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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

Lipopolysaccharide (LPS)-sensitized hypoxic-ischemic (HI) injury induced up-regulation of neuroinflammation, blood–brain barrier damage and cell apoptosis. (A) P5 mouse pups received normal saline (NS) or LPS (0.05 mg/kg) injection before 30-minute HI. Neuropathology was performed on P17. Compared to the control group (n = 6), the LPS + HI group (n = 10) but not the NS + HI group (n = 10) had significantly increased cortical damage (Nissl staining, upper panel), markedly reduced myelination (MBP, middle panel), and increased astrogliosis in the ipsilateral hemisphere (GFAP, lower panel). (B) At 24 hours post-insult, immunohistochemistry revealed that the LPS + HI group (n = 10) had significant increases in Iba1-positive microglia and IgG extravasation than the NS + HI (n = 10) and control (n = 6) groups. (C) LPS injection before HI did not up-regulate TNF-α expression compared to NS injection (upper panel). TNF-α levels were significantly increased at 3 hours, and especially at 24 hours after LPS-sensitized HI (lower panel). n = 4 experiments. (D) The LPS + HI group had significantly higher levels of TNF-α (upper panel) and cleaved caspase-3 (lower panel) than the NS + HI group at 24 hours post-insult. n = 6 experiments. Scale bar = 200 μm for MBP and = 100 μm for others. Inset scale bar = 10 μm in (B). Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; MBP, myelin basic protein.
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Fig2: Lipopolysaccharide (LPS)-sensitized hypoxic-ischemic (HI) injury induced up-regulation of neuroinflammation, blood–brain barrier damage and cell apoptosis. (A) P5 mouse pups received normal saline (NS) or LPS (0.05 mg/kg) injection before 30-minute HI. Neuropathology was performed on P17. Compared to the control group (n = 6), the LPS + HI group (n = 10) but not the NS + HI group (n = 10) had significantly increased cortical damage (Nissl staining, upper panel), markedly reduced myelination (MBP, middle panel), and increased astrogliosis in the ipsilateral hemisphere (GFAP, lower panel). (B) At 24 hours post-insult, immunohistochemistry revealed that the LPS + HI group (n = 10) had significant increases in Iba1-positive microglia and IgG extravasation than the NS + HI (n = 10) and control (n = 6) groups. (C) LPS injection before HI did not up-regulate TNF-α expression compared to NS injection (upper panel). TNF-α levels were significantly increased at 3 hours, and especially at 24 hours after LPS-sensitized HI (lower panel). n = 4 experiments. (D) The LPS + HI group had significantly higher levels of TNF-α (upper panel) and cleaved caspase-3 (lower panel) than the NS + HI group at 24 hours post-insult. n = 6 experiments. Scale bar = 200 μm for MBP and = 100 μm for others. Inset scale bar = 10 μm in (B). Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; MBP, myelin basic protein.

Mentions: Therefore, 0.05-mg/kg LPS was chosen for the following LPS-sensitized HI experiments on brain injury. On P17, the LPS + HI group had significantly more cortical damage, white matter injury with decreased MBP expression, and increased astrogliosis than the control and NS + HI groups (Figure 2A).Figure 2


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)

Lipopolysaccharide (LPS)-sensitized hypoxic-ischemic (HI) injury induced up-regulation of neuroinflammation, blood–brain barrier damage and cell apoptosis. (A) P5 mouse pups received normal saline (NS) or LPS (0.05 mg/kg) injection before 30-minute HI. Neuropathology was performed on P17. Compared to the control group (n = 6), the LPS + HI group (n = 10) but not the NS + HI group (n = 10) had significantly increased cortical damage (Nissl staining, upper panel), markedly reduced myelination (MBP, middle panel), and increased astrogliosis in the ipsilateral hemisphere (GFAP, lower panel). (B) At 24 hours post-insult, immunohistochemistry revealed that the LPS + HI group (n = 10) had significant increases in Iba1-positive microglia and IgG extravasation than the NS + HI (n = 10) and control (n = 6) groups. (C) LPS injection before HI did not up-regulate TNF-α expression compared to NS injection (upper panel). TNF-α levels were significantly increased at 3 hours, and especially at 24 hours after LPS-sensitized HI (lower panel). n = 4 experiments. (D) The LPS + HI group had significantly higher levels of TNF-α (upper panel) and cleaved caspase-3 (lower panel) than the NS + HI group at 24 hours post-insult. n = 6 experiments. Scale bar = 200 μm for MBP and = 100 μm for others. Inset scale bar = 10 μm in (B). Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; MBP, myelin basic protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Lipopolysaccharide (LPS)-sensitized hypoxic-ischemic (HI) injury induced up-regulation of neuroinflammation, blood–brain barrier damage and cell apoptosis. (A) P5 mouse pups received normal saline (NS) or LPS (0.05 mg/kg) injection before 30-minute HI. Neuropathology was performed on P17. Compared to the control group (n = 6), the LPS + HI group (n = 10) but not the NS + HI group (n = 10) had significantly increased cortical damage (Nissl staining, upper panel), markedly reduced myelination (MBP, middle panel), and increased astrogliosis in the ipsilateral hemisphere (GFAP, lower panel). (B) At 24 hours post-insult, immunohistochemistry revealed that the LPS + HI group (n = 10) had significant increases in Iba1-positive microglia and IgG extravasation than the NS + HI (n = 10) and control (n = 6) groups. (C) LPS injection before HI did not up-regulate TNF-α expression compared to NS injection (upper panel). TNF-α levels were significantly increased at 3 hours, and especially at 24 hours after LPS-sensitized HI (lower panel). n = 4 experiments. (D) The LPS + HI group had significantly higher levels of TNF-α (upper panel) and cleaved caspase-3 (lower panel) than the NS + HI group at 24 hours post-insult. n = 6 experiments. Scale bar = 200 μm for MBP and = 100 μm for others. Inset scale bar = 10 μm in (B). Values are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor molecule-1; MBP, myelin basic protein.
Mentions: Therefore, 0.05-mg/kg LPS was chosen for the following LPS-sensitized HI experiments on brain injury. On P17, the LPS + HI group had significantly more cortical damage, white matter injury with decreased MBP expression, and increased astrogliosis than the control and NS + HI groups (Figure 2A).Figure 2

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