12/15-lipoxygenase expression is increased in oligodendrocytes and microglia of periventricular leukomalacia

Abstract

Oxidative stress involving premyelinating oligodendrocytes (OLs) is a major factor in the pathogenesis of preterm white matter injury. In animal and cell culture studies, activation of the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15-LOX) plays a central role as an inflammatory mediator in the pathology of oxidative stress and OL cell death, as well as ischemia and neuronal death. The role of 12/15-LOX, however, is unclear in the developing human brain. The mechanism of 12/15-LOX involves the production of reactive oxygen species through the metabolism of arachidonic acid, as well as direct detrimental effects on organelle membranes. Here we tested the hypothesis that the density of 12/15-LOX-expressing cells is increased in periventricular leukomalacia (PVL). Using immunocytochemistry (ICC) in human paraffin-embedded tissue, 12/15-LOX expression was seen in macrophages of the focally necrotic lesions in the periventricular white matter, as well as in glial cells throughout the surrounding white matter with reactive gliosis. Interestingly, no significant 12/15-LOX expression was detected in neurons in the cerebral cortex overlying the damaged white matter. Using a scoring system from 0 to 3, we assessed the density of 12/15-LOX-expressing cells in diffusely gliotic white matter from 20 to 43 postconceptional (PC) weeks in 19 PVL cases (median = 36 PC weeks) and 10 control (non-PVL) cases (median = 34 PC weeks). The density of 12/15-LOX-positive cells was significantly increased in the diffuse component of PVL (score = 1.17 ± 0.15) compared to controls (score = 0.48 ± 0.21; p = 0.014). Using double-label ICC, 12/15-LOX was observed in PVL in OLs of the O4 and O1 premyelinating stages, as well as in mature OLs as determined with the mature OL marker adenomatous polyposis coli (APC). In addition, 12/15-LOX expression was present in a population of CD68-positive activated microglia. There was no 12/15-LOX expression in reactive astrocytes. Finally we observed terminal deoxynucleotide transferase dUTP nick end-labeling-positive cells within the white matter of PVL that expressed 12/15-LOX and/or within close proximity of 12/15-LOX-positive cells. Our data support a role for 12/15-LOX activation as an inflammatory mediator of injury in PVL, with a contribution of 12/15-LOX to PVL-induced damage to or cell death of OLs, including those at the O1 and O4 stages.

Figure 1

12/15-LOX expression in controls. A) 12/15-LOX expression shows co-localization with microglial marker tomato lectin in the white matter of a control case at 34 PC weeks (40X magnification). B) A rare 12/15-LOX expressing-O4 cell is detected in the white matter of a control case at 37 PC weeks (40X magnification). C) and (D) Scattered neuronal cells throughout all layers express 12/15-LOX in a control case at 40 PC weeks. Neurons are shown at (C) low magnification (scale bar = 200 μm) and at (D) high magnification (scale bar = 100 μm). 12/15-LOX expression is shown in pyramidal neurons (arrow) and granular neurons (arrowhead) (C).

Figure 2

12/15-LOX expression in macrophages of the focal necrotic lesion of PVL. A) Shown is a representative hemotoxylin and eosin stained section of a posterior frontal section of PVL with a macrocyst (arrow) and microcystic lesions (marked by asterisks) in the periventricular white matter. B) 12/15-LOX is expressed in the macrophages and/or amoeboid microglia of the microcystic lesion as seen by DAB staining at 20X magnification (scale bar = 200 μm). C) Macrophages/amoeboid microglia within the focal lesion show co-localization with macrophage/microglia marker CD68 and 12/15-LOX (40X magnification).

Figure 3

12/15-LOX expression in the diffusely gliotic lesion of PVL. A) 12/15-LOX is expressed in glial cells of the diffusely gliotic lesion in a 40 PC week case with PVL. Arrows indicate cells with the morphological appearance of microglia. The arrowhead indicates a cell with the appearance of an oligodendrocyte. B) 12/15-LOX expression is detected in scattered nuclei (*) of cells in the gliotic white matter of a PVL case at 39 PC weeks. C) There is no detectable expression of 12/15-LOX in the white matter of a control case at 40 PC weeks. All images are 40X magnification. The representative scale bar is 100 μm.

Figure 4

12/15-LOX cell density in PVL and controls. Semi-quantitative analysis using a standardization density score for 12/15-LOX expressing cells indicates an increase in 12/15-LOX density in PVL cases compared to controls (p = 0.014). The graph shown indicates the number of cases and controls given a specific density score of 0–3.

Figure 5

12/15-LOX expression in reactive microglia and reactive astrocytes of the diffuse component. A) 12/15-LOX is expressed in a population of CD68 positive activated microglia in the diffuse component of PVL. B) There is a population of CD68 positive activated microglia in the diffuse component of PVL that does not show co-localization with 12/15-LOX. Based on morphology the 12/15-LOX positive/CD68 negative cell is likely to be a mature OL. Both A and B are from a PVL case at 40 PC weeks. C) There is no co-localization of 12/15-LOX with the reactive astrocyte marker GFAP in a PVL case at 38 PC weeks. All images are 40X magnification

Figure 6

12/15-LOX expression in OL of the premyelinating (O4) and immature (O1) stages. A) There is co-localization of 12/15-LOX with the O4 marker of premyelinating OLs in a PVL case at 30 PC weeks. B) There is co-localization of 12/15-LOX with the O1 marker of immature OLs in a PVL case at 30 PC weeks.

Figure 7

12/15-LOX expression in mature OLs. A) and B) 12/15-LOX is expressed in cells with the morphological appearance of mature OLs as determined by DAB staining in a PVL case at 38 PC weeks. C. 12/15-LOX is co-localized with the mature OL marker APC in a PVL case at 33 PC weeks (40X magnification).

Figure 8

Relationship between TUNEL positive cells and 12/15-LOX expression. A) In a case at 39 PC weeks, 2 TUNEL-positive cells show colocalization with cytoplasmic 12/15-LOX expression. Also shown is a 12/15-LOX positive cell negative for TUNEL. (40X magnification) B) In a case at 41 PC weeks, a low power (20X) magnification shows cells positive for TUNEL-only proximal to cells positive for 12/15-LOX only. Also shown are 2 TUNEL positive cells with colocalization of cytoplasmic 12/15-LOX expression. C) In a case at 39 PC weeks, a TUNEL positive cell is shown with nuclear colocalization of 12/15-LOX. Also shown is a 12/15-LOX positive cell negative for TUNEL (40X magnification).