Male C57BL/6 mice aged 6-8 weeks were purchased from the Chongqing Medical University Animal Experiment Center (Chongqing, China). Mice were randomly allocated into three groups including sham group, TBI group, and melatonin treatment group. 30 mice were used for MRI and cerebral blood flow (CBF) monitoring at the 3rd and 7th day, with 5 in each group; 15 mice were used for EEG detection at the 14th and 30th days, training and testing inwater mazeduring the 25th-30th days, with 5 in each group. 15 mice were used for brain water content determination on the 3rd day, with 5 in each group. 9 mice were used for RNA sequencing, with 3 in each group; 75 mice were used for brain tissue acquisition, with 5 in each group with 5 time points (1st, 3rd, 7th, 14th, and 30th day after the operation).

Male C57BL/6 mice aged 6-8 weeks were purchased from the Chongqing Medical University Animal Experiment Center (Chongqing, China). Mice were randomly allocated into three groups including sham group, TBI group, and melatonin treatment group. 30 mice were used for MRI and cerebral blood flow (CBF) monitoring at the 3rd and 7th day, with 5 in each group; 15 mice were used for EEG detection at the 14th and 30th days, training and testing inwater mazeduring the 25th-30th days, with 5 in each group. 15 mice were used for brain water content determination on the 3rd day, with 5 in each group. 9 mice were used for RNA sequencing, with 3 in each group; 75 mice were used for brain tissue acquisition, with 5 in each group with 5 time points (1st, 3rd, 7th, 14th, and 30th day after the operation).

Rats were injected with 4 mL/kg of chloral hydrate for anesthesia and then put on a stereotactic apparatus. Subsequently, the needle was tilted at 55 in the sagittal plane and fixed anterior to the bregma (7.5 mm). The needle tip was toward the right and lowered the anterior to the chiasma (2 mm). Finally, the nonheparinized autologous femoral arterial blood (0.3 mL) was injected into a prechiasmatic cistern using a syringe pump. Rat temperature was maintained at 37 ± 0.5 during the surgery. The rats in the sham group were injected with the same dose of saline into a prechiasmatic cistern. At last, rats were monitored for recovery and then returned to cages.

Rats were injected with 4 mL/kg of chloral hydrate for anesthesia and then put on a stereotactic apparatus. Subsequently, the needle was tilted at 55 in the sagittal plane and fixed anterior to the bregma (7.5 mm). The needle tip was toward the right and lowered the anterior to the chiasma (2 mm). Finally, the nonheparinized autologous femoral arterial blood (0.3 mL) was injected into a prechiasmatic cistern using a syringe pump. Rat temperature was maintained at 37 ± 0.5 during the surgery. The rats in the sham group were injected with the same dose of saline into a prechiasmatic cistern. At last, rats were monitored for recovery and then returned to cages.

Healthy male Sprague-Dawley rats (weighing 370 g-420 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. The rats were randomized into five groups. Rats in sham group (n = 6) underwent the same anesthetic and surgical procedures, excluding cardiac arrest and CPR. All of the remaining four groups were given the interventions within 10 minutes of ROSC. Rats in CPR group (n = 6) received an intraperitoneal injection of 0.9% saline (1 mL/kg). Rats in baicalein group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein at the same time, based on previous studies. Rats in tunicamycin group (n = 6) were intraperitoneally injected with tunicamycin (2 mg/kg body weight) (22-24). Rats in baicalein + tunicamycin group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein and 2 mg/kg (body weight) tunicamycin. All groups were given the same volume of normal saline solvent (2 mL/kg).

Healthy male Sprague-Dawley rats (weighing 370 g-420 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. The rats were randomized into five groups. Rats in sham group (n = 6) underwent the same anesthetic and surgical procedures, excluding cardiac arrest and CPR. All of the remaining four groups were given the interventions within 10 minutes of ROSC. Rats in CPR group (n = 6) received an intraperitoneal injection of 0.9% saline (1 mL/kg). Rats in baicalein group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein at the same time, based on previous studies. Rats in tunicamycin group (n = 6) were intraperitoneally injected with tunicamycin (2 mg/kg body weight) (22-24). Rats in baicalein + tunicamycin group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein and 2 mg/kg (body weight) tunicamycin. All groups were given the same volume of normal saline solvent (2 mL/kg).

Healthy male Sprague-Dawley rats (weighing 370 g-420 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. The rats were randomized into five groups. Rats in sham group (n = 6) underwent the same anesthetic and surgical procedures, excluding cardiac arrest and CPR. All of the remaining four groups were given the interventions within 10 minutes of ROSC. Rats in CPR group (n = 6) received an intraperitoneal injection of 0.9% saline (1 mL/kg). Rats in baicalein group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein at the same time, based on previous studies. Rats in tunicamycin group (n = 6) were intraperitoneally injected with tunicamycin (2 mg/kg body weight) (22-24). Rats in baicalein + tunicamycin group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein and 2 mg/kg (body weight) tunicamycin. All groups were given the same volume of normal saline solvent (2 mL/kg).

