Recent evidence supports the hypothesis that repetitive mild traumatic brain injuries (rmTBIs) culminate in neurological impairments and chronic neurodegeneration, which have wide-ranging implications for patient management and return-to-play decisions for athletes. Adolescents show a high prevalence of sports-related head injuries and may be particularly vulnerable to rmTBIs due to ongoing brain maturation. However, it remains unclear whether rmTBIs, below the threshold for acute neuronal injury or symptomology, influence long-term outcomes. To address this issue, we first defined a very mild injury in adolescent mice (postnatal day 35) as evidenced by an increase in Iba-1- labeled microglia in white matter in the acutely injured brain, in the absence of indices of cell death, axonal injury, and vasogenic edema. Using this level of injury severity and Avertin (2,2,2-tribromoethanol) as the anesthetic, we compared mice subjected to either a single mTBI or 2 rmTBIs, each separated by 48 h. Neurobehavioral assessments were conducted at 1 week and at 1 and 3 months postimpact. Mice subjected to rmTBIs showed transient anxiety and persistent and pronounced hypoactivity compared to sham control mice, alongside normal sensorimotor, cognitive, social, and emotional function. As isoflurane is more commonly used than Avertin in animal models of TBI, we next examined long-term outcomes after rmTBIs in mice that were anesthetized with this agent. However, there was no evidence of abnormal behaviors even with the addition of a third rmTBI. To determine whether isoflurane may be neuroprotective, we compared the acute pathology after a single mTBI in mice anesthetized with either Avertin or isoflurane. Pathological findings were more pronounced in the group exposed to Avertin compared to the isoflurane group. These collective findings reveal distinct behavioral phenotypes (transient anxiety and prolonged hypoactivity) that emerge in response to rmTBIs. Our findings further suggest that selected anesthetics may confer early neuroprotection after rmTBIs, and as such mask long-term abnormal phenotypes that may otherwise emerge as a consequence of acute pathogenesis.

Concussions, or mild traumatic brain injuries (mTBIs), are particularly common during adolescence, accounting for 5-13% of all reported sports injuries in athletes of high school age [1,2]. By the start of high school, half of all student athletes have reported a history of mTBIs [3], while approximately 6% of high school football players sustain a concussion each season [4]. A single concussion is typically not associated with the presence of early structural damage on standard computed tomography [5], and symptoms are usually transient, resolving within days to weeks in most young athletes [6,7]. However, a subset of young patients report persistent symptoms including sleep dysregulation, cognitive deficits and emotional disturbances up to at least 1 month after single or repetitive mTBIs (rmTBIs) [8,9,10,11].

Based upon reports of poorer outcomes after mTBIs sustained during adolescence compared to older athletes [3,12,13,14], it is postulated that the adolescent brain shows age-dependent vulnerability to injury, with repetitive injuries in particular having the potential to interfere with ongoing brain maturation [15]. Clinically, greater impairments are often seen after repeated injuries compared to a single mTBI, suggesting that a second impact while recovering from the first may result in prolonged and/or more severe symptoms [16,17,18]. Supporting this concept, rmTBIs have been associated with persistent neurocognitive changes [5,19] and may be a contributing factor in long-term neurodegeneration including chronic traumatic encephalopathy [20,21]. Even asymptomatic or ‘subconcussive' impacts, below the threshold of force required to cause detectable tissue damage or the clinical hallmarks of concussions [22], may accrue when repetitive in nature, and have been postulated to result in significant long-term consequences [23,24,25] associated with alterations in neurochemistry, cerebral metabolism, and functional connectivity [26,27,28,29,30].

Despite the recent proliferation of experimental models to examine the effects of mTBIs in adult animals, there remains a paucity of studies designed to characterize the consequences of mTBIs to the child or adolescent brain. The risk of adverse long-term consequences after cumulative mTBIs, and the potential for age-dependent vulnerability at this time, highlights the need for age-appropriate models to better understand the response of the adolescent brain to injury.

Here, we employed a controlled cortical impactor device to generate a closed-skull impact to the mouse brain at postnatal day 35 (p35), an age that approximates adolescence in humans [31]. Establishing a model of very mild or subconcussive injury severity, we observed an early microglial response after a single mTBI in the absence of acute hemorrhage, edema, neuronal damage, or axonal injury. Using an extensive battery of neurobehavioral assessments across an extended time course postimpact we found anesthetic-dependent transient anxiety and long-term hypoactivity after rmTBIs to the adolescent brain.

Animals

Male C57Bl/6J pups at p28-30 were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and group-housed in the Laboratory Animal Resource Center at the University of California San Francisco (UCSF) Parnassus for habituation prior to surgery. A total of 155 mice were included in this study, with analysis of 131 after several animals were excluded from analysis (fig. 1). Study exclusions were related to mortality at the time of impact, euthanasia due to skull fracture or malocclusion, or morbidity associated with the development of intestinal ileus in a subset of mice anaesthetized by Avertin (2,2,2-tribromoethanol), a side effect previously reported by others [32,33]. Standard bedding, rodent chow, and water were available ad libitum, and housing was maintained on a 12-hour light/dark cycle at approximately 20°C. All surgical and behavioral procedures were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and approved by the UCSF Institutional Animal Care and Use Committee. The animals were randomized and the investigators were blinded during data collection and analysis in line with best practice guidelines [34].

Fig. 1

Study design. Adolescent mice (p35) were anesthetized with either Avertin or isoflurane for sham surgery, single mTBI, or rmTBI as indicated. rmTBIs were delivered 48 h apart. A subset of mice that underwent long-term behavioral assessments (3 or 6 months) were randomly allocated for histological analyses. Superscript annotations indicate exclusion justification: (1) surgery-related death, (2) euthanized due to morbidity, (3) euthanized due to malocclusion and (4) euthanized due to skull fracture at impact.

Fig. 1

Study design. Adolescent mice (p35) were anesthetized with either Avertin or isoflurane for sham surgery, single mTBI, or rmTBI as indicated. rmTBIs were delivered 48 h apart. A subset of mice that underwent long-term behavioral assessments (3 or 6 months) were randomly allocated for histological analyses. Superscript annotations indicate exclusion justification: (1) surgery-related death, (2) euthanized due to morbidity, (3) euthanized due to malocclusion and (4) euthanized due to skull fracture at impact.

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Single mTBIs and rmTBIs to Adolescent Mice

Our first objective was to define a subconcussive level of mTBI by adjusting the parameters of the controlled cortical impact and assessing early histological changes. This model was then used to conduct two studies to evaluate the long-term consequences of rmTBIs at adolescence, using (1) Avertin (2,2,2-tribromoethanol) anesthesia and (2) isoflurane anesthesia during the mTBI or sham surgery. The experimental design, including outcomes specific to each anesthetic, are summarized in figure 1.

mTBIs were induced in mice at ∼p35 using a controlled cortical impactor device [35]. The mice were anesthetized with either 2% Avertin in isotonic saline administered intraperitoneally at 0.025 ml/g body weight or 4% isoflurane with maintenance at 2% isoflurane throughout the surgical procedure. The animal groups were (1) sham (anesthesia and surgical preparation, but no impact), (2) single impact (mTBI ×1), (3) 2 repeat impacts spaced 48 h apart (mTBI ×2) or (4) 3 repeat impacts spaced 48 h apart (mTBI ×3; isoflurane study only).

