Animals and TBI model
All surgical procedures and animal experiments were performed according to the protocols approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee (IACUC). Experiments were conducted using young adult male C57BL/6 mice (10–12 weeks old) or aged male mice (18 months old) from Charles River. After being fully anesthetized with isoflurane, mice were subjected to either controlled cortical injury (CCI) or sham surgery [57, 58]. Briefly, a midline incision of approximately 10 mm in length was made over the skull, with the skin and fascia retracted, and a 4-mm craniotomy was made on the central aspect of the left parietal bone. An injury of moderate severity was induced by a TBI-0310 Head Impactor (Precision Systems and Instrumentation) with a 3.5-mm diameter tip followed by impact velocity of 3.9 m/s and a displacement depth of 1.2 mm. After surgery, all mice were assigned to one of four groups based on surgery (sham or TBI) and treatment (trehalose or sucrose control) according to a randomized block experimental design. The surgical procedures were performed by the same investigator and all behavioral tests were carried out with the same equipment by blinded experimenters.
Experimental design
Study 1
To investigate whether age affects neurological function and pathological changes after TBI, young or aged C57BL/6 male mice were subjected to either moderate CCI or sham surgery (Fig. 1A). All mice underwent a battery of neurobehavioral tasks which consisted of open field (OF), catwalk (CW), grip strength (GS), and rotarod for assessment of motor function; Y-maze (YM) and novel object recognition (NOR) for cognitive function; novelty suppressed feeding (NSF) and social recognition (SR) for depressive-like behaviors; and hot plate for pain sensitivity. The OF, YM, CW, GS, rotarod, and HP tests were performed starting at 4 days before CCI for establishment of functional performance baseline between young and old mice and repeatedly at 1 week, 3, 6, and 9 weeks after CCI. At 12 weeks post-injury, a final round of behavior experiments was conducted to assess motor and cognitive function, in addition to depressive-like behaviors. After completion of all behavioral tests, ipsilateral brain tissue was collected at 16 weeks post-injury and processed for NanoString analysis, flow cytometry assays, and histological outcome measures.
Study 2
To assess whether autophagy enhancer mitigates age-exacerbated functional recovery after TBI, aged C57BL/6 mice were fed trehalose or sucrose as control starting on the date of the injury until the time of sacrifice. Based on prior studies [51, 52, 74] and our pilot data, a single dose of 5% trehalose (Sigma-Aldrich, Cat# T9449) or 5% sucrose was injected subcutaneously into each mouse immediately after CCI surgery. Five percent trehalose or sucrose was also administered into their drinking water for the first 7 days followed by continuous administration of trehalose or sucrose solution at 2.5% of concentration in their drinking water. Trehalose and sucrose stocks were made fresh on a weekly basis using autoclaved water provided by our animal facility. Previous reports using this concentration demonstrated enhancement in autophagic function associated with neuroprotection in aging and disease [30, 39, 41]. These studies reported no effect on body weight or obvious adverse effects in WT mice [34, 62, 68, 76]. The OF, YM, CW, GS, and rotarod tests were performed starting at 4 days before CCI and repeatedly at 2 and 4 weeks after CCI. At 6 weeks post-injury, a final round of behavior experiments was conducted to fully assess the effects of trehalose on functional recovery after TBI. After completion of all behavioral tests, ipsilateral brain tissue was collected at 9 weeks post-injury and processed for flow cytometry assays.
Neurological behavioral tests
The following behavior tests were performed with mice and group information blinded to the operators. To minimize stress and fatigue, each test was performed on a different day.
Motor function
Open field (OF) test
Spontaneous locomotor activity was examined in the OF apparatus [72]. Each test subject mouse was individually placed in a corner while facing towards the chamber wall of the apparatus (22.5 × 22.5 cm). The mice were allowed to freely explore the chambers for 10 min. Parameters on the distance travelled, average speed, time immobile, and percentage of time spent in the center of the chamber were recorded by the computer-based ANY-maze automated video-tracking system (Stoelting Co).
