Cheng-Yi Chang

Prognostic and clinical implication of IL-6 expression in glioblastoma multiforme

Interleukin-6 (IL-6) is frequently produced in gliomas and has been implicated as a mediator of growth control in several human neoplasms. In this study, IL-6 expression was examined in 11 surgically resected glioblastomas and a cell line U87MG by immunohistochemical staining and quantitative real-time RT-PCR. The relationships between IL-6 expression level and clinical presentation, survival, imaging findings, age and preoperative Karnofsky performance status were analyzed. The median survival times were 16 months in patients with negative IL-6 expression and 7 months in those with positive IL-6 expression (P = 0.075). Three of these patients with a relatively longer survival time (> 1 year) did not express IL-6 in the tumor. Relatively more severe peri-focal edema on imaging was also noted in the glioblastomas with IL-6 expression. IL-6 was also found in the cytoplasm of endothelial cells of newly formed vessels and infiltrating inflammatory cells. These preliminary results implicate IL-6 expression as a possible prognostic indicator in glioblastoma. This cytokine may also play a role in tissue edema, angiogenesis and inflammation of this tumor, but whether IL-6 expression promotes malignancy is uncertain.

Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats.

Tetramethylpyrazine (TMP) has been used to treat ischemic stroke. However, scientific evidence related to its effectiveness or precise modes of neuroprotective action is largely unclear. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of TMP on intracerebral cellular inflammatory response in a rat model of permanent cerebral ischemia. TMP exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, and edema. The results of immunohistochemistry, enzymatic assay, Western blot, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometric analysis revealed that TMP reduced the percentages of activated macrophages/microglia and infiltrative lymphocytes, neutrophils, and macrophages and pro-inflammatory cytokine expression after cerebral ischemia. In parallel with these immunosuppressive phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules and transcription factors. Another finding in this study was that the anti-inflammatory and neuroprotective effects of TMP were accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in ipsilateral neurons and macrophages/microglia after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous defense capacity and the attenuation of the extent and composition percentage of the major cellular inflammatory responses via targeting of macrophages/microglia by elevating Nrf2/HO-1 expression might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-α signaling and contributes to neuronal death

The substantial activation of microglia in Japanese encephalitis virus (JEV)‐induced Japanese encephalitis found in numerous studies demonstrates that the disease pathogenesis involves bystander damage caused by microglia‐released mediators. Previously, we reported that microglia synthesized and secreted bioactive mediators with neurotoxic potential into the cultured supernatants in response to JEV infection. In this study, we found that the supernatants of JEV‐infected microglia caused MK801‐inhibitable neuronal damage in cultured neurons, indicating a potential excitotoxic mechanism. Infection with JEV was found to elicit the extracellular glutamate accumulation from microglia but not from neuron and astrocyte cultures. The glutaminase inhibitor 6‐diazo‐5‐oxo‐L‐norleucine, cystine/glutamate antiporter inhibitor α‐aminoadipic acid, and the gap junction inhibitor carbenoxolone reduced JEV infection‐induced microglial glutamate release and neurotoxicity. We further demonstrated that tumor necrosis factor‐alpha (TNF‐α) was a key cytokine which stimulated extensive microglial glutamate release by up‐regulating glutaminase expression via signals involving protein kinase C, cAMP responsive element‐binding protein, and CAAT‐enhancer‐binding protein‐beta. Although the elevated expression of excitatory amino acid transporter 1 and 2 was observed in JEV‐infected cells, the glutamate uptake activity was significantly inhibited by TNF‐α. The JEV infection‐induced alterations, such as the extracellular glutamate release and glutamate‐mediated excitoneurotoxicity, also occurred in neuron/glia cultures. Our findings support a potential link between neuroinflammation and the development of excitotoxic neuronal injury in Japanese encephalitis. The link between neuroinflammation and excitotoxic death may involve a mechanism in which TNF‐α released by microglia plays a facilitory role in glutamate excitoneurotoxicity via up‐regulation of glutamate synthesis and down‐regulation of glutamate uptake.

Docosahexaenoic acid reduces cellular inflammatory response following permanent focal cerebral ischemia in rats.

