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Fibronectin and leucine-rich transmembrane (FLRT) proteins are necessary for various developmental processes and in pathological conditions. FLRT2 acts as a homophilic cell adhesion molecule, a heterophilic repulsive ligand of Unc5/Netrin receptors, and a synaptogenic molecule; the last feature is mediated by binding to latrophilins. Although the function of FLRT2 in regulating cortical migration at the late gestation stage has been analyzed, little is known about the expression pattern of FLRT2 during postnatal central nervous system (CNS) development. In this study, we used Flrt2-LacZ knock-in (KI) mice to analyze FLRT2 expression during CNS development. At the early postnatal stage, FLRT2 expression was largely restricted to several regions of the striatum and deep layers of the cerebral cortex. In adulthood, FLRT2 expression was more prominent in the cerebral cortex, hippocampus, piriform cortex (PIR), nucleus of the lateral olfactory tract (NLOT), and ventral medial nucleus (VM) of the thalamus, but lower in the striatum. Notably, in the hippocampus, FLRT2 expression was confined to the CA1 region and partly localized on pre- and postsynapses whereas only few expression was observed in CA3 and dentate gyrus (DG). Finally, we observed temporally limited FLRT2 upregulation in reactive astrocytes around lesion sites 7 days after thoracic spinal cord injury. These dynamic changes in FLRT2 expression may enable multiple FLRT2 functions, including cell adhesion, repulsion, and synapse formation in different regions during CNS development and after spinal cord injury.

Background Microglial cells are activated in response to changes in brain homeostasis during aging, dementia, and stroke. Type 2 endocannabinoid receptors (CB2) and translocator protein 18 kD (TSPO) are considered to reflect distinct aspects of microglia-related neuroinflammatory responses in the brain. CB2 activation is considered to relate to the neuroprotective responses that may occur predominantly in the early stage of brain disorders such as Alzheimer’s disease, while an increase in TSPO expression tends to occur later during neuroinflammation, in a proinflammatory fashion. However, this information was deduced from studies with different animal samples under different experimental settings. In this study, we aimed to examine the early microglial status in the inflammation occurring in the brains of senescence-accelerated mouse prone 10 (SAMP10) mice, using positron emission tomography (PET) with CB2 and TSPO tracers, together with immunohistochemistry. Methods Five- and 15-week-old SAMP10 mice that undergo neurodegeneration after 7 months of age were used. The binding levels of the TSPO tracer (R)-[11C]PK11195 and CB2 tracer [11C]NE40 were measured using PET in combination with immunohistochemistry for CB2 and TSPO. To our knowledge, this is the first study to report PET data for CB2 and TSPO at the early stage of cognitive impairment in an animal model. Results The standard uptake value ratios (SUVRs) of [11C]NE40 binding were significantly higher than those of (R)-[11C]PK11195 binding in the cerebral cortical region at 15 weeks of age. At 5 weeks of age, the [11C]NE40 SUVR tended to be higher than the (R)-[11C]PK11195 SUVR. The (R)-[11C]PK11195 SUVR did not significantly differ between 5- and 15-week-old mice. Consistently, immunostaining analysis confirmed the upregulation of CB2, but not TSPO. Conclusions The use of the CB2 tracer [11C]NE40 and/or an immunohistochemical approach allows evaluation of the role of microglia in acute neuroinflammatory processes in the early stage of neurodegeneration. The present results provide in vivo evidence of different responses of two types of microglia to senescence-accelerated neuroinflammation, implying the perturbation of microglial balance by aging. Specific treatment for CB2-positive microglia might help ameliorate senescence-related neuroinflammation and the following neurodegeneration.

Osteoclasts are multinucleated giant cells derived from myeloid progenitors. Excessive bone resorption by osteoclasts can result in serious clinical outcomes for which better treatment options are needed. Here, we identified fibronectin leucine-rich transmembrane protein 2 (Flrt2), a ligand of the Unc5 receptor family for neurons, as a novel target associated with the late/maturation stage of osteoclast differentiation. Flrt2 expression is induced by stimulation with receptor activator of nuclear factor-kB ligand (RANKL). Flrt2 deficiency in osteoclasts results in reduced hyper-multinucleation, which could be restored by RNAi-mediated knockdown of Unc5b. Treatment with Netrin1, another ligand of Unc5b which negatively controls osteoclast multinucleation through down regulation of RANKL-induced Rac1 activation, showed no inhibitory effects on Flrt2-deficient cells. In addition, RANKL-induced Rac1 activation was attenuated in Flrt2-deficient cells. Taken together, these results suggest that Flrt2 regulates osteoclast multinucleation by interfering with Netrin 1-Unc5b interaction and may be a suitable therapeutic target for diseases associated with bone remodeling. [BMB Reports 2019; 52(8): 514-519].

BMP6, a member of the TGF-β superfamily, is known to be involved in many diseases, such as Alzheimer's disease, suggesting that BMP6 plays pivotal roles in the central nervous system (CNS), however, there's no information about the distribution of BMP6 in the adult CNS. Therefore, we investigated BMP6 expression in the CNS using immunohistochemistry. BMP6 was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes, astrocytes and ependymal cells also express BMP6 protein. These data indicate that BMP6 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that BMP6 plays important roles in the adult brain.

In mammals, excitatory cortical neurons develop from the proliferative epithelium and progenitor cells in the ventricular zone and subventricular zone, and migrate radially to the cortical plate, whereas inhibitory GABAergic interneurons are born in the ganglionic eminence and migrate tangentially. The migration of newly born cortical neurons is tightly regulated by both extracellular and intracellular signaling to ensure proper positioning and projections. Non-cell-autonomous extracellular molecules, such as growth factors, axon guidance molecules, extracellular matrix, and other ligands, play a role in cortical migration, either by acting as attractants or repellents. In this article, we review the guidance molecules that act as cell–cell recognition molecules for the regulation of neuronal migration, with a focus on netrin family proteins, their receptors, and related molecules, including neogenin, repulsive guidance molecules (RGMs), Down syndrome cell adhesion molecule (DSCAM), fibronectin leucine-rich repeat transmembrane proteins (FLRTs), and draxin. Netrin proteins induce attractive and repulsive signals depending on their receptors. For example, binding of netrin-1 to deleted in colorectal cancer (DCC), possibly together with Unc5, repels migrating GABAergic neurons from the ventricular zone of the ganglionic eminence, whereas binding to α3β1 integrin promotes cortical interneuron migration. Human genetic disorders associated with these and related guidance molecules, such as congenital mirror movements, schizophrenia, and bipolar disorder, are also discussed.

Bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2 (GDF2), is a member of the transforming growth factor β (TGF β) superfamily. Although BMP9 plays pivotal roles during development, including angiogenesis, hematopoiesis, hepatogenesis, osteogenesis, and glucose metabolism, little information is available for BMP9 expression in the central nervous system (CNS). We, thus, investigated BMP9 expression in the adult rat CNS using immunohistochemistry. BMP9 was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes and ependymal cells also express BMP9 protein. These data indicate that BMP9 is widely expressed throughout the adult CNS, and this abundant expression strongly supports the idea that BMP9 also plays important roles in the adult brain.

In the adult mouse brain, neurogenesis mainly occurs in the ventricular-subventricular zone (V-SVZ) and the subgranular zone of the hippocampal dentate gyrus. Neuroblasts generated in the V-SVZ migrate to the olfactory bulb via the rostral migratory stream in response to guidance molecules, such as netrin-1. We previously showed that the related netrin-5 (NTN5) is expressed in Mash1-positive transit-amplifying cells and doublecortin-positive neuroblasts in the granule cell layer of the olfactory bulb, the rostral migratory stream, and the subgranular zone of the adult mouse brain. However, the precise role of NTN5 in adult neurogenesis has not been investigated. In this study, we show that neurogenic proliferation is impaired in NTN5 knockout mice. The number of proliferated (EdU-labeled) cells in the NTN5 KO mice was significantly lower in the V-SVZ, whereas the number of Ki67-positive proliferating cells were unchanged, suggesting that longer cell cycle and decreased cell division in NTN5 KO mice. The number of EdU-labeled cells in the rostral migratory stream (RMS) and olfactory bulb were unchanged. By contrast, the number of EdU-labeled cells in the cortex, basal ganglia/lateral septal nucleus, and corpus callosum/anterior commissure was increased, which largely represented oligodendrocyte lineage cells. Lastly, we found that the chain-migration in the RMS of NTN5 KO mice was disorganized. These findings suggest that NTN5 may play important roles in promoting neurogenic proliferation in the V-SVZ, organizing proper chain-migration in the RMS and suppressing oligodendrogenesis in the brain.

Introduction Many researchers have assumed that neurovascular compression of the facial nerve at the site covered by central myelin sheath causes hemifacial spasm. However, some cases do not correspond to this hypothesis. The aim of this study was to clarify the myelin histology in the facial nerve. Materials and methods Histological analyses were conducted on 134 facial nerves from 67 cadavers. Three dimensions were measured in these sections: the length from the upper border of the medullopontine sulcus to the boundary between the central and peripheral myelin sheath along the anterior side; the length from the detachment point of the brain stem to the boundary along the posterior side; and the length of the transitional zone (TZ), known as the Obersteiner-Redlich zone. Results Of the 134 facial nerves, 41 were available for study. The length of the central myelin segment ranged from 4.62 to 12.6 mm (mean 8.06 mm; median 7.98 mm) along the anterior side and from 0.00 to 4.58 mm (mean 1.68 mm; median 1.42 mm) along the posterior side of the facial nerve, and the length of the TZ ranged from 0.00 to 2.76 mm (mean 1.51 mm; median 1.42 mm). Conclusions In this study, the length of the central myelin segment in the facial nerve was found to be longer than that previously reported.

Bone morphogenetic protein-7 (BMP7), a member of the transforming growth factor-β (TGF-β) superfamily, has various effects in many biological events. However, there is little information on BMP7 expression in the adult central nervous system (CNS). Therefore, we investigated BMP7 levels in the adult rat CNS using immunohistochemistry. Abundant BMP7 expression was seen in astrocytes throughout the CNS and strong BMP7 expression was also observed in neuropils of the gray matter. Furthermore, BMP7 expression was observed in several kinds of neurons, including oxytocin, dopaminergic and noradrenergic neurons. These data suggest that BMP7 is widely expressed throughout the adult CNS, and support the idea that BMP7 plays pivotal roles in the adult brain, as well as in the developing brain., Highlights • BMP7 is expressed throughout the adult CNS, and abundantly expressed in astrocytes. • BMP7 is also expressed in some kinds of neurons and axons.

Primary sensory neurons are usually situated in ganglia outside the brain, while the mesencephalic nucleus of the trigeminal nerve (Me5) is situated inside the brain. However, it remains unknown why only Me5 situated inside the brain is. The neurons of Me5 are the cell bodies of primary afferent fibers concerned with the muscles of mastication and periodontal receptors of both maxillary and mandibular teeth. Interestingly, there was no Me5 till the evolution level of the agnatha, vertebrates which lack jaws, while Me5 appeared with the evolution of jawed vertebrates, the gnathostomes. Thus, I speculate that the appearance of jaws necessitated the emergence of a novel sensory system including newly-made primary sensory neurons to co-ordinate jaw movement and this need was met by the appearance of Me5. Although primary sensory neurons are usually generated from the neural crest or the neurogenic placodes, primary sensory neurons in Me5 are derived from neuroepithelium of the dorsal midline of the midbrain. Taken together, I propose the following hypothesis; (1) Me5 did not exist till the evolution level of agnatha, which lacks jaw. (2) When jawed vertebrates evolved, a new sensory system including new primary sensory neurons for mastication was needed. (3) At that point, there was no capacity for the neural crest and neurogenic placodes to make primary sensory neurons. (4) However, there remained capacity only for the neuroepithelium of the midbrain to make primary sensory neurons. (5) Thus, Me5 was newly made inside the CNS.

Follistatin was initially cloned as a monomeric polypeptide that inhibits the release of follicle-stimulating hormone. Although follistatin also plays pivotal roles in skeletal muscle hypertrophy and immunoregulation in the epididymis, little information is available regarding follistatin function in the adult central nervous system (CNS). Hence, we investigated follistatin expression in the adult rat CNS using immunohistochemistry. Follistatin was intensely expressed in most neurons and their axons. Furthermore, oligodendrocytes, ependymal cells, and some astrocytes also expressed follistatin protein. These data indicate that follistatin is widely expressed throughout the adult CNS. The abundant expression of follistatin in the adult brain suggests that this protein plays important roles in the CNS.

Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Glycine is an important amino acid in the central nervous system. The aberrant conditions of glycine concentrations cause sever neurological disorders, such as nonketotic-hyperglycinemia (NKH), also known as glycine encephalopathy. Therefore, a better understanding of its relative abundance and distribution in the developing and adult brains would provide insights into the pathogeneses of this kind of disorders. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging has been used for direct molecular-specific compound detection, distribution mapping, and identifying molecular species in tissue sections. Although a few reports have already shown the imaging of glycine using MALDI-MS in the adult mouse brain, they lack detailed neuroanatomical and developmental evaluations. We, thus, investigated the detailed distribution and abundance of glycine not only in the adult mouse brain but also in the developing mouse brain using this technique. In both brains, we detected derivatized glycine throughout the mouse brain. Interestingly, in both brains, derivatized glycine was abundantly detected in the brain stem. The other areas showed relatively lower signal intensities. As many model mice are used for glycine-related diseases, MALDI-MS is a suitable technique to analyze the pathogenesis of these diseases.

Rhododendrol is a phenolic compound that shows a tyrosinase-dependent toxicity for melanocytes and occasionally induces a vitiligo-like skin depigmentation. The post-tyrosinase mechanisms determining melanocyte death or survival, however, are far from clear. Here, we find that rhododendrol treatment leads to a reduction in the levels of cellular glutathione but also induces a cellular antioxidant response that eventually increases glutathione levels. We further find that rhododendrol toxicity is enhanced when glutathione levels are experimentally reduced and alleviated when glutathione levels are increased. Hence, it appears that the size of the preexisting glutathione pool along with the capacity to supply glutathione via the antioxidant response determines whether melanocytes survive or die after rhododendrol exposure. It is conceivable, therefore, that rhododendrol-induced leukoderma depends on the capacity to maintain appropriate glutathione levels and that enhancement of glutathione levels may preserve a patient's melanocytes and potentially help in repigmentation.

Bone morphogenetic protein-3 (BMP3) is a very unique member of the TGF-β superfamily, because it functions as an antagonist to both the canonical BMP and activin pathways and plays important roles in multiple biological events. Although BMP3 expression has been described in the early development of the kidney, intestine and bone, little information is available for BMP3 expression in the central nervous system (CNS). We, thus, investigated BMP3 expression in the adult rat CNS using immunohistochemistry. BMP3 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express BMP3 protein. These data indicate that BMP3 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that BMP3 plays important roles in the adult brain.

Vasa vasorum (VV) and lymphatic vasa vasorum (LVV) form their own networks in the adventitia. VV supply the aorta with nutrition and oxygen; however, the distribution and role of LVV remains to be determined. The purpose of this study was to investigate differences in the distribution of VV and LVV along the aorta.Aortic samples were obtained from 22 autopsy cases without medical history of aortic diseases. Aortic segments were classified as arch (Ar), descending thoracic (De), suprarenal abdominal (S-Ab), and infrarenal abdominal (I-Ab). Adventitial VV and LVV were identified immunohistochemically.VV were most dense in the arch aorta, becoming less dense along the aorta in more distal segments, with the lowest density occurring in the infrarenal abdominal aorta. There was a significant correlation between the numbers of VV and medial thickness in the total aortic segments (r = 0.518, p0.01). In contrast, there was no significant correlation between the number of LVV and medial thickness in any aortic segments. However, there was a significant correlation between the number of LVV and intimal thickness in I-Ab (r = 0.425, p0.05).The distributions of adventitial VV and LVV were characteristic along the aortic segments. Differences in the distributions may imply the prevalence of aortic diseases such as dissection, abdominal aortic aneurysm, and atherosclerotic occlusive disease in each aortic segment.

Several studies have suggested that vascular compression of more distal portions of the trigeminal nerve (Vth cranial nerve: VN) may cause trigeminal neuralgia (TN). However, neurosurgeons performing microvascular decompression intraoperatively cannot identify which type of myelin is being compressed by blood vessels. The aim of this study was to clarify the histological anatomy of central and peripheral myelin in the human VN. Histological analyses were conducted using photomicrographs from 134 cisternal segments of the VN from the brains of 67 cadavers. The three dimensions of the VN were measured in these sections: distance from the point at which the lateral-most pontine VN merges with the boundary between central and peripheral myelin (line-a), distance along the medial aspect (line-b), and the length of the transitional zone (TZ), known as the Obersteiner-Redlich zone. Twenty-nine of 134 VNs were available for study. The length of central myelin ranged from 0.69 to 8.66 mm (mean, 3.56 mm; median, 3.10 mm) along the lateral aspect and from 0.36 to 5 mm (mean, 1.81 mm; median, 1.40 mm) along the medial aspect of the VN. The length of the TZ ranged from 0.31 to 3.37 mm (mean, 1.75 mm; median, 1.63 mm). We report here, for the first time, that some individuals had much longer spans of central myelin than those reported previously. Some cases of TN may thus be caused by vascular compression of VN peripheral myelin, especially in cases where central myelin is extended to an unprecedented degree. Clin. Anat. 32:541-545, 2019. © 2019 Wiley Periodicals, Inc.

Myostatin (also called as growth and differentiation factor 8 or GDF8), a member of the transforming growth factor β (TGF-β) superfamily of secreted differentiation and growth factors, is a potent inhibitor of skeletal muscle mass in mammals. Although myostatin also plays pivotal roles in cardiac growth and metabolism, postnatal glucose metabolism and adipogenesis, little information is available for myostatin function in the adult central nervous system (CNS). We, thus, investigated myostatin expression in the adult rat CNS using immunohistochemistry. Myostatin was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes, astrocytes and ependymal cells also express myostatin protein. These data indicate that myostatin is widely expressed throughout the adult CNS, and its abundant expression in the adult brain suggests the idea that myostatin plays important roles in the CNS.

Glycine is an important amino acid in the central nervous system. Interestingly, the content of glycine is about 9 times higher in the spinal cord grey matter than in the telencephalon. And this kind of caudal to rostral gradient is never seen in any other neurotransmitters. However, the cause of this phenomenon remains unknown. In the present report, I, thus, postulate the following theory. Glycine has dual roles as a neurotransmitter, one is the agonist for inhibitory glycine receptors (GlyRs), and the other is a co-agonist for excitatory NMDA receptors (NMDARs). Inhibitory GlyRs are concentrated in the lower brain and the affinity of glycine to GlyRs is low, leading to the high content of glycine in the lower brain. In contrast, in the upper brain, there are little glycinergic neurons and the affinity of glycine to NMDARs is very high, leading to the low content of glycine in the forebrain. These different roles of glycine as a neurotransmitter between in the upper brain and in the lower brain make this steep caudal-rostral gradient in glycine content.