After the positioning of the head in a stereotaxic frame (David Kopf Instruments, Tujunga, Calif., USA), the skull was exposed by a midline skin incision and reflection of the soft tissues. The mice randomly allocated for concussive injury were then positioned beneath the injury device (eCCI-6.3; Custom Design and Fabrication) and subjected to a closed-skull impact using a 3.0-mm convex rubber impactor tip [36,37]. The impact was generated to specified velocity, depth, and dwell time parameters and delivered to the left midparietal bone, centered 2 mm posterior to the bregma and 2 mm lateral to the midline. Following the impact, the scalp was closed with sutures, and ∼1 ml of isotonic saline was administered subcutaneously to prevent postoperative dehydration. All mice were maintained on a water-circulating heating pad for a similar period of time throughout the procedure and recovery. The mice receiving repetitive closed-skull impacts were allowed to recover from the initial impact and were reanesthetized 48 h later for impact at the same location as the first. Body weights were monitored immediately prior to impact and at days 1, 3, and 7 postimpact, and then weekly thereafter, as a measure of general health. Anesthesia at euthanasia was by overdose with 2.5% Avertin administered intraperitoneally. For the rmTBI groups, ‘time postimpact' is defined as the time following the final impact.

Righting Reflex

In mice that received isoflurane anesthesia during surgical procedures, righting reflex was evaluated as the latency to return upright from a supine position [38].

Edema Measurements

Vasogenic edema was evaluated at 24 h postimpact in a subset of mice after Avertin anesthesia and sham, mTBI ×1 or mTBI ×2 (n = 5/group), using the wet weight-dry weight method as previously described [39]. Water content in each brain hemisphere was calculated as [(wet weight - dry weight)/wet weight] ×100.

Immunohistochemistry

Tissue from a subset of mice was collected at 24 h or 3 or 6 months postsurgery, following transcardial perfusion with ice-cold saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline. Brains were postfixed overnight in 4% PFA and then transferred into a 30% sucrose solution for 72 h and embedded in Neg50™ (Richard-Allan Scientific, Thermo Fisher Scientific, Kalamazoo, Mich., USA) for storage at -80°C. Coronal sections of 20 or 40 μm spanning the entire cortex were collected.

Immunohistochemistry was performed on evenly spaced sections per brain, between bregma 0.3 and -3.3 mm, to assess tissue integrity (by cresyl violet, CV), axonal injury by accumulation of β-amyloid precursor protein (β-APP), microglia labeled with ionized calcium-binding adaptor molecule 1 (Iba-1), and astrocytes labeled with glial fibrillary associated protein (GFAP). Sections were immersed in hydrogen peroxide (0.3% in methanol) followed by 1 h of incubation in a blocking solution (10% species-appropriate serum, 0.2% Triton-X100, and 0.1% bovine serum albumin). Sections were then incubated overnight at 4°C in serum-containing blocking solution containing either rabbit polyclonal β-APP (1:500; Invitrogen), goat polyclonal Iba-1 (1:500; Abcam), or mouse monoclonal GFAP (1:500; Abcam). A subsequent 1 h of incubation with either biotinylated goat anti-rabbit IgG (for β-APP; Vector Laboratories Inc., Burlingame, Calif., USA) or horse anti-goat IgG (for Iba-1) was followed by detection with the Vectastain ABC kit (Vector Laboratories Inc.) and visualization with nickel-enhanced 3,3′-diaminobenzidine tetrahydrochloride. Fluorescently labeled secondary antibodies were used to detect GFAP (Cy3-conjugated goat anti-mouse IgG; Jackson ImmunoResearch). Blinded evaluations of tissue stained with CV or β-APP were performed at 20× magnification using a Brightfield microscope.

Microglial Cell Counts

Numbers of Iba-1-positive microglia in the dorsal cortex and corpus callosum were estimated using the optical fractionator method at 24 h and 3 months postimpact (n = 4-5/group. Regions of interest (ipsilateral and contralateral dorsal cortex and the corpus callosum from the midline to the cingulum) were contoured using a 2× or 4× objective. The sectioning interval was 24 or 8 (for 24 h and 3 months postimpact, respectively), with quantification performed on 6-10 sections per brain. For the cortex, analyzed sections ranged from bregma 0.74 to -3.46 mm (24 h postimpact) or -1.34 to -3.40 mm (3 months postimpact). For corpus callosum measurements, analyzed sections ranged from -1.34 to -2.46 mm. A 20× objective was used for cell counting. Systematic random sampling was achieved by counting cells at a regular predetermined interval (grid size of 600 × 600 µm for cortex and 120 × 120 µm for corpus callosum), within a disector counting frame (8 µm in height) superimposed upon the contoured region of interest (180 × 180 µm for cortex; 60 × 60 µm for corpus callosum). The Gundersen mean coefficient of error (m = 1) for individual estimates was maintained at ≤0.10. Using Stereo Investigator software (v10.21.1; MicroBrightField, Williston, Vt., USA), the total number of cells per contoured region was estimated as previously described [40,41].

Quantification of β-APP Cells

Cells that stained positive for β-APP accumulation, an indicator of axonal injury, were quantified within the corpus callosum/external capsule at 60× magnification using Stereo Investigator software. Cell numbers were averaged across 6-8 sections per brain (n = 4-5/group), between bregma -0.7 and -3.64 mm. One brain was excluded from analysis due to a high number of damaged or missing sections.

GFAP Expression

GFAP expression in the ipsilateral external capsule at 24 h postimpact was imaged at 20× magnification on a Nikon Eclipse 80i fluorescence microscope for semiquantitative analysis using Metamorph Software (Molecular Devices, LLC.). Preliminary assessment of a subset of samples yielded a minimum threshold for detecting GFAP expression, which was used to determine the integrated intensity of GFAP expression for all sections. The percent GFAP intensity [(the integrated intensity with minimum threshold divided by the integrated intensity without minimum threshold) multiplied by 100] was calculated per section and averaged per brain [42].

Assessment of Cell Death

To quantify dead and dying cells at 24 h postimpact (n = 4/group), terminal deoxynucleotidyl transferase-mediated dUTP nick 3′-end labeling (TUNEL) was performed on 3 sections per brain (480 µm apart, between bregma -1.2 and -2.8 mm) using the in situ Cell Death Detection kit (Roche Diagnostics) according to the manufacturer's instructions. Slides were then incubated overnight with a rabbit polyclonal antibody against active (cleaved) caspase-3 (1:500; Millipore) followed by detection with a Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch). Stained sections were examined at 10× and 20× magnification under a fluorescence microscope, and positively labeled cells were identified by colabeling with the nuclear stain 4',6-diamidino-2-phenylindole.

Cortical and Corpus Callosum/External Capsule Volume Measurements

An estimation of intact cortical and corpus callosum/external capsule volumes was performed on 40-µm CV-stained coronal sections collected at 3 or 6 months postimpact (n = 4-6/group). The unbiased Cavalieri method was performed with Stereo Investigator software [40]. Measurements were performed at 3 months postimpact between bregma 1.5 and -3.8 mm, with an average of 15-20 sections (for cortex) or 10-13 sections (for corpus callosum/external capsule), using a sampling interval of 8 and a grid size of 200 µm (for cortex) or 50 µm (for corpus callosum/external capsule). Measurements at 6 months postimpact were between bregma 1.1 and 2.46 mm, with 15-18 sections per brain for the corpus callosum/external capsule, using a sampling interval of 6 and a grid size of 50 µm. Three brains were excluded from the 6-month analysis due to sectioning or staining artifacts. Cortical measurements were confined to the dorsal hemispheres, which contained the impacted region, and the inferior boundary was defined by a horizontal line underlining the most ventromedial point of the corpus callosum. Corpus callosum/external capsule measurements were confined to when the corpus callosum joined at the midline to when it split at the midline. The Gundersen mean coefficient of error (m = 1) for individual estimates maintained ≤0.10.

