Cardiovascular Organ Damage in Clinical Subtypes of Systemic Sclerosis: Arterial Stiffness and Echocardiography Might Not Be the Ideal Tools for Patient Risk Stratification.

BACKGROUND: Vascular damage is recognized as a diagnostic landmark in systemic sclerosis (SSc), both in its limited and diffuse subtypes. Early detection at a subclinical stage with transthoracic echocardiography (TTE) and carotid femoral pulse wave velocity (cfPWV) may be helpful in therapeutic planning and management. Aim of the Study. The aim of the study was to evaluate presence of subclinical cardiovascular damage in patients with limited and diffuse SSc in comparison with a cohort of healthy individuals. METHODS: Consecutive patients with limited and diffuse SSc underwent complete TTE and cfPWV and a complete review of clinical data. As controls, 23 healthy subjects with similar hemodynamic profiles were selected. RESULTS: 41 patients (35 female, aged 56.9 years), 21 with diffuse and 20 with limited SSc, were recruited. Past medical history, cardiovascular risk factors, gender distribution, and disease duration were similar in the two groups as well as TTE parameters and hemodynamic indexes-cfPWV (6.5 [6-6.8] vs. 7.0 [6.2-8.5], p=0.24) and augmentation index (145.6 ± 14.2 vs. 149 ± 20.6, p=0.52). Patients with limited SSc were 10 years older than patients with diffuse SSc. In the multiple regression analysis, only age (p=0.0154) and disease duration (p=0.0467) resulted as the significant determinant of cfPWV. When compared to healthy controls, no significant difference emerged in TTE or hemodynamic indexes. CONCLUSION: In SSc, cfPWV increases with age, with no additional impact of pathology or subtype. Vascular damage in the SSc population is not accurately reflected in increased arterial stiffness, as evaluated with cfPWV, or classically defined echocardiographic findings of organ damage (i.e., left ventricular concentric remodelling and increased filling pressures).

Neuroprotective effects of the MAO-B inhibitor, PF9601N, in an in vivo model of excitotoxicity.

BACKGROUND: PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] is an inhibitor of monoamine oxidase B (MAO-B), which has shown to possess neuroprotective properties in several in vitro and in vivo models of Parkinson's disease (PD). As there is evidence that excitotoxicity may be implicated in the pathophysiology of several neurodegenerative diseases, the aim of the present work was to investigate the effects of PF9601N in an acute in vivo model of excitotoxicity induced by the local administration of kainic acid during striatal microdialysis in adult rats. METHODS: The basal and evoked release of neurotransmitters was monitored by HPLC analysis of microdialysate samples and tissue damage was evaluated histologically "ex vivo." RESULTS: PF9601N (40 mg/kg, single i.p. administration) reduced the kainate-evoked release of glutamate and aspartate and increased taurine release, but it had no effect on the release of dopamine, DOPAC, and HVA. PF9601N pretreatment also resulted in a significant reduction in the kainate-induced astrocytosis, microgliosis, and apoptosis. CONCLUSIONS: The results suggest PF9601N to be a good candidate for the treatment of neurodegenerative diseases mediated by excitotoxicity.

Highly reactive oxygen species: detection, formation, and possible functions.

The so-called reactive oxygen species (ROS) are defined as oxygen-containing species that are more reactive than O(2) itself, which include hydrogen peroxide and superoxide. Although these are quite stable, they may be converted in the presence of transition metal ions, such as Fe(II), to the highly reactive oxygen species (hROS). hROS may exist as free hydroxyl radicals (HO·), as bound ("crypto") radicals or as Fe(IV)-oxo (ferryl) species and the somewhat less reactive, non-radical species, singlet oxygen. This review outlines the processes by which hROS may be formed, their damaging potential, and the evidence that they might have signaling functions. Since our understanding of the formation and actions of hROS depends on reliable procedures for their detection, particular attention is given to procedures for hROS detection and quantitation and their applicability to in vivo studies.

SNARE protein redistribution and synaptic failure in a transgenic mouse model of Parkinson's disease.

The pre-synaptic protein alpha-synuclein is the main component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinson's disease and dementia with Lewy bodies. Mutations in the alpha-synuclein gene cause familial forms of Parkinson's disease and dementia with Lewy bodies. We previously described a transgenic mouse line expressing truncated human alpha-synuclein(1-120) that develops alpha-synuclein aggregates, striatal dopamine deficiency and reduced locomotion, similar to Parkinson's disease. We now show that in the striatum of these mice, as in Parkinson's disease, synaptic accumulation of alpha-synuclein is accompanied by an age-dependent redistribution of the synaptic SNARE proteins SNAP-25, syntaxin-1 and synaptobrevin-2, as well as by an age-dependent reduction in dopamine release. Furthermore, the release of FM1-43 dye from PC12 cells expressing either human full-length alpha-synuclein(1-140) or truncated alpha-synuclein(1-120) was reduced. These findings reveal a novel gain of toxic function of alpha-synuclein at the synapse, which may be an early event in the pathogenesis of Parkinson's disease.

