Animal
In the present study, we used adult male wild-type C57BL/6J, Fos-cre ERT2 (TRAP2), Gad2-ires-cre, GFP-Mito tagfloxed, and Gad2-Mito-GFP mice. C57BL/6J mice were purchased from the Fourth Military Medical University Animal Center (Xi’an, China). All mice were housed in a 12-hour light (8am)/dark (8pm) environment. To further control for differences due to animal sex differences, we used male mice throughout the study. All mice were randomly assigned to each group. All experiments in this study were performed in accordance with the ethical guidelines of the International Association for the Study of Pain, and approved by the Pain Research Committee of the Fourth Military Medical University. All mice were randomly assigned to the experimental groups.
Fos-cre ERT2 (TRAP2) mice were purchased from Jackson Laboratory (Fostm2.1(icre/ERT2) Luo/J; stock #030323), which was designed to have expression of a tamoxifen(TM) - inducible, improved Cre recombinase (icre/ERT2) from the Fos promoter/enhancer elements, without disrupting endogenous Fos expression. TRAP2 mice were a useful Cre-lox tool allowing inducible iCre recombination in Fos-expressing cells/tissues and Fos positive expression (Fos+) represented the neurons activated by specific stimuli. This TRAP2 mice, combined with the infection by viral vectors containing double floxed sites (DIO) (AAV8-hSyn-DIO-mCherry) marked by mCherry fluorescence could make Fos + neurons expressing mCherry.
Gad2-ires-cre mice were purchased from Jackson Laboratory, and had an internal ribosome entry site and cre recombinase in the 3' UTR of the glutamic acid decarboxylase 2 locus (Gad2). As such, the endogenous Gad2 promoter/enhancer elements direct cre expression to Gad2 positive neurons, which represented GABAergic population in the central nervous system. When the Gad2-ires-cre mice were infected with viral vector containing double-floxed inverse open reading frames (DIO) including pAAV-DIO-mCherry-NC-WPRE (Ucp2 NC); pAAV-DIO-mCherry-shRNA(Ucp2)-WPRE (Ucp2 KD); pAAV-DIO-mCherry-P2A-Ucp2-WPRE (Ucp2 OE), the Gad2-expressing GABA population could achieve the overexpression (OE) or knock-down (KD) regulation.
Furthermore, when the Gad2-ires-cre mice were infected with viral vector containing the elements of designer receptors exclusively activated by designer drugs (DREADDs) including hM4D or hM3Dq (AAV8-hSyn-DIO-hM4Di(Gi)-mCherry, AAV8-hSyn-DIO-hM3Dq(Gq)-mCherry), the Gad2-expressing GABA population in the special area could achieve chemogenetic inhibition (Gi) or activation (Gq), respectively.
GFP-Mito tagfloxed mice were generated by GemPharmatech Co., Ltd (Nanjing, China), in which a Mito-Tag cassette of CAG-LSL-GFP-Mito tag was floxed in according to the report 16. In detailed, a construct carrying CAG-loxP-STOP-loxP-Kozak-GFP-Mito-TGA-pA was targeted to Rosa26 of the mouse Gt (ROSA) 26S. When GFP-Mito tagfloxed mouse crossed with Gad2-iris-cre drivers, the resulting mouse line, referred to as Gad2-Mito-GFP, exhibited bright mito-GFP fluorescence localized specifically to the mitochondrial compartment of all Gad2-expressing GABAergic population in the central nervous system.
Materials And Methods
Establishment of minimal hepatic encephalopathy (MHE) model
A toxin thioacetamide (TAA) (Sigma, USA) -induced minimal hepatic encephalopathy (MHE) model was established according to previous studies15, 17. Briefly, intraperitoneal (i.p.) injections of TAA (150 mg per kg of body weight) were administered once daily for consecutive 3 days. The Sham control group was administered with the same volume of saline. To prevent hypoglycemia and electrolyte imbalance subcutaneous injection of 0.45% NaCl, 5% dextrose and 20 mM KCl was given 12 hours after TAA injection in the first time.
Stereotaxic Aav Viral Vectors Injection
The viral vectors used in the present study included: pAAV-hSyn-DIO-mCherry, AAV8-hSyn-DIO-mCherry, AAV8-hSyn-DIO-hM4Di(Gi)-mCherry, AAV8-hSyn-DIO-hM3Dq(Gq)-mCherry, pAAV-DIO-mCherry-NC-WPRE (Ucp2 NC), pAAV-DIO-mCherry-shRNA(Ucp2)-WPRE (Ucp2 KD), and pAAV-DIO-mCherry-P2A-Ucp2-WPRE (Ucp2 OE). The mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p), placed in stereotaxic apparatus (RWD, China). The fiber tip placement was mapped according to the mouse brain atlas (from bregma, anteroposterior: -3.4 mm; mediolateral: ± 1.5 mm; dorsoventral: -4.55 mm). All mice were injected bilaterally with 1 µL viral vector (1.0 × 1012 viral genomes [VG] / mL) at a rate of 100 nl / min using an injector (Hamilton Co., Reno, NV) connected with a 5-µL Hamilton syringe. After injection, the needle was kept in place for 10 minutes allowing for diffusion of the virus and slowly withdrawn to avoid possible leakage from the needle track.