Bone morphogenetic protein-5 (BMP5), a member of the transforming growth factor-β (TGF-β) superfamily, has many effects in several biological events. Although BMP5 expression has been well reported in the early development of the central nervous system (CNS), there is little information about its expression in the adult CNS. Thus, we analyzed BMP5 expression in the adult rat CNS by immunohistochemistry. Abundant BMP5 expression was observed in most neurons, and their dendrites and axons. Furthermore, strong BMP5 expression was also detected in the neuropil of the gray matters with high plasticity, such as the molecular layer of the cerebellum, locus coeruleus, and nucleus of the solitary tract. In addition, we showed BMP5 expression also in astrocytes, ependymal cells and meninges. Our data suggest that BMP5 is widely expressed throughout the adult CNS, and this abundant expression in the adult brain strongly supports the idea that BMP5 plays important roles not only in the developing brain but also in the adult brain.

We investigated the expressions of bone morphogenetic protein-4 (BMP4) and its receptors, bone morphogenetic protein receptor IA (BMPRIA), bone morphogenetic protein receptor IB (BMPRIB) and bone morphogenetic protein receptor II (BMPRII) in the adult rat eye. Interesting differences in expression profile were observed between BMPRIA and BMPRIB in the retina. BMPRIA-like immunoreactivity (IR) was very intensely seen in the photoreceptor layer, while BMPRIB-IR was mainly observed in the other layers. In the cornea, BMP4, BMPRIA, BMPRIB and BMPRII-IRs were abundantly seen in the cell body of basal cells in the corneal epithelium, and endothelium. In the lens, BMP4, BMPRIA, BMPRIB and BMPRII-IRs were observed in epithelial cells, lens cortical fiber cells, however they were not seen in the capsule and the central region of the lens. In the iris and ciliary body, strong BMP4 and BMPRIB-IRs were observed in nonpigmented epithelium. These results suggest that different kinds of BMP signaling should be needed in different areas in the adult eye to keep the shapes, differentiation levels, and functions of various cells.

Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a member of the transforming growth factor β (TGF-β) superfamily. Although GDF11 plays pivotal roles during development, including anterior/posterior patterning, formation of the kidney, stomach, spleen and endocrine pancreas, little information is available for GDF11 expression in the adult central nervous system (CNS). We, thus, investigated GDF11 expression in the adult rat CNS using immunohistochemistry. GDF11 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express GDF11 protein. These data indicate that GDF11 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that GDF11 plays important roles in the adult brain.

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.

Rhododendrol, an inhibitor of melanin synthesis developed for lightening/whitening cosmetics, was recently reported to induce a depigmentary disorder principally at the sites of repeated chemical contact. Rhododendrol competitively inhibited mushroom tyrosinase and served as a good substrate, while it also showed cytotoxicity against cultured human melanocytes at high concentrations sufficient for inhibiting tyrosinase. The cytotoxicity was abolished by phenylthiourea, a chelator of the copper ions at the active site, and by specific knockdown of tyrosinase with siRNA. Hence, the cytotoxicity appeared to be triggered by the enzymatic conversion of rhododendrol to active product(s). No reactive oxygen species were detected in the treated melanocytes, but up-regulation of the CCAAT-enhancer-binding protein homologous protein gene responsible for apoptosis and/or autophagy and caspase-3 activation were found to be tyrosinase dependent. These results suggest that a tyrosinase-dependent accumulation of ER stress and/or activation of the apoptotic pathway may contribute to the melanocyte cytotoxicity.

Bone morphogenetic proteins (BMPs) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators. Chordin is an extracellular BMP antagonist that binds BMP-2, 4, and 7 with high affinity and thus interferes with binding to BMP receptors. Although chordin expression has been well described in the early development of the CNS, little information is available for its expression in the adult CNS. We, thus, investigated chordin expression in the adult rat CNS using immunohistochemistry. Chordin was intensely expressed in most neurons, and their dendrites and axons. In addition, abundant chordin expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes and ependymal cells also express chordin protein. These data indicate that chordin is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that chordin plays pivotal roles also in the adult brain.

Bone morphogenetic proteins (BMP) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators, such as chordin and noggin. Although the deep involvement of BMP signaling in the development and functions of the trigeminal nuclei has been postulated, little information is available for its expression in the trigeminal nuclei. We, thus, investigated chordin and noggin expression in the adult rat trigeminal nuclei using immunohistochemistry. Chordin and noggin were intensely expressed throughout the trigeminal nuclei. In addition, interesting differences are observed between chordin expression and noggin expression. For example, chordin prefers dendritic expression than noggin, suggesting that chordin is involved in the regulation of dendritic morphology and synaptic homeostasis. Furthermore, chordin and noggin were differentially expressed in the neuropil of the trigeminal nuclei. Since BMP signaling is known to play a pivotal role to make precise neural network, theses differences might be important to keep precise interneuronal connections by regulating local BMP signaling intensity in each region. Interestingly, we also detected chordin and noggin expression in axons of the trigeminal nerves. These data indicate that chordin and noggin play pivotal roles also in the adult trigeminal system.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily. BMPs, such as BMP2 and BMP4, exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators such as noggin. Although BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII expressions have been well described in the central nervous system, little information is available for their expressions in the spinal cord. We, thus, investigated these protein expressions in the adult rat spinal cord using immunohistochemistry. Here, we show that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII are widely and differentially expressed in the spinal cord. Besides abundant BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII protein expressions in neurons, we detected them also in astrocytes, oligodendrocytes, and ependymal cells. In addition, we found BMPRIA, BMPRIB, and BMPRII protein expressions in microglia. Interestingly, we also observed that these proteins are strongly expressed in many kinds of axons in both ascending and descending tracts. These data indicate that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII proteins are more widely expressed in the adult spinal cord than previously reported, and their continued abundant expressions in the adult spinal cord strongly support the idea that BMP signaling plays pivotal roles in the adult spinal cord.

Bone morphogenetic proteins (BMP) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators. Noggin is an extracellular BMP antagonist that binds BMP-2/4 with high affinity and thus interferes with binding to BMP receptors. Although noggin expression has been well described in the early development of the CNS, little information is available on its expression in the adult CNS. We, thus, investigated noggin expression in the adult rat CNS using immunohistochemistry. Noggin was intensely expressed in most neurons, and their axons. In addition, strong noggin expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes and ependymal cells also express noggin protein. These data indicate that noggin is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that noggin plays pivotal roles also in the adult brain.