Regional differential effects of the novel histamine H3 receptor antagonist 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) on histamine release in the central nervous system of freely moving rats.

After oral administration, the nonimidazole histamine H(3) receptor antagonist, 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254), increased histamine release from the tuberomammillary nucleus, where all histaminergic somata are localized, and from where their axons project to the entire brain. To further understand functional histaminergic circuitry in the brain, dual-probe microdialysis was used to pharmacologically block H(3) receptors in the tuberomammillary nucleus, and monitor histamine release in projection areas. Perfusion of the tuberomammillary nucleus with GSK189254 increased histamine release from the tuberomammillary nucleus, nucleus basalis magnocellularis, and cortex, but not from the striatum or nucleus accumbens. Cortical acetylcholine (ACh) release was also increased, but striatal dopamine release was not affected. When administered locally, GSK189254 increased histamine release from the nucleus basalis magnocellularis, but not from the striatum. Thus, defined by their sensitivity to GSK189254, histaminergic neurons establish distinct pathways according to their terminal projections, and can differentially modulate neurotransmitter release in a brain region-specific manner. Consistent with its effects on cortical ACh release, systemic administration of GSK189254 antagonized the amnesic effects of scopolamine in the rat object recognition test, a cognition paradigm with important cortical components.

Neuro-inflammation induced in the hippocampus of 'binge drinking' rats may be mediated by elevated extracellular glutamate content.

The neuropathological and immune changes induced in the brain by 'binge drinking' have been investigated in a rat model. Evidence of neuro-inflammation was identified in the 'binge drinking' rat model of alcohol abuse after 3 weeks of administration of 2 or 3 g/kg ethanol (EtOH), three times per day for two consecutive days, followed by 5 days of abstinence: Firstly, alveolar macrophages, isolated from these animals, showed significant increases in inducible nitric oxide synthase, as assayed by nitrite release, both before and after lipopolysaccaharide stimulation. Secondly, significant numbers of activated microglia were present in the dentate gyrus region of the hippocampus of the 'binge drinking' model, after major histocompatibility complex class II staining, by comparison with the control. Microdialysis studies in the ventral hippocampus identified a significant increase in the basal extracellular concentration of glutamate, in both the 2 and 3 g/kg administered 'binge drinking' rats. In contrast, no changes in the hippocampal extracellular concentrations, of GABA and taurine, or the dopamine and serotonin metabolites were observed under basal conditions. A further dose of EtOH induced a significant decrease in the concentrations of both 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid, whereas glutamate, taurine and GABA levels were unaffected. There was no evidence that EtOH preference was initiated by the 'binge drinking' regimen. Our results suggest that the possible toxicity associated with 'binge drinking' maybe directed by the elevated glutamate levels, which in turn, activate phagocytic cells to release their inflammatory cytokines and chemokines, ultimately leading to neuro-inflammation.

Stimulation by neurotensin of dopamine and 5-hydroxytryptamine (5-HT) release from rat prefrontal cortex: possible role of NTR1 receptors in neuropsychiatric disorders.

The modulation of cortical dopaminergic and serotonergic neurotransmissions by neurotensin (NT) was studied by measuring the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) from the prefrontal cortex (PFC) of freely moving rats. The samples were collected via transversal microdialysis. Dopamine and 5-HT levels in the dialysate were measured using high-performance liquid chromatography (HPLC) with an electrochemical detector. Local administration of neurotensin (1microM or 0.1microM) in the PFC via the dialysis probe produced significant, long-lasting, and concentration-dependent increase in the extracellular release of DA and 5-HT. The increase produced by 1microM neurotensin reached a maximum of about 210% for DA and 340% for 5-HT. A high-affinity selective neurotensin receptor (NTR1) antagonist {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3yl)carbonylamino tricyclo (3.3.1.1.(3.7)) decan-2-carboxylic acid} (SR 48692), perfused locally at a concentration of 0.1microM and 0.5microM in the PFC antagonized the effects of 1microM neurotensin. Our in vivo neurochemical results indicate, for the first time, that neurotensin is able to regulate cortical dopaminergic and serotonergic neuronal activity in freely moving rats. These effects are possibly mediated by interactions of neurotensin with neurons releasing DA or 5-HT, projecting to the PFC from the ventrotegmental area (VTA) and from the dorsal raphe nuclei (DRN), respectively. The potentiating effects of neurotensin on DA and 5-HT release in the PFC are regulated by NTR1 receptors, probably located on dopaminergic and serotonergic nerve terminals or axons.

