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Cited by 119Neonatal and fetal brain pathologyNeuroinflammation and Neurodegeneration MechanismsTraumatic Brain Injury and Neurovascular Disturbances

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JNK signaling is the shared pathway linking neuroinflammation, blood-brain barrier disruption and oligodendroglial apoptosis in the white matter injury of the immature brain

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Wang, Lan-Wan, Tu, Yi-Fang, Huang, Chao-Ching, Ho, Chien-Jung·2012·Journal of Neuroinflammation
DOI 10.1186/1742-2094-9-175PMC PMC3414763
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Abstract

BACKGROUND: White matter injury is the major form of brain damage in very preterm infants. Selective white matter injury in the immature brain can be induced by lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) in the postpartum (P) day 2 rat pups whose brain maturation status is equivalent to that in preterm infants less than 30 weeks of gestation. Neuroinflammation, blood-brain barrier (BBB) damage and oligodendrocyte progenitor apoptosis may affect the susceptibility of LPS-sensitized HI in white matter injury. c-Jun N-terminal kinases (JNK) are important stress-responsive kinases in various forms of insults. We hypothesized that LPS-sensitized HI causes white matter injury through JNK activation-mediated neuroinflammation, BBB leakage and oligodendroglial apoptosis in the white matter of P2 rat pups. METHODS: P2 pups received LPS (0.05 mg/kg) or normal saline injection followed by 90-min HI. Immunohistochemistry and immunoblotting were used to determine microglia activation, TNF-α, BBB damage, cleaved caspase-3, JNK and phospho-JNK (p-JNK), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) expression. Immunofluorescence was performed to determine the cellular distribution of p-JNK. Pharmacological and genetic approaches were used to inhibit JNK activity. RESULTS: P2 pups had selective white matter injury associated with upregulation of activated microglia, TNF-α, IgG extravasation and oligodendroglial progenitor apoptosis after LPS-sensitized HI. Immunohistochemical analyses showed early and sustained JNK activation in the white matter at 6 and 24 h post-insult. Immunofluorescence demonstrated upregulation of p-JNK in activated microglia, vascular endothelial cells and oligodendrocyte progenitors, and also showed perivascular aggregation of p-JNK-positive cells around the vessels 24 h post-insult. JNK inhibition by AS601245 or by antisense oligodeoxynucleotides (ODN) significantly reduced microglial activation, TNF-α immunoreactivity, IgG extravasation, and cleaved caspase-3 in the endothelial cells and oligodendrocyte progenitors, and also attenuated perivascular aggregation of p-JNK-positive cells 24 h post-insult. The AS601245 or JNK antisense ODN group had significantly increased MBP and decreased GFAP expression in the white matter on P11 than the vehicle or scrambled ODN group. CONCLUSIONS: LPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.