Cellular inflammatory response plays an important role in ischemic brain injury and anti-inflammatory treatments in stroke are beneficial. Dietary supplementation with docosahexaenoic acid (DHA) shows anti-inflammatory and neuroprotective effects against ischemic stroke. However, its effectiveness and its precise modes of neuroprotective action remain incompletely understood. This study provides evidence of an alternative target for DHA and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of 3 consecutive days of DHA preadministration on circulating and intracerebral cellular inflammatory responses in a rat model of permanent cerebral ischemia. DHA exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, edema and blood-brain barrier disruption. The results of enzymatic assay, Western blot, real-time reverse transcriptase polymerase chain reaction and flow cytometric analysis revealed that DHA reduced central macrophages/microglia activation, leukocyte infiltration and pro-inflammatory cytokine expression and peripheral leukocyte activation after cerebral ischemia. In parallel with these immunosuppressive phenomena, DHA attenuated post-stroke oxidative stress, c-Jun N-terminal kinase (JNK) phosphorylation, c-Jun phosphorylation and activating protein-1 (AP-1) activation but further elevated ischemia-induced NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) expression. DHA treatment also had an immunosuppressive effect in lipopolysaccharide/interferon-γ-stimulated glial cultures by attenuating JNK phosphorylation, c-Jun phosphorylation and AP-1 activation and augmenting Nrf2 and HO-1 expression. In summary, we have shown that DHA exhibited neuroprotective and anti-inflammatory effects against ischemic brain injury and these effects were accompanied by decreased oxidative stress and JNK/AP-1 signaling as well as enhanced Nrf2/HO-1 expression.

Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

ABSTRACT Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.

Endothelial Japanese encephalitis virus infection enhances migration and adhesion of leukocytes to brain microvascular endothelia via MEK-dependent expression of ICAM1 and the CINC and RANTES chemokines

Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis‐associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV‐infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule‐1, cytokine‐induced neutrophil chemoattractant‐1, and regulated‐upon‐activation normal T‐cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal‐regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up‐regulating cytosolic phospholipase A2, NF‐κB, and cAMP response element‐binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier‐disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.

TNF-α and IL-1β mediate Japanese encephalitis virus-induced RANTES gene expression in astrocytes

Infection with Japanese encephalitis virus (JEV) causes neuroinfection and neuroinflammation characterized by profound neuronal destruction/dysfunction, concomitant microgliosis/astrogliosis, and production of various molecules that initiate the recruitment of immune cells to the sites of infection. Previously, we reported that glial cells expressed RANTES (regulated upon activation, normal T cell expressed and secreted) with chemotactic activity in response to JEV infection. In this study, we further demonstrated that JEV-infected microglia had an additional activity in regulating RANTES production. Both astrocytes and microglia responded to JEV infection by releasing RANTES through a process likely related to viral replication. Independent of infectious virus, supernatants of JEV-infected microglia, but not JEV-infected astrocytes, caused additional RANTES production from astrocytes. Antibody neutralization studies suggested the potential involvement of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in mediating additional RANTES production. Treatment of astrocyte cultures with TNF-α and IL-1β caused activation of several signaling molecules and transcription factors crucial to RANTES gene expression, including reactive oxygen species, extracellular signal-regulated kinase, NF-κB, and NF-IL6, increased RANTES gene promoter activity, and provoked RANTES production. As with RANTES, neutralization of bioactive TNF-α and IL-1β caused an attenuation of chemotactic activity from supernatants of mixed glia containing astrocytes and microglia during the course of JEV infection. In conclusion, TNF-α and IL-1β produced by JEV-infected microglia might trigger another mechanism which induces a secondary wave of RANTES gene expression by activating astrocytes. The released RANTES from glial cells might play a role in the recruitment of immune cells during JEV infection.

