracellular Ca2+ or reducing membrane Ca2+ channel opening induces apoptosis in a wide range of cell types [reviewed in ]. Many signaling pathways transduce the ability of moderate Ca2+ levels to promote cell survival, including the activation of PI3K/Akt/mTOR, Ras/Raf/ERK, and AMPK pathways, as well as modulation of gene expression by CREB and NFAT family transcription factors downstream of Ca2+ / calmodulin.

28Supporting the premise of Ca2+ starvation in neurons undergoing ischemic apoptosis, [Ca2+]i was abnormally low in neurons dying after exposure to blocked OGD, and normalizing [Ca2+]i with the voltage-gated Ca2+ channel opener, S)--BayK-8644, or even low concentrations of kainate improved neuronal survival. 1–2 days NMDAR blockade alone reduced cultured neuronal [Ca2+]i below baseline levels and triggered / enhanced apoptosis; injection of a single dose of MK-801 induced neuronal apoptosis within hours in the developing rat brain. Consistent with a benefit of low level glutamate receptor activation in vivo, delayed administration of the partial NMDA agonist, D-cycloserine, improved functional outcome in rats after traumatic brain injury or focal ischemia. Figure 3 illustrates in broad brush strokes how neuronal [Ca2+]i, apoptosis and necrosis might interrelate after excitotoxic / ischemic insults.

29In sum, whether NMDA antagonists are beneficial or harmful in the ischemic brain may depend on multiple insult and treatment specifics, with key issues being the relative proportion of necrosis vs. apoptosis taking place; and the timing, location and amount of [Ca2+]i lowering induced by the drugs. A dynamic that might slow excitotoxic necrosis and thus increase the overall contribution of apoptosis to ischemic brain damage is a reduction of extracellular Na+ and Ca2+, as documented with ion-sensitive electrodes; also see below under Potassium). Furthermore, ischemic apoptosis might be expected to be more prominent in human stroke, with its sometimes stuttering onset and potentially large penumbral areas across complex gyral anatomy, than after surgical artery occlusion in some lissencephalic rat stroke models. Another reason why early subtype-unspecific NMDA antagonists might have disappointed in human stroke, despite their routine effectiveness in rat stroke.

30Returning to the excitotoxicity story, the next major advance in the 1990s was appreciating that Ca2+ source makes a difference. Noting that elevations of [Ca2+]i mediated by voltage-gated Ca2+ channels lacked the toxicity associated with NMDAR activation, proposed that NMDARs were preferentially linked to downstream mediators of excitotoxic injury. The discovery that the PDZ domains of both NR2B and nNOS interacted with PDZ domains of the postsynaptic density protein, PSD-95, fit the bill, and furthermore placed NR2B in the foreground of excitotoxicity. Suppressing PSD-95 protein expression in cultured neurons attenuated both NMDA-induced NO production and neuronal death, without affecting NMDA currents or NMDA-induced Ca2+ loading.

31Extension of the Ca2+ source specificity hypothesis was pursued in studies assessing the relative contributions of extrasynaptic and synaptic subpopulations of NMDARs to excitotoxic death. Synaptic and extrasynaptic NMDA receptors exhibit similar single channel properties but have been reported to trigger different transcription factor and gene expression changes; activation of extrasynaptic but not synaptic NMDARs caused rapid loss of mitochondrial membrane potential and neuronal death. Mediation of excessive Ca2+ influx into mitochondria was postulated to be favored by extrasynaptic NMDAR activation, possibly due to spatial proximity. However, the distinction between synaptic and extrasynaptic receptors may not be sharp, as burst stimulation of inputs to hippocampal CA1 neurons activates both populations. And arguing against synaptic localization per se altering NMDAR contributions to excitotoxicity, delocalizing synaptic NMDARs by depolymerizing F-actin did not alter NMDAR-induced current, Ca2+ loading, or cell death. The delocalization did attenuate death after OGD, consistent with synaptic localization increasing exposure to the glutamate released by nerve terminals.

32Comparing cell death after intense activation of extrasynaptic vs synaptic NMDARs, some investigators have proposed that these subpopulations have opposing effects on excitotoxic death: extrasynaptic receptors promoting death, and synaptic receptors promoting survival. Consistent with that proposal, synaptic activity repressed expression of the mitochondrial Ca2+ uniporter, Mcu and reduced neuronal vulnerability to excitotoxic death, a plausible neuroprotective adaptive mechanism. However, while supported by careful experiments, the receptor location hypothesis is best considered to be still under test. It does not fit easily with the prominence of PSD-95/nNOS signaling in NMDAR-mediated excitotoxicity, since association with PSD-95 is a hallmark of synaptic localization. Furthermore, as outlined above, the ability of NMDAR activation to reduce apoptosis at lower levels and yet drive excitotoxic apoptosis or necrosis at higher levels can be explained in terms of net Ca2+ fluxes and Ca2+ setpoints, without invoking this additional level of source specificity. The experimental necessity of utilizing different paradigms to activate synaptic vs extrasynaptic receptors (eg, bicuculline + 4-aminopyridine to stimulate the former, bath glutamate + prior MK-801 to stimulate the latter) leaves open the possibility that outcome differences primarily reflect differences in ionic current envelopes or even just overall Ca2+ influx, rather than differences in the fundamental properties of extrasynaptic vs. synaptic receptors. The equivalence of cellular Ca2+ loading achieved by the extrasynaptic vs. synaptic activation paradigms is not assured by measuring peak [Ca2+]i, especially if high affinity indicators like Fluo-3 are used, as these may saturate below micromolar excitotoxic elevations. Additionally, modulatory influences present differentially at synapses vs elsewhere, e.g., pH changes or Zn2+ released by nerve terminals (see below), or differential levels of cell injury may affect outcomes. The receptor location hypothesis might not matter much in stroke anyway, as a large buildup of extracellular glutamate would probably overstimulate both synaptic and extrasynaptic receptors.

33In the strongest formulation of Ca2+ source specificity, NMDAR subtypes have been assigned both to distinct locations and opposing roles, with NR2A assigned to synaptic locations and pro-survival roles, and NR2B assigned to extrasynaptic locations and pro-death roles. However, electrophysiological examination of ifenprodil sensitivity in cultured hippocampal neurons suggested that NR2A and NR2B can both be located in either synaptic or extrasynaptic compartments. This formulation will also need adjustment to accommodate other subunit compositions of NMDARs, as many are likely triheteromeric, containing both NR2A and NR2B subunits.

