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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.

Hernandez-Rabaza V, Cabrera-Pastor A, Taoro-Gonzalez L, Gonzalez-Usano A, Agusti A, Balzano T, Llansola M, Felipo V - J Neuroinflammation (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

Hyperammonemia induces and treatment with sulforaphane reduces activation of astrocytes, which express IL-1b and GAT-3. Immunohistochemistry was performed as indicated in methods with DAB staining using antibodies against GFAP (a–d), IL-1b (e–h), or GAT-3 (i–l). Hyperammonemic rats show an altered morphology of astrocytes stained with GFAP (indicated by arrows), indicating activation (c). Treatment with SFN reduces astrocyte activation and normalizes the morphology (d). Activated astrocytes in hyperammonemic rats show increased labeling of IL-1b (g) and GAT-3 (k), which are normalized by treatment with SFN (h, l). m–p Double immunofluorescence staining of GFAP (red) and Iba-1 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GFAP and Iba-1 is observed
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Fig6: Hyperammonemia induces and treatment with sulforaphane reduces activation of astrocytes, which express IL-1b and GAT-3. Immunohistochemistry was performed as indicated in methods with DAB staining using antibodies against GFAP (a–d), IL-1b (e–h), or GAT-3 (i–l). Hyperammonemic rats show an altered morphology of astrocytes stained with GFAP (indicated by arrows), indicating activation (c). Treatment with SFN reduces astrocyte activation and normalizes the morphology (d). Activated astrocytes in hyperammonemic rats show increased labeling of IL-1b (g) and GAT-3 (k), which are normalized by treatment with SFN (h, l). m–p Double immunofluorescence staining of GFAP (red) and Iba-1 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GFAP and Iba-1 is observed

Mentions: Immunohistochemistry analysis showed that GAT-3 is expressed mainly in astrocytes surrounding Purkinje cells (Fig. 4b), and the increase in GAT-3 expression in hyperammonemia occurs mainly in activated astrocytes (Fig. 6k). No co-localization of GAT-3 and the neuronal marker Neun was observed when analyzed by double immunofluorescence labeling (Fig. 4c). This is in agreement with the literature showing that GAT-3 is expressed in astrocytes [34, 35].Fig. 6


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.

Hernandez-Rabaza V, Cabrera-Pastor A, Taoro-Gonzalez L, Gonzalez-Usano A, Agusti A, Balzano T, Llansola M, Felipo V - J Neuroinflammation (2016)

Hyperammonemia induces and treatment with sulforaphane reduces activation of astrocytes, which express IL-1b and GAT-3. Immunohistochemistry was performed as indicated in methods with DAB staining using antibodies against GFAP (a–d), IL-1b (e–h), or GAT-3 (i–l). Hyperammonemic rats show an altered morphology of astrocytes stained with GFAP (indicated by arrows), indicating activation (c). Treatment with SFN reduces astrocyte activation and normalizes the morphology (d). Activated astrocytes in hyperammonemic rats show increased labeling of IL-1b (g) and GAT-3 (k), which are normalized by treatment with SFN (h, l). m–p Double immunofluorescence staining of GFAP (red) and Iba-1 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GFAP and Iba-1 is observed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835883&req=5

Fig6: Hyperammonemia induces and treatment with sulforaphane reduces activation of astrocytes, which express IL-1b and GAT-3. Immunohistochemistry was performed as indicated in methods with DAB staining using antibodies against GFAP (a–d), IL-1b (e–h), or GAT-3 (i–l). Hyperammonemic rats show an altered morphology of astrocytes stained with GFAP (indicated by arrows), indicating activation (c). Treatment with SFN reduces astrocyte activation and normalizes the morphology (d). Activated astrocytes in hyperammonemic rats show increased labeling of IL-1b (g) and GAT-3 (k), which are normalized by treatment with SFN (h, l). m–p Double immunofluorescence staining of GFAP (red) and Iba-1 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GFAP and Iba-1 is observed
Mentions: Immunohistochemistry analysis showed that GAT-3 is expressed mainly in astrocytes surrounding Purkinje cells (Fig. 4b), and the increase in GAT-3 expression in hyperammonemia occurs mainly in activated astrocytes (Fig. 6k). No co-localization of GAT-3 and the neuronal marker Neun was observed when analyzed by double immunofluorescence labeling (Fig. 4c). This is in agreement with the literature showing that GAT-3 is expressed in astrocytes [34, 35].Fig. 6

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.

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.

No MeSH data available.


Related in: MedlinePlus