title: Neuroinflammation increases GABAergic tone and impairs cognitive and motor function in hyperammonemia by increasing GAT-3 membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia.
abstract: BACKGROUND
  Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them.
  
  
  METHODS
  We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3.
  
  
  RESULTS
  Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination.
  
  
  CONCLUSIONS
  Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Hernandez-Rabaza
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cabrera-Pastor
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Lucas
authors.initials: L
authors.lastname: Taoro-Gonzalez
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Alba
authors.initials: A
authors.lastname: Gonzalez-Usano
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Agusti
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Tiziano
authors.initials: T
authors.lastname: Balzano
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Llansola
authors.affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain. vfelipo@cipf.es.
authors.forename: Vicente
authors.initials: V
authors.lastname: Felipo
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum; D004195:Disease Models, Animal; D050485:GABA Plasma Membrane Transport Proteins / Q000096:biosynthesis*; D006501:Hepatic Encephalopathy / Q000150:complications / Q000378:metabolism*; D022124:Hyperammonemia / Q000209:etiology / Q000378:metabolism* / Q000503:physiopathology; D007150:Immunohistochemistry; D007249:Inflammation / Q000378:metabolism; D017879:Isothiocyanates / Q000494:pharmacology; D008297:Male; D018782:Maze Learning / Q000187:drug effects / Q000502:physiology; D017551:Microdialysis; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D013454:Sulfoxides; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D050485:GABA Plasma Membrane Transport Proteins; D017879:Isothiocyanates; D013454:Sulfoxides; D005680:gamma-Aminobutyric Acid; C016766:sulforaphane
keywords: GABA; GAT-3; Hepatic encephalopathy; Hyperammonemia; Learning; Microglia; Motor in-coordination; Neuroinflammation; Sulforaphane