Someone suffers a stroke every 53 seconds – and every 3.3 minutes somebody dies of one.

Stroke is a sudden-onset disturbance in brain activity resulting when blood supply to the brain is either compromised or altogether blocked (1). More commonly known as a cerebrovascular accident (CVA), stroke can be caused by arteriosclerotic disease, hypertension, embolism and hemorrhaging, and often results in debilitating paralysis, coma, convulsions, amnesia, dizziness, unsteadiness, weakness, impaired speech and vision, as well as other sensory and motor deficits. Stroke is the third leading cause of death in the United States and is the leading cause of long-term disability, accounting for an estimated $40 billion each year in health care costs and lost productivity (2). According to the American Heart Association approximately 500,000 strokes occur annually (2,3). Stroke morbidity is about equal for both men and women, but its mortality is not. More than half of total stroke deaths occur in women (3).

Two types of cerebrovascular disorders lead to stroke, a cerebral hemorrhage (hemorrhagic stroke) and cerebral ischemia (ischemic stroke). Ischemic strokes, which account for 80 percent of all strokes, begin with atherosclerotic disease, a process in which fatty deposits build on the inner walls of blood vessels and narrow the space through which blood can flow (4). Thrombi, or blood clots, form at the site of an atherosclerotic deposit because the deposit causes blood to flow in a turbulent and disorderly fashion. Turbulent blood flow can potentially nick off pieces of the calcified deposit, which then travel and land in a blood vessel too small to allow them to pass (emboli). When either thrombi or emboli develop and obstruct cerebral blood flow, regions of the brain that are deprived of oxygen and nutrition experience a slow, cascading death. Lack of blood flow to any part of the brain results in the signs and symptoms of stroke, a cerebral infarction, or permanent damage, that interferes with the functions of the affected part of the brain. Specific effects of a stroke, though, vary greatly depending on what part of the brain was deprived of oxygen and nutrition. If, for example, blood flow is reduced or cut off completely to part of the brain controlling speech (Broca’s area in the frontal lobes), the stroke may well result in a speech disability – either the ability to express oneself through speech, or writing, or the ability to understand speech or writing may be affected. On the other hand, if the motor cortex becomes damaged the stroke may result in difficulties walking, moving an arm or main-taining balance (2,5).

Despite improved imaging techniques of the brain and improved or new thrombolytic and neuroprotective agents, stroke continues to be one of the most ravaging and least understood cardiovascular events. A possible explanation is the unforgiving, and as yet cryptic, cycle of events that occurs following disruption of blood supply to parts of the

brain. Until recently care of stroke patients was largely supportive. There was little in the way of therapy to alter the course of the stroke itself, and therefore little cause for urgency in transport or therapy (6). Today, early recognition of signs of stroke, rapid transport and hospital triage can save and/or dramatically improve the lives of stroke victims. The recognition of signs and symptoms of stroke can lead to early diagnosis and treatment within 30 to 60 minutes of onset (7). Realistically, most victims of stroke deny symptoms and delay access for several hours after symptom onset. Delays in care often eliminate any possibility of innovative care. Patients who delay care do not improve and their prognosis is poor. Breakthroughs in biochemistry and medicine have shown that, almost paradoxically, some of the

Not so long ago, it was common to attribute neuronal damage that occurred during a stroke to a lack of oxygen and glucose. However, researchers now believe a toxic cascade of glutamate spreads chaos to other parts of the brain, resulting in the devastating and sometimes irreversible effects of stroke and a transient ischemic attack (TIA). When a blood vessel becomes blocked in the brain by an emboli or thrombi, oxygen and nutrition are not allowed to get to neurons downstream of the blockage. Neurons starved of oxygen and glucose release excessive amounts of glutamate from their synaptic bulbs. The glutamate then binds to NMDA (N-methyl-D-aspartate) receptors and triggers excessive influx of sodium and calcium ions, along with water, into the postsynaptic neurons. The neurons swell and neuronal toxicity and apoptotic death are initiated (9). Before glutamate-poisoned neurons die they too release excessive amounts of glutamate and the process of poisoning continues in a cascade-like fashion. Calcium released en bloc triggers release of calmodulin, which then stimulates nitric oxide synthase, an enzyme needed for the synthesis of nitric oxide. This acts as a retrograde messenger that travels back to the presynaptic neuron where it activates guanylyl cyclase, initiating greater calcium and sodium influx (10). Some scientists believe that, along with glutamate, excessive release of NO contributes to neuronal damage. Most agree, however, that glutamate plays the central and starring role in ischemia-induced brain damage.

