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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

Effects of hypoxic-ischemia (HI) or lipopolysaccharide (LPS) alone on brain injury. Mouse pups were exposed to different duration of HI or different doses of LPS on P5, and neuropathological examinations were performed on P17. (A) Compared to the control groups (n = 6), pups exposed to 40 minute-HI (n = 9), but not to 30-minute HI (n = 10), had significant injury in the cortex (upper panel) and decreased myelin basic protein (MBP) expression in the white matter of the ipsilateral hemispheres (middle panel). There were no significant differences in MBP expression in the contralateral white matter among the control, 30-minute HI and 40-minute HI groups (lower panel). (B) Compared to the normal saline (NS) group (n = 5), the 0.05-mg/kg LPS (n = 5) and 0.5-mg/kg LPS (n = 5) groups showed no evident cortical (Nissl staining, upper panel) or white matter injury (MBP staining, middle and lower panels). Scale bar = 200 μm. Values are means ± SEM. **P < 0.01, ***P < 0.001.
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Fig1: Effects of hypoxic-ischemia (HI) or lipopolysaccharide (LPS) alone on brain injury. Mouse pups were exposed to different duration of HI or different doses of LPS on P5, and neuropathological examinations were performed on P17. (A) Compared to the control groups (n = 6), pups exposed to 40 minute-HI (n = 9), but not to 30-minute HI (n = 10), had significant injury in the cortex (upper panel) and decreased myelin basic protein (MBP) expression in the white matter of the ipsilateral hemispheres (middle panel). There were no significant differences in MBP expression in the contralateral white matter among the control, 30-minute HI and 40-minute HI groups (lower panel). (B) Compared to the normal saline (NS) group (n = 5), the 0.05-mg/kg LPS (n = 5) and 0.5-mg/kg LPS (n = 5) groups showed no evident cortical (Nissl staining, upper panel) or white matter injury (MBP staining, middle and lower panels). Scale bar = 200 μm. Values are means ± SEM. **P < 0.01, ***P < 0.001.

Mentions: The cortical and white matter injuries in P5 mouse pups exposed to different HI durations (30 and 40 minutes) were first determined. Neuropathologic examinations performed on P17 showed that the pups exposed to 40-minute (40% mortality) HI, but not to 30-minute (5.6% mortality) HI, had significant brain area loss (Figure 1A, upper panel) and reduced MBP expression (Figure 1A, middle panel) in the ipsilateral hemisphere compared to the control pups without HI exposure. The 30- and 40-minute HI and the control groups had similar MBP expression in the contralateral hemispheres (Figure 1A, lower panel). Thus, 30-minute HI was used for the LPS-sensitized HI experiments.Figure 1


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)

Effects of hypoxic-ischemia (HI) or lipopolysaccharide (LPS) alone on brain injury. Mouse pups were exposed to different duration of HI or different doses of LPS on P5, and neuropathological examinations were performed on P17. (A) Compared to the control groups (n = 6), pups exposed to 40 minute-HI (n = 9), but not to 30-minute HI (n = 10), had significant injury in the cortex (upper panel) and decreased myelin basic protein (MBP) expression in the white matter of the ipsilateral hemispheres (middle panel). There were no significant differences in MBP expression in the contralateral white matter among the control, 30-minute HI and 40-minute HI groups (lower panel). (B) Compared to the normal saline (NS) group (n = 5), the 0.05-mg/kg LPS (n = 5) and 0.5-mg/kg LPS (n = 5) groups showed no evident cortical (Nissl staining, upper panel) or white matter injury (MBP staining, middle and lower panels). Scale bar = 200 μm. Values are means ± SEM. **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4300587&req=5

Fig1: Effects of hypoxic-ischemia (HI) or lipopolysaccharide (LPS) alone on brain injury. Mouse pups were exposed to different duration of HI or different doses of LPS on P5, and neuropathological examinations were performed on P17. (A) Compared to the control groups (n = 6), pups exposed to 40 minute-HI (n = 9), but not to 30-minute HI (n = 10), had significant injury in the cortex (upper panel) and decreased myelin basic protein (MBP) expression in the white matter of the ipsilateral hemispheres (middle panel). There were no significant differences in MBP expression in the contralateral white matter among the control, 30-minute HI and 40-minute HI groups (lower panel). (B) Compared to the normal saline (NS) group (n = 5), the 0.05-mg/kg LPS (n = 5) and 0.5-mg/kg LPS (n = 5) groups showed no evident cortical (Nissl staining, upper panel) or white matter injury (MBP staining, middle and lower panels). Scale bar = 200 μm. Values are means ± SEM. **P < 0.01, ***P < 0.001.
Mentions: The cortical and white matter injuries in P5 mouse pups exposed to different HI durations (30 and 40 minutes) were first determined. Neuropathologic examinations performed on P17 showed that the pups exposed to 40-minute (40% mortality) HI, but not to 30-minute (5.6% mortality) HI, had significant brain area loss (Figure 1A, upper panel) and reduced MBP expression (Figure 1A, middle panel) in the ipsilateral hemisphere compared to the control pups without HI exposure. The 30- and 40-minute HI and the control groups had similar MBP expression in the contralateral hemispheres (Figure 1A, lower panel). Thus, 30-minute HI was used for the LPS-sensitized HI experiments.Figure 1

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