White Matter Abnormalities in Multiple Sclerosis Evaluated by Quantitative Synthetic MRI, Diffusion Tensor Imaging, and Neurite Orientation Dispersion and Density Imaging.

BACKGROUND AND PURPOSE: A number of MR-derived quantitative metrics have been suggested to assess the pathophysiology of MS, but the reports about combined analyses of these metrics are scarce. Our aim was to assess the spatial distribution of parameters for white matter myelin and axon integrity in patients with relapsing-remitting MS by multiparametric MR imaging. MATERIALS AND METHODS: Twenty-four patients with relapsing-remitting MS and 24 age- and sex-matched controls were prospectively scanned by quantitative synthetic and 2-shell diffusion MR imaging. Synthetic MR imaging data were used to retrieve relaxometry parameters (R1 and R2 relaxation rates and proton density) and myelin volume fraction. Diffusion tensor metrics (fractional anisotropy and mean, axial, and radial diffusivity) and neurite orientation and dispersion index metrics (intracellular volume fraction, isotropic volume fraction, and orientation dispersion index) were retrieved from diffusion MR imaging data. These data were analyzed using Tract-Based Spatial Statistics. RESULTS: Patients with MS showed significantly lower fractional anisotropy and myelin volume fraction and higher isotropic volume fraction in widespread white matter areas. Areas with different isotropic volume fractions were included within areas with lower fractional anisotropy. Myelin volume fraction showed no significant difference in some areas with significantly decreased fractional anisotropy in MS, including in the genu of the corpus callosum and bilateral anterior corona radiata, whereas myelin volume fraction was significantly decreased in some areas where fractional anisotropy showed no significant difference, including the bilateral posterior limb of the internal capsule, external capsule, sagittal striatum, fornix, and uncinate fasciculus. CONCLUSIONS: We found differences in spatial distribution of abnormality in fractional anisotropy, isotropic volume fraction, and myelin volume fraction distribution in MS, which might be useful for characterizing white matter in patients with MS.

Subunit- and site-specific pharmacology of the NMDA receptor channel.

N-Methyl-D-aspartate (NMDA) receptor channels play important roles in various physiological functions such as synaptic plasticity and synapse formation underlying memory, learning and formation of neural networks during development. They are also important for a variety of pathological states including acute and chronic neurological disorders, psychiatric disorders, and neuropathic pain syndromes. cDNA cloning has revealed the molecular diversity of NMDA receptor channels. The identification of multiple subunits with distinct distributions, properties and regulation, implies that NMDA receptor channels are heterogeneous in their pharmacological properties, depending on the brain region and the developmental stage. Furthermore, mutation studies have revealed a critical role for specific amino acid residues in certain subunits in determining the pharmacological properties of NMDA receptor channels. The molecular heterogeneity of NMDA receptor channels as well as their dual role in physiological and pathological functions makes it necessary to develop subunit- and site-specific drugs for precise and selective therapeutic intervention. This review summarizes from a molecular perspective the recent advances in our understanding of the pharmacological properties of NMDA receptor channels with specific references to agonists binding sites, channel pore regions, allosteric modulation sites for protons, polyamines, redox agents, Zn2+ and protein kinases, phosphatases.

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

The sensitivity of AMPA-selective glutamate receptor channels to pentobarbital is determined by a single amino acid residue of the alpha 2 subunit.

Clinical concentrations of pentobarbital inhibit the alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor (GluR) channels. Recently, the AMPA-selective GluR channels that contained the alpha 2 subunit were shown to be more sensitive to pentobarbital block than those without the alpha 2 subunit. Here we demonstrated that replacement by glutamine of the arginine residue in putative transmembrane segment M2 of the alpha 2 subunit (mutation alpha 2-R586Q) drastically reduced the pentobarbital sensitivity of the alpha 2 heteromeric channel to the level comparable to those of the alpha 1 and alpha 2-R586Q homomeric channels. These results suggest that the arginine residue in segment M2 of the alpha 2 subunit is the critical determinant of the sensitivities of the AMPA-selective GluR channels to pentobarbital.

Involvement of presurgical pain in preemptive analgesia for orthopedic surgery: a randomized double blind study.

