The International Journal of Biochemistry & Cell Biology 55 (2014) 252–263
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Elevated dopamine induces minimal hepatic encephalopathy by activation of astrocytic NADPH oxidase and astrocytic protein tyrosine nitration Saidan Ding a,1 , Jianjing Yang b,1 , Leping Liu a , Yiru Ye c , Xuebao Wang d , Jiangnan Hu b , Bicheng Chen a , Qichuan Zhuge b,∗ a Zhejiang Provincial Key Laboratory of Aging and Neurological Disease Research, Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China b Neurosurgery Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c Department of Computer, Wen Zhou Medical University, Wenzhou 325000, China d Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325000, China
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 14 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Minimal hepatic encephalopathy Dopamine Protein tyrosine nitration NADPH oxidase
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
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,
∗ Corresponding author at: The First Affiliated Hospital of Wenzhou Medical University, Shangcai Village, Wenzhou City, Zhejiang Province, China. Tel.: +86 13676768666. E-mail address: fi[email protected] (Q. Zhuge). 1 Saidan Ding and Jianjing Yang contributed equally to this work. http://dx.doi.org/10.1016/j.biocel.2014.09.003 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
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
S. Ding et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 252–263
(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; Pani et 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.
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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 × 30 cm, 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.
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S. Ding et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 252–263
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 75 cm2 tissue culture flasks at a concentration of 15 × 106 cells in 11 ml of 1% serumcontaining DMEM/F12 medium, incubating for 72 h. 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 75 cm2 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) Na2 S2 O5 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-[3 H]-glucose (OMG) method as described previously (Kletzien et al., 1975) (Norenberg et al., 1991). Briefly, cultured astrocytes were incubated with [3 H] OMG (1 mM 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 NO2 Tyr 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: NO2 Tyr (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-NO2 Tyr 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-NO2 Tyr (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-NO2 Tyr 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 10 l of protein A- and 10 l 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).
S. Ding et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 252–263
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% H2 O2 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 (NO2 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.
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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
2.14. Determination of nitric oxide synthase (NOS) activity in PCAs The conversion of [14 C] arginine to [14 C] citrulline was determined as described by Kiedrowski et al. (1992). Ten days after seeding, PCAs were washed twice with Locke’s solution without magnesium and [14 C] arginine (1.7 M, 0.25 Ci) 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 H3 ClO4 . After centrifugation, [14 C] citruline was determined in the supernatant and protein in the pellet. [14 C] Citruline was separated from [14 C] arginine by chromatography using Dowex AG50WX-8 (Na+ form) column. For each sample, a blank control treated with 100 M nitroarginine to inhibit NOS was conducted. NOS activity is expressed as the difference between [14 C] citruline formed in absence and presence of nitroarginine.
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
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 KH2 PO4 , 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 [14 C] arginine (1.7 M, 0.25 Ci) was added. After 5 min, 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 H3 ClO4 . After centrifugation (14,000× g, 5 min) [14 C] citruline was determined for astrocytes. 2.16. Detection of ROS production Slices or PCAs were incubated with 5 M of 5-(and-6)-carboxy20,70 dichlorodihydrofluorescein diacetate (Carboxy-H2 DCFDA) 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-H2 DCFDA, slices were transferred on the inverted fluorescence microscope (Zeiss) and mounted by fixation with a platinum wire. DCF-fluorescence was measured at room
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
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Elevated dopamine induces minimal hepatic encephalopathy by activation of astrocytic NADPH oxidase and astrocytic protein tyrosine nitration Saidan Ding a,1 , Jianjing Yang b,1 , Leping Liu a , Yiru Ye c , Xuebao Wang d , Jiangnan Hu b , Bicheng Chen a , Qichuan Zhuge b,∗ a Zhejiang Provincial Key Laboratory of Aging and Neurological Disease Research, Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China b Neurosurgery Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c Department of Computer, Wen Zhou Medical University, Wenzhou 325000, China d Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325000, China
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 14 August 2014 Accepted 1 September 2014 Available online 8 September 2014 Keywords: Minimal hepatic encephalopathy Dopamine Protein tyrosine nitration NADPH oxidase
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
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,
∗ Corresponding author at: The First Affiliated Hospital of Wenzhou Medical University, Shangcai Village, Wenzhou City, Zhejiang Province, China. Tel.: +86 13676768666. E-mail address: fi[email protected] (Q. Zhuge). 1 Saidan Ding and Jianjing Yang contributed equally to this work. http://dx.doi.org/10.1016/j.biocel.2014.09.003 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
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
S. Ding et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 252–263
(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; Pani et 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.
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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 × 30 cm, 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.
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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 75 cm2 tissue culture flasks at a concentration of 15 × 106 cells in 11 ml of 1% serumcontaining DMEM/F12 medium, incubating for 72 h. 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 75 cm2 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) Na2 S2 O5 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-[3 H]-glucose (OMG) method as described previously (Kletzien et al., 1975) (Norenberg et al., 1991). Briefly, cultured astrocytes were incubated with [3 H] OMG (1 mM 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 NO2 Tyr 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: NO2 Tyr (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-NO2 Tyr 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-NO2 Tyr (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-NO2 Tyr 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 10 l of protein A- and 10 l 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).
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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% H2 O2 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 (NO2 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.
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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
2.14. Determination of nitric oxide synthase (NOS) activity in PCAs The conversion of [14 C] arginine to [14 C] citrulline was determined as described by Kiedrowski et al. (1992). Ten days after seeding, PCAs were washed twice with Locke’s solution without magnesium and [14 C] arginine (1.7 M, 0.25 Ci) 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 H3 ClO4 . After centrifugation, [14 C] citruline was determined in the supernatant and protein in the pellet. [14 C] Citruline was separated from [14 C] arginine by chromatography using Dowex AG50WX-8 (Na+ form) column. For each sample, a blank control treated with 100 M nitroarginine to inhibit NOS was conducted. NOS activity is expressed as the difference between [14 C] citruline formed in absence and presence of nitroarginine.
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
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 KH2 PO4 , 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 [14 C] arginine (1.7 M, 0.25 Ci) was added. After 5 min, 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 H3 ClO4 . After centrifugation (14,000× g, 5 min) [14 C] citruline was determined for astrocytes. 2.16. Detection of ROS production Slices or PCAs were incubated with 5 M of 5-(and-6)-carboxy20,70 dichlorodihydrofluorescein diacetate (Carboxy-H2 DCFDA) 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-H2 DCFDA, slices were transferred on the inverted fluorescence microscope (Zeiss) and mounted by fixation with a platinum wire. DCF-fluorescence was measured at room
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
