Rats were anaesthetised through intraperitoneal injection of pentobarbital (40 mg/kg) and placed onto a stereotaxic instrument (RWD Life Science Co., Ltd.). A 1-cm midline incision was performed in the rat scalp to expose the intersection point. Then, a hole 3.2 mm lateral and 1.4 mm anterior to the right bregma was produced. Next, 1.0 ul collagenase type IV (0.25 IU/ul; C5138; Sigma-Aldrich, USA) was injected into the basal ganglia via a microinjection pump (4.2 mm depth below the endocranium) at a rate of 0.2 ul/min. The needle was maintained for 5 min after injection to prevent backflow. Thereafter, the skin incision was closed using sutures. Rats in the sham group received 1.0 ul saline instead of collagenase type IV.

The rats underwent surgery using an ultraclean table and were fixed in a stereotaxic frame. The scalp was opened to expose the anterior brain region. A dental drill was used to drill a 1-mm-diameter hole in the skull surface. Blood (100 ul) was collected from the rat tail vein and injected into the rat striatum with a microsyringe (stereotaxic coordinates; 2 mm lateral to the midline, 0.2 mm posterior to bregma, and 5.5 mm deep below the skull). First, 60 ul of autogenous blood were injected at a rate of 2 ul/min, and the next 40 ul of blood were injected at 5 ul/min. Finally, the needle was left for 10 min before being removed.

Six-to-eight week old male ICR mice were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). Herein, a total of 51 mice were randomly divided into 3 groups: (i) sham group (n = 15), (ii) ICH group (n = 18), and (iii) ICH + Rot group (n = 18). All mice were euthanized at 3 d after operation and brain samples were harvested, as per our previously described reports.

Adult male C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME USA). Mice at 10-12 weeks of age were anesthetized by 1-3% isoflurane inhalation and ventilated with oxygen-enriched air (20% O2:80% air). The right striatum of mice was injected with 0.5 ul of 0.075 U collagenase VII-S (MilliporeSigma, St. Louis, MO, USA) at 0.1 ul per minute. Injections were administered at 0.5 mm anterior and 2.2 mm lateral of the bregma, and 3.0 mm in depth, as previously described. Sham-operated mice received the same treatment, including needle insertion, but were not injected with collagenase. Mice that died before the end of the surgery or shortly thereafter were excluded.

A total of 60 male C57BL/6 mice (10weeks old, 25-28g) were purchased from Guangzhou University of Chinese Medicine Experimental Animal Center (Guangzhou, China). The mice were maintained with enough food and water at 24, 60% relative humidity and 12/12h light/dark cycle. The mice were randomly divided into three groups: sham operation group (Sham), ICH model group (Mod) and baicalin group (Bai) (n = 20/group). Baicalin was suspended in 0.5% carboxymethylcellulose sodium solution. Given the extremely low solubility of baicalin, the concentration of baicalin solution was 0.5 mg/ml. To achieve 20 mg/kg/day dosage, the baicalin solution was administered to the mice in the Bai group by oral route twice at an interval of 1 h within 2 h after ICH injury onset. The remaining two groups received an equal volumes of saline through oral gavage. Since the second day after ICH, mice in the Bai group received 20 mg/kg of baicalin solution while those in the remaining two groups received equal volumes of saline once a day for three consecutive days.

Adult male C57BL/6 mice weighing 20-28 g were maintained in the specific pathogen-free (SPF) facility to be used in this study. Mice were subjected to a 12-h light/dark cycle at a constant ambient temperature (22 ± 1 ). Mice were randomly assigned into the following five groups based on random numbers generated using SPSS. The sham group(n = 20, of which 20 survived) was subjected to mock surgery (craniotomy without collagenase) and treated with 0.1 mL 0.9% saline. The intracerebral hemorrhage(ICH) group (n = 29, of which 23 survived) was subjected to ICH surgery, then treated with 0.9% saline. The low Dauricine(Dau) group (n = 24, of which 20 survived) was subjected to ICH surgery, then immediately treated with 5 mg/kg Dau via tail vein injection. The medium Dau group (n = 25, of which 22 survived)was subjected to ICH surgery, then treated with 10 mg/kg Dau. The high Dau group (n = 24, of which 22 survived)was subjected to ICH surgery, then immediately treated with 15 mg/kg Dau.

C57BL/6 J pregnant mice (E15 ~ E16 day) were disinfected with 75% alcohol and then decapitated. The brain was collected and washed in pre-cold D-Hanks solution with the midbrain and hippocampus being removed. The cortex was collected and the meninx was removed. Then the brain tissues were made into 1 mm³ blocks for digestion with 1 ~ 2 mL lysis at 37 for 15 min. The tissues were cultured in high glucose DMEM to terminate the digestion and made into single-cell suspension. The suspension was filtered through a 70 um mesh screen for centrifugation for 5 min with the supernatant being removed. High glucose DMEM was used to re-suspended cells and the concentration of cells was adjusted for cells were seeded into the plate. About 4 h later, the high glucose DMEM was replaced with Neurobasal medium (containing 2% B27). The culture medium was half refreshed every 2 ~ 3 d. The cells were cultured for 8 ~ 10 d before following experiments.