Extracellular levels of brain aspartate, glutamate and GABA during an inhibitory avoidance response in the rat.

The extracellular levels of aspartate, glutamate and GABA were measured by microdialysis, coupled with an HPLC method, in rat prefrontal cortex (mPFC) and ventral hippocampus (VH) before and during the performance of a step-down inhibitory task. The basal levels of glutamate were about 50% higher than those of aspartate, and GABA levels were about 20-folds smaller than those of the excitatory amino acids. There were no significant differences in the basal levels of any of the three amino acids between the two brain regions. The extracellular levels of aspartate increased during acquisition and recall trials in both VH and mPFC, whereas those of glutamate increased in the VH during acquisition only. A significant increase in GABA levels was also detected during acquisition but only in the mPFC. The neuronal origin of the increased extracellular levels of aspartate, glutamate and GABA was demonstrated by administering tetrodotoxin directly into the mPFC or VH by reverse dialysis. These findings, together with previous evidence from our and other laboratories, indicate a differential release of aspartate and glutamate from excitatory neurons during the performance of behavioral responses, and therefore, distinct roles for the two excitatory amino acids should be envisaged.

The detection of hydroxyl radicals in vivo.

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound "crypto" radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress. Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis.

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA2-) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/muL. Derivatization of samples with o-phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA2- in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death 'ex vivo' showed TA2- to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2- forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.

Differential effect of cannabinoid agonists and endocannabinoids on histamine release from distinct regions of the rat brain.

Cannabinoids exert complex actions on neurotransmitter systems involved in cognition, locomotion, appetite, but no information was available so far on the interactions between the endocannabinoid system and histaminergic neurons that command several, similar behavioural states and memory. In this study, we investigated the effect of cannabimimetic compounds on histamine release using the microdialysis technique in the brain of freely moving rats. We found that systemic administration of the cannabinoid receptors 1 (CB1-r) agonist arachidonyl-2'chloroethylamide/N-(2chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA; 3 mg/kg) increased histamine release from the posterior hypothalamus, where the histaminergic tuberomamillary nuclei (TMN) are located. Local infusions of ACEA (150 nm) or R(+)-methanandamide (mAEA; 1 microm), another CB1-r agonist, in the TMN augmented histamine release from the TMN, as well as from two histaminergic projection areas, the nucleus basalis magnocellularis and the dorsal striatum. When the endocannabinoid uptake inhibitor AM404 was infused into the TMN, however, increased histamine release was observed only in the TMN. The cannabinoid-induced effects on histamine release were blocked by co-administrations with the CB1-r antagonist AM251. Using double-immunofluorescence labelling and confocal laser-scanning microscopy, CB1-r immunostaining was found in the hypothalamus, but was not localized onto histaminergic cells. The modulatory effect of cannabimimetic compounds on histamine release apparently did not involve inhibition of gamma-aminobutyric acid (GABA)ergic neurotransmission, which provides the main inhibitory input to the histaminergic neurons in the hypothalamus, as local infusions of ACEA did not modify GABA release from the TMN. These profound effects of cannabinoids on histaminergic neurotransmission may partially underlie some of the behavioural changes observed following exposure to cannabinoid-based drugs.

Species-specific chemosignals evoke delayed excitation of the vomeronasal amygdala in freely-moving female rats.

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species-specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species-specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO-mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species-specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.

Differential autoinhibition of 5-hydroxytryptamine neurons by 5-hydroxytryptamine in the dorsal raphe nucleus.

5-Hydroxytryptamine neurons in the dorsal raphe nucleus are under autoinhibitory control by endogenous 5-hydroxytryptamine. Tonic activation of 5-hydroxytryptamine 1A autoreceptors was demonstrated in awake animals, but was inconsistently observed in anaesthetized animals and slice preparations, leading to questioning of its physiological significance. We re-evaluated autoinhibition in single-unit recordings from deeply seated 5-hydroxytryptamine neurons in slices in which endogenous 5-hydroxytryptamine bioavailability was restored by supplementing its precursor L-tryptophan. In these conditions, the application of the neutral 5-hydroxytryptamine 1A receptor antagonist WAY-100635 markedly increased 5-hydroxytryptamine neuron firing. Responses to WAY-100635 in single experiments ranged from a lack of effect to a several-fold increase in firing rate, suggesting that 5-hydroxytryptamine neurons in the dorsal raphe nucleus represent a heterogeneous population regarding their susceptibility to autoinhibition by endogenous 5-hydroxytryptamine.