Auditory evoked BOLD responses in awake compared to lightly anaesthetized zebra finches.

Functional magnetic resonance imaging (fMRI) is increasingly used in cognitive neuroscience and has become a valuable tool in the study of auditory processing in zebra finches, a well-established model of learned vocal communication. Due to its sensitivity to head motion, most fMRI studies in animals are performed in anaesthetized conditions, which might significantly impact neural activity evoked by stimuli and cognitive tasks. In this study, we (1) demonstrate the feasibility of fMRI in awake zebra finches and (2) explore how light anaesthesia regimes affect auditory-evoked BOLD responses to biologically relevant songs. After an acclimation procedure, we show that fMRI can be successfully performed during wakefulness, enabling the detection of reproducible BOLD responses to sound. Additionally, two light anaesthesia protocols were tested (isoflurane and a combination of medetomidine and isoflurane), of which isoflurane alone appeared to be the most promising given the high success rate, non-invasive induction, and quick recovery. By comparing auditory evoked BOLD responses in awake versus lightly anaesthetized conditions, we observed overall effects of anaesthetics on cerebrovascular reactivity as reflected in the extent of positive and negative BOLD responses. Further, our results indicate that light anaesthesia has limited effects on selective BOLD responses to natural versus synthetic sounds.

In Vivo Longitudinal Monitoring of Changes in the Corpus Callosum Integrity During Disease Progression in a Mouse Model of Alzheimer's Disease.

The corpus callosum is the largest commissural fiber connecting left and right hemisphere of the brain. Emerging evidence suggests that a variety of abnormalities detected in the microstructure of this white matter fiber can be an early event in Alzheimer's disease (AD) pathology. However, little is known about tissue characteristics of these abnormalities and how these abnormalities evolve during AD progression. In this study, we measured in vivo magnetic resonance transverse relaxation times (T2) to longitudinally monitor changes in tissue integrity and abnormalities related to myelination and demyelination processes in corpus callosum of AD mouse models. The most striking finding of our study was a significant elongation of T2 values in the corpus callosum at 10, 14, 16 and 18 months of age compared to age-matched wild-type mice. In contrast, the gray matter regions surrounding the corpus callosum, such as the cortex and hippocampus, showed a significant T2 decrease compared to wild-type mice. Histological analyses clearly revealed demyelination, gliosis and amyloid-plaque deposition in the corpus callosum. Our results suggest that demyelinating and inflammatory pathology may result in prolonged relaxation time during AD progression. To our knowledge, this is the first in vivo T2 study assessing the microstructural changes with age in the corpus callosum of the Tg2576 mouse model and it demonstrates the application of T2 measurement to noninvasively detect tissue integrity of the corpus callosum, which can be an early event in disease progression.

Cigarette smoke-induced CXCR3 receptor up-regulation mediates endothelial apoptosis.

Endothelial monocyte-activating polypeptide II (EMAP II) and interferon-inducible protein (IP)-10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke-exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke-induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II-induced and IP-10-induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II-induced and IP-10-induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II-induced and IP-10-induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II-induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema.

Inhibition of HIV-1 entry by extracts derived from traditional Chinese medicinal herbal plants.

BACKGROUND: Highly active anti-retroviral therapy (HAART) is the current HIV/AIDS treatment modality. Despite the fact that HAART is very effective in suppressing HIV-1 replication and reducing the mortality of HIV/AIDS patients, it has become increasingly clear that HAART does not offer an ultimate cure to HIV/AIDS. The high cost of the HAART regimen has impeded its delivery to over 90% of the HIV/AIDS population in the world. This reality has urgently called for the need to develop inexpensive alternative anti-HIV/AIDS therapy. This need has further manifested by recent clinical trial failures in anti-HIV-1 vaccines and microbicides. In the current study, we characterized a panel of extracts of traditional Chinese medicinal herbal plants for their activities against HIV-1 replication. METHODS: Crude and fractionated extracts were prepared from various parts of nine traditional Chinese medicinal herbal plants in Hainan Island, China. These extracts were first screened for their anti-HIV activity and cytotoxicity in human CD4+ Jurkat cells. Then, a single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms of these extracts. RESULTS: Two extracts, one from Euphorbiaceae, Trigonostema xyphophylloides (TXE) and one from Dipterocarpaceae, Vatica astrotricha (VAD) inhibited HIV-1 replication and syncytia formation in CD4+ Jurkat cells, and had little adverse effects on host cell proliferation and survival. TXE and VAD did not show any direct inhibitory effects on the HIV-1 RT enzymatic activity. Treatment of these two extracts during the infection significantly blocked infection of the reporter virus. However, pre-treatment of the reporter virus with the extracts and treatment of the extracts post-infection had little effects on the infectivity or gene expression of the reporter virus. CONCLUSION: These results demonstrate that TXE and VAD inhibit HIV-1 replication likely by blocking HIV-1 interaction with target cells, i.e., the interaction between gp120 and CD4/CCR5 or gp120 and CD4/CXCR4 and point to the potential of developing these two extracts to be HIV-1 entry inhibitors.

