Stereotaxic apparatus

Wild-type adult C57BL/6 mice were first anesthetized and placed on a stereotaxic apparatus (RWD Life Science, Shanghai, China), then drug was injected into the right lateral ventricle (anteroposterior, −1.0 mm; mediolateral, −0.5 mm, dorsoventral, −2.0 mm) in a duration of 5 min. After injection, the needle was left in place for 10 min before being withdrawn to reduce backflow. Mice were allowed to recover in a dark warm place.

The ICH model was conducted according to our previously developed method (Yuan et al., 2019 (link)). Briefly, mice were immobilized on the stereotaxic apparatus (RWD Life Science Co, Shenzhen, China) after being anesthetized with 3% isoflurane for induction and 1.5% for maintenance. Whole blood (20 μl) without anticoagulant was obtained from its tail and then injected into the left striatum (0.8 mm anterior and 2 mm lateral of bregma, at a depth of 3.5 mm) using a syringe pump (Hamilton, Bonaduz, AG) at 2.0 μl/min. The microinjector was detained for 10 min. The sham group was injected with 20 μl saline using the same procedures. Unsuccessful ICH models that were asymptomatic or dead were excluded from this study. Perihematomal cerebral tissues were collected for oxylipins quantification at 0.5, 1, and 3 days after ICH or 1 day after saline injection.

A commercialized Vivid E9 diagnostic ultrasound system (GE Healthcare, Milwaukee, WI, USA) was used in this study. An ultrasound beam was generated by a M5S-D phased array transducer operating in the second harmonic mode (transmit: 1.5 MHz, receive: 3.0 MHz). The transducer was positioned using a stereotaxic apparatus (RWD Life Science Co., Ltd, Shenzhen, China) to ensure the acoustic beam targeted the brain precisely. The transducer was submersed in a water tank containing deionized and degassed water whose bottom was sealed by a polyurethane membrane. Focal depth was set at 5 cm, which is approximately 3 mm below the dorsal surface of the skull.

We established a rat ICH model by inducing ICH in the striatum (caudate + putamen), according to the method of Ni et al. (2015 (link)). Briefly, rats were anesthetized with 3.5 and 1.5% isoflurane for induction and maintenance, respectively, and then immobilized on a stereotaxic apparatus (RWD Life Science Co., Shenzhen, China), followed by the drilling of a 1-mm diameter burr hole, 0.2 mm anterior and 3.5 mm lateral to the bregma. We then injected a total of 75 μl of autologous blood into the striatum using a Hamilton needle attached to a micropump (RWD Life Science Co.), 5.5 mm ventral to the skull surface, as described in our previous study (Yuan et al., 2019 (link)).

The application of the Cre-dependent Rosa26 Cas9 knockin mice for the in vivo gene editing was described previously^{[15 (link)]}. The Cas9 mice with C57BL/6 background were used for gene knockout in the hypothalamus. For the stereotactic surgery, eight-week-old Cas9 mice were deeply anesthetized with isoflurane and fixed on a stereotaxic apparatus (RWD Life Science, China) with ear bars. After exposing the skull via a small incision, an injection needle was inserted into the brain, and the Epha3 sgRNA AAVs or control viruses (AAV without sgRNA) was injected at a rate of 25 nL per minute using a micro syringe pump. After injection, the pipette was left in position for another 5 min to allow enough absorption and spreading of AAVs before being withdrawn. The coordinates and injection volume used in the study were as follows: the ARC (anterior-posterior [AP], −1.60 mm; dorsal-ventral [DV], −5.80 mm; left-right [LR], ±0.30 mm, 500 nL/side). The AAV-injected mice were allowed to recover for two weeks and then feed with either chow or high-fat diet. All stereotaxic injection sites in the hypothalamus were verified by sequencing section and imaging. All 'missed' or 'partially injected' animals were excluded from data analysis.

Mice were placed in a stereotaxic apparatus (RWD Life Science, San Diego, CA, USA) under isoflurane (3%, inhalation) anesthesia, and the skull was exposed. A small hole was then made using a dental drill. Virus was bilaterally injected into the nucleus accumbens core (from the bregma: AP + 1.4 mm, ML ± 1.5 mm, DV -3.6 mm from the brain surface at an angle of 10°: 1.0 μL was applied to each side using a Hamilton syringe), the lateral shell (from the bregma: AP + 1.0 mm, ML ± 1.8 mm, DV -4.9 mm from the skull at an angle of 0°: 300 nL was applied to each side using a Nanoject III (Drummond Scientific Company, Broomall, PA, USA)) and the medial shell (from the bregma: AP + 1.5 mm, ML ± 0.5 mm, DV − 4.7 mm from the skull at an angle of 0°: 300 nL was applied to each side using a Nanoject III (Drummond Scientific Company)).