Proliferation of endothelial cells in the choroid plexus of normal and hydrocephalic mice.

The choroid plexus (CP), located at the walls of the brain ventricles, produces and secretes cerebrospinal fluid (CSF). Hydrocephalus is a neurological disorder in which the CP abnormally secretes excess amounts of CSF into the ventricles. There is currently no information on the vascular dynamics of the CP in adult brains under normal and hydrocephalic conditions. In the present study, we reported the continuous proliferation of endothelial cells in the CP of normal mice, which depended on vascular endothelial cell growth factor (VEGF). The proliferation of endothelial cells increased in mice with intraventricular hemorrhage, which was attenuated by a pretreatment with the toll-like receptor 4 (TLR4) inhibitor VIPER. Moreover, the intracerebroventricular infusion of the TLR4 agonist, lipopolysaccharide, increased endothelial cell proliferation in the CP and induced ventriculomegaly. The present results provide insights into the importance of the TLR4-initiated and VEGF-dependent proliferation of endothelial cells in the pathogenesis of hydrocephalus.

Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression and hypothermia in the adult mouse.

Toll-like receptor 2 (TLR2) recognizes a wide range of microbial molecules and plays critical roles in the initiation of innate immune responses. In the present study, we aimed to investigate whether the depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) attenuates the activation of mouse brain circuits for TLR2-mediated inflammation and hypothermia. The peripheral administration of the TLR2 agonist zymosan induced nuclear factor-κB activation in microglia and macrophages and Fos expression in astrocytes/tanycytes and neurons in the circumventricular organs (CVOs). The depletion of microglia and macrophages with Clod-Lips markedly decreased zymosan-induced Fos expression in astrocytes/tanycytes and neurons in the CVOs. The treatment with Clod-Lips significantly attenuated zymosan-induced hypothermia. These results indicate that microglia and macrophages in the CVOs participate in the initiation and transmission of inflammatory responses after the peripheral administration of zymosan.

Activation of microglia and macrophages in the circumventricular organs of the mouse brain during TLR2-induced fever and sickness responses.

Toll-like receptor 2 (TLR2) recognizes cell wall components from Gram-positive bacteria. Until now, however, little has been known about the significance of brain TLR2 in controlling inflammation and thermoregulatory responses during systemic Gram-positive bacterial infection. In the present study, the TLR2 immunoreactivity was seen to be prominent in the microglia/macrophages of the circumventricular organs (CVOs) of the mouse brain. The intraperitoneal injection of Pam3CSK4, a TLR2 agonist, induced nuclear factor-κ B activation in the microglia/macrophages of the CVOs. The injection of Pam3CSK4 also produced the expression of Fos at astrocytes and neurons in the CVOs and the regions neighboring the CVOs. The Pam3CSK4 injection induced fever and sickness responses. Pretreatment with lipopolysaccharide, a TLR4 agonist, augmented the Pam3CSK4-induced fever together with the increased TLR2 immunoreactivity. These results indicate that the TLR2 in microglia/macrophages of the CVOs are possibly associated with initiating and transmitting inflammatory responses in the brain.

VEGF- and PDGF-dependent proliferation of oligodendrocyte progenitor cells in the medulla oblongata after LPC-induced focal demyelination.

The myelin sheath is critical in maintaining normal functions of the adult central nervous system (CNS) and the loss of the myelin sheath results in various neurological diseases. Although remyelination is the intrinsic repair system against demyelination that new myelin sheath is formed around axons in the adult CNS, little has been reported on remyelination system in the medulla oblongata. In the present study, we showed that the proliferation of oligodendrocyte progenitor cells (OPCs) was increased in the medulla oblongata by lysophosphatidylcholine (LPC)-induced focal demyelination, but that of NSCs was not changed. The inhibition of vascular endothelial growth factor (VEGF)- and platelet-derived growth factor (PDGF)-signaling suppressed the proliferation of OPCs by LPC-induced demyelination. Thus, the present study indicates that resident OPCs contribute to focal remyelination and VEGF and PDGF signaling is required for the proliferation of OPCs in the medulla oblongata of the adult mouse.

Microglia are continuously activated in the circumventricular organs of mouse brain.

