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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Exp Neurol. 2021 Apr 5;341:113714. doi: 10.1016/j.expneurol.2021.113714

Differential Neuropathology and Functional Outcome After Equivalent Traumatic Brain Injury in Aged Versus Young Adult Mice

Mecca BAR Islam 1,#, Booker T Davis IV 1,#, Mary J Kando 2, Qinwen Mao 3,4, Daniele Procissi 5,6, Craig Weiss 2, Steven J Schwulst 1
PMCID: PMC8276249  NIHMSID: NIHMS1695222  PMID: 33831399

Abstract

The CDC estimate that nearly 3 million Americans sustain a traumatic brain injury (TBI) each year. Even when medical comorbidities are accounted for, age is an independent risk factor for poor outcome after TBI. Nonetheless, few studies have examined the pathophysiology of age-linked biologic outcomes in TBI. We hypothesized that aged mice would demonstrate more severe neuropathology and greater functional deficits as compared to young adult mice after equivalent traumatic brain injuries. Young adult (14-week-old) and aged (80-week-old) C57BL/6 male mice underwent an open-head controlled cortical impact to induce TBI or a sham injury. At 30-days post-injury groups underwent behavioral phenotyping, magnetic resonance imaging, and histologic analyses. Contrary to our hypothesis, young adult TBI mice exhibited more severe neuropathology and greater loss of white matter connectivity as compared to aged mice after TBI. These findings correlated to differential functional outcomes in anxiety response, learning, and memory between young adult and aged mice after TBI. Although the mechanisms underlying this age-effect remain unclear, attenuated signs of secondary brain injury in aged TBI mice point towards different inflammatory and repair processes between age groups. These data suggest that age may need to be an a priori consideration in future clinical trial design.

Keywords: Traumatic Brain Injury, Age, Neurodegeneration, Behavior, Fear, Trauma, Controlled Cortical Impact

1. Introduction:

Trauma is the leading cause of death and disability in patients 1 to 44 years of age. Traumatic brain injury (TBI) contributes to over a third of these injury-related deaths (Corso et al., 2006; M., 2010; Pearson WS, 2012; Whitlock and Hamilton, 1995). Healthcare expenditures related to TBI approach 80 billion dollars annually with average costs of 4 million dollars per survivor of a severe TBI (Corso et al., 2006; Pearson et al., 2012; Whitlock and Hamilton, 1995). The clinical impacts of TBI are highlighted by a high mortality rate as well as significant long-term complications suffered by its survivors with the progressive development of motor, cognitive, and behavioral disorders. Even subconcussive events, those resulting in subclinical brain dysfunction without the typical symptoms of concussion, have been linked to long-term neurologic impairment (Belanger et al., 2016; Carman et al., 2015). Taken together, it is estimated that 2% of the U.S. population currently lives with a physical disability or behavioral deficits, related to TBI (Oliveira et al., 2008; Selassie et al., 2013; Thurman et al., 1999).

TBI follows a bimodal distribution primarily afflicting young-adults (15–24yrs) and the aged (>65yrs). The highest rate of TBI is in patients 75 years of age or older (Centers for Disease Control and Prevention, 2016). These aged patients have been shown to have higher rates of both hospitalization and cognitive decline as well as a decline in functional outcome in comparison to younger patients (Fraser et al., 2019; M., 2010; Rozzenbeek B, 2013). The relationship between advanced age and cognitive decline post-TBI has been described in detail in both cross-sectional and longitudinal analyses (Fraser et al., 2019; Himanen et al., 2006; Senathi-Raja et al., 2010). These differences are often attributed to higher rates of medical comorbidities, decreased pre-injury functional status, and inflammaging in aged patients as compared to younger patients (Thompson et al., 2006). However, even when these variables are accounted for, age remains an independent risk factor for poor outcome after TBI (Coronado et al., 2005; Hukkelhoven et al., 2003).

Despite much preclinical and clinical research, no effective therapies exist for TBI. Promising results of clinical trials to date have been hindered by design, lack of characterization of multiple pathophysiologic processes, and the heterogeneity of TBI (Prince and Bruhns, 2017a; Saatman et al., 2008). Although, there have been successes in preclinical interventions, these have not translated to clinical success due to the paucity of studies parsing the effect of age and other demographic differences on TBI (Iboaya et al., 2019; Sun et al., 2020) Variations in clinical management, broad inclusion criteria, disparate injury patterns, and different outcome measures all remain important variables(Marmarou et al., 2007). These factors likely contribute to the high failure rate when promising treatments are applied clinically.

It is well known that age is an independent risk factor for a negative prognosis with TBI (Coronado et al., 2005; Hukkelhoven et al., 2003). However, the extent of this age-effect remain unclear. To this end, we aimed to determine the age-specific differences in the pathophysiology of TBI in a murine model TBI. We hypothesized that aged mice would demonstrate more severe neuropathology, greater loss of connectivity between brain regions, and marked behavioral deficits as compared to young mice after TBI.

