Region-specific reductions in brain apparent diffusion coefficient in cytomegalovirus-infected fetuses.

OBJECTIVE: To evaluate the effects of cytomegalovirus (CMV) infection on apparent diffusion coefficient (ADC) values of the fetal brain in utero. METHODS: In this retrospective analysis we compared 58 fetal head magnetic resonance imaging (fhMRI) scans of PCR-verified CMV-infected fetuses, obtained in 2008-2012, with those of a normal control group of 36 gestational age (GA)-matched uninfected fetuses scanned between 2006 and 2012. Estimated GA at infection ranged from 1 to 32 weeks, and fhMRI was performed at 24 to 38 weeks. The frontal, parietal, temporal and occipital lobes (mainly white matter), basal ganglia, thalamus, pons and cerebellum were analyzed by assessing ADC values. Two pregnancies were terminated and postmortem confirmation was available in these cases. RESULTS: ADC values of CMV-infected fetuses correlated significantly and negatively with GA in all brain regions except the basal ganglia. The cerebellum had the greatest reduction (r = -0.52, P < 0.0001). Maternal age correlated positively with ADC in the frontal lobe (P < 0.05). GA at infection and overt pathological changes did not affect ADC significantly. Compared with non-infected fetuses, ADC values of affected fetuses were significantly reduced in the frontal (P < 0.0001), parietal (P < 0.0001), occipital (P = 0.0005) and temporal (P = 0.001) lobes and thalamus (P = 0.006). CONCLUSION: CMV infection of the fetal brain results in a highly significant, region-dependent reduction of ADC values in the frontal, parietal, occipital and temporal lobes and thalamus, probably reflecting hypercellularity and inclusion bodies in damaged areas. Further studies are needed to determine if reduction in ADC values may serve as a prognostic factor in CMV-infected fetuses. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.

Critical role of the embryonic mid-hindbrain organizer in the behavioral response to amphetamine and methylphenidate.

The embryonic mid-hindbrain organizer, which is composed of a transient cell population in the brainstem, controls the development of dopaminergic and serotonergic neurons. Different genes determining the position and activity of this embryonic structure have been implicated in dopamine- and serotonin-associated disorders. Mouse mutants with a caudally shifted mid-hindbrain organizer, are hyperactive, show increased numbers of dopaminergic neurons and a reduction in serotonergic cells. In the present study we used these mutants to gain insights into the genetic and developmental mechanisms underlying motor activity and the response to psychostimulants. To this end, we studied the motor activity of these animals after exposure to methylphenidate and amphetamine and characterized their dopaminergic and serotonergic innervation. Saline-treated mutants showed increased locomotion, more stereotypic behavior and a decrease in rearing compared to wild-type mice. This baseline level of activity was similar to behaviors observed in wild-type animals treated with high doses of psychostimulants. In mutants methylphenidate (5 or 30 mg/kg) or amphetamine (2 or 4 mg/kg) did not further increase activity or even caused a decrease of locomotor activity, in contrast to wild-type mice. Fluoxetine (5 or 10 mg/kg) reduced hyperactivity of mutants to levels observed in wild-types. Transmitter measurements, dopamine and serotonin transporter binding assays and autoradiography, indicated a subtle increase in striatal dopaminergic innervation and a marked general decrease of serotonergic innervation in mutants. Taken together, our data suggest that mice with an aberrantly positioned mid-hindbrain organizer show altered sensitivity to psychostimulants and that an increase of serotonergic neurotransmission reverses their hyperactivity. We conclude that the mid-hindbrain organizer, by orchestrating the formation of dopaminergic and serotonergic neurons, is an essential developmental parameter of locomotor activity and psychostimulant response.

Fat halo sign in the bowel wall of patients with Crohn's disease.