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Figures

Figure 1 Upregulation of neuroinflammation, blood–brain barrier damage and cell apoptosis in association with white matter injury in P2 rat pups after lipopolysaccharide-sensitized hypoxic-ischemia. On P11 in the LPS + HI group, Nissl staining (A) showed no significant injury in the cortex (gross picture in the upper panel; microscopic picture in the lower panel photographed from the cortex marked with an asterisk). (B) Immunohistochemical staining demonstrated that the LPS + HI group had markedly decreased MBP expression and increased GFAP-positive astrogliosis in the white matter of the ipsilateral hemisphere compared to the control and NS + HI groups. (C) Immunohistochemistry 24 h post-insult showed that the LPS + HI but not the NS + HI group had significant increases in ED1-positive microglia, TNF-α immunoreactivities, IgG extravasation, and cleaved caspase 3-positive apoptotic cells in the white matter. Microscopic pictures of (B,C) were taken from the white matter area marked with a circle in (A). ED1, microglia marker; GFAP, glial fibrillary acidic protein; HI, hypoxic-ischemia; LPS, lipopolysaccharide; MBP, myelin basic protein; NS, normal saline; P, postpartum. Scale bar = 200 μm for MBP, 50 μm for cleaved caspase 3, and 100 μm for the others.
Figure 1 Upregulation of neuroinflammation, blood–brain barrier damage and cell apoptosis in association with white matter injury in P2 rat pups after lipopolysaccharide-sensitized hypoxic-ischemia. On P11 in the LPS + HI group, Nissl staining (A) showed no significant injury in the cortex (gross picture in the upper panel; microscopic picture in the lower panel photographed from the cortex marked with an asterisk). (B) Immunohistochemical staining demonstrated that the LPS + HI group had markedly decreased MBP expression and increased GFAP-positive astrogliosis in the white matter of the ipsilateral hemisphere compared to the control and NS + HI groups. (C) Immunohistochemistry 24 h post-insult showed that the LPS + HI but not the NS + HI group had significant increases in ED1-positive microglia, TNF-α immunoreactivities, IgG extravasation, and cleaved caspase 3-positive apoptotic cells in the white matter. Microscopic pictures of (B,C) were taken from the white matter area marked with a circle in (A). ED1, microglia marker; GFAP, glial fibrillary acidic protein; HI, hypoxic-ischemia; LPS, lipopolysaccharide; MBP, myelin basic protein; NS, normal saline; P, postpartum. Scale bar = 200 μm for MBP, 50 μm for cleaved caspase 3, and 100 μm for the others.CC BY · PubMed Central
Figure 2 Upregulation of JNK activation in lipopolysaccharide-sensitized hypoxic-ischemic white matter injury. (A) Immunoblotting of white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed there was an early rise of phospho-c-Jun N-terminal kinase (p-JNK) expression at 1 h, which peaked at 6 h and persisted at 24 h post-insult. The JNK expression did not differ between the control and LPS + HI groups at various time points post-insult. (B) p-JNK immunohistochemistry at 6 and 24 h post-insult showed the LPS + HI group (n = 10) had significantly higher p-JNK immunoreactivities in the white matter of the ipsilateral hemisphere than the control (n = 7) groups. Scale bar =100 μm in (B). Values are means ± SEM. ***P < 0.001, **P < 0.01.
Figure 2 Upregulation of JNK activation in lipopolysaccharide-sensitized hypoxic-ischemic white matter injury. (A) Immunoblotting of white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed there was an early rise of phospho-c-Jun N-terminal kinase (p-JNK) expression at 1 h, which peaked at 6 h and persisted at 24 h post-insult. The JNK expression did not differ between the control and LPS + HI groups at various time points post-insult. (B) p-JNK immunohistochemistry at 6 and 24 h post-insult showed the LPS + HI group (n = 10) had significantly higher p-JNK immunoreactivities in the white matter of the ipsilateral hemisphere than the control (n = 7) groups. Scale bar =100 μm in (B). Values are means ± SEM. ***P < 0.001, **P < 0.01.CC BY · PubMed Central
Figure 3 JNK activation in microglia, vascular endothelial cells and oligodendrocyte progenitors at 6 h post-insult. Immunofluorescence of the ipsilateral white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed increased phospho-c-Jun N-terminal kinase (p-JNK) expression in (A) ED1-positive microglia, (B) RECA-positive endothelial cells and (C) O4-positive oligodendrocyte progenitors. Scale bar = 25 μm. Inset scale bar = 5 μm in (A), 10 μm in (B) and 2.5 μm in (C).
Figure 3 JNK activation in microglia, vascular endothelial cells and oligodendrocyte progenitors at 6 h post-insult. Immunofluorescence of the ipsilateral white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed increased phospho-c-Jun N-terminal kinase (p-JNK) expression in (A) ED1-positive microglia, (B) RECA-positive endothelial cells and (C) O4-positive oligodendrocyte progenitors. Scale bar = 25 μm. Inset scale bar = 5 μm in (A), 10 μm in (B) and 2.5 μm in (C).CC BY · PubMed Central
Figure 4 Activated microglia expressed p-JNK, p-c-Jun and TNF-α. Immunofluorescence of the ipsilateral white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group 24 h post-insult showed that ED1-positive activated microglia expressed phospho-c-Jun N-terminal kinases (p-JNK) (A) and TNF-α (C), and had nuclear translocation of p-c-Jun (B). Scale bar = 25 μm. Inset scale bar = 5 μm.
Figure 4 Activated microglia expressed p-JNK, p-c-Jun and TNF-α. Immunofluorescence of the ipsilateral white matter in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group 24 h post-insult showed that ED1-positive activated microglia expressed phospho-c-Jun N-terminal kinases (p-JNK) (A) and TNF-α (C), and had nuclear translocation of p-c-Jun (B). Scale bar = 25 μm. Inset scale bar = 5 μm.CC BY · PubMed Central
Figure 5 JNK activation-mediated apoptosis in cerebral vascular endothelial cells and oligodendrocyte progenitors in the white matter after lipopolysaccharide-sensitized hypoxic-ischemia. Immunofluorescence of the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group 24 h post-insult showed numerous phospho-c-Jun N-terminal kinase (p-JNK)-positive cells attached to or located around the microvessels in the white matter (A). RECA-positive endothelial cells (A) and O4-positive oligodendrocyte progenitors (B) co-expressed p-JNK. Many p-JNK-positive cells (C), RECA-positive endothelial cells (D) and O4-positive oligodendrocyte progenitors (E) expressed cleaved caspase 3. Scale bar = 25 μm. Inset scale bar = 2.5 μm.
