Spectral Modification by Operant Conditioning of Cortical Theta Suppression in Rats

OBJECTIVE: Brain activity is known to be voluntarily controllable by neurofeedback, a kind of electroencephalographic (EEG) operant conditioning. Although its efficacy in clinical effects has been reported, it is yet to be uncovered whether or how a specific band activity is controllable. Here, we examined EEG spectral profiles along with conditioning training of a specific brain activity, theta band (4–8 Hz) amplitude, in rats. METHODS: During training, the experimental group received electrical stimulation to the medial forebrain bundle contingent to suppression of theta activity, while the control group received stimulation non-contingent to its own band activity. RESULTS: In the experimental group, theta activity gradually decreased within the training session, while there was an increase of theta activity in the control group. There was a significant difference in theta activity during the sessions between the two groups. The spectral theta peak, originally located at 7 Hz, shifted further towards higher frequencies in the experimental group. CONCLUSION: Our results showed that an operant conditioning technique could train rats to control their specific EEG activity indirectly, and it may be used as an animal model for studying how neuronal systems work in human neurofeedback.

Axon Guidance Molecules Guiding Neuroinflammation

Axon guidance molecules (AGMs), such as Netrins, Semaphorins, and Ephrins, have long been known to regulate axonal growth in the developing nervous system. Interestingly, the chemotactic properties of AGMs are also important in the postnatal period, such as in the regulation of immune and inflammatory responses. In particular, AGMs play pivotal roles in inflammation of the nervous system, by either stimulating or inhibiting inflammatory responses, depending on specific ligand-receptor combinations. Understanding such regulatory functions of AGMs in neuroinflammation may allow finding new molecular targets to treat neurodegenerative diseases, in which neuroinflammation underlies aetiology and progression.

Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases.

Deficiency of Lipocalin-2 Promotes Proliferation and Differentiation of Osteoclast Precursors via Regulation of c-Fms Expression and Nuclear Factor-kappa B Activation

BACKGROUND: Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development. METHODS: Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system. RESULTS: Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IkappaBalpha) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions. CONCLUSIONS: Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.

Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways

Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 microg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91(phox), which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.

Involvement of Endoplasmic Reticulum Stress Response in Orofacial Inflammatory Pain

Endoplasmic reticulum (ER) stress is involved in many neurological diseases and inflammatory responses. Inflammatory mediators induce neuronal damage and trigger the neuropathic or inflammatory pain. But there is very little data on the role of the ER stress response in pain mechanisms. In this study, we explored whether the ER stress response is involved in orofacial inflammatory pain by using a complete Freund's adjuvant (CFA)-injected rat model. The thermal pain hypersensitivity increased significantly after CFA injection. We found that the protein and mRNA levels of ER stress response genes, GRP78/Bip and p-eIF2alpha, increased significantly in trigeminal ganglion (TG) of CFA-injected rats compared to control animals. In immunofluorescence analysis, a significant increase of GRP78 and p-eIF2alpha immunopositive neurons was observed in CFA-injected TG compared to control TG. When we administered an ER stress modulator, salubrinal, CFA-induced thermal pain hypersensitivity was temporally reduced. Thus, our study suggests that ER stress responses in TG neurons contribute to CFA-induced inflammatory pain, and may comprise an important molecular mechanism underlying the orofacial inflammatory pain pathway.

Delayed and Prolonged Local Brain Hypothermia Combined with Decompressive Craniectomy: A Novel Therapeutic Strategy That Modulates Glial Dynamics

Hypothermia is considered a useful intervention for limiting pathophysiological changes after brain injury. Local hypothermia is a relatively safe and convenient intervention that circumvents many of the complications associated with systemic hypothermia. However, successful hypothermia treatment requires careful consideration of several factors including its practicality, feasibility, and associated risks. Here, we review the protective effects-and the cellular mechanisms that underlie them-of delayed and prolonged local hypothermia in rodent and canine brain injury models. The data show that the protective effects of therapeutic hypothermia, which mainly result from the modulation of inflammatory glial dynamics, are limited. We argue that decompressive craniectomy can be used to overcome the limitations of local brain hypothermia without causing histological abnormalities or other detrimental effects to the cooled area. Therefore, delayed and prolonged local brain hypothermia at the site of craniectomy is a promising intervention that may prove effective in the clinical setting.