Behavioral Assessments

All behavioral testing was conducted within the Neurobehavioral Core for Rehabilitation Research at UCSF. For the Avertin anesthetic long-term study, an extensive battery of behavioral assessments was performed in the following order: open field test, elevated plus maze (EPM), and rotarod test at 1 week, with the addition of the novel object recognition (NOR) test and radial arm water maze (RAWM) at 1 and 3 months postimpact, and the further addition of the cylinder test at 3 months postimpact (n = 10-12 per group). In the subsequent long-term study with isoflurane as the anesthetic, initial behavior assessment was conducted at 1 month after impact as follows: open field test, EPM, rotarod test, and then RAWM. Additional testing in the open field and EPM was performed at 3 and 6 months after impact. The mice were placed into individual clean cages 24-72 h prior to the commencement of testing for isolation and habituation, and all testing was conducted between 9.00 a.m. and 6.00 p.m. daily.

Open Field Test. Exploratory behaviors were assessed over a 10-min session in an automated open field arena (40.64 × 40.64 cm; Kinder Scientific, Poway, Calif., USA). Interfaced Motor Monitor software allowed for calculation of parameters including total distance traveled and relative time spent in the center versus the periphery [35]. Time spent rearing and the number of vertical rearing events was also recorded.

Elevated Plus Maze. The EPM (Kinder Scientific) was used to assess anxiety based upon the natural tendency of rodents to avoid the open arms in preference for enclosed areas. The mice were placed individually on the apparatus and allowed free access for a 10-min period. Time spent in the open versus closed arms, total distance moved, and time spent at rest were assessed as previously described [35].

Rotarod Test. The accelerating rotarod test (Ugo Basile 7650; Comerio, Italy) was performed to assess general motor function, coordination, and motor learning as previously described [35]. The latency to fall was recorded in seconds, and the mice were tested across 2 consecutive days, 3 times per session with an intersession interval of approximately 1 h. One cohort (11 mice; 2-3 per impact group) was excluded from rotarod analysis in study 3 due to malfunctioning of the rotarod equipment on day 1 of testing.

Novel Object Recognition. The NOR task takes advantage of a rodent's spontaneous preference to explore novel objects relative to familiar objects and thereby measures hippocampal-dependent recognition memory [43]. During the habituation phase, an animal was placed in the open field arena (40.64 × 40.64 cm) for 10 min and then returned to its home cage. The following morning, the animal was placed into the arena for a 10-min familiarization phase with 2 identical objects positioned in the center of the arena 15 cm apart and equidistant from the arena walls. Objects included a beige-colored, rubber faucet nozzle attachment (5 × 6 cm) positioned spout down and a small silver cone (6 × 7 cm). The animal was returned to the arena 4 h later for a 5-min testing phase, when it was presented with one object previously encountered during the familiarization phase alongside one novel object. Sessions was recorded with an overhead SANYO Xacti video camera for analysis using Stopwatch+© software (Center for Behavioral Neuroscience, Georgia State University). Object exploration was defined as an animal being orientated towards the object when ≤2 cm away from the object.

Cylinder Test. To examine forepaw usage, the mice were individually placed in an open-topped plexiglass cylinder (7 × 15 cm) for a 5-min period. Behaviors were video-recorded from one side, with a mirror positioned behind to allow for 360° visualization of the cylinder walls. Videos were analyzed at half speed, and the number of paw placements onto the walls when rearing was quantified (right paw first, left paw first, or both simultaneously [44].

Radial Arm Water Maze. The RAWM was employed to measure spatial learning and memory [45]. In this task, the goal arm location containing the platform remained constant, while the start arm was changed during each trial. On day 1 (the training phase), the mice were trained for 15 trials, with trials alternating between a visible (cued) and hidden platform for the first 12 trials, followed by a final 3 hidden trials (60 s maximum per trial). On day 2 (the testing phase), the mice were tested for 15 trials with the hidden platform. Errors were defined as entry into an incorrect arm prior to finding the target platform or not moving from the same arm or center for >15 s, and errors were averaged over visible/hidden training blocks (3 consecutive trials). At the end of day 2, an open pool task was conducted to assess all mice for swimming and visual abilities [45].

Magnetic Resonance Imaging

At 6 months postimpact, a subset of mice (n = 4/group) underwent ex vivo magnetic resonance imaging (MRI). The mice were transcardially perfused with saline followed by 4% PFA containing 2 mM gadoteridol (Gd) solution (ProHance®; Bracco Imaging, Princetown, N.J., USA) to facilitate white-gray matter susceptibility contrast and reduce acquisition time [46,47]. Whole brains were collected and postfixed overnight in 4% PFA containing 2 mM Gd and then transferred to PBS containing 2 mM Gd for 5-7 days prior to imaging. Following MRI, the brains were rinsed in fresh PBS and then incubated in a 30% sucrose solution for 3-5 days before processing for histology.

Imaging was performed on a 600-MHz NMR spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA) with a 20-mm quadrature coil. 3D gradient echo was used with TE/TR = 15/ 75 ms, 4 averages, field of view 12.8 mm isotropic, resolution of 50 × 50 × 50 μm, and a total scan time of 3 h. Acquired T1-weighted images were converted to DICOM format on the Agilent console and realigned into the same orientation as the histology sections (coronal plane view) using the open-source image viewer OsiriX (Pixmeo, Geneva, Switzerland). The corpus callosum/external capsule white matter was manually delineated and outlined on all images where callosal fibers were evident at the midline (n = 66-78 images per brain; thickness 50 µm). Custom-built software in Matlab® (MathWorks, Natick, Mass., USA) was used to calculate the overall volume estimation for each brain.

Statistical Analysis

Statistical analyses were performed using Prism v.5.0 (GraphPad Software Inc., La Jolla, Calif., USA), with a significance level of p < 0.05. One-way ANOVAs were used where appropriate to compare between impact groups, with Dunnett's post hoc analyses for comparison of individual groups to sham-operated controls. Two-way repeated-measures (RM) ANOVAs were used to compare histological data and edema (CV and Iba-1 quantification) with factors of brain hemisphere and impact group. Significant interactions between these factors are reported as such; where the interaction was nonsignificant, only main effects are reported. For behavioral assessments that were repeated across several time points after impact (e.g. day 7 and 1 and 3 months), data were analyzed using two-way RM ANOVAs, with main effect of time and impact. Tukey's multiple comparisons were then used to identify individual differences between specific groups when one or both main effects were statistically significant. Normally distributed data are presented as means + SEM. Nonparametric data are presented as medians and analyzed by nonparametric statistical tests (e.g. Kruskal-Wallis or Mann-Whitney tests) as appropriate.

Characterization of mTBI in Adolescent Mice

In order to determine the optimal impact parameters to induce mTBIs in mice at adolescence, we first generated a range of impact severities under Avertin anesthesia and observed neuropathological markers of axonal injury (β-APP) and cell death (TUNEL and caspase-3) at 24 h after a single impact (fig. 2a). Here, the objective was to define a subthreshold level of injury by titrating the impact parameters to a level with no immediate indication of gross pathology or mortality. The different impacts were generated using (1) 4.5 m/s velocity and 1.5 mm depth of penetration, (2) 4.5 m/s velocity and 0.5 mm depth, (3) 3.5 m/s velocity and 0.5 mm depth and (4) 2.5 m/s velocity and 0.5 mm depth.