2. Materials and Methods:

2.1. Study Design

A 2×2 study design was employed to assess the interaction between age (aged vs. young) and traumatic brain injury (TBI vs. sham injury). A power analysis was used to determine the minimum number of mice per experimental group necessary to detect a statistical difference (utilizing the conventional values of 0.05 and 0.2 for α and β, respectively). The resultant animal numbers per experimental group are detailed in each subsection below. Experimental groups were randomly assigned, and all experiments were performed in duplicate. Investigators blinded to experimental groups performed the data analysis.

2.2. Mice

Male C57BL/6 mice were used in all experiments. All mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed within a barrier facility at the Northwestern University Center for Comparative Medicine, Chicago IL. Young mice underwent TBI vs. sham injury at 14 weeks of age (n =22) and aged mice underwent TBI vs. sham injury at 78–80 weeks of age (n=26). Animals were housed in cages based on group with an average of 3 animals per cage. All procedures were approved by the Northwestern University Institutional Animal Care and Use Committee and are reported in accordance with the ARRIVE guidelines on in vivo experimentation.

2.3. Controlled Cortical Impact

TBI or sham injury was induced via a controlled cortical impact (CCI) as previously described by our laboratory. In brief, all mice were size and weight matched prior to injury to ensure equivalent degrees of injury between groups. All mice were anesthetized with 50mg/kg Ketamine (Ketaset, Fort Dodge, IA) and 2.5mg/kg Xylazine (Anased, Shenandoah IA) via intraperitoneal injection. All mice underwent a 1cm longitudinal scalp incision placed in the midline of the scalp exposing the sagittal and coronal sutures of the skull. TBI mice underwent a 5mm craniectomy located 2mm left of the sagittal suture and 2mm rostral to the coronal suture. The dura mater was left intact. The mice were then secured in a stereotaxic operating frame and a commercially available impactor (Leica Biosystems, IL) was rotated into the field. TBI was administered with a 3mm impacting rod at 2.5 m/s to a depth of 2mm and a 0.1s dwell time. Sham-injured mice underwent all the same procedures except for craniectomy and CCI. Scalp incisions were sealed with VetBond 3M (Santa Cruz Animal Health, Dallas, TX). All mice received 0.1mg/kg buprenorphine SR for post-operative analgesia (SR Veterinary Technologies, Windsor, CO). Mice were allowed to recover in their home cages over a warming pad and observed until they regained their righting reflex. There were 3 immediate mortalities related following CCI, all within the aged TBI group. Tissues from these mice were excluded from study. Mice were euthanized at experimental endpoints via carbon dioxide inhalation and cervical dislocation per AVMA guidelines(Makinde et al., 2017b; Makinde HM, 2018; Schwulst and Islam, 2019; Trahanas et al., 2015).

2.4. Histopathology and Immunohistochemistry

Animals (n=5/per group) were euthanized and perfused with 4 °C 1x Hank’s Balanced Salt Solution (HBSS) followed by 4% paraformaldehyde in PBS. Brains were excised and fixed in 4% paraformaldehyde. After fixation the brains were paraffin-embedded and 4μm brain sections were cut. Sections were processed and stained with hematoxylin and eosin (H&E) and neuronal nuclei (NeuN) at the Northwestern University Mouse Histology and Phenotyping Laboratory. Slides were imaged using an Olympus BX41 microscope equipped with an Olympus DP21 camera and photographed at 1.25x and 5x magnification. To quantify cerebral edema we assessed the percentage of vessels showing perivascular vacuolation/enlargement. 5 random 10x microscopic fields per slide were assessed and given scores according to edema level (0, none, 1, mild – 30 % or less, 2, moderate, -- 60 % or less, 3, severe -- more than 60%. (Sobel, 2015). Sections were scored for neuronal degeneration and results are reported as the mean objects per unit area (mm2). All samples were analyzed by a neuropathologist blinded to the experimental groups and scored from 0–3, with 0 representing no neurodegeneration and 3 representing “severe” neurodegeneration as previously published by our group (Mao et al., 2017; Mao et al., 2019).

2.5. MRI Acquisition and Image Processing Methods

At 30 days post-TBI or sham injury mice (n=3–4/per group) underwent magnetic resonance imaging (MRI) examination. Mice were scanned in a 7T Clinscan MRI (Bruker, Billerica, MA) at the Northwestern University Center for Translational Imaging. Each mouse was anesthetized using isoflurane with 100% O2 and secured within a stereotaxic frame within the dedicated animal holder. During the procedure the animals received continuous anesthesia delivered via a nose cone. Their body temperature and respiration were monitored during the procedure using a physiological monitoring system (SAI, New Jersey, USA). A four-channel mouse brain surface coil was positioned over the mouse head and brain images were acquired, MR images processed, and region of interest selected as we previously published (Makinde HM, 2018). A 3D multi gradient echo sequence (mGRE) with the following parameters was used to obtain high isotropic resolution of each mouse brain (150 micrometers in each direction) as well as 3D R2* maps with the following MR timing and acquisition parameters: TR=80msec, and 12 echoes with TE=2.5, 6.3, 10.1, 13.9, 17.7, 21.5, 25.3, 29, 32, 36.7, 40.4, and 44.2 milliseconds) and FA=20. A 2D T1 map was generated using a gradient echo sequencing (GRE) sequence with multiple flip angles (5, 10, 15, 30, 40, 50, 60, 70, and 90). Finally a 2D echo planar sequence with 3 orthogonal directions and multiple -values was used to obtain diffusion weighted images and to generate modified fractional anisotropy (FA) patterns as described in(Makinde HM, 2018). Contrast agent (Agliozzo et al., 2012) and a 3D scan was repeated using the same 3D sequence as described with the aim of enhancing possible lesions associated with blood-brain-barrier disruption. Analysis of morphological data and extraction of quantitative parameters was accomplished using several different software packages: ImageJ (Xinapse) for generating quantitative maps and ITK-SNAP for threshold-based segmentation.