Lipocalin-2 Acts as a Neuroinflammatogen in Lipopolysaccharide-injected Mice

Lipocalin-2 (LCN2) is a key mediator of various cellular processes. Recent studies have indicated that LCN2 also plays an important role in central nervous system (CNS) injuries and neurological diseases, such as spinal cord injury, stroke, experimental autoimmune encephalomyelitis, and neurodegenerative diseases. Here, we investigated the role of LCN2 in a rodent model of lipopolysaccharide (LPS)-induced neuroinflammation. At 24 hours after intraperitoneal injection of LPS, LCN2 expression was strongly induced in the brain; LCN2 was mainly expressed in endothelial cells, astrocytes, and microglia. Next, we used LCN2-deficient mice to further investigate the role of LCN2 in neuroinflammation. LCN2 deficiency attenuated LPS-induced glial activation in the brain. In a mechanistic study employing glia/neuron co-cultures, LCN2 deficiency reduced glial neurotoxicity. Our results indicate that LCN2 plays a central role in the neuroinflammatory responses following LPS administration, and that LCN2 might contribute to the uncontrolled neurotoxic glial activation under excessive and chronic inflammatory conditions.

Pyruvate Dehydrogenase Kinase as a Potential Therapeutic Target for Malignant Gliomas

Metabolic aberrations in the form of altered flux through key metabolic pathways are the major hallmarks of several life-threatening malignancies including malignant gliomas. These adaptations play an important role in the enhancement of the survival and proliferation of gliomas at the expense of the surrounding normal/healthy tissues. Recent studies in the field of neurooncology have directly targeted the altered metabolic pathways of malignant tumor cells for the development of anti-cancer drugs. Aerobic glycolysis due to elevated production of lactate from pyruvate regardless of oxygen availability is a common metabolic alteration in most malignancies. Aerobic glycolysis offers survival advantages in addition to generating substrates such as fatty acids, amino acids and nucleotides required for the rapid proliferation of cells. This review outlines the role of pyruvate dehydrogenase kinase (PDK) in gliomas as an inhibitor of pyruvate dehydrogenase that catalyzes the oxidative decarboxylation of pyruvate. An in-depth investigation on the key metabolic enzyme PDK may provide a novel therapeutic approach for the treatment of malignant gliomas.

Acute Phase Protein Lipocalin-2 Is Associated with Formalin-induced Nociception and Pathological Pain

Lipocalin-2 (LCN2) is an acute-phase protein induced by injury, infection, or other inflammatory stimuli. LCN2 binds small hydrophobic ligands and interacts with cell surface receptor to regulate diverse cellular processes. The role of LCN2 as a chemokine inducer in the central nervous system (CNS) has been previously reported. Based on the previous participation of LCN2 in neuroinflammation, we investigated the role of LCN2 in formalin-induced nociception and pathological pain. Formalin-induced nociceptive behaviors (licking/biting) and spinal microglial activation were significantly reduced in the second or late phase of the formalin test in Lcn2 knockout mice. Likewise, antibody-mediated neutralization of spinal LCN2 attenuated the mechanical hypersensitivity induced by peripheral nerve injury in mice. Taken together, our results suggest that LCN2 can be therapeutically targeted, presumably for both prevention and reversal of acute inflammatory pain as well as pathological pain.

Apoptosis of Human Islet Cells by Cytokines

FasL, perforin, TNFalpha, IL-1 and NO have been considered as effector molecule(s) leading to beta-cell death in autoimmune diabetes. However, the real culprit(s) of beta-cell destruction have long been elusive despite intense investigation. Previously we have suggested IFNgamma/TNFalpha synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFNgamma and TNFalpha but neither cytokine alone, induced classical caspase-dependent apoptosis in murine insulinoma and pancreatic islet cells. IFNgamma treatment conferred susceptibility to TNFalpha-induced apoptosis on otherwise resistant murine insulinoma cells by STAT1 activation followed by IRF-1 induction. Here we report that IFNgamma/TNFalpha synergism induces apoptosis of human pancreatic islet cells. We also observed STAT1 activation followed by IRF-1 induction by IFNgamma treatment in human islet cells. Taken together, we suggest that IFNgamma/TNFalpha synergism could be involved in human islet cell death in type 1 diabetes, similar to murine type 1 diabetes.

Glia as a Link between Neuroinflammation and Neuropathic Pain

Contemporary studies illustrate that peripheral injuries activate glial components of the peripheral and central cellular circuitry. The subsequent release of glial stressors or activating signals contributes to neuropathic pain and neuroinflammation. Recent studies document the importance of glia in the development and persistence of neuropathic pain and neuroinflammation as a connecting link, thereby focusing attention on the glial pathology as the general underlying factor in essentially all age-related neurodegenerative diseases. There is wide agreement that excessive glial activation is a key process in nervous system disorders involving the release of strong pro-inflammatory cytokines, which can trigger worsening of multiple disease states. This review will briefly discuss the recent findings that have shed light on the molecular and cellular mechanisms of glia as a connecting link between neuropathic pain and neuroinflammation.

