Hyperammonemia Induces Neuroinflammation That Contributes to Cognitive Impairment in Rats With Hepatic Encephalopathy

doi:10.1053/j.gastro.2010.03.040

Gastroenterology

Volume 139, Issue 2, August 2010, Pages 675-684

https://doi.org/10.1053/j.gastro.2010.03.040 Get rights and content

Background & Aims

Hyperammonemia and inflammation cooperate to induce neurological alterations in hepatic encephalopathy. Recent studies in animal models suggest that chronic hyperammonemia and neuroinflammation impair learning ability by the same mechanism. Chronic hyperammonemia might induce inflammatory factors in the brain that impair cognitive function. We sought to determine whether hyperammonemia itself induces neuroinflammation, whether ammonia-induced neuroinflammation mediates cognitive impairment, and whether neuroinflammation also occurs in rats with bile duct ligation (BDL rats)—a model of chronic liver injury that results in hyperammonemia and hepatic encephalopathy.

Methods

Chronic moderate hyperammonemia was induced by feeding male Wistar rats an ammonium-containing diet or performing BDL. Rats that received a standard diet or a sham operation were used as controls. Neuroinflammation was assessed by measuring activation of microglia and inflammatory factors. Brain samples were collected from hyperammonemic and BDL rats; microglial activation was determined by immunohistochemistry and quantification of inflammatory markers (ie, inducible nitric oxide synthase, interleukin-1β, and prostaglandin E2). Learning ability and motor activity were assessed in hyperammonemic and BDL rats given ibuprofen as an anti-inflammatory agent.

Results

Chronic moderate hyperammonemia or BDL activated the microglia, especially in cerebellum; increased inducible nitric oxide synthase, interleukin-1β, and prostaglandin E2 levels; and impaired cognitive and motor function, compared with controls. Ibuprofen reduced microglial activation and restored cognitive and motor functions in the hyperammonemic and BDL rats.

Conclusions

Chronic hyperammonemia is sufficient to induce microglial activation and neuroinflammation; these contribute to the cognitive and motor alterations that occur during hepatic encephalopathy.

Section snippets

Model of Pure Chronic Hyperammonemia in Rats Without Liver Failure

Male Wistar rats (120–140 g) were made hyperammonemic by feeding them an ammonium-containing diet for 1, 7, or 28 days, as in Felipo et al.⁹

Model of BDL in Rats

Male Wistar rats (175–200 g) were used. Liver injury was induced by common BDL, as in Jover et al.¹¹ Control rats were sham-operated. Animal experiments were approved by the Center and carried out in accordance with the European Communities Council Directive (86/609/EEC).

Treatment With Ibuprofen

Rats were treated daily with S-(+)ibuprofen (Fluka, Seelze, Germany) or saline.

Chronic Hyperammonemia Induces Activation and Migration of Microglial Cells Selectively in Some Brain Areas

The presence, amount, localization, and grade of activation of microglia were assessed by immunohistochemitry, using MHCII as a marker. Under normal conditions, microglial cells are ramified, showing a large number of processes. Activated microglial cells take an ameboid shape, reducing the length and number of ramifications.

The observation of the sections from different brain areas showed that hyperammonemia induces microglial activation selectively in some areas.

The area most affected was the

Discussion

Data reported show that chronic moderate hyperammonemia in rats, similar to that present in patients with liver cirrhosis, induces, in the absence of liver failure, activation of microglial cells and neuroinflammation.

Moreover, chronic treatment with an anti-inflammatory (ibuprofen) reduces neuroinflammation and restores motor and cognitive function in hyperammonemic rats, indicating that neuroinflammation mediates the deleterious effects of hyperammonemia on learning ability and motor activity.

Acknowledgments

Drs Rodrigo and Cauli contributed equally to this work.

