Developmental changes in diffusion anisotropy coincide with immature oligodendrocyte progression and maturation of compound action potential

Abstract

Disruption of oligodendrocyte lineage progression is implicated in the white-matter injury that occurs in cerebral palsy. We have previously published a model in rabbits consistent with cerebral palsy. Little is known of normal white-matter development in perinatal rabbits. Using a multidimensional approach, we defined the relationship of oligodendrocyte lineage progression and functional maturation of axons to structural development of selected cerebral white-matter tracts as determined by diffusion tensor imaging (DTI). Immunohistochemical studies showed that late oligodendrocyte progenitors appear at gestational age 22 [embryonic day 22 (E22)], whereas immature oligodendrocytes appear at E25, and both increase rapidly with time (approximately 13 cells/mm2/d) until the onset of myelination. Myelination began at postnatal day 5 (P5) (E36) in the internal capsule (IC) and at P11 in the medial corpus callosum (CC), as determined by localization of sodium channels and myelin basic protein. DTI of the CC and IC showed that fractional anisotropy (FA) increased rapidly between E25 and P1 (E32) (11% per day) and plateaued (<5% per day) after the onset of myelination. Postnatal maturation of the compound action potential (CAP) showed a developmental pattern similar to FA, with a rapid rise between E29 and P5 (in the CC, 18% per day) and a slower rise from P5 to P11 (in the CC, <5% per day). The development of immature oligodendrocytes after E29 coincides with changes in FA and CAP area in both the CC and IC. These findings suggest that developmental expansion of immature oligodendrocytes during the premyelination period may be important in defining structural and functional maturation of the white matter.

Figure 1.

A, The CAP was measured in the corpus callosum using stimulating (left) and recording (right) electrodes. B, The onset time of the first positive peak and area under the curves was measured at various distances between electrodes (250-900 μm). C, The onset of the CAP (ordinate) was plotted to the distance between electrodes (abscissa). Linear regression was significant (p < 0.05) with R² = 0.98. The CV of the CAP was determined as the slope of the line depicted. D, The amplitude and area of the CAP increased proportionally to the stimulation intensity and saturated at certain threshold levels for each age. A step current stimulation was used, and measurements of CV and CAP area were finally done at a suprathreshhold level (arrow).

Figure 2.

Cell counts were obtained from 50-μm-thick adjacent sections that were alternately stained with the O4 or O1 antibody A, The population pool of O4+ cells in the corpus callosum (open circles and dashed lines) and internal capsule (squares and solid lines) generally increases with age (ANOVA; p < 0.05). B, The O1+ cells exhibit a steady increase with age (ANOVA; p < 0.05). Note the later onset of immature oligodendrocytes in the corpus callosum.

Figure 3.

Frozen 5-6 μm coronal sections of brains were stained with a pan-isoform-specific polyclonal antibody against voltage-gated NaCh (first and third rows). MBP staining was done on 20 μm paraformaldehyde-fixed sections (second and fourth rows). Rectangles on the diagrams and the T2 images at the panel corners outline the position of the panel on the slice. The internal capsule is shown in A, C, E, and G; the corpus callosum is shown in B, D, F, and H. The images are from P5 pups (A-D) and P11 pups (E-H). There were no visible NaCh clusters at P1 (data not shown). In B and F, there are areas of higher-intensity staining that represent diffuse staining from NaCh (on higher power). Note that the increased diffuse intensity is not representative of clustering of NaCh. NaCh clusters appear as bright puncta first in the internal capsule (A, arrow) and dorsolateral corpus callosum at P5, along with MBP staining (C, D). There was a marked increase in the number of clusters in the internal capsule and dorsolateral corpus callosum at P11. Even at P11, there are very few NaCh clusters (arrowheads) in the center of the corpus callosum (F). Each panel has a drawing in the corner, indicating the location on the slice where the images were taken.

Figure 4.

A, Frozen brain slices (6 μm) were incubated with a pan-isoform-specific monoclonal antibody against voltage-gated sodium channels (NaCh) and were then incubated with an HRP-labeled secondary antibody. NaCh clusters were counted per 100 μm on six FOVs per slice. There was a fivefold to sixfold increase in NaCh clusters (ordinate) in the internal capsule (IC) compared with the corpus callosum (CC) from P5 to P11, reflecting the contribution of the process of myelination. The center of the CC remains unmyelinated at P11. Note that the dorsolateral region of the CC behaves like the IC with earlier myelination than that in the center of the CC. Measurements of NaCh clusters were taken on coronal slices at the level of the hippocampus (1 mm posterior to bregma). Data are presented as mean ± SEM (n = 3 per age per tract). B, The methodology of obtaining the CAP area is given in Figure 1. Measurements from the CC were taken on coronal slices at the level of the hippocampus and from the IC on horizontal slices at the level of the anterior commissure and 1 mm posterior to bregma. Stimulation intensity was fixed to a maximal threshold for P1, and distance between electrodes for the CAP area was between 700 ± 10 μm. The CAP area (ordinate) increased significantly with age (abscissa) in both the CC and IC (ANOVA; p < 0.01). C, Calculation of the CV is given in Figure 1. CV (ordinate) increases differently with age (abscissa) in the CC compared with the IC. In the CC, there is an increase from E29 to P5 and then plateau is reached by P11. In contrast, there is a continued increase in CV at P11, probably reflecting the effect of myelinated fibers. There is an increased variability of CV in the IC at P11, reflecting the contribution of both myelinated and unmyelinated fibers. Myelination and the corresponding increase in CV occurs later in the CC. Adult values are 10 times that at birth in both tracts. Data are presented as mean ± SEM (n = 3-5 per age per tract).

