Background Animal stroke models demonstrate excitatory amino acid (EAA) release in ischemic tissue, as measured by microdialysis. Currently glutamate antagonist drugs are being developed to protect brain tissue after ischemic events. However, the role of EAAs in human occlusive stroke is not well known. We therefore measured glutamate and aspartate release in a patient after occlusive stroke.
Case Description We describe a case of occlusive stroke in a 50-year-old man. A partial temporal lobectomy was done to remove infarcted tissue and to prevent brain stem compression as well as uncal herniation. A microdialysis probe was placed into the cortex to measure EAAs. Massively increased levels of glutamate and aspartate were detected in the extracellular fluid in this patient (>300 times normal levels 6 days after infarction).
Conclusions These findings indicate that EAAs are tremendously increased in brain tissue after occlusive stroke. The time course of the release of EAAs is much longer than animal studies have suggested previously. Administration of EAA antagonists to patients with ischemic stroke may therefore be beneficial.
Currently no effective therapy exists for human occlusive stroke. However, major interest is currently focused on the development and evaluation of effective strategies for pharmacological therapy of this condition.1 At present the two most promising avenues of intervention appear to be blockade of free radical–induced brain damage after ischemia and blockade of excitatory amino acid (EAA) release. For both these mechanisms, promising drugs are available that are highly effective in the laboratory.1 2 Phase II and phase III trials are currently under way in human stroke with both free radical scavengers and glutamate N-methyl-d-aspartate (NMDA) antagonists.3
The use of glutamate antagonists is based on the premise that release of EAAs persists for at least several hours after the ictal event in human stroke. However, this has never been demonstrated. Release of EAAs, as measured by microdialysis, has been a brief and transient phenomenon in a variety of small animal stroke models.4 5 6 In this case report we document extremely high levels of extracellular glutamate release, persisting for 8 days, after severe occlusive stroke.
This study was approved by the Committee on the Conduct of Human Research at the Medical College of Virginia, Virginia Commonwealth University.
A 10-mm flexible, custom-built, commercial microdialysis probe with an external diameter of 0.5 mm was used (CMA 20 custom probe, CMA/Microdialysis). The probe was inserted intraoperatively into the cortex. In the intensive care unit the probe was perfused at 2 μL/min with the use of sterile 0.9% saline. Every 30 minutes we collected 60-μL dialysates into sealed glass tubes using a refrigerated (4°C) collector system (CMA 170 system, CMA/Microdialysis). The microdialysis probe was removed after 60 hours, and a total of 118 samples were collected during this period. In vitro calibration of the probe, by perfusion in a bath solution of known EAA concentration, after removal revealed a recovery rate for EAAs of 43%. Glutamate, aspartate, and threonine were measured with the use of high-performance liquid chromatography (HPLC).7 Fluorimetric detection was used after precolumn derivatization with ortho-pthaldialdehyde, according to the method of Lindroth and Mopper.8 Using an autosampler, we injected a panel of amino acid standards of known concentration into the HPLC system after each 10 samples. Amino acid concentrations in samples were then measured by peak integration. Clinical events and the sampling times of the dialysate were logged into a mainframe computer, together with arterial blood pressure, intracranial pressure, and end-tidal CO2.
A 50-year-old man presented to a community hospital with sudden onset of left hemiplegia and drowsiness on September 23, 1994. He had sustained a right internal carotid artery occlusion after transient ischemic attacks, without significant brain infarction, in 1992. No carotid surgery was performed. Four days after this admission he rapidly worsened. He became comatose, with increased muscle tone in the left extremities. A repeated CT scan showed extensive infarction in the territory of the right middle cerebral and anterior cerebral arteries. There was also a 10-mm right-to-left midline shift and uncal herniation. After intubation, he was transferred to the stroke service of the Medical College of Virginia and placed on sodium warfarin, heparin, and phenytoin. Mannitol was given to minimize brain swelling, and a myocardial infarct was excluded.
The patient underwent a left ventriculostomy to reduce intracranial pressure after his coagulation status was corrected. He was then taken to the operating room for temporal lobectomy and partial frontal lobe resection of infarcted tissue because intracranial pressure remained high (>25 mm Hg). A 2×2-mm fragment of the infarcted tissue, 15 mm from the site of microdialysis probe placement, was sent for electron microscopy. The surgery was uneventful. A microdialysis probe was placed intraoperatively within infarcted tissue in the right parietal lobe, at least 2 cm from the resection site (Fig 1⇓). Intracranial pressure remained below 25 mm Hg postoperatively. During the first postoperative day, the patient obeyed commands and opened both eyes. He had a persistent dense left hemiplegia. After extubation, speech was slow and slurred for the first 3 days. The microdialysis probe was removed and saved for calibration 60 hours after implantation. The patient improved clinically, and by the third month he was partially caring for himself and was ambulant at home with the use of a quadripod cane.
