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9.4 Tesla in vivo Quantitative Susceptibility Mapping (QSM) detects thalamic calcium influx associated with repeated mild traumatic brain injury (mTBI)
Ferdinand Schweser1,2, Austin Poulsen3, Dhaval Shah1, Nicola Bertolino1, Marilena Preda1,2, Jenni Kyyriäinen4, Asla Pitkänen4, Robert Zivadinov1,2, and David J Poulsen3

1Buffalo Neuroimaging Analysis Center, Department of Neurology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY, United States, 2MRI Clinical and Translational Research Center, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY, United States, 3Department of Neurosurgery, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, NY, United States, 4Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland

Synopsis

This work investigated if Quantitative Susceptibility Mapping (QSM) can detect thalamic Ca2+ influx associated with an alteration of the N-methyl-D-aspartate receptor in a rodent model of mild TBI (mTBI). We found significant concentrations of calcium after repeated mTBI, but not after single mTBI, suggesting that persistent calcium deposits represent a primary pathology of repeated injury.

Introduction

A key event in the pathophysiology of traumatic brain injury (TBI) is the dynamic molecular alteration of the N-methyl-D-aspartate receptor structure and function1, with the concomitant thalamic influx of substantial amounts of Ca2+.2 Detection of this calcium influx in vivo would provide a window into the biochemical mechanisms of TBI that has significant clinical implications for the objective assessment of injury severity and drug development.

In the present work, we investigated if the novel, calcium-sensitive9 technique Quantitative Susceptibility Mapping (QSM)3-6 can detect diamagnetic calciumphosphates7-9 associated with Ca2+ influx in mild TBI (mTBI).

Methods

Animal model: In this IACUC-approved study, we employed the lateral fluid percussion injury (FPI) model10, which replicates clinical TBI without skull fracture. We performed a 5mm craniotomy (right hemisphere) in 12 adult, male Wistar rats. Application of a single 1.7atm pressure pulse modeled single mTBI (s-mTBI, four rats), repeated pressure pulses once/week over four weeks modeled repeated mTBI (r-mTBI, five rats); 3 rats were taken as shams; 2 control rats had no surgery. We performed Neurological Severity Score (NSS) assessments at 24 hours post-injury to confirm negligible functional impairment.

Data acquisition and analysis: We performed MRI at 9.4T (four-channel Rx-array; Isoflurane/O2) at one week and one month after the first injury using a multi-echo gradient echo sequence (TE1=2.31ms, ΔTE=3.10ms, TR=100ms, tip=19°, 16 echoes, BW=50kHz, FOV=30x30x14mm3, matrix=135x208x97). Susceptibility maps were reconstructed with best-path unwrapping11, multi-echo multi-channel phase combination12, R2*-weighted field-mapping13, V-SHARP14-16, and QUASAR-HEIDI17. We assessed volumes of lateral ventricles and bilateral hippocampus and segmented focal diamagnetic lesions in the thalamus on QSM. After the second MRI, rats were perfused with gadobutrol (Gadavist, Bayer) for MR-microscopy18 (MRM; FLASH, TE=4.14ms, TR=18.6ms, averages=8, 36µm isotropic) with a cryogenic coil (CryoProbe, Bruker Biospin) and histology (calcium: Alizarin red; iron: Perl’s Prussian Blue; neurons: Nissel). We used paired and two-sample t-test, respectively, for comparisons.

Results

QSM visualized FPI-induced hemorrhages at gray-white matter junctions (Figs. 1 and 2). No differences in ventricular space were detected between s-mTBI and r-mTBI (p>0.4) at either time point. Hippocampal volumes of shams and s-mTBI (but not r-mTBI; p=0.06) were decreased (sclerosis) at 5 weeks compared to controls (p<0.014) and were higher (swelling) in r-mTBI than in s-mTBI (p=0.036). We identified focal hypointensities in the ipsilateral thalamus (Figure 4) in 100% of r-mTBI animals (0.41±0.26mm3; -66±14ppb; Figs. 1-right, 2 and 3), 50% of s-mTBI (0.014±0.009mm3; -56±56ppb), and 33% of sham (0.021mm3; -41ppb). Iron and calcium stains were distinct from one another and correlated with QSM (Figure 5); Nissl-stain sections revealed neuronal loss in deep layers of the ipsilateral cortex, thalamus and dentate hilus in r-mTBI. MRM indicated calcification in both thalamic gray and adjacent white matter tracts (Figure 3).

Discussion

Using 45Ca-autoradiography, Osteen et al.2 reported a delayed focal accumulation of Ca2+ in the thalamus beginning 2-4 days post-injury and progressing for up to 2 weeks. Using QSM of the FPI model, we successfully detected this clinically relevant pathology in vivo. Conventional magnitude-based MRI was not able to differentiate calcium from other susceptibility inclusions, such as hemorrhages (Figures 1 and 2). While we also detected hypointense focal spots in s-mTBI and shams, those were of substantially lower volume than in r-mTBI (Figure 4), suggesting a different biophysical or imaging/reconstruction-related origin and indicating excellent separation from r-mTBI-induced calcium deposits. In particular, the finding in shams indicates that these small lesions were not related to mTBI pathology (false-positive).

Conclusion

The unique ability of QSM to differentiate between diamagnetic (calcium phosphate) and paramagnetic (heme-iron) substances unambiguously revealed thalamic calcium influx concomitant to mTBI. Hence, the technique has the potential to become a sensitive tool for studying mTBI pathophysiology and in drug development. In particular, our finding of significant concentrations of calcium in the r-mTBI model, but not in s-mTBI (Figure 4), suggests that persistent calcium deposits represent a primary pathology of repeated injury.

Acknowledgements

Research reported in this publication was funded by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001412. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

References

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Figures

Figure 1. MRI of rats that received s-mTBI (left) and r-mTBI (right), respectively. Representative echo-averaged magnitude images are shown in the left columns and susceptibility maps in the right columns. Images were collected at one week (top) and one month (bottom) after the first injury. The hypointense spots on the magnitude images in the thalamus of r-mTBI at one month correspond to diamagnetic substances (red circles). Hemorrhage appears hyperintense on susceptibility maps.

Figure 2. MRI images of r-mTBI corresponding to the stains in Fig. 5 (marked by red circles). Shown are echo-averaged magnitude image, R2*-map (0…100/s), SHARP-corrected phase image, and the susceptibility map (-80…+150ppb).

Figure 3. Minimum intensity projections (mIPs) over 50µm of MR-microscopy (MRM) images of an r-mTBI animal. The yellow inserts are magnifications of the MRM-mIPs showing the calcifications (arrows). The red inserts are mIPs over 1.16mm of the in vivo susceptibility map of the same animal at a similar anatomical location.

Figure 4. Volume over average magnetic susceptibility for all hypointense lesions on QSM found in this study (4 in r-mTBI, 2 in s-mTBI, and 1 in sham; one data point per lesion).

Figure 5. Histological stains of regions indicated by red circles in Figure 2. Top: Calcium (Alizarin) and neuronal stains (Thionin) of the cortex (top row) and thalamus (bottom row). Bottom: Iron staining (Perl’s Russian blue). Note intensive labeling at gray /white matter junction under the impact site (left panel).

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)
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