Animals
This study was conducted in accordance with the National Institutes of Health’s Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Adult male mice weighing 26-30g were obtained from the Animal Experiment Center at Southern Medical University. The mice were housed in a controlled environment with a temperature of 22 ± 1℃, humidity of 60 ± 5%, a 12h light/dark cycle, and ad libitum access to food and water.
Experimental design
Experiment 1.
The mice were randomly allocated into seven groups: Sham, SAH-3h, SAH-6h, SAH-12h, SAH-24h, SAH-48h, and SAH-72h. Proteins was extracted from the left cerebral cortex of mice in each group, and then western blot analysis was used to detect the expression level of p97, K48 poly-ubiquitinated proteins, P-eIF2α, and CHOP. To facilitate subsequent experiments, Immunofluorescence staining and TUNEL staining was used to explore the damaged site in the mice’s brain. Additionally, double immunofluorescence staining was performed to investigate the cellular localization of p97 and K48 poly-ubiquitinated proteins in the Sham and SAH-24 h group.
Experiment 2.
To explore the effect of p97 on neuronal apoptosis, mice were injected with the AAV9 virus using a stereotactic technique to achieve overexpress of p97 proteins 28 days before SAH. Immunofluorescence staining and western blot analysis were used to confirm the successful overexpression of p97. Eighteen mice were randomly divided into three groups: SAH, SAH + AAV9-NC, and SAH + AAV9-p97.
To assess the effect of p97 on EBI and long-term prognosis after SAH, mice were randomly divided into four groups, including Sham, SAH, SAH + AAV9-NC, and SAH + AAV9-p97. The expression of p97 and its downstream proteins, as well as neuronal apoptosis was assessed after 24 hours of SAH using western blot, immunofluorescence staining, and TUNEL staining. The long-time prognosis of mice was assessed at 21 days using the open field test, Golgi staining, and FJC staining.
Experiment 3.
To determine the appropriate dose of the selective p97 inhibitor NMS-873 (MCE, HY-15713), mice were randomly divided into six groups: Sham, SAH, SAH + Vehicle, SAH + NMS-873 (0.25mg/kg), SAH + NMS-873 (0.5mg/Kg), SAH + NMS-873 (1mg/kg). The optimal dose of NMS-873 (0.5mg/Kg) was selected for the subsequent studies based on the modified Garcia score and beam balance test.
To investigate whether inhibition of p97 would aggravate neuronal injury, mice were divided into four groups: Sham, SAH, SAH + Vehicle1, SAH + NMS-873. Western blot analysis was performed to evaluate the ratio of Bcl-2 to Bax. Immunofluorescence staining and TUNEL staining was performed to evaluate the number of apoptotic neurons.
Experiment 4.
To explore the role of the proteasome in p97-mediated anti-apoptosis pathway, the specific proteasome inhibitor, PS-341 (MCE, HY-10227), was administered via tail vein at 30 min prior to SAH. Mice were randomly divided into four groups: Sham, SAH + AAV9-p97, SAH + AAV9-Vehicle2, and SAH + PS-341. Western blot and Immunofluorescence staining were performed to evaluate the related molecules. FJC was conducted at 24h after SAH.
Experiment 5.
To study the relationship between ISR and p97, the selective inhibitor of ISR, ISRIB (MCE, HY-12495), was administered (via i.p.) at 1h after SAH induction. Mice were randomly divided into four groups: Sham, SAH + NMS-873, SAH + NMS-873 + Vehicle3, SAH + NMS-873 + ISRIB. Western blot, immunohistochemistry staining, and Nissl staining were conducted at 24 h after SAH.
Experiment 6.
To assess the effects of proteasome on ISR, ISRIB was used at 1h after SAH. Mice were randomly divided into four groups: Sham, SAH + PS-341, SAH + PS-341 + Vehicle3, and SAH + PS-341 + ISRIB. The left hemisphere of the mice were collected for western blot analysis at 24 h after SAH.
SAH models of mouse
The well-recognized endovascular perforation SAH model was performed in this study. Mice were anesthetized with 2.5% isoflurane in pure oxygen for 3 min and were maintained with 1.5% isoflurane. The mechanically ventilated maintained throughout the operation. Then, a sharpened 1.0 monofilament nylon suture was inserted into the left external carotid artery, and eventually used to perforated the bifurcation of the internal carotid artery. Mice underwent the same procedures except for perforation in the Sham group.