34Additional to activation of nNOS, other NR2B-linked signaling pathways have been proposed to be mediators of excitotoxic death, including death-associated protein kinase 1 (DAPK1), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and NOX2 (see below). DAPK1 was recruited to the NR2B protein complex after transient middle cerebral artery occlusion (tMCAO), binding to a unique region of the NR2B C-terminal region, phosphorylating NR2B and upregulating channel current; administration of a peptide uncoupling DAPK1 from NR2B or genetic deletion of DAPK1 reduced infarction after tMCAO and selective neuronal death after TGI. However, another study did not find evidence of DAPK1 modifying NR2B after excitotoxic insults, or reduced neuronal death after TGI in Dapk1–/– mice. Downregulating PTEN expression with antisense oligodeoxynucleotides enhanced Akt signaling and reduced the death of vulnerable hippocampal CA1 neurons after TGI.

35Likely many signaling pathways affecting cell death are triggered by glutamate receptor overactivation, not only via direct signaling linkages, but also unspecifically consequent to cellular damage. Example of the latter are ROS/RNS-induced DNA single strand breaks activating PARP, or mitochondrial damage releasing ROS and cytochrome c. Another example of an unspecific link would be damage to ER and accumulation of misfolded proteins, activating the PERK/eIF2 pathway, increasing levels of ATF4 and CHOP, and promoting apoptosis. Furthermore, whether a given downstream signaling pathway or event is ultimately responsible for excitotoxic death, or even whether it promotes death vs survival, may depend on quantitative specifics and cellular state. As discussed above, raising [Ca2+]i can be either survival-promoting or death-promoting, depending. PI3K/Akt signaling is often survival promoting, but in some settings can enhance apoptosis, and cytoplasmic PI3K activation enhances superoxide production by NOX2 (see below). JNK signaling is usually pro-apoptotic but anti-apoptotic functions have been described; and Jnk3 –/– mice exhibited reduced kainate-induced and ischemic apoptosis. Yet another example of context-dependent pathway effects on cell survival would be upregulation of BDNF expression by glutamate receptor-induced Ca2+ influx or ischemia. While BDNF is classically anti-apoptotic, it accentuated excitotoxic neuronal necrosis and glutathione depletion-induced death of immature neurons.

36NADPH oxidase (NOX) comprises a family of 7 membrane-active protein complexes, NOX1 through NOX5, DUOX1, and DUOX2, that catalyze the transfer of electrons from NADPH to molecular oxygen, primarily generating superoxide. While long recognized as a key component of neutrophil antimicrobial defenses, NOX was later found in many tissues, including brain, and implicated in acute and chronic neurodegeneration. NOX1, NOX2 and NOX4 are the main brain isoforms, and mice with genetic deletion of the gp91phox catalytic unit of NOX2 exhibited reduced brain infarction after tMCAO, even if neutrophil NOX2 was rescued with a bone marrow transplant. Subsequent studies showed that ischemia raised the expression of NOX2 and NOX4 in multiple brain cell types over ensuing hours-days, and that amelioration of ischemic infarction could also be achieved by genetic disruption of NOX4. Increased expression of endothelial and neuronal NOX4, which is constitutively active, likely contributes to blood-brain barrier (BBB) breakdown and neuronal death, respectively.

37identified NOX2 as a specific source of ROS production after NMDAR overactivation, demonstrating that both NMDA-induced ROS production and neuronal death in cortical cultures were blocked by the NOX2 inhibitor, apocynin, the hexose monophosphate pathway inhibitor, 6-aminonicotinamide (reducing NADPH), or deletion of the NOX2 subunit gene, p47phox, as well as by removal of extracellular Ca2+ or inhibition of PKCζ, the latter responsible for phosphorylating and activating p47phox. Subsequent study implicated NR2B and phophoinositide-3 kinase (PI3K) upstream of PKCζ. PI3K activation is likely mediated by Ca2+ / calmodulin, as well as possibly through the NR2B C-terminal / PSD-95 signalosome. Another way this signalosome might facilitate NOX2 activation is via Src and Panx1 channel opening; loss of Zn2+ homeostasis may also contribute (see below). Importantly, the NR2B/NOX2 pathway is strongly inhibited by small drops in intracellular pH, so its contribution to ischemic injury may be limited to onset, the outer penumbra and reperfusion (see Protons, below).

38As noted above, AMPA receptors (AMPARs) mediate Na+ influx and hence can contribute to excitotoxic Ca2+ overload and neuronal death. Although these receptors lack direct linkage to NOS/NOX, they participate substantially in brain damage after focal and global ischemia, in the latter setting typically contributing more than NMDARs to the delayed death of selectively vulnerable neurons. This prominent contribution to delayed selective neuronal death may be largely due to upregulated expression of Ca2+-permeable AMPA receptors.

39The Ca2+ (and Zn2+, see below) permeability of central AMPARs during development and in response to synaptic activity is regulated by the expression of an RNA-edited form of AMPA subunit GluR2 that contains an arginine residue instead of the genetically-coded glutamine in a key position within the second transmembrane domain. While most central AMPARs contain such edited GluR2 subunits and exhibit low Ca2+ permeability, certain telencephalic and cerebellar neurons, including aspiny interneurons and neurons with high levels of nNOS, express Ca2+ -permeable AMPARs and are highly vulnerable to AMPA-induced neuronal death. Ischemia enhances the expression of Ca2+ -permeable AMPARs in vulnerable neuronal populations, which then constitute a dominant route for toxic Ca2+ /Zn2+ entry, limiting the protective reach of NMDA antagonists. This enhancement is mediated by turning on expression of the gene silencer REST, reducing GluR2 promoter activity.

40While less studied than other glutamate receptor due to a historical paucity of selective pharmacological tools, kainate receptors (KARs) are expressed postsynaptically at glutamatergic synapses and presynaptically on glutamatergic and GABAergic nerve terminals, modifying transmitter release. Postsynaptic KARs contribute to synaptic excitation, more at some synapses, such as mossy fiber inputs to hippocampal CA3 neurons, than others; and likewise participate variably in synaptic plasticity. Analogous to AMPARs, a minority of KARs containing unedited GluK1 or GluK2 subunits can be Ca2+ permeable and convey heightened vulnerability to glutamate excitotoxicity where heavily expressed, for example on somatostatin-containing interneurons. Administration of the GluK1R antagonist, LY377770, 2 h after endothelin-1-induced focal ischemia in rats reduced infarction and produced a surprisingly large reduction in extracellular glutamate levels in ipsilateral striatum.

