John J. Woodward

Continuous Monitoring of Cerebral Substrate Delivery and Clearance: Initial Experience in 24 Patients with Severe Acute Brain Injuries

OBJECTIVE Current neuromonitoring techniques in severe human head injury often fail to detect the causes of clinical deterioration. A sensor is now available for continuous monitoring of brain oxygen tension, carbon dioxide tension, and pH values. In this study, brain tissue oxygen tension was used to differentiate patients at risk for brain ischemia and to predict outcome. METHODS The multiparameter sensor was inserted into brain tissue, along with a standard ventriculostomy catheter and a microdialysis probe, in 24 patients. Lactate and glucose were measured by high-pressure liquid chromatography in hourly dialysate samples. RESULTS Patients who experienced a good recovery (n = 8) sustained a mean brain partial oxygen pressure of 39 +/- 4 mm Hg, brain partial carbon dioxide pressure (PCO2) of 50 +/- 8 mm Hg, and a brain pH of 7.14 +/- 0.12. Patients with moderate to severe disability (n = 6) sustained a mean brain partial oxygen pressure of 31 +/- 5 mm Hg, brain PCO2 of 47 +/- 2 mm Hg, and a brain pH of 7.11 +/- 0.12. Ten patients who died or remained vegetative sustained a mean brain partial oxygen pressure of 19 +/- 8 mm Hg, a brain PCO2 of 64 +/- 21 mm Hg, and a brain pH of 6.85 +/- 0.41. Mean brain PCO2 levels of 90 to 150 mm Hg were consistently observed after cerebral circulatory arrest or brain death. Dialysate lactate and glucose were less clearly correlated to outcome than brain oxygen tension. Dialysate glucose was extremely low in all patients and zero in most patients who died. CONCLUSION Brain oxygen pressure, brain carbon dioxide pressure, and brain pH measurements, as well as a microdialysis probe for glucose and lactate analysis, may optimize the management of comatose neurosurgical patients by allowing a fuller understanding of the dynamic factors affecting brain metabolism.

Massive Persistent Release of Excitatory Amino Acids Following Human Occlusive Stroke

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.

Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene

Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N‐methyl‐D‐aspartate (NMDA), γ‐aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non‐NMDA receptors. We utilized two‐electrode voltage‐clamp and whole cell patch‐clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 μM to 10 mM) produced a reversible, concentration‐dependent inhibition of acetylcholine‐induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The α4β2 and α3β2 subunit combinations were significantly more sensitive to toluene inhibition than the α4β4, α3β4 and α7 receptors. Receptors composed of α4 and β2(V253F) subunits showed α4β4‐like toluene sensitivity while those containing α4 and β4(F255V) subunits showed α4β2‐like sensitivity. In hippocampal neurons, toluene (50 μM to 10 mM) dose‐dependently inhibited ACh‐mediated responses with an IC50 of 1.1 mM. Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit‐dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene.

Effects of volatile solvents on recombinant N‐methyl‐D‐aspartate receptors expressed in Xenopus oocytes

We have previously shown that toluene dose‐dependently inhibits recombinant N‐methyl‐D‐aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC50 value for toluene of 0.17 mM. We now report on the effects of other commonly abused solvents (benzene, m‐xylene, ethylbenzene, propylbenzene, 1,1,1‐trichlorethane (TCE) and those of a convulsive solvent, 2,2,2‐trifluoroethyl ether (flurothyl), on NMDA‐induced currents measured in Xenopus oocytes expressing NR1/2A or NR1/2B receptor subtypes. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA‐induced currents that was dose‐ and subunit‐dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.

Effects of nitric oxide on reactive oxygen species production and infarction size after brain reperfusion injury.

