The cerebral cortex overlying periventricular leukomalacia: analysis of pyramidal neurons

Abstract

The role of the cerebral cortex in the cognitive deficits in preterm survivors is poorly understood. Periventricular leukomalacia (PVL), the key feature of encephalopathy of prematurity, is characterized by periventricular necrotic foci and diffuse gliosis in the surrounding cerebral white matter. Here, we tested the hypothesis that reductions in the density of layer I neurons and/or pyramidal neurons in layers III and/or V are associated with PVL, indicating cortical pathology potentially associated with cognitive deficits in long-term survivors. In controls (23 gestational weeks to 18 postnatal months) (n = 15), a lack of significant differences in pyramidal density among incipient Brodmann areas suggested that cytoarchitectonic differences across functional areas are not fully mature in the fetal and infant periods. There was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases (n = 17) compared to controls (n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six-layer cortex is visually distinct (P < 0.024). This may reflect a dying-back loss of somata complicating transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the potential role of secondary cortical injury in the encephalopathy of prematurity.

Figure 1

The Brodmann area was determined by comparison of the region counted to standard available maps. A. The cortex is subdivided for analysis into regions based upon cytoarchitectonic patterns: koniocortex (sensory related) with stripes (primary sensory) and dots (secondary and tertiary sensory); agranular (motor and limbic related), light gray; and homotypical (association), dark gray. The specific Brodmann area is defined from standard maps (courtesy of the Harvard Brain Bank) (B) in the tissue section (C). Examination of the area 24 (cingulate gyrus) is exemplified in a representative control at 72 postconceptional weeks (2 postnatal months).

Figure 2

The Neurolucida software program (MicroBrightField) was used to quantify the density of layer I neurons and pyramidal neurons in layers III and V with MAP2 immunostaining in a two‐dimensional sampling column from the pial surface to the gray‐white border. A. We first selected the Brodmann area for study, as demonstrated in area 24 (cingulate gyrus). At low magnification (×40), we outlined the boundary of the entire section and then the boundary of the entire cortex in the section (Figure 1). Next, we defined three rectangular “boxes” of the cortex that were each 600 µm wide in the Brodmann area. B. Within this defined cortical region, the boundaries of the six cortical laminae were outlined, as illustrated in a representative control cases at 40 postconceptional weeks (term). Once the outlines were set in place, the selected neurons in layers I, III and VI were counted at high magnification (×200) in the MAP2‐immunostained section. The circles identify the neurons counted in each lamina of interest.

Figure 3

Selected neurons in the frontal association (homotypical) cortex are illustrated in representative controls at 43 postconceptional weeks (A), 53 weeks (B and D) and 23 weeks (C). These neurons are: A. MAP2‐immunostained horizontal neurons in layer I which are putative Cajal–Retzius cells; B. MAP2‐immunostained pyramidal neurons in layer III; C. MAP2‐immunostained pyramidal neurons in layer V at 23 weeks; and D. MAP2‐immunostained pyramidal neurons in layer V at 53 weeks. The pyramidal neurons in layer III (B) tend to be smaller than those in layer V (D) at the same age. The pyramidal neurons in layer V increase in size and process complexity from 23 weeks (C) to 53 weeks (D). Scale bar = 50 µm.

Figure 4

There is a developmental change in the appearance of MAP2‐immunopositive neurons in the frontal association cortex from 20 postconceptional weeks to 54 weeks in the control group. At 20 weeks, MAP2‐immunopositive neurons are not identified, but are present in the incipient layer V at 23 weeks. At 30 weeks, MAP2‐immunopositive neurons are prominent in layers II–V, and in layers II–VI thereafter. Scale bar = 1 mm.

Figure 5

There is a striking change in the laminar pattern between 23 (A) and 43 (C) postconceptional weeks, with differentiation of layers III–VI appearing around 30 weeks (B), as illustrated in representative controls in the frontal association cortex. Over this time course, layers III–VI are increasingly detected, first appearing around 30 weeks. At 23 weeks, layers I and II are discernible; the latter characterized by dense cellular packing. The change in the laminar pattern is caused by an increase in the neuropil and differentiation of neuronal subtypes. The dotted line represents the border between layer VI and underlying white matter. Scale bar = 1 mm.

Figure 6

There is a normative significant decrease in the density of pyramidal neurons in layer V with increasing postconceptional age for all layers cortical regions combined (control cases) (P = 0.048).

Figure 7

The cerebral cortex overlying periventricular leukomalacia (PVL) is relatively intact histologically, as illustrated in the PVL case of an infant dying at 39 postconceptional weeks. A. A low‐power histological section provides orientation, with the microcysts of focally necrotic lesions in the deep (periventricular) white matter (arrow) illustrated in high magnification (B) and with the overlying cerebral cortex (arrowhead) (C). Scale bar = 200 µm.

Figure 8

Between 22% and 39% of the periventricular leukomalacia cases demonstrated mild and/or non‐specific histopathological changes in the cerebral cortex that were not significantly different in incidence or features from controls. These changes included: (A) astrogliosis characterized by naked glial nuclei (arrows) (H & E); (B) acutely necrotic, hyper‐eosinophilic neurons with pyknotic nuclei (arrows) (H & E); (C) reactive astrocytes diffusely present throughout all layers (arrows) (glial fibrillary acid protein immunocytochemistry); and (D) diffusely activated microglia (arrows) (CD68 immunocytochemistry). Scale bar = 100 µm.