Takahiro Katayama

Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor α in mice

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors which belong to the nuclear receptor family. We examined whether PPARalpha agonists and resveratrol, a polyphenol contained in grapes, protect the brain against ischemia. To investigate whether resveratrol activates PPARs, we performed a cell-based transfection activity assay using luciferase reporter plasmid. PPARalpha and PPARgamma were activated by resveratrol in primary cortical cultures and vascular endothelial cells. Resveratrol (20 mg/kg, 3 days) reduced infarct volume by 36% at 24 h after middle cerebral artery occlusion in wild-type mice. The PPARalpha agonists fenofibrate (30 mg/kg, 3 days) and Wy-14643 (30 mg/kg, days) exerted similar brain protection. However, resveratrol and fenofibrate failed to protect the brain in PPARalpha knockout mice. The data indicate that PPARalpha agonists protect the brain through PPARalpha.

Activation of the β-Adrenoceptor–Protein Kinase A Signaling Pathway within the Ventral Bed Nucleus of the Stria Terminalis Mediates the Negative Affective Component of Pain in Rats

Pain is an unpleasant sensory and emotional experience. The neural systems underlying the sensory component of pain have been studied extensively, but we are only beginning to understand those underlying its affective component. The bed nucleus of the stria terminalis (BNST) has been implicated in stress responses and negative affective states, such as anxiety, fear, and aversion. Recently, we demonstrated the crucial role of the BNST in the negative affective component of pain using the conditioned place aversion (CPA) test. In the present study, we investigated the involvement of the β-adrenoceptor–protein kinase A (PKA) signaling pathway within the BNST, in particular, within the ventral part of the BNST (vBNST), in pain-induced aversion in male Sprague Dawley rats. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST were significantly increased by intraplantar formalin injection. Using the CPA test, we found that intra-vBNST injection of timolol, a β-adrenoceptor antagonist, dose-dependently attenuated the intraplantar-formalin-induced CPA (F-CPA) without reducing nociceptive behaviors. Experiments with subtype-selective antagonists demonstrated the essential role of β2-adrenoceptors in F-CPA. Intra-vBNST injection of isoproterenol, a β-adrenoceptor agonist, dose-dependently produced CPA even in the absence of noxious stimulation. This isoproterenol-induced CPA was reversed by the coinjection of Rp-cyclic adenosine monophosphorothioate (Rp-cAMPS), a selective PKA inhibitor. Furthermore, intra-vBNST injection of Rp-cAMPS dose-dependently attenuated the F-CPA. Together, these results suggest that PKA activation within the vBNST via the enhancement of β-adrenergic transmission is important for the negative affective component of pain.

Mesenchymal stem cells transmigrate across brain microvascular endothelial cell monolayers through transiently formed inter-endothelial gaps.

Mesenchymal stem cells (MSCs) hold much promise for cell therapy for neurological diseases such as cerebral ischemia and Parkinsons disease. Intravenously administered MSCs accumulate in lesions within the brain parenchyma, but little is known of the details of MSC transmigration across the blood-brain barrier (BBB). To study MSC transmigration across the BBB, we developed an in vitro culture system consisting of rat brain microvascular endothelial cells (BMECs) and bone marrow-derived MSCs using Transwell or Millicell culture inserts. Using this system, we first investigated the influence of the number of MSCs added to the upper chamber on BMEC barrier integrity. The addition of MSCs at a density of 1.5 × 10⁵ cells/cm² led to disruption of the BMEC monolayer structure and decreased barrier function as measured by the transendothelial electrical resistance (TEER). When applied at a density of 1.5 × 10⁴ cells/cm², neither remarkable disruption of the BMEC monolayers nor a significant decrease in TEER was observed until at least 12 h. After cultivation for 24 h under this condition, MSCs were found in the subendothelial space or beneath the insert membrane, suggesting that MSCs transmigrate across BMEC monolayers. Time-lapse imaging revealed that MSCs transmigrated across the BMEC monolayers through transiently formed intercellular gaps between the BMECs. These results show that our in vitro culture system consisting of BMECs and MSCs is useful for investigating the molecular and cellular mechanisms underlying MSC transmigration across the BBB.

TAK-779, a nonpeptide CC chemokine receptor antagonist, protects the brain against focal cerebral ischemia in mice.

The effect of a nonpeptide CC chemokine receptor antagonist, TAK-779, on ischemic brain injury resulting from 1-hour middle cerebral artery occlusion followed by 48-hour reperfusion was examined in ddY mice. On intracerebroventricular injection of vehicle or TAK-779, infarct volume in the vehicle-treated group was 44.2 ± 13.2% of the contralateral hemispheric volume, and TAK-779 (25 and 250 ng/mouse) dose-dependently reduced the infarct volume to 35.0 ± 12.2% and 31.1 ± 12.9%, respectively. On intravenous injection, infarct volume in the vehicle-treated group was 32.0 ± 16.1%, and TAK-779 (5 μg per 20 g body weight) significantly reduced this to 22.0 ± 10.5%. The results showed for the first time that a nonpeptide chemokine receptor antagonist is protective against ischemic brain injury.