After generating FA Index maps for each subject, analysis and extraction of FA Index values was done using a threshold approach. Brain FA maps were loaded in ITK-SNAP and segmentation of high FA regions was obtained using IT-SNAP’s semi-automated 3D segmentation approach based on a threshold value (i.e. only brain regions with highest FA values were included in the final segmentation volume). The volume of the segmented high FA regions were then divided by the total brain volume (for each subject) and recorded as an index % value (FA Index). Higher FA index values correspond to subjects with more defined FA patterns while subjects with lower FA index are reflective of white matter loss.

2.6. Behavioral Phenotyping

All mice (n=9–13/group) underwent behavioral phenotyping starting at 30 days post-injury in the Northwestern University Behavioral Phenotyping Core. Studies took place over the course of 3 months and were conducted during the light phase cycle.

2.7. Elevated Zero Maze

The elevated zero maze (ZM) began 31 days post injury (DPI) and was used to evaluate the anxiety response of mice (Crawley, 2009). The maze is grey and has a 45cm inner diameter with a 6cm wide track and is elevated 30cm from the floor (Phenome Technologies Skokie, IL). The ZM has 2 open quadrants and 2 closed quadrants with 13.3cm tall acrylic walls. The mice were placed inside the center of the closed quadrant and were exposed to white/yellow light. The animals were recorded for 10 minutes using Limelight 4 software (Actimetrics, Wilmette, IL) to track their movements. The maze was cleaned between trials using 70% ethanol.

2.8. Morris Water Maze

Morris water maze (MWM), conducted 32 DPI, was used to evaluate spatial learning and memory (Oh et al., 2003). In this test, mice were trained to find a hidden platform in a 5ft diameter pool of opaque water (~25 degrees C, 152cm diameter pool). The mice underwent 4 days of training with a visible platform(Watase et al., 2002). Each day of training consisted of 8 trials in which the platform was placed in a different quadrant for each trial. Each trial lasted a maximum of 60s and animals were allowed to stay on the platform for 15s before being dried and returned to their home cage. 48 hours after last day of training the animals underwent 5 days of testing using a hidden platform. Each day had 6 trials. The time spent to reach the platform and the distance swam to reach the platform were recorded. Additionally, a probe test was administered without a platform at the end of days 3–5 of testing. During this probe trial the animals were placed into the center of the maze and allowed 60s to swim freely about the maze with learning indicated by close proximity to where the escape platform had been located. All trials were recorded using the Actimetrics WaterMaze software (Actimetrics, Wilmette, IL).

2.9. Open Field

Open field (OF) testing, conducted 67 DPI, was used to measure anxiety, exploratory behavior, and locomotive activity (Crawley, 2009; Todd D. Gould, 2009). Mice were placed in the center of an enclosed 54.5 × 54.5 cm square box with 30cm tall walls and exposed to white/yellow light. Each trial lasted 5 minutes and movement was recorded and tracked using LimeLight 4 software (Actimetrics, Wilmette, IL). The box was cleaned with 70% ethanol in between trials to minimize olfactory cues. Behavior was recorded with LimeLight 4 software.

2.10. Fear Conditioning

The last assay performed was contextual and cued fear conditioning at 93 DPI(Shors et al., 1992; Weiss et al., 2020). The mice were trained in a 29.5 × 25 cm x 29.5cm chamber equipped with rods that were sized and spaced for use with mice (Coulbourn Instruments, Holliston, MA). During the training the animals received 3 tone-shock stimuli over the span of 15 minutes. 24 hours post-training the animals were placed back into the training chamber and the contextual fear in the form of freezing behavior was assessed for 6 minutes. ~4 hours after contextual fear was assessed, cued fear was sassed by placing the animals into a novel 34 × 28 × 18.5 cm chamber for 8 minutes. 180s into the test the tone from training was played for 60s. Data were recorded using FreezeFrame software (Actimetrics, Wilmette, IL).

2.11. Statistical Methods

Statistical analyses were performed using the statistical software package Prism (GraphPad Software, San Diego, CA). One-way ANOVA with Holm-Šídák’s multiple comparison test was used to assess interaction across groups while two-way ANOVA with a Tukey’s multiple comparison test was used to analyze the interaction between age and TBI. Data are reported as mean ± SEM, unless otherwise stated. Data points were categorized as outliers and excluded if the values were more than two standard deviations away from the mean.