The Experimental Animal Center at Southern Medical University provided the wild-type (WT) adult male and female C57BL/6 mice (body weight, 22-25 g). Briefly, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg), and mounted on a stereotaxic frame. A midsagittal incision was made in the scalp and a circular craniotomy (4.5 mm diameter) was made over the left parietotemporal cortex.

The Experimental Animal Center at Southern Medical University provided the wild-type (WT) adult male and female C57BL/6 mice (body weight, 22-25 g). Briefly, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg), and mounted on a stereotaxic frame. A midsagittal incision was made in the scalp and a circular craniotomy (4.5 mm diameter) was made over the left parietotemporal cortex.

Sixty-four male Sprague-Dawley rats (2 weeks), weighing 20-30 g. All animals were brought from the Institute of Medical Laboratory Animals at the Chinese Academy of Medical Sciences and were kept in the same unit in a temperature-controlled environment [(22 ± 1) ]. The rats were fasted for 12 h before the experiment and drank water freely. After being numbered according to body weight, the rats were randomly divided into four groups using the random number table. The number of rats in each group was 16. The experimental groups were as follows: sham-operated group (S group, n = 16), the model group receiving HIR (HIR group, n = 16), sevoflurane group treated (HIR + Sev group, n = 16), and desferrioxamine treated group [deferoxamine (HIR + Sev + DFO) group, n = 16]. In HIR+Sev and HIR + Sev + DFO groups, rats were placed in an anesthetizing chamber and exposed to 3.6% sevoflurane (Cayman, 23996, USA) with complete oxygen for 2 h, and sham and HIR group rats were conducted with the same procedure without sevoflurane exposure. DFO (100 mg/kg, MCE, HY-B0988, China) was administered continuously daily for 6 days before surgery in the HIR + Sev + DFO group. Other groups were given equal amounts of saline.

Adult male C57BL/6J mice (6-8 weeks, weighting 20-25 g) were used for all experiments. Mice were housed in pairs in a cage with access to food and water ad libitum. Mice were anesthetized with 4% chloral hydrate (0.4 mg/g) and mounted in a stereotaxic system (David Kopf Instruments, Tujunga, California). A midline skin incision was performed on the scalp to expose the skull, and a 5-mm craniotomy was made lateral to the sagittal suture and centered between bregma and lambda. The skull cap was then removed carefully to avoid destroying the dura mater.

Male Sprague-Dawley (SD) rats were introduced into research, for the present SAH model a total of 383 rats, weighing 250-300 g, were purchased from the Animal Center of Chongqing Medical University. The adult male SD rats assigned to SAH model procedures were randomly divided into several groups. The rats assigned to SAH model procedures were randomly divided into the groups, first to determine the expression of hepcidin, DMT1, FPN1, and GPX4, the main regulator of ferroptosis, and to subsequently select the most suitable timing for drug injections. Second, adult male SD rats were randomly divided into the groups to determine the significant preoperative doses of ebselen, heparin and OSM in terms of their effects on hepcidin, DMT1, FPN1, and GPX4 for further study. Lastly, male SD rats were randomly divided into the groups to determine the effects of hepcidin and DMT1 on iron metabolism, ferroptosis, and EBI, by using heparin, ebselen and OSM as the experimental interventions.

Adult male C57BL/6J mice (6-8 weeks, weighting 20-25 g) were used for all experiments. Mice were housed in pairs in a cage with access to food and water ad libitum. Mice were anesthetized with 4% chloral hydrate (0.4 mg/g) and mounted in a stereotaxic system (David Kopf Instruments, Tujunga, California). A midline skin incision was performed on the scalp to expose the skull, and a 5-mm craniotomy was made lateral to the sagittal suture and centered between bregma and lambda. The skull cap was then removed carefully to avoid destroying the dura mater.

Adult male C57BL/6J mice, aged 10-12 weeks and weighing 20 to 24 g. Briefly, following anesthesia with isoflurane (4% for induction and 12% for maintenance), mice were mounted on a stereotaxic frame. A midsagittal incision was performed in the scalp under sterile conditions and a 4.5 mm diameter circular craniotomy was made over the left parietotemporal cortex with a burr drill. Then, the skullcap was gently removed and a 3.0 mm diameter round and flat tip was carefully placed vertically to the dural surface. The electromagnetic controlled cortical impact device was set to 5.0 m/s for strike velocity, 2.0 mm for strike depth and 100 ms for dwell time. A sterile plastic film covered the bone window and intermittent sutures of the skin, and disinfection with iodophor were performed. The entire procedure required 15-30 min per mouse.

