Animal care and experiments were conducted following ethical approvals provided by the Small Animal Protection Board of Tianjin Medical University.
Animals
Adult male C57BL/6 mice weighing 22 - 25 g (8 - 10 weeks old) at the time of surgery were purchased from the Experimental Animal Laboratories of the Academy of Military Medical Sciences (Beijing, China). All mice were housed individually in a temperature- (20 ± 2 °C) and humidity-controlled (55 ± 5%) vivarium and maintained on a standard 12-hr light/dark cycle (7:00 a.m. to 7:00 p.m) with access to food and water ad libitum. All efforts were made to minimize the number of mice used and their suffering. In all experiments, the data were obtained by investigators blinded to the experimental design.
Experimental design
In the present study, the following separate experiments were conducted:
In Experiment 1, we investigated the dynamic changes in neuronal ER-mitochondrion physical contacts after TBI. Thirty-six mice were randomly assigned to the following six groups: sham, TBI 1 hr, TBI 3 hr, TBI 6 hr, TBI 12 hr, and TBI 24 hr. We used transmission electron microscopy (TEM) to examine the time profile of ER-mitochondrion physical tethering, as described below.
In Experiment 2, we investigated altered MAM-resident protein expression and mitochondrial ROS production after TBI. One hundred eighty mice were randomly allocated to the following six groups: sham, TBI 1 hr, TBI 3 hr, TBI 6 hr, TBI 12 hr, and TBI 24 hr. The ER-mitochondrion tethering proteins phosphofurin acidic cluster sorting protein 2 (PACS2) and mitofusin-2 (MFN2) were analyzed by immunofluorescence staining and western blot. The expression levels of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), glucose-regulated protein 75 (GRP75), voltage-dependent anion channel 1 (VDAC1), and Sigma 1 receptor (Sigma-1R) were also measured by western blotting. The mitochondrial ROS content was detected by MitoSOX staining as described below.
In Experiment 3, we studied ER stress, UPR signaling, and neuroinflammatory response after TBI. Thirty-six mice were randomly allocated into the following six groups: sham, TBI 1 hr, TBI 3 hr, TBI 6 hr, TBI 12 hr and TBI 24 hr. The expression levels of GRP78, phosphorylated protein kinase (PKR)-like ER kinase (p-PERK), PERK, p-eIF2α, eIF2α, activating transcription factor 4 (ATF4), cleaved interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNFα) were determined by western blot.
In Experiment 4, we examined cleaved Caspase 12-dependent ER stress-mediated apoptosis after TBI. Thirty mice were randomly assigned to the following five groups: sham, TBI 6 hr, TBI 12 hr, TBI 24 hr, and TBI 72 hr. Immunoblot assays were used to detect the expression of cleaved Caspase 12, cleaved Caspase 3, cleaved Poly(ADP-Ribose) Polymerase 1 (PARP1), C/EBP homologous protein (CHOP), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (BAX), and cytochrome C (Cytc).
In Experiment 5, we studied the effects of Pacs2 silencing on MAM-resident protein expression, ROS production, ER stress and UPR activation, and the neuroinflammatory response at 6 hr after TBI. Forty-eight mice were randomly assigned to the following four groups: sham, TBI, TBI + PACS2 siRNA, and TBI + NC siRNA. The protein expression of PACS2, Mfns, IP3R1, GRP75, VDAC1, Sigma-1R, GRP78, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, cleaved IL-1β, and TNFα was assayed by immunoblot, and mitochondrial ROS production was detected by MitoSOX staining.
In Experiment 6, we investigated the potential effects of PACS2 knockdown on BBB functions, ER stress-mediated apoptosis, and neurological outcomes. One hundred twenty mice were randomly allocated into the following four groups: sham, TBI, TBI + PACS2 siRNA, and TBI + NC siRNA. Neurological scores were used to evaluate the neurological functions of the mice at preinjury and at postinjury days 1, 3, 5, 7, and 14. At 72 hr post injury, extravasation of Evans blue (EB) and the brain water content (BWC) were measured in all groups. cleaved Caspase 12, cleaved Caspase 3, cleaved PARP1, CHOP, Bcl-2, BAX, and Cytc expression levels were also determined by western blot. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and immunofluorescence staining were performed in each group at 72 hr after injury.
All mice in the sham group were subjected to the same surgical procedure but were not treated with the controlled cortical impact (CCI) device.
