5.1 Animal Experiments
All animal experiments were approved by the Ethics Committee of The First Hospital of China Medical University, and all experiments were performed in accordance with relevant guidelines and regulations.
5.1.1 Laboratory Animals and Groups
Forty-seven male Sprague-Dawley (SD) rats, about 8 weeks old, weighing 250±20 g, were ordered (China Medical University animal laboratory, SYXK (Liao)2008-0005, Shenyang, China) to SPF animal laboratory. Before the experiment, the rats were fed freely and given 12h-12h light-dark cycle.
Ischemic models were established by injecting ET-1 (MERCK, USA) into the left thalamus, the steps in detail were described in the previous study. The model of sham operation group was established: the specific method was the same as above, but ET-1 was replaced by normal saline. On the first day after operation, nicotine (Sigma Aldrich), DHβE (ApexBio, USA), AG490 (SELLECK, USA) or saline were administered according to different grouping: nicotine group (nicotine, 1.5 mg/kg/d), nicotine+DHβE group (nicotine, 1.5mg/kg/d; DHβE, 3mg/kg/d), DHβE group (DHβE, 3mg/kg/d), nicotine+AG490 group (nicotine, 1.5mg/kg/d; AG490, 3mg/kg/d), AG490 group (AG490, 3 mg/kg/d), ischemia group (saline, 0.8 ml/kg/d) and sham operation group (saline, 0.8 ml/kg/d). Each group consisted of 6 rats and all 42 rats received the intervention by intraperitoneal injection for nine consecutive days.
The other 3 SD rats did not receive any intervention neither operation nor drugs and they were used as a control group for PCR test. Another two rats were sacrificed 24 hours after surgery only for histological analysis with HE staining to make sure the success of ischemic model (Fig. 7), one from the sham operation group, the other from ischemia group.
5.1.2 Morris Water maze experiment
On the fourth day of drug intervention, Morris water maze (MWM) (endocrinology laboratory, China Medical University) test was performed to test the spatial learning and memory ability of rats. Rats of different groups were subjected to six consecutive days of experiments, which were divided into the following two parts:
Directional navigation experiment: the experiment lasted for 5 days. On the first day, a directional navigation program was set up. Rats were put into the pool from any two quadrants to familiarize themselves with the water maze environment. Platform was placed in the center of a quadrant of the pool. The training tests were conducted at the same time every day for the next four days. The software automatically recorded the swimming trajectory and swimming time of each rat in the pool.
The space exploration experiment: on the sixth day of the experiment, after setting up the space exploration program, the platform was removed, and the rats were put into any quadrant (except the target quadrant where the original platform was located). The software automatically recorded and analyzed the times that the rats crossed the target quadrant and the activity time in target quadrant in 120 seconds.
5.1.3 2-[18F]-A-85380 Micro PET imaging
After the MWM test, rats in each group underwent 2-[18F]-A-85380 micro-PET imaging (Shandong Madic Technology Co., Ltd, China) on the 6th day of MWM. 2-[18F]-A-85380 was synthesized according to the previous report, which included the synthesis program, the information of the average yield, specific activity, chemistry and radiochemistry necessary for the synthesis. The radiochemical purity of the product 2-[18F]-A-85380 was 98.94%, which can meet the imaging requirements of 2-[18F]-A-85380.
Micro PET imaging was performed with a spatial resolution of 1.3 mm in the transverse and axial directions and field of vision (Transaxial: 128 mm, axial:59 mm). 37 MBq 2-[18F]-A-85380 was injected by tail vein of rats. Head scan was performed for 10 minutes after 120 minutes.
Quantitative analysis: Drawing ROI was based on standard rat brain atlas by Paxinos. The cerebellum was used as the reference area, and the ratio of the average SUV (SUVave) in each area to the SUVave of the cerebellum was calculated. The distribution of 2-[18F]-A-85380 in the left thalamus and the whole brain of each group was quantitatively analyzed.