Changing body temperature affects the T2* signal in the rat brain and reveals hypothalamic activity.

This study was designed to determine brain activity in the hypothalamus-in particular the thermoregulatory function of the hypothalamic preoptic area (PO). We experimentally changed the body temperature in rats within the physiological range (37-39 degrees C) and monitored changes in blood oxygenation level-dependent (BOLD) MR signal. To explore PO activity we had to deal with general signal changes caused by temperature-dependent alterations in the affinity of oxygen for hemoglobin, which contributes to BOLD contrast because it is partly sensitive to the amount of paramagnetic deoxyhemoglobin in the voxel. To reduce these overall temperature-induced effects, we corrected the BOLD data using brain-specific correction algorithms. The results showed activity of the PO during body warming from 38 degrees C to 39 degrees C, supported by an increased BOLD signal after correction. This is the first fMRI study on the autonomous nervous system in which hypothalamic activity elicited by changes in the internal environment (body temperature) was monitored. In this study we also demonstrate 1) that any fMRI study of anesthetized small animals should guard against background BOLD signal drift, since animals are vulnerable to body temperature fluctuations; and 2) the existence of a link between PO activity and the sympathetically-mediated opening of the arteriovenous anastomoses in a parallel study on the rat tail, a peripheral thermoregulatory organ.

In vivo dynamic ME-MRI reveals differential functional responses of RA- and area X-projecting neurons in the HVC of canaries exposed to conspecific song.

HVC (nidopallial area, formerly known as hyperstriatum ventrale pars caudalis), a key centre for song control in oscines, responds in a selective manner to conspecific songs as indicated by electrophysiology. However, immediate-early gene induction cannot be detected in this nucleus following song stimulation. HVC contains neurons projecting either towards the nucleus robustus archistriatalis (RA; motor pathway) or area X (anterior forebrain pathway). Both RA- and area X-projecting cells show auditory responses. The present study analysed these responses separately in the two types of HVC projection neurons of canaries by a new in vivo approach using manganese as a calcium analogue which can be transported anterogradely and used as a paramagnetic contrast agent for magnetic resonance imaging (MRI). Manganese was stereotaxically injected into HVC and taken up by HVC neurons. The anterograde axonal transport of manganese from HVC to RA and area X was then followed by MRI during approximately 8 h and changes in signal intensity in these targets were fitted to sigmoid functions. Data comparing birds exposed or not to conspecific songs revealed that song stimulation specifically affected the activity of the two types of HVC projection neurons (increase in the sigmoid slope in RA and in its maximum signal intensity in area X). Dynamic manganese-enhanced MRI thus allows assessment of the functional state of specific neuronal populations in the song system of living canaries in a manner reminiscent of functional MRI (but with higher resolution) or of 2-deoxyglucose autoradiography (but in living subjects).

Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features.

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.

Effect of seasonal allergen exposure on mucosal IL-16 and CD4+ cells in patients with allergic rhinitis.

BACKGROUND: CD4+ T cells constitute a major source of cytokines in allergic diseases such as allergic rhinitis. Interleukin (IL)-16 selectively recruits CD4+ cells. METHODS: We evaluated the effect of natural allergen exposure during a grass-pollen season on IL-16 expression and number of CD4+ cells in nasal mucosa. Patients with allergic rhinitis (n=16) were treated with either a nasal glucocorticoid beclomethasone (BDP; 400 microg/day) or placebo, and gave nasal biopsies prior to and during the grass-pollen season. The evaluated markers in allergic rhinitis patients were also compared to those in healthy control subjects (n=5). RESULTS: Prior to the pollen season, the expression of IL-16, but not the number of CD4+ cells, was significantly higher in patients with allergic rhinitis than in healthy control subjects. The grass-pollen season further increased IL-16 expression and also increased the number of CD4+ cells in placebo-treated, but not in BDP-treated, allergic rhinitis patients. The pollen-season-induced change in IL-16 expression and in CD4+ cells was significantly more pronounced in placebo- than in BDP-treated patients. There was a significant correlation between the change in IL-16 expression and the number of CD4+ cells. CONCLUSIONS: These data suggest that local upregulation of IL-16 expression contributes to the inflammation observed in seasonal allergic rhinitis. Hypothetically, inhibition of IL-16 expression can be one of several mechanisms by which nasal glucocorticoids achieve their anti-inflammatory effect in allergic rhinitis.