AIM: To evaluate the prevalence and localization of the deposition of submucosal fat, the "fat halo sign" (FHS), using computed tomography (CT) in the bowel wall of patients with Crohn's Disease, and to assess its relation to the duration of the disease. MATERIALS AND METHODS: The abdominal CT examinations of 100 consecutive patients were reviewed for the presence of the FHS in the bowel wall. A measurement of less than -10HU was regarded as indicative of fat. CT findings were correlated with the duration of the disease and statistically tested by simple regression analysis. The patients were divided into two groups: group A included 26 patients with a disease duration of less than 1 year and group B included 73 with a longer disease duration. In one patient disease duration was unknown. To test the relationship between disease duration and FHS the cumulative number of FHS positive and negative patients was plotted against disease duration. RESULTS: The FHS was present in 17 of the 100 patients in 20 bowel segments, mainly in the ileum (10) and the ascending colon (8). The FHS was present in 3.8% in group A and in 21.9% in group B (p<0.0375). CONCLUSION: The FHS was present in 17% of patients with CD. Its location was mainly in the terminal ileum and ascending colon, typical sites of the disease. Its prevalence was significantly duration dependent.

In vivo occupancy of female rat brain estrogen receptors by 17beta-estradiol and tamoxifen.

Estrogens or antiestrogens are currently used by millions of women, but the interaction of these hormonal agents with brain estrogen receptors (ER) in vivo has not been characterized to date. Our goal was to assess, in vivo, the extent and regional distribution of brain ER occupancy in rats chronically exposed to 17beta-estradiol (E(2)) or tamoxifen (TAM). For that purpose, female ovariectomized Sprague-Dawley rats were implanted with subcutaneous pellets containing either placebo (OVX), E(2), or TAM for 3 weeks. ER occupancy in grossly dissected regions was quantified with 16alpha-[(18)F]fluoroestradiol ([(18)F]FES). Both E(2) and TAM produced significant decreases in radioligand uptake in the brain although the effect of E(2) was larger and more widespread than the effect of TAM. Detailed regional analysis of the interaction was then undertaken using a radioiodinated ligand, 11beta-methoxy-16alpha-[(125)I]iodo-estradiol ([(125)I]MIE(2)), and quantitative ex vivo autoradiography. E(2) treatment resulted in near-complete (86.6 +/- 17.5%) inhibition of radioligand accumulation throughout the brain, while ER occupancy in the TAM group showed a marked regional distribution such that percentage inhibition ranged from 40.5 +/- 15.6 in the ventrolateral part of the ventromedial hypothalamic nucleus to 84.6 +/- 4.5 in the cortical amygdala. These results show that exposure to pharmacologically relevant levels of TAM produces a variable, region-specific pattern of brain ER occupancy, which may be influenced by the regional proportion of ER receptor subtypes. These findings may partially explain the highly variable and region-specific effects observed in neurochemical, metabolic, and functional studies of the effects of TAM in the brain of experimental animals as well as human subjects.

Increase in peripheral benzodiazepine receptors and loss of glutamate NMDA receptors in a mouse model of closed head injury: a quantitative autoradiographic study.

Increases in peripheral type benzodiazepine receptors (PTBR) have been utilized for the detection of neuroinflammation and neurotoxicity in the brain. We have investigated the relationship between PTBR and NMDA receptor binding density in mice with closed head injury (CHI) using quantitative autoradiography. CHI was induced by a weight drop in nine mice, four of which received a single injection of the rat sarcoma (Ras) inhibitor famesyl thiosalicylate (FTS) 1 h after the insult. Sham controls received anesthesia but no contusion. The neurological status of the mice was evaluated at 1 h, and hence up to 7 days using a neurological severity score (NSS). Animals were killed 7 days after CHI and consecutive brain sections were incubated with [3H]PK11195, a PTBR antagonist, or [3H]MK801, an n-methyl-d-aspartate receptor (NMDAR) use-dependent antagonist. CHI produced large (two- to threefold), widespread increases in PK11195 binding in the traumatized hemisphere and a significant decrease (20%-40%) in NMDAR binding limited to regions at close proximity to the lesion. Histologically, these regions were characterized by glial proliferation and neuronal loss. Significant increases in PTBR binding, but no concomitant decrease in NMDAR, were identified in several regions remote from the lesion, including the contralateral ventrolateral striatum and the ipsilateral ventral thalamus. Drug treatment significantly improved the neurological deficits but had only a marginal effect on PTBR. These results support a complex role for glial activation and PTBR increases in the context of CHI.

Region-selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain.

Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl-tert-butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post-injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open-channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two- to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl-tert-butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide-induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide-induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimer's disease.

Non-MAO A binding of clorgyline in white matter in human brain.

