Animals
12–16 weeks old C57BL/6 mice and 5xFAD mice were used (Additional File 1: Fig. 1A). 5xFAD mouse model is a well-characterized mouse model of AD, with expression of human APP and PSEN1 transgenes with a total of five AD-linked mutations: the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1 [22]. All procedures are approved by the Institutional Animal Care and Use Committee at the University of Southern California using the US National Institutes of Health (NIH) guidelines.
Randomization and blinding
All animals were randomized for their genotype information and surgical procedures, and included in the analysis. The operators responsible for the experimental procedures and data analysis were blinded and unaware of group allocation throughout the experiments.
Closed-head mTBI mouse model
Mice received a mild closed-head impact following the procedures previously reported with minor modifications [23]. Briefly, for a precise impact mimicking mTBI, we utilized a stereotactic frame with adjustable angle (Stereotaxic Alignment System, KOPF) to ensure the impacted surface (the impact central point at Bregma − 2 mm and lateral 2.5 mm) of the skull was presented perpendicular to the impactor (Additional File 1: Fig. 1b, Brain and Spinal Cord Impactor, #68099, RWD Life Science). We also custom-made plastic flat-tip impactor (RWD Life Science) to reduce the impact. Mice were anesthetized with ketamine/xylazine (90 mg/kg, 9 mg/kg; i.p.), then the head was fixed with a 15° angle, and a gel pad was placed under the head in order to maximally avoid the skull fractures and intracerebral bleeding. An incision was made to expose skull surface after anesthesia and then the mice were subjected to an impact using a 4 mm plastic flat-tip impactor. The velocity was 3 m/s, the depth was 1.0 mm and the impact duration was 180 ms (see Additional File 1: Fig. 1c–d). After the procedure, the mice were put back to their cages with heating to recover from the anesthesia. For sham-operated mice, the same procedure was performed as the mTBI group except the tip was set 2.0 mm above the skull.
Open-head severe TBI (sTBI) mouse model
Mice received the sTBI following the procedures previously reported [24]. Briefly, mice were anesthetized with ketamine/xylazine (90 mg/kg, 9 mg/kg; i.p.), and the mouse head was then fixed in the stereotactic frame (Stereotaxic Alignment System, KOPF). A craniotomy was made using a drill and 4.5-mm trephine in the center between Lambda and Bregma, and then the bone flap was removed. Mice were subjected to an impact using a 4 mm metal flat-tip impactor (Brain and Spinal Cord Impactor, #68099, RWD Life Science). The impact central point was Bregma − 2.5 mm and lateral 2.5 mm. The velocity was 3 m/s, the depth was 1 mm and the impact duration was 180 ms. Then the scalp was closed with suture, and the mice were put back to their cages to recover from the anesthesia. For sham-operated mice, the same procedure was performed as the sTBI group except the tip was set 2.0 mm above the skull.
Behavioral tests
Rotarod test
Mice were trained on an accelerating (5–20 rpm) rotating rod (rotarod) for 3 days before TBI. Test sessions consist of six trials at a variable speed (an initial velocity of 5 rpm was used for the first 10 s, a linear increase from 5 to 10 rpm for the next 30 s, and a linear increase from 10 to 20 rpm between 40 and 80 s). The final score was determined as the mean time that a mouse was able to remain on the rod over six trials.
Foot fault test
The foot-fault test was performed as previously described [25]. Mice were placed on hexagonal grids of different sizes. Mice placed their paws on the wire while moving along the grid. With each weight-bearing step, the paw may fall or slip between the wire. This was recorded as a foot fault. The total number of steps (movement of each forelimb) that the mouse used to cross the grid was counted, and the total number of foot faults for each forelimb was recorded.
Contextual fear conditioning
A hippocampus-dependent fear conditioning test was performed as previously described [26]. The experiments were performed using standard conditioning chambers housed in a soundproof isolation cubicle and equipped with a stainless-steel grid floor connected to a solid-state shock scrambler. The scrambler was connected to an electronic constant-current shock source that was controlled via Freezeframe software (Coulbourne Instruments). A digital camera was mounted on the steel ceiling and behavior was monitored. During training, mice were placed in the conditioning chamber for 4 min and received two-foot shocks (0.25 mA, 2 s) at 1-min interval starting 2 min after placing the mouse in the chamber. Contextual memory was tested the next day in the chamber without foot shocks. Hippocampus-dependent fear memory formation was evaluated by scoring freezing behavior (the absence of all movement except for respiration). For the contextual fear conditioning paradigms, the automated Freezeframe system was used to score the percentage of total freezing time with a threshold set at 10% and minimal bout duration of 0.25 s.
Determination of lesion volume
Mouse brains were cut into serial 20 µm cryostat sections. Every 10th section was stained with cresyl violet and the lesion area was determined using the Image J analysis software. Sections were digitized and converted to gray scale, and the border between TBI and non-TBI tissue was outlined. The lesion volume was calculated by subtracting the volume of the non-lesioned area in the ipsilateral hemisphere from the volume of the whole area in the contralateral hemisphere and expressed in mm3 as previously described [27].
