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. Author manuscript; available in PMC: 2025 Aug 25.
Published in final edited form as: Neuroimage. 2025 Feb 28;310:121103. doi: 10.1016/j.neuroimage.2025.121103

UTE MRI for Assessing Demyelination in an mTBI Mouse Model: An Open-field Low-intensity Blast Study

Yajun Ma 1, Qingbo Tang 1,2, Xin Cheng 1,2, Jiyo S Athertya 1, David Coughlin 3, Eric Y Chang 1,2, Catherine E Johnson 4, Jiankun Cui 5,6, Zezong Gu 5,6, Jiang Du 1,2,7
PMCID: PMC12372958  NIHMSID: NIHMS2102237  PMID: 40024556

Abstract

Mild traumatic brain injury (mTBI) is a leading cause of long-term disability. Following mTBI, secondary chemical cascades and neuroinflammation can result in myelin damage, significantly impairing cognitive function. This study aims to assess demyelination in mice with mTBI induced by open-field low-intensity blast (LIB) using a novel three-dimensional short repetition time adiabatic inversion recovery UTE (3D STAIR-UTE) magnetic resonance imaging (MRI) sequence. Thirty male C57BL/6 mice, with 15 experiencing mTBI and 15 serving as sham controls, were included in this study. Behavioral tests were performed starting at 5 days post-injury to assess motor activity and anxiety-like responses followed by STAIR-UTE imaging using a pre-clinical 3T MRI scanner. Additionally, a proton density-weighted UTE sequence was scanned alongside the STAIR-UTE for quantification of myelin proton fraction (MPF). Luxol fast blue (LFB) staining was performed to evaluate myelin changes between the mTBI group and the control group. The behavioral tests indicated decreased motor activity in the center zone and increased anxiety-like response in the mTBI mice compared to sham controls. The STAIR-UTE sequence revealed significantly lower MPFs in the corpus callosum of mTBI mice (8.4±0.4% vs. 8.7±0.4%; P = 0.003), consistent with the myelin reduction observed in the LFB staining (0.77±0.22 vs. 1.09±0.15; P = 0.004). Our findings demonstrate that the STAIR-UTE sequence facilitates quantitative myelin imaging at 3T MRI, enabling the detection of demyelination in the white matter of the mouse brain associated with alterations in motor and anxiety domains post-LIB exposure.

Keywords: mTBI, mice, demyelination, MRI, UTE

INTRODUCTION

Mild traumatic brain injury (mTBI) is a significant cause of long-term disability, affecting approximately 1.7 million Americans annually 1,2. This leads to over one million emergency room visits and incurs total annual healthcare costs exceeding $60 billion 2. Despite its prevalence, mTBI is notoriously difficult to evaluate 1,3–5. While conventional magnetic resonance imaging (MRI) techniques are widely used in the clinical diagnosis of various neuroinflammatory and neurodegenerative diseases, they often fail to reveal abnormalities in mTBI cases 3,6,7. Typically, individuals with mTBI do not exhibit conventional imaging findings, such as hyperintense lesions, making accurate diagnosis challenging.

It has been recognized that myelin is particularly susceptible to secondary damage, such as decompaction and demyelination, due to secondary chemical cascades and neuroinflammation that occur both acutely and chronically after TBI 5,8,9. Both linear and rotational brain accelerations can cause severe damage to the myelin sheath 4. Myelin facilitates the rapid propagation of action potentials across nerve cells, thereby supporting cognitive function and behavior. Myelin alteration disrupts axonal transport, integrity, and structural plasticity, potentially leading to physical, cognitive, emotional, and behavioral deficits in mTBI 10–12.

Various forms of myelin damage have been associated with mTBI in both animal and human brains 13–18. Studies have reported myelin loss in white matter tracts following mTBI in rats, with widespread myelin loss observed 3-21 days post-injury 13. Reduced Luxol Fast Blue (LFB) and Rip staining 21 days post-mTBI in some white matter regions suggest remyelination may occur. Myelin damage has also been observed in cadaveric human brains post-mTBI, with long tract demyelination recognized as a long-term sequela of mTBI and myelin degeneration seen as acute lesions in diffuse axonal injury (DAI) 17,18. Autopsy investigations have identified small myelin globoids, likely indicative of secondary myelin damage due to axonal disruption 17.

