Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain

BACKGROUND: Nitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx(-)) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used. RESULTS: We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx(- )levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx(- )levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. CONCLUSION: Concomitant variations occurring in NO levels derived from nNOS and iNOS at an early age constitute a major factor of risk for sleep and/or memory impairments in SAMP8

Cerebral and Peripheral Changes Occurring in Nitric Oxide (NO) Synthesis in a Rat Model of Sleeping Sickness: Identification of Brain iNOS Expressing Cells

International audienceBACKGROUND: The implication of nitric oxide (NO) in the development of human African trypanosomiasis (HAT) using an animal model, was examined. The manner by which the trypanocidal activity of NO is impaired in the periphery and in the brain of rats infected with Trypanosoma brucei brucei (T. b. brucei) was analyzed through: (i) the changes occurring in NO concentration in both peripheral (blood) and cerebral compartments; (ii) the activity of nNOS and iNOS enzymes; (iii) identification of the brain cell types in which the NO-pathways are particularly active during the time-course of the infection. METHODOLOGY/PRINCIPAL FINDINGS: NO concentration (direct measures by voltammetry) was determined in central (brain) and peripheral (blood) compartments in healthy and infected animals at various days post-infection: D5, D10, D16 and D22. Opposite changes were observed in the two compartments. NO production increased in the brain (hypothalamus) from D10 (+32%) to D16 (+71%), but decreased in the blood from D10 (-22%) to D16 (-46%) and D22 (-60%). In parallel with NO measures, cerebral iNOS activity increased and peaked significantly at D16 (up to +700%). However, nNOS activity did not vary. Immunohistochemical staining confirmed iNOS activation in several brain regions, particularly in the hypothalamus. In peritoneal macrophages, iNOS activity decreased from D10 (-83%) to D16 (-65%) and D22 (-74%) similarly to circulating NO. CONCLUSION/SIGNIFICANCE: The NO changes observed in our rat model were dependent on iNOS activity in both peripheral and central compartments. In the periphery, the NO production decrease may reflect an arginase-mediated synthesis of polyamines necessary to trypanosome growth. In the brain, the increased NO concentration may result from an enhanced activity of iNOS present in neurons and glial cells. It may be regarded as a marker of deleterious inflammatory reactions

Analyses spectroscopiques du liquide céphalo-rachidien de rat en ex vivo et du noyau du raphé dorsal in vivo

Les propriétés d'absorption et de fluorescence du liquide céphalo-rachidien (LCR) ponctionné au niveau de la cisterna magna du rat, sont analysées puis comparées à l'émission mesurée in situ dans le noyau du raphe dorsal du rat libre de tous mouvements. Les mesures de fluorescence en ex vivo du LCR et in vivo du noyau raphé dorsal, ont été réalisées par la mise en œuvre d'un microcapteur à fibre optique (FOCS). La fluorescence mesurée in vivo sous excitation à 337 nm, présente 2 pics d'émission situés vers 410 et 460 nm. Les spectres d'absorption, d'émission en fluorescence statique et en fluorescence induite par laser sont rapportés. Avec des domaines de longueur d'onde d'excitation de 300-315 nm, 320-355 nm et 360-470 nm, les spectres d'émission du LCR en ex vivo montrent respectivement des pics centrés vers 340 nm, 390 nm et 530 nm. Malgré les limites liées aux différences de localisation anatomique, ces approches ainsi que celles de la littérature permettent de suggérer que le signal de fluorescence mesuré in vivo à 460nm pourrait dépendre pour une grande partie du NADH intracellulaire

Fractionated Stereotactic Radiotherapy with Helical Tomotherapy for Brain Metastases: A Mono-Institutional Experience

