Chien Jung Ho

cAMP response element-binding protein activation in ligation preconditioning in neonatal brain

Perinatal hypoxic‐ischemic (HI) brain injury is a major cause of permanent neurological dysfunction in children. An approach to study the treatment of neonatal HI encephalopathy that allows for neuroprotection is to investigate the states of tolerance to HI. Twenty‐four‐hour carotid‐artery ligation preconditioning established by delaying the onset of hypoxia for 24 hours after permanent unilateral carotid ligation rats markedly diminished the cerebral injury, however, the signaling mechanisms of this carotid‐artery ligation preconditioning in neonatal rats remain unknown. Ligation of the carotid artery 24 hours before hypoxia provided complete neuroprotection and produced improved performance on the Morris water maze compared with ligation performed 1 hour before hypoxia. Carotid artery ligation 6 hours before hypoxia produced intermediate benefit. The 24‐hour carotid‐artery ligation preconditioning was associated with a robust and sustained activation of a transcription factor, the cAMP response element–binding protein (CREB), on its phosphorylation site on Ser133. Intracerebroventricular infusions of antisense CREB oligodeoxynucleotides significantly reduced the 24‐hour carotid‐artery ligation–induced neuroprotection effects by decreasing CREB expressions. Pharmacological activation of the cAMP‐CREB signaling with rolipram 24 hours before hypoxia protected rat pups at behavioral and pathological levels by sustained increased CREB phosphorylation. This study suggests that 24‐hour carotid‐artery ligation preconditioning provides important mechanisms for potential pharmacological preconditioning against neonatal HI brain injury. Ann Neurol, 2004

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

BackgroundWhite 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 30u2009weeks 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.MethodsP2 pups received LPS (0.05u2009mg/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.ResultsP2 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 24u2009h 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 24u2009h 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 24u2009h 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.ConclusionsLPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.

Overweight worsens apoptosis, neuroinflammation and blood-brain barrier damage after hypoxic ischemia in neonatal brain through JNK hyperactivation.

BackgroundApoptosis, neuroinflammation and blood-brain barrier (BBB) damage affect the susceptibility of the developing brain to hypoxic-ischemic (HI) insults. c-Jun N-terminal kinase (JNK) is an important mediator of insulin resistance in obesity. We hypothesized that neonatal overweight aggravates HI brain damage through JNK hyperactivation-mediated upregulation of neuronal apoptosis, neuroinflammation and BBB leakage in rat pups.MethodsOverweight (OF) pups were established by reducing the litter size to 6, and control (NF) pups by keeping the litter size at 12 from postnatal (P) day 1 before HI on P7. Immunohistochemistry and immunoblotting were used to determine the TUNEL-(+) cells and BBB damage, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and phospho-JNK and phospho-BimEL levels. Immunofluorescence was performed to determine the cellular distribution of phospho-JNK.ResultsCompared with NF pups, OF pups had a significantly heavier body-weight and greater fat deposition on P7. Compared with the NF-HI group, the OF-HI group showed significant increases of TUNEL-(+) cells, cleaved levels of caspase-3 and PARP, and ED1-(+) activated microglia and BBB damage in the cortex 24 hours post-HI. Immunofluorescence of the OF-HI pups showed that activated-caspase 3 expression was found mainly in NeuN-(+) neurons and RECA1-(+) vascular endothelial cells 24 hours post-HI. The OF-HI group also had prolonged escape latency in the Morris water maze test and greater brain-volume loss compared with the NF-HI group when assessed at adulthood. Phospho-JNK and phospho-BimEL levels were higher in OF-HI pups than in NF-HI pups immediately post-HI. JNK activation in OF-HI pups was mainly expressed in neurons, microglia and vascular endothelial cells. Inhibiting JNK activity by AS601245 caused more attenuation of cleaved caspase-3 and PARP, a greater reduction of microglial activation and BBB damage post-HI, and significantly reduced brain damage in OF-HI than in NF-HI pups.ConclusionsNeonatal overweight increased HI-induced neuronal apoptosis, microglial activation and BBB damage, and aggravated HI brain damage in rat pups through JNK hyperactivation.