Figure 5 JNK activation-mediated apoptosis in cerebral vascular endothelial cells and oligodendrocyte progenitors in the white matter after lipopolysaccharide-sensitized hypoxic-ischemia. Immunofluorescence of the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group 24 h post-insult showed numerous phospho-c-Jun N-terminal kinase (p-JNK)-positive cells attached to or located around the microvessels in the white matter (A). RECA-positive endothelial cells (A) and O4-positive oligodendrocyte progenitors (B) co-expressed p-JNK. Many p-JNK-positive cells (C), RECA-positive endothelial cells (D) and O4-positive oligodendrocyte progenitors (E) expressed cleaved caspase 3. Scale bar = 25 μm. Inset scale bar = 2.5 μm.CC BY · PubMed Central
Figure 6 AS601245 significantly reduced neuroinflammation, blood–brain-barrier damage and cell apoptosis after lipopolysaccharide-sensitized hypoxic-ischemic white matter injury. (A) In vitro kinase assay of c-Jun N-terminal kinase (JNK) in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed that AS601245 (40 mg/kg) effectively blocked JNK activity at 6 and 24 h post-insult compared with vehicle. (B) AS601245 (40 mg/kg) treatment (n = 8) significantly reduced upregulation of ED1-positive activated microglia, TNF-α immunoreactivities, IgG extravasation and cleaved caspase 3-positive cells in the white matter 24 h post-insult compared to vehicle (n = 7). Scale bar =100 μm for ED1, TNF-α and IgG; 50 μm for cleaved caspase 3. Values are means ± SEM. ***P <0.001, **P < 0.01, *P < 0.05.
Figure 6 AS601245 significantly reduced neuroinflammation, blood–brain-barrier damage and cell apoptosis after lipopolysaccharide-sensitized hypoxic-ischemic white matter injury. (A) In vitro kinase assay of c-Jun N-terminal kinase (JNK) in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed that AS601245 (40 mg/kg) effectively blocked JNK activity at 6 and 24 h post-insult compared with vehicle. (B) AS601245 (40 mg/kg) treatment (n = 8) significantly reduced upregulation of ED1-positive activated microglia, TNF-α immunoreactivities, IgG extravasation and cleaved caspase 3-positive cells in the white matter 24 h post-insult compared to vehicle (n = 7). Scale bar =100 μm for ED1, TNF-α and IgG; 50 μm for cleaved caspase 3. Values are means ± SEM. ***P <0.001, **P < 0.01, *P < 0.05.CC BY · PubMed Central
Figure 7 AS601245 significantly reduced perivascular attachment of p-JNK-positive cells and cell apoptosis in the white matter 24 h post-insult. Immunofluorescent staining in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed that, compared with vehicle, AS601245 (40 mg/kg) significantly attenuated perivascular phospho-c-Jun N-terminal kinase (p-JNK)-positive cell attachment (A), and also decreased cleaved caspase 3-positive endothelial (B) and oligodendroglial cells (C) in the white matter. Scale bar =25 μm.
Figure 7 AS601245 significantly reduced perivascular attachment of p-JNK-positive cells and cell apoptosis in the white matter 24 h post-insult. Immunofluorescent staining in the lipopolysaccharide (LPS) + hypoxic-ischemic (HI) group showed that, compared with vehicle, AS601245 (40 mg/kg) significantly attenuated perivascular phospho-c-Jun N-terminal kinase (p-JNK)-positive cell attachment (A), and also decreased cleaved caspase 3-positive endothelial (B) and oligodendroglial cells (C) in the white matter. Scale bar =25 μm.CC BY · PubMed Central
Figure 8 Pharmacological inhibition of c-Jun N-terminal kinase (JNK) activity using AS601245 significantly attenuated white matter injury. AS601245 (40 mg/kg) (n = 8) but not AS601245 (20 mg/kg) (n = 8) treatment had significantly higher myelin basic protein (MBP) (A) and lower glial fibrillary acidic protein (GFAP) (B) expression in the white matter than vehicle (n = 10) on P11 after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) on P2. Scale bar = 200 μm in (A), 100 μm in (B). Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 8 Pharmacological inhibition of c-Jun N-terminal kinase (JNK) activity using AS601245 significantly attenuated white matter injury. AS601245 (40 mg/kg) (n = 8) but not AS601245 (20 mg/kg) (n = 8) treatment had significantly higher myelin basic protein (MBP) (A) and lower glial fibrillary acidic protein (GFAP) (B) expression in the white matter than vehicle (n = 10) on P11 after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) on P2. Scale bar = 200 μm in (A), 100 μm in (B). Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.CC BY · PubMed Central
Figure 9 JNK antisense oligodeoxynucleotide significantly reduced neuroinflammation, blood–brain-barrier damage and apoptosis in the white matter after lipopolysaccharide-sensitized hypoxic-ischemia. (A) Immunoblotting of the white matter showed that intracerebroventricular infusion of c-Jun N-terminal kinase (JNK) antisense oligodeoxynucleotides (ODN) (n = 3) effectively suppressed JNK expression compared with scrambled ODN (n = 3) at 3, 6 and 12 h post-insult. (B) Antisense ODN treatment (n = 8) significantly attenuated upregulation of ED1-positive activated microglia, TNF-α immunoreactivities, IgG extravasation and cleaved caspase 3-positive cells in the white matter 24 h post-insult compared with scrambled oligodeoxynucleotide (n = 8). Scale bar =100 μm for ED1, TNF-α and IgG; 50 μm for cleaved caspase 3. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 9 JNK antisense oligodeoxynucleotide significantly reduced neuroinflammation, blood–brain-barrier damage and apoptosis in the white matter after lipopolysaccharide-sensitized hypoxic-ischemia. (A) Immunoblotting of the white matter showed that intracerebroventricular infusion of c-Jun N-terminal kinase (JNK) antisense oligodeoxynucleotides (ODN) (n = 3) effectively suppressed JNK expression compared with scrambled ODN (n = 3) at 3, 6 and 12 h post-insult. (B) Antisense ODN treatment (n = 8) significantly attenuated upregulation of ED1-positive activated microglia, TNF-α immunoreactivities, IgG extravasation and cleaved caspase 3-positive cells in the white matter 24 h post-insult compared with scrambled oligodeoxynucleotide (n = 8). Scale bar =100 μm for ED1, TNF-α and IgG; 50 μm for cleaved caspase 3. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.CC BY · PubMed Central
Figure 10 c-Jun N-terminal kinase (JNK) antisense oligodeoxynucleotide significantly attenuated white matter injury. Antisense oligodeoxynucleotide (ODN) treatment (n = 8) markedly increased myelin basic protein (MBP) (A) and decreased glial fibrillary acidic protein (GFAP) (B) expression in the white matter compared with scrambled ODN (n = 8) on P11 after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) on P2. Scale bar = 200 μm in (A), 100 μm in (B). Values are means ± SEM. ***P < 0.001, *P < 0.05.
Figure 10 c-Jun N-terminal kinase (JNK) antisense oligodeoxynucleotide significantly attenuated white matter injury. Antisense oligodeoxynucleotide (ODN) treatment (n = 8) markedly increased myelin basic protein (MBP) (A) and decreased glial fibrillary acidic protein (GFAP) (B) expression in the white matter compared with scrambled ODN (n = 8) on P11 after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) on P2. Scale bar = 200 μm in (A), 100 μm in (B). Values are means ± SEM. ***P < 0.001, *P < 0.05.CC BY · PubMed Central

