Regional brain volumes, diffusivity, and metabolite changes after electroconvulsive therapy for severe depression.

OBJECTIVE: To investigate the role of hippocampal plasticity in the antidepressant effect of electroconvulsive therapy (ECT). METHOD: We used magnetic resonance (MR) imaging including diffusion tensor imaging (DTI) and proton MR spectroscopy (1 H-MRS) to investigate hippocampal volume, diffusivity, and metabolite changes in 19 patients receiving ECT for severe depression. Other regions of interest included the amygdala, dorsolateral prefrontal cortex (DLPFC), orbitofrontal cortex, and hypothalamus. Patients received a 3T MR scan before ECT (TP1), 1 week (TP2), and 4 weeks (TP3) after ECT. RESULTS: Hippocampal and amygdala volume increased significantly at TP2 and continued to be increased at TP3. DLPFC exhibited a transient volume reduction at TP2. DTI revealed a reduced anisotropy and diffusivity of the hippocampus at TP2. We found no significant post-ECT changes in brain metabolite concentrations, and we were unable to identify a spectral signature at ≈1.30 ppm previously suggested to reflect neurogenesis induced by ECT. None of the brain imaging measures correlated to the clinical response. CONCLUSION: Our findings show that ECT causes a remodeling of brain structures involved in affective regulation, but due to their lack of correlation with the antidepressant effect, this remodeling does not appear to be directly underlying the antidepressant action of ECT.

Ventricular dilation and elevated aqueductal pulsations in a new experimental model of communicating hydrocephalus.

In communicating hydrocephalus (CH), explanations for the symptoms and clear-cut effective treatments remain elusive. Pulsatile flow through the cerebral aqueduct is often significantly elevated, but a clear link between abnormal pulsations and ventriculomegaly has yet to be identified. We sought to demonstrate measurement of pulsatile aqueductal flow of CSF in the rat, and to characterize the temporal changes in CSF pulsations in a new model of CH. Hydrocephalus was induced by injection of kaolin into the basal cisterns of adult rats (n = 18). Ventricular volume and aqueductal pulsations were measured on a 9.4 T MRI over a one month period. Half of the animals developed ventricular dilation, with increased ventricular volume and pulsations as early as one day post-induction, and marked chronic elevations compared to intact controls (volume: 130.15 +/- 83.21 microl vs. 15.52 +/- 2.00 microl; pulsations: 114.51 nl +/- 106.29 vs. 0.72 +/- 0.13 nl). Similar to the clinical presentation, the relationship between ventricular size and pulsations was quite variable. However, the pulsation time-course revealed two distinct sub-types of hydrocephalic animals: those with markedly elevated pulsations which persisted over time, and those with mildly elevated pulsations which returned to near normal levels after one week. These groups were associated with severe and mild ventriculomegaly respectively. Thus, aqueductal flow can be measured in the rat using high-field MRI and basal cistern-induced CH is associated with an immediate change in CSF pulsatility. At the same time, our results highlight the complex nature of aqueductal pulsation and its relationship to ventricular dilation.

Metabolomics of neural progenitor cells: a novel approach to biomarker discovery.

Finding biomarkers of human neurological diseases is one of the most pressing goals of modern medicine. Most neurological disorders are recognized too late because of the lack of biomarkers that can identify early pathological processes in the living brain. Late diagnosis leads to late therapy and poor prognosis. Therefore, during the past decade, a major endeavor of clinical investigations in neurology has been the search for diagnostic and prognostic biomarkers of brain disease. Recently, a new field of metabolomics has emerged, aiming to investigate metabolites within the cell/tissue/ organism as possible biomarkers. Similarly to other "omics" fields, metabolomics offers substantial information about the status of the organism at a given time point. However, metabolomics also provides functional insight into the biochemical status of a tissue, which results from the environmental effects on its genome background. Recently, we have adopted metabolomics techniques to develop an approach that combines both in vitro analysis of cellular samples and in vivo analysis of the mammalian brain. Using proton magnetic resonance spectroscopy, we have discovered a metabolic biomarker of neural stem/progenitor cells (NPCs) that allows the analysis of these cells in the live human brain. We have developed signal-processing algorithms that can detect metabolites present at very low concentration in the live human brain and can indicate possible pathways impaired in specific diseases. Herein, we present our strategy for both cellular and systems metabolomics, based on an integrative processing of the spectroscopy data that uses analytical tools from both metabolomic and spectroscopy fields. As an example of biomarker discovery using our approach, we present new data and discuss our previous findings on the NPC biomarker. Our studies link systems and cellular neuroscience through the functions of specific metabolites. Therefore, they provide a functional insight into the brain, which might eventually lead to discoveries of clinically useful biomarkers of the disease.