Role of enhanced noradrenergic transmission within the ventral bed nucleus of the stria terminalis in visceral pain-induced aversion in rats

Pain is an unpleasant sensory and emotional experience. We demonstrated the crucial role of the bed nucleus of the stria terminalis (BNST) in the negative affective component of somatic and visceral pain induced by intraplantar formalin and intraperitoneal acetic acid injections, respectively, in rats. Recently, we reported the involvement of enhanced noradrenergic transmission via beta-adrenoceptors within the ventral BNST (vBNST) in formalin-induced aversion. Here, we examined the role of intra-vBNST noradrenergic transmission in the negative affective component of visceral pain induced by intraperitoneal acetic acid injection. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST significantly increased after intraperitoneal acetic acid injection. Using a conditioned place aversion (CPA) test, we found that intra-vBNST injection of timolol, a beta-adrenoceptor antagonist, dose-dependently attenuated the acetic acid-induced CPA without reducing nociceptive behaviors. These results suggest that enhanced noradrenergic transmission via beta-adrenoceptors within the vBNST plays a pivotal role in the negative affective, but not sensory, component of visceral pain.

ATP induces leukemia inhibitory factor mRNA in cultured rat astrocytes

Leukemia inhibitory factor (LIF) is a cytokine involved in the survival and differentiation of the neural cells in the central and peripheral nervous systems. In the present study, we examined the effects of various neurotransmitter receptor agonists on LIF mRNA expression in cultured rat astrocytes, microglia and neurons to elucidate the cell types producing LIF and to clarify the neurotransmitter(s) regulating the mRNA expression. The results demonstrated that the expression of LIF mRNA was intensely induced by ATP in the cultured astrocytes. Experiments using ATP, UTP and related compounds showed the involvement of P2Y2 and P2Y4 purinoceptors in the expression induced by ATP.

Accumulating Microglia Phagocytose Injured Neurons in Hippocampal Slice Cultures : Involvement of p38 MAP Kinase

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.

Excitotoxic injury induces production of monocyte chemoattractant protein-1 in rat cortico-striatal slice cultures

The effect of excitotoxic injury on the production of monocyte chemoattractant protein-1 (MCP-1) was examined in rat cortico-striatal slice cultures. Treatment with 50 microM N-methyl-D-aspartate (NMDA) for 4 h, which caused severe damage in neurons, induced the production of MCP-1 in astrocytes. Production levels were markedly elevated immediately after the treatment, peaked at 4-8 h, and had decreased to nearly the basal level by 72 h. Since the treatment promoted the release of MCP-1 in the slice cultures, but not in the enriched astrocyte cultures, it is unlikely that NMDA directly acted on the astrocytes. These results suggest that information on neuronal injury induced by NMDA is transmitted to astrocytes to induce the production of MCP-1. Organotypic slice cultures are useful for investigating the inflammatory responses of astrocytes in the process of neuronal injury.

Cyclopropane-based conformational restriction of GABA by a stereochemical diversity-oriented strategy: identification of an efficient lead for potent inhibitors of GABA transports.

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC50=23.9μM) and betaine-GABA transporter1 (5.48μM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.

Kainic acid induces leukemia inhibitory factor mRNA expression in the rat brain: differences in the time course of mRNA expression between the dentate gyrus and hippocampal CA1/CA3 subfields.

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which affects the survival and differentiation of various types of cells both in the hematopoietic and nervous systems. In this study, the time course and localization of LIF mRNA expression following kainic acid-induced seizures were examined by northern blot analyses and in situ hybridization. Northern blot analyses demonstrated that intraperitoneal injection of kainic acid at a convulsive dose induced LIF mRNA expression intensely in the hippocampus and moderately to weakly in the cerebral cortex, thalamus and hypothalamus. The expression peaked at 8-24 h after the injection in the hippocampus and cerebral cortex and at 8 h in the thalamus and hypothalamus. In situ hybridization revealed different time courses of LIF mRNA expression depending on the area of the hippocampus; that is, the expression peaked at 10 h in the granule cell layer of the dentate gyrus, then at 12 h in the polymorph and molecular layers of the dentate gyrus, and finally at 12-24 h in the strata oriens and radiatum of the CA1 and CA3 subfields. It is worth noting that the expression of LIF mRNA was intense in the dentate gyrus, the region where neurogenesis and aberrant network reorganization have been shown to be induced by seizures. The upregulation of LIF mRNA expression in the dentate granule cell layer followed by that in the dentate polymorph and molecular layers may be involved in activity-dependent neurogenesis in the granule cell layer and ectopic migration of granule cells to the polymorph and molecular layers in the dentate gyrus.