41Glutamate released from nerve terminals and astrocytes will activate G-protein coupled mGluRs on neurons and glia concurrently with ionotropic glutamate receptors. There are 8 major mGluRs and several splice variants, divided into 3 groups based on structure, function, and pharmacology: Group I (mGluR1, mGluR5) linked to activation of phospholipase C (PLC); and Groups II (mGluR2, mGluR3) and III (mGluR4, mGluR6, mGluR7, mGluR8) linked to inhibition of adenylate cyclase. mGluRs modulate synaptic transmission and plasticity; although they probably do not play a primary role in mediating acute excitotoxic brain injury, they influence this injury and are thus worth keeping in view for possible secondary therapeutic targeting in stroke.

42Group I mGluR-activated PLC generates inositol 1,4,5-trisphosphate, triggering Ca2+ release from ER, and diacylglycerol, which together through PKC can enhance excitotoxic Ca2+ entry through NMDARs, NCXs and NHEs (see below); and activate phospholipase A2, promoting ROS formation and lipid peroxidation. Possibly through PLC / diacylglycerol signaling, mGluR1 also opens plasmalemmal TRP (TRPC3) cation channels (see below). As expected from these actions, and the typically net excitatory effects of Group I agonists on neural circuits, activation of Group I mGluRs enhanced NMDA-induced neuronal death; and inhibition, especially of mGluR1, reduced that death in vitro as well as ischemic injury in vivo. Besides canonically activating PLC, Group I mGluRs could potentially have neuroprotective effects through G-protein mediated downmodulation of NMDARs and neuroprotective Akt signaling via Homer, PIKE-L and nuclear PI3K. However at least the latter action may be quickly abrogated by calpain. Surprisingly though, interfering with the C-terminal interactions between mGluR1 and NR2A with cell-permeable peptides was recently reported to attenuate NMDA-induced neuronal death in vitro and infarction after tMCAO.

43In contrast, Group II and III mGluRs most often have inhibitory effects on neural circuits. and anti-excitotoxic effects of Group II or Group III agonists have been observed in vitro. The Group III agonist ACPT-1 reduced infarction in rats after tMCAO. Within Group II however, neuronal mGluR2 activation has been proposed to enhance excitotoxicity, perhaps by limiting GABA release. A novel selective mGluR2 negative allosteric modulator, ADX92639, reduced selective neuronal death after TGI.

44In addition to Na+ and Ca2+, another cation participating in excitotoxicity is H+. Rapid local changes in extracellular pH accompany physiological neuronal activity and modulate the behavior of many receptors, channels and transporters, including NMDARs, which are inhibited by H+ around physiological pH. In the ischemic brain, tissue and extracellular pH typically drops within minutes toward 6.5 or lower, due to reliance on anerobic glycolysis for ATP resynthesis, an increase in extracellular H+ sufficient to attenuate NMDAR channel openings and NOX2 activity (see above). Expectedly NMDAR-mediated excitotoxicity is also reduced.

45Despite reducing NMDAR and NOX activation, acidosis is still likely a net contributor to ischemic brain damage as long postulated, as movements of H+ or H+ equivalents amplify excitotoxicity. Ischemic acidosis enhances neurotoxic Ca2+ overload via the gating of acid-sensing ion channels (ASICs – see below). Furthermore, several studies have shown that glutamate- or NMDA-induced Ca2+ influx into hippocampal neurons is accompanied by rapid intracellular acidification, in part due to Ca2+ displacement of H+ from intracellular binding sites. NOX2 activity and electroneutral operation of the plasma membrane calcium ATPase (PMCA) may also contribute, the latter importing 2 H+ ions for every Ca2+ ion exported. Intracellular pH dropped initially to around 6.5 during excitotoxic glutamate exposure (500 μM × 5 m) before progressively recovering and overshooting to pH 7.5–8.0 as a result of acid-extrusion mechanisms, especially membrane Na+/H+ exchangers (NHEs). Sustained operation of NHEs loads intracellular Na+, which then raises intracellular Ca2+ via NCXs. Consistent with this sequence contributing to excitotoxicity, inhibition of NHE-1 with cariporide reduced glutamate-induced cortical neuronal death, as well as three predicted intermediate steps: a later increase in [Ca2+]i, loss of mitochondrial membrane potential, and intracellular production of ROS. NHE–/– mice exhibited reduced infarction after tMCAO.

46In addition, ischemic acidosis is likely itself cytotoxic. Exposure to pH 6.5 for 6 h is lethal to both neurons and glia. Astrocytes are especially vulnerable to proton-induced death, and can be killed after only 15–20 m exposure to pH 6.6 if other extracellular ions are altered to levels found in ischemic brain (low Ca2+ and Na+, high K+, hypoxia), conditions likely to impair cellular pH homeostasis. Sustained intracellular acidity likely becomes lethal due to myriad disturbances in protein conformation and essential cellular processes, including energy failure. If cellular H+ favors release of ferrous iron, the Fenton reaction may facilitate hydroxy radical formation, lipid peroxidation, and a “ferroptosis” form of regulated cell death if necrosis does not supervene. Proton-induced death of cerebellar neurons is accompanied by an increase in [Zn2+]i and reduced by Zn2+ chelation, raising the possibility that disturbance in Zn2+ storage or other homeostatic mechanisms may also contribute to H+ cytotoxicity.

47A core property of the cation channels gated by ionotropic glutamate receptors, besides permeability to Na+, and in certain cases Ca2+/Zn2+, is high permeability to K+. Although glutamate receptor-mediated K+ movement has received less attention than Na+ or Ca2+ movement, it may contribute significantly to excitotoxic apoptosis after mild insults or under other conditions where excitotoxic necrosis is blunted.

48Noting that the hallmark cell volume loss occurring during apoptosis would require K+ to exit, my laboratory examined K+ currents in cortical neurons undergoing serum deprivation-induced apoptosis and observed early enhancement of the delayed rectifier current, IK. Attenuating this current with tetraethylammonium (TEA) or raising extracellular K+ inhibited apoptosis, even if [Ca2+]i was kept at resting levels with gadolinium. This implication of K+ efflux in neuronal apoptosis converged with observations implicating that efflux in other cell types, and in particular two findings: 1) lymphocyte apoptosis required enough K+ efflux to lower [K+]i to less than 50 mM; and 2) in those cells, activation of pro-caspase-3 by cytochrome c and apoptotic nuclease activity was [K+]i -dependent, inhibited by normal [K+]i and released by lowered [K+]i.