Evolution of a focal brain lesion produced by interlaced microplanar X-rays.

Stereotactic radiosurgery has led to advances in the treatment of central nervous system disease. It relies upon the principle of delivering relatively high dose irradiation to a precise target, while exposing surrounding tissues to extremely low doses. We describe a novel radiosurgical approach using interlaced microplanar X-rays which we have termed "microradiosurgery." The use of microbeams allows for 1,000-times greater precision than current clinically employed techniques. As a demonstration of this new method, we produced a approximately 3.8 mm (3) lesion in the rat brain. The lesion was followed over a period of 216 days using 9.4 Tesla magnetic resonance imaging. Our results show a gradually developing lesion at the site of the interlaced beams. The lesion began as a high T2 signal only, but advanced to include a central area of low T1 and mixed T2 signal within 2 months. No lesion was observed in the other side of the brain which was exposed to non-interlaced microbeams only. Interlaced microbeams is an effective method to create focal brain microlesions. This technique may allow the future treatment of pathology not accessible by surgical or more traditional radiosurgical means.

A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy.

A comprehensive three-dimensional digital atlas database of the C57BL/6J mouse brain was developed based on magnetic resonance microscopy images acquired on a 17.6-T superconducting magnet. By using both manual tracing and an atlas-based semi-automatic segmentation approach, T2-weighted magnetic resonance microscopy images of 10 adult male formalin-fixed, excised C57BL/6J mouse brains were segmented into 20 anatomical structures. These structures included the neocortex, hippocampus, amygdala, olfactory bulbs, basal forebrain and septum, caudate-putamen, globus pallidus, thalamus, hypothalamus, central gray, superior colliculi, inferior colliculi, the rest of midbrain, cerebellum, brainstem, corpus callosum/external capsule, internal capsule, anterior commissure, fimbria, and ventricles. The segmentation data were formatted and stored into a database containing three different atlas types: 10 single-specimen brain atlases, an average brain atlas and a probabilistic atlas. Additionally, quantitative group information, such as variations in structural volume, surface area, magnetic resonance microscopy image intensity and local geometry, were computed and stored as an integral part of the database. The database augments ongoing efforts with other high priority strains as defined by the Mouse Phenome Database focused on providing a quantitative framework for accurate mapping of functional, genetic and protein expression patterns acquired by a myriad of technologies and imaging modalities.

Progress in high field MRI at the University of Florida.

In this article we report on progress in high magnetic field MRI at the University of Florida in support of our new 750MHz wide bore and 11.7T/40cm MR instruments. The primary emphasis is on the associated rf technology required, particularly high frequency volume and phased array coils. Preliminary imaging results at 750MHz are presented. Our results imply that the pursuit of even higher fields seems warranted.

Tracking brain volume changes in C57BL/6J and ApoE-deficient mice in a model of neurodegeneration: a 5-week longitudinal micro-MRI study.

