Efficacy of a music-based intervention in a preclinical model of traumatic brain injury: an initial foray into a novel and non-pharmacological rehabilitative therapy

Abstract

Traumatic brain injury (TBI) causes neurobehavioral and cognitive impairments that negatively impact life quality for millions of individuals. Because of its pernicious effects, numerous pharmacological interventions have been evaluated to attenuate the TBI-induced deficits or to reinstate function. While many such pharmacotherapies have conferred benefits in the laboratory, successful translation to the clinic has yet to be achieved. Given the individual, medical, and societal burden of TBI, there is an urgent need for alternative approaches to attenuate TBI sequelae and promote recovery. Music based interventions (MBIs) may hold untapped potential for improving neurobehavioral and cognitive recovery after TBI as data in normal, non-TBI, rats show plasticity and augmented cognition. Hence, the aim of this study was to test the hypothesis that providing a MBI to adult rats after TBI would improve cognition, neurobehavior, and histological endpoints. Adult male rats received a moderate-to-severe controlled cortical impact injury (2.8 mm impact at 4 m/s) or sham surgery (n=10–12 per group) and 24 h later were randomized to classical Music or No Music (i.e., ambient room noise) for 3 h/day from 19:00 to 22:00 h for 30 days (last day of behavior). Motor (beam-walk), cognitive (acquisition of spatial learning and memory), anxiety-like behavior (open field), coping (shock probe defensive burying), as well as histopathology (lesion volume), neuroplasticity (BDNF), and neuroinflammation (Iba1, and CD163) were assessed. The data showed that the MBI improved motor, cognitive, and anxiety-like behavior vs. No Music (p’s < 0.05). Music also reduced cortical lesion volume and activated microglia but increased resting microglia and hippocampal BDNF expression. These findings support the hypothesis and provide a compelling impetus for additional preclinical studies utilizing MBIs as a potential efficacious rehabilitative therapy for TBI.

Introduction

Traumatic brain injury (TBI) is estimated to impact sixty-nine million individuals globally each year (Dewan et al., 2019) and of those approximately 2.8 million reside in the USA (Selassie et al., 2008; Babikian and Asarnow, 2009; Faul et al, 2010; Shiller et al., 2012; Hyder et al., 2017; Taylor et al., 2017). The majority of moderate-to-severe TBI survivors endure persistent disturbances in motor, cognitive, and emotional health domains (e.g., anxiety and coping) that negatively impact their long-term academic, occupational, and social functioning (Shaklai et al., 2014; Williams et al., 2019). Despite the prolific research on pharmacological interventions to promote recovery after TBI, there are no FDA-approved therapies as none have successfully translated to the hospital or rehabilitative facilities (Doppenberg et al., 2004; Menon, 2009). The dire lack of beneficial pharmacotherapies strongly advocates for rigorous empirical evaluation of novel alternative strategies in the continued pursuit to achieve measurable therapeutic success after TBI.

Music-based intervention (MBI) is a non-invasive approach that integrates musical elements to create an enriched and multimodal rehabilitative paradigm that exerts psychophysiological effects across neurological disorders. MBIs, including rhythmic auditory stimulation, can effectively entrain movement evidenced by improved gait and balance in patients with movement disorders (Raglio, 2015) and in pre-clinical models of stroke (Xu et al., 2022). MBIs also exert positive effects on anxiety and depressive symptoms and can enhance cognition such as fluency, working memory, and recognition. In normal uninjured rats, classical music enhances spatial learning and memory (Korsos et al., 2018). In pre-clinical models of epilepsy, cerebral ischemia, and maternal separation, MBIs attenuate cognitive deficits (Xing et al., 2016a; Chen et al., 2021; Papadakakis et al., 2019).

Neural plasticity is important for recovery after TBI, and rodent models provide a unique opportunity to explore the mechanistic pathways by which MBIs promote plasticity and improve functional outcomes. Preclinical TBI studies reveal a chronic downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus and cortex, while non-pharmacological therapeutic strategies like environmental enrichment (EE), a pre-clinical model of neurorehabilitation, or exercise increase BDNF expression (Griesbach et al., 2004; Chen et al., 2005). Concomitant with increased BDNF expression are improvements in functional and cognitive recovery. These correlative findings support the notion that post-injury multimodal rehabilitative paradigms can enhance therapeutic efficacy through BDNF upregulation. Hence, MBIs may hold an untapped potential for enhancing neurobehavioral, cognitive, and affective recovery after TBI by inducing upregulation of BDNF-TrkB signaling.

The few studies reporting on the effects of MBIs discussed are encouraging but empirical research is needed in preclinical TBI models as the data reported derive from normal uninjured rats. We seek to expand on those scant findings by determining whether the benefits of MBI can be verified in a well-established model of TBI in adult male rats. The hypothesis is that classical music provided for 3 h/day commencing 24 h after a controlled cortical impact (CCI) injury will promote significant neurobehavioral and histological benefit relative to only ambient room noise exposure.

Materials and methods

Subjects

Forty-four adult (3 months old) male Sprague-Dawley rats (Envigo RMS, Inc., Indianapolis, IN) weighing 300–325 g on the day of surgery were pair-housed in ventilated polycarbonate cages and maintained in a temperature (21 ± 1°C) and light-controlled (on 07:00 to 19:00) environment with ad libitum access to rat chow and water. After 1 week of acclimatization, training to traverse a narrow elevated wooden beam, on which the rats would subsequently be tested for motor agility, was commenced. The training protocol consisted of 3–5 trials to reach criterion performance defined as traversing the length of the beam with less than 2 hindlimb slips. At the completion of training, the rats were randomly assigned to one of the following conditions: TBI (No Music), n=12; TBI (Music), n=12; Sham (No Music), n=10; or Sham (Music), n=10 (see Fig. 1. for experimental paradigm during the MBI). All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and were conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals. Every attempt was made to limit the number of rats used and to minimize suffering.

Fig. 1. Experimental paradigm.

