Shun Fen Tzeng

Tumor necrosis factor-α and interleukin-18 modulate neuronal cell fate in embryonic neural progenitor culture

Neural progenitor cells (NPCs) in developing and adult CNS are capable of giving rise to various neuronal and glial cell populations. Neurogenesis in the adult hippocampus has been found to be inhibited by a proinflammatory cytokine, interleukin-6 (IL-6), suggesting that activated microglia in the inflamed brain may control neurogenesis. Yet, little is known about the effect of microglia-derived factors on the cell fate of embryonic NPCs. In this study, we show that neurons with betaIII-tubulin immunoreactivity in the NPC culture were reduced by the condition media collected from microglia treated with endotoxin lipopolysaccharide (LPS/M-CM). Treatment with pentoxifylline (PTX), an inhibitor for tumor necrosis factor-alpha (TNF-alpha) secretion from LPS-activated microglia, blocked the reduction of betaIII-tubulin+ cells in NPC culture. Furthermore, treatment of NPCs with interleukin-18 (IL-18), a recently discovered proinflammatory cytokine, also decreased the number of betaIII-tubulin+ cells in a dose- and time-dependent manner. Surprisingly, we also observed that the remaining betaIII-tubulin+ cells in the LPS/M-CM-treated culture exhibited more branching neurites. Thus, the activated microglia-derived cytokines, TNF-alpha and IL-18, may either inhibit the neuronal differentiation or induce neuronal cell death in the NPC culture, whereas these cells may also produce factors to improve the neurite branching in the NPC culture.

Prostaglandins and Cyclooxygenases in Glial Cells During Brain Inflammation

Many brain disorders such as Parkinsons disease, Alzheimers disease, amyotrophic lateral sclerosis (ALS), Huntington, stroke, head trauma, and infection, are associated with inflammation that is involved in neuropathologenesis and hyperalgesis. Microglia and astrocytes act as immune cells in the inflamed brain. Both cell types, but especially microglia, are thought to contribute to the onset of inflammation in many brain diseases by producing deleterious proinflammatory mediators. Prostaglandins (PGs), which are critical mediators of physiologic processes and inflammation, are largely produced by activated microglia and reactive astrocytes during brain inflammation. These compounds are converted from arachnoidic acid (AA) by two isoforms of the cyclooxygenase (COX) enzyme, namely COX-1 and COX-2. In particular, the action of COX-2 and PGs in CNS inflammation has gained much attention recently. PGs have been found to act neuroprotectively by elevating intracellular cAMP levels in neurons. These molecules also function as anti-inflammatory molecules to reduce the production of nitric oxide and proinflammatory cytokines, and to increase the expression of anti-inflammatory cytokines. However, accumulating evidence also shows that COX inhibitors alleviate various types of brain damage via suppressing inflammatory reactions. Accordingly, the roles of two COX enzymes in mediating inflammation and anti-inflammation have recently been debated. We provide here a review of recent findings indicating that the reciprocal interaction of glial cell activation, COX enzymes and PGs mediates neurodegeneration and neuroprotection during brain inflammation. In addition, the mechanism by which PGs mediate signaling is discussed.

Neuroprotection of glial cell line-derived neurotrophic factor in damaged spinal cords following contusive injury

