Developmental changes and injury induced disruption of the radial organization of the cortex in the immature rat brain revealed by in vivo diffusion tensor MRI

Abstract

During brain development, morphological changes modify the cortex from its immature radial organization to its mature laminar appearance. Applying in vivo diffusion tensor imaging (DTI), the microstructural organization of the cortex in the immature rat was analyzed and correlated to neurohistopathology. Significant differences in apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were detected between the external (I-III) and deep (IV-VI) cortical layers in postnatal day 3 (P3) and P6 pups. With cortical maturation, ADC was reduced in both cortical regions, whereas a decrease in FA was only seen in the deep layers. A distinct radial organization of the external cortical layers with the eigenvectors perpendicular to the pial surface was observed at both ages. Histology revealed maturational differences in the cortical architecture with increased neurodendritic density and reduction in the radial glia scaffolding. Early DTI after hypoxia-ischemia at P3 shows reduced ADC and FA in the ipsilateral cortex that persisted at P6. Cortical DTI eigenvector maps reveal microstructural disruption of the radial organization corresponding to regions of neuronal death, radial glial disruption, and astrocytosis. Thus, the combined use of in vivo DTI and histopathology can assist in delineating normal developmental changes and postinjury modifications in the immature rodent brain.

Figure 1

Cortical regions of interest for ADC and FA measures. ADC and FA were measured in the external (light gray circle) and internal layers (dark gray circle) of the parietal cortex at the level of the striatum (A), the dorsal hippocampus (B), and the ventral hippocampus (C).

Figure 2

Cortical ADC and FA during development: (A) Boxplot of ADC values measured in the external and deep cortex at P3 and P6. (B) Boxplot of FA values measured in the external and deep cortex at P3 and P6. * = P < 0.05.

Figure 3

Eigenvectors overlaid on coronal DTI from a P3 and P6 rat. The orientation and the density of the fibers are shown by the indicated vectors, and the degree of anisotropy is symbolized by the vector length. Fibers that are perpendicular to the image plane are distinguished by the clear dots. At both ages, the radial organization of the cortex is depicted by the eigenvectors being perpendicular to the pial surface. The insets show a magnification of the parietal cortex. At P3 (left panel), the radial organization is clearly present, whereas at P6, (right panel) the radial orientation of the eigenvectors is less prominent, especially in the deep layers. The white and gray arrows correspond, respectively, to the external layers and to the deeper layers of the cortex where ADC and FA measures were made (see Fig. 1).

Figure 4

Immunohistochemical analysis of the cortex at P3 and P6: (A, B) Parietal cortex at P3 (A) and P6 (B) in the regions of interest of ADC and FA measurements stained with MAP2 to identify neurons and their processes. A higher cellular density is seen in the external layers (yellow arrows) compared with the deeper layers (red arrow) at both ages (scale bar = 500 μm). Radial organization of neuronal processes could be seen in the external layers at P3, with a reduction of this pattern at P6. (C) Boxplot showing the OD of MAP2 immunostaining in the deep and external cortical layers at both ages analyzed (* = P < 0.05). (D, E) Nestin immunostaining of radial glial cells in the parietal cortex at P3 (D) and P6 (E). The photomicrographs represent the full thickness of the cortex. At P3, a dense and regular pattern of radially oriented glial cells is present. At P6, the organization of the radial glia appears less dense and regular (scale bar = 100 μm). (F) Boxplot of nestin-positive cells in the deep and external layers of the cortex at P3 and P6 (* = P < 0.05).

Figure 5

Combined ADC and FA of the external and deep cortical layers after HI injury: (A) Boxplot of cortical ADC values measured 24 and 72 h after HI. (B) Boxplot of cortical FA values measured 24 and 72 h after HI. * = P < 0.05.

Figure 6

DTI and histological changes in the same animal 24 h after HI injury: (A) A disrupted pattern of eigenvectors 24 h after HI injury in the parietal cortex. (B) Degenerating neurons stained with FJB appear bright and correspond to the area of disorganized eigenvectors. Note that the area where the eigenvectors remain with a regular and radial pattern corresponds to a region with no neuronal degeneration detected with FJB.

Figure 7

Immunostaining of nestin 24 h after HI injury (A, B): (A) Representative nestin immunostaining in the left, uninjured parietal cortex and in the right, injured cortex (B). Similar changes in radial glia were present at 72 h. Confocal imaging of nestin and GFAP 24 h after HI injury (C–E): (C) Disrupted nestin-positive radial glia; (D) Increased GFAP immunostaining in the area of injury with hypertrophic foamy astrocytes; (E) Colocalization of nestin and GFAP (scale bars: 50 μm). Quantification of radial glia morphometric changes (F, G): (F) Boxplots of the number of radially orientated nestin-positive radial glial fibers in the left and right hemispheres at 24 and 72 h following HI injury. (G) Boxplots of the total cumulative length of radially orientated nestin-positive glial fibers in the left and right hemispheres at 24 and 72 h following HI injury. (* = P < 0.05).