Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization

Abstract

Children who survive preterm birth exhibit persistent unexplained disturbances in cerebral cortical growth with associated cognitive and learning disabilities. The mechanisms underlying these deficits remain elusive. We used ex vivo diffusion magnetic resonance imaging to demonstrate in a preterm large-animal model that cerebral ischemia impairs cortical growth and the normal maturational decline in cortical fractional anisotropy (FA). Analysis of pyramidal neurons revealed that cortical deficits were associated with impaired expansion of the dendritic arbor and reduced synaptic density. Together, these findings suggest a link between abnormal cortical FA and disturbances of neuronal morphological development. To experimentally investigate this possibility, we measured the orientation distribution of dendritic branches and observed that it corresponds with the theoretically predicted pattern of increased anisotropy within cases that exhibited elevated cortical FA after ischemia. We conclude that cortical growth impairments are associated with diffuse disturbances in the dendritic arbor and synapse formation of cortical neurons, which may underlie the cognitive and learning disabilities in survivors of preterm birth. Further, measurement of cortical FA may be useful for noninvasively detecting neurological disorders affecting cortical development.

Fig. 1

High-field MRI studies demonstrate progressive disturbances in cortical growth and dysmaturation of cortical microstructure. Coronal MRI images of one hemisphere at the level of the frontal periventricular white matter are presented at 4 weeks. (A to C) Representative cortical segmentation (A) (light gray shading) used to analyze cortical volume, (B) FA, and (C) ADC. Orientation marker indicates superior (S), inferior (I), medial (M), and lateral (L). (D to F) Changes in (D) cortical volume, (E) FA, and (F) ADC at 1, 2, and 4 weeks of recovery in control (Con; black bars) and ischemia (HI; white bars) groups: 1 week (control, n = 9; ischemia, n = 7); 2 weeks (control, n = 5; ischemia, n = 6); 4 weeks (control, n = 6; ischemia, n = 5). Data are shown as 25th, 50th, and 75th percentiles, and minimum and maximum data points. *P < 0.05, **P < 0.01 versus age-matched controls.

Fig. 2

Cerebral ischemia did not cause a loss of cortical neurons. (A to C) Representative NeuN-stained brain tissue showing (A) delineation of cortical boundaries (dotted line), (B) laminar distribution of neurons, and (C) nuclear labeling (white arrows). Scale bar, 5 μm. (D and E) Unbiased stereological estimates of neuron number (D) and neuron density in the cerebral cortex (E) of control (Con; black bars) and ischemia (HI; white bars) groups: 1 week (control, n = 6; ischemia, n = 6); 2 weeks (control, n = 5; ischemia, n = 6). Data are means ± SEM.

Fig. 3

Pyramidal neuron complexity increases with gestation in the fetal cerebral cortex. (A and B) Golgistained pyramidal neurons in the cerebral cortex at (A) 0.65 gestation (0 weeks) and (B) 4 weeks later. Scale bars, 20 μm. (C to E) Total number of basal dendritic branches (C), total basal dendritic length (D), and total number of nodes in control animals (E) at 0 weeks (black bars) and 4 weeks (white bars). (F) Sholl analysis of the number of basal dendritic intersections at 0 weeks (closed circles) and 4 weeks (open circles). (G to I) Branch order analysis of (G) total number of basal dendritic branches, (H) total basal dendritic length, and (I) total number of nodes at 0 weeks (black bars) and 4 weeks (white bars). Analyses were performed independent of cortical location: 0 weeks, n = 48 cells; 4 weeks, n = 100 cells. Data are means ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001.

Fig. 4

Abnormal development of basal dendritic arborization of cortical pyramidal neurons was seen in the cerebral cortex at 4 weeks after ischemia. (A) Example of Golgi-stained pyramidal neurons in the control cortex. Scale bar, 20 μm. (B) Example of computer-assisted reconstructions of representative neurons in the control (Con) and ischemia (HI) groups. (C to E) Total number of basal dendritic branches (C), total basal dendritic length (D), and total number of nodes (E) in control (black bars) and ischemia (white bars) groups. (F) Sholl analysis of the number of basal dendritic intersections in control (closed circles) and ischemia (open circles) groups. (G to I) Branch order analysis of total number of basal dendritic branches (G), total basal dendritic length (H), and total number of nodes (I) in control (black bars) and ischemia (white bars) groups. Analyses were performed independent of cortical location. n = 100 cells per group. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

Abnormal spine density on cortical pyramidal neurons was noted at 4 weeks after ischemia. For the same population of pyramidal neurons that were sampled for dendritic morphology, spine density was quantified on second-order terminal dendritic branches, which were commonly identified for both groups of neurons. (A to C) Examples of second-order terminal branches (arrows) on (A) Neurolucida tracing (white lines) and (B and C) the corresponding Golgi-impregnated neuron. In (A), the yellow, white, pink, green, and blue lines represent first-, second-, third-, fourth-, and fifth-order branches, respectively. (D) Dendritic spines (arrowheads) visualized on a Golgi-impregnated neuron. Scale bars, 10 μm (B), 5 μm (C), and 2 μm (D). (E) Spine density in control (Con; black bars) and ischemia (HI; white bars) groups at 4 weeks of recovery. Analyses were performed independent of cortical location. Control, n = 72 cells; ischemia, n = 73 cells. Data are means ± SEM. *P < 0.05.

Fig. 6

Anisotropy of the basal dendrites of cortical pyramidal neurons in the cerebral cortex was increased at 4 weeks after ischemia. (A to D) Neurolucida (A and A′) and Matlab 3D reconstructions (B and B′) of a Golgi-impregnated cortical neuron were used to generate a set of orthogonal distance regression line segments (C and C′) of the basal dendrites, from which (D) the anisotropy of the dendritic processes (FAn) was calculated. Images in A′, B′, and C′ are rotated 90° relative to the corresponding images in A, B, and C. (D) FAn in control (Con; black bars) and ischemia (HI; white bars) groups at 4 weeks of recovery. n = 100 cells per group. Data are means ± SEM. *P < 0.01.