Adult male rats were randomly assigned to either a controlled cortical impact injury or Sham surgery. Music (MBI) or No Music (ambient room noise) was initiated 24 h after surgery and provided for 3 h (19:00–22:00) every night for 30 days. Motor (Beam), cognitive (MWM), anxiety-like (OFT), and coping (SPDB) behaviors were assessed during the day on post-surgery days 1–5, 14–20, 29, and 30, respectively. (CCI, controlled cortical impact; MBI, music-based intervention; MWM, Morris water maze; OFT, open field test; SPDB, shock probe defensive burying).

Surgery

A controlled cortical impact (CCI) injury or sham surgery was performed as previously described (Cheng et al., 2008; Cheng et al., 2012; Bondi et al., 2014a; de la Tremblaye et al., 2021; Moschonas et al., 2021; Moschonas et al., 2023). Briefly, a surgical level of anesthesia was induced, and maintained, with 4% and 2% concentrations of isoflurane, respectively, in 2:1 N₂O:O₂. After endotracheal intubation the rats were secured in a stereotaxic frame and ventilated mechanically. Using aseptic procedures, a midline scalp incision was made, the skin and fascia were reflected to expose the skull, and a craniectomy (6-mm in diameter) was made in the right hemisphere with a high-speed dental drill. The bone flap was removed and the craniectomy encompassing bregma and lambda and between the sagittal suture and coronal ridge was enlarged further. The impact tip was centered and lowered through the craniectomy until it touched the dura mater, then the rod was retracted, and the impact tip was advanced 2.8 mm farther to produce a moderate-to-severe brain injury. Core temperature was monitored and maintained at 37 ± 0.5°C with a rectal thermistor and heating pad. Anesthesia was discontinued immediately after the impact and the incision was promptly sutured. The rats were extubated and assessed for acute neurological outcomes. Sham rats underwent all surgical procedures, except the impact and removal of anesthesia during suturing.

Acute neurological assessments

After the cessation of anesthesia, hindlimb reflexive ability was assessed by gently squeezing the right and left paw every 5 s and recording the latency to elicit a withdrawal response. Additionally, return of the righting reflex was determined by the latency to turn from the supine to the prone position (Kline et al., 2010; de Witt et al., 2011; Cheng et al., 2012; Bondi et al., 2014a; Bondi et al., 2014b).

Music intervention

Music or ambient noise was initiated 24 h after TBI or sham surgery and was provided for 3 h per night during the rat’s natural active state (19:00 to 22:00) for 30 days (last day of behavior). Specifically, prior to the onset of music or ambient noise, the rats were transferred from the animal colony to a normal lit room where the treatment would take place for 1 h (18:00 to 19:00) so that the rats could acclimate before the initiation of the music. At 19:00 h the room lights were turned off and either the music or ambient-noise condition was initiated.

The TBI and Sham groups randomized to the MBI were exposed to a classical playlist that contained pieces from the classical baroque period with composers including, but not limited to, Mozart, Bach, and Handel (see breakdown, Fig. 2A). The classical baroque style embodies stark juxtapositions between fast and slow movements and structurally is dynamic with repeated motifs between solo instruments and the orchestra and simultaneous harmonies with contrasting timbre, pitch, and rhythm. To ensure variability in overall pitch, movements were selected across various keys, including A, B, C, D, E, F, and G major (see breakdown, Fig. 2B). Tempo is defined as the speed of the music and is measured according to the beats per minute (BPM). It can be classified across a range from Prestissimo, the fastest tempo ranging from 200–206 BPM to Largo, the slowest tempo, with a range of 40 to 60 BPM (Fig. 2C). Musical tempo has been previously linked to evoking positive emotion and influence movement like gait (Rose et al., 2019). In our experiment, we examined a range of tempos and thus covered a sufficient range of standard beats (Fig. 2D). Variability in composers, key, and tempo were necessary to ensure novelty and reflect the diversity by which typical music-listening occurs rather than the same musical piece or composer repeated, thereby holding greater clinical translatability. The music was played through an MP3 player connected to a surround sound speaker system. Using an established playlist, all songs were played in order and consecutively (i.e., no silent pause between pieces). The No Music control group was exposed to normal ambient room noise. Both conditions were kept at 75 decibels.

Fig. 2. Music intervention playlist.

Schematic representation of musical categories that compose the daily playlist. (A) The percentage of musical pieces categorized by composer. Mozart represents the highest percentage (27.78%) of the represented pieces, followed closely by Handel and Corelli with 25% and 16.67%, respectively. (B) The percentage of pieces categorized by musical key, a group of pitches or notes that form the harmonic foundation of a musical piece. (C) Percentage of pieces categorized by the tempo marking which translates to the overall tempo or beats per minute (BPM) that form the speed of a musical piece. The slowest tempo being largo (40–60 BPM) and andante (76–108 BPM) to a medium or walking pace, allegro (120–156 BPM), presto (168–200 BPM) and Prestissimo (200–208 BPM) which are the fastest tempos. (D) The violin plot illustrates the distribution of BPM. The width of the shaded area represents the proportion of pieces of BPM. The median BPM for the music playlist represented by the dashed line is 126.5 BPM with the first and third quartile range of 87.25 and 138.75 BPM.

Motor performance

Motor function was assessed using well-established beam-walk tasks. Briefly, the beam-walk test, modified from that originally devised by Feeney and colleagues (Feeney et al., 1982) and used extensively in our laboratory (Kline et al., 2002; Kline et al., 2012; Olsen et al., 2012; Monaco et al., 2014; Tapias et al., 2022) consists of training/assessing rats using a negative-reinforcement paradigm to escape bright light and white noise by traversing an elevated narrow beam (90 cm above floor level, 2.5 cm wide, and 100 cm in length) and entering a darkened goal box. Performance is measured by time to traverse the beam and quality of ambulation, with the latter quantified using a rating scale from 1 to 7, where 1 reflects an inability to get up onto the beam (severe deficit) and 7 represents two or fewer foot slips (no deficit), while scores of 2–6 reflect various degrees of motor agility (Feeney et al., 1982; Goldstein and Davis, 1990, Kline et al., 1994). Rats were assessed on the beam prior to surgery to establish a baseline measure and again on post-operative days 1–5. Testing consisted of a single trial (60 s allotted time) per day on each task. If a rat was unable to traverse the beam, the maximum time of 60 s was recorded. The daily scores for each rat were used in the statistical analyses.

