Neuroimaging researchers interested in microstructural changes of neurological disease models that affect the mouse spinal cord.

Axons and dendrites, which are collectively known as neurites, are cellular building units of the central nervous system (CNS)¹. Axonal damage due to injury, autoimmune or neurodegenerative diseases can affect sensory and/or motor functions which result in a dramatic impact on patient quality of life and health outcomes ². Clinically, white matter (WM) pathology has been investigated by MR diffusion tensor imaging (DTI) to assess microstructural changes. However, DTI indices alone are not adequate to describe changes in the complex architecture of CNS tissue. Novel high-order diffusion MRI techniques, such as Neurite Orientation Dispersion and Density Imaging (NODDI) and AxCaliber have been developed to provide distinct information to complement DTI. NODDI provides neurite dispersion, neurite density and CSF measurements that are important to characterise changes in GM^1,3. AxCaliber provides information about the axon diameter distribution of WM tracts⁴. Until recently, such information was only accessible through histological examination using invasive tissue biopsy.

This project aims to apply NODDI and AxCaliber techniques to the in-vivo and ex-vivo imaging of mouse lumbar spinal cord at ultrahigh magnetic fields. This study also assesses the feasibility and the sensitivity of these techniques as a first step in the study of disease models that affect the spinal cord.

MRI acquisition: Four control adult male mice (7 weeks old) were imaged in-vivo using a 30cm 9.4T MRI scanner (Bruker Biospin, Ettlingen, Germany) equipped with a rat head phase array receive/quadrature 86 mm coil and BGA-S12 gradient (440mT/m). Ex-vivo imaging was performed using a Bruker 16.4T vertical wide-bore microimaging system equipped with a Micro2.5 gradient (1.5T/m) and 20 mm volume coil (M2M Imaging, Brisbane, Australia). In-vivo NODDI experiments were acquired using the Stejskal-Tanner diffusion Echo Planar Imaging (DWI-EPI), and DWI-Spin Echo images were acquired ex-vivo. AxCaliber acquisition used stimulated-echo diffusion EPI (ST-DWI-EPI). Three b-values of 750, 1500 and 3000 s/mm² were used for NODDI, whereas AxCaliber employed 10-12 b-values with six incremented diffusion times from 15-100ms. Details of acquisition parameters are shown in Table 1. The same slice positions were used for in-vivo and ex-vivo NODDI and AxCaliber experiments starting at vertebral level T11 and ending at L1 which is equal to the spinal level L1-L6 ⁴.

Image processing: NODDI was processed using the NODDI Matlab toolbox developed by UCL ³. DTI parameters were calculated using the b=1500 s/mm² dataset. NODDI/DTI data was analysed using ROIs manually drawn based on Sengul and Watson ⁵ gross anatomy of the mouse spinal cord (Figure 3). AxCaliber data was processed using a custom program written in Matlab as described by Assaf et al ⁶. ROIs were drawn manually using regions similar to those described by Assaf et al ⁷.

NODDI: The relative amount of orientation diffusion dispersion (ODI), axonal density (represented by intracellular volume fraction (ICVF)) and the estimated isotropic diffusivity (ISO) within the extra axonal space are shown in Figure 1. ODI showed high WM/GM contrast, in which GM ODI values are three times the value of WM across all subjects (Figure 2), indicating a high degree of neurite dispersion in the GM compared to the WM.

AxCaliber: Our protocol determined major peaks of axon diameter populations of 0.25, 1.5 and 2.5 microns (in-vivo) and 0.5, 1.25 and 1.75 microns (ex-vivo) for various ROIs of the mouse lumbar spinal cord. The axon diameter average in these ROIs were 1.55±0.15mm (in-vivo) and 1.37±0.20 mm (ex-vivo) (Figure 4 bottom row). This difference may be resulted from physiological motion in-vivo or fixation effects for ex-vivo samples. Overall, our results appear similar to those reported for an ex­-vivo q-space imaging (QSI) study performed to measure the mouse spinal cord axon diameter (Figure 4 top row) ⁸. At the cervical level C6 – C7, QSI measurements showed high correlation with axon diameters obtained from histology in the range 0.81-1.82 microns (ex-vivo).

We have successfully applied AxCaliber and NODDI protocols to measure axons diameter distribution and tissue microstructure in-vivo and ex-vivo in the mouse lumbar spinal cord. These measurements will be compared to histology to further validate these­­­­­­ findings.