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TNFR1-JNK signaling is the shared pathway of neuroinflammation and neurovascular damage after LPS-sensitized hypoxic-ischemic injury in the immature brain.

Wang LW, Chang YC, Chen SJ, Tseng CH, Tu YF, Liao NS, Huang CC, Ho CJ - J Neuroinflammation (2014)

Bottom Line: Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury.The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI.TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chi Mei Medical Center, Tainan, 710, Taiwan. lanwan@ms67.url.com.tw.

ABSTRACT

Background: Hypoxic-ischemia (HI) and inflammation are the two major pathogenic mechanisms of brain injury in very preterm infants. The neurovascular unit is the major target of HI injury in the immature brain. Systemic inflammation may worsen HI by up-regulating neuroinflammation and disrupting the blood-brain barrier (BBB). Since neurons and oligodendrocytes, microvascular endothelial cells, and microglia may closely interact with each other, there may be a common signaling pathway leading to neuroinflammation and neurovascular damage after injury in the immature brain. TNF-α is a key pro-inflammatory cytokine that acts through the TNF receptor (TNFR), and c-Jun N-terminal kinases (JNK) are important stress-responsive kinases.

Objective: To determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain.

Methods: Postpartum (P) day-5 mice received LPS or normal saline (NS) injection before HI. Immunohistochemistry, immunoblotting and TNFR1- and TNFR2-knockout mouse pups were used to determine neuroinflammation, BBB damage, TNF-α expression, JNK activation, and cell apoptosis. The cellular distribution of p-JNK, TNFR1/TNFR2 and cleaved caspase-3 were examined using immunofluorescent staining.

Results: The LPS + HI group had significantly greater up-regulation of activated microglia, TNF-α and TNFR1 expression, and increases of BBB disruption and cleaved caspase-3 levels at 24 hours post-insult, and showed more cortical and white matter injury on P17 than the control and NS + HI groups. Cleaved caspase-3 was highly expressed in microvascular endothelial cells, neurons, and oligodendroglial precursor cells. LPS-sensitized HI also induced JNK activation and up-regulation of TNFR1 but not TNFR2 expression in the microglia, endothelial cells, neurons, and oligodendrocyte progenitors, and most of the TNFR1-positive cells co-expressed p-JNK. Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury. The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI.

Conclusion: TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

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Related in: MedlinePlus

Tumor necrosis factor receptor 1 (TNFR1) deficiency reduced c-Jun N-terminal kinase (JNK), microglial activation and blood–brain barrier (BBB) damage, and attenuated injury after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) At 24 hours post-insult, the TNFR1- (n = 12) but not the TNFR2-KO (n = 12) pups had significant reduction in p-JNK expression and decreased Iba1-positive microglia, IgG extravasation, and cleaved caspase 3-positive cells than the WT pups (n = 10). (B) The TNFR1-KO but not the TNFR2-KO mice also had significantly reduced cortical injury (Nissl stain, upper panel), preserved myelination (MBP, middle panel), and decreased astrogliosis (GFAP, lower panel) than the WT mice on P17. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; KO, knockout; MBP, myelin basic protein; WT, wild-type. Scale bar = 100 μm in (A), and 200 μm for MBP, 100 μm for GFAP in (B). Inset scale bar = 10 μm. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
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Fig7: Tumor necrosis factor receptor 1 (TNFR1) deficiency reduced c-Jun N-terminal kinase (JNK), microglial activation and blood–brain barrier (BBB) damage, and attenuated injury after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) At 24 hours post-insult, the TNFR1- (n = 12) but not the TNFR2-KO (n = 12) pups had significant reduction in p-JNK expression and decreased Iba1-positive microglia, IgG extravasation, and cleaved caspase 3-positive cells than the WT pups (n = 10). (B) The TNFR1-KO but not the TNFR2-KO mice also had significantly reduced cortical injury (Nissl stain, upper panel), preserved myelination (MBP, middle panel), and decreased astrogliosis (GFAP, lower panel) than the WT mice on P17. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; KO, knockout; MBP, myelin basic protein; WT, wild-type. Scale bar = 100 μm in (A), and 200 μm for MBP, 100 μm for GFAP in (B). Inset scale bar = 10 μm. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.

Mentions: The TNFR1-KO pups, but not the TNFR2-KO pups, had significantly reduced p-JNK expression, with decreased activated microglia, BBB breakdown, and cleaved caspase-3-positive cells compared to the wild-type pups at 24 hours post-insult (Figure 7A). On P17, the TNFR1-KO mice, but not the TNFR2-KO mice, had significant attenuation of cortical and white matter injury with decreased astrogliosis compared to the wild-type mice (Figure 7B).Figure 7


TNFR1-JNK signaling is the shared pathway of neuroinflammation and neurovascular damage after LPS-sensitized hypoxic-ischemic injury in the immature brain.

