L-NMMA | Ckit Signal

Elevated dopamine induces minimal hepatic encephalopathy by activation of astrocytic NADPH oxidase and astrocytic protein tyrosine nitration

Saidan Dinga,1, Jianjing Yangb,1, Leping Liua, Yiru Yec, Xuebao Wangd, Jiangnan Hub, Bicheng Chena, Qichuan Zhugeb,∗

Abstract

Background: We previously demonstrated that dopamine (DA) overload may be a key mechanism behind development of minimal hepatic encephalopathy (MHE) in rats. It has been shown that low-grade cerebral oedema and oxidative stress play important roles in the pathogenesis of MHE. In the current study, DA-triggered oxidative injury in cerebral cortex was studied.
Methods: An MHE rat model was used. DA was injected intracerebroventricularly (i.c.v.) into rats and added to primary cortical astrocytes (PCAs). Immunoblotting, immunoprecipitation and immunostaining were conducted after DA injection.
Results: Cognitive impairment and cerebral edema were observed in MHE rats and rats injected with 10 g
DA. Astrocyte swelling was increased by DA. Astrocytic protein tyrosine nitration (PTN) was induced by DA. DA-induced PTN was insensitive to l-NMMA but was blunted by apocynin, superoxide dismutase, catalase and uric acid. Exposure to DA substantially increased levels of astrocytic NADPH oxidase subunits and induced p47phox phosphorylation and reactive oxygen species production but decreased the expression and activity of neuronal-type nitric oxide synthase (nNOS).
Conclusions: PTN induced by DA, which was attributed to NADPH oxidase and not to nNOS, may alter astrocyte function and thereby contribute to the precipitation of MHE episodes.

Keywords:
Minimal hepatic encephalopathy
Dopamine
Protein tyrosine nitration NADPH oxidase

1. Introduction

Minimal hepatic encephalopathy (MHE) is a neurocognitive disorder that affects up to 80% of cirrhotic patients (Montgomery and Bajaj, 2011). Subtle changes in cognitive function, electrophysiological parameters, cerebral neurochemical/neurotransmitter homeostasis, cerebral blood flow, metabolism, and fluid homeostasis can be observed in cirrhosis patients without hepatic encephalopathy (HE) (Dhiman and Chawla, 2009). Morphological abnormalities of the brain have been identified in this population, such as mild brain edema, hyperintensity of the globus pallidus and other subcortical nuclei observed in cerebral MR studies, and the central and cortical atrophy observed in neural imaging studies; however, these morphological abnormalities are unlikely to have diagnostic utility. Similarly to overt HE, oxidative stress plays key roles in the pathogenesis of MHE (Montgomery and Bajaj, 2011). However, the exact pathogenesis of MHE remains unknown (Torres et al., 2013).
Intracellular glutamine accumulation caused by increased ammonia detoxification leads to astrocyte swelling (Willard-Mack et al., 1996), which is a recognized early pathogenic event in MHE in cirrhotic patients (Häussinger, 2006) and may contribute to the severe rise in intracranial pressure in patients with fulminant hepatic failure (Blei and Larsen, 1999). Under pathophysiological circumstances, the interplay of multiple factors may account for astrocyte swelling. For example, in MHE, with liver disease and portosystemic shunting, inefficiently detoxified gut-derived toxins (eg, ammonia, benzodiazepine-like substances) will accumulate in the blood, cross the blood-brain barrier (BBB), and result in altered neurotransmission and astrocyte swelling (Prakash et al., 2013). It was hypothesized that dopamine (DA), a confirmed MHEprecipitating factor (Ding et al., 2013), contributes at least in part to astrocyte swelling.
Astrocytes are involved in water homeostasis and edema formation (Gill et al., 1973). Astrocyte swelling can increase reactive oxygen species (ROS) and NO production, which cause protein tyrosine nitration (PTN); this is induced, for example, by toxins relevant for HE in quantitites sufficient to produce oxidative stress and PTN and thus contributing to altered astrocytic and neuronal function (Görg et al., 2013; Lachmann et al., 2013). ROS mediate astrocyte swelling induced by glutamate (Bender et al., 1998; Dombro et al., 2000) or ammonia (Norenberg et al., 2005). PTN in astrocytes, the consequence of oxidative/nitrosative stress, is induced by hypoosmotic astrocyte swelling (Schliess et al., 2002; Görg et al., 2003, 2006; Schliess et al., 2004). Therefore, we assumed that astrocyte swelling may account for cerebral PTN caused by DA overload in MHE.
Oxidative/nitrosative stress has a variety of functional consequences, which are considered to be crucial in the pathogenesis of HA. Examples include PTN attributed to ONOO-production (Görg et al., 2013). The respective production of superoxide anion radical (O2−) and NO leading to ONOO-synthesis is triggered through activation of NADPH oxidase and neuronal-type nitric oxide synthase (nNOS) (Schliess et al., 2002). NADPH oxidase and nitric oxide synthase (NOS) are major contributors to early ROS and NO formation (Kruczek et al., 2009; Reinehr et al., 2007). NADPH oxidase is composed of a catalytic moiety (gp91), which is activated by assembly with regulatory proteins including p47phox,p67phox, and Rac (Bokoch and Diebold, 2002; Nauseef, 2004; Paniet al., 2001; Vignais, 2002). Serine phosphorylation of the cytosolic subunit p47phox relieves its inhibitory intramolecular interaction and is critical for p47phox-dependent NADPH oxidase activation (Groemping et al., 2003; Johnson et al., 1998; Park and Babior, 1997).
Our previous study found that the pathogenesis of MHE may be associated with elevated DA of cirrhotic livers: excessive DA from livers crosses the BBB and inhibits learning and memory formation (Ding et al., 2013). In the current study, we investigated the effect of DA on activation of NADPH oxidase, NO production, and PTN both in rat brain in vivo and in cultured rat astrocytes.

2. Materials and methods

2.1. MHE models

A total of 50 Sprague–Dawley rats (Experimental Animal Center of The Chinese academy of sciences in shanghai) weighing 220–250 g were used. All animals were subjectred to series of behavioral tests: Y-maze (YM), open-field tests (OF), elevatedplus maze (EPM), and water-finding task (WFT). Rats were then randomly divided into 2 groups: control group (n = 10) and thioacetamid (TAA) group (n = 40). MHE was induced by intraperitoneal (i.p.) injection of TAA (200 mg/kg in normal saline, Sigma–Aldrich) twice per week for 8 weeks (Jia and Zhang, 2005). After 8 weeks, the behavioral tests were performed for all rats again. Criteria of MHE: (a) values of YM were lower than 3/4-fold average normal values, (b) values of WFT were more than 3/2-fold average value normal values, (c) EEG showed no typical slow wave of hepatic encephalopathy (HE) (Jia and Zhang, 2005). If TAA-treated rats met the criteria of either (a) + (c) or (b) + (c), rats were included in the MHE group. Liver/serum/cerebral cortex were collected for Fluorescent staining, immunoblotting and determination of DA.

2.2. DA-treated rat models

Intracerebroventricular (i.c.v.) injection of dopamine hydrochloride (1 g/3 l and 10 g/3 l in saline) was stereotaxically performed in the left lateral ventricles of rats (anterior–posterior, +0.3 mm; lateral, 1.0 mm; horizontal, 3.0 mm from the bregma) (n = 15). At 7 days after injection, rats were performed for an OF test, a YM, an EPM test and a WFT test.