Amino Acid Release
As shown in the top panel of Fig 2⇓, glutamate and aspartate in the dialysate were massively increased. Levels of 250 μmol/L for glutamate and 120 μmol/L for aspartate were seen in the first 5 hours after implantation (day 6 after occlusive stroke). Thereafter, there was a gradual decline in both EAAs, which declined to 3 to 5 times above normal levels after 50 hours of measurement (day 8 after initial event). Absolute extracellular fluid levels of glutamate were thus at least 500 μmol/L and possibly much higher, allowing for tortuosity factors. The normal values for glutamate and aspartate in human extracellular fluid are less than 2 μmol/L and 0.2 to 0.6 μmol/L, respectively.9 10 Structural amino acid release into extracellular fluid may be an indicator of diffuse cellular breakdown and autolysis. However, threonine levels were much lower than those of EAAs in this patient (Fig 2⇓, bottom panel).
Fig 3⇓ demonstrates the cytoarchitectural changes in the infarcted tissue. Massive astrocyte swelling and neuronal pyknosis are seen.
There are currently at least four trials with competitive and noncompetitive glutamate NMDA antagonists, either in progress or about to commence, in Europe and the United States.1 3 Before the construction of these trials, there was debate regarding issues such as the “window of opportunity” after the ischemic event, the optimal duration of therapy, and appropriate dosing. In animal middle cerebral artery occlusion stroke models, release of EAAs has been brief and transient, lasting only 1 to 2 hours. Thus, if information obtained from these animal studies with microdialysis is extrapolated into the clinic, the “enrollment window” after the ischemic event and before administration of drug would need to be kept as short as possible, and a single dose of drug (competitive NMDA antagonist) or a brief infusion may be sufficient. In our patient, however, glutamate and aspartate appear to be elevated to levels at least 300 times higher than normal for several days after the ischemic event.
Although it is necessary to interpret these data with caution because of the effects of resectional surgery on EAA release as detected by microdialysis, the implication of these data is clear: EAA release appears to persist much longer in ischemically damaged human tissue than in animal models, and the magnitude of EAA release appears to be very much higher. This is consistent with our findings in severe human head injury.1 11 Patients who have sustained global posttraumatic ischemic brain damage and focal contusions demonstrate prolonged increases in EAAs for up to 4 days at levels 50 to 70 times higher than normal.1 10 It is also consistent with the findings of Persson et al7 10 in grade IV subarachnoid hemorrhage patients with significant focal tissue damage.
We speculate that this persistence of EAA release and the greater magnitude of EAAs in humans may be due to the very much larger volume of ischemically damaged tissue relative to the dialyzing surface of the microdialysis probe in humans in comparison to rodent studies. The persistent but less marked increase in the structural amino acid threonine (Fig 2⇑, bottom panel), which was also seen in this patient, suggests that the release of EAAs is probably chiefly due to vesicular release of neurotransmitter glutamate and aspartate. Clearly, however, autolysis of infarcted tissue or phagocytic activity of leukocytes may also release EAAs from the damaged tissue, and breakdown of the blood-brain barrier after infarction may allow egress of EAAs from plasma into the cerebral extracellular fluid space.
Nevertheless, this persistent EAA release may constitute a potent mechanism for delayed brain swelling by exposure of penumbral astrocytes and neurons to increased levels of EAAs, which would in turn induce ionic leak, calcium entry, cell swelling, and consequent infarct recruitment, with swelling of the tissue, as shown in Fig 3⇑. This study strongly suggests that such a mechanism may be important in the delayed deterioration that is seen in at least one third of human stroke patients.12 This study supports the hypothesis that EAA antagonists may be beneficial in ameliorating outcome after severe human stroke, and it further argues in favor of a longer duration of therapy with these agents than has hitherto been considered, at least in patients with severe ischemic lesions such as ours.
Review of this manuscript was directed by Guest Editor Richard J. Traystman, PhD.
- Received April 10, 1995.
- Revision received August 17, 1995.
- Accepted August 17, 1995.
- Copyright © 1995 by American Heart Association
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