Asymptomatic middle cerebral artery stenosis diagnosed by magnetic resonance angiography.

We reviewed 1440 MRA studies to identify patients with middle cerebral artery stenosis (MCAS). We identified 99 cases, and after reviewing the clinical records, classified 28 as asymptomatic MCAS (AMCAS), a prevalence of 2%. Suspected stroke was the most frequent indication for MRA. Follow-up was available for 21, mean 46.7 months (range 2.4-75.6 months). One stroke occurred in the AMCAS territory (5%), other strokes in five patients (24%). There were five deaths in patients with MCAS; age > 69 (P = 0.045) was the only associated risk factor. This study suggests that patients in whom MRA is performed and shows AMCAS may be at increased risk of strokes in any vascular distribution or of death.

Benefits of perfusion MR imaging relative to diffusion MR imaging in the diagnosis and treatment of hyperacute stroke.

BACKGROUND AND PURPOSE: The development of thrombolytic agents for use with compromised cerebral blood flow has made it critical to quickly identify those patients to best treat. We hypothesized that combined diffusion and perfusion MR imaging adds vital diagnostic value for patients for whom the greatest potential benefits exist and far exceeds the diagnostic value of diffusion MR imaging alone. METHODS: The cases of patients with neurologic symptoms of acute ischemic stroke who underwent ultra-fast emergent MR imaging within 6 hours were reviewed. In all cases, automatic processing yielded isotropic diffusion images and perfusion time-to-peak maps. Images with large vessel distribution ischemia and with mismatched perfusion abnormalities were correlated with patient records. All follow-up images were reviewed and compared with outcomes resulting from hyperacute therapies. RESULTS: For 16 (26%) of 62 patients, hypoperfusion was the best MR imaging evidence of disease distribution, and for 15 of the 16, hypoperfusion (not abnormal diffusion) comprised the only imaging evidence for disease involving large vessels. For seven patients, diffusion imaging findings were entirely normal, and for nine, diffusion imaging delineated abnormal signal in either small vessel distributions or in a notably smaller cortical branch in one case. In all cases, perfusion maps were predictive of eventual lesions, as confirmed by angiography, CT, or subsequent MR imaging. CONCLUSION: If only diffusion MR imaging is used in assessing patients with hyperacute stroke, nearly one quarter of the cases may be incorrectly categorized with respect to the distribution of ischemic at-risk tissue. Addition of perfusion information further enables better categorizing of vascular distribution to allow the best selection among therapeutic options and to improve patient outcomes.

Creation and characterization of 5-fluorodeoxyuridine-resistant Arg50 loop mutants of human thymidylate synthase.

Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase. Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y. Tong et al., J. Biol. Chem. 273: 11611-11618, 1998). We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy. A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR. E. coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E. coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants. Four 5-FdUR-resistant mutants were purified to apparent homogeneity. Kinetic studies indicate that these enzymes are highly efficient. Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme. These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.

Predictors of clinical improvement, angiographic recanalization, and intracranial hemorrhage after intra-arterial thrombolysis for acute ischemic stroke.

BACKGROUND AND PURPOSE: We sought to evaluate predictors of clinical outcome, angiographic success, and adverse effects after intra-arterial administration of urokinase for acute ischemic stroke. METHODS: We designed a Brain Attack program at University Hospitals of Cleveland for diagnosis and treatment of patients presenting within 6 hours of onset of neurological deficit. Patients with ischemia referable to the carotid circulation were treated with intra-arterial urokinase. Angiographic recanalization was assessed at the end of medication infusion. Intracerebral hemorrhage was investigated immediately after and 24 hours after treatment. Stroke severity was determined, followed by long-term outcome. RESULTS: Fifty-four patients were treated. There was improvement of >/=4 points on the National Institutes of Health Stroke Scale from presentation to 24 hours after onset in 43% of the treated patients, and this was related to the severity of the initial deficit. Forty-eight percent of patients had a Barthel Index score of 95 to 100 at 90 days, and total mortality was 24%. Cranial CT scans revealed intracerebral hemorrhage in 17% of patients in the first 24 hours, and these patients had more severe deficits at presentation. Eighty-seven percent of patients received intravenous heparin after thrombolysis, and 9% of them developed a hemorrhage into infarction. Angiographic recanalization was the rule in complete occlusions of the horizontal portion of the middle cerebral artery, but distal carotid occlusions responded less well to thrombolysis. CONCLUSIONS: The intra-arterial route for thrombolysis allows for greater diagnostic precision and achievement of a higher concentration of the thrombolytic agent in the vicinity of the clot. Disadvantages of this therapy lie in the cost and delay. Severity of stroke and site of angiographic occlusion may be important predictors of successful treatment.

Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.

Hyperacute stroke: ultrafast MR imaging to triage patients prior to therapy.