OBJECTIVEDeleterious effects of strokes may be ameliorated when thrombolysis (i.e., with recombinant tissue plasminogen activator) restores circulation. However, reperfusion injury, mediated by oxygen free radicals (reactive oxygen species [ROS]), may limit the benefits of recombinant tissue plasminogen activator treatment. We hypothesized that, during reperfusion, exogenous nitric oxide (NO) would reduce stroke size by quenching ROS. METHODSTo investigate this hypothesis, we used two in vivo ischemia-reperfusion models, i.e., autologous cerebral embolism in rabbits and filament middle cerebral artery occlusion in rats. Using these models, we measured ROS levels (rabbit model) and stroke volumes (rat model) in response to transient ischemia, with and without intracarotid administration of ultrafast NO donor proline NO (proliNO). RESULTSIn the rabbit cerebral embolism model, intracarotid administration of proliNO (10−6 mol/L) (n = 6) during reperfusion decreased free radical levels from 538 ± 86 nmol/L in the vehicle-treated group (n = 7) to 186 ± 31 nmol/L (2,3′-dihydroxybenzoic acid;P < 0.001) and from 521 ± 86 nmol/L (n = 7) to 201 ± 39 nmol/L (2,5′-dihydroxybenzoic acid;P < 0.002). In the rat middle cerebral artery occlusion model, intracarotid administration of proliNO (10−5 mol/L) (n = 10) during reperfusion reduced the brain infarction volume from 256 ± 48 mm3 in the vehicle-treated group (n = 8) to 187 ± 41 mm3 (P < 0.005). In both experimental groups, intracarotid infusion of proliNO did not affect regional cerebral blood flow, mean arterial blood pressure, or brain and body temperatures. CONCLUSIONThe beneficial effects of early restoration of cerebral circulation after cerebral ischemia were enhanced by intracarotid infusion of proliNO, most likely because of ROS scavenging by NO. These findings suggest the possibility of preventive treatment of reperfusion injury using NO donors.

Measurement of nitric oxide and brain tissue oxygen tension in patients after severe subarachnoid hemorrhage

OBJECTIVENitric oxide (NO), one of the most powerful endogenous vasodilators, is thought to play a major role in the development of delayed vasospasm in patients with subarachnoid hemorrhage (SAH). However, the role of the production of cerebral NO in patients with SAH is not known. In other SAH studies, NO metabolites such as nitrite and nitrate have been demonstrated to be decreased in cerebrospinal fluid and in plasma. METHODSIn this study, a microdialysis probe was used, along with a multiparameter sensor, to measure NO metabolites, brain tissue oxygen tension, brain tissue carbon dioxide tension, and pH in the cortex of patients with severe SAH who were at risk for developing secondary brain damage and vasospasm. NO metabolites, glucose, and lactate were analyzed in the dialysates to determine the time course of NO metabolite changes and to test the interrelationship between the analytes and clinical variables. RESULTSBrain tissue oxygen tension was strongly correlated to dialysate nitrate and nitrite (r2 = 0.326;P < 0.001); however, no correlation was noted between brain tissue oxygen tension and NO metabolites in cerebrospinal fluid (r2 = 0.018;P = 0.734). No significant correlation between NO production, brain tissue carbon dioxide tension, and dialysate glucose and lactate was observed. CONCLUSIONCerebral ischemia and compromised substrate delivery are often responsible for high morbidity rates and poor outcomes after SAH. The relationship between brain tissue oxygen and cerebral NO metabolites that we demonstrate suggests that substrate delivery and NO are linked in the pathophysiology of vasospasm after SAH.

Intracellular calcium enhances the ethanol sensitivity of NMDA receptors through an interaction with the C0 domain of the NR1 subunit

Abstract: Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit‐dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild‐type NR1‐1a (NR1011) subunit with the calcium‐impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium‐impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent‐free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high‐calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2‐bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA‐induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1‐4a (NR1000), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium‐dependent enhancement of ethanols inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild‐type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch‐clamp electrophysiology was significantly reduced by expression of the C‐terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium‐dependent process that requires the C0 domain of the NR1 subunit.