Preemptive analgesia (PA) is effective in animal models but its clinical effectiveness remains controversial. We examined the effect of preexisting pain on PA. Subjects were recruited from patients needing orthopedic surgery. Some had presurgical pain (fracture surgery and arthritic surgery), while others had no presurgical pain (removal surgery for a tumor, nail or plate). Epidural morphine or a saline control was given preemptively before surgery and maintained until skin closure. Following skin closure, naloxone or placebo was injected intravenously to erase the aftereffects of the morphine. After total recovery, the PCA pump was set to inject epidural morphine. Pain intensity after surgery was measured by a visual analogue scale (VAS), and the amount of morphine used within 48h after surgery. PA was significantly effective for removal surgery, but ineffective for fracture or arthritic surgery. For the fracture and arthritic surgery PA treatment groups, there was a significant correlation between pre- and postsurgical (6h) spontaneous pain, while the corresponding control groups showed no significant correlation. Postsurgical VAS values in the fracture and arthritic surgery control groups increased significantly compared with presurgical VAS values. PA was effective when presurgical pain was absent, but ineffective when presurgical pain was present. We propose that central sensitization is already established by presurgical pain, and preserved until the termination of surgery. The ineffectiveness of PA did not depend on whether the pain was acute (fracture surgery) or chronic (arthritic surgery).

Sensitivity of the N-methyl-D-aspartate receptor channel to butyrophenones is dependent on the epsilon2 subunit.

The effects of three kinds of butyrophenones, haloperidol, droperidol and spiperone, on the N-methyl-D-aspartate (NMDA) receptor channel were examined on the epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1 and epsilon4/zeta1 heteromeric NMDA receptor channels, expressed in Xenopus oocytes. Micromolar concentrations of haloperidol selectively inhibited the epsilon2/zeta1 channel, whereas the epsilon1/zeta1, epsilon3/zeta1 and epsilon4/zeta1 channels were enhanced or minimally affected by higher concentrations of haloperidol. Similarly, droperidol and spiperone inhibited the epsilon2/zeta1 channel more strongly than the other epsilon/zeta channels, although sensitivities of the epsilon2/zeta1 channel to droperidol and spiperone were lower than those to haloperidol. These results suggest that the sensitivities of the NMDA receptor channels to butyrophenones are dependent on the epsilon2 subunit. Furthermore, the replacement with glutamine of the conserved asparagine residue in segment M2, which constitutes the Mg2+ block sites, of the epsilon2 and zeta1 subunits (the mutations epsilon2-N589Q and zeta1-N598Q, respectively) reduced the sensitivities to haloperidol. The mutation zeta1-N598Q reduced the sensitivities to haloperidol more effectively than the mutation epsilon2-N589Q. These results, together with previous findings, suggest that the haloperidol block sites of the NMDA receptor channel partially overlap the Mg2+ block sites.

Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.

Synthesis and 5-lipoxygenase inhibitory activities of eicosanoid compounds.

Ten eicosanoid compounds (3, 6, 9, 11, 12, 15, 18, 21, 23, and 25), methyl (6E,8Z,11Z,14Z)-5-hydroxy-6,8,11,14-eicosatetraenoate (5-HETE, 10), leukotriene A4 (26), and (5S,6E,8E,10E,12RS,14E)-5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE, 27) were prepared and their inhibitory activities against the 5-lipoxygenase from guinea pig polymorphonuclear leukocytes (PMNL) were tested. 5,6-Methanoleukotriene A4 (18) was especially a potent and specific inhibitor of the 5-lipoxygenase without inhibiting the cyclooxygenase and the 12-lipoxygenase. Leukotriene A4, 5-HETE, and 5,12-diHETE also have inhibitory activities against the 5-lipoxygenase at micromolar concentrations, which can regulate the formation of slow-reacting substance of anaphylaxis intracellulary.

Regionally different elevation of intracellular free calcium in hippocampus of septic rat brain.

