Magnetic resonance microimaging of intraaxonal water diffusion in live excised lamprey spinal cord

Abstract

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on a priori reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20-40 microm in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 +/- 0.06 (10(-3) mm2 sec) and 0.97 +/- 0.11 (10(-3) mm2 sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system.

Fig 1.

Scheme of perfusion system for micro diffusion-weighted imaging. Larval sea lampreys (P. marinus, 4–5 years old) were anesthetized in tricaine methanesulfonate (0.1%) for 3–5 min. The spinal cords in all animals were excised in Tris-buffered lamprey Ringer's solution. The cords were placed immediately into a capillary micropipet (0.9-mm i.d.) for MRI and perfused with cold buffer. All procedures were performed in an ice bath. The RF probe is a 400-MHz solenoidal transmit/receive RF coil, tuned and impedance-matched via a standard capacitor network that interfaced with the spectrometer's RF transmit/receive chain (coil size: 2.5-mm i.d., 6 mm long, seven turns of 0.5-mm o.d. copper wire).

Fig 2.

Histological appearance of the sea lamprey spinal cord. (a) The lamprey spinal cord in transverse section immunostained for neurofilaments. Axons stain darkly. White ellipsoids represent a selection of the dorsal, dorsolateral, and ventral columns, respectively. GM, gray matter; Ma, Mauthner axon; Mü, Müller axons. (b) For comparison, a T1-weighted micro-MR image of a fixed spinal cord at 9 × 9-μm resolution.

Fig 3.

Transverse diffusion MR images of the living spinal cord. The motion-sensitive gradients are applied perpendicular to the orientation of the axons. Images were obtained with the gradients off (a) and on (b). Increasing the b value from 728.2 to 1,551.2 sec/mm² causes rapid loss of signal intensity, particularly in the axon, enhancing the contrast between the Mauthner axon and the surrounding WM tissue. (Scale bar, 100 μm.)

Fig 4.

Anisotropy of water diffusion in axon tracts but not within axons. t-ADC (a) and l-ADC (b) for water within the Mauthner axon and in different regions of the WM are shown. The degree of anisotropy is shown in c. The data represent mean ± standard deviation, which is treated in the multiple-comparison test (Tukey–Kramer).

Fig 5.

Preservation of histological structure of the spinal cord after MR microimaging. (a) A diffusion-weighted MR microimage (19 × 19 × 250 μm³) with the motion-sensitive gradients on read direction (b = 1,551 sec/mm²). (b) A neurofilament-immunostained transverse histological section from the same piece of spinal cord after imaging. Note that the micro-DWI provides structural information almost as detailed as a histological section. Aside from slight deformity of the cord due to confinement in the micropipet, the histological appearance showed no postmortem changes.