Gait analysis with catwalk XT (CW)
Analysis of gait and posture was performed with the catwalk XT automated system as mentioned in our previous publications (Noldus; RRID:SCR_004074) [43, 57, 58]. Acquisition of data took place in a darkened room with red light. A single researcher blinded to the grouping of each mouse was tasked with handling the subjects. The catwalk apparatus records print position, gait postures, and weight distribution through its green illuminated walkway. A minimum of 3 valid runs, complete crossings with no turns or scaling of sidewalls, were obtained for each tested mouse. Runs that did not comply to the preset standards were excluded from the final analysis.
Rotarod test
The accelerating rotarod was used to assess locomotor function and coordination at baseline and various timepoints post-injury [11]. The mouse was placed on a rotarod device (IITC Life Science, Inc.), and their latency to falling off the accelerating rotarod was recorded. The acceleration settings for the device were 4 to 40 rpm over 90 s, with each trial lasting for a maximum of 300 s. Individual scores from three trials were averaged and evaluated relative to their baseline latencies.
Grip strength (GS) test
Grip strength was measured using a digital grip strength meter (Bioseb BP, In Vivo Research Instruments, France), as previously described [27]. Forelimb grip strength was measured from the mouse using both the ipsilateral and contralateral forepaws together. The mouse was held by its tail, the forelimbs were placed on the grasping metal wire grid, and the mouse gripped the wire grid attached to the force transducer. Once the grip was secured, the animal was slowly pulled away from the bar. The maximal average force exerted on the grip strength meter by both forepaws was averaged from 10 trials per day for each mouse at each timepoint.
Cognitive function
Y-maze (YM) test
YM was used to assess the hippocampus-dependent spatial working memory of mice as described previously [57, 58]. The percentage of spontaneous alterations was calculated using the following equation: total alternations × 100/(total arm entries—2). If a mouse had a percentage of alternation above 50% (the chance level for randomly choosing the unfamiliar arm), it was indicative of a functional spatial memory.
Novel object recognition (NOR)
For testing non-hippocampal mediated memory, mice underwent NOR according to procedures described in previous studies [57, 58]. Mice were tested in an open field apparatus after a 5-min habituation period on the first day. The time spent with two identical objects was recorded on the second day of testing, and one of the familiar objects was switched out with a novel object on the third day. Testing stopped after each mouse went through a sum total of 30-s exploration time. Since mice would inherently prefer to explore novel objects, a preference for the novel object with an exploration time of more than 15 s was considered as having intact learning and memory skills.
Depressive-like behaviors
Novelty-suppressed feeding (NSF) test
The NSF was performed on subject mice to assess a rodent’s aversion to eating in a novel environment. After fasting for 24 h, mice were placed in an open field apparatus designed to model the introduction of a novel environment. Several food pellets were placed in the center of the apparatus and latency time for each mouse to reach the food and bite into it was recorded. If the mouse failed to eat within the maximum time of 10 min, a latency time of 600 s was recorded for this subject.
Social recognition (SR) test
This task is based on rodent’s innate tendency to investigate a novel congener over a familiar one [46, 57, 58]. Using a three-chambered rectangular apparatus made of Plexiglas with each one at equal size (20 × 40 × 23 cm). An opening between the walls allows for free access to each chamber, which contains two identical wire mesh cup containers. Before testing, each mouse was single housed overnight. On the first day, the tested mice were placed in the apparatus with two empty cups for a 10-min habituation period. On the second day, a stranger mouse was introduced and randomly placed inside one of the empty cups in either the left- or right-side chamber while the other cup was left empty. The tested mouse started from the middle chamber and allowed to freely explore all three chambers for an exploration period of 10 min. Afterwards, a second unfamiliar, stranger was placed inside the previously empty cup. The test subject was once again allowed to freely explore all three chambers for a period of 10 min. Exploration time that the subject mice spent with each cup versus stranger mouse was recorded. Since a socially functional mouse would naturally seek out unfamiliar mice for interaction, the test subject was considered capable of social recognition if index for novel mouse scored higher than 50%.