The functional and structural unit of the nervous system is the neuron. There are an estimated 100 billion neurons in the human brain, and the number of possible interconnections that exist among them has been estimated to be greater than the total number of atomic particles in the known universe (11). The role of the neuron is to receive, process and transmit nerve impulses that can travel at speeds up to 120 meters per second and affect a chemical or electrical change downstream of the original stimulus. A typical neuron consists of a cell body, dendrites which receive chemical signals from other cells, and an axon, which carries an electrical signal from the cell body to the synaptic knobs [Fig 2].

In 1957 researchers Lucas and Newhouse first established neurotoxicity in rodent retinal tissue following intense exposure to extracellular glutamate (14). Twenty years later John Olney reported that low oxygen and glucose neuro-toxicity in the brains of animal stroke models brought about a huge increase in glutamate. He further found CAI hippo-campal neurons were especially sensitive to higher than normal glutamate concentrations since they appeared to degenerate and die faster (15). (The hippocampus is a region of the brain believed responsible for long-term retention of spatial memories.) Without fully understanding the mechanism by which in vitro neurotoxicity occurred, Olney termed the glutamate-poisoning phenomenon "excitotoxicity."

There are neuronal uptake systems in place to remove synaptically released glutamate from the extracellular space before a toxic build-up occurs. In a laboratory setting, where glutamate exposure could be measured and controlled, it was possible to examine directly the effects of glutamate on neuron degeneration. Choi and others found that exposure to 100 µM glutamate for five minutes destroyed large numbers of cultured cortical neurons (16). Steven Rothman observed that over-exposure to glutamate produced swelling and degeneration in cortical and hippocampal neurons in cell culture - swelling and degeneration that could be reduced and, in some cases, prevented with the controlled removal of extracellular calcium and sodium (14). To that end, Rothman suggested glutamate toxicity was somehow mediating an uncontrolled influx of calcium and sodium ions into postsynaptic neurons in hypoxia-induced laboratory conditions.

Similar results were observed in the late nineties when John Werth investigated excitotoxic swelling in oxygen and glucose deprived brain slices in both human and rodent models. The goal was to monitor the integrity of tissue (and thus cells) by directly monitoring the membrane potential of individual neurons in determining the effect hypoxia had on membrane depolarization. Werth and others examined human neocortical slices from eleven patients undergoing neural resections (removal of brain tissue) for intractable epilepsy. They also examined neocortical slices in rats. Earlier studies with neurons had shown that 70-75% of current flow in tissues was through the extracellular space (9). Tissue resistance was found to be inversely related to interstitial volume. So the higher the interstitial volume, the less resistance a current would experience. Werth reasoned that if cells were to swell and thereby shrink the extracellular space, the electrical resistance would be elevated in the tissues, given that the current experienced a higher resistance due to the decreased interstitial volume. Simulating hypoxia and hypoglycemia in the laboratory, Werth found that resistance increased as he passed a current across the tissue slices. The result was that the extracellular space shrunk since neurons were swelling and decreasing the extracellular space. Werth subsequently measured the resistance after adding a glutamate antagonist (5 µM of MK-801) to the tissue slices and found, to his amazement, decreased resistance in the tissues. The result was that MK-801 delayed and diminished cell swelling in both rat and human slices by binding at the NMDA site and preventing ion influx and consequent swelling (9). Werth showed that, upon oxygen and glucose deprivation of no more than ten minutes, neuronal swelling, degeneration and death were equal in both human and rodent tissue slices. Werth was able to reduce swelling and degeneration in tissue slices by administering MK-801, a chemical analog that competed with extracellular glutamate for the NMDA receptors and blocked the glutamate-gated receptor-ionophore. This decreased calcium and sodium entry and forestalled the neuro-toxic cascade. Although Werth and his team were not able to stop complete neuron swelling via antagonism with MK-801, they were able to show that by aggravating and manipulating the NMDA receptors, it was possible to delay early swelling that accompanied oxygen and glucose-deprived neurons. Despite shortcomings with sample tissue (namely the human cortical samples from patients suffering from severe epilepsy) theirs was the first experiment to validate the excitotoxic model of ischemia in the human brain (9). Perhaps most significant of all, Werth’s experiments antagonizing the glutamate receptors helped to determine the efficacy of neuroprotection, the idea that drugs could be administered (time-appropriate) as prophylaxis following ischemic injury in hopes of protecting what tissue was initially spared – in essence stopping the neurotoxic cascade dead in its tracks and preventing further cell death.