Cognitive performance: acquisition of spatial learning

Spatial learning was assessed using a Morris water maze (MWM) task (Morris, 1984) that is sensitive to TBI-induced deficits and rehabilitative effects on recovery (Hoffman et al., 2008; Sozda et al., 2010; Kline et al., 2012; Monaco et al., 2014; Tapias et al., 2022). Briefly, the maze consisted of a plastic pool (180 cm diameter; 60 cm high) filled with tap water (26 ± 1°C) to a depth of 28 cm. The platform was a clear Plexiglas stand (10 cm diameter, 26 cm high) positioned 26 cm from the maze wall in the southwest quadrant and held constant for each rat. The maze was situated in a quiet and brightly lit room with salient visual cues. Acquisition of spatial learning began on post-operative day 14 and consisted of providing a block of four daily trials for 6 consecutive days (14–19) to locate the escape platform, which was submerged 2 cm below the water surface. On day 20 the platform was raised 2 cm above the water surface (i.e., visible to the rat) as a control procedure to determine the contributions of non-spatial factors on cognitive performance. For each daily block of trials, the rats were placed in the pool facing the wall at each of the four possible start locations (north, east, south, and west) in a quasi-randomized manner. Each trial lasted until the rat climbed onto the platform or until 120 s had elapsed, whichever occurred first. The rats that failed to locate the escape platform within the allotted time were manually guided to it. All rats remained on the platform for 30 s before being placed in a heated incubator between trials for a 4-min intertrial interval. The times of the four daily trials for each rat were averaged and used in the statistical analyses. All data were obtained using ANY-maze video tracking software.

Cognitive performance: probe trial (memory retention)

On day 20, prior to the visible platform test, a single trial was provided to probe memory retention (Kline et al., 2012; Monaco et al., 2014; Tapias et al., 2022; Moschonas et al., 2023). Briefly, the platform was removed from the pool and the rat was placed in the maze at the most distal point from the target zone (i.e., quadrant where the platform was previously located) and allowed to freely explore the pool for 30 s. The percentage of time spent in the target zone was used in the statistical analysis. The data were obtained using ANY-maze video tracking software.

Open field test (OFT; anxiety-like behavior)

On post-operative day 29, the rats were placed individually in a random corner of an open field arena and allowed to explore freely for 5 min. Locomotion was recorded using ANY-maze video tracking software. Anxiety-like behavior was operationally defined and quantified as the total time spent in the center zone during the 5 min session. Our laboratory has previously demonstrated that the OFT is sensitive to increased anxiety-like behavior after CCI in adult male rats (de la Tremblaye et al., 2021).

Shock probe defensive burying test (SPDB; coping strategies)

On post-operative day 30, the shock probe defensive burying (SPDB) test was administered to assess passive avoidant coping vs. adaptive active coping strategies. The SPDB is a preclinical test that utilizes a brief 2 mA noxious shock in a test cage equipped with bedding to measure time spent immobile and burying, which are behavioral correlates of passive and active coping, respectively (Bondi et al., 2007; Lapiz-Bluhm et al., 2008; Fucich and Morilak, 2018). Briefly, 5 cm of Sani-Chips^® bedding was evenly distributed throughout a modified plastic home cage (42 × 20 × 20 × 20 cm). The shock probe, a 5 mm diameter × 200 mm length glass rod with two uninsulated copper wires spiraling to the end, was inserted 2 cm above the bedding through a pre-drilled center hole on the short wall of the cage. The shock probe extended 6 cm into the cage and was connected to a constant current generator (Coulbourn, model H13–15). At the beginning of the test, rats were placed into the middle of the modified home cage facing away from the shock probe. The rat was allowed to explore the cage and upon contact with the probe was delivered a brief 2 mA shock. Following the shock, the 15 min session was started, and the probe now discharged was left in the cage. Following the completion of the task, that rat was removed and placed back into the original home cage. During the task, the total time spent immobile and burying the probe was recorded, as well as the number of rearings investigating the roof of the enclosure. The dependent measures were active coping, defined as the amount of time (s) the rat spent burying (i.e., displacement of bedding material) directed at the probe, or passive coping, defined as the amount of time (s) spent immobile. All data were obtained using ANY-maze video tracking software.

Histology: cortical lesion volume

After the completion of behavioral testing (i.e., post-operative day 31), the rats were anesthetized with Fatal-plus ^® (0.3 mL, i.p.) and perfused transcardially with 200 mL 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 300 mL 4% paraformaldehyde (PFA). The extracted brains were post-fixed in 4% PFA for one week, dehydrated, and embedded in paraffin. 7-μm thick coronal sections were cut at 1-mm intervals through the lesion on a rotary microtome and mounted on Superfrost ^®/Plus glass microscope slides. The sections were deparaffinized, rehydrated, and stained with Cresyl violet. Cortical lesion volumes (mm³) were assessed by an observer blinded to experimental conditions by calculating the area of the lesion (mm²), which is done by outlining the inferred area of missing cortical tissue for each section taken at 1-mm intervals and then by summing the lesions obtained from each section as previously reported (Kline et al., 2007; Cheng et al., 2008; Bondi et al., 2014a).