Magnetic resonance imaging (MRI)-based volume measurements of brain structures are useful indicators of pending cognitive decline in humans suffering from neurodegenerative diseases. Transgenic mouse models that mimic the clinical conditions of these disorders have been developed. Noninvasive methods that can follow progression and regression of relevant pathology in these mice are therefore in great demand. In this study we tested whether high-resolution MRI (micro-MRI) in a mouse model of neurodegeneration (cerebral ischemia) could reliably track development of brain atrophy. We first established that diffusion imaging at a spatial resolution of 1.6 x 10(-3) mm(3) allowed superior visualization of forebrain, ventricles, and dorsal hippocampus in the mouse brain compared to either T2*- or T1-weighted MR imaging. Using this predetermined protocol we subsequently scanned C56BL/6J (C57) and ApoE-deficient (ApoE(-/-)) mice before and after ischemia. Four groups were studied: C57/sham (n = 9), ApoE(-/-)/sham (n = 9), C57/ischemia (n = 9), and ApoE(-/-)/ischemia (n = 11). All mice received a baseline 3D diffusion scan. One week later C57/ischemia and ApoE(-/-)/ischemia mice were exposed to 10 min of ischemia and scanned again on the 3rd and 30th postischemic day. The C57/sham and ApoE(-/-)/sham mice served as controls and were scanned at corresponding time points. Diffusion images of ApoE(-/-)/ischemia mice on the 3rd postischemic day revealed multiple localized high signal intensity areas. An increase in ventricle and a decrease in dorsal hippocampal volumes (which included the associated cortex laterally) at 30 days confirmed brain atrophy in C57 mice after ischemia. Excessive mortality of ApoE(-/-)/ischemia mice restricted statistical analysis, but ventricle enlargement postischemia was demonstrated. Our results show that volume changes in the brain of a 30-g mouse can be tracked by micro-MRI in a model of neurodegeneration. Clearly the ability to follow progression of pathology in mice will greatly aid our understanding of neurodegenerative diseases and facilitate the many possibilities to intervene pharmacologically.

Magnetic resonance microscopy of the C57BL mouse brain.

With the rapid progression in gene technologies, transgenic, targeted, and chemically induced mutations in mice are continually created. The major goal of these studies is to understand and characterize the effects of genotype on anatomy, physiology, and behavior and ultimately the role of genotype in development of disease. The demand for imaging techniques with high spatial resolution potential is rising because such imaging tools would expedite anatomical phenotyping in the genetically altered mice. Magnetic resonance microscopy (MRM) is a noninvasive, inherently three-dimensional (3D) imaging technique capable of visualizing several anatomical structures in the small mouse. The 3D nature of MRM also allows for interpretation of complex spatial relationships between substructures, which is important when phenotyping anatomically. The goal of this paper is to systematically describe three major brain regions in the C57BL/6J mouse at microanatomical spatial resolution ranges using in vitro MRM. We explore different MR contrast parameters, voxel sizes, and signal-to-noise ratios to best characterize C57BL/6J mouse brain microstructure by MRM. Further, we compare all MRM images with Nissl-stained brain sections. Major findings were as follows: T2* MR images visualized several gross anatomical regions in the mouse brain but not, for example, subregions within the hippocampus. Diffusion proton stains on the other hand were superior to T2* MR images and delineated many subregions within the hippocampus proper. Finally, contrast enhancement facilitated visualization of hippocampal anatomy on the T2* MR images. The results of this study are part of an ongoing initiative at our Center focused on creating a complete C57BL/6J mouse anatomical 3D image database by MRM.

Cerebral hemorrhage and edema following brain biopsy in rats: significance of mean arterial blood pressure.

OBJECT: It is taken for granted that patients with hypertension are at greater risk for intracerebral hemorrhage during neurosurgical procedures than patients with normal blood pressure. The anesthesiologist, therefore, maintains mean arterial blood pressure (MABP) near the lower end of the autoregulation curve, which in patients with preexisting hypertension can be as high as 110 to 130 mm Hg. Whether patients with long-standing hypertension experience more hemorrhage than normotensive patients after brain surgery if their blood pressure is maintained at the presurgical hypertensive level is currently unknown. The authors tested this hypothesis experimentally in a rodent model. METHODS: Hemorrhage and edema in the brain after needle biopsy was measured in vivo by using three-dimensional magnetic resonance (MR) microscopy in the following groups: WKY rats, acutely hypertensive WKY rats, spontaneously hypertensive rats (SHR strain), and SHR rats treated with either sodium nitroprusside or nicardipine. Group differences were compared using Tukey's studentized range test followed by individual pairwise comparisons of groups and adjusted for multiple comparisons. There were no differences in PaCO2, pH, and body temperature among the groups. The findings in this study indicated that only acutely hypertensive WKY rats had larger volumes of hemorrhage. Chronically hypertensive SHR rats with MABPs of 130 mm Hg did not have larger hemorrhages than normotensive rats. There were no differences in edema volumes among groups. CONCLUSIONS: The brains of SHR rats with elevated systemic MABPs are probably protected against excessive hemorrhage during surgery because of greater resistance in the larger cerebral arteries and, thus, reduced cerebral intravascular pressures.