3. Results

3.1. Neuropathology: Histology and MRI show divergent, age-dependent, anatomic outcomes after TBI

Aged Mice Show Attenuated Edema, Neuronal Loss, and Regeneration after TBI

Cerebral edema was assessed on H&E stained brain sections. Young TBI mice demonstrated significantly more edema in the hippocampus (F3,14 =6.10, p =0.007). Sham injured mice did not demonstrate any notable edema. NeuN stained brain sections were used to asses neuronal loss. Young mice demonstrated severe and extensive edema, neuron loss, and gliosis within the cortex, hippocampus, and subcortical grey matter (mean score of 3) as compared to aged mice which demonstrated moderate and variable edema and neuronal loss (2.60±0.25 vs. 0 ±0.0, p<0.001).

NeuN stained brain sections were used to assess neuronal loss. These sections demonstrated severe neuronal loss within hippocampus of the young TBI mice while there was only moderate and variable loss within the hippocampus of aged TBI mice (2.60±0.25 vs. 0 ±0.0, p<0.001).These sections demonstrated severe neuronal loss and decreased NeuN staining, an indicator of neuron maturity within hippocampus of the young TBI mice. Aged TBI mice demonstrated only moderate and variable loss within the hippocampus.

MRI

Aged Mice Demonstrate Attenuated Tissue Loss and Increased White Matter Connectivity after TBI

Increased ventricular volume is used as a surrogate, or biomarker, for brain matter loss after TBI as we have previously published (Makinde et al., 2018). Representative axial MRI images with their corresponding 3D renderings are shown in Figure 3_Image. Ventricular space is color coded and superimposed on the 3D brain outline. The average ratio of ventricular volume to whole brain volume for each group is shown in the graph (bottom panel). As expected, both TBI groups demonstrated increased ventricle size as compared to sham injury (Figure 3_graph, F1,10=10.26, p=0.0094). However, the young TBI group demonstrated significantly greater ventricle enlargement than the aged TBI group despite undergoing identical biomechanical injury patterns (Figure 3_graph, 3.77±0.24% vs. 2.703 ±0.20%, p=0.035).

Figure 3. Contrast-Enhanced T1 MRI Reveals Increased Tissue Loss in Young Animals After TBI.

Figure 3.

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Representative axial MRI gradient echo images with corresponding 3D renderings. Ventricular volume is color coded and superimposed on the 3D brain outline. Percent ventricle volume versus total brain volume is expressed as a ratio. Higher ratio indicates increased tissue loss and replacement with cerebral spinal fluid (ventricle volume). Both TBI groups demonstrate increased tissue loss compared to sham injury (F1,10=10.26, p=0.0094). Young TBI mice exhibit a greater volume of tissue loss and replacement with ventricular space( F3,10=15.50, p=0.0004). The young TBI demonstrated significantly greater ventricular enlargement than aged TBI mice (3.77±0.24% vs. 2.703 ±0.20%, p=0.035). *, *** = P<0.05 & 0.001 respectively.

Comparing the FA index parameter for all cohorts we saw a decrease in the FA index parameter, an indicator of overall white matter loss and consequent altered brain connectivity Figure 4_Image. The young TBI demonstrated the greatest decrease in FA index in comparison to the other groups (Figure 4_Graph, 6.99 ± 0.16% F3,10 =31.69, p<0.0001). Both the images and the statistical comparison of the averaged values suggests a TBI effect on the overall FA parameters consistent with what found in the literature. (Figure 4_Graph F1,10 =32.57, p = 0.0002) This diffusion MRI derived data also indicates a more pronounced effect on FA in the young versus old mice (i.e. greater loss of connectivity). This corresponds to the 3D rendering visualizations shown for a mouse in each cohort (Figure 4_Image).

Figure 4. Aged Mice Demonstrate Preserved White Matter Connectivity as Compared to Young Adult Mice After TBI.

Figure 4.

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Average fractional anisotropy (FA) index. Fractional anisotropy maps were extracted from contrast-enhanced T1 MRI images to reveal white matter connectivity. Aged mice maintained higher connectivity as compared to young mice after TBI (Figure 4_Graph 11.52 ± 0.42%, p = 0.005). Also shown are representative set of rendered 3D images depicting the FA patterns obtained through thresholding from diffusion MRI images (color coded) and superimposed on corresponding 3D brain outlines for each group. **, *** = P< 0.01, & 0.001 respectively.

3.2. Behavioral Phenotyping: Young Adult and Aged Mice Demonstrate Disparate Functional Outcomes After TBI

Anxiety-like behavior

Anxiety-like behavior is regulated by the limbic system, specifically the hippocampus (Popovitz et al., 2019). Prior research has shown that the zero maze (ZM) is a test ideally structured to identify post-TBI anxiety-like behavior in mice (Tucker et al., 2017). This behavior is identified when a mouse spends more time in the closed region than in the open region of the maze (Pellow et al., 1985). In our study, young adult TBI mice show an obvious qualitative disinhibition of normal anxiety-like behavior when compared to the other groups (Figure 5).

