Oxidative injury in the cerebral cortex and subplate neurons in periventricular leukomalacia

Abstract

We previously identified immunocytochemical evidence of nitrative and oxidative injury in premyelinating oligodendrocytes in periventricular leukomalacia (PVL). Here, we tested the hypothesis that free radical injury occurs in the overlying cerebral cortex and subplate neurons in PVL. We immunostained for nitrotyrosine, malondialdehyde, and hydroxynonenal adducts and scored neuron staining density in PVL (n = 11) and non-PVL (n = 15) cases (postconceptional ages from 34 to 109 weeks). Analysis of covariance controlled for age. Mean malondialdehyde scores in PVL cases were increased over controls (p = 0.005). Hydroxynonenal scores increased with age only in PVL cases (diagnosis vs age interaction; p = 0.024). Nitrotyrosine scores were not significantly increased. In 11 PVL and 23 control cases between 20 and 183 postconceptional weeks, cells morphologically consistent with subplate and Cajal-Retzius neurons showed qualitatively increased free radical modification in PVL over control cases with statistically significant odds ratios for hydroxynonenal and nitrotyrosine in both subplate neurons and Cajal-Retzius cells. Glial fibrillary acidic protein and CD68 scores for reactive astrocytes and microglia, respectively, were not significantly increased, suggesting a minimal inflammatory response. Thus, oxidative/nitrative damage to cortical and "pioneer" neurons, although mild overall, may contribute to cortical volume loss and cognitive/behavioral impairment in survivors of prematurity.

Figure 1

Immunostaining of cerebral cortex with markers of free radical injury and cellular response. (A) Malondialdehyde (MDA) adduct in cytoplasm of scattered cerebral cortical neurons, PVL case aged 95 postconceptional weeks. (B) HNE adduct in neuronal cytoplasm, PVL case aged 95 postconceptional weeks. (C) Nitrotyrosine (NT) in neuronal nucleus and cytoplasm, PVL case aged 40 postconceptional weeks. (D) Hydroxynonenal (HNE) immunostain showing no immunoreactivity in cortical neurons of a non-PVL case aged 54 postconceptional weeks. (E) HNE adduct in a segmental cortical infarct in a “positive control” case aged 44 postconceptional weeks. (F) HNE adduct in the white matter of a PVL case aged 51 weeks. (G) GFAP in astrocytes, PVL case aged 51 postconceptional weeks. (H) CD68 for macrophages, PVL case aged 35 postconceptional weeks. Original magnification: x200 for each. Note the variable immunostaining of the background neuropil, interpreted as free radical adducts involving plasma membranes of cell processes; as expected, this is strongest in the cortical infarct (E), but also notable in (A–C).

Figure 2

Mean age-adjusted marker scores (see text for score criteria) in cerebral cortex of PVL cases (red bars) and controls (black bars). Note the overall low scores for all markers (each less than 1.0) and that statistical significance is achieved only for MDA, although NT, CD68, and GFAP scores showed a trend toward significance (Table 1).

Figure 3

Relationship between age and HNE marker scores in PVL (XXX dots) versus controls (XXX dots) across 34 to 109 postconceptional weeks. With increasing age, HNE adduct formation increases significantly in the cerebral cortex in PVL but not in controls (see text for discussion).

Figure 4

Immunostaining of subplate neurons for HNE (A, 40 postconceptional weeks PVL case) and Cajal-Retzius cells for MDA (B, 40 postconceptional weeks PVL case). Original magnification: x400 for each.