SAN DIEGO —Despite the significant advances that have been made over the past decade in delineating the cellular mechanisms of ischemic brain injury, researchers have not been able to translate these gains in basic research into clinically effective neuroprotective treatments. One major line of investigation has involved the theory of excitotoxicity, which is based on the observation that large concentrations of glutamate can destroy neurons. The failure of multiple clinical trials to demonstrate the neuroprotective efficacy of several glutamate or N-methyl-D-aspartate (NMDA) receptor antagonists has led investigators to search for other potential causative mechanisms.

Now investigators have identified new, glutamate-independent targets for neuroprotection in brain ischemia. The findings may have important implications for the development of a new clinical paradigm for neuroprotection, according to Roger P. Simon, MD. Dr. Simon is Director of Neurobiology Research and Chair in Neurology at the Robert S. Dow Neurobiology Laboratories in Portland, Oregon.

Currently, he said, the central investigative focus regarding ischemic brain injury continues to involve calcium toxicity, which is seen as the trigger for apoptosis as well as necrosis. "However, the ineffectiveness of the glutamate/NMDA receptor antagonists in terms of protecting the brain from stroke has clouded our understanding of the process by which toxic calcium loading of CNS cells is injurious," he noted.

Newly identified glutamate-independent targets—called acid-sensing ion channels—are widespread in the central and the peripheral nervous systems of mammals. Because acidosis, a common metabolic feature of ischemic brain, is thought to play a key role in brain injury and because acid-sensing ion channels exhibit calcium permeability, investigators hypothesized that acid-sensing ion channels have a pathologic function in ischemic injury, Dr. Simon explained at the 130th Annual Meeting of the American Neurological Association.

"Acid-sensing ion channels are present in brain, so keep your eye out for them," he said. "They can flux calcium via the ASIC-1a subunit. They are activated by the simplest of molecules—the hydrogen ion—but they also sense, in the setting of acidosis, substrate depletion. Thus, they’re ischemia receptors as well.

"They cause injury, but they can also modulate injury—at least in the paradigm that global ischemia gives us—via up-regulation of the acid-2a subunits," he continued. "So I offer to you acid-sensing ion channels as new, glutamate-independent targets for neuroprotection in brain ischemia."

Dr. Simon also offered his audience a visual representation of this new neuroprotective paradigm, involving the lipid bilayers in a normal cell and an ischemic cell. In this model of ischemia, hydrogen ions accumulate in the extracellular compartment, and when the pH is around 6, the hydrogen ions open the acid-sensing ion channels and calcium fluxes through. However, with the introduction of a peptide antagonist blocking the acid-1a channel subunit, the calcium entry and injury can both be blocked.

"We’ll have better drugs to modulate this system in the future, I hope," Dr. Simon said, "but that’s the [current] neuroprotective paradigm for ischemic brain."

If Dr. Simon and his colleagues are correct about these glutamate-independent targets, then it may not be long before others in the field subscribe to the view expressed in the informal alternative title of his lecture: "Acidotoxicity trumps excitotoxicity."

A FIRST STEP IN THE SEARCH FOR NEUROPROTECTION

In the 1970s, J. B. Brierley and colleagues hypothesized that the development of vacuoles in the cytoplasm of neurons was the earliest morphologic change one could see in ischemic brain. Dr. Brierley hypothesized that if one knew what caused the vacuolization, one might have a handle on the pathogenesis of brain ischemia and then might be able to intervene pharmacologically.

Dr. Simon recounted research conducted in 1984 with his colleague, Terry Griffiths, following up on Dr. Brierley’s observation and involving a then new experimental process called the oxalate/ pyroantimonate procedure. "If calcium is the toxic element in brain, one can find out where the calcium is by perfusing the brain with potassium oxalate, which is soluble," he said. "But calcium oxalate is not soluble and will precipitate in situ. If you then react those sections with pyroantimonate, a radiodense complex (calcium-pyroantimonate) is formed and one can see where the calcium is in brain.

"The vacuoles are loaded with calcium deposits," he continued. "So one step that we saw, in 1984, was that calcium loading was an early step in the pathogenesis of ischemic brain injury."

On closer examination, Dr. Simon and his colleagues found that almost all of the vacuoles were dilated mitochondria with the cristae destroyed, "and thus it turned out that mitochondrial failure was the answer to Dr. Brierley’s question about what caused microvacuole change in ischemic brain." This finding helped lay the foundation for the early work done by Dr. Simon and Brian Meldrum involving calcium flux and NMDA receptors, which were already known in some systems to have the ability to gate a calcium channel.