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This can lead to cerebral edema and increased gamma-aminobutyric acid-ergic (GABAergic) activity.17,23 Chronic hyperammonemia is sufficient to induce astrocyte and microglial activation, resulting in brain-derived proinflammatory tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1β (Fig. 1).24–27 In an azoxymethane (AOM) induced HE mouse model, TNF-α receptor knockout (KO) mice had decreased brain swelling as opposed to the wild-type mice, further showing the impact of TNF-α on brain inflammation.28
Over 20% of mortality during acute liver failure is associated with the development of hepatic encephalopathy (HE). Thus, HE is a complication of acute liver failure with a broad spectrum of neuropsychiatric abnormalities ranging from subclinical alterations to coma. HE is caused by the diversion of portal blood into systemic circulation through portosystemic collateral vessels. Thus, the brain is exposed to intestinal-derived toxic substances. Moreover, the strategies to prevent advancement and improve the prognosis of such a liver-brain disease rely on intestinal microbial modulation. This is supported by the findings that antibiotics such as rifaximin and laxative lactulose can alleviate hepatic cirrhosis and/or prevent HE. Together, the significance of the gut-liver-brain axis in human health warrants attention. This review paper focuses on the roles of bacteria metabolites, mainly ammonia and bile acids (BAs) as well as BA receptors in HE. The literature search conducted for this review included searches for phrases such as BA receptors, BAs, ammonia, farnesoid X receptor (FXR), G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5), sphingosine-1-phosphate receptor 2 (S1PR2), and cirrhosis in conjunction with the phrase hepatic encephalopathy and portosystemic encephalopathy. PubMed, as well as Google Scholar, was the search engines used to find relevant publications.
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The role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the pathogenesis of hepatic encephalopathy (HE) is unclear. Mitochondrial reactive oxygen species (mtROS) is a signal for NLRP3 inflammasome activation. Therefore, we aimed to determine whether mtROS-dependent NLRP3 inflammasome activation is involved in HE, using in vivo and in vitro models.
Bile duct ligation (BDL) in C57/BL6 mice was used as an in vivo HE model. NLRP3 activation was assessed in the hippocampus. Immunofluorescence staining was performed to determine the cellular source of NLRP3 in the hippocampal tissue. For the in vitro experiment, BV-2 microglial cells were primed with lipopolysaccharide (LPS), followed by ammonia treatment. NLRP3 activation and mitochondrial dysfunction were measured. Mito-TEMPO was used to suppress mtROS production.
BDL mice showed cognitive impairment with hyperammonemia. Both the priming and activation steps of NLRP3 inflammasome activation were processed in the hippocampus of BDL mice. Moreover, intracellular ROS levels increased in the hippocampus, and NLRP3 was mainly expressed in the microglia of the hippocampus. In LPS-primed BV-2 cells, ammonia treatment induced NLRP3 inflammasome activation and pyroptosis, with elevation of mtROS and altered mitochondrial membrane potential. Pretreatment with Mito-TEMPO suppressed mtROS production and the subsequent NLRP3 inflammasome activation and pyroptosis under LPS and ammonia treatment in BV-2 cells.
Hyperammonemia in HE may be involved in mtROS overproduction and subsequent NLRP3 inflammasome activation. Further studies using NLRP3-specific inhibitor or NLRP3 knockout mice are needed to elucidate the important role of NLRP3 inflammasome in HE development.
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Fifty-one participants (PBC group – 38 patients, all patients but one Child-Pugh A; control group – 13 healthy controls) were included in our prospective, single center study. We assessed the plasma levels of sGPV, plasma procoagulant phospholipids (PPL) and rotational thromboelastometry (ROTEM) profiles in all study participants. Porto-systemic encephalopathy syndrome test was used to assess MHE.
The sGPV levels were higher in the PBC group compared to the controls: 36.07 ± 11.32 ng/mL vs 27.04 ± 11.72 ng/mL, p = 0.031. The PPL level was lower in the PBC group compared to controls resulting in increased clotting time in a factor Xa-based coagulation assay: 54.65 (47.83–58.83) sec. vs 45.90 (43.3–50.5) sec., p = 0.0065. PPL levels were correlated with platelet count (rho = −0.46, p = 0.001). ROTEM parameters did not differ significantly between groups. Coagulation parameters did not differ significantly between patients with and without MHE.
We have showed increased levels of sGPV - a plasma marker of platelet activation by thrombin in patients with early stage PBC compared to healthy controls. We found no relationship between the coagulation disorders and the occurrence of MHE. The PPL level was lower in the PBC group.
The Protective Effect of Ultrasonicated Ginseng Berry Extract on Rat Hepatic Encephalopathy Model Induced by Mild Bile Duct Ligation
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: Conflicts of interest The authors disclose no conflicts.

: Funding This work was supported by grants from Ministerio de Ciencia e Innovacion (SAF2005-06089, SAF2008-00062, CSD2008-00005) and from Consellería de Educacion (ACOMP06/005, ACOMP-2009-025, Prometeo/2009/027) and AP005/06, EVES 034/2007, AP-024/08 and A-01/08 from Conselleria de Sanitat of Generalitat Valenciana and by European Regional Development Fund. R. Rodrigo has a Contrato-Investigador SNS Miguel Servet (CP06/242) from Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación.

View full text

Basic—Liver, Pancreas, and Biliary TractHyperammonemia Induces Neuroinflammation That Contributes to Cognitive Impairment in Rats With Hepatic Encephalopathy

Background & Aims

Methods

Results

Conclusions

Section snippets

Model of Pure Chronic Hyperammonemia in Rats Without Liver Failure

Model of BDL in Rats

Treatment With Ibuprofen

Chronic Hyperammonemia Induces Activation and Migration of Microglial Cells Selectively in Some Brain Areas

Discussion

Acknowledgments

J Hepatol

Mol Genet Metab

Exp Neurol

Brain Res

Neurosci Lett

Hepatology

Neurobiol Aging

Brain Res Bull

Neurobiol Aging

Neurochem Int

IL-6 and IL-18 in blood may discriminate cirrhotic patients with and without minimal hepatic encephalopathy

J Clin Gastroenterol

Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunt

Hepatology

Basic—Liver, Pancreas, and Biliary Tract
Hyperammonemia Induces Neuroinflammation That Contributes to Cognitive Impairment in Rats With Hepatic Encephalopathy