Immunohistochemistry: Iba1+, CD163

Paraffin-embedded coronal brain sections were used for immunohistochemistry (IHC) staining with 3,3’-diaminobenzidine (DAB) chromogen. Deparaffinization was performed by placing sections in xylene for 5 min, repeated twice, followed by rehydration with 100%, 95%, and 70% ethanol (EtOH) for 5 min each and washing with 0.1 M TBS (0.1 M tris buffer, pH 7.4 ) for 5 min. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide in 0.1 M TBS for 30 min. Sections allocated for ionized calcium-binding adaptor molecule 1 (Iba1) staining were incubated in a citrate antigen retrieval buffer (pH = 6) heated to 90°C for 10 min, then cooled to room temperature (RT). Sections allocated for CD163 staining were incubated in Tris-EDTA (pH = 9) antigen retrieval buffer heated to 90°C for 10 min, followed by cooling to RT. Non-specific binding was blocked by incubating sections in 3% normal serum in TBS-T (0.1 M tris buffer, pH 7.4, and 0.5% triton x-100) for 30 min at RT. Sections were incubated with primary antibody in blocking solution at 4°C. Specifically, sections were stained with anti-Iba1 (1:1000) for microglia/macrophages, and cluster of differentiation 163 (CD163, 1:1000) for anti-inflammatory macrophages (Zhang et al., 2012; Pedragosa et al., 2018). After washing with TBS-T 3x for 5 min, sections were incubated with secondary antibody (conjugated to horseradish peroxidase) diluted in blocking solution for 1 h at RT. After washing with TBS for 3× 5 min, sections were incubated with avidin-biotin complex (ABC) solution for 1 h at RT. Sections were then washed with TBS 3x for 5 min. Staining was developed by incubating sections with DAB chromogen solution until the desired staining intensity was achieved. Sections were then dehydrated with 70%, 95%, 100% ethanol and 100% xylene for 5 min each. Lastly, sections were mounted and coverslipped with Permount mounting medium.

For CD163 analysis, DAB-stained cells in the perilesional space of the dorsal hippocampus were analyzed using ImageJ software. The perilesional space was selected as CD163 is restricted to perivascular macrophages (Zang et al., 2012; Fabriek et al., 2015). Briefly, images were captured using a Nikon i90 optical microscope at 20x and loaded into ImageJ. The color threshold was adjusted to capture all DAB+ stained particles while excluding any background staining or artifact. The same threshold was applied to every micrograph. Particle analysis was performed to obtain the mean gray value, which represents the intensity of the DAB particles. The same region of interest and area (pixels) was selected for each image that was analyzed. The mean intensity of DAB+ stained cells was subtracted from the background intensity of the tissue (i.e., without any DAB+ stain). For Iba1+ microglial analyses, 7-μm thick coronal sections of the ipsilateral and contralateral dorsal hippocampus were randomly selected. All Iba1+ cells were quantified in 2 different optical frames per section in the hilus of the dentate gyrus of the hippocampus and captured at 20x magnification. Therefore, a total of 4 counting frames were assessed per rat. As previously described, Iba1+ microglial cells were classified into two morphological conditions: resting glia exhibited a small soma with long and thin ramifications and activated glia presented a large soma with retracted and thick ramifications (Roque et al., 2019; Diaz-Chávez et al., 2020). The percentage of resting and activated Iba1+ cells were proportionate to the total Iba1+ cells in the optical frame. Mean intensity of CD163+ cells (EHM, VJV) and morphometric analysis of Iba1+ cells were performed by two investigators (EHM, JPC) blinded to the experimental groups.

Western blot: brain derived neurotrophic factor (BDNF)

At 31-days post-injury, we examined BDNF expression in the ipsilateral and contralateral hippocampus of the TBI (Music) and TBI (No Music) groups. Rats received an overdose of Fatal-Plus (Henry Schein Animal Health, Columbus, OH; 0.25 mL, i.p.) and decapitated. The ipsilateral and contralateral dorsal hippocampus were rapidly dissected on ice, immediately frozen in liquid nitrogen, and stored at −80°C. Samples were homogenized by sonication in lysis buffer (50 mM Tris Base, 150 mM sodium chloride, 5 mM EDTA, pH 7.4) with protease inhibitor (Thermo Scientific). The homogenized whole brain samples were centrifuged at 14,000 RPM for 30 min at 4°C, and the supernatants were collected. Total protein concentration was determined in the supernatant by a BSA protein assay kit (Thermo Scientific, Pittsburgh, PA) using a 96-well microplate reader (Biotek, Winooski, VT). Equal amounts of protein [10 μg] were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Novex) in 10% Tris-glycine polyacrylamide gels and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen, Carlsbad, CA). The membranes were then blocked in 5% non-fat milk (Lab scientific bioKEMIX, Inc.) in 0.1% Tween-20/TBS buffer (TBST) for 1 h at RT. To detect BDNF the blot membranes were incubated overnight at 4°C in rabbit anti-BDNF antibody (1:1000, Abcam) in blocking solution. The following day, the membranes were washed 3x in 0.1% TBST for 5 min at RT and then incubated in anti-rabbit secondary antibody (1:5000, Abcam) diluted in 1% blocking solution containing for 1 h at RT. Protein bands were detected using a chemiluminescence detection system. Membranes were incubated in stripping buffer (Thermo Scientific) for 10 min at RT, blocked, and then probed with mouse anti-β-actin antibody (1:5000, Abcam) in blocking solution, which was used as a loading control. Membranes were imaged with the Chemidoc Imager (BioRad). To directly compare the effect of MBI vs. No Music, one hemisphere from a single brain for TBI (Music) and TBI (No Music) was loaded in the same gel for comparison. Optical density (OD) of each protein was measured using ImageJ (National Institutes of Health). The OD of β-actin was measured for each lane as used for normalization as a loading control for each sample.

Statistical analyses

Analyses of the data were performed by an investigator blinded to experimental conditions using StatView 5.0.1 (Abacus Concepts, Inc., Berkeley, CA). Only after the analyses were concluded and the code was broken did the statistician know the composition of the groups. The motor and cognitive data were acquired over multiple days and thus were analyzed by repeated-measures analysis of variance (rmANOVA). The acute neurological assessment, probe trial, visible platform, swim speed, OFT, SPDB, cortical lesion volume, IHC, and Western blots were assessed on only one day and therefore were analyzed by ANOVA. When the ANOVAs revealed an overall statistically significant effect, the Newman-Keuls post-hoc test was utilized to determine specific group differences. The results are expressed as the mean ± standard error of the mean (S.E.M.) and are considered significant when p values are ≤ 0.05.

Results

There were no significant differences in any of the behavioral outcomes between the Sham control groups regardless of treatment and thus their data were pooled into one group and designated Sham (No Music / Music).