5.1.4 Real-Time PCR Detection of Receptor Subunits of nAChRs
The RNA extraction was performed firstly, followed by the reverse transcription synthesis of cDNA. The mixture was homogenized and the sample was placed on a reverse transcription instrument at 37℃, 15 min; 85℃, 5 s; 4℃, 10 min. Real-time PCR amplification curve and dissolution curve were confirmed after the reaction, and the CT value of the sample was calculated automatically by software. RNAiso plus (NO.9108/9109), SYBR Premix Ex Taq II (RR820A), Prime Script RT Reagent Kit (#RR037A) were purchased from TaKaRa company, Japan. Primers used for the study were as follows:
α4-nAChR: F: 5′-ATGGATGAAACCTACCTGATGAGCA-3′
β2-nAChR: F: 5′-CGGGAAGCAGTGGATGGCGTA-3′
β-Actin: F: 5′-CATCCTGCGTCTGGACCTGG-3′
5.1.5 Western blot
The left thalamic tissue was taken and stored in an Eppendorf tube in a refrigerator at - 80℃. The tissues of each group were added with 1 ml RIPA buffer and PMSF mixture (RIPA: PMSF=100:1). After homogenizing on ice by tissue homogenizer, the tissues were placed on ice for 30 minutes, centrifuged, 4℃, 15,000 rpm for 15 min. The supernatant was then taken and stored at - 80℃. According to BCA protein quantitative standard curve, different samples were adjusted to the same concentration with lysis buffer (3-5µg/µl for each sample). All the samples were denatured by heating at 95℃ for 10 mins, and stored at -20℃. Finally, 15-20 µl sample was added into each lane to ensure the same amount of protein. Equal amounts of proteins were fractionated by SDS–PAGE electrophoresis apparatus, transferred to PVDF membranes. Immunoblotting was carried out with antibodies against α4-nAChRs(1:500), β2−nAChRs(1:500), IL-1β(1:2000), IL-6(1:2000), JAK2(1:1000), STAT3(1:1000), p-JAK2(1:1000), p-STAT3(1:2000), β-actin (1:1000) at 4℃ for 24h. The membranes were incubated with corresponding secondary antibodies at room temperature for 1h. Western blots were developed by enhanced chemiluminescence detection system. Image Lab software was used for gel electrophoresis image analysis. The gray value of the panel was used for quantitative analysis, and the histogram was obtained to show the protein expression of each group in each index. Quantitative analysis was performed basing on three individual samples preparation following with western bloting.
Primary antibodies (α4-nAChR, ab41172; β2-nAChR, ab189174; IL-1β, ab2105; IL-6, ab9324; p-JAK2, ab32101) were purchased from ABCAM company, USA; Primary antibodies (JAK2, D2E12; STAT3,124H6; p-STAT3, D3A7) were from Cell Signaling Technology, USA; BCA Protein Assay Kit was from Beyotime Biotechnology Research Institute, China; RIPA Lysis Buffer was from Beyotime Biotechnology Research Institute, China; PMSF was from Beijing Solarbio Science & Technology company, China.
5.2 Cell Experiment
5.2.1 Cell Culture
HEK-293T cells were cultured at 37℃ in condition of 5% CO2 and 95% oxygen in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (100 units/mL). The cells were divided into two groups: (1) the control (Con) group: cells without lentiviral infection; (2) the overexpression (OE) group: cells infected with two lentivirus that up-regulated CHRNA4 and CHRNB2 expression.
5.2.2 Construction of a Stable Cell Line with Up-regulated CHRNA4 and CHRNB2 Expression
For lentivirus production, HEK-293T cells were co-transfected with PGMLV-CMV-H_CHRNA4-EF1-ZsGreen1-T2A-Puro or PGMLV-CMV-H_CHRNB2-EF1-mCheery-T2A-Blasticidin. The plasmids were amplified in E. coli DH5 cells, purified using a Plasmid Maxi Kit (Tiangen, Beijing, China), and transfected into 70% confluent 293T cells using HG transgene reagent (Genomeditech, Shanghai, China). Lentiviral particles were harvested from the supernatant 72 h after the transfection and purified by ultracentrifugation. These particles are hereafter referred to as LEN-CHRNA4 and LEN-CHRNB2. Stably infected 293T cells with CHRNB2 were selected using blasticidin (10 µg/ml; Beyotime Biotechnology, CA), which killed the uninfected cells. Real-time PCR and western blotting confirmed that the LEN-CHRNB2-infected 293T cells stably over-expressed CHRNA4.
5.2.3 RNA Extraction and Quantitative RT-PCR
The total RNA from cells was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesize by RevertAid Reverse Transcriptase (TaKaRa, Tokyo, Japan). Real-time PCR was performed on StepOnePlus Real Time System (Applied Biosystems, Foster City, CA, USA), and the reaction steps are as follows: 95°C for 30 s followed by 40 cycles at 95°C for 5 s, and then 60°C for 30 s. The primers for RT-PCR to amplify α4-nAChR and β2-nAChR is mentioned above. The primers for GAPDH (internal standard) are as follows.
GAPDH: F: 5′-GAGTCAACGGATTTGGTCGT-3′
The 2–ΔΔCt method was used to quantify mRNA expression relative to GAPDH.
5.2.4 Protein Extraction and Western Blot Analysis
HEK-293T cells were seeded in 6-well plates. After treatments, the total extracts were extracted according to the protocols supplied by the manufacturer (Sangon, Shanghai, China). Next, identical amounts of proteins were separated by SDS–PAGE and then blotted onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk as described elsewhere. They were then incubated with conjugated goat antirabbit antibodies at 4°C overnight. Thereafter, the membranes were incubated with the secondary antibody for 1 h at room temperature. Finally, the membranes were detected using the EZ-ECL.