Thermal stimulation
Hot plate (HP) test
To test pain sensitivity of the hindpaws, mice were placed on the contact probe of computerized thermal stimulator on an Incremental Hot/Cold Plate Analgesia Meter (PE34, IITC Life Science, Woodland Hills, CA). The temperature was increased from 30 to 50 °C with the incremental rate at 10 °C per minute. When the tested mouse licked either one of its hindpaws, the test was stopped, and the threshold temperature was recorded. The test was conducted twice with the interval of 3 h [73].
RNA extraction and qPCR
Following completion of all behavioral tests, mice were first euthanized with Euthasol (0.1 mL/mouse) and then transcardially perfused with 40 mL ice-cold saline. RNA samples were obtained from the ipsilateral cerebral cortex surrounding the injury site and the ipsilateral hippocampus. Total RNA was extracted from flash frozen tissue samples using a cordless motorized homogenizer with RNAse-free pellet pestles (FisherBrand) followed by the miRNeasy Mini Kit (Qiagen, Cat# 74104). Complementary DNA (cDNA) was synthesized with the Verso cDNA RT kit (Thermo Scientific, Cat# AB1453B). Both kits were used according to the manufacturer’s instructions included in the kit box. Quantitative PCR for all target RNAs (see Supplemental Table S1) was performed with the TaqMan Gene Expression assay kit (Applied Biosystems). Each sample was run in duplicates with 3 stages of 40 cycles, 2 min at 50 °C, 10 s at 95 °C (denaturing step) followed by a final transcription step of 1 min at 60 °C. Gene expression was normalized by the transcription counts of GAPDH and final relative expression levels were calculated with the 2–ΔΔCt method [36, 37].
NanoString neuroinflammation panel analysis
Isolated RNA was eluted in a 40 µL volume and tested using an Agilent 2100 Bioanalyzer to ensure it met specifications for purity (RNA integrity number ≥ 9) and concentration (≥ 12.5 ng/µl). Total RNA (20 ng/ul) was run on a NanoString nCounter system using the Mouse Neuroinflammation v1.0 panel (NanoString Technologies, Seattle, WA) to profile RNA transcript counts for 757 genes and 13 housekeeping genes [36, 37]. The transcription counts were normalized prior to downstream analysis and pairwise differential expression analysis with the NanoString nSolver software Version 4.0. All statistical analysis of NanoString data was performed in R language software RStudio Version 1.2.5033. Principal component analysis (PCA) was performed with the command “prcomp” and a Euclidean distance measurement method was used for clustering. Three-dimensional plotting of the PCA was performed with the pca.3d package in RStudio. All pairwise comparisons of “A vs. B” should be interpreted as “A relative to B” in the text and figures. Volcano plots depicting the fold change and p value of the genes were performed with the EnhancedVolcano package in R. Differentially expressed genes with a raw p value of equal to or less than 0.01 were further grouped into subsets based on their annotations provided by NanoString and plotted into heatmaps with the ComplexHeatmap package in R.
Lesion volume, immunohistochemistry (IHC), and quantification
A subset of tested mice was perfused intracardially with normal saline followed by 4% paraformaldehyde. The brain was extracted and embedded in Tissue-Tek OCT compound (Sakura, Cat# 4583). Serial sections of 20-μm and 60-μm thicknesses were placed on Superfrost Plus slides (ThermoFisher, Cat# 4951PLUS). Lesion volume was measured on 60-μm coronal sections that were stained with cresyl violet (FD NeuroTechnologies, Cat# PS102-02). Quantification of the lesion volume was performed with the Stereoinvestigator software (MBF Biosciences). By outlining the missing tissue on the injured hemisphere, the software was able to estimate lesion volume with the Cavalieri method at a grid spacing of 0.1 mm [57].
