Stereotactic frame

Prior to surgery, each mouse was anesthetized with 2% sodium pentobarbital and mounted on a stereotactic frame (RWD Life Science Co, Shenzhen, China). Eye drops were applied to prevent corneal drying and a heat pad (RWD, Shenzhen, China) was used to maintain the core body temperature of the mice at 37 °C [20 (link)]. To knockdown the expression of STING, the rAAV2/9-U6-shRNA(STING1)-CMV-EGFP (2.0 × 10¹² vg/ml, 5′-CCAACAGCGTCTACGAGA-3′) was used. And rAAV2/9-U6-shRNA(Scramble)-CMV-EGFP (2.0 × 10¹² vg/ml, 5′-CCTAAGGTTAAGTCGCCCTCG-3′) was used as a negative control. To overexpress the expression of STING, rAAV9-hSyn-STING1-P2A-EGFP (1.0 × 10¹² vg/ml, NM_001289591.1) was used. And rAAV9-hSyn-EGFP (1.0 × 10¹² vg/ml) was used as a negative control. The above virus was all purchased from BrainVTA (Wuhan, China). A volume of 350 nl virus was injected into the target coordinate (AP: − 1.5 mm, ML: 2.0 mm, DV: − 1.0 mm) at a speed of 50 nl/min, using the Hamilton syringe (65,460–02, Stoelting, Wood Dale, IL, USA). And the barrier inner diameter of needle is 0.343 mm. After the virus injection, the needle was placed in the target brain region for an additional 10 min to prevent backflow. Twenty-one days later, the mice were subjected to the controlled cortical impact (CCI). The mice of the sham group underwent the same surgical procedures except for the virus injection.

The surgical procedure for implanting the EEG/EMG electrodes was as described previously (Xu et al., 2014 (link); Oishi et al., 2017b (link)). Using aseptic techniques, the mice were anesthetized and fixed to a stereotactic frame (RWD Life Science, Shenzhen, China). Two stainless steel screws served as the EEG electrodes, which were inserted into the skull over the right frontal cortex and right parietal cortex. Two insulated Teflon-coated, stainless steel wires (Cooner Wire Co., Los Angeles, CA, United States) placed bilaterally into the neck muscles served as the EMG electrodes (Figure 1A). An implant for sleep-wake recordings was affixed to the skull using self-curing dental cement. The mice were allowed to recover for 14 days after the EEG/EMG electrode implantation surgery.

Ten weeks old male Sprague-Dawley rats weighing 180–220 g were performed MCAo. The next day, animals were randomly divided into the following three groups (n = 10/group): Lentivirus miR-148b inhibitor (LV-148b-inhibitor, CMV-ZsGREEN1-148b-miRNAinhibitor-PGK-puromycin, 3.0 × 10⁶ IU in 3 μL saline, Genomeditech, China), LV-GFP or saline vehicle control. Rats were anesthetized with 10% chloral hydrate, and fixed on a stereotactic frame (RWD, China). The liquid was injected into the right striatum at a rate of 0.2 μL/min via a mini-pump (WPI, USA). Behavioral tests, including modified neurological severity score (mNSS) test, foot-fault test, and adhesive patch removal test were performed on day 1, day 4, day 7, and day 14 after MCAo (Chen et al., 2001 (link); Shen et al., 2007 (link)). On day 14, all the animals were perfused using saline, followed by 4% paraformaldehyde solution. Brains were removed and fixed in 4% formalin, dehydrated, and embedded in paraffin separately. To measure lesion volume, brain slices were stained with hematoxylin and eosin (HE), and the infarct lesion volume (mm³) was measured as appropriate area (mm²) multiply section interval thickness (2 mm).

After deep anesthesia, C57BL/6 mice (or Kunming) were positioned in a stereotactic frame (RWD, Shenzhen, China). Punch a small hole with a 25-gauge needle behind right bregma and 2.5–3 mm away from the midline. Then, the GL261-luc cells (or G422-luc) (10⁵ cells) were stereotactically injected into the brain parenchyma at a depth of 3 mm.

The mice were anesthetized with sodium pentobarbital (1%, 100 mg/kg; IP), and erythromycin eye ointment was applied to each eye. After disinfection with iodophor, in the prone position, the back skin was cut open at the highest point of the dorsal eminences to expose the vertebrae. The spine was fixed on a stereotactic frame (RWD Life Science Inc., Shenzhen, China), and the muscles and dura covering the intervertebral space were removed with forceps to reveal the white spinal cord. About 500 nL of pAV-U6-RFP-Mapt-shRNA or scrambled adeno-associated virus pAV-U6-RFP-shRNA (WZ Biosciences Inc. Jinan, China), and 500 nL of pAV-CMV-Mapt-RFP or scrambled adeno-associated virus pAV-CMV- RFP (WZ Biosciences Inc. Jinan, China) were injected into the lumbar spinal cord with a glass micropipette. The insertion depth of the needle tip was 400 μm below the surface of the spinal cord and the speed was 50 nL/min. After the injection, the needle was left for 5 min before removal.

All viruses in the present study were packaged by BrainVTA (China). The male mice were fixed in a stereotactic frame (RWD, China) under 2% isoflurane anesthesia. A heating pad was used to maintain the body temperature of the mice at 37 °C. Unless otherwise noted, a volume of 100 nl virus was injected per side. The injections were given over 5 min at a rate of 20 nl/min by an infusion pump (Drummond, USA) and left in place for 10 min. The stereotaxic coordinates for the eLPB are used as following: AP, - 5.07/5.19/5.33 mm, ML, ± 1.45 mm and DV, - 3.30 mm. The stereotaxic coordinates of the lCeA are used as following: AP, - 1.31 mm; ML, ± 2.90 mm; DV, - 4.50 mm. The stereotaxic coordinates for the ovBNST are used as following: AP, + 0.13 mm, ML, ± 1.1mm, and DV, - 4.05 mm. For anterograde tracing, the rAAV2/9-EF1α-DIO-EGFP (PT-0795, 2.04E+12 vg/ml, BrainVTA, China) virus was injected into the unilateral eLPB of ChAT-Cre mice. For retrograde tracing, the CTB-555 (CTB-02, 1 μg/μl, BrainVTA, China) was injected into the unilateral lCeA and ovBNST of WT mice. After 1-week (retrograde tracing) and 3/4-week (anterograde tracing) transfection, male mice were perfused with 0.9% saline, followed by 4% PFA and then images of the CTB-555 or EGFP signals were visualized to assess the virus-injected positions. Animals with missed injections were excluded from the study.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University School of Medicine. For GL261 cell transplantation, we utilized 4-week-old C57BL/6 mice that contained an intact immune system. GL261 cells expressing either the empty vector or YKL-40 (3 × 10⁵ cells in 10 μL PBS) (n = 6/group) were injected into mouse brains at a controlled flow rate of 2 μL/min by a stereotactic frame (RWD Life Science, San Diego, CA, USA). Mice displayed labored breath and difficulty moving between weeks 3 and 4 after transplantation, at which point they were sacrificed, and brain tumors were collected.