MHE models and treatment
A total of 40 Sprague-Dawley (SD) rats (Experimental Animal Center of the Chinese Academy of Sciences in Shanghai, China), weighing 220–250 g, were housed under controlled conditions of temperature (24 ± 1°C) and light (12 h light starting at 07:00 a.m.). All experiments were carried out in accordance with the guidelines laid down by the Ethics Committees of the Affiliated Hospital of Wenzhou Medical University regarding the care and use of animals for experimental procedures [11].
Before experiments, all animals underwent a series of behavioral tests: Y-maze (YM) and water-finding task (WFT). The normal values of these behavioral tests were obtained. Then, the rats were then randomly divided into two groups: control (n = 20) and thioacetamid (TAA) (n = 30). Liver cirrhosis was induced by intraperitoneal injection (i.p.) of TAA (200 mg/kg in normal saline, Sigma–Aldrich,Darmstadt, Germany) twice per week for 8 weeks. TAA-treated rats with symptoms were diagnosed as HE. The symptoms of HE were as follows: a subsequent decrease in motor activity, lethargy, and an eventual progression to coma. TAA-treated rats with no hepatic encephalopathy (HE) symptoms were again subjected to behavioral tests to confirm MHE. If TAA-treated rats met the criteria: a) values of YM were lower than mean ± 1.96 (SD); b) values of WFT were more than mean ± 1.96 (SD), the animals were included in the MHE group.
MHE rats were microinjected with TSP1 overexpression plasmid into hippocampus for 24 h.
YM test
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 the 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 two entries could not be evaluated) [12].
WFT test
A rat was placed at the near-right corner of the apparatus and allowed to explore freely for 3 min. The rats that could not find the tube within 3 min were omitted from the analysis. After the training session, the animals were deprived of water for 24 h. In the trial session, they were again individually placed at the same corner of the apparatus and allowed to find and drink the water in the alcove. The elapsed time until the entry into the alcove (entry latency, EL) until the touching/sniffing/licking of the water tube (contacting latency, CL) and until the initiation of drinking from the water tube (drinking latency, DL) was measured [13].
TSP1 knockdown mice
WT (wild-type) mice (Balb/c) were anesthetized with intramuscular xylazine (16 mg/kg), followed by ketamine (100 mg/kg). TSP1 small interfer ribonucleic acid (siRNA) was (10 or 40 µg/5µL) injected into the left hippocampus of mice for 24 h (anterior -posterior, + 0.3 mm; lateral, 1.0 mm; horizontal, 3.0 mm from the bregma) (n = 15).
Primary hippocampal neurons culture
Primary hippocampal rats neurons (PHNs) were prepared from 1-day-old SD rat pups and dissociated from freshly dissected hippocampus or cerebral cortex by mechanical disruption in the presence of trypsin and DNase and then plated in poly-L-lysine-precoated six-well plates. The cells were seeded at a density of 2×106 cells/well in Neurobasal® Medium (1X) supplemented with 0.5 mM GlutaMAX™-I and B-27® and incubated at 37°C, 5% CO2 .
PC12 cell culture
The rat pheochromocytoma PC12 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM with 2 mM glutamine, 10% horse serum, and 5% heat-inactivated fetal bovine serum (FBS) that was passaged one time per week.
Cells treatment
The cells were stimulated with TSP1 (1, 5, or 20 µg/mL), human Wnt7a (100 ng/ml), human TNFα (20 ng/ml), Wnt inhibitor XAV939 (1 µmol/L), p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 (SB, 50 µmol/L), p38MAPK activator anisomycin (Ani, 10 µmol/L), and TNFα inhibitor lenalidomide (len, 50 µmol/L) for 3 h. Wells received 0.25 µg of p38/TNFα/Wnt7a/CNTF Lentiviral Activation Particles (Santa Cruz, CA, USA) along with 10 ng of the control plasmid pCMV-Tag2A for normalization of transfection efficiency.
Each well of cells received 0.25 µg of p38/TNFα/Wnt7a/CNTF siRNA (Santa Cruz, CA, USA) using Lipofectamine 2000 reagent (Invitrogen,Carlsbad, CA,USA), according to the manufacturer’s protocol. An equivalent of 10 ng of The Silencer Negative Control number 1 siRNA (scrambled siRNA) was used as a control (Santa Cruz).
Real-time quantitative PCR (RT/q-PCR)
For RT-PCR, total RNA was isolated using the Qiagen RNA-Easy kit according to the manufacturer’s protocol. Complementary deoxyribonucleic acid (cDNA) was synthesized using oligo (dT), deoxy-ribonucleoside triphosphate (dNTP), 0.1 M Dithiothreitol (DTT), Moloney murine leukemia virus reverse transcriptase, RNaseOUT (all from Invitrogen). The amplification was carried out using the PCR Master Mix (Promega,Wisconsin, USA). The amplified products were analyzed by electrophoresis on 2% agarose gels, visualized by EtBr staining, and quantitated against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qPCR was performed on the ABI-Prism7700 sequence detection system (Applied Biosystems༌ Foster City, CA) using iTaq™ Fast Supermix with ROX (Bio-Rad༌Hercules, CA, USA) and 6-carboxyfluorescein-labeled TSP1, p38, TNFα, Wnt7a, CNTF, β-catenin, and GAPDH primers (Integrated DNA Technologies, Coralville, IA, USA). The mRNA expression was analyzed using the relative 2−∆∆CT method. The following primers (Invitrogen) for murine genes:
TSP1, Forward: 5’- TTGCCAGCGTTGCCA-3’,
Reverse: 5’- TCTGCAGCACCCCCTGAA-3’;
P38, Forward: 5’-AGTGGCTGACCCTTATGAC-3’,
Reverse: 5’-CACAGTGAAGTGGGATGGA-3’;
TNFα, Forward: 5’-AGGTCTACTTTGGAGTCATTG − 3’,
Reverse: 5’- TTCTGAGCAT-CGTAGTTGTTG − 3’;
Wnt7a, Forward: 5’- CTGTGGCTGCGACAAAGAGAA-3’,
Reverse: 5’- GCCGTGGCACTTACATTCC-3’;
CNTF, Forward: 5’-AGGAAGATTCGTTCAGACCT-3’,
Reverse: 5’-CCAGTGGCAAGCACTGATC-3’;
GAPDH, Forward: 5’-TGTCATCAACGGGAAGCCCA-3’,
Reverse: 5’-TTGTCATGGATGACCTTGGC-3’.