PURPOSE: To test diffusion- and perfusion-weighted MR imaging techniques within the extreme time constraints of stroke evaluation before therapy, and then, with MR imaging, stratify patients into those without ischemia, those with noncortical ischemia, and those with cortical ischemia. MATERIALS AND METHODS: T2-weighted turbo gradient- and spin-echo images and echo-planar diffusion- and perfusion-weighted images were obtained. Trace diffusion-weighted images and time-to-peak perfusion maps were automatically postprocessed and immediately available for interpretation. RESULTS: Forty-one patients with acute stroke symptoms underwent imaging within 6 hours of symptom onset; 35 were eligible for the therapy protocol. The mean time from entering the emergency department to beginning MR imaging was 45 minutes; the mean total MR imaging time was less than 15 minutes. Immediate image analysis directly affected individual clinical management. Four patients showed evidence of no infarct; seven, of lacunar infarct; and 24, of acute cortical infarct. Sixteen patients underwent angiography, thirteen had large-vessel occlusion, eleven were treated intraarterially, and in seven, recanalization was achieved. CONCLUSION: Echo-planar diffusion- and perfusion-weighted MR imaging for acute stroke is feasible and applicable before therapy decisions. Ultrafast MR imaging permitted immediate triage of 35 patients with symptoms of hyperacute stroke and thus helped avoid the risks from angiography and thrombolytic agents in some or spurred the judicious use of more aggressive intervention in others.

Current patterns and future possibilities in the care of acute ischemic stroke.

Zack Hall, Director of the NINDS, succinctly described the present situation: "This is the golden age of neuroscience research" (verbal presentation at the annual meeting of the American Neurological Association, 1997). Recent trials in clinical research have demonstrated the power of thrombolytic therapy in acute ischemic stroke, and investigators across the country are now refining that therapy. The advantages of thrombolytic intervention can be provided to a much larger proportion of the population as we defined techniques to slow the rate of neuronal cell death after ischemia. However, the most exciting future opportunities for care of individuals of acute ischemic stroke arises from two somewhat unsuspected avenues. First, neuroscientists are learning in an extraordinarily rapid fashion the potentials of replacement of neural cell populations using progenitor cells. Second, the incredible explosion of genetic information has created an opportunity to identify genes responsible for atherosclerosis and other cerebrovascular disease. This, in turn, could lead to precise therapies that prevent or diminish the disease. Exploiting these opportunities requires that neural clinicians continue their cooperative efforts and, also, learn to work together with neuroscientists and geneticists. With such cooperation, we are poised to translate the golden age of neuroscience research into a genuine benefit for individuals with cerebrovascular disorders.

Improving enzymes for cancer gene therapy.

New techniques now make it feasible to tailor enzymes for cancer gene therapy. Novel enzymes with desired properties can be created and selected from vast libraries of mutants containing random substitutions within catalytic domains. In this review, we first consider genes for the ablation of tumors, namely, genes that have been mutated (or potentially can be mutated) to afford enhanced activation of prodrugs and increased sensitization of tumors to specific chemotherapeutic agents. We then consider genes that have been mutated to provide better protection of normal host tissues, such as bone marrow, against the toxicity of specific chemotherapeutic agents. Expression of the mutant enzyme could render sensitive tissues, such as bone marrow, more resistant to specific cytotoxic agents.

Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases.

Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.

Brain attack: emergency treatment of ischemic stroke.

Thrombolysis has been demonstrated to be an effective treatment for ischemic stroke. The major obstacles to more widespread use of this therapy are lack of awareness that the treatment is possible and the short (less than three hours) therapeutic window. Indiscriminant use of this therapy can lead to an unacceptably high rate of intracerebral hemorrhage. Early recognition of the onset of stroke. Immediate transfer to a suitably equipped treatment facility and careful screening of a computed tomographic scan of the head for signs of early infarction are necessary for the safe administration of intravenous thrombolysis.

Thrombolysis for acute ischemic stroke.

The use of thrombolytic agents to restore cerebral blood flow is one of the most notable advances in the treatment of ischemic stroke. This article reviews thrombolytic therapy, its limitations, and the techniques by which thrombolytic agents can be delivered.

Evidence for independent feedback control of horizontal and vertical saccades from Niemann-Pick type C disease.

We measured the eye movements of three sisters with Niemann-Pick type C disease who had a selective defect of vertical saccades, which were slow and hypometric. Horizontal saccades, and horizontal and vertical pursuit and vestibular eye movements were similar to control subjects. The initial movement of oblique saccades was mainly horizontal and most of the vertical component occurred after the horizontal component ended; this resulted in strongly curved trajectories. After completion of the horizontal component of an oblique saccade, the eyes oscillated horizontally at 10-20 Hz until the vertical component ended. These findings are best explained by models that incorporate separate feedback loops for horizontal and vertical burst neurons, and in which the disease selectively affects vertical burst neurons.

Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists.