The mineralocorticoid receptor has been implicated in the pathogenesis of chronic cardiorenal disease. Statins improve renal remodeling and dysfunction in patients with proteinuric kidney diseases. We aimed to clarify the beneficial effects and mechanisms of action of statins in renal insufficiency.Dahl salt-sensitive rats fed a high-salt diet were treated from 12 to 20 weeks of age with vehicle, the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin, the synthetic cathepsin inhibitor E64d, or a low or high dosage of pitavastatin (1 or 3 mg/kg daily). Rats fed a low-salt diet served as controls. Rats on the high-salt diet developed massive proteinuria and glomerulosclerosis; these changes were attenuated by both doses of pitavastatin. The amounts of mRNAs or proteins for mineralocorticoid receptor, angiotensin-converting enzyme, angiotensin II type 1 receptor (AT1R), monocyte chemoattractant protein-1, osteopontin, macrophage infiltration, and NADPH subunits (gp91phox, p22phox, and Rac1) were significantly higher in the failing kidneys of vehicle-treated rats than in the kidneys of control rats. Either dose of pitavastatin significantly attenuated these changes. These effects of pitavastatin were mimicked by those of apocynin and E64d. Pretreatment with pitavastatin and apocynin inhibited mRNA and protein of mineralocorticoid receptor induced by angiotensin II in cultured podocytes.The beneficial effects of pitavastatin are likely attributable, at least in part, to attenuation of the mineralocorticoid receptor-dependent inflammatory mediator, matrix protein, and cathepsin expressions induced by AT1R-mediated NADPH oxidase activation in the kidneys of a salt-induced hypertensive Dahl salt-sensitive rat model.

CLP36, a member of the α-actinin-associated LIM protein (ALP)/enigma protein family, plays a role in neurite outgrowth in the peripheral nervous system. However, the underlying molecular mechanisms are not known. In this study, we performed yeast two-hybrid screening of an E18 mouse whole-body cDNA library with CLP36 as the bait and isolated palladin as a CLP36-binding protein. Palladin is an actin-binding protein and it was shown to have a role in the extension of cortical neurons. A coimmunoprecipitation study showed that CLP36 and palladin formed a complex in the dorsal root ganglion (DRG). In addition, CLP36 and palladin were colocalized in the neurites and cell bodies of primary DRG neurons. Furthermore, sciatic nerve transection increased the expression of both CLP36 and palladin mRNAs in DRG neurons, with the increase in CLP36 mRNA being more prominent. This implies that CLP36 has a more specific role in nerve regeneration than palladin. Our results suggest that CLP36 may interact with palladin to influence neurite outgrowth during sciatic nerve regeneration.

Although it has been demonstrated that statins stabilize atherosclerotic lesions in animal models of advanced atherosclerosis, there is little evidence to suggest that statins have a preventive effect on plaque rupture itself. In the present study, we examined the effect of fluvastatin on plaque disruption using a simple and quick method of plaque disruption in carotid artery lesions in apolipoprotein E-deficient mice. Male apolipoprotein E-deficient mice received normal chow and underwent ligation of the left common carotid artery just proximal to its bifurcation. Four weeks later, a polyethylene cuff was placed around the artery immediately proximal to the ligation site. Fluvastatin (10 mg/kg per day) was given by oral gavage every day starting at 3 days before cuff placement. The administration of fluvastatin suppressed atherosclerotic plaque disruption accompanied by luminal thrombi by 31.5% compared with controls at 4 days after the cuff was placed at the ligated carotid artery. Fluvastatin administration decreased matrix metalloproteinase-9 expression, gelatinolytic activity, endothelial adhesion molecules expression and neutrophil infiltration, and increased type I collagen content in the cuffed region. In summary, fluvastatin was found to prevent plaque disruption through pleiotropic effect on acute inflammation in an animal model using apolipoprotein E-deficient mice.

Phosphatidylcholines (PCs) are the most abundant constituents of lipid in the brain. PCs function as major structural components of cell membranes and as important sources for signaling molecules. In the brain, three kinds of PCs, dipalmitoyl PC, palmitoyloleoyl PC, and stearoyloleoyl PC have been reported to be major species. They have different chemical and biological characteristics depending on the length of alkyl chains and the degree of saturation, suggesting that the abundance of PCs might be important to keep specialized membrane structures in the brain, such as myelin and synaptic membranes. However, detailed imaging of PCs in the total rat brain has not done yet. Thus, using imaging technology by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), we investigated the total distribution of PC32:0, PC34:1, and PC36:1 in the rat brain. PC32:0 and PC34:1 were more abundantly observed in the gray matter areas than in the white matter areas throughout the central nervous system (CNS), while PC36:1 was evenly seen at low levels in both areas. In addition, we found that PC32:0 and PC34:1 were detected at very high levels in the granular layer of the olfactory bulb, piriform cortex, insular cortex, and molecular layer of the cerebellum, which are known for areas showing high neuronal plasticity. The present imaging data clearly show that various PCs are differentially distributed throughout the rat CNS, and suggest that these differential distributions of various PCs are necessary to keep normal brain functions.

The aortic wall is perfused by the adventitial vasa vasorum (VV). Tissue hypoxia has previously been observed as a manifestation of enlarged abdominal aortic aneurysms (AAAs). We sought to determine whether hypoperfusion of the adventitial VV could develop AAAs. We created a novel animal model of adventitial VV hypoperfusion with a combination of a polyurethane catheter insertion and a suture ligation of the infrarenal abdominal aorta in rats. VV hypoperfusion caused tissue hypoxia and developed infrarenal AAA, which had similar morphological and pathological characteristics to human AAA. In human AAA tissue, the adventitial VV were stenotic in both small AAAs (30-49 mm in diameter) and in large AAAs (> 50 mm in diameter), with the sac tissue in these AAAs being ischemic and hypoxic. These results indicate that hypoperfusion of adventitial VV has critical effects on the development of infrarenal AAA.

The pathophysiology underlying abdominal aortic aneurysms (AAAs) remains unknown. In this study, we applied imaging mass spectrometry (IMS) to analyze the pathophysiology of the aneurysmal wall. Comparisons were performed between the tissue samples from the neck and the sac of the AAA, at a single time point, in 30 patients who underwent elective surgery of their AAAs. The localization of each lipid molecule in the aortic wall was assessed by IMS. Histopathological examination and IMS revealed a characteristic distribution of triglycerides (TGs) specifically in the aneurismal adventitia of the sac. This characteristic TG distribution was derived from an ectopic appearance of adipocytes in the adventitia. Furthermore, ectopic adipocyte accumulation in the aortic wall leads to the loss of the collagen fiber network subsequent to the wall rupture. The underlying mechanism of adipocyte accumulation involves the presence of adipose-derived stem cells (ADSCs) in the aneurismal adventitia and the expression of peroxisome proliferator-activated receptor gamma 2, a master regulator of adipocyte differentiation by some ADSCs. This study reveals new, previously overlooked aspects of AAA pathology.