Acute neurological evaluations

No statistical differences were observed between the TBI groups in hind limb withdrawal reflex after a brief paw pinch (left range, 166.7 ± 7.9 s to 173.1 ± 4.5 s, p > 0.05; right range, 162.6 ± 8.1 s to 168.7 ± 4.6 s, p > 0.05) or return of righting reflex (range 360.7 ± 13.4 s to 364.3 ± 10.5 s, p > 0.05) after the termination of anesthesia. The lack of differences with these acute neurological indices suggests that all rats received equivalent injuries and anesthesia. Sham rat reflexes were significantly shorter than TBI rats: paw pinch (left mean = 23.5 ± 2.3 s; right mean = 18.6 ± 2.0 s, p < 0.0001) and righting reflex mean (132.7 ± 5.8 s, p < 0.0001).

Motor performance

Pre-injury baseline assessment of time to traverse the beam revealed no differences among the groups (p > 0.05) indicating that all rats traversed the length of the beam and entered the goal box similarly in under 10 s (Fig. 3A). After TBI, the rmANOVA revealed significant Group [F_2,41 = 298.994, p < 0.0001] and Day [F_5,205 = 137.495, p < 0.0001] differences, as well as significant Group x Day [F_10,205 = 38.572, p < 0.0001] interaction. The post-hoc revealed that the TBI groups, regardless of treatment, took longer to traverse the beam than the Sham (No Music / Music) group (p < 0.05). Regarding the TBI groups, the TBI (Music) group traversed the beam significantly faster than the TBI (No Music) group (p < 0.05). Similar outcomes were observed for the beam-walk score (Fig. 3B). The rmANOVA revealed significant Group [F_2,41 = 422.406, p < 0.0001] and Day [F_5,205 = 227.966] differences, as well as a significant Group x Day interaction [F_10,205 = 55.916]. The post-hoc test showed that both TBI groups scored lower than the Sham group (p < 0.05) and the TBI (Music) group scored better than the TBI (No Music) group (p < 0.05).

Fig. 3. Motor performance.

(A) Time (s) to traverse an elevated narrow beam prior to, and after, TBI or sham injury on post-operative days 1–5. ^#p < 0.05 vs. TBI (No Music) and TBI (Music). *p < 0.05 vs. TBI (No Music). No statistical difference was revealed between the Sham groups exposed to ambient room noise (i.e., No Music) or the music-based intervention (MBI) and thus the data were pooled and are depicted as Sham (No Music / Music). (B) Beam-walk scores prior to, and after, TBI or sham injury on post-operative days 1–5. ^#p < 0.05 vs. TBI (No Music) and TBI (Music). *p < 0.05 vs. TBI (No Music). All data were analyzed by repeated measures ANOVA followed by the Newman-Keuls multiple comparisons post-hoc test. The results are expressed as the mean ± S.E.M. TBI (No Music), n=12; TBI (Music), n=12; pooled Sham (No Music / Music), n=20.

Cognitive performance: acquisition of spatial learning

The rmANOVA of the spatial learning data revealed significant Group [F_2,41 = 57.478, p < 0.0001] and Day [F_5,205 = 25.470, p < 0.0001] differences. The Sham (No Music / Music) group required less time to find the escape platform relative to the TBI groups (p < 0.05). Moreover, the TBI (Music) group located the hidden platform quicker over time vs. the TBI (No Music) group (p < 0.05; Fig. 4A, C). Analysis of the probe data (i.e., memory retention) revealed a significant Group effect for the percentage of time spent in the target zone [F_2,41 = 8.668, p = 0.0007]. Specifically, the post-hoc revealed that the Sham (No Music / Music) and TBI (Music) groups did not differ from each other (p > 0.05) and spent more time (34.8 ± 1.9% and 30.8 ± 1.8%, respectively) in the target zone relative to the 23.6 ± 1.7% of the TBI (No Music) group (p < 0.05; Fig. 4B, D).

Fig. 4. Cognitive performance.

(A) Time (s) to locate the submerged (i.e., hidden) platform in the Morris water maze on post-operative days 14–19. ^#p < 0.05 vs. TBI (Music) and TBI (No Music). *p < 0.05 vs. TBI (No Music). No statistical difference (p > 0.05) was revealed between the Sham groups exposed to ambient room noise (i.e., No Music) or the MBI and thus the data were pooled and are depicted as Sham (No Music / Music). (B) Percent of time spent in the target zone (i.e., quadrant where the platform was previously located) following a single probe trial at day 20. ^#p < 0.05 vs. TBI (No Music). *p < 0.05 vs. TBI (No Music). No statistical differences were revealed between the Sham (No Music / Music) and TBI (Music) groups. No differences were noted among the TBI groups in time to the visible platform (p > 0.05), but both required more time than the Sham controls (p < 0.05). Dotted line represents performance at the chance level (25%). (C) and (D) are representative swim paths showing a decrease in navigation to the escape platform on day 19 vs. day 14, as well as differences in the target zone. All data were analyzed by repeated measures ANOVA followed by the Newman-Keuls multiple comparisons post-hoc test. The results are expressed as the mean ± S.E.M. TBI (No Music), n=12; TBI (Music), n=12; pooled Sham (No Music / Music), n=20.

Analysis of time to locate the visible platform showed a significant Group [F_2,41 = 7.903, p = 0.0012]. The Sham (No Music / Music) group required less time to reach the escape platform vs. the TBI groups, which did not differ from one another (p > 0.05). There were no differences in swim speed among the groups (mean range = 29.7 ± 0.7 to 31.9 ± 1.8).

Open field test (OFT; anxiety-like behavior)

Analysis of the OFT data showed a significant Group effect for time spent in the center of the arena [F_2,39 = 3.828, p = 0.0303]. The post-hoc confirmed that the Sham (No Music / Music) and TBI (Music) groups, which did not differ from one another (p > 0.05) spent more time in the center than the TBI (No Music) group (p < 0.05; Fig. 5). There was no difference in distance traveled among the groups [F_2,39 = 1.377, p = 0.2644], suggesting that locomotor ability was intact (data not graphed due to non-significance).

