Operating microscope

The detailed mice MCAO model was described previously [35 (link), 36 (link)]. Adult male ICR mice weighting 26-32 grams (SLAC laboratory animal, Shanghai, China) were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. The body temperature was maintained at 37.0±0.3°C during surgery by a heating pad (RWD life science, Shenzhen, China). A midline incision was made on the neck under an operating microscope (Leica, Wetzlar, Germany). The left common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were isolated. A silicone-coated round top 6-0 suture (Dermalon, 1756-31, Covidien, OH) was gently inserted from the ECA stump to the ICA to induce MCA occlusion. The suture inserted distance from the bifurcation to the opening of MCA was 10.5±0.5 mm. The success of occlusion was characterized as the reduction of cerebral blood down to 20%, which was verified by a laser Doppler flowmetry (Moor Instruments, Devon, UK). After 60 minutes of occlusion, the suture was withdrawal, the CCA was restored and blood flow returned to at least 70% of the baseline blood flow. Sham mice underwent the same procedure without inserting the suture.

According to our previous study [8 (link)], the sudden density jump at air-tissue interface can introduce significant phase contrast for X-rays. Thus, in our ex vivo mouse brain imaging, we made an air-filled mouse brain whose vascular system was filled with air, making “air-tissue” interfaces.
The experiment followed the standard laboratory animal use procedure and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Bioethics Committee of School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China. Male adult CD-1 mouse (Sppir-BK Inc., Shanghai, China) weighing 30 grams was anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Then, the chest and abdominal cavities were opened under an operating microscope (Leica, Wetzlar, Germany). 20 mL of heparinized saline was manually perfused transcardially, followed by 20 mL of 4% paraformaldehyde perfusion. Thereafter, the brain was taken out and placed in 4% paraformaldehyde for 24 hours at 4°C. The brain was further dehydrated at room temperature using graded ethanol. For imaging, the brain was air dried naturally after dehydration. For hematoxylin and eosin (H&E) staining, the brain was embedded in paraffin. Then, coronary paraffin sections with 4 μm thickness of the brain tissue were prepared for H&E staining.

All interventions were performed at day-time under inhalation of 1.5–2.0% isoflurane mixed with pure oxygen at a flow of 0.3 L/min (isoflurane vaporizer, Sigma Delta, UK) in a dedicated S1 operation room. All procedures were done under an operating microscope (Leica, magnification 10–25×, Germany) to ensure the preservation of the branches of the hepatic artery and portal vein. We induced total biliary occlusion in 36 male mice (C57BL/6N, 25–28 g) as described before.¹³ (link) We performed a total bile duct ligation and transection of the ligated main bile duct between the two distal ligatures (tBDT). We used three ligatures for ligation of the main bile duct and transected the ligated bile duct between the two distal ligatures. The transection of the ligated main bile duct prevents the formation of biliary collaterals or recanalization due to loosening of ligatures, compared to bile duct ligation using single or double ligation of the main bile duct without transection (BDL).14 (link), 15 (link), 16 (link), 17 , 18 (link)We used 12 mice for each experiment. No mice died before the intended sacrifice date. Detailed description of the surgical procedure and postoperative observation and analgetic treatment of the animals is given in the supplement.

8-week-old mice were anaesthetized with an intraperitoneal injection of a mixture of tiletamine and zolazepam (1:1, 2.25 mg per kg body weight) and xylazine hydrochloride (0.7 mg per kg body weight). AAV9-CjCas9 (2 × 10¹⁰ viral genome in 2 μl) was intravitreally injected using a Nanofil syringe with a 33 G blunt needle (World Precision Instruments Inc.) under an operating microscope (Leica Microsystems Ltd.).