Figure 5. Young TBI Mice Show Disinhibition of Anxiety-Like Behavior.

Figure 5.

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Zero Maze tracings show the roaming preferences of mouse groups within the Zero Maze. Increased preference for roaming in the open regions, which are highlighted by white boxes, indicate lower anxiety-like behavior. Young adult TBI mice spend significantly more time in the open regions than do aged TBI mice indicating a marked disinhibition of normal anxiety-like behavior (BOTTOM) (16.08 ± 2.3% vs. 5.70 ± 1.43% time, p =0.0007). *** = P<0.001.

Young adult TBI Mice Exhibit Disinhibition and Increased Locomotion with TBI.

A quantitative interaction based on the percent time the mice spent in the open region of the ZM (Figure 5 Bottom) confirmed theses obvious qualitative observations (Figure 5 TOP). The young adult TBI group spent significantly more time in the open regions indicating disinhibition of the normal anxiety-like behavior seen in mice as compared to young adult sham (16.08 ± 2.3% vs. 5.70 ± 1.43% time, p =0.0007), aged sham (16.08 ± 2.3% vs. 9.2 ± 0.87% time, p <0.0400), and aged TBI cohorts (16.08 ± 2.3% vs. 5.85 ± 1.34% time, p =0.0008). Multiple comparison post-testing did not show any significant interactions between the other groups. Importantly, there was a marked interaction between both age and injury (F1,30 =16.26, p =0.0003).

Aged Mice Demonstrate Greater Anxiety After TBI

Open field testing (OF) was an additional test for an anxiety-like phenotype in mice as well as for a generalized assessment of exploratory behavior. Anxiety is determined by the percent time spent and total distance traveled within the box (Figure 6_Image). OF testing demonstrated that aged TBI mice spent more time in the outer-edge region as compared to other groups indicating inhibition of anxiety-like behavior( F1,10 =32.57, p = 0.0002). The most significant difference was between aged TBI and aged sham (Figure 6 Bottom_Right, 7.77 ± 0.62 %time vs. 10.72 ± 0.98 %time, p =0.048), indicating a hypervigilant state. A significant interaction between age and injury was noted (F1,30=7.522, p=0.0102). Upon analysis of the distance travelled, aged TBI mice were also less explorative. The aged TBI group traveled less and demonstrated an inhibition of exploratory behavior as compared to the young adult TBI group ( 351.12 ± 26.67 cm vs. 545.93 ± 29.69 cm, p =0.0162) and young sham group (351.12 ± 26.67 cm vs. 558.79 ± 78.35 cm, p =0.0274). The greatest difference was between the aged TBI and aged sham groups (Figure 6 Bottom_Left, 351.12 ± 26.67 cm vs. 643.17 ± 45.87 cm, p =0.0003). Again, a group interaction between both age and injury was seen (F1,29 =9.883, p =0.0038). These behavioral observations in Figure 6 demonstrate a marked disinhibition of the aged TBI group compared to young adult TBI. These data are consistent with clinical data showing increased generalized aggression and disinhibition in human patients post-TBI (Wang et al., 2011)

Figure 6. Aged TBI Mice Experience an Increase in Anxiety-Like Behavior in Open Field Testing.

Figure 6.

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Anxiety levels was measured using the percent time the mice spent in the center region (top schematic). While comparisons of young adult groups were unremarkable in open field tests, aged TBI mice spent less time(7.77 ± 0.62 %time vs. 10.72 ± 0.98 %time, p =0.048) and travelled less distance (351.12 ± 26.67 cm vs. 643.17 ± 45.87 cm, p =0.0003)in the center area than their aged sham counterparts indicating a significant inhibition in exploratory behavior. *, ** = P<0.05, & 0.01 respectively.

Associative and Working Memory

TBI Impairs Spatial Learning and Memory in Both Young and Aged Groups

The Morris Water Maze (MWM) is frequently used to asses spatial learning and reference memory. In this test, learning is measured by a decrease in the distance traveled, and proximity to, the hidden platform. Both injury groups (young TBI and aged TBI) demonstrated an increase in the distance traveled as compared to both sham groups (F1,26 =11.1, p= 0.0026). This indicates spatial memory impairment after TBI. Aged animals also traveled more than younger animals (F1,26 =2.82, p =0.069). Proximity to the hidden platform was also measured and demonstrated a greater improvement in spatial learning and memory in young TBI mice as compared to aged TBI mice (54.68 ± 3.56 cm vs. 66.58 ± 5.50 cm, p =0.028)).