Experiments such as these lead to the theory that hypoxic-ischemic brain damage could be explained as over-stimulation of glutamate NMDA receptors and calcium and sodium incursion gone awry. A mechanism of neuronal degeneration and death brought about by hypoxia was quickly elucidated: 1) A blood vessel is blocked and hypoxic-ischemic conditions soon develop; 2) neurons affected downstream of the blockage release excessive glutamate; 3) glutamate binds to NMDA receptors, triggering excessive influx of calcium and sodium ions; 4) uncontrolled influx of calcium and sodium poisons postsynaptic neurons which, as they are dying, release even more glutamate into the extracellular space, spreading the toxicity and furthering the viscous cycle of neuronal death and destruction (17).

The theory was that ischemia-induced brain damage involved three noteworthy factors: 1) time between ischemic attack and damage; 2) the extent of damage; and 3) the mechanism of damage (17,18). In all stroke animal models it was found that, after a cerebral ischemic episode of no more than ten minutes, there was little or no evidence of brain damage. However, substantial neuron damage was detected days later when animal models were sacrificed and brain tissue was examined. The second finding was that brain damage didn’t occur equally in all parts. Neurons in the hippocampus are particularly susceptible to glutamate overload. Investigators in two different laboratories selectively lesioned the glutaminergic inputs and nerve tracts to the hippocampi in laboratory animals (18). They then simulated ischemic-hypoxic conditions by ligating the carotid arteries in the animals, reducing or halting blood flow to the brain and allowing sufficient time for ischemic damage to occur. Upon later examination the animals showed dramatically preserved and undamaged hippocampi. Another group of animals was given the same carotid ligation as the first group, but this time, the glutaminergic inputs and nerve tracts were left untouched by investigators. Later examination revealed substantial assault to the hippocampi.

A powerful correlation between glutamate and hippocampal damage was made when it was discovered that hippo-campi are particularly rich in glutamate NMDA receptors - explaining why hippocampal neurons possessing NMDA receptors are more vulnerable to elevated glutamate under conditions of global hypoxia or anoxia. A link between the presence of glutamate NMDA receptors and vulnerability to brain ischemia was made: the hippocampus is most susceptible to ischemic injury in laboratory animals due to the diffuse nature of the glutaminergic pathways in the hippocampi. The third and final factor in ischemia-induced brain damage was that mechanisms of damage vary somewhat from structure to structure, despite glutamate’s central and starring role in neuronal death and degeneration (17,18).

