Hyperoxia causes maturation-dependent cell death in the developing white matter - PubMed (original) (raw)
Comparative Study
Hyperoxia causes maturation-dependent cell death in the developing white matter
Bettina Gerstner et al. J Neurosci. 2008.
Abstract
Periventricular leukomalacia is the predominant injury in the preterm infant leading to cerebral palsy. Oxygen exposure may be an additional cause of brain injury in these infants. In this study, we investigated pathways of maturation-dependent oligodendrocyte (OL) death induced by hyperoxia in vitro and in vivo. Developing and mature OLs were subjected to 80% oxygen (0-24 h). Lactate dehydrogenase (LDH) assay was used to assess cell viability. Furthermore, 3-, 6-, and 10-d-old rat pups were subjected to 80% oxygen (24 h), and their brains were processed for myelin basic protein staining. Significant cell death was detected after 6-24 h incubation in 80% oxygen in pre-OLs (O4+,O1-), but not in mature OLs (MBP+). Cell death was executed by a caspase-dependent apoptotic pathway and could be blocked by the pan-caspase inhibitor zVAD-fmk. Overexpression of BCL2 (Homo sapiens B-cell chronic lymphocytic leukemia/lymphoma 2) significantly reduced apoptosis. Accumulation of superoxide and generation of reactive oxygen species (ROS) were detected after 2 h of oxygen exposure. Lipoxygenase inhibitors 2,3,5-trimethyl-6-(12-hydroxy-5-10-dodecadiynyl-1,4-benzoquinone and N-benzyl-N-hydroxy-5-phenylpentamide fully protected the cells from oxidative injury. Overexpression of superoxide dismutase (SOD1) dramatically increased injury to pre-OLs but not to mature OLs. We extended these studies by testing the effects of hyperoxia on neonatal white matter. Postnatal day 3 (P3) and P6 rats, but not P10 pups, showed bilateral reduction in MBP (myelin basic protein) expression with 24 h exposure to 80% oxygen. Hyperoxia causes oxidative stress and triggers maturation-dependent apoptosis in pre-OLs, which involves the generation of ROS and caspase activation, and leads to white matter injury in the neonatal rat brain. These observations may be relevant to white matter injury observed in premature infants.
Figures
Figure 1.
Hyperoxia causes cell death in pre-OLs. Primary oligodendrocyte cytotoxicity, as measured by LDH release, after 0, 6, 12, and 24 h incubation with 80% oxygen (means ± SEM of 3 independent experiments). *p < 0.05, ***p < 0.001 in one-way ANOVA followed by Tukey–Kramer test; comparison between 0 and 6, 12, and 24 h of 80% oxygen exposure.
Figure 2.
Representative phase contrast photomicrographs of primary oligodendrocytes after 12 h of incubation with either 21 or 80% oxygen. A–D, After incubation with 21% oxygen, pre-OLs (A) and mature OLs (C) are intact without signs of apoptosis. Hyperoxia-treated pre-OLs (B) show complete loss of processes, cell shrinkage, plasma membrane blebbing, and nuclear condensation. Mature OLs do not show signs of apoptosis after incubation in 80% oxygen for 12 h (D). E, Pre-OL and mature OL cell viability, as measured by LDH release, after 12 h of incubation with 80% oxygen (means ± SEM of 3 independent experiments). ***p < 0.001, Student's t test; comparison of hyperoxia group (black bar) versus the normoxia group (white bar) for each maturation stage.
Figure 3.
Involvement of caspases in cell toxicity by hyperoxia. A–F, Representative phase contrast photomicrographs of pre-OLs after 12 h of incubation with either 21% (A, C, E) or 80% oxygen (B, D, F). After incubation with 21% oxygen, pre-OLs (A) or cells treated with zVAD-fmk (40 μ
m
) (C) do not display signs of apoptosis. E, Staurosporine (1 μ
m
) was used as a positive control for inducing apoptosis in pre-OLs. B, After 12 h of 80% oxygen exposure, pre-OLs (B) showed signs of apoptosis, whereas zVAD-fmk (40 μ
m
)-treated cells (D) were protected. E, F, Staurosporine-induced apoptosis in pre-OLs was increased after incubation with hyperoxia. G, Pre-OLs were treated with hyperoxia and/or with the pan-caspase inhibitor zVAD-fmk (40 μ
m
), and/or with staurosporine (1 μ
m
) for induction of apoptosis. Cells were incubated under normoxic (21% O2, white bars) or hyperoxic (80% O2, black bars) conditions for 12 h, and cell viability was determined via LDH release assay (bars represent mean ± SEM). ***p < 0.001 in two-way ANOVA, comparison between 21 and 80% oxygen-treated cells. ##p ≤ 0.01, Student's t test; comparison of staurosporine (1 μ
m
)-treated pre-OLs between 21 and 80% oxygen.
Figure 4.
BCL2 overexpression decreased cell death in pre-OLs. A, Western blot analysis of pre-OLs (O4) overexpressing BCL2 showing that BCL2 protein was detectable with HSV-BCL2 (BCL2) but not with control reporter gene HSV-GFP (GFP) infection. B, C, Pre-OLs, either infected with virus containing the reporter gene GFP (HSV-GFP) (B) or BCL2 (HSV- BCL2) (C) were cultured for 16 h under hyperoxic (80% oxygen) conditions and then assayed for cell survival using LDH release. D, HSV-BCL2-infected OLs demonstrated an increased resistance to hyperoxia-induced oxidative stress compared with HSV-GFP-infected or control cells. Values shown are mean ± SEM from three independent experiments that were performed. *p ≤ 0.05, ***p ≤ 0.001, in two-way ANOVA; comparison of hyperoxia group (black bar) versus the normoxia group (white bar) for each group. ###p ≤ 0.01; comparison of hyperoxia-treated HSV-GFP-infected OLs versus HSV-BCL2-infected OLs.