Therefore, the development of advanced myelin imaging techniques holds the potential to significantly improve health outcomes and quality of life by enabling more accurate diagnoses and more effective therapeutic monitoring of mTBI, particularly through non-invasive MRI methods. However, conventional MRI techniques, such as gradient recalled echo (GRE) or fast spin echo (FSE) sequences, are unable to directly detect myelin signals due to the ultrashort T2 relaxation time of myelin (~0.2 ms) 19–23.

Recently, ultrashort echo time (UTE) sequences, with echo times (TEs) less than 0.1 ms, have been developed and applied to image and quantify short T2 tissues, such as myelin, cortical bone, tendon, and ligaments 24,25. Myelin loss has been successfully detected in mouse brains and in both ex vivo and in vivo human brains affected by multiple sclerosis 26–32. However, to the best of our knowledge, no UTE myelin imaging study has yet been conducted to investigate mTBI. In this study, we aim to evaluate the potential of a novel 3D short TR adiabatic inversion recovery UTE (STAIR-UTE) sequence in detecting demyelination correlated to behavioral outcomes in mTBI mice 28. We employed a well-established murine model of mTBI, specifically an open-field low-intensity blast (LIB) injury model 33–35. Previous studies have identified myelin sheath abnormalities, including split layers, pronounced degeneration, myelin ballooning, disruption, and detachment, within the corpus callosum (CC) of these mTBI mice 34. Behavioral tests were performed to assess motor activity and anxiety-like responses before MRI 34,36. Myelin proton fractions (MPFs) in the CC region were measured using the STAIR-UTE technique to compare myelin contents between mTBI and control groups. Histological staining was also performed to evaluate myelin changes between the mTBI group and the control group.

METHODS AND MATERIALS

Open Field Repetitive LIB Injury Mouse Model

A total of 30 male C57BL/6J mice, approximately eight weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). The study adhered to the guidelines of the Harry S. Truman Memorial VA Hospital Subcommittee for Animal Studies (protocol #1676549-5, approved on 07/28/2022). The animals were randomly assigned into two groups: the mTBI group (n=15) and the sham control group (n=15), with the study being double-blinded to the investigators analyzing the experimental outcomes.

The mTBI group was subjected to a highly reproducible open-field LIB murine model at the Missouri University of Science & Technology, as previously described 34–38. In this model, anesthetized mice were positioned in animal holders in a prone position, facing the explosive source with their head and body longitudinally oriented along the direction of shock wave propagation. The mice were exposed to three blasts, one each day for three consecutive days. They were located three meters from a 350g high-energy explosive C4 detonation, with both the mice and the explosive being one meter above the ground. Mice in the sham control group underwent identical anesthesia procedures without LIB exposure.

Behavioral Tests

Behavioral tests were performed starting at 5 days post-injury (5 DPI, counting from the final blast exposure) 36. Locomotor/exploratory activity and anxiety-like behavior were tested in the open field arenas during the light phase 34,36. The open-field arena (Noldus Information Technology), measuring 40 × 40 cm, was placed on a gray color platform. The animals were placed in the center of the square arena for 10 minutes. After each trial, the box was cleaned with a 10% ethanol solution and air-dried. The animals’ movements were tracked and analyzed using EthoVision TX software (v14. Noldus Information Technology; Windows XP OS). A 10 × 10 cm in the center of the arena was defined as the center zone to evaluate each mouse’s activity tracks and the arena’s remaining area as the peripheral zone. The total distance traveled and time spent in different zones (center vs. periphery, as a measure of anxiety-like response) of the field were determined.

Testing anxiety-like behaviors in response to aversive spotlight stimulation in the dark phase (LightSpot test) for spontaneous behaviors in a home-cage environment was performed in the evening 5 DPI. Figure 1 shows the experimental setup and timeline of the spontaneous home-cage monitoring system 36. For the LightSpot test to investigate the responses of each mouse to a mild aversive stimulus, a yellow light-emitting diode light (2000 lux, comparable to the light of a typical overcast day in the absence of heat production) was introduced without generating explicit pain or discomfort. The LightSpot test followed pre-calibrated experimental protocols in the absence of human intervention.

Figure 1.

Figure 1

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Experimental setup and timeline of the spontaneous home-cage monitoring system. (A) Overview of the experimental setup for spontaneous behavioral testing within the home-cage monitoring system. (B) Timeline outlining the spontaneous behavioral tests conducted using the home-cage monitoring system.