Severe traumatic brain injury model
CCI-induced brain injury is an extensively characterized and broadly used preclinical model of head injury[21, 23, 24]. In brief, a digital electromagnetic CCI device (eCCI Model 6.3; Custom Design, Richmond, VA, USA) was used to establish the sTBI model in male C57BL/6 mice. The mice were allowed to adapt to their new environment for 1 week before surgery and then anesthetized with 10% chloral hydrate. The depth of anesthesia was assessed by monitoring the pedal withdrawal reflex and respiratory rate. Then, the mice were placed in a stereotaxic apparatus, and the surgical site was clipped and cleaned with Nolvasan scrubs. A 4.0-mm hole was drilled into the right parietal bone to expose the dura. The CCI device subsequently impacted the skull at a depth of 2.5 mm and a velocity of 5 m/s over a period of 200 ms. The incision was closed immediately following injury, and the mice were then placed in heated cages to allow recovery from anesthesia at room temperature (RT). Fig. 1 shows the hematoxylin and eosin (H&E) staining of the cerebral cortex at 24 hr after CCI, which confirmed the severe injury of the mice used in this study.
Transmission electron microscopy and MAM quantification
Tissue (1 mm × 1 mm) samples were obtained from the perilesional cortex of TBI and sham mice and fixed in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M PBS (pH 7.4) overnight at 4°C for 1 hr. The tissues were then washed in 0.1 M phosphate buffer (pH 7.4) and postfixed in 1% osmium tetroxide in the same buffer for 30 min at RT. The specimens were dehydrated through a graded series of ethanol and embedded in a mixture of Epon resin. Serial sections were cut on an ultratome and double stained with uranyl acetate and lead citrate. Finally, the ultrathin sections were examined by TEM at 80 kV.
ER-mitochondrion imaging and quantification were performed according to a previous study[25]. Briefly, 60 images of mice in each experimental group (6 mice from each group) were obtained at ×6,800 magnification, and ImageJ (National Institutes of Health, Bethesda, Maryland, USA) was used to analyze ER-mitochondrion contacts and mitochondrial morphology. We delineated the mitochondria and ER membranes using the free-hand tool. Two independent investigators blinded to the experimental design calculated the ratio of ER adjacent to mitochondria to mitochondrial perimeter, and the total number and area of mitochondria.
Immunofluorescence and image analysis
At designated time points, mice were sacrificed with an overdose of 10% chloral hydrate and then immediately perfused through the heart with PBS followed by 4% paraformaldehyde. The brain was rapidly dissected and embedded in OCT medium (Sakura, Oakland, CA, USA). Coronal sections of 8-μm thickness were cut on a cryostat at -20°C and imprinted on poly-L-lysine-coated slides. The sections were stained for NeuN (neuronal marker), glial fibrillary acidic protein (GFAP, astrocyte marker), and PACS2 and Mfn2 (markers of ER-mitochondrion contacts).
In brief, the sections were fixed with 2% paraformaldehyde lysine periodate (PLP), rinsed three times with PBS (pH 7.4), and blocked with 1% normal donkey serum in PBS containing 0.1% Triton X-100 PBST at RT for 1 hr. The sections were then incubated with a rabbit anti-PACS-2 antibody diluted at 1:1000 (Abcam, Cambridge, MA, USA), a rabbit anti-Mfn2 antibody diluted at 1:1000 (Abcam), a mouse anti-NeuN antibody diluted 1:100 (Cell Signaling Technology, Danvers, MA, USA), and a mouse anti-GFAP antibody diluted at 1:1000 (Abcam, Cambridge, MA, USA) in PBST containing 1% normal donkey serum at 4°C overnight followed by extensive washing with PBS. Finally, the sections were incubated with Alexa Fluor-conjugated anti-mouse or anti-rabbit IgG (1:1000, Invitrogen, Grand Island, NY, USA) for 3 hr at RT. The nuclei were counterstained with Hoechst for 5 min.
Images of each section were captured using a fluorescence microscope (Olympus IX81, Tokyo, Japan), and the data were analyzed from 15 randomly selected microscopic fields (five fields per section x three sections per mouse) with ImageJ (National Institutes of Health, Bethesda, Maryland, USA).
Western blotting
Mice were sacrificed by transcardiac perfusion with cold PBS to eliminate the proteins expressed by blood cells at designated time points. Their brain tissues were homogenized in ice-cold RIPA buffer (Beyotime) containing phenylmethylsulfonyl fluoride (PMSF, 1 mM final) for 30 min and then centrifuged for 10 min (12,000 rpm, 4℃). After centrifugation, the supernatants were collected and boiled with 4x sample buffer at 95℃ for 10 min. The total protein content was determined by the BCA protein assay kit (Thermo). Proteins (8 μg per lane) and prestained molecular weight markers (Thermo) were separated by SDS/PAGE and transferred to PVDF membranes (Roche, Canada), which were then blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 2 hr at RT. After blocking, the blots were incubated overnight at 4℃ with primary antibodies (Table 1), rinsed with TBS, incubated with the appropriate HRP-conjugated secondary IgG for 1 hr at RT and then developed with the ECL system (Millipore, Billerica, MA, USA). Protein expression was quantified by ImageJ (National Institutes of Health, Bethesda, Maryland, USA) according to the mean pixel density of each protein band, and β-actin was employed as a loading control.
