2.1. Animals
Male adult C57BL/6 mice (6–8 weeks, weighting 23-28g) were utilized for all experiments in this study, which approved by the laboratory animal centre of Xuzhou Medical University. The mice were acclimatized for at least a week before surgery in a given temperature (22–25°C) and a controlled humidity (50% relative humidity), and a 12h dark and light cycle. According to animal ethical requirements, all mice were allowed to access to food and water freely, except for a 12-hour period preceding the surgery.
2.2. Traumatic Brain Injury model and drug administration
TBI models was established using the modified Feeney’s weight drop injury (WDI) model, and operational details are recorded in previous literature[22, 23]. briefly, the mice were anesthetized initially with 3% isofluranemaintain, followed by maintenance of anesthesia with 1.5% isoflurane. Subsequently, they were secured in a stereotaxic apparatus, and their hair was removed using a razor. The scalp was incised longitudinally to reveal the skull, and a 5 mm diameter circular craniotomy was performed on the left skull between the bregma and lambda. The bone flap was gently removed, taking care to avoid damaging the dura mater. The weight dropped in this study was 200 g from a height of 10 cm, inducing a deformation depth of 1.0 mm with a dwell time of 180 ms. Post-surgery, the bone flap was replaced and the incision sutured. Anesthesia was then discontinued, and the mice were placed on pure oxygen for 5–10 minutes to facilitate rapid recovery. Mice in the sham group underwent the same procedure as those in the TBI group, but without the weight drop injury.
Before administering the drug, Fer-1, a specific inhibitor of ferroptosis (HY-100579, MCE, USA), was prepared by diluting it in 0.01% DMSO in saline to achieve a final concentration of 1 mmol/L. Following the WDI, either 3 µL of Fer-1 or a vehicle solution was injected into the right lateral ventricle (coordinates: AP -0.5mm, lateral 1.0mm, and 2.0mm beneath the pia mater) 30 minutes post-injury. These procedures were informed by protocols established in prior research. All surgical interventions were conducted on a constant temperature heating pad to maintain the mice's body temperature at 37°C.
2.3. Transmission electron microscopy (TEM)
Mice were perfused with 4% paraformaldehyde dissolved in Phosphate Buffer Saline (PBS). Subsequently, brain samples from the left hippocampus CA1 region were post-fixed in 0.1 M phosphate-buffered paraformaldehyde (4%) and glutaraldehyde (2.5%). The brain samples were then stained with 2% uranyl acetate (UA), dehydrated in graded acetone solutions, and embedded in epoxide resin. Next, sections (70 nm) were stained with uranyl acetate and lead citrate. Through this method, the ultrastructural details of the cells were visualized using transmission electron microscopy (Teneo VS, FEI, Czech Republic).
2.4. Slice preparation
Mice were anesthetized with 3% isoflurane and their limbs immobilized before undergoing transcardial perfusion with 0.9% saline at 4°C, followed by perfusion with 4% paraformaldehyde at the same temperature. Subsequently, the brains were carefully extracted in their entirety and post-fixed in 4% paraformaldehyde for 24 hours at 4°C. They were then dehydrated successively in 20% and 30% sucrose solutions for 24 hours each at 4°C. Following dehydration, the brain tissues were sectioned coronally into 13 µm slices and stored at -80°C until further use.
2.5. PI staining
Mice received intraperitoneal injections of propidium iodide (PI, C0080, Solarbio, China) at a concentration of 0.4 mg/ml (100 µl) one hour prior to euthanasia. Subsequently, the mice were euthanized, and their brain tissues were harvested and immediately cooled to 4°C. These tissues were then rapidly frozen for 30 seconds at -80°C. Continuous coronal sections of the hippocampus were prepared using a cryostat and stored at -80°C in a light-protected environment. The sections were immersed in anhydrous ethanol for 5 minutes and counterstained with DAPI for 30 minutes in preparation for microscopic examination. Three slices from each animal were observed and photographed using a fluorescence microscope (MG607CCool, ZEISS, Germany). PI-positive cells in both the CA1 and CA3 regions were quantified at 200x magnification using microscopic fields. Cell degeneration counts were performed using NIH Image J software (Bethesda, MD, USA).
2.6. Perl’s staining
Iron deposition in hippocampal tissues was assessed using Perl's Prussian blue staining (G1428, Solarbio, China), with slight modifications from previously described methods[24]. Initially, slices were equilibrated to room temperature (25–30°C). They were then incubated in Perl's solution (5% potassium ferrocyanide mixed with 5% hydrochloric acid) for 20 minutes, followed by three washes in PBS. Counterstaining was performed using 3,3'-diaminobenzidine and hematoxylin. Iron-positive cells within the CA1 and CA3 regions of the hippocampus were identified and quantified under a microscope (MG607CCool, ZEISS, Germany) at 200x magnification.