Wide Variance in Associative Learning and Memory After TBI with age

Contextual and Cued Fear Conditioning are examinations of associative learning and memory. Contextual fear is hippocampal dependent and is assessed by observing the freezing response of a mouse when placed into the same environment that it previously received an aversive stimulus in. As expected, the TBI groups demonstrated decreased freezing activity as compared to sham group in context ( 35.55 ± 7.80% vs. 49.28 ± 1.56% freezing, F1,25 =62.69, p<0.0001) and cue (37.77 ± 9.90% vs. 53.47 ± 9.31% freezing, F1,25 =13.40, p =0.0012) i.e. they exhibited impaired learning. However, aged TBI mice showed a greater deficit in contextual learning as comparted to young adult TBI mice (30.03 ± 1.84 vs. 41.06 ± 0.97 %freezing; p =0.0003), Figure 7 Top_Right, Bottom Right). This resulted in both an age and injury interaction (F1,25 =14.57, p =0.0008)

Figure 7. TBI Results in Disparate Patterns of Learning and Memory Impairment Between Young Adult and Aged Mice.

Figure 7.

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Measurements of contextual and cued fear were used as a proxy for associative learning and memory between groups. Young animals demonstrated significant decreases in contextual (41.07 ± 0.97% vs. 48.17 ± 1.33% freezing, p=0.045) and cued (27.88 ± 3.57% vs. 44.16 ± 5.42% freezing, p=0.0412) fear post-TBI indicating a decrease in reaction to an aversive stimulus due to injury. Aged animals showed a significant change in contextual fear (30.03 ± 1.84% vs. 50.38 ± 2.21% freezing, p<0.0001) but little effect on cued fear (47.68 ± 4.45% vs. 62.78 ± 3.62% freezing, p=0.0318) indicating a preservation of learning. *,*** = P<0.05 & 0.001 respectively.

Cued fear, on the other hand, assesses the connectivity between the amygdala, hippocampus, and prefrontal cortex. In this test, the mouse is placed into a new environment and subjected to tone associated with an adverse stimulus for 60s. The young TBI mice were the found to be the most disinhibited of all groups spending the least amount of time freezing ( F3,25 =12.72, p <0.001, Figure 7, Figure 7 Top_Left, Bottom_Left). More specifically the young adult TBI group was significantly more disinhibited than the aged TBI group ( 27.88± 3.57% freezing vs. 47.68 ± 4.45% freezing, p =0.0114). This demonstrates a greater preservation of connectivity between the amygdala, hippocampus, and prefrontal cortex in the aged TBI mice as compared to young adult TBI mice. These data are consistent with the fractional anisotropy data showing relative preservation of white matter connectivity in aged TBI mice as compared to young adult TBI mice.

4. Discussion

A number of acute and chronic neurocognitive disorders have been linked to mild-to-severe injuries to the brain (Prince and Bruhns, 2017b). TBI-induced disruptions in neuronal networks can manifest as deficits in memory, attention, cognition, impulsivity, and mobility (Rabinowitz and Levin, 2014). Much of the data guiding our understanding of these post-traumatic neurocognitive outcomes comes from preclinical research in adolescent-aged mice. This is despite the well-known structural and functional changes that occur within the aged brain, and TBI-related deficits have not been well characterized in aged subjects (Peters, 2006). Given the lack of prior research on the interaction between TBI and age, we hypothesized that aged mice would have marked behavioral, anatomic, and neuropathologic deficits after TBI as compared to young mice after TBI. Counter to our hypothesis, we observed larger structural abnormalities and greater degree of neuropathologic injury in young adult mice after TBI as compared to aged animals. This corresponded to differential deficits in learning, memory, and anxiety response between young adult and aged mice after TBI. The significance of these results is the finding of a divergent pathophysiology of injury in aged versus young subjects.

To interrogate the interaction between age and TBI we examined functional, anatomic, and pathologic outcome measures. We found that aged mice had markedly less secondary tissue injury after TBI. In our model, H&E stained sections show extension of the controlled cortical impact lesion through the left cortical hemisphere and into the left hippocampus in both young adult and aged TBI mice demonstrating an equivalent injury severity between groups. However, 30 days post-injury, extensive edematous vacuolization was identified in the young adult TBI brains demonstrating significant post-injury edema in the young injury cohort as compared to the aged injury cohort (Figure 1_H&E). In stark contrast, young adult TBI mice demonstrated a decrease in the number of mature neurons 30 days post-injury while only mature neurons were observed in the brains of aged TBI mice. Neuronal immaturity may be suggestive of an active neurogenesis in the young adult TBI cohort that was absent in the aged TBI cohort (Figure 1_NeuN and Figure 2). Interestingly, recent analysis of microglia in rats and mice with TBI has shown evidence of immunosenecence and impaired phagocytic activity in aged microglia, a finding that could explain the lack of tissue clearing that we see in the aged TBI brains (Ritzel et al., 2019; Sun et al., 2019). These data support a distinctly different injury response between young and aged subjects.

Figure 1. Low Magnification (1.25x and 5x) Reveal Disparate Structural Injury Patterns Between Young and Aged Mice 30-Days Post-TBI.

Figure 1.