Direct Local High Ammonia Stimulation Into Snr
NH4Cl 1 µL was injected at a concentration of 10 mM into bilateral SNr areas at a rate of 100 nL/min and the same dose of saline was as control.
c-Fos (+)- neuron labeling (TRAP)
To capture neurons that were active at MHE situation or local hogh ammonia stimulation, TRAP2 mice were used according to previous reports, combined with tamoxifen (TM, sigma) dissolved in corn oil (100 mg/ml) i.p and cre-inducible viral vectors carrying the gene encoding the mCherry fluorescent reporter and expressed in a cre-dependent manner. The the pamming of Fos + neurons were observed and analyzed by a confocal fluorescence microscope (FV-1000, Olympus, Japan).
Designer Receptors Exclusively Activated By Designer Drugs (Dreadds) Manipulation
To precisely regulate Gad2-expressing GABA population, viral vector carrying Designer receptors exclusively activated by designer drugs (DREADDs) elements (Gi or Gq) were injected into the bilateral SNr areas of Gad2-ires-cre mice, and combined with the injection i.p. of ligand clozapine N-oxide (CNO, Sigma) dissolved in saline (0.9% NaCl) at a concentration of 2 mg/ml.
Electrophysiological Slice Recordings
Mouse brain slice electrophysiology recording was performed as described. In brief, to obtain SNr slices, Gad2-expressing GABA population in SNr (mCherry/Gi/Gq) were decapitated and the brains were dissected rapidly and placed in ice-cold oxygenated (95% O2 and 5% CO2) solution containing the following (in mM): NaCl 88, KCl 2.5, MgCl2 7, CaCl2 0.5, NaH2PO4 1.25, NaHCO3 25, sucrose 75, pH 7.4. Brain slices with a thickness of 300 µm were cut with a vibrating microtome (Leica VT1200S, Germany) and transferred into artificial cerebrospinal fluid (ACSF) containing the following (in mM): NaCl 126, KCl 2.5, NaH2PO4 1.25, NaH2PO4 1.25, NaHCO3 25, CaCl2 2, MgSO4 2, glucose 10. Whole-cell patch-clamp recording was performed by a patch-clamp amplifier (Axon 700B, MD, USA). The tip diameter of the electrode used for recording cells was generally about 2 µm, and the impedance was about 4–5 MΩ. To record the spontaneous firing frequency of Gad2-expressing GABA population expressing mCherry fluorescence in SNr, the following perfusion electrode solutions were routinely administered (mM): K-gluconate 133, NaCl 8, EGTA 0.6, Mg ATP 2, Na3•GTP 0.3, HEPES 10, The pH was adjusted to 7.2–7.4 with KOH and the osmolarity was 280–290 mOsm. The spontaneous action potential was stably recorded for 1 min before and after CNO (5 µM) application. Used the Clampfit 11.0.3 software to analyze the frequency of spontaneous neuron firing within 30 s.
Mitochondrial Network Analysis (MiNA)
Mitochondrial Network Analysis (MiNA) uses the freely available FIJI distribution of the ImageJ platform and amalgamates open source tools into a simple macro toolset. Mitochondria in Gad2 neurons were observed by Gad2-Mito-GFP mice brain sections as described above, and imaging was conducted using the inverted laser scanning confocal microscope (Olympus FV1000). The number of mitochondria were evaluated by ImageJ FiJi with the mitochondrial analyzer plug to analyze the individual mitochondria.
Determination Of Reactive Oxygen Species (Ros)
Determination of reactive oxygen species (ROS) in the SNr was assessed using ROS fluorescent probe - (BBoxiProbe®O13, China) staining. BBoxiProbe®O13, a cell-permeable oxidative-sensitive fluorescent dye, is oxidized to ethidium by superoxide, which, subsequently binds to DNA in the nucleus and emits red fluorescence. The frozen brains were cut at a thickness of 15 µm using a CM 3050 cryostat (Leica Microsystems), after which the sections were incubated with A liquid in PBS for 10 min at 37℃ in a humidified chamber protected from light. The images of each section were captured with a confocal fluorescence microscope (FV-1000) and quantification of positive cells in each animal was analyzed using the Image J program.