Fig. 2

Model characterization and experimental timeline to examine acute and chronic outcomes after rmTBIs under Avertin anesthesia. EPM = Elevated plus maze; RAWM = radial arm water maze; NOR = novel object recognition; OF = open field test. a, b During model characterization, the impact parameters were altered to determine subconcussive injury severity based upon minimal pathological markers at 24 h after a single mTBI. The presence of β-APP-positive cells/axons was considered an indication of axonal injury, as in representative images (a) from the ipsilateral corpus callosum/external capsule from each impact severity. The highest severity (4.5 m/s, 1.5 mm) was associated with considerable β-APP accumulation (black arrows), as well as intraparenchymal hemorrhage. b Quantification revealed that the degree of axonal injury was dependent upon impact severity and identified the lowest severity (2.5 m/s, 0.5 mm) as below the threshold to detect β-APP accumulation. c To examine the acute and chronic effects of rmTBIs, using Avertin as the anesthetic agent, adolescent mice received either 0, 1, or 2 mTBI impacts spaced 48 h apart, for evaluation at either 24 h (grey arrow; for histology) or up to 3 months for neurobehavioral testing.

Fig. 2

Model characterization and experimental timeline to examine acute and chronic outcomes after rmTBIs under Avertin anesthesia. EPM = Elevated plus maze; RAWM = radial arm water maze; NOR = novel object recognition; OF = open field test. a, b During model characterization, the impact parameters were altered to determine subconcussive injury severity based upon minimal pathological markers at 24 h after a single mTBI. The presence of β-APP-positive cells/axons was considered an indication of axonal injury, as in representative images (a) from the ipsilateral corpus callosum/external capsule from each impact severity. The highest severity (4.5 m/s, 1.5 mm) was associated with considerable β-APP accumulation (black arrows), as well as intraparenchymal hemorrhage. b Quantification revealed that the degree of axonal injury was dependent upon impact severity and identified the lowest severity (2.5 m/s, 0.5 mm) as below the threshold to detect β-APP accumulation. c To examine the acute and chronic effects of rmTBIs, using Avertin as the anesthetic agent, adolescent mice received either 0, 1, or 2 mTBI impacts spaced 48 h apart, for evaluation at either 24 h (grey arrow; for histology) or up to 3 months for neurobehavioral testing.

Close modal

At the greatest impact severity (4.5 m/s velocity, 1.5 mm depth), an initial cohort of mice (n = 5) showed a 20% rate of skull fracture. By histological evaluation, all brains were positive for β-APP in the dorsal external capsule ipsilateral to the impact site, between bregma -0.1 and -3.8 mm (fig. 2b). Blood products were identified on immunostained tissue in the external capsule just above the lateral ventricle. Cell death evidenced by sporadic TUNEL and caspase-3 staining was also observed at this severity, primarily within the granular and subgranular layer of the ipsilateral hippocampus dentate gyrus (data not shown). Based upon the mortality rate, risk of skull fracture, and evidence of acute cell death, we determined that these parameters generated an injury greater than our clinically based definition of concussive-like mTBI [6] and did not pursue it further.

We next evaluated mice subjected to lower impact severities, of 4.5, 3.5, or 2.5 m/s velocity, all to a reduced impact depth of 0.5 mm (n = 4-5/group). Here, we determined that the presence of β-APP was velocity and depth dependent, with minimal detectable staining in the mildest impact severities (3.5 and 2.5 m/s) and an intermediate degree of β-APP labeling in the 4.5 m/s group. Quantification confirmed the presence of fewer β-APP-positive cells after impact at 3.5 m/s (0.5 mm) compared to 4.5 m/s (1.5 mm depth; Kruskal-Wallis test: p = 0.0038, with Dunn's post hoc p < 0.05; fig. 2c). A few scattered TUNEL- and caspase-3 labeled cells were detected in the 4.5 m/s, 0.5 mm group, and there was no evidence of cell death after the 2 mildest impact severities (3.5 or 2.5 m/s, 0.5 mm).

Based upon this lack of overt neuropathology after a single mTBI, the lowest impact severity (2.5 m/s, 0.5 mm) was chosen for subsequent long-term studies evaluating behavioral consequences after rmTBIs at adolescence.

Assessment of Acute and Long-Term Changes after Single mTBIs or rmTBIs in Mice Anesthetized with Avertin

Acute Pathological Assessment after rmTBIs at Adolescence

We sought to characterize the acute consequences of either single mTBIs or rmTBIs at this defined mild severity in adolescent mice (fig. 2). Avertin was chosen as the anesthetic agent based on our previous experience with this agent [35,48]. Adolescent mice received either sham surgery, a single impact (mTBI ×1), or 2 impacts timed 48 h apart (mTBI ×2), and a subset of brains were collected at 24 h to identify acute pathological markers. No edema was detected after injury, as the water content was equivalent in the sham, mTBI ×1, and mTBI ×2 groups (two-way RM ANOVA, p = 0.2148) in both the ipsilateral and contralateral hemispheres (p = 0.2842). The mild nature of this injury paradigm was confirmed by the absence of macroscopic bleeding or gross tissue distortion observed by CV staining of all impact groups (fig. 3a).

Fig. 3

Acute pathology after rmTBIs at adolescence under Avertin anesthesia. a-c At 24 h postimpact (2.5 m/s, 0.5 mm), intact tissue integrity was observed by CV staining in the brains of sham (1st row), mTBI ×1 (2nd row) and mTBI ×2 (3rd row) mice (a). No β-APP accumulation was detected (b), and Iba-1-positive microglia were not visibly altered after mTBI ×1 or mTBI ×2 (c). Positive control tissues for comparison and validation of staining were obtained from a moderate-severe CCI model in pediatric (p21) mice [49]. Iba-1-positive microglia were quantified in the dorsal cortex (d) and corpus callosum white matter (e). Volumetric analysis of the dorsal cortex (f) and corpus callosum/external capsule (g) was performed at 3 months postimpact on CV-stained sections. * p < 0.05; ** p < 0.01, two-way ANOVA with Tukey's post hoc analysis (n = 4-5/group). Scale bars = 100 µm.

Fig. 3

Acute pathology after rmTBIs at adolescence under Avertin anesthesia. a-c At 24 h postimpact (2.5 m/s, 0.5 mm), intact tissue integrity was observed by CV staining in the brains of sham (1st row), mTBI ×1 (2nd row) and mTBI ×2 (3rd row) mice (a). No β-APP accumulation was detected (b), and Iba-1-positive microglia were not visibly altered after mTBI ×1 or mTBI ×2 (c). Positive control tissues for comparison and validation of staining were obtained from a moderate-severe CCI model in pediatric (p21) mice [49]. Iba-1-positive microglia were quantified in the dorsal cortex (d) and corpus callosum white matter (e). Volumetric analysis of the dorsal cortex (f) and corpus callosum/external capsule (g) was performed at 3 months postimpact on CV-stained sections. * p < 0.05; ** p < 0.01, two-way ANOVA with Tukey's post hoc analysis (n = 4-5/group). Scale bars = 100 µm.

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At the cellular level, neither single mTBIs nor rmTBIs resulted in elevated cell death compared to sham animals, with fewer than 5 TUNEL- or caspase-3 labeled cells detected across 4 sections per brain. This impact severity did not result in detectable axonal damage following single mTBIs or rmTBIs, as indicated by a lack of immunolabeling for β-APP (fig. 3b). Together, these data confirm that mTBI at this impact severity, delivered either once or repeated after a 48-hour time window, is below the threshold required to induce hemorrhage, skull fracture, cell death, oxidative stress, or axonal damage.