Spontaneous behaviors were assessed in sham controls and mTBI mice using the PhenoTyper home cages (Model 3000; Noldus Information Technology, Wageningen, The Netherlands), as previously described 39–41. Briefly, the PhenoTyper home cages (L = 30 × W = 30 × H = 35 cm) were composed of a top control unit, four semitransparent Perspex walls, an opaque Perspex floor, a shelter, and food and water stations. Mice were housed individually with bedding (Bed-o’Cobs, Laboratory Animal Bedding; The Andersons Laboratory Animal Bedding, Maumee, OH) and provided food and water ad libitum. Mouse behaviors were automatically recorded through a 24/7, infrared-sensitive automated video-tracking system controlled by EthoVision XT software (v14; Noldus Information Technology), sampling at 15 frames per second (FPS). The LightSpot program started at 7:00 PM, and an average spotlight shined for 1 h, from 7:15 PM to 8:15 PM after the acquisition in the dark phase.

Data were uploaded to the Web-based AHCODA-DB (Sylics, Bilthoven, The Netherlands) for meta-analysis. Briefly, movements were classified into short movements, such as turning or rearing against the wall, and long movements, such as traveling from one location to the next. Activity bouts stopped at the encounter of a long arrest segment or a shelter visit that exceeded the short shelter visit threshold.

MRI Scans

This animal research was also approved by the Institutional Animal Care and Use Committee and Research Safety and Security Committee of the VA San Diego Healthcare System (protocol #1223488, approved on 07/07/2021). After the behavioral tests, the mice were transported to the Radiology Service unit at VA San Diego Healthcare System for MRI, which was performed with a DPI of 10.8±1.5, ranging from 8 to 13.

The STAIR-UTE sequence was implemented on a 3T pre-clinical MRI scanner (BioSpec 3T, Bruker, Billerica, MA, USA), equipped with a gradient strength of 450 mT/m and a slew rate of 4200 T/m/s. Before MRI, the mice were euthanized by cervical dislocation following anesthesia with an intraperitoneal injection of phenobarbital at a dose of 200 mg/kg, after which their brains were extracted. Each brain was then washed with saline, coated with Fomblin (34807PI, Solvay Specialty Polymers, Bollate, Italy), positioned in the prone orientation within the cap of a Fisherbrand™ 15 ml conical centrifuge tube (05-539-12, Fisher Scientific, Pittsburgh, USA), and covered with a round glass coverslip. A 1-cm surface coil was placed on top of the coverslip, and the brain sample along with the surface coil was positioned at the isocenter of an 82-volume coil for MRI.

The major features of the 3D STAIR-UTE sequence can be seen in Figure 2. Ensuring robust suppression of long T2 signals necessitates the utilization of a short TR in the STAIR-UTE sequence 28. In this study, employing a short TR of 150 ms guaranteed effective suppression of the broad spectrum of long T2 components with varying T1 values. The optimal TI was determined by numerical optimization (as detailed in Eq. [E10] in Ref. 28). A proton density-weighted UTE (PD-UTE) sequence was scanned alongside the STAIR-UTE to facilitate myelin proton fraction (MPF) quantification. The MPF is defined as the ratio of myelin proton density (i.e., M0,m) to total proton density (i.e., M0,all, myelin proton density + water proton density) 42, taking into account the myelin T1 weighting term from the STAIR-UTE sequence:

MPF=M0,mM0,all [9]

Figure 2.

Figure 2

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3D STAIR-UTE sequence diagram. This sequence incorporates an adiabatic full passage (AFP) pulse to achieve the inversion of longitudinal magnetization primarily associated with long T2 water components, concurrently saturating signals from the ultrashort T2 myelin components. The TI is defined as the temporal interval between the center of the AFP pulse and the center of acquisition spokes. Data acquisition is carried out using a 3D radial UTE sequence with a TE of 20 μs. To enhance scanning efficiency, data acquisition is achieved through a series of UTE spokes within each TR. Selective myelin imaging could be achieved by the synergistic use of a short TR and an optimized TI in STAIR-UTE.

Myelin T1 was set to 380 ms in this study 28,42,43.

The sequence parameters for the mouse brain scans are as follows: 1) 3D STAIR-UTE: TR/TI = 150/64.8 ms, TE = 0.015/1.6 ms, field of view (FOV) = 12×12×36 mm3, resolution = 167×167×500 μm3, flip angle (FA) = 40°, number-of-spokes per TR = 7, interspoke TR = 4.4 ms, bandwidth = 50 kHz, number of excitations (NEX) = 59, and scan time = 5 h 42 min; 2) 3D PD-UTE: TR = 12 ms, TE = 0.015/1.6 ms, FOV = 12×12×36 mm3, resolution = 167×167×500 μm3, FA = 2°, bandwidth=50 kHz, NEX = 6, and scan time = 19 min.