Moderate Dietary Restriction Reduces p53-Mediated Neurovascular Damage and Microglia Activation After Hypoxic Ischemia in Neonatal Brain

Background and Purpose— Neurovascular damage, including neuronal apoptosis and blood–brain barrier (BBB) damage, and microglia activation account for the hypoxic-ischemia (HI) susceptibility in neonatal brain. The p53 upregulation is involved in apoptosis, endothelial cell damage, and microglia activation. We hypothesized that underweight induced by dietary restriction (DR) protects against HI in rat pups by attenuating p53-mediated neurovascular damage. Methods— Male rat pups were grouped as normal litter (NL) size (12 pups/dam), DR (18 pups/dam), and extreme DR (24 pups/dam) from postnatal day 1 and subjected to HI on postnatal day 7. Immunohistochemistry and immunoblotting were used to determine p53, phospho-murine double minute-2, caspases, BBB damage and microglia activation, and immunofluorescence to determine the cellular distribution of p53. Pharmacological approaches were used to regulate p53. Results— The NL, DR, and extreme DR pups had similar TUNEL-positive cells and caspases on postnatal day 7 and comparable learning performance at adulthood. After HI, the DR-HI, but not extreme DR-HI, pups had significantly lower p53, higher phospho-murine double minute-2, lower cleaved caspases, less BBB damage and microglia activation, and less brain volume loss than NL-HI pups. In NL-HI pups, p53 expression was located mainly in the neurons, endothelial cells, and microglia. The p53 blockage by pifithrin-&agr; in NL-HI pups decreased apoptosis, BBB damage, and microglia activation, and was neuroprotective. In contrast, upregulating p53 by nutlin-3 in DR-HI pups increased apoptosis, BBB damage, and microglia activation, and worsened brain damage. Conclusions— Moderate DR, but not extreme DR, reduces p53-mediated neurovascular damage after HI and confers long-term protection in neonatal brain.

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

BackgroundHypoxic-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.ObjectiveTo determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain.MethodsPostpartum (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.ResultsThe LPSu2009+u2009HI 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 24xa0hours post-insult, and showed more cortical and white matter injury on P17 than the control and NSu2009+u2009HI 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.ConclusionTNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

Background and Purpose— The neurovascular unit is a major target of hypoxia-ischemia (HI) injury in the neonatal brain. Although neurons are the cellular target of ischemic preconditioning (IP), vessel tolerance also contributes greatly to protection. Nerves and vessels cross-talk and use common signals during development. Cellular inhibitor of apoptosis 1 (cIAP1) is an important regulator that inhibits apoptosis. This study hypothesized that cIAP1 is a shared molecule underlying IP-mediated neurovascular protection against HI in the neonatal brain. Methods— In vivo IP was induced by 2-hour reversible occlusion of right carotid artery 24 hours before HI on postpartum day 7 in rat pups. In vitro oxygen–glucose deprivation (OGD) preconditioning was established in SH-SY5Y neuronal cells and in human microvascular endothelial cell-1 vascular endothelial cells. cIAP1 expression was inhibited by cIAP1 small interfering RNA in vivo or by lentivirus-mediated short hairpin RNA in vitro, or was upregulated by the lentiviral expression system. Results— IP reduced apoptosis, selectively increased cIAP1 in neurons and vascular endothelial cells, and provided long-term neuroprotection against HI. Intracerebroventricular delivery of cIAP1 small interfering RNA significantly attenuated IP-mediated cIAP1 upregulation and neuroprotection in vivo. In vitro, OGD preconditioning induced cIAP1 and protected against OGD cell death in SH-SY5Y neuronal and human microvascular endothelial cells-1. Knockdown of cIAP1 by lentivirus-mediated short hairpin RNA decreased the protective effect of OGD preconditioning in SH-SY5Y and human microvascular endothelial cell-1, whereas overexpression of cIAP1 by lentivirus protected against OGD in these cells. Conclusions— cIAP1 is a shared molecule underlying IP-induced protection in neurons and vascular endothelial cells against HI in the neonatal brain.

Cerebral microvascular damage occurs early after hypoxia-ischemia via nNOS activation in the neonatal brain

Microvascular injury early after hypoxic ischemia (HI) may contribute to neonatal brain damage. N-methyl-D-aspartate receptor overstimulation activates neuronal nitric oxide synthases (nNOS). We hypothesized that microvascular damage occurs early post-HI via nNOS activation and contributes to brain injury. Postpartum day-7 rat pups were treated with 7-nitroindazole (7-NI) or aminoguanidine (AG) before or after HI. Electron microscopy was performed to measure neuronal and endothelial cell damage. There were vascular lumen narrowing at 1u2009hour, pyknotic neurons at 3u2009hours, and extensive neuronal damage and loss of vessels at 24u2009hours post HI. Early after reoxygenation, there were neurons with heterochromatic chromatin and endothelial cells with enlarged nuclei occluding the lumen. There was also increased 3-nitrotyrosin in the microvessels and decreased cerebral blood perfusion. 7-NI and AG treatment before hypoxia provided complete and partial neuroprotection, respectively. Early post-reoxygenation, the AG group showed significantly increased microvascular nitrosative stress, microvascular interruptions, swollen nuclei that narrowed the vascular lumen, and decreased cerebral perfusion. The 7-NI group showed significantly decreased microvascular nitrosative stress, patent vascular lumen, and increased cerebral perfusion. Our results indicate that microvascular damage occurs early and progressively post HI. Neuronal nitric oxide synthases activation contributes to microvascular damage and decreased cerebral perfusion early after reoxygenation and worsens brain damage.

