Patients
Human brain tissue was obtained from the tissue bank of the Institutional Review Board-approved Department of Neurosurgery at the First Affiliated Hospital of Nanjing Medical University (NMU; Nanjing China) (STable 1). Brain tissue resected from the pericontusional area of the temporal or frontal cortices of severe TBI patients who underwent decompressive craniectomy. Brain tissue resected from the temporal cortices of epilepsy patients who underwent surgical treatment was used as a control.
Mice and protocols
This study encompassed male C57BL/6 mice (12 weeks of age) weighing 24±5 g. RIPK1−/− mice were obtained from the Model Animal Resource Center (MARC; Nanjing, China), beclin1 transgenic mice from the JAX lab, GFP-LC3 transgenic mice from Beijing CasGene Biotech, and Drd1-/- and Drd2-/-mice from the lab of Dr. Zhou [21]. All transgenic mice were of C57BL/6J background. Standard laboratory animal food and water were provided by the Animal Center of NMU. Mice were maintained in a pathogen-free environment and housed on 12-h day/night cycles at an ambient temperature of 22°C ± 2°C. All animal experiments were approved by the Animal Care and Use Committee of NMU.
Cortical lesion volume and brain water content were measured on day 3 after brain injury, and behavioral tests were performed on post-TBI days 7–13. Fourteen days after brain injury, mice were sacrificed and samples were obtained for other designated experiments.
Traumatic brain injury
As previously described, controlled cortical impact (CCI) was applied to cause cerebral contusion [5]. Briefly, injury to the right cortex was performed using a 3-mm flat-tip impounder with a velocity of 6 m/s, a depth of 0.6 mm, and a duration of 100 ms.
Primary cortical neurons and stretch injury (SI)
Primary cortical neurons were isolated from 14-day-old embryonic mouse cortex and used for designated experiments on 14 day in vitro, according to the study protocol. TBI was produced via mechanical stretch of the neurons cultured on a flexible silastic membrane using a pneumatic pressure pulse as described previously [22].
Oxygen–glucose deprivation (OGD)
In brief, the medium was changed to Dulbecco’s modified Eagle’s medium (DMEM) without glucose and serum. Hypoxia was induced by placing the cells in a hypoxic chamber (Billups–Rothenberg, Inc., San Diego, CA, US) that we infused with 90% N2, 5% H2, and 5% CO2 for 30 min to obtain <0.5% O2. The chamber was then sealed and kept at 37 °C, and primary neurons were incubated at 37 °C for 2 h prior to reperfusion.
Drug treatment
DA, A-68930, bafilomycin A (BAF), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). We administered A-68930 intraperitoneally to mice at a dosage of 0.5 mg/100 g/day for 14 consecutive days, starting 8 h before brain injury. BAF and 3-MA were used according to our previous study [22]. Saline (vehicle) was used in the sham group.
siRNA-Mediated Gene Silences
siRNA was chemically synthesized by GenePharma (Shanghai,China).The siRNA sequences were: siDrd1 (5′-GGGAGACUAAAGUCCUGAAdTdT-3′), siDrd2 (5′-GGCCAUGCCUAUGUUGUAUdTdT-3′), siDrd3 (5′-GGUGGAGUCUGGAAUUUCAdTdT-3′), siDrd4 (5′-CCUGGAGAACCGAGACUAUdTdT-3′), siDrd5 (5′-CUGCCUAUGUCCACAUGAUdTdT-3′), siChip (5′-CCTATGACCGCAAGGAATT-3′), siFbxo10 (5′-GGATCCTTCGCGGGACGTCCT-3′), siMarch7 (5′-GGACUUAUGUAGAAUUUGUdTdT-3′).
Lactate dehydrogenase (LDH) release assay
LDH release was done with a commercial assay kit (catalog no. ab102526, Abcam) according to the manufacturer’s instructions.
Cortical lesion volume and brain edema
An MRI apparatus for small animals at 11.7T (Bruker, AVANCE 500WB) at the Animal Core Facility of NUM was used to measure cortical lesion volume. As previously described, we calculated lesion volume from the summation of defective areas of on each slice, and multiplied it by slice thickness [23]. The brain water content was measured as water content: (%) = ([wet weight-dry weight]/wet weight) x 100% [5].
Immunoblot Analysis
The level of target protein was measured by immunoblot analysis as the previous study [5]. The follow primary antibodies were used: p62 (catalog no. ab56416, Abcam), LC3 (catalog no. 2775s, Cell Signaling Technology), COXIV (catalog no. 11967, Cell Signaling Technology), NeuN (catalog no. ab104224, Abcam), β-actin (catalog no. 3700, Cell Signaling Technology).
Reactive oxygen species (ROS)
Dihydroethidium (DHE; catalog no. D11347; Thermo Fisher) staining was applied to assess the production of ROS in brain tissues, and samples were incubated with DHE and DAPI according to the manufacturer’s instructions. We observed the frozen slices under confocal microscopy and assessed expression with Image J (NIH, USA).
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-1β, IL-6, and TNF-α in the brain tissues were measured by ELISA kits (Elabscience, China) according to the manufacturer’s instructions. The absorbance value was evaluated with a microplate spectrophotometer (Tecan, Switzerland).
TUNEL staining
A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit was purchased from Roche (catalog no. 12156792910, Roche, Germany). The mice were sacrificed and perfused with 0.1 mM PBS (pH 7.4). In brief, coronal frozen sections (7 mm in thickness) were incubated with TUNEL reaction mixture (red) and then stained with DAPI (blue), and the apoptosis was assessed as the number of red positive cells/DAPI-stained cells.
Immunohistochemistry Analysis
We executed immunohistochemistry to assess CCI-induced inflammation with IL-1b (ab9787, abcam) antibody. Each section was observed and imaged randomly according to the previous study [24].
Neurological severity score (NSS)
In brief, a 10-point NSS was applied to assess motor function, beam balance, and startle reflexes [5]. One point was recorded for failing to complete each task.
Novel object recognition (NOR)
On test day 1, mice were placed on an empty surface for 5 min; on day 2, we placed the mice on the same surface that included two identical objects for 5 min; on day 3, we placed the mice on this same surface that included an “old” object and a “new” object for 5 min. The percentage of time spent in probing the “new” object was recorded to evaluate the extent of learning and memory.
Morris water maze (MWM)
The MWM was implemented by the same experimenter at the identical time with a tracking device and software (Chromotrack 3.0, San Diego Instruments). In a series of daily trials, we placed mice at a random locations (north, south, east, and west), and a probe trial was done 2 h after the last hidden-platform trial.
Statistical analysis
All data are expressed as the means ± standard deviation (SD). We used the unpaired Student’s t test was used to compare the differences between two groups with Prism Software 6.04 (GraphPad Software, Inc.). NSS and MWM data were analyzed by two-way ANOVA for an overall statistic, followed by Tukey’s test for between-group comparisons. Significance was defined as a P value <0.05.