2.3. Behavioral tests

Open-field tests (OF) were performed as described (Kawasumi et al., 2004). Briefly, rats were individually placed at the center of a 10 × 10 cm gray plastic field (with 20-cm interval black grids) surrounded by a 20-cm wall, and allowed to move freely for 3 min. Ambulation was measured as the total grid line crossing.
The apparatus for Y-maze (YM) was made of gray plastic, with each arm 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top (Kawasumi et al., 2004; Yamada et al., 2005). The three arms were connected at an angle of 120◦. Rats were individually placed at the end of an arm and allowed to explore the maze freely for 8 min. Total arm entries and spontaneous alternation percentage (SA%) were measured. SA% was defined as a ratio of the arm choices that differed from the previous two choices (‘successful choices’) to total choices during the run (‘total entry minus two’ because the first two entries could not be evaluated). For example, if a rat made 10 entries, such as1-2-3-2-3-1-2-3-2-1, there are 5 successful choices in 8 total choices (10 entries minus 2).
The elevated-plus maze (EPM) apparatus was made of four crossed arms (Kawasumi et al., 2004; Itoh et al., 1990). Two arms were open (50 × 10 cm grey plastic floor plate without wall), whereas the other two were closed (same floor plates with 20-cmhigh transparent acrylic wall). The maze was set at 100 cm above the floor. Rats were allowed to explore the maze freely for 90 s. Examined parameters were: (1) transfer latency (the time elapsed until the first entry to a closed arm); (2) duration of the first stay in a closed arm (the time from the first entry to a closed arm to the first escape from the arm); (3) cumulative time spent in the open/closed arms.
Water-finding task (WFT) was performed to analyze latent learning or retention of spatial attention of the rats (Kawasumi et al., 2004; Ichihara et al., 1989; Mamiya et al., 1998). The testing apparatus consisted of a grey plastic rectangular open field (50×30cm, with a black 10-cm2 grid) with a15-cm wall, and a cubic alcove (10 × 10 × 10 cm) was attached to the center of one longer wall. A drinking tube was inserted through a hole at the center of the alcove ceiling, with the tip of the tube placed at 5 cm for training or at 7 cm for the trial from the floor. A mouse was first placed at the near-right corner of the apparatus and allowed to explore it freely for 3 min. Rats were omitted from the analysis when they could not find the tube within the 3-min exploration. After the training session, rats were deprived of water for 24 h. In the trial session, rats were again individually placed at the same corner of the apparatus and allowed to find and drink the water in the alcove. The elapsed times until the first entry into the alcove (entry latency, EL), until the first touching/sniffing/licking of the water tube (contacting latency, CL) and until the initiation of drinking from the water tube (drinking latency, DL) were measured.

2.4. Histopathology

Liver tissues were fixed in 10% formalin for 24 h and then paraffin-embedded in an automated tissue processor; 5 m sections were stained with Hematoxylin and Eosin (H&E) or Masson and subjected to histopathological examination.

2.5. Brain slice preparation and treatments

MHE and DA (10 0)-treated rats were anesthesized by intraperitoneal injection of 0.7 ml phenobarbital, respectively. 400–500 m thick horizontal slices were cut from the cerebral cortex of normal, MHE and DA (10 no)-treated rats and immediately placed in ice-cold DMEM containing 1000 mg/l d-Glucose (Gibco BRL, Life-Sciences, Gaithersburg, MD). Slices were incubated with NADPH oxidase inhibitor apocynin (300 M), NOS inhibitor NG-Monomethyl-l-arginine,Monoacetate Salt (l-NMMA) (1 mM), superoxide dismutase (SOD) (300 units/ml), catalase (8000 units/ml) and peroxynitrite scavenger uric-acid (200 M) for 6 h.

2.6. Cell culture and treatments

Primary cortical astrocytes (PCAs) were prepared from 1-dayold Sprague–Dawley rat pups (Bernabeu et al., 1996). Tissues of cerebral cortex were dissociated into a cell suspension using mechanical digestion. Cells were plated in 75cm2 tissue culture flasks at a concentration of 15×106 cells in 11ml of 1% serum- containing DMEM/F12 medium, incubating for 72h. The medium was changed at this time and every 72 h. After incubating the primary cultures for 7 days, the medium was changed completely (11 ml). Flasks were placed on a shaker platform in a horizontal position, and were shaken at 200× g for 18 h at 37◦C to separate the oligodendrocytes from the astrocytes. Cells were then poured into a new 75cm2 flask, and incubated for 7 days, and plated in six-well plates. PCAs were exposed to DA (final concentration of 1, 5, 10 M, 24 h) with and without 6-h pretreatment with NH4Cl (100 M), apocynin (300 M), l-NMMA (1 mM), SOD (300 units/ml), catalase (8000 units/ml) and uric-acid (200 M).

2.7. Determination of DA levels

Liver/serum/cerebral cortex/PCAs samples were homogenized in 300–800 l of 0.4 M HClO4 containing 0.1% (w/v) Na2S2O5 by sonication (Labsonic-U-Braun). The homogenates were centrifuged for 15 min at 20,000× g at 4◦C and aliquots of supernatants were taken for analysis of DA level using a high performance liquid chromatography (HPLC) technique with electrochemical detection with modifications in the mobile phase (Colado et al., 1993).

2.8. Assessment of cerebral edema

Brain water content (BWC), a sensitive measure of cerebral edema, was quantified using the wet–dry method, as described previously (Hayakata et al., 2004; Laird et al., 2010). BWC was measured in 3 mm coronal sections of the cortex surrounding the injection site. Tissue was weighed immediately after dissection (wet weight), and then dehydrated at 65◦C. The tissue was reweighed 48 h later to obtain a dry weight. The percentage of tissue water content was calculated using the following formula: BWC = [(wet weight) − (dry weight)/wet weight] × 100.

2.9. Monitoring of astrocyte volume changes

Cell volume (intracellular water space) was determined using the 3-O-methyl-[3H]-glucose (OMG) method as described pre- viously (Kletzien et al., 1975) (Norenberg et al., 1991). Briefly, cultured astrocytes were incubated with [3H] OMG (1mM containing 1 Ci of radioactive OMG) (Sigma–Aldrich), and at the end of incubation, a small aliquot of medium was saved for specific activity determination. Cultures were washed three times with icecold buffer containing 290 mM sucrose, 1 mM Tris–nitrate (pH7.4), 0.5 mM calcium nitrate and 0.1 mM phloretin. Cells were harvested in 0.5 ml of 1 N sodium hydroxide. Radioactivity was converted to intracellular water space and expressed as l/mg cell protein. Protein content was determined by the BCA method (Amresco).

2.10. Immunoblotting

For gp91phox/p47phox/p67phox/Rac/nNOS assays, rat cerebral cortex tissues or PCAs were harvested in the lysis buffer (10 mM Tris/HCl buffer (pH7.4) containing 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 ug/ml aprotinin, 2 ug/ml leupeptin, 1 mM EGTA and 0.5% Nonidet-P40).
For NO2Tyr assays, rat cerebral cortex tissues/slices or PCAs were harvested in the lysis buffer (10 mM Tris/HCl buffer (pH7.4) containing 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 ug/ml aprotinin, 2 ug/ml leupeptin, 1 mM EGTA, 20 mM NaF, and 0.5% Nonidet-P40). The total amount of protein was determined by BCA protein assay (Amresco). Samples (50 g protein) were separated by 10% SDS-PAGE and electroblotted to PVDF membrane, which were blocked by incubation in 5% non-fat dry milk dissolved in TBS-T (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Following transfer, proteins were probed using a primary antibody: NO2Tyr (Abcam), 1:1000; gp91phox (Abcam), 1:100; p47phox (Cell Signaling Technology), 1:1000; p67phox (Abcam), 1:1000; Rac (Abcam), 1:10; nNOS (Abcam), 1:500; GAPDH (Abcam), 1:3000. Then horseradish peroxidase-conjugated anti-rabbit secondary antibody was used. After extensive washing, protein bands detected by antibodies were visualized by ECL reagent (Thermo) after exposure on Kodak BioMax film (Kodak). The films were subsequently scanned, and band intensities were quantified using Quantity One software.