Figure 5.
A, B, Mitochondrial dysfunction during hyperoxia in pre-OLs. Pre-OLs were cultured for 6 h in normoxic (21% oxygen) or hyperoxic (80% oxygen) conditions and then stained using MitoSOX red, a mitochondrial superoxide indicator and derivate of ethidium bromide. Once in the mitochondria, MitoSOX reagent is oxidized by superoxide and exhibits red fluorescence after binding to nucleic acids. If pre-OLs were kept under normoxic conditions, only a small number of cells displayed red fluorescence staining (A), whereas pre-OLs that were exposed to 80% oxygen showed an increased number of cells with red labeling of mitochondria indicating increased superoxide production (B). C, D, Generation of ROS in the cytoplasm of premyelinating OLs. Pre-OLs were cultured for 6 h in normoxic (21% oxygen) (C) or hyperoxic (80% oxygen) (D) conditions and then stained using carboxy-H2DCFDA, which serves as an indicator of the overall degree of intracellular oxidative stress. If pre-OLs were kept under normoxic conditions, only a small number of cells showed green fluorescence staining (C), whereas pre-OL cultures that were exposed to 80% oxygen showed an increased number of cells with detection of ROS in the cytoplasm (D). E–H, Higher magnification with confocal microscopy (60×) showed the distribution of the red fluorescent-stained mitochondria (E) and the distribution of the green fluorescent-stained ROS in the cytoplasm (F, H) of O4 cells exposed to 80% oxygen for 2 h. G and H show a combined light and phase contrast photomicrograph of an O4 cell that was exposed to 80% oxygen for 2 h and then stained with red MitoSOX, green carboxy-H2DCFDA, and blue Hoechst nuclear stain (H). Note the typical morphological signs of apoptotic cell death: cell shrinkage, loss of processes, nuclear condensation, and plasma membrane blebbing. Scale bar, 5 μm.
Figure 6.
Inhibition of hyperoxia-induced oxidative stress by extracellular superoxide dismutase, catalase, and the GPx mimic ebselen. Pre-OLs were exposed to 80% oxygen for 12 h and treated with or without SOD (100 U/ml, 1000 U/ml), catalase (100 U/ml, 1000 U/ml), SOD/catalase (1000 U/ml each), or the GPx mimic ebselen (10 μ
m
). A second plate with the same treatments was kept under normoxic conditions and used as control. There was decreased cell death within the treatment groups compared with untreated cells under normoxic conditions. Values shown are means ± SEM from three independent experiments (***p ≤ 0.001 in one-way ANOVA followed by Tukey–Kramer test; comparison of drug-treated vs vehicle-treated cells).
Figure 7.
SOD1 overexpression increased cell death in pre-OLs. A, HSV-SOD1-infected pre-OLs demonstrated increased toxicity compared with HSV-GFP-infected control cells, when cells were kept under room air conditions (###p ≤ 0.001, Student's t test; comparison of HSV-GFP-infected cells vs HSV-SOD1-infected pre-OLs). After exposure to 80% oxygen, cell death increased significantly in HSV-GFP- or HSV-SOD1-infected pre-OLs. Values shown are means ± SEM from six independent experiments that were performed [**p < 0.01, ***p ≤ 0.001, in two-way ANOVA; comparison of hyperoxia group (black bar) vs the normoxia group (white bar)]. B, C Pre-OLs, either infected with virus containing the reporter gene GFP (HSV-GFP) (B) or SOD1 (HSV-SOD1) (C) were cultured for 24 h under normoxic conditions and then assayed for cell survival using LDH release. D, Toxicity from SOD1 overexpression was found in pre-OLs but not mature OLs. Values shown are mean ± SEM from three independent experiments that were performed [***p ≤ 0.001, Student's t test; comparison of hyperoxia group (black bar) vs the normoxia group (white bar) for each maturation stage].
Figure 8.
The 12-LOX inhibitors, AA-861, BMD-122, and baicalein, all at 5 μ
m
, protected against hyperoxia-induced toxicity in pre-OLs. The graph represents means ± SEM of three independent experiments. There was no difference between the three treatments. ***p < 0.001, one-way ANOVA; comparison of drug-treated versus vehicle-treated cells.
Figure 9.
Hyperoxia caused decrease in MBP expression in the developing rat brain. A, C, E, Normal expression of MBP in the white matter tracts of P11 rats that were kept under room air conditions at P3, P6, and P10 (controls; n = 4 each group). B, D, I, Loss of MBP expression is seen in the P11 pup in the medial white matter, after 24 h of oxygen exposure (80%) at P3 (n = 4) (B) or P6 (n = 5) (D). E–I, No difference in MBP expression was detected at P11 (n = 4) (F) nor at P14 (n = 4) (H) if the animal was subjected to 80% oxygen at P10. ***p < 0.001, using the nonparametric Wilcoxon test.
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