The microglial response was examined by Iba-1 immunoreactivity at 24 h postimpact, focusing on the dorsal cortex and corpus callosum as the regions most likely to be affected by the impacts. Qualitatively, no overt differences in morphology were observed in mTBI brains compared to shams (fig. 3c). Quantification of Iba-1-positive microglia by unbiased stereological counts in the dorsal cortex (fig. 3d) confirmed the lack of an impact effect (two-way RM ANOVA, p = 0.0845) and similar cell numbers in both the hemispheres ipsilateral and contralateral to the impact site (p = 0.1623).

In the white matter (fig. 3e), however, both the degree of impact and proximity to the impact site influenced the magnitude of the microglia response (two-way RM ANOVA effect of impact, p = 0.0130; effect of hemisphere, p = 0.0071). Specifically, post hoc analyses revealed an increased number of Iba-1-positive microglia in the ipsilateral corpus callosum after both single mTBIs and rmTBIs compared to sham controls (p < 0.05 and p < 0.01, respectively). Contralateral to the impact site, more Iba-1-positive microglia were detected in the corpus callosum of the mTBI ×2 group compared to sham controls (p < 0.05). This impact-dependent increase was specific to microglial cells rather than a broader inflammatory response, as neither of the impact groups showed an increase in astrocytic GFAP expression in the corpus callosum compared to sham controls (data not shown; p = 0.8194). At 3 months postimpact, neither single mTBIs nor repeat mTBIs affected the number of microglial cells in either the dorsal cortex (two-way RM ANOVA effect of impact, p = 0.6406) or corpus callosum (p = 0.6359; data not shown).

We lastly considered the possibility that mTBIs may induce a degree of progressive neurodegeneration by conducting unbiased estimations of tissue volume from CV-stained sections at 3 months postimpact (fig. 3f, g). The volume of the ipsilateral and contralateral dorsal cortices were comparable (two-way RM ANOVA, p = 0.4122) and independent of the number of mTBIs (p = 0.1354). Similarly, no differences were observed when comparing white matter volumes across groups (p = 0.1669).

Normal Long-Term Sensorimotor Function after Single mTBIs and rmTBIs

The accelerating rotarod test was used to assay gross motor function and coordination after mTBIs with Avertin anesthesia (fig. 4a). This task was performed across 2 consecutive days at 1 week and 1 and 3 months postimpact. No improvement in rotarod performance was seen over time (two-way RM ANOVA, p = 0.2228), and no impairments were noted in this task as a result of either single mTBIs or rmTBIs (p = 0.5591).

Fig. 4

Assessments of sensorimotor, activity, and anxiety measures after rmTBIs at adolescence in animals under Avertin anesthesia reveal hypoactivity and reduced exploratory behavior. All mice showed normal sensorimotor performance on the rotarod from 1 week to 3 months postimpact (a), as well as equivalent forepaw usage when rearing in the cylinder test at 3 months (b, c). In the open field, mTBI mice traveled a reduced distance at 1 week postimpact compared to sham controls, which resolved by 1 month (d, e). f The number of rearing events in the open field arena was significantly lower in both mTBI ×1 and mTBI ×2 mice compared to sham controls at 1 and 3 months postimpact. g In the EPM, mTBI mice traveled a reduced distance compared to sham controls at 1 month postimpact. h This was reflected in an increased percent time spent at rest. i Anxiety-like behavior, quantified as the percent time spent in the open arms of the maze, was reduced in mTBI mice at 1 week postimpact, indicating a transient anxiolytic phenotype. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way RM ANOVAs with post hoc analyses (n = 10-12/group).

Fig. 4

Assessments of sensorimotor, activity, and anxiety measures after rmTBIs at adolescence in animals under Avertin anesthesia reveal hypoactivity and reduced exploratory behavior. All mice showed normal sensorimotor performance on the rotarod from 1 week to 3 months postimpact (a), as well as equivalent forepaw usage when rearing in the cylinder test at 3 months (b, c). In the open field, mTBI mice traveled a reduced distance at 1 week postimpact compared to sham controls, which resolved by 1 month (d, e). f The number of rearing events in the open field arena was significantly lower in both mTBI ×1 and mTBI ×2 mice compared to sham controls at 1 and 3 months postimpact. g In the EPM, mTBI mice traveled a reduced distance compared to sham controls at 1 month postimpact. h This was reflected in an increased percent time spent at rest. i Anxiety-like behavior, quantified as the percent time spent in the open arms of the maze, was reduced in mTBI mice at 1 week postimpact, indicating a transient anxiolytic phenotype. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way RM ANOVAs with post hoc analyses (n = 10-12/group).

Close modal

Due to the unilateral nature of the brain impact, the complementary cylinder test was employed at 3 months postimpact to probe for possible unilateral deficits. The total number of rearing events was equivalent between the impact groups (one-way ANOVA, p = 0.8725), indicating normal motor ability to reach an upright position for forepaw placement on the cylinder walls (fig. 4b). All mice showed a strong preference for initiating the rearing posture by placing a single paw compared to both paws simultaneously (two-way RM ANOVA, p < 0.0001; fig. 4c); however, the impact did not affect this preference, and use of the contralateral forepaw was similar across all groups (p = 0.4780). Together, these data indicate normal motor function after single mTBIs and rmTBIs.

rmTBIs Result in Chronic Hypoactivity and Reduced Exploratory Behavior

To assess anxiety, general activity, and exploratory behavior, the mice were tested in the open field and EPM paradigms at 1 week and 1 and 3 months after 0, 1, or 2 mTBIs under Avertin anesthesia. General activity was highest when the mice were tested at 1 week postimpact and reduced over time as the mice matured to adulthood, in both the open field arena (two-way RM ANOVA, p < 0.0001) and the EPM (p < 0.0001). In the open field paradigm (fig. 4d), mTBI mice tended to move around less than sham-operated mice, with an overall trend towards traveling a reduced distance (p = 0.0544). Of note, the mTBI ×2 group showed hypoactivity at 1 week postimpact compared to sham controls (Tukey's post hoc, p < 0.05). However, time spent at rest was not significantly different between impact groups (p = 0.2459; fig. 4e).

Exploratory behavior was also quantified as rearing up to place forepaws on the arena walls in the open field arena (fig. 4f). Sham controls showed an increase in rearing behavior over the time course (two-way RM ANOVA, p < 0.0001), consistent with previous evidence that this behavior is developmentally regulated [49]. At 1 week post-impact, rearing behavior appears normal in mTBI mice; however, a striking reduction becomes apparent over time, with both single mTBI and rmTBI mice rearing significantly fewer times at 1 and 3 months postimpact (Tukey's post hoc, p < 0.05 and p < 0.001, respectively). As mTBI mice show normal sensorimotor function, this reduction in exploratory rearing behavior is unlikely to be attributed to a physical deficit.

In the EPM (fig. 4g), mTBI mice typically moved less overall (two-way RM ANOVA, p = 0.0160), particularly at 1 month postimpact, when mTBI ×1 mice traveled a significantly smaller distance compared to sham controls (Tukey's post hoc, p < 0.01). Similarly, mTBI mice spent more time at rest overall (p = 0.0108; fig. 4h), particularly at 1 month postimpact, when both the mTBI ×1 and mTBI ×2 groups spent more time immobile compared to sham controls (Tukey's post hoc, p < 0.01 and p < 0.05, respectively).