SAH grade
The severity of SAH were evaluated independently by two experimenters who did not know the group status of the mice in advance. The basal cistern of mice was divided into six sections, with each section assigned a score from 0 to 3 based on the presence of the subarachnoid blood clot. Mice with a total score of 7 or less were excluded from the experiment.
Assessment neurological performance
To assess the neurological function scores of mice at 24 h after SAH, the modified Garcia scoring system and beam balance test were performed by two independent researchers who were blind to the experiment previously. The modified Garcia scoring system is the same as in our laboratory’s previous study, with a total score of 3–18. In the beam balance test, mice were placed on a rough cylindrical stick with a diameter of 1.2cm, and their walking posture and the time of grip on the stick were evaluated. The higher the score on a scale of 0–5, the better the neurological function of the mice.
Open field test
We performed open field tests to detect voluntary movement in mice at 14 days after SAH and evaluate the long-term neurobehavior of mice. The trained mice before surgery were placed gently in a clean box of 50*50cm, and the activities of the mice within 10min were filmed. EthoVision XT 15 software was used to analyze the video of mouse activity and calculate the total distance of horizontal movement.
Drug administration and AAV9 injection
NMS-873 (6.25mg/ml) was administered at 30 min prior to SAH by intracerebroventricular injection, and the dosage of the drug was as described above. The vehicle1 solution consists of 10% DMSO (MCE, HY-Y0320), 5% Tween-80 (MCE, HY-Y1891), 40% PEG300 (MCE, HY-Y0873), and 45% saline. PS-341 (0.5mg/ml) was administered 30 min prior to SAH via the tail vein, and the injection dosage was 0.5mg/kg[28]. The vehicle2 solution consists of 1% DMSO and 99% saline. ISRIB (0.1mg/ml) was administered at 1 h after SAH via intraperitoneal injection, and injections were at 2.5mg/Kg[29]. The vehicle3 solution consists of 5% DMSO, 2% Tween-80, 20% PEG400 (MCE, HY-Y0873A), and 73% dextrose. We injected the AAV9 virus into the left cerebral cortex of mice at 28 days before SAH with the aid of the stereotaxic apparatus. We used the fontanelle as the origin of the coordinate, and the coordinate of virus injection is X = 3.3 mm, Y = 1.9 mm, Z= -2.5 mm. Each mouse was injected with 1µl AAV9 virus with a titer of 1E + 13 GC/ml. The construction diagram of the virus vector is shown in the supplemental materials.
Western blot analysis
After 24 hours of SAH, the anesthetized mouse brain tissue was separated and stored in a refrigerator at -80℃. The left cerebral cortex of the brain tissue was separated, and the lysate containing protease inhibitors and phosphatase inhibitors was added. The protein in the tissue was then extracted by full grinding. The supernatant was obtained by centrifugation and the protein concentration of the sample was measured using the BCA kit. Proteins totaling 30µg were electrophoresed using 10–12%SDS-PAGE gel and then transferred to PVDF membranes. PVDF membranes were blocked in 5% BSA solution at room temperature for 2h and incubated overnight at 4℃ with the following antibodies: anti-p97 (1:10000, ab109240, Abcam), anti-K48-ubiquitin (1:5000, ab140601, Abcam), anti-GRP78 (1:2000, 11587-1-AP, Proteintech), anti-P-eIF2α (1:1000, 9721s, CST), anti-eIF2α (1:1000, 9722s, CST), anti-CHOP (1:1000, 15204-1-AP, Proteintech), anti-Bcl-2 (1:1000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 50599-2-Ig, Proteintech), anti-β-actin (1:1000, 20536-1-AP, Proteintech). The PVDF membranes were incubated in an appropriate HRP conjugated secondary antibody (1:10000, S0001, Affinity) solution at room temperature for 1h. The proteins were visualized with an ECL chemiluminescence reagent kit (WBKLS0500, Merck) and analyzed with the ImageJ 1.5 software (National Institutes of Health, USA).