2.11. Determination of tyrosine nitration of GS and GAPDH

Rats cerebral cortex slices and PCAs were lysed at 4◦C using 10 mM Tris/HCl buffer (pH7.4) containing 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 ug/ml aprotinin, 2 ug/ml leupeptin, 1 mM EGTA, 20 mM NaF and 0.5% Nonidet-P40. Analysis of anti-NO2Tyr precipitates from brain lysates for the presence of GS was performed according to Schliess et al. (2002). Cell lysates containing defined protein amounts were incubated with 1.5 g anti-NO2Tyr (Abcam, 1:1000) and anti-glutamine synthetase (Abcam, 1:2000)/GAPDH (Abcam, 1:2500), respectively. The immune complexes were collected by using protein A/G sepharose (Santa Cruz), washed five times, and then subjected to SDS-polyacrylamide gel electrophoresis. Tyrosine nitration of GS/GAPDH was detected using anti-glutamine synthetase/GAPDH antibody and anti-NO2Tyr antibody, respectively.

2.12. Determination of p47phox serine phosphorylation

PCAs or cerebral cortex tissues were lysed at 4◦C using 20 mM Tris/HCl buffer (pH7.4) containing 1% Triton X-100, 140 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2ug/ml Aprotinin, 2ug/ml Leupeptin, 1 mM EGTA, 10 mM NaF, 10 mM Na-pyrophosphate, 1 mM sodium vanadate, 20 mM -glycerophosphate. Equal protein amounts (200 g) of each sample were incubated for 2 h at 4◦C with a polyclonal rabbit anti-p47phox (Cell Signaling Technology; 1:1000) to immunoprecipitate p47phox. Then 10l of protein A- and 10l of protein-G-agarose (Santa Cruz, CA, USA) were added and the incubation was continued at 4◦C overnight. Immunoprecipitates were washed 3 times and then subjected to Western blot analysis as published recently (Reinehr et al., 2005). p47phox serine phosphorylation was detected using an anti-phosphoserine antibody (Abcam, 1:125) (Bataller et al., 2003) and p47phox loading was monitored using the anti-p47phox antibody (Cell Signaling Technology, 1:1000).

2.13. Double-labeled fluorescent staining

For tissues assay, four-micron frozen cerebral cortex sections fixed in acetone or 4% formaldehyde were blocked for endogenous peroxidase activity with 0.03% H2O2 if appropriate. For cells assay, PCAs, cultured on glass coverslips precoated with 0.01% poly-llysine (Sigma–Aldrich), were fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 for 10 min at room temperature.
Blocking was achieved with PBS containing 5% normal goat serum for 1 h at room temperature. Sections were then incubated overnight at 4◦C with the following primary antibodies: 3nitrotyrosine (NO Tyr, Abcam), 1:1000; gp91phox (Abcam), 1:100; p47phox (Cell Signaling Technology), 1:1000; p67phox (Abcam), 1:500; Rac (Abcam), 1:10; neuronal nitric oxide synthase (nNOS) (Abcam), 1:500; glial fibrillary acidic protein (GFAP) (Abcam), 1:100. Binding of primary antibodies was detected by incubating the sections for 30 min with FITC (green)/Alexa Fluor 594 (red) conjugated secondary antibody. Imaging was performed with a Leica TCS SP2 confocal laser scanning microscope. The image data were analyzed and quantified using Imagepro Plus software.

2.14. Determination of nitric oxide synthase (NOS) activity in PCAs

The conversion of [14C] arginine to [14C] citrulline was deter- mined as described by Kiedrowski et al. (1992). Ten days after seeding, PCAs were washed twice with Locke’s solution without magnesium and [14C] arginine (1.7M, 0.25Ci) was added for 10 min. The medium was removed and the astrocytes washed three times with 2 ml cold Locke’s solution and resuspended in 1 ml 0.3 M H ClO . After centrifugation, [14C] citruline was determined in the supernatant and protein in the pellet. [14C] Citruline was separated from [14C] arginine by chromatography using Dowex AG50WX-8 (Na+ form) column. For each sample, a blank control treated with100 M nitroarginine to inhibit NOS was conducted. NOS activity is expressed as the difference between [14C] citruline formed in absence and presence of nitroarginine.

2.15. Determination of NOS activity in cortical slices

Cerebral cortex were dissected and transversal slices (400 m) were obtained using a manual chopper, transferred to incubation wells and incubated for 30 min at 35.5◦C in Krebs buffer (119 mM NaCl, 2.5 mM KCl, 1 mM KH2PO4, 26.2 mM NaHCO3, 2.5 mM CaCl2 and 11 mM glucose, aerated with 95% O2 and 5% CO2 at pH7.4). Cortical slices (400 m) were incubated for 30 min at 35.5◦C in Krebs buffer and [14C] arginine (1.7M, 0.25Ci) was added. After 5min,0.3 mM NMDA was added and the incubation continued for 5 min. The buffer was removed and slices washed three times with 2 ml cold Krebs buffer and homogenized in 1 ml 0.3 M H3ClO4. After centrifugation (14,000× g, 5min) [14C] citruline was determined forastrocytes.

2.16. Detection of ROS production

Slices or PCAs were incubated with 5 M of 5-(and-6)-carboxy20,70 dichlorodihydrofluorescein diacetate (Carboxy-H2DCFDA) in DMEM equilibrated with O2/CO2 (95/5; v/v). After 5 min, 300 M apocynin was added and the incubation continued for 25 min. During the entire dye loading period slices were kept on a nylon cell strainer (BD Biosciences) in a bathing chamber to ensure proper oxygenation of the slice. After washing with DMEM to remove excess Carboxy-H2DCFDA, slices were transferred on the inverted fluorescence microscope (Zeiss) and mounted by fixation with a platinum wire. DCF-fluorescence was measured at room temperature by using an excitation wavelength of 488 nm generated by a mono-chromator collecting the emission at 515–565 nm using a CCD camera provided by the QuantiCell 2000-calcium imaging setup (VisiTech). All experiments were carried out between 3 and 4 h after slice preparation.

2.17. Detection of superoxide production

Cellular localization of superoxide production in brain slices was examined by monitoring dihydroethidium (Het) fluorescence (Bindokas et al., 1996). Brain slices were kept in a bathing chamber containing DMEM, 1000 mg/l d-Glucose without phenolred equilibrated with O2/CO2 (95/5; v/v). Immediately before experimental treatment HEt was added to the chamber (final concentration: 100 M). At the end of the incubation slices were washed with DMEM to remove excess HEt, then placed in ice-cold paraformaldehyde and fixed overnight at 4◦C. After fixation slices were washed three times in PBS before processed for immunohistochemistry and fluorescence monitoring by confocal microscopy using an excitation wavelength of 543 nm collecting the emission at 560–615 nm.