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Metadata

Authors
Wang, L.-W., Tu, Y.-F., Huang, C.-C., & Ho, C.-J.
Affiliations
National Cheng Kung University (TW)
Corresponding author
Huang, Chao-Ching
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2012-07-17
Volume
9
Issue
1
Pages
175-175
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-9-175
PMC
PMC3414763
OpenAlex
W1971748792
Cited by
119
References
72
Citations by year
2026: 4 · 2025: 8 · 2024: 6 · 2023: 7 · 2022: 5 · 2021: 11 · 2020: 8 · 2019: 9 · 2018: 10 · 2017: 10 · 2016: 11 · 2015: 11 · 2014: 8 · 2013: 9 · 2012: 1
FWCI
8.15
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Science Council
  • National Health Research Institutes
  • Chi Mei Medical Center
Topics
  • Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Neonatal and fetal brain pathology
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Traumatic Brain Injury and Neurovascular Disturbances
Keywords
JNK signaling, white matter injury, neuroinflammation, blood-brain barrier disruption, hypoxia-ischemia, preterm infants, microglial activation, TNF-α, IgG extravasation, JNK inhibition, oligodeoxynucleotides
MeSH terms
Animals, Animals, Newborn, Blood-Brain Barrier, Brain, Inflammation, Nerve Fibers, Myelinated, Oligodendroglia, Rats, Sprague-Dawley, Apoptosis, Hypoxia-Ischemia, Brain, MAP Kinase Signaling System, Rats

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