Diffusion tensor imaging detects early cerebral cortex abnormalities in neuronal architecture induced by bilateral neonatal enucleation: an experimental model in the ferret

Abstract

Diffusion tensor imaging (DTI) is a technique that non-invasively provides quantitative measures of water translational diffusion, including fractional anisotropy (FA), that are sensitive to the shape and orientation of cellular elements, such as axons, dendrites and cell somas. For several neurodevelopmental disorders, histopathological investigations have identified abnormalities in the architecture of pyramidal neurons at early stages of cerebral cortex development. To assess the potential capability of DTI to detect neuromorphological abnormalities within the developing cerebral cortex, we compare changes in cortical FA with changes in neuronal architecture and connectivity induced by bilateral enucleation at postnatal day 7 (BEP7) in ferrets. We show here that the visual callosal pattern in BEP7 ferrets is more irregular and occupies a significantly greater cortical area compared to controls at adulthood. To determine whether development of the cerebral cortex is altered in BEP7 ferrets in a manner detectable by DTI, cortical FA was compared in control and BEP7 animals on postnatal day 31. Visual cortex, but not rostrally adjacent non-visual cortex, exhibits higher FA than control animals, consistent with BEP7 animals possessing axonal and dendritic arbors of reduced complexity than age-matched controls. Subsequent to DTI, Golgi-staining and analysis methods were used to identify regions, restricted to visual areas, in which the orientation distribution of neuronal processes is significantly more concentrated than in control ferrets. Together, these findings suggest that DTI can be of utility for detecting abnormalities associated with neurodevelopmental disorders at early stages of cerebral cortical development, and that the neonatally enucleated ferret is a useful animal model system for systematically assessing the potential of this new diagnostic strategy.

Keywords: Golgi; brain; diffusion tensor imaging; enucleation; ferret; interhemispheric callosal connections; magnetic resonance imaging; visual system.

Figure 1

Effect of bilateral enucleation on postnatal day 7 (BEP7) on the distribution of interhemispheric visual callosal connections in the ferret. The distribution of callosal connections in one hemisphere of adult BEP7 and control ferrets were studied following multiple intracortical injections of the tracer HRP in the contralateral hemisphere. Green areas in (A) and (B) include regions of visual cortex analyzed. Approximate locations of visual areas described in previous reports are indicated in (B); the blue line marks the representation of the horizontal meridian. Red dots indicate the crown of the suprasylvian and ectosylvian and gyri, and the dorsal/caudal edge of the occipital lobe, which were marked directly on the brains before flattening. (C) Flattened brain before sectioning, area outlined by green line contains visual areas analyzed. Labeled callosal connections (labeled somas and axon terminations) appear as dark areas in (D) and (F); and as colored areas in the thresholded versions (E, G). The percent area occupied by callosal connections was significantly (p < 0.05) greater in BEP7 ferrets than in Control ferrets (H). LG, lateral gyrus; PPc, posterior parietal caudal area; PPr, posterior parietal rostral area; SSG, suprasylvian gyrus; SSV, suprasylvian visual areas; as, ansate sulcus; ls, lateral sulcus, sss; suprasylvian sulcus. Scale = 5 mm.

Figure 2

Anisotropy in water diffusion within the developing cerebral cortex is oriented parallel to apical dendrites of pyramidal neurons. Corresponding coronal views of DTI data (A) and Golgi-stained tissue (B) obtained from brain BEP7-1. The rostrocaudal level of the coronal plane is represented by the dashed line in the inset. (C,D) Close-up views of the region of the suprasylvian gyrus indicated by the rectangles in A and B, respectively. Diffusion tensor primary eigenvector (C) and apical dendrites (D) are both oriented perpendicular to the pial surface. In (D), yellow arrows indicate two apical dendrites, and red arrows indicate the associated cell bodies. The approximate size of the field in (D) is shown as a rectangle in (C). The size of an individual DTI voxel is illustrated as a square in (D). Abbreviations are as in Figure 1. Scale bars = 0.5 mm in panels (C) and (D).

Figure 3

Post mortem DTI measurements of two BEP7 hemispheres show increased visual cortical FA relative to two control hemispheres at P31. Coronal views of FA parameter maps are shown in (A) for two control animals (left) and two BEP7 animals (right) according to the gray color scale. In (B), cortical FA is presented on cortical surface models for animals Cntrl-1 and BEP7-1, according to the red/yellow color scale. In A and B, increased FA is evident in visual areas of BEP7 animals compared to controls. Blue dots in (B) indicate the approximate location of the representation of the horizontal meridian (see Figure 1 for comparison). Red dots indicate crown of gyri, as in Figure 1. Histograms reflecting data from all four animals are shown in (C) for visual cortex [encircled by black dots in (B)] and a rostrally located control area in (D) [encircled by blue-green dots in (B)].

Figure 4

Characterization of the distributions of neuronal process orientations. Golgi-stained tissue visualized at 10× magnification from the Cntrl-1 hemisphere is shown in (A). Line segments (red) representing neuronal processes throughout a region of the cerebral cortex (corresponding to location 3 in Figure 5) are overlaid on the skeletonized image and original image in (B) and (C), respectively. Line segments derived from the corresponding location in BEP7-1 are overlaid on the Golgi image in (D). The polar angle for each line segment was determined as described in the text, and in (E), histograms representing the distribution of polar angles are shown for the Cntrl-1 (black data points) and BEP7-1 (red data points) Golgi fields. Solid lines in (E) represent the results of approximating the data points as a von Mises distribution. To estimate 95% confidence intervals κ, a bootstrap procedure was used in which κ was determined 1000 times from randomly sampled subsets of the measured polar angles for each field. Histograms, with associated 95% confidence regions, of the resulting κ values are shown in (F) for the set of angles derived from Cntrl-1 (black) and BEP7-1 (red).

Figure 5

Comparison of the distributions of orientations of neuronal processes in visual and non-visual areas of BEP7 and control ferrets at P31. The value of the parameter κ decreases as the distribution of orientations broadens (less concentrated). In (A), filled circles represent locations of regions analyzed in coronal sections of Cntrl-1 and BEP7-1, with red indicating visual cortical areas, and orange a non-visual area. Open circles represent locations of regions analyzed in axial sections of Cntrl-2 and BEP7-2, with red and orange indicating visual and non-visual areas, respectively. (B) Cortical locations 1–4 and non-visual area 1 are shown in montages of Golgi sections (C) Concentration parameters, κ, of von Mises distributions associated with the sets of neuronal process orientations are shown for case BEP7-1 (visual locations, red filled bars; non-visual location, filled orange bar) and case Cntrl-1 (visual locations, black filled bars; non-visual location, filled gray bar), and case BEP7-2 (visual location, red open bar; non-visual location, open orange bar) and case Cntrl-2 (visual location, black open bar; non-visual location, open gray bar). Error bars represent 95% confidence intervals for κ. For visual locations indicated with asterisks, 95% confidence intervals for BEP7 and control regions do not overlap.