LFB Staining and Histological Analysis

After MRI, each brain sample was immediately fixed in zinc-formalin (Anatech, Battle Creek, MI) at 4 °C for 48 hours, then paraffin-embedded and coronally sectioned at 5 μm thicknesses. Slides were collected with 50 μm intervals, and three slides were stained overnight in LFB (StatLab, McKinney, TX) at 60 °C, followed by brief differentiation with 0.05% lithium carbonate (Poly Scientific R&D C, Bay Shore, NY). 10 control and 10 mTBI mouse brains were stained for comparison. To ensure the staining consistency across different samples, continuous sections from the same sample were collected and stained as controls for each batch. Brightfield image digitization was performed using an Axio slide scanner at 0.22 μm/pixel (AxioScan.Z1, ZEISS, Thornwood, NY, USA). The average optical density (AOD) of the entire corpus callosum (CC) was measured using HALO AI (v3.6.4134, Indica Labs, Albuquerque, NM, USA).

Data Analysis

Velocity, movements, and time spent in the center during the open field test, as well as time in the shelter (TIS) and time outside the shelter (TOS) during the Light Spot test, were compared between the mTBI and control groups using independent t-tests and two-way ANOVA, with P values < 0.05 considered statistically significant. The medial corpus callosum (MCC) region was selected for MRI data analysis, as previous studies have reported myelin changes in this area 34. This region can also provide more reliable MPF measurements due to its relatively large structure, thereby reducing measurement error due to the partial volume effect. The region of interest (ROI) was manually delineated for each mouse brain. To assess the ability of the STAIR-UTE technique to longitudinally detect remyelination, we analyzed the correlation between MPF and days post-injury (DPI) in both the mTBI and control groups.

For LFB-stained slides, the entire CC region was included in the analysis. Specifically, as the control slides for each batch staining are from the same region of the sample, their AODs are assumed to be consistent. The AOD value of each control slide, divided by the AOD of the first control slide, was used as the coefficient to normalize the staining across different batches for comparison. An independent t-test was performed to compare the MPFs between the mTBI and control groups. The correlation between LFB and MRI measurements was also performed using linear regression. ROI delineation and MPF calculation were performed using MATLAB 2022a software (MathWorks Inc., Natick, MA, USA). All statistical analyses were conducted using SPSS 28.0 software (IBM, Armonk, NY, USA).

RESULTS

Figure 3 shows the results of the open-field test to assess anxiety behavior in mice at 5 DPI. There was a trend towards decreased movements in the center and reduced time spent in the center for mTBI mice. Interestingly, mTBI mice also moved faster in the center.

Figure 3.

Figure 3

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Open field test of mTBI mice at 5 DPI. P-values were obtained by paired t-test to compare sham and blast mice. There was a trend toward decreased movements in the center of the square arena and less time spent in the center, accompanied by faster movement in the center by mTBI mice.

Figure 4 presents behavioral observations combining spotlight-evoked behaviors from 7:15 PM to 8:15 PM and subsequent spontaneous behaviors from 8:15 PM to 9 AM in the next day in the home-cage monitoring system. At 5 DPI, mice were individually placed in the home-cage monitoring system in the evening, exposed to the spotlight for 60 minutes, and their behaviors were monitored until the next morning. While there was no difference in the behavioral indices between mTBI and sham mice during the lighting period, mTBI mice spent more time in the shelter and less time outside the shelter from 10 PM to 1 AM in early dark phase. The number of movements, time spent moving, distance moved, total arrest time, and number of arrests observed outside the shelter were also lower, likely due to the decreased time spent by mTBI mice outside the shelter. There was also an increase in the average size of shelter segments. Thus, mTBI mice exhibited a preference for sheltering even in a dark environment during the period from 10 PM to 1 AM.

Figure 4.

