2.1. Experimental animals
All experimental adult Sprague Dawley rats (280–350 g) were obtained from the Experimental Animal Center of Shanghai Jiaotong University (license number SYXK (Shanghai) 2018-0028). The rats were raised in a specific pathogen-free laboratory at suitable temperature, with a 12-h light/dark cycle, proper humidity and free access to food and water. All surgical operations were performed under deep anesthesia to minimize the pain of animals.
A total of 126 rats were assigned to sham group (n = 48) and SAH group (n = 78). Nine of them died during or immediately after the operation, and 11 of them were excluded because of their low SAH scores. At 24 and 72 hours after operation, rats were euthanized by intraperitoneal injection of pentobarbital (800 mg/kg) [13, 14] and brain tissues were removed according to different experimental requirements, or embedded in paraffin or ground into homogenate or other treatments. In each group, 6 brain tissues were ground into homogenate, 6 samples were used for staining, 3 were observed by transmission electron microscopy (TEM), and 6 were for detection of blood-brain barrier (BBB) and brain water content.
2.2. Establishment of SAH in rats
Rats were anesthetized with 60 mg/kg pentobarbital and 7.5 mg/kg xylazine intraperitoneally. Rats spontaneously breathed room air and were supplemented with oxygen to maintain 90% < O2 < 98% using pulse oximetry. The rearing temperature was maintained at 37 °C using a rectal temperature sensor (Harvard Apparatus, Holliston, MA, USA). Surgical operations were carried out with aseptic techniques.
A sharpened 4 − 0 nylon suture was inserted into the right external carotid artery and passed through the intracranial internal carotid artery; when confronted with resistance, the nylon suture was pushed 3 mm further to make the internal carotid artery wall perforated. Next, the suture was withdrawn, and the internal carotid artery was reperfused. Meanwhile, the filaments were introduced without arterial perforation in the sham controls. The severity of SAH was blindly evaluated as previously described [15]. The sub-arachnoid cistern was split into 6 segments, and each one was graded from 0 to 3. Grade 0: no sub-arachnoid blood; grade 1: a small amount of sub-arachnoid blood; grade 2: moderate blood clot with recognizable arteries; and grade 3: blood clot in all arteries within the segment. The total score of each rat ranged from 0 to 18.
2.3. Isolation and culture of rat neurons
After euthanasia with pentobarbital, the fetuses of 18-day pregnant rats were removed, and the hippocampus of the fetuses was stripped and detached with 0.25% trypsin. The hippocampus of fetal rats was added with neuron preculture media (modified Eagle’s medium, 10% fetal bovine serum, 1 mmoL/L sodium pyruvate, 100 U penicillin and streptomycin double antibody, 1 × GlutaMAX) to stop the enzyme reaction. After repeated trituration, the hippocampus tissue was dispersed into single cells, counted and seeded into the culture dishes. After 3–5 hours, the media were changed into neuron media (neurobasal medium, 1 × B27, 1 × GlutaMAX, 100 U penicillin and streptomycin), and the media were changed every 3 days.
The hippocampal neurons were allocated into blank group (normal cultured), FIN56 group (added with FIN56, a ferroptosis inducer), FIN56 + NC group (FIN56-induced neurons were transfected with adenovirus negative control), FIN56 + OTUB1 group (FIN56-induced neurons were transfected with adenovirus ubiquitin aldehyde binding 1 (OTUB1), a deubiquitylase), and FIN56 + TAK-243 group (FIN56-induced neurons were added with TAK-243, an ubiquitination inhibitor).
The recombinant adenovirus vector Ad-OTUB1 and the control Ad-NC were purchased from Hanbio Biotechnology Co., Ltd. China (Shanghai, China). FIN56 and TAK-243 were purchased from MedChemExpress Co., Ltd (Monmouth Junction, NJ, USA).
According to the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), Ad-OTUB1 and Ad-NC were transfected into neurons. FIN56 (5 µm) [16] and TAK-243 (1 nm) [17] were applied to different groups of hippocampal neurons.
2.4. Assessment of neurologic score, SAH grade and mortality
Neurological scores were assessed at 24 and 72 hours in a blinded manner as previously reported [15]. An 18-point scoring system with 6 subsets (spontaneous activity, spontaneous movements of 4 limbs, forelimb outstretching, wire cage wall climb, trunk touch reaction and vibrissae touch response) was used. After that, the rats were sacrificed to quantify the severity of SAH [15]. Rats with score < 5 points were excluded, and the mortality after the operation was recorded.
2.5. Assessment of BBB permeability and brain water content
BBB permeability was assessed using Evans blue extrapolation [18]. In each group, 6 rats were injected with 4% Evans blue dye (2.5 mL/kg) into the left femoral vein under deep anesthesia 24 hours after operation to circulate for 60 minutes. The Evans blue dye was quantified using a spectrophotometer at 620 nm excitation wavelength, 680 nm emission wavelength and 10 nm bandwidth. To measure the water content in the brain, the brain was quickly divided into different parts under deep anesthesia, then weighed immediately (wet weight), and placed in the oven at 105℃ (dry weight) for 72 hours. Brain water content = (wet weight - dry weight)/wet weight × 100%.