The other key outcome measure of the EPM is anxiety behavior. Percentage time in the open arms (fig. 4i) was reduced across the time course (two-way RM ANOVA, p < 0.0001), consistent with previous evidence from our laboratory of an increase in baseline anxiety at adulthood compared to adolescence [49]. Mice that received mTBIs generally spent less time in the open arms across the time course (two-way RM ANOVA, p = 0.0193), indicating an increase in anxiety. Specifically, mTBI ×1 mice spent significantly less time in the open compared to sham-operated mice at 1 week postimpact (Tukey's post hoc, p < 0.05), which was resolved by 1 and 3 months postimpact.

Normal Cognition after Single mTBIs and rmTBIs at Adolescence

Hippocampal recognition memory was assessed at 1 month postimpact in the NOR test. During familiarization (fig. 5a), all mice spent ∼50% of their exploratory time with each of two identical objects (one-way ANOVA, p = 0.5749). After a 4-hour interval, the mice were returned to the arena for 5 min and presented with one familiar and one novel object (fig. 5b). All groups exhibited recognition memory by spending more time than chance investigating the novel object. However, this preference for the novel object over a now familiar one was not affected by mTBI (p = 0.6529). The total time spent investigating objects was also equivalent across groups (p = 0.2984 and p = 0.8163 for the familiarization and testing phases, respectively).

Fig. 5

rmTBIs do not impair cognition at 1 month postimpact in animals anesthetized with Avertin. During the familiarization stage of the NOR task (a), all groups spent equivalent time with the left- and right-positioned identical objects, as expected. During the test stage (b), all groups showed evidence of object recognition, as evident by increased time spent with the introduced novel object compared to the familiar one (∼60%). Data are expressed as means + SEM. During the RAWM, all groups demonstrated learning over time during trial blocks (3 trials) when the platform was visible (c) and hidden (d). One- or two-way ANOVAs (n = 10-12/group).

Fig. 5

rmTBIs do not impair cognition at 1 month postimpact in animals anesthetized with Avertin. During the familiarization stage of the NOR task (a), all groups spent equivalent time with the left- and right-positioned identical objects, as expected. During the test stage (b), all groups showed evidence of object recognition, as evident by increased time spent with the introduced novel object compared to the familiar one (∼60%). Data are expressed as means + SEM. During the RAWM, all groups demonstrated learning over time during trial blocks (3 trials) when the platform was visible (c) and hidden (d). One- or two-way ANOVAs (n = 10-12/group).

Close modal

Hippocampal spatial memory was assessed using the RAWM at 1 month postimpact. During the visible trials (fig. 5c), all mice showed learning of the task over time (two-way RM ANOVA, p = 0.0230). mTBI did not affect this learning, with mTBI ×1 and mTBI ×2 mice making a comparable number of errors to sham mice (p = 0.6827). During the hidden platform trials (fig. 5d), a learning curve was again evident across all groups (p < 0.0001). mTBI mice displayed normal spatial memory for the platform location during this testing phase, with all groups making a similar number of errors (p = 0.6827). These findings were also reflected by latency to reach the platform (data not shown). An open pool task comprised of 6 consecutive trials was conducted at the end of day 2, when the maze arm inserts were removed and latency to reach the visible platform was recorded. A lack of difference between sham and injured groups in this task (p = 0.2482) indicates normal swimming abilities by all mice.

Both the NOR and RAWM tests were repeated in the same mice at 3 months postimpact. At this time, however, sham control mice did not exhibit recognition memory in the NOR, spending an average of just 53% of total investigative time with the novel object (data not shown). In the RAWM, a low number of errors made during the first trials resulted in a lack of learning across subsequent trials (data not shown). As these observations may have resulted from confounding prior exposure to these tests, these data were not interpreted further.

Assessment of Long-Term Changes after Single mTBIs or rmTBIs in Mice Anesthetized with Isoflurane

Next, we determined the reproducibility of our long-term findings, when an alternative anesthetic, isoflurane, was substituted for Avertin. This evaluation included an additional group that received a third consecutive rmTBI (mTBI ×3), as well as additional tests of home cage activity and measures of depressive-like behavior, to explore the hypoactivity phenotype seen in those animals that had been anesthetized with Avertin.

No Additive Effect from a Third Consecutive mTBI at Adolescence

The mice received 0, 1, 2, or 3 mTBIs during adolescence followed by behavioral assessments at 1, 3, and 5-6 months after impact (fig. 6a). The mean righting time across all groups was 134 ± 5 s after the removal of anesthesia, and time to right was not affected by repetitive injuries (p = 0.3175).

Fig. 6

a Adolescent mice received 0, 1, 2, or 3 mTBI impacts under isoflurane anesthesia and were evaluated over a 6-month period. OF = Open field test. In contrast to the long-term outcomes observed after Avertin anesthesia (fig. 3), mTBI mice after isoflurane anesthesia showed equivalent activity levels and exploratory rearing compared to sham controls during the open field paradigm (b-d). In the EPM, rmTBI also failed to significantly impact either activity level (e) or anxiety (g). However, there was an overall effect of impact on time spent at rest (f; p = 0.0301). Two-way ANOVAs (n = 12-14/group).

Fig. 6

a Adolescent mice received 0, 1, 2, or 3 mTBI impacts under isoflurane anesthesia and were evaluated over a 6-month period. OF = Open field test. In contrast to the long-term outcomes observed after Avertin anesthesia (fig. 3), mTBI mice after isoflurane anesthesia showed equivalent activity levels and exploratory rearing compared to sham controls during the open field paradigm (b-d). In the EPM, rmTBI also failed to significantly impact either activity level (e) or anxiety (g). However, there was an overall effect of impact on time spent at rest (f; p = 0.0301). Two-way ANOVAs (n = 12-14/group).

Close modal

At 1 month postimpact, all groups showed equivalent sensorimotor performance on the accelerating rotarod task, averaging a latency to fall of 208 s (one-way ANOVA, p = 0.4715). Also at 1 month, in the RAWM, all groups demonstrated comparable learning over time in both the visible and hidden platform blocks (visible: two-way RM ANOVA effect of time, p = 0.0198; effect of impact, p = 0.1681; hidden: effect of time, p < 0.0001; effect of impact, p = 0.6273; data not shown). These findings are consistent with our observations after rmTBIs with Avertin anesthesia, and confirm that up to 3 rmTBIs at adolescence do not result in persistent cognitive or sensorimotor deficits.

At 1, 3, and 6 months postimpact the mice were evaluated using the open field and EPM tests for general activity and anxiety. In the open field (fig. 6b-d), behaviors changed over time as expected in all measures (distance, percent time at rest, and rearing events: two-way RM ANOVA, p < 0.001). However, contrary to observations after anesthesia with Avertin, injuries generated under isoflurane anesthesia did not influence these behaviors, even with the addition of the mTBI ×3 group (distance: p = 0.4865; percent time at rest: p = 0.5646; rearing events: p = 0.7976).

In the EPM, key behavioral measures, including the time at rest and time in the open arms, changed across time (two-way RM ANOVA, p = 0.0016 and p < 0.0001, respectively), whereas the total distance traveled did not (p = 0.1904). rmTBIs appeared to modestly reduce the distance traveled in the EPM (fig. 6e), particularly at the 1-month time point compared to sham controls; however, this did not reach statistical significance (p = 0.0638). In alignment, mTBI mice (×1, ×2, and ×3) spent more time at rest at 1 month postimpact compared to sham controls (p = 0.0301; fig. 6f), although Tukey's post hoc multiple comparisons did not identify differences between specific groups. Lastly, the percent time spent in the open arms, a measure of anxiety, was not affected by up to 3 rmTBIs (p = 0.2475).