Bone morphogenetic protein-4 (BMP4) is a member of the transforming growth factor-beta (TGF-beta) superfamily and plays important roles in multiple biological events. Although BMP4 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. Therefore, we investigated BMP4 expression in the adult rat CNS by using immunohistochemistry. BMP4 is intensely expressed in most neurons and their dendrites. In addition, intense BMP4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes also express BMP4 protein. These data indicate that BMP4 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP4 plays pivotal roles also in the adult brain.

Olfactory receptors are G-protein coupled receptors and are encoded by an extremely large and diverse family of genes in mammals. There is increasing evidence that olfactory receptors are widely distributed in many organs, suggesting that olfactory receptors do not only recognize airborne odorants but also play important roles in chemotaxis or organ construction in embryo. In this study, we investigated whether olfactory receptors and their transduction molecule, Golf are expressed in the rat placenta. By RT-PCR, we identified 11 different olfactory receptor genes, which are all members of class II, in the rat placenta cDNAs, and our results suggested that particular members of the olfactory receptor gene family might be preferentially expressed in the placenta. By western blot analysis, we demonstrated that Golf protein is expressed in the placenta and its expression levels are developmentally regulated. We found that Golf immunoreactivity is exclusively localized to giant cell trophoblasts and spongiotrophoblast cells. These findings raised a possibility that a particular subset of olfactory receptors might be coupled with Golf and function in giant cell trophoblasts and spongiotrophoblast cells.

The rat olfactory bulb is anatomically immature at birth, and considerable neurogenesis and synaptogenesis are known to take place postnatally. In addition, significant physiological changes have also been reported in this period. For example, granule cell-mediated inhibition following electrical stimulations to the lateral olfactory tract is robust during the first postnatal week, and then decreases abruptly after the second week. However, the mechanism underlying this enhanced inhibition remains to be elucidated. To know the cause of this phenomenon, we investigated the expression patterns of cation-Cl(-) co-transporters (KCC1, KCC2 and NKCC1) mRNAs, which are responsible for the regulation of [Cl(-)](i). In addition, responses to gamma-aminobutyric acid (GABA) were measured by gramicidin-perforated patch-clamp recordings and Ca(2+) imaging using fura-2. We found that in the early postnatal period, mitral cells expressing KCC2 mRNA were inhibited by GABA, while granule cells lacking KCC2 mRNA expression were depolarized or excited by GABA. These results indicate that transient GABA-mediated excitation on granule cells might be the main cause of the enhanced inhibition on mitral cells, and suggest that these differential GABA responses between relay and intrinsic neurons play pivotal roles in the early postnatal rat olfactory bulb.

Nogo-A and Nogo-receptor have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We investigated the total distribution of Nogo-A and Nogo-receptor mRNAs in the adult rat central nervous system using in situ hybridization histochemistry. Nogo-A is abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system. Interestingly, we could not find any neuron which lacks Nogo-A mRNA expression, indicating that Nogo-A mRNA is universally expressed in all neurons. In contrast, Nogo-R mRNA expression was very restricted. Nogo-R mRNA was expressed in the olfactory bulb, hippocampus, tentia tecta, some amygdala nuclei, cerebral cortex, some thalamic nuclei, medial habenular, whereas we could not detect it in the other regions. Interestingly, we did not detect Nogo-R mRNA in monoaminergic neurons, which are known to have high regenerative capacity, in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, Nogo-R mRNA was not detected there. These data indicate that Nogo-A and Nogo-R mRNAs were differentially expressed in the central nervous system, and suggest that the lack of Nogo-R expression in a given neuron might be necessary to keep its high regenerative capacity.

A novel model to induce occlusive thrombus formation was developed in mice in vivo. Mice were simultaneously treated with ligation and cuff placement at the left carotid artery. At 7 days after the treatment, occlusive thrombus was observed at the intracuff region, but not in the distal and proximal regions of the cuff, and not induced by a single treatment of ligation or cuff placement. The plasma levels of von Willebrand factor (vWF), which represent the endothelial status, were significantly increased in combined treatment of ligation and cuff placement 1 day after the operation. Whereas no significant changes in plasma vWF were observed in either single treatment of ligation or cuff placement. The expression of vWF, considered to be the endothelial marker, was detected on the luminal surface distal and proximal to the cuff and the carotid artery in the single treatment groups treated with either ligation or cuff placement, but was not detected in the intracuff region. Furthermore, the binding of Griffolia Simplicifolia Lectin-I (GSL-I) and endothelial nitric oxide synthase (eNOS) expression indicating the endothelial integrity was not detected in the intracuff region. Intermittent injections of ancrod, which decreases the plasma fibrinogen, inhibited occlusive thrombus formation in the intracuff region. The expression of eNOS was detected at the distal and proximal but not the intracuff region of the carotid artery treated with ancrod. Daily administration of aspirin significantly suppressed the thrombus formation in this model. These results indicate that occlusive thrombus formation accompanied by endothelial damage or dysfunction is induced by the combined application of ligation and cuff placement at the carotid artery, and suggest that this endothelial damage or dysfunction may be one pathogenesis of thrombogenesis in this model.

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.

The tricarboxylate carrier protein catalyzes an electroneutral exchange across the mitochondrial inner membrane of tricarboxylate, dicarboxylate or phosphoenolpyruvate. We examined expression and localization of mitochondrial tricarboxylate carrier TCC mRNA and protein in the rat brain. TCC mRNA was ubiquitously expressed in all rat tissues examined and was abundant in brain, liver and kidney. TCC protein as well as mRNA was widely expressed in brain, and the protein expression was strong in neuronal cells in the hippocampus, the olfactory bulb, the corpus mamillare and the cerebellum. Our results suggest that this tricarboxylate carrier protein may contribute to biosynthesis and bioenergetics in neuronal cells in brain.