The effect of sepsis on cellular calcium homeostasis in the central nervous system (CNS) was investigated using hippocampal slices of rats in which sepsis was induced by cecal ligation and puncture (CLP). Hippocampal slices were prepared from septic or sham-operated rats at 24 h after abdominal surgery. The basal intracellular calcium ([Ca2+]i) and its response to oxygen-glucose deprivation in hippocampal slices were measured for assessing cellular calcium homeostasis using fura-2 fluorescent imaging technique. The levels of [Ca2+]i were estimated by the fluorescence ratio (R340/380). Twenty-four hours after CLP, spontaneous movement was reduced and plasma lactate was increased in the septic rats in comparison with the sham-operated rats in which laparotomy was performed without CLP. Basal level of R340/380 in the CA4 ara (.72 +/- .07) was significantly higher (p < .001) in the septic group than that in the sham-operated group (.55 +/- (.06). The fluorescence ratio of septic vs. sham-operated in other hippocampal regions were .55 +/- .09 vs. .48 +/- .06 in CA1 (not significant) and .65 +/- .10 vs. .59 +/- .08 (not significant) in CA3, respectively. Increase in [Ca2+]i due to oxygen-glucose deprivation was significant in CA1 and CA3 of the septic group and in all hippocampal regions of sham-operated group. However, it was not significantly increased in CA4 of the septic group. These results suggest that regional deregulation of cellular calcium occurs in the CNS following CLP. Cellular calcium deregulation may be one of the pathogeneses occurred in clinically observed septic encephalopathy.

Ischemic tolerance preserves propagation of membrane depolarization.

The functional integrity of the synaptic connections within the hippocampus in gerbils that had acquired ischemic tolerance was investigated. The propagation of membrane depolarization across the hippocampus in response to electrical stimulation of CA1 was monitored with the use of a high speed optical recording technique. In comparison to control slices, propagation was significantly depressed and depolarization was shortened in slices from gerbils subjected to 5 min of ischemia. Hippocampal slices from gerbils who were preconditioned with prior sublethal ischemia demonstrated only a slight reduction in propagation. The duration of depolarization was longer than that of ischemia group. These findings suggest that ischemia induces a functional disturbance of synaptic transmission and membrane depolarization. Ischemic preconditioning significantly reduced the extent of this functional disturbance.

Different sensitivities of NMDA receptor channel subtypes to non-competitive antagonists.

Four kinds of heteromeric N-methyl-D-aspartate (NMDA) receptor channels, the epsilon 1/zeta 1, epsilon 2/zeta 1, epsilon 3/zeta 1 and epsilon 4/zeta 1 channels, were expressed in Xenopus oocytes and their sensitivities to various non-competitive antagonists were examined. The epsilon 1/zeta 1 and epsilon 2/zeta 1 channels were more sensitive to (+)MK-801 (dizocilpine) than the epsilon 3/zeta 1 and epsilon 4/zeta 1 channels, whereas the sensitivities to phencyclidine (PCP), ketamine and N-allylnormetazocine (SKF-10,047) were only slightly variable among the four epsilon/zeta channels. Furthermore, the replacement by glutamine or arginine of the conserved asparagine residue in segment M2 of the epsilon 2 and zeta 1 NMDA receptor channel subunits reduced the sensitivities to PCP, ketamine and SKF-10,047, though to different extents. These results, together with previous findings, suggest that these non-competitive antagonists as well as (+)MK-801 and Mg2+ act on a common site.

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice.

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.

Brain injury improves survival of mice following brain ischemia.

Minor brain injury was inflicted with a small needle at 4 sites one week before the production of incomplete brain ischemia in the mouse. A bilateral carotid clamp was applied for 60 min under pentobarbital anesthesia, and the number of survivors at one week after the ischemic insult was compared with those in animals anesthetized only and those in a sham-operated group. The number of survivors in the brain-injured group was significantly higher than in the other two groups. The results suggest that anti-ischemic factors are released by the injured brain or that certain unknown protective mechanisms against ischemia become active following brain injury.

Halothane-induced hyperpolarization and depression of postsynaptic potentials of guinea pig thalamic neurons in vitro.