Healthy male Sprague-Dawley rats (weighing 370 g-420 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. The rats were randomized into five groups. Rats in sham group (n = 6) underwent the same anesthetic and surgical procedures, excluding cardiac arrest and CPR. All of the remaining four groups were given the interventions within 10 minutes of ROSC. Rats in CPR group (n = 6) received an intraperitoneal injection of 0.9% saline (1 mL/kg). Rats in baicalein group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein at the same time, based on previous studies. Rats in tunicamycin group (n = 6) were intraperitoneally injected with tunicamycin (2 mg/kg body weight) (22-24). Rats in baicalein + tunicamycin group (n = 6) were intraperitoneally injected with 50 mg/kg (body weight) baicalein and 2 mg/kg (body weight) tunicamycin. All groups were given the same volume of normal saline solvent (2 mL/kg).

Male Sprague-Dawley (SD) rats were introduced into research, for the present SAH model a total of 383 rats, weighing 250-300 g, were purchased from the Animal Center of Chongqing Medical University. The adult male SD rats assigned to SAH model procedures were randomly divided into several groups. The rats assigned to SAH model procedures were randomly divided into the groups, first to determine the expression of hepcidin, DMT1, FPN1, and GPX4, the main regulator of ferroptosis, and to subsequently select the most suitable timing for drug injections. Second, adult male SD rats were randomly divided into the groups to determine the significant preoperative doses of ebselen, heparin and OSM in terms of their effects on hepcidin, DMT1, FPN1, and GPX4 for further study. Lastly, male SD rats were randomly divided into the groups to determine the effects of hepcidin and DMT1 on iron metabolism, ferroptosis, and EBI, by using heparin, ebselen and OSM as the experimental interventions.

Male Sprague-Dawley (SD) rats were introduced into research, for the present SAH model a total of 383 rats, weighing 250-300 g, were purchased from the Animal Center of Chongqing Medical University. The adult male SD rats assigned to SAH model procedures were randomly divided into several groups. The rats assigned to SAH model procedures were randomly divided into the groups, first to determine the expression of hepcidin, DMT1, FPN1, and GPX4, the main regulator of ferroptosis, and to subsequently select the most suitable timing for drug injections. Second, adult male SD rats were randomly divided into the groups to determine the significant preoperative doses of ebselen, heparin and OSM in terms of their effects on hepcidin, DMT1, FPN1, and GPX4 for further study. Lastly, male SD rats were randomly divided into the groups to determine the effects of hepcidin and DMT1 on iron metabolism, ferroptosis, and EBI, by using heparin, ebselen and OSM as the experimental interventions.

Sixty-four male Sprague-Dawley rats (2 weeks), weighing 20-30 g. All animals were brought from the Institute of Medical Laboratory Animals at the Chinese Academy of Medical Sciences and were kept in the same unit in a temperature-controlled environment [(22 ± 1) ]. The rats were fasted for 12 h before the experiment and drank water freely. After being numbered according to body weight, the rats were randomly divided into four groups using the random number table. The number of rats in each group was 16. The experimental groups were as follows: sham-operated group (S group, n = 16), the model group receiving HIR (HIR group, n = 16), sevoflurane group treated (HIR + Sev group, n = 16), and desferrioxamine treated group [deferoxamine (HIR + Sev + DFO) group, n = 16]. In HIR+Sev and HIR + Sev + DFO groups, rats were placed in an anesthetizing chamber and exposed to 3.6% sevoflurane (Cayman, 23996, USA) with complete oxygen for 2 h, and sham and HIR group rats were conducted with the same procedure without sevoflurane exposure. DFO (100 mg/kg, MCE, HY-B0988, China) was administered continuously daily for 6 days before surgery in the HIR + Sev + DFO group. Other groups were given equal amounts of saline.

Eight-week-old male mice were subjected to severe CCI. Anesthesia was induced with 3% isoflurane in nitrous oxide: oxygen (7:3) and maintained with 1.5% isoflurane via nose cone. Temperature was maintained at 37 ± 0.5 using a heating blanket. After anesthesia, mice were placed in a stereotaxic frame (R.W.D. Shenzhen, China). A 4-mm-diameter craniotomy was performed over the left parietal bone and the bone flap was removed for trauma. A vertically directed CCI was applied (6.0 ± 0.2 m/s, 50 ms dwell time, 1.4 mm depth) using an impactor (R.W.D., Shenzhen, China). After the injury, the skin incision was closed. Mice were monitored with supplemental oxygen (100%) for 1h before returning to their cages. Fer-1 (1 mg/kg per day) was given i.p. at 7 days before CCI and once daily until euthanasia or the MWM test. Before the brain tissues were obtained, mice were perfused intracardially with 4 phosphate-buffer saline (PBS) solution.

Rats were injected with 4 mL/kg of chloral hydrate for anesthesia and then put on a stereotactic apparatus. Subsequently, the needle was tilted at 55 in the sagittal plane and fixed anterior to the bregma (7.5 mm). The needle tip was toward the right and lowered the anterior to the chiasma (2 mm). Finally, the nonheparinized autologous femoral arterial blood (0.3 mL) was injected into a prechiasmatic cistern using a syringe pump. Rat temperature was maintained at 37 ± 0.5 during the surgery. The rats in the sham group were injected with the same dose of saline into a prechiasmatic cistern. At last, rats were monitored for recovery and then returned to cages.