The pathogenesis of abdominal aortic aneurysm (AAA) is characterized to be inflammation-associated degeneration of vascular wall. Neovascularization is regularly found in human AAA and considered to play critical roles in the development and rupture of AAA. However, little is known about lymphangiogenesis in AAA. The purpose of this study was to demonstrate both angiogenesis and lymphangiogenesis in AAA. Abdominal aortic tissue was harvested either from autopsy (control group) and during open-repair surgery for AAA (AAA group). Adventitial lymphatic vasa vasorum was observed in both groups, but seemed to be no significant morphological changes in AAA. Immunohistochemical studies identified infiltration of lymphatic vessel endothelial hyaluronan receptor (LYVE) −1, vascular endothelial growth factor (VEGF)-C, and matrix metalloproteinase (MMP)-9-positive macrophages and podoplanin and Prox-1-positive microvessels in the intima/media in AAA wall, where hypoxia-inducible factors (HIF)-1α was expressed. VEGF-C and MMP-9 were not expressed in macrophages infiltrating in the adventitia. Intraoperative indocyanine green fluorescence lymphography revealed lymph stasis in intima/medial in AAA. Fluorescence microscopy of the collected samples also confirmed the accumulation of lymph in the intima/media but not in adventitia. These results demonstrate that infiltration of macrophages in intima/media is associated with lymphangiogenesis and angiogenesis in AAA. Lymph-drainage appeared to be insufficient in the AAA wall.

The glycine cleavage system (GCS) is a mitochondrial multienzyme system consisting of four individual proteins, three specific components (P-, T-, and H-proteins) and one house-keeping enzyme, dihydrolipoamide dehydrogenase. Inherited deficiency of the GCS causes nonketotic hyperglycinemia (NKH), an inborn error of glycine metabolism. NKH is characterized by massive accumulation of glycine in serum and cerebrospinal fluids and severe neuronal dysfunction in neonates. To elucidate the neuropathogenesis of NKH, we cloned cDNAs encoding three specific components of the GCS and studied the gene expression in rat central nervous system. P-, T-, and H-protein cDNAs encoded 1024, 403, and 170 amino acids, respectively. In situ hybridization analysis revealed that P-protein mRNA was expressed mainly in glial-like cells, including Bergmann glias in the cerebellum, while T- and H-protein mRNAs were detected in both glial-like cells and neurons. T- and H-protein mRNAs, but not P-protein mRNA, were expressed in the spinal cord. Primary astrocyte cultures established from cerebral cortex had higher GCS activities than hepatocytes whereas those from spinal cord expressed only H-protein mRNA and had no enzymatic activity. An important role of glycine as inhibitory neurotransmitter has been established in the brainstem and spinal cord and another role of glycine as an excitation modulator of N-methyl-D-aspartate receptor is suggested in the hippocampus, cerebral cortex, olfactory bulbus, and cerebellum. Our results suggest that the GCS plays a major role in the forebrain and cerebellum rather than in the spinal cord, and that N-methyl-D-aspartate receptor may participate in neuropathogenesis of NKH.

Glutamate is the main neurotransmitter in the olfactory bulb. Therefore, glutamate transporters, which regulate the concentration of extracellular glutamate, might play pivotal roles in odor processing. In this study, we examined expressions of three glutamate transporters (GLAST, GLT1 and EAAC1) in the olfactory bulb using in situ hybridization and immunohistochemistry. EAAC1 mRNA was expressed in neurons, such as periglomerular cells, tufted cells, mitral cells and granule cells as shown before in other brain areas. In contrast, GLAST and GLT1 were found in glial cells throughout the olfactory bulb, with intenser expressions in the glomerular layer, external plexiform layer and internal plexiform layer where glutamatergic synapses are concentrated. In addition, using double staining immunohistochemistry we clearly showed that GLAST and GLT1 were expressed in astrocytes. Furthermore, we found that GLAST was also intensely expressed in the subependymal layer where precursor cells exist. These results suggest each glutamate transporter plays its unique role not only in glutamatergic neurotransmission but also in cell differentiation and migration in the olfactory bulb.

Erythropoietin (EPO) prevents the ischemia-induced delayed neuronal death in the hippocampal CA1 field in gerbils. EPO receptor (EPOR) is also expressed in the cerebral cortex but its function is not known. To examine whether EPO has a neuroprotective action in the cortex, EPO was infused into the cerebroventricles of stroke-prone spontaneously hypertensive rats with permanent occlusion of the left middle cerebral artery. Morris water maze test indicated that EPO infusion alleviated the ischemia-induced place navigation disability. The left (ischemic)-to-right (contralateral nonischemic) (L/R) ratio of cerebrocortical area in the EPO-infused ischemic group was larger than that in the vehicle-infused ischemic group. The occlusion caused secondary thalamic degeneration but infusion of EPO prevented the decrease in the L/R ratio of thalamic area and supported neuron survival in the ventroposterior thalamic nucleus. In situ hybridization indicated that EPOR mRNA was upregulated in the periphery (ischemic penumbra) of a cerebrocortical infarct after occlusion of the middle cerebral artery, suggesting that an increased number of EPOR in neurons facilitates the EPO signal transmission, thereby preventing the damaged area from enlarging.