Western Blot Analysis
Protein isolated from homogenized tissues of bilateral SNr areas with modified RIPA buffer supplemented with halt protease and Phosphatase Inhibitor (Thermo Fisher Scientific). Protein concentrations were measured using Assay Reagent Kit. Protein was run on 12.5% SDS-PAGE and transferred to a 4.5 um PVDF membrane, followed blocked with 5% skim milk in TBST incubate at room temperature for 2 h. The primary antibodies used were SOD1 monoclonal antibody (1:1000, Abclonal) and Gpx1 (1:1000, Abclonal) for oxidative stress; LC3 (1:1000, CST) and Pink1 (1:1000, Abcam) for autophagy; Drp1 (1:1000, CST), phospho Ser616 Drp1 (1:1000, CST), Mfn2 (1:1000, CST), Fis1 (1:1000, Abclonal), Mff (1:1000, CST), and Ucp2 (1:1000, CST) for mitochondrial function; anti-β-Actin (1:5000, Abclonal) as internal reference; overnight incubation at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, abbkine) were used. Membranes were scanned by the Fusion FX EDGE chemiluminescence imaging and gray value analysis was performed using ImageJ FiJi software.
Tissue Preparation And Immunofluorescence Analysis
The mice were anesthetized and transcardially perfused with the 50 mL of 0.01 M phosphate buffer saline (PBS, pH 7.3) and then fixed by 4% paraformaldehyde (pH 7.3). The brains were them collected and subsequently immersed in 0.1 M PB containing 30% sucrose at 4°C. Coronal sections through the SNr area with a thickness of 30 µm were cut on a cryostat (Leica CM1800; Heidelberg, Germany). Sections were incubated in a blocking PBS buffer containing 0.3% Triton X-100 and 0.05% sodium azide, 10% bovine serum albumin for 30 min at room temperature. Then primary antibodies included mouse anti-Gad1 (1:100, abcam), rabbit anti-Gad2 (1:50, CST), rabbit anti-PV (1:200, CST), rabbit anti-vGlut1 (1:500, Abclonal), and rabbit anti-GFAP (1:200, CST). The secondary antibodies anti-rabbit or anti-mouse 488 (1:500, abbkine) were used, together adding DAPI at room temperature for 12 min. All images were taken under FV-1000 confocal fluorescence microscope.
Analysis of locomotor activities and coordination.
Open Field (OF) test: Locomotor activities of mice were analyzed with automatically via Video Tracking in Open Field (OF) test. Half an hour after CNO injection, mice were allowed to explore an open-field arena (45 cm × 45 cm × 45 cm) for 10 min. Before each test, mice were acclimated to the behavior room for at least 1 h. Analysis of the animal's trajectory, including the animal's total distance traveled, the distance traveled to the center, and the number of times the animal entered the center and stood up. After open field test, the locomotor capacity of the mice was assessed again using the elevated plus maze test. To begin the test, mice were placed in the neutral area of the equipment and is unremitting recorded through a video connection for 5 minutes. Total distance traveled and the central distance were monitored by tracking movement from the mice’s body.
Rotarod and CatWalk test: The motor defect and coordination was evaluated by the Rotarod test (BYZ-007, China) and CatWalk analysis (Noldus). All mice were trained for three consecutive days before formal behavior testing. In the test, mice were placed on the device and started to rotate from 5 revolutions per minute (rpm), and increased to the maximum speed of 40 rpm within 150 s, for a total of 5 minutes. Record the time when the mouse fall, and each mouse was tested 3 times. Mouse was placed on the CatWalk XT and displaced after at least 3 runs that were evaluated by the software (Noduls) with its load standard defaults. Each paw identification was identified automatically by the software and manually checked to confirmed the accuracy.
Haematoxylin And Eosin (H&E) Staining
Mice were euthanized, and liver tissue were removed for histology verification of acute liver injury. In brief, the liver tissues were immersed in 10% formalin, dehydrated with ethanol gradient, subsequently permeabilized with xylene and paraffin embedded. Sample blocks were cut into 4 µm thickness and stained with hematoxylin and eosin (H&E). Finally, observed the slide under a brightfield microscope (Olympus, Japan).
Determination Of Ammonia Levels In Blood And Brain
The blood sample was obtained by removing the eyeball, and then the mouse killed. Blood was collected and centrifuged at 3500 g at 4°C for 10 min, stored at -80℃ for testing. Blood ammonia were detected using a commercial kit (Mlbio, China). The steps were strictly performed in accordance with the instructions of the kits
Enzyme Linked Immunosorbent Assay (Elisa)
To observe indicators of changes in liver function, we observed the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and TBiL, which were measured by commercial kits (Nanjing Jiancheng Bioengineering Institute). superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured with commercial kits (Nanjing Jiancheng, China) were used for detection of oxidative stress levels in peripheral blood.
Statistical analysis
All data in this paper were represented as mean ± SD, with error bars representing SD. SPSS software version 26.0 and GraphPad Prism 8.02 were used for all statistical analyses. For data with a normal distribution, Paired or independent t tests Student's t test used to compare two groups to analyze. For more than three groups, statistical analysis was performed using one-way ANOVA, followed by the Tukey or Dunnett post analysis, as appropriate. P < 0.05 was considered significant.