Flavonoid, iridoid, and lignan glycosides from Putoria calabrica.

From the aerial parts of Putoria calabrica, two new flavonol triglycosides were isolated and their structures were elucidated as quercetin-3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside]-7-O-beta-D-glucopyranoside (1, calabricoside A) and quercetin-3-O-[4' "-O-caffeoyl-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside]-7-O-beta-D-glucopyranoside (2, calabricoside B). Additionally, seven iridoid and three lignan glycosides were isolated and characterized. Radical scavenging activities of all compounds were determined by quantifying their effects on luminol-enhanced chemiluminescence in formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated human polymorphonuclear neutrophils (PMNs). Calabricoside A and B showed strong radical scavenging activity with IC(50) values of 0.25 and 0.3 microM, respectively.

Increased expression of neuronal Src and tyrosine phosphorylation of NMDA receptors in rat brain after systemic treatment with MK-801.

We have observed that systemic treatment with the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 increases Src expression and NMDA receptor phosphorylation in rat brain. A partial cDNA encoding rat neuronal Src was isolated and its sequence was used to design specific oligonucleotide probes. Systemically administered MK-801 (5 mg/kg for 4 h) increased by 28+/-4% mRNA expression of neuronal Src in the superficial layers of the parietal cortex. This effect was observed at doses as low as 0.2 mg/kg. A similar, although more modest, induction was observed 6 h after phencyclidine (15 mg/kg) administration, but not after high doses of memantine and ketamine. The MK-801-induced effect was not blocked by pretreatment with clozapine. Consistent with the increase in mRNA levels, cortical Src protein was increased to 186 +/- 24% of control 24 h after MK-801 treatment. Total cellular Src activity was also increased in parietal cortex homogenates 4 h after MK-801 (5 mg/kg). Moreover, MK-801 treatment (0.5 mg/kg and 5 mg/kg for 4 h) increased tyrosine phosphorylation, but not protein levels, of the NMDA receptor subunit NR2A. These results provide evidence for a contribution of Src and tyrosine phosphorylation of NMDA receptors in the pharmacological actions of uncompetitive NMDA receptor antagonists.

Uncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptors alter the mRNA expression of proteins associated with the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptor function appears to be under complex control during physiological and pharmacological states. We have investigated the effects of acute administration of uncompetitive NMDA receptor antagonists on mRNA levels of NMDA receptor subunits and on molecules known to cluster or phosphorylate the receptor utilizing in situ hybridization on rat brain sections. A high dose (5 mg/kg; 4 hr) of dizocilpine (MK-801) decreased mRNA levels of NMDA receptor subunits NR2C and NR2B in the entorhinal and parietal cortices, respectively. MK-801 increased mRNA levels of synapse-associated protein-90/postsynaptic density-95 (SAP90/PSD-95) and a gamma-isoform of protein kinase C (PKCgamma) in cortical regions. Synapse-associated protein-97 (SAP97) mRNA levels were increased in the entorhinal cortex layer III after MK-801 or after relatively high doses of other uncompetitive NMDA receptor antagonists: phencyclidine (15 mg/kg; 6 hr) and memantine (50 mg/kg; 6 hr). Memantine also increased SAP97 mRNA expression in other cortical regions, but this effect was not observed with MK-801 or phencyclidine. NMDA receptor uncompetitive antagonists alter the expression of multiple receptor components and such events may ultimately play a role in adaptation or toxic responses.

Non invasive in vivo anatomical studies of the oscine brain by high resolution MRI microscopy.