2.18. Determination of nitrites and nitrates

PCAs and cortical slices were incubated for 10 min in the presence or the absence of 300 M apocynin, collected and homogenized in 300 l of acetate buffer (Verdon et al., 1995). Samples were centrifuged (14,000× g, 5 min) and nitrites +nitrates were measured in the supernatant as above. Pellets were resuspended in 300 l of 0.25 M NaOH and protein was measured by the bicinchoninic acid (BCA) method.

2.19. Statistical analysis

The statistical significance between group comparisons was determined by one-way analysis of variance (ANOVA). All data are presented as mean ± SD (standard deviation). P < 0.05 or P < 0.01 was considered statistically significant.

3. Results

3.1. Memory impairment and elevation of intracranial DA levels in MHE models

We established a rat hepatic cirrhosis model by chronic TAA injection, and the degree of liver cirrhosis was assessed by H&E and Masson staining. As shown in Fig. 1a and b, regenerating hepatic nodules were present based on HE staining (Fig. 1a) and fibrous septa formation was observed by Masson staining (Fig. 1b) in the liver of TAA-treated rats.
Rats were then subjected to a series of behavioral tests: an OF test, a YM, an EPM test and a WFT. In the OF test, ambulation was significantly increased in 24 of the TAA-treated rats compared with the control group (Fig. 1c). The SA% in the YM in 26 TAA-treated rats was significantly lower (P < 0.01) than that of control rats (Fig. 1d). In the EPM, for 21 TAA-treated rats, cumulative time spent in the open arms was longer than that of controls, while the cumulative time spent in the closed arms was shorter than that of controls (Fig. 1e). In the WFT, a significant delay in EL, CL and DL was detected in 27 TAA-treated rats compared with the controls (Fig. 1f). We found that at least one of the values of behavioral tests for 31 TAAtreated rats was significantly different from values of the control group. In TAA-treated rats, 26 out of 31 with abnormal behavior displayed an alpha (8–13 Hz) band in the EEG tests, whereas a slow wave (theta: 4–7 Hz or delta <4 Hz wave) was observed in the other 5 (Fig. 1g). We also examined the concentrations of DA in the liver, serum and cerebral cortex of MHE rats. Increased levels of DA in the liver (Fig. 1h), serum (Fig. 1i) and cerebral cortex (Fig. 1j) were observed compared with control rats. We propose that elevated DA levels in the liver caused DA to cross the BBB and enter the brains of MHE rats, as previously observed. This suggests that DA may be one of the factors precipitating MHE.

3.2. DA treatment causes memory impairment

To examine whether memory impairment in MHE rats is associated with the elevation of DA in the brain, we injected DA into normal rats. Then the rats were again subjected to behavioral tests: an OF test, a YM, an EPM test and a WFT. In OF, increased ambulation was observed in DA (10 g)-treated rats (Fig. 2a). In YM, 10 g DAtreated rats displayed significantly different SA% values (Fig. 2b). The results of EPM showed a longer time spent in the open arms and a shorter time spent in the closed arms for 10 g DA-treated rats (Fig. 2c). In WFT, 10 g DA treatment led to a significant delay in EL, CL and DL (Fig. 2d). An alpha (8–13 Hz) band in the EEG tests was detectable in DA-treated rats (Fig. 2e). This confirmed that elevation of DA in the brain induced cognitive impairment.

3.3. The effect of DA on cerebral edema in MHE

Low-grade cerebral edema is considered to be pathognomonic of MHE in cirrhotic patients (Qi et al., 2013). To examine whether elevated DA induced brain edema in MHE, the water content was tested in the cerebral cortex of MHE rats and DA (i.c.v.)-treated rats. We found that water content was significantly increased in the cortex of MHE rats (Fig. S1a). In the i.c.v. injection groups, there was no brain edema induced by low-dose DA (1 g) treatment, while a 43 ± 4% brain edema was observed in rats treated with a high dose of DA (10 g) (Fig. S1b).

3.4. The effect of DA on PCA cell volume

Astrocyte swelling has been implicated as a major process responsible for cytotoxic edema (Martinez-Hernandez et al., 1977; Norenberg, 1994, 1996). We found that DA treatment increased PCA cell volume in a dose-dependent manner (Fig. S1c): cell swelling was not significant in cultures treated with 1 M DA, while 5 M treatment resulted in a significant increase in PCA cell volume, and there was a further increase in the degree of PCA swelling when cultures were treated with 10 M DA. We further examined whether there was any additive or synergistic effects of NH4Cl and DA in the induction of cell swelling. We found that the increase of cell volume induced by 5 M DA was similar to that induced by 100 M NH4Cl (Fig. S1c). Co-treatment of cultures with NH4Cl and 5 M DA for 24 h showed additive effects on PCA swelling (Fig. S1c).
Treatment of cultures with 5 M DA resulted in a timedependent increase in PCAs swelling: at 3 h, no PCA swelling was observed, while at 6 h, a 22 ± 3% cell swelling was identified, which persisted for up to 12 h, and cultures continued to display cell swelling for up to 24 h (38 ± 5%) (Fig. S1d). These results suggest that astrocyte swelling might contribute to the cerebral edema induced by DA.

3.5. Cerebral DA overload increases PTN

Low-grade cerebral edema is one of the characteristics of MHE (Goel et al., 2010). We examined astrocytic PTN in MHE and found that PTN was significantly increased in the cerebral cortex of MHE rats based on western blotting with an antibody raised against NO2Tyr (Fig. 3a). PTN-positive astrocytes were observed in the cerebral cortex of MHE rats (Fig. 3b). Among the tyrosine-nitrated proteins, glutamine synthetase (GS) and GAPDH were identified. Elevated levels of tyrosine nitration of GS and GAPDH were found in the cerebral cortex of MHE rats compared with sham-operated controls (Fig. 3c–e).
To examine whether DA overload increases PTN in the brain, we analyzed PTN in the cerebral cortex of DA-treated rats using double immunofluorescence and immunoblotting. As shown in Fig. 4f, DA increased tyrosine nitration of distinct proteins in the range of 25–170 kD in a dose-dependent fashion in DA-treated rats. Little PTN was detected in 1 g DA-treated rats by immunoblotting, while 10 g DA treatment significantly increased 3-nitration of protein tyrosine residues (Fig. 4a). Double immunofluorescence staining revealed that the number of PTN-positive astrocytes in the cerebral cortex of 10 g DA-treated rats was significantly higher than those of control rats (Fig. 4b). In addition, 10 g DA induced a significant increase in tyrosine nitration of GS and GAPDH (Fig. 4c–e).
We next examined PTN in PCAs exposed to DA. We detected very little PTN under control and 1 M DA treatments, while PTN was moderately increased by 5 M DA and strongly increased by 10 M DA (24 h; Fig. S2a). Some heterogeneity of the PCA population was observed with respect to DA-induced PTN, which was more obvious by superimposition of GFAP and NO2Tyr staining (Fig. S2b). DA at a concentration of 5 M induced similar PTN as 100 M NH4Cl (Fig. S2a and b). When PCAs were co-treated with 5 M DA and 100 M NH4Cl, PTN was further increased (Fig. S2a and b). Exposure of PCAs to DA for 24 h induced a dose-dependent tyrosine nitration of GS and GAPDH (Fig. S2c–e) and 10 M DA significantly increased nitration of GS and GAPDH (Fig. S2c-e). DA at 5 M also had a similar effect to 100 M NH4Cl with respect to tyrosine nitration of GS and GAPDH; additivity was also observed for 5 M DA plus NH4Cl (Fig. S2c–e). Moreover, increased nitration of a protein larger than 25 kD was observed only in presence of both stimuli (Fig. S2c–e). These results suggested that elevated DA in the brain might contribute to PTN in MHE.