Microglia are the primary resident immune cells of the brain parenchyma and transform into the amoeboid form in the "activated state" under pathological conditions from the ramified form in the "resting state" under physiologically healthy conditions. In the present study, we found that microglia in the circumventricular organs (CVOs) of adult mice displayed the amoeboid form with fewer branched cellular processes even under normal conditions; however, those in other brain regions showed the ramified form, which is characterized by well-branched and dendritic cellular processes. Moreover, microglia in the CVOs showed the strong protein expression of the M1 markers CD16/32 and CD86 and M2 markers CD206 and Ym1 without any pathological stimulation. Thus, the present results indicate that microglia in the CVOs of adult mice are morphologically and functionally activated under normal conditions, possibly due to the specialized features of the CVOs, namely, the entry of blood-derived molecules into parenchyma through fenestrated capillaries and the presence of neural stem cells.

TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic pathways in the mouse brain.

Toll-like receptor 4 (TLR4) recognizes bacteria-derived lipopolysaccharide (LPS). In the present study, we found that intraperitoneal LPS activated nuclear factor-κ B (NF-κB) in TLR4-expressing neural stem cells (NSCs) in the circumventricular brain regions of mice. Intracerebroventricular preadministration of low-dose TLR4 inhibitors significantly augmented hyperthermia together with the inhibition of NF-κB activation in circumventricular NSCs of LPS-inflamed animals. Moreover, intracerebroventricular administration of high-dose TLR4 inhibitors induced hyperthermia and Fos activation in circumventricular NSCs and hypothalamic neurons. These results suggest that TLR4 on circumventricular NSCs functions as a central regulator for thermogenesis under inflamed and normal conditions.

Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1.

The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu). Immunofluorescence microscopy with an anti-EF-Tu polyclonal antibody showed that Colpoda EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with Congo red, which is bound to the stacked ß-sheets of amyloid protofibrils. In the presence of puromycin, no cyst wall components including lepidosomes were formed, indicating that cyst wall formation requires synthesis of proteins including EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.

Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse.

Although evidence has accumulated that neurogenesis and gliogenesis occur in the subventricular zone (SVZ) and subgranular zone (SGZ) of adult mammalian brains, recent studies indicate the presence of neural stem cells (NSCs) in adult brains, particularly the circumventricular regions. In the present study, we aimed to determine characterization of NSCs and their progenitor cells in the sensory circumventricular organs (CVOs), including organum vasculosum of the lamina terminalis, subfornical organ, and area postrema of adult mouse. There were two types of NSCs: tanycyte-like ependymal cells and astrocyte-like cells. Astrocyte-like NSCs proliferated slowly and oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) actively divided. Molecular marker protein expression of NSCs and their progenitor cells were similar to those reported in the SVZ and SGZ, except that astrocyte-like NSCs expressed S100ß. These circumventricular NSCs possessed the capacity to give rise to oligodendrocytes and sparse numbers of neurons and astrocytes in the sensory CVOs and adjacent brain regions. The inhibition of vascular endothelial growth factor (VEGF) signaling by using a VEGF receptor-associated tyrosine kinase inhibitor AZD2171 largely suppressed basal proliferation of OPCs. A single systemic administration of lipopolysaccharide attenuated proliferation of OPCs and induced remarkable proliferation of microglia. The present study indicates that sensory circumventricular NSCs provide new neurons and glial cells in the sensory CVOs and adjacent brain regions.

Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

Structural characterization and subcellular localization of Drosophila organic solute carrier partner 1.

BACKGROUND: Organic solute carrier partner 1 (OSCP1) is known to facilitate the transport of various organic solutes into cells and reported to play a role in cell growth and cell differentiation. Moreover, OSCP1 is known as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas and acute myeloid leukemia. However, the underlying mechanisms of action remain unclear and the subcellular localization of OSCP1 has yet to be determined in detail. RESULTS: Drosophila contains a single orthologue of OSCP1 (dOSCP1) that shares 58% homology with its human counterpart. To study the expression pattern and subcellular localization of dOSCP1, we prepared a specific antibody. Subcellular localization analyses of dOSCP1 with these revealed localization in the plasma membrane, endoplasmic reticulum, Golgi apparatus and mitochondria, but no detection in cytosol. dOSCP1 signals were also detected in the nucleus, although at weaker intensity than in plasma membranes and subcellular organelles. In addition, native polyacrylamide gel electrophoresis analysis with and without ß-mercaptoethanol treatment revealed that recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 could also be detected by Western immunoblot analyses in third instar larval extracts. CONCLUSIONS: The data revealed that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells, providing clues to understanding the functions of dOSCP1 during Drosophila development.

VEGF-dependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice.