It is important to remember that glutamate may not be the be-all and end-all in neuronal death. Glutamate may not necessarily be the sole contributor and may, in fact, have little importance in different conditions (14). Other neuro-physiological variables – including focal versus global ischemia, different brain structures, integrity of the blood-brain barrier (BBB), animal models versus human models, brain temperature, brain edema, effects of other neuro-transmitters and/or chemicals as well as a myriad of other unknown factors – could easily account for, if not directly bring about, ischemia-induced brain damage. Still, with a decade full of supporting research and data, it remains clear that L-glutamate, released in excess of 100 µM, either initiates or accelerates a neurotoxic cascade that is not self-limiting.

Another mechanism in delayed brain injury (and a new target for therapeutic intervention) is the molecular cascade in neuronal apoptosis or programmed cell death (PCD). Cerebral ischemia tends to be followed by an inflammation reaction that promotes migration of leukocytes into areas of greatest oxygen deprivation (19). Inflammation chemicals such as cytokines, chemokines, and adhesion molecules appear to play a role in toxicity, by either plugging up the extracellular space or promoting a build-up of the neuronal toxins that cannot otherwise diffuse and escape into the extracellular space. Elevated levels of tumor necrosis factor-a (TNF-a) appear to be activated by a calpain, a proteolytic enzyme that becomes active once cytosolic calcium reaches a high enough level (as it would during hypoxic conditions when NMDA receptors are over-stimulated by glutamate and cannot prevent calcium rushing in) (9,13,14,19). Calpains initiate irreversible breakdown of key proteins within the cell, chewing away at parts of the cytoskeleton and proteins anchored to the plasma membrane, in effect causing the cell to destroy itself in a controlled manner.

Researchers for stroke therapies are now trying to manipulate, among other things, the neuron’s apoptotic pathway in hopes of developing drugs that aim to intervene in specific steps in programmed cell death. One novel approach is caspase inhibition. In vitro and in vivo studies performed by Gotton and Choi (1999) indicate that caspase inhibition reduced neuronal apoptosis and infarction volume when a caspase inhibitor N-benzyloxcarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK) was introduced to cultured rat neurons in a oxygen and glucose-deprived environment (9,19). The negative effects of the excitotoxic cascade were not observed and neuroprotection was assured. However, the extent of protection was limited to a therapeutic window of no more than six hours following a brief ischemic attack (one lasting no more than 30 minutes) (19). If z-VAD.FMK was administered at a later juncture (say, 8 or 10 hours after initial onset) infarction volume did not show appreciable reduction and the effectiveness of the inhibition was duly compromised. In both animal and human models, the first few hours after onset seem to offer the best hope of productive therapy when neuroprotecting and thrombolytic agents could have their greatest and most beneficial effects, saving what brain tissue remained and restoring blood flow where there was not any.

As of April 2001 there were over 800 experimental and clinical trials underway – the most important of which involved NMDA antagonists (MK-801), GABA agonists, AMPA agonists, calcium channel blockers, reducers of intracellular calcium, inhibitors of nitric oxide, free radical scavengers, glutamate release inhibitor, along with many others (9,14,21). Although not a neuroprotector t-PA continues to offer the best hope for patients and their families. This is of clinical significance because the average time patients seek medical attention is usually within 13 hours of the attack, and most patients are likely to arrive at the Emergency Department after the hyperacute stage (6 hour therapeutic window) (22). t-PA can significantly halt the cascade by restoring blood flow to damaged areas before toxicity develops. But if too much time has elapsed between onset and medical attention, neuroprotection and t-PA therapy will offer no substantial benefit. Recognition of warning signs is extremely important. Rapid recognition of stroke onset – including symptoms such as altered level of consciousness (ALOC), hypertension (HTN), hemiparesis (asymmetrical weakness and/or one-sided weakness), facial hemiparesis (facial drooping), dysphasia (impairment of speech), aphasia (absence of speech), unequal pupils, drooling, inability to swallow, and incontinence for stool and urine – as well as rapid activation of EMS, rapid transport, rapid triage and diagnosis and definitive care (using thrombolytics and other emerging therapies) can save and dramatically improve lives (6).