3.6. Pharmacologic characterization of DA-induced PTN

We pharmacologically characterized PTN, tyrosine nitration of GS (NO2Tyr-GS) and tyrosine nitration of GAPDH (NO2Tyr-GAPDH) induced by DA. Exposure of the NADPH oxidase inhibitor apocynin to cortical mouse brain slices from MHE rats significantly decreased PTN, NO2Tyr-GS, and NO2Tyr-GAPDH (Fig. 5a and b). In contrast, NOS inhibitor l-NMMA did not affect PTN, NO2Tyr-GS, or NO2TyrGAPDH in cortical rat brain slices from MHE rats (Fig. 5a and b). PTN was largely absent in cortical brain slices from MHE rats following exposure to catalase or SOD (Fig. 5a and b). Induction of NO2TyrGS and NO2Tyr-GAPDH in MHE rats was sensitive to peroxynitrite scavenger uric acid (Fig. 5a and b). PTN, NO2Tyr-GS and NO2Tyr-GAPDH induced by 10 g DA were abolished in the presence of apocynin (Fig. 5c and d). l-NMMA had no effect on DA-induced PTN, NO2Tyr-GS and NO2Tyr-GAPDH (Fig. 5c and d). Addition of catalase or SOD to the cortical brain slices from DA (10 g)-treated rats also significantly suppressed PTN, NO2Tyr-GS and NO2Tyr-GAPDH (Fig. 5c and d). Uric acid strongly decreased PTN, NO2Tyr-GS and NO2Tyr-GAPDH in response to 10 g DA treatment (Fig. 5c and d).
Apocynin abolished PTN induced by 10 M DA (Fig. 5e), indicating a critical involvement of NADPH oxidase. l-NMMA, which abolished NH4Cl-induced PTN, failed to decrease the amount of PTN, NO2Tyr-GS, or NO2Tyr-GAPDH induced by 10 M DA (Fig. 5e), indicating that NOS is not involved in DA-induced tyrosine nitration. However, catalase and SOD strongly inhibited PTN induced by 10 M DA (Fig. 5e), suggesting the involvement of superoxide. Uric acid abolished PTN induced by 10 M DA (Fig. 5e). Our data suggests that PTN induced by DA is associated with activation of the NADPH oxidase-ROS signaling pathway, but not the activation of the nNOS-NO signaling pathway.

3.7. Increased expression of NADPH oxidase by DA in MHE

Activation of NADPH oxidase and nNOS triggers PTN (Kruczek et al., 2009; Reinehr et al., 2007). Then, we investigated the effect of DA on the expression of NADPH oxidase and nNOS in vivo and in vitro. First, we examined the expression of NADPH oxidase and nNOS in MHE rats. We found increased expression of NADPH oxidase subunits gp91phox, p47phox, p67phox, and Rac, and decreased expression of nNOS in the cerebral cortex of MHE rats by western blot analysis (Fig. 6a and b). Consistently, the cerebral cortex of MHE rats displayed an increased number of astrocytes positive for gp91phox,p47phox,p67phox andRac(Fig.6c–f)andadecreasednumber of nNOS-positive astrocytes in double immunofluorescence staining (Fig. 6 g). Then we examined the expression of subunits of NADPH oxidase and nNOS in DA (10 g)-treated rats. Immunoblotting showed that DA induced a dose-dependent elevation of expression of gp91phox, p47phox, p67phox and Rac in the cerebral cortex (Fig. 7a and b). However, nNOS expression was decreased in response to 10 g DA (Fig. 7a and b). Strong immunoreactivity of gp91phox, p47phox, p67phox and Rac was observed in the cerebral cortex of 10g DA-treated rats, whereas very little gp91phox, p47phox, p67phox or Rac was detectable under 1g DA-treated conditions (Fig. 7c–f). However, slightly decreased immunoreactivity of nNOS was detected in response to 10 g DA (Fig. 9g).
We further examined the expression of subunits of NADPH oxidase and nNOS in PCAs. The levels of gp91phox, p47phox, p67phox and Rac were significantly increased and the level of nNOS was decreased in response to 5 and 10 M DA based on immunoblotting (Fig. S3a and b). Consistently, immunocytochemistry indicated that the expression of gp91phox, p47phox, p67phox and Rac were increased in PCAs (Fig. S3c–f) and nNOS expression was decreased (Fig. S3g) by 5 and 10 M DA treatment. DA at a concentration of 5 M was similarly effective to 100 M NH4Cl in regulating the expression of gp91phox, p47phox, p67phox, Rac and nNOS (Fig. S3a and b). Co-treatment with 5 M DA and 100 M NH4Cl produced a more dramatic increase in gp91phox, p47phox, p67phox and Rac expression and a decrease in nNOS expression (Fig. S3a and b). Taken together, these data indicate that PTN due to DA was attributable to the activation of the NADPH oxidase-ROS signaling pathway rather than to the activation of the nNOS-NO signaling pathway.

3.8. Activation of NADPH oxidase by DA in MHE

NADPH oxidase activation is dependent on serine phosphorylation of the cytosolic subunit p47phox (Groemping et al., 2003; Johnson et al., 1998; Park and Babior, 1997). We examined the effect of DA on p47phox serine phosphorylation and nNOS activity in vivo and in vitro. The induction of p47phox serine phosphorylation was significantly increased (Fig. 8a and b), while the activation of nNOS was reduced in the cerebral cortex of MHE rats (Fig. 8c). p47phox serine phosphorylation was significantly increased (Fig. 8d and e), and the inactivation of nNOS was confirmed in the cerebral cortex of 10g DA-treated rats (Fig. 8f). An increase in p47phox serine phosphorylation was detected in PCAs exposed to 10 M DA (Fig. 8g and h), and the activity of nNOS was lower in PCAs exposed to 10 M DA than in controls (Fig. 8i), indicating the effectiveness of activation of NADPH oxidase on PTN by DA and the ineffectiveness of its activity on nNOS.

3.9. ROS production by DA in PCAs

We next examined the effect of DA on the content of products of NADPH oxidase (ROS) and nNOS (NO). To examine the potential contribution of astrocytes to ROS production by DA, PCAs and brain slices were loaded with carboxy-H2DCFDA and HEt. DA (1, 5 and 10 M) in PCAs induced an almost instantaneous dose-dependent increase in DCF fluorescence, indicating rapid ROS production (Fig. 9a and b). The NADPH oxidase inhibitor apocynin (Muijsers et al., 2000) almost completely prevented induction of DCF fluorescence by 10 M DA (Fig. 9a and b). An increase in ROS production in DA (1, 5 and 10 M)-treated PCAs was confirmed by HEt (Fig. 9c). Exposure of PCAs to 10 M DA significantly increased cellular HEt fluorescence, which was inhibited by apocynin (Fig. 9c).
A significant increase in DCF fluorescence was also observed in cortical slices of MHE rats, which was inhibited by apocynin (Fig. 9d). Increased cellular HEt fluorescence was detected in cortical slices of MHE rats, and decreased cellular HEt fluorescence was detectable on addition of apocynin (Fig. 9e). DA (10 g) treatment significantly increased DCF fluorescence in cultured rat cortical slices, which was abolished by apocynin (Fig. 9f). HEt fluorescence largely colocalized with GFAP positive astrocytes in DA (10 g)-treated rat cortical slices (Fig. 9g). The increase in HEt fluorescence of DA-treated rat cortical slices was abolished by apocynin (Fig. 9g), indicating an involvement of ROS production. We also analyzed production of nitrites and nitrates. The content of nitrites and nitrates was 2.58 ± 0.5 nmol/mg protein in control rats, and addition of 10 M DA decreased nitrites and nitrates to 1.46 ± 0.1 nmol/mg protein (Fig. 9h). The content of nitrites and nitrates was decreased in cerebral cortex of MHE rats to 63 ± 3% of the control (Fig. 9i). The nitrite and nitrate level was decreased by 38 ± 3% in 10 g DA-treated rats (Fig. 9j), indicating the ineffectiveness of NO production.