A predictable pattern of selective neuronal cell death occurs in organotypic slice cultures of neonatal rat hippocampus during the second and third weeks in vitro. We serially examined organotypic cultures at three, four, seven, 14, 21 and 28 days in vitro, using uptake of the fluorescent dye propidium iodide to identify degenerating cells. After seven days in vitro, the cell degeneration that accompanies the slicing procedure appears to have ended. However, at 14 days in vitro, degenerating neurons could be identified in area CA3. When many degenerating cells were present in a slice, they were distributed in the dentate hilus (CA4) and proximal portions of CA1 as well. Neuronal degeneration involving mainly CA1 pyramidal cells was still apparent at 21 days in vitro, but was much less marked than at 14 days. Study of fixed cultures with light and electron microscopy methods confirmed the presence of degenerating neurons with a pyknotic or vacuolated appearance. Spontaneous neuronal degeneration at 14 and at 21 days in vitro was almost entirely prevented by the addition of 10.5 mM Mg2+ or 3 mM kynurenic acid (a glutamate receptor antagonist), beginning at seven days in vitro. Cell death was markedly decreased by treatment with 100 microM DL-2-amino-5-phosphonovaleric acid (a selective antagonist of N-methyl-D-aspartate glutamate receptors). Removal of the blocking agents by returning cultures to control media at 28 days in vitro induced widespread neuronal degeneration, involving all the regions of the hippocampal slice cultures. The inhibition of spontaneous neuronal cell death by glutamate receptor antagonists and by blockade of glutamate release at synapses suggests that the mechanism of cell death involves glutamate receptors. The time course of degeneration suggests that the vulnerability to glutamate excitotoxicity is an aspect of developmentally regulated components of glutamatergic synapses acquired in the hippocampal organotypic cultures after the first week in vitro.

Effects of strength training on bone mineral density: hormonal and bone turnover relationships.

The effects of a 16-wk strength-training program on bone mineral density (BMD) was assessed by dual-energy X-ray absorptiometry in 21 men [age 61 +/- 1 (SE) yr]. Sixteen men (age 59 +/- 2 yr) served as control subjects. To investigate the possible hormonal relationships underlying the effects on BMD, serum concentrations of growth hormone, insulin-like growth factor I, and testosterone were determined before and after training. In addition, osteocalcin and skeletal alkaline phosphatase (markers of bone formation) and tartrate-resistant acid phosphatase (a marker of bone resorption) were measured before and after training to assess bone turnover. The training program resulted in a 2.8 +/- 0.6% increase in femoral neck BMD (1.004 +/- 0.037 vs. 1.031 +/- 0.037 g/cm2; P < 0.001). However, there were no significant changes in total body, anterioposterior spine, lateral spine, Ward's triangle, or greater trochanter BMD. Moreover, there were no significant changes in growth hormone, insulin-like growth factor I, testosterone, osteocalcin, or skeletal alkaline phosphatase. There were no changes in the control group. Thus, strength training can increase femoral neck BMD, and this effect does not appear to be accompanied by changes in anabolic hormones or markers of bone formation and resorption.

Cytoplasmic structure in organotypic cultures of rat hippocampus prepared by rapid freezing and freeze-substitution fixation.

We have compared rapid freezing followed by freeze-substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 microns without visible ice crystals. Freeze-substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze-substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques.

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.

A novel epitope expressed on the surface of developing and mature astrocytes.

Plasmalemmal fractions from cultured astrocytes have been used as the immunogen in generating a monoclonal antibody, termed 8C10, which binds to the surface of cultured astrocytes of the rat. 8C10 immunoreactivity is present on the membrane surface of cultured type 1 astrocytes, type 2 astrocytes, oligodendrocytes, meningeal cells, and 02A progenitor cells, and it persists after aldehyde fixation. The antibody also stains aldehyde-fixed central nervous system, in a pattern which suggests that the plasma membranes of fine astrocytic processes in adult neuropil express the epitope. Astrocytic perikarya and processes in white matter are also stained, but there is no immunoreactivity present in neuronal processes or perikarya. Astrocytic processes in developing cerebellar cortex are stained at postnatal ages when some of these processes are guiding the migration of neuronal perikarya.

Effects of dexamethasone on the differentiation of membrane structure in cultured astrocytes.

Astrocytic processes investing vascular structures or forming the surface of mammalian brain have large numbers of orthogonally packed aggregates of intramembrane particles, termed "assemblies." Similar particle aggregates are expressed by astrocytes derived from neonatal rat forebrain in secondary culture, but they are much more uniformly distributed across the membranes of the cultured cells. Dexamethasone, a potent glucocorticoid, affects the differentiation of astrocyte membrane structure in two patterns, depending on the rate of proliferation in the culture. When confluent secondary cultures of astrocytes are exposed to 5 microM dexamethasone, the densities of assemblies increase, and in some cells approach the values present in the glial limitans in vivo. However, when rapidly proliferating astrocytes are exposed to dexamethasone during the first week of secondary culture, most of the astrocytes fail to express any assemblies. The rate of astrocyte proliferation is slowed, and a lower cell density is reached during the first 2 weeks of secondary culture in dexamethasone. The suppression of assemblies is transient: as the cultures approach confluence, the proportion of cells expressing assemblies increases to nearly control levels, and the density of assemblies increases to greater than control values in some astrocytes. Certain of the effects of dexamethasone on cultured astrocytes may have relevance for understanding the mechanism(s) of its action in treating cerebral edema.