Fig. 5. Anxiety-like behavior.

Mean (± S.E.M.) time (s) in the center zone of the open field test on post-surgical day 29. ^#p < 0.05 vs. TBI (No Music). *p < 0.05 vs. TBI (No Music). No statistical differences were revealed between the Sham (No Music / Music) and TBI (Music) groups. TBI (No Music), n=12; TBI (Music), n=12; pooled Sham (No Music / Music), n=20.

Shock probe defensive burying test (coping strategies)

The ANOVA for rearing indicated significant group differences [F_2,41 = 5.765, p = 0.0062]. The post-hoc revealed no differences between the Sham and TBI (Music) groups (p > 0.05) but both displayed a greater number of rears compared to the TBI (No Music) group (p < 0.05; Fig. 6A). Burying behavior was significantly reduced after TBI as demonstrated by negligible time spent burying in the TBI (No Music) group compared to the TBI (Music) group (t (22) = 2.368, p = 0.027). Burying time did not differ between the Sham (No Music / Music) and both TBI groups (p > 0.05; Fig. 6B). As depicted in Fig. 6C, there were no immobility differences among the groups (F_2,41 = 0.094, p = 0.916).

Fig. 6. Adaptive vs. maladaptive coping.

(A) Mean (± S.E.M.) number of rears in the shock probe defensive burying test (SPDB). ^#p < 0.05 vs. TBI (No Music). *p < 0.05 vs. TBI (No Music). No statistical difference was revealed between the Sham (No Music / Music) and TBI (Music) groups. (B) Mean (± S.E.M.) burying time (s) in the SPDB test. *p < 0.05 vs. TBI (No Music). No statistical differences were revealed among the Sham (No Music / Music) and the TBI groups, regardless of treatment. (C) Mean (± S.E.M.) immobility (s) in the SPDB test. No differences in immobility were observed among the groups (p > 0.05). TBI (No Music), n=12; TBI (Music), n=12; pooled Sham (No Music / Music), n=20.

Histology: cortical lesion volume

Analysis of cortical lesion volume demonstrated a significant Group effect [F_1,4 = 8.575, p = 0.00429], which was attributed to the TBI (Music) group exhibiting a smaller lesion (50.4 ± 4.9 mm³) relative to the TBI (No Music) group which had a mean lesion volume of 67.8 ± 3.3 mm³ (Fig. 7).

Fig. 7. Histology.

Mean (± S.E.M.) cortical lesion volume (mm³) at day 31 after controlled cortical impact injury. *p < 0.05 vs. TBI (No Music). Inserts in bars depict average size lesions at the level of the dorsal hippocampus. TBI (No Music), n=3; TBI (Music), n=3. No lesion was present in the Sham groups.

Immunohistochemistry: Iba1+ and CD163

IBA1+

Analysis of microglia morphology in the ipsilateral dentate gyrus (DG) of the hippocampus demonstrated significant Group effects for resting [F_2,6 = 96.933, p < 0.0001] and activated [F_2,6 = 96.933, p < 0.0001] states (Fig. 8A–H). The post-hoc analysis revealed that the Sham (No Music / Music) group had an increase in the total percent of resting microglia compared to the TBI (Music) and TBI (No Music) groups (p < 0.05). Between the TBI groups, the MBI increased total percent of resting microglia relative to the ambient noise group (p < 0.05). With respect to the total percent of activated microglia comparing the Sham group to the TBI groups, the Sham exhibited less compared to the MBI and ambient noise groups (p < 0.05), but between the TBI groups MBI reduced activated microglia relative to ambient noise (p < 0.05). Examination of resting microglia in the contralateral DG demonstrated a significant Group effect [F_2,8 = 4.574, p = 0.0474], which was attributed by the post-hoc test to be a difference between the TBI (No Music) and Sham groups with the former exhibiting significantly less resting state microglia than the latter (p < 0.05). No other comparisons were different as the MBI did not significantly impact contralateral hemisphere DG resting microglia (p > 0.05). Analysis of activated microglia morphology in the contralateral DG revealed a significant Group effect [ F_2,8 = 4.775, p = 0.0432] and again the post-hoc analysis revealed the effect to be between the TBI (No Music) and Sham groups, with the former exhibiting an increase in activated state microglia (p < 0.05). The TBI MBI group did not differ from the TBI ambient room noise or Sham groups (p > 0.05).

Fig. 8. Immunohistochemistry, Iba1:

Representative photomicrographs (10x) of Iba-1+ cells in the ipsilateral (A, C, E) and contralateral (B, D, F) dentate gyrus (DG) of the hippocampus. Black arrows in the wide field point to activated Iba1+ cells in the DG subfield that are magnified in the inserts. As depicted in Fig. 8G, total percent of resting microglia in the ipsilateral DG was increased in the TBI (Music) group vs. the TBI (No Music) group (p < 0.05), and the Sham (No Music / Music) had significantly more than both TBI groups (p < 0.05). Regarding total percent of activated microglia, the TBI (Music) exhibited less than the TBI (No Music) group, but the Sham control showed less than both TBI groups (p < 0.05). As shown in Fig. 8H, total percent of resting, and activated, microglia in the contralateral DG were increased, and decreased, respectively, in Sham relative to the TBI (No Music) (p < 0.05). The TBI (Music) group did not differ from either TBI (No Music) or Sham (p > 0.05). TBI (No Music), n=3; TBI (Music), n=3; pooled Sham (No Music / Music), n=3.

CD163

Analysis of CD163+ cells also revealed a significant Group effect [F_1,6 = 11.863, p = 0.0137]. The post hoc analysis confirmed a significant increase of perilesional CD163+ cells in the TBI (Music) group (Fig. 9B, D, F, G) compared to the TBI (No Music) group (Fig. 9A, C, E, G).