Measurement of TNFα/Wnt7a/CNTF release
Extracellular TNFα/Wnt7a/CNTF levels in the culture medium of primary neurons was measured using enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Plates were analyzed spectrophotometrically on a Thermo-Fisher Multiskan MCC plate reader(Thermo Fisher Scientific, Waltham, MA).).
Immunoblotting (IB) analysis
The total amount of protein in the lysates was determined by the BCA protein assay (Amresco,USA). An equivalent of 50 µg protein was resolved by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and electroblotted to Polyvinylidene Fluoride (PVDF) membrane that was blocked with 5% non-fat dry milk in Tris buffer saline and Tween 20 (TBST) (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Subsequently, the membranes were probed with primary antibodies, TSP1, p38MAPK, TNFα, TNFR1, Wnt7a, frizzled2 (FZD2), CNTF, CNTFR, spinophilin, and β-actin (Abcam༌Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody(Abcam, Cambridge, UK). After extensive washing, the immunoreactive bands were visualized by ECL reagent (Thermo༌USA) after exposure on Kodak BioMax film (Kodak). The band intensities were quantified using QuantityOne software. The intensities were expressed as fold-change relative to the GAPDH levels.
For coimmunoprecipitation, the lysates of tissues were incubated with antibodies overnight (4°C) and subsequently with protein G-agarose beads (Millipore, USA) for 5 h (4°C). Followed by washes with lysis buffer, the eluent was separated by SDS-PAGE and electroblotted to PVDF membrane using primary and secondary antibodies.
Dendritic spine density analysis in primary neurons
For dendritic spine analysis, we used immunocytochemistry with anti-microtubule-associated protein 2 (anti-MAP2) and anti-vesicular glutamate transporter 1 antibodies. After fixation, the primary antibodies used were 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). Primary antibodies were applied overnight at 4°C, followed by incubation with AlexaFluor-conjugated secondary antibody for 1 h (1:500; Life Technologies, Waltham, MA, USA) and glass cover slipping. A z stack of the optical sections was visualized on a confocal laser scanning microscope (FV10i-w; Olympus Corporation, Tokyo, Japan). At least 10 cultured neurons from two batches of cultures per group were used for quantitative analysis.
Functional labeling of presynaptic boutons with FM4-64
FM4-64 staining (Invitrogen) was performed according to the manufacturer’s instructions. Briefly, primary neurons were incubated with 5 mg/mL FM4-64 (Invitrogen) and 50 mM KCl in Hank’s balanced salt solution for 1 min at 4°C, and then washed with the solution to remove free FM4-64.
Double-labeled fluorescence staining
Frozen brain sections or neurons (4mm) cultured on glass coverslips were fixed with 4% paraformaldehyde for 30min and then treated with 0.1% Triton X-100 for 10 min at room temperature. Subsequently, the sections were blocked with 5% normal goat serum in phosphate-buffered saline (PBS) for 1 h at room temperature. Then, the sections were incubated overnight at 4°C with the following primary antibodies: TSP1, TNFR1, FZD2, CNTFR, spinophilin, and MAP2 (Abcam) that were detected by incubation for 30 min with fluoresceine isothiocyanate (FITC) (green)/Alexa Fluor 594 (red)-conjugated secondary antibody. The images were captured using a Leica TCS SP2 (Leica Microsystems, Heerbrugg, Switzerland) confocal laser scanning microscope. The imaging data were analyzed and quantified using Image Pro Plus software.
Electrophysiological Analysis
Rats were anesthetized with isoflurane, decapitated, and the hippocampi were cut into 400-mm thick transverse slices with a vibratome. After incubation at room temperature in a-CSF for 60–90 min, the slices were placed in a recording chamber on the stage of an upright microscope (Olympus CX-31) and perfused at a rate of 3 mL/min with a-CSF (containing 1 mM MgCl2) at 23–24°C. A 0.1-MΩ tungsten monopolar electrode was used to stimulate the Schaffer collaterals. The field excitatory postsynaptic potentials (fEPSPs) were recorded in CA1 stratum radiatum by a glass microelectrode filled with a-CSF with resistance of 3–4 MΩ. Field potential input-output curves were constructed by measuring fEPSP slope responding to the stimulus intensity increased from 1 to 7 V, with a 0.5 V increment. The long-term potentiation (LTP) of fEPSPs was induced by three theta-burst stimulations (TBSs), i.e., four pulses at 100 Hz, repeated three times at a 200-ms interval. Paired-pulse facilitation (PPF) was examined by applying pairs of pulses, separated by 20–500-ms intervals. The magnitudes of LTP were expressed as the mean percentage of the baseline fEPSP initial slope.
Statistical analysis
Data are presented as mean ± SD. The statistical significance between-group comparisons was determined by one-way analysis of variance (ANOVA). P < 0.05 or P < 0.01 was considered to be statistically significant.