Simultaneously, Benveniste and Diemer, at the Institute of Neuropathology in Copenhagen, were using another technique, also new at the time—microdialysis—and demonstrated a marked increase of extracellular glutamate during brain ischemia. As glutamate is the agonist for the NMDA receptor, "the glutamate hypothesis, which, of course, John Olney and colleagues had shown in the retina some years ago [and] called excitotoxicity, was all laid out in ischemic brain in vivo in 1984."

The in vivo experiments, performed with Dr. Meldrum, in rat global ischemia models using antagonists of the NMDA receptor, demonstrated marked attenuation of injury from ischemic damage in the brain—to such an extent that Dr. Simon thought much of the needed basic research work for a stroke therapy had been accomplished.

"Later, it was shown that the non-NMDA receptor—at least the AMPA receptor—could gate calcium flux as well. And so the pharmacologic approach—the translational approach in regard to treating brain ischemia—was laid out."

By the 1990s, Dr. Simon continued, several ways to attenuate NMDA receptors had been found in experimental studies, and research teams such as Dr. Simon’s set out to bring these basic science observations to the clinical trial setting. Regardless of which drug was being tested, which technique was used, or at what laboratory, the results were essentially the same, confirming efficacy. "The experimental results were so consistent that the first time an NMDA receptor antagonist was studied in a human clinical trial and failed, there was at least one person who read the study and knew it spelled problems for future trials," Dr. Simon observed. "They were all going to be the same no matter which technique you used," he said, citing Fred Plum, MD, who noted in a 2001 JAMA editorial regarding the clinical trial failure of a glycine antagonist: "‘This was no surprise to me.’"

THREE-HOURS-PLUS OF PROTECTION

The search for glutamate-independent targets for neuroprotection in brain ischemia was on, and Dr. Simon proceeded to describe a series of studies involving acid-sensing ion channels, culminating in a set of experiments he termed "the blockbuster." In the NMDA receptor experiments that he had worked on, any positive effect seen early on (eg, within the first 15 minutes) was totally lost by the first hour. This new set of experiments, conducted by Giuseppe Pignataro, MD, and Dr. Simon, involved the acid-sensing ion channel–1a receptor. Dr. Pignataro and his colleagues realized, from the beginning of the experiments, that the finding of another one-hour time window would have relegated the study drug to the heap of interesting laboratory curiosities that went nowhere clinically. But something different occurred, Dr. Simon recounted.

"The first experiment was to give this antagonist (a peptide toxin) 15 minutes after the MCA [middle cerebral artery] occlusion," he said. "Then we waited an hour after the beginning of the MCA occlusion to give the toxin at the maximum effective dose, and we got the same result. So already we’re much better than any NMDA antagonist that I’ve ever seen. It’s the same effect at 15 minutes and an hour, and there’s really no step-down of the effect.

"Then we waited for two hours after the stroke, and the effect’s the same. And then we waited for three hours after the stroke, and the effect’s the same—three hours, the magic number for thrombolytics.

"Then we waited for five hours and you do lose some efficacy, but it’s still effective. But it doesn’t work at 12 hours," Dr. Simon said. "However, this [agent] has a time window that’s a real time window for clinical translational research, I suggest."

NR

—Fred Balzac

Suggested Reading
Benveniste H, Dreger J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem. 1984;43:1369-1374.
Brown AW, Brierley JB. The earliest alterations in rat neurons and astrocytes after anoxia-ischaemia. Acta Neuropathol (Berl). 1973;23:9-22.
Plum F. Neuroprotection in acute ischemic stroke. JAMA. 2001;285:1760-1761.
Simon RP, Griffiths T, Evans MC, et al. Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischemia: an electron microscopy study in the rat. J Cereb Blood Flow Metab. 1984;4:350-361.
Simon RP, Swan JH, Griffiths T, Meldrum BS. Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain. Science. 1984;226:850-852.
Xiong ZG, Zhu XM, Chu XP, et al. Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels. Cell. 2004;118:687-698.
Young C, Tenkova T, Dikranian K, Olney JW. Excitotoxic versus apoptotic mechanisms of neuronal cell death in perinatal hypoxia/ischemia. Curr Mol Med. 2004;4:77-85.

Return to table of contents