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4um sections were principally stained with Hematoxylin and Eosin (Ott et al., 2010) and Neuronal Nuclear Antigen (NeuN) (BOTTOM). H&E staining of the hippocampal region, highlighted by the black boxes, demonstrates more edema and structural deformation of the hippocampus in the young TBI group (F3,14 =6.10, p =0.007). The greatest statistical significance was seen between young TBI vs. young sham (2.00±0.45 vs. 0.60 ±0.25, p=0.013). NeuN staining reveals significantly more neuronal loss in the hippocampus of young TBI mice (F3,14 =94.93, p <0.0001). The greatest difference seen in young TBI vs. aged TBI mice (2.60±0.25 vs. 0 ±0.0, p<0.001)

Figure 2. Higher Magnification Demonstrates Decreased Neurodegeneration in Aged Groups Post-TBI.

Figure 2.

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Histological analysis of hippocampal regions in young and aged mice was performed by a neuropathologist blinded to the experimental group. These analyses revealed more neurodegeneration and decreased NeuN staining in the dentate gyrus (DG) and CA3 in both hemispheres of young mice post TBI compared to aged. Histological reveals complete ablation of ipsilateral DG and CA3 in young TBI animal. This obliteration of the tissue can be seen as an absence of stained tissue in the young TBI image panel.

These post-mortem findings were also observed in-vivo utilizing magnetic resonance imaging (MRI). MRI was used to extract ventricle volume size and generate fractional anisotropy (FA) maps. Enlargement of the ventricles post-TBI was used as a marker of tissue loss as we have previously published (Figure 3) (Makinde et al., 2018). These data demonstrated significantly greater ventricular volume in young adult TBI mice as compared to aged TBI mice representing relative preservation of brain matter in the aged subject post-TBI. Similarly, FA maps (measurement of white matter connectivity via preservation of axonal diameter, fiber density, and myelin structure) showed that aged TBI mice had a significantly higher threshold of volumetric FA as compared to young adult mice post TBI (Figure 4). Again, these data suggest a markedly different pathophysiology of injury in the aged subject as compared to the young adult subject after TBI.

These neuropathologic and anatomic changes were then found to correlate with marked differences in functional outcomes between young adult and aged TBI mice. We examined several different neurocognitive outcome measures including learning, memory, and anxiety-like behavior. With regard to anxiety-like behavior, mice normally display a situationally dependent level of protective, anxiety-like, behavior to novel objects and spaces. In our study, we noted a marked disinhibition of this normal anxiety-like behavior in the young adult TBI group as compared to sham TBI and aged TBI mice. This behavioral phenotype suggests a disruption in the connections between the hippocampus, basolateral amygdala, and prefrontal cortex— areas of the brain associated with the regulation of anxiety and aggression (Corder et al., 2018; Johnson et al., 2018; Wang et al., 2011). The disinhibited behavior seen in the young adult TBI group is analogous to the behavior seen in human patients after TBI and has also been used as a confirmatory indicator of anxiety and depression like behavior in mice (Wang et al., 2011).

The marked behavioral differences between young and aged mice after TBI were underscored by the disinhibited behavior of young adult mice post-TBI compared to the higher level of anxiety-like behavior and lower exploratory behavior in aged post-injury mice (Figure 5). These opposing effects of age on functional outcome after TBI were also observed in open field testing. In open field testing, young adult TBI mice were observed to spend significantly less time in the center of the field. This again indicates disinhibition of normal anxiety-like behavior. Aged TBI animals, on the other hand, demonstrated a significant reduction in exploratory behavior as measured by total distance traveled, a result that is congruous to the depressed activity seen in up to 42% of post-TBI adults ≥65 years (Figure 6) (Albrecht et al., 2015). These data, in conjunction with the associated literature, suggest commonalities in the behavioral changes observed in both preclinical models as well as human patients after TBI. Furthermore, they both demonstrate a marked interaction between age and injury. While further study is required to improve the rigor of these observations, the current data points towards an age-related preservation of connectivity between brain regions. This data correlates well with the marked difference in FA index between young adult and aged mice after TBI.

Next, age-related differences in learning and memory were tested post TBI. For this analysis associative learning and memory were tested with contextual (Context) and cued (Cue) fear conditioning while spatial learning and memory were assessed with the Morris Water Maze (MWM). In keeping with behavior phenotypes discussed above, these tests also revealed significant, age-related, differences in learning and memory after TBI. While both young adult and aged TBI mice demonstrated increased freezing in response to a cued stimulus, deficits in associative learning and memory were more pronounced in aged mice as compared to young adult mice (Figure 7). Meanwhile, differences in spatial learning and memory were largely unremarkable between groups. First, the observed similarities in spatial learning and memory deficits may be secondary to the asymmetric nature of spatial memory regulation in the rodent brain. In rodents, the left hippocampus is required for acquisition of long-term spatial memory engrams during learning (Shipton et al., 2014). However, in our model of controlled cortical impact, the left hippocampus is mechanically obliterated. Associative learning and memory, on the other hand, is a bilateral process relying on preserved thalamo-cortical networks (Pergola and Suchan, 2013). Tests of associative learning and memory in our model demonstrated relative preservation of these networks in aged TBI mice as compared to young adult TBI mice, again contributing to the different pathophysiology of injury in aged subjects as compared to young subjects.