Lack of Long-Term White Matter Pathology after Three rmTBIs

A subset of brains (n = 4/group) underwent ex vivo MRI and subsequent histological analysis to specifically examine potential changes in white matter at 6 months after impact (fig. 7a). The volume of predominant white matter structures in the mouse brain, including the corpus callosum/external capsule, was calculated from area measurements made on coronal T1-weighted images throughout the structure (fig. 7b). From MRI, the mean volume of the corpus callosum/external capsule was unaffected by repetitive impacts (Kruskal-Wallis test, p = 0.2204). This was consistent with volumetric analyses from CV-stained sections (p = 0.6912; fig. 7c).

Fig. 7

There was no evidence of chronic neuropathology by MRI and histological analysis after rmTBIs during adolescence in animals anesthetized with isoflurane. CC/EC = Corpus callosum/external capsule. At 6 months postimpact, tissue integrity of a subset of mice was evaluated by ex vivo MRI and histology. a Representative images are illustrated for T1-weighted and CV-stained coronal sections. The volume of the corpus callosum/external capsule was quantified from MRI data (b) and histology (c). Kruskal-Wallis tests (n = 3-4/group).

Fig. 7

There was no evidence of chronic neuropathology by MRI and histological analysis after rmTBIs during adolescence in animals anesthetized with isoflurane. CC/EC = Corpus callosum/external capsule. At 6 months postimpact, tissue integrity of a subset of mice was evaluated by ex vivo MRI and histology. a Representative images are illustrated for T1-weighted and CV-stained coronal sections. The volume of the corpus callosum/external capsule was quantified from MRI data (b) and histology (c). Kruskal-Wallis tests (n = 3-4/group).

Close modal

Isoflurane as an Acute Neuroprotectant after rmTBI

Using isoflurane as the surgical anesthetic for equivalent rmTBIs to the adolescent mouse, we did not replicate the hypoactive phenotype observed with Avertin. We hypothesized that the differential results may be attributed to the choice of anesthetic agents, based upon previous studies identifying isoflurane as a neuroprotectant [50,51,52]. To test this hypothesis, neuropathological markers of injury were compared in the brains of Avertin- versus isoflurane-anesthetized mice at 24 h after an equivalent impact severity. A new cohort of animals (n = 5), anesthetized with isoflurane, were subjected to a single mTBI (2.5 m/s, 0.5 mm) and evaluated 24 h later for comparison with Avertin-anesthetized mice that likewise sustained a single mTBI in the initial characterization experiments. Iba-1-positive microglia were quantified in the ipsilateral corpus callosum by an investigator blinded to the anesthetic. Quantification revealed significantly higher microglial reactivity in Avertin- versus isoflurane-treated mice after mTBI (Mann-Whitney test, p = 0.0159; fig. 8a, b). We found a median of 12,418 Iba-1-positive microglia in Avertin-anesthetized mice (consistent with previous quantification by a different investigator) compared to a median of 9,859 Iba-1-positive cells in isoflurane-anesthetized mice - only marginally higher than sham-operated, Avertin-anesthetized mice (fig. 3e).

Fig. 8

Comparison of acute neuropathology after mTBI with Avertin vs. isoflurane anesthesia. Immunohistochemistry for Iba-1 (a) revealed higher numbers of microglia in the corpus callosum/external capsule at 24 h after TBI in Avertin-anesthetized mice compared to isoflurane-anesthetized mice. b Mann-Whitney test, p = 0.0159. Staining for β-APP (c) revealed higher numbers of labeled cells at 24 h after TBI in Avertin-anesthetized mice. d Mann-Whitney test, p = 0.0029.

Fig. 8

Comparison of acute neuropathology after mTBI with Avertin vs. isoflurane anesthesia. Immunohistochemistry for Iba-1 (a) revealed higher numbers of microglia in the corpus callosum/external capsule at 24 h after TBI in Avertin-anesthetized mice compared to isoflurane-anesthetized mice. b Mann-Whitney test, p = 0.0159. Staining for β-APP (c) revealed higher numbers of labeled cells at 24 h after TBI in Avertin-anesthetized mice. d Mann-Whitney test, p = 0.0029.

Close modal

To determine whether axonal injury after mTBI was similarly sensitive to the anesthetic choice, β-APP was evaluated in the corpus callosum/external capsule in Avertin- versus isoflurane-anesthetized mice. As the model parameters utilized for the long-term studies (including the above microglial counts) were deliberately below the threshold necessary to induce β-APP pathology, a higher impact severity was chosen for this comparison (4.5 m/s, 1.5 mm; as described during model characterization). Quantification of β-APP reactivity revealed significantly more axonal injury in Avertin-anesthetized mice (Mann-Whitney test, p = 0.0029, 6-8 sections per brain; fig. 8c, d). Together, these findings suggest that isoflurane provided early neuroprotection after mTBI, thus potentially masking the emergence of long-term changes in behaviors such as hypoactivity.

Summary of Results

Here we define a model of closed-skull mTBI at adolescence that produced no mortality, skull fractures, or acute evidence of neuronal injury, and then used this model to compare single versus a limited number of repeat impacts in terms of long-term consequences. We hypothesized that mTBIs to the adolescent brain would negatively affect long-term outcomes when repeated within an acute postimpact window of vulnerability. To test this hypothesis the impact was titrated below the threshold required to induce cell death or axonal injury either acutely or chronically after a single impact. Repetitive impacts were then applied at 48-hour intervals during a postimpact period of metabolic depression [53,54,55,56] to evaluate the hypothesis that repetitive injuries at adolescence have additive or cumulative effects and result in chronic impairments. Initial studies using Avertin as the surgical anesthetic revealed a modest but significant increase in white matter microglial numbers in acutely injured brains after 1 or 2 rmTBIs compared to sham controls. While longer-term behavioral analyses revealed normal cognitive and sensorimotor behaviors, both single mTBIs and rmTBIs resulted in a consistent hypoactive phenotype. In contrast, there was no evidence of either early pathology or long-term abnormal behaviors, even after 3 rmTBIs at adolescence, when isoflurane was used as the anesthetic. Collectively, these studies demonstrate resilience of the adolescent mouse brain to rmTBIs, with an anesthetic-dependent increase in microglia in the acutely injured brain and hypoactivity serving as a signature long-term outcome.

rmTBIs during Adolescence

Adolescence is a time of ongoing brain maturation, with continuing synaptic pruning, myelination, and network connectivity underlying developmental changes in both structure and function [57]. Injuries to the brain during this time are likely to have age-dependent consequences, although whether this manifests as selective vulnerability or the capacity for enhanced plasticity remains unclear. There is some clinical evidence suggesting that adolescents may have poorer outcomes after mTBIs compared to older athletes [3,7,58], and it has been postulated that even subtle behavioral and/or functional deficits may have profound effects on academic performance at this age. A rising interest in rmTBIs at adolescence has culminated in consensus and evidence-based guidelines identifying key risk factors, screening methods, and management practices in this population [6,59,60].

There is considerable variability across different laboratories in terms of the biomechanical and physical forces used to model concussive-like impacts, including differences in impact severity, location, number, and frequency [61,62]. The complexity of attempting to scale size and time to the human scenario renders it difficult to generate an optimal model. Of note, there remains a paucity of studies designed to specifically characterize the effects of mTBIs on the adolescent brain. The risk of adverse long-term cumulative consequences after rmTBIs, and the potential for age-dependent vulnerability or plasticity at this time, necessitates the employment of age-specific models combined with clinically relevant outcome measures to better understand the mechanisms and manifestations of mTBIs at different developmental stages.