Modulation of Glial and Neuronal Migration by Lipocalin-2 in Zebrafish

BACKGROUND: Glial cells are involved in immune and inflammatory responses in the central nervous system (CNS). Glial cells such as microglia and astrocytes also provide structural and functional support for neurons. Migration and morphological changes of CNS cells are associated with their physiological as well as pathological functions. The secreted protein lipocalin-2 (LCN2) has been previously implicated in regulation of diverse cellular processes of glia and neurons, including cell migration and morphology. METHODS: Here, we employed a zebrafish model to analyze the role of LCN2 in CNS cell migration and morphology in vivo. In the first part of this study, we examined the indirect effect of LCN2 on cell migration and morphology of microglia, astrocytes, and neurons cultured in vitro. RESULTS: Conditioned media collected from LCN2-treated astrocytes augmented migration of glia and neurons in the Boyden chamber assay. The conditioned media also increased the number of neuronal processes. Next, in order to further understand the role of LCN2 in the CNS in vivo, LCN2 was ectopically expressed in the zebrafish spinal cord. Expression of exogenous LCN2 modulated neuronal cell migration in the spinal cord of zebrafish embryos, supporting the role of LCN2 as a cell migration regulator in the CNS. CONCLUSION: Thus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.

Cell to Cell Interaction Can Activate Membrane-bound APRIL Which Are Expressed on Inflammatory Macrophages

BACKGROUND: APRIL, originally known as a cytokine involved in B cell survival, is now known to regulate the inflammatory activation of macrophages. Although the signal initiated from APRIL has been demonstrated, its role in cellular activation is still not clear due to the presence of BAFF, a closely related member of TNF superfamily, which share same receptors (TACI and BCMA) with APRIL. METHODS: Through transfection of siRNA, BAFF-deficient THP-1 cells (human macrophage-like cells) were generated and APRIL-mediated inflammatory activities were tested. The expression patterns of APRIL were also tested in vivo. RESULTS: BAFF-deficient THP-1 cells responded to APRIL-stimulating agents such as monoclonal antibody against APRIL and soluble form of TACI or BCMA. Furthermore, co-incubation of the siBAFF-deficient THP-1 cells with a human B cell line (Ramos) resulted in an activation of THP-1 cells which was dependent on interactions between APRIL and TACI/BCMA. Immunohistochemical analysis of human pathologic samples detected the expression of both APRIL and TACI in macrophage-rich areas. Additionally, human macrophage primary culture expressed APRIL on the cell surface. CONCLUSION: These observations indicate that APRIL, which is expressed on macrophages in pathologic tissues with chronic inflammation, may mediate activation signals through its interaction with its counterparts via cell-to-cell interaction.

The Stimulation of CD147 Induces MMP-9 Expression through ERK and NF-kappaB in Macrophages: Implication for Atherosclerosis

BACKGROUND: CD147, as a cellular receptor for cyclophilin A (CypA), is a multifunctional protein involved in tumor invasion, inflammation, tissue remodeling, neural function, and reproduction. Recent observations showing the expression of CD147 in leukocytes indicate that this molecule may have roles in inflammation. METHODS: In order to investigate the role of CD147 and its ligand in the pathogenesis of atherosclerosis, human atherosclerotic plaques were analyzed for the expression pattern of CD147 and CypA. The cellular responses and signaling molecules activated by the stimulation of CD147 were then investigated in the human macrophage cell line, THP-1, which expresses high basal level of CD147 on the cell surface. RESULTS: Staining of both CD147 and CypA was detected in endothelial cell layers facing the lumen and macrophage-rich areas. Stimulation of CD147 with its specific monoclonal antibody induced the expression of matrix metalloproteinase (MMP)-9 in THP-1 cells and it was suppressed by inhibitors of both ERK and NF-kappaB. Accordingly, the stimulation of CD147 was observed to induce phosphorylation of ERK, phosphorylation-associated degradation of IkappaB, and nuclear translocation of NF-kappaB p65 and p50 subunits. CONCLUSION: These results suggest that CD147 mediates the inflammatory activation of macrophages that leads to the induction of MMP-9 expression, which could play a role in the pathogenesis of inflammatory diseases such as atherosclerosis.