Glial cell line‐derived neurotrophic factor (GDNF) acts as a potent survival factor for many neuronal populations, including spinal motoneurons, indicating the therapeutic promise of GDNF for neurological disorders. Injury to spinal cord (SCI) triggers processes destructive to ascending sensory and descending motor conduction and extends tissue loss, thereby leading to permanent behavioral dysfunction. In this study, we attempted to examine whether GDNF protects neurons from SCI and subsequently lessens locomotor deficit in SCI rats. We utilized the NYU weight‐drop device developed at New York University to induce spinal cord contusion at the T9–10 spinal segment. After SCI, GDNF was administrated into the cord 1–2 mm rostral and caudal to the epicenter. Animals receiving GDNF treatment showed significant improvement over phosphate‐buffered saline (PBS)‐treated controls on the Basso Beattie Bresnahan (BBB) locomotor rating scale (P < 0.01‐0.001). GDNF treatment increased the remaining neuronal fibers with calcitonin gene‐related peptide, neurofilament, and growth‐associated protein 43 immunoreactivity in injured spinal tissues compared with PBS‐treated controls. Moreover, treatment with GDNF caused approximately 50% cell survival in the contused spinal cord tissues. Examination of signal transduction triggered by GDNF indicated that GDNF injection transiently induced activation of the mitogen‐activated protein (MAP) kinase pathway in the spinal cord. Additionally, an up‐regulation of anti‐apoptotic Bcl‐2 levels in the contusive center of the damaged spinal cord was observed 24 hr post‐GDNF injection. Together our results show that GDNF exerts behavioral and anatomic neuroprotection following SCI. Additionally, GDNF‐activated MAP kinase and Bcl‐2 signaling may contribute to neuronal survival after spinal cord contusion.

Capnellene, a natural marine compound derived from soft coral, attenuates chronic constriction injury-induced neuropathic pain in rats

Background and purpose:  Natural compounds obtained from marine organisms have received considerable attention as potential sources of novel drugs for treatment of human inflammatory diseases. Capnellene, isolated from the marine soft coral Capnella imbricate, 4,4,6a‐trimethyl‐3‐methylene‐decahydro‐cyclopenta[]pentalene‐2,3a‐diol (GB9) exhibited anti‐inflammatory actions on activated macrophages in vitro. Here we have assessed the anti‐neuroinflammatory properties of GB9 and its acetylated derivative, acetic acid 3a‐hydroxy‐4,4,6a‐trimethyl‐3‐methylene‐decahydro‐cyclopenta[]pentalen‐2‐yl ester (GB10).

Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury

Glial cell line derived neurotrophic factor (GDNF) induces neuronal survival and tissue repair after spinal cord injury (SCI). A continuous GDNF supply is believed to gain greater efficacy in the neural restoration of the injured spinal cord. Accordingly, nanovehicle formulation for their efficient delivery and sustained release in injured spinal cord was examined. We first used fluorescence-labeled bovine serum albumin (FBSA) loaded in biodegradable poly(lactic acid-co-glycolic acid) (PLGA) for intraspinal administration after SCI and for in vitro study. Our results showed that the preservation of PLGA-FBSA was observed in the injured spinal cord at 24h, and PLGA-FBSA nanoparticles were well absorbed by neurons and glia, indicating that PLGA as a considerable nanovehicle for the delivery of neuroprotective polypeptide into injured spinal cord. Furthermore, intraspinal injection of GDNF encapsulated in PLGA (PLGA-GDNF) nanoparticles into the injured spinal cord proximal to the lesion center had no effect on gliosis when compared to that observed in SCI rats receiving PLGA injection. However, local administration of PLGA-GDNF effectively preserved neuronal fibers and led to the hindlimb locomotor recovery in rats with SCI, providing a potential strategy for the use of PLGA-GDNF in the treatment of SCI.

Tumor necrosis factor-alpha modulates the proliferation of neural progenitors in the subventricular/ventricular zone of adult rat brain.

Little is known about the response of neural progenitors to inflammation following injuries of the central nervous system. In combination with bromodeoxyuridine (BrdU) intraperitoneally (i.p.) injected, tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that increased ED1+ activated microglia/macrophage population at injured sites, was administrated into adult rat brains. No difference in the immunostaining for proliferating cell nuclear antigen (PCNA) was observed in the subventricular/ventricular zone (SVZ/VZ) between TNF-alpha injected sites and controls. However, BrdU+ cells were apparently observed in the SVZ/VZ proximal to TNF-alpha injected site, and the number of BrdU+ cells increased at 6 and 24 h post injection. Since cell apoptosis was rarely found in the SVZ/VZ after TNF-alpha injection, these observations suggest that the diffusible TNF-alpha may directly and/or indirectly modulate the proliferation of neural progenitors.