49Supporting the notion that K+ efflux may promote apoptosis after mild excitotoxic insults, in a cortical culture model of NMDA-induced neuronal apoptosis (achieved by using a low concentration of NMDA, lowering extracellular Na+ and Ca2+ to levels found in the ischemic brain and extending exposure time), NMDAR activation markedly reduced cellular K+ content. Furthermore, intraventricular injection of TEA reduced infarction in rats after tMCAO, a finding contrary to prediction from a classic excitation / excitotoxicity standpoint, but consistent with participation of K+ efflux in ischemic apoptosis.

50Yet another less-studied cation that participates in excitotoxicity is zinc. Zn2+ is a dietary requirement and has many essential roles in cell biology, including service as a central neurotransmitter/neuromodulator regulating circuit behavior and synaptic plasticity. Reactive Zn2+ is stored in presynaptic vesicles within a subset of glutamatergic nerve terminals throughout CNS and co-released with glutamate in a Ca2+-dependent fashion upon nerve terminal firing. Synaptically released Zn2+ modifies the behavior of multiple moieties on postsynaptic membranes, including voltage- and agonist-gated channels, with primary signaling actions likely on NMDA and AMPA receptors. It can also enter (and likely leave) postsynaptic target neurons though Ca2+ routes that might equally well be called Ca2+/ Zn2+ routes, including voltage-gated Ca2+ channels, NMDARs, Ca2+-permeable AMPARs, TRP channels, ASIC1a and in depolarized cells, reverse operation of NCXs, the last augmented by intracellular H+ and NHE operation. Ischemic acidosis may also favor Zn2+ entry over Ca2+ entry through voltage-gated Ca2+ channels. Gating of unselective Panx1 channels, on neurons by NMDAR-Src signaling, and on astrocytes and oligodendrocytes by P2X7 signaling, might be another route for cellular Zn2+ entry or egress (see below).

51Once “translocated” from afferent terminals into postsynaptic target cells, Zn2+ participates in myriad intracellular signaling pathways including the activation of a metabotropic Zn2+ receptor, mZnR/GPR39, and inducing intracellular Ca2+ release. Additional to translocated Zn2+, a major source of intracellular signaling Zn2+ is mobilization from intracellular stores, especially metallothioneins, by oxidation reactions.

52Cellular [Zn2+]i is normally tightly regulated, but in ischemia this homeostasis is disrupted. Release from depolarized nerve terminals and oxidized intracellular stores, together with failure of energy dependent transport, produces increases in extracellular and intracellular Zn2+ that can be cytotoxic, especially to neurons. Zn2+ like glutamate induces apoptosis at lower toxic exposures and necrosis at higher exposures. The parallel between glutamate and Zn2+ neurotransmission thus extends to excitotoxicity: both are potential transmitter-killers in the ischemic brain.

53Evidence that Zn2+ dysregulation contributes to acute brain injury was provided by observations of apparent Zn2+ translocation from afferent terminals to the cytoplasm of neurons degenerating after sustained seizures or transient global ischemia, and supported by the finding that intraventricular CaEDTA, which chelates extracellular Zn2+, blocked both cytoplasmic Zn2+ accumulation and degenerative changes in vulnerable neurons after TGI. At the time we thought CaEDTA was only chelating Zn2+ released from nerve terminals, but later studies indicated that CaEDTA can also pull Zn2+ from intracellular stores. determined that the major accumulation of Zn2+ in vulnerable hippocampal CA1 neurons occurred more than 48 h after TGI, and that early injection of CaEDTA attenuated later appearance of Ca2+-permeable AMPARs, consistent with the possibility that loss of cellular Zn2+ homeostasis constituted a trigger for reducing GluR2 expression. Contribution of Zn2+ to infarction after short duration tMCAO was similarly detected: intraventricular CaEDTA reduced infarct volume 3 days later, but this protective effect was lost after full maturation of the infarct 11 days later, suggesting that an early Zn2+-triggered component of ischemic infarction was eventually overtaken by other injuries.

54Zn2+ like Ca2+ also serves as a downstream mediator of excitotoxicity – the latter’s shadow, unleashed and dysregulated by glutamate receptor overactivation. The relationship between Zn2+ and Ca2+ in mediating excitotoxicity is complex, with target overlap and reciprocal influences; and delineating roles has been further challenged by lack of specificity in historical Ca2+ assay tools such as fura-2. Studies with Zn2+ selective dyes indicated that OGD induced an early increase in [Zn2+]i within hippocampal slice CA1 neurons; a Zn2+ chelator blocked this increase and attenuated neuronal death. Further examination revealed that an OGD-induced increase in neuronal [Zn2+]i preceded mitochondrial depolarization, Ca2+ deregulation and membrane failure, with Zn2+ likely entering mitochondria and contributing to loss of mitochondrial membrane potential. This meshed with earlier demonstration that elevated intracellular Zn2+, like Ca2+, can enter and damage mitochondria, leading to their swelling, loss of membrane potential and, at high levels of Zn2+, increased ROS generation, the last likely a consequence of disturbances in mitochondrial electron transport as well as Zn2+ induction of p47PHOX and increased NOX activity. Downstream ROS/RNS-induced release of Zn2+ from intracellular stores thus drives further oxidative stress. Other injury-promoting events linked to Zn2+ overload include activation of nNOS, PARP and cyclin-dependent kinase 5 (Cdk5 –; see; ) for links to excitotoxicity). Neuronal Zn2+ overload together with Ca2+-activated CaMKII increase insertion of delayed rectifier Kv2.1 channels into the plasma membrane, increasing K+ efflux and facilitating apoptosis. Zn2+ may also upregulate NMDAR activity through a Src-dependent mechanism.