Detection of neuritic plaques in Alzheimer's disease by magnetic resonance microscopy.

Magnetic resonance microscopy (MRM) theoretically provides the spatial resolution and signal-to-noise ratio needed to resolve neuritic plaques, the neuropathological hallmark of Alzheimer's disease (AD). Two previously unexplored MR contrast parameters, T2* and diffusion, are tested for plaque-specific contrast to noise. Autopsy specimens from nondemented controls (n = 3) and patients with AD (n = 5) were used. Three-dimensional T2* and diffusion MR images with voxel sizes ranging from 3 x 10(-3) mm(3) to 5.9 x 10(-5) mm(3) were acquired. After imaging, specimens were cut and stained with a microwave king silver stain to demonstrate neuritic plaques. From controls, the alveus, fimbria, pyramidal cell layer, hippocampal sulcus, and granule cell layer were detected by either T2* or diffusion contrast. These structures were used as landmarks when correlating MRMs with histological sections. At a voxel resolution of 5.9 x 10(-5) mm(3), neuritic plaques could be detected by T2*. The neuritic plaques emerged as black, spherical elements on T2* MRMs and could be distinguished from vessels only in cross-section when presented in three dimension. Here we provide MR images of neuritic plaques in vitro. The MRM results reported provide a new direction for applying this technology in vivo. Clearly, the ability to detect and follow the early progression of amyloid-positive brain lesions will greatly aid and simplify the many possibilities to intervene pharmacologically in AD.

In vivo diffusion-weighted magnetic resonance microscopy of rat spinal cord: effect of ischemia and intrathecal hyperbaric 5% lidocaine.

BACKGROUND AND OBJECTIVES: Pathophysiologic mechanisms underlying persistent neurologic deficits after continuous spinal anesthesia using hyperbaric 5% lidocaine are still not well understood. It has been suggested that high-dose intrathecal lidocaine induces irreversible conduction block and even ischemia in white matter tracts by breakdown of the blood-nerve barrier. In this study, we use diffusion-weighted magnetic resonance microscopy to characterize the effect of intrathecal hyperbaric 5% lidocaine in rat spinal cord. The parameter measured with DWM, is an "apparent diffusion coefficient," (ADC), which can be used to exclude the presence of ischemia. METHODS: Female Fischer CDF rats were used. Group 1 (n = 5) was exposed to ischemia, group 2 (n = 7) was exposed to intrathecal 5% hyperbaric lidocaine, and group 3 (n = 5) was exposed to intrathecal 7.5% glucose. Diffusion-weighted MR images in group 1 were acquired before and after ischemia induced by cardiac arrest and in groups 2 and 3 rats prior to and during perfusion of the spinal catheter with either 5% hyperbaric lidocaine or 7.5% glucose. RESULTS: Ischemia decreased the ADC by 40% in gray matter and by 30% in white matter of spinal cord. Continuous intrathecal anesthesia with hyperbaric 5% lidocaine did not affect the spinal cord ADC. Further, 7.5% intrathecal glucose had no effect on ADCs in gray or white matter of spinal cord. CONCLUSIONS: Ischemia reduced the ADC in both spinal cord white and gray matter. Hyperbaric 5% lidocaine did not affect the spinal cord ADC during the first 1.5 hours. We suggest that 5% hyperbaric lidocaine does not induce irreversible neurologic deficits by causing spinal cord ischemia.

Anxiety, vocalization, and agitation following peripheral nerve block with ropivacaine.

BACKGROUND AND OBJECTIVES: Central nervous system (CNS) and cardiovascular toxicity are potential side effects of local anesthetics. However, ropivacaine has been reported to be less CNS toxic than bupivacaine in human volunteers. METHODS: We describe three cases of peripheral nerve blockade with ropivacaine that resulted in unusual symptoms of CNS toxicity. RESULTS: In three patients, unexpected behavioral changes occurred during administration of ropivacaine. The patients became extremely agitated, anxious, and screamed, and they did not respond to verbal commands. CONCLUSION: This case report shows that ropivacaine may cause CNS toxicity that differs from classical signs of local anesthetic-induced toxicity. This effect might be related to the unique structure of ropivacaine, which is formulated in an S-enantiomer preparation. It has been shown that S-enantiomers bind differently to receptors in both the CNS and cardiovascular systems. This property may account for the disinhibition of select neural pathways that are specifically involved in mediation of anxiety and aggression.