Mitochondrial reactive oxygen species content
MitoSOX-based assays were used to detect mitochondrial ROS production according to the manufacturer’s instructions (Thermo Fisher, Waltham, USA) and previous studies [26-28]. Perilesional cortex tissues were dispersed into a single-cell suspension and washed with 10 mM PBS twice. A 5 mM Mito SOXTX reagent stock solution in HBSS/Ca/Mg buffer was diluted to a 5 μM MitoSOX reagent working solution, in which the cells were incubated for 10 min at 37°C. Fluorescence signals were read and independently and blindly quantified by ImageJ. All experimental procedures were performed in the dark.
siRNA transfection
siRNA transfection was performed in vivo according to the method of Zhao L. et al. [29]. To knockdown Pacs2, a total of 1.32 μg/5 μL PACS-2 siRNA (Sangon Biotech, Shanghai, China) or NC siRNA (Sangon Biotech, Shanghai, China) was diluted with an equal volume of EntransterTM-in vivo transfection reagent (Engreen, Beijing, China). The solution was mixed gently and injected intracerebroventricularly (i.c.v) into mice 48 hr prior to TBI. In terms of the i.c.v injection[30], a 1-mm cranial burr hole was drilled into the skull, and a 30-gauge needle on a Hamilton syringe was implanted into the lateral ventricle using the following stereotactic coordinates: 1.5 mm posterior to bregma, 1.0 mm right lateral to the midline, 2 mm in depth, with an injection speed of 1μL/min (total volume=5μL). The transfection rate is approximately 85 ± 5%.
Modified neurological severity scores
The modified neurological severity score (mNSS) was used to evaluate neurological function, as described previously[31]. Neurological assessments were performed at baseline before the injury and at post injury days 1, 3, 5, 7, and 14 using the mNSS. The assessments included motor, sensory, reflex, and balance tests. These scores were used 1) to ensure the relative uniformity in injury severity and 2) to compare neurological impairments among mice receiving different treatments. The tests were performed by two independent observers who were blinded to the experimental conditions and treatments.
Brain water content
Hemispheric cerebral edema was determined by measuring the BWC, as previously described[31]. Mice were sacrificed with an overdose of chloral hydrate at 72 hr post injury. Their brains were promptly removed, and the hemispheres were immediately weighed (wet weight) and then placed in an incubator at 100°C for 24 hr. The samples were weighed again to determine the dry weight, and the BWC was calculated as follows: (wet weight - dry weight)/wet weight x 100%.
Evans blue dye extravasation
The BBB permeability of the cerebral hemispheres was assessed by measuring the extravasation of EB dye 72 hr after TBI. EB dye injected intravenously binds instantaneously to albumin and other plasma proteins and serves as a marker for plasma exudation. In brief, EB (2% in PBS, Sigma) was injected slowly through the jugular vein (4 ml/kg) and allowed to circulate for 1.5 hr. Then, mice were sacrificed and transcardially perfused with PBS followed by 0.9% saline. The hemispheres were removed, frozen in -55°C isopentane and freeze-dried. The freeze-dried specimens were homogenized in formamide (1:20) and incubated at 60°C overnight, and the homogenates were then centrifuged at 14000 rpm for 30 min to collect the supernatant. The EB content in the supernatant was determined spectrophotometrically at OD 620 nm (Thermo Scientific). The tissue EB concentration was quantified using a standard linear curve and expressed as micrograms per gram of brain tissue.
Terminal deoxynucleotidyl transferase dUTP nick end labeling assay
The TUNEL assay is a well-defined method for detecting apoptotic DNA fragmentation in a cell[32]. We used the In Situ Cell Death Detection Kit, POD (Roche, Germany) to detect apoptosis in the perilesional cortex of the mouse brain at 72 hr after TBI. According to the manufacturer’s instructions, the sections were fixed in acetone for 8 min at 4°C. After being rinsed with PBS, the brain sections were treated with 3% BSA for 30 min at 37°C and then incubated with the TUNEL reaction mixture in the dark for 90 min at 37°C. The nuclei were counterstained with Hoechst for 5 min. Images of each section were captured using a fluorescence microscope (Olympus IX81, Tokyo, Japan), and the data were analyzed from 15 randomly selected microscopic fields (five fields per section x three sections per mouse) with ImageJ (National Institutes of Health, Bethesda, Maryland, USA).
Data analysis
The data are presented as the mean ± standard deviation (SD) and were analyzed using Prism 8.3.1 (GraphPad Software, San Diego, CA). Parameters were compared by ANOVA followed by Tukey’s multiple comparisons test. Pearson's correlation coefficients (r) were calculated to assess the strength of relationships. P values < 0.05 were considered statistically significant.