Assessment of edema
Brain water content is a sensitive measure of cerebral edema which was determined using the wet/dry method as previously described [27]. Mice were decapitated and brains were rapidly removed from the skull. The fresh brain was weighed in 3 mm coronal sections of the ipsilateral cortex, centered upon the impact site, then dehydrated for 24 h at 110 °C and reweighed. The percentage of brain water content was calculated using the following formula: (wet weight − dry weight)/(wet weight) × 100.
Tracer injection to detect BBB leakage
Mice were injected via the tail vein with Alexa Fluor 555-cadaverine (6 µg/g; Invitrogen, A30677) [28] dissolved in saline 1, 3 and 8 days after mTBI. After 2 h mice were anesthetized and perfused with phosphate-buffered saline (PBS) at pH 7.4, and the brains were collected.
Evaluation of BBB permeability
BBB permeability was assessed by measuring the extravasation of Evans Blue dye as described previously [29]. 24 h after TBI injury, the mice were anesthetized, and Evans Blue dye (2% in saline) was injected slowly through the jugular vein (4 mL/kg) and allowed to circulate for 1.5 h before sacrifice.
Laser speckle contrast imaging (LSCI)
LSCI is based on the blurring of interference patterns of scattered laser light by the flow of blood cells to visualize blood perfusion in the microcirculation instantaneously [30]. Briefly, mice were anesthetized with gas anesthesia (isoflurane 2% in oxygen), with the head fixed in a stereotaxic frame (Kopf Instruments) and placed under an RFLSI Pro (RWD life sciences) and maintained at 1.2% isoflurane throughout the experiment. The surface of the skull is illuminated with a 784-nm 32-mW laser (RWD life science) at a 30° angle with a beam expander and light intensity controlled by a polarizer. Blood flow is detected by a CCD camera and the image acquisition is performed using custom software (RWD life science). Three hundred frames are acquired at 10 Hz with 10-ms exposure time. For the assessment of speckle contrast over time, regions of interest (ROIs) were selected and centered at the site where the laser is targeted on the cortex.
Immunohistochemistry and confocal microscopy analysis
At endpoint, mice were anesthetized and transcardially perfused with PBS, and mouse brains were extracted and fixed for 12 h in 4% paraformaldehyde (PFA) in PBS at 4 °C. 20 µm-thick coronal cryosections were used for immunohistochemistry in this study. After washing with PBS, brain sections were permeabilized and incubated in 5% Donkey Serum for 1 h for blocking. Then the brain tissues were incubated in primary antibody overnight at 4 °C. The primary antibody information is as following: anti-Fibrinogen antibody (1:200, Dako, A0080), anti-Iba1 antibody (1:500, Wako, 019-19741), anti-NeuN antibody (1:200, Abcam, ab177487), anti-Glial fibrillary acidic protein (GFAP) antibody (1:200, Invitrogen, 13-0300), anti-SMI-32 antibody (1:1000, Biolegend, 801709), anti-Cluster of differentiation (CD13) antibody (1:200, R&D Systems, AF2335), anti-β amyloid antibody (1:200, #D54D2, Cell Signaling). After washing off the primary antibody with PBS, the brain sections were then incubated with secondary antibodies (1:500; Jackson Immunoresearch Laboratories) for 1 h at room temperature. Dylight 488 conjugated-lectin (1:200, Vector, DL1174) was used to label brain vasculature, and Alexa Fluor 647 Donkey anti-Mouse IgG (1:500; Invitrogen, A-31571) was used to detect extravasation of IgG. After that, the sections were rinsed with PBS and covered with fluorescence mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, #H-1200).
Detection of apoptosis
TUNEL assay was performed according to the manufacturer's protocol (CAS7791-13-1, Roche). Tissues were incubated with the TUNEL reaction mixture in a humidified chamber for 30 min at 37 °C in the dark.
Detection of Aβ plaques
After PFA fixation, the sections were incubated in 1% aqueous Thioflavin S (T1892; Sigma) for 5 min and rinsed in 80% ethanol, 95% ethanol, and distilled water. All images were taken with the Nikon A1R confocal microscopy and analyzed using NIH ImageJ software. In each animal, 4 randomly selected fields in the cortex and hippocampus in TBI-affected hemisphere were analyzed in 4 non-adjacent sections (~ 100 µm apart) and averaged per mouse.
Cresyl violet staining
The brain tissue sections were firstly fixed by methanol for 5 min, then stained with the Cresyl Echt Violet staining kit (American MasterTech, catalog # AHC0443) and incubated for 5 min; next, the slides were rinsed in 2 changes of distilled water (15 s each) to remove the excess stain; then the slides were rinsed using 100% ethanol (10 s). At last, the slides were coverslipped with mounting medium.