2.6. TEM observation
The mitochondria in the brain of rats were observed by TEM. The rats were euthanized by pentobarbital at 24 h and 72 h after the operation respectively. The brain tissue of 3 rats in each group were removed quickly, first fixed at 4 °C for 2 hours in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and then fixed for 1 hour in 1% osmium tetroxide on ice. Thereafter, tissues were embedded in Epon 12 after dehydration in a graded acetone series (up to 100%), and sectioned using a Reichert ultramicrotome (Leica, Wetzlar, Germany) and stained with 3% uranyl acetate and lead citrate. Then, the sections were visualized under TEM (Technai G2 Spirit TWIN, FEI, USA) at 80 kV.
2.7. Histological staining
At 24 hours or 72 hours after the operation, the brain sections of rats were stained to observe the iron content and nerve injury.
After SAH for 24 hours or 72 hours, brain tissue sections were dewaxed. After staining with Nissl dyeing (Beyotime Biotechnology Co., Ltd, Shanghai, China), they were washed with distilled water, cleared in xylene, and sealed with neutral gum.
After SAH for 24 hours, the brain tissue sections were dewaxed and then put into Prussian blue dyeing (80 mL 20% HCl and 80 mL 10% potassium ferrocyanide) for 30 minutes. After washing with distilled water, the sections were restained with eosin and observed under the microscope.
2.8. Detection of ROS
At 24 hours and 72 hours after the SAH operation, rats in each group were injected with 5 mmoL/L dihydroetorphine (DHE) working solution by tail vein. After 30 minutes, rats were killed and fresh brain tissues were taken for frozen sections. After dripping with anti-fluorescence quenching agent, the sections were blocked. At the same time, the nucleus was located using Hoechst staining. The neurons were incubated with DHE for 90 minutes at 37℃ in the dark, and then washed with fresh media and observed under the fluorescence microscope.
2.9. Detection of GSH and malondialdehyde (MDA) content
The content of GSH and MDA in brain homogenate or neurons of SAH rats were determined in strict accordance with the instructions of the total glutathione test kit (S0052, Beyotime) and MDA test kit (S0131, Beyotime).
2.10. Western blot analysis
At 24 and 72 hours after the operation, the brain homogenates of rats in each group were taken, and the homogenates or neurons treated with different methods were lysed in cold radio-immunoprecipitation assay containing protease inhibitor cocktail (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 30 minutes. Then, the lysate was centrifuged at 4℃ for 20 minutes at 16000 g, and the supernatant was collected. The protein concentration was quantified using a Pierce bicinchoninic acid protein assay kit (Beyotime). Equal amount of proteins (20 µL/well) were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Next, the membranes were blocked with 5% skimmed milk powder for 1 hour, and probed with the primary antibodies and secondary antibody immunoglobulin G (IgG, 1:2000, ab205718, Abcam Inc., Cambridge, MA, USA). The gray values of target bands were analyzed with Image J software, with β-actin as the internal reference. The used antibodies were p65 (1:1000, ab16502, Abcam), p-p65 (1:2000, ab86299, Abcam), FTH1 (1:1000, ab183781, Abcam), GPX4 (1:1000, ab125066, Abcam), β-actin (1:1000, ab8227, Abcam), SLC7A11 (1:1000, PA1-16893, Invitrogen), SLC3A2 (1:1000, MA5-31209, Invitrogen) and glutamate cysteine ligase catalytic (GCLC) (1:1000, ab190685, Abcam).
2.11. Enzyme-linked immunosorbent assay (ELISA)
The levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β and IL-18 in brain homogenates or neurons treated with different methods were detected in strict accordance with the instructions of the rat IL-6 quantikine ELISA kit (R6000B, R&D Systems, Minneapolis, MN, USA), rat TNF-α quantikine ELISA kit (RTA00, R&D Systems), rat IL-1β/IL-1F2 quantikine ELISA kit (RLB00, R&D systems), and mouse IL-18 ELISA (7625, R&D systems).
2.12. Co-immunoprecipitation (Co-IP) assay
Co-IP assay was performed according to the instructions provided by the supplier (Thermo Fisher, Waltham, MA, USA). The cells or tissues were dissolved in IP buffer supplemented with protease inhibitor. The supernatant was incubated with antibody against SLC3A2 or IgG antibody at 4℃ with protein A/G agarose beads (Fast Flow, Beyotime) for 3 hours. The agarose beads were rinsed in cold phosphate-buffered saline, and then the binding proteins were eluted. The precipitated protein was measured by Western blot analysis and anti-ubiquitin. IgG was a control. Similarly, immunocoprecipitation of SLC7A11, GPx4 and ubiquitin C (UBC), SLC3A2 and OTUB1, GPX4 and OTUB1 was carried out in a similar way.
2.13. Statistical analysis
Statistical analysis was conducted by SPSS 21.0 (IBM Corp. Armonk, NY, USA). All the data were in normality distribution checked by the Kolmogorov-Smirnov test. Measurement data were expressed as mean ± standard deviation. The t test was used for comparisons between two groups. One-way or two-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and Tukey's multiple comparisons test was used for post-hoc test. The p value was obtained by a two-tailed test and p < 0.05 indicated a significant difference.