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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The membranes of mammalian astrocytic processes apposed to blood vessels or forming the surface of the brain contain high concentrations of a characteristic intramembrane particle aggregate, termed "assemblies." In order to identify developmental processes which contribute to this remarkable regional specialization of membrane structure, we have devised culture conditions which support the differentiation of assemblies in secondary cultures of astrocytes derived from neonatal rat forebrain. We report here that different lots of fetal calf serum vary dramatically in their capacity to support the differentiation of assemblies. Fetal calf serum thus appears to exert two distinct influences on astrocyte development: it promotes the differentiation of type 2 astrocytes from bipotential precursor cells, as shown by others, and it influences the density of assemblies in type 1, flat, GFAP-immunoreactive astrocytes in our secondary cultures. Horse serum and defined media also support the appearance of assemblies in flat, GFAP-immunoreactive astrocytes. The separate effects of serum supplementation upon cell lineage and membrane differentiation have to be carefully considered in studies designed to examine factors influencing astrocytic development in vitro.

An improved method of preparing central nervous system tissue for examination in a scanning electron microscope is described. In cerebellar cortex, the precise distribution of synaptic junctions on Purkinje cell bodies, as well as complex arrangements of neurites in the glomeruli of the granule cell layer, are directly visualized. The method also provides views of glial cell processes investing brain surfaces, blood vessels and neurons. The principal advantage of a scanning electron microscopic study is that relationships between cells in the brain can be seen directly instead of inferred from laborious serial reconstructions or other more complex and indirect anatomical methods.

In tissue prepared by rapid freezing, freeze fracture, and shallow etching, the postsynaptic density of Purkinje cell dendritic spines has a substructure consisting of fine filaments and irregular, globular adherent proteins. The number and packing density of the globular proteins vary from region to region within a single density and are even more variable when different junctions are compared. Whereas actinlike microfilaments and spectrinlike filaments are juxtaposed to the postsynaptic density, they do not appear to be continuous with the constituent filaments of the density. We suggest that the postsynaptic density at this class of synapse is composed of fine filamentous proteins that insert on the postsynaptic membrane and serve as a supporting framework for a variety of globular proteins. The globular proteins may vary qualitatively and quantitatively from junction to junction, and are positioned in the region of the spine that has the greatest concentration of ionized calcium entering with the synaptic current, and the greatest extent of postsynaptic depolarization.

The distribution of somatostatin-like immunoreactivity in the caudate, putamen, globus pallidus and ventral mesencephalon of the normal human brain has been studied with immunocytochemical techniques, and compared to that seen in Huntington's disease. Within the normal striatum, sparsely distributed varicose fibers and a population of medium-sized neurons were stained. In Huntington's disease, somatostatin immunoreactive striatal neurons appear to degenerate in proportion to the loss of striatal tissue, but there is an increase in the density of immunostained varicose fibers. In contrast, the pattern and amount of fiber staining in the substantia nigra appeared virtually unchanged from that seen in the normal brain. The morphology of striatal perikarya containing somatostatin-like immunoreactivity and the patterns of fiber staining in normal and Huntington's disease pallidum and substantia nigra suggest that striatal neurons containing somatostatin-like immunoreactivity are local circuit neurons.

We used light and electron microscopic immunocytochemical methods to examine the structure of neuronal perikarya and processes containing cholecystokinin-like immunoreactivity (CCK-IR) in area CA1 of the rat hippocampus. The morphology of stained perikarya, their positions within all laminae, and the orientation of their dendrites indicate that CCK-IR is located in interneurons. These cells were seen in the electron microscope to have deeply folded nuclei and to receive both symmetric and asymmetric synaptic junctions on their cell somata and dendritic shafts. Their dendrites are essentially spine-free, but form bulges at the site of some asymmetric synaptic junctions. Axonal varicosities containing CCK-IR make symmetric synaptic junctions with cell somata and dendritic shafts of both pyramidal and non-pyramidal neurons. In addition, CCK-IR varicosities form symmetric junctions with unstained non-pyramidal neurons and with CCK-IR cells, suggesting either recurrent innervation of one cell on itself or interaction between interneurons. The presence of CCK-IR varicosities and synaptic junctions on pyramidal cells is in agreement with physiological data which indicate that CCK has a direct postsynaptic action. The observation of CCK-IR varicosities forming synaptic junctions on non-pyramidal cells suggests that CCK might also modify the response of interneurons.