Background: The present study reports on the outcomes of our mono-institutional experience of Helical Tomotherapy (HT)-based SRT for brain metastases. The use of this linac is less frequently reported for this kind of treatment. Methods: This retrospective study displays a series of patients treated with HT-SRT. The eligibility of using SRT for brain metastases was defined by a Karnofsky performance status of >70, a life expectancy of >6 months, and controlled extra-cranial disease; no SRT was allowed in the case of a number of brain metastases larger than 10. All the cases were discussed by a multidisciplinary board. Toxicity assessments were performed based on CTCAE v5.0. Survival endpoints were assessed using the Kaplan–Meier method, and univariate and multivariate analyses were carried out to identify any potential predictive factor for an improved outcome. Results: Sixty-four lesions in 37 patients were treated using HT-SRT with a median total dose of 30 Gy in five fractions. The median follow-up was 7 months, and the 1- and 2-year LC rates were both 92.5%. The IPFS rates were and 56.75% and 51.35%. The OS rates were 54% and 40%. The UA showed better IPFS rates significantly related to male sex (p = 0.049), a BED12 of ≥42 Gy (p = 0.006), and controlled extracranial disease (p = 0.03); in the MA, a favorable trend towards LC (p = 0.11) and higher BED (p = 0.11) schedules maintained a correlation with improved IPFS rates, although statistical significance was not reached. Conclusions: HT-based SRT for brain metastases showed safety and efficacy in our monoinstiutional experience. Higher RT doses showed statistical significance for improved outcomes of LC and OS

Impact of Serotonin Transporter Absence on Brain Insulin Receptor Expression, Plasma Metabolome Changes, and ADHD-like Behavior in Mice fed a Western Diet

The impaired function of the serotonin transporter (SERT) in humans has been linked to a higher risk of obesity and type 2 diabetes, especially as people age. Consuming a “Western diet” (WD), which is high in saturated fats, cholesterol, and sugars, can induce metabolic syndrome. Previous research indicated that mice carrying a targeted inactivation of the Sert gene (knockout, KO) and fed a WD display significant metabolic disturbances and behaviors reminiscent of ADHD. These abnormalities might be mediated via a dysfunction in insulin receptor (IR) signaling, which is also associated with adult ADHD. However, the impact of Sert deficiency on IR signaling and systemic metabolic changes has not been thoroughly explored. In this study, we conducted a detailed analysis of locomotor behavior in wild-type (WT) and KO mice fed a WD or control diet. We investigated changes in the blood metabolome and examined, via PCR, the expression of insulin receptor A and B isoforms and key regulators of their function in the brain. Twelve-month-old KO mice and their WT littermates were fed a WD for three weeks. Nuclear magnetic resonance spectroscopy analysis of plasma samples showed that KO mice on a WD had higher levels of lipids and lipoproteins and lower levels of glucose, lactate, alanine, valine, and isoleucine compared to other groups. SERT-KO mice on the control diet exhibited increased brain levels of both IR A and B isoforms, accompanied by a modest increase in the negative regulator ENPP. The KO mice also displayed anxiety-like behavior and reduced exploratory activity in an open field test. However, when the KO animals were fed a WD, the aberrant expression levels of IR isoforms in the KO mice and locomotor behavior were ameliorated indicating a complex interaction between genetic and dietary factors that might contribute to ADHD-like symptoms. Overall, our findings suggest that the lack of Sert leads to a unique metabolic phenotype in aged mice, characterized by dysregulated IR-related pathways. These changes are exacerbated by WD in the blood metabolome and are associated with behavioral abnormalities

Altered Behaviour, Dopamine and Norepinephrine Regulation in Stressed Mice Heterozygous in TPH2 Gene

Gene-environment interaction (GxE) determines the vulnerability of an individual to a spectrum of stress-related neuropsychiatric disorders. Increased impulsivity, excessive aggression, and other behavioural characteristics are associated with variants within the tryptophan hydroxylase-2 (Tph2) gene, a key enzyme in brain serotonin synthesis. This phenotype is recapitulated in naïve mice with complete, but not with partial Tph2 inactivation. Tph2 haploinsufficiency in animals reflects allelic variation of Tph2 facilitating the elucidation of respective GxE mechanisms. Recently, we showed excessive aggression and altered serotonin brain metabolism in heterozygous Tph2-deficient male mice (Tph2+/−) after predator stress exposure. Here, we sought to extend these studies by investigating aggressive and anxiety-like behaviours, sociability, and the brain metabolism of dopamine and noradrenaline. Separately, Tph2+/− mice were examined for exploration activity in a novel environment and for the potentiation of helplessness in the modified swim test (ModFST). Predation stress procedure increased measures of aggression, dominancy, and suppressed sociability in Tph2+/− mice, which was the opposite of that observed in control mice. Anxiety-like behaviour was unaltered in the mutants and elevated in controls. Tph2+/− mice exposed to environmental novelty or to the ModFST exhibited increased novelty exploration and no increase in floating behaviour compared to controls, which is suggestive of resilience to stress and despair. High-performance liquid chromatography (HPLC) revealed significant genotype-dependent differences in the metabolism of dopamine, and norepinephrine within the brain tissue. In conclusion, environmentally challenged Tph2+/− mice exhibit behaviours that resemble the behaviour of non-stressed null mutants, which reveals how GxE interaction studies can unmask latent genetically determined predispositions. © 2020 The Authors.The authors' work reported here was supported by Deutsche Forschungsgemeinschaft (DFG:CRC TRR58A1/A5), DAAD (to ES), the European Union's Seventh Framework Programme (FP7/2007–2013) under Grant No.602805 (Aggressotype) and the Horizon 2020 Research and Innovation Programme under Grant No.728018 (Eat2beNICE) (to KPL and TS) and the President's program of PhD Exchange of RF-2017 (to TS and DA). We appreciate the valuable technical help of Natalia Bazhenova, Drs. Alexander Trofimov and Natalia Markova with this project