4. Discussion

An increased turnover of serotonin and possibly DA in the brain has been observed in patients with fulminant hepatic failure and in encephalopathic rats (Knell et al., 1974; Yurdaydin et al., 1990; Record et al., 1976). Our previous study showed that elevated DA in the brain is involved in the pathogenesis of MHE (Ding et al., 2013). In our present study, we have demonstrated that DA induces astrocyte swelling and oxidative stress.
Recently, ammonia was identified to induce PTN in cultured astrocytes and in rat models of acute and chronic liver failure (Schliess et al., 2002). As shown in the present study, DA induced PTN in astrocytes in vitro and in vivo. We assumed that DA and ammonia may act synergistically on PTN because both compounds, when added at suboptimal concentrations, tend to strengthen the overall PTN response and contribute to the tyrosine nitration of some individual proteins in astrocytes.
Liver–brain signaling mechanisms in liver failure include altered permeability of the BBB (Butterworth, 2013). The development of acute liver failure occurs together with BBB breakdown (Martins and Daniel-Ribeiro, 2013). A number of substances such as ammonia, serotonin, bradykinin, adenosine, purine nucleotides, interleukins, free radicals, NO, and steroids may influence brain endothelium function and tightness of the BBB (Skowronska and Albrecht, 2012). In acute liver failure, inflammation-related BBB permeability changes (Chastre et al., 2013). Hepatitis C virus (HCV)related neurocognitive dysfunction has been believed to be a consequence of cirrhosis-associated HA. Recently, HCV sequences have been detected in cerebrospinal fluid and brain tissue in chronically infected individuals, suggesting that HCV might cross the BBB and could affect cognitive functions (Abdel Rahman et al., 2014). Therefore, BBB disturbances are considered to be involved in the development of liver cirrhosis. The mechanism behind impaired permeability of the BBB appears to be important for the passage of DA into the brain in the development of MHE.
Numerous studies have described specific but inconsistent effects of DA manipulation on learning. Some studies showed that learning and retention of memory, required for optimal response choice, rely significantly on DA in Alzheimer’s disease (Coulthard et al., 2012; Shiner et al., 2012). However, some studies found that antibody in cerebral cortex of DA (1 and 10 g)-treated rats following densitometry (e). (f) The activity of NOS in cerebral cortex of control and DA (1 and 10 g)-treated rats. (g) Immunoblot analysis of p47phox serine phosphorylation using an anti-phosphoserine antibody and overall p47phox using an anti-p47phox antibody in PCAs treated with in Parkinsonism or MHE, DA impairs or has no effect on stimulusresponse learning and working memory (Ding et al., 2013; Kobza et al., 2012; Moustafa et al., 2013). Similarly, in our present study, we observed that overproduction of DA is a feature of memory impairment in MHE.
There have also been numerous inconsistent reports from studies of changes in DA level in HE. Chronic HE patients exhibit abnormalities in the dopaminergic systems, including an increased brain content of DA metabolites (Bergeron et al., 1989). An increased catabolism of DA causes alteration of DA reuptake in HE (Weissenborn et al., 2000). An increased turnover of serotonin and possibly DA (Knell et al., 1974) and the accumulation of DA metabolites was found in the brain during HE (Borkowska et al., 1999) and in vivo (Borkowska et al., 1997). In our previous study, we found that memory impairment of MHE is associated with elevated DA of cirrhotic livers (Ding et al., 2013); the results in our present study were consistent. Inconsistences in various studies can be explained by an evolution of MHE to HE going along with a decrease in DA, followed by compensatory elevation of DA in liver cirrhosis with MHE.
Nitration of GS is associated with a loss of activity (Schliess et al., 2002); however, the functional impact of this for MHE is unclear. On the one hand, inhibition of GS may alleviate astrocyte swelling. On the other hand, a reduced capacity of the astrocytes for ammonia clearance could favor direct toxicity to neurons.
The mechanisms underlying PTN by DA were associated with apocynin-sensitive ROS production in vivo and in vitro. DA-induced PTN does not involve NOS activity (Fig. 5). Consistently, Cu/Zn-SOD and catalase, enzymes reducing free radical levels, significantly attenuated DA-induced PTN. The present findings suggest that NADPH oxidase-mediated ROS production represents a major route toward PTN by DA.
Further studies are required to precisely delineate the mechanism of PTN triggered by DA in astrocytes. More interestingly, certain subunits of NADPH oxidase were rapidly induced after injurious DA exposure. Furthermore, in addition to being induced, NADPH oxidase appears to be functionally activated after DA exposure. As shown in this study, serine phosphorylation of p47phox, critical for NADPH oxidase activation, was rapidly induced by DA.
In the present study, which focused on cerebral cortex tissues and cortical cultures, DA was sufficient to induce swelling of cultured astrocytes and NADPH oxidase-dependent ROS production and did not induce nNOS-dependent NO production (Fig. 9). The contribution of NADPH oxidase to DA-induced oxidative injury was directly supported by the findings that inhibitors of NADPH oxidase attenuate superoxide generation after DA exposure. Combined with the evidence that DA overload is one of the mechanisms triggering MHE (Ding et al., 2013), our study confirms the idea that the induction and activation of NADPH oxidase in astrocytes by DA overload may play a role in PTN in MHE. This again supports the idea that oxidative stress is a significant mechanism behind the neurotoxicity of DA overload. Further, astrocytic ROS may impair GS activity (Schliess et al., 2002; Görg et al., 2006; Knorpp et al., 2006), which may protect the brain from excessive glutamine accumulation and astrocyte swelling and/or increase the direct neurotoxicity of ammonia. Finally, astrocytic ROS promotes the loss of organic osmolytes (Brand et al., 1999). Osmolyte depletion predisposes astrocytes to swelling under MHE-precipitating conditions, which in cirrhotic patients is closely related to the severity of neuropsychological deterioration.
As shown in the present study, PTN was most pronounced in the perivascular area and colocalized in part with GFAP-positive cells. Expression of subunits of NADPH oxidase and ROS production in rat brain appear accentuated in astrocytes located in proximity to blood vessels, which are important constituents of the BBB. A similar localization of PTN was found in brains of acutely ammonia-loaded rats and portocaval anastomized rats (Schliess et al., 2002; Suárez et al., 2006; Vaquero et al., 2003). Astrocytes are known to be important constituents of the BBB. It is tempting to speculate that PTN, a marker of oxidative stress in perivascular astrocytes, may correspond to an altered BBB permeability, which has been observed in some animal models of HE (Bassett et al., 1990) and in patients (Lockwood et al., 1991), and perivascular astrocytes may be a major source of swelling-dependent, NADPH oxidase-catalyzed ROS production under MHE-relevant conditions. The induction-activation of NADPH oxidase because of astrocyte swelling by DA and related protein modifications such as tyrosine nitration could correspond to the known specific alterations of BBB permeability in MHE.
Together, the present results suggest that, in MHE conditions in which DA neurotoxicity contributes, the resultant NADPH oxidase induction-activation may play a significant role in causing PTN, astrocyte swelling and cognitive impairment. The question as to which outcome induced by PTN contributes to clinical symptoms of MHE cannot be answered at present. Further studies are required to settle this issue.