C57BL/6 J pregnant mice (E15 ~ E16 day) were disinfected with 75% alcohol and then decapitated. The brain was collected and washed in pre-cold D-Hanks solution with the midbrain and hippocampus being removed. The cortex was collected and the meninx was removed. Then the brain tissues were made into 1 mm³ blocks for digestion with 1 ~ 2 mL lysis at 37 for 15 min. The tissues were cultured in high glucose DMEM to terminate the digestion and made into single-cell suspension. The suspension was filtered through a 70 um mesh screen for centrifugation for 5 min with the supernatant being removed. High glucose DMEM was used to re-suspended cells and the concentration of cells was adjusted for cells were seeded into the plate. About 4 h later, the high glucose DMEM was replaced with Neurobasal medium (containing 2% B27). The culture medium was half refreshed every 2 ~ 3 d. The cells were cultured for 8 ~ 10 d before following experiments.

C57BL/6 J pregnant mice (E15 ~ E16 day) were disinfected with 75% alcohol and then decapitated. The brain was collected and washed in pre-cold D-Hanks solution with the midbrain and hippocampus being removed. The cortex was collected and the meninx was removed. Then the brain tissues were made into 1 mm³ blocks for digestion with 1 ~ 2 mL lysis at 37 for 15 min. The tissues were cultured in high glucose DMEM to terminate the digestion and made into single-cell suspension. The suspension was filtered through a 70 um mesh screen for centrifugation for 5 min with the supernatant being removed. High glucose DMEM was used to re-suspended cells and the concentration of cells was adjusted for cells were seeded into the plate. About 4 h later, the high glucose DMEM was replaced with Neurobasal medium (containing 2% B27). The culture medium was half refreshed every 2 ~ 3 d. The cells were cultured for 8 ~ 10 d before following experiments.

The rats underwent surgery using an ultraclean table and were fixed in a stereotaxic frame. The scalp was opened to expose the anterior brain region. A dental drill was used to drill a 1-mm-diameter hole in the skull surface. Blood (100 ul) was collected from the rat tail vein and injected into the rat striatum with a microsyringe (stereotaxic coordinates; 2 mm lateral to the midline, 0.2 mm posterior to bregma, and 5.5 mm deep below the skull). First, 60 ul of autogenous blood were injected at a rate of 2 ul/min, and the next 40 ul of blood were injected at 5 ul/min. Finally, the needle was left for 10 min before being removed.

Male C57BL/6 mice (8-10 weeks old) were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). Briefly, mice were anesthetized and placed in a prone position with head stabilization in a stereotaxic frame. A dental drill was then utilized to generate a 1 mm burr hole at 2.0 mm to the lateral right of the bregma and 3.5 mm deep of the brain. Next, acute ICH was induced by slowly injecting 0.1U of type IV collagenase into this hole.

Male 8-week-old Sprague-Dawley rats were chosen for this study. These animals were randomized to 3, 4, or 5 groups: Design for 3 groups: Sham group, rats underwent sham operation; ICH group, rats underwent ICH operation; ICH + Ac group, rats underwent ICH operation and treated with acupuncture at target acupoints. Design for 4 groups: Sham group; ICH group; ICH + Ac group; ICH + sAc group, rats underwent ICH operation and treated with acupuncture at non-acupoints located 1 cm posterior and parallel to Baihui-penetrating-Qubin needling as previously described. Another design for 4 groups: Sham group; ICH group; ICH + NC group, rats underwent ICH operation and injected with antagomiR-NC; ICH + antagomiR group, rats underwent ICH operation and injected with antagomiR-23a-3p.

C57BL/6 mice (aged 8-10 weeks, Vital River, Beijing, China) were housed in SPF conditions. The animal study was performed according to the National Institutes of Health Guide and approved by the Ethics Committees of Affiliated Jiangmen Traditional Chinese Medicine Hospital of Jinan University.

C57BL/6 mice (aged 8-10 weeks, Vital River, Beijing, China) were housed in SPF conditions. The animal study was performed according to the National Institutes of Health Guide and approved by the Ethics Committees of Affiliated Jiangmen Traditional Chinese Medicine Hospital of Jinan University.

Adult male Sprague-Dawley rats (SD rats, weighing 250-300 g) aged 11-12 weeks were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All 96 rats were randomly divided into four groups of 24 rats each: Sham group, Sham + IRN (30 mg/Kg) group, ICH group, and ICH + IRN (30 mg/Kg) group. The rats in sham group were injected with PBS solution, and the Sham + IRN (30 mg/Kg) group was received an equal amount of 30 mg/Kg IRN solution (intra-peritoneal injection) after the sham operation. After ICH, the rats in ICH group were injected with PBS solution, and the ICH + IRN (30 mg/Kg) group was received an equal amount of 30 mg/Kg IRN solution (intra-peritoneal injection).