Fig. 9. Immunohistochemistry, CD163:

The MBI increased CD163+ cells in the ipsilateral perilesional space after adult CCI injury. (A-F) Representative photomicrographs (10x) of CD163+ cells in the ipsilateral perilesional space of the TBI (No Music) and TBI (Music) groups. (A, B) Representative photomicrograph of CD163+ cells counterstained with Cresyl-violet to better visualize perilesional space in the TBI (No Music) and TBI (Music) groups, respectively. (C, D) Serial sections were DAB stained to observe CD163+ cells in the ipsilateral perilesional space in the TBI (No Music) and TBI (Music) groups, respectively. Black arrows designate CD163+ cells in panels A, B, C, and D. (E, F) Inverted photomicrograph to better visualize CD163+ cells. (G) MBI significantly increased CD163+ cells in the perilesional space compared to the TBI (No Music) group, suggesting increased expression of anti-inflammatory cytokines (p < 0.05). TBI (No Music), n=4; TBI (Music), n= 4. (CCI, controlled cortical impact; CD163, cluster of differentiation 163; MBI, music-based intervention).

Western blot: BDNF

Quantification of BDNF in the ipsilateral dorsal hippocampus demonstrated a significant Group effect [F_1,4 =22.452, p = 0.009], which was attributed to increased BDNF expression in the TBI (Music) group compared to the TBI (No Music) group (Fig. 10A, C). There were no significant differences in contralateral BDNF expression between the groups (p > 0.05; Fig. 10B, D).

Fig. 10. Western blot, BDNF:

(A) MBI increased BDNF expression in the ipsilateral dorsal hippocampus relative to the TBI (No Music) group at day 31 after CCI injury (p < 0.05). (B) No difference in BDNF expression was observed between the groups in the contralateral dorsal hippocampus (p > 0.05). (C, D) Representative immunoblots of hippocampal BDNF expression. Optical density is normalized to β-actin. TBI (No Music), n=3, and TBI (Music), n=3. (CCI, controlled cortical impact; BDNF, brain derived neurotrophic factor; MBI, music-based intervention).

Discussion

Preclinical evaluations of pharmacological interventions for TBI have been robust with researchers working tirelessly to find a treatment that can help the millions of survivors recover from disturbances in neurobehavior and cognition. Regretfully, no pharmaceutical has successfully translated to the hospital in a manner that warrants FDA approval, which strongly advocates for the consideration of nonpharmacological therapeutic strategies. Our goal with this novel experiment was to determine the efficacy of a music-based intervention (MBI) initiated acutely after a controlled cortical impact injury on motor, cognition, and affect, which are all impaired after TBI. In this first of its kind study, we used classical music as that was the genre that showed increased markers of plasticity and cognition in normal rats (Xing et al., 2016a, 2016b; Chen et al., 2021; Rizzolo et al., 2021).

Supporting our hypothesis, post-TBI MBI attenuated deficits in motor performance as evidenced by decreased time to traverse the beam and less hindlimb slips relative to the non-music group. The MBI also facilitated the acquisition of spatial learning and memory in the water maze task relative to the TBI group exposed solely to ambient room noise. Specifically, the MBI group located the escape platform significantly faster and took a much shorter path to the target compared to the No Music control suggesting that daily MBI improves spatial navigational strategies. Additionally, MBI enhanced spatial memory as demonstrated by increased time spent in the target zone searching for the previously situated escape platform. Assessment of control measures, like swimming ability and visual acuity revealed no significant differences between the TBI groups as both displayed similar swim speeds and time to locate the visible platform, confirmed that no extraneous variables precluded the accurate assessment of cognitive ability.

Contrary to the MBI studies in normal uninjured rats, (Xing et al., 2016a; Xing et al., 2016b; Korsós et al., 2018) we did not see an enhancement in cognitive performance in the Sham controls. A likely explanation is that the rats in the current study were pretrained before the surgical manipulation, regardless of surgical grouping, and thus our control rats, which do not exhibit deficits after surgery, are at their maximum performance during music listening, whereas in the basic science studies learning occurred solely during music or no music (Xing et al., 2016a; Xing et al., 2016b; Korsós et al., 2018; Rizzolo et al., 2021).

The MBI also attenuated anxiety-like behavior as measured by the OFT. Specifically, the TBI (No Music) group spent significantly less time in the center of the arena, reflecting an increase in anxiety-like behavior, while the TBI (Music) group and the Sham controls, which did not differ from one another, spent a greater percentage of time in the center zone, which is indicative of non-anxious behavior.

With respect to the SPDB test, the data showed that the active coping response to a stressor was obliterated by TBI as demonstrated by a considerable decrease in time spent burying in the TBI (No Music) group. Burying did not differ between the TBI (Music) group and Sham controls, which suggests an active, adaptive coping response in the MBI group. Although stress hormones were not evaluated in this initial study, burying the probe has been associated with a reduction in stress hormone levels (Bondi et al., 2007; Lapiz-Bluhm et al., 2008). Despite not quantifying stress hormones, our OFT data demonstrate that the MBI reduced stress and anxiety. Post-injury MBI also increased the number of rears indicating increased vigilance. Increased vigilance can be beneficial in detecting and responding to potential threats in the environment. Moreover, there were no differences in immobility among the groups, which suggests that freezing is not why the TBI (No Music) group did not rear or bury. Taken together, these SPDB findings are indicative of adaptive responses to the aversive stimulus via increased defensive responses and alertness. Our results corroborate previous studies reporting that classical music induces anxiolytic effects in disease rodent models (Papadakakis et al., 2019) and clinical trials (Hirokawa et al., 2003; Leardi et al., 2007; Koyama et al., 2009).