2.7. Western blotting
Mice were initially euthanized using carbon dioxide, after which the hippocampus were carefully dissected under ice-cold conditions. The hippocampal tissue samples were homogenized in RIPA lysis buffer (P0013B, Beyotime, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF, ST507, Beyotime, China). Following complete lysis, the homogenates were centrifuged at 13,000 rpm for 20 minutes at 4°C, and the supernatants were collected. The total protein concentration was then determined using a BCA protein assay kit (KGB2101-100, KeyGEN BioTECH, China). Based on their molecular weights, the protein samples were subjected to SDS-PAGE on either a 10% or a 15% polyacrylamide gel. Subsequently, the proteins were electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (ISEQ00010, Millipore, USA). The membrane was blocked with 5% skim milk at room temperature for 2 hours and then incubated with primary antibodies overnight at 4°C. Following three washes in Tris-buffered saline with Tween-20 (TBST) for 10 minutes each, the polyvinylidene difluoride (PVDF) membrane was incubated with the secondary antibody at room temperature for two hours. Detection of the signals was performed using enhanced chemiluminescence (ECL) substrate (P10200, NCM, China). The intensity of the protein bands was quantified using NIH Image J software (Bethesda, MD, USA), with the results normalized to GAPDH levels. The primary antibodies employed in this study included polyclonal rabbit anti-GPX4 (1:1000, Beyotime, China), monoclonal rabbit anti-TFRC (1:1000, Bioss, China), polyclonal rabbit anti-SLC40A1 (1:1000, Proteintech, USA), and mouse anti-GAPDH (1:1000, Proteintech, USA).
2.8. Measurement of malondialdehyde(MDA)
To assess lipid peroxidation levels in the mouse hippocampus at various time points following TBI, we harvested the ipsilateral hippocampal tissue from the injured groups and the equivalent region from the control group. The MDA levels were then measured using a commercial assay kit (S0131S, Beyotime, China).
2.9. Measurement of ROS
ROS levels were quantified using a ROS assay kit following the manufacturer's instructions (BB-470512, BestBio, China). Hippocampal samples were collected and homogenized in ice-cold conditions. For each sample, 200 µl of the solution, containing 2 µl DCFH-DA, was incubated at 37°C in the dark for 30 minutes. Subsequently, the samples were transferred to a fluorescence microplate (PerkinElmer VICTOR Nivo, Revvity, Finland), with an argon laser (488–535 nm) for immediate analysis.
2.10. Behavioral test
The open field test (OFT), novel object recognition test (NOR), Y-maze test and Morris water maze test (MWM) were performed 21 days post-injury as described previously [25, 26].
For the OFT test, briefly, the open field (40cm length × 40cm width) was placed in the middle of the room, free of other noises and unexpected interruptions. And before each trial, the field was cleaned with 75% alcohol and left to air dry completely. Then the mice were placed in the centre of the field, and recorded the free movement of the mice in 5 minutes by the camera overhead. The movement distance in centre region and the time spent in centre region were automatically analyzed by an behavioral tracking system (Any-maze, Stoelting, USA).
For the NOR test, the mice were placed in the open field (40cm length × 40cm width) with two identical objects of similar surface and size, and recorded for 5 minutes. Then 2 hours later, the mice were reinsert into the field, and one of a familiar object was replaced by a novel object, and allowed to explore the field for 5 minutes. An normal mouse has the ability to remember and distinguish objects, spend more time on exploring the novel object than the familiar one. But an injuried mouse took the same amount of time on novel and familiar objects. The exploration of the mice of objects was manifested in touch and sniff at a distance of less than 2cm. The exploration of the novel object discrimination index was calculated as (time spent in novel object region)/(time spent in novel object region + time spent in familiar object region).
For the Y-maze test, the ice were placed in the centre of the Y-maze with three arms (each length 30cm, width 6cm,height 15cm). The mice were allowed to free explore the maze for 10 minutes and with video recorded. The alternation value was the number of times the mouse entered into three arms in a row. The percentage of spontaneous alternation performance (SAP%) was calculated as (number of spontaneous alternation)/(number of total arm entries − 2) ×100%. In a Y-maze test, a lower SAP%, which reminded us that spatial memory ability of the mouse impaired.
For the MWM test, the experiment was conducted in a round pool, which diameter was 120cm and depth was 50cm. A white platform, which diameter was 6cm and depth was 30cm, lay in the centre of southwest quadrant of the pool. The pool was filled with water and milk powder was added to make the water white, the temperature of the pool was 24°C. And the platform placed 1cm beneath the water surface. In each trail, each mouse was released from one of four directions (east, west, north, south), and allocated 60 seconds to search for the hidden platform. Each trail ended with the mice has finding the platform. If the mouse failed to find the platform in 60 seconds, the mouse was guided to the platform and allowed to remain on it for 15 seconds. After each trail, the mice were placed in a dry cage. In the training phase, each mouse was tested in four trails per day for four consecutive days. On the fifth day, each mouse was tested from one direction to access cognitive function. The movement of each mouse was recorded by camera above the pool. And the results, including latency time, swimming distance and the number of times it crossed the original platform, were calculated by an behavioral tracking system (Any-maze, Stoelting, USA).
2.11. Statistical analysis
In the experiment, all data were expressed as mean ± standard deviation. Statistical analysis of the data was performed using Prism 9 software. Multiple group comparisons were conducted using one-way ANOVA, and intergroup comparisons were made using Dunnett's T-test. Differences were considered statistically significant at P < 0.05.