55In addition to promoting neuronal death, excitotoxic Zn2+ dysregulation may contribute to the death of adjacent non-neuronal cells in the ischemic brain. Zn2+-induced death of astrocytes was potentiated by concurrent hypoxia and reduced by PARP knockdown. Intracellular Zn2+ release was implicated in mediating peroxynitrite-induced death of oligodendrocytes, through activation of ERK42/44, 12-lipoxygenase and further ROS generation, rather than immediately through hydroxyl radical formation. OGD-induced death of cultured oligodendrocytes was attenuated by Zn2+ chelation or PARP inhibition. Hydrogen peroxide-induced death of endothelial cells and astrocytes was also attenuated by Zn2+ chelators At sublethal levels, Zn2+ can upregulate ICAM-1 expression in vascular endothelial cells, thereby promoting leukocyte attraction and microvascular leakage.

56Together, these observations on glia and endothelial cells suggest that reduction of downstream Zn2+ toxicity may help account for the observed ability of glutamate antagonists to reduce brain infarction, not just neuronal death, in animal models of stroke.

57Several other membrane channels, likely activated in part consequential to glutamate receptor overstimulation, can contribute to toxic Ca2+/ Zn2+ overload and other ionic derangements in the ischemic brain:

58Transient receptor potential channels. These are variably selective cation channels, subdivided into 7 groups and including receptor-, second messenger- and store-operated members. Two members of the melastatin group, TRPM7 and TRPM2 are plasma membrane channels highly expressed in brain and implicated in the pathogenesis of ischemic brain damage. TRPM7 is activated in the ischemic brain by ROS/RNS and augments Ca2+ overload and oxidative stress. Suppressing TRPM7 expression in cortical cultures subjected to either OGD or blocked OGD attenuated Ca2+ uptake and neuronal death, leading to suggest that TRPM7 might be a foreground pathway for neuronal Ca2+ influx into anoxic neurons. Notably, the divalent cation most permeable through TRPM7 is Zn2+.

59TRPM2 is opened by ADP-ribose accumulating secondary to PARP activity downstream of oxidative DNA damage. Knockdown of TRPM2 expression or drug inhibitors protected male but not female cortical neurons in culture from OGD-induced death, and similarly reduced infarction after focal ischemia in male but not female mice.

60Acid-sensing ion channels are part of the Epithelial Na channel/degenerin (ENaC/DEG) family of monovalent plasma membrane cation channels widely expressed throughout the nervous system and gated by extracellular protons. found that pH 6.0 activation of ASIC1a, which is permeable to divalent cations, mediated Ca2+-dependent neuronal injury and augmented the neuronal death induced in cortical cultures by blocked OGD. The ability of ischemic acidosis to activate ASICs is potentiated by arachidonic acid liberated by phospholipase A2, as well as by NO, extracellular Zn2+, and Ca2+ signaling through CaMKII. Mice lacking ASIC1a or pretreated with blocking PcTX venom developed smaller infarcts after tMCAO, and the protective effect of PcTX added to that of the NMDA antagonist, memantine (see below). In a combined pH 6.0 + blocked OGD paradigm, reducing TRP7 activation by adding an antioxidant or NOS inhibitor was not neuroprotective – perhaps another example of one pathway masking another in a parallel race to death.

61Pannexin ion channels constitute 3 new members of the gap junction superfamily, first cloned from mammalian tissue 20 years ago. Panx1 is widely expressed on the plasma membrane of central neurons, glia, and endothelial cells. While constitutively permeable to Cl–, when activated Panx1 becomes a large conductance pore unspecifically permeable to ions as well as to some larger molecules such as ATP and dyes. It can be activated downstream of NMDARs, primarily through a Src kinase signalosome, but possibly additionally through Ca2+ or NO signaling. Panx1 can also be activated by P2X7 purinergic receptors and irreversibly activated by C-terminal cleavage via caspases, an event that likely contributes to the execution of apoptosis.

62NMDAR-Src activation of Panx1 contributes heavily to anoxic depolarization. This linkage positions Panx1 as an excitotoxicity amplifier, operating in parallel with the NMDA channel to augment disturbances in Ca2+, Zn2+, Na+, and K+. Administration of a novel peptide interfering with NMDAR-Src activation of Panx1, TAT-Panx308, reduced OGD-induced elevation in neuronal [Ca2+]i, mitochondrial membrane permeabilization and neuronal death in hippocampal cultures, as well as infarction after tMCAO in rats.

63Store-operated Ca2+ entry provides Ca2+ entry and replenishment of ER Ca2+ stores in both non-excitable cells and excitable cells, and has been implicated in the pathogenesis of excitotoxicity and ischemic brain damage. The system consists of Orai1-3, plasma membrane channels selective for Ca2+ [blocked by Zn2+] and gated by the ER Ca2+ sensors, stomal interaction molecule isoforms, STIM1-2. Release of ER Ca2+ induces STIM oligomerization and migration to ER-plasma membrane junctions, where these open Orai to permit cytoplasmic Ca2+ entry, which is then taken up by ER via SERCA ATP pumps. As noted above, Group I mGluR activation likely contributes to excitotoxic Ca2+ overload by triggering ER Ca2+ release, placing SOCE in an enabling role. STIM2 is prominently expressed in brain, and stim2–/– mice exhibited reduced neuronal vulnerability to hypoxia in vitro and infarction after tMCAO in vivo.

64Although excitotoxicity was originally described as specific to neurons, oligodendrocytes express a full array of glutamate receptors and are highly vulnerable to excitotoxic injury and death. Young cultured oligodendrocytes can be killed by 24 h exposure to kainate or glutamate; this toxicity was blocked by an AMPAR/KAR antagonist and enhanced by cyclothiazide, which reduces AMPAR desensitization. Vulnerability to a non-glutamate receptor-mediated mechanism involving inhibition of cystine update by high concentrations of glutamate was also demonstrated. More prominent AMPAR/KAR-mediated, extracellular Ca2+-dependent excitotoxicity was demonstrated on cultured optic nerve oligodendrocytes, and a 5–7 days infusion of kainate destroyed optic nerve oligodendrocytes in vivo. Similarly, after maturing 3–5 weeks on a monolayer of astrocytes, oligodendrocyte vulnerability to AMPAR/KAR-mediated excitotoxicity was comparable to that of neurons, with death induced by exposure to as little as 30 μM AMPA for 3 h. OGD triggered AMPAR/KAR-mediated oligodendrocyte death in cultures or in adult brain slices. Ca2+ -permeable AMPA receptors are expressed on oligodendrocytes and may substantially mediate this excitotoxic vulnerability. Loss of cellular Zn2+ homeostasis may contribute, as oligodendrocytes exposed to OGD developed increased [Zn2+]i and subsequent death was reduced by a Zn2+ chelator.