Hypothalamo-neurohypophysial system (HNS) releases arginine vasopressin (AVP) and oxytocin (OXT) from axonal terminals of the neurohypophysis (NH) into blood circulation for controlling body fluid homeostasis and lactation. Chronic osmotic and suckling stimulations have been shown to cause neurovascular and neuroglial reconstruction in the NH of adult mammals and no study has been reported for vascular dynamics. The aim of this study was to elucidate the occurrence of continuous angiogenesis and growth factor-dependent neurovascular reconstruction in the NH of adult mice. Active proliferation of endothelial cells and oligodendrocyte progenitor cells (OPCs) was observed using the immunohistochemistry of bromodeoxyuridine and Ki-67. Vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2 (KDR)) were highly expressed at pituicytes and endothelial cells respectively. Moreover, prominent expression of platelet-derived growth factor B (PDGFB) and PDGF receptor beta was observed at OXT-containing axonal terminals and pericytes respectively. Administration of the selective tyrosine kinase inhibitor AZD2171 for VEGFRs and STI571 for PDGFRs significantly decreased proliferation of endothelial cells and OPCs. Moreover, AZD2171 treatment decreased vascular density by facilitating apoptosis of endothelial cells and the withdrawal of its treatment led to remarkable rebound proliferation of endothelial cells, so that vascular density rapidly returned to normal levels. AZD2171 decreased the density of both AVP- and OXT-containing axonal terminals, whereas STI571 selectively decreased the density of AVP-containing ones. Thus, this study demonstrates that the signaling pathways of VEGF and PDGF are crucial mediators for determining proliferation of endothelial cells and OPCs and the density of AVP- and OXT-containing axonal terminals in the HNS.

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation.

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.

Lack of both α2-antiplasmin and plasminogen activator inhibitor type-1 induces high IgE production.

AIMS: We investigated the pathophysiological changes in mice lacking α2-antiplasmin (α2-AP) and plasminogen activator inhibitor type-1 (PAI-1) genes, and elucidated the involvement of these inhibitors for fibrinolysis in immune response. MAIN METHODS: The pathophysiological changes induced by a lack of both α2-AP and PAI-1 were investigated using double knockout (KO) mice. The lung, liver, kidney and spleen tissues from α2-AP/PAI-1-double KO mice were compared with those from wild-type (WT) mice. Furthermore, the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, and then the effects of the transplantation were studied. KEY FINDINGS: Plasma IgE levels in the α2-AP/PAI-1-double KO mice increased with age and exceeded 1000 ng/mL after 6 months of age. The plasma cells that produced IgE were detected in perivascular assembled lymphocytes. In the α2-AP/PAI-1-double KO mice, perivascular lymphocyte infiltration was observed in the lung, liver, and kidneys and peribronchial lymphocyte infiltration was present in the lung. When the bone marrow cells from α2-AP/PAI-1-double KO mice were transplanted into 10-Gy X ray irradiated WT mice, the phenotypes of the recipients were similar to those of α2-AP/PAI-1-double KO mice. SIGNIFICANCE: The simultaneous expression of both the α2-AP and PAI-1 genes contributes to the maintenance of immunological functions that are related to IgE. Moreover, it is suggested that both α2-AP and PAI-1 are involved in the recruitment of lymphocytes in the peripheral tissues.

Tumor sólido pseudopapilar do pâncreas (Tumor de Frantz): estudo retrospectivo e revisão da literatura / Tumor solid pseudopapilary of the pancreas Frantz Tumor: retrospective study and literature review

Introdução: o Tumor de Frantz ou tumor sólido pseudopapilar do pâncreas (TSPPP) foi primeiramente descrito em 1959. O escopo do presente estudo é relatar quatro casos e revisar a literatura afim. Métodos: estudo retrospectivo de quatro casos de TSPPP, operados e acompanhados de novembro de 2002 a março de 2010 e revisão da literatura relacionada. Resultados: todas as pacientes eram do sexo feminino, idade de 20 a 33 anos, com predomínio de queixas inespecíficas; 50% dos tumores foram encontrados no processo uncinado. O tamanho das lesões variou de 6,0 a 14,0cm em seu maior eixo. Foram realizadas duas enucleações, uma duodenopancreatectomia e uma pancreatectomia corporal com anastomose pancreatojejunal. O seguimento clínico das quatro pacientes não evidenciou sinais clínicos ou radiológicos de recidiva tumoral. O TSPPP é composto por células relativamente monomórficas que expressam marcadores epiteliais, mesenquimais e endócrinos. O tumor apresenta distribuição aleatória no pâncreas, sem predileção por topografia específica. Discussão: o TSPPP é um tumor raro, que tem sido mais descrito nas últimas décadas. Apresenta agressividade local, com baixo grau de malignidade, e tende a um prognóstico favorável após tratamento cirúrgico. Acredita-se que sua origem envolve um fator hormonal, e as alterações genéticas a ele relacionadas diferem daquelas dos adenocarcinomas de pâncreas.

