White Matter Swelling Masked Axonal Loss Detected by Diffusion Basis Spectrum Imaging  (DBSI)
Tsen-Hsuan Lin1, Mitchell Hallman1,2, Fay Hwang1, Yong Wang1,3,4,5, Sheng-Kwei Song1,4,5, and Peng Sun1

1Radiology, Washington University School of Medicine, St. Louis, MO, United States, 2Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States, 3Obstertic and Gynecology, Washington University School of Medicine, St. Louis, MO, United States, 4The Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, United States, 5Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, United States

Synopsis

The extent of axonal loss plays a significant role in irreversible neurological impairment in spinal cord injury (SCI). We detected a 15% axonal loss in SCI mice using diffusion basis spectrum imaging (DBSI) that was masked by injury induced white matter swelling.

Introduction

Axonal loss is the primary cause of permanent neurological disability in spinal cord injury.1,2 Diffusion tensor imaging (DTI) derived fractional anisotropy (FA) has been widely sued to non-invasively and longitudinally assess the severity of SCI. However, co-existing axonal injury/loss, demyelination, and inflammation significantly interfere with the accuracy of FA assessed SCI severity. In this study, we applied diffusion basis spectrum imaging (DBSI)3,4 to detect, distinguish, and quantify axonal pathologies in a mouse model of SCI in vivo, followed by immunohistochemistry (IHC) validation. Our results support that DBSI may serve as outcome measure to quantitatively reflect irreversible damage in SCI.

Materials and Methods

Animal model: A contusive SCI at T9 vertebral level was performed in eleven of seventeen 10-week-old female C57BL/6 mice. The remaining six mice (sham) underwent laminectomy without contusion. The injury group received contusion injury at 0.1 m/s with 0.8 mm impact displacement using 1.3-mm diameter rounded tip. After impact, the site was closed in layers followed by subcutaneous administration of Baytril (2.5mg/kg) and lactated Ringers (5mL). Standard postoperative care was performed.5 DBSI: A pair of 8-cm diameter volume and 15 × 8 mm2 surface active-decoupled coils was used to cover T8 – T11 vertebrae. DBSI was performed at day 3 post contusion on a 4.7-T Agilent small-animal MR scanner utilizing a multiple-echo spin-echo diffusion-weighted sequence.6 A 25-direction diffusion scheme was performed.7 All images were obtained with following acquisition parameters: TR = 0.3 (with respiratory gating) s, TE = 43 ms, inter-echo delay = 16 ms, Δ = 25 ms, δ = 6 ms, maximal b-value = 2,200 s/mm2, slice thickness = 1.8 mm, FOV (field of view) = 10 × 10 mm2, in-plane resolution = 104 × 104 µm2 (before zero-filled). Data analysis: A lab-developed DBSI code was performed on diffusion weighted MR data to estimate $$$\lambda$$$$$$\parallel$$$, $$$\lambda$$$$$$\perp$$$, and FA derived by DBSI and DTI, and DBSI specific fiber, restricted (putative cellularity) and non-restricted isotropic (putative edema) diffusion tensor fractions. Histology: mice were perfusion fixed immediately after the in vivo diffusion weighted MR measurements for immunohistochemical (IHC) staining of SMI-312, MBP, DAPI, and SMI-31.

Results

DTI and DBSI derived $$$\lambda$$$$$$\parallel$$$, $$$\lambda$$$$$$\perp$$$, and FA (Fig. 1 and 2) revealed that DBSI specifically reflected axonal pathologies without confounding effects from inflammation and axonal loss. The extent of inflammation, i.e., increased cellularity and edema, and axonal loss was reflected by DBSI derived restricted and non-restricted isotropic, and anisotropic (injured and non-injured axonal fibers) tensor fractions, respectively (Fig. 3, and 4). White matter volume estimated using diffusion weighted anatomic images increased at 3 days after SCI (Fig. 4C). In contrast, axonal fiber fraction (Fig. 4D) derived by DBSI significantly decreased in SCI group. A new metric “axon volume” was derived, i.e., fiber fraction × white matter volume, to quantitatively estimate the extent of axonal loss. DBSI derived “axon volume” significantly decreased in SCI (Fig. 4E). IHC confirmed the DBSI detected/quantified co-exiting pathologies, including axon and myelin damage, inflammation, and axon loss in mouse SCI.

Conclusion

DBSI detected, distinguished, and quantified the co-exiting axonal pathologies in SCI mice 3 days after contusion, confirmed by post-MRI quantitative IHC. Results demonstrated that DBSI can “see through” confounding co-existing pathologies, e.g., inflammation and tissue loss, for quantifying the extent of axonal injury, demyelination, inflammation, and the irreversible axonal loss.

Acknowledgements

Supported in part by NIH R01-NS047592, P01-NS059560, and NMSS RG 5258-A-5

References

1. Wujek JR, Bjartmar C, Richer E, et al. Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. Journal of neuropathology and experimental neurology 2002;61:23-32.

2. Kornek B, Storch MK, Weissert R, et al. Multiple sclerosis and chronic autoimmune encephalomyelitis - A comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. American Journal of Pathology 2000;157:267-276.

3. Wang Y, Sun P, Wang Q, et al. Differentiation and quantification of inflammation, demyelination and axon injury or loss in multiple sclerosis. Brain : a journal of neurology 2015;138:1223-1238.

4. Wang Y, Wang Q, Haldar JP, et al. Quantification of increased cellularity during inflammatory demyelination. Brain : a journal of neurology 2011;134:3587-3598.

5. Kim JH, Loy DN, Wang Q, et al. Diffusion Tensor Imaging at 3 Hours after Traumatic Spinal Cord Injury Predicts Long-Term Locomotor Recovery. Journal of neurotrauma 2010;27:587-598.

6. Tu TW, Budde MD, Xie M, et al. Phase-aligned multiple spin-echo averaging: a simple way to improve signal-to-noise ratio of in vivo mouse spinal cord diffusion tensor image. Magnetic resonance imaging 2014;32:1335-1343.

7. Chiang CW, Wang Y, Sun P, et al. Quantifying white matter tract diffusion parameters in the presence of increased extra-fiber cellularity and vasogenic edema. NeuroImage 2014;101:310-319.

Figures

Figure 1 Representative DTI and DBSI metric maps were overlaid on gray-scale diffusion-weighted images from one control and one SCI mouse at T9 vertebra. DTI metrics reflect the “averaged” effects of co-existing axon/myelin pathologies and inflammation that could exaggerate or underestimate the severity of SCI.

Figure 2 Box plots suggest significant axonal damage in SCI group. Significant exaggeration of decreased DTI-FA (C) due to co-existing inflammation and axonal loss is apparent comparing with DBSI-FA (F). * indicates p < 0.05

Figure 3 Representative DBSI maps of restricted and non-restricted isotropic and anisotropic diffusion tensor fractions maps reveal the increased cellularity and edema/axonal loss in SCI mice.

Figure 4 The significant cell infiltration (A) and edema (B) were shown in SCI cords. Diffusion-weighted anatomic image (DWI)-based white matter volume suggested tissue “swelling” (C) with lower fiber fraction (D) in SCI group. DBSI-derived axon volume excluded inflammation contamination quantified axon loss in SCI (E). * p < 0.05

Figure 5 DBSI-detected co-existing axonal pathologies (including axon and myelin injury, inflammation, axon loss) was seen in the representative triple IHC staining images of total neurofilaments (SMI-312, green, axons), myelin basic protein (MBP, red, myelin sheaths), and 4’, 6-dianidino-2-phenylindole (DAPI, blue, nuclei) displayed.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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