Extravascular IgG, fibrinogen and fibrin deposits
We used antibodies that detected IgG, fibrinogen and fibrinogen-derived fibrin polymers (see Immunohistochemistry). Ten-micron maximum projection z-stacks were reconstructed, and the IgG, fibrinogen and fibrin-positive perivascular signals on the abluminal side of lectin-positive endothelial profiles on microvessels ≤ 6 µm in diameter were analyzed using ImageJ. In each animal, 4–5 randomly selected fields in the cortex in both mTBI-affected hemisphere and unaffected contralateral hemisphere were analyzed in 4 non-adjacent sections (~ 100 µm apart) and averaged per mouse.
Analysis for SMI-32
Ten-micron maximum projection z-stacks were reconstructed, and the SMI-32 signals were analyzed using ImageJ. In each animal, 4 randomly selected fields in the cortex and hippocampus in TBI-affected hemisphere were analyzed in 4 non-adjacent sections (~ 100 µm apart) and averaged per mouse.
GFAP-positive astrocytes and Iba1-positive microglia counting
For quantification, GFAP-positive astrocytes or Iba1-positive microglia that also co-localized with DAPI-positive nuclei were quantified by using the Image J Cell Counter analysis tool. In each animal 5 randomly selected fields from the cortex were analyzed in 4 nonadjacent sections (~ 100 µm apart). The number of GFAP-positive astrocytes was expressed per mm2 of brain tissue. The activated microglia undergo morphological changes including retraction of the processes and acquisition of a phagocytotic shape, but the quiescent microglia exhibit a ramified morphology [31].
NeuN-positive neuronal nuclei counting
For quantification, NeuN-positive neurons were quantified by using the Image J Cell Counter analysis tool. In each animal, 5 randomly selected fields from the cortex were analyzed in 4 nonadjacent sections (~ 100 µm apart). The number of NeuN-positive neurons colocalized with TUNEL was counted followed by the same procedure.
Quantification of pericyte numbers and coverage
The quantification analysis of pericyte numbers and coverage was restricted to CD13-positive perivascular mural cells that were associated with brain capillaries defined as vessels with ≤ 6 µm in diameter. For pericyte numbers, ten-micron maximum projection z-stacks were reconstructed, and the number of CD13-positive perivascular cell bodies that co-localized with DAPI-positive nuclei on the abluminal side of lectin-positive endothelium on vessels ≤ 6 µm counted using ImageJ Cell Counter plug-in. In each animal, 4–5 randomly selected fields in the cortex regions were analyzed in 4 non-adjacent sections (~ 100 µm apart) and averaged per mouse. The number of pericytes was expressed per mm2 of tissue. For pericyte coverage, ten-micron maximum projection z-stacks were reconstructed, and the areas occupied by CD13-positive (pericyte) and lectin-positive (endothelium) fluorescent signals on vessels ≤ 6 µm were subjected separately to threshold processing and analyzed using ImageJ as previously described [32]. In each animal, 4–5 randomly selected fields in the cortex regions were analyzed in 4 nonadjacent sections (~ 100 µm apart) and averaged per mouse.
Microvascular capillary length
Ten-micron maximum projection z-stacks were reconstructed, and the length of lectin-positive capillary profiles (≤ 6 µm in diameter) was measured using the ImageJ plugin “Neuro J” length analysis tool. In each animal, 4–5 randomly selected fields in the cortex were analyzed from 4 nonadjacent sections (~ 100 µm apart) and averaged per mouse. The length was expressed in mm of lectin-positive vascular profiles per mm3 of brain tissue.
Aβ deposits calculation
Aβ-positive areas were determined using ImageJ software. Briefly, the images were taken on a BZ9000 fluorescent microscope in single plain at 20× and subjected to threshold processing (Otsu) using ImageJ, and the percent area occupied by the signal in the image area was measured as described previously [26]. In each animal, four randomly selected fields from the cortex and hippocampus were imaged and analyzed in four nonadjacent sections (~ 100 µm apart).
Western blot
The total proteins from cortical tissue of TBI-affected hemisphere in sham-operated, sTBI and mTBI mice were extracted, and Synapsin I was analyzed by Western blot. Briefly, protein samples were separated by electrophoresis on a 10% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. Nonspecific binding sites were blocked in TBS, overnight at 4 °C, with 2% BSA and 0.1% Tween-20. Membranes were rinsed for 10 min in a buffer (0.1% Tween-20 in TBS) and then incubated with anti-synapsin I (1:1500, Sigma) followed by anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology). After rinsing with buffer, the immunocomplexes were developed with G:BOX Chemi XX6 gel doc system (Syngene) and analyzed in Image Lab software. β-Actin was used as an internal control for Western blot.
Statistical analysis
Sample sizes were calculated using nQUERY assuming a two-sided alpha-level of 0.05, 80% power, and homogeneous variances for the 2 samples to be compared, with the means and common standard deviation for different parameters predicted from published data and pilot studies. For comparison between two groups, the F test was conducted to determine the similarity in the variances between the groups that are statistically compared, and statistical significance was analyzed by Student’s t-test. For multiple comparisons, Bartlett's test for equal variances was used to determine the variances between the multiple groups and one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test was used to test statistical significance. All analyses were performed using GraphPad Prism 8 software by an investigator blinded to the experimental conditions. A p-value < 0.05 was considered as statistically significant.