A Comparison of the Effects of Ethanol and the Competitive Glycine Antagonist 7‐Chlorokynurenic Acid on N‐Methyl‐d‐Aspartic Acid‐Induced Neurotransmitter Release from Rat Hippocampal Slices

Abstract: N‐Methyl‐d‐aspartate (NMDA; 500 μM) stimulated the net release of preloaded tritiated norepinephrine from rat hippocampal slices. Both ethanol and the competitive glycine antagonist 7‐chlorokynurenic acid (7‐CK) dose‐dependently inhibited NMDA‐stimulated release without affecting basal, nonstimulated efflux. These inhibitory effects were readily reversed upon washout of the drugs. Over the concentration range tested (25–200 mM), ethanol inhibited ∼65% of NMDA‐stimulated release with an estimated IC50 of ∼70 mM. In contrast, 7‐CK fully inhibited release (>95%) at a concentration of 30 μM with half‐maximal inhibition occurring at ∼2 μM. The combination of 7‐CK (1–30 μM) and ethanol (25–100 mM) had an additive inhibitory effect on NMDA‐stimulated release but did not alter the inhibitory potency of 7‐CK. Calculated IC50values for 7‐CK in the presence of 25, 50, or 100 mM ethanol were (mean × SEM; μM) 2.33 (0.11), 2.38 (0.23), and 1.99 (0.30), respectively. 7‐CK (3 μM) inhibited NMDA‐stimulated [3H]norepinephrine release by ∼50%. This inhibition was fully attenuated by the addition of the glycine agonistserine with complete reversal occurring at 30 μMd‐serine. Increasing the 7‐CK concentration to 10 μM shifted the d‐serine dose‐effect curve to the right in a parallel fashion as expected for a competitive antagonist. In contrast, the inhibitory effects of ethanol or the combination of 7‐CK (3 μM) and ethanol (25 or 50 mM) were not reversed by the addition of d‐serine (0.1–1,000 μM). Together, these results suggest that ethanols inhibition of NMDA‐stimulated [3H]norepinephrine release from hippocampal slices is not due to a simple competitive interaction with the glycine site on the NMDA receptor.

Ethanol sensitivity of recombinant human N-methyl-D-aspartate receptors.

In this study, the ethanol sensitivity of human N-methyl-D-aspartate (NMDA) receptors stably expressed in L(tk-) cells, or transiently expressed in HEK 293 cells and Xenopus oocytes was determined. NMDA receptor function was measured using fura-2 calcium imaging for L(tk-) cells, whole cell voltage-clamp for HEK 293 cells, and two-electrode voltage clamp for oocytes. Ethanol inhibited NMDA receptor function in all three expression system, but was less potent for receptors expressed in L(tk-) cells. NMDA receptors composed of NR1a/2B subunits were inhibited to a greater extent by ethanol than NR1a/2A receptors when expressed in L(tk-) cells and HEK 293 cells, but not in oocytes. These results suggest that the method of receptor expression and assay system used may influence the degree of ethanol inhibition of recombinant NMDA receptors.

Patterns of Excitatory Amino Acid Release and Ionic Flux After Severe Human Head Trauma

In many of the conditions that result in acute cerebral damage, the role of excitatory amino acids has become the focus of current investigation [1, 2]. The demonstration of excitatory amino acid release has been shown in several animal models of brain trauma, and has been associated with both structural damage and metabolic abnormalities [3–5]. These changes may be reversed by administration of excitatory amino acid antagonists, and this group of compounds has shown a greater magnitude of neuroprotective effect than any other series of drugs in the laboratory [3, 6]. As part of an ongoing study, we have recently measured release of excitatory amino acids (EAAs) and ions in 17 acutely head-injured patients in our institution, with the aim of determining the circumstances that are responsible for glutamate release and their possible role as an exacerbating factor in causing secondary brain damage after severe human head injury.