2.1 MHE models and treatment
A total of 40 Sprague-Dawley (SD) rats (220–250 g) were used, and all guidelines of the care and use of animals for experimental procedures were laid down by the Ethics Committees of the Affiliated Hospital of Wenzhou Medical University regarding .
Before experimenting, all animals were subjected to behavioural tests: Y-maze (YM) and water-finding task (WFT). Normal values of YM and WFT these behavioural tests were obtained. Rats were then intraperitoneally (i.p.) injected with TAA (200 mg/kg in normal saline, Sigma-aldrich) to induce liver cirrhosis twice per week for 8 weeks. TAA-treated rats with symptoms of decreased movement, lethargy, and coma were diagnosed as HE. Then behavioral tests were conducted for TAA-treated rats with no HE symptoms. If values of YM were decreased or values of WFT were increased compared to mean ± 1.96·SD of normal values, rats were included in the MHE group.
MHE Rats were anaesthetized with pentobarbital (40 mg/kg, i.p.) and subjected to stereotaxic surgery. Two 23-gauge, stainless-steel, thin-wall cannulae were implanted to the right lateral ventricles at the following coordinates: 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 2.0 mm from the surface of the skull. MHE rats were injected with DA (10 µg / 3 µl) 3 times per week for 1 week through guide cannula, following by the intraperitoneal injection with β-asarone (βASA, 20 µg) 3 times per week for 1 week. Then Rats were conducted with a YM and a WFT test.
2.2 Behavioral test
Rats were individually placed at the end of one of arm of the three arms in the apparatus for YM, and were allowed to find the arm entries freely for 8 min. Total arm entries and spontaneous alternation percentage (SA%, a ratio of successful choices to total choices minus two) were measured .
Rats were deprived of water for 24 h and individually placed at the near-right corner of the testing apparatus for WFT and allowed to explore the tube and drink the water in the cubic alcove freely for 3 min. The latency times of the first entry into the alcove (entry latency, EL), of the first touching/sniffing/licking of the water tube (contacting latency, CL) and of the initiation of drinking from the water tube (drinking latency, DL) were measured .
2.3 Primary rats neurons culture and treatments
Primary hippocampal rats neurons (PHNs) were obtained from freshly dissected hippocampus and cerebral cortex from 1-day-old SD rat pups by mechanical disruption in the presence of trypsin and DNase and then seeded in poly-L-lysine-precoated six-well plates at a density of 2 × 106 cells/well in Neurobasal® Medium (1X) supplemented with 0.5 mM GlutaMAX™-I, B-27®. Then neurons remained untreated or were stimulated with DA (1, 10 or 20 µmol/l) alone or after preincubation with JNK1 inhibitor SP600125, ASK1 inhibitor K811, 50 µM Notch inhibitor MK-0752, 100 ng/ml Notch activator Jagged-1 or anti-TNFα antibody (ATAB), or treated with βASA in the presence or absence of DA together with MK-0752/TNFα.
2.4 PC12 cells culture and treatments
The rat pheochromocytoma PC12 cell line were achieved from the American Type Culture Collection (ATCC, Manassas, VA). PC12 cells were routinely cultured in DMEM with 2 mM glutamine, 10% horse serum, and 5% heat-inactivated fetal bovine serum (FBS) and passaged one time per week. Then PC12 cells remained untreated or were stimulated with DA (1, 10 or 20 µmol/l) alone or after preincubation with JNK1 inhibitor SP600125, ASK1 inhibitor K811, 50 µM Notch inhibitor MK-0752, 100 ng/ml Notch activator Jagged-1 or anti-TNFα antibody (ATAB), or treated with βASA in the presence or absence of DA together with MK-0752/TNFα.
2.5 Transfection for PC12 cells
PC12 cells were transfected with Notch1/JNK1/ASK1/TNFα/ the Silencer Negative Control siRNA (Scrambled siRNA) (Santa Cruz, CA, USA) or Notch cDNA (activated) /empty vector plasmid (Millipore) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the instructions suggested by the manufacture.
2.6 Measurement of TNFα release
TNFα level in the culture medium of neurons was measured using enzyme-linked immunosorbent assay (ELISA) kit (R&D systems) in the light of the manufacturer’s protocols and a Thermo-Fisher Multiskan MCC plate reader.
2.7 Immunoblotting (IB) analysis
The total amount of protein in the lysates were separated by 10% SDS-PAGE and electroblotted to PVDF membrane, the membrane was blocked with 5% non-fat dry milk dissolved in TBS-T (150 mM NaCl, 50 mM Tris, 0.05% Tween 20), and incubated with following primary antibodies: DA, TNFα, GDNF, NGF, Apoptosis signal-regulating kinase1 (ASK1), c-Jun N-Terminal Kinase1 (JNK1), pASK1, pJNK1, Notch Intracellular Domain1 (NICD1), Hairy and enhancer of split1 (HES1), Homer, synaptotagmin, β-actin (Abcam) overnight at 4℃, then probed with horseradish peroxidase-conjugated secondary antibody for 1 h. After extensive washing, membrane was visualized by ECL reagent (Thermo) and exposed on Kodak BioMax film (Kodak). Intensities of protein bands were analysed with Quantity One software. Densitometry was indicated as ratio of proteins to β-actin levels.
2.8 Functional labeling of presynaptic boutons with FM4-64
FM4-64 staining (Invitrogen) was followed by the manufacturer’s instructions. Briefly, neurons were incubated with 5 mg/ml FM4-64 and 50 mM KCl-contained Hank’s balanced salt solution for 1 minute at 4 ℃.
2.9 Double-labeled fluorescent staining
Frozen brain sections or neurons cultured on glass coverslips were fixed with 4% paraformaldehyde for 30 min and treated with 0.1% Triton X-100 for 10 min, then blocked with bovine serum albumin (BSA) for 1 h. Sections were then incubated with the following primary antibodies: DA, TNFα, GDNF, NGF, pASK1, pJNK1, NICD1, HES1, MAP2 (Abcam) overnight at 4 ℃, following with FITC (green)/Alexa Fluor 594 (red) conjugated secondary antibody for 30 min. A Leica TCS SP2 confocal laser scanning microscope was used for imaging.
2.10 Dendritic spine density analysis in primary neurons
After fixation, neurons cultured on glass coverslips were incubated with primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA), and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA), then with 1-hour secondary antibody-conjugated AlexaFluor (1:500; Life Technologies, Waltham, MA, USA). A z stack of optical section was imaged on a confocal laser scanning microscope (FV10i-W, Olympus, Japan).
2.11 Statistical analysis
All of the data were expressed as mean ± SD. Data comparisons were analyzed using one-way analysis of variance (ANOVA). All of the data were detected in normal distribution and equal variances, when Back-of-the-envelope test was used to verify the normal distribution, and F-test was applied for determining the equality of variances. Dunnett’s post hoc multiple comparison test was applied when significant differences were detected by the ANOVA model. Then P values were made for adjustment by Bonferroni correction. The level of significance was determined for P < 0.05 or P < 0.01. All analyses were performed with SPSS 18.0 (PASW Statistics 18.0).