Figure 4

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Spotlight-evoked behavioral tests in the automated Home Cage Monitoring (aHCM) system. At 5 DPI, mice were individually placed in the home-cage monitoring system in the evening. They were exposed to the spotlight for the first 60 minutes, and their general behaviors during lighting and afterward were monitored until the next morning. The time in the shelter (A), time spent outside of the shelter (B), number of movements (C), time spent moving (D), distance moved (E), total arrest time (F), number of arrests (G) observed outside the shelter; and average size of shelter segments (H) were plotted against time. Two-way ANOVA was performed for data collected from 10 PM to 1 AM (shaded box) with p-values depicted.

Figure 5 shows representative brain STAIR-UTE images at TE = 0.015 ms and TE = 1.6 ms, PD-UTE images, and corresponding quantitative MPF maps from an adult control C57BL/6 mouse. In the STAIR-UTE images, myelin signals in white matter regions are selectively imaged at the first echo but decay to near zero at the second echo, consistent with their short T2* relaxation times 28. The MPF values are much higher in white matter regions (e.g., corpus callosum) than in grey matter regions (~8.7% vs. ~5.7%), largely consistent with the literature 44–46.

Figure 5.

Figure 5

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Representative STAIR-UTE images at TE = 0.015 ms (first row) and TE = 1.6 ms (second row), along with PD-UTE images (third row) and corresponding quantitative MPF maps (fourth row), from an adult control C57BL/6 mouse. In the STAIR-UTE images, myelin signals in white matter regions are selectively imaged at the first echo but decay to near zero at the second echo, consistent with their short T2 relaxation times. The MPF values are significantly higher in white matter regions (e.g., CC, indicated by a blue arrow) compared to grey matter regions (~8.7% vs. ~5.7%).

Figure 6 shows representative brain MPF maps from a control mouse and an mTBI mouse as well as the summarized MPF measurements for MCC in 13 controls and 13 mTBI. Two mice from each group were excluded from data analysis due to scanner malfunction. The measured MPF values in the MCC region for the mTBI mice are significantly lower than those for the control mice (8.4±0.4% vs. 8.7±0.4%; P=0.003). Figure 7 displays representative brain LFB staining images from a control mouse and an mTBI mouse, along with summarized staining measurements for the MCC in the control and mTBI groups. The LFB staining values in the MCC region for the mTBI mice are significantly lower than those for the control mice (0.77±0.22 vs. 1.09±0.15; P=0.004). These results indicate that demyelination in mTBI mouse brains can be detected using the STAIR-UTE technique, which is consistent with findings from histological staining.

Figure 6.

Figure 6

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Representative brain MPF maps from a control mouse (A) and an mTBI mouse (B) as well as bar plots of the summarized MPF measurements for 13 controls and 13 mTBI in the MCC region (e.g., white matter region inside of the dashed rectangle in panel A, indicated by black arrows) (C). The measured MPF values in the MCC region for the mTBI mice (MPF=8.4±0.4%) are significantly lower than those for the control mice (MPF=8.7±0.4%) (P=0.003). “**” in panel C indicates a P-value lower than 0.01.

Figure 7.

Figure 7

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Representative brain LFB staining images from a control mouse (A) and an mTBI mouse (B) as well as bar plots of the summarized histology measures in the MCC regions for the control and mTBI groups (C). The high magnification of MCC regions in the boxes of panels A and B were shown in A1 and B1 (indicated by black arrows), respectively. The histology measures in the MCC regions for the mTBI mice (0.77±0.22) are significantly lower than those for the control mice (1.09±0.15) (P = 0.004). “**” in panel C indicates a P-value lower than 0.01.

As shown in Figure 8, a significant positive correlation is observed between LFB and STAIR-UTE measurements (R = 0.61, P < 0.05), validating the capability of the STAIR-UTE technique for myelin quantification. Figure 9 presents the correlation between MPF and DPI for both the mTBI and control groups. In mTBI mice, a significant increase in myelin is observed with a longer DPI (R = 0.80, P = 0.001), suggesting remyelination between 8 and 13 DPI. In contrast, no significant myelin changes are detected in the control group during the same period (P > 0.05). These findings suggest that the STAIR-UTE technique has the potential to longitudinally monitor remyelination.

Figure 8.

Figure 8

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Correlation between STAIR-UTE and LFB optical density measurements. A significant positive correlation between MPF and LFB optical density measurements is observed in mouse brains (R = 0.61, P = 0.012).

Figure 9.

Figure 9

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Correlation between MPF and DPI. A significant positive correlation between MPF and DPI is observed in mTBI mice (A), indicating remyelination between 8 and 13 DPI (R = 0.80, P = 0.001). In contrast, no significant correlation is observed in control mice (P >0.05) (B).