Introduction: Frantzs Tumor or solid pseudopapillary tumor of the pancreas (SPPTP) was first described in 1959. The aim of this study is to present four cases of SPPTP and a review of the literature. Methods: retrospective analysis of four cases of SPPTP, which were operated and then followed from November 2002 to March 2010, and review of the related literatures. Results: all patients were female, aged from 20 to 33 years, with predominantly unspecific symptoms. 50% of the tumors were located on the uncinated process. Size ranged from 6,0 to 14,0cm. There were performed two enucleations, one pancreaticoduodenectomy and one body pancreatectomy with pancreato-jejunal anastomosis. Follow up of all four patients has not shown any clinical or radiologic signs of tumoral recurrence. SPPTP are composed of mainly monomorphic cells, which express ephitelial, mesenquimal and endocrine markers. The tumor presents a random distribution, without preference for a specific topography. Discussion: SPPTP is a rare tumor, which has been more described in the last decades. It is locally aggressive, has a low degree of malignancy, and tends to a favorable prognosis after surgical treatment. There?s supposed to be an hormonal factor, and its genetic alterations differ from those of pancreatic adenocarcinoma.

Tissue plasminogen activator and plasminogen are critical for osmotic homeostasis by regulating vasopressin secretion.

Systemic osmotic homeostasis is regulated mainly by neuroendocrine system of arginine-vasopressin (AVP) in mammalians. In the present study, we demonstrated that the immunoreactivity of tissue plasminogen activator (tPA) was observed specifically at neurosecretory granules of AVP-positive magnocellular terminals and that of plasminogen was seen at astrocytes in the neurohypophysis (NH). Both tPA and plasminogen knockout (KO) mice revealed higher plasma osmolarity upon water deprivation, a chronic osmotic stimulation, as compared with their wild-type (WT) animals, indicating abnormal osmotic control in these KO mice. tPA KO mice but not plasminogen ones revealed lower ability in secreting AVP into the blood circulation upon an acute osmotic stimulation. Both tPA and plasminogen KO animals showed lower ability in secreting AVP into the blood circulation upon a chronic osmotic stimulation. The recombinant tPA was able to promote the release of AVP from isolated NH. Chronic osmotic stimulation decreased the laminin expression level of neurohypophysial microvessel in WT mice but not in plasminogen KO ones. We suggest that AVP secretion is critically regulated by tPA-dependent facilitation of AVP release from terminals and plasminogen-dependent increase of AVP permeability across microvessels possibly via laminin degradation.

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori.

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.

Polarized targeting of IgLON cell adhesion molecule OBCAM to dendrites in cultured neurons.

Opioid-binding cell adhesion molecule (OBCAM) belongs to the immunoglobulin superfamily CAMs and shows a dendritically polarized distribution in hypothalamic magnocellular neurons. In the present study, the cellular localization of OBCAM was monitored in cultured cortical and hippocampal neurons to examine its polarized distribution. Double labeling immunofluorescence microscopy after fixation showed only faint OBCAM immunoreactivity in the neuronal somata during the early stages of culture, whereas the immunoreactivity was strong in MAP2-positive somata and dendrites of fully polarized neurons after longer culture. Moreover, the immunoreactivity for OBCAM showed a punctate pattern in the dendrites similar to the immunostaining pattern of synapsin I. High resolution revealed close apposition with only a partial overlap of synapsin I and OBCAM immunoreactivities, suggesting the synaptic localization of OBCAM to the dendrites. When the fully polarized neurons were reacted with anti-OBCAM antibody before fixation, OBCAM immunoreactivity became stronger on the dendritic surface than the somatic surface. Extracellular immunoreactivity was eliminated with phosphatidylinositol-specific phospholipase C and this immunoreactivity resisted extraction with the nonionic detergent Triton X-100 at 4 degrees C, indicating that OBCAM is attached to the rafts via a glycosylphosphatidyl inositol anchor. These results indicate that OBCAM is efficiently targeted to the dendritic surface of fully polarized cortical and hippocampal neurons. OBCAM is, hence, concluded to be a dendrite-associated CAM in cortical and hippocampal neurons as in hypothalamic magnocellular neurons.