Extracellular Vesicles from Hyperammonemic Rats Induce Neuroinflammation and Motor Incoordination in Control Rats

Abstract

Minimal hepatic encephalopathy is associated with changes in the peripheral immune system which are transferred to the brain, leading to neuroinflammation and thus to cognitive and motor impairment. Mechanisms by which changes in the immune system induce cerebral alterations remain unclear. Extracellular vesicles (EVs) seem to play a role in this process in certain pathologies. The aim of this work was to assess whether EVs play a role in the induction of neuroinflammation in cerebellum and motor incoordination by chronic hyperammonemia. We characterized the differences in protein cargo of EVs from plasma of hyperammonemic and control rats by proteomics and Western blot. We assessed whether injection of EVs from hyperammonemic to normal rats induces changes in neuroinflammation in cerebellum and motor incoordination similar to those exhibited by hyperammonemic rats. We found that hyperammonemia increases EVs amount and alters their protein cargo. Differentially expressed proteins are mainly associated with immune system processes. Injected EVs enter Purkinje neurons and microglia. Injection of EVs from hyperammonemic, but not from control rats, induces motor incoordination, which is mediated by neuroinflammation, microglia and astrocytes activation and increased IL-1β, TNFα, its receptor TNFR1, NF-κB in microglia, glutaminase I, and GAT3 in cerebellum. Plasma EVs from hyperammonemic rats carry molecules necessary and sufficient to trigger neuroinflammation in cerebellum and the mechanisms leading to motor incoordination.

Sensitivity of the Natriuretic Peptide/cGMP System to Hyperammonaemia in Rat C6 Glioma Cells and GPNT Brain Endothelial Cells

“…Our findings confirm the production of extracellular vesicles from C6 cells and astrocytes [66,67], and represent the first demonstration that hyperammonaemia can increase extracellular vesicle production from C6 cells. This complements in vivo studies, using a dietary manipulation approach to induce chronic hyperammonaemia, that showed peripheral production of extracellular vesicles that promote neuroinflammation in astrocytes [68]. Our functional data demonstrate that hyperammonaemia-induced extracellular vesicles from C6 cells are inhibitory to CNP-stimulated cGMP accumulation in GPNT cells.…”

“…Extracellular vesicle production is associated with various metabolic, cardiovascular and endocrine pathologies [ 58 , 65 ], and their production has been demonstrated from astrocytes and C6 cells [ 66 , 67 ]. In addition, rats fed a manipulated diet to induce chronic hyperammonaemia, peripherally produce extracellular vesicles that promote neuroinflammation in astrocytes [ 68 ]. Initially, to establish whether extracellular vesicles production from C6 cells was affected by conditions of hyperammonaemia, we stimulated C6 cells in serum-free media containing 0 or 10 mM NH 4 Cl for up to 72 h, before harvesting the spent media, removing cellular debris, and centrifuging to pellet extracellular vesicles.…”

The Role of Intestinal Bacteria and Gut–Brain Axis in Hepatic Encephalopathy

“…activation of blood cytokine receptors in endothelial cells ( Dantzer et al, 2008 ). Chronic hyperammonemia can also cause neuroinflammatory reactions ( Rodrigo et al, 2010 ; Hernández-Rabaza et al, 2016 ; Balzano et al, 2020 ) Injecting extracellular vesicles of hyperammonemic rats into the control group causes neuroinflammatory reactions and dyskinesia ( Izquierdo-Altarejos et al, 2020 ) The use of anti-TNFa therapy, which does not pass the BBB, prevents the neuroinflammatory responses induced by hyperammonemia ( Balzano et al, 2020 ). Therefore, the pro-inflammatory effect of hyperammonemia may be mediated by peripheral inflammation.…”

CNS-Draining Meningeal Lymphatic Vasculature: Roles, Conundrums and Future Challenges

“…Hepatic encephalopathy is a neurological complication that results from cirrhosis and causes cognitive, psychiatric and motor impairment ( Weissenborn, 2019 ). Aberrant neuroinflammation is a common feature observed in animal models of hepatic encephalopathy ( Liu R. et al, 2020 ; Izquierdo-Altarejos et al, 2020 ). Rats subjected to bile duct ligation, a hepatic encephalopathy disease model, presented increase meningeal lymphangiogenesis that was further stimulated by increasing VEGF-C levels in the CSF ( Hsu et al, 2020 ).…”