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

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

Nitric oxide (NO) and experimental african trypanosomiasis in the rat

Grâce à un modèle expérimental de la trypanosomose humaine Africaine (THA ou maladie du sommeil), le rat infecté par Trypanosoma brucei brucei, nous avons examiné l’implication du monoxyde d’azote (NO) dans le développement de cette pathologie. Des variations opposées de la concentration de ce composé ont été observées chez les animaux infectés, au niveau des compartiments périphérique et central : le NO diminue au niveau du sang et augmente au niveau cérébral. Ces changements sont dépendants de la NO-synthase inductible (iNOS). Au niveau périphérique, la diminution du NO qui survient favorise l’installation du parasite car la pression trypanocide de ce composé est diminuée. Dans cette situation, la L-arginine, le substrat à la base de la synthèse du NO, est utilisée pour la synthèse de polyamines, des composés nécessaires à la croissance du parasite. Ces mécanismes sont très probablement déclenchés par le trypanosome via ses facteurs solubles. Au niveau cérébral, la synthèse du NO est aussi soumise à des régulations qui impliquent l’arginase et la NG, NG-diméthylarginine diméthylaminohydrolase (DDAH). Tandis que l’activité de l’arginase demeure constante, celle de la DDAH augmente au cours de l’infection en accord avec les données des western-blot et des amino-acides. Cette augmentation, qui dépend essentiellement de l’isoforme DDAH-2, conduit à une augmentation du NO cérébral dont les propriétés sont trypanocides. Ces changements, contraires à ceux observés en périphérie, sont défavorables à la survie du trypanosome au niveau du cerveau. Ils pourraient constituer une protection supplémentaire contre l’entrée des trypanosomes dans cet organeBy way of an experimental model of human African trypanosomiasis (HAT or sleeping sickness), the rat infected by Trypanosoma brucei brucei, we examined the involvement of nitric oxide (NO) in the development of this pathology. In the infected animals, opposite variations in NO concentration were observedeither at peripheral or brain compartments: NO decreases in blood but increases in brain. These changes are dependent on the activity of the inducible NO-synthase (iNOS). In periphery, the decrease observed in NO concentration favors the parasite entrance because the trypanocidal pressure exerted by NO is decreased. In such a situation, L-arginine, the substrate conducing to the synthesis of NO, is employed for the synthesis of polyamines, a category of compounds necessary for the parasite growth. It is likely that above mechanisms might be triggered by parasites. In brain, NO synthesis is submitted to additive complex regulatory processes implying arginase and NG, NG-dimethylarginine dimethylaminohydrolase (DDAH). While the arginase activity remains constant, that of DDAH increases throughout the infection process in keeping with western-blot and amino acids data. This increase, depending mainly on DDAH-2 isoform, lasts in a brain NO increase which enhance the trypanocidal pressure. Above changes, opposite to those observed in periphery, are not favorable to the parasite survival in brain. They might constitute an additive protection against the parasite entry in this orga

Nitric oxide in the regulation of the sleep-wake states

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Cerebral inducible nitric oxide synthase protein expression in microglia, astrocytes and neurons in Trypanosoma brucei brucei-infected rats.

To study the anatomo-biochemical substrates of brain inflammatory processes, Wistar male rats were infected with Trypanosoma brucei brucei. With this reproducible animal model of human African trypanosomiasis, brain cells (astrocytes, microglial cells, neurons) expressing the inducible nitric oxide synthase (iNOS) enzyme were revealed. Immunohistochemistry was achieved for each control and infected animal through eight coronal brain sections taken along the caudorostral axis of the brain (brainstem, cerebellum, diencephalon and telencephalon). Specific markers of astrocytes (anti-glial fibrillary acidic protein), microglial cells (anti-integrin alpha M) or neurons (anti-Neuronal Nuclei) were employed. The iNOS staining was present in neurons, astrocytes and microglial cells, but not in oligodendrocytes. Stained astrocytes and microglial cells resided mainly near the third cavity in the rostral part of brainstem (periaqueductal gray), diencephalon (thalamus and hypothalamus) and basal telencephalon. Stained neurons were scarce in basal telencephalon, contrasting with numerous iNOS-positive neuroglial cells. Contrarily, in dorsal telencephalon (neocortex and hippocampus), iNOS-positive neurons were plentiful, contrasting with the marked paucity of labelled neuroglial (astrocytes and microglial) cells. The dual distribution between iNOS-labelled neuroglial cells and iNOS-labelled neurons is a feature that has never been described before. Functionalities attached to such a divergent distribution are discussed

Dual molecular regulations of nitric oxide at the peripheral and central levels in experimental African sleeping sickness

International audienceThe two-stage trypanosomiasis exists in rats infected with Trypanosoma brucei brucei, the neurological phase appearing after 9 to 12 days of infection. NO was determined in rats before and after infection (15 and 21 days) using a specific voltammetric sensor in the central nervous system (CNS) and peripheral (blood) compartments. Opposite changes were observed in the two compartments: a decrease in the periphery, an increase in the CNS (13 days after infection). This duality was analyzed in both healthy and infected Wistar rats using a molecular approach of the metabolic pathways of L-arginine, as it is the substrate of NOS, arginase and protein synthesis. In the peripheral compartment of infected animals, the decrease in inducible iNOS activity is counterbalanced by the rise in arginase activity resulting in an overproduction of related aminoacids (arginine, proline, glutamine, glutamate). Contrarily in the CNS, iNOS activity increased while that of arginase did not vary (no variation in related aminoacid concentrations). Concomitantly, there was an increased activity of dimethylarginine dimethyl aminohydrolase (DDAH), a regulatory enzyme metabolizing asymmetric dimethylarginine (ADMA), an end product of proteolysis inhibiting iNOS activity. In conclusion, the duality of NO behavior in the CNS and periphery is backed by different molecular regulations. In the peripheral compartment of rats, mice and humans (a decrease in blood NO was also observed in patients), the reduced NO production reflects the strategy deployed by the trypanosome to impair the immune processes The increased brain NO certainly accounts for blood-brain barrier and CNS pathophysiological alterations

Presence of trypanosomes in the CSF of <i>T. b. brucei</i>-infected Wistar rats.

*<p>Three infected animals per day.</p><p><i>T. b. brucei</i> = <i>Trypanosoma brucei brucei</i>.</p><p>CSF = Cerebrospinal fluid.</p

Immunostaining of iNOS in the brain of healthy and <i>T. b. brucei</i>-infected Wistar rats.

<p>(A) Immunohistochemistry for iNOS observed in different regions of the hypothalamus and thalamus. Perifornical nucleus (PeF); supraoptic nucleus; retrochiasmatic (SOR); magnocellular nucleus of the lateral hypothalamus (MCLH); lateral hypothalamic area (LH); periventricular hypothalamic nucleus (Pe); ventromedial hypothalamic nucleus (VMH); paraventricular thalamic nucleus, posterior (PVP); habenular nucleus (Hb). (B) Controls of iNOS labelling: peritoneal macrophages were incubated with (B-c, e) or without (B-b, d) LPS during 8 h (B-b, c) or 24 h (B-d, e). Specificity of secondary antibody was verified in incubating peritoneal macrophages without anti-iNOS antibody (B-a). Number of animals, n = 4 per group. The microscope used was an Olympus BX51 equipped with Olympus DP50 camera (objective×40). Abbreviations: iNOS, inducible NO-synthase; LPS, lipopolysaccharide.</p