Immunofluorescence imaging was performed on 20-μm coronal brain sections at around − 1.70 to 1.90-mm from bregma using standard immunostaining protocol, as described previously [37, 72]. Briefly, sections were blocked with 5% goat or guinea pig serum containing 0.3% Triton X-100. After incubation with primary and secondary antibodies (see Table S1), sections were counterstained with 4′,6-diamidino-2-phenylinodole (DAPI, Sigma-Aldrich, Cat# MBD0015) and mounted with glass coverslips using an anti-fade Hydromount solution (National Diagnostics, Cat# HS106100ML). Images from the perilesional cortex (n = 6 sections/location/mouse for 5–6 mice/group) were acquired using a fluorescent Nikon Ti-E inverted microscope, at 20 × (CFI Plan APO VC 20X NA 0.75 WD 1 mm) magnification, and the background of each image was subtracted using background ROI [35, 38]. The number of NeuN+, LC3+, p62+, and Iba-1+ cells was normalized to the total imaged area (mm2) using the NIH Image J software (1.43; NIH). Myelin images were acquired using a Leica TCS SP5 II Tunable Spectral Confocal microscope system (Leica Microsystems, Bannockburn, IL). All IHC staining experiments were performed with appropriate positive control tissue, as well as primary/secondary only negative controls.
Flow cytometry and ex vivo functional assays
Mice were perfused with 40 mL of cold saline, and the ipsilateral (i.e., craniotomy-side) hemisphere was isolated [37, 57]. The olfactory bulb and cerebellum were removed, brains were halved along the interhemispheric fissure, and the ipsilateral hemisphere was placed separately in complete Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen, Cat# 22400105) medium and mechanically and enzymatically digested in collagenase/dispase (1 mg/ml, Roche Diagnostics, Cat# 10269638,001), papain (5U/ml, Worthington Biochemical, Cat# LS003119), 0.5 M EDTA (1:1000, Invitrogen, Cat# 15575020), and DNAse I (10 mg/ml, Roche Diagnostics, Cat# 10104159001) for 1 h at 37 °C on a shaking incubator (200 rpm). The cell suspension was washed twice with RPMI, filtered through a 70-μm filter, and RPMI was added to a final volume of 5 mL/hemisphere and kept on ice. Cells were then transferred into FACS tubes and washed with FACS buffer. Cells were then incubated with TruStain FcX Block (Biolegend, Cat# 101320), for 10 min on ice, and stained for the following surface antigens (see Table S1): CD45-eF450 (eBioscience, Cat# 48–0451-82), CD11b-APCeF780 (eBioscience, Cat# 47–0112-82), Ly6C-AF700 (Biolegend, Cat# 128024), and MHCI-PECy7 (Biolegend, Cat# 114616). The fixable viability dye Zombie Aqua was used for live/dead discrimination (Biolegend, Cat# 423102). Cells were then washed in FACS buffer, fixed in 2% paraformaldehyde for 10 min, and washed once more prior to adding 500 ul FACS buffer. Intracellular staining for Ki67-PECy7 (Biolegend, Cat# 652426), PCNA-AF647 (Biolegend, Cat# 307912), CD68-PerCPCy5.5 (Biolegend, Cat# 137010), NeuN-PE (Millipore Sigma, Cat# FCMAB317PE), Vglut1-APC (StressMarq, Cat# SMC-394D-APC), Lamp1-PerCPCy5.5 (Biolegend, Cat# 121626), Lamp2-PE (Biolegend, Cat# 108506), Sqstm1/p62-AF647 (Novus Biologicals, Cat# NBP1-42822AF647), ATG5-AF647 (Biolegend, Cat# 847410), ATG7-AF700 (R&D Systems, Cat# FAB6608N), Ubiquitin-AF647 (Biolegend, Cat# 838710), H3-AF647 (Cell Signaling Technology, Cat# 12230S), Acetylated (Ac) Lysine (Lys)-PECy7 (Biolegend, Cat# 623408), H3-Ac-Lys9-AF488 (Cell Signaling Technology, Cat# 9683S), H3-Ac-Lys18-AF488 (Cell Signaling Technology, Cat# 73508S), H3-Ac-Lys27-AF647 (Cell Signaling Technology, Cat# 39030S), H3-Ac-Lys36-AF647 (Cell Signaling Technology, Cat# 84061S), Phospho(ser149)-H2A.X-PECy7 (Biolegend, Cat# 613420), p16-APC (StressMarq, Cat# SPC-1280D-APC), and p21-AF488 (RND-NBP2-43697AF488) was performed using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat# 554714) according to manufacturer’s instructions and as described previously [11]. Cytokine staining for TNF-PECy7 (Biolegend, Cat# 506324, and IL-1β-PerCPeF710 (eBioscience, Cat# 46–7114-82) was performed after 3-h incubation with Brefeldin A (Biolegend, Cat# 420601) at 37 °C followed by fixation/permeabilization.
The following commercially available dyes (see Table S1) were used for ex vivo cell staining according to the manufacturer’s instructions: H2dcfda (DCF, ThermoFisher, Cat# D399), LysoTracker Deep Red (ThermoFisher, Cat# L12492), Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Cat# ENZ-51031-K200), BODIPY 493/503 (ThermoFisher, Cat# D3922), Lipi-Blue (Dojindo, Cat# LD01-10), FluoroMyelin Red (ThermoFisher, Cat# F34652), FerroOrange (Dojindo, Cat# F374-12), ProteoStat Aggresome Detection Kit (Enzo Life Sciences, Cat# ENZ-51035-K100), MitoSpy Red CMXRos (Biolegend, Cat# 424802), Glucose Uptake Assay Kit (Cayman Chemical Company, Cat# 600470), BioTracker ATP-Red (Millipore Sigma, Cat# SCT045), BODIPY-Pepstatin A (ThermoFisher, Cat# P12271), and LysoLive™ Lysosomal Acid Lipase Assay Kit (Abcam, Cat# ab253380).
Data were acquired on a BD LSRFortessa cytometer using FACSDiva 6.0 (BD Biosciences) and analyzed using FlowJo (Treestar Inc.). At least 5–10 million events were collected for each sample. CountBright Absolute Counting Beads (ThermoFisher, Cat# C36950) were used to estimate cell counts per the manufacturer’s instructions. Data were expressed as counts/hemisphere. Leukocytes were first gated using a splenocyte reference (SSC-A vs FSC-A). Singlets were gated (FSC-H vs FSC-W), and live cells were gated based on Zombie Aqua exclusion (SSC-A vs Zombie Aqua-Bv510). Resident microglia were identified as the CD45int CD11b+Ly6C− population, whereas peripheral leukocytes were identified as CD45hiCD11b+ myeloid cells or CD45hiCD11b− lymphocytes. Cell type–matched fluorescence minus one (FMO) controls were used to determine the positivity of each antibody and indicator dye [55].
Statistical analysis
All quantitative data are plotted as mean ± standard error of mean. Statistical analysis was performed with Sigmaplot Version 12 (Systat software) or Graphpad Prism Version 4 for Windows (Graphpad Software, Inc). Kaplan–Meier survival curves were analyzed using the log-rank Mantel-Cox test. When comparing between two individual samples/groups, statistical significance was evaluated with the Student’s unpaired t-tests (detailed in figure legends). Comparisons within each surgery group were analyzed with 2-way ANOVA group analysis followed by multiple comparisons Dunnett’s or Tukey’s post-hoc test. For non-parametric data, the Mann Whitney test was used. A p value of ≤ 0.05 was considered as statistically significant.