Spinal cord neural anatomy in rats examined by in vivo magnetic resonance microscopy.

BACKGROUND AND OBJECTIVES: Magnetic resonance microscopy (MRM) is a technique that is worthwhile for anesthesiologists because it allows spinal cord and plexus anatomy to be visualized three dimensionally and followed over time in the same animal. For example, the long-term effect of indwelling intrathecal or plexus catheters can be studied in situ, and convective and diffusive forces within intrathecal, epidural, or nerve sheath spaces can be investigated. Further, diffusion-weighted MRM, which measures an "apparent diffusion coefficient" (ADC), can be used to track the presence of ischemia, hypoperfusion, or cytotoxic edema. This study investigates problems associated with the use of in vivo MRM for spinal cord and peripheral nerve studies in the rat. METHODS: Twenty-one anesthetized female Fisher CDF rats were used. Group 1 (n=7) was used for anatomic three-dimensional studies. Groups 2 (n=4), 3 (n=4), and 4 (n=6) were used for measurements of the ADC. Group 2 served as controls, group 3 received lumbar intrathecal catheters, and group 4 received cervical intrathecal catheters. RESULTS: Cervical spine, lumbar spine, and spinal nerves and ganglia were accurately visualized with MRM. As a rule, spinal cord gray and white matter were better demonstrated using diffusion-weighted proton stains. By contrast, T2-weighted proton staining superiorly demonstrated structures surrounding the spinal cord. In groups 3 and 4, indwelling intrathecal catheters did not affect the spinal cord ADC, indicating normal blood flow and no cytotoxic edema. Contrast studies revealed nonhomogeneous distribution of contrast predominately in the lateral and ventral intrathecal space. CONCLUSION: Three-dimensional diffusion-weighted MRM displays cervical and lumbar spine anatomy accurately in vivo. Apparent diffusion coefficients measurements are feasible in rat cervical spinal cord with intrathecal catheters. Spinal cord ADCs are unaffected by intrathecal catheters, indicating normal spinal cord perfusion.

Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons.

The L1 CAM family of cell adhesion molecules and the ankyrin family of spectrin-binding proteins are candidates to collaborate in transcellular complexes used in diverse contexts in nervous systems of vertebrates and invertebrates. This report presents evidence for functional coupling between L1 and 440-kD ankyrinB in premyelinated axons in the mouse nervous system. L1 and 440-kD ankyrinB are colocalized in premyelinated axon tracts in the developing nervous system and are both down-regulated after myelination. AnkyrinB (-/-) mice exhibit a phenotype similar to, but more severe, than L1 (-/-) mice and share features of human patients with L1 mutations. AnkyrinB (-/-) mice exhibit hypoplasia of the corpus callosum and pyramidal tracts, dilated ventricles, and extensive degeneration of the optic nerve, and they die by postnatal day 21. AnkyrinB (-/-) mice have reduced L1 in premyelinated axons of long fiber tracts, including the corpus callosum, fimbria, and internal capsule in the brain, and pyramidal tracts and lateral columns of the spinal cord. L1 was evident in the optic nerve at postnatal day 1 but disappeared by postnatal day 7 in mutant mice while NCAM was unchanged. Optic nerve axons of ankyrinB (-/-) mice become dilated with diameters up to eightfold greater than normal, and they degenerated by day 20. These findings provide the first evidence for a role of ankyrinB in the nervous system and support an interaction between 440-kD ankyrinB and L1 that is essential for maintenance of premyelinated axons in vivo.

Progression of a focal ischemic lesion in rat brain during treatment with a novel glycine/NMDA antagonist: an in vivo three-dimensional diffusion-weighted MR microscopy study.

Stroke was induced in two groups of anesthetized rats by occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery. Group 1 (control) received vehicle and group 2 received the glycine N-methyl-D-aspartate (NMDA) antagonist ZD9379. Stroke volume was assessed by three-dimensional diffusion-weighted MR microscopy at 2.5 and 6 hours of MCA occlusion. At 2.5 hours, stroke volumes were identical in the two groups. At 6 hours, stroke volumes had increased by 15% in the control group; in contrast, the treated group showed a 40% reduced stroke volume. Conclusions from this in vivo study were as follows: (a) our technique allows more efficient and accurate measurement of stroke volume with an improvement in resolution over a previous method; (b) the ability to measure stroke volume at multiple time points shows volume change and assessment of time dependency of drug treatment; (c) at 6 hours, the glycine antagonist ZD9379 reduced stroke volume by 40%.