Using radioactive in situ hybridization histochemistry, we examined the topographical patterns of expression of the messenger RNA encoding a glutamate-binding protein ( N -methyl- d -aspartate receptor glutamate-binding protein in rat; NMDARgbs) in the central nervous system of the rat. Expression patterns of N -methyl- d -aspartate receptor glutamate-binding protein were compared with those of N -methyl- d -aspartate receptor subunit 1 (NMDAR1) of the N -methyl- d -aspartate receptor on adjacent sections. N -methyl- d -aspartate receptor glutamate-binding protein is not expressed in glial cells. The expression of both N -methyl- d -aspartate receptor glutamate-binding protein and N -methyl- d -aspartate receptor subunit 1 was observed in virtually all neurons throughout the central nervous system. The mean level of N -methyl- d -aspartate receptor subunut 1 expression was higher than that of N -methyl- d -aspartate receptor glutamate-binding protein. Similar topographical patterns of expression of N -methyl- d -aspartate receptor glutamate-binding protein and N -methyl- d -aspartate receptor were observed in most regions, except in discrete thalamic, hypothalamic and brainstem nuclei. Concomitantly for N -methyl- d -aspartate receptor glutamate-binding protein and N -methyl- d -aspartate receptor subunit 1, the highest expression levels were distributed in the mitral layer of main and accessory olfactory bulbs, granule cell layer of the dentate gyrus, polymorphic and pyramidal layers of CA1–3 fields of Ammon's horn. A slightly less prominent expression was observed in the glomerular and granule cell layers of main and accessory olfactory bulbs, anterior olfactory nucleus, layer 2 of piriform cortex, olfactory tubercle and taenia tecta. In the cerebellum, the prominent level of N -methyl- d -aspartate receptor glutamate-binding protein expression was slightly higher in the Purkinje cell layer than in the granule cell layer, an opposite pattern being observed for N -methyl- d -aspartate receptor subunit 1. A moderately high expression level of both messenger RNAs was observed in the medial septal nucleus, nucleus of the diagonal band of Broca, dorsal part of the endopiriform nucleus, and in the anteroventral and anterolateral parts of the bed nucleus of the stria terminalis. In the neocortex, the mean expression level of N -methyl- d -aspartate receptor glutamate-binding protein is moderate, while the mean level of N -methyl- d -aspartate receptor subunit 1 expression is high. With both probes, layer IV is slightly less labeled than the other layers. In layer V, N -methyl- d -aspartate receptor subunit 1 expression is slightly lower than in layers II and III, whereas N -methyl- d -aspartate receptor glutamate-binding protein expression is slightly higher. A moderately high intensity of signal obtained with both probes was observed in the anterodorsal thalamic nucleus, medial habenular nucleus, in the magnocellular, ventromedial and lateral mammillary nuclei of the hypothalamus, and in the pars compacta of the substantia nigra, red nucleus, pontine nuclei, tegmental and lateral reticular nuclei, oculomotor and motor trigeminal nuclei, nucleus of the solitary tract, hypoglossal nucleus, dorsal raphe´, nucleus of the trapezoid body, external cuneate nucleus and motorneurons of the ventral horn. Weak to moderate levels of expression were observed with both probes in the other regions of the central nervous system. In the hypothalamus, a high expression level of N -methyl- d -aspartate receptor subunit 1 contrasted with a low expression level for N -methyl- d -aspartate receptor glutamate-binding protein in the suprachiasmatic, arcuate and tuberomammillary nuclei. Conversely, as a unique feature in the rat central nervous system, a moderately high level of expression of N -methyl- d -aspartate receptor glutamate-binding protein and a low expression level of N -methyl- d -aspartate receptor subunit 1 were observed in the parafascicular nucleus of the thalamus. These findings show that the N -methyl- d -aspartate receptor glutamate-binding protein is expressed throughout the rat central nervous system, and that the topographical patterns of expression of this protein are closely similar but not identical to those of N -methyl- d -aspartate receptor. The high level of expression of both N -methyl- d -aspartate receptor glutamate-binding protein and N -methyl- d -aspartate receptor subunit 1 in brain areas that display high sensitivity to N -methyl- d -aspartate is consistent with the involvement of N -methyl- d -aspartate receptor glutamate-binding protein in mechanisms of N -methyl- d -aspartate receptor-mediated excitotoxicity.

Although trigeminal neuropathic pain is one of the most common chronic pain syndromes, the etiology is still unknown. Here, a rat model was generated using chronic constrictive injury (CCI) with ligation of the infraorbital nerve to test the hypothesis that collapse of chloride homeostasis in trigeminal neurons causes impairment of γ-aminobutyric acid-ergic (GABAergic) inhibition and induces trigeminal allodynia. Rats showed a reduction and increase in pain threshold and pain response scores, respectively, to mechanical stimulation, 1 and 3weeks after CCI. In situ hybridization and immunohistochemical analysis showed that inward-directed Na(+), K(+)-2Cl(-) cotransporter (NKCC1) mRNA and protein were upregulated in the small-sized and large-sized primary neurons in the injured side of the trigeminal ganglion and in the peripherin-positive terminal, respectively, for the first 2weeks, while outward-directed K(+)-Cl(-) cotransporter (KCC2) mRNA and protein were downregulated in secondary relay neurons on the injured side of the spinal trigeminal nucleus caudalis (Sp5C). Optical imaging of evoked synaptic responses using a voltage-sensitive dye revealed that pre- and post-synaptic GABA actions were disinhibited and excitatory in the injured side, respectively, but inhibited in the sham-operated side of the Sp5C. This downregulation of KCC2 in the Sp5C may result in an excitatory switch by impairing postsynaptic GABA inhibition. GABA-mediated presynaptic disinhibition was attenuated by bumetanide, suggesting that NKCC1 upregulation in primary neurons may facilitate pain transmission by presynaptic GABAergic depolarization. Such Cl(-) homeostatic disruption resulting in perturbation of the inhibitory system possibly increases pain transmission, which may underlie the pathophysiology of trigeminal neuropathic pain.

Abdominal aortic aneurysm (AAA) is a common disease among elderly individuals. However, the precise pathophysiology of AAA remains unknown. In AAA, an intraluminal thrombus prevents luminal perfusion of oxygen, allowing only the adventitial vaso vasorum (VV) to deliver oxygen and nutrients to the aortic wall. In this study, we examined changes in the adventitial VV wall in AAA to clarify the histopathological mechanisms underlying AAA. We found marked intimal hyperplasia of the adventitial VV in the AAA sac; further, immunohistological studies revealed proliferation of smooth muscle cells, which caused luminal stenosis of the VV. We also found decreased HemeB signals in the aortic wall of the sac as compared with those in the aortic wall of the neck region in AAA. The stenosis of adventitial VV in the AAA sac and the malperfusion of the aortic wall observed in the present study are new aspects of AAA pathology that are expected to enhance our understanding of this disease.

Objective Matrix metalloproteinases (MMPs) have been implicated in the process of vascular calcification. However, the exact roles of individual MMPs in vascular calcification are poorly understood. To study the putative role of MMP-2 in atherogenic calcification in vivo and in vitro , we investigate whether or not MMP-2 deficiency affects aortic atherosclerotic calcification in apolipoprotein E-deficient (Apoe −/− ) mice and cultured smooth muscle cell (SMC) calcification. Methods and results The area of calcified lesions in aortic intima was significantly larger in MMP-2 +/+ Apoe −/− mice at 45 and 60 weeks of age than in MMP-2 −/− Apoe −/− mice. In these aortic calcified atherosclerotic lesions, the expression of type II collagen, osteoprotegerin, bone morphogenetic protein (BMP)-2 and osteocalcin which are chondrocyte maker and bone-related proteins, was observed. MMP-2 deficiency reduced BMP-2 and osteocalcin expression in aortae in Apoe −/− mice at 30 weeks and 45–60-weeks-old, respectively. The expressions of MMP-2 and that of α-SMC actin were observed in chondrocyte-like cells of calcified lesions. Beta-glycerophosphate administration induced calcium deposition in MMP-2 +/+ aorta-derived cultured SMCs, and this calcium deposition was significantly suppressed in MMP-2 −/− aorta-derived cultured SMCs. Conclusions These results suggest that MMP-2 may contribute to the mechanisms of calcification associated with atherosclerosis.