65In the presence of AMPAR/KAR blockade, OGD induced extracellular Ca2+-dependent damage to oligodendrocyte myelinating processes, likely mediated by process-specific expression of an unusual NR3-containing NMDAR relatively insensitive to Mg2+ block. Another contributor to ischemic oligodendrocyte damage may be the their Panx1 channels, possibly activated by P2X7 receptors and ATP released from nearby dying or permeabilized cells.

66Most astrocytes express AMPARs and mGluRs, but NMDARs and Ca2+-permeable AMPARs are generally not abundant. In contrast to oligodendrocytes, astrocytes appear to be relatively insensitive to excitotoxicity, although as noted above they are vulnerable to Zn2+ or H+-induced damage, and they express Panx1 channels. Hence their death could be enhanced secondary to excitotoxicity occurring in nearby neurons or oligodendrocytes.

67It is time to get back in the water.

68Failure of the first wave of NMDA antagonists in stroke trials, viewed with the easy clarity of informed hindsight, does not come close to excluding excitotoxicity as a major driver of ischemic brain damage or the feasibility of reducing that damage with anti-excitotoxic approaches. Those drugs needed to be in the brain at sufficient levels to ameliorate excitotoxicity at stroke onset, not > 3 h later when efficacy can no longer be demonstrated in animal models. Patients with slowly progressive vascular occlusions might still have benefited, but in many patients the drugs would have entered brain after cells were largely en route to death in a glutamate receptor-independent fashion. Neurons in the outer stroke penumbra with less severe NMDAR overactivation would have been the slowest to die, but there pan NMDA receptor blockade may have sometimes lowered [Ca2+]i too far and exacerbated regulated cell deaths.

69Over the last 25 years much has been added to the basic NMDAR-Ca2+ overload scenario that served as the basis for initial NMDA antagonist/stroke trials. Returning to an earlier process framework for excitotoxicity (induction, amplification, expression -, I would highlight the following events in an updated four-stage working scenario:

70Induction. Excitotoxicity in the ischemic brain is predominantly triggered by overactivation of neuronal NR2B receptors at stroke onset, and in the ischemic penumbra later, raising [Ca2+]i, activating nNOS and NOX2, and generating ROS/RNS. Concurrent overactivation of other NMDARs, AMPARs, KARs, and mGluR1 augments neuronal Ca2+ overload. Ca2+-permeable AMPARs, KARs, and NR3-containing NMDARs mediate Ca2+ overload in oligodendrocytes; reverse operation of NCXs convert Na+ entry to Ca2+ overload in axons. Zn2+ overload shadows Ca2+ overload everywhere. Intracellular pH falls and intracellular K+ begins to leave.

71Amplification. Excitotoxic Ca2+/Zn2+ overload is promoted by the activation of other plasmalemmal channels, including TRPM7, TRPM2, ASIC1, Panx1 and Orai; NCXs enhanced by Na+ import via NHEs; oxidative Zn2+ release from intracellular metallothioneins; and in some neurons, increasing expression of Ca2+/Zn2+-permeable AMPARs. Astrocytic swelling and oxidative stress activate the volume-regulated anion channel (VRAC – see below), facilitating glutamate release and intensifying excitotoxic induction.

72Expression. An expanding array of concurrent injury and signaling pathways, activated by Ca2+/Zn2+ overload, K+ efflux, intracellular H+ and oxidative stress head toward regulated cell deaths over the next hours to days. DNA damage and PARP activation, membrane ATPases, and mitochondrial failure deplete energy stores. Catabolic enzymes, especially phospholipase A2 and calpains, damage cellular structures and promote ROS/RNS formation. Increasing intracellular H+ synergizes with elevated [Zn2+]i to damage all cells. In severely ischemic cells (core, inner penumbra), fulminant energy and structural failure culminate in necrosis before a regulated cell death can complete. Oxidative damage is enhanced if reperfusion occurs, which it does with increasing frequency in human stroke today due to interventions.

73Later events. Excitotoxicity per se triggers and augments subsequent inflammatory processes to continue destroying brain tissue. The neurovascular unit (neurons, glia, vascular elements) releases cytokines and chemokines, recruiting leukocytes to the evolving infarct over hours to days, advancing microvascular damage and oxidative stress. In particular, iNOS is expressed in infiltrating neutrophils and endothelial cells 6–96 h after MCAO in rats and in human cerebral infarcts, adding NO fuel to the fire and synergizing oxidatively with superoxide emanating from neutrophil NOX2 and endothelial NOX4. Remarkably, inhibition of iNOS with aminoguanidine reduced infarct volume after pMCAO in rats when given 24 h later. Cellular release of lysosomal cathepsin B may also contribute to remote cell damage and later secondary degeneration.

74There have also been significant advances in relevant drug development methodologies and clinical trial capabilities. In the wake of the first NMDA antagonist failures, academic and industry investigators met to devise guidelines for improving the testing of candidate neuroprotective treatments in animal models and human trials. Vitally, in the interim stroke-is-untreatable nihilism has departed from medicine, as both medical (tissue plasminogen activator, tPA) thrombolysis and, more recently, endovascular thrombectomy improve outcomes in selected patients, even if the latter is delayed up to 24 h after stroke onset. Major hospitals now deploy specialized stroke teams, capable of completing brain imaging and delivering drugs within minutes of a patient’s arrival, and emergency medical services are tuned to getting patients to stroke centers as quickly as possible. In some communities, mobile stroke vans can image and treat patients in remote locations.

75Neuroprotective treatment aimed at reducing ischemic excitotoxicity could be in theory directed at points anywhere along the causality chain from excessive extracellular accumulation of glutamate to the downstream activation of regulated cell death pathways and destruction of cellular structures. Upstream targeting has the general advantage of gaining leverage on multiple divergent downstream pathways but requires early intervention and could be problematic in terms of side effects or interference with beneficial downstream events. Downstream targeting has the advantage of a potentially longer therapeutic time window, greater specificity and less side effects, but risks lower efficacy if unblocked pathways still reach cell death.