We describe in this paper an in vivo Magnetic Resonance Imaging (MRI) procedure that allows one to obtain three-dimensional high quality images of the entire brain of small passerine birds such as the canary with a slice thickness of 58 micron and an image resolution of 78 microns. This imaging procedure was completed in 70 min on anaesthetised birds that later recovered uneventfully and could be reused for subsequent additional imaging. To illustrate the high resolution and anatomical detail that can be achieved, examples of coronal images through the entire hypothalamus are provided in the same sectioning plane as the previously published canary brain atlas. The data set can be used to create sections in any desired plane and the entire data set can be viewed from any point of view in a volume rendered image. This provides a useful tool in understanding the three-dimensional organisation of the brain. Similar procedures can also be applied on fixed brains and might allow an even better anatomical resolution of images because time constrains no longer limit the duration of image acquisition. The non-invasive MRI technique enables to study neuroanatomical features with a high resolution and without killing the animal subjects so that measures can be obtained in a same individual both before and after an experimental treatment.

Antiplasmodial and cytotoxic metabolites from the Maltese sponge Agelas oroides.

From a Maltese sample of the marine sponge Agelas oroides, five compounds: oroidin (1), 2-cyano-4,5-dibromopyrrole (2), 4,5-dibromopyrrole-2-carboxylic acid (3), 4,5-dibromopyrrole-2-carboxylic acid methyl ester (4), and 4 alpha-methyl-5 alpha-cholest-8-en-3 beta-ol (5) have been isolated. For compounds 1-5, completely assigned 1H- and 13C-NMR data are reported for the first time. For 2 a single crystal X-ray crystallographic analysis proved its structure unambiguously. The X-ray analysis of 2 indicated it to crystallise in an unexpected polar space group. Biological activity assessment of all isolates indicate 5 to have moderate antiplasmodial activity, as well as being cytotoxic, and 2 to be moderately cytotoxic towards several cancer cell lines.

Complementary use of T2-weighted and postcontrast T1- and T2*-weighted imaging to distinguish sites of reversible and irreversible brain damage in focal ischemic lesions in the rat brain.

The evolution of a photochemically induced cortical infarct was monitored using T2-, postcontrast (GdDOTA) T1-, and postcontrast (DyDTPA-BMA) T2*-weighted NMR imaging techniques. Data acquired with these different NMR imaging types were compared, both qualitatively and quantitatively. The T2*-weighted NMR images after spordiamide injection (DyDTPA-BMA) were perfusion-weighted images that allowed the differentiation between several infarct-related areas in terms of different degrees of perfusion deficiency. No quantitative information on cerebral blood flow (CBF) was obtained. A clear distinction was made between areas with a complete lack of CBF located in the core of the lesion and temporary CBF insufficiencies in the rim surrounding this core. Concomitant observations on T2-weighted and postcontrast T1-weighted images revealed the same temporary rim characterized by an increased water content, and an intact blood-brain barrier (BBB), as well as by reduced perfusion. This rim appeared within the first hours after infarct induction, reached a maximum 24 h later, and lasted between 3-5 days, when its size gradually decreased until complete disappearance. These observations suggest the existence of an area at risk. Only on postcontrast T1-weighted images, the core of the lesion remained visible during the whole experimental period (10 days) and reflected in all likelihood the irreversibly damaged ischemic central core. The combined application of different NMR imaging techniques when studying focal cerebral infarctions in the rat brain allowed us to distinguish, in terms of NMR characteristics, zones of reversible from irreversible brain damage and to estimate the severity of the damage. This might offer an appropriate experimental setup for the screening of cerebroprotective compounds.

Evidence of grass-pollen allergenic activity in the smaller micronic atmospheric aerosol fraction.

In June 1988, during the grass-pollen season in Leiden, The Netherlands, outdoor airborne particulate matter was collected and separated into fractions according to aerodynamic sizes (greater than or equal to 10 microns, 4.9-10 microns, 2.7-4.9 microns, 1.3-2.7 microns, 0.6-1.3 microns, less than or equal to 0.6 microns), with a cascade impactor mounted on top of a high volume sampler. The different fractions were tested for the presence of grass-pollen allergenic activity using a RAST-inhibition assay: specific IgE-antibody-containing patient serum was applied on the particle-loaded impaction strips, and the serum was recovered by descending elution for further analysis in the RAST. Simultaneously, continuous measurements were made of the airborne grass-pollen concentration using a volumetric pollen trap. Sampling observations lasting 7-9 hr during a period with relatively high airborne grass-pollen concentrations showed reliably detectable amounts of grass-pollen allergen, not only in the first impaction stage where intact pollen were collected, but also in the lower stages collecting the smaller, paucimicronic and submicron atmospheric aerosol fraction. It is evident that this result has serious implications for the understanding of the bronchial symptoms frequently seen in hay fever patients on days with high pollen concentrations in the air.