The distribution of substance P and leucine-enkephalin in selected regions of brain obtained postmortem from patients with Huntington's disease and from neurologically normal persons has been studied with immunocytochemical techniques. In the normal brain, substance P immunoreactivity was identified in medium-sized neurons in the neostriatum, in neurons of the external segment of the globus pallidus, and in fine fibers in teh neostriatum, inner segment of the globus pallidus, and in the pars reticulata of the substantia nigra. Huntington's disease brains all exhibited a marked decrease in substance P fiber density in the substantia nigra and globus pallidus. A few medium-sized neurons with substance P immunoreactivity remained in the neostriatal remnant. Leucine-enkephalin immunoreactive processes were present throughout the neostriatum of normal brain, and were densely packed in the external segment of the globus pallidus and in the substantia nigra. A uniform population of medium-sized neurons containing immunoreactive leucine-enkephalin was present in the caudate and putamen. By contrast, in the Huntington's disease brains there was a marked diminution of fiber staining in the globus pallidus and substantia nigra. A few medium-size neurons and sparse fibers with leucine-enkephalin immunoreactivity persisted in the atrophic neostriatum. These observations are consistent with previous reports of regional peptide concentrations in both normal and Huntington's disease brain. Cells containing substance P and leucine-enkephalin immunoreactivity persist in the basal ganglia in brains from patients with Huntington's disease, and we have no evidence that cellular content of one or the other peptide is associated with disproportionate cell death or survival.

The application of freeze-fracture techniques to studies of brain structure has led to the recognition of two unsuspected specializations of membrane structure, each distributed in a specific pattern across the surface of astrocytes. 'Assemblies' (aggregates of uniform, small particles packed in orthogonal array into rectangular or square aggregates) are found to characterize astrocytic plasma membranes apposed to blood vessels or to the cerebrospinal fluid at the surface of the brain. These particle aggregates are much less densely packed in astrocytic processes in brain parenchyma. Assemblies are not fixation artifacts, have been shown to extend to the true outer surface of the membrane, are remarkably labile in the setting of anoxia, and are at least in part protein. The function of assemblies is unknown, but their positioning suggests that they may have a role in the transport of some material into or out of the blood and cerebrospinal fluid compartments. A second specialization of intramembrane particle distribution, the polygonal particle junction, links astrocytic processes at the surface of the brain, and also links proximal, large caliber astrocytic processes in brain parenchyma. The function of this membrane specialization also is unknown, but it may subserve a mechanical role.

Huntington's disease is accompanied by severe neuronal death in the striatum, but despite this cell loss, there is a marked increase in the striatal concentration of somatostatin-like immunoreactivity (SLI). We attempted to examine the mechanism of this increase by using kainic or ibotenic acid to effect a unilateral lesion in the rat neostriatum. Graded doses of toxin cause a proportional decrease in the concentration of somatostatin-like immunoreactivity to a maximum of 50% of control, which is stable over an interval of 3 months. The increased somatostatin-like immunoreactivity in Huntington's disease is not mimicked by the excitotoxin lesions in rats. In addition we find that unilateral kainic acid lesions in the striatum reduce SLI in the contralateral striatum as well, although histologic evidence and assay of choline acetyltransferase activity indicate that damage is confined to the injected side. Immunocytochemistry demonstrates a loss of somatostatin-containing neurons on the lesioned side but no discernible loss in the contralateral striatum. The bilateral decrease in SLI following unilateral lesions suggests damage to a somatostatin projection to the contralateral striatum or a compensatory interaction between the two striatal nuclei.