References

Abdel Rahman TT, Abou Hashem RM, Abdel Guaad MA, Mortagy AK. Executive functions in chronic hepatitis C virus-infected elderly patients. Geriatr Gerontol Int 2014., http://dx.doi.org/10.1111/ggi.12187.
Bassett ML, Mullen KD, Scholz B, Fenstermacher JD, Jones EA. Increased brain uptake of gamma-aminobutyric acid in a rabbit model of hepatic encephalopathy. Gastroenterology 1990;98:747–57.
Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, et al. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest 2003;112(9):1383–94.
Bender AS, Schousboe A, Reichelt W, Norenberg MD. Ionic mechanisms in glutamateinduced astrocyte swelling: role of K+ influx. J Neurosci Res 1998;52:307–21.
Bergeron M, Reader TA, Layrargues GP, Butterworth RF. Monoamines and metabolites in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Neurochemical Res 1989;14:853–9.
Bernabeu R, Schmitz P, Faillace MP, Izquierdo I, Medina JH. Hippocampal cGMP and cAMP are differentially involved in memory processing of inhibitory avoidance learning. NeuroReport 1996;7:585–8, PMID.
Bindokas VP, Jordán J, Lee CC, Miller RJ. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J Neurosci 1996;16:1324–36.
Blei AT, Larsen FS. Pathophysiology of cerebral edema in fulminant hepatic failure. J Hepatol 1999;31:771–6.
Bokoch GM, Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 2002;100:2692–6.
Borkowska HD, Oja SS, Saransaari P, Albrecht J. Release of [3H]dopamine from striatal and cerebral cortical slices from rats with thioacetamide-induced hepatic encephalopathy: different responses to stimulation by potassium ions and agonists of ionotropic glutamate receptors. Neurochem Res 1997;22:101–6.
Borkowska HD, Oja SS, Oja OS, Saransaari P, Hilgier W, Albrecht J. N-methyl-daspartate-evoked changes in the striatal extracellular levels of dopamine and its metabolites in vivo in rats with acute hepatic encephalopathy. Neurosci Lett 1999;268:151–4.
Brand A, Leibfritz D, Richter-Landsberg C. Oxidative stress-induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy. J Neurosci Res 1999;58:576–85.
Butterworth RF. The liver-brain axis in liver failure: neuroinflammation and encephalopathy. Nat Rev Gastroenterol Hepatol 2013;10:522–8, http://dx.doi.org/10.1038/nrgastro.2013.99.
Chastre A, Belanger M, Nguyen BN, Butterworth RF. Lipopolysaccharide precipitates hepatic encephalopathy and increases blood-brain barrier permeability in mice with acute liver failure. Liver Int 2013., http://dx.doi.org/10.1111/liv.12252.
Colado MI, Ormazabal MJ, Alfaro MJ, Martin MI. Effect of Bay K 8644 on the synthesis and metabolism of dopamine and 5-hydroxytryptamine in various brain areas of the rat. J Pharm Pharmacol 1993;45:220–2.
Coulthard EJ, Bogacz R, Javed S, Mooney LK, Murphy G, Keeley S, et al. Distinct roles of dopamine and subthalamic nucleus in learning and probabilistic decision making. Brain 2012;135:3721–34.
Dhiman RK, Chawla YK. Minimal hepatic encephalopathy. Indian J Gastroenterol 2009;28:5–16, http://dx.doi.org/10.1007/s12664-009-0003-6.
Ding SD, Liu LP, Jing HJ, Xie JY, Wang XB, Mao JP, et al. Dopamine from cirrhotic liver contributes to the impaired learning and memory ability of hippocampus in minimal hepatic encephalopathy. Hepatol Int 2013;7:923–36, http://dx.doi.org/10.1007/s12072-12013-19431-12076.
Dombro RS, Bender AS, Norenberg MD. Association between cell swelling and glycogen content in cultured astrocytes. Int J Dev Neurosci 2000;18:161–9.
Gill TH, Young OM, Tower DB. The uptake of 36C1 into astrocytes in tissue culture by a potassium-dependent, saturable process: implications for epilepsy and cortical edema. Trans Am Neurol Assoc 1973;98:262–4.
Goel A, Yadav S, Saraswat V, Srivastava A, Thomas MA, Pandey CM, et al. Cerebral oedema in minimal hepatic encephalopathy due to extrahepatic portal venous obstruction. Liver Int 2010;30:1143–51, http://dx.doi.org/10.1111/j.1478-3231.2010.02289.x.
Görg B, Foster N, Reinehr R, Bidmon HJ, Höngen A, Häussinger D, et al. Benzodiazepine-induced protein tyrosine nitration in rat astrocytes. Hepatology 2003;37:334–42.
Görg B, Bidmon HJ, Keitel V, Foster N, Goerlich R, Schliess F, et al. Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes. Arch Biochem Biophys 2006;449:104–14.
Görg B, Schliess F, Häussinger D. Osmotic and oxidative/nitrosative stress in ammonia toxicity and hepatic encephalopathy. Arch Biochem Biophys 2013;536:158–63, http://dx.doi.org/10.1016/j.abb.2013.03.010.
Groemping Y, Lapouge K, Smerdon SJ, Rittinger K. Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 2003;113: 343–55.
Häussinger D. Low grade cerebral edema and the pathogenesis of hepatic encephalopathy in cirrhosis. Hepatology 2006;43:1187–90.
Hayakata T, Shiozaki T, Tasaki O, Ikegawa H, Inoue Y, Toshiyuki F, et al. Changes in CSF S100B and cytokine concentrations in early-phase severe traumatic brain injury. Shock 2004;22:102–7.
Ichihara K, Nabeshima T, Kameyama T. Differential effects of pimozide and SCH 23390 on acquisition of learning in mice. Eur J Pharmacol 1989;164:189–95.
Itoh J, Nabeshima T, Kameyama T. Utility of an elevated plus-maze for the evaluation of memory in mice: effects of nootropics, scopolamine and electroconvulsive shock. Psychopharmacology (Berl) 1990;101:27–33.
Jia L, Zhang MH. Comparison of probiotics and lactulose in the treatment of minimal hepatic encephalopathy in rats. World J Gastroenterol 2005;11:908–11.
Johnson JL, Park JW, Benna JE, Faust LP, Inanami O, Babior BM. Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity. J Biol Chem 1998;273:35147–52.
Kawasumi M, Chiba T, Yamada M, Miyamae-Kaneko M, Matsuoka M, Nakahara J, et al. Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer’s disease in aged mice. Eur J Neurosci 2004;19:2826–38.
Kiedrowski L, Costa E, Wroblewski JT. Glutamate receptor agonists stimulate nitric oxide synthase in primary cultures of cerebellar granule cells. J Neurochem 1992;58:335–41.
Kletzien RF, Pariza MW, Becker JE, Potter VR. A method using 3-O-methyl-d-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture. Anal Biochem 1975;68:537–44.
Knell AJ, Davidson AR, Williams R, Kantamaneni BD, Curzon G. Dopamine and serotonin metabolism in hepatic encephalopathy. Br Med J 1974;1:549–51.