76Safely reducing glutamate release from depolarized/de-energized neurons seems a stretch goal, although observations noted above with a GluK1R antagonist are worth following up, and reverse operation of neuronal transporters has been proposed to be a major mediator. Ischemic glutamate release from astrocytes may be a more promising target, as this appears substantially mediated by druggable volume-regulated anion channels (VRACs). VRACs are activated by cell swelling and oxidative stress, and permeable to certain organic anions including glutamate. Astrocyte-specific deletion of the obligate VRAC subunit gene, Swell1, reduced brain infarction in mice subjected to tMCAO. This excitotoxicity amplification mechanism might be specifically targeted by VRAC inhibitors, or unspecifically and collaterally targeted by antioxidant approaches.

77Blocking all glutamate receptors at stroke onset would likely have great anti-excitotoxic efficacy even against initial excitotoxic necrosis, but widespread loss of fast synaptic excitation would likely be poorly tolerated. Pan block of NMDARs could be tried again, achieved with a short-acting drug that could be withdrawn in time to avoid later Ca2+ starvation. However, as cells in an evolving stroke are unlikely to be synchronously in the same state, getting this timing right might be challenging. Future advances in methods for staging the progression of stroke pathophysiology or for location-specific drug delivery might bring this approach back into active consideration.

78Block of NR2B is an appealing upstream approach, especially given experience suggesting that this is doable with an acceptable level of side effects. If the strong Ca2+ source specificity hypothesis turns out correct, then full block should be the goal to interdict all death signaling. The Ca2+ setpoint perspective (and the complication of triheteromeric NR2A + NR2B receptors) would favor a more conservative goal of partial block with a low affinity antagonist that would spare some potentially anti-apoptotic Ca2+ signaling. A South Korean biotechnology company, GNT Pharma Co., has developed such a new molecular entity NR2B antagonist, Neu2000 / Nelonemdaz, which also has potent antioxidant properties. Nelonemdaz has a favorable profile in animal stroke model paradigms including delayed treatment of tMCAO and pMCAO, and is currently in phase II clinical trials in patients undergoing endovascular thrombectomy (SONIC) or presenting with acute ischemic stroke (ENIS I). Another way to improve the therapeutic index of NR2B antagonists may be to dial in pH sensitivity, so that greater block would occur in brain regions with severe ischemia and low pH, and lesser block in brain regions with more normal pH.

79Moving one step downstream, one might seek to decouple NR2B from signalosome interactions mediating toxicity, including PSD-95-nNOS, Src-Panx1 and possibly PI3K-NOX2, while leaving NR2B channel gating intact. Tymianski and colleagues have explored this latter approach, using a novel eicosapeptide, Tat-NR2B9c that disrupts the interaction of the NR2B C-terminal region with PSD-95. Tat-NR2B9c did not block NMDAR-mediated Ca2+ influx, but reduced NMDA-induced neuronal death even if applied 1 h after NMDA, and also reduced infarction when applied 3 h after tMCAO or 1 h after pMCAO. Interestingly, another group has proposed that the Tat targeting sequence of Tat-NR2B9c, pulled from a human immunodeficiency virus protein, may have unexpected anti-excitotoxic properties independent of its NR2B9c payload, perhaps mediated by inducing endocytic internalization of membrane ion channels.

80A Toronto-based biotech company, NoNO Inc. has taken Tat-NR9B2c (NA-1 / Nerinetide) into clinical development. In a phase II trial (ENACT), 185 patients undergoing endovascular repair of intracranial aneurysms received NA-1 or placebo at the end of their procedure, and the NA-1 treated group sustained fewer ischemic infarcts. The results of a just-completed phase III trial of Nerinetide (ESCAPE-NA1) in 1105 patients with acute ischemic stroke were less encouraging, with no difference in clinical outcome 3 m after stroke. However an exploratory analysis suggested that drug treatment within a 12 h window was associated with improved outcome in the subset of patients that did not receive tPA. The investigators have hypothesized that plasmin generated by tPA may have cleaved Nerinetide.

81Inhibiting many individual steps implicated in augmenting excitotoxic induction (e.g., blocking mGluR1, AMPAR, or KAR activation), or downstream in amplifying or expressing excitotoxicity has been demonstrated to be neuroprotective in animal stroke models, but taking such selective targeting forward into the clinic bears significant risk of being bypassed by concurrent unblocked pathways, especially given the huge variability of human stroke presentations. To achieve a robust protective effect in the human wild, it may prove necessary to inhibit multiple excitotoxic mechanisms. The idea of combination therapies has been endorsed for a long time by many workers in the stroke neuroprotection field. My personal view, after time spent working in the pharmaceutical industry, is that a historical focus on single drug, single mechanism therapies is a central reason why the development of innovative drugs has had a lower success rate in neuroscience than in other areas. Several important drugs, especially in the infectious disease or oncology areas, would disappoint if tested as monotherapies.

82An anti-excitotoxic drug combination approach would ideally be configured to ameliorate multiple processes and pathways at both early and late time points. The development and approval process for combination drugs is more complicated than for monotherapies, but not insurmountably so; indeed the number of combination drugs approved by the US FDA has increased each decade since the 1950s, with over 400 combination drugs approved to date. Development of an innovative combination drug can be eased if only one constituent is a new molecular entity and other(s) are previously developed drugs that are well-understood, well-tolerated, and easily available. A growing number of such drugs have useful anti-excitotoxic properties that might boost the efficacy or improve the side effect profile (by permitting lower dosing) of a co-administered new molecular entity anti-excitotoxic therapy. Some examples of candidates for service as a background “partner” (or two) in anti-excitotoxic drug combinations:

83Memantine. The prototype low-affinity NMDA antagonist, approved since 2003 to treat cognitive deficiency in patients with Alzheimer’s disease and now off-patent. Although NMDA receptor subtype non-selective, it may preferentially block extrasynaptic receptors. It is a low-affinity blocker of the NMDA channel with a fast off-time and use-dependence, properties that likely explain its low side effect profile. It reduces NMDAR-mediated excitotoxicity in cultures and brain infarction after focal ischemia.

84Perampanel. Non-competitive AMPA antagonist approved since 2012 as an anticonvulsant. It is still under US patent protection, but this begins to lapse in 2021. The basis for its favorable side effect profile is unclear but perhaps reflects partial antagonism of central AMPA receptors, analogous to memantine on NMDA receptors. It reduces infarction in rats after focal ischemia, and even if dosed too low to achieve neuroprotection on its own, it might aid the survival of neurons and oligodendrocytes expressing Ca2+/Zn2+-permeable AMPA receptors.