Intralaminar thalamic nuclei have been considered to be a component of the non-specific sensory system which is involved in physiological functions related to consciousness and pain sensation. The effect of halothane on membrane potentials and synaptic properties of neurons of the parafascicular (Pf) nucleus in guinea pig brain slices was investigated using intracellular recording methods. Halothane at concentrations of 0.4-1.0 mM, which are in the range of clinical concentrations, produced hyperpolarizations of 2-8 mV in approximately 50% of the cells. The halothane-induced hyperpolarization was nullified at a membrane potential close to the K+ equilibrium potential. The amplitude of the hyperpolarization was dependent on the external K+ concentration, and was decreased by either Ba2+, or 4-aminopyridine, or intracellular injection of Cs+. All these results indicate that the hyperpolarization was due to an increase in K+ conductance. Halothane at clinical concentrations depressed both excitatory and inhibitory postsynaptic potentials in a concentration-dependent manner. On the other hand hyperpolarizing responses to exogenous gamma-aminobutyric acid (GABA) in the presence of bicuculline were suppressed by halothane, but depolarizing responses to L-glutamate were not altered. The results indicate that the depressant action of the anesthetic on the excitatory postsynaptic potential (EPSP) may occur presynaptically, whereas the blocking action on the inhibitory postsynaptic potential (IPSP) may occur postsynaptically.

Anesthetics block excitation with various effects on inhibition in MRF neurons.

The effects of isoflurane and halothane on the behavior of mesencephalic reticular neurons in the rat were studied by long-term extracellular microelectrode recording. Both anesthetics always suppressed the excitatory responses with simultaneous blocking or augmenting effects on the inhibitory responses of the reticular neurons. The blocking effects on the inhibitory responses were more frequently noticed during light anesthesia than during the deep stage.

Selective depression of hippocampal inhibitory postsynaptic potentials and spontaneous firing by volatile anesthetics.

The effects of halothane, isoflurane and enflurane on the rat hippocampal CA1 neurons in in vitro preparations were studied by intracellular recordings. All volatile anesthetics, at the concentrations which are likely to be in the range of clinical doses, depressed the IPSPs and the spontaneous firing without affecting the resting membrane properties and the EPSPs. The results suggest that the simultaneous blocking effects of the anesthetics on both the spontaneous firing and IPSPs are responsible for production of general anesthesia rather than their blocking actions on the EPSPs in terms of behavior of hippocampal neurons.

Role of A delta afferent fibers in modulation of primary afferent input to the adult rat spinal cord.

To address the question of whether fine myelinated and unmyelinated primary afferent fibers contribute to the mechanism of presynaptic inhibition in the spinal cord, we studied dorsal root-evoked dorsal root potentials (DR-DRPs) using a newly developed longitudinal spinal cord slice preparations in the adult rat. Single stimuli applied to the L6 dorsal root elicited a DR-DRP in the L5 dorsal root which had an amplitude of 50-150 microV and had a half decay time of 20-66 ms. The DR-DRP was depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-20 microM), while DL-2-amino-5-phosphonovaleric acid (APV, 50-100 microM) had no significant effect. DR-DRP was markedly depressed by bicuculline or picrotoxin. The evoked DR-DRP was unchanged in rats treated with capsaicin which eliminated the majority of unmyelinated C afferent fibers. Taken together with the higher voltages (> or = 1.9 V) required to elicit DR-DRP, this observation strongly suggests that the A delta afferent fibers are primarily responsible for producing and receiving the DR-DRP. The present study shows that the DR-DRP mediated by the A delta fibers in the slice preparation is analogous to those described for larger myelinated fibers in vivo. This pathway may contribute importantly to synaptic modulation of somatosensory information, including nociception at the superficial dorsal horn through an interneuronal connection which are mediated by the non-NMDA and GABAA receptors.

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca2+ and glycolysis.

To elucidate the mechanism of pHi changes induced by membrane depolarization, the variations in pHi and [Ca2+]i induced by a number of depolarizing agents, including high K+, veratridine, N-methyl-D-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pHi and an elevation of intracellular calcium ([Ca2+]i) in the CA1 pyramidal cell layer. The increases in [Ca2+]i caused by the depolarizing agents almost completely disappeared in the absence of Ca2+ (0 mM Ca2+ with 1 mM EGTA). In Ca2+ free media, pHi acid shifts produced by high K+, veratridine or NMDA were attenuated by 10-25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pHi acid shifts induced by both high K+ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K+, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na+-K+ ATPase activity. A Ca2+-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H+ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.