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats.

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive regardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.

A new in vivo method for quantitative analysis of stroke lesions using diffusion-weighted magnetic resonance microscopy.

Using three-dimensional diffusion-weighted MR microscopy and a rat model of focal cerebral ischemia, we evaluated the statistical characteristics of two parameters: absolute stroke volumes and change in stroke volumes over 6 h of middle cerebral artery (MCA) occlusion. In all rats, the absolute stroke volumes increased linearly over the 6-h MCA occlusion time period. On average, stroke volume growth rate was 2.1 +/- 0.5%/h. Sample size power analysis of our data demonstrated that to demonstrate a 10% reduction of the 6-h volumes, sample size per group would require 29 animals (these calculations are based on alpha = 0.05, beta = 0.20 using normal approximation). A similar 30% reduction of stroke volume at 6 h poststroke (from approximately equal to 200 to 140 mm3) would, in our "slope model," translate into a reduction of stroke growth rate from the normal + 11.25 mm3/h (150 to 200 mm3 over 4 h) to 7 mm3/h (150 to 178 mm3 over 4 h); power analysis in this case demonstrated that sample size is reduced to 15 animals per group (these calculations are based on alpha = 0.05, beta = 0.20 using normal approximation). We conclude that from a statistical standpoint our study demonstrates that stroke growth rate might be a more suitable parameter for evaluating the effect of treatment in both clinical and experimental stroke trials.

Nitric oxide synthase inhibition and extracellular glutamate concentration after cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Transient cerebral ischemia in rats results in selective loss of neuronal viability, eg, hippocampal CA1 neurons. The neurochemical variables responsible for this selective vulnerability to ischemia/reperfusion (IR) appear to involve excitatory amino acids. In brain IR, excitatory amino acid toxicity may be modulated by endogenous nitric oxide (NO.) gas. To investigate NO. in global brain IR, we measured the effects of NO. synthase (NOS) inhibition on interstitial excitatory amino acids in rats. Changes in postischemic cerebral blood flow and blood-brain barrier function also were evaluated. METHODS: Forebrain ischemia was produced by systemic hypotension and occlusion of both carotid arteries for 15 minutes. Blood flow was restored for 60 minutes by unclamping the carotids and reinfusing with blood. A microdialysis probe was placed into the cortex and hippocampus using a stereotaxic device. Interstitial glutamate concentration was measured during IR with high-performance liquid chromatography. A competitive NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), was given intraperitoneally 30 minutes before ischemia in doses of 1, 4, and 20 mg/kg. Changes in cerebral blood flow and blood-brain barrier during IR were determined using laser-Doppler flowmetry and microdialysis with sodium fluorescein. RESULTS: Glutamate in the dialysate during IR increased transiently 10-fold and returned to baseline levels by 30 minutes of reperfusion. Animals treated with L-NAME 30 minutes before ischemia also showed increases in glutamate concentration during ischemia, but glutamate remained elevated during reperfusion. The increase in glutamate concentration during reperfusion caused by L-NAME was prevented by L-arginine. The administration of L-arginine and L-NAME together decreased extracellular glutamate concentration during ischemia. Cerebral blood flow decreased to about 5% of baseline values during ischemia but increased approximately fourfold relative to control values on reperfusion. The hyperemic responses after ischemia were not different between IR groups treated with or without L-NAME. Brain ischemia increased the permeability of the blood-brain barrier to fluorescein; however, this change was attenuated by L-NAME administration at 20 mg/kg. CONCLUSIONS: NOS inhibition did not attenuate extracellular glutamate accumulation during ischemia and increased its concentration on reperfusion. The elevated glutamate concentration after IR in L-NAME-treated rats did not appear to be due to either a decrease in cerebral blood flow response after ischemia or increases in local blood-brain barrier permeability. For the most part, the blood-brain barrier was spared in the immediate postischemic period by L-NAME treatment. These data suggest that NO. production may oppose synaptic excitatory amino acid accumulation and presumably excitotoxicity during IR.