DISCUSSION

In this study, we applied the novel STAIR-UTE MRI technique to assess myelin changes in mice subjected to mTBI. Fifteen mice were exposed to open-field LIB to induce mTBI, while another fifteen mice served as controls without being subjected to LIB. Behavioral tests were performed starting 5 DPI before the MRI scans. All mouse brains were scanned using a high-performance pre-clinical 3T MRI scanner. The behavioral tests indicated decreased motor activity and increased anxiety in the mTBI mice compared to controls. The STAIR-UTE sequence revealed significantly lower MPFs in the MCC regions of mTBI mice, which was consistent with the histological findings. Our results demonstrate the capability of the 3D STAIR-UTE technique to detect demyelination in mice subjected to open-field LIB injury-induced mTBI.

Demyelination is well-documented in TBI 5,8,9. However, state-of-the-art methods for detecting demyelination, such as histology and transmission electron microscopy 47,48, are not applicable for clinical diagnosis or treatment monitoring. There is a lack of non-invasive imaging techniques for direct quantitative mapping of myelin loss associated with mTB. Myelin signal decays too fast for conventional clinical MRI to detect 19–23. On the other hand, myelin loss is small in mTBI, which makes it technically challenging to detect. Several conventional MRI techniques have been proposed for indirect myelin mapping, but the quantitative measurements are subject to errors due to edema, iron accumulation, and other factors 49–51. In this study, we propose using our newly developed MR imaging technique, STAIR-UTE, to investigate demyelination in mTBI. This technique has previously been successfully applied to detect myelin loss in cuprizone-treated mice and patients with multiple sclerosis 26. To our knowledge, this is the first time UTE imaging techniques have been applied to study mTBI.

Compared with sham mice, 5 DPI mTBI mice exhibited a trend of spending less time and moving less in the center of the square arena in the open-field test. This was accompanied by faster movement in the center, resulting in no difference in the total distance traveled in the center. These findings differ slightly from those observed in single-blast mTBI mice, which displayed anxiety-like behaviors characterized by decreased distance traveled in the center in the open-field test 34. A surprising result was obtained from a study combining the light-evoked response with subsequent general behavioral observation in a home-cage monitoring system. mTBI mice exhibited a preference for sheltering even in darkness during the period from 10 PM to 1 AM. It is not known if this difference would still exist if the mice had not been exposed to the spotlight. The meaning of this preference is unclear but could potentially be an indicator of anxiety.

Even though mTBI can lead to sustained physical, cognitive, emotional, and behavioral deficits, mild structural or compositional changes in mTBI brains are often undetectable using clinical MRI 3,6,7. Myelin facilitates faster propagation of action potentials across nerve cells and provides crucial support to cognitive function and behavior, playing a significant role in mTBI pathology 10–12. However, clinical MRIs lack specificity for myelin and can be challenging to interpret. Advances in UTE MRI have enabled whole-brain myelin imaging and quantification. Specifically, the recently developed STAIR-UTE technique allows for selective myelin imaging by suppressing signals from long T2 water components with a broad range of T1 values 28,42. This technique has successfully detected demyelination in patients with multiple sclerosis 28,42.

In this study, the STAIR-UTE technique was applied to investigate demyelination in mTBI mice. The signal-to-noise ratio (SNR) performance of STAIR-UTE imaging in mouse brains was significantly lower than in human brains. This is due to the much smaller structure of white matter in mouse brains, which requires much higher image resolution to visualize and quantify accurately. To improve SNR performance, a large number of NEX (up to 59) was utilized in the STAIR-UTE imaging. In addition, the mouse brains were extracted from the skull for MRI. This procedure helps mitigate Gibbs ringing artifacts caused by the skull which has much higher signal than myelin. The STAIR-UTE-measured MPFs of mTBI brains are only slightly lower than those of controls (8.5±0.4% vs. 8.8±0.4%), despite the significant difference. This demonstrates that brain myelin damage in this open-field LIB injury model is relatively minor, suggesting the possibility of remyelination 5,14.

