Neuron deficit in the white matter and subplate in periventricular leukomalacia

Abstract

Objective: The cellular basis of cognitive abnormalities in preterm infants with periventricular leukomalacia (PVL) is uncertain. One important possibility is that damage to white matter and subplate neurons that are critical to the formation of the cerebral cortex occurs in conjunction with oligodendrocyte and axonal injury in PVL. We tested the hypothesis that the overall density of neurons in the white matter and subplate region is significantly lower in PVL cases compared to non-PVL controls.

Methods: We used a computer-based method for the determination of the density of microtubule-associated protein 2-immunolabeled neurons in the ventricular/subventricular region, periventricular white matter, central white matter, and subplate region in PVL cases and controls.

Results: There were 5 subtypes of subcortical neurons: granular, unipolar, bipolar, inverted pyramidal, and multipolar. The neuronal density of the granular neurons in each of the 4 regions was 54 to 80% lower (p≤0.01) in the PVL cases (n=15) compared to controls adjusted for age and postmortem interval (n=10). The overall densities of unipolar, bipolar, multipolar, and inverted pyramidal neurons did not differ significantly between the PVL cases and controls. No granular neurons expressed markers of neuronal and glial immaturity (Tuj1, doublecortin, or NG2).

Interpretation: These data suggest that quantitative deficits in susceptible granular neurons occur in the white matter distant from periventricular foci, including the subplate region, in PVL, and may contribute to abnormal cortical formation and cognitive dysfunction in preterm survivors.

Figure 1

Strategy for the quantitation of subcortical neuronal density in PVL cases and controls, as demonstrated in a control case at 33 postconceptional weeks. A. We used a computer-based method (Neurolucida) for cell counting consisting of a grid over the ventricular/subventricular zone (Box 1), periventricular white matter (Box 2), central white matter (Box 3), and subplate region (defined “ribbon” of tissue directly beneath cortex). B. The corresponding histological section stained with hematoxylin-and-eosin illustrates the regions sampled, including the subplate region which is designated by a dotted line directly beneath the cortex. Abbreviations: CC, corpus callosum; CG, cingulate gyrus; vent, ventricle.

Figure 2

Five subtypes of subcortical neurons based upon morphological criteria with MAP2 immunostaining at all ages studied, i.e., 23 to 43 postconceptional weeks. These subtypes are: granular (A), unipolar (B), bipolar (C), multipolar (D), and inverted pyramidal (E). All images are at x40. Scale bar = 25 microns

Figure 3

The mean percentages of each morphological subtype of the total subcortical neuronal population of all controls combined. The main subtype is the granular neuron.

Figure 4

Double-label immunocytochemistry with MAP2 to characterize the phenotype of the granular neurons. The neuronal migration marker DCX (green) is not expressed by the MAP2 neuron shown (red) and is rarely expressed by MAP2 neurons of any phenotype, including granular. We show the typical, non-granular, appearance of the DCX expressing cell with a small oval soma and a delicate thin trailing process.

Figure 5

A. Focal necrosis is found in the periventricular region in a representative PVL case at 29 postconceptional week. Microcysts reflective of resolving foci of necrosis are present in the periventricular white matter [x10; scale bar = 200 microns] (B) surrounded by reactive gliosis [x40; scale bar = 25 microns] (C). Reactive astrocytes are indicated with arrows.

Figure 6

The significantly lower granular density in PVL (white diamonds) exemplified in the central white matter (Box 3) in PVL cases (age- and postmortem interval-adjusted mean, 0.51±0.31 neurons/mm²) compared to controls (black diamonds) (mean, 3.02±0.37 neurons/mm²)(p<0.001).