Knorpp T, Robinson SR, Crack PJ, Dringen R. Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress. J Neural Transm 2006;113:1145–55.
Kobza S, Ferrea S, Schnitzler A, Pollok B, Sudmeyer M, Bellebaum C. Dissociation between active and observational learning from positive and negative feedback in Parkinsonism. PLoS ONE 2012;7:e50250.
Kruczek C, Görg B, Keitel V, Pirev E, Kröncke KD, Schliess F, et al. Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes. Glia 2009;57:79–92, http://dx.doi.org/10.1002/glia.20737.
Lachmann V, Görg B, Bidmon HJ, Keitel V, Häussinger D. Precipitants of hepatic encephalopathy induce rapid astrocyte swelling in an oxidative stress dependent manner. Arch Biochem Biophys 2013;536:143–51, http://dx.doi.org/10.1016/j.abb.2013.05.004.
Laird MD, Sukumari-Ramesh S, Swift AE, Meiler SE, Vender JR, Dhandapani KM. Curcumin attenuates cerebral edema following traumatic brain injury in mice: a possible role for aquaporin-4? J Neurochem 2010;113:637–48, http://dx.doi.org/10.1111/j.1471-4159.2010.06630.x [Epub 2010 Jan 20].
Lockwood AH, Yap EW, Wong WH. Cerebral ammonia metabolism in patients with severe liver disease and minimal hepatic encephalopathy. J Cereb Blood Flow Metab 1991;11:337–41.
Mamiya T, Noda Y, Nishi M, Takeshima H, Nabeshima T. Enhancement of spatial attention in nociceptin/orphanin FQ receptor-knockout mice. Brain Res 1998;783:236–40.
Martinez-Hernandez A, Bell KP, Norenberg MD. Glutamine synthetase: glial localization in brain. Science 1977;195:1356–8.
Martins YC, Daniel-Ribeiro CT. A new hypothesis on the manifestation of cerebral malaria: the secret is in the liver. Med Hypotheses 2013;81:777–83, http://dx.doi.org/10.1016/j.mehy.2013.08.005.
Montgomery JY, Bajaj JS. Advances in the evaluation and management of minimal hepatic encephalopathy. Curr Gastroenterol Rep 2011;13:26–33, http://dx.doi.org/10.1007/s11894-010-0150-z.
Moustafa AA, Herzallah MM, Gluck MA. Dissociating the cognitive effects of levodopa versus dopamine agonists in a neurocomputational model of learning in Parkinson’s disease. Neuro-Degen Dis 2013;11:102–11.
Muijsers RB, van Den Worm E, Folkerts G, Beukelman CJ, Koster AS, Postma DS,et al. Apocynin inhibits peroxynitrite formation by murine macrophages. Br J Pharmacol 2000;130:932–6.
Nauseef WM. Assembly of the phagocyte NADPH oxidase. Histochem Cell Biol 2004;122:277–91.
Norenberg MD. Astrocyte responses to CNS injury. J Neuropathol Exp Neurol 1994;53:213–20.
Norenberg MD. Astrocytic–ammonia interactions in hepatic encephalopathy. Semin Liver Dis 1996;16:245–53.
Norenberg MD, Baker L, Norenberg LO, Blicharska J, Bruce-Gregorios JH, NearyJT. Ammonia-induced astrocyte swelling in primary culture. Neurochem Res 1991;16:833–6.
Norenberg MD, Rao KV, Jayakumar AR. Mechanisms of ammonia-induced astrocyte swelling. Metab Brain Dis 2005;20:303–18.
Pani G, Bedogni B, Colavitti R, Anzevino R, Borrello S, Galeotti T. Cell compartmentalization in redox signaling. IUBMB Life 2001;52:7–16.
Park JW, Babior BM. Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox. Biochemistry 1997;36:7474–80.
Prakash RK, Kanna S, Mullen KD. Evolving concepts: the negative effect of minimal hepatic encephalopathy and role for prophylaxis in patients with cirrhosis. Clin Ther 2013;35:1458–73, http://dx.doi.org/10.1016/j.clinthera.2013.07.421.
Qi R, Zhang LJ, Zhong J, Zhu T, Zhang Z, Xu C, et al. Grey and white matter abnormalities in minimal hepatic encephalopathy: a study combining voxel-based morphometry and tract-based spatial statistics. Eur Radiol 2013;23:2370–8.
Record CO, Buxton B, Chase RA, Curzon G, Murray-Lyon IM, Williams R. Plasma and brain amino acids in fulminant hepatic failure and their relationship to hepatic encephalopathy. Eur J Clin Invest 1976;6:387–94.
Reinehr R, Becker S, Eberle A, Grether-Beck S, Häussinger D. Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. J Biol Chem 2005;280:27179.
Reinehr R, Görg B, Becker S, Qvartskhava N, Bidmon HJ, Selbach O, et al. Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices. Glia 2007;55:758–71.
Schliess F, Görg B, Fischer R, Desjardins P, Bidmon HJ, Herrmann A, et al. Ammonia induces MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes. FASEB J 2002;16:739–41.
Schliess F, Foster N, Görg B, Reinehr R, Häussinger D. Hypoosmotic swelling increases protein tyrosine nitration in cultured rat astrocytes. Glia 2004;47: 21–9.
Shiner T, Seymour B, Wunderlich K, Hill C, Bhatia KP, Dayan P, et al. Dopamine and performance in a L-NMMA reinforcement learning task: evidence from Parkinson’s disease. Brain 2012;135:1871–83.
Skowronska M, Albrecht J. Alterations of blood brain barrier function in hyperammonemia: an overview. Neurotox Res 2012;21:236–44, http://dx.doi.org/10.1007/s12640-011-9269-4.
Suárez I, Bodega G, Arilla E, Felipo V, Fernández B. The expression of nNOS, iNOS and nitrotyrosine is increased in the rat cerebral cortex in experimental hepatic encephalopathy. Neuropathol Appl Neurobiol 2006;32:594–604.
Torres DS, Abrantes J, Brandão-Mello CE. Cognitive assessment of patients with minimal hepatic encephalopathy in Brazil. Metab Brain Dis 2013;28:473–83, http://dx.doi.org/10.1007/s11011-013-9405-3.
Vaquero J, Chung C, Cahill ME, Blei AT. Pathogenesis of hepatic encephalopathy in acute liver failure. Semin Liver Dis 2003;23:259–69.
Verdon CP, Burton BA, Prior RL. Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. Anal Biochem 1995;224:502–8.
Vignais PV. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci 2002;59:1428–59.
Weissenborn K, Berding G, Kostler H. Altered striatal dopamine D2 receptor density and dopamine transport in a patient with hepatic encephalopathy. Metab Brain Dis 2000;15:173–8.
Willard-Mack CL, Koehler RC, Hirata T, Cork LC, Takahashi H, Traystman RJ, et al. Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat. Neuroscience 1996;71:589–99.
Yamada M, Chiba T, Sasabe J, Nawa M, Tajima H, Niikura T, et al. Implanted cannula-mediated repetitive administration of Abeta25-35 into the mouse cerebral ventricle effectively impairs spatial working memory. Behav Brain Res 2005;164:139–46.
Yurdaydin C, Hörtnagl H, Steindl P, Zimmermann C, Pifl C, Singer EA, et al. Increased serotoninergic and noradrenergic activity in hepatic encephalopathy in rats with thioacetamide-induced acute liver failure. Hepatology 1990;12:695–700.