Fluoro-Jade C staining
We used Fluoro-Jade C (FJC) staining kit (Biosensis, USA) to identify the degenerating neurons. The samples used for staining were paraffin brain sections with a thickness of 4µm, and the procedures were carried out in strict accordance with the instructions. Finally, the DAPI solution was incubated at room temperature for 12min under dark conditions. Images were collected using the Nikon microscope, and FJC-positive cells were counted using ImageJ software.
Immunofluorescence staining
We used frozen sections (10 µm thickness) for immunofluorescence staining, and the samples were placed in boiling citric acid repair buffer (PH 6.0, Servicebio, China) for antigen retrieval. No antigenic retrieval was performed on the sections during co-staining with GFP. The samples were treated with 0.25% Triton-100 solution for 20min. The sections were then blocked at room temperature for 1h with 10% donkey serum solution and incubated overnight at 4℃ with the following antibodies: anti-p97 (1:800, ab109240, Abcam), anti-GRP78 (1:50, 11587-1-AP, Proteintech), anti-K48-ubiquitin (1:500, ab140601, Abcam), anti-NeuN (1:200, ab104224, Abcam). After the sections were fully cleaned with phosphate-buffered saline (PBS) solution, the sections were incubated at room temperature for 1h with immunofluorescent secondary antibodies as follows: Donkey anti-Rabbit IgG H + L (diluted 1:500, Alexa Fluor Plus 555, A31572, Invitrogen), Donkey anti-Mouse IgG H + L (diluted 1:500, Alexa Fluor Plus 488, A32766, Invitrogen). Images were captured by Nikon microscope.
Immunohistochemistry staining
Paraffin-embedded sections (4µm) were used for immunohistochemical staining and the sections were stained by using relevant kits (SP-9001, ZSGB-BIO). The process of antigen retrieval and membrane breaking of the samples was the same as IF. The samples were treated with 0.3% H2O2 in the kit for 10min. Samples were blocked by 5% goat serum at room temperature for 20min. CHOP primary antibody working solution was added and incubated at room temperature for 1h. After the samples were cleaned, biotin-labeled antibodies were added to the slices and incubated at room temperature for 15min. Then the samples were incubated with an enzyme-labeled working solution at room temperature for 15min. Finally, freshly configured DAB solution was added for color reaction. The images are captured using a light microscope system (Leica, DM2500).
TUNEL Immunofluorescence co-staining
TUNEL staining kit (G1502-50T, Servicebio) was used to detect apoptotic cells in paraffin-embedded sections of brain tissue, and the staining process was carried out according to the instructions. After the samples were digested by protease K and the membrane was broken, the appropriate amount of TUNEL reaction working solution was added to the samples and incubated at room temperature for 1h. The samples were then blocked with donkey serum for 1h. The remaining steps were the same as the immunofluorescence staining steps described above.
Nissl staining
Nissl staining can be used to evaluate the degree of cortical neuron damage. Fresh paraffin-embedded brain tissue sections were used for Nissl staining, which was done according to the kit instructions. In simple terms, the sample was incubated at room temperature with methyl violet solution for 15min and then rinsed with distilled water and treated with Nissl differentiation solution for about 30s. Images were obtained using the light microscope system (Leica, DM2500).
Golgi staining
Golgi staining was conducted as previously described[30]. The newly obtained mouse brain tissues were fixed in 0.1 M PBS and 4% paraformaldehyde solution at room temperature for 72h, and the brain tissues were cut into 1mm thick slices. The slices were immersed in Golgi stain solution (G1069, Servicebio) and left for 2 weeks at room temperature, away from light. The samples were placed in glacial acetic acid solution at 4℃ for 14h and were cut into 100µm thickness by a cryostat system (CRYOSTAR NX50, Thermo), and was then attached to a gelatin-coated glass slide, and finally developed with a developer-solution. Images information was obtained by using a 3D HISTECH bright field scanner (Pannoramic MIDI, China).
Statistical Analysis
For all data in this experiment, we used mean ± standard deviations (SD) to present. The GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA) was used for data statistics and analysis. Statistical analysis among multiple groups was performed using one-way ANOVA followed by Tukey's post hoc test. p ༜ 0.05 indicated a statistically significant difference.