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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

Up-regulation of c-Jun N-terminal kinase (JNK) activation in microglia, endothelial cells, neurons, and oligodendrocyte progenitors after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) Immunoblotting showed that the LPS + HI group (n = 4), but not the NS + HI group (n = 4) had increased p-JNK expression at 6 and 24 hours post-insult compared to the control group (n = 3). (B) Immunofluorescence in the LPS + HI group 24 hours post-insult showed up-regulation of p-JNK expression in Iba1-positive microglia, IB4-positive microvascular endothelial cells, NeuN-positive neurons, and O4-positive oligodendrocyte progenitors. Arrowheads indicate many p-JNK-positive cells attached to or were located around the IB4-positive microvessels. Scale bar = 50 μm for Iba1 and IB4, and 25 μm for others. Inset scale bar = 2.5 μm. Values are means ± SEM. *P < 0.05, ***P < 0.001.
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Fig4: Up-regulation of c-Jun N-terminal kinase (JNK) activation in microglia, endothelial cells, neurons, and oligodendrocyte progenitors after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) Immunoblotting showed that the LPS + HI group (n = 4), but not the NS + HI group (n = 4) had increased p-JNK expression at 6 and 24 hours post-insult compared to the control group (n = 3). (B) Immunofluorescence in the LPS + HI group 24 hours post-insult showed up-regulation of p-JNK expression in Iba1-positive microglia, IB4-positive microvascular endothelial cells, NeuN-positive neurons, and O4-positive oligodendrocyte progenitors. Arrowheads indicate many p-JNK-positive cells attached to or were located around the IB4-positive microvessels. Scale bar = 50 μm for Iba1 and IB4, and 25 μm for others. Inset scale bar = 2.5 μm. Values are means ± SEM. *P < 0.05, ***P < 0.001.

Mentions: Immunoblotting demonstrated persistent JNK activation at 6 and 24 hours post-insult in the LPS + HI group, but not in the NS + HI group (Figure 4A). Immunofluorescence study in the LPS + HI group further revealed up-regulation of p-JNK expression in activated microglia, microvascular endothelial cells, neurons, and oligodendrocyte progenitors at 24 hours post-insult (Figure 4B). In addition, there were many p-JNK-positive cells (green light) attached to or located around the IB4-positive microvessels (arrowheads in Figure 4B).Figure 4


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)

Up-regulation of c-Jun N-terminal kinase (JNK) activation in microglia, endothelial cells, neurons, and oligodendrocyte progenitors after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) Immunoblotting showed that the LPS + HI group (n = 4), but not the NS + HI group (n = 4) had increased p-JNK expression at 6 and 24 hours post-insult compared to the control group (n = 3). (B) Immunofluorescence in the LPS + HI group 24 hours post-insult showed up-regulation of p-JNK expression in Iba1-positive microglia, IB4-positive microvascular endothelial cells, NeuN-positive neurons, and O4-positive oligodendrocyte progenitors. Arrowheads indicate many p-JNK-positive cells attached to or were located around the IB4-positive microvessels. Scale bar = 50 μm for Iba1 and IB4, and 25 μm for others. Inset scale bar = 2.5 μm. Values are means ± SEM. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300587&req=5

Fig4: Up-regulation of c-Jun N-terminal kinase (JNK) activation in microglia, endothelial cells, neurons, and oligodendrocyte progenitors after lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI). (A) Immunoblotting showed that the LPS + HI group (n = 4), but not the NS + HI group (n = 4) had increased p-JNK expression at 6 and 24 hours post-insult compared to the control group (n = 3). (B) Immunofluorescence in the LPS + HI group 24 hours post-insult showed up-regulation of p-JNK expression in Iba1-positive microglia, IB4-positive microvascular endothelial cells, NeuN-positive neurons, and O4-positive oligodendrocyte progenitors. Arrowheads indicate many p-JNK-positive cells attached to or were located around the IB4-positive microvessels. Scale bar = 50 μm for Iba1 and IB4, and 25 μm for others. Inset scale bar = 2.5 μm. Values are means ± SEM. *P < 0.05, ***P < 0.001.
Mentions: Immunoblotting demonstrated persistent JNK activation at 6 and 24 hours post-insult in the LPS + HI group, but not in the NS + HI group (Figure 4A). Immunofluorescence study in the LPS + HI group further revealed up-regulation of p-JNK expression in activated microglia, microvascular endothelial cells, neurons, and oligodendrocyte progenitors at 24 hours post-insult (Figure 4B). In addition, there were many p-JNK-positive cells (green light) attached to or located around the IB4-positive microvessels (arrowheads in Figure 4B).Figure 4

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