Disruption of in vitro endothelial barrier integrity by Japanese encephalitis virus‐Infected astrocytes

Blood–brain barrier (BBB) characteristics are induced and maintained by crosstalk between brain microvascular endothelial cells and neighboring cells. Using in vitro cell models, we previously found that a bystander effect was a cause for Japanese encephalitis‐associated endothelial barrier disruption. Brain astrocytes, which neighbor BBB endothelial cells, play roles in the maintenance of BBB integrity. By extending the scope of relevant studies, a potential mechanism has been shown that the activation of neighboring astrocytes could be a cause of disruption of endothelial barrier integrity during the course of Japanese encephalitis viral (JEV) infection. JEV‐infected astrocytes were found to release biologically active molecules that activated ubiquitin proteasome, degraded zonula occludens‐1 (ZO‐1) and claudin‐5, and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. JEV infection caused astrocytes to release vascular endothelial growth factor (VEGF), interleukin‐6 (IL‐6), and matrix metalloproteinases (MMP‐2/MMP‐9). Our data demonstrated that VEGF and IL‐6 released by JEV‐infected astrocytes were critical for the proteasomal degradation of ZO‐1 and the accompanying disruption of endothelial barrier integrity through the activation of Janus kinase‐2 (Jak2)/signal transducer and activator of transcription‐3 (STAT3) signaling as well as the induction of ubiquitin–protein ligase E3 component, n‐recognin‐1 (Ubr 1) in endothelial cells. MMP‐induced endothelial barrier disruption was accompanied by MMP‐mediated proteolytic degradation of claudin‐5 and ubiquitin proteasome‐mediated degradation of ZO‐1 via extracellular VEGF release. Collectively, these data suggest that JEV infection could activate astrocytes and cause release of VEGF, IL‐6, and MMP‐2/MMP‐9, thereby contributing, in a concerted action, to the induction of Japanese encephalitis‐associated BBB breakdown. GLIA 2015;63:1915–1932

Prenatal buprenorphine exposure decreases neurogenesis in rats.

Perinatal opioid exposure has a negative effect on neurogenesis and produces neurological consequences. However, its mechanisms of action are incompletely understood. Buprenorphine, a mixed opioid agonist/antagonist, is an alternative medication for managing pregnant opioid addicts. This study provides evidence of decreased neurogenesis and depression-like consequences following prenatal exposure to buprenorphine and sheds light on mechanisms of action in a rat model involving administration of intraperitoneal injection to pregnant rats starting from gestation day 7 and lasting for 14 days and a cultured neurosphere model. Results of forced swimming test and tail suspension test showed that pups at postnatal day 21 had worse parameters of depression-like neurobehaviors, independent of gender. Neurobehavioral changes were accompanied by reduction of neuronal composition, biochemical parameters of neural stem/progenitor cells, brain-derived neurotrophic factor (BDNF) expression, tropomyosin-related kinase receptor type B phosphorylation, protein kinase A (PKA) activity, and cAMP response element-binding protein phosphorylation. Results of parallel cell studies further demonstrated a negative impact of buprenorphine on cultured neurospheres, including proliferation, differentiation, BDNF expression and signaling, and PKA activity. Taken together, our results suggest that prenatal exposure to buprenorphine might result in depression-like phenotypes associated with impaired BDNF action and decreased neurogenesis in the developing brain of weanlings.

Signaling cascades mediate astrocyte death induced by zinc.

Zinc overload is known to cause the death of neural cells. Although the activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)) have been implicated in zinc-induced astrocyte death, the detailed mechanisms of their activation and upstream regulatory cascades are incompletely understood. Here, we report that protein kinase C (PKC)- and Src-related Ras/Raf/ERK cascades and ERK-associated cPLA(2) participate in astrocyte death caused by ZnCl(2). Sustained exposure to ZnCl(2) caused damage to astrocytes in a time- and concentration-dependent manner. The cell death caused by ZnCl(2) was accompanied by increased reactive oxygen species (ROS) generation, PKC-α membrane association, Src phosphorylation, Ras membrane association, Raf phosphorylation, ERK phosphorylation, and cPLA(2) activation, and decreased protein phosphatase activity. Pharmacological studies revealed that these activations/inactivations all contributed to ZnCl(2)-induced astrocyte death. ROS, such as superoxide, appear to be a key trigger in response to ZnCl(2) treatment in astrocytes because of the attenuations in protein phosphatase inhibition, signaling activation, and cell death by antioxidant treatments. Mechanistic studies had suggested that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK were potential signals linking zinc and cPLA(2). These observations indicated that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK signaling and cPLA(2) were actively involved in zinc-induced astrocyte damage.