Many animal models have been developed to replicate various aspects of human mTBI to better understand the underlying pathophysiology and explore potential treatments 52. The most widely used models include the fluid percussion injury (FPI) model, the controlled cortical impact (CCI) model, the weight drop-impact acceleration closed-head injury (CHI) model, and the open-field blast injury model 34,53–56. Unfortunately, many of these animal models show uncontrollable adverse effects, such as skull fracture, intracerebral hemorrhage, axonal injury, neuronal cell death, and brain tissue loss, which are not typically present following a concussion in humans 52,56. In other words, most TBI animal models escalate the mild brain injury to the level of moderate or acute injury, making them unsuitable for the study of mTBI. The open-field LIB injury model used in this study is well-suited for studying mTBI, particularly for evaluating military personnel and veterans. Further STAIR-UTE imaging studies are warranted with this model to evaluate remyelination.

It is technically challenging to obtain reliable results due to the partial volume effect arising from the thin structure of white matter regions in mouse brains and the limited image resolution used in this study. Additionally, the ultrashort T2 properties of myelin (T2 ~ 0.2 ms) further blurred the STAIR-UTE images, making it difficult to map myelin in small white matter bundles reliably. The relatively large MCC regions provided the most consistent measurements across different observers, and were therefore chosen for this study. Moreover, we did not observe significant cortical demyelination in mTBI mice, likely due to the generally much lower myelin content in cortical areas. Subtle changes in gray matter may fall beyond the sensitivity of the STAIR-UTE imaging technique with the current hardware setup. However, we recently upgraded our MRI system with the installation of a cryoprobe, which has improved the image SNR by approximately fivefold. This enhanced setup shows great promise for studying cortical demyelination in future research.

Recently, a promising technique called Rapid Estimation of Myelin for Diagnostic Imaging (REMyDI) has been developed to map myelin water volume in the human brain 57,58. It utilizes a fast multi-parametric quantitative MRI sequence combined with a four-compartment model to decompose unique brain components. Multiple sclerosis studies have highlighted its clinical potential 59. In contrast, our STAIR-UTE technique directly detects short T2 myelin signals, eliminating the need for complex modeling. A future study comparing the myelin quantities measured by these two techniques would be highly informative.

There are several limitations in this study. First, the number of mice for the mTBI and control groups is relatively small, which might affect the statistical significance, especially in behavior tests. Second, the scan time of the STAIR-UTE sequence is relatively long, making it unsuitable for in vivo mouse imaging. Third, our results suggest that the open-field LIB injury model induces minor myelin loss. A longitudinal study using this murine model could provide valuable insights into the processes of demyelination and remyelination in mTBI and assess their detectability with the STAIR-UTE sequence. Although such a study is highly significant, it is currently limited by the requirement for long scan times. Recent advancements in deep learning-based denoising techniques, along with hardware upgrades such as the use of a cryoprobe, are expected to enhance SNR performance and improve scanning efficiency. Fourth, we did not observe significant correlations between MRI measurements and behavioral test outcomes. This may be attributed to the subtle differences observed between the small mTBI and control groups in both MRI and behavioral tests. Future studies with larger animal cohorts could offer greater statistical power to uncover more significant correlations.

CONCLUSION

The 3D STAIR-UTE sequence enables quantitative myelin imaging in the murine brain using a pre-clinical 3T MRI scanner, facilitating the detection of demyelination within the mouse brain’s white matter following open-field LIB exposure. This innovative STAIR-UTE technique has great promise for in vivo mTBI diagnosis and treatment monitoring.

FUNDING

The authors acknowledge grant support from the National Institutes of Health (R01AR079484, RF1AG075717, and F32AG082458), and VA Research and Development Services (Merit Awards I01CX002211 and I01BX006480).

Footnotes

COMPETING INTERESTS

The authors report no competing interests.

CRediT Authorship Contribution Statement

Yajun Ma: Writing – original draft, Methodology, Formal analysis, Supervision, Conceptualization, Funding acquisition. Qingbo Tang: Writing – original draft, Methodology, Investigation, Formal analysis. Xin Cheng: Writing – review & editing, Methodology, Formal analysis. Jiyo S. Athertya: Writing – review & editing, Formal analysis. David Coughlin: Writing – review & editing. Eric Y. Chang: Writing – review & editing. Catherine E. Johnson: Writing – review & editing, Methodology. Jiankun Cui: Writing – review & editing, Methodology. Zezong Gu: Writing – review & editing, Methodology, Supervision, Project administration, Funding acquisition. Jiang Du: Writing – review & editing, Methodology, Supervision, Project administration, Conceptualization, Funding acquisition.

DATA AVAILABILITY

The data that support the findings of this study are available on request from the corresponding author.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

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