Microglia retard dengue virus-induced acute viral encephalitis

Patients with dengue virus (DENV) infection may also present acute viral encephalitis through an unknown mechanism. Here, we report that encephalitic DENV-infected mice exhibited progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days post-infection. These symptoms were accompanied by CNS inflammation, neurotoxicity, and blood-brain barrier destruction. Microglial cells surrounding the blood vessels and injured hippocampus regions were activated by DENV infection. Pharmacologically depleting microglia unexpectedly increased viral replication, neuropathy, and mortality in DENV-infected mice. In microglia-depleted mice, the DENV infection-mediated expression of antiviral cytokines and the infiltration of CD8-positive cytotoxic T lymphocytes (CTLs) was abolished. DENV infection prompted the antigen-presenting cell-like differentiation of microglia, which in turn stimulated CTL proliferation and activation. These results suggest that microglial cells play a key role in facilitating antiviral immune responses against DENV infection and acute viral encephalitis.

Human Umbilical Vein Endothelial Cells Protect Against Hypoxic-Ischemic Damage in Neonatal Brain via Stromal Cell-derived Factor 1/C-X-C Chemokine Receptor Type 4

Background and Purpose— Agents that protect against neurovascular damage provide a powerful neuroprotective strategy. Human umbilical vein endothelial cells (HUVECs) may be used to treat neonates with hypoxic-ischemia (HI) because of its autologous capability. We hypothesized that peripherally injected HUVECs entered the brain after HI, protected against neurovascular damage, and provided protection via stromal cell–derived factor 1/C-X-C chemokine receptor type 4 pathway in neonatal brain. Methods— Postpartum day 7 rat pups received intraperitoneal injections of low-passage HUVEC-P4, high-passage HUVEC-P8, or conditioned medium before and immediately after HI. HUVECs were transfected with adenovirus-green fluorescent protein for cell tracing. Oxygen–glucose deprivation was established by coculturing HUVEC-P4 with mouse neuroblastoma neuronal cells (Neuro-2a) and with mouse immortalized cerebral vascular endothelial cells (b.End3). Results— HUVEC-P4–treated group had more blood levels of green fluorescent protein–positive cells than HUVEC-P8–treated group 3 hours postinjection. Intraperitoneally injected HUVEC-P4, but not HUVEC-P8, entered the cortex after HI and positioned closed to the neurons and microvessels. Compared with the condition medium–treated group, the HUVEC-P4–treated but not the HUVEC-P8–treated group showed significantly less neuronal apoptosis and blood–brain barrier damage and more preservation of microvessels in the cortex 24 hours after HI. On postpartum day 14, the HUVEC-P4–treated group showed significant neuroprotection compared with the condition medium–treated group. Stromal cell–derived factor 1 was upregulated in the ipsilateral cortex 3 hours after HI, and inhibiting the stromal cell–derived factor 1/C-X-C chemokine receptor type 4 reduced the protective effect of HUVEC-P4. In vitro transwell coculturing of HUVEC-P4 also significantly protected against oxygen–glucose deprivation cell death in neurons and endothelial cells. Conclusions— Cell therapy using HUVECs may provide a powerful therapeutic strategy in treating neonates with HI.

Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury

Perinatal cerebral hypoxic-ischemic (HI) injury damages the architecture of neurovascular units (NVUs) and results in neurological disorders. Here, we differentiated adipose-derived stem cells (ASCs) toward the progenitor of endothelial progenitor cells (EPCs) and neural precursor cells (NPCs) via microenvironmental induction and investigated the protective effect by transplanting ASCs, EPCs, NPCs, or a combination of EPCs and NPCs (E+N) into neonatal HI injured rat pups. The E+N combination produced significant reduction in brain damage and cell apoptosis and the most comprehensive restoration in NVUs regarding neuron number, normal astrocytes, and vessel density. Improvements in cognitive and motor functions were also achieved in injured rats with E+N therapy. Synergistic interactions to facilitate transmigration under in vitro hypoxic microenvironment were discovered with involvement of the neuropilin-1 (NRP1) signal in EPCs and the C-X-C chemokine receptor 4 (CXCR4) and fibroblast growth factor receptor 1 (FGFR1) signals in NPCs. Therefore, ASCs exhibit great potential for cell sources in endothelial and neural lineages to prevent brain from HI damage.