TNF‐α/IFN‐γ‐induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2‐evoked cAMP/PKA and intracellular calcium signaling

Astrocytes, the most abundant glia in the central nervous system (CNS), produce a large amount of prostaglandin E2 (PGE2) in response to proinflammatory mediators after CNS injury. However, it is unclear whether PGE2 has a regulatory role in astrocytic activity under the inflamed condition. In the present work, we showed that PGE2 increased inducible nitric oxide synthase (iNOS) production by tumor necrosis factor‐α and interferon‐γ (T/I) in astrocytes. Pharmacological and RNA interference approaches further indicated the involvement of the receptor EP2 in PGE2‐induced iNOS upregulation in T/I‐treated astrocytes. Quantitative real‐time polymerase chain reaction and gel mobility shift assays also demonstrated that PGE2 increased iNOS transcription through EP2‐induced cAMP/protein kinase A (PKA)‐dependent pathway. Consistently, the effect of EP2 was significantly attenuated by the PKA inhibitor KT‐5720 and partially suppressed by the inhibitor (SB203580) of p38 mitogen‐activated protein kinase (p38MAPK), which serves as one of the downstream components of the PKA‐dependent pathway. Interestingly, EP2‐mediated PKA signaling appeared to increase intracellular Ca2+ release through inositol triphosphate (IP3) receptor activation, which might in turn stimulate protein kinase C (PKC) activation to promote iNOS production in T/I‐primed astrocytes. By analyzing the expression of astrocytic glial fibrillary acidic protein (GFAP), we found that PGE2 alone only triggered the EP2‐induced cAMP/PKA/p38MAPK signaling pathway in astrocytes. Collectively, PGE2 may enhance T/I‐induced astrocytic activation by augmenting iNOS/NO production through EP2‐mediated cross‐talk between cAMP/PKA and IP3/Ca2+ signaling pathways.

Inhibition of cadmium‐induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd2+) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS‐supporting cells, are resistant to Cd2+‐induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl2 for 24 h at concentrations higher than 20 µM substantially induced astrocytic cytotoxicity, which also resulted from long‐term exposure to 5 µM of CdCl2. Intracellular calcium levels were found to rapidly increase after the addition of CdCl2 into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd2+‐treated astrocytes. Adenovirus‐mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd2+‐exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd2+ exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd2+‐induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd2+‐primed astrocytes. This information provides a useful approach for treating Cd2+‐induced CNS neurological disorders. J. Cell. Biochem. 103: 825–834, 2008.

Early-life fluoxetine exposure reduced functional deficits after hypoxic-ischemia brain injury in rat pups ☆

Neuroplasticity after perinatal programming may allow for neuroprotection against hypoxic-ischemia (HI) at birth. The cAMP response element-binding protein (CREB) is a key mediator of stimulus-induced nuclear responses that underlie survival, memory and plasticity of nervous system. Chronic treatment of fluoxetine, a selective serotonin reuptake inhibitor, can upregulate CREB activation in the hippocampus. We examined whether fluoxetine administration before HI may protect against neonatal HI brain injury through CREB-mediated mechanisms. We found that low-dose fluoxetine pretreatment in a neonatal HI brain injury model significantly reduced functional deficits at adulthood. The neuroprotective mechanisms were associated with increased CREB phosphorylation and increased brain-derived neurotrophic factor and synapsin I mRNA expression in the hippocampus. Neurogenesis also increased because of greater precursor cell survival in the hippocampal dentate gyrus. These findings suggest that functional deficits after HI in the developing brain can be reduced by agents that enhance neural plasticity and neurogenesis through CREB activation.

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9-T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.