Figure 5.

A, A hysterotomy was performed at E22, and fetal brains were extirpated. Brains were immersion fixed with 4% paraformaldehyde and underwent ex vivo DTI on a 4.7 T magnet: TR/TE, 2500/38 ms; b = 0 and 1815 s/mm²; slice thickness/in-plane resolution, 0.5/0.165 mm; 12 averages. Color intensity on the directionally encoded FA map is proportional to the FA value. The predominant diffusion direction is depicted by colored arrows at the bottom left. Most of the white-matter structures are formed by E22, except for the corpus callosum (white arrow). B, A similar procedure was done at E25, and the directionally encoded FA map shows the appearance of the corpus callosum (white arrow). C, P11 pup measured in vivo on a 4.7 T magnet: TR/TE, 2000/35 ms; b = 0 and 780 s/mm²; slice thickness/in-plane resolution, 1.5/0.195 mm; eight averages. The directionally encoded FA map shows that the volume and anisotropy of white-matter tracts have significantly increased compared with earlier gestation (A, B). D, Various white-matter tracts and gray-matter structures were outlined, as shown on the P5 pup map. Automatic segmentation was used to define the corpus callosum (CC) and internal capsule (IC) (see Materials and Methods), and manual segmentation was used for the rest of the structures. Scale bars, 2 mm. Ac, Anterior commissure; Ec, external capsule; Fi, fimbria hippocampi; Cr, corona radiata; Cx, cerebral cortex; Pu, putamen; Hc, hippocampus.

Figure 6.

A, DTI, consisting of a set of diffusion-weighted images in six noncollinear directions, was obtained from live rabbit pups in a 4.7 T magnet. The ADC of tissue water was calculated as a trace of the diffusion matrix. Postnatally, the ADC decreased with age in the corpus callosum (CC) and significantly in the internal capsule (IC) (ANOVA; p < 0.05). There is a different rate of change in the IC compared with the CC. Measurements in the CC and IC were taken on coronal slices at the level of the hippocampus. B, A T2 parametric map was obtained by monoexponential fitting of signal intensity from a multi-echo T2-weighted sequence. T2 times were decreased in the CC and IC. Note the difference between the CC and IC. C, DTIs were performed as described above on live pups ranging in age from P1 to P11 and on fixed fetal brains from E22 to E29. The diffusion matrix was diagonalized to obtain principal eigenvectors. The corresponding eigenvalues were used to calculate an index reflecting directional anisotropy water diffusion in tissue (FA). FA in the CC and IC increased significantly with age (ANOVA; p < 0.05). The greatest increase was between E25 and P5 (E36) for the CC and between E29 and P1 for the IC. Subsequently, the rate of increase in FA reaches a plateau. Data are presented as mean ± SEM (n = 5-9 per age per tract).

Figure 7.

A, We measured the directional change in water diffusivity in white-matter tracts. Axial diffusivity (λ_∥, ordinate), along the course of white-matter tracts, increases with age (abscissa) in the corpus callosum and decreases in the internal capsule. B, Radial diffusivity (λ_⊥, ordinate), across the course of white-matter tracts, decreases with age (abscissa) in both the corpus callosum and internal capsule, resulting in an increase in FA (Fig. 6 D). Data are presented as mean ± SEM (n = 5-9 per age per tract).

Figure 8.

A, B, Coincidental increase in cells, electrophysiology, and MRI parameters in the corpus callosum (A) and internal capsule (B). Because E29 was the earliest time point measured for all three parameters, curves are superimposed using E29 as a common starting point. The y-axis was adjusted so that the maximum mean value was always 85% of the maximum ordinate value. The increase in immature oligodendrocytes (OL; gray squares and gray dashed line) coincided with the increase in the CAP (open circles and dotted line) and FA (filled circles and solid lines) in the corpus callosum (A) and, to a lesser degree, in the internal capsule (B). Note that O1+ cells cannot be detected at P5 in the internal capsule and at P11 in the corpus callosum as immature oligodendrocytes mature into a myelinating phenotype. Comparison of three different slopes (with three different units) were done by normalizing the curve to the range of values, adjusting for the decreased rate of change by converting the days to the logarithmic value, performing a linear regression of the normalized percentage of change from E29 with log days and comparing the slopes by confidence intervals. Slopes of FA (803, 605-1001) and CAP (797, -92 to 1685) were not different from that of O1+ cells (957, 732-1182 normalized percentage of change per log day; mean, 5th to 95th percentiles) between E29 and P5 for the corpus callosum. The abscissa is the same for A and B (postconceptional age).