Clorgyline is an irreversible inhibitor of monoamine oxidase (MAO A) which has been labeled with carbon-11 (C-11) and used to measure human brain MAO A with positron emission tomography (PET). In this study we compared [11C]clorgyline and deuterium-substituted [11C]clorgyline ([11C]clorgyline-D2) to better understand the molecular link between [11C]clorgyline binding and MAO A. In PET studies of five normal healthy volunteers scanned with [11C]clorgyline and [11C]clorgyline-D2 2 h apart, deuterium substitution generally produced the expected reductions in the brain uptake of [11C]clorgyline. However, the reduction was not uniform with the C-11 binding in white matter being significantly less sensitive to deuterium substitution than other brain regions. The percentages of the total binding attributable to MAO A is largest for the thalamus and smallest for the white matter and this is clearly seen in PET images with [11C]clorgyline-D2. Thus deuterium-substituted [11C]clorgyline selectively reduces the MAO A binding component of clorgyline in the human brain revealing non-MAO A binding which is most apparent in the white matter. The characterization of the non-MAO A binding component of this widely used MAO A inhibitor merits further investigation.

Autoradiographic study of pre- and postnatal distribution of cannabinoid receptors in human brain.

Cannabinoid receptors have been characterized and localized in the brain of several species, including human. The pre- and postnatal distribution of human brain CB1 receptors was investigated using quantitative autoradiography with [(3)H]CP55,940 as a ligand. Normal fetal brains (N = 8, gestational age 14-24 weeks)were obtained from voluntary abortions. Normal (drug and pathology free) adult human brains (N = 16, age 18-78) were obtained from the medical examiner's offices in New York City and Jaffa, Israel. Brains were stored frozen at -70 degrees C and sectioned (40 microm) at -15 degrees C. The radioligand (5 nM) was incubated with the sections for 3 h at room temperature. Washed and dried sections were exposed to tritium-sensitive film along with standards for 7-28 days and autoradiograms quantitated using NIH Image software. In the fetal human brain, low densities of THC-displaceable, region-specific binding could be observed as early as 14 weeks gestation. Receptor density increased slowly with gestational age but did not reach adult levels by the end of the second trimester (24 weeks gestation). In addition, the distribution pattern in the fetal brains was markedly different from the adult pattern. The most striking difference was the very low density of binding in the fetal caudate and putamen. In contrast, the globus pallidus pars medialis has almost-adult levels of cannabinoid receptors by 17-18 weeks gestation. The relatively low and regionally selective appearance of cannabinoid receptors in the fetal human brain may explain the relatively mild and selective nature of postnatal neurobehavioral deficits observed in infants exposed to cannabinoids in utero.

Scyllo-inositol in post-mortem brain of bipolar, unipolar and schizophrenic patients.

Inositol levels measured in postmortem brain of unipolar, bipolar and schizophrenic patients, suicide victims and normal controls showed no difference in scyllo-inositol levels in frontal or occipital cortex between any of the groups. We could not replicate previous reports of low myo-inositol levels in the frontal cortex of unipolar, bipolar and schizophrenic patients and suicide victims. There was no correlation between myo-inositol levels and estimated chlorpromazine equivalents in neuroleptic-treated subjects, and no effect of chronic haloperidol treatment on rat brain myo-inositol levels.

Unaltered alpha(2)-noradrenergic/imidazoline receptors in suicide victims: a postmortem brain autoradiographic analysis.

In vitro quantitative autoradiography of alpha(2)-adrenergic/imidazoline receptors, using [(125)I]iodoclonidine as a ligand, was performed on 24 human brains postmortem. Twelve brains were obtained from suicide victims and 12 from matched controls. We found no significant, region-dependent alterations in the density of alpha(2)-adrenergic receptors in brains of suicide victims as compared to matched controls. We also report age-dependent reductions in binding in the prefrontal cortex and hippocampus, as well as significant recent alcohol ingestion-dependent reductions in binding in the prefrontal cortex. Sex and time from death to autopsy did not affect iodoclonidine binding in our sample.

Dexanabinol (HU-211) effect on experimental autoimmune encephalomyelitis: implications for the treatment of acute relapses of multiple sclerosis.