Prins et al. [63] pioneered work in this arena, also using a CCI device to generate concussive-like, closed-skull impacts delivered to rats at p35. The model is characterized by apnea, delayed righting, and toe pinch responses in the absence of a skull fracture, and 2-4 impacts with an interimpact interval of 24 h [53,63,64,65]. Acute cognitive deficits were reported up to 3 days after repetitive impacts, alongside acute glial reactivity and axonal injury. Four rmTBIs resulted in neuroendocrine dysfunction and abnormal sociosexual behavior over several months postimpact [64,65], suggesting that mTBIs during adolescence may disrupt the process of pubertal maturation. One caveat of this model is the reported 10% mortality rate after 2 repetitive impacts [63], provoking the possibility that this impact is more severe than the concussive and subconcussive mTBI injuries that frequently occur during adolescence. A second juvenile model has been developed by adapting the Wayne State method, whereby p28-30 rats undergo horizontal rotation upon impact [62,66,67]. Using this model, a single mTBI results in changes in cognitive and psychosocial behavior [38,67], and a succession of 10 daily impacts impaired vestibulomotor function acutely, which was resolved by 1 month postimpact [62]. Together, these existing models, while limited in scope in terms of evaluation of acute and chronic neuropathology and longer-term outcomes, represent an important first step in understanding how the adolescent brain responds to mTBI.

Here, we sought to evaluate the potential long-term consequences of repetitive head impacts below the threshold required to induce evidence of axonal injury and cell death from a single impact. The detection of a microglial response acutely after impact in our model, in the absence of a delayed righting reflex or neurocognitive deficits, is consistent with subconcussive injury in humans [25]. However, the behavioral and histopathological consequences of subconcussive hits remain controversial, and there is not yet a consensus about what defines a pathological impact to the head.

Our chosen impact severity may be deemed ‘too mild'. Indeed, not every blow to the head is necessarily pathological. This issue highlights the importance of research attempts such as these to explore and delineate the threshold for long-term consequences of brain injuries in an age-specific manner. In addition, adaption of the CCI device as used in the current experiments typically involves stabilization of the head, so that movement upon impact is minimized or restricted to a linear motion. Accumulating evidence suggests that rotational and/or acceleration forces may contribute considerably to concussive-like pathology, and these parameters may need to be included in future modeling of injuries at adolescence [62]. Future work using this mTBI model and others will yield a better understanding of the number, types, and severity of impacts necessary to reach the threshold which produces enduring adverse behavioral consequences.

White Matter Microglia in rmTBI

Microglia are highly motile, rapid responders to perturbations in brain homeostasis known to undergo morphological transformation, migration, and proliferation in response to brain injuries [68,69]. At 24 h after both single mTBI and 2 repeat mTBIs under Avertin anesthesia, we observed an increase in local microglial numbers, which may be attributed to either proliferation or migration, or both. This change, specifically in the corpus callosum, is consistent with recent hypotheses that white matter is particularly vulnerable to both mTBIs [70] and repetitive subconcussive impacts characterized by the lack of a clinical diagnosis of concussion [71,72]. Contrary to a typical inflammatory response, few cells exhibited an amoeboid morphology, with the vast majority of Iba-1-positive cells displaying a ramified ‘resting' appearance and maintaining alignment of key processes alongside white matter axons. While studies have reported the persistence of chronic glial activation for weeks to months after experimental concussive-like injuries [73], this was not the case in our model whereby microglial numbers were equivalent to sham controls at 3 months postimpact. Of note, although we did not see an upregulation of GFAP-positive astrocytes, future studies incorporating other markers of the inflammatory response (e.g. cytokines and chemokines) may provide a more comprehensive characterization of the inflammatory response in this model.

Hypoactivity and mTBI

Hypoactivity, detected as reduced distance traveled, reduced exploratory rearing, and increased time spent at rest, was a consistent finding in Avertin-anesthetized mice after single mTBI or 2 rmTBIs. This phenotype, in the absence of cognitive or sensorimotor abnormalities, appeared to emerge over time after impact, with the greatest difference from sham-operated mice observed at 3 months postimpact. In the clinical arena, reduced activity may reflect depression and/or fatigue, both common consequences of mTBI in patients [8,10,11]. Thus, future studies are needed to determine whether hypoactivity is consistent with indices of depression and/or fatigue.

Influence of Anesthesia on Outcomes after rmTBI

The pathological findings and long-term evidence of hypoactivity, as seen after rmTBIs in mice anesthetized with Avertin, were not replicated in mice that had been anesthetized with isoflurane. In fact, increasing the number of rmTBIs beyond what was studied in those mice anesthetized with Avertin likewise yielded no long-term hypoactivity. This lack of reproducibility may be attributed to one of several reasons. Firstly, it is possible that the phenotype exhibited by rmTBI mice with Avertin anesthesia was not sufficiently robust to be reproducible in the presence of isoflurane. However, as noted above, the abnormal hypoactivity seen in Avertin-anesthetized mice was consistent across several assays and multiple time points, supporting the reproducibility and validity of this observation.

Secondly, Avertin use was associated with ileus or peritonitis in a subset of mice, a side effect previously reported by others [33,74]. While these mice were excluded from the analyses (fig. 1), it is plausible that undetectable Avertin-related complications in the surviving mice could have synergized with rmTBI-related pathology to produce the observed behavioral changes. This explanation seems unlikely given that there were no signs of morbidity in these animals, including weight loss, throughout the duration of the study. Thirdly, the absence of long-term hypoactivity may have been due to isoflurane acting as an acute neuroprotectant. Supporting this reasoning, both axonal damage, as evidenced by β-APP reactivity, and white matter Iba-1-positive microglia numbers were significantly less in mice that had been anesthetized with isoflurane. In line with these findings, others have demonstrated that exposure to isoflurane at or near the time of experimental TBI protects against apoptosis, excitotoxicity, inflammation, and vascular damage compared to several other anesthetics [51,75]. Modulation of secondary injury mechanisms by volatile anesthetics can yield long-term consequences in terms of histological and neurological outcomes after TBI [52], with some investigators even advocating for their potential application as neuroprotective agents in the clinical setting [76]. Our findings warrant caution and careful consideration of future study designs, particularly for those involving mTBIs where certain anesthetics may mask subtle adverse events that give rise to long-term changes in behaviors.

A key objective of this study was to define a model of closed-skull mTBI at adolescence that produced no mortality, skull fractures, or acute evidence of neuronal injury, and then to use this model to compare single versus a limited number of repeat impacts in terms of the long-term consequences. At this magnitude of impact and using Avertin as the anesthetic, we found an increase in microglial numbers that corresponded to long-term hypoactivity in mice that received either a single mTBI or 2 rmTBIs, but a largely normal phenotype of sensorimotor and neurocognitive outcomes. These findings speak to the resilience of the brain to mTBIs, when rotational and/or acceleration forces of the head are not components of the impact. A follow-up experiment using isoflurane as the anesthetic found neither acute pathology nor abnormal long-term behaviors after rmTBIs, a finding that is consistent with this anesthetic functioning as a neuroprotectant.

This project was conducted with the generous financial support of Mr. and Mrs. D. Wong. B.D. Semple was supported by a CJ Martin Early Career Fellowship from the National Health and Medical Research Council of Australia. Research in the Noble-Haeusslein Laboratory and the Xu Laboratory was additionally supported by the National Institutes of Health (grants R01 NS050159 and NS077767 and grant R01EB009756, respectively). The authors would also like to thank S.A. Canchola (Neurobehavioral Core for Rehabilitation Research, UCSF) for assistance with behavioral assays.

The authors declare no competing financial interests.

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