The mean cortical lesion volume was significantly smaller in the TBI (Music) group relative to the TBI (No Music) group. The larger size of the lesion in the TBI (No Music) group also impacted the hippocampus and that could partly explain the continued deficits in cognition. Several studies from our laboratory and others have shown correlations between cortical lesion and hippocampal size and its negative influence on learning and memory (Kline et al., 2007; Kline et al., 2010; Kline et al., 2012; Sozda et al., 2010; Monaco et al., 2013). Moreover, the histological benefits extended by MBI are robust and may be multifaceted, which suggest that alternative mechanisms may have also contributed to the observed finding. Several experimental studies have shown that concurrent with enhanced cognition, classical music upregulates BDNF expression in behaviorally relevant brain regions, such as the motor cortex (Chen et al., 2021) and the hippocampus (Xing et al., 2016a; Xing et al., 2016b; Rizzolo et al., 2021). Accordingly, the elevated expression of BDNF revealed in the TBI (Music) group may have served as a neuroprotectant, which resulted in reduced cortical lesion volumes. The increase in BDNF is like that reported in MBI studies utilizing normal uninjured rats (Xing et al., 2016a; Xing et al., 2016b; Chen et al., 2021). Moreover, following TBI, long-term pathological neuroinflammation, including microglial dysregulation and upregulation of pro-inflammatory molecules contribute, in part, to long-term neurodegeneration and neurobehavioral dysfunction. Seminal studies suggest that overtime, TBI-induced lesions lead to progressive tissue loss thereby providing insight into the functional outcomes associated with TBI severity (Bramlett et al., 1997; Dixon et al., 1999; Bramlett and Dietrich, 2002; Kochanek et al., 2002; Bondi et al., 2015). The decreased lesion volume in the MBI group may also be attributed to underlying anti-inflammatory mechanisms.

We therefore assessed perilesional expression of CD-163, an anti-inflammatory specific marker (Zhang et al., 2012; Pedragosa et al., 2018). Our results demonstrate that the MBI significantly increase CD-163+ in the perilesional space compared to the No Music group. These findings suggest that MBI may promote an anti-inflammatory response after injury, as indicated by the increase in CD-163+ cells. Data from clinical and pre-clinical studies suggest that MBIs hold psychoneuroimmunological effects albeit, the mechanisms are poorly understood (Fancourt et al., 2014). Clinically, various forms of MBIs such as passive music listening to participatory music therapy reduced levels of leukocytes (Bittman et al., 2001; Hirokawa and Ohira, 2003; Leardi et al., 2007; Koyama et al., 2009) and cytokines (Stefano et al., 2004; Conrad et al., 2007; Okada et al., 2009; Koyama et al., 2009). Experimental studies demonstrate that music reduced levels of cytokines and histamines in models of anaphylaxis leading to reduced mortality rate (Kim et al., 2015) and enhanced production of anti-inflammatory cytokines and regulatory T-cells in a murine allograft cardiac model (Uchiyama et al., 2012).

We utilized a MBI paradigm with differential musical elements. Rhythm, a time-based pattern of music, can be defined in terms of tempo (i.e., the speed of the beat), meter (i.e., organization of beats), and rhythmic patterns (i.e., arrangement of beats and accents) and can influence motor ability (Rose et al., 2019). Passively listening to rhythmic stimulation simultaneously recruits auditory and motor systems and can synchronize physical movement to the rhythm (Rose et al., 2019; Ito et al., 2022) as exemplified by the subconscious tapping of the foot in synchrony to the beat of the music. Clinical studies demonstrate that rhythmic auditory stimulation, an auditory cueing technique in music therapy, improves gait and balance in stroke and Parkinson’s disease (PD) patients (Rose et al., 2019). Furthermore, medium and fast tempo, defined as approximately 116 and 140 beats per minute (BPM), respectively, enabled better rhythmic entrainment and less asynchrony in PD patients (Rose et al., 2019). Similarly, rats also exemplify a susceptibility to spontaneous rhythmic entrainment as demonstrated by beat synchronization within 120–140 BPM with neural recordings in the auditory cortex (Ito et al., 2022). In our study, we employed classical movements of varying tempos with the greatest proportion (i.e., 47.22%; Fig. 2 C, D) between 109–132 BPM falling within the beat synchronization range of rats. In support of our findings, Chen and colleagues (2019) demonstrated that daily classical music (12 h/day) alleviated motor dysfunction across a battery of motor, sensory, balance, and reflection assessments in a model of focal cerebral ischemia-reperfusion injury in conjunction with upregulated BDNF in the motor cortex. Therefore, we postulate that post-TBI daily MBI enhanced performance in part, through the physiological, neural, and molecular mechanisms underlying rhythmic entrainment.

The behavioral and histological benefits conferred by the MBI are like those observed with environmental enrichment (EE), a preclinical model of neurorehabilitation, that utilizes an expansive environment consisting of various stimuli and social interaction (Hamm et al., 1996; Passineau et al., 2001; Sozda et al., 2010; Matter et al., 2011; Monaco et al., 2013; Bondi et al., 2014b; Moschonas et al., 2021; Moschonas et al., 2023). Like EE, our MBI provides auditory enrichment through various musical elements such as melody, rhythm, and harmony with pieces shuffled daily to mimic the daily rearranging of EE objects to maintain environmental novelty. Moreover, we utilized pieces from the classical baroque period as it stylistically embodies stark juxtapositions between fast and slow movements, structurally, dynamic with repeated motifs between solo instruments and the orchestra, and simultaneous harmonies with contrasting timbre, pitch, and rhythm. This is the first study to demonstrate the efficacy of a MBI in a preclinical model of TBI via controlled cortical impact and together the data support the notion that music-listening is a form of enrichment that imparts the physical-and cognitive-stimulating components necessary to enhance recovery.

Conclusion

The intent of the study was to evaluate a novel, nonpharmacological therapeutic paradigm that could improve the many sequelae of TBI. The findings revealed that a MBI can serve that purpose, at least in the current model of TBI as music listening enhanced motor, cognitive, and affective recovery through modulation of immunological mechanisms and protected against histological damage. Despite the limitation of small groups for the histology, immunohistochemistry, and Western blot assays in this initial foray, we believe that the many benefits provided by the MBI in a well-established model of TBI can be the impetus in establishing a novel therapeutic strategy of music listening as a pre-clinical model of auditory enrichment after preclinical TBI. Future studies will evaluate MBI in females as well as molecular markers and potential mechanisms in larger groups.

Highlights.

• Music-based intervention (MBI) after TBI improved motor, cognition, and affect

• Cortical lesion volume and activated microglia were reduce by MBI

• Resting microglia and BDNF expression were increased by MBI

• The data support MBIs as an efficacious rehabilitative therapy for TBI