Dexanabinol (HU-211) is a synthetic non-psychotropic cannabinoid which suppresses TNF-alpha production in the brain and peripheral blood. The effects of dexanabinol in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses, modes of administration and time regimes. Dexanabinol, 5 mg/kg i.v. given once after disease onset (day 10), significantly reduced maximal EAE score. Increasing the dose or treatment duration resulted in further suppression of EAE. Drug administration at earlier phases during disease induction was not effective. Histological studies supported the clinical findings demonstrating reduction in the inflammatory response in the brain and spinal cord in animals treated with dexanabinol. The results suggest that dexanabinol may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis (MS).

In vitro and in vivo study of water-soluble prodrugs of dexanabinol.

Trialkylammonium acetoxymethyl esters of dexanabinol were synthesized and evaluated as water-soluble prodrugs. Syntheses were performed by conventional methods; solubility in water and stability in buffers and human plasma were determined by HPLC, and in vivo tissue distribution studies were performed in a rat model. Most of the new derivatives were soluble in water (approximately 50 mg/mL). They were relatively stable in water, while rapidly hydrolyzed in human plasma. Distribution studies indicated that peak concentrations of drug both in blood (30 microg/mL) and brain (2 microg/mL) were rapidly (5 min) achieved after iv administration of a selected prodrug to rats. The blood concentration decreased faster than brain levels which were detectable even after 24 h. Some of the examined esters could be further developed as water soluble prodrugs of dexanabinol.

The suicide brain: a review of postmortem receptor/transporter binding studies.

The present review summarizes the last 15 years of research involving postmortem receptor/transporter binding studies on brains of suicide victims. It is our working hypothesis, on the basis of psychological, behavioral and epidemiological studies, that suicidal behavior is an independent unique behavioral entity with specific neurochemical characteristics. This review tries to test this hypothesis at the level of neurotransmitter receptors by using a different approach to data analysis. We suggest that this statistical approach, involving multivariate analyses, can contribute to the formulation of new hypotheses at the level of molecular biology and genetics. Such studies if undertaken in the future, would help define suicidal behavior as a psycho-neuro-pathological entity.

Distribution of glutamate transporter subtypes during human brain development.

In the mature brain, removal of glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of glutamate. This requirement is handled by a family of glutamate transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of glutamate also in neuronal development, it is believed that glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four glutamate transporters during human brain development using human pre- and postnatal brain tissue. Regional analysis showed that each transporter subtype has a unique distribution during development. EAAT2 was the most prominent glutamate transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for glutamate transporters in the developing brain.

Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production by dexanabinol (HU-211), a nonpsychotropic cannabinoid.

Dexanabinol, HU-211, a synthetic cannabinoid devoid of psychotropic effects, improves neurological outcome in models of brain trauma, ischemia and meningitis. Recently, HU-211 was found to inhibit brain tumor necrosis factor (TNFalpha) production after head injury. In the present study, we demonstrate the ability of HU-211 to suppress TNFalpha production and to rescue mice and rats from endotoxic shock after LPS (Escherichia coli 055:B5) inoculation. In BALB/c mice, a dose of 10 mg/kg LPS, injected i.p., caused 57% and 100% mortality, at 24 and 48 hr, respectively. HU-211, administered i.p. 30 min before lipopolysaccharide (LPS), reduced lethality to 9 and 67% at these time points (P < .05). When coinjected with D-galactoseamine (i.p.), LPS was 100% lethal within 24 hr, whereas eight hourly injections of HU-211 caused mortality of C57BL/6 mice to drop to 10% (P < .001). Administration of LPS to Sprague-Dawley rats resulted in a 30% reduction in the mean arterial blood pressure within 30 min, which persisted for 3 hr. HU-211, given 2 to 3 min before LPS, completely abolished the typical hypotensive response. Furthermore, the drug also markedly suppressed in vitro TNFalpha production and nitric oxide generation (by >90%) by both murine peritoneal macrophages and rat alveolar macrophage cell line exposed to LPS. HU-211 may, therefore, have therapeutic implications in the treatment of TNFalpha-mediated pathologies.

Clinical pharmacokinetics of escalating i.v. doses of dexanabinol (HU-211), a neuroprotectant agent, in normal volunteers.