Cerebral Changes Occurring in Arginase and Dimethylarginine Dimethylaminohydrolase (DDAH) in a Rat Model of Sleeping Sickness

Involvement of nitric oxide (NO) in the pathophysiology of human African trypanosomiasis (HAT) was analyzed in a HAT animal model (rat infected with Trypanosoma brucei brucei). With this model, it was previously reported that trypanosomes were capable of limiting trypanocidal properties carried by NO by decreasing its blood concentration. It was also observed that brain NO concentration, contrary to blood, increases throughout the infection process. The present approach analyses the brain impairments occurring in the regulations exerted by arginase and N(G), N(G)-dimethylarginine dimethylaminohydrolase (DDAH) on NO Synthases (NOS). In this respect: (i) cerebral enzymatic activities, mRNA and protein expression of arginase and DDAH were determined; (ii) immunohistochemical distribution and morphometric parameters of cells expressing DDAH-1 and DDAH-2 isoforms were examined within the diencephalon; (iii) amino acid profiles relating to NOS/arginase/DDAH pathways were established.Arginase and DDAH activities together with mRNA (RT-PCR) and protein (western-blot) expressions were determined in diencephalic brain structures of healthy or infected rats at various days post-infection (D5, D10, D16, D22). While arginase activity remained constant, that of DDAH increased at D10 (+65%) and D16 (+51%) in agreement with western-blot and amino acids data (liquid chromatography tandem-mass spectrometry). Only DDAH-2 isoform appeared to be up-regulated at the transcriptional level throughout the infection process. Immunohistochemical staining further revealed that DDAH-1 and DDAH-2 are contained within interneurons and neurons, respectively.In the brain of infected animals, the lack of change observed in arginase activity indicates that polyamine production is not enhanced. Increases in DDAH-2 isoform may contribute to the overproduction of NO. These changes are at variance with those reported in the periphery. As a whole, the above processes may ensure additive protection against trypanosome entry into the brain, i.e., maintenance of NO trypanocidal pressure and limitation of polyamine production, necessary for trypanosome growth

The neuronal insulin sensitizer dicholine succinate reduces stress-induced depressive traits and memory deficit: possible role of insulin-like growth factor 2.

BACKGROUND: A number of epidemiological studies have established a link between insulin resistance and the prevalence of depression. The occurrence of depression was found to precede the onset of diabetes and was hypothesized to be associated with inherited inter-related insufficiency of the peripheral and central insulin receptors. Recently, dicholine succinate, a sensitizer of the neuronal insulin receptor, was shown to stimulate insulin-dependent H2O2 production of the mitochondrial respiratory chain leading to an enhancement of insulin receptor autophosphorylation in neurons. As such, this mechanism can be a novel target for the elevation of insulin signaling. RESULTS: Administration of DS (25 mg/kg/day, intraperitoneal) in CD1 mice for 7 days prior to the onset of stress procedure, diminished manifestations of anhedonia defined in a sucrose test and behavioral despair in the forced swim test. Treatment with dicholine succinate reduced the anxiety scores of stressed mice in the dark/light box paradigm, precluded stress-induced decreases of long-term contextual memory in the step-down avoidance test and hippocampal gene expression of IGF2. CONCLUSIONS: Our data suggest that dicholine succinate has an antidepressant-like effect, which might be mediated via the up-regulation of hippocampal expression of IGF2, and implicate the neuronal insulin receptor in the pathogenesis of stress-induced depressive syndrome.journal articleresearch support, non-u.s. gov't2012 Sep 182012 09 18importe