Adult male Sprague-Dawley rats (SD rats, weighing 250-300 g) aged 11-12 weeks were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All 96 rats were randomly divided into four groups of 24 rats each: Sham group, Sham + IRN (30 mg/Kg) group, ICH group, and ICH + IRN (30 mg/Kg) group. The rats in sham group were injected with PBS solution, and the Sham + IRN (30 mg/Kg) group was received an equal amount of 30 mg/Kg IRN solution (intra-peritoneal injection) after the sham operation. After ICH, the rats in ICH group were injected with PBS solution, and the ICH + IRN (30 mg/Kg) group was received an equal amount of 30 mg/Kg IRN solution (intra-peritoneal injection).

A total of 60 male C57BL/6 mice (10weeks old, 25-28g) were purchased from Guangzhou University of Chinese Medicine Experimental Animal Center (Guangzhou, China). The mice were maintained with enough food and water at 24, 60% relative humidity and 12/12h light/dark cycle. The mice were randomly divided into three groups: sham operation group (Sham), ICH model group (Mod) and baicalin group (Bai) (n = 20/group). Baicalin was suspended in 0.5% carboxymethylcellulose sodium solution. Given the extremely low solubility of baicalin, the concentration of baicalin solution was 0.5 mg/ml. To achieve 20 mg/kg/day dosage, the baicalin solution was administered to the mice in the Bai group by oral route twice at an interval of 1 h within 2 h after ICH injury onset. The remaining two groups received an equal volumes of saline through oral gavage. Since the second day after ICH, mice in the Bai group received 20 mg/kg of baicalin solution while those in the remaining two groups received equal volumes of saline once a day for three consecutive days.

Male C57BL/6 mice, aged 8-10 weeks and weighing 22-25 g, were purchased from the Hubei Provincial Center for Disease Control and Prevention in Wuhan, China. Mice were initially administered with 1% sodium pentobarbital (70 mg/kg; Sinopharm Chemical Agent Co., Ltd., Shanghai, China) intraperitoneally. Subsequently, they were secured on a stereotaxic device (RWD Life Science Co., Shenzhen). 0.6 ul of collagenase (1 ku/ml) was injected into the caudate nucleus of the mice (bregma 0: 0.5 mm anterior, 2 mm left lateral and 3.5 mm deep) with the help of a brain stereotactic sampling needle (Hamilton).

Six-to-eight week old male ICR mice were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All animal procedures have been approved by the Institutional Animal Care and Use Committee of Ruijin hospital, Shanghai Jiao Tong University (Shanghai, China). Efforts were made as much as possible to reduce the number of mice used and to minimize suffering. Herein, a total of 51 mice were randomly divided into 3 groups: (i) sham group (n = 15), (ii) ICH group (n = 18), and (iii) ICH + Rot group (n = 18). To be specific, the current study was divided into two parts of the experimental design. Part 1: to observe the effects of Rot on the mitochondria-related genes, iron levels, MDA levels, SOD activity, hematoma volume, brain edema, and ultrastructural changes of mitochondria, animals were randomly divided into the sham group (n = 9), ICH group (n = 12), and ICH + Rot group (n = 12). All mice were euthanized at 3 d after operation and brain samples were harvested, as per our previously described reports. Part 2: to observe the effect of Rot on neurological deficits following ICH, 18 mice were randomly divided into additional 3 groups (sham group, ICH group, and ICH + Rot group). We evaluated mNSS scores at 1, 3, 7, and 14 days after ICH. We also assessed the memory function with the MWM test at 14 days after ICH.

Male C57BL/6 mice (8-10 weeks old) were purchased from Charles River Laboratories. For intraperitoneal injections, mice received daily intraperitoneal injections of 25 mg/kg U0126 or vehicle for 7 d (n = 13 for sham+vehicle, n = 17 for ICH+vehicle, n = 16 for ICH + 25 mg/kg U0126). Four ICH and six ICH+U0126 animals died from the surgery. For intracerebroventricular injections, mice received a single intracerebroventricular injection of 12 ug U0126 or vehicle (n = 16 per group) 2 h after ICH.

Fpn-floxed (Fpnflox/flox) mice were obtained from Dr. N.C. Andrews and transferred into a C57bl/6 background. Mice were treated with a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immobilized on a stereotaxic apparatus (RWD Life Science Co., Shenzhen). All experimental mice received a total of 20 ul autologous blood injected successively into the caudate nucleus (bregma 0: 0.8 mm anterior, 2 mm left lateral and 3.5 mm deep).