The pharmacokinetics of dexanabinol (HU-211), a synthetic, nonpsychotropic cannabinoid with neuroprotectant action, was evaluated in a phase I clinical trial. The compound was administered at doses of 48 mg, 100 mg, and 200 mg as short i.v. infusions in a Cremophor-ethanol vehicle diluted with saline. All administrations were well-tolerated and no compound-related side-effects were observed. Plasma concentrations of dexanabinol were quantitated using a GC/MS/MS technique which provided a limit of quantitation of 100 pg/ml. The elimination of dexanabinol was best fitted to a 3-compartment model with a rapid distribution half-life (< 5 min), an intermediate phase half-life of approximately 90 min, and a slow terminal elimination half-life (approximately 9 h). The pharmacokinetics were linear over the evaluated dose range. The plasma clearance of the drug was high (1,700 ml/min) and the volume of distribution approximately 15 l/kg. These data are similar to those reported for naturally occurring cannabinoids such as delta 9-tetrahydrocannabinol and cannabidiol.

Interaction of dexanabinol (HU-211), a novel NMDA receptor antagonist, with the dopaminergic system.

The interaction of 7-hydroxy-delta6-tetrahydrocannabinol 1,1-dimethylheptyl (Dexanabinol: HU-211), a novel NMDA receptor antagonist, with the dopaminergic system was examined using in vitro and in vivo systems. HU-211 (50 or 100 microM) inhibited the binding of [3H]R(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol hydrochloride ([3H]SCH-23390), a dopamine D1 receptor antagonist, by 29.7 +/- 1.8% and 52.7 +/- 6.3%, respectively. HU-211 10 microM, like the dopamine D1 receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393), enhanced the conversion of [3H]adenine to cyclic AMP (cAMP) (51.8 +/- 29.7% and 35.6 +/- 21.5% over control, respectively). The HU-211-induced increase was not inhibited by SCH-23390. HU-211 together with the dopamine D1 receptor agonist caused a synergistic elevation (314.7 +/- 14.3%). HU-211 reduced the catalepsy induced by dopamine receptor antagonists. At 10 mg/kg, HU-211 significantly (P < 0.001) reduced the catalepsy time induced by D1, D2 and non-selective dopamine receptor antagonists. Overall, the results of the present study demonstrate that HU-211 interacts with the dopaminergic system and enhances activity at the dopamine D1 receptor level. This activity may have implications in diseases involving the dopaminergic system, such as Parkinson's disease.

A permanently charged tamoxifen derivative displays anticancer activity and improved tissue selectivity in rodents.

A quaternized form of tamoxifen (TAM), tamoxifen methiodide (TMI), was shown to demonstrate very low brain uptake compared to TAM and, unexpectedly, was considerably less estrogenic than TAM in the uterus. The agonist activity of TMI in the bone was similar to that of TAM. TMI manifested significant dose-dependent tumoricidal activity with a rapid onset of action against MCF-7 human breast cancer implants in nude mice and a mean reduction in tumor size of 60% over six weeks.

Iodine-123-5-iodo-6-nitroquipazine: SPECT radiotracer to image the serotonin transporter.

UNLABELLED: Because serotonergic function has been implicated in the pathophysiology of a number of diseases of the nervous system, efforts to image this system in vivo have received considerable recent attention. Promising preliminary results with the tracer 5-iodo-6-nitroquipazine (INQUIP) have prompted us to perform further studies designed to validate the use of the tracer as an in vivo ligand for the serotonin transporter. METHODS: We studied six adult macaca mulatta in eight experiments which involved SPECT imaging at 17 to 24 hr post-tracer injection, including three experiments with coinjection of the 123I-and 125I-radiolabeled tracer for direct comparison of autoradiography and SPECT, and three experiments in which animals were lesioned with the serotonergic neurotoxin (+/-)3,4-methyl-enedioxymethamphetamine (MDMA). In addition, we evaluated the metabolism of the tracer in the brain and periphery. RESULTS: SPECT images obtained at 17 and 24 hr reflected the known pattern of distribution of serotonin transporters and also showed close correspondence to the autoradiograms. Ratios of binding in the brain-stem to binding in the cerebellum were close to 3 at 17 hr. autoradiograms from an MDMA-treated animal showed up to 95% reductions of binding, while the SPECT data showed smaller reductions. Virtually all of the tracer in the brain stem was in the form of unmetabolized parent compound, but plasma showed rapid peripheral metabolism of the tracer. CONCLUSION: These results demonstrate that INQUIP SPECT images are sensitive measures of in vivo binding to the serotonin transporter, and support the further development of the tracer as a method for the in vivo study of serotonergic neurons in humans.