Our study’s findings demonstrated significant differences in neuropathology, anatomy, and functional outcome between young and aged mice after TBI. Our data also correspond with recent findings in aged rats revealing worsened behavioral deficits and an altered immune response in aged animals after TBI(Sun et al., 2019; Sun et al., 2020) This provides strong evidence that injury propagates differently in young and aged subjects. TBI follows a bimodal distribution primarily afflicting young-adults (15–24yrs) and the aged (>65yrs). In fact, the highest rate of TBI is in patients 75 years of age or older (Centers for Disease Control and Prevention, 2016). Advanced age is associated with higher rates of hospitalization, decreased functional outcome, and higher rates of cognitive decline after TBI as compared to younger patients(Faul, 2010; Roozenbeek et al., 2013; Willemse-van Son et al., 2007). In fact, the association between older age and cognitive decline after TBI has been well described across both cross-sectional and longitudinal analyses (Fraser et al., 2019; Himanen et al., 2006; Senathi-Raja et al., 2010). These differences are often attributed to higher rates of medical comorbidities, decreased pre-injury functional status, and inflammaging in aged patients as compared to younger patients(Thompson et al., 2006). However, even when these variables are accounted for, age remains an independent risk factor for poor outcome after TBI (Coronado et al., 2005; Hukkelhoven et al., 2003). Our data suggests a markedly different pathophysiology of injury after TBI in young adult versus aged subjects which may account for some of these age-related differences seen in human patients. Furthermore, different pathophysiology of injury between the young and aged may underlie the failure of clinical trials to date to demonstrate clinical efficacy.

The authors acknowledge the limitations of the present study. The first are the limits of generalizability of an animal study to human subjects and the utilization of the CCI model. The chosen model of TBI provides the benefit of delivery of a focal injury while limiting diffuse effects. Admittedly, the CCI model’s tight control of injury parameters and consistent reproducibility between experiments may not fully recapitulate the disparate nature of TBI in human patients. Further, deeper analysis of neuro-regeneration would provide insight that is more mechanistic. Additionally, in focusing on the influence of age on traumatic brain injury we were required to exclude female mice from this study. Significant size differences between male and female animals would have resulted in greater mechanical injury in the smaller animals. Furthermore, there are multiple reports of sex-based differences in TBI. These differences require detailed, in-depth analysis outside the scope of the current study.

5. Conclusions

The current study presents novel evidence demonstrating divergent pathophysiology of injury between young adult and aged subjects after TBI. The results demonstrate marked differences in neuropathology, neuroanatomy, and functional outcome after TBI in size and weight matched young vs. aged mice after equivalent biomechanical traumatic brain injuries. Young mice demonstrated a marked disinhibition of normal anxiety-like behavior after TBI while aged mice demonstrated relative preservation of associative learning and memory. These differences in functional outcome correlated with relative preservation of white matter connectivity between different regions of the brain in aged TBI mice as compared to young adult TBI mice. This preserved connectivity matches the post-mortem neuropathology in that brain sections from aged TBI mice showed markedly attenuated signs of secondary brain injury as compared to young adult TBI mice. At the same time, the young adult TBI cohort demonstrated evidence of new, immature, neurons at 30-days post injury suggesting a more active repair and regeneration process. Taken together these data show a significantly different pathophysiology of injury in young adult versus aged mice after TBI. These data suggest that age may need to be an a priori consideration in future clinical trial design. Moreover, explicit attention paid to intrinsic biological variability may allow previously promising preclinical interventions to be successfully translated to age-specific groups of TBI patients.

Highlights:

  • Recent reviews implicate unrefined research for the current lack of clinically viable TBI treatments.

  • Here we show the novel finding that a matching TBI leads to contrasting pathophysiology of injury in aged vs young mice.

  • Identical brain injuries induced different age-dependent behavioral and cognitive outcomes.

  • MRI and histopathologic analyses revealed divergent age-based effects on white matter connectivity.

  • Explicit attention to divergent age-related pathophysiology of injury may allow age-targeted use of previously unsuccessful TBI treatments.

Acknowledgements:

Funding: This work was supported by the National Institutes of Health 1R01GM13066 and 3R01 GM130662–01S1

Abbreviations:

TBI

Traumatic Brain Injury

CCI

Controlled Cortical Impact

FA

Fractional Anisotropy

MRI

Magnetic Resonance Imaging

GRE

Gradient Echo Sequence

OF

Open Field

ZM

Elevated Zero Maze

MWM

Morris WaterMaze

FC

Contextual and Cued Fear Conditioning

ARRIVE

Animal Research: Reporting of In Vivo Experiments

Footnotes

Declarations:

-Competing interests

The authors of this work declare no competing interests.

Ethical Approval and Consent to participate

Animals were treated and cared for in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals. The Northwestern University Institutional Animal Care and Use Committee approved the experimental protocol.

Consent for publication

All authors of the manuscript have read and agreed to its content and are accountable for all aspects of the accuracy and integrity of the manuscript in accordance with ICMJE criteria

This article is original, has not already been published in a journal, and is not currently under consideration by another journal

Availability of supporting data

N/A

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