Nitrosative stress and inducible nitric oxide synthase expression in periventricular leukomalacia

Abstract

Periventricular leukomalacia (PVL) is a lesion of the immature cerebral white matter in the perinatal period and associated predominantly with prematurity and cerebral ischemia/reperfusion as well as inflammation due to maternofetal infection. It consists of focal necrosis in the periventricular region and diffuse gliosis with microglial activation and premyelinating oligodendrocyte (pre-OL) injury in the surrounding white matter. We previously showed nitrotyrosine in pre-OLs in PVL, suggesting involvement of nitrosative stress in this disorder. Here we hypothesize that inducible nitric oxide synthase (iNOS) expression is increased in PVL relative to controls. Using immunocytochemistry in human archival tissue, the density of iNOS-expressing cells was determined in the cerebral white matter of 15 PVL cases [29-51 postconceptional (PC) weeks] and 16 control cases (20-144 PC weeks). Using a standardization score of 0-3, the density of iNOS-positive cells was significantly increased in the diffuse component of PVL (score of 1.8 +/- 0.3) cases compared to controls (score of 0.7 +/- 0.3) (P = 0.01). Intense iNOS expression occurred in reactive astrocytes in acute through chronic stages and in activated microglia primarily in the acute stage, suggesting an early role for microglial iNOS in PVL's pathogenesis. This study supports an important role for iNOS-induced nitrosative stress in the reactive/inflammatory component of PVL.

Fig. 1

Localization of iNOS expression in PVL. The expression of iNOS is seen in macrophages of the focal necrosis of a subacute PVL lesion in a 39-PC week infant (a, b). In this same case, iNOS expression is seen in the diffusely gliotic white matter surrounding the focal necrosis in cells morphologically identified as reactive astrocytes (c). The number of iNOS-expressing cells is significantly reduced in a control case at 37 PC weeks (d). Binding of the iNOS antibody was blocked in the PVL case from above using an excess of the peptide from which the antibody was produced (e)

Fig. 2

The expression of iNOS in PVL and controls. The standardization score of iNOS-expressing cells adjusted for PC age is significantly increased in PVL relative to controls (P = 0.01). The score of iNOS-expressing cells adjusted for PN age as well as GA is also significantly increased in PVL relative to controls (data not shown)

Fig. 3

Cell-specific expression of iNOS in the diffuse component of PVL. The expression of iNOS is seen in CD68-positive microglia (a) as well as in GFAP-positive reactive astrocytes (b) in the diffuse component of an acute PVL case at 40 PC weeks. A PVL case of the subacute (organizing) phase at 39 PC weeks shows no iNOS expression in CD68-positive microglia (c), but continues to show strong iNOS expression in GFAP-positive reactive astrocytes (d)

Fig. 4

Astrocytic expression of iNOS in PVL and controls. The percentage of GFAP-positive reactive astrocytes that express iNOS was determined in the diffuse component of PVL and in the corresponding periventricular white matter of control cases. In the overlapping ages of 35–54 PC weeks, there is no significant difference in the percentage of GFAP-positive astrocytes that express iNOS, although the overall density of iNOS positive glia (Fig. 2) as well as GFAP positive astrocytes [16] in control tissue is significantly less than in PVL