Wang LW, Chang YC, Chen SJ, Tseng CH, Tu YF, Liao NS, Huang CC, Ho CJ - J Neuroinflammation (2014)

Tumor necrosis factor receptor 1 (TNFR1) deficiency reduced c-Jun N-terminal kinase (JNK), microglial activation and blood–brain barrier (BBB) damage, and attenuated injury after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) At 24 hours post-insult, the TNFR1- (n = 12) but not the TNFR2-KO (n = 12) pups had significant reduction in p-JNK expression and decreased Iba1-positive microglia, IgG extravasation, and cleaved caspase 3-positive cells than the WT pups (n = 10). (B) The TNFR1-KO but not the TNFR2-KO mice also had significantly reduced cortical injury (Nissl stain, upper panel), preserved myelination (MBP, middle panel), and decreased astrogliosis (GFAP, lower panel) than the WT mice on P17. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; KO, knockout; MBP, myelin basic protein; WT, wild-type. Scale bar = 100 μm in (A), and 200 μm for MBP, 100 μm for GFAP in (B). Inset scale bar = 10 μm. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
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Fig7: Tumor necrosis factor receptor 1 (TNFR1) deficiency reduced c-Jun N-terminal kinase (JNK), microglial activation and blood–brain barrier (BBB) damage, and attenuated injury after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) At 24 hours post-insult, the TNFR1- (n = 12) but not the TNFR2-KO (n = 12) pups had significant reduction in p-JNK expression and decreased Iba1-positive microglia, IgG extravasation, and cleaved caspase 3-positive cells than the WT pups (n = 10). (B) The TNFR1-KO but not the TNFR2-KO mice also had significantly reduced cortical injury (Nissl stain, upper panel), preserved myelination (MBP, middle panel), and decreased astrogliosis (GFAP, lower panel) than the WT mice on P17. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; KO, knockout; MBP, myelin basic protein; WT, wild-type. Scale bar = 100 μm in (A), and 200 μm for MBP, 100 μm for GFAP in (B). Inset scale bar = 10 μm. Values are means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
Mentions: The TNFR1-KO pups, but not the TNFR2-KO pups, had significantly reduced p-JNK expression, with decreased activated microglia, BBB breakdown, and cleaved caspase-3-positive cells compared to the wild-type pups at 24 hours post-insult (Figure 7A). On P17, the TNFR1-KO mice, but not the TNFR2-KO mice, had significant attenuation of cortical and white matter injury with decreased astrogliosis compared to the wild-type mice (Figure 7B).Figure 7

Bottom Line: Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury.The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI.TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chi Mei Medical Center, Tainan, 710, Taiwan. lanwan@ms67.url.com.tw.

ABSTRACT

Background: Hypoxic-ischemia (HI) and inflammation are the two major pathogenic mechanisms of brain injury in very preterm infants. The neurovascular unit is the major target of HI injury in the immature brain. Systemic inflammation may worsen HI by up-regulating neuroinflammation and disrupting the blood-brain barrier (BBB). Since neurons and oligodendrocytes, microvascular endothelial cells, and microglia may closely interact with each other, there may be a common signaling pathway leading to neuroinflammation and neurovascular damage after injury in the immature brain. TNF-α is a key pro-inflammatory cytokine that acts through the TNF receptor (TNFR), and c-Jun N-terminal kinases (JNK) are important stress-responsive kinases.

Objective: To determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain.

Methods: Postpartum (P) day-5 mice received LPS or normal saline (NS) injection before HI. Immunohistochemistry, immunoblotting and TNFR1- and TNFR2-knockout mouse pups were used to determine neuroinflammation, BBB damage, TNF-α expression, JNK activation, and cell apoptosis. The cellular distribution of p-JNK, TNFR1/TNFR2 and cleaved caspase-3 were examined using immunofluorescent staining.

Results: The LPS + HI group had significantly greater up-regulation of activated microglia, TNF-α and TNFR1 expression, and increases of BBB disruption and cleaved caspase-3 levels at 24 hours post-insult, and showed more cortical and white matter injury on P17 than the control and NS + HI groups. Cleaved caspase-3 was highly expressed in microvascular endothelial cells, neurons, and oligodendroglial precursor cells. LPS-sensitized HI also induced JNK activation and up-regulation of TNFR1 but not TNFR2 expression in the microglia, endothelial cells, neurons, and oligodendrocyte progenitors, and most of the TNFR1-positive cells co-expressed p-JNK. Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury. The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI.

Conclusion: TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

Show MeSH
Related in: MedlinePlus