85Minocycline. Minocycline is a second generation, brain-penetrant tetracycline antibiotic in clinical use since 1971. Aside from antibiotic properties, it has unexpected neuroprotective effects in a wide range of acute and chronic injury models, including infarct reduction after focal ischemia in nearly 20 animal studies. This ischemic neuroprotection has been largely attributed to inhibition of matrix metalloproteinases (MMPs) at standard antimicrobial doses. Brain MMP-9 cellular expression and release to the extracellular space is increased after ischemia, likely contributing to inflammation and BBB breakdown. However minocycline also inhibits PARP at lower concentrations (10–100 nM) than needed for MMP-9 inhibition. It has been tested as a monotherapy in human stroke in several small pilot studies, and shown suggestions of benefit; a third study was negative, cooling enthusiasm.

86Pyruvate. The glycolytic metabolite, pyruvate, is sold as a human dietary supplement, typically with suggestions that it may aid weight loss or improve athletic performance. High (mM) concentrations of pyruvate reduce Zn2+ neurotoxicity in vitro, a protective effect that may be due to intracellular Zn2+ chelation by accumulating citrate and isocitrate, additional to benefits on cellular NAD+ and ATP stores. Anti-inflammatory effects have also been described. Large doses of pyruvate (500 mg/kg) powerfully blocked selective neuronal death in rat hippocampus after TGI and 30 m-1 h delayed treatment with lower doses (62.5–125 mg/kg) reduced infarction after tMCAO or pMCAO. Chronic doses of oral pyruvate (22–44 g/d × 4 weeks) were well tolerated in a human dietary trial, except for some diarrhea.

87Hydrogen. While the physicochemical properties of H2 have been long known, recent studies have highlighted how these position H2 favorably for use as a biological protectant. H2 efficiently quenches toxic hydroxyl and peroxynitrite radicals, but has little effect on the superoxide or hydrogen peroxide molecules that participate in normal cellular signaling, a profile partially shared with edaravone. H2 readily diffuses through tissues and penetrates cell membranes, quenching hydroxyl radicals generated even in cell nuclei. H2 exposure is not associated with any known toxicity and hence environmental concerns focus on explosivity (occurring at 41,000 ppm with ambient O2). Humans are routinely exposed to H2 as a trace gas in air at about 0.5 ppm, and this can reach 7,500 ppm (0.75%) in closed environments such as within submerged submarines. Inhalation of 49% H2 (with 50.2% helium and 0.8% O2) in a gas mixture called Hydreliox has been used by human divers.

88Administration of H2 has been reported to produce protective and anti-inflammatory effects in multiple experimental injury settings, including cardiac ischemia, organ transplantation, sepsis, and 1-methl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal loss. Inhalation of 2% H2 gas reduced infarction in rats after tMCAO and brain injury in swine after TGI. Besides radical scavenging, indirect mechanisms, e.g., activation of transcription factor Nrf2, have been suggested to contribute to protective effects. A small randomized controlled trial of H2 gas inhalation in stroke patients reported good safety and a hint of benefit. My colleagues at Stony Brook University have initiated a pilot therapeutic trial of H2 in patients with acute ischemic stroke in combination with a 5 days course of minocycline (H2M treatment – ClinicalTrials.gov Identifier NCT03320018).

89Another potential partner drug is the endogenous purine metabolite and plasma antioxidant, uric acid. Uric acid reduces excitotoxicity in culture and infarction after tMCAO in rats; further reviewed in, and a Phase IIb clinical trial (URICO-ICTUS) examining the effect of a 1 g dose administered together with tPA showed a trend toward better outcomes at 90 days. However uric acid can have pro-oxidant effects in membrane and intracellular compartments, promoting NOX activity and itself forming radicals.

90Non-pharmacological partner approaches should also be considered. Mild hypothermia attenuates a broad swath of injury cascades, including early and late excitotoxicity pathways, and protects the human brain after TGI (cardiac arrest/resuscitation). Implementation challenges and side effects have slowed efforts to bring hypothermia forward as a monotherapy for stroke, but these issues would recede if it were implemented leniently without need to reach monotherapy effectiveness. As noted by, ischemic brain injury lies on a temperature-dependence continuum: even mild hyperthermia is clearly bad for the injured brain.

91There are of course a daunting – factorial, if order matters – number of possible rational drug and dosage combinations. If only a highly optimized combination will succeed, the discovery and development road ahead could end up lengthy, but everything we know at present leaves open the more favorable possibility that many combination approaches now on the table would end up working well enough to be clinically useful. Perhaps Radicava or Nerinetide are already there as monotherapies.

92To systematically identify the most promising and robust combinations for clinical testing, the animal efficacy bar might be further raised to the point that most monotherapies would fail. This has been done historically by increasing treatment delay after tMCAO or pMCAO, but it may be that commitment points to various deaths will constitute a hard assay ceiling, limiting ability to differentiate among multiple promising approaches. And a long treatment window per se is no longer a requirement for clinical value, as treatment delays are becoming progressively shorter in today’s stroke centers. Other ways to raise the laboratory animal efficacy bar might be to raise core temperature, raise pre-stroke glucose (increasing ischemic acidosis), add concurrent organ stressors, or utilize specific genetic backgrounds prone to severe strokes. Adequate survival times to permit delayed infarction to complete, efficacy in both male and female animals, and demonstration of white matter protection in animal models larger than rodents should also be sought.

93Stroke remains today a leading cause of death and disability throughout the world, and hypoxic-ischemic CNS injury occurs additionally in other common settings such as head trauma, spinal cord injury, cardiac arrest and surgery. The unabridged strength of the central hypothesis that excitotoxicity damages brain in human stroke, expanded understandings of how excitotoxicity works and where it might be interdicted, readiness of the health care system to manage stroke patients with alacrity (finally), and the accessibility of several promising partner treatments for combination therapies, all suggest that it is time to recharge the global effort to develop anti-excitotoxic neuroprotective drugs for stroke. Unfortunately, in recent years large pharmaceutical companies have substantially shifted investment away from discovering and developing innovative neuroscience drugs, based on return-on-investment assessments. Alzheimer’s disease R&D may still be on the table, but stroke R&D is certainly not - the failure of earlier stroke neuroprotection trials contributed palpably to corporate de-investment decisions. Thus the recharge will need to be led, at least initially, by academic investigators and biotechnology companies.

94DC wrote the work and approved it for